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

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Contains No Confidential Business Information Petition for Determination of Nonregulated Status for Herbicide Tolerant DAS - 444Ø6 - 6 Soybean OEC D Unique Identifier: DAS - 444Ø6 - 6 The undersigned submits this petition under 7 CFR 340.6 to request that the Administrator, Animal and Plant Health Ins pection 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 Submitte d 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 1 7 , 2011USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 p art of the regulatory process. By submit ting 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 F OIA request covering all or some of the information in this submission, DAS and MS Tech expec t 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, confidentiali ty and/or competitive concerns. DAS and MS Tech expec t that no information that has been identified as CBI (confidential business information) will be provi ded 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 par t 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 .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Certification The undersigned certifies that, to the best k nowledge 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. Kri eger, Ph.D. Regulatory Leader Dow AgroSciences LLC 9330 Zionsville Road Indianapolis, Indiana 46268 Telephone: (317) 337 - 3458 Fax: (317) 337 - 4649 mkrieger2@dow.comUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Summary Dow AgroSciences LLC (herein referred to as ―DAS‖) and M.S. Technologies LLC (her ein 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 t ransformation 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 herbici des 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 ap plication 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 di oxygenase - 12 (AAD - 12) , doub le mutant 5 - enol pyruvylshikimate - 3 - phosphate synthase (2mEPSPS), and phosphinothricin ace tyltransferase (PAT) proteins. The AAD - 12 protein is an enzyme with an alpha ketoglutarate - dependent dioxygenase activity which results in m etabolic inactivation of the herbicides o f 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 herbic ide 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 phosphino thricin 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 gen e expression cassettes was stably integrated into the soybean genome. Southern blot analyses also confirmed the absence of the plasmid backbone DNA in DAS 444Ø6
- 6 soybean. T he integrity of the inserted DNA was demonstrated in five different breeding generations . Data from segregating generations confirmed the predicted Mende lian inh eritance 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 link ed immunosorb ent assays (ELISA). Protein expression wa s 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 combi nation , or not treated with any of these her bicide s . 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 ef fects from the AAD - 12 protein. Bioinformatic analyses revealed no meaningful homologies with known or putative allergens or toxins for t he AAD - 12 amino acid sequence. The AAD - 12 protein hydrolyze d rapidly in simulated gastric fluid . There was no evidence of acute to xicity in mice at a dose of 2000 mg/kg b ody 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. T he low level expression of the AAD - 12 protei n 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 po tential adverse effects to humans and animals resulting from the environmental release of crops c ontaining the 2mEPSPS protein. A step - wise, weight - of - evidence approach was used to assess the potential for toxic or allergenic effects from the 2mEPSPS prote in. Bioinformatic analyses revealed no meaningful homologies to known or putative allergens or toxins for the 2mEPSPS amino acid sequence. The 2mEPSPS protein hydrolyze d rapid ly 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. T he low level expression of the 2mEPSPS protein presents a l ow 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 prev iously 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 cro ps containing the PAT protein. A step - wise, weight - ofevidence a pproach 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 hydrolyze d rap id ly in simulated gastric fluid. There was no evidence of acute toxicity in mice at a dos e of 5000 mg/kg body weight of PAT protein . T he low level expression of the PAT protein presents a low exposure risk to humans and animals, and the results of the over all 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, soyb eans, 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 a ssessments were conducted on DAS - 444Ø6
- 6 soybean in multi - site field studies to measure characteristics such as emergence, seedling vigor, pla nt height, lodging, and yield. All field trials were also observed for opportunistic disease or insect stressors a s 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 w eediness potential of DAS - 444Ø6
- 6 soybean.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Nutrient composition analyses of forage and grain were conducted to compare the composition of DAS - 444Ø6
- 6 soybean with the compositio n 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 compositiona l 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 product s. 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 add r ess 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 pr esented 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 u nlikely to increase the weediness potential of any other cul tivated 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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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 - 444 Ø 6 - 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 - 444 Ø 6 - 6 Soybean ................................ ................................ ........ 21 3.1. Descriptio n 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 t he 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 Molecula r 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 ................................ ........... 73USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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. Bioc hemical 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 Desi gn ................................ ................................ ................................ 90 8.2. Compositional Analysis ................................ ................................ ........................ 91 8.3. Statistical Analysis ................................ ................................ ................................ 93 8.4. Composition Analysis Results ................................ ................................ .............. 94 8.4.1. Proximate, F iber, 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 A nalysis 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. Po tential 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 ................................ ................................ ..... 149USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 C haracterization 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 We eds ...................... 200 Appendix 9. Stewardship of Herbicide Tolerant DAS - 444Ø6
- 6 Soybean ............................ 211 Appendix 10. References ................................ ................................ ................................ ....... 219USDA Petition for Nonregulated Status of DAS - 444Ø6
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List of Figures Figure 1. Plasmid map of pDAB8264. ................................ ................................ .......................... 22 Figure 2. Diagram o f 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 DAS 444Ø6
- 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 Hind III and Msc I digested DAS - 444Ø6
- 6 soybean: RB7 probe. ................................ ................................ ................................ ........................ 42 Figure 9. Southern blot analysis of Hind III and Msc I digested DAS - 444Ø6
- 6 soybean: Histone H4A748 UTR probe. ................................ ................................ ........................... 43 Figure 10. Southern blot analysis of Xho I and Msc I digested DAS - 444Ø6
- 6 soybean: Histone H4A748 Promoter probe. ................................ ................................ .................... 44 Figure 11. Southern blot analysis of Xho I and Msc I digested DAS - 444Ø6
- 6 soybean: AtUbi10 Promoter probe. ................................ ................................ ................................ .. 45 Figure 12. Southern blot analysis of Xho I and Hind III digested DAS - 444Ø6
- 6 soybean: AtuORF23 UTR probe. ................................ ................................ ................................ ..... 46 Figure 13. Southern blot analysis of Xho I and Hind III digested DAS - 444Ø6
- 6 soybean: CsVMV probe. ................................ ................................ ................................ .................. 47 Figure 14. Southern blot analysis of Xho I and Hind III digested DAS - 444Ø6
- 6 soybean: AtuORF1 UTR probe. ................................ ................................ ................................ ....... 48 Figure 15. Southern blot analysis of Xho I, Hind III and Msc I digested DAS - 444Ø6
- 6 soybean: 2mepsps probe. ................................ ................................ ................................ .. 49 Figure 16. Southern blot analysis of Xho I, Hind III and Msc I digested DAS - 444Ø6
- 6 soybean: aad - 12 probe. ................................ ................................ ................................ ..... 50 Figure 17. Southern blo t analysis of Xho I, Hind III, and Msc I digested DAS - 444Ø6
- 6 soybean: pat probe. ................................ ................................ ................................ ........... 51 Figure 18. Southern blot analysis of Msc I/ Eco RI digested DAS - 444Ø6
- 6 soybean: Histone H4A748 Promoter, 2mepsps and Histone H4A748 UTR probes. ....................... 52 Figure 19. Southern blot analys is of Pst I/ Xho I digested DAS - 444Ø6
- 6 soybean: AtUbi10 Promoter, aad - 12 , and AtuORF23 UTR probes. ................................ .............................. 53 Figure 20. Southern blot anal ysis of Pst I/ Xho I digested DAS - 444Ø6
- 6 soybean: CsVMV, pat , and AtuORF1 UTR probes. ................................ ................................ ....................... 54 Figure 21. Southern blot analysis of Msc I/ Ec oRI and Hind III digested DAS - 444Ø6
- 6 soybean: Backbone 3 and Backbone 4 probes. ................................ ................................ . 55 Figure 22. Southern blot analysis of Hind III and Pst I/ Xho I digested DAS - 444Ø6
- 6 soybean: Ori - Rep probe. ................................ ................................ ................................ ... 56 Figure 23. Southern blot analysis of Hind III and Pst I/ Xho I digested DAS - 444Ø6
- 6 soybean: Backbone 1 probe. ................................ ................................ ............................. 57 Figure 24. Southern blot analysis of Hind III and Pst I/ Xho I digested DAS - 444Ø6
- 6 soybean: Backbone 2 probe. ................................ ................................ ............................. 58USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 25. Southern blot analysis of Hind III and Pst I/ Xho I 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 CH 3 ). ................................ .............. 63 Figure 29. Amino acid sequence of the 2mEPSPS protein. ................................ .......................... 68 Figure 30. Amino acid sequence of the PAT protein. ................................ ................................ ... 74 Fig ure 31. Mode of action of the PAT protein. ................................ ................................ ............. 74 Figure 32. Proximate and fiber (% dry weight for all proximate and fiber except moi sture (% 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 s eed. ................................ ................................ ................................ ........... 102 Figure 35. Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non - transgenic (Isoline), E vent DAS - 444Ø6
- 6, and reference line soybean seed. ................................ ................................ ................................ .................. 107 Figure 36. Amino acids (% dry weight) in non - transgenic (Isoline), Event DA S - 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. ................................ ................................ ..................... 13 4 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 - deriv ed AAD - 12 protein. ................................ ................................ .................. 163 Figure 47. Glycosylation analysis of DAS - 444Ø6
- 6 soybean - and microbe - derived AAD 12 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 TOF TOF. ................................ ................................ ................................ ................................ 167USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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. ................................ ................................ ................................ ................... 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. ................................ ................................ ................................ ................................ 206USDA Petition for Nonregulated Status of DAS - 444Ø6
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List of Tables Table 1. Genetic elements of the T - DNA insert from plasmid pDAB8264. ................................ 26 Table 2. List of pr obes 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 generatio n. ................................ ................................ .... 60 Table 5. Results of BC1F2 individual plants from DAS - 444Ø6
- 6 soybean tested for AAD - 12 and 2mEPSPS protein expression withi n 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. Combine d - 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 proximat e 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. Sum mary 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. Sum mary 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 soybe an 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 sp ecific weed species in soybeans. ................................ .......... 143 Table 28. Number of herbicide resistant weeds reported globally by herbicide group and mode o f 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. ................................ ................................ ............. 168USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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. S ummary 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. ................................ ................ 209USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 Gen e from Delftia acidovorans which encodes the AAD - 12 protein AAD - 12 Aryloxyalkanoate d ioxygenase - 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 A ctive 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 Atu ORF1 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 eve nt expressing the AAD - 12, 2mEPSPS, and PAT proteins DCP 2,4 - Dichlorophenol dmmg Same as 2mepsps DNA Deoxyribonucleic acid ELISA Enzyme - linked immun osorbent 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 HectareUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information HRAC Herbici de 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 l aser desorption/ionization time - of - flight mass spectrometry Maverick Publicly available soybean line used in transformat ion to produce DAS - 444Ø6
- 6 soybean mEPSPS Same as 2mEPSPS MOA Mode of action MS Tech M.S. Technologies LLC NDF Neutral detergent fi ber OECD Organisation for Economic Co - operation and Development pat Gene from Streptomyces viridochromogenes which encodes the PAT protein PAT Phosphinothricin N - acetyl transferase protein PBN US FDA Pre - market Biotechnology Notice PCR Polymerase chain reaction pDAB 8264 DNA vector carrying the aad - 12 , 2mepsps and pa t 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 S tates Department of Agriculture UTR Untranslated region WSSA Weed Science Society of AmericaUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 1. Rational e for the Development of DAS - 444Ø 6 - 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 int o 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 reas on to believe are plant pests. The APHIS regulations at 7 CFR 430.6 provide that an applicant may petition APHIS to evalu ate 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 conside red regulated articles under 7 CFR 340. 1.2. Benefits of DAS - 444 Ø 6 - 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 fro m Streptomyces viridochromogenes into soybean. The aad - 12 gene encodes the aryloxyalkanoate dioxygenase - 12 (AAD - 12) enzyme which, when expres sed in plants, degrades 2,4 - D to herbicidally - inactive 2,4 - dichlorophenol (DCP). The 2mepsps gene encodes the 2mEPS PS protein, which has a decreased sensitivit y 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 acetyl transf erase 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 add ress their weed control needs. The availability of DAS - 444Ø6
- 6 s oybean will allow growers to proactively manage weed populations while avoiding adverse population shifts of troublesome weeds or the development of resistance, particularly g lyphosate - resistance in weeds. With the introduction of genetically engineered, g lyphosate - 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 wa s 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,USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information tank mixing, and incorporation of mechanical with chemi cal 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 glyphosate tolerant crops ( e.g., wheat, rice, turf, etc. ) are poised for introduction pen ding 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 l ike 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 we ed 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 propag ation 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 ide ntified in the past 5 - 8 years. Resistant wee ds 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 artemisiif olia (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 n ow 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 diffi cu lt - 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 - to control weed species, growers can c ompensate by tank mixing or alternating with other herbicides that wil l control the surviving weeds. One popular and efficacious tank mix active ingredient for controlling broadleaf escapes has been 2,4 - dicl orophenoxyacetic acid (2,4 - D). 2,4 - D has been use d agronomically and in non - crop situations for broad spectrum, broadleaf weed c ontrol for more than 60 years. Individual cases of more tolerant weed species have been reported, but 2,4 - D remains one of the most wi dely used herbicides globally. The developm ent 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 - t olerance trait with a glyphosate - tolerance trait give s growers the ability to use tank mixes of glyphosate/2,4 - D over the - top of the tolerant pla nts to control the glyphosate - resistant broadleaf species. DAS - 444Ø6
- 6 soybeans also provide tolerance to glufosinate herbicid es. Glufosinate is a non - selective, contact herbicide that controls a broad spectrum of annual and perennia l grasses and broadleaf weeds. The tolerance to glufosinate allows use of an additional mode of action as part of effective herbicide resistance ma nagement strategies. Glufosinate herbicides can also beUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information used as selection agents in breeding nurseries to select herbicide - tolerant plants to maintain seed trait purity. The commercial introduction of transgenic soybean exhibiting tolerance to 2,4 - D , glyphosate and glufosinate will bring new weed con trol alternatives to growers. This new weed management tool will allow for the improve d 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 incide nce of glyphosate - and acetolactate s ynthase (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 product s 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 regul atory 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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, an d 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 ) : Vegeta tive 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 i ts 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 po ds 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 3. Development of DAS - 444 Ø 6 - 6 Soybean 3.1. Description of the Transfor mation 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 2m epsps within the T - DNA region, was u sed to initiate transfor mation. Agrobacterium - mediated transformation was carried out using a modified procedure of Zeng et al . ( 2004 ) . Briefly , s oybean 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 v ancomycin to inhibit the growth of Agrobacterium . Glufosinate select ion was employed to inhibit the gro wth of non - transformed shoots. Selected shoots were transferred to rooting medium for root development and then transferred to soil mix fo r acclimatization of plantlets. Terminal leaflets of selected plantlets were painte d with glufosinate to screen for putative transformants. The glufosinate - resistant plantlets were transferred to the greenhouse, allowed to acclimate and then painted with glufo sinate to reconfirm tolerance. Surviving plantlets were deemed to be putative t ransformants. The screened plants were sampled and analyzed at the molecular level for the presence of the T - DNA insert and the abse nce of the vector backbone DNA. Specifically, for T0 plants, PCR analysis was performed to verify the absence of the spectin omycin resistance gene in the vector backbone as well as the presence of the aad - 12 coding region and 2m epsps plant transcription unit (PTU) . A PCR - based zygosity assay was conducted for copy number detection for pat , aad - 12 , and 2m epsps genes. Selected T0 plants were allowed to self

- fertilize in the gree nhouse 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 show s a plasmid map of pDAB8264 . Figure 2 shows a diagram of the T - DNA insert in plasmid pDAB8264. Figure 3 outlines the development of D AS - 444Ø6
- 6 soybean. Figure 4 shows a breeding diagram for DAS - 444Ø6
- 6 soybean including identification of the generations used in var ious safety assessment studies.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 1 . Plasmid m ap of pDAB8264.

