CONFIDENTIAL BUSINESS INFORMATION DELETED BASF Plant Science, L.P. Petition for the Determination of Nonregulated Status for Imidazolinone-tolerant Soybean BPS-CV127-9 The purpose of this petition is to request a determination that the article should not be regulated under 7 CFR Part 340 Submitting Company BASF Plant Science, L.P. 26 Davis Drive Research Triangle Park, NC 27709 Representing BASF Agrochemical Products B.V. Groningensingel 1, NL-6835 EA Arnhem, The Netherlands Submitted by James M. Ligon, Ph.D. BASF Plant Science, L.P. 26 Davis Drive Research Triangle Park, NC 27709 Prepared by James M. Ligon, Ph.D., Andrew Reed, Ph.D., Bruce Luzzi, Ph.D. OECD Unique Identifier BPS-CV127-9 January 13, 2009 BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 1 of 375 CONFIDENTIAL BUSINESS INFORMATION DELETED Release of Information As part of the regulatory requirement in the United States, BASF Plant Science, L.P. is submitting the information in this petition for review by the USDA. The submission of this information by BASF Plant Science, L.P. does not constitute an authorization to release any information contained within it to any third party. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of this information, BASF Plant Science, L.P. expects that USDA will provide BASF Plant Science, L.P. with a copy of the material proposed to be released and the opportunity to object to the release of any information based on appropriate legal grounds, e.g. responsiveness, confidentiality, and/or competitive concerns in advance of the release of the document(s). BASF Plant Science, L.P. authorizes the posting of the petition on the USDA Biotechnology Regulatory Services website when the Plant Pest Risk Assessment and the Environmental Assessment are published in the Federal Register for public comment. BASF Plant Science, L.P. understands that a copy of this information may be made available to the public by individual request, as part of a public comment period. Except in accordance with the foregoing, BASF Plant Science, L.P. does not authorize the release, publication or other distribution of this information (including website posting) without prior notice and consent by BASF Plant Science, L.P. © 2009 BASF Plant Science, L.P. All Rights Reserved. This document is protected under copyright law. This document is for use only by the regulatory authority to which it has been submitted by BASF Plant Science, L.P. and only in support of actions requested by BASF Plant Science, L.P. Any other use or citation of this material, without prior written consent of BASF Plant Science, L.P., is strictly prohibited. By submitting this document, BASF Plant Science, L.P. does not grant any party or entity any right to license or to use the information or intellectual property described in this document. Clearfield, Cultivance, OnDuty, Aramo, Arsenal, Clarity, Distinct, Fastac, Klap, Nomolt, Opera, Pirate, Pursuit, Raptor, Scepter, and Talcord are trademarks or registered trademarks of BASF or its affiliates. STS is a trademark of DuPont or its affiliates. Plenish is a trademark of Pioneer Hi-Bred. Roundup Ready is a registered trademark of Monsanto Technology L.L.C. Engeo and NK are registered trademarks and AAtrex is a trademark of Syngenta Group Company. Hornet, Lorsban, Vexter, and Widematch are registered trademarks of Dow AgroSciences LLC. Bronate, Connect, Folicur, Sevin, and Tamaron are registered trademarks and Bronate Advanced is a trademark of Bayer. Sumithion is a registered trademark of Sumitomo Chemical Co. Impact is a registered trademark of Cheminova. Metafos is a registered trademark of Milenia Agrociencias. Folisuper is a registered trademark and Rival is a trademark of NuFarm. BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 2 of 375 CONFIDENTIAL BUSINESS INFORMATION DELETED Certification The undersigned certifies that, to the best knowledge 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. _________________________________ James M. Ligon, Ph.D. Regulatory Affairs Manager Address: BASF Plant Science, L.P. 26 Davis Drive Research Triangle Park, NC 27709 Tel: (919)-547-2169 Fax: (919)-547-2463 Email: [email protected] BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 3 of 375 CONFIDENTIAL BUSINESS INFORMATION DELETED Table of Contents Page RELEASE OF INFORMATION 2 CERTIFICATION 3 TABLE OF CONTENTS 4 LIST OF FIGURES 7 LIST OF TABLES 8 ABBREVIATIONS AND DEFINITIONS 11 SUMMARY 13 I. RATIONALE FOR THE DEVELOPMENT OF IMIDAZOLINONE- 18 TOLERANT SOYBEAN BPS-CV127-9 A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR 18 Part 340.6 B. Rationale for the Development of Imidazolinone-tolerant Soybean CV127 18 C. Submissions to Other Regulatory Agencies 22 II. PRODUCTION AND BIOLOGY OF SOYBEAN 24 A. Soybean as a Commercial Crop 24 B. The History of Soybean 24 C. The Taxonomy and Genetics of Soybean 25 D. Morphology of Cultivated Soybean 26 E. Reproductive Biology and Hybridization with Cultivated Soybean and Related 29 Species F. Weediness Potential of Cultivated Soybean 30 G. Characteristics of the Recipient Soybean Cultivar 31 H. Soybean Comparators Used in this Petition 31 III. DESCRIPTION OF THE TRANSFORMATION AND DEVELOPMENT 33 OF CV127 SOYBEAN IV. DONOR GENES AND REGULATORY SEQUENCES 36 A. Vector pAC321 36 B. The csr1-2 Gene Coding Sequence and the AtAHAS Protein 39 C. The Arabidopsis thaliana SEC61 Gamma Subunit Protein 41 D. Regulatory Sequences 41 E. Genetic Elements of pAC321 Outside of the Transformation Fragment 41 V. GENETIC ANALYSIS OF THE INSERTION IN CV127 SOYBEAN 42 A. Copy Number and Insert Integrity 44 B. Analysis for Plasmid pAC321 Backbone 48 C. Stability of the Insert Across Multiple Generations 48 D. Organization of the Genetic Elements in the Insert of CV127 51 E. A. thaliana SEC61γ Subunit Gene 55 F. Inheritance of the Imidazolinone-Tolerance Trait in CV127 59 G. Conclusions of the Molecular Characterization 60 BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 4 of 375 CONFIDENTIAL BUSINESS INFORMATION DELETED Page VI. CHARACTERIZATION OF PROTEINS EXPRESSED IN CV127 62 SOYBEAN A. Biochemistry of the AtAHAS Protein and the Imidazolinone Herbicide 63 Tolerance B. Characterization of the AtAHAS Protein Produced in CV127 Soybean and 65 Equivalence to the E. coli-Produced Protein Used in Safety Studies C. A Summary of the Food, Feed and Environmental Safety of the AtAHAS 69 Protein D. Levels of the AtAHAS Protein in CV127 Soybean 71 E. Levels of the AtSEC61γ Subunit Protein in CV127 Soybean 78 VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENTS OF CV127 82 SOYBEAN A. Grain Composition 84 B. Forage Composition 102 C. Poultry Feeding Study 105 VIII. AGRONOMIC, PHENOTYPIC AND ECOLOGICAL EVALUATIONS 109 OF CV127 A. Seed Germination, Dormancy and Quality Characteristics 111 B. Pollen Number and Germination Characteristics 114 C. Agronomic and Phenotypic Evaluations in Replicated Field Trials 117 D. Ecological Evaluations 124 D.1. Disease Susceptibility 124 D.2. Insect Interactions 127 D.3. Nitrogen Fixation Characteristics 131 E. Conclusions from Agronomic, Phenotypic and Ecological Evaluations of 133 CV127 Soybeans IX. ENVIRONMENTAL ASSESSMENT OF CV127 SOYBEANS 134 A. Safety of the Imidazolinone-tolerance Trait 134 B. Ecological Characteristics of CV127 Soybean 136 B.1. Weediness Potential of CV127 Soybean 136 B.2. Interactions of CV127 Soybean with Other Organisms in the 137 Environment B.3. Potential for Gene Flow in Soybean 138 C. Cultivation of Soybean 139 C.1. Cultivation of Soybean in Brazil 140 C.2. Cultivation of Soybean in Argentina 141 C.3. Cultivation of Soybean in the U.S. 141 C.4. Comparison of the Cultivation of Soybean in Brazil and the U.S. 145 D. Imidazolinone Herbicide Use in the U.S. 156 E. Potential Impact of Introduction of CV127 Soybean on Agronomic Practices 158 F. Herbicide Resistance Management for Imidazolinone-tolerant CV127 Soybean 162 BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 5 of 375 CONFIDENTIAL BUSINESS INFORMATION DELETED Page G. Summary of the Agronomic and Environmental Assessments of CV127 163 Soybean X. ADVERSE CONSEQUENCES OF INTRODUCTION 165 XI. REFERENCES 166 XII. APPENDICES 184 A. Materials and Methods for the Molecular Characterization of CV127 Soybean 185 B. Characterization of AtAHAS Protein Produced in Imidazolinone-tolerant 195 CV127 Soybean and Comparison with AtAHAS Protein Expressed in Recombinant Escherichia coli C. Methods for Determining Levels of the AHAS and SEC61γ Subunit Proteins in 213 Soybean Tissues D. Methods Used and Results of the Grain and Forage Composition Analyses 217 E. Methods and Results of Seed Germination, Dormancy and Seed Quality 269 Studies of CV127 Soybean F. Materials and Methods and Results from the Agronomic, Phenotypic, and 277 Ecological Interaction Evaluations of CV127 Soybean G. Best Management Practice (BMP) Program for the Imidazolinone-tolerant 347 CV127 Soybean Production System H. Digestive Fate of the AtAHAS Produced in CV127 Soybean and in the 364 Recombinant E. coli Expression System BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 6 of 375 CONFIDENTIAL BUSINESS INFORMATION DELETED List of Figures Figure Page II-1 Partial Pedigree for Soybean Variety