Soybean Event FG72 Petition - Revised 

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Petition for the Determination of Non-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 

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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 companies 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 

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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 requested 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. Technologies, 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 information herein must not be used, reproduced or 

disclosed to third parties without the written consent of Bayer CropScience and M.S. Technologies, LLC. 
Soybean Event FG72 Petition - Revised 

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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: 919-549-3929 
Soybean Event FG72 Petition - Revised 

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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 protein with two amino acid substitutions 

(2mEPSPS). This modification confers a decreased 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 resistant weeds currently found in soybean fields. IFT 

has the flexibility to be applied pre-plant, pre-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 plant 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 

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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 

LD50 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 

T1, T2, etc generations after T0 (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, corn

 
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TABLE OF CONTENTS 

TITLE PAGE ................................................................................................................................................. 1

COMPANY NAMES ...................................................................................................................................... 2

RELEASE OF INFORMATION ..................................................................................................................... 3

CERTIFICATION........................................................................................................................................... 4

SUMMARY.................................................................................................................................................... 5

ACRONYMS AND SCIENTIFIC TERMS...................................................................................................... 6

TABLE OF CONTENTS................................................................................................................................ 7

LIST OF TABLES........................................................................................................................................ 11

LIST OF FIGURES...................................................................................................................................... 13

I. RATIONALE 

I.A.Basis for the request for determination of non-regulated status .............................................. 15 

I.B.Double-herbicide-tolerant soybean event FG72....................................................................... 15 

I.C.Rationale for the development of event FG72 and benefits..................................................... 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 soybean .................................................................................................................. 18 

II.B.Characteristics of the recipient cultivar.................................................................................... 18 

III. DEVELOPMENT OF DOUBLE-HERBICIDE-TOLERANT SOYBEAN EVENT FG72 

III.A.Description of the transformation system ............................................................................... 19

III.B.Parent line............................................................................................................................... 19 

III.C.Breeding diagram ................................................................................................................... 19 

III.D.Generations used for analysis................................................................................................ 19 

IV. GENETIC MATERIAL USED FOR TRANSFORMATION OF EVENT FG72 

IV.A.Construction of the plasmid used for transformation ............................................................. 21 

IV.B.Donor genes and associated regulatory regions ................................................................... 22 

IV.B.1. The 2mepsps gene expression cassette................................................................. 22 

IV.B.2. The hppdPfW336 gene expression cassette ......................................................... 22 

 IV.C. Identity and source of the genetic material ........................................................................... 23 

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 sequence(s) ................................................................. 27 

V.E.Mendelian inheritance ............................................................................................................. 27 

V.F.Stability across and within generations ................................................................................... 28 

V.G.Conclusion .............................................................................................................................. 29 
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VI. CHARACTERIZATION OF THE INTRODUCED PROTEINS 

VI.A.The 2mEPSPS protein ........................................................................................................... 30 

VI.A.1. History and background .......................................................................................... 30 

VI.A.2. Characterization of the 2mEPSPS protein .............................................................. 30 

VI.A.2.1. Biochemistry and mode of action ............................................................ 30 

VI.A.2.2. 2mEPSPS protein safety......................................................................... 31 

VI.B.The HPPD protein .................................................................................................................. 33 

VI.B.1. History and background .......................................................................................... 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 use of the source organism............................................. 36 

VI.B.2.4. Familiarity of the gene product................................................................ 38 

VI.C.Expression of the 2mEPSPS and HPPD W336 proteins in event FG72............................... 38 

VI.C.1. Expression in grain.................................................................................................. 38 

VI.C.2. Expression in plant parts and during the life cycle.................................................. 40 

VI.D.Verification of the biochemical and functional equivalence of the expressed proteins.......... 42 

VI.D.1. 2mEPSPS 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 protein .......................................................................................... 44

VI.E.2. Potential allergenicity .............................................................................................. 44 

VI.E.2.1. Homology search to known allergens ..................................................... 44 

 VI.E.2.2. Potential N-glycosylation sites................................................................. 45 

 VI.E.2.3. In vitro stability to human simulated gastric fluid..................................... 45 

 VI.E.2.4. In vitro stability to human simulated intestinal fluid ................................. 46 

 VI.E.2.5. In vitro stability to heat............................................................................. 46 

VI.E.3. Homology search to known toxins........................................................................... 47 

VI.E.4. Acute toxicity study in the mouse............................................................................ 47 

VI.F.Summary of the food and feed safety assessment of the HPPD protein............................... 47 

VI.F.1. Familiarity to the protein .......................................................................................... 47

VI.F.2. Potential allergenicity...............................................................................................48 

VI.F.2.1. Homology search to known allergens ..................................................... 48 

VI.F.2.2. Potential N-glycosylation sites................................................................. 48 

VI.F.2.3. In vitro stability to human simulated gastric fluid..................................... 49 

VI.F.2.4. In vitro stability to human simulated intestinal fluid ................................. 49 

VI.F.2.5. In vitro stability to heat............................................................................. 50 

VI.F.3. Homology search to known toxins........................................................................... 51 

VI.F.4. Acute toxicity study in the mouse ............................................................................ 52 

VII. AGRONOMIC AND PHENOTYPIC EVALUATION 

VII.A.History of field activities ........................................................................................................ 53 

VII.B.Agronomic and phenotypic evaluation .................................................................................. 55 

VII.C.Agronomic performance of event FG72................................................................................ 57 

VII.C.1. Agronomic evaluation of event FG72 (2003 data) ............................................... 57 

VII.C.1.1.Yield evaluation of event FG72 under 

 conventional weed control practices....................................................... 57 

VII.C.1.2. Intended herbicide tolerance evaluations of event FG72 ....................... 58 

VII.C.2. Agronomic evaluation of event FG72 (2008 data) ............................................... 59 

VII.C.2.1. Agronomic performance and yield evaluation of event FG72 under 

conventional weed control practices....................................................... 59 

VII.C.2.2. Objective variety description .................................................................. 62 
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VII.C.2.3. Seed characteristics............................................................................... 63 

VII.D.Biotic and abiotic stress characteristics ................................................................................ 65 

VII.E.Equivalence between Jack-derived and commercial varieties ............................................. 65 

VII.F.Seed dormancy evaluation.................................................................................................... 67 

VII.G.Composition analysis............................................................................................................ 69 

VII.G.1. Introduction ............................................................................................................ 69 

VII.G.2. Nutritional composition of soybean grain............................................................... 69 

VII.H.Poultry feeding study ............................................................................................................ 75 

VII.I.Conclusion for agronomic evaluation of event FG72 ............................................................. 75 

 

VIII. ENVIRONMENTAL SAFETY AND IMPACT ON AGRONOMIC PRACTICES 

VIII.A.Environmental assessment of the introduced proteins ........................................................ 76 

VIII.B.Potential for horizontal or vertical gene transfer .................................................................. 76 

VIII.C.Weediness potential of double-herbicide-tolerant soybean event FG72............................. 77 

VIII.D.Current agronomic practices for soybean............................................................................77 

VIII.E.Potential impact on agricultural practices for soybean......................................................... 80 

VIII.F.Weed resistance management ............................................................................................ 81 

VIII.G.Potential impact on farming practices.................................................................................. 82 

VIII.H.Potential effects on non-target organisms, including beneficial organisms......................... 82 

VIII.I.Threatened and endangered species considerations ........................................................... 82 

VIII.J Potential impact on biodiversity ............................................................................................ 83 

VIII.K.Conclusion............................................................................................................................ 83 

IX. STATEMENT OF GROUNDS UNFAVORABLE.................................................84

X. REFERENCES....................................................................................................85

Appendix 1.FIELD TRIAL TERMINATION REPORTS 2001-2008...............................92

Appendix 2. MATERIALS AND METHODS- PRODUCT CHARACTERIZATION 

2.A. Materials and methods for molecular characterization ................................................................ 116 

2.B. Materials and methods for protein characterization tests ............................................................ 118 

2.C. Materials and methods for protein levels in seeds....................................................................... 122 

2.D. Materials and methods for protein levels in plant parts and during the life cycle ........................ 127 

2.E. Materials and methods for agronomic studies ............................................................................. 131 

2.F. Materials and methods for seed germination studies .................................................................. 133 

2.G. Materials and methods for composition analysis ......................................................................... 134

Appendix 3. MOLECULAR CHARACTERIZATION OF EVENT FG72 

3.A Verification of the insert................................................................................................................ 140 

 3.B. Absence of vector backbone........................................................................................................ 154 

 3.C. Stability across and within generations........................................................................................ 159 

 3.D. Demonstration of protein equivalence ......................................................................................... 171 

 3.D.1. 2mEPSPS protein.............................................................................................................. 171 

3.D.2. HPPD W336 protein .......................................................................................................... 176 
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Appendix 4.AGRONOMIC DATA ............................................................................... 180 

Appendix 5. HERBICIDE RESISTANCE AND STEWARDSHIP 

5.A. Evolution of herbicide resistant weeds......................................................................................... 186 

5.B. Managing herbicide resistant weeds............................................................................................ 187 

5.C. Evolution of resistance to glyphosate and isoxaflutole herbicides .............................................. 190 

5.D. Characteristics of glyphosate and isoxaflutole herbicides ......................................................... 191 

5.D.1. Glyphosate herbicide......................................................................................................... 191 

5.D.2. Isoxaflutole herbicide......................................................................................................... 191 

5.E. Stewardship of double-herbicide-tolerant soybean event FG72.................................................. 192 

5.E.1. Customer outreach ............................................................................................................ 193 

5.E.2. Additional customer support.............................................................................................. 194 

5.E.3. Monitoring of effectiveness of the stewardship plan.......................................................... 195 

Appendix 6. REFERENCES - APPENDICES 1-5....................................................... 196 
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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.............................................................................................................................. 73 

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 quantification of 2mEPSPS and HPPD W336 proteins in 

fortified non-transgenic soybean grain............................................................................. 125 
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LIST OF TABLES (continued) 

Table 33. Amounts of 2mEPSPS 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............................................................................................. 181 
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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....................................................................................................................... 168 
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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 microbially-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 ............................................ 187 
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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 environment 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 soybeans 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 efficacious weed control systems when utilized 

with FG72 herbicide tolerant soybean. Broad spectrum 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 time during the development of the soybean plant. 
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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 practices 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 retention, 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_planting/342.genetically_modified_soybean_global_

area_under_cultivation.html). 

