Soybean Event FG72 Petition - Revised Page 1 of 198

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## Petition for the Determination of No n-regulated Status for Event FG72 OECD Unique Identifier MST-FGØ72-2

The undersigned submits this petition under 7 CFR 340.6 to request that the
Administrator make a determination that the article should not be Regulated under 7 CFR 340.

Submitted by:

Isabelle S. Coats, Ph.D. US Registration Manager Bayer CropScience

For:

Bayer CropScience LP
2 T.W. Alexander Dr. Research Triangle Park, NC 27709 Telephone: 919-549-2599
FAX: 919-549-3929

and

M.S. Technologies LLC
103 Avenue D West Point, Iowa 52656

Contributors (BCS): Jayla Allen, Dean Bushey, Jean-Marc Ferullo Martine Freyssinet, Penny Hunst, Princy Jesudason, Donna Mitten, Martha Poe, and Mike See

Contributors (MSTech): William H. Eby, Bruce M. Held, Justin T. Mason and Vaithilingam Sekar



### Submitted: November 20, 2009 (Revised)Soybean Event FG72 Petition - Revised Page 2 of 198

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### COMPANY NAMES

On June 3, 2002, Bayer CropScience was formed by the acquisition of Aventis CropScience by Bayer AG. From that date, Bayer CropScience is the agricultural business unit of Bayer that is engaged in the research, development, and marketing of crop protection, seed technology, turf and ornamentals, professional pest and vector control, and home and garden products.

On December 15, 1999, Aventis S.A. was formed by the completion of the merger between Hoechst AG and Rhône-Poulenc S.A. Aventis CropScience was formed as part of a worldwide merger between Rhone-Poulenc S.A. and Hoechst AG. A portion of that merger created Aventis CropScience Holding S.A. that included interests from Hoechst AG and Schering AG. Hoechst AG and Schering AG were the parent co mpanies of AgrEvo USA Company which were all merged into the Aventis companies.

Some of the activities described in this petition were undertaken before the merger and acquisition. Consequently, the names Aventis CropScience, AgrEvo USA Company, AgrEvo, and Hoechst Schering AgrEvo GmbH may appear throughout this petition.

M.S. Technologies, LLC, is an Iowa limited liability company, with offices at 103 Avenue D, West Point, Iowa 52656, U.S.A. The FG72 transformation event is owned by M.S. Technologies, LLC.

In November of 2007, M.S. Technologies, LLC and Bayer CropScience AG entered into an agreement for the joint development of herbicide tolerant soybeans, including the FG72
transformation event.

Some of the activities described in this report were undertaken in the context of the agreement between Bayer CropScience AG and M.S. Technologies, LLC. For example, some of the field activities, described in this petition were conducted by M.S. Technologies, LLC.Soybean Event FG72 Petition - Revised Page 3 of 198

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### RELEASE OF INFORMATION

The information in this petition is being submitted by Bayer CropScience and M.S. Technologies, LLC for review by the USDA as part of the regulatory process. By submitting this information, Bayer CropScience and M.S. Technologies, LLC do not authorize its release to any third party except to the extent it is request ed under the Freedom of Information Act (FOIA), 5 U.S.C., Section 552 and 7 CFR 1, covering all or some of this information. Except in accordance with FOIA, Bayer CropScience and M.S. Technologies, LLC do not authorize the release, publication or other distribution of this information without Bayer CropScience’s and M.S. Technologies’ prior notice and consent.

©2009 Bayer CropScience and M.S. Technol ogies, LLC. All rights reserved.

This document is property of Bayer CropScience and M.S. Technologies, LLC and is for use by the regulatory authority to which it has been submitted, and only in support of actions requested by Bayer CropScience and M.S. Technologies. All data and in formation herein must not be used, reproduced or disclosed to third parties without the written cons ent of Bayer CropScience and M.S. Technologies, LLC.Soybean Event FG72 Petition - Revised Page 4 of 198

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### CERTIFICATION

The undersigned certifies, that to the best knowledge and belief of the undersigned, this petition includes all information and views on which to base a determination, and that it includes relevant data and information known to the petitioner which is unfavorable to the petition.

Isabelle S. Coats, Ph.D. US Registration Manager

Bayer CropScience
2 T.W. Alexander Dr.
P.O. Box 12014 Research Triangle Park, NC 27709

Phone: 919-549-2599
FAX: 919-549-3929Soybean Event FG72 Petition - Revised Page 5 of 198

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### SUMMARY

Bayer CropScience (BCS) and M.S. Technologies, LLC (MSTech) are submitting a Petition for the Determination of Non-regulated Status under 7 CFR 340 to USDA Animal and Plant Health Inspection Service (APHIS) for double-herbicide- tolerant soybean event FG72 (Event FG72), any progeny, and crosses of this event with other non-regulated soybean lines.

Transformation event FG72 contains the stably integrated 2mepsps gene which confers tolerance to the herbicide glyphosate and the hppdPfW336 gene which confers tolerance to HPPD inhibitors such as the herbicide isoxaflutole (IFT). The genes were introduced into soybean via direct-gene transfer. Southern blot analyses show soybean event FG72 contains two copies of the 2mepsps and hppdPfW336 genes.

The 2mepsps gene was generated by introducing mutations into the wild-type epsps ( wt epsps ) gene from maize, leading to a modified EPSPS prot ein with two amino acid substitutions (2mEPSPS). This modification confers a decre ased binding affinity of the protein for glyphosate, allowing it to maintain sufficient enzymatic activity in the presence of the herbicide. Therefore, the plants expressing the 2mEPSPS protein (encoded by the 2mepsps gene) are tolerant to glyphosate herbicides.

The wild-type (wt) hppd gene was isolated and cloned from Pseudomonas fluorescens. A single amino acid substitution introduced in the wt hppd gene resulted in the modified hppdPf
W336 gene. The expressed protein, HPPD W336, has a molecular weight of 40 kDa and is made of up of 358 amino acids. The modified protein possesses greater than 99.5% homology to the native HPPD protein from P. fluorescens and is tolerant to isoxaflutole (IFT).

Planting double-herbicide-tolerant soybean varieties, containing transformation event FG72, provides growers with new options for weed control using IFT herbicide in combination with a glyphosate herbicide. Glyphosate is widely used in herbicide-tolerant soybean and other agricultural production systems. IFT herbicide offers an alternative weed control option for the soybean grower. IFT controls weeds via a new herbicide mode of action for soybeans that is efficacious against many of the herbicide resist ant weeds currently found in soybean fields. IFT has the flexibility to be applied pre-plant, pr e-emergence, or post emergence to FG72 soybeans.

Event FG72 has been field tested beginning in 2001 in adapted growing regions of the U.S.
These tests have occurred at more than 10 locations under USDA APHIS field release authorizations. Data and results collected from these trials as well as laboratory analyses presented herein demonstrate that FG72 soybeans: 1) exhibit no plant pathogenic properties; 2) are no more likely to become a weed than non-modified soybeans; 3) are unlikely to increase the weediness potential of any other cultivated pl ant or native wild species; 4) do not cause damage to processed agricultural commodities; and 5) are unlikely to harm other organisms that are beneficial to agriculture.Soybean Event FG72 Petition - Revised Page 6 of 198

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### ACRONYMS AND SCIENTIFIC TERMS

ai active ingredient
A acre ADF Acid Detergent Fiber ANOVA Analysis Of Variance APHIS Animal and Plant Health Inspection
Service BCS Bayer CropScience BLASTP Basic Local Alignment Search Tool BLASTx BLAST search of protein databases using a translated nucleotide query
BLOSUM BLOcks SubstitUtion Matrix bp base pairs bu/ac bushels/acre
CAC Codex Alimentarius Commission DAD DDBJ Amino acid sequence Database DDBJ DNA Data Bank of Japan dw Dry weight DNA DeoxyriboNucleic Acid
E. coli Escherichia coli ELISA Enzyme Linked Immunosorbent Assay EMBOSS European Molecular Biology Open
Software Suite EPSPS 5-enolpyruvylshikimate-3-phosphate synthase 2mEPSPS modified 5-enolpyruvylshikimate-3- phosphate synthase 2mepsps modified 5-enolpyruvylshikimate-3- phosphate synthase gene FAO Food and Agriculture Organization of the
United Nations FDA Food and Drug Administration FGENESH Find GENES using Hidden markov
model FIFRA Federal Insecticide Fungicide and
Rodenticide Act FRAC Fractionated Raw Agricultural Commodity fw Fresh weight
INCAP Institution Of Nutrition Of Central
America And Panama

g gram GetORF EMBOSS database for ORFs G. max Glycine max GM Genetically Modified GLY glyphosate HPPD p-hydroxyphenylpyruvate dioxygenase HPPD W336 modified p-hydroxyphenylpyruvate dioxygenase hppdPfW336 modified p-hydroxyphenylpyruvate dioxygenase gene
HRP Horseradish Peroxidase
ID identification IFT isoxaflutole
kDa kiloDalton kg kilogram
L liter
LB Left Border lb pound (1 pound = 0,454 kg) LC/MS Liquid Chromatography/Mass Spectroscopy
LD 50 lethal dose for 50% of animals
LOQ Limit of Quantitation
M million mg milligram
mL milliter μg microgram NA Not Applicable

ng nanogram ND Not Detectable: Below the limit of
detection NDF Neutral Detergent Fiber

nm nanometer
nt nucleotide OECD Organization for Economic Co operation and Development ORF Open Reading Frame P. fluorescens, Pf Pseudomonas fluorescens PCR Polymerase Chain Reaction
PDB Protein DataBase
PIR Protein Identification Resources RAC Raw Agricultural Commodity RB Right Border RCB Randomized complete block RBS Ribosome Binding Site RR Roundup Ready
SD Standard Deviation SDS-PAGE Sodium Dodecyl Sulfate PolyAcrylamide Gel Electrophoresis
SGF Simulated Gastric Fluid
SIF Simulated Intestinal Fluid SIM Selected Ion Monitoring Subsp. Subspecies
T 1 , T 2 , etc generations after T 0 (transformation) T-DNA transfer DNA from Agrobacterium TDN Total Digestible Nutrients
TEP Total Extractable Protein TrEMBL Translated Sequences from the European Molecular Biology Laboratory Nucleotide Sequence Database
US United States of America USDA United States Department of Agriculture WHO World Health Organization wt Wild type Z. mays Zea mays, cornSoybean Event FG72 Petition - Revised Page 7 of 198

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### TABLE OF CONTENTS

TITLE PAGE ..................................................................................................................... ............................ 1
COMPANY NAMES .................................................................................................................. .................... 2
RELEASE OF IN FORMATION ......................................................................................................... ............ 3
CERTIFICATION.................................................................................................................. ......................... 4
SUMMARY........................................................................................................................ ............................ 5
ACRONYMS AND SCIE NTIFIC TERMS.................................................................................................. .... 6
TABLE OF CONTENTS.............................................................................................................. .................. 7
LIST OF TABLES................................................................................................................. ....................... 11
LIST OF FIGURES................................................................................................................ ...................... 13



### I. RATIONALE I.A.Basis for the request for deter mination of non-regu lated status .............................................. 15
I.B.Double-herbicide-tolerant soybean ev ent FG 72....................................................................... 15 I.C.Rationale for the development of event FG72 and benefit s..................................................... 15 I.D.Adoption of event FG72 ..................................................................................................... ...... 16
I.E.Submissions to other regulatory agencies ............................................................................... 17



### II. THE BIOLOGY OF SOYBEAN II.A.Biology of so ybean ........................................................................................................ .......... 18
II.B.Characteristics of t he recipient cultivar................................................................................. ... 18



### III. DEVELOPMENT OF DOUBLE-HERBIC IDE-TOLERANT SO YBEAN EVENT FG72 III.A.Description of the tr ansformation system ............................................................................... 19
III.B.Parent line.............................................................................................................. ................. 19 III.C.Breeding diagram ......................................................................................................... .......... 19
III.D.Generations us ed for analysis............................................................................................ .... 19



### IV. GENETIC MATERIAL USED FOR TRANSFORMATION OF EVENT FG72 IV.A.Construction of the plasmi d used for tran sformation ............................................................. 21 IV.B.Donor genes and associated regulatory region s ................................................................... 22
IV.B.1. The 2mepsps gene expression cassette................................................................. 22 IV.B.2. The hppdPfW336 gene expression ca ssette ......................................................... 22 IV.C. Identity and source of the genetic material ........................................................................... 2 3



### V. GENETIC CHARACTERIZATION OF EVENT FG72

V.A.Overview ................................................................................................................... .............. 25 V.B.Copy number and Insertion.................................................................................................. ... 25
V.C.Absence of vector backbone................................................................................................. .. 25 V.D. The flanking regions of the inserted se quence(s) ................................................................. 27
V.E.Mendelian inheritance ...................................................................................................... ....... 27 V.F.Stability across and within genera tions ................................................................................... 2 8
V.G.Conclusion ................................................................................................................. ............. 29Soybean Event FG72 Petition - Revised Page 8 of 198

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### VI. CHARACTERIZATION OF TH E INTRODUCED PROTEINS

VI.A.The 2mEPSPS protein ....................................................................................................... .... 30
VI.A.1. Histor y and back ground .......................................................................................... 30
VI.A.2. Characterization of the 2mEPSPS protei n .............................................................. 30 VI.A.2.1. Biochemistry and mode of action ............................................................ 30 VI.A.2.2. 2mEPSPS pr otein sa fety......................................................................... 31
VI.B.The HPPD protein .......................................................................................................... ........ 33
VI.B.1. Histor y and back ground .......................................................................................... 33
VI.B.2. The function of the gene product............................................................................. 33 VI.B.2.1. Biochemistry and mode of action ............................................................ 33
VI.B.2.2. Source of the gene.................................................................................. 35 VI.B.2.3. History of safe us e of the source organism............................................. 36 VI.B.2.4. Familiarity of the gene pr oduct................................................................ 38 VI.C.Expression of the 2mEPSPS and HPPD W336 proteins in event FG72............................... 38 VI.C.1. Expressi on in grain.................................................................................................. 3 8 VI.C.2. Expression in plant par ts and during t he life cyc le.................................................. 40
VI.D.Verification of the biochemical and functio nal equivalence of the expressed proteins.......... 42
VI.D.1. 2mE PSPS protein ................................................................................................... 42 VI.D.2. HPPD W336 protein ............................................................................................... 43 VI.E.Summary of the food and feed safety assessment of the 2mEPSPS protein ....................... 44 VI.E.1. Familiarity to the pr otein .......................................................................................... 44
VI.E.2. Potentia l allergenicity .............................................................................................. 4 4 VI.E.2.1. Homology search to known al lergens ..................................................... 44
VI.E.2.2. Potential N-glycosylation sites................................................................. 45
VI.E.2.3. In vitro stability to human simula ted gastric fluid..................................... 45
VI.E.2.4. In vitro stability to human simulat ed intestinal fluid ................................. 46
VI.E.2.5. In vitro stability to heat ............................................................................. 46 VI.E.3. Homology sear ch to know n toxi ns........................................................................... 47 VI.E.4. Acute toxicity study in t he mous e............................................................................ 47 VI.F.Summary of the food and feed safety assessment of the HPPD prot ein............................... 47 VI.F.1. Familiarity to the pr otein .......................................................................................... 47
VI.F.2. Potentia l allergenicity............................................................................................... 48 VI.F.2.1. Homology search to known al lergens ..................................................... 48
VI.F.2.2. Potential N- glycosylation sites................................................................. 48
VI.F.2.3. In vitro stability to human simula ted gastric fluid..................................... 49
VI.F.2.4. In vitro stability to human simulat ed intestinal fluid ................................. 49
VI.F.2.5. In vitro stability to heat ............................................................................. 50 VI.F.3. Homology sear ch to know n toxi ns........................................................................... 51 VI.F.4. Acute toxicity study in t he mous e ............................................................................ 52



### VII. AGRONOMIC AND PHENOTYPIC EVALUATION VII.A.History of fi eld activi ties ............................................................................................. ........... 53 VII.B.Agronomic and phenot ypic eval uation .................................................................................. 55 VII.C.Agronomic performa nce of event FG72................................................................................ 57 VII.C.1. Agronomic evaluati on of event FG72 (2003 data) ............................................... 57
VII.C.1.1.Yield evaluation of event FG72 under
conventional weed co ntrol prac tices....................................................... 57
VII.C.1.2. Intended herbicide toleranc e evaluations of event FG72 ....................... 58 VII.C.2. Agronomic evaluati on of event FG72 (2008 data) ............................................... 59 VII.C.2.1. Agronomic performance and yi eld evaluation of event FG72 under
conventional weed co ntrol prac tices....................................................... 59 VII.C.2.2. Objective variety description .................................................................. 62Soybean Event FG72 Petition - Revised Page 9 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION VII.C.2.3. Seed ch aracteristics............................................................................... 63
VII.D.Biotic and abiotic st ress characteristics ................................................................................ 65 VII.E.Equivalence between Jack-der ived and commercia l varieties ............................................. 65
VII.F.Seed dormanc y evaluation................................................................................................. ... 67 VII.G.Compositi on analysis..................................................................................................... ....... 69
VII.G.1. In troduction ......................................................................................................... ... 69 VII.G.2. Nutritional compos ition of soyb ean grai n............................................................... 69 VII.H.Poultry feeding study .................................................................................................... ........ 75 VII.I.Conclusion for agronomic evaluation of event FG 72 ............................................................. 75



### VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTICES

VIII.A.Environmental assessment of the introduce d protei ns ........................................................ 76
VIII.B.Potential for horizontal or vertical gene transf er .................................................................. 76 VIII.C.Weediness potential of double-herbic ide-tolerant soy bean event FG72............................. 77 VIII.D.Current agronomic practices fo r soy bean............................................................................ 77 VIII.E.Potential impact on agricul tural practices for soybean......................................................... 80
VIII.F.Weed resistan ce managem ent ............................................................................................ 8 1 VIII.G.Potential impact on farming practices.................................................................................. 8 2
VIII.H.Potential effects on non- target organisms, including beneficia l organisms......................... 82
VIII.I.Threatened and endangered species co nsiderat ions ........................................................... 82 VIII.J Potential impac t on biodiversity ........................................................................................ .... 83
VIII.K.Conc lusion.............................................................................................................. .............. 83



### IX. STATEMENT OF GROUNDS UNFAVORABLE

................................................. 84



### X. REFERENCES

.................................................................................................... 85



### Appendix 1.FIELD TRIAL TE RMINATION REPORTS 2001-2008

............................... 92



### Appendix 2. MATERIALS AND METHOD S- PRODUCT CHARACTERIZATION

2.A. Materials and methods for molecular charac terizati on ................................................................ 116 2.B. Materials and methods for pr otein characteri zation te sts ............................................................ 118
2.C. Materials and methods for protein levels in seeds....................................................................... 12 2 2.D. Materials and methods for protein levels in plant parts and dur ing the life cycle ........................ 127
2.E. Materials and methods for agronomic studies ............................................................................. 13 1
2.F. Materials and methods fo r seed germina tion studi es .................................................................. 133
2.G. Materials and methods for compositio n analys is ......................................................................... 134



### Appendix 3. MOLECULAR CHARACT ERIZATION OF EVENT FG72

3.A Verification of the insert................................................................................................. ............... 140
3.B. Absence of vector backbone............................................................................................... ......... 154 3.C. Stability across a nd within gene rations.................................................................................. ...... 159
3.D. Demonstration of protein e quivalence ..................................................................................... .... 171
3.D.1. 2m EPSPS protein....................................................................................................... ....... 171
3.D.2. HPPD W3 36 protein ....................................................................................................... ... 176Soybean Event FG72 Petition - Revised Page 10 of 198

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### Appendix 4.AGRO NOMIC DATA ............................................................................... 180



### Appendix 5. HERBICIDE R ESISTANCE AND STEWARDSHIP

5.A. Evolution of herbi cide resistant weeds.................................................................................... ..... 186 5.B. Managing herbicid e resistan t weeds........................................................................................ .... 187 5.C. Evolution of resistance to gly phosate and isoxaflu tole herbi cides .............................................. 190 5.D. Characteristics of glyphos ate and isoxaflutole herbici des ......................................................... 191 5.D.1. Glyphosate herbicide.................................................................................................... ..... 191
5.D.2. Isoxaflu tole herbicide.................................................................................................. ....... 191 5.E. Stewardship of double-herbicid e-tolerant soy bean event FG72.................................................. 192
5.E.1. Custom er outre ach ....................................................................................................... ..... 193
5.E.2. Additional cu stomer su pport............................................................................................. . 194 5.E.3. Monitoring of effectivenes s of the stewar dship pl an.......................................................... 195



### Appendix 6. REFERENC ES - APPENDI CES 1-5....................................................... 196Soybean Event FG72 Petition - Revised Page 11 of 198

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### LIST OF TABLES

Table 1. Generations used for analysis of event FG72 .................................................................... 19

Table 2. Genetic elements located on the insert .............................................................................. 23

Table 3. Segregation data for progeny rows and individuals of self-pollinated event FG72 ....... 28

Table 4. Segregation data for individual F2 progeny of the FG72 x conventional line cross

using zygosity PCR probes designed to identify the FG72 insert ................................... 28

Table 5. Risk group classification of P. fluorescens ........................................................................ 36

Table 6. Protein expression levels in event FG72 soybean seeds ................................................. 39

Table 7. HPPG W336 protein expression in different tissues and growth stages ........................ 41

Table 8. 2mEPSPS protein expression in different tissues and growth stages ............................ 41

Table 9. Criteria and methodology of demonstrating protein equivalence ................................... 42

Table 10. Summary of field activities under USDA notifications for event FG72 ............................ 53

Table 11. Summary of field activities under CFIA notifications for event FG72 ............................. 54

Table 12. Summary of field activities under CONABIA permits for event FG72 ............................. 54

Table 13. Summary of performance characteristics evaluated for event FG72 (2008) .................. 56

Table 14. Yield means for event FG72 – US (2003) ............................................................................ 57

Table 15. Yield of event FG72 and Jack for selected herbicide treatments ..................................... 59

Table 16. Trial site locations for the equivalence field tests ............................................................. 60

Table 17. Treatments for the 2008 season field studies .................................................................... 60

Table 18. Summary of agronomic performance for event FG72 and Jack ....................................... 61

Table 19. General plant descriptors comparison for event FG72 and Jack .................................... 63

Table 20. Seed phenotype characteristics .......................................................................................... 64

Table 21. Paired comparison of variety characteristics ..................................................................... 64

Table 22. Plant diseases and syndromes observed ........................................................................... 65

Table 23. Summary of agronomic performance for event FG72 and commercial varieties ........... 66

Table 24. Seed germination test results .............................................................................................. 68

Table 25. Proximate and fiber components ........................................................................................ 71

Table 26. Amino acids ........................................................................................................................... 72

Table 27. Fatty acids .............................................................................................................................. 7 3

Table 28. Anti-nutrients ......................................................................................................................... 74

Table 29. Isoflavones ............................................................................................................................. 74

Table 30. Sources of genetic based herbicide tolerance ................................................................... 80

Table 31. Validation of sample extraction with fortified non-transgenic soybean grain .............. 125

Table 32. Limits of detection and quantifica tion of 2mEPSPS and HPPD W336 proteins in

fortified non-transgenic soybean grain ............................................................................. 125Soybean Event FG72 Petition - Revised Page 12 of 198

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### LIST OF TABLES (continued)

Table 33. Amounts of 2m EPSPS and HPPD W336 proteins ............................................................ 127

Table 34. Results of dry weight determination on FG72 samples .................................................. 129

Table 35. Final dilutions for protein extracts of the soybean FG72 tissues used for the HPPD

and the EPSPS ELISA ......................................................................................................... 129

Table 36. LOQ for the HPPD W336 ELISA in different soybean tissues ......................................... 130

Table 37. LOQ for the EPSPS ELISA in different soybean tissues ................................................. 130

Table 38. Treatment schedule for agronomic field tests in 2008 .................................................... 132

Table 39. Sample identification .......................................................................................................... 135

Table 40. Methods used for the analysis of soybean grain ............................................................. 136

Table 41. Methods used for the analysis of soybean hulls ............................................................. 136

Table 42. Methods used for the analysis of soybean meal and toasted meal ............................... 137

Table 43. Methods used for the analysis of soybean protein isolate ............................................. 137

Table 44. Methods used for the analysis of soybean crude oil and RBD oil ................................. 138

Table 45. Methods used for the analysis of soybean lecithin ......................................................... 138

Table 46. Probes used in Southern hybridization of event FG72 ................................................... 140

Table 47. Expected and obtained hybridization fragments ............................................................. 142

Table 48. Probes used for demonstration of absence of vector backbone ................................... 154

Table 49. Expected and obtained hybridization fragments ............................................................. 156

Table 50. Overview of the tested seed lots of event FG72 ............................................................... 160

Table 51. Probes used for stability studies ....................................................................................... 160

Table 52. Expected and obtained hybridization fragments ............................................................. 170

Table 53. Electrospray LC/MS peptide mapping of the 2mEPSPS protein .................................... 174

Table 54. Amino acid coverage of the 2mEPSPS protein ................................................................ 175

Table 55. Electrospray LC/MS peptide mapping of the HPPD W336 protein ................................. 178

Table 56. Amino acid coverage of the HPPD W336 protein ............................................................. 179

Table 57. Raw agronomic data for 2008 ............................................................................................. 181Soybean Event FG72 Petition - Revised Page 13 of 198

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### LIST OF FIGURES

Figure 1. Rise in Glyphosate resistance among different biotypes ................................................. 17

Figure 2. Breeding Diagram for event FG72 ....................................................................................... 20

Figure 3. Map of plasmid pSF10 ........................................................................................................... 21

Figure 4. Event FG72 insert diagram ................................................................................................... 26

Figure 5. Biochemical pathways of HPPD proteins ........................................................................... 34

Figure 6. Interaction of HPPD and isoxazole herbicides ................................................................... 35

Figure 7. The effect of temperature on the activity of the HPPD W336 enzyme ............................. 50

Figure 8. Yield of event FG72 – Event FG72 and location means .................................................... 58

Figure 9. Growth of herbicide tolerant traits ...................................................................................... 78

Figure 10. Comparison of herbicide use for the years 1996 and 2007 ............................................... 79

Figure 11. Schematic drawing of the event FG72 insert indicating restriction enzymes, probes,

and expected fragment lengths ......................................................................................... 141

Figure 12. Southern blot analysis of event FG72 – 3’histonAt probe .............................................. 145

Figure 13. Southern blot analysis of event FG72 – 3’ nos probe ..................................................... 146

Figure 14. Southern blot analysis of event FG72 – hppdPfW336 probe .......................................... 147

Figure 15. Southern blot analysis of event FG72 – 5’tev+TPotp Y probe ....................................... 148

Figure 16. Southern blot analysis of event FG72 – Ph4a748 probe ................................................ 149

Figure 17. Southern blot analysis of event FG72 – Ph4a748B probe .............................................. 150

Figure 18. Southern blot analysis of event FG72 – intron1 h3At probe .......................................... 151

Figure 19. Southern blot analysis of event FG72 – 2mepsps probe ............................................... 152

Figure 20. Southern blot analysis of event FG72 – insert DNA probe ............................................ 153

Figure 21. Schematic drawing of plasmid pSF10 indicating the relevant restriction sites and

probes

............................................................................................................................... .... 155

Figure 22. Southern blot analysis of event FG72 – Absence of vector backbone- PT056-1 probe

and insert DNA probe .......................................................................................................... 157

Figure 23. Southern blot analysis of event FG72 – Absence of vector backbone- PT057-1 probe

and insert DNA probe .......................................................................................................... 158

Figure 24. Environment Adel ................................................................................................................ 161

Figure 25. Environment Osborn ........................................................................................................... 162

Figure 26. Environment Sharpsville .................................................................................................... 163

Figure 27. Environment Fithian ............................................................................................................ 164

Figure 28. Background 3068115-48 x Jack ......................................................................................... 165

Figure 29. Background 3066617-48 x Jack ......................................................................................... 166

Figure 30. Generation T2 ....................................................................................................................... 167

Figure 31. Generation T7 ....................................................................................................................... 168Soybean Event FG72 Petition - Revised Page 14 of 198

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### LIST OF FIGURES (continued)

Figure 32. Schematic drawing of hybridization strategy for the demonstration of structural

stability

............................................................................................................................... .. 169

Figure 33. Comparison of the plant-produced and microbially-produced 2mEPSPS protein ....... 171

Figure 34. Standard curve of electrophoretic mobility versus molecular weight ........................... 172

Figure 35. Western blot comparison of the plant-produced and micr obially-produced 2mEPSPS

protein

............................................................................................................................... .... 173

Figure 36. Comparison of the plant-produced and microbially-produced HPPD W336 protein ... 176

Figure 37. Western blot comparison of the plant-produced and microbially-produced HPPD W336

protein

............................................................................................................................... .... 177

Figure 38. Timeline of the development of herbicide resistant weeds ............................................ 187Soybean Event FG72 Petition - Revised Page 15 of 198

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### I. RATIONALE

I.A. Basis for the Request for Determination of Non-regulated status

The United States Department of Agriculture (UDSA) Animal and Plant Health Inspection Services (APHIS) is responsible for protection of the US agricultural infrastructure against noxious pests and weeds. Under the Plant Protection Act (7 USC § 7701-7772) APHIS considers plants altered or produced by genetic engineering as restricted article under 7 CFR
340 which cannot be released into the environm ent without appropriate approvals. APHIS provides that petitions may be filed under 7 CFR §340.6 to evaluate data to determine that a particular regulated article does not present a risk as a noxious pest or weed to agricultural infrastructure. Should APHIS determine that the submitted article does not present a plant pest risk, the article may be deregulated and released without further restrictions.

I.B. Double-herbicide-tolerant soybean event FG72

Bayer CropScience (BCS) and M.S.Technologies, LLC (MSTech) have developed doubleherbicide-tolerant soybean event FG72 which produces the 2mEPSPS and HPPD W336 proteins which confer tolerance to the herbicides glyphosate and isoxaflutole (IFT), respectively. The combination of the two herbicide tolerances in a single plant provides an effective, broad spectrum weed control option using glyphosate and IFT.

I.C. Rationale for the development of event FG72 and benefits

Planting double-herbicide-tolerant soybean FG72 varieties, containing transformation event FG72, provides growers with new options for weed control using IFT herbicide (registered in
North America as Balance Pro ® 

### ) in combination with glyphosate herbicide. Glyphosate is widely used in herbicide-tolerant soybean and other agricultural production systems and over the years, resistance to the chemical has been reported for certain weed species (Heap, 2011). IFT herbicide offers an alternative weed control option for the soybean grower to assist in controlling weeds already resistant to glyphosate as well as providing growers an alternative herbicide for soybean production, thus providing a tool for weed resistance management. IFT controls weeds via a new herbicide mode of action for soy beans that is efficacious against many of the herbicide resistant weeds currently found in soybean fields as well as offering an alternative time of application. IFT has the flexibility to be applied pre-plant incorporation, pre-emergence, or post emergence to FG72 soybeans which allows more flexibility to the user. When used as a pre-emergent herbicide, along with glyphosate as a post-emergent herbicide, the combination potentially offers a one-pass application for season long control of weeds.

Efficacious Weed Control Both IFT and glyphosate have been shown to be e fficacious weed control systems when utilized with FG72 herbicide tolerant soybean. Broad spectr um weed control is usually achieved with a single co-application that controls the weeds in the field as well as germination of new weeds for 6-8 weeks (Loux et al ., 2011). This flexibility is key in timing herbicide sprays at the ideal time in weed development, rather than at a specific ti me during the development of the soybean plant.Soybean Event FG72 Petition - Revised Page 16 of 198

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### Simplicity and Convenience FG72 herbicide-tolerant soybean provides an easy-to-use system that allows a highly efficient weed control in the crop. High efficiency allows for the cultivation of additional acreage and expansion of production operations with the existing level of infrastructure. Additionally, some equipment costs and labor may be eliminated in situations where cultivation equipment is no longer necessary, such as no-till practices (Carson, 2008).

Economic Benefit to Growers BCS believes that the use of glyphosate-tolerant soybeans will increase grower returns in the form of higher yields and reduced overhead production costs.

Environmental Benefits The main environmental benefit of FG72 herbicide-tolerant soybean is the use of reduced and no-till production systems. These cultivation practi ces contribute to reductions in soil erosion from water and wind. Reduced tillage also contributes to reduced fossil fuel use, less air pollution from dust, improved soil moisture rete ntion, and reduced soil compaction (Reicosky, 2008; Duiker and Myers, 2006).

I.D. Adoption of event FG72

Soybeans are an important oilseed crop. In 2010, soybeans represented 58% of the world oilseed production, with 35% of those soybeans being produced in the U.S. (http://www.soystats.com/2011/Default-frames.htm)

. In 2009, 77% of the world's soybean crop was genetically modified, a higher percentage than for any other crop. The United States (91%)and Argentina (99%) produce almost exclusively GM soybeans (http://www.gmocompass.org/eng/agri_biotechnology/gmo_plant ing/342.genetically_modified_soybean_global_ area_under_cultivation.html).

Adoption of this technology is expected to progr ess in a manner similar to the adoption rate for other genetically modified soybean technologies. Where allowed around the world, growers are expected to adopt FG72 soybean at a swift rate based on the real problem of glyphosate resistant weeds as well as proactive crop management to avoid the eventual development of glyphosate resistance. The rise in glyphos ate resistance among different biotypes is demonstrated in Figure 1.

This strategy will prolong the effective use of these excellent herbicides well into the future.Soybean Event FG72 Petition - Revised Page 17 of 198

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### Figure 1. Rise in glyphosate resistance among different biotypes

I.E. Submissions to other regulatory agencies

Food and Drug Administration FG72 soybean is within the scope of the 1992 US FDA policy statement concerning regulation of products derived from new plant varietie s, including those developed through biotechnology (FDA, 1992). In compliance with this policy, BCS and MSTech will submit a food and feed safety and nutritional assessment summary for FG72 soybean to the US FDA.

Environmental Protection Agency The United States Environmental Protection Agency (US EPA) has authority over the use of pesticidal substances under the Federal Insecticide Fungicide and Rodenticide Act (FIFRA) as amended (7 USA §136 et. Seq .). A submission for the use of an isoxaflutole formulation to be used on FG72 soybean will be presented to the Agency.

Foreign Governments BCS and MSTech intend to submit dossiers to request import of FG72 soybean to the proper regulatory authorities of foreign governments that have regulatory processes in place. These may include submissions to the relevant Regulatory Authorities in Canada, Mexico, EU, Japan, China and others. FG72 soybean has been, or is cu rrently, in field trials in soybean growing regions around the world.Soybean Event FG72 Petition - Revised Page 18 of 198

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### II. THE BIOLOGY OF SOYBEAN

II.A. Biology of soybean

The scientific name of soybean is Glycine max (L.) Merr. The genus Glycine is classified under
the tribe Phaseoleae , subfamily Papilionoideae , and the family Leguminosae (Fabaceae)

. The OECD consensus document (OECD, 2000) and the CFIA biology document (CFIA, 1996) provide information pertaining to the following aspects of soybean biology:

 General description, including taxonomy and morphology and use as a crop plant;

 Agronomic practices;

 Centers of origin of the species;

 Reproductive biology;

 Cultivated Glycine max as a volunteer weed;

 Inter-species/genus crosses, introgression into relatives

 Interactions with other organisms;

 Summary of the ecology of Glycine max

.

II.B. Characteristics of the recipient soybean cultivar

The publicly available cultivar, Jack, was used as the recipient line for the generation of soybean event FG72. The variety was originally developed at the Illinois Agricultural Experimental Station and commercially released in 1989 (Nickell et al ., 1990). Jack is classified as maturity group II and is best adapted to approximately 40 to 42 degrees of Northern latitude. It has white flowers, gray pubescence, brown pods at maturity, and seeds with dull yellow coat and yellow hila. Jack was developed and released because of its resistance to soybean cyst nematode (Races 3 and 4) and higher yield when com pared with cultivars of similar maturity. It is susceptible to Phytophthora sp. rot (Races 1, 4, and 7) (Nickell et al ., 1990).

Jack is extensively used in soybean transform ation because of its high embryogenic capacity (Stewart et al ., 1996; Santarem et al ., 1998; Yan et al ., 2000). Somatic embryos can be induced from immature cotyledons, proliferated, and maintained in liquid medium until
transformation.Soybean Event FG72 Petition - Revised Page 19 of 198

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### III. DEVELOPMENT OF DOUBLE-HERBI CIDE-TOLERANT SOYBEAN EVENT
FG72

III.A. Description of the transformation system

Soybean ( Glycine max (L.) Merr.) plants of the variety Jack were genetically modified by means of direct gene transfer of the purified Sal I fragment from plasmid pSF10 into an embryogenic cell line. The transformed cells were selected on IFT, and after a round of multiplication cycles, regenerated into embryos and shoots in the absence of the selective agent. The regenerated plantlets were then transferred to the greenhouse and sprayed with glyphosate for evaluation. Surviving plantlets were allowed to flower and set seeds.

III.B. Parent line

The publicly available cultivar, Jack, was used as the recipient line for the generation of event FG72 soybean (See Section II.B).

III.C. Breeding Diagram

The breeding diagram of soybean event FG72 is shown in Figure 2.

III.D. Generations Used for Analysis

The generations used for the studies to analyze soybean event FG72 are described in Table 1.

Table 1. Generations used for analysis of event FG72

Generation Study

T2, F4 Inheritance study

T2, T7, T9, F4 Structural stability study

T5 Yield, agronomic, herbicide evaluations

T7 Absence of vector backbone sequences, detailed insert characterization, full expression analysis

T8 Nutrient composition assessment, protein expression in grain, phenotypic and agronomic assessment, grain production

F4 Full DNA sequenceSoybean Event FG72 Petition - Revised Page 20 of 198

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### Figure 2. Breeding Diagram of event FG72



### T 1



### T 4



### T 5 

### T 2



### T 7



### T 8 

### T 6 

### T 3 

### T 0



### F 1



### F 2



### F 3



### Commercial Candidate Evaluations T 9 

### F 4 

### Initial Transformation in ‘Jack’

self

self

self

self

self

self

self

self

self self

self

self Cross with commercial varietiesSoybean Event FG72 Petition - Revised Page 21 of 198

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### IV. GENETIC MATERIAL USED FOR TRANSFORMATION OF EVENT FG72

IV.A. Construction of the plasmid used for transformation

The double-herbicide tolerance was introduced to the plant by direct DNA integration of a linear DNA fragment isolated from the plasmid pSF10 (Figure 3). Plasmid pSF10 was constructed by inserting the 2mepsps and hppdPfW336 genes into an E.coli pBR322-derived cloning plasmid (Bolivar et al. , 1977). The fragment of interest was cleaved and isolated from the plasmid by means of the restriction enzyme Sal I and HPLC purification.



### Figure 3. Map of plasmid pSF10



# pSF10

10398 bp 2000

4000

6000 8000 10000

Sal I Sal I

ORI ColE1

bla

ORI f1

3'nos

hppdPf W336

TPotp Y 5'tev Ph4a748 ABBC Ph4a748 intron1 h3At TPotp C 2mepsps 3'histonAtSoybean Event FG72 Petition - Revised Page 22 of 198

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### IV.B. Donor genes and associated regulatory regions

IV.B.1. The 2mepsps gene expression cassette

The 2mepsps gene expression cassette borne by pSF10 is represented by the following string: “Ph4a748-intron1 h3At-TPotpC:: 2mepsps ::3’histonAt”. In this cassette, the 2mepsps gene coding sequence is under the control of the H4 promoter of Arabidopsis thaliana (Ph4A748,
Chabouté et al. , 1987), followed by the first intron of gene II of the histone H3.III variant of Arabidopsis thaliana (Chaubet et al ., 1992), and by the optimized transit peptide as described by Lebrun et al . (1996), and terminated by the 3’ untranslated region of the histone H4 gene of Arabidopsis thaliana (Chabouté et al. , 1987).

Ph4a748 promoter and intron 1 h3At The Ph4a748 promoter sequence is derived from the histone H4 gene of Arabidopsis thaliana (Chabouté et al ., 1987) and controls expression of the 2mepsps gene. The Ph4a748 promoter, combined with the first intron of gene II of the histone H3.III variant of Arabidopsis thaliana (Chaubet et al ., 1992) directs high level constitutive expression, especially in rapidly growing plant tissues.

TPotp C transit peptide The optimized transit peptide, which contains sequences from the RuBisCO small subunitgenes of corn and sunflower, targets the mature protein to the plastids, which is where the wildtype protein would be located (Lebrun et al ., 1996).

2mepsps coding sequence The wild type epsps gene isolated from maize ( Zea mays ) was mutated using site-directed mutagenesis. Two point mutations resulted in the double mutant 2mepsps gene (Lebrun et al

., 1997). A methionine codon is added to the N-terminal of the 2mEPSPS protein sequence in order to restore the cleavage site of the optimized plastid transit peptide. The 2mepsps gene encodes a 47 kDa protein consisting of 445 amino acids.

3’histonAt terminator The 3’ untranslated region of the histone H4 gene of Arabidopsis thaliana (Chabouté et al

., 1987) is a polyadenylation signal.

IV.B.2. The hppdPfW336 gene expression cassette

The hppdPfW336 gene expression cassette is represented by the following string: “Ph4a748 ABBC-5’tev -TPotpY:: hppdPfW336 ::3’nos”. In this cassette, the hppdPfW336 gene coding sequence is under the control of the duplicated H4 promoter of Arabidopsis thaliana (Ph4A748,
Chabouté et al ., 1987), followed by the enhancer sequence of the tobacco etch virus (Carrington and Freed, 1990), and by the optimized transit peptide as described by Lebrun et al. (1996), and terminated by the 3’ untranslated region of the nopaline synthase from Agrobacterium tumefaciens

.Soybean Event FG72 Petition - Revised Page 23 of 198

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### Ph4a748 ABBC promoter and 5’tev enhancer The same Ph4a748 promoter was used to drive the expression of the hppdPfW336 gene, but an internal portion of the promoter sequence (referred to as “B”) was duplicated to increase the promoter activity in plant cells. In combination with the leader sequence of the tobacco etch virus (5’tev), this promoter brings the level of expression of hppdPfW336 gene to an appropriate level that enables tolerance at agronomic doses of IFT.

TPotp Y transit peptide The optimized transit peptide, which contains sequences from the RuBisCO small subunitgenes of corn and sunflower, targets the mature protein to the plastids, which is where the wildtype protein would be located (Lebrun et al ., 1996).

hppdPfW336 gene coding sequence The wild type hppd gene isolated from Pseudomonas fluorescens was mutated using sitedirected mutagenesis. A point mutation resulted in the hppdPfW336 gene (Boudec et al. , 2001).
The hppdPfW336 gene encodes a 40 kDa protein consisting of 358 amino acids.

3' nos terminator The 3’ untranslated region of the nopaline synthase from Agrobacterium tumefaciens is a polyadenylation signal.

IV.C. Identity and source of the genetic material

Table 2 summarizes the identity and source of the genetic elements of the Sal I insert excised from plasmid pSF10 (Figure 3) and their regulatory sequences.

