USDA Petition for Nonregulated Status of DAS-444Ø6-6 Soybean Page 1 of 228

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Petition for Determination of Nonregulated Status for

Herbicide Tolerant DAS-444Ø6-6 Soybean

OECD Unique Identifier: DAS-444Ø6-6

The undersigned submits this petition under 7 CFR 340.6 to request that the

Administrator, Animal and Plant Health Inspection Service, make a determination that the article 

should not be regulated under 7 CFR 340.

Submitting Companies:

Dow AgroSciences LLC M.S. Technologies LLC

9330 Zionsville Road 103 Avenue D

Indianapolis, IN 46268 West Point, IA 52656

Submitted by:

__________________________________

Mark S. Krieger, Ph.D.

Regulatory Leader

Regulatory Sciences & Government Affairs

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

Telephone: (317) 337-3458

FAX: (317) 337-4649

mkrieger2@dow.com

August 18, 2011

Revised October 17, 2011
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Release of Information

Dow AgroSciences LLC (DAS) and M.S. Technologies LLC (MS Tech) are submitting the 

information in this petition for deregulation to USDA APHIS as part of the regulatory process. 

By submitting this information, DAS and MS Tech do not authorize release of this information 

to any third party except to the extent the information is requested under the Freedom of 

Information Act (FOIA), 5 U.S.C., Section 522. In the event that USDA receives a FOIA request 

covering all or some of the information in this submission, DAS and MS Tech expect that, in 

advance of the release of the document(s), USDA will provide DAS and MS Tech with a copy of 

the material proposed to be released and the opportunity to object to the release of any 

information based upon appropriate legal grounds, e.g. responsiveness, confidentiality and/or 

competitive concerns. DAS and MS Tech expect that no information that has been identified as 

CBI (confidential business information) will be provided to any third party. DAS and MS Tech

understand that a CBI-deleted copy of this information may be made available to the public in a 

reading room and by individual request, as part of the public comment period. Except in 

accordance with the foregoing, DAS and MS Tech do not authorize the release, publication or 

other distribution of this information (including website posting) without prior notice and 

consent from DAS and MS Tech.
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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 all 

relevant data and information known to the petitioner that are unfavorable to the petition.

Mark S. Krieger, Ph.D.

Regulatory Leader

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, Indiana 46268

Telephone: (317) 337-3458

Fax: (317) 337-4649

mkrieger2@dow.com
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Summary

Dow AgroSciences LLC (herein referred to as ―DAS‖) and M.S. Technologies LLC (herein 

referred to as ―MS Tech‖) are submitting a Petition for Determination of Nonregulated Status for 

Herbicide Tolerant DAS-444Ø6-6 Soybean. DAS and MS Tech request a determination from 

USDA Animal and Plant Health Inspection Service (APHIS) that soybean transformation event 

DAS-444Ø6-6 and any soybean lines derived from crosses with DAS-444Ø6-6 soybeans no 

longer be considered regulated articles under 7 CFR Part 340.

DAS-444Ø6-6 soybean is a transgenic soybean product that provides tolerance to the herbicides 

2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate and glufosinate. This herbicide-tolerant 

soybean will provide growers with greater flexibility in selection of herbicides for the improved 

control of economically important weeds; allow an increased application window for effective 

weed control; and provide an effective weed resistance management solution to the increased 

incidence of glyphosate resistant weeds.

DAS-444Ø6-6 soybean plants have been genetically modified to express the aryloxyalkanoate 

dioxygenase-12 (AAD-12), double mutant 5-enolpyruvylshikimate-3-phosphate synthase

(2mEPSPS), and phosphinothricin acetyltransferase (PAT) proteins. The AAD-12 protein is an 

enzyme with an alpha ketoglutarate-dependent dioxygenase activity which results in metabolic 

inactivation of the herbicides of the aryloxyalkanoate family. The aad-12 gene, which expresses 

the AAD-12 protein, was derived from Delftia acidovorans, a gram-negative soil bacterium. The 

2mEPSPS protein has a decreased sensitivity to the herbicide glyphosate, allowing the enzyme to 

function in the presence of the herbicide and thereby making the plant tolerant to glyphosate. The 

2mEPSPS protein is encoded by a modified version of the epsps gene from corn (Zea mays). The 

PAT enzyme acetylates the primary amino group of phosphinothricin rendering it inactive. The

pat gene expressing the PAT protein was derived from Streptomyces viridochromogenes.

The aad-12, 2mepsps and pat genes were introduced into DAS-444Ø6-6 soybean using 

Agrobacterium-mediated transformation. Molecular characterization by Southern blot analyses 

of DAS-444Ø6-6 soybean confirmed that a single, intact DNA insert containing the aad-12, 

2mepsps and pat gene expression cassettes was stably integrated into the soybean genome.

Southern blot analyses also confirmed the absence of the plasmid backbone DNA in DAS444Ø6-6 soybean. The integrity of the inserted DNA was demonstrated in five different breeding 

generations. Data from segregating generations confirmed the predicted Mendelian inheritance 

pattern. These data confirmed the stability of DAS-444Ø6-6 soybean during traditional breeding 

procedures.

The AAD-12, 2mEPSPS and PAT proteins in DAS-444Ø6-6 soybean were characterized 

biochemically and measured using protein-specific enzyme linked immunosorbent assays 

(ELISA). Protein expression was analyzed in leaf, root, whole-plant and grain tissues collected 

throughout the growing season from DAS-444Ø6-6 soybean plants treated with 2,4-D, 

glyphosate, glufosinate, all three herbicides in combination, or not treated with any of these

herbicides. The results showed a low level of expression of the AAD-12, 2mEPSPS, and PAT 

proteins across herbicide treatments and environments, indicating a low exposure to humans and 

animals.
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The AAD-12 protein was assessed for any potential adverse effects to humans or animals 

resulting from the environmental release of crops containing the AAD-12 protein. A step-wise, 

weight-of-evidence approach was used to assess the potential for toxic or allergenic effects from

the AAD-12 protein. Bioinformatic analyses revealed no meaningful homologies with known or 

putative allergens or toxins for the AAD-12 amino acid sequence. The AAD-12 protein 

hydrolyzed rapidly in simulated gastric fluid. There was no evidence of acute toxicity in mice at 

a dose of 2000 mg/kg body weight of AAD-12 protein. Glycosylation analysis revealed no 

detectable covalently linked carbohydrates in the AAD-12 protein expressed in DAS-444Ø6-6

soybean plants. The low level expression of the AAD-12 protein presents a low exposure risk to 

humans and animals, and the results of the overall safety assessment of the AAD-12 protein 

indicate that it is unlikely to cause allergenic or toxic effects in humans or animals.

The 2mEPSPS protein was assessed for any potential adverse effects to humans and animals 

resulting from the environmental release of crops containing the 2mEPSPS protein. A step-wise, 

weight-of-evidence approach was used to assess the potential for toxic or allergenic effects from 

the 2mEPSPS protein. Bioinformatic analyses revealed no meaningful homologies to known or 

putative allergens or toxins for the 2mEPSPS amino acid sequence. The 2mEPSPS protein 

hydrolyzed rapidly in simulated gastric fluid. There was no evidence of acute toxicity in mice at

a dose of 5000 mg/kg body weight of 2mEPSPS protein. Glycosylation analysis revealed no 

detectable covalently linked carbohydrates in the 2mEPSPS protein expressed in DAS-444Ø6-6

soybean plants. The low level expression of the 2mEPSPS protein presents a low exposure risk 

to humans and animals, and the results of the overall safety assessment of the 2mEPSPS protein 

indicate that it is unlikely to cause allergenic or toxic effects in humans or animals. The safety of 

the 2mEPSPS protein has been assessed previously and it has been approved for use in corn and

cotton.

The PAT protein was assessed for any potential adverse effects to humans and animals resulting 

from the environmental release of crops containing the PAT protein. A step-wise, weight-ofevidence approach was used to assess the potential for toxic or allergenic effects from the PAT

protein. Bioinformatic analyses revealed no meaningful homologies to known or putative 

allergens or toxins for the PAT amino acid sequence. The PAT protein hydrolyzed rapidly in 

simulated gastric fluid. There was no evidence of acute toxicity in mice at a dose of 5000 mg/kg 

body weight of PAT protein. The low level expression of the PAT protein presents a low 

exposure risk to humans and animals, and the results of the overall safety assessment of the PAT

protein indicate that it is unlikely to cause allergenic or toxic effects in humans or animals. The 

safety of the PAT protein has been assessed previously and it has been approved for use in 

canola, corn, cotton, rice, soybeans, and sugar beets.

DAS-444Ø6-6 soybean has been field tested in the major soybean growing regions of the 

continental United States as well as Puerto Rico. All field tests were conducted under field 

permits granted by USDA APHIS. Agronomic performance assessments were conducted on 

DAS-444Ø6-6 soybean in multi-site field studies to measure characteristics such as emergence, 

seedling vigor, plant height, lodging, and yield. All field trials were also observed for 

opportunistic disease or insect stressors as well as normal phenotypic characteristics. There were 

no meaningful differences observed between DAS-444Ø6-6 soybean and the non-transgenic 

control for plant pest characteristics and no indication of a selective advantage that would result 

in increased weediness potential of DAS-444Ø6-6 soybean.
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Nutrient composition analyses of forage and grain were conducted to compare the composition 

of DAS-444Ø6-6 soybean with the composition of a non-transgenic control. Compositional 

analyses were used to evaluate any changes in the levels of key nutrients and anti-nutrients in 

DAS-444Ø6-6 soybean which was treated with 2,4-D, glyphosate, glufosinate, all three 

herbicides in combination, or not treated with any herbicide. Along with the agronomic data, the 

compositional analyses indicate that DAS-444Ø6-6 soybean is substantially equivalent to 

conventional soybean and will not exhibit unexpected or unintended effects with respect to plant 

pest risk.

Since DAS-444Ø6-6 soybean is agronomically and nutritionally similar to conventional soybean, 

and the safety of the AAD-12, 2mEPSPS and PAT proteins has been demonstrated, no 

significant impact is expected on current crop production practices, non-target or endangered 

species, crop rotation, volunteer management, or commodity food and feed soybean products. 

The availability of DAS-444Ø6-6 soybean is expected to have a beneficial impact on weed 

control practices by providing growers with another tool to address their weed-control needs. 

The use of DAS-444Ø6-6 soybean will allow growers to proactively manage weed populations 

while avoiding adverse population shifts of troublesome weeds or the development of resistance, 

particularly glyphosate-resistance in weeds.

Information collected during field trials and laboratory analyses presented herein demonstrate 

that DAS-444Ø6-6 soybean exhibits no plant pathogenic properties or weediness characteristics. 

DAS-444Ø6-6 soybean is no more likely to become a plant pest than conventional soybean, and 

the AAD-12, 2mEPSPS and PAT proteins are unlikely to increase the weediness potential of any 

other cultivated plant or wild species.

DAS and MS Tech hereby request a determination from APHIS that herbicide-tolerant 

DAS-444Ø6-6 soybean and all progeny derived from the conventional breeding of this line no 

longer be considered regulated articles under 7 CFR Part 340.
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Table of Contents

Release of Information ................................................................................................................. 2

Certification................................................................................................................................... 3

Summary........................................................................................................................................ 4

Table of Contents.......................................................................................................................... 7

List of Figures.............................................................................................................................. 10

List of Tables............................................................................................................................... 13

Acronyms and Scientific Terms................................................................................................. 15

1. Rationale for the Development of DAS-444Ø6-6 ......................................................... 17

1.1. Basis for the Request for Nonregulated Status..................................................... 17

1.2. Benefits of DAS-444Ø6-6 Soybean...................................................................... 17

1.3. Submission to Other Regulatory Agencies........................................................... 19

2. The Biology of Soybean .................................................................................................. 20

2.1. Overview of Soybean Biology.............................................................................. 20

2.2. Characterization of the Recipient Soybean Cultivar............................................. 20

3. Development of DAS-444Ø6-6 Soybean........................................................................ 21

3.1. Description of the Transformation System........................................................... 21

3.2. Selection of Comparators for DAS-444Ø6-6 Soybean......................................... 25

4. Donor Genes and Regulatory Sequences...................................................................... 26

4.1. Identity and Source of Genetic Material in pDAB8264 ....................................... 26

5. Genetic Characterization ............................................................................................... 30

5.1. Overview of Molecular Analysis.......................................................................... 30

5.2. Analysis of the Insert and Its Genetic Elements................................................... 38

5.2.1. Number of Insertion Sites............................................................................... 38

5.2.2. Structure of the Insert and Genetic Elements ................................................. 39

5.3. Absence of Vector Backbone DNA...................................................................... 40

5.4. Stability of the Insert across Generations ............................................................. 40

5.5. Segregation Analysis ............................................................................................ 60

5.5.1. Genetic and Molecular Analysis of a Segregating Generation....................... 60

5.5.2. Segregation Analysis of Breeding Generations.............................................. 60

5.6. Summary of the Genetic Characterization............................................................ 61

6. Characterization of the Introduced Proteins ............................................................... 62

6.1. AAD-12................................................................................................................. 62

6.1.1. Identity of the AAD-12 Protein ...................................................................... 62

6.1.2. Mode of Action of the AAD-12 Protein ......................................................... 62

6.1.3. Biochemical Characterization of the AAD-12 Protein ................................... 63

6.1.4. Expression of the AAD-12 Protein in Plant Tissues....................................... 64

6.1.5. Food and Feed Safety Assessment for AAD-12 Protein ................................ 66

6.1.6. Summary of AAD-12 Protein Characterization.............................................. 67

6.2. 2mEPSPS.............................................................................................................. 68

6.2.1. Identity of the 2mEPSPS Protein.................................................................... 68

6.2.2. Mode of Action of the 2mEPSPS Protein....................................................... 68

6.2.3. Biochemical Characterization of the 2mEPSPS Protein................................. 69

6.2.4. Expression of the 2mEPSPS Protein in Plant Tissues.................................... 69

6.2.5. Food and Feed Assessment for 2mEPSPS Protein ......................................... 72

6.2.6. Summary of 2mEPSPS Protein Characterization ........................................... 73
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6.3. PAT....................................................................................................................... 74

6.3.1. Identity of the PAT Protein............................................................................. 74

6.3.2. Mode of Action of the PAT Protein................................................................ 74

6.3.3. Biochemical Characterization of the PAT Protein.......................................... 74

6.3.4. Expression of the PAT Protein in Plant Tissues............................................. 75

6.3.5. Food and Feed Safety Assessment for PAT Protein....................................... 77

6.3.6. Summary of PAT Protein Characterization .................................................... 77

7. Agronomic Performance ................................................................................................ 78

7.1. Phenotypic and Agronomic Characteristics.......................................................... 78

7.1.1. Agronomic Study............................................................................................ 78

7.1.2. Agronomic Results.......................................................................................... 82

7.1.3. Conclusions..................................................................................................... 85

7.2. Ecological Evaluations.......................................................................................... 86

7.3. Germination and Dormancy Evaluations.............................................................. 88

7.4. Summary of Agronomic, Disease, and Pest Characteristics................................. 89

8. Grain and Forage Composition ..................................................................................... 90

8.1. Field Study Design................................................................................................ 90

8.2. Compositional Analysis........................................................................................ 91

8.3. Statistical Analysis................................................................................................ 93

8.4. Composition Analysis Results.............................................................................. 94

8.4.1. Proximate, Fiber, and Mineral Analysis of Forage......................................... 94

8.4.2. Proximate and Fiber Analysis of Seed.......................................................... 100

8.4.3. Mineral Analysis of Seed.............................................................................. 104

8.4.4. Amino Acid Analysis of Seed....................................................................... 110

8.4.5. Fatty Acid Analysis of Seed ......................................................................... 119

8.4.6. Vitamin Analysis of Seed ............................................................................. 125

8.4.7. Bioactive Analysis of Seed ........................................................................... 131

8.5. Composition Summary ....................................................................................... 137

8.6. Conclusions......................................................................................................... 137

9. Environmental Consequences and Impact on Agronomic Practices ....................... 138

9.1. Field Efficacy...................................................................................................... 138

9.2. Weediness Potential............................................................................................ 140

9.3. Gene Flow Assessment....................................................................................... 140

9.3.1. Vertical Gene Flow....................................................................................... 140

9.3.2. Horizontal Gene Flow................................................................................... 140

9.4. Current US Agronomic Practices for Soybeans.................................................. 141

9.4.1. Soybean Production ...................................................................................... 141

9.4.2. Weeds in Soybean......................................................................................... 141

9.4.3. Weed Management in Soybean .................................................................... 143

9.4.4. Crop Rotation and Tillage Practices............................................................. 144

9.5. Potential Impact on Agronomic Practices........................................................... 144

9.5.1. Potential Impact on Cultivation and Management Practices........................ 144

9.5.2. Potential Impact on Weed Control Practices................................................ 145

9.5.3. Potential Impact on Volunteer Management ................................................ 147

9.5.4. Potential Impact on Non-Target Organisms and Endangered Species......... 148

9.6. Herbicide Resistance Management..................................................................... 149
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9.6.1. Herbicide Resistance..................................................................................... 149

9.6.2. Factors Impacting Development of Resistance............................................. 152

9.6.3. Herbicide Resistance Management............................................................... 153

9.7. Summary of Environmental Consequences and Impact on Agronomic 

Practices.............................................................................................................. 154

10. Adverse Consequences of Introduction ...................................................................... 155

11. Appendices..................................................................................................................... 156

Appendix 1. Methods for Molecular Characterization of DAS-444Ø6-6 Soybean............... 157

Appendix 2. Methods and Results for Characterization of AAD-12 Protein ........................ 160

Appendix 3. Methods and Results for Characterization of 2mEPSPS Protein...................... 172

Appendix 4. Methods and Results for Characterization of PAT Protein............................... 187

Appendix 5. Methods for AAD-12, 2mEPSPS and PAT Protein Expression Analysis........ 191

Appendix 6. USDA Notifications for DAS-444Ø6-6 Soybean ............................................. 194

Appendix 7. Literature Ranges for Compositional Analysis ................................................. 195

Appendix 8. Glyphosate, 2,4-D, Glufosinate and Herbicide Resistant Weeds...................... 200

Appendix 9. Stewardship of Herbicide Tolerant DAS-444Ø6-6 Soybean ............................ 211

Appendix 10. References....................................................................................................... 219
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List of Figures

Figure 1. Plasmid map of pDAB8264........................................................................................... 22

Figure 2. Diagram of intended T-DNA insert in plasmid pDAB8264. ........................................ 22

Figure 3. Event sorting and selection process for DAS-444Ø6-6 soybean. ................................. 23

Figure 4. Breeding diagram of DAS-444Ø6-6 soybean. .............................................................. 24

Figure 5. Location of probes on pDAB8264 used in Southern blot analysis of DAS444Ø6-6 soybean. ............................................................................................................. 32

Figure 6. Plasmid map of pDAB8264 with selected restriction enzyme sites used for 

Southern analysis. ............................................................................................................. 37

Figure 7. Linearized intended T-DNA insert from pDAB8264 with restriction enzymes 

used for DNA digestion and the expected hybridization bands........................................ 38

Figure 8. Southern blot analysis of HindIII and MscI digested DAS-444Ø6-6 soybean: 

RB7 probe. ........................................................................................................................ 42

Figure 9. Southern blot analysis of HindIII and MscI digested DAS-444Ø6-6 soybean: 

Histone H4A748 UTR probe. ........................................................................................... 43

Figure 10. Southern blot analysis of XhoI and MscI digested DAS-444Ø6-6 soybean: 

Histone H4A748 Promoter probe. .................................................................................... 44

Figure 11. Southern blot analysis of XhoI and MscI digested DAS-444Ø6-6 soybean: 

AtUbi10 Promoter probe................................................................................................... 45

Figure 12. Southern blot analysis of XhoI and HindIII digested DAS-444Ø6-6 soybean: 

AtuORF23 UTR probe...................................................................................................... 46

Figure 13. Southern blot analysis of XhoI and HindIII digested DAS-444Ø6-6 soybean: 

CsVMV probe................................................................................................................... 47

Figure 14. Southern blot analysis of XhoI and HindIII digested DAS-444Ø6-6 soybean: 

AtuORF1 UTR probe........................................................................................................ 48

Figure 15. Southern blot analysis of XhoI, HindIII and MscI digested DAS-444Ø6-6 

soybean: 2mepsps probe. .................................................................................................. 49

Figure 16. Southern blot analysis of XhoI, HindIII and MscI digested DAS-444Ø6-6 

soybean: aad-12 probe...................................................................................................... 50

Figure 17. Southern blot analysis of XhoI, HindIII, and MscI digested DAS-444Ø6-6 

soybean: pat probe. ........................................................................................................... 51

Figure 18. Southern blot analysis of MscI/EcoRI digested DAS-444Ø6-6 soybean: 

Histone H4A748 Promoter, 2mepsps and Histone H4A748 UTR probes. ....................... 52

Figure 19. Southern blot analysis of PstI/XhoI digested DAS-444Ø6-6 soybean: AtUbi10 

Promoter, aad-12, and AtuORF23 UTR probes. .............................................................. 53

Figure 20. Southern blot analysis of PstI/XhoI digested DAS-444Ø6-6 soybean: CsVMV, 

pat, and AtuORF1 UTR probes. ....................................................................................... 54

Figure 21. Southern blot analysis of MscI/EcoRI and HindIII digested DAS-444Ø6-6 

soybean: Backbone 3 and Backbone 4 probes.................................................................. 55

Figure 22. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 

soybean: Ori-Rep probe. ................................................................................................... 56

Figure 23. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 

soybean: Backbone 1 probe. ............................................................................................. 57

Figure 24. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 

soybean: Backbone 2 probe. ............................................................................................. 58
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Figure 25. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 

soybean: SpecR probe....................................................................................................... 59

Figure 26. Amino acid sequence of the AAD-12 protein. ............................................................ 62

Figure 27. Degradation reaction of 2,4-D catalyzed by AAD-12................................................. 63

Figure 28. General reaction catalyzed by AAD-12 (R=H or CH3)............................................... 63

Figure 29. Amino acid sequence of the 2mEPSPS protein........................................................... 68

Figure 30. Amino acid sequence of the PAT protein.................................................................... 74

Figure 31. Mode of action of the PAT protein.............................................................................. 74

Figure 32. Proximate and fiber (% dry weight for all proximate and fiber except moisture 

(% fresh weight)) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference 

line soybean forage. .......................................................................................................... 97

Figure 33. Minerals (mg/100g dry weight) in non-transgenic (Isoline), Event 

DAS-444Ø6-6, and reference line soybean forage. .......................................................... 99

Figure 34. Proximate and fiber (% dry weight for all proximate and fiber except moisture 

(% fresh weight)) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference 

line soybean seed. ........................................................................................................... 102

Figure 35. Minerals (mg/100g dry weight for all minerals except selenium (ppb dry 

weight)) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line 

soybean seed. .................................................................................................................. 107

Figure 36. Amino acids (% dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, 

and reference line soybean seed...................................................................................... 114

Figure 37. Fatty acids (% total fatty acid) in non-transgenic (Isoline), Event 

DAS-444Ø6-6, and reference line soybean seed. ........................................................... 123

Figure 38. Vitamins (mg/kg dry weight) in non-transgenic (Isoline), Event 

DAS-444Ø6-6, and reference line soybean seed. ........................................................... 128

Figure 39. Bioactives (% dry weight (DW) for all bioactives except lectin (H.U./mg 

protein DW, H.U. = hemagglutination unit) and trypsin inhibitor (TIU/mg DW, 

TIU = trypsin inhibitor unit)) in non-transgenic (Isoline), Event DAS-444Ø6-6, 

and reference line soybean seed...................................................................................... 134

Figure 40. Bioactives: isoflavones (mcg/g dry weight) in non-transgenic (Isoline), Event 

DAS-444Ø6-6, and reference line soybean seed. ........................................................... 136

Figure 41. Adoption of genetically engineered crops in the U.S................................................ 145

Figure 42. 2,4-D herbicide application timing and rates for conventional and 

DAS-444Ø6-6 soybeans. ................................................................................................ 146

Figure 43. Glyphosate application rates in U.S. corn and soybeans from 1998 -2010............... 147

Figure 44. Resistant weed biotypes per herbicide mode of action. ............................................ 152

Figure 45. SDS-PAGE of DAS-444Ø6-6 soybean- and microbe-derived AAD-12. ................. 162

Figure 46. SDS-PAGE and western blot analysis of DAS-444Ø6-6 soybean- and 

microbe-derived AAD-12 protein................................................................................... 163

Figure 47. Glycosylation analysis of DAS-444Ø6-6 soybean- and microbe-derived AAD12 proteins....................................................................................................................... 164

Figure 48. Theoretical trypsin (top panel) and chymotrypsin (bottom panel) cleavage of 

the AAD-12 protein. ....................................................................................................... 166

Figure 49. Overall sequence coverage of trypsin and chymotrypsin digests for 

DAS-444Ø6-6-derived AAD-12 protein by MALDI-TOF MS and MALDI TOFTOF................................................................................................................................. 167
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Figure 50. Overall sequence coverage of trypsin, chymotrypsin, Arg-C, Asp-N, and GluC digests for P. fluorescens-derived AAD-12 protein by MALDI-TOF MS and 

ISD MS/MS. ................................................................................................................... 171

Figure 51. SDS-PAGE of DAS-444Ø6-6- and microbe-derived 2mEPSPS. ............................. 176

Figure 52. Western blot analysis of DAS-444Ø6-6 soybean- and microbe-derived 

2mEPSPS protein............................................................................................................ 178

Figure 53. Glycosylation analysis of DAS-444Ø6-6 soybean- and microbe-derived 

2mEPSPS protein............................................................................................................ 179

Figure 54. Theoretical trypsin (top panel) and chymotrypsin (bottom panel) cleavage of 

the 2mEPSPS protein...................................................................................................... 181

Figure 55. Sequence coverage of trypsin and chymotrypsin digests for DAS-444Ø6-6-

derived 2mEPSPS protein by MALDI-TOF MS and MALDI TOF-TOF...................... 182

Figure 56. Sequence coverage of trypsin, chymotrypsin, Arg-C, Asp-N, and Glu-C 

digests for P. fluorescens-derived 2mEPSPS protein by MALDI-TOF MS and 

ISD MS/MS. ................................................................................................................... 183

Figure 57. SDS-PAGE of DAS-444Ø6-6 and non-transgenic Maverick soybean extracts, 

microbe-derived PAT protein. ........................................................................................ 188

Figure 58. Western blot analysis of DAS-444Ø6-6 and Maverick soybean extracts. ................ 189

Figure 59. U.S. adoption rates of glyphosate-tolerant soybean and corn. .................................. 201

Figure 60. Number of glyphosate-resistant weeds reported globally by year from 1996 to 

2010................................................................................................................................. 206
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List of Tables

Table 1. Genetic elements of the T-DNA insert from plasmid pDAB8264. ................................ 26

Table 2. List of probes and their positions in plasmid pDAB8264. ............................................. 31

Table 3. Predicted and observed sizes of hybridizing fragments in Southern blot....................... 33

Table 4. Results of F2 individual plants tested for PAT expression and event-specific 

PCR within a single segregating generation. .................................................................... 60

Table 5. Results of BC1F2 individual plants from DAS-444Ø6-6 soybean tested for 

AAD-12 and 2mEPSPS protein expression within a single segregating generation........ 61

Table 6. Expression of AAD-12 in DAS-444Ø6-6 soybean......................................................... 65

Table 7. Expression of 2mEPSPS in DAS-444Ø6-6 soybean. ..................................................... 71

Table 8. Expression of PAT in DAS-444Ø6-6 soybean............................................................... 76

Table 9. Agronomic characteristics. ............................................................................................. 80

Table 10. Combined-site analysis: summary of agronomic characteristics.................................. 83

Table 11. Analysis of disease incidence and insect damage......................................................... 87

Table 12. Disease and insect stressors observed in trials of DAS-444Ø6-6 and 

conventional soybean........................................................................................................ 88

Table 13. Percentage emergence (number of seeds emerged divided by number of seeds 

planted x 100) for DAS-444Ø6-6 soybean and control soybean...................................... 89

Table 14. Composition analytes.................................................................................................... 92

Table 15. Summary of the proximate and fiber analysis of DAS-444Ø6-6 soybean forage 

from all sites, and associated literature range. .................................................................. 95

Table 16. Summary of the mineral analysis of DAS-444Ø6-6 soybean forage from all 

sites, and associated literature range................................................................................. 96

Table 17. Summary of the proximate and fiber analysis of DAS-444Ø6-6 soybean seed 

from all sites, and associated literature range. ................................................................ 101

Table 18. Summary of the mineral analysis of DAS-444Ø6-6 soybean seed from all sites, 

and associated literature range. ....................................................................................... 105

Table 19. Summary of the amino acid analysis of DAS-444Ø6-6 soybean seed from all 

sites, and associated literature range............................................................................... 111

Table 20. Summary of the fatty acid analysis of DAS-444Ø6-6 soybean seed from all 

sites, and associated literature range............................................................................... 120

Table 21. Summary of the vitamin analysis of DAS-444Ø6-6 soybean seed from all sites, 

and associated literature range. ....................................................................................... 126

Table 22. Summary of the bioactive analysis of DAS-444Ø6-6 soybean seed from all 

sites, and associated literature range............................................................................... 132

Table 23. DAS-444Ø6-6 soybean tolerance to application of 2,4-D (Weedar 64)..................... 138

Table 24. DAS-444Ø6-6 soybean tolerance to application of glyphosate (Durango DMA)...... 139

Table 25. DAS-444Ø6-6 soybean tolerance to application of glufosinate (Ignite 280SL). ....... 139

Table 26. Common troublesome weeds in soybeans in 2006-2008............................................ 142

Table 27. Yield reduction from specific weed species in soybeans. .......................................... 143

Table 28. Number of herbicide resistant weeds reported globally by herbicide group and 

mode of action................................................................................................................. 151

Table 29. Assessment of resistance risk by evaluation of cropping systems.............................. 153

Table 30. Glyphosate- and ALS-resistant weeds controlled by 2,4-D. ...................................... 154

Table 31. Summary of N-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived AAD-12 protein.............................................................................. 168
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Table 32. Summary of C-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived AAD-12 protein.............................................................................. 170

Table 33. Composition of extraction buffer for soybean-derived 2mEPSPS. ............................ 173

Table 34. Western blot extraction buffer. ................................................................................... 174

Table 35. Summary of N-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived 2mEPSPS........................................................................................ 184

Table 36. Summary of C-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived 2mEPSPS........................................................................................ 185

Table 37. Literature ranges reported for soybean seed: proximates, fiber, and minerals. .......... 195

Table 38. Literature ranges reported for soybean seed: amino acids.......................................... 196

Table 39. Literature ranges for soybean seed: fatty acids........................................................... 197

Table 40. Literature ranges reported for soybean seed: vitamins. .............................................. 198

Table 41. Literature ranges for soybean seed: bioactives. .......................................................... 199

Table 42. Herbicide-tolerant crops available to farmers in North America in 2005. ................. 200

Table 43. Weed species with reported glyphosate-resistant biotypes......................................... 205

Table 44. Global reports of glyphosate-resistant weed biotypes with resistance to other 

herbicide modes of action. .............................................................................................. 207

Table 45. Potential weed shifts with use of glyphosate in U.S. corn and soybeans. .................. 208

Table 46. Weed species with reported 2,4-D-resistant biotypes................................................. 209
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Acronyms and Scientific Terms

2,4-D 2,4-Dichlorophenoxyacetic acid

2mepsps Gene encoding the double mutant 5-enolpyruvylshikimate-3-

phosphate synthase (2mEPSPS) from Zea mays

2mEPSPS Mutant 5-enolpyruvylshikimate-3-phosphate synthase

A Acre

aad-12 Gene from Delftia acidovorans which encodes the AAD-12 protein

AAD-12 Aryloxyalkanoate dioxygenase-12 protein

ACCase Acetyl CoA carboxylase

ADF Acid detergent fiber

ae Acid equivalent

ae/A Acid equivalent per acre

ae/ha Acid equivalent per hectare

ai Active ingredient

ai/A Active ingredient per acre

ai/ha Active ingredient per hectare

ALS Acetolactate synthase

ANOVA Analysis of variance

APHIS Animal and Plant Health Inspection Service, USDA

AtUbi10 Ubiquitin promoter from Arabidopsis thaliana

AtuORF1 3′ untranslated region from Agrobacterium tumefaciens

AtuORF23 3′ untranslated region from Agrobacterium tumefaciens

bp Base pair

bu Bushel

CFIA Canadian Food Inspection Agency

CFSAN Center for Food Safety and Nutrition, US FDA

CsVMV Promoter from cassava vein mosaic virus

DAS Dow AgroSciences LLC

DAS-444Ø6-6 OECD identifier for the soybean event expressing the AAD-12, 

2mEPSPS, and PAT proteins

DCP 2,4-Dichlorophenol

dmmg Same as 2mepsps

DNA Deoxyribonucleic acid

ELISA Enzyme-linked immunosorbent assay

EPA Environmental Protection Agency (US)

epsps Gene encoding the wild-type 5-enolpyruvylshikimate-3-phosphate 

synthase (EPSPS)

EPSPS Wild-type 5-enolpyruvylshikimate-3-phosphate synthase

ESA Endangered Species Act

ESI-LC/MS Electrospray ionization-liquid chromatography mass spectrometry

FDA Food and Drug Administration (US)

FDR False Discovery Rate

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FWS Fish and Wildlife Service

GS Glutamine synthetase

ha Hectare
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HRAC Herbicide Resistance Action Committee

IAA Indole acetic acid

ILSI International Life Sciences Institute

IWM Integrated weed management

Kb Kilobase pair

kDa Kilodalton

L Liter

LOD Limit of Detection

LOQ Limit of Quantitation

MALDI-TOF MS Matrix assisted laser desorption/ionization time-of-flight mass 

spectrometry

Maverick Publicly available soybean line used in transformation to produce 

DAS-444Ø6-6 soybean

mEPSPS Same as 2mEPSPS

MOA Mode of action

MS Tech M.S. Technologies LLC

NDF Neutral detergent fiber

OECD Organisation for Economic Co-operation and Development

pat Gene from Streptomyces viridochromogenes which encodes the 

PAT protein

PAT Phosphinothricin N-acetyltransferase protein

PBN US FDA Pre-market Biotechnology Notice

PCR Polymerase chain reaction

pDAB8264 DNA vector carrying the aad-12, 2mepsps and pat expression 

cassettes

PPO Protoporphyrinogen oxidase

PPT Phosphinothricin

PTU Plant transcription unit consisting of promoter, gene, and terminator 

sequences

RB7 MAR Matrix attachment region (MAR) from Nicotiana tabacum

RCB Randomized complete block

SCN Soybean cyst nematode

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SGF Simulated gastric fluid

spp species

subsp subspecies

T-DNA Transfer DNA

USDA United States Department of Agriculture

UTR Untranslated region

WSSA Weed Science Society of America
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1. Rationale for the Development of DAS-444Ø6-6

1.1. Basis for the Request for Nonregulated Status

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

(USDA) has responsibility, under the Plant Protection Act (7 U.S.C. 7701-7772) and the Plant 

Quarantine Act (7 U.S.C. 151-167), to prevent the introduction or dissemination of plant pests 

into or within the United States. Part 340 regulates introduction of organisms altered or produced 

through genetic engineering which are plant pests or for which there is a reason to believe are 

plant pests. The APHIS regulations at 7 CFR 430.6 provide that an applicant may petition 

APHIS to evaluate submitted data on the genetically engineered crop to determine that a 

regulated article does not present a plant pest risk and therefore should no longer be regulated.

Dow AgroSciences LLC (herein referred to as ―DAS‖) and M.S. Technologies LLC (herein

referred to as ―MS Tech‖) are submitting data for genetically engineered herbicide-tolerant 

DAS-444Ø6-6 soybean and request a determination from APHIS that DAS-444Ø6-6 soybean

and crosses of DAS-444Ø6-6 with nonregulated soybean lines no longer be considered regulated 

articles under 7 CFR 340.

1.2. Benefits of DAS-444Ø6-6 Soybean

DAS and MS Tech have developed transgenic soybean plants that are tolerant to the herbicides 

2,4-dichlorophenoxyacetic acid (2,4-D), glyphosate and glufosinate. The unique identifier for 

these plants, in accordance with the Organisation for Economic Co-operation and Development‘s 

(OECD) ―Guidance for the Designation of a Unique Identifier for Transgenic Plants‖ (OECD, 

2004), is DAS-444Ø6-6.

DAS-444Ø6-6 soybean was developed using Agrobacterium-mediated transformation to stably 

incorporate the aad-12 gene from Delftia acidovorans, the 2mepsps gene from Zea mays, and the 

pat gene from Streptomyces viridochromogenes into soybean. The aad-12 gene encodes the 

aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme which, when expressed in plants, degrades 

2,4-D to herbicidally-inactive 2,4-dichlorophenol (DCP). The 2mepsps gene encodes the 

2mEPSPS protein, which has a decreased sensitivity to the herbicide glyphosate. This allows the 

enzyme to function in the presence of the herbicide and thereby makes the plant tolerant to 

glyphosate. The pat gene encodes the enzyme phosphinothricin acetyltransferase that inactivates 

glufosinate. 

The availability of DAS-444Ø6-6 soybean is expected to have a beneficial impact on weed 

control practices by providing growers with an advanced tool to address their weed control 

needs. The availability of DAS-444Ø6-6 soybean will allow growers to proactively manage 

weed populations while avoiding adverse population shifts of troublesome weeds or the 

development of resistance, particularly glyphosate-resistance in weeds.

With the introduction of genetically engineered, glyphosate-tolerant crops in the mid-1990‘s, 

growers were enabled with a simple, convenient, flexible, and inexpensive tool for controlling a 

wide spectrum of broadleaf and grass weeds that was unparalleled in agriculture. Consequently, 

producers were quick to adopt glyphosate-tolerant crops, and in many instances, abandon many 

of the accepted best agronomic practices such as crop rotation, herbicide mode of action rotation, 
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tank mixing, and incorporation of mechanical with chemical and cultural weed control. Currently 

glyphosate-tolerant soybean, cotton, corn, alfalfa, sugar beets, and canola are commercially 

available in the United States and elsewhere in the Western Hemisphere. More glyphosatetolerant crops (e.g., wheat, rice, turf, etc.) are poised for introduction pending global market 

acceptance. Many other glyphosate-tolerant species are in experimental or development stages 

(e.g., sugar cane, sunflower, beets, peas, carrot, cucumber, lettuce, onion, strawberry, tomato, 

and tobacco; forestry species like poplar and sweetgum; and horticultural species like marigold, 

petunia, and begonias) (Information Systems for Biotechnology, 2011). Additionally, the cost of 

glyphosate has dropped dramatically in recent years to the point that few conventional weed 

control programs can effectively compete on price and performance with glyphosate-tolerant 

crops systems.

Extensive use of glyphosate-only weed control programs is resulting in the selection of 

glyphosate-resistant weeds, and is selecting for the propagation of weed species that are 

inherently more tolerant to glyphosate than most target species (i.e., weed shifts) (Heap, 2011). 

Although glyphosate has been widely used globally for more than 30 years, only a handful of 

weeds have been reported to have developed resistance to glyphosate; however, most of these 

have been identified in the past 5-8 years. Resistant weeds in the U.S. include both grass and 

broadleaf species—Lolium rigidum (Rigid ryegrass), Lolium multiflorum (Italian ryegrass),

Sorghum halepense (Johnsongrass), Amaranthus palmeri (Palmer amaranth), Amaranthus rudis 

(Common waterhemp), Ambrosia artemisiifolia (Common ragweed), Ambrosia trifida (Giant 

ragweed), Conyza canadensis (Horseweed), and Conyza bonariensis (Hairy fleabane). 

Additionally, weeds that had previously not been an agronomic problem prior to the wide use of 

glyphosate-tolerant crops are now becoming more prevalent and difficult to control in the 

context of glyphosate-tolerant crops, which now comprise >90% of U.S. soybean acres and 

>60% of U.S. corn and cotton acres (USDA ERS, 2011a). These weed shifts are occurring 

predominantly, but not exclusively, with difficult-to-control broadleaf weeds. Some examples 

include Ipomoea, Amaranthus, Chenopodium, Taraxacum, and Commelina species. 

In areas where growers are faced with glyphosate-resistant weeds or a shift to more difficult-tocontrol weed species, growers can compensate by tank mixing or alternating with other 

herbicides that will control the surviving weeds. One popular and efficacious tank mix active 

ingredient for controlling broadleaf escapes has been 2,4-diclorophenoxyacetic acid (2,4-D). 

2,4-D has been used agronomically and in non-crop situations for broad spectrum, broadleaf 

weed control for more than 60 years. Individual cases of more tolerant weed species have been 

reported, but 2,4-D remains one of the most widely used herbicides globally. The development 

of 2,4-D-tolerant soybeans provides an excellent option for controlling glyphosate-resistant (or 

highly tolerant and shifted) broadleaf weed species for in-crop applications, allowing the grower 

to focus applications at the critical weed control stages and extending the application window 

without the need for specialized sprayer equipment. Combining the 2,4-D-tolerance trait with a 

glyphosate-tolerance trait gives growers the ability to use tank mixes of glyphosate/2,4-D overthe-top of the tolerant plants to control the glyphosate-resistant broadleaf species.

DAS-444Ø6-6 soybeans also provide tolerance to glufosinate herbicides. Glufosinate is a 

non-selective, contact herbicide that controls a broad spectrum of annual and perennial grasses 

and broadleaf weeds. The tolerance to glufosinate allows use of an additional mode of action as 

part of effective herbicide resistance management strategies. Glufosinate herbicides can also be 
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used as selection agents in breeding nurseries to select herbicide-tolerant plants to maintain seedtrait purity.

The commercial introduction of transgenic soybean exhibiting tolerance to 2,4-D, glyphosate and 

glufosinate will bring new weed control alternatives to growers. This new weed management 

tool will allow for the improved control of key broadleaf and grassy weeds which affect the vigor 

and yield of the crop, allow an increased herbicide application window for effective weed 

control, and provide an effective resistance management/prevention solution to the increased 

incidence of glyphosate- and acetolactate synthase (ALS)-resistant weeds.

1.3. Submission to Other Regulatory Agencies

DAS-444Ø6-6 falls within the scope of the FDA policy statement, published in the Federal 

Register on May 29, 1992, concerning regulation of products derived from new plant varieties, 

including those developed via biotechnology. DAS and MS Tech intend to submit a pre-market 

biotechnology notification (PBN) to FDA in 2011.

The regulation and use of herbicides on DAS-444Ø6-6 soybean is governed by the US under the 

Federal Insecticide, Fungicide and Rodenticide Act (FIFRA). The use of herbicides on 

DAS-444Ø6-6 soybean will be consistent with either currently authorized uses or uses that are 

currently in review by US EPA, therefore there are no new regulatory submissions to US EPA 

associated with DAS-444Ø6-6 soybean.

DAS and MS Tech intend to submit dossiers beginning in 2011 to the regulatory authorities of 

trade partners for import clearance and production approval which may include Canada, Japan, 

Korea, Taiwan, European Union, Australia/New Zealand, South Africa, Brazil, Argentina and 

Mexico.
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2. The Biology of Soybean

2.1. Overview of Soybean Biology

Refer to the OECD Consensus Document on the Biology of Glycine max (L.) Merr. (Soybean) 

(OECD, 2000), for information related to the following aspects of soybean biology:

 general description, including taxonomy, morphology, and the uses of soybean as a crop 

plant

 agronomic practices

 centers of origin 

 reproductive biology

 cultivated Glycine max as a volunteer weed

 ability to cross inter-species/genus, introgressions into relatives, and interactions with 

other organisms

 summary of the ecology of Glycine max

The vegetative and reproductive stages of a soybean plant are described using the following 

nomenclature (Pedersen, 2004; Gaska, 2006):

Vegetative Stages Reproductive Stages

VE Emergence R1 Beginning bloom

VC Unrolled unifoliate leaves R2 Full bloom

V1 First-trifoliate R3 Beginning pod

V2 Second-trifoliate R4 Full pod

V3 Third-trifoliate R5 Beginning seed

V(n) n

th

-trifoliate R6 Full seed

R7 Beginning maturity

R8 Full maturity

2.2. Characterization of the Recipient Soybean Cultivar

The publicly available cultivar  ̳Maverick‘ was used as the recipient line for the generation of 

DAS-444Ø6-6 soybean.

Maverick was originally developed by the Missouri and Illinois Agricultural Experiment Stations 

at the Universities of Missouri and Illinois, respectively, and released in 1996 (Sleper et al., 

1998). Maverick was developed because of its resistance to the soybean cyst nematode (SCN) 

and higher yield compared with SCN-resistant cultivars of similar maturity. Maverick is 

classified as a late Group III maturity (relative maturity 3.8). Maverick has purple flowers, grey 

pubescence, brown pods at maturity, and dull yellow seed with buff hila. Maverick is resistant to 

phytophthora rot but is susceptible to brown stem rot and sudden death syndrome. 
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3. Development of DAS-444Ø6-6 Soybean

3.1. Description of the Transformation System

DAS-444Ø6-6 soybean was generated through Agrobacterium-mediated transformation of 

soybean (Glycine max) cotyledonary node explants. The disarmed Agrobacterium tumefaciens

strain EHA101 (Hood et al., 1986), carrying the binary vector with the pat, aad-12 and 2mepsps

within the T-DNA region, was used to initiate transformation.

Agrobacterium-mediated transformation was carried out using a modified procedure of Zeng et 

al. (2004). Briefly, soybean seeds (cv Maverick) were germinated on basal media and 

cotyledonary nodes were isolated and infected with Agrobacterium. Shoot initiation, shoot 

elongation, and rooting media were supplemented with cefotaxime, timentin and vancomycin to 

inhibit the growth of Agrobacterium. Glufosinate selection was employed to inhibit the growth 

of non-transformed shoots. Selected shoots were transferred to rooting medium for root 

development and then transferred to soil mix for acclimatization of plantlets.

Terminal leaflets of selected plantlets were painted with glufosinate to screen for putative 

transformants. The glufosinate-resistant plantlets were transferred to the greenhouse, allowed to 

acclimate and then painted with glufosinate to reconfirm tolerance. Surviving plantlets were 

deemed to be putative transformants. The screened plants were sampled and analyzed at the 

molecular level for the presence of the T-DNA insert and the absence of the vector backbone 

DNA. Specifically, for T0 plants, PCR analysis was performed to verify the absence of the 

spectinomycin resistance gene in the vector backbone as well as the presence of the aad-12

coding region and 2mepsps plant transcription unit (PTU). A PCR-based zygosity assay was 

conducted for copy number detection for pat, aad-12, and 2mepsps genes. Selected T0 plants 

were allowed to self-fertilize in the greenhouse to give rise to T1 seed. For T1 plants, PCR 

analysis, zygosity assay, and Southern blot analysis were performed to detect copy number, 

number of integration sites, and PTU integrity.

Figure 1 shows a plasmid map of pDAB8264.

Figure 2 shows a diagram of the T-DNA insert in plasmid pDAB8264.

Figure 3 outlines the development of DAS-444Ø6-6 soybean.

Figure 4 shows a breeding diagram for DAS-444Ø6-6 soybean including identification of the 

generations used in various safety assessment studies.
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Figure 1. Plasmid map of pDAB8264.

Figure 2. Diagram of intended T-DNA insert in plasmid pDAB8264.

pDAB8264

16018 bp

2mepsps

aad-12

SpecR

trfA

pat

TPotp C

Histone H4A748 promoter

AtUbi10 promoter

CsVMV promoter

Ori Rep

Histone H4A748 3' UTR

AtuORF23 3' UTR

AtuORF1 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR

T-DNA Insert pDAB8264

10658 bp

aad-12

pat

2mepsps

TPotp C

AtUbi10 promoter

Histone H4A748 promoter

CsVMV promoter

Histone H4A748 3' UTR

AtuORF23 3' UTR

AtuORF1 3' UTR

T-DNA Border A

T-DNA Border B T-DNA Border A

RB7 MAR

T-DNA Border A
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Figure 3. Event sorting and selection process for DAS-444Ø6-6 soybean.

Assembled Agrobacterium binary vector pDAB8264 carrying the aad-12, 2mepsps 

and pat gene expression cassettes.