Adoption of this technology is expected to progress 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 glyphosate resistance among different biotypes is 

demonstrated in Figure 1. 

This strategy will prolong the effective use of these excellent herbicides well into the future. 
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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 varieties, 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 currently, in field trials in soybean growing 

regions around the world. 
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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 compared 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 transformation 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. 
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III. DEVELOPMENT OF DOUBLE-HERBICIDE-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 SalI 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 sequence 
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Figure 2. Breeding Diagram of event FG72 

T1

T4

T5

T2

T7

T8

T6

T3

T0 

F1 

F2 

F3 

Commercial Candidate Evaluations 

T9 F4 

Initial Transformation in ‘Jack’ 

self

self

self

self

self

self

self

self

self

self

self

self

Cross with commercial varieties
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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 SalI and HPLC purification. 

Figure 3. Map of plasmid pSF10 

pSF10

10398 bp

2000

4000

6000

8000

10000

SalI

SalI

ORI ColE1

bla

ORI f1

3'nos

hppdPf W336

TPotp Y 5'tev

Ph4a748 ABBC

Ph4a748

intron1 h3At

TPotp C

2mepsps

3'histonAt

 
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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 subunit 

genes 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. 
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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 subunit 

genes 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 SalI 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) 
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Table 2. Genetic elements located on SalI 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 al., 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) 
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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 region 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 FG72 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.
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Figure 4. Event FG72 insert diagram 

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

3’histon At

3’histonAt

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

3’histon At

3’histonAt

5’ flanking seq. 

FG72 – TR1

FG72 – TR2

FG72 – TR3

FG72 – TR4

FG72 

Pre-insertion 

locus 

d c b a 

25 bp 

deletion 

2 bp 

deletion 

d b c a 

FG72 

Insert 

Seq. 

FG72 

locus 

FG72 

insert 

Ph4a748 fragment 

FG72 – TL2 

Junction 1 Junction 6 Junction 7 Junctions 8 and 9 

JACK – WT1 JACK – WT2 JACK – WT3 

Junction 2 Junction 3 Junction 4 

FG72 – TL1 

FG72 – TR 
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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 T0 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 T1 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 (T2 ) 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 T4 generation which was determined to be 

homozygous for transformation event FG72 and selected for core seed production in the fourth 

generation. In 2004, T5 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. F1 hybrid plants 

(FG72 x conventional lines) were grown to maturity and the F2 seed was planted. Leaf samples 

of 901 F2 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). 
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Table 3. Segregation data for progeny rows and individuals of self-pollinated event FG72 

Fully 

Toleranta

Rows/ 

Partially 

Tolerant 

Parents and zygosity for Rows/ 

the FG72 locus Progeny 

Tolerant 

Plants 

Sensitive 

Plants 

Expected 

Ratio 

χ2 

calculatedb

Tolerant T1 progenies of the 

self-pollinated T0 

transformants 

(1/4 FG72/FG72 ; 

2/4 FG72/-) 

T2 Rows 1 5 1 to 2 0.485 

Hemizygous T1 plants 

(FG72/-) resulting in the 

partially resistant rows 

T2

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 resulta 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 valueb = 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 event 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 progeny of transgenic plants cultivated at 4 

different field locations. The isolated DNA was digested with the restriction enzyme HindIII, 

which provides a unique pattern for event FG72. 
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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 stable and shows a typical Mendelian segregation 

pattern. Bioinformatics analyses of the junctions between the original genome and inserted 

sequences did not reveal any potential creation of unintended genes or any disruption of preexisting genes. 
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VI. CHARACTERIZATION OF THE INTRODUCED PROTEINS 

VI.A. The 2mEPSPS protein 

VI.A.1. History and background 

In the early 1970s, it was demonstrated that inhibitors 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 tyrosine (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 (Herrmann, 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 synthase (EPSPS) is involved in the shikimic 

acid pathway (Williams et al., 2000). This pathway produces an important branch point 

intermediate, 
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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 nature, 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 following 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 tolerance 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 the 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 Alimentarius 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 publicly 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: 
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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 2mepsps 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 and 

functionalities 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 biochemical 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 food/feed use in many countries (CERA, 2011). 

Lack of allergenic potential

The 2mEPSPS protein has no amino acid sequence similarity to known allergens, as 

demonstrated by overall amino acid and epitope homology searches (refer to Section VI.E.2.1. 

of this petition for details). 

As expected, the 2mEPSPS protein has high structural 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). 
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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 high 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 could 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 the 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 modified 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., 
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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 benzoylisoxazoles 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 diketonitrile (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) 
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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

SO2CH3 O O

CN

CF3

Arogenate

Tyrosine

Hydroxyphenylpyruvate

Homogentisate

Tocopherols

CH 2 CH COOH

OH

O

HPPD enzyme

CH 2 COOH

OH

OH

O 2

CO2

Plastoquinones

Chorismate Prephenate
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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-aspc.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/index.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). 
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Allergenicity

In general fluorescent pseudomonads have not been described 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 δendotoxins (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). 
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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. 
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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 Carolina 

at 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 W336 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 observed 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 μg/g fw in seeds treated with a conventional 

herbicide (mean value: 130 μg/g fw) and between 87 and 240 μg/g fw in the seeds treated with 

GLY + IFT (mean value: 140 μg/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 μg/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 μg/g fw in the seeds from soybean 

plants treated with a conventional herbicide (mean value: 0.85 μg/g fw) and from 0.41 to 1.31 

μg/g fw in the seeds from soybean plants treated with GLY + IFT (mean value: 0.8 μg/g fw). 

When converted to a dry weight basis, the expression levels were 0.62 to 1.26 μg/g dw (mean 

value: 0.94 μg/g dw) and 0.54 to 1.40 μg/g dw (mean value: 0.89 μg/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 Proteinb

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

HPPD Range 0.46– 1.32 

W336 

Conventional 

Treated/B Mean ± SD 0.85 ± 0.20 

0.94 c

 0.00024 c

HPPD Range 0.41 – 1.31 

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 fresh weight protein content in grain samples from the 

10 sites. 

b

 The dry weight (dw) and % crude protein analyte amounts were calculated using the average 2mEPSPS value of 

samples from each plot. 

c The dry weight (dw) and % crude protein analyte amounts were determined using the average of four individual 

results per sample, therefore no standard deviation or range is given for these amounts.
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In summary, the mean concentration of 2mEPSPS protein measured in soybean seeds was 150 

μg/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 soybean (Habex and Debaveye, 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 HPPD 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. Expressions 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. 
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Table 7. HPPD W336 protein expression in different tissues and growth stages 

HPPD W336 protein content 

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

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 

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

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 – 81.2 
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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.coliproduced, 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 Methodologya

Confirm identity of 2mEPSPS and HPPD W336 proteins Edman degradation 

Comparable molecular weight Protein mobility in SDS-PAGE 

Comparable immuno-reactivity Western blot analysis 

Comparable peptide masses HPLC/Electrospray 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 small 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 amino 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. 
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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, western 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 spectrometry (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. 
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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 6th 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 ubiquitous 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 that 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 acid 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 the 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 sequence 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, the 
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epitope search showed no identity between all the 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 glycosylation 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 the 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. 
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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 immunodetection 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 intensity 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 partially heat-stable up to 90°C for 60 minutes. 
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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 sequence 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 was 

BLOSUM62. 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 2mEPSPS 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 LD50 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 protein 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), mouse 
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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 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, 2009a). 
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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 western 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 Coomassie blue staining and western blot analyses. 

The immunodetection was performed using a polyclonal 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. 
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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 through 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 was developed which monitored the production of 

homogentisate and CO2 from 4-HPP and O2 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 conditions

Amount converted HPP / mg HPPD W336 *

45°C

60°C

95°C
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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 45o

C and is completely lost at higher temperatures (60oC 

and above) after 2.5 minutes. 

VI.F.3. Homology search to known toxins 

An overall amino acid sequence identity search 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 the 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 proteins 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. 
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These observations support the hypothesis that the VLLY and LLY proteins are HPPDs and 

therefore, share the typical HPPD structure with 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 clinical 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 toxicity in the OF1 female mouse. 
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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 FL 
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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) 
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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 factors. 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 the 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 action 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 T8 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 resistance, 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. 
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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 Soybean 
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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 commercial development. The planting seed was derived 

from homozygous lines in the T4 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 did not differ at any of the 9 locations. Event 

FG72 had a very consistent performance across 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 

FG72b Jackb Location Meanc

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. 
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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 extreme 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/Ac
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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 plots of event FG72 soybeans (Regimen B), and 

three 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 applications 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. 
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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-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). b

 In one location (Trial #09), a foliar application of imazethapyr (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 the 
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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 Unita 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 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 representing that plant stems are 45 degrees to the ground, and 9 representing that 

all plant 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 approximately 2 weeks after maturity (on border rows). 
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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, Regimen 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 target evaluation intervals were 6-10 days, 11-18 

days and 25-44 days following the foliar herbicide 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 reproductive 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 the 
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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 parameters 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. 
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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 qualityd 7.3 ± 0.9 7.0 ± 0.9 

Seed shapee 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 replicate 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 visual appearance of the seed (9 = best quality, 1 = worst 

quality) e The seed shape is measured as the length 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 spherical 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% 
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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 characteristics 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 commercial 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 of 
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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 Unita 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 date 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 representing that plant stems are 45 degrees to the 

ground, and 9 representing that all plant 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 approximately 2 weeks after maturity (on border rows). 
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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 small 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. 
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Table 24. Seed germination test results 

ID Location Germinationa 

(%) 

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 

StDevb 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

StDevb 2.1 0.9 0.0 1.8 1.7 0.7

a

 Warm germination – 8 reps of 50 seed 

b

 StDev; standard deviation
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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 T8 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). 
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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 glucosides 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. 
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Table 25. Proximate and fiber components 

Component Jack FG72 FG72 

Treated 

Commercial 

Lines b

Literature 

Referencec

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) 
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Table 26. Amino acids 

Amino acid 

% dw Jack FG72 FG72 Treated 

Commercial 

Lines b

Literature 

Referencec

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) 
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Table 27. Fatty acids 

Fatty Acid % relative Jack FG72 FG72 Treated 

Commercial 

Lines b

Literature 

Referencec

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) 
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Table 28. Anti-nutrients 

Anti-nutrients 

(dw) Jack FG72 FG72 Treated 

Commercial 

Lines b

Literature 

Referencec

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 

Referencec

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 LOQ 
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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, Jack 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, survival, 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 differences 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 assess 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 soybean 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. 
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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 proteins introduced in FG72 soybean will not 

present adverse environmental effects, as both 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 recent studies conducted by BCS have confirmed the 

safety profile of the 2mEPSPS protein (Section VI). 