Table 2. Genetic elements located on insert

Nt Positions Orientation Origin

3262 - 3553 Counter
clockwise 3 ́nos: sequence including the 3’ untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al. , 1982)

3554 - 4630 Counter
clockwise hppdPfW336 : the coding sequence of the 4- hydroxyphenylpyruvate dioxygenase of Pseudomonas
fluorescens strain A32 modified by the replacement of the amino acid glycine with a tryptophane, as described by
Boudec et al . (2001)

4631 - 5002 Counter
clockwise TPotp Y: coding sequence of an optimized transit peptide derivative (position 55 changed into tyrosine), containing sequence of the RuBisCO small subunit genes of Z. mays (corn) and Helianthus annuus (sunflower), as described by
Lebrun et al. (1996)Soybean Event FG72 Petition - Revised Page 24 of 198

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### Table 2. Genetic elements located on Sal I insert (continued)

Nt Positions Orientation Origin

5003 - 5143 Counter
clockwise 5'tev: sequence including the leader sequence of the tobacco etch virus as described by Carrington and Freed (1990)

5144 - 6433 Counter
clockwise Ph4a748 ABBC: sequence including the promoter region of the histone H4 gene of Arabidopsis thaliana , containing an internal duplication (Chabouté et al ., 1987)

6434 - 7448 Clockwise Ph4a748: sequence including the promoter region of the histone H4 gene of Arabidopsis thaliana (Chabouté et al

., 1987)

7449 - 7929 Clockwise intron1 h3At: first intron of gene II of the histone H3.III
variant of Arabidopsis thaliana (Chaubet et a l., 1992)

7930 - 8301 Clockwise TPotp C: coding sequence of the optimized transit peptide, containing sequence of the RuBisCO small subunit genes of Z. mays (corn) and Helianthus annuus (sunflower), as described by Lebrun et al. (1996)

8302 - 9639 Clockwise 2mepsps : the coding sequence of the double-mutant 5- enol-pyruvylshikimate-3-phosphate synthase gene of Z.

mays (corn) (Lebrun et al ., 1997)

9640 - 10326 Clockwise 3 ́histonAt: sequence including the 3 ́ untranslated region of the histone H4 gene of Arabidopsis thaliana (Chabouté et
al ., 1987)Soybean Event FG72 Petition - Revised Page 25 of 198

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### V. GENETIC CHARACTERIZATION OF EVENT FG72

V.A. Overview

In order to obtain the double-herbicide-tolerant soybean event FG72, a linear fragment of DNA originating from the plasmid pSF10 and bearing the 2mepsps and the hppdPfW336 expression cassettes was introduced into the soybean genome by means of direct gene transfer. Event FG72 contains a single insert of two consecutive complete copies of the linear fragment. These two copies were integrated into the soybean genome and are flanked on their 5’ side by two partial fragments corresponding to the 3’histonAt element. At the insertion point of the two full copies, a stretch of soybean genomic DNA was excised and translocated downstream of the 3’end of the insertion point in association with another partial fragment corresponding to the H4 promoter. This molecular modification is genetically stable and shows a typical Mendelian segregation pattern. The materials and methods for the molecular characterization of event FG72 are provided in Appendix 2.A.

V.B. Copy number and Insertion

The inserted transgenic sequence in soybean event FG72 consists of two partial 3’histonAt sequences in a head to head orientation followed by 2 complete copies of the linear fragment excised from plasmid pSF10 arranged in a head to tail orientation. Upon integration of the FG72 insert into the soybean genome, a genomic r egion translocated to a new position, which is joined at the 3’ junction by 158 bases of Ph4a748 promoter sequences (Figure 4).

Genomic DNA prepared from FG72 soybean plants was subjected to Southern blot analysis using eight probes, each containing a single genetic element present in the pSF10 vector used for the transformation ( 2mepsps , 3’histonAt, 3’nos, Ph4a748, Intron1 h3At, 5’tev + TPotp Y, Ph4a748B, and hppdPfW336 ), and the complete insert probe. The expected and observed hybridization fragments, as well as the hybridization strategy, are described in Appendix 3, Tables 44 and 45. Hybridization results support the model of the FG72 insert organization as described above (Appendix 3, Figures 12-20).

V.C. Absence of vector backbone

For the molecular verification of the absence of pSF10 vector backbone sequences in event FG72 soybean, Southern blot analysis was performed using two overlapping probes covering the complete vector backbone sequences of the pSF10 transformation vector. Afterwards, the membranes were stripped of the vector backbone probes and re-hybridized with an insert probe, in order to demonstrate that ample FG 72 soybean genomic DNA was loaded on the gels. Information on the probes and a schematic overview of the Southern blot strategy is presented in Appendix 3, Tables 46 and 47.

Since both vector backbone probes contain a number of regions also present in the insert sequence, several fragments originating from inserted transgenic DNA hybridize with the vector backbone probes. Based on sequence homology, only the expected fragments were obtained when hybridizing with vector backbone probes, demonstrating the absence of vector backbone sequences in event FG72 soybean (Appendix 3, Figures 21-23). The hppdPf W366 gene was used as the selectable marker, therefore the same gene of interest acts as a marker. No other marker genes were present.Soybean Event FG72 Petition - RevisedPage 26 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONFigure 4. Event FG72 insert diagramhppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAt3’histon At3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAt3’histon At3’histonAt5’ flanking seq.3’ flanking seq.FG72 – TR1FG72 – TR2FG72 – TR3FG72 – TR4FG72 – TR5FG72 – TR6Filler DNAPre-insertionlocusabcd25 bpdeletion2 bpdeletionacbdFG72InsertSeq.FG72locusFG72insertPh4a748 fragmentFG72 – TL2Junction 1Junction 6Junction 7Junctions 8 and 9JACK – WT1JACK – WT3JACK – WT2Junction 2Junction 3Junction 4Junction 5FG72 – TL1FG72 – TRSoybean Event FG72 Petition Page 27 of 91

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### V.D. The flanking regions of the inserted sequence(s)

The flanking regions of the insert and the translocated fragment have been determined by means of PCR-based sequencing experiments. The corresponding sequences have been determined in parallel on the original, non-transformed genome of the parent line Jack. The obtained sequences are exactly identical, which demonstrates that the flanking sequences of the inserted DNA correspond to the original soybean genome in its original organization. For a diagram of event FG72 and its flanking regions refer to Figure 4 above.

The junctions between the inserted sequences and the original genome, as well as the junction created at the translocation point, have been analyzed by means of bioinformatics tools (open reading frames, promoter and gene predictions). No evidence of any potential unintended genes or any disruption of pre-existing genes was found.

V.E. Mendelian inheritance

Transformation event FG72 was derived from the transformation of soybean cells. Seed
harvested from T 0 

### plants in the greenhouse in Lyon, France was sent to Puerto Rico for the winter season of 2001-2002. Three blocks were planted and sprayed with 0, 2, or 4 kg/ha glyphosate (GLY). Seed was harvested from plants demonstrating the desired level of tolerance to the glyphosate herbicide.

Seed harvested from T 1 

### plants grown in Puerto Rico (2001-2002 season) were planted “plant to row” in the US (Benton, IN) in 2002. Six progeny rows of event FG72 (T 2 

### ) were planted and the census of surviving plants was taken in August, 2002. Of the six rows, one row had no plants sensitive to glyphosate. Of the 172 individual plants, 124 were tolerant and 48 were scored as sensitive to glyphosate. The expected ratio for a single locus is 1 fully resistant to 2 partially resistant rows and for the individual plants, the expected ratio is 3 tolerant for each sensitive plant. Chi square analysis of segregation data for rows (fully or partially tolerant) and of individual plants within rows (tolerant or sensitive) demonstrates the expected Mendelian inheritance of a single insertion (Table 3).

Selection and seed increase continued until the T 4 

### generation which was determined to be homozygous for transformation event FG72 and selected for core seed production in the fourth generation. In 2004, T 5 

### generation seed of event FG72 was transferred to MSTech as a candidate for variety development. In the summer 2007 season, plants in the sixth generation were crossed with conventional soybean breeding lines in the introgression program designed to move event FG72 into a broader base of commercial soybean germplasm. F 1 

### hybrid plants (FG72 x conventional lines) were grown to maturity and the F 2 

### seed was planted. Leaf samples
of 901 F 2 

### plants were analyzed by PCR probes designed to identify the zygosity of the event FG72 insert. The expected ratio of 1:2:1 for a single insertion segregating by the rules of Mendel was observed (Table 4).Soybean Event FG72 Petition - Revised Page 28 of 198

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### Table 3. Segregation data for progeny rows and individuals of self-pollinated event FG72

Fully
Tolerant a
Rows/ Partially
Tolerant Rows/ Parents and zygosity for
the FG72 locus Progeny

Tolerant
Plants Sensitive
Plants Expected
Ratio χ 2

calculated b

Tolerant T 1 progenies of the self-pollinated T 0 transformants (1/4 FG72/FG72 ; 2/4 FG72/-) T 2 Rows 1 5 1 to 2 0.485

Hemizygous T 1 plants (FG72/-) resulting in the partially resistant rows T 2 Individual
Plants 124 48 3 to 1 0.194

a Based upon survival to herbicide (glyphosate) application.

b Assumes a single locus model. There was no significant difference (p=0.05) for the χ 2 goodness-of-fit test for the hypothesis of a single locus. To reject the null hypothesis, the χ 2 value must be greater than 3.84, with one degree of freedom.



### Table 4. Segregation data for individual F2 progeny of the FG72 x conventional line cross using zygosity PCR probes designed to identify the FG72 insert

Zygosity of the FG72 locus PCR result a total Ratio Expected ratio

Homozygous (null / null) nn 212 0.24 0.25

Heterozygous (FG72 / null) pn 471 0.52 0.5

Homozygous (FG72 / FG72) pp 218 0.24 0.25

# plants tested: 901 χ 2 value b = 0.172

a PCR result; n = negative, p = positive

b Assumes a single locus model. There was no significant difference (p=0.05) for the χ 2 goodness-of-fit test for the hypothesis of a single locus. To reject the null hypothesis, the χ 2 value must be greater than 3.84, with one degree of freedom.



### V.F. Stability across and within generations

In order to demonstrate the stability of ev ent FG72, genomic DNA was prepared from several individual plants of three generations and three different genetic backgrounds. The impact of environment was assessed by analyzing the pro geny of transgenic plants cultivated at 4 different field locations. The isolated DNA was digested with the restriction enzyme Hin dIII, which provides a unique pattern for event FG72.Soybean Event FG72 Petition - Revised Page 29 of 198

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### Successive hybridization of these samples with the Ph4a748B probe and the insert probe revealed the expected profile in all tested samples (Appendix 3, Tables 49 and 50). These findings demonstrate the stability of event FG72 at the genomic level in different environments (Appendix 3, Figures 24-27), different backgrounds (Appendix 3, Figures 28 and 29), and different generations (Appendix 3, Figures 30 and 31).

V.G. Conclusion

The double-herbicide-tolerant soybean event FG72 contains two consecutive complete copies of the pSF10 DNA fragment that bears the 2mepsps and the hppdPfW336 expression cassettes. The two copies integrated in the soybean genome at a single locus and are flanked on their 5’ side by two partial fragments of the 3’histonAt element. At the insertion point of the two copies, a stretch of soybean genomic DNA was excised and translocated downstream of the 3’ end of the insertion point, in association with a partial fragment originating from the H4 promoter. The absence of vector backbone was demonstrated by Southern blot analysis.

The molecular modification is genetically st able and shows a typical Mendelian segregation pattern. Bioinformatics analyses of the junctions between the original genome and insertedsequences did not reveal any potential creation of unintended genes or any disruption of preexisting genes.Soybean Event FG72 Petition - Revised Page 30 of 198

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### VI. CHARACTERIZATION OF TH E INTRODUCED PROTEINS

VI.A. The 2mEPSPS protein

VI.A.1. History and background

In the early 1970s, it was demonstrated that inhibito rs of the aromatic amino acid biosynthetic pathway can have an herbicidal activity (Jaworski, 1972; Baillie et al. , 1972). In particular, the work published by Jaworski’s group opened the path for the development of herbicides, such as glyphosate.

In plants, as much as 20% of all fixed carbon flows through the shikimate pathway leading to the formation of the aromatic amino acids tyrosi ne (tyr), phenyalanine (phe) and tryptophan (trp), as well as tetrahydrofolate, ubiquinone, and vitamins K and E (Haslam, 1993; Franz et al ., 1997). The aromatic amino acids, in turn, serve as precursors for an array of secondary metabolites including lignin, flavanoids and alkaloids (He rrmann, 1995). The shikimate pathway exists exclusively in plants and microorganisms including fungi. In contrast, mammals, fish, birds, reptiles, and insects must derive their aromatic compounds from their diet. For this reason, there has been interest over the last three decades in the shikimate pathway enzymes as potential targets for non-toxic herbicides and anti-microbial compounds.

Glyphosate is the active ingredient of a non-selective, broad-spectrum, systemic, postemergence herbicide that has been used extensively throughout the world over the past three decades. It has a very low mammalian toxicity and low soil persistence. It is used to inhibit weeds in conservation tillage systems just prior to planting (Rueppel et al ., 1977; Williams et al

., 2000; Andréa et al ., 2003). It is also applied as a non-selective herbicide with direct spraying in orchards (Pacific Northwest Weed Management Handbook, 2010; Willamette Valley Pest Management Guide, 2011). Given the importance of this compound, considerable effort has been made in attempts to engineer glyphosate tolerance in various crops.

Study of the shikimate pathway led to the discovery of 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) by Amrhein et al. , (1980). The mode of action of glyphosate [N- (phosphonomethyl)glycine], a simple amino acid analog, was determined to be the selective inhibition of EPSP synthase (EPSPS; EC 2.5.1.19), the sixth and penultimate enzyme of the shikimate pathway (Steinrücken and Amrhein, 1980). The reaction catalyzed by EPSPS is the reversible transfer of the phosphoenolpyruvate (PEP) to shikimate-3-phosphate (S3P), leading to the formation of 5-enolpyruvyl-3-shikimate phosphate (EPSP). Substrate binding to the enzyme is sequential, with S3P binding first, followed by PEP (Boocock and Coggins, 1983). The reaction catalyzed by EPSPS proceeds via C-O bond cleavage of PEP (Walsh et al. , 1996).

VI.A.2. Characterization of the 2mEPSPS protein

VI.A.2.1. Biochemistry and mode of action

The enzyme 5-enolpyruvylshikimate-3-phosphate synt hase (EPSPS) is involved in the shikimic acid pathway (Williams et al ., 2000). This pathway produces an important branch point intermediate,Soybean Event FG72 Petition - Revised Page 31 of 198

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### chorismate, for aromatic amino acid and aromatic metabolites biosynthesis in plants and microorganisms (Williams et al ., 2000; Steinruecken and Amrhein, 1980). The shikimate pathway is not present in animals, a fact that contributes to the selective effect of glyphosate to plants (Hermann, 1995)



### .

The family of EPSPS proteins is wide-spread in natur e, specifically in plant, fungi and microbial sources. In higher plants, EPSPS is synthesized from a nuclear gene in the form of a cytoplasmic precursor and then imported into the plastids where it accumulates in its mature form (Kishore and Shah, 1988; Forlani et al. , 1994; Lebrun et al. , 1997). Transit peptides are typically cleaved from the mature protein fo llowing delivery to the plastids (Della-Cioppa et al

., 1986).

Since the 1980s, several attempts have been made to identify and characterize glyphosateinsensitive EPSPS enzyme variants from various organisms with the ultimate aim to engineer glyphosate tolerance in crop plants (Kishore and Shah, 1988). Lebrun et al. (1997) selected the 2mepsps gene, a double mutant gene from maize which, when fused to a chimeric optimized transit peptide, generates optimal glyphosate toler ance in various crops without any pleiotropic
effects. The 2mepsps gene has been introduced as the source of glyphosate tolerance in the maize transgenic event GA21 which has been approved by different agencies worldwide for environment, food, and feed (OECD unique identifier MON-ØØØ21-9) (CERA, 2011). Glyphosate tolerance was also achieved in rice by mutagenesis of the rice epsps gene (Zhou et
al. , 2006).

VI.A.2.2. 2mEPSPS protein safety

The 2mepsps gene was generated by introducing mutations into the wt epsps gene from maize ( Z. mays L.), leading to a double mutant EPSPS protein with two amino acid substitutions (2mEPSPS). These modifications confer to t he protein a decreased binding affinity for glyphosate, allowing it to maintain sufficient enzymatic activity in the presence of the herbicide. Therefore, the plants bearing this gene become tolerant to glyphosate herbicides (Lebrun et al.

, 1997).

In order to assess any potential adverse effects to humans or animals resulting from the environmental release of crops containing the 2mEPSPS protein, BCS has conducted a detailed safety evaluation based on the Codex Alimentar ius Commission (Codex; Alinorm 03/34A). As a basis, BCS performed a series of safety studies with the 2mEPSPS protein, including homology searches of the amino acid sequence to all known allergens and toxins from large public databases, an in vitro digestibility assay of the protein, and an acute toxicity test in the mouse. As reviewed in ILSI (2010), several public ly available documents issued by regulatory authorities indicate that similar EPSPS protein family members are safe. The results of studies conducted by BCS are consistent with the published information, confirming that the crops containing this protein can be safely used as food or feed.

Assessments of the maize ( Zea mays L.) source organism, the 2mepsps gene, and the 2mEPSPS protein indicate that they are not pathogenic, allergenic, or toxic to mammals:Soybean Event FG72 Petition - Revised Page 32 of 198

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### History of safe use The maize source organism is a safe crop plant widely used for food and feed with little pathogenic, toxic, or allergenic effects on humans and animals (OECD, 2003).

The 2 mepsps gene is composed of the same essential nucleic acids found in any food or feed DNA, which is commonly consumed as part of human or animal diets. Decades of research have indicated that dietary DNA poses no direct toxicity to human health.

The EPSPS proteins are ubiquitous in nature, widely expressed in food and feed crops ( e.g

. soybean, tomato, maize) (ILSI, 2010). No health-related adverse effects have been associated with these proteins. Since the 2mEPSPS protein is derived from maize and has only two amino acid modifications, the safety profile of the novel protein is expected to remain unchanged relative to its wild-type counterpart.

The 2mEPSPS protein is highly homologous to, and shares similar molecular weight andfunctionalities with other shikimate synthase proteins which have been demonstrated to be nontoxic and non-allergenic over the years through consumption. Its identity with the wt EPSPS enzyme is greater than 99.5% (Herouet-Guicheney, 2009).

The EPSPS proteins have a very well known and specific biochemical role in plants. The biochemical properties of the 2mEPSPS enzyme have been well characterized in comparison to the wt EPSPS protein. Except for the insensitivity to glyphosate herbicide, the change in the two amino acids results in comparable biochemic al properties. The metabolic effects of the 2mEPSPS activity in plants are comparable to those of endogenous EPSPS proteins except for the insensitivity to glyphosate (Herouet-Guicheney, 2009).

The 2mEPSPS protein is present in glyphosate tolerant maize event GA21 (MON-ØØØ21-9), which has been approved for cultivation and for f ood/feed use in many countries (CERA, 2011).

Lack of allergenic potential The 2mEPSPS protein has no amino acid sequenc e similarity to known allergens, as demonstrated by overall amino acid and epitope homol ogy searches (refer to Section VI.E.2.1. of this petition for details).

As expected, the 2mEPSPS protein has high stru ctural similarity only to the non-allergenic Z.

mays wt EPSPS protein and other non-allergenic EPSPS enzymes (refer to Section VI.D.1. of this petition for details).

The 2mEPSPS shares the same potential N-glycosylation sites as the endogenous Z. mays EPSPS enzyme, and both proteins are targeted to the same plastid cellular compartment. Therefore, it is unlikely that post-translational glycosylation occurs on the 2mEPSPS protein, which could potentially lead to allergenic characteristics different from the wild-type enzyme (refer to Section VI.E.2.2. of this petition for details).

The 2mEPSPS protein is rapidly and completely degraded in human simulated gastric and
intestinal fluids. This minimizes the likelihood that this protein could survive in the human digestive tract and be absorbed (refer to Sections VI.E.2.3. and VI.E.2.4. of this petition for details).Soybean Event FG72 Petition - Revised Page 33 of 198

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### Lack of toxic potential The 2mEPSPS protein has no amino acid sequence similarity to known toxins, as demonstrated by overall amino acid and epitope homology searches (refer to Section VI.E.3. of this petition for details).

As expected, the 2mEPSPS protein only has hi gh structural similarity to the non-toxic Z. mays wt EPSPS protein and other non-toxic EPSPS enzymes (refer to Section VI.D.1. of this petition for details).

The 2mEPSPS protein is rapidly and completely degraded in human simulated gastric and intestinal fluids (refer to Sections VI.E.2.3. and VI.E.2.4. of this petition for details). This minimizes the likelihood that this protein c ould survive in the human digestive tract and be
absorbed.

There were no mortalities, clinical signs, or treatment-related effects on OF1 mice after an acute oral administration by gavage of 2mEPSPS protein at 2,000 mg protein/kg body weight (refer to Section VI.E.4. of this petition for details).

In conclusion, it is considered that the source organism, maize, is non-pathogenic and the 2mepsps gene as well as the 2mEPSPS protein are not toxic to mammals and do not possess any of the characteristics associated with food allergens. Therefore, no adverse effects on animal and human health are to be expected from the consumption of the 2mepsps gene and the 2mEPSPS protein.

VI.B. The HPPD protein

VI.B.1. History and background

The coding sequence of the 4-hydroxyphenylpyruvate dioxygenase (HPPD) protein was isolated
from the Pseudomonas fluorescens strain A32 by a DNA amplification technique (PCR). The primers in the amplification were based on t he amino acid sequence of the HPPD protein present in Pseudomonas strain P.J. 874. The isolated DNA sequence was modified to improve the tolerance against HPPD inhibitors. The m odified protein is designated as HPPD W336.

VI.B.2. The function of the gene product

VI.B.2.1. Biochemistry and mode of action

The biochemical pathways in which HPPD is involved differ between plants and nonphotosynthetic organisms. In bacteria and animals , it merely serves catabolic purposes by catalyzing the first committed step in tyrosine degradation that in the end yields energetically exploitable glucogenic and ketogenic products (Brownlee et al. , 2004). In plants, however, it is also involved in several anabolic pathways; its reaction product homogentisate (2,5- dihydroxyohenylacetate) being the aromatic precursor of tocopherol and plastoquinone, which are essential to the photosynthetic transport chain and antioxidative systems (Fritze et al

.,Soybean Event FG72 Petition - Revised Page 34 of 198

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### 2004). Figure 5 shows a diagram of the different metabolic pathways in plants and nonphotosynthetic organisms.

HPPD enzymes require a α -keto acid and molecular oxygen to oxidize or oxygenate a third
molecule. The activity of HPPD is suppressed by benz oylisoxazoles bleaching herbicides, such

as IFT, and by β -triketones such as sulcotrione and mesotrione (Pallett et al. , 2001; Dayan et
al. , 2007). The inhibitor of HPPD is the diketoni trile (DKN) derivative of IFT formed by the opening of the isoxazole ring. DKN is formed rapidly in plants following uptake of IFT by roots
and shoots. HPPD enzyme inhibition results in the disruption of the biosynthesis of carotenoids, which destabilizes photosynthesis and leads to bleaching of the foliage and death of the plant (Figure 6).

In order to create a form of the HPPD enzyme with tolerance to IFT herbicide, a single amino acid substitution, glycine (G) to tryptophan (W) at position 336, was introduced to the native HPPD protein from Pseudomonas fluorescens (Boudec et al. , 2001), resulting the modified IFTtolerant HPPD W336 protein.

Several different HPPD variants, including the wild type HPPD and modified HPPD W336 enzymes were tested for their activity in the presence or absence of the inhibitor IFT. When compared to the wild type HPPD enzyme, HPPD W336 enzyme was significantly less inhibited by IFT (Fischer, 2008).

Figure 5. Biochemical pathways of HPPD proteins

a) catabolism of tyrosine, b) biosynthesis of plastoquinone (plants) c) biosynthesis of tocopherol (plants)Soybean Event FG72 Petition - Revised Page 35 of 198

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### Figure 6. Interaction of HPPD and isoxazole herbicides

VI.B.2.2. Source of the gene

The hppd gene was isolated from the bacterium Pseudomonas fluorescens , strain A32 – Genebank A69533 (McKellar, 1982). P. fluorescens is a non-pathogenic bacterium, ubiquitous in nature, with a good history of safe use.

P. fluorescens (Migula 1895, type strain ATCC 13525; taxonomy ID: 136843) belongs to the
Pseudomonadaceae family, order of Pseudomonadales , class of Gammaproteobacteria (Skerman, 1980). P. fluorescens, P. putida and P. chlororaphis are closely related to each other and are seen as forming a complex within the fluorescent subgroup of the Pseudomonas genus. In addition, P. fluorescens is a heterogeneous species comprising several biovars, each of which may deserve species rank, but which are so interconnected that adequate methods have not been devised to clearly separate them (OECD, 1997).

P. fluorescens is a Gram-negative, rod-shaped, motile, asporogenous, aerobic bacteria. This species produces fluorescent pigments and is catalase and oxidase-positive. P. fluorescens strains are generally not able to grow above 42°C, but grow at 5°C (OECD, 1997, Palleroni, 1981). This organism is a nonpathogenic saprophyte which inhabits soil, water and plant surface environments. It is able to produce a soluble, greenish fluorescent pigment, which
relates to its name. Isoxazoles

DKN SO 2 CH 3 O O

CN

CF 3 Arogenate

Tyrosine

Hydroxyphenylpyruvate

Homogentisate

Tocopherols CH 2 CH COOH

OH O



### HPPD enzyme

CH 2 CO O H

OH O H O 2

CO 2

Plastoquinones Prephenate ChorismateSoybean Event FG72 Petition - Revised Page 36 of 198

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### VI.B.2.3. History of safe use of the source organism

Risk Group Classification
P. fluorescens ( Pf ) strains are generally classified as non-pathogenic bacteria in several national classifications for microorganisms (Table 5).

Table 5. Risk group classification of P. fluorescens

USA Not classified. http://www.cdc.gov/OD/ohs/biosfty/bmbl5/bmbl5toc.htm, accessed on March 02, 2009.

Canada Non-pathogenic organism. http://www.phac-a spc.gc.ca/ols-bsl/pathogen/organismeng.php, accessed on March 02, 2009.

European
Union Not classified. Directive 2000/54/EC

Belgium Risk Group 2 plant pathogen. Belgian Monitor 01.04.2004 18362-18442. 2004

Switzerland 1 + opportunistic pathogen. http://www.bafu.admin.ch/publikationen/i ndex.html?action=show_publ&lang=de&id_t hema=6&series=VU&nr_publ=4401; accessed on March 02, 2009.

France Not classified. Commission de Genie Génétique

Germany Risk Group 1 + - opportunistic pathogen. Classification of bacteria and archaea bacteria into risk groups – TRBA 466. 2005



### Pathogenicity to humans
P. fluorescens can be an opportunistic pathogen in immunocompromised patients (McKellar, 1982). Some cases of septicemia have been reported due to P. fluorescens contamination of transfused blood and blood products, given its ability to grow at 5°C (Gibb et al ., 1995, Puckett
et al ., 1992). Some P. fluorescens strains were also reported to create biofilms on compounded sterile products like catheters and have led to rare infections in immunocompromised populations (Gershman et al., 2008). However, the general virulence of P fluorescens is low, due to its inability to multiply rapidly at body temperature and having to compete with defense mechanisms of the host (Liu, 1964).

Pathogenicity to animals
P. fluorescens can infect a wide range of animals including horses, chickens, marine turtles, and many fish and invertebrate species. However, since it is unable to grow at elevated temperatures, it is probably only an opportunistic pathogen for warm-blooded animals (OECD, 1997).

Pathogenicity to plants Generally P. fluorescens is considered saprophytic but it may be an opportunistic pathogen causing soft rot in plants (OECD, 1997).Soybean Event FG72 Petition - Revised Page 37 of 198

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### Allergenicity In general fluorescent pseudomonads have not been de scribed as allergens. However, they do possess an endotoxin (lipopolysaccharide) which may induce an allergic response in some individuals (OECD, 1997).

History of safe use
P. fluorescens is a ubiquitous bacterium frequently present in water, soil and the plant rhizosphere (Bossis et al ., 2000). It can be isolated from water, animals, human clinical specimens, the hospital environment, and spoiled foodstuffs such as fish and meat. The
survival of P. fluorescens is affected by number of biotic and abiotic factors such as soil density, temperature, pH, humidity (OECD, 1997).

P. fluorescens is used in agriculture as growth-promoting agent (Fliessbach et al ., 2009; OECD, 1997). It can enhance plant growth through production of siderophores, which efficiently complex environmental iron, making it unavailable to other components of the soil microflora. In addition, P. fluorescens is used as a biopesticide on certain crops and fruits to prevent the growth of frost-forming bacteria on leaves and blossoms (Compant et al ., 2005; Raaijmakers et
al ., 2006; EPA, 2011). It is also used as seed treatment agent for damping off diseases caused by fungi (Haas and Defago, 2005; Thrane et al ., 2001; Voisard et al ., 1989) and nematodes (Hamid et al ., 2003). This pesticide activity of P. fluorescens is attributed to three mechanisms: competition for an ecological niche or a substrate, production of inhibitory chemicals, and induction of systemic resistance in host plants to a broad spectrum of pathogens (Compant et
al ., 2000; Haas and Defago, 2005).

Naturally occurring strains of P. fluorescens have been registered commercially for the control of frost injury and fire blight on pear (Wilson and Lindow, 1993). Since 1992, 4 end products containing P. fluorescens strains as active ingredients have been approved by US-EPA (EPA, 2011). US-EPA has recognized that this bacterial active ingredient is not expected to have any adverse health effects on humans, based on various studies that found no evidence that these
P. fluorescens strains are harmful to mammals (EPA, 2011). In addition, US-EPA has established a tolerance exemption for residues of P. fluorescens in or on raw agricultural commodity mushrooms (EPA, 1994).

Moreover, strains of P. fluorescens have been genetically modified to encapsulate crystal δ

endotoxins (Cry proteins) from the bacterium Bacillus thuringiensis ( Bt ) (Downing et al ., 2000, Peng et al ., 2003). The Cry proteins encapsulated by P. fluorescens showed high insecticidal activity and retained their activity for two to three times longer than conventional Bt formulations (Peng et al. , 2003).

In pharmaceutical uses, P. fluorescens produces the antibiotic pseudomonic acid (also called mupirocin), which is used to prevent Staphylococcus aureus infections (Hothersall et al ., 2007;
Tacconelli et al ., 2003).

Finally, due to the metabolic diversity of P. fluorescens , it may be used in bioremediation applications. P. fluorescens is able to degrade a wide variety of compounds, including 3- chlorobenzoic acid, naphthalene, phenathrene, fluorene and fluoranthene, chlorinated aliphatic hydrocarbons, styrene, pure hydrocarbons and crude oil (OECD, 1997).Soybean Event FG72 Petition - Revised Page 38 of 198

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### The source organism of the hppd gene, P. fluorescens

, is ubiquitous in the environment, including soil, water and food. It has many beneficial uses in agriculture, human health and bioremediation. Despite this widespread presence, it is not described as allergenic, toxic or pathogenic to healthy humans and animals and has an overall history of safe use.

VI.B.2.4. Familiarity of the gene product

History of safe consumption HPPD is ubiquitous in nature across all kingdoms: bacteria, fungi, plants and animals including mammals. For instance, HPPD amino acid sequences have been determined in bacteria such

as Streptomyces avermitilis (Accession number Q53586), in fungi such as Aspergillus fumigatus (Accession number Q4WPV8), in plants such as Arabidopsis thaliana (Accession number P93836), and in animals such as Caenorhabditis elegans (Accession number Q22633), mouse ( Mus musculus , Accession number P49429), and human ( Homo sapiens , Accession number P32754).

In particular, HPPD has been characterized in organisms present in human food, such as carrot ( Daucus carota , Accession number O23920; Garcia et al ., 1997), barley ( Hordeum vulgare Accession number O48604; Falk et al ., 2002), pork ( Sus scrofa , Accession number Q02110; Endo, 1992) and beef ( Bos Taurus , Accession number Q5EA20; Harhay, 2005).

No toxicity or allergenicity findings were found associated with HPPD proteins.

In conclusion, HPPD proteins are present in food from plant, fungal or animal origin, with good safety records. Therefore, HPPD have a history of safe consumption.

VI.C. Expression of the 2mEPSPS and HPPD W336 proteins in event FG72

VI.C.1. Expression in grain

Expression of the 2mEPSPS (Currier and Harbin, 2011) and HPPD W366 (Poe, 2009) proteins was determined from field-grown event FG72 soybean plants. The trial design applied at all 10 locations was a randomized complete block (RCB) design including three plots planted with the non-transgenic variety Jack and six plots planted with event FG72. Three additional plots were planted with the non-transgenic commercial soybean lines Stine® 2686-6, Stine® 2788 and Stine® 3000-0. There were a total of 12 plots at each of the 10 trial locations.

The plants were grown under conditions typical of production practices. Transgenic and nontransgenic plants were treated identically, except for the IFT and GLY treatments of some transgenic plots. Fertilization and normal cultural practices were carried out by MSTech test site personnel.

Treatment with IFT (at a target rate of 70 grams ai/hectare) and GLY herbicide (at 1060 grams ai/hectare) was done as a foliar spray at about the V4-V5 growth stage. Ammonium sulfate at 2850 grams/hectare was added to the spray mixture. Plots not treated with these herbicides were conventionally treated.Soybean Event FG72 Petition - Revised Page 39 of 198

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### Soybean seed samples were harvested from the center two rows of each six-row plot for each field trial. The soybean plots were harvested at normal maturity by mechanical means. The samples were stored after harvest and shipped to the BCS, BioAnalytics, RTP, North Carolinaat ambient temperatures. Within one week of arrival at BCS, the seed samples were subsampled and transferred to frozen storage. Appendix 2.C. describes the materials and methods for the 2mEPSPS and HPPD W336 protein levels in grain.

The expression results of the 2mEPSPS and HPPD W 336 protein levels in soybean seeds were obtained for the conventionally treated and GLY + IFT herbicide treated entries. The across location averages as well as the range of protein expression are presented in Table 6.

A fairly broad range of expression was observ ed for the 2mEPSPS protein in the FG72 soybean grain from entries B (conventional treated) and C (treated with GLY + IFT herbicides). The expression level was between 87 and 180 μ g/g fw in seeds treated with a conventional herbicide (mean value: 130 μ g/g fw) and between 87 and 240 μ g/g fw in the seeds treated with GLY + IFT (mean value: 140 μ g/g fw).

The dry weight and % crude protein analyte amounts were calculated using the average 2mEPSPS value of samples from each plot. The respective amounts for the 2mEPSPS protein expressed on a dry weight basis were150 μ g/g dw in seeds from conventionally treated and in seeds from GLY + IFT treated soybean plants.

The HPPD W336 protein content ranged from 0.46 to 1.32 μ g/g fw in the seeds from soybean plants treated with a conventional herbicide (mean value: 0.85 μ g/g fw) and from 0.41 to 1.31

μ g/g fw in the seeds from soybean plants treated with GLY + IFT (mean value: 0.8 μ g/g fw). When converted to a dry weight basis, the expression levels were 0.62 to 1.26 μ g/g dw (mean
value: 0.94 μ g/g dw) and 0.54 to 1.40 μ g/g dw (mean value: 0.89 μ g/g dw).

Table 6. Protein expression levels in event FG72 soybean seeds

Protein Treatment/Entry Fresh Weight (μg/g) a 

### Dry Weight (μg/g) Content as %
Crude Protein b 

### Range 87 – 180 2mEPSPS Conventional
Treated/B Mean ± SD 130 ± 22 150 b 

### 0.039 b



### Range 87 – 240 2mEPSPS GLY + IFT
Treated/C Mean ± SD 140 ± 33 150 b 

### 0.041 b



### Range 0.46– 1.32 HPPD
W336 Conventional
Treated/B Mean ± SD 0.85 ± 0.20 0.94 c 

### 0.00024 c



### Range 0.41 – 1.31 HPPD
W336 GLY + IFT
Treated/C Mean ± SD 0.80 ± 0.22 0.89 c 

### 0.00023 c

a Range and overall mean ± standard deviation in μg/g for the fr esh weight protein content in grain samples from the
10 sites.

b The dry weight (dw) and % crude protein analyte amou nts were calculated using the average 2mEPSPS value of samples from each plot.

c The dry weight (dw) and % crude protein analyte am ounts were determined us ing the average of four individual results per sample, therefore no standard deviation or range is given for these amounts.Soybean Event FG72 Petition - Revised Page 40 of 198

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### In summary, the mean concentration of 2mEPSPS protein measured in soybean seeds was 150

μ g/g dw for both entries B an C. The mean concentration of HPPD W336 measured in soybean seeds on a dry weight basis was 0.94 μg/g dw and 0.89 μg/g dw for entries B an C, respectively.

VI.C.2. Expression in plant parts and during the life cycle

The HPPD W336 and 2mEPSPS protein expression levels were determined in different tissues at different growth stages of event FG72 so ybean (Habex and Debavey e, 2009). Event FG72 and wt plants were grown under greenhouse conditions. Leaf samples were taken at three different growth stages (V4, V6 and V8) and samples of stem and root were taken at two different growth stages (V4 and V8). Samples of the wt soybean line (Jack) were also sampled at the same stages for the same tissues. Seeds were collected for both event FG72 and wt soybean plants. Samples were analyzed for HP PD W336 and 2mEPSPS protein content by ELISA. HPPD W336 and 2mEPSPS proteins were detected in all event FG72 samples for all growth stages and tissue types analyzed. Ex pressions levels for HPPD W336 and 2mEPSPS proteins are summarized in Tables 7 and 8, respectively. Appendix 2.D. describes the materials and methods for the 2mEPSPS and HPPD W336 protein levels in different plants parts and during the life cycle.Soybean Event FG72 Petition - Revised Page 41 of 198

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### Table 7. HPPD W336 protein expression in different tissues and growth stages

HPPD W336 protein content

μg/g fresh weight (fw) μg/g dry weight (dw) Matrix Growth stage

Average ± SD Range Average ± SD Range

V4 6.10 ± 2.78 2.65 – 10. 4 38.4 ± 17.5 16.7 – 65.7

V6 6.48 ± 4.08 2.31 – 17. 4 35.8 ± 22.5 12.8 – 96.0 Leaf

V8 4.69 ± 1.87 2.00 – 8. 91 27.2 ± 10.9 11.6 – 51.8

V4 1.48 ± 0.42 0.74 – 2. 20 16.6 ± 4.65 8.29 – 24.6 Stem V8 0.69 ± 0.35 0.29 – 1. 49 6.04 ± 3.10 2.49 – 13.0

V4 0.87 ± 0.35 0.45 – 1. 66 5.81 ± 2.30 2.98 – 11.0
Root V8 0.84 ± 0.50 0.20 – 1. 64 6.42 ± 3.82 1.51 – 12.4

Seed NA 1.27 ± 0.42 0.71 – 2.68 1.41 ± 0.47 0.79 – 2.96



### Table 8. 2mEPSPS protein expression in different tissues and growth stages

2mEPSPS protein content

μg/g fresh weight (fw) μg/g dry weight (dw) Matrix Growth stage

Average ± SD Range Average ± SD Range

V4 90.4 ± 26.1 44.9 – 152 569 ± 164 283 – 958

V6 79.1 ± 29.6 39.2 – 136 437 ± 163 216 – 753 Leaf

V8 115 ± 38.2 60.5 – 203 668 ± 222 351 – 1180

V4 18.8 ± 6.16 6.08 – 31.3 211 ± 68.9 68.0 – 350 Stem V8 13.4 ± 2.62 8.71 – 17.3 117 ± 22.9 76.1 – 151

V4 4.89 ± 1.99 1.63 – 8.21 32.5 ± 13.2 10.8 – 54.5
Root V8 5.75 ± 2.31 2.62 – 10.7 43.7 ± 17.6 19.9 – 81.2Soybean Event FG72 Petition - Revised Page 42 of 198

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### VI.D. Biochemical and functional equivalence of the expressed proteins

The 2mEPSPS and HPPD W336 proteins were produced in E. coli for use in studies to investigate the toxicity and potential allergenicity of the protein, since it is not feasible to produce an adequate amount of the respective proteins for these studies from event FG72 soybean plants. Therefore, it was necessary to demonstrate the equivalence between the E.coli produced proteins and the plant-produced proteins in order to utilize the safety data. The E.coli

produced, and plant-produced 2mEPSPS and HPPD W336 proteins, were compared, using the 5 following criteria and associated methods listed in Table.

Table 9. Criteria and methodologies for demonstrating protein equivalence

Equivalence criteria Methodology a

Confirm identity of 2mEPSPS and HPPD W336 proteins Edman degradation

Comparable molecular weight Protein mobility in SDS-PAGE

Comparable immuno-reactivit y Western blot analysis

Comparable peptide masses HPLC/Ele ctrospray Mass Spectrometry (LC/MS) of peptides Comparable biological activity Enzymatic activity

a N-terminal amino acid sequencing and enzyme activity analyses could not be performed on the HPPD W336 protein produced in plants because of the sma ll amount of protein isolated from the plant.



### VI.D.1. 2mEPSPS protein

Equivalence of E.coli produced 2mEPSPS protein and plant produced protein was established via SDS-PAGE, western blot, N-terminal ami no acid sequencing, enzyme activity and LC/MS
methods.

The SDS-PAGE (Appendix 3, Figures 33 and 34) and western blot (Appendix 3, Figure 35) demonstrated that the molecular weight and mobility of the two proteins are the same. The western blot also indicated that both proteins have the same immuno-reactivity.

The activity assay indicated that both proteins were active.

The N-terminal amino acid sequence data suggests that both the N-terminal of the E. coli produced 2mEPSPS protein and the FG72 soybean produced 2mEPSPS protein is missing the N-terminal methionine. The loss of a methionine is not unusual, post-translational modifications, such as removal of a methionine are often found in proteins from both prokaryotic and eukaryotic organisms (Bradshaw et al ., 1998). Only a small amount of the N-terminal peptide
with the methionine was detected for the E. coli produced 2mEPSPS protein by N-terminal amino acid sequencing. The N-terminal amino acid sequence data also suggests that some of the plant produced 2mEPSPS protein may be blocked at the N-terminus. The mass spectrometry data confirms the N-terminal amino acid sequencing data.Soybean Event FG72 Petition - Revised Page 43 of 198

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### The mass spectrometry results indicate that 98% of the E. coli- produced 2mEPSPS sequence coverage and 71% sequence coverage of the FG72 soybean produced 2mEPSPS protein was detected (Appendix 3, Tables 51 and 52). The mass spectrometry data presents direct evidence that 72% of the protein sequence of the FG72 soybean produced 2mEPSPS protein is identical to the protein sequence of the E. coli produced 2mEPSPS protein

. Taken together, the analytical results demonstrate equivalence of the FG72 soybean produced 2mEPSPS protein to
the E. coli produced 2mEPSPS protein.

VI.D.2. HPPD W336 protein

Equivalence of E. coli produced HPPD W336 protein and the plant produced HPPD W336 protein was established via SDS-PAGE, wester n blot and LC/MS methods. N-terminal amino acid sequencing and enzyme activity analyses could not be performed on the plant produced HPPD W336 protein because of the small amount of protein isolated from the plant.

The SDS-PAGE (Appendix 3, Figure 36) and western blot (Appendix 3, Figure 37) demonstrated that the molecular weight and mobility of the two proteins are the same. The western blot also indicated that both proteins have the same immuno-reactivity.

The activity assay indicates that the protein from the E. coli -produced protein was active; but the concentration of the protein in event FG72 soybean leaves was below the limit of detection of the assay.

An N-terminal amino acid sequence analysis could not be performed on the plant produced HPPD W336 protein because the amount of protein isolated from the leaves was insufficient for analysis. However, an N-terminal amino acid sequence analysis was performed on the E. coli - produced HPPD W336 protein. The N-terminal amino acid sequencing results indicated that the methionine was missing from the N-terminal of the E. coli produced HPPD W336 protein. Mass spectrometry analysis results of the E. coli produced HPPD W336 protein confirmed that the methionine was missing from the N-terminal peptide.

Mass spectrometry analysis results of the plant produced HPPD W336 protein also indicate that the methionine is missing from the N-terminal peptide. A 95.2 % sequence coverage of the E.
coli produced HPPD W336 protein and 70.1% sequence coverage of the plant produced HPPD W336 protein was detected by mass spectromet ry (Appendix 3, Tables 53 and 54). The mass spectrometry results indicate that 71% of the protein sequence of plant produced HPPD W336 protein is equivalent to the protein sequence of E. coli produced HPPD W336 protein

. Taken together, the analytical results demonstrate equivalence of the plant produced HPPD W336 protein to the E. coli produced HPPD W336 protein.Soybean Event FG72 Petition - Revised Page 44 of 198

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### VI.E. Summary of the Food and Feed Safety Assessment of the 2mEPSPS Protein

VI.E.1. Familiarity to the protein

EPSPS is the 6 th 

### enzyme of the shikimate pathway, the metabolic pathway for the biosynthesis of aromatic compounds found in microorganisms and in plants (Herrmann et al ., 1995). As such, it has been shown that EPSPS enzymes are ubi quitous in nature and are present in foods derived from plant and microbial sources.