Introduced the aad-12, 2mepsps and pat gene expression cassettes into the soybean 

line Maverick via Agrobacterium tumefaciens-mediated transformation. 

Screened transformed plants based on in vitro tolerance to glufosinate herbicide. 

Selected T0 soybean plants for regeneration. 

Screened regenerated T0 plants for the absence of vector backbone, presence of genes 

of interest, copy number and tolerance to glufosinate herbicide. Allowed self 

pollination of the T0 plants that met the desired criteria.

Continued self pollination. Evaluated transgenic plants via molecular characterization 

and agronomic performance and herbicide efficacy.

Selected homozygous plants and carried out crossing and backcrossing for product 

development.

Selected DAS-444Ø6-6 as the lead commercial candidate. 
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Analysis Petition Section(s) DAS-444Ø6-6 Soybean 

Generations Used

Control

Molecular Analysis 5.1, 5.2, 5.3, 5.4 T2, T3, T4, T6, F2 Maverick

Segregation Analysis 5.5 F2, BC1F2 Maverick

Protein Characterization 6.1.3, 6.2.3, 6.3.3 T4 Maverick

Protein Expression 6.1.3, 6.2.3, 6.3.3 T4 Maverick

Agronomics 7 T4 Maverick

Germination/Dormancy 7.3 T5 Maverick

Composition 8 T4 Maverick

Efficacy 9.1 T4 Maverick

Figure 4. Breeding diagram of DAS-444Ø6-6 soybean.

T0

Self

T1

Self

T2

Self

T3 F1

Self Self

T4 F2

Self

T5

Self

T6

Backcross to recurrent parents

Cross to elite lines

BC1F1

Self

BC1F2
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3.2. Selection of Comparators for DAS-444Ø6-6 Soybean

To ensure the accurate assessment of the impact of transgene insertion on various characteristics 

of DAS-444Ø6-6 soybean, a proper selection of comparator plants is important.

The control plants should have a genetic background similar to that of DAS-444Ø6-6 soybean, 

but lack the transgenic insert. In all cases, the non-transgenic variety Maverick was used as the 

control. Maverick is the recipient variety that was transformed to generate DAS-444Ø6-6 

soybean. The T0 plants and all subsequent self-pollinated generations derived posttransformation were essentially genetically identical to Maverick soybean with the exception of 

the transgenic insert DNA.
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4. Donor Genes and Regulatory Sequences

4.1. Identity and Source of Genetic Material in pDAB8264

DAS-444Ø6-6 soybean was generated by Agrobacterium-mediated transformation using the 

plasmid pDAB8264 (Figure 1). The T-DNA insert in the plasmid contains the 2mepsps gene 

from Zea mays, a synthetic, plant-optimized sequence of the aad-12 gene from Delftia 

acidovorans, and the pat gene from Streptomyces viridochromogenes (Figure 2). A summary of 

the genetic elements is given in Table 1.

Table 1. Genetic elements of the T-DNA insert from plasmid pDAB8264.

Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

T-DNA 

Border B 1 24 24 Required for transfer of T-DNA insert from Agrobacterium tumefaciens into plant cells (Barker et al., 1983)

Intervening 

sequence

25 160 136 Non-specific DNA sequences necessary for cloning

RB7 MAR 161 1326 1166 Matrix attachment region from the Nicotiana tabacum rb-7-5A 

gene (Hall et al., 1991)

Intervening 

sequence

1327 1365 39 Non-specific DNA sequences necessary for cloning

Histone 

H4A748 3′

UTR 

1366 2026 661

3′ untranslated region (UTR) comprising the transcriptional 

terminator and polyadenylation site of the histone H4A748 gene 

from Arabidopsis thaliana (Chaboute et al., 1987)

Intervening 

sequence

2027 2049 23 Non-specific DNA sequences necessary for cloning

2mepsps 2050 3387 1338

Native 5-enolpyruvylshikimate-3-phosphate synthase gene from 

Zea mays with two mutations providing glyphosate tolerance 

(Lebrun et al., 1996; Lebrun et al., 2003)

TPotp C 3388 3759 372 Optimized chloroplast transit peptide derived from maize and 

sunflower RuBisCO (Lebrun et al., 1996; Lebrun et al., 2003)

Intervening 

sequence

3760 3763 4

Non-specific DNA sequences necessary for cloning

Histone 

H4A748 

promoter 

3764 5193 1430

Promoter along with the 5′ untranslated region of the Histone 

H4A748 gene from Arabidopsis thaliana including an intron 

from the Histone 3 gene from Arabidopsis thaliana (Chaboute

et al., 1987)

Intervening 

sequence

5194 5285 92 Non-specific DNA sequences necessary for cloning
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Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

AtUbi10 

promoter 5286 6607 1322

Promoter along with the 5' untranslated region and intron from 

the Arabidopsis thaliana polyubiquitin 10 (UBQ10) gene 

(Norris et al., 1993)

Intervening 

sequence

6608 6615 8

Non-specific DNA sequences necessary for cloning

aad-12 6616 7497 882

Plant-optimized version of an aryloxyalkanoate dioxygenase 

gene from Delftia acidovorans encoding an enzyme with an 

alpha ketoglutarate-dependent dioxygenase activity which 

results in metabolic inactivation of the herbicide(s) (Wright et 

al., 2009; Wright et al., 2010)

Intervening 

sequence

7498 7599 102 Non-specific DNA sequences necessary for cloning

AtuORF23 3′

UTR 7600 8056 457

3′ untranslated region (UTR) comprising the transcriptional 

terminator and polyadenylation site of open reading frame 23 

(ORF23) of plasmid pTi15955 from Agrobacterium tumefaciens

(Barker et al., 1983)

Intervening 

sequence

8057 8170 114 Non-specific DNA sequences necessary for cloning

CsVMV 

promoter 8171 8687 517 Promoter along with the 5′ untranslated region derived from the Cassava Vein Mosaic virus (Verdaguer et al., 1996)

Intervening 

sequence

8688 8694 7

Non-specific DNA sequences necessary for cloning

pat 8695 9246 552

Plant-optimized version of phosphinothricin acetyltransferase 

(PAT) gene, isolated from Streptomyces viridochromogenes, 

encoding a protein that confers tolerance to glufosinate 

(Wohlleben et al., 1988)

Intervening 

sequence

9247 9348 102 Non-specific DNA sequences necessary for cloning

AtuORF1 3′

UTR 9349 10052 704

3′ untranslated region (UTR) comprising the transcriptional 

terminator and polyadenylation site of open reading frame 1 

(ORF1) of plasmid pTi15955 from Agrobacterium tumefaciens

(Barker et al., 1983)

Intervening 

sequence

10053 10280 228 Sequence from Ti plasmid C58 (Zambryski et al., 1982; Wood

et al., 2001)

T-DNA 

Border A 10281 10304 24 Required for transfer of T-DNA insert from Agrobacterium tumefaciens into plant cells (Barker et al., 1983)

Intervening 

sequence

10305 10323 19 Sequence from Ti plasmid C58 (Zambryski et al., 1982; Wood

et al., 2001)
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Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

T-DNA 

Border A 10324 10347 24

Required for transfer of T-DNA insert from Agrobacterium 

tumefaciens into plant cells, aiming to prevent vector DNA 

being transferred into plant genome (Barker et al., 1983)

Intervening 

sequence

10348 10634 287 Sequence from Ti plasmid pTi15955 (Barker et al., 1983)

T-DNA 

Border A 10635 10658 24

Required for transfer of T-DNA insert from Agrobacterium 

tumefaciens into plant cells, aiming to prevent vector DNA 

being transferred into plant genome (Barker et al., 1983)

Three gene expression cassettes, also referred to as plant transcription units or PTUs, were 

present in the pDAB8264 vector for insertion into soybeans. The 2mepsps expression cassette is 

designed to express a double mutant maize 5-enolpyruvylshikimate-3-phosphate synthase gene 

that encodes the 2mEPSPS protein. The 2mepsps gene was originally isolated from Zea mays

and fused with an optimized chloroplast transit peptide derived from maize and sunflower 

ribulose-1,5-bisphosphate carboxylase-oxygenase (RuBisCO) at its N-terminus (Lebrun et al., 

1996; Lebrun et al., 2003). The 2mepsps gene (also referred to as dmmg, mEPSPS) has been 

introduced as the source of glyphosate tolerance in the maize transgenic event GA21(OECD 

unique identifier MON-ØØØ21-9), which has been approved by different agencies worldwide 

for environment, food, and feed (USDA, 1997) and in GlyTolTM cotton (OECD unique identifier 

BCS-GHØØ2-5), which was deregulated by USDA APHIS in 2009 (USDA, 2009). It is also 

present in soybean event FG-72 (OECD unique identifier MST-FGØ72-3) which is currently in 

review at USDA APHIS as petition 09-328-01p. The 2mepsps gene encodes a protein of 445

amino acids that has a molecular weight of approximately 47.5 kDa. The encoded protein is 

insensitive to glyphosate, thus providing tolerance to glyphosate in plants expressing the 

2mEPSPS protein.

Expression of 2mepsps in the T-DNA insert of pDAB8264 is controlled by the Histone H4A748

promoter from Arabidopsis thaliana and Histone H4A748 3′ UTR sequence from Arabidopsis 

thaliana. The Histone H4A748 promoter is known to drive constitutive expression of the genes it 

controls (Chaboute et al., 1987).

The aad-12 expression cassette is designed to express the plant-optimized aryloxyalkanoate 

dioxygenase (aad-12) gene that encodes the AAD-12 protein. The aad-12 gene was isolated 

from Delftia acidovorans and the synthetic version of the gene was optimized to modify the G+C 

codon bias to a level more typical for plant expression. The native and plant-optimized DNA 

sequences of aad-12 are 79.7% identical. The aad-12 gene encodes a protein of 293 amino acids 

that has a molecular weight of approximately 32 kDa. The insertion of aad-12 into soybean 

plants confers tolerance to herbicides such as 2,4-D by production of the aryloxyalkanoate 

dioxygenase-12 enzyme (AAD-12).

Delftia acidovorans, which has previously been identified as Pseudomonas acidovorans and 

Comamonas acidovorans, is a non glucose-fermenting, gram-negative, non spore-forming rod 
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present in soil, fresh water, activated sludge, and clinical specimens (Von Graevenitz, 1985;

Tamaoka et al., 1987; Wen et al., 1999). Delftia acidovorans can be used to transform ferulic 

acid into vanillin and related flavor metabolites (Rao and Ravishankar, 2000; Shetty et al., 2006). 

This utility has led to a history of safe use for Delftia acidovorans in the food processing 

industry. For example, US Patent 5,128,253 ―Bioconversion process for the production of 

vanillin‖ was issued on July 7, 1992 to Kraft General Foods (Labuda et al., 1992).

Expression of aad-12 in the T-DNA insert of pDAB8264 is controlled by the AtUbi10 promoter 

from Arabidopsis thaliana and AtuORF23 3′ UTR sequence from Agrobacterium tumefaciens

plasmid pTi15955. The AtUbi10 promoter is known to drive constitutive expression of the genes 

it controls (Norris et al., 1993). The function of AtuORF23 (GenBank Accession Number:

CAA25184) in pTi15955 (GenBank Accession Number: X00493) was not identified (Barker et 

al., 1983). A search of its translated amino acid sequence returned no significant similarity with 

known functional proteins.

The pat expression cassette is designed to express the plant-optimized phosphinothricin N-acetyl 

transferase (pat) gene that encodes the PAT protein. The pat gene was isolated from 

Streptomyces viridochromogenes and the synthetic version of the gene was optimized to modify 

the G+C codon bias to a level more typical for plant expression. The insertion of the pat gene 

into the soybean genome confers tolerance to glufosinate and was used as a selectable marker 

during the soybean transformation. The pat gene has been widely used both as a selectable 

marker and herbicide tolerance trait in previously deregulated products (USDA, 1996, 2001, 

2004, 2005).

Expression of the pat gene is controlled by the CsVMV promoter from cassava vein mosaic virus

and AtuORF1 3′ UTR sequence from Agrobacterium tumefaciens plasmid pTi15955. The 

cassava vein mosaic virus is a circular double-stranded DNA virus which infects cassava plants 

(Manihot esculenta Crantz) and has been characterized as a plant pararetrovirus belonging to the 

caulimovirus subgroup. The CsVMV promoter is known to drive constitutive expression of the 

genes it controls (Verdaguer et al., 1996). The function of AtuORF1 (GenBank Accession

Number: CAA25163) in pTi15955 (GenBank Accession Number: X00493) was not identified

(Barker et al., 1983), but its translated amino acid sequence has a significant similarity with an 

indole-3-lactate synthase (GenBank Accession Number: AAK90967) from Agrobacterium 

tumefaciens str. C58.

A matrix attachment region (MAR) of RB7 from Nicotiana tabacum was included at the 5′ end 

of the 2mepsps PTU to potentially facilitate transgene expression in the plant. Matrix attachment

regions are natural and abundant regions found in genomic DNA that are thought to attach to the 

matrix or scaffold of the nucleus. When positioned on the flanking ends of gene cassettes, some 

MARs have been shown to increase expression of transgenes and to reduce the incidence of gene 

silencing (Han et al., 1997; Abranches et al., 2005; Verma et al., 2005).
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5. Genetic Characterization

5.1. Overview of Molecular Analysis

Molecular characterization of DAS-444Ø6-6 soybean was conducted by Southern blot analysis. 

The results demonstrate that the transgene insert in DAS-444Ø6-6 soybean occurred as a simple 

integration of the T-DNA insert from plasmid pDAB8264, including a single, intact copy of each 

of the 2mepsps, aad-12, and pat expression cassettes. The insert is stably integrated and inherited 

across and within breeding generations, and no plasmid backbone sequences are present in DAS444Ø6-6 soybean.

Detailed Southern blot analysis was conducted using probes specific to the gene coding 

sequences, promoters, terminators, and other regulatory elements contained in the pDAB8264

plasmid. The locations of the probes on the pDAB8264 plasmid are described in Table 2 and 

shown in Figure 5. The expected and observed fragment sizes with specific digest and probe 

combinations, based on the known restriction enzyme recognition sites of the pDAB8264

plasmid are shown in Table 3 and Figure 7, respectively. The Southern blot analyses described 

here made use of two types of restriction fragments: a) internal fragments in which known 

restriction enzyme recognition sites are completely contained within the T-DNA insert of 

pDAB8264 and b) border fragments in which one known restriction enzyme recognition site is 

located within the T-DNA insert and another site is located in the soybean genome flanking the 

insert. Border fragment sizes vary by event because they rely on the location of the restriction 

enzyme recognition sites in the DNA sequence of flanking genomic region. Since integration 

sites are unique for each event, border fragments provide a means to determine the number of 

DNA insertions and to specifically identify the event.

Genomic DNA for Southern blot analysis was prepared from leaf material of individual 

DAS-444Ø6-6 soybean plants from five distinct breeding generations. Genomic DNA from 

leaves of non-transgenic variety Maverick was used as the control material. Plasmid DNA of 

pDAB8264 added to genomic DNA from the conventional control Maverick served as the 

positive control for the Southern blot analysis. Materials and methods used for the Southern

analyses are further described in Appendix 1.

The expected restriction fragments of the inserted DNA are shown in Table 3 and Figure 7, and 

the Southern blot results are shown in Figure 8 through Figure 25. Southern blot analysis showed 

that DAS-444Ø6-6 soybean contains a single intact copy of the 2mepsps, aad-12, and pat

expression cassettes integrated at a single locus (Section 5.2). No vector backbone sequences 

were detected in DAS-444Ø6-6 soybean (Section 5.3). The hybridization patterns across five

generations of DAS-444Ø6-6 soybean (T2, T3, T4, T6, and F2) were identical, indicating that 

the insertion is stably integrated in the soybean genome (Section 5.4). The inheritance of DAS444Ø6-6 soybean insert in segregating generations was investigated using event-specific PCR, 

detection of the PAT and/or AAD-12 protein, and detection of the aad-12 gene (Section 5.5). All 

results confirmed the predicted inheritance pattern of the transgene at a single locus (Section 

5.6).
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Table 2. List of probes and their positions in plasmid pDAB8264.

Probe Name Position in

pDAB8264

Size 

(bp)

RB7 306-1315 1010

Histone H4A748 UTR 1356-1907 552

2mepsps 2048-3759 1712

Histone H4A748 Promoter 3682-5197 1516

AtUbi10 Promoter 5347-6659 1313

aad-12 6616-7497 882

AtuORF23 UTR 7637-8049 413

CsVMV 8172-8703 532

pat 8676-9284 609

AtuORF1 UTR 9257-10055 799

Backbone 3 10670-10990 321

Ori-Rep 10971-12057 1087

Backbone 2 12038-13751 1714

Backbone 1 13721-14974 1254

SpecR 14955-15749 795

Backbone 4 15724-16015 292
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Figure 5. Location of probes on pDAB8264 used in Southern blot analysis of DAS-444Ø6-6 

soybean.

pDAB8264

16018 bp

SpecR

trfA

TPotp C

2mepsps

pat

aad-12

RB7 Probe

Histone UTR Probe

2mEPSPS Probe

Histone Promoter Probe

AtUbi10 Promoter Probe

aad-12 Probe

CsVMV Probe

pat Probe

AtuORF1 UTR Probe

Backbone 3 Probe

Ori-Rep Probe

Backbone 2 Probe

Backbone 1 Probe

SpecR Probe

Backbone 4 Probe

AtuORF23 UTR Probe

Histone H4A748 promoter

AtUbi10 promoter

CsVMV promoter

Ori Rep

AtuORF23 3' UTR

AtuORF1 3' UTR

Histone H4A748 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR
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Table 3. Predicted and observed sizes of hybridizing fragments in Southern blot 

analyses of DAS-444Ø6-6 soybean.

Probe/ 

Feature

Enzyme Sample Expected 

Result (bp)1

Observed 

Result (bp)2

Figure 

Number

RB7

pDAB8264 9322 ~9300

Figure

8A

HindIII Maverick None None

DAS-444Ø6-6 >4261 ~4700

pDAB8264 5929 ~5900

Figure

8B

MscI Maverick None None

DAS-444Ø6-6 >1330 ~3400

Histone 

H4A748 

UTR

pDAB8264 9322 ~9300

Figure

9A

HindIII Maverick None None

DAS-444Ø6-6 >4261 ~4700

pDAB8264 10089 ~10100

Figure

9B

MscI Maverick None None

DAS-444Ø6-6 >9328 ~15000

Histone 

H4A748 

Promoter

pDAB8264 16018 ~16000

Figure

10A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 10089 ~10100

Figure

10B

MscI Maverick None None

DAS-444Ø6-6 >9328 ~15000

AtUbi10 

Promoter

pDAB8264 16018 ~16000

Figure

11A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 10089 ~10100

Figure

11B

MscI Maverick None None

DAS-444Ø6-6 >9328 ~15000

AtuORF23 

UTR

pDAB8264 16018 ~16000

Figure

12A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 4731 ~4700

Figure

12B

HindIII Maverick None None

DAS-444Ø6-6 >4432 ~7000
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Probe/ 

Feature

Enzyme Sample Expected 

Result (bp)1

Observed 

Result (bp)2

Figure 

Number

CsVMV

pDAB8264 16018 ~16000

Figure

13A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 4731 ~4700

Figure

13B

HindIII Maverick None None

DAS-444Ø6-6 >4432 ~7000

AtuORF1 

UTR

pDAB8264 16018 ~16000

Figure

14A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 4731 ~4700

Figure

14B

HindIII Maverick None None

DAS-444Ø6-6 >4432 ~7000

2mepsps 

pDAB8264 16018 ~16000

Figure

15A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 9322 ~9300

Figure

15B

HindIII Maverick None None

DAS-444Ø6-6 >4261 ~4700

pDAB8264 10089 ~10100

Figure

15C

MscI Maverick None None

DAS-444Ø6-6 >9328 ~15000

aad-12

pDAB8264 16018 ~16000

Figure

16A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 4731 ~4700

Figure

16B

HindIII Maverick None None

DAS-444Ø6-6 >4432 ~7000

pDAB8264 10089 ~10100

Figure

16C

MscI Maverick None None

DAS-444Ø6-6 >9328 ~15000
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Probe/ 

Feature

Enzyme Sample Expected 

Result (bp)1

Observed 

Result (bp)2

Figure 

Number

pat

pDAB8264 16018 ~16000

Figure

17A

XhoI Maverick None None

DAS-444Ø6-6 >10093 ~12000

pDAB8264 4731 ~4700 Figure

HindIII Maverick None None 17B

DAS-444Ø6-6 >4432 ~7000

pDAB8264 10089 ~10100 Figure

MscI Maverick None None 17C

DAS-444Ø6-6 >9328 ~15000

Histone

H4A748

Promoter

pDAB8264 4469 ~4500

Figure

Maverick 18ANone None

DAS-444Ø6-6 4469 ~4500

2mepsps 

MscI/EcoRI pDAB8264 4469 ~4500

Figure

18B

(Release Maverick None None

PTU) DAS-444Ø6-6 4469 ~4500

Histone 

H4A748 

UTR

pDAB8264 4469 ~4500

Figure

Maverick 18CNone None

DAS-444Ø6-6 4469 ~4500

AtUbi10 

Promoter

pDAB8264 2868 ~2900

Figure

Maverick 19ANone None

DAS-444Ø6-6 2868 ~2900

aad-12

PstI/XhoI pDAB8264 2868 ~2900

Figure

19B

(Release Maverick None None

PTU) DAS-444Ø6-6 2868 ~2900

AtuORF23 

UTR

pDAB8264 2868 ~2900

Figure

Maverick 19CNone None

DAS-444Ø6-6 2868 ~2900

CsVMV

pDAB8264 1928 ~1900

Figure

Maverick 20ANone None

DAS-444Ø6-6 1928 ~1900

pat

PstI/XhoI pDAB8264 1928 ~1900

Figure

20B

(Release Maverick None None

PTU) DAS-444Ø6-6 1928 ~1900

AtuORF1 

UTR

pDAB8264 1928 ~1900

Figure

Maverick 20CNone None

DAS-444Ø6-6 1928 ~1900
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Probe/ 

Feature

Enzyme Sample Expected 

Result (bp)1

Observed 

Result (bp)2

Figure 

Number

pDAB8264 1049, 5929 ~1000, ~5900

Figure

21A

MscI/EcoRI Maverick None None

Backbone 3/ DAS-444Ø6-6 None None

Backbone 4 pDAB8264 4731, 9322 ~4700, ~9300

Figure

21B

HindIII Maverick None None

DAS-444Ø6-6 None None

Ori-Rep

pDAB8264 9322 ~9300

Figure

22A

HindIII Maverick None None

DAS-444Ø6-6 None None

pDAB8264 9288 ~9300

Figure

22B

PstI/XhoI Maverick None None

DAS-444Ø6-6 None None

Backbone 1

pDAB8264 9322 ~9300

Figure

23A

HindIII Maverick None None

DAS-444Ø6-6 None None

pDAB8264 9288 ~9300

Figure

23B

PstI/XhoI Maverick None None

DAS-444Ø6-6 None None

Backbone 2

pDAB8264 9322 ~9300

Figure

24A

HindIII Maverick None None

DAS-444Ø6-6 None None

pDAB8264 9288 ~9300

Figure

24B

PstI/XhoI Maverick None None

DAS-444Ø6-6 None None

SpecR

pDAB8264 9322 ~9300

Figure

25A

HindIII Maverick None None

DAS-444Ø6-6 None None

pDAB8264 9288 ~9300

Figure

25B

PstI/XhoI Maverick None None

DAS-444Ø6-6 None None

1. Expected fragment sizes are based on the plasmid map of pDAB8264 (Figure 6) and the linearized T-DNA map 

(Figure 7).

2. Observed fragment sizes are considered approximations from these analyses and are based on the indicated sizes 

of the DIG-labeled DNA Molecular Weight Marker fragments. Due to the incorporation of DIG molecules for 

visualization, the Marker fragments typically run approximately 5-10% larger than their actual indicated 

molecular weight.
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Figure 6. Plasmid map of pDAB8264 with selected restriction enzyme sites used for 

Southern analysis.

pDAB8264 

16018 bp

SpecR

trfA

TPotp C

2mepsps

aad-12

pat

Histone H4A748 promoter

AtUbi10 promoter

CsVMV promoter

Ori Rep

Histone H4A748 3' UTR

AtuORF23 3' UTR

AtuORF1 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR

XhoI (10093)

MscI (1330)

MscI (11419)

EcoRI (5799)

EcoRI (9322)

EcoRI (10370)

HindIII (4261)

HindIII (5281)

HindIII (6226)

HindIII (10957)

PstI (3363)

PstI (5207)

PstI (5297)

PstI (8165)
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Figure 7. Linearized intended T-DNA insert from pDAB8264 with restriction enzymes used 

for DNA digestion and the expected hybridization bands.

5.2. Analysis of the Insert and Its Genetic Elements

5.2.1. Number of Insertion Sites

The restriction enzymes MscI, XhoI and HindIII were chosen to determine the number of 

insertions in DAS-444Ø6-6 soybean (Table 3 and Figure 6). Probes derived from the DNA 

sequences for 2mepsps, Histone H4A748 Promoter, Histone H4A748 UTR, aad-12, pat, RB7, 

AtUbi10 Promoter, AtuORF23 UTR, CsVMV, and AtuORF1 UTR were then hybridized to the 

digested genomic DNA to determine the number of insertion sites in DAS-444Ø6-6 soybean.

When digested with the MscI restriction enzyme and independently probed with the Histone 

H4A748 UTR, Histone H4A748 Promoter, AtUbi10 Promoter, 2mepsps, aad-12 and pat probes, 

a single hybridization band of ~15000 bp was observed in all DAS-444Ø6-6 samples, consistent 

with the predicted size of >9328 bp for the MscI fragment as indicated in Figure 7 (Figure 9B, 

Figure 10B, Figure 11B, Figure 15C, Figure 16C, Figure 17C respectively). The same enzyme 

digestion was also used for characterization of the RB7 MAR feature. The resulting Southern 

1844bp

>1330 bp

pDAB8264 T-DNA

10658 bp

2mepsps

aad-12

pat

TPotp C

Histone H4A748 promoter

AtUbi10 promoter

CsVMV promoter

Histone H4A748 3' UTR

AtuORF23 3' UTR

AtuORF1 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border B T-DNA Border A

RB7 MAR

Msc I (1330)

Xho I (10093)

Eco RI (5799)

Eco RI (9322)

Eco RI (10370)

Hin dIII (4261)

Hin dIII (5281)

Hin dIII (6226)

Pst I (3363)

Pst I (5207)

Pst I (5297)

Pst I (8165)

MscI >9328 bp

HindIII >4261 bp >4432 bp

XhoI >10093 bp

PstI/ XhoI >3363 bp 2868bp 1928bp

MscI/ EcoRI 4469bp 3525bp
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analysis indicated a single band of ~3400 bp in DAS-444Ø6-6 samples which is consistent with 

the expected size of >1330 bp (Figure 8B).

For additional characterization of the T-DNA insert, genomic DNA samples were digested with 

the XhoI restriction enzyme and independently probed with the Histone H4A748 Promoter, 

AtUbi10 Promoter, AtuORF23 UTR, CsVMV, AtuORF1 UTR, 2mepsps, aad-12, and pat 

probes. In each case, a single hybridization band of ~12000 bp was observed in all

DAS-444Ø6-6 samples, consistent with the predicted size of >10093 bp as indicated in Figure 7

(Figure 10A, Figure 11A, Figure 12A, Figure 13A, Figure 14A, Figure 15A, Figure 16A, and 

Figure 17A respectively).

Moreover, the restriction enzyme HindIII was also used to provide further characterization of 

DAS-444Ø6-6 soybean. Digestion of the genomic DNA with this enzyme followed by 

independently probing with the RB7, Histone H4A748 UTR, or 2mepsps probe resulted in a 

single hybridization band of ~4700 bp across all DAS-444Ø6-6 samples, which is consistent 

with the expected size of >4261 bp as indicated in Figure 7 (Figure 8A, Figure 9A, and Figure 

15B respectively). Digestion of the genomic DNA with HindIII and probed independently with 

the AtuORF23 UTR, CsVMV, AtuORF1 UTR, aad-12, or pat probe resulted in a single 

hybridization band of ~7000 bp for all DAS-444Ø6-6 samples. This hybridization pattern is 

consistent with the expected size of >4432 bp for all probe combinations (Figure 12B, Figure 

13B, Figure 14B, Figure 16B, and Figure 17B respectively).

All these data indicate that there is a single insertion of the T-DNA containing all the expected

elements in DAS-444Ø6-6 soybean genome.

5.2.2. Structure of the Insert and Genetic Elements

According to the restriction map of the T-DNA insert in pDAB8264 in Figure 6 and Figure 7, the 

plant transcription unit (PTU) for aad-12 and pat could be released by restriction digestion with

PstI/XhoI, while the 2mepsps PTU could be released with MscI/EcoRI digestion. These 

digestions were performed to verify the presence of intact PTUs in DAS-444Ø6-6 soybean.

When digested with MscI/EcoRI and hybridized with the 2mepsps probe, each individual 

DAS-444Ø6-6 plant across the five generations along with the pDAB8264 positive control 

resulted in a single hybridization band of ~4500 bp, which is consistent with the predicted size of 

4469 bp for the 2mepsps PTU (Figure 18B). When the same genomic DNA samples were probed 

with the Histone H4A748 Promoter or Histone H4A748 UTR probe, the same hybridization band 

of ~4500 bp was detected in the same sample set (Figure 18A and Figure 18C). These data 

indicate that an intact 2mepsps PTU is present in all generations of DAS-444Ø6-6 soybean 

tested.

When digested with PstI/XhoI and hybridized with an aad-12 probe, each individual 

DAS-444Ø6-6 plant across the five generations along with the pDAB8264 positive control 

resulted in a single hybridization band of ~2900 bp, which is consistent with the predicted size of 

2868 for the aad-12 PTU (Figure 19B). When the same genomic DNA samples were hybridized 

with the AtUbi10 Promoter or AtuORF23 UTR probes, the same hybridization band of ~2900 bp 

was also detected in the same sample set (Figure 19A and Figure 19C). These data indicate that 

an intact aad-12 PTU is present in all generations of DAS-444Ø6-6 soybean tested.
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When digested with PstI/XhoI and hybridized with pat probe, each individual DAS-444Ø6-6 

plant across the five generations along with the pDAB8264 positive control resulted in a single 

hybridization band of ~1900 bp, which is consistent with the predicted size of 1928 bp for the 

pat PTU (Figure 20B). When the same genomic DNA samples were hybridized with the CsVMV 

or AtuORF1 UTR probes, the same hybridization band of ~1900 bp was detected in the same 

sample set (Figure 20A and Figure 20C). These data indicate that an intact pat PTU is present in 

all generations of DAS-444Ø6-6 soybean tested.

As expected, no specific hybridization bands were detected in the negative control samples in 

any of the restriction enzyme and probe combinations.

Taken together, the Southern blot analyses indicate that the single insert in DAS-444Ø6-6 

soybean contains an intact single copy of each of the PTUs for 2mepsps, aad-12, and pat.

5.3. Absence of Vector Backbone DNA

To confirm that no plasmid vector backbone sequences exist in DAS-444Ø6-6 soybean, six 

probes covering nearly the entire backbone region of pDAB8264 were used to hybridize the 

Southern blots containing genomic DNA samples digested with MscI/EcoRI, HindIII, and 

PstI/XhoI (Figure 21 - Figure 25). Based on the expected fragment sizes of MscI/EcoRI and 

HindIII digestions, the Backbone 3 and Backbone 4 probes were mixed at an approximate 1:1 

molar ratio for hybridization, while the Ori-Rep, Backbone 1, Backbone 2, and SpecR probes 

were hybridized independently on separate blots of HindIII and PstI/XhoI digestions. The results 

demonstrated that no specific hybridization bands were detected in any samples tested, except for 

the positive controls, as expected (Figure 21 - Figure 25). When hybridized with the Backbone 4 

and Backbone 3 probes, the MscI/EcoRI digested positive control sample had an observed 

hybridization band of ~5900 bp for Backbone 4 and ~1000 bp for backbone 3 probes,

respectively, which is consistent with the expected bands of 5929 bp and 1049 bp, respectively 

(Figure 21A). When hybridized with the same two probes, the HindIII digested positive control 

samples had an observed hybridization band of ~9300 bp for the Backbone 4 probe and an 

observed band of ~4700 bp for the Backbone 3 probe, which is consistent with the expected 

bands of 9322 bp and 4731 bp, respectively (Figure 21B). When hybridized with the individual 

Ori-Rep, Backbone 1, Backbone 2, and SpecR probes, the HindIII and PstI/XhoI digested 

positive control samples resulted in a single hybridization band of ~9300 bp and ~9300 bp,

respectively, which is consistent with the expected bands of 9322 bp and 9288 bp, respectively 

(Figure 22 - Figure 25). Taken together, these data confirm that no vector backbone sequences 

from pDAB8264 were integrated into DAS-444Ø6-6 soybean.

5.4. Stability of the Insert across Generations

Southern blot hybridizations were conducted with samples across five distinct generations (T2, 

T3, T4, T6, and F2) of DAS-444Ø6-6 soybean. Prior to initiation of Southern blot analysis, all 

plants were tested for PAT protein expression using a lateral flow strip test kit to allow for 

confirmation of PAT expression-positive plants. All of the genetic element probes: 2mepsps, 

Histone H4A748 promoter, Histone H4A748 UTR, aad-12, AtUbi10 promoter, AtuORF23 UTR, 

CsVMV promoter, pat, AtuORF1 UTR, and RB7 MAR, as well as the probes covering the 

vector backbone sequences of plasmid pDAB8264, were hybridized with the DAS-444Ø6-6 

soybean samples. As described above, results across all five generations of DAS-444Ø6-6 

soybean were consistent with what was expected (Table 3, Figure 8 - Figure 25), indicating 
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stable integration and inheritance of the intact, single copy transgene insert across multiple 

generations of DAS-444Ø6-6 soybean.
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Figure 8. Southern blot analysis of HindIII and MscI digested DAS-444Ø6-6 soybean: RB7 probe.

Approximately 10 g of genomic DNA was digested with HindIII (Panel A) and MscI (Panel B) and hybridized with the RB7 probe. Plasmid pDAB8264 served 

as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II and VII are 

indicated adjacent to image; 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 9. Southern blot analysis of HindIII and MscI digested DAS-444Ø6-6 soybean: Histone H4A748 UTR probe.

Approximately 10 g of DAS-444Ø6-6 genomic DNA was digested with HindIII (Panel A) and MscI (Panel B) and hybridized with the Histone UTR probe. 

Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight 

Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 10. Southern blot analysis of XhoI and MscI digested DAS-444Ø6-6 soybean: Histone H4A748 Promoter probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A) and MscI (Panel B) and hybridized with the Histone Promoter probe. Plasmid 

pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II 

and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 11. Southern blot analysis of XhoI and MscI digested DAS-444Ø6-6 soybean: AtUbi10 Promoter probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A) and MscI (Panel B) and hybridized with the AtUbi10 Promoter probe. Plasmid 

pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II 

and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 12. Southern blot analysis of XhoI and HindIII digested DAS-444Ø6-6 soybean: AtuORF23 UTR probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A) and HindIII (Panel B) and hybridized with the AtuORF23 UTR probe. Plasmid 

pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II 

and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 13. Southern blot analysis of XhoI and HindIII digested DAS-444Ø6-6 soybean: CsVMV probe.

Approximately 10 g genomic DNA was digested with XhoI (Panel A) and HindIII (Panel B) and hybridized with the CsVMV probe. Plasmid pDAB8264 served 

as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II and VII are 

indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 14. Southern blot analysis of XhoI and HindIII digested DAS-444Ø6-6 soybean: AtuORF1 UTR probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A) and HindIII (Panel B) and hybridized with the AtuORF1 UTR probe. Plasmid 

pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II 

and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 15. Southern blot analysis of XhoI, HindIII and MscI digested DAS-444Ø6-6 soybean: 2mepsps probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A), HindIII (Panel B) and MscI (Panel C) followed by hybridization with the 2mepsps v1

probe. Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular 

Weight Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B Panel C

Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B Panel C
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Figure 16. Southern blot analysis of XhoI, HindIII and MscI digested DAS-444Ø6-6 soybean: aad-12 probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A), HindIII (Panel B) and MscI (Panel C) followed by hybridization with the aad-12

probe. Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular 

Weight Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B Panel C

Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B Panel C
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Figure 17. Southern blot analysis of XhoI, HindIII, and MscI digested DAS-444Ø6-6 soybean: pat probe.

Approximately 10 g of genomic DNA was digested with XhoI (Panel A), HindIII (Panel B) and MscI (Panel C) followed by hybridization with the Pat probe. 

Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight 

Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B Panel C

Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B Panel C
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Figure 18. Southern blot analysis of MscI/EcoRI digested DAS-444Ø6-6 soybean: Histone H4A748 Promoter, 2mepsps and 

Histone H4A748 UTR probes.

Approximately 10 g of genomic DNA was digested with MscI/EcoRI followed by hybridization with the Histone Promoter (Panel A), 2mepsps (Panel B) and 

Histone UTR (Panel C) probes. Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIGlabeled DNA Molecular Weight Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B Panel C

Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-3 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2 2 Maverick-3 + pDAB8264 12 4406-T3-2

3 Maverick-3 13 4406-T3-3 3 Maverick-1 13 4406-T3-3 3 Maverick-3 13 4406-T3-3

4 Maverick-2 14 4406-T4-1 4 Maverick-2 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B Panel C
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Figure 19. Southern blot analysis of PstI/XhoI digested DAS-444Ø6-6 soybean: AtUbi10 Promoter, aad-12, and AtuORF23 

UTR probes.

Approximately 10 g of genomic DNA was with digested PstI/XhoI followed by hybridization with the AtUbi10 Promoter (Panel A), aad-12 (Panel B) and 

AtuORF23 UTR (Panel C) probes. Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the 

DIG-labeled DNA Molecular Weight Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B Panel C

Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-1 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2

3 Maverick-1 13 4406-T3-3 3 Maverick-1 13 4406-T3-3 3 Maverick-1 13 4406-T3-3

4 Maverick-2 14 4406-T4-1 4 Maverick-2 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B Panel C
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Figure 20. Southern blot analysis of PstI/XhoI digested DAS-444Ø6-6 soybean: CsVMV, pat, and AtuORF1 UTR probes.

Approximately 10 g of genomic DNA was digested with PstI/XhoI followed by hybridization with the CsVMV (Panel A), Pat (Panel B) and AtuORF1 UTR 

(Panel C) probes. Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA 

Molecular Weight Marker II and VII are indicated adjacent image. 4406= DAS-444Ø6-6.

Panel A Panel B Panel C

Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-1 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2

3 Maverick-1 13 4406-T3-3 3 Maverick-1 13 4406-T3-3 3 Maverick-1 13 4406-T3-3

4 Maverick-2 14 4406-T4-1 4 Maverick-2 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B Panel C
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Figure 21. Southern blot analysis of MscI/EcoRI and HindIII digested DAS-444Ø6-6 soybean: Backbone 3 and Backbone 4 

probes.

Approximately 10 g of genomic DNA was digested with MscI/EcoRI (Panel A) and HindIII (Panel B) and hybridized with the Backbone 3 and Backbone 4

probes. Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular 

Weight Marker II and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-1 + pDAB8264 12 4406-T3-2 2 Maverick-5 + pDAB8264 12 4406-T3-2

3 Maverick-1 13 4406-T3-3 3 Maverick-5 13 4406-T3-3

4 Maverick-2 14 4406-T4-1 4 Maverick-4 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 22. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 soybean: Ori-Rep probe.

Approximately 10 g of genomic DNA was digested with HindIII (Panel A) PstI/XhoI and (Panel B) and hybridized with the Ori-Rep probe. Plasmid pDAB8264 

served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II and VII are 

indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-1 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 23. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 soybean: Backbone 1 probe.

Approximately 10 g of genomic DNA was digested with HindIII (Panel A) PstI/XhoI and (Panel B) and hybridized with the Backbone 1 probe. Plasmid 

pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II 

and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-1 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 24. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 soybean: Backbone 2 probe.

Approximately 10 g of genomic DNA was digested with HindIII (Panel A) PstI/XhoI and (Panel B) and hybridized with the Backbone 2 probe. Plasmid 

pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II 

and VII are indicated adjacent to image. 4406= DAS-444Ø6-6.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-1 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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Figure 25. Southern blot analysis of HindIII and PstI/XhoI digested DAS-444Ø6-6 soybean: SpecR probe.

Approximately 10 g of genomic DNA was digested with HindIII (Panel A) Pst I/Xho I and (Panel B) and hybridized with the SpecR probe. Plasmid pDAB8264 

served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG-labeled DNA Molecular Weight Marker II and VII are 

indicated adjacent to image. 4406= DAS-444Ø6-6. Note: The splotch at ~4000 bp between lanes 9 and 10 in panel B is non-specific background signal since it falls between the lanes.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1 DIG MWM II 11 4406-T3-1 1 DIG MWM II 11 4406-T3-1

2 Maverick-5 + pDAB8264 12 4406-T3-2 2 Maverick-1 + pDAB8264 12 4406-T3-2

3 Maverick-5 13 4406-T3-3 3 Maverick-1 13 4406-T3-3

4 Maverick-4 14 4406-T4-1 4 Maverick-2 14 4406-T4-1

5 4406-T2-1 15 4406-T4-4 5 4406-T2-1 15 4406-T4-4

6 4406-T2-2 16 4406-T4-5 6 4406-T2-2 16 4406-T4-5

7 4406-T2-3 17 4406-T6-1 7 4406-T2-3 17 4406-T6-1

8 4406-F2-1 18 4406-T6-2 8 4406-F2-1 18 4406-T6-2

9 4406-F2-3 19 4406-T6-3 9 4406-F2-3 19 4406-T6-3

10 4406-F2-4 20 DIG MWM VII 10 4406-F2-4 20 DIG MWM VII

Panel A Panel B
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5.5. Segregation Analysis

5.5.1. Genetic and Molecular Analysis of a Segregating Generation

The inheritance pattern of the transgene insert within a segregating generation was demonstrated 

with event-specific PCR analysis of individual plants from a F2 population of DAS-444Ø6-6 

soybean. The F1 generation was generated by crossing T2 plants of DAS-444Ø6-6 soybean with 

a conventional proprietary soybean line. The F1 plants were self pollinated to produce the F2 

seeds.

A total of 119 F2 plants were tested by event-specific PCR to determine the presence or absence 

of the DAS-444Ø6-6 transgene insert. Genomic DNA samples from each of the 119 plants, along 

with DNA samples from the non-transgenic control Maverick, were analyzed by DAS-444Ø6-6 

soybean event-specific PCR. Of the 119 plants tested, 96 plants were positive for the presence of 

DAS-444Ø6-6 transgene insert, and the remaining 23 plants were negative (segregated null). In 

addition, leaves of the individual plants were tested for the presence or absence of the PAT 

protein using a PAT-specific lateral flow strip test kit. All of the plants that tested positive for 

DAS-444Ø6-6 transgene insert displayed positive result for PAT protein expression, and all

plants that were negative for DAS-444Ø6-6 transgene insert displayed negative result for PAT 

protein expression (Table 4), confirming that the phenotypic segregation matched the genotypic 

makeup of the tested F2 population. Statistical analysis using a 2 goodness of fit test indicated 

the genotypic segregation ratio of the plants with positive transgene insert versus negative is 

consistent with the 3:1 segregation ratio characteristic of the Mendelian inheritance pattern of a 

single dominant trait.

Table 4. Results of F2 individual plants tested for PAT expression and event-specific PCR 

within a single segregating generation.

Tested Method Total plants 

tested

Positive Negative Expected 

ratio

P-valuea

Event-Specific PCR 119 96 23 3:1 0.153 PAT Expression 119 96 23 3:1

a Based on a chi-squared goodness of fit test

5.5.2. Segregation Analysis of Breeding Generations

Chi-square analysis of trait inheritance data from three populations of the BC1F2 breeding 

generation was conducted to determine the Mendelian inheritance of aad-12 and 2mepsps in 

DAS-444Ø6-6 soybeans. The presence or absence of aad-12 and 2mepsps was determined using 

a herbicide spray (2,4-D + glyphosate), which is specific for AAD-12 and 2mEPSPS protein 

expressing soybeans. The expected segregation ratio of 3:1 for plants expressing AAD-12 and 

2mEPSPS versus plants that do not express AAD-12 nor 2mEPSPS proteins was observed

(Table 5).
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Table 5. Results of BC1F2 individual plants from DAS-444Ø6-6 soybean tested for AAD-12 

and 2mEPSPS protein expression within a single segregating generation.

Generation/

Source ID

Total 

plants 

tested

aad-12 and

2mepsps gene 

positive (resistant to

2,4-D + glyphosate)

aad-12 and 2mepsps

gene negative 

(susceptible to 2,4-D 

+ glyphosate)

Expected 

ratio

P-valuea

BC1F2 39 27 12 3:1 0.4054

BC1F2 58 46 12 3:1 0.4484

BC1F2 31 26 5 3:1 0.2540

a Based on a chi-squared goodness of fit test

5.6. Summary of the Genetic Characterization

DAS-444Ø6-6 soybean was produced using Agrobacterium-mediated transformation with the 

plasmid pDAB8264. The T-DNA insert of pDAB8264 consists of 1) the 2mepsps gene, 

controlled by the Histone H4A748 promoter and Histone H4A748 3′ UTR regulatory sequences; 

2) the aad-12 gene, controlled by the AtUbi10 promoter and AtuORF23 3′ UTR regulatory 

sequences; 3) the pat gene, controlled by the CsVMV promoter and AtuORF1 3′ UTR regulatory 

sequences. In addition, a RB7 MAR element is located at the 5′ end of the T-DNA insert. 

Various breeding generations were developed and used to examine the integrity, stability, and 

inheritance of the transgenic insert in DAS-444Ø6-6 soybean.

Molecular characterization of DAS-444Ø6-6 soybean by Southern blot analysis confirmed the 

single insertion of the T-DNA insert from pDAB8264 containing a single intact copy of each of 

the 2mepsps, aad-12 and pat PTUs. No additional DNA fragments from the 2mepsps, aad-12

and pat expression cassettes were identified in DAS-444Ø6-6 and no plasmid backbone 

sequences were present. The T-DNA insert for DAS-444Ø6-6 was shown to be stably integrated 

across five breeding generations (T2, T3, T4, T6, and F2) tested. Moreover, DAS-444Ø6-6

displayed the expected Mendelian inheritance pattern for a single independent insert/locus in a 

segregating generation (F2).
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6. Characterization of the Introduced Proteins

6.1. AAD-12

6.1.1. Identity of the AAD-12 Protein

The aryloxyalkanoate dioxygenase (AAD-12) protein was derived from Delftia acidovorans, a 

gram-negative soil bacterium. The amino acid sequence is identical to the native enzyme 

sequence except for the addition of an alanine at position number 2. The additional alanine codon 

encodes part of an NcoI restriction enzyme recognition site (CCATGG) spanning the ATG

translational start codon. This additional codon serves the dual purpose of facilitating subsequent 

cloning operations and improving the sequence context surrounding the ATG start codon to 

optimize translation initiation. The proteins encoded by the native and plant-optimized coding 

regions are 99.3% identical, differing only at amino acid number 2. The AAD-12 protein is 

comprised of 293 amino acids and has a molecular weight of ~32 kDa (Figure 26).

001 MAQTTLQITPTGATLGATVTGVHLATLDDAGFAALHAAWLQHALLIFPGQ

051 HLSNDQQITFAKRFGAIERIGGGDIVAISNVKADGTVRQHSPAEWDDMMK

101 VIVGNMAWHADSTYMPVMAQGAVFSAEVVPAVGGRTCFADMRAAYDALDE

151 ATRALVHQRSARHSLVYSQSKLGHVQQAGSAYIGYGMDTTATPLRPLVKV

201 HPETGRPSLLIGRHAHAIPGMDAAESERFLEGLVDWACQAPRVHAHQWAA

251 GDVVVWDNRCLLHRAEPWDFKLPRVMWHSRLAGRPETEGAALV

Figure 26. Amino acid sequence of the AAD-12 protein.