Figure 2 . Diagram of intended T - DNA insert in plasmid pDAB8264.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 a nd 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. Screen ed 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Analysis Pet ition Section(s) DAS - 444Ø6
- 6 Soybean Generations Use d Control Molecular Analysis 5.1 , 5.2 , 5.3 , 5.4 T2, T3, T4, T6, F2 Maverick Segregatio n 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 3.2. Selection of Comparators for DAS - 444Ø6 - 6 Soybean To ensure the accurate assessment of the impact of transgene insertion on various chara cteristics 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 Ma verick 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 post transformation were essentially genetically identical to Maver ick soybean with the exceptio n of the transgenic insert DNA.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 transform ation 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 viridochrom ogenes ( Figure 2 ). A summary of the genetic elements is given in Table 1 . Table 1 . Genetic elements of the T - DNA insert from p lasmid pDAB8264 . Featu re 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 taba cum 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 20 26 661 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 2m epsps 2050 3387 1338 Native 5 - enolpyruvylshikimate - 3 - phosphate synthase gene from Zea mays with two mutations p roviding glyphosate tolerance ( Lebrun et al. , 1996 ; Lebrun et al. , 2003 ) TPotp C 3388 3759 372 Optimized chloroplas t transit peptide derived from maize and sunflower RuBisC O ( 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′ u ntranslated 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 cloningUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Featu re 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 polyubi quitin 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 - optimiz ed 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 sequ ences 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 necessar y for cloning pat 8695 9246 552 Plant - optimized ver sion of phosphinothricin acetyl transferase (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 pla smid 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 )USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Featu re 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 sequen ce 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 ca sset tes , also referred to as plant transcription units or PTUs, were present in the pDAB8264 vecto r for insertion into soybeans. The 2m epsps expression cassette is designed to express a double mutant maize 5 - enolpyruvylshikimate - 3 - phosphate synthase gene t hat encodes the 2mEPSPS protein. The 2m epsps 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 2m epsps 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 2m epsps gene encodes a protein of 445 amino acids that has a molecular w eight of approximately 47.5 kDa. The encoded protein is insensitive to glyphosate, thus providing tolerance to g lyphosate in plants ex pressing the 2mEPSPS protein. Expression of 2m epsps 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 H4A74 8 promoter is known to drive constitutive expression of the genes it controls ( Chaboute et al. , 1987 ) . The aad - 12 expressio n 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 co don bias to a level more t ypical 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 w eight of approximately 32 kDa. The insertion of aa d - 12 into soybean plants confers tole rance to herbicides such as 2,4 - D by production of the a ryloxyalkanoate 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 rodUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information present in soil, fresh water, activated sludge, and clinical specimens ( Von Graevenitz, 1985 ; Tamaoka et al. , 1987 ; Wen et al. , 1999 ) . D elftia 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 D elftia acidovorans in the food processing industry. For example, US Patent 5,128,253 ―Bioconversion process for the producti on of vanillin‖ was issued on July 7, 1992 to Kraft General Foods ( Labuda et al. , 1992 ) . Exp ression of aad - 12 in the T - DNA insert of pDAB 8264 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 fun c tion of AtuORF23 (GenBank Accession Number: CAA25184) in pTi15955 (Gen B ank 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 ) gen e that encodes the PAT protein. The pat gene was isolated from Streptomyces viridochromogenes and the synthetic version of the gene was optimi zed 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 soybea n transformation. The pat gene has be en 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 AtuO RF1 (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 Agrobac terium 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 mat rix or scaffold of the nucleus. When positioned on t he 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 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 5. Genetic Characterization 5.1. Overview of Molecular Analysis M olecular characterization of DAS - 444Ø6
- 6 soybean wa s conduc ted by Southern blot analysis. The results demonstrate that the t ransgene 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 DAS 444Ø6
- 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 location s of the probe s 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 fr agments in which known restriction enzyme recognition sites are comp letely contained within the T - DNA insert of pDAB8264 an d b) border fragments in which one known restriction enzyme recognition site is located with in the T - DNA insert and another site is located in the soybe an genome flanking the insert. Border fragment si zes vary by event because they rely on the location of the restriction enzyme recognition sites in the DNA sequen ce of flanking genomic region. Since integration sites are unique for each event, border fragments provide a means to determine the number of D NA 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 wa s 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 a re 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 ( Sectio n 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 , T 3 , T 4, T6, and F2 ) were identical, indicating that the insertion is stably integrated in the soybean genome ( Section 5.4 ) . The inheritance of DAS 444Ø6
- 6 soybean insert in segregating generations was investig ated using event - specific PCR , detection of the PAT and/or AAD - 12 protein, and detection of the aad - 12 gene (Section 5.5 ) . A ll results confirmed the predicted inheritance pattern of the transgene at a single locus ( Section 5.6 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 171 2 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 292USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 5 . Location of probes on pDAB8264 used in Southern blot analysis of DAS - 444Ø6 - 6 soybean.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 8 A Hind III Maverick None None

DAS - 444Ø6
- 6 >4261 ~4700 pDAB8264 5929 ~5900 Figure 8 B Msc I Maverick None None

DAS - 444Ø6
- 6 >1330 ~3400 Histone H4A748 UTR pDAB8264 9322 ~93 00 Figure 9 A Hind III Maverick None None

DAS - 444Ø6
- 6 >4261 ~4700 pDAB8264 10089 ~10100 Figure 9 B Msc I Maverick None None

DAS - 444Ø6
- 6 >9328 ~15000 Hist one H4A748 Promoter pDAB8264 16018 ~16000 Figure 10 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 10089 ~10100 Figure 10 B Msc I Maverick None None

DAS - 444Ø6
- 6 >9328 ~15000 AtUbi10 Promoter pDAB8264 16018 ~16000 Figure 11 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 10089 ~10100 Figure 11 B Msc I Maverick None None

DAS - 444Ø6
- 6 >9328 ~15000 AtuORF 23 UTR pDAB8264 16018 ~16000 Figure 12 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 4731 ~4700 Figure 12 B Hind III Maverick None None

DAS - 444Ø6
- 6 >4432 ~7000USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Probe/ Feature Enzyme Sample Expected Result (bp) 1 Observed

Result (bp) 2 Figure

Number

CsVMV pDAB8264 16018 ~16000 Figure 13 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 4731 ~4700 Figure 13 B Hind III Maverick None None

DAS - 444Ø6
- 6 >4432 ~7000 AtuORF1 UTR pDAB8264 16018 ~16000 Figure 14 A Xh o I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 4731 ~4700 Figure 14 B Hind III Maverick None None

DAS - 444Ø6
- 6 >4432 ~7000

2mepsps pDAB8264 16018 ~16000 Figure 15 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 9322 ~9300 Figure 15 B Hind III Maverick None None

DAS - 444Ø6
- 6 >4261 ~4700 pDAB8264 10089 ~10100 Figure 15 C Msc I Maverick None None

DAS - 444Ø6
- 6 >9328 ~15000

aad - 12 pDAB8264 16018 ~16000 Figure 16 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 4731 ~4700 Figure 16 B Hind III Maverick None None

DAS - 444Ø6
- 6 >4432 ~7000 pDAB8264 10089 ~10100 Figure 16 C Msc I Maverick None None

DAS - 444Ø6
- 6 >9328 ~1 5000USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Probe/ Feature Enzyme Sample Expected Result (bp) 1 Observed

Result (bp) 2 Figure

Number

pat pDAB8264 16018 ~16000 Figure 17 A Xho I Maverick None None

DAS - 444Ø6
- 6 >10093 ~12000 pDAB8264 4731 ~4700 Figure 17 B Hind III Maverick None None

DAS - 444Ø6
- 6 >4432 ~7000 pDAB8264 10089 ~10100 Figure 17 C Msc I Maverick None None

DAS - 444Ø6
- 6 >9328 ~15000 Histone H4A748 Promoter pDAB8264 4469 ~450 0 Figure 18 A Maverick None None DAS - 444Ø6
- 6 4469 ~4500 2mepsps Msc I/

Eco RI pDAB8264 4469 ~4500 Figure 18 B (Release Maverick None None PTU) DAS - 444Ø6
- 6 4469 ~4500 Histone H4A748 UTR pDAB8264 4469 ~4500 Figure 18 C Maverick None None DAS - 444Ø6
- 6 4469 ~4500 AtUbi10 Promoter pDAB8264 2868 ~2900 Figure 19 A Maverick None None DAS - 444Ø6
- 6 2868 ~2900 aad - 12 Pst I/

Xho I pDAB8264 2868 ~2900 Figure 19 B (Release Maverick None None PTU) DAS - 444Ø6
- 6 2868 ~2900 AtuORF23 UTR pDAB8264 2868 ~2900 Figure 19 C Maverick None None DAS - 444Ø6
- 6 2868 ~2900 CsVMV pDAB8264 1928 ~1900 Figure 20 A Maverick None None DAS - 444Ø6
- 6 1928 ~1900 pat Pst I/

Xho I pDAB8264 1928 ~1900 Figure 20 B (Release Maverick None None PTU) DAS - 444Ø6
- 6 1928 ~1900 AtuORF1 UTR pDAB8264 1928 ~1900 Figure 20 C Maverick None None DAS - 444Ø6
- 6 1928 ~1900USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Probe/ Feature Enzyme Sample Expected Result (bp) 1 Observed

Result (bp) 2 Figure

Number pDAB826 4 1049, 5929 ~1000, ~5900 Figure 21 A Msc I / Eco RI Maverick None None Backbone 3/ DAS - 444Ø6
- 6 None None Backbone 4 pDAB8264 4731, 9322 ~4700, ~9300 Figure 21 B Hin d III Maverick None None

DAS - 444Ø6
- 6 None None
Ori - Rep pDAB8264 9322 ~9300 Figure 22 A Hin dIII Maverick None None

DAS - 444Ø6
- 6 None None pDAB8264 9288 ~9300 Figure 22 B Pst I / Xho I Maverick None None

DAS - 444Ø6
- 6 None None
Backbone 1 pDAB8264 9322 ~9300 Figure 23 A Hind III Maverick None None

DAS - 444Ø6
- 6 None None pDAB8264 9288 ~9300 Figure 23 B Pst I/ Xho I Maverick None None

DAS - 444Ø6
- 6 None None
Backbone 2 pDAB8264 9322 ~9300 Figure 24 A Hind III Maverick None None

DAS - 444Ø6
- 6 None None pDAB 8264 9288 ~9300 Figure 24 B Pst I / Xho I Maverick None None

DAS - 444Ø6
- 6 None None SpecR pDAB8264 9322 ~9300 Figure 25 A Hind III Maverick None None

DAS - 444Ø6
- 6 None None pDAB8264 9288 ~9300 Figure 25 B Pst I/ Xho I 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 si zes are considered approximations from these analyses and are based on the indicated sizes of the DIG - labeled DNA Molecular Weight Ma rke r 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 6 . Plasmid map of pDAB8264 with sel ected restriction enzyme sites used for Southern analysis.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 an d Its Genetic Elements 5.2.1. Number of Insertion Sites The restriction enzymes Msc I, Xho I and Hin d III 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 Msc I 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 Msc I fragment as indicated in Figure 7 ( Figure 9 B, Figure 10 B, Figure 11 B, Figure 15 C, Figure 16 C, Figure 17 C r espectively ). The same enzyme digestion was also used for characterization of the RB7 MAR feature. The resulting SouthernUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information analysis indicated a single band of ~3400 bp in DAS - 444Ø6
- 6 samples which is consistent with the expected size of >1330 bp ( Figure 8 B ). For additional characterization of the T - DNA insert, genomic DNA samples w ere digested with the Xho I restriction enzyme and independently probed with the Histone H4A748 Promoter, AtUbi10 Promoter, AtuORF23 UTR, C sVMV, 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 10 A, Figure 11 A, Figure 12 A, Figure 13 A, Figure 14 A, Figure 15 A, Figure 16 A, and Figure 17 A respectively). Moreover, the restriction enzyme Hin d III was also use d 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 8 A, Figure 9 A, and Figure 15 B respectively). Digestion of the genomic DNA with Hin d III 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 combina tions ( Figure 12 B, Figure 13 B, Figure 14 B, Figure 16 B, and Figure 17 B 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 i n Figure 6 and Figure 7 , the pla nt transcription unit (PTU) for aad - 12 and pat could be released by restriction digestion with Pst I/ Xho I, while the 2mepsps PTU could be released with Msc I/ Eco RI digestion. These di gestions were performed to verify the presence of intact PTUs i n DAS - 444Ø6
- 6 soybean. When digested with Msc I/ Eco RI 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 18 B). When the same genomic DNA samples were probed with the Histone H4A748 Promoter or Histone H4A748 UTR probe, the same hybridization ba nd of ~4500 bp was detected in the same sample set ( Figure 18 A and Figure 18 C). These data indicate that an intact 2mepsps PTU is present in all generations of DAS - 444Ø6
- 6 soybean tested. When digested with Pst I/ Xho I 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 consisten t with the predicted size of 2868 for the aa d - 12 PTU ( Figure 19 B). When the same genomic DNA samples were hybridized with the AtUbi10 Promoter or AtuORF23 UTR probes, the same hybridization band of ~2900 bp was als o detected in the same sample set ( Figure 19 A and Figure 19 C ). These data indicate that an intact aad - 12 PTU is present in all generations of DAS - 444Ø6
- 6 soybean tested.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Wh en digested with Pst I/ Xho I and hybridized with pat probe, each individual DAS - 444Ø6
- 6 plant across the five generation s along with the pDAB8264 positive control resulted in a single hybridization band of ~1900 bp, which is consistent with the predicted siz e of 1928 bp for the pat PTU ( Figure 20 B). 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 20 A and Figure 20 C). 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 w ere 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 2 mepsps, aad - 12 , and pat . 5.3. Absence of Vector Backbone DNA T o 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 containi ng genomic DNA samples digested with Msc I/ Eco RI, Hin d III , and Pst I/ Xho I ( Figure 21 - Figure 25 ). Based on the expected fragment sizes of Msc I/ Eco RI and Hin d III digestion s , the Backbone 3 and Backbone 4 probes were mixed at an approximate 1:1 molar rat io for hybridization, while the Ori - Rep, Backbone 1, Backbone 2 , and SpecR probes were hybridized independently on separate blots of Hin d III and Pst I/ Xho I digestion s . The result s demonstrated that no specific hybridization bands were detected in any samples tested, except for the positive controls, as expected ( Figure 21 - Figure 25 ). When hybridi zed with the Bac kbone 4 and Backbone 3 probes, the Msc I/ Eco RI 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 59 29 bp and 1049 bp , respectively ( Figure 21 A). When hybridized with the same two probes, the Hin d III digested positive control samples had an observed hybridization band of ~9300 bp for the Backbone 4 probe and an o bserved band of ~4700 bp for the Backbone 3 probe, which is consistent with the expected bands of 9322 bp and 4731 bp , respectively ( Figure 21 B). When hybridized with the individual Ori - Rep, Backbone 1, Backbone 2, and SpecR probes, the Hin d III and Pst I/ Xho I digested positive control sample s 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 ). Take n 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 Genera tions Southern blot hybridizations were conducted with samples across five distinct generations (T2, T 3 , T 4, 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 latera l flow strip test kit to allow for confirmation of PAT expression - positive plants. All of the genetic element probes: 2mepsps , Histone H4A748 p romoter, Histone H4A748 UTR, aad - 12 , AtUbi 10 promoter, AtuORF23 UTR , CsVMV promoter, pat , AtuORF1 UTR , and RB7 MA R, as well as the pro bes covering the vector backbone sequences of plasmid pDAB8264 , were hybridized with the DAS - 444Ø6
- 6 soybean samples . As described above, r esults across all five generations of DAS - 444Ø6
- 6 soybean were consistent with what was expected ( Table 3 , Figure 8 - Figure 25 ), indicatingUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information stable integration and inheritance of the intact, single copy transgene insert across multiple generations of DAS - 444Ø6
- 6 soybean.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 8 . Southern blot a nalysis of Hin d III and Msc I d ig ested DAS - 444Ø6 - 6 soyb ean: RB7 p robe . Approximately 10  g of genomic DNA was digested with Hin d III (Panel A) and Msc I (Pa nel B) and hybridized with the RB7 probe. Plasmid pDAB8264 served as the positive control at approximatel y 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 - 444Ø 6 - 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 Pan el BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 9 . Southern blot a nalysis of Hin d III and Msc I d igested DA S - 444Ø6 - 6 soybean: H istone H4A748 UTR p robe . Approximately 10  g of DAS - 444Ø6
- 6 genomic DNA was digested with Hin d III (Panel A) and Msc I (Panel B) and hybridized with the Histone UTR probe. Plasmid pDAB8264 served as the positive control at approximatel y 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 DI G MWM VII Panel A Panel BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 10 . Southern blot a nalysis of Xho I and Msc I d igested DAS - 444 Ø6 - 6 soybean: Histone H4A748 Promoter p robe . Approximately 10  g of genomic DNA was digested with Xho I (Panel A) and Msc I (Panel B) and hybridized with the Histone Promoter probe. Plasmid pDAB8264 served as the positive control at approximatel y 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 . Pan el 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 Mave rick - 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 4 406 - 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 11 . Southern blot a nalysis of Xho I and Msc I d igested DAS - 444 Ø6 - 6 soybean: AtUbi10 Pro moter p robe . Approximately 10  g of genomic DNA was digested with Xho I (Panel A) and Ms c I (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 4 406 - 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 12 . Southern blot a nalysis of Xho I and Hin d III d igested DAS - 444Ø6 - 6 soybean: AtuORF23 UTR p robe . Approximately 10  g of genomic DNA was digested with Xho I (Panel A) and Hin d III (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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 13 . Southern blot a nalysis of Xho I and Hin d III d iges ted DAS - 444Ø6 - 6 soybean: CsVMV p robe . Approximately 10  g genomic D NA was digested with Xho I (Panel A) and Hin d III (Panel B) and hybridized with the CsVMV probe. Plasmid pDAB8264 served as the positive control at approximatel y one copy per soybean genome. Fragment sizes of the DIG - labeled DNA Molecular Weight Marker II an d 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 17 440 6 - 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 14 . Southern blot a nalysi s of Xho I and Hin d III d igested DAS - 444Ø6 - 6 soybean: AtuORF1 UTR p robe . Approximately 10  g of genomic DNA was digested with Xho I (Panel A) and Hin d III (Panel B) and hybridized with the AtuORF1 UTR probe. Plasmid pDAB8264 served as the positive control at a pproximate ly 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 15 . Southern blot a nalysis of Xho I , Hin d III and Msc I d igested DAS - 444Ø6 - 6 soybean: 2mepsps p robe . Approximately 10  g of genomic DNA was digested with Xho I (Panel A), Hin d III (Panel B) and Msc I (Panel C) followed by hybridization with the 2mepsps v1 probe. Plasmid pDAB8264 served as the positive control at approximately one co py 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 + pDAB82 64 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 44 06 - 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 CUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 16 . South ern blot a nalysis of Xho I , Hin d III and Msc I d igested DAS - 444Ø6 - 6 soybean: aad - 12 p robe . Approximately 10  g of genomic DNA was digested with Xho I (Panel A), Hin d III (Panel B) and Msc I (Panel C) followed by hybridization with the aad - 12 probe. Plasmid pDAB8 264 served as the positive control at approximatel y 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 Maveri ck - 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 9 440 6 - 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 CUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 17 . Southern blot a nalysis of Xho I , Hin d III , and Msc I d igested DAS - 444Ø6 - 6 soybean: p at p robe . Approximatel y 10  g of genomic DNA was digested with Xho I (Panel A), Hin d III (Panel B) and Msc I (Panel C) followed by hybridization with the Pat probe. Plasmid pDAB8264 served as the positive control at approximatel y 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 M averick - 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 44 06 - 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 VI I Panel A Panel B Panel CUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 18 . Southern blot a nalysis of Msc I/ Eco RI d igested DAS - 444Ø6 - 6 soybean: Histone H4A748 Promoter, 2mepsps and Histone H4A748 UTR p robes . Approximately 10  g of genomic DNA was digested with Msc I/ Eco RI followed by hybridization with the Histone Promoter (Panel A), 2mepsps (Panel B) and Histone UTR (Panel C) probe s . Plasmid pDAB8264 served as the positive control at approximately one copy per soybean genome. Fragment sizes of the DIG labeled DNA Molecular Weight Mar ker 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 M averick - 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 1 4 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 CUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 19 . Southern blot a nalysis of Pst I/ Xho I d igested DAS - 444Ø6 - 6 soybean: AtUbi10 Promoter, aad - 12 , and AtuORF23 UTR probes. Approximately 10  g of genomic DNA was with digested Pst I/ Xho I followed by hybridization with the AtUbi10 Promoter (Panel A), aad - 12 (Panel B) and AtuORF23 UTR (Panel C) probe s . Plasmid pDAB8264 served as the positive control at approximatel y 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 + pDA B8264 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 44 06 - 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 CUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 20 . Southern blot a nalysis of Pst I/ Xho I d ige sted DAS - 444Ø6 - 6 soybean: CsVMV, pat , and AtuORF1 UTR p robes . Approximately 10  g of genomic DNA was digested with Pst I/ Xho I followed by hybridization with the CsVMV (Panel A), Pat (Panel B) and AtuORF1 UTR (Panel C) probe s . Plasmid pDAB8264 served as the positive control at approximate ly 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 L ane 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 M averick - 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 1 0 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 CUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 21 . Southern blot a nalysis of Msc I/ Ec o R I and Hin d III d igested DAS - 444Ø6 - 6 soybean: Backbone 3 and Backbone 4 probes. Approximately 1 0  g of genomic DNA was digested with Msc I/ Eco RI (Panel A) and Hin d III (Panel B) and hybridized with the Backbone 3 and Backbone 4 probe s . Plasmid pDAB8264 served as the positive control at approximatel y one copy per soybean genome. Fragment sizes of the D IG - 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 M averick - 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 22 . Southern blot a nalysis of Hin d III and Pst I/ Xho I d igeste d DAS - 444Ø6 - 6 soybean: Ori - Rep p robe . Approximately 10  g of genomic DNA was digested with Hin d III ( Panel A) Pst I/