In addition, insensitivity of some EPSPS enzymes to glyphosate also exists in nature at various levels and has been specifically studied for the development of the glyphosate tolerance trait in plants (Van der Klis et al ., 2006).

It is apparent that these proteins have a long history of safe use as endogenous components of food and feed. There is no evidence suggesting t hat these proteins may be related to any type of allergenicity or toxicity to humans or other animals (Herouet-Guicheney, 2009). Thus, exposure to the known EPSPS proteins can be deemed as innocuous as exposure to other naturally occurring proteins without inducing adverse effects (ILSI, 2011).

The 2mEPSPS, which contains only two amino ac id substitutions of the maize wt EPSPS protein, was modified in such a way that the enzymatic characteristics remain as much as possible unchanged with the exception of t he insensitivity to glyphosate (Schultz et al ., 1985). Therefore, it is expected to have the same safety profile as the wild-type protein.

VI.E.2. Potential allergenicity

VI.E.2.1. Homology search to known allergens

The overall amino acid sequence homology search was carried out by using FASTA algorithm, which compares the complete amino acid sequ ence of the 2mEPSPS protein with all protein sequences present in the public allergen database AllergenOnline (www.allergenonline.com; release 8.0, 1313 sequences) (Capt, 2008 a and b). The criterion indicating potential allergenicity was 35% identity over at least 80 consecutive amino acids with an allergenic protein.

In addition, an allergenic identity search (80-mer amino acid sequence homology) was performed to compare the query sequence subdivided into 80 amino acid blocks, with all known allergens present in the AllergenOnline database. The criterion indicating potential allergenicity was 35 % identity with an allergenic protein.

Furthermore, the amino acid sequence of the 2mEPSPS protein, subdivided into 8 amino acid blocks, was compared with all known allergens present in the allergen database (epitope search). The algorithm used was FindPatterns and the criterion indicating potential allergenicity was 100 % identity on a window of 8 amino acids with an allergenic protein.

The overall and 80-mer identity searches showed no relevant similarity between the 2mEPSPS sequences and any known allergenic sequences from the allergen database. In addition, theSoybean Event FG72 Petition - Revised Page 45 of 198

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### epitope search showed no identity between all t he blocks (8 amino acids) of the 2mEPSPS protein and known allergens.

Although very conservative, this homology search confirms that it is unlikely that the 2mEPSPS protein possesses any allergenic properties.

VI.E.2.2. Potential N-glycosylation sites

Potential N-glycosylation sites were determined using in silico search of the 2mEPSPS protein sequence for the presence of the consensus epitope Asn-Xaa-Ser/Thr (N-X~P-S/T), where Xaa = any amino acid except Pro (P), and Asn-Xaa-Cys (N-X-C) (Capt, 2008 a, Larsen et al ., 1998).

Two potential N-glycosylation sites were identified on the amino acid sequence of the 2mEPSPS protein. However, the biological relevance of those potential N-glycosylations in eliciting allergenic response is not proven. The 2mEPSPS protein is not expected to be glycosylated, since chloroplastic proteins targeted directly to the chloroplast do not transit through the Endoplasmic Reticulum (ER) where gl ycosylation occurs in eukaryotes (Mousdale and Coggins, 1985, Pattison and Amtmann, 2008)

. In bacteria, protein glycosylation is rare (Sherlock et al ., 2006).

Furthermore, in the specific case of event FG72, it has been shown that the 2mEPSPS protein is not glycosylated (see Section D.1.). Therefore, potential allergenicity triggered by the presence of N-glycosylation sites is a remote possibility.

VI.E.2.3. In vitro digestibility in human simulated gastric fluid

The 2mEPSPS protein was assayed for digestibility in SGF containing pepsin at pH 1.2 for incubation times from 0.5 to 60 minutes (Rouquié, 2006a).

The test protein was incubated at 37°C in SGF with pepsin at a final concentration of 10 units of pepsin per μg test protein, at pH 1.2, and samples were taken for analysis at time-points of 0, 0.5, 2, 5, 10, 20, 30 and 60 minutes. The resulting protein solution was analyzed for presence of the test proteins and potential stable protein fragments by SDS-PAGE followed by Coomassie blue staining. Appropriate controls included the test protein at pH 1.2 without pepsin and SGF without the test protein.

Coomassie blue staining analysis showed that t he 2mEPSPS protein was very rapidly digested in pepsin at pH 1.2, within 30 seconds of incubation. No fragment bands were found to result from digestion of the 2mEPSPS protein.

In conclusion, the 2mEPSPS protein is very rapidly degraded in SGF. This minimizes the likelihood that this protein could survive in the human digestive tract and cause an allergic
reaction.Soybean Event FG72 Petition - Revised Page 46 of 198

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### VI.E.2.4. In vitro digestibility in human simulated intestinal fluid

The 2mEPSPS protein was further tested for stability in SIF with pancreatin at pH 7.5 for incubation times from 0.5 to 60 minutes, using a protocol adapted from the SGF assay (Rouquié, 2006b). A solution of the test protein was incubated with SIF, a porcine pancreatin solution at pH 7.5, at approximately 37°C. Then samples were analyzed at time-points of 0, 0.5, 2, 5, 10, 20, 30 and 60 minutes for the presence of the 2mEPSPS protein or potential stable protein fragments by western blot. The imm unodetection was performed using a polyclonal antibody directed against the 2mEPSPS protein. Appropriate controls included 2mEPSPS protein in buffer without pancreatin and SIF without 2mEPSPS protein.

Western blot analysis showed that the 2mEPSPS protein band was not visible anymore at time 0 and all subsequent incubation times, indicating that the 2mEPSPS protein was degraded within a few seconds in the presence of pancreatin.

In conclusion, a complete digestion of the 2mEPSPS protein was observed within a few seconds of incubation with SIF, in presence of pancreatin, at pH 7.5.

Rapid degradation of the 2mEPSPS protein in the SGF and SIF indicates a minimal likelihood that the protein could survive and be absorbed through the gastrointestinal system. In case the protein survives in the stomach, 2mEPSPS would be rapidly degraded in the intestine.

VI.E.2.5. In vitro stability to heat

Highly purified (>99%) 2mEPSPS protein produced in E. coli (batch LEJ5837) was tested for structural stability at temperatures of 60, 75 or 90°C for periods of 10, 30 or 60 minutes. The protein was examined by SDS-PAGE followed by Coomassie blue staining or by western blot analysis (Rouquié, 2007). The immunodetection was performed using a polyclonal antibody directed against 2mEPSPS protein.

The Coomassie blue-stained SDS-PAGE showed no visible changes of the band intensity at 60°C and 75°C from 10 up to 60 minutes. After 30 minutes of incubation at 90°C, the band was visible with a lower intensity than other heated samples at 60°C and 75°C. After 60 minutes at 90°C, the band was still visible, with a marked decrease in intensity compared to all other samples, including the unheated sample.

The western blot analysis showed an unchanged in tensity of the intact 2mEPSPS band after incubation at 60°C or 75°C for 10 up to 60 minutes. At 90°C, the intensity of the intact 2mEPSPS band was unchanged after 10 and 30 minutes, but was decreased after 60 minutes, in accordance with the results obtained by SDS-PAGE analysis after Coomassie blue staining.

In conclusion, the 2mEPSPS protein is parti ally heat-stable up to 90°C for 60 minutes.Soybean Event FG72 Petition - Revised Page 47 of 198

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### VI.E.3. Homology search to known toxins

The overall amino acid sequence identity search was carried out by using BLASTP algorithm, which compared the complete amino acid sequ ence of the 2mEPSPS protein with all protein sequences present in the following large reference databases: Uniprot_Swissprot, Uniprot_TrEMBL, PDB, DAD and GenPept (Capt, 2008c). The scoring matrix used wasBLOSUM62. The overconservative criterion for selecting similar proteins was a threshold Evalue of 1.0. Matched sequence proteins were further examined for potential toxicity records in literature in order to assess their biological relevance.

The results showed no sequence identity of the 2mEPSPS protein with known toxins.

In conclusion, it is unlikely that the 2mEP SPS protein would exhibit any toxic properties.

VI.E.4. Acute toxicity study in the mouse

A group of 5 female OF-1 mice were treated by oral gavage with the 2mEPSPS protein produced in E. coli (>99% purity) at a dose level of 2000 mg/kg body weight (Rouquié, 2006c). Another group of 5 female OF-1 mice were treated by oral gavage with bovine serum albumin at the same dose level as the negative control. All animals were observed for clinical signs daily for 15 days, with special attention given during the first 4 hours. Their body weights were measured weekly. At study termination, animals were subjected to a necropsy including a macroscopic examination and the spleen, liver, kidney and brain were weighed.

There were no mortalities, no clinical signs or treatment-related effects in female OF1 mice.

In conclusion, a single administration of the 2mEPSPS protein at 2000 mg/kg body weight via the oral route did not produce signs of systemic toxicity in the OF1 female mouse. The acute
oral LD 50 

### of 2mEPSPS was found to be greater than 2000 mg/kg body weight in mice.

These results taken together with the results of the homology search with known toxins indicate that it is unlikely that the 2mEPSPS pr otein would exhibit any toxic properties.

VI.F. Summary of the Food and Feed Safety Assessment of the HPPD Protein

VI.F.1. Familiarity to the protein

HPPD is ubiquitous in nature across all kingdoms: bacteria, fungi, plants and animals including mammals. For instance, HPPD amino acid sequences have been determined in bacteria such

as Streptomyces avermitilis (Accession number Q53586), in fungi such as Aspergillus fumigatus (Accession number Q4WPV8), in plants such as Arabidopsis thaliana (Accession number P93836), and in animals such as Caenorhabditis elegans (Accession number Q22633), mouseSoybean Event FG72 Petition - Revised Page 48 of 198

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### ( Mus musculus , Accession number P49429), and human ( Homo sapiens , Accession number P32754).

In particular, HPPD has been characterized in organisms present in human food, such as carrot ( Daucus carota , Accession number O23920; Garcia et al ., 1997), barley ( Hordeum vulgare Accession number O48604; Falk et al ., 2002), pork ( Sus scrofa , Accession number Q02110; Endo, 1992)) and beef ( Bos Taurus , Accession number Q5EA20; Harhay, 2005).

In conclusion, HPPD proteins are present in food from plant, fungal or animal origin, with good safety records. Therefore, HPPD proteins have a history of safe consumption.

VI.F.2. Potential allergenicity

VI.F.2.1. Homology search to known allergens

The overall amino acid sequence homology search was carried out by using FASTA algorithm, which compares the complete amino acid sequence of the HPPD W336 protein with all protein sequences present in the public allergen database AllergenOnline (www.allergenonline.com; release 9.2, 1386 sequences) (Capt, 2009a). The criterion indicating potential allergenicity was 35% identity over at least 80 consecutive amino acids with an allergenic protein.

In addition, an allergenic identity search (80-mer amino acid sequence homology) was performed to compare the query sequence subdivided into 80 amino acid blocks, with all known allergens present in the AllergenOnline database. The criterion indicating potential allergenicity was 35 % identity with an allergenic protein.

Furthermore, the amino acid sequence of the HPPD W336 protein, subdivided into 8 amino acid blocks, was compared with all known allergens present in the AllergenOnline allergen database (epitope search). The algorithm used was FindPatterns and the criterion indicating potential allergenicity was 100 % identity on a window of 8 amino acids with an allergenic protein.

The overall and 80-mer identity searches showed no relevant similarity between the HPPD W336 sequences and any known allergenic sequences from the allergen database. In addition, the epitope search showed no identity between all the blocks (8 amino acids) of the HPPD W336 protein and known allergens.

Although very conservative, this homology search confirms that it is unlikely that the HPPD W336 protein possesses any allergenic properties.

VI.F.2.2. Potential N-glycosylation sites

Potential N-glycosylation sites were determined using an in silico search of the HPPD W336 protein sequence for the presence of the cons ensus epitope Asn-Xaa-Ser/Thr (N-X~P-S/T), where Xaa = any amino acid except Pro (P ), and Asn-Xaa-Cys (N-X-C) (Capt, 2009a).Soybean Event FG72 Petition - Revised Page 49 of 198

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### No potential N-glycosylation sites were identified on the amino acid sequence of the HPPD W336 protein.

VI.F.2.3. In vitro digestibility in human simulated gastric fluid

The test protein was incubated at 37°C in SGF with pepsin at a final concentration of 10 units of pepsin per μg test protein, at pH 1.2, and samples were taken for analysis at time-points of 0, 0.5, 2, 5, 10, 20, 30 and 60 minutes (Rascle, 2009a). The resulting protein solution was analyzed for presence of the test proteins and potential stable protein fragments by SDS-PAGE followed by Coomassie blue staining or by we stern blot. The immunodetection was performed using a polyclonal antibody directed against HPPD W336 protein. Appropriate controls included the test protein at pH 1.2 without pepsin, SGF without the test protein, and a 10% loading control (1/10 dilution of the test protein). This 10% loading control was used to estimate the time to reach 90% digestion of the protein, i.e. the first sample time having less than 10% residual protein.

Both Coomassie blue staining and western blot analysis showed that the HPPD W336 protein was very rapidly digested in pepsin at pH 1.2, with more than 90% of the protein being digested in less than 30 seconds. No fragments were found as a result of the digestion of the HPPD W336 protein in SGF.

In conclusion, the HPPD W336 protein is very rapidly degraded in simulated gastric fluid. This minimizes the likelihood that this protein could survive in the human digestive tract and cause an allergic reaction.

VI.F.2.4. In vitro digestibility in human simulated intestinal fluid

The HPPD W336 protein was further tested for stability in SIF with pancreatin at pH 7.5 for incubation times from 0.5 to 60 minutes, using a protocol adapted from the SGF assay (Rascle, 2009b). A solution of the test protein was incubated with SIF, a porcine pancreatin solution at pH 7.5, at approximately 37°C. Samples were analyzed at time-points of 0, 0.5, 2, 5, 10, 20, 30 and 60 minutes for the presence of the HPPD W336 protein or potential stable protein fragments by SDS-PAGE coupled with Coomassi e blue staining and western blot analyses. The immunodetection was performed using a poly clonal antibody directed against the HPPD W336 protein. Appropriate controls included HPPD W336 protein in buffer without pancreatin, the corresponding 10% loading control (to verify the sensitivity of the detection procedure) and SIF without HPPD W336 protein.

Both Coomassie blue staining and western blot analysis showed that the HPPD W336 protein band was not visible anymore at time 0 and all subsequent incubation times, indicating that more than 90% of the HPPD W336 protein was degraded within a few seconds in presence of pancreatin.

In conclusion, a complete digestion of the HPPD W336 protein was observed within a few seconds of incubation with SIF, in presence of pancreatin, at pH 7.5.Soybean Event FG72 Petition - Revised Page 50 of 198

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### Rapid degradation of the HPPD W336 protein in the SGF and SIF indicates a minimal likelihood that the protein could survive and be absorbed thr ough the gastrointestinal system. In case the protein survives in the stomach, HPPD W336 would be rapidly degraded in the intestine.

VI.F.2.5. In vitro stability to heat

The effect of heat on the enzyme activity and structural stability of the protein was investigated. For enzymatic activity, an absorbance assay wa s developed which monitored the production of homogentisate and CO 2 

### from 4-HPP and O 2 

### catalyzed by HPPD (Habex, 2009).

To assess the temperature stability of the HPPD W336 protein, the protein was incubated at 45°C, 60°C and 95°C for 2.5, 5, 10, 20 and 60 minutes (Habex, 2009). Subsequently the activity of the protein was assessed under standard conditions (room temperature). The activity drops below 50% after the protein was incubated at 45°C for 20 minutes. At more elevated temperatures (60°C and 95°C) HPPD activity is abolished after 2.5 minutes.

Figure 7. The effect of temperature on the activity of the HPPD W336 enzyme



### Temperature stability

0.000 2.000 4.000 6.000 8.000 10.000 12.000 14.000 16.000 18.000

0 10 20 30 40 50 60

Storage time (min)

* under the used conditionsAmount converted HPP / mg HPPD W336 *

45°C

60°C

95°CSoybean Event FG72 Petition - Revised Page 51 of 198

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### Highly purified HPPD W336 protein produced in E. coli (96% purity) was also tested for structural stability at temperatures of 60, 75 or 90°C for periods of 10, 30 or 60 minutes (Rascle, 2009c). The protein was examined by SDS-PAGE followed by Coomassie blue staining and by western blot analysis. The immunodetection was performed using a polyclonal antibody directed against HPPD W336 protein. A 10% loading control was included in gels, to verify the sensitivity of the staining procedures.

The Coomassie blue-stained SDS-PAGE showed no significant changes in the HPPD W336 protein after heat treatment at 60, 75 or 90°C from 10 to 60 minutes, with intensities similar to the unheated sample. Similar results were obtained with western blot analyses.

These findings illustrate that while the HPPD W336 protein may retain its structural stability, its enzymatic activity decreases rapidly at 45 o 

### C and is completely lost at higher temperatures (60 o 

### C and above) after 2.5 minutes.

VI.F.3. Homology search to known toxins

An overall amino acid sequence identity sear ch was carried out by using BLASTP algorithm, which compared the complete amino acid sequence of the HPPD W336 protein with all protein sequences present in the following large reference databases: Uniprot_Swissprot, Uniprot_TrEMBL, PDB, DAD and GenPept (Capt, 2009b). The scoring matrix used was BLOSUM62 (block substitution matrix at ≥ 62% alignment). The overconservative criterion for selecting similar proteins was a threshold E-value of 0.1. Matched sequence proteins were further examined for potential toxicity records in the scientific literature in order to assess their biological relevance.

As expected, the query sequence matched with t he HPPD proteins (also called HPPDase; MelA; enzyme classification E.C. 1.13.11.27) from P. fluorescens and other bacterial origins. No records were found on the potential toxicity associated with these proteins.

An identity of 54% was observed with the VLLY protein (Uniprot_SwissProt entry: O06695) from
Vibrio vulnificus , a pathogenic bacterium present in sea waters and able to infect humans who consume seafood (Chang et al ., 1997). Similarities with lower percentages of identity (49-50%) were also found between HPPD W336 and LLY, also called legiolysin, from strains of Legionella pneumophila . Both VLLY protein and LLY are suspected to be hemolysins, and are also annotated to belong to the HPPD family.

Because VLLY and LLY proteins are also annotated as HPPD proteins, the hypothesis was raised that the similarity between these prot eins and the HPPD W336 protein was caused by the presence of HPPD domains on the VLLY and LLY proteins. To test this hypothesis, a multiple alignment of the HPPD W336, VLLY, LLY and 27 other HPPD sequences extracted from the Uniprot_Swissprot database from bacteria, plants, vertebrates, invertebrates and fungi was performed. Refined analysis of this multiple alignment showed that similarities between HPPD W336 and either VLLY or LLY proteins correspond to the domains conserved among all HPPD proteins.Soybean Event FG72 Petition - Revised Page 52 of 198

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### These observations support the hypothesis that the VLLY and LLY proteins are HPPDs and therefore, share the typical HPPD structure wi th HPPD W336. This is corroborated by several authors who demonstrated the HPPD activity of the LLY protein (Wintermeyer et al ., 1994;
Steinert et al ., 2001). In addition, although VLLY or LLY protein expression was shown to be necessary for the hemolytic activity of bacteria, the direct hemolytic activity of these proteins was not observed (Wintermeyer et al. , 1994; Chang et al. , 1997; Steinert et al. , 2001).

In conclusion, the HPPD W336 is similar to other HPPD proteins from various origins. The list includes the HPPD-like hemolysin (e.g. from Vibrio vulnificus ), which is described as a bacterial toxin. However, a refined bioinformatics analysis demonstrated that this specific homology was unlikely to be relevant. This match is due to the domains of proteins that possess the HPPD activity and that are conserved between all the HPPD proteins.

Since all other HPPD proteins from various organisms, although sharing the typical HPPD conserved domains, display no toxic properties, and since no limitation cut-off criteria have been determined for defining relevant homologies to toxins (Delaney et al ., 2008), this homology is highly unlikely to be biologically relevant.

VI.F.4. Acute toxicity study in the mouse

A group of 5 female OF1 mice were administered a single dose of purified E.coli produced HPPD W336 protein (97% purity) by oral gavage at the dose level of 2000 mg/kg body weight (Rascle, 2009d). A similarly constituted group of 5 female mice received bovine serum albumin (BSA) at the same dose level and acted as a negative control. Both proteins were administered in two doses of 1000 mg/kg body weight each administered within a 4 hours period on the day
of treatment. All animals were observed for c linical signs daily for fifteen days. Their body weights were measured weekly. At termination of the study period, all animals were subjected to a necropsy including a macroscopic examination, and the spleen, liver, kidney and brain were weighed. Microscopic examination of the spleen was performed.

There were no mortalities, no clinical signs or treatment-related effects on body weight, body weight gain, organ weights, gross and microscopic examinations.

In conclusion, an acute oral dose of 2000 mg/kg body weight of the HPPD W336 protein did not induce any evidence of systemic toxi city in the OF1 female mouse.Soybean Event FG72 Petition - Revised Page 53 of 198

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### VII. AGRONOMIC AND PHENOTYPIC EVALUATION

VII.A. History of field activities

Event FG72 has been field tested in adapted growing regions of the United States and Canada, and in winter nurseries (Argentina, Brazil, Chile and Puerto Rico). The field activities were managed by BCS from 2001 to 2003 and by MSTech beginning in 2007. Field activities in Canada are managed by BCS. Table 10 presents a summary of the field trials and associated USDA notifications (see Appendix 1 for field trial termination reports). Table 11 lists the field trials and associated CFIA permits granted for Canada. The activities in Argentina are
summarized in Table 12.

Table 10. Summary of field activities under USDA notifications for event FG72

USDA
Notification # Planting /
Harvest Dates Number of
Locations Type of Trial Locations

01-268-11n Dec 2001 / April 2002 1 Herbicide efficacy, Progeny advanced PR

02-071-03n June 2002 /
Nov 2002 1 Herbicide efficacy, Progeny advanced IN

02-071-03n July 2002 /
November 2002 1 Herbicide efficacy, Progeny advanced PR

02-274-09n Jan 2003 / May 2003 1 Herbicide efficacy,
Seed increase PR

03-080-05n May 2003 /
Oct 2003 7 Herbicide efficacy, Yield, Breeding IL, IA, NE

07-065-121n May 2007 / Sept-Nov 2007 1 Breeding,
Seed increase IA

07-211-105n Dec 2007 / May 2008 1 Breeding FL

08-057-116n May 2008 /
Oct 2008 10 Registration studies and Breeding AR, IA, IL, IN, MI, MN, MO

08-219-102n Oct 2008/
Feb 2009 1 Breeding,
Seed increase FLSoybean Event FG72 Petition - Revised Page 54 of 198

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### Table 11. Summary of field activities under CFIA permits for event FG72

CFIA permit # Year Trial # (Type) Location in Canada

08-MTS1-3110SOY01-0864-ON101-01 2008 HD08NARKBP (Efficacy & Tolerance) Rockwood,
Ontario

2009-MST1-387-SOY01-0864-ON016-01 2009 HD09NARKBN (Efficacy & Tolerance) Breslau, Ontario

HD09NARKBO (Efficacy & Tolerance)

2009-MST1-387-SOY01-0864-ON101-01 2009 HD08NARKBP (Efficacy & Tolerance) Rockwood,
Ontario
HD09NARKBN (Efficacy & Tolerance)
HD09NARKBO (Efficacy & Tolerance)
RAISP006 (Residue Analysis on RAC’s)
RAISP008 (Residue Analysis on RAC’s)
RAISP010 (Residue Analysis on RAC’s)



### Table 12. Summary of field activities under CONABIA permits for event FG72

CONABIA
Permit # Planting /
Harvest Dates Number of
Locations Type of Trial Location in Argentina
(Facility)

800-9541/01 Dec 2001 / April 2002 1 Basic observations, progeny advanced Balcarce (Agrar del Sur)

247377/02 16 Dec 2002 / 3 April 2003 1 Basic observations, progeny advanced Chacabuco (Don Mario)

182500/03 31 Dec 2003/ 25 April 2004 Chacabuco (Don Mario)

182500/03 12 Jan 2004/ 26 April 2004 Chacabuco (Don Mario)

295033/07 19 Dec 2007 / 5 April 2008 Seed increase and breeding Chacabuco (Don Mario)

107155/08 10 Dec 2008 /
27 March 2009 Seed increase and breeding Chacabuco (Don Mario)Soybean Event FG72 Petition - Revised Page 55 of 198

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### VII.B. Agronomic and phenotypic evaluation

Agronomic evaluations of the event FG72 were conducted in field tests in 2003 and 2008. Evaluations included key agronomic parameters to assess the growth habit and phenotype of the transformed lines, their reactions to biotic and abiotic stressors in their respective environments, and analyze soybean meal and oil fact ors. These parameters were designed to evaluate event FG72 in soybean plants to ensure commercial herbicide tolerance and agronomic performance. Other field activities, such as rating herbicide tolerance and efficacy, planting seed increases and breeding allowed for the material to be assessed for stability and performance of the introduced trait and t he agronomic characteristics of event FG72.

Event FG72 was created by transformation of the soybean variety Jack (maturity group II) to express the 2mEPSPS protein which will convey tolerance to the herbicide glyphosate (GLY) and the HPPD W336 protein which will impart tolerance to the herbicide IFT. Event FG72 was selected based on demonstrated tolerance to the herbicide glyphosate, the herbicide IFT and agronomic performance.

Glyphosate is widely used in herbicide-tolerant soybean and other agricultural production systems. IFT herbicide offers an alternative weed control option for the soybean grower. It
controls weeds via a new herbicide mode of acti on for soybeans that is effective against many of the herbicide resistant weeds currently found in soybean fields. IFT has the flexibility to be applied pre-plant, pre-emergence, or post emergence to event FG72 soybeans.

Event FG72 was evaluated by comparison to the non-transgenic counterpart Jack in different growing regions of the mid-western United States (Table 10). Agronomic performance field studies were managed in a manner representative of normal agricultural practices, including; conventional herbicide applications, both pre- and post- planting. In addition, event FG72 was evaluated for herbicide tolerance to GLY and IFT. Thus, comparisons of agronomic properties and performance of event FG72 were made under conventional herbicide and GLY and/or IFT herbicide regimens. Appendix 2.E. describes the materials and methods for the agronomics
studies.

Event FG72 was evaluated at the T 8 

### generation with Jack for equivalence testing (Table 13) to assess seed characteristics that may contribute to weediness potential of a plant such as increased seed dormancy, disease susceptibility or pest preference. No changes in fitness characteristics (reproduction, disease resistan ce, fecundity, seed dispersal, dormancy, and persistence) were found that could contribute to increased weediness potential of event FG72. Comparison of pollen from FG72 and Jack found no difference in viability or germination (Haas, 2009).

Morphological parameters necessary for the filing of a Plant Variety Protection (PVP) certificate were measured. These parameters are described in the objective description of the variety for soybean and include plant and seed descriptive characteristics, oil profile, and seed protein content among others. In all these parameters, event FG72 and Jack were found to be similar.Soybean Event FG72 Petition - Revised Page 56 of 198

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### Table 13. Summary of performance characteristics evaluated for event FG72 – US (2008)

Type of study Parameters analyzed Comparator Findings

Self and cross pollination Segregation analysis for herbicide
tolerance Jack No change in self or cross fertility

Plant phenotype comparison using PVP a

standards Flower color, pubescence color, pod color, seed coat, hilum color, canopy architecture, leaf shape, growth habit Jack,
Stine ® 2686-6, Stine 2788,
Stine 3000-0 FG72 was identical to Jack,
but some differences compared to the other
varieties

Seed phenotype comparison using PVP a

standards Seed coat color and luster, hilum color, seed coat mottling Shape class defined by L/W, L/T
and T/W ratios Jack, Stine 2686-6, Stine 2788,
Stine 3000-0 FG72 was identical to Jack,
but some differences compared to the other
varieties

Comparison of grain characters using PVP a

standards Fatty Acid profile
Total Oil content
Protein content Jack, Stine 2686-6, Stine 2788,
Stine 3000-0 No change in grain qualities

Field performance Emergence, stand, vigor, height, yield Jack, Stine 2686-6, Stine 2788,
Stine 3000-0 FG72 and the other varieties are shorter at maturity than Jack. Plant stand and yield
were reduced in FG72.

Reproduction Pollen viability, date of emergence, date of 50% flowering, date of maturity Jack No change in reproductive potential

Disease
resistance Severity rating for naturally occurring pathogens Jack No change in susceptibility

Fecundity 100 seed weight Jack No change

Seed dispersal Pod shattering, lodging rating of mature plants Jack FG72 has less lodging than
Jack

Dormancy Germination ,survival of imbibed
seed Jack No difference

Persistence Census of volunteers in subsequent

season Jack No difference observed

a Characters described by the Plant Variety Protection office of the USDA for SoybeanSoybean Event FG72 Petition - Revised Page 57 of 198

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### VII.C. Agronomic performance of event FG72

VII.C.1. Agronomic evaluation of event FG72 (2003 data)

VII.C.1.1. Yield evaluation of event FG72 under conventional weed control practices

After field trial evaluations performed in 2003 in the US, BCS and MSTech selected event FG72 as the event best suited for potential commerc ial development. The planting seed was derived from homozygous lines in the T 4 

### generation. The evaluation included agronomic performance under conventional weed control practices and tolerance evaluation to GLY and IFT herbicides. As a result, event FG72 was selected for tolerance to both herbicides and acceptable agronomic characteristics.

For the 2003 yield data summarized below (Table 14), fields were planted and managed using conventional weed control at nine locations. There were six sites, three of which had an early and a late planting. All trial locations were grown with conventional weed control (commercial rate of imazethapyr, 196 g ai/ha). Data provided are the mean of three replicated plots at each
location.

The yield of event FG72 and the parent line Jack di d not differ at any of the 9 locations. Event FG72 had a very consistent performance acro ss locations when compared to all entries receiving the conventional herbicide treatment. The yield of event FG72 follows closely the mean yield of all the entries (location mean) at each of the nine locations (Figure 8).

Table 14. Yield means for FG72 -US (2003)

Location County/State Loc # a Event
FG72 b Jack b Location Mean c

Minburn (Late) Dallas/IA 1 25.5 24.0 25.1

Minburn (Early) Dallas/IA 2 24.4 26.2 27.8

Winterset (Late) Madison/IA 3 31.4 32.8 33.5

Winterset (Early) Madison/IA 4 33.6 36.8 37.0

Linden (Late) Dallas/IA 5 36.5 38.7 39.1

Linden (Early) Dallas/IA 6 37.6 42.3 40.7

West IA Carroll/IA 7 47.2 48.8 47.5

South East IA Des Moines/IA 8 51.8 54.4 52.2

Marshalltown Marshall/IA 9 52.6 48.3 53.4

a Loc #; location # corresponds to x axis of Figure 8.

b Yield is reported as bu/ac

c Location mean is the average yield of all the entries in the trial, including other conventional soybean varieties.Soybean Event FG72 Petition - Revised Page 58 of 198

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### Figure 8. Yield of event FG72 – Event FG72 and location means

Yield data is ranked by location mean, and best fit regression line for location mean yield of all entries (individual data points not shown).

VII.C.1.2. Intended herbicide tolerance evaluations of event FG72

Field tests at three locations (Champaign, IL; Seymour, IL; and Adel, IA ) 

### were designed to evaluate tolerance to the intended herbicides; GLY and IFT in 13 different herbicide treatments. Event FG72 performed well as illustrated by the data in Table 15 which summarizes the mean yield data (bu/ac) from the three locations with three replicates at each location. The conventional soybean variety, Jack, showed ex treme crop injury when sprayed with the intended herbicides for the event, GLY, IFT, and IFT + GLY, which resulted in a substantial yield loss. The crop injury values for FG72 were recorded as 0.7% in the GLY treatment and 5.7% in the IFT+GLY treatment. Crop injury is a standard weed science rating, where 0% indicates no injury and a 100% indicates complete plant death. 0.0 10.0 20.0 30.0 40.0 50.0 60.0

0 1 2 3 4 5 6 7 8 9 10 Series1

Series2
Linear (Series2) FG72

Mean yield

for all entries Soybean
Yield as
Bu/AcSoybean Event FG72 Petition - Revised Page 59 of 198

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### Table 15. Yield of event FG72 and Jack for selected herbicide treatments

Conventional

herbicide GLY IFT GLY+ IFT IFT Pre

Jack 47.6 0.5 18.5 0.5 3.2

FG72 43.1 34.1 38.0 30.7 37.5
Standard error for comparison = 3.4 GLY; glyphosate at 2800grams of ai/Ha IFT; isoxaflutole at 210 grams ai/Ha GLY+IFT; tank mix of the same herbicide amounts IFT Pre; IFT applied pre-emergence at 315 grams ai/Ha



### In the 2003 field season, FG72 soybean demonstrated agronomic performance equivalent to an appropriate comparator, the parent line, Jack, and was tolerant to more than twice the anticipated commercial application rates of both glyphosate and IFT herbicides.

VII.C.2. Agronomic evaluation of event FG72 (2008 data)

VII.C.2.1. Agronomic performance and yield evaluation of event FG72 under conventional weed control practices

Field trials were conducted in the states of Iowa, Illinois, Indiana and Missouri, which are typical soybean growing regions of the Midwestern United States (Table 16). The plants in this study were grown under conditions typical of production practices for Group II maturity soybeans. Plants were observed through out the season, harvested at maturity and samples were reserved for the analysis of nutritional composition.

The trial design was a RCB design (Kowite, 2009a). Each trial contained three replicate plots of Jack soybean (Regimen A), three replicate pl ots of event FG72 soybeans (Regimen B), andthree replicate plots of sprayed (IFT + GLY) event FG72 soybeans (Regimen C). Three nonreplicated plots of commercial conventional soybean lines were also grown at each trial location. The non-replicated commercial soybean plots are not included in the statistical analysis of the quantitative agronomic data. These lines were Stine ® 

### 2686-6 (Regimen D), Stine ® 

### 2788 (Regimen E) and Stine ® 

### 3000-0 (Regimen F). Thus there were a total of 12 plots (9 randomized) established at each field trial site. The plot sizes were 15 x 20 feet, with 6 rows per plot and 30 inch row spacing.

The entire trial site received a conventional, pre-emergence soil herbicide application of pendimethalin (1060 gm ai /Ha) with the exception of trial #05, which was treated with metolachlor (1880 gm ai/Ha). Regimen C represents the intended weed control practice in which event FG72 plots were sprayed with IFT at a target rate of 70 grams ai/Ha and GLY at a target rate of 1060 grams ai/Ha. Herbicide appl ications were made to the Regimen C plants as a foliar spray at about the V4-V5 plant growth stage. The trial treatments are summarized in
Table 17.Soybean Event FG72 Petition - Revised Page 60 of 198

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### Table 16. Trial site location for the equivalence field tests

Trial
Number County State Sowing
date Seedling

emergence Harvest date

01 Cherokee IA May 17 May 30 November 3

02 Hardin IA May 20 June 1 November 2

03 Greene IA May 19 May 30 October 10

04 Dallas IA May 21 June 3 October 3

05 Dallas IA May 8 May 18 October 3

06 Madison IA May 20 June 1 October 6

07 Clinton MO May 29 June 5 October 5

08 Vermillion IL June18 June 24 October 22

09 Tipton IN May 28 June 6 September 28

10 Des Moines IA May 22 June 4 October 4



### Table 17. Treatments for the 2008 season field studies

Regimen Designation rDNA status Soil herbicide

treatment a Foliar
herbicide

treatment b Reps

A Jack Non-transgenic Conventional - 3

B FG72 Transgenic Conventional - 3

C FG72 Transgenic Conventional GLY + IFT 3

D Stine 2686-6 Non-transgenic Conventional - 1

E Stine 2788 Non-transgenic Conventional - 1

F Stine 3000-0 Non-transgenic Conventional - 1

a The entire trial site received a conventional, pre-emer gence soil herbicide application of pendimethalin (1060 gm ai /Ha) with the exception of trial #05, whic h was treated with metolachlor (1880 gm ai/Ha).

b In one location (Trial #09), a foliar application of imaz ethapyr (68.6 gm ai/Ha) was made for Regimen A, B, D, E and F at the same time as the Regimen C application.



### Agronomic observations were made in the early, mid and late season (Table 18). To evaluate some of the parameters important for soybean yield, a census of all the plants in two 10 ft sections of each plot were made. The plant density (parameter; stand count) was higher for the Jack and the conventional line plots than in the event FG72 plots. The early stand count difference may be the result of seed lot quality, as the FG72 planting seed was produced at the counter season nursery in South America, while the seed of the conventional lines were produced in the US during the normal production season. However, the number of days to achieve 50% emergence and the plant vigor ratings were the same, indicating that the seed lots were comparable for these performance parameters. In late season stand counts, Jack and theSoybean Event FG72 Petition - Revised Page 61 of 198

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### conventional lines remained different from event FG72. Plot yields of FG72 event plants were also lower than those of Jack, perhaps a result of the lower plant density of the FG72 event plots. The yield for the conventional lines was higher than for Jack; a demonstration of the advancement in yield potential to be found in the current commercial varieties. Agronomic raw data for all 10 locations of the 2008 season is provided in Appendix 4.

Table 18. Summary of agronomic performance for event FG72 and Jack

Regimen

Jack FG72 FG72 Treated

Agronomic characteristic Unit a MEAN ± STD MEAN ± STD MEAN ± STD

Early Season

Emergence (50% of plants) N of days 9.7 ± 2.4 10.0 ± 2.3 10.0 ± 2.4

Stand count (20 ft section of row in each plot) N of plants 111.1 ± 21.4 82.1 ± 21.7 81.2 ± 20.6

Plant vigor rating 1-9 8.1 ± 0.6 7.8 ± 0.5 7.8 ± 0.5

Plant health at V4-5 rating 0-5 4.8 ± 0.5 4.6 ± 0.7 3.7 ± 0.7

Mid Season Flowering date (50% of plants) N of days 46.5 ± 5.3 46.7 ± 5.2 47.4 ± 5.6

Plant health at R1 rating 0-5 4.9 ± 0.3 4.6 ± 0.8 3.6 ± 0.8

Late Season at Crop Maturity
Days to maturity (95% of pods turning color) N of days # 127.7 ± 7.2 127.7 ± 7.0 128.8 ± 7.1

Yield bu/ac 53.18 ± 8. 96 46.41 ± 11.00 45.24 ± 10.79

Plant height (mean from 12 points in each plot) inch 42.03 ± 5.94 37.20 ± 6.08 32.52 ± 5.47

Plant lodging rating 1-9 4.4 ± 1.7 5.1 ± 1.6 6.1 ± 1.2

Final Stand count (20 ft section of row in each plot) N of plants 93.0 ± 19.1 73.5 ± 19.0 73.6 ± 21.4

Pod shattering rating 1-9 8.3 ± 0.5 8.0 ± 0.6 7.9 ± 0.6

Plant health at maturity rating 0-5 5.0 ± 0.0 5.0 ± 0.0 5.0 ± 0.0

Mean values and standard deviations are over all trial sites.

a N; Number of days from the date of planting or number of plants Plant vigor rating; 1 is no emer gence and 9 is complete emergence Plant health rating, 0 is death, 5 is no injury Plant lodging score; 1 is plants are flat on the ground, 5 repres enting that plant stems are 45 degrees to the ground, and 9 re presenting that all plant stems are straight up and down with minimal br anching. Lodging scores were taken just prior to harvest. Pod shattering score; 1 is all pods are open, 5 being 50% of pods are open, and 9 representing no pods are open. Shattering sc ores were taken at maturity and approximately 2 weeks after maturity (on border rows).Soybean Event FG72 Petition - Revised Page 62 of 198

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### Field agronomists made plant health ratings at three growth stages; V4-5, R1 and full maturity. The first evaluation was shortly after the intended herbicide application. In Regimen C, the health ratings reflect the bleaching characteristic of the IFT herbicidal action. By the final plant health evaluation, the plants in Regimen C had the same score as Regimen A (Jack) and B (FG72). In ratings by the agronomic staff, R egimen C (FG72 treated) plants received a health rating of 3-4 (moderate injury) at the V4-5 and R1 plant growth stages. Plants in the other Regimen were rated as 4.6-4.8 (rating of 5 indicates no injury). At the final plant health rating, all plots received the same rating of 5 (no injury).

Independent ratings were made by experienced weed scientists at 8 of the 10 locations using a standard rating for herbicide-related crop injury in which 0% is no injury and 100% is complete plant death within the experimental plot. The ta rget evaluation intervals were 6-10 days, 11-18 days and 25-44 days following the foliar herbi cide treatment. The 2008 season was one of exceptional rainfall and crop injury in the event FG72 plants following the intended herbicide was more obvious than observed in previous seasons. The crop damage ratings ranged from 10% to 30% in Regimen C, where the event FG72 plots were treated with IFT + GLY. Unlike the experience in the 2003 season, event FG72 plants consistently showed crop injury following foliar application of the intended herbicides.

The field evaluations included monitoring of the fitness characters (reproduction, disease resistance, fecundity, seed dispersal, dormancy, persistence) that could contribute to increased weediness potential in soybean. For the reproducti ve characteristics; days to emergence, days to 50% flowering and days to 95% pods maturing, the event FG72 and Jack plants were not different. The Jack plots were higher in seed production than those plots containing event FG72. Reaction to natural infestations of plant diseases and insect pests were monitored, and no differences were noted (see Section VII.D.). Although event FG72 produced less ultimate yield than Jack, no difference in fecundity (100 seed weight) was found. The assessment of seed dispersal parameters (pod shattering and plant lodging) found that event FG72 and Jack have the same pod shattering score, but found event FG72 to be less prone to lodging. Evaluation of seed harvested from the 10 locations found no concerns raised by germination or dormancy testing (see Section VII.F.). Volunteer monitoring found no evidence of persistence in the following season.

In the 2008 field season, a 10 location study was designed to compare the agronomic performance of event FG72 soybean to the parent line, Jack. Using a RCB design, event FG72 soybean was grown in replicated plots with either conventional weed control or with the intended herbicides, GLY and IFT. The final yield of event FG72 soybean, regardless of the weed control treatment, was less than the yield of Jack. Crop injury (bleaching in 10-30% of the crop area) was reported for event FG72 plots up to six weeks following foliar application of the GLY and IFT herbicides. However, at time of maturity, “no injury” plant health ratings were assigned to all plots.

VII.C.2.2. Objective variety description

USDA Plant Variety Protection Office has published a guidance for the description of soybean varieties (Objective Description of Variety, Soybean, Glycine max (L.) Merr.). It includes theSoybean Event FG72 Petition - Revised Page 63 of 198

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### following qualitative traits of flower color, pubescence color, pod color, hilum color, canopy architecture, leaf shape and growth habit, which were recorded for the plots from ten field trials in 2008 (Table 19). For these characters, observations of the variety Jack were in agreement with the variety description and event FG72 was not different in any of these traits (Nickell et al

., 1990).

Table 19. General plant descriptors comparison for event FG72 and Jack

Morphology characters 

### Jack FG72

Flower color White White

Pubescence color Gray Gray

Pod color Brown Brown

Seed coat Dull Yellow Dull Yellow

Hilum color Yellow Yellow Canopy architecture Medium Medium Leaf shape Oval Oval

Growth habit Indeterminate Indeterminate



### VII.C.2.3. Seed characteristics

Observations of the phenotypic characteristics of soybean event FG72 and the parent variety Jack included seed characteristics that are commonly used to describe soybean varieties (Objective Description of Variety, Soybean, Glycine max (L.) Merr.). Following the convention described by the USDA, National Genetic Resources Program, the following observations for event FG72 and Jack seed were made: seed size hilum color, mottling score, seed coat color, seed coat luster, seed quality and seed shape. The measurement of seed size was made using four independent samples of 100 seed each from each of the ten locations. The four independent samples were also examined for other seed characteristics. In all locations and for all characters, Jack and FG72 are identical (Table 20).