Moreover, the safety of the 2mEPSPS protein, which is also present in the genetically modified 

herbicide tolerant maize Event GA21 and in GlyTolTM 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 completely 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 is 
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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 climatic conditions are permissive, volunteer soybeans 

provide minimal interference in the rotational crop and are not recognized as an economic 

problem in soybean production. Volunteer soybeans 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 actioni

; 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 soybean 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 glyphosate enabling the grower to simply, effectively 

and economically manage their weeds. 

Glyphosate herbicide is exceptionally effective 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 which 
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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. Additional 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 restricted-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 practice 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 herbicide 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 2009

Planted 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 2001-2009 
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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 herbicide 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 glyphosate 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, IWM 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 reluctant 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 will 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. 
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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 program 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 available 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 only 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. LibertyLink 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 and 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 tolerant 
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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 difficult 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 with 

FG72 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 overall weed control, it provides additional 

benefits such as improving the overall level and consistency 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 sustainable and socially acceptable weed 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 the 

same field in a two-year period. One method to accomplish this is to rotate herbicidetolerant trait systems. 
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5) Apply recommended rates of herbicides to actively 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 commercial 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 glyphosate 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 soybean 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 horizontal 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. 
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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 growers 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 crosses 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. 
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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 relevant phenotypic difference between Event FG72 

soybean and conventional soybean is the expression of the 2mEPSPS and HPPD W336 

proteins which provide tolerance to the application 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 IFT 

herbicide in combination with a glyphosate herbicide. Glyphosate is widely used in herbicidetolerant soybean and other agricultural production systems. IFT herbicide offers an alternative 

weed control option for the soybean grower via a new herbicide mode of action for soybeans 

that is efficacious against many of the herbicide resistant weeds currently found in soybean 

fields. 
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1 Roundup is a registered trademark of Monsanto.
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Appendix 1 

FIELD TRIAL TERMINATION REPORTS 2001-2008 
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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 established for breeding, efficacy testing and 

the generation of analytical data. 

General Field Observations

Experienced personnel qualified in soybean cultivation performed 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 counts 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 respective levels of herbicide tolerance. 

Insect pest species recorded from the Puerto Rico plot were bean leaf beetles (Coleoptera: Chrysomelidae), whitefly 

(Homoptera: Aleyrididae), and loopers (Lepidoptera: Noctuidae). Only bean leaf beetles 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 either site. Both sites listed the growing season as typical. 

Final Disposition and 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. 
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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. 
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Purpose of Release: The trials were established for the purpose evaluating efficacy, 

generating seed and providing analytical data. The performance of the 

herbicide-tolerant soybean with respect 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 District. 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 beneficials were noted among the plots, 

as was phytopathology. No difference 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. 
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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 was in the desired trait – tolerance to 

glyphosate herbicide – in the transgenic 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 inspected 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. 
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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 were 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. Additionally, 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.
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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 performance 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. Damage 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. 
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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 and 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-tolerant transgenic soybean plants as 

compared to nontransgenic soybean plants. 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 soybean 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. 
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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 & 12/3/03 
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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 

(1st 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 

(2nd 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 

(3rd 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 9/20/03
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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 

(1st and 2nd 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 

(3rd 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 

(1st 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 

(2nd 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 flowering, pod development and seed 

fill. The Champaign Co. site reported delayed emergence due to dry soil 

conditions. A killing frost occurred 10/2/03. 
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County or Observations/Dates 

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 

(1st and 2nd

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 

(3rd 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 

(1st 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 

(2nd and 3rd

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 nontransgenic plants in terms of the level of 

tolerance to glyphosate and isoxazole herbicide treatments. 
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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 inspected for volunteer soybean plants 

during the following growing season. The table below summarizes 

observations made and actions taken 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 None 
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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 

(1st and 2nd

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 

(3rd 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 

(1st 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 

(2nd & 3rd

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. 
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Weediness Characteristics: There was no evidence of change in characteristics that would enhance 

survival of the transgenic soybean plants as compared to the 

nontransgenic soybean plants. No difference 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 separated 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. 
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Appendix 2 

MATERIALS AND METHODS- PRODUCT CHARACTERIZATION 
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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 genomic copies equivalent) was mixed with Jack 

genomic DNA (negative control) and separated by electrophoresis on agarose gels. Phage 

Lambda (PstI or HindIII 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-custody 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 amount 

representing 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 performed under conditions allowing hybridization of the 

probe with target sequences. Phage Lambda DNA digested with PstI or HindIII was included as

a size standard. 
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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 

ExpandTM 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 [α32P]-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). 
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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 HPPD 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 proteins 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 amino 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, the 
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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 2mEPSPS 

protein. 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 the 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 soybean 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-enolpyruvylshikimate-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. 
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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-terminal 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 purification 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 second 
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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. 
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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-herbicide-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 herbicide 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. 
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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 showed 

that 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 ‘2mEPSPS 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. 
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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. 
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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 1b 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 expressed as the average of 10 data points from duplicate 

extracts of 5 samples at each fortification 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 fortified 
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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 units 

were 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

/ %

crude protein x

g analyte g analyte as crude protein  

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 analyte dry weight content g g  

A summary of the results is given in Table 33. 
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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 Proteinb

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

HPPD Range 0.46– 1.32 

W336 

Conventional 

Treated/B Mean ± SD 0.85 ± 0.20 

0.94 c

 0.00024 c

HPPD Range 0.41 – 1.31 

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 fresh weight protein content in grain samples from the 

10 sites. 

b

 The dry weight (dw) and % crude protein analyte amounts were calculated using the average 2mEPSPS value of 

samples from each plot. 

c The dry weight (dw) and % crude protein analyte amounts were determined using 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 expression 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 tissues 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 zygosity (homozygous) was confirmed by means of 

PCR. As control items, the HPPD W336 protein produced in bacteria (Batch no. LB020309) and 
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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 μg/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 buffer. 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. 
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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. 
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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. 
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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 representative 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 necessary 
Soybean Event FG72 Petition - Revised 

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All trials received similar agronomic treatments for the care and upkeep of the plots. Field 

studies utilized an experimental treatment regime which compared the transformed event FG72 

sprayed and unsprayed compared to the non-transformed 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 grown 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 the agronomic parameters observed eight determined 

qualitative measurement characteristics and 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 hectare 
Soybean Event FG72 Petition - Revised 

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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 

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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. Three 

additional 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 herbicide 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 transgenic 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, USA 
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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 first, 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. 
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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 B1 (Thiamin) BIDE AOAC 942.23, 953.17 and 957.17 

Vitamin B2 (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. 379 
Soybean Event FG72 Petition - Revised 

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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 982.30 
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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 

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Appendix 3 

CHARACTERIZATION OF EVENT FG72 SOYBEAN 
Soybean Event FG72 Petition - Revised 

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3.A. Verification of the insert 

Several aliquots of soybean event FG72 genomic DNA were digested with the restriction 

enzymes HincII, SacI, HindIII, BspHI, ApaI, StuI, NcoI, ScaI, EcoRI and Bsu36I. Wild type 

genomic DNA (variety Jack) digested with HindIII was used as negative control, and an aliquot 

was supplemented with HindIII 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 ApaI shows an additional fragment of 8570 bp (Table 

45). This fragment is also visible after hybridization with the 3’ nos probe, indicating that it 

comes from incomplete digestion of the ApaI restriction site between the internal 558 bp 

fragment and the 7900 bp 3’ integration fragment. Hybridization results of Bsu36I 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 presentation 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 → 4612 
Soybean Event 

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

lengths 

ApaI

EcoRI

SacI

EcoRI

StuI

ApaI

BspHI

EcoRI (2x)

NcoI

EcoRI

HincII

HincII

HindIII Bsu36I

HindIII

NcoI ScaI

HincII

ApaI

EcoRI

HincII

SacI

EcoRI

StuI

ApaI

BspHI

EcoRI (2x)

NcoI

EcoRI

HincII

HincII

HindIII Bsu36I

HindIII

NcoI ScaI

HincII

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’

hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’

3’histon At

3’histonAt

3’histon At probe (PT016)hppdPf W336 probe (PT063)3’nos probe (PT024) 5’tev + TPotpY probe (PT061)

Ph4a748 probe (PT059)Ph4a748B probe (PT062)

Intron1 h3At probe (PT060)2mepsps probe (PT015)T-DNA probe (PT058)

ApaI x x x xBspHI x xBsu36I x xEcoRI x x x x x x x xHincII x x x x x x xHindIII x x x xNcoI x x x x NcoI x x x xSacI x xScaI x xStuI x x

> 621 3010 713 2476 4091 713 2476 

> 685 7280

72807280

7280

7280

> 4021 947 6333 947 

> 1382 > 665 558 6722 558 

> 1903 > 2543 2929 4351 2929 

> 6389 > 675> 4989

528 915 551 5277 9 528 9 915 551
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Table 47 . Expected and obtained hybridization fragments 

PT016-1: 

3’histonAt 

PT024-2: 

3’nos 

PT063-1: 

hppdPfW336 

PT061-1: 

5’tev+TPotp Y 

PT059-1: 

Ph4a748 

PT062-1: 

Ph4a748B 

PT060-1

Intron1 h3

Digest Description Expected fragment 

sizes (bp) 

Obtained 

fragment 

sizes (bp) 

Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. O

5’ integration fr. > 621 5250 Yes Yes No No No No No No No No No No No N

internal fr. 3010 3010 No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Ninternal fr. 4091 4091 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No Ninternal fr. 713 713 No No No No No No No No Yes Yes No No No Ninternal fr. 2476 2476 No No No No No No Yes Yes Yes Yes Yes Yes Yes Y