Xho I and (Panel B) and hybridized with the Ori - Rep probe. Plasmi d pDAB8264 served as the positive control at approximatel y 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 44 06 - 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 44 06 - 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 23 . Southern blot a nalysis of Hin d III and Pst I/ Xho I d igested D AS - 444Ø6 - 6 soybean: Backbone 1 p robe . Approximately 10  g of ge nomic DNA was digested with Hin d III ( Panel A) Pst I/

Xho I and (Panel B) and hybridized with the Backbone 1 probe. Plasmid pDAB8264 served as the positive control at approximate ly one copy per soybean genome. Fragment sizes of the DIG - labeled DNA Molecular We ight 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 12 440 6 - 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 24 . Sout hern b lot a nalysis of Hin d III and Pst I/ Xho I d igested D AS - 444Ø6 - 6 soybean: Backbone 2 p robe . Approximately 10  g of genomic DNA was digested with Hin d III ( Panel A) Pst I/

Xho I and (Panel B) and hybridized with the Backbone 2 probe. Plasmid pDAB8264 served as the positive control at approximatel y 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 Lan e 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 BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 25 . Southern blot a nalysis of Hin d III and Pst I/ Xho I d iges ted DAS - 444Ø6 - 6 soybean: SpecR p robe . Approximately 10  g of genomic DNA was digested wit h Hin d III ( 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 in dicated 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 1 0 4406 - F2 - 4 20 DIG MWM VII 10 4406 - F2 - 4 20 DIG MWM VII Panel A Panel BUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 P CR 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 polli nated to produce the F2 se eds. 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 . G enomic 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 w ere negative (segregated null). In addition, leaves of the indivi dual plan ts were tested for the presence or absence of the PAT protein using a PAT - specifi c lateral flow strip test kit. All of the plants that tested positive for DAS - 444Ø6
- 6 transgene insert displayed positive result for PA T protein expression, and all plants tha t were negative for DAS - 444Ø6
- 6 transgene insert displayed negative result for PAT protein expression ( Table 4 ), confirm ing that the phenotypic segregation matched the genotypic makeup of the tested F2 population . Statistical analysis using a  2 g oodness of fit test indicated the geno typic 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 i ndividual plants tested for PAT e xpres sion and event - specific PCR within a single s egregating g eneration. Tested Method Total plants tested P ositive N egative Expected ratio P - value a Event - Specific PCR 119 96 23 3:1 0.153 PAT Expression 119 96 23 3:1 a Based on a chi - squared g oodness of fit test

5.5.2. Segregation Analysis of Breeding Generations Chi - square analysis o f trait inheritance data from three populations of the BC1F2 breeding gen eration was conducted to determine the Mendelian inheritance of aad - 12 and 2m epsps in DAS - 444 Ø 6 - 6 soybeans. The presence or absence of aad - 12 and 2m epsps 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 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 5 . R esults of BC 1F2 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 2m epsps gene negative (susceptible to 2,4 - D + glyphosate ) Expected

ratio P - value a 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 Characteriza tion DAS - 444Ø6
- 6 soybean was produced using Agrobacterium - mediated transfor mat ion with the plasmid pDAB8264. The T - DNA insert of pDAB8264 consists of 1) the 2m epsps gene, controlled by the Histone H4A748 promoter and Histone H4A748 3′ UTR regulatory sequen ces; 2) the aad - 12 gene, controlled by the AtUbi10 promoter and AtuOR F23 3′ UTR regulatory sequences; 3) the pat gene, controlled by the CsVMV promoter and AtuOR F1 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 t he single insertion of the T - DNA inse rt from pDAB8264 containing a single intact copy of each of the 2m epsps , aad - 12 and pat PTUs . No additional DNA fragments from the 2m epsps , aad - 12 and pat expression cassettes were identified in DAS - 444Ø6
- 6 and no plasm id ba ckbone sequences were present. The T - DNA insert for DAS - 444Ø6
- 6 was shown to be stably integrated across five breeding generations ( T2, T 3 , T 4, 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) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 6. Characterization of the Introduced Proteins 6.1. AAD - 12 6.1.1. Identity of the AAD - 12 Protein The aryl oxy alkanoate dioxygenase (AAD - 12) protein was derived from Delftia acidov orans , a gram - negativ e 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 Nco I 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 op timize translation initiation. The prote ins encoded by the native and plant - optimized coding regions are 99.3% identical, differin g 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 MAQ TTLQITPTGATLGATVTGVHLATLDDAGFAALHAAWLQHALLIFPGQ 051 HLSNDQQITFAKRFGAIERIGGGDIVAISNVKADGTVRQHSPAEWDDMMK 101 VIVGNMAWHADSTYMPVMAQGAVFSAEVVPAVGGRTCFADMRAAYDALDE 151 ATRALVHQRSARHSLVYSQSKLGHVQQAGSAYIGYGMDTTATPLRPLVKV 201 HPETGRPSLLIGRHAHAIPGMDAAESERFLEGLVDWACQ APRVHAHQWAA 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 c atalyzing 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 degra dation of the (R) - enantiomers ( Kohler, 1999 ; Schleinitz et al. , 2004 ) . T he R - enantiomers are herbic idally active; therefore , AAD - 12 does not pr ovide tolerance to commercially - available chiral phenoxy acid herbicides.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 27 . Degradation reaction of 2,4 - D catalyzed by AAD - 12.

Figure 28 . General reaction catalyzed by AAD - 12 (R=H or CH 3 ) . 6.1.3. Biochemical Characterization of the AAD - 12 Protein Large quantities of purified AAD - 12 protein are required to perf orm safety assessment studies. Because it is technically infeasible to extract and purify sufficient amounts of recombinant protein fro m transgenic plants ( Evans, 2004 ) , the AAD - 12 protein was microbially - produced in Pseudomonas fluorescens . Characterization studies were performed to confirm the e quivalency 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 (SDS PAGE), western blot, glycoprotein detection, and protein sequence analysis by matrix assisted laser desorption/ionization t ime - 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. fluor escens 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 mi crobe - 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 i mmunoreactive to AAD - 12 protein - specific antibo dies by western blot analysis. There was no evidence of glycosylation of the DAS - 444Ø6
- 6 so ybean -USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information derived AAD - 12 protein. Greater than 84% of the soybean - derived protein a mino acid sequence was confirmed by e ither 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 acet ylation, 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 s oybean 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 20 10. Ten sites ( Georgia, Iowa (2 sites), Illinois (2 sites), Indiana, Michiga n, Missouri, and Nebraska (2 sites) ) were planted with DAS - 444Ø6
- 6 soybean and the c onventional 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 a pplication rates and timings). Pla nt tissues sampled included leaf, grain, root, and forage. Leaf tissues were collected at the V5 and V10 - 12 stage s , while root and forage w ere collected at the R3 stage. G rain was collected at the R8 st age of development ( Gaska, 2006 ) . The soluble, extractable AAD - 1 2 protein was measured using a validated enzyme - linked immunosorbent assay (ELISA) method . AAD - 12 protein levels for all tissue types were calcula ted 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 i n Table 6 . Average expression values ranged from 23 . 52 ng/mg dry weight in R3 stage root to 121.22 ng/mg dry we ight in V 10 - 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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/ G lyp 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/ Gly p 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 - 44 4Ø6
- 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 sa mple 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 1.0USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 unli kely to cause an allergic rea ction 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 - negati ve, non spore forming 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 - o f - evidence approach ( Codex Alimentari us Commission, 2009 ) was used to assess the allergenic p otential 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 seq uence 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, o r >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 condi tions (0.32% pepsin, pH 1.2; 37 o C) as demonstrated by both SDS - PAGE and western blot analyses.  The AAD - 12 protein is not pr esent 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 do es not share meaningful amino acid sequence s imilarities 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 - de pendent sulfonate dioxygenase, ketoglutarate dehyronase, t aurine catabolism dioxygenase , taurine dioxyge nase , d ioxygenase , oxidoreductase, pyoverdine biosynthesis protein, and hypothetical ( putative) or unnamed proteins. AAD - 12 (aryloxyalkanoate dioxygenase - 12) itself is an alpha - ketogl utarate dependent dioxygenase. Hypothetical and unnamed proteins are deri ved from conceptual translation of DNA sequences generated from massive genomeUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information sequencing projects of various fungi and bacteria. Those proteins have functional annotations such as ―p robable taurine catabolism dioxygenase ‖, ―c lavaminic 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 t reatment - 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 aryl oxy alkanoate dioxygenase (AAD - 12) protein was derived from Delftia acid ovorans , 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 mi crobial sources was conducted. Additio nally, characterization of AAD - 12 p rotein 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 herbi cides in combination , and non sprayed . A step - wise, weight - of

- evidence approach was used to assess the potential for toxic or allergenic e ffects from the AAD - 12 protein. Bioinformatic analyses revealed no meaningful homologies to known or putative allergen s or toxins for the AAD - 12 amino acid sequenc e. The AAD - 12 protein hydrolyzed rapid ly 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 dete ctable covalently linked carbohydrates in AAD - 12 protein express ed in DAS - 444Ø6
- 6 soybean . T he 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 pro tein indicate that it is unlikely to cause allergenic or toxi c effects in humans or animals.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 : Un iProt Accession number: O24566 ) . The mutation s 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 2 mEPSPS protein is comprised of 445 amino acids a nd has a molecular weight of ~47.5 kDa. The N - term inal 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 EEVQLFLGNAG I AMR S LTAAVTAAGGNATY 120 121 VLDGVPRMRERPIGDLVVGLKQLGADVDCF 150 151 LGTDCPPVRVNGIGGLPGGKVKLSGSISSQ 180 181 YLSALLMAAPLALGDVEIEIIDKLISIPYV 210 211 EMTLRLMERFGV KAEHSDSWDRFYIKGGQK 240 241 YKSPKNAYVEGDASSASYFLAGAAITGGTV 270 271 TVEGCGTTSLQGDVKFAEVLEMMGAKVTWT 300 301 ETSVTVTGPPREPFGRKHLKAIDVNMNKMP 330 331 DVAMTLAVVALFADGPTAIRDVASWRVKET 360 361 ERMVAIRTELTKLGASVEEGPDYCIITPPE 390 391 KLNVTAIDTYDDHRMAMAFSLAACAEVP VT 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 Glyp h osate normally exerts herbicidal activity by binding an d 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 ) line s have been genetically modified for tolerance to gly phosate herbicides by expressing in the plant a modi fied epsps gene from corn ( Zea mays ), 2mepsps , which introduce d two amino acid change s 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 prese nce of the herbicide ( Herouet - Guicheney et al. , 2009 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 6.2.3. Biochemical Characterization of the 2mEPSPS Protein Large quantities of purified 2mEPSPS protein are required to perf orm safety assessment studies. As it is technically infeasible to extract and pu rify sufficient amounts of recombinant protein from transgenic plants ( Evans, 2004 ) , the 2mEPSPS protein was produced in Pseudomonas fluorescens . Characterization studies were performed to confi rm the equivalency of the 2mEPSPS protein expressed in DAS - 444Ø6
- 6 soybean with the P . fluorescens - derived 2m EPSPS 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 - T OF MS/MS, and electrospray ionization - liquid chromatography mass spectrometry (ESI - LC/MS) were used to characterize the biochemical properties of the protein s. 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 i n detail in App endix 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 a mino acid sequence (including the N - and C - termini) was confirmed by enzymatic peptide ma ss fingerprinting usi ng 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 20 10 . 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 c onventional control (Maverick). The test sites represented regions of diverse agronomic practices and environmental conditions f or 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 a pplication rates and timings). Plant tissues sampled included leaf, grain, root, and forage. Leaf tissues were collected at the V5 and V10 - 12 stage s , while root and forage were collected at the R3 stage. G rain was collected at the R8 stage of dev elopment ( 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 calcul ated 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 dr yUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information weight in grain to 2583.46 ng/mg dry weight in V 10 - 12 stage leaf tissue. Expression levels were comparable for all treatments . No 2mEPSPS protein was detected in the control tissues across the ten locations.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 7 . Expression o f 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

– 36 00.00 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 330.0 2 109.78 189.20

– 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) a nd 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 8.0USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 re actions in humans or be toxic to humans or animals. History of Safe Use  The donor organism, Z ea mays , (commonly referred to as corn or maize) i s 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 previo usly 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 G HB614 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 co mpared 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% ident ity 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 an alyzed 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 pro tein 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 pathwa y associa ted proteins including 3 - p hosphoshikimate 1 - carboxyvinyltransferase enzyme 5 - enolpyruvyl shikimate 3 - phosphate synthase (EPSPS) enzyme , and UDP - N - acetylglucosamine 1 - carboxyvinyltransferase enzyme , andUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information dehydroquinate synthase . None of those protei ns is associated with known protein toxins that are harmful to humans and animals.  In acute mouse toxicity testi ng, 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. S ummary of 2mEPSPS Protein Characterization The 2 mepsps 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 bind ing 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 a nd has a molecular weight of ~47 .5 kDa. Detailed biochemica l characterization of the 2mEPSPS protein d erived from DAS - 444Ø6
- 6 soybean and microbial sources were conducte d. Additionally, characterization of 2mEPSPS p rotein 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 hydrolyze d rapi dly in simulated gastric fluid. There was no evidence of acute toxic ity 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 toxi c eff ects in humans or animals.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 N umber : Q57146 ) . PAT is co mprised of 183 amino acids a nd has a molecular weight of ~21 kDa ( Figure 30 ) .