In addition to the morphological characteristics, the USDA Plant Variety Protection Office requests information concerning variety maturity, height, fatty acid profile and total oil and protein content when applying for registration of a new soybean variety. The information compiled in Table 21 follows the template provided by the USDA Plant Variety Protection (PVP) Office. The comparison demonstrates that Jack and event FG72 are similar in all characteristics, with the exception of height. There have been eight generations of selection of
lines and seed increase since the transformation of Jack to create event FG72. Modern soybean varieties are shorter than the older variety Jack, and we can see evidence of the breeder’s eye at work in the 12 cm difference in plant height.

FG72 soybean is similar to Jack for all parameter s considered by Objective Variety Description, with the exception of plant height. The only distinction between event FG72 and Jack is the
addition of the 2mepsps and hppd genes to confer double herbicide tolerance to GLY and IFT.Soybean Event FG72 Petition - Revised Page 64 of 198

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### Table 20. Seed phenotypic characteristics

Seed Characteristics FG72 (mean ± std) a Jack (mean ± std) a

Seed size b 13.1 ± 1.4 12.3 ± 1.0

Hilum color Yellow Yellow

Mottling c 1.9% ± 1.6 1.5% ±1.7

Seed coat color Yellow Yellow

Seed coat luster Dull Dull

Seed quality d 7.3 ± 0.9 7.0 ± 0.9

Seed shape e Spherical Spherical

L/W 1.1 1.2

L/T 1.0 1.1

T/W 1.1 1.1

a Mean and standard deviation for the replicat e measurements from 10 locations (2008).

b Seed size was recorded as the weight in grams of 100 seed.

c Mottling is the number of mottled seeds in each 100 seed reported as %

d Seed quality rating is a numerical score of 1-9 based on vi sual appearance of the seed (9 = best quality, 1 = worst

quality)

e The seed shape is measured as the le ngth and width of the seed with the hilum facing up (L and W) and the width

with the hilum on the side (T). A seed shape is scored as s pherical when the L/W, L/T and T/W ratio is less than or equal to 1.2.



### Table 21. Paired comparison of variety characteristics

Paired comparison # Days

to maturity Plant height in

cm %
Linoleic acid (18:2) %
Oleic acid (18:1) %
Linolenic acid (18:3) %
Palmitic
acid (16:0) Total
oil
% dw Crude protein
% dw

Jack 128 106 54% 22% 8% 10% 19% 38%

FG72 128 94 54% 24% 8% 9% 19% 38%Soybean Event FG72 Petition - Revised Page 65 of 198

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### VII.D. Biotic and abiotic stress characteristics

Insect pests of soybean encountered at the trial sites included aphid and bean leaf beetle. The presence of beneficial or non-pest insects, such as lady beetles and leafhoppers was noted at some of the sites. In all cases, no preference was observed by the insects for any of the soybean varieties or herbicide regimen (Kowite, 2009b).

Natural infestations of common soybean plant diseases were observed at most of the sites (Table 22). In most cases, the symptoms were uniform in all the plots. In locations where the incidence (% of plants with symptoms) and severity (% of plant tissue showing symptoms) could be scored, ratings by plot were recorded (Kowite, 2009b).

Table 22. Plant diseases and syndromes observed

Phytopathology observed Causal agent

Downy mildew Peronospora manshurica

Bacterial blight Pseudomonas syringae pv. Glycinea

Cercosopora leaf blight Cercospora kikuchii

Brown spot Septoria glycines

Frogeye leafspot Cercospora sojina

Powdery mildew Microsphaera diffusa

Top die back cause unknown, syndrome described by Iowa State University as plants dying from the top down

Sudden death syndrome Fusarium virguliforme



### The variety registration of Jack, claims resistance to soybean cyst nematode (SCN) (Races 3 and 4) (Heterodera glycines Ichinohe) and susceptibility to Phytophthora rot (Races 1, 4, and 7) caused by Phytophthora megasperma (Drechs.) f. sp. glycinea T. Kuan & D.C. Erwin. Neither of these phytopathologies was observed consistently in the trials, so it was not possible to confirm expression of these variety traits. At the Perry location, one plant of event FG72 was presumed to have died of Phytophthora root rot.

No insect susceptibility or disease susceptibility or resistance differences were observed between the event FG72 soybean plants and Jack.

There was no evidence of a change in characteri stics that would enhance survival of event FG72 soybean plants when compared to Jack.

VII.E. Equivalence between event FG72 and commercial varieties

A comparison of event FG72 soybean and the comme rcial varieties were used to evaluate any potential agronomic effect of the transformation on the plant parameters of event FG72. The early plant density and plant vigor ratings were lower in event FG72 plots than in the commercial variety plots (Table 23). These early season differences may again be the result ofSoybean Event FG72 Petition - Revised Page 66 of 198

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### seed lot quality (see Section 

### VII.C.2.1.) . In the late season stand counts, event FG72 and the commercial varieties remained different. Plot yields of event FG72 plants were also lower than those of the commercial varieties, perhaps a result of the lower plant density of the event FG72 plots. Agronomic raw data for all 10 locations of the 2008 season is provided in Appendix 4.

Plant health ratings showed similar trends, as the at the three growth stages; V4-5, R1 and full maturity. At the final plant health rating, all the plots received the same rating of 5.

Table 23. Summary of agronomic performance for FG72 and commercial varieties

FG72 unsprayed Commercial
varieties

Agronomic characteristic Unit a MEAN ± STD MEAN ± STD

Early Season

Stand count (20 ft section of row in each plot) N of plants 82.1 ± 21.7 113.0 ± 22.4

Plant vigor rating 1-9 7.8 ± 0.5 8.1 ± 0.6

Plant health at V4-5 rating 0-5 4.6 ± 0.7 4.9 ± 0.3

Mid Season
Flowering date (50% of plants) N of days 46.7 ± 5.2 48.5 ± 6.1

Plant health at R1 rating 0-5 4.6 ± 0.8 4.8 ± 0.5

Late Season at Crop Maturity

Days to maturity (95% of pods turning color) N of days # 127.7 ± 7.0 127.3 ± 6.7

Yield bushel/acre 46.41 ± 11.00 60.36 ± 11.00

Plant lodging rating 1-9 5.1 ± 1.6 7.0 ± 0.9

Final Stand count (20 ft section of row in each plot) N of plants 73.5 ± 19.0 104.4 ± 21.1

Pod shattering rating 1-9 8.0 ± 0.6 8.8 ± 0.4

Plant health at maturity rating 0-5 5.0 ± 0.0 5.0 ± 0.0

Mean values and standard deviations are over all trial sites.

a N; Number of days from the dat e of planting or number of plants Plant vigor rating; 1 is no emergence and 9 is complete emergence Plant health rating, 0 is death, 5 is no injury Plant lodging score; 1 is plants are flat on the ground, 5 repr esenting that plant stem s are 45 degrees to the ground, and 9 representing that all pl ant stems are straight up and down with minimal branching. Lodging scores were taken just prior to harvest. Pod shattering score; 1 is all pods are open, 5 being 50% of pods are open, and 9 representing no pods are open. Shattering scores were taken at maturity and approx imately 2 weeks after maturity (on border rows).Soybean Event FG72 Petition - Revised Page 67 of 198

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### For the reproductive characteristics; days to 50% flowering were shorter for event FG72 than for the conventional varieties, but for days to 95% pods maturing, event FG72 and commercial variety plants were not different. The conventional plots were higher in seed production than event FG72, probably due to the higher plant stand count. The assessment of seed dispersal parameters (pod shattering and plant lodging) found event FG72 and the commercial varieties to have the same pod shattering score, but found the commercial varieties to be less prone to lodging.

The yield of the conventional varieties was higher than that of event FG72; a demonstration of the advancement in yield potential to be found in the current varieties.

In the 2008 field season, double-herbicide-tolerant soybean event FG72 was compared to a series of commercial varieties. Overall, no differences were observed that could be attributed to any pleiotropic effects of the transformation process, or the presence of the introduced proteins, 2mEPSPS and HPPD W336, but rather to the advancement of the new varieties.

VII.F. Seed dormancy evaluation

To provide an evaluation of seed dormancy potential of event FG72 and the variety Jack, measurements of seed germination and dormancy were conducted. The hypothesis was to test seed samples harvested from plants grown to maturity at 10 locations. These seed samples would represent the physiological state of seed that might fall into a field at the end of the season. The seed tests were completed by the Iowa State Seed Lab using the standard test (warm germination) which is used for seed lot evaluations of field emergence under favorable conditions. Seeds are rolled into germination towels, placed into germination trays and incubated at 25°C and 90%relative humidity for five days. Germination is scored on day 6. A minimum of 400 seed were evaluated from each location. Appendix 2.F. describes the materials and methods for the germination studies.

A very small difference in germination (94% vs. 96%) at day 6 was observed (Table 24). In cases where hard seed were observed on day 6, the germination study was extended to 13 days, and in every case, the hard seed germinated. The percent of viable seed from each of the samples (total viable; 95% vs. 96%) was the same.

No dormant seed were identified. Although sm all difference was observed between the event FG72 and the parent line Jack, the differences were not more than one standard deviation. No impact of the production environment or harvest conditions was observed.Soybean Event FG72 Petition - Revised Page 68 of 198

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### Table 24. Seed germination test results

ID Location Germination a (%) Hard
seed Dormant
seed Total
viable Abnormal seedlings Dead
seed Days
tested FG72 Mediapolis 92 2 0 94 3 3 13 FG72 Perry 89 3 0 92 8 0 13 FG72 Sharpsville 82 6 0 88 10 2 13

FG72 Adel 97 1 0 98 2 0 6

FG72 Fithian 90 0 0 90 9 1 13

FG72 Marcus 97 0 0 97 3 0 6

FG72 Glidden 95 0 0 95 5 0 13

FG72 Winterset 99 0 0 99 0 1 6

FG72 Osborn 97 2 0 99 1 0 13

FG72 Iowa Falls 99 0 0 99 1 0 7

Mean 93.7 1.4 0 95.1 4.2 0.7

StDev b 5.4 2.0 0.0 4.0 3.6 1.1 Jack Mediapolis 97 0 0 97 1 2 Jack Perry 96 0 0 96 3 1 6 Jack Sharpsville 93 3 0 96 4 0 13

Jack Adel 94 0 0 94 5 1 6

Jack Fithian 94 0 0 94 6 0 6

Jack Marcus 98 0 0 98 2 0 6

Jack Glidden 98 0 0 98 2 0 6

Jack Winterset 99 0 0 99 1 0 6

Jack Osborn 98 0 0 98 2 0 6

Jack Iowa Falls 95 0 0 95 4 1 6

Mean 96.2 0.3 0 96.5 3 0.5

StDev b 2.1 0.9 0.0 1.8 1.7 0.7

a Warm germination – 8 reps of 50 seed

b StDev; standard deviationSoybean Event FG72 Petition - Revised Page 69 of 198

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### VII.G. Composition analysis

VII.G.1. Introduction

Analysis of the nutritional composition of the double-herbicide-tolerant soybean event FG72 was performed for soybean grain harvested from 10 different locations in the soybean growing areas of North America (Mackie, 2009). The study was conducted during the 2008 growing season using seed of the T 8 

### generation. Planted at each of the 10 locations were three entries:

Entry A; the control counterpart variety Jack, which was treated with conventional herbicides registered for use on soybean; (designated as Jack in Tables 25-29) Entry B; the test entry event FG72 treated with conventional herbicides (designated as FG72 in Tables 25-29) Entry C; the test entry event FG72 treated with the intended herbicides (IFT + GLY) (designated as FG72 treated in Tables 25-29).

Each of the three entries was planted in a RBC design with three replications per location. Three commercial soybean varieties were planted along side the test and control entries at the same locations. These three commercial soybean varieties provided reference values to establish ranges of natural variation for the nutritional components analyzed in this study.

The nutritional composition analysis conducted was based on the OECD guidance document for soybean (OECD, 2001). The nutritional endpoints selected were proximates, fiber compounds, total amino acids, fatty acids, anti-nutrients and isoflavones.

For comparative purposes, the values obtained for the commercial reference lines were used to establish in-study ranges in addition to the ranges reported in the published literature (OECD 2001; ILSI 2007). Together, these two sets of ranges were used to evaluate the nutritional composition results of event FG72 soybean. Nutrient component means that fell within the limits of the commercial or literature reference ranges were considered to be within the normal variation for commercial soybeans.

The test plots were each 15 ft by 20 ft in size and contained 6 rows spaced 30 inches apart. At maturity, grain samples were harvested from the two interior rows of each plot.

VII.G.2. Nutritional composition of soybean grain

Tables 25-29 show the comparisons of the pooled results of the two test and counterpart control entries from all locations, with reference ranges calculated from three commercial soybean varieties. Appendix 2.G. describes the materials and methods for the composition analysis. All mean values typically fell within the respective commercial variety or literature reference ranges and are not considered to be of biological concern or due to the intended modification of event FG72 (Rattemeyer, 2009).

The analysis of proximates and fiber between the test and the counterpart control entries were similar. All mean values were within the calculated commercial variety and literature reference ranges (Table 25).Soybean Event FG72 Petition - Revised Page 70 of 198

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### Analysis of the total amino acid profile for all 18 amino acids between the two test and counterpart control entries were found to be similar (Table 26).

Levels of 24 fatty acids were measured for the two test entries, the counterpart control Jack, and the three commercial varieties. Seventeen of these fatty acids; C08:0, C10:0, C12:0, C14:0, C14:1, C15:0, C15:1, C16:1, C17:1, C18:3 (gamma), C18:4, C20:2, C20:3, C20:4, C20:5, C22:1, C22:5 and C22:6 were below the limit of quantification (LOQ = 0.02 % fw) in all soybean seed samples. These minor fatty acids of soybean were not statistically analyzed and are not reported in Table 26.

The results of the fatty acid analysis of the two test entries and Jack are shown in Table 27. All mean values for the fatty acids listed in Table 27 fell within both the calculated commercial variety and literature reference ranges. The sum of all detected fatty acids was 99.9% and accounts for nearly all fatty acids present in the oil.

The level of the anti-nutrient phytic acid in Jack and event FG72 soybean grain entries fell within the commercial variety and the literature reference ranges (Table 28).

The levels of the two low molecular weight carbohydrates raffinose and stachyose in the two test and control entries were within the range of the commercial lines tested (Table 28).

The analysis of the levels of lectins in the two test and isoline control entries were found to be similar, and fell within the commercial variety and literature reference ranges (Table 28).

The trypsin inhibitor mean values for the two test and control entries fell within the commercial variety and reference literature ranges (Table 28).

Soybeans contain isoflavones which are glucosi des and esters of three aglycones (daidzein, genistein and glycitein). The mean values and range reported for isoflavone content in the test and counterpart entries were very similar in numerical value, and all mean values fell within the ranges for the commercial lines and literature references (Table 29).

In summary, no safety related issues were identified in the analysis of the nutrient composition of event FG72 soybean grain. All components measured were comparable to either the commercial soybean varieties grown at the same locations as the test and control entries, or were within the cited literature reference ranges.Soybean Event FG72 Petition - Revised Page 71 of 198

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### Table 25. Proximate and fiber components

Component Jack FG72 FG72
Treated Commercial
Lines b Literature
Reference c

Moisture % fw Mean a 9.51 9.65 9.45 Range 6.57-10.50 7.90-11.50 6. 51-10.90 8.00 – 10.60 5.6-12
Protein % dw Mean 38.2 38.2 38.1 Range 36.2-40.3 36.8-39.8 36.5-39.6 35.8 – 40.1 32 – 45.5
Fat % dw Mean 19.3 18.9 19.2 Range 17.9-21.4 16.6-21.0 17.1-21.6 15.1 – 21.4 8.1 – 24.7
Ash % dw Mean 5.24 5.07 5.06 Range 4.38-6.07 4.17-5.56 4. 50-5.68 4.89 – 5.73 3.9 – 7.0
Carb. % dw Mean 37.3 37.9 37.6 Range 34.3-39.3 35.6-39.7 35.3-40.0 34.8 – 41.6 29.6- 50.2
ADF % dw Mean 17.8 18.1 17.9 Range 14.2-22.4 14.1-23.5 15.2-21.4 13.6 – 23.5 7.8 – 18.6
NDF % dw Mean 19.8 20.3 20.0 Range 16.8-24.5 16.9-25.4 17.4-23.0 16.1 – 24.8 5.0 – 21.3

a Least square mean

b Reference ranges of the 3 analyzed commercial soybean lines

c Literature ranges from OECD (2001) and ILSI (2007)Soybean Event FG72 Petition - Revised Page 72 of 198

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### Table 26. Amino acids

Amino acid
% dw Jack FG72 FG72
Treated Commercial
Lines b Literature
Reference c

Alanine Mean a 1.68 1.68 1.68 Range 1.60-1.75 1.60-1.74 1.60-1.72 1.55 – 1.78 1.51 – 2.10 Arginine Mean 2.94 2.97 2.95 Range 2.71-3.20 2.77-3.14 2. 74-3.10 2.69 – 3.13 2.17 – 3.40 Aspartic acid Mean 4.40 4.38 4.37 Range 4.15-4.70 4.08-4.60 4. 13-4.55 4.06 – 4.67 3.81 – 5.12 Cystine Mean 0.58 0.58 0.59 Range 0.53-0.63 0.51-0.62 0. 49-0.63 0.50 – 0.63 0.37 – 0.81
Glutamic acid Mean 6.75 6.77 6.74 Range 6.30-7.24 6.30-7.21 6. 34-7.03 6.32 – 7.23 5.84 – 8.20 Glycine Mean 1.68 1.68 1.68 Range 1.60-1.76 1.60-1.75 1. 60-1.74 1.53 – 1.76 1.46 – 2.27
Histidine Mean 1.05 1.05 1.05 Range 1.00-1.10 0.99-1.09 0. 98-1.09 0.93 – 1.07 0.84 – 1.22
Isoleucine Mean 1.81 1.80 1.79 Range 1.73-1.92 1.69-1.87 1. 67-1.86 1.62 – 1.96 1.54 – 2.32
Leucine Mean 2.99 2.99 2.98 Range 2.84-3.18 2.84-3.13 2. 81-3.09 2.71 – 3.13 2.2 – 4.0 Lysine Mean 2.48 2.48 2.47 Range 2.37-2.62 2.34-2.58 2. 33-2.56 2.34 – 2.64 1.55 – 2.84
Methionine Mean 0.54 0.54 0.54 Range 0.49-0.60 0.49-0.58 0. 46-0.58 0.50 – 0.58 0.43 – 0.76
Methionine Mean 0.54 0.54 0.54 Range 0.49-0.60 0.49-0.58 0. 46-0.58 0.50 – 0.58 0.43 – 0.76 Phenylalanine Mean 1.97 1.98 1.96 Range 1.89-2.13 1.87-2.09 1. 83-2.05 1.83 – 2.08 1.60 – 2.39
Proline Mean 1.82 1.83 1.82 Range 1.68-1.97 1.72-1.98 1. 65-1.94 1.71 – 1.94 1.69 – 2.33
Serine Mean 1.97 1.98 1.99 Range 1.82-2.14 1.75-2.10 1. 83-2.11 1.77 – 2.13 1.11 – 2.48
Methionine Mean 0.54 0.54 0.54 Range 0.49-0.60 0.49-0.58 0. 46-0.58 0.50 – 0.58 0.43 – 0.76 Phenylalanine Mean 1.97 1.98 1.96 Range 1.89-2.13 1.87-2.09 1. 83-2.05 1.83 – 2.08 1.60 – 2.39
Proline Mean 1.82 1.83 1.82 Range 1.68-1.97 1.72-1.98 1. 65-1.94 1.71 – 1.94 1.69 – 2.33
Serine Mean 1.97 1.98 1.99 Range 1.82-2.14 1.75-2.10 1. 83-2.11 1.77 – 2.13 1.11 – 2.48
Threonine Mean 1.55 1.54 1.53 Range 1.48-1.66 1.45-1.61 1. 44-1.62 1.44 – 1.62 1.14 – 1.89 Tryptophan Mean 0.45 0.44 0.44 Range 0.40-0.50 0.38-0.48 0. 39-0.50 0.39 – 0.54 0.36 – 0.67 Tyrosine Mean 1.40 1.40 1.40 Range 1.28-1.49 1.33-1.46 1. 30-1.46 1.32 – 1.48 0.10 – 1.61
Valine Mean 1.89 1.88 1.87 Range 1.80-2.01 1.78-1.98 1. 75-1.95 1.66 – 2.03 1.50 – 2.44

a Least square mean; b Reference ranges of the 3 analyzed commercial soybean lines, c Reference ranges from OECD (2001) and ILSI (2007)Soybean Event FG72 Petition - Revised Page 73 of 198

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### Table 27. Fatty acids

Fatty Acid % relative Jack FG72 FG72
Treated Commercial
Lines b Literature
Reference c

Saturated

C16:0 (palmitic) Mean a 10.1 9.34 9.38 9.78 – 11.40 7 – 16 Range 9.75-10.9 9.02-9.58 9.03-10.4 C18:0 (stearic) Mean 4.28 4.52 4.51 Range 4.07-4.70 4.23-5.05 3.80-5.08 3.49 – 4.81 2 – 5.9 C20:0 (arachidic) Mean 0.31 0.32 0.32 Range 0.28-0.36 0.30-0.37 0.27-0. 38 0.25 – 0.35 < 0.10 - 0.48 C22:0 (behenic) Mean 0.32 0.33 0.33 Range 0.30-0.34 0.31-0.35 0. 26-0.36 0.25 – 0.35 0.28 – 0.60 C24:0 (lignoceric) Mean 0.113 0.119 0.122 Range < 0.10 -0.16 < 0.10 -0.17 < 0.10 -0.17 < 0.10 – 0.15 0.15
Sum of the saturated 14.9 14.5 14.5 13.8 – 17.2 9.43 – 23.55

Mono-unsaturated

C18:1 (oleic) Mean 21.97 24.65 24.12 Range 20.10-25.00 23.20-27.20 22.40- 26.30 21.10 –24.10 14 – 34 C20:1 (eicosenoic) Mean 0.16 0.16 0.17 Range 0.14- 0.19 0.15-0.19 0.15- 0.19 < 0.10 – 0.18 0.14 – 0.35
Sum of mono-unsaturated 22.13 24.81 24.29 21.10 – 24.28 14.14 – 34.83

Poly-unsaturated

C18:2 (linoleic) Mean 54.56 52.65 53.08 Range 51.70-55.90 50.60-53.70 51.20- 54.70 51.50 –55.40 48 – 60 C18:3 ( α -linolenic) Mean 8.27 7.94 8.01 Range 7.37-9.14 7.24-8.65 7.22-8.82 7.59– 10.30 2 – 10 Sum of poly-unsaturated 62.83 60.59 61.09 59.09 – 65.70 50 - 70

Sum of all the fatty acids 99.93 99.91 99.92

a Least mean square

b Reference ranges of the 3 analyzed commercial soybean lines

c Reference ranges from OECD (2001) and ILSI (2007)Soybean Event FG72 Petition - Revised Page 74 of 198

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### Table 28. Anti-nutrients

Anti-nutrients (dw) Jack FG72 FG72
Treated Commercial
Lines b Literature
Reference c Phytic Acid (%) Mean a 1.40 1.37 1.35 Range 1.03-1.70 0.89-1.91 0. 79-1.87 0.96 – 1.50 0.63 – 2.74 Raffinose (%) Mean 0.361 0.378 0.379 Range 0.286-0.428 0. 280-0.526 0.295-0.511 0.290 – 0.504 0.11 – 1.28 Stachyose (%) Mean 2.49 2.42 2.50 Range 2.04-2.91 2.09-2.88 2. 06-2.90 2.23 – 2.96 1.21 – 6.30 Lectin (HU/mg) Mean 1.74 1.40 1.54 Range 0.91-4.29 0.66-3.08 0.88-2.63 0.46 – 8.63 0.11 - 129 Trypsin inhibitor Mean 33.0 30.1 33.9 Range 23.3-47.6 19.6-42.4 23.6-43.4 23.5 – 60.1 19.59-118

a Least mean square

b Reference ranges of the 3 analyzed commercial soybean lines

c Reference ranges from OECD (2001) and ILSI (2007)



### Table 29. Isoflavones

Isoflavones mg/kg dw Jack FG72 FG72
Treated Commercial
Lines b Literature
Reference c

Daidzin Mean 1035 1034 994 Range 480-1850 416-1690 400-1810 568 – 2530 60.0 – 2454
Genistin Mean 1817 1682 1640 Range 839-2760 627-2460 609-2400 1130 – 3290 144 – 2837 Glycitin Mean 365 414 400 Range 298-445 345-511 169-492 142 – 315 15.3 – 1070
Daidzein e Mean -------- -- ---------- -- -------- Range d < 10 – 17.5 < 10 – 15.1 < 10 – 14.6 < 10 – 14.0 5 – 35
Genistein e Mean -------- -- ---------- -- -------- Range d < 10 – 17.2 < 10 – 15.7 < 10 – 12.2 < 10 – 20.6 0.3 – 46 Glycitein Mean <LOQ <LOQ <LOQ <LOQ Range d < 10 < 10 < 10 < 10 1.1 – 80
Total Mean a 2010 1953 1891 Isoflavones Range 1040-3130 930-2860 881-2890 1160 - 3390 679 – 3733

a Least mean square

b Reference ranges of the 3 analyzed commercial soybean lines

c Reference ranges from ILSI (2007)

d some or all values reported below the limit of quantification

e Mean not calculated as some samples were below LOQSoybean Event FG72 Petition - Revised Page 75 of 198

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### VII. H. Poultry feeding study

A 42-day broiler chicken feeding study was conducted using diets containing 20% toasted soybean meal from event FG72 soybean, Ja ck soybean and a non-commercial soybean line (Stafford, 2009). Broiler chicken is very sensitive to minor differences in nutrient quality, since it undergoes an approximate 15-fold increase in body weight during the first 21 days of life.

All chickens were monitored at least daily for health status, overt signs of toxicity, and mortality. Effects of diets on health, surviv al, live body weight, total weight gain, feed consumption, food conversion, marketable carcass weight and muscle tissue weight and yield (breast, thigh, leg, wing), and abdominal fat pad weight were compared among groups. Gross post-mortem examination findings were reported as appropriate.

After 42 days of daily exposure, no differenc es were observed between the event FG72 group and the control groups. Minor statistical differences were recorded and were considered not treatment-related. Overall, the growth and health of chickens were similar in all groups.

In conclusion, there was no evidence that the group of broiler chickens fed event FG72 soybean toasted meal were adversely affected in any manner. The toasted meal with event FG72 soybean incorporated at 20 % was as safe and nutritious as the meals made with 20 % of control group soybeans.

VII.I. Conclusion for agronomic evaluation of event FG72

A thorough review of double-herbicide-tolerant soybean event FG72 was conducted over the 2003 and 2008 crop seasons. During these field studies, more than 20 different agronomic parameters were identified and evaluated to as sess the impact of event FG72 on the soybean plant. Development and maturity, environmental susceptibility to biotic and abiotic stressors, and the yield potential and quality of the soybean grain were all evaluated to determine if event FG72 differed from the parent line Jack and other conventional soybean varieties of the same type.

In addition to the agronomic evaluation, event FG72 was analyzed for its main nutritional components and compared to the parent line Jack and commercial soybean varieties. The compositional analysis demonstrated that the intended modification in event FG72 did not change the compositional make-up and the nutritional profile of event FG72 is similar to that of the Jack and within the range of commercial soybeans lines and the established literature

ranges.

The overall conclusion is that there are no agronomically meaningful differences between the transformed double-herbicide-tolerant soy bean event FG72 and other soybean varieties evaluated. The resulting conclusion is that the introduction of event FG72 soybean poses no new agronomic plant pest risks.Soybean Event FG72 Petition - Revised Page 76 of 198

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### VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTICES

VIII.A. Environmental assessment of the introduced proteins

The presence of the 2mEPSPS and HPPD W336 prot eins introduced in FG72 soybean will not present adverse environmental effects, as bot h are derived from common, naturally occurring proteins and differ by only 2 and 1 amino acid substitutions, respectively.

The 2mepsps gene was generated by introducing two point mutations into the wild-type epsps (wt epsps ) gene cloned from maize ( Zea mays ). These changes to the gene result in the production of a double mutant EPSPS (2mEPSPS) protein which has a lower binding affinity for glyphosate, thus allowing sufficient enzyme activity for the plants to grow normally in the presence of this herbicide.

The naturally occurring EPSPS protein is universally expressed in plants and microorganisms, and has been a safe component of food and feed for a long history of consumption. In addition, the substitution of two amino acids was not expected to change the protein’s safety or its potential for toxicity and allergenicity. And rec ent studies conducted by BCS have confirmed the safety profile of the 2mEPSPS protein (Section VI).

Moreover, the safety of the 2mEPSPS protein, wh ich is also present in the genetically modified herbicide tolerant maize Event GA21 and in GlyTol TM 

### cotton, has been evaluated by several regulatory agencies.

HPPD proteins have been isolated from many different organisms, such as bacteria, fungi, plants, animals, and humans. HPPD proteins from diverse origins share a common structure and have several amino acids that are complete ly conserved among all HPPD proteins. They are present in food of plant, fungal and animal origin and have a history of safe consumption.

In plants, the inhibition of HPPD by IFT herbicide leads to the disruption of photosynthesis and subsequent bleaching of foliage and eventually plant death. To take advantage of this interaction for purposes of weed control, an HPPD protein with increased tolerance to IFT, was developed by introducing a single amino acid change to the native HPPD protein isolated from
Pseudomonas fluorescens . A simple mutant has been made to introduce a glycine (G) to tryptophan (W) substitution at position 336 of the native enzyme, resulting in the HPPD W336 protein. This mutation reduces the sensitivity of the enzyme to the herbicide isoxaflutole. A single mutation is not expected to change the safety aspects of the HPPD protein. Studies conducted by BCS have established the safety of the HPPD protein (Section VI).

VIII.B. Potential for horizontal or vertical gene transfer

Soybean is a self pollinating crop. Anthers mature in the floral buds and directly pollinate the stigma of the same flower (cleistogamy). Natural cross-pollination with near-by soybean plants is reported to be less than 1% (OECD 2000). The extent of outcrossing can be influenced by the distance between individual plants, floral characteristics of different varieties, environmental conditions and insect activities. In seed production fields, the occurrence of cross pollination isSoybean Event FG72 Petition - Revised Page 77 of 198

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### so low that the standards of certified seed production require isolation distances to prevent mechanical mixture (7CFR 201.76) and are based upon the width of the harvest machinery.

Soybean is a non-native crop of the Americas. The origin of the Glycine species is Asia and there are no wild or native soybean relatives in the Americas which could be considered to be potential targets for gene flow.

VIII.C. Weediness potential of double-herbicide-tolerant soybean event FG72

Commercial soybean varieties in the United States are neither problematic volunteer weeds in other cropping systems nor are they found as feral populations on unmanaged lands (OECD 2000). The potential fate of soybean seeds remaining in the field after harvest includes; rot, predation, herbicides from rotational crops, and winter weather. Soybeans generally do not
survive over the winter season. When climat ic conditions are permissive, volunteer soybeans provide minimal interference in the rotational crop and are not recognized as an economic problem in soybean production. Volunteer so ybeans are not competitive and can be managed by existing agronomic practices.

Double-herbicide-tolerant soybean transformation event FG72 is tolerant to two herbicides with
different modes of action i 

### ; class G (glyphosate) and class F (isoxaflutole), and remains sensitive to herbicides registered for pre-plant and pre-emergence use for weed control in soybean and other crops which are common in rotation with soybean. Volunteer soybeans can be treated with a pre-emergence or post-emergence herbicides such as 2,4-D, atrazine, glufosinate, mesotrione, acetochlor, dicamba, and others. These products are also widely used for weed control in the rotational crops of soybean.

As soybean are not difficult to control as volunteers in a subsequent crop, and as FG72 has been shown to be no different from cultivated soy bean in any of the traits that might impact weediness, the current practice to control volunteers will be effective.

VIII.D. Current agronomic practices for soybean

The introduction of glyphosate tolerant soybeans in 1996 significantly changed the way growers manage weed control in soybeans. Glyphosate tolerant soybeans were rapidly adopted by growers due to many unique properties of glyphosat e enabling the grower to simply, effectively and economically manage their weeds.

Glyphosate herbicide is exceptionally effectiv e for controlling a broad spectrum of weeds, including many difficult to control weeds, in glyphosate tolerant soybeans with virtually no crop damage. Weed management with glyphosate is also exceptionally simple as the application technique (spray nozzles, spray pattern, carrier volume, and speed of application) has little impact on weed control. Glyphosate herbicide has flexible use rates and patterns and now is more economical since becoming available from generic manufacturers. The use of glyphosate has also resulted in reduction in tillage both prior to planting and in crop cultivation due to its effective weed control (Boerboom and Owen, 2006). This allows growers to forgo tillage whichSoybean Event FG72 Petition - Revised Page 78 of 198

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### improves soil conservation, saves labor, and reduces fuel consumption. In addition, the glyphosate tolerant system allows growers to reduce field scouting as effective herbicide applications can be made to large weeds with a wide application window. Furthermore, late season weed control methods for weed escapes such as hand labor, rope wicking, and spot spraying have been virtually eliminated. Addi tional weed management benefits with glyphosate include no carryover concerns to the following crop, no replanting restrictions, low environmental
and human health risks and it is not a restrict ed-use pesticide (Boerboom and Owen 2006). The glyphosate tolerant trait is widely available and can be found in the highest yielding soybean
varieties available on the market.

The simple, effective and economical management of weeds with glyphosate in soybean has led to the adoption of a solitary chemical weed control pr actice at the sacrifice of chemical diversity. By 2007, more than 90% of the US soybean production area was planted to glyphosate herbicide tolerant soybean (USDA 2009). The grower of glyphosate tolerant soybeans makes an average of 1.7 applications of glyphosate to the crop per growing season (USDA 2007). The rapid adaptation of this new technology and the exclusion of other weed control measures set the stage for weed population shifts and the evolution of weeds resistant to glyphosate herbicide. With the application of glyphosate herbicide over most of the soybean production areas, many weeds were exposed to the her bicide and resistant biotypes were enriched

The extensive use of glyphosate was encouraged by the availability of glyphosate tolerance in other crops. Glyphosate tolerant corn is grown on 68% of the 87 million acres of corn in the US. Glyphosate tolerant cotton is grown on 71% of the 9.5 million acres of cotton (Figure 9, USDA 2009). Also available in the market are glyphosate tolerant canola and sugar beet varieties thus, creating unprecedented selection pressure for resistant biotypes. In 1996, seventeen herbicides composed 90% of the market. In 2007, only three herbicides comprised 90% of the market with glyphosate at 80% (Figure 10) for use in soybean production in the USA.

Figure 9. Growth of herbicide tolerant traits

0 20 40 60 80 100 120 140

2000 2001 2002 2003 2004 2005 2006 2007 2008 2009Planted HT Acres in Millions

0% 10% 20% 30% 40% 50% 60% 70% 80% 90% 100%% of planted acres HT for each crop

HT Corn Acres HT Cotton Acres HT Soybean Acres % HT cotton % HT soybean % HT corn Adapted from USDA, NASS Acreage 2001-2009Soybean Event FG72 Petition - Revised Page 79 of 198

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### Figure 10. Comparison of herbicide use for the years 1996 and 2007

0% 50% 100%

1 3 5 7 9 11 13 15 17 19 21 23 25 1996

2007 (RR Varieties only)

Number of AI ranked by market share 90%

Glyphosate (G)

Pendimethalin (K1)

Trifluralin (K1) Acifluorfen (E)

Imazaquin (B)

Chlorimuron (B) Fluazifop (A)

Imazethapyr (B) Thifensulfuron (B) Metolachlor (K3) 2,4-D (O)

Glyphosate (G) Chlorimuron (B)

2,4-D (O) Quizalofop (A) Sethoxydim (A) Lactofen (E) Bentazone (C3) Metribuzin (C1) Clethodim (A)

The declining use of herbicide options lead to the loss of her bicide diversity in soybean weed control in the USA. Indicated in the figure by the red diamond line, seventeen herbicides composed 90% of the market in 1996. In 2007, only three herbicides comprised 90% of the market with gl yphosate at 80% and 2,4-D and chlorimuron in minor use. Letters in brackets indicate the mode of action (HRAC 2009).



### The over-reliance on a single weed control method can lead to the eventual development of resistant weeds and consequential loss of that particular production system and perhaps even eventually jeopardize the ability to grow a specific crop in a specific field. Because of cost considerations and the additional workload, IW M tactics generally have not been employed until herbicidal efficacy starts to fail and herbicide resistance becomes a problem threatening the economic viability of the farmer. Working preemptively through incorporating integrated weed management measures can lead to the successful prevention of the development of a resistant weed population.

There are currently 189 species of resistant weeds worldwide, 16 of which are resistant to glyphosate (Heap 2009). Growers have been relu ctant to alter their weed management practices and return to conventional herbicides in light of the advent of glyphosate resistant weeds. Even with weed resistance, growers w ill continue to produce glyphosate tolerant crops as glyphosate herbicide remains effective for a number of weeds that are difficult to control with
other herbicides. Conventional herbicides generally have a narrower application window, narrower weed spectrum, increased risk of crop injury, various application techniques required, additional time for sprayer cleanout between fields, carryover concerns, replanting restrictions
and are considered to be less economical.Soybean Event FG72 Petition - Revised Page 80 of 198

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### However, growers are beginning to alter their farming practices to gain better control of glyphosate resistant weeds. Some growers are utilizing tankmixes with conventional herbicides to help control herbicide-resistant weeds. Conventional tankmix partners have limitations however such as increased cost, increase the risk of injury to the soybeans and limit the application window due to weed size restrictions. Some growers have moved away from reduced or no-till practices as their burndown progr am no longer provides effective control of glyphosate resistant marestail. Uncontrolled weeds result in soybean yield loss as the weeds compete for soil nutrients, moisture, and sunlight. The impact of glyphosate-resistant weeds firmly impacts a grower’s avail able time and financial resources.

There is an urgency to produce viable alternatives to glyphosate weed control programs in soybeans. There are several HT soybean products available to the US soybean grower (Table 3) however most provide crop tolerance to herbicides for which herbicide resistant weeds are already identified (ALS inhibitors and glyphosate).

Today, the LibertyLink soybean system is the onl y nonselective alternative to the glyphosate system available for growers. Launched in 2009, there were more than 300,000 acres of LibertyLink soybeans planted in the US. Liberty Link soybeans also must be managed correctly to prevent the development of herbicide-resistant weeds.

VIII.E. Potential impact on agricultural practices for soybean

In the near future, soybean growers will have additional options. Table 30 identifies several new soybean events in the process of development a nd registration. A new herbicide mode of action 4-hydroxy-phenyl-pyruvate-deoxygenase enzyme by specific inhibitors (HPPD inhibitors) was developed during the 1980’s. The double-herbicide-tolerant soybean event, FG72 which combines glyphosate tolerance with isoxaflutole tolerance is the first genetic source of crop tolerance to the HPPD inhibiting herbicides.

Table 30. Sources of genetic-based herbicide tolerance

Applicant(s) Event / Trade Name Trait Description(s) Bayer CropScience 98-014-01p A5547-127 / LibertyLinkTM Glufosinate tolerant

Pioneer Hi-Bred

International 06-271-01p 356042 / OptimumTM GATTM Glyphosate and ALS inhibitor
tolerant

BASF Plant Science 09-015-01p BPS-CV127-9 ALS tolerant

Bayer CropScience
and MS Tech

09-328-01p FG72/ Double-HerbicideTolerant soybean Glyphosate and HPPD tolerant

Monsanto Company Not announced Dicamba tolerantSoybean Event FG72 Petition - Revised Page 81 of 198

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### The use of isoxaflutole, based on the FG72 tolerance trait, will enable growers to proactively manage resistant weeds with a new herbicide mode of action for soybeans. Growers will still be able to use glyphosate with FG72, but they will not be dependent upon it as their only broad spectrum herbicide choice. Managed appropriately, glyphosate will be able to maintain its utility for post-emergence control of many diffi cult to control weeds in soybeans.

FG72 will offer growers unmatched flexibility in their weed control programs for both conservation and conventional till acres. IFT herbicide can be applied either pre-emergence to the soybean or in a post-emergence tankmix. IFT can provide control of a broad spectrum of weeds including herbicide-resistant weed species. IFT not only introduces a new MOA into soybean production for resistance management, it also provides a residual tool to prevent early season weed competition. Early season weed control is key in preventing soybean yield loss due to weed competition.

Growers utilizing conservation tillage practices will be able to tankmix isoxaflutole with their burndown program prior to planting. A post-emergence application of glyphosate can be applied if needed. Growers in conventional tillage practices have two base options for weed control withFG72 soybeans. Isoxaflutole can be applied prior to planting, pre-plant incorporated, or preemergence to the soybean crop. Again, a post-emergence application of glyphosate can be applied if needed. Growers that prefer a total post-emergence weed control program due to the critical time management needed at planting will be able to apply glyphosate for emerged weed
control in a tankmix with isoxaflutole for residual weed control.

VIII.F. Weed resistance management

Weed scientists agree that adopting and implementing best management practices that reduce weed resistance to herbicides is critical (Boerboom and Owen, 2006). We have developed detailed methods for integrated weed management that includes diverse farming practices. Integrated weed management not only improved over all weed control, it provides additional benefits such as improving the overall level and c onsistency of weed control, adding flexibility in scheduling applications and reducing the risk of yield loss due to weed competition

Ideally integrated weed management should utilize all available tools including herbicides in a well balanced program as the lower the diversity of weed control tools, the higher the risk of selecting resistant biotype becomes. To ensure diversification is maintained in weed control methods, we will also encourage growers to keep detailed records of weed management practices for each field. Our integrated weed management guidelines promote an economically viable, environmentally sustai nable and socially acceptable w eed control program is fully detailed in Appendix 5. The highlights of our integrated weed management include:

1) Correctly identify weeds and look for trouble areas within field to identify resistance
indicators. 2) Rotate crops. 3) Start the growing season with clean fields. 4) Rotate herbicide modes of action by using multiple modes of action during the growing season and apply no more than two applications of a single herbicide mode of action to thesame field in a two-year period. One method to accomplish this is to rotate herbicidetolerant trait systems.Soybean Event FG72 Petition - Revised Page 82 of 198

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### 5) Apply recommended rates of herbicides to active ly growing weeds at the correct time with the right application techniques. 6) Control any weeds that may have escaped the herbicide application. 7) Thoroughly clean field equipment between fields.

VIII.G. Potential impact on farming practices

Although more than 90% of the soybean acres planted today are glyphosate-tolerant, conventional and organic farming continue to be an important sect of the soybean market. Conventional and organic soybean growers will find no adverse effect on their farming practices with the introduction of FG72 soybeans.

It is not likely that organic farmer or other farmers who choose not to grow FG72 soybeans will be significantly impacted by the expected co mmercial use of this product. Nontransgenic soybeans varieties will still be available for conventional and organic soybean producers. Soybean is mostly a self-fertilized plant and therefore limits the chance of hybridization to conventional soybean varieties. In addition to the National Organic Program administered by USDA’s Agricultural Marketing Service which requires organic production operations to have distinct, defined boundaries and buffer zones to prevent unintended contact with prohibited substances from adjoining land that is not under organic management.

VIII.H. Potential effects on non-target organisms, including beneficial organisms

No adverse effect on non-target organisms from either the transgenic or non-transgenic plants was observed during any of the trials. Refer to Section VII D for biotic and abiotic stress
characteristics.

The FDA issued a finding of “No Concern” for gly phosate tolerant soybeans. As the presence of the 2mEPSPS and the HPPD proteins are the only difference found in FG72 that is not found in conventional soybean, FG72 and its progeny should have no indirect or direct plant pest effects.