3’ integration fr. > 1063 1130 Yes Yes No No No No No No No No No No No N

HincII 

3' junction 

translocation > 158 1300 No No No No No No No No Yes Yes Yes Yes No N

5’ integration fr. > 685 6060 Yes Yes No No No No No No No No No No No N

internal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

3’ integration fr. > 7198 >14 kb Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

SacI 

3' junction 

translocation > 158 >14 kb No No No No No No No No Yes Noa Yes Yes No N

5’ integration fr. > 4021 9550 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No N

internal fr. 6333 6333 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yinternal fr. 947 947 No No No No No No No No Yes Yes Yes Yes No N

3’ integration fr. > 2915 5500 Yes Yes No No No No Yes Yes Yes Noa No No Yes Y

HindIII 

3' junction 

translocation > 158 1480 No No No No No No No No Yes Yes Yes Yes No N

5’ integration fr. > 1382 3200 Yes Yes Yes Yes Yes Yes No No No No No No No N

internal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

3’ integration fr. > 6501 7480 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

BspHI 

3' junction 

translocation > 158 4260 No No No No No No No No Yes Yes Yes Yes No N

5’ integration fr. > 665 10760 Yes Yes No No No No No No No No No No No N

internal fr. 6722 6722 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

ApaI 

internal fr. 558 558 No No Yes Yes Yes Yes No No No No No No No N
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PT016-1: 

3’histonAt 

PT024-2: 

3’nos 

PT063-1: 

hppdPfW336 

PT061-1: 

5’tev+TPotp Y 

PT059-1: 

Ph4a748 

PT062-1: 

Ph4a748B 

PT060-1

Intron1 h3

Digest Description Expected fragment 

sizes (bp) 

Obtained 

fragment 

sizes (bp) 

Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. O

3’ integration fr. > 6660 7900 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

Partial fr. / 8570 No Yes No Yes No Yes No Yes No Yes No Yes No Y

3' junction 

translocation > 158 >14 kb No No No No No No No No Yes Noa Yes Yes, weak No N

5’ integration fr. > 1903 4710 Yes Yes Yes Yes Yes Yes No No No No No No No N

internal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

3’ integration fr. > 5980 6210 Yes Yes No No Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

StuI 

3' junction 

translocation > 158 >14 kb No No No No No No No No Yes Noa Yes Yes, weak No N

5’ integration fr. > 2543 7660 Yes Yes Yes Yes Yes Yes Yes Yes No No No No No N

internal fr. 2929 2929 No No No No No No Yes Yes Yes Yes Yes Yes Yes Yinternal fr. 4351 4351 Yes Yes Yes Yes Yes Yes Yes Yes No No No No Yes N

3’ integration fr. > 2411 10270 Yes Yes No No No No Yes Yes No No No No Yes N

NcoI 

3' junction 

translocation > 158 >14 kb No No No No No No No No Yes Yes, weak Yes Yes No N

5’ integration fr. > 6389 9900 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

internal fr. 7280 7280 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

3’ integration fr. > 1494 4730 Yes Yes No No No No No No No No No No No N

ScaI 

3' junction 

translocation > 158 11430 No No No No No No No No Yes Noa Yes Yes No N

5’ integration fr. > 675 5110d Yes Yes No No No No No No No No No No No N

internal fr. 5277 5277d Yes Yes No No No No Yes Yes Yes Yes Yes Yes Yes Yinternal fr. 528c 528c No No Yes Yes Yes Yes No No No No No No No Ninternal fr. 915 915 No No No No Yes Yes No No No No No No No Ninternal fr. 9b / No No No No No No No No No No No No No Ninternal fr. 551c 551c No No No No Yes Yes Yes Yes No No No No No N

3’ integration fr. > 5205 9610 Yes Yes No No No No Yes Yes Yes Yes Yes Yes Yes Y

EcoRI 

3' junction 

translocation > 158 4670 No No No No No No No No Yes Yes Yes Yes No N

Bsu36I 5’ integration fr. > 4989 7650e Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes No N
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PT016-1: 

3’histonAt 

PT024-2: 

3’nos 

PT063-1: 

hppdPfW336 

PT061-1: 

5’tev+TPotp Y 

PT059-1: 

Ph4a748 

PT062-1: 

Ph4a748B 

PT060-1

Intron1 h3

Digest Description Expected fragment 

sizes (bp) 

Obtained 

fragment 

sizes (bp) 

Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. O

internal fr. 7280 7280e Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Y

3’ integration fr. > 2894 10350 Yes Yes No No No No Yes Yes No No No No Yes Y

3' junction 

translocation > 158 7670e No No No No No No No No Yes Yes Yes Yes No N

Partial fr. / 11590 No No No No No No No No No Yes, weak No Yes, weak No NPartial fr. / >14 kb No No No No No No No No No Yes, weak No Yes, weak No N

WT 

genomic 

DNA-HindIII 

 / / / / / / / / / / / / / 

Positive control 3420 3420 No No Yes Yes Yes Yes Yes Yes, weak Yes Yes Yes Yes No NPositive control 947 947 No No No No No No No No Yes Yes Yes Yes No NPositive control 2961 2961 Yes Yes No No No No Yes Yes, weak Yes Noa No No Yes Y

WT 

genomic 

DNA - 

HindIII + 

pSF10 - 

HindIII 

Positive control 3070 / No No No No No No No No No No No No No N

a Not visualized due to the small overlap between the fragments and the T-DNA probe. b This fragment is too small to be visualized. c,d, e These 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 – HindIII digested Lane 10: Event FG72 – NcoI digested 

Lane 2: Phage Lambda – PstI digested Lane 11: Event FG72 – ScaI digested 

Lane 3: empty Lane 12: Event FG72 – EcoRI digested 

Lane 4: Event FG72 – HincII digested Lane 13: Event FG72 – Bsu36I digested 

Lane 5: Event FG72 – SacI digested Lane 14: WT variety Jack – HindIII digested 

Lane 6: Event FG72 – HindIII digested Lane 15: WT variety Jack – HindIII digested + half of an 

Lane 7: Event FG72 – BspHI digested equimolar amount of pSF10 – HindIII digested 

Lane 8: Event FG72 – ApaI digested Lane 16: Phage Lambda – PstI digested 

Lane 9: Event FG72 – StuI digested Lane 17: Phage Lambda – HindIII digested 
Soybean 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 different restriction enzymes and probed with the 3’nos 

probe (PT024-2: 214 bp MDB355-MDB055 fragment of pTDL004). 

Lane 1: Phage Lambda – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + an equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested

Lane 17: Phage Lambda – HindIII digested
Soybean 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 – HindIII digested 

 Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + half of an equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindII 
Soybean 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 – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + an equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindIII digested 
Soybean 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 – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + half of an equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindIII digested 
Soybean Event FG72 Petition - Revised 

Page 150 of 198 

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

Figure 17. Southern blot analysis of event 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 different restriction enzymes and probed with the 

Ph4a748B probe (PT062-1: 430 bp JDB018-JDB019 fragment of pTEM2). 

Lane 1: Phage Lambda – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + half of an equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindIII digested 
Soybean 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 – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + an 

equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindIII digested 
Soybean Event FG72 Petition 

Page 152 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

Figure 19. Southern blot analysis of event FG72 – 2mepsps 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 – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + pSF10 – 

HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindIII digested 
Soybean Event FG72 Petition 

Page 153 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

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 – HindIII digested 

Lane 2: Phage Lambda – PstI digested 

Lane 3: empty 

Lane 4: Event FG72 – HincII digested 

Lane 5: Event FG72 – SacI digested 

Lane 6: Event FG72 – HindIII digested 

Lane 7: Event FG72 – BspHI digested 

Lane 8: Event FG72 – ApaI digested 

Lane 9: Event FG72 – StuI digested 

Lane 10: Event FG72 – NcoI digested 

Lane 11: Event FG72 – ScaI digested 

Lane 12: Event FG72 – EcoRI digested 

Lane 13: Event FG72 – Bsu36I digested 

Lane 14: WT variety Jack – HindIII digested 

Lane 15: WT variety Jack – HindIII digested + an 

equimolar amount of pSF10 – HindIII digested 

Lane 16: Phage Lambda – PstI digested 

Lane 17: Phage Lambda – HindIII digested 
Soybean Event FG72 Petition 

Page 154 of 198

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

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 HindIII and HincII. The resulting 

DNA fragments were separated by agarose gel electrophoresis, transferred to a membrane 

and subjected to Southern blot analysis using 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 demonstration 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 number 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 hybridizing with vector backbone probes. 

After hybridization with the insert-DNA probe, the expected internal 947 bp HindIII 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 HindIII 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. This 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 using 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. 
So

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

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

Vector backbone T-DNA

pSF10

HincII

HindIII

HincII

HincII

HindIII

HincII

HindIII HincII

HincII

HindIII

ORi ColE1 bla ORI f1

PT058 PT056 PT057 PT056

x x 947 bp 2961 bp x 3420 bp x 3070 bp pSF10 - HindIII

FG72 - HincII x x x x x x x

FG72 - HindIII x x x x

> 621 bp 3010 bp 713 bp 2476 bp 4091 bp 713 bp 2476 bp >1063 bp

> 4021 bp 947 bp 947 bp 6333 bp >2915 bp

PT058

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBCPh4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBCPh4a748

Intron1 h3At

2mepsps 3’histonAt 3’histon At

3’histonAt

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBCPh4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBCPh4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBCPh4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBCPh4a748

Intron1 h3At

2mepsps 3’histonAt 3’histon At

3’histonAt

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt

FG72

SalI

SalI

HindIIIHindIII HindIII HindIII

HincII HincII HincII HincII HincII HincII HincII
So

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Table 49. Expected and obtained hybridization fragments 

Figure 1 Figure 2 PT056-1 PT058-6 PT057-1 PTVector backbone probe T-DNA probe Vector backbone probe T-

Sample 

Expected TDNA or plasmid fragment sizes 

Fragment description 

Exp. Obt. Exp. Obt. Exp. Obt. Ex

9550 bp c 5' integration fr. Yes d Yes Yes Yes Yes d Yes Y947 bp internal fragment Yes d Yes Yes Yes Yes d No Y6333 bp internal fragment Yes d Yes Yes Yes Yes d Yes Y5500 bp c 3' integration fr. No No Yes Yes No No Y