001 MSPERRPVEIRPATAADMAAVCDIVNHYIETSTVNFRTEPQTPQEWIDDL 051 ERLQDRYPWLVAEVEGVVAGIAYAGPWKARNAYDWTVESTVYVSHRHQRL 101 GLGSTLYTHLLKSMEAQGFKSV VAVIGLPNDPSVRLHEALGYTARGTLRA 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 syn thetase (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 o f the plant cell ( Duan et al. , 2009 ) . The pat gene which encodes phosphinothricin acetyltransferase (PAT) acetylates the free NH 2 group of PPT ( in t he presence of acetyl coenzyme A) and thereby prevents autotoxicity in the producin g 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 result s 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 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 6.3.4. Expression of the PAT Protein in Plant Tissues A field expression study was conducted in the U.S. during 20 10 . Ten sites ( Georgia, Iowa (2 sites), Illinois (2 sites), Indiana, Michigan, Missouri, and Nebraska (2 sites) ) were planted with DAS - 444Ø6
- 6 soybe an 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 appli cation rates and timings). Plant tissues sampled included leaf, grain, root, and forage. Leaf tissues were collected at the V5 and V10 - 12 stage s , while root and forage w ere collected at the R3 stage. G rain 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 calcula ted 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 soybe an 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 V 10 - 12 s tage leaf tissue. Expression levels were comparable for all treatme nts . No PAT protein was detected in the control tissues across the ten locations.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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

DA S - 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 2.1 1 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 PA T 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 0.12USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 6.3.5. Food and Feed Safety Assessment for PAT Protein T he PAT protein pr oduced 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 significa nt 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. populati on, 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 bacte rium. 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 immunorea ctivity as the native protein. Characterization of PAT 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, glufosinate, all t hree herbicides in combination , and non - sprayed. T he 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 toxi c effects in humans or animals.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 7. Agronomic Performance 7.1. Phenotypic and Agronomic Characteristics Field trials with DAS - 444Ø6
- 6 soybean , a non - transgen ic control, and reference lines were conducted in 2010 at ten sites l ocated 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 non transgenic near - isogenic control (Maverick) and DAS - 444Ø6
- 6 soybean plots. Results from this study demonstrate agronomic equival ence between DAS - 444Ø6
- 6 soybean (unsprayed and sprayed) and non - transgenic soybean (Maverick) .

7.1.1. Agronomic Study An agronomic study with DAS - 444 Ø6
- 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 l ines. At each of the ten sites, all entries were arranged in a randomized complete block design with four blocks. Across all sites, e ach control and DAS - 444Ø6
- 6 entry was represented by a total of 40 plots (10 sites, 4 replicate plots per entry at each si te). Three of the six r eference 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 cultiv ar of similar maturity. Appropriate insect, weed, and disease control practices were applied to produce an agronomically acceptable crop. Unsprayed DAS - 444 Ø6
- 6 soybean plots and DAS - 444 Ø6
- 6 soybean plots treated with one of four herbicide regimes were incl uded 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 2,4 - D only Treatment: 2,4 - D (Weedar 64) was applied as three broadcas t applications to DAS - 444Ø6
- 6. Application timing was at planting / pre - emergence, and a pproximately V3 and R2 stages. Individual target application rates were 1.0 lb ae (acid equivalent)/A f or Weedar 64, or 1 120 g ae/ha. Glufosinate only Treatment: Glufosinate (Liberty) was applied as two broadcast applications to DAS - 444Ø6
- 6 . Application timing was at a pproximately V5 and R1 stages. The target application rate at V5 was 0.33 lb ai/A for Liberty , or 374 g ai/h a. 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 broadcas t applications to DAS - 444Ø6
- 6. Individual applications were at planting / pre - emergence, and approxim ately 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 broadcas t app lications to DAS - 444Ø6
- 6. Individual applications were at planting / pre - emergence, and a pproximately V3 and R2 stages. Individual target application rates were 1.0 lb ae/A f or Weedar 64, or 1120 g ae/ha. Individual target application rates were 1.1 l b ae/A for Durango DMA, or 1260 g ae/ha. Glufosinate (Liberty) was also applied as two broadcas t applications to DAS - 444Ø6
- 6. Application timing was at a pproximately V5 and R1 stages. The target application rate was 0.33 lb ai/A for Liberty, or 374 g ai/h a . The following agronomic characteristics were measured and recorded for all test entries at each location on a per plot basis ( Table 9 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 9 . Ag r onomic characteristics. Trait Evaluation

Time Descript ion 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 plant s 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 wh en »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 th at 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 lodgedUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Trait Evaluation

Time Descript ion 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 plan ts 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 gra ms 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 sampleUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 effec ts. Significant differences were declare d 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 stu dy, multiplicity was an issue. Multiplicity is an issue when a large number of comparisons are made in a single study t o look for unexpected effects. Under these conditions, the probability of falsely declaring differences base d on comparison - wise P - values is very high (1 - 0.95 number 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. There fore, the probability of declaring one or more false differences based on unadjusted P - values was >97% for agronomics (1 - 0.95 70 ) . One method to account for multiplicity is to adjust P - val u es to control the experiment - wise error rate, but when many comparis ons 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 decla red difference is significant. This can be accomplished u sing 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 discrimina tion of true differences among treatments from ran dom 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 - t ransgenic near - isogenic Maverick , unsprayed DAS - 444Ø6
- 6 and sprayed DAS - 444 Ø6
- 6 entries was conducted. For each agronomic character and entry, the least square means, standard error, and minimum and maxim um sample values are reported. Also for comparison, the minimum and maximum value s from reference lines across all sites (reference ranges) for each a gronomic character are rep orted. 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, shatterin g, yield, and 100 seed weight. Unadjusted P - values were significant at the 0.0 5 level for paired t - tests for one comparison each for seedling vigor, lodging, final population, and n umber of pods per five plants. For each significant unadjusted P - value, mean differences between transgenic and control entries were negligible and trans genic means were within the range observed for reference va rieties included in the study. Results from this study demonstrate agronomic equivalence between DAS - 444Ø6
- 6 (unsprayed and sprayed) and non - transgenic Maverick soybean.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 10 . Combined - site analysis: summary of agronomic characteristics.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 7.1.3. Conclusions Field agronomic characteristics of DAS - 444Ø6
- 6 (unsprayed or sprayed with 2,4 - D, glyphosate, glufosinate, or al l three herbicides) were eva luated 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 agronomi c effects were obs erved for DAS - 444Ø6
- 6 soybean. Results from this study demonstrate agronomic equival ence between DAS - 444Ø6
- 6 soybean and non - transgenic Maverick soybean.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 7.2. Ecological Evaluations The DAS - 444Ø6
- 6 soybean field trials were monitored and ob served by personnel familiar with soybean cultivation practices (breeders, field station managers, field a gronomists, field associates). The personnel conducting the field tests visually monitored the incidence of plant disease and pests on DAS - 444Ø6
- 6 soy beans compared to the conventional soybean varieties , including Maverick and six reference lines (Section 7.1.1), in the same trials. D isease and insect damage was rated on a numerical scale of 0 - 100%, with 0% representing no damage due to diseas e incidenc e 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 interact ions with diseases and insects.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 Agro nomic 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) (0.57 4, 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 D iscovery Rate (FDR) procedure; P - values < 0.05 were considered significant.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Ecological observations were also made from all USDA APHIS notifie d field trials conducted in 2009 - 2011 ( Appendix 6 ) . Incidence of dise ase and insect p resence in trials of DAS - 444Ø6
- 6 soybeans was recorded and differences in incidence or response of DAS - 444Ø6
- 6 soybeans compared with the conve ntional control Maverick were examined. Table 12 summar izes 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 t he 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 - 07 7 - 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, frogey e 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, S tark), 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 Do rmancy Evaluations T he 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, Metro TM 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 t o 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 germinationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information experiment were placed in a growth chamber where they were incubated at 1 0° C f or seven days followed by an additional seven days at 25° C. Flats for the warm emergence experiment were plac ed in the growth chamber at 25° C for seven days immediately after planting. For both experiments, the emerged seedlings in each half - flat were coun ted 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 emer ged 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 e valuating 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 soybean s across diverse environments. Therefore, these data support the conclusi on 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8. Grain and Forage Composition Field trials with DAS - 444Ø6
- 6 soybean , the non - transgen ic 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 expr ession ( Appendix 5 ) and agronomic characterization ( Section 7 ) studies. No biologically meaningful unintended compositional differences were observed between the non - transgenic near - isogenic control Ma verick 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 - 444 Ø6
- 6 soybean ( unspra yed 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) w as 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 t reatments), 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, e ach control and DAS - 444Ø6
- 6 en try 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 lin es 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, wi th 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 s eeding 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 b y 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 - 444 Ø6
- 6 soybean plots and DA S - 444 Ø6
- 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 s ulfate (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 a pproximately V3 andUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information R2 stages. Individual tar get application rates were 1.0 lb ae (acid equivalent)/A for Weedar 64, o r 1120 g ae/ha. Glufosinate only Treatment: Glufosinate (Liberty) was applied as two broadcast applications to DAS - 444Ø6
- 6 . Application timing was at a pproximately V5 and R1 stages. T he 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 t o DAS - 444Ø6
- 6. Individual applications were at planting / pre - emergence, and a pproximately 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 a pproximately V3 and R2 stages. Individual target application rates were 1.0 lb ae/A fo r 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 a pproximately V5 and R1 stag es. 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 .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 14 . Composition analytes.

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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8.3. Statistical Analysis An alysis of variance was conducted across field sites (combined - site a nalysis) 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 effec ts. Signi ficant differences were declare d 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) usin g t - tests. Due to the large number of contrasts made in this stu dy, multiplicity was an issue. Multiplicity is an issue when a large number of comparisons are made in a single study t o look for unexpected effects. Under these conditions, the probability of falsely declaring differences based on comparison - wise P - values is very high (1 - 0.95 number of comparisons ). In th is study there were five comparisons per analyte (71 analyzed analytes for composition), resulting in 355 comparisons made in the combined - sit e composition analysis. Therefore, the probability of declaring one or more false differences based on unadjusted P - values was >99.99% (1 - 0.95 355 ) . One method to account for multiplicity is to adjust P - val u es 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 decla red difference is significant. This ca n 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 e t 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8.4. Composition Analysis Results A statistical analysis of compositio n 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 an d sprayed DAS - 444 Ø6
- 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 erro r, and minimum and maxim um sample values are reported. Also for comparison, the minimum and maximum value s from the six reference lines across all sites (reference range) and literature range for each ana lyte are reported. M inimum and maximum value s are fo r individual plot results as are literature ranges ( except where noted ) . Arithmetic mea ns for each analyte from e ach 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. Proxima te, Fiber, and Mineral Analysis of Forage S oybean forage samples from the control (Maverick) , reference, and DAS - 444Ø6
- 6 entries were analyzed for proximate content ( protein, fat, ash, moisture, and carbohydrate s) , fiber ( acid detergent fiber (ADF), neutr al detergent fiber (NDF) ) , and minerals (calcium and phosphorus ) . A summary of the r esults 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 referenc e lines included in the study. No statistical differences were observed in the combined site 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 biolog ically meaningful as means were within literature ranges and within ranges for reference lines included in the study.USDA Petition for Nonregulated Stat us of DAS - 444Ø6
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Contains No Confidential Business Information Table 15 . Summary of the proximate and fiber analysis of DAS - 444Ø6 - 6 soybean forage from all sites, and associate d literature range.USDA Petition for Nonregulated Stat us of DAS - 444Ø6
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Contains No Confidential Business Information Table 16 . Summary of the mineral analysis of DAS - 444Ø6 - 6 soybean forage from all sites, and associated literature range.USDA Petition for Nonregulated Stat us of DAS - 444Ø6
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Contains No Confidential Business Information 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 rang e is shaded for each analyte.USDA Petition for Nonregulated Stat us of DAS - 444Ø6
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Contains No Confidential Business Information Figure 32 . (Cont). 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 = N E1, filled diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Stat us of DAS - 444Ø6
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Contains No Confidential Business Information Figure 33 . Mine rals (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 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8.4.2. Proximate and Fiber Analysis of Seed Soybean seed samples from the control (Maverick) , reference, and DAS - 444Ø6
- 6 entries we re 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 prese nted in Table 17 and Figure 34 . All mean results were within literature ranges (when available) and within ranges for reference lines include d in the study. Statistically significant overall treatment effects were found for pro tein and carbohydrates, where s ome DAS - 444Ø6
- 6 entries contained more protein and less carbohydrate than the control. Similarly, variations in fat, ash, and moisture were also observed for s ome pair - wise contrasts between DAS - 444Ø6
- 6 entries and the contro l . 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 part ial 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.US DA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 17 . Summary of the proximate and fiber analysis of DAS - 444Ø6 - 6 soybean seed from all sites, and assoc iated literature range.US DA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 s oybean 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 f or each analyte (when available).US DA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 34 (Cont). Proximate and fiber (% dry weight for all proximate and fiber except moisture (% fresh weight)) in non transgenic (Isoline), Event DAS - 444Ø6 - 6, and refer ence 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 i s shaded for each analyte (when available).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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, selen ium, 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 refer enc e 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 - 44 4Ø6
- 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 negligi ble and not biologically meaningful as means were within literature ranges and /or within ranges for reference lines included in the study.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 18 . Summary of the mineral analysis of DAS - 444Ø6 - 6 soybean seed from all sites, and a ssociated literature range.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 35 . Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, a n d 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).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 35 (Cont). Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, an d referen ce 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 d iamond = NE2. Literature range is shaded for each analyte (when available).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 35 (Cont). Minerals (mg/100g dry weight for all minerals except selenium (ppb dry weight)) in non - transgenic (Isoline), Event DAS - 4 44Ø6 - 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 d iamond = NE2. L iterature range is shaded for each analyte (when available).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 locat ions is presented in Table 19 and Figu re 36 . All mean results were within literature ranges and within ranges for referenc e lines included in the study. No statistical differences were observed in the com bined site 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 wer e 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 19 . Summary of the amino acid analysis of DAS - 444Ø6 - 6 soybean seed from all sites, and associated literature range.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figu re 36 . Amino acids (% dry weight) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, an d reference line soybean seed. Symbols for each location shown: open circle = GA,  = IA1, + = IA2, open triangle = IL1, open square = IL2, open di amond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figu re 36 (Cont). Amino acids (% dry weight) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, an d 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, f illed diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figu re 36 (Cont). Amino acids (% dry weight) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, and reference line soybean seed. Symbols for ea ch 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figu re 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, op en square = IL2, open diamond = IN, filled circle = MI, filled triangle = MO, filled squar e = NE1, filled diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figu re 36 (Cont). Amino acids (% dry weight) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, an d 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, fill ed squar e = NE1, filled diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8.4.5. Fatty Acid Analysis of Seed Soybean seed samples from the control (Maverick) , reference, and DAS - 444Ø6
- 6 entries were an alyzed for fatty acid content. A summary of the re sults across all locations is presented in Table 20 and Figure 37 . All mean results were within literature ranges (when available) and within ranges for referenc e lines included in the study. S tatistical analysis was not performed for the following analytes since greater than 50% of the samples were found to be below the LO Q: caprylic (8:0), capric (10:0), lauric (12:0), myristic (14:0), myristoleic (14:1), pentadecanoic (15:0), pentadecenoic (15:1), palmi toleic (16:1), heptadecanoic (17:0), heptadecenoic (17:1), γ linolenic (18:3), eicosadienoic (20:2), eicosatrienoic (20:3), and arachid onic (20:4). No statisti cal differences were observed in the combined - site analysis between the control and DAS - 444Ø6
- 6 e ntries 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 c ontrol, 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 20 . Summary of the fatty acid analysis of DAS - 444Ø6 - 6 soybean seed from all sites, and associated literature range.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 r ange is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 .USDA Petiti on for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8.4.6. Vitamin Analysis of Seed Soybean seed sa mples 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 res ults were within literature ranges (when available) and /or within ranges for referenc e lines included in the study. For Vitamin A and β - Tocopherol, s tatistical analysis was not performed since greater than 50% of the samples were found to be below the LO Q. No statisti cal differences were observed in the combined - site analysis between the control and DAS - 444Ø6
- 6 entries for Vitamins B 3 , B 5 , B 6 , and δ - Tocopherol. Statistically significant differences were observed for Vitamins B 1 , B 2 , B 9 , 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 21 . Summary of the vitamin analysis of DAS - 444Ø6 - 6 soybean seed from all sites, and asso ciated literature range.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 38 . Vitamins (mg/kg dry weight) in non - transgenic (Isoline), Event DAS - 444Ø6 - 6, and reference line soybean seed. Symbols for each locat ion 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).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 (w hen available).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 8.4.7. Bioactive Analysis of Seed Soybean seed samples from the control (Maverick) , reference, and D AS - 444Ø6
- 6 entries were analyzed f

or 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 referenc e lines included in the study. No statisti cal differences were observed in the combined - site analysis between the control and DAS - 444Ø6
- 6 entries for phytic acid, stachyose, a nd total glycitei n 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 neglig ible and not biologically meaningful as means were within literature ranges and /or within ranges for referenc e 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 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 22 . Summary of the bioactive analysis of DAS - 444Ø6 - 6 soybean seed from all sites, and associated literature range.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business InformationUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 - 4 44Ø6 - 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. L iterature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 39 (Cont). Bioactives (% dry weight (DW) for all bioactives except lectin (H.U./mg protein DW, H.U. = hemagglutination unit) and tr ypsin 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 di amond = IN, filled circle = MI, filled triangle = MO, filled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 40 . Bioactives: isoflavones (mcg/g dry weigh t) 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, fi lled square = NE1, filled diamond = NE2. Literature range is shaded for each analyte.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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, gluf osinate, 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 fou nd 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, glufosi nate, or all three herbicides) was eval uated 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, indicatin g that no unintended compositional effects were observed for DAS - 444Ø6
- 6 soybean. Results from this study demonstrate compositional equivalen ce between DAS - 444Ø6
- 6 and non - transgenic soybean.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 9. Environmental Consequences and Impact on Agronomic Practices 9.1. Field Efficacy Field studies were conducted with DAS - 444Ø 6 - 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 (U S). 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 a part 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 applicati ons 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 an alyzed 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 - 444Ø 6 - 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 in jury 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 non transgenic near - isogenic s oybean (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 toleranc e to application of 2,4 - D (Weeda r 64). Herbicide Rate a Application Stage b Percent Plant Injury c