VIII.I. Threatened and endangered species considerations

The US Fish & Wildlife Service (FWS) has accountability for endangered species under the Endangered Species Act (ESA), (16 USC 1531). Section 6 of the ESA requires federal agencies who conduct activities which may affect listed species to consult with the FWS to ensure that listed species are protected should there be a potential impact.

It is not anticipated that the use of FG72 soy bean will impact any currently listed species of concern. Species of concern that may inhabit areas close to commercial soybean operations would not be impacted by the use of FG72 soybean. Commercial agriculture routinely disturbs the ground in which crops are currently planted. As a result, perennial vegetative species would not grow in these areas. Additionally, because hori zontal gene flow to sexually incompatible species is not an issue, there is negligible potential for exposure to the transgenes contained in FG72 soybean through sexual reproduction.Soybean Event FG72 Petition - Revised Page 83 of 198

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### Isoxaflutole is currently registered for weed control use in corn in 18 of the primary corn producing states in the US. Collectively, these states represent approximately 75 % of the planted acres for corn and soybean (a four year average through 2009 - http://www.doane.com/research.php). End use products containing the active ingredient isoxaflutole are listed as “Restricted Use” and are for sale and use only by certified applicators. The approved and proposed end use product labels (e.g., tolerant soybean) also have extensive precautionary and restrictive language statements addressing handling and use of the product including specific endangered species protection requirements. The EPA “Registration Review” process for isoxaflutole is scheduled to be initiated in fiscal year 2011.

Glyphosate is currently supplied to US grower s by numerous generic sources of the active ingredient and generic end use products. This active is registered for use on tolerant soybean, corn and cotton as well as on specific non-tolerant crops and non-crop uses. The current Bayer understanding is that future endangered species assessments will be addressed in the “Registration Review” process for this active which was initiated by EPA in July of 2009.

VIII.J. Potential impact on biodiversity

Soybean is considered a self-pollinated species, propagated commercially by seed. The soybean flower stigma is receptive to pollen approximately 24 hours before anthesis and remains receptive 48 hours after anthesis. The anthers mature in the bud and directly pollinate the stigma of the same flower. As a result, soybeans exhibit a high level of self-fertilization and cross pollination is usually less than one percent (Beckie 2007, Palmer et al ., 2001).

There is no evidence of genetic transfer and exchange with organisms other than those with which soybean is able to produce fertile cros ses through sexual reproduction (Beckie 2007,
Stewart et al ., 2003). There are no wild Glycine species in the United States, nor are their wild or weedy species with which soybeans can produce fertile crosses.

VIII.K. Conclusion

It has been demonstrated that the presence of the 2mEPSPS and HPPD W336 proteins introduced in FG72 soybean will not present adverse environmental effects. The lack of wild type soybean species or relatives in the Americas in addition to the self pollinating nature of soybean prevents gene transfer into unintended targets. The current practice to control volunteer soybean plants will not be altered by FG72. Current agronomic practices limit weed control diversity tactics. The introduction of FG72 will provide a new mode of action for weed control in soybean to improve resistant weed management. It is expected that growers who choose not to grow FG72 will not be impacted by the commercial use of this product. It is also not anticipated that the commercial use of FG72 will have any potential impacts on non-target organisms or on threatened or endangered species.Soybean Event FG72 Petition - Revised Page 84 of 198

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### IX. STATEMENT OF GROUNDS UNFAVORABLE

Bayer CropScience and M.S. Technologies know of no study data and/or observations associated with Event FG72 soybean that will result in adverse environmental consequences for its introduction. The only biologically rele vant phenotypic difference between Event FG72 soybean and conventional soybean is the ex pression of the 2mEPSPS and HPPD W336 proteins which provide tolerance to the applic ation of glyphosate herbicide and isoxaflutole herbicide, respectively. Planting double-herbicide-tolerant soybean varieties, containing transformation event FG72, will provide growers with new options for weed control using IFTherbicide in combination with a glyphosate herbicide. Glyphosate is widely used in herbicidetolerant soybean and other agricultural production sy stems. IFT herbicide offers an alternative weed control option for the soybean grower vi a a new herbicide mode of action for soybeans that is efficacious against many of the herbi cide resistant weeds currently found in soybean
fields.Soybean Event FG72 Petition - Revised Page 85 of 198

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188:1798-1807. Skerman, V.B.D. 1980. Approved lists of bacterial names. Int. J. Syst. Bacteriol. 30:225-420. Available at http://www.ncbi.nlm.n ih.gov/books/bv.fcgi?rid=bacname Stafford, J. 2009. Broiler chicken feeding study with FG72 soybeans. Springborn and Smithers Laboratories. Unpublished report. Steinrücken H.C. and Amrhein N. 1980. The herbicide glyphosate is a potent inhibitor of 5- enolpyruvylshikimic acid-3-phosphate synthase. Biochem. Biophys. Res. Comm. 94(4): 1207-1212 Steinert, M., Flugel, M., Schuppler, M., Helbig, J.H ., Supriyono, A., Proksch, P. and Luck, P.C. 2001. The Lly protein is essential for p-hydr oxyphenylpyruvate dioxygenase activity in Legionella pneumophila

. FEMS Microbiol. Lett. 203:41-47. Stewart, C.N., Adang, M.J., All, J.N., Boerma, H.R., Cardineau, G., Tucker, D., and Parrott, W.A. 1996. Genetic transformation, recovery, and characterization of fertile soybean transgenic for a synthetic Bacillus thuringiensis cry1Ac gene. Plant Phys. 112:121-129. Stewart, C.N., Halfhill, M.D., and Warwick, S.I. 2003. Transgene introgression from genetically modified crops to their wild relatives. Nat. Rev. Gen. 4:806-817. Tacconelli, E., Carmeli, Y., Aizer, A., Ferreira, G., Foreman, M.G. and D'Agata, E.M. 2003. Mupirocin prophylaxis to prevent Staphylococcus aureus infection in patients undergoing dialysis: a meta-analysis. Clin. Infect. Dis. 37:1629-1638.Soybean Event FG72 Petition - Revised Page 91 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Thrane, C., Nielsen, M.N., Sorensen, J. and Olsson, S. 2001. Pseudomonas fluorescens DR54 reduces sclerotia formation, biomas s development, and disease incidence of
Rhizoctonia solani causing damping-off in sugar beet. Microb. Ecol. 42:438-445. USDA. 2007. Agricultural Chemical Usage 2006 Field Crops Summary. http://usda.mannlib.cornell.edu/usda/na ss/AgriChemUsFC//2000s/2007/AgriChemUsF C-05-16-2007_revision.pdf USDA. 2009. Adoption of Genetically Engineered Crops in the US. NASS, Acreage 2001-2009 http://www.ers.usda.gov/data/biotechcrops/ Van der Klis, R-J., Hendrickx, K., Herouet-G uicheney, C., and Rouan, D. 2006. The double mutant 5-enol-pyruvylshikimate-3-phosphate synthase gene product: 2mEPSPS description and characterization. Bayer CropScience. Unpublished report. Voisard, C., Keel, C., Haas, D. and Defago, G. 1989. Cyanide production by Pseudomonas
fluorescens helps suppress black root rot of tobacco under gnotobiotic conditions.
Embo J. 8:351-358. Walsh, C.T., Benson, T.E., Kim, D.H., and Lees, W.J. 1996. The versatility of phosphoenolpyruvate and its vinyl ether products in biosynthesis. Chemistry & Biology . 3: 83-91. Willamette Valley Pest Management Guide, 2011. http://ir.library.oregonstate.edu/xmlui/bitstream/handle/1957/20058/em8418.pdf Williams, G.M., Kroes, R., and Munro, I.C. 2000. Safety evaluation and risk assessment of the herbicide Roundup 1 

### and its active ingredient, glyphosate, for humans. Regul. Toxicol.
Pharmacol. 31:117-165. Wilson, M. and Lindow, S.E. 1993. Release of recombinant microorganisms. Annu. Rev.
Microbiol. 47:913-944. Wintermeyer, E., Flugel, M., Ott, M., Steinert, M., Rdest, U., Mann, K.H. and Hacker, J. 1994. Sequence determination and mutational analysis of the lly locus of Legionella pneumophila

. Infect. Immun. 62:1109-1117. Yan B., Srinivasa Reddy, M.S. and Collins, G.B. 2000. Agrobacterium tumefaciens -mediated transformation of soybean ( Glycine max (L.) Merr.) using immature zygotic cotyledon explants. Plant Cell Reports 19:1090-1097. Zhou, M., Xu, H., Wei, X., Ye, Z., Wei, L., Gong, W., Wang, Y., and Zhu, Z. 2006. Identification of a glyphosate-resistant mutant of rice 5- enolpyruvylshikimate-3-phosphate synthase using a directed evolution strategy. Plant Physiol . 140:184-195.

1 Roundup is a registered trademark of Monsanto.Soybean Event FG72 Petition - Revised Page 92 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 1

FIELD TRIAL TERMINATION REPORTS 2001-2008Soybean Event FG72 Petition - Revised Page 93 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 2001 USDA Termination Report for Herbicide Tolerant Soybean

Bayer CropScience LP

Trials Conducted: State (County)

01-087-08n: IL (Champaign) 01-268-11n: PR (Juana Diaz)

1. PLANTING DATES

May 25, 2001 (Champaign Co., IL) through December 15, 2001 (Juana Diaz, PR).

2. HARVEST/DESTRUCT DATES

September 28, 2001 (Champaign Co., IL) through April 12, 2002 (Juana Diaz, PR) with all plants tilled and disked
under at the termination of the studies.

3. PURPOSE

Field trials utilizing herbicide tolerant transgenic soybean lines were estab lished for breeding, efficacy testing and the generation of analytical data.

General Field Observations

Experienced personnel qualified in soybean cultivation perf ormed all plot observations. Records for the transgenic and non-transgenic control plots were provided from VE (emergence) through final stand counts prior to harvest and crop destruction.

Plant emergence measured at four to five days averaged 50% at both sites. Stand counts taken at the V2 and V3 stages ranged from 70% (IL) to 80% (PR). Final stand coun ts of transgenic plants at the V3 and V4 stages of development at both sites ranged from 40 - 60% while non- transgenic controls average greater than 70 – 80%.

The only phenotypic difference recorded between the plant types were their resp ective levels of herbicide tolerance.

Insect pest species recorded from the Puerto Rico plot were bean leaf bee tles (Coleoptera: Chry somelidae), whitefly (Homoptera: Aleyrididae), and loopers (Lepidoptera: Noctui dae). Only bean leaf bee tles were recorded at the
Illinois site.

Beneficial species recorded in Illinois were ladybugs ( Hippodamia convergens ), earthworms ( Lumbricus sp. ), and honey bees ( Apis melifera ). The Puerto Rico plot had ladybugs ( Hippodamia convergens ) and honey bees ( Apis melifera ). No differences in density or diversity of these species were noted between the two plot types.

No diseases were found affecting any of the plants at e ither site. Both sites listed the growing season as typical.

Final Disposition an d Volunteer Monitoring

All plant materials remaining at the completion of the studies were tilled under. Volunteer plants were found at the Puerto Rico study site on May 20, June 15, and June 28, 2002. A second “clean” count for this plot was achieved by August 5, 2002.Soybean Event FG72 Petition - Revised Page 94 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## USDA Field Termination Report

Notification No .: 02-071-03n

Applicant No .: GLY/ISX-2A-Soybean-MR

Permittee : Aventis CropScience (Now Bayer CropScience LP) Research Triangle Park, NC; 919-549-2655

Regulated Article : Herbicide-tolerant, glyphosate and isoxazole-tolerant, Soybean ( Glycine max )

Site Release Information : As shown below, five (5) of seven (7) sites were planted:

County/Parish/Distri

ct State or Territory Release Status

Hamilton IA Planted

Webster IA Not Planted Champaign IL Planted

Benton IN Planted

York NE Planted

Juana Diaz PR Planted

Sabana Grande PR Not Planted



### Information on each release follows:

County/

State Acreage

Planted Date
Planted Germination Data Transgenic vs. Non-transgenic Date
Terminated

Hamilton/IA 0.248 6/6/02 % Emergence/Seedling Vigor Not provided

Initial Stand Count Percentage

80.28% vs >88.06% on 6/28/02

Final Stand Count Percentage
80.28% vs >88.06% on 7/17/02 Harvested
11/1/02. Plant
material was chopped
11/4/02.

Champaign/IL 0.5 6/7/02 % Emergence/Seedling Vigor
>70% vs >75% on 6/14/02

Initial Stand Count Percentage
>60% vs >70% on 7/3/02

Final Stand Count Percentage
0-60+% vs 0-70+% on 7/22/02 Harvested
10/14/02 and
10/15/02. Plot
area disked
11/15/02.Soybean Event FG72 Petition - Revised Page 95 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Purpose of Release : The trials were established for the purpose evaluating efficacy, generating seed and providing analyti cal data. The performance of the herbicide-tolerant soybean with resp ect to the nontransgenic counterpart was also evaluated, as were the overall agronomic characteristics.

Observations : The test sites were inspected multiple times during the growing season for agronomic growth characteristics and disease and insect pest
infestation.

Plant emergence patterns were uniform and vigorous within the transgenic and nontransgenic plots for the plots located in Hamilton Co., Benton Co., York Co. and Juana Diaz Di strict. Plant emergence of the transgenic soybeans at the Champaign Co. site was not as good as the non-transgenic counterpart due to the poor seed quality produced in
Puerto Rico.

Observations were recorded at various stages throughout the growing season, ranging from plant emergence through full maturity. Insect species categorized as pests and benefi cials were noted among the plots, as was phytopathology. No diffe rence in susceptibility was noted between transgenic and nontransgenic plants. County/

State Acreage
Planted Date
Planted Germination Data Transgenic vs. Non-transgenic Date
Terminated

Benton/IN 0.3 6/8/02 % Emergence/Seedling Vigor
>70% vs >70% on 6/14/02

Initial Stand Count Percentage
>60% vs >60% on 7/3/02

Final Stand Count Percentage
0-60+% vs 0-60+% on 7/22/02 Harvested
10/21/02.
Plot area
mowed and
disked
10/25/02.

York/NE 0.37 6/10/02 % Emergence/Seedling Vigor % not provided. Good vigor.

Initial Stand Count Percentage Not provided

Final Stand Count Percentage Not provided Harvested
10/21/02.
Plot area
tilled
11/8/02.

Juana Diaz/PR 0.33 7/18/02 % Emergence/Seedling Vigor
>50% vs >50% on 7/23/02

Initial Stand Count Percentage
>80% vs >80% on 8/1/02

Final Stand Count Percentage

40-60% vs >80% on 8/15/02 Harvested
11/11/02 and
11/12/02.
Plot area mechanically
cultivated
12/4/02.Soybean Event FG72 Petition - Revised Page 96 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Observations/Dates County/State Fungi/Diseases Insect Pests Beneficial Insects

Hamilton/IA Iron chlorosis was evident 7/19/02 on both plant
types. Bean leaf beetles and occasional grasshoppers were noted 6/17/02, 7/19/02, 8/19/02 on both plant types. Damage was very minor. Ladybugs were observed
7/19/02 and 8/19/02 on both plant types.

Champaign/IL Bacterial blight and brown spot were observed
7/22/02 and 8/9/02 on both plant types. Damage
was minor. Woolly bear, caterpillar, grasshoppers and bean
leaf beetles were observed
7/22/02 and 8/28/02 on both plant types. Damage
was mild. Ladybugs were seen
7/19/02 and 7/30/02 on both plant types.

Benton/IN Bacterial blight and brown spot were noted 7/22/02 and 8/9/02 on both plant types. Damage was
minor. The Japanese beetle was seen during flowering on 7/31/02 on both plant types. Damage was
minor. Ladybugs were seen on
7/16/02 and 7/31/02 on both plant types.

York/NE No phytopathology noted 6/17/02, 6/27/02, 7/18/02 or 10/21/02 on either plant
type. No pests were observed on 6/17/02, 6/27/02,
7/18/02 or 10/21/02 on either plant type. Ladybugs were seen
6/27/02 and 7/18/02 on both plant types.

Juana Diaz/PR No phytopathology noted 9/3/02, 9/6/02 or 9/11/02 on either plant type. Whiteflies and loopers were evident 7/20/02,
8/15/02 and 9/3/02 on both plant types. Damage levels ranged from mild to moderately severe. Ladybugs were seen
8/15/02 and 9/6/03 on both plant types.



### Results : No agronomic, insect susceptibility or disease susceptibility or resistance
differences were observed between the transgenic soybean plants and the nontransgenic counterpart. The only difference noted between the transgenic and nontransgenic plants w as in the desired trait – tolerance to glyphosate herbicide – in the tran sgenic soybean plants, whereas the nontransgenic plants were susceptible to treatment with glyphosate.

Plant Disposition : Crops were harvested from mid October (10/14/02) until about mid November (11/12/02). Methods used for the destruction of remaining plant material included chopping, mowing, mechanical cultivation and under-tilling.

Volunteer Monitoring : The plot areas were visually insp ected for volunteer soybean plants during the following growing season. The table below summarizes observations made and remedial action( s) taken, if needed, at each site.Soybean Event FG72 Petition - Revised Page 97 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Post-Season Volunteer Monitoring County/State Date No. Plants Observed/Stage Method of Destruction

Hamilton/IA 6/5/03 None
7/1/03 None
7/30/03 None
8/27/03 None Champaign/IL 7/3/03 1 to 10 plants/V5-V6 Tillage
8/8/03 None
8/19/03 None
8/25/03 None

Benton/IN 6/25/03 None
7/25/03 None
8/12/03 None
8/26/03 None York/NE 5/17/03 11 to 50 plants/V1 Mechanically cultivated 6/20/03 11 to 50 plants/V1-V2 Hand-weeded
7/18/03 None
7/31/03 None
8/28/03 None Juana Diaz/PR 12/18/02 >50 plants/V2 Mechanically cultivated 1/3/03 >50 plants/V3 Mechanically cultivated 1/17/03 11 to 50 plants/V3 Mechanically cultivated 1/31/03 None Field mechanically cultivated 2/7/03 None Field mechanically cultivated



### Weediness Characteristics: There was no evidence of change in characteristics that would enhance
survival of the herbicide-tolerant soybean plants as compared to nontransgenic soybean plants. No difference in weediness characteristics between the transgenic and nontransgenic soybean lines
was observed.

Field Management: Conventional field practices we re utilized at all sites.

Non-Target Organisms: No adverse effect on non-target organisms from either the transgenic or nontransgenic plants was observed in any of the trials.

Weather Synopsis: Weather notations indicate the Hamilton Co., Benton Co. and Juana Diaz sites experienced normal/typical climatic conditions. The Champaign and York Co. sites were noted as being hotter and drier
than normal.

Containment Measures: Isolation methods included the separation of soybean plants by
distances of 5 to 10 feet. Additiona lly, border rows were used to prevent the flow of pollen. The border rows were not harvested but destroyed at the conclusion of the trial. The test plots and border areas were monitored the following growing season for volunteer soybean plants.Soybean Event FG72 Petition - Revised Page 98 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## USDA Field Termination Report

Notification No .: 02-274-09n

Applicant No .: GLY/ISX-2E-Soybean-MR

Permittee : Bayer CropScience LP Research Triangle Park, NC; 919-549-2655

Regulated Article : Herbicide-tolerant, glyphosate and isoxazole-tolerant, Soybean ( Glycine max )

Site Release Information :

As follows:

District/ Territory Acreage

Planted Date
Planted Germination Data Transgenic vs . Non-transgenic Date
Terminated

Sabana
Grande/

PR 1.10 1/5/03 % Emergence/Seedling Vigor
>50% vs >50% on 1/10/03

Initial Stand Count Percentage
>80% vs >80% on 1/20/03

Final Stand Count Percentage

40-60% vs >80% on 2/4/03 Harvest
occurred on
5/6/03 and
5/12/03. The
field was mechanically
cultivated on
5/16/03.



### Purpose of Release : The purpose of the release was to test the efficacy as well as breeding of transgenic soybean plants. The pe rformance of the herbicide-tolerant soybean with respect to the nontransgenic counterpart plant was also evaluated, as were the overall agronomic characteristics.

Observations : The test site was inspected eleven (11) times during the growing season (1/10/03, 1/20/03, 2/4/03, 3/3/03, 3/24/03, 4/1/03, 4/16/03, 5/1/03, 5/5/03, 5/6/03 and 5/12/03) for agronomic growth characteristics and disease and insect pest infestation.

Plant emergence patterns were uniform and vigorous within the transgenic and nontransgenic plots; germination rate was noted as >80%. Observations were recorded from first node through beginning pod stages. Two (2) species of insect pests were noted: whiteflies and loopers. Whiteflies were seen on 1/20/03, 2/4/03, 3/24/03 and 5/1/03. Loopers were observed on 3/24/03. Da mage levels ranged from mild to moderately severe. Two beneficial insect species were observed: ladybug and ant. The ladybugs were seen on 2/4/03 and 4/1/03. The
ants were observed on 3/24/03. Observations were made on both the transgenic and nontransgenic plots.Soybean Event FG72 Petition - Revised Page 99 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### No disease susceptibility was noted on the transgenic or nontransgenic plants during any of the visits made on 3/3/03, 3/24/03, 4/16/03 and
5/5/03.

Results : No agronomic, insect susceptibility or disease susceptibility or resistance
differences were observed between the transgenic soybean plants and its nontransgenic counterpart.

Plant Disposition : The crop was harvested on 5/6/03 a nd 5/12/03. All remaining vegetative material was mechanically cultivated on 5/16/03.

Volunteer Monitoring : The plot area was visually inspected for volunteer soybean plants five (5) times during the following growing season, until no volunteers had been observed for two (2) consecutive monitoring visits.

Post-Season Volunteer Monitoring
Date No. Plants Observed/Stage Method of Destruction 5/22/03 >50 plants/V2 Mechanically Cultivated 6/6/03 >50 plants/V3 Mechanically Cultivated 6/27/03 11 to 50 plants/V3 Mechanically Cultivated 7/6/03 None Field Mechanically Cultivated 7/21/03 None Field Mechanically Cultivated



### Weediness Characteristics: There was no evidence of change in characteristics that would enhance
survival of the herbicide-tolera nt transgenic soybean plants as compared to nontransgenic soybean pl ants. No difference in weediness characteristics between the transgenic and non-transgenic soybean lines
was observed.

Non-Target Organisms: No adverse effect on non-target organisms from either the transgenic or nontransgenic plants was observed in the trial.

Weather Synopsis: Weather notations indicate the site experienced normal/typical climatic
conditions.

Containment Measures: The Sabana Grande test site is a 59-acre farm. The site produces no commercial crops. Transgenic soyb ean plants were separated by a distance of 10 feet. Border rows surrounded the perimeter of the test plot to minimize the flow of pollen. Border rows were not harvested but destroyed at trial conclusion. The test plot and border area were monitored for volunteer soybean plants during the period that
followed.Soybean Event FG72 Petition - Revised Page 100 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## USDA Field Termination Report

Notification No .: 03-080-05n

Applicant No .: GLY/ISX-3C-Soybean-MR

Permittee : Bayer CropScience LP Research Triangle Park, NC; 919-549-2655

Regulated Article : Herbicide-tolerant, Glyphosate and Isoxazole-tolerant, Soybean ( Glycine

max )

Site Release Information : Eight (8) of twenty (20) sites were planted:

County/Parish/District State or Territory Release Status

Boone IA Not Planted

Carroll IA Planted

Dallas IA Planted

Des Moines IA Planted

Madison IA Planted

Marshall IA Planted

Polk IA Not Planted

Webster IA Not Planted Champaign (Bayer) IL Planted Champaign (Mertec) IL Not Planted

Hamilton IL Not Planted

Logan IL Not Planted

Warren IL Not Planted

Boone IN Not Planted

Clinton IN Not Planted Tipton IN Not Planted

Clinton MO Not Planted

Saline MO Not Planted

York NE Planted

Sabana Grande PR Planted



### Acreage and planting and termination dates follow:

County or District/ State or Territory Acreage

Planted Date Planted Date Terminated

Carroll/IA 0.45 5/22/03 10/1/03 Dallas/IA 4.19 5/19/03, 5/20/03, 5/29/03 & 6/4/03 10/4/03

Des Moines/IA 0.45 5/17/03 9/28/03

Madison/IA 1.59 5/23/03 & 6/5/03 10/7/03

Marshall/IA 0.45 5/17/03 10/5/03 Champaign/IL 3.25 5/22/03, 5/23/03, 8/14/03 10/10/03

York/NE 2.04 5/28/03 & 5/30/03 9/4/03 Sabana Grande/PR 4.93 5/24/03, 7/11/03 & 8/21/03 9/1/03 & 12/3/03Soybean Event FG72 Petition - Revised Page 101 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Purpose of Release : The trials were established for the purpose of breeding, evaluating efficacy and agronomic characteristics, and to obtain analytical data on the herbicide-tolerant soybean plants . The performance of the transgenic soybean with respect to the nontransgenic counterpart was also evaluated, as were the overall agronomic characteristics.

Observations : The test sites were visually inspected multiple times during the growing season for agronomic growth characteristics and disease and insect pest infestation. Observations were recorded for the transgenic and nontransgenic plants from emergence through harvest.

County or District/ State or Territory Germination Data Transgenic vs. Non-transgenic

Carroll/IA % Emergence/Seedling Vigor
88.5% vs. 88.5% on 6/3/03 Initial Stand Count Percentage
88.5% vs. 88.5% on 6/21/03 Final Stand Count Percentage

88.5% vs. 88.5% on 10/1/03

Dallas/IA (1 st Location) % Emergence/Seedling Vigor
88.5% vs . 88.5% on 5/31/03 Initial Stand Count Percentage
88.5% vs. 92.0% on 6/14/03 Final Stand Count Percentage

96.4% vs . 88.5% on 10/2/03

Dallas/IA (2 nd Location) % Emergence/Seedling Vigor
76.9% vs . 76.9% on 5/31/03 Initial Stand Count Percentage

76.9% vs . 76.9% on 6/14/03 Final Stand Count Percentage

76.9% vs . 76.9% on 10/3/03

Dallas/IA (3 rd Location) % Emergence/Seedling Vigor
88.5% vs . 88.5% on 6/10/03 Initial Stand Count Percentage
88.5% vs . 88.5% on 6/25/03 Final Stand Count Percentage

88.5% vs . 88.5% on 10/4/03

Des Moines/IA % Emergence/Seedling Vigor
84.6% vs . 84.6% on 5/29/03 Initial Stand Count Percentage
84.6% vs . 84.6% on 6/16/03 Final Stand Count Percentage

84.6% vs. 84.6% on 9/28/03

Madison/IA % Emergence/Seedling Vigor
92.3% vs . 92.3% on 6/5/03 Initial Stand Count Percentage

92.3% vs . 92.3% on 6/20/03 Final Stand Count Percentage

~92.3% vs. 92.3% on 9/20/03Soybean Event FG72 Petition - Revised Page 102 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION County or District/ State or Territory Germination Data Transgenic vs. Non-transgenic

Marshall/IA % Emergence/Seedling Vigor
88.5% vs . 88.5% on 6/1/03 Initial Stand Count Percentage
88.5% vs . 88.5% on 6/18/03 Final Stand Count Percentage

88.5% vs . 88.5% on 7/19/03

Champaign/IL (1 st and 2 nd Plantings) % Emergence/Seedling Vigor
>60% vs . >60% on 6/4/03 Initial Stand Count Percentage
>60% vs . >60% on 6/16/03 Final Stand Count Percentage
60% vs . 0% on 7/14/03

Champaign/IL (3 rd Planting) % Emergence/Seedling Vigor
>70% vs . >70% on 9/3/03 Initial Stand Count Percentage
>70% vs . >70% on 9/18/03 Final Stand Count Percentage

>70% vs . >70% on 10/2/03

York/NE % Emergence/Seedling Vigor
50-90% vs . 50-90% on 6/17/03 Initial Stand Count Percentage

50-90% vs . 50-90% on 6/17/03 Final Stand Count Percentage

50-90% vs . 0-90% on 9/4/03

Sabana Grande/PR (1 st Planting) % Emergence/Seedling Vigor
>50% vs . >50% on 6/3/03 Initial Stand Count Percentage
>80% vs . >80% on 6/9/03 Final Stand Count Percentage

40-60% vs . >80% on 6/23/03

Sabana Grande/PR (2 nd and 3rd Plantings) % Emergence/Seedling Vigor
>50% vs . >50% on 7/16/03 & 8/27/03

Initial Stand Count Percentage
>80% vs . >80% on 7/28/03 & 9/8/03

Final Stand Count Percentage
40-60% vs . >80% on 8/11/03 & 9/22/03



### All sites reported normal growth and development of the soybean plants from seedling emergence through flow ering, pod development and seed fill. The Champaign Co. site reporte d delayed emergence due to dry soil conditions. A killing frost occurred 10/2/03.Soybean Event FG72 Petition - Revised Page 103 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Observations/Dates County or
District/State or Territory Fungi/Diseases Insect Pests Beneficial Insects

Carroll/IA None observed 6/3/03, 6/21/03, 7/18/03 and 10/1/03. Dallas/IA-Loc.1 None observed 5/31/03, 6/14/03, 7/10/03, 9/3/03 and 10/2/03. Dallas/IA-Loc.2 None observed 5/31/03, 6/14/03, 7/10/03, 9/3/03
and 10/3/03. Light infestation of bean
leaf beetle observed
5/31/03. None observed 5/31/03, 6/14/03, 7/10/03, 9/3/03
and 10/3/03.
Dallas/IA-Loc.3 Moderate levels of iron deficiency chlorosis seen 6/25/03, 7/14/03, 9/7/03 and 10/4/03 on both plant
types. None observed 6/25/03, 7/14/03, 9/7/03 and 10/4/03.

Des Moines/IA None observed 5/29/03, 6/16/03, 7/6/03, 8/27/03 and 9/28/03.

Madison/IA None observed 6/20/03, 7/10/03, 9/20/03 and
10/7/03. None observed 6/5/03, 6/20/03, 9/20/03 and 10/7/03.

Marshall/IA None observed 6/1/03, 6/18/03, 7/19/03 and 10/5/03. Champaign/IL

(1 st and 2 nd Plantings) Light infestation of brown spot and bacterial blight
observed 7/23/03 and 8/21/03 on both plant
types. Light to moderate
infestations of leafhoppers, bean leaf beetles, Japanese beetles and soybean aphids observed 6/16/03, 7/23/03
and 8/21/03. Ladybugs and spiders
observed 7/23/03 and
8/21/03.

Champaign/IL (3 rd Planting) None observed 9/3/03,
9/18/03 and 10/2/03. Bean leaf beetles and

Western corn rootworms observed on both plant types 9/18/03 and 10/2/03 but no damage evident. Ladybugs observed 9/3/03, 9/18/03 and
10/2/03.

York/NE None observed 6/17/03, 7/8/03, 8/1/03 and 9/4/03. Light populations of bean leaf beetles, loopers, grasshopper and blister beetles observed 6/17/03, 7/8/03, 8/1/03 and 9/4/03. Ladybugs observed 7/8/03, 8/1/03 and
9/4/03.

Sabana
Grande/PR (1 st Planting) None observed 6/9/03, 6/23/03, 7/15/03 and
8/14/03. Light populations of loopers and whiteflies seen 6/23/03, 7/9/03 and
8/15/03. Ladybugs seen 7/15/03.

Sabana
Grande/PR (2 nd and 3 rd Plantings) None observed 7/28/03, 8/11/03, 9/4/03, 9/8/03,
9/22/03 and 11/17/03. Light populations of loopers and whiteflies observed 8/11/03, 8/29/03, 9/22/03, 10/9/03 and
11/17/03. Ladybugs observed 8/11/03, 9/10/03, 9/22/03
and 10/21/03.



### Results : No insect susceptibility or disease susceptibility or resistance differences were observed between the transgenic soybean plants and its nontransgenic counterpart. Some phenotypic differences were observed between the transgenic and nontransgeni c plants in terms of the level of
tolerance to glyphosate and isoxazole herbicide treatments.Soybean Event FG72 Petition - Revised Page 104 of 198

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### Plant Disposition : See below:

County or District/ State or Territory Harvest or Crop Destruct Date(s) Disposition of Remaining
Plant Material Carroll/IA 10/1/03 Plot area mechanically cultivated. Dallas/IA 10/2/03, 10/3/03 &
10/4/03 Plot area disked under 10/4/03.

Des Moines/IA 9/28/03 Plot area mechanically cultivated.

Madison/IA 10/7/03 Plot area mechanically cultivated.

Marshall/IA 10/5/03 Plot area disked under 10/5/03. Champaign/IL 9/15/03, 10/2/03,
10/7/03 & 10/10/03 Trial areas mowed and disked under
10/10/03.

York/NE 9/4/03 Plot area disked under. Sabana Grande/PR 8/15/03, 11/17/03,
11/20/03 & 12/3/03 Trial areas mechanically cultivated
9/1/03 and 12/3/03.



### Volunteer Monitoring : The plot areas were visually insp ected for volunteer soybean plants during the following growing season. The table below summarizes
observations made and actions take n to eliminate volunteer plants.

Post-Season Volunteer Monitoring County or
District/State or Territory Date No. Plants Observed/Stage Method of Destruction

Carroll/IA 4/15/04 None 5/12/04 11 to 50 plants Mechanically cultivated 6/9/04 >50 plants/V1 Removed plants by hand.
7/6/04 None
8/11/04 None

Dallas/IALoc.1 4/17/04 None

5/15/04 1 to 10 plants Mechanically cultivated 6/11/04 >50 plants Atrazine applied.
7/6/04 None
8/11/04 None

Dallas/IALoc.2 4/17/04 None

5/15/04 1 to 10 plants Mechanically cultivated 6/11/04 11 to 50 plants Atrazine applied.
7/6/04 None
8/11/04 None

Dallas/IALoc.3 4/17/04 None

5/15/04 1 to 10 plants Mechanically cultivated 6/11/04 11 to 50 plants Atrazine applied.
7/6/04 None
8/11/04 None

Des Moines/IA 4/19/04 None 5/17/04 1 to 10 plants Mechanically cultivated 6/10/04 11 to 50 plants Removed plants by hand.
7/8/04 None
8/12/04 NoneSoybean Event FG72 Petition - Revised Page 105 of 198

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Post-Season Volunteer Monitoring County or
District/State or Territory Date No. Plants Observed/Stage Method of Destruction

Madison/IA 4/16/04 1 to 10 plants Removed plant by hand. 5/14/04 11 to 50 plants Mechanically cultivated 6/11/04 >50 plants Removed plants by hand.
7/6/04 None
8/11/04 None

Marshall/IA 4/15/04 None 5/12/04 1 to 10 plants Removed plants by hand and field mechanically cultivated. 6/9/04 11 to 50 plants Removed plants by hand.
7/8/04 None
8/12/04 None *Champaign/IL

(1 st and 2 nd Plantings) 5/20/04 >50 plants/V3 Callisto herbicide applied and field mechanically cultivated.

6/28-30/04 >50 plants/V1-V5 Removed plants by hand.
7/13/04 None
9/7/04 None 9/13/04 >50 plants/V1-R1 Removed plants by hand Champaign/IL (3 rd Planting) 5/28/04 None Callisto herbicide applied and the field was mechanically cultivated, despite no presence of volunteers.
6/11/04 None
6/25/04 None
6/29/04 None
7/13/04 None

York/NE 5/3/04 None
6/4/04 None
7/1/04 None
7/30/04 None

Sabana
Grande/PR (1 st Planting) 9/16/03 >50 plants/V2 Mechanically cultivated

9/30/03 11 to 50 plants/V3 Mechanically cultivated 10/17/03 1 to 10 plants/V4 Mechanically cultivated
10/31/03 None
11/14/03 None

Sabana
Grande/PR (2 nd & 3 rd Plantings) 11/24/03 >50 plants/V2 Mechanically cultivated

12/10/03 >50 plants/V2 Mechanically cultivated 12/22/03 11 to 50 plants/V2 Mechanically cultivated
1/2/04 None
1/16/04 None
2/6/04 None
2/26/04 None
3/24/04 None



### *Volunteer monitoring of this plot will continue in 2005 to ensure the elimination of all volunteer soybean plants.Soybean Event FG72 Petition - Revised Page 106 of 198

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### Weediness Characteristics: There was no evidence of change in characteristics that would enhance survival of the transgenic soyb ean plants as compared to the nontransgenic soybean plants. No di fference in weediness characteristics was observed between the transgenic and nontransgenic soybean lines.

Non-Target Organisms: No adverse effect on non-target organisms from either the transgenic or nontransgenic plants was observed during any of the trials.

Field Management/Experimental Treatments: As follows:

County or District/ State or Territory Field Management/Experimental

Treatments Carroll/IA Pursuit herbicide applied ~6/20/03. Dallas/IA For two locations, Pursuit herbicide was applied to half of the plot, while

Roundup was applied to the other half. Only Pursuit herbicide was

applied at the third location. Des Moines/IA Pursuit herbicide applied ~6/20/03.

Madison/IA Pursuit herbicide was applied to half of the plot, while Roundup was applied to the other half on 6/20/03. Marshall/IA Pursuit herbicide applied ~6/20/03. Champaign/IL Made multiple herbicide treatments containing glyphosate and HPPD inhibiting herbicides. York/NE No special field management techniques. Sabana Grande/PR Roundup applied.



### Weather Synopsis: Climatic conditions were as follows:

County or District/ State or Territory Weather Conditions

Carroll/IA
Dallas/IA
Des Moines/IA

Madison/IA
Marshall/IA Spring was wet and cool, while August was very hot and dry.

Champaign/IL Initially dry, but later becoming
normal York/NE Weather was dry and hot. Sabana Grande/PR Typical



### Containment Measures: Transgenic soybean plants were sepa rated from other soybean plants not a part of the trial by distances up to 100 feet. In addition, some sites planted border rows that were destroyed at the conclusion of the trial.

The test plots including border areas were monitored the next growing season for volunteer soybean plants



### .Soybean Event FG72 Petition - Revised Page 107 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 108 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 109 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 110 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 111 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 112 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 113 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 114 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONSoybean Event FG72 Petition - Revised Page 115 of 198

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### Appendix 2

MATERIALS AND METHODS- PRODUCT CHARACTERIZATIONSoybean Event FG72 Petition - Revised Page 116 of 198

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### 2.A. Materials and methods for molecular characterization - DNA tests

Materials

DNA for the analyses was isolated from leaves of event FG72 soybean and the control (Jack) produced in the greenhouse. The references included the plasmid pSF10 that was used to produce soybean event FG72. For Southern blot analysis of soybean genomic DNA, digested plasmid pSF10 DNA (approximately 0.1 or 1 genom ic copies equivalent) was mixed with Jack genomic DNA (negative control) and separated by electrophoresis on agarose gels. Phage Lambda ( Pst I or Hind III digested) was used as a molecular weight marker for size estimation of the DNA fragments.

Identity of the materials

The identity of the greenhouse produced material was confirmed by PCR prior to use in order to verify the presence or absence of FG72, as appropriate. The zygosity status of the harvested plants was determined by means of zPCR when necessary. The integrity of the isolated DNA was verified in each Southern analysis by observation of the DNA samples on an ethidium bromide stained agarose gel. The identity of the materials used in generational stability analyses was confirmed by chain-of-c ustody documents and by PCR analysis.

DNA preparation for Southern blot and PCR analyses

Harvested plant tissues were directly transferred and frozen in liquid nitrogen, then stored in an ultralow freezer until DNA extraction. Genomic DNA was isolated following standard procedures, and stored at 4°C. Plasmid DNA was prepared from an E. coli cell strain containing plasmid pSF10. Concentration of the DNA extracts was determined by measurement with PicoGreen®. DNA was digested with restriction enzymes following the procedure indicated by
the manufacturer.

Southern blot analysis of genomic DNA

Digested genomic DNA samples were subjected to electrophoresis on 1% TAE agarose gels and transferred to nylon membranes following standard procedures (Sambrook et al ., 1989).

An appropriate dilution of the restriction enzyme digested pSF10 was prepared, and an amountrepresenting 0.1 or 1 plasmid copies per soybean genome was added to a digested nontransgenic DNA sample. This reconstitution sample served as a positive control and was used to show that the hybridizations were perform ed under conditions allowing hybridization of the probe with target sequences. Phage Lambda DNA digested with Pst I or Hind III was included as
a size standard.Soybean Event FG72 Petition - Revised Page 117 of 198

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### DNA probe preparation

The T-DNA probe template was prepared by means of a SacI/SmaI restriction digestion of the pSF10 plasmid DNA, according to the conditions proposed by the manufacturer. To purify the probe template, the complete reaction mixture of the restriction digestion was loaded on a 1% TAE agarose gel, the fragment of 7204 bp was cut out of the gel and the probe template was isolated from the gel slice using the QIAquick Gel Extraction Kit (Qiagen).

The Ph4a748B probe template was synthesized by means of PCR amplification using the Expand TM 

### High Fidelity PCR system and pSF10 plasmid. Each primer has two priming sites in plasmid pSF10; therefore, two amplicons were obtained. After loading the complete reaction mixture on a 1% TAE agarose gel, the 430 bp amplicon was cut out of the gel and the probe template was isolated from the gel slice using the QIAquick Gel Extraction Kit (Qiagen).

The DNA templates were labeled with [ α

32 

### P]-dCTP using the Ready-to-go DNA labeling beads(GE Healthcare). Unincorporated nucleotides were removed by separation on a micro BioSpin ® 

### 30 column (Bio-Rad).Soybean Event FG72 Petition - Revised Page 118 of 198

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### 2.B. Materials and methods for protein characterization tests

Studies on potential toxicology and allergenicity for food, feed and the environment are conducted with purified 2mEPSPS and H PPD W336 proteins expressed by Escherichia coli ( E.coli ). In order to utilize the safety data of the proteins produced in the microorganism for the safety assessment of the same protein produced in a genetically modified plant, it is important to confirm that the protein produced in a microorganism is representative of the protein produced in the modified plant. The analytical tests show that the proteins produced in E.coli are representative of 2mEPSPS and HPPD W336 prot eins produced in FG72 soybean plants.

2mEPSPS

Equivalency of E.coli -produced 2mEPSPS protein and plant-produced protein was established via SDS-PAGE, western blot, N-terminal ami no acid sequencing, enzyme activity and LC/MS
methods.

Materials

The plant-produced 2mEPSPS protein was isolated from greenhouse-grown plants of doubleherbicide-tolerant soybean. The identity of the plants was confirmed by PCR of the planted seed. Leaf extract was purified on an antibody affinity column, and the purified protein solution was stored at -20°C or lower until further analyses were performed. The antibody affinity column used for this purification was purchased from Pierce (Rockford, IL, product number 44894), and was prepared using a covalently attached polyclonal antibody specific for
2mEPSPS.

The 2mEPSPS protein reference standard (BCS reference standard, Batch N° LEJ5837, purity >99%) was produced in E. coli , and purified following a modification of the method of Priestman
et al. (2005). The protein solution is stored in an Ultrafreezer

.

Analysis by N-terminal sequencing

The affinity purified FG72 soybean produced 2mEPSPS protein, the non-transgenic (NT) Jack soybean extract and the E.coli produced 2mEPSPS protein were loaded onto the PVDF membrane at Catalent in San Diego, CA for analysis of the N-terminal amino acid sequence of the proteins by Edman degradation. The protein bands of interest were excised from the PVDF membranes and placed into labeled Eppendorf tubes. The PVDF strips of E. coli produced 2mEPSPS protein, FG72 soybean produced 2mEPSPS and NT Jack soybean controls were placed into the center of the Procise Sequencing cartridge blocks. Sequencing was performed on the first 5 residues using a pulsed liquid method for PVDF on an Applied Biosystems Procise 494 protein sequencer. Amino acids were identified by comparison with the retention times of PTH amino-acid standards analyzed at the beginning of the sequence.

Analysis by SDS-PAGE

SDS-PAGE was performed using a Novex Bis-Tris 10% polyacrylamide gel (InVitrogen, CA, product number NP0301) and a MOPS SDS running buffer (cat# NP0001) according to the
manufacturer’s instructions. The 2mEPSPS protein isolated from FG72 soybean leaves, theSoybean Event FG72 Petition - Revised Page 119 of 198

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### corresponding protein from E.coli and the NT Jack soybean sample were denatured and analyzed by electrophoresis on a denaturing polyacrylamide gel where mobility can be correlated to molecular weight. The gel was then stained with Pierce ImperialTM Stain to visualize the protein bands. The gel was stained with Pierce ImperialTM Protein Stain (cat# 24615) for 1 hour and then destained in water over-night. For the first 2 hours of destaining, the water was changed every 30 minutes. BenchMarkTM molecular weight markers from InVitrogen Life Technologies (product number 100747-012) were used.

Analysis by western blotting

Western blotting was performed in the same electrophoresis system used for SDS-PAGE and the gel was blotted to a PVDF membrane (Bio-Rad, Immun-Blot PVDF Membrane, cat# 162- 0174) according to the instructions provided by Bio-Rad and InVitrogen. The proteins in the gel were transferred out of the gel perpendicular to the direction of the first electrophoresis. They were adsorbed to the membrane giving an exact replica of the positions of all the proteins in the gel. The membrane was washed with TBS (Tris buffer saline, Bio-Rad, cat # 170-6435) for 10
minutes and blocked for 1 hour with 5% non-fat milk in TBS. The membrane was washed twice with TTBS (Tween Tris-buffer saline, Bio-Rad, cat #170-6531 and cat #170-6435) and once with TBS and incubated for 2 hours with rabbit polyclonal antibodies raised against the 2mEPSPSprotein. The membrane was washed twice with TTBS and once with TBS and incubated overnight with the enzyme conjugate, a second antibody with a horse radish peroxidase (HRP) linked anti-rabbit antibody. After the second antibody incubation, the membrane was washed with TTBS and TBS. The color was developed according to the manufacture's instructions (BIO-RAD, LIT178RevC). All reagents except t he rabbit polyclonal anti-2mEPSPS antibodies used for western blotting were obtained from Bio-Rad as the HRP Conjugate Substrate kit (cat # 170-6431) which included the blotting grade goat anti-rabbit IgG HRP conjugate (cat# 170- 6515). The rabbit polyclonal anti-2mEPSPS antibodies were obtained from Bayer BioScience in Gent, Belgium.

Analysis by HPLC/Electrospray Mass Spectrometry

This analysis was performed at Catalent in San Diego, CA, USA.The 2mEPSPS protein from E.
coli and the 2mEPSPS protein from the NT Jack so ybean extracts were denatured for 1 hour at 37 °C in Rapid Gest (Waters Corporation, 1 mg/mL) containing 10 mM dithiothreitol (DTT). The denatured protein disulfide bonds were alkylated with iodoacetamide for 30 minutes in the dark and digested with trypsin for 1 hour at 37°C. A full scan analysis was performed on the E. coli produced 2mEPSPS protein trypsin digest

, the trypsin digest of the NT extract and the trypsin digest of the FG72 soybean produced 2mEPSPS protein to determine the location and identification of the peptides.

Analysis of enzymatic activity

The enzymatic activity of the FG72 soybean produced 2mEPSPS protein was determined using the Gent SOP BBS 07/74/02 with modifications to adjust for sample concentration. The activity is determined by measuring the release of inorganic phosphate using a malachite green dye at a wavelength of 660 nm. 2mEPSPS catalyzes the reaction of shikimate-3-phospate and phosphoenolpyruvate to form 5-enolpyruvylshi kimate-3-phosphate and phosphoric acid. The 5- enolpyruvylshikimate-3-phosphate then eliminates phosphoric acid to form chorismate. An increase in the detection of inorganic phosphate release above 10% indicates the protein is
active.Soybean Event FG72 Petition - Revised Page 120 of 198

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### HPPD W366

Equivalence of E.coli -produced HPPD W336 protein and plant-produced protein was established via SDS-PAGE, western blot, N-term inal amino acid sequencing, enzyme activity and HPLC/Electrospray Mass Spectrometry methods.

Materials

The plant-produced HPPD W336 protein was isolated from greenhouse-grown plants of double herbicide-tolerant-soybean for event FG72. The identity of the plants was confirmed by PCR. Leaf extract was purified using HPPD W336 monoclonal antibody affinity column, and the purified protein solution was stored at 4°C or lower until further analyses were performed. The antibody affinity column used for this purificat ion was purchased from Pierce (Rockford, IL, product number 44894), and was prepared using a covalently attached monoclonal antibody specific for HPPD W336.

The HPPD W336 protein reference standard (BCS reference standard, Batch N° LB18050, purity >96%) was produced in E. coli , and purified following a modification of the method of
Priestman et al. (2005). The protein solution is stored in an Ultralowfreezer.

Analysis by N-terminal sequencing

The affinity purified E.coli produced HPPD W336 protein was loaded onto the PVDF membrane of a sample preparation cartridge according to manufacturer’s instructions. The completed analysis of the N-terminal amino acid sequence of the protein by Edman degradation was performed at Catalent, San Diego, CA, USA.

Analysis by SDS-PAGE

SDS-PAGE was performed using a InVitrogen 10% BisTris polycarylamide gel (InVitrogen, CA, cat# NP0001) and a MOPS SDS running buffer according to the manufacturer’s instructions

. The gel was stained with Pierce ImperialTM stain (cat# 24615) for 1 hour and then destained in water over-night. For the first 2 hours of destaining, the water was changed every 30 minutes. BenchMarkTM molecular weight markers from InVitrogen Life Technologies (product number 100747-012) were used.

Analysis by western blotting

Western blotting was performed in the same electrophoresis system as used for SDS-PAGE and the gel was blotted to PVDF membranes (Bio-Rad, Immun-Blot PVDF Membrane, cat#162- 0174) according to the instructions provided by BioRad. The proteins in the gel were transferred out of the gel perpendicular to the direction of the first electrophoresis using a 10mM CAP buffer, pH 11. The proteins were adsorbed to the membrane giving an exact replica of the positions of all the proteins in the gel. The membrane was washed with TBS (Tris buffer saline, Bio-Rad, cat # 170-6435) for 10 minutes and blocked for 1 hour with 5% non-fat milk in TBS. The membrane was washed twice with TTBS (Tween Tris-buffer saline, Bio-Rad, cat #170-6531 and cat #170-6435) and once with TBS and incubated for 2 hours with mouse monoclonal antibodies raised against the HPPD W336 protein. The membrane was washed twice with TTBS and once with TBS and incubated over-night with the enzyme conjugate, a second antibody with a horse radish peroxidase (HRP) linked anti-mouse antibody. After the secondSoybean Event FG72 Petition - Revised Page 121 of 198

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### antibody incubation, the membrane was washed twice with TTBS and once with TBS. The color was developed according to the manufacture's instructions (BIO-RAD, LIT178RevC). All reagents except the mouse monoclonal anti-HPPD antibodies used for western blotting were obtained from BIO-RAD as the HRP Conjugate Substrate kit (cat # 170-6431) which contains the blotting grade goat anti-mouse IgG HRP conjugate (cat# 170-6516). The mouse monoclonal anti-HPPD antibody was obtained from M.S. Technologies (US Patent application USSN 12/609,200)

Analysis by HPLC/Electrospray Mass Spectrometry

The E. coli produced HPPD W336 protein, the NT Jack soybean extract and the FG72 soybean produced HPPD W336 protein were denatured for 1 hour at 37 °C in Rapid Gest (Waters Corporation, 1mg/mL) containing 10 mM dithiothreitol at Catalent in San Diego, CA, USA. The denatured protein disulfide bonds were alkylated with iodoacetamide for 30 minutes in the dark and digested with trypsin for 2 hour at 37°C. A full scan analysis was performed on the E. coli produced HPPD W336 protein trypsin digest

, the trypsin digest of the NT extract and the trypsin digest of the FG72 soybean produced HPPD W336 protein to determine the location and identification of the peptides.

Analysis of enzymatic activity

HPPD catalyzes the reaction of 4-hydroxyphenylpyruvate (HPP) with oxygen to form homogentisate. The activity assay, which is a colorimetric method, measures the amount 2,4- dinitrophenylhydrazine (DNP) derivatized HPP remaining in the assay mixture at the end of the incubation and derivitization period. The wavelength monitored for the derivatized HPP is 405 nm. An activity of at least 10% is considered to be active.Soybean Event FG72 Petition - Revised Page 122 of 198

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### 2.C. Materials and methods for protein levels in grain

Seed samples analyzed in this study were produced under field conditions in 2008. The ten field trials supplying samples for this study were performed under M.S. Technologies, LLC

. The seed for all soybean grown, analyzed, and reported herein was supplied by M.S. Technologies LLC, Adel, Iowa. The field-produced seed samples were assayed by PCR testing to confirm their identity.

An E.coli produced 2mEPSPS protein standard (batch # LEJ5838) and HPPD W336 standard (batch# LB020309) were used as reference materials for analysis, and to fortify non-transgenic samples for validation and recovery studies.

Field design

Soybean plants containing the double-herbici de-tolerant soybean event FG72 and soybean plants representing the non-transgenic (non-transformed) counterpart “Jack” were grown in the field for Bayer CropScience by M.S. Technologies, LLC

. Trials were conducted in EPA Regions V in the following locations: Trial number 01-Cherokee county, Iowa; Trial number 02-Hardin County, Iowa; Trial number 03-Greene county, Iowa, Trial number 04-Dallas County, Iowa; Trial number 05-Dallas County, Iowa; Trial number 06-Madison County, Iowa; Trial number 07- Clinton County, Missouri; Trial number 08-Vermillion County, Illinois;

Trial number 09-Tipton County, Indiana and Trial Number 10-Des Moines County, Iowa, which are typical soybean growing regions of the United States. The plants in this study were grown under conditions typical of production practices for Group II maturity soybeans. There were three non-transgenic plots (Regimen A) and six transgenic plots (Regimen B and C) at each test site. The plots were randomized at each trial site. The Regimen A plots were planted with the non-transgenic counterpart variety “Jack” soybeans. The Regimen B and C plots were planted with the transgenic event FG72 soybeans. Three additional plots (Regimen D, E and F) were planted with commercial conventional (non-transgenic) soybeans for reference (Stine® 2686-6, Stine 2788, and Stine 3000-0, respectively). Thus there were 12 plots total at each trial site.

The Regimen C transgenic event FG72 plots were sprayed with isoxaflutole herbicide at a target rate of 70 grams ai/Ha and with glyphosate herbici de at a target rate of 1060 grams ai/Ha, and the other plots were not treated with these herbicides. Ammonium sulfate at 2850 grams/Ha was added to the spray mixture for the Regimen C herbicides. Application of the herbicides was made to the Regimen C plants as a foliar spray at about the V4-V5 growth stage. The herbicide applications were made by Bayer CropScience personnel according to Bayer CropScience protocol HD08NARJX5. To keep the site weed-free, a conventional soil-applied herbicide was sprayed after planting but prior to soybean emergence to all the plots. This herbicide treatment was pendimethalin (1060 grams ai per ha), except for trial 05 in which metolachlor (1880 gm ai/Ha) was used.

Soybean grain was obtained from each test plot at maturity by M.S. Technologies, LLC field personnel. A representative sample of grain from each plot was shipped to the BioAnalytics laboratories of Bayer CropScience in Research Triangle Park, NC, USA.Soybean Event FG72 Petition - Revised Page 123 of 198

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### Certificates of analysis (COA) were produced by the BCS BioAnalytics Laboratory in Research Triangle Park, NC for seed shipped to the nine field test sites for planting. The data showedthat the transgenic soybean seed that was planted in the field was indeed double-herbicidetolerant soybean event FG72.

Sample preparation

Grain samples were ground in a blender pre-chilled with dry ice. Small amounts of dry ice were added to the blender periodically to ensure the samples remained frozen during preparation. A separate blender was used for each sample. The ground samples were stored in a freezer at approximately -20  C for overnight or longer to allow the dry ice to sublimate before extraction.

Protein extraction

2mEPSPS

Total protein was extracted from the raw agricultural product of soybean grain using an optimized extraction procedure using ‘2mEPSP S Extraction Buffer’ (0.2% SDS in EPSPS (SDIX Catalog number 7000102)) as the extraction/dilution buffer.

A representative fraction (approximately 0.1 g) of ground sample was mixed with 4 mL extraction buffer in a 50 mL polypropylene centrifuge tube, and then shaken for 30 minutes at ~ 4°C on a shaker (IKA-SCHÜTTLER MTS 4) at 250 rpm. The liquid extract was transferred to a clean centrifuge tube and centrifuged at approximately 18000 x g for 10 minutes at ~ 4°C. The clear supernatant was used for 2mEPSPS analyses. Duplicate extracts were prepared for each sample. Total extractable protein was determined as a relative measure of extraction efficiency.

HPPD W336

PBST Extraction/Dilution Buffer, Agdia, Inc. Catalog Number ACC 00501A, was used. A representative fraction (approximately 0.1 g) of ground sample was mixed with 4 mL extraction buffer in a 50 mL polypropylene centrifuge tube, and then shaken for 30 minutes at ~ 4°C on a shaker (IKA-SCHÜTTLER MTS 4) at 250 rpm. The liquid extract was transferred to a clean centrifuge tube and centrifuged at approximately 18000 x g for 10 minutes at ~ 4°C. The clear supernatant was then used for HPPD W336 analyses. Duplicate extracts were prepared for each sample. Total extractable protein was determined as a relative measure of extraction efficiency.

Bioassay

All quantitative determinations of 2mEPSPS protein were conducted at Bayer CropScience, Morrisville, NC. All quantitative determinations of HPPD W336 protein were conducted at Bayer CropScience, Research Triangle Park, NC.

The levels of HPPD W336 and 2mEPSPS proteins were determined by an Enzyme Linked ImmunoSorbent Assay (ELISA) using antibodies specific for each protein.Soybean Event FG72 Petition - Revised Page 124 of 198

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### 2mEPSPS

The levels of 2mEPSPS protein were determined by an Enzyme Linked ImmunoSorbent Assay (ELISA) using antibodies specific for the protein. The sample to buffer ratio was 0.1 g of matrix per 4 mL extraction buffer. All ELISA assays were conducted at Morrisville, NC.

Protein standards were included in duplicate on each 2mEPSPS ELISA plate at the following concentrations: 32, 16, 8, 4, 2, 1, and 0.5 ng/mL.

HPPD W336

The amount of HPPD W336 in the total protein extracts was measured using a quantitative ELISA developed by M.S. Technologies (US Patent application USSN 12/609,200). Before the analysis was performed the Limit of Detection (LOD) was determined for seeds.

Serially-diluted sample extracts were applied to ELISA plates at 100 μL/well. This was followed by about 2 hours of incubation on a shaker at 200 rpm at room temperature. After the first incubation, the plate was washed four times using the BIO-TEK EL404 microplate washer. One hundred μL of diluted (1:12500) HPPD antibody conjugate was added to each well and incubated on ELISA plate shaker at approximately 200 rpm for about 2 hours at room temperature. This allowed the protein that was present in the samples to bind to the capture antibody. Unbound material was removed by rinsing the wells 4 times with wash solution. One hundred μL of substrate solution per well was added and the plate was incubated on a shaker at 200rpm for about 30 minutes.

Validation

The ELISA procedures were validated with a non-transgenic sample “Jack”. The standards were added to the extraction buffer at the indicated concentrations prior to extraction in five replicates. Each replicate was analyzed using duplicate wells. A summary of the validation data for each analyte is shown in Table 31.Soybean Event FG72 Petition - Revised Page 125 of 198

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### Table 31. Validation of sample extraction with fortified non-transgenic soybean grain

2mEPSPS ELISA Validation HPPD W336 ELISA Validation

2mEPSPS
fortified (ng/mL) 2mEPSPS
detected (ng/mL) a

Mean ± SD %
2mEPSPS Recovery
Mean ±
SD 2mEPSPS Recovery
%CV HPPD W336
fortified (ng/mL) HPPD W336
detected (ng/mL) a

Mean ± SD % HPPD W336 Recovery
Mean ± SD HPPD W336 Recovery
%CV

100 85.3 ± 3.4 85.3 ± 3.4 3. 93 100 123 ± 4 123 ± 4 3.01

32 26.6 ± 1.5 83.1 ± 4.7 5.63 32 31.4 ± 1.3 98.2 ± 3.9 4.01

16 11.9 ± 1.2 74.3 ± 7.7 10.3 16 18.0 ± 0.9 112 ± 6 5.26

8 6.41 ± 0.71 80.1 ± 8.9 11.1 8 8.34 ± 0.54 104 ± 7 6.47

4 3.13 ± 0.37 78.2 ± 9.2 11.7 4 3.51 ± 0.24 87.8 ± 6.0 6.78

2 b 0.09 ± 0.14 4.68 ± 7.0 151 2 1.43 ± 0.26 71.6 ± 13.1 18.2

1 b -0.16 ± 0.17 -15.8 ± 17 -109 1 b 0.43 ± 0.19 43.0 ± 19.2 44.6

0.5 b -0.03 ± 0.33 -5.36 ± 66 -1230

a The protein analyte detected and its recovery are expr essed as the average of 10 data points from duplicate

extracts of 5 samples at each fort ification level using non-transgenic matrix. Averages, standard deviations and %CV values are calculated with full precision and then rounded to 2 or 3 significant figures.

b Validity criteria were not met at indicated concentration.



### Limit of detection and limit of quantification

The limit of detection (LOD) is determined for each matrix using the average standard curve and the concentration derived from the background optical density (OD) of the negative control samples. The LOD is the concentration corresponding to an OD value three standard deviations above the mean background OD.

The LOD is expressed in the unit of concentration (ng/mL) and the unit of weight ratio (ng/g matrix, i.e. ppb) calculated based on the extraction of an amount of the matrix with a known volume of extraction buffer, e.g. , 0.1 g of matrix per 4 mL extraction buffer. The estimated LOD are summarized in Table 32 below. An absorbance reading giving rise to a protein analyte concentration above the LOD is assumed to be greater than the zero dose reading.

Table 32. Limits of detection and quantification of 2mEPSPS and HPPD W336 proteins in fortified non-transgenic soybean grain

Protein Analyte LOD LOQ

2mEPSPS 1.50 ng/mL 60.1 ng/g a 4 ng/mL 160 ng/g a
HPPD W336 0.52 ng/mL 20.8 ng/g a 2 ng/mL 80 ng/g a

a Calculated based on the extraction of 0.1 g matrix per 4 mL of extraction buffer.



### The limit of quantification (LOQ) is defined as the lowest concentration of the standard that meets the validity criteria for the LOQ. Validity criteria are a) analyte recoveries from fortifiedSoybean Event FG72 Petition - Revised Page 126 of 198

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### matrix samples are  60 % and  130 % and b) the coefficient of variance (relative standard deviation) is less than 25%. When a lower recovery is caused by the nature of a specific matrix or the effect of a process, the lowest concentration of the standard that gives a coefficient of variance equal to or less than 25% is used as the LOQ. Values below the LOD are reported as zero and values below the LOQ but above the LOD are reported at the LOQ. In calculations, values below the LOD are treated as zero. Values below the LOQ but above the LOD are assumed to be at the LOQ.

Protein Analyte Content

Protein determinations were made in order to confirm that protein was extracted from the samples. SoftMax ProTM software (Molecular Devices, Version 4.0) was used to derive the concentration of 2mEPSPS and HPPD W336 proteins from the ELISA data. Absorbance unitswere adjusted for the buffer blank. A set of wells containing samples of the corresponding nontransgenic matrix was included on each plate for background subtraction. The appropriate background corrections for the transgenic grain samples were obtained from background values of a non-transgenic grain sample, which was diluted on the same plate and to the same extent as the transgenic grain sample. Thus the dilution of the non-transgenic sample used for background subtraction was the same as the dilution of the transgenic sample that was required in order to place the OD reading in the center portion of the standard curve. The absorbance readings corrected for both buffer blank and non-transgenic background were converted to the protein concentration using the standard curve.

The ELISA assays give results in units of ng of analyte per milliliter of extract that are then converted into μg of analyte per gram of fresh sample. As different tissues have different protein and water contents, the results are also expressed in dry weight concentrations as well as percent of crude protein. Percent moisture and crude protein (fresh and dry weight basis) data for these samples were obtained from study DQ08B009. Results presented are rounded to 2 or 3 significant figures.

Analyte as percent crude protein is obtained by dividing the fresh weight content of analyte by the % crude protein value, then dividing by 100 for unit conversion, as shown in the following
formula.



### 100 % / %

x protein crude g analyte g protein crude as analyte 



Dry weight analyte content is obtained by dividing the fresh weight analyte content by the calculated percent dry matter and multiplying by 100. Percent dry matter is obtained by subtracting the percent moisture from 100. The following is the formula used.



#   100 % 100 / /         

  moisture g analyte g g g content weight dry analyte  

A summary of the results is given in Table 33.Soybean Event FG72 Petition - Revised Page 127 of 198

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### Table 33. Amounts of 2mEPSPS and HPPD W336 proteins

Protein Treatment/Entry Fresh Weight (μg/g) a 

### Dry Weight (μg/g) Content as %
Crude Protein b 

### Range 87 – 180 2mEPSPS Conventional
Treated/B Mean ± SD 130 ± 22 150 b 

### 0.039 b



### Range 87 – 240 2mEPSPS GLY + IFT
Treated/C Mean ± SD 140 ± 33 150 b 

### 0.041 b



### Range 0.46– 1.32 HPPD
W336 Conventional
Treated/B Mean ± SD 0.85 ± 0.20 0.94 c 

### 0.00024 c



### Range 0.41 – 1.31 HPPD
W336 GLY + IFT
Treated/C Mean ± SD 0.80 ± 0.22 0.89 c 

### 0.00023 c

a Range and overall mean ± standard deviation in μg/g for the fr esh weight protein content in grain samples from the
10 sites.

b The dry weight (dw) and % crude protein analyte amou nts were calculated using the average 2mEPSPS value of samples from each plot.

c The dry weight (dw) and % crude protein analyte am ounts were determined us ing the average of four individual results per sample, therefore no standard deviation or range is given for these amounts.



### Statistical analysis

Descriptive statistics (mean, standard deviation, and coefficient of variance) were calculated for each sample matrix and treatment (Devore and Peck, 1986).

Results

The 2mEPSPS and HPPD W336 proteins were detected in all transgenic soybean grain samples. The results show that the expre ssion of the 2mEPSPS and HPPD W336 proteins is similar between sprayed and unsprayed FG72 soybean plants.

2.D. Materials and methods for protein levels in plant parts and during the life cycle

FG72 transgenic and Jack wild type plants were grown under greenhouse conditions. Leaf samples taken at three different growth stages (V4, V6 and V8), stem and root samples taken at two different growth stages (V4 and V8) and seeds used to grow the plants were analyzed for the HPPD W336 and 2mEPSPS protein content.

The transgenic plants were selected by spraying with glyphosate before harvesting. The test items used in this study are different ti ssues from FG72 plants, the reference items are different tissues from WT soybean plants, variety Jack. The samples were directly frozen in liquid nitrogen and shipped on dry ice. Samples were stored at -70°C. Seeds were collected from the same seedlots and were stored at room temperature until crushing.

For all FG72 plants, the FG72 identity and zygosit y (homozygous) was confirmed by means of PCR. As control items, the HPPD W336 protein produced in bacteria (Batch no. LB020309) andSoybean Event FG72 Petition - Revised Page 128 of 198

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### the 2mEPSPS protein produced in bacteria (Batch no. LEJ5838) were used to generate a
standard curve for the ELISAs.

In order to analyze the presence of HPPD W336 and 2mEPSPS protein in these tissues, samples were crushed, extracted and the total amount of extractable protein (TEP) was determined using the Bradford method. The amount of HPPD W336 in the protein extracts was measured using a quantitative ELISA developed by Agdia (Elkhart, IN 46514, USA). The amount of 2mEPSPS in the protein extracts was measured using an ELISA developed by Strategic Diagnostics Inc. (SDI, Newark, DE, USA).

Extraction

For the expression analysis of HPPD W336 and 2mEPSPS protein, ten separate samples per tissue and per growth stage were chosen randomly and the complete samples were crushed with dry ice in a blender, resulting in homogeneous tissue samples. The powders were collected in pre-cooled 50 mL Falcon tubes, stored at -20 °C overnight to let the dry ice evaporate, and subsequently stored at -70 °C.

The amount of approximately 100 mg crushed material was weighed in pre-cooled 15 mL tubes. A volume of 4 mL extraction buffer was added. Samples were vortexed for 15 seconds or until all powder was in suspension, incubated on a rotary mixer at 30 rpm for 10 minutes at 4 °C and centrifuged at 4000 g for 10 minutes at 4°C. Supernatants were partly transferred to a 1.5 mL eppendorf tube and centrifuged at 20000 g for 5 minutes at 4°C. Clear supernatants were used as such or in dilutions to load on the ELISA plate.

Determination of Total Extractable Protein

After extraction, the total extractable protein (TEP) was measured using the Bradford protein assay with bovine serum albumin (BSA) as reference protein and by measuring the optical density (OD) at 595 nm. Clear supernatants of the extracts were diluted in MQ water so that the total protein concentration of all samples could be fit to the standard curve. The BSA concentrations used to generate the standard curve (36-25-20-15-12-10-5 μ g/mL) were prepared using PBST diluted with MQ water to the same extend as the samples, in order to compensate background signal from the PBST bu ffer. The value of the TEP measurement was used as an internal control to quality check the protein extraction process. The value was not used in any calculation of ELISA results.

The TEP data, determined to assess the quality of the extractions, was per tissue for all samples always in the same range. Therefore, no ELISA results coming from samples having a poor TEP content were excluded.

Determination of Dry Weight

Per soybean tissue and per growth stage, 2 pooled samples were used to determine dry weight. The analysis was performed by SGS Belgium, Agro Food Services, CTS, Antwerpen. Crushed samples were weighed, dried overnight in an oven at 103 ± 2 °C and weighed again. This weight data was used to calculate the % moisture and the % dry weight of the samples. The conversion of the expression data from fresh weight to dry weight was done using the
values shown in Table 34.Soybean Event FG72 Petition - Revised Page 129 of 198

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### Table 34 : Results of dry weight determination on FG72 samples

HPPD and 2mEPSPS ELISA

The HPPD ELISA kit from Agdia and the EPSPS ELISA kit commercialized by Strategic Diagnostics Inc. were used to determine respectively the HPPD W336 and the 2mEPSPS content in the protein extracts of FG72 tissues.

Per tissue and growth stage, each of the 10 samples was assayed 3 times on the ELISA plate. In order to fit the concentrations of HPPD W336 and 2mEPSPS to the respective standard curves, the protein extracts had to be diluted as described in Table 35.

Table 35. Final dilutions for protein extracts of the soybean FG72 tissues used for the HPPD and the EPSPS ELISA

Validation

To validate an ELISA for a certain matrix, different concentrations of a standard dilution series are spiked into extraction buffer, which is subsequently used to prepare 5 independent extracts of each soybean WT tissue. Each of the 5 extracts is loaded on the ELISA plate in triplicate, resulting in 15 values for each concentration of the standard curve. An average of all 15 values per spike, the recovery and the coefficient of variance (CV) are calculated.Soybean Event FG72 Petition - Revised Page 130 of 198

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### Validity criteria for the determination of the LOQ are a) analyte recoveries from spiked matrix samples are  60 % and  130 % and b) the coefficient of variance is less than 25%. The LOQ is defined as the lowest spiked
concentration that meets these criteria.

LOD values were not determined since the expression levels of HPPD W336 and 2mEPSPS were in all transgenic soybean tissues and developmental stages higher than the LOQ. The OD values of the non-transgenic controls were in the range of the buffer blanks. Therefore, the measured absorbances for all samples were corrected with the average signal of at least 2 buffer-only samples (blanks).

The LOQs were expressed as the concentration in ELISA of HPPD W336and 2m EPSPS (ng/ml) and as the amount of HPPD W336 and 2m EPSPS per gram fresh weight (ng/g fw) and per gram dry weight (ng/g dw). The LOQ results are summarized in Tables 36 and 37.

Table 36. LOQ for the HPPD W336 ELISA in different soybean tissues

Table 37. LOQ for the EPSPS ELISA in different soybean tissues

An ELISA absorbance resulting in a HPPD W336 or 2mEPSPS concentration equal to or above the LOQ level is assumed to represent a reliable concentration. Reliable results for the HPPD ELISA were only obtained at concentrations between 1 ng/ml and 8 ng/ml. Concentration above 8 ng/ml had to be diluted before measurement.

The HPPD ELISA plate validation indicated that the measurement of HPPD W336 protein using these plates had an LOQ of 40 ng/g fw for leaf, stem, root and seed. This corresponds to an LOQ of 252 ng/g dw for leaf, 447 ng/g dw for stem, 266 ng/g dw for root and 44 ng/g dw for
seed.Soybean Event FG72 Petition - Revised Page 131 of 198

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### The 2mEPSPS ELISA plate validation indicated that the measurement of 2m EPSPS protein using these plates had an LOQ of 75.2 ng/g fw for soybean leaf, stem and seed and 37.6 ng/g fw for root tissue. This corresponds to an LOQ of 474 ng/g dw for leaf, 841 ng/g dw for stem, 250 ng/g dw for root and 83 ng/g dw for seed.

Results

The averages and the range of the HPPD W336 and the 2mEPSPS protein contents per gram fresh weight and per gram dry weight in the different transgenic tissues are given per growth stage in Tables 7 and 8 (Section VI.C.2.). The ranges as well as the averages and standard
deviations were derived and calculated from all 30 data points. All values were calculated with full precision and then rounded to 2 or 3 significant figures. The average and SD were calculated on triplicate measurements of the individual samples.

2.E. Materials and methods for agronomic studies

Materials

Materials for efficacy evaluation were created at field sites in 2008 in the mid-western region of the United States. Ten locations in the states of Iowa, Illinois, Indiana and Missouri were used to produce the reference material for seed characteristic analysis, and the plants used in agronomic performance. Material was obtained from the three treatment regimes of the transformed soybean and their corresponding non-transgenic counterpart in the Jack variety.

Characterization of the materials

Identity of the materials was preserved through chain of custody documentation. Chain of custody documentation was utilized to identify the materials shipped to their respective field sites for proper identification of the evaluated plots in the field. Harvested materials contained chain of custody documentation for samples sent from the field to analytical laboratories to preserve identity.

Performing facility and experimental methods

Trials in 2008 were utilized to characterize and evaluate agronomic performance of the selected event, and develop materials for nutritional and compositional testing. Trials were conducted in one geographic region of the United States.

Field studies were managed in a manner repres entative of normal agricultural practices for inputs including, but not limited to:

 Conventional herbicide treatments, both pre- and post- planting

 Granular insecticide and/or fungicide application at planting

 Fertilizer applications

 Necessary in-season insecticide and/or fungicide applications

 Irrigation if needed to maintain adequate moisture

 Additional hand weeding or cultivation as necessarySoybean Event FG72 Petition - Revised Page 132 of 198

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### All trials received similar agronomic treatments for the care and upkeep of the plots. Field studies utilized an experimental treatment regi me which compared the transformed event FG72 sprayed and unsprayed compared to the non-tr ansformed counterpart of the Jack soybean variety.

Trials were performed using a randomized complete block design using six row plots with three replications and three treatments (Table 38). In addition, three non-replicated plots of conventional non-transgenic soybeans were grow n at each location to provide comparative data. A total of 17 agronomic parameters were used to measure the growth and development of the plant, and provide visual observations on the effect of any biotic and abiotic stressors upon the field plots across locations. Of t he agronomic parameters observed eight determined qualitative measurement characteristics an d nine determined quantitative measurement characteristics. These parameters were selected as key indicators of commercial and agronomic importance to commercial soybean growers, and the ability of the crop to perform under a variety of stresses from the different growing locations within the region.

Table 38. Treatment schedule for agronomic field tests in 2008

Evaluation of seed phenotypic characteristics of seed produced at the 10 different locations was conducted. Following the convention described by the USDA, National Genetic Resources Program, the following observations for FG72 and Jack seed were collected: seed size, hilum color, mottling score, seed coat color, seed coat luster, seed quality and seed shape.

Genotypic traits that can be assessed using qualitative measures, including flower color, pubescence color, pod color, hilum color and shapes, canopy architecture, leaf shape, growth habit, and susceptibility to pests and diseases were collected and analyzed.

Agronomic traits that can be measured quantitatively, including emergence, stand count, plant vigor and health rating, flowering date, plant height, days to maturity, plant lodging, pod shattering and yield were recorded and evaluated statistically.

Statistical analysis

Analysis of variance (ANOVA) between groups was calculated to analyze data for significant differences. All treatments were analyzed in comparison to their non-transgenic counterpart across locations and locally. Data were reviewed using a confidence interval of 95%. Label Treatment Description UTC Not sprayed Non-transgenic unsprayed Control Not sprayed Transgenic FG72 unsprayed

Sprayed Glyphosate and
Isoxaflutole 1060 g a.i. glyphosate per hectare and 70 g a.i. isoxaflutole per hectareSoybean Event FG72 Petition - Revised Page 133 of 198

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### 2.F. Materials and methods for seed germination studies

Materials

Materials were created in efficacy trials conducted at ten locations in 2008. Samples were taken from each plot replicate from locations in the mid-western United States.

Characterization of the materials

Identity of the materials was preserved through chain of custody documentation. Chain of custody documentation was utilized to identify the materials shipped to their respective field sites for proper identification of the evaluated plots in the field. Harvested materials contained chain of custody documentation for samples sent from the field to analytical laboratories to preserve identity.

Performing facility and experimental methods

To provide an evaluation of seed dormancy potential of event FG72 and the variety Jack, measurement of seed germination and dormancy were conducted. Seed samples from plants grown to maturity at 10 difference locations were taken. These seed represent the physiological state of seed that might fall into a field at the end of the season. The seed tests were conducted by the Iowa State Seed Lab using the standard test (warm germination) which is used for seed lot evaluations of field emergence under favorable conditions. A minimum of 400 seed were evaluated from each location. A very small difference in germination (94% vs. 96%) at day 6
was observed. In cases where hard seed were observed on day 6, the germination study was extended to 13 days, and in every case, the hard seed germinated so that the Total Viable score (95% vs. 96%) was the same. No dormant seed were identified.

Statistical analysis

The mean and standard deviation for each of the measured parameters was determined.Soybean Event FG72 Petition - Revised Page 134 of 198

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### 2.G. Materials and methods for composition analysis

Field design

Field trials were conducted by M.S. Technologies, LLC in EPA Region V in Iowa, Illinois, Indiana and Missouri, which are typical soybean growing regions of the Midwestern United States. The plants in this study were grown under conditions typical of production practices for Group II maturity soybeans. There were three non-transgenic plots (Regimen A) and six transgenic plots (Regimen B and C) at each test site. The plots were randomized at each trial site. The Regimen A plots were planted with the non-transgenic counterpart variety “Jack” soybeans. The Regimen B and C plots were planted with the transgenic event FG72 soybeans. Threeadditional plots (Regimen D, E and F) were planted with commercial conventional (nontransgenic) soybeans for reference (Stine® 2686-6, Stine 2788, and Stine 3000-0, respectively). Thus there were 12 plots total at each trial site.

The Regimen C transgenic event FG72 plots were sprayed with isoxaflutole herbicide at a target rate of 70 grams ai/Ha and with glyphosate herbici de at a target rate of 1060 grams ai/Ha, and the other plots were not treated with these herbicides. Ammonium sulfate at 2850 grams/Ha was added to the spray mixture for the Regimen C herbicides. Application of the herbicides was made to the Regimen C plants as a foliar spray at about the V4-V5 growth stage. The herbicide applications were made by Bayer CropScience personnel. To keep the site weed-free, a conventional soil-applied herbicide was sprayed after planting but prior to soybean emergence to all the plots. This herbicide treatment was pendimethalin (1060 grams ai per ha), except for one trial in which metolachlor (1880 gm ai/Ha) was used.

One sample of soybean grain was obtained from each test plot at maturity by M.S. Technologies, LLC field personnel. A representative sub-sample of grain from each plot was shipped to the BioAnalytics laboratories of Bayer CropScience in Research Triangle Park, NC, USA. Samples of the received soybean grain were shipped in a frozen state from Bayer CropScience to the analytical laboratory, Covance Laboratories Inc., 3301 Kinsman Blvd, Madison, WI, USA.

Characterization of the material

A Certificate of Analysis (COA) was prepared by MS Technologies, LLC for the FG72 seed lots used for planting in this study. The Jack seed used was certified seed lot number 1297. Identity and purity of the transgenic event FG72 seed and the corresponding non-transgenic seed were confirmed to be acceptable.

Processed fractions

Portions of the sprayed and unsprayed tr ansgenic event FG72 soybeans and non-transgenic “Jack” soybeans were shipped from Adel, IA by M.S. Technologies to the processing facility. Processing of the soybeans into toasted meal was performed by GLP Technologies, with Dick Dusek serving as the Processing Principal Investigator. The samples were processed at: GLP Technologies, 22723 State Highway 6, South, Navasota, TX 77868, USASoybean Event FG72 Petition - Revised Page 135 of 198

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### After receipt at the processing facility, the soybeans were placed into frozen storage. The processing of the soybean samples took place between June 23, 2009, and July 14, 2009. The non-transgenic “Jack” grain was processed firs t, followed by the unsprayed transgenic event FG72 grain, and finally the GLY and IFT herbicide sprayed transgenic event FG72 grain. Processing of each Regimen was done in cleaned equipment using simulated industry standard protocols. The nutritional composition of the soybean processed commodities (fractions) was determined at Covance Laboratories Inc. The following samples were obtained in the processing study for composition analysis and are identified in Table 39.

Table 39. Sample identification

Sample Regimen A Regimen B Regimen C Total Sample Code: HT08SOY001-01- Soybean Seed 01 02 03 3 (Grain used for processing) Sample Code: DQ09B002 - Hulls (H) Meal (M) AH
AM BH
BM CH
CM 3
3 Toasted Meal (T) AT BT CT 3 Protein Isolate (P) AP BP CP 3 Crude Oil (C) Refined, Bleached, Deodorized (RBD) Oil (D) Crude Lecithin (L) AC
AD
AL BC
BD
BL CC
CD
CL 3
3
3

Total Samples 8 8 8 24



### Analytical procedures

The analyses performed and methods used are detailed in Tables 40 to 45.Soybean Event FG72 Petition - Revised Page 136 of 198

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### Table 40. Methods used for analysis of soybean grain

Parameter (Analyte) Method
Mnemonic Covance Method Reference

Proximates
Ash ASHM AOAC 923.03
Fat FSOX AOAC 960.39 and 948.22
Moisture M100 AOAC 926.08 and 925.09
Protein PGEN AOAC 955.04 and 979.09 Carbohydrate (Calculated) CHO Difference between 100 and the sum of moisture, crude protein, fat and ash. Agric. Handbook No.
74 Acid detergent fiber ADF Agric. Handbook No. 379 Neutral detergent fiber NDFE AACC 32.20 and Agric. Handbook No. 379 Calcium, Iron, Magnesium, Phosphorus, Potassium, Sodium ICPS AOAC 984.27 and 985.01

Vitamin A ( 

- carotene) BCLC AOAC 941.15

Vitamin B 1 (Thiamin) BIDE AOAC 942.23, 953.17 and 957.17
Vitamin B 2 (Riboflavin) B2FV AOAC 940.33 and 960.46
Folic Acid FOAN AOAC 960.46 and 992.05
Vitamin K VKLC AOAC 992.27 Tocopherols (single and total) TTLC/TOIL HPLC method (see references) Raffinose and Stachyose SUGT Gas-Liquid Chromatography (see references) Phytic Acid PHYT HPLC method (see references) Trypsin Inhibitor TRIP AOCS Ba 12-75 Lectins LECT Photometric methods (see references)
Isoflavones ASOF AOAC 2001.10
Total Amino Acids TAA5 AOAC 982.30 Total Fatty Acids FALC AOCS Ce 1-62 and Ce 1b-89



### Table 41. Methods used for analysis of soybean hulls

Analyte Method
Mnemonic Covance Method Reference

Proximates
Ash ASHM AOAC 923.03
Fat FSOX AOAC 960.39 and 948.22
Moisture M100 AOAC 926.08 and 925.09
Protein PGEN AOAC 955.04 and 979.09 Carbohydrate (Calculated) CHO Difference between 100 and the sum of moisture, crude protein, fat and ash. Agric. Handbook No.
74 Acid detergent fiber ADF Agric. Handbook No. 379 Neutral detergent fiber NDFE AACC 32.20 and Agric. Handbook No. 379Soybean Event FG72 Petition - Revised Page 137 of 198

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### Table 42. Methods used for analysis of soybean meal and toasted meal

Analyte Method
Mnemonic Covance Method Reference

Proximates
Ash ASHM AOAC 923.03
Fat FSOX AOAC 960.39 and 948.22
Moisture M100 AOAC 926.08 and 925.09
Protein PGEN AOAC 955.04 and 979.09 Carbohydrate (Calculated) CHO Difference between 100 and the sum of moisture, crude protein, fat and ash. Agric.
Handbook No. 74 Acid detergent fiber ADF Agric. Handbook No. 379 Neutral detergent fiber NDFE AACC 32.20 and Agric. Handbook No. 379 Raffinose and Stachyose SUGT Gas-Liquid Chromatography (see references) Phytic Acid PHYT HPLC method (see references) Trypsin Inhibitor TRIP AOCS Ba 12-75 Lectins LECT Photometric methods (see references)
Isoflavones ASOF AOAC 2001.10
Total Amino Acids TAA5 AOAC 982.30

Table 43. Methods used for analysis of soybean protein isolate

Analyte Method
Mnemonic Covance Method Reference

Proximates
Moisture M100 AOAC 926.08 and 925.09
Protein PGEN AOAC 955.04 and 979.09 Trypsin Inhibitor TRIP AOCS Ba 12-75 Lectins LECT Photometric methods (see references)
Total Amino Acids TAA5 AOAC 982.30Soybean Event FG72 Petition - Revised Page 138 of 198

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### Table 44. Methods used for analysis of soybean crude oil and RBD oil

Analyte Method
Mnemonic Covance Method Reference

Vitamin A ( 

- carotene) BCLC AOAC 941.15

Vitamin K VKLC/VKLP AOAC 992.27 and 999.15 Tocopherols (single and total) TTLC/TOIL HPLC method (see references) Total Fatty Acids FALC AOCS Ce 1-62 and Ce 1b-89

Table 45. Method used for analysis of soybean lecithin

Analyte Method
Mnemonic Covance Method Reference

Phosphatides LPLC HPLC method (see reference)Soybean Event FG72 Petition - Revised Page 139 of 198

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### Appendix 3

CHARACTERIZATION OF EVENT FG72 SOYBEANSoybean Event FG72 Petition - Revised Page 140 of 198

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### 3.A. Verification of the insert

Several aliquots of soybean event FG72 genomic DNA were digested with the restriction

enzymes Hinc II, Sac I, Hin dIII, Bsp HI, Apa I, Stu I, Nco I, Sca I, Eco RI and Bsu36 I. Wild type genomic DNA (variety Jack) digested with Hin dIII was used as negative control, and an aliquot was supplemented with Hin dIII digested pSF10 plasmid DNA for use as a positive control. After separation by agarose gel-electrophoresis, the DNA was transferred to a nylon membrane and hybridized with eight probes, each containing a single genetic element present in the pSF10 vector used for the transformation, and the complete insert probe (Table 44). Figure 11 provides a schematic drawing of the event FG72 insert indicating restriction enzymes, probes, and expected fragment lengths. The membranes were visualized by autoradiography, and electronic scans (Figures 12 through 17).

The expected and obtained hybridization fragments for each of the 10 different restriction patterns are listed in Table 45. Some expected fragments were not visualized on the membranes due to the small overlap between the DNA fragments and the probe used.