FG72 - HindIII 

1480 bp c 3' junction translocation No No Yes b No No No Y

5250 bp c 5' integration fr. Yes d No Yes b No No No Y3010 bp internal fragment Yes d Yes Yes Yes Yes d Yes Y713 bp internal fragment Yes d Yes Yes Yes, weak Yes d No Y2476 bp internal fragment No No Yes Yes No No Y4091 bp internal fragment Yes d Yes Yes Yes Yes d Yes Y1130 bp c 3' integration fr. No No Yes Yes No No Y

FG72 – HincII 

1300 bp c 3' junction translocation No No Yes b No No No Y

WT - HindIII / / / / / / / / / 

3420 bp positive control Yes d No Yes No a Yes b No Ye947 bp positive control Yes d No Yes No a Yes d No Ye2961 bp positive control Yes b No Yes No a No No Ye

WT - HindIII + 0.1 

equimolar amount pSF10 - HindIII 

3070 bp positive control Yes Yes Yes b No Yes Yes Y

3420 bp positive control Yes d No Yes Yes Yes b No Y947 bp positive control Yes d No Yes No a Yes d No Ye2961 bp positive control Yes b No Yes Yes No No Y

WT - HindIII + 1 

equimolar amount pSF10 - HindIII 

3070 bp positive control Yes Yes Yes b No Yes Yes Y

a These 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 b The overlap between the probe and the fragment can be too small to visualize this fragment. c Expected fragment sizes as determined in the detailed insert characterization study. d Since part of the sequence of both vector backbone probes is present in the T-DNA sequences, fragments originating from inserted transgenic sequences hybridiz
Soybean Event FG72 Petition 

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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, SacI/SmaI fragment of pSF10). 

Lane 1: λ-PstI digested (not shown) 

Lane 2: λ-HindIII digested (not shown) 

Lane 3: Event FG72 – HindIII digested 

Lane 4: Event FG72 – HincII digested 

Lane 5: WT variety Jack – HindIII digested 

Lane 6: WT variety Jack – HindIII digested + a 10-fold dilution of an equimolar amount of pSF10 – HindIII digested 

Lane 7: WT variety Jack – HindIII digested + an equimolar amount of pSF10 – HindIII digested 

Lane 8: λ-HindIII digested (not shown) 

Lane 9: λ-PstI digested (not shown) 
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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, SacI/SmaI fragment of pSF10). 

Lane 1: λ-PstI digested (not shown)

Lane 2: λ-HindIII digested (not shown)

Lane 3: Event FG72 – HindIII digested 

Lane 4: Event FG72 – HincII digested 

Lane 5: WT variety Jack – HindIII digested 

Lane 6: WT variety Jack – HindIII digested + a 10-fold dilution of an equimolar amount of pSF10 – HindIII

digested 

Lane 7: WT variety Jack – HindIII digested + an equimolar amount of pSF10 – HindIII digested 

Lane 8: λ-HindIII digested (not shown)

Lane 9: λ-PstI digested (not shown)
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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 three different genetic backgrounds (Table 48). The 

impact of different environments was assessed by analyzing the progeny of transgenic plants 

cultivated at 4 different field locations (Table 50). The isolated DNA was digested with the 

restriction enzyme HindIII, 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. 
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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 SacI/SmaI 3142 bp → 10345 bp 7204 bp 

JDB018 

Ph4a748B 

JDB019 6866 bp → 7295 bp 430 bp 
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Figure 24. Environment Adel 

Ph4a748B probe 

T-DNA probe 

A: event FG72 samples; B: Negative control; C: positive control 

A B C

A B C
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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 

A B C 

A
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Figure 26. Environment Sharpsville 

Ph4a748B probe 

T-DNA probe 

A: event FG72 samples; B: Negative control; C: positive control 

A B C

A B C
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Figure 27. Environment Fithian 

Ph4a748B probe

T-DNA probe 

A: event FG72 samples; B: Negative control; C: positive control 

A B C

A B C 
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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 

A B C

A B C 
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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 

A B C 

A B C 
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Figure 30. Generation T2 

Ph4a748B probe 

T-DNA probe 

A: event FG72 samples; B: Negative control; C: positive control 

A B C 

A B C 
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Figure 31. Generation T7 

Ph4a748B probe 

T-DNA probe 

A: event FG72 samples; B: Negative control; C: positive control 

A B C

A B C
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Figure 32. Schematic drawing of hybridization strategy for the demonstration of structural stability 

pSF10

HindIII HindIII

HindIII HindIII

ORi ColE1 bla ORI f1

x x 947 bp 2961 bp x 3420 bp x 3070 bp pSF10 - HindIII

FG72 - HindIII x x x x 9550 bp 947 bp 947 bp 6333 bp 5500 bp

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748

Ph4a748 ABBC

Intron1 h3At

2mepsps

3’histonAt hppdPf

W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748

Ph4a748 ABBC

Intron1 h3At

2mepsps 3’histo

3’histon At

3’histonAt

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt

FG72

SalI

SalI

T-DNA probe

Ph4a748B probe

T-DNA probe

Ph4a748B probe

HindIIIHindIII HindIII HindIII

pSF10

HindIII HindIII

HindIII HindIII

ORi ColE1 bla ORI f1

x x 947 bp 2961 bp x 3420 bp x 3070 bp pSF10 - HindIII

FG72 - HindIII x x x x 9550 bp 947 bp 947 bp 6333 bp 5500 bp

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748

Ph4a748 ABBC

Intron1 h3At

2mepsps

3’histonAt hppdPf

W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748

Ph4a748 ABBC

Intron1 h3At

2mepsps 3’histo

3’histon At

3’histonAt

hppdPfW336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt hppdPf W336 3’nos TPotp Y TPotp C

5’tev

Ph4a748 ABBC Ph4a748

Intron1 h3At

2mepsps 3’histonAt

FG72

SalI

SalI

T-DNA probe

Ph4a748B probe

T-DNA probe

Ph4a748B probe

HindIIIHindIII HindIII HindIII
Soybe

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Table 52. Expected and obtained hybridization fragments 

Phpr

Samples Condition tested Seed lot N° of plants

Expected T-DNA or plasmid fragment sizes 

Fragment description 

Ex

Location Adel, Iowa HT08SOY002-05-32 22 Location Osborn, Missouri HT08SOY002-07-32 22 Location Fithian, Illinois HT08SOY002-08-32 21 9550 bp d 5' integration fr. YeLocation Sharpsville, Indiana HT08SOY002-09-32 16 947 bp internal fragment Ye

Background 3068115-48 x Jack 7BD60008 21 6333 bp internal fragment YeBackground 3066617-48 x Jack 7BD60018 22 5500 bp d 3' integration fr. No 

Generation T2 FG72a-T2, FG72b-T2, FG72c-T2, FG72d,T2 22 1480 bp d 3' junction translocation Ye

FG72 - HindIII 

Generation T7 FG72-x-x-14-5-1-6-x T7 13 

Non transgenic Jack - HindIII 

Non-transgenic variety Jack / Negative control / 

3420 bp positive control Ye947 bp positive control Ye2961 bp positive control No 

Non transgenic Jack - HindIII + pSF10 - HindIII

Non-transgenic variety Jack + equimolar amount pSF10 

3070 bp positive control No 

a In some hybridizations this fragment is very weak, but present for all samples b The overlap between the probe and the fragment can be too small to visualize this fragment c Not always visible after hybridization with T-DNA probe but presence is confirmed after hybridization with probe Ph4a748B. d Expected fragment sizes as determined in the detailed insert characterization study 
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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 microbially-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 Protein Stain. Lane 2 contains approximately 300 ng of 

2mEPSPS protein produced in E. coli. Lane 3 contains the non-transgenic Jack soybean leaves control sample. 

Lane 4 contains approximately 430 ng 2mEPSPS protein isolated from FG72 soybean leaves. 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 from FG72 soybean leaves 

Lane 5. BenchMark Protein Ladder 

12 3 4

kD

220- 

160- 

70- 

60- 

100- 

50- 

40- 

30- 
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Figure 34. Standard curve of electrophoretic mobility versus molecular weight 

y = -1.2374x + 86.777

R2

 = 0.9834

1

10

100

1000

1 10 100

mobility (mm)

Molecular weight (kDa)

The electrophoretic mobility of the protein standards for the SDS-PAGE gel shown in Figure 3.23 were plotted against 

their respective molecular weights bracketing the 2mEPSPS protein. The equation defining the curve is y = -1.2374x 

+ 86.777. The R2 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. 
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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 2mEPSPS 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 protein. 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 methionine 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-translational 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- 
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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

1 to 20 MAGAEEIVLQPIKEISGTV ND

K

705.4 [M+3H] ND

ND

406 to 429 MAMAFSLAACAEVPVTIRDPGC

TR (Cys_CAM mod) 875.9 [M+3H] 875.9 [M+3H] 876.3 [M+3H]

330 to 357 MPDVAMTLAVVALFADGPTAIR

DVASWR 992.5 [M+3H] ND

174 to 233 ND

LSGSISSQYLSALLMAAPLALGD

VEIEIIDKLISIPYVEMTLRLMERF

GVKAEHSDSWDR

1114.5 [M+6H] ND

ND

445 to 445 N ND** ND** ND

431 to 444 TFPDYFDVLSTFVK 841 [M+2H] 840.9 [M+2H]

ND

430 to 430 K ND** ND** ND

369 to 373 TELT

K

592.4 [M+H] 592.3 [M+H]

ND

364 to 368 MVAIR 590.4 [M+H] 590.3 [M+H] ND

360 to 363 ETER 535.3 [M+H] 534.8 [M+H]

ND

358 to 359 VK 246.8 [M+H] 247.2 [M+H] 246.2 [M+H]

352 to 357 DVASWR 733.9 [M+H] 734.3 [M+H]

ND

330 to 351 MPDVAMTLAVVALFADGPTAIR ND** ND ND

322 to 329 AIDVNMNK 453.6 [M+2H] 453.4 [M+2H]

ND

319 to 321 HLK 398.3 [M+H] 398.2 [M+H] ND

318 to 318 K ND ND

ND

313 to 317 EPFGR ND ND ND

298 to 312 VTWTETSVTVTGPPR 817 [M+2H] 816.9 [M+2H]

1310 [M+3H]

287 to 297 FAEVLEMMGAK 614.6 [M+2H] 614.3 [M+2H] ND

247 to 286 NAYVEGDASSASYFLAGAAITG

GTVTVEGCGTTSLQGDVK 1309.8 [M+3H] 1309.8 [M+3H]

ND

244 to 246 SPK 332.3 [M+H] 332.2 [M+H] ND

242 to243 YK 310.8 [M+H] 310.7 [M+H]

ND

238 to 241 GGQK 390.3 [M+H] 390.1 [M+H] ND

234 to 237 FYIK 286.2 [M+2H] 286.3 [M+2H]

ND

225 to 233 AEHSDSWDR ND** ND** ND

221 to 224 FGVK ND** ND**

ND

217 to 220 LMER ND** ND ND

205 to 216 LISIPYVEMTL

R

ND** ND

ND

174 to 204 LSGSISSQYLSALLMAAPLALGD

VEIEIIDK ND** ND ND

172 to 173 VK 246.8 [M+H] 247.2 [M+H]

ND

161 to 171 VNGIGGLPGGK 485.6 [M+2H] 485.5 [M+2H] ND

143 to 160 QLGADVDCFLGTDCPPVR 1011.7 [M+2H] 1011.7 [M+2H]

ND

131 to 142 ERPIGDLVVGLK 649.2 [M+2H] 649.4 [M+2H] ND

129 to 130 MR 307.2 [M+H] 307.1 [M+H]

ND

107 to 128 SLTAAVTAAGGNATYVLDGVPR 702.9 [M+3H] 702.8 [M+3H] 702.7 [M+3H]

92 to 106 EEVQLFLGNAGIAMR ND** ND**

ND

85 to 91 FPVEDAK ND** ND** ND

76 to 84 AVVVGCGG

K

424.5 [M+2H] 424.5 [M+2H]

289.9[M+H]

75 to 75 R ND** ND ND

72 to 74 AAK 289.5 [M+H] 289.7 [M+H]

ND

62 to 71 TLGLSVEAD

K

517.6 [M+2H] 517.5 [M+2H] ND

31 to 61 ILLLAALSEGTTVVDNLLNSEDV

HYMLGALR 1115.3 [M+3H] ND

ND

26 to 30 SLSNR 577.4 [M+H] 577.5 [M+H] ND

21 to 25 LPGSK 502.4 [M+H] 502.3 [M+H]

ND

14 to 20 EISGTVK 734.4 [M+H] 734.4 [M+H] ND

2 to 13 AGAEEIVLQPI

K

635.4 [M+2H] 635.3 [M+2H]

71% cov. FG72 

Soybean Leaf* Non-transgenic Jack 

Soybean Leaf*

ND ND

98% cov. 

E. coli 2mEPSPS 

protein*

1 to 13 MAGAEEIVLQPIK 700.9 [M+2H]

2mEPSPS Residue Sequence Theoretical 

[M+H]

ND

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 DPGCTR ND** ND** ND

406 to 423 MAMAFSLAACAEVPVTIR ND** ND**

85 to 106 FPVEDAKEEVQLFLGNAGIAMR 812.4 [M+3H] 812.5 [M+3H] ND
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Table 54. Amino acid coverage of the 2mEPSPS protein 

Calculation of % Number of Amino Acids Not Detected

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

NAa  

a

 NA = Not Applicable 
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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 proteins. The plant-produced HPPD W336 protein had other bands 

due to non-specific binding of proteins from the soybean matrix as shown in the SDS-PAGE 

(Figure 36). These non-specifically bound matrix proteins 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 

-10 
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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 soybean produced HPPD W336 protein by full scan 

analysis obtained 70.1% sequence coverage 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 

-20 
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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

PSVCGMAFR (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 

Soybean 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]

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

Calculation of % Number of Amino Acids Not Detected 

Amino Acid overage E. coli HPPD W336 Plant-produced HPPD W336 

Residue Number

1 1 1-38 

2b 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 

NAa

a

 NA = Not Applicable. bThe 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. 
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Appendix 4 

RAW AGRONOMIC DATA FOR 2008 
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Table 57. Raw agronomic data for 2008 

Trial 

Number Description

Early 

SC

Plant 

Vigor

Days to 

flowering

Flower 

Color

Leaf 

Shape

PH 

V4-5 PH R1 PH M Pb Pd Hilum Canopy

Days to 

Maturity

Yield 

bu/acre Lodgin

01 Jack 109 8 51 W Oval 5 5 5 G B Y Medium 138 50.17 5 01 Jack 100 8 52 W Oval 5 5 5 G B Y Medium 136 50.31 5 01 Jack 96 8 52 W Oval 5 5 5 G B Y Medium 138 52.78 5 02 Jack 110 8 52 W Oval 5 5 5 G B Y Medium 134 64.40 4 02 Jack 112 8 52 W Oval 5 5 5 G B Y Medium 135 67.52 4 02 Jack 117 8 52 W Oval 5 5 5 G B Y Medium 136 69.62 5 03 Jack 113 8 45 W Oval 5 5 5 G B Y Medium 131 71.87 2 03 Jack 106 8 45 W Oval 5 5 5 G B Y Medium 131 59.75 2 03 Jack 115 8 45 W Oval 5 5 5 G B Y Medium 131 67.23 2 04 Jack 150 8 45 W Oval 5 5 5 G B Y Medium 128 57.43 3 04 Jack 147 9 45 W Oval 5 5 5 G B Y Medium 128 47.77 3 04 Jack 146 9 45 W Oval 5 5 5 G B Y Medium 129 50.38 2 05 Jack 100 8 54 W Oval 5 5 5 G B Y Medium 136 60.84 3 05 Jack 98 8 54 W Oval 5 5 5 G B Y Medium 136 56.70 4 05 Jack 114 8 54 W Oval 5 5 5 G B Y Medium 137 56.27 4 06 Jack 90 8 48 W Oval 5 5 5 G B Y Medium 128 44.36 4 06 Jack 81 8 50 W Oval 5 5 5 G B Y Medium 129 40.51 4 06 Jack 95 8 48 W Oval 5 5 5 G B Y Medium 130 51.98 4 07 Jack 90 8 40 W Oval 5 5 5 G B Y Medium 119 44.21 6 07 Jack 106 9 40 W Oval 5 5 5 G B Y Medium 120 51.69 5 07 Jack 89 8 40 W Oval 5 5 5 G B Y Medium 119 48.64 5 08 Jack 123 8 36 W Oval 5 5 5 G T Y Medium Bushy 118 58.37 4 08 Jack 135 7 37 W Oval 4 5 5 G T Y Medium Bushy 118 53.29 4 08 Jack 129 9 36 W Oval 4 5 5 G T Y Medium Bushy 118 45.88 3 09 Jack 52 6 46 W Oval 3 4 5 G T Y Medium Bushy 118 39.78 9 09 Jack 101 8 46 W Oval 5 4 5 G T Y Medium Bushy 118 8 09 Jack 127 8 46 W Oval 5 5 5 G T Y Medium Bushy 119 39.78 8 10 Jack 118 8 46 W Oval 4 5 5 G B Y Medium 124 46.54 5 10 Jack 139 9 46 W Oval 4 5 5 G B Y Medium 124 45.81 5 10 Jack 126 8 46 W Oval 4 5 5 G B Y Medium 124 48.35 5 

Mean 111.1 8.1 46.47 4.8 4.9 5 127.7 53.18 4.4 

SD 21.4 0.6 5.28 0.5 0.3 0 7.2 8.96 1.7 
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Trial 

Number Description

Early 

SC

Plant 

Vigor

Days to 

flowering

Flower 

Color

Leaf 

Shape

PH 

V4-5 PH R1 PH M Pb Pd Hilum Canopy

Days to 

Maturity

Yield 

bu/acre Lodging 

01 FG72 unsprayed 99 8 52 W Oval 5 5 5 G B Y Medium 137 42.11 4 01 FG72 unsprayed 91 8 53 W Oval 5 5 5 G B Y Medium 137 45.59 5 01 FG72 unsprayed 97 8 53 W Oval 5 5 5 G B Y Medium 136 46.61 5 02 FG72 unsprayed 89 8 52 W Oval 5 5 5 G B Y Medium 133 55.10 5 02 FG72 unsprayed 86 8 53 W Oval 5 5 5 G B Y Medium 134 59.97 4 02 FG72 unsprayed 79 8 52 W Oval 5 5 5 G B Y Medium 134 59.82 4 03 FG72 unsprayed 95 8 45 W Oval 5 3 5 G B Y Medium 131 59.90 4 03 FG72 unsprayed 101 8 45 W Oval 5 5 5 G B Y Medium 131 58.01 4 03 FG72 unsprayed 92 8 45 W Oval 5 5 5 G B Y Medium 130 60.91 4 04 FG72 unsprayed 94 8 46 W Oval 5 3 5 G B Y Medium 128 44.14 4 04 FG72 unsprayed 88 8 45 W Oval 5 5 5 G B Y Medium 127 55.76 4 04 FG72 unsprayed 95 8 45 W Oval 5 5 5 G B Y Medium 128 48.86 4 05 FG72 unsprayed 95 8 54 W Oval 5 5 5 G B Y Medium 137 51.33 5 05 FG72 unsprayed 87 8 54 W Oval 5 5 5 G B Y Medium 138 63.23 4 05 FG72 unsprayed 109 8 54 W Oval 5 5 5 G B Y Medium 138 70.13 3 07 FG72 unsprayed 23 7 40 W Oval 3 3 5 G B Y Medium 120 31.07 6 07 FG72 unsprayed 26 7 41 W Oval 3 3 5 G B Y Medium 120 33.32 6 07 FG72 unsprayed 39 7 42 W Oval 3 3 5 G B Y Medium 120 34.05 6 06 FG72 unsprayed 70 8 50 W Oval 3 5 5 G B Y Medium 131 43.49 6 06 FG72 unsprayed 87 8 48 W Oval 5 5 5 G B Y Medium 129 49.88 5 06 FG72 unsprayed 79 8 49 W Oval 5 5 5 G B Y Medium 129 48.06 5 08 FG72 unsprayed 102 8 37 W Oval 5 5 5 G T Y Medium Bushy 118 40.66 4 08 FG72 unsprayed 95 9 37 W Oval 5 4 5 G T Y Medium Bushy 119 36.66 5 08 FG72 unsprayed 103 7 37 W Oval 5 5 5 G T Y Medium Bushy 118 39.35 4 09 FG72 unsprayed 89 7 46 W Oval 4 4 5 G T Y Medium Bushy 118 41.09 9 09 FG72 unsprayed 89 7 46 W Oval 4 4 5 G T Y Medium Bushy 118 35.14 9 09 FG72 unsprayed 76 9 46 W Oval 5 5 5 G T Y Medium Bushy 119 27.15 9 10 FG72 unsprayed 70 8 45 W Oval 4 5 5 G B Y Medium 124 36.66 6 10 FG72 unsprayed 52 7 45 W Oval 4 5 5 G B Y Medium 124 37.75 5 10 FG72 unsprayed 67 8 45 W Oval 4 5 5 G B Y Medium 124 36.45 4 