6.1.2. Mode of Action of the AAD-12 Protein

Expression of the AAD-12 protein in transgenic crops has been shown to provide tolerance to the 

herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) by catalyzing the conversion of 2,4-D to 

2,4-dichlorophenol (DCP) a herbicidally inactive compound (Figure 27) (Müller et al., 1999; 

Westendorf et al., 2002; Westendorf et al., 2003; Wright et al., 2009; Wright et al., 2010). 

AAD-12 is also able to degrade related achiral phenoxyacetate herbicides such as MCPA 

((4-chloro-2-methylphenoxy) acetic acid) and pyridyloxyacetate herbicides such as triclopyr and 

fluroxypyr to their corresponding inactive phenols and pyridinols, respectively (Figure 28).

AAD-12 has selectivity for (S)-enantiomers of the chiral phenoxy acid herbicides (e.g., 

dichlorprop and mecoprop), but does not catalyze degradation of the (R)-enantiomers (Kohler, 

1999; Schleinitz et al., 2004). The R-enantiomers are herbicidally active; therefore, AAD-12 

does not provide tolerance to commercially-available chiral phenoxy acid herbicides.
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O

O

OH

Cl Cl

2,4-D

OH

Cl Cl AAD-12

DCP

Figure 27. Degradation reaction of 2,4-D catalyzed by AAD-12.

Figure 28. General reaction catalyzed by AAD-12 (R=H or CH3).

6.1.3. Biochemical Characterization of the AAD-12 Protein

Large quantities of purified AAD-12 protein are required to perform safety assessment studies. 

Because it is technically infeasible to extract and purify sufficient amounts of recombinant 

protein from transgenic plants (Evans, 2004), the AAD-12 protein was microbially-produced in

Pseudomonas fluorescens. Characterization studies were performed to confirm the equivalency 

of the AAD-12 protein produced in P. fluorescens with the AAD-12 protein produced in planta 

in DAS-444Ø6-6 soybean. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDSPAGE), western blot, glycoprotein detection, and protein sequence analysis by matrix assisted 

laser desorption/ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF 

MS/MS) were used to characterize the biochemical properties of the protein. Using these 

methods, the AAD-12 protein isolated from P. fluorescens and DAS-444Ø6-6 soybean were 

shown to be biochemically equivalent, thereby supporting the use of the microbe-derived protein 

in safety assessment studies.

The methods and results of the biochemical characterization of the DAS-444Ø6-6 soybean- and 

microbe-derived AAD-12 proteins are described in detail in Appendix 2. Briefly, both the plant 

and P. fluorescens-derived AAD-12 proteins were observed at the expected molecular weight of 

~32 kDa by SDS-PAGE and were immunoreactive to AAD-12 protein-specific antibodies by 

western blot analysis. There was no evidence of glycosylation of the DAS-444Ø6-6 soybean-
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derived AAD-12 protein. Greater than 84% of the soybean-derived protein amino acid sequence 

was confirmed by either enzymatic peptide mass fingerprinting or MS/MS sequence analysis by

MALDI-TOF MS/MS. The N-terminal methionine was found to be cleaved from both protein 

sources and a portion of the N-terminal peptide of the plant-derived AAD-12 was determined to 

be acetylated after the N-terminal methionine was cleaved [two forms of the N-terminal peptide 

were detected (both acetylated and non-acetylated forms)]. These two post-translational 

processes, cleavage of the N-terminal methionine residue and N-terminal acetylation, are 

common modifications that have been found to occur on the vast majority (~85%) of eukaryotic 

proteins (Polevoda and Sherman, 2003). The C-terminal peptides from DAS-444Ø6-6 soybean 

and P. fluorescens were intact and determined to be identical.

6.1.4. Expression of the AAD-12 Protein in Plant Tissues

A field expression study was conducted in the U.S. during 2010. Ten sites (Georgia, Iowa (2 

sites), Illinois (2 sites), Indiana, Michigan, Missouri, and Nebraska (2 sites)) were planted with 

DAS-444Ø6-6 soybean and the conventional control (Maverick). The test sites represented 

regions of diverse agronomic practices and environmental conditions for soybean in North 

America. Five treatments of the DAS-444Ø6-6 soybean (unsprayed, sprayed with 2,4-D, sprayed 

with glufosinate, sprayed with glyphosate, or sprayed with 2,4-D, glufosinate, and glyphosate) 

were tested (see Appendix 5 for application rates and timings). Plant tissues sampled included 

leaf, grain, root, and forage. Leaf tissues were collected at the V5 and V10-12 stages, while root 

and forage were collected at the R3 stage. Grain was collected at the R8 stage of development 

(Gaska, 2006).

The soluble, extractable AAD-12 protein was measured using a validated enzyme-linked 

immunosorbent assay (ELISA) method. AAD-12 protein levels for all tissue types were 

calculated on ng/mg dry weight basis. Methods used for tissue sampling and quantification of 

protein expression by ELISA are detailed in Appendix 5.

A summary of the AAD-12 protein concentrations (averaged across sites) in the various soybean 

matrices is shown in Table 6. Average expression values ranged from 23.52 ng/mg dry weight in 

R3 stage root to 121.22 ng/mg dry weight in V10-12 stage leaf tissue. Expression levels were 

comparable for all treatments. No AAD-12 protein was detected in the control tissues across the 

ten locations.
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Table 6. Expression of AAD-12 in DAS-444Ø6-6 soybean.

AAD-12 ng/mg Tissue Dry Weight

Tissue Treatment Mean Std. Dev. (n=10) Min/Max Range

Leaf V5

DAS-444Ø6-6 112.61 34.05 42.00 – 179.50

DAS-444Ø6-6 w/ 2,4-D 111.32 27.48 58.62 – 190.50

DAS-444Ø6-6 w/ Gluf 107.75 29.91 60.00 – 179.50

DAS-444Ø6-6 w/ Glyp 101.93 29.54 36.58 – 179.50

DAS-444Ø6-6 w/ All 103.67 34.25 52.50 – 196.50

Leaf V10-12

DAS-444Ø6-6 118.57 36.34 68.00 – 312.00

DAS-444Ø6-6 w/ 2,4-D 121.22 36.61 58.40 – 279.00

DAS-444Ø6-6 w/ Gluf 109.29 25.94 64.50 – 170.00

DAS-444Ø6-6 w/ Glyp 114.73 27.75 62.82 – 193.50

DAS-444Ø6-6 w/ All 119.83 46.45 54.00 – 240.00

Forage R3

DAS-444Ø6-6 73.47 20.77 35.00 – 122.00

DAS-444Ø6-6 w/ 2,4-D 72.53 22.59 37.00 – 117.50

DAS-444Ø6-6 w/ Gluf 73.75 20.39 37.00 – 123.50

DAS-444Ø6-6 w/ Glyp 76.04 19.36 40.00 – 121.00

DAS-444Ø6-6 w/ All 70.73 21.88 38.50 – 118.00

Root R3

DAS-444Ø6-6 23.52 10.81 0.77 – 52.80

DAS-444Ø6-6 w/ 2,4-D 24.62 10.16 0.67 – 67.60

DAS-444Ø6-6 w/ Gluf 24.35 11.12 ND – 67.60

DAS-444Ø6-6 w/ Glyp 29.03 7.86 2.19 – 67.40

DAS-444Ø6-6 w/ All 27.21 9.44 6.00 – 50.60

Grain

DAS-444Ø6-6 27.37 9.70 6.99 – 45.40

DAS-444Ø6-6 w/ 2,4-D 27.34 10.35 8.03 – 43.00

DAS-444Ø6-6 w/ Gluf 27.34 10.02 9.77 – 47.20

DAS-444Ø6-6 w/ Glyp 25.77 6.79 10.04 – 46.60

DAS-444Ø6-6 w/ All 25.83 6.51 12.60 – 42.00

Gluf = glufosinate; Glyp = glyphosate; All = 2,4-D + glufosinate + glyphosate

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the AAD-12 ELISA in all

tissue matrices were as follows:

AAD-12 (ng/mg

sample dry weight)

Matrix LOD LOQ

Leaf V5 0.5 1.0

Leaf V10-12 0.5 1.0

Root 0.5 1.0

Forage 0.5 1.0

Grain 0.5 1.0
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6.1.5. Food and Feed Safety Assessment for AAD-12 Protein

Results of the overall safety assessment of the AAD-12 protein indicate that it is unlikely to 

cause an allergic reaction in humans or be a toxin to humans or animals.

History of Safe Use

 The donor organism, Delftia acidovorans (formerly designated as Pseudomonas acidovorans

and Comamonas acidovorans) is a non glucose-fermenting, gram-negative, non sporeforming rod present in soil, fresh water, activated sludge, and clinical specimens (Von 

Graevenitz, 1985; Tamaoka et al., 1987; Wen et al., 1999).

 Delftia acidovorans can be used to transform ferulic acid into vanillin and related flavor 

metabolites (Toms and Wood, 1970; Rao and Ravishankar, 2000; Shetty et al., 2006). This 

utility has led to a history of safe use for Delftia acidovorans in the food processing industry. 

For example, see US Patent 5,128,253 ―Bioconversion process for the production of vanillin‖ 

issued on July 7, 1992 to Kraft General Foods (Labuda et al., 1992).

Lack of allergenic potential

 The step-wise, weight-of-evidence approach (Codex Alimentarius Commission, 2009) was 

used to assess the allergenic potential of the AAD-12 protein.

 The AAD-12 protein does not share meaningful amino acid sequence similarities with known 

allergens. No significant homology was identified when the AAD-12 protein sequence was 

compared with known allergens in the FARRP (Food Allergy Research and Resource 

Program) version 11.00 allergen database (Released in February, 2011), using the search 

criteria of either a match of eight or more contiguous identical amino acids, or >35% identity 

over 80 amino acid residues.

 The AAD-12 protein is rapidly degraded below the level of detection in simulated gastric 

fluid (SGF). The AAD-12 protein was readily digested, i.e., not detectable after 30 seconds, 

under in vitro SGF conditions (0.32% pepsin, pH 1.2; 37oC) as demonstrated by both 

SDS-PAGE and western blot analyses.

 The AAD-12 protein is not present in a glycosylated state. No glycosylation of the AAD-12 

protein was detected using SDS-PAGE and a glycosylation detection system (Appendix 2).

Lack of toxic potential

 The AAD-12 protein does not share meaningful amino acid sequence similarities with 

known toxins. Amino acid homologies with the AAD-12 protein sequence were evaluated 

using BLASTp search algorithm against the GenBank non-redundant protein sequences (up 

to date as of February 18, 2011 containing 13,473,798 sequences with 4,621,495,809 amino 

acids). By their annotations, the proteins returned by BLASTp search can be grouped into 

the following 11 categories: 2,4-D/alpha-ketoglutarate dioxygenase, alkylsulfatase AtsK, 

alpha-ketoglutarate (dependent) dioxygenase, alpha-ketoglutarate-dependent sulfonate 

dioxygenase, ketoglutarate dehyronase, taurine catabolism dioxygenase, taurine 

dioxygenase, dioxygenase, oxidoreductase, pyoverdine biosynthesis protein, and 

hypothetical (putative) or unnamed proteins. AAD-12 (aryloxyalkanoate dioxygenase-12) 

itself is an alpha-ketoglutarate dependent dioxygenase. Hypothetical and unnamed proteins 

are derived from conceptual translation of DNA sequences generated from massive genome 
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sequencing projects of various fungi and bacteria. Those proteins have functional 

annotations such as ―probable taurine catabolism dioxygenase‖, ―clavaminic acid synthetase 

(CAS) –like‖, and ―putative alpha-ketoglutarate dependend dioxygenase‖. None of the 

proteins returned by the BLASTp search are associated with protein toxins that are harmful 

to humans or animals.

 In acute mouse toxicity testing, there were no mortalities or treatment-related clinical signs in 

CD-1 mice after oral administration by gavage of AAD-12 protein at 2000 mg protein/kg 

body weight.

6.1.6. Summary of AAD-12 Protein Characterization

The aryloxyalkanoate dioxygenase (AAD-12) protein was derived from Delftia acidovorans, a

gram-negative soil bacterium. AAD-12 is comprised of 293 amino acids and has a molecular 

weight of ~32 kDa. Detailed biochemical characterization of the AAD-12 protein derived from 

DAS-444Ø6-6 soybean and microbial sources was conducted. Additionally, characterization of 

AAD-12 protein expression in DAS-444Ø6-6 soybean over the growing season was 

determined by analyzing leaf, root, whole plant, and grain tissues from DAS-444Ø6-6 soybean

sprayed with 2,4-D, glufosinate, glyphosate, all three herbicides in combination, and nonsprayed.

A step-wise, weight-of-evidence approach was used to assess the potential for toxic or 

allergenic effects from the AAD-12 protein. Bioinformatic analyses revealed no meaningful 

homologies to known or putative allergens or toxins for the AAD-12 amino acid sequence. The 

AAD-12 protein hydrolyzed rapidly in simulated gastric fluid. There was no evidence of acute 

toxicity in mice at a dose of 2000 mg/kg body weight of AAD-12 protein. Glycosylation 

analysis revealed no detectable covalently linked carbohydrates in AAD-12 protein expressed 

in DAS-444Ø6-6 soybean. The low level expression of the AAD-12 protein presents a low 

exposure risk to humans and animals, and the results of the overall safety assessment of the 

AAD-12 protein indicate that it is unlikely to cause allergenic or toxic effects in humans or 

animals.
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6.2. 2mEPSPS

6.2.1. Identity of the 2mEPSPS Protein

The double mutant 5-enolpyruvylshikimate-3-phosphate synthase (2mEPSPS) protein is encoded 

by a modified epsps gene from corn (Zea mays) (Herouet-Guicheney et al., 2009). The 2mepsps

transgene in DAS-444Ø6-6 encodes a protein sequence that is the wild-type EPSPS carrying two 

substitutions at amino acids 102 and 106 (Figure 29: UniProt Accession number: O24566). The 

mutations are known as the TIPS mutation where threonine was substituted by isoleucine at 

amino acid 102 and proline was substituted by serine at amino acid 106. The 2mEPSPS protein 

is comprised of 445 amino acids and has a molecular weight of ~47.5 kDa. The N-terminal 

methionine is cleaved from the mature protein in vivo and is not included in Figure 29.

 1 AGAEEIVLQPIKEISGTVKLPGSKSLSNRI 30

 31 LLLAALSEGTTVVDNLLNSEDVHYMLGALR 60

 61 TLGLSVEADKAAKRAVVVGCGGKFPVEDAK 90

 91 EEVQLFLGNAGIAMRSLTAAVTAAGGNATY 120

121 VLDGVPRMRERPIGDLVVGLKQLGADVDCF 150

151 LGTDCPPVRVNGIGGLPGGKVKLSGSISSQ 180

181 YLSALLMAAPLALGDVEIEIIDKLISIPYV 210

211 EMTLRLMERFGVKAEHSDSWDRFYIKGGQK 240

241 YKSPKNAYVEGDASSASYFLAGAAITGGTV 270

271 TVEGCGTTSLQGDVKFAEVLEMMGAKVTWT 300

301 ETSVTVTGPPREPFGRKHLKAIDVNMNKMP 330

331 DVAMTLAVVALFADGPTAIRDVASWRVKET 360

361 ERMVAIRTELTKLGASVEEGPDYCIITPPE 390

391 KLNVTAIDTYDDHRMAMAFSLAACAEVPVT 420

421 IRDPGCTRKTFPDYFDVLSTFVKN 444

Figure 29. Amino acid sequence of the 2mEPSPS protein.

* Note: The N-terminal Met is not shown, as it is cleaved from the 

mature protein.

6.2.2. Mode of Action of the 2mEPSPS Protein

Glyphosate normally exerts herbicidal activity by binding and inactivating EPSPS 

(5-enolpyruvylshikimate-3-phosphate synthase), an essential enzyme in the shikimic acid 

pathway which is found only in plants and certain microorganisms (Sikorski and Gruys, 1997).

Soybean (Glycine max) lines have been genetically modified for tolerance to glyphosate 

herbicides by expressing in the plant a modified epsps gene from corn (Zea mays), 2mepsps, 

which introduced two amino acid changes in the enzyme. The amino acids changed in the 

2mEPSPS protein significantly lower the sensitivity to glyphosate, allowing the enzyme to 

continue to function in the presence of the herbicide (Herouet-Guicheney et al., 2009).
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6.2.3. Biochemical Characterization of the 2mEPSPS Protein

Large quantities of purified 2mEPSPS protein are required to perform safety assessment studies. 

As it is technically infeasible to extract and purify sufficient amounts of recombinant protein 

from transgenic plants (Evans, 2004), the 2mEPSPS protein was produced in Pseudomonas 

fluorescens. Characterization studies were performed to confirm the equivalency of the 

2mEPSPS protein expressed in DAS-444Ø6-6 soybean with the P. fluorescens-derived 

2mEPSPS protein. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), 

western blot, glycoprotein detection, and protein sequence analysis by matrix assisted laser 

desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), MALDI-TOF 

MS/MS, and electrospray ionization-liquid chromatography mass spectrometry (ESI-LC/MS)

were used to characterize the biochemical properties of the proteins. Using these methods, the 

2mEPSPS protein from P. fluorescens and DAS-444Ø6-6 soybean were shown to be 

biochemically equivalent, thereby supporting the use of the microbe-derived protein in safety 

assessment studies.

The methods and results of the biochemical characterization of DAS-444Ø6-6 soybean- and 

microbe-derived 2mEPSPS proteins are described in detail in Appendix 3. Briefly, both the plant 

and P. fluorescens-derived 2mEPSPS proteins showed the expected molecular weight of ~47

kDa by SDS-PAGE and were immunoreactive to 2mEPSPS protein-specific antibodies by 

western blot analysis. There was no evidence of any post-translational modifications (i.e. 

glycosylation) of the DAS-444Ø6-6 soybean-derived 2mEPSPS protein. The amino acid 

sequence (including the N- and C-termini) was confirmed by enzymatic peptide mass 

fingerprinting using MALDI-TOF MS and MALDI-TOF MS/MS and was shown to be as 

expected and was identical to the protein expressed in P. fluorescens. The 2mEPSPS protein of 

both sources did not contain the methionine residue at its N terminus. The result is consistent 

with those for the 2mEPSPS protein expressed in other systems (Herouet-Guicheney et al., 

2009).

6.2.4. Expression of the 2mEPSPS Protein in Plant Tissues

A field expression study was conducted in the U.S. during 2010. Ten sites [Georgia, Iowa (2 

sites), Illinois (2 sites), Indiana, Michigan, Missouri, and Nebraska (2 sites)] were planted with 

DAS-444Ø6-6 soybean and the conventional control (Maverick). The test sites represented 

regions of diverse agronomic practices and environmental conditions for soybean in North 

America. Five treatments of the DAS-444Ø6-6 soybean (unsprayed, sprayed with 2,4-D, sprayed 

with glufosinate, sprayed with glyphosate, or sprayed with 2,4-D, glufosinate, and glyphosate) 

were tested (see Appendix 5 for application rates and timings). Plant tissues sampled included 

leaf, grain, root, and forage. Leaf tissues were collected at the V5 and V10-12 stages, while root 

and forage were collected at the R3 stage. Grain was collected at the R8 stage of development

(Gaska, 2006).

The soluble, extractable 2mEPSPS protein was measured using a validated enzyme-linked 

immunosorbent assay (ELISA) method. 2mEPSPS protein levels for all tissue types were 

calculated on ng/mg dry weight basis. Methods used for tissue sampling and quantification of 

protein expression by ELISA are detailed in Appendix 5. 

A summary of the 2mEPSPS protein concentrations (averaged across sites) in the various 

soybean matrices is shown in Table 7. Average expression values ranged from 21.86 ng/mg dry 
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weight in grain to 2583.46 ng/mg dry weight in V10-12 stage leaf tissue. Expression levels were 

comparable for all treatments. No 2mEPSPS protein was detected in the control tissues across 

the ten locations.
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Table 7. Expression of 2mEPSPS in DAS-444Ø6-6 soybean.

2mEPSPS ng/mg Tissue Dry Weight

Tissue Description Mean Std. Dev. (n=10) Min/Max Range

Leaf V5

DAS-444Ø6-6 2368.16 973.22 585.00 – 7250.00

DAS-444Ø6-6 w/ 2,4-D 2261.10 1009.75 850.00 – 7400.00

DAS-444Ø6-6 w/ Gluf 2062.07 962.71 262.00 – 5150.00

DAS-444Ø6-6 w/ Glyp 1846.04 975.50 353.00 – 4715.00

DAS-444Ø6-6 w/ All 2100.96 784.83 680.00 – 4860.00

Leaf V10-12

DAS-444Ø6-6 2583.46 825.47 961.40 – 4999.85

DAS-444Ø6-6 w/ 2,4-D 2203.83 584.92 256.57 – 3600.00

DAS-444Ø6-6 w/ Gluf 2188.12 543.24 1335.25 – 3405.00

DAS-444Ø6-6 w/ Glyp 2512.58 1259.06 511.74 – 8650.00

DAS-444Ø6-6 w/ All 2131.73 726.92 412.94 – 3210.00

Forage R3

DAS-444Ø6-6 357.09 146.12 182.40 – 862.22

DAS-444Ø6-6 w/ 2,4-D 330.02 109.78 189.20 – 680.15

DAS-444Ø6-6 w/ Gluf 321.92 74.69 173.46 – 539.08

DAS-444Ø6-6 w/ Glyp 400.47 140.66 167.21 – 1150.00

DAS-444Ø6-6 w/ All 367.32 125.39 154.04 – 1196.00

Root R3

DAS-444Ø6-6 89.71 32.33 ND – 200.4

DAS-444Ø6-6 w/ 2,4-D 93.54 20.51 4.96 – 174.40

DAS-444Ø6-6 w/ Gluf 103.48 47.88 ND – 200.40

DAS-444Ø6-6 w/ Glyp 112.27 30.26 7.10 – 233.60

DAS-444Ø6-6 w/ All 104.97 43.24 16.59 – 195.60

Grain

DAS-444Ø6-6 21.97 6.28 8.68 – 35.80

DAS-444Ø6-6 w/ 2,4-D 22.17 6.95 8.94 – 34.90

DAS-444Ø6-6 w/ Gluf 22.22 7.43 8.52 – 35.02

DAS-444Ø6-6 w/ Glyp 22.80 6.87 8.24 – 46.80

DAS-444Ø6-6 w/ All 21.86 6.81 8.66 – 39.85

Gluf = glufosinate; Glyp = glyphosate; All = 2,4-D + glufosinate + glyphosate

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the 2mEPSPS ELISA in all

tissue matrices were as follows:

2mEPSPS (ng/mg 

sample dry weight)

Matrix LOD LOQ

Leaf V5 4.0 8.0

Leaf V10-12 4.0 8.0

Root 4.0 8.0

Forage 4.0 8.0

Grain 4.0 8.0
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6.2.5. Food and Feed Assessment for 2mEPSPS Protein

Results of the overall safety assessment of the 2mEPSPS protein indicate that it is unlikely to 

cause allergic reactions in humans or be toxic to humans or animals.

History of Safe Use

 The donor organism, Zea mays, (commonly referred to as corn or maize) is a major cereal

crop grown for food and feed. The 2mEPSPS contains two point mutations compared 

with the wild-type epsps gene (Herouet-Guicheney et al., 2009).

 The 2mEPSPS protein is expressed in other events and crops that have previously been 

deregulated by the USDA and have received authorizations in numerous countries, for 

example: 

o GA21 corn (OECD Unique Identifier MON-ØØØ21-9) was deregulated by 

USDA in 1997 and was approved by the Canadian Food Inspection Agency 

(CFIA) in 1998.

o GHB614 cotton (OECD Unique Identifer BCS-GHØØ2-5) was deregulated by 

USDA in 2008 and was approved by CFIA in 2008.

Lack of allergenic potential

 The step-wise, weight-of-evidence approach was used to assess the allergenic potential of 

the 2mEPSPS protein (Codex Alimentarius Commission, 2009).

 The 2mEPSPS protein does not share meaningful amino-acid sequence similarities with 

known allergens. No significant homology was identified when the 2mEPSPS protein 

sequence was compared with known allergens in the FARRP (Food Allergy Research and 

Resource Program) version 11.00 allergen database (Released in February, 2011), using 

the search criteria of either a match of eight or more contiguous identical amino acids, or 

>35% identity over 80 amino-acids residues.

 The 2mEPSPS protein is rapidly degraded below the level of detection in simulated 

gastric fluid (SGF). The 2mEPSPS protein was readily digested (i.e., not detectable at 1 

minute) in SGF (0.32% pepsin, pH 1.2; 37oC) when analyzed by both SDS-PAGE and 

western blot analyses.

 The 2mEPSPS protein is not present in a glycosylated state. No glycosylation of the 

2mEPSPS protein was detected using SDS-PAGE and a glycosylation detection system.

Lack of toxic potential

 The 2mEPSPS protein does not share meaningful amino-acid sequence similarities with 

known toxins that would present any safety concerns. Amino acid homologies with the 

2mEPSPS protein sequence were evaluated using BLASTp search algorithm against the 

GenBank non-redundant protein sequences (up to date as of March 29, 2011 containing 

13,254,464 sequences with 4,535,100,774 amino acids). By their annotations, the 

majority of proteins returned by BLASTp with statistically significant alignments are 

related to shikimate pathway associated proteins including 3-phosphoshikimate 

1-carboxyvinyltransferase enzyme 5-enolpyruvyl shikimate 3-phosphate synthase 

(EPSPS) enzyme, and UDP-N-acetylglucosamine 1-carboxyvinyltransferase enzyme, and 
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dehydroquinate synthase. None of those proteins is associated with known protein toxins 

that are harmful to humans and animals.

 In acute mouse toxicity testing, there were no mortalities or clinical signs in CD-1 mice 

after oral administration by gavage of 2mEPSPS at 5000 mg protein/kg body weight.

6.2.6. Summary of 2mEPSPS Protein Characterization

The 2mepsps coding sequence was produced by introducing two point mutations to the wild-type 

epsps gene cloned from corn (Zea mays) through in vitro DNA technologies. The resultant

2mEPSPS protein has a lower binding affinity for glyphosate, thus allowing sufficient enzyme 

activity for the plants to grow in the presence of glyphosate herbicide. 2mEPSPS is comprised of 

445 amino acids and has a molecular weight of ~47.5 kDa. Detailed biochemical characterization 

of the 2mEPSPS protein derived from DAS-444Ø6-6 soybean and microbial sources were 

conducted. Additionally, characterization of 2mEPSPS protein expression in DAS-444Ø6-6

soybean over the growing season was determined by analyzing leaf, root, whole plant, and grain 

tissues from DAS-444Ø6-6 soybean sprayed with 2,4-D, glyphosate, and glufosinate, all three 

herbicides in combination, and non-sprayed.

A step-wise, weight-of-evidence approach was used to assess the potential for toxic or allergenic 

effects from the 2mEPSPS protein. Bioinformatic analyses revealed no meaningful homologies 

to known or putative allergens or toxins for the 2mEPSPS amino acid sequence. The 2mEPSPS

protein hydrolyzed rapidly in simulated gastric fluid. There was no evidence of acute toxicity in 

mice at a dose of 5000 mg/kg body weight of 2mEPSPS protein. Glycosylation analysis revealed 

no detectable covalently linked carbohydrates in 2mEPSPS protein expressed in DAS-444Ø6-6

soybean plants. Therefore, the low level expression of the 2mEPSPS protein presents a low 

exposure risk to humans and animals, and the results of the overall safety assessment of the 

2mEPSPS protein indicate that it is unlikely to cause allergenic or toxic effects in humans or 

animals.
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6.3. PAT

6.3.1. Identity of the PAT Protein

The phosphinothricin acetyltransferase (PAT) protein was derived from Streptomyces 

viridochromogenes, a gram-positive soil bacterium (Strauch et al., 1988; OECD, 1999). The pat

transgene in DAS-444Ø6-6 encodes a protein sequence that is identical to the native PAT protein 

(UniProt Accession Number: Q57146). PAT is comprised of 183 amino acids and has a 

molecular weight of ~21 kDa (Figure 30).

001 MSPERRPVEIRPATAADMAAVCDIVNHYIETSTVNFRTEPQTPQEWIDDL

051 ERLQDRYPWLVAEVEGVVAGIAYAGPWKARNAYDWTVESTVYVSHRHQRL

101 GLGSTLYTHLLKSMEAQGFKSVVAVIGLPNDPSVRLHEALGYTARGTLRA

151 AGYKHGGWHDVGFWQRDFELPAPPRPVRPVTQI

Figure 30. Amino acid sequence of the PAT protein.

6.3.2. Mode of Action of the PAT Protein.

The L-isomer of phosphinothricin (PPT) is a potent inhibitor of glutamine synthetase (GS) in 

plants and is used as a non-selective herbicide (OECD, 1999). Inhibition of GS by PPT causes 

rapid accumulation of intracellular ammonia which leads to cessation of photorespiration and 

results in the death of the plant cell (Duan et al., 2009). The pat gene which encodes 

phosphinothricin acetyltransferase (PAT) acetylates the free NH2 group of PPT (in the presence 

of acetyl coenzyme A) and thereby prevents autotoxicity in the producing organism (Figure 31, 

(Duke, 1996)).

Figure 31. Mode of action of the PAT protein.

6.3.3. Biochemical Characterization of the PAT Protein

Characterization of the biochemical properties of the plant-derived PAT protein was 

accomplished through the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis 

(SDS-PAGE), ELISA and western blot analysis. The methods and results are described in detail 

in Appendix 4. Using these methods the PAT protein produced in DAS-444Ø6-6 soybean was 

shown to be equivalent to that produced in other transgenic crops (USDA, 1996, 2001, 2004, 

2005).
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6.3.4. Expression of the PAT Protein in Plant Tissues

A field expression study was conducted in the U.S. during 2010. Ten sites (Georgia, Iowa (2 

sites), Illinois (2 sites), Indiana, Michigan, Missouri, and Nebraska (2 sites)) were planted with 

DAS-444Ø6-6 soybean and the conventional control (Maverick). The test sites represented 

regions of diverse agronomic practices and environmental conditions for soybean in North 

America. Five treatments of the DAS-444Ø6-6 soybean (unsprayed, sprayed with 2,4-D, sprayed 

with glufosinate, sprayed with glyphosate, or sprayed with 2,4-D, glufosinate, and glyphosate) 

were tested (see Appendix 5 for application rates and timings). Plant tissues sampled included 

leaf, grain, root, and forage. Leaf tissues were collected at the V5 and V10-12 stages, while root 

and forage were collected at the R3 stage. Grain was collected at the R8 stage of development 

(Gaska, 2006).

The soluble, extractable PAT protein was measured using a validated enzyme-linked 

immunosorbent assay (ELISA) method. PAT protein levels for all tissue types were calculated 

on ng/mg dry weight basis. Methods used for tissue sampling and quantification of protein 

expression by ELISA are detailed in Appendix 5.

A summary of the PAT protein concentrations (averaged across sites) in the various soybean 

matrices is shown in Table 8. Average expression values ranged from 1.56 ng/mg dry weight in 

R3 stage root to 10.59 ng/mg dry weight in V10-12 stage leaf tissue. Expression levels were 

comparable for all treatments. No PAT protein was detected in the control tissues across the ten

locations.
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Table 8. Expression of PAT in DAS-444Ø6-6 soybean.

PAT ng/mg Tissue Dry Weight

Tissue Description Mean Std. Dev. (n=10) Min/Max Range

Leaf V5

DAS-444Ø6-6 8.98 4.03 3.00 – 19.70

DAS-444Ø6-6 w/ 2,4-D 9.20 3.24 4.07 – 15.80

DAS-444Ø6-6 w/ Gluf 8.46 4.01 0.42 – 21.10

DAS-444Ø6-6 w/ Glyp 8.14 3.58 0.44 – 17.60

DAS-444Ø6-6 w/ All 8.47 3.23 4.29 – 17.60

Leaf V10-12

DAS-444Ø6-6 10.59 2.86 5.80 – 17.23

DAS-444Ø6-6 w/ 2,4-D 9.95 3.75 2.18 – 21.20

DAS-444Ø6-6 w/ Gluf 10.42 2.74 3.10 – 17.60

DAS-444Ø6-6 w/ Glyp 9.64 3.16 0.59 – 19.40

DAS-444Ø6-6 w/ All 10.49 3.09 3.88 – 16.80

Forage R3

DAS-444Ø6-6 6.19 1.79 3.55 – 10.45

DAS-444Ø6-6 w/ 2,4-D 5.90 1.40 3.50 – 9.65

DAS-444Ø6-6 w/ Gluf 6.72 1.67 2.90 – 11.20

DAS-444Ø6-6 w/ Glyp 6.48 1.87 3.65 – 10.35

DAS-444Ø6-6 w/ All 6.33 1.54 4.25 – 9.55

Root R3

DAS-444Ø6-6 1.56 0.68 ND – 3.04

DAS-444Ø6-6 w/ 2,4-D 1.71 0.67 0.37 – 3.34

DAS-444Ø6-6 w/ Gluf 1.77 0.77 ND – 3.10

DAS-444Ø6-6 w/ Glyp 1.80 0.45 0.10 – 2.94

DAS-444Ø6-6 w/ All 1.86 0.60 0.62 – 3.60

Grain

DAS-444Ø6-6 2.12 0.49 1.36 – 3.19

DAS-444Ø6-6 w/ 2,4-D 2.13 0.36 1.38 – 2.82

DAS-444Ø6-6 w/ Gluf 2.11 0.44 1.21 – 3.23

DAS-444Ø6-6 w/ Glyp 2.15 0.39 1.30 – 3.05

DAS-444Ø6-6 w/ All 2.11 0.38 1.26 – 3.04

Gluf = glufosinate; Glyp = glyphosate; All = 2,4-D + glufosinate + glyphosate

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the PAT ELISA in all tissue 

matrices were as follows:

PAT (ng/mg

sample dry weight)

Matrix LOD LOQ

Leaf V5 0.06 0.12

Leaf V10-12 0.06 0.12

Root 0.06 0.12

Forage 0.06 0.12

Grain 0.06 0.12
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6.3.5. Food and Feed Safety Assessment for PAT Protein

The PAT protein produced in DAS-444Ø6-6 soybean was shown to be equivalent to that 

produced in other transgenic crops that have been previously deregulated by USDA (USDA, 

1996, 2001, 2004, 2005). The food and feed safety of PAT was assessed in these products and 

shown to present no significant food or feed safety risk.

Additionally, the US EPA has concluded, after reviewing data on the acute toxicity and 

digestibility of the PAT protein, that there is a reasonable certainty that no harm will result from 

aggregate exposure to the U.S. population, including infants and children, to the PAT protein and 

the genetic material necessary for its introduction (US EPA, 1997). US EPA has consequently 

established an exemption from tolerance requirements pursuant to FFDCA section 408(j)(3) for 

PAT and the genetic material necessary for its production in all plants.

6.3.6. Summary of PAT Protein Characterization

The phosphinothricin acetyltransferase (PAT) protein was derived from Streptomyces 

viridochromogenes, a gram-positive soil bacterium. PAT is comprised of 183 amino acids and 

has a molecular weight of ~21 kDa (OECD, 1999, 2002). Western blot analysis demonstrated

that the PAT protein expressed in DAS-444Ø6-6 soybean had the same molecular weight and 

immunoreactivity as the native protein. Characterization of PAT protein expression in DAS444Ø6-6 soybean over the growing season was determined by analyzing leaf, root, whole plant, 

and grain tissues from DAS-444Ø6-6 soybean sprayed with 2,4-D, glyphosate, glufosinate, all 

three herbicides in combination, and non-sprayed. The low level expression of the PAT protein

in DAS-444Ø6-6 soybean presents a low exposure risk to humans and animals, and the results of 

the overall safety assessment of the PAT protein indicate that it is unlikely to cause allergenic or 

toxic effects in humans or animals.
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7. Agronomic Performance

7.1. Phenotypic and Agronomic Characteristics

Field trials with DAS-444Ø6-6 soybean, a non-transgenic control, and reference lines were 

conducted in 2010 at ten sites located in the U.S. This study used the same plots that were used 

for protein expression (sections 6.1.4, 6.2.4 and 6.3.4) and nutrient composition (section 8) 

studies. No agronomically meaningful unintended differences were observed between the nontransgenic near-isogenic control (Maverick) and DAS-444Ø6-6 soybean plots. Results from this 

study demonstrate agronomic equivalence between DAS-444Ø6-6 soybean (unsprayed and 

sprayed) and non-transgenic soybean (Maverick).

7.1.1. Agronomic Study

An agronomic study with DAS-444Ø6-6 soybean (unsprayed or sprayed with 2,4-D, glyphosate, 

glufosinate, or all three herbicides), a near-isogenic non-transgenic control (Maverick) and six 

non-transgenic reference lines (Dairyland Seed (DSR) 75213-72, 98860-71, 99914N, and 99915; 

Porter 75148; Williams 82) was conducted in 2010 at ten sites located in Sycamore, Georgia;

Richland, Iowa; Bagley, Iowa; Carlyle, Illinois; Wyoming, Illinois; Sheridan, Indiana; Deerfield, 

Michigan; Fisk, Missouri; Brunswick, Nebraska; and York, Nebraska. 

An entry in this study was defined as a soybean line with herbicide treatment, if applicable. For 

example, DAS-444Ø6-6 with 2,4-D treatment-only was one entry. Each trial site included nine 

entries, including five entries of DAS-444Ø6-6 (one untreated and four different herbicide 

treatments), one entry of control (Maverick), and three entries of non-transgenic reference lines. 

At each of the ten sites, all entries were arranged in a randomized complete block design with 

four blocks. Across all sites, each control and DAS-444Ø6-6 entry was represented by a total of 

40 plots (10 sites, 4 replicate plots per entry at each site). Three of the six reference lines were 

included at each site by randomizing across sites in a balanced incomplete-block design. Each 

of the six reference lines was assigned to five sites; therefore, each reference line was 

represented by a total of 20 plots across sites (5 sites per reference line, 4 replicate plots per entry 

at each site). 

At each site, four replicate plots of each entry were established, with each plot consisting of four 

25 ft (7.62 m) rows. Herbicide applications and observations of agronomic characteristics were 

conducted on the center two rows of each plot; row number one and four were included as 

additional border rows. Soybean seeds were planted at a seeding rate of approximately 125 seeds 

per row with seed spacing within each row of approximately 2.4 inches (6 cm). Each soybean 

plot was bordered by two rows of a non-transgenic soybean cultivar of similar maturity. The 

entire trial site was surrounded by a minimum of four rows (10 ft or 3.0 m) of a non-transgenic 

soybean cultivar of similar maturity. Appropriate insect, weed, and disease control practices 

were applied to produce an agronomically acceptable crop.

Unsprayed DAS-444Ø6-6 soybean plots and DAS-444Ø6-6 soybean plots treated with one of 

four herbicide regimes were included as separate entries. Herbicides were applied in a spray 

volume of approximately 20 gallons per acre (187 L/ha). Herbicide applications included 

approximately 2% v/v Ammonium sulfate (AMS) for Weedar 64, Durango DMA, and Liberty.
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2,4-D only Treatment: 2,4-D (Weedar 64) was applied as three broadcast applications to 

DAS-444Ø6-6. Application timing was at planting / pre-emergence, and approximately V3 and 

R2 stages. Individual target application rates were 1.0 lb ae (acid equivalent)/A for Weedar 64, 

or 1120 g ae/ha.

Glufosinate only Treatment: Glufosinate (Liberty) was applied as two broadcast applications to 

DAS-444Ø6-6. Application timing was at approximately V5 and R1 stages. The target 

application rate at V5 was 0.33 lb ai/A for Liberty, or 374 g ai/ha. The target application rate at 

R1 was 0.41 lb ai/A for Liberty, or 454 g ai/ha.

Glyphosate only Treatment: Glyphosate (Durango DMA) was applied as three broadcast 

applications to DAS-444Ø6-6. Individual applications were at planting / pre-emergence, and

approximately V3 and R2 stages. Individual target application rates were 1.1 lb ae/A for

Durango DMA, or 1260 g ae/ha.

2,4-D + Glufosinate + Glyphosate Treatment: 2,4-D (Weedar 64) + Glyphosate (Durango 

DMA) as a tank mixture was applied as three broadcast applications to DAS-444Ø6-6. 

Individual applications were at planting / pre-emergence, and approximately V3 and R2 stages. 

Individual target application rates were 1.0 lb ae/A for Weedar 64, or 1120 g ae/ha. Individual 

target application rates were 1.1 lb ae/A for Durango DMA, or 1260 g ae/ha. Glufosinate 

(Liberty) was also applied as two broadcast applications to DAS-444Ø6-6. Application timing 

was at approximately V5 and R1 stages. The target application rate was 0.33 lb ai/A for Liberty, 

or 374 g ai/ha.

The following agronomic characteristics were measured and recorded for all test entries at each 

location on a per plot basis (Table 9).
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Table 9. Agronomic characteristics.

Trait Evaluation 

Time

Description Scale

Early 

Population 

(Stand 

Count)

V2 Number of plants in 

a representative 1 

meter section of one 

row per plot

Number of emerged plants in 1 meter

Seedling 

Vigor

V2 Visual estimate of 

average vigor of 

plants in each plot

1-9 Rating Scale, 5 = average, 9 = high 

vigor; e.g. 1 = short plants with small, thin 

leaves; 9 = tall plants with large, robust 

leaves; Not based on growth of the control 

entries; Germination/ emergence (stand 

count) not considered

Days to 50% 

Flowering

R1/R2 Date at which »50% 

of plants were 

flowering

Date recorded when »50% of the plants in 

each plot were flowering; Days since 

planting calculated

Disease 

Incidence

R6 Visual estimate of 

disease incidence

0-100%; Estimated % plant tissue/leaf area 

diseased over all plants in plot; Did not 

record % of plants in plot that had 

detectable disease; 100% = all plant tissues 

in plot were diseased; Recorded type of 

disease if incidence was greater than 30%

Insect 

Damage

R6 Visual estimate of 

insect damage

0-100%; Estimated % plant tissue/leaf area 

damaged over all plants in plot; Did not 

record % of plants in plot that had 

detectable damage; 100% = all plant tissues 

had feeding damage; Recorded type of 

damage, e.g. chewing, stippling, distortion 

if damage was greater than 30%; Recorded 

type of insect(s) if present 

Days to 

Maturity

R8 Date at which »95% 

of plants had 

reached 

physiological 

maturity/dry down 

color

Recorded the date when »95% of the plants 

in each plot reached physiological 

maturity/dry down color; Days since 

planting calculated

Lodging R8 Visual estimate of 

lodging severity

0-100%; Estimated % of plants lodged in 

plot; 100% = all plants in plot were lodged
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Trait Evaluation 

Time

Description Scale

Plant Height R8 Average plant 

height: from soil 

surface to growing 

tip (at senescence / 

after leaf shed)

Recorded the average height of all plants in 

plot (stand) in centimeters (cm); One value 

for each plot; If plot was lodged, a 

representative group of plants was held up 

to obtain a measurement

Final 

Population 

(Stand 

Count)

R8 Number of plants in 

a representative 1 

meter section of one 

row per plot 

Number of plants in 1 meter; Did not 

sample a section where plants were 

removed during previous sampling

Number of 

Seeds

R8 

(prior to 

harvest)

Number of pods and 

seeds from 5 plants 

collected from each 

plot

Recorded the number of pods and seeds 

present on 5 plants collected from each plot

Shattering R8 

(prior to 

harvest)

Visual estimate of 

pod shattering 

0-100%; Estimated % of shattered pods for 

each plot; 100% = all pods shattered

Yield R8 Weight of grain 

harvested from each 

plot

Recorded the weight in grams of grain 

harvested from each plot

100 seed 

weight

R8 Weight in grams for 

100 representative 

seeds from bulk 

yield sample

Recorded the weight in grams for 100 

representative seeds taken from the bulk 

yield sample
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Analysis of variance was conducted across field sites (combined-site analysis) for agronomic 

data using a mixed model (SAS Institute Inc., 2009). Entry was considered a fixed effect, and 

location, block within location, and location-by-entry, were designated as random effects. 

Significant differences were declared at the 95% confidence level. The significance of an overall 

treatment effect was estimated using an F-test. Paired contrasts were made between 

DAS-444Ø6-6 (sprayed or unsprayed) entries and the control entry (Maverick) using t-tests.

Due to the large number of contrasts made in this study, multiplicity was an issue. Multiplicity is 

an issue when a large number of comparisons are made in a single study to look for unexpected 

effects. Under these conditions, the probability of falsely declaring differences based on 

comparison-wise P-values is very high (1-0.95number of comparisons). In this study there were five 

comparisons per endpoint (14 analyzed observation types for agronomics), resulting in 70 

comparisons made in the combined-site agronomic analysis. Therefore, the probability of 

declaring one or more false differences based on unadjusted P-values was >97% for agronomics 

(1-0.9570).

One method to account for multiplicity is to adjust P-values to control the experiment-wise error 

rate, but when many comparisons are made in a study, the power for detecting specific effects

can be reduced significantly. An alternative with much greater power is to adjust P-values to 

control the probability that each declared difference is significant. This can be accomplished 

using False Discovery Rate (FDR) control procedures (Benjamini and Hochberg, 1995); FDR

methods are commonly applied in studies examining transgenic crops (Herman et al., 2007; Coll

et al., 2008; Huls et al., 2008; Jacobs et al., 2008; Stein et al., 2009; Herman et al., 2010). 

Therefore, the P-values from the agronomics evaluations were each adjusted using the FDR

method to improve discrimination of true differences among treatments from random effects 

(false positives). Differences were considered significant if the FDR-adjusted P-value was less 

than 0.05.

7.1.2. Agronomic Results

A statistical analysis of the agronomic data collected from the non-transgenic near-isogenic

Maverick, unsprayed DAS-444Ø6-6 and sprayed DAS-444Ø6-6 entries was conducted. For each 

agronomic character and entry, the least square means, standard error, and minimum and 

maximum sample values are reported. Also for comparison, the minimum and maximum values 

from reference lines across all sites (reference ranges) for each agronomic character are reported.

Each minimum and maximum value is an individual data point reported for a single plot.

For the combined-site analysis (Table 10), no statistically significant differences were observed 

following False Discovery Rate (FDR) adjustment of P-values for all of the agronomic 

characteristics evaluated: early population, seedling vigor, days to flowering, disease incidence, 

insect damage, days to maturity, lodging, plant height, final population, number of pods per five 

plants, number of seeds per five plants, shattering, yield, and 100 seed weight. Unadjusted 

P-values were significant at the 0.05 level for paired t-tests for one comparison each for seedling 

vigor, lodging, final population, and number of pods per five plants. For each significant 

unadjusted P-value, mean differences between transgenic and control entries were negligible and 

transgenic means were within the range observed for reference varieties included in the study. 

Results from this study demonstrate agronomic equivalence between DAS-444Ø6-6 (unsprayed 

and sprayed) and non-transgenic Maverick soybean.
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 Table 10. Combined-site analysis: summary of agronomic characteristics.