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 be tween sprayed and unsprayed DAS - 444Ø6
- 6 at 95% confidence level.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Tolera nce 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 near isog enic soybean (Maverick) ranged from 80 to 99 % at two weeks after treatment when sprayed at 1X the proposed use rate of gl yphosate . Table 24 . DAS - 444Ø6 - 6 soybean tolerance to application of glyphosate (Durango DMA). Herbicide Rate a A pplication Stage b Percent Plant Injury c

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 1 120 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 be tween sprayed and unsprayed DAS - 444Ø6
- 6 at 95% confidence lev el. 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 propos ed 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 Rate a Application Stage b Percent Plant Injury c

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 g rowth 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 pl ants. ns indicates no statistical differences be tween sprayed and unsprayed DAS - 444Ø6
- 6 at 95% confidence level.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Summary of Herbicide Tolerance The outcome of the field studies demonstrates that DAS - 444Ø6
- 6 soybean provides excellent tolerance to applicati ons 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 w eeds and are not effective at in vading 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 pl ant 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 o r primary colonizers ( OECD, 2000 ) . The introduction of aryloxyalkanoate herbicide , glufosinate, and glyphosate - tolerance into soybean will not alter the weedine ss characteristics of soybean. Agr onomic 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 iden tical to conventional Maverick soybean. If DAS - 444Ø 6 - 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, fluomet uron in cotton ( Hayes, 2000 ) , and atrazine in corn. The refore, the se findi ngs demonstrate that DAS - 444Ø6
- 6 soybean do not have increased weediness potential relative to conventional soybean. 9.3. Gene Flow Assessment 9.3.1. Vert ical Gene Flow Soybean is considered to be a self - pollinated species, although natural crossing can occur at low rates ( OECD, 2000 ) . The level of c ross 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 Ge ne Flow There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to microbes ( Conner et a l. , 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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. soyb ean production in 200 8 /0 9 was $2 9.6 billion, the second highest value among U.S. - produ ced crops, trailing only corn. Soybean and soybean product exports accounted for 54 percent of U.S. soybean production in 200 8 /0 9. Soybeans are about 90 percent of U.S. t otal oilseed production, while other oilseeds

— such as cottonseed, sunflower seed, canola, and peanuts

— account for the remainder ( USDA ERS, 2010 ) . A total of 7 7 . 4 million acres were pl anted to soybeans in the United States in 20 10 ( USDA NASS, 2011 ) . Of these planted acres, about 7 6 .6 million acres were harvested, valued at $ 38.9 billion. The majority of these soybeans were grown wi thin t he 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 t he crit ical period of competition in soybeans as the point of time when weeds that emerge with th e 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 . T he crit ical 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 alr eady 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 th e 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 s ince they can reproduce from seed or underground root buds or rhizomes.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 26 . Common troublesome weeds in soybeans in 2006 - 2008. ( Third Party Proprietary Data ). Weed Species Total Soybean Acres Treated 1 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 18,3 99,609 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 Pig weed, 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 7,61 0,855 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 her bicide - treated acreage is much more, due to multiple sprays on each acre.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 dif ficulties, 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 condi tions, and other weeds present. Generally, broadleaf weeds are more damaging to a broadleaf crop, while grass weeds are mor e 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 reduction 1 1 2 4 6 8 10

Weeds per 100 feet of row Cocklebur 1 2 4 6 8 10 Pigweed or Lam

bsquarters 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 fr om ( Knake and Slife, 1962 ; Stoller et al. , 1987 ; Werner and C urran, 1995 ) .

9.4.3. Weed Management in Soybean In 200 6 , USDA NASS surveyed 1 9 states ( Arkansas, Illinois, Indiana, Iowa, Kansas, Kentucky, Louisiana, Michigan, Minnesota, Mississippi, Missouri, Nebraska, North Carolina, North Dakota, Ohio, South Dakota, Tenn essee, Virginia, and Wisconsin ; about 9 6 % 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 i n U.S. soybeans ( USDA NASS, 2007 ) . In 2006, 97.5 % of the planted soybean acres received an average of 2.2 herbicide applications to contro l weeds ( USDA ERS ARMS, 2006 ) . Many herbicides are registered for pre - plant, pre - emergent and/or post - emergent application to selectively control most weed speci es commonly found in soybeans. In general, soybeans sometimes receive a soil applied or burndown her

bicide prior to planting or at plant, but almost always receive a post - em ergence herbicide application. In 200 6 , the most widely used herbicide in soybeans was glyphosate, driven by the high adoption o f glyphosate tolerant soybeans ( USDA N ASS, 2007 ) . S oybeans received an average of 1. 7 applications of glyphosate on 9 6 % of the acres planted in 200 6. Herbicides such as 2,4 - D ( 10 % of acres), chlorimuron (4% of acres), and trifluralin (2% of acres) were applied on significantly fewer acres. A ll 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 bel ow threshold levels that reduce yield and profits ( Knezevic, 2010 ) . Herbicides are only one of several tools available for growersUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information to consider using in an IWM approach. Herbicide - toleran t 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 herbicide tolerant crops as part of an I WM 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 th e 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 contr ol 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 up on 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 corn soybean 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 fu el use and fuel cost strongly support the premise that soybean produced with conservation tillage in any production system is more economically a nd environmentally sustainable ( CAST, 2009 ) . In fact, considering the generally accepted 30% minimum res idue cover and the 3 to 5 tons/acre/year tolerable soil loss for a sustain able soybean production system ( CAST, 2009 ) , conservation tillage is the only system that seems to support sustainable soybean production from an environmental standpoint. 9.5. Po tential 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 theUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 9 3 percent in 20 10 ( 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 9 3 % of soybean acres in the U.S. in 20 10 planted to herbicide tolerant ( glyphosate - tolerant ) soybeans, typical cultivation and management practices used by growers today already t ake into account the management of herbicide - tolerant traits . DAS - 444Ø 6 - 6 soybeans are comparable to conventional soybeans phenotypically and agronomically ( Section 7.4 ), and are not expected to alter the geographic range or se aso nality 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 c ontrol 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 o f 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 - em ergence applications of 2,4 - D control a broa d 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 late r germinating broadleaf weeds. Post - emergence applica tions of glyphosateUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 w ould 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 t he 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 t o 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 1 2 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 - 4 4 4Ø 6 - 6 soybeans will allow growers to apply 2,4 - D from burndown or pre - emergence up through R2 stage soybean s 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 o n DAS - 4 4 4Ø 6 - 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 - 444Ø 6 - 6 soybeans will be consistent with the current use pattern of glyphosate on other soybean products that are tolerant to glyphosate or glufosinate.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information While 2,4 - D is currently registered as a pesticide, supporting information on proposed label changes for its use with DAS - 444Ø 6 - 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 appl ication 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 techno logies. P roducts 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 - emer gence weed control programs. T hey 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 - 444Ø 6 - 6 soybeans , which will enable the use of 2,4 - D, will allow use of a low cost, hi gh 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 199 8 - 20 10 . ( Third Party Proprietary Data ) 9.5.3. Potential I mpact on Volunteer Management Cultivated soybean seed rarely displays any dormancy characteristics , and only under certain environmental conditions grows as a volunteer in the year foll owing 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 no t found outside of cultivation. In managed ecosystems, soybean does not effecti vely compete with other cultivated plants or primary coloni z ers. DAS - 444 Ø 6 - 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 effective ly controlled with other herbicides ( York et al. , 2005 ) . Lb. a.i./Acre

Corn Soybeans

0.9USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Soybean is considered a self - p ollinated species ( OECD, 2000 ) . Cros sing does not general ly occur. Soybean can only cross with other members of Glycine subgenus Soja . Approximately 9 3 % of all soybeans planted in the U.S. in 20 10 were glyphosate - tolerant soybeans ( USDA NASS, 2010 ) . If DAS - 444Ø 6 - 6 soybeans were to cross with soybean varieties expressing to lerance 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, fluo meturon 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 activi ty 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 protein s 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 allergen s or toxin s ( Sections 6.1.5 , 6.2.5 and 6.3.5 ) and DAS - 444Ø 6 - 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 soybea n ( 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 shoul d 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 wi ll cause new soybean acres to be planted in areas that are no t already in agricultural use. Commercial cropping systems routinely disturb the gr ound in which crops are grown. Habitat disruption within DAS - 444Ø6
- 6 soybean fields would be comparable to other no - till or herbic ide - 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 gene s 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 agricult ural 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. TheUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information environmental fate and ecological effec ts 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 pre plant burn down prior t o p lanting 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 al so 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 c onsistency 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 soyb ean. 9.6. Herbicide Resistance Management 9.6.1. Herbicide Resistance Herbicides have revolutionized weed control in mo st countries around the world. Herbicides are often the most reliable and least expensive method of weed control available, but reliance upon herbic ides 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 res istance 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, b ut in extremely small numbers. Such plants rema in 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 pl ants to survive and reproduce. The number of resista nt 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 suscepti ble to being resistant. Individual plants do not change from being susceptible to being resistant; rather, the proportion of resistant individuals within the p opulation 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 herbici des ( Bandeen et al. , 1982 ) . Today, more than 3 55 weed biotypes around the world have been reported to have some populations that are resistant to one or more herbicides (Heap, 20 11). A weed biotype is a sub - type or sub - population of a weed species, in this case one that has developed resista nce to one or mor e 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 beenUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 28 . Number of herbicide resistant weeds reported globally by herbicide group and mode of action . ( Heap, 2011 ) Herbicide Group Mode of Action HRAC Gr oup a WSSA Group a Example Herbicide

Total Count
ALS Inhibitors Inhibition of acetolactate synthase ALS (acetohydroxyacid synthase AHAS) B 2 Chlorsulfuron 10 9 Photosystem II Inhibitors Inhibition of photosynthesis at photosystem II C1 5 Atrazine 6 9 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 2 8 Bipyridiliums Photosystem - I - electron diversion D 24 Paraquat 2 5 Ureas and Amides Inhibition o f 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 O xy fluorfen 4 Nitriles and others Inhibition of photosynthesis at photosystem II C3 6 Bromoxynil 3 Carotenoid Biosynthesis Inhibitors Bleaching: Inhibition of carotenoid s 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 22 7 (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 th e 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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, rese archers, 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 - resista nt weeds based on cropping system practices in shown in Table 29 . This table assesses the risk of herbicide resistance development for each management practice as eith er ―low‖, ―medium‖, or ―high‖. The greatest chance for resistan ce development occurs when several of these management practices fall into the ―high‖ category.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 29 . Assessment of resistance risk by evaluation of cropping systems. ( Nevill et al. , 19 98 ) 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 Che mical 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 P oor *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 c aused 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 t he sole method of weed control. Under these conditions, the risk of weeds developing herbicide - resistance is greatest and th e 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), c otton (1997), and corn (1998). This evolution of glyp hosate - resistant weed populations threatens the ongoing sustainability of glyphosate and its contributions to world food produ ction ( 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 - cont rol 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 u sed to control glyphosate - resistant weed populations and reduce selection pressure for additional gly phosate - resistant weed species. Table 30 shows that several common weeds in U.S. corn and soybeans which are resistant to or diff icult to control with glyphosate or ALS herbicides can be effectively controlled with 2,4 - D.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Introduction of DAS - 444Ø 6 - 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, in cluding the use of glyphosate. Importantly, because DAS - 444Ø 6 - 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 manageme nt. 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 biotype s that is superior to glyphosate alone. DAS - 444Ø 6 - 6 soybeans will allow use of 2,4 - D to control glyphosate - resistant weeds and significantly delay the selection for glyphosate - res istance in other weed species. The same benefits of DAS - 444Ø 6 - 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 Resistan t (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 herbicide tolerant 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 glufosinat e 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 relat ives 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 s oybean 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 manageUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information weed populations while avoiding adve rse 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 - isoge nic 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 c onsequences from introduc tion.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 11. Appendices Appendix 1 . Methods for Molecular Characterization of DAS - 444Ø6
- 6 Soybean Appendix 2 . Methods and Results for Characterization of AAD - 12 Protein App endix 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 E xpression 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 Appen dix 9 . St ewardship of Herbicide Tolerant DAS - 444Ø6
- 6 Soybean Appendix 10. ReferencesUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Appendix 1 . Methods for Molecular Characterization of DAS - 444Ø6 - 6 Soybean DAS - 44 4Ø6 - 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 protei n expression using a rapid lateral flow test strip according to th e 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 t he greenhouse. The Maverick seeds had a genetic background representative of the transgenic seeds but did not contain the 2mepsps, aad - 12, and pat gene s . Reference Materials DNA of the plasmid pDAB8264 was added to samples of the Maverick control genomic D NA 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 (P CR) 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 gre en house for DNA extrac tion 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 mill iliter CTAB extraction buffer: gram leaf tissu e) and RNase - A (>10 μL) (Qiagen ). After approximate ly 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 precipi tated by mixing the supernatant with equal volume of preci pitation buffer (1% CTAB, 50 mM Tris - HCl, pH 8.0, 10 mM EDTA ). The precipitated DNA was diss olved in high salt TE buffer (1 × TE pH 8.0 , 1.0 M NaCl ) followed by preci pitati on with i sopropyl alcohol. The precipitated DNA was rinsed with 70% ethanol, air - dried, then dissol ved 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 electrophoretic ally separated on a 1% agarose gel containing eth idium bromide (~1 μg/mL) with 1× TBE buffer (89 mM Tri s - 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 rem oved by the RNase - A. The concentration of DNA in solution was determined by a PicoGreen kit (Invitrogen ) using a spectro fluorometer (Bio - TEK, FLX800).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information DNA Digestion and Electrophoretic Separation of the DNA Fragments Genomic DNA extracted from the soybean leaf tissue was digested with restriction enzy mes by combining approximately 10  g of ge nomic 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 37 o C overnig ht. The positive control sample wa s 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 res tr iction 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 BioS ystems) and re suspended 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 mem branes 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 c itrate) 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 us ing a PCR - based incorporation of a digoxigenin (DIG) labeled nucleotide, [DIG - 11] - dUTP, into the DNA fragments generated by primers specific to the gene tic elements and ot her regions from plasmid pDAB8264 . The PCR synthesis o f the probes was performed usin g 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 Diagnostic s). DIG - labeled DNA molecular weight marker II and VII were used to determine the size of the hybridizing fragment s 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 f or ~1 hr at room temperature. The anti - DIG antibody specifically bound to the probes was then visualized using CDP - Star Chemiluminescent Nucleic Acid Detecti on System (Roche Diagnostics). Blots we re exposed to chemiluminescent film for one or more time points to detect the hybridizing fragments and toUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information visualize t he 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 la beled probes from the membranes, allowing them to be re hybridized with a different DNA probe.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Appendix 2 . Methods and Results for Characterization of AAD - 12 Protein DAS - 444Ø6 - 6 transgenic soybean material Greenhouse - grown DAS - 444 Ø 6 - 6 soybean plants ( T 4 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 a n immunospecific rapid lateral flo w test strip according to th e 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 back ground representative of the DAS - 444 Ø 6 - 6 soybean plants, but did not contain the aad - 12 gene. Absence of AAD - 12 expression in t he 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 - 444 Ø 6 - 6 soybean. Reference material Recombinant AAD - 12 protein was produced in Pseudomonas fluorescens and purified to a lyophilized powder . The microb e - deri ved 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 e xtracted 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 pla nt material by centrifugation. The supernatant was slowly adjusted to 1 M ammonium sulfate and insoluble proteins w ere 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 EL I SA ) were pooled. The pooled fraction was then incubated with an anti AAD - 12 immunoaffinity resin which had been conjugated with an AAD - 12 specific monoclonal antibody using a crosslinked immuno - pre cipitation kit (Thermo - Pierce). Bound proteins were eluted according to the manufacturer‘s protocols. The unbound fraction was re - in cubated with the resin and a second pool of protein was collected. Eluted fractions were analyzed by SDS PAGE (stained with GelCode Blue total protein stain from Thermo - Pierce), western blot, and ELISA . Fractions containing the soybean - derived AAD - 12 prot ein were pooled, desalted into 50 mM ammonium bicarbonate utilizing a PD - 10 desalting column (GE Healthcar e), aliquoted, and lyophilized. SDS - PAGE and western blot analysis of crude soybean leaf extracts Fresh leaf tissue from event DAS - 444 Ø 6 - 6 and Maveric k was mixed with a Tris - based buffer containing ~3 % protease inhibitor cocktail (Sigma) and the soluble protein s were extracted byUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information grindi ng with ball bearings in a Geno/ Grinder (Certiprep, Metuchen, NJ) for 3 minutes . The samples were centrifuged and the s upernatants were mixed with Laemmli sample buffer, heated at 95 °C for 5 minutes , and briefly centrifuged. The positive and negative reference standard s , microb e - derived AAD - 12 and bovine serum albumin, respectively , were also mixed with sample buffer and p repared the same as the plant extracts . The samples were loaded directly on to two separate Bio - Rad Criterion SDS - PAGE gel s and the proteins were electrophoresed with MES running buffer (Bio - Rad) for ~ 60 minutes at 150 volts . Following electrophoresis, on e 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, t he nitrocellulose membrane was blocked with 5% dry milk in PB ST and probed with an AAD - 12 sp ecific 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 s amples were adjusted to concentrations approximately equal to that of the plant - derived AAD - 12 protein and mixed w ith Laemmli buffer. The proteins were heated at 95 °C for 5 minutes , centrifuged, and applied directly to a B io - Rad Criterion SDS - PAGE gel. Fol lowing electrophor esis, the gel was cut in half. One gel half was stained with Thermo Pierce GelCode Blue stai n for total protein. The remaining half of the gel was stained with GelCode Glycoprotein Sta in to visualize glycoproteins. The glycoproteins prese nt on the gel were visualized as magenta ba nds on a light pink background. Mass spectrometry peptide mass fingerprinting and sequence analysis of plant - and microbe derived AAD - 12 protein The immunoaffinity purified AAD - 12 plant - deriv ed protein was subject ed 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 microb e - derived pr otein . 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 approx imately 32 kDa ( Figure 45 ). As expected, the corresponding plant derived AAD - 12 protein was identical in size t o the microbe - derived protein. Predictably, the protein fractions purified from DAS - 444Ø6
- 6 tissue cont ained a minor amount of non immunoreactive impurities in a ddition 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 - 444 Ø 6 - 6 pla nt tissue extract showed a positive signal of the expe cted size on the w estern blot using an anti - AAD - 12 polyclonal antibody ( Fi gure 46 ). InUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information the AAD - 12 w estern 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 samp les from the transgenic plant. Lack of alternate size proteins in the AAD - 12 sample derived from DAS - 444Ø6
- 6 soybean tissue in the western analys is 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 - 444 Ø 6 - 6 soybean - and mic rob e - derived AAD - 12. Note: Affinity - purified soybean - derived AAD - 12 and microbe - derived AAD - 12 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. The protein fractions purified from DAS - 444Ø6
- 6 tissue contained a minor amount of non - immunoreactive impurities in a ddition 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 ngUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Panel A. SDS - PAGE Panel B. Western blot.

Fi gure 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 - der ived AAD - 12 and bovine serum albumin. Following electrophoresis the gel was cut in half, one half was stained with Thermo Pierce GelCo de Blue stain for total protein and the remaining half was electro - blotted onto a nitrocellulose membrane. The nitrocellul ose 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 Amo unt Loaded M Novex Sharp Unstained MW Marker 10 μL kDa Novex Pre - stained MW Marker 1 Non - transgenic (Maverick ) Leaf Extract 30 μL 1 Microb e - derived AAD - 12 (TSN030732 ) 1 ng 2 DAS - 444Ø6
- 6 Soybean Leaf Extract 30 μL 2 Bovine Serum Albumin (BSA) 1 ng 3 Microb e - 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 μLUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 h orseradish peroxidase, a glycoprotein, was used as a positive indicator for glycopr otein 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 - 444 Ø 6 - 6 , microb e - 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 μLUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Results of MALDI

- TOF MS and MALDI

- TOF MS/MS t ryptic and chymotryptic peptide mass fingerprints of DAS - 444Ø6 - 6 - derived AAD - 12 p rotein Following digestion of the DAS - 444 Ø 6 - 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 t he trypsin and chym otrypsin digestion s 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 (L 40 to L 45 ), two in the middle of the protein (Q 89 to Y 114 and S 125 to F 138 ), and one near the C - termi nus (I 211 to R 213 ) . 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 ) ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 differe ntiate the potential peptides after protease digestion. The numbers on the left and right sides indicate the amino acid residue numbers.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 49 . Overall sequence coverage of trypsin and chymotrypsin digests for DAS - 444 Ø 6 - 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 - t erminal peptide was detected with Met 1 intact and removed by an aminopeptidase. The peptide with Met 1 missing was acetylated at Ala 2 . 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 o f the soybean - derived AAD - 1 2 protein (immunoaffinity purified from DAS - 444Ø6
- 6 soybean ) were determined and compared with the sequence of the microb e - derived protein . The soybean - derived AAD - 12 pro tein sequences were determined by MALDI - TOF MS/MS . The chym otrypsin 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 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 31 . Summary of N - terminal sequence data of DAS - 444Ø6 - 6 soybean - and P. fluorescens - derived AAD - 12 protein . Source Expected N - terminal Sequence a 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 Sequence b,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 - Ac 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 4 Notes : a Expected N - terminal sequence of the first 27 amino acid residues of P. fluorescens - and soybean - derived AAD - 12. b Detected N - terminal sequences of P. fluorescens - and soybean - derive d AAD - 12 ( Appendix 2 ). c Numbers in superscript (R x ) indicate the amino acid residue number in the sequence. The N terminal amino acid sequence was confirmed by peptide mass fingerprinting and MS/MS sequencing. d Th e 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 - Ac etyl - 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: valineUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information These results demonstrate that the N - terminus of the AAD - 12 protein was intact and the amino acid sequence was as predict ed ( 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 fre quently with eukaryotic (plant) expressed proteins as approximately 80 - 90 % of the N - terminal residue s 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 m ethionine 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 acetyl ated ( Polevoda and Sherman, 2002 ) . The two co - translati onal processes, cleavage o f N - terminal methionine residue and N - terminal acetylation, are by far the most common modifications and occur on the vast majority (~85%) of eu karyotic proteins ( Polevoda and Sherman, 2002 ) . However, e xamples demonstrating biological significance associated with N ter minal acetylation are rare ( Polevoda and Sherman , 2000 ) . The C - terminal sequence of the soybean - and microbe - derived AAD - 1 2 protein s were determined essentially as described above and compared with the expected amino acid sequence s. The results indicated the detected sequences were identical to the ex pected 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 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 32 . Summary of C - terminal sequence data of DAS - 444 Ø 6 - 6 soybean - and P. fluorescens - derived AAD - 12 protein . Source Expected C - terminal Sequence a P. fluorescens 281 L A G R P E T E G A A L V 293 Soybean Event DAS - 444 Ø 6 - 6 281 L A G R P E T E G A A L V 293

Source Detected C - terminal Sequence b P. fluorescens 281 L A G R P E T E G A A L V 293 Soybean Event 281 L A G R P E T E G A A L V 293 DAS - 444 Ø 6 - 6

a Expected C - terminal sequence of the last 13 amino acid residue s of P. fluorescens - and soybean - derived AAD - 12. b Detected C - terminal sequences of P. fluorescens - and DAS - 444 Ø 6 - 6 - derived AAD - 12 . Notes : Numbers in superscript (R x ) indicate amino acid residue numbers in the sequence. Am ino acid residue abbreviations: A: alanine E: glutamate G: glycine L: leucine P: proline R: arginine T: threonine V: valineUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 Met 1 ) with peptide mass fingerprint data and 94.2% by in source decay MS/MS dat a. The N - terminal Met 1 was removed by an aminopeptidase. Conclusions The biochemical characteristics of mi crobe - derived AAD - 12 protein were equivalent to the protein purified from root tissue of event DAS - 444 Ø 6 - 6 . The plant - and microbe - derived AAD - 12 prot eins showed the expected molecular weight of ~32 kDa by SDS - PAGE and were immunoreactive to AAD - 12 protein specific antibodies by w estern blot analysis. The amino acid sequence of both proteins was confirmed by enzymatic pepti de mass fingerprinting by MALD I - 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 - 444 Ø 6 - 6 soybean are biochemically equivalent.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information App endix 3 . Methods and Results for Characterization of 2mEPSPS Protein DAS - 444Ø6 - 6 transgenic soybean material Greenhouse - grown DAS - 444 Ø 6 - 6 soybean plants ( T 4 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 r apid 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 h ad a genetic background representative of the DAS - 444 Ø 6 - 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 pla nts 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 powd er 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 e xtract 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 Hi Trap 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 weste rn 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 assayedUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information for 2mEPSPS content by Coomassie staining a SDS - PAGE gel. The fraction co ntaining the highest level of 2mEPSPS by visual inspection was concentrated to ~130 μ L 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 analy ses . 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 - 444 Ø6
- 6 and nontransgenic isoline were harvested fresh from the greenhouse on the day of testing . Extracts were prepared by grinding the tissue wit h 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 refere nce standard (microbe - derived 2mEPSPS - TSN0331 71 - 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 fitt ed 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 GelC ode 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 (Lo t #: 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 i mmunoreactive 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 34 . Western blot extraction buffe r . 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 i soline 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 resultin g supernatants were loaded directly on the gel with a reference standard, 2mEPSPS (TSN033171 - 0001), and control sta ndard, BSA (Thermo - Pierce). SDS - PAGE and western blot analysis of the recombinant 2mEPSPS, BSA, non - transgenic Maverick, and DAS - 444Ø6
- 6 soyb ean 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 nitrocellul ose membrane and probed with a 2mEPSPS - specific mouse monoclonal antib ody (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 Healthc are 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. S oybean trypsin inhibitor, bovine serum albumin, an d 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 -USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information PAGE gel and electrophoresed as described above. Following electrophor esis, the gel was cut in half and o ne half of the gel was stained with Thermo Pierce GelCo de 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 ba nds on a light pink backgr ound. Mass spectrometry peptide mass fingerprinting and sequence analysis of plant - and microbe derived 2mEPSPS proteins The purified plant - deriv ed 2mEPSPS protein was subjected to in - gel and in - solution digestion by trypsin and chymotrypsin followed by ma trix 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 m icrobe - derived pr otein . Results of the SDS - PAGE and western b lot a nalysis 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 t o the microbe - derived protein. Predictably, the plant purified fractions contained a minor amount of non immunoreactive 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 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 51 . SDS - PAGE of DAS - 444Ø6 - 6 - and microbe - derived 2mEPSPS. Note: The p urified soy bean - 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 Nove x 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 ngUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Western blot The microbe - derived 2mEPSPS and DAS - 444 Ø 6 - 6 plant tissue extract showed a positive signal of the expected size on the western blot using an anti - 2mEPSPS polyclonal ant ibody ( 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 immunoreactiv e 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 bot h the polyclonal and monoclonal western blot analyses, no alternate size proteins (aggregates or degradation products) were s een 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.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Panel A. Polyclonal antibody western blot Panel B. Monoclonal antibody western blot

Figure 52 . Western blo t analysis of DAS - 444Ø6 - 6 soybean - and microbe - derived 2mEPSPS protein. Note: Crude extracts from leaf tissue of event DAS - 444 Ø 6 - 6 and Maverick were separated by SDS - PAGE along with microbe derived 2mEPSPS and bovine serum albumin. The nitrocellulose membrane s w ere then probed with 2mEPSPS spe cific polyclonal and monoclonal antibodies and detecte d with HRP - labeled antibodies. A chemiluminescent substrate was used to visua lize 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 D AS - 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 μLUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Results of detection of glycosylation o f 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 simulta neously 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 microb e - derived 2mEPSPS proteins had no detectable covalently linked carbohydrates ( Figure 53 ).

Note: The Novex Sharp prot ein 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 - 444 Ø 6 - 6 - deriv ed 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 staine d 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) ~50 0 ng 4 DAS - 444Ø6
- 6 derived 2mEPSPS ~500 ng 5 P. fluorescens derived 2mEPSPS ~500 ng P N ovex Pre - stained MW Marker 10 μLUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 mixture s were analyzed by MALDI - TOF and MALDI - TOF MS /MS to determine the peptide sequenc es 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) fr eeware 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 (S 65 to K 70 and A 75 to K 83 ), three in the middle of the protein (V 249 to Y 258 , A 287 to K 296 , and A 321 to K 328 ) , and one near the C - terminus (M 405 to R 422 ) . 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 ) a t 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 t he amino acid sequence of the soybean - derived 2mEPSPS protein was equivalent to the P. fluorescens - expressed protein characterized earlier.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Digestion at K (lysine) and R (arginine)

Digestion at F (phenylalanine), L (leucine), W (tryptophan), and Y (ty rosine)

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 t o differentiate the potential peptides after protease digestion. The numbers on the left and right sides indicate the amino acid residue numbers.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 55 . S equence coverage of trypsin and chymotrypsin digests for DAS - 444 Ø 6 - 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 .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 compar ed with the sequence of the previously characterized microbe - derived protein . The soybean - derived 2mEPSPS pro tein sequences were determined by MALDI - TOF MS/MS. The chymotrypsin and trypsin digestions were performed on the soybean - derived 2mEPSPS protein fo llowed 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 ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 35 . Summary of N - terminal sequenc e data of DAS - 444Ø6 - 6 soybean - and P. fluorescens - derived 2mEPSPS . Source Expected N - terminal Sequence a 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 Sequence b 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. b Detected N - terminal sequences of P. fluorescens - and soybean - derived 2mEP SPS. Numbers in superscript (R x ) indicate amino acid r esidue numbers in the sequence. Am ino 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: va lineUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information The C - terminal sequence of the soybean - and microbe - derived 2mEPSPS protein s was determined essentially as described above and compared with the expected amino acid sequence s. The results indicated the measured sequences were identical to the expect ed sequences, and both the soybean - and microbe - derived 2mEPSPS proteins were indistinguishable and unaltered at the C - t erminu s ( Table 36 and Figure 54 ). Table 36 . Summary of C - terminal sequence d ata of DAS - 444Ø6 - 6 soybean - and P. fluorescens - derived 2mEPSPS . Source Expected C - terminal Sequence a

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 Sourc e Detected C - terminal Sequence b

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. b Detected C - terminal sequences of P. fluorescens - and soybean - derived 2mEPSPS . Numbers in superscript (R x ) indicate amino acid r esidue numbers in the sequence. Am ino acid residue abbreviations: D: aspartic acid F: phenylalanine K: lysin e L: leucine N: a sparagine 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. f luorescens . 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 antibo dies by western blot analysis. T he amino acid sequence of both proteins was co nfirmed 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 2m EPSPS protein provided additional evidence thatUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information the 2mEPSPS protein produced by P. fluorescens and DAS - 444 Ø 6 - 6 soybean are biochemically equivalent.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 c ontrol 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 a bsence 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 homogen eity by GeneScript (Piscataway, NJ

– Identification number: 55238) . The E . coli - derived PAT protein preparation was aliquoted and stored at - 80 oC to maintain activity . SDS - PAGE and Western Blot Analysis of Crude Extracts Lyophilized tissue from event DAS - 4 44Ø6
- 6 and Maverick was mixed with a PBST based buffer containing ~2.0% protease inhibitor cocktail (Sigma) and the soluble protein s were extracted by grinding with ba ll 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) . T he positive reference standard ( E. coli - derived PAT ) and negative reference s tandard (BSA) were also mixed with sample buffer and t he resulting supernatants were loaded directly on to a Bio - Rad Criterion SDS - PAGE gel and e lectrophoresis was conducted with T ris/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 o nto a nitrocellulose membrane. The nitrocellulose membrane was then cut in half with one half probed with a PAT specific polyclonal rabbit antibo dy and the remaining half probed with a PAT specific monoclonal ant ibody. The antibodies were detected with HRP labeled secondary antibodies. A chemiluminescent substrate (GE Healthcare) was used to visua lize the immunoreactive bands. Results of the SDS - PA GE 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 soy bean - derived PAT protein was visualized by immunospecific polyclonal and monoclonalUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information antibodies and showed the expected band at approximately 21 kDa ( Figure 58 ). In the PAT western blot analysis, no immunoreactive p roteins were observed in the control Maverick extract and no alternate size proteins (aggregates or degradation products) were seen in t he transgenic soybean extract. The monoclonal antibody did detect a small amount of a dimer in the mi crobe derived PAT p reparation. These result s 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 P AT protein standard (TSN031116 - 0001) 1.2 μ g 4 Bovine serum albumin (BSA) 1.5 μ gUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Figure 58 . Western blot analysis of DAS - 444Ø6 - 6 and Maverick soybean extracts. Note: Crude extracts from lyophilized leaf tissu e 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 detecte d 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 N on - 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 μLUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 ) .USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Appendix 5 . Methods for AAD - 12, 2mEPSPS and PAT Protein E xpression 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 a s 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 uniq ue ra ndomization at each site. Each soybean plot was bordered by 2 rows of a non - transgeni c soybean of similar maturity. The entire trial site was surrounded by a minimum of 1 0 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 rate s : 2,4 - D only Treatment: 2,4 - D (Weedar 64) was applied as three b roadcas t applications to DAS - 444Ø6
- 6. Application timing was at planting / pre - emergence, and a pproximately V3 and R2 stages. Individual target application rates were 1.0 lb ae (acid equivalent)/A f or 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 a pproximately 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 broadcas t applications to DAS - 444Ø6
- 6. Individual applications were at planting / pre - emergence, and approxim ately V3 and R2 stages. Individ ual 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 app lications to DAS - 444Ø6
- 6. Individua l applications were at planting / pre - emergence, and a pproximately V3 and R2 stages. Individual target application rates were 1.0 lb ae/A f or 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 broadcas t applications to DAS - 444Ø6
- 6. Application timing was at a pproximately V5 and R1 stages. The target application rate was 0.33 lb ai/A for Liberty, or 374 g ai/h a. Sample Collection Samples were shipped to Dow AgroSciences Regulatory Science s and Government Affairs laboratories an d 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 (Ce rtip rep, Metuchen, NJ ).USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information 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 f

or each test and control entry. Each leaf sample was the youngest set of fully expanded trif

oliate 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 fro m each test and control entry. d. Grain (R8

– Maturity) One individual sample was collected from each plot o f each test and control entry. Eac h sample contained approximately 500 gram s 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 centrif uged; the aqueous supernatant was collected, diluted with appropriate buffer if necessary, and analyzed using an AAD - 1 2 ELISA kit. Briefly, an aliquot of the diluted sample and a horseradish peroxidase (HRP)/anti - AAD - 12 monoclonal antibody conjugate were i ncubated 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 a nd the immobilized antibodies. The unbound samples and excess conjugate were then removed from the plate by washing with PBST. Subsequent addition of an enzyme substrat e 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 resul t 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 s amples was calculated from the polynomial regression equation using Soft - MAX ProTM or Triturus Version 4.01B software which was com patible 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 bufferedUSDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information s aline 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 aliq uot 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 s pecific 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 incubatio n period, the unbound reagents we re removed from the plate by washing with PBST. Subsequent addition of an enzyme substrat e 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 th e absorbance of the solution, was related to the concentration of 2mEPSPS in the sample (i.e., lower protein concentrations resul t in lower color development). The absorbance at 450 nm nm with a background subtraction at 650 or 620 nm was measured using a Molec ular 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 Pro TM or Triturus Version 4.01B software which was com patible 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 met hod 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 w as 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 enzyme conjugated anti - PAT monoclonal antibody and anti - PAT polyclonal antibod ies coated in the wells of a 96 - well pla te in a sandwich ELISA format. At the end of the incubation period, the unbound reagents were remo ved from the plate by washing. Subsequent addition of an enzyme substrate generated a colored produ ct. The reaction wa s 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 residu e concentrations resu lt in lower color development). The absorbance at 450 nm with a background subtraction at 650 or 620 nm was measured usi ng a Molecular Devices Spectra M ax M2 plate reader or a Grifols Triturus Automated Immunoassay Analyzer. A calibrat ion 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 duplica te wells with the average concentration of the duplicate wells being reported.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Appendix 6 . USDA Notifications for DAS - 444Ø6 - 6 Soybean USDA Notification Number Notification

Authorization Date Notific ation

Expiration Date State(s) Total Number of Trials Planted Status of

Trial 1 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 noti fication expiration date.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Appendix 7 . Literature Ranges for Compositional Analysis Table 37 . Literature ranges reported for soybean seed: proximates, fiber, and minerals.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Tabl e 38 . Literature ranges reported for soybean seed: amino acids.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 39 . Literature ranges for soybean seed: fatty acids.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 40 . Literature ranges reported for soybean seed: vitamins.USDA Petition for Nonregulated Status of DAS - 444Ø6
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Contains No Confidential Business Information Table 41 . Literature ranges for soybean seed: bioactives.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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 a nd for capturing market share. This was soon realized with the development of glyphosate - a nd glufosinate - tolerant crops. S ince the transgenic crops would tolerate the application of thos e 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 herbici de. 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 - tole rant crops are no longer s old. 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. Herbi cide Crop Year Available Bromoxynil Cotton b 1995 Canola b 2000 Cyclohexanediones (sethoxydim) a , b Corn 1996 Glufosinate Canola 1995 Corn 1997 Cotton 2004 Glyphosate Soybean 1996 Canola 1996 Cotton 1997 Corn 1998 Imidazolinones a Corn 1993 C anola 1997 Wheat 2002 Rice 2002 Sulfonylureas a Soybean 1994 Triazines a Canola 1984 a Not transgenic, b No longer available by 2005USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Growers choose glyphosate - tolerant crops because they make weed control easier and more effective, increase profit, req uire less tillage, and do not restrict crop rotations ( Green, 2009 ) . Thus, glyphosate - tol erant corn and soybeans have experienced an unprecedented rapid adoption rat e by U.S. farmers ( Fig ure 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 . ).

Fig ure 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 f or 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 veget ation control is desired (aquatics, rights of way, industrial sites, etc.) ( US EPA, 1993 ) . When applied at lower rates, glyphosate als o 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 aro und the globe, including alfalfa, barley, buckwheat, corn, dry beans, grassUSDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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 c rops, 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 s usceptibility between species. Once in the plant, glyphosate moves in the phloem wi t h 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 s edge weeds in agronomic crops. The mode of action for glyphosate is unique in that it is the only her bicide 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 tryptopha n, 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 environmentall y 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 (Monsant o, 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 metab olized 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 ani mals ( Duke and Powles, 2008a ) . The enzyme, EPSP synthase, is not present in humans or animals, which contributes to the l ow risk to human health when glyphosate is used according to labe l 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 w idely 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 mill ion pounds (34%) used in non - agriculture settings such as pasture/rangeland a nd 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 herbic ides because it economically enhances the weed control spectrum of many other herbicides such as glyphosate, dicamba, mecoprop, ALS h erbicides, etc ( US EPA, 2006 ) . 2,4 - D controls many broadleaf weeds including carpetweed, dandelion, cocklebur, horseweed, morning glory,USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information pigweed sp., lambsquarters, ragwee d spp., sh epherd‘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 f

or health and safety as 2,4 - D. There have been more than 40,0 00 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 re regi stration assessment of 2,4 - D. T he EPA concluded that 2,4 - D does not present risks of concern to human health when users follow its product instructio ns ( 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 rate s increase, they can cause various growth abno rmalities 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 fo rmati on, 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 a uxin 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 facto r (ARF) dependent transcrip tion 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). P lant metabolism of 2,4 - D typica lly 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 -USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information selective herbicide and is the active ingredient of the commercial herbicide formulations, Liberty and Ignite. The L - isome r of glufosinate is a structural analogue of glutamate and, therefore, is a competitive inhibitor of the enzyme glutamine synthetas e (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 - met hylphosphinico butanoic acid). N - acetyl - L - glufosinate does not inhibit glutamine synthetase. Therefore, no phytotoxic physiol ogical 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 perennia l grasses and broadleaf weeds. Due to its limited systemic action, there is no endu ring 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 CO 2 and natural phosphorus compounds. Evolution of Resistance to Glyphosate, 2,4 - D, and Glufos inate 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 m et abolic 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), c otton (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.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Table 43 shows a summary of the twenty - one glyphosate - resistant weed species that have been reported from 1996 to November 20 10 ( Heap, 2011 ) . The data clearly show s that glyphosate resistance in w eeds 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 a lready become a significant problem for farmers a cross 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 co rn 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 Reports Common Name Species Name Year Co untry** 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 multifloru m 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 trifi da 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 P araguay 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 Su matran 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.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Figure 60 . Number of glyphosate - resistant weeds reported globally by year from 1996 to 20 10 . (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 2 0 # of Gly - Res Weed SpeciesUSDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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 c anadensis 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

– C hile 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, U reas, I soxazolidiones 2010

– Australia Bip yridiliums 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 field s 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 g lyphosate - 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 p revalent. 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 .USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Table 45 . Potential weed shifts with use of glyphosate in U.S. co rn 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 Sesban ia 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 Trida x 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 w eed 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. mai nland ( Heap, 2011 ) . Wild carrot, yellow bur - head, wild radish, musk thistle, and corn poppy are the o nly 2,4 - D resistant weeds that have reported infestat ions 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 s ome ALS - inhibiting herbicides. It is notable th at most of these resistant species do not appear to be spreading, as indicated by few reports of additional sites after the initial report.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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 Michiga n 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 U nited 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 arv ensis 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, bensulfuron methyl 1998 Malaysia Italian thistle Carduus pycnocephalus 2,4 - D 1997 New Ze aland 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, metsulfuron methyl 2005 Australia Prickly lettuce Lactuca serriola 2,4 - D, dicamba, MCPA 2 007 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 wi th 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 res is tance 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 ) T he mechanism of resistance to auxinic herbicides has been investigated, in varying degrees for only a fe w of these resistant biotypes. Resistance mechanisms in these biotypes have proven toUSDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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 herbicide s are not yet well understood. Further r esearch is needed across a range of resistant species and biotypes to identify the potentially numerous gene mu tations that cause resistance. It is also possible, but less likely, that a biotype might be resistant to all of these auxin herbicides. Due to t he 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 structur e) or clopyralid (picolinate structu re). 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 be en found in corn fields to date. Use of 2,4 - D in DAS - 444Ø 6 - 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 herbicide s 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.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Appen dix 9 . St ewardship of Herbicide Tolerant DAS - 444Ø6 - 6 Soybean 1. Introduction DAS - 444Ø6
- 6 s oybean is a transgenic soybean product that provides tolerance to 2,4 - dichlorophenoxyacetic acid (2,4 - D) glyphosate and glufosinate. This herb icide - 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 appropria te 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 responsi ble 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 Spec ific goals of Dow AgroSciences‘ c omprehensive 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, r etailers, 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 t his 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 he rbicide or herbicides that would nor mally 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 b ecomes dominant in a given area, and 3). Hard - to - Control Weed : A weed species that is inherently able to withstan d treatment with a herbicide as a result of natural factors not involving herbicide selection or genetic mutation.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information To meet the above - described stewardship goals, Dow AgroScienc es 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 technolog ies,  Compre hensive 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 g rower a greement that provides the terms and condi tions for the authorized use of the technology. The g rower a greement 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 develo ped 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 prod ucts with ultra low volatility and minimiz ed potential for physical drift. An exceptionally low volatility of 2,4 - D choline salt has been demonstrated in both laboratory studies and preliminary field studie s. 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 a t 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 ( Hill ger 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 .USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information ( Wilson et al. , 2010 ) . This premix product will reduce the potential for off - target movement from both particle drift and volatility. 3.3 Comprehen sive 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 p lanting 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 h erbicide (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. Certa in 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 population s 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 an d/or other cultural prac tices. 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 reco mmends the following practices for her bicide selection, crop selection and cultural practices:USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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, tan k 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, s uch as dela yed 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 lea ving 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 r epeated 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 O ff - Target Movement The new 2,4 - D choline containing he rbicide 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 minimi ze 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 a pplicators to evaluate factors and make ap propriate adjustments when applying 2,4 - D choline salt for use with DAS - 444Ø6
- 6 soybean consistent with the final productUSDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information label approved by EPA. Dow AgroSciences is conducting significant research on nozzles for spray application. The product use guide wi ll 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 Produ ct 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 - tar get 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 Bag s and Tags Seed b ags 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 g rower a greement, as well as a c ustomer i nformation t elephone 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 gui de 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 el ements of the herbicide label. Dow AgroSciences wi ll 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 Lite rature and Direct Mailings Dow AgroSciences will also p osition the herbicide tolerant trait technology and p romote its responsible use in its product literature by including crop agronomy, weed management, herbicide use and application information . Dow Agr oSciences commonly uses direct mailings to customers, and will promote responsible stewardship of the herbicide tolerant trait technology through n ewsletters and correspondence. These direct mailings will emphasize responsible useUSDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information practices, label complian ce, weed resistance management, and application guidance to minimize the pote ntial for off

- target movement. 3.3.5 Internet Resources Information on the technology including agronomic and proper product placement, specific guidelines for planting transgenic trait s, and herbicide use and application will be made available on technology provider websites. Relevant i ndustry and university links may also be included to provide the user with additional technical information. Dow AgroSciences provides product stewardshi p 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 a nd training retailers, growers and applicators on the proper use of the technolo gy 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 e quipment for field conditions. Dow AgroSciences will communicate to customers the importance of stewardshi p and responsible product use. Dow AgroSciences will utilize a variety of approaches to ed ucate and train retail ers, 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 pla ns 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 bulle tins, sales literature, direct mailings, and websites. Education topics for r etailers, g rowers and a pplica tors will include: DAS - 444Ø6
- 6 soybean trait technology (including herbicide tolerance and agronomic practices), 2,4 - D choline herbicide solutions (in cluding label education, weed resistance management and herbicide application practices to minimize off - t arget movement). As an example, w eed 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  Appropri ate cultural and mechanical practices for weed control and weed resistance management.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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 DAS 444Ø6
- 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 resp onsible 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 grow er‘s access to the technology. The grower agreemen t 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 her bicide applied to these fields. In addition, the grower w ill 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 thi s product stewardship program. Dow AgroScience s is consulting with retailers, growers and applicators to understand and address their needs in order to develop an effective and comp rehensive 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 processor s of non - target crops. These efforts will support implementation of stewardship practices for this technology as broadly as possible throughout the value chain. Dow AgroScien ce s will evaluate additional feedback from r etailers, g rowers and a pplicators for the continuous improvement of th is s tewardship p rogram following the launch of DAS - 444Ø6
- 6 soybean.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 5. Industry Commitment and Involvement Dow AgroSciences participates i n several organizations and associations globally to promote the safe research and development, production, distribution, and responsible use of agricultural chemic al 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/resi stance_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 pr ivate researchers, especially in the areas of problem identification and devising and implementing he rbicide management strategies. Dow AgroSciences personnel interact with academic weed scientists in addressing w eed r esistance m anagement [ http://www.wssa.net/Weeds/Resistance/ ] issues. Dow AgroSciences conducts joint trials at university sites as well as seeking input from university research ers 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 go vernment 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 produc t‘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 t echnology, 2,4 - D choline salt. The stewardship program will focus on educating and training retailers, growers and applicators on the appropria te use of this new technology. This will be accomplished by using a multi - faceted approach, includ ing use of a variety of tools and delivery methods, and working with customers, stakeholders and industry organizations to promote responsible use of the technology .USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information 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. Ahrent, D.K., Caviness, C.E., 1994. Natural cross - pollination of twelve soybean cultivars in Arka nsas. Crop Science 34, 376 - 378. Bandeen, J.D., Stephenson, G.R., Cowett, E.R., 1982. Discovery and distribution of herbicide resistance weeds in North America. In: LeBaron, H.M., Gressel, J. (Eds.), Herbicide resistance in plants. John Wiley & Sons, Rehovo t, Israel, pp. 9 - 30. Barker, R.F., Idler, K.B., Thompson, D.V., Kemp, J.D., 1983. Nucleotide - sequence of the T DNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Molecular Biology 2, 335 - 350. Beard, B.H., Knowles, P.F., 1971. Frequency of cross - pollination of soybeans after seed irradiation. Crop Science 11, 489 - 492. Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate - a practical and powerful approach to multiple testing. Journal of the Royal Statistical Society Series B Methodological 57, 289 - 300. Berman, K.H., Harrigan, G.G., Riordan, S.G., Nemeth, M.A., Hanson, C., Smith, M., Sorbet, R., Zhu, E., Ridley, W.P., 2009. Compositions of seed, forage, and processed fractions from insect - protected soybean MON 87701 are equivalent to those of conventional soybean. Journal of Agricultural and Food Chemistry 57, 11360 - 11369. Berman, K.H., Harrigan, G.G., Riordan, S.G., Nemeth, M.A., Hanson, C., Smith, M., Sorbet, R., Zhu, E., Ridley, W.P., 2010. Compositions of fo rage and seed from second - generation glyphosate - tolerant soybean MON 89788 and insect - protected soybean MON 87701 from brazil are equivalent to those of conventional soybean ( Glycine max ). Journal of Agricultural and Food Chemistry 58, 6270 - 6276. Bilyeu, K .D., Zeng, P., Coello, P., Zhang, Z.J., Krishnan, H.B., Bailey, A., Beuselinck, P.R., Polacco, J.C., 2008. Quantitative conversion of phytate to inorganic phosphorus in soybean seeds expressing a bacterial phytase. Plant Physiology 146, 468 - 477. Bourdot, G .W., Saville, D.J., Hurrell, G.A., 1996. Ecological fitness and the decline of resistance to the herbicide MCPA in a population of ranunculus acris. Journal of Applied Ecology 33, 151 - 160. Bradshaw, L.D., Padgette, S.R., Kimball, S.L., Wells, B.H., 1997. P erspectives on glyphosate resistance. Weed Technology 11, 189 - 198. CAST, 2009. Sustainability of U.S. Soybean production: Conventional, transgenic, and organic production systems. Council for Agricultural Science and Technology. http://www.cast science.org/publications/ . Caviness, C.E., 1966. Estimates of natural cross - pollination in Jackson Soybeans in Arkansas. Crop Science 6, 211 - 212. Chaboute, M. - E., Chaubet, N., Philipps, G., Ehling, M., Gigot, C. , 1987. Genomic organization and nucleotide sequences of two histone H3 and two histone H4 genes of Arabidopsis thaliana . Plant Molecular Biology 8, 179 - 191. Coble, H.D., Warren, L.S., 1997. Weed control investigations in corn, cotton, crop rotations, soyb ean, small grain. Annual Report. Department of Crop Science. North Carolina State University, NC. 28, 103 - 113.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Codex Alimentarius Commission, 2009. Foods derived from modern biotechnology. www. fao.org/docrep/011/a1554e/a1554e00.htm . Coll, A., Nadal, A., Palaudelmàs, M., Messeguer, J., Melé, E., Puigdomènech, P., Pla, M., 2008. Lack of repeatable differential expression patterns between MON810 and comparable commercial varieties of maize. Plant Molecular Biology 68, 105 - 117. Conner, A.J., Glare, T.R., Nap, J.P., 2003. The release of genetically modified crops into the environment. Part II. Overview of ecological risk assessment. The Plant Journal 33, 19 - 46. Cranston, H.J., Kern, A.J., Hackett, J. L., Miller, E.K., Maxwell, B.D., Dyer, W.E., 2001. Dicamba resistance in kochia. Weed Science 49, 164 - 170. Culpepper, A.S., Whitaker, J.R., MacRae, A.W., York, A.C., 2008. Distribution of glyphosate resistant palmer amaranth ( Amaranthus palmeri ) in Georgia and North Carolina during 2005 and 2006. The Journal of Cotton Science 12, 306 - 310. Curran, B., Sprague, C., Stachler, J., Loux, M., 2007. The glyphosate, weeds, and crops series: Biology and management of common lambsquarters (GWC - 11). http://www.glyphosateweedscrops.org/Info/GWC - 11.pdf . Dalley, C.D., Renner, K.A., Kells, J.J., 2002. Weed competition in Roundup Ready soybeans and corn. http://msue.anr .msu.edu/ . Dietz, K., 2010. DHT - managing the performance, minimizing the risk. Proceedings of the 2010 North Central Weed Science Society 65, http://ncwss.org . Duan, J.J., Lundgren, J.G., Naranjo, S., Marvier, M., 2009. Ex trapolating non - target risk of Bt crops from laboratory to field. Biology Letters 6, 74 - 77. Duke, S.O. (Ed), 1996. Herbicide - resistant crops: Agricultural, environmental, economic, regulatory, and technical aspects. CRC Press, Inc., Boca Raton. Duke, S.O., 2005. Taking stock of herbicide - resistant crops ten years after introduction. Pest Management Science 61, 211 - 218. Duke, S.O., Powles, S.B., 2008a. Editorial: Glyphosate - resistant weeds and crops. Pest Management Science 64, 317 - 318. Duke, S.O., Powles, S .B., 2008b. Glyphosate: A once - in - a - century herbicide. Pest Management Science 64, 319 - 325. Dyer, W.E., 1994. Resistance to glyphosate. In: Powles, S.B., Holtum, J.A.M. (Eds.), Herbicide resistance in plants: Biology and biochemistry. CRC Lewis Publishers, New York, New York, pp. 229 - 241. Evans, S.L., 2004. Producing proteins derived from genetically modified organisms for toxicology and environmental fate assessment of biopesticides. In: Parekh, S.R. (Ed.), The GMO Handbook: Genetically modified animals, m icrobes, and plants in biotechnology. Humana Press, Inc., Totowa, NJ, pp. 53 - 83. FAO, 1997. Pesticide residues in food - glyphosate report. http://www.fao.org/docrep/W8141E/w8141e0u.htm . Foresman, C., Glasgow, L., 2008. US grower perceptions and experiences with glyphosate resistant weeds. Pest Management Science 64, 388 - 391. Franz, J.E., Mao, M.K., Sikorski, J.A., 1997. Glyphosate: A unique global herbicide. American Chemical Society, Washington, DC. Gaska, J., 2006. Soybean growth and development. http://soybean.uwex.edu/library/soybean/morphology/documents/04_soy_growth_develo pment.pdf .USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Givens , W.A., Shaw, D.R., Kruger, G.R., Johnson, W.G., Weller, S.C., Young, B.G., Wilson, R.G., Owen, M.D.K., Jordan, D., 2009. Survey of tillage trends following the adoption of glyphosate - resistant crops. Weed Technology 23, 150 - 155. Green, J.M., 2009. Evoluti on of glyphosate - resistant crop technology. Weed Science 57, 108 - 117. Gustafson, D.I., 2008. Sustainable use of glyphosate in north american cropping systems. Pest Management Science 64, 409 - 416. Hall, G., Jr., Allen, G.C., Loer, D.S., Thompson, W.F., Spik er, S., 1991. Nuclear scaffolds and scaffold - attachment regions in higher - plants. Proceedings of the National Academy of Sciences of the United States of America 88, 9320 - 9324. Hamby, S., Hirst, J., 2008. Prediction of glycosylation sites using random fore sts. BMC Bioinformatics 9, 500 - 512. Hammond, B.G., Jez, J.M., 2011. Impact of food processing on the safety assessment for proteins introduced into biotechnology - derived soybean and corn crops. Food and Chemical Toxicology 49, 711 - 721. Han, K.H., Ma, C.P., Strauss, S.H., 1997. Matrix attachment regions (MARs) enhance transformation frequency and transgene expression in poplar. Transgenic Research 6, 415 - 420. Harrigan, G.G., Glenna, K.C., Ridley, W.P., 2010. Assessing the natural variability in crop composit ion. Regulatory Toxicology and Pharmacology 58, S13 - S20. Harrigan, G.G., Ridley, W.P., Riordan, S.G., Nemeth, M.A., Sorbet, R., Trujillo, W.A., Breeze, M.L., Schneider, R.W., 2007. Chemical composition of glyphosate - tolerant soybean 40 - 3 - 2 grown in europe remains equivalent with that of conventional soybean ( Glycine max L.). Journal of Agricultural and Food Chemistry 55, 6160 - 6168. Hartwig, E.E., Kilen, T.C., 1991. Yield and composition of soybean seed from parents with different protein, similar yield. Cro p Science 31, 290 - 292. Hartzler, B., Pringnitz, B., 2005. Early - season weed competition. Department of Agronomy, Iowa State University, IA. http://www.ipm.iastate.edu/ipm/icm/2000 /5 - 8 - 2000/earlyweed.html . Hayes, R.M., 2000. Herbicide resistant crops and weed species shifts. http://www.btinternet.com/~nlpWESSEX/Documents/cottonrrproblems.htm . Heap, I. M., 1997. The occurrence of herbicide - resistant weeds worldwide. Pesticide Science 51, 235 - 243. Heap, I.M., 2011. International survey of herbicide resistant weeds. http://www.weedscience.org/In.asp . Herman, R.A., Phillips, A.M., Lepping, M.D., Fast, B.J., Sabbatini, J., 2010. Compositional safety of event DAS - 40278 - 9 (AAD - 1) herbicide - tolerant maize. GM Crops 1, 294 - 311. Herman, R.A., Storer, N.P., Phillips, A.M., Prochaska, L.M., Windels, P., 2007. Compositi onal assessment of event DAS - 59122 - 7 maize using substantial equivalence. Regulatory Toxicology and Pharmacology 47, 37 - 47. Herouet - Guicheney, C., Rouquié, D., Freyssinet, M., Currier, T., Martone, A., Zhou, J., Bates, E.E., Ferullo, J.M., Hendrickx, K., R ouan, D., 2009. Safety evaluation of the double mutant 5 - enol pyruvylshikimate - 3 - phosphate synthase (2mEPSPS) from maize that confers tolerance to glyphosate herbicide in transgenic plants. Regulatory Toxicology and Pharmacology 54, 143 - 153.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Hillger, D.E., Havens, P.L., Simpson, D.M., Braxton, B., 2010. Field methods for evaluation of herbicide volatility. Proceedings of the 2010 North Central Weed Science Society 65, http://ncwss.org . Hite, G.A., King, S.R., Hagood, E.S., Hol tzman, G.I., 2008. Differential response of a Virginia common lambsquarters ( Chenopodium album ) collection to glyphosate. Weed Science 56, 203 - 209. Holroyde, M.J., Chesher, J.M.E., Trayer, I.P., Walker, D.G., 1976. Studies on use of sepharose N - (6 - aminohex anoyl) - 2 - amino - 2 - deoxy - d - glucopyranose for large - scale purification of hepatic glucokinase. Biochemical Journal 153, 351 - 361. Hood, E.E., Helmer, G.L., Fraley, R.T., Chilton, M.D., 1986. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T - DNA. Journal of Bacteriology 168, 1291 - 1301. HRAC, 1998. Herbicide resistance action committee: Guidelines to the management of herbicide resistance. http://www.hracglobal.com/Publications/ManagementofHerbicideResistance/tabid/225/D efault.aspx#background . Huls, T.J., Erickson, G.E., Klopfenstein, T.J., Luebbe, M.K., Vander Pol, K.J., Rice, D.W., Smith, B ., Hinds, M., Owens, F., Liebergesell, M., 2008. Effect of feeding DAS - 59122 - 7 corn grain and nontransgenic corn grain to individually fed finishing steers. The Professional Animal Scientist 24, 572 - 577. ILSI, 2011. International Life Sciences Institute cr op composition database. www.cropcomposition.org . Industry Task Force II, 2005. 2,4 - D research data. Issue backgrounder: What is 2,4 - D? http://www.24d.or g/backgrounders/whatis.aspx . Information Systems for Biotechnology, 2011. A national resource in agbiotech information. http://www.isb.vt.edu/data.aspx . Iskander, F.Y., 1987. Maturation stage and mineral conte nt in soybeans. Food Chemistry 24, 29 - 35. Jacobs, C.M., Utterback, P.L., Parsons, C.M., Rice, D., Smith, B., Hinds, M., Liebergesell, M., Sauber, T., 2008. Performance of laying hens fed diets containing DAS - 59122 - 7 maize grain compared with diets containi ng nontransgenic maize grain. Poultry Science 87, 475 - 479. Jaehnig, K.C., 2005. Farmers facing rise of herbicide - resistant weeds. Southern Illinois University Carbondale. http://news.siuc.edu/ news/April04/042704kj4032.jsp . Jasieniuk, M., Brule - Babel, A.L., Morrison, I.N., 1996. The evolution and genetics of herbicide resistance in weeds. Weed Science 44, 176 - 193. Kelley, K.B., Riechers, D.E., 2007. Recent developments in auxin biology and new opportunities for auxinic herbicide research. Pesticide Biochemistry and Physiology 89, 1 - 11. Kennedy, J.F., Barnes, J.A., 1983. Immunochemical studies of the non - specific interactions of cyanogen - bromide activated macroporous agarose based immunoadsorbent s. Journal of Chromatography 281, 83 - 93. Kern, A.J., Chaverra, M.E., Cranston, H.J., Dyer, W.E., 2005. Dicamba - responsive genes in herbicide - resistant and susceptible biotypes of kochia ( Kochia scoparia ). Weed Science 53, 139 - 145. Knake, E.L., Slife, F.W., 1962. Competition of Setaria faberii with corn and soybeans. Weeds 10, 26 - 29.USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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Contains No Confidential Business Information Thill, C.D., Lemerle, D., 2001. World wheat and herbicide resistance. In: Powles, S.B., Shaner, D.L. (Eds.), Herbicide resistance and world grains. CRC Press LLC, Lawrenceville, NJ, pp. 165 - 194. Toms, A., Wood, J.M., 1 970. The degradation of trans - ferulic acid by Pseudomonas acidovorans . Biochemistry 9, 337 - 343. Tu, M., Hurd, C., Randall, J.M., 2001. Weed control methods handbook: Tools & techniques for use in natural areas. http://www.invasive.org/gist/handbook.html . US EPA, 1993. Glyphosate reregistration eligibility decision (RED). EPA - 738 - F - 93 - 011. www.epa.gov/oppsrrd1/REDs/factsheets/0178fac t.pdf . US EPA, 1997. Phosphinothricin acetyltransferase and the genetic matetial necessary for its production in all plants; exemption from the requirement of a tolerance on all raw agricultural commodities. Federal Register 62 (70): 17717 - 17720. http://www.gpo.gov/fdsys/pkg/FR - 1997 - 04 - 11/html/97 - 9373.htm . US EPA, 2006. Pesticide reregistration 2,4 - D RED facts. EPA - 738 - f - 05 - 002. http://www.epa.gov/oppsrrd1/REDs/factsheets/24d_fs.htm . USDA, 1996. Availability of determination of nonregulated status for soybeans genetically engineered for glufosinate herbicide tolerance. Federal Register 61 (160): 42581 - 42582. http://federalregister.gov/a/96 - 20921 . USDA, 1997. Availability of determination of nonregulated status for genetically engineered corn line. Federal Register 62 (234): 64350 - 64351. www.aphis.usda.gov/brs/aphisdocs2/97_09901p_com.pdf . USDA, 2001. Availability of determination of nonregulated status for corn genetically engineered for insect resistance and glufosinate herbicide tolerance. Fede ral Register 66 (157): 42624 - 42625. http://www.gpo.gov/fdsys/pkg/FR - 2001 - 08 - 14/html/01 - 20307.htm . USDA, 2004. Availability of determination of nonregulated status for cotton lines g enetically engineered for insect resistance. Federal Register 69 (156): 50154 - 50155. http://edocket.access.gpo.gov/2004/pdf/04 - 18524.pdf . USDA, 2005. Availability of determination of nonregu lated status for genetically engineered corn (corn line DAS - 59122 - 7). Federal Register 70 (194): 58663 - 58664. http://www.gpo.gov/fdsys/pkg/FR - 2005 - 10 - 07/html/05 - 20194.htm . USDA, 200 9. Determination of nonregulated status for cotton genetically engineered for glyphosate herbicide tolerance. Federal Register 74 (98): 23987 - 23988. http://edocket.access.gpo.gov/2009/E9 - 11972.ht m . USDA ERS, 2010. USDA Economic Research Service. Soybeans and oil crops. http://www.ers.usda.gov/Briefing/SoybeansOilCrops/ . USDA ERS, 2011a. USDA Economic Research Service. Adoption of ge netically engineered crops in the U.S. http://www.ers.usda.gov/Data/BiotechCrops/ . USDA ERS, 2011b. USDA Economic Research Service. Adoption of genetically engineered crops in the U.S.: Soybean varie ties. http://www.ers.usda.gov/Data/BiotechCrops/ExtentofAdoptionTable3.htm . USDA ERS ARMS, USDA Economic Research Service - agricultural resource management survey. http://www.ers.usda.gov/Data/ARMS/CropOverview.htm . USDA ERS ARMS, 2006. USDA Economic Research Service. Agricultural resource management survey. Crop production practices for soybeans: All survey st ates. http://www.ers.usda.gov/Data/ARMS/app/default.aspx?survey_abb=CROP .USDA Petition for Nonregulated Sta tus of DAS - 444Ø6
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