Mean 82.1 7.8 46.7 4.6 4.6 5.0 127.7 46.4 5.1 

SD 21.7 0.5 5.2 0.7 0.8 0.0 7.0 11.0 1.6 
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Trial 

Number Description

Early 

SC

Plant 

Vigor

Days to 

flowering

Flower 

Color

Leaf 

Shape

PH 

V4-5 PH R1 PH M Pb Pd Hilum Canopy

Days to 

Maturity

Yield 

bu/acre Lodging 

01 FG72 sprayed 79 8 53 W Oval 3 3 5 G B Y Medium 137 43.05 6 01 FG72 sprayed 99 8 54 W Oval 3 3 5 G B Y Medium 138 45.96 6 01 FG72 sprayed 95 8 53 W Oval 3 3 5 G B Y Medium 138 48.28 6 02 FG72 sprayed 109 8 54 W Oval 3 3 5 G B Y Medium 134 57.50 6 02 FG72 Sprayed 82 8 54 W Oval 3 3 5 G B Y Medium 137 49.95 6 02 FG72 sprayed 72 8 58 W Oval 3 3 5 G B Y Medium 137 51.40 7 03 FG72 sprayed 85 8 45 W Oval 3 3 5 G B Y Medium 133 52.49 6 03 FG72 sprayed 78 8 46 W Oval 3 3 5 G B Y Medium 133 56.70 6 03 FG72 sprayed 84 8 45 W Oval 3 3 5 G B Y Medium 133 60.19 6 04 FG72 sprayed 89 8 45 W Oval 4 3 5 G B Y Medium 127 49.08 6 04 FG72 sprayed 69 8 46 W Oval 3 3 5 G B Y Medium 129 43.12 5 04 FG72 sprayed 74 8 47 W Oval 3 3 5 G B Y Medium 130 44.94 6 05 FG72 sprayed 89 8 54 W Oval 4 5 5 G B Y Medium 138 58.30 5 05 FG72 sprayed 85 8 54 W Oval 4 5 5 G B Y Medium 137 62.80 6 05 FG72 sprayed 99 8 54 W Oval 4 5 5 G B Y Medium 137 63.31 4 06 FG72 sprayed 89 8 49 W Oval 5 3 5 G B Y Medium 130 44.58 5 06 FG72 sprayed 91 8 49 W Oval 5 3 5 G B Y Medium 130 50.75 6 06 FG72 sprayed 92 8 49 W Oval 5 3 5 G B Y Medium 131 42.40 6 07 FG72 sprayed 33 7 41 W Oval 3 3 5 G B Y Medium 121 29.04 6 07 FG72 sprayed 39 8 42 W Oval 3 3 5 G B Y Medium 121 34.78 6 07 FG72 sprayed 40 7 42 W Oval 3 3 5 G B Y Medium 121 31.00 6 08 FG72 sprayed 127 7 37 W Oval 4 4 5 G T Y Medium Bushy 120 43.34 5 08 FG72 sprayed 106 6 37 W Oval 4 4 5 G T Y Medium Bushy 120 55.54 5 08 FG72 sprayed 95 7 37 W Oval 4 4 5 G T Y Medium Bushy 119 36.30 5 09 FG72 sprayed 80 8 46 W Oval 5 5 5 G T Y Medium Bushy 119 9 09 FG72 sprayed 72 7 46 W Oval 4 4 5 G T Y Medium Bushy 119 28.10 9 09 FG72 sprayed 93 8 46 W Oval 4 4 5 G T Y Medium Bushy 119 33.83 9 10 FG72 sprayed 68 8 46 W Oval 4 5 5 G B Y Medium 125 32.89 6 10 FG72 sprayed 54 8 46 W Oval 4 3 5 G B Y Medium 125 27.23 6 10 FG72 sprayed 68 8 46 W Oval 4 5 5 G B Y Medium 125 35.21 7 

Mean 81.2 7.8 47.4 3.7 3.6 5.0 128.8 45.2 6.1 

SD 20.6 0.5 5.6 0.7 0.8 0.0 7.1 10.8 1.2 
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Trial 

Number Description

Early 

SC

Plant 

Vigor

Days to 

flowering

Flower 

Color

Leaf 

Shape

PH 

V4-5 PH R1 PH M Pb Pd Hilum Canopy

Days to 

Maturity

Yield 

bu/acre Lodging 

01 2686-6 123 8 57 P Ovate 5 5 5 G B Y Medium Bushy 135 58.59 6 01 2788 139 8 55 P Ovate 5 5 5 T B Bl Medium Bushy 136 66.43 6 01 3000-0 127 8 59 P Ovate 5 5 5 t T Br Medium Bushy 138 73.62 6 02 2686-6 135 8 53 P Ovate 5 5 5 G B Y Medium Bushy 129 53.51 7 02 2788 109 8 54 P Ovate 5 5 5 T B Bl Medium Bushy 135 71.00 7 02 3000-0 100 8 57 P Ovate 5 5 5 t T Br Medium Bushy 139 78.84 7 03 2686-6 132 8 47 P Ovate 5 5 5 G B Y Medium Bushy 127 57.79 7 03 2788 129 8 47 P Ovate 5 5 5 T B Bl Medium Bushy 128 66.07 7 03 3000-0 106 8 50 P Ovate 5 5 5 t T Br Medium Bushy 131 67.81 7 04 2686-6 100 8 47 P Ovate 5 5 5 G B Y Medium Bushy 129 65.12 8 04 2788 121 9 46 P Ovate 5 5 5 T B Bl Medium Bushy 128 66.21 5 04 3000-0 118 8 48 P Ovate 5 5 5 t T Br Medium Bushy 129 65.63 6 05 2686-6 137 8 55 P Ovate 5 5 5 G B Y Medium Bushy 134 71.00 7 05 2788 101 8 55 P Ovate 5 5 5 T B Bl Medium Bushy 134 75.14 6 05 3000-0 103 8 57 P Ovate 5 5 5 t T Br Medium Bushy 134 79.93 6 06 2686-6 115 8 50 P Ovate 5 5 5 G B Y Medium Bushy 128 56.63 8 06 2788 124 8 51 P Ovate 5 5 5 T B Bl Medium Bushy 130 56.85 7 06 3000-0 116 8 53 P Ovate 5 5 5 t T Br Medium Bushy 131 71.15 6 07 2686-6 93 8 40 P Ovate 5 5 5 G B Y Medium Bushy 116 48.93 8 07 2788 54 8 40 P Ovate 5 3 5 T B Bl Medium Bushy 118 51.11 8 07 3000-0 65 8 42 P Ovate 5 3 5 t T Br Medium Bushy 120 63.89 7 08 2686-6 134 7 36 P Ovate 5 5 5 G T Y Medium Bushy 118 49.01 8 08 2788 133 9 37 P Ovate 5 5 5 T B Bl Bushy 120 34.85 6 08 3000-0 123 6 44 P Ovate 5 4 5 T B Br Bushy 120 58.08 7 09 2686-6 128 9 46 W Ovate 5 5 5 G T Y Medium Bushy 119 47.55 9 09 2788 133 9 46 W Ovate 5 5 5 T B Bl Bushy 119 58.23 8 10 3000-0 120 8 46 W Ovate 4 5 5 T B Br Bushy 119 48.35 8 10 2686-6 124 9 45 P Ovate 4 5 5 G B Y Medium Bushy 123 53.72 8 10 2788 65 8 46 P Ovate 4 5 5 T B Bl Medium Bushy 125 41.75 8 10 3000-0 84 8 47 P Ovate 4 5 5 t T Br Medium Bushy 127 54.16 7 

Mean 113.0 8.1 48.5 4.9 4.8 5.0 127.3 60.4 7.0 

SD 22.4 0.6 6.1 0.3 0.5 0.0 6.7 11.0 0.9 

Legends

Early SC = Early Stand Count; PH V4-5 = Plant Health Rating Stage V4-5; PH R1 = Plant Health Rating Stage R1; PH M = Plant Health Rat

Pb = Pubescence color; Pd = Pod color; Final SC = Final Stand Count
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Appendix 5 

HERBICIDE RESISTANCE AND STEWARDSHIP 
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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 targeting and inhibiting specific plant biochemical 

processes or pathways. The process of specific activity is termed “mode of action” (MOA). 

Herbicides are classified into groups 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 levels including combining smaller farms to form 

larger farms. The resulting economic pressures have 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 cultural control practices has resulted in the use 

of herbicides as the only weed control tactic. With this decline in use of alternative weed control 

methods, extensive use of herbicides with a single MOA has not only resulted in weed shifts but 

also high selection pressure for herbicide resistant weeds. Plants have the ability to adapt to 

ensure survival, which includes adapting to survive an herbicide application. The development 

of herbicide resistance is a function of time and exposure and also the genetic capability of the 

weed population present in a field. 