Overall 

Isoline DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference Range

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Early Population - V2 14 ± 1 13 ± 1 13 ± 1 13 ± 1 14 ± 1 14 ± 1

(number of plants in a 6 - 22 3 - 19 1 - 19 3 - 20 3 - 24 3 - 23 2 - 27

 1 m section of row) 0.556 (0.202, 0.692) (0.596, 0.817) (0.189, 0.692) (1.000, 1.000) (0.940, 1.000)

Seedling Vigor - V2 6.2 ± 0.3 6.0 ± 0.3 5.9 ± 0.3 6.0 ± 0.3 6.1 ± 0.3 5.8 ± 0.3

(1-9 scale 5 - 8 4 - 8 3 - 8 4 - 8 4 - 8 3 - 8 3 - 9

 1 = low vigor, 9 = high vigor) 0.269 (0.408, 0.746) (0.102, 0.682) (0.249, 0.692) (0.508, 0.781) (0.024, 0.669)

Days to Flowering - R1/R2 42 ± 3 43 ± 3 42 ± 3 43 ± 3 42 ± 3 42 ± 3

(days since planting) 22 - 54 22 - 56 24 - 56 28 - 56 22 - 55 22 - 54 22 - 57

0.664 (0.317, 0.692) (0.859, 0.986) (0.317, 0.692) (0.953, 1.000) (0.859, 0.986)

Disease Incidence - R6 5 ± 2 5 ± 2 4 ± 2 5 ± 2 5 ± 2 5 ± 2

(0-100% scale, 0% = no disease 0 - 20 0 - 25 0 - 15 0 - 15 0 - 20 0 - 15 0 - 20

 100% = all plants diseased) 0.661 (0.968, 1.000) (0.400, 0.746) (0.574, 0.817) (0.422, 0.746) (1.000, 1.000)

Insect Damage - R6 12 ± 8 14 ± 8 13 ± 8 13 ± 8 13 ± 8 14 ± 8

(0-100% scale, 0% = no damage 0 - 80 0 - 90 0 - 80 0 - 90 0 - 80 0 - 90 0 - 90

 100% = all plants damaged) 0.664 (0.136, 0.692) (0.524, 0.781) (0.326, 0.692) (0.299, 0.692) (0.144, 0.692)

Days to Maturity - R8 115 ± 5 114 ± 5 114 ± 5 115 ± 5 114 ± 5 115 ± 5

(days since planting) 97 - 143 95 - 143 96 - 143 97 - 143 97 - 143 97 - 143 96 - 143

0.323 (0.293, 0.692) (0.139, 0.692) (0.660, 0.839) (0.255, 0.692) (0.895, 0.999)

Lodging - R8 10 ± 4 12 ± 4 16 ± 4 12 ± 4 12 ± 4 13 ± 4

(0-100% scale, 0% = no lodging 0 - 30 0 - 60 0 - 70 0 - 70 0 - 50 0 - 50 0 - 35

 100% = all plants lodged) 0.428 (0.437, 0.746) (0.038, 0.669) (0.431, 0.746) (0.614, 0.817) (0.367, 0.739)

Plant Height - R8 94 ± 4 96 ± 4 94 ± 4 95 ± 4 93 ± 4 91 ± 4

(cm) 58 - 122 70 - 123 48 - 123 68 - 120 61 - 120 64 - 116 22 - 112

0.242 (0.218, 0.692) (1.000, 1.000) (0.478, 0.781) (0.641, 0.831) (0.266, 0.692)

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

Agronomic Component (Units)a
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Overall 

Isoline DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference Range

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Final Population - R8 13 ± 1 13 ± 1 12 ± 1 12 ± 1 12 ± 1 12 ± 1

(number of plants in a 4 - 20 5 - 20 4 - 18 3 - 18 4 - 19 3 - 18 2 - 20

 1 m section of row) 0.317 (0.220, 0.692) (0.150, 0.692) (0.027, 0.669) (0.081, 0.682) (0.083, 0.682)

Number of Pods - R8 361 ± 42 384 ± 42 364 ± 42 387 ± 42 404 ± 42 379 ± 42

(number of pods on 5 plants) 176 - 743 197 - 698 151 - 683 184 - 786 188 - 1163 189 - 765 133 - 1008

0.298 (0.265, 0.692) (0.899, 0.999) (0.199, 0.692) (0.038, 0.669) (0.370, 0.739)

Number of Seeds - R8 802 ± 81 826 ± 81 771 ± 81 815 ± 81 881 ± 81 772 ± 82

(number of seeds from 5 plants) 295 - 1187 407 - 1183 390 - 1329 380 - 1472 152 - 1824 319 - 1416 258 - 1882

0.170 (0.588, 0.817) (0.499, 0.781) (0.774, 0.939) (0.086, 0.682) (0.514, 0.781)

Shattering - R8 3 ± 3 4 ± 3 4 ± 3 3 ± 3 4 ± 3 3 ± 3

(0-100% scale, 0% = no shattering 0 - 30 0 - 35 0 - 50 0 - 45 0 - 75 0 - 50 0 - 60

 100% = all pods shattered) 0.801 (0.318, 0.692) (0.318, 0.692) (0.948, 1.000) (0.500, 0.781) (0.760, 0.939)

Yield - R8 33 ± 3 30 ± 3 32 ± 3 30 ± 3 31 ± 3 30 ± 3

(bushels per acre) 1.15 - 53.78 2.05 - 41.87 3.59 - 49.94 2.05 - 52.95 8.45 - 52.5 4.1 - 49.94 4.99 - 55.44

0.423 (0.106, 0.682) (0.433, 0.746) (0.063, 0.682) (0.187, 0.692) (0.107, 0.682)

100 Seed Weight - R8 13.5 ± 0.4 13.2 ± 0.4 13.5 ± 0.4 13.6 ± 0.4 13.6 ± 0.4 13.5 ± 0.4

(grams) 9.4 - 16.3 10.85 - 16.3 10.17 - 15.2 11.3 - 16.4 10.3 - 15.6 10.76 - 16.2 9.4 - 20.7

0.507 (0.241, 0.692) (0.818, 0.971) (0.619, 0.817) (0.575, 0.817) (0.778, 0.939)

a

 Unit of measure for yield was converted from grams per plot to bushels per acre prior to analysis; conversion formula:

(X g/125 ft 2 plot)  (43560 ft 2/A)  (bu./27.2155 kg)  (kg/1000 g), where X is the individual sample value.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

Agronomic Component (Units)a
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7.1.3. Conclusions

Field agronomic characteristics of DAS-444Ø6-6 (unsprayed or sprayed with 2,4-D, glyphosate, 

glufosinate, or all three herbicides) were evaluated in field trials in 2010. DAS-444Ø6-6 

agronomic results were all statistically indistinguishable from the control (Maverick) and/or 

within reference ranges for non-transgenic soybean, indicating that no unintended agronomic 

effects were observed for DAS-444Ø6-6 soybean. Results from this study demonstrate 

agronomic equivalence between DAS-444Ø6-6 soybean and non-transgenic Maverick soybean.
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7.2. Ecological Evaluations

The DAS-444Ø6-6 soybean field trials were monitored and observed by personnel familiar with 

soybean cultivation practices (breeders, field station managers, field agronomists, field 

associates). The personnel conducting the field tests visually monitored the incidence of plant 

disease and pests on DAS-444Ø6-6 soybeans compared to the conventional soybean varieties, 

including Maverick and six reference lines (Section 7.1.1), in the same trials. Disease and insect 

damage was rated on a numerical scale of 0-100%, with 0% representing no damage due to 

disease incidence or insect pests (Section 7.1). Table 11 summarizes the results obtained from 

the field trials conducted in 2010 at ten sites as described in Section 7.1. The results showed that 

there were no statistically significant differences between the isoline and DAS-444Ø6-6 (with or 

without herbicide treatment) in susceptibility to and interactions with diseases and insects.
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Table 11. Analysis of disease incidence and insect damage.

Isoline DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference Range

Agronomic Component 

(Units)a

Overall 

Treatment

Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

(Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Disease Incidence - R6 5 ± 2 5 ± 2 4 ± 2 5 ± 2 5 ± 2 5 ± 2

(0-100% scale, 0% = no 

disease

0 - 20 0 - 25 0 - 15 0 - 15 0 - 20 0 - 15 0 - 20

100% = all plants 

diseased)

0.661 (0.968, 1.000) (0.400, 0.746) (0.574, 0.817) (0.422, 0.746) (1.000, 1.000)

Insect Damage - R6 12 ± 8 14 ± 8 13 ± 8 13 ± 8 13 ± 8 14 ± 8

(0-100% scale, 0% = no 

damage

0 - 80 0 - 90 0 - 80 0 - 90 0 - 80 0 - 90 0 - 90

100% = all plants 

damaged)

0.664 (0.136, 0.692) (0.524, 0.781) (0.326, 0.692) (0.299, 0.692) (0.144, 0.692)

a Unit of measure was not converted prior to analysis.

b Overall treatment effect estimated using an F-test. 

c Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 

were considered significant.
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Ecological observations were also made from all USDA APHIS notified field trials conducted in 

2009-2011 (Appendix 6). Incidence of disease and insect presence in trials of DAS-444Ø6-6 

soybeans was recorded and differences in incidence or response of DAS-444Ø6-6 soybeans 

compared with the conventional control Maverick were examined. Table 12 summarizes the 

disease and insect stressors observed in trials of DAS-444Ø6-6 and conventional Maverick 

soybeans. In all cases, no differences between DAS-444Ø6-6 and conventional Maverick 

soybeans were observed in any of the trials. These observations support the conclusion that the 

response of DAS-444Ø6-6 soybeans to ecological stressors does not differ from that of 

conventional soybeans (Maverick).

Table 12. Disease and insect stressors observed in trials of DAS-444Ø6-6 and conventional

soybean.

Year USDA 

Notification 

Number

State (County) Diseases Insects

2009 08-254-109n PR (Santa Isabel) None None

2009-

2010

09-068-103n IN (Benton), PR (Santa 

Isabel)

None None

2009-

2010

09-259-108n PR (Santa Isabel) Carlavirus, rust None

2010 10-077-107n GA (Turner), IL 

(Champaign, Clinton, 

Ford, Jackson, Madison, 

Shelby, Stark), IN 

(Benton, Boone), IA 

(Jefferson, Shelby, Story), 

MD (Queen Anne), MO 

(Adair, Butler), NE (Polk, 

York), OH (Fulton), PR 

(Santa Isabel)

Carlavirus, rust, 

brown spot, frogeye 

leaf spot, Sudden 

Death Syndrome

aphids, bean leaf 

beetles, grasshoppers, 

ladybugs, leaf 

hoppers, green 

cloverworm, stink 

bugs

2010 10-083-105n IN (Benton), IA (Story), 

MS (Washington), MO 

(Platte)

None None

2010 10-085-103n GA (Turner), IL (Clinton, 

Stark), IN (Boone), IA 

(Guthrie, Jefferson), MI 

(Lenawee), MO (Butler), 

NE (Antelope, York)

brown spot, brown 

leaf spot, frogeye 

leaf spot

aphids, brown leaf 

beetles, grasshoppers, 

stink bugs, lady bugs, 

ground beetles, 

Lepidoptera spp,

7.3. Germination and Dormancy Evaluations

The germination of DAS-444Ø6-6 seed compared with the non-transgenic control (Maverick) 

under warm and cold conditions was evaluated to determine any impact on seed dormancy 

characteristics.

To conduct the germination studies, MetroTM Mix potting soil was placed in plastic flats (10.5 × 

21 × 2.5 in.), and flats were sub-irrigated with 3 L of distilled water. Seeds of DAS-444Ø6-6 

soybean (T5 generation) and non-transgenic, near-isogenic control (Maverick) soybean were 

planted in the flats to a depth of 1 in. on the day following irrigation, and each half-flat contained 

100 seeds (200 seeds total per flat). Immediately after planting, flats for the cold germination 
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experiment were placed in a growth chamber where they were incubated at 10°C for seven days 

followed by an additional seven days at 25°C. Flats for the warm emergence experiment were 

placed in the growth chamber at 25°C for seven days immediately after planting. For both 

experiments, the emerged seedlings in each half-flat were counted following the seven day 25°C 

incubation period.

The experimental design was a completely randomized design with four replicates of 100 seeds 

per replicate. Data were transformed using the arcsine of the square root of the decimal fraction 

of seeds emerged per replicate and subjected to analysis of variance (ANOVA) (SAS Institute 

Inc., 2009).

Emergence data and P-values for the significance of the effect of event on emergence are 

provided in (Table 13). Emergence of DAS-444Ø6-6 soybean did not differ significantly (α = 

0.05) from that of the non-transgenic, near-isogenic control Maverick under cold (P = 0.7989) 

and warm (P = 0.8947) conditions. These results indicate that the seed dormancy characteristics 

have not been changed in DAS-444Ø6-6 soybeans.

Table 13. Percentage emergence (number of seeds emerged divided by number of seeds 

planted x 100) for DAS-444Ø6-6 soybean and control soybean.

Soybean emergence (%)

Temperature Event Rep 1 Rep 2 Rep 3 Rep 4 Mean (±SE) P

Cold DAS-444Ø6-6 93 94 93 99 94.8 (±1.4)

0.7989

Cold Control 90 91 95 99 93.8 (±2.1)

Warm DAS-444Ø6-6 98 98 92 97 96.3 (±1.4)

0.8947

Warm Control 99 99 92 95 96.3 (±1.7)

7.4. Summary of Agronomic, Disease, and Pest Characteristics

Agronomic data evaluating plant growth characteristics throughout the growing season 

demonstrate the equivalence of DAS-444Ø6-6 soybean with the non-transgenic near-isogenic 

soybean Maverick. Plant growth and phenotypic characteristics, response to ecological stressors 

as indicated by susceptibility to disease and insect pressure, and germination and dormancy 

characteristics were unchanged between DAS-444Ø6-6 soybeans and conventional Maverick 

soybeans across diverse environments. Therefore, these data support the conclusion that 

agronomic, disease, and pest characteristics of DAS-444Ø6-6 soybean are not significantly 

different from that of conventional soybeans, and there is no indication that DAS-444Ø6-6

soybeans will pose an increased plant pest risk.
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8. Grain and Forage Composition

Field trials with DAS-444Ø6-6 soybean, the non-transgenic control Maverick, and reference 

lines were conducted in 2010 at ten sites located in the U.S. This study used the same plots that 

were used for protein expression (Appendix 5) and agronomic characterization (Section 7) 

studies. No biologically meaningful unintended compositional differences were observed 

between the non-transgenic near-isogenic control Maverick and DAS-444Ø6-6 soybean plots. 

Results from this study demonstrate compositional equivalence between event DAS-444Ø6-6 

(unsprayed and sprayed) and non-transgenic soybean.

8.1. Field Study Design

A crop composition study with DAS-444Ø6-6 soybean (unsprayed or sprayed with 2,4-D, 

glyphosate, glufosinate, or all three herbicides), a near-isogenic non-transgenic control 

(Maverick) and six non-transgenic reference lines (Dairyland Seed (DSR) 75213-72, 98860-71, 

99914N, and 99915; Porter 75148; Williams 82) was conducted in 2010 at ten sites located in 

Sycamore, Georgia; Richland, Iowa; Bagley, Iowa; Carlyle, Illinois; Wyoming, Illinois;

Sheridan, Indiana; Deerfield, Michigan; Fisk, Missouri; Brunswick, Nebraska; and York,

Nebraska. 

An entry in this study was defined as a soybean line with herbicide treatment, if applicable. For 

example, DAS-444Ø6-6 with 2,4-D treatment-only was one entry. Each trial site included nine 

entries, including five entries of DAS-444Ø6-6 (one untreated and four different herbicide 

treatments), one entry of control (Maverick), and three entries of non-transgenic reference lines. 

At each of the ten sites, all entries were arranged in a randomized complete block design with 

four blocks. Across all sites, each control and DAS-444Ø6-6 entry was represented by a total of 

40 plots (10 sites, 4 replicate plots per entry at each site). Three of the six reference lines were 

included at each site by randomizing across sites in a balanced incomplete-block design. Each 

of the six reference lines was assigned to five sites; therefore, each reference line was 

represented by a total of 20 plots across sites (5 sites per reference line, 4 replicate plots per entry 

at each site). 

At each site, four replicate plots of each entry were established, with each plot consisting of four 

25 ft (7.62 m) rows. Herbicide applications and crop composition sampling were conducted on 

the center two rows of each plot; row number one and four were included as additional border 

rows. Soybean seeds were planted at a seeding rate of approximately 125 seeds per row with 

seed spacing within each row of approximately 2.4 inches (6 cm). Each soybean plot was 

bordered by two rows of a non-transgenic soybean cultivar of similar maturity. The entire trial 

site was surrounded by a minimum of four rows (10 ft or 3.0 m) of a non-transgenic soybean 

cultivar of similar maturity. Appropriate insect, weed, and disease control practices were applied 

to produce an agronomically acceptable crop.

Unsprayed DAS-444Ø6-6 soybean plots and DAS-444Ø6-6 soybean plots treated with one of 

four herbicide regimes were included as separate entries. Herbicides were applied in a spray 

volume of approximately 20 gallons per acre (187 L/ha). Herbicide applications included 

approximately 2% v/v Ammonium sulfate (AMS) for Weedar 64, Durango DMA, and Liberty.

2,4-D only Treatment: 2,4-D (Weedar 64) was applied as three broadcast applications to 

DAS-444Ø6-6. Application timing was at planting / pre-emergence, and approximately V3 and 
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R2 stages. Individual target application rates were 1.0 lb ae (acid equivalent)/A for Weedar 64, 

or 1120 g ae/ha.

Glufosinate only Treatment: Glufosinate (Liberty) was applied as two broadcast applications to 

DAS-444Ø6-6. Application timing was at approximately V5 and R1 stages. The target 

application rate at V5 was 0.33 lb ai/A for Liberty, or 374 g ai/ha. The target application rate at 

R1 was 0.41 lb ai/A for Liberty, or 454 g ai/ha.

Glyphosate only Treatment: Glyphosate (Durango DMA) was applied as three broadcast 

applications to DAS-444Ø6-6. Individual applications were at planting / pre-emergence, and 

approximately V3 and R2 stages. Individual target application rates were 1.1 lb ae/A for 

Durango DMA, or 1260 g ae/ha.

2,4-D + Glufosinate + Glyphosate Treatment: 2,4-D (Weedar 64) + Glyphosate (Durango 

DMA) as a tank mixture was applied as three broadcast applications to DAS-444Ø6-6. 

Individual applications were at planting / pre-emergence, and approximately V3 and R2 stages. 

Individual target application rates were 1.0 lb ae/A for Weedar 64, or 1120 g ae/ha. Individual 

target application rates were 1.1 lb ae/A for Durango DMA, or 1260 g ae/ha. Glufosinate 

(Liberty) was also applied as two broadcast applications to DAS-444Ø6-6. Application timing 

was at approximately V5 and R1 stages. The target application rate was 0.33 lb ai/A for Liberty, 

or 374 g ai/ha.

8.2. Compositional Analysis

Samples of soybean forage and seed were analyzed at Covance Laboratories Inc. for nutrient 

content. The analytes examined are presented in Table 14.
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Table 14. Composition analytes.

A. Forage

Proximates and Fiber Minerals

Protein Calcium

Fat Phosphorus

Ash

Moisture

Carbohydrates

Acid Detergent Fiber (ADF)

Neutral Detergent Fiber (NDF)

B. Seed

Proximates and Fiber Minerals

Protein Calcium Alanine Lysine 8:0 Caprylic 18:0 Stearic

Fat Copper Arginine Methionine 10:0 Capric 18:1 Oleic

Ash Iron Aspartic acid Phenylalanine 12:0 Lauric 18:2 Linoleic

Moisture Magnesium Cystine Proline 14:0 Myristic 18:3 Linolenic

Carbohydrates Manganese Glutamic acid Serine 14:1 Myristoleic 18:3 γ-Linolenic

Acid Detergent Fiber (ADF) Phosphorus Glycine Threonine 15:0 Pentadecanoic 20:0 Arachidic

Neutral Detergent Fiber (NDF) Potassium Histidine Tryptophan 15:1 Pentadecenoic 20:1 Eicosenoic

Total Dietary Fiber Selenium Isoleucine Tyrosine 16:0 Palmitic 20:2 Eicosadienoic

Sodium Leucine Valine 16:1 Palmitoleic 20:3 Eicosatrienoic

Zinc 17:0 Heptadecanoic 20:4 Arachidonic

17:1 Heptadecenoic 22:0 Behenic

Vitamin A (β-Carotene) Vitamin C (Ascorbic acid) Total Daidzein Equivalent Lectin

Vitamin B1 (Thiamine HCl) Vitamin E (α-Tocopherol) Total Genistein Equivalent Phytic acid

Vitamin B2 (Riboflavin) β-Tocopherol Total Glycitein Equivalent Raffinose

Vitamin B3 (Niacin) γ-Tocopherol Stachyose

Vitamin B5 (Pantothenic acid) δ-Tocopherol Trypsin Inhibitor

Vitamin B6 (Pyridoxine HCl) Total Tocopherol

Vitamin B9 (Folic acid)

Amino Acids Fatty Acids

Vitamins Bioactives

The results of the compositional analysis for soybean forage and seed were compared with 

values reported in literature (Iskander, 1987; Hartwig and Kilen, 1991; Padgette et al., 1996; 

Taylor et al., 1999; OECD, 2001; McCann et al., 2005; Harrigan et al., 2007; Bilyeu et al., 2008; 

Lundry et al., 2008; Berman et al., 2009, 2010; Harrigan et al., 2010; ILSI, 2011). A summary of 

the compositional data used for comparison can be found in Appendix 7.
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8.3. Statistical Analysis

Analysis of variance was conducted across field sites (combined-site analysis) for composition 

data using a mixed model (SAS Institute Inc., 2009). Entry was considered a fixed effect, and 

location, block within location, and location-by-entry, were designated as random effects. 

Significant differences were declared at the 95% confidence level. The significance of an overall 

treatment effect was estimated using an F-test. Paired contrasts were made between 

DAS-444Ø6-6 (sprayed or unsprayed) entries and the control entry (Maverick) using t-tests.

Due to the large number of contrasts made in this study, multiplicity was an issue. Multiplicity is 

an issue when a large number of comparisons are made in a single study to look for unexpected 

effects. Under these conditions, the probability of falsely declaring differences based on 

comparison-wise P-values is very high (1-0.95number of comparisons). In this study there were five 

comparisons per analyte (71 analyzed analytes for composition), resulting in 355 comparisons 

made in the combined-site composition analysis. Therefore, the probability of declaring one or 

more false differences based on unadjusted P-values was >99.99% (1-0.95355).

One method to account for multiplicity is to adjust P-values to control the experiment-wise error 

rate, but when many comparisons are made in a study, the power for detecting specific effects

can be reduced significantly. An alternative with much greater power is to adjust P-values to 

control the probability that each declared difference is significant. This can be accomplished 

using a False Discovery Rate (FDR) control procedure (Benjamini and Hochberg, 1995); FDR 

methods are commonly applied in studies examining transgenic crops (Herman et al., 2007; Coll

et al., 2008; Huls et al., 2008; Jacobs et al., 2008; Stein et al., 2009; Herman et al., 2010). 

Therefore, the P-values from the composition contrasts were each adjusted using the FDR 

method to improve discrimination of true differences among treatments from random effects 

(false positives). Differences were considered significant if the FDR-adjusted P-value was less 

than 0.05.
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8.4. Composition Analysis Results

A statistical analysis of composition data from the non-transgenic near-isogenic Maverick 

(referred to as isoline in Table 15 to Table 22, Figure 32 to Figure 40), unsprayed DAS-444Ø6-6 

and sprayed DAS-444Ø6-6 entries was conducted. A summary of the compositional results 

across locations is presented in Table 15 to Table 22. For each analyte and entry, the least square 

means, standard error, and minimum and maximum sample values are reported. Also for 

comparison, the minimum and maximum values from the six reference lines across all sites 

(reference range) and literature range for each analyte are reported. Minimum and maximum 

values are for individual plot results as are literature ranges (except where noted). Arithmetic 

means for each analyte from each field site are plotted for the non-transgenic control Maverick, 

DAS-444Ø6-6 (sprayed and unsprayed), and reference line entries (Figure 32 to Figure 40). 

Literature ranges are depicted as the shaded area on each plot. Literature ranges reported as not 

detected (ND) or less than the limit of quantitation (<LOQ) were plotted as zeros.

8.4.1. Proximate, Fiber, and Mineral Analysis of Forage 

Soybean forage samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for proximate content (protein, fat, ash, moisture, and carbohydrates), fiber (acid 

detergent fiber (ADF), neutral detergent fiber (NDF)), and minerals (calcium and phosphorus). A 

summary of the results across all locations is presented in Table 15, Table 16, Figure 32 and 

Figure 33. All mean values were within literature ranges (when available) and within ranges for 

reference lines included in the study. No statistical differences were observed in the combinedsite analysis between the control and DAS-444Ø6-6 entries for protein, fat, ash, carbohydrates, 

ADF, NDF, calcium, and phosphorus. Statistically significant differences were observed between 

the control and DAS-444Ø6-6 entries for moisture, where mean differences were negligible and 

not biologically meaningful as means were within literature ranges and within ranges for 

reference lines included in the study.
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Table 15. Summary of the proximate and fiber analysis of DAS-444Ø6-6 soybean forage from all sites, and associated 

literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Proximate

Protein 19.7 ± 0.5 19.7 ± 0.5 19.4 ± 0.5 19.2 ± 0.5 19.6 ± 0.5 19.7 ± 0.5

13.7 - 23.4 16 - 25.3 15.5 - 23.4 14.8 - 24.1 14.5 - 23.9 15.2 - 25.2 13 - 29.1 11.2 - 24.71

(% DW) 0.425 (0.890, 0.958) (0.227, 0.440) (0.103, 0.275) (0.655, 0.806) (0.990, 0.996)

Fat 2.87 ± 0.1 2.75 ± 0.1 2.81 ± 0.1 2.85 ± 0.1 2.68 ± 0.1 2.68 ± 0.1

1.95 - 4.11 0.769 - 4.01 1.6 - 3.88 1.14 - 3.79 1.91 - 3.52 1.47 - 3.69 1.69 - 4.63 1.01 - 9.87

(% DW) 0.443 (0.337, 0.549) (0.648, 0.804) (0.886, 0.956) (0.122, 0.297) (0.121, 0.297)

Ash 9.4 ± 0.8 9.2 ± 0.8 8.9 ± 0.8 9.7 ± 0.8 8.9 ± 0.8 8.9 ± 0.8

7.13 - 28.3 5.96 - 31 6.14 - 19 6.57 - 24 6.42 - 21.4 6.85 - 18.7 5.86 - 36.6 4.68 - 10.782

(% DW) 0.523 (0.766, 0.894) (0.388, 0.604) (0.542, 0.726) (0.367, 0.582) (0.301, 0.514)

Moisture 78.7 ± 0.6 77.6 ± 0.6 77.6 ± 0.6 77.6 ± 0.6 77.2 ± 0.6 77.4 ± 0.6

75.6 - 82.3 71.8 - 80.5 71 - 81 69.9 - 81.1 69.1 - 80.7 69.8 - 81.1 70.9 - 81.4 32.05 - 84.60

(% FW) 0.002 (0.003, 0.023) (0.003, 0.019) (0.003, 0.020) (<0.001, 0.002) (<0.001, 0.005)

Carbohydratese68.0 ± 1 68.4 ± 1 68.9 ± 1 68.2 ± 1 68.8 ± 1 68.8 ± 1

55.8 - 74.3 49.8 - 76.3 59.1 - 73 53.8 - 74.4 56.3 - 74.4 59.5 - 73.8 48.8 - 74.7 59.8 - 80.18

(% DW) 0.534 (0.548, 0.729) (0.128, 0.303) (0.763, 0.894) (0.174, 0.372) (0.196, 0.400)

Fiber

Acid Detergent 31.6 ± 0.9 31.5 ± 0.9 31.7 ± 0.9 31.7 ± 0.9 32.3 ± 0.9 30.6 ± 0.9

Fiber (ADF) 22.9 - 38.7 24.3 - 44.2 23 - 42.2 25 - 44.7 26 - 43.6 23.7 - 39.3 21.5 - 57.2 22.72 - 59.03

(% DW) 0.653 (0.933, 0.976) (0.882, 0.956) (0.903, 0.963) (0.478, 0.692) (0.301, 0.514)

Neutral Detergent 37.6 ± 1.2 37.4 ± 1.2 38.1 ± 1.2 37.3 ± 1.2 37.5 ± 1.2 37.6 ± 1.2

Fiber (NDF) 29.1 - 46.5 27.2 - 51.3 27.3 - 50 28.4 - 50.4 24.7 - 50 21.8 - 52.3 24.9 - 63.1 19.61 - 73.05

(% DW) 0.974 (0.840, 0.941) (0.609, 0.778) (0.759, 0.894) (0.924, 0.970) (0.951, 0.976)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

e

 % Carbohydrates = 100 % - (% Protein + % Fat + % Ash + % Moisture)

Analytical 

Component 

(Units)a
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Table 16. Summary of the mineral analysis of DAS-444Ø6-6 soybean forage from all sites, and associated literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Mineral

Calcium 1240 ± 63 1236 ± 63 1208 ± 63 1211 ± 63 1227 ± 63 1263 ± 63

880 - 1770 652 - 1590 817 - 1560 650 - 1540 858 - 1600 762 - 1760 695 - 1860 NR

(mg/100 g dry wt.) 0.222 (0.859, 0.949) (0.187, 0.384) (0.233, 0.443) (0.581, 0.758) (0.345, 0.554)

Phosphorus 271 ± 13 266 ± 13 266 ± 13 264 ± 13 265 ± 13 265 ± 13

190 - 384 177 - 381 197 - 374 186 - 385 197 - 399 170 - 394 175 - 427 NR

(mg/100g dry wt.) 0.690 (0.253, 0.458) (0.239, 0.449) (0.118, 0.294) (0.182, 0.378) (0.231, 0.443)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was converted from % dry wt. to mg/100g dry wt. prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Figure 32. Proximate and fiber (% dry weight for all proximate and fiber except moisture (% fresh weight)) in non-transgenic 

(Isoline), Event DAS-444Ø6-6, and reference line soybean forage.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 32. (Cont). Proximate and fiber (% dry weight for all proximate and fiber except moisture (% fresh weight)) in nontransgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean forage. Symbols for each location shown: open circle = GA,  = IA1, + 

= IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. 

Literature range is shaded for each analyte.
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Figure 33. Minerals (mg/100g dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean forage.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = 

MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte (when available).
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8.4.2. Proximate and Fiber Analysis of Seed 

Soybean seed samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for proximate content (protein, fat, ash, moisture, and carbohydrates) and fiber (acid 

detergent fiber (ADF), neutral detergent fiber (NDF), and total dietary fiber). A summary of the 

results across all locations is presented in Table 17 and Figure 34. All mean results were within 

literature ranges (when available) and within ranges for reference lines included in the study.

Statistically significant overall treatment effects were found for protein and carbohydrates, where 

some DAS-444Ø6-6 entries contained more protein and less carbohydrate than the control. 

Similarly, variations in fat, ash, and moisture were also observed for some pair-wise contrasts 

between DAS-444Ø6-6 entries and the control. Carbohydrate composition is calculated from 

values for protein, fat, ash, and moisture (Table 17). Therefore, an increase in protein and related 

proximate components is expected to result in a partial decrease in carbohydrates. Statistical 

differences were also found for neutral detergent fiber (NDF) and total dietary fiber, where 

values were slightly lower in some DAS-444Ø6-6 entries compared with the control. No 

biologically meaningful differences were detected as all results for proximate content and fiber 

were within literature ranges and within ranges for reference lines included in the study.
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Table 17. Summary of the proximate and fiber analysis of DAS-444Ø6-6 soybean seed from all sites, and associated literature 

range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference 

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Proximate

Protein 37.8 ± 0.3 38.0 ± 0.3 38.5 ± 0.3 38.2 ± 0.3 38.2 ± 0.3 38.6 ± 0.3

29.7 - 40.3 35.6 - 39.7 37.1 - 40.4 34 - 40.6 36 - 40.9 36.1 - 42.5 35.1 - 44.9 32 - 48.4

(% DW) 0.039 (0.323, 0.537) (0.012, 0.065) (0.106, 0.279) (0.086, 0.246) (0.002, 0.019)

Fat 18.9 ± 0.6 19.5 ± 0.6 19.2 ± 0.6 19.3 ± 0.6 19.2 ± 0.6 19.1 ± 0.6

13.6 - 23.5 16.9 - 23.5 15.8 - 23.4 16.7 - 23.4 16.5 - 23.1 16.2 - 22.6 15.3 - 22.9 8.104 - 24.7

(% DW) 0.069 (0.003, 0.023) (0.152, 0.346) (0.028, 0.109) (0.144, 0.333) (0.246, 0.453)

Ash 5.15 ± 0.09 5.23 ± 0.09 5.24 ± 0.09 5.24 ± 0.09 5.21 ± 0.09 5.22 ± 0.09

4.49 - 5.86 4.66 - 6.34 4.59 - 5.99 4.55 - 6.87 4.49 - 5.78 4.48 - 6.42 4.45 - 6.3 3.885 - 6.994

(% DW) 0.278 (0.060, 0.188) (0.035, 0.128) (0.041, 0.140) (0.158, 0.357) (0.128, 0.303)

Moisture 10.6 ± 0.7 10.2 ± 0.7 10.0 ± 0.7 9.9 ± 0.7 9.9 ± 0.7 9.9 ± 0.7

7.58 - 20.4 7.1 - 22.1 7.19 - 13.8 6.54 - 14.1 7.13 - 14.5 6.87 - 12.9 7.26 - 17.2 4.7 - 34.4

(% FW) 0.072 (0.160, 0.358) (0.026, 0.104) (0.018, 0.085) (0.010, 0.059) (0.010, 0.060)

Carbohydratese38.13 ± 0.75 37.22 ± 0.76 37.11 ± 0.75 37.21 ± 0.75 37.38 ± 0.75 37.04 ± 0.75

32.6 - 47.7 32.5 - 40.8 31.3 - 41.3 31.2 - 40.6 32.1 - 41.5 32.2 - 40.7 28.7 - 43 29.3 - 50.2

(% DW) 0.002 (0.002, 0.014) (<0.001, 0.005) (0.001, 0.012) (0.008, 0.049) (<0.001, 0.002)

Fiber

Acid Detergent 15.5 ± 0.5 15.2 ± 0.5 15.5 ± 0.5 15.0 ± 0.5 15.6 ± 0.5 14.9 ± 0.5

Fiber (ADF) 9.84 - 24.1 7.68 - 20.7 8.71 - 18.7 10.3 - 18.2 11.7 - 19.6 11.3 - 20.3 8.02 - 20.9 7.81 - 26.26

(% DW) 0.577 (0.435, 0.648) (0.911, 0.966) (0.284, 0.491) (0.940, 0.976) (0.174, 0.372)

Neutral Detergent 17.7 ± 0.3 17.0 ± 0.3 17.1 ± 0.3 16.7 ± 0.3 17.3 ± 0.3 16.5 ± 0.3

Fiber (NDF) 14.6 - 24.1 9.41 - 20.9 10.9 - 21.4 13.1 - 20.1 13.2 - 22 13.4 - 19.7 13.3 - 22.2 8.53 - 23.90

(% DW) 0.030 (0.088, 0.249) (0.111, 0.284) (0.012, 0.065) (0.317, 0.535) (0.002, 0.014)

Total Dietary 22.4 ± 0.5 21.6 ± 0.5 21.7 ± 0.5 21.5 ± 0.5 21.9 ± 0.5 21.4 ± 0.5

Fiber 16.7 - 27.8 18 - 25.4 17.9 - 26.1 17.3 - 26.5 16.9 - 25.8 16 - 25.6 16.2 - 27.7 NR

(% DW) 0.144 (0.048, 0.159) (0.092, 0.256) (0.033, 0.120) (0.252, 0.458) (0.012, 0.065)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

e

 % Carbohydrates = 100 % - (% Protein + % Fat + % Ash + % Moisture)

Analytical 

Component 

(Units)a
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Figure 34. Proximate and fiber (% dry weight for all proximate and fiber except moisture (% fresh weight)) in non-transgenic 

(Isoline), Event DAS-444Ø6-6, and reference line soybean seed. 

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte (when available). 
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Figure 34 (Cont). Proximate and fiber (% dry weight for all proximate and fiber except moisture (% fresh weight)) in nontransgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean seed. Symbols for each location shown: open circle = GA,  = IA1, + = 

IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature 

range is shaded for each analyte (when available).
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8.4.3. Mineral Analysis of Seed

Soybean seed samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, 

selenium, sodium, and zinc). A summary of the results across all locations is presented in Table 

18 and Figure 35. All mean results were within literature ranges (when available) and/or within 

ranges for reference lines included in the study. For sodium, statistical analysis was not 

performed since greater than 50% of the samples were found to be below the LOQ. No statistical 

differences were observed in the combined-site analysis between the control and DAS-444Ø6-6

entries for copper, iron, magnesium, manganese, phosphorus, and selenium. Statistically 

significant differences were observed for calcium, potassium, and zinc for some DAS-444Ø6-6

entries compared with the control, where mean differences were negligible and not biologically 

meaningful as means were within literature ranges and/or within ranges for reference lines 

included in the study.
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Table 18. Summary of the mineral analysis of DAS-444Ø6-6 soybean seed from all sites, and associated literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Mineral

Calcium 301 ± 13 324 ± 13 318 ± 13 304 ± 13 320 ± 13 306 ± 13

235 - 403 261 - 425 252 - 404 241 - 398 249 - 413 243 - 404 174 - 383 116.55 - 510

(mg/100 g DW) <0.001 (<0.001, <0.001) (<0.001, 0.001) (0.407, 0.623) (<0.001, <0.001) (0.184, 0.381)

Copper 1.32 ± 0.05 1.34 ± 0.05 1.35 ± 0.05 1.32 ± 0.05 1.35 ± 0.05 1.33 ± 0.05

0.995 - 1.68 1.01 - 1.71 1.03 - 1.93 1.04 - 1.74 1.15 - 1.7 1.09 - 1.71 0.91 - 1.77 0.632 - 1.092

(mg/100 g DW) 0.310 (0.222, 0.436) (0.057, 0.182) (0.780, 0.901) (0.082, 0.242) (0.324, 0.537)

Iron 8.2 ± 0.5 8.3 ± 0.6 7.7 ± 0.5 8.6 ± 0.5 7.8 ± 0.5 8.5 ± 0.5

6.51 - 14.2 6.33 - 26.2 6.18 - 9.64 6.55 - 41.9 6.42 - 12.3 6.54 - 24.5 5.35 - 87.9 3.734 - 10.954

(mg/100 g DW) 0.650 (0.906, 0.963) (0.395, 0.609) (0.547, 0.729) (0.537, 0.725) (0.674, 0.822)

Magnesium 229 ± 6 231 ± 6 230 ± 6 227 ± 6 230 ± 6 229 ± 6

207 - 279 205 - 283 206 - 287 202 - 276 203 - 279 200 - 284 195 - 317 219.40 - 312.84

(mg/100 g DW) 0.226 (0.281, 0.490) (0.331, 0.543) (0.285, 0.491) (0.347, 0.554) (0.974, 0.985)

Manganese 2.99 ± 0.53 3.10 ± 0.54 3.27 ± 0.53 3.09 ± 0.53 3.18 ± 0.53 3.14 ± 0.53

2.11 - 7.83 1.69 - 8.27 1.78 - 10.4 2.03 - 8.57 1.89 - 10.8 2.08 - 9.46 1.9 - 9.53 2.52 - 3.876

(mg/100 g DW) 0.620 (0.483, 0.695) (0.084, 0.243) (0.532, 0.725) (0.231, 0.443) (0.335, 0.548)

Phosphorus 557 ± 21 561 ± 21 558 ± 21 554 ± 21 558 ± 21 557 ± 21

400 - 640 394 - 661 384 - 681 377 - 657 403 - 645 388 - 660 360 - 659 506.74 - 935.24

(mg/100 g DW) 0.856 (0.474, 0.690) (0.905, 0.963) (0.526, 0.723) (0.935, 0.976) (0.866, 0.949)

Potassium 1730 ± 20 1780 ± 20 1790 ± 20 1770 ± 20 1770 ± 20 1770 ± 20

1580 - 1850 1610 - 1930 1640 - 1940 1630 - 1930 1620 - 1890 1610 - 1940 1530 - 2030 1868.01 - 2510

(mg/100 g DW) <0.001 (<0.001, <0.001) (<0.001, <0.001) (<0.001, 0.002) (<0.001, 0.005) (0.001, 0.011)

Selenium 451 ± 108 438 ± 108 489 ± 108 389 ± 108 420 ± 108 469 ± 108

66.7 - 1980 77.7 - 1770 63.8 - 2320 77 - 1770 84 - 1670 72.3 - 2360 59.5 - 3380 NR

(ppb DW) 0.552 (0.831, 0.934) (0.496, 0.707) (0.278, 0.488) (0.592, 0.767) (0.741, 0.885)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Mineral

Sodium NA NA NA NA NA NA

< LOQ < LOQ - 15.7 < LOQ - 15 < LOQ - 16.7 < LOQ - 13.4 < LOQ - 11.7 < LOQ - 18.5 4.05 - 30

(mg/100 g DW) NA

Zinc 4.17 ± 0.12 4.34 ± 0.12 4.34 ± 0.12 4.25 ± 0.12 4.3 ± 0.12 4.3 ± 0.12

3.62 - 4.7 3.45 - 5.02 3.53 - 5.59 3.62 - 5.01 3.56 - 5.05 3.67 - 6.01 3.34 - 5.82 4.98 - 7.578

(mg/100 g DW) 0.077 (0.010, 0.058) (0.010, 0.058) (0.205, 0.414) (0.036, 0.128) (0.042, 0.143)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Figure 35. Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non-transgenic (Isoline), Event 

DAS-444Ø6-6, and reference line soybean seed.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte (when available).
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Figure 35 (Cont). Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non-transgenic (Isoline), 

Event DAS-444Ø6-6, and reference line soybean seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open 

triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. 

Literature range is shaded for each analyte (when available).
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Figure 35 (Cont). Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non-transgenic (Isoline), 

Event DAS-444Ø6-6, and reference line soybean seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open 

triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. 

Literature range is shaded for each analyte (when available).
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8.4.4. Amino Acid Analysis of Seed

Soybean seed samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for amino acid content. A summary of the results across all locations is presented in

Table 19 and Figure 36. All mean results were within literature ranges and within ranges for 

reference lines included in the study. No statistical differences were observed in the combinedsite analysis between the control and DAS-444Ø6-6 entries for alanine, arginine, aspartic acid, 

glutamic acid, glycine, isoleucine, leucine, methionine, phenylalanine, proline, serine, threonine, 

and valine. Statistically significant differences were observed for cystine, histidine, lysine, 

tryptophan, and tyrosine for some DAS-444Ø6-6 entries compared with the control, where mean 

differences were negligible and not biologically meaningful as means were within literature 

ranges and within ranges for reference lines included in the study.
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Table 19. Summary of the amino acid analysis of DAS-444Ø6-6 soybean seed from all sites, and associated literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Amino Acid

Alanine 1.68 ± 0.01 1.67 ± 0.01 1.67 ± 0.01 1.68 ± 0.01 1.67 ± 0.01 1.68 ± 0.01

1.56 - 1.76 1.58 - 1.76 1.6 - 1.74 1.6 - 1.74 1.6 - 1.81 1.59 - 1.88 1.55 - 1.9 1.43 - 2.10

(% DW) 0.734 (0.282, 0.490) (0.453, 0.667) (0.980, 0.988) (0.616, 0.784) (0.763, 0.894)

Arginine 2.74 ± 0.02 2.71 ± 0.02 2.75 ± 0.02 2.74 ± 0.02 2.74 ± 0.02 2.75 ± 0.02

2.55 - 2.94 2.46 - 2.88 2.6 - 2.95 2.6 - 2.99 2.55 - 3 2.58 - 3.16 2.59 - 3.45 2.15 - 3.46

(% DW) 0.440 (0.182, 0.378) (0.620, 0.786) (0.856, 0.949) (0.762, 0.894) (0.603, 0.776)

Aspartic Acid 4.18 ± 0.03 4.19 ± 0.03 4.23 ± 0.03 4.22 ± 0.03 4.22 ± 0.03 4.24 ± 0.03

3.85 - 4.45 3.9 - 4.4 4.03 - 4.49 4.02 - 4.59 3.94 - 4.64 3.89 - 4.82 3.58 - 4.94 3.81 - 6.04

(% DW) 0.231 (0.959, 0.977) (0.093, 0.256) (0.165, 0.363) (0.168, 0.365) (0.055, 0.177)

Cystine 0.532 ± 0.010 0.556 ± 0.010 0.547 ± 0.010 0.560 ± 0.010 0.549 ± 0.010 0.560 ± 0.010

0.458 - 0.647 0.493 - 0.661 0.474 - 0.645 0.489 - 0.672 0.482 - 0.652 0.487 - 0.66 0.429 - 0.71 0.37 - 0.81

(% DW) 0.002 (0.001, 0.011) (0.032, 0.120) (<0.001, 0.002) (0.015, 0.075) (<0.001, 0.002)

Glutamic Acid 6.26 ± 0.06 6.22 ± 0.06 6.26 ± 0.06 6.27 ± 0.06 6.26 ± 0.06 6.28 ± 0.06

5.75 - 6.74 5.65 - 6.78 5.82 - 6.76 5.8 - 6.95 5.85 - 6.92 5.74 - 7.18 5.85 - 7.77 5.84 - 9.15

(% DW) 0.837 (0.429, 0.646) (0.960, 0.977) (0.709, 0.851) (0.964, 0.978) (0.602, 0.776)

Glycine 1.64 ± 0.01 1.63 ± 0.01 1.65 ± 0.01 1.64 ± 0.01 1.65 ± 0.01 1.65 ± 0.01

1.53 - 1.75 1.51 - 1.73 1.58 - 1.73 1.55 - 1.75 1.56 - 1.84 1.54 - 1.87 1.55 - 1.89 1.41 - 2.00

(% DW) 0.592 (0.511, 0.720) (0.378, 0.591) (0.761, 0.894) (0.391, 0.607) (0.434, 0.648)

Histidine 1.02 ± 0.01 1.02 ± 0.01 1.04 ± 0.01 1.02 ± 0.01 1.03 ± 0.01 1.03 ± 0.01

0.943 - 1.1 0.91 - 1.1 0.957 - 1.11 0.964 - 1.13 0.935 - 1.17 0.946 - 1.19 0.197 - 1.18 0.86 - 1.24

(% DW) 0.299 (0.945, 0.976) (0.045, 0.152) (0.896, 0.960) (0.551, 0.730) (0.669, 0.819)

Isoleucine 1.81 ± 0.02 1.81 ± 0.02 1.83 ± 0.02 1.81 ± 0.02 1.83 ± 0.02 1.82 ± 0.02

1.66 - 1.94 1.64 - 1.94 1.67 - 2.02 1.58 - 1.93 1.68 - 2.12 1.64 - 2.06 1.68 - 2.18 1.49 - 2.08

(% DW) 0.458 (0.815, 0.928) (0.108, 0.281) (0.608, 0.778) (0.116, 0.291) (0.421, 0.639)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Amino Acid

Leucine 2.84 ± 0.02 2.83 ± 0.02 2.87 ± 0.02 2.85 ± 0.02 2.86 ± 0.02 2.86 ± 0.02

2.65 - 3.02 2.62 - 2.99 2.72 - 3.06 2.67 - 3.06 2.69 - 3.2 2.67 - 3.25 2.68 - 3.32 2.2 - 4.0

(% DW) 0.151 (0.522, 0.723) (0.059, 0.186) (0.483, 0.695) (0.209, 0.418) (0.255, 0.460)

Lysine 2.46 ± 0.03 2.48 ± 0.03 2.53 ± 0.03 2.48 ± 0.03 2.49 ± 0.03 2.49 ± 0.03

2.1 - 2.77 2.11 - 2.76 2.14 - 2.76 2.22 - 2.8 2.06 - 2.97 2.12 - 3.03 2 - 3.04 2.19 - 3.32

(% DW) 0.369 (0.522, 0.723) (0.029, 0.111) (0.502, 0.713) (0.344, 0.554) (0.320, 0.535)

Methionine 0.504 ± 0.006 0.502 ± 0.006 0.505 ± 0.006 0.501 ± 0.006 0.499 ± 0.006 0.507 ± 0.006

0.447 - 0.577 0.435 - 0.595 0.449 - 0.567 0.456 - 0.555 0.445 - 0.564 0.442 - 0.621 0.418 - 0.596 0.39 - 0.68

(% DW) 0.873 (0.821, 0.931) (0.922, 0.970) (0.694, 0.838) (0.450, 0.666) (0.647, 0.804)

Phenylalanine 1.9 ± 0.02 1.89 ± 0.02 1.91 ± 0.02 1.9 ± 0.02 1.91 ± 0.02 1.91 ± 0.02

1.76 - 2.04 1.72 - 2.02 1.8 - 2.04 1.77 - 2.05 1.77 - 2.13 1.77 - 2.18 1.8 - 2.28 1.6 - 2.44

(% DW) 0.471 (0.633, 0.795) (0.248, 0.454) (0.622, 0.786) (0.356, 0.567) (0.225, 0.439)

Proline 1.96 ± 0.02 1.95 ± 0.02 1.95 ± 0.02 1.95 ± 0.02 1.96 ± 0.02 1.95 ± 0.02

1.77 - 2.12 1.81 - 2.13 1.84 - 2.09 1.67 - 2.12 1.76 - 2.19 1.77 - 2.34 1.78 - 2.41 1.63 - 2.28