Event FG72 genomic DNA digested with Apa I shows an additional fragment of 8570 bp (Table 45). This fragment is also visible after hybridiz ation with the 3’ nos probe, indicating that it comes from incomplete digestion of the Apa I restriction site between the internal 558 bp fragment and the 7900 bp 3’ integration fragment. Hybridization results of Bsu 36I digested event FG72 genomic DNA with probes PT059-1 and PT062-1 show the presence of very weak bands of 11590 bp and >14 kb (Figure 16 lane 13, Figure 17 lane 13, Table 45), also from incomplete digestion.

The inserted sequence in event FG72 consists of two partial 3’histonAt sequences in a head to head orientation, followed by two complete T-DNA copies arranged in a head to tail orientation. Upon integration of the event FG72 insert into the Glycine max genome, a non-transgenic region translocated to a new position, which is joined at the 3’ junction by 158 bases of Ph4a748 promoter sequences. Figure 11 shows a schematic pr esentation of this model with indication of the restriction enzymes, probes used, and the expected hybridization fragments. The hybridization results support this model of the event FG72 insert organization.

Table 46. Probes used in Southern hybridization of event FG72

Probe template ID Description Size (bp) Position in pSF10 (bp)

PT015-1 2mepsps 1351 8309 → 9659

PT016-1 3’histonAt 753 9614 → 10366

PT024-2 3’nos 214 3265 → 3478

PT058-4 insert probe 7204 3142 → 10345

PT059-1 Ph4a748 959 6491 → 7449

PT060-1 Intron1 h3At 507 7446 → 7952

PT061-1 5‘tev + TPotp Y 460 4650 → 5109

PT062-1 Ph4a748B 430 6866 → 7295

PT063-1 hppdPf W336 1055 3558 → 4612Soybean Event FG72 Petition - RevisedPage 141of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONFigure 11. Schematic drawing of the event FG72 insert indicating restriction enzymes, probes, and expected fragmentlengthsApaIEcoRISacIEcoRIStuIApaIBspHIEcoRI (2x)NcoIEcoRIHincIIHincIIHindIIIBsu36IHindIIINcoIScaIHincIIApaIEcoRIHincIISacIEcoRIStuIApaIBspHIEcoRI (2x)NcoIEcoRIHincIIHincIIHindIIIBsu36IHindIIINcoIScaIHincIIhppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAt3’histon At3’histonAt3’histon At probe (PT016)hppdPf W336probe (PT063)3’nos probe (PT024)5’tev + TPotpY probe (PT061)Ph4a748 probe (PT059)Ph4a748B probe (PT062)Intron1 h3At probe (PT060)2mepspsprobe (PT015)T-DNA probe (PT058)ApaIxxxxBspHIx xBsu36Ix xEcoRIxxx xxxx xHincIIxxxx xxxHindIIIxxxxNcoIxxxxNcoIxxxxSacIx xScaIx xStuIx xPh4a748 B> 6213010 713 2476 4091 713 2476 >1063>161> 68572807280728072807280>7198> 40216333947947>2915> 1382>6501> 6655585586722>6660> 1903>5980> 2543 292929294351>2411> 6389>1494> 675> 4989>5205>28945277528 91555195289915551Soybean Event FG72 Petition - RevisedPage 142of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 47 . Expected and obtained hybridization fragmentsPT016-1:3’histonAtPT024-2:3’nosPT063-1:hppdPfW336PT061-1:5’tev+TPotp YPT059-1:Ph4a748PT062-1:Ph4a748BPT060-1:Intron1 h3AtPT015-1:2mepspsPT058-4:T-DNA probeDigestDescriptionExpectedfragmentsizes (bp)Obtainedfragmentsizes (bp)Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.5’ integration fr. > 621 5250 Yes Yes No No No No No No No No No No No No No No Yes Noainternal fr. 3010 3010 No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes Yesinternal fr. 4091 4091 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yes Yes Yesinternal fr. 713 713 No No No No No No No No Yes Yes No No No No No No Yes Noainternal fr. 2476 2476 No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes3’ integration fr. > 1063 1130 Yes Yes No No No No No No No No No No No No Yes Yes Yes YesHincII3' junctiontranslocation> 158 1300 No No No No No No No No Yes Yes Yes Yes No No No No Yes Noa5’ integration fr. > 685 6060 Yes Yes No No No No No No No No No No No No No No YesYes,weakinternal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes3’ integration fr. > 7198 >14 kb Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesSacI3' junctiontranslocation> 158 >14 kb No No No No No No No No Yes NoaYes Yes No NoNo No Yes Noa5’ integration fr. > 4021 9550 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes Yesinternal fr. 6333 6333 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yesinternal fr. 947 947 No No No No No No No No Yes Yes Yes Yes No No No No Yes Yes3’ integration fr. > 2915 5500 Yes Yes No No No No Yes Yes Yes NoaNo No Yes Yes Yes Yes Yes YesHindIII3' junctiontranslocation> 158 1480 No No No No No No No No Yes Yes Yes Yes No No No No Yes Noa5’ integration fr. > 1382 3200 Yes Yes Yes Yes Yes Yes No No No No No No No No No No Yes Yesinternal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes3’ integration fr. > 6501 7480 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesBspHI3' junctiontranslocation> 158 4260 No No No No No No No No Yes Yes Yes Yes No No No No Yes Noa5’ integration fr. > 665 10760 Yes Yes No No No No No No No No No No No No No No YesYes,weakinternal fr. 6722 6722 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesApaIinternal fr. 558 558 No No Yes Yes Yes Yes No No No No No No No No No No Yes YesSoybean Event FG72 Petition - RevisedPage 143of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONPT016-1:3’histonAtPT024-2:3’nosPT063-1:hppdPfW336PT061-1:5’tev+TPotp YPT059-1:Ph4a748PT062-1:Ph4a748BPT060-1:Intron1 h3AtPT015-1:2mepspsPT058-4:T-DNA probeDigestDescriptionExpectedfragmentsizes (bp)Obtainedfragmentsizes (bp)Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.3’ integration fr. > 6660 7900 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesPartial fr. / 8570 No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes No Yes3' junctiontranslocation> 158 >14 kb No No No No No No No No Yes NoaYesYes,weakNo No No No Yes Noa5’ integration fr. > 1903 4710 Yes Yes Yes Yes Yes Yes No No No No No No No No No No Yes Yesinternal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes3’ integration fr. > 5980 6210 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesStuI3' junctiontranslocation> 158 >14 kb No No No No No No No No Yes NoaYesYes,weakNo No No No Yes Noa5’ integration fr. > 2543 7660 Yes Yes Yes Yes Yes Yes Yes Yes No No No No No No No No Yes Yesinternal fr. 2929 2929 No No No No No No Yes Yes Yes Yes Yes Yes Yes Yes No No Yes Yesinternal fr. 4351 4351 Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes NoaYes Yes Yes Yes3’ integration fr. > 2411 10270 Yes Yes No No No No Yes Yes No No No No Yes NoaYes Yes Yes YesNcoI3' junctiontranslocation> 158 >14 kb No No No No No No No No YesYes,weakYes Yes No NoNo No Yes Noa5’ integration fr. > 6389 9900 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yesinternal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes3’ integration fr. > 1494 4730 Yes Yes No No No No No No No No No No No No Yes Yes Yes YesScaI3' junctiontranslocation> 158 11430 No No No No No No No No Yes NoaYes Yes No NoNo No Yes Noa5’ integration fr. > 675 5110dYes Yes No No No NoNo No No No No NoNo No No No Yes Yesinternal fr. 5277 5277dYes Yes No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yesinternal fr. 528c528cNo No Yes Yes Yes YesNo No No No No NoNo No No No Yes Yesinternal fr. 915 915 No No No No Yes Yes No No No No No No No No No No Yes Yesinternal fr. 9b/ No No No No No No No No NoNo No No No No No No No Nointernal fr. 551c551cNo No No No Yes YesYes Yes No No No NoNo No No No Yes Yes3’ integration fr. > 5205 9610 Yes Yes No No No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes YesEcoRI3' junctiontranslocation> 158 4670 No No No No No No No No Yes Yes Yes Yes No No No No Yes NoaBsu36I5’ integration fr. > 4989 7650eYes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes YesSoybean Event FG72 Petition - RevisedPage 144of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONPT016-1:3’histonAtPT024-2:3’nosPT063-1:hppdPfW336PT061-1:5’tev+TPotp YPT059-1:Ph4a748PT062-1:Ph4a748BPT060-1:Intron1 h3AtPT015-1:2mepspsPT058-4:T-DNA probeDigestDescriptionExpectedfragmentsizes (bp)Obtainedfragmentsizes (bp)Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.internal fr. 7280 7280eYes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes3’ integration fr. > 2894 10350 Yes Yes No No No No Yes Yes No No No No Yes Yes Yes Yes Yes Yes3' junctiontranslocation> 158 7670eNo No No No No No No No YesYes Yes Yes No No No No Yes YesPartial fr. / 11590 No No No No No No No No NoYes,weakNoYes,weakNo No No No No NoPartial fr. / >14 kb No NoNo No No No No No NoYes,weakNoYes,weakNo No No No No NoWTgenomicDNA-HindIII/ / / / / / / / / / / / / / / / / /Positive control 3420 3420 No No Yes Yes Yes Yes YesYes,weakYes Yes Yes Yes No No No No Yes YesPositive control 947 947 No No No No No No No No Yes Yes Yes Yes No No No No Yes NoaPositive control 2961 2961 Yes Yes No No No No YesYes,weakYes NoaNo No Yes Yes Yes Yes Yes YesWTgenomicDNA -HindIII +pSF10 -HindIIIPositive control 3070 / No No No No No No No No No No No No No No No No No NoaNot visualized due to the small overlap between the fragments and the T-DNA probe.bThis fragment is too small to be visualized.c,d, eThese fragments can appear as a single fragment.Soybean Event FG72 Petition Page 145 of 136



### Figure 12. Southern blot analysis of event FG72 – 3’histonAt probe

4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the 3’histonAt probe (PT016-1: 753 bp DPA207-SMP086 fragment of pTEM2).

Lane 1: Phage Lambda – Hind III digested Lane 10: Event FG72 – Nco I digested Lane 2: Phage Lambda – Pst I digested Lane 11: Event FG72 – Sca I digested Lane 3: empty Lane 12: Event FG72 – Eco RI digested
Lane 4: Event FG72 – Hinc II digested Lane 13: Event FG72 – Bsu36 I digested
Lane 5: Event FG72 – Sac I digested Lane 14: WT variety Jack – Hind III digested
Lane 6: Event FG72 – Hind III digested Lane 15: WT variety Jack – Hind III digested + half of an
Lane 7: Event FG72 – BspH I digested equimolar amount of pSF10 – Hind III digested
Lane 8: Event FG72 – Apa I digested Lane 16: Phage Lambda – Pst I digested
Lane 9: Event FG72 – Stu I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition - Revised Page 146 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 13. Southern blot analysis of event FG72 – 3’nos probe

4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with diffe rent restriction enzymes and probed with the 3’nos probe (PT024-2: 214 bp MDB355-MDB055 fragment of pTDL004).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested
Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition - Revised Page 147 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 14. Southern blot analysis of event FG72 – hppdPfW336 probe

2459 - 2443 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp 2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 2459 - 2443 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp 2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp 2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the hppdPf W336 probe (PT063-1: 1055 bp SMP083-SMP082 fragment of pSF10).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested
Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + half of an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind IISoybean Event FG72 Petition - Revised Page 148 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 15. Southern blot analysis of event FG72 – 5’tev+TPotp Y probe

4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp
14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp 1700 bp

1159 bp 1093 bp

805 bp 11501 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp
14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp 1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp
14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp 1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the 5’tev+TPotp Y probe (PT061-1: 460 bp MLD123-STV129 fragment of pSF10).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested
Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition - Revised Page 149 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 16. Southern blot analysis of event FG72 – Ph4a748 probe

23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the Ph4a748 probe (PT059-1: 959 bp SMP131-KM008 fragment of pSF10).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested
Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + half of an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition - Revised Page 150 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 17. Southern blot analysis of even t FG72 – Ph4a748B probe

23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

Genomic DNA samples (8 μg) were digested with di fferent restriction enzymes and probed with the Ph4a748B probe (PT062-1: 430 bp JDB 018-JDB019 fragment of pTEM2).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested
Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + half of an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition Page 151 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 18. Southern blot analysis of event FG72 – Intron1 h3At probe

4 5 6 7 8 9 10 11 12 13 14 15

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 4 5 6 7 8 9 10 11 12 13 14 15

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the Intron1 h3At probe (PT060-1: 507 bp MLD148-SMP052 fragment of pSF10).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition Page 152 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 19. Southern blot analysis of event FG72 – 2 mepsps probe

23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 23130 bp 14057 bp 9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 4 5 6 7 8 9 10 11 12 13 14 15

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the 2mepsps probe (PT015-1: 1351 bp SMP084-MLD090 fragment of pTEM2).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + pSF10 –
Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition Page 153 of 198

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### Figure 20. Southern blot analysis of event FG72 – insert-DNA probe

4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp
14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 11501 bp 4 5 6 7 8 9 10 11 12 13 14 15

23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp 23130 bp 14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp
2459 - 2443 bp 2322 bp 2140 bp 1986- 2027 bp

1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp
14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 11501 bp

2459 - 2443 bp 23130 bp
14057 bp

9416 bp

6557 bp

4507- 4361 bp 5077 bp 4749 bp

2838 bp

2322 bp 2140 bp 1986- 2027 bp
1700 bp

1159 bp 1093 bp

805 bp 11501 bp

Genomic DNA samples (8 μg) were digested with different restriction enzymes and probed with the insert-DNA probe (PT058-4: 7204bp SacI/SmaI fragment of pSF10).

Lane 1: Phage Lambda – Hind III digested Lane 2: Phage Lambda – Pst I digested Lane 3: empty
Lane 4: Event FG72 – Hinc II digested
Lane 5: Event FG72 – Sac I digested
Lane 6: Event FG72 – Hind III digested
Lane 7: Event FG72 – BspH I digested
Lane 8: Event FG72 – Apa I digested
Lane 9: Event FG72 – Stu I digested Lane 10: Event FG72 – Nco I digested
Lane 11: Event FG72 – Sca I digested
Lane 12: Event FG72 – Eco RI digested
Lane 13: Event FG72 – Bsu36 I digested Lane 14: WT variety Jack – Hind III digested Lane 15: WT variety Jack – Hind III digested + an equimolar amount of pSF10 – Hind III digested Lane 16: Phage Lambda – Pst I digested Lane 17: Phage Lambda – Hind III digestedSoybean Event FG72 Petition Page 154 of 198

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### 3.B. Absence of Vector Backbone

For the molecular verification of the absence of pSF10 vector backbone, event FG72 genomic DNA was digested with the restriction enzymes Hin dIII and Hinc II. The resulting DNA fragments were separated by agarose gel electrophoresis, transferred to a membrane and subjected to Southern blot analysis usi ng two overlapping vector backbone probes covering the complete vector backbone sequences of the pSF10 transformation vector. Afterwards, the vector backbone probes were removed from the membranes and they were re-hybridized with the insert DNA probe.

Information on the probes used is presented in Table 48. A schematic overview of the Southern blot strategy is presented in Figure 21.

Table 48. Probes used for demonstratio n of absence of vector backbone sequences

Probe template ID Description Size (bp) Probe Overlap (bp)

PT056 Vector backbone probe 1730

PT057 Vector backbone probe 1982 573

PT058 T-DNA probe 7204 N.A.



### An overview of the expected and obtained hybridization fragments is presented in Table 49. The obtained Southern blot results are presented in Figures 22 and 23.

Since both vector backbone probes contain a num ber of regions also present in the T-DNA sequence, several fragments originating from inserted transgenic DNA hybridize with the vector backbone probes. However, based on sequence homology, only the expected fragments were obtained when hybridizi ng with vector backbone probes.

After hybridization with the insert-DNA probe, the expected internal 947 bp Hin dIII fragment was visible for the genomic event FG72 DNA samples, while this fragment was not visible in the positive control. Also the 0.1x equimolar amount of Hin dIII digested pSF10 plasmid DNA could not be visualized after hybridization with the insert-DNA probe while it could be visualized after hybridization with the vector backbone probes. However, these results have no influence on the final interpretation of the Southern blot analyses.

The expected Southern blot profile was obtained in the event FG72 samples after hybridization with the insert-DNA probe. Th is demonstrated that an adequate amount of a sufficient quality of event FG72 genomic DNA was loaded on the gels for the detection of the presence of vector backbone sequences in event FG72.

In conclusion, Southern blot analysis us ing two overlapping vector backbone probes covering the complete vector backbone sequence of the pSF10 transformation vector confirmed the absence of vector backbone sequences in the genome of the soybean
transformation event FG72.Soybean Event FG72 PetitionPage 155 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONFigure 21. Schematic drawing of plasmid pSF10indicating the relevant restriction sites and probesVector backboneT-DNApSF10HincIIHindIIIHincIIHincIIHindIIIHincIIHindIIIHincIIHincIIHindIIIORi ColE1ORI f1blaPT058PT056PT057PT056xx2961 bp947 bpx3420 bpx3070 bppSF10 -HindIIIFG72 -HincIIxxxx xxxFG72 -HindIIIxxxx> 621 bp3010 bp 713 bp 2476 bp 4091 bp713 bp2476 bp>1063 bp> 4021 bp6333 bp947 bp 947 bp>2915 bpPT058hppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAt3’histon At3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAt3’histon At3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAtFG72SalISalIHindIIIHindIIIHindIIIHindIIIHincIIHincIIHincIIHincIIHincIIHincIIHincIISoybean Event FG72 PetitionPage 156 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 49. Expected and obtained hybridization fragmentsFigure 1Figure 2PT056-1PT058-6PT057-1PT058-6Vector backbone probeT-DNA probeVector backbone probeT-DNA probeSampleExpected TDNA orplasmidfragmentsizesFragmentdescriptionExp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.9550 bpc5' integration fr. YesdYes Yes Yes YesdYes Yes Yes947 bpinternal fragment YesdYes Yes Yes YesdNo Yes Yes6333 bpinternal fragment YesdYes Yes Yes YesdYes Yes Yes5500 bpc3' integration fr. No No Yes Yes No No Yes YesFG72 -HindIII1480 bpc3'junction translocation No No YesbNo No No YesbNo5250 bpc5' integration fr. YesdNo YesbNo No No YesbNo3010 bpinternal fragment YesdYes Yes Yes YesdYes Yes Yes713 bpinternal fragment YesdYes Yes Yes,weak YesdNo Yes Yes,weak2476 bpinternal fragment No No Yes Yes No No Yes Yes4091 bpinternal fragment YesdYes Yes Yes YesdYes Yes Yes1130 bpc3' integration fr. No No Yes Yes No No Yes YesFG72 –HincII1300 bpc3'junction translocation No No YesbNo No No YesbNoWT -HindIII/ /////////3420 bppositive control YesdNo Yes NoaYesbNo Yes Noa947 bppositive control YesdNo Yes NoaYesdNo Yes Noa2961 bppositive control YesbNo Yes NoaNo No Yes NoaWT -HindIII + 0.1equimolar amountpSF10 -HindIII3070 bppositive control Yes Yes YesbNo Yes Yes YesbNo3420 bppositive control YesdNo Yes Yes YesbNo Yes Yes947 bppositive control YesdNo Yes NoaYesdNo Yes Noa2961 bppositive control YesbNo Yes Yes No No Yes YesWT -HindIII + 1equimolar amountpSF10 -HindIII3070 bppositive control Yes Yes YesbNo Yes Yes YesbNoaThese fragments of the positive control were weak or could not be visualized after hybridization with the T-DNA probe. This has no impact on the interpretation of the results.bThe overlap between the probe and the fragment can be too small to visualize this fragment.cExpected fragment sizes as determined in the detailed insert characterization study.dSince part of the sequence of both vector backbone probes is present in the T-DNA sequences, fragments originating from inserted transgenic sequences hybridize with vector backbone probes.Soybean Event FG72 Petition Page157 of 197

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### Figure 22. Southern blot analysis of event FG72 – Absence of vector backbone – PT056-1 probe and insert-DNA probe

bp 34567

bp

2459 - 2443 23130
14057

9416

6557

4507- 4361 5077 4749

2838

2322 2140 1986- 2027

1700

1159 1093

805

564 514 23130

14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564
514



### Panel A: PT056-1 Probe 34567

23130

14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564 514 23130

14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564 514 bp bp



### Panel B: PT058-6 Probe

Genomic DNA samples (10 μg) were digested with different restriction enzymes and hybridized sequentially with a vector backbone probe (PT056-1: 1730 bp, KM033 – DPA010 fragment of pSF10) and with the insert-DNA probe (PT058-6: 7204 bp, Sac I/ Sma I fragment of pSF10).

Lane 1: λ

- Pst I digested (not shown)
Lane 2: λ

- Hin dIII digested (not shown)
Lane 3: Event FG72 – Hin dIII digested
Lane 4: Event FG72 – Hin cII digested Lane 5: WT variety Jack – Hin dIII digested Lane 6: WT variety Jack – Hin dIII digested + a 10-fold dilution of an equimolar amount of pSF10 – Hin dIII digested Lane 7: WT variety Jack – Hin dIII digested + an equimolar amount of pSF10 – Hin dIII digested
Lane 8: λ

- Hin dIII digested (not shown)
Lane 9: λ

- Pst I digested (not shown)Soybean Event FG72 Petition Page 158 of 198

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### Figure 23. Southern blot analysis of event FG72 – Absence of vector backbone – PT057-1 probe and insert-DNA probe

23130
14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564 23130

14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564 34567

bp bp



### Panel A: PT057-1 Probe 23130 14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564 bp
23130

14057

9416

6557

4507- 4361 5077 4749

2838

2459 - 2443 2322 2140 1986- 2027

1700

1159 1093

805

564

bp 34 56 7



### Panel B: PT058-6 Probe

Genomic DNA samples (10 μg) were digested with different restriction enzymes and hybridized sequentially with a vector backbone probe (PT056-1: 1982 bp, VH055 – STV039 fragment of pSF10) and with the insert-DNA probe (PT058-6: 7204 bp, Sac I/ Sma I fragment of pSF10).

Lane 1: λ

- Pst I digested (not shown) Lane 2: λ

- Hin dIII digested (not shown) Lane 3: Event FG72 – Hin dIII digested
Lane 4: Event FG72 – Hin cII digested Lane 5: WT variety Jack – Hin dIII digested Lane 6: WT variety Jack – Hin dIII digested + a 10-fold dilution of an equimolar amount of pSF10 – Hin dIII digested Lane 7: WT variety Jack – Hin dIII digested + an equimolar amount of pSF10 – Hin dIII digested
Lane 8: λ

- Hin dIII digested (not shown) Lane 9: λ

- Pst I digested (not shown)Soybean Event FG72 Petition Page 159 of 198

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### 3.C. Stability across and within generations

To demonstrate the structural stability of event FG72, genomic DNA was prepared from several individual plants of three generations and thr ee different genetic backgrounds (Table 48). The impact of different environments was assess ed by analyzing the progeny of transgenic plants cultivated at 4 different field locations (Tabl e 50). The isolated DNA was digested with the restriction enzyme Hin dIII, which provides a unique pattern for transformation event FG72.

Successive hybridization of these samples with the Ph4a748B probe and the insert-DNA probe revealed the expected profile in all tested samples. These findings demonstrated the stability of event FG72 at the genomic level in different generations, different environments and different backgrounds.

Information on the probes is given in Table 51. A schematic presentation of the hybridization strategy is shown in Figure 32. Hybridization results are shown in Figures 24 – 31 and are
summarized in Table 52.Soybean Event FG72 Petition Page 160 of 198

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### Table 50. Overview of the tested seed lots of event FG72

Different locations for seed lot production Generation Seed lot n°

Adel, Iowa T9 HT08SOY002-05-32 Osborn, Missouri T9 HT08SOY002-07-32 Fithian, Illinois T9 HT08SOY002-08-32 Sharpsville, Indiana T9 HT08SOY002-09-32

Different generations Generation Seed lot n°

T2 T2 FG72a-T2, FG72b-T2, FG72c-T2, FG72d-T2 **
T7 T7 FG72-x-x-14-5-1-6-x T7
T9 T9 All seed lots of different locations
Different backgrounds Generation Seed lot n°

3068115-48 x Jack * F4 7BD60018
3066617-48 x Jack * F4 7BD60008

Jack All seed lots of different locations and generations tested

* Conventional lines 3068115-48 and 3066617-48 were crossed with event FG72 in Jack genetic background one
time and then selfed 3 times.

** Seeds lots FG72a-T2, FG72b-T2, FG72c -T2 and FG72d-T2 are each the progeny of 1 individual seed of event FG72 T1 generation. Leaf material grown from these T2 seeds was randomly collected from individual plants and used in this study as T2 generation.



### Table 51. Probes used for stability studies

Probe Restriction digest or Primer pair Position in pSF10 Probe size

FG72 T-DNA Sac I/ Sma I 3142 bp → 10345 bp 7204 bp

JDB018 Ph4a748B JDB019 6866 bp → 7295 bp 430 bpSoybean Event FG72 Petition Page 161 of 198

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### Figure 24. Environment Adel

Ph4a748B probe

T-DNA probe

A: event FG72 samples; B: Negative control; C: positive control B C A

B C ASoybean Event FG72 Petition Page 162 of 198

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### Figure 25. Environment Osborn

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C

B C A ASoybean Event FG72 Petition Page 163 of 198

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### Figure 26. Environment Sharpsville

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C A

B C ASoybean Event FG72 Petition Page 164 of 198

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### Figure 27. Environment Fithian

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C A

B C

ASoybean Event FG72 Petition Page 165 of 198

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### Figure 28. Background 3068115-48 X Jack

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C A

B C

ASoybean Event FG72 Petition Page 166 of 198

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### Figure 29. Background 3066617-48 X Jack

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C A

B C ASoybean Event FG72 Petition Page 167 of 198

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### Figure 30. Generation T2

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C A

B C ASoybean Event FG72 Petition Page 168 of 198

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### Figure 31. Generation T7

Ph4a748B probe

T-DNA probe



### A: event FG72 samples; B: Negative control; C: positive control B C A

B C ASoybean Event FG72 PetitionPage 169 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONFigure 32. Schematic drawing of hybridization strategy for the demonstration of structural stabilitypSF10HindIIIHindIIIHindIIIHindIIIORi ColE1ORI f1blaxx2961 bp947 bpx3420 bpx3070 bppSF10 -HindIIIFG72 -HindIIIxxxx9550 bp6333 bp947 bp 947 bp5500 bphppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAt3’histon At3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAtFG72SalISalIT-DNA probePh4a748B probeT-DNA probePh4a748B probe1480 bpPh4a748BHindIIIHindIIIHindIIIHindIIIpSF10HindIIIHindIIIHindIIIHindIIIORi ColE1ORI f1blaxx2961 bp947 bpx3420 bpx3070 bppSF10 -HindIIIFG72 -HindIIIxxxx9550 bp6333 bp947 bp 947 bp5500 bphppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1h3At2mepsps3’histonAt3’histon At3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAthppdPfW336TPotp C3’nos TPotp Y5’tevPh4a748Ph4a748 ABBCIntron1 h3At2mepsps3’histonAtFG72SalISalIT-DNA probePh4a748B probeT-DNA probePh4a748B probe1480 bpPh4a748BHindIIIHindIIIHindIIIHindIIISoybean Event FG72 PetitionPage 170 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 52. Expected and obtained hybridization fragmentsPh4a748BprobeT-DNAprobeSamples Condition tested Seed lotN° ofplantsExpectedT-DNA orplasmidfragmentsizesFragment descriptionExp. Obt. Exp. ObtLocation Adel, Iowa HT08SOY002-05-32 22Location Osborn, Missouri HT08SOY002-07-32 22Location Fithian, Illinois HT08SOY002-08-32 21 9550 bpd5' integration fr. Yes Yes Yes YesLocation Sharpsville, Indiana HT08SOY002-09-32 16 947 bp internal fragment YesYes Yes YesBackground 3068115-48 x Jack 7BD60008 21 6333 bp internal fragment Yes Yes Yes YesBackground 3066617-48 x Jack 7BD60018 22 5500 bpd3' integration fr. No No Yes YesGeneration T2FG72a-T2, FG72b-T2,FG72c-T2, FG72d,T222 1480 bpd3' junction translocation Yes YesaYesbNocFG72 -HindIIIGeneration T7 FG72-x-x-14-5-1-6-x T7 13NontransgenicJack -HindIIINon-transgenic variety Jack / Negative control / / / /3420 bp positive control Yes Yes Yes Yes947 bp positive control Yes Yes Yes Noc2961 bp positive control No No Yes YesNontransgenicJack -HindIII +pSF10 -HindIIINon-transgenic variety Jack + equimolar amount pSF103070 bp positive control No No No NoaIn some hybridizations this fragment is very weak, but present for all samplesbThe overlap between the probe and the fragment can be too small to visualize this fragmentcNot always visible after hybridization with T-DNA probe but presence is confirmed after hybridization with probe Ph4a748B.dExpected fragment sizes as determined in the detailed insert characterization studySoybean Event FG72 Petition Page 171 of 198

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### 3.D. Demonstration of protein equivalence

3.D.1 2mEPSPS protein

The SDS-PAGE and western blots demonstrated that the molecular weight, mobility, and immuno-reactivity of the plant-produced and mi crobially-produced 2mEPSPS proteins are the same. The western blot also indicated that the non-transgenic Jack soybean control sample did not have immunoreactive proteins. The band appearing below the 2mEPSPS band in the plant-produced protein did not appear on the western blot, indicating that the band is not related to the 2mEPSPS protein.

Figure 33. Comparison of the plant-produced and microbially-produced 2mEPSPS protein

The SDS-PAGE gel was stained with Pierce Imperial Protei n Stain. Lane 2 contains approximately 300 ng of 2mEPSPS protein produced in E. coli. Lane 3 cont ains the non-tr ansgenic Jack soybean leaves control sample. Lane 4 contains approximately 430 ng 2mEPSPS protein isol ated from FG72 soybean leav es. Lanes 1 and 5 contain molecular weight markers of 220, 160, 120, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 and 10 kDa. Underlined molecular weights are shown on the gel. A band below the FG72 soybean produced 2mEPSPS band in lane 4 appears to be coming from the soybean matrix.

Lane 1. BenchMark Protein Ladder
Lane 2. E.coli produced 2mEPSPS Protein (Batch LEJ5838) Lane 3. Non-transgenic Jack soybean leaves control sample
Lane 4. 2mEPSPS isolated fr om FG72 soybean leaves
Lane 5. BenchMark Protein Ladder 

### 12 3 4
kD

220-

160-

70-

60- 100-

50-

40-

30-Soybean Event FG72 Petition Page 172 of 198

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### Figure 34. Standard curve of electrophoretic mobility versus molecular weight



# y = -1.2374x + 86.777

R 2 

# = 0.9834

1 10 100 1000

1 10 100

mobility (mm)Molecular weight (kDa)

The electrophoretic mobility of the protein standards for th e SDS-PAGE gel shown in Figure 3.23 were plotted against their respective molecular weights brac keting the 2mEPSPS protein. The equation defining the curve is y = -1.2374x
+ 86.777. The R 2 value for this curve is 0.9834. The equation defining this curve was used to calculate an approximate molecular weight of 50.3 kDa for 2mEPSPS isolated from soybean, event FG72. The molecular weight
of the E. coli produced 2mEPSPS protein calculated from the equation is 49.7 kDa.Soybean Event FG72 Petition Page 173 of 198

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### Figure 35. Western blot comparison of the plant-produced and microbially-produced 2mEPSPS protein

Lane 2 contains the 2mEPSPS protein produced in E. coli. Lane 3 contains the Jack soybean leaves control sample. Lane 4 contains the 2m EPSPS protein isolated from FG72 soybean leaves. Lanes 1 and 5 contain molecular weight markers of 250 , 150 , 100 , 75, 50 , 37 , 25 and 20 kDa. Underlined molecular weights are shown on the gel.

Lane 1. Kaleidoscope Precision Plus Protein Standards
Lane 2. E. coli produced 2mEPSPS Protein (Batch LEJ5838) Lane 3. Non-transgenic Jack soybean leaves control sample
Lane 4. 2mEPSPS isolated from FG72 soybean leaves Lane 5. Kaleidoscope Precision Plus Protein Standards



### The full scan mass spectrometry results and amino acid coverage are given in Tables 53 and 54. The full scan analysis obtained 98% sequence coverage of the E. coli produced 2mEPSPS protein. The analysis of the tryptic digest of the plant-produced 2mEPSPS protein by full scan analysis obtained 71% sequence coverage of the pr otein. The peptides not being detected by full scan mass spectrometry could be caused by a missed cleavage, modification or matrix. The mass spectrometry results indicate that the me thionine is missing from the N-terminal peptide, residue 1 to 13, for both the E. coli produced 2mEPSPS protein and the plant-produced 2mEPSPS protein. The loss of a methionine is not unusual, as post-transl ational modifications
such as removal of a methionine are often found in proteins from both prokaryotic and eukaryotic organisms (Bradshaw et al. , 1998). 

### 1 2 3 4

kDa

250-

150-

100-

50-

37-

25-

20-Soybean Event FG72 Petition Page 174 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 53. Electrospray LC/MS peptide mapping of the 2mEPSPS protein

* = Average mass reported. ND= Not detected. ND** = Missed cleavage, peptide not detected by full scan analysis. 1268.7

733.8

2113.2

501.6

576.6

3342.9

1033

289

175

790

805.9

1648.9

2434.2

2105

306

1296

1907

969

246

3219.8

1435.8

548.7

450.6

1103

6681.8

570.7

389

310

331

3870

1226

1631.8

605.6

147

397

905

2260.7

733.8

2975.5

246

534

589.8

591.7

2019

1533.6

1882

648.7

2624.2

147

1679.9

133 N D 1 to 20 MAGAEEIVLQPIKEISGTV

K 705.4 [M+3H] N D

N D

406 to 429 MAMAFSLAACAEVPVTIRDPGC TR (Cys_CAM mod) 875.9 [M+3H] 875.9 [M+3H] 876.3 [M+3H] 330 to 357 MPDVAMTLAVVALFADGPTAIR
DVASW R 992.5 [M+3H] N D N D 174 to 233 LSGSISSQYLSALLMAAPLALGD

VEIEIIDKLISIPYVEMTLRLMERF
GVKAEHSDSWD R 1114.5 [M+6H] N D

N D

445 to 445 N N D** N D** N D 431 to 444 TFPDYFDVLSTFVK 841 [M+2H] 840.9 [M+2H] N D

430 to 430 K N D** N D** N D 369 to 373 TELT

K 592.4 [M+H] 592.3 [M+H] N D

364 to 368 MVAIR 590.4 [M+H] 590.3 [M+H] N D 360 to 363 ETE R 535.3 [M+H] 534.8 [M+H] N D

358 to 359 VK 246.8 [M+H] 247.2 [M+H] 246.2 [M+H] 352 to 357 DVASW R 733.9 [M+H] 734.3 [M+H] N D

330 to 351 MPDVAMTLAVVALFADGPTAIR N D** N D N D 322 to 329 AIDVNMNK 453.6 [M+2H] 453.4 [M+2H] N D

319 to 321 HLK 398.3 [M+H] 398.2 [M+H] N D 318 to 318 K N D N D N D

313 to 317 EPFGR N D N D N D 298 to 312 VTWTETSVTVTGPP R 817 [M+2H] 816.9 [M+2H] 1310 [M+3H]

287 to 297 FAEVLEMMGAK 614.6 [M+2H] 614.3 [M+2H] N D 247 to 286 N AYVEGDASSASYFLAGAAITG GTVTVEGCGTTSLQGDVK 1309.8 [M+3H] 1309.8 [M+3H] N D
244 to 246 SPK 332.3 [M+H] 332.2 [M+H] N D 242 to243 YK 310.8 [M+H] 310.7 [M+H] N D 238 to 241 GGQK 390.3 [M+H] 390.1 [M+H] N D 234 to 237 FYIK 286.2 [M+2H] 286.3 [M+2H] N D

225 to 233 AEHSDSWD R N D** N D** N D 221 to 224 FGVK N D** N D** N D

217 to 220 LMER N D** N D N D 205 to 216 LISIPYVEMTL

R N D** N D N D

174 to 204 LSGSISSQYLSALLMAAPLALGD

VEIEIIDK N D** N D N D 172 to 173 VK 246.8 [M+H] 247.2 [M+H] N D

161 to 171 VNGIGGLPGGK 485.6 [M+2H] 485.5 [M+2H] N D 143 to 160 QLGADVDCFLGTDCPPVR 1011.7 [M+2H] 1011.7 [M+2H] N D

131 to 142 ERPIGDLVVGLK 649.2 [M+2H] 649.4 [M+2H] N D 129 to 130 M R 307.2 [M+H] 307.1 [M+H] N D

107 to 128 SLTAAVTAAGGNATYVLDGVPR 702.9 [M+3H] 702.8 [M+3H] 702.7 [M+3H] 92 to 106 EEVQLFLGNAGIAMR N D** N D** N D

85 to 91 FPVEDAK N D** N D** N D 76 to 84 AVVVGCGG

K 424.5 [M+2H] 424.5 [M+2H] 289.9[M+H]

75 to 75 R N D** N D N D 72 to 74 AAK 289.5 [M+H] 289.7 [M+H] N D

62 to 71 TLGLSVEAD

K 517.6 [M+2H] 517.5 [M+2H] N D 31 to 61 ILLLAALSEGTTVVDNLLNSEDV

HYMLGALR 1115.3 [M+3H] N D N D

26 to 30 SLSNR 577.4 [M+H] 577.5 [M+H] N D 21 to 25 LPGSK 502.4 [M+H] 502.3 [M+H] N D

14 to 20 EISGTVK 734.4 [M+H] 734.4 [M+H] N D 2 to 13 AGAEEIVLQPI

K 635.4 [M+2H] 635.3 [M+2H] 71% cov. FG72 Soybean Leaf* Non-transgenic Jack Soybean Leaf*

N D N D 98% cov.

E . coli 2mEPSPS protein*

1 to 13 MAGAEEIVLQPIK 700.9 [M+2H] 2mEPSPS Residue Sequence Theoretical
[M+H]

N D

393 to 405 LNVTAIDTYDDHR 767.9 [M+2H] 768.1 [M+2H] 767.4 [M+2H] 374 to 392 LGASVEEGPDYCIITPPEK 1039.2 [M+2H] 1039.2 [M+2H]

876.3 [M+3H]

424 to 429 DPGCT R N D** N D** N D 406 to 423 MAMAFSLAACAEVPVTIR N D** N D** N D 85 to 106 FPVEDAKEEVQLFLGNAGIAMR 812.4 [M+3H] 812.5 [M+3H]Soybean Event FG72 Petition Page 175 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 54. Amino acid coverage of the 2mEPSPS protein

Number of Amino Acids Not Detected Calculation of %

Amino Acid coverage E. coli 2mEPSPS Plant

‐ produced 2mEPSPS Residue Number

1 1

‐ 13

31 31

‐ 61

1 1 75

‐ 75

60 174

‐ 233

5 5 313

‐ 317

1 1 318

‐ 318

28 330

‐ 357

1 1 430

‐ 430

1 1 445

‐ 445

Total 9 129

Total number amino

Acids 445 445

% Amino Acid Not

Detected or

Analyzed 2 29

% Amino Acid

Sequence Coverage 98 71

% Amino Acid

Coverage to

2mEPSPS

from E. coli. 100 72 NA a

a NA = Not ApplicableSoybean Event FG72 Petition Page 176 of 198

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### 3.D.2. HPPD W366 protein

The SDS-PAGE and western blots demonstrated that the molecular weight, mobility, and immuno-reactivity of the microbially-produced and plant-produced HPPD W336 proteins are the same. The western blot also indicated that the non-transgenic Jack soybean sample did not contain immunoreactive protei ns. The plant-produced HPPD W336 protein had other bands due to non-specific binding of proteins from the soybean matr ix as shown in the SDS-PAGE (Figure 36). These non-specifically bound matrix pr oteins are not immunoreactive and were not detected in the western blot (Figure 37). The E. coli- produced HPPD W336 protein batch LB020309 had higher molecular weight bands appearing in the western blot, most likely due to protein dimers. A band below the E. coli produced HPPD W336 protein was also detected and is believed to be due to protein degradation.

Figure 36. Comparison of the plant-produced and microbially-produced HPPD W336 protein

The SDS-PAGE gel was stained with Pierce Imperial Protein Stain. Lane 1 contains approximately 300 ng of HPPD W336 batch LB020309 protein produced in E. coli. Lane 2 contains the non-transgenic Jack soybean leaves control sample. Lane 3 contains the HPPD W336 protein isolated from FG72 soybean leaves. Lane 4 contains molecular weight markers of 220, 160, 120, 100, 90, 80, 70, 60, 50, 40, 30, 25, 20, 15 and 10 kDa. Underlined molecular weights are shown on the gel.

Lane 1. E. coli produced HPPD W336 Protein (Batch LB020309) Lane 2. Non-transgenic Jack soybean leaves control sample Lane 3. HPPD W336 isolated from FG72 soybean leaves
Lane 4. BenchMark Protein Ladder 

### 1 2 3

kDa

-220 -160

-100

-80

-70

-60

-50

-40

-30

-25

-20

-15 -10Soybean Event FG72 Petition Page 177 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 37. Western blot comparison of the plant-produced and microbially-produced HPPD W336 protein

Lane 2 contains the HPPD W336 batch LB020309 protein produced in E. coli. Lane 3 contains the nontransgenic Jack soybean leaves control sample. Lane 4 contains the HPPD W336 protein isolated from FG72 soybean leaves. Lane 5 contains molecular weight markers of 250, 150, 100, 75, 50, 37, 25 and 20 kDa. Underlined molecular weights are shown on the gel.

Lane 1. Kaleidoscope Precision Plus Protein Standard
Lane 2. E. coli produced HPPD W336 Protein (Batch LB020309) Lane 3. Non-transgenic Jack soybean leaves control sample Lane 4. HPPD W336 isolated from FG72 soybean leaves Lane 5. Kaleidoscope Precision Plus Protein Standard



### The full scan mass spectrometry results are shown in Table 55. The full scan analysis obtained 95.2% sequence coverage of the E. coli produced HPPD W336 protein (Table 56). The analysis of the tryptic digest of the FG72 soy bean produced HPPD W336 protein by full scan analysis obtained 70.1% sequence cove rage of the protein (Table 56) . Peptides not identified may be due to matrix effects or incomplete digestion. 

### 1 2 3 4

-250 

### kD

-100

-75

-50

-37

-25

-20Soybean Event FG72 Petition Page 178 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 55. Electrospray LC/MS peptide mapping of the HPPD W336 protein

4274

4143

583.7

234.3

801.9

3609

246.3

477.5

523.6

2543

1245.5

2296.5

755.9

1367.5

232.3

1351.6

696.8

375.4

685.8

956.0

1049.2

1017.1

3208.6

733.8

147.2

862.0

476.6

1922.2

1575.7

1290.4

175.2

1446.6

2530.0425

147.1968

1272.3154

965.0936

517.5621

175.2102

673.7496

575.6391 ND** 962.2 [M+2H] ND 2 to 38 ADLYENPMGLMGFEFIEFASPT
PGTLEPIFEIMGFTK 1382 [M+3H] 1381.6 [M+3H]

ND

353 to 358 GVLTAD 576.3 [M+H] 576.3 [M+H] ND 352 to 352 R ND** ND** ND 340 to 347 ALFESIER 483.5 [M+2H] 483.2 [M+2H]

348 to 351 DQVR ND** ND** ND 328 to 328 K

ND ND ND 329 to 339 GDDGFGEWNFK 637.1 [M+2H] ND ND

307 to 327 LLLQIFSETLMGPVFFEFIQR ND ND ND 306 to 306 R

ND ND** ND** ND

ND

279 to 292 LPDHGEPVDQLQAR 526.3 [M+3H] 526.1 [M+3H] ND 263 to 278 FMTAPPDTYYEMLEGR 1070.8 [M+3H] ND

ND

259 to 262 IGMR 477.3 [M+H] ND ND 258 to 258 K 525.8 [M+2H] 525.7 [M+2H]

ND

252 to 257 TWDALK ND** ND ND 223 to 251 GAGQIEEFLMQFNGEGIQHVAF 343.7 [M+2H] 343.4 [M+2H]

ND 214 to 222 IPLNEESSK 509.5 [M+2H] 509.2 [M+2H] ND 204 to 213 AMSAPDGMIR ND 697.4 [M+H]

ND

195 to 203 GEYTGLTSK 479.0 [M+2H] 478.7 [M+2H] ND 190 to 194 YFDIK 232.6 [M+H] 232.6 [M+H]

ND 187 to 189 EAR 375.7 [M+H] 375.5 [M+H] ND 182 to 186 LFNFR 378.7 [M+2H] ND

ND 172 to 181 MVYWANFYEK 676.9 [M+2H] 676.5 [M+2H] ND 170 to 171 GR 623.9 [M+2H] 623.6 [M+2H]

ND

159 to 169 VIDHLTHNVYR 456.9 [M+3H] 456.5 [M+3H] ND 151 to 158 NPVGAGLK 524.3 [M+H] 524.2 [M+H]

ND 131 to 150 FGEGSSIYDIDFVYLEGVER 1149.4 [M+2H] ND ND 119 to 130 GIGGAPLYLIDR 247.2 [M+H] ND

ND

95 to 118 ALELGAQPIHIDTGPMELNLPAI

K 848.8 [M+3H] 848.7 [M+3H] ND 91 to 94 AYNR 402 [M+2H] 401.7 [M+2H]

ND

87 to 90 DSQK ND ND ND 85 to 86 VK 584.4 [M+H] 584.3 [M+H]

ND

52 to 84 QGEINLILNNEPNSIASYFAEHG
PSV C GMAFR (cys_CAM mod) 1203.7 [M+3H] 1204 [M+3H] ND 46 to 51 NVHLYR ND ND

ND

44 to 45 SK ND 234.1 [M+H] ND 39 to 43 VATHR ND 95.2% cov

E. coli HPPD W336
Protein *

1 to 38 MADLYENPMGLMGFEFIEFASP
TPGTLEPIFEIMGFTK ND HPPD W336 Residue Sequence Theoretical [M+H] 70.1% cov. FG72 Soybean Leaf* Non-transgenic Jack
So y bean Leaf*

ND 252 to 258 TWDALKK 432.0 [M+2H] ND ND

293 to 305 GILLDGSSVEGDK ND** ND**

ND 293 to 306 GILLDGSSVEGDKR 724.4 [M+2H] 724.1 [M+2H]

348 to 352 DQVRR 337.7 [M+2H] 337.6 [M+2H] NDSoybean Event FG72 Petition Page 179 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 56. Amino acid coverage of the HPPD W336 protein

Number of Amino Acids Not Detected Calculation of %

Amino Acid overage E. coli HPPD W336 Plant-produced HPPD W336 Residue Numbe r

1 1 1-38

2 b 44-45

2 85-86

4 4 87-90

20 131-150

8 151-158

5 b 182-186

29 223-251

7 252-258

4 259-262

16 263-278

11 329-339

4 4 348-351

1 1 352-352

Total 17 107

Total number amino

Acids 358 358

% Amino Acid Not

Detected or

Analyzed 5 30

% Amino Acid

Sequence Coverage 95 70

% Amino Acid

Coverage to

HPPD W336

from E. coli 100 71 NA a

a NA = Not Applicable. b The amino acids were detected in the FG72 soybean produced HPPD W336 sample protein; but not
in the E. coli produced HPPD W336 protein. c The sequence coverage was adjusted to account for the aminoacids 44-45 and 182-186 that were detected in the FG72 HPPD W336 sample; but not in the E. coli produced HPPD W336 protein.Soybean Event FG72 Petition Page 180 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 4

RAW AGRONOMIC DATA FOR 2008Soybean Event FG72 PetitionPage 181 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 57. Raw agronomic data for 2008TrialNumberDescriptionEarlySCPlantVigorDays tofloweringFlowerColorLeafShapePHV4-5 PH R1 PH M Pb Pd HilumCanopyDays toMaturityYieldbu/acre Lodging Final SCPodShatterGrowthHabit01 Jack 109 8 51 W Oval 5 5 5 G B Y Medium 138 50.17 5 97 8 I01 Jack 100 8 52 W Oval 5 5 5 G B Y Medium 136 50.31 5 86 8 I01 Jack 96 8 52 W Oval 5 5 5 G B Y Medium 138 52.78 5 92 8 I02 Jack 110 8 52 W Oval 5 5 5 G B Y Medium 134 64.40 4 90 8 I02 Jack 112 8 52 W Oval 5 5 5 G B Y Medium 135 67.52 4 104 8 I02 Jack 117 8 52 W Oval 5 5 5 G B Y Medium 136 69.62 5 94 8 I03 Jack 113 8 45 W Oval 5 5 5 G B Y Medium 131 71.87 2 89 8 I03 Jack 106 8 45 W Oval 5 5 5 G B Y Medium 131 59.75 2 94 8 I03 Jack 115 8 45 W Oval 5 5 5 G B Y Medium 131 67.23 2 92 8 I04 Jack 150 8 45 W Oval 5 5 5 G B Y Medium 128 57.43 3 97 8 I04 Jack 147 9 45 W Oval 5 5 5 G B Y Medium 128 47.77 3 98 7 I04 Jack 146 9 45 W Oval 5 5 5 G B Y Medium 129 50.38 2 90 8 I05 Jack 100 8 54 W Oval 5 5 5 G B Y Medium 136 60.84 3 74 8 I05 Jack 98 8 54 W Oval 5 5 5 G B Y Medium 136 56.70 4 77 9 I05 Jack 114 8 54 W Oval 5 5 5 G B Y Medium 137 56.27 4 89 9 I06 Jack 90 8 48 W Oval 5 5 5 G B Y Medium 128 44.36 4 82 9 I06 Jack 81 8 50 W Oval 5 5 5 G B Y Medium 129 40.51 4 60 9 I06 Jack 95 8 48 W Oval 5 5 5 G B Y Medium 130 51.98 4 85 9 I07 Jack 90 8 40 W Oval 5 5 5 G B Y Medium 119 44.21 6 78 8 I07 Jack 106 9 40 W Oval 5 5 5 G B Y Medium 120 51.69 5 77 9 I07 Jack 89 8 40 W Oval 5 5 5 G B Y Medium 119 48.64 5 69 9 I08 Jack 123 8 36 W Oval 5 5 5 G T Y Medium Bushy 118 58.37 4 124 9 I08 Jack 135 7 37 W Oval 4 5 5 G T Y Medium Bushy 118 53.29 4 134 8 I08 Jack 129 9 36 W Oval 4 5 5 G T Y Medium Bushy 118 45.88 3 129 9 I09 Jack 52 6 46 W Oval 3 4 5 G T Y Medium Bushy 118 39.78 9 51 8 I09 Jack 101 8 46 W Oval 5 4 5 G T Y Medium Bushy 118 8 101 8 I09 Jack 127 8 46 W Oval 5 5 5 G T Y Medium Bushy 119 39.78 8 128 8 I10 Jack 118 8 46 W Oval 4 5 5 G B Y Medium 124 46.54 5 104 8 I10 Jack 139 9 46 W Oval 4 5 5 G B Y Medium 124 45.81 5 106 8 I10 Jack 126 8 46 W Oval 4 5 5 G B Y Medium 124 48.35 5 100 8 IMean 111.1 8.1 46.47 4.8 4.9 5 127.7 53.18 4.4 93.0 8.3SD 21.4 0.6 5.28 0.5 0.3 0 7.2 8.96 1.7 19.1 0.5Soybean Event FG72 PetitionPage 182 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTrialNumberDescriptionEarlySCPlantVigorDays tofloweringFlowerColorLeafShapePHV4-5 PH R1 PH M Pb Pd HilumCanopyDays toMaturityYieldbu/acre Lodging Final SCPodShatterGrowthHabit01 FG72 unsprayed 99 8 52 W Oval 5 5 5 G B Y Medium 137 42.11 4 94 8 I01 FG72 unsprayed 91 8 53 W Oval 5 5 5 G B Y Medium 137 45.59 5 86 8 I01 FG72 unsprayed 97 8 53 W Oval 5 5 5 G B Y Medium 136 46.61 5 87 8 I02 FG72 unsprayed 89 8 52 W Oval 5 5 5 G B Y Medium 133 55.10 5 84 8 I02 FG72 unsprayed 86 8 53 W Oval 5 5 5 G B Y Medium 134 59.97 4 79 8 I02 FG72 unsprayed 79 8 52 W Oval 5 5 5 G B Y Medium 134 59.82 4 70 8 I03 FG72 unsprayed 95 8 45 W Oval 5 3 5 G B Y Medium 131 59.90 4 81 8 I03 FG72 unsprayed 101 8 45 W Oval 5 5 5 G B Y Medium 131 58.01 4 79 8 I03 FG72 unsprayed 92 8 45 W Oval 5 5 5 G B Y Medium 130 60.91 4 83 8 I04 FG72 unsprayed 94 8 46 W Oval 5 3 5 G B Y Medium 128 44.14 4 71 7 I04 FG72 unsprayed 88 8 45 W Oval 5 5 5 G B Y Medium 127 55.76 4 76 7 I04 FG72 unsprayed 95 8 45 W Oval 5 5 5 G B Y Medium 128 48.86 4 71 7 I05 FG72 unsprayed 95 8 54 W Oval 5 5 5 G B Y Medium 137 51.33 5 74 8 I05 FG72 unsprayed 87 8 54 W Oval 5 5 5 G B Y Medium 138 63.23 4 67 8 I05 FG72 unsprayed 109 8 54 W Oval 5 5 5 G B Y Medium 138 70.13 3 74 8 I07 FG72 unsprayed 23 7 40 W Oval 3 3 5 G B Y Medium 120 31.07 6 23 7 I07 FG72 unsprayed 26 7 41 W Oval 3 3 5 G B Y Medium 120 33.32 6 30 8 I07 FG72 unsprayed 39 7 42 W Oval 3 3 5 G B Y Medium 120 34.05 6 35 8 I06 FG72 unsprayed 70 8 50 W Oval 3 5 5 G B Y Medium 131 43.49 6 63 8 I06 FG72 unsprayed 87 8 48 W Oval 5 5 5 G B Y Medium 129 49.88 5 74 8 I06 FG72 unsprayed 79 8 49 W Oval 5 5 5 G B Y Medium 129 48.06 5 79 8 I08 FG72 unsprayed 102 8 37 W Oval 5 5 5 G T Y Medium Bushy 118 40.66 4 102 9 I08 FG72 unsprayed 95 9 37 W Oval 5 4 5 G T Y Medium Bushy 119 36.66 5 93 9 I08 FG72 unsprayed 103 7 37 W Oval 5 5 5 G T Y Medium Bushy 118 39.35 4 102 9 I09 FG72 unsprayed 89 7 46 W Oval 4 4 5 G T Y Medium Bushy 118 41.09 9 88 9 I09 FG72 unsprayed 89 7 46 W Oval 4 4 5 G T Y Medium Bushy 118 35.14 9 88 9 I09 FG72 unsprayed 76 9 46 W Oval 5 5 5 G T Y Medium Bushy 119 27.15 9 76 8 I10 FG72 unsprayed 70 8 45 W Oval 4 5 5 G B Y Medium 124 36.66 6 68 7 I10 FG72 unsprayed 52 7 45 W Oval 4 5 5 G B Y Medium 124 37.75 5 47 7 I10 FG72 unsprayed 67 8 45 W Oval 4 5 5 G B Y Medium 124 36.45 4 62 8 IMean 82.1 7.8 46.7 4.6 4.6 5.0 127.7 46.4 5.1 73.5 8.0SD 21.7 0.5 5.2 0.7 0.8 0.0 7.0 11.0 1.6 19.0 0.6Soybean Event FG72 PetitionPage 183 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTrialNumberDescriptionEarlySCPlantVigorDays tofloweringFlowerColorLeafShapePHV4-5 PH R1 PH M Pb Pd HilumCanopyDays toMaturityYieldbu/acre Lodging Final SCPodShatterGrowthHabit01 FG72 sprayed 79 8 53 W Oval 3 3 5 G B Y Medium 137 43.05 6 78 8 I01 FG72 sprayed 99 8 54 W Oval 3 3 5 G B Y Medium 138 45.96 6 95 8 I01 FG72 sprayed 95 8 53 W Oval 3 3 5 G B Y Medium 138 48.28 6 87 8 I02 FG72 sprayed 109 8 54 W Oval 3 3 5 G B Y Medium 134 57.50 6 99 8 I02 FG72 Sprayed 82 8 54 W Oval 3 3 5 G B Y Medium 137 49.95 6 76 8 I02 FG72 sprayed 72 8 58 W Oval 3 3 5 G B Y Medium 137 51.40 7 75 8 I03 FG72 sprayed 85 8 45 W Oval 3 3 5 G B Y Medium 133 52.49 6 74 8 I03 FG72 sprayed 78 8 46 W Oval 3 3 5 G B Y Medium 133 56.70 6 74 8 I03 FG72 sprayed 84 8 45 W Oval 3 3 5 G B Y Medium 133 60.19 6 83 8 I04 FG72 sprayed 89 8 45 W Oval 4 3 5 G B Y Medium 127 49.08 6 70 7 I04 FG72 sprayed 69 8 46 W Oval 3 3 5 G B Y Medium 129 43.12 5 58 7 I04 FG72 sprayed 74 8 47 W Oval 3 3 5 G B Y Medium 130 44.94 6 50 7 I05 FG72 sprayed 89 8 54 W Oval 4 5 5 G B Y Medium 138 58.30 5 74 8 I05 FG72 sprayed 85 8 54 W Oval 4 5 5 G B Y Medium 137 62.80 6 69 8 I05 FG72 sprayed 99 8 54 W Oval 4 5 5 G B Y Medium 137 63.31 4 81 8 I06 FG72 sprayed 89 8 49 W Oval 5 3 5 G B Y Medium 130 44.58 5 63 8 I06 FG72 sprayed 91 8 49 W Oval 5 3 5 G B Y Medium 130 50.75 6 83 8 I06 FG72 sprayed 92 8 49 W Oval 5 3 5 G B Y Medium 131 42.40 6 81 8 I07 FG72 sprayed 33 7 41 W Oval 3 3 5 G B Y Medium 121 29.04 6 28 8 I07 FG72 sprayed 39 8 42 W Oval 3 3 5 G B Y Medium 121 34.78 6 32 7 I07 FG72 sprayed 40 7 42 W Oval 3 3 5 G B Y Medium 121 31.00 6 38 7 I08 FG72 sprayed 127 7 37 W Oval 4 4 5 G T Y Medium Bushy 120 43.34 5 127 9 I08 FG72 sprayed 106 6 37 W Oval 4 4 5 G T Y Medium Bushy 120 55.54 5 109 9 I08 FG72 sprayed 95 7 37 W Oval 4 4 5 G T Y Medium Bushy 119 36.30 5 92 9 I09 FG72 sprayed 80 8 46 W Oval 5 5 5 G T Y Medium Bushy 119 9 78 8 I09 FG72 sprayed 72 7 46 W Oval 4 4 5 G T Y Medium Bushy 119 28.10 9 72 8 I09 FG72 sprayed 93 8 46 W Oval 4 4 5 G T Y Medium Bushy 119 33.83 9 92 8 I10 FG72 sprayed 68 8 46 W Oval 4 5 5 G B Y Medium 125 32.89 6 57 8 I10 FG72 sprayed 54 8 46 W Oval 4 3 5 G B Y Medium 125 27.23 6 54 7 I10 FG72 sprayed 68 8 46 W Oval 4 5 5 G B Y Medium 125 35.21 7 60 7 IMean 81.2 7.8 47.4 3.7 3.6 5.0 128.8 45.2 6.1 73.6 7.9SD 20.6 0.5 5.6 0.7 0.8 0.0 7.1 10.8 1.2 21.4 0.6Soybean Event FG72 PetitionPage 184 of 198CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTrialNumberDescriptionEarlySCPlantVigorDays tofloweringFlowerColorLeafShapePHV4-5 PH R1 PH M Pb Pd HilumCanopyDays toMaturityYieldbu/acre Lodging Final SCPodShatterGrowthHabit01 2686-6 123 8 57 P Ovate 5 5 5 G B Y Medium Bushy 135 58.59 6 112 8 I01 2788 139 8 55 P Ovate 5 5 5 T B Bl Medium Bushy 136 66.43 6 126 8 I01 3000-0 127 8 59 P Ovate 5 5 5 t T Br Medium Bushy 138 73.62 6 112 8 I02 2686-6 135 8 53 P Ovate 5 5 5 G B Y Medium Bushy 129 53.51 7 124 8 I02 2788 109 8 54 P Ovate 5 5 5 T B Bl Medium Bushy 135 71.00 7 109 8 I02 3000-0 100 8 57 P Ovate 5 5 5 t T Br Medium Bushy 139 78.84 7 90 8 I03 2686-6 132 8 47 P Ovate 5 5 5 G B Y Medium Bushy 127 57.79 7 114 9 I03 2788 129 8 47 P Ovate 5 5 5 T B Bl Medium Bushy 128 66.07 7 115 9 I03 3000-0 106 8 50 P Ovate 5 5 5 t T Br Medium Bushy 131 67.81 7 100 9 I04 2686-6 100 8 47 P Ovate 5 5 5 G B Y Medium Bushy 129 65.12 8 79 9 I04 2788 121 9 46 P Ovate 5 5 5 T B Bl Medium Bushy 128 66.21 5 99 9 I04 3000-0 118 8 48 P Ovate 5 5 5 t T Br Medium Bushy 129 65.63 6 97 9 I05 2686-6 137 8 55 P Ovate 5 5 5 G B Y Medium Bushy 134 71.00 7 117 9 I05 2788 101 8 55 P Ovate 5 5 5 T B Bl Medium Bushy 134 75.14 6 92 9 I05 3000-0 103 8 57 P Ovate 5 5 5 t T Br Medium Bushy 134 79.93 6 93 9 I06 2686-6 115 8 50 P Ovate 5 5 5 G B Y Medium Bushy 128 56.63 8 104 9 I06 2788 124 8 51 P Ovate 5 5 5 T B Bl Medium Bushy 130 56.85 7 117 9 I06 3000-0 116 8 53 P Ovate 5 5 5 t T Br Medium Bushy 131 71.15 6 103 9 I07 2686-6 93 8 40 P Ovate 5 5 5 G B Y Medium Bushy 116 48.93 8 84 9 I07 2788 54 8 40 P Ovate 5 3 5 T B Bl Medium Bushy 118 51.11 8 55 9 I07 3000-0 65 8 42 P Ovate 5 3 5 t T Br Medium Bushy 120 63.89 7 60 9 I08 2686-6 134 7 36 P Ovate 5 5 5 G T Y Medium Bushy 118 49.01 8 134 9 I08 2788 133 9 37 P Ovate 5 5 5 T B Bl Bushy 120 34.85 6 132 9 I08 3000-0 123 6 44 P Ovate 5 4 5 T B Br Bushy 120 58.08 7 124 9 I09 2686-6 128 9 46 W Ovate 5 5 5 G T Y Medium Bushy 119 47.55 9 127 9 I09 2788 133 9 46 W Ovate 5 5 5 T B Bl Bushy 119 58.23 8 128 9 I10 3000-0 120 8 46 W Ovate 4 5 5 T B Br Bushy 119 48.35 8 120 9 I10 2686-6 124 9 45 P Ovate 4 5 5 G B Y Medium Bushy 123 53.72 8 115 8 I10 2788 65 8 46 P Ovate 4 5 5 T B Bl Medium Bushy 125 41.75 8 68 9 I10 3000-0 84 8 47 P Ovate 4 5 5 t T Br Medium Bushy 127 54.16 7 82 9 IMean 113.0 8.1 48.5 4.9 4.8 5.0 127.3 60.4 7.0 104.4 8.8SD 22.4 0.6 6.1 0.3 0.5 0.0 6.7 11.0 0.9 21.1 0.4LegendsEarly SC = Early Stand Count; PH V4-5 = Plant Health Rating Stage V4-5; PH R1 = Plant Health Rating Stage R1; PH M= Plant Health Rating Mature plants;Pb = Pubescence color; Pd = Pod color; Final SC = Final Stand CountSoybean Event FG72 Petition Page 185 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 5

HERBICIDE RESISTANC E AND STEWARDSHIPSoybean Event FG72 Petition Page 186 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 5.A. Herbicide resistant weeds

Herbicides are the most economical, effective and reliable method of weed control in most crop production systems. Herbicides act by target ing and inhibiting specif ic plant biochemical processes or pathways. The process of specific activity is termed “m ode of action” (MOA). Herbicides are classified into gr oups based on their MOA (HRAC 2009).

During the past several decades, diversity in weed control methods has been declining. Consolidation of agriculture has occurred at all le vels including combining smaller farms to form larger farms. The resulting economic pressures ha ve led to the selection of the most profitable crops and have driven the adoption of monocultures. Tillage, a key cultural practice contributing to a diversified weed management program, has also been severely reduced through the adoption of conservation tillage systems such as no-till and minimum tillage to combat the widespread problem of soil erosion (Anderson, 1996).

Weed control in the absence of complementary cult ural control practices has resulted in the use of herbicides as the only weed cont rol tactic. With this decline in use of alternative weed control methods, extensive use of herbicide s with a single MOA has not only resulted in weed shifts but also high selection pressure for herbicide resist ant weeds. Plants have the ability to adapt to ensure survival, which includes adapting to su rvive an herbicide applicat ion. The development
of herbicide resistance is a function of time and exposure and also the ge netic capability of the weed population present in a field.

Herbicide resistance is the natur ally-occurring inheritable ability of some weed biotypes within a given population to survive an herbicide treatment that should, under normal use conditions, effectively control that weed population (HRAC, 2009).

The first herbicide resistant weed was identified in 1964 (HRAC, 2009). An increase in the
number of documented herbicide resistant weeds began a steep incline after the ALS inhibiting
herbicides were introduced in the 1980’s. ALS herb icides inhibit the plant enzyme acetolactate synthase (ALS) and provide effective contro l of many grass and broadleaf weed species (Anderson, 1996; Whaley et al. , 2007). ALS herbicides were available for a broad number of crops for both post-emergence and residual weed contro l. Farming practices shifted, as use of ALS inhibitors reduced the amount of tillage needed for weed control. The lack of diversified weed control methods lead to the selection of populations of ALS herbicide-resistant weed species or biotypes.

There are 39 weed species resistant to the ALS class of chemistry in the US today and more than 90 resistant weed species reported world wide (Heap, 2009). Virtually all waterhemp ( Amaranthus rudis ) is considered by university weed scient ists to be resistant to ALS inhibiting herbicides, resulting in the conclusion that AL S inhibiting herbicides ar e considered “obsolete” technology for weed control in soybean (Nordby et al ., 2007). In addition to weeds resistant to ALS inhibiting herbicides, resistance has also developed too many ot her herbicide modes of action as evident in Figure 38.Soybean Event FG72 Petition Page 187 of 198

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### Figure 38. Timeline of the development of herbicide resistant weeds

5.B. Managing herbicide resistant weeds

Ideally integrated weed management should utilize all available t ools including herbicides in a well balanced program as the lower the diversity of weed control tools, the higher the risk of selecting a resistant biotype becomes. To ensure diversification is maintained in weed control methods, we will also encourage growers to keep detailed records of weed management practices for each field. The following ar e our integrated weed management guidelines to promote an economically viable , environmentally sustainable, and socially acceptable weed control program:

Know your weeds, know your fields Today’s herbicides control a broad spectrum of weed species, mi nimizing the importance of weed identification to a grower. However, identif ication of weed species will help identify an herbicide program that works best for every acre . Equally important is for the grower to understand the weed pressure and history within each field. Problematic ar eas like difficult-tocontrol weeds or dense weed popul ations should be closely monitored. There are several indications for a grower to consider with weed escapes to identify resistant weeds.

Resistance Indicators

- The field has been sprayed repeatedly with the same herbicide (mode of action), particularly if there was no mode of acti on diversity in the weed management system.

- A patch of weeds occurs in the same area year after year and is spreading.

- Many weed species are managed, but one particular weed species is no longer controlled. For example, following a glyphosate application, actively growing marestail can still be seen, in the absence of other weeds.Soybean Event FG72 Petition Page 188 of 198

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### - Surviving weeds of the problem species may be in a patch where some are dead and some exhibit variable symptoms, but all are approximately the same age.

Crop rotation Crop rotation is one of the most important fa ctors in an IWM program. Crop rotation adds weed management diversity through the inherent use of herbi cides with different m odes of action. In addition, crops vary in their ab ility to compete for sunlight, water and nutrients with weeds. Different planting times and seedbed preparation tec hniques can lead to a variety of cultural methods which employ diversity in a weed m anagement program. Reliance on a monoculture crop leads to weed population shifts to fewer weed species but to overall higher densities, which increases the selection pressure for herbicide resistant weeds.

Start with clean fields Yields can be significantly r educed by early season weed competit ion. Proper tillage or the use of a burndown herbicide program should be used to c ontrol all emerged weeds prior to planting. Not only does the control of weeds prior to planting aid in the ease of planting, it also eliminates weed competition for soil moisture, light and nutrients.

Regardless of the tillage system (conventional, minimal, or no-till ), a pre or early post-emergent soil-applied residual herbicide s hould be a part of every spray program. A soil-applied herbicide provides residual weed control allowing the cr op to get a head start. Residual herbicides minimize the weed pressure and allow a wider post-emergent herbici de application window. Generally, soil-applied herbicides can be in cluded in the burndown herbicide program for
residual weed control on no-till acres. A re sidual herbicide also introduces another mode of action into weed resistance management programs (Nordby et al ., 2007).

Rotate herbicide modes of action There are three key factors in using herbicides to promote good resistant weed management:

1. Use multiple modes of action during the growing season The use of multiple modes of action during the growing season increases the diversity within the weed control program by reducing th e selection pressure of a single mode of action. A planned two pass herbicide (pre followed by post-emergence) program implements multiple modes of action in weed management systems for delaying weed
resistance.

2. Apply no more than two applications of a single herbicide mode of action to the same field in a two-year period Repeated, successive use of herbicides with the same mode of action increases the likelihood that resistant plants will repr oduce and become dominant in the population. The best way to manage resistant weeds is to prevent them from spreading or populating. Herbicide-resistant weeds become problematic due to overuse of a single herbicide mode of action. To pr eserve an herbicide’s efficacy, maintain its use and reap its benefits, growers should not use more than two applications of a single herbicide
mode of action on the same field in a two- year period (Boerboom et al. 2006). In addition, rotating crops generally allows addit ional modes of actions to be used in a weed management program.Soybean Event FG72 Petition Page 189 of 198

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### 3. Rotate herbicide-tolerant trait systems To ensure the viability of all traits for the future, rotate the herbicide tolerant trait used in each field each year to increase the c hemical diversity used in each field.

Correct herbicide application Product efficacy can be influenced by a multitude of factors. Ensuring correct use rates, weed stage and crop growth, and application techniqu e will maximize weed control (Boerboom et al., 2006).

Apply to Actively Growing Weeds Herbicides provide peak performance when appl ied to actively growing weeds. Weeds that are actively growing absorb more herbi cide. Conditions that provide peak growing environment for weeds are adequate soil moistu re, sunlight and optimal soil nutrients.

Timing The use of pre-emergent residual herbicides wi ll provide key control of early season weeds that result in the greatest crop yield reduction and open a wider application window for post-emergence appl ications. Post emergence herbicides should be applied after crop emergence when weeds are 3 inches to 4 inches tall for optimal performance. Applying post emergence herbicides to sm aller weeds increases crop yield again by eliminating early season weed competition.

Application Technique Herbicides differ in the optimal application technique. Read and follow all label instructions to ensure proper application tech nique is achieved. Factors affecting weed control include: spray coverage, carrie r volume, application speed, adjuvants, and tankmix partners.

Product Rate The rate listed on the product label has been researched and tested by manufacturers and university researchers to provide the optimal control of the weeds at the height listed on the label. The application of an herbicide at a rate less than listed on the label can
result in insufficient control and will have a significant impact on the immediate weed control and therefore the weed seed bank by allowing partially controlled weeds to reproduce and set seed.

Control weed escapes Problematic weeds that escape the herbicide applicat ions should be controlled to reduce weed seed production. A grower should co nsider spot herbicide applications, row wi cking, cultivation or hand removal of weeds to improve weed management for the subsequent growing seasons.

Clean equipment To prevent the spread of herbicide-resistant weeds and potentially introduce new invasive weeds on to the farm, avoid moving equipm ent that has not been thoroughly cleaned.Soybean Event FG72 Petition Page 190 of 198

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### 5.C. Evolution of herbicide resistant weeds

There are currently 9 glyphosate-resistant weeds in the United States. These weeds include palmer amaranth ( Amaranthus palmeri ), marestail (Conyza Canadensis) , waterhemp

( Amaranthus rudis ), giant ragweed (Ambrosia trifida) , common ragweed ( Ambrosia
artemisiifolia ), Johnsongrass ( Sorghum halepense ), Italian ryegrass ( Lolium multiflorum ), hairy fleabane ( Conyza bonariensis ), and rigid ryegrass ( Lolium rigidum ). There are an additional 7 glyphosate resistant weeds that can be found in other parts of the world (Heap 2009). Giant ragweed, common ragweed, and waterhemp are 3 of the top 10 most frequently sprayed for weeds in soybeans (Bayer CropScience, 2009).

Marestail, also known as horseweed, is the most widely spread glyphosate-resistant weed in the U.S. Marestail can produce up to 200,000 seeds per plant. In a management study conducted in Michigan, soybean yields could be reduced up to 83% by marestail in untreated check treatments (Bruce and Kells, 1990). Some populations of marestail have become resistant to other available herbicides including atrazines, si mazines, diurons, and ALS inhibiting herbicides (Loux et al., 2006; Heap, 2009).

Another glyphosate-resistant weed of concern is giant ragweed. Glyphosate-resistant giant ragweed isn’t as widespread today as glyphosate-resistant waterhemp; however, it can be just
as difficult to control with al ternative herbicides. Giant ragw eed can grow up to 17 feet tall and produces allergenic pollen. One giant ragw eed plant per 110 square foot can reduce soybean yield 50%. There are also populations of giant ragweed that are resistant to ALS inhibiting herbicides (Johnson et al. , 2007).

Waterhemp is likely the weed of most concern in terms of control to soybean growers and university researchers. Waterhemp can produce more than 1 million seeds per plant. Waterhemp can reduce soybean yields by 37 to 44% in 7.5” and 30” rows, respectively (Nordby
et al. , 2007). Moreover, nearly all populations of wa terhemp are also resistant to ALS inhibiting herbicides and some populations are resistant to triazines and PPO inhibiting herbicides (Boerboom and Owen, 2006; Heap,2009).

Today there are few choices for conventional herbicides that are rated as “good” by University Extension programs for waterhemp control in gly phosate tolerant soybeans. Of those that are rated as good, their use is complicated as described in the following discussion.

Soil-applied residual herbicides Growers applied pre-emergence or pre-plant in corporated herbicides on less than 5% of soybean acres in 2006 (USDA, 2007). The use of residual herbicides declined due to the efficacy and ease of glyphosate us e. The seedling growth inhibito rs or microtubule inhibitors such as pendimethalin (Prowl ® 

### ), trifluralin (Treflan ® 

### ), and ethalfluralin (Sonalan ® 

### ) which inhibit cell division, provide residual control of waterhemp, however, these herbicides need to be incorporated into the soil for maximum effi cacy. Products that c ontain chloroacetamide herbicides and control waterhemp in so ybean include s- metolachlor (Dual II Magnum ® 

### ), smetolachlor + fomesafen, dimethenamid-P (Outlook ® 

### ) and alachlor (Intrro ® 

### , Micro-tech ® 

### ). Another pre-emergence herbicide for the control of waterhemp is metribuzin (Sencor ® 

### ), which is in the triazinone family but can result in cr op damage in certain environmental conditions.Soybean Event FG72 Petition Page 191 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Post-applied herbicides The only conventional herbicide mode of action that provides “good” control of waterhemp postemergence as rated by Universi ty Weed Scientists are the PPO inhibitors. These herbicides inhibit the protoporphyrinogen oxid ase (PPO) enzyme which is involved in the heme-pigment synthesis pathway. Products that contai n PPO inhibitors, such as lactofen (Cobra ® 

### , Phoenix TM 

### ), fomesafen (Flexstar ® 

### , Reflex ® 

### ) and s-metolachlor + fomesafen (Prefix TM 

### ) have potential to injure the soybean crop. Applying PPO inhibitors under high temperature and humidity increases the potential crop injury. Also, there are populations of waterhemp that are resistant to PPO inhibiting herbicides (Boerboom and Owen 2006; Heap 2009). In addition, there conventional herbicides have stringent limitations on the size of waterhemp and other weeds that they can control. Environmental situations prevent ti mely application of conventional herbicides, weed
control will be sacrificed.

5.D. Characteristics of glyphosate and isoxaflutole herbicides

5.D.1. Glyphosate herbicide

Glyphosate is a non-selective, broad spectrum systemic herbicide introduced to the marketplace in the 1970’s. Glyphosate can be formulated in mu ltiple ways: glyphosate isopropylamine salt (Roundup ® 

### ), glyphosate trimethylsulfonium salt (Touchdown ® 

### ), or glyphosate diammonium salt (Touchdown ® 

### 4 or Touchdown Pro ® 

### ). Glyphosate is the only member of the glycine herbicide family. Glyphosate inhibits the biosysnthesis of the aromatic am ino acids in the shikimic acid pathway by inhibiting the enz yme 5-enolpyruvylshikimate-3 -phosphate synthase (EPSPS) (Anderson, 1996; Vencill, 2002).

Glyphosate is labeled for the control of 113 annual broadleaf and grass weeds and additional 62 perennial weeds (Roundup Weathermax ® 

### label 2006). Glyphosate is likely the most broad spectrum herbicide available today for weed control in row crops. The effectiveness of glyphosate is established; more than 90% of the soybean acres in 2006 were treated with glyphosate at an average use rate of 0.802 lb/A with an average of 1.7 applications per season (USDA, 2007). The lack of effective alternatives is illustrated by the fact that the second most commonly used herbicide in 2006 was 2,4-D 2-EHE which was sprayed on only 7% of the US soybean acres (USDA, 2007). In addition to so ybean, glyphosate was applied to 85% of the planted cotton acres in 2007 (USDA, 2008) although resistance to glyphosate has developed in several weed species, the chemical is extrem ely effective on the vast array of weeds in commercial crops.

5.D.2. Isoxaflutole herbicide

Isoxaflutole (IFT), the base active ingredient for several broad spectrum residual herbicides, is an isoxazole which inhibits the enzyme p-hy droxylphenyl pyruvate deoxygenase (HPPD) in plants resulting in inhibition of carotenoid biosynthesis which gi ves rise to a bleached appearance of new growth (Vencill, 2002). Isoxaflu tole has been commercially available in the
US since 1999 as Balance ® 

### Pro registered for use in field corn. Other herbicides registered for use in the US based upon the acti ve ingredient IFT include Balance ® 

### Flexx (IFT + cyprosulfamide), Corvus TM 

### (IFT + thiencarbazone + cyprosulfamide) and Radius TM 

### (IFT + flufenacet). There are two IFT containing product s available for use on field corn in Canada: Converge ® 

### Pro (IFT) and Converge ® 

### XT (IFT + cyprosulfamide).Soybean Event FG72 Petition Page 192 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### To date, there is no documented resistance of any weed species to isoxaflutole or any other HPPD inhibitor in the world (Heap, 2009). The her bicidal mode of action of IFT, class F (4-hppd inhibitors), is unique to soybean production systems. Soil app lied herbicides, like IFT, increase the consistency of weed control, reduce ear ly season weed competition which results in significant yield losses, and introduces a new mo de of action for decreased selection pressure for glyphosate resistance (Nordby et al. , 2007).

IFT herbicide can be applied either pre-emerge nce to the soybean or post emergence in a tankmix with a post-emergence her bicide. IFT can provide control of a broad spectrum of weeds including herbicide-resistant weed species. IFT provides pre-emergence control of 54 grass and broadleaf weed species important to soybean production including weeds resistant to glyphosate such as marestail, palmer amarant h, waterhemp, giant ragweed, common ragweed, and seedling Johnsongrass (Balance ® 

### Pro herbicide label).

5.E. Stewardship of double-herbicide-tolerant soybean event FG72

Bayer CropScience (BCS) places a high importance on the sustai nability of its technology and has adopted a life-cycle approach to product stew ardship. This means that appropriate stewardship principles are applied at every stage of biotechnology deve lopment from research through to product discontinuation and as a founding member of Excellence Through Stewardship ® 

### , BCS is helping advance stewardship best practices throughout the industry. BCS commitment to stewardship extends to our co rporate relationships and is evidenced by the stewardship and quality assurance standards that are required in those relationships and is also indicated in the following clause that is now included in third party agreements related to BCS biotechnology traits:

“ BAYER is committed to the proper stewardshi p of its products and expects those with
whom it contracts to handle material c ontaining BAYER technology in an appropriate
manner. This includes without limitation adherence to the stewardship and quality assurance provisions of this Agreement. BAYER supports and has affirmed its commitment to the Excellence Through Stewardship ® 

### industry stewardship initiative. Further information relating to this initiative can be found at www.excellencethroughstewardship.org .”

In the BCS organization, our crop market ar ea teams are committed to BCS stewardship principles and are aware of procedures to comm unicate appropriate information within the BCS crop team matrix to rapidly respond to issues t hat may develop from use of our technologies. Field development and market support teams ar e provided the tools necessary to serve the grower as a local and direct contact for any ques tions related to BCS technologies with regards to product performance or impacts on hum an and environmental health and safety.Soybean Event FG72 Petition Page 193 of 198

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### BCS participates in several industry and profes sional initiatives in support of stewardship:

 Herbicide Resistance Action Committee (HRAC) HRAC is an industry initiative which fost ers co-operation between plant protection manufacturers, government, researchers, ad visors and farmers. The objective of the working group is to facilitate the effective management of herbicide resistance. Weed scientists employed by Bayer CropScience participate as members of the Herbicide
Resistance Action Committee and BCS supports the work of this group.

 CropLife America - US BCS is active in CropLife, serving on committees and working groups that develop industry-wide approaches to regulatory and technology management issues.

 American Seed Trade Association (ASTA) M.S. Technologies, LLC (MSTech) and BCS ar e active in serving on committees and working groups that set industry standards for seed quality and purity, and product stewardship.

 BIO BCS is active in the Biotechnology Industr y Organization, serving on committees and working groups that develop industry-w ide approaches to regul atory and technology management issues.

 Excellence Through Stewardship BCS is active in Excellence Through Stewards hip, serving on the board of directors, committees and working groups that develop indu stry best practices for stewardship.

 Weed Science professional societies BCS is active participant in a number of organi zations. We maintain active memberships in the Weed Science Society of America, North Central Weed Science Society, Northeastern Weed Science Society, Sout hern Weed Science Society, and Western Weed Science Society, all of which are profes sional, non-profit societies, established to promote research, education, and extension outr each activities related to weeds; provide science-based information to the public and policy makers; and foster awareness of weeds and their impacts on managed and natural ecosystems (WSSA 2009).

5.E.1. Customer outreach

BCS and MSTech have a commitment to stewardshi p of all of our products, including herbicidetolerant trait (HTT) technology. We strive to provide best management practices of HTT technology which includes integrated weed man agement to our customers (see section 5.B.). Education of integrated weed management is th e only practical method for its success. Education starts internally with our own field development, technical service, chemical sales representatives, and seed salesmen. Externally, we collaborate with key influencers to help growers understand the long term economic vi ability of integrated weed management. Those key influencers include university extension agents, agronomists, consultants, and local retail seed and chemical salesmen. In addition, we directly provide the integrated weed management message to growers through grower meetings, tr ade shows, and web and mail communications.Soybean Event FG72 Petition Page 194 of 198

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### A Technology Use Agreement or similar agreement will be developed that will provided to each grower at the time of seed purchase. By sign ing the agreement, the grower will agree to best management strategies that are indicated in the agreement. The agreement will contain company contact information including a w ebsite for the best management practices and product information. In addition, a toll free hotline for grower s to obtain live technical product support will be provided. BCS and MSTech ar e committed to stewardship principles and procedures, and to communicating appropriate inform ation in order to rapidly respond to any issues that may develop.

Growers may also contact the seed compan y for product support. The seed company name and contact information will be provided on the l abel of each bag of seed sold. Each grower purchase of FG72 Soybeans will be recorded by seed company part ners. This information will be provided to MSTech whic h will enable MSTech to maintain a database of all growers utilizing event FG72 products. This database could be used to disseminate updated stewardship
information.

5.E.2. Additional customer support

Product information There are a number of ways that a grower can ob tain product information. The product label is the formal legal method of communicating directi ons for use of an herbicide. BCS’s history of including recommendations on pr oduct labels for integrated weed management. Here is an example of a BCS product label on this topic.

BALANCE ® FLEXX Herbicide is also recommended as the fi rst herbicide applied in an integrated weed control program that includes sequential post-emergence herbicide applications.

CORVUS TM Herbicide may be applied as the first herbicide in an Integrated weed control program that includes sequential post-emergence herbicide applications with products such as LAUDIS TM Herbicide, or
IGNITE ® 280 SL Herbicide or glyphosate in transgenic field corn.



### BCS is committed to supporting research by univ ersity institutions to generate local grower recommendations. University Ex tension Weed Control Handbooks ( 2008 Guide for Iowa Corn and Soybean Production, Illinois Agricultural Pest Management Handbook, 2009 Weed Control
Guide for Ohio and Indiana ) contain use directions and pr oduct information on many BCS
herbicides.

Screening for Herbicide Resistance Currently, confirmation of weed resistance is commonly conducted by collecting seed of suspected resistant plants. Those seeds are replanted in a greenhouse environment and sprayed with various rates of the herbicide to whic h resistance is suspected. The survival of the
weeds confirms resistance.

BCS invests a significant amount of resour ces to inform and train our own employees, customers and stakeholders so that they can develop sustainable programs to manage both their resistant and susceptible weed populations. Modern testing conducted in the laboratory such as those employed by Bayer CropScience wi ll in the future allow faster and more reliable herbicide resistance diagnosis. Such methods include testing for metabolic resistance by following the degradation of an active substance in a plant and testing for target-site resistance through PCR analysis coupled with pyrosequencing.Soybean Event FG72 Petition Page 195 of 198

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### 5.E.3. Monitoring of effectiveness of the stewardship plan

Each grower purchase of event FG72 soy beans will be recorded by the individual seed company making the sale. This information w ill be provided to MSTec h which will enable MSTech to maintain a database of all grower s utilizing event FG72 pr oducts. BCS regularly utilizes market research surveys to determi ne market share and adaptation of technology.

Seed company partners will have direct contact with growers and will be able to provide feed back to MSTech regarding the stewardship effectiv eness. BCS field representatives will also interact with growers and will be a source of information.

BCS will continue to support ongoing efforts to understand weed resistance to herbicides, to apply up-to-date information to product labels, and to provide in formation to growers.Soybean Event FG72 Petition Page 196 of 198

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### Appendix 6

REFERENCES – APPENDICES 1-5Soybean Event FG72 Petition Page 197 of 198

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### References

Anderson, W.P. 1996. Weed Science: Principles and Applications. West Publishing, St. Paul, MN. pg. 128, 219-220, 256-257.

Balance ® 

### Pro Herbicide product label. Bayer CropScience. 2005.

Bayer CropScience. 2009. 2009 Trait and Technol ogy Use Manual.

Boerboom, C. and Owen, M. 2006. The Glyphosate, Weeds, and Crop Series: Facts About Glyphosate-Resistant Weeds. www.glyphosateweedscrops.org

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Bradshaw, R.A., Brickey, W.W. and Walker, K. W. 1998. N-terminal processing: the methionine amino peptidase and N-acetyl tr ansferase families. Trends Biochem. Sci. 23: 263-267.

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