Herbicide resistance is the naturally-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 herbicides inhibit the plant enzyme acetolactate 

synthase (ALS) and provide effective control 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 control. 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 scientists to be resistant to ALS inhibiting 

herbicides, resulting in the conclusion that ALS inhibiting herbicides are 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 other herbicide modes of 

action as evident in Figure 38. 
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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 tools 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 are 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, minimizing the importance of 

weed identification to a grower. However, identification 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 areas like difficult-tocontrol weeds or dense weed populations 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 action 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. 
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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 factors in an IWM program. Crop rotation adds weed 

management diversity through the inherent use of herbicides with different modes of action. In 

addition, crops vary in their ability to compete for sunlight, water and nutrients with weeds. 

Different planting times and seedbed preparation techniques can lead to a variety of cultural 

methods which employ diversity in a weed management 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 reduced by early season weed competition. Proper tillage or the use 

of a burndown herbicide program should be used to control 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 should be a part of every spray program. A soil-applied herbicide 

provides residual weed control allowing the crop to get a head start. Residual herbicides 

minimize the weed pressure and allow a wider post-emergent herbicide application window. 

Generally, soil-applied herbicides can be included in the burndown herbicide program for 

residual weed control on no-till acres. A residual 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 the 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 reproduce 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 preserve 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 additional modes of actions to be used in a 

weed management program. 
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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 chemical 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 technique will maximize weed control (Boerboom et al.,

2006). 

Apply to Actively Growing Weeds 

Herbicides provide peak performance when applied to actively growing weeds. Weeds 

that are actively growing absorb more herbicide. Conditions that provide peak growing 

environment for weeds are adequate soil moisture, sunlight and optimal soil nutrients. 

Timing 

The use of pre-emergent residual herbicides will provide key control of early season 

weeds that result in the greatest crop yield reduction and open a wider application 

window for post-emergence applications. 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 smaller 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 technique is achieved. Factors affecting weed 

control include: spray coverage, carrier 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 applications should be controlled to reduce weed 

seed production. A grower should consider spot herbicide applications, row wicking, 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 equipment that has not been thoroughly cleaned. 
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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, simazines, 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 alternative herbicides. Giant ragweed can grow up to 17 feet tall and 

produces allergenic pollen. One giant ragweed 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 waterhemp 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 glyphosate 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 incorporated 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 use. The seedling growth inhibitors 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 efficacy. Products that contain chloroacetamide 

herbicides and control waterhemp in soybean 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 crop damage in certain environmental conditions. 
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Post-applied herbicides

The only conventional herbicide mode of action that provides “good” control of waterhemp postemergence as rated by University Weed Scientists are the PPO inhibitors. These herbicides 

inhibit the protoporphyrinogen oxidase (PPO) enzyme which is involved in the heme-pigment 

synthesis pathway. Products that contain PPO inhibitors, such as lactofen (Cobra®, PhoenixTM), 

fomesafen (Flexstar®, Reflex®) and s-metolachlor + fomesafen (PrefixTM) 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 timely 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 multiple 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 amino acids in the shikimic acid 

pathway by inhibiting the enzyme 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 soybean, 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 extremely 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-hydroxylphenyl pyruvate deoxygenase (HPPD) in 

plants resulting in inhibition of carotenoid biosynthesis which gives rise to a bleached 

appearance of new growth (Vencill, 2002). Isoxaflutole 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 active ingredient IFT include Balance® Flexx (IFT + 

cyprosulfamide), CorvusTM (IFT + thiencarbazone + cyprosulfamide) and RadiusTM (IFT + 

flufenacet). There are two IFT containing products available for use on field corn in Canada: 

Converge® Pro (IFT) and Converge® XT (IFT + cyprosulfamide). 
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To date, there is no documented resistance of any weed species to isoxaflutole or any other 

HPPD inhibitor in the world (Heap, 2009). The herbicidal mode of action of IFT, class F (4-hppd 

inhibitors), is unique to soybean production systems. Soil applied herbicides, like IFT, increase 

the consistency of weed control, reduce early season weed competition which results in 

significant yield losses, and introduces a new mode of action for decreased selection pressure 

for glyphosate resistance (Nordby et al., 2007). 

IFT herbicide can be applied either pre-emergence to the soybean or post emergence in a 

tankmix with a post-emergence herbicide. 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 amaranth, 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 sustainability of its technology and 

has adopted a life-cycle approach to product stewardship. This means that appropriate 

stewardship principles are applied at every stage of biotechnology development 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 corporate 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 stewardship of its products and expects those with 

whom it contracts to handle material containing 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 area teams are committed to BCS stewardship 

principles and are aware of procedures to communicate appropriate information within the BCS 

crop team matrix to rapidly respond to issues that may develop from use of our technologies. 

Field development and market support teams are provided the tools necessary to serve the 

grower as a local and direct contact for any questions related to BCS technologies with regards 

to product performance or impacts on human and environmental health and safety. 
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BCS participates in several industry and professional initiatives in support of stewardship: 

 Herbicide Resistance Action Committee (HRAC)

HRAC is an industry initiative which fosters co-operation between plant protection 

manufacturers, government, researchers, advisors 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 are 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 Industry Organization, serving on committees and 

working groups that develop industry-wide approaches to regulatory and technology 

management issues. 

 Excellence Through Stewardship 

BCS is active in Excellence Through Stewardship, serving on the board of directors, 

committees and working groups that develop industry best practices for stewardship. 

 Weed Science professional societies 

BCS is active participant in a number of organizations. We maintain active memberships 

in the Weed Science Society of America, North Central Weed Science Society, 

Northeastern Weed Science Society, Southern Weed Science Society, and Western 

Weed Science Society, all of which are professional, non-profit societies, established to 

promote research, education, and extension outreach 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 stewardship of all of our products, including herbicidetolerant trait (HTT) technology. We strive to provide best management practices of HTT 

technology which includes integrated weed management to our customers (see section 5.B.). 

Education of integrated weed management is the 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 viability 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, trade shows, and web and mail communications. 
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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 signing 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 website for the best management practices and 

product information. In addition, a toll free hotline for growers to obtain live technical product 

support will be provided. BCS and MSTech are committed to stewardship principles and 

procedures, and to communicating appropriate information in order to rapidly respond to any 

issues that may develop. 

Growers may also contact the seed company for product support. The seed company name 

and contact information will be provided on the label of each bag of seed sold. Each grower 

purchase of FG72 Soybeans will be recorded by seed company partners. This information will 

be provided to MSTech which 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 obtain product information. The product label is 

the formal legal method of communicating directions for use of an herbicide. BCS’s history of 

including recommendations on product 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 first herbicide applied in an integrated weed 

control program that includes sequential post-emergence herbicide applications. 

CORVUSTM 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 LAUDISTM Herbicide, or 

IGNITE® 280 SL Herbicide or glyphosate in transgenic field corn. 

BCS is committed to supporting research by university institutions to generate local grower 

recommendations. University Extension 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 product 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 which resistance is suspected. The survival of the 

weeds confirms resistance. 

BCS invests a significant amount of resources 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 will 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. 
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5.E.3. Monitoring of effectiveness of the stewardship plan 

Each grower purchase of event FG72 soybeans will be recorded by the individual seed 

company making the sale. This information will be provided to MSTech which will enable 

MSTech to maintain a database of all growers utilizing event FG72 products. BCS regularly 

utilizes market research surveys to determine 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 effectiveness. 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 information to growers. 
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Appendix 6 

REFERENCES – APPENDICES 1-5 
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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 Technology Use Manual. 

Boerboom, C. and Owen, M. 2006. The Glyphosate, Weeds, and Crop Series: Facts About 

Glyphosate-Resistant Weeds. www.glyphosateweedscrops.org. 

Bradshaw, R.A., Brickey, W.W. and Walker, K. W. 1998. N-terminal processing: the methionine 

amino peptidase and N-acetyl transferase families. Trends Biochem. Sci. 23: 263-267. 

Bruce, J.A. and Kells, J.J. 1990. Horseweed (Conyza canadensis) control in no-tillage 

soybeans (Glycine max) with preplant and preemergence herbicides. Weed 

Technology. 4:642-647. 

Devore, J. and Peck, R. 1986. Statistics. West Publishing Company, St.Paul. 

Heap, I. 2009. The International Survey of Herbicide Resistant Weeds. Online. 

Internet. September 15, 2009. Available www.weedscience.com 

HRAC, 2009. Herbicide Resistance Action Committee www.hracglobal.com

Johnson, B., Loux, M., Nordby, D., Sprague, C., Nice, G., Westhoven, A., and Stachler, J. 

2007. The Glyphosate, Weeds, and Crops Series: Biology and Management of Giant 

Ragweed. www.glyphosateweedscrops.org. 

Loux, M., Stachler, J., Johnson, B., Nice, G., Davis, V., and Nordby, D. 2006. The Glyphosate, 

Weeds, and Crops Series: Biology and Management of Horseweed. 

www.glyphosateweedscrops.org. 

Nordby, D., Hartzler, B. and Bradley, K. 2007. The Glyphosate, Weeds, and Crop Series: 

Biology and Management of Waterhemp. www.glyphosateweedscrops.org. 

Priestman, M.A., Funke, T., Singh, I.M., Crupper, S.S., and Schoenbrunn, E. 2005. 5-

Enolpyruvylshikimate-3-phosphate synthase from Staphylococcus aureus is insensitive 

to glyphosate. FEBS 579:728-732. 

Roundup Weathermax® Herbicide product label. Monsanto. 2006. 

Sambrook, J., Fritsch, E.F., and Maniatis, T. 1989. Molecular cloning, a laboratory manual. 

Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY. 
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USDA. 2007. Agricultural Chemical Usage 2006 Field Crops Summary.

http://usda.mannlib.cornell.edu/usda/nass/AgriChemUsFC//2000s/2007/AgriChemUsF

C-05-16-2007_revision.pdf

USDA. 2008. Agricultural Chemical Usage 2007 Field Crops Summary.

http://usda.mannlib.cornell.edu/usda/current/AgriChemUsFC/AgriChemUsFC-05-21-

2008.pdf

Vencill, W.K., ed. 2002. Herbicide Handbook: Eighth Edition. Weed Science Society of 

America, Lawrence KS. pg. 231-234, 267-268. 

Weed Science Society of America (WSSA). 2009. www.wssa.net

Whaley C.M., H.P. Wilson, and Westwood, J.H. 2007. A New Mutation in Plant Als Confers 

Resistance to Five Classes of Als-inhibiting Herbicides. Weed Science 55(2):83-90.