(% DW) 0.986 (0.530, 0.725) (0.782, 0.901) (0.537, 0.725) (0.854, 0.949) (0.772, 0.899)

Serine 1.79 ± 0.02 1.80 ± 0.02 1.79 ± 0.02 1.82 ± 0.02 1.82 ± 0.02 1.83 ± 0.02

1.62 - 1.98 1.58 - 1.96 1.62 - 1.97 1.62 - 2 1.64 - 1.97 1.6 - 2.1 1.56 - 2.2 1.11 - 2.48

(% DW) 0.264 (0.701, 0.843) (0.883, 0.956) (0.114, 0.290) (0.234, 0.443) (0.093, 0.256)

Threonine 1.52 ± 0.01 1.51 ± 0.01 1.52 ± 0.01 1.52 ± 0.01 1.53 ± 0.01 1.53 ± 0.01

1.43 - 1.58 1.38 - 1.59 1.42 - 1.59 1.43 - 1.6 1.46 - 1.6 1.42 - 1.75 1.4 - 1.75 1.14 - 1.89

(% DW) 0.171 (0.374, 0.590) (0.524, 0.723) (0.487, 0.697) (0.205, 0.414) (0.109, 0.281)

Tryptophan 0.574 ± 0.01 0.588 ± 0.01 0.589 ± 0.01 0.575 ± 0.01 0.583 ± 0.01 0.583 ± 0.01

0.512 - 0.667 0.521 - 0.739 0.515 - 0.676 0.512 - 0.641 0.505 - 0.699 0.517 - 0.645 0.495 - 0.704 0.30 - 0.67

(% DW) 0.091 (0.030, 0.112) (0.021, 0.092) (0.860, 0.949) (0.140, 0.325) (0.165, 0.363)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Amino Acid

Tyrosine 1.44 ± 0.01 1.44 ± 0.01 1.46 ± 0.01 1.45 ± 0.01 1.45 ± 0.01 1.45 ± 0.01

1.36 - 1.51 1.35 - 1.52 1.39 - 1.54 1.38 - 1.55 1.38 - 1.61 1.35 - 1.62 1.34 - 1.64 0.79 - 1.61

(% DW) 0.148 (0.872, 0.953) (0.024, 0.098) (0.242, 0.449) (0.178, 0.377) (0.219, 0.432)

Valine 1.85 ± 0.01 1.85 ± 0.01 1.86 ± 0.01 1.85 ± 0.01 1.86 ± 0.01 1.85 ± 0.01

1.72 - 1.98 1.71 - 1.96 1.68 - 2.03 1.61 - 1.96 1.71 - 2.14 1.7 - 2.01 1.71 - 2.16 1.5 - 2.44

(% DW) 0.737 (0.921, 0.970) (0.241, 0.449) (0.954, 0.976) (0.459, 0.670) (1.000, 1.000)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Figure 36. Amino acids (% dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean seed.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 36 (Cont). Amino acids (% dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled 

circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 36 (Cont). Amino acids (% dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled 

circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 36 (Cont). Amino acids (% dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled 

circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 36 (Cont). Amino acids (% dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled 

circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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8.4.5. Fatty Acid Analysis of Seed

Soybean seed samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for fatty acid content. A summary of the results across all locations is presented in 

Table 20 and Figure 37. All mean results were within literature ranges (when available) and 

within ranges for reference lines included in the study. Statistical analysis was not performed for 

the following analytes since greater than 50% of the samples were found to be below the LOQ: 

caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), myristoleic (14:1), pentadecanoic 

(15:0), pentadecenoic (15:1), palmitoleic (16:1), heptadecanoic (17:0), heptadecenoic (17:1), γlinolenic (18:3), eicosadienoic (20:2), eicosatrienoic (20:3), and arachidonic (20:4). No statistical 

differences were observed in the combined-site analysis between the control and DAS-444Ø6-6

entries for stearic (18:0) and eicosenoic (20:1). Statistically significant differences were observed 

for palmitic (16:0), oleic (18:1), linoleic (18:2), linolenic (18:3), arachidic (20:0), and behenic 

(22:0) for some DAS-444Ø6-6 entries compared with the control, where mean differences were 

negligible and not biologically meaningful as means were within literature ranges and within

ranges for reference lines included in the study.
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Table 20. Summary of the fatty acid analysis of DAS-444Ø6-6 soybean seed from all sites, and associated literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference 

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Fatty Acid

8:0 Caprylic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.148

(% total fatty acid) NA

10:0 Capric NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ ND - 0.27

(% total fatty acid) NA

12:0 Lauric NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.132

(% total fatty acid) NA

14:0 Myristic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.238

(% total fatty acid) NA

14:1 Myristoleic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.125

(% total fatty acid) NA

15:0 Pentadecanoic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ ND

(% total fatty acid) NA

15:1 Pentadecenoic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ ND

(% total fatty acid) NA

16:0 Palmitic 10.9 ± 0.1 10.7 ± 0.1 10.7 ± 0.1 10.7 ± 0.1 10.7 ± 0.1 10.6 ± 0.1

10.4 - 12.55 10.21 - 11.02 10.24 - 11.11 10.07 - 11.06 10.25 - 11.2 10.05 - 11.03 9.5 - 11.31 9.55 - 15.77

(% total fatty acid) <0.001 (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Fatty Acid

16:1 Palmitoleic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.194

(% total fatty acid) NA

17:0 Heptadecanoic NA NA NA NA NA NA

< LOQ - 0.11 < LOQ - 0.136 < LOQ - 0.136 < LOQ - 0.126 < LOQ - 0.135 < LOQ - 0.142 < LOQ < LOQ - 0.146

(% total fatty acid) NA

17:1 Heptadecenoic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.087

(% total fatty acid) NA

18:0 Stearic 4.51 ± 0.07 4.47 ± 0.07 4.47 ± 0.07 4.48 ± 0.07 4.48 ± 0.07 4.52 ± 0.07

3.88 - 5 3.96 - 4.93 4.05 - 4.89 4.07 - 5.04 4.08 - 4.96 4.11 - 4.95 3.28 - 4.98 2.59 - 5.88

(% total fatty acid) 0.391 (0.174, 0.372) (0.159, 0.358) (0.343, 0.554) (0.261, 0.468) (0.785, 0.901)

18:1 Oleic 23.5 ± 0.5 21.4 ± 0.5 21.5 ± 0.5 21.8 ± 0.5 21.7 ± 0.5 21.9 ± 0.5

20.8 - 28.3 18.4 - 23.7 18.8 - 25.9 18.9 - 26 19.1 - 26.3 19.2 - 26.3 18.1 - 27.9 14.3 - 45.68

(% total fatty acid) <0.001 (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001)

18:2 Linoleic 53.0 ± 0.3 54.8 ± 0.3 54.6 ± 0.3 54.4 ± 0.3 54.5 ± 0.3 54.2 ± 0.3

50.9 - 54.5 53.4 - 56.8 52.6 - 56.5 52.5 - 56.1 52.1 - 56.2 51.7 - 56.2 50.1 - 56.7 35.36 - 58.8

(% total fatty acid) <0.001 (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001)

18:3 Linolenic 7.32 ± 0.33 7.77 ± 0.33 7.79 ± 0.33 7.86 ± 0.33 7.76 ± 0.33 7.92 ± 0.33

5.03 - 8.88 5.56 - 9.38 5.33 - 9.29 5.48 - 9.47 5.46 - 9.42 5.38 - 9.48 4.83 - 9.82 3 - 12.52

(% total fatty acid) <0.001 (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001) (<0.001, <0.001)

18:3 γ-Linolenic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ ND

(% total fatty acid) NA

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Fatty Acid

20:0 Arachidic 0.328 ± 0.005 0.323 ± 0.005 0.323 ± 0.005 0.325 ± 0.005 0.323 ± 0.005 0.327 ± 0.005

0.298 - 0.39 0.29 - 0.353 0.293 - 0.358 0.289 - 0.366 0.29 - 0.359 0.296 - 0.357 0.254 - 0.427 0.163 - 0.57

(% total fatty acid) 0.055 (0.015, 0.075) (0.018, 0.083) (0.084, 0.243) (0.010, 0.058) (0.401, 0.616)

20:1 Eicosenoic 0.169 ± 0.009 0.171 ± 0.009 0.171 ± 0.009 0.171 ± 0.009 0.172 ± 0.009 0.168 ± 0.009

< LOQ - 0.254 < LOQ - 0.239 < LOQ - 0.254 < LOQ - 0.247 < LOQ - 0.24 < LOQ - 0.239 < LOQ - 0.272 < LOQ - 0.350

(% total fatty acid) 0.194 (0.131, 0.307) (0.181, 0.378) (0.123, 0.297) (0.072, 0.215) (0.865, 0.949)

20:2 Eicosadienoic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ - 0.245

(% total fatty acid) NA

20:3 Eicosatrienoic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ ND

(% total fatty acid) NA

20:4 Arachidonic NA NA NA NA NA NA

< LOQ < LOQ < LOQ < LOQ < LOQ < LOQ < LOQ ND

(% total fatty acid) NA

22:0 Behenic 0.326 ± 0.004 0.332 ± 0.004 0.331 ± 0.004 0.332 ± 0.004 0.328 ± 0.004 0.335 ± 0.004

0.273 - 0.365 0.303 - 0.368 0.298 - 0.371 0.299 - 0.367 0.294 - 0.365 0.309 - 0.371 0.29 - 0.454 0.277 - 0.595

(% total fatty acid) 0.009 (0.018, 0.083) (0.029, 0.112) (0.014, 0.075) (0.237, 0.448) (<0.001, 0.003)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Figure 37. Fatty acids (% total fatty acid) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean seed.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = 

MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 37 (Cont). Fatty acids (% total fatty acid) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled 

circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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8.4.6. Vitamin Analysis of Seed

Soybean seed samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for vitamin content. A summary of the results across all locations is presented in Table 

21 and Figure 38. All mean results were within literature ranges (when available) and/or within 

ranges for reference lines included in the study. For Vitamin A and β-Tocopherol, statistical 

analysis was not performed since greater than 50% of the samples were found to be below the 

LOQ. No statistical differences were observed in the combined-site analysis between the control 

and DAS-444Ø6-6 entries for Vitamins B3, B5, B6, and δ-Tocopherol. Statistically significant 

differences were observed for Vitamins B1, B2, B9, C, E, γ-Tocopherol, and total tocopherol for 

some DAS-444Ø6-6 entries compared with the control, where mean differences were negligible

and not biologically meaningful as means were within literature ranges and/or within ranges for 

reference lines included in the study.
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Table 21. Summary of the vitamin analysis of DAS-444Ø6-6 soybean seed from all sites, and associated literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Vitamin

Vitamin A NA NA NA NA NA NA

(β-Carotene) 0 - 0.894 0 - 0.803 0 - 0.928 0 - 0.762 0 - 1.1 0 - 0.78 0 - 0.871 NR

(mg/kg DW) NA

Vitamin B1 3.64 ± 0.20 3.42 ± 0.20 3.46 ± 0.20 3.32 ± 0.20 3.38 ± 0.20 3.55 ± 0.20

(Thiamine) 2.28 - 5.87 2.2 - 4.75 2.35 - 4.5 1.14 - 5.12 2.03 - 5.01 2.13 - 5.54 1.65 - 5.48 1.01 - 2.54

(mg/kg DW) 0.241 (0.126, 0.303) (0.215, 0.428) (0.026, 0.105) (0.067, 0.202) (0.521, 0.723)

Vitamin B2 3.99 ± 0.09 3.90 ± 0.09 3.88 ± 0.09 3.99 ± 0.09 3.88 ± 0.09 3.77 ± 0.09

(Riboflavin) 3.04 - 4.97 2.99 - 4.81 3.03 - 4.91 2.98 - 4.71 2.64 - 5.1 2.32 - 4.88 2.72 - 4.76 1.90 - 3.21

(mg/kg DW) 0.199 (0.318, 0.535) (0.244, 0.451) (0.943, 0.976) (0.219, 0.432) (0.022, 0.095)

Vitamin B3 26.5 ± 1.0 26.2 ± 1.0 26.5 ± 1.0 26.3 ± 1.0 26.6 ± 1.0 26.4 ± 1.0

(Niacin) 22.5 - 33.8 19.2 - 32.8 22.9 - 34.3 21.8 - 34.1 22.9 - 36.8 22.6 - 35.4 20.1 - 33 NR

(mg/kg DW) 0.765 (0.328, 0.541) (0.993, 0.996) (0.557, 0.735) (0.650, 0.804) (0.880, 0.956)

Vitamin B5 15.4 ± 0.6 15.9 ± 0.6 15.6 ± 0.6 15.3 ± 0.6 15.6 ± 0.6 15.8 ± 0.6

(Pantothenic Acid) 12.5 - 20.1 12.3 - 20.5 8.29 - 20.4 13 - 19.8 12.3 - 21.2 12.9 - 20.3 9.55 - 18.1 NR

(mg/kg DW) 0.277 (0.123, 0.297) (0.446, 0.662) (0.579, 0.758) (0.646, 0.804) (0.178, 0.377)

Vitamin B6 4.89 ± 0.09 4.85 ± 0.09 4.86 ± 0.09 4.79 ± 0.09 4.94 ± 0.09 4.93 ± 0.09

(Pyridoxine) 3.95 - 5.81 3.68 - 5.7 3.98 - 5.95 4.19 - 5.85 4.15 - 5.88 4.08 - 6.19 2.77 - 6.2 NR

(mg/kg DW) 0.268 (0.587, 0.763) (0.689, 0.834) (0.166, 0.363) (0.428, 0.646) (0.536, 0.725)

Vitamin B9 4.29 ± 0.19 4.09 ± 0.19 4.03 ± 0.19 4.02 ± 0.19 4.07 ± 0.19 3.92 ± 0.19

(Folic Acid) 2.7 - 5.78 2.72 - 5.46 2.63 - 5.85 2.5 - 5.84 2.88 - 6.02 2.57 - 5.28 2.35 - 5.98 2.386 - 4.709

(mg/kg DW) 0.029 (0.063, 0.195) (0.016, 0.076) (0.015, 0.075) (0.039, 0.137) (0.001, 0.009)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Vitamin

Vitamin C 121.4 ± 12.7 107.8 ± 12.7 111.1 ± 12.7 112.1 ± 12.7 112.8 ± 12.7 113.6 ± 12.7

(Ascorbic Acid) 0 - 198 16.5 - 181 17.6 - 194 16.8 - 173 23.8 - 193 25.3 - 171 0 - 141 NR

(mg/kg DW) 0.154 (0.008, 0.051) (0.040, 0.139) (0.064, 0.196) (0.083, 0.243) (0.117, 0.294)

Vitamin E 18.6 ± 3.9 22.2 ± 3.9 22.4 ± 3.9 22.2 ± 3.9 21.9 ± 3.9 22.2 ± 3.9

(α-Tocopherol) 10.5 - 46 10.9 - 69 11 - 56.8 10.1 - 106 10.9 - 55.7 8.85 - 76.2 6.43 - 49.9 0.108 - 61.693

(mg/kg DW) 0.115 (0.023, 0.096) (0.016, 0.076) (0.020, 0.092) (0.035, 0.128) (0.023, 0.096)

β-Tocopherol NA NA NA NA NA NA

0 - 0 0 - 0 0 - 0 0 - 0 0 - 0 0 - 0 0 - 6.42 NR

(mg/kg DW) NA

γ-Tocopherol 174 ± 5 185 ± 5 184 ± 5 179 ± 5 183 ± 5 181 ± 5

88.4 - 208 157 - 224 154 - 220 99 - 214 153 - 217 116 - 227 116 - 215 NR

(mg/kg DW) 0.005 (0.001, 0.007) (0.001, 0.010) (0.097, 0.261) (0.002, 0.015) (0.015, 0.075)

δ-Tocopherol 73.3 ± 5.2 72.0 ± 5.2 71.5 ± 5.2 73.8 ± 5.2 72.1 ± 5.2 72.2 ± 5.2

22.5 - 96.8 40.4 - 94.3 35.5 - 94.6 40.5 - 99.6 40 - 98.2 31.5 - 96.1 40 - 114 NR

(mg/kg DW) 0.317 (0.262, 0.468) (0.109, 0.281) (0.687, 0.834) (0.269, 0.475) (0.315, 0.535)

Total Tocopherol 266 ± 4 279 ± 4 278 ± 4 275 ± 4 277 ± 4 276 ± 4

132 - 305 244 - 316 252 - 306 161 - 375 248 - 308 196 - 330 199 - 321 NR

(mg/kg DW) 0.035 (0.003, 0.023) (0.005, 0.034) (0.040, 0.139) (0.008, 0.049) (0.024, 0.100)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Figure 38. Vitamins (mg/kg dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean seed.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte (when available).
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Figure 38 (Cont). Vitamins (mg/kg dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, 

filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte (when available).
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Figure 38 (Cont). Vitamins (mg/kg dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line soybean 

seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled 

circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte (when available).
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8.4.7. Bioactive Analysis of Seed

Soybean seed samples from the control (Maverick), reference, and DAS-444Ø6-6 entries were 

analyzed for bioactive chemical content. A summary of the results across all locations is 

presented in Table 22, Figure 39 and Figure 40. All mean results were within literature ranges 

and/or within ranges for reference lines included in the study. No statistical differences were 

observed in the combined-site analysis between the control and DAS-444Ø6-6 entries for phytic 

acid, stachyose, and total glycitein equivalent. Statistically significant differences were observed 

for lectin, raffinose, trypsin inhibitor, total daidzein equivalent, and total genistein equivalent for 

some DAS-444Ø6-6 entries compared with the control, where mean differences were negligible 

and not biologically meaningful as means were within literature ranges and/or within ranges for 

reference lines included in the study. Additionally, bioactive components including lectin and 

trypsin inhibitor are inactivated during standard processing of soybean seed prior to consumption

(Rackis, 1974; Padgette et al., 1996; Hammond and Jez, 2011).
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Table 22. Summary of the bioactive analysis of DAS-444Ø6-6 soybean seed from all sites, and associated literature range.

Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Bioactive

Lectin 79 ± 8 107 ± 8 94 ± 8 94 ± 8 92 ± 8 99 ± 8

(H.U./mg protein 27.9 - 153 60.6 - 228 39.4 - 188 56.3 - 146 44.8 - 151 31 - 196 18.5 - 144 37 - 323

 DW) 0.004 (<0.001, 0.001) (0.021, 0.092) (0.021, 0.092) (0.049, 0.161) (0.004, 0.026)

Phytic Acid 1.19 ± 0.07 1.18 ± 0.07 1.19 ± 0.07 1.18 ± 0.07 1.18 ± 0.07 1.19 ± 0.07

0.513 - 1.53 0.679 - 1.53 0.65 - 1.59 0.603 - 1.51 0.707 - 1.46 0.651 - 1.55 0.55 - 1.54 0.41 - 2.74

(% DW) 0.958 (0.789, 0.903) (0.866, 0.949) (0.511, 0.720) (0.632, 0.795) (0.824, 0.932)

Raffinose 0.82 ± 0.06 0.80 ± 0.06 0.80 ± 0.06 0.80 ± 0.06 0.80 ± 0.06 0.79 ± 0.06

0.497 - 1.29 0.556 - 1.22 0.581 - 1.18 0.569 - 1.22 0.438 - 1.3 0.478 - 1.23 0.569 - 1.4 0.212 - 1.62

(% DW) 0.200 (0.151, 0.345) (0.092, 0.256) (0.050, 0.162) (0.104, 0.275) (0.012, 0.065)

Stachyose 3.88 ± 0.06 3.88 ± 0.06 3.87 ± 0.06 3.86 ± 0.06 3.83 ± 0.06 3.89 ± 0.06

2.98 - 4.22 3.38 - 4.11 3.21 - 4.26 2.95 - 4.29 2.77 - 4.18 3.08 - 4.38 2.92 - 4.48 1.21 - 6.1

(% DW) 0.905 (0.952, 0.976) (0.816, 0.928) (0.779, 0.901) (0.375, 0.590) (0.827, 0.932)

Trypsin Inhibitor 30.8 ± 3.0 35.0 ± 3.0 35.5 ± 3.0 36.6 ± 3.0 33.6 ± 3.0 34.2 ± 3.0

18.4 - 54.6 19 - 56 17.4 - 71 21.1 - 78.9 21.3 - 51.7 21.8 - 62.6 15.6 - 59.7 18.14 - 118.68

(TIU/mg DW) 0.025 (0.015, 0.075) (0.007, 0.047) (0.001, 0.010) (0.095, 0.260) (0.047, 0.159)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Unit of measure was not converted prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

e

 Combined range for Stachyose includes individual and mean values.

Analytical 

Component 

(Units)a
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Overall 

Isoline

DAS-444Ø6-6 

unsprayed

DAS-444Ø6-6 

sprayed w/ 2,4-D

DAS-444Ø6-6 

sprayed w/ 

Glufosinate

DAS-444Ø6-6 

sprayed w/ 

Glyphosate

DAS-444Ø6-6 

sprayed w/ 

Three Herbicides

Reference

Range

Combined 

Literature 

Ranged

Trt Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE Mean ± SE

 Effect Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max Min - Max

 (Pr > F)b(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c(P-value, Adj.P)c

Bioactive

Total Daidzein 809 ± 114 777 ± 114 799 ± 114 800 ± 114 771 ± 114 781 ± 114

Equivalent 186 - 1450 175 - 1420 179 - 1470 182 - 1510 124 - 1490 149 - 1430 153 - 1710 25 - 2453.5

(mcg/g DW) 0.176 (0.067, 0.202) (0.540, 0.726) (0.581, 0.758) (0.029, 0.111) (0.097, 0.261)

Total Genistein 890 ± 155 863 ± 155 870 ± 155 877 ± 155 848 ± 155 855 ± 155

Equivalent 267 - 1670 300 - 1730 251 - 1690 264 - 1720 215 - 1770 186 - 1700 205 - 1980 28 - 2837.2

(mcg/g DW) 0.216 (0.133, 0.311) (0.269, 0.475) (0.458, 0.670) (0.021, 0.092) (0.057, 0.181)

Total Glycitein 453 ± 107 459 ± 107 465 ± 107 452 ± 107 448 ± 107 443 ± 107

Equivalent 222 - 1300 237 - 1250 223 - 1290 223 - 1340 197 - 1250 212 - 1270 85.2 - 1630 15.3 - 349.19

(mcg/g DW) 0.736 (0.656, 0.806) (0.415, 0.632) (0.948, 0.976) (0.760, 0.894) (0.521, 0.723)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation); ND (Not Detected) = < LOQ; NR = Not Reported.

a

 Aglycone and glycone forms of each isoflavone were summed to produce a total aglycone equivalent prior to analysis.

b

 Overall treatment effect estimated using an F-test. 

c

 Comparison to the control using t-tests (P-value); P-values adjusted (Adj. P) using a False Discovery Rate (FDR) procedure; P-values < 0.05 were considered significant.

d

 Combined range from Appendix 7.

Analytical 

Component 

(Units)a
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Figure 39. Bioactives (% dry weight (DW) for all bioactives except lectin (H.U./mg protein DW, H.U. = hemagglutination unit) 

and trypsin inhibitor (TIU/mg DW, TIU = trypsin inhibitor unit)) in non-transgenic (Isoline), Event DAS-444Ø6-6, and 

reference line soybean seed.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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Figure 39 (Cont). Bioactives (% dry weight (DW) for all bioactives except lectin (H.U./mg protein DW, H.U. = 

hemagglutination unit) and trypsin inhibitor (TIU/mg DW, TIU = trypsin inhibitor unit)) in non-transgenic (Isoline), Event 

DAS-444Ø6-6, and reference line soybean seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, 

open square = IL2, open diamond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is 

shaded for each analyte.
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Figure 40. Bioactives: isoflavones (mcg/g dry weight) in non-transgenic (Isoline), Event DAS-444Ø6-6, and reference line 

soybean seed.

Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open diamond = IN, filled circle = MI, filled 

triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.
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8.5. Composition Summary

All overall mean values for the non-transgenic isogenic control Maverick and DAS-444Ø6-6 

entries (unsprayed or sprayed with 2,4-D, glyphosate, glufosinate, or all three herbicides) were 

within literature ranges (when available) for soybean and/or within ranges for non-transgenic 

reference soybean lines included in the study. A limited number of statistically significant 

differences between DAS-444Ø6-6 entries (unsprayed and/or sprayed) and the control

(Maverick) were observed, but the differences were not biologically meaningful as the results 

were within ranges found for non-transgenic soybean. In conclusion, the compositional results 

for DAS-444Ø6-6 soybean, unsprayed or sprayed with 2,4-D, glyphosate, glufosinate, or all 

three herbicides, confirm equivalence to non-transgenic soybean lines.

8.6. Conclusions

Nutrient composition of DAS-444Ø6-6 soybean (unsprayed or sprayed with 2,4-D, glyphosate, 

glufosinate, or all three herbicides) was evaluated in field trials in 2010. DAS-444Ø6-6 soybean 

composition samples were all statistically indistinguishable from the control line and/or within 

literature or reference ranges for non-transgenic soybean, indicating that no unintended 

compositional effects were observed for DAS-444Ø6-6 soybean. Results from this study 

demonstrate compositional equivalence between DAS-444Ø6-6 and non-transgenic soybean.
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9. Environmental Consequences and Impact on Agronomic Practices

9.1. Field Efficacy

Field studies were conducted with DAS-444Ø6-6 soybean in 2010 to evaluate herbicide injury in 

three trials which differed by the timing of application (V2, V6, or R2). All three trials were 

conducted at Greenville, MS (US) and Weston, MO (US). The trial applied at V2 was also 

conducted at Huxley, IA (US), and the trials applied at V6 and R2 were also conducted at 

Fowler, IN (US) and Cambridge, IA (US). Seeds were planted between May 24 and June 6, 2010 

in 2-row plots with rows spaced 30 cm apart and seed placed at ~26 seeds per meter in the row. 

Rows were 5.3 m long with a 76 cm alley between plots. Therefore, each plot included ~275 

seeds. Plots were arranged in a randomized complete block design with 3 blocks per trial. 

Herbicide applications were applied 140 L/ha at between 21 and 32 PSI. Glyphosate (Durango 

DMA) and 2,4-D (Weedar 64) were applied at 1120 (1X) or 2240 (2X) g ae/ha. Glufosinate 

(Ignite 280 SL) was applied at 411 (1X) or 822 (2X) g ae/ha. Visual injury ratings were taken on 

a 0 to 100 scale, which reflects a visual composite of all injury symptoms observed across all 

plants in a plot. A rating of zero indicates no injury as compared to untreated plots and a rating of 

100 indicates death of all plants. Experimental data was analyzed across locations by ANOVA 

with treatment, replication and experiment being factors. A single replicate of the non-transgenic 

near-isogenic control Maverick was also planted separately at each location and sprayed with the 

1X rate of each herbicide. Injury ratings were collected to verify the negative effects of the 

herbicide sprays on non-transgenic soybean (Maverick).

Tolerance to 2,4-D

DAS-444Ø6-6 soybean displayed a high level of tolerance to applications of 2,4-D (Table 23).

When 2,4-D was sprayed at 1X or 2X, the proposed maximum use rate at the V2 growth stage, 

plant injury was 1% at both 1X and 2X rates two weeks after application. The same herbicide 

treatment was applied at the V6 or R2 stage. Plant injury was zero for treatment given at the V6 

stage. For treatment at the R2 stage, plant injury after one week of application was zero at 1X, 

the proposed maximum use rate, and 2% at 2X the proposed maximum use rate. Injury to nontransgenic near-isogenic soybean (Maverick) ranged from 80 to 99% at two weeks after 

treatment when sprayed at 1X, the proposed use rate of 2,4-D.

Table 23. DAS-444Ø6-6 soybean tolerance to application of 2,4-D (Weedar 64).

Herbicide Ratea

Application 

Stageb

Percent Plant Injuryc

Unsprayed DAS-444Ø6-6 Statistical Analysis

2,4-D amine 1120 g ae/ha V2 0.0 1.0 ns

2,4-D amine 2240 g ae/ha V2 0.0 1.0 ns

2,4-D amine 1120 g ae/ha V6 0.0 0.0 ns

2,4-D amine 2240 g ae/ha V6 0.0 0.0 ns

2,4-D amine 1120 g ae/ha R2 0.0 0.0 ns

2,4-D amine 2240 g ae/ha R2 0.0 2.0 ns

a

ae/ha = acid equivalent/hectare

b Application stage in terms of soybean plant growth development.

c Ratings were taken two weeks after application for treatment at V2 or V6 stage. Ratings were taken one 

week after application for treatment at R2 stage. A rating of zero indicates no injury and a rating of 100 

indicates death of all plants. ns indicates no statistical differences between sprayed and unsprayed 

DAS-444Ø6-6 at 95% confidence level.
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Tolerance to Glyphosate

DAS-444Ø6-6 soybean displayed a high level of tolerance to applications of glyphosate (Table 

24). Plant injury was zero under all of the experimental conditions. Injury to non-transgenic nearisogenic soybean (Maverick) ranged from 80 to 99% at two weeks after treatment when sprayed 

at 1X the proposed use rate of glyphosate.

Table 24. DAS-444Ø6-6 soybean tolerance to application of glyphosate (Durango DMA).

Herbicide Ratea

Application 

Stageb

Percent Plant Injuryc

Unsprayed DAS-444Ø6-6 Statistical Analysis

glyphosate 1120 g ae/ha V2 0.0 0.0 ns

glyphosate 2240 g ae/ha V2 0.0 0.0 ns

glyphosate 1120 g ae/ha V6 0.0 0.0 ns

glyphosate 2240 g ae/ha V6 0.0 0.0 ns

glyphosate 1120 g ae/ha R2 0.0 0.0 ns

glyphosate 2240 g ae/ha R2 0.0 0.0 ns

a

ae/ha = acid equivalent/hectare

b Application stage in terms of soybean plant growth development.

c Ratings were taken two weeks after application for treatment at V2 or V6 stage. Ratings were taken one 

week after application for treatment at R2 stage. A rating of zero indicates no injury and a rating of 100 

indicates death of all plants. ns indicates no statistical differences between sprayed and unsprayed 

DAS-444Ø6-6 at 95% confidence level.

Tolerance to Glufosinate

DAS-444Ø6-6 soybean displayed a high level of tolerance to applications of glufosinate (Table 

25). Plant injury was 1% for applications at the V6 and R2 growth stages at 2X, the proposed 

maximum use rate. Under all other conditions plant injury was zero. Injury to non-transgenic 

near-isogenic soybean (Maverick) ranged from 65 to 99% at two weeks after treatment when 

sprayed at 1X, the proposed use rate of glufosinate.

Table 25. DAS-444Ø6-6 soybean tolerance to application of glufosinate (Ignite 280SL).

Herbicide Ratea

Application 

Stageb

Percent Plant Injuryc

Unsprayed DAS-444Ø6-6 Statistical Analysis

glufosinate 411 g ae/ha V2 0.0 0.0 ns

glufosinate 822 g ae/ha V2 0.0 0.0 ns

glufosinate 411 g ae/ha V6 0.0 0.0 ns

glufosinate 822 g ae/ha V6 0.0 1.0 ns

glufosinate 411 g ae/ha R2 0.0 0.0 ns

glufosinate 822 g ae/ha R2 0.0 1.0 ns

a

ae/ha = acid equivalent/hectare

b Application stage in terms of soybean plant growth development.

c Ratings were taken two weeks after application for treatment at V2 or V6 stage. Ratings were taken one 

week after application for treatment at R2 stage. A rating of zero indicates no injury and a rating of 100 

indicates death of all plants. ns indicates no statistical differences between sprayed and unsprayed 

DAS-444Ø6-6 at 95% confidence level.
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Summary of Herbicide Tolerance

The outcome of the field studies demonstrates that DAS-444Ø6-6 soybean provides excellent 

tolerance to applications of 2,4-D, glyphosate and glufosinate, at rates of up to two times the 

proposed maximum use rates.

9.2. Weediness Potential

Commercial soybean varieties in the United States are not considered weeds and are not effective 

at invading established ecosystems.

Cultivated soybean seed rarely displays any dormancy characteristics and only under certain 

environmental conditions grows as a volunteer in the year following cultivation. If this should 

occur, volunteers do not compete well with the succeeding crop, and can easily be controlled 

mechanically or chemically. The soybean plant is not weedy in character. In North America, 

Glycine max is not found outside of cultivation. In managed ecosystems, soybean does not 

effectively compete with other cultivated plants or primary colonizers (OECD, 2000).

The introduction of aryloxyalkanoate herbicide, glufosinate, and glyphosate-tolerance into 

soybean will not alter the weediness characteristics of soybean. Agronomic properties of 

DAS-444Ø6-6 soybean related to weediness, such as germination (Section 7.3), emergence

(Section 7.1.2), seedling vigor (Section 7.1.2), and response to environmental stressors (Section 

7.1.2), have been shown to be identical to conventional Maverick soybean. If DAS-444Ø6-6

soybeans were to overwinter, they can still be effectively controlled mechanically or with other 

herbicide modes of action; such as paraquat in burndown programs, fluometuron in cotton 

(Hayes, 2000), and atrazine in corn. Therefore, these findings demonstrate that DAS-444Ø6-6

soybean do not have increased weediness potential relative to conventional soybean.

9.3. Gene Flow Assessment

9.3.1. Vertical Gene Flow

Soybean is considered to be a self-pollinated species, although natural crossing can occur at low 

rates (OECD, 2000). The level of cross pollination can vary as a function of growing conditions, 

spatial arrangement of plants, and genotype. Cross pollination levels ranging from 0.09% up to 

3.6% in adjacent rows have been reported (Beard and Knowles, 1971; Ahrent and Caviness, 

1994), but outcrossing frequency rapidly declines with distance. Cross pollination rates have 

been reported to be <0.1% at distances of >5 m (Caviness, 1966; Ray et al., 2003). Cultivated 

soybeans can cross only with members of its subgenus Soja. Wild soybean species of the 

subgenus Soja are native to Korea, Japan, Taiwan, northeastern China, and areas of the former 

USSR, but do not exist naturally in the United States (OECD, 2000). Therefore, there is no 

potential for gene flow from DAS-444Ø6-6 soybeans to wild soybean relatives in the United 

States.

9.3.2. Horizontal Gene Flow

There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to 

microbes (Conner et al., 2003). Even if such a transfer were to take place, transfer of the aad-12, 

2mepsps and pat genes from DAS-444Ø6-6 soybean would not present a human health or plant 

pest risk, based on the safety data presented in this petition.
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9.4. Current US Agronomic Practices for Soybeans

9.4.1. Soybean Production

Processed soybeans are the largest source of protein feed and the second largest source of 

vegetable oil in the world. The United States is the world's leading soybean producer and 

exporter. Farm value of U.S. soybean production in 2008/09 was $29.6 billion, the secondhighest value among U.S.-produced crops, trailing only corn. Soybean and soybean product 

exports accounted for 54 percent of U.S. soybean production in 2008/09. Soybeans are about 90 

percent of U.S. total oilseed production, while other oilseeds—such as cottonseed, sunflower 

seed, canola, and peanuts—account for the remainder (USDA ERS, 2010).

A total of 77.4 million acres were planted to soybeans in the United States in 2010 (USDA 

NASS, 2011). Of these planted acres, about 76.6 million acres were harvested, valued at $38.9 

billion. The majority of these soybeans were grown within the North Central states. About 80% 

of the planted soybean acres and 80% of the harvested soybean acres were concentrated in 11 

states – Iowa, Illinois, Minnesota, Indiana, Missouri, Nebraska, Ohio, South Dakota, North 

Dakota, Kansas, and Arkansas.

9.4.2. Weeds in Soybean

Weeds in soybean compete with the crop for light, nutrients and soil moisture. When weeds are 

uncontrolled for the entire season, yield losses can exceed 68 to 75% (Krausz et al., 2001; Dalley

et al., 2002). Hartzler and Pringnitz (2005) define the critical period of competition in soybeans 

as the point of time when weeds that emerge with the crop begin to impact yields. Most studies 

have found that soybean yields are protected if weeds are controlled before they reach a height of 

6 to 8 inches. The critical period varies widely, depending upon weed species and densities, 

environmental conditions, and cultural practices.

To maximize soybean yields, weeds must be removed or controlled when they are no more than 

6 inches tall (Loux et al., 2006). Field trials conducted in Ohio in 2001 found that applying 

glyphosate when weeds are already 9 to 12 inches tall resulted in a 6-10% yield loss due to weed 

competition prior to control of the weeds (Loux et al., 2006). Dalley et al. (2002) studied the 

effect of row width and weed competition on soybean yield in 1998-2001. They found that weed 

competition could reduce soybean yields by the time weeds reached 6, 6, and >12 inches in 

height in 7.5, 15, and 30 inch rows, respectively.

The most common weed problems in soybeans are the annual grass and broadleaf weeds (Table 

26). Some fields are also infested with perennial weeds (quackgrass, Johnsongrass, field 

bindweed, Canada thistle, others) which are more difficult to control since they can reproduce 

from seed or underground root buds or rhizomes.
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Table 26. Common troublesome weeds in soybeans in 2006-2008.

(Third Party Proprietary Data).

Weed Species

Total Soybean Acres Treated1

2006 2007 2008

 Annual Broadleaf Weeds

Lambsquarters, Common 21,859,614 24,459,895 28,242,972

Velvetleaf 23,820,731 23,373,573 26,786,349

Pigweed, Redroot 21,093,224 21,788,121 26,715,150

Cocklebur, Common 23,657,980 22,389,376 23,962,063

Waterhemp, Common 18,399,609 15,970,794 21,364,980

Ragweed, Giant 13,369,296 14,684,000 16,565,209

Marestail 4,044,060 5,382,190 11,257,267

Morningglory Spp. 10,711,087 11,432,904 11,011,185

Ragweed, Common 9,417,252 9,438,871 9,518,051

Sunflower, Wild 5,558,526 5,759,216 5,709,292

Kochia 4,859,759 3,671,795 5,317,528

Smartweed Pennsylvania 2,366,851 1,835,825 3,529,114

Waterhemp, Tall 2,301,380 2,926,358 3,826,647

Horseweed 2,188,359 3,159,712 3,470,274

Mustard, Wild 2,019,346 1,975,291 2,688,590

Sicklepod 2,024,031 1,650,086 2,535,829

Sida, Prickly 1,639,261 1,567,275 2,432,701

Sunflower, Volunteer 1,089,460 1,007,691 1,913,860

Chickweed 1,652,712 1,259,096 1,823,638

Nightshade, Black 1,766,649 1,277,416 1,385,751

Buckwheat, Wild 1,167,746 855,879 1,331,675

Pigweed, Smooth 188,160 801,569 1,322,732

 Annual Grass Weeds

Foxtail Spp. 24,409,043 18,489,746 18,446,420

Foxtail, Giant 11,817,612 17,513,493 17,804,622

Foxtail, Yellow 10,870,761 11,217,512 13,947,018

Foxtail, Green 5,629,880 7,109,316 7,610,855

Crabgrass 5,170,684 5,928,919 7,424,879

Barnyardgrass 4,189,156 3,967,425 3,805,391

Corn, Volunteer 2,292,705 2,088,371 3,704,330

Oat, Wild 1,792,389 1,478,890 2,886,300

Cupgrass, Woolly 1,765,244 2,470,437 2,108,135

Shattercane 2,408,592 2,715,388 1,879,416

Panicum, Fall 2,251,014 2,241,088 1,852,417

 Perennial / Biennial Weeds

Johnsongrass 10,152,393 11,057,825 10,368,155

Thistle, Canada 4,123,437 3,584,676 4,840,383

Quackgrass 2,628,187 2,570,688 2,786,633

Dandelion 1,578,579 1,528,332 2,154,008

Thistle 1,479,038 647,315 1,513,566

1 Total soybean acres in 2006, 2007, and 2008 was 75.5, 64.7, and 75.7 million acres, respectively (USDA 

NASS, 2011). However, the total soybean herbicide-treated acreage is much more, due to multiple sprays 

on each acre.
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The economic threshold for weeds is the density of a weed population at which control is 

economically justified due to the potential for yield reduction, quality loss, harvesting 

difficulties, or other problems that weeds may cause (Penn State Agronomy Guide, 2011). 

Broadleaf and grass weeds compete at different levels of intensity depending upon the 

competitiveness of the crop, tillage system, environmental conditions, and other weeds present.

Generally, broadleaf weeds are more damaging to a broadleaf crop, while grass weeds are more 

competitive in a grass crop. Crop yield loss information is available for certain single weed 

species growing with soybeans in the U.S. Midwest (Table 27).

Table 27. Yield reduction from specific weed species in soybeans.

Weed Species

Percent soybean yield reduction1

1 2 4 6 8 10

Weeds per 100 feet of row

Cocklebur 1 2 4 6 8 10

Pigweed or Lambsquarters 2 4 6 10 15 20

Shattercane (5-8/clump) 2 5 8 11 14 17

Giant foxtail 15 25 80 300 400 600

Velvetleaf 1 3 6 10 13 16

Smartweed 2 4 6 10 15 20

Volunteer corn 1 2 3 4 5 6

1

Source (Penn State Agronomy Guide, 2011). Interference data are from (Knake and Slife, 

1962; Stoller et al., 1987; Werner and Curran, 1995).

9.4.3. Weed Management in Soybean

In 2006, USDA NASS surveyed 19 states (Arkansas, Illinois, Indiana, Iowa, Kansas, Kentucky, 

Louisiana, Michigan, Minnesota, Mississippi, Missouri, Nebraska, North Carolina, North 

Dakota, Ohio, South Dakota, Tennessee, Virginia, and Wisconsin; about 96% of the total U.S. 

soybean acreage) and found that 98% of the planted soybean acreage was treated with 

herbicides, an indication of the intensive weed management that is used in U.S. soybeans (USDA 

NASS, 2007). In 2006, 97.5% of the planted soybean acres received an average of 2.2 herbicide 

applications to control weeds (USDA ERS ARMS, 2006). Many herbicides are registered for 

pre-plant, pre-emergent and/or post-emergent application to selectively control most weed 

species commonly found in soybeans. In general, soybeans sometimes receive a soil applied or 

burndown herbicide prior to planting or at plant, but almost always receive a post-emergence 

herbicide application. In 2006, the most widely used herbicide in soybeans was glyphosate, 

driven by the high adoption of glyphosate tolerant soybeans (USDA NASS, 2007). Soybeans 

received an average of 1.7 applications of glyphosate on 96% of the acres planted in 2006. 

Herbicides such as 2,4-D (10% of acres), chlorimuron (4% of acres), and trifluralin (2% of acres) 

were applied on significantly fewer acres. All other herbicides applied in soybeans that year 

comprised 3% or less of the total acres planted.

Integrated weed management (IWM) programs advocate the use of a combination of preventive, 

cultural, mechanical and chemical tools to keep weed pressure below threshold levels that reduce 

yield and profits (Knezevic, 2010). Herbicides are only one of several tools available for growers 
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to consider using in an IWM approach. Herbicide-tolerant soybeans, including DAS-444Ø6-6 

soybean, represent another tool that can be used in an IWM program.

As with any technology, some concerns have been raised about using herbicides and herbicidetolerant crops as part of an IWM program (Knezevic, 2010). Some of these concerns include 

selection for herbicide-resistant weeds, shifts in weed species, drift to sensitive non-target crops 

or non-agricultural habitats, herbicide persistence in the soil resulting in carryover to the next 

crop, herbicide misapplications, and over-reliance on herbicides to control weeds.

Weed management decisions are difficult decisions for growers since no one tool will effectively 

control all possible weed problems. In general, growers need to implement management 

practices that limit the introduction and spread of weeds, help the crop to compete with weeds,

and not allow weeds to adapt. The combination of weed control practices that a grower chooses 

is dependent upon the weed spectrum, level of infestation, soil type, cropping system, weather, 

and time and labor available for the treatment option.

9.4.4. Crop Rotation and Tillage Practices

In 2006, 71% of U.S. soybean acres were grown in rotation after corn, 13% were grown after 

soybeans, and 16% were grown after small grains, cotton, fallow or other crops (USDA ERS 

ARMS). Crop rotation is a widespread management practice that has been recognized and 

exploited for centuries to increase crop yields (Lauer, 2007). In the Midwestern U.S., a cornsoybean rotation produces at least 10% greater yields in both crops, and sometimes as much as

19% higher corn yields. The exact mechanism for the rotation effect is unknown, but may be 

influenced by increases in organic matter and soil fertility, as well as management of diseases, 

insects, and weeds.

The introduction of numerous herbicides in the decades following the launch of 2,4-D in the 

mid-1940s allowed reduced and conservation tillage systems to become more feasible and 

popular (Givens et al., 2009). The introduction of glyphosate-resistant (GR) crops in 1996 

brought a technology that enabled many producers to adopt reduced-tillage production systems. 

By 2002, only 17% of U.S. soybeans were produced under conventional tillage (<15% residue) 

systems (USDA ERS ARMS). Some form of conservation tillage was being used on the other 

83% of U.S. soybean acres, as follows: 18% reduced tillage (15-30% residue), 32% mulch till, 1-

2% ridge till, and 31% no-till. Increased yields coupled with decreases in erosion and water 

runoff and lower fuel use and fuel cost strongly support the premise that soybean produced with 

conservation tillage in any production system is more economically and environmentally 

sustainable (CAST, 2009). In fact, considering the generally accepted 30% minimum residue 

cover and the 3 to 5 tons/acre/year tolerable soil loss for a sustainable soybean production system 

(CAST, 2009), conservation tillage is the only system that seems to support sustainable soybean 

production from an environmental standpoint.

9.5. Potential Impact on Agronomic Practices

9.5.1. Potential Impact on Cultivation and Management Practices

Soybean lines that contain herbicide-tolerance traits (glyphosate) have been on the market since 

1996 and have experienced broad adoption (Figure 41). The adoption track record of 

biotechnology-derived soybean represents the most rapid case of technology adoption in the 
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history of agriculture (Sankula and Blumenthal, 2004). Based on USDA survey data, 

herbicide-tolerant soybeans (glyphosate-tolerant) went from 17 percent of U.S. soybean acreage 

in 1997 to 68 percent in 2001 and 93 percent in 2010 (USDA ERS, 2011b). Glyphosate has 

provided simple, inexpensive, and highly effective weed control and has resulted in an increase 

in no-till soybean production, a practice that is now accepted as improving soil health and 

agricultural sustainability.

With 93% of soybean acres in the U.S. in 2010 planted to herbicide tolerant (glyphosate-tolerant)

soybeans, typical cultivation and management practices used by growers today already take into 

account the management of herbicide-tolerant traits. DAS-444Ø6-6 soybeans are comparable to 

conventional soybeans phenotypically and agronomically (Section 7.4), and are not expected to 

alter the geographic range or seasonality of soybean cultivation. Furthermore, ecological 

observations during field testing have shown no changes in insect susceptibility of DAS-444Ø6-

6 soybeans (Section 7.2) and therefore, no impacts are expected on insect control practices for 

DAS-444Ø6-6 soybeans. It is anticipated that the same management practices used today for 

soybeans with the glyphosate-tolerance trait will also be appropriate for DAS-444Ø6-6 soybeans.

Figure 41. Adoption of genetically engineered crops in the U.S.

(USDA ERS, 2011a)

9.5.2. Potential Impact on Weed Control Practices

DAS-444Ø6-6 soybeans confer tolerance to the herbicides 2,4-D, glyphosate, and glufosinate, all

of which will provide expanded weed management options in soybeans.

Post-emergence applications of 2,4-D control a broad spectrum of broadleaf weeds. 2,4-D also 

has some short-lived soil residual activity (4-10 day soil half life) which provides limited 

residual control of later germinating broadleaf weeds. Post-emergence applications of glyphosate 
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will control a broad spectrum of grass and broadleaf weeds. Thus, DAS-444Ø6-6 soybeans will 

provide flexibility to farmers over glyphosate alone in weed management systems. Inclusion of 

2,4-D (and possibly glufosinate) with glyphosate would control the already glyphosate-resistant 

and hard to control broadleaf weeds, plus slow down the selection for more glyphosate-resistant 

broadleaf weeds (Powles, 2008a).

Currently, for soybeans without the aad-12 gene, 2,4-D can be applied only as a burndown or 

pre-emergence application at up to 1.0 lb ae/A (1120 g ae/ha). 2,4-D currently cannot be applied 

at burndown or pre-emergence to conventional soybeans any later than 7-15 days 

(0.5 - 1.0 lbs ae/A, or 560 - 1120 g ae/ha of ester formulations) or 15-30 days (0.5 – 1.0 lbs ae/A, 

or 560 - 1120 g ae/ha of amine formulations) prior to planting, due to potential for crop injury.

In DAS-444Ø6-6 soybeans, the proposed use pattern will be to allow application of 2,4-D at 

burndown or pre-emergence (1 lb ae/A or 1120 g ae/ha) without plant back restrictions, followed 

by one or two post-emergence (0.5 – 1.0 lb ae/A or 560-1120 g ae/ha) applications at least 12

days apart over-the-top of the soybeans up to the R2 stage (full flower) of development (Figure 

42). Thus, the proposed maximum seasonal rate of 2,4-D on soybeans will increase from 1.0 lb 

ae/A to 3.0 lbs ae/ha. DAS-444Ø6-6 soybeans will allow growers to apply 2,4-D from burndown 

or pre-emergence up through R2 stage soybeans without risk of crop injury. This will provide 

new options for improved weed control during the soybean development period when weeds 

have the greatest potential yield impact. The proposed use pattern for 2,4-D on DAS-444Ø6-6 

soybeans is identical to that for DAS-68416-4 soybeans.

Figure 42. 2,4-D herbicide application timing and rates for conventional and DAS-444Ø6-6

soybeans.

The use pattern for glyphosate and glufosinate on DAS-444Ø6-6 soybeans will be consistent 

with the current use pattern of glyphosate on other soybean products that are tolerant to 

glyphosate or glufosinate.
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While 2,4-D is currently registered as a pesticide, supporting information on proposed label 

changes for its use with DAS-444Ø6-6 soybeans is being provided by Dow AgroSciences to U.S. 

EPA for review, and will be provided to the PMRA shortly. Dow AgroSciences is also 

developing an extensive stewardship program that will include technological advancements in 

application to reduce potential for off-target movement, as well as utilizing several media venues 

to educate and facilitate adoption of the technology and decision management tools to ensure the 

proper use and stewardship of both the trait and chemical technologies.

Products such as DAS-444Ø6-6 that provide tolerance to multiple modes of action will improve 

weed control by allowing use of herbicide combinations or mixtures which can provide more 

consistent performance in post-emergence weed control programs. They counteract glyphosate 

―rate-creep‖ (steady increase in rates needed over time to obtain effective weed control; Figure 

43) on hard-to-control weeds (Jaehnig, 2005). DAS-444Ø6-6 soybeans, which will enable the 

use of 2,4-D, will allow use of a low cost, high performance solution to reduce the escalation of 

glyphosate- and ALS-resistance in weed populations.

Figure 43. Glyphosate application rates in U.S. corn and soybeans from 1998 -2010.

(Third Party Proprietary Data)

9.5.3. Potential Impact on Volunteer Management

Cultivated soybean seed rarely displays any dormancy characteristics, and only under certain 

environmental conditions grows as a volunteer in the year following cultivation (OECD 2000). If 

this should occur, volunteers do not compete well with the succeeding crop, and can easily be 

controlled mechanically or chemically. The soybean plant is not weedy in character. In North 

America, Glycine max is not found outside of cultivation. In managed ecosystems, soybean does 

not effectively compete with other cultivated plants or primary colonizers.

DAS-444Ø6-6 soybeans are tolerant to 2,4-D, glyphosate and glufosinate. In the unlikely event 

that they would grow as volunteers in the year following cultivation, they can still be effectively 

controlled with other herbicides (York et al., 2005). Lb. a.i./Acre

Corn Soybeans

0.9
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Soybean is considered a self-pollinated species (OECD, 2000). Crossing does not generally 

occur. Soybean can only cross with other members of Glycine subgenus Soja. Approximately 

93% of all soybeans planted in the U.S. in 2010 were glyphosate-tolerant soybeans (USDA 

NASS, 2010). If DAS-444Ø6-6 soybeans were to cross with soybean varieties expressing 

tolerance to herbicides with different modes of action to produce soybean volunteers with 

multiple herbicide-tolerance, they can still be effectively controlled mechanically or with other 

herbicide modes of action; such as paraquat in burndown programs, fluometuron in cotton 

(Hayes, 2000), and atrazine in corn. Additionally, agronomic practices such as appropriate 

variety selections, crop rotation, and rotation of herbicides with different modes of action can be 

used to avoid or manage volunteer soybeans tolerant to one or a few herbicides.

9.5.4. Potential Impact on Non-Target Organisms and Endangered Species

Based on substrate specificity of the aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme 

activity and the biological activity of the 2mEPSPS enzyme, no effect on non-target organisms 

or endangered species is anticipated for DAS-444Ø6-6 soybean. The aad-12 gene and expressed 

protein are present in nature in the soil bacterium Delftia acidovorans, while the 2mepsps and pat

genes, as well as the expressed proteins are expressed in other crops grown in the United States 

with no effect on non-target organisms or endangered species. AAD-12, 2mEPSPS, and PAT are

not potential food allergens or toxins (Sections 6.1.5, 6.2.5 and 6.3.5) and DAS-444Ø6-6

soybean has been shown to be substantially equivalent to conventional soybean based on the 

compositional analysis of grain and forage (Section 8). Observations made during field testing of 

DAS-444Ø6-6 soybean revealed no effects on invertebrate populations (Section 7.2) and 

agronomic characteristics were found to be equivalent to conventional soybean (Section 7.1).

The Endangered Species Act (ESA) (16 USC 1531) is administered by the U.S. Fish & Wildlife 

Service (FWS). Under ESA, Section 6 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 DAS-444Ø6-6 soybean will impact 

any currently listed species of concern since it is not anticipated that DAS-444Ø6-6 soybean will 

cause new soybean acres to be planted in areas that are not already in agricultural use. 

Commercial cropping systems routinely disturb the ground in which crops are grown. Habitat 

disruption within DAS-444Ø6-6 soybean fields would be comparable to other no-till or 

herbicide-tolerant cropping systems.

Soybean is not an invasive or weedy species, there are no invasive or weedy sexually compatible 

relatives of soybean in the US, and these properties are not anticipated to be altered by the 

insertion of the genes conferring tolerance to herbicides. It is therefore reasonable to conclude 

that DAS-444Ø6-6 soybean will not affect threatened or endangered species or adversely affect 

or change designated critical habitats as compared to current commercial agricultural practices.

2,4-D is currently registered for use in soybean production for pre-plant and burndown herbicide 

treatment. The environmental fate and ecological effects on non-target organisms for the use of 

2,4-D in soybeans will be addressed by the EPA as part of their review process.

Glyphosate and glufosinate are currently registered for use in soybean production as a post 

emergent herbicide treatment in glyphosate- and glufosinate-tolerant soybean varieties. The 
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environmental fate and ecological effects of these herbicides on non-target organisms and 

endangered species has been addressed by the EPA as part of its review process.

Corn and soybean are typically planted in rotation in the US. In corn, 2,4-D is used both as a preplant burn down prior to planting and post-emergence. The post-emergent and seasonal use 

patterns for 2,4-D in DAS-444Ø6-6 soybeans are consistent with those currently approved for 

use in conventional corn, and are consistent with those proposed for use in DAS-40278-9 corn 

and DAS-68416-4 soybean that also provide tolerance to 2,4-D. Therefore, no significant new 

geography will be treated with 2,4-D beyond what is already available through pre-emergent 

applications in soybeans and pre- and post-emergent applications in corn. This consistency in the 

application rates, timings, and the maximum seasonal rate with currently registered use patterns 

indicates that there should be no change in the ecological risk assessments or endangered species 

assessments for 2,4-D with DAS-444Ø6-6 soybean.

9.6. Herbicide Resistance Management

9.6.1. Herbicide Resistance

Herbicides have revolutionized weed control in most countries around the world. Herbicides are 

often the most reliable and least expensive method of weed control available, but reliance upon 

herbicides as the primary method of weed control can have unintended consequences. The 

widespread use of herbicides can lead to weed populations that are no longer susceptible to the 

herbicide being used.

The Weed Science Society of America defines herbicide resistance as "the inherited ability of a 

plant to survive and reproduce following exposure to a dose of herbicide normally lethal to the 

wild type‖(WASS, 1998). Target plants with genes conferring resistance to a given herbicide can 

occur naturally within a population, but in extremely small numbers. Such plants remain 

reproductively compatible with the wild-type and can pass resistance genes on to their progeny 

(Mallory-Smith, 2010). Repeated use of the herbicide may allow these resistant plants to survive 

and reproduce. The number of resistant plants then increases in the population until the herbicide 

no longer effectively controls the weed. Thus, this is an evolutionary process (Moss, 2002), 

whereby a population changes from being susceptible to being resistant. Individual plants do not 

change from being susceptible to being resistant; rather, the proportion of resistant individuals 

within the population increases over time.

Herbicide-resistant weeds have been a problem for growers for decades (Heap, 1997). The 

earliest documented reports of herbicide-resistant weeds were resistance to 2,4-D in wild carrot 

(Daucus carota) in 1952 and spreading dayflower (Commelina diffusa) in 1957 (Heap, 2011). 

During the 1970s, up to 30 different weed species were reported to be resistant to the triazine 

herbicides (Bandeen et al., 1982). Today, more than 355 weed biotypes around the world have 

been reported to have some populations that are resistant to one or more herbicides (Heap, 2011).

A weed biotype is a sub-type or sub-population of a weed species, in this case one that has 

developed resistance to one or more herbicides. Report of a resistant biotype for a given weed 

species does not mean that weed resistance is common, widespread, or persistent in that species. 

There are generally many other options available to control these resistant biotypes. Table 28

shows a tabular summary of the total number of resistant species for each herbicide mode of 

action as of May 2011. Figure 44 shows the number of resistant weed biotypes that have been 
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reported over time for each herbicide mode of action. Additional information on glyphosate, 

2,4-D, glufosinate, and the evolution of herbicide resistant weeds can be found in Appendix 8.
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Table 28. Number of herbicide resistant weeds reported globally by herbicide group and 

mode of action.

(Heap, 2011)

Herbicide Group Mode of Action

HRAC 

Group

a

WSSA 

Group

a

Example 

Herbicide

Total 

Count

ALS Inhibitors

Inhibition of acetolactate synthase 

ALS (acetohydroxyacid synthase 

AHAS)

B 2 Chlorsulfuron 109

Photosystem II 

Inhibitors

Inhibition of photosynthesis at 

photosystem II C1 5 Atrazine 69

ACCase Inhibitors Inhibition of acetyl CoA carboxylase 

(ACCase) A 1 Diclofop-methyl 40

Synthetic Auxins Synthetic auxins (action like 

indoleacetic acid) O 4 2,4-D 28

Bipyridiliums Photosystem-I-electron diversion D 24 Paraquat 25

Ureas and Amides Inhibition of photosynthesis at 

photosystem II C2 7 Chlorotoluron 21

Glycines Inhibition of EPSP synthase G 9 Glyphosate 21

Dinitroanilines and 

others Microtubule assembly inhibition K1 10 Trifluralin 10

Thiocarbamates and 

others

Inhibition of lipid synthesis – not

ACCase inhibition N 8 Triallate 8

Chloroacetamides and 

others

Inhibition of cell division (inhibition 

of very long chain fatty acids) K3 15 Butachlor 5

Triazoles, Ureas, 

Isoxazolidiones

Bleaching: Inhibition of carotenoids 

biosynthesis (unknown target) F3 11 Amitrole 4

PPO Inhibitors Inhibition of protoporphyrinogen 

oxidase (PPO) E 14 Oxyfluorfen 4

Nitriles and others Inhibition of photosynthesis at 

photosystem II C3 6 Bromoxynil 3

Carotenoid 

Biosynthesis Inhibitors

Bleaching: Inhibition of carotenoids 

biosynthesis at the phytoene 

desaturase

F1 12 Flurtamone 2

Arylaminopropionic 

Acids Unknown Z 25 Flamprop-methyl 2

Mitosis Inhibitors Inhibition of mitosis / microtubule 

polymerization inhibitor K2 23 Propham 1

Cellulose Inhibitors Inhibition of cell wall (cellulose) 

synthesis L 27 Dichlobenil 1

Unknown Unknown N 8 (Butylate 1

Unknown Unknown Z 227 (chloro)-flurenol 1

Total Number of 

Unique Herbicide 

Resistant Biotypes

355

a Two different classification systems have been developed independently by HRAC (alphabetical) and 

the WSSA (numerical) to communicate the mode of action of herbicides. Weeds which have developed 

resistance to one herbicide may also be resistant to other herbicides which have the same mode of action.
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Figure 44. Resistant weed biotypes per herbicide mode of action.

9.6.2. Factors Impacting Development of Resistance

There are several factors to consider when assessing the risk for herbicide resistance in a weed 

species. Some of these relate to the biology of the weed species in question, others relate to

particular farming practices. The key factors influencing a plant‘s potential to develop resistance 

have been outlined by the Herbicide Resistance Action Committee (HRAC), an industry 

initiative that fosters cooperation between plant protection manufacturers, government, 

researchers, advisors and farmers. These key factors include the number or density of weeds, 

natural frequency of resistant plants in the population, seed soil dormancy potential, frequent use

of herbicides with a similar mode of action, cropping rotations with reliance primarily on 

herbicides for weed control, and lack of non-chemical weed control practices (HRAC, 1998).

A matrix that can be used to evaluate the risk of selection for herbicide-resistant weeds based on 

cropping system practices in shown in Table 29. This table assesses the risk of herbicideresistance development for each management practice as either ―low‖, ―medium‖, or ―high‖. The 

greatest chance for resistance development occurs when several of these management practices 

fall into the ―high‖ category.
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Table 29. Assessment of resistance risk by evaluation of cropping systems.

(Nevill et al., 1998)

Management Option

Risk of Resistance

Low Moderate High

Herbicide mix or rotation in 

cropping system

>2 modes of action 2 modes of action 1 mode of action

Weed control in cropping 

system

Cultural, mechanical and 

chemical

Cultural* and 

chemical

Chemical only

Use of same MOA per season Once More than once Many times

Cropping system Full rotation Limited rotation No rotation

Resistance status to MOA Unknown Limited Common

Weed infestation Low Moderate High

Control in last 3 years Good Declining Poor

*Cultural control can be by using cultivation, stubble burning, competitive crops, stale seedbeds, etc. See 

HRAC guidelines for more details.

Development of herbicide-resistance is often thought of as a problem caused by the herbicide 

itself. However, it is well documented that resistance results from management practices that 

have relied too heavily on a particular herbicide as the sole method of weed control. Under these 

conditions, the risk of weeds developing herbicide-resistance is greatest and the best defense is 

diversity in weed management practices.

9.6.3. Herbicide Resistance Management

Although no cases of glyphosate-resistant weeds were documented for 20 years after the launch 

of glyphosate (Dyer, 1994), glyphosate-resistant biotypes of several weed species have now been 

reported in the United States (Powles, 2008b). This may be attributed to increased reliance on 

glyphosate for weed control after the launch of glyphosate-tolerant soybeans (1996), cotton 

(1997), and corn (1998). This evolution of glyphosate-resistant weed populations threatens the 

ongoing sustainability of glyphosate and its contributions to world food production (Duke and 

Powles, 2008a).

As the number of glyphosate-resistant weed species increases, it becomes increasingly important 

for growers to introduce greater diversity into their weed management programs (Powles, 

2008a). This diversity could be achieved with herbicide rotations/sequences, mixtures of robust 

herbicides with different modes of action, and use of non-herbicide weed control tools. 

Glyphosate is increasingly being mixed with effective doses of other herbicides to manage these 

hard-to-control and resistant weed species. New herbicide-tolerant traits that allow selective use 

of additional herbicides with a wider weed control spectrum compared to conventional 

herbicides can be used to control glyphosate-resistant weed populations and reduce selection 

pressure for additional glyphosate-resistant weed species. Table 30 shows that several common 

weeds in U.S. corn and soybeans which are resistant to or difficult to control with glyphosate or 

ALS herbicides can be effectively controlled with 2,4-D.
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Introduction of DAS-444Ø6-6 soybeans will give farmers one more tool for use in their weed 

management programs which will help insure the long term sustainability of weed management 

programs, including the use of glyphosate. Importantly, because DAS-444Ø6-6 will provide 

tolerance to 2,4-D and glyphosate, these herbicides can be tank mixed to provide an effective, 

flexible and cost effective approach to weed management. 2,4-D controls several key broadleaf 

weed species that are resistant to, or difficult to control with, glyphosate and ALS herbicides 

(Table 30). Mixtures of 2,4-D with glyphosate provide control of glyphosate-resistant biotypes 

that is superior to glyphosate alone.

DAS-444Ø6-6 soybeans will allow use of 2,4-D to control glyphosate-resistant weeds and 

significantly delay the selection for glyphosate-resistance in other weed species. The same 

benefits of DAS-444Ø6-6 soybeans can be extended to other herbicide-tolerant cropping 

systems, such as those with tolerance to glufosinate or ALS-inhibiting herbicides.

Table 30. Glyphosate- and ALS-resistant weeds controlled by 2,4-D.

(Heap, 2011)

Weed Species Glyphosate ALS Herbicides 2,4-D

Common 

lambsquarters

Difficult: Suspected Resistant (2004) Resistant (2001) Susceptible

Common ragweed Confirmed Resistant (2004) Resistant (1998) Susceptible

Eastern black 

nightshade

Difficult (2004) Resistant (1999) Susceptible

Giant ragweed Confirmed Resistant (2004) Resistant (1998) Susceptible

Marestail 

(horseweed)

Confirmed Resistant (2000) Resistant (2000) Susceptible

Palmer amaranth Confirmed Resistant (2005) Resistant (1991) Susceptible

Waterhemp spp. Confirmed Resistant (2005) Resistant (1993) Susceptible

Prickly sida Difficult (2004) Resistant (1993) Susceptible

DAS-444Ø6-6 soybeans will expand the range of herbicides that can be used in herbicidetolerant soybean production systems, improving the ease and effectiveness of managing resistant 

and hard-to-control weeds and delaying the evolution of resistance to glyphosate and other 

herbicides.

9.7. Summary of Environmental Consequences and Impact on Agronomic Practices

Field testing results confirm that AAD-12, 2mEPSPS, and PAT proteins expressed in 

DAS-444Ø6-6 soybean provides robust tolerance to 2,4-D, glyphosate, and glufosinate 

respectively. There are no new phenotypic characteristics in DAS-444Ø6-6 soybean to indicate it 

is any different from conventional soybean in weediness potential, and like conventional 

soybean, the risk of gene flow from DAS-444Ø6-6 soybean to wild relatives in the U.S. is 

negligible.

No significant impact is expected on current crop management practices, non-target or 

endangered species, crop rotation, or volunteer management from the introduction of 

DAS-444Ø6-6 soybean. The availability of DAS-444Ø6-6 soybean will have a beneficial impact 

on weed control practices by providing growers with another tool to address their weed control 

needs. The availability of DAS-444Ø6-6 soybean will allow growers to proactively manage 
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weed populations while avoiding adverse population shifts of troublesome weeds or the 

development of resistance.

10. Adverse Consequences of Introduction

Field and laboratory testing of DAS-444Ø6-6 soybean has demonstrated that it has no significant 

differences from the non-transgenic near-isogenic control soybean Maverick apart from the 

intended change of herbicide-tolerance. DAS and MS Tech know of no study results or other 

observations associated with DAS-444Ø6-6 soybean that would be anticipated to result in 

adverse consequences from introduction.
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11. Appendices

Appendix 1. Methods for Molecular Characterization of DAS-444Ø6-6 Soybean

Appendix 2. Methods and Results for Characterization of AAD-12 Protein

Appendix 3. Methods and Results for Characterization of 2mEPSPS Protein

Appendix 4. Methods and Results for Characterization of PAT Protein

Appendix 5. Methods for AAD-12, 2mEPSPS and PAT Protein Expression Analysis

Appendix 6. USDA Notifications for DAS-444Ø6-6 Soybean

Appendix 7. Literature Ranges for Compositional Analysis

Appendix 8. Glyphosate, 2,4-D, Glufosinate and Herbicide Resistant Weeds

Appendix 9. Stewardship of Herbicide Tolerant DAS-444Ø6-6 Soybean

Appendix 10. References
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Appendix 1. Methods for Molecular Characterization of DAS-444Ø6-6 Soybean

DAS-444Ø6-6 Soybean Material

Transgenic soybean seeds from five distinct generations of soybean containing event 

DAS-444Ø6-6 were planted in the greenhouse. After at least one week of growth, leaf punches 

were taken from each plant and were tested for PAT protein expression using a rapid lateral flow 

test strip according to the manufacturer‘s instructions. Each plant was given a ―+‖ or ―-‖ for the 

presence or absence of the PAT protein.

Control Soybean Material

Seeds from the unmodified Maverick were planted in the greenhouse. The Maverick seeds had a 

genetic background representative of the transgenic seeds but did not contain the 2mepsps,

aad-12, and pat genes.

Reference Materials

DNA of the plasmid pDAB8264 was added to samples of the Maverick control genomic DNA at 

a ratio approximately equivalent to 1 copy of the transgene per soybean genome and used as the 

positive control for the Southern hybridization.

DNA Probe Preparation

DNA probes specific to the genetic elements in the T-DNA insert of pDAB8264 and the vector 

backbone sequences were produced via polymerase chain reaction (PCR) amplification using 

pDAB8264 plasmid DNA as a template, followed by purification from agarose gel.

Sample Collection and DNA Extraction

Labeled leaf samples were collected from green house for DNA extraction or stored in -80°C 

freezer for future use. Genomic DNA was extracted with the modified CTAB method. Briefly, 

leaf samples were individually ground in liquid nitrogen followed by the addition of extraction 

buffer (~5:1 ratio milliliter CTAB extraction buffer: gram leaf tissue) and RNase-A (>10 μL) 

(Qiagen). After approximately 2 hours of incubation at ~65°C with gentle shaking, samples were 

spun down and the supernatants were extracted with equal volume of chloroform:octanol = 24:1 

(Sigma). DNA was precipitated by mixing the supernatant with equal volume of precipitation 

buffer (1% CTAB, 50 mM Tris-HCl, pH 8.0, 10 mM EDTA). The precipitated DNA was 

dissolved in high salt TE buffer (1 × TE pH 8.0, 1.0 M NaCl) followed by precipitation with 

isopropyl alcohol. The precipitated DNA was rinsed with 70% ethanol, air-dried, then dissolved 

in appropriate volume of 1 × TE buffer (pH 8.0).

To determine the quality of the resultant genomic DNA, an aliquot of the DNA samples was 

electrophoretically separated on a 1% agarose gel containing ethidium bromide (~1 μg/mL) with 

1× TBE buffer (89 mM Tris-Borate, 20 mM EDTA, pH 8.3). The gel was visualized under 

ultraviolet (UV) light to confirm that the DNA was not degraded and that the RNA had been 

removed by the RNase-A. The concentration of DNA in solution was determined by a PicoGreen 

kit (Invitrogen) using a spectrofluorometer (Bio-TEK, FLX800).
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DNA Digestion and Electrophoretic Separation of the DNA Fragments

Genomic DNA extracted from the soybean leaf tissue was digested with restriction enzymes by 

combining approximately 10 g of genomic DNA with approximately 5-10 units of the selected 

restriction enzyme per g of DNA in the corresponding reaction buffer. Each sample was 

incubated at 37oC overnight. The positive control sample was prepared by combining 

pDAB8264 plasmid DNA with genomic DNA from the Maverick control (at a ratio 

approximately equivalent to 1 copy of the transgene per soybean genome) and was digested 

using the same procedures and restriction enzymes as the test samples. DNA from the Maverick 

control was digested using the same procedures and restriction enzymes as the test samples to 

serve as the negative control.

The digested DNA samples were precipitated with Quick-Precip (Edge BioSystems) and resuspended to achieve the desired volume for gel loading. The DNA samples and molecular size 

markers were then electrophoresed through 0.8% agarose gels with 1 TBE buffer (89 mM Tris, 

89 mM Boric acid, 2 mM EDTA) at 55-65 V for 18-22 hours to achieve fragment separation.

The gels were stained with ethidium bromide and the DNA was visualized under UV light. A 

photographic record was made of each stained gel.

Southern Transfer

The DNA fragments on the agarose gels were transferred to nylon membranes via Southern 

transfer, essentially as described by Memelink et al. (1994). The agarose gels were depurinated, 

denatured, neutralized in situ and transferred to a nylon membrane in 10 SSC buffer (3 M NaCl, 

0.3 M Na citrate) using a wicking system. Following transfer to the membrane, the DNA was 

bound to the membrane by crosslinking through UV treatment.

Probe Synthesis and Hybridization

The hybridization probes were generated using a PCR-based incorporation of a digoxigenin 

(DIG) labeled nucleotide, [DIG-11]-dUTP, into the DNA fragments generated by primers 

specific to the genetic elements and other regions from plasmid pDAB8264. The PCR synthesis 

of the probes was performed using a PCR DIG Probe Synthesis Kit (Roche Diagnostics) 

following the manufacturer‘s recommended procedures.

Labeled probes were hybridized to the target DNA on the nylon membranes using the DIG Easy 

Hyb Solution according to manufacturer‘s instructions (Roche Diagnostics). DIG-labeled DNA 

molecular weight marker II and VII were used to determine the size of the hybridizing fragments

on the Southern blots.

Detection

DIG-labeled probes bound to the nylon membranes after stringent washing were incubated with 

AP (Alkaline Phosphatase)-conjugated anti-Digoxigenin antibody for ~1 hr at room temperature.

The anti-DIG antibody specifically bound to the probes was then visualized using CDP-Star 

Chemiluminescent Nucleic Acid Detection System (Roche Diagnostics). Blots were exposed to 

chemiluminescent film for one or more time points to detect the hybridizing fragments and to 
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visualize the molecular weight standards. The images were then scanned and stored. The number 

and size of each of the detected bands were documented for each digest and for each probe.

Once the data was recorded, membranes were rinsed with milli-Q water and then stripped of the 

bound probe in a solution containing 0.2 M NaOH and 1.0% SDS. The alkali-based stripping 

procedure successfully removes the labeled probes from the membranes, allowing them to be rehybridized with a different DNA probe.
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Appendix 2. Methods and Results for Characterization of AAD-12 Protein

DAS-444Ø6-6 transgenic soybean material

Greenhouse-grown DAS-444Ø6-6 soybean plants (T4 generation) were used as the plant source 

of the AAD-12 protein. Prior to use, individual plants were leaf tested to confirm expression of 

the AAD-12 protein using an immunospecific rapid lateral flow test strip according to the 

manufacturer‘s instructions. Tissues from AAD-12 expressing plants were harvested, 

lyophilized, ground to a fine powder, and stored frozen until needed.

Control soybean material

Control soybean line Maverick had a genetic background representative of the DAS-444Ø6-6

soybean plants, but did not contain the aad-12 gene. Absence of AAD-12 expression in the 

control plants was confirmed using the same AAD-12 specific rapid lateral flow test strip as 

previously mentioned. Tissues of control plants were harvested, lyophilized, ground and stored 

under the same conditions as the DAS-444Ø6-6 soybean.

Reference material

Recombinant AAD-12 protein was produced in Pseudomonas fluorescens and purified to a 

lyophilized powder. The microbe-derived AAD-12 protein preparation was stored dry and 

resuspended in a Tris-based buffer to maintain activity prior to use.

Protein purification of AAD-12 from DAS-444Ø6-6 soybean plant tissue

A soybean crude protein extract containing the AAD-12 protein was extracted from lyophilized 

root tissue in a Tris-based buffer, pH 8.0 with added stabilizers, filtered though cheesecloth and 

the soluble proteins were separated from the insoluble plant material by centrifugation. The 

supernatant was slowly adjusted to 1 M ammonium sulfate and insoluble proteins were removed 

by centrifugation. The remaining soluble protein fraction was loaded onto a 5 mL Phenyl 

Sepharose HP Hi-Trap Column (GE Healthcare) and the unbound proteins were washed from the 

column with 50 mM Tris (pH 8.0), 1 M ammonium sulfate. Proteins retained on the column were 

eluted with a decreasing gradient of ammonium sulfate and fractions containing AAD-12 protein 

(as determined by ELISA) were pooled. The pooled fraction was then incubated with an antiAAD-12 immunoaffinity resin which had been conjugated with an AAD-12 specific monoclonal 

antibody using a crosslinked immuno-precipitation kit (Thermo-Pierce). Bound proteins were 

eluted according to the manufacturer‘s protocols. The unbound fraction was re-incubated with 

the resin and a second pool of protein was collected. Eluted fractions were analyzed by SDSPAGE (stained with GelCode Blue total protein stain from Thermo-Pierce), western blot, and 

ELISA. Fractions containing the soybean-derived AAD-12 protein were pooled, desalted into 50 

mM ammonium bicarbonate utilizing a PD-10 desalting column (GE Healthcare), aliquoted, and 

lyophilized.

SDS-PAGE and western blot analysis of crude soybean leaf extracts

Fresh leaf tissue from event DAS-444Ø6-6 and Maverick was mixed with a Tris-based buffer 

containing ~3% protease inhibitor cocktail (Sigma) and the soluble proteins were extracted by 
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grinding with ball bearings in a Geno/Grinder (Certiprep, Metuchen, NJ) for 3 minutes. The 

samples were centrifuged and the supernatants were mixed with Laemmli sample buffer, heated

at 95°C for 5 minutes, and briefly centrifuged. The positive and negative reference standards, 

microbe-derived AAD-12 and bovine serum albumin, respectively, were also mixed with sample 

buffer and prepared the same as the plant extracts. The samples were loaded directly on to two 

separate Bio-Rad Criterion SDS-PAGE gels and the proteins were electrophoresed with MES 

running buffer (Bio-Rad) for ~ 60 minutes at 150 volts. Following electrophoresis, one gel was

stained with Thermo Pierce GelCode Blue protein stain for total protein and the other gel was 

electro-blotted onto a nitrocellulose membrane for western blot analysis. After transfer, the 

nitrocellulose membrane was blocked with 5% dry milk in PBST and probed with an AAD-12 

specific polyclonal antibody. A chemiluminescent substrate (GE Healthcare) was used to 

visualize the immunoreactive bands on X-ray films (Thermo Pierce).

Detection of post-translational glycosylation

The immunoaffinity-purified, plant-derived AAD-12 protein was analyzed for evidence of 

glycosylation by SDS-PAGE along with microbe-derived AAD-12 protein, soybean trypsin 

inhibitor, bovine serum albumin, and horseradish peroxidase used as reference controls. The 

reference protein samples were adjusted to concentrations approximately equal to that of the 

plant-derived AAD-12 protein and mixed with Laemmli buffer. The proteins were heated at 

95°C for 5 minutes, centrifuged, and applied directly to a Bio-Rad Criterion SDS-PAGE gel. 

Following electrophoresis, the gel was cut in half. One gel half was stained with Thermo Pierce 

GelCode Blue stain for total protein. The remaining half of the gel was stained with GelCode 

Glycoprotein Stain to visualize glycoproteins. The glycoproteins present on the gel were 

visualized as magenta bands on a light pink background.

Mass spectrometry peptide mass fingerprinting and sequence analysis of plant- and microbederived AAD-12 protein

The immunoaffinity purified AAD-12 plant-derived protein was subjected to in-gel digestion by 

trypsin and chymotrypsin followed by matrix assisted laser desorption/ionization time-of-flight 

mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS-MS. The peptide fragments of the 

plant-derived AAD-12 protein (including the N- and C-termini) were analyzed and compared 

with the sequence of the microbe-derived protein.

Results of the SDS-PAGE and western blot analysis

In the microbe-derived AAD-12, the major protein band, as visualized on the Coomassie stained 

SDS-PAGE gel, was approximately 32 kDa (Figure 45). As expected, the corresponding plantderived AAD-12 protein was identical in size to the microbe-derived protein. Predictably, the 

protein fractions purified from DAS-444Ø6-6 tissue contained a minor amount of nonimmunoreactive impurities in addition to the AAD-12 protein. The co-purified proteins were 

likely retained on the column by weak interactions with the column matrix (Holroyde et al., 

1976; Kennedy and Barnes, 1983; Williams et al., 2006).

The microbe-derived AAD-12 and DAS-444Ø6-6 plant tissue extract showed a positive signal of 

the expected size on the western blot using an anti-AAD-12 polyclonal antibody (Figure 46). In 
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the AAD-12 western blot analysis, no immunoreactive proteins were detected in the control

Maverick extract and no alternate size proteins (aggregates or degradation products) were 

detected in the samples from the transgenic plant. Lack of alternate size proteins in the AAD-12 

sample derived from DAS-444Ø6-6 soybean tissue in the western analysis indicates the absence 

of any glycosylated form of AAD-12. This result is consistent with the outcome of the 

glycosylation study on the AAD-12 protein of DAS-444Ø6-6 soybean.

Figure 45. SDS-PAGE of DAS-444Ø6-6 soybean- and microbe-derived AAD-12.

Note: Affinity-purified soybean-derived AAD-12 and microbe-derived AAD-12 were separated by SDSPAGE. Following electrophoresis, the gel was stained with Thermo-Pierce GelCode Blue stain for total 

protein according to the manufacturer‘s protocol. The protein fractions purified from DAS-444Ø6-6 

tissue contained a minor amount of non-immunoreactive impurities in addition to the AAD-12 protein. 

The co-purified proteins were likely retained on the column by weak interactions with the column matrix.

Lane Sample Amount Loaded

M Novex Sharp MW Markers 5 μL

1 DAS-444Ø6-6 derived AAD-12 20 μL

2 DAS-444Ø6-6 derived AAD-12 20 μL

3 Microbe-derived AAD-12 780 ng

M 1 2 3

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

AAD-1 2
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Panel A. SDS-PAGE Panel B. Western blot.

Figure 46. SDS-PAGE and western blot analysis of DAS-444Ø6-6 soybean- and microbe-derived AAD-12 protein.

Note: Crude extracts from fresh leaf tissue of event DAS-444Ø6-6 and Maverick were separated by SDS-PAGE along with microbe-derived 

AAD-12 and bovine serum albumin. Following electrophoresis the gel was cut in half, one half was stained with Thermo Pierce GelCode Blue 

stain for total protein and the remaining half was electro-blotted onto a nitrocellulose membrane. The nitrocellulose membrane was then probed 

with AAD-12 specific polyclonal antibodies and detected with HRP-labeled antibodies. A chemiluminescent substrate was used to visualize the 

immunoreactive bands.

Panel A. Panel B.

Lane Sample Amount Loaded Lane Sample Amount Loaded

M Novex Sharp Unstained MW Marker 10 μL kDa Novex Pre-stained MW Marker

1 Non-transgenic (Maverick) Leaf Extract 30 μL 1 Microbe-derived AAD-12 (TSN030732) 1 ng

2 DAS-444Ø6-6 Soybean Leaf Extract 30 μL 2 Bovine Serum Albumin (BSA) 1 ng

3 Microbe-derived AAD-12 (TSN030732) 1.1 μg 3 DAS-444Ø6-6 Soybean Leaf Extract 10 μL

4 Microbe-derived 2mEPSPS (TSN033171) 1.0 μg 4 Non-transgenic (Maverick) Leaf Extract 10 μL

5 Bovine Serum Albumin (BSA) 1.1 μg

P Novex Pre-stained MW Marker 10 μL
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Results of detection of glycosylation of AAD-12 protein

Glycoproteins were not detected in the DAS-444Ø6-6 soybean-derived or microbe-derived 

AAD-12 samples (Figure 47). The result indicates that the AAD-12 protein was not modified 

with covalently bound carbohydrate moieties. In this study horseradish peroxidase, a 

glycoprotein, was used as a positive indicator for glycoprotein staining. Soybean trypsin inhibitor 

and bovine serum albumin, both non-glycoproteins, served as negative controls.

Note: The Novex Sharp protein standard does not appear on the 

glycoprotein stained gels as they do not contain glycoproteins.

Figure 47. Glycosylation analysis of DAS-444Ø6-6 soybean- and microbe-derived AAD-12 

proteins.

Note: The affinity-purified AAD-12 derived from event DAS-444Ø6-6, microbe-derived AAD-12, 

soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were diluted to a similar 

concentration prior to separation by SDS-PAGE. After electrophoresis, the gel was cut in half and one 

half was stained with Thermo Pierce GelCode Blue stain for total protein, and the other half of the gel 

was stained with a Thermo Pierce GelCode Glycoprotein Staining Kit to visualize the glycoproteins.

Lane Sample Amount Loaded

M Novex Sharp Protein Standard 10 μL

1 Horseradish peroxidase (+ control) ~500 ng

2 Soybean trypsin inhibitor (- control) ~500 ng

3 Bovine serum albumin (- control) ~500 ng

4 DAS-444Ø6-6 derived AAD-12 ~200 ng

5 P. fluorescens derived AAD-12 ~500 ng

P Novex Pre-stained MW Marker 10 μL

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

M 1 2 3 4 5 P M 1 2 3 4 5 P

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

- 3.5

kDa

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

kDa

total protein stain - 3.5 glycoprotein stain
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Results of MALDI-TOF MS and MALDI-TOF MS/MS tryptic and chymotryptic peptide mass 

fingerprints of DAS-444Ø6-6-derived AAD-12 protein

Following digestion of the DAS-444Ø6-6-derived AAD-12 protein by trypsin and chymotrypsin, 

the masses of the detected peptides were compared with those deduced based on potential 

cleavage sites in the sequence of the AAD-12 protein. Figure 48 illustrates the theoretical peptide 

cleavage which was generated in silico using PAWs software (Proteometrics LLC).

In the trypsin and chymotrypsin digestions of soybean-derived AAD-12 protein, the peptide 

sequence coverage was excellent (84.3%) and 76.1% of the peptide primary sequence was 

confirmed by MS/MS analysis (Figure 49). The detected peptide fragments covered nearly the 

entire protein sequence lacking only four peptide fragments (Figure 49), one near the N-terminus 

(L40 to L45), two in the middle of the protein (Q89 to Y114 and S125 to F138), and one near the 

C-terminus (I211 to R213). The peptide sequence that was missed did not contain sequence motif 

that are typically required for glycosylation (Asn-Xxx-Ser/Thr (Hamby and Hirst, 2008)).
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Digestion at K (lysine) and R (arginine)

Digestion at F (phenylalanine), L (leucine), W (tryptophan), and Y (tyrosine)

Figure 48. Theoretical trypsin (top panel) and chymotrypsin (bottom panel) cleavage of the 

AAD-12 protein.

Note: Alternating blocks of upper (black) and lower (red) case letters within the amino acid 

sequence are used to differentiate the potential peptides after protease digestion. The numbers on 

the left and right sides indicate the amino acid residue numbers.
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Figure 49. Overall sequence coverage of trypsin and chymotrypsin digests for 

DAS-444Ø6-6-derived AAD-12 protein by MALDI-TOF MS and MALDI TOF-TOF.

Cys residues were alkylated with iodoacetamide. Overall sequence coverage was 84.3% with 

peptide mass fingerprint data and 76.1% by tandem MS data. Two forms of the N-terminus were 

detected. The N-terminal peptide was detected with Met1intact and removed by an 

aminopeptidase. The peptide with Met1 missing was acetylated at Ala2.

Results of MALDI-TOF MS/MS N- and C-terminal sequence analysis of AAD-12

The amino acid residues at the N-and C-termini of the soybean-derived AAD-12 protein 

(immunoaffinity purified from DAS-444Ø6-6 soybean) were determined and compared with the 

sequence of the microbe-derived protein. The soybean-derived AAD-12 protein sequences were 

determined by MALDI-TOF MS/MS. The chymotrypsin and trypsin digestions were performed 

on the soybean-derived AAD-12 protein followed by mass spectrometry analysis and two forms 

of the N-terminus were determined (Table 31).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M A Q T T L Q I T P T G A T L G A T V T G V H L A T L D D A 30 30 27

31 G F A A L H A A W L Q H A L L I F P G Q H L S N D Q Q I T F 60 24 20

61 A K R F G A I E R I G G G D I V A I S N V K A D G T V R Q H 90 28 22

91 S P A E W D D M M K V I V G N M A W H A D S T Y M P V M A Q 120 6 0

121 G A V F S A E V V P A V G G R T C F A D M R A A Y D A L D E 150 16 16

151 A T R A L V H Q R S A R H S L V Y S Q S K L G H V Q Q A G S 180 30 30

181 A Y I G Y G M D T T A T P L R P L V K V H P E T G R P S L L 210 30 30

211 I G R H A H A I P G M D A A E S E R F L E G L V D W A C Q A 240 30 27

241 P R V H A H Q W A A G D V V V W D N R C L L H R A E P W D F 270 30 28

271 K L P R V M W H S R L A G R P E T E G A A L V 293 23 23

247 223

A = Tryptic MS coverage

A = Tryptic MS-MS data = 84.3 %

A = Chymotryptic MS coverage = 76.1 %

A = Chymotryptic MS-MS data

Peptides 

covered by 

MS-MS

MALDI MS-MS sequence coverage

Tot. a.a. covered =

MALDI PMF sequence coverage

Peptides 

covered 

by PMF
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Table 31. Summary of N-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived AAD-12 protein.

Source Expected N-terminal Sequencea

P. fluorescens M A Q T T L Q I T P T G A T L G A T V T G V H L A T L

Soybean Event M A Q T T L Q I T P T G A T L G A T V T G V H L A T L

DAS-444Ø6-6

Source Detected N-terminal Sequenceb,c,d

P. fluorescens A Q T T L Q I T P T G A T L G A T V T G V H L A T

Soybean Event M A Q T T L Q I T P T G A T L G A T V T G V H L A T

DAS-444Ø6-6

Soybean Event N-AcA Q T T L Q I T P T G A T L G A T V T G V H L A T

DAS-444Ø6-6

4

Notes:

a

Expected N-terminal sequence of the first 27 amino acid residues of P. fluorescens- and 

soybean-derived AAD-12.

bDetected N-terminal sequences of P. fluorescens- and soybean-derived AAD-12 (Appendix 2).

cNumbers in superscript (Rx

) indicate the amino acid residue number in the sequence. The Nterminal amino acid sequence was confirmed by peptide mass fingerprinting and MS/MS 

sequencing.

d

The MALDI-TOF MS/MS data for the N-terminal peptide revealed that the soybean-derived 

AAD-12 protein had a portion of the peptide that was acetylated (N-Acetyl-AQTTL).

Amino acid residue abbreviations:

A: alanine G: glycine H: histidine

I: isoleucine L: leucine M: methionine

P: proline Q: glutamine S: serine

T: threonine V: valine
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These results demonstrate that the N-terminus of the AAD-12 protein was intact and the amino 

acid sequence was as predicted (Table 31 and Figure 49). In addition, a portion of the protein 

extracts was missing the N-terminal methionine and the second amino acid, alanine, was 

acetylated (Table 31). This result is encountered frequently with eukaryotic (plant) expressed 

proteins as approximately 80-90% of the N-terminal residues are modified in such a way 

(Wellner et al., 1990; Polevoda and Sherman, 2003). This result indicates that during or after 

translation in soybean and P. fluorescens, the N-terminal methionine was cleaved by a 

methionine aminopeptidase (MAP). MAPs cleave methionyl residues rapidly when the second 

residue on the protein is small, such as Gly, Ala, Ser, Cys, Thr, Pro, and Val (Walsh, 2005).

Also, it has been shown that proteins with serine and alanine at the N-termini are most frequently 

acetylated (Polevoda and Sherman, 2002). The two co-translational processes, cleavage of 

N-terminal methionine residue and N-terminal acetylation, are by far the most common 

modifications and occur on the vast majority (~85%) of eukaryotic proteins (Polevoda and 

Sherman, 2002). However, examples demonstrating biological significance associated with Nterminal acetylation are rare (Polevoda and Sherman, 2000).

The C-terminal sequence of the soybean- and microbe-derived AAD-12 proteins were

determined essentially as described above and compared with the expected amino acid 

sequences. The results indicated the detected sequences were identical to the expected sequences, 

and both the soybean- and microbe-derived AAD-12 proteins were identical and unaltered at the 

C-terminus (Table 32, Figure 49, and Figure 50).
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Table 32. Summary of C-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived AAD-12 protein.

Source Expected C-terminal Sequencea

P. fluorescens 281L A G R P E T E G A A L V293

Soybean Event

DAS-444Ø6-6

281L A G R P E T E G A A L V293

Source Detected C-terminal Sequenceb

P. fluorescens 281L A G R P E T E G A A L V293

Soybean Event 281L A G R P E T E G A A L V293

DAS-444Ø6-6

a

Expected C-terminal sequence of the last 13 amino acid residues of P. fluorescens- and 

soybean-derived AAD-12.

bDetected C-terminal sequences of P. fluorescens- and DAS-444Ø6-6-derived AAD-12.

Notes:

Numbers in superscript (Rx) indicate amino acid residue numbers in the sequence. 

Amino acid residue abbreviations:

A: alanine E: glutamate G: glycine

L: leucine P: proline R: arginine

T: threonine V: valine
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Figure 50. Overall sequence coverage of trypsin, chymotrypsin, Arg-C, Asp-N, and Glu-C 

digests for P. fluorescens-derived AAD-12 protein by MALDI-TOF MS and ISD MS/MS.

Cys residues were alkylated with iodoacetamide. Overall sequence coverage was 100% (taking into 

account the post-translational removal of Met1) with peptide mass fingerprint data and 94.2% by insource decay MS/MS data. The N-terminal Met1 was removed by an aminopeptidase.

Conclusions

The biochemical characteristics of microbe-derived AAD-12 protein were equivalent to the 

protein purified from root tissue of event DAS-444Ø6-6. The plant- and microbe-derived 

AAD-12 proteins showed the expected molecular weight of ~32 kDa by SDS-PAGE and were 

immunoreactive to AAD-12 protein specific antibodies by western blot analysis. The amino acid 

sequence of both proteins was confirmed by enzymatic peptide mass fingerprinting by 

MALDI-TOF MS, MALDI-TOF MS/MS, and ESI-LC/MS. In addition, the lack of glycosylation 

of the plant-derived AAD-12 protein provided additional evidence that the AAD-12 protein 

produced by P. fluorescens and DAS-444Ø6-6 soybean are biochemically equivalent.

Microbe derived AAD-12 overall peptide mapping

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M A Q T T L Q I T P T G A T L G A T V T G V H L A T L D D A 30 29 29

31 G F A A L H A A W L Q H A L L I F P G Q H L S N D Q Q I T F 60 30 21

61 A K R F G A I E R I G G G D I V A I S N V K A D G T V R Q H 90 30 30

91 S P A E W D D M M K V I V G N M A W H A D S T Y M P V M A Q 120 30 30

121 G A V F S A E V V P A V G G R T C F A D M R A A Y D A L D E 150 30 30

151 A T R A L V H Q R S A R H S L V Y S Q S K L G H V Q Q A G S 180 30 22

181 A Y I G Y G M D T T A T P L R P L V K V H P E T G R P S L L 210 30 30

211 I G R H A H A I P G M D A A E S E R F L E G L V D W A C Q A 240 30 30

241 P R V H A H Q W A A G D V V V W D N R C L L H R A E P W D F 270 30 30

271 K L P R V M W H S R L A G R P E T E G A A L V 293 23 23

292 275

A = PMF MS coverage (MS)

A = ISD Coverage (MS/MS) = 100.0 %

= 94.2 %

Peptides 

covered 

by PMF

Peptides 

covered by 

MS-MS

Tot. a.a. covered =

MALDI PMF sequence coverage

MALDI MS-MS sequence coverage
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Appendix 3. Methods and Results for Characterization of 2mEPSPS Protein

DAS-444Ø6-6 transgenic soybean material

Greenhouse-grown DAS-444Ø6-6 soybean plants (T4 generation) were used as the plant source 

of the 2mEPSPS protein. Prior to use, individual plants were analyzed by a lateral flow strip 

assay (American Bionostica Inc.) to confirm the presence of the AAD-12 protein (through 

inference, the plants were considered to contain 2mEPSPS as it is part of the molecular stack)

using a rapid lateral flow test strip according to the manufacturer‘s instructions. Tissues from 

2mEPSPS expressing plants were harvested, lyophilized, ground to a fine powder, and stored 

frozen until needed.

Control soybean material

Control soybean line Maverick had a genetic background representative of the DAS-444Ø6-6

soybean plants, but did not contain the 2mepsps gene. Seeds of the Maverick soybean line were 

planted, grown, harvested, tested, processed, and stored under the same conditions as the 

transgenic plants described above.

Reference material

Recombinant 2mEPSPS protein was produced in Pseudomonas fluorescens and purified to a 

lyophilized powder. The microbe-derived 2mEPSPS protein preparation (Lot Number: 

DMMG_033110) was stored dry as a lyophilized powder and resuspended in a HEPES based 

buffer to maintain activity prior to use.

Protein purification of 2mEPSPS from DAS-444Ø6-6 soybean plant tissue

The 2mEPSPS protein was extracted from lyophilized leaf tissue with a HEPES-based buffer 

(see Table 33 for buffer components). The tissue was blended and the extract was filtered 

through cheesecloth and the filtrate was collected and clarified by centrifugation at 10,000 ×g. 

Ammonium sulfate was added to the clarified extract to a final concentration of 2 M and the 

solution was then centrifuged, filtered and loaded onto a 1 mL Phenyl HP Hi-trap column (GE 

Healthcare) equilibrated with 50 mM HEPES, 2.0 M ammonium sulfate, pH 7.0. After loading, 

the column was washed with the same buffer and the bound proteins were eluted with a gradient 

to 100% 50 mM HEPES, pH 7.0. The eluted proteins were collected and assayed for 2mEPSPS 

content by western blot using a mouse monoclonal antibody raised against the microbe-derived 

2mEPSPS protein. Fractions containing 2mEPSPS were pooled and desalted into 50 mM 

HEPES, pH 7.0 buffer using a PD-10 desalting column (GE Healthcare) according to 

manufacturer‘s protocol. The Phenyl HP Pool was then loaded onto a 1 mL Q Sepharose FF HiTrap Column (GE Healthcare) equilibrated with 50 mM HEPES, pH 7.0. After loading, the 

column was washed and eluted with a gradient to 100% 50 mM HEPES, 500 mM NaCl, pH 7.0. 

The eluted proteins were collected and the fractions were assayed for 2mEPSPS content by 

western blot as described above. Fractions containing 2mEPSPS were pooled and desalted into 

50 mM HEPES, pH 7.0 buffer using a PD-10 desalting column. The desalted Q-pool was then 

loaded onto a 5 mL Blue Sepharose HP Hi-Trap column (GE Healthcare) equilibrated with 50 

mM HEPES, pH 7.0 buffer. The column was washed and the bound protein was eluted with a 

gradient to 100% 50 mM HEPES, 500 mM NaCl, pH 7.0. Fractions were collected and assayed 
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for 2mEPSPS content by Coomassie staining a SDS-PAGE gel. The fraction containing the 

highest level of 2mEPSPS by visual inspection was concentrated to ~130 μL using a 10 kDa 

MWCO centrifugal filter device (Amicon) according to the manufacture‘s protocol. The final 

purified sample was held at 4°C until used for subsequent analyses.

Table 33. Composition of extraction buffer for soybean-derived 2mEPSPS.

Ingredient Supplier Final Concentration

HEPES Fisher 50 mM

Sodium Ascorbate Sigma 20 mM

Sodium Metabisulfite Sigma 10 mM

Polyvinylpyrrolidone Sigma 1.0%

Protease Inhibitor Cocktail Sigma 0.5%

Notes:

a. The buffer pH was adjusted to 7.0 before bringing buffer up to final volume.

b. The buffer was made fresh the day of use.

SDS-PAGE and polyclonal antibody western blot analysis of crude soybean leaf extracts

The soybean leaf tissues of the transgenic event DAS-444Ø6-6 and nontransgenic isoline were 

harvested fresh from the greenhouse on the day of testing. Extracts were prepared by grinding

the tissue with steel ball bearings in a Tris-based buffer (Table 34) using a Geno/Grinder 

(Certiprep, Metuchen, NJ). The supernatants collected were mixed with Laemmli sample buffer 

(containing -mercaptoethanol) heated/denatured and loaded directly on the gel with a positive 

reference standard (microbe-derived 2mEPSPS - TSN033171-0001), and control standard BSA 

(Thermo-Pierce). SDS-PAGE separation of the recombinant 2mEPSPS, BSA, non-transgenic 

Maverick, and DAS-444Ø6-6 soybean extracts was performed with Bio-Rad Criterion gels fitted 

in a Criterion Cell gel module with XT-MES running buffer (Bio-Rad). Two identical gels were 

prepared and the electrophoresis was conducted at a constant voltage of 150 V for ~60 minutes. 

After separation, one gel was stained with Thermo Scientific GelCode Blue protein stain and 

scanned with a densitometer (GE Healthcare) to obtain a permanent record of the image. The 

remaining gel was electro-blotted to a nitrocellulose membrane (Bio-Rad) and probed with a 

2mEPSPS-specific rabbit polyclonal antibody (Lot #: G2874, 3.3 mg/mL). A conjugate of goat 

anti-rabbit IgG (H+L) antibody and horseradish peroxidase (Thermo-Pierce) was used as the 

secondary detection antibody. GE Healthcare chemiluminescent substrate was used for 

development and visualization of the immunoreactive protein bands. The membranes were 

exposed to CL-XPosure detection film (Thermo Scientific) for various time points and 

subsequently developed with an All-Pro 100 film developer.
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Table 34. Western blot extraction buffer.

Ingredient Supplier Final Concentration

1M Tris-HCl Sigma 50 mM

Sodium Ascorbate Sigma 20 mM

Sodium Metabisulfite Sigma 10 mM

NaCl N/A 250 mM

PVP Sigma 0.7%

2-mercaptoethanol Bio-Rad 0.2%

Protease Inhibitor Cocktail Sigma 3.3%

Notes:

a. The buffer pH was adjusted to 8.0 before bringing buffer up to final volume.

b. The buffer was made fresh the day of use.

SDS-PAGE and monoclonal antibody western blot analysis of the 2mEPSPS protein

The soybean leaf tissues of the transgenic event and non-transgenic isoline were harvested fresh 

from the greenhouse and stored at -80°C until used for testing. On the day of analysis, soybean 

leaf material was ground in liquid nitrogen, transferred to a micro-centrifuge tube, and the 

soluble proteins were extracted using a Geno/Grinder (Certiprep, Metuchen, NJ). The 

supernatants were clarified by centrifugation, mixed with Laemmli sample buffer (Bio-Rad, 

containing freshly added  -mercaptoethanol) and heated at 95°C for 5 minutes. After a brief 

centrifugation, the resulting supernatants were loaded directly on the gel with a reference 

standard, 2mEPSPS (TSN033171-0001), and control standard, BSA (Thermo-Pierce). 

SDS-PAGE and western blot analysis of the recombinant 2mEPSPS, BSA, non-transgenic 

Maverick, and DAS-444Ø6-6 soybean extracts were performed with Bio-Rad Criterion gels 

fitted in a Criterion Cell gel module with XT-MES running buffer (Bio-Rad). One gel was 

prepared and the electrophoresis was conducted at a constant voltage of 150 V for ~60 minutes. 

After separation the gel was cut in half and one half of the gel was stained with Thermo 

Scientific GelCode Blue protein stain and scanned with a densitometer (GE Healthcare) to obtain 

a permanent record of the image. The remaining gel half was transferred to a nitrocellulose 

membrane and probed with a 2mEPSPS-specific mouse monoclonal antibody (Lot #: 609.48A-2-

4, 2.1 mg/mL). A conjugate of goat anti-mouse IgG (H+L) antibody and horseradish peroxidase 

(Thermo-Pierce) was used as the secondary detection antibody. GE Healthcare 

chemiluminescent substrate was used for development and visualization of the immunoreactive 

protein bands. The membrane was exposed to CL-XPosure detection film (Thermo Scientific) 

for various time points and subsequently developed with an All-Pro 100 film developer.

Detection of post-translational glycosylation

The purified, plant-derived 2mEPSPS protein was analyzed for evidence of glycosylation by 

SDS-PAGE along with microbe-derived 2mEPSPS protein. Soybean trypsin inhibitor, bovine 

serum albumin, and horseradish peroxidase were added as reference controls. The reference

protein samples were adjusted to concentrations approximately equal to that of the plant-derived 

2mEPSPS protein and mixed with Laemmli buffer. The proteins were heated at 95°C for 5 

minutes, centrifuged at 20,000×g for 2 minutes, and applied directly to a Bio-Rad Criterion SDS-
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PAGE gel and electrophoresed as described above. Following electrophoresis, the gel was cut in 

half and one half of the gel was stained with Thermo Pierce GelCode Blue stain for total protein. 

The remaining half was stained with GelCode Glycoprotein Stain to visualize glycoproteins

according to the manufacture‘s protocol. The glycoproteins present on the gel were visualized as 

magenta bands on a light pink background.

Mass spectrometry peptide mass fingerprinting and sequence analysis of plant- and microbederived 2mEPSPS proteins

The purified plant-derived 2mEPSPS protein was subjected to in-gel and in-solution digestion by 

trypsin and chymotrypsin followed by matrix assisted laser desorption/ionization time-of-flight 

mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS-MS. The peptide fragments of the 

plant-derived 2mEPSPS protein (including the N- and C-termini) were analyzed and compared 

with the sequence of the microbe-derived protein.

Results of the SDS-PAGE and western blot analysis

SDS-PAGE

In the microbe-derived 2mEPSPS sample, the major protein band, as visualized on the 

Coomassie stained SDS-PAGE gel, was approximately 47 kDa (Figure 51). As expected, the 

corresponding plant-derived 2mEPSPS protein was identical in size to the microbe-derived 

protein. Predictably, the plant purified fractions contained a minor amount of nonimmunoreactive impurities in addition to the 2mEPSPS protein. The co-purified proteins were 

likely retained on the column by weak interactions with the column matrix (Holroyde et al., 

1976; Kennedy and Barnes, 1983; Williams et al., 2006).
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Figure 51. SDS-PAGE of DAS-444Ø6-6- and microbe-derived 2mEPSPS.

Note: The purified soybean-derived 2mEPSPS and microbe-derived 2mEPSPS were separated 

by SDS-PAGE. Following electrophoresis, the gel was stained with Thermo-Pierce GelCode 

Blue stain for total protein according to the manufacturer‘s protocol.

Lane Sample Amount Loaded

M Novex Sharp Protein Standard 10 μL

1 DAS-444Ø6-6 derived 2mEPSPS ~500 ng

2 DAS-444Ø6-6 derived 2mEPSPS ~500 ng

3 P. fluorescens derived 2mEPSPS 520 ng

2mEPSPS

M 1 2 3

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -
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Western blot

The microbe-derived 2mEPSPS and DAS-444Ø6-6 plant tissue extract showed a positive signal 

of the expected size on the western blot using an anti-2mEPSPS polyclonal antibody (Figure 52, 

Panel A). In the 2mEPSPS western blot analysis, the native soybean EPSPS protein (48.3 kDa) 

was also observed in the control Maverick extract as well as in the transgenic event 

DAS-444Ø6-6 extract. This result was expected as the native soybean endogenous EPSPS has 

76% homology to the 2mEPSPS protein and likely cross-reacted with the polyclonal antibody. 

To prove this hypothesis, a monoclonal antibody (lot #: 609.48A-2-4) was used to probe the 

soybean leaf extracts. As a result only the recombinant 2mEPSPS protein was detected in the 

microbe-derived 2mEPSPS preparation and DAS-444Ø6-6 tissue, with no immunoreactive 

proteins observed in the Maverick control extract (Figure 52, Panel B). This result indicated that 

the polyclonal antibody was reacting to the native EPSPS protein at the expected molecular 

weight (48.3 kDa). In addition, in both the polyclonal and monoclonal western blot analyses, no 

alternate size proteins (aggregates or degradation products) were seen in the transgenic samples. 

These results add to the evidence that the protein expressed in soybean is not glycosylated or 

post-translationally modified which would add to the overall protein molecular weight.
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Panel A. Polyclonal antibody western blot Panel B. Monoclonal antibody western blot

Figure 52. Western blot analysis of DAS-444Ø6-6 soybean- and microbe-derived 2mEPSPS protein.

Note: Crude extracts from leaf tissue of event DAS-444Ø6-6 and Maverick were separated by SDS-PAGE along with microbederived 2mEPSPS and bovine serum albumin. The nitrocellulose membranes were then probed with 2mEPSPS specific polyclonal and 

monoclonal antibodies and detected with HRP-labeled antibodies. A chemiluminescent substrate was used to visualize the 

immunoreactive bands.

Panel A. Panel B.

Lane Sample Amount Loaded Lane Sample Amount Loaded

kDa Novex Pre-stained MW Marker kDa Novex Pre-stained MW Marker

1 Microbe-derived 2mEPSPS (TSN033171) ~1 ng 1 Bovine Serum Albumin (BSA) ~1 ng

2 Bovine Serum Albumin (BSA) ~1 ng 2 Microbe-derived 2mEPSPS (TSN033171) ~1 ng

3 DAS-444Ø6-6 Soybean Leaf Extract 10 μL 3 Non-transgenic (Maverick) Leaf Extract 10 μL

4 Non-transgenic (Maverick) Leaf Extract 10 μL 4 DAS-444Ø6-6 Soybean Leaf Extract 10 μL
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Results of detection of glycosylation of 2mEPSPS protein

Detection of carbohydrates, possibly covalently linked to soybean-derived 2mEPSPS proteins, 

was assessed by the GelCode Glycoprotein Staining Kit from Thermo-Pierce. The purified 

soybean-derived 2mEPSPS protein was electrophoresed simultaneously with a set of control and 

reference protein standards. A glycoprotein, horseradish peroxidase, was loaded as a positive 

indicator for glycosylation, and non-glycoproteins; microbe-derived 2mEPSPS, soybean trypsin 

inhibitor, and bovine serum albumin, were employed as negative reference controls. The results 

showed that the soybean- and microbe-derived 2mEPSPS proteins had no detectable covalently 

linked carbohydrates (Figure 53).

Note: The Novex Sharp protein standard does not appear on the 

glycoprotein stained gels as they do not contain glycoproteins.

Figure 53. Glycosylation analysis of DAS-444Ø6-6 soybean- and microbe-derived 

2mEPSPS protein.

Note: The purified DAS-444Ø6-6-derived 2mEPSPS, microbe-derived 2mEPSPS, soybean trypsin 

inhibitor, bovine serum albumin, and horseradish peroxidase were diluted to a similar concentration prior 

to separation by SDS-PAGE. After electrophoresis, the gel was cut in half and one half was stained with 

Thermo Pierce GelCode Blue stain for total protein, and the other half of the gel was stained with a 

Thermo Pierce GelCode Glycoprotein Staining Kit to visualize the glycoproteins.

Lane Sample Amount Loaded

M Novex Sharp Protein Standard 10 μL

1 Horseradish peroxidase (+ control) ~500 ng

2 Soybean trypsin inhibitor (- control) ~500 ng

3 Bovine serum albumin (- control) ~500 ng

4 DAS-444Ø6-6 derived 2mEPSPS ~500 ng

5 P. fluorescens derived 2mEPSPS ~500 ng

P Novex Pre-stained MW Marker 10 μL

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

M 1 2 3 4 5 P M 1 2 3 4 5 P

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

kDa

- 3.5

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

- 3.5

total protein stain glycoprotein stain
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Results of MALDI-TOF MS and MALDI-TOF MS/MS tryptic and chymotryptic peptide mass 

fingerprints of DAS-444Ø6-6-derived 2mEPSPS protein

The 2mEPSPS protein derived from the tissue of the transgenic soybean event DAS-444Ø6-6

was separated by SDS-PAGE (Figure 51). The band corresponding to the size of 2mEPSPS was 

excised and subjected to in-gel digestion by trypsin and chymotrypsin. In addition, the protein 

was subject to in solution digestion with the same enzymes. The resulting peptide mixtures were

analyzed by MALDI-TOF and MALDI-TOF MS/MS to determine the peptide sequences and 

protein identity. The masses of the detected peptides were compared to those deduced based on 

potential trypsin or chymotrypsin cleavage sites in the sequence of the soybean-derived 

2mEPSPS protein. Figure 54 illustrates the theoretical cleavage of the 2mEPSPS protein when 

subjected to endoprotease digestion in silico using Protein Analysis Worksheet (PAWS) 

freeware from Proteometrics LLC.

The theoretical and observed amino acid digest (and molecular weights) of the soybean-derived 

2mEPSPS protein are shown in Figure 54 and Figure 55. The 2mEPSPS protein, once denatured, 

is readily digested by endoproteases and will generate numerous peptide peaks.

In the endoproteinase digest of the transgenic-soybean-derived 2mEPSPS protein, the peptide 

sequence coverage was excellent (86.3%) and 70.0% of the peptide primary sequence was 

confirmed by MS/MS analysis (Figure 55). The detected peptide fragments covered nearly the 

entire protein sequence lacking only six peptide fragments (Figure 55), two near the N-terminus 

(S65 to K70 and A75 to K83), three in the middle of the protein (V249 to Y258, A287 to K296, and A321

to K328), and one near the C-terminus (M405 to R422). The peptide fragments that were not 

detected in this study did not contain sequence motifs that are typically required for 

glycosylation (Asn-Xxx-Ser/Thr, (Hamby and Hirst, 2008)).

This analysis confirmed the soybean-derived protein amino acid sequence matched that of the 

microbe-derived 2mEPSPS protein (Figure 56) at both the N- and C-terminus as well as a major 

portion of the internal sequence. In the MS chromatograms, there were several unidentified 

peptides detected in the enzyme digest preparations (as discussed earlier). Results of these 

analyses indicate that the amino acid sequence of the soybean-derived 2mEPSPS protein was 

equivalent to the P. fluorescens-expressed protein characterized earlier.
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Digestion at K (lysine) and R (arginine)

Digestion at F (phenylalanine), L (leucine), W (tryptophan), and Y (tyrosine)

Figure 54. Theoretical trypsin (top panel) and chymotrypsin (bottom panel) cleavage of the 

2mEPSPS protein.

Note: Alternating blocks of upper (black) and lower (red) case letters within the amino acid 

sequence are used to differentiate the potential peptides after protease digestion. The numbers on 

the left and right sides indicate the amino acid residue numbers.
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Figure 55. Sequence coverage of trypsin and chymotrypsin digests for DAS-444Ø6-6-

derived 2mEPSPS protein by MALDI-TOF MS and MALDI TOF-TOF.

Cys residues were alkylated with iodoacetamide. Overall sequence coverage was 86.3% with 

peptide mass fingerprint data and 70.0% by tandem MS data.

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 A G A E E I V L Q P I K E I S G T V K L P G S K S L S N R I 30 30 30

31 L L L A A L S E G T T V V D N L L N S E D V H Y M L G A L R 60 30 29

61 T L G L S V E A D K A A K R A V V V G C G G K F P V E D A K 90 15 15

91 E E V Q L F L G N A G I A M R S L T A A V T A A G G N A T Y 120 30 30

121 V L D G V P R M R E R P I G D L V V G L K Q L G A D V D C F 150 30 30

151 L G T D C P P V R V N G I G G L P G G K V K L S G S I S S Q 180 30 27

181 Y L S A L L M A A P L A L G D V E I E I I D K L I S I P Y V 210 30 30

211 E M T L R L M E R F G V K A E H S D S W D R F Y I K G G Q K 240 30 30

241 Y K S P K N A Y V E G D A S S A S Y F L A G A A I T G G T V 270 20 8

271 T V E G C G T T S L Q G D V K F A E V L E M M G A K V T W T 300 20 7

301 E T S V T V T G P P R E P F G R K H L K A I D V N M N K M P 330 22 20

331 D V A M T L A V V A L F A D G P T A I R D V A S W R V K E T 360 30 14

361 E R M V A I R T E L T K L G A S V E E G P D Y C I I T P P E 390 30 23

391 K L N V T A I D T Y D D H R M A M A F S L A A C A E V P V T 420 14 2

421 I R D P G C T R K T F P D Y F D V L S T F V K N 444 22 16

383 311

A = Tryptic MS coverage

A = Tryptic MS-MS data = 86.3 %

A = Chymotryptic MS coverage = 70.0 %

A = Chymotryptic MS-MS data

Peptides 

covered by 

MS-MS

MALDI MS-MS sequence coverage

Tot. a.a. covered =

MALDI PMF sequence coverage

Peptides 

covered 

by PMF
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Figure 56. Sequence coverage of trypsin, chymotrypsin, Arg-C, Asp-N, and Glu-C digests 

for P. fluorescens-derived 2mEPSPS protein by MALDI-TOF MS and ISD MS/MS.

Cys residues were alkylated with iodoacetamide. Overall sequence coverage was 98.2% with 

peptide mass fingerprint data and 93.2% by tandem MS data.

Results of MALDI-TOF MS/MS N- and C-terminal sequence analysis of 2mEPSPS

The amino acid residues at the N-and C-termini of the soybean-derived 2mEPSPS protein 

(purified from DAS-444Ø6-6 soybean) were determined and compared with the sequence of the 

previously characterized microbe-derived protein. The soybean-derived 2mEPSPS protein 

sequences were determined by MALDI-TOF MS/MS. The chymotrypsin and trypsin digestions 

were performed on the soybean-derived 2mEPSPS protein followed by mass spectrometry 

analysis and the N-terminus was determined to be identical to the expected sequences, and both 

the soybean- and microbe-derived 2mEPSPS proteins were indistinguishable and unaltered

(Table 35 and Figure 54).

Microbe derived 2m-EPSPS overall peptide mapping

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 A G A E E I V L Q P I K E I S G T V K L P G S K S L S N R I 30 30 30

31 L L L A A L S E G T T V V D N L L N S E D V H Y M L G A L R 60 30 30

61 T L G L S V E A D K A A K R A V V V G C G G K F P V E D A K 90 27 27

91 E E V Q L F L G N A G I A M R S L T A A V T A A G G N A T Y 120 30 30

121 V L D G V P R M R E R P I G D L V V G L K Q L G A D V D C F 150 30 30

151 L G T D C P P V R V N G I G G L P G G K V K L S G S I S S Q 180 28 28

181 Y L S A L L M A A P L A L G D V E I E I I D K L I S I P Y V 210 30 30

211 E M T L R L M E R F G V K A E H S D S W D R F Y I K G G Q K 240 26 26

241 Y K S P K N A Y V E G D A S S A S Y F L A G A A I T G G T V 270 25 6

271 T V E G C G T T S L Q G D V K F A E V L E M M G A K V T W T 300 30 27

301 E T S V T V T G P P R E P F G R K H L K A I D V N M N K M P 330 30 30

331 D V A M T L A V V A L F A D G P T A I R D V A S W R V K E T 360 30 30

361 E R M V A I R T E L T K L G A S V E E G P D Y C I I T P P E 390 30 30

391 K L N V T A I D T Y D D H R M A M A F S L A A C A E V P V T 420 30 30

421 I R D P G C T R K T F P D Y F D V L S T F V K N 444 30 30

436 414

A = PMF MS coverage (MS)

A = ISD Coverage (MS/MS) = 98.2 %

= 93.2 %

Peptides 

covered by 

MS-MS

MALDI MS-MS sequence coverage

Tot. a.a. covered =

MALDI PMF sequence coverage

Peptides 

covered 

by PMF
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Table 35. Summary of N-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived 2mEPSPS.

Source Expected N-terminal Sequencea

P. fluorescens A G A E E I V L Q P I K E I S G T V K L P G S K S L S

Soybean Event A G A E E I V L Q P I K E I S G T V K L P G S K S L S

DAS-444Ø6-6

Source Detected N-terminal Sequenceb

P. fluorescens A G A E E I V L Q P I K E I S G T V K L P G S K S L S

Soybean Event A G A E E I V L Q P I K E I S G T V K L P G S K S L S

DAS-444Ø6-6

Notes:

a

Expected N-terminal sequence of the first 27 amino acid residues of P. fluorescens- and 

soybean-derived 2mEPSPS.

bDetected N-terminal sequences of P. fluorescens- and soybean-derived 2mEPSPS.

Numbers in superscript (Rx) indicate amino acid residue numbers in the sequence.

Amino acid residue abbreviations:

A: alanine E: glutamic acid G: glycine

I: isoleucine K: lysine L: leucine

P: proline Q: glutamine S: serine

T: threonine V: valine
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The C-terminal sequence of the soybean- and microbe-derived 2mEPSPS proteins was

determined essentially as described above and compared with the expected amino acid 

sequences. The results indicated the measured sequences were identical to the expected 

sequences, and both the soybean- and microbe-derived 2mEPSPS proteins were 

indistinguishable and unaltered at the C-terminus (Table 36 and Figure 54).

Table 36. Summary of C-terminal sequence data of DAS-444Ø6-6 soybean- and P. 

fluorescens-derived 2mEPSPS.

Source Expected C-terminal Sequencea

P. fluorescens K T F P D Y F D V L S T F V K N

Soybean Event

DAS-444Ø6-6 K T F P D Y F D V L S T F V K N

Source Detected C-terminal Sequenceb

P. fluorescens K T F P D Y F D V L S T F V K N

Soybean Event

DAS-444Ø6-6 K T F P D Y F D V L S T F V K N

Notes:

a

Expected C-terminal sequence of the last 16 amino acid residues of P. fluorescens- and 

soybean-derived 2mEPSPS.

bDetected C-terminal sequences of P. fluorescens- and soybean-derived 2mEPSPS.

Numbers in superscript (Rx) indicate amino acid residue numbers in the sequence.

Amino acid residue abbreviations:

D: aspartic acid F: phenylalanine K: lysine

L: leucine N: asparagine P: proline

S: serine T: threonine V: valine

Y: tyrosine

Conclusions

The biochemical analyses confirmed that the 2mEPSPS derived from the leaf tissue of soybean 

event DAS-444Ø6-6 was equivalent to 2mEPSPS purified from P. fluorescens. The plant- and 

microbe-derived 2mEPSPS proteins showed the expected molecular weight of ~47 kDa by 

SDS-PAGE and were immunoreactive to 2mEPSPS protein specific antibodies by western blot 

analysis. The amino acid sequence of both proteins was confirmed by enzymatic peptide mass 

fingerprinting by MALDI-TOF MS. The N- and C-terminus of the protein from the 2 different 

sources were shown to be identical via MALDI-TOF MS/MS and ESI-LC/MS. In addition, the 

lack of glycosylation of the plant-derived 2mEPSPS protein provided additional evidence that 
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the 2mEPSPS protein produced by P. fluorescens and DAS-444Ø6-6 soybean are biochemically 

equivalent.
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Appendix 4. Methods and Results for Characterization of PAT Protein

DAS-444Ø6-6 Transgenic Soybean Material

Greenhouse-grown DAS-444Ø6-6 soybean plants (T4) were used as the source of the PAT 

protein. Prior to use, the plant tissue was tested to confirm expression of the PAT protein using a 

commercially available ELISA kit according to the manufacturer‘s instructions (EnviroLogix 

Inc.). Leaves (and some stems) from the PAT expressing plants were harvested, lyophilized, 

ground to a fine powder, and stored frozen until needed.

Control Soybean Material

The control soybean line (Maverick) had a genetic background representative of the 

DAS-444Ø6-6 soybean plants, but did not contain the pat gene. Prior to use, the absence of the 

PAT protein in the control plants was confirmed by immunoassay using a commercially 

available PAT specific ELISA kit. Leaves (and some stems) of control plants were grown, 

harvested, lyophilized, ground, and stored under the same conditions as the DAS-444Ø6-6

soybean.

Reference Material

Recombinant PAT protein was produced in Escherichia coli and purified to homogeneity by 

GeneScript (Piscataway, NJ – Identification number: 55238). The E. coli-derived PAT protein 

preparation was aliquoted and stored at -80oC to maintain activity.

SDS-PAGE and Western Blot Analysis of Crude Extracts

Lyophilized tissue from event DAS-444Ø6-6 and Maverick was mixed with a PBST based 

buffer containing ~2.0% protease inhibitor cocktail (Sigma) and the soluble proteins were

extracted by grinding with ball bearings in a Geno/Grinder (Certiprep, Metuchen, NJ). The 

samples were centrifuged at 4°C for 5 minutes at 20,000×g and the supernatants were mixed 

with Laemmli sample buffer, heated at 100°C for 5 minutes, and briefly centrifuged (20,000×g 

for 2 minutes at 4°C). The positive reference standard (E. coli-derived PAT) and negative 

reference standard (BSA) were also mixed with sample buffer and the resulting supernatants

were loaded directly on to a Bio-Rad Criterion SDS-PAGE gel and electrophoresis was 

conducted with Tris/glycine/SDS buffer. Following separation, the gel was cut in half, with one 

half stained with Thermo Scientific GelCode Blue protein stain and the other gel half was 

electro-blotted onto a nitrocellulose membrane. The nitrocellulose membrane was then cut in 

half with one half probed with a PAT specific polyclonal rabbit antibody and the remaining half 

probed with a PAT specific monoclonal antibody. The antibodies were detected with HRPlabeled secondary antibodies. A chemiluminescent substrate (GE Healthcare) was used to 

visualize the immunoreactive bands.

Results of the SDS-PAGE and Western Blot Analysis

The extracts of lyophilized soybean tissue, microbe-derived PAT protein, and bovine serum 

albumin (BSA) were separated by SDS-PAGE and visualized by Coomassie stain (Figure 57).

The soybean-derived PAT protein was visualized by immunospecific polyclonal and monoclonal 
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antibodies and showed the expected band at approximately 21 kDa (Figure 58). In the PAT 

western blot analysis, no immunoreactive proteins were observed in the control Maverick extract 

and no alternate size proteins (aggregates or degradation products) were seen in the transgenic 

soybean extract. The monoclonal antibody did detect a small amount of a dimer in the microbederived PAT preparation. These results add to the evidence that the PAT protein expressed in 

soybean is not post-translationally modified which would have added to or subtracted from the 

overall protein molecular weight.

Figure 57. SDS-PAGE of DAS-444Ø6-6 and non-transgenic Maverick soybean extracts, 

microbe-derived PAT protein.

Lane Sample Amount Loaded

M Novex Prestained MW Markers 10 μL

1 Non-transgenic soybean Maverick 40 μL

2 DAS-444Ø6-6 soybean 40 μL

3 PAT protein standard (TSN031116-0001) 1.2 μg

4 Bovine serum albumin (BSA) 1.5 μg

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

1 2 3 4

BSA

PAT

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

1 2 3 4

BSA

PAT

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

1 2 3 4

BSA

PAT

M
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Figure 58. Western blot analysis of DAS-444Ø6-6 and Maverick soybean extracts.

Note: Crude extracts from lyophilized leaf tissue of event DAS-444Ø6-6 and Maverick were 

separated by SDS-PAGE along with microbe-derived PAT and bovine serum albumin. Following 

electrophoresis the gel was cut in half, one half was stained with Thermo Scientific GelCode Blue 

stain for total protein (Figure 57) and the remaining half was electro-blotted onto a nitrocellulose 

membrane (Figure 58). The nitrocellulose membrane was then probed with PAT specific polyclonal 

and monoclonal antibodies and detected with HRP-labeled antibodies. A chemiluminescent 

substrate (GE Healthcare) was used to visualize the immunoreactive bands.

Lane Sample Amount Loaded

Probed with Monoclonal Antibody

kDa Novex Pre-stained MW Markers 10 μL

1 Non-transgenic soybean Maverick 40 μL

2 DAS-444Ø6-6 soybean 40 μL

3 PAT protein standard (TSN031116-0001) 12.1 ng

Probed with Polyclonal Antibody

M Novex Pre-stained MW Markers (not labeled) 10 μL

4 Non-transgenic soybean Maverick 40 μL

5 DAS-444Ø6-6 soybean 40 μL

6 PAT protein standard (TSN031116-0001) 12.1 ng

kDa Novex Pre-stained MW Markers 10 μL

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

kDa

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

1 2 3 M 4 5 6

Monoclonal Ab Polyclonal Ab

10 -

3.5 -

- 1 0

- 3.5

*Arrow denotes a PAT protein dimer

*

PAT
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Conclusions

The PAT protein produced in DAS-444Ø6-6 soybean was shown to be equivalent to that 

produced in E. coli and the characterization results are consistent with the protein expressed in 

other transgenic crops (USDA, 1996, 2001, 2004, 2005).
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Appendix 5. Methods for AAD-12, 2mEPSPS and PAT Protein Expression Analysis

Experimental Design

Samples were collected from a field study conducted in the US in 2010 that included ten (10) 

field sites; Georgia, Iowa (2 sites), Illinois (2 sites), Indiana, Michigan, Missouri, and Nebraska 

(2 sites) (referred to as GA, IA1, IA2, IL1, IL2, IN, MI, MO, NE1, and NE2). Each site consisted 

of one plot of each treatment per block, with 4 blocks per location. Plot size was 4 rows by 25 

feet. Plots were arranged in a randomized complete block (RCB) design, with a unique 

randomization at each site. Each soybean plot was bordered by 2 rows of a non-transgenic 

soybean of similar maturity. The entire trial site was surrounded by a minimum of 10 feet of a 

non-regulated soybean of similar relative maturity. At each location, all blocks were used for 

collection of samples for protein expression, agronomic properties, and nutrient composition 

analysis.

Herbicide treatments were designed to replicate maximum label rates:

2,4-D only Treatment: 2,4-D (Weedar 64) was applied as three broadcast applications to 

DAS-444Ø6-6. Application timing was at planting / pre-emergence, and approximately V3 and 

R2 stages. Individual target application rates were 1.0 lb ae (acid equivalent)/A for Weedar 64, 

or 1120 g ae/ha.

Glufosinate only Treatment: Glufosinate (Liberty) was applied as two broadcast applications to 

DAS-444Ø6-6. Application timing was at approximately V5 and R1 stages. The target 

application rate at V5 was 0.33 lb ai/A for Liberty, or 374 g ai/ha. The target application rate at 

R1 was 0.41 lb ai/A for Liberty, or 454 g ai/ha.

Glyphosate only Treatment: Glyphosate (Durango DMA) was applied as three broadcast 

applications to DAS-444Ø6-6. Individual applications were at planting / pre-emergence, and 

approximately V3 and R2 stages. Individual target application rates were 1.1 lb ae/A for

Durango DMA, or 1260 g ae/ha.

2,4-D + Glufosinate + Glyphosate Treatment: 2,4-D (Weedar 64) + Glyphosate (Durango 

DMA) as a tank mixture was applied as three broadcast applications to DAS-444Ø6-6. 

Individual applications were at planting / pre-emergence, and approximately V3 and R2 stages. 

Individual target application rates were 1.0 lb ae/A for Weedar 64, or 1120 g ae/ha. Individual 

target application rates were 1.1 lb ae/A for Durango DMA, or 1260 g ae/ha. Glufosinate 

(Liberty) was also applied as two broadcast applications to DAS-444Ø6-6. Application timing 

was at approximately V5 and R1 stages. The target application rate was 0.33 lb ai/A for Liberty, 

or 374 g ai/ha.

Sample Collection

Samples were shipped to Dow AgroSciences Regulatory Sciences and Government Affairs 

laboratories and maintained frozen until use. Samples of soybean tissues were prepared for 

expression analysis by coarse grinding, lyophilizing and/or fine-grinding with a Geno/Grinder 

(Certiprep, Metuchen, NJ).
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a. Leaf (V5 and V10-12)

One leaf sample per plot, each sample containing 8 trifoliate set of leaves collected from separate 

plants, was collected for each test and control entry. Each leaf sample was the youngest set of 

fully expanded trifoliate leaves.

b. Root (R3)

One root sample (representing 3 plants) per plot was collected for each test and control entry at 

the R3 stage by cutting a circle around the base of the plant. The root ball was removed and 

cleaned.

c. Forage (R3)

One forage sample (representing 3 plants) per plot, each consisting of the aerial portion (no 

roots) of 3 whole plants was collected from each test and control entry.

d. Grain (R8 – Maturity)

One individual sample was collected from each plot of each test and control entry. Each sample 

contained approximately 500 grams of grain.

Determination of AAD-12 Protein in Soybean Tissue Samples

ELISA method GRM08.04 was used to determine AAD-12 protein concentration in soybean 

tissue samples. The AAD-12 protein was extracted from soybean tissues except grain with a 

phosphate buffered saline solution with 0.05% Tween-20 (PBST) and 0.75% ovalbumin (OVA).

For grain, the protein was extracted with a PBST buffer containing 0.1% (v/v) Triton-100. The 

plant tissue and grain extracts were centrifuged; the aqueous supernatant was collected, diluted 

with appropriate buffer if necessary, and analyzed using an AAD-12 ELISA kit. Briefly, an 

aliquot of the diluted sample and a horseradish peroxidase (HRP)/anti-AAD-12 monoclonal 

antibody conjugate were incubated in the wells of a microtiter plate coated with an immobilized 

anti-AAD-12 polyclonal antibody. These antibodies bind with AAD-12 protein in the wells and 

form a "sandwich" with AAD-12 protein bound between soluble and the immobilized antibodies. 

The unbound samples and excess conjugate were then removed from the plate by washing with 

PBST. Subsequent addition of an enzyme substrate generated a colored product. The reaction 

was stopped by adding a dilute acid solution. Since the AAD-12 was bound in the antibody 

sandwich, the level of color development, determined by measuring the absorbance of the 

solution, was related to the concentration of AAD-12 in the sample (i.e., lower protein 

concentrations result in lower color development). The absorbance at 450 nm nm with a 

background subtraction at 650 or 620 nm was measured using a Molecular Devices Spectra Max 

M2 plate reader or a Grifols Triturus Automated Immunoassay Analyzer. A calibration curve 

was generated and the AAD-12 concentration in unknown samples was calculated from the 

polynomial regression equation using Soft-MAX ProTM or Triturus Version 4.01B software 

which was compatible with the plate reader. Samples were analyzed in duplicate wells with the 

average concentration of the duplicate wells being reported.

Determination of 2mEPSPS Protein in Soybean Tissue Samples

ELISA method 101768 was used to determine 2mEPSPS protein concentration in soybean tissue 

samples. The 2mEPSPS protein was extracted from soybean tissues with a phosphate buffered 
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saline solution with 0.05% Tween-20 (PBST) and 2X Casein (PBST/Casein). The plant tissue 

extracts were centrifuged; the aqueous supernatant was collected, diluted with appropriate buffer 

if necessary, and analyzed using a 2mEPSPS ELISA kit. Briefly, an aliquot of the diluted sample 

is incubated in the wells of a microtiter plate coated with an immobilized anti-2mEPSPS

polyclonal antibody. After a washing step, an enzyme-conjugated monoclonal antibody specific 

to the 2mEPSPS protein is added to the microtiter plate. These antibodies bind with 2mEPSPS

protein in the wells and form a "sandwich" with 2mEPSPS protein bound between soluble and 

the immobilized antibodies. At the end of an incubation period, the unbound reagents were 

removed from the plate by washing with PBST. Subsequent addition of an enzyme substrate 

generated a colored product. The reaction was stopped by adding a dilute acid solution. Since 

the 2mEPSPS was bound in the antibody sandwich, the level of color development, determined 

by measuring the absorbance of the solution, was related to the concentration of 2mEPSPS in the 

sample (i.e., lower protein concentrations result in lower color development). The absorbance at 

450 nm nm with a background subtraction at 650 or 620 nm was measured using a Molecular 

Devices Spectra Max M2 plate reader or a Grifols Triturus Automated Immunoassay Analyzer. 

A calibration curve was generated and the 2mEPSPS concentration in unknown samples was 

calculated from the polynomial regression equation using Soft-MAX ProTM or Triturus Version 

4.01B software which was compatible with the plate reader. Samples were analyzed in duplicate 

wells with the average concentration of the duplicate wells being reported.

Determination of PAT Protein in Soybean Tissue Samples

ELISA method GRM08.05 was used to determine PAT protein concentration in soybean tissue 

samples. The PAT protein was extracted from soybean tissues with a phosphate buffered saline 

solution with 0.05% Tween-20 (PBST) and 1% polyvinylpyrrolidone (PVP). The extract was 

centrifuged; the aqueous supernatant was collected, diluted with PBST/1% PVP, and analyzed 

using a PAT ELISA kit. Briefly, an aliquot of the diluted sample was incubated with enzymeconjugated anti-PAT monoclonal antibody and anti-PAT polyclonal antibodies coated in the 

wells of a 96-well plate in a sandwich ELISA format. At the end of the incubation period, the 

unbound reagents were removed from the plate by washing. Subsequent addition of an enzyme 

substrate generated a colored product. The reaction was stopped by adding a dilute acid solution. 

Since the PAT was bound in the antibody sandwich, the level of color development, determined 

by measuring the absorbance of the solution, was related to the concentration of PAT in the 

sample (i.e., lower residue concentrations result in lower color development). The absorbance at 

450 nm with a background subtraction at 650 or 620 nm was measured using a Molecular 

Devices Spectra Max M2 plate reader or a Grifols Triturus Automated Immunoassay Analyzer.

A calibration curve was generated and the PAT concentration in unknown samples was 

calculated from the polynomial regression equation using Soft-MAX ProTM or Triturus Version 

4.01B software which was compatible with the plate reader. Samples were analyzed in duplicate 

wells with the average concentration of the duplicate wells being reported.
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Appendix 6. USDA Notifications for DAS-444Ø6-6 Soybean

USDA 

Notification 

Number

Notification 

Authorization 

Date

Notification 

Expiration 

Date

State(s) Total 

Number of 

Trials 

Planted

Status of 

Trial1

11-095-105n 4/29/2011 4/29/2012 MS 2 Pending

11-087-114n 4/20/2011 4/20/2012 AL, AR, GA, 

IL, IN, MD, NE 34 Pending

11-067-105n 3/30/2011 3/30/2012

AR, CA, IA, IN, 

IL, LA, MN,

MO, MS, OH, 

WI

28 Pending

10-243-104n 9/30/2010 9/30/2011 PR 9 Pending

10-085-103n 4/19/2010 4/19/2011 GA, IA, IN,IL, 

MI, MO, NE 18 Submitted

10-083-105n 4/22/2010 4/22/2011 IA, IN, MO,MS 8 Submitted

10-077-107n 4/14/2010 4/14/2011

GA, IA, IN,IL, 

MD, MO,NE, 

OH, PR

27 Submitted

09-259-108n 10/5/2009 10/5/2010 PR 3 Submitted

09-068-103n 4/1/2009 4/1/2010 IN, PR 4 Submitted

08-254-109n 9/30/2008 9/30/2009 PR 1 Submitted

1

Pending reports as of June 21, 2011 to be submitted within six months of the notification 

expiration date.
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Appendix 7. Literature Ranges for Compositional Analysis

Table 37. Literature ranges reported for soybean seed: proximates, fiber, and minerals.

Analyte Units Min Max Min Max Min Max Min Max Min Max

Protein % Dry weight 3 2 48.4 3 2 43.6 33.19 45.48 32.54 48.4 Harrigan et al. 2007 Hartwig and Kilen 1991

Total Fat % Dry weight 8.104 24.7 15.5 24.7 8.104 23.562 14.10 23.67 Padgette et al. 1996 Berman et al. 2010

Ash % Dry weight 3.885 6.994 4.5 6.4 3.885 6.994 4.29 6.44 Padgette et al. 1996 Harrigan et al. 2007

Moisture % Fresh weight 4.7 34.4 NR NR 4.7 34.4 4.71 14.30 Harrigan et al. 2007 Taylor et al. 1999

Carbohydrates % Dry weight 29.3 50.2 31.7 31.8 29.6 50.2 29.3 44.35 Padgette et al. 1996 Harrigan et al. 2007

Acid Detergent Fiber (ADF) % Dry weight 7.81 26.26 9 11.1 7.81 18.61 9.22 26.26 Lundry et al. 2008 Lundry et al. 2008

Neutral Detergent Fiber (NDF) % Dry weight 8.53 23.90 1 0 14.9 8.53 21.25 10.79 23.90 Lundry et al. 2008 Lundry et al. 2008

Total Dietary Fiber % Dry weight NR NR NR NR NR NR NR NR NR NR

Calcium mg/100g Dry weight 116.55 510 NR NR 116.55 307.1 258 510 Iskander 1987 Bilyeu et al. 2008

Copper mg/100g Dry weight 0.632 1.092 NR NR NR NR 0.632 1.092 Bilyeu et al. 2008 Bilyeu et al. 2008

Iron mg/100g Dry weight 3.734 10.954 NR NR 5.536 10.954 3.734 6.624 Bilyeu et al. 2008 Bilyeu et al. 2008

Magnesium mg/100g Dry weight 219.40 312.84 NR NR 219.40 312.84 261 280 Iskander 1987 Bilyeu et al. 2008

Manganese mg/100g Dry weight 2.52 3.876 NR NR NR NR 2.52 3.876 Iskander 1987 Bilyeu et al. 2008

Phosphorus mg/100g Dry weight 506.74 935.24 NR NR 506.74 935.24 770 790 Bilyeu et al. 2008 Bilyeu et al. 2008

Potassium mg/100g Dry weight 1868.01 2510 NR NR 1868.01 2316.14 1910 2510 Iskander 1987 Bilyeu et al. 2008

Selenium ppb Dry weight NR NR NR NR NR NR NR NR NR NR

Sodium mg/100g Dry weight 4.05 3 0 NR NR NR NR 4.05 3 0 Iskander 1987 Bilyeu et al. 2008

Zinc mg/100g Dry weight 4.98 7.578 NR NR NR NR 4.98 7.578 Iskander 1987 Bilyeu et al. 2008

NR = Not Reported.

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Table 38. Literature ranges reported for soybean seed: amino acids.

Analyte Units Min Max Min Max Min Max Min Max Min Max

Alanine % Dry weight 1.43 2.10 NR NR 1.51 2.10 1.43 1.93 Berman et al. 2009 Berman et al. 2009

Arginine % Dry weight 2.15 3.46 2.45 3.1 2.29 3.4 2.15 3.46 Berman et al. 2009 Padgette et al. 1996

Aspartic Acid % Dry weight 3.81 6.04 NR NR 3.81 5.12 3.90 6.04 Harrigan et al. 2007 Berman et al. 2010

Cystine % Dry weight 0.37 0.81 0.45 0.67 0.37 0.81 0.41 0.71 Berman et al. 2009 Berman et al. 2009

Glutamic Acid % Dry weight 5.84 9.15 NR NR 5.84 8.2 5.97 9.15 Harrigan et al. 2007 Berman et al. 2010

Glycine % Dry weight 1.41 2.00 NR NR 1.46 2.00 1.41 1.99 Berman et al. 2009 Berman et al. 2009

Histidine % Dry weight 0.86 1.24 1 1.22 0.88 1.18 0.86 1.24 Berman et al. 2009 Berman et al. 2009

Isoleucine % Dry weight 1.49 2.08 1.76 1.98 1.54 2.08 1.49 2.02 Berman et al. 2009 Berman et al. 2009

Leucine % Dry weight 2.2 4.0 2.2 4.0 2.59 3.62 2.39 3.42 Berman et al. 2009 Lundry et al. 2008

Lysine % Dry weight 2.19 3.32 2.5 2.66 2.29 2.84 2.19 3.32 Berman et al. 2009 Berman et al. 2010

Methionine % Dry weight 0.39 0.68 0.5 0.67 0.43 0.68 0.39 0.65 Berman et al. 2009 Berman et al. 2009

Phenylalanine % Dry weight 1.6 2.44 1.6 2.08 1.63 2.35 1.62 2.44 Berman et al. 2009 Berman et al. 2009

Proline % Dry weight 1.63 2.28 NR NR 1.69 2.28 1.63 2.25 Berman et al. 2009 Berman et al. 2009

Serine % Dry weight 1.11 2.48 NR NR 1.11 2.48 1.63 2.42 Berman et al. 2009 Lundry et al. 2008

Threonine % Dry weight 1.14 1.89 1.4 1.89 1.14 1.86 1.28 1.74 Berman et al. 2009 Berman et al. 2009

Tryptophan % Dry weight 0.30 0.67 0.51 0.67 0.356 0.502 0.30 0.63 Lundry et al. 2008 Padgette et al. 1996

Tyrosine % Dry weight 0.79 1.61 NR NR 1.02 1.61 0.79 1.59 Berman et al. 2009 Padgette et al. 1996

Valine % Dry weight 1.5 2.44 1.5 2.44 1.6 2.2 1.57 2.13 Berman et al. 2009 Berman et al. 2009

NR = Not Reported.

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Table 39. Literature ranges for soybean seed: fatty acids.

Analyte Units Min Max Min Max Min Max Min Max Min Max

8:0 Caprylic % of total fatty acid <LOQ 0.148 NR NR <LOQ 0.148 ND ND Harrigan et al. 2007 Harrigan et al. 2007

10:0 Capric % of total fatty acid ND 0.27 NR NR ND ND ND 0.27 Harrigan et al. 2007 Berman et al. 2009

12:0 Lauric % of total fatty acid <LOQ 0.132 NR NR <LOQ 0.132 ND ND Harrigan et al. 2007 Harrigan et al. 2007

14:0 Myristic % of total fatty acid <LOQ 0.238 NR NR <LOQ 0.238 ND 0.097 Harrigan et al. 2007 Berman et al. 2009

14:1 Myristoleic % of total fatty acid <LOQ 0.125 NR NR <LOQ 0.125 ND ND Harrigan et al. 2007 Harrigan et al. 2007

15:0 Pentadecanoic % of total fatty acid ND ND NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

15:1 Pentadecenoic % of total fatty acid ND ND NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

16:0 Palmitic % of total fatty acid 9.55 15.77 NR NR 9.55 15.77 9.80 12.63 Berman et al. 2009 Berman et al. 2009

16:1 Palmitoleic % of total fatty acid <LOQ 0.194 NR NR <LOQ 0.194 ND 0.14 Harrigan et al. 2007 Berman et al. 2009

17:0 Heptadecanoic % of total fatty acid <LOQ 0.146 NR NR <LOQ 0.146 ND 0.13 Harrigan et al. 2007 Berman et al. 2009

17:1 Heptadecenoic % of total fatty acid <LOQ 0.087 NR NR <LOQ 0.087 ND 0.064 Harrigan et al. 2007 Berman et al. 2009

18:0 Stearic % of total fatty acid 2.59 5.88 NR NR 2.70 5.88 2.59 5.50 Berman et al. 2010 Berman et al. 2009

18:1 Oleic % of total fatty acid 14.3 45.68 NR NR 14.3 32.2 15.80 45.68 Harrigan et al. 2007 Berman et al. 2010

18:2 Linoleic % of total fatty acid 35.36 58.8 NR NR 42.3 58.8 35.36 57.72 Berman et al. 2010 Berman et al. 2009

18:3 Linolenic % of total fatty acid 3 12.52 NR NR 3 12.52 4.27 9.60 Berman et al. 2009 Berman et al. 2009

18:3 γ-Linolenic % of total fatty acid ND ND NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

20:0 Arachidic % of total fatty acid 0.163 0.57 NR NR 0.163 0.482 0.25 0.57 Harrigan et al. 2007 Berman et al. 2009

20:1 Eicosenoic % of total fatty acid <LOQ 0.350 NR NR <LOQ 0.350 0.13 0.35 Berman et al. 2009 Berman et al. 2010

20:2 Eicosadienoic % of total fatty acid <LOQ 0.245 NR NR <LOQ 0.245 ND 0.065 Harrigan et al. 2007 Berman et al. 2010

20:3 Eicosatrienoic % of total fatty acid ND ND NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

20:4 Arachidonic % of total fatty acid ND ND NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

22:0 Behenic % of total fatty acid 0.277 0.595 NR NR 0.277 0.595 0.28 0.59 Harrigan et al. 2007 Berman et al. 2009

 <LOQ = Less than Limit of Quantitation; ND = Not Detected; NR = Not Reported.

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Table 40. Literature ranges reported for soybean seed: vitamins.

Analyte Units Min Max Min Max Min Max Min Max Min Max

Vitamin A (β-Carotene) mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

Vitamin B1 (Thiamine HCl) mg/kg Dry weight 1.01 2.54 NR NR 1.01 2.54 NR NR NR NR

Vitamin B2 (Riboflavin) mg/kg Dry weight 1.90 3.21 NR NR 1.90 3.21 NR NR NR NR

 Vitamin B3 (Niacin) mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

Vitamin B5 (Pantothenic Acid) mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

Vitamin B6 (Pyridoxine HCl) mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

Vitamin B9 (Folic Acid) mg/kg Dry weight 2.386 4.709 NR NR 2.386 4.709 NR NR NR NR

Vitamin C (Ascorbic Acid) mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

Vitamin E (α-Tocopherol) mg/kg Dry weight 0.108 61.693 NR NR 1.934 61.693 0.108 48.0 Berman et al. 2010 Lundry et al. 2008

β-Tocopherol mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

γ-Tocopherol mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

δ-Tocopherol mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

Total Tocopherol mg/kg Dry weight NR NR NR NR NR NR NR NR NR NR

NR = Not Reported.

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Table 41. Literature ranges for soybean seed: bioactives.

Analyte Units Min Max Min Max Min Max Min Max Min Max

Lectin HU/mg Protein 

Dry weight

3 7 323 3 7 323 NR NR NR NR NR NR

Phytic Acid % Dry weight 0.41 2.74 1 2.74 0.634 1.96 0.41 2.68 Lundry et al. 2008 Berman et al. 2010

Raffinose % Dry weight 0.212 1.62 NR NR 0.212 0.661 0.22 1.62 Harrigan et al. 2007 Berman et al. 2009

Stachyose % Dry weight 1.21 6.1b NR NR 1.21 3.5 1.52 6.1b Harrigan et al. 2007 Harrigan et al. 2010

Trypsin Inhibitor TIU/mg Dry weight 18.14 118.68 NR NR 19.59 118.68 18.14 75.5 Berman et al. 2009 McCann et al. 2005

Total Daidzein Equivalent mcg/g Dry weight 2 5 2453.5 NR NR 6 0 2453.5 2 5 2099.75 McCann et al. 2005 Berman et al. 2010

Total Genistein Equivalent mcg/g Dry weight 2 8 2837.2 NR NR 144.3 2837.2 2 8 2600.70 McCann et al. 2005 Harrigan et al. 2007

Total Glycitein Equivalent mcg/g Dry weight 15.3 349.19 NR NR 15.3 310.0 4 5 349.19 McCann et al. 2005 Harrigan et al. 2007

NR = Not Reported.

* Maximum value for stachyose is a mean value reported from the literaure, all other records are individual values.

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Appendix 8. Glyphosate, 2,4-D, Glufosinate and Herbicide Resistant Weeds

Herbicide Tolerant Crops

Soon after the first weeds evolved resistance to herbicides, scientists began to consider altering 

crops to make them resistant to herbicides (Duke, 2005). Initially, non-transgenic methods were 

used until the early 1980s when the tools for producing transgenic crops were becoming 

available. The first transgenic herbicide-tolerant crops included bromoxynil resistant cotton and 

canola. However, transgenic crops with resistance to broad-spectrum, non-selective herbicides 

were perceived as a better approach for weed management and for capturing market share. This 

was soon realized with the development of glyphosate- and glufosinate-tolerant crops. Since the

transgenic crops would tolerate the application of those broad spectrum herbicides, they could 

survive and prosper while reducing the amount and number of applications of herbicides by the 

growers.

Herbicide-tolerance (often called resistance) in plants employs one of two strategies (or a 

combination) to make the plant tolerant to the applied herbicide:

 the plant produces a new protein which detoxifies the herbicide, or

 the protein in the plant which is normally the target of the herbicide‘s action is replaced 

by a new protein which is unaffected by the herbicide.

Herbicide-tolerant crops which were made available to farmers up to 2005 are listed in Table 42

(Duke, 2005). Transgenes were only used to confer tolerance to bromoxynil, glufosinate, and 

glyphosate. The bromoxynil-tolerant crops are no longer sold. This leaves only glyphosate- and 

glufosinate-tolerant transgenic crops, and of those, glyphosate has had the strongest impact on 

weed management (Duke, 2005).

Table 42. Herbicide-tolerant crops available to farmers in North America in 2005.

Herbicide Crop Year Available

Bromoxynil Cottonb

1995

Canolab2000

Cyclohexanediones (sethoxydim)a,b Corn 1996

Glufosinate

Canola 1995

Corn 1997

Cotton 2004

Glyphosate

Soybean 1996

Canola 1996

Cotton 1997

Corn 1998

Imidazolinonesa

Corn 1993

Canola 1997

Wheat 2002

Rice 2002

SulfonylureasaSoybean 1994

Triazinesa Canola 1984

aNot transgenic, bNo longer available by 2005
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Growers choose glyphosate-tolerant crops because they make weed control easier and more 

effective, increase profit, require less tillage, and do not restrict crop rotations (Green, 2009). 

Thus, glyphosate-tolerant corn and soybeans have experienced an unprecedented rapid adoption 

rate by U.S. farmers (Figure 59). The planting of glyphosate-tolerant crops has increased steadily 

since their introduction in 1996 (glyphosate-tolerant soybean plantings are currently >90% of all 

soybean planting in the U.S.).

Figure 59. U.S. adoption rates of glyphosate-tolerant soybean and corn.

Sources: (Duke and Powles, 2008b; USDA NASS, 2010; USDA ERS, 2011a)

Many growers now rely only on glyphosate for their weed control in these crops (Foresman and 

Glasgow, 2008; Gustafson, 2008). This has significantly increased selection pressure for 

glyphosate-resistant weeds. Thus, introduction of products which are tolerant to glyphosate plus 

at least one other class of herbicides is needed as a tool to delay selection for glyphosate-resistant 

weed species.

Characteristics of Glyphosate, 2,4-D, and Glufosinate Herbicides

Glyphosate

Glyphosate is a nonselective, foliar applied herbicide which is registered for use on over one 

hundred food and feed crops, several non-food field crops (fallow, fencerows, woody 

ornamentals, golf courses, etc.), forestry uses (conifer release and reforestation), and non-crop 

areas where total vegetation control is desired (aquatics, rights of way, industrial sites, etc.) (US 

EPA, 1993). When applied at lower rates, glyphosate also is a plant growth regulator. Pre-plant 

or pre-emergence uses of glyphosate in food and feed crops include most of the major 

agricultural crops around the globe, including alfalfa, barley, buckwheat, corn, dry beans, grass 

0%

10%

20%

30%

40%

50%

60%

70%

80%

90%

100%

19961997199819992000200120022003200420052006200720082009

Percent of Planted Acres

Soybeans Corn
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forage/fodder/hay, lentils, millet, oats, pastures, rye, sorghum, soybeans, and wheat. In addition, 

it can be foliar applied over-the-top of recent glyphosate-tolerant transgenic crops, including 

corn, soybeans, cotton, canola, sugarbeets and alfalfa.

Glyphosate is absorbed relatively rapidly through plant surfaces (Duke and Powles, 2008a). Leaf 

uptake rates vary considerably between species, accounting for at least some of the difference in 

glyphosate susceptibility between species. Once in the plant, glyphosate moves in the phloem 

with sugar to the growing points. Foliar post-emergence applications of glyphosate at 560 to

1120 g ae/ha control a broad spectrum of grass, broadleaf, and sedge weeds in agronomic crops.

The mode of action for glyphosate is unique in that it is the only herbicide that is highly effective 

at inhibiting an essential plant enzyme called EPSPS (5-enolpyruvylshikimate-3-phosphate 

synthase), which produces EPSP from shikimate-3-phosphate and phosphoenolpyruvate in the 

shikimic acid pathway (Vencill, 2002). Many assume that this EPSPS inhibition leads to 

depletion of the aromatic amino acids tryptophan, tyrosine, and phenylalanine (Duke and 

Powles, 2008a). However, others believe that the deregulation of the pathway through EPSPS 

inhibition increases carbon flow to the shikimate pathway, which results in shortages of carbon 

for other essential pathways (Siehl, 1997). The EPSPS of all higher plants appears to be inhibited 

by glyphosate (Duke and Powles, 2008a), which makes glyphosate a nonselective herbicide with 

activity on a wide range of plant species.

In general, glyphosate is an environmentally benign molecule (Franz et al., 1997). It is degraded 

microbially in soil and water. Glyphosate binds tightly to most types of soil, so it is not available

for uptake by roots of nearby plants (Monsanto, 2005). Therefore, even though glyphosate has a 

typical field half life of 47 days, crops can be planted immediately after application due to its 

strong adsorption to soil. Glyphosate is not appreciably metabolized in plants when applied at 

normal use rates for weed control. It is slowly metabolized to amino methylphosphonic acid 

(AMPA) (FAO, 1997; Vencill, 2002). Glyphosate is also one of the least toxic pesticides to 

animals (Duke and Powles, 2008a). The enzyme, EPSP synthase, is not present in humans or 

animals, which contributes to the low risk to human health when glyphosate is used according to 

label directions (Monsanto, 2005).

2,4-Dichlorophenoxyacetic Acid (2,4-D)

2,4-Dichlorophenoxyacetic acid (2,4-D) was introduced in 1946 as the first selective herbicide,

and rapidly became the most widely used herbicide in the world (Industry Task Force II, 2005). 

Today, it is still the third most widely used herbicide in the U.S. and Canada, as well as the most 

widely used herbicide globally. Approximately 46 million pounds of 2,4-D is used in the U.S. 

annually, with 30 million pounds (66%) used by agriculture and 16 million pounds (34%) used in 

non-agriculture settings such as pasture/rangeland and lawn/garden (US EPA, 2006). 2,4-D is an 

ingredient in approximately 660 agricultural and home use products as a sole active ingredient 

and in conjunction with other active ingredients. Agriculturally, it is used on a variety of crops 

including corn, rice, sorghum, sugar cane, wheat, rangeland and pasture as well as being used on 

rights-of-way, roadsides, non-crop areas, forestry, lawn and turf care and on aquatic weeds

(Industry Task Force II, 2005). A major use of 2,4-D is in combination with other herbicides 

because it economically enhances the weed control spectrum of many other herbicides such as 

glyphosate, dicamba, mecoprop, ALS herbicides, etc (US EPA, 2006). 2,4-D controls many 

broadleaf weeds including carpetweed, dandelion, cocklebur, horseweed, morning glory, 
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pigweed sp., lambsquarters, ragweed spp., shepherd‘s-purse and velvetleaf. It has little to no 

activity on grasses (Industry Task Force II, 2005).

In the over 60 years since its discovery, few other compounds have been as thoroughly and 

extensively evaluated for health and safety as 2,4-D. There have been more than 40,000 research 

studies conducted and more than 140 peer-reviewed published epidemiologic studies specific to 

2,4-D. In August, 2005, the U.S. Environmental Protection Agency (EPA) completed its reregistration assessment of 2,4-D. The EPA concluded that 2,4-D does not present risks of 

concern to human health when users follow its product instructions (US EPA, 2006). 2,4-D data 

has been reviewed by more than a dozen government and expert panels since 1986 and not one 

regulatory agency has ever identified 2,4-D as a human carcinogen.

The mode of action of 2,4-D is described as an ―auxin mimic‖, meaning that it kills the target 

weed by mimicking auxin plant growth hormones like indole acetic acid (IAA). Auxins and 

synthetic auxinic herbicides all regulate virtually every aspect of plant growth and development

(Mockaitis and Estelle, 2008). At low doses, auxinic herbicides possess similar hormonal 

properties to natural auxin (Kelley and Riechers, 2007). However, as rates increase, they can 

cause various growth abnormalities in sensitive dicots. Observable plant responses to 2,4-D can 

include epinasty, root growth inhibition, meristematic proliferation/callusing, leaf 

cupping/narrowing, stem cracking, adventitious root formation, senescence, and chlorosis. This 

uncontrolled and disorganized plant growth eventually leads to plant death when applied at 

effective doses (Tu et al., 2001).

IAA and auxin herbicides work through stimulation of the ubiquination and degradation of the 

Aux/IAA family of transcriptional regulators. Degradation of these Aux/IAA proteins results in 

derepression of auxin-regulated genes that in turn leads to the physiological and morphological 

events associated with auxin action (Walsh et al., 2006; Kelley and Riechers, 2007; Mockaitis 

and Estelle, 2008). Stated another way, high concentrations of IAA or auxin herbicides (like 

2,4-D) promote ubiquitin mediated degradation of Aux/IAA protein repressors, which permits 

auxin response factor (ARF) dependent transcription of auxin-regulated genes. This results in 

―uncontrolled‖ growth which leads to plant death in susceptible species.

In the environment, 2,4-D is mainly degraded by soil microorganisms. Once it contacts soils, all 

2,4-D forms are rapidly converted to the acid form and thus, the rate of soil dissipation is often 

the same as for the acid (Tu et al., 2001). 2,4-D has a relatively short soil half-life and no 

significant carryover effects to subsequent crops are encountered, adding to 2,4-D‘s herbicidal 

utility. 2,4-D has different levels of selectivity on certain plants, i.e., dicots are more sensitive 

than monocots. Differential metabolism of 2,4-D by different plants is one explanation for

varying levels of selectivity. In general, plants metabolize 2,4-D slowly, so varying plant 

response to 2,4-D may be more likely explained by different activity at the target site(s). Plant 

metabolism of 2,4-D typically occurs via a two-phase mechanism of hydroxylation followed by 

conjugation with amino acids or glucose (Vencill, 2002).

Glufosinate

Glufosinate was first reported as a herbicide in 1981(Vencill, 2002). Glufosinate 

(phosphinothricin; DL-homoalanin-4-yl-(methyl)-phosphinic acid) is a racemic phosphinic 

amino acid (Vencill, 2002). Its ammonium salt (glufosinate-ammonium) is widely used as a non-
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selective herbicide and is the active ingredient of the commercial herbicide formulations, Liberty

and Ignite. The L-isomer of glufosinate is a structural analogue of glutamate and, therefore, is a 

competitive inhibitor of the enzyme glutamine synthetase (GS) of bacteria and plants. The 

D-isomer is not a GS inhibitor and is not herbicidally active.

Due to the inhibition of GS, non-tolerant plant cells accumulate large amounts of toxic ammonia 

produced by nitrate assimilation and photorespiration and the level of available glutamine drops 

(OECD, 2002). Damage to cell membranes and inhibition of photosynthesis are followed by 

plant cell death.

In genetically modified glufosinate-tolerant plants (OECD, 2002), the L-isomer of glufosinate is 

rapidly metabolized by the action of the enzyme phosphinothricin acetyltransferase (PAT) into 

the non-phytotoxic stable metabolite N-acetyl-L-glufosinate (2-acetamido-4-methylphosphinicobutanoic acid). N-acetyl-L-glufosinate does not inhibit glutamine synthetase. Therefore, no 

phytotoxic physiological effects are observed in genetically modified glufosinate-tolerant plants.

Glufosinate is a contact herbicide which is taken up by the plant primarily through the leaves. 

There is no uptake from the soil through the roots, presumably because of the rapid degradation 

of glufosinate by soil microorganisms. There is limited translocation of glufosinate within the 

plant.

Glufosinate is a nonselective herbicide (Vencill, 2002). It controls a broad spectrum of annual 

and perennial grasses and broadleaf weeds. Due to its limited systemic action, there is no 

enduring effect on perennial weeds. Weeds which emerge after herbicide application are not 

affected.

Glufosinate is rapidly broken down in soil due to microbial degradation (Vencill, 2002). At 

20°C, the soil half life is less than 7 days. The end products of microbial degradation are CO2

and natural phosphorus compounds.

Evolution of Resistance to Glyphosate, 2,4-D, and Glufosinate

Glyphosate Resistance and Weed Shifts

Glyphosate Resistance

It was initially thought that evolution of glyphosate-resistant weeds would be very slow, and the 

levels of resistance would be very low (Bradshaw et al., 1997). This was based on the amount of 

glyphosate applied over many years, the repeated applications made to many perennial crops, the 

high level of herbicidal activity that it had demonstrated, and the uniqueness of its metabolic 

activity in the plant. More than twenty years after the launch of glyphosate, rigid ryegrass in 

Australia was reported as the first glyphosate-resistant weed in 1996 (Powles et al., 1998). About 

the same time, sales of glyphosate began to increase dramatically in the U.S. due to the launch of 

glyphosate-tolerant transgenic soybeans (1996), cotton (1997), and corn (1998). Rapid adoption 

of this new technology drove dramatic increases in the use of glyphosate-only weed control, 

which resulted in increased selection pressure for glyphosate-resistant weeds.
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Table 43 shows a summary of the twenty-one glyphosate-resistant weed species that have been 

reported from 1996 to November 2010 (Heap, 2011). The data clearly shows that glyphosateresistance in weeds is expanding around the globe. Most notably, there have been reports of ten

new weed species with some biotypes resistant to glyphosate in the U.S. since 2000 (Figure 60). 

Two of these glyphosate-resistant weed species have already become a significant problem for 

farmers across a large geographic area. Conyza canadensis infests at least two million hectares of 

glyphosate-tolerant crops in the U.S. (Main et al., 2004) and glyphosate-resistance in Palmer 

amaranth has serious ramifications for future weed management in the Southeast U.S. due to its 

rapid growth rate, extremely competitive nature, and resistance to other herbicide modes of 

action (Culpepper et al., 2008). Researchers have also reported that individual biotypes of ten of 

the 21 glyphosate-resistant species are also resistant to herbicides with other modes of action 

(Table 44). The Palmer amaranth, common waterhemp, and horseweed/marestail biotypes were 

reported in the corn and soybean growing states. Although herbicide options to control these 

biotypes with multiple herbicide resistance will be more limited, 2,4-D is still a viable control 

option for the broadleaf weeds.

Table 43. Weed species with reported glyphosate-resistant biotypes.

First Confirmed 

Report

Later 

Confirmed 

Common Name Species Name Year Country** Reports

Rigid ryegrass Lolium rigidum 1996 Australia USA, S.Africa, 

France, Spain

Goosegrass Eleusine indica 1997 Malaysia Colombia

Horseweed/Marestail* Conyza Canadensis 2000 USA Brazil, China, 

Spain, Czech 

Republic

Italian ryegrass Lolium multiflorum 2001 Chile Brazil, USA, 

Spain, Argentina

Hairy fleabane Conyza bonariensis 2003 S.Africa Spain, Brazil, 

Colombia, USA

Buckhorn plantain Plantago lanceolata 2003 S.Africa

Common ragweed* Ambrosia artemisiifolia 2004 USA

Giant ragweed* Ambrosia trifida 2004 USA

Ragweed parthenium Parthenium hysterophorus 2004 Colombia

Palmer amaranth* Amaranthus palmeri 2005 USA

Common waterhemp* Amaranthus rudis 2005 USA

Johnsongrass* Sorghum halepenses 2005 Argentina USA

Sourgrass Digitaria insularis 2006 Paraguay Brazil

Wild poinsettia* Euphorbia heterophylla 2006 Brazil

Junglerice Echinochloa colona 2007 Australia

Kochia Kochia scoparia 2007 USA

Liverseedgrass Urochloa panicoides 2008 Australia

Perennial ryegrass Lolium perenne 2008 Argentina

Sumatran fleabane Coryza sumatrensis 2009 Spain

Australian fingergrass Chloris truncate 2010 Australia

Annual bluegrass Poa annua 2010 USA

* Important weeds in US corn, soybean, and cotton production.

** Ten new species confirmed resistant in US since 2000.
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Figure 60. Number of glyphosate-resistant weeds reported globally by year from 1996 to 

2010.

(Compiled from (Heap, 2011))

0

2

4

6

8

10

12

14

16

18

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008 2009 2010

20

# of Gly-Res Weed Species
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Table 44. Global reports of glyphosate-resistant weed biotypes with resistance to other 

herbicide modes of action.

(Heap, 2011)

Common Name Species Name Year – Country 

(State)

Multiple Resistance to 

Other Herbicide 

MOAs

Palmer amaranth Amaranthus palmeri 2008 – US (MS) ALS

Common waterhemp Amaranthus rudis 2005 – US (MO) ALS, PPO

2006 – US (IL) ALS

Common ragweed Ambrosia artemisiifolia 2006 – US (OH) ALS

Hairy fleabane Conyza bonariensis 2009 – US (CA) Bipyridiliums

Horseweed Conyza canadensis 2003 – US (OH) ALS

2007 – US (MS) Bipyridiliums

Giant ragweed Ambrosia trifida 2006 – US (OH) ALS

2008 – US (MN) ALS

Goosegrass Eleusine indica 1997 – Malaysia ACCase

Wild poinsettia Euphorbia heterophylla 2006 – Brazil ALS

Italian ryegrass Lolium multiflorum 2002 – Chile ALS

2006 – Chile ACCase

2008 – Chile ACCase, ALS

Rigid ryegrass Lolium rigidum 1999 - Australia ACCase, ALS, 

Dinitroanilines

2003 – S. Africa ACCase, Bipyridiliums

2008 – Australia Triazoles, Ureas, 

Isoxazolidiones

2010 – Australia Bipyridiliums

In addition, researchers in Virginia have been testing a biotype of common lambsquarters that 

survived 1.0 lb ae/acre (1120 g ae/ha) glyphosate, and thus appears to have low level resistance 

to glyphosate (Hite et al., 2008). Weed scientists in Ohio and Indiana have also identified a 

biotype of common lambsquarters in at least a dozen fields that appears to have low-level 

glyphosate-resistance (Curran et al., 2007). The increased reports of glyphosate-resistant species, 

plus the geographic spread of their infestations, have caused some to raise concerns about the 

long term sustainability for glyphosate. Some researchers have stated that applying glyphosate 

alone over wide areas on highly variable and prolific weeds made the evolution of resistant 

weeds inevitable (Owen, 2001; Thill and Lemerle, 2001).

Weed Shifts

When glyphosate-tolerant crops are grown intensively with high reliance on glyphosate for weed 

control, species which possess some level of natural tolerance to glyphosate will become more 

prevalent. These ―weed shifts‖ can occur more rapidly than selection for glyphosate-resistance 

(Shaner, 2000). Coble and Warren (1997) demonstrated that continuous use of glyphosate caused 

an increase in the infestation of morningglory (Ipomoea spp.) species over a three year period 

compared with other herbicide programs. Some common hard to control weed species that could 

become ―weed shifts‖ in U.S. corn and soybeans are listed below in Table 45.
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Table 45. Potential weed shifts with use of glyphosate in U.S. corn and soybeans.

(Duke and Powles, 2008b; Owen, 2008)

Common Name Species Name

Asiatic dayflower Commelina communis

Brazil callalily Richardia brasiliensis

Broadleaf buttonweed Spermacoce latifolia

Common waterhemp Amaranthus rudis

Common lambsquarters Chenopodium album

Eastern black nightshade Solanum ptycanthum

Giant ragweed Ambrosia trifida

Hemp sesbania Sesbania exaltata

Kochia Kochia scoparia

Marestail / Horseweed Conyza canadensis

Morningglory spp. Ipomoea spp.

Nutsedge spp. Cyperus spp.

Prickly sida Sida spinosa

Russian thistle Salsola iberica

Tall waterhemp Amaranthus tuberculatus

Tridax daisy Tridax procumbens

Tropical spiderwort Commelina benghalensis

Velvetleaf Abutilon theophrasti

2,4-D Resistance

The earliest documented reports of herbicide-resistant weeds were for resistance to 2,4-D in wild 

carrot (Daucus carota) (observed in 1952 but not reported until 1957) and spreading dayflower 

(Commelina diffusa) in 1957 (Heap, 2011). Today, a total of sixteen weed species have 

documented reports of 2,4-D resistant biotypes someplace around the globe (Table 46). Wild 

carrot in soybeans and roadsides, field bindweed in cropland, and prickly lettuce in cereals are 

the only ones reported on the U.S. mainland (Heap, 2011). Wild carrot, yellow bur-head, wild 

radish, musk thistle, and corn poppy are the only 2,4-D resistant weeds that have reported 

infestations in more than 1,000 acres. Some of these 2,4-D resistant biotypes have documented 

cross resistance to other auxin herbicides or multiple resistance to some ALS-inhibiting 

herbicides. It is notable that most of these resistant species do not appear to be spreading, as 

indicated by few reports of additional sites after the initial report.
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Table 46. Weed species with reported 2,4-D-resistant biotypes.

(Heap, 2011)

Common Name Species Name Herbicide(s) Year Country 

or State

Wild carrot Daucus carota 2,4-D 1952 Ontario

2,4-D 1993 Michigan

2,4-D 1994 Ohio

Dayflower Commelina diffusa 2,4-D 1957 Hawaii

Field bindweed Convolvulus arvensis 2,4-D 1964 Kansas

Musk thistle Carduus nutans 2,4-D, MCPA 1981 New Zealand

Scentless chamomile Matricaria perforate 2,4-D 1975 France

2,4-D 1975 United 

Kingdom

Gooseweed Spenoclea zeylanica 2,4-D 1983 Philippines

2,4-D 1995 Malaysia

2,4-D 2000 Thailand

Canada thistle Cirsium arvense 2,4-D, MCPA 1985 Hungary

Globe fringerush Fimbristylis miliacea 2,4-D 1989 Malaysia

Wild mustard Sinapsi arvensis 2,4-D, most other 

auxins

1990 Manitoba

Corn poppy Papaver rhoeas 2,4-D, tribenuron 1993 Spain

Yellow bur-head Limnocharis flava 2,4-D 1995 Indonesia

2,4-D, bensulfuronmethyl1998 Malaysia

Italian thistle Carduus pycnocephalus 2,4-D 1997 New Zealand

Wild radish Raphanus raphanistrum 2,4-D 1999 Australia

Marshweed Limnophila erecta 2,4-D, ALS 2002 Malaysia

Indian hedge mustard Sisymbrium orientale 2,4-D, metsulfuronmethyl2005 Australia

Prickly lettuce Lactuca serriola 2,4-D, dicamba, 

MCPA

2007 Washington

Few of these auxin resistant weeds have had a significant economic impact due to the wide array 

of alternatives that successfully control these resistant weeds (Heap, 1997). The overall incidence 

of auxinic herbicide-resistance after more than 60 years of use is low compared with other 

herbicide families such as the ALS inhibitors (imidazolinones, sulfonylureas, and sulfonamides), 

triazines, and ACCase herbicides in a much shorter period of use (Section 9.6). Furthermore, 

there is no widespread resistance to auxinic herbicides. It has been suggested by various 

researchers that the rarity of auxinic herbicide-resistant biotypes in the field is due to: a) a 

commonly held belief that these herbicides have multiple sites of action in the plant (Jasieniuk et 

al., 1996), b) redundancy in auxin receptors (AFBs) and other components of the auxin signal 

response (Walsh et al., 2006), c) moderate selection pressure and their use in mixtures with other 

herbicides (Kern et al., 2005), d) fitness penalties (Bourdot et al., 1996), and e) quantitative 

inheritance of the resistance trait (Cranston et al., 2001)

The mechanism of resistance to auxinic herbicides has been investigated, in varying degrees for 

only a few of these resistant biotypes. Resistance mechanisms in these biotypes have proven to 
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be difficult to elucidate. A lack of differences between biotypes in auxinic herbicide absorption, 

translocation, and metabolism has led to the hypothesis that auxinic herbicide-resistance is most 

often likely due to differences at the target site or differences along the signal transduction 

pathway (Van Eerd et al., 2005).

Patterns and mechanisms of cross resistance in auxin herbicide-resistant biotypes to other classes 

of auxin herbicides are not yet well understood. Further research is needed across a range of 

resistant species and biotypes to identify the potentially numerous gene mutations that cause 

resistance. It is also possible, but less likely, that a biotype might be resistant to all of these auxin 

herbicides. Due to the diversity of chemistry representing the synthetic auxin mode of action, it 

is unlikely plants will derive a single metabolic mechanism for tolerance to this class broadly: 

2,4-D (phenoxy auxins), fluroxypyr (pyridyloxy auxin), dicamba (benzoate structure) or 

clopyralid (picolinate structure).

To summarize, selection for auxin resistant weed bioytpes after more than 60 years of use has 

been slow; none show significant spread from initial sites, none are of significant economic 

importance, and none have been found in corn fields to date. Use of 2,4-D in DAS-444Ø6-6

soybeans should not result in 2,4-D resistant weeds becoming a significant issue in soybeans. 

2,4-D will likely be used in a mixture with one or more other herbicides. Other alternative 

herbicides which are effective on the same weeds can be used to control any 2,4-D resistant 

weeds that might occur.

Glufosinate Resistance

There is currently only one report of a weed biotype that has developed resistance to glufosinate, 

Goosegrass (Eleusine indica) in Malaysia (Heap, 2011). Thus, glufosinate is an excellent tool to 

include in a weed management program.
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Appendix 9. Stewardship of Herbicide Tolerant DAS-444Ø6-6 Soybean

1. Introduction

DAS-444Ø6-6 soybean is a transgenic soybean product that provides tolerance to 

2,4-dichlorophenoxyacetic acid (2,4-D) glyphosate and glufosinate. This herbicide-tolerant

soybean will provide growers with greater flexibility in selection of herbicides for the improved 

control of economically important weeds; allow an increased application window for effective 

weed control; and provide an effective resistance management prevention solution to the 

increased incidence of hard-to-control and glyphosate resistant weeds.

1

Dow AgroSciences is committed to promoting the responsible use and stewardship of this new 

herbicide tolerant trait technology and will implement a comprehensive stewardship program for 

DAS-444Ø6-6 soybean and its associated herbicide technology containing a new 2,4-D choline 

salt. The stewardship program will focus on educating and training retailers, growers and 

applicators on the appropriate use of this new technology. This will be accomplished by using a 

multi-faceted approach, including use of a variety of tools and delivery methods, and working 

with customers, stakeholders and industry organizations to promote responsible use of the 

technology (Dietz, 2010).

The stewardship program for DAS-444Ø6-6 soybean will be identical to that associated with 

DAS-68416-4 soybean.

2. Stewardship Goals

Specific goals of Dow AgroSciences‘ comprehensive stewardship program for DAS-444Ø6-6 

soybean and 2,4-D choline salt include:

 Promoting the responsible use of this new technology to sustain its viability,

 Promoting compatibility with other crops and cropping systems,

 Providing distributors, retailers, growers, and applicators comprehensive guidance on 

responsible technology use, and

 Supporting compliance with state and federal regulatory requirements.

3. Stewardship Program Components

 

1

The following definitions are applied throughout this appendix: 1). Herbicide Tolerant Crop: A crop plant that 

has been developed (using techniques such as genetic engineering or selection of variants produced by tissue 

culture or mutagenesis) to survive and reproduce after treatment with a specific herbicide or herbicides that would 

normally damage an unmodified crop; 2). Herbicide Resistant Weed: A herbicide-resistant weed is a member of a 

population within a species that has an inherited ability to survive and reproduce following exposure to a dose of a 

herbicide normally lethal to susceptible populations of the species. Through repeated herbicide selection, the 

resistant population becomes dominant in a given area, and 3). Hard-to-Control Weed: A weed species that is 

inherently able to withstand treatment with a herbicide as a result of natural factors not involving herbicide 

selection or genetic mutation.
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To meet the above-described stewardship goals, Dow AgroSciences is developing a 

comprehensive stewardship program based on its experience and input from stakeholders. Key 

components of the stewardship program include:

 Authorized use through grower agreements,

 New 2,4-D choline based herbicide technologies, 

 Comprehensive product use information,

 Education and training, and

 Compliance monitoring.

3.1 Authorized Use Through Grower Agreements 

Growers who purchase and plant DAS-444Ø6-6 soybeans will be required to sign a grower 

agreement that provides the terms and conditions for the authorized use of the technology. The 

grower agreement will include a provision requiring them to follow the product use guide and all 

pesticide label requirements.

3.2 2,4-D Choline Herbicide Technology

Dow AgroSciences has discovered and developed a novel (non-ester or non-amine) 2,4-D 

choline salt herbicide product for use with DAS-44406-6 soybean. New herbicide formulations 

containing the active ingredient, 2,4-D choline salt,

2

are focused on improving non-target crop 

and environmental safety by offering growers and applicators herbicide products with ultra low 

volatility and minimized potential for physical drift.

An exceptionally low volatility of 2,4-D choline salt has been demonstrated in both laboratory 

studies and preliminary field studies. For example, in a laboratory study, wheat plants treated 

with rates ten times higher than specified on the label of 2,4-D choline were placed in an 

enclosed chamber with grape and tomato plants (Ouse et al., 2010). After 24 hours of exposure 

in the chamber at 104°F, neither sensitive crop showed any symptoms of injury. The lack of any 

symptoms on the grape and tomato plants is a clear indication that there was no detectable 

volatility following application of 2,4-D choline salt. Preliminary field results are consistent with 

these laboratory findings (Hillger et al., 2010).

Dow AgroSciences is also developing an innovative premix formulation that combines 2,4-D 

choline with glyphosate for use with DAS-444Ø6-6 soybean.3 As compared to tank mixes of 

traditional 2,4-D products and commercial glyphosate, this premix uses formulation technologies 

that will result in a significant reduction in herbicide off-target movement from particle drift

 

2

The United States Environmental Protection Agency (EPA) has accepted and is currently reviewing DAS’s 

submission for a wide range of crop and non-crop herbicide uses for 2,4-D choline salt. 

3 An application for 2,4-D choline and glyphosate premix use with DAS-44406-6 soybean will be submitted to EPA 

in 2012.
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(Wilson et al., 2010). This premix product will reduce the potential for off-target movement from 

both particle drift and volatility.

3.3 Comprehensive Product Use Information

3.3.1 Product Use Guides

Dow AgroSciences will establish product use guidelines and educate the technology users on 

responsible use via channel partner standards and agreements, grower agreements and product 

use guides. The product use guide will require that growers only use authorized 2,4-D choline 

products for use with DAS-444Ø6-6 soybeans. It will include information on herbicide 

resistance management, application and management of off-target herbicide movement, as well 

as seed planting directions and grain stewardship.

3.3.1.1 Herbicide Resistance Management

Based on the mode-of-action classification system of the Weed Science Society of America, 

2,4-D is a Group 4 growth regulator herbicide (synthetic auxin). Some naturally occurring weed 

biotypes that are tolerant (resistant) to 2,4-D may exist due to genetic variability in a weed 

population. Where resistant biotypes exist, the repeated use of herbicides with the same mode of 

action can lead to the selection for resistant weeds. Certain agronomic practices reduce the 

likelihood that resistant weed populations will develop and can be utilized to manage weed 

resistance once it occurs. 

Proactively implementing diversified weed control strategies to minimize selection for weed 

populations resistant to one herbicide or more is recommended. A diversified weed management 

program may include the use of multiple herbicides with different modes of action and 

overlapping weed spectrum with or without tillage operations and/or other cultural practices. 

Research has demonstrated that using the labeled rate and directions for use is important to delay 

selection for resistance. 

To aid in the prevention of developing 2,4-D resistant weeds, Dow AgroSciences recommends 

the following practices for herbicide selection, crop selection and cultural practices:
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Herbicide Selection

 Rotate the use of 2,4-D choline salt with non-auxin (non-Group 4) herbicides. 

 Utilize a broad spectrum soil-applied herbicide as a foundation treatment. 

 Utilize premixes, tank mixes or sequential applications of herbicides with alternative 

modes of action. 

 Avoid using more than two applications of a Group 4 herbicide, such as 2,4-D choline 

salt, within a single growing season unless mixed with another mode of action herbicide

with overlapping spectrum. 

 Apply full rates of 2,4-D choline salt at the specified time (correct weed size) to minimize 

escapes of tolerant weeds.

Crop Selection and Cultural Practices 

 Incorporate additional weed control practices whenever possible, such as delayed 

planting, crop rotation, mechanical cultivation and weed-free crop seeds, as part of an 

integrated weed control program. 

 Do not allow weed escapes to produce seeds, roots or tubers. 

 Thoroughly clean plant residues from equipment before leaving fields suspected to 

contain resistant weeds. 

 Scout fields after application to detect weed escapes or shifts in weed species. 

 If resistance is suspected, treat weed escapes with an alternate mode of action or 

cultivation. 

 Report any incidence of repeated non-performance of this product against a particular 

weed species to the local retailer, county extension agent, or Dow AgroSciences 

representative. 

3.3.1.2 Herbicide Application and Management of Off-Target Movement

The new 2,4-D choline containing herbicide products will minimize off target movement by 

reducing volatility and, in the case of the glyphosate + 2,4-D choline premix, off target 

movement will be minimized by reducing volatility and reducing the number of driftable fines.

To further minimize the potential for off-target movement with the new 2,4-D choline containing 

herbicide products, the product use guide will include recommendations on application 

equipment and methods such as following proper equipment maintenance, calibration and use. 

A variety of factors including weather conditions (e.g., wind direction, wind speed, temperature, 

and relative humidity) and method of application can influence pesticide drift. Product use 

guides will require applicators to evaluate factors and make appropriate adjustments when 

applying 2,4-D choline salt for use with DAS-444Ø6-6 soybean consistent with the final product 
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label approved by EPA. Dow AgroSciences is conducting significant research on nozzles for 

spray application. The product use guide will include instructions on using specific nozzles that 

minimize driftable spray droplets, specified environmental conditions such as wind speed and 

field temperature that minimize the potential for off-target movement due to particle or vapor 

drift.

3.3.2 Product Labels, Seed Bags and Tags

3.3.2.1 Product Labels

Dow AgroSciences has submitted a product label to EPA that includes comprehensive 

information and requirements for responsible use of 2,4-D choline with DAS-444Ø6-6 soybean. 

Such label directions are designed to minimize the potential for weed resistance development 

and off-target movement of 2,4-D choline. In addition, the submitted label does not allow 

herbicide application through any type of irrigation equipment and prohibits aerial application. 

3.3.2.2 Seed Bags and Tags

Seed bags and tags of soybean seed containing this new herbicide tolerant trait technology will 

have tags that provide stewardship information such as where to find the product use guide and 

grower agreement, as well as a customer information telephone number. Herbicide icons have 

been developed and will be included in marketing materials to clearly communicate applicable 

herbicide requirements to growers. When growers order seed, the icons and information will be 

included in the product use guide and on product fact sheets. Icons will appear on seed bag tags 

to remind growers of herbicide requirements at the time of planting.

3.3.3 Technical Bulletins

Dow AgroSciences will create comprehensive technical bulletins on these products including 

elements that address weed resistance management strategies, herbicide application technologies, 

responsible application practices to reduce the potential for off-target herbicide movement, and 

education on other elements of the herbicide label. Dow AgroSciences will make these available 

to channel and grower customers. They will also be made available to university cooperators, 

agronomists, crop consultants, other technical professionals and the general public through 

publication on internet websites.

3.3.4 Product Literature and Direct Mailings

Dow AgroSciences will also position the herbicide tolerant trait technology and promote its 

responsible use in its product literature by including crop agronomy, weed management, 

herbicide use and application information. Dow AgroSciences commonly uses direct mailings to 

customers, and will promote responsible stewardship of the herbicide tolerant trait technology 

through newsletters and correspondence. These direct mailings will emphasize responsible use 
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practices, label compliance, weed resistance management, and application guidance to minimize 

the potential for off-target movement.

3.3.5 Internet Resources

Information on the technology including agronomic and proper product placement, specific 

guidelines for planting transgenic traits, and herbicide use and application will be made available 

on technology provider websites. Relevant industry and university links may also be included to 

provide the user with additional technical information. Dow AgroSciences provides product 

stewardship information on its corporate and product websites.

3.4 Education and Training for Retailers, Growers and Applicators

An extensive network of Dow AgroSciences sales representatives, field scientists, and 

agronomists will play an important role in educating and training retailers, growers and 

applicators on the proper use of the technology and application guidelines. Dow AgroSciences 

field personnel, in addition to product suppliers, will work with growers to select the seed and 

herbicide products appropriate to their needs, growing conditions, and proper application 

equipment for field conditions.

Dow AgroSciences will communicate to customers the importance of stewardship and 

responsible product use. Dow AgroSciences will utilize a variety of approaches to educate and 

train retailers, growers, and applicators. These will include written communications, face-to-face 

meetings, field visits, television, radio, and/or computer-based training such as webinars and 

self-paced learning modules. Product stewardship plans for the herbicide tolerant trait technology 

will be based on applicator, grower and grain channel education, with reinforcement through 

written and verbal communications, including grower agreements, product use guides, product 

profiles, technical bulletins, sales literature, direct mailings, and websites.

Education topics for retailers, growers and applicators will include: DAS-444Ø6-6 soybean trait 

technology (including herbicide tolerance and agronomic practices), 2,4-D choline herbicide 

solutions (including label education, weed resistance management and herbicide application 

practices to minimize off-target movement). As an example, weed resistance management 

training will include information on: 

 The value of rotating herbicides with different modes of action, 

 How to use herbicides with different modes of action to achieve effective weed control,

 Recommendations to follow all labeled application rates and recommendations, 

 How to apply herbicides at the proper weed size or growth stage, and

 Appropriate cultural and mechanical practices for weed control and weed resistance 

management.
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As part of the various delivery methods and tools to educate retailers, growers, and applicators 

on the responsible use of this technology, Dow AgroSciences will use an online, interactive 

Learning Management System that will include training modules and information targeted to the 

users of the technology.

3.5 Compliance Monitoring

Dow AgroSciences is committed to implementing a compliance monitoring program for DAS444Ø6-6 soybean and its associated new 2,4-D choline herbicide technology. The monitoring 

program will be developed for retailers, growers, and applicators to promote responsible use of 

the technology.

Compliance with the grower agreement and product use guides will be tracked and monitored 

through surveys, communications, and on-farm visits. Failure to follow the requirements set 

forth in the grower agreement and product use guide may result in the loss of a grower‘s access 

to the technology. The grower agreement to be signed by the grower will require that upon 

request by Dow AgroSciences, the grower must provide information indicating the location of all 

fields planted with DAS-444Ø6-6 soybean and the herbicide applied to these fields. In addition, 

the grower will be required to cooperate with any on-farm visits and field inspections.

4. Stakeholder Outreach and Input

Dow AgroSciences is using a variety of methods to obtain stakeholder input in the development 

of this product stewardship program. Dow AgroSciences is consulting with retailers, growers and 

applicators to understand and address their needs in order to develop an effective and 

comprehensive stewardship program. In addition, Dow AgroSciences is proactively seeking

input from recognized experts with a diverse range of experience in U.S. agriculture as well as 

producers and processors of non-target crops. These efforts will support implementation of 

stewardship practices for this technology as broadly as possible throughout the value chain.

Dow AgroSciences will evaluate additional feedback from retailers, growers and applicators for 

the continuous improvement of this stewardship program following the launch of DAS-444Ø6-6 

soybean.
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5. Industry Commitment and Involvement 

Dow AgroSciences participates in several organizations and associations globally to promote the 

safe research and development, production, distribution, and responsible use of agricultural 

chemical and biotechnology products.

Dow AgroSciences is a participant in the Herbicide Resistance Action Committee (HRAC) 

[http://www.hracglobal.com], an industry-based group supported by CropLife International 

[http://www.croplife.org/public/resistance_management]. HRAC focuses on encouraging 

responsible herbicide usage, communicating herbicide resistance management strategies and 

supporting their implementation through practical guidelines. HRAC engages in active 

collaboration with public and private researchers, especially in the areas of problem 

identification and devising and implementing herbicide management strategies.

Dow AgroSciences personnel interact with academic weed scientists in addressing weed 

resistance management [http://www.wssa.net/Weeds/Resistance/] issues. Dow AgroSciences 

conducts joint trials at university sites as well as seeking input from university researchers 

regarding weed management. Dow AgroSciences also participates in a wide range of 

professional organizations including agronomy societies, seed trade groups, weed science 

societies, and crop commodity groups.

In addition to managing the technology in accordance with the applicable requirements of federal 

and state government agencies, Dow AgroSciences is a Founding Member of the biotechnology 

industry‘s Excellence Through Stewardship®organization which encourages effective and 

comprehensive stewardship programs and quality management systems throughout a trait 

product‘s life cycle. 

6. Conclusion

Dow AgroSciences is committed to promoting the responsible use and stewardship of this new 

herbicide tolerant trait technology and will implement a comprehensive stewardship program for 

DAS-444Ø6-6 soybean and its associated new herbicide technology, 2,4-D choline salt. The 

stewardship program will focus on educating and training retailers, growers and applicators on 

the appropriate use of this new technology. This will be accomplished by using a multi-faceted 

approach, including use of a variety of tools and delivery methods, and working with customers, 

stakeholders and industry organizations to promote responsible use of the technology.
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Appendix 10. References

Abranches, R., Shultz, R.W., Thompson, W.F., Allen, G.C., 2005. Matrix attachment regions and 

regulated transcription increase and stabilize transgene expression. Plant Biotechnology 

Journal 3, 535-543.

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