CONFIDENTIAL BUSINESS INFORMATION DELETED

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# 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 Re ed, Ph.D., Bruce Luzzi, Ph.D.

OECD Unique Identifier BPS-CV127-9

January 13, 2009CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 2 of 375 

### Release of Information

As part of the regulatory requirement in th e 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 part y. In the event the USDA receives a Freedom of Information Act request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, cove ring 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 opportuni ty to object to the release of any information based on appropr iate 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 US DA Biotechnology Regulatory

Services website when the Plant Pest Risk Assessment and the Environmental Assessment are published in the Federal Regist er for public comment. BASF Pl ant Science, L.P. understands that a copy of this information may be made ava ilable to the public by indi vidual request, as part of a public comment period. Except in accordance with the foregoing, BASF Plant Science, L.P. does not authorize the release, pub lication 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 gr ant any party or entity any right to license or

to use the information or intellectua l property described in this document.

Clearfield, Cultivance, OnDuty, Aram o, Arsenal, Clarity, Distinct, Fastac, Klap, Nomolt, Opera, Pirate, Pursuit, Raptor, Scepter, and Talcord are trademarks or re gistered 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 trademar k 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. Sumith ion is a registered trademark of Sumitomo Chemical Co. Impact is a registered trademark of Cheminova. Me tafos is a registered trademark of Milenia Agrociencias. Folisuper is a registered tradem ark and Rival is a trademark of NuFarm.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 3 of 375 

### Certification

The undersigned certifies that , to the best knowledge of the undersigned, th is petition includes all

information and views on which to base a determinat ion, and that it includes all relevant data and information known to the petitioner th at 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: james.ligon@basf.comCONFIDENTIAL BUSINESS INFORMATION DELETED

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### 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 IMIDAZOLINONETOLERANT SOYBEAN BPS-CV127-9 18

A. Basis for the Request for a Determinati on of Nonregulated Status under 7 CFR

Part 340.6 18

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 Species 29

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 TRANSFO RMATION AND DEVELOPMENT

OF CV127 SOYBEAN 33

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 Elem ents of pAC321 Outsid e of the Transformation Fragment 41

V. GENETIC ANALYSIS OF THE IN SERTION 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 Elem ents in the Insert of CV127 51

E. A. thaliana SEC61 γ Subunit Gene 55

F. Inheritance of the Imidazoli none-Tolerance Trait in CV127 59

G. Conclusions of the Molecular Characterization 60CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 5 of 375 Page

VI. CHARACTERIZATION OF PRO TEINS EXPRESSED IN CV127

SOYBEAN 62

A. Biochemistry of the AtAHAS Protei n and the Imidazolinone Herbicide

Tolerance 63

B. Characterization of th e AtAHAS Protein Produced in CV127 Soybean and Equivalence to the E. coli -Produced Protein Used in Safety Studies 65

C. A Summary of the Food, Feed and Environmental Safety of the AtAHAS

Protein 69

D. Levels of the AtAHAS Protein in CV127 Soybean 71

E. Levels of the AtSEC61 γ Subunit Protein in CV127 Soybean 78

VII. COMPOSITIONAL AND NUTRITIONAL ASSESSMENTS OF CV127

SOYBEAN 82

A. Grain Composition 84

B. Forage Composition 102

C. Poultry Feeding Study 105

VIII. AGRONOMIC, PHENOTYPIC AND ECOLOGICAL EVALUATIONS

OF CV127 109

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, Phe notypic and Ecological Evaluations of CV127 Soybeans 133

IX. ENVIRONMENTAL ASSESSME NT OF CV127 SOYBEANS 134

A. Safety of the Imidazolinone-tolerance Trait 134

B. Ecological Characteris ti cs of CV127 Soybean 136

B.1. Weediness Potential of CV127 Soybean 136

B.2. Interactions of CV127 Soybean with Other Organisms in the

Environment 137

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 162CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 6 of 375 Page

G. Summary of the Agronomic and Envi ronmental Assessments of CV127 Soybean 163

X. ADVERSE CONSEQUENCES OF INTRODUCTION 165

XI. REFERENCES 166

XII. APPENDICES 184

A . Materials and Methods for the Mo lecular Characterization of CV127 Soybean 185

B . Characterization of At AHAS Protein Produced in Imidazolinone-tolerant CV127 Soybean and Comparison with AtAHAS Protein Expressed in

Recombinant Escherichia coli 195

C. Methods for Determining Levels of the AHAS and SEC61 γ Subunit Proteins in Soybean Tissues 213

D. Methods Used and Results of the Grain and Forage Composition Analyses 217

E. Methods and Results of Seed Germ ination, Dormancy and Seed Quality Studies of CV127 Soybean 269

F. Materials and Methods and Results fr om the Agronomic, Phenotypic, and Ecological Interaction Ev aluations of CV127 Soybean 277

G . Best Management Practice (BMP) Pr ogram for the Imidazolinone-tolerant CV127 Soybean Production System 347

H. Digestive Fate of the AtAHAS Pr oduced in CV127 Soybean and in the

Recombinant E. coli Expression System 364CONFIDENTIAL BUSINESS INFORMATION DELETED

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## List of Figures

Figure Page

II-1 Partial Pedigree for Soybean Variety Conquista 32 III-1 Breeding History of CV127 Soybean 34 III-2 Schematic Diagram Depicting the Process Followed in the Development of Imidazolinone-Tolerant CV127 Soybeans 35

IV-1 Circular Map of Plasmid pAC 321 and the Linear Map of the DNA Fragment Derived from the Plasmid Used for Plant Transformation 37

IV-2 The Deduced Amino Acid Se quence of the AtAHAS Protein in CV127 Soybean Including the Predicte d Chloroplast Transit Peptide (Underlined) 40

V-1 Alignment of pAC321 Transforma tion Fragment with the Transgene Insert in CV127 Soybean 43

V-2 Southern Blot Analysis of In sert Copy Number in CV127 Soybean 46 V-3 Southern Blot Analysis for the Absence of Vector Backbone Sequence in CV127 Soybean 49

V-4 Southern Blot Analysis Demonstr ating Intergenerational Stability of the Insert in CV127 Soybean 50

V-5 Diagram of Overlapping PCR Am plicons Used to Demonstrate the Integrity of the Insert in CV127 Soybean 52

V-6 RT-PCR Analysis of a 501 bp ORF Created by Insertion of a 376 bp

Portion of the csr1-2 Coding Sequence at the 3’ Flanking Sequence Junction in CV127 Soybean 53

V-7 PCR Analysis of Genomic DN A from CV127 Soybean Confirming

the Presence of ORF 501 54

V-8 RT-PCR Analysis of Tr anscription of the At SEC61 γ Subunit Gene in CV127 Soybean 57

V-9 RLM-5’-RACE Secondary PCR Analysis of the At SEC61 γ Subunit Gene in CV127 Soybean 58

VI-1 SEC61 γ Subunit Western Blot Analysis of Leaf and Grain Microsomal Membrane Protein Prep arations from CV127 and Control (Conquista) Soybeans 80

VI-2 Cytochrome c Reductase Activity in Various Fractions Prepared from Leaves of CV127 and Control (Conquista) Soybeans 81

VI-3 Alignment of the Deduced Amino Acid Sequences of the SEC61 γ Subunit Proteins from Soybean (Gmax) and Arabidopsis thaliana (Athal) 82

VIII-1 Counting the Pollen Grains (A) and Germinated Pollen Grains with Pollen Tubes (B) 115

IX-1 Map Showing Regions in the Con tinental United States and Southern Canada where Various Soybean Ma turity Groups (MG) Are Adapted

for Full-Season Growth 146CONFIDENTIAL BUSINESS INFORMATION DELETED

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## List of Tables

Table Page II-1 List of Species in the Genus Glycine Willd. 28

IV-1 DNA Components of Plasmid pAC321 38

V-1 Predicted and Observed Hybridizing Bands on So uthern Blots of CV127 Soybean Genomic DNA 47

V-2 Chi-Square Analysis Demonstrat ing Single-Gene Mendelian Inheritance (1:2:1 Segregation) of the csr1-2 Gene in Two F 3 Families, Each Derived from an Individual Hemizygous F 2 Plant, Based on a Quantitative PCR Assay 60

V-3 Chi-Square Analysis Demonstrat ing Single-Gene Mendelian Inheritance (3:1 Segregation) of the csr1-2 Phenotype in Two F 4 Progeny Derived from Hemizygous Plants from Two F 3 Families Based on a Herbicide Tolerance Assay 60

VI-1 The Calculated Limit of Detec tion (LOD) and Limit of Quantification (LOQ) for AHAS Protein by ELISA 74

VI-2 AHAS Protein Levels ( ± Std. Dev.) on a Fresh- and Dry-Weight Basis in Different Tissues and Growth Stages of CV127 Soybean and the Isoline

Control 75

VI-3 AHAS Protein Levels ( ± Std. Dev.) on a Fresh- and Dry-Weight Basis in Leaves (V2 stage) and Grain (R8 stage) of CV127 Soybean and the

Isoline Control 77

VII-1 Grain Proximate Composition 92

VII-2 Grain Fiber Composition 93

VII-3 Grain Amino Acid Composition 94

VII-4 Grain Fatty Acid Composition 96

VII-5 Grain Mineral Composition 97

VII-6 Grain Vitamin Composition 98

VII-7 Grain Isoflavone Composition 99

VII-8 Grain Phospholipid Composition 100

VII-9 Grain Antinutrient Composition 101

VII-10 Proximate and Fiber Composition of Soybean Forage 104

VII-11 Performance of Broilers Fed CV127, Conquista, Monsoy 8001 or Coodetec Soybean Meal 108

VIII-1 Means per Treatment Across Loca tions for Seed Quality, Germination and Dormancy Traits of CV127, the Isoline Control and Two Conventional Soybean Varieties 113

VIII-2 Pollen Grain Count of Isoline Control Soybean and CV127 Soybean 115CONFIDENTIAL BUSINESS INFORMATION DELETED

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VIII-3 Number and Percentage of Germinated Pollen Grains of the Isoline Control and CV127 Soybeans using the Neubauer Chamber 116

VIII-4 Pollen Tube Length (microns) of Germinating Pollen of the Isoline Control Soybean and CV127 Soybean 116

VIII-5 Locations, Planting Dates, and Harvest Dates for each Field Trial Location in Brazil During Two Growing Seasons 117

VIII-6 Agronomic Characteristics Eval uated in the Brazilian Field Trials During the 2006/2007 and 2007 Growing Seasons 119

VIII-7 Mean Values for Phenotypic and Agronomic Characteristics of Each

Combination of Treatment and Locati on for all Trials Conducted in the

2006/2007 Season in Brazil 122

VIII-8 Mean Values for Phenotypic and Agronomic Characteristics of Each

Combination of Treatment and Locati on for all Trials Conducted in the

2007 Season in Brazil 123

VIII-9 Disease Assessments for CV127 Soybean, the Isoline Control, and Conventional Soybean Varieties (Monsoy 8001 and CD217) during the

2006/2007 Field Season 125

VIII-10 Disease Assessments for CV127 Soybean, the Isoline Control, and Conventional Soybean Varieties (Monsoy 8001 and CD217) during the

2007 Field Season 126

VIII-11 The Mean Number of Coleopteran (Col), Lepidopteran (Lep), and Hemipteran (Hem) Insects Collected in Each Plot During Field Trials in

Brazil in the 2006/2007 Season 129

VIII-12 The Mean Number of Coleopteran (Col), Lepidopteran (Lep), and Hemipteran (Hem) Insects Collected in Each Plot During Field Trials in

Brazil in the 2007 Season 130

VIII-13 Effect of Different Treatments on Nitrogen Fixation Parameters in the 2006/2007 Growing Season 132

VIII-14 Effect of Different Treatments on Nitrogen Fixation Parameters in the 2007 Growing Season 132

IX-1 Commercialization Status of Genetically-Modified Soybeans in the

United States in 2011 142

IX-2 Planted Acres of Soybeans and of Biotechnology-Derived Soybeans in

the United States in 2001 thr ough 2011 (USDA ERS, 2011; USDA NASS, 2011) 143

IX-3 Herbicide Use in Soybean in the United States in 2010 143

IX-4 Comparison of the Average Monthl y Temperatures (Degrees Fahrenheit) during the Soybean Growing Season 147

IX-5 Average Monthly and Total Rainfall (Inches) during the Soybean Growing Season 148

IX-6 Important Viral Diseases of Soyb ean in the U.S. and South America (from Tolin and Lacy, 2004) 154

IX-7 Causal Organisms of Commonly-O ccurring Soybean Diseases in the Top Ten Soybean-Producing Countries and Soybean Yield Losses Caused by Them in the U.S. and Brazil in 1998 (Wrather et al ., 2001) 155CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 10 of 375 Table Page

IX-8 List of Crops in the U.S. That Are Treated with Imidazolinone Herbicides for Weed Control and Total Area Treated (Acres) in the

Years 2006-2010 158

IX-9 Identity, Year of Market Launc h and the CFIA Decision Document

Number for Imidazolinone-tolerant Clearfield ® Crops 161CONFIDENTIAL BUSINESS INFORMATION DELETED

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### Abbreviations and Definitions

A.D. Anno Domini ADF Acid Detergent Fiber

AHAS Acetohydroxyacid synthase gene

AHAS Acetohydroxyacid synthase large subunit

ai Active ingredient ANOVA Analysis of variance

At Arabidopsis thaliana AtAHAS Acetohydroxyacid synthase from Arabidopsis thaliana

AtSEC61 γ SEC 61 gamma subunit protein from Arabidopsis thaliana

B.C. Before Christ bp Base-pair

ca Circa cDNA Complementary DNA CDS Coding sequence CFIA Canadian Food Inspection Agency

CIP Calf intestinal alkaline phosphatase

csr1-2 Gene from Arabidopsis thaliana encoding an imidazolinonetolerance-conferring AHAS enzyme

CTAB Cetyl-trimethyl ammonium bromide CTP Chloroplast transit peptide dNTP Deoxynucleotide triphosphate EDTA Ethylenediaminetetraacetic acid

FAD Flavin adenine dinucleotide

FDA United States Food and Drug Administration

Fn nth filial generation gDNA Genomic DNA GM Genetically modified

GmSEC61 γ SEC 61 gamma subunit protein from Glycine max

ha Hectare

HC Health Canada

ILSI International Life Sciences Institute

kb Kilobase pairs

LD 50 Dose that is lethal to 50% of treated animals

LOD Limit of detection

M Molar

MAFF Ministry of Agriculture, Fo restry and Fisheries of Japan MHLW Ministry of Health, La bour and Welfare of Japan

MOA Ministry of Agriculture of China mRNA Messenger RNA

MT Million metric tons

N Normal (chemical concentration equal to 1 g/L)

NDF Neutral Detergent Fiber

nt Nucleotide

OECD Organisation for Economic Co-operation and DevelopmentCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 12 of 375 ORF Open reading frame

PCR Polymerase chain reaction PVP Polyvinylpyrrolidone

R272K Mutation resulting in a substitution of an arginine residue (R) with a lysine (K) at amino acid 272 re lative to the am ino acid sequence of the AHAS protein of Arabidopsis thaliana RLM-5’-RACE RNA ligase-mediated rapid amplification of 5’ complementary

DNA ends RT-PCR Reverse transcriptase-polymerase chain reaction

S653N Mutation resulting in a substitution of a serine residue (S) with an asparagine (N) at amino acid 653 relative to the amino acid sequence of the AHAS protein of Arabidopsis thaliana

SDS Sodium dodecyl sulfate

SSC Sodium chloride-sodium citrate buffer

T 0 generation resulting from regenerated transformed cells

T 4 Fourth transgenic generation

TAP Tobacco acid phosphatase

TE Tris-EDTA buffer

Tn nth self-pollinated generation post-transformation Tris Tris(hydroxymethyl)aminomethane USDA-APHIS United States Department of Agriculture-Animal and Plant Health Inspection Service

UTR Untranslated region w/v Weight per volumeCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 13 of 375 

### Summary

BASF Plant Science is submitting this request to USDA-APHIS for a determination of nonregulated status for imidazolinone-tolerant BPS-CV127-9 soybean and for any progeny of BPS-CV127-9 soybean that are derived by conven tional breeding practices with other soybeans, including conventional varieties and genetically modified variet ies that have been granted nonregulated status under 7 CFR Part 340.

Soybean ( Glycine max L.) plants that are tolerant to the imidazolinone class of agricultural herbicides have been developed by BASF Plan t Science, L.P. (BPS) and EMBRAPA (Empresa Brasileira de Pesquisa Agropecuaria, Brasil). The herbicide-tolerant soybean plants BPSCV127-9 (also referred to as CV127 in this pet ition) are derived from a single transformation event and were produced by introduction of the imidazolinone-tol erant acetohydroxyacid synthase large subunit ( AHAS ) gene csr1-2 1 with its native promoter from Arabidopsis thaliana into the soybean plant genome via bio listics transforma tion technology. The csr1-2 gene from A.

thaliana encodes an acetohydroxyacid synthase large s ubunit (AtAHAS) enzyme that is tolerant

to imidazolinone herbicides due to a point mutation that re sults in a single amino acid substitution in which the serine residue at position 653 is replaced by asparagine (S653N). The AtAHAS catalytic subu nit encoded by the csr1-2 gene has altered herb icide binding properties

such that imidazolinone herbic ides do not bind to the enzyme while retaining its normal biosynthetic function in the plant (Pang et al ., 2002).

The Arabidopsis AHAS (AtAHAS) is a member of the class of AHAS proteins found ubiquitously in plants. The AHAS enzyme catalyzes the first step in the biosynthesis of the branched-chain amino acids, valine, leucine, an d isoleucine. Typically, inhibition of the AHAS enzyme by imidazolinone herbicides leads to a de ficiency in branched-chain amino acids and other compounds derived from this pathway that are needed for plant growth and survival, and results in plant death. The herbicide tolerance in CV127 soybean will allo w growers to treat the soybean crop with imidazolinone herbicides fo r weed control without causing injury to the soybean plant at normal field application rate s. Soybean CV127 was developed for cultivation primarily in Brazil and Argentin a, and the introducti on of CV127 soybean varieties will offer soybean growers an additional tool for controlling weeds, as well as an important option for weed resistance management. Imidazolinone herb icides control a wide spectrum of grass and broadleaf weeds. The growing use of glyphosate with glyphosate-tolerant soybeans in Argentina and Brazil has led to a shift in the species of prev alent weeds with those that are more tolerant to glyphosate predominating. The most common weed s in this category in clude Benghal dayflower ( Commelina benghalensis L.)

, morning glory ( Ipomoea spp.), Brazil pusley ( Richardia

brasiliensis ), and winged false buttonweed ( Spermacoce alata ). These weeds are sensitive to imidazolinone herbicides and the product concept of an imidazolinone-tolera nt soybean fits well

with the commercial needs for weed control in Sout h America. Furthermore, it is expected that

1 It should be noted that throughout this petition, the gene that resides within the genome of CV127 soybean that encodes the

imidazolinone-tolerance trait is referred to as the csr1-2 gene which is derived from A. thaliana ; it is recognized that the csr1-2 gene in CV127 soybean differs from the csr1-2 gene in A. thaliana by a single nucleotide change that results in an R272K amino acid replacement. By convention, the am ino acid numbering of plant AHAS enzymes are cross-referenced to the corresponding

amino acid numbers of the AHAS from Arab idopsis, including the re sidues comprising the chlor oplast trans it peptide.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 14 of 375 growers planting CV127 soybeans will be able to reduce the number of herbicides used to control weeds in their soybean fields and benef it from reduced weed control costs. During the 12-year period from 1996 through 2008, biotechnology-derived herbicid e-tolerant soybeans alone accounted for a 50.45 million kg decrease in the amount of active ingredient applied (Brookes and Barfoot, 2010). The reduction in herb icide use is also expe cted to benefit the

environment.

Several AHAS genes encoding AHAS enzymes that are tole rant to imidazolinon e herbicides have been discovered in plants as naturally o ccurring mutations and through the process of chemically-induced mutagenesis. The S653N mutation in the csr1-2 gene is among the five most common single-point mutations in AHAS genes that result in tolerance to imidazolinone herbicides in plants (Tan et al ., 2005). For example, imidazolinone-tolerant maize ( Zea mays L.), rice ( Oryza sativa L.), bread wheat ( Triticum aestivum L.), and oilseed rape ( Brassica napus

and B. juncea L. Czern.), were developed through mu tagenesis, selection, and conventional breeding technologies and have been commercialized under the Clearfield ® brand name since 1992, 2003, 2002, and 1996, respectively. Therefore, th ere has been a long history of safe production of crops containing an imidazolinone- tolerant AHAS with the same S653N amino acid substitution as found in the AtAHAS encoded by the csr1-2 gene present in CV127 soybeans (Tan et al., 2005). In addition, these crops have been used to produce food and feed products that have proven to be as nutritious and as safe as similar products produced from conventional crops.

The environmental as well as food and feed safe ty of CV127 soybeans was confirmed based on results of a series of inter-related safety assessment studies. The detailed molecular

characterization of CV127 s oybean confirmed that CV127 c ontains a single functional csr1-2 gene cassette integrated in the soybean ge nome. Biochemical characterization of the imidazolinone-tolerant AtAHAS pr otein expressed in CV127 soybean showed that the AtAHAS protein is typical of other AHAS pr oteins in this diverse protein family as well as most dietary proteins with a history of safe use in food and feed products and lacks any of the characteristics associated with known allergenic or toxic proteins. Extensive phenotypic and agronomic

evaluations as well as ecological interactions of CV127 show that cultivation of CV127 soybean poses no different plant pest or weediness risk and no greater potentia l environmental impact than cultivation of conventional soybeans. Fi nally, the composition and nutritional equivalence of CV127 soybean compared to conventional s oybeans was demonstrated by analysis of key

nutrients and antinutrients in bot h grain and forage, a nd nutritional equivale nce to conventional soybeans was further confirmed in a poultry feedi ng study. The results of these studies show that CV127 soybeans are as safe as conventional soybeans for environmental release as well as

for food and feed uses.

Molecular characterization of CV12 7 soybean demonstrated that the csr1-2 gene cassette that was inserted into the soybean genome by particle bombardment transformation is present as a single, intact copy. Furthermore, it was demonstr ated that there are no additional fragments of DNA derived from the backbone of the plasmid used to produce the DNA transformation fragment inserted within the ge nome of CV127 soybeans. In the insert, there is also a 376 basepair (bp) duplicati on of a portion of the csr1-2 coding sequence directly be fore the 3’ integration point. This duplicated 376 bp segment creates a 501 bp open reading frame (ORF) that extendsCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 15 of 375 into the 3’ flanking sequence. Reverse transcri ption-polymerase chain re action (RT-PCR) results showed that this 501 bp ORF is not transcribed. It was also discovered that in addition to the

csr1-2 native gene promoter, the regi on upstream of (i.e. 5’ to) the csr1-2 coding sequence contains the complete coding sequence of the A. thaliana SEC61 (AtSEC61) gamma ( γ

) subunit protein, which is a component of the DNA fragment used for transfor mation. This protein is part of a multi-subunit secretory complex that is ubiquitous in all eukaryotes. The At SEC61 γ 5’ UTR, as annotated by The Arabidopsis Information Resource, begins 18 nucleotides downstream from the 5’ transgene integration site. As such, it is extremely unlikely that the insert contains the complete native promoter for the At SEC61 γ gene. Protein expression studies demonstrated that

no detectable levels of the A. thaliana SEC61 gamma protein are produced in CV127 soybean leaf tissue or grain.

Southern blot analysis showed that the transgen e insert in CV127 is stably integrated into the soybean genome across the breeding generations studied. This conclusion was confirmed in a study of the inheritance of the imidazolinone-to lerance trait over multiple breeding generations of CV127 soybean. Results of this study demonstrated that the trai t is stably inherited according to classical Mendelian genetics , and results were c onsistent with the presence of a single dominant imidazolinone-tolerance ge ne in the soybean genome.

The environmental safety of CV127 was shown to be comparable to an isoline control and other conventional soybean varietie s through evaluation of phenotypi c, agronomic and ecological

interaction characteristics of CV 127 and control comparator plan ts. Field trials with CV127 soybeans were conducted at seven different lo cations in Brazil duri ng the 2006/2007 growing season and at six trial locations in Brazil during the 2007 growing season. In all field trials, the CV127 soybeans were compared to a near-isogeni c null segregant soybean line (referred to as the isoline control) that does not contain the csr1-2 gene cassette and to two other conventional soybean varieties. The field tria l sites were representative of the different regions within Brazil where soybean is commercially cul tivated and each trial location consisted of four replications of CV127, the isoline control, and two conventional soybean varieties that were organized in a randomized block design. During the growing season, important agronomic characteristics including seed germination rate, days to reach key developmenta l stages, plant height, disease and insect susceptibility, and grain yield were evaluated. In addition, the impact of cultivating CV127 soybeans on factors relevant to th e nitrogen fixation symbiosis with Bradyrhizobium was assessed and compared between CV127, the isolin e control and two conventional comparator soybean varieties. Furthermore, laboratory and greenhouse-based studies of seed germination as well as pollen number and germination were also conducted and comparisons of these characteristics were made between CV127 soybean, the isoline control, and the other conventional soybean varieties. The results of th ese comparative studies demonstrated that other

than tolerance to imidazolinone herbicides, there are no biologi cally meaningful differences

between CV127 and the isoline control and/or th e other conventional soybean varieties with respect to the parameters measured as describe d above. Therefore, these results reinforce the conclusion that the cultivation of CV127 soybeans poses no different plan t pest or weediness potential and will have no different environmenta l impact than the cultivation of conventional soybean varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 16 of 375 The environmental as well as food and feed safe ty of the AtAHAS protein expressed in CV127 soybeans was demonstrated in studies that confir med that the AtAHAS prot ein is equivalent to other AHAS proteins found ubiquitously among plant species that have a history of environmental safety as well as safe use in food and feed products. Tissue samples were collected from CV127, the isoline control, and two conventional s oybean varieties at different growth stages at two field locations in the Braz ilian field trials during two growing seasons. In addition, leaf samples and grain samples were collected from all tria l locations from young plants at the vegetative growth stage V2 and at harvest, respectively. These samples were analyzed by enzyme-linked immunosorbent assa y (ELISA) to determine the quantity of AHAS enzyme in the tissues. As expected, the leve ls of AHAS in CV127 were higher than in the

isoline control since the former contains the csr1-2 gene that encodes the imidazolinone-tolerant AtAHAS as well as the genes encoding the endo genous soybean AHAS proteins. Generally, the level of AHAS was higher in young, actively-growi ng tissues than in older, mature tissues and very low or undetectable amounts were measured in grain. In all cases the levels of AHAS protein were less than 1 ppm (part per million) on a tissue dry weight basis. The results of this study demonstrate that total AHAS levels in ti ssues of CV127 are rela tively very low.

The safety of the AtAHAS protein expressed in CV127 soybean was demonstrated in several

different studies. The AtAHAS protein is structurally homologous to endogenous AHAS proteins found ubiquitously among plan t species, including cr op plants with a history of safe use. The AtAHAS protein was readily degraded in si mulated mammalian digestive conditions, typical of most dietary proteins with a history of safe use in food and feed products. Also, the AtAHAS protein expressed in CV127 soybean does not share immunologically relevant amino acid

sequence segments or structure with known allergens or have significant sequence homology to known protein toxins. Also, the AtAHAS protein s howed no acute toxicity when administered to

mice. Results of these studies show that the AtAHAS protein expr essed in CV127 soybean tissues does not possess any attrib utes of known protein food allerg ens, is not toxic to mammals,

and therefore confirm the environmental as well as food and feed safety of AtAHAS.

Composition analyses of the grain and forage demonstrated that CV127 is compositionally and nutritionally equivalent to and as safe as the isoline contro l as well as other conventional soybeans. Samples of grain or forage were ha rvested from CV127, the is oline control, and two conventional varieties from multi-location replicat ed field trials conducted in Brazil. Grain samples were collected from trials conducted in two separate growing seas ons and analyzed for a comprehensive range of important nutrients and an tinutrients of soybean. Forage samples were collected from trials in one growing season and were analyzed for proximates and fiber content. Statistical analysis of compositi on data from grain and forage samples demonstrated that the composition of grain and forage from CV127 soyb eans is comparable to that of the isoline control and conventional soybean va rieties. Furthermore, the nut ritional equivalence of CV127 soybean to the isoline control and conventional soybean varietie s was confirmed in a poultry feeding study. A 42-day feedin g trial with broiler chickens was conducted to compare the performance of the animals fed with soybean meal from CV127 soybeans to those fed soybean meal from conventional soybean grain. Results of this study demonstrated that the performance

of the animals fed a feed cont aining soybean meal from CV 127 soybean was comparable to those fed a feed containing s oybean meal from the grain of the isoline control and two conventional soybean varie ties. Collectively, these results demonstrate that the grain and forageCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 17 of 375 produced by CV127 soybean are compositionally equivalent to, and as nutritious as, grain and forage produced by the isoline control a nd other conventional s oybean varieties.

In summary, an extensive range of studies of the agronomic and environmental safety characteristics of CV127 soybeans and the safety and nutritional qualities of grain and forage produced by CV127 soybean have been conducte d. In each of these studies, CV127 soybean was compared to the isoline control and in most cases to two other conventional soybean

varieties. The results of th ese studies demonstrated that the agronomic and environmental qualities of CV127 soybeans are comparable to t hose of conventional soyb eans that have a long history of safe cultivation and consump tion. The grain produced by CV127 was also

demonstrated to be as nutritious and as safe as the soybean grain produced by conventional varieties of soybeans. Collectively, these stud ies demonstrate that, other than tolerance to imidazolinone herbicides, CV127 soybeans po ssess the same agricultural and nutritional characteristics as conventional soybeans that have a long history of culti vation and safe use in food and feed products.

There are many similarities in agronomic practices used in soybean production between the U.S. and Brazil, including weed, insect and disease control practices. The Maturity Groups of Brazilian soybean cultivars are common to those appropriate for cultivation in the southern U.S. Furthermore, environmental conditions durin g the growing season, including average temperatures and rainfall, are comparable be tween U.S. and Brazilian soybean production.

Details of these similarities are presented in section IX-C.4 of th is petition. Ther efore, in the event that CV127 soybean were to be introduced in to the U.S. environment, the data generated from field studies conducted in Brazil to supp ort the environmental as well as food and feed safety of CV127 soybeans are equally applicable to the environmental, food and feed safety

assessment of CV127 in the U.S. environment.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 18 of 375 

### I. Rationale for the Development of Imidazolinone-tolerant Soybean BPSCV127-9

A. Basis for the Request for a Determination of Nonregulated Status under 7 CFR Part

340.6

The Animal and Plant Health Inspection Service (A PHIS) of the U.S. Department of Agriculture (USDA) is responsible under the Plant Protec tion Act (7 U.S.C. § 7701-7772) for preventing the introduction or dissemination of plant pests into the United States. Under APHIS regulation 7 CFR § 340.6 an applicant may petition APHIS to ev aluate submitted data to determine that a particular regulated article does not present a plant pest risk a nd should no longer be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted and unrestricted introduction of the article is permitted.

BASF Plant Science, L.P. is submitting safety assessment data for imidazolinone herbicidetolerant soybean BPS-CV127-9, hereafter refe rred to as CV127 soybean, and requests a determination from APHIS that CV127 soybean and all progeny derived from CV127 soybean by traditional breeding methods with nonregulated s oybean varieties be considered nonregulated

articles under 7 CFR 340.

B. Rationale for the Development of Im idazolinone-tolerant Soybean CV127

Soybean is the leading oilseed crop produced a nd consumed in the world today. The current world production of soybean greatly exceeds that of all other edible oilseed crops (Wilcox, 2004; Soy Stats, 2011). In 2011, 258.4 million metric tons (MMT) of soybeans were produced worldwide representing 58% of th e world’s total oilseed producti on (Soy Stats, 2011). Soybean

has been the dominant oilseed cr op produced in the world since the 1960s (Smith and Huyser, 1987) and in the past twenty years (1989 th rough 2009) soybean production has increased by 116 MMT (FAOSTAT, 2011). For the past 50 years, th e United States (U.S.) has been the world’s leading producer of soybeans and in 2010 approx imately 90.6 MMT were produced in the U.S.,

or about 35% of the world’s to tal production (Soy Stats, 2011). The second and third largest producers of soybean are Brazil and Argentin a who produced 70.0 and 49.5 MMT of soybeans, respectively, in 2010 (Soy Stats, 2011). Soybean production in Brazil has increased dramatically in recent years with a 31 MMT increase in production between 2000 and 2010 (Soy Stats, 2008). The two largest soybean producers in Asia ar e the People’s Republic of China (15.2 MMT in 2011) and India (9.6 MMT). Soybean yield can greatly impact the economic value of the crop, and increased yield can be achieved by recomme nded weed control and other crop management practices and by using soybean varieties that have e nhanced yield potential.

For many years, genetically modified (GM) s oybean varieties have constituted the largest percentage of genetically modifi ed crops planted worldwide. In 2010 the area planted to GM soybeans was 73.3 million hectares or 50% of the global area planted to soybeans (James, 2010). The vast majority of this acreage was planted with herbicide-tolerant soybean varieties. Herbicide-tolerant soybeans were planted on 30.0 million of the total 31.6 million hectares planted to soybeans in the U.S. in 2010 (James, 2010). In Argentina, virt ually the entire area planted to soybeans in 2010, equal to 19.5 million h ectares, was planted with herbicide-tolerantCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 19 of 375 GM soybeans (James, 2010). Cultivation of herbic ide-tolerant GM soybeans is also increasing rapidly in Brazil accounting for about 75% of the total area planted to soybean in 2010 (James, 2010). The rapid and widespread adoption of he rbicide-tolerant soybean s by growers worldwide has been due to increased yields, the reduced cost of effective weed cont rol, and a simplified, more flexible weed control program (Reddy, 2001; Gianessi, 2005) that is offered by the use of herbicide-tolerant crops.

The primary herbicide-tolerant soybeans grown t oday are tolerant to the herbicide glyphosate. Glyphosate-tolerant soybeans were first grown co mmercially in 1996 and the cultivation of this crop has steadily increased every year since introduction (Gianessi and Reigner, 2006). In recent years, several weed species have evolved resistance to glyphosat e (Heap, 2011; Nandula et al

., 2005). It should be noted that most of the ca ses of glyphosate-resistan t weeds occurred in nontransgenic crops. Exposure to glyphosate st arted well before the introduction of glyphosatetolerant crops and thus attribut ing the development of resistance to glyphosate in weed species solely to the use of glyphosate-tolerant crops is not warranted (Dill, 2005).

In addition, soybeans that are tolerant to sulfonylurea herbicides due to a mutation in the acetohydroxyacid synthase ( AHAS ) large subunit gene have b een developed by conventional mutagenesis procedures (Sebastian et al ., 1989). The STSTM, or su lfonylurea-tolerant soybean, technology does not provide cross- tolerance to imidazolinone herb icides and it is currently

marketed in the United States as a stacked trait together with gl yphosate tolerance by NK ®

, among others. Use of STSTM soybeans allows eff ective weed control by sulfonylurea herbicides in soybean production areas where glyphos ate-tolerant weeds are present.

The introduction of imidazolinone herbicide-tolera nt soybeans will provide the grower with the opportunity to cultivate soybeans th at are tolerant to a different herbicide with a different mode of action than glyphosate as a mean s to control weeds that may be tolerant to glyphosate and to help prevent the continued evolution of more w eed species that are gl yphosate tolerant. The rotation of herbicide-tolerant crops that are resistant to different herbicide classes in combination with the application of the co rresponding herbicides is an e ffective strategy for managing the development of herbicide to lerance in weed populations.

Although soybeans are naturally tole rant to some imidazolinone herbicides due to an ability to metabolize specific imidazolinones (Tecle et al ., 1993), there are certain imidazolinone compounds, for example imazapyr and imazapic, that are active ingredients in a number of imidazolinone herbicide products and are not read ily metabolized in soybeans. As a result, conventional soybeans are very sensitive to imazapyr and imazapic and the product concept for CV127 soybeans includes the use of imidazolinone he rbicides containing th ese active ingredients

for effective weed control. Imidazolinone herb icide tolerance has been developed in several different crops by either conventional seed mutage nesis procedures or by natural mutation. For example, imidazolinone-tolerant bread wheat, canol a, rice, lentils, and sunflower are marketed by BASF under the Clearfield ® brand name. However, multiple attempts by BASF to introduce

commercial levels of toleran ce to postemergence applications of imidazolinone herbicides containing imazapic by seed mutagenesis methods were unsuccessful in soybeans (unpublished data). Therefore, BASF used plant gene tic engineering techno logy to introduce the imidazolinone herbicide-tolerant gene csr1-2 from Arabidopsis thaliana into soybeans toCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 20 of 375 develop the imidazolinone-tolerance trait. BASF is developing the imidazolinone herbicidetolerant soybean CV127 for cultiv ation primarily in Brazil and Ar gentina. The major weeds in soybean cultivation in these c ountries are sensitive to the im idazolinone herbicides containing imazapyr and imazapic, making this product c oncept an attractive proposition for soybean cultivation in South America. Therefore, regu latory approvals for th is product are sought in Brazil and Argentina for production as well as for food and feed us es, and in the U.S. and other countries for importation of grain from CV127 soybean for food, feed, and processing uses.

CV127 soybeans have been compared to an is oline control and two conventional soybean varieties with respect to phenotypi c and agronomic characteristics as well as specific ecological parameters in multi-location field trials in Br azil in two separate growing seasons (Section VIII,

page 109). Results of these studies show that CV127 soybeans pose no greater weediness or plant pest risk and no different potential environm ental impact than conventional soybean

varieties.

The introduction of CV127 soybeans in Argentina and Brazil will offer gr owers excellent weed control options which in turn is expected to provi de enhanced soybean yield potential. Furthermore, cultivation of CV127 soybean is expected to further build on the environmental

benefits realized as a result of cultivation of herbicide-tolerant soybeans. The imidazolinone herbicides possess several environmentally benefici al characteristics compared to other herbicide classes (Tan et al ., 2005). Imidazolinone herbicides co ntrol a wide spectrum of grass and broadleaf weeds. The growing use of glyphosate with glyphosate-tolerant soybeans in Argentina and Brazil has led to a shift in the species of prev alent weeds with those that are more tolerant to glyphosate predominating. The most common weed s in this category in clude Benghal dayflower ( Commelina benghalensis L.)

, morning glory ( Ipomoea spp.), Brazil pusley ( Richardia

brasiliensis ), and winged false buttonweed ( Spermacoce alata ). These weeds are sensitive to

imidazolinone herbicides. Imidazolinone herbicid es are effective at low application rates (Shaner and Singh, 1998) and possess residual activity in the soil resulting in fewer herbicide applications for effective weed control (Bovey a nd Senseman, 1998). For example, in Brazil, the recommended application rate and frequency th at are proposed for imidazolinone herbicides used with CV127 soybean is one application pe r season at a rate of approximately 70 g

ai/hectare. The imidazolinone herbicides are rapidly absorbed by both roots and shoots and subsequently rapidly translocated, accumulating in actively-growing ti ssue (Shaner and Singh, 1998); they are among the most poten t herbicides on the market, with effective weed control at application rates of 32 to 125 g ai/ha (Shaner an d Singh, 1998). As a reference, the application

rate of other conventional he rbicides ranges from 10 to 1300 g ai/ha with glyphosate commonly applied at a rate of 750 g ai/ha (Bonny, 2008).

In addition, imidazolinone herbicides have a very favorable toxicology profile. Due to the fact

that im idazolinones are rapidly excreted before they can accumulate in blood or tissues (Gagne et
al. , 1991) and since animals do not possess the AHAS enzyme that is the target for imidazolinones, imidazolinone herbicides have very low mammalian toxi city. Subchronic and chronic toxicity tests with rats showed no adverse effect s at doses up to 10,000 mg/kg body weight (Gagne et al. , 1991). At the highest levels tested, imidazolinone herbicides have shown no toxicity to birds (LD 50 >2,150 mg/kg), fish (LD 50 >100 mg/liter), or honey bees (LD 50 >100 μg/bee) (Gagne et al ., 1991). Imidazolinone herbicides are readily degraded in the soil to non-CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 21 of 375 toxic, naturally occu rring compounds by the ac tivity of soil microbe s (Basham and Lavy, 1987; Flint and Witt, 1997). Furthermore, due to the relatively mild climate during the winters in Brazil and Argentina, there is incr eased microbial activity in the soils that leads to a shorter halflife and lower carry-over ac tivity from the application of imidazolinones (Ulbrich et al. , 2005).

Due to the characteristics of the imidazolinone herb icides described above, it is anticipated that

the use of imidazolinone herbicides with imid azolinone-tolerant CV127 soybeans will provide a safe and environmentally beneficial system of weed control for soybean growers.

Grower adoption of imidazolinone-tolerant CV127 soyb eans is expected to ha ve other benefits to the environment. The adoption of herbicide-to lerant soybeans has resulted in a significant reduction in the adverse environmental impacts of intensive agricultural practices (Fawcett and Towery, 2002; Brookes and Barfoot, 2008; Brooke s and Barfoot, 2010; Holland, 2004). One of the greatest negative environmental impacts from agriculture comes from the practice of tilling the soil. Soil tillage is practiced to reduce weed s in crops but it leads to reductions in topsoil by promoting erosion from wind and water, loss of soil moisture, and soil compaction (Holland, 2004). The use of a broad-spectrum herbicide to control weeds without affecting the crop has facilitated the adoption of reduced- or zero-tillage practices in which seeds are planted directly into unplowed soil. A survey conducted by the American Soybean Association (ASA, 2001) demonstrated that in a five-year period in the U. S. when the use of herbicide-tolerant soybeans increased from 1.7% of the U.S. soybean acres to 74%, there was a dramatic increase in the adoption of no-tillage and reduced -tillage practices (ASA, 2001). Reduced tillage practices have resulted in improved soil structure, reduced so il erosion, and reduced risk from runoff and pollution of surface waters with sediment, nutrients, and pesticides (Fawcett and Towery, 2002; Holland, 2004). This survey also found that 53% of U.S. soybean farmers made fewer tillage passes per year through th eir soybean fields since the adoption of herbicide-to lerant soybeans, with an average reduction of 1.8 tillage passes across all farmers (ASA , 2001), resulting in an estimated savings of $385 million per year in re duced tillage costs (Duke and Cerdeira, 2005). In Argentina there has also been a documented in crease in the use of no-tillage agriculture in soybeans concomitant with the cultivation of herbic ide-tolerant soybeans. This has been shown to reduce the loss of topsoil from erosion from 10 tons of topsoil lost per hectare per year in conventional soybeans to 2.5 tons lost annually in fi elds cultivated in herbicide-tolerant soybean varieties using reduced tillage pr actices (Penna and Lema, 2003). It is anticipated that grower adoption of imidazolinone-tolerant CV127 soybeans will similarly promote the adoption of notillage and reduced-tillage pr actices by soybean growers.

The use of herbicide tolerant crops, includ ing soybean, has also resulted in a documented reduction in the consumption of tractor fuel (Brookes and Barfoot, 2010). The amount of time spent by farmers on their tractors in fields of herbicide-tolerant crops compared to fields of conventional crops has been reduc ed since fewer herbicides are being applied and due to the adoption of reduced- or no-tillage practices (Bonny, 2008). During the 12-year period from 1996 through 2008, Brookes and Barfoot (2010) reported an 8.4% reduction in pesticide use due to the cultivation of biotechnology-derived crops. Biotechnology-derived herbic ide-tolerant soybeans alone accounted for a 50.45 million kg decrease in th e amount of active ingredient applied over this same time period (Brookes and Barfoot, 2010).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 22 of 375 The adoption of no-till farming practices has been estimated to save 3.9 gallons of fuel per acre compared to conventional tillage (CTIC, 2002; Jasa et al. , 1991). The reduced fossil fuel usage due to no-till and low-till farming translates into an estimated reduction of carbon dioxide release into the environment of 88.81 kg/ ha and 35.66 kg/ha, respectivel y, relative to conventional practices (Brookes and Barfoot, 2010). Carbon dioxi de is one of the greenhouse gases that have been implicated in the global warming phenomenon. In addition, no-tillage and reduced-tillage farming utilizes less plowing and results in an increase in organic matter and carbon sequestration in the field, which also leads to less carbon dioxide releas e into the atmosphere (Holland, 2004). Brookes and Barfoot (2010) es timated that the increase in soil carbon sequestration in the United States due to re duced tillage soybean production prevented the atmospheric release of a maximum of 38,057 m illion kg of carbon dioxide over the period of 1996 through 2008. In 2008, the permanent combined reduction in carbon dioxide emissions due

to fuel reduction and increas ed soil carbon storage was approxi mately 15.6 billion kg which is equivalent to removing 6.9 million cars from the road for a year (Brookes and Barfoot, 2010). Similarly, grower adoption of imidazolinone herbic ide-tolerant soybeans is expected to promote reductions in fuel use and tillage with conc omitant reductions in carbon dioxide emissions.

C. Submissions to Other Regulatory Agencies

Imidazolinone herbicide-tolerant CV127 soybean is within the scope of the 1992 Food and Drug Administration’s (FDA) policy stat ement addressing the regulation of products derived from new plant varieties, including those developed through biotech nology (FDA, 1992). Therefore, BASF Plant Science, L.P. has submitted a summary of food and feed safety and nutritional assessment for CV127 soybean to the FDA. Al so, an import tolerance petition and supporting

residue data were submitted to the U.S. Envi ronmental Protection Agency (EPA) on September 29, 2010 for the use of imazapyr and imazapic on imported CV127 soybeans (Federal Register volume 76, number 24; Fede ral Register volume 76, number 60, respectively).

Regulatory submissions for the cultivation, as well as for food and feed uses, of CV127 soybeans were made in Brazil and Argentina where cu ltivation of CV127 is an ticipated. Regulatory approval for CV127 was obtained in Brazil in December 2009. Regulatory submissions for the importation of CV127 soybeans and processed soyb ean products for food and feed uses have been or will be made in countries that impor t significant quantities of soybeans or processed soybean products from Brazil or Argentina and that have implemented functional regulatory review processes. These include submissions to a num ber of governmental re gulatory agencies including, but not limited to, the Ministry of Health, Labour and Welfare (MHLW) and the Ministry of Agriculture, Forestry and Fisheries (MAFF) of Japan, the Canadian Food Inspection Agency (CFIA) and Health Canada (HC), the European Commission of the European Union, the Ministry of Agriculture (MOA) of China, Food Standards Austra lia New Zealand (FSANZ), and the Taiwanese Department of Health (DOH). Su bmissions have also been completed to the Rural Development Administration (RDA) of th e Republic of Korea, for determination of environmental and feed safety of CV127 s oybean, and to the other Korean Regulatory Authorities including the Korea Food and Drug Administration (KFDA), th e National Institute of Environmental Research (NIER), the National Fi sheries Research and Development Institute (NFRDI), and the Korean Center for Disease C ontrol and Prevention (KCDC). The Bureau of Plant Industry, Republic of the Philippines, gran ted regulatory approval for food, feed, andCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 23 of 375 processing uses of CV127 soybeans in October 2010. The Comisión Federa l para la Protección contra Riesgos Sanitarios (COFEPRIS), Mexic o, granted approval for CV127 import for food and feed uses as well as processing in May 2011.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 24 of 375 

### II. Production and Biology of Soybean

The biology of soybean described herein is based upon the consensus documents for Glycine

max (L.) Merr. prepared by the Organisation for Economic Co-operation and Development (OECD, 2000), and a biology document published by the CFIA Plant Biosafety Office (CFIA, 1996), as well as recent literature on the topic.

A. Soybean as a Commercial Crop

Soybean is an important crop for the production of oil and protein. The current world production of soybean greatly exceeds that of all other edible oilseed cr ops (Wilcox, 2004; Soy Stats, 2011). In 2011, 258.4 million metric tons (MMT) of soyb eans were produced worldwide representing

58% of the world’s total oilseed production (Soy Stats, 2011). Soybean has been the dominant oilseed produced in the world since the 1960s (Smith and Huyser , 1987) and in the past twenty years soybean production has in creased by 116 MMT (FAOSTAT, 2011 ). Soybean is grown as a commercial crop in over 35 different countr ies (OECD, 2001). For the past 50 years, the United States (U.S.) has been the world’s leading producer of soybeans and in 2010 approximately 90.6 MMT were produced in the U.S., or about 35% of the world’s total production (Soy Stats, 2011). The second and third largest producers of soybean are Brazil and Argentina who produced 70.0 and 49.5 MMT of s oybeans, respectively, in 2010 (Soy Stats, 2011). The fourth and fifth largest soybean prod ucers in the world are the People’s Republic of China (15.2 MMT in 2010) and India (9.6 MMT), respectively (Soy Stats, 2011). In addition to being the world’s largest producers of soybeans, the U.S., Brazil, and Argentina lead the world in the export of soybeans (43.3, 32.5, and 11.0 MMT, respectively in 2010), (Soy Stats, 2011).

Palm oil and soybean oil are the leading vegetabl e oils consumed in the world today with 2010 production of 48.7 and 42.1 MMT, resp ectively, representin g 33 and 29 percent of the world’s vegetable oil production (Soy Stats, 2011). Soyb eans also dominate th e production of protein meals for animal feed. Worldwide consumption of soybean meal in 2010 was 178.6 MMT, representing 69% of the total protein meal consumed. Due to its large animal production industry, the U.S. exported only 8.4 MMT of soybean meal in 2010, ranking third worldwide behind Argentina (29.35 MMT) and Brazil (13.9 MMT).

The major commodity products of soybean incl ude the seeds and the processed products, soybean oil and meal. There are only limited food and animal feed uses for unprocessed soybeans due to their anti-nutrient content, incl uding trypsin inhibitors a nd lectins. However,

heat treatment inactivates the an ti-nutrient factors and allows th e use of soybeans to produce soy sprouts, roasted soybeans and th e traditional soy foods, for exam ple miso, soy milk, soy sauce, and tofu (OECD, 2001). It is estimated that so ybean oil accounts for 94% of soybean ingredients in the human diet (OECD, 2001). Examples of th e use of soybean oil in food products include purified oil for use in margarin es, shortenings, and cooking and sa lad oils (USDA-ERS, 2006a). Soybean oil is the second largest source of ve getable oil worldwide (Soy Stats, 2011). In addition, fatty acids, sterols and lecithin which are generated from soybean are used in various food products. Soybean meal is the most valuab le processed product of soybean, accounting for roughly 50-75% of its overall value (USDA-ERS, 2006a ). Most soybean meal (97%) is used in feed rations for livestock, includi ng poultry, swine, and dairy and beef cattle, a nd is also used inCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 25 of 375 pet food (OECD, 2001). The utility of soybean meal for animal feed is partly due to its high content of essential amino acids, particularly lysi ne, leucine, and isoleucine, which are required supplements in animal feed (OECD, 2001).

Soybeans are used to manufacture soaps, inks, paints and disinfectants (CFIA, 1996). Industrial uses of soybean have been summarized by Ca hoon (2003), and the United Soybean Board (USB, 2003). Aside from human consumption, refined soybean oil is used in numerous technical applications (OECD, 2001).

B. The History of Soybean

Soybeans are native to North and Central Ch ina (Hymowitz, 1970; OECD, 2000). Historic and archeological evidence sugge sts that the soybean [ Glycine max (L.) Merr.] first emerged as a domesticate during the Zhou dynasty in nor theastern China (Hymowitz, 2004). Soybean domestication most likely occurre d during the Shang dynasty (ca. 1500-1100 B.C.) or earlier. It is thought that by the first century A.D., the soyb ean had spread to central and south China and Korea. The subsequent movement of soybean germplasm within the primary gene center was probably associated with the development and cons olidation of territories and the collapse of the Chinese dynasties (Ho, 1 969; Hymowitz, 1970).

Due to the development of land races, the cultivati on of soybeans spread to other regions in East Asia, including Japan, Indonesia, the Philippines, Vietnam, Tha iland, Malaysia, Burma, Nepal, and north India from the first century A.D. to the 15 th to 16 th centuries (Hymowitz, 1990). The establishment of trade routes over sea and land during this time facilitated the movement of soybeans from China and the rapid acceptance of seeds as a staple food by other cultures (Hymowitz and Newell, 1981; Hymowitz , 1990). Beginning in the late 16 th century and during

the 17 th century, European visitors to China and Japan noted the various food products produced from soybeans (Hymowitz, 1990). Soy sauce was commonly traded between the East and Europe by the late 1600s (Hymowitz, 1990).

The soybean was first introduced into North America in 1765 by Samuel Bowen who obtained the soybean seed while he was a seaman empl oyed by the East India Company on a trade ship sailing between China and London (Hymowitz, 1990) . He enabled the planting of the first soybeans in North America on a farm near Savanna h, Georgia. Nearly a century later, in 1851, soybean was first introduced into Illinois and from there it spread throughout the Midwestern U.S. (Hymowitz, 1990).

Soybeans were first introduced from the U.S. in to Brazil in 1882 (Dall’Agnol, 2004). However, the germplasm that was introduced in the state of Bahia at latitude 12°S was not adapted to such low latitudes and did not perform well (Dall’ Agnol, 2004). A decade later and after the development of later-maturing varieties, soybeans were planted further south in the state of Sao Paulo at 23°S and improved production was realized. In the first years of the 20 th century, soybeans were grown at 30°S in Rio Grande do Su l, the most southerly state in Brazil, with

climatic conditions similar to t hose in the southern U.S. Co mmercial production of soybeans in Brazil increased dramatically in the mid-1950’s following a government decision that provided incentives to grow wheat. Since soybeans are a good rotational crop with wheat and use similarCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 26 of 375 farm machinery and infrastructure, this policy al so led to increased cul tivation of soybeans in Brazil. Soybean cultivation has expanded and is now possible in all regions of Brazil from 5°N to 33°S latitude except in the Amazon and Pant anal regions due to environmental protection

restrictions and in the eastern areas due to th e mountainous terrain (Gui delines for GAP, 2002). The primary centers of soybean cultivation in Brazil range from southern Brazil to the savannas (Cerrado) of central Brazil. Soybeans are typica lly planted from mid-October to mid-December with harvest from mid-March to late April, depending on latitude. To day Brazil is the second largest producer of soybeans behind the U.S.

C. The Taxonomy and Genetics of Soybean

Glycine max (L.) Merr. is a diploidized tetraploid (2n=40) that bel ongs to the family Fabaceae and is further classified taxonomically as follows:

Kingdom Plantae

-- Plants Subkingdom Tracheobionta -- vascular plants Division Magnoliophyta -- angi osperms, flowering plants

Class Magnoliopsida -- dicots

Subclass Rosidae

Order Fabales Family Fabaceae Genus Glycine Willd. -- soybean Species Glycine max (L.) Merr. -- soybean

The above taxonomic information for soybean was obtained from the Integrated Taxonomic Information System ( http://www.itis.gov/ ) and soybean is assigned the taxonomic serial number

26716.

The genus Glycine Willd. contains two subgenera, Glycine and Soja (Moench) F.J. Herm. The subgenus Glycine comprises 22 wild perennial species that are indigenous to Australia, islands in the west, central and southern Pacific Ocean, Chin a, Russia, Japan, Indonesia, Korea, Papua New Guinea, the Philippines, and Taiwan (Hymowitz, 2004) A list of the recognized species of the

genus Glycine Willd. is presented in Table II-1.

The cultivated soybean, G. max (L.) Merr. and its wild a nnual relatives from Asia, G. soja Sieb. and Zucc. are classified in the subgenus Soja

. Glycine soja is an annual that gr ows in the wild in fields, hedgerows, roadsides, and riverbanks in many countries of East As ia (OECD, 2000). This plant has a slender build with na rrow trifoliate leaves. The flowers are purple, or on rare instances white, and are found on short, slende r racemes. The pods are short and tawny with hirsute pubescence and contain ova l-oblong seeds (Hermann, 1962).

In addition to G. max and G. soja , the subgenus Soja also contains a form known as G. gracilis

. This semi-cultivated or weedy plant is found onl y in northeast China and is intermediate in morphology between G. max and G. soja

. G. gracilis is a variant of G. max (Hermann, 1962; Wang, 1976; Shoemaker et al ., 1986). The three sp ecies of the subgenus Soja are capable of cross-pollination and the hybrid seed that is produced can germinate normally and produce plantsCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 27 of 375 with fertile pollen and seed (Singh and Hymo witz, 1989). The wild, weedy relatives of G. max, G. soja and G. gracilis are indigenous to Asia and do not occur naturally in North America (CFIA, 1996) or Brazil. Therefore, there is no potential for outcrossing of the imidazolinone

herbicide-tolerant trait from CV127 soybean to weedy relatives in Brazil where the crop will be

cultivated nor in the U.S.

Within the tribe Phaseoleae, the genus Glycine is the only genus contai ning species that have diploid chromosome numbers of 40 and 80 and not 20 (Lackey, 1980). Based on taxonomic, cytological, and molecular systematics evidence, it has been proposed that the unique

chromosome number of Glycine is most likely derived from an unknown progenitor species with

a chromosome base number of 11. From this ancient progenitor, a putative an cestor of Glycine

arose in Southeast Asia with 2n=20 (Kumar and Hymowitz, 1989; Singh and Hymowitz, 1999; Lee and Hymowitz, 2001; Singh et al ., 2001). Tetraploidization (2n = 2x = 40) through auto- or allopolyploidy of this ancestor sp ecies occurred at some time to produce a species in which 2n = 40. The sequence of events in the development of G. max from the ancient progenitor species is proposed by Singh et al . (2001) to be the following: wild perennial (2n = 4x = 40; unknown or extinct) to wild a nnual (2n = 4x = 40; G. soja ) to soybean (2n = 4x = 40; G. max ). Soybean is regarded as a stable tetraploid with a diploidized genome (Gurley et al ., 1979; Lee and Verma, 1984; Skorupska et al ., 1989).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 28 of 375 Table II-1. List of Species in the Genus Glycine Willd.

Includes 2n Chromosome Number , Genome Symbol, and Distri bution (from Hymowitz, 2004).

2n Genome † Distribution

Subgenus Glycine

1. G. albicans Tind. & Craven 40 Il Australia

2. G. aphyonota B. Pfeil 40 ? Australia

3. G. arenaria Tind. 40 HH Australia

4. G. argyrea Tind. 40 A2A2 Australia

5. G. canescens F.J. Herm. 40 AA Australia

6. G. clandestina Wendl. 40 A1A1 Australia

7. G. curvata Tind. 40 C1C1 Australia

8. G. cyrtoloba Tind. 40 CC Australia

9. G. dolichocarpa Tateishi & Ohashi 80 ? (Taiwan)

10. G. falcate Benth. 40 FF Australia

11. G. hirticaulis Tind. & Craven 40

80 H1H1

? Australia

12. G. lactovirens Tind. & Craven. 40 IlIl Australia

13. G. latifolia (Benth.) Newell & Hymowitz 40 B1B1 Australia

14. G. latrobeana (meissn.) Benth. 40 A3A3 Australia

15. G. microphylla (Benth.) Tind. 40 BB Australia

16. G. peratosa B. Pfeil & Tind. 40 ? Australia

17. G. pindanica Tind. & Craven 40 H3H2 Australia

18. G. pullenii B. Pfeil, Tind. & Craven 40 ? Australia

19. G. rubiginosa Tind. & B. Pfeil 40 ? Australia

20. G. stenophita B. Pfeil & Tind. 40 B3B3 Australia

21. G. tabacina (Labill.) Benth. 40

80 B2B2 Complex ‡ Australia Australia, West Central and

South Pacific Islands

22. G. tomentella Hayata 38

40

78

80 EE

DD Complex ¶

Complex § Australia Australia, Papua New Guinea Australia, Papua New Guinea Australia, Papua New Guinea, Indonesia, Philippines, Taiwan

Subgenus Soja (Moench) F.J. Herm.

23. G. soja Sieb. & Zucc. 40 GG China, Russia, Taiwan, Japan, Korea (Wild Soybean)

24. G. max (L.) Merr. 40 GG Cultigen (Soybean)
† Genomically similar species ca rry the same letter symbols. ‡ Allopolyploids (A and B genomes) and segmental allopolyploids (B genomes).
¶ Allopolyploids (D and E, A and E, or any other unknown combination).
§ Allopolyploids (A and D genomes, or any other unknown combination).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 29 of 375 D. Morphology of Cultivated Soybean

The following section dealing with the morphol ogy of cultivated soybean draws heavily on the 2000 OECD consensus document on the biology of Glycine max (L.) Merr. (soybean).

The cultivated soybean is an annual that has an erect, sparsely branched, bushy growth habit and can reach a height of 1.5 meters (OECD, 2000). The primary leaves are unifoliate, opposite and ovate, the secondary leaves are tr ifoliate and alternate, and com pound leaves with four or more leaflets are occasionally present. The nodulated root system consists of a taproot from which emerges a lateral root system. The plants of most cultivars are covered with fine trichomes, but glabrous types also exist (OECD, 2000). The purp le, pink, or white flowers are situated on short axillary racemes or reduced peduncles. The flower consists of a tubular calyx of five sepals, a corolla of five petals (one banner, two wings and two keels), one pistil and nine fused stamens with a single separate posterior stamen (OECD, 2000). The pod is straight or slightly curved, varies in length from two to seven centimeters, and consists of two halv es of a single carpel which are joined by a dorsal and ventral suture (OECD, 2000). Th e shape of the seed, usually oval, can vary amongst cultivars from almost s pherical to elongate and flattened (OECD, 2000). The pods typically produce one to three seeds.

There are three types of growth habit found amongst G. max (L.) Merr. soybean cultivars: determinate, semi-determinate and indeterm inate (OECD, 2000). Determinate growth is characterized by the cessation of vegetative activity of the terminal bud when it becomes an inflorescence at both axillary and terminal racemes (OECD, 2000). Determinate genotypes are typically grown in Brazil and the southern United States (Maturity Groups V to X). Indeterminate genotypes continue vegetative ac tivity throughout the flowering period and are grown primarily in central and northern regions of North America (Maturity Groups 000 to IV), [OECD, 2000]. Semi-determinate types have indeterminate stems that terminate vegetative growth abruptly after the fl owering period (OECD, 2000).

E. Reproductive Biology and Hybridization wi th Cultivated Soybea n and Related Species

Soybean is a self-pollinating species that is propagated by seed (OECD, 2000). The flowers consist of a tubular calyx of five sepals, a corolla of five petals, one pistil, and nine fused stamens with a single separate posterior stamen (OECD, 2000). One day before pollination, the stamens elongate and form a ring around the stigma that is receptive to pollen for a period from approximately 24 hours before to 48 hours afte r flowering (OECD, 2000). The anthers, following maturation in the bud, directly pollinate the stigma within the same flower (OECD, 2000). Pollination typically occu rs on the same day that the flower opens and depending on environmental conditions, anthesis normally occurs in the late morning. Pollen typically comes in contact with the stigma during the process of anthesis. Pollen viability lasts for a short time of

two to four hours and no viable pollen is present by late afternoon. Natural or artificial crosspollination is only possible during the short time when the pollen is viable. Accordingly, soybean exhibits a very low leve l of cross-pollination (below one percent) and is primarily selfpollinating (Caviness, 1966; Yoshimura et al ., 2006).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 30 of 375 Due to the strong propensity for self-fertilization described above, the frequency of crosspollination is very low. This is true even under conditions designed to optimize crossfertilization including bringing synchronous flowers into close proximity to one another. Between plants in the field located in adjacent rows, outcrossing has been measured in the range of 0.03 to 3.62% (Caviness, 1966; B eard and Knowles, 1971; Yoshimura et al ., 2006). When plants are located more than 4.5 meters from one another, natural cross-pollination in soybean is undetectable or extremely low (less than 0.02%), [Caviness, 1966]. More recent studies of soybean cross-pollination were conducted by Ray et al . (2003) and Yoshimura et al . (2006) and they report similar findings. Plants grown in cl ose proximity to each other (15 cm) were found to have average outcrossing rate s of 1.8%, while plants separa ted by distances of 0.9 m and 5.4 m had outcrossing rates of 0.41 and 0.03%, respectively (Ray et al ., 2003). Soybeans are generally not a preferred plant for insect pollinators and insect activity has been found not to increase the outcrossing rate (Erickson, 1975; Erickson, 1984). The regulations governing the production of certified Foundation soybean seed ar e consistent with the low outcrossing rate recognized for soybean. These regulations place no restriction on the separation distance between different cultivars in the field provide d that the distance is sufficient to prevent mechanical mixing during harvest.

Due to the low level of genomic si milarity among species of the genus Glycine

, G. max is unable to cross with other plants outside of the subgenus Soja and so intergeneric hybridization does not

occur. Since G. max is the only Glycine species found in either Brazil or in the United States, there is no possibility fo r cross pollination between G. max and G. soja in these countries.

F. Weediness Potential of Cultivated Soybean

Soybean plants are not weedy and are not found outside of cultivation (O ECD, 2000). Soybeans are annuals that reproduce solely from seeds. Cultivated soybean rarely displays any dormancy characteristics (TeKrony et al ., 1987) and are sensitive to cold temperatures (Raper and Kramer, 1987); thus, their potential to survive in the U.S. from one gr owing season to the next is very low. Soybean seeds normally germinate quickly under the appropriate environmental conditions that include adequate moisture and moderate temp eratures and thus, could potentially grow as a volunteer. However, any volunteers that grow af ter harvest would be destroyed by the low and freezing temperatures encountered during the followi ng winter. In the even t that volunteers were to become established, they would not compet e well with succeeding crops and they could be controlled by either mechanical or chemical means (OECD, 2000).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 31 of 375 G. Characteristics of the Recipient Soybean Cultivar

The soybean variety that was used as a recipient in transforma tion experiments to create the imidazolinone-tolerant CV127 soybean was th e commercial variety ‘Conquista’. Conquista (also known as MG BR 46) is a highly productive, conventional Brazilia n soybean variety in maturity group VIII. It is well adapted to cultiv ation in regions of less than 25 degrees latitude and was developed by Empresa Brasileira de Pes quisa Agropecuaria (EMBRAPA) with release

for commercial cultivati on in Brazil in 1995 (Pardey et al ., 2004). A partial pedigree of Conquista (Figure II-1) shows that a significant portion of its gene tic background is derived from varieties developed in the U.S. From the 1950s to the 1970s, a substa ntial amount of breeding was conducted by USDA researchers using soybean va rieties adapted to the southern U.S. states to develop commercially successful soybean varieties (Pardey et al ., 2004). These varieties were also adapted to the tropical latitudes of Brazil such as the Cerrados region (Pardey et al ., 2004) where many soybeans are cultivated today. The soybean variety Conquista was a product of this breeding effort and, as a result, it is closely related to soybean varieties that are currently

cultivated in the U.S.

H. Soybean Comparators Used in this Petition

In the studies and expe riments conducted to support the safe ty assessments of CV127 presented in this petition, appropriate cont rol soybean varieties were used as comparators. In field and laboratory studies, near-i sogenic null segregants or, in some instances, the parental control variety Conquista were used as co mparator varieties in order to minimize all differences except for the presence of the csr1-2 gene cassette in CV127 soybean which conferred imidazolinoneherbicide tolerance. For thos e studies that used the AtAHAS protein expressed in CV127 soybean, this protein was derive d either from CV127 soybean tissu es or from protein produced and purified from an E. coli fermentation process. In the fi eld studies, two other conventional varieties of soybean that are also typical of commercial varieties cultivated in Brazil were also

used as reference varieties to es tablish a range of responses typical of soybean varieties that are

cultivated in Brazil.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 32 of 375 Figure II-1. Partial Pedigree for Soybean Variety Co nquista (largely derived from Pardey

et al ., 2004) . Where available, information on the name of the breedi ng line (in italics), institution, and year of release are included.

Volstate

Tennessee

1942 Palmetto

USDA

1927 S-100

Missouri

1945 CNS

Clemson

1927 Roanoke

North

Carolina

1946

Jackson

USDA/NC

1953 D49-2491

USDA/MS

Bragg

USDA/MS

1962 Bragg

USDA/MS

1962

LO 76-4484

CNPSo N45-1497

USDA/NC Ralsoy

Arkansas

1940 Ogden

Tennessee

1942

D49-2573

USDA/NC N45-1497

USDA/NC

Davis

Arkansas

1965 IAC 71-1113

IAC

Numbaira

CNPSo

1980

MG BR 46 Conquista

CNPSo Hill

USDA/MS

1959 PI 240664 Philippines Volstate

Tennessee

1942 Palmetto

USDA

1927 S-100

Missouri

1945 CNS

Clemson

1927 Roanoke

North

Carolina

1946

Jackson

USDA/NC

1953 D49-2491

USDA/MS

Bragg

USDA/MS

1962 Bragg

USDA/MS

1962

LO 76-4484

CNPSo N45-1497

USDA/NC Ralsoy

Arkansas

1940 Ogden

Tennessee

1942

D49-2573

USDA/NC N45-1497

USDA/NC

Davis

Arkansas

1965 IAC 71-1113

IAC

Numbaira

CNPSo

1980

MG BR 46 Conquista

CNPSo Hill

USDA/MS

1959 PI 240664 PhilippinesCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 33 of 375 

### III. Description of the Transformation and Development of CV127 Soybean

A DNA fragment of plasmid pAC321 containing the csr1-2 gene that confers tolerance to

imidazolinone herbicides was used to transform embryogenic axis tissue derived from the apical meristem of a single soybean seed of the commer cial variety ‘Conquista’. This variety and tissue type was chosen for insertion of the csr1-2 gene because it responds well to particle

bombardment transformation and tissue culture re generation. In addition, Conquista is a highly productive Brazilian soybean variety with wide adaptation to different Brazilian growing environments. Biolistic transformation (also known as microprojectile or particle bombardment) (Aragão et al ., 1996) was used to produce soybean transformation events containing the csr1-2 gene. This DNA delivery system is well documen ted to transfer and integrate new DNA into a plant genome (Klein et al., 1987; Sanford et al., 1993; Lee et al ., 1996). Prior to bombardment, a purified DNA fragment containing the csr1-2 gene was precipitated onto microscopic gold particles. The precipitated DNA and particles we re then placed onto a plastic macrocarrier and accelerated at high velocity. A stopping screen in the flight path of the particles retained the macrocarrier while the particles with DNA were permitted to conti nue their flight and eventual penetration and incorporation into the soybean pl ant cells. These cells were transferred to a selective media containing the equivalent of 50 g ai/ha imazapyr (i.e. 500 nM, Aragão et al

., 2000), an imidazolinone herbicide, and onl y those cells transformed with the csr1-2 gene continued to grow. From this process a tolerant T 0 generation plant was identified and named Soybean Event 127 (CV127).

The T 0 generation plants resulting from the transformation of Conqui sta were advanced to the fourth transgenic generation (T 4 ) via four cycles of self-pollination (Figure III-1). Some T 4 plants were then backcrossed to Conquista to re duce the number of copi es of the transgenic insert to a single locus (Figure V-4). The resulting progeny, the first filial (F 1 ) generation, were advanced to the eighth filial (F 8 ) generation by successive rounds of self-pollination and selection. The homozygous F 8 line developed from this pro cess was identified as CV127 line 603 (Line 603), (Figure III-1).

Line 603 was crossed to a conventional soybean breeding line which was known only by its pedigree, Conquista 3 x BRI98-641. One of the parental lines in the pedigree, BRI98-641, is a conventional soybean line with insect tolerance. BRI98-641 was never released in Brazil as a commercial variety, but it was used as a parental line in Embrapa’s commercial soybean breeding program. The other pa rental line in the Conquista 3 x BRI98-641 pedigree, Conquista, is the commercial variety used in the original transf ormation to develop Li ne 603. As indicated by the superscript (3 ) in the pedigree, breeding line Conquista 3 x BRI98-641 was developed in a backcrossing program in which Conquista was used as the recurrent parent for three backcrosses

to transfer the insect toleran ce trait from BRI98-641 in to the high-performing Conquista variety. Thus, the genetic background of breeding line Conquista 3 x BRI98-641 is approximately 94% Conquista. The cross of Line 603 with this breedi ng line, Conquista 3 x BRI98-641, produced a line identified as CV127 line 127 (Line 127) whos e genetic background is approximately 97% Conquista (Figure III-1).

A schematic diagram depicting the development of CV127 soybeans is shown in Figure III-2.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 34 of 375 Figure III-1. Breeding History of CV127 Soybean

. The original transformation event is designated as the T 0 generation and the fourth selfpollinated generation as T 4 . Key molecular and inheritance da ta was collected at the points in the history as indicated in the legend. Tw o elite lines are shown. CV127 line 603 was the product of a cross between the T 4 generation and the conventional soybean variety Conquista. CV127 line 127 is the product of a cross betw een CV127 line 603 and a backcross-derived conventional soybean line known as ‘Conquista 3 x BRI98-641’.

x

C

o n q

u i s t a

x ( C

o n q u i s t a 3

x B

R

I 9 8

- 6 41

) T 0

T 1

T 4 T 2

T 3 F 2

F 4

F 8

F 9 F 1

F 3

F 5

F 7 F 6 F 2

F 4 F 1

F 3

F 5 

### Development of

Line 127

= Self-pollinated

= Intergeneration stability

= DNA sequence

= Southern blot analysis

F 6

F 7 

### Development of

Line 603

x

C

o n q

u i s t a

x ( C

o n q u i s t a 3

x B

R

I 9 8

- 6 41

) T 0

T 1

T 4 T 2

T 3 T 0

T 1

T 4 T 2

T 3 F 2

F 4

F 8

F 9 F 1

F 3

F 5

F 7 F 6 F 2

F 4

F 8

F 9 F 1

F 3

F 5

F 7 F 6 F 2

F 4 F 1

F 3

F 5 

### Development of

Line 127

= Self-pollinated

= Intergeneration stability

= DNA sequence

= Southern blot analysis

F 6

F 7 

### Development of

Line 603CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 35 of 375 Figure III-2. Schematic Diagram Depicting th e Process Followed in the Development of Imidazolinone-Tolerant CV127 Soybeans.

Constructed plasmid pAC321 containing the csr1-2 AHAS gene from Arabidopsis. The DNA fragment used in transformation, a 6.2 kb PvuII fragment, was released from pAC321 by restriction digestion with PvuII and purified.

The 6.2 kb PvuII fragment containing the csr1-2 gene was precipitated onto metal microprojectile beads that were used to transform embryogenic axis tissue derived from the apical meristem of a soybean seed of the commercial variety ‘Conquista’ by biolistic or microprojectile bombardment technology.

Transformants were selected on agar medium containing imidazolinone herbicide followed by the generation of shoots from the transformed meristem tissues.

Transformation events were evaluated for tolerance to imidazolinone herbicide. Tolerant

events were selected and selfed to the T 4 generation.

CV127 soybean was selected for development based on agricultural field performance, herbicide tolerance and the presence of a stable, single copy of the csr1-2 gene insert.

Breeding development of CV127 soybean progressed and included backcrossing the T 4 generation to Conquista followed by selfing to the F 8 generation and crossing to a second conventional soybean variety (Conquista 3 xBRI98-641) followed by selfing to the F 7 generation.

Laboratory and field studies were conducted to assess the food, feed and environmental safety of CV127 imidazolinone-tolerant soybeans.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 36 of 375 IV. 

### Donor Genes and Regulatory Sequences

This section describes the donor genes and regulatory elements used in the development of CV127 and the deduced amino acid sequence of the AtAHAS protein produced in CV127.

A. Vector pAC321

Molecular maps of the DNA fragment containing the csr1-2 gene that was used in the transformation of soybean tissues and the plasmid pAC321 from which it was derived are shown in Figure IV-1. The genetic elements of plasmi d pAC321 are described in Table IV-1. Plasmid pAC321 is approximately 8.7 kb and the DNA fragment derived from it that was used in the transformation of soybean tissues is an approximately 6.2 kb PvuII fragment. This fragment was cleaved from plasmid pAC321 by restriction with the restric tion endonuclease PvuII and was purified prior to use in transformation.

Plasmid pAC321 consists primarily of the E. coli cloning plasmid pBlues cript SK(-) (Stratagene, La Jolla, CA) with a 5.7 kb XbaI fragment containing the csr1-2 gene cassette from Arabidopsis

thaliana cloned into it. The csr1-2 gene cassette consists of the csr1-2 gene encoding the acetohydroxyacid synthase large subunit (AtAHAS) protein which is responsible for the

imidazolinone herbicide tolerance trait in CV127 soybeans, with it s native promoter and 5’- and 3’-untranslated regions (UTR) from Arabidopsis thaliana . In addition to the csr1-2 native gene promoter, the 5’ region upstream of the csr1-2 coding sequence contains the complete coding sequence of the A. thaliana SEC61 (AtSEC61) gamma subunit protein, which is a component of the DNA fragment used for transformation. Characterization of this coding sequence is

discussed in more detail in Sec tions IV.C. and VI.E. of this petition. The backbone section of plasmid pAC321, (the section outside of the borders of the transformation fragment) consists of genes and genetic elements derived from the E. coli cloning plasmid pBluescript SK(-).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 37 of 375 Figure IV-1. Circular Map of Plasmid pAC3 21 and the Linear Map of the DNA Fragment

Derived from the Plasmid Used for Plant Transformation

. A) Circular map of plasmid pAC321. B) Linear map of th e PvuII fragment of pAC321 containing the At AHAS promoter and 5’ UTR, csr1-2 coding sequence and At AHAS 3’ UTR that was used for transformation. The restriction sites of the enzymes (NcoI, XbaI, SpeI) and DNA probes used for Southern blot analyses of copy number, absence of backbone and intergenerational stabil ity are indicated.

pAC321

8669 bp

csr1-2 CDS bla CDS At3g48570 CDS

lacZ' CDS, interrupted lacZ' CDS, interrupted

At3g48570 intron 1

At3g48570 intron 2

Arabidopsis gDNA, unannotated Arabidopsis gDNA, unannotated

AtAHASL 5'UTR & putative promoter lacZ promoter T3 promoter

T7 promoter phage f1 (-) ori ColE1 ori

AtAHASL 3' UTR pBluescript SK(-) (5718)

At3g48570 3' UTR At3g48570 5' UTR

S653N Nco I (2483) Pvu II (5916) Pvu II (8429) Xba I (2)

Xba I (5713)

Spe I (5342) Spe I (5622) Spe I (5719)



## B

A

Fragment of pAC321 with probes csr1-2 CDS At3g48570 CDS

At3g48570 intron 1 At3g48570 intron 2

Arabidopsis gDNA, unannotated

Southern probe: 5' UTR Southern probe: At AHAS

Southern probe: 3' UTR At

AHAS 5'UTR & putative promoter

At

AHAS 3' UTR At3g48570 3' UTR

At3g48570 5' UTR

N co I (2483) P vu P

vu II (8429) X ba I (2)

X ba I (5713) S pe I (5342)

S pe I (5622) S pe I (5719 ) II (5916) Arabidopsis gDNA, unannotatedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 38 of 375 Table IV-1: DNA Components of Plasmid pAC321

Genetic Element Range (bp) Function Arabidopsis gDNA,

unannotated 1-1051 Arabidopsis thaliana genomic DNA: no genes currently annotated in this region Arabidopsis locus At3g48570 1052-2119 Coding and regulatory sequences for protein translocation complex SEC61 GAMMA CHAIN-LIKE protein from Arabidopsis thaliana At3g48570 5’ UTR 1052-1113 5’ untranslated region for putative Arabidopsis SEC61 GAMMA

CHAIN At3g48570 coding sequence (CDS) 1114-1207,

1307-1422 Putative Arabidopsis SEC61 GAMMA CHAIN coding sequence

At3g48570 intron 1 1208-1306 Putative Arabidopsis SEC61 GAMMA CHAIN intron 1, interrupts CDS

At3g48570 3’ UTR 1423-1442,

1916-2119 3’ untranslated region for putative Arabidopsis SEC61 GAMMA

CHAIN At3g48570 intron 2 1443-1915 Putative Arabidopsis SEC61 GAMMA CHAIN intron 2

At AHAS putative promoter and 5’

UTR 2120-2483 Putative promoter and 5’ untranslated region for Arabidopsis

ACETOHYDROXYACID SYNTHASE LARGE SUBUNIT

csr1-2 CDS 2484-4496 Coding sequence for Arabidopsis thaliana acetohydroxyacid synthase large subunit with (S653N) point mutation (csr1-2) which confers tolerance to imidazolinones (Sathasivan et al ., 1990)

At AHAS 3’ UTR 4497-4714 3’ untranslated region for Arabidopsis ACETOHYDROXYACID

SYNTHASE LARGE SUBUNIT Arabidopsis gDNA,

unannotated 4715-5717 Arabidopsis thaliana genomic DNA: no genes currently annotated in this region 1
pBluescript SK(-) phagemid 5718-8669 Stratagene Corporation; La Jolla, CA. (Short et al ., 1988) 2

T7 promoter transcription

initiation site 5805 Bacteriophage T7 promoter transcription initiation site; allows in vitro synthesis of RNA from DNA cloned in phagemid by T7 RNA polymerase phage f1 (-) ori 5986-6442 Bacteriophage f1 orig in of replication; allows single-strand DNA production in E. coli strains containing the F’ episome when a helper phage is present

bla CDS 6573-7433 E. coli β -lactamase coding sequence; confers resistance to β -lactam antibiotics such as ampicillin and carbenicillin

ColE1 ori 7581-8248 E. coli plasmid replication origin ColE1; derived from pUC19

lacZ promoter 8468-8589 E. coli lacZ promoter; drives transcription of the alpha fragment of β

galactosidase ( lacZ’ ).

lacZ’ CDS, interrupted 8590-8669,

5718-5994 E. coli β -galactosidase alpha fragment co ding sequence, interrupted by Arabidopsis genomic DNA in pAC321; allows blue-white screeningfor DNA insertions in pBluescript SK(-) multiple cloning site by alphacomplementation T3 promoter transcription

initiation site 8632 Bacteriophage T3 promoter transcription initiation site; allows in vitro synthesis of RNA from DNA cloned in phagemid by T3 RNA polymerase

1 The sequence of pAC321 differs from the Arabidopsis genome sequence data available at www.Arabidopsis.org

by a single nucleotide within the unannotated region at nucleotide 5073 of pAC321. pAC321 is missing an A residue relative to the public sequence.

2 The pBluescript SK(-) sequen ce in pAC321 differs from acce ssion number X52324 at nu cleotide 7751 of pAC321.

Nucleotide 7751 of pAC321 is T (in ag reement with the pUC19 origin of re plication sequence, accession number L09137) while the X52324 sequence contains a C residue at this position. This difference does not occur within the region of pAC321 that was used for transformation (the 6156 bp PvuII fragment).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 39 of 375 B. The csr1-2 Gene Coding Sequence and the AtAHAS Protein

The mechanism of action of imidazolinone herbic ides on weeds and non-tole rant plants is by inhibition of the AHAS enzyme and the resulti ng inhibition of branch ed-chain amino acid biosynthesis. AHAS catalyses the first common st ep in branched-chain amino acid biosynthesis that is specific to plants and microorganism s (Stidham and Singh, 1991). The enzyme catalyzes

the condensation of two molecule s of pyruvate to form acetolactate, the precursor of valine and leucine, or the condensation of a molecule of pyr uvate with a molecule of 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate, an intermediate in isoleucine biosynthesis (Delfourne et al ., 1994; Singh and Shaner, 1995; Duggleby and Pang, 2000). AHAS is the key control enzyme within the biosynthetic pathway and is regulated by f eedback inhibition by the end-product amino acids, valine, leucine and isoleucine. Imidazolinone herbicides readily i nhibit the activity of AHAS by binding to the active site of the protein resu lting in plant death (Duggleby and Pang, 2000; Tan et
al ., 2005). Studies have shown that specif ic single-nucleotide mutations in the AHAS genes confer tolerance to imidazolinone herbicides by altering the binding site for these herbicides on the mutant AHAS enzymes (Tan et al ., 2005), but have no effect on feedback regulation by

branched-chain amino acids and the normal bi osynthetic function of the enzyme (Newhouse et
al ., 1992).

CV127 soybean contains the csr1-2 gene from Arabidopsis thaliana that encodes an imidazolinone-tolerant AHAS enzyme (AtAHAS) (Sathasivan et al ., 1990). The AtAHAS protein encoded by csr1-2 gene is functionally identical to the native AtAHAS, except for its

tolerance to imidazolinone herbicides. The herbic ide tolerance is due to a single point mutation of a guanine to adenine in the coding sequence for AtAHAS that results in a substitution of a serine with an asparagine at residue 653 (S 653N). This amino acid change in plant AHAS proteins is known to prevent th e binding of imidazolinone herbicid es and thereby to result in

tolerance to these herbicides with no effect on feedback regulation by branched-chain amino acids or the enzyme’s normal biosynthetic function (Newhouse et al ., 1992). In addition, a

second mutation was discovered in the csr1-2 gene integrated in the genome of CV127 soybean. This mutation, in which an arginine residue at position 272 is replaced by lysine, does not impact the enzymatic function of the A HAS enzyme or its herbicide to lerance properties. A study was conducted to compare the AtAHAS large subunit protein, containing both the S653N and R272K mutations, as expressed in CV127 with the AtAHAS large subunit protein encoded by the csr1-2 gene that contains only the S653N mutation. In th is study it was demonstrated that both AtAHAS enzymes had equivalent levels of catal ytic activity and tole rance to imidazolinone herbicides. Feedback regulation of AHAS activ ity by the branched-chain amino acids is effected through the AHAS small subunit (AHASS) (Weinstock et al ., 1992; Hershey et al ., 1999). The AtAHAS enzyme encoded by the csr1-2 gene in CV127 interacts w ith the endogenous soybean AHAS small subunit protein to achiev e this regulation. Therefore, the feedback regulation of the AtAHAS encoded by the csr1-2 gene in CV127 is expected to be identical to that of the endogenous soybean AHAS.

The csr1-2 gene encodes a single polypeptide of 670 amino acids that includes the A. thaliana native chloroplast transit peptide, predic ted to consist of 85 amino acids (Mazur et al ., 1987). During transport into the chloroplast, it is pr edicted that the chloroplast transit peptide isCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 40 of 375 removed to produce the mature AtAHAS enzyme th at is approximately 64 kD and consists of 585 amino acids (Mazur et al ., 1987). The amino aci d residues of AtAHAS are counted from the N-terminus of the 670 amino acid full-length protei n containing the chloropl ast transit peptide.

The csr1-2 gene is expressed from the native A. thaliana promoter and it also contains the native 3’-untranslated region (UTR) that contains the polyadenylation signal. The deduced amino acid sequence of the AtAHAS enzyme encoded by the csr1-2 gene in CV127 is presented in Figure

IV-2.

Figure IV-2. The Deduced Amino Acid Se quence of the AtAHAS Protein in CV127 Soybean Including the Predicted Chlo roplast Transit Peptide (Underlined)

. The lysine (R272K) and asparagi ne residues (S653N) that are changed from the wildtype AtAHAS protein are in bold text.

1 10 20 30 40 50 60

MAAATTTTTT SSSISFSTKP SPSSSKSPLP ISRFSLPFSL NPNKSSSSSR RRGIKSSSPS

70 80 90 100 110 120

SISAVLNTTT NVTTTPSPTK PTKPE TFISR FAPDQPRKGA DILVEALERQ GVETVFAYPG

130 140 150 160 170 180

GASMEIHQAL TRSSSIRNVL PRHEQGGVFA AEGYARSSGK PGICIATSGP GATNLVSGLA

190 200 210 220 230 240

DALLDSVPLV AITGQVPRRM IGTDAFQETP IVEVTRSITK HNYLVMDVED IPRIIEEAFF

250 260 270 280 290 300

LATSGRPGPV LVDVPKDIQQ QLAIPNWEQA M **K** LPGYMSRM PKPPEDSHLE QIVRLISESK

310 320 330 340 350 360

KPVLYVGGGC LNSSDELGRF VELTGIPVAS TLMGLGSYPC DDELSLHMLG MHGTVYANYA

370 380 390 400 410 420

VEHSDLLLAF GVRFDDRVTG KLEAFASRAK IVHIDIDSAE IGKNKTPHVS VCGDVKLALQ

430 440 450 460 470 480

GMNKVLENRA EELKLDFGVW RNELNVQKQK FPLSFKTFGE AIPPQYAIKV LDELTDGKAI

490 500 510 520 530 540

ISTGVGQHQM WAAQFYNYKK PRQWLSSGGL GAMGFGLPAA IGASVANPDA IVVDIDGDGS

550 560 570 580 590 600

FIMNVQELAT IRVENLPVKV LLLNNQHLGM VMQWEDRFYK ANRAHTFLGD PAQEDEIFPN

610 620 630 640 650 660

MLLFAAACGI PAARVTKKAD LREAIQTMLD TPGPYLLDVI CPHQEHVLPM IP **N** GGTFNDV

670

ITEGDGRIKYCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 41 of 375 C. The Arabidopsis thaliana SEC61 Gamma Subunit Protein

The linear DNA fragment used in the transforma tion contains a 2.5 kb segment upstream of the

csr1-2 coding region that was orig inally annotated as the AHAS promoter and 5’ untranslated region (UTR). More-recen t sequence analysis has revealed that this segment also contains a previously unannotated Arabidopsis gene encoding the gamma subunit of SEC61 (AtSEC61 γ ) , a multimeric transport protein of the endoplasmic re ticulum that is ubiquitous in all plants and other eukaryotes (Hartmann et al ., 1994). The CV127 insert cont ains the majority of the

At SEC61 γ subunit gene including the complete coding sequence. The At SEC61 γ 5’ UTR, as annotated by The Arabidopsis Information Resour ce, begins 18 nucleotides downstream from the 5’ junction of the transgene and soybean genomic DNA flanking the integration site. As such, it is extremely unlikely that the insert contains the complete native promoter for the At SEC61 γ gene. The possible production of the Arabidopsis AtSEC61 γ subunit protein in CV127 was

evaluated and results showed that no AtSEC61 γ subunit protein was detected in tissues of CV127 soybean above the levels of de tection of the assay. (No AtSEC61 γ subunit protein was detected at levels > 30 or >110 ppb, respectively, for leaf and grain). These studies are described

in more detail in Section VI.E.

D. Regulatory Sequences

Transcriptional re gulation of the csr1-2 gene in CV127 is under th e control of its native A.

thaliana promoter that is located immediately 5’ to the csr1-2 5’ UTR and coding sequence and

3’ to the At SEC61 γ gene. Termination of transcription of the csr1-2 gene is also effected by the

native A. thaliana csr1-2 gene transcription termination se quences contained within the 3’- untranslated region located downstream of the csr1-2 gene.

E. Genetic Elements of pAC321 Outside of the Transformation Fragment

The plasmid pAC321 consists primarily of the transformation fragment cloned into the E. coli cloning plasmid pBluescript SK(-) (S tratagene, La Jolla, CA). This plasmid contains the ColE1 origin of replication for ma intenance of the plasmid in E. coli and the E. coli gene encoding β

– lactamase which confers ampicillin resistance and is used as a selectable marker for the plasmid

in E. coli . In addition, the plasmid enc odes the alpha fragment of the E. coli lacZ gene whose expression is regulated by the lacZ gene promoter. The lacZ gene encodes the E. coli β

galactosidase enzyme. The lacZ alpha fragment gene in plas mid pAC321 is disrupted by the insertion of the 5.7 kb XbaI fragment comprising the csr1-2 gene cassette. The genetic elements within the plasmid pAC321 that are not part of the transformation fragment are listed and

described in Table IV-1. Since only the 6.2 kb Pvu II fragment containing the csr1-2 gene cassette was used in the transformation, very fe w of the sequences derived from the plasmid backbone of pAC321 were included within the transformation fragment. No plasmid backbone DNA was detectable in Southern blot analyses with probes specific for these fragments (Section V of this petition).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 42 of 375 

### V. Genetic Analysis of the Insertion in CV127 Soybean

A complete genetic analysis of the transgene insert and flanking regi on of soybean CV127 has been conducted and the results of these analyses are presented in this section. The results of these analyses demonstrated that the csr1-2 gene expression cassette was integrated at a single genetic locus in the soybean genome and that no DNA sequences from the backbone of the

transformation vector were de tected in the genome. The csr1-2 gene cassette in CV127 soybean contains three point mutations relative to the transformation plasmid with one mutation in the

At AHAS coding sequence and the other two downstream of the 3’ untranslated region (UTR) of

the csr1-2 gene. The G to A mutation in the csr1-2 coding sequence, which caused an amino acid change of R 272 to K 272 does not affect the desired herbic ide tolerance phe notype conferred by the csr1-2 gene. Southern blot analysis and se quence verification of the point mutation indicate that the insert is stably integrated in the soybean genome across the nine breeding generations studied. Also, tra it inheritance studies across breed ing generations confirmed the expected trait segregation ratios , further demonstrating that imid azolinone herbicid e tolerance in CV127 soybean is the result of a single functional dominant csr1-2 gene stably integrated in the soybean genome.

In the transgene insert, there is also a 376 ba se-pair (bp) duplication of a portion of the csr1-2 coding sequence directly before th e 3’ integration point of the in sert. This duplicated 376 bp segment creates a 501 bp open reading frame (ORF) th at extends into the 3’ flanking sequence. The results of reverse transcription-polymerase chain reaction (RT-PCR) analyses of total RNA extracted from leaf, root, leaf bud, and seed fr om BPS-CV127-9 soybean showed the absence of a detectable ORF 501-specific PCR product, sugges ting that this 501 bp ORF is not expressed. The insert also contains the majority of the Arabidopsis SEC61 γ subunit gene locus (refer to Table IV-1 for genetic element At3g 48570) , which is a component of the DNA fragment used for transformation. This gene is discussed in mo re detail in section V.E. A more detailed description of these analyses and the results foll ows. The materials and methods used in these analyses are presented in Appendix A.

The genetic elements contained within the 5.7 kb XbaI fragment that was derived from the genome of Arabidopsis and integrat ed into plasmid pAC321 are li sted in Table IV-1 beginning with nucleotide 1 and continuing to nucleotid e 5717. The hybridization probes used in the Southern blot analyses and th e map of plasmid pAC321 containing the transformation fragment used to generate CV127 soybean are presented in Figure IV-1. The DNA sequence data (not presented) and other results from the molecular characterization of the insert were used to construct a linear map of the transgene insert and the flanking soybean genomic DNA (Figure V1). The pAC321 transformation fragment is shown aligned with the DNA insert in CV127 soybean to identify the portions of the fragment th at are present in the soybean CV127 insert in Figure V-1.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 43 of 375Figure V-1.Alignment of pAC321 Transformation Fragmentwith the Transgene Insert in CV127 Soybean1.1The PvuII fragment from plasmid pAC321 that was used to transform soybean is shown in the upper portion of the figure. Parts of this fragment that are notcontained within the transgene insert in CV127 soybean are indicated by boxes filled with diagonal stripes. Characteristics ofthe transgene insert and theflanking genomic soybean DNA in CV127 soybean are shown in thelower portion of the figure. The DNA between the vertical dotted lines (drawn betweenthe maps of the transformation fragment and the transgene insertion region) is common to DNA from both sources. Restriction sites relevant to the Southernblot analysis are indicated. The numbering system of the PvuII transformation fragment corresponds to that of the pAC321 plasmid map in Figure IV-1. Thenumbering system for the CV127 soybean DNA insert region begins with the first nucleotide at the 5’ end of the soybean genomicflanking sequence (flankingsequences indicated by gray boxes).pAC321 PvuII fragment used for transformationcsr1-2 CDSAt3g48570 CDSArabidopsis gDNA, unannotatedArabidopsis gDNA, unannotatedAtAHAS5'UTR & putativepromoterAtAHAS3' UTRNcoI(2483)PvuII(5916)PvuII(8429)XbaI(2)XbaI(5713)SpeI(5622)SpeI(5719)SpeI(1268)SpeI(5342)csr1-2 CDSAt3g48570 CDSArabidopsis gDNA, unannotatedArabidopsis gDNA, unannotated376 bp from csr1-2 CDS5' flanking sequence3' flanking sequenceAtAHAS5'UTR & putative promoterAtAHAS3' UTRNcoI(2761)XbaI(410)XbaI(10652)SpeI(1268)SpeI(5620)SpeI(6505)CV127 SoybeanCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 44 of 375 A. Copy Number and Insert Integrity

The copy number and integrity of the DNA insert in CV127 soybean were evaluated by Southern blot analysis of genomic DNA from plants of the F 8 generation of line 603 digested with NcoI, SpeI and XbaI restriction enzymes. (For a description of specific generations of CV127, a breeding diagram is presented in Figure III-1.) Blots were hybridized separately with three different probes (the At AHAS 5’ UTR, the csr1-2 coding sequence for the AtAHAS protein, and

the At AHAS 3’ UTR) that spanned the entire DNA frag ment used for transformation (Figure IV1). The csr1-2 coding sequence probe is referred to as the “AHAS” probe in Figure V-2, Figure V-4, and Table V-1. Genomic DNA from the non-transgenic cultivar Conquista as well as Conquista genomic DNA spiked with one- and two-genome copy equi valents of pAC321 plasmid DNA was included in the Southern blots as controls.

Non-transgenic Conquista DNA digested with all th ree restriction enzymes and hybridized with the three probes did not show any hybridizing signal, indicating that neither the endogenous soybean AHAS nor the endogenous soybean SEC61 γ subunit genes are detect ed at the Southern blot stringency conditions used (Figure V-2, la nes 1, 5 and 9). The pAC321 DNA, spiked at one- and two-genome copy equivalents into non-tr ansgenic Conquista DNA (Figure V-2, lanes 2, 3, 6, 7, 10 and 11), was detected at the expected band sizes (Table V-1) with a fairly strong intensity suggesting that the Southern method em ployed was sensitive enough to detect a singlecopy insert in the transgenic event. DNA samples from the CV127 soybean F 8 generation treated with the different restriction enzyme and prob e combinations all gave single hybridizing bands (Figure V-2, lanes 4, 8 and 12) except fo r the SpeI digest hybridized with the csr1-2 coding sequence probe, which had an additional small band of approximately 800 to 900 bp (Figure V2B, lane 8). This small csr1-2 -hybridizing SpeI fragme nt is consistent with the observation that a small fragment of the csr1-2 coding sequence was duplicated at the 3’ flanking sequence junction in CV127 soybean (see Section V.D.) ther eby creating an 885 bp SpeI fragment. All major bands had signal intensities similar to the one-genome copy equivalent of pAC321. Due to the homozygous state of the material analyzed, it would be reasonable to expect band intensities closer to the two genome copy equiva lent control. The quantification of plasmid DNA versus genomic DNA coupled with the larg e dilution required for loading the plasmid DNA, can result in variations of intensity

. However, the DNA sequence information for the insert as well as genomic flanking DNA (data no t presented), and the number of bands observed

in the Southern blot substantiate the conclusion that a single copy of the insert is present in CV127 soybean.

Genomic DNA from CV127 soybean that was di gested with NcoI and probed with At AHAS 5’ UTR produced a hybridizing band approximately 4.5 kb in size (Figure V-2). The size of this band is consistent with the produc tion of a single DNA fragment de fined by the NcoI site within the insert (nt 2761, Figure V-2 pane l D) and an NcoI site approximately 4.5 kb upstream in the 5’ genomic soybean flanking DNA sequence. Th e same digest probed with either the csr1-2 coding sequence or At AHAS 3’UTR produced a hybridizing band appr oximately 9.0 kb in size. The size of this band is consistent with a single DNA frag ment defined by the NcoI site in the insert (nt 2761, Figure V-2 panel D) and an NcoI site appr oximately 9.0 kb downstream in the 3’ soybean genomic flanking DNA sequence.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 45 of 375 Digestion of CV127 genomic DNA with SpeI and probed with At AHAS 5’ UTR produced a hybridizing band of approximate size 4.4 kb. This is consistent with th e production of a single DNA fragment from a SpeI site in the insert (n t 5620, Figure V-2, panel D) and a SpeI restriction site approximately 4.4 kb upstream in the 5’ DNA flanking sequence of the soybean genome (nt 1268, Figure V-2, panel D). The presence of this upstream SpeI site was confirmed in the analysis of the 5’ DNA flanking sequence of CV127 soybean (data not presented). The same digest probed with either the csr1-2 coding sequence or the At AHAS 3’ UTR also produced a 4.4 kb hybridizing band corresponding to the same fragment described above. In addition, a hybridizing band of approximate size 800 to 900 bp was detected when the SpeI digest was probed with the csr1-2 coding sequence, consistent with a single SpeI DNA fragment containing the duplicated 376 bp segment of the csr1-2 gene at the 3’ flanki ng DNA sequence junction. This hybridizing fragment was produced from the Sp eI site in the DNA insert and a SpeI site 885 bp downstream in the soybean genome (nt 56 20 – 6505, Figure V-2, pa nel D). The 885 bp hybridizing band was not detected by the At AHAS 3’ UTR probe, indicating that At AHAS 3’ UTR DNA was not included in the 885 bp fragment, a nd the SpeI nt 5620 site in the insert is adjacent to the duplicated 376 bp segment of the csr1-2 gene. Therefore, SpeI restriction enzyme sites at nucleotide 5622 and 5719 in the lin ear PvuII fragment of plasmid pAC321 used for transformation (shown in Figure V-1) were not included in the DNA insert in the CV127 genome. This was confirmed by DNA sequence an alysis of the DNA insert (refer to Section V.D.). The smaller predicted SpeI fragments of 280 and 97 bp in the pAC321-spiked controls were not detected because these fragments were expected to produce low-intensity hybridizing signals below the level of detection of the analysis.

CV127 soybean genomic DNA when digest ed with XbaI and probed with At AHAS 5’ UTR shows a single hybridizing band of approximate si ze 10 kb. Based on the positions of the XbaI

restriction sites in the linear DNA used for tr ansformation (Figure V-1), a hybridizing band of approximately 5.7 kb was expected to be produ ced from within the DNA insert in the CV127 soybean genome. However, DNA sequence analys is of the DNA insert in CV127 showed that

neither of the XbaI restriction sites in the linear transformation DNA were included in the DNA insert. Therefore, the 10 kb hybridizing band wa s produced from XbaI re striction sites within the 5’ and 3’ DNA sequences flanking the in sert (nt 410 and 10652, Figure V-2, panel D). Accordingly, the same dige st probed with either the csr1-2 coding sequence or At AHAS 3’ UTR produced the same 10 kb hybrid izing band corresponding to the same DNA fragment described

above.

Analysis of the number and size of all hybridizing bands on the Southern blots shown in Figure V-2 is consistent with the integration of a single DNA insert in the CV127 soybean genome containing a single functional copy of the csr1-2 gene, as well as c oding sequence for the

AtSEC61 γ subunit protein on the 5’ end of the csr1-2 gene, and a single DNA fragment containing a 376 bp duplicated segment of the cs r1-2 gene at the 3’ end of the transgene insert.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 46 of 375 Figure V-2. Southern Blot Analysis of Insert Copy Number in CV127 Soybean

.

Genomic DNA of non-transgenic soybean variety Conquista (lanes 1, 5 and 9); Conquista spiked with 1- (lanes 2, 6 and 10); or 2-genome copy equivalents of pAC321 (lanes 3, 7 and 11); and genomic DNA of CV127 soybean from the F 8 generation (lanes 4, 8 and 12) was digested with NcoI (1-4), SpeI (5-8) and XbaI (9-12) restriction enzymes. Blots were hybridized with probe 5’ UTR (panel A), probe AHAS (panel B) and probe 3’ UTR (panel C). The first and last lanes (labeled M) contain a λ /HindIII ladder; band sizes are indicated in kilobases. Panel D indicates regions of homology between the Southern hybridization probes and the CV127 soybean insert. The arrow in panel B indicates a 885 bp SpeI fragment containing an additional 376 bp fragment of csr1-2 present in CV127 at the 3’ flanking sequence junction.

Note: The 5’ UTR probe and the 3’ UTR probe each overlap a XbaI site in pAC321 and therefore hybridize to both XbaI fragments of the plasmid. These two bands are marked with a dot to the right of them in lane 11 (Panels A and C).

D 23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

9.4

6.6

4.4

2.3

2.0 M 1 2 3 4 5 6 7 8 9 10 11 12 M

Probe 5’UTR

Probe AHAS

Probe 3’UTR 

### A

B

C Nco I Spe I Xba I

23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

9.4

6.6

4.4

2.3

2.0 M 1 2 3 4 5 6 7 8 9 10 11 12 M

Probe 5’UTR

Probe AHAS

Probe 3’UTR 

### A

B

C Nco I Spe I Xba I

23.1 kb

9.4

6.6

4.4

2.3

2.0 23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

9.4

6.6

4.4

2.3

2.0 23.1 kb

9.4

6.6

4.4

2.3

2.0 23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

9.4

6.6

4.4

2.3

2.0 23.1 kb

9.4

6.6

4.4

2.3

2.0 23.1 kb

9.4

6.6

4.4

2.3

2.0 M 1 2 3 4 5 6 7 8 9 10 11 12 M

Probe 5’UTR

Probe AHAS

Probe 3’UTR 

### A

B

C Nco I Spe I Xba I

csr1-2 CDS At3g48570 CDS
At3g48570 intron 1 At3g48570 intron 2

Arabidopsis gDNA, unannotated Arabidopsis gDNA, unannotated
376 bp from csr1-2 CDS 5' flanking sequence 3' flanking sequence

Southern probe: AHAS Southern probe: 5' UTR

Southern probe: 3' UTR Hybridizes with AHAS probe AtAHASL 5'UTR & putative promoter At3g48570 3' UTR

AtAHASL 3' UTR At3g48570 5' UTR

Nco I (2761)

Xba I (410) Xba I (10652) Spe I (1268) Spe I (5620) Spe I (6505)

Regions of homology to hybridization probesCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 47 of 375 Table V-1. Predicted and Observed Hybridizing Bands on Southern Blots of CV127 Soybean Genomic DNA.

Figure Probe Restriction Enzyme Predicted Fragment

Size from

CV127 Insert (bp) a Observed

CV127 Fragment

Size Predicted Fragment

Size from

Plasmid pAC321

(bp) Observed

Plasmid Fragment

Size

NcoI >2760 ~4500 8669 ~9000
SpeI 4352 ~4400 8292 ~8500 V-2A 5’ UTR

XbaI 10242 ~10000 5711 b

2958 ~5500

~3000

NcoI >7896 ~9000 8669 ~9000

4352 SpeI 885 ~4400

~800 c 8292 ~8500 V-2B AHAS

XbaI 10242 ~10000 5711 ~5500

NcoI >7896 ~9000 8669 ~9000

SpeI 4352 ~4400 8292

280

97 ~8500 V-2C 3’ UTR

XbaI 10242 ~10000 5711 b

2958 ~5500

~3000

NcoI none none 8669 ~9000
SpeI none none 8292 ~8500 V-3A VP1

XbaI none none 2958 ~3000

NcoI none none 8669 ~9000
SpeI none none 8292 ~8500 V-3B VP2

XbaI none none 2958 ~3000

a The predicted fragment size is estimated based on the cloned insert and flanking sequences in CV127.

b The 5’ UTR probe and the 3’ UTR probe each overlap a XbaI site and theref ore hybridize to both XbaI fragments of the plasmid.

c Sequence analysis of the CV127 insert indicates that a small portion of the csr1-2 coding region was duplicated immediately upstream of the 3’ transg ene integration site, conf irming the identity of the 885 bp band observed in

these Southern blots.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 48 of 375 B. Analysis for Plasmid pAC321 Backbone

Although the transformation was carr ied out with the PvuII restri ction fragment of pAC321 that included minimal vector backbone DNA, Southern blot studies were conducted to confirm the absence of plasmid pAC321 vector backbone DNA in the CV127 soybean genome. In order to determine whether there was any vector backbone integrated in CV127, the same set of blots used for Southern blot analysis described abov e (Figure V-2) was hybridized with two vector backbone-specific probes designate d VP-1 and VP-2 (Figure V-3, panel C). As expected, no hybridizing bands were detected in lanes cont aining non-transgenic C onquista genomic DNA. Non-transgenic Conquista genomic DNA spiked w ith one- or two-genome copy equivalents of transformation plasmid pAC321 showed hybridizi ng bands of the expected sizes (Figure V-3 panels A and B, and Table V-1). No hybridiz ing bands were detected in CV127 soybean DNA, clearly indicating that no vect or backbone DNA was integrated into the genome of CV127 soybean.

C. Stability of the Insert Across Multiple Generations

In order to determine the stab ility of the insert in CV127, DNA samples from four different generations, T 4 , F 4 , F 8 and F 9 , were subjected to Southern blot analysis. For reference, the breeding history of CV127 is presented in Figur e III-1. Genomic DNA samples were digested with NcoI and SpeI and probed with either the At AHAS 5’ UTR, csr1-2 coding sequence or

At AHAS 3’ UTR probes spanning the entire DNA frag ment used for transformation (Figure IV1). The combination of these restriction enzy mes and probes provides a unique fingerprint for the DNA insert in CV127 (Figure V-2). Non-tran sgenic Conquista genomic DNA was used as a negative control and Conquista spik ed with one- and two-genome copy equivalents of pAC321 was used as a positive control. Multiple bands from CV127 T 4 generation DNA digested with either NcoI or SpeI were detected with all three probes, indicating that the T 4 generation contains multiple copies of the csr1-2 gene cassette (Figure V-4) . However, DNA from the F 4 , F 8 and F 9 generations all showed the same Southern pattern (Figure V-4) pr eviously observed in the insert and copy number analyses (Figure V-2). This resu lt clearly indicates that the multiple copies of

the insert in the T 4 generation segregated in the progeny of the cross between T 4 and Conquista and that only a single copy is retained in th e segregant selected. Moreover, these results demonstrate that this single copy is stably inte grated in the soybean genome and is inherited across subsequent breeding generations.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 49 of 375 Figure V-3. Southern Blot Analysis for th e Absence of Vector Backbone Sequence in CV127 Soybean

. Genomic DNA of non-transgenic soybean variety Conquist a (lanes 1, 5 and 9); Conquista spiked with 1- (lanes 2, 6 and 10); or 2-genome copy equivalents of pAC321 (lanes 3, 7 and 11); and genomic DNA of

CV127 from the F 8 generation (lanes 4, 8 and 12) were digested with NcoI (lanes 1-4), SpeI (lanes 5-8) and XbaI (lanes 9-12) restriction enzymes. The blot was hybridized with probe VP1 (panel A) and probe VP2 (panel B). The first and last lanes (labeled M) contain a λ /HindIII ladder; sizes are indicated in kilobases. Panel C indicates the origin of probes VP1 and VP2 in the sequence of pAC321.

A

B

C 

### M 1 2 3 4 5 6 7 8 9 10 11 12 M

23.1 kb

9.4

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### M 1 2 3 4 5 6 7 8 9 10 11 12 M M 1 2 3 4 5 6 7 8 9 10 11 12 M

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6.6

4.4

2.3

2.0 Nco I Spe I Xba I

Fragment of pAC321 with probes bla CDS

Southern probe: VP1

Southern probe: VP2 lacZ promoter phage f1 (-) ori

ColE1 ori Pvu II (5916) Pvu II (8429)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 50 of 375 23.1 kb

9.4

6.6

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6.6

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b

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2.0 NcoI SpeI

Probe 5’UTR 

### A

Probe AHAS 

### B

Probe 3’UTR 

### C Figure V-4. Southern Blot Analysis Demonstrat ing Intergenerational Stability of the Insert in CV127 Soybean.

Genomic DNA of non-transgenic soybean variety Conquista (lanes 1 and 8); Conquista spiked with 1-copy (lan es 2 and 9), or 2-copy equivalents of pAC321 (lane 3 and 10); and genomic DNA of CV127 of either the T 4 (lanes 4 and 11), F 4 (lanes 5 and 12), F 8 (lanes 6 and 13) or F 9 (lanes 7 and 14) generation was digested with either NcoI (lanes 1- 7) or SpeI (lanes 8- 14) restriction enzymes. The blot was hybridized with either probe 5’ UTR (panel A); probe AHAS (panel B); or probe 3’ UTR (panel C). The first and last lanes (labeled M) contain

a λ /HindIII ladder; sizes are indicated in

kilobases. Panel D shows the Southern hybridization probes that were used in this experiment and their relationship to the transgene insertion. The arrow in panel B indicates the 885 bp SpeI fragment containing the 376 bp fragment of csr1-2 coding sequence inserted near the 3’ flanking sequence junction.

D

csr1-2 CDS At3g48570 CDS
At3g48570 intron 1 At3g48570 intron 2

Arabidopsis gDNA, unannotated Arabidopsis gDNA, unannotated
376 bp from csr1-2 CDS 5' flanking sequence 3' flanking sequence

Southern probe: AHAS Southern probe: 5' UTR

Southern probe: 3' UTR Hybridizes with AHAS probe AtAHASL 5'UTR & putative promoter At3g48570 3' UTR

AtAHASL 3' UTR At3g48570 5' UTR

Nco I (2761)

Xba I (410) Xba I (10652) Spe I (1268) Spe I (5620) Spe I (6505)

Regions of homology to hybridization probesCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 51 of 375 D. Organization of the Genetic Elements in the Insert of CV127

Although Southern blot analysis demonstrated that the transgene insert in CV127 soybean contains the complete csr1-2 expression cassette, cloning a nd sequencing of the insert was performed to confirm insert integrity. The co mplete sequence of the inserted DNA was obtained by PCR amplification of six overlapping amplicons with Taq DNA polymerase (Figure V-5). The complete soybean CV127 insert sequence is 4758 bp in length and other than the insertion of the 376 bp fragment from csr1-2 at the 3’ integration point, the sequence is identical to the sequence of the transformation fragment except for three point mutations. One of the point

mutations is a G to A mutation in the AHAS coding sequence, which results in an amino acid change from R 272 to K 272. This is a conservative amino aci d substitution and has no impact on the herbicide tolerance or enzymatic properties of the AtAHAS protein. The other two mutations

include a G to A mutation and a G to C mutati on, both of which are located downstream of the

3’UTR of the csr1-2 gene and so are genetically silent. These results confirm that the organization of the genetic elements within the DNA insert in CV127 soybean is identical to the organization of the genetic elements in the P vuII linear DNA fragment of pAC321 (Figure IV-1) used to produce CV127 soybean, except for th e insertion of the 376 bp fragment from csr1-2 at the 3’ integration point.

The insertion of a 376 bp portion of the csr1-2 coding sequence near the 3’ flanking sequence junction (Figure V-1) created a 501 bp ORF. Th e possible transcription of this ORF was also investigated by RT-PCR analys is. RT-PCR was carried out with 500 ng of RNA template. CV127 genomic DNA was also used in a positive control reaction with ORF-specific primers (Figure V-7). Primers specific for the soybean endogenous cyclophilin gene were used in positive control reactions to confirm the quality of the template RNA (Figure V-6). The ORFspecific primers amplified a 435 bp fragment from CV127 genomic DNA (Figure V-7). However, no detectable RT-PCR pr oduct was observed using total RNA from leaf, root, leaf bud and seed tissue as a template, suggesting th at the ORF is not expressed in CV127 soybean (Figure V-6).CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 52 of 375Figure V-5.Diagram of Overlapping PCR Amplicons Used to Demonstrate the Integrity of the Insert in CV127 Soybean.The insert and flanking sequencesare displayed. Six amplicons used for sequencingof the insert that span the length of the insertfrom the 5’ to 3’ flankingregions are also indicated.csr1-2 CDSAt3g48570 CDSAt3g48570 intron 1At3g48570 intron 2Arabidopsis gDNA, unannotatedArabidopsis gDNA, unannotated376 bp from csr1-2 CDS5' flanking sequence3' flanking sequencePCR1PCR2PCR3PCR4PCR5PCR6AtAHASL 5'UTR & putative promoterAt3g48570 3' UTRAtAHASL 3' UTRAt3g48570 5' UTRNcoI (2761)XbaI (410)XbaI (10652)SpeI (1268)SpeI (5620)SpeI (6505)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 53 of 375 Figure V-6. RT-PCR Analysis of a 501 bp ORF C reated by Insertion of a 376 bp Portion of

the csr1-2 Coding Sequence at the 3’ Flanking Sequence Junction in CV127 Soybean.

Total RNA was extracted from l eaf, root, leaf bud a nd seed tissue from CV127 soybean and nontransgenic commercial variety Conquista with Qiagen RNeasy Mini Kit and then treated with DNase. RT-PCR was conducted using primers speci fic for the ORF 501 (a nticipated amplicon 435 bp). Primers specific for the soybean endogenous cyclophilin gene, that is constitutively expressed, were used in positive control reac tions (anticipated amplicon 315 bp). Panel A:

Seed. Panel B: Leaves. Pane l C: Leaf buds. Panel D: R oots. Lanes 1, 3, 5 and 7, ORF 501 reactions; lanes 2, 4, 6, and 8, cyclophilin reacti ons; Lanes 1, 2, 5 and 6 used Conquista RNA template; Lanes 3, 4, 7 and 8 used CV127 RNA as template. Lanes 1 – 4 reactions included reverse transcriptase whereas it was omitted in reactions shown in lanes 5 – 8 to serve as a

control. The entire 25 μ l reaction was loaded on the gel. Mo lecular weight markers (1 kb Plus DNA Ladder, Invitrogen) are in the lanes labeled M.



# A. B.

M 1 2 3 4 5 6 7 8 M

500 bp 400 300 200 500 bp 400 300 200



# C. D.

M 1 2 3 4 5 6 7 8 M

500 bp 400 300 200 500 bp 400 300 200 M 1 2 3 4 5 6 7 8 M

500 bp 400 300 200 500 bp 400 300 200

M 1 2 3 4 5 6 7 8 M

500 bp 400 300 200 500 bp 400 300 200CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 54 of 375 Figure V-7. PCR Analysis of Genomic DNA from CV127 Soybea n Confirming the

Presence of ORF 501. Genomic DNA from CV127 soybean and Conquista was evaluated for the presence of the ORF 501 and the cyclophilin gene to test the primers for ORF501 and verify the robustness of amplification c onditions. PCR primers used were identical to those applied in the RT-PCR experiments in Figure V-6. Panel A: lanes 1 – 4, s eed genomic DNA; lanes 5 – 8, leaf genomic DNA. Panel B: lanes 1 – 4, root genomic DNA; lanes 5 – 8, leaf bud genomic DNA. Lanes 1, 3, 5, and 7, ORF 501 PCR; lane s 2, 4, 6, and 8, cyclophilin PCR. Lanes 1, 2, 5 and 6, Conquista genomic DNA, Lanes 3, 4, 7, and 8, CV127 genomic DNA. The entire 25 μ l reaction was loaded on the gel. Molecular we ight markers (1 kb Plus DNA Ladder) are in the

lanes labeled M.



# A.

B. M 1 2 3 4 5 6 7 8 M

500 bp 400 300 200 500 bp 400 300 200

M 1 2 3 4 5 6 7 8 M

500 bp 400 300 200 500 bp 400 300 200CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 55 of 375 E . A. thaliana SEC61 γ

Subunit Gene

The linear DNA used to produce soybean CV127 cont ains a 2.5 kb segment that was originally

annotated as the csr1-2 gene promoter and 5’ untranslated region (UTR). More-recent sequence analysis has revealed that this segment also co ntains a previously unannotated Arabidopsis gene encoding the gamma subunit of SEC61 (At SEC61 γ ) , a small (69 amino acid) multimeric transport protein of the endoplas mic reticulum that is ubiquitous in all plants and other eukaryotes (Hartmann et al ., 1994). The CV127 soybean insert sequence contains the majority

of the At SEC61 γ subunit gene including the complete coding sequence. The At SEC61 γ 5’ UTR, as annotated by The Arabidopsis Information Resource, begins 18 nucleotides downstream from the 5’ junction of the transgene and soybean ge nomic DNA flanking the in tegration site. As such, it is extremely unlikely that the insert contains the complete native promoter for the

At SEC61 γ subunit gene.

The possible transcription of the Arabidopsis At SEC61 γ subunit gene found in the insert in CV127 soybean was evaluated using RT-PCR. RT-PCR was carried out using DNase-treated

total RNA extracted from the F 7 generation of CV127 soybean as a template. Primers specific to two endogenous soybean genes, Iota and Gm SEC61 γ, were used as positive controls to confirm the quality of the template RNA. Total RNA fr om Arabidopsis leaf and root tissues without DNase treatment was also used as a positive contro l. Results showed that a very low amount of mRNA specific to the At SEC61 γ subunit gene in the CV127 F 7 generation was present, indicating that the gene is only weakly transcribed (Fi gure V-8; for more de tails on the methods used see Appendix A). The amplified 393 bp At SEC61 γ subunit DNA band amplified by RTPCR is the same size as that amplified from Ar abidopsis leaves and roots (Figure V-8). The same set of primers also amplified a band of the expected size, 965 bp, from contaminating genomic DNA in Arabidopsis leaf and root samples (Figure V-8).

To confirm the identity of the soybean CV127 At SEC61 γ subunit gene transcript, the 5’ end of the Arabidopsis SEC61 γ subunit transcript in CV127 soyb ean was determined by RNA-ligase mediated rapid amplification of 5’ comple mentary DNA ends (RLM-5’-RACE). (For more

details on the methods used in the RLM-5’-R ACE experiment see Appendix A.) Figure V-9

shows the AtSec61 γ RLM-5’-RACE secondary PCR result for CV127. Secondary PCR amplifications us ing soybean Sec61 γ subunit-specific primer served as a positive control to confirm the ability to amplify endogenous soybean transcripts in the RLM-5’-RACE experiment. Due to the lack of a published full-length c DNA sequence, it was difficult to calculate an expected size for the endogenous soybean Sec61 γ subunit secondary PCR product. Using an expressed sequence tag (GenBank accession BM731537) which shows 100% identity to both the primary and secondary PCR primers, the endogenous soybean Sec61 γ subunit secondary PCR product could be roughly estimated to be 199 bp, taking into account that 30 bp of the PCR product are derived from the Gene Racer RNA Oligo. The soybean pos itive control lanes in both conventional cultivar Conquista and CV127 show PCR products with similar intensity at the estimated size for the endogenous soybean Sec61 γ subunit (Figure V- 9). This proportional amplification of the positive c ontrol indicates that the band obs erved with Arabidopsis SEC61 γ

specific primers in CV127, but not in conventi onal Conquista, is not due to non-specific background amplification.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 56 of 375 CV127 RLM-5’-RACE secondary PCR products amplified with Arabidopsis SEC61 γ subunitspecific primer RS42 are not a single distinct band, indicating multiple tr anscript sizes (Figure V-9). The presence of PCR products in the co rresponding “minus tobacco acid pyrophosphatase (TAP)” control lane indicates that some of these PCR produc ts are derived from partial transcripts. However, because the largest PCR product in this “minus TAP” control lane is clearly smaller than the larges t PCR products observed in the corresponding “plus TAP” lane, it is expected that the largest CV127 secondary PCR products amp lified with Arabidopsis SEC61 γ subunit-specific primers repres ent full-length transcripts. The largest CV127 RLM-5’-RACE secondary PCR product amplified with Arabidopsis SEC61 γ subunit

− specific primers is clearly larger than the PCR product amplified from Arabidopsis ecotype Col-0 with Arabidopsis

SEC61 γ subunit-specific primers s uggesting a transcriptional fu sion in CV127 (Figure V-9).

The resulting RLM-5’-RACE PC R product was sequenced and compared with the predicted mRNA sequence of the At SEC61 γ subunit gene. The sequence data obtained indicated that the Arabidopsis SEC61 γ -subunit transcript in CV127 soybean is a transcriptional fusion with a short segment (89 nucleotides) of adjacent soybean fla nking sequence. However, the first “ATG” start codon in the sequence coincides with the predicte d translation initiation site of the native Arabidopsis SEC61 γ -subunit gene. As there are no changes at the nucleotide level, the predicted protein sequence of the Arabidopsis SEC61 γ -subunit protein in soybean CV127 does not differ

from the native SEC61 γ -subunit protein in Arabidopsis. Furt her experiments were conducted to

determine if detectable amounts of the AtSEC61 γ -subunit protein are produc ed in CV127 tissues. These experiments demonstrated that ther e are no detectable amounts of the SEC61 γ -subunit protein present in CV127 tissues (LOD = 30 ng/g fr esh weight for leaf tissue and 110 ng/g grain tissue). Further details of these experi ments are presented in Section VI.E.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 57 of 375 Figure V-8. RT-PCR Analysis of Transcription of the At SEC61 γ Subunit Gene in CV127 Soybean

. Total RNA from CV127 F 7 generation plant leaf tissue was ex tracted with Qiagen RNeasy Mini

Kit and treated with DNase and total RNA from Arabidopsis leaf (At-L) and root (At-R) was extracted with TRIzol reagent without DNase treatment. Primers specific to the soybean proteasomal Iota subunit gene and the endogenous soybean SEC61 γ subunit gene (Gm SEC61 γ ) are used in positive contro l reactions. Reactions without template RNA using At SEC61

γ subunit-specific primers and Gm SEC61 γ subunit-specific primers are used as negative controls.

M: 1 kb DNA ladder. The arrow indicates the faint RT-PCR product correspondi ng to the

At SEC61 γ subunit gene fragment amplified from soybean CV127.

298 506, 517 At-L MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

1018 bp

396 1018 bp

506, 517
396

344

298 344 298 506, 517 At-L MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

1018 bp

396 1018 bp

506, 517
396

344

298 298 506, 517 At-L MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

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506, 517
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298 506, 517 At-L MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

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396 1018 bp

506, 517
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298 At-L MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

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506, 517
396

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298 At-L MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

1018 bp

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506, 517
396

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298 MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

1018 bp

396 1018 bp

506, 517
396

344

298 MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

1018 bp

396 1018 bp MIOTAGmsec61Atsec61Atsec61Gmsec61Atsec61

Event 127 F7 At-R MAtsec61

1018 bp

396 1018 bp

506, 517
396

344

298 344CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 58 of 375 Figure V-9. RLM-5’-RACE Seconda ry PCR Analysis of the At SEC61 γ Subunit Gene in CV127 Soybean. Messenger RNA extracted from CV127 (“127” in figure below), conventional soybean variety Conquista (“C” in figure below), and Arabidopsis ecotype Col-0 (“At” in figure below) leaf tissue was used as a template for RLM-5’-RAC E. In the RLM-5’-RACE secondary PCR, the GeneRacer 5’ Nested Primer (Invitrogen) was paired with either a primer expected to specifically amplify endogenous soybean Sec61 γ (“Gm” in figure below) or a primer expected to specifically amplify Arabidopsis SEC61 γ (“At” in figure below). Lanes 7 through 12 are identical to lanes 1 to 6, respectively, with th e exception that lanes 7 th rough 12 were not treated with tobacco acid pyrophosphatase (TAP) during the RLM-5’-RACE procedure. Samples in lanes 7 through 12 serve as contro ls: any products observed in th ese lanes are derived from nonfull-length transcripts. Lanes labeled “M ” each contain 700 ng of 1 kb Plus DNA Ladder (Invitrogen).

Summary of Expected and Observed Re sults for RLM-5’-RACE Secondary PCR

Lane in

Gel mRNA Source Gene-Specific Secondary PCR

Primer Expected

Band Size Expected

Result

Observed? 1 Conquista leaf Sec61 γ , soybean ~199 bp a yes 2 Conquista leaf SEC61 γ , Arabidopsis none yes

3 CV127 leaf Sec61 γ , soybean ~199 bp a yes 4 CV127 leaf SEC61 γ , Arabidopsis ? smeared band 5 Arabidopsis leaf Sec61 γ , soybean none yes 6 Arabidopsis leaf SEC61 γ , Arabidopsis 342 bp b yes

a This “expected” band size is estimated: 30 bp are deri ved from the GeneRacer RNA Oligo and an estimated 169 bp

are derived from soybean Sec61 γ

. b A 342 bp band is expected: 30 bp are derived from the GeneRacer RNA Oligo and 312 bp are derived from

Arabidopsis SEC61 γ

. PCR Primer:

mRNA Source:

M 1 2 3 4 5 6 7 8 9 10 11 12 M

1,000 Base pairs
3,000

1,650

500

100 300 400

200 650 M 1 2 3 4 5 6 7 8 9 10 11 12 M

1,000 Base pairs
3,000

1,650

500

100 300 400

200 650 1,000 Base pairs
3,000

1,650

500

100 300 1,000 Base pairs
3,000

1,650

500

100 300 Base pairs
3,000

1,650

500

100 300 400

200 650 Gm At Gm At Gm At Gm At Gm At Gm At

C C 127 127 At At C C 127 127 At At + TAP

− TAP (control reactions)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 59 of 375 F. Inheritance of the Imidaz olinone-Tolerance Trait in CV127.

The inheritance of the imid azolinone herbicide tolerance trait in CV127 soybeans was determined by evaluating the F 3 and F 4 progeny from the cross of CV127 line CV603 (F 8 generation, homozygous for the csr1-2 gene) with a conventional, imidazolinone-sensitive line with the pedigree Conquista *3 x BRI98-641 (Figure III-1). Individual F 1 seed, hemizygous for

the csr1-2 gene, were planted in a greenhouse and th e plants were self-pollinated to produce individual populations of F 2 seed segregating for the csr1-2 gene (one F 2 population per each F 1 plant). Seed from one F 2 population were planted in the greenhouse and each plant was selfpollinated to produce individual F 3 families (one F 3 family per each F 2 plant).

Seed from each F 2 -derived F 3 family (F 2:3 ) were planted in a greenhous e and six to eight plants within each family were sprayed with imazapyr he rbicide (100 g ai/ha) to identify families that were segregating for the csr1-2 gene. The number of homozygous, hemizygous, or null plants present within each of two fa milies, ‘family 12’ and ‘family 13’, segregating for the csr1-2 gene was determined by a quantitative PCR assay. Th e proportion of individuals in families 12 and 13 that were homozygous, hemizygous, or null for the csr1-2 gene and the results of the chisquare test for the 1:2:1 segreg ation pattern expected for a singl e gene are presented in Table V2. The results of the chi-square test (P<0.05) support the single-gene hypothesis and indicate

that tolerance to the imidazoli none herbicide is expressed by a single gene that is inherited according to the principles of Mendelian genetics.

Each F 3 plant within families 12 and 13 which was identified as hemizygous based on the PCR assay, was self-pollinated. The F 4 seed from each hemizygous F 3 plant were bulke d together, by family, and planted in a greenhouse. After spraying each F 4 plant with imazapyr (100 g ai/ha), each plant was classified ‘resistant’ (no foliar sy mptoms) or ‘sensitive’ (f oliar symptoms and/or plant death) to the herbicide. The proportion of individuals in families 12 and 13 that were resistant (homozygous or hemizygous) or sensitiv e (null) to the imidazolinone herbicide and the results of the chi-square test for the 3:1 segr egation pattern expected for a single gene are presented in Table V-3. The results of the chi- square test (P<0.05) again support the conclusion

that tolerance to an imidazolinone herbicide is due to a single dominant gene that is inherited in a typical Mendelian fashion. Further, the results of these genetic studies ar e consistent with the

conclusions of the molecular characterization studies conduc ted with CV127 soybean which showed that a single functional csr1-2 gene is integrated in the genome of CV127.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 60 of 375 Table V-2. Chi-Square Analysis Demonstrating Single -Gene Mendelian Inheritance (1:2:1 Segregation) of the csr1-2 Gene in Two F 3 Families, Each Derive d from an Individual Hemizygous F 2 Plant, Based on a Quantitative PCR Assay.

Family 12

PCR result Observed number of plants Expected number of plants Chi-square value

for 1:2:1 hypothesis Hemizygous 30 28.50 Homozygous (positive) 18 14.25 Homozygous (negative) 9 14.25 X 2 = 3.00

P = 0.22

Total 57 57 Family 13 Hemizygous 23 26 Homozygous (positive) 14 13 Homozygous (negative) 15 13 X 2 = 0.73

P = 0.69

Total 52 52

Table V-3

. Chi-Square Analysis Demonstrating Sing le-Gene Mendelian Inheritance (3:1 Segregation) of the csr1-2 Phenotype in Two F 4 Progeny Derived from Hemizygous Plants

from Two F 3 Families Based on a Herbicide Tolerance Assay.

Family 12

Herbicide result Observed number of plants Expected number of plants Chi-square value for 3:1 hypothesis Tolerant (homozygous or hemizygous) 493 508 Sensitive (null) 185 169 X 2 = 1.89

P = 0.17

Total 678 678 Family 13 Tolerant (homozygous or hemizygous) 330 340 Sensitive (null) 124 113 X 2 = 1.30

P = 0.26

Total 454 454

G. Conclusions of the Mo lecular Characterization

Molecular analyses were performed to charac terize the integrated DNA insert in CV127 soybean. Southern blot analyses were used to determine the number of DNA insertions in the soybean genome, the number of copies of the insert, the intactness of the csr1-2 gene cassette,
and to establish the abse nce of plasmid backbone elements in the genome. The stability of the DNA insert across multiple generati ons was also demonstrated by Southern blot analysis. Finally, the complete nucleotide sequence of the insert was determined to confirm the organization and integrity of the gene tic elements within the insert.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 61 of 375 The results of these analyses demonstrated that one intact copy of the csr1-2 gene cassette was integrated at a single locus in the chromosome. No elements derived from the backbone of the plasmid pAC321 either linked or un linked to the insert were detected in the genome of CV127. Southern blot analyses of DNA isolated from multiple generations of CV127 soybean demonstrated that the transgene insert is stably integrated in the soybean genome over multiple breeding generations. In addition, determination of the nucleotide sequence of the entire insert

confirmed the intactness of the insert and demonstrated that the organization of the genetic elements are identical to their organization in plasmid pAC321. Also, trait inheritance studies across breeding generations confir med the expected trait segregat ion ratios, further confirming

that the imidazolinone herbicid e tolerance trait in CV127 soyb ean is conferred by a single functional copy of the csr1-2 gene and that the DNA insert in CV127 soybean is stably integrated in the plant genome across multiple breeding generations.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 62 of 375 

### VI. Characterization of Protei ns Expressed in CV127 Soybean

CV127 soybeans were produced by introduction of the imidazolinone-tolerant acetohydroxyacid synthase large subunit gene csr1-2 from Arabidopsis thaliana into the soybean plant genome.

The csr1-2 gene encodes an AtAHAS protei n that is tolerant to imid azolinone herbicides due to a single nucleotide mutation that results in a sing le amino acid substitution in which the serine residue at position 653 of the protein is repl aced by asparagine (S653N). This amino acid change in plant AHAS proteins is known to prev ent the binding of imid azolinone herbicides and thereby to result in toleran ce to these herbicides (Tan et al. , 2005).

As part of the food, feed and environmental sa fety assessment of CV 127 soybean, studies were conducted to confirm that the AtAHAS protein produced in CV127 soybeans is equivalent to other AHAS proteins that are found ubiquitously am ong plant species and have a history of safe use in food and feed products, as well as safety to the environment. These safety assessments of the AtAHAS protein included a mouse acute gava ge study and a digestive fate study conducted with purified AtAHAS protein. Because the At AHAS protein is expressed at extremely low levels in tissues of CV127 soybean plants (refer to Section VI.D. of this petition), it was not technically feasible to extract sufficient quantit ies of the AtAHAS protein from soybean tissues for the safety assessments of the protein. Therefore, the same AtAHAS coding sequence introduced in the CV127 soybean genome was introduced into Escherichia coli for overexpression of the AtAHAS protein. Gram quantities of the AtAHA S protein were purified from

the Escherichia coli over-expression system for the prot ein safety assessment studies. Key biochemical and functional parameters of the CV127-produced and Escherichia coli -produced AtAHAS proteins were characte rized to demonstrate that th e plant-produced protein is equivalent to the microbially- produced AtAHAS protein, and ther efore justify the use of the microbially-produced protein for safety assessm ent studies of the AtAHAS protein produced in CV127 soybean.

This section of the petition presents the fo llowing characterization studies of the AtAHAS protein expressed in CV127 soybeans: A. Biochemistry of the AtAHAS protein and imidazolinone herb icide tolerance;

B. Characterization of the At AHAS protein produced in CV127 soybean and equivalence to

the E. coli -produced protein used in safety studies; C. A summary of the food, feed and envir onmental safety of the AtAHAS protein; D. Expression levels of the AtAHA S protein in CV127 soybean tissues.

In addition, studies are presen ted in this section of the pe tition that address whether the

AtSEC61 γ subunit protein is expressed in CV127 s oybean tissues, and a summary of the food, feed and environmental safety of the AtSEC61 γ subunit protein is included.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 63 of 375 A. Biochemistry of the AtAHAS Protein and Imidazolinone Herbicide Tolerance

The mechanism of action of imidazolinone herbic ides on weeds and non-tole rant plants is by inhibition of the AHAS enzyme and branched -chain amino acid biosynthesis. The AHAS enzyme is ubiquitous in plants and microbes and cat alyzes the first step in the biosynthesis of the essential branched-chain amino acids valine, le ucine, and isoleucine (Stidham and Singh, 1991). The AHAS enzyme of eukaryotes is composed of a large catalytic s ubunit (AHAS) and a small regulatory subunit. The enzyme catalyzes the condensation of two mol ecules of pyruvate to form acetolactate, the precursor of valine and le ucine, or the condensa tion of a molecule of pyruvate with a molecule of 2-ketobutyrate to form 2-aceto-2-hydroxybutyrate, an intermediate in isoleucine biosynthesis (Delfourne et al ., 1994; Singh and Shaner, 1995; Duggleby and Pang, 2000). AHAS is the key control enzyme within the branched-chain amino acid biosynthetic pathway and is regulated by feedback inhibition by the end-product amino ac ids, valine, leucine and isoleucine. Regulation is mediated through binding of these amino acids to the AHAS small (regulatory) subunit.

Imidazolinone herbicides readily inhibit the activ ity of AHAS by binding to the active site of the protein resulting in plant death (Duggleby and Pang, 2000; Tan et al ., 2005). Molecular modeling of the AHAS–imidazolinone interaction sugge sts that the herbicide binding pocket is at the entry site for the substrate of the AHAS en zyme, and imidazolinones may inhibit the enzyme by impeding substrate binding to AHAS (Tan et al ., 2005). Studies have shown that specific single-nucleotide mutations in the AHAS genes result in single amino acid substitutions in the AHAS protein, and these amino acid substitutions confer tolerance to imidazolinone herbicides by altering the binding site for these herbic ides on the mutant AHAS enzymes (Tan et al ., 2005).

Imidazolinone herbicide-tolerant amino acid substitu tions are located in all three domains of the AHAS protein, but protein folding places all the amino acid substitu tions that confer herbicide tolerance at the proposed herb icide-binding site of the pr otein. While these amino acid

substitutions confer herbicide tolerance to the AHAS protein, th ey have no effect on feedback regulation by branched-chain amino acids and the normal biosynthetic function of the enzyme (Newhouse et al ., 1992).

CV127 soybean contains the csr1-2 gene from Arabidopsis thaliana that encodes an imidazolinone-tolerant AHAS enzyme (Sathasivan et al ., 1990). The csr1-2 gene encodes a single polypeptide of 670 ami no acids that includes the A. thaliana native chloroplast transit peptide (CTP) which is predicted to consist of 85 amino acids (Mazur et al ., 1987). During transport into the chloroplast, it is predicted th at the chloroplast transit peptide is removed to produce the mature AtAHAS enzyme that is approximately 64,000 mo lecular weight and consists of 585 amino acids (Mazur et al ., 1987). The amino acid residues of AtAHAS are

counted from the N-terminus of the 670 amino aci d full-length protein containing the chloroplast transit peptide. The csr1-2 gene is expressed from its native A. thaliana promoter and it also contains the native 3’-untranslated region (UTR) bearing the polya denylation signal.

The AtAHAS protein encoded by the csr1-2 gene in CV127 soybean is functionally identical to the native AtAHAS, except for its tolerance to imidazolinone herb icides. The mutation in the

csr1-2 gene responsible for imidazolinone herbicide tolerance is a single nucleotide change ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 64 of 375 guanine to adenine, which results in a codon ch ange from AGT to AAT and a single amino acid substitution of serine to asparagine at posit ion 653 of the AtAHAS protein. This amino acid change in plant AHAS proteins is known to conf er imidazolinone herbicide tolerance, but have no effect on feedback regulati on by branched-chain amino acids or normal biosynthetic function of the enzyme (Newhouse et al ., 1992; Tan et al ., 2005). In addition, a second mutation was

discovered in the csr1-2 gene integrated in the genome of CV127 soybean. This mutation, in which an arginine residue at position 272 is re placed by lysine, does not impact the enzymatic function of the AHAS enzyme or herbicide tolerance properti es. A study was conducted to compare the AtAHAS subunit protein expressed in CV127 that contains both the S653N and R272K mutations with the AtAHAS encoded by the csr1-2 gene that contains only the S653N mutation. In this study it was demonstrated that both AtAHAS enzymes had equivalent levels of catalytic activity and toleran ce to imidazolinone herbicides. Feedback regulation of AHAS activity by the branched-chain amino acids is e ffected through the AHAS small subunit. The AtAHAS enzyme encoded by the csr1-2 gene in CV127 is expected to interact with the endogenous soybean AHAS small subunit protein (AHASS) to achieve this regulation. Therefore, the feedback regulation of the AtAHAS encoded by the csr1-2 gene in CV127 was expected to be identical to that of the e ndogenous soybean AHAS. This was confirmed in enzyme activity studies with the CV127-produced AtAHAS protein (section VI.B.). Also, this conclusion was supported by results of grain com positional analyses that showed levels of branched-chain amino acids in CV127 soybean are comparable to levels in the control soybean (refer to section VII. of this petition), indicatin g that regulation of this biosynthetic pathway is equivalent between CV127 soybean and its conventional comparators.

Several imidazolinone herbicide-tolerant crops that produce AHAS enzymes with the same serine to asparagine substitution at residue 653 have been commercialized and cultivated for many years without any adverse environmental or health effects. Th ese crops have been

marketed under the Clearfield ® brand name and include ( Zea mays L.), rice ( Oryza sativa L.), bread wheat ( Triticum aestivum L.), and oilseed rape ( Brassica napus L.). Therefore, there has been a long history of safe production of crops containing an imidazolinone herbicide-tolerant AHAS protein with the same S 653N amino acid substitution as that in the AtAHAS encoded by

the csr1-2 gene in CV127 soybeans. These crops have been used to produce food and feed products that have proven to be as nutritious and as safe as similar products produced from conventional crops, and have posed no different environmental impact than the corresponding conventional crop.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 65 of 375 B. Characterization of the At AHAS Protein Produced in CV127 Soybean and Equivalence

to the E. coli -Produced Protein Used in Safety Studies

As described above in this sectio n of the petition, it was not tec hnically feasible to purify gram quantities of the AtAHAS protei n from CV127 soybean to conduc t protein safety assessment studies. Therefore, the same coding sequence for the predicted mature AtAHAS protein in CV127 soybean was introduced into Escherichia coli for over-expression of the AtAHAS protein, and the microbially-produced protein was used for the protein safety studies, including digestive fate and an acute mouse gavage st udy. Therefore, the evaluation of the AtAHAS protein produced in CV127 soybean is presented in this section and the results are compared to

those of a similar evaluation of the protein produced in the Escherichia coli expression system in order to establish equivalence between these AtAHAS proteins from different sources, and justify use of the protein safe ty data generated from the Escherichia coli -produced protein to confirm the safety of the AtAHAS produced in CV127 soybean.

The AtAHAS produced in the Escherichia coli expression system lacks the predicted 85 amino acid N-terminal chloroplast transit peptid e (CTP) sequence that targets the protein in planta to the chloroplast (Mazur et al ., 1987). The Escherichia coli -produced AtAHAS protein contains the S653N amino acid substitution presen t in the AtAHAS prot ein encoded by the A. thaliana

csr1-2 gene, as well as the R272K amino acid substitution that was discovered in the csr1-2 gene in CV127 soybean. AHAS protein was purified fr om young leaf tissue of CV127 soybean plants

and the biochemical characteristics of the protein were assessed and compared to that of the E.

coli -produced AtAHAS protein. The biochemical characteristics that were assessed included: 1) molecular weight, 2) immunoreactivity with AHAS-specific antibody, 3) enzymatic activity and the inhibition of the activity by imidazolinone he rbicide and feedback inhibition by branchedchain amino acids, 4) glycosylation, and 5) determination of the amino acid sequence of several peptide fragments derived from purified AHAS pr otein preparations. Th e results from these

evaluations show that the CV 127 soybean-produced AtAHAS protein is equivalent to the AtAHAS protein produced in Escherichia coli . A summary of the findings of these analyses are presented below and the materials and methods and a detailed description of the studies are presented in Appendix B of this petition.

The molecular weight and immunoreactivity of the AHAS produced in CV127 (AtAHAS and endogenous soybean AHAS) were shown to be similar to that of the endogenous AHAS from the nontransgenic isoline control soybean a nd the reference AtAHAS produced in the Escherichia

coli over-expression system. The microbially-pr oduced AtAHAS and the plant-produced AHAS proteins demonstrated the expected enzymatic activity with the substr ate pyruvate, confirming the same functional activity of the AHAS protei ns from both microbial and plant sources. The specific activity of the AHAS produced in CV 127 soybean was very sim ilar to the endogenous soybean AHAS and was higher than the AHAS in AtAHAS produced in Escherichia coli

. AHAS is a very unstable protein and readil y loses activity upon pur ification (Chang and Duggleby, 1997). Therefore, it was not unexpected that the AHAS activity in the relatively impure state purified from plant tissues was highe r than that retained in the much more pure material produced in Escherichia coli . Both CV127-produced and microbially-produced AtAHAS proteins showed tolerance to inhibi tion by the imidazolinone herbicide imazethapyrCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 66 of 375 compared to the control plant-produced protein. An intermediate level of inhibition of the CV127-produced AHAS activity was observed, and this was as expected due to the mixture of sensitive (endogenous) and tolerant (transgenic) AHAS proteins present in CV127. Feedback inhibition by branched-chain amino acids, leuc ine and valine, was very similar for both the AHAS proteins in CV127 and the nontransgenic is oline control soybean. No feedback inhibition was observed for the microbially-produced AtAHAS as expected. Acetohydroxyacid synthase in

Escherichia coli is composed of two subunits. However, pr evious studies have demonstrated that

when the A. thaliana acetohydroxyacid synthase catalytic (large) subunit (AtAHAS) is expressed

in E. coli , the E. coli small (regulatory) subunit is incapable of interacting with the Arabidopsis

thaliana catalytic or “large” subunit to confer the feedback inhibition observed in Arabidopsis

thaliana seedlings (Singh et al ., 1992; Chang and Duggleby, 1997). This result also confirms that the endogenous soybean AHASS must inte ract with the AtAHAS in CV127 soybean to effect feedback regulation by the branched-cha in amino acids. There was no evidence of glycosylation associated w ith either the AtAHAS in CV 127 soybean leaves or the Escherichia

coli -produced protein.

The amino acid sequence of th e AtAHAS protein produced in CV127 soybean was compared with that produced in the Escherichia coli expression system. Attempts to obtain N-terminal amino acid sequence of the plant-produced prot ein using Edman degradation methods were unsuccessful. Therefore, SDS-PAGE followed by liquid chromatography coupled with tandem mass spectroscopy (LC/MS/MS) was used to obtain amino acid sequence data. Peptides derived from trypsin-treated AtAHAS protein prepared from CV127 soybean were isolated and their amino acid sequence determined by liquid LC/MS/MS. The amino acid sequences of peptides derived from CV127 soybean were compared to the deduced amino acid sequence of the AtAHAS protein encoded by the csr1-2 gene in CV127 soybean. The amino acid sequence obtained covered approximately 23% of the en tire predicted AtAHAS amino acid sequence of the CV127-produced protein. All amino acid se quence data obtained from the AtAHAS of CV127 soybeans was identical to the co rresponding amino acid sequence of the Escherichia

coli -produced AtAHAS with the exception of a region near the N-terminus th at was predicted to contain the CTP. Based on a sequence comparison of the deduced amino acid sequence of plant and microbial AHAS proteins, Mazur et al. (1987) predicted that the CTP of the AtAHAS is

cleaved at the C-terminal side of residue 85. However, because AHAS proteins are present at very low levels in plant tissues, the amino aci d sequence of the mature AtAHAS has not been determined and the precise location of the cl eavage site for the CTP is unknown. Amino acid sequence analysis of the AtAHAS protein pr oduced in CV127 showed a peptide fragment corresponding to amino acid residue s 52 to 73. Therefore, the CT P cleavage site of the AtAHAS protein produced in CV127 is most likely not at the pr edicted site at the C-terminal side of residue 85, but is located at least at the C-terminal side of residue 51.

Even though the data in this study suggest that the mature AtAHAS protein produced in CV127 consists of the predicted matu re protein (585 amino acids) plus additional amino acids in the CTP predicted by Mazur et al . (1987), the m icrobially-produced AtAHAS protein is considered equivalent to the CV127 protein for the following reasons. The plant-produced and E. coli

produced proteins should show structural, f unctional, and biochemical equivalence (Codex, 2003). The CV127-produced AtAHAS and E. coli -produced AtAHAS demonstrated the same expected enzymatic activity with the substrat e pyruvate, thus confirmi ng that AtAHAS fromCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 67 of 375 both sources possessed the same functional activit y. Both are resistant to inhibition by the imidazolinone herbicide imazethapyr (Figure B-2), a characteristic produc ed by the serine to asparagine amino acid substitution at residue 653 (relative to the Arabidopsis thaliana sequence).

CV127 leaf extracts contain a mixture of both AtAHAS (R272K, S653N) and endogenous GmAHAS and exhibited similar sensitivity to feedback inhibition by the metabolic pathway products, branched–chain amino acids, as leaf extr acts from the isoline control soybeans (Figure B-3). Due to the absence of the acetohydroxyacid synthase small (regulatory) subunit in the E.

coli expression system, the E. coli -produced Arabidopsis thaliana acetohydroxyacid synthase large (catalytic) subunit did not display feedb ack inhibition by leucine and valine. This observation is not surprising given that the feed back inhibition is mediated by the regulatory (small) subunit. In planta , it is believed that the endogenous Glycine max acetohydroxyacid synthase regulatory (small) subuni t is capable of inte racting with the AtAHA S large (catalytic) subunit to impart the branched-chain amino acid f eedback inhibition charac teristic to both the AtAHAS and GmAHAS catalytic subunits, thus preserving normal biochemical control mechanisms for a crucial anabolic pathway in CV127 soybean plants.

In addition, the microbially-produced AtAHAS protein and CV127-produced AHAS proteins had similar immunoreactivity to antibodies specifi c to the AHAS protein based upon results of both western blot and ELISA analyses. Both the microbially-produced AtAHAS protein and the CV127-produced protein showed no ev idence of glycosylation. Als o, both proteins shared the same amino acid sequence of th e predicted mature protein of 585 amino acids, based upon amino acid sequence analysis of overlappi ng peptide fragments. Finally, in vitro digestibility and lability studies also suppor ted the equivalence of th e CV127-produced AtAHAS and E. coli

produced AtAHAS proteins. Because some food allergens show resistance to digestion by the gastric enzyme pepsin, the CV127-produced AtAHAS and E. coli -produced AtAHAS were tested for susceptibility to digestion by pepsinin simulated mammalian gastric fluid (Figure H1). Both the E. coli -produced AtAHAS as well as AtAHAS from grain and leaf extracts of CV127 were rapidly degraded in simulated mammalian gastric fluid. Thus, AtAHAS from CV127 was not shown to display this characteristic of a llergenic proteins and was equivalent to

the E. coli -produced AtAHAS in this respect.

Furthermore, other studies confirm the food, f eed and environmental safety of the AtAHAS protein expressed in CV127 soybean.

History of safe use The acetohydroxyacid synthase large subunit protein is ubiquitous in the plant world. As such, AHAS has a long history of safe use in the diet of humans and animals. While AtAHAS (R272K, S653N), as expressed in CV127, and the microbi ally-produced AtAHAS sequences do differ at the N-terminal end, numerous other dicoty ledon AHAS sequences also show significant sequence divergence at the N-terminal end in th e region corresponding to the transit peptide. Although Arabidopsis thaliana is not a commonly-consumed crop, the AtAHAS sequence shows extremely high protein sequence identity to AH AS from commonly-consumed crop species: 90% identity to Brassica napus AHASL1A (access ion number P27819), 79% identity to Glycine max AHASL2 (JGI locus Glyma13g31470.1), 77% identity to Helianthus annuus AHASL1 (AAT07322), 77% identity to Phaseolus vulgaris AHASL (ACV84152), and 77% identity to

Solanum tuberosum AHASL (ADI56521.1). More over, the model plant Arabidopsis thaliana isCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 68 of 375 closely related to Brassica oleracea , a species which includes the widely consumed cultivars broccoli, cabbage, brussel sprouts, cauliflower, collard greens, and kohlra bi (Zhang and Wessler, 2004). Thus, while Arabidopsis thaliana itself does not have a histor y of safe use, it shares a

common ancestor with a relative with a record of safe consumption.

The AtAHAS (R272K, S653N) prot ein expressed in CV127 soyb ean differs from the AtAHAS (P197S) protein expressed in su lfonylurea herbicide-tolerant Linum usitatissimum L. (flax) CDC Triffid (FP967) by only three ami no acids (McHughen, 1989; Nakamura et al ., 2010). As is the case in CV127, the AtAHAS (P197S) coding sequence in flax line FP967 is under the control of

the native Arabidopsis thaliana AHAS large subunit promoter and would be expected to display an expression pattern similar to that of AtAHAS in CV127. The environmental safety of the flax line FP967 was reviewed by the USDA and approved for unconfined environmental release in the United States 12 years ago (Uni versity of Saskatchewan, 1999).

Bioinformatics analyses The full-length deduced amino acid sequence of AtAHAS (R272K, S653N), as expressed in CV127 prior to cleavage of the transit peptide, wa s subjected to bioinformatics analyses to assess its allergenicity and toxicity potential. The CV127 full-length AtAHAS deduced amino acid sequence, including the entire transit peptide, did not show significant homology to a known protein toxin as defined in the United States Code of Federal Regulations ( 40 CFR Part 725.421 ). The CV127 full-length AtAHAS dedu ced amino acid sequence did no t show greater than 35% identity over 80 amino acids to a potential allergen and did not share a sequence of eight or more

consecutive identical amino acids with a potential allergen. Thus , the bioinformatics analyses of the full-length AtAHAS (R272K, S653N), as expre ssed in CV127 soybean prior to cleavage of the transit peptide, do not provide any indication of a potential allergenicity or toxicity concern due to sequence homology to known protein toxins or known allergens.

Expression level and dietary intake AtAHAS in CV127 utilizes its native plant promot er which expresses low levels of AtAHAS, consistent with typical native AHAS expression pa tterns. The resulting low level of dietary exposure is a key consideration since acu te toxicity is related to dosage.

The level of exposure to a given protein is also a factor in pot ential allergen icity since the majority of major food allergens are expressed at a high level, gr eater than 1% of total protein (Lehrer et al. , 2002; Metcalfe et al. , 1996). AtAHAS protein levels in CV127 grain were shown to be detectable, but no t quantifiable, in enzyme-linked im munosorbent assays (ELISAs) using AHAS-specific antibodies (section VI.D.). The AtAHAS protein is very unstable and would be expected to rapidly degrade under the extreme c onditions present during standard soybean grain processing, further reducing the already-low levels of AtAHAS in CV127 in human food and

animal feed.

Additional safety a ssessment studies A 42-day broiler feeding study was conducted to compare the performance of poultry fed soybean meal from CV127 soybeans and broilers fed meal from conventional soybean grain (Table VII-11). This study dem onstrated that there was no evidence of a significant difference in the performance of the broilers fed a feed containing soybean me al from CV127 soybeanCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 69 of 375 compared to those fed a feed containing soybean meal from the grain of conventional soybeans. Thus, the nutritional equivale nce of CV127 and its isoline c ontrol were confirmed and no adverse effects were observed in broilers fed meal containing CV127 soybeans.

Therefore, results of these studies justify the use of the microbially-pr oduced AtAHAS protein as an appropriate substitute for the AtAHAS produced in CV127 in safety assessment studies of the AtAHAS protein (see Appendices B and H for details of these studies).

C. A Summary of the Food, Feed and Enviro nmental Safety of the AtAHAS Protein

The data and information relevant to a safety assessment of the AtAHAS protein produced in CV127 soybean are summarized and reviewed in this section. More detailed information relating to the safety of this protein is repo rted in other sections of this petition. A comprehensive food and feed safety and nutri tional assessment of CV127 soybean has been submitted to the FDA and it includes the following conclusions:

1. The source organism of the csr1-2 gene that encodes the AtAHAS protein in CV127 soybean, Arabidopsis thaliana , is not a known human or anim al pathogen and is not known to cause allergic reactions in humans. Similarly, A. thaliana is not known to produce toxic

substances.

2. The AtAHAS protein expressed in CV127 soybean is structura lly and biologically closely related to the AHAS proteins in all plants. For example, there is 79% amino acid identity between the AtAHAS and endogenous soyb ean AHASL2 (JGI locus Glyma13g31470.1) proteins. Similar levels of amino acid iden tity and homology are observed between AtAHAS and the AHAS proteins from corn and wheat. Therefore, the AtAHAS protein expressed in CV127 soybean is highly homologous to the AHA S proteins present in these crops with a history of safe production and use in food and feed products.

3. Several imidazolinone-tolerant crops that pr oduce AHAS enzymes with the same serine to asparagine substitution at residue 653 that is present in CV127 soybeans have been commercialized and cultivated for many years w ithout any adverse environmental or health effects. These crops have been marketed under the Clearfield ® brand name and include maize ( Zea mays L.), rice ( Oryza sativa L.), bread wheat ( Triticum aestivum L.), and oilseed rape ( Brassica napus L.).

4. An acute oral toxicity study with mice was conducted with purified AtAHAS protein and the

results demonstrated no adverse effects in mice with a No Observable Effect Level (NOEL) of equal to or greater than 2620 mg AtAHAS protei n/kg body weight, the highest dose level

tested.

5. Bioinformatic analyses showed no biologi cally relevant amino acid sequence homology between the AtAHAS protein expressed in CV 127 soybean and known toxins. In addition, analyses were conducted to confirm that the AtAHAS ami no acid sequence did not share potentially immunologically relevant amino acid sequence segments or structure with known allergens. Results of these analyses show ed that the amino acid sequence of the AtAHASCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 70 of 375 protein expressed in CV127 soybean did not sh are either 35% or gr eater identity over 80 amino acids or a sequence of eight or more consecutive identical amino acids with any potential allergens. Furthermore, the AtAHA S protein is rapidly degraded in simulated digestive fluids and is rapidly inactivat ed by temperatures a bove 60°C, which are characteristics typical of other dietary proteins with a history of safe use in food and feed products.

6. The AtAHAS protein is present at extr emely low levels in CV127 soybean grain, approximately less than 1.5 x 10 -6 % on a dry weight basis (refer to section VI.D. of this petition). Based on this level of protein expr ession in the grain, as well as average U.S. consumption of soybean food products, it was estimated that the average U.S. dietary exposure to the AtAHAS protei n expressed in CV127 soybean would be no greater than

0.5x10 -10 g/kg body weight/day. Compared to the NOEL of >2620 mg/kg body weight, the exposure level represents a human dietar y safety factor of greater than 10 10 for the AtAHAS protein in CV127 soybeans.

7. The AtAHAS produced in CV127 soybean is an enzyme, all enzymes are proteins, and all proteins have a primary structur e that consists of their amino acid sequence. Since proteins are regarded as nutrients and are degraded in the vertebrate digestive system to their constituent amino acids, the main basis for toxicity of a protein is due to its biological mode of action. The mode of action of AHAS enzy mes is well known and there is no scientific basis to suggest that its activity would result in toxicity.

Therefore, results of these st udies show that the AtAHAS prot ein expressed in CV127 soybean does not possess any attributes of known food allergens, is not toxic to mammals, and supports

the environmental as well as food and feed sa fety of the AtAHAS protein expressed in CV127 soybean.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 71 of 375 D. Levels of the AtAHAS Protein in CV127 Soybean

To assess the level of production (or expression) of the AtAHAS enzyme in CV127 soybean that is encoded by the csr1-2 gene, the amount of AHAS protein wa s determined in tissues of CV127 soybean and the isoline control that were grow n in Brazil in two different growing seasons.

Seven field trials in the 2006/ 2007 growing season and six in the 2007 growing season were conducted in geographically distinct locations th at were representative of commercial soybean production areas in Brazil. The fiel d sites were located in or near the locations listed as follows:

Field Trial Locations for the AHAS Expression Study

Location Trial Season 1

Oct 2006-Mar 2007 Trial Season 2 Feb –July 2007

Santo Antônio de Posse – SP 

### √

Ponta Grossa – PR/South 

### √

Londrina – PR/North 

### √

Uberaba – MG 

### √ √

Brasília – DF 

### √ √

Santo Antônio de Goiás – GO 

### √ √

Sete Lagoas-MG 

### √ √

Teresina - PI 

### √

Vilhena - RO 

### √

A detailed description of these field trials is presented in Section VIII.C. The results of the AHAS protein expression study are summarized here and the materials and methods used in the study are presented in Appendix C.

Each trial location had two replications each of CV127 soybean and the isoline control soybean for this study. All plots of the CV127 soybean we re sprayed with an im idazolinone herbicide for weed control, whereas all plots of the isoline control were treated with a conventional herbicide. In each of the two field trial seasons, leaf and gr ain samples were collected at all field trial sites at the V2 and R8 growth stages, respectively. In addition, six whole plants per plot, including roots, were collected at two of the trial sites at three differe nt developmental st ages, including the V2 (plants 15-20 cm tall with three nodes and tw o unfolded leaflets), R2 (plants in full bloom), and R8 (full maturity) stages. Three whole plan ts were maintained as such, and the remaining three whole plants were dissected into plant parts, including l eaves, stems, roots, flowers and pods, depending on the stag e of plant development

. Because of a late planting date coupled with the southerly location of the Ponta Grossa fiel d site in the 2006/2007 growing season, plants of

all treatments failed to reach fu ll maturity and grain was not harvested from treatments at this field site. Therefore, there is no AHAS expression data for grain from either CV127 or the

isoline control at th is field location.

Expression levels of AHAS enzyme in differenttissue samples were determined by enzymelinked immunosorbent assay (ELI SA) using AHAS-specific antibodies . Due to the high level ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 72 of 375 amino acid homology between the AtAHAS and the endogenous soybean AHAS enzymes, the AHAS-specific antibodies used in the ELISA assa y were not capable of distinguishing between the AtAHAS and endogenous soybean AHAS proteins , and the total AHAS levels of soybean tissues were measured in this study. Therefore, it was expected that tissues from CV127 soybean that are expressing both the AtAHAS protein and the endogenous soybean AHAS proteins would have higher levels of total AHAS protein compared to the isoline control th at only expresses the endogenous soybean AHAS proteins. The diffe rence between the AHAS amount in CV127 plants and the isoline control is attributed to the expression of the AtAHAS protein. The limit of quantification (LOQ) and limit of detection (LOD) for AHAS by the ELISA assay were determined experimentally for e ach tissue type in each experiment and these are listed in Table VI-1. The limit of detection (LOD) is define d as the lowest amount of AHAS that can be detected with suitable confidence by the assay, but not necessarily reliably quantified. The LOD is determined by taking the results of at l east 24 separately-determined “blank” assays and calculating two times the standard deviation. The limit of quantitation (LOQ) is the lowest amount of AHAS that can be accurately quan tified by the assay with a defined degree of confidence. The ELISA used in the analyses of AHAS protein levels in plant tissues yields a sigmoidal response curve to different concentrati ons of the AHAS protein. The assay is accurate only in the linear portion of this curve. Values which ar e above zero, but below the linear portion of the standard response curve are consid ered to be non-quantifiable since the accuracy of the standard curve in this region is low. Therefore, these values may be considered to be detectable (if above the LOD) but not quantifiable (below the LOQ).

Amount of AHAS enzyme in CV127 soybean and isoline control tissues. AHAS protein levels were generally higher at all field locations in CV127 soybean than in the isoline control, which is

consistent with the fact that the ELISA assa y detects both the AtAHAS protein encoded by the

csr1-2 gene and the endogenous soybean AHAS en zymes. AHAS enzymatic activity is known to be highest in growing tissues where the need for branched-chain and other amino acids is greatest due to the higher level of de novo protein synthesis (Sti dham and Singh, 1991). The AHAS protein expression results from both trial seas ons reflect this since the highest levels of

AHAS were detected in leaves and whole plants at the V2 growth stage (Tables VI-2 and VI-3). During the 2006/2007 season AHAS protein levels in leaves of CV127 soybean plants from all sites at the V2 stage ranged from 53 to 128 ng AHAS/g tissue and 335 to 714 ng AHAS/g tissue on a fresh and dry weight basis, respectively (Tab les VI-2 and VI-3). AHAS levels of 18 to 59 ng AHAS/g fresh tissue and <80 to 254 ng AHAS/g dr y weight were determined for the leaves of CV127 soybean plants at the V2 stage during the 2007 season (Tables VI-2 and VI-3). In the isoline control leaf tissues at the V2 stage, the AHAS protein levels on a fr esh weight basis at all sites ranged from below the limit of quantifi cation (LOQ = 13 ng/g) to 16 ng/g during the 2006/2007 season and were equal to or below the L OQ (14 ng/g) at all locations during the 2007 season (Tables VI-2 and VI-3).

Expression levels of the AHAS pr otein decreased with age of th e plant and AHAS protein levels were either barely detectable or below the limit of detection of the assay in roots, older leaves, reproductive tissues and grain, although season one CV127 tissues at Santo Antônio de Posse did have higher AHAS levels on a dry-we ight basis than were observed at other sites and seasons at this same growth stage. Plant tissues collected from CV127 and isoline control plants at two locations in the R2 and R8 growth stages dur ing the 2006/2007 season had low levels of AHASCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 73 of 375 protein ranging from below the LOQ (13 ng/g fresh weight) to 34 ng/g fresh weight (Table VI2). Similar results were determined during the 2007 season with AHAS leve ls below or near the LOQ. It is noteworthy that AHAS levels in all grain samples from CV127 soybean and the isoline control from both growing seasons were at or below the LOQ (Tables VI-2 and VI-3). Even though the expression levels of the AtAHAS protein in CV127 soybean are extremely low, especially at later stages of pl ant growth and development, these levels were sufficient to confer herbicide tolerance to CV127 soybean plants.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 74 of 375 Table VI-1. The Calculated Limit of Detection (LOD ) and Limit of Quantification (LOQ) for AHAS Protein by ELISA

. Data are presented for different tissues derived from soybeans during the fi eld trials conducted in Brazil during the 2006/2007 (Season 1) and 2007 (Season 2) growing seasons.

Season 1 Season 2 LOQ (ng AHAS/g Fresh Wt.) 13 14 LOQ (ng AHAS/g Dry Wt.) LOD (ng AHAS/g Dry Wt.) Tissue Growth Stage Season 1 Season 2 Season 1 Season 2

V2 68 78 16 17

R2 65 66 15 14 Whole

Plants

R8 15 28 3 6

V2 70 76 16 16 Leaves

R2 62 59 14 13

V2 41 51 9 11

R2 38 41 9 9 Roots

R8 17 37 4 8

Flowers R2 77 83 18 18

Pods R8 15 17 3 4

Grain R8 15 15 3 3CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 75 of 375Table VI-2.AHAS Protein Levels (±Std. Dev.) on a Fresh- and Dry-Weight Basis in Different Tissuesand Growth Stages ofCV127 Soybean and the Isoline Control.Plants were cultivated at Londrina (Lond),Santo Antônio de Posse (SAP), Brasília (Bras), and Santo Antônio de Goiás (SAG) duringthe 2006/2007 and 2007 growing seasons (Season 1 and Season 2, respectively).Mean ng AHAS/g Fresh Wt (±Std Dev)Mean ng AHAS/g Dry Wt1(±Std Dev)TissueSeason 1Season 2Season 1Season 2GrowthStageSoybeanLineLocationLondSAPBrasSAGLondSAPBrasSAGWhole Plant6±10 40±6<14 <14314±59 214±37<78 <78Leaves103±24 128±47 27±9 18±4 511±116 714±284 133±51 113±31Roots <132<13 <14 <14 <41 <41 <51 <51CV1271st Trifoliate55±5 60±12 22±8 21±4 278±27 300±47 111±40 126±23Whole Plant <13 <13 <14 <14 <68 <68 <78 <78Leaves15±3 14±1<14 <1478±17 73±8<76 <76Roots <13 <13 <14 <14 <41 <41 <51 <51V2IsolineControl1st Trifoliate <13 <13<14 <14 <70 <70 <76 <76Whole Plant ND334±6<14 <14 ND160±33<66 <66Leaves <1324±4<14 <14 <62106±19<59 <59Roots <1317±4<14 <14 <3850±13<41 <41CV127Flowers <1322±9<14 -4<77125±45<83 -Whole Plant ND <13 <14 <14 ND <65 <66 <66Leaves ND <13 <14<14 ND <62 <59 <59Roots <13 <13 <14 <14 <38 <38 <41 <41R2IsolineControlFlowers <13 <13 <14 - <77 <77 <83 -CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 76 of 375Table VI-2. continued.Mean ng AHAS/g Fresh Wt (±Std Dev)Mean ng AHAS/g Dry Wt1(±Std Dev)TissueSeason 1Season 2Season 1Season 2GrowthStageSoybeanLineLocation Lond SAP BrasSAG Lond SAP Bras SAGWhole Plant <13 <13 <14 <14 <15 <15 <28 <28Pods24±1 26±2<14 <1426±1 30±2<17 <17Roots <13 <1315±5 17±8<17 <1742±13 48±27CV127Grain <13 <13 <14 <14 <15 <15 <15 <15Whole Plant <13 <13 <14 <14 <15 <15 <28 <28Pods <13 <13 <14 <14 <15 <15 <17 <17Roots ND ND15±3<14 ND ND41±8<37R8IsolineControlGrain <13 <13 <14 <14 <15 <15 <15 <151The limit of quantification (LOQ) on a dry-weight basis varied for each tissue type and growth stage based on the moisture content of the fresh tissues.Values below the LOQ are reported as less than the LOQ for thattissue and growth stage. LOQ values for different tissues areshown in Table VI-1.2Sample had detectable but non-quantifiable amounts of AHAS. That is, value was below the limit of quantification (13 or 14 ngAHAS/g fresh weight in the2006/2007 and 2007 seasons, respectively), but detectable.3ND = AHAS was considered non-detectable because the result generated by ELISA was below the limit of detection (LOD) of the method. LOD values fordifferent tissues are shown in Table VI-1.4Not tested due to insufficient amount of flower tissues at this site.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 77 of 375 Table VI-3. AHAS Protein Levels ( ± Std. Dev.) on a Fresh- and Dry-Weight Basis in Leaves (V2 stage) and Grain (R8 stage) of CV127 Soybean and the Isoline Control

. Plants were cultivated at field locations in Brazil duri ng the 2006/2007 (Season 1) and 2007 (Season 2) growing seasons.

Mean ng AHAS/g

Fresh Wt (± Std Dev) Mean ng AHAS/g Dry Wt (± Std Dev) Growth Stage /

Tissue Location Soybean

Line Season 1 Season

2 Season 1 Season 2

CV127 80 ± 12 59 ± 57 427 ± 73 254 ± 227 Uberaba Isoline 16 ± 0 <14 86 ± 9 <76

CV127 53 ± 31 40 ± 23 335 ± 189 220 ± 141 Sete Lagoas Isoline <13 1 <14 <70 <76 2

CV127 61 ± 13 - 3 337 ± 53 - Sto. Ant. de

Goiás Isoline <13 - <70 -

CV127 61 ± 42 - 363 ± 257 - Brasília Isoline <13 - <70 -

CV127 92 ± 29 - 478 ± 136 - Ponta Grossa Isoline <13 - <70 -

CV127 - <14 - <80 4 Teresina Isoline - <14 - <76

CV127 - 39 ± 10 - 207 ± 56 V2 /

Leaves

Vilhena Isoline - 14 - <83 4

CV127 <13 <14 <15 <15 Uberaba Isoline <13 <14 <15 <15

CV127 13 ± 0 <14 <15 <15 Sete Lagoas Isoline <13 <14 <15 <15

CV127 <13 - <15 - Sto. Ant. de

Goiás Isoline <13 - <15 -

CV127 <13 - <15 - Brasília Isoline <13 - <15 -

CV127 - <14 - <15 Teresina Isoline - <14 - <15

CV127 - <14 - <15 R8 /

Grain

Vilhena Isoline - <14 - <15

1 Sample had detectable but non-quantifiable amounts of AHAS. That is, AHAS levels were below the limit of

quantification (13 or 14 ng AHAS/g fresh weight in the 2006/2007 and 2007 seasons, respectively), but

detectable.

2 Values below the LOQ are reported as less than the L OQ for that tissue and growth stage. LOQ values for

different tissues are shown in Table VI-1.

3 Sample was not tested at this location.

4 Value shown is the mean of the values determined for the two replicates at the location. One of these values was

determined to be <LOQ. Thus, the mean presented here is the average of the LOQ and value which exceeded the LOQ.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 78 of 375 E. Levels of the AtSEC61 γ Subunit Protein in CV127 Soybean

As described in section V.E of this pe tition, the complete c oding sequence of the A. thaliana gene encoding the Arabidopsis SEC61 γ (AtSEC61 γ) subunit protein resides in the insert of CV127 soybean near the 5’ junction with genomic soybean DNA. Although the At SEC61 γ gene in CV127 soybean appears to lack the majority of its native promoter, experiments were performed to evaluate its possi ble expression. These demonstrat ed that a very low amount of mRNA is produced from this gene that, if translated, would encode the native

AtSEC61 γ− subunit protein. Since the transcript was highly amplified by RT-PCR to maximize detection capability, and only a faint band corresponding to the At SEC61 γ subunit transcript was detected, it was presumed that either no AtSEC61 γ subunit protein is produced in CV127, or if produced the protein would be pres ent at extremely low levels in the plant, acknowledging that transcript and protein levels may not be correlated.

To determine if AtSEC61 γ subunit protein is expressed in CV127 soybean tissues, western blot analysis of microsomal membrane protein fracti ons prepared from leaf and grain samples of CV127 was conducted (methods for thes e studies are presented in Appendix C of this petition). Identity of the microsomal membrane fractio n was confirmed by measuring the activity of cytochrome c reductase, a marker enzyme for the endoplasmic reticulum (found in the microsomal membrane fraction), (Figure VI-2). Protein samples were derived from leaf and grain of CV127 soybean plants as well as leaf and grain tissues from the parental soybean variety Conquista. Both CV127 soybean and control (Conquista) plants were grown under greenhouse conditions. Arabidopsis leaf and seed tissue were also harveste d from greenhouse-grown plants.

The AtSEC61 γ subunit protein used as a standard in these studies was produced and purified

from an Escherichia coli expression system. An ECL Plus Western Blotting Detection method was used for protein detection on the western blot (Figure V I-1). The rabbit anti-AtSEC61 γ polyclonal antibody can detect ap proximately 0.4 ng of SEC61 γ subunit protein by the western blot development method utilized.

The E. coli -produced SEC61 γ subunit protein was readily detected by western blot analysis in the standard protein lanes at a molecular weight of appr oximately 7000 (Figure VI-1). Furthermore, expression of the endogenous AtSEC61 γ subunit protein in Arabidopsis was detected in microsomal membrane protein prep arations from seed but was not detected in Arabidopsis leaf tissue (Fi gure VI-1). This result showed that the AtSEC61 γ subunit protein can

be detected in tissues of the plant species which se rved as the donor of the At SEC61 γ subunit coding sequence introduced into the genome of CV127 soybean; thus, the Arabidopsis seed tissue served as a positive control for this study. In contrast, west ern blot analysis detected no

AtSEC61 γ subunit protein in leaf or grain microsomal membrane pr otein preparations of CV127 (Figure VI-1, panels A and B). Because of the high amino acid sequence homology of SEC61 γ subunit proteins across species (e.g., 86% homology between soyb ean and Arabidopsis) it was expected that the an tibody would detect the endogenous soybean SEC61 γ subunit protein; however, no protein band corresponding to the SEC61 γ subunit protein was detected in tissue extracts of the control soybean Conquista. Therefore, AtSEC61 γ subunit protein was not detectable at levels greater than 30 ng/g leaf i ssue or 110 ng/g grain tissue or greater than 30 and 110 ppb for leaf and grain of CV127, respectively. This result is no t unexpected given theCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 79 of 375 extremely low levels of the tr anscript corresponding to the At SEC61 γ subunit gene detected in

CV127.

These results demonstrate that no detectable amounts of the AtSEC61 γ subunit protein are produced in either leaf tissue or grain of CV127 soybean. However, in the unlikely event that

the AtSEC61 γ subunit protein is present in tissues of CV127 at extremely low levels, below the limit of detection of the assay, the following summ ary confirms the food, feed and environmental safety of the AtSEC61 γ subunit protein.

1. The source organism of the AtSEC61 γ subunit protein, Arabidopsis thaliana , is not a known human or animal pathogen and is not known to cause allergic reac tions in humans. Similarly, A. thaliana is not known to produce toxic substances.

2. The SEC61 γ subunit is part of the protein transl ocation complex asso ciated with the endoplasmic reticulum and is ubiquitous and highly conserved in eukaryotes as well as being structurally related to analogous proteins in prokaryotes (Hartmann et al ., 1994). There is a high degree of amino acid sequence homology (86%) be tween the soybean SEC61 γ subunit and that from Arabidopsis (see Figure VI-3 for the amino acid alignment). The amino acid sequence of the Arabidopsis SEC61 γ subunit protein is 86% identical and 93% similar to the corresponding protein in soybean. Therefore the AtSEC61 γ subunit protein is highly homologous to the same protein produced in a crop with a history of safe use in food and feed products, as well as safety to the environment.

3. Bioinformatic analyses showed no biologi cally relevant amino acid sequence homology

between the AtSEC61 γ subunit protein and known toxins. In addition, bioinformatic analyses were conducted to confirm that the AtSEC61 γ subunit protein amino acid sequence did not share potentially immunologically re levant amino acid sequence segments or structure with known allergens. Results of these analyses showed that the amino acid sequence of the AtSEC61 γ subunit protein did not share eith er 35% or greater identity over 80 amino acids or a sequence of eight or more consecutive identical amino acids with any potential allergens. Furthermore, the AtSEC61 γ subunit protein is rapidly degraded in simulated digestive fluids which is a characteri stic typical of other di etary proteins with a history of safe use in food and feed products.

Therefore, results of these studies show that the AtSEC61 γ subunit protein does not possess the attributes of known food allergens, is not hom ologous to known protein toxins, and supports the environmental as well as food and feed safety of the AtSEC61 γ subunit protein.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 80 of 375 Figure VI-1. SEC61 γ Subunit Western Blot Analysis of Leaf and Gr ain Microsomal Membrane Protein Preparations from CV 127 and Control (Conquista) Soybeans Microsomal membrane proteins were prepared from leaf and grain samples and subjected to electrophoresis on an 8 – 16% poly acrylamide gel. Western blot analysis was conducted using

rabbit anti-AtSEC61 γ subunit polyclonal antibodies and developed using a chemiluminescent

substrate. The molecu lar weight of AtSEC61 γ subunit protein is approximately 7000. All samples except for the AtSEC61 γ subunit protein standard are from microsomal membrane protein preparations. Panel A. Lanes 1, and 5, purified SEC 61 γ subunit protein standard, 2.4

and 1.2 μ g, respectively; lane 2, 20 μ g protein from Arabidopsis leaf microsomal membrane protein preparation; lane 3, 20 μ g protein from Conquista soybean leaf microsomal membrane protein preparation; lane 4, 20 μ g protein from CV127 leaf mi crosomal membrane protein preparation; lane 6, 15 μ g protein from Arabidopsis seed microsomal membrane protein preparation; lane 7, 15 μ g protein from Conquista soybean s eed microsomal membrane protein preparation and lane 8, 15 μ g from CV127 soybean seed microsomal membrane protein preparation. Panel B. Longer exposure of same sa mples (different blot) from Panel A, lanes 5 – 8. Molecular weight standards are shown to the left of Panel A.



### 1 2 3 4 5 6 7 8

148

98

64

50

36

22

16

6 SEC61 γ subunit 

### 1 2 3 4 A. B.

1 2 3 4 5 6 7 8

148

98

64

50

36

22

16

6 

### 1 2 3 4 5 6 7 8

148

98

64

50

36

22

16

6 SEC61 γ subunit 

### 1 2 3 4 A. B.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 81 of 375 Figure VI-2. Cytochrome c Reductase Activi ty in Various Fractions Prepared from Leaves of CV127 and Control (Conquista) Soybeans The crude extract (supernatant after low speed centrifugation, 12,000 x g) and the microsomal membrane fraction (pellet after high speed cen trifugation, 100,000 x g) as well as the high-speed centrifugation supernatant were assayed for the endoplasmic reticulum enzyme marker, cytochrome c reductase.

Conquista

Crude Extract 100K Super 100K pellet 0.0 2.5 5.0 7.5 10.0

FractionCyt. c red. activity (ΔA547/min)

CV127

Crude Extract 100K Super 100K pellet 0.0 2.5 5.0 7.5 10.0

FractionCyt. c Red. Activity (ΔA547/min)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 82 of 375 Figure VI-3. Alignment of the Deduced Amino Acid Sequences of the SEC61 γ Subunit Proteins from Soybean (Gmax) and Arabidopsis thaliana (Athal). The amino acid sequence for soybean SEC61 γ ( G. max gene Glyma02g42660) was obtained from the Joint Genome Institute Glyma 1.0 annot ation available throu gh Phytozome (release version 7.0), http://www.phytozome.net/s oybean. The amino acid sequence for A. thaliana

SEC61 γ was deduced from the sequence of the gene encoding the AtSEC61 γ subunit protein in CV127 soybeans. Identical amino acids are shad ed in grey, conservati ve replacements are presented as white text in a black background, and non-conservative ami no acid differences are

unshaded.

10 20 30 40 50 .........|.........|.........|.........|.........|

**Gmax M D**  **AIDSVFDPLR E**  **FAKDSVRLVKRCHKPDRKEF S**  **KVAVRTAIGFVVMGFV**

**Athal M E**  **AIDSAIDPLR D**  **FAKSSVRLVQRCHKPDRKEF T**  **KVAVRTAIGFVVMGFV**

60 70 .........|.........|

**Gmax GFFVK II**  **FIPINNIIVGSG**

**Athal GFFVK LV**  **FIPINNIIVGSS**



### VII. Compositional and Nutritional Assessments of CV127 Soybean

Soybean has many uses in human and animal nutriti on. The grain is typica lly processed into two commodity products, oil and meal. The major f ood uses for soybean include the production of soybean oil that is utilized in margarines, shortenings, and sala d and cooking oils. Soybeans are also used in various food produc ts that include tofu, soya sau ce, and simulated milk and meat products. Also, the isolated soybean protein is used in the production of infant formula and other food products as a source of amino acids. Soybean meal is used as a source of protein for animal feed. Other components of the soybean plant, in cluding the forage, are also fed to a limited extent to animals, primarily to cattle. Therefore, the purpose of th is section of the petition was to demonstrate that the grain and fo rage of CV127 is substantially equivalent in composition to grain and forage from the isoline control and other conventional soybean varieties, and that CV127 grain is appropriate for use in human food and animal feed.

Compositional analyses were conducted on grain and forage to confirm that the nutrient and antinutrient levels in the grain and the proximate and fiber levels in forage derived from CV127 soybean are comparable to those from the nontrans genic, isoline control s oybean that is closely related to CV127 soybean but that lacks the csr1-2 gene. Two additional conventional soybean varieties that are typical of soybean varieties commonly cultiv ated in Brazil, Monsoy 8001 and Coodetec 217, were also included in these analyses to establish a range of natural variability for each analyte for soybeans grown in Brazil. Furthe rmore, results were compared to analytical data for soybean grain in the International Life Sciences Institute Crop Composition Database (ILSI, 2008), with comparis ons to globally-derived values as well as da ta produced in Brazil, where available. A summary of the results of th ese comparisons are presented in this section of the petition, and detailed results for grain and forage analyses at each field trial location areCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 83 of 375 presented in Appendix D. Results showed that CV127 soybean is compositionally and nutritionally equivalent to convent ional soybean varietie s that are currently cultivated in Brazil as well as globally.

Grain samples of CV127 soybean, the isoline contro l, and the two conventi onal soybean varieties were harvested from soybeans grown in four replicat ed plots at each of six field trial locations in Brazil during the 2006/2007 season and from four fiel d trial locations in the 2007 short season. Forage tissues were harvested at the R2 growth stage from four re plicated plots at each of six

field trial locations in Brazil during the 2007/2008 growing season (a ll field trial locations for the production of grain and forage fo r compositional analyses are pres ented in Appendix D). Since CV127 soybeans are intended to be cultivated using imidazolinone herbicides for weed control, the grain and forage composition data for CV127 s oybeans that is presented in this petition are derived from plants treated with imidazolinone herbicide (70g ai/ha). In the case of grain compositional analyses, an additional treatment of CV127 treated with conventional herbicide (same herbicide application as applied to controls ) was included in the study. However, because

this treatment will not be used in the commer cial production of CV127 soybean, the means and ranges of analyte values across field trial locations for this treatment are not presented in this section of the petition. Data for this treatment at each fiel d trial location are presented in Appendix D. The samples that were obtained fro m the isoline control and the two conventional soybeans varieties were derived from plants th at were treated with bentazon and acifluorfensodium (commercial name Volt ® ) at the rate of 1.0 liters/ha for th e control of weeds. The field sites used in this study were located in regions of Brazil where soybeans are typically cultivated and were representative of locations wher e soybeans are cultivat ed commercially. The cultivation practices and a descrip tion of the protocol for the fiel d trials that produced the grain used in this composition study ar e presented in Appendix F.

Components analyzed for grain included proxima tes (protein, fat, ash, carbohydrates, calories, total dietary fiber [TDF], and moisture), crude fiber, acid detergent fiber (ADF), neutral detergent fiber (NDF), amino acids, fatty acid s (C14-C24), minerals (calcium, iron, phosphorus, magnesium, and potassium), vitamins ( α -, β -, γ -, δ -, and total tocopherols, vitamins E and B 1 , and folic acid), isoflavones (daidzin, malonyl -daidzin, daidzein, glycitin, malonyl glycitin, glycitein, genistin, malonyl ge nistin, and genistein), phospholip ids (phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl inosito l, and phosphatidyl choline) , and antinutrients (phytic acid, raffinose and stachyose, lectin s, urease and trypsin inhibito r). Components analyzed in forage samples included proximates (protein, fa t, ash, moisture, carbohydr ates and ca lories), crude fiber, ADF and NDF. Compositional anal yses were conducted at the Instituto de Tecnologia de Alimentos (ITAL)

, Campinas, Brazil on grain and forage samples, and complete methods of analyses are pr esented in Appendix D.

In addition to the grain and forage compositiona l analyses, the nutritiona l equivalence of CV127 soybean to the isoline control and conventional soybean varietie s was confirmed in a poultry feeding study. A 42-day feedin g trial with broiler chickens was conducted to compare the performance of the animals fed with soybean meal from CV127 soybeans to those fed soybean meal from conventional soybean grain. This study demonstrated that there was no evidence of a significant difference in the perfor mance of the animals fed a feed containing soybean meal from CV127 soybean compared to those fed a feed c ontaining soybean meal from the grain ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 84 of 375 conventional soybeans, thereby showing that meal derived from CV127 grain is as wholesome as meal derived from grain of the isoline control or conve ntional soybean varie ties for animal feed.

Collectively, results of the above studies as well as the poultry feeding study demonstrate that the grain and forage produced by CV127 soybean are compositionally equivalent to, and as nutritious as, grain and forage produced by the isoline control and other conventional soybean

varieties.

The generations of CV127 and comparators used in each of these studies described above are

shown below.

Comparative Analyses Performed, Generations Used for Analyses, and Comparators Used as Controls (Refer to Figure III-1 for Identi ty of CV127 and Isoline Control Generations).

Analysis CV127 Soybean

Generation Used Control Used

Compositional analysis (Grain)

2006/2007 F5 (CV127 line 127) Isoline control (F5 null) Monsoy 8001

Coodetec 217

Compositional analysis (Grain)

2007 F6 (CV127 line 127) Isoline control (F6 null) Monsoy 8001

Coodetec 217

Compositional analysis (Forage)

2007/2008 F7 (CV127 line 127) Isoline control (F7 null) Monsoy 8001

Coodetec 217

Poultry feeding study

2006/2007 F9 (CV127 line 603) Conquista Monsoy 8001

Coodetec 217

A. Grain Composition

Field trials for grain production and compositi onal analyses were conducted under standard agronomic practices in a comple te randomized block design with four replicate blocks per

location. For the CV127 and isolin e control treatments, grain was harvested separately from each replicate block at the conclusion of the gr owing season and approximately 2 kg of each replicate grain sample was separated for com positional analyses. For the two conventional soybean varieties, the same grain harvest and separation procedures were followed, but approximately 500 g of grain wa s further sub-sampled from each 2 kg replicate grain sample, and the four 500 g replicate samples for each conve ntional soybean variety from each field trial location were pooled to make a single sample from each location for compositional analyses. Therefore, statistical analyses of the compos itional data were only conducted for the CV127 and isoline control treatments, and data from the tw o conventional soybean varieties were used for comparative purposes to establish a range of na tural variability for e ach analyte for soybeans grown in Brazil.

Analysis of variance was carried out on the gr ain composition data using SAS Version 9.1 (SAS Institute Inc., Cary, North Carolina) following tw o procedures, the General Linear Model and the Mixed Model. With the exception of moisture content, all data were expressed on a dry-weightCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 85 of 375 basis for statistical analyses. Differences were assessed across locatio n and by location. The model for across-location analyses is the following:

y = variety + location + variety x location + block(location) + e Random effects: location, vari ety x location, block(location). where y is the response variable (any analyte measured)

The model for separate analyses by location is the following:

y = variety + block + e where e is the response error

For grain composition, contrasts were carried out to compare CV127 soybean with the isoline control. For forage composition, contrasts were carried out to compare the isoline control as well as the conventional soybean varieties to CV127. Values we re considered statistically significantly different at the p < 0.05 level.

Proximates. Grain samples from the 2006/2007 (season 1) and 2007 (seas on 2) field trial seasons were analyzed for moisture, ash, protein, crude fat, total dietar y fiber, carbohydrate and calorie content. The carbohydrate composition and calorie values were calculated. The means and ranges of values for proximates in CV127 soybean, the isoline control, and the two conventional soybean varieties in two growing s easons and across all locations in comparison with the means and ranges published in the ILSI Crop Composition Da tabase (ILSI, 2008) are

shown in Table VII-1. These data show that in both seasons there were no statistically significant differences in the ash, total dietary fiber, or carbohydrate content between CV127 and the isoline control soyb eans. In addition, in season 2 th ere was no significant difference for calories or crude fat between grain from the is oline control and CV127 soybeans. Slight grain

moisture differences were observed between CV 127 and the isoline control in both seasons, but the differences were inconsistent, since CV127 grai n moisture content was lower than the isoline control in season 1, but higher than the isoline control in season 2. Therefore, these differences

were not considered to be biologically meaningful. Furtherm ore, the means and ranges of grain moisture levels in CV127 were comparable to the mean and range levels in the two conventional soybean varieties cultivated in the same trials, and were within the range of moisture values reported for soybeans globally as well as for soyb eans produced in Brazil. In season 1, grain from CV127 soybean was statistically significantly higher for crude fat and caloric content and statistically significantly lower fo r protein content compared to grain from the isoline control soybeans. In season 2, there was no statistically significant difference in grain crude fat levels between the two treatments, but the protei n content of grain from CV127 soybean was statistically significantly higher than that for grai n from the isoline control. However, all means and ranges of values for these nutrients in CV127 grain from both seasons were either within or comparable to the range of values for the conve ntional soybean varieties cultivated in the same trials as well as to the range reported for soyb eans globally and for soybeans produced in Brazil. Overall, these results show that proximate levels (moisture, ash, protein, crude fat, total dietary fiber, carbohydrates and calories) in CV127 grain are equivalent to levels in the isoline control grain and comparable to and in the same range as grain proximate content of other conventional soybean varieties with a history of safe food and feed uses as well as safety to the environment.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 86 of 375 Fiber composition. Grain samples were analyzed for cr ude fiber (CF), acid detergent fiber (ADF) and neutral detergent fibe r (NDF). The means and range s of values for CF, ADF and NDF in CV127 soybean, the isoline control and tw o conventional soybean control treatments in

two field trial seasons and across all locatio ns in comparison with the means and ranges published in the ILSI Crop Composition Database (ILSI, 2008) are shown in Table VII-2. In seasons 1 and 2, there were no sta tistically significant differences in crude fiber values between the isoline control and CV127 soybeans, and no stat istically significant diffe rences in grain ADF values in season 2 between CV127 soybeans and the isoline control. However, the ADF values

in season 1 and NDF values in both seasons in CV127 grain were statis tically significantly different (higher) than those in grain from the isoline control (Table VII-2). The CF, ADF and NDF means and ranges of values obtained for CV127 in the two seasons were either within or comparable to the means and ranges of values of the conventional soybean varieties cultivated in the same trials as well as to the ranges of values reported globally for soybeans and for soybeans produced in Brazil (ILSI, 2008). These results indicate that grain fiber content of CV127 soybean is equivalent to the grai n fiber content of the isoline c ontrol and comparable to and in the same range as the grain fiber content of othe r conventional soybean vari eties with a history of

safe food and feed uses as well as safety to the environment.

Amino Acids. Grain samples from the two field tria l seasons were analyzed for amino acid content. The means and ranges of values fo r all amino acids in CV127 soybean, the isoline control and the two conventional soybean varie ties in two field trial seasons and across all locations in comparison with the means and ranges published in the ILSI Crop Composition Database (ILSI, 2008) are shown in Table VII-3. For grain obtained from season 1, there were no statistically significant differences detected in amino acid content of the grain between

CV127 and the isoline cont rol soybeans, except for small but st atistically significant differences in values for cysteine and meth ionine between the two treatments . In the grain from season 2, there were no statistically si gnificant differences in amino aci d content between CV127 and the isoline control soybeans, except for alanine, cyst eine, histidine, methionine, and tyrosine where statistically significant differences were observe d between the two treatments. The means and ranges of values for the amino acid content of CV 127 grain from both seasons were either within or comparable to the range of values of the c onventional soybean varieties cultivated in the same trials as well as to the ranges of values repor ted globally for soybeans and for soybeans produced in Brazil. These results demonstrate that the amino acid content of CV127 soybean grain is equivalent to the content of the isoline control gr ain and is comparable to, and in the same range as, grain amino acid content of conventional soyb ean varieties with a history of safe food and feed uses as well as safety to the environment.

Results of the amino acid analyses in grain al so confirm that the amino acid mutation in the AtAHAS protein responsible for conferring imidazolinone herbicide tolerance in CV127 soybean has no impact on the feedback regulation of this enzyme by branched-chain amino acids. The AHAS enzyme catalyzes the first common step in branched-chain amino acid biosynthesis (leucine, isoleucine and valine) . The data presented in this section show there were no statistically significant differen ces in levels of these amino acids between gr ain of CV127 and the isoline control. Therefore, these data confirm that the mutation in the AtAHAS enzyme hasCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 87 of 375 no effect on the feedback regulation of the enzy me by the branched-chain amino acids or the biosynthetic function of the enzyme.

Fatty Acids. Grain samples from the two field trial seas ons were analyzed for fatty acid content. The eight most prevalent fatty acids in the grain from both seasons included myristic, palmitic, stearic, oleic, linoleic, linolenic, arachidic, an d behenic acids. In addition, low levels of eicosenoic acid were detected in grain of th e different soybean treatments from season 1, but were not detected in the grain from season 2. Th e means and ranges of valu es for fatty acids in grain from CV127, the isoline control soybeans a nd two conventional soybean varieties in two field trial seasons and across all locations in comparison with the means and ranges for fatty acids in soybean grain published in the ILSI Crop Composition Database (ILSI, 2008) are shown in Table VII-4. In grain from s eason 1 there were no statistically significant differences in levels of myristic, stearic, arachidic, eicosenoic, and behenic acids between the grain from CV127 and the isoline control soybeans (Table VII-4). Two fa tty acids (palmitic and oleic) were statistically significantly higher and two others (linoleic and li nolenic) were statistically significantly lower in grain from CV127 soybean compared to the isolin e control soybean in season 1. In grain from season 2, there were no statistically significant di fferences in the levels of myristic, palmitic,

arachidic and behenic acids be tween the grain from CV127 a nd the isoline control soybeans (Table VII-4). Statistically significant differen ces in levels of stearic, oleic, linoleic, and linolenic fatty acids were observe d between grain of CV127 and th e isoline control in season 2. However, for all fatty acids, the means and ra nges of fatty acid values for CV127 soybean grain

were either within or comparable to the range of values of the conventional soybean varieties

cultivated in the same trials as well as to the ranges of values reported globally for soybeans and for soybeans produced in Br azil (Table VII-4). These results demonstrate that the fatty acid content of CV127 grain is equivalent to the content in is oline control grain a nd is comparable to and in the same range as the grain fatty acid cont ent of other conventional soybean varieties with a history of safe food and feed use as well as safety to the environment.

Minerals. Soybeans are considered a significant source of potassium and magnesium and a

source of bioavailable iron in th e animal feed diet (Baker, 2000). Soybeans are also a source of phosphorus and calcium in the animal feed diet; how ever, the bioavailability of these minerals is typically limited by chelation by phytate. Therefore, these fi ve minerals (calcium, iron, magnesium, phosphorous and potassium) were quantif ied in soybean grain harvested from the different treatments from two growing seasons. The means and ranges of values for minerals in grain from CV127, the isoline control soybeans a nd two conventional soybean varieties in two

field trial seasons and across all locations in co mparison with the means and ranges for minerals in soybean grain published in the ILSI Crop Composition Database (ILSI, 2008) are shown in Table VII-5. In season 1, the mean values for calcium and phosphorus in grain from CV127 soybean were not statistically significantly diffe rent from those of the isoline control, while levels of iron (lower), magnesium (higher), and potassium (lower) were st atistically significantly

different from the isoline contro l. All mineral mean values determined for grain from CV127 soybean in season 2 were statistic ally significantly different from the mean values obtained for grain from the isoline control soybean, except for grain iron content. However, for each of the five minerals measured, the mean and range of mineral values for CV127 soybean grain were either within or comparable to the range of values of the conventional soybean varieties

cultivated in the same trials as well as to the range of values reported globally for soybeansCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 88 of 375 (Table VII-5). These results demonstrate that the mineral content in grain produced from CV127 soybean is comparable to, and in the same range as, the mineral content of the isoline control as well as other conventional soybean varieties with a history of safe food and feed us e as well as safety to the environment.

Vitamins. The tocopherols ( α, β, γ, δ and total tocopherols) together with vitamins E, B1, folic acid, niacin, and riboflavin were analyzed in the soybean grain samples obtained from trials

conducted in two field trial seasons. Levels of niacin and riboflavin in the grain samples were below the level of detection for the assays and so these analytes are not included in the data table. The means and ranges of values for vita mins in grain from CV127, the isoline control soybeans and two conventional soybean varieties in two field trial seasons and across all locations in comparison with the means and rang es for vitamins in soybean grain published in the ILSI Crop Composition Database (ILSI, 2008) are shown in Table VII-6. The mean vitamin content of CV127 grain produced in season 1 was found to be statistica lly significantly lower than that in the grain of the isoline control for folic acid, γ− tocopherol and vitamin B1 and significantly higher for α

- , β -, and δ -tocopherol, and vitamin E. The mean values for total tocopherols in the grain of CV127 and the isol ine control soybeans from season 1 were not statistically significantly di fferent. There were no statistically significant differences in the mean vitamin content of grain from CV127 soybean in season 2 compared to those from the isoline control except for α - and γ− tocopherols. However, for each of the different vitamins measured, the means and ranges of vitamin values for CV 127 soybean grain were either within or comparable to the range of values of the conve ntional soybean varieties cultivated in the same field trials. Global ranges for ty pical vitamin values in soybean grain are only available for folic
acid, vitamin B1 and α -tocopherol. The mean va lues of folic acid and α -tocopherol in grain from CV127 soybeans in both seasons were within the ranges reported globally for soybeans in the ILSI Crop Composition Database. In the case of vitamin B1, the grain from CV127 and the isoline control soybeans in both seasons had highe r values compared to the typical global range of values for this vitamin, and this may be a characteristic of soybean germplasm adapted for Brazilian growing conditions. Overall, these resu lts demonstrate that vitamin levels in grain produced by CV127 soybean are comp arable to and in the same ra nge as vitamin levels in the

isoline control as well as other conventional soybean varieties with a long history of safe food and feed use as well as safety to the environment.

Isoflavones. Grain samples from two field trial seasons were an alyzed for their isoflavone composition, including daidzin, mal onyl daidzin, daidzein, glyciti n, malonyl glycitin, glycitein, genistin, malonyl genistin, and ge nistein. The mean and range of values for the total isoflavones (total daidzein, total genistein, and total glyc itein) in grain from CV127, the isoline control soybeans and two conventional soybean varieties in two field trial seasons and across all locations in comparison with the mean and range of values for isoflavones in soybean grain published in the ILSI Crop Composition Database (ILSI, 2008) are shown in Table VII-7. There was no statistically significant difference in the m ean values f or total glycitein between the grain

from CV127 and the isoline c ontrol soybeans in season 1, but levels of glycitein were statistically significantly lower in grain of CV127 compared to levels in the grai n of the isoline

control in season 2. Levels of total daidzein an d genistein were statistically significantly lower in grain of CV127 compared to leve ls in the isoline control in both field trial seasons. However, for each of the isoflavones, the mean and rang e of isoflavone values for CV127 soybean grainCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 89 of 375 were either within or comparable to the range of values of the conventional soybean varieties

cultivated in the same field trials as well as to the range of values re ported globally for soybeans and for soybeans produced in Brazil (Table VII-7). These results demonstrate that the isoflavone content in grain produced from CV 127 soybean is comparable to, and in the same range as, the

isoflavone content of the isoline control as well as conventional s oybean varieties with a history

of safe food and feed use as well as safety to the environment.

Phospholipids. Phospholipids are common contaminan ts during soybean oi l processing, often referred to as gums in the solvent-extracted oil. If not removed, the gum material typically settles out and can cause significa nt losses in oil refining. Theref ore, phospholipid levels were analyzed in grain samples and the mean and range of values for phosphatidyl ethanolamine, phosphatidic acid, phosphatidyl inos itol and phosphatidyl cholin e in grain from CV127, the isoline control soybeans and two conventional soyb ean varieties in two field trial seasons and

across all locations are shown in Table VII-8. Data were not available for these analytes in the ILSI Crop Composition Database. In season 1, th e mean values for all measured phospholipids in grain from CV127 soybean were statistically significantly lower compared to the corresponding values from the grain of the isolin e control. Conversely, in season 2, there were no statistically significan t differences in the mean phospholipid values of grain from CV127 and the isoline control soybeans, except for levels of phosphatidic acid levels that were slightly but statistically significantly higher in grain of CV127 compared to levels in grain of the isoline control. Therefore, the differences observed from the first field trial season were not considered biologically meaningful since they were not consistently observed. In general, the values for the phospholipids obtained from grain of CV127 soybean were comparable to the values obtained from grain produced by the isoline control at all locations. Since phospholipid content of soybeans is not included in th e ILSI Crop Composition Database, comparison of these values to this database was not possible. However, compar isons to the means and ranges of values for the two conventional soybean varieties that were included in this study demonstrated that levels of phospholipids in grain from CV127 soybean were comparable to those in grain produced by conventional soybean varieties th at are commonly cultivated in Brazil and that have a long history of safe food and feed use an d safety to the environment.

Antinutrients. Grain samples were analyzed for antinut rient content, and the mean and range of values for phytic acid, raffinose, stachyose, lectin s, urease and trypsin i nhibitor in grain from CV127, the isoline control and two conventional soybean varieties in two field trial seasons and across all locations in comparison with the mean and range of valu es for antinutrients in soybean grain published in the ILSI Crop Composition Data base (ILSI, 2008) are shown in Table VII-9. There were no statistically significant differences in the levels of phytic acid between grain from CV127 and the isoline control soybe ans in either field trial season, and the mean and range of phytic acid values for CV127 grain were either within or comparab le to the range of values for the conventional soybean varieties included in this study. However, the mean and range of phytic acid values for CV127, the is oline control and the conventi onal soybean varieties values were lower than the rang e that is typical for s oybeans cultivated globally (Table VII-9), and this most likely reflects a germplasm characteristic of soybean varieties adapted for cultivation in

Brazil.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 90 of 375 In the case of raffinose, the mean value for gr ain from CV127 soybeans produced in season 1 was slightly, but statistically significantly higher than the grain content of the isoline control, whereas in season 2 there was no statistically significant difference in grain raffinose levels

between CV127 and the isoline control. Theref ore the difference in grain raffinose levels

observed between the two treatment s in season 1 was not consid ered biologically meaningful since it was not observed consistently. Alt hough the mean values for raffinose in both CV127 and the isoline control soybeans in grain from both seasons were higher than the range for raffinose in soybeans cultivated globally (ILSI Crop Composition Database), the mean and range

of raffinose levels in both CV127 and the isoline control were comparable to the mean and range

of raffinose values obtained for the two conventiona l soybean varieties include d in the field trials (Table VII-9). Therefore the difference in raffi nose levels in grain of CV127, the isoline control and the conventional soybean varieties from the range of raffinose levels that are typical for soybeans cultivated globally, most likely reflects a characteristic of soybean varieties adapted for

cultivation in Brazil.

The mean values for stachyose content in grai n of CV127 soybeans in bot h field trial seasons were slightly, but statistically significantly lowe r than the mean values for the grain from the isoline control. Although the mean and range of values for stachyose in grain from both CV127 and the isoline control soybeans in both seasons were higher th an the range for stachyose in soybeans cultivated globally, these means and ranges of values were comparable to the mean and range of stachyose values obtained for the two conventional soybean varieties included in the field trials (Table VII-9). Again, this suggests that higher stachyose levels observed in grain of CV127, the isoline control and the conventional soybean varieties co mpared to levels recorded for soybeans cultivated globally, is a characteristic of soybean varieties ad apted for cultivation in

Brazil.

The mean values for lectins in season 1, and ur ease and trypsin inhibitor in both seasons for CV127 were not statistically significantly different fr om levels of the same analytes in the isoline control soybeans. The mean valu e for lectin levels in grain fr om CV127 soybeans from season 2 was statistically significantly lower than the values obtained from the grain of the isoline control. The mean and range of values for lectins in CV127 were either with in or comparable to the range of values of the conventional soybean varieties cultiv ated in the same field trials as well as to the range of values reported globally for soybeans a nd for soybeans produced in Brazil in the ILSI Crop Composition Database (Table VII-9). In the case of trypsin inhibitor, the mean and range of values in grain of both CV127 and the isoline control soybeans were comparable to the mean and range of trypsin inhibitor values obtained for the two conve ntional soybean varieties, but values were below the range repor ted globally (Table VII-9).

Urease activity is typically used as an indicator of reduction of trypsin inhibitor activity in meal

as a result of heat treatment during soybean processing. This ha s been a reliable method because trypsin inhibitor is read ily reduced by heat as is urease ac tivity. Urease is not harmful to nonruminant animals but can cause ammonia toxicity in cattle fed urea-suppl emented diets (Dr. G. Weigel, personal communication). The means a nd ranges of urease values in grain of both

CV127 and the isoline control so ybeans were comparable to the means and ranges of urease values obtained for the two conventional soybean varieties. Overall, comparing antinutrient levels in CV127 soybeans to levels in the is oline control and the conventional BrazilianCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 91 of 375 commercial varieties, these results show that antinutrient levels in grain produced by CV127 soybeans are comparable to and in the same range as antinutrient levels in the isoline control as well as conventional soybean varie ties cultivated in Brazil and/or globally that have a long history of safe food and feed uses and safety to the environment.

Conclusions. The results of the compositional analyses conducted with grain produced in Brazil in two different growing seasons dem onstrate that the introduction of the csr1-2 gene into the soybean genome does not impact the nutriti onal composition of grain produced by CV127 soybeans. In summary, results of these analyses demonstrate that grain from CV127 soybeans is compositionally equivalent to, and as nutritious as, grain from the isoline control well as other conventional soybean varieties typi cally cultivated in Brazil. Therefore, grain derived from CV127 soybean is appropriate for use in hu man food and animal feed products. Minor

differences in amounts of individua l nutritional constituents that we re detected between the grain of CV127 soybean and that of the isoline contro l soybean are likely due to the natural genetic heterogeneity that exists between these two. Also, analyte differences from the global composition database most likely reflect char acteristics of soybean germplasm bred and developed for production under tropical growing c onditions in Brazil. The complete results of these composition analyses are presented in Appendix D.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 92 of 375Table VII-1. Grain Proximate Composition.Comparison of the proximate composition of grain derived fromCV127 soybean with the isoline control and two conventionalsoybean varieties grown in two seasons as well as with globaland Brazilian ranges of values from the ILSI Crop CompositionDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217,were combined to provide asingle mean value (Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)Analyte (unit)IsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazilN = 24N = 12N = 15N = 8N = 80 – 323N = 69Mean (range)Moisture (%)10.1 a†(9.2 – 10.9)9.4 b(8.8 – 9.9)9.8(9.4-10.5)7.6 b(7.1 – 8.2)7.9 a(7.6 – 8.2)7.7(7.0-8.2)10.1(4.7 – 34.4)9.8(7.6 – 11.2)Ash(g/100 g DW)5.0 a(4.6 – 5.3)4.9 a(4.5 – 5.4)4.9(4.5-5.3)5.2 a(4.8 – 5.7)5.1 a(4.7 – 5.6)5.2(4.9-5.5)5.32(3.89 – 6.99)5.00(4.58 – 5.47)Protein(g/100 g DW)40.3 a(38.1 – 42.2)39.4 b(37.3 – 41.9)37.6(36.4-39.6)39.2 b(36.4 – 41.6)39.7 a(36.7 – 41.6)39.2(36.8-42.0)39.47(33.19 – 45.48)40.15(37.19 – 44.85)Crude Fat(g/100 g DW)21.7 b(20.0 – 23.3)22.7 a(20.1 – 24.2)22.8(20.2-24.8)20.2 b(17.7 – 23.8)20.5 ab(18.9 – 24.1)20.6(16.9-25.1)16.68(8.10 – 23.56)18.85(14.44 – 23.56)Total DietaryFiber(g/100 g DW)24.59 a(21.93 – 26.90)24.70 a(22.13 – 28.11)25.43(22.29-28.03)24.54 a(18.94 – 28.01)24.66 a(21.37 – 28.43)25.0(21.9-27.3)NA* NACarbohydrate1(g/100 g DW)33.1 a(31.6 – 34.4)32.9 a(31.9 – 34.9)34.7(27.39-42.03)35.4 a(25.2 – 44.9)34.7 a(24.2 – 43.3)35.0(26.7-39.8)38.2(29.6 – 50.2)36.0(29.6 – 41.6)Calories(kcal/100 g DW)390 b(379 – 402)395 a(384-405)393(377-400)382 a(362 – 403)383 a(365 – 413)382(374-395)NA NA†Values in the same season and row followed by the same letter are not significantly different at P<0.05.*NA = not available1Carbohydrates including total dietary fiberCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 93 of 375Table VII-2. Grain Fiber Composition.Comparison of the fiber composition of grain derived from CV127 soybean with the isoline control and two conventional soybeanvarieties grown in two seasons as well as with global and Brazilian ranges of values from the ILSI Crop Composition Database.Themean values for the conventional soybean varieties, Monsoy 8001and Coodetec 217, were combinedto provide a single mean value(Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)AnalyteIsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazil(g/100 gDW)N = 24N = 12N = 15N = 8N = 80 – 323N = 69Mean (range)Crude Fiber8.5 a†(6.9 – 11.0)8.1 a(6.4 – 11.3)8.2(6.7-9.3)7.9 a(6.7 – 10.6)8.2 a(6.2 – 14.7)8.2(7.2-12.1)7.81(4.12 – 13.87)8.46(6.42 – 10.93)ADF11.39 b(8.84 – 14.95)13.76 a(10.96 – 19.00)11.76(9.32-14.43)10.25 ab(8.53 – 12.05)10.49 a(7.35 – 13.40)11.11(8.89-12.59)11.97(7.81 – 18.61)11.34(7.81 – 16.39)NDF14.98 b(12.04 – 17.32)17.52 a(14.06 – 20.01)14.85(10.63-16.92)14.08 a(11.32 – 15.93)15.13 b(12.53 – 18.17)14.11(12.26-16.18)12.33(8.53 – 21.25)12.39(8.53 – 21.25)†Values in the same season and row followed by the same letter are not significantly different at P<0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 94 of 375Table VII-3. Grain Amino Acid Composition.Comparison of the amino acid composition ofgrain derived from CV127 soybean withthe isoline control and two conventionalsoybean varieties grown in two seasons as well as with globaland Brazilian ranges of values from the ILSI Crop CompositionDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217,were combined to provide asingle mean value (Conv Stds) for each analyte.Season 1(2006/2007)Season 2(2007)AnalyteIsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazil(g/100 gDW)N = 24N = 12N = 15N = 8N = 80 – 323N = 69Mean (range)Alanine1.64 a†(1.45 – 1.921.63 a(1.27 – 1.76)1.56(1.39-1.68)1.51 b(1.29 – 1.71)1.62 a(1.38 – 1.86)1.58(1.41-1.79)1.72(1.51 – 2.10)1.73(1.62 – 1.85)Arginine3.03 a(2.64 – 3.09)3.01 a(2.36 – 3.33)2.71(2.34-2.97)2.84 a(2.37 – 3.34)2.84 a(2.53 – 3.15)2.95(2.49-3.45)2.84(2.29 – 3.40)2.93(2.59 – 3.28)Aspartate4.64 a(4.02 – 5.37)4.61 a(3.66 – 5.17)4.32(3.94-4.72)4.17 a(3.50 – 4.66)4.29 a(3.85 – 4.65)4.45(3.70-5.06)4.49(3.81 – 5.12)4.59(4.23 – 5.12)Cysteine0.54 a(0.51 – 0.58)0.52 b(0.48 – 0.56)0.52(0.49–0.56)0.51 a(0.43–0.55)0.52 b(0.43 – 0.56)0.52(0.44 – 0.59)0.59(0.37 – 0.81)0.57(0.50 – 0.81)Glutamate7.61 a(6.59 – 8.92)7.54 a(5.83 – 8.45)6.98(6.16-7.68)6.74 a(5.72 – 7.41)6.78 a(6.17 – 7.46)7.18(5.90-8.55)7.09(5.84 – 8.20)7.29(6.58 – 8.09)Glycine1.65 a(1.45 – 1.87)1.65 a(1.32 – 1.81)1.56(1.41-1.71)1.50 a(1.28 – 1.68)1.50 a(1.34 – 1.83)1.56(1.35-1.72)1.69(1.46 – 2.00)1.70(1.56 – 1.82)Histidine0.87 a(0.78 – 1.03)0.85 a(0.67 – 0.97)0.79(0.72-0.86)1.11 b(0.80 – 1.32)1.37 a(0.91 – 2.13)1.10(0.73-1.34)1.04(0.88 – 1.18)1.06(0.98 – 1.18)Isoleucine1.61 a(1.42 – 1.94)1.61 a(1.24 – 1.75)1.54(1.38-1.71)1.46 a(1.22 – 1.62)1.43 a(1.08 – 1.97)1.50(1.35-1.65)1.81(1.54 – 2.08)1.85(1.59 – 2.04)Leucine2.89 a(2.51 – 3.40)2.87 a(2.24 – 3.16)2.80(2.45-3.65)2.56 a(2.19 – 2.86)2.61 a(2.44 – 2.85)2.73(2.39-3.15)3.04(2.59 – 3.62)3.07(2.81 – 3.38)Lysine2.48 a(2.12 – 2.85)2.46 a(1.93 – 2.68)2.33(2.08-2.54)2.24 a(1.97 – 2.47)2.27 a(1.99 – 2.62)2.32(2.03-2.58)2.56(2.29 – 2.84)2.58(2.42 – 2.82)CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 95 of 375Table VII-3. continued.Season1(2006/2007)Season 2(2007)AnalyteIsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazil(g/100 g DW)N = 24N = 12N = 15N = 8N = 80 – 323N = 69Mean(range)Methionine0.63 a(0.60–0.67)0.62 b(0.59–0.68)0.60(0.55 – 0.65)0.60 b(0.53 – 0.66)0.61 a(0.51 – 0.66)0.60*(0.53–0.69)0.55(0.43 – 0.68)0.55(0.50 – 0.68)Phenylalanine1.99 a(1.77 – 2.37)1.98 a(1.52 – 2.19)1.87(1.64-2.09)1.76 a(1.52 – 1.94)1.78 a(1.65 – 1.90)1.87(1.64-2.12)1.98(1.63 – 2.35)2.06(1.82 – 2.24)Proline1.98 a(1.74 – 2.32)1.91 a(1.49 – 2.12)1.82(1.58-2.03)1.74 a(1.50 – 1.95)1.73 a(1.11 – 1.89)1.86(1.60-2.05)2.00(1.69 – 2.28)2.06(1.86 – 2.28)Serine2.09 a(1.83 – 2.39)2.07 a(1.63 – 2.29)1.96(1.78-2.16)1.82 a(1.54 – 2.05)1.83 a(1.65 – 1.97)1.92(1.67-2.21)2.02(1.11 – 2.48)2.17(1.96 – 2.48)Threonine1.56 a(1.34 – 1.80)1.55 a(1.25 – 1.72)1.48(1.36-1.55)1.37 a(1.21 – 1.50)1.37 a(1.17 – 1.67)1.40(1.23-1.55)1.47(1.14 – 1.86)1.40(1.28 – 1.52)Tryptophan0.73 a(0.59 – 0.84)0.76 a(0.60 – 1.03)0.77(0.57-1.16)0.64 a(0.53 – 0.74)0.67 a(0.51 – 0.84)0.65(0.56-0.73)0.43(0.36 – 0.50)0.44(0.38 – 0.49)Tyrosine1.34 a(1.17 – 1.55)1.31 a(1.06 – 1.42)1.27(1.12-1.39)1.18 a(0.98 – 1.33)1.14 b(1.02 – 1.38)1.25(1.14-1.45)1.32(1.02 – 1.61)1.38(1.27 – 1.56)Valine1.66 a(1.36 – 1.98)1.64 a(1.26 – 1.79)1.58(1.40-1.67)1.56 a(1.35 – 1.83)1.62 a(1.41 – 1.79)1.62(1.31-1.87)1.91(1.60 – 2.20)1.91(1.63 – 2.08)†Values in the same season and row followed by the same letter are not statistically significantly different at P<0.05.*N = 7CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 96 of 375Table VII-4. Grain Fatty Acid Content.Comparison of the fatty acid composition ofgrain derived from CV127 soybean withthe isoline control and two conventionalsoybean varieties grown in two seasons as well as with globaland Brazilian ranges of values from the ILSI Crop CompositionDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217,were combined to provide asingle mean value (Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)Analyte (unit)IsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazil(%Total FA)N = 24N = 12N = 15N = 8N = 80 – 323N = 69Mean (range)Myristic 14:0<0.09 a†(nd‡ – 0.10)<0.09 a(nd – 0.10)0.10(0.9-0.11) N=70.11 a N=5(0.09 – 0.12)0.11 a N=7(0.10 – 0.11)0.09 N=4(0.06 – 0.12)NA* NAPalmitic 16:09.77 b(9.12 – 10.44)9.98 a(9.35 – 10.59)10.17(9.08-11.16)10.32 a(9.67 – 10.82)10.18 a(9.75 – 10.64)9.77(8.31 – 10.67)11.12(9.55 – 15.77)11.27(10.28 – 12.73)Stearic 18:03.39 a(2.84 – 3.87)3.30 a(2.77 – 3.61)3.46(2.97-3.92)4.04 b(3.14 – 5.05)4.21 a(3.50 – 4.80)3.99(3.04 – 5.01)4.01(2.70 – 5.88)3.95(2.70 – 5.52)Oleic 18:120.07 a(18.47 – 21.02)22.07 b(20.10 – 25.76)20.06(18.54-21.38)24.38 b(21.72 – 33.15)27.79 a(22.65 – 43.63)23.39(20.13 – 28.06)20.7(14.3 – 32.2)22.6(18.7 – 28.9)Linoleic 18:245.87 a(44.01 – 47.68)45.00 b(42.60 – 46.62)53.54(52.36-54.40)48.86 a(42.54 – 50.83)45.65 b(31.97 – 49.57)50.10(46.78 – 52.80)53.3(42.3 – 58.8)52.6(48.2 – 55.5)Linolenic 18:35.65 a(5.05 – 6.10)5.10 b(4.59 – 5.72)7.23(6.31-8.15)6.62 a(4.20 – 8.11)6.32 b(3.42 – 8.12)7.06(4.76 – 8.52)8.34(3.00 – 12.52)7.06(5.92 – 8.18)Arachidic 20:00.37 a(0.32 – 0.44)0.34 a(0.26 – 0.39)0.31(0.26-0.40)0.39 a(0.32 – 0.49)0.42 a(0.27 – 0.55)0.33(0.26 – 0.48)0.32(0.16 – 0.48)0.37(0.28 – 0.48)Eicosenoic 20:10.13 a(0.09 – 0.19)0.13 a(0.08 – 0.18)0.14(0.09-0.20)nd nd nd0.20(0.14 – 0.35)0.22(0.17 – 0.28)Behenic 22:00.46 a(0.37 – 0.52)0.43 a(0.39 – 0.53)0.39(0.37-0.50)0.51 a(0.40 – 0.75)0.50 a(0.38 – 0.80)0.44(0.37 – 0.51)0.40(0.28 – 0.60)0.45(0.37 – 0.57)†Values in the same season and row followed by the same letter are not significantly different at P<0.05.‡nd = not detected, *NA = not availableCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 97 of 375Table VII-5. Grain Mineral Composition.Comparison of the mineral composition of grain derived from CV127 soybean with the isoline control and two conventional soybeanvarieties grown in two seasons as well as with global and Brazilian ranges of values from the ILSI Crop Composition Database.Themean values for the conventional soybean varieties, Monsoy 8001and Coodetec 217, were combinedto provide a single mean value(Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)Analyte (unit)IsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazil(mg/100 g DW)N = 24N = 12N = 15N = 8N = 80 – 323Mean (range)Calcium268 a†(221 – 330)266 a(214 – 318)254(205-313)246 b(172 – 359)286 a(198 – 484)234(167-329)217(117 – 307)NAIron8.50 a(6.01 – 10.43)7.75 b(5.79 – 10.48)8.57(6.54-10.35)9.16 a(7.98 – 10.90)9.15 a(7.80 – 11.40)10.16(7.93-14.13)7.81(5.54 – 10.95)NAMagnesium246 b(204 – 266)266 a(227 – 304)269(225-308)238 b(211 – 266)287 a(268 – 309)255(223-293)264(219 – 313)NAPhosphorus687 a(541 – 834)667 a(546 – 760)670(527-875)768 a(655 – 845)732 b(580 – 871)745(614-808)715(507 – 935)NAPotassium1928 a(1782 – 2071)1881 b(1703 – 2069)1961(1772-2103)1744 b(1593 – 2061)1864 a(1599 – 2021)1758(1595-2033)2061(1868 – 2316)NA†Values in the same season and row followed by the same letter are not significantly different at P<0.05.*NA = not availableCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 98 of 375Table VII-6. Grain Vitamin Composition.Comparison of the vitamin composition of grain derived from CV127 soybean with the isoline control and two conventional soybeanvarieties grown in two seasons as well as with global and Brazilian ranges of values from the ILSI Crop Composition Database.Themean values for the conventional soybean varieties, Monsoy 8001and Coodetec 217, were combinedto provide a single mean value(Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)Analyte (unit)IsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazilN = 24N = 12N = 15N = 8N = 80–323N = 69Mean (range)Folic Acid(μg/100 g DW)330 a†(216 – 456)270 b(183 – 338)291(205-403)365 a(249 – 462)374 a(320 – 464)323(261-438)360(240 – 470)NA*α-tocopherol(mg/100 g DW)3.04 b(2.33 – 4.44)3.49 a(2.63 – 4.69)3.22(2.45-4.61)3.21 b(1.74 – 8.17)3.67 a(1.96 – 9.23)3.54(2.07-7.51)1.91(0.19 – 6.17)3.44(1.36 – 6.17)β-tocopherol(mg/100 g DW)0.60 b(0.20 – 1.01)0.90 a(0.58 – 1.22)0.70(0.12-1.08)0.84 a(0.54 – 1.15)0.96 a(0.68 – 1.37)0.88(0.53-1.34)NA NAγ-tocopherol(mg/100 g DW)16.51 a(11.62 – 20.83)15.81 b(12.31- 18.84)16.28(12.68-20.88)16.41 a(13.79 – 21.63)14.96 b(12.28 – 18.46)15.68(11.65-22.27)NA NAδ-tocopherol(mg/100 g DW)6.18 b(4.41 -7.53)6.56 a(4.9 – 7.99)6.09(5.31-7.42)7.65 a(4.52 – 9.72)8.15 a(3.31 – 11.35)6.99(5.16-8.94)NA NATotal tocopherol(mg/100 g DW)26.19 a(19.26 – 31.54)26.75 a(21.95 – 31.12)26.32(21.68-31.03)28.12 a(25.02 – 34.43)27.41 a(23.38 – 31.40)27.09(21.56-33.23)NA NAVitamin B1(mg/100 g DW)0.65 a(0.44 – 0.86)0.52 b(0.34 – 0.78)0.58(0.42-0.71)0.52 a(0.34 – 0.80)0.55 a(0.28 – 0.75)0.48(0.31-0.71)0.20(0.10 – 0.25)NAVitamin E(mg/100 g DW)5.51 b(4.11 – 7.21)5.91 a(4.68 – 7.16)5.70(4.95 - 7.05)5.75 a(3.94 – 10.79)6.05 a(4.35 – 11.21)5.98(4.64-9.92)NANA†Values in the same season and row followed by the same letter are not significantly different at P<0.05.*NA = not availableCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerantSoybean BPS-CV127-9Page 99 of 375Table VII-7. Grain Isoflavone Composition.Comparison of the isoflavone composition of grain derived from CV127 soybean with the isoline control and two conventionalsoybean varieties grown in two seasons as well as with globaland Brazilian ranges of values from the ILSI Crop CompositionDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217,were combined to provide asingle mean value (Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)AnalyteIsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazil(mg/100 gDW)N = 24N = 12N = 15N = 8N = 80 – 323N = 69Means(range)TotalDaidzein72.2 a†(48.4 – 106.6)52.4 b(41.4 – 72.4)81.3(66.2-96.2)114.6 a(19.7 – 237.8)79.0 b(14.7 – 161.0)100.2(30.8-186.4)86.3(6.0 – 245.4)51.0(6.0 – 112.9)TotalGenistein101.7 a(57.1 – 153.4)83.4 b(60.2 – 121.0)134.5(102.8-166.6)144.5 a(26.1 – 270.1)114.6 b(11.8 – 246.8)144.6(54.3-255.1)97.9(14.4 – 283.7)65.2(14.4 – 135.7)TotalGlycitein22.3 a(14.9 – 31.4)21.7 a(14.6 – 30.2)49.2(36.3-75.4)19.0 a(7.3 – 30.2)16.5 b(7.4 – 23.9)35.3(27.9-43.0)16.1(1.5 – 31.0)13.3(1.5 – 26.4)†Values in the same season and row followed by thesame letter are not significantly different at P<0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 100 of 375Table VII-8. Grain Phospholipid Composition.Comparison of the phospholipid composition of grain derived fromCV127 soybean with the isoline control and two conventionalsoybean varieties grown in two seasons as well as with globaland Brazilian ranges of values from the ILSI Crop CompositionDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217,were combined to provide asingle mean value (Conv Stds) for each analyte.Season1(2006/2007)Season 2(2007)AnalyteIsolineCV127Conv StdsIsolineCV127Conv Stds(mg/g fat)N = 24N = 12N = 15N = 8Means(range)Phosphatidylethanolamine101.9 a†(89.7 – 127.9)90.9 b(57.9 – 108.9)98.2(79.4-113.7)109.4 a(65.7 – 156.8)106.0 a(52.1 – 139.5)100.5(51.1-133.7)Phosphatidic acid4.0 a(1.8 – 6.9)2.9 b(1.0 – 4.7)4.1(2.1-7.5)2.1 b(0.7 – 6.7)2.6 a(0.5 – 9.9)3.0(0.8-7.2)Phosphatidyl inositol11.8 a(10.1 – 14.2)9.6 b(6.1 – 11.2)12.3(11.4-13.5)10.4 a(8.6 – 14.8)9.7 a(8.0 – 11.0)10.8(8.1-12.4)Phosphatidyl choline29.3 a(24.9 – 38.5)26.9 b(15.9 – 32.3)30.7(25.6-38.2)32.9 a(20.6 – 45.0)32.5 a(17.5 – 40.2)33.4(17.0-42.0)†Values in the same season and row followed by the same letter are not significantly different at P<0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 101 of 375Table VII-9. Grain Antinutrient Composition.Comparison of the antinutrient composition ofgrain derived from CV127 soybean withthe isoline control and two conventionalsoybean varieties grown in two seasons as well as with globaland Brazilian ranges of values from the ILSI Crop CompositionDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217,were combined to provide asingle mean value (Conv Stds) for each analyte.Season 1(2006/2007)Season 2(2007)Analyte (unit)IsolineCV127Conv StdsIsolineCV127Conv StdsGlobalBrazilN = 24N = 15N = 8N = 80 – 323N = 69Means(range)Phytic Acid(mg/g DW)2.95 a†(1.43 – 6.09)2.54 a(0.71 – 5.27)2.89(1.47-7.39)3.36 a(1.89 – 6.00)3.81 a(2.63 – 4.77)4.06(2.37-5.44)11.21(6.34 – 19.60)NA*Raffinose(g/100 g DW)1.1 b(0.9 – 1.5)1.3 a(1.0 – 1.7)1.1(0.8-1.6)1.30 a(1.00 – 1.50)1.2 a(0.9 – 1.7)1.3(1.1-1.4)0.355(0.212 – 0.661)NAStachyose(g/100 g DW)3.7 a(3.0 – 4.2)3.6 b(3.1 – 4.1)3.8(3.1-4.6)4.0 a(3.0 – 4.6)3.6 b(2.4 – 4.1)4.1(3.1-4.8)2.19(1.21 – 3.50)NALectinsHU‡/mg DW2.24 a(1.35 – 3.35)2.20 a(1.27 – 3.49)2.03(1.38-3.53)1.70 a(0.85 – 3.43)0.92 b(0.11 – 3.43)1.65(0.67-2.67)1.718(0.105 – 9.038)0.815(0.299 – 1.892)Urease(ΔpH)1.93 a(1.27 – 2.12)1.91 a(0.90 – 2.21)1.93(1.43-2.16)1.53 a(0.28 – 2.06)1.58 a(0.49 – 2.03)1.63(0.27-2.02)NA NATrypsin Inhibitor(TIU/mg DW)12.29 a(6.03 – 16.4)12.01 a(9.14 – 15.13)11.45(5.03-19.64)13.16 a(8.48 – 17.97)13.80 a(7.82 – 18.03)14.02(9.84-16.76)48.33(19.59 – 118.68)NA†Values in the same season and row followed by the same letter are not significantly different at P<0.05.‡HU = Hemagglutinating units*NA = not availableCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 102 of 375 B. Forage Composition

Soybean has many uses in animal and human nutrition. The main soybean product fed to animals is the defatted/toasted soybean meal (OECD, 2001). However, other components of the soybean plant, including the forage, are also fed to a limited extent to animals, primarily to cattle. Soybean forage is typically harvested between th e time the plants reach the sixth node stage to the beginning of pod formation. Therefore, the purpose of the current study was to demonstrate that the forage of CV127 soybeans, treated with an imidazolinone herbicide, is substantially equivalent in composition to forage from the isoline control and other conventional soybean varieties, and that CV127 forage is appropriate for use in animal feed. The forage was produced from plants grown in replicated field trials (three replicate plots per treatm ent at each location) at six locations in Brazil during the summer of 2007/2008 (field trial locations included Santo Antônio de Goiás, GO; Uberaba, MG; Sete Lagoas, MG; Londrina, PR; Brasília, DF; and Santo Antônio de Posse, SP and are listed in Appendi x D). The components analyzed included: proximates (moisture, fat, ash, protein, carbohydrat es and calories) and fibe r (crude fiber, acid detergent fiber and neutral deterg ent fiber). The results of anal yses for CV127 were compared to

the isoline control and to tw o conventional soybean varietie s, Monsoy 8001 and Coodetec 217, grown in the same field trials.

At each field trial location, the above-ground portion of three plants from three replicate plots for each soybean treatment was harvested when the plants were at the R2 growth stage. The forage samples were shipped on wet ice to the Instituto de Tecnologia de Alimentos (ITAL), Campinas, Brazil where they were processe d and analyzed for proximate a nd fiber composition. The three plants from each replicate plot were pooled t ogether prior to analyses, and analyses were conducted for each replicate treatm ent at each field site. The methods used for this study are presented in Appendix D. Also, the forage com position data from each individual field site are presented in Appendix D of this pe tition. Results are reported on a dry-weight basis. Statistical analysis was conducted using the dry-weight data. In this st udy, statistical analyses of the compositional data included all treatments: CV127 + imi, isoline control, and the two conventional soybean varieties. Statistical anal ysis methods were as described for compositional analyses of the grain, except that for forage composition, contrast s were carried out to compare

the isoline control as well as th e conventional soybean varieties to CV127 soybean. Values were considered statistically significantl y different at the P < 0.05 level.

The proximate and fiber composition results calcu lated and statistically analyzed across all locations for forage samples of CV127 soybean treated with imidazolinone herbicide are compared in Table VII-10 to those of the isol ine control and the two conventional varieties treated with conventional herbicide. Proximate analyses incl uded moisture, ash, fat, protein, carbohydrates, and calories. The fiber constituents included crude fiber, acid detergent fiber (ADF) and neutral detergent fiber (NDF). There were no statistical ly significant differences in levels of moisture, ash, fat, pr otein, carbohydrates, calories, crude fiber, ADF or NDF in forage samples produced from CV127 soybean compared to levels of these anal ytes in the isoline control soybean. In addition, comparison of th e forage composition of CV127 soybean and the

isoline control with that of th e two conventional soybean varieties demonstrat ed that there were no statistically significant differences among thes e treatments for fat, calories, ADF or NDF.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 103 of 375 Where small differences in analyte values we re observed between CV127 and the conventional soybean varieties, this was attributed to germplasm differences, since the same differences were

observed between the isoline control and the conventional varieties.

The results of this study confirm that forage derived from CV127 soybeans contains the same

level of nutrients as the isoline control and similar levels as c onventional soybean varieties that are currently cultivated. Therefore, forage de rived from CV127 soybeans is appropriate for use

in animal feed. These results further su pport the conclusion that CV127 soybean is compositionally equivalent to and as nutritious as conventional varieties with a long history of

safe use in animal feed.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 104 of 375 Table VII-10. Proximate and Fiber Composition of Soybean Forage. Forage composition of CV127 soybean was compared to composition of the isoline control and two conventional soybean varietie s (Conv Std 1 and Conv Std 2) acros s six locations in Brazil in

the 2007/2008 Season.

Analyte/Unit Isoline CV127 Conv Std 1 Conv Std 2

N=18**

Proximates Mean (range)

Moisture 81.4 ab* 81.1 b 82.3 a 81.4 a g/100 g FW (79.0 – 84.3) (78.4 – 85.3) (78.4 – 85.3) (73.1 – 84.2)

Ash 8.5 c 8.6 bc 9.0 a 9.0 ab g/100 g DW (7.1 – 10.3) (7.0 – 10.7) (6.4 – 11.2) (6.8 – 10.5)

Fat 2.5 a 2.6 a 2.6 a 2.5 a g/100 g DW (1.7 – 3.5) (1.6 – 3.4) (1.9 – 3.5) (1.7 – 4.1)

Protein 17.7 b 17.3 b 19.0 a 19.1a g/100 g DW (15.1 – 19.5) (15.3 – 19.5) (15.9 – 23.1) (17.0 – 22.4)

Carbohydrates 71.0 a 71.6 a 69.5 b 69.6 b g/100 g DW (66.9 – 73.1) (66.7 – 75.6) (62.4 – 74.9) (64.8 – 73.5)

Calories 378 a 379 a 377 a 378 a kcal/100 g DW (367 – 386) (371 – 387) (368 – 385) (372 – 387)

Fiber

Crude Fiber 29.8 a 29.8 a 28.8 b 29.6 a g/100 g DW (27.5 – 33.2) (27.9 – 32.7) (25.1 – 32.3) (27.0 – 33.1)

ADF 36.57 a 36.41 a 35.68 a 36.00 a g/100 g DW (31.79 – 42.81) (33.39 – 42.16) (28.94 – 41.82) (30.43 – 44.58)

NDF 45.28 a 45.36 a 44.45 a 44.68 a g/100 g DW (39.71 – 50.90) (40.07 – 52.85) (39.33 – 50.67) (38.93 – 52.28) *Numbers followed by the same letter are not sta tistically significantly different at P < 0.05.

**N=17 for Conv Std 1 and Conv Std 2 for ADF and NDFCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 105 of 375 C. Poultry Feeding Study

Soybeans are used primarily to pr oduce oil and high protein soybean meal. Soybean meal is the predominant protein component of animal feeds w ith 97% of soybean meal being used in animal feeds and accounting for nearly 65% of the world’s protein in animal feed. In order to assess the wholesomeness of soybean meal from CV127 soyb eans and to compare it with that of soybean meals from conventional soybean varieties, a 42-day f eeding study with rapidly growing broiler

chickens was conducted. The broiler chicken consumes high amounts of feed per unit of body weight and is a well-recognized model animal for assessing the whol esomeness of feed ingredients (ILSI, 2003).

In order to produce the grain for this feeding study, CV127 s oybeans and three conventional soybean varieties, including Conquista, Monsoy 8001, and Coodetec 217, were grown at a field

trial location near Santo Antônio de Po sse, Brazil during the 2006/2007 growing season. Conquista is the conventional variety that was originally tr ansformed with the csr1-2 gene encoding an imidazolinone-tolerant AtAHAS enzyme and it is closely related genetically to CV127 soybean. The soybeans were cultivated accord ing to standard agricultural practices for soybean production in Brazil, and cultivation prac tices were the same as used in the 2006/2007 agronomy field trials described in Appendix F. The generation of CV127 soybean used as a grain source in this feeding study was designated as the F 9 generation of imidazolinone-tolerant CV127 Line 603 (refer to Figure III-1). The CV127 plants were treated with 70 g imazapyr/ha, while the conventional soybean varieties Conquista, Monsoy 8001, and Coodetec 217 were

treated with the conventi onal soybean herbicide Volt ®

.

Grain from each treatment was harvested from the field trial and was processed separately into toasted soybean meal. The soybean grain processing was conducted by the Instituto de Tecnologia de Alimentos (ITAL) in Campinas, Brazil, using a pilot-scale processing method

. First, the soybeans were cleaned of impurities su ch as dust, stones, branches and weed seeds using manual separation and sieves. The soybeans were flaked in an expeller type press with a 40 kg/h processing capacity. Approximately 5% of the oil was removed during this preparation. The flaked soybean material was extracted in a ba tch-type extractor, using indirect steam to heat the n-hexane solvent to 45-50oC. In the producti on of the defatted and toasted soybean meal, the

extractors were heated with indirect steam for 20 minutes in order to evaporate the solvent in the meal. Subsequently, direct steam (30 psi) wa s applied for 30 minutes through an orifice plate with a 2.5 mm aperture. The residual solvent wa s removed by treating with direct steam for 30 minutes under vacuum (250 mm Hg). To finish the process, the m eal was subjected to a pressure of 0.2 kg/cm 2 for 10 minutes. The meal moisture was re duced to less than 12% (w/w) in a flash dryer with a temperature of 200-250 oC for 54 seconds.

The identity of test and contro l soybean grain used to produce the meal as well as the soybean meal was confirmed using an event-sp ecific PCR for the detection of the csr1-2 gene in CV127. These analyses were conducted by GeneScan, Brazil. Of the four grain and four related soybean meal samples, only the CV127 soybean grain and soybean meal tested positive by PCR, thereby verifying the identity of these samples and demo nstrating that there was no cross-contamination of the conventional soybean grain or meal with gr ain or meal from CV127 soybean. In addition, the grain samples and the corresponding soybean m eal samples were tested for the presence ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 106 of 375 important mycotoxins, including aflatoxins B1, B2, G1, and G 2, zearalenone, and ochratoxin A by the Instituto de Tecnologia de Alimentos (ITAL). None of these mycotoxi ns were detected in any of the soybean grain or meal samples. The grain samples were also tested by Bioensaios (São Paulo, Brazil) for the presence of residues of pesticides that were used in the cultivation of the soybeans. For all pesticides tested, residues were either not detectable or were well below the levels of concern for broiler performance studies.

The poultry feeding experiments were conducted by the Embrapa Suínos e Aves (Embrapa Swine and Poultry) at their animal feeding f acilities in Concórdia, Brazil. Preliminary experiments were conducted with feeds contai ning the soybean meals from CV127 soybean and the three conventional soybean varieties to determine the a pparent metabolizable energy corrected for nitrogen and the true digestibility va lues of the amino acids. The results of these experiments were used to produce nutritionally-b alanced feeds for the feeding study intended to assess the wholesomeness of soybean meal fr om CV127 soybeans. Five hundred and seventysix broiler chickens of the lineag e AgRoss 508, half male and half female, were used in the study. The birds were organized randomly in blocks by body weight with 12 re plications with 12 broilers (six males and six females) per replica tion for a total of 144 broilers per treatment.

Feeds were formulated for each of the four diff erent treatments that included the soybean meal and all feeds were formulated to be isoenergetic and isoproteic. The expe riment was divided into four phases, the initial (one to ten days of age) , growth (11 to 28 days), and two final stages (29 to 35 and 36 to 42 days, respectively), and the feed was balanced for each phase to meet the changing nutritional requirements of the animal s. The feeds contained varying amounts of soybean meal that were approximately 40% of th e feed in the initial stage and declined to approximately 30% in the final stage. During the study the initial weight, body weight, weight gain, feed intake, and feed conversion were assessed at the end of each experimental stage (at 10, 28, 35, and 42 days of age). Statistical analysis of the resulting data was performed using the Dunnett Test (SAS software, 2003) to compare the results from the animals fed feed containing soybean meal from CV127 soybean with those resu lts from the animals fed feed containing the soybean meals from the conventional soybean varieties.

Analysis of the resulting data demonstrated that there was no interaction between the different

feed treatments and the sex of the animals, so the statistica l analysis was conducted without segregation of the sexes. The results demonstrat ed that there were no statistically significant differences (P>0.05) in body weight, weight gai n, feed intake or feed conversion between animals fed feed containing soybean meal from CV127 soybeans and thos e fed feeds containing soybean meal from the conventional varietie s Conquista and Monsoy 8001 (Table VII-11). Animals fed diets containing soybean meal from Coodetec 217 soybeans had statistically significantly lower body weights and weight gain in all growth stages compared to animals fed diets containing soybean meal from CV127 soybean. Based on the results of this study, it can be concluded that soybean meal derived from CV127 soybean is nutritionally comparable to soybean meals derived from conventional soybean varieties that are cultivated commercially. This study also confirms that meal produced fr om CV127 grain is appropri ate for use in animal

feed.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 107 of 375 Conclusions:

Collectively, results of compositional analyses of whole grain and forage as well as a poultry feeding study demonstrate that the grain and forage produced by CV127 soybean are compositionally equivalent to, a nd as nutritious as, grain and forage produced by the isoline control and other conventional soyb ean varieties with a history of safe use in food and feed products, as well as safety to the environment.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 108 of 375 Table VII-11. Performance of Broilers Fed CV127, Conquista, Monsoy 8001 or Coodetec 217 Soybean Meal. Measurements included body weight (BW), weight gain (WG), feed intake (FI), and feed conversion (FC) with their respective average st andard errors for broiler chickens during the periods studied.

Treatments

T1 T2 T3 T4 Performance

CV127 CONQUISTA MONSOY 8001 COODETEC 217

Initial weight (g) 44.44 ± 0.05 44.40 ± 0.05 44.40 ± 0.05 44.43 ± 0.05

Period from 1 to 10 days of age

BW (g) 289 ± 4 295 ± 4 292 ± 4 273 ± 4 *

WG (g) 244 ± 4 251 ± 4 247 ± 4 228 ± 4 *

FI (g) 272 ± 3 273 ± 3 271 ± 3 258 ± 3*

FC 1.11 ± 0.01 1.09 ± 0.01 1.10 ± 0.01 1.13 ± 0.01

Period from 1 to 28 days of age

BW (g) 1480 ± 10 1503 ± 10 1507 ± 10 1443 ± 10 *

WG (g) 1436 ± 10 1459 ± 10 1463 ± 10 1398 ± 10 *

FI (g) 1955 ± 15 1983 ± 15 1970 ± 15 1915 ± 15

FC 1.36 ± 0.01 1.36 ± 0.01 1.35 ± 0.01 1.37 ± 0.01

Period from 1 to 35 days of age

BW (g) 2068 ± 18 2106 ± 18 2101 ± 18 2004 ± 18 *

WG (g) 2024 ± 18 2062 ± 18 2057 ± 18 1959 ± 18 *

FI (g) 3023 ± 18 3055 ± 18 3037 ± 18 2975 ± 18

FC 1.50 ± 0.01 1.49 ± 0.01 1.48 ± 0.01 1.52 ± 0.01

Period from 1 to 42 days of age

BW (g) 2620 ± 15 2644 ± 15 2666 ± 15 2567 ± 15 *

WG (g) 2576 ± 15 2600 ± 15 2621 ± 15 2522 ± 15 *

FI (g) 4183 ± 22 4187 ± 22 4210 ± 22 4125 ± 22

FC 1.63 ± 0.01 1.62 ± 0.01 1.61 ± 0.01 1.64 ± 0.01

* Averages with an asterisk are significantly different (P<0.05) as compared with the CV127 soybean meal

treatment.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 109 of 375 

### VIII. Agronomic, Phenotypic and Ecological Evaluations of CV127

The environmental safety of CV127 was shown to be comparable to an isoline control and other conventional soybean varietie s through the evaluation of va rious agronomic, phenotypic, phenologic, and ecological in teraction characteristic s of CV127 compared to an isoline control as well as other conventional soybean plants. The studies were conducted either as part of multilocation field trials or under laboratory or gr eenhouse conditions. Field trials with CV127 soybeans were conducted at seven different lo cations in Brazil duri ng the 2006/2007 growing season and at six trial locations in Brazil during the 2007 growing season. In all field trials, the CV127 soybeans were compared to a near-isogenic, null segregant soybean (referred to as the isoline control) that does not contain the csr1-2 gene cassette and to two other conventional soybean varieties commonly cultivated in Brazil. The field trial locations were representative of the different regions within Brazil where soyb ean is commercially cult ivated and each trial location consisted of four replications of the test, isoline control and two conventional soybean varieties that were organized in a randomized complete block design. Details on field trial locations, trial management practices, experimental methods and results are presented in detail in Appendix F.

There are many similarities in agronomic prac tices used in soybean production between the United States and Brazil, includi ng Maturity Groups of soybean cul tivars as well as weed, insect and disease control practices. There are some si milarities in the climatic conditions between the regions in the southeastern Unit ed States where soybeans of matu rity group VIII are cultivated and Brazil, such as the average temperatures during the growing season (see section IX and Table IX-4), and there are climatic differences, in cluding rainfall (Table IX-5). A more detailed comparison of the agronomic practices used in the cultivation of soybeans and of the environmental and climatic conditions in the United States and Brazil is presented in section IX.C.4 of this petition. Therefor e, in the event that CV127 soybean s were to be introduced into the United States environment, the data genera ted in the field trials conducted in Brazil to support the environmental as well as food and feed safety of CV127 soybeans are equally applicable to the environmental, food and feed safety assessme nt of CV127 for the United States

environment.

CV127 soybean was compared to the isoline control and tw o other conven tional soybean varieties with respect to key vegetative and reproductive developm ent characteristics associated with the competitiveness and survival of plant species. In field trials, characteristics including seed germination rate, seedling vigor, days to reach key developmental st ages, plant height, and grain yield, as well as susceptibility to and interactions with diseas es and insects were evaluated. In addition, the impact of cultiv ating CV127 soybeans on factors re levant to the nitrogen-fixing symbiosis with Bradyrhizobium japonicum were assessed and compared between CV127, the

isoline control and conventional soybean varieties. Furtherm ore, laboratory- and greenhousebased studies on seed germination as well as pollen number and pollen germination were also conducted and comparisons of these characteristics made between CV127 soybeans and the isoline control and, for the seed germinatio n studies, the two other conventional soybean

varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 110 of 375 Results of these studies demonstrated that, ex cept for herbicide tolerance, soybean CV127 is agronomically, phenotypically, an d phenologically equivalent to the isoline control and other conventional soybean varieties. Further, these studies showed no different biological effect of CV127 compared to the isolin e control soybeans with resp ect to interaction with Bradyrhizobium
japonicum and nitrogen fixation capacity or interaction with various diseases or insects. Therefore, these results reinforce the conclusion that the cultivation of CV127 soybeans poses no different plant pest or weediness potential and will have no different environmental impact than

the cultivation of conventi onal soybean varieties.

The treatments included in the seed germination, dormancy and quality studi es, as well as in the field trials used for phenotypic, agronomic, a nd ecological evaluations are listed below:

Treatment Genotype Herbicide application Treatment 1 (T1) CV127 Imazapyr (70 g ai/ha) Treatment 2 (T2) CV127 Volt ® (570 g ai/ha) Treatment 3 (T3) Isoline control Volt ® (570 g ai/ha) Treatment 4 (T4) Monsoy 8001 Volt ® (570 g ai/ha) Treatment 5 (T5) Coodetec 217 (CD 217) Volt ® (570 g ai/ha)

The methods as well as results for all treatments are included in Appendices E and F to this petition. However, because the obj ectives of the evaluations pres ented in this section of the petition were to compare the intrinsic agronomic, phenotypic and ecological characteristics of

CV127 to those of the isoline c ontrol and the conventional soybean varieties in the absence of potential effects of imidazolinone herbicide application to the plant, Treatment 1 is not included in the summary data presented in this section, but is included in the detailed data presented in Appendices E and F. Statistical analyses of the data included all of the above five treatments.

The generations of CV127 and controls us ed in these studies are shown below.

Comparative Analyses Performed, Generation s Used for Analyses, and Comparators Used

as Controls.

Analysis CV127 Soybean

Generation Used Comparator Used

Seed Quality, Germination and Dormancy:

Seed Derived from 2007 Field Trials F 7 (CV127 line 127) Isoline Control (F 7 null) Monsoy 8001

Coodetec 217

Pollen Number and Germination: Greenhouse-Grown, Londrina, Brazil, 2007. F 5 (CV127 line 127) Isoline Control (F 5 null)

Agronomic, Phenotypic and Ecological Evaluation:

Field Trials 2006/07 F 5 (CV127 line 127) Isoline Control (F 5 null) Monsoy 8001

Coodetec 217 Agronomic, Phenotypic and Ecological Evaluation:

Field Trials 2007 F 6 (CV127 line 127) Isoline Control (F 6 null) Monsoy 8001

Coodetec 217CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 111 of 375 A. Seed Germination, Dormancy and Quality Characteristics

Assessments of seed germination, dormanc y and quality were compared between CV127 soybean, the isoline cont rol and two conventional soybean va rieties. Seed germination and quality are important agronomic ch aracteristics used in comparisons of different varieties within a crop species, and seed dormancy or “hard seed” in plants is an important characteristic often associated with plants that are weeds (Anderson, 1996; Li ngenfelter and Hartwig, 2003). An assessment of seed dormancy is often used to a ssess the weediness potential of different plant species (Baker, 1974). Although soybeans do not typically display seed dormancy characteristics (OECD, 2000), the potential fo r seed dormancy in CV127 soybean was assessed and compared to that of the isoline control as well as two conventi onal soybean varieties (Monsoy 8001, Coodetec 217 [CD 217]).

Data are presented in this section for compar isons made between CV127 seed produced from plants cultivated using conventional herbicide (570 g Volt ® ai/ha) application for weed control,

and seed of the isoline contro l and the two conventional soybean varieties produced with the same conventional herbicide application. Seed sa mples from all treatments were harvested from

field trials conducted in six different locations (listed in Table VIII-5) in Brazil in the 2007 short growing season. Two hundred seed per experiment al unit (four sub-sample s of 50 seed derived from each replicate treatment at each field tr ial location) were tested for germination and dormancy, following standard procedures (AOSA, 2007), and for other seed quality parameters by tetrazolium staining (França Neto et al ., 1998). Prior to the initi ation of the tests, the identities of the test (CV127), control (iso line control), and reference (Monsoy 8001 and Coodetec 217) seed materials were verified by testing for the presence or absence of the csr1-2 transgene by event-specific PCR analyses; chain-of-custody docume ntation for the identity of the

control and reference materials was maintained.

To assess seed germination, each sub-sample of 50 seed was placed in rolled, moist paper towels and incubated at 100% humidity in an incubator at 20/30oC with the lower temperature maintained for 16 hours and the higher temperature for eight hours. Germination was assessed at five and eight days after planting. Seed that failed to germinate in this test were evaluated for developmental abnormalities and hard or firm seed coat characteristics to determine the cause of lack of germination. Hard seed typically remain ed unimbibed after the test, whereas firm seed imbibed water and were swollen but did not germinate. The hard or firm seed coat characteristic is related to seed dormancy in s oybean. Any seed that did not ge rminate and did no t fall into the “abnormal”, “hard” or “firm” seed coat categorie s were designated as “dead” seed. Hard seed was determined at the final reading of the germin ation test and the percentage of hard seed was added to the germination percenta ge, based on the fact that hard -seeded soybean is considered viable (Potts et al ., 1978).

Tetrazolium staining for determining seed viability and quality was performed with two subsamples of 50 seed each from all treatments from each field trial location. Initially, seed were kept overnight in a moist germination paper towe l at 25oC for 16 h in an environment with a relative humidity of 100%. After conditioning, the s eed were placed in plastic cups, and covered

with 0.075% tetrazolium solution. The cups were incubated in an oven at 40oC for 150 minutes.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 112 of 375 After staining, the seed were rinsed with tap wa ter several times to st op the staining reaction. Seed were kept submerged in water to avoid deh ydration. Individual seed were examined with a magnifying lens (6X) under fluorescent light. S eed were dissected long itudinally through the midsection of the embryonic axis. After the seed was sectioned, the seed halves were separated and the seed coat was removed to expose the out er surface of the coty ledons. The inner and outer surfaces of the cotyledons were observed to identify seed defects that are revealed by the staining procedure. Damage was attributed to either mechanical , weathering or stink bug causes, and was recorded for each sample. Special care was applied during the ev aluation of the radiclehypocotyl axis and the vascular region on the cotyledons, which are structures of major importance for the viability of the seed. After th e seed was sectioned, its internal surfaces were inspected and percent viability was recorded for each sample. Live cotyledons are normally white, due to the lack of diffusion of the tetrazolium solution to the inner tissues of the cotyledons. Data from the germination and tetraz olium-staining tests were statistically analyzed by Tukey’s test (Steel and Torrie, 1980) to compar e means of sources of variation determined by analysis of variance (ANOVA) to have a significant effect on each of the measured traits. In order to compare treatments cultivated similarly, only the data from the treatments treated with the conventional herbicide are presented. In th e tetrazolium staining test , only the most serious seed damage (i.e. ratings between 6 and 8) in the tetrazolium test results was included in the statistical analysis and reported in Table VIII-1. A complete de scription of the experimental methods and results of this study are presented in Appendix E.

The results of the tetrazolium and germination tests demonstrated that there were no statistically significant differences between CV127 soybean a nd the isoline control or the conventional soybean varieties across field trial locations in seed damage caused by either mechanical means, weathering or stink bugs. In addition there were no statistically signifi cant differences between

the treatments across field trial lo cations for seed viability, seed germination, or occurrence of abnormal, dead, hard and firm seed (Table VIII-1 ). Seed damage levels were relatively low across all treatments. Percent viability and germ ination within each treatment were comparable,

with the lowest levels associated with conven tional variety Coodetec 217 and the highest values found for CV127. The lack of germination of some seed in all treatments was primarily attributed to seed abnormalities, since firm and hard seed percentages were zero or nearly zero, respectively, and CV127 soybean did not differ from the isoline or conventional control varieties in these characteristics. Together, these result s demonstrate that seed germination, dormancy and other seed quality parameters of CV127 soybean are not differe nt from those of conventional soybean varieties. Furthermore, these result s support the conclusion that CV127 soybean does not pose any greater weediness po tential or any different potenti al environmental impact than conventional soybean varieties.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 113 of 375Table VIII-1. Means per Treatment Across Locations for Seed Quality, Germination and Dormancy Traits of CV127, theIsoline Control and Two Conventional Soybean Varieties.TraitsAssessed1Tetrazolium Test2Germination TestDmecDweathDstinkViabGermAbnorDeadHardFirm(6-8)(6-8)(6-8)CV127 3.17 ab32.65 a 3.09 a 91.00 a 88.48 a 8.22 a 3.30 a 0.04 a 0.00 aIsoline 4.91 a 2.48 a 1.83 a 90.78 a 87.43 a 9.13 a 3.43 a 0.72 a 0.00 aMonsoy 8001 2.71 ab 2.79 a 4.63 a 89.83 a 85.13 a 9.48 a 5.40 a 0.04 a 0.00 aCD 217 2.63 ab 7.33 a 3.29 a 86.75 a 81.71 a 10.90 a 7.40 a 0.90 a 0.00 a1Dmec: mechanical damage (%); Dweath: weathering damage (%); Dstink: damage by stink bugs (%); Viab: seed viability (%); Germ: seed germination (%);Abnor: abnormal seedling (%); Dead: dead seed (%);Firm: firm seed (%); Hard: hard seed (%).2Seed receiving a rating of 6 to 8 in the assessment are considered nonviable (Franca Netoet al., 1998). The cause of the nonviability of these seed was furtherinvestigated and the results are reported here.3Means in columns followed by the same letter do not differ statistically significantly at the 5% probability level (Tukey’s test).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 114 of 375 B. Pollen Number and Germination Characteristics

Soybean is a self-pollinating plant species that is commercially propagated by seed. Artificial hybridization techniques are used for breeding purposes. The stig ma of the soybean flower is receptive to pollen about 24 hours prior to anthesis and remains receptive for 48 hours thereafter. The anthers in the bud directly pol linate the stigma of the same flower and as a result, soybeans exhibit a high degree of self-fertilization and cr oss-pollination is usually less than one percent (Caviness, 1966). The purpose of the current study was to demonstrate that CV127 soybean produces pollen with similar characteristics to the pollen produced by a near-isogenic control soybean. Flowers from CV127 soybean and the iso line control plants that were grown in the greenhouse were collected and used to determine the number of pollen grains in each flower. In addition, the pollen was incubated under conditi ons to stimulate germination and pollen germination was assessed. The results of this study demonstrate that th ere is no difference in pollen number or pollen germination between th e imidazolinone-tolerant CV127 soybean or its isogenic, non-transgenic count erpart (isoline control).

Plants of CV127 soybean (F 5 generation of CV127, line 127, refer to Figure III-1) and the corresponding isoline control were grown in pots in a greenhouse in Londrina, PR, Brazil. Plants were watered daily and grown under a regime of 28°C day and 20°C night (12 h day/12 h night).

Mature anthers were collected from five flower s from each of five different plants one day

before anthesis from both CV127 and isoline cont rol plants to determine the number of pollen grains produced per flower. The anthers were coll ected in the morning using a pair of forceps to

remove them from the flowers and the anthers from five flowers from each plant were bulked and were placed immediately into separate E ppendorf tubes containing 0 .2 ml distilled water.

Pollen germination was determined by in vitro germination using flowers that were collected randomly from five plants of each treatment and the anthers from the five flowers from each plant were bulked and were placed in a sucros e solution (0.45 M) and shaken slightly. The solution containing the pollen grains from each pl ant was allowed to stand for approximately 3 hours. A grain of pollen was considered germinat ed when the germination tube length was equal to the grain diameter. Germinated pollen grai ns were counted in a Neubauer chamber and the number of pollen grains that pr oduced a pollen tube and the mean number of germinated pollen grains were determined (Figure VIII-1). In ad dition, the size of the pollen tube was measured for 20 pollen tubes of both CV127 soybean and the isoline control that were randomly selected in the Neubauer chamber using a microscope. Statis tical analysis of the data was by the t-test (Steel and Torrie, 1980).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 115 of 375 Figure VIII-1. Counting the Pollen Grains (A) and G erminated Pollen Grains with Pollen Tubes (B). A Neubauer chamber and 20X magnification were used.

Table VIII-2 shows the quantity of pollen grains in the samples from five plants in each treatment. Results showed no statistically significant difference (at the 5% level) between the number of pollen grains produced by CV127 so ybean compared to the isoline control.

Table VIII-2. Pollen Grain Count of Isoline Control Soybean and CV127 Soybean.

Isoline Control Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Average Pollen per flower* 8200 7933 8000 7933 8400 8093

CV127 Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Average Pollen per flower* 7800 7866 8000 7866 8733 8053

*Mean of 5 flowers

T test: t value d.f. 1 P 2

0.2046 8 0.84

1 d.f.=Degrees of freedom

2 P=Probability

The number and percentage of germinated pollen grains was determined for each sample of the CV127 and isoline control soybean pl ants, and results are presented in Table VIII-3. Statistical analysis of the data was by the t-test (Steel and Torri e, 1980). Results s howed no evidence of a statistically significant difference at the 5% level between the number of germinated pollen grains produced by CV127 soybean and its isoline control.

The length of the polle n tube was measured for 20 pollen tubes of both CV127 soybean and the isoline control and results are presented in Ta ble VIII-4. Results showed no statistically significant difference at the 5% level between polle n tube length of CV127 soybean compared to

the isoline control. 

### A BCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 116 of 375 Results of these studies showed no evidence of statistically significant differences in pollen number and pollen germination or tube length between CV127 soybean and the isoline control. Therefore, the production of CV127 soybean by the introduction of the imidazolinone-tolerant acetohydroxyacid synthase large subunit gene ( csr1-2 ) into the soybean genome confers herbicide tolerance to the soybean plant but has no effect on polle n production or on the viability of the pollen produced.

Table VIII-3. Number and Percentage of Germinated Pollen Grains of the Isoline Control and CV127 Soybeans using the Neubauer Chamber.

Isoline Control Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Average Total pollen* 8000 8200 8000 8266 7866 8067 Germinated pollen* 2933 2800 3000 2800 2733 2853 Germination (%) 36.66 34.15 37.50 33.87 34.74 35.37

CV127 Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Average Total pollen* 8133 8266 7866 8066 9200 8307 Germinated pollen* 2933 3000 2600 2666 3400 2920 Germination (%) 36.06 36.29 33.05 33.05 36.96 35.15

*Mean of 5 flowers

T test: t value d.f. 1 P 2

0.4437 8 0.6690

1 d.f.=Degrees of freedom

2 P=Probability

Table VIII-4. Pollen Tube Length (microns) of Germinating Pollen of the Isoline Control Soybean and CV127 Soybean.

Isoline

Control Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Average Average 101.5 102.5 104.5 103.0 102.0 102.7

CV127 Plant 1 Plant 2 Plant 3 Plant 4 Plant 5 Average Average 102.5 104.5 101.0 105.5 102.0 103.1

T test: t value d.f 1 P 2

0.4104 8 0.6849

1 d.f.=Degrees of freedom

2 P=ProbabilityCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 117 of 375 C. Agronomic and Phenotypic Evalua tions in Replicated Field Trials

Plant growth, development and yi eld characteristics were assess ed under field conditions for CV127 soybean compared to the is oline control and two conventi onal soybean varieties. The purpose of these evaluations was to confirm that the introduction of the imidazolinone herbicide

tolerance trait in CV127 soybean did not affect any phenotypic or agronomic characteristics of CV127 relative to the comparators.

Replicated field trials were conducted in Brazil at 13 field trial locations: seven locations in the 2006/2007 growing season and six locations in the 2007 short growing season. The short season is typically planted in February after the main season in the norther n regions of Brazil that have a longer growing season. A similar cultivation practic e occurs in the southern United States where soybeans are sometimes planted as a double crop in July following the harvest of winter wheat. According to a survey conducted by the Conservation Technology Information Center, in 2004 through 2008 approximately 7% of the acres plan ted to soybeans in the U.S. followed this practice (CTIC, 2008). The locations and planti ng and harvest dates for the field trials are presented in Table VIII-5. Details of field trial management practices, data collection and results are presented in Appendix F.

Table VIII-5. Locations, Planting Dates, and Harvest Dates for each Field Trial Location in Brazil During Two Growing Seasons.

Location – State 2006/2007 Season 2007 Season

Santo Antônio de Posse – SP Oct. 25 - Mar. 16

Ponta Grossa – PR Oct. 10 1

Londrina – PR Oct. 18 - Mar. 14

Uberaba – MG Nov. 21 - Mar. 29 Mar. 02 - Jul. 03

Brasília – DF Nov. 16 - Mar. 29 Mar. 01 - Jul. 09

Santo Antônio de Goiás – GO Nov. 7 - Mar. 15 Feb. 27 - Jul. 08 Sete Lagoas – MG Nov. 9 - Mar. 22 Mar. 06 - Jul. 11

Teresina – PI Mar. 14 - Jul. 04

Vilhena – RO Mar. 12 - Jul. 10

1 Poor germination of initial planting on 10 October prompted the need to replant on 31 October, 2006. The trial was destroyed just prior to harvest on 20 April 2007.

In this section of the petition, results from the treatments listed below are presented across locations by year. In all field trial locations , each treatment was replicated four times in a randomized complete block design.

 CV127 soybean (CV127, line 127 F 5 and F 6 generations used in the 2006/2007 and 2007 seasons, respectively; refer to breeding di agram in Figure III-1) with weed control provided by the conventional soybean herbicide Volt ® [ a combination of bentazon (400 g ai/ha) and acifluorfen (170 g ai/ha)], sprayed at a rate of 570 g ai /ha was included in the

trial.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 118 of 375  An isoline control that is a null se gregant of CV127 soybean and lacks the csr1-2 gene cassette but is from the same stage of breed ing as the CV127 soybean was included in the trial. Weed control was accomplished usi ng the conventional soybean herbicide Volt ® as described above.

 Two conventional soybean varieties were us ed in these field trials: Monsoy 8001 and Coodetec 217 (CD 217). Both of these va rieties are common nontransgenic Brazilian commercial varieties with a similar maturity classification as CV127 soybean (Maturity Groups VIII-IX). Weed control was by a pplication of the conventional soybean

herbicide Volt ® as described above.

As mentioned earlier, an additional treatment of CV127 soybean grown using an imidazolinone

herbicide for weed contro l was included in the st udy and the associated st atistical analysis, and phenotypic and agronomic evaluations of this treat ment are presented in Appendix F. However, to compare only the treatments cultivated in a si milar manner, only the data for those treatments

treated with the conve ntional herbicide Volt ® are presented in this section of the petition.

Important agronomic characteristics of plants of each treatment, including twelve different characteristics of growth and development were evaluated as presented in Table VIII-6. Evaluation parameters included germination, final plant stand, seedling vi gor, plant height, green stem, degree of shattering and lodging, days to fu ll flower, days to full maturity, seed size and grain yield. Statistical analysis of the resulting data from all trea tments was conducted by Tukey’s test (Steel and Torrie, 1980) to compare means of sources of va riation determined by ANOVA to have a significant eff ect on each measured trait.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 119 of 375 Table VIII-6. Agronomic Characteristics Evaluated in the Brazilian Field Trials During the 2006/2007 and 2007 Growing Seasons.

Characteristic

Evaluated Growth Stage at

Evaluation Evaluation Definitions Germination V2 Percent emerged plant seedlings relative to the quantity

of seed sown. Seedling Vigor V2 Overall seedling a ppearance based on a rating scale: 1=Plant emerged, no trifoliate leaves 2=One trifoliate leaf, unhealthy plant

3=One trifoliate leaf, healthy plant 4=Two trifoliate leaves, unhealthy plant 5= Two trifoliate leaves, healthy plant

Initial Plant

Stand V2 Number of plants per plot at the V2 stage

Days to Flowering R2 Number of days from planting to full flower

Days to Maturity R8 Number of days from plan ting to full plant maturity

Plant Height R8 Average height of fi ve plants in a plot measured from soil level to the top of the plant Green Stem R8 Percent plants in a plot that were green relative to the total number of plants in the plot

Shatter R8 Percent plants in a plot that show premature dehiscence of pods Lodging R8 Average standability (l odging) of plants in a plot according to the scale: 1=All plants sta nding erect (80-90°) 2=Most plants have a slight lean (60-70°) 3=Most plants have a moderate lean (40-50°) 4=Most plants have a severe lean (20-30°) 5=All plants are prostrate (0-10°)

Final Plant

Stand R8 Number of plants per plot at the R8 stage

Yield R8 Total weight of all gr ain harvested (adjusted to 12% moisture)

100-Seed Weight R8 Weight (g) of 100 seedsCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 120 of 375 Due to the naturally different environmental char acteristics (temperature, rainfall, humidity, day length, etc.) among the trial locations , location had a significant effe ct on all assessed traits in

both the 2006/2007 and the 2007 field trial seasons. For field trials in the 2006/2007 growing

season, at Ponta Grossa, the most s outherly location (latitude 25 o S) and the location for which the Conquista soybean variety is least adapted, pl ants were taller and the number of days to flower and to maturity were higher than at a ll other locations during the 2006/2007 season (Table VIII-7). Yield was not recorded from this site du e to a necessary late replanting of the study at this site and subsequent unchara cteristic plant growth and late maturity of the CV127, the isoline control, and the conventional s oybean varieties included in the study. In the 2006/2007 season, seed size and yield were highest at Londrina (Table VIII-7). In fact, during this season grain yield was above the Brazilian average yield at all locations except Brasília. At Brasília, seedling vigor was slightly less than at ot her locations and yield appeared to be more adversely affected by a high and late incidence of Asian rust infestation than other locations similarly infected (refer to section VIII.D for disease incidence). In general, within each location in the 2006/2007 season, the performance of CV127 soybean treated with Volt ® and the performance of the isoline control were not significantly different for germination, final stand, seed ling vigor, plant height, green stem, lodging, or yield (Table VIII-7). Only at some locations were significant differences found between CV127 soybean treated with Volt ® and the isoline control for days to flowering (3 of 7 locations) and days to maturity (2 of 7 locations), (Table VIII-7). Shattering was not identified in any plot at any loca tion during either season and so data for this characteristic is not presented.

Some statistically significant differences between the conventional soybean varieties and CV127 soybean treated with Volt ® and the isoline contro l were observed for plan t height, lodging, days to full flower, days to maturity and yield. Du e to the different genetic backgrounds of CV127 soybean and the isoline control compared to th e two conventional soyb ean varieties, these differences were not unexpected.

In the trials of the 2007 season, plant density was nearly ideal at all loca tions, except Teresina,

where the final stand was lower due to low seed germination (Table VIII-8) caused by excessive rainfall shortly after planting. As expected for the short-season growing conditions , plant height was lower compared to the 2006/2007 season, excep t at Sete Lagoas, where plants reached a mean stature of 82 cm and the lodging reached th e higher index of 3.5 (Table VIII-8). The mean for seed size ranged from 16.4 g at Uberaba to 19 g at Vilhena. At the end of the vegetative cycle, flowering occurred before 40 days after pl anting at Vilhena and Teresina as a consequence of the lower latitudes of these sites. Correlated to vegetative cycle (days to flowering), the total cycle (days to maturity) was rela tively short (less than 115 days) in these two sites and also in Uberaba and Sete Lagoas. The yield was relativel y high at Brasilia and Sete Lagoas, medium at

Teresina and Uberaba and lower at Santo Antoni o de Goiás and Vilhena (Table VIII-8). The wide-ranging performance obtained for grain yiel d among the different sites is expected and

considered normal for the short-season in Brazil. However, the coefficients of variation for all

traits were considered low a nd within an acceptable range.

In general, within locations, the perfor mance of CV127 soybean treated with Volt ® and the

isoline control in the 2 007 trials was not significantly diff erent for germination, final stand, seedling vigor, lodging, days to flower, and days to maturity (Table VIII-8). CV127 soybeanCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 121 of 375 treated with Volt ® and the isoline control plants only diffe red significantly for plant height at Sete Lagoas, percent green stem at Uberaba and Vilhena, seed size at Uberaba, Sete Lagoas and Vilhena and yield at Vilhena (Table VIII-8). Some significant differences between the conventional soybean varieties (Monsoy 8001 and Coodetec 217) and CV127 soybean treated

with Volt ® and the isoline control were observed for plant height, green stem, seed size, yield and days to flowering and matu rity. The conventional soybean varieties matured one to four days earlier in some locations and 100-seed we ights were generally le ss in relation to CV127 soybean and the isoline control. Due to the different genetic backgr ounds of CV127 soybean and the isoline control compar ed to the two conventional soybean varieties, these differences were not unexpected.

Although some statistically significant differe nces did exist between CV127 soybean and the isoline control for specific phenotypic and agronomi c traits at specific locations in the trials conducted during the 2006/2007 and 2007 growing seas ons, none of the trait differences were consistent across all locations and years, an d therefore were not c onsidered biologically significant. Furthermore, all trait values for CV 127 soybeans and the isoline control were within the range for soybeans commercially cultivated in Brazil and elsewhere, including the conventional soybean varieties in cluded in these studies. These results support the conclusion

that the cultivation of CV127 soybeans presents no different environmental impact than that presented by the cultivation of c onventional soybean varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 122 of 375 Table VIII-7. Mean Values for Phenotypic and Agronomic Characteristics of Each

Combination of Treatment and Location for all Trials Conducted in the 2006/2007 Season

in Brazil.

Trait †
Location Treatment G FS V PH GS L DF DM 100SW Yield

CV127 86.8 103.1 5.0 56.9 ab ‡ 0.0 1.0 43 b 119 a 19.1 3275

Isoline 92.5 109.4 5.0 57.9 a 0.0 1.0 43 b 119 a 17.1 3131

M8001 94.3 111.1 5.0 56.5 ab 0.0 1.0 43 b 114 b 14.2 3397 Sto.

Antônio

de Goiás
CD217 91.1 108.4 5.0 53.0 b 0.0 1.0 44 a 119 a 13.1 3007

CV127 80.1 94.1 4.0 57.4 0.0 1.0 44 a 126 a 18.0 1553 b

Isoline 81.9 96.3 4.0 58.0 0.0 1.0 44 a 126 a 15.6 1435 b

M8001 79.0 92.9 4.0 58.1 0.0 1.0 42 b 118 b 12.8 2622 a Brasília

CD217 82.3 96.9 4.0 57.3 0.0 1.0 43 ab 126 a 12.3 2347 ab

CV127 89.6 99.8 5.0 87.3 ab 2.0 ab 2.3 ab 43 c 112 a 18.3 4028

Isoline 90.6 108.0 5.0 91.9 a 0.0 b 3.5 a 43 c 112 a 16.8 3829

M8001 85.5 101.5 5.0 84.2 ab 0.0 b 1.0 b 45 b 112 a 12.2 3830 Sete Lagoas

CD217 87.6 101.9 5.0 80.7 b 0.3 b 3.0 a 47 a 108 b 12.9 4033

CV127 91.5 89.0 5.0 100.3 a 3.5 1.8 46 b 129 b 22.1 5171 a

Isoline 92.1 90.3 5.0 101.9 a 3.0 1.8 47 a 130 a 19.5 4463 ab

M8001 89.6 86.4 5.0 91.3 b 4.6 1.0 44 c 125 c 15.0 3799 b Londrina

CD217 90.1 89.0 5.0 83.5 c 2.8 2.3 44 c 125 c 16.9 4143 ab

CV127 89.9 101.0 5.0 75.9 a 0.0 1.0 44 b 111 a 17.2 3404

Isoline 92.4 105.0 5.0 79.5 a 0.3 1.0 44 b 111 a 16.1 3107

M8001 92.5 104.3 5.0 61.6 b 0.0 1.0 44 b 111 a 11.9 3303 Uberaba

CD217 90.9 99.6 5.0 62.4 b 0.0 1.0 47 a 108 b 12.4 3276

CV127 82.4 79.5 5.0 102.3 4.3 1.8 ab 47 a 130 a 18.1 3202

Isoline 88.4 88.4 5.0 105.2 1.0 2.3 a 45 b 126 b 16.1 2788

M8001 87.5 84.9 5.0 98.2 4.0 1.0 b 41 c 123 c 13.0 3182 Sto.

Antônio

de Posse CD217 79.8 78.0 5.0 91.2 3.0 2.8 a 41 c 123 c 13.4 3392

CV127 80.1 67.1 5.0 104.0 ab 4.5 2.8 b 49 c 154 b . .

Isoline 80.5 64.6 5.0 107.8 a 3.5 2.8 b 51 b 154 b . .

M8001 86.8 73.6 5.0 103.5 ab 2.3 2.0 bc 53 a 148 c . . Ponta

Grossa

CD217 79.3 66.0 5.0 94.9 bc 0.0 4.0 a 53 a 143 d . .

† G = initial germination (%); FS = final plant stand; V = seedling vigor; PH = plant height (cm); GS = green stem (%); L = degree of lodging; DF = days to full flower (vegetative cycle); DM = days to full maturity (total cycle); 100SW = seed size (weight of 100 seeds in grams); and Yield = grain yield (kg/ha).

‡ Means within locations followed by the same letter or not followed by a letter do not differ significantly by the Tukey test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 123 of 375Table VIII-8. Mean Values for Phenotypic and Agronomic Characteristics of Each Combination of Treatment and Locationfor all Trials Conducted in the 2007 Season in Brazil.Trait†LocationVarietyGISFSVPHGSLDFDM100SWYieldCV12770.25 84.00 76.50 5.00 a 48.80 ab 47.50 b 1.00 a 45.00 b 109.00 a 19.53 a 1863.50 aIsoline73.75 88.75 77.50 5.00 a 54.05 a 25.00 c 1.00 a 45.00 b 109.00 a 17.60 b 2593.50 aM800178.00 93.50 88.00 5.00 a 46.90 b 22.50 c 1.00 a 46.00 a 106.00 b 14.28 c 2019.25 aUberabaCD21776.50 92.00 79.25 5.00 a 38.50 c 1.00 d 1.00 a 46.00 a 106.25 b 14.35 c 2091.75 aCV12777.75 93.25 86.00 5.00 a 81.85 bc 35.00 bc 4.00 a 48.50 ab 114.00 a 20.23 a 2446.00 aIsoline77.25 93.00 85.75 5.00 a 91.75 a 23.75 c 4.00 a 49.00 ab 114.00 a 18.15 b 2742.50 aM800178.00 93.50 90.75 5.00 a 85.55 ab 42.50 b 2.75 a 49.25 ab 112.25 b 16.10 c 2509.50 aSeteLagoasCD21778.75 94.75 90.25 5.00 a 74.35 d 5.50 d 3.00 a 49.75 a 112.00 b 14.95 c 2939.50 aCV12794.50 113.25 113.13 5.00 a 58.00 a 10.00 a 1.00 a 44.00 a 131.00 a 19.18 a 1928.75 aIsoline93.25 111.63 110.38 5.00 a 58.00 a 10.00 a 1.00 a 44.00 a 130.50 a 18.53 a 2179.50 aM800192.50 110.88 109.50 5.00 a 48.50 b 10.00 a 1.00 a 44.00 a 129.25 b 16.28 b 1985.50 aSto. Ant.de GoiásCD21793.00 111.38 109.88 5.00 a 48.00 b 10.00 a 1.00 a 44.00 a 129.00 b 15.90 b 1631.50 aCV12790.00 108.00 107.63 5.00 a 59.00 a 5.00 a 1.00 a 49.00 a 128.00 a 19.70 ab 3385.50 aIsoline87.25 104.88 103.50 5.00 a 58.75 a 5.00 a 1.00 a 49.00 a 127.25 a 18.88 ab 2898.25 abM800187.75 105.50 104.25 5.00 a 45.75 b 0.00 b 1.00 a 49.00 a 125.00 b 16.60 c 2874.00 abBrasíliaCD21791.50 109.75 108.38 5.00 a 45.00 b 0.00 b 1.00 a 49.00 a 124.75 b 17.63 bc 2771.75 bCV12783.25 86.50 77.00 5.00 a 60.00 a 60.00 b 1.00 a 37.50 ab 99.50 bc 21.58 a 1800.50 bIsoline88.00 92.00 86.25 5.00 a 58.25 a 21.25 c 1.00 a 38.00 ab 98.00 bc 18.58 b 2371.25 aM800196.50 100.50 93.25 5.00 a 54.25 a 90.00 a 1.00 a 35.25 c 104.75 a 18.20 b 1518.75 bVilhenaCD21789.25 93.25 87.25 5.00 a 41.25 b 3.25 c 1.00 a 39.00 a 97.00 c 15.25 c 2487.75 aCV12741.50 49.88 49.50 3.75 a 56.50 a 10.00 a 1.00 a 40.00 a 111.00 a 19.10 a 2278.67 aIsoline48.75 58.25 56.88 4.25 a 55.00 a 10.00 a 1.00 a 40.00 a 111.00 a 17.33 ab 2197.00 aM800170.25 84.25 82.88 4.25 a 49.75 b 0.00 b 1.00 a 38.00 b 107.00 b 17.05 ab 2045.50 aTeresinaCD21770.50 84.50 81.00 4.00 a 50.25 b 0.00 b 1.00 a 38.00 b 107.00 b 14.63 b 2446.75 a†G = initial germination (%); IS= initial stand; FS = final plant stand; V = seedling vigor; PH = plant height (cm); GS = greenstem (%); L = degree of lodging;DF = days to full flower (vegetative cycle); DM = days to full maturity (total cycle); 100SW = seed size (weight of 100 seed, grams); Yield = grain yield (kg/ha).‡Means followed by the same letter do not differ significantly by the Tukey test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 124 of 375 D. Ecological Evaluations

Assessments of interactions be tween CV127 soybeans and the envir onment were conducted at all

thirteen field trial locations in Brazil during the 2006/2007 and 2007 growing seasons, as

described in section VIII.C. Treatments were as described in section VIII.C. Evaluations included interactions with plant di seases, insect pests and characte ristics related to the biological nitrogen fixing symbiosis with Bradyrhizobium japonicum . The purpose of these evaluations was to assess whether any differences in environmental interactions exist between imidazolinone-tolerant CV127 soybean and the is oline control and other conventional soybean

varieties.

D.1. Disease Susceptibility Disease susceptibility of CV127 soybeans relative to the isoline control and conventional soybean varieties was determined at different stages of plant growth and development throughout the growing season. Diseases that were assessed included Asian soybean rust ( Phakopsora pachyrhizi ), Downy mildew ( Peronospora manshurica ), Powdery mildew ( Erysiphe diffusa ), and end-of-cycle diseases (DC) caused mainly by Septoria glycines (brown spot or Septoriose)

and Cercospora kikuchii (soybean leaf spot) that can cause serious losses to soybean yield in commercial production in Brazil.

The incidence of all diseases present at the time of assessment was recorded for each plot as the percent of affected leaf area. The dominant dise ases causing the infection were identified at each

assessment. Assessments were made at or near vegetative stage V4 and reproductive stages R1, R5, and R7. However, no disease infestations we re observed at the V4 stage with the exception of powdery mildew in Santo Antônio de Goiás in 2007. Due to the poten tial in Brazil for fungal diseases to devastate the soybean crop and there by compromise the goals of this study, all plots were uniformly treated with fungicides in order to maintain plant health. Evaluations of disease incidence in each plot were typically made prio r to disease control treatments. Data were analyzed by ANOVA on a per location, per disease, and per time of assessment basis. In those cases where differences among treatments were signi ficant, Tukey’s procedur e (Steel and Torrie, 1980) was used to make pair-wise comparisons of treatment means.

Other than a high incidence of Asian rust at seve ral locations, the incidenc e of other diseases was generally low during both growi ng seasons (Tables VIII-9 and V III-10). Although a few isolated differences in disease susceptibi lity were noted among the treatments, these were not consistent across field locations and in general there were no differences in susceptibility between CV127 soybean and the isoline control. Where suscep tibility differences were observed, they were generally between varieties with the Conquista genetic ba ckground (CV127 soybean and the isoline control) and the two conventional soybean varieties with different genetic backgrounds. Therefore, disease susceptibility of CV127 soybean was found to be no different from that of the isoline control plants. This study dem onstrated that the insertion of the csr1-2 gene into the genome of CV127 soybean did not affect the disease susceptibility of CV127 soybean. A complete description of the disease assessm ent methodology is included in Appendix F.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 125 of 375Table VIII-9. Disease Assessments for CV127 Soybean, the Isoline Control, and Conventional Soybean Varieties (Monsoy8001 and CD217) during the 2006/2007 Field Season. Evaluations were conducted at seven field trial locations in Brazil andpresented for three growth stages1(R1, R5 and R7). Values are mean percent affected leaf area.R1R5R7LocationTreat-mentPM†DMDCARPMDMDCARPMDMDCARCV1276 a‡2-§-- - 5 6.0 ab- - 5 43 abIsoline 7 a 2 - -- - 5 6.0 ab- - 5 40 abM8001 16 b 2 - -- - 5 5.3 b- - 5 30 bPontaGrossaCD217 8 a 2 - -- - 5 4.8 b- - 5 36 abCV127 - 1.5 - 2.0 c2 - - 51 a- - 5 60 aIsoline - 1.8 - 2.0 c2 - - 51 a- - 5 60 aM8001 - 1.8 - 2.8 b2 - - 44 b- - 5 51 bLondrinaCD217 - 1.8 - 4.0 a2 - - 30 c- - 5 40 cCV127 - - - 6.8- 2.0 - 45 a- - 5 58 aIsoline - - - 7.3- 1.5 - 50 a- - 5 60 aM8001 - - - 7.5- 1.8 - 30 b- - 5 40 bSto. Ant.de PosseCD217 - - - 6.9- 2.8 - 43 a- - 5 52 aCV127 - - - -1.5 - - 2.0- - - 2.5Isoline - - - -1.5 - - 2.0- - - 2.5M8001 - - - -1.5 - - 2.0- - - 2.5UberabaCD217 - - - -1.5 - - 2.2- - - 2.5CV127 - - - 1.5- - - 2.5- - - 2.5Isoline - - - 1.5- - - 2.5- - - 2.5M8001 - - - 1.5- - - 2.5- - - 2.5SeteLagoasCD217 - - - 1.5- - - 2.5- - - 2.5CV127 - - - 2.0- - 0.6 ab 42 a- - 0.5 55 aIsoline - - - 1.7- - 0.4 ab 40 a- - 0.1 45 bM8001 - - - 1.9- - 1.1 a 19 b- - 0.3 23 cBrasiliaCD217 - - - 1.8- - 0.0 b 19 b- - 0.6 23 cCV127 - - - 1.0- - 0.5 17- - 2.9 22Isoline - - - 0.9- - 0.1 19- - 2.4 22M8001 - - - 0.6- - 0.3 18- - 3.6 21Sto. Ant.de GoiásCD217 - - - 0.8- - 0.6 18- - 3.1 221Disease assessments were also conductedat the V4 growth stage butno diseases were observed at any location.†Diseases: PM = Powdery mildew (Erysiphe diffusa); DM = Downy mildew (Peronospora manshurica); DC = end of cycle diseases (Septoria glycines,Cercospora kikuchii); AR = Asian rust (Phakopsora pachyrhizi). -§= Disease not observed.‡Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 126 of 375Table VIII-10. Disease Assessments for CV127 soybean, the Isoline Control, and Conventional Soybean Varieties (Monsoy8001 and CD217) during the 2007 Field Season. Evaluations were conductedat six field trial locationsin Brazil and presented forgrowth stages V4, R1, R5 and R7. Values are mean percentaffected leaf area.V4R1R5R7LocationTreat-mentPM†PMDMDCARPMDMDCARPMDMDCARCV127 -§- - - 3.7- - - 8.6- -5.0 20.6Isoline -- - - 3.6- - - 7.6- -4.4 20.2M8001 -- - - 3.6- - - 8.0- -5.0 19.8VilhenaCD217 -- - - 2.1- - - 5.2- -5.0 10.5CV127 -- - - -- - - -- - - -Isoline -- - - -- - - -- - - -M8001 -- - - -- - - -- - - -TeresinaCD217 -- - - -- - - -- - - -CV127 -- - - 1.5- -1.1 ab0.7- -1.4 b 0.6Isoline -- - - 1.5- -0.9 ab0.6- -1.3 b 0.5M8001 -- - - 1.5- -1.2 a1.0- -1.5 a 0.6UberabaCD217 -- - - 1.5- -0.8 ab0.8- -1.5 a 0.5CV127 -0.60.8 b‡- -0.8- - -1.1Isoline -0.60.7 b - - 1.0- - - 1.0M8001 -0.61.7 a - - 0.7- - - 1.1Sete LagoasCD217 -0.50.6 b - - 0.6- - - 1.0CV127 -1.6 - - -- - - 0.9- - - 1.6Isoline -1.3 - - -- - - 0.7- - - 1.6M8001 -1.6 - - -- - - 0.9- - - 1.3BrasiliaCD217 -1.6 - - -- - - 1.4- - - 1.6CV12757.1 - - -- - - 2.2- - - 1.9Isoline56.6 - - -- - - 2.5- - - 0.6M800156.3 - - -- - - 2.1- - - 1.3Sto. Ant. deGoiásCD21756.4 - - -- - - 3.1- - - 0.9†Diseases: PM = Powdery mildew (Erysiphe diffusa); DM = Downy mildew (Peronospora manshurica); DC = end of cycle diseases (Septoria glycines,Cercospora kikuchii); AR = Asian rust (Phakopsora pachyrhizi).‡Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.-§= Disease not observed.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 127 of 375 D.2. Insect Interactions Populations of several different insect orders are common in commercial soybean production fields in Brazil and include the Coleopterans (Diabrotica and Aracanthus spp.), Lepidopterans (Anticarsia, Agrotis, Pseudoplusia, and Spodoptera spp.), and Hemipterans (Euschistus, Piezodorus, and Nezara spp.). Insect pests in th ese orders can cause serious losses to soybean yield in commercial production in Brazil. Resistance to insect damage and effects on in-field insect populations of CV127 soybean relative to the isoline control and conventional soybean varieties were assessed at different stages of plant growth and development throughout the growing season during the 2006/2007 and 2007 growing se asons in Brazil. Insects on the plants were collected in the field by pl acing a shake cloth on the ground be tween the center two rows of each plot. The plants in the two adjacent rows were bent over the cl oth and were shaken vigorously to remove all insects within the canop y. All insects thus coll ected were counted and identified by order. In a ddition, insect feeding damage on the leaves, stems and pods was evaluated at different stages of plant development according to th e following rating scale. Insect populations as well as insect feed ing damage were assessed on all pl ots at each field site at the V1, R1, R5 and R7 stages of plant developmen t. Data were analyzed by ANOVA on a per location, per insect order, and pe r time of assessment basis. In those cases where differences among treatments were significant, Tukey’s pro cedure (Steel and Torrie, 1980) was used to make pair-wise comparisons of treatment means.

Rating scale used to evaluate each plot for damage caused by insect feeding.

Insect damage rating Percent of leaves with insect damage

0 0

1 1 – 10

2 11 – 20

3 21 – 30

4 31 – 40

5 41 – 50

6 51 – 60

7 61 – 70

8 71 – 80

9 81 – 90

10 91+

The numbers of Coleopteran, Lepidopteran, and Hemipteran insects detected on CV127 soybean plants were not significantly diffe rent from numbers detected on pl ants of the isoline control or on the conventional soybean varieties at any of four sampling dates during the 2006/2007 (Table VIII-11) and 2007 (Table VIII-12) growing seasons (e xcept for one instance at the Teresina field site in 2007 for Coleopterans at the R5 growth stage, where le vels on the conve ntional soybean variety Monsoy 8001 were statistica lly significantly higher compared to the other treatments). Leaf, stem, or pod damage from insect feeding wa s below 2% for all soybean varieties across all treatments and locations during both growing s easons. This damage was considered minimal and no differences were evident among the different soybean treatments. Therefore the insect damage data was not statistically analyzed nor is it presented. Based upon the results of theseCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 128 of 375 evaluations, in-field in sect populations and resistance to in sect damage of CV127 soybeans was

found to be no different from that of the iso line control soybean or the conventional soybean

varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 129 of 375 Table VIII-11. The Mean Number of Coleopteran (Col), Lepidopteran (Lep), and Hemipteran (Hem) Insects Collected in Each Plot During Field Trials in Brazil in the

2006/2007 Season . No significant differences were determined for any of the mean values for any treatment across all locat ions and growth stages.

Growth Stage

V1 R1 R5 R7

Insect Order Location Treatment

Col Lep Hem Col Lep Hem Col Lep Hem Col Lep Hem

CV127 0.25 0.25 0 2 2 0 5.75 3 2 2.25 0 0.75

Isoline 0.75 0.25 0 2.25 1.25 0 5.5 2.75 2.5 2.75 0 0.75

M8001 0.25 0.25 0 3.75 2.25 0 4.75 3.25 2.75 2 0 1.25 Ponta

Grossa

CD217 0.5 0.25 0 3.25 2.25 0 6 3 2.25 2.25 0 1

CV127 1 0 0.25 2.25 2.25 0 1 2.25 1.25 0 0.25 2.25

Isoline 1 0 1 1.75 1.75 0 1.25 1.25 0.25 0.75 0.25 1.5

M8001 1 0 0.25 2.25 2.25 0 0.75 2.25 1.25 0.25 0.75 1.25 Londrina

CD217 1.25 0 0.5 1.75 1.75 0 1.25 1 2.25 0.25 0.25 2.75

CV127 0.25 0 0.25 0 0 0 2.25 3.75 4 0 4.25 3

Isoline 0.5 0.5 0.25 0 0 0.5 1.75 4 3.75 0 3.25 2.75

M8001 0.5 0.5 0.25 0 0 0.5 2.25 4 3.75 0 4.25 3.25 Sto. Ant.

de Posse

CD217 0.5 0.5 0.25 0 0 0.25 2.75 4.5 4.25 0 4.25 3.25

CV127 0.25 0 0 0 0 0 0 0.5 0 0 0 0.25

Isoline 0 0 0 0 0 0 0 0.25 0 0 0.25 0.5

M8001 0.75 0 0 0 0.25 0 0 0 0.25 0 0 0 Uberaba

CD217 0.5 0 0 0 0.25 0.25 0 0.75 0.25 0 0.75 0.25

CV127 0.25 0 0 0 0.25 0.25 0 0.25 0 0.5 1 0

Isoline 0.5 0 0 0 0.25 0 0 0.25 0 0 1.25 0.5

M8001 0.5 0 0 0 0.5 0 0.25 0 0 0.25 0.75 0.25 Sete Lagoas

CD217 0.75 0 0 0 0.5 0 0.75 0.25 0 0.75 0.5 0

CV127 0.25 0 0 0 0 0 0 0 0.5 0 0 0.5

Isoline 0.5 0 0 1 0 0 0 0 0.25 0 0 0.25

M8001 1.25 0 0 0.75 1.25 0 0 0 0 0 0 0 Sto. Ant.

de Goiás

CD217 0.25 0 0 0 0.5 0 0 0 0.25 0 0 0.25

CV127 0 0.25 0 0.5 0 0 0 0 0 0 0 0

Isoline 0.25 0 0 0.25 0.25 0 0 0.25 0.5 0 0 0

M8001 0.25 0.25 0 0 1 0 0 0 0.5 0 0 0.25 Brasilia

CD217 0.5 0 0 0.25 0 0 0 0 0 0 0 0.25CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 130 of 375 Table VIII-12. The Mean Number of Coleopt eran (Col), Lepidopteran (Lep), and Hemipteran (Hem) Insects Collected in Each Plot During Field Trials in Brazil in the 2007

Season. No significant differences were determin ed for any of the mean values for any

treatment across all locations and growth stages, except for one instance at the Teresina field site for Coleopterans at the R5 growth stage, wh ere numbers followed by different letters are statistically significantly di fferent at the 5% level.

Growth Stage

V1 R1 R5 R7

Insect Order

Location Treatment Col Lep Hem Col Lep Hem Col Lep Hem Col Lep Hem

CV127 1.56 0.71 0.71 0.97 0.84 0.71 0.71 0.93 0.84 0.71 0.84 0.71

Isoline 1.48 0.71 0.71 1.06 0.84 0.71 0.71 1.06 0.84 0.71 0.84 0.71

M8001 1.56 0.71 0.71 0.84 0.71 0.71 0.71 0.84 0.84 0.71 0.71 0.71 Uberaba

CD217 1.40 0.71 0.71 0.97 0.97 0.71 0.71 0.97 0.71 0.71 0.71 0.71

CV127 1.06 0.71 0.71 0.84 1.06 0.71 0.71 1.31 0.84 0.71 0.84 0.97

Isoline 1.18 0.71 0.71 1.06 1.06 0.71 0.71 1.10 0.71 0.71 0.84 0.71

M8001 1.22 0.71 0.71 0.97 1.18 0.71 0.71 0.84 0.97 0.71 0.97 0.71 Sete Lagoas

CD217 1.10 0.71 0.71 0.97 0.97 0.71 0.71 1.06 0.97 0.71 1.06 0.71

CV127 1.06 0.84 0.71 0.84 0.71 0.71 0.71 0.71 0.84 0.71 0.71 0.71

Isoline 1.06 0.71 0.71 0.84 0.84 0.71 0.71 0.71 0.84 0.71 0.71 0.84

M8001 0.93 0.84 0.71 0.84 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 Sto. Ant.

de Goiás

CD217 0.93 0.71 0.71 0.71 0.84 0.71 0.71 0.71 0.84 0.71 0.71 0.71

CV127 0.71 0.84 0.71 0.71 0.84 0.71 0.71 0.71 0.71 0.71 0.71 0.84

Isoline 0.84 0.71 0.71 0.84 0.71 0.71 0.71 0.84 0.97 0.71 0.71 0.84

M8001 0.84 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 Brasilia

CD217 0.97 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71

CV127 0.97 0.71 0.71 1.49 0.71 0.71 1.61 0.84 1.27 0.71 0.71 1.22

Isoline 1.10 0.71 0.71 1.65 0.71 0.71 1.31 1.10 1.40 0.71 0.71 1.10

M8001 1.06 0.71 0.71 1.49 0.71 0.71 1.79 1.13 1.44 0.71 0.71 1.18 Vilhena

CD217 0.97 0.71 0.71 1.40 0.71 0.71 1.59 0.84 1.49 0.71 0.71 1.06

CV127 0.71 2.04 0.71 0.71 0.84 0.71 0.71 b 0.71 0.84 0.71 0.71 0.71

Isoline 0.71 1.90 0.71 0.71 0.97 0.71 0.71 b 0.71 0.71 0.71 0.71 0.71

M8001 0.71 1.83 0.71 0.71 0.71 0.71 1.79 a 1.13 0.71 0.71 0.71 0.84 Teresina

CD217 0.71 1.63 0.71 0.71 0.71 0.71 0.71 b 0.71 0.71 0.71 0.71 0.71CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 131 of 375 D.3. Nitrogen Fixation Characteristics

An important agronomic characteristic of soyb eans is the ability to support a nitrogen fixing symbiosis with the soil bacterium Bradyrhizobium japonicum

. B. japonicum cells in the soil stimulate the development of root nodules on soybean plants. The B. japonicum cells populate the interior of the nodule and the plant create s an environment within the nodule that is conducive for nitrogen fixation by the e ndosymbiont. Through this symbiosis, B. japonicum is able to convert atmospheric ni trogen to fixed forms of nitroge n and reduce the soybean host’s dependency on nitrogen fertilizer. It is a common practice in Br azil and Argentina for farmers to inoculate soybean seed with B. japonicum prior to planting to ensure the development of

effective root nodulation and st rong nitrogen fixing activity. The purpose of the nitrogen fixation study was to confirm that CV127 soybean is equivalent to the isoline control and to conventional soybean varieties with respect to nitrogen fixation and nitrogen accumulation in the plant. Characteristics of nitrogen fixation in CV127 soybean were evaluated and compared with

the characteristics of the iso line control and conventional s oybean varieties, including measurements of soil populations of B. japonicum , plant growth and nodulation and total nitrogen and ureide nitrogen accu mulation in the plants. These determinations were made on plants grown in field studies during the 2006/2007 growing season at seven geographicallydistinct field locations in Braz il which are representative of areas of commercial soybean production and to which the soybean varieties in cluded in the study ar e adapted. Identical

evaluations of the B. japonicum nitrogen fixing symbiosis were also made at six trial locations in the 2007 short growing season in Braz il. The field trial locations are the same as described in section VIII.C. (“Agronomic and Phenotypic Evalua tions in Replicated Field Trials”). Plants were harvested at the R2 stage of plant development for analysis of nitrogen fixation parameters. Statistical analyses were conducted by ANOVA, and means comparisons across treatments at each location were determined using Least Signif icant Difference (LSD) statistical procedures, and across locations using the Tuke y's test (Steel and Torrie, 1980)

The results of these studies showed that CV127 s oybean is equivalent to the isoline control with respect to biological nitrogen fixation, including nodule nu mber, nodule dry weight, shoot dry weight, total N content and the contribution of nitrogen fixation to the total nitrogen (N) accumulated in tissues as evaluated by the N-ur eide content of the shoot (Tables VIII-13 and VIII-14). Comparison of CV127 soybean and the isoline control as a group with the conventional soybean varieties showed some diffe rences in nitrogen fixation parameters, which were attributed to basic genetic differences between these soybean va rieties in biological nitrogen fixation capabilities. A more detailed description of the methodology used in the study and the results obtained are pr esented in Appendix F.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 132 of 375 Table VIII-13. Effect of Different Treatments on Nitrogen Fixation Parameters in the 2006/2007 Growing Season. Measurements included nodule number (NN, number plant -1 ), nodule dry weight (NDW, mg plant -1 ), shoot dry weight (SDW, g plant -1 ), concentration of N in shoots (CNS, g N kg -1 plant -1 ), total N accumulated in shoots (TNS, mg N plant -1 ), % N as Nureide (%NU) 2 and total N as N-ureide (TNU, mg N-ureide plant -1 ) 3 in soybean harvested at R2 in the seven trials performe d in the summer of 2006/2007 1

.

Parameter Treatment

NN NDW SDW CNS TNS %NU TNU

CV127 52.6 a 168.4 20.78 a 48.13 ab 1031.0 a 75.04 a 772.6 a

Isoline 46.2 ab 163.6 21.84 a 50.66 ab 1153.4 a 76.70 a 894.9 a

M8001 36.7 b 158.9 16.95 b 52.82 a 928.8 b 67.49 b 626.7 b

CD217 33.8 b 124.2 17.16 b 48.78 ab 886.8 b 66.26 b 598.6 b

1 The data represent the mean of 20 repli cates, with ten plants per replicate. Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.

2 N-ureides were measured in stem and petiole tissues only and expressed as a percent (%NU) of total N (N-ureides plus nitrate) in these tissues. N-ureides are an estim ate of the contribution of nitrogen fixation to total N accumulated by the plant.

3 The N-ureide concentration in the stem and petiole plant tissues was tran sformed to total N-ureide content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes.

Table VIII-14. Effect of Different Treatments on Nitrogen Fixation Parameters in the 2007 Growing Season. Measurements included nodule number (NN, number plant -1 ), nodule dry weight (NDW, mg plant -1 ), shoot dry weight (SDW, g plant -1 ), concentration of N in shoots (CNS, g N kg -1 plant -1 ), total N accumulated in the shoots (TNS, mg N plant -1 ), % N as N-ureide (%NU) 2 and total N as ureides (TNU, mg ureide-N plant -1 ) 3 in soybean at the R2 stage in field

trials conducted in Brazil at six loca tions in the 2007 short growing season 1

.

Parameters Treatment

NN NDW SDW CNS TNS %NU TNU

CV127 29.4 150.0 a 1 9.06 ab 42.5 396 69.1 ab 293 ab

Isoline 34.6 165.4 a 10.01 a 42.0 421 74.3 a 324 a

M8001 21.4 120.2 ab 9.12 ab 40.9 388 71.0 a 297 ab

CD217 21.8 112.9 b 7.96 b 39.4 313 67.0 b 212 b

1 Data are means of 24 replicates and analyses in duplicate. Means followe d by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.

2 N-ureides were measured in stem and petiole tissues only and expressed as a percent (%NU) of total N (N-ureides plus nitrate) in these tissues. N-ureides are an estim ate of the contribution of nitrogen fixation to total N accumulated by the plant.

3 The N-ureide concentration in the stem and petiole plant tissues was tran sformed to total N-ureide content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 133 of 375 E. Conclusions from Agronomic, Phenotypic and Ecological Evaluations of CV127 Soybeans

The purpose of the studies summari zed in this section was to: 1) Evaluate the agronomic, phenotypic, an d phenologic characteristics of CV127 soybean

relative to an isoline control and to ot her conventional soyb ean varieties; 2) Determine the ecological impact (interaction w ith diseases, insects a nd factors relevant to nitrogen fixation) of cultivating CV127 soybean re lative to cultivating the isoline control and the conventional soybean varieties in the plant and soil environments.

CV127 soybean was compared to the isoline control and tw o other conven tional soybean varieties with respect to ke y vegetative and reproductive developmental characteristics associated with competitiveness and survival of plant species. In field trials, characteristics including seed germination rate, seedling vigor, days to reach key developmental stages, plant height, and grain yield, as well as susceptibility to and interact ions with diseases and insects were evaluated. In addition, the impact of cultivating CV127 soybeans on factors relevant to the nitrogen fixing symbiosis with Bradyrhizobium japonicum were assessed and compared between CV127, the isoline control and co nventional soybean varieties. Furthermore, laboratory and greenhouse-based studies on seed germination, pollen number and pollen germination were also conducted and comparisons of these characteristics made between CV127 soybeans, the isoline control and the two other conventional soybean varieties.

Data from these studies demonstrate that CV127 soybean has no different agronomic or phenotypic characteristics, disease or insect in teractions, seed germination, pollen number and pollen germination characteristics, or abilit y to support a nitrogen fixing symbiosis with Bradyrhizobium japonicum than do the isoline c ontrol or the comparator conventional soybean varieties. The only trait for which there was a trend toward a difference between CV127 and the

isoline control was seed size. For all other traits measured there were no consistent statistically significant differences between CV127 and the isoline control. Furthe r, where seed size

differences were observed between CV127 and the isol ine control, this did not confer a selective advantage with respect to ge rmination, seedling vigor, plant stand or any other agronomic, phenotypic or ecological interaction trait.

In addition to agronomic, phenotypic and ecological interaction evaluations , extensive analyses were conducted on the key nutrients and antinutri ents of the grain and forage derived from CV127 soybean and the isoline control and conventi onal soybean varieties (section VII). Results of these studies demonstrated that grain an d forage from CV127 soybean is substantially equivalent in its nutrient and antinutrient content to the grain and forage from conventional soybean varieties that have a long history of safe use for the production of food and feed products. Collectively, from results of all the above studies it is concluded that the cultivation of imidazolinone-tolerant CV127 soybean poses no great er environmental or plant pest risk than

does the cultivation of conventional soybeans.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 134 of 375 IX. Environmental Assessment of CV127 Soybeans

The potential for CV127 soybean to exhibit plant pe st or weediness characteristics as well as an

evaluation of interactio ns of CV127 soybean with both pe st and non-pest organisms in the

environment are discussed in this section. Th e potential for gene fl ow from CV127 and the impact of the cultivation of CV127 soybeans in the environment are also addressed.

CV127 soybean was developed for cultivation prim arily in Brazil and Argentina. The major weeds in soybean cultivation in these countries are sensitive to the imidazolinone herbicides containing imazapyr and imazapic, making this product concept an attr active proposition for soybean cultivation in South America. Therefore, regula tory approvals for this product are sought in Brazil and Argentina for production as we ll as for food and feed uses, and in the U.S. and other countries for importati on of grain from CV127 soybean for food, feed, and processing uses. However, the purpose of this discussion is to establish that, in the unlikely event that CV127 soybeans were to be intr oduced in to the U.S. environment, CV127 soybeans would present no different environmenta l impact compared to conven tional soybean varieties with regard to cultivation practices, interactions with pest and non-pest organisms, and weed control measures, other than the ability to use imidazo linone herbicides for weed control in the CV127

crop.

A. Safety of the Imidazolinone-tolerance Trait

The imidazolinone herbicides inhibit plant growth due to their inhibiti on of a plant enzyme, acetohydroxyacid synthase (AHAS), which has an im portant function in the biosynthesis of the branched-chain amino acids valine, leucine, and isoleucine. CV127 soybean is tolerant to the

imidazolinone class of herbicides as a result of the introduction of the csr1-2 gene from Arabidopsis thaliana encoding an AHAS enzyme that is tolerant to imidazolinone herbicides.

Several AHAS genes that encode AHAS enzymes that are tolerant to imidazolinone herbicides have been discovered in plants as naturally-occurring mutati ons or through the process of chemically-induced mutagenesis. The mutation in the csr1-2 gene which results in the substitution of a serine residue with an aspara gine at position 653 [S653N] (relative to the amino acid sequence of the AHAS enzyme from Arabidopsis thaliana ) is one of the five most common single-point mutations in AHAS genes that have been found to result in tolerance to imidazolinones in plants (Tan et al ., 2005). This mutation confers tolerance to imidazolinone

herbicides with no cross-toleran ce to other AHAS inhibitors such as the sulfonylurea herbicides (Lee et al ., 1999). To date, imid azolinone-tolerant maize ( Zea mays L.), rice ( Oryza sativa L.), bread wheat ( Triticum aestivum L.), oilseed rape ( Brassica napus and B. juncea L. Czern.), lentil ( Lens culinaris ) and sunflower ( Helianthus annuus L.) have been develo ped through mutagenesis or introgression from natural sources, selec tion, and conventional breeding technologies and

have been commercialized under the Clearfield ® brand name since 1992, 2003, 2002, 1996, 2006, and 2003, respectively. Of these, Clearfield ® varieties of maize, rice, bread wheat, and oilseed rape have been commercialized that contain the same S653(At)N AHAS amino acid substitution that is pres ent in CV127 soybean. These crops ha ve been cultivated for many years without adverse environmental impact and withou t the development of weediness or other plant pest characteristics (Tan et al ., 2005). Furthermore, the grain and other food or feed componentsCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 135 of 375 produced from the Clearfield ® crops described above have been demonstrated to be as nutritious and as safe as the same food or feed compone nts produced from their respective imidazolinonesensitive counterparts. Therefore, there is a history of safe production as well as safe food and feed use of products containing the same S653 (At)N AHAS amino acid substitution that is present in CV127 soybean.

In two separate growing seasons in field trials in Brazil, CV 127 soybean was compared to the

isoline control and two other conv entional soybean varieties with respect to key vegetative and reproductive development characteristics associat ed with the competitiveness and survival of plant species (section VIII of this petition). Al so, CV127 was assessed for disease susceptibility

and interactions with different orders of ins ects, as well as the impact of cultivating CV127 soybeans on factors relevant to th e nitrogen fixing symbiosis with Bradyrhizobium japonicum.

Data from these studies demonstrated that CV127 soybean has no different agronomic or phenotypic characteristics, disease or insect intera ctions or abil ity to support a nitrogen fixing symbiosis with Bradyrhizobium japonicum than do conventional s oybean varieties. In combination, all of these stud ies support a conclusion that CV127 soybean poses no different plant pest or weediness risk or environmental impact compared to that from the cultivation of conventional soybean varie ties. Also, extensive an alyses of the nutrient and antinutrient content of the grain and forage produced from CV127 soybean s described in section VII of this petition confirmed that these products are as nutritious and safe as grain and forage produced by conventional soybean varieties.

All plants possess a biochemically -active AHAS enzyme that is necessary for the production of the branched-chain amino acids required for pr otein synthesis. The imidazolinone-tolerant AtAHAS enzyme encoded by the csr1-2 gene that was introduced into CV127 soybeans encodes an AtAHAS enzyme that has the same biochemical characteristics as other plant AHAS enzymes except for its tolerance to imidazolinone herbic ides. The AtAHAS protein expressed in CV127 soybean is structurally and biolog ically closely related to the AHAS proteins found in all plants. For example, there is 79% amino acid identi ty between the AtAHAS and endogenous soybean AHASL2 (JGI locus Glyma13g31470.1) proteins. Also, the At AHAS protein e xpressed in CV127 soybean does not share any biologically relevant amino acid sequence homology or biological characteristics with known toxins or al lergens. Finally, the source organism of the

csr1-2 gene that encodes the At AHAS protein in CV127 soybean, Arabidopsis thaliana , is not a known human or animal pathogen and is not known to cause alle rgic reactions in humans. Similarly, A. thaliana is not known to produce toxic substan ces. Therefore, results of these studies show that neither the AtAHAS protei n nor CV127 soybeans are likely to pose any increased safety risk to other plants and organisms in the envi ronment compared to other plantproduced AHAS proteins or othe r conventional soybean plants.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 136 of 375 B. Ecological Characteristics of CV127 Soybean

B.1. Weediness Potential of CV127 Soybean

The commercial varieties of soybean that are cultivated in the U.S. do not possess any

characteristics that are commonly associated with weeds, such as seed dormancy or persistence of seed in the soil, or the ability to widely disp erse, invade diverse habitats, compete with native plant species, and become a dominant species in different habitats (CFIA, 1996). Cultivated soybean seed rarely display any dormancy char acteristics and only under certain environmental conditions will they grow as a volunteer in the year following cu ltivation (Raper and Kramer, 1987; CFIA, 1996; OECD, 2000). In manage d ecosystems, soybean does not compete effectively with other cultivated plants or primary colonizers. Further, soybeans are not frosttolerant and do not typically survive freezing temperatures during winter.

Volunteer soybean management is normally not a significant management concern in North America because of environmental conditions, crop rotation, the numerous herbicide control options available, and the fact that soybean s do not compete well with the succeeding crop (CFIA, 1996; OECD, 2000). Since soybean seed does not survive the freezing temperatures of winter, soybean seed do not overwinter and are typically not viable in the following season (CFIA, 1996). When a rotational cr op such as winter wheat is plan ted after soybean harvest, it is possible for soybean seed to be incorporated in to the soil and to germinate that fall. These volunteer plants will be contro lled by freezing temperatures in the fall and do not become a competitive consideration. Therefore, normally volunteer soybean is not a target for weed

control measures in winter wheat.

When corn is the rotational crop with soybean, the first option availabl e to contro l volunteer soybeans the following spring is pre-plant tillage or the use of a pre-plant burn-off herbicide such as glyphosate. The most common herbicide activ e ingredients applied to corn in the United States in 2010 are listed in Appe ndix G, Table G-1; there were 58 active ingredient s reported as used on corn. Herbicides from group 4 (auxi nic herbicides), group 5 (triazines), group 10 (phosphorylated amino acids) and group 27 or group 28 (4-hydroxyphenyl pyruvate dioxygenase or HPPD inhibitors) all will control volunteer soybeans in corn. Seventeen of the 58 active ingredients applied to corn (29% of the total) are members of one of these herbicide groups.

Therefore there are numerous tools available to control volunteer soybeans in corn. Thus, in the event that CV127 soybean volunteer s were to become established, they would not compete well with succeeding crops and they co uld be controlled by either mech anical means or by herbicides other than the group tw o herbicides that ta rget the AHAS enzyme.

In Roundup Ready ® (RR) corn, volunteer conventional or imidazolinone-tolerant soybeans will be controlled by any of the gl yphosate products (group 9) applied. In some cases the glyphosate is tank-mixed or used in sequence with anothe r broadleaf herbicide. Any of the broadleaf herbicide products from groups 4, 5 or 27 will also control vol unteer soybeans. Therefore, virtually all the herbicide options available in Roundup Ready ® corn will control volunteer soybeans.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 137 of 375 The agronomic evaluations that were conducted with CV127 soybean in two growing seasons suggest no different characterist ics compared to conventional soybeans that would impart the ability to be invasive or wee dy. CV127 soybean was equivalent to the isoline control and to two conventional soybean varieties w ith respect to seed germination, quality and dormancy, as well as pollen number and pollen germination and ke y phenotypic characteristics including seedling vigor, days to flowering and to maturity a nd seed production. Also, no differences in the frequency of volunteers between CV127 soybean, th e isoline control, and the two conventional soybean varieties were observed in the post-harvest monitoring of the field trials described in this petition, providing further suppor t for the conclusion that CV127 soybean has no different survivability or persistence characteristics than co nventional soybean varietie s. Also, since wild species of Glycine are not indigenous to, or known to ex ist in, the U.S., it is biologically improbable that CV127 soybean would out-cross to weedy plants and transfer its imidazolinone herbicide tolerance trait to compatible weedy spec ies. Based upon the results of these studies it was concluded that CV127 soybean has no incr eased weediness potential compared to conventional soybean.

B.2. Interactions of CV127 Soybean with Other Organisms in the Environment

Soybean like every other plant is known to interact with other organisms in the environment including microorganisms, viruse s, insects, birds, and mammals. A list with examples of potential interactions of soybean with pathogens, consumers, symbionts, and others is given in

the consensus document for Glycine max (L.) Merr. ( OECD, 2000). Also, soybeans develop

sym biosis with Bradyrhizobium japonicum , a plant-associated nitroge n-fixing bacterium. When soybeans are grown in new production areas, seeds are normally inoculated with B. japonicum prior to planting.

CV127 soybean was evaluated for susceptibility to a number of important soybean diseases in Brazilian agriculture, including Asian soybean rust ( Phakopsora pachyrhizi ), Downy mildew ( Peronospora manshurica ), Powdery mildew ( Erysiphe diffusa ), and end-of-cycle diseases (DC) caused mainly by Septoria glycines (brown spot or Septoriose) and Cercospora kikuchii (soybean leaf spot). CV127 soybean was also evaluated for intera ctions with several different insect orders common in commercial soybean production fields in Brazil that included the Coleopterans ( Diabrotica and Aracanthus spp.), Lepidopterans ( Anticarsia

, Agrotis

, Pseudoplusia , and Spodoptera spp.), and Hemipterans ( Euschistus

, Piezodorus , and Nezara spp.). Finally, the impact of cultivating CV127 soybeans on factors relevant to the nitrogen fixing symbiosis with Bradyrhizobium japonicum were assessed. In all of these studies, CV127 soybean was compared to the isoline control and two conventional s oybean varieties.

Results of these studies demonstrated that CV127 soybean has no different interaction with various diseases, insects, or Bradyrhizobium japonicum nor any difference in nitrogen fixation capacity compared to the isoline control. Th erefore, these results further confirmed that cultivation of CV127 soybeans poses no different en vironmental impact than the cultivation of conventional soybean varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 138 of 375 B.3. Potential for Gene Flow in Soybean

Soybean is considered to be a self-pollinating sp ecies that is propagated commercially by seed. Commercial cultivars are developed through breedi ng by artificial hybridization. The longevity of soybean pollen is generally two to four hour s at most (Andersson a nd de Vicente, 2010). The soybean flower stigma is receptive to pollen for a period that begins 24 hours prior to anthesis and ends 48 hours thereafter (C arlson and Lersten, 1987; Carlson and Lersten, 2004). Typically

the anthers mature in the bud and di rectly pollinate the s tigma of the same fl ower. As a result, soybeans exhibit a high percentage of self-fertilization and cross pollination is usually less than 0.5% (Caviness, 1966; Ray et al ., 2003; Yoshimura et al ., 2006). In addition, the seed pod and individual seed do not possess mo rphological characteristics that encourage the spread of the seed by animals.

For a trait to become incorporated in to a species’ genome, recurrent backcrosses of plants of the species with hybrid intermediates and survival and fertility of the resulting offspring are necessary. The wild relatives of G. max include G. soja (Sieb. and Zucc.) (2n=40) and G. gracilis Skvortz. (2n=40) that are native to Asia. G. soja is a wild viny annual with small, narrow trifoliate leaves, purple flowers, and small round black-brown seeds. It grows wild in Korea, Taiwan, Japan, and the Ya ngtze river valley in northeast China and areas near the border

with Russia. G. gracilis has been observed growing in northeast China (Skvortzow, 1927). Interspecific fertil e hybrids between G. max and G. soja (Sieb. and Zucc.) (Hadley and Hymowitz, 1973; Ahmad et al ., 1977) and between G. max and G. gracilis (Karasawa, 1952) are readily obtained.

In addition to the subgenus Soja , the genus Glycine also contains the subgenus Glycine . The subgenus Glycine contains 22 wild pere nnial species, including G. clandestine Wendl., G. falcata Benth., G. latifolia Benth., G. latrobeana Meissn. Benth., G. canescens F.J. Herm., G.

tabacina Labill. Benth., and G. tomentella Hayata. These members of the subgenus Glycine are indigenous to Australia, the South Pacific Island s, China, Papua New Guinea, the Philippines, and Taiwan (Hermann, 1962; Newell and Hymow itz, 1978; Hymowitz and Newell, 1981; Grant, 1984; Tindale, 1984 and 1986). Hybridizat ion between the diploid perennial Glycine species produces normal meiosis and fertile offspring.

Attempts at hybridization between members of the annual subgenus Soja and the perennial subgenus Glycine have proven unsuccessful. Although hybridization results in the initiation of pod development, the pods eventually abort and abscise (Ladizinksy et al ., 1979; Hood and Allen, 1980). Later, inte rsubgeneric hybrids between G. max and G. clandestine Wendl. and

between G. max and G. tomentella Hayata were obtained in vitro through embryo rescue (Singh and Hymowitz, 1985; Singh et al ., 1987). Successful hybr idization between G. max and G.

canescens was also achieved using transplanted endosperm as a nurse layer (Broué et al ., 1982). In all cases, the progeny of th ese intersubgeneric hybrids were obt ained with difficulty and only

with human intervention and all resu lting progeny were sterile. Soyb ean is not able to cross with other plants outside of the Glycine subgenus Soja and so intergeneric hybridization does not

occur.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 139 of 375 The potential for the genetic tran sfer of the imidazolinone-toleran ce trait from CV127 soybean is limited to members of the subgenus Soja due to biological barriers. The wild relatives of the subgenus Soja do not exist in North or S outh America. Consequently, the possibility of natural gene transfer between CV127 soybeans and compatib le relatives or wild species of the subgenus Soja does not exist in North or South America. In the unlikely event transfer of the trait to weedy species should occur, th e acquisition of the imidazolinone -tolerance trait would not be expected to enhance the weedin ess of the recipient plants. Such hypothetical imidazolinonetolerant weedy plants could be controlled by ma ny other herbicides other than those of the imidazolinone class or by cultivation.

There is no evidence that gene tic information can transfer from soybean to other organisms that cannot sexually interbreed with soybean (horizontal gene tran sfer). Therefore, genetic information in CV127 soybean will not be transfe rred by horizontal gene flow to other organisms

in the environment.

C. Cultivation of Soybean

Soybean is the leading oilseed crop produced a nd consum ed in the world today. The current world production of soybean greatly exceeds that of all other edible oilseed crops (Wilcox, 2004; Soy Stats, 2011). In 2011, 258.4 MMT of soybeans were produced worldwide representing 58% of the world’s total oilseed production (Soy Stats, 2011). Soybean has been the dominant oilseed produced in the world since the 1960s (Smith a nd Huyser, 1987) and in the past twenty years (1989 through 2009) soybean production has increased by 116 MMT (FAOSTAT, 2011). For the past 50 years, the United States has been the world’s leading produ cer of soybeans and in 2010 it produced 90.6 MMT, or about 35% of the wo rld’s total production (Soy Stats, 2011). The second and third largest producers of soybean are Brazil and Argentina who produced 70.0 and 49.5 MMT of soybeans, respectively, in 2010 (Soy Stats, 2011). The two largest soybean producers in Asia are the People’ s Republic of China (15.2 MMT in 2011) and India (9.6 MMT).

Due to the ability of soybean to s upport a nitrogen fixing symbiosis with Bradyrhizobium
japonicum and to convert atmospheric nitrogen to fixed forms of nitrogen, soybeans are often planted in rotation with corn, wheat, cotton, rice a nd other crops. Studies have demonstrated that the rotation of soybean with other crops generall y leads to increased yields for the soybean and subsequent crops (Johnson, 1987; Wesley, 1999). Other benefits for the farmer from rotation with soybeans include reduced nitrogen input in the following crop, increased residue cover, and reduction of pest and weed cycles.

For many years, genetically modified (GM) s oybean varieties have constituted the largest percentage of GM crops planted worldwide. In 2010 the area planted to GM soybeans was 73.3 million hectares or 50% of the global area plante d to soybeans (James, 2010). The vast majority of this acreage was planted in he rbicide-tolerant soybean varieties. In 2 010, herbicide-tolerant soybeans comprised about 93% of the soybean acreage planted in the United States, almost 100% in Argentina, and 75% in Brazil (James, 2010). The rapi d and widespread adoption of herbicide-tolerant soybeans by gr owers worldwide has been due to increased yields, the reduced cost of effective weed control, and a simplifie d, more flexible weed control program (Reddy, 2001; Gianessi, 2005) that is offere d by the cultivation of herbicide- tolerant crops. Perhaps moreCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 140 of 375 importantly to the grower, the benefits from th e cultivation of herbicide-tolerant soybeans have increased the income of those growers that have planted them instead of conventional varieties.

It has been estimated that the cultivation of he rbicide-tolerant soybeans increased the income of farmers by $2.84 billion in 2005 (Brookes and Barfoot, 2006). Given that the U.S. soybean crop value in 2005 was $17,337 million (Soy Stats, 2011), this represents an increase equal to approximately 16% of the total value of the soybean crop.

The primary herbicide-tolerant soybeans grown t oday are tolerant to the herbicide glyphosate. Glyphosate-tolerant soybeans were first grown co mmercially in 1996 and the cultivation of this crop has steadily increased every year since introduction (Gianessi and Reigner, 2006). In recent years, several weed species have evolved resistance to glyphosat e (Heap, 2011; Nandula et al

., 2005). It should be noted that most of the ca ses of glyphosate-resistan t weeds occurred in nontransgenic crops. Exposure to glyphosate st arted well before the introduction of glyphosatetolerant crops and thus, attributing the developm ent of glyphosate-resistant weed species to the use of glyphosate-tolerant crops is not warranted (Dill, 2005). The introduction of imidazolinone-tolerant soybeans will provide the gr ower with the opport unity to cultivate soybeans that are tolerant to a herbicide with a different mo de of action compared to glyphosate as a means to control weeds that may be tolerant to glyphosate and to help prevent the continued evolution of more weed species that are glyphosate-tolerant. The rotation of herbicide-tolerant crops that are resistant to different herbicide cl asses in combination with the application of the corresponding herbicides is an effective strategy for managing the development of herbicide tolerance in weed populations.

C.1. Cultivation of Soybean in Brazil. Brazil is the largest producer of soybeans in South America and the second largest producer in the wo rld behind the U.S. The first soybean varieties produced in Brazil were varieties that were br ed for production and co mmercialization in the U.S. However, after the early introduction of U. S. varieties, research and breeding programs in Brazil, including the public agricultural research organization ‘Empresa Brasileira de Pesquisa Agropecuaria’ (Embrapa), began to develop varieties with increased yield potential and resistance to pathogens specifically adapted to a ll major agricultural regions of Brazil. Today, many of the most productive Brazilia n varieties developed in these programs contain an ancestor that was developed in the US. As a result of the Brazilian breeding effort, soybean production in Brazil has steadily increased from about 0.5 MMT in 1965 to 31 MMT in 1999 and 70 MMT in 2010 (Wilcox, 2004; Soy Stats, 2011) and yield on a per hectare basis has doubled from 1.2 to 2.4 tons/ha (Wilcox, 2004).

Soybeans may be planted in any region of Brazil as a full-season crop in late October or early November with harvest occurring in March. In some tropical and sub-tropical regions, soybean may be planted as a short-season crop in late Febr uary or early March with harvest in July. The cultivation of soybean plants in Brazil is similar to the cultivatio n of soybean plants in the U.S. as the plant’s requirements fr om the environment are not di fferent. However, the input requirements to produce a crop in Brazil may be different from those requi red to produce a crop

in the U.S. due to environmenta l differences that may exist betw een the two countries. Soybeans are commonly grown in rotations with grain crop s such as maize or winter wheat. The use of reduced tillage that has been facilitated by the introduction of herbicide- tolerant varieties ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 141 of 375 soybean and the correction of so il acidity and improved seed quali ty have all contributed to increased soybean production in Brazil.

C.2. Cultivation of Soybean in Argentina. Argentina ranks second to Brazil in the production of soybeans in South America and third worl dwide (Soy Stats, 2011). There are many similarities in soybean production in Argentina a nd Brazil, including the f act that soybeans were also first introduced into Argentina from the U.S. However, since Argentina is located at

latitudes that are more similar to those in the regions of soybean cultivation in the U.S., the American cultivars were better adapted for cultivation in Argentina. Soybean varieties in Maturity Groups III to IX are cult ivated from latitude 23 to 39°S. As in Brazil, production of soybeans in the past few decades has increased tremendously in Argentina, from 1.4 MMT in 1977, to 19.9 MMT in 1999, and 49.5 MMT in 2010 (Wilcox, 2004; Soy Stats, 2011). The use of herbicide-tolerant soybean varieties has been common practice for many years and this has led to the widespread adoption of minimum- or no -tillage cultivation practices. About 65% of soybeans that are cultivated in Argentina are gr own as a full-season crop with the remaining 35% being double cropped with wheat. Soybean is al so commonly rotated with maize, sorghum, pasture, peanut, and flax (Wilcox, 2004).

C.3. Cultivation of Soybean in the U.S. The U.S. is the world’s larg est producer of soybeans. As in the other major soybean producing coun tries of Brazil and Argentina, production has increased dramatically in the U.S. in recent ye ars. While production of soybean has increased 70% in the U.S. since 1985, from 51 to 87 MMT in 2006, during this period the area planted to soybean increased only about 14%, from 25.6 to 29.3 million hectares (Wilcox, 2004). From 1985 to 2010, soybean yields on a per-acre basis increased from 34.1 bushels per acre to 43.5 bushels per acre in the U.S. (Soy Stats, 2011). Soybean is grown pr imarily in the eastern half of

the U.S. from the Gulf of Mexico to the Canadi an border where rainfall is sufficient to sustain the crop. This area includes 31 states in which at least one million acres of soybean were planted in 2011, including the following states: IA, IL , MN, IN, MO, NE, OH, SD, AR, ND, KS, MI, MS, WI, NC, KY, TN, and LA (USDA-NASS, 2011) . The most common rotational crops for soybeans in the United States are corn, other row crops, and small grains. In 2002 approximately 80% of the soybeans were grown in rotation with corn (USDA-ERS, 2006b). Soybean is normally grown as a full-season cr op that is seeded in the spri ng, but a small percentage of the crop is planted mid-June to early July as a se cond crop following winter wheat, rice or winter

canola in the more southerl y regions of the U.S.

Soybeans with the non-recombinant DNA-derived STSTM trait are tolerant to sulfonylurea

herbicides. The STSTM tr ait does not provide cro ss-tolerance to imidazolinone herbicides and it is currently marketed by NK ® , among others, in the United States as a stacked trait together with glyphosate tolerance. Use of STSTM soybeans allows effective weed control by sulfonylurea herbicides in soybean production areas where glyphosate-to lerant weeds are present. [

]

All of the GM soybeans commercially planted to date in the U.S. contain herbicide tolerance traits (Table IX-1, Ta ble IX-2). In 2011 approximately 94% of the U.S. soybean crop was genetically modified for herbic ide tolerance (USDA-ERS, 2011a). Since 2007 over 90% of the [CBI]

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 142 of 375 soybean cultivated in the U.S. in recent years contains one or more bi otechnology-derived traits and if CV127 soybean were to be cultivated in the U.S., it would not be expected to significantly alter the area of biotechnology-deri ved soybeans planted in the U.S.

As in other regions of the worl d where herbicide-tolerant soybe ans have been widely adopted, including Brazil and Argentina, the practice of minimum or no-till cultivation practices have

increased in the U.S. Minimum- and no-till cultiv ation practices have lead to reduced: i) soil erosion, ii) contamination of surf ace waters with agricultural fer tilizers and chemicals, iii) fuel consumption, and iv) release of gree nhouse gases into the atmosphere.

Table IX-1. Commercialization Status of Genetically-Modified Soybeans in the United

States in 2011

OECD Unique

Identifier Soybean Trait Applicant USDA Approval

Date U.S.

Commercial

Cultivation in

2011 1 ?

MON-04032-6 Glyphosate tolerant Monsanto May 1994 yes ACS-GMØØ5-3 Glufosinate tolerant Bayer CropScience July 1996 yes ACS-GMØØ4-2 Glufosinate tolerant Bayer CropScience July 1996 no ACS-GMØØ2-9 Glufosinate tolerant Bayer CropScience July 1996 no ACS-GMØØ1-8 Glufosinate tolerant Bayer CropScience July 1996 no

DD-Ø26ØØ5-3 Increased oleic acid

content Du Pont May 1997 no ACS-GMØØ6-4 Glufosinate tolerant Bayer CropScience June 1998 no

ACS-GMØØ3-1 Glufosinate tolerant Bayer CropScience November

1998 no
MON-89788-1 Glyphosate tolerant Monsanto July 2007 yes

DP-356Ø43-5 Glyphosate and acetolactate synthase

tolerant Pioneer July 2008 no DP-3Ø5423-1 High oleic acid content Pioneer June 2010 no 2

BPS-CV127-9 Imidazolinone tolerant BASF Plant

Science L.P. n.a. no
MON-87701-2 Lepidopteran resistant Monsanto n.a. no

MON-87769-7 Stearidonic acid produced Monsanto n.a. no

MON-877Ø5-6 Improved fatty acid profile Monsanto n.a. no

MST-FGØ72-3 Glyphosate and

isoxaflutole tolerant Bayer CropScience

and MS Tech n.a. no

DAS-68416-4 2,4-D and glufosinate

tolerant Dow AgroSciences n.a. no MON-877Ø8-9 Dicamba tolerant Monsanto n.a. no
MON-87712-4 Increased yield Monsanto n.a. no

1 Source: www.biotradestatus.com , accessed August 31, 2011.

2 PlenishTM high oleic soybeans will be grown under contract in 2011 for ongoing field and oil testing.

Commercialization of PlenishTM high oleic soybean s is anticipated in 2012, upon full regulatory approval . Source: http://www.plenish.com/about plenish.aspx , accessed September 3, 2011.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 143 of 375 Table IX-2. Planted Acres of Soybeans and of Biotechnology-Derived Soybeans in the United States in 2001 through 2011 (US DA-ERS, 2011a; USDA-ERS, 2011b; USDA-NASS, 2011).

Year Acres Planted Biotech Percentage of

Planted Acres* Acres of Biotech Soybeans Planted*

2011 75208000 0.94 70695520

2010 77404000 0.93 71985720

2009 77451000 0.91 70480410

2008 75718000 0.92 69660560

2007 64741000 0.91 58914310

2006 75522000 0.89 67214580

2005 72032000 0.87 62667840

2004 75208000 0.85 63926800

2003 73404000 0.81 59457240

2002 73963000 0.75 55472250

2001 74075000 0.68 50371000 * Since there are not currently any non-herbic ide tolerant biotechnology-derived products commercialized, the acreage of biotechnology- derived soybeans presented here is synonymous with the acreage of biotechnology- derived herbicide-tolerant soybeans.

Since a large percentage of the s oybeans that are cultivated currently in the U.S. are tolerant to glyphosate, this herbicide is the l eading herbicide used on soybeans in the U.S. (Table IX-3). In addition, soybean is naturally resistant to some imidazolinone herbicides, including imazamox, imazethapyr, and imazaquin due to lower uptake a nd higher metabolism of these herbicides by soybean. These three imidazolinone herbicides are registered for use on soybeans in the United States under brand names such as Raptor ® , Pursuit ® , and Scepter ® , among others. The 2010 usage of all herbicides on soybeans in the U.S. is presented in Table IX-3.

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 145 of 375 C.4. Comparison of the Cultivation of Soybean in Brazil and the U.S.

The following section reviews the similarities between soybean genot ype, environment, and agronomic practices used in soybean production in Brazil and the U.S. This comparison demonstrates that the data generated in fi eld trials conducted in Brazil to support the environmental and food and feed safety of CV127 soybean are equally applicable to the environmental and food and feed safety assessmen ts of CV127 in the U.S. environment.

Soybean genotype comparison

Based on their photosensitivity, soybean varieties are divided into maturity groups (MG) 000 to

X. Varieties in the ea rliest maturity group (000 to II) are adapted to the northernmost soybean production regions of North Ameri ca and the southernmost production regions of South America. Varieties in the latest MG (V II-X) are adapted to the southernmost soybean production regions of North America and the northernmost production regions of South America. Thus, specific varieties are grown at certain latitudes because they flower and mature within an optimal time period required for commercial production at that latitude. A recent study conducted in Brazil using soybean varieties developed in Brazil and other varieties de veloped in the U.S. concluded

that the maturity classification system used in North America was an efficient method for describing relative maturity in Brazil on a broad environmental basis (Alliprandini et al. , 2009).

Differences between early maturing (MG 000 to IV) and late maturing (MG V to X) soybean varieties with respect to time to flowering and maturity are controlled by at least five genes at the

‘E’ locus: E1 and E2 (Bernard, 1971), E3 (Buzzell, 1971), E4 (Buzzell and Voldeng, 1980), and

E5 (McBlain and Bernard, 1987). Three of these genes ( E1

, E3

, E4 ) are also involved in photoperiod sensitivity (Cober et al. , 1996). Soybean breeders, us ing classical methodologies, routinely recombine genes at th e E locus to modify time to ma turity and develop varieties adapted to specific environments. Another main genetic difference normally observed between early maturing and late maturing varieties occurs at the locus (dt) for stem determinancy. Early maturing varieties generally have an indeterminat e growth habit with genotype dt1/dt1 and late maturing varieties generally have a determinate growth habit with genotype Dt1/Dt1 (Bernard, 1972). This common relationship between maturity and determinancy is a result of selection for the best combination of genes required to maxi mize production in a specific region. For example, in a study which included later-maturing determinate and indeterminate vari eties adapted to the southern U.S., it was conclude d that the indeterminate grow th habit may confer a yield advantage over the determinate growth habit in 10 to 15% of the environments with limited yield and growth potential, but that the determinate growth habit provided a yield advantage over a larger percentage of the environments. Thus, while indeterminate late-maturing varieties are genetically possible and available, the yield advantage an indeterminate may have over a determinate growth habit in a late-maturing va riety is small (Kilgore-Norquest and Sneller, 2000).

In response to the variation in day length and climatic conditions in soybean production regions of the U.S., varieties grown in the U.S. range from MG I to MG IX (Figure IX-1). In Brazil, the

variation in environmental c onditions across the soybean pr oduction regions supports the production of varieties in MG V to X (Alliprandini et al ., 2009). In general, the soybeanCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 146 of 375 genotypes produced in Brazil are similar to those produced in the midsouthern and southeastern U.S. where variety maturities range from IV to VIII. In fact, the orig inal soybean varieties cultivated in Brazil during the early 20 th century were varieties developed for cultivation in the southern U.S (refer to Figure II-1 as an exam ple of the breeding lineage of the commercial Brazilian variety Conquista). Later, these U.S.-developed varieties became the parental genotypes used in Brazilian-based breeding pr ograms to develop modern Brazilian soybean varieties. Therefore, soybean varieties grown in the U.S. and Brazil share common genetic backgrounds.

The lines of CV127 soybean that were evaluated in the field trials describe d in this petition are MG VIII. These lines are adapted to the nort hern soybean production regions of Brazil and the southern soybean production re gions of the U.S. However, using convent ional breeding methods, it is possible to use lines of CV127 soybean to develop early (MG I to IV) or late (MG V to VIII) maturing soybean vari eties with determinate or inde terminate growth habits. The specific production environment ultimately dictates which combination of traits is required in that environment to enable the plant to be most productive.

Figure IX-1. Map Showing Regions in the Cont inental United States and Southern Canada where Various Soybean Maturity Groups (MG) Are Adapted for Full-Season Growth. (Willis, 1989).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 147 of 375 Environmental comparison

Climate: The varieties of CV127 soybean that have been developed for cultivation in Brazil are in maturity group VIII. In the U.S., soybeans in maturity group VIII are recommended for cultivation from the eastern coastal plains regions of the Southeastern states to Texas. The average monthly temperatures in this region during the growing s eason are shown in Table IX-4. Also shown for comparison are the average monthly temperatures at locations near the individual

locations where CV127 field trials were conducted in Brazil. Th e average monthly temperatures during the soybean growing season in Brazil ar e less variable across the season compared to those in the soybean growing regions of the sout heastern U.S. (Table IX-4). In the U.S. locations, the average monthly temperatures are comparable to those in Brazil.

Table IX-4. Comparison of the Average Monthly Temperatures (Degrees Fahrenheit) during the Soybean Growing Season. Different locations in the southeastern United States where soybeans in maturity group VIII are cultivat ed and cities closest to the individual field

trial locations in Brazil where CV 127 soybean was evaluated are shown.

US Locations 1 Lat. Apr. May June July Aug. Sep. Charleston, SC 33° N 65 74 78 82 81 76
Macon, GA 33° N 64 72 78 81 80 75
Houston, TX 30° N 70 76 81 84 84 80 Mobile, AL 31° N 68 75 80 82 82 78 Jackson, MS 32° N 65 72 78 82 81 76 Baton Rouge, LA 30° N 68 75 81 82 82 78 Gainesville, FL 30° N 68 74 78 81 81 78

Brazil Locations 2 Lat. Oct. Nov. Dec. Jan. Feb. Mar. Ponta Grossa, PR 25° S 64 66 69 71 71 69

Sto. Ant. de Posse (Campinas), SP 23° S 71 73 75 75 77 75

Uberaba, MG 19° S 75 75 75 75 77 75 Sete Lagoas, MG 20° S 71 71 71 73 73 73

Sto. Ant. de Goiás (Goiânia), GO 16° S 73 73 73 71 73 73

Brasília, DF 16° S 69 69 69 69 69 69

1 Source: http://countrystudies.us/u nited-states/weather/ , accessed September 21, 2011.

2 Source: http://www.weatherreports.com/ /Brazil , accessed September 22, 2011. Average monthly temperature over five to 36 year s, depending on location, is reported.

There are differences in the total amount of annua l rainfall and in the di stribution of rainfall throughout the year between the regions in Brazil where soybeans are cultivated and the southeastern U.S. where maturity group VIII soyb eans are cultivated (Table IX-5). Soybeans in Brazil are cultivated primarily in the high plateau or savannah region in central Brazil and in the southern, more temperate region. The average rain fall in this area ranges from 26 to 60 inchesCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 148 of 375 per year. In the savannah, rainfall is highe st during the growing se ason (November through March) and is much lower during the winter months (May through September). As rainfall is plentiful during the growing seas on in Brazil, irrigation is not typically required for soybean

cultivation. In the southeastern U.S. where rainfall can occasionally be inadequate during the soybean growing season, irrigation, if available, is used during dry pe riods to insure that the crop receives adequate water for grow th. A similar comparison of th e monthly average rainfall during the growing season in the southeastern U.S. wi th locations near the individual Brazilian CV127

field trial locations is shown in Table IX-5. In general, rain fall during the soybean growing season in the primary soybean cultivation regions in Brazil is higher compared to the rainfall in the soybean-producing southern U.S. However, with the use of supplemental irrigation for soybean production in the southern U.S., the total amount of water available to the crop grown in the two countries is comparable. In areas of s oybean cultivation in the southeastern U.S. where irrigation is not available, soyb ean yield can be reduced during periods of inadequate rainfall.

Table IX-5. Average Monthly and Total Ra infall (Inches) during the Soybean Growing

Season. Different locations in the southeastern Unite d States where soybeans in maturity group

VIII are cultivated and cities closest to the indi vidual field trial locations in Brazil where CV127 soybean was evaluated are shown.

U.S. Locations 1 Apr. May June July Aug. Sep. Total Charleston, SC 2.7 4.0 6.4 6.8 7.3 4.7 31.9
Macon, GA 3.5 3.6 3.6 4.3 3.6 2.8 21.4
Houston, TX 3.1 5.3 6.4 4.8 4.5 5.5 29.6 Mobile, AL 4.5 5.7 5.0 6.9 7.0 5.9 35.0 Jackson, MS 5.6 5.1 3.3 4.5 3.8 3.6 25.9 Baton Rouge, LA 5.4 4.9 4.5 6.7 6.0 4.9 32.4 Gainesville, FL 2.6 3.8 6.8 6.8 8.0 5.3 33.3

Brazil Locations 2 Oct. Nov. Dec. Jan. Feb. Mar. Total Ponta Grossa, PR 5.25 3.32 4.08 6.07 4.64 3.25 26.61

Sto. Ant. de Posse (Campinas), SP 4.33 6.42 9.35 12.23 7.98 6.01 46.32

Uberaba, MG 4.13 4.88 6.98 8.81 7.11 5.47 37.38 Sete Lagoas, MG 2.58 5.79 9.34 7.63 4.81 4.85 35.00

Sto. Ant. de Goiás (Goiânia), GO 4.25 9.79 13.78 13.50 10.57 8.22 60.11

Brasília, DF 2.94 6.40 6.71 5.04 5.10 5.75 31.94

1 Source: http://countrystudies.us/u nited-states/weather/ , accessed Septem ber 21, 2011.

2 Source: http://weather.uk.ms n.com/region.aspx? we alocations=Brazil , accessed September 22,

2011.

In summary, the climatic conditions for soybean production in the major growing regions of Brazil are comparable to those in the growing re gions of the U.S., and where differences between

environments in Brazil and the US were observe d, these were generally not different from the range of environments encounter ed across the soybean production regions within the southernCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 149 of 375 US. In any environment, the soybean plant’s requirements for resources from the environment are not significantly different. However, in th ose environments in whic h certain resources are limiting, the input requirements to pr oduce the crop may be different.

Soil: Most of the soils in the new agricultural areas in Brazil are classified as tropical soils, or

oxisols. The oxisols contain on ly hydrated oxides of iron a nd aluminum, are highly weathered with low pH and low in fertilit y and organic matter. The low pH of the soils often limits the availability of phosphorus to the soybean plan t. Because soybeans require relatively large amounts of phosphorus and a soil pH in the range of 5.0 to 5.5 (Guidelines for GAP, 2002) for optimal growth and development compared to ot her crops such as corn or wheat, Brazilian soybean growers typically add lime and phosphorus to the soils, and with this treatment oxisol soils can be very productive for soybean cultivation (Leibold et al . 2001). Also, addition of lime and phosphorus helps minimize potential aluminum toxicity to growth and development of the soybean plant. Similarly, in the U.S., soybean growers often apply phosphorus as well as potash to increase crop production, but this practice is highly variable from state to state. In both Brazil and the U.S., the full nitrogen needs of soybean can be supplied through the nitrogen fixing, root nodule symbiosis with Bradyrhizobium japonicum and nitrogen applicati on is not necessary nor does it usually result in increased yi eld (Guidelines for GAP, 2002; Whitaker et al ., 2011).

Agronomic comparison

Cultivation Practices. The planting dates for soybean vary depending on the latitude and region. In Brazil, soybeans are typically planted from mid-October to mid-December and in the more northern areas, in late December. The optim um planting rate and row-spacing for soybean cultivation in Brazil varies depe nding on the climate of the various soybean cultivating regions. A planting rate of 220,000 to 320,0 00 seed/ha with row spacing of 0.4 to 0.5 m is typical for soybean cultivation in Brazil (Guidelines for GA P, 2002). Planting depth for soybeans in Brazil is typically 3 to 5 cm deep. Planting deeper decreases germination while planting more shallow results in losses due to high surface-soil temperat ures and water stress. Harvest of soybeans in Brazil is at the R8 growth stage (full maturity) when the soybeans have a moisture content of 13 to 15%. In the southeastern U.S. where soybean s in MG VII to VIII ar e planted (e.g., Georgia), the optimal planting date for soybeans is May 10 to June 10 (Whitaker et al ., 2011). Seed should be planted at a depth of 2.5 to 3 cm (1 to 1.25 inches) in moist soil. The recommended planting rate and row spacing for soybean cultivation in the U.S. is 10-20 seed/m of row (3-6/foot of row) with a row spacing of 0.5 – 0.9 m (20 – 36 inch es) to achieve a final stand of 200,000 to 250,000 plants/ha (85,000 to 100,000 plants/acre). In order to help minimize disease and insect pests, it

is recommended in both Brazil and the U.S. that soybeans are rotated with grain crops such as

wheat and corn.

Weeds and Weed Control Practices : The major pest problems in the cultivation of soybeans in the U.S. are weeds followed by insects and di seases (Aref and Pike, 1998). Since weeds compete with soybeans for light, nu trients and moisture, weed control is an important factor in optimizing yield. The main factors affecting yi eld loss due to weeds are the predominant weed species, the weed density, and the length of the competition by weeds during the growing season. If weeds are allowed to grow with a soybean cr op for the entire growi ng season, yield losses can be as high as 75% of the crop (Dalley et al ., 2001). In addition, the impact of different weedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 150 of 375 species on the yield of soybeans has shown that yield losses vary with the predominant weed species and level of infestation (Coble et al ., 1981; Conley et al ., 2003). Soybeans grow quickly to close the crop canopy, after which they are ve ry competitive with weeds. However, soybeans are very sensitive to weed competition in the firs t weeks after emergence from the soil. It is recommended by weed specialists that weeds be controlled in s oybeans within three to five weeks after emergence in order to prevent irreversible yield losse s due to early competition from weeds. The practice of narrow row planting has been used to shorten the time for soybean to close the canopy and thereby gain an advantage over weeds. Howeve r, weeds that are taller than soybeans can still break through the canopy and cause yield losses. Common weeds in soybean

cultivation in the U.S. include the annual grasses and br oad-leaf weeds such as foxtail, ragweed and lambsquarter, winter annuals such as marestail and biennial wormwood, and perennials such as quackgrass and Johnsongrass. With the adop tion of no-till or redu ced-tillage cultivation systems, the weed spectrum has shifted to favor the winter annual, biennial, a nd perennial weeds.

Brazilian soybean farmers tend to have fewer weed control problems than farmers in the U.S. This is partly due to the fact that the agricultural la nds in Brazil do not have as long a history of farming as those in the U.S. However, the prac tice of no-till farming in Brazil is common, just as it is in the U.S., and weed pressure is expect ed to increase over time in Brazil. For example, soybean farmers in the southern regions of Braz il are faced with greater weed pressure than farmers in other soybean growing regions of Brazil because this area has a longer history of crop cultivation. Another challenge to soybean producti on in Brazil is that te mperatures stay warm after the soybean leaf canopy has fallen, which encourages weeds to start growing before the crop is harvested. While this late season weed growth has little impact on soybean yield, it can be a challenge to harvest crops with young green weeds growing. However, it is not common for either Brazilian or U.S. farmers to apply herbicid es at this late stage of crop development. The fact that it rarely freezes in Brazil during the winter months means that perennial weeds pose a greater weed problem in Brazil compared to th e weed spectrum in soybeans in the U.S.

Prior to the use of herbicides, the primary method of weed control in all crops was by mechanical

cultivation. The first herbicid es used in soybean were soil -incorporated and pre-emergence

herbicides were introduced in the 1960’s (Carpenter and Gianessi , 1999). These were replaced in the 1980’s by post-emergent herbicides th at were not active on soybeans and their use increased dramatically until recently. The us e of post-emergence herbicides has greatly facilitated the adoption of reduced- or no-tillage practices since farmers could effectively control weeds with one or two early treatments. This has both economic and environmental advantages, including more effective weed control leading to enhanced yield and reduc tions in tractor passes, fuel consumption, soil erosion a nd the release of greenhouse gases. The more recent advent of genetically-modified (GM) soybeans that are tole rant to post-emergent treatment with broadspectrum herbicide has further facilitated the adoption of reduced- or no-tillage practices (Gianessi, 2005).

The cultivation of glyphosate-tole rant soybeans treated with glyphosate has been widely adopted in the U.S., Argentina, and Brazi l. In 2010, 60% of the United Stat es treated acres were treated with glyphosate (Table IX-3). Similarly, nearly 100% of the soybean crop in Argentina is glyphosate-toleran t and the percenta ge of cultivation of glyphosate- tolerant soybeans cultivated in Brazil is rapidly increasing since regulatory approval in 2005 (approxima tely 75% in Brazil inCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 151 of 375 2010, [James, 2010]). Therefore, the uses of gly phosate herbicide to control weeds in soybeans is the most common practice in bot h the U.S. and Brazil. These tr ends suggest that there is a clear need for tolerance in soybeans to herbicides with different modes of action as a means to better manage the development of herbicide resistance. Chemical carryover is less of a problem in Brazil compared to the U.S. due to prolonged wa rmer soil temperatures and higher rainfall in Brazil. However, there is still a need to rotate crops and chemical modes of action to minimize the probability of developing herbicide-resistan t weeds in Brazil. In addition to glyphosate,

other herbicides used for weed control in soybeans are similar between the U.S. and Brazil (Leibold et al ., 2001).

The broad use of any class of herbicide with a specific mode of action is known to eventually lead to the development of resistance to the herbicide in weed popul ations. Some weeds commonly found in soybeans have developed re sistance to glyphosate , namely horseweed

( Conyza canadensis) , common waterhemp ( Amaranthus rudis ), common ragweed ( Ambrosia artemisiifolia ), and giant ragweed ( Ambrosia trifida ) (Heap 2011). Also, the growing use of glyphosate with glyphosate-tolerant soybeans in Br azil has led to a shift in the species of prevalent weeds with those that are more tole rant to glyphosate pred ominating. The most common weeds in this category include Benghal dayflower ( Commelina benghalensis L.)

, morning glory ( Ipomoea spp.), Brazil pusley ( Richardia brasiliensis ), and winged false buttonweed ( Spermacoce alata ). These weeds are sensitiv e to imidazolinone herbicides. An effective method of managing and reducing the de velopment of resistance is the rotation of herbicides with different modes of action (Loux et al . 2004; Loux and Stachler 2006). Therefore, the introduction of imidazolinone-tolerant CV127 soybeans will provide soybean growers an additional tool for the management of herbicide resistance in weeds.

Insect Control: In the U.S., the control of insects in soybean with insecticid es is seco nd only to weed control in total pesticide use. In 2006, insecticides were applied to 16% of the U.S. soybean acreage (USDA-NASS, 2007). The thr ee most commonly-used insecticides included lambda-cyhalothrin, chlorpyrifos, and esfenvalerate that were applied to 6, 5, and 3% of the planted soybean acres, respectively (USDA-NASS, 2007). In the U.S., the number of soybean insect pest species and their densities follows a north-south gradient with a greater insect pressure in the southern states bordering the Gulf of Mexico and the Atlantic Ocean (Way, 1994). This is the same region in which s oybeans in maturity group VIII such as CV127 are adapted for cultivation. The major insect pests of soybean in the southern U.S. include the lepidopterous defoliators, velvetbean caterpillar ( Anticarsia gemmatalis ) and soybean looper ( Pseudoplusia includens ); the coleopterous defoli ator, bean leaf beetle ( Cerotoma trifurcata ); the pod-feeding stink bugs, complex- southern green stink bug ( Nezara viridula ) and green stink bug ( Acrosternum hilare ); and the corn earworm ( Helicoverpa zea ) that attacks foliage and pods. The midwestern U.S. suffers only sporadically fro m severe outbreaks of insect pests in soybeans (Boethel, 2004). Some of the insect pests that occasionally are problematic in soybeans in the Midwest include the green cloverworm ( Plathypena scabra ), Mexican bean beetle ( Epilachna

varivestis ) and bean leaf beetle. More recently, the soybean aphid ( Aphis glycines ) that is a native of Asia has become a significant soybean in sect pest since its disc overy in the U.S. in

2000.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 152 of 375 In Brazil the warmer winter temperatures result in a larger number of insects, typical of the southern U.S. The main insect pests of soybean in Brazil, as in the so uthern U.S., include the velvetbean caterpillar and th e green stink bug (Turnipseed an d Kogan, 1976). The lepidopterous

cutworm Agrotis ipsilon is known to cause sporadic problems in soybean production in Brazil (Turnipseed and Kogan, 1976). The lesser cornstalk borer, Elasmopalpus fignosellus , is widely distributed from the southern U.S. through Mexic o, to southern Brazil and Argentina where stand reductions frequently necessitate replanting in la te-planted soybean in sandy soils, particularly under drier conditions (Turnipseed and Kogan, 1976). While the simila rities in the insect pest species between the U.S. and Brazil are greater for the southern U. S., the insect pests of soybean

are fewer in the midwestern U.S. due to colder winters in this region.

The imidazolinone-tolerant CV127 soybean has been assessed in multi-location, replicated field trials conducted in two growing seasons in Brazil and has been s hown to be no different in its agronomic characteristics or environmental inte ractions compared to conventional soybeans. The susceptibility of CV127 to insects was found to be no diff erent in CV127 soybean compared to conventional soybean va rieties. Protease inhib itors produced by plants have been shown to play a role in the defense against insect predation and disease infestation (Hilder et al ., 1987; Boulter, 1993; Williamson and Hussey, 1996; Joshi et al . 1998). Levels of soybean trypsin

inhibitor were measured in the grain of CV127 soybean and the is oline control in field trials conducted during two growing seasons in Brazil and it was determined that there were no differences in the level of trypsin inhibitor in the grain from these soybeans (Section VII, Table VII-9).

A key component of the protection of crop plants from insects is the development of genetic

resistance to insects that has been introduced into modern soyb ean varieties through breeding. The strong genetic similarity between soybeans cultiv ated in the U.S. and Brazil suggests that the same insect resistance traits are incorporated into both. The commercial cultivation of CV127 soybean in the U.S. would require the imidazolinone tolerance trait to be transferred by breeding into soybean cultivars that are adapted to the various climatic and environmental conditions of the soybean cultivating regions of the U.S. Based on the a bove considerations and on the extensive experience with many different imidazolinone-tolerant Clearfield ® crops that have notdemonstrated different sensitivity to insects compared to their corresponding imidazolinonesensitive conventional c ounterparts and on the extensive know ledge of the biological mode of action of the AHAS enzyme, it is not expect ed that CV127 soybean would demonstrate a different response to insect s were it to be cultiv ated in the U.S. compared to soybean varieties that are currently cultivated in the U.S.

Disease Control : Diseases caused by viruses and fungi can occasionally cause serious damage leading to significant loss of soybean yield in both the U.S. and Brazil. A list of the major viral diseases of soybean that occur in the U.S. and Brazil is presented in Table IX-6 . Of the 14 major viral diseases listed, seven are commonly f ound in both the U.S. and Argentina, Brazil, or South America (Tolin and Lacy, 2004). Of the ot hers, five are present in the U.S. but not in South America and only two are pr esent in South America but not in the U.S. (Table IX-6).

Bacterial pathogens are widespread on soybean but do not generally cause serious problems that result in significant yield reduc tions (Tolin and Lacy, 2004).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 153 of 375 Over 40 fungal pathogens of s oybean are reported to cause significant disease problems worldwide. The environment and crop management practices have a large impact on the prevalence and severity of th ese diseases (Yang and Feng, 2001). A key component in the management of soybean diseases is the incor poration of genetic resistance to important pathogens through breeding. Howe ver, resistance genes are not known for some pathogens and for these proper management practices are impor tant. Due to economic considerations, the widespread application of fo liar fungicides on soybeans in the U.S. is not common, but fungicides are commonly applied as seed treatments. In Br azil where Asian soybean rust ( Phakopsora pachyrhizi ) is especially problematic, foliar f ungicides are commonly used to treat this pathogen. The major soybean pathogens in the top ten soybean-p roducing countries are listed in Table IX-7. Also presented in Table IX -7 are the soybean yield losses in the U.S. and Brazil in 1998 that were caused by these diseases (from Wrather et al ., 2001). It should be noted that soybean rust ( Phakopsora pachyrhizi ) was not a major disease of soybeans in South or North America in 1998 but it has since developed into a major disease problem in Brazil in recent years. To date, effectiv e genetic resistance to Asian rust has not been identified and so

control relies on the use of foliar fungicides where infestations are severe. Asian rust has not developed into a significant disease of soybean in the U.S. or Argentina. All of the major soybean diseases that caused significant soybean yield losses in Brazil in 1998 also caused significant losses of soybean yield in the U. S. with the exception for powdery mildew ( Microsphaera diffusa ) and target spot ( Corynespora cassiicola ). Both of these diseases are found in the U.S. (Grau et al ., 2004), but were not the source of significant losses in the U.S. in

1998.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 154 of 375 Table IX-6. Important Viral Diseases of So ybean in the U.S. and South America (from Tolin and Lacy, 2004). Viruses that occur in both the U. S. and South America are denoted with a grey background.

Family, Genus or

Virus Distribution Major Plant Symptoms Genetic

Resistance in Soybean Tospovirus

Tomato spotted wilt

virus U.S. (WI), Brazil,

Iran Systemic chlorosis and

necrosis NR †

Comoviridae

Bean pod mottle U.S., South

America Chlorotic mottle, green stems after pod maturity None found

Bean rugose mosaic Brazil, Argentina,

Central America Puffy areas along veins,

chlorotic blotches NR

Cowpea severe

mosaic Brazil, Puerto Rico, U.S. (AR, LA, IL) Severe necrosis and bud blight NR

Tomato ringspot

virus U.S., China Bud blight, curved

terminal bud and lateral bud proliferation Observed

Potyvirus

Bean yellow mosaic Worldwide Yellow mottling, rusty necrotic spotting Observed

Peanut mottle U.S., China, Thailand, Argentina Mosaic with green islands; line pattern Rpv1; Rpv1, rpv1

Soybean mosaic virus Worldwide Green to yellow mosaic,

leaf curl Rsv1, Rsv3, Rsv4

Bromoviridae

Alfalfa mosaic Worldwide Bright yellow mosaic and

mottle NR

Cowpea chlorotic

mottle U.S. (AR, GA, SC) Stunting, chlorosis, yellow stipple Single dominant

gene Peanut stunt U.S. (IL, KY, VA), Japan Mild mottle and leaf

crinkle Observed

Tobacco streak U.S. (IA, OH, OK, WI, VA) Bud blight; axillary branches, stunting NR

Others

Cowpea mild mottle Brazil, S.E. Asia,

Africa Vein clearing, leaf roll;

vein necrosis NR

Tobacco mosaicsoybean U.S., Eastern Europe Very mild vein clearing,

chlorotic mosaic NR

† No report of a search for resistanceCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 155 of 375 Table IX-7. Causal Organisms of Commonly- Occurring Soybean Diseases in the Top Ten Soybean-Producing Countries and Soybean Yiel d Losses Caused by Them in the U.S. and Brazil in 1998 (Wrather et al ., 2001).

Common Name Causal Organism Yield Loss (‘000 metric tons)

US Brazil

Anthracnose Colletotrichum truncatum 188.9 2.0

Bacterial diseases Pseudomonas savastanoi pv. glycinea and Xanthomonas axonopodis pv. glycines 27.0 —

Brown spot Septoria glycines 167.0 2194.9

Brown stem rot Phialophora gregata 398.8 —

Charcoal rot Macrophomina phaseolina 1036.8 750.0 Diaporthe–phomopsis complex Diaporthe and Phomopsis spp. 24.1 — Downy mildew Peronospora manshurica 106.3 — Frog-eye leaf spot Cercospora sojina 86.6 —

Fusarium root rot Fusarium spp. 243.8 2.5 Phytophthora root and stem rot Phytophthora sojae 1149.0 — Pod and stem blight Diaporthe phaseolorum var. sojae 143.6 627.1 Powdery mildew Microsphaera diffusa — 156.8 Purple stain of seed and cercospora leaf blight Cercospora kikuchii 112.4 940.7

Rhizoctonia foliar blight Rhizoctonia solani 111.5 14.4 Rhizoctonia–pythium root rot Rhizoctonia solani and Pythium spp. 128.0 313.6

Sclerotinia stem rot Sclerotinia sclerotiorum 509.0 1.5

Seed diseases Alternaria spp., Cercospora spp.

, Corynespora, Cladosporium

, Phomopsis , and Fusarium spp. 45.8 2.0

Seedling diseases Rhizoctonia

, Pythium , and Fusarium

spp. 776.9 1.5

Southern blight Sclerotium rolfsii 9.2 —

Stem canker Diaporthe phaseolorum var. caulivora and var. meridionalis 30.0 10.0

Sudden death syndrome Fusarium solani f. sp. glycines 900.6 200.0
Target Spot Corynespora cassiicola — 130.0 Note: Soybean rust ( Phakopsora pachyrhizi ) was not a major soybean pathogen in Brazil in 1998, but has developed into a majo r pathogen in recent years.

Differences in environmental conditions, whethe r between the U.S. a nd Brazil or between different regions within Brazil or the U.S., may result in di fferences in how the soybean crop is managed. For example, in regions of the U.S. or Brazil where rainfall is insufficient, irrigation is used to provide sufficient water for the crop and to prevent yield losses. Similarly, if soils are acidic, limestone may be applied. If weeds are a problem in soybean production, herbicides areCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 156 of 375 applied. In cases of he avy insect infestations, insecticides may be applied. Whether soybean is cultivated in the U.S. or in Brazil, it has the sa me general needs for growth and development. In

some environments these needs ar e met naturally and in others they must be met through human intervention. There are many similarities in environmental conditions and agronomic practices used in soybean production betwee n the southern U.S. and Brazil, including temperatures and rainfall, the maturity groups of soybean cultivat ed, as well as weed, insect and disease control practices. Furthermore, some environmental conditions during the growing season such as average temperatures are comparable between U.S. and Brazilian soybean production. In the event that CV127 soybean was cultivated in the U.S. environment, due to the close genetic relationship between the s oybean varieties cultivated in the U.S. and Brazil as well as similarities in environmental conditions and agronomic prac tices that exist between the U.S.and Brazil, CV127 soybean would not be expected to present any new environmental ri sks relative to the cultivation of soybean varieties that are currently grown in th e U.S. The long history of

cultivation and safe use of Clearfield ® crops also supports the conclu sion that the introduction of

the imidazolinone-tolerance trait in to CV127 soybean will have no impact on its interaction with disease pathogens or the environment. Based on th ese considerations, the data generated in field

studies conducted in Brazi l to support the environmental as well as food and feed safety of CV127 soybeans are equally applicab le to the environmen tal, food and feed safety assessment of

CV127 in the U.S. environment.

D. Imidazolinone Herbicide Use in the U.S.

Imidazolinone-tolerant crops have been cultivated in the U.S. since the introduction of

imidazolinone-tolerant corn in 1992 a nd are currently comme rcialized under the Clearfield ®

brand name. Clearfield ® crops are developed by seed mu tagenesis or other non-recombinant DNA techniques and contain mutations in the AHAS gene(s) that result in the production of an imidazolinone-tolerant AHAS. [

] As previo usly noted, soybean is naturally tolerant to some imid azolinone herbicides (imazaquin, imazethapyr, and imazamox), (Tecle et al. , 1993; Nelson et al. , 1998) and these herbicides are applied to soybeans in the U.S. (Table IX-3). In addition, some ot her crops - field peas, snow peas, dry beans, snap beans, adzuki beans, fenugreek, seedling and establis hed alfalfa, bird’s foot trefoil and chickling vetch- possess a natural tolerance to individual imidazolinone herbicides and imidazolinones are used on these crops for weed control (Health Canada Pest Manageme nt Regulatory Agency, http://pr-rp.hc-sc.gc.ca/ls-re/index-eng.php ). Soybeans are sensitive to imazapyr and imazapic and CV127 soybean was developed to be tolerant to these compounds. Imazapyr and imazapic are registered in the U.S. for weed control in pastures, rangeland, and other noncrop areas. Imazapic is registered in the United States fo r use in the control of weeds on pastures and peanuts, while imazapyr is registered for use on past ures and corn. A list of the crops in the U.S.

for which the use of different imidazolinone herbicid es is registered and th e total area treated is presented in Table IX-8. [CBI]

deletedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 157 of 375 BASF developed CV127 soybean for commercial cu ltivation in Brazil and Argentina to address weed control challenges facing soybean farmers in these countries. However, in the event that CV127 soybean may be introduced into the U.S ., it is not anticipated that CV127 soybean

varieties will cause an increase in the total number of soybean acres planted in the U.S., and it is not expected that the herbicide tolerance trait wi ll confer an incentive to grow the crop outside of its normal geographic range or habitat (e.g. cultivate d agricultural lands). Furthermore, based on

the use of imidazolinone herbicides for weed control on the differe nt crops listed in Table IX-8, introduction of CV127 soybean in th e U.S. is unlikely to have any significant effect on the total use of imidazolinone herbicides for weed control in U.S. agriculture.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 158 of 375 E. Potential Impact of Introduction of CV127 Soybean on Agronomic Practices

The introduction of soybean CV127 in Argentina and Brazil is expected to provide enhanced soybean yield potential and offer growers excelle nt weed control options in addition to the

environmental benefits that result from the cultivation of imidazolinone herbicide-tolerant [CBI]

deletedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 159 of 375 soybeans. The imidazolinone herbicides po ssess several environmentally-beneficial characteristics compared to other herbicide classes (Tan et al ., 2005). Imidazolinone herbicides control a wide spectrum of grass and broadleaf weeds. The growing use of glyphosate with glyphosate-tolerant soybeans in Arge ntina and Brazil has led to a shif t in the species of prevalent weeds with those that are more tolerant to glyphosate predominating. The most common weeds in this category incl ude Benghal dayflower ( Commelina benghalensis L.)

, morning glory ( Ipomoea spp.), Brazil pusley ( Richardia brasiliensis ), and winged false buttonweed ( Spermacoce alata ). These weeds are sensitive to imid azolinone herbicides, so introduction of CV127 soybean will provide growers an effective m eans to control these weeds at relatively low herbicide application rates.

Furthermore, with increased grower adoption of glyphosate-tolerant crops and use of glyphosate for weed control purposes, it has been reported that several weed species have evolved resistance to glyphosate (Heap, 2011; Nandula et al ., 2005). Therefore introduction of CV127 soybeans will provide growers with the opportunity to cultiv ate soybeans that are tolerant to a herbicide with a different mode of action compared to glyp hosate as a means to control weeds that may be tolerant to glyphosate and to help prevent the continued evolution of more weed species that are

herbicide tolerant. The introducti on of CV127 soybean varieties is not only expected to offer soybean growers an additional tool for controllin g weeds, but also provide growers an important option for weed resistance management.

The environmental safety of CV127 was shown to be comparable to conventional soybean varieties which do not exhibit plant pest or w eediness characteristics. CV127 soybean was compared to conventional soybean varieties with respect to impor tant agronomic characteristics including seedling vigor, days to reach key develo pmental stages, plant height, disease and insect susceptibility, and grain yield. In addition, the impact of cul tivating CV127 soybeans on factors relevant to the nitroge n fixing symbiosis with Bradyrhizobium was assessed. Furthermore, laboratory- and greenhouse-based studies on seed germination as well as pollen number and pollen germination were conducted and comparis ons of these characteristics made between CV127 soybeans and the isoline c ontrol and, for the seed germ ination studies, two other conventional soybean varieties. The results of th ese comparative studies demonstrated that other

than tolerance to imidazolinone herbicides, there are no biologi cally meaningful differences

between CV127 and the conventional soybean varieties with respect to the parameters measured

as described above.

Although it is not anticipated th at CV127 soybean will be cultivated in the U.S., cultivation of CV127 soybean in the U.S. would not be expected to present any different environmental risks compared to the cultivation of herbicide-sensi tive, conventional or glyph osate-tolerant soybean

varieties. As noted above and elsewhere in th is petition, extensive assessments of agronomic characteristics of CV127 soybean and its interaction with the environment have demonstrated that CV127 soybean is not significantly different from conventional soybean varieties in these characteristics. In addition, th e Brazilian soybean variety, Conquista, that was transformed with

the csr1 -2 gene cassette to produce CV127 soybean is cl osely related to soybean varieties that are currently cultivated in the U.S. (refer to Figure II-1). Therefore, it is expected that if CV127 soybeans were cultivated in the U.S., they woul d interact with the environment in a similar manner to commercial soybeans that are currently cultivated in the U.S.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 160 of 375 Under the Canadian regulatory system, crops that are developed to be tolerant to herbicides for which the parental variety is susceptible, are c onsidered novel, regard less of the methods by which they are developed. These Plants with N ovel Traits require revi ew and approval by the Canadian Food Inspection Agency (CFIA) and/or Health Canada for environmental release and

food and feed use. All Clearfield ® crops have undergone the re quired regulatory review in

Canada and have been assessed to be safe for food and feed uses. In addition, in each case, after review of pertinent environmental data, the CFIA has concluded that Clearfield ® crops do not present an altered risk to the environment comp ared to the herbicide-sensitive crop from which they were developed. A list of the Clearfield ® crops that have been co mmercialized to date is presented in Table IX-9. In the more than 19 years in which Clearfield ® crops have been cultivated in North America, other than their to lerance to imidazolinone he rbicides, none of these crops have demonstrated any different envir onmental characteristics compared to their imidazolinone-sensitive counterparts. Similarl y, there is no reason to believe that the imidazolinone-tolerant CV127 soybean will present an altered risk to th e environment compared to conventional soybeans.

The agronomic and environmental assessments and the results and data fr om other studies of CV127 soybean that are described in this peti tion were generated using CV127 soybean lines 127 and 603 which are from maturity group VIII. Currently there are breeding programs in Brazil and Argentina that are breeding the imid azolinone-tolerance trait of CV127 soybean into genetic backgrounds that are earlier maturing and better-adapted for cultivation in Argentina and the southern regions of Brazil which have shor ter growing seasons. B ecause of their earlier maturity, these varieties will also be adapted to soybean-cultivating areas of the midwestern U.S. (Figure IX-1). Given the common ancestry that exists among Brazilian and U.S. soybean varieties (Pardey et al ., 2004), it is unlikely that the earlier-maturing varieties developed in Brazil or Argentina would interact differently in the U.S. environment or present any different environmental risk compared to soybean varieties that are currently cultiv ated in the midwestern U.S. In addition, the experience with th e safe cultivation of the many different Clearfield ® crops in the U.S. and other countries supports the c onclusion that CV127 soybeans that are adapted for cultivation in the midwestern U.S. would not pr esent any different environmental risks compared to soybean varieties currently cultivated in this region.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 161 of 375 Table IX-9. Identity, Year of Market Launc h and the CFIA Decision Document Number

for Imidazolinone-tolerant Clearfield ® Crops.

Crop First Market

Introduction for Crop Line Name Decision

Document 1

XA-17 DD96-10 Corn 1992 XI-12 DD98-29

NS1471 DD95-03 Canola 1996
S006 DD2008-73

SWP 965001 DD1999-31

AP602CL DD2003-44

AP205CL DD2004-47

Teal 11A DD2004-48

BW255-2

BW238-3 DD2006-60

DW1 DD2006-63

DW2

DW6

DW12 DD2007-64 Wheat 2002

BW7 DD2007-66

X81359 DD2005-50 Sunflower 2003

CLHA-PLUS DD2010-80

Lentil 2006 RH44 DD2004-46

CL121

CL141

CFX51 DD2002-40

PWC16 DD2002-42 Rice 2003

IMINTA 1

IMINTA 4 DD2006-62

1 Decision documents are available on the Canadian Food Inspection Agency (CFIA) website at the following address: http://www.inspection.gc.ca/en glish/plaveg/bio/dde.shtm l

.

Based on the above characterizations of CV127 soybean, introduction of CV127 soybean is not expected to have any negative impact on current agronomic practices in soybean production in Brazil and Argentina, or in the U.S. for the following reasons:CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 162 of 375 a) CV127 soybean will offer growers an add itional tool for weed control as well as herbicide resistance management in weeds as part of an integrat ed weed management program, and is not expected to change w eed control practices currently followed by soybean growers. b) CV127 soybean exhibited no different disease susceptibility or insect interactions compared to the conventional soybean variet ies, so no change in disease and insect control practices in each diffe rent country is expected. c) Conquista, the parental soybean variety that was used to develop CV127 soybean, is derived from and closely related to, soybean varieties cultivated in the southern U.S. and therefore possesses agronomic characteristics similar to U.S. soybean varieties. d) A large variety of imidazolinone-toler ant crops currently commercialized under the

Clearfield ® brand name have been cultivated in North America since 1992 (Table IX-9). Each of these has been evalua ted and reviewed by the Canadian Food Inspection Agency (CFIA) and has been determined to present no different environmental risk compared to thei r conventional, im idazolinone-sensitive counterparts. The collectiv e experience of cultivating Clearfield ® crops in North America and other regions of the world has confirmed that these crops do not present any additional environmental risks compar ed to corresponding conventional crops. Likewise, there is no reason to believe that imidazolinone-tolerant CV127 soybean will present any different environmental risk compared to imidazolinone-sensitive soybean varieties. e) Volunteer soybean management is normally not a significant management concern in

North America because of environmenta l conditions, crop rotation, the numerous herbicide control options avai lable, and the fact that soybeans do not compete well with the succeeding crop. However, the cu rrent practices used for management of volunteer soybeans are equall y applicable to CV127 soyb ean. In the event that CV127 soybean volunteers were to become es tablished, they could be controlled by either mechanical means or by herbicides other than those that target the AHAS

enzyme. f) As outlined in section IX.F. of this petition and detailed in Appendix G, BASF is committed to the stewardship or Best Management Practice (BMP) program for management of weed resistance to imidazoli none herbicides in CV 127 soybean crops. As detailed in Appendix G, gr ower compliance with this program is essential to delay

the onset of imidazolinone he rbicide tolerance in weed s in CV127 soybean crops. The guiding principles of the BMP for CV127 soybeans require that growers do not exceed a maximum of two exclusive Group 2 herbicide applications on any one field in any four-year period. Therefore, the introduction of CV127 soybeans is expected to expand the options to growers for inte grated weed management programs and not have any negative impact on integrated weed management programs that growers currently follow that include the use of a wi de range of herbicides, cultural practices and crop rotations to manage weed populations.

F. Herbicide Resistance Management fo r Imidazolinone-tolerant CV127 Soybean

The objectives of herbicide resist ance management are to achiev e weed control while preserving

the value of each herbicide and each herbicid e group for the longer term. An integratedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 163 of 375 approach to weed control is the Best Management Practice to dela y the onset of weed resistance to herbicides. Integrated weed management invol ves the use of a range of methods available to the grower in order to provide effective weed control in the crop. The use of herbicides is one of

a number of useful tools available to growers.

BASF is committed to maintaining the efficacy of all of its herbicides in order to provide growers with effective, high-performance, envi ronmentally-sound products for many years. The key to the performance of the Imidazolinone-to lerant Soybean Production System is effective weed resistance management. BASF is committe d to delivering sustainable cropping systems that incorporate best practic e principles. The Imidazolinone-tolerant Soybean Production System provides alternative options for growers within a well-manag ed rotation. As part of its weed resistance management program, BASF ha s developed a detailed weed resistance management plan for implementation with im idazolinone-tolerant CV127 soybean. An outline of this product stewardship plan is presented below and the complete details of the plan are presented in Appendix G.

Key Points of the Imidazolinone-tolera nt CV127 Soybean Resistance Management

Recommen dations:

• DO NOT exceed a maximum of two exclusive A HAS-inhibiting herbicides on any one field, in any four-year period.

• ALWAYS follow an Integrated Weed Manage ment (IWM) program that includes a wide range of herbicides, cultural practices and crop rotations in order to manage weed populations and minimize weed seed development.

• ALWAYS control volunteers in the season following an imidazolinone-tolerant soybean

crop.

• USE practices which minimize the likelihood of out-crossing to similar crops or related

weeds.

• Scout fields for weeds or volunteer cr ops that are uncontro lled by herbicides.

• FOLLOW the Best Management Practices outlined in the Imidazolinone-tolerant Soybean Production System Stewardship Guid e (presented in Appendix G).

G. Summary of the Agronomic and Environmental Assessments for CV127 Soybean

The agronomic characteristics and environm ental interactions of CV127 soybean were thoroughly evaluated in Brazil during two different growing seasons. In addition, the molecular insert in CV127 soybean was completely character ized, the level of AHAS protein in various tissues of CV127 soybean was analyzed, and the nutrient and antinutrient composition of grain and forage was evaluated. Review of the data from these studies supports the conclusion that CV127 soybean presents no different plant pest pote ntial or environmental ri sk compared to that of conventional soybean va rieties. This conclusi on is further supported by the long history of theCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 164 of 375 cultivation of other imidazoli none-tolerant crops containing the same S653N AHAS amino acid substitution as found in CV127 soybeans. None of these crops have demonstrated enhanced plant pest tendencies or envi ronmental risks compared to their corresponding imidazolinonesensitive counterparts.

The AHAS enzyme is ubiquitous in plants and its biological mode of action is well studied and is not known to be toxic or allergenic. The f ood safety of the imidazolinone-tolerant AtAHAS introduced into CV127 soybean is further supported by the long hi story of safe consumption of food and feed products derive d from the many different Clearfield ® crops that contain the S653N AHAS mutation and by the grain com position studies of CV127 soybean that demonstrate that the grain from CV127 soybean is as nutritious as gr ain from conventional soybeans.

The environmental safety of CV127 was shown to be comparable to conventional soybean varieties which do not exhibit plant pest or w eediness characteristics. CV127 soybean was compared to conventional soybean varieties with respect to impor tant agronomic characteristics including seedling vigor, days to reach key develo pmental stages, plant height, disease and insect susceptibility, and grain yield. In addition, the impact of cul tivating CV127 soybeans on factors relevant to the nitrogen fixing symbiosis with Bradyrhizobium were assessed. Furthermore, laboratory- and greenhouse-ba sed studies on seed germina tion, pollen number and pollen germination were conducted and comparisons of these characteristics made between CV127 soybeans and the isoline control and, for the se ed germination studies, two other conventional soybean varieties The results of these compar ative studies demonstrated that other than tolerance to imidazolinone herbicides, there are no biologically meaningful differences between CV127 and the conventional soybean varieties with respect to the parameters measured as described above. Therefore, these results reinforce the conclusion that the cultivation of CV127 soybean poses no different plant pest or weediness potential and will have no different environmental impact than the cultivation of conventional soybean varieties.

Other than the additional option to apply imidazo linone herbicides for weed control in CV127 soybeans, the commercial introduc tion of CV127 soybeans is not ex pected to change the typical agronomic practices used for soybean produc tion, will have no different environmental interactions compared to other commercially-pr oduced soybean varieties, or any impact on the nutritional quality and safety of soybean food and feed products compared to those from conventional soybean varieties. The introducti on of imidazolinone-tolerant CV127 soybeans is expected to expand the tools available to grower s for the management of herbicide resistance in weed populations in s oybean cultivation.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 165 of 375 X. Adverse Consequences of Introduction

BASF Plant Science L.P. is not aware of any information, study results or observations that would indicate that CV127 soybean poses any differe nt weediness or plant pest potential and no different environmental impact than conventio nal soybean varieties. There are no adverse environmental consequences anticipated w ith the introduction of CV127 soybean.

The environmental as well as food and feed safe ty of CV127 soybeans was confirmed based on

the results of a series of interrelated safe ty assessment studies. The detailed molecular

characterization of CV127 s oybean confirmed that CV127 c ontains a single functional csr1-2 gene cassette integrated in the soybean ge nome. Biochemical characterization of the imidazolinone-tolerant AtAHAS pr otein expressed in CV127 soybean showed that the AtAHAS protein is typical of other AHAS proteins in this ubiquitous pr otein family as well as most dietary proteins with a history of safe use in food and feed products and lacks any of the characteristics associated with known allergenic or toxic proteins. Extensive phenotypic and agronomic evaluations as well as ecological interactions of CV127 show that cultivation of CV127 soybean poses no different plant pest or weediness risk and no greater potential environmental impact than cultivation of c onventional soybean vari eties. Finally, the composition and nutritional equiva lence of CV127 soybean compared to conventional soybean varieties was demonstrated by analysis of key nutrients and antinutrients in both grain and forage, and nutritional equivalen ce to conventional soybeans was further confirmed in a poultry feeding study. The results of these studies show that CV127 soybeans are as safe as conventional soybeans for environmental rel ease as well as for food and feed uses.

CV127 soybean was developed for cultivation primar ily in Brazil and Argentina. Introduction of CV127 soybean will allow growers to treat the soybean crop with imidazolinone herbicides for weed control without causing inju ry to the soybean plant at nor mal field application rates. Introduction of CV127 soybean varie ties is expected to offer soybean growers an additional tool for controlling weeds, as well as an importa nt option for weed resistance management. Furthermore, it is expected that growers pl anting CV127 soybeans will be able to reduce the

number of herbicides used to control weeds in their soybean fields and benefit from reduced

weed control costs. The reduction in herbicide use is also expected to benefit the environment.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 166 of 375 XI. References

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 184 of 375 XII. Appendices

Appendix A: Materials and Methods for th e Molecular Characterization of CV127 Soybean

Appendix B: Characterization of AtAHAS Protein Produced in Imidazolinonetolerant CV127 Soybean and Comparison with AtAHAS Protein Expressed in Recombinant Escherichia coli

Appendix C: Methods for Determinin g Levels of the AHAS and SEC61 γ Subunit Proteins in Soybean Tissues

Appendix D: Methods Used and Results of the Grain and Forage Composition Analyses

Appendix E: Methods and Results of Seed Germination, Dormancy and Seed Quality Studies of CV127 Soybean

Appendix F: Materials and Methods and Re sults from the Agronomic, Phenotypic, and Ecological Interaction Evaluations of CV127 Soybean

Appendix G: Best Management Practice (BMP) Program for the Imidazolinonetolerant CV127 Soybean Production System

Appendix H: Digestive Fate of the AtAHAS Produced in CV127 Soybean and in the

Recombinant E. coli Expression SystemCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 185 of 375 Appendix A

Materials and Methods for the Molecula r Characterization of CV127 Soybean

Source of plant materials for DNA analyses

. Young leaf tissue of CV127 soybean was provided to DNA Landmarks, Inc. (Montreal , Canada) by BASF SA for DNA isolation and

characterization. Control DNA wa s isolated from leaf tissue of the non-transgenic soybean variety Conquista that is cl osely related to CV127 soybean. All plants were grown under greenhouse conditions in Londrin a, Brazil, and young, fully-expande d trifoliate leaves were harvested for molecular analyses.

DNA isolation and quantification methods

. DNA was isolated from soybean leaf tissue via a modified cetyl trimethyl ammonium bromide (CTAB) method (Carlson et al ., 1991). Silica gel-desiccated leaf tissue wa s frozen with liquid nitrogen and ground with an Autogrinder (Autogen; Holliston, MA). The ground tissue was incubated with preheated extraction buffer consisting of 2% (w/v) CTAB, 100 mM Tris-HCl, 1.4 M NaCl, 1% (w/v) polyvinylpyrrolidone (PVP), 20 mM ethylenedi amine tetraacetic acid (EDTA), pH 9.5 (5 ml/60 mg dried leaf tissue) and β -mercaptoethanol (10 μ l/ml buffer) at 74 ° C for 20 min. After centrifugation at 2440 x g for 10 min, the supernatant was extracted twice with an equal volume of chloroform/isoamyl alcohol (24:1) . DNA was precipitated with 0.7 volume of isopropanol and dissolved in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 0.5 mg/ml RNase A (Invitrogen; Carlsbad, CA) adde d to a final concentration of about 500 ng/ μ l

. The isolated DNA was quantified w ith Hoechst 33258 dye (Invitrogen) with calf thymus DNA (Invitrogen) used as the DNA standard on a Packard FluoroCountTM BF10000 Microplate Fluorometer ( Packard Instrument Company; Meriden, CT) according to the

fluorometer user manual.

Probe isolation and labeling methods

. The DNA fragments used as transgene-specific probes are indicated in Figure IV -1 of the applica tion. The vector backbone probes are indicated in Figure V-3 of the application. Together these 5 overlapping probes span the entire plasmid. Specifically, probe 1 spans the At AHAS promoter region, probe 2 the csr1-2 coding sequence (CDS), probe 3 the At AHAS terminator region, and probes 4 and 5 together cover the complete vector backbone (VB). The probe DNA fragments were generated by polymerase chain reaction (PCR) amplification us ing plasmid pAC321 as a template (Table A-1). The probes (25 – 50 ng each) were radiolabeled with 50 μ Ci of ( α

32 P)-dCTP (3000 Ci/mmol) (MP Biomedicals; Irvine, CA) using the Redipr imeTM II DNA Labeling System (Amersham; Piscataway, NJ) according to th e manufacturer’s instru ctions. The labeled probes were purified with a Spin-X ® Centrifuge Tube Filter (C orning Costar Corporation; Acton, MA).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 186 of 375 Table A-1. Primers Used to Generate Probes for Southern Blot Analysis

Purpose Direction Primer Sequence Position in pAC321

Probe 1 Forward TGCGTTATCCCCTGATTCTG 8261-8280

5' UTR Reverse TGTTGGGGTTTAGGGAG 2597-2613

Probe 2 Forward CGAAGGCTCAATCACAAATAC 2269-2289

At AHAS CDS Reverse AGCAGGCAGATCAACAAC 4604-4621

Probe 3 Forward GAACATGTGTTGCCGATGAT 4416-4435

3' UTR Reverse CGCAACTGTTGGGAAGGG 5949-5966

Probe 4 Forward GTTTT ACAACGTCGTGACTG 5839-5858

VP1 Reverse CGGTTAGCTCCTTCGGTC 6997-7014

Probe 5 Forward CACTGCGGCCAACTTACT 6962-6979

VP2 Reverse CTTGGCGTAATCATGGTC 8592-8609

Restriction digestions an d Southern blot analyses

. Southern blot anal yses were used to determine the number of copies and the integrity of the csr1-2 expression cassette as well as to confirm the absence of plasmid backbone in CV127. Restriction en zymes NcoI, SpeI and XbaI were used to digest the genomic DNA of CV127 and non-transgenic control Conquista. The pAC321 PvuII transformation fragment is aligned with the CV127 insert in Figure V-1 of the application. A single NcoI restriction site in the csr1-2 cassette is located at the 5’ end

of the csr1-2 coding sequence and digestion of genomic DNA of CV127 with NcoI was expected to generate two frag ments that contain DNA from the csr1-2 cassette. Both fragments are defined by the NcoI site in the csr1-2 cassette and by the nearest NcoI sites in the flanking soybean genomic sequence. There is one SpeI restriction site in the 5’ flanking soybean genomic sequence and two SpeI re striction sites downstream of the At AHAS 3’ UTR

in CV127. The XbaI restricti on sites flank the complete csr1-2 expression cassette. The number and sizes of the DNA fragments expected to be detected by S outhern hybridization are listed in Table V-1 of the petition.

Genomic DNA (7 μ g) from the F 8 generation of CV127 line 603 (Fi gure III-1 of the petition) and from the non-transgenic c ontrol Conquista was di gested overnight in a volume of 40 μ l with the restriction enzymes listed above (8 units/ μ g DNA) under the conditions specified by the enzyme manufacturers (New England Biol abs; Ipswich, MA; or Amersham). Two additional non-transgenic Conquista genomic D NA samples were spiked with one- and twogenome copy equivalents 2 of pAC321 plasmid DNA (27 and 54 pg, respectively) and used as

2 Calculation of copy number equivalents

Assumptions:

• The haploid content of the soybean genome is 1.115 X 10 9 bp (Arumuganathan and Earle, 1991).

• Plasmid pAC321 is 8669 bp.

Since the insert in Cultivance ® Event 127 is homozygous and 7 μ g of soybean DNA is used per digest in the Southern blot analysis, the mass of one copy equivalent of pAC321 is:

mass of pAC321 DNA = 8669 bp transgene DNA

7 μ g genomic DNA (1.115 X 10 9 bp genomic DNA) x 2

mass of pAC321 DNA = 27 pgCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 187 of 375 positive controls. Restriction digests were separated by electrophoresis in 10 cm long 0.8% agarose gels. The DNA was further fragmented by soaking the gels in 0.25 N HCl for about

20 min and was denatured with 0.4 N NaOH for about 30 min. The gels were rinsed with 2X NaCl/sodium citrate solution (SSC) and the denatured DNA was transferred onto Hybond N+ nylon membrane (Amersham) using 0.4 N NaOH as a transfer buffer. Southern hybridization was carried out according to Sambrook et al . (1989). The membranes were prehybridized at

65 ° C for 2-4 h and hybridized at 65 ° C overnight in 20 – 30 ml (about 0.2 ml/cm 2 ) of hybridization buffer (2x SSC, 0.6% SDS, 50 mM Na 2 HPO 4, 1x Denhardt’s solution, 2.5 mM EDTA, 5% dextran sulfate, pH 7.2) in a Hybaid MAXI 14 Hybridization Oven (Thermo Electron Corporation). After hybridization, the membranes were washed with 2x SSC, 0.5% SDS (1 ml/cm 2 ) at room temperature for 15 min, 2x SSC, 0.1% SDS (4 ml/cm 2 ) at 65 ° C for 30 min, and finally with 0.1x SSC, 0.1% SDS (4 ml/cm 2 ) at 65 ° C for 15 min. After washing, the membranes were wrapped in plastic wr ap and exposed to HyperfilmTM MP film (Amersham) for 2-5 days, depending on the radi oactive signal intensity, in cassettes with intensifying screens at –80oC.

Stability of the DNA insert across breeding generations

. Southern blot analyses were also

conducted as described above to monitor the stability of the insert across multiple generations. Plant material was obtained from the T 4 , F 4 , F 8 and F 9 generations of CV127 line 603 (Figure III-1). Genomi c DNA from these samples was digested with NcoI and SpeI (as described above) and Southern blot anal ysis was carried out as described above.

Complete sequence of the DNA insert

. Six PCR-generated amplicons were designed to span

the entire insert as well as th e junctions with the adjacent soybean genomic sequences (Figure V-5). The primer pairs used to generate thes e six amplicons are described in Table A-2. The complete sequence of the inserted DNA was ob tained by PCR amplification of these six overlapping fragments followed by DNA sequ ence analysis. PCR amplicons containing sequence discrepancies relative to the sequenc e of the transformation fragment were reamplified with rTth DNA polymerase XL. PCR products were purified with the Zymo DNA Clean & ConcentratorTM-5 and were sequenced on both strands to a quality level of Phred 40 by direct sequencing and primer walking. DNA sequencing was performed as described

above.

Table A-2. Primers Used for CV127 DNA Insert Amplification

Purpose Direction Primer Sequence Position in pAC321

Forward GCTTGATATGCCTTTTGGTTC 5265-5285 PCR1 Reverse TTGTCTTCCCTCATTGGAC 6150-6168

Forward GACGAGATATTCCCGAAC 4544-4561 PCR2 Reverse GTCTGATTAGTGCTTCTGG 5525-5543

Forward CCCTGTTGCGAGTACGTTGA 3739-3758 PCR3 Reverse CTTCCGTTATGACATCGTTG 4732-4751

Forward AACCACTCCCTCTCCAAC 2980-2997 PCR4 Reverse CTGATGATAGCCACTGCC 4266-4283

Forward TTCGTTCGCTCTGGTGTC 2062-2079 PCR5 Reverse ACGGTTTCTACGCCTTG 3089-3105

Forward GAAAATAGGAAGTTTAGGCTTG 1000-1021 PCR6 Reverse GGGCTGATAATGTCGTTTG 2229-2247CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 188 of 375 Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the At SEC61 γ subunit transcript. Young leaf tissue of silica gel-desi ccated young leaves derived from F 7 generation plants of CV127 soybean and from le aves of the non-transgen ic parental soybean variety Conquista was provided to DNA Landmarks, Inc. (Montre al, Canada), by BASF SA

for RNA isolation and characterization. All plants were grown under greenhouse conditions in Londrina, Brazil, and young, fully-expanded trifo liate leaves were harvested for molecular analyses.

Total RNA was extracted from silica gel-desi ccated young leaves w ith the Qiagen RNeasy Mini Kit (Qiagen; Valencia, CA). About 25 mg of s ilica gel-desiccated l eaf tissue was frozen with liquid nitrogen and ground w ith an Autogrinder. The total RNA isolation procedure was carried out according to the manufacturer’s di rections. On-column DNase digestion was performed with RNase-Free DNase (Qiagen) to eliminate any soybean genomic DNA from the total RNA preparation according to the recommendation in the RNeasy Mini Kit user manual. The isolated RNA was quantitated by measuring the absorbance at 260 nm using a BioMateTM 3 spectrophotometer (Thermo Electron Corporation; Waltham, MA).

RT-PCR analysis was conducted to determine if the At SEC61 γ subunit gene present in CV127 was transcribed. The primers used for th is RT-PCR analysis ar e described in Table A-3. Total RNA was used as template for RT-PCR using the Qiagen OneStep RT-PCR Kit (Qiagen). For RT-PCR analysis of the At SEC61 γ subunit coding sequence, Arabidopsis total RNA samples from leaves and roots were also used as positive controls. The Arabidopsis total RNA samples were provided by BASF Plan t Science (Research Tr iangle Park, NC).

The Arabidopsis total RNA samples were prepared with TRIzol reagent (Invitrogen) without

DNase treatment. The RT-PCR reactions cont ained 1x Qiagen OneStep RT-PCR Buffer, 400

μ M of each dNTP, 0.6 μ M of each primer, 2 μ l of Qiagen OneStep RT-PCR Enzyme mix, and 500 ng or 125 ng of total RNA in a total volume of 50 μ l. The RT-PCR was conducted using the GeneAmp PCR System 9700. Followi ng a 30 min reverse transcription step at

50 ° C, PCR amplification was carried out un der the following conditions: one 15 min denaturation step at 95oC; 30 cycles at 94oC for 30 sec, 64oC for 30 sec, and 72oC for one min; and one ten-min extension at 72oC.

The endogenous soybean SEC61 γ subunit (Gm SEC61 ) and Iota genes were used as positive controls. The soybean Iota subunit gene is expressed constitutively and ubiquitously in soybean (Yamamoto and Knap, 2001).

Table A-3. Primers Used for RT-PCR Analysis of the At SEC61 γ subunit transcript

Purpose Direction Primer Sequence Position in pAC321

Forward ACGAACCTGCTGAAACCCTAAT 1338-1359 RT-PCR

At SEC61 Reverse TAAGAATGGAGAATTTGGCTACA 2280-2302

Forward TGAAGCAGCAGCTGAGTTTCGC N/A* RT-PCR

Iota Reverse GGCAGTCTGAACCGTCTCCTC N/A

Forward GCTTGGGAGACAGAGAAAGAGA N/A* RT-PCR

Gm SEC61 Reverse CCTTTTGCTTGACAACCTGAAT N/A
Forward TTGGAATGCATGGGACTGT 3807-3825, 5733-5751 RT-PCR

ORF501 Reverse TGTCTTCCCTCATTGGACTG 6148-6167 *N/A – not applicable. This is a positive control; primer set is expected to amplify cDNA derived from an endogenous soybean tran script unrelated to the CV127 insert.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 189 of 375 RNA ligase-mediated rapid amplification of 5’ complementary DNA ends (RLM-5’-RACE) of the At SEC61 γ subunit transcript

. The test material for this study was RNA isolated from greenhouse-grown CV127 soybean plant material. The control material for this study was RNA isolated from greenhouse-grown conventional soybean variety Conquista plant material. The reference material for this study was RNA isolated from Arabidopsis thaliana ecotype Columbia (Col-0)

. Leaf tissue from the CV127 eighth filial (F 8 ) generation and its comparator non-transgenic line, Conquist a, was obtained from young (25-day-old) greenhouse-grown plants. Plant tissue from greenhouse-grown Arabidopsis was also used.

Messenger RNA (mRNA) was isolated from Arab idopsis and soybean l eaf tissue using the

FastTrack MAG Maxi mRNA Is olation Kit (Invitrogen GmbH; Karlsruhe, Germany). Approximately 200 mg of leaf ti ssue was ground in liquid nitrogen using a mortar and pestle. Pre-mixed Lysis Buffer L4/Protein Degrader (510 μ l) was added to each sample and samples were suspended by vortexing. Samples were cent rifuged for five minutes at approximately 16,100 x g at room temperature. Supernatants were transferred to ne w tubes and incubated

for 15 minutes at 45  ̊ C. FastTrack MAG Beads (200 μ l per sample) were pre-washed and bound to each sample in accordance with the ma nufacturer’s instructions. The recommended wash procedure was followed with the inclus ion of an optional DNase I digestion. Two additional washes (beyond the standard protoc ol) with Wash Buffer W7 were performed immediately before elution of the mRNA . Messenger RNA was eluted under the

recommended conditions in 20 μ l RNase-free water followed by a second elution in 10 μ l

RNase-free water. The two mRNA eluate s were combined and stored at –80  ̊ C.

Messenger RNA was diluted in 1 M Tris-HCl, pH 7.5 and qua ntitated by measuring the absorbance at 260 nm using a BioPhoto meter (Eppendorf AG; Hamburg, Germany).

RNA ligase-mediated rapid amplification of 5’ cDNA ends (RLM-5’-RACE) was conducted using the GeneRacer Kit (Invitrogen) in accord ance with the manufactu rer’s instructions unless otherwise noted. Messenger RNA (250 ng ) was dephosphorylated with calf intestinal phosphatase (CIP) to remove the 5’ phosphate from any truncated mRNA or non-mRNA present in the mRNA prepara tion. Because CIP has no effect on capped full-length mRNA, this dephosphorylation step was expected to eliminate, or at least reduce, the contribution from non-full-length messages in later step s of the RLM-5’-RACE procedure.

This dephosphorylated RNA was then treated with tobacco acid pyrophosphatase (TAP) to remove the 5’ cap structure from full-length mR NA. In order to determine the contribution from non-full-length messages in this RLM-5 ’-RACE experiment, a second set of samples was processed identically to the experimental samples with the exception that TAP was

excluded from these control reactions. If th e RLM-5’-RACE experiment is only amplifying capped full-length message, then no PCR product s would be evident in these “minus TAP”

controls.

TAP treatment generates a free 5’ phosphate wh ich is required for the subsequent step: ligation of the GeneRacer RNA Oligo to the 5’ end of the de-capped full-length mRNA with T4 RNA ligase. The ligated mRNA was reve rse transcribed into first-strand cDNA using SuperScript III RT (Invitrogen) and the GeneRacer Oligo dT Prim er in a 75 minute reaction. The region of the first-strand cDNA correspon ding to the GeneRacer RNA Oligo contains binding sites for the GeneRacer 5’ Primer and th e GeneRacer 5’ Nested Primer which will be used in the primary and secondary PCR amplifications, respectively.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 190 of 375 Primary PCR amplification was performed using 2 μ l of each reverse transcription reaction as a template for amplification by Platinum Taq DNA Polymerase High Fidelity (Invitrogen) under the reaction conditions specifi ed in the GeneRacer Kit inst ructions. In this primary PCR, the GeneRacer 5’ Primer was paired with either gene-specific primer RS41 (Integrated DNA Technologies; Coralville, Iowa) or gene-specific primer RS46 (BASF AG Oligonucleotide Synthesis Facility ; Ludwigshafen, Germany). Gene-specific primer RS41,

when used in combination with the GeneRacer 5’ Primer, was expected to amplify the 5’ end of cDNAs derived from endogenous soybean SEC61 γ subunit transcripts. Due to the lack of a published full-length cDNA sequence, soybean SEC61 γ subunit-specific primers were designed based on sequences from The Institute for Genomic Research’s Glycine max gene index of likely genes and their variants. Gene-specific primer RS46, when used in combination with the GeneRacer 5’ Primer, wa s expected to amplify the 5’ end of cDNAs derived from Arabidopsis SEC61 γ subunit (locus At3g48570) transcripts. Although there are two additional putative SEC61 γ subunit genes in Arabidopsis, primer RS46 was designed to bind specifically within the 3’ untranslate d region (UTR) of only the locus At3g48570 cDNA to reduce background amplificatio n in control RLM-5’-RACE experiments using Arabidopsis

mRNA.

Table A-4. Gene-Specific Primers Used in RLM-5’-RACE Experiment: Primary PCR

Name Primer Sequence and Description

RS41 CATCCTAACCAGATCCGACGATGATGTTG

SEC61 γ subunit, soybean. Reverse primer

RS46 CAAAGGGCTGATAATGTCGTTTGGTTCGTTCTTC

SEC61 γ subunit, Arabidopsis. Reverse prim er

Primary PCR amplification was carried out on a PrimusHT thermal cycler (MWG-Biotech AG; Ebersberg, Germany) using the following thermal cycling conditions:

Table A-5. Primary PCR Thermal Cycling Parameters

Temperature Time Number of Cycles

94  ̊ C 2 minutes 1

94  ̊ C 30 seconds

72  ̊ C 2 minutes 5

94  ̊ C 30 seconds

70  ̊ C 2 minutes 5

94  ̊ C 30 seconds

60  ̊ C 30 seconds

68  ̊ C 2 minutes 25

68  ̊ C 10 minutes 1

8  ̊ C ∞

In order to increase both yield and specificity, a secondary P CR amplification was performed using nested primers. The secondary PCR utilized 1 μ l of each primary amplification reaction as a template for amplification in 50 μ l reactions containing final concentrations (or quantities) of 2.5 units of Platinum Taq DNA Polymerase High Fidelity (Invitrogen), 1X High Fidelity PCR Buffer, 2 mM magnesium sulfate, 0.2 μ M of each primer, and 0.2 mM ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 191 of 375 each dNTP (Invitrogen). In this secondary PCR, the GeneRacer 5’ Nested Primer was paired with either nested gene-specific primer RS 43 (Integrated DNA Technologies) or nested genespecific primer RS42 (Integrated DNA Technologi es). Nested gene-specific primer RS43,

when used in combination with the GeneRacer 5’ Nested Primer, was expected to amplify the 5’ end of cDNAs derived from endogenous soybean SEC61 γ subunit transcripts. Nested gene-specific primer RS42, when used in combination with the GeneRacer 5’ Nested Primer, was expected to amplify the 5’ end of cDNAs derived from Arabidopsis SEC61 γ subunit (locus At3g48570) transcripts. Although there are two additional putative SEC61 γ subunit genes in Arabidopsis, primer RS42 was de signed to bind specifically within the 3’ untranslated region (UTR) of only the locus At3g48570 cDNA to reduce background amplification in control RLM-5’-RACE experiments using Arabidopsis mRNA.

Table A-6. Gene-Specific Primers Used in RLM-5’-RACE Experiment: Secondary

PCR

Name Primer Sequence and Description

RS42 CCTACTCCTCGGAGCATTGCCTCGTA

SEC61 γ subunit, Arabidopsis. Reverse primer, nested. Spans intron.

RS43 CAGTACGGACGGCAACCTTGGAGAAT

SEC61 γ subunit, soybean. Reverse prim er, nested.

Secondary PCR amplification was carried out on a PrimusHT thermal cycler (MWG-Biotech AG) using the following thermal cycling conditions:

Table A-7. Secondary PCR Thermal Cycling Parameters

Temperature Time Number of Cycles

94  ̊ C 2 minutes 1

94  ̊ C 30 seconds

65  ̊ C 30 seconds

68  ̊ C 2 minutes 20

68  ̊ C 10 minutes 1

8  ̊ C ∞

Secondary PCR products (20 μ l) were separated by electrophores is at 10 V/cm in a 1% (w/v) agarose gel containing 0.2 μ g/ml ethidium bromide in TAE (40 mM Tris-acetate, 1 mM EDTA).

The CV127 RLM-5’-RACE secondary PCR products generated with Arabidopsis SEC61 γ subunit-specific primers were cloned usi ng the TOPO TA Cloning Kit for Sequencing (Invitrogen). TOPO vector pCR4-TOPO (1 μ l) was incubated with an aliquot of the secondary PCR (4 μ l) and 1 μ l of a 1.2 M sodium chloride, 0.06 M magnesium chloride

solution for 30 minutes at 23  ̊ C. An aliquot of the cloning reaction (2 μ l) was transformed into One Shot TOP10 Chemically Competent Escherichia coli (Invitrogen) according to

manufacturer’s instructions and plated on Luria-Bertani agar plates containing 50 μ g/ml kanamycin.

Twelve transformants were sc reened for positive clones by co lony PCR. Each colony was suspended in 4 μ l of sterile water. One microliter of this suspension was added to a 25 μ l reaction containing final conc entrations of 1X Eppendor f MasterMix (Eppendorf AG;CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 192 of 375 Hamburg, Germany) and 0.2 μ M of each primer. Due to the bidirectional nature of the cloning, a gene-specific primer (R S42) was paired with either vector-specific primer RS7 (Integrated DNA Technologies) or vector-s pecific primer RS 8 (Integrated DNA Technologies) to determine th e insert orientation.

Table A-8. Primers Used in PCR Screen for Positive Clones

Name Primer Sequence Description RS7 CGCCAGGGTTTTCCCAGTCACGAC M13 forward (-41) primer
RS8 AGCGGATAACAATTTCACACAGG M13 reverse (-48) primer

RS42 CCTACTCCTCGGAGCATTGCCTCGTA SEC61 γ subunit, Arabidopsis. Reverse primer, nested. Spans

intron.

PCR amplification was carried out on a Primus HT thermal cycler (MWG-Biotech AG) using the following thermal cycling conditions:

Table A-9. PCR Screen Thermal Cycling Parameters

Temperature Time Number of Cycles

95  ̊ C 5 minutes 1

95  ̊ C 15 seconds

55  ̊ C 15 seconds

72  ̊ C 30 seconds 30

72  ̊ C 7 minutes 1

8  ̊ C ∞

Aliquots of each reaction (5 μ l) were separated by electrophores is at 10 V/cm in a 1% (w/v) agarose gel containing 0.2 μ g/ml ethidium bromide in TAE (40 mM Tris-acetate, 1 mM EDTA).

All transformants identified by the PCR screen as containing positive clones were cultured in

2.5 ml of Luria-Bertan i broth containing 50 μ g/ml kanamycin. Plasmids were purified from these cultures using the QIAprep Spin Minipr ep Kit (Qiagen GmbH; Hilden, Germany) in

accordance with manufactu rer’s instructions.

Double-stranded sequencing of selected clone s was performed at the BASF AG Sequencing Facility (Ludwigshafen, Germany). Sequenc ing data was assembled using Sequencher, version 4.5 (Gene Codes Corporat ion; Ann Arbor, Michigan).

RT-PCR analysis of the 501 bp open reading frame (ORF). RT-PCR was conducted to determine if the 376 bp dupli cation of a portion of the csr1-2 coding sequence present in CV127 was transcribed. Tissues (roots, l eaves, and leaf buds) were collected from greenhouse-grown CV127 and Conquista plants and, together with seed, were used as the source of RNA and genomic DNA samples for this study.

Genomic DNA for PCR analysis was isolated from leaf, root, leaf buds a nd seed tissue using the NucleoSpin ® Plant II kit (Macherey-Nagel; Beth lehem, PA) in accordance with the manufacturer’s protocol with the following modifications. Powdered tissue sample (100 mg)

was mixed with 400 μ l Buffer PL1. Following vortexing, 10 μ l RNase A was added and afterCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 193 of 375 mixing, the sample was incubated at 65oC for 30 minutes. The lysate of each sample was loaded onto a single NucleoSpin Plant II Colu mn and centrifuged for 1 minute at 12,000 rpm at room temperature. The fl ow-through was mixed with 450 μ l Buffer PC and loaded onto another NucleoSpin Plant II Column and centrifuged for 1 minute at 12,000 rpm. The flowthrough from this spin was discarded and th e column was washed consecutively with 400 μ l PW1, 700 μ l PW2 and 200 μ l PW2. Finally, the DNA was eluted with 50 μ l Elution Buffer PE. All genomic DNA samples were stored at 4oC.

Genomic DNA was quantitated using the Quant-iT PicoGreen dsDNA A ssay Kit (Invitrogen; Carlsbad, CA) in accordance with the manufact urer’s instructions. Lambda DNA provided with the kit was used to generate a standard curve from 1 ng/ml to 1000 ng/ml. Fluorescence was measured with a Turner Quantech Digita l Filter Fluorometer (Barnstead International; Dubuque, IA), λ

ex = 490 nm, λ

em = 515 nm. All genomic DNA sa mples were quantitated in duplicate.

RNA was isolated from 100 mg of powdered ti ssue using the Qiagen RNeasy Plant Mini Kit (Qiagen Inc., Valencia, CA) and tr eated with DNase as described in the Mini Kit instructions. Powdered tissue (100 mg) was mixed with Buffer RLT and the lysate was transferred to a QIAshredder spin column. The flowthrough wa s mixed with 0.5 ml et hanol, loaded onto the RNeasy mini spin column, centrifuged at 11,000 rpm for 1 min and washed with 350 μ l Buffer RW1. DNase I was added directly onto th e column and incubated at room temperature

for 15 min. The column was washed with 350 μ l Buffer RW1 followed by two washes with

500 μ l Buffer RPE. RNA was eluted in 35 μ l RNase free water and quantitated using 1.5 μ l of the RNA sample on the Na noDrop ND-1000 spectrophotometer.

To monitor the presence of the ORF 501 transcript in the CV127 soybean plants, RT-PCR was conducted using primers specific for this open reading frame (anticipated size 435 bp) and control primers to amplify the transcri pt of an endogenous, constitutively-expressed soybean gene, cyclophilin (Jian et al ., 2008, anticipated amplicon size 315 bp). As an additional control, reactions in which the reve rse transcriptase was omitted were included to confirm that amplification was not occu rring from contaminating genomic DNA.

Table A-10. Primers Used for RT-P CR Analysis of the 501 bp ORF

Purpose Direction Primer Sequence (5’ → 3’) Position in pAC321

Forward TTGGAATGCATGGGACTGT 3807 – 3825

5733 - 5751 ORF 501

Reverse TGTCTTCCCTCATTGGACTG 6148 - 6167

Forward AGCTCTATGCCGACGTGACTCC N/A* Cyclophilin Reverse ACTCCGTCTTCGTCGTGCAGAT N/A
*N/A – not applicable. Cyclophili n was used as a positive control; the primer set is expected to amplify cDNA derived from an endogenous s oybean transcript unre lated to the CV127

insert.

Total RNA (500 ng) was used as a template fo r RT-PCR using the Superscript First Strand Synthesis System for RT-PCR (Invitrogen; Carl sbad, CA). RNA was converted to cDNA in the first step that included a denaturation of RNA in the presence of random hexamers and

dNTPs at 65oC for 5 minutes a nd 4oC for 5 minutes. Each 20 μ l RT reaction contained finalCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 194 of 375 concentrations of 1.25 ng/ μ l random hexamers, 0.5 mM dNTP mix, 1x RT Buffer, 5 mM magnesium chloride, 10 μ M DTT, 2 U/ μ l RNaseOUT, and 10 U/ μ l Superscript III Reverse Transcriptase. The reaction wa s incubated at 25oC for 10 minut es, 50oC for 50 minutes, 85oC for 5 minutes and then stored at 4oC to cool down followed by addition of 1 μ l of RNase H

and incubation at 37oC for 20 minutes. The seco nd step is the PCR amplification using 1 μ l of the cDNA template generated in the first step. Each 25 μ l reaction contained final concentrations of 1x AmpliTaq ® Gold 360 Buffer, 2.5 mM magnesium chloride, 0.2 mM of dNTP mix, 0.6 U of AmpliTaq ® Gold 360 DNA Polymerase and 0.2 μ M of each primer. All primers were synthesized by Integrated DNA T echnologies (Coralville, IA). Following a 30 minute reverse transcription step at 50oC, PCR amplification was carried out under the following cycling conditions: 95oC for 10 minu tes, 30 cycles at 95oC for 30 seconds, 64oC for 30 seconds, and 72oC for one minute, followed by 72oC for 7 minutes.

PCR of genomic DNA (50 ng) using the same primers as were used in the RT-PCR amplifications was conducted as a positive control. Genomic DNA template was amplified with both the ORF 501 primers and the cy clophilin primers described above.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 195 of 375 Appendix B

Characterization of AtAHAS Protein Prod uced in Imidazolinone-tolerant CV127 Soybean and Comparison with AtAHAS Protein Expressed in Recombinant Escherichia

coli

The herbicide tolerance in CV127 soybean is the result of a single amino acid substitution of serine to asparagine at position 653 of the AtAHA S protein. In addition, a second amino acid substitution of an arginine replaced by lysi ne was identified at position 272 of the AtAHAS protein; this amino acid subs titution does not impact the en zymatic function of the AtAHAS

enzyme.

The full-length AtAHAS protein consists of 670 amino acids and includes the A. thaliana native chloroplast transit peptide (CTP) that is predicted to consist of 85 amino acids on the N-terminus (Mazur et al ., 1987). During transport into the ch loroplast, the chloroplast transit peptide is removed to produce the mature AtAHAS enzyme that has a molecular weight of approximately 64,000 and is predicted to consist of 585 amino acids.

As part of the food, feed and environmental safety assessment of CV127 soybean, studies were conducted to confirm that the AtAHAS pr otein is equivalent to other AHAS proteins that are found ubiquitously among plant species a nd have a history of safe use in food and feed products. These safety assessments of the AtAHAS protein included a mouse acute gavage study and a digestive fate study condu cted with purified AtAHAS protein. The AtAHAS protein is expre ssed at extremely low levels in tissues of CV127 soybean plants. For example, the AHAS protein is present in soybean grain at levels less than 1.5 x 10 -6 % on a dry-weight basis (Schwerz, 2007 and 2008). Therefore, it was not technically feasible to extract sufficient quantities of the AtAHAS pr otein from soybean tissues for the safety assessment studies of the protein. The sa me AtAHAS coding sequence introduced in the CV127 soybean genome was introduced into Escherichia coli for over-expre ssion of the AtAHAS protein. The At AHAS protein, as encoded in this expression system, lacks the predicted 85-amino acid N-terminal CT P sequence that targets the protein in planta to the chloroplast. The microbially-produced At AHAS protein contained the same S653N and

R272K amino acid substitutions as f ound in the plant-produced protein.

Microbial fermentation provided sufficient st arting material for purification of gram quantities of the AtAHAS protei n required for the safety asse ssment studies. The purpose of the following study was to characterize the AtAHAS protein produced in the transgenic imidazolinone-tolerant CV127 soybean to ev aluate key biochemical and functional parameters and to demonstrate that it is subs tantially similar to the microbially-produced AtAHAS protein which has been used in various safety assessment studies.

The AHAS characterized in this study was obtained from extrac ts of young leaves of fieldgrown CV127 plants. In some studies, immuno-purified AHA S was used, and in others, ammonium sulfate precipitated AHAS-enriched extr act was utilized. These materials served as the test substances. As a control, the endogenous soybean AHAS from an ammonium sulfate precipitated AHAS-enriched extract of l eaves from the isoline control soybean was

included in some of the studies. The AtAHAS protein purified from the Escherichia coli over-expression system was used as a reference substance. This prot ein is identified as AtAHAS-0107. Soybean-expressed AH AS protein was evaluated for its molecular weight, immunoreactivity, enzymatic activity, sensitivit y to branched-chain amino acid feedbackCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 196 of 375 inhibition and imidazolinone herb icide inhibition. In additi on, the soybean- expressed AHAS protein was evaluated for pos t-translational modificati ons and amino acid sequence

confirmation.

Western blot analyses using antibodies specifi c for AHAS showed the same molecular weight of approximately 64,000 and immunoreactivity for both the plant-produced and the microbially-produced proteins.

The functionality of the AtAHAS protein produ ced in CV127 and the microbially-produced protein was demonstrated by monitoring the enzy matic activity, its lack of sensitivity to inhibition by imidazolinone herbicide, and f eedback inhibition by branched-chain amino acids. While both the plant-produced and micr obially-produced proteins demonstrated the expected enzymatic activity with the substrat e pyruvate, the specific activity of the AHAS protein in CV127 was found to be higher compared to the microbially-p roduced protein, most likely a result of enzyme activity losses incurred during extensive purification of the microbially-produced protein. Both CV 127-produced AHAS and microbially-produced AtAHAS proteins showed tolerance to inhibi tion by the imidazolinone herbicide imazethapyr compared to the control plant-produced protein. Sensitivity to feedback inhibition by

branched-chain amino acids was retained in th e plant extracts but not in the microbiallyproduced material. Acetohydroxyacid synthase in Escherichia coli is composed of two subunits. However, previous studies have demonstrated that when the A. thaliana acetohydroxyacid synthase catalytic (large ) subunit (AtAHAS) is expressed in E. coli , the E.

coli small (regulatory) subunit is incapable of interacting with the Arabidopsis thaliana catalytic or “large” subunit to confer the feedback inhibition observed in Arabidopsis

thaliana seedlings (Singh et al ., 1992; Chang and Duggleby, 1997).

No evidence of glycosylation associated with either the plant- or microbially-produced AtAHAS was detected. Mass spectral analys is of the immuno-purified AHAS from young leaves of CV127 plants confirmed the expected amino acid sequence of the protein for 23% of the entire amino sequence of the protein.

This study confirmed that the AHAS protein produced in CV127 plants had the expected size, immunoreactivity, and f unctionality and was substan tially similar to the AtAHAS produced in a microbial over-exp ression system for use in safety assessment studies. Therefore, this study demonstrates the f unctional and chemical equivalence of the microbially-produced AtAHAS and the plant- produced AHAS proteins (AtAHAS and the endogenous soybean AHAS proteins) and therefore justifies the use of the microbialproduced protein for safety assessment studies of the AtAHAS protein produced in CV127 soybean.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 197 of 375 Preparation of test substance . AHAS expressed in CV127 used in these experiments was either from an ammonium sulfate precipi tated AHAS-enriched fr action or it was immunopurified from young leaves (collected from the F 6 generation of CV127 line 127, grown in regulatory field trials in Sant o Antônio de Goiás, GO, Brazil in 2007). Crude extracts were prepared by homogenizing frozen powdered leav es (1:3 w:v) in ex traction buffer [50 mM potassium phosphate, pH 7.2, 100 mM sodium pyruvate, 5 mM MgCl 2 , with HALTTM protease inhibitor cock tail (Pierce Biotechnology, Inc., Ro ckford, IL)] using a PolytronTM (Brinkmann, Westbury, NY). Extracts were filte red through miracloth and subjected to centrifugation for 15 min at appr ox. 10,000 x g. The resulting supe rnatant was considered to

be the crude extract. The ammonium sulf ate precipitated AHAS-enriched fraction was obtained by adding an equal volume of saturated ammonium sulfate to th e crude extract with stirring on ice for 30 min, centrifugation for 15 min at 10,000 x g, and resuspension of the pellet with extraction buffer. Further purif ication was achieved by using goat anti-AtAHAS antibody (which had been purified using Prot ein G affinity chromatography followed by chromatography on an AtAHAS affinity column).

Preparation of the control substance . Endogenous AHAS from the isoline control soybean used in these experiments was from an am monium sulfate precipitated AHAS-enriched fraction prepared from young leaves [collect ed from the isoline control soybean (F 6 null) corresponding to the F 6 generation of CV127 line 127, and grow n in regulatory field trials in Santo Antônio de Goiás, GO, Brazil in 2007], as described above for the test substance.

Table B-1. Reference Substance Description

.

Identification Lot Number Gene

Source Expression System Physical Description Acetohydroxyacid Synthase R272K,

S653N 3 AtAHAS-0107 Arabidopsis

thaliana Escherichia coli

strain BL21(DE3)pLysS Light yellow proteinaceous powder

AtAHAS was purified by Invitrogen, Inc. and sent as an ammoni um sulfate pellet to the Regulatory Science Laboratory at BASF Plant Sc ience, Research Triangle Park, NC, where it was received on February 27, 2007. AtAHAS protein was purified from 2,400 g cell paste after lysis in 20 L buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM β -mercaptoethanol, 30 mM imidazole) using an Em ulsiflex cell disruptor (Avestin, Ontario, Canada). Insoluble material was remove d by centrifugation. The His-tagged AtAHAS present in the supernatant was purified by chromatography using Talon ® cobalt resin (Clontech, Inc., Mountain View, CA). Once el uted, the fractions containing AtAHAS were pooled and immediately precipitated by ammonium sulfate at 40% saturation. The ammonium sulfate pellet was resuspended in 100 mM Tris-HCl, pH 7.0 and dialyzed vs. 20

3 The AtAHASL gene was cloned into the inducible, over-expression pTrcHis A® vector (Invitrogen, Madison, WI) and expressed in E. coli strain BL21(DE3)pLysS. AtAHAS protein as encoded by this vector lacks the 85- amino acid N-terminal leader sequ ence that targets the protein in planta to the chloroplast. This leader has been replaced in this vector with 38 amino acids including a 6x His tag to allow for ease in purification, a gene 10 leader sequence to enhance solubility and an XpressTM tag for detection. The remainder of the protein is identical in amino acid sequence to that produced by the native gene from Arabidopsis thaliana (Mazur et al

., 1987) except for the mutation at amino acid 653 that results in asparagine replacing serine and a mutation at amino acid 272 that results in lysine replacing arginine. The S653N mutation renders a decreased binding of the herbicide imparting the tolerance. The R272K mutation was found in the AtAHAS protein produced in CV127 soybean but has no apparent effect on activity or sensitivity to imidazolinone herbicide.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 198 of 375 mM ammonium bicarbonate buffer, pH 7.9, containing 20 μ M FAD. The dialyzed material was then lyophilized and designated AtAHAS-0107. This reference substance was characterized (Privalle, 2007a), and resu lts are summarized in Table B-2.

Table B-2. Characterizati on of Sample AtAHAS-0107

AtAHAS Concentration Specific Activity

Date of Analysis AtAHAS (g/g sample) % AtAHAS by weight AtAHAS as % total protein Mean units/mg protein

± standard deviation April 12, 2007 0.524 52.4 90.6 0.790 ± 0.216

Protein quantification

. Protein in the AtAHAS referen ce substance and a mmonium sulfatetreated test and control AHAS-enriched fractio ns from plant tissues was quantified by the BCATM procedure (bicinchonin ic acid procedure; Pierce) in accordance with the manufacturer’s instructions, us ing bovine serum albumin as the standard (SOP BPS 510.04). Samples were prepared such that the expected concentration of protein would be within the standard curve. Samples (25 μ l) were loaded onto a multiwell pl ate in triplicate, reacted with

200 μ l of a 1:50 (B:A) mixture of BCA reagent, incubated at 37o C for 30 min and allowed to cool at room temperature for 10 min. Th e absorbance at 550 nm was measured using a Multiskan Ascent V1.24 multiwell pl ate reader (Therma Labsystems , Helsinki, Finland). The results were analyzed using DeltaSoft PC software (Ver sion 1.71.4, Biometallics, Inc.; Princeton, NJ) using the linea r regression curve fit.

AHAS quantification

. Samples were quantitatively analyzed for AHAS protein by a sandwich enzyme-linked immunosorben t assay (ELISA) [Tijssen, 1985] using immunoaffinity-purified polyclonal rabbit an ti-AtAHAS peptide 2 antibody and Protein Gpurified goat antibodies specific for AHAS. Nunc 96-well plates (VWR; West Chester, PA) were coated with rabbit anti-peptide 2 and inc ubated at 4oC overnight. The plate was washed

two times with wash buffer and then blocked with 1% BSA in Tris- buffered saline (25 mM Tris-HCl, 3 mM KCl, 0.14 M NaCl) with 0.05% Tween for 2 hours at 37o C. After washing twice, samples and standards were applied in triplicate. Plates were incubated at 4oC for 1.5 hr and 45 min at room temperature, and then wa shed five times prior to the addition of the goat anti-AtAHAS followed by incubation for 1 hr at 37oC. Plates were then washed three times and donkey anti-goat-horseradish peroxida se (HRP) was added. After incubation at 37oC for 1 hr, the plates were washed three tim es and HRP substrate was added (1 Step Ultra TMB; Pierce). After 20 min at room temper ature the absorbance at 620 nm was measured using a Multiskan Ascent V1.24 multiwell plate reader

. The results were analyzed using DeltaSoft PC software (Version 1.71.4; Biomet allics, Inc.; Princeton, NJ). The fourparameters algorithm was used to generate a curve. The A HAS component of the samples was quantified from the standard concentr ation curve generate d from highly purified AtAHAS protein.

Molecular weight and immunoreactivity determination

. To confirm that the AtAHAS produced in CV127 soybean plants had the predicted molecular weight of AtAHAS ( ca.64,000), aliquots of the sample preparations were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-P AGE, SOP BPS 510.02), using an 8 – 16% polyacrylamide gradient Tris-glycine gel (I nvitrogen) followed by el ectroblotting onto PVDF membrane (Invitrogen; SOP BPS 510.03). Samples of the ammonium sulfate AHAS-CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 199 of 375 enriched preparations and At AHAS-0107 preparations were lo aded onto the gel. After electroblotting the membrane was probed with rabbit anti-AtAHAS peptide 2 polyclonal antibody. Donkey anti-rabbit IgG linked to horse radish peroxidase (Pierce), diluted 1:3,000 in blocking buffer (3% nonfat dry milk in 0.1% Tween-20, 10 mM Tris -HCl, 150 mM NaCl, pH 7.5), was used to bind to the primary anti body and was visualized by development with ECL Plus Western Blotting Detection R eagents (Amersham, GE Healthcare).

Western blot analysis, using rabbit anti-AtAHAS peptide 2 poly clonal antibodies, of extracts prepared from both CV127 and the isoline c ontrol soybean contained immuno-reactive bands at a molecular weight of a pproximately 64,000 (Figure B-1). This was the same apparent molecular weight as the microbially-produ ced AtAHAS reference s ubstance, AtAHAS-0107, indicating that the AHAS produced in the CV 127 and isoline control soybeans was of the same anticipated size and of similar immunore activity to the microbia lly-produced protein. Both plant extracts included a smaller immunoreactive band at approximately 47,000 molecular weight, which is most likely a degrad ation product of the matu re AtAHAS protein. The molecular weights of the microbially -produced AtAHAS and plant-produced AHAS proteins were also shown to be similar usi ng SDS-PAGE and Coomassi e blue staining. The approximate molecular weights of the imm uno-purified AHAS protein from CV127 soybean plants and the AtAHAS in the AtAHAS-0107 test substance were shown to be approximately 64,000 (Figure B-5).

Based on a sequence comparison of the deduced amino acid sequences of AHAS proteins from several different plant and microbial sources, Mazur et al . (1987) predicted that the CTP

of the AtAHAS is cleaved at the C-terminal side of residue 85 to produce a mature AtAHAS enzyme that consists of 585 amino acids. In the current study, th e microbially-produced AtAHAS protein consisted of the predicted mature AtAHAS (585 amino acids) plus an Nterminal addition of 38 amino aci d to facilitate protein purification, folding and solubility. Therefore, it was expected that the molecula r weight of the microbi ally-produced protein would be slightly greater than that of the AHAS proteins produced in either CV127 soybean

or the isoline control soybean. Because the apparent mole cular weights of the plant-produced AHAS and microbially-produced At AHAS proteins were equivalent, results of these studies suggest that the mature AtAHAS protein produced in plant tissues contains more than the predicted 585 amino acid residue s of the mature AtAHAS protein. These additional amino acids are most likely derived from the defined CTP component at the N-terminal end of the protein due to a different cleavage site in soybean s compared to the predicted cleavage site in Arabidopsis. This conclusion is supported by da ta presented in the la ter section of Appendix B entitled “Amino acid sequence analysis”.

Enzymatic activity

. The enzymatic activity of AHAS was assayed according to Singh et al

. (1988) and SOP BPS 510.09. AHAS catalyzes th e synthesis of acetolactate (an acetohydroxy acid) by the condensati on of two molecules of pyruvate. The acetolactate produced by AHAS in this assay is converted to acetoin in the presence of acid an d acetoin is detected colorimetrically (A 530 nm ) after interaction with creatine and naphthol. One unit of AHAS activity is defined as 1 μ mole acetoin produced per minute. The ammonium sulfate AHASenriched preparations of CV127 and the is oline control soybean as well as AtAHAS-0107, dissolved in 50 mM Tris-HCl, pH

∼ 7.0 and diluted to the desire d concentration, were mixed with an equal volume of 2x assay buffer [ 100 mM potassium phosphate, pH 7.2, 10 mM MgCl 2 , 200 mM sodium pyruvate, with 20 μ M FAD and 2 mM thiamine pyrophosphate (TPP)] and incubated at 37oC for 90 mi nutes prior to the addition of 20 μ l 5% H 2 SO 4 followed by a 15 min incubation at 60oC. Background absorbance was determined byCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 200 of 375 immediately quenching the reac tions prior to incubation at 37oC. Acetoin color was developed by incubating the quenched reactions with creatine (0.17%, final concentration) and 1-naphthol (1.7% in 4 N NaOH, fi nal concentration) for 15 min at 60 ° C and the

absorbance was measured at 550 nm. Absorb ance values were compared to an acetoin

standard curve and corrected for background absorbance. Seve ral dilutions of a single dissolved sample of AtAHAS were prepared an d each dilution was assayed in triplicate; the mean corrected value is reported.

Feedback inhibition of AHAS enzymatic activ ity by leucine and valine was confirmed by including leucine and valine in the assay mix. Leucine and valin e solutions were prepared in 2x assay buffer at twice the desired fina l assay concentration (1 mM). Fifty μ l of 20-fold diluted AtAHAS-0107, the ammonium sulfate AHAS- enriched preparation of CV127 and the

ammonium sulfate AHAS-enriched preparation of isoline cont rol soybean were incubated

with 50 μ l of 2x assay buffer or leucine and valine solution for 120 min at 37 ° C and AHAS enzymatic assays were performed in triplicate.

AHAS enzymatic activity sensitivity to inhibition by an imidazolinone herbicide was examined using a stock solution (50 mM) of imazethapyr, a commercial imidazolinone herbicide, diluted in 2x AHAS assay buffer to tw o times the desired final assay concentration,

where the final concentration of he rbicide ranged from 0 to approx. 500 μ M. Fifty μ l of the sample was incubated with 50 μ l of 2x assay buffer or imazethapyr solution for 90 min at

37 ° C. The absorbance was measured at 550 nm. Background samples were generated by pre-quenching with acid prior to assay incubation and the abso rbances thus generated were subtracted from the test samples. All assays were conducted in triplicate and results are presented as mean values. The amount of enzymatic activity obtained for each sample in the

absence of inhibitor was assumed to be 100%.

The microbially-produced AtAHAS and the plan t-produced AHAS proteins demonstrated the expected enzymatic activity with the substr ate pyruvate, confirming the same functional activity of the AHAS proteins from both microbi al and plant sources. However, the specific activity of ammonium su lfate AHAS-enriched plant extracts wa s considerably higher than the highly purified microbially-produc ed AtAHAS protein (Table B-3) . AHAS is a highly labile protein of low abundance in plants and has been demonstrated to be extremely difficult to purify from plant preparations (Durner and Boger, 1988; Muhitch et al ., 1987; Singh et al.

, 1991; Chang and Duggleby, 1997). In these leaf preparations, AHAS wa s present at very similar levels of 0.516 ng/ml and 0.384 ng/ml in the CV127 and the isoline control soybean extracts, respectively. This corresponds to AHAS representing only 0.06 ng/mg total protein or 0.000006% of the total protein. The AHAS protein is stable in ammonium sulfate fractions but further purification results in greatly reduced enzymatic activity. The microbially-produced AtAHAS was purified exte nsively for the protein safety studies, whereas the plant AHAS used in these enzyma tic studies was derived from an ammonium sulfate purification step. Hence, these differences in the level of purification accounted for the difference in specific activity between the microbially-produced material, AtAHAS-0107,

and the ammonium sulfate AHAS-enriched leaf ex tracts (Table B-3). Activity was very similar between CV127 soybean and the iso line control soybean, 0.33 and 0.30 Units AHAS activity/mg protein, respectively.

Both CV127-produced and microbially-produced AtAHAS proteins showed tolerance to inhibition by the imidazolinone herbicide imazetha pyr compared to the isoline control plantproduced protein (Figure B-2). An intermedia te level of inhibition of the CV127-producedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 201 of 375 AtAHAS protein was observed, an d this was as expected due to the mixture of sensitive (endogenous) and tolerant (transgenic) AHAS pr esent in CV127 soybean. In general, the degree of herbicide inhibition observed for the AHAS proteins in the test (i.e. CV127) and isoline control soybeans was lowe r than anticipated but may refl ect the tolerance that soybean naturally has to this particular im idazolinone herbicide, imazethapyr.

Feedback inhibition by the bran ched-chain amino acids leucine and valine was very similar for both the CV127 soybean AHAS and the isolin e control soybean AHAS (Figure B-3). No

feedback inhibition was observed for the At AHAS present in the microbially-produced reference substance as expecte d. Acetohydroxyacid synthase in Escherichia coli is composed of two subunits. However, previous studi es have demonstrated that when the A. thaliana acetohydroxyacid synthase catalytic (large ) subunit (AtAHAS) is expressed in E. coli , the E.

coli small (regulatory) subunit is incapable of interacting with the Arabidopsis thaliana catalytic or “large” subunit to confer the feedback inhibition observed in Arabidopsis

thaliana seedlings (Singh et al ., 1992; Chang and Duggleby, 1997).

Glycosylation analysis. The DIG Glycan Detection kit (Roche Diagnostics GmbH, Mannheim, Germany) was used in accordance w ith the manufacturer’s instructions (method B, described below) to monitor for glycosyl ation associated with CV127 soybean-expressed AtAHAS following the SDS-PAGE/electroblotting-to-membrane procedure. This method takes advantage of the adjacent hydroxyl residues in sugars of glyco-conjugates by oxidizing them to aldehyde groups by mild periodate tr eatment. The spaced-linked steroid hapten digoxigenin (DIG) is then covalently attach ed to these aldehydes via a hydrazide group. Digoxigenin-labeled glycoconjugates are then subsequently detect ed by western blot analysis using a digoxigenin-specific an tibody conjugated with alkaline phosphatase. Samples were

run on SDS-PAGE and transferred to PVDF membrane. After washing with phosphatebuffered saline, PBS (50 mM potassium phosphate , pH 6.5, 150 mM NaCl), the membrane is

treated with 10 mM sodium meta periodate (in sodium acetate buffer, pH 5.5) for 20 minutes at room temperature (oxidation). The membrane is then washed three times with PBS and incubated with DIG-3-O-succinyl- ε -aminocaproic acid hydrazide for 1 hour at room temperature to label the glycoproteins. The membrane was washed three times with TBST (25 mM Tris, 3 mM KCl, 0.14 M NaCl, 0.05% Tween -20, pH 7.4) and blocked for at least 30 minutes, followed by incubation with anti-digoxigenin-AP for 1 hour. The chromagenic

reaction with nitroblue tetrazolium was used to monitor the alkaline phosphatase bound to the membrane. Transferrin and creatinase were used as the positive and the negative controls, respectively. All incubations were conducted at room temperature w ith gentle agitation except for color development (room temperature, no agitation). The limit of detection for this method was determined to be 2 – 4 molecules of glucose equivalents per molecule of

AtAHAS under these conditions.

No evidence of glycosylation was detected for either the CV127-produced or microbiallyproduced AtAHAS protein (Figure B-4). As shown in Figure B-5, there are only four Nglycosylation site motifs [NX(S/T)X, where X ca n be any amino acid except for proline] in the full-length AtAHAS polypeptide. At least one N-glycosylation site motif is most likely present in the chloroplast transit sequence and w ould not be present in the mature protein. There are, however, numerous potential O-glycosyl ation sites (any serine or threonine). It was not anticipated that AHAS would be glycosylated as it does not enter the secretory pathway, a prerequisite of glycos ylation, but rather it is des tined to be lo calized in the chloroplast where proteins are not typically glycosylated. Likewise, the AtAHAS produced in the microbial expression system should not be glycosylated as gl ycosylation does notCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 202 of 375 occur in prokaryotes. The results shown here de monstrate that no glyc osylation was detected above the limit of detection (2 – 4 molecules of glucose equivalents/molecule of AtAHAS).

Amino acid sequence analysis

. To further confirm that th e AtAHAS protein produced in CV127 soybean was the protein expected, am ino acid sequence was determined. The attempts to obtain N-terminal amino acid se quence using Edman degradation methods were unsuccessful. Therefore, a combination of SDS-PAGE and liquid chromatography coupled with tandem mass spectroscopy (LC/MS/MS) was used to obtain amino acid sequence data. Immunoaffinity-purified AtAHAS from CV127 soybean young leaves was subjected to SDSPAGE and stained with Coomassie blue. The ba nds were excised and transferred into 0.5 ml Eppendorf cups. The gel pieces we re washed consecutively with 100 μ l of acetonitrile/ water (1:1, v:v), acetonitrile and acetoni trile/water (1:1). For each of the wash steps, the samples were shaken at 800 rpm for 5 min at 30°C, subj ected to 1 min of sonication in an ultrasonic bath, centrifuged at 13,000 x g and the respective supernatants were discarded. Following

addition of 10 μ l of 100 mM ammonium bicarbonate, the gel pieces were shaken for 5

minutes at 30°C. Then 50 μ l of the trypsin solution [0.76 μ g trypsin, (Roche Mannheim),

0.12% CaCl 2 , 8% acetonitrile, 100 mM ammonium bi carbonate] were added and the samples

were incubated at 37°C for 16 hours with shak ing (300 rpm). After stopping the reaction by

addition of 90 μ l stop solution (75% acetonitrile, 0.2% trifluoroacetic acid), shaking was continued for 1 hour followed by centrifugation for 1 min at 13,000 rpm. The supernatants

were transferred to a fresh tube and 10 μ l acetonitrile were added, shaken for 2 min and centrifuged as before. The wash was repeated a second time and the supernatants combined. The combined supernatants containing the peptid es were concentrated in a Speed Vac until dry and used for analysis.

The peptide fragments were then subjected to nanoHPLC/ESI-MS/MS in the MS-laboratory of BASF Central Research, Ludwigshafen, Germ any. MS-analysis of the trypsin-digested protein samples was carried out on a quadru pole ion-trap mass-spectrometer (LCQ, Fa. Thermofinnigan) with a gold-platinated spray capillary (Fa. New Objektive, Inc.). The spectrometer is directly linked to a μHPLC uni t (Fa. SunChrom GmbH), allowing separation of the peptide mix prior to the MS-analysis.

The peptides were separated on the μHPLC un it using a reverse-phase column (Fa. LCPackings) with 75 μm i.d. and a length of 15 cm. The column was packed with PepMapTM

C18 [ 4] material of 100 Å pore diameter and 3 μm part icle size. A gradient of 5 – 50% solvent A and B (A: 95% H 2 O, 5% ACN, 0.08% HCOOH ; B: 95% ACN, 5% H 2 O, 0.08% HCOOH) in 60 min was selected for sepa ration of the trypsin-digested peptides. The 10 μl/min flow from an Eldex HPLC pump (Fa. SunChrome) wa s reduced using a split-system to achieve a

column flow-rate of 180 nl/min.

The separated peptide mix was directly eluted into the mass spectrometer. MS-experiments

were run in an alte rnating sequence of " full MS-scan // MS/MS

- scan" . The full scan experiments provided an overview of the ions present at the respective elution time. In this study, these were mostly intact mono-, di-, and tri-protonated peptide ions. The most

abundant ions of the full scan were then se lected for MS/MS-analyses. In an MS/MSexperiment a specific ion is sele cted (by its m/z value) and subj ected to collision with helium atoms. This collision brings about a collision-induced dissociation (CID) of the selected peptide ion into fragment ions. In the chosen experimental set-up, diss ociation occurs mainly

[4] C18 material, i.e. covalently-bonded octadecylsilane, is used as bonded phase in the column.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 203 of 375 at the peptide bonds, with an idea lly statistical distri bution. Two series of ions are formed: b-ions, where the charge is retained on the N-terminal fragment, a nd y-ions, carrying the charge on the C-terminus of the fragmented peptide. The m/z-difference between two adjacent signals of one ion series corresponds to one amino acid residue. In theory, the MS/MS-spectra feature the full amino acid sequen ces of the analyzed proteolytic peptides. But usually not every expected signal is retrieved, so that instead of full-length sequences, sequence tags are obtained. A sequence tag is a data combination, consisting of the m/zvalue of the entire peptide, and a partial, ac curately localized amino acid sequence of the peptide. Depending on the length/sequence coverage of the tag, a sequence tag of a proteolytic peptide can be highl y specific for the originating pr otein. In summary, the MSexperiments provide the molecular masses of th e proteolytic peptides and either full-length sequences or specific sequence tags.

In order to identify the origina ting protein of the peptide mix, the MS/MS-data were analyzed using the MASCOT MS/MS-ions search. In short, MASCOT compares the experimental MS/MS-data against a comprehensive primary sequence database. For this comparison, the entries of the sequence database are subjected to ion cleavage rules, thus yielding calculated fragment-ion mass-values. The quality of the obtained matches is evaluated by an appropriate scoring algorithm. Depending on the contents of the database, the originating protein or a protein w ith close homology can be identified. The MASCOT search was carried out without specifying a diges ting enzyme. All MASCOT re sults were checked manually against the experimental data to assure appropriate evaluation. In addition to the automated

database search with MASCOT , amino-acid sequences were retrieved by hand from the MS/MS-spectra as far as possible.

Amino acid sequence data was obtained fo r approximately 23% of the AHAS protein produced in CV127 soybean, and approximately 73% of the microbially-produced AtAHAS. The results (Figure B-5) confirm that th e major protein band at approximately 64,000 molecular weight in CV127 soybean is AtAHA S since the amino acid sequences obtained from peptide fragments derived from it were id entical to regions of the deduced amino acid sequence from the coding sequence of the csr1-2 gene in the CV127 soybean genome. Similarly, sequences obtained from peptide frag ments derived from the microbially-produced AtAHAS were identical to regions of the de duced amino acid sequence from the coding sequence of the csr1-2 gene in the Escherichia coli over-expression system (data not presented). Also, where AtAHAS peptide frag ments overlapped between the plant-produced and microbially-produced sources, the ami no acid sequences were the same (data not presented). Since the same DNA coding se quence for the AtAHAS protein was introduced into the genome of CV127 soybean and into the Escherichia coli over-expression system, these data confirm that the amino acid sequenc es of peptides produced from the predicted

mature AtAHAS from the Escherichia coli over-expression system and from CV127 soybean are the same and correspond to the predicted amino sequen ce of AtAHAS with the S653N

and R272K amino acid substitutions.

Two of the four potential N-glycosylation site s were included in the amino acid sequence obtained, providing confirmatory support for the l ack of glycosylation at these sites. If glycosylation had been present, fragments of different masses would have been obtained.

The peptide fragment corresponding to residues 52 to 73 was not expected to be present in

the m ature AtAHAS protein pr oduced in CV127 soybean. The csr1-2 gene from Arabidopsis

thaliana encodes a single polype ptide of 670 amino ac ids that includes a chloroplast transitCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 204 of 375 peptide (CTP) on the N-terminus that has been hypothesized, but not confirmed, to consist of 85 amino acids (Mazur et al ., 1987; Chang and Duggleby, 1997). This hypothesized CTP was deduced by comparing the amino acid seque nces of known AHAS proteins and looking

for the first conserved residue (i.e. threonine 86). An id entical CTP cleavage site was experimentally determined for the AHAS enzyme in maize (B. K. Singh, personal communication). During transport into the chlo roplast, the chloroplast transit peptide is removed to produce the mature AtAHAS enzyme . For this reason, the peptide fragment corresponding to residues 52 to 73 was not exp ected to be present in CV127 soybean. The microbially-produced AtAHAS protein was designe d to represent the ma ture form of the AtAHAS protein and lacks the 85-amino acid N-terminal sequence corresponding to the predicted chloroplast transit peptide sequenc e. The microbial pr otein does contain an additional 38 amino acids (see footnote on page 197) to aid in bacterial expression and purification. The molecula r weights of the CV127-produced and microbially-produced proteins were shown to be similar and, based on this information, represent 623 amino acids

in the Escherichia coli -produced AtAHAS and potentially 619 amino acids in CV127- produced AtAHAS. Furthermore, the N-term inal amino acid for one of the endogenous soybean AHAS proteins (AHAS1) has been shown to be the serine 48; thus, the chloroplast transit peptide for that protein is appa rently only 47 amino acids in length (Rood et al ., 2006).

Conclusions. Key biochemical and functional paramete rs were evaluated to demonstrate that the AtAHAS protein produced in CV127 soybean is substantially simila r to the microbiallyproduced AtAHAS protein which has been used in various safety assessment studies. The molecular weight and immunoreactivity of the AHAS produced in CV127 soybean was

shown to be similar to that of the endogenous AHAS from the isoline control soybean and the reference AtAHAS produced in a microbial over- expression system. The specific activity of the AHAS produced in CV127 soybean was very similar to the endogenous soybean AHAS and was higher than the microbially-produced At AHAS. This was attributed to AHAS being a very unstable protein that loses activity readily upon purification (Chang and Duggleby, 1997). Therefore, it is not unexpected that the AHAS specific activity in an impure state such

as in the ammonium sulfate AHAS-enriched fraction is higher than that retained in the much more highly purified microbially-produced mate rial. Both CV127-produced and microbiallyproduced AtAHAS proteins showed tolerance to inhibition by the imidazolinone herbicide imazethapyr compared to the control plant-produced protein. Sensitivity to feedback inhibition by branched-chain amino acids was re tained in the plant extracts but not in the microbially-produced material as ex pected. Acetohydroxyacid synthase in Escherichia coli is composed of two subunits. However, previ ous studies have demonstrated that when the A.

thaliana acetohydroxyacid synthase catal ytic (large) subunit (AtAHAS) is expressed in E.

coli , the E. coli small (regulatory) subunit is incap able of interacting with the Arabidopsis

thaliana catalytic or “large” subunit to confer the feedback inhibition observed in Arabidopsis thaliana seedlings (Singh et al ., 1992; Chang and Duggleby, 1997). No evidence of glycosylation was found associat ed with the AtAHAS in CV127 leaves or the microbially-produced AtAHAS.

To com pare the amino acid sequence of th e AtAHAS protein produced in CV127 soybean with that produced in the E. coli expression system, the amin o acid sequence of the AtAHAS protein of CV127 soybean was investigated. Attempts to obtain N-terminal amino acid sequence using Edman degradation methods were unsuccessful. This was most likely due to the low abundance of the AtAHAS protein in plan t tissues, coupled with the repetitive amino acid content of the putative CTP. The high conten t of serine, threonine and proline within the first 85 amino acids of the full-length AtAHAS (the putative CTP segment of the protein)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 205 of 375 contributes to the difficultie s in obtaining unambiguous resu lts. Serines and threonines negatively impact the yield of amino acid sequ ence and have high carryover into subsequent chromatograms, leading to ambiguous interpreta tion of results of the amino acid sequence analysis. Proline is not efficiently cleav ed (only 50 – 60%) by the Edman degradation chemistry. This also contributes to the high carryover into subsequent chromatograms. These three amino acids represent 60% (51/85) of the putative CTP. Therefore, the resulting data gave the appearance of N-termin al blockage of the AtAHAS protein.

Because the above approach was not successful in determining the N-terminus of the mature AtAHAS protein, a combination of SDS-PAGE and liquid chromatography coupled with tandem mass spectroscopy (LC/MS/MS) was perf ormed. Peptides were derived from CV127 soybean and compared to the deduced amino acid sequence of the AtAHAS gene in the E.

coli expression vector. The ami no acid sequence obtained covered approximately 23% of the entire AtAHAS amino acid sequence. All amino acid sequence data obtained from the AtAHAS of CV127 soybeans was identical to the corresponding amino acid sequence of the

E. coli -produced AtAHAS with the exception of a region near the N-terminus that is predicted to contain the chloroplast transit pe ptide. Based on a sequence comparison of the deduced amino acid sequence of plan t and microbial AHAS proteins, Mazur et al. (1987) predicted that the chloro plast transit peptide of AtAHAS is cl eaved at the C-terminal side of residue 85. Amino acid sequence analysis of the AtAHAS protei n produced in CV127 soybean showed a peptide fragment corres ponding to amino acid residues 52 to 73. Therefore, the CTP cleavage site of the AtAHA S protein produced in CV127 soybean is most likely not at the pred icted site at the C-terminal side of residue 85, but is located at least at the

C-terminal side of residue 51. This conclusi on was further confirmed because the apparent molecular weights of the CV127-produced At AHAS protein and the microbially-produced AtAHAS protein were equivale nt, yet the microbially-produc ed protein included an

additional 38 amino acids attached to the 585 amino acids of the predicted mature AtAHAS protein.

However, even though the data in this st udy suggest that the mature AtAHAS protein produced in CV127 soybean consists of the predicted mature prot ein (585 amino acids) plus approximately 34 additional amino acids from the CTP as predicted by Mazur et al . (1987), the microbially-produced AtAHAS protein is cons idered equivalent to the CV127 protein for the following reasons. The microbially-pr oduced AtAHAS protein wa s functionally active and had the expected enzymatic activity with the substrate pyruvate. Furthermore, the microbially-produced protein lacked sensitivit y to inhibition by the imidazolinone herbicide imazethapyr, similar to the lack of sensitivity of the CV127 protein to the same herbicide. In addition, the microbially-pr oduced AtAHAS protein and CV 127-produced AHAS proteins had similar immunoreactivity to antibodies specific to the AHAS protein based on results of both western blot and ELISA analyses. Both the microbially-produced AtAHAS protein and the CV127-produced protein showed no evidence of glycosylation. Fina lly, both proteins had the same amino acid sequence of the predicte d mature protein of 585 amino acids, based on amino acid sequence analysis of overlapping peptide fragments.

Results of these studies justify the use of the microbially-produced AtAHAS protein as an appropriate substitute for the AtAHAS produ ced in CV127 soybean in safety assessment studies of the AtAHAS protein.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 206 of 375 Standard Operating Procedures

BPS 510.02 SDS-Polyacrylamide Gel Electrophoresis

BPS 510.03 Western Blot Analysis

BPS 510.04 Protein Determina tion Using the BCA Procedure

BPS 510.09 Enzymatic Assay for Acetohydroxyacid Synthase (AHAS)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 207 of 375 Table B-3. Specific Activity of AHAS in CV127 Soybean, the Isoline Control Soybean,

and the Reference Substance AtAHAS-0107

Source of

AHAS ng AHAS/ml

extract mg protein/

ml

extract AHAS activity

nmole/min/ml

extract AHAS specific activity nmole/min/mg protein AHAS specific activity

Units/ μ g

AHAS

CV127 0.516 8.16 2.697 0.33 5.227

Isoline Control 0.384 8.13 2.435 0.30 6.341

AtAHAS-0107 85411.000 0.32 436.003 1379.43 0.005CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 208 of 375 Figure B-1. Western Blot Analysis of AHAS Pr otein in CV127 Soybean, the Isoline Control Soybean and Reference Substance AtAHAS-0107. The immunoreactivity and integr ity of AHAS from CV127 soybean and the isoline control, as well as in sample AtAHAS-0107, was evaluated by western blot analysis. Lane 1, 0.4 ng
AtAHAS from AtAHAS-0107; lane 2, 76 μ g protein from the ammonium sulfate AHASenriched fraction from CV127 soybean; and lane 3, 81 μ g protein from the ammonium sulfate

AHAS-enriched fraction of the isoline control s oybean. Blots were prob ed with rabbit antiAHAS peptide 2 antibody. The molecular weight of intact AHAS corresponds to ca. 64,000 mol. wt. Molecular weight (x 10 -3 ) markers are indicated.



# 1 2 3



# AHAS 148

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# 1 2 3



# AHAS 148

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 209 of 375 Figure B-2. Sensitivity of the Enzymatic Activity of AHAS from CV127 Soybean, the Isoline Control Soybean and Reference Substa nce AtAHAS-0107 to Imazethapyr, an

Imidazolinone Herbicide

. AHAS enzymatic activity was measured in the presence of increasing concentrations of the imidazolinone herbicide imazethapyr in AHAS ex tracts enriched by ammonium sulfate precipitation, prepared fr om CV127 soybean leaves (- ▲ -) and isoline control soybean leaves (- ■ -), as well as in the reference AHAS micr obially-produced test substance, AtAHAS-0107 (- ▼ -).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 210 of 375 Figure B-3. Sensitivity of the Enzymatic Activi ty of AHAS from CV127 Soybean, Isoline Control Soybean and Reference Substance AtAHAS-0107 to Feedback Inhibition by Leucine and Valine

. AHAS enzymatic activity was measured in the presence of increasing concentrations of the

branched-chain amino acids leucine and va line in AHAS-enriched ammonium sulfate precipitation extracts prepared from CV127 soybean leaves (- ▲ -) and the isoline control soybean leaves (- ■ -), as well as in the reference AHAS microbially-produced test substance, AtAHAS-0107 (- ▼ -).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 211 of 375 Figure B-4. Examination of Glycosylation of AHAS from CV127 Soybean and

Reference Substance AtAHAS-0107. Samples were subjected to SDS-PAGE on a single 8 – 16% polyacrylamide gel and

electroblotted onto a PVDF membrane. The membra ne was divided so that lane 1 and part of the marker lane (M) were developed by wester n blot analysis usi ng goat anti-AtAHAS, Lanes 2 – 4 were developed in the absence of oxidati on, and lanes 5 – 10 with oxidation. Lanes 1 and 5, 25 - 50 ng AHAS from the AHAS-enriched CV127 soybean leaf preparation; lane “M”, SeeBluePlusTM protein markers; lanes 2 and 10, 100 ng transferrin each and lanes 8 and 9, 5 and 50 ng of transferrin (positive control), respectively; lanes 3 and 6, 2 ng AtAHAS-0107; lane 7, 100 ng creatinase (n egative control). The AtAHAS position is indicated by arrows.

148

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## 1 M 2 3 4 M 5 6 7 8 9 10

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## 1 M 2 3 4 M 5 6 7 8 9 10CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 212 of 375 Figure B-5. AtAHAS Amino Acid Sequence

. Panel A . SDS-PAGE with Coomassie blue st aining of immuno-purif ied AtAHAS from CV127 soybean (lane 2, arrow). Lanes 3 through 5, microbially-produced AtAHAS loaded at 25, 50, and 250 ng AHAS, respectively. Molecular weight (x 10 -3 ) markers are indicated.

Panel B . The predicted amino acid sequence of AtAHAS encoded by the gene transformed into soybean resulting in th e event designated CV127. The am ino acid sequences underlined in Panel B were obtained by LC/MS/MS analysis of the protein band indicated by the arrow in Panel A, lane 2. The four potential N-glyc osylation sites [motif NX(S/T)X] are indicated by shading. The two amino acids that are different from wild -type AtAHAS are indicated in bold (K 272 , N 653 )



## A.

B.

1 MAAATTTTTT SSSISFSTKP SPSSSKSPLP ISRFSLPFSL NPNKSSSSSR

51 RRGIKSSSPS SISAVLNTTT NVT TTPSPTK PTKPETFISR FAPDQPRKGA
101 DILVEALERQ GVETVFAYPG GASMEIHQAL TRSSSIRNVL PRHEQGGVFA

151 AEGYAR SSGK PGICIATSGP GATNLVSGLA DALLDSVPLV AITGQVPRRM
201 IGTDAFQETP IVEVTR SITK HNYLVMDVED IPRIIEEAFF LATSGRPGPV
251 LVDVPKDIQQ QLAIPNWEQA M **K** LPGYMSRM PKPPEDSHLE QIVRLISESK

301 KPVLYVGGGC LNSSDELGKF VELTGIPVAS TLMGLGSYPC DDELSLHMLG

351 MHGTVYANYA V EHSDLLLAF GVRFDDRVTG KLEAFASRAK IVHIDIDSAE
401 IGKNKTPHVS VCGDVKLALQ GMNKVLENRA EELKLDFGVW RNELNVQKQK
451 FPLSFKTFGE AIPPQYAIKV LDELTDGKAI ISTGVGQHQM WAAQFYNYKK
501 PRQWLSSGGL GAMGFGLPAA IGASVANPDA IVVDIDGDGS FIMNVQELAT

551 IRVENLPVKV LLLNNQHLGM VMQWEDRFYK ANRAHTFLGD PAQEDEIFPN
601 MLLFAAACGI PAARVTKKAD LREAIQTMLD TPGPYLLDVI CPHQEHVLPM

651 IP **N** GGTFNDV ITEGDGRIKY 148

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### 1 2 3 4 5

AHAS

6 148

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### 1 2 3 4 5

AHAS

6CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 213 of 375 Appendix C

Methods for Determining Levels of the AHAS and SEC61 γ Subunit Proteins in Soybean

Tissues

A. Determining the level of AHAS protein in soybean tissues

To assess the level of production (or expression) of th e AtAHAS enzyme in CV127 soybean that is encoded by the csr1-2 gene, the amount of AHAS protein was determined in tissues of CV127 soybean and the isoline control that were grown in Brazil in two different growing

seasons. Seven field trials in the 2006/2007 growing season and six in the 2007 short growing season were conducted in geographically distinct locations that were representative of commercial soybean production ar eas in Brazil. The field sites were located in or near the

locations listed as follows:

Field Trial Locations for the AHAS Expression Study

Location Trial Season 1

Oct. 2006–Mar. 2007 Trial Season 2 Feb.–July 2007

Sto. Antônio de Posse – SP 

### √

Ponta Grossa – PR/South 

### √

Londrina – PR/North 

### √

Uberaba – MG 

### √ √

Brasília – DF 

### √ √

Sto. Antônio de Goiás – GO 

### √ √

Sete Lagoas-MG 

### √ √

Teresina - PI 

### √

Vilhena - RO 

### √

A detailed description of these field trials is presented in Section VIII.C, and Appendix F.

Tissue sample collection

. Plant tissues were collected from two replicate plots of the CV127 soybean plants treated with the imidazolinone herbicide imazapyr (treatment T1) and from two replicate plots of the isoline control that were treated with the conventional soybean

herbicide Volt ® (bentazon + acifluorfen-sodium at a ra te of 1.0 liters/ha; treatment T3) as described in Section VIII.C and Appendix F. Le af samples were collected at the V2 growth stage at all field trial locations in the 2006/2007 and 2007 growing seasons, as described in Section VI.D. Also, approximately 500 g of grai n was harvested from each plot at all of the

field test locations in the 2006/2007 and 2007 growing seas ons, except for Ponta Grossa in the 2006/2007 growing seas on. In addition, six whol e plants per plot, in cluding roots, were

collected at two of the seven field trial locations in the 2006/2007 growing season (Sto. Antônio de Posse and Londrina) and two sites in the 2007 growing se ason (Brasília and Sto. Antônio de Goiás ) at three different devel opmental stages, including the V2 (plants 15-20

cm tall with three nodes and two un folded leaflets), R2 (plants in full bloom), and R8 (full maturity) stages. Each whole plant was place d in a separate, labele d plastic bag and was shipped on ice within 24 hr to the test facility:CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 214 of 375 BASF S.A. Global Environmental and Consumer Safety Laboratory Avenida Brasil, 791 – Bairro Engenheiro Neiva Guaratinguetá – São Paulo – Brasil

CEP: 12521-1802

Upon receipt at the test facility, three of the six plants were dissected into plant parts, including leaves, stems, roots, and flowers a nd pods, if present. The plant parts from each plant were maintained as a single sample excep t for the flowers at the R2 stage and roots at the V2 stage that were pooled to form a single sample in order to obtain sufficient material for analysis. The other three whole plants from each plot were maintained and processed as a whole plant and formed a single sample. Th e whole plant and leaf tissue samples were shipped on wet ice within 24 hrs to the test faci lity where they were stored at -80±6°C until they were processed.

Sample processing and extraction

. Leaves, grain, whole plants and plant part samples were ground in a mill with dry ice to produce a homogenous, powdered sample. The samples were lyophilized in a freeze drier and stored at -80±6°C until anal ysis. A 0.1 g aliquot (±0.001 g) of each sample was weighed and placed in a 10 mL centrifuge tube. Three mLs of extraction solution (14.8 mM KH 2 PO 4 , 35.2 mM K 2 HPO 4 , 100 mM Na-Pyruvate, 5 mM MgCl 2 ) were added to each sample followed by incubation on ice for 30 min. The tissues were extracted with a tissue homogenizer at 25,000 rpm with several up and down movements of the homogenizer probe over 10 sec. The extracts were centrifuged at 3000 rpm at 5°C for 10 min. The liquid phase was removed and filtered thro ugh miracloth and was centrifuged again at 14,000 rpm at 5°C for 15 min. Th e liquid phase was recovered and stored at 5°C prior to analysis by ELISA.

Sample analysis by ELISA

. The amount of AHAS enzyme in th e different plant tissues was analyzed using an Enzyme-Linked Immunosorbent Assay (ELISA) protoc ol that used a goat anti-AHAS IgG antibody. Due to the high amino acid similarity between the AtAHAS enzyme encoded by the csr1-2 AHAS gene and the homologous endogenous soybean AHAS enzymes, the antibody used in the ELISA is not capable of disti nguishing between these enzymes but rather measures the total AHAS prot ein in the samples. Therefore, expression levels of the AHAS protein in CV127 soybean include levels of the endogenous soybean AHAS proteins as well as levels of the AtAHAS enzyme encoded by the csr1-2 gene. Expression levels of the endogenous AHAS proteins in the isoline control soybean were also determined. The ELISA was carried out in flat-bottom, 96-well microtiter plates and the reaction was quantified using a spectrophotomete r with a microplate reader at 450 nm. A dilution series of purified At AHAS protein was included in every microtiter plate for calibration purposes. The limit of quantification (LOQ) for this assay with soybean tissues is 13 ng AHAS enzyme/g fresh-weight tissue. The limit of detection (LOD) for this assay with soybean tissues is 3 ng AHAS enzyme/g fresh-wei ght tissue. The laboratory portion of this study was conducted under Good Laboratory Practices (GLP).

B. Determining the level of SEC61 γ subunit protein in soybean tissues

Source of plant materials. Leaf tissue was harvested fro m greenhouse-grown plants of CV127 soybean (grown from F 9 generation seed) and the c onventional soybean variety Conquista for microsomal membrane protein is olation. The soybean seeds used to produce the plants from which the leaf material was harvested were used as the grain samples.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 215 of 375 Arabidopsis thaliana leaves and seed were produced in greenhouses in Research Triangle Park, North Carolina, United States.

Test material. The test materials for this study were microsomal membrane protein preparations from leaf ti ssues and grain derived from CV127 soybean. Microsomal membranes include the endoplasmic reticulum and were selected to enrich the SEC61 γ subunit in the sample s to be analyzed.

Control material. The control materials for this st udy were microsomal membrane protein preparations from leaf tissues and grain de rived from the conventional soybean variety Conquista.

Reference material

. The reference materials for this study were microsomal membrane protein preparations from Arabidopsis thaliana leaves and seed

.

AtSEC61 γ

protein standard. The At3g48570 gene, encoding the AtSEC61 γ subunit protein, was cloned into the expression cassette pGEX -6P (GE Healthcare Bio-Sciences Corp, Piscataway, NJ) and transformed into Escherichia coli strain Origami B (DE3)pLysS (EMD Biosciences, San Diego, CA). This expressi on cassette encodes a glut athione S-transferase (GST)–AtSEC61 γ subunit fusion protein (mol. wt. ca . 35,000). The GST-AtSEC61 γ subunit fusion protein was purified from E. coli using an immobilized glutathione column according to the manufacturer’s instructions (GE Health care). On-column digestion using PreScission ProteaseTM (GE Healthcare) was pe rformed to release the AtSEC61 γ subunit protein (mol. wt.

ca . 7000). This was the AtSEC61 γ subunit protein that was used as a standard on the western

blots.

Antibody generation and sensitivity. Rabbit polyclonal antibodie s used in western blot analysis were generated against two synthetic peptides which t ogether spanned the entire 69-

amino acid AtSEC61 γ subunit protein. These peptides were conjugated to a carrier protein and injected into rabbits. IgG was purified from the sera using Protein A affinity chromatography. A 2.6 mg/ml stock was routin ely diluted 1:3000 in blocking buffer for use in western blot analysis as described below.

Microsomal membrane protein preparations. Arabidopsis and gr eenhouse-grown soybean leaf extracts were prepared by homogenizing 6 g frozen powdered leaves in 18 ml extraction buffer [8 mM sodium phosphate, 2 mM potassi um phosphate, 0.14 M s odium chloride, 10 mM potassium chloride, pH 7.4 (PBS) with prot ease inhibitor cocktail (Thermo Scientific, Inc., Rockford, IL) and 2 mM MgCl 2 ]. The homogenates were filtered through miracloth and centrifuged at 12,000 x g for 15 minutes. The s upernatant was then centrifuged at 100,000 x g for 1.5 hours. The resulting microsomal membrane pellet was resuspended in 250 μ l PBS. For soybean grain and Arabidopsis seed extracts , 1 g powdered grain/seed was extracted with

10 ml extraction buffer. The extracts were filtered through miracl oth and microsomal membranes were isolated as described above for leaf extracts. The pe llets were resuspended

in 250 μ l PBS.

Cytochrome c reductase assay. The activity of cytochrome c reductase, a marker enzyme of the endoplasmic reticulum (microsomal membra ne fraction), was measured in the crude extract, 100,000 x g supernatant and pellet (mic rosomal membrane fraction) by monitoring the rate of reduction of cytochrome c (the ch ange in absorbance at 547 nm with time) to provide assurance that the fraction being an alyzed for the presence of the SEC61 gammaCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 216 of 375 subunit protein was indeed the microsomal fr action containing the endoplasmic reticulum. The samples were assayed using the cytochrome c reductase (NADPH) assay kit (Sigma, Saint Louis, MO). The total reaction was 230 μ l and contained 300 mM potassium phosphate, pH 7.8, 0.1 mM EDTA, 85 μ g NADPH, and 36 μ M cytochrome c. The reaction was initiated by the addition of NADPH and th e change in absorbance at 547 nm was monitored using a Tecan Sunrise ® multiwell plate reader. The results were analyzed using DeltaSoft PC software (Version 1.71.4, BioMetal lics, Inc., Princeton, NJ) using the kinetic

mode to calculate the rate.

Western blot analysis. Aliquots of the microsomal membra ne preparations were subjected to SDS-polyacrylamide gel electrophoresis on an 8 – 16% polyacrylamide gradient gel followed by electroblotting onto a PVDF membrane (In vitrogen; SOPs BPS 510.02 and 510.03). The membrane was probed with rabbit anti-AtSEC61 γ polyclonal antibody which can detect approximately 0.4 ng SEC61 γ subunit protein. Donkey anti-ra bbit IgG linked to horseradish peroxidase (Jackson ImmunoResearch Laborator ies, Inc., West Grove, PA), diluted 1:20,000 in blocking buffer (3% nonfat dry milk in 0.05% Tween-20, 25 mM Tris-HCl, 140 mM NaCl, 3 mM KCl, pH 7.4), was used to bind to the primary an tibody and was visualized by development with the SuperSignal ® West Femto Maximum Sensitivity Substrate (Thermo Fisher). The limit of detection of this de velopment method with this antibody was found to be approximately 0.4 ng SEC61 γ subunit protein.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 217 of 375 Appendix D

Methods Used and Results of the Grai n and Forage Composition Analyses

The purpose of these studies was to analyze and compare the composition of soybean grain and forage derived from CV127 soybean, the is oline control, and two conventional soybean varieties. For weed control, CV127 soybean plants were treated with imidazolinone herbicide at 70 g ai/ha (treatment presented as “CV127+imi”) and the isoline control and two other conventional soybean va rieties commonly grown in Br azil (Monsoy 8001 and Coodetec 217 presented as “Std1” and “Std2”, respective ly, in the data tables) were treated with bentazon + acifluorfen-sodium (commercial name Volt ® ) at the rate of 1.0 liter/ha. For grain composition analyses only, an additional treatm ent of CV127 treated with the conventional

herbicide Volt ® was included in these studies, and the data are reported in the following data tables in this Appendix for this treatment. Ho wever, because this treat ment will not be used in the commercial production of CV127 soybean, th is treatment was not included in the mean and range analyte data determined across field tr ial locations and presented in Section VII of the petition.

a) Grain Composition Analyses

For grain production, plants were grown at six locations in Brazil during the 2006/2007 growing season and four locations during the short growing season of 2007. The field trial locations and cultivation practices are described in detail in Appendix F, and are summarized in Table D-1. The plants were grown under st andard agronomic prac tices in a complete randomized block design with four replicate blocks per location. Fo r the CV127 and isoline control treatments, grain was harvested separately from each replicate block at the conclusion of the growing season and approximately 2 kg of each replicate grain sample was separated for compositional analyses. For the two convent ional soybean varieties (“Std1” and “Std2”), the same grain harvest and separation procedur es were followed, but approximately 500 g of grain was further sub-sampled from each 2 kg replicate grain sample, and the four 500 g replicate samples for each conventional soybean variety from each field location were pooled to make a single sample from each location for compositional analyses. Therefore, statistical analyses of the compositional data were only conducted for the CV127 and isoline control treatments, and data from the two conventional soybean varieties were used for comparative purposes to establish a range of natural vari ability for each analyte for soybeans grown in Brazil. All compositional analys es were conducted using methods listed in Table D-2 by the Instituto de Tecnologia de Alimentos (ITAL) in Campinas, Brazil or, for cysteine and methionine analyses only, Eurofins (Des Moines , IA). Proximate resu lts are reported on both a fresh- and dry-weight basis; all other analytes are re ported on a dry-weight basis unless otherwise stated. All results from grain produced in the 2006/2007 season and the 2007 short season are presented in the follo wing tables of this Appendix (D-3 to D-22). Statistical analysis was conducted usi ng the dry-weight data.

b) Forage Composition Analyses

Imidazolinone-treated CV127 plants together with the isoline cont rol, and two other conventional soybean varieties [ Monsoy 8001 (“Std 1”) and Coode tec 217 (“Std 2”)] were grown at six locations in Brazil during the 2007/2008 growi ng season. The field trial locations are presented in Table D-1. The plants were grown under standard agronomic practices in a complete randomized block desi gn with three replicate blocks per location.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 218 of 375 With the exception of the CV127 plants treated with the imidazolinone herbicide at 70 g ai/ha, all other entries in the study were treated with bentazon + acifluorfen-sodium (commercial name Volt ® ) at the rate of 1.0 liter/ha. Three pl ants at the full-flowering stage of development in each replicate plot were harveste d by cutting the plants at the base of the stem near ground level. The three plants from each plot or treatment replicate were pooled together in a plastic bag and shipped on wet ice to the Instituto de Tecnologia de Alimentos

( ITAL) in Campinas, Brazil, for compositiona l analysis. At ITAL, the pooled samples consisting of three plants from each replic ate plot were chopped and mixed to produce a composite sample. After partial drying and we ighing, the samples were reduced to a fine powder using a Waring blender. Aliquots (2 5 g each) of each sample were removed to separate air-tight containers an d were shipped on dry ice to Eurofins (Des Moines, IA) for analysis of cysteine and methionine. Sample s were stored in air-tight glass bottles until analyzed. Results were recorded on a fresh-we ight (FW) basis and adjusted for moisture content to calculate the corresponding dry-wei ght (DW) value. Statistical analysis was conducted using the dry-weight data. Details of the methods of compositional analyses are presented below and summarized in Table D-2. Results of forage composition analyses are presented in Tables D-23 and 24. Reference values for forage composition analyses from OECD (2001) are presented in Table D-25.

Table D-1. Field Trial Locations in Br azil for Grain and Forage Compositional Analyses

Location 2006-07

Season - Grain 2007

Season - Grain 2007-08 Season - Forage

Santo Antônio

de Posse – SP 

### √ √

Londrina –

PR/North 

### √ √

Uberaba – MG 

### √ √

Brasília – DF 

### √ √ √

Santo Antônio

de Goiás – SG 

### √ √ √

Sete Lagoas-MG 

### √ √

Teresina - PI 

### √

Vilhena - RO 

### √CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 219 of 375 c) Analytical Methods

The specific methods used for composition analyses of the grain and the forage are listed in Table D-2. Below is a genera l description of the methods used for measurement of each analyte.

Ash

. The sample was placed in an electric furn ace at 550°C and ignited to drive off all volatile organic matter. The nonvolatile matter remaining was quantitated gravimetrically and calculated to determine percent ash. Th e limit of quantitation for this study was 0.1%

FW.

Carbohydrates and Calories

. The total carbohydrate level was calculated by difference using the fresh weight-derived data and th e following equation: % carbohydrates = 100% - (% protein + % fat + % moisture + % ash). Ca lories were calculated using the following equation on fresh-weight derived data: (kcal/100 g) + (4 x % protein) + (9 x % fat) + (4 x % carbohydrates). The limit of quantitati on for carbohydrates for this study was 0.1%

FW.

Fat by Butt Extraction . The sample was weighed into a cellulose thimble. Petroleum ether was dripped through the sample to remove the fat. The extract was then evaporated, dried, and weighed. The limit of quantitation for this study was 0.1% FW.

Moisture. The sample was dried in a forced-draft oven at 130°C to a constant weight. The moisture weight loss was determined and converted to percent moisture. The limit of quantitation for this study was 0.1% FW.

Protein

. Nitrogenous compounds in the sample were reduced in the presence of boiling

sulfuric acid and a CuSO 4 + K 2 SO 4 + Se mixture to form ammonia. The acid digest was

made alkaline. The ammonia was distilled and then titrated with a standard acid. The percent nitrogen was calculated and converted to protein using the factor 6.25. The limit of quantitation for this study was 0.100%.

Amino Acid Composition

. The methods of analysis estimate the levels of 18 amino acids in the sample: alanine, arginine, aspartic acid (including asparagine), cystine (including cysteine), glutamic acid (including glutamin e), glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalan ine, proline, serine, threonine, tryptophan, tyrosine, and valine. This was accomplished for all ami no acids except cysteine, methionine, and tryptophan through direct acid hydrolysis w ith 6N hydrochloric acid. Once hydrolyzed, the individual amino acids were then quantif ied using an automated amino acid analyzer and detected at 520 nm. The limit of quan titation for this study was 0.1 mg/g. The

reference standards were 2.5 μ mol/mL per amino acid with the exception of cystine

which was 1.25 μ mol/mL (Pierce, Rockford IL). Cysteine (including cystine) and methionine levels were determined by first converting these residues in protein to cysteic acid and methionine sulfone, respectively, by oxidation with performic acid. Following acid hydrolysis to release cysteic acid and methionine sulfone, these residues were quantified by ion exchange chromatography with an o- phthalaldehyde post-column reaction. The limit of quantification for th is method is 0.01% on an “as is” basis. Tryptophan levels were determined by measur ing the absorbance at 590 nm following direct enzymatic hydrolysis with pronase at 40o C for 24 hours. The reference standardCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 220 of 375 was L-tryptophan, >99% (used as 100%, Sigma Chemical Co., Saint Louis, MO). The limit of quantitation for this method was 0.1 mg/g.

Crude Fiber

. Crude fiber was measured as the loss on ignition of dried residue remaining after digestion of the sample with 1.25% solutions of sulfuric acid and sodium hydroxide. The limit of quantitation for this study was 0.1 g/100 g FW sample.

Acid Detergent Fiber (ADF). The sample was placed in a fr itted vessel and washed with an acidic boiling detergent solution that dissolved the protein, carbohydrate, and ash. After an acetone wash to remove the fats and pigments, the lignocellulose fraction was collected on the frit and quantitated gravimetrically. The limit of quantitation for this study was 0.1g/100g FW.

Neutral Detergent Fiber (NDF)

. Samples were placed in a fritted vessel and washed with a neutral boiling detergent solution that dissolved the protein, carbohydrate, enzyme, and ash. After an acetone wash to remove the fats and pigments, the hemicellulose, cellulose, and lignin fractions were collected on the frit and quantitat ed gravimetrically. The limit of quantitation for this study was 0.1g/100g FW.

Total Dietary Fiber . The finely ground sample was gelatinized with Termamyl and then enzymatically digested with protease and amyl oglucosidase to remove protein and starch. Four volumes of 95% ethyl alc ohol were added to precipitate soluble dietary fiber. The

total residues were filtered and washed consecutively with 78% ethyl alco hol, 95% ethyl alcohol, and acetone. After dr ying, the residue was weighe d. One duplicate was then analyzed for protein, and the other was incine rated at 525o C for ash determination. Total dietary fiber = weight residue – weight (protein + ash) – blank (containing enzymes only). The limit of quantitation for this study was 0.1g/100g FW.

Fatty Acids

. The lipids were extracted and saponi fied with 0.5 N sodium hydroxide in

methanol. The mixture was met hylated with a solution of NH 4 Cl and H 2 SO 4 in methanol based on Hartman and Lago (1973). The resulting methyl esters were extracted with hexane. The methyl esters of the fatty aci ds were analyzed by gas chromatography using area normalization for quant itation. The 37-Component FAME mix from Supelco (Sigma, Saint Louis, MO) was used for refere nce standards. The limit of quantitation for this study was 0.01% FW.

Isoflavones

. The defatted sample was extracted using an aqueous solution of 70%

ethanol with 0.1% acetic acid. The extract wa s centrifuged and filtered. The sample was analyzed on a high-performance liquid chro matography (HPLC) system with a diode array detector. Quantification was conducted against an external standard curve of known standards. The limit of quantifi cation for each component was 0.3 mg/100g.

Phospholipids

. Lipids were extracted from the sample by a mixture of chloroformmethanol (2:1). Lecithins were purified by solid phase extraction on a silica column. Separation and quantification of lecith ins were conducted by normal phase liquid chromatography with ultraviolet detection. The limit of quantitation for phospholipids was 0.1 mg/g oil.

Lectin

. The sample was suspended in phosphate-buffered saline (PBS), shaken, and filtered. An aliquot of the resulting extract was serially diluted in 10 cuvettes containing PBS. A 2% erythrocyte (from dog blood) su spension was added to an equal volume ofCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 221 of 375 the sample and the mixture incubated at 37o C for 30 minutes followed by another 30- minute incubation at room temperature. The la st well to show visi ble agglutination was considered the point of equivalence. One hemagglutinating unit (HU) was defined as the reciprocal of dilution at the poi nt of equivalence, the specif ic activity bei ng given as one hemagglutinating unit per μg or mg of pr otein (HU) in the und iluted sample. The

minimum dose was defined as the minimum concentration necessary to show visible agglutination. These assays are only semiqua ntitative and should be regarded as liable to

an error of 25%.

Minerals. The sample was placed in an electric furnace at 450°C and ignited to drive off all volatile organic matter. The minerals remaining were quantitated by inductively coupled plasma-optical emission spectroscopy (ICP-OES). The lim it of quantitation for this study was 0.01 mg/kg for all minerals.

Phytic Acid

. The sample was extracted using 2.4% HCl. Purification and concentration was conducted on an anion exchange column (Dowex-1 AGX-4, 100-200 mesh). Sample

and standards were submitted to a color reaction with Wade reagent, with the absorbance measured at 500 nm. Inositol hexaphosphoric acid was used as a standard.

Sugars (Raffinose and Stachyose)

. Sugars were extracted from the sample with ethanol + deionized water (1:1). Pr oteins and lipids which co-ext racted were eliminated by precipitation, followed by filtration. Ra ffinose and stachyose were separated and quantified by HPLC with a refractive index dete ctor. The limit of quantification for this study was 0.2 g/100 g.

Trypsin Inhibitor

. Trypsin inhibitor units (TIU) were determined by photometrically measuring the inhibition of the trypsin clea vage of benzoyl-DL-ar ginine-p-nitroanalide hydrochloride. The sample was ground and/or defatted with petroleum ether, if necessary. A sample of matrix was extrac ted for 3 hours with 0.1 N sodium hydroxide. Varying aliquots of the sample suspension were exposed to a known amount of trypsin and benzoyl-DL-arginine-p-nitr oanalide hydrochloride. The sample was allowed to react for 10 minutes at 37° C. After 10 minutes, the reaction was quenched by the addition of

30% trichloroacetic acid. The solution was f iltered or centrifuged and the absorbance at 410 nm was measured. The limit of quantitation for this study was 1.00 TIU/mg.

Urease Activity

. This assay is based on an increase in pH as ammonia is released from urea by residual urease enzyme in the soy m eal. The urease activity was assayed by measuring the hydrolysis of 3% urea at pH 7.0 at 37o C. A difference between the pH of the test sample and the pH of the blank is an indication and index of urease activity. The optimum pH increase has generally been considered to be 0.05–0.30.

Folates. The VitaFast Folic Acid kit from R- Biopharm was used. The method is based on a microbiological assay whic h uses microplates and the Lactobacillus casei subspecies

rhamnosus – ATCC 7469. The antioxidants 2-mer captoethanol and ascorbic acid were used in all preparation stages of the extract. The samples we re hydrolyzed in an autoclave with potassium dihydrogen phosphate 0.1 M pH 6.8, followed by consecutive enzymatic hydrolysis steps. Each enzyme was inactivat ed by boiling the sample before addition of the next enzyme. The enzymes used were: α -amylase for 3 hours, protease for 3 hours, and folate conjugase for 5 hours. After the ne cessary dilutions, the extracts were applied

to the m icroplates. After the addition of the samples, the plate was kept in a chamber at 37o C for 44-48 hours and the quantification of the turbidity in the microplates wasCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 222 of 375 measured at 630 nm in a microplate read er. Quantitation was by comparison to a standard curve. The limit of quantitation for this study was 0.1 μ g/g FW sample.

Vitamin E (Tocopherols)

. The samples were saponified at 90-95o C with reflux using nitrogen forced through th e condenser, with potassi um hydroxide and with the antioxidant, ascorbic acid. The extraction of the non-saponifiable material was conducted with diethyl ether. The diethyl ether extract was concentrated in a rotary evaporator at 40o C, dried under nitrogen and the residue was dissolved in n-hexane. The tocopherols were separated by HPLC over a Lichros pher Si60 (125 x 4 mm) column, (Merck, Germany). The mobile phase consisted of n- hexane, ethyl acetate, and n-propyl alcohol in an isocratic system. The detection and quantification was accomplished using a

fluorescence detector with excitation at 294 nm and emission at 326 nm. The limit of quantitation for this study was 0.05 mg/g FW sample.

Vitamin Niacin

. For the extractio n of niacin, the samp les were first hydrolyzed by 1 N sulfuric acid hydrolysis in an autoclave for 30 minutes. Then the pH was adjusted to 4.5 with 10 N sodium hydroxide, as reco mmended by AOAC (2005) method 961.14. The extracts were passed over an ion-exchange re sin prior to HPLC on a Lichrospher 100 RP18 (250 x 4 mm) column (Merck, Germany). The mobile phase consisted of heptanesulfonic acid sodium salt, triet hylamine, potassium dihydrogen phosphate, and methanol in a gradient system. Niacin leve ls were monitored at 265 nm. The limit of quantification for this stu dy was 0.50 mg/100 g FW sample.

Vitamins B1 (Thiamin) and B2 (Riboflavin)

. For the extraction of the vitamins, the samples were hydrolyzed with 0.01 N hydrochlor ic acid in an autoclave for 15 minutes; the pH was adjusted to 4.5 with sodium acetat e. Enzymatic hydrolysis using diastase and papain was carried out for 12 hours at room temperature. Extracts were subjected to HPLC using a Lichrospher 100 RP-18 (250 x 4 mm) column (Merck, Germany). The mobile phase consisted of potassium chloride, methanol, and water in an isocratic system.

A fluorescence detector was used to monito r the natural fluorescence of the riboflavin (excitation 432 nm and emission 545 nm) and thiochrome, after oxidation of thiamine to thiochrome (362 nm for excitation and 464 nm for emission). The limit of quantitation for this study for Vitamin B1 was 0.03 mg/g fresh weight sample. The limit of quantification for this study for v itamin B2 was 0.02 mg/100 g FW sample.

Statistical analysis

. Analysis of variance was carried out on the data using SAS Version 9.1 (SAS Institute Inc., Cary, NC) following two procedures, the General Linear Model and the Mixed Model. With the exception of moisture content, all data were expressed on a dryweight basis for statistical analyses. Di fferences were assessed across location and by

location. The model for across-locati on analysis was the following:

y = variety + location + variety x location + block(location) + e Random effects: location, vari ety x location, block(location). Where y is the response variable (any analyte measured)

The model for separate analyses by location was the following:

y = variety + block + e where e is the response errorCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 223 of 375 Contrasts were carried out to compare each of the sprayed and unsprayed CV127 treatments

with the isoline contro l. Differences were considered statistically significant at the 0.05

confidence level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 224 of 375 Table D-2. Methods for Compositional Anal yses of Grain Harvested from 2006-07 and 2007 Field Trial Seasons in Brazil and for Forage Harvested from the 2007-08 Field

Trial Season. An “X” is used to identify the specific method of analysis for each analyte.

Analyte Method Grain Comp.

2006-07 Grain Comp.

2007 Forage

2007-08

Moisture AOCS (1998) method Bc 2-49 X X AOAC (2005) method 934.01 X

Ash AOAC (2000) method 945.38 C X X AOAC (2005) method 942.05 X

Fat AOAC (2000) method 945.38 F X X ISO (1973) method 34 B X Crude Fiber Beythien and Diemair (1963) X X X Protein AOAC (2000) method 979.09 X X AOAC (2000) method 945.39 AOAC (2005) method 954.01 X Carbohydrates

and calories USDA (1963) X X X

Total Dietary

Fiber AOAC (2000) method 985.29 X X

ADF AOAC (1995) method 920.85 X AOAC (1975) method 7.057 X X

NDF AOAC (1995) method 920.85 and AOAC (2000)

method 973.18 X

AOAC (1995) method 920.85 and

Van Soest et al . (1991) X X

Fatty Acids AOCS (1998) methods Ce 1F-96, Ce 1-62, Ce2-66 AOAC (2000) method 996.06 Hartman and Lago (1973) X X

Raffinose and Stachyose Cicek (2001) and Kennedy et al. (1985) X X

Ca, Fe, P, Mg,

K AOAC (2005) methods 985.35, 984.27 X X

Vitamin E Brubacher et al . (1985) X X Vitamin B1 AOAC (2005) methods 942.23 and 970.65 X X Vitamin B2 AOAC (2005) method 970.65 and 942.23 X X Niacin AOAC (2005) method 961.14 X X Folate AOAC (2005) method 2004.05 X X Phytate Latta and Eskin (1980) X X Isoflavones Berhow (2002) X X

Amino Acids (except Cys, Met, Trp) Spackman et al . (1958) X X

Cysteine and

Methionine AOAC (2000) method 994.12 mod. X X

Tryptophan Spies (1967) X X Trypsin

Inhibitor Rackis et al . (1974) X X

Urease AOCS (1998) method Ba 9-58 X X Phospholipids Beare-Rogers et al. (1992) X X Lectins Padgette et al . (1996) and Wititsuwannakul et al. (1998) X XCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 225 of 375Table D-3. Proximate Composition of Grain on a Fresh-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 SeasonLocationTreatmentNMoisture(g/100 g FW)Total Dietary Fiber(g/100 g FW)Protein(g/100 g FW)Fat(g/100 g FW)Ash(g/100 g FW)Carbohydrates(g/100 g FW)Calories(kcal/100 g FW)Mean±Standard Deviation (range)Isoline 410.2±0.1(10.1 – 10.3)22.00±1.12(21.30 – 23.66)35.8±1.2(35.1 – 37.5)19.6±0.2(19.3 – 19.8)4.4±0.2(4.2 – 4.5)8.0±1.3(6.9 – 9.5)352±5(344 – 356)SantoAntôniode GoiasCV127 49.1±0.3*(8.8 – 9.4)22.73±0.79(21.90 – 23.70)35.9±0.3(35.6 – 36.3)21.2±0.2(21.0 – 21.5)4.4±0.1(4.2 – 4.5)6.7±1.0(5.4 – 7.9)362±4(356 – 366)CV127+ imi49.6±0.1*(9.4 – 9.7)23.99±2.30(20.23 – 25.41)36.6±0.3(36.3 – 36.9)20.5±0.5(19.9 – 20.9)4.2±0.1(4.2 – 4.3)5.6±2.3(3.4 – 8.8)353±9(347 – 366)Std 1 1 10.5 23.29 35.4 18.1 4.4 8.3 338Std 2 1 9.9 22.84 33.5 21.2 4.2 8.4 359Uberaba Isoline 49.9±0.4(9.6 – 10.4)21.45±1.68(20.05 – 23.74)37.3±0.9(35.9 – 38.0)19.2±0.9(18.5 – 20.5)4.3±0.1(4.2 – 4.3)8.0±1.7(6.0 – 9.6)354±8(342 – 360)CV127 48.9±0.2*(8.7 – 9.1)21.55±1.35(20.15 – 22.80)35.9±0.3(35.4 – 36.1)20.3±0.4(19.9 – 20.4)4.3±0.1(4.2 – 4.3)9.2±1.7(7.3 – 11.0)363±4(358 – 367)CV127+ imi49.0±0.2*(8.8 – 9.1)22.03±1.43(20.19 – 23.52)36.3±0.1(36.2 – 36.4)20.6±0.3(20.1 – 20.9)4.2±0.1(4.1 – 4.3)7.9±1.7(6.0 – 10.1)362±7(356 – 370)Std 1 1 9.5 22.55 34.9 19.1 4.1 9.9 351Std 2 1 9.6 24.19 35.0 21.8 4.1 5.3 357SeteLagoasIsoline 410.6±0.2(10.5 – 10.6)21.47±1.39(20.13 – 23.42)37.0±0.2(36.6 – 37.1)19.4±0.1(19.2 – 19.5)4.4±0.1(4.3 – 4.4)7.3±1.6(5.0 – 8.9)351±5(344 – 356)CV127 49.7±0.3*(9.3 – 10.0)21.56±0.97(20.56 – 22.74)36.0±0.2(35.8 – 36.2)20.3±0.5(19.8 – 20.9)4.5±0.1(4.3 – 4.6)8.0±1.0(6.8 – 9.0)359±5(354 – 366)CV127+ imi49.8±0.1*(9.7 – 9.9)22.17±1.46(20.73 – 23.55)35.6±0.3(35.2 – 35.9)20.9±0.3(20.7 – 21.3)4.4±0.1(4.4 – 4.5)7.1±1.4(5.9 – 8.6)359±5(353 – 364)Std 1 1 9.8 23.31 34.0 19.4 4.4 9.1 347Std 2 1 9.8 22.80 33.7 21.7 4.3 7.7 361CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 226 of 375Table D-3. continued.*Statistically significantly different from the isoline control at P < 0.05.LocationTreatmentNMoisture(g/100 g FW)Total Dietary Fiber(g/100 g FW)Protein(g/100 g FW)Fat(g/100 g FW)Ash(g/100 g FW)Carbohydrates(g/100 g FW)Calories(kcal/100 g FW)Mean±Standard Deviation (range)Londrina Isoline 49.4±0.2(9.2 – 9.7)24.04±1.01(22.64 – 25.03)34.7±0.2(34.4 – 34.9)21.0±0.2(20.7 – 21.1)4.6±0(4.6)6.3±1.1(5.3 – 7.7)353±3(350 – 357)CV127 49.1±0.1(9.0 – 9.2)23.06±1.93(20.27 – 24.48)34.1±0.4(33.7 – 34.5)21.3±0.1(21.2 – 21.3)4.6±0.1(4.6 – 4.7)7.9±2.0(6.3 – 10.7)359±8(354 – 370)CV127+ imi49.4±0.1(9.3 – 9.5)22.53±1.30(21.37 – 24.38)34.4±0.4(33.8 – 34.6)21.4±0.2(21.2 – 21.5)4.6±0.1(4.5 – 4.7)7.8±0.8(6.6 – 8.4)361±6(352 – 366)Std 1 1 9.9 21.30 32.8 20.5 4.7 10.8 359Std 2 1 9.4 24.81 34.1 22.5 4.6 4.6 358Brasília Isoline 410.1±0.1(9.9 – 10.2)22.05±1.60(19.74 – 23.43)36.7±0.9(35.7 – 37.9)18.6±0.5(18.0 – 19.1)4.7±0.1(4.6 – 4.7)8.0±2.1(6.3 – 10.9)346±5(342 – 352)CV127 49.1±0.1*(9.0 – 9.2)21.76±1.47(19.67 – 23.01)36.9±0.9(35.8 – 38.1)19.2±0.6(18.4 – 19.7)4.6±0.1(4.5 – 4.6)8.6±1.4(6.8 – 10.3)355±8(345 – 364)CV127+ imi49.0±0.1*(8.9 – 9.2)20.75±0.46(20.31 – 21.37)37.1±0.7(36.4 – 38.0)18.8±0.7(18.3 – 19.9)4.6±0(4.6)9.7±1.2(8.6 – 11.2)357±3(354 – 361)Std 1 1 9.5 22.63 33.7 19.9 4.5 9.8 353Std 2 19.5 22.30 32.9 21.5 4.6 9.2 362Isoline 410.3±0.2(10.0 – 10.4)21.58±1.21(19.77 – 22.29)36.1±0.4(35.7 – 36.6)19.3±0.3(19.1 – 19.7)4.7±0.1(4.5 – 4.8)8.2±1.1(7.3 – 9.7)351±6(347 – 360)SantoAntôniode PosseCV127 410.1±0.1(9.9 – 10.2)22.90±0.37(22.39 – 23.29)34.7±0.3(34.5 – 35.1)20.9±0.9(20.4 – 22.2)4.7±0.2(4.6 – 4.9)6.7±1.1(5.1 – 7.4)354±5(350 – 360)CV127+ imi49.6±0.3*(9.3 – 9.9)23.27±0.73(22.32 – 23.85)34.4±0.5(33.8 – 34.9)21.3±0.6(20.7 – 21.9)4.8±0.1(4.7 – 4.9)6.7±0.8(6.0 – 7.8)357±6(351 – 364)Std 1 1 10.1 20.04 33.2 19.6 4.5 12.6 360Std 2 1 9.7 25.31 33.5 21.6 4.8 5.1 349CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 227 of 375Table D-4. Proximate Composition of Grain on a Fresh-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocationTreatmentNMoisture(g/100 g FW)Total Dietary Fiber(g/100 g FW)Protein(g/100 g FW)Fat(g/100 g FW)Ash(g/100 g FW)Carbohydrates(g/100 g FW)Calories(kcal/100 g FW)Mean±Standard Deviation (range)Isoline 47.7±0.3(7.6 – 8.1)19.68±1.96(17.51 – 21.73)37.8±0.5(37.2 – 38.2)16.7±0.3(16.4 – 17.2)4.8±0.2(4.7 – 5.0)13.3±1.8(11.9 – 15.6)355±7(346 – 363)SantoAntônio deGoiásCV127 48.1±0.1*(8.0 – 8.2)22.20±0.66(21.36 – 22.96)37.8±0.5(37.0 – 38.1)17.6±0.4(17.3 – 18.1)4.9±0.1(4.8 – 4.9)9.5±0.6(8.9 – 10.2)325±45(258 – 351)CV127+ imi48.1±0.2*(7.9 – 8.2)23.23±1.54(22.14 – 25.49)37.6±0.4(37.1 – 38.0)17.9±0.2(17.7 – 18.2)4.8±0.1(4.7 – 4.8)8.4±1.5(6.3 – 9.6)345±7(335 – 350)Std1 1 7.9 20.16 38.1 15.6 4.8 13.4 346Std2 1 7.7 21.35 37.6 17.8 4.8 10.8 354Teresina Isoline 37.4±0.2(7.2 – 7.5)22.55±1.62(20.78 – 23.95)36.5±1.1(35.5 – 37.7)21.7±0.5(21.2 – 22.0)5.2±0.1(5.1 – 5.3)6.7±1.0(5.8 – 7.8)368±5(363 – 373)CV127 37.6±0.2(7.4 – 7.8)22.98±1.84(20.92 – 24.45)35.8±1.3(34.3 – 36.8)22.3±0.6(21.7 – 22.9)5.1±0.2(4.9 – 5.3)6.2±1.6(4.9 – 8.0)369±8(363 – 378)CV127+ imi37.8±0.2*(7.6 – 7.9)24.08±3.45(20.09 – 26.19)37.5±0.9(36.6 – 38.3)20.9±1.2(19.9 – 22.3)5.1±0.2(4.9 – 5.2)4.7±3.3(2.6 – 8.5)357±21(344 – 382)Std1 1 7.5 22.02 36.9 20.7 5.1 7.8 365Std2 1 7.0 25.36 34.7 23.3 5.1 4.5 367CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 228 of 375Table D-4. continued.LocationTreatmentNMoisture(g/100 g FW)Total Dietary Fiber(g/100 g FW)Protein(g/100 g FW)Fat(g/100 g FW)Ash(g/100 g FW)Carbohydrates(g/100 g FW)Calories(kcal/100 g FW)Mean±Standard Deviation (range)Vilhena Isoline 47.4±0.2(7.1 – 7.5)24.29±1.47(22.44 – 25.94)36.6±0.4(36.2 – 36.9)19.5±0.2(19.2 – 19.8)4.7±0.1(4.7 – 4.8)7.6±1.4(6.0 – 9.3)352±4(347 – 357)CV127 47.8±0.1*(7.7 – 7.9)22.70±0.79(22.14 – 23.82)36.1±0.6(35.5 – 36.8)19.6±0.4(19.2 – 20.0)4.8±0.1(4.7 – 4.8)9.1±0.9(7.7 – 9.7)357±4(351 – 361)CV127+ imi47.8±0*(7.8)20.98±1.47(19.70 – 23.10)37.0±0.7(36.2 – 37.6)19.2±0.4(18.7 – 19.5)4.7±0.1(4.6 – 4.7)10.4±1.3(8.7 – 11.6)363±4(356 – 365)Std1 1 7.8 22.33 38.7 16.8 4.8 9.6 345Std2 1 7.4 24.60 35.6 20.8 4.6 7.0 358Brasília Isoline 48.1±0.1(7.9 – 8.2)24.12±1.02(23.3 – 25.61)33.9±0.4(33.4 – 34.4)17.5±0.2(17.3 – 17.7)4.7±0.2(4.4 – 4.9)11.8±1.0(10.6 – 12.9)340±6(332 – 344)CV127 47.9±0.1*(7.8 – 8.0)23.34±0.75(22.72 – 24.43)34.9±0.5(34.5 – 35.7)17.5±0.2(17.3 – 17.7)4.6±0.2(4.4 – 4.8)11.8±0.8(10.7 – 12.6)344±4(339 – 347)CV127+ imi47.9±0*(7.9)22.90±1.77(21.09 – 25.30)34.5±0.6(33.8 – 35.3)17.9±0.4(17.4 – 18.2)4.6±0.2(4.3 – 4.7)12.3±1.6(10.0 – 13.7)349±7(339 – 357)Std1 1 8.2 23.90 33.8 18.1 4.5 11.5 344Std2 1 8.0 24.70 34.3 18.7 4.7 9.6 344*Statistically significantly different from the isoline control at P <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 229 of 375Table D-5. Proximate Composition of Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 SeasonLocationTreatmentNTotal Dietary Fiber(g/100 g DW)Protein(g/100 g DW)Fat(g/100 g DW)Ash(g/100 g DW)Carbohydrates(g/100 g DW)Calories(kcal/100 g DW)Mean±Standard Deviation (range)Isoline 424.65±1.22(23.75 – 26.34)39.8±1.3(39.1 – 41.8)21.8±0.2(21.5 – 22.0)4.9±0.2(4.7 – 5.0)9.0±1.4(7.7 –10.6)392±6(383 – 397)SantoAntôniode GoiásCV127 425.00±0.92(24.07 – 26.15)39.5±0.4(39.2 – 40.1)23.3±0.2*(23.2 – 23.6)4.8±0.2(4.6 – 5.0)7.4±1.1(6.0 –8.7)398±4(393 – 401)CV127+ imi426.08±2.53*(22.40 – 28.11)40.5±0.3(40.1 – 40.8)22.7±0.5*(22.0 – 23.1)4.7±0.1*(4.6 – 4.8)6.1±2.5(3.4 –8.8)391±10(384 – 405)Std 1 1 26.02 39.6 20.24.9 9.3 377Std 2 1 25.34 37.2 23.54.7 9.3 398Uberaba Isoline 423.79±1.87(22.18 – 26.28)41.4±0.9(40.1 – 42.1)21.3±1.1(20.5 – 22.9)4.8±0.1(4.6 – 4.8)8.8±1.8(6.6 –10.6)392±9(379 – 399)CV127 423.66±1.53(22.07 – 25.08)39.4±0.3(38.9 – 39.6)22.3±0.5(21.8 – 22.9)4.7±0.1(4.6 – 4.7)10.0±1.9(8.0 – 12.0)398±4(394 – 402)CV127+ imi424.20±1.59*(22.13 – 25.87)39.9±0.1*(39.7 – 40.0)22.6±0.4*(22.0 – 23.0)4.6±0.1(4.5 – 4.7)8.7±1.8(6.6 –11.1)398±7(392 – 406)Std 1 1 24.91 38.6 21.14.5 10.9 388Std 2 1 26.75 38.7 24.14.5 5.9 395SeteLagoasIsoline 424.02±1.56(22.49 – 26.19)41.4±0.2(41.1 – 41.5)21.7±0.2(21.5 – 21.8)4.9±0.1(4.8 – 4.9)8.1±1.8(5.6 –9.9)393±6(385 – 398)CV127 423.87±1.07(22.84 – 25.18)39.9±0.3(39.6 – 40.1)22.5±0.4*(22.0 – 23.0)4.9±0.2(4.7 – 5.1)8.8±1.1(7.5 –10.0)397±5(392 – 403)CV127+ imi424.57±1.61*(23.01 – 26.07)39.5±0.3*(39.1 – 39.8)23.2±0.3*(22.9 – 23.6)4.9±0.1(4.9 – 5.0)7.9±1.5(6.6 –9.5)398±6(391 – 403)Std 1 1 25.84 37.7 21.54.9 10.1 384Std 2125.28 37.4 24.14.8 8.5 400CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 230 of 375Table D-5. continued.*Statistically significantly different from the isoline control at P < 0.05.LocationTreatmentNTotal Dietary Fiber(g/100 g DW)Protein(g/100 g DW)Fat(g/100 g DW)Ash(g/100 g DW)Carbohydrates(g/100 g DW)Calories(kcal/100 g DW)Mean±Standard Deviation (range)Londrina Isoline 426.53±1.06(25.07 – 27.57)38.3±0.2(38.1 – 38.5)23.1±0.2(22.8 – 23.3)5.1±0(5.1)7.0±1.2(5.8 – 8.5)389±4(385 – 395)CV127 425.37±2.11(22.32 – 26.90)37.5±0.4(37.0 – 38.0)23.4±0.1(23.3 – 23.5)5.1±0.1(5.1 – 5.2)8.7±2.2(6.9 – 11.8)395±8(389 – 407)CV127+ imi424.85±1.45(23.58 – 26.94)37.9±0.4(37.3 – 38.2)23.6±0.2*(23.4 – 23.7)5.1±0.1(5.0 – 5.2)8.6±0.9(7.3 – 9.3)398±6(389 – 404)Std 1 1 23.63 36.4 22.8 5.2 12.0 398Std 2 1 27.38 37.6 24.8 5.1 5.1 395Brasília Isoline 424.51±1.79(21.93 – 26.06)40.8±1.1(39.6 – 42.2)20.7±0.6(20.0 – 21.2)5.2±0.1(5.1 – 5.2)8.9±2.4(7.0 – 12.1)385±5(381 – 391)CV127 423.92±1.64(21.61 – 25.34)40.6±1.1(39.3 – 42.0)21.1±0.6(20.3 – 21.6)5.0±0.1(4.9 – 5.1)9.5±1.6(7.5 – 11.4)390±8(380 – 400)CV127+ imi422.80±0.51(22.29 – 23.46)40.8±0.9(40.0 – 41.9)20.7±0.8(20.1 – 21.8)5.1±0.1(5.0 – 5.1)10.7±1.3(9.4 – 12.3)393±3(390 – 396)Std 1 1 25.00 37.2 22.0 5.0 10.8 391Std 2 124.64 36.4 23.8 5.1 10.2 400Isoline 424.04±1.32(22.06 – 24.78)40.2±0.4(39.8 – 40.8)21.5±0.4(21.2 – 22.0)5.2±0.1(5.0 – 5.3)9.1±1.2(8.1 – 10.9)391±8(385 – 402)SantoAntôniode PosseCV127 425.46±0.42(24.90 – 25.93)38.6±0.3(38.4 – 39.0)23.2±0.9*(22.7 – 24.6)5.3±0.2(5.1 – 5.5)7.5±1.3(5.6 – 8.3)394±5(390 – 400)CV127+ imi425.72±0.86*(24.60 – 26.46)38.0±0.6*(37.4 – 38.5)23.6±0.7*(22.8 – 24.2)5.3±0.1(5.2 – 5.4)7.4±0.9(6.6 – 8.6)394±6(388 – 401)Std 1 122.29 36.9 21.8 5.0 14.0 400Std 2 128.03 37.1 23.9 5.3 5.6 386CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 231 of 375Table D-6. Proximate Composition of Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocationTreatmentNTotal Dietary Fiber(g/100 g DW)Protein(g/100 g DW)Fat(g/100 g DW)Ash(g/100 g DW)Carbohydrates(g/100 g DW)Calories(kcal/100 g DW)Mean±Standard Deviation (range)Isoline 421.33±2.11(18.94 – 23.52)41.0±0.6(40.3 – 41.6)18.1±0.4(17.7 – 18.6)5.2±0.2(5.1 – 5.4)14.5±1.9(12.9 – 16.9)385±9(374 – 393)SantoAntônio deGoiásCV127 424.14±0.70*(23.26 – 24.95)41.1±0.6(40.2 – 41.5)19.1±0.4*(18.8 – 19.7)5.3±0.1(5.2 – 5.3)10.3±0.8*(9.3 – 11.1)354±48(281 – 382)CV127+ imi425.27±1.69*(24.12 – 27.76)40.9±0.4(40.4 – 41.3)19.5±0.2*(19.3 – 19.8)5.2±0.1(5.1 – 5.2)9.2±1.6*(6.9 – 10.5)375±7(365 – 381)Std1 1 21.88 41.4 16.9 5.2 14.6 377Std2 1 23.13 40.7 19.3 5.2 11.7 384Teresina Isoline 324.34±1.75(22.44 – 25.89)39.4±1.2(38.4 – 40.7)23.5±0.5(22.9 – 23.8)5.6±0.1(5.5 – 5.7)7.2±1.1(6.3 – 8.4)398±6(392 – 403)CV127 324.86±1.99(22.62 – 26.40)38.7±1.5(37.0 – 39.9)24.1±0.6(23.5 – 24.7)5.5±0.2(5.3 – 5.7)6.7±1.7(5.3 – 8.6)399±8(394 – 409)CV127+ imi326.12±3.79(21.74 – 28.43)40.6±1.0(39.6 – 41.6)22.7±1.3(21.6 – 24.1)5.5±0.2(5.3 – 5.6)5.0±3.6(2.8 – 9.2)388±22(376 – 374)Std1 1 23.80 39.9 22.4 5.5 8.4 394Std2 1 27.26 37.3 25.1 5.5 4.8 395CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 232 of 375Table D-6. continued.LocationTreatmentNTotal Dietary Fiber(g/100 g DW)Protein(g/100 g DW)Fat(g/100 g DW)Ash(g/100 g DW)Carbohydrates(g/100 g DW)Calories(kcal/100 g DW)Mean±Standard Deviation (range)Vilhena Isoline 426.21±1.56(24.26 – 28.01)39.5±0.4(39.1 – 39.9)21.1±0.2(20.8 – 21.4)5.1±0.1(5.1 – 5.2)8.2±1.5(6.5 – 10.1)380±5(378 – 386)CV127 424.61±0.84(24.01 – 25.80)39.2±0.7(38.5 – 39.9)21.3±0.4(20.8 – 21.7)5.2±0.1(5.1 – 5.2)9.9±1.0(8.3 – 10.5)387±5(380 – 391)CV127+ imi422.76±1.59*(21.37 – 25.05)40.1±0.8(39.3 – 40.8)20.8±0.4(20.3 – 21.1)5.1±0.1(5.0 – 5.1)11.3±1.4*(9.4 – 12.6)393±5*(386 – 396)Std1 1 24.22 42.0 18.2 5.2 10.4 374Std2 1 26.57 38.4 22.5 5.0 7.6 387Brasília Isoline 426.23±1.14(25.29 – 27.89)36.9±0.5(36.4 – 37.4)19.0±0.2(18.8 – 19.2)5.1±0.2(4.8 – 5.3)12.8±1.0(11.5 – 14.0)370±6(362 – 374)CV127 425.34±0.83(24.66 – 26.55)37.9±0.6*(37.4 – 38.8)19.0±0.2(18.8 – 19.2)5.0±0.2(4.8 – 5.2)12.7±0.8(11.6 – 13.4)374±4(368 – 376)CV127+ imi424.86±1.92(22.90 – 27.46)37.4±0.7(36.7 – 38.3)19.5±0.4*(18.9 – 19.8)5.0±0.2(4.7 – 5.1)13.4±1.7(10.9 – 14.9)379±8(368 – 388)Std1 1 26.03 36.8 19.7 4.9 12.5 375Std2 1 26.84 37.3 20.3 5.1 10.4 374*Statistically significantly different from the isoline control at P <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 233 of 375 Table D-7. Fiber Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 + imi), the Isoline Control and Tw o Conventional Soybean Varieties (Std 1 and Std 2) Grow n at Six Locations in Brazil in the 2006/07

Season

Location Treatment N Crude Fiber Acid Detergent Fiber Neutral Detergent Fiber

Mean ± Standard Deviation (range) g/100 g dry weight

Isoline 4 9.6 ± 1.2
(8.1 – 11.0) 10.71 ± 1.37
(9.07 – 12.08) 14.82 ± 0.94
(13.95 – 16.10) Santo

Antônio

de Goiás CV127 4 7.3 ± 0.3*
(6.9 – 7.6) 10.90 ± 0.94
(9.75 – 11.79) 13.97 ± 0.97
(12.72 – 15.07)
CV127

+ imi 4 7.0 ± 0.2*
(6.8 – 7.2) 13.11 ± 1.25*
(11.73 – 14.76) 16.91 ± 1.91*
(14.06 – 18.12)
Std 1 1 8.4 11.51 14.82

Std 2 1 9.0 12.14 16.36

Uberaba Isoline 4 8.3 ± 0.3
(7.9 – 8.6) 12.06 ± 2.44
(9.27 – 14.95) 15.12 ± 1.14
(13.57 – 16.16)
CV127 4 8.7 ± 1.3
(7.5 – 10.5) 13.77 ± 1.41
(12.08 – 15.20) 17.50 ± 1.27*
(15.61 – 18.31)
CV127

+ imi 4 10.2 ± 1.2*
(9.0 – 11.3) 12.34 ± 1.30
(10.96 – 14.06) 16.76 ± 1.60*
(15.07 – 18.15)
Std 1 1 8.5 9.32 11.71

Std 2 1 9.3 10.64 10.63

Sete Lagoas Isoline 4 8.7 ± 0.2
(8.5 – 8.9) 10.22 ± 1.05
(8.84 – 11.20) 13.60 ± 1.05
(12.04 – 14.26)
CV127 4 7.8 ± 0.6
(7.2 – 8.4) 14.40 ± 0.43*
(14.15 – 15.04) 18.24 ± 1.09*
(16.84 – 19.26)
CV127

+ imi 4 7.2 ± 0.6
(6.4 – 7.9) 13.69 ± 1.49*
(12.07 – 15.62) 18.21 ± 1.30*
(17.01 – 19.96)
Std 1 1 7.6 12.74 16.57

Std 2 1 8.4 10.96 12.72

Londrina Isoline 4 7.8 ± 0.6
(7.3 – 8.6) 11.70 ± 0.58
(11.18 – 12.51) 14.27 ± 0.52
(13.76 – 14.81)
CV127 4 9.8 ± 1.3*
(8.0 – 11.1) 12.68 ± 0.97
(11.41 – 13.59) 17.33 ± 1.77*
(15.25 – 19.34)
CV127

+ imi 4 7.9 ± 1.3
(6.8 – 9.7) 14.24 ± 1.25
(12.42 – 15.20) 18.01 ± 1.04*
(16.73 – 19.15)
Std 1 1 7.7 11.69 14.36

Std 2 1 7.6 11.45 13.11

Brasília Isoline 4 8.3 ± 1.4
(6.9 – 10.2) 11.66 ± 0.38
(11.10 – 11.94) 16.31 ± 1.10
(15.05 – 17.32)
CV127 4 8.9 ± 1.0
(7.6 – 9.9) 12.94 ± 0.65
(12.28 – 13.81) 19.60 ± 0.97*
(18.27 – 20.55)
CV127

+ imi 4 8.5 ± 0.7
(7.8 – 9.2) 14.86 ± 2.84
(12.51 – 19.00) 16.89 ± 1.45
(15.59 – 18.61)
Std 1 1 9.2 11.75 16.71

Std 2 1 6.7 12.59 17.73

Isoline 4 8.4 ± 0.3
(8.0 – 8.7) 12.03 ± 1.42
(11.05 – 14.11) 15.77 ± 0.83
(14.91 – 16.73) Santo

Antônio

de Posse CV127 4 7.5 ± 0.4
(7.1 – 8.0) 14.12 ± 1.48
(12.51 – 15.92) 18.11 ± 1.50*
(16.48 – 20.05)
CV127

+ imi 4 7.7 ± 0.5
(7.0 – 8.2) 14.32 ± 1.53
(12.54 – 16.13) 18.32 ± 1.16*
(17.49 – 20.01)
Std 1 1 8.1 14.43 16.92

Std 2 1 8.0 11.88 16.51
*Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 234 of 375 Table D-8. Fiber Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 + imi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grow n at Four Locations in Brazil in the 2007

Season

Location Treatment N Crude Fiber Acid Detergent Fiber Neutral Detergent Fiber

Mean ± Standard Deviation (range) g/100 g dry weight

Isoline 4 8.1 ± 1.7 (6.9 – 10.6) 9.63 ± 0.95 (8.53 – 10.73) 14.84 ± 0.85 (13.58 – 15.44) Santo

Antônio

de Goiás CV127 4 7.6 ± 0.5 (7.1 – 8.3) 10.21 ± 0.56 (9.61 – 10.93) 14.66 ± 0.55 (14.02 – 15.35)

CV127

+ imi 4 9.1 ± 3.7 (6.8 – 14.7) 11.01 ± 0.73* (9.96 – 11.65) 16.17 ± 0.83* (15.17 – 16.98)

Std 1 1 12.1 11.73 15.23

Std 2 1 7.2 11.61 16.18

Teresina Isoline 3 7.6 ± 0.8 (6.7 – 8.1) 10.29 ± 0.59 (9.61 – 10.70) 15.10 ± 0.97 (14.03 – 15.93)

CV127 3 8.9 ± 0.7* (8.5 – 9.7) 10.58 ± 0.66 (9.88 – 11.19) 15.68 ± 1.07 (15.04 – 16.91)

CV127

+ imi 3 9.0 ± 0.1* (8.9 – 9.1) 13.00 ± 0.52* (12.41 – 13.40) 16.75 ± 1.64 (14.96 – 18.17)

Std 1 1 7.8 9.94 15.59

Std 2 1 7.4 12.08 12.26

Vilhena Isoline 4 7.6 ± 1.0 (6.7 – 8.5) 10.94 ± 1.21 (9.26 – 12.05) 12.32 ± 1.80 (11.32 – 15.02)

CV127 4 7.8 ± 0.4 (7.3 – 8.3) 8.92 ± 0.90* (8.09 – 9.78) 12.67 ± 0.63 (11.86 – 13.32)

CV127

+ imi 4 7.4 ± 0.9 (6.2 – 8.1) 8.57 ± 1.06* (7.35 – 9.81) 13.16 ± 0.49 (12.53 – 13.66)

Std 1 1 8.4 8.89 12.72

Std 2 1 7.6 12.59 12.70

Brasília Isoline 4 8.1 ± 0.8 (7.2 – 8.9) 10.14 ± 1.23 (8.73 – 11.70) 14.30 ± 1.19 (12.67 – 15.50)

CV127 4 7.8 ± 0.7 (7.2 – 8.5) 9.16 ± 0.63 (8.37 – 9.86) 14.32 ± 0.55 (13.73 – 15.05)

CV127

+ imi 4 7.3 ± 0.4 (6.7 – 7.6) 10.03 ± 0.48 (9.44 – 10.57) 14.85 ± 1.36 (13.43 – 16.68)

Std 1 1 7.7 10.84 15.00

Std 2 1 7.3 11.22 13.23

* Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 235 of 375Table D-9. Amino Acid Composition of Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 SeasonLocation/TreatmentAspThrSerGluProGlyAlaCysValSanto Antôniode GoiásMean±Standard Deviation (range)g/100 g dry weightIsoline4.53±0.33(4.25 – 5.00)1.55±0.09(1.46 – 1.68)2.07±0.13(1.93 – 2.25)7.47±0.48(7.20 – 8.19)2.06±0.10(1.92 – 2.16)1.59±0.11(1.49 – 1.74)1.62±0.08(1.57 – 1.74)0.53 ± 0.01(0.51 – 0.53)1.59±0.17(1.36 – 1.76)CV1274.50±0.13(4.33 – 4.64)1.53±0.04(1.47 – 1.56)2.04±0.05(1.97 – 2.09)7.51±0.29(7.10 – 7.79)1.91±0.06(1.82 – 1.95)1.63±0.05(1.56 – 1.68)1.58±0.05(1.52 – 1.63)0.53 ± 0.01(0.52 – 0.54)1.60±0.11(1.44 – 1.70)CV127+ imi4.85±0.34(4.36 – 5.12)1.63±0.11(1.47 – 1.72)2.17±0.14(1.95 – 2.26)8.01±0.54(7.22 – 8.42)2.02±0.13(1.83– 2.11)1.72±0.10(1.57 – 1.79)1.67±0.09(1.54 – 1.74)0.53 ± 0.01(0.52 – 0.54)1.68±0.10(1.53 – 1.74)Std 1 4.72 1.55 2.16 7.68 1.99 1.71 1.67 0.54 1.63Std 2 4.13 1.40 1.90 6.58 1.78 1.46 1.50 0.52 1.50UberabaIsoline5.02±0.16(4.79 – 5.16)1.63±0.03(1.60 – 1.66)2.28±0.08(2.17 – 2.34)8.17±0.37(7.63 – 8.46)2.12±0.06(2.04 – 2.17)1.78±0.09(1.65 – 1.86)1.75±0.05(1.68 – 1.78)0.54 ± 0.01(0.53 – 0.56)1.76±0.04(1.70 – 1.79)CV1274.36±0.45*(3.70 – 4.66)1.46±0.14(1.25 – 1.56)1.98±0.19*(1.71 – 2.12)7.17±0.73*(6.17 – 7.82)1.81±0.17*(1.65 – 1.96)1.57±0.14*(1.37 – 1.67)1.56±0.14*(1.36 – 1.67)0.50 ± 0.01*(0.48 – 0.51)1.59±0.13(1.42 – 1.71)CV127+ imi4.28±0.37*(3.83 – 4.61)1.46±0.14(1.30 – 1.61)1.96±0.19*(1.76 – 2.16)7.01±0.64*(6.20 – 7.56)1.87±0.13*(1.67 – 1.94)1.55±0.14*(1.39 – 1.71)1.53±0.14*(1.37 – 1.68)0.51 ± 0.01*(0.50 – 0.52)1.57±0.16(1.41 – 1.74)Std 1 4.44 1.50 2.05 7.24 1.87 1.66 1.62 0.51 1.59Std 2 4.64 1.52 2.14 7.52 1.97 1.68 1.68 0.49 1.67Sete LagoasIsoline4.75±0.61(4.02 – 5.37)1.59±0.21(1.34 – 1.80)2.12±0.24(1.83 – 2.39)7.77±1.02(6.59 – 8.92)2.01±0.24(1.74 – 2.32)1.66±0.18(1.45 – 1.87)1.69±0.20(1.45 – 1.92)0.52 ± 0.01(0.51 – 0.53)1.73±0.22(1.46 – 1.98)CV1274.44±0.22(4.20 – 4.65)1.48±0.07(1.38 – 1.54)1.96±0.08(1.87 – 2.03)7.33±0.31(6.95 – 7.60)1.91±0.08(1.85 – 2.01)1.60±0.06(1.52 – 1.67)1.59±0.08(1.48 – 1.66)0.51 ± 0.02(0.50 – 0.54)1.59±0.08(1.47 – 1.66)CV127+ imi4.86±0.33(4.43 – 5.17)1.62±0.07(1.57 – 1.70)2.15±0.12(2.02 – 2.29)8.04±0.42(7.53 – 8.45)2.04±0.09(1.90 – 2.12)1.72±0.07(1.64 – 1.81)1.71±0.04(1.67 – 1.76)0.51 ± 0.01(0.50 – 0.52)1.73±0.05(1.68 – 1.79)Std 1 4.33 1.51 1.96 7.05 1.84 1.56 1.61 0.53 1.64Std 2 4.14 1.43 1.90 6.83 1.77 1.50 1.52 0.50 1.63CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 236 of 375Table D-9. continued.Location/TreatmentAspThrSerGluProGlyAlaCysValLondrinaMean±Standard Deviation (range)g/100 g dry weightIsoline4.42±0.09(4.32 – 4.50)1.53±0.05(1.48 – 1.59)2.01±0.06(1.95 – 2.08)7.35±0.20(7.13 – 7.59)1.87±0.04(1.84 – 1.93)1.60±0.05(1.55 – 1.66)1.57±0.04(1.52 – 1.62)0.54 ± 0(0.54)1.59±0.04(1.55 – 1.63)CV1274.23±0.40(3.67 – 4.52)1.49±0.15(1.26 – 1.58)1.97±0.20(1.70 – 2.18)7.01±0.66(6.10 – 7.57)1.82±0.17(1.56 – 1.93)1.57±0.16(1.33 – 1.69)1.55±0.16(1.32 – 1.64)0.52 ± 0.01*(0.51 – 0.53)1.59±0.17(1.34 – 1.68)CV127+ imi4.50±0.15(4.36 – 4.72)1.55±0.01(1.54 – 1.56)2.07±0.07(2.01 – 2.16)7.48±0.27(7.19 – 7.85)1.92±0.05(1.86 – 1.99)1.65±0.05(1.60 – 1.72)1.65±0.04(1.61 – 1.70)0.52 ± 0.01*(0.52 – 0.54)1.66±0.05(1.60 – 1.72)Std 14.27 1.52 1.92 6.98 1.76 1.56 1.57 0.53 1.58Std 24.43 1.51 2.00 7.25 1.86 1.58 1.59 0.52 1.61BrasíliaIsoline4.61±0.20(4.44 – 4.86)1.55±0.07(1.48 – 1.63)2.04±0.08(1.95 – 2.13)7.51±0.34(7.21 – 7.92)1.89±0.10(1.78 – 2.00)1.63±0.08(1.54 – 1.71)1.62±0.07(1.56 – 1.70)0.57 ± 0.01(0.55 – 0.58)1.65±0.05(1.59 – 1.70)CV1274.51±0.10(4.42 – 4.65)1.50±0.02(1.48 – 1.51)2.02±0.02(1.99 – 2.04)7.29±0.16(7.10 – 7.47)1.74±0.03(1.69 – 1.77)1.63±0.02(1.61 - 1.66)1.59±0.03(1.56 – 1.63)0.55 ± 0.01*(0.54 – 0.56)1.56±0.04(1.52 – 1.61)CV127+ imi4.64±0.10(4.50 – 4.74)1.54±0.05(1.49 – 1.60)2.09±0.05(2.02 – 2.14)7.49±0.16(7.30 – 7.69)1.80±0.05(1.76 – 1.86)1.68±0.03(1.64 – 1.71)1.63±0.04(1.57 – 1.66)0.55 ± 0.01*(0.54 – 0.56)1.64±0.06(1.56 – 1.71)Std 14.26 1.49 1.88 6.93 1.76 1.55 1.55 0.53 1.58Std 23.94 1.36 1.78 6.16 1.58 1.41 1.39 0.56 1.40Santo Antôniode PosseIsoline4.54±0.28(4.32 – 4.94)1.52±0.07(1.47 – 1.63)2.02±0.13(1.92 – 2.20)7.39±0.42(7.02 – 7.99)1.94±0.23(1.81 – 2.28)1.64±0.11(1.57 – 1.81)1.62±0.12(1.54 – 1.79)0.56 ± 0.01(0.55 – 0.58)1.64±0.11(1.54 – 1.79)CV1274.74±0.51(4.14 – 5.24)1.54±0.26(1.27 – 1.70)2.17±0.27(1.79 – 2.41)7.52±1.06(6.33 – 8.46)2.01±0.28(1.70 – 2.31)1.70±0.24(1.45 – 1.94)1.69±0.21(1.48 – 1.89)0.53 ± 0.01*(0.52 – 0.53)1.69±0.27(1.42 – 1.94)CV127+ imi4.54±0.59(3.66 – 4.91)1.53±0.19(1.25 – 1.67)2.01±0.26(1.63 – 2.19)7.21±0.96(5.83 – 8.00)1.86±0.27(1.49 – 2.11)1.60±0.20(1.32 – 1.74)1.58±0.22(1.27 – 1.73)0.53 ± 0.01*(0.52 – 0.54)1.58±0.22(1.26 – 1.76)Std 14.17 1.46 1.88 6.66 1.64 1.50 1.50 0.53 1.46Std 24.32 1.51 1.94 6.93 2.05 1.51 1.56 0.53 1.64*Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 237 of 375Table D-9.continued.Location/TreatmentMetIleLeuTyrPheLysHisArgTrpSanto Antôniode GoiásMean±Standard Deviation (range)g/100 g dry weightIsoline 0.60 ± 0.01(0.60 – 0.61)1.57±0.15(1.42 – 1.77)2.86±0.20(2.68 – 3.14)1.33±0.10(1.25 – 1.47)2.02±0.12(1.92 – 2.20)2.43±0.16(2.24 – 2.64)0.88±0.07(0.79 – 0.96)2.91±0.30(2.64 – 3.34)0.77±0.12(0.65 – 0.89)CV127 0.60 ± 0.01(0.59 – 0.61)1.57±0.08(1.46 – 1.63)2.87±0.14(2.66 – 2.97)1.31±0.05(1.25 – 1.36)1.95±0.10(1.80 – 2.01)2.45±0.10(2.30 – 2.52)0.88±0.02(0.86 – 0.91)2.93±0.13(2.75 – 3.04)0.78±0.08(0.69 – 0.86)CV127+ imi0.61 ± 0.01(0.60 – 0.62)1.67±0.13(1.48 – 1.75)3.02±0.21(2.70 – 3.13)1.36±0.09(1.23 – 1.42)2.07±0.15(1.86 – 2.19)2.57±0.18(2.30 – 2.68)0.88±0.07(0.78 – 0.94)3.16±0.16(2.92 – 3.29)0.79±0.14(0.61 – 0.94)Std 1 0.61 1.64 2.97 1.39 2.04 2.54 0.84 2.97 0.82Std 2 0.55 1.50 3.65 1.20 1.83 2.22 0.79 2.57 0.70UberabaIsoline 0.65 ± 0.02(0.63 – 0.67)1.76±0.06(1.67 – 1.80)3.18±0.10(3.03 – 3.22)1.46±0.04(1.40 – 1.49)2.18±0.06(2.09 – 2.23)2.70±0.10(2.55 – 2.78)0.94±0.02(0.92 – 0.96)3.28±0.13(3.10 – 3.35)0.74±0.12(0.59 – 0.87)CV127 0.60 ± 0.02*(0.59 – 0.63)1.50±0.13*(1.31 – 1.58)2.71±0.24*(2.37 – 2.88)1.25±0.12*(1.11 – 1.36)1.87±0.17*(1.64 – 2.00)2.33±0.19*(2.08 – 2.49)0.81±0.05*(0.74 – 0.87)2.82±0.20*(2.57 – 2.98)0.69±0.02(0.67 – 0.71)CV127+ imi0.62 ± 0.02*(0.59 – 0.64)1.52±0.13*(1.38 – 1.68)2.71±0.24*(2.48 – 2.98)1.23±0.11*(1.11 – 1.33)1.88±0.16*(1.71 – 2.07)2.36±0.19*(2.14 – 2.57)0.83±0.09*(0.75 – 0.95)2.87±0.24*(2.62 – 3.15)0.79±0.16(0.67 – 1.03)Std 1 0.60 1.58 2.83 1.35 1.96 2.46 0.81 2.82 0.57Std 2 0.55 1.71 3.03 1.38 2.09 2.54 0.86 2.94 0.82Sete LagoasIsoline 0.64 ± 0.01(0.63 – 0.65)1.67±0.22(1.43 – 1.94)2.96±0.38(2.51 – 3.09)1.35±0.16(1.17 – 1.55)2.06±0.26(1.77 – 2.37)2.50±0.32(2.12 – 2.85)0.90±0.10(0.79 – 1.03)3.19±0.42(2.75 – 3.69)0.75±0.08(0.65 – 0.83)CV127 0.63 ± 0.03(0.61 – 0.68)1.56±0.06(1.55 – 1.64)2.75±0.13(2.57 – 2.87)1.23±0.06(1.15 – 1.28)1.92±0.10(1.78 – 2.01)2.40±0.11(2.27 – 2.53)0.84±0.06(0.79 – 0.91)3.02±0.21(2.76 – 3.23)0.75±0.09(0.63 – 0.83)CV127+ imi0.63 ± 0.01(0.62 – 0.63)1.69±0.05(1.63 – 1.74)3.02±0.13(2.87 – 3.16)1.35±0.04(1.32 – 1.39)2.09±0.08(1.98 – 2.16)2.57±0.08(2.46 – 2.65)0.89±0.04(0.86 – 0.943.23±0.10(3.10 – 3.33)0.79±0.16(0.70 – 1.02)Std 1 0.65 1.60 2.75 1.32 1.92 2.38 0.82 2.86 0.94Std 2 0.59 1.55 2.73 1.20 1.88 2.31 0.80 2.74 0.77CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 238 of 375Table D-9.continued.Location/TreatmentMetIleLeuTyrPheLysHisArgTrpLondrinaMean±Standard Deviation (range)g/100 g dry weightIsoline0.64 ± 0.01(0.63 – 0.64)1.52±0.05(1.48 – 1.59)2.72±0.08(2.64 – 2.82)1.28±0.03(1.25 – 1.32)1.85±0.05(1.79 – 1.92)2.39±0.05(2.33 – 2.44)0.80±0.01(0.79 – 0.81)2.80±0.08(2.69 – 2.89)0.72±0.09(0.63 – 0.83)CV1270.62 ± 0.01*(0.60 – 0.63)1.51±0.15(1.28 – 1.59)2.69±0.26(2.31 – 2.83)1.27±0.13(1.08 – 1.35)1.85±0.18(1.59 – 1.97)2.35±0.23(2.00 – 2.49)0.79±0.08(0.69 – 0.86)2.76±0.27(2.35 – 2.92)0.70±0.07(0.62 – 0.76)CV127+ imi0.62 ± 0.01*(0.62 – 0.63)1.60±0.03(1.58 – 1.64)2.88±0.08(2.82 – 2.99)1.35±0.02(1.33 – 1.38)1.98±0.04(1.95 – 2.03)2.47±0.06(2.40 – 2.54)0.85±0.03(0.83 – 0.89)2.94±0.05(2.90 – 3.01)0.73±0.10(0.60 – 0.81)Std 10.631.48 2.59 1.27 1.78 2.31 0.75 2.63 0.72Std 20.601.53 2.76 1.27 1.88 2.36 0.79 2.75 0.72BrasíliaIsoline0.64 ± 0.02(0.62 – 0.66)1.56±0.06(1.51 – 1.65)2.82±0.11(2.72 – 2.96)1.30±0.05(1.23 – 1.34)1.92±0.09(1.82 – 2.03)2.43±0.10(2.31 – 2.54)0.86±0.07(0.78 – 0.94)3.00±0.18(2.78 – 3.22)0.75±0.08(0.67 – 0.84)CV1270.64 ± 0.02(0.62 – 0.66)1.57±0.03(1.54 – 1.61)2.73±0.04(2.70 – 2.79)1.25±0.01(1.24 – 1.26)1.89±0.04(1.87 – 1.95)2.31±0.03(2.28 – 2.35)0.77±0.03(0.75 – 0.82)2.87±0.07(2.82 – 2.97)0.75±0.13(0.60 – 0.92)CV127+ imi0.63 ± 0.01(0.63 – 0.64)1.62±0.05(1.57 – 1.68)2.80±0.07(2.71 – 2.89)1.28±0.04(1.24 – 1.32)1.96±0.05(1.90 – 2.01)2.39±0.06(2.32 – 2.46)0.84±0.01(0.83 – 0.84)2.96±0.04(2.90 – 2.99)0.75±0.13(0.64 – 0.94)Std 10.631.49 2.60 1.22 1.77 2.30 0.81 2.65 0.66Std 20.601.38 2.45 1.12 1.64 2.08 0.72 2.34 0.77Santo Antôniode PosseIsoline0.62 ± 0.01(0.61 – 0.63)1.59±0.09(1.51 – 1.71)2.81±0.14(2.73 – 3.01)1.33±0.06(1.29 – 1.42)1.93±0.09(1.87 – 2.06)2.45±0.18(2.29 – 2.70)0.87±0.04(0.83 – 0.93)3.03±0.18(2.84 – 3.28)0.66±0.07(0.59 – 0.72)CV1270.61 ± 0.02(0.59 – 0.63)1.64±0.25(1.36 – 1.88)2.93±0.44(2.45 – 3.36)1.32±0.17(1.15 – 1.50)2.00±0.30(1.66 – 2.29)2.58±0.37(2.17 – 2.93)0.91±0.14(0.77 – 1.04)3.06±0.45(2.56 – 3.51)0.67±0.07(0.61 – 0.77)CV127+ imi0.60 ± 0.01(0.59 – 0.61)1.56±0.22(1.24 – 1.70)2.78±0.37(2.24 – 3.04)1.28±0.16(1.06 – 1.40)1.89±0.26(1.52 – 2.08)2.40±0.33(1.93 – 2.66)0.85±0.13(0.67 – 0.97)2.90±0.39(2.36 – 3.20)0.73±0.12(0.63 – 0.91)Std 10.601.39 2.51 1.22 1.69 2.18 0.74 2.49 0.64Std 20.561.61 2.76 1.32 1.97 2.31 0.76 2.75 1.16*Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 239 of 375Table D-10. Amino Acid Composition of Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocation/TreatmentAspThrSerGluProGlyAlaCysValMean±Standard Deviation (range)g/100 g dry weightSanto Antôniode GoiásIsoline4.50±0.17(4.26 – 4.66)1.46±0.05(1.41 – 1.50)1.95±0.09(1.83 – 2.05)7.30±0.16(7.06 – 7.41)1.85±0.09(1.76 – 1.95)1.62±0.06(1.55 – 1.68)1.69±0.02(1.67 – 1.71)0.54 ± 0.01(0.53 – 0.55)1.77±0.05(1.72 – 1.83)CV1274.48±0.20(4.23 – 4.68)1.47±0.11(1.35 – 1.62)1.92±0.06(1.85 – 1.99)7.18±0.28(6.84 – 7.52)1.86±0.09(1.80 – 1.99)1.59±0.08(1.50 – 1.69)1.67±0.16(1.52 – 1.90)0.55 ± 0.01*(0.55 – 0.56)1.72±0.09(1.63 – 1.84)CV127+ imi4.50±0.11(4.41 – 4.65)1.50±0.12(1.39 – 1.67)1.93±0.05(1.86 – 1.97)7.23±0.19(7.00 – 7.46)1.67±0.38(1.11– 1.88)1.65±0.13(1.53 – 1.83)1.69±0.13(1.54 – 1.86)0.55 ± 0.01*(0.54 – 0.56)1.73±0.11(1.56 – 1.79)Std 14.77 1.51 2.05 7.54 1.87 1.72 1.74 0.53 1.83Std 24.93 1.55 2.21 8.02 2.05 1.72 1.79 0.54 1.87TeresinaIsoline4.43±0.20(4.20 – 4.57)1.40±0.05(1.36 – 1.45)1.90±0.05(1.85 – 1.95)7.00±0.28(6.68 – 7.22)1.80±0.09(1.70 – 1.86)1.53±0.03(1.50 – 1.55)1.51±0.06(1.45 – 1.56)0.44 ± 0.01(0.43 – 0.44)1.41±0.03(1.37 – 1.43)CV1274.05±0.14*(3.89 – 4.15)1.17±0.03*(1.15 – 1.21)1.75±0.05*(1.70 – 1.80)6.35±0.16*(6.20 – 6.51)1.73±0.08(1.64 – 1.79)1.42±0.02*(1.41 – 1.44)1.45±0.06(1.39 – 1.51)0.46 ± 0.04(0.43 – 0.51)1.51±0.02*(1.50 – 1.53)CV127+ imi4.38±0.17(4.19 – 4.52)1.20±0.03*(1.17 – 1.23)1.74±0.09*(1.65 – 1.82)6.46±0.27*(6.17 – 6.69)1.75±0.14(1.62 – 1.89)1.44±0.04*(1.40 – 1.47)1.66±0.18(1.46 – 1.82)0.47 ± 0.02(0.46 – 0.50)1.54±0.02*(1.51 – 1.55)Std 15.06 1.34 1.91 8.55 2.05 1.60 1.58 0.45 1.38Std 24.03 1.31 1.78 6.47 1.69 1.41 1.41 0.44 1.31CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 240 of 375Table D-10. continued.Location/TreatmentAspThrSerGluProGlyAlaCysValMean±Standard Deviation (range)g/100 g dry weightVilhenaIsoline4.20±0.23(3.91 – 4.41)1.40±0.10(1.29 – 1.50)1.85±0.09(1.73 – 1.93)6.82±0.35(6.40 – 7.18)1.77±0.07(1.70 – 1.85)1.52±0.09(1.44 – 1.64)1.48±0.04(1.43 – 1.51)0.54 ± 0.01(0.53 – 0.54)1.58±0.05(1.50 – 1.61)CV1274.12±0.16(3.91 – 4.28)1.37±0.06(1.31 – 1.44)1.78±0.06(1.71 – 1.86)6.73±0.33(6.34 – 7.11)1.79±0.06(1.72 – 1.85)1.42±0.08(1.34 – 1.52)1.69±0.09*(1.58 – 1.79)0.55 ± 0.01(0.53 – 0.56)1.66±0.06(1.61 – 1.74)CV127+ imi4.27±0.25(4.01 – 4.59)1.39±0.05(1.31 – 1.43)1.83±0.10(1.71 – 1.95)6.83±0.29(6.55 – 7.22)1.84±0.04(1.79 – 1.88)1.45±0.16(1.34 – 1.68)1.66±0.05*(1.58 – 1.70)0.55 ± 0.01(0.54 – 0.56)1.62±0.06(1.53 – 1.66)Std 1 4.66 1.47 2.00 7.51 2.03 1.61 1.57 0.59 1.65Std 2 4.32 1.42 1.90 6.86 1.81 1.55 1.54 0.56 1.63BrasíliaIsoline3.63±0.11(3.50 – 3.73)1.25±0.03(1.21 – 1.28)1.61±0.05(1.54 – 1.66)5.90±0.16(5.72 – 6.07)1.56±0.04(1.50 – 1.60)1.33±0.05(1.28 – 1.39)1.37±0.07(1.29 – 1.45)0.51 ± 0.01(0.50 – 0.51)1.45±0.09(1.35 – 1.54)CV1273.93±0.41(3.32 – 4.18)1.33±0.12(1.14 – 1.40)1.76±0.13*(1.56 – 1.84)6.29±0.54(5.49 – 6.63)1.68±0.10*(1.56 – 1.80)1.45±0.14(1.24 - 1.55)1.50±0.12(1.33 – 1.59)0.51 ± 0.01(0.50 – 0.51)1.64±0.12*(1.49 – 1.76)CV127+ imi4.03±0.15(3.85 – 4.17)1.36±0.06(1.29 – 1.43)1.79±0.04*(1.74 – 1.82)6.53±0.08*(6.45 – 6.63)1.69±0.03*(1.65 – 1.72)1.47±0.06(1.40 – 1.53)1.49±0.10(1.38 – 1.61)0.51 ± 0.01(0.50 – 0.51)1.56±0.13(1.41 – 1.73)Std 1 4.11 1.38 1.84 6.57 1.75 1.53 1.58 0.51 1.72Std 2 3.70 1.23 1.67 5.90 1.60 1.35 1.41 0.54 1.53CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 241 of 375Table D-10. continuedLocation/TreatmentMetIleLeuTyrPheLysHisArgTrpMean±Standard Deviation (range)g/100 g dry weightSanto Antôniode GoiásIsoline 0.63 ± 0.02(0.62 – 0.66)1.54±0.05(1.50 – 1.62)2.79±0.06(2.72 – 2.86)1.30±0.04(1.24 – 1.33)1.89±0.05(1.82 – 1.94)2.38±0.06(2.31 – 2.46)1.29±0.02(1.27 – 1.32)3.20±0.11(3.11 – 3.34)0.66±0.07(0.57 – 0.74)CV127 0.66 ± 0.01*(0.65 – 0.66)1.60±0.16(1.46 – 1.83)2.66±0.15(2.45 – 2.79)1.22±0.09(1.12 – 1.33)1.93±0.16(1.78 – 2.16)2.43±0.22(2.27 – 2.76)1.23±0.18(0.99 – 1.38)3.10±0.10(2.98 – 3.22)0.63±0.09(0.50 – 0.70)CV127+ imi0.66 ± 0.01*(0.65 – 0.66)1.65±0.22(1.45 – 1.97)2.75±0.10(2.66 – 2.85)1.23±0.11(1.10 – 1.38)1.84±0.06(1.78 – 1.90)2.43±0.13(2.34 – 2.62)1.21±0.27(0.91 – 1.52)3.11±0.03(3.07 – 3.15)0.62±0.12(0.51 – 0.76)Std 1 0.62 1.57 2.92 1.39 1.96 2.45 1.29 3.45 0.61Std 2 0.60 1.65 3.15 1.45 2.12 2.58 1.34 3.34 0.65TeresinaIsoline 0.54 ± 0.01(0.53 – 0.55)1.55±0.04(1.51 – 1.58)2.53±0.07(2.46 – 2.60)1.25±0.03(1.22 – 1.28)1.82±0.06(1.77 – 1.88)2.26±0.08(2.18 – 2.33)0.82±0.02(0.80 – 0.83)2.93±0.11(2.80 – 3.00)0.61±0.01(0.60 – 0.62)CV127 0.56 ± 0.05(0.51 – 0.61)1.46±0.03(1.43 – 1.48)2.49±0.04(2.46 – 2.54)1.09±0.03*(1.05 – 1.11)1.76±0.02(1.74 – 1.78)2.16±0.04(2.12 – 2.20)1.27±0.08*(1.18 – 1.34)2.62±0.11*(2.51 – 2.73)0.60±0.04(0.56 – 0.63)CV127+ imi0.58 ± 0.01(0.58 – 0.60)1.45±0.07(1.37 – 1.51)2.50±0.06(2.44 – 2.55)1.11±0.03*(1.09 – 1.14)1.77±0.07(1.69 – 1.81)2.13±0.13(1.99 – 2.25)1.18±0.10*(1.10 – 1.29)2.81±0.09(2.71 – 2.88)0.61±0.08(0.55 – 0.70)Std 1 0.56 1.35 2.85 1.24 1.90 2.29 0.83 2.85 0.63Std 2 0.53 1.47 2.39 1.16 1.71 2.09 0.73 2.65 0.64CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 242 of 375Table D-10. continued.Location/TreatmentMetIleLeuTyrPheLysHisArgTrpMean±Standard Deviation (range)g/100 g dry weightVilhenaIsoline 0.62 ± 0.02(0.60 – 0.64)1.49±0.07(1.43 – 1.58)2.59±0.09(2.49 – 2.67)1.17±0.04(1.13 – 1.22)1.78±0.06(1.70 – 1.85)2.32±0.14(2.16 – 2.47)1.17±0.08(1.07 – 1.26)2.80±0.11(2.66 – 2.90)0.63±0.05(0.60 – 0.70)CV127 0.64 ± 0.01(0.63 – 0.66)1.20±0.11*(1.11 – 1.35)2.69±0.14(2.54 – 2.86)1.08±0.04*(1.05 – 1.14)1.78±0.09(1.68 – 1.90)2.21±0.06(2.13 – 2.28)1.85±0.32*(1.40 – 2.09)2.61±0.08*(2.50 – 2.69)0.69±0.08(0.60 – 0.76)CV127+ imi0.64 ± 0.02(0.62 – 0.66)1.21±0.19*(1.08 – 1.48)2.59±0.03(2.56 – 2.62)1.09±0.05*(1.02 – 1.14)1.77±0.06(1.70 – 1.85)2.27±0.11(2.17 – 2.41)1.80±0.43*(1.17 – 2.13)2.68±0.14(2.53 – 2.86)0.72±0.11(0.58 – 0.84)Std 1 0.69 1.57 2.77 1.18 1.93 2.51 1.14 3.24 0.73Std 2 0.62 1.53 2.72 1.19 1.90 2.36 1.23 2.84 0.72BrasíliaIsoline 0.58 ± 0.01(0.58 – 0.59)1.29±0.06(1.22 – 1.35)2.31±0.09(2.19 – 2.41)1.03±0.06(0.98 – 1.11)1.58±0.05(1.52 – 1.63)2.03±0.04(1.97 – 2.06)1.08±0.03(1.04 – 1.12)2.45±0.06(2.37 – 2.50)0.65±0.08(0.53 – 0.71)CV127 0.59 ± 0.01(0.58 – 0.60)1.40±0.20(1.10 – 1.55)2.55±0.17*(2.30 – 2.69)1.10±0.06(1.01 – 1.16)1.71±0.11*(1.55 – 1.78)2.34±0.18*(2.23 – 2.61)1.43±0.49(0.93 – 2.11)2.71±0.18*(2.44 – 2.85)0.66±0.10(0.54 – 0.79)CV127+ imi0.58 ± 0.01(0.58 – 0.59)1.41±0.08(1.34 – 1.52)2.58±0.10*(2.47 – 2.69)1.12±0.06(1.06 – 1.21)1.74±0.07*(1.65 – 1.80)2.21±0.06*(2.12 – 2.27)1.22±0.19(0.96 – 1.39)2.74±0.14*(2.54 – 2.84)0.73±0.09(0.59 – 0.79)Std 1 0.59 1.53 2.63 1.25 1.79 2.28 1.17 2.72 0.56Std 2 0.57 1.37 2.43 1.14 1.64 2.03 1.05 2.49 0.69*Statistically significantly different from the isoline control at P <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 243 of 375Table D-11. Fatty Acid Composition of Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 SeasonLocation/TreatmentC14:0MyristicC16:0PalmiticC18:0StearicC18:1OleicC18:2LinoleicC18:3LinolenicC20:0ArachidicC20:1EicosenoicC22:0BehenicSanto Antôniode GoiásMean±Standard Deviation (range)g/100 g DWIsoline<0.02(nd^ – 0.02)2.36±0.03(2.31 – 2.40)0.75±0.09(0.69 – 0.88)4.70±0.21(4.42 – 4.93)11.39±0.13(11.23 – 11.55)1.40±0.07(1.30 – 1.46)0.08±0.01(0.08 – 0.09)0.03±0.01(0.02 – 0.03)0.10±0.01(0..09 – 0.10)CV1270.02±0(0.02)2.54±0.07*(2.45 – 2.61)0.76±0.02(0.74 – 0.79)5.43±0.14*(5.27 – 5.61)11.85±0.18*(11.64 – 12.05)1.38±0.06(1.32 – 1.45)0.08±0.01(0.07 – 0.09)0.03±0.01(0.02 – 0.04)0.10±0.01(0.09 – 0.11)CV127+ imi<0.02(nd – 0.02)2.44±0.04(2.40 – 2.48)0.71±0.01(0.70 – 0.73)5.97±0.57*(5.40 – 6.57)11.02±0.17*(10.86 – 11.26)1.25±0.07*(1.21 – 1.35)0.07±0.01(0.07 – 0.09)0.04±0*(0.04)0.11±0.01(0.10 – 0.11)Std 10.02 2.08 0.66 4.06 10.75 1.56 0.06 0.02 0.08Std 20.02 2.21 0.70 4.94 12.71 1.65 0.07 0.02 0.09UberabaIsoline0.02±0(0.02)2.21±0.13(2.08 – 2.39)0.76±0.02(0.74 – 0.80)4.66±0.19(4.49 – 4.90)11.07±0.67(10.68 – 12.06)1.37±0.08(1.31 – 1.48)0.08±0.01(0.08 – 0.09)0.03±0.01(0.02 – 0.03)0.10±0.01(0.10 – 0.11)CV1270.02±0(0.02)2.43±0.07*(2.35 – 2.53)0.84±0.03*(0.81 – 0.87)5.23±0.14*(5.06 – 5.35)11.23±0.22*(11.03 – 11.53)1.27±0.02*(1.25 – 1.29)0.09±0*(0.09)0.02±0.01(0.02 – 0.03)0.11±0(0.11)CV127+ imi<0.02(nd – 0.02)2.47±0.05*(2.41 – 2.53)0.84±0.01*(0.83 – 0.85)5.50±0.08*(5.39 – 5.59)11.26±0.26*(10.95 – 11.57)1.24±0.03*(1.21 – 1.28)0.09±0*(0.09)0.02±0*(0.02)0.10±0.01(0.10 – 0.11)Std 10.02 2.18 0.76 3.99 11.35 1.65 0.06 0.03 0.08Std 2nd 2.19 0.76 5.07 13.09 1.68 0.07 0.03 0.09Sete LagoasIsoline<0.02(nd – 0.02)2.23±0.04(2.20– 2.28)0.89±0.04(0.84 – 0.93)5.02±0.09(4.93 – 5.11)11.01±0.18(10.85 – 11.26)1.27±0.04(1.23 – 1.32)0.09±0.01(0.08 – 0.10)0.03±0.01(0.02 – 0.04)0.11±0.01(0.10 – 0.12)CV127<0.02(nd – 0.02)2.36±0.10*(2.26 – 2.47)0.93±0.02(0.89 – 0.94)5.56±0.08*(5.44 – 5.62)11.14±0.34(10.77 – 11.53)1.23±0.04(1.19 – 1.27)0.09±0(0.09)0.02±0*(0.02)0.10±0.01(0.09 – 0.11)CV127+ imi<0.02(nd – 0.02)2.42±0.03*(2.39– 2.45)0.91±0.02(0.89 – 0.92)6.03±0.12*(5.86 – 6.15)11.29±0.30(11.05 – 11.73)1.21±0.07(1.16 – 1.31)0.09±0(0.09)0.02±0.01(0.02 – 0.03)0.11±0(0.11)Std 1nd 2.28 0.84 4.28 11.39 1.54 0.08 0.03 0.08Std 2nd 2.34 0.84 5.14 12.94 1.52 0.07 0.03 0.09CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 244 of 375Table D-11. continued.Location/TreatmentC14:0MyristicC16:0PalmiticC18:0StearicC18:1OleicC18:2LinoleicC18:3LinolenicC20:0ArachidicC20:1EicosenoicC22:0BehenicLondrinaMean±Standard Deviation (range)g/100 g DWIsoline <0.02(nd – 0.02)2.58±0.05(2.52 – 2.64)0.79±0.02(0.77 – 0.81)5.17±0.18(4.95 – 5.32)11.86±0.12(11.73 – 11.98)1.48±0.06(1.41 – 1.53)0.09±0(0.09)0.03±0.01(0.02 – 0.04)0.12±0.01(0.11 – 0.13)CV1270.02±0(0.02)2.61±0.03(2.57 – 2.63)0.78±0.01(0.77 – 0.78)5.41±0.19(5.18 – 5.63)11.87±0.14(11.66 – 11.96)1.37±0.03*(1.33 – 1.41)0.09±0(0.09)0.03±0.01(0.02 – 0.04)0.10±0.01*(0.10 – 0.11)CV127+ imi0.02±0(0.02)2.62±0.03(2.58 – 2.64)0.80±0.01(0.78 – 0.82)5.50±0.19*(5.24 – 5.67)11.91±0.12(11.76 – 12.06)1.36±0.02*(1.35 – 1.39)0.09±0(0.09)0.03±0.01(0.02 – 0.04)0.10±0.01*(0.09 – 0.11)Std 1 nd 2.49 0.79 4.22 12.21 1.85 0.07 0.02 0.09Std 2 nd 2.42 0.81 5.41 13.22 1.63 0.07 0.03 0.10BrasíliaIsoline0.02±0(0.02)2.37±0.06(2.29 – 2.41)0.86±0.04(0.81 – 0.89)4.69±0.24(4.43 – 4.97)10.18±0.22(9.97 – 10.38)1.32±0.02(1.31 – 1.35)0.09±0.01(0.09 – 0.10)0.04±0.01(0.03 – 0.04)0.11±0.01(0.10 – 0.12)CV1270.02±0(0.02)2.41±0.08(2.29 – 2.47)0.84±0.01(0.82 – 0.85)5.10±0.20(4.96 – 5.38)10.25±0.42(9.63 – 10.52)1.25±0.06*(1.17 – 1.31)0.09±0.01(0.08 – 0.10)0.03±0.01(0.02 – 0.04)0.10±0.01(0.09 – 0.12)CV127+ imi0.02±0(0.02)2.41±0.07(2.36 – 2.51)0.80±0.03*(0.77 – 0.82)4.76±0.35(4.44 – 5.25)10.26±0.30(10.09 – 10.71)1.26±0.01(1.25 – 1.28)0.08±0.01*(0.08 – 0.09)0.02±0*(0.02)0.10±0.01(0.10 – 0.11)Std 1 0.02 2.45 0.85 4.20 11.51 1.69 0.09 0.04 0.11Std 2 0.02 2.42 0.86 4.82 12.63 1.69 0.09 0.02 0.09Santo Antôniode PosseIsoline0.02±0(0.02)2.38±0.05(2.34 – 2.44)0.85±0.01(0.84 – 0.86)4.79±0.07(4.74 – 4.89)10.84±0.22(10.64 – 11.15)1.32±0.02(1.30 – 1.34)0.10±0.01(0.09 – 0.10)0.03±0.01(0.02 – 0.04)0.12±0(0.12)CV1270.02±0(0.02)2.61±0.08*(2.57 – 2.73)0.90±0.04*(0.88 – 0.97)5.35±0.20*(5.23 – 5.65)11.69±0.49*(11.40 – 12.43)1.31±0.07(1.26 – 1.41)0.09±0.01(0.09 – 0.11)0.04±0.01(0.02 – 0.04)0.11±0.01(0.11 – 0.12)CV127+ imi0.02±0(0.02)2.63±0.11*(2.48 – 2.73)0.90±0.03*(0.87 – 0.94)5.45±0.19*(5.19 – 5.62)11.89±0.33*(11.59 – 12.28)1.32±0.02(1.29 – 1.34)0.09±0.01(0.09 – 0.10)0.04±0*(0.04)0.12±0.01(0.11 – 0.13)Std 1 0.02 2.38 0.78 4.05 11.72 1.65 0.08 0.03 0.09Std 2 0.02 2.34 0.80 4.83 13.01 1.61 0.07 0.04 0.09*Statistically significantly different from the isoline control at P < 0.05.^ nd = not detectedCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 245 of 375Table D-12. Fatty Acid Composition of Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocation/TreatmentC14:0MyristicC16:0PalmiticC18:0StearicC18:1OleicC18:2LinoleicC18:3LinolenicC20:0ArachidicC22:0BehenicC24:0TetracosanoicMean±Standard Deviation (range)g/100 g DWSanto Antôniode GoiásIsoline0.02±0(0.02) N=21.83±0.05(1.79 – 1.89)0.85±0.05(0.80 – 0.91)4.43±0.07(4.33 – 4.49)8.71±0.24(8.48 – 9.04)1.23±0.06(1.18 –- 1.30)0.08±0.01(0.06 – 0.09)0.09±0.01(0.09 – 0.10)0.03±0(0.03)CV1270.02±0(0.02)1.91±0.04*(1.85 – 1.95)0.89±0.03(0.86 – 0.94)4.85±0.07*(4.77 – 4.91)9.05±0.36(8.79 – 9.57)1.30±0.06(1.23 – 1.38)0.08±0.01(0.08 – 0.09)0.10±0.01(0.09 – 0.11)0.03±0(0.03)CV127+ imi0.02±0(0.02) N=31.96±0.02*(1.93 – 1.98)0.91±0.04(0.87 – 0.95)5.20±0.10*(4.98 – 5.20)9.09±0.20(8.83 – 9.31)1.30±0.03(1.27 – 1.34)0.09±0.01(0.08 – 0.10)0.10±0.01(0.09 – 0.10)0.03±0(0.03)Std 1 0.01 1.80 0.83 3.87 8.10 1.37 0.07 0.08 0.01Std 2 NA 1.77 0.89 4.82 9.44 1.27 0.08 0.10 0.03TeresinaIsoline0.02±0(0.02) N=22.37±0.14(2.21 – 2.48)0.80±0.05(0.74 – 0.85)6.45±0.99(5.88 – 7.59)11.27±1.32(9.74 – 12.05)1.09±0.12(0.96 – 1.19)0.09±0.02(0.08 – 0.11)0.15±0.02(0.14 – 0.17)0.07±0.02(0.05 – 0.09)CV1270.02±0(0.02)2.58±0.11(2.48 – 2.70)0.87±0.01(0.85 – 0.88)6.86±0.78(6.07 – 7.62)11.34±1.16(10.16 – 12.47)1.02±0.11(0.92 – 1.13)0.10±0.01(0.10 – 0.11)0.14±0.01(0.13 – 0.15)0.08±0.01(0.06 – 0.09)CV127+ imiNA2.33±0.23(1.56 – 2.31)0.89±0.05*(0.84 – 0.93)8.42±1.12*(7.21 – 9.42)8.75±2.12(6.91 – 11.07)0.84±0.14*(0.74 – 1.00)0.11±0.01(0.10 – 0.12)0.16±0.02(0.14 – 0.17)0.10±0.03(0.06 – 0.12)Std 1 NA 2.14 0.70 5.48 11.49 1.26 0.06 0.11 0.04Std 2 0.02 2.09 0.76 7.04 12.44 1.19 0.06 0.12 0.08CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 246 of 375Table D-12. continued.Location/TreatmentC14:0MyristicC16:0PalmiticC18:0StearicC18:1OleicC18:2LinoleicC18:3LinolenicC20:0ArachidicC22:0BehenicC24:0TetracosanoicMean±Standard Deviation (range)g/100 g DWVilhenaIsoline NA2.24±0.05(2.18 – 2.28)0.87±0.01(0.86 – 0.88)5.09±0.08(4.99 – 5.18)10.28±0.22(10.00 – 10.54)1.38±0.03(1.33 – 1.39)0.08±0.01(0.08 – 0.09)0.11±0.01(0.10 – 0.12)0.04±0(0.04)CV1270.02±0(0.02) N=22.18±0.04(2.14 – 2.22)0.91±0.01*(0.90 – 0.91)5.57±0.04*(5.53 – 5.60)10.06±0.32(9.74 – 10.35)1.31±0.04*(1.27 – 1.34)0.09±0(0.09)0.11±0.01(0.10 – 0.12)0.04±0(0.04)CV127+ imi0.02N=12.12±0.06*(2.03 – 2.16)0.90±0.01*(0.90 – 0.91)5.59±0.06*(5.50 – 5.63)9.72±0.24*(9.41 – 9.97)1.30±0.03*(1.26 – 1.32)0.09±0.01(0.08 – 0.10)0.10±0.01(0.10 – 0.12)0.04±0(0.04)Std 1 0.02 1.88 0.91 4.52 8.51 1.33 0.09 0.09 0.03Std 2 NA 2.21 0.81 4.70 11.88 1.64 0.06 0.09 0.04BrasíliaIsoline 0.02N=11.97±0.03(1.94 – 2.00)0.72±0.02(0.69 – 0.73)4.20±0.06(4.13 – 4.25)9.55±0.14(9.47 – 9.70)1.52±0.03(1.48 – 1.54)0.07±0.01(0.07 – 0.08)0.08±0.01(0.08 – 0.09)0.02±0.01(0.02 – 0.03)CV1270.02±0(0.02)1.93±0.02(1.91 – 1.96)0.73±0.01(0.72 – 0.74)4.41±0.09*(4.29 – 4.48)9.35±0.06(9.28 – 9.39)1.54±0.03(1.51 – 1.58)0.08±0(0.08)0.08±0(0.08)0.03±0.01(0.02 – 0.03)CV127+ imi0.02±0(0.02) N=32.00±0.07(1.90 – 2.05)0.75±0.01*(0.74 – 0.76)4.52±0.05*(4.48 – 4.58)9.56±0.27(9.19 – 9.82)1.56±0.05(1.51 – 1.61)0.07±0.01(0.05 – 0.08)0.08±0(0.08)0.02±0.01(0.02 – 0.03)Std 1 0.02 2.10 0.78 3.97 10.10 1.68 0.05 0.08 0.02Std 2 NA 1.97 0.74 4.24 10.61 1.67 0.05 0.08 0.03NA=Not Available*Statistically significantly different from the isoline control at P <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 247 of 375 Table D-13. Mineral Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 + imi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grow n at Six Locations in Brazil in the 2006/07

Season

Location Treatment N Calcium Iron Phosphorus Magnesium Potassium

Mean ± Standard Deviation (range) mg/100 g dry weight

Santo

Antônio Isoline 4 258 ± 30
(222 – 296) 8.45 ± 1.48
(6.40 – 9.85) 618 ± 20
(594 – 643) 252 ± 20
(222 – 265) 1878 ± 35
(1837 – 1912)
de Goiás CV127 4 269 ± 24
(238 – 290) 8.18 ± 0.44
(7.68 – 8.69) 686 ± 60
(617 – 759) 290 ± 10*
(281 – 304) 1938 ± 46
(1908 – 2006)
CV127

+ imi 4 230 ± 16*
(214 – 247) 7.78 ± 0.68
(7.20 – 8.58) 618 ± 42
(588 – 680) 285 ± 13*
(274 – 303) 1844 ± 63
(1778 – 1918)
Std 1 1 216 9.07 580 264 2044

Std 2 1 278 10.35 559 308 1772

Uberaba Isoline 4 266 ± 19
(238 – 280) 6.84 ± 1.25
(6.01 – 8.69) 557 ± 19
(541 – 584) 226 ± 25
(204 – 260) 1857 ± 68
(1782 – 1935)
CV127 4 294 ± 23
(274 – 327) 5.96 ± 0.35
(5.56 – 6.42) 552 ± 45
(515 – 611) 231 ± 9
(218 – 240) 1803 ± 57
(1720 – 1850)
CV127

+ imi 4 296 ± 17*
(276 – 318) 5.96 ± 0.19
(5.79 – 6.22) 576 ± 32
(546 – 611) 241 ± 12
(227 – 257) 1797 ± 37
(1751 – 1833)
Std 1 1 256 6.54 541 225 1960

Std 2 1 279 7.35 527 257 1822

Sete Lagoas Isoline 4 237 ± 4
(232 – 241) 7.82 ± 0.14
(7.71 – 8.01) 703 ± 64
(666 – 799) 230 ± 7
(219 – 235) 1905 ± 81
(1785 – 1959)
CV127 4 249 ± 13
(231 – 261) 6.97 ± 0.17
(6.84 – 7.22) 728 ± 14
(712 – 745) 236 ± 5
(229 – 240) 1783 ± 21*
(1756 – 1806)
CV127

+ imi 4 240 ± 8
(231 – 248) 7.09 ± 0.22
(6.94 – 7.40) 723 ± 19
(712 – 752) 247 ± 7
(240 – 256) 1791 ± 104*
(1703 – 1907)
Std 1 1 205 7.23 674 240 2020

Std 2 1 301 8.24 719 273 1945

Londrina Isoline 4 236 ± 16
(221 – 257) 9.76 ± 0.47
(9.41 – 10.43) 740 ± 53
(677 – 805) 249 ± 6
(240 – 255) 1977 ± 56
(1902 - 2035)
CV127 4 284 ± 33*
(242 – 322) 10.27 ± 0.78
(9.18 – 10.91) 725 ± 76
(618 – 799) 306 ± 26*
(268 – 326) 2109 ± 94*
(1969 – 2164)
CV127

+ imi 4 266 ± 15*
(251 – 280) 9.92 ± 0.47
(9.41 – 10.48) 691 ± 32
(644 – 717) 289 ± 16*
(267 – 304) 2040 ± 35*
(1989 – 2065)
Std 1 1 221 9.50 729 255 2048

Std 2 1 244 9.46 742 297 2011

Brasília Isoline 4 314 ± 11
(305 – 330) 9.02 ± 0.47
(8.64 – 9.71) 786 ± 34
(763 – 834) 262 ± 5
(256 – 266) 1902 ± 29
(1874– 1936)
CV127 4 276 ± 14*
(265 – 295) 7.18 ± 0.59*
(6.67 – 8.02) 675 ± 20*
(655 – 703) 267 ± 1
(266 - 269) 1815 ± 33
(1775 – 1850)
CV127

+ imi 4 275 ± 9*
(265 – 284) 7.27 ± 0.43*
(6.78 – 7.76) 694 ± 21*
(679 – 724) 267 ± 4
(264 – 273) 1848 ± 11
(1833 – 1857)
Std 1 1 246 8.22 733 257 1911

Std 2 1 313 9.54 875 306 1933CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 248 of 375 Table D-13. continued

.

Location Treatment N Calcium Iron Phosphorus Magnesium Potassium

Mean ± Standard Deviation (range) mg/100 g dry weight

Isoline 4 296 ± 12
(281 – 309) 9.15 ± 0.16
(8.93 – 9.28) 722 ± 54
(672 – 789) 256 ± 5
(248 – 259) 2050 ± 26
(2013 – 2071) Santo

Antônio

de Posse CV127 4 291 ± 23
(261 – 312) 8.78 ± 0.28
(8.47 – 9.13) 694 ± 59
(619 – 751) 265 ± 12
(251 – 281) 1936 ± 132
(1822 – 2100)
CV127

+ imi 4 288 ± 14
(271 – 305) 8.46 ± 0.39
(8.03 – 8.97) 701 ± 48
(655 – 760) 266 ± 4
(261 – 269) 1964 ± 119
(1794 – 2069)
Std 1 1 209 8.04 620 258 2103

Std 2 1 286 9.25 745 283 1964

*Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 249 of 375Table D-14. Mineral Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 + imi), the IsolineControl and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocationTreatmentNCalciumIronPhosphorusMagnesiumPotassiumMean±Standard Deviation (range)mg/100 g dry weightSantoAntônio deGoiásIsoline 4235±14(223 – 252)8.67±0.44(8.24 – 9.24)831±14(817 – 845)240±5(235 – 245)1643±37(1593 – 1676)CV127 4267±13*(255 – 279)9.63±1.08(8.64 – 10.93)739±21*(719 – 769)268±9*(258 – 279)1794±18*(1775 – 1810)CV127+ imi4256±9*(244 – 264)9.03±0.25(8.70 – 9.26)706±41*(654 – 743)275±10*(268 – 289)1760±118*(1599 – 1875)Std1 1 192 8.68 793 230 1631Std2 1 213 9.98 808 255 1595Teresina Isoline 3354±8(345 – 359)8.44±0.41(7.98 – 8.77)758±20(735 – 771)258±7(252 – 266)1983±76(1910 – 2061)CV127 3377±48(332 – 427)8.44±0.32(8.23 – 8.81)711±63(672 – 784)277±14(261 – 288)2018±56(1970 – 2079)CV127+ imi3448±61*(377 – 484)8.45±0.49(7.90 – 8.81)797±70(733 – 871)296±14*(280 – 308)1933±95(1832 – 2021)Std1 1 329 7.93 734 264 2033Std2 1 323 8.27 703 279 1911CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 250 of 375Table D-14. continued.LocationTreatmentNCalciumIronPhosphorusMagnesiumPotassiumMean±Standard Deviation (range)mg/100 g dry weightVilhena Isoline 4234±5(230 – 242)9.90±0.84(8.96 – 10.90)704±52(655 – 752)243±12(231 – 258)1693±32(1666 – 1739)CV127 4273±13*(257 – 287)12.10±0.99*(10.70 – 13.00)708±35(679 – 759)287±18*(270 – 309)1821±101*(1713 – 1945)CV127+ imi4275±17*(260 – 297)10.33±0.99(9.00 – 11.40)753±14(741 – 770)304±6*(296 – 309)1894±22*(1875 – 1926)Std1 1 231 14.13 790 246 1815Std2 1 224 12.23 614 293 1630Brasília Isoline 4188±11(172 – 194)9.47±0.26(9.23 – 9.80)778±47(708 – 809)218±7(211 – 227)1715±80(1645– 1831)CV127 4205±17(190 – 228)9.01±0.49(8.60 – 9.70)707±38(654 – 736)276±3*(271 - 279)1911±16*(1894 – 1930)CV127+ imi4206±11(198 – 223)8.63±0.60*(7.80 – 9.20)690±90(580 – 799)277±9*(270 – 289)1887±96*(1796 – 2001)Std1 1 167 9.43 754 223 1708Std2 1 195 10.60 755 253 1738*Statistically significantly different from the isoline control at p <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 251 of 375Table D-15. Selected Vitamin Compositionof Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi),the Isoline Control and Two Conventional Soybean Varieties (Std1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 SeasonLocation/Treatmentα-tocopherol(mg/100 g DW)β-tocopherol(mg/100 g DW)γ-tocopherol(mg/100 g DW)δ-tocopherol(mg/100 g DW)Total Tocopherol(mg/100 g DW)Vitamin E(IU/100 g DW)Vitamin E(mg/100 g DW)Vitamin B1(mg/100 g DW)Folic Acid(μg/100 gDW)Santo Antôniode GoiásMean±Standard Deviation (range)Isoline2.87±0.26(2.62 – 3.19)0.40±0.21(0.20 – 0.61)18.31±1.69(17.19 – 20.83)7.04±0.71(6.01 – 7.53)27.94±3.24(23.73 – 31.54)6±1(6 – 7)5.71±0.33(5.22 – 5.96)0.61±0.12(0.48 – 0.74)362±44(320 – 424)CV1273.05±0.15(2.86 – 3.22)0.72±0.12*(0.58 – 0.86)16.70±1.07*(15.19 – 17.69)7.64±0.44(7.11 – 8.15)28.11±1.48(26.10 – 29.61)6±0(6)5.60±0.25(5.37 – 5.84)0.51±0.10(0.40 – 0.63)290±13*(276 – 304)CV127+ imi2.81±0.28(2.43 – 3.08)0.69±0.12*(0.58 – 0.81)15.88±1.45*(14.55 – 17.64)7.21±0.43(6.62 – 7.65)26.53±1.62(25.13 – 26.97)6±1(5 – 6)5.21±0.44(4.68 – 5.53)0.48±0.15(0.34 – 0.67)299±15*(282 – 316)Std 13.29 0.85 14.22 6.31 24.666 5.51 0.66 277Std 22.45 0.12 20.88 7.42 30.866 5.40 0.66 350UberabaIsoline2.82±0.25(2.54 – 3.13)0.59±0.20(0.34 – 0.82)16.51±1.09(15.48 – 18.04)6.35±0.38(6.08 – 6.90)26.31±1.61(25.05 – 28.58)6±1(5 – 6)5.17±0.20(4.91 – 5.40)0.70±0.17(0.44 – 0.79)431±23(402 – 456)CV1273.18±0.07*(3.11 – 3.25)0.93±0.09*(0.83 – 1.04)15.48±0.56(14.83 – 16.11)6.98±0.22*(6.81 – 7.30)26.57±0.65(25.60 – 26.96)6±0*(6)5.61±0.13*(5.41 – 5.70)0.46±0.05*(0.40 – 0.52)276±30*(235 – 305)CV127+ imi3.30±0.14*(3.21 – 3.51)0.98±0.17*(0.88 – 1.22)16.49±0.49(16.06 – 17.03)7.38±0.09*(7.27 – 7.47)28.16±0.53(27.66 – 28.90)7±1*(6 – 7)5.88±0.18*(5.77 – 6.15)0.47±0.08*(0.37 – 0.54)303±28*(277 – 338)Std 12.98 0.71 12.68 5.31 21.685 4.95 0.45 381Std 22.53 0.55 17.89 6.06 27.046 5.18 0.42 403Sete LagoasIsoline2.53±0.05(2.48 – 2.59)0.49±0.07(0.40 – 0.57)16.20±0.36(15.90 – 16.71)6.64±0.21(6.40 – 6.85)25.81±0.39(25.26 – 26.16)5±0(5)4.92±0.08(4.81 – 4.97)0.62±0.14(0.49 – 0.78)367±14(352 – 379)CV1273.04±0.09*(2.93 – 3.12)0.90±0.20*(0.69 – 1.17)15.98±0.78(14.82 – 16.49)6.83±0.23(6.64 – 7.16)26.57±1.04(25.06 – 27.29)6±0*(6)5.49±0.18(5.26 – 5.65)0.50±0.03(0.47 – 0.54)284±43*(242 – 327)CV127+ imi2.95±0.21*(2.49 – 3.23)0.81±0.09*(0.68 – 0.88)15.95±0.65(15.33 – 16.68)6.86±0.47(6.37 – 7.50)26.50±1.34(25.18 – 28.26)6±0*(6)5.39±0.31(5.12 – 5.80)0.59±0.13(0.49 – 0.78)297±27*(282 – 337)Std 13.27 0.47 14.50 6.04 24.286 5.44 0.63 317Std 22.48 0.49 17.83 6.47 27.056 5.11 0.49 205CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 252 of 375Table D-15. continued.Location/Treatmentα-tocopherol(mg/100 g DW)β-tocopherol(mg/100 g DW)γ-tocopherol(mg/100 g DW)δ-tocopherol(mg/100 g DW)Total Tocopherol(mg/100 g DW)Vitamin E(IU/100 g DW)Vitamin E(mg/100 g DW)Vitamin B1(mg/100 g DW)Folic Acid(μg/100 g DW)LondrinaMean±Standard Deviation (range)Isoline3.42±0.19(3.23 – 3.66)0.93±0.08(0.83 – 1.01)16.31±0.24(16.01 – 16.33)6.93±0.50(6.22 – 7.38)27.51±0.46(26.87 – 27.95)7±1(6 – 7)5.94±0.15(5.78 – 6.12)0.63±0.04(0.58 – 0.68)266±37(234 – 319)CV1273.68±0.23(3.46 – 3.91)1.01±0.16(0.81 – 1.20)17.26±0.56(16.43 – 17.58)6.81±0.14(6.64 – 6.93)28.54±0.37*(28.11 – 28.99)7±0(7)6.27±0.27*(5.97 – 6.54)0.55±0.04(0.51 – 0.58)260±23(229 – 279)CV127+ imi3.91±0.23*(3.64 – 4.20)0.97±0.10(0.85 – 1.08)17.79±0.76(17.18 – 18.84)7.37±0.42(7.11 – 7.99)30.03±1.15*(28.79 – 31.12)7±1(7 – 8)6.66±0.31*(6.28 – 7.01)0.50±0.04(0.45 – 0.55)260±30(234 – 302)Std 13.89 0.99 13.99 6.26 25.157 6.13 0.71 228Std 23.39 0.73 20.02 6.48 31.037 6.51 0.53 247BrasíliaIsoline2.93±0.40(2.33 – 3.21)0.70±0.10(0.55 – 0.77)13.73±2.06(11.62 – 16.03)4.78±0.28(4.41 – 5.08)22.0±2.55(19.26 – 24.61)6±1(5 – 6)5.00±0.63(4.11 – 5.48)0.63±0.17(0.48 – 0.86)244±32(216 – 290)CV1273.24±0.20*(3.07 – 3.52)1.03±0.22*(0.81 – 1.32)13.39±1.02(12.50 – 14.86)5.49±0.93*(4.85 – 6.87)23.08±2.36*(21.59 – 26.57)6±1*(6 – 7)5.38±0.41(5.05 – 5.97)0.46±0.05*(0.40 – 0.52)234±32(201 – 274)CV127+ imi3.47±0.21*(3.16 – 3.62)1.05±0.15*(0.89 – 1.19)13.59±1.05(12.31 – 14.86)5.42±0.36*(4.96 – 5.78)23.57±1.39*(21.95 – 25.35)6±1*(6 – 7)5.65±0.35*(5.13 – 5.92)0.48±0.05*(0.45 – 0.55)196±9*(183 – 205)Std 13.21 0.91 13.44 5.65 23.206 5.34 0.71 314Std 22.74 0.67 16.81 5.51 25.736 5.27 0.56 254Santo Antôniode PosseIsoline3.65±0.55(3.20 – 4.44)0.49±0.03(0.45 – 0.52)18.04±0.85(17.00 – 19.00)5.32±0.35(5.04 – 5.84)27.51±1.60(25.95 – 29.74)7±1(6 – 8)6.29±0.65(5.70 – 7.21)0.70±0.10(0.59 – 0.81)310±28(272 – 333)CV1274.48±0.26*(4.29 – 4.86)0.82±0.11*(0.68 – 0.94)16.14±0.86*(15.29 – 17.08)5.18±0.32(4.95 – 5.66)26.56±0.75(25.44 – 27.01)8±1*(7– 8)6.93±0.27*(6.69 – 7.30)0.62±0.06(0.54 – 0.67)258±17*(235 – 272)CV127+ imi4.48±0.23*(4.20 – 4.69)0.93±0.14*(0.82 – 1.13)15.20±1.48*(13.84 – 17.16)5.12±0.27(4.90 – 5.49)25.74±1.91(24.04 – 27.89)8±1*(7 – 8)6.69±0.32(6.42 – 7.16)0.60±0.02(0.57 – 0.62)266±8*(260 – 277)Std 14.61 1.08 15.31 6.03 27.008 7.05 0.62 291Std 23.80 0.80 17.77 5.76 28.147 6.48 0.58 227*Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 253 of 375Table D-16. Selected Vitamin Compositionof Grain on a Dry-WeightBasis of CV127 Soybean Treatments (CV127 and CV127 + imi),the Isoline Control and Two Conventional Soybean Varieties (Std1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocation/Treatmentα-tocopherol(mg/100 g DW)β-tocopherol(mg/100 g DW)γ-tocopherol(mg/100 g DW)δ-tocopherol(mg/100 g DW)Total tocopherol(mg/100 g DW)Vitamin E(IU/100 g DW)Vitamin E(mg/100 g DW)Vitamin B1(mg/100 g DW)Folic Acid(μg/100 g DW)Mean±Standard Deviation (range)Santo Antôniode GoiásIsoline2.50±0.36(2.20 – 3.00)0.81±0.16(0.63 – 1.03)14.32±0.47(13.79 – 14.88)7.84±0.28(7.58 – 8.12)25.49±0.44(25.05 – 25.90)5±1(5 – 6)4.75±0.37(4.42 – 5.26)0.64±0.13(0.52 – 0.80)444±19(428 – 462)CV1272.81±0.27(2.51 – 3.17)1.12±0.17*(0.99 – 1.35)15.04±0.39*(14.77 – 15.61)8.97±0.26*(8.71 – 9.32)27.93±0.26*(27.71 – 28.27)6±1(5 – 6)5.25±0.30*(4.90 – 5.64)0.66±0.05(0.61 – 0.72)443±12(432 – 460)CV127+ imi2.53±0.18(2.37 – 2.73)0.79±0.16(0.68 – 1.02)14.17±0.32(13.80 – 14.46)7.95±0.34(7.75 – 8.46)25.44±0.86(24.63 – 26.66)5±1(5 – 6)4.75±0.23(4.52 – 5.05)0.58±0.02(0.55 – 0.60)452±15(430 – 464)Std 12.67 1.24 11.65 7.51 23.07 5 4.67 0.71 401Std 22.53 0.66 15.44 7.18 25.81 5 4.88 0.59 438TeresinaIsoline6.61±1.42(5.38 – 8.17)0.69±0.13(0.54 – 0.77)19.24±2.17(17.38 – 21.63)5.05±0.65(4.52 – 5.77)31.58±2.56(29.48 – 34.43)10±2(9 – 12)9.46±1.34(8.12 – 10.79)0.43±0.06(0.37 – 0.48)291±36(249 – 315)CV1276.85±0.12(6.71 – 6.94)0.79±0.05(0.73 – 0.82)19.67±1.93(17.46 – 21.05)5.79±1.00(4.67 – 6.58)33.10±2.95(29.77 – 35.38)10±1(10 – 11)9.80±0.28(9.53 – 10.09)0.40±0.07(0.35 – 0.48)348±47(302 – 396)CV127+ imi7.84±1.38(6.47 – 9.23)0.94±0.11*(0.81 – 1.00)15.66±3.13(12.28 – 18.46)4.57±1.18(3.31 – 5.66)29.00±2.87(25.82 – 31.40)11±1(10 – 12)10.27±0.98(9.26 – 11.21)0.37±0.15(0.28 – 0.55)367±26*(352 – 397)Std 17.51 1.34 14.62 5.61 29.09 11 9.92 0.41 261Std 25.23 0.53 22.27 5.16 33.19 9 8.46 0.35 267CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 254 of 375Table D-16. continued.Location/Treatmentα-tocopherol(mg/100 g DW)β-tocopherol(mg/100 g DW)γ-tocopherol(mg/100 g DW)δ-tocopherol(mg/100 g DW)Total tocopherol(mg/100 g DW)Vitamin E(IU/100 g)Vitamin E(mg/100 g DW)Vitamin B1(mg/100 g DW)Folic Acid(μg/100 g DW)Mean±Standard Deviation (range)VilhenaIsoline2.77±0.36(2.37 – 3.16)0.98±0.10(0.90 – 1.12)17.30±1.26(16.32 – 19.14)8.67±0.93(7.73 – 9.72)29.72±2.40(27.40 – 32.93)6±1(5 – 7)5.47±0.51(4.93 – 6.11)0.43±0.06(0.34 – 0.47)320±17(302 – 339)CV1273.21±0.14*(3.09 – 3.40)0.96±0.14(0.76 – 1.09)15.88±0.89(14.99 – 17.11)8.75±0.76(7.63 – 9.31)28.80±1.72(26.72 – 30.91)6±1(6 – 7)5.72±0.28(5.48 – 6.11)0.49±0.08(0.40 – 0.60)311±7(303 – 320)CV127+ imi3.17±0.25(2.79 – 3.32)0.94±0.26(0.68 – 1.26)15.45±1.10*(14.34 – 16.59)8.70±1.06(7.51 – 9.77)28.25±2.24(25.90 – 30.27)6±0(6)5.61±0.33(5.16 – 5.89)0.56±0.06(0.50 – 0.62)337±12(320 – 349)Std 1 3.43 1.04 12.63 7.04 24.14 6 5.50 0.34 329Std 2 2.69 0.66 20.93 8.94 33.23 6 5.81 0.31 285BrasíliaIsoline1.80±0.08(1.74 – 1.92)0.85±0.21(0.69 – 1.15)15.51±1.09(14.18 – 16.85)8.38±0.26(8.13 – 8.73)26.54±1.06(25.02 – 27.46)5±1(4 – 5)4.23±0.20(3.94 – 4.40)0.55±0.07(0.47 – 0.64)389±46(323 – 427)CV1272.09±0.14*(1.96 – 2.26)1.23±0.27(0.95 – 1.49)14.55±0.86(13.91 – 15.81)10.49±1.02*(9.62 – 11.72)28.34±1.73(26.96 – 30.82)5±0(5 )4.50±0.20(4.35 – 4.80)0.63±0.05(0.57 – 0.70)346±14(326 – 355)CV127+ imi2.20±0.16*(1.96 – 2.33)1.18±0.24(0.85 – 1.37)14.74±0.60(14.21 – 15.59)10.49±0.63*(9.86 – 11.35)27.36±2.98(23.38 – 30.55)5±0(5 )4.62±0.20*(4.35 – 4.84)0.65±0.07(0.59 – 0.75)338±13*(324 – 354)Std 1 2.07 0.65 11.98 6.86 21.56 4 3.95 0.53 309Std 2 2.16 0.89 15.94 7.63 26.61 5 4.64 0.60 294*Statistically significantly different from the isoline control at P <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 255 of 375Table D-17. Isoflavone Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 SeasonLocation/TreatmentDaidzinMalonyl-DaidzinDaidzeinGlycitinMalonyl-glycitinGlyciteinGenistinMalonyl-genistinGenisteinSanto Antôniode GoiásMean±Standard Deviation (range)mg/100 g dry weightIsoline13.2±2.8(9.3 – 15.9)54.9±3.5(51.7 – 59.9)1.1±0.4(0.8 – 1.6)4.6±1.0(3.9 – 6.1)14.0±1.6(11.9 – 15.8)<0.8(nd^ – 0.9)16.2±3.0(11.7 – 18.0)85.7±4.0(82.1 – 91.1)1.1±0.4(0.7 – 1.7)CV12711.7±0.5(11.0 – 12.3)40.3±1.9*(38.5 – 42.7)1.0±0.3(0.6 – 1.3)5.0±0.4(4.5 – 5.3)14.0±0. 9(12.8 – 14.7)<0.8(nd – 0.8)15.4±0.8(14.5 – 16.4)69.9±2.6*(68.2 – 73.8)0.6±0.1*(0.5 – 0.7)CV127+ imi11.2±0.3(10.9 – 11.6)35.8±1.9*(34.1 – 37.7)1.1±0. 1(1.1 – 1.2)4.9±0.5(4.3 – 5.4)12.5±1.4(11.0 – 14.3)0.7±0(0.7)15.1±0.3(14.8 – 15.5)62.9±2.8*(59.5 – 65.5)0.7±0.1*(0.7 – 0.8)Std 1 14.2 60.1 1.3 9.4 26.3 0.6 18.1 103.4 0.6Std 2 11.5 55.5 1.7 12.7 36.2 1.6 15.1 87.1 0.6UberabaIsoline12.1±2.4(9.3 – 15.0)58.2±7.7(50.0 – 66.6)1.1±0.3(0.7 – 1.5)5.2±0.4(4.8 – 5.6)17.5±2.9(13.6 – 20.3)<1(nd – 1.1)13.1±2.2(11.2 – 16.2)86.0±9.1(75.4 – 93.6)0.9±0.4(0.6 – 1.5)CV12710.9±0.7(9.9 – 11.3)46.3±2.8*(42.5 – 49.1)1.1±0.08(1.0 – 1.2)5.8±0.1(5.6 – 5.9)17.6±1.5(15.8 – 19.2)0.8±0. 1(0.7 – 0.9)13.1±1.1(11.6 – 14.1)80.5±4.1(75.8 – 85.7)0.7±0.2(0.5 – 0.9)CV127+ imi11.4±1.8(9.0 – 13.2)48.6±6.8*(42.0 – 58.0)1.2±0.2(1.0 – 1.4)6.3±0.9(5.2 – 7.3)18.1±3.0(14.4 – 21.5)0.7±0.1(0.5 – 0.8)13.9±0.7(13.2 – 14.5)82.7±5.5(78.7 – 90.8)0.8±0.2(0.7 – 1.1)Std 1 12.2 70.5 1.7 10.3 34.5 1.4 15.6 132.7 1.2Std 2 12.6 65.0 0.8 12.6 34.6 1.5 16.2 115.9 1.5Sete LagoasIsoline15.7±1.4(14.3 – 17.2)52.4±5.9(47.3 – 59.3)2.0±0.4(1.5 – 2.4)5.1±1.0(3.8 – 5.9)13.6±2.2(10.4 – 15.1)<0.7(nd – 0.7)17.7±1.6(16.0 – 19.4)83.0±7.5(76.1 – 92.2)2.2±0.4(1.7 – 2.7)CV12711.7±1.5*(10.2 – 13.3)37.2±6.9(29.7 – 45.3)1.5±0.4(0.9 – 1.8)5.9±0.9(5.3 – 7.2)15.3±2.0(13.6 – 18.1)0.8±0.3*(0.4 – 1.0)14.6±2.9(11.9 – 18.7)61.7±13.7*(49.6 – 81.3)1.9±0.1(1.8 – 2.0)CV127+ imi12.5±1.6*(11.3 – 14.7)38.6±5.6(34.0 – 46.3)1.7±0.3(1.2 – 1.9)5.7±1.0(4.5 – 6.7)14.5±2.8(10.7 – 17.4)0.5±0.3*(0.3 – 0.9)15.7±1.3(14.3 – 17.3)64.6±4.0*(61.6 – 70.4)2.0±0.2(1.7 – 2.1)Std 1 15.3 57.0 2.8 12.1 31.9 1.1 21.0 100.3 1.9Std 2 15.2 53.9 2.7 15.3 39.7 1.8 24.3 97.0 3.2CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 256 of 375Table D-17. continued.Location/TreatmentDaidzinMalonyl-DaidzinDaidzeinGlycitinMalonyl-glycitinGlyciteinGenistinMalonyl-genistinGenisteinLondrinaMean±Standard Deviation (range)mg/100 g dry weightIsoline19.6±1.4(18.5 – 21.7)74.5±6.0(70.8 – 83.4)2.0±0.4(1.5 – 2.3)5.5±0.2(5.2 – 5.7)15.9±0.9(15.2 – 17.2)<0.9(nd – 1.0)22.9±1.5(21.8 – 25.0)112.6±9.7(105.5 – 126.9)1.4±0.1(1.3 – 1.5)CV12713.1±1.2*(12.2 – 14.9)45.8±3.7*(41.8 – 50.8)1.5±0.5*(0.9 – 1.7)4.9±0.6(4.1 – 5.5)12.9±1.4*(11.1 – 14.5)<0.7(nd – 0.8)20.5±3.3(17.8 – 25.3)94.7±11.5(86.1 – 110.8)1.6±0.3(1.3 – 1.9)CV127+ imi12.6±0.6*(12.1 – 13.4)41.1±3.1*(37.8 – 45.2)1.6±0.4*(1.0 – 1.8)5.7±1.0(4.2 – 6.2)14.4±2.7(10.4 – 16.3)0.7±0.3(0.3 – 0.9)17.7±2.5*(16.0 – 21.4)79.4±13.1*(67.5 – 97.9)1.7±0.1(1.5 – 1.8)Std 1 18.3 72.7 2.7 10.4 29.5 1.3 26.7 130.7 1.9Std 2 19.3 70.8 2.9 11.5 28.5 1.5 30.7 128.7 2.9BrasíliaIsoline14.2±1.6(12.5 – 15.9)57.0±8.8(46.0 – 67.3)2.5±0.4(2.2 – 3.0)5.9±0.9(5.2 – 7.0)18.1±3.1(15.5 – 22.7)0.9±0.1(0.8 – 1.0)14.2±1.9(11.7 – 15.8)78.5±11.8(61.7. – 89.5)1.6±0.2(1.3 – 1.8)CV12710.8±0.7*(10.2 – 11.6)39.1±4.9*(33.7 – 43.6)2.0±0.3(1.6 – 2.4)5.9±1.0(5.2 – 7.3)15.2±2.3(13.8 – 18.6)0.8±0.1(0.7 – 0.8)12.5±2.0(9.5 – 13.6)59.5±10.0*(45.7 – 69.5)1.4±0.4(0.9 – 1.7)CV127+ imi11.7±0.3*(11.5 – 12.2)39.1±2.5*(36.4 – 42.2)2.2±0.3(1.9 – 2.5)6.4±0.3(6.1 – 6.7)16.7±1.2(15.3 – 18.3)0.9±0.1(0.8 – 0.9)11.6±1.7(10.3 – 14.1)54.2±8.2*(48.5 – 66.4)1.7±0.3(1.4 – 1.9)Std 1 14.4 67.3 1.7 11.7 33.8 1.4 20.1 109.6 1.2Std 2 15.1 70.7 1.2 12.8 34.8 0.6 20.4 118.7 1.1Santo Antôniode PosseIsoline12.5±1.4(10.6 – 13.7)38.8±5.7(33.4 – 43.8)1.6±0.5(0.9 – 2.0)5.8±0.6(5.2 – 6.4)15.6±0.5(15.2 – 16.2)0.7±0.2(0.4 – 0.9)12.5±2.7(9.2 – 15.5)59.7±11.3(46.7 – 72.1)1.3±0.2(1.2 – 1.6)CV12711.2±0.5(10.8 – 11.8)31.0±1.4*(29.9 – 33.0)1.2±0.1(1.0 – 1.3)5.9±0.9(5.1 – 7.1)13.2±1.4*(12.0 – 14.9)0.6±0.2(0.3 – 0.7)12.7±1.6(11.1 – 14.8)54.5±7.2(46.7 – 63.7)1.3±0.2(1.1 – 1.6)CV127+ imi11.1±1.6(9.6 – 13.4)31.5±4.9*(27.2 – 38.4)1.3±0.2(1.1 – 1.6)5.1±1.2(3.9 – 6.5)11.9±2.4*(8.9 – 14.5)0.7±0.2(0.3 – 0.8)14.4±2.4(12.3 – 17.5)60.2±10.6(49.4 – 73.7)1.4±0.1(1.2 – 1.5)Std 1 15.2 54.9 2.4 14.0 38.8 0.7 23.9 102.7 1.9Std 2 20.6 68.1 2.9 22.9 50.7 1.8 27.6 106.6 3.1*Statistically significantly different from the isoline control at P < 0.05.^nd = not detectedCONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 257 of 375Table D-18. Isoflavone Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 + imi), theIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 SeasonLocation/TreatmentDaidzinMalonyl-DaidzinDaidzeinGlycitinMalonyl-glycitinGlyciteinGenistinMalonyl-genistinGenisteinMean±Standard Deviation (range)mg/100 g dry weightSanto Antôniode GoiásIsoline14.2±1.4(12.2 – 15.3)91.4±2.9(87.2 – 94.0)0.8±0.1(0.7 – 0.9)3.7±0.5(3.1 – 4.2)13.3±0.3(13.0 – 13.8)0.6±0.2(0.3 – 0.8)17.2±3.7(12.0 – 20.8)116.6±9.0(107.8 – 126.8)ndCV1279.2±0.5*(8.6 – 9.8)56.5±3.8*(51.7 – 59.8)<0.5*(nd – 0.5)3.3±0.5(2.7 – 3.9)11.2±1.8(8.8 – 13.1)<0.8(nd^ – 0.8)10.0±1.9*(8.4 – 12.1)84.2±1.8*(82.3 – 86.6)ndCV127+ imi9.3±0.5*(8.7 – 9.9)58.6±3.0*(56.1 – 62.3)0.6±0*(0.5 – 0.6)3.6±0.5(3.0 – 4.2)11.8±2.3(9.6 – 14.2)nd11.8±0.6*(11.2 – 12.4)83.6±3.6*(78.5 – 86.6)ndStd 113.9 94.1 0.7 7.526.9 0.7 17.7 119.6 ndStd 210.4 71.5 0.8 6.623.4 1.8 16.0 108.1 ndTeresinaIsoline6.2±1.4(4.9 – 7.7)16.8±3.7(13.9 – 20.9)0.7±0.2(0.4 – 0.8)2.8±1.2(1.6 – 3.9)7.7±3.0(4.7 – 10.7)1.5±0.3(1.1 – 1.7)5.6±1.6(3.8 – 6.8)30.0±8.0(21.7 – 37.7)0.5±0.2(0.3 – 0.7)CV1276.9±0.8(6.3 – 7.8)20.0±4.2(15.3 – 23.4)0.7±0.1(0.6 – 0.8)4.0±0.8(3.1 – 4.5)10.2±2.4(7.5 – 12.0)< 0.8(nd – 0.8)6.7±2.5(4.1 – 9.1)31.2±9.3(22.1 – 40.6)0.5±0.2(0.4 – 0.8)CV127+ imi5.7±1.5(4.0 – 6.7)16.7±6.4(9.7 – 22.3)<1.0(nd – 1.2)3.3±1.2(1.9 – 4.2)7.8±3.0(4.4 – 10.2)<0.4*(nd – 0.4)4.0±2.0(2.2 – 6.2)17.0±9.0(8.9 – 26.6)< 0.4(nd – 0.4)Std 19.4 28.0 1.8 9.620.6 1.4 15.9 65.1 1.9Std 28.0 22.1 1.0 10.8 23.4 1.4 10.2 43.8 1.4CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 258 of 375Table D-18. continued.Location/TreatmentDaidzinMalonyl-DaidzinDaidzeinGlycitinMalonyl-glycitinGlyciteinGenistinMalonyl-genistinGenisteinMean±Standard Deviation (range)mg/100 g dry weightVilhenaIsoline14.5±1.7(12.1 – 15.8)61.1±7.3(51.9 – 67.0)1.3±0.2(1.0 – 1.5)3.9±0.6(3.1 – 4.6)11.0±1.4(9.6 – 12.7)<0.9(nd – 0.9)17.3±0.7(16.7 – 18.3)101.6±10.6(87.9 – 111.2)0.7±0.2(0.5 – 0.9)CV1279.7±1.0*(8.7 – 10.7)40.1±4.5*(35.3 – 45.8)0.8±0.1*(0.7 – 0.9)4.0±0.4(3.7 – 4.4)11.8±1.6(10.6 – 14.1)< 0.7(nd – 0.7)14.6±2.2*(12.3 – 17.4)71.6±9.1*(62.1 – 82.8)0.6±0.1(0.5 – 0.8)CV127+ imi9.8±0.6*(9.0 – 10.5)40.7±3.2*(38.1 – 45.4)0.9±0*(0.9 – 1.0)4.1±0.7(3.6 – 5.1)10.7±0.5(10.2 – 11.2)<0.4*(nd – 0.5)14.0±1.1*(12.6 – 15.3)70.7±5.1*(65.1 – 75.4)0.6±0.1(0.5 – 0.6)Std 1 15.7 73.9 1.2 7.5 22.2 0.8 14.9 102.2 0.5Std 2 15.5 63.4 2.0 11.0 26.2 1.5 20.4 118.0 1.1BrasíliaIsoline29.6±0.5(29.1 – 30.2)196.1±6.3(191.1 – 205.0)1.0±0.2(0.8 – 1.2)5.5±0.5(5.2 – 6.2)18.4±1.6(16.9 – 20.0)1.0±0.2(0.6 – 1.1)34.4±7.2(28.1 – 40.6)224.4±3.4(220.9 – 227.3)<0.4(nd – 0.4)CV12719.6±1.2*(18.6 – 21.2)131.7±5.4*(125.2 – 137.5)0.6±0.1*(0.5 – 0.7)4.4±0.6*(3.8 – 5.2)14.9±2.0*(12.8 – 17.2)<0.8(nd – 0.8)27.8±4.6(22.9 – 32.0)194.8±3.3*(191.7 – 198.2)< 0.3(nd – 0.3)CV127+ imi20.6±0.6*(19.7 – 21.1)134.5±1.1*(133.8 – 136.2)0.7±0.1*(0.5 – 0.8)4.6±0.5*(4.2 – 5.3)14.2±1.4*(12.4 – 15.9)<0.6(nd – 0.6)30.8±5.2(23.2 – 34.3)200.4±8.9*(192.9 – 210.8)<0.4(nd – 0.4)Std 1 26.9 157.5 1.4 8.0 28.0 1.2 29.0 216.7 0.3Std 2 25.1 156.5 1.0 10.5 30.2 1.4 35.7 217.5 0.4*Statistically significantly different from the isoline control at P <0.05.^nd = not detectedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 259 of 375 Table D-19. Phospholipid Composition of Grain of CV127 Soybean Treatments (CV127 and CV127 + imi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in Brazil in the 2006/07 Season

Location Treatment N Phosphatidyl

ethanolamine Phosphatidic

acid Phosphatidyl

inositol Phosphatidyl

choline

Mean ± Standard Deviation (range) mg/g fat

Santo Antônio

de Goiás Isoline 4 107.1 ± 4.1 (101.0 – 109.4) 3.8 ± 1.3 (2.0 – 5.1) 11.7 ± 0.5 (11.1 – 12.1) 31.6 ± 0.9 (30.4 – 32.7)

CV127 4 102.3 ± 5.5 (96.1 – 107.6) 2.8 ± 0.3 (2.5 – 3.1) 10.1 ± 0.4 (9.7 – 10.6) 29.5 ± 1.9 (27.7 – 32.1)

CV127

+ imi 4 103.3 ± 4.7 (96.8 – 107.3) 3.1 ± 0.1 (2.9 – 3.2) 10.1 ± 0.5 (9.7 – 10.7) 30.7 ± 1.2 (29.5 – 32.3)

Std 1 1 104.5 3.7 12.3 32.6

Std 2 1 91.3 5.6 11.5 30.4

Uberaba Isoline 4 114.5 ± 16.1 (92.2 – 127.9) 2.3 ± 0.5 (1.8 – 2.9) 12.2 ± 1.3 (10.5 – 13.5) 33.5 ± 5.4 (25.9 – 38.5)

CV127 4 98.5 ± 7.9 (93.5 – 110.3) 1.0 ± 0.1 (0.8 – 1.1) 10.0 ± 0.9 (8.9 – 11.0) 30.4 ± 2.5 (28.6 – 34.1)

CV127

+ imi 4 103.5 ± 3.6* (101.0 – 108.9) 1.4 ± 0.4 (1.0 – 1.9) 9.8 ± 1.0* (8.3 – 10.4) 30.1 ± 1.0 (28.8 – 30.7)

Std 1 1 108.7 2.1 12.2 38.2

Std 2 1 103.4 3.4 12.9 27.0

Sete Lagoas Isoline 4 97.9 ± 2.6 (94.6 – 100.9) 5.7 ± 1.0 (4.6 – 6.9) 12.0 ± 0.5 (11.5 – 12.4) 26.9 ± 1.1 (25.4 – 27.8)

CV127 4 93.6 ± 3.5 (90.1 – 98.4) 3.7 ± 0.5* (3.2 – 4.3) 9.8 ± 0.8* (9.3 – 10.9) 25.9 ± 0.9 (24.8 – 26.7)

CV127

+ imi 4 78.5 ± 21.7* (57.9 – 99.2) 2.8 ± 0.4* (2.5 – 3.2) 8.2 ± 2.3* (6.1 – 10.7) 21.3 ± 5.3* (15.9 – 26.7)

Std 1 1 113.7 4.8 13.1 33.2

Std 2 1 79.4 7.5 11.4 25.6

Londrina Isoline 4 96.3 ± 9.0 (89.7 – 109.6) 2.5 ± 0.5 (2.0 – 3.2) 10.4 ± 1.3 (9.2 – 12.2) 25.8 ± 0.8 (24.9 – 26.7)

CV127 4 94.9 ± 5.3 (90.2 – 100.7) 2.9 ± 0.5 (2.4 – 3.5) 10.0 ± 0.7 (9.2 – 10.9) 28.7 ± 1.8 (26.7 – 30.3)

CV127

+ imi 4 91.1 ± 0.3 (90.9 – 91.5) 3.2 ± 0.2 (3.1 – 3.4) 10.6 ± 0.7 (9.7 – 11.2) 27.0 ± 0.3 (26.6 – 27.4)

Std 1 1 100.8 2.3 12.6 31.3

Std 2 1 94.4 3.5 12.1 29.4

Brasília Isoline 4 95.5 ± 5.1 (91.7 – 102.9) 4.6 ± 0.7 (4.0 – 5.5) 11.7 ± 1.0 (10.7 – 13.0) 28.8 ± 1.6 (27.6 – 31.1)

CV127 4 92.4 ± 4.8* (88.5 – 98.6) 3.0 ± 0.7 (2.3 – 3.7) 9.5 ± 1.2* (7.9 – 10.7) 27.2 ± 1.1 (25.8 – 28.6)

CV127

+ imi 4 93.6 ± 7.3* (84.8 – 102.6) 3.5 ± 1.3 (1.6 – 4.7) 10.4 ± 0.5* (9.9 – 10.9) 27.5 ± 2.5 (25.1 – 30.6)

Std 1 1 97.4 2.5 11.6 30.6

Std 2 1 98.1 2.9 11.4 31.8

Santo Antônio

de Posse Isoline 4 100.1 ± 6.9 (95.0 – 110.0) 5.2 ± 0.6 (4.4 – 5.9) 12.9 ± 1.0 (11.8 – 14.2) 29.0 ± 2.1 (27.3 – 31.9)

CV127 4 74.0 ± 16.3* (51.0 – 87.6) 3.8 ± 0.6* (3.3 – 4.6) 9.3 ± 1.7* (6.8 – 10.6) 20.6 ± 4.2 (14.9 – 23.9)

CV127

+ imi 4 75.2 ± 18.2* (49.4 – 88.5) 3.6 ± 0.8* (2.7 – 4.4) 8.8 ± 1.8* (6.2 – 10.0) 21.9 ± 5.5 (13.9 – 25.8)

Std 1 1 101.4 4.6 13.5 32.0

Std 2 1 84.9 6.0 12.6 26.8 *Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 260 of 375 Table D-20. Phospholipid Composition of Grain of CV127 Soybean Treatments (CV127 and CV127 + imi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil in the 2007 Season

Location Treatment N Phosphatidyl

ethanolamine Phosphatidic

acid Phosphatidyl

inositol Phosphatidyl

choline

Mean ± Standard Deviation (range) mg/g fat

Santo Antônio

de Goiás Isoline 4 136.5 ± 20.7
(113.8 – 156.8) 0.9 ± 0.2
(0.7 – 1.1) 12.1 ± 2.3
(10.1 – 14.8) 39.5 ± 5.0
(34.7 – 45.0)
CV127 4 135.6 ± 14.6
(120.6 – 155.5) 1.0 ± 0.2
(0.7 – 1.1) 11.3 ± 1.3
(10.2 – 13.2) 37.4 ± 1.8
(35.4 – 39.4)
CV127

+ imi 4 126.1 ± 9.9
(115.9 – 139.5) 0.9 ± 0.2
(0.6 – 1.1) 10.6 ± 0.4
(10.1 – 11.0) 38.1 ± 1.8
(35.8 – 40.2)
Std 1 1 132.2 0.9 11.7 39.1

Std 2 1 133.7 1.4 12.4 42.0

Teresina Isoline 3 68.6 ± 3.7
(65.7 – 72.7) 5.6 ± 1.0
(4.9 – 6.7) 8.8 ± 0.1
(8.7 – 8.9) 21.6 ± 1.5
(20.6 – 23.4)
CV127 3 63.6 ± 1.8
(61.7 – 65.2) 5.0 ± 0.9
(4.3 – 6.1) 8.7 ± 0.8
(8.1 – 9.6) 21.2 ± 1.8
(19.8 – 23.2)
CV127

+ imi 3 61.8 ± 14.0
(52.1 – 77.8) 8.1 ± 1.6*
(7.0 – 9.9) 8.7 ± 0.9
(8.0 – 9.8) 19.2 ± 2.8
(17.5 – 22.4)
Std 1 1 70.1 5.3 10.6 24.3

Std 2 1 51.1 7.2 8.1 17.0

Vilhena Isoline 4 98.0 ± 6.4
(88.9 – 103.8) 2.2 ± 0.2
(1.8 – 2.3) 9.6 ± 0.7
(8.6 – 10.1) 30.9 ± 1.9
(28.6 – 33.1)
CV127 4 102.7 ± 4.9
(96.0 – 107.8) 2.4 ± 0.6
(2.0 – 3.2) 9.8 ± 1.0
(9.2 – 11.3) 31.5 ± 1.5
(30.2 – 33.5)
CV127

+ imi 4 107.0 ± 10.5
(98.1 – 120.7) 2.1 ± 0.2
(1.9 – 2.4) 9.6 ± 0.9
(8.4 – 10.2) 32.0 ± 0.5
(31.3 – 32.4)
Std 1 1 116.2 2.8 12.1 39.0

Std 2 1 86.6 4.7 10.6 30.4

Brasília Isoline 4 124.2 ± 13.1
(114.7 – 142.7) 0.8 ± 0.1
(0.7 – 0.9) 10.6 ± 0.8
(9.7 – 11.7) 36.8 ± 3.1
(32.9 – 40.4)
CV127 4 123.4 ± 10.7
(112.4 – 135.8) 0.8 ± 0.1
(0.6 – 0.9) 10.0 ± 0.3
(9.7 – 10.4) 36.1 ± 2.1
(33.7 – 38.2)
CV127

+ imi 4 117.9 ± 6.2
(110.2 – 124.8) 0.8 ± 0.2
(0.5 – 0.9) 9.8 ± 0.5
(9.2 – 10.3) 37.5 ± 1.7
(35.0 – 39.0)
Std 1 1 107.2 0.9 10.8 37.2

Std 2 1 106.7 0.8 10.5 38.5

* Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 261 of 375Table D-21. Antinutrient Composition ofGrain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 +imi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown atSix Locations in Brazil in the2006/07 SeasonLocationTreatmentNRafffinose(g/100 g dry wt.)Stachyose(g/100 g dry wt.)Trypsin Inhib.(TIU/mg dry wt.)Urease(ΔpH)Lectin(HU/mg dry wt.)Phytate(mg/g dry wt.)Mean±Standard Deviation (range)Santo Antôniode GoiásIsoline 41.1±0.1(1.0 – 1.2)4.0±0.2(3.8 – 4.2)11.45±1.66(9.28 – 12.95)2.05±0.05(2.01 – 2.12)2.35±0.68(1.36 – 2.79)2.85±0.86(2.12 – 4.01)CV127 41.1±0.2(1.0 – 1.3)3.8±0.1(3.6 – 3.9)15.56±0.73*(14.98 – 16.58)2.10±0.04(2.05 – 2.13)2.27±0.62(1.54 – 3.01)2.89±0.70(1.87 – 3.41)CV127+ imi41.1±0.1(1.0 – 1.2)3.6±0.1*(3.5 – 3.8)13.29±1.20*(12.95 – 14.73)2.12±0.03(2.08 – 2.15)2.60±0.81(1.57 – 3.49)3.16±1.44(2.06 – 5.27)Std 1 1 1.24.16.112.053.531.47Std 2 1 1.03.79.592.101.522.65UberabaIsoline 41.1±0.1(1.0 – 1.1)4.1±0.1(3.9 – 4.1)7.92±3.36(6.03 – 12.95)2.05±0.05(2.01 – 2.12)2.28±0.74(1.75 – 3.35)2.31±0.63(1.89 – 3.23)CV127 41.2±0.1(1.1 – 1.3)3.8±0.2(3.6 – 4.0)10.15±1.41(8.69 – 12.01)2.09±0.04(2.05 – 2.13)2.01±0.49*(1.46 – 2.57)2.47±0.86(1.25 – 3.22)CV127+ imi41.2±0.1(1.1 – 1.3)3.9±0.2(3.7 – 4.1)9.72±0.91(8.97 – 10.98)2.02±0.14(1.81 – 2.13)1.37±0.16*(1.27 – 1.56)2.10±1.05(0.93 – 3.36)Std 1 1 1.14.65.402.071.382.19Std 2 1 1.03.75.032.061.582.02SeteLagoasIsoline 41.4±0.1(1.3 – 1.5)3.2±0.1(3.0 – 3.2)11.71±0.36(11.30 – 12.14)1.64±0.25(1.27 – 1.80)2.65±0.41(2.20 – 3.15)3.19±1.59(1.43 – 4.94)CV127 41.7±0.1*(1.6 – 1.8)3.1±0.2(2.9 – 3.4)12.65±2.60(10.28 – 16.08)1.21±0.49*(0.67 – 1.74)2.45±0.94(1.68 – 3.70)3.02±0.71(2.38 – 3.89)CV127+ imi41.5±0.2*(1.3 – 1.7)3.2±0.1(3.1 – 3.2)11.62±0.80(10.44 – 12.17)1.47±0.40*(0.90 – 1.79)2.83±0.34(2.42 – 3.25)2.22±0.49(1.60 – 2.77)Std1 1 1.6 3.7 10.73 1.43 2.13 2.28Std2 1 1.0 3.1 11.06 1.65 1.68 2.05CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 262 of 375Table D-21. continued.LocationTreatmentNRafffinose(g/100 g dry wt.)Stachyose(g/100 g dry wt.)Trypsin Inhib.(TIU/ mg dry wt.)Urease(ΔpH)Lectin(HU/mg dry wt.)Phytate(mg/g dry wt.)Mean±Standard Deviation (range)LondrinaIsoline 41.1±0.1(1.0 – 1.2)3.8±0.2(3.6 – 4.0)14.58±1.82(12.08 – 16.40)1.74±0.13(1.66 – 1.93)1.74±0.26(1.52 – 2.10)3.56±1.43(2.22 – 5.37)CV127 41.3±0.1(1.2 – 1.3)3.7±0.1(3.6 – 3.7)13.14±1.63(11.23 – 15.21)1.68±0.15(1.55 – 1.89)2.19±0.76(31.86 – 65.25)2.64±0.91(1.45 – 3.51)CV127+ imi41.2±0.1*(1.1 – 1.3)3.8±0.1(3.7 – 3.9)12.92±1.66(11.32 – 15.13)1.78±0.25(1.60 – 2.15)2.52±0.74(1.49 – 3.24)1.72±0.80(0.71 – 2.50)Std 1 1 1.24.113.431.781.853.65Std 2 1 0.83.617.401.832.172.38BrasíliaIsoline 41.0±0.1(0.9 – 1.0)3.6±0.2(3.3 – 3.8)15.74±0.65(14.94 – 16.39)2.09±0.01(2.09 – 2.10)2.21±0.85(1.35 – 3.20)3.22±1.98(1.73 – 6.09)CV127 41.2±0.1*(1.2 – 1.3)3.6±0.2(3.3 – 3.8)11.08±0.82(10.48 – 12.25)2.06±0.11(1.90 – 2.14)2.97±0.43(2.59 – 3.47)2.86±1.33(1.45 – 4.52)CV127+ imi41.2±0.1*(1.1 – 1.2)3.6±0.1(3.5 – 3.7)11.64±0.75(10.54 – 12.25)2.17±0.04(2.13 – 2.21)1.81±0.70(1.44 – 2.85)2.96±0.66(2.42 – 3.91)Std 1 1 1.04.214.542.151.603.99Std 2 10.9 3.8 19.64 2.16 2.48 7.39Santo Antôniode PosseIsoline 41.2±0.1(1.1 – 1.3)3.7±0.1(3.5 – 3.8)12.37±0.63(11.66 – 13.10)1.99±0.03(1.96 – 2.02)2.20±0.73(1.60 – 3.16)2.58±0.67(1.80 – 3.31)CV127 41.7±0.1*(1.6 – 1.7)3.4±0.1*(3.3 – 3.5)11.73±1.73(10.34 – 14.24)1.96±0.16(1.79 – 2.18)1.49±0.05*Z(1.43 – 1.54)2.62±0.90(1.53 – 3.39)CV127+ imi41.5±0.2*(1.2 – 1.7)3.4±0.1*(3.3 – 3.4)12.88±1.17(11.76 – 14.49)1.91±0.02(1.88 – 1.94)1.87±0.84*(1.36 – 3.12)3.08±0.97(1.75 – 3.87)Std1 1 1.3 4.1 10.88 1.96 2.35 2.09Std2 1 1.0 3.3 13.58 1.94 2.13 2.53*Statistically significantly different from the isoline control at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 263 of 375Table D-22. Antinutrient Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CV127 and CV127 +imi), the Isoline Control and Two Conventional Soybean Varieties(Std 1 and Std 2) Grown at Four Locations in Brazil in the2007 SeasonLocationTreatmentNRaffinose(g/100 g dry wt.)Stachyose(g/100 g dry wt.)Trypsin Inhib.(TIU/mg dry wt.)Urease(ΔpH)Lectin(HU/mg dry wt.)Phytate(mg/g dry wt.)Mean±Standard Deviation(range)Isoline 41.4±0.1(1.2 – 1.5)4.2±0.1(4.1 – 4.2)13.38±1.15(11.88 – 14.68)1.81±0.09(1.71 – 1.92)1.31±0.03(1.28 – 1.34)2.70±0.85(1.89 – 3.72)SantoAntôniode GoiásCV127 41.2±0.1*(1.1 – 1.2)3.9±0.1*(3.7 – 4.0)13.84±0.52(13.05 – 14.13)1.78±0.10(1.69 – 1.91)0.94±0.46(0.33 – 1.29)4.29±1.05*(3.06 – 5.62)CV127+ imi41.2±0.1*(1.1 – 1.3)3.9±0.1*(3.8 – 4.0)13.27±0.67(12.46 – 14.05)1.84±0.09(1.75 – 1.95)0.57±0.49*(0.22 – 1.29)3.81±0.74(2.75 – 4.32)Std1 1 1.4 4.8 13.62 1.92 0.67 5.04Std2 1 1.2 4.0 16.76 1.74 1.66 2.52Teresina Isoline 31.4±0.1(1.3 – 1.4)3.4±0.3(3.0 – 3.6)10.02±1.33(8.48 – 10.82)2.04±0.02(2.03 – 2.06)1.46±0.22(1.33 – 1.72)4.57±1.83(2.50 – 6.00)CV127 31.3±0(1.3)3.2±0.2(3.0 – 3.4)9.64±1.47(8.16 – 11.09)1.95±0.02*(1.94 – 1.97)1.71±0(1.71)3.13±0.39(2.90 – 3.58)CV127+ imi31.5±0.3(1.2 – 1.7)2.6±0.3*(2.4 – 3.0)10.31±2.53(7.82 – 12.87)1.96±0.02*(1.94 – 1.97)2.29±0.99(1.71 – 3.43)3.09±0.41(2.63 – 3.38)Std1 1 1.5 3.8 9.84 1.98 1.34 2.48Std2 1 1.4 3.1 12.56 2.02 2.67 4.73CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 264 of 375Table D-22. continued.LocationTreatmentNRaffinose(g/100 g dry wt.)Stachyose(g/100 g dry wt.)Trypsin Inhib.(TIU/mg dry wt.)Urease(ΔpH)Lectin(HU/mg dry wt.)Phytate(mg/g dry wt.)Mean±Standard Deviation(range)Vilhena Isoline 41.3±0.2(1.2 – 1.5)4.0±0.3(3.7 – 4.3)14.25±2.52(12.44 – 17.97)0.40±0.15(0.28 – 0.61)1.50±0.43(0.85 – 1.71)3.23±0.55(2.81 – 4.01)CV127 41.2±0.1(1.1 – 1.3)3.8±0.3(3.4 – 4.0)17.30±0.99*(16.28 – 18.20)0.67±0.25(0.41– 0.91)0.17±0*(0.17)3.92±1.17(2.66 – 5.49)CV127+ imi41.2±0.1(1.1 – 1.3)3.9±0.1(3.7 – 4.0)17.49±0.36*(17.22 – 18.03)0.65±0.17(0.49 – 0.87)0.13±0.03*(0.11 – 0.17)4.57±0.31*(4.12 – 4.77)Std1 1 1.2 4.4 14.61 1.16 1.71 4.79Std2 1 1.4 3.7 16.66 0.27 1.71 2.37Brasília Isoline 41.1±0.1(1.0 – 1.1)4.4±0.2(4.2 – 4.6)14.21±0.39(13.90 – 14.73)2.00±0.02(1.98 – 2.03)2.47±1.13(1.29 – 3.43)3.25±0.90(2.19 – 4.19)CV127 41.0±0.1(0.9 – 1.1)4.0±0.1*(3.9 – 4.2)13.08±0.48(12.45 – 13.57)1.98±0.02(1.96 – 2.00)0.75±0.54*(0.26 – 1.37)4.31±1.80(2.57 – 6.22)CV127+ imi41.0±0.1(0.9 – 1.1)3.9±0.1*(3.8 – 4.1)13.26±1.35(11.35 – 14.44)1.98±0.05(1.93 – 2.03)1.03±0.84*(0.34 – 2.06)3.59±0.33(3.17 – 3.98)Std1 1 1.1 4.8 14.91 1.98 1.72 5.44Std2 1 1.3 4.3 13.18 2.02 1.72 5.14*Statistically significantly different from the isoline control at P <0.05.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 265 of 375Table D-23. Proximate Composition of Forage of CV127 Soybean (CV127 + Imi), the Isoline Control and Two ConventionalSoybean Varieties (Std 1 and Std 2), Grown atSix Locations in Brazil in the 2007/2008 SeasonLocationTreatmentNMoisture(g/100 g FW)Ash(g/100 g DW)Fat(g/100 g DW)Protein(g/100 g DW)Carbohydrates(g/100 g DW)Calories(kcal/100 g DW)Mean ± Standard Deviation(range)Santo Antôniode GoiásIsoline382.0 ± 1.0(81.2 – 83.1)9.1 ± 0.9(8.2 – 9.9)2.3 ± 0.6(1.7 – 2.7)17.9 ± 0.4(17.6 – 18.3)70.8 ± 1.0(69.8 – 71.8)375 ± 8(367 – 383CV127 + imi381.8 ± 0.8(81.1 – 82.7)9.3 ± 1.1(8.3 – 10.4)3.0 ± 0.2(2.7 – 3.1)17.9 ± 1.3(16.5 – 19.0)69.7 ± 0.9(68.8 – 70.5)380 ± 4(376 – 383)Std 1382.9 ± 0.7(82.2 – 83.5)10.9 ± 0.3*(10.6 – 11.2)2.7 ± 0.3(2.5 – 3.1)21.3 ± 3.2*(17.6 – 23.1)65.1 ± 3.6*(62.4 – 69.2)370 ± 2*(368 – 371)Std 2382.0 ± 1.3(80.9 – 83.5)10.2 ± 0.4(9.7 – 10.5)2.6 ± 0.4(2.3 – 3.0)19.3 ± 1.4(17.9 – 20.7)68.2 ± 2.1(66.1 – 70.2)375 ± 3(372 – 377)UberabaIsoline379.8 ± 0.8(79.0 – 80.6)7.9 ± 0.7(7.1 – 8.5)2.5 ± 0.3*(2.1 – 2.7)18.4 ± 0.2(18.3 – 18.6)71.3 ± 0.6(70.8 – 71.9)381 ± 5(376 – 386)CV127 + imi379.1 ± 0.3(78.8 – 79.3)7.3 ± 0.3(7.0 – 7.5)3.1 ± 0.3(2.9 – 3.4)17.5 ± 1.1(16.6 – 18.7)72.0 ± 1.4(70.3 – 72.9)385 ± 2(383 – 387)Std 1380.0 ± 1.7(78.4 – 81.8)8.4 ± 0.9*(7.6 – 9.3)2.5 ± 0.2*(2.3 – 2.7)18.6 ± 0.3(18.3 – 18.9)70.5 ± 0.6(69.8 – 70.9)378 ± 2(376 – 380)Std 2380.1 ± 0.7(79.3 – 80.5)8.4 ± 0.1(8.3 – 8.5)2.3 ± 0.2*(2.1 – 2.5)18.8 ± 0.5*(18.4 – 19.3)70.6 ± 0.5(70.0 – 70.9)376 ± 5*(372 – 382)Sete LagoasIsoline380.8 ± 0.7(80.2 – 81.6)8.1 ± 0.4(7.6 – 8.3)2.4 ± 0.3*(2.1 – 2.7)17.9 ± 0.5(17.5 – 18.5)71.9 ± 0.8(71.2 – 72.7)380 ± 1(379 – 381)CV127 + imi379.4 ± 1.1(78.4 – 80.5)8.6 ± 0.2(8.4 – 8.8)1.9 ± 0.1(1.9 – 2.0)17.3 ± 0.4(16.9 – 17.7)72.2 ± 0.4(71.8 – 72.6)376 ± 2(375 – 379)Std 1381.7 ± 2.3(79.2 – 83.7)8.5 ± 0.4(8.0 – 8.7)2.2 ± 0.3(1.9 – 2.4)19.3 ± 0.6(18.6 – 19.7)70.0 ± 0.7(69.3 – 70.7)378 ± 4(373 – 380)Std 2377.9 ± 4.6(73.1 – 82.2)8.3 ± 1.3(6.8 – 9.1)2.2 ± 0.2(1.9 – 2.3)19.4 ± 2.1(17.0 – 20.7)70.1 ± 2.9(68.4 – 73.5)379 ± 6(375 – 386)CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 266 of 375Table D-23. continuedLocationTreatmentNMoisture(g/100 g FW)Ash(g/100 g DW)Fat(g/100 g DW)Protein(g/100 g DW)Carbohydrates(g/100 g DW)Calories(kcal/100 g DW)Mean±Standard Deviation(range)LondrinaIsoline380.7 ± 0.7(80.1 – 81.4)8.2 ± 0.5(7.8 – 8.8)2.0 ± 0.3(1.7 – 2.2)16.2 ± 0.9(15.1 – 16.9)71.3 ± 1.6(70.1 – 73.1)374 ± 3(371 – 376)CV127 + imi381.2 ± 1.1(80.3 – 82.4)8.8 ± 1.1(7.6 – 9.8)2.3 ± 0.5(1.9 – 2.8)17.0 ± 2.2(15.3 – 19.5)73.4 ± 2.7(70.3 – 75.6)377 ± 8(371 – 386)Std 1381.8 ± 0.6(81.3 – 82.5)8.4 ± 1.8(6.4 – 9.7)2.2 ± 0.2(2.0 – 2.4)17.5 ± 1.4(15.9 – 18.7)72.5 ± 3.5(68.4 – 74.9)377 ± 5(372 – 381)Std 2382.4 ± 1.1(81.7 – 83.7)9.1 ± 0.2(8.9 – 9.3)2.1 ± 0.1(2.0 – 2.2)17.8 ± 0.5(17.3 – 18.3)71.5 ± 0.5(71.0 – 71.8)377 ± 3(374 – 380)BrasiliaIsoline381.9 ± 1.1(80.9 – 83.1)9.2 ± 1.1(8.1 – 10.3)3.4 ± 0.2(3.2 – 3.5)18.8 ± 1.0(17.6 – 19.5)68.5 ± 2.2(66.9 – 71.0)381 ± 2(379 – 383)CV127 + imi381.4 ± 1.1(80.7 – 82.6)9.3 ± 1.2(8.6 – 10.7)3.3 ± 0.1(3.2 – 3.4)17.4 ± 1.4(16.6 – 19.0)70.0 ± 2.9(66.7 – 71.9)377 ± 3(374 – 380)Std 1382.1 ± 0.3(81.8 – 82.3)8.7 ± 0.6(8.1 – 9.3)3.4 ± 0.1(3.4 – 3.5)19.3 ± 1.8(17.6 – 21.1)68.6 ± 2.4(65.9 – 70.6)383 ± 3*(380 – 385)Std 2381.9 ± 0.5(81.4 – 82.4)9.0 ± 0.3(8.7 – 9.2)3.8 ± 0.3*(3.6 – 4.1)20.5 ± 1.6*(19.5 – 22.4)66.8 ± 1.8(64.8 – 68.3)387 ± 1*(386 – 387)Santo Antôniode PosseIsoline383.3 ± 0.9(82.7 – 84.3)8.6 ± 0.4(8.2 – 8.9)2.2 ± 0.1(2.2 – 2.3)16.9 ± 0.4(16.5 – 17.3)72.4 ± 0.5(71.8 – 72.8)378 ± 3(376 – 382)CV127 + imi383.6 ± 1.5(82.5 – 85.3)8.6 ± 0.3(8.4 – 8.9)2.1 ± 0.4(1.6 – 2.4)16.7 ± 1.7(15.3 – 18.6)72.5 ± 1.6(70.9 – 74.1)377 ± 4(374 – 381)Std 1385.0 ± 0.3(84.8 – 85.3)9.2 ± 0.3(8.9 – 9.4)2.3 ± 0.2(2.2 – 2.5)18.1 ± 0.3(17.8 – 18.3)70.4 ± 0.3*(70.1 – 70.7)374 ± 1(373 – 375)Std 2384.0 ± 0.3(83.7 – 84.2)8.9 ± 0.2(8.8 – 9.1)1.9 ± 0.3(1.7 – 2.2)18.6 ± 0.8(17.7 – 19.1)70.4 ± 0.7*(69.8 – 71.2)375 ± 2(373 – 377)*Statistically significantly different from CV127 + imi at P < 0.05.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 267 of 375 Table D-24. Fiber Composition of Forage of CV127 Soybean (CV127 + imi), the Isoline Control and Two Conventional Soybean Vari eties (Std 1 and Std 2), Grown at Six

Locations in Brazil in the 2007/2008 Season

Location Treatment N Crude Fiber ADF NDF

Mean ± Standard Deviation (range) g/100 g DW

Isoline 3 29.0 ± 0.9
(28.4 – 30.0) 34.87 ± 0.81
(34.04 – 35.65) 44.36 ± 3.25
(40.61 – 46.31) Santo Antônio

de Goiás

CV127 + imi 3 29.7 ± 0.6
(29.1 – 30.3) 33.66 ± 0.27
(33.39 – 33.92) 41.32 ± 1.27
(40.07 – 42.60)
Std 1 3^ 26.9 ± 2.6*
(25.1 – 29.9) 32.33 ± 1.92
(30.97 – 33.69) 42.56 ± 3.79
(39.88 – 45.24)
Std 2 3^ 28.8 ± 1.3
(27.9 – 30.3) 33.45 ± 0.28
(33.25 – 33.65) 44.67 ± 0.92
(44.02 – 45.32)
Uberaba Isoline 3 30.4 ± 1.4
(30.3 – 31.9) 36.93 ± 1.42
(35.56 – 38.39) 46.40 ± 1.89
(44.88 – 48.52)
CV127 + imi 3 29.6 ± 1.1
(28.6 – 30.7) 35.44 ± 1.68
(33.50 – 36.47) 46.30 ± 1.93
(44.19 – 47.96)
Std 1

3 30.1 ± 1.1
(29.4 – 31.4) 36.72 ± 1.23
(35.51 – 37.97) 44.66 ± 1.70
(42.94 – 46.33)
Std 2 3 30.5 ± 0.6
(29.9 – 31.0) 36.05 ± 1.31
(34.65 – 37.25) 44.88 ± 0.23
(44.67 – 45.13) Sete Lagoas Isoline 3 28.7 ± 1.1
(27.5 – 29.7) 35.40 ± 1.15
(34.33 – 36.62) 44.77 ± 1.84
(42.69 – 46.20)
CV127 + imi 3 28.7 ± 0.7
(27.9 – 29.1) 35.03 ± 1.24
(34.13 – 36.45) 44.56 ± 1.75
(43.11 – 46.50)
Std 1 3 27.0 ± 0.2*
(26.8 – 27.2) 35.76 ± 1.28
(34.29 – 36.66) 43.74 ± 1.71
(42.11 – 45.52)
Std 2 3 28.8 ± 0.6
(28.3 – 29.5) 35.34 ± 0.73
(34.91 – 36.18) 43.84 ± 1.73
(42.13 – 45.58)
Londrina Isoline 3 29.4 ± 1.6
(27.6 – 30.4) 35.91 ± 2.70
(34.34 – 39.03) 44.70 ± 3.37
(42.21 – 48.54)
CV127 + imi 3 30.2 ± 0.9
(29.2 – 30.8) 37.26 ± 1.09
(36.08 – 38.23) 45.31 ± 0.40
(44.84 – 45.56)
Std 1 3 29.1 ± 2.5
(26.3 – 30.6) 36.71 ± 3.93
(32.31 – 39.88) 44.78 ± 4.85
(39.33 – 48.61)
Std 2 3 29.7 ± 1.0
(28.6 – 30.5) 36.83 ± 2.03
(35.48 – 39.17) 45.44 ± 1.79
(43.79 – 47.34)
Brasilia Isoline 3 28.3 ± 0.4
(27.9 – 28.6) 35.08 ± 2.85
(31.79 – 36.82) 42.49 ± 2.77
(39.71 – 45.25)
CV127 + imi 3 28.8 ± 0.6
(28.2 – 29.3) 35.97 ± 1.13
(34.75 – 36.97) 43.97 ± 0.53
(43.38 – 44.40)
Std 1 3 27.6 ± 0.6
(26.9 – 28.1) 31.00 ± 2.81*
(28.94 – 34.20) 41.68 ± 1.80
(39.68 – 43.18)
Std 2 3 27.8 ± 1.2
(27.0 – 29.1) 32.17 ± 1.97
(30.43 – 34.30) 40.28 ± 1.21*
(38.93 – 41.28)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 268 of 375 Table D-24. continued

Location Treatment N Crude Fiber ADF NDF

Mean ± Standard Deviation (range) g/100 g DW

Santo Antônio

de Posse Isoline 3 32.6 ± 0.7 (31.9 – 33.2) 41.23 ± 1.50 (39.83 – 42.81) 48.99 ± 1.96 (46.99 – 50.90)

CV127 + imi

3 31.9 ± 1.2 (30.6 – 32.7) 41.07 ± 1.31 (39.62 – 42.16) 50.69 ± 2.18 (48.49 – 52.85)

Std 1

3 31.8 ± 0.4 (31.6 – 32.3) 40.45 ± 2.25 (37.86 – 41.82) 48.66 ± 2.29 (46.16 – 50.67)

Std 2

3 32.2 ± 1.3 (30.8 – 33.1) 41.33 ± 3.00 (38.67 – 44.58) 48.95 ± 2.88 (47.26 – 52.28) *Statistically significantly different from CV127 + imi at P < 0.05.

^ N= 2 for ADF and NDF.

Table D-25. Proximate and Fiber Content of Soybean Forage (% of Dry Matter) as Reported in OECD (2001).

Nutrient Soybean Forage Content (% of Dry Matter)

Moisture 74 - 79
Protein 11.2 - 17.3

Fat 3.1 - 5.1
Ash 8.8 - 10.5

NDF 34 - 40

ADF 32 - 38CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 269 of 375 Appendix E

Methods and Results of Seed Germination, Dormancy and Seed Quality Studies of CV127 Soybean

Summary

Soybean ( Glycine max L. Merrill) plants have been de veloped that are tolerant to the imidazolinone class of ag ricultural herbicides a nd are referred to as CV127 soybean. Seed quality and germination parameters are importa nt characteristics used in comparisons of agronomic performance among different varieties of a crop species and seed dormancy is an important biological characteristic used to as sess the weediness potential of different plant species. Therefore, seed quality (measured by a tetrazolium staining method), seed germination and dormancy (measured as hard seed) parameters of CV127 soybean plants (grown with either

imidazolinone herbicide or conventional herbicide tr eatment for weed control) were compared to

the isoline control and two conve ntional soybean varieties to confirm that CV127 soybean is equivalent to the isoline contro l and other conventional soybean varieties with respect to these biological characteristics. Seed samples from all treatments were harvested from field trials

conducted in six different locations in Brazil du ring the 2007 short growing season. The results of seed quality, germination and dormancy tests de monstrated that there were no statistically significant differences across field trial locations in seed quality parameters, seed germination or seed dormancy between CV127 soybean and the is oline control or the conventional soybean

varieties that were also included in the tests. These results demonstrate that the seed quality, germination and dormancy of CV127 soybeans are not different from that of the isoline control or other conventional soybean varieties. Furthe rmore, these results support the conclusion that CV127 soybean does not pose any greater weedines s potential or any diffe rent environmental impact than conventional soybean varieties.

Introduction

Assessments of seed germination, dormancy and quality were compared between CV127 soybean, the isoline control and two conventiona l soybean varieties. Seed germination and quality are important agronomic characteristics used in comparisons of different varieties within a crop species, and seed dormancy or “hard seed” in plants is an important characteristic often associated with plants that are weeds (Ande rson, 1996; Lingenfelter a nd Hartwig, 2003). An assessment of seed dormancy is often used to assess the weediness potential of different plant species (Baker, 1974). Therefore, seed quality and germination parameters of CV127 soybeans were compared to the isoline control as well as to two conventional so ybean varieties [Monsoy 8001 and Coodetec 217 (CD 217)] to confirm that CV127 soybean is equiva lent to conventional soybean varieties with re spect to these biological characteris tics. Also, even though soybeans do not typically display seed dormancy characte ristics (OECD, 2000), th e potential for seed dormancy in CV127 soybeans was assessed and compared to that of the isoline control as well as the two conventional soybean varieties. There are scientifically recognized tests that are used in assessing seed quality and germination character istics. These include tetrazolium staining for seed quality and the standard germination test (S GT). Seed dormancy was assessed by hard seedCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 270 of 375 determinations. The purpose of these studies was to confirm that CV127 soybean does not pose any greater weediness potential or any different environmental impact than conventional soybean

varieties.

Materials and Methods

Test, Control and Reference Seeds. The soybean seed materials used in this study were produced at six different field tr ial locations (Uberaba, MG; Sete Lagoas, MG; Santo Antônio de Goiás, GO; Brasília, DF; Vilhena, RO; Teresina, PI) within Brazil in the regulatory field trials of the 2007 short season that are described in Appendix F of this pe tition, and are listed below. Test, control and reference seeds tested included the following:

1. Test Seed. (Treatments T1 and T2) The test seed was harvested from CV127 plants grown in the regulat ory field trials in

Brazil in the 2007 short-season. The test seed was harvested from two different treatments, T1 and T2. T1 seed was harv ested from CV127 plants treated with an imidazolinone herbicide (imazapyr, 70 g ai/ha), whereas T2 seed was harvested from CV127 plants treated with conventional he rbicide (bentazon + acifluorfen-sodium,

commercial name Volt ® , 570 ai g/ha). Grain produc tion of treatment T1 was compromised in Uberaba and Sete Lagoas due to misapplication of imidazolinone herbicide, and data were not recorded for this treatment at these sites.

2. Near-Isogenic (Isoline) Cont rol Seed. (Treatment T3)

The isoline control seed was ha rvested from plants grown in the regulatory field trials

in Brazil in the 2007 short-s eason. The isoline control s eed was harvested from the near-isogenic (null segregant) soybean that is genetically similar to, and derived from, imidazolinone-tolerant CV127 soybean by br eeding, but which does not contain the imidazolinone-tolerance trait. The isoline control is included to provide germination

characteristic values to which the test seed can be compared. T3 plants were treated

with the conventional herbicide Volt ® as described above.

3. Reference Seed. (Treatments T4 and T5)

The reference seeds were obtained from tw o different conventional soybean varieties that are typical commercial varieties grow n in Brazil and these were cultivated and

harvested in the same 2007 fi eld trials as the CV127 see d. The reference soybean varieties were Monsoy 8001 (T 4) and Coodetec 217 (T5). Th ese reference seeds will provide a range of background values for dormancy and germination characteristics that are common to soybean. T4 and T5 pl ants were treated with the conventional

herbicide Volt ® as described above.

Prior to the initiation of the tests, the identities of the test, isoline control, and reference seed materials were verified by testing for the presence or absence of the csr1-2 transgene by PCR analyses, and by chain-of-custody records to verify identities of the isoline control and conventional soybean varieties.

Seed Quality. Tetrazolium testing for determining seed viability was performed following the procedures described by França Neto et al . (1998). The tetrazolium dye reacts withCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 271 of 375 dehydrogenase enzymes in living tissues to form a red pigment, there by distinguis hing living from nonliving tissues. Two sub-sa mples of 50 seed each were test ed for all treatments (T1, T2, T3, T4, and T5) harvested from each of the six fi eld trial locations. Initially, seed were kept overnight in a moist germination paper towel at 25oC for 16 hours in a saturated environment with a relative humidity of 100% . After conditioning, the seeds we re placed in plastic cups, and

covered with a 0.075% tetrazolium solution. The c ups were incubated in an oven at 40oC for 150 minutes. After staining, the seed s were rinsed with tap water several times to stop the staining reaction. Seeds were kept submerged in wate r to avoid dehydration. Individual seeds were examined with a magnifying lens (6X) unde r fluorescent light. Seed were dissected longitudinally through the midsection of the embr yonic axis with a single-edge razor blade. After the seed was sectioned, the seed halves we re separated and the seed coat was removed to expose the outer surface of the cotyledons. The inner and outer surfaces of the cotyledons were observed to identify seed defects that are reve aled by the staining proce dure and attributed to either mechanical, weathering, or stink bug damage for each sample. Special care was applied during the evaluation of the radicle-hypocotyl ax is and the vascular region on the cotyledons, which are structures of major importance for th e viability of the seed. After the seed was sectioned, the internal surfaces of live cotyledons are normally white, due to the lack of diffusion

of the tetrazolium solution to the inner tissues of the cotyle dons, and viability was measured accordingly.

Seeds were rated for each type of damage (mec hanical, weathering, or stink bug) according to the following scale (Franca Neto et al ., 1998; AOSA, 2007):

Rating Description 1 very high vigor, no seed damage, viable 2 high vigor, only minor seed damage, viable 3 medium vigor, low seed damage, viable 4 low vigor, moderate seed damage, viable 5 very low vigor, high seed damage, viable 6 nonviable, some living tissue but with moderate damage that is lethal 7 nonviable, some living tissue but with extensive damage that is lethal 8 dead, no living tissue

Seed Germination. Two hundred seed per experimental unit [four sub-samples of 50 seed each derived from each treatment (T1, T2, T3, T4, a nd T5) harvested from each of six field trial locations] were tested for germination, followi ng the procedures prescribed by the Rules for Testing Seeds (AOSA, 2007). To assess seed germination, each sub-sample of 50 seed was placed in rolled, moist paper toweling and inc ubated at 100% humidity in an incubator at 20/30oC with the lower temperature maintained for 16 hours and the higher temperature for 8

hours. Germination was assessed at five and eight days after planting. S eeds that failed to germinate in this test were evaluated for developmental abnormalities and hard or firm seed coat

characteristics to determine the cause of lack of germination. Hard seed did not imbibe during the test period, whereas firm seed imbibed wate r and were swollen but did not germinate during the test period. The hard or firm seed coat char acteristic is related to seed dormancy in soybean. Any seeds that did not germinate and did not fall into the abnormal and hard or firm seed coat categories were designated as dead seeds. Hard seed was determined at the final reading of theCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 272 of 375 germination test and the percenta ge hard seed was added to the germination percentage, based on the fact that hard-seeded soybean is considered viable (Potts et al ., 1978).

Seed Dormancy. Seeds that failed to germinate in the germination study and were classified as

hard seed as described above we re evaluated as dormant seeds.

Statistical Methods. In all field locations, five treatments were replicated four times in a randomized complete block design. Tukey’s test (Steel and Torrie, 1980) was used to compare means of sources of variation determined by AN OVA to have a significant effect on each of the

measured traits.

Results

Seed Quality and Seed Germination. In the analysis of variance (ANOVA) of data collected for traits associated with tetrazolium staining and seed germination (Table E-1), location (“Location” in Table E-1) had a statistically signif icant (P<0.01) effect on a ll the traits except for firm seed (which was zero for all treatments in all locations). Given th e vast geographical and environmental differences among the trial loca tions, significant differences among locations were expected. In contrast to location, and despite genetic di fferences between the CV127 lines and the commercial varieties, there was no stat istically significant treatment (“Treatment” in Table E-1) effect for any of the traits.

In general, seed damage (due to mechanical, we athering, and stink bugs), s eed viability, and seed germination levels were very good for seed of al l treatments produced at a ll field trial locations with the exception of seeds produced in Teresina (Table E-2). Seeds harvested from Teresina had the highest amount of seed damage (from w eathering and stink bugs) and the highest percent

abnormal and dead seed. In addi tion, seed produced in Teresina had the lowest percent seed viability and germination. It is likely that the quality of s eed produced in Teresina was negatively affected by the high temperatures and large amounts of rainfall that commonly occur at this location during the production season.

A statistically significant intera ction between treatment and location (“L x T” in Table E-1), was observed for all variables except stink bug damage and percent abnormal seedlings. Differences among treatments were observed for mechanical damage and percent hard seeds at Santo Antônio de Goiás; for viability, germination, and percent dead seed s at Vilhena; fo r viability and percent hard seeds at Teresina; for percent dead seeds at Sete Lagoas; and for percent hard seeds at Brasilia (Table E-4). Differences among T1 , T2, and T3, were significant for mechanical damage at Santo Antônio de Goiá s, for viability and percent hard seeds at Teresina, for percent dead seeds at Sete Lagoas, and for percent hard seeds at Brasilia (Table E-4).

Seed Dormancy. Seed dormancy in soybeans can be expressed as hard seed. Based on the germination test, the percentages of hard seed of each treatment, as a mean across locations, were not significantly different (Table E-1). The tr eatments means ranged from 0.9% to 0.04% across the six locations (Table E-3). Based on the location by treatment interaction (Table E-4), statistically significant differen ces among CV127 treatments and/or the isoline (T1, T2, and/or T3) for percent hard seed were evident at Brasilia, where the T1 treatment was higher than eitherCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 273 of 375 treatments T2 or T3 and at Tere sina, where the T3 treatment (iso line) was higher than the T1 or T2 (CV127) treatments. In eith er case, the values are low enough to pose no environmental risk.

These results were consistent with the fact that soybean typically does not display dormancy

characteristics.

Conclusions

The results of seed quality (seed damage and viability), seed ge rmination (percent germination, abnormal, dead, hard, or firm seeds), and dormanc y tests (percent hard seeds) demonstrated that there were statistically signifi cant differences among location m eans for all measured traits (“Location” in Table E-1), but th ere were no statistically signif icant differences among treatment means for any of the measured traits (“Trea tment” in Table E-1). The difference among locations was expected given the environmental differences among the locations. However, lack of significant treatment differences demonstr ates that the seed quality, germination and dormancy characteristics of CV127 s oybean are not different from that of the isoline control or other conventional soybean variet ies. These results support th e conclusion that CV127 does not pose any greater weediness potential or any diffe rent environmental imp act than conventional soybean varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 274 of 375 Table E-1. Mean Squares and Significance of Sources of Variation Considered in the

Statistical Model for Traits Assessed in the Tetrazolium and Germination Tests.

Traits Assessed (%) 1

Tetrazolium Test 2 Germination Test

Dmec Dweath Dstink Viab Germ Abnor Dead Hard Firm Sources

of

Variation (6-8) (6-8) (6-8) (1-5)

Location (L) 19.82** 2362.23** 126.36** 3007.83** 6494.27** 835.06** 2838.46** 7.23** -

Treatment (T) 24.60 178.12 21.59 77.34 80.08 34.96 87.12 3.61 -

L x T 6.20* 153.10** 9.45 ns 110.58** 102.64** 15.30 ns 91.27** 3.93** -

Mean 3.21 4.91 3.28 88.61 84.96 9.36 5.77 0.40 0

CV% 54.81 117.45 75.98 5.42 6.77 33.74 87.87 163.91 -

1 Dmec: mechanical damage; Dweath: weathering damage; Dstink: damage by stink bugs; Viab: seed viability; Germ: seed germination; Abnor: abnormal seedling; Dead: dead seed; Firm: firm seed; Hard: hard seed.

2 Seeds receiving a rating of 6 to 8 are considered nonviable. *,** = significant at P<0.05 and P<0.01, respectively. ns = not significant

Table E-2. Means of Each Location Across Treatments for Traits Assessed in the

Tetrazolium and Germination Tests

.

Traits Assessed 1, 4

Tetrazolium Test 2 Germination Test

Dmec 3 Dweath Dstink Viab Germ Abnor Dead Hard 3 Firm Locations (6-8) (6-8) (6-8) (1-5)

Uberaba 3.13 a 4 0.19 b 1.19 c 95.31 a 93.88 a 5.91 bc 0.22 b 0.00 a 0.00

Sete Lagoas 2.63 a 0.63 b 0.13 c 96.63 a 95.38 a 4.09 c 0.53 b 0.00 a 0.00

Santo

Antônio

de Goiás 4.40 a 0.20 b 5.40 ab 90.00 a 89.15 a 9.40 b 1.45 b 0.73 a 0.00

Brasília 4.40 a 0.30 b 2.05 c 93.25 a 91.65 a 7.80 bc 0.55 b 0.08 a 0.00

Vilhena 2.30 a 0.85 b 3.15 bc 93.70 a 92.55 a 6.05 bc 1.40 b 0.03 a 0.00

Teresina 2.12 a 29.12 a 7.35 a 61.71 b 45.06 b 23.23 a 32.29 a 1.59 a 0.00

1 Dmec: mechanical damage (%); Dweath: weathering damage (%); Dstink: damage by stink bugs (%); Viab: seed viability (%); Germ: seed germination (%); Abnor: abnormal seedling; Dead: dead seed; Firm: firm seed; Hard: hard

seed.

2 Seeds receiving a rating of 6 to 8 are considered nonviable.

3 Differences among location means for mechanical damage and hard seed were not detected by Tukey’s test.

4 Means in a column followed by the same letter do not differ statistically significantly at th e 5% level (Tukey’s test).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 275 of 375 Table E-3. Mean of Each Treatment Across Locations for the Traits Assessed in the

Tetrazolium and Germination Tests.

Traits Assessed 1

Tetrazolium Test 2 Germination Test

Dmec Dweath Dstink Viab Germ Abnor Dead Hard Firm Trt (6-8) (6-8) (6-8) (1-5)

T1 2.40 3 11.60 3.67 82.67 80.73 8.80 11.13 0.23 0.00

T2 3.17 2.65 3.09 91.00 88.48 8.22 3.30 0.04 0.00

T3 4.91 2.48 1.83 90.78 87.43 9.13 3.43 0.72 0.00

T4 2.71 2.79 4.63 89.83 85.13 9.48 5.40 0.04 0.00

T5 2.63 7.33 3.29 86.75 81.71 10.90 7.40 0.90 0.00

1 Dmec: mechanical damage (%); Dweath: weathering damage (%); Dstink: damage by stink bugs (%); Viab: seed viability (%); Germ: seed germination (%); Abnor: abnormal seedling; Dead: dead seed; Firm: firm seed; Hard: hard

seed.

2 Seeds receiving a rating of 6 to 8 are co nsidered nonviable. The cause of the nonviability of these seeds was further assessed and the results are reported here.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 276 of 375 Table E-4. Means of Each Combination of Lo cation and Treatment for the Traits Assessed

in the Tetrazolium and Germination Tests.

Traits Assessed 1, 4

Tetrazolium Test 2 Germination Test

Dmec Dweath Dstink Viab Germ Abnor Dead Hard Firm L ocation Trt (6-8) (6-8) (6-8) (1-5)

T1 * 3 * * * * * * * *

T2 3.25 a 4 0.50 a 0.50 95.50 a 93.50 a 6.25 0.25 a 0.00 a 0.00 a

T3 4.50 a 0.25 a 0.75 94.50 a 93.25 a 6.50 0.25 a 0.00 a 0.00 a

T4 1.50 a 0.00 a 1.75 96.25 a 95.00 a 4.75 0.25 a 0.00 a 0.00 a U beraba

T5 3.25 a 0.00 a 1.75 95.00 a 93.75 a 6.12 0.13 a 0.00 a 0.00 a

T1 * 3 * * * * * * * *

T2 2.00 a 1.25 a 0.00 96.75 a 96.75 a 3.25 0.00 b 0.00 a 0.00 a

T3 3.00 a 0.25 a 0.00 96.75 a 96.00 a 3.75 0.25 ab 0.00 a 0.00 a

T4 2.75 a 0.50 a 0.50 96.25 a 94.50 a 4.12 1.38 a 0.00 a 0.00 a Sete

L agoas

T5 2.75 a 0.50 a 0.00 96.75 a 94.25 a 5.25 0.50 ab 0.00 a 0.00 a

T1 3.00 b 0.75 a 4.75 91.50 a 91.00 a 7.75 1.25 a 0.25 b 0.00 a

T2 5.75 ab 0.00 a 6.00 88.25 a 88.25 a 10.50 1.25 a 0.25 b 0.00 a

T3 7.75 a 0.00 a 2.75 89.25 a 86.50 a 12.25 1.25 a 0.13 b 0.00 a

T4 2.75 b 0.00 a 5.25 92.00 a 91.50 a 7.38 1.13 a 0.25 b 0.00 a Santo

A ntônio

de Goiás

T5 2.75 b 0.25 a 8.25 89.00 a 88.50 a 9.13 2.38 a 2.75 a 0.00 a

T1 3.50 a 0.75 a 2.50 93.25 a 93.00 a 6.25 0.75 a 0.38 a 0.00 a

T2 4.00 a 0.00 a 1.75 94.25 a 92.25 a 7.50 0.25 a 0.00 b 0.00 a

T3 7.50 a 0.50 a 1.75 90.25 a 88.75 a 10.50 0.75 a 0.00 b 0.00 a

T4 3.50 a 0.00 a 2.00 94.50 a 92.25 a 7.38 0.38 a 0.00 b 0.00 a B rasília

T5 3.50 a 0.25 a 2.25 94.00 a 92.00 a 7.38 0.63 a 0.00 b 0.00 a

T1 2.00 a 1.50 a 2.25 94.25 ab 94.75 a 4.50 0.75 ab 0.13 a 0.00 a

T2 1.00 a 1.00 a 2.75 95.25 ab 94.25 a 4.25 1.50 ab 0.00 a 0.00 a

T3 3.00 a 0.00 a 1.00 96.00 a 94.75 a 5.00 0.25 b 0.00 a 0.00 a

T4 2.00 a 0.00 a 7.75 90.25 b 90.25 ab 7.25 2.50 a 0.00 a 0.00 a V ilhena

T5 3.50 a 1.75 a 2.00 92.75 ab 88.75 b 9.25 2.00 ab 0.00 a 0.00 a

T1 0.67 a 54.00 a 5.67 41.33 b 32.00 a 19.33 52.00 a 0.17 b 0.00 a

T2 3.00 a 16.67 a 9.00 71.00 ab 58.33 a 20.67 21.00 a 0.00 b 0.00 a

T3 3.33 a 17.67 a 5.67 73.67 a 58.00 a 19.33 22.67 a 5.33 a 0.00 a

T4 3.75 a 16.25 a 10.50 69.75 ab 47.25 a 26.00 26.75 a 0.00 b 0.00 a Teresina

T5 0.00 a 41.25 a 5.50 53.00 ab 33.00 a 28.25 38.75 a 2.63 ab 0.00 a

1 Dmec: mechanical damage (%); Dweath: weathering damage (%); Dstink: damage by stink bugs (%); Viab: seed viability (%); Germ: seed germination (%); Abnor: abnormal seedling; Dead: dead seed; Firm: firm seed; Hard: hard

seed.

2 Seeds receiving a rating of 6 to 8 are considered nonviable.

3 Seed were not harvested from this treatment at these fiel d sites because of a misapplication of imazapyr herbicide.

4 Means in a column followed by the same letter do not di ffer statistically significantly at the 5% level (Tukey’s test).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 277 of 375 Appendix F

Materials and Methods and Results from th e Agronomic, Phenotypic, and Ecological Interaction Evaluations of CV127 Soybean

Introduction

Evaluations of the agronomic, phenotypic and ec ological interactions of CV127 soybeans were

conducted at a total of thirteen field trial lo cations in Brazil, seven in the 2006/2007 growing

season and six in the 2007 short growing season. In all field trials, the CV127 soybeans were compared to a near-isogenic, null segregant soybean (referred to as the isoline control) that does

not contain the csr1-2 gene cassette and to two other c onventional soybean varieties commonly

cultivated in Brazil. The field tr ial sites were representative of the different regions within Brazil where soybean is commercially cul tivated and each trial location consisted of four replications of the two CV127 treatments, isoline control and tw o conventional soybean varieties that were organized in a randomized block design. The methodology for the trials conducted in the two different seasons was similar. The methodology used in the 2006/2007 trials is presented here and, for the 2007 season, only differences in the methodology from the 2006/2007 season are noted. The complete results from the evaluations of both growing seasons are also presented.

Field observations included seed germinatio n rate, seedling vigor, days to reach key developmental stages, plant height, and grain yield, as well as susceptibility to and interactions with diseases and insects. In addition, th e impact of cultivating CV127 soybeans on factors relevant to the nitrogen fixing symbiosis with Bradyrhizobium japonicum was assessed.

The treatments included in the field trials us ed for phenotypic, agronomic, and ecological

evaluations are listed below:

Treatment Genotype Herbicide application Treatment 1 (T1) CV127 Imazapyr (70 g ai/ha) Treatment 2 (T2) CV127 Volt ® (570 g ai/ha) Treatment 3 (T3) Isoline control Volt ® (570 g ai/ha) Treatment 4 (T4) Monsoy 8001 Volt ® (570 g ai/ha) Treatment 5 (T5) Coodetec 217 (CD 217) Volt ® (570 g ai/ha)

Results of these studies demonstrated that, ex cept for herbicide tolerance, CV127 soybean is phenotypically, phenologically and agronomically equivalent to the isoline control and other conventional soybean varieties. Further, these studies showed no different biological effect of CV127 compared to the isolin e control soybeans with resp ect to interaction with Bradyrhizobium
japonicum and nitrogen fixation capacity or interaction with various diseases or insects. Therefore, these results reinforce the conclusion that the cultivation of CV127 soybeans poses no different plant pest or weediness potential and will have no different environmental impact than

the cultivation of conventi onal soybean varieties.

The methods and results presented in th is Appendix are organized as follows:CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 278 of 375 1.0. Agronomic and phenotypic evaluations in the a) 2006/2007 and b) 2007 growing seasons 2.0. Disease susceptibility in the a) 2006/2007 and b) 2007 growing seasons 3.0. Insect interactions in the a) 2006/2007 and b) 2007 growing seasons 4.0. Nitrogen fixation characte ristics in the a) 2006/2007 and b) 2007 growing seasons

1.0 Agronomic and Phenotypic Evaluations

a) 2006/2007 growing season.

Materials and Methods

Field Locations . The experiments were planted dur ing the 2006/2007 growi ng season at seven experimental stations in Brazil. One station is ow ned and operated by BA SF S.A. and six by Embrapa. The stations are loca ted in regions that are represen tative of areas of commercial soybean production and areas in wh ich the soybean trial entries ar e adapted. All locations had a Certificate of Quality in Biosafety (CQB), an es tablished infrastructure, laboratory facilities and field equipment, and personnel experienced in ag ricultural research a nd trained in biosafety (Table F-1).

Table F-1. Experimental Station Loca tions with Experiment Codes, 2006/2007.

Research Station City, State Location (Experiment) Code

BASF Experimental Station Santo Antônio de Posse, SP EEA (011) Embrapa SNT Ponta Grossa, PR SNT (008) Embrapa Soybean Londrina, PR CNPSO (010) Embrapa / EPAMIG Uberaba, MG CTTP (012) Embrapa Horticulture Brasília, DF CNPH (016) Embrapa Rice and Beans Santo Ant ônio de Goiás, GO CNPAF (014) Embrapa Corn and Sorghum Sete Lagoas, MG CNPMS (013)

The predominant soil type, the tillage system (c onventional disc tillage or no tillage), and the cropping history varied by location (Table F-2). All but one lo cation (CNPMS) had been used for crop production within the previous two years.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 279 of 375 Table F-2. A Description of the S oybean Trial Field Locations, 2006/2007.

Cropping history

Location Soil type Tillage system Summer

2004/05 Short

Season 2005 Summer

2005/06 Short Season

2006
SNT Muddy sand Conventional Soybean Oats Soybean Oats
CNPSo Clay Conventional Soybean Oats Soybean Oats

EEA Clay sand Conventional Soybean Fallow Corn and soybean Potato

CTTP Sandy No till Soybean Fallow Soybean Fallow
CNPMS Clay Conventional Fallow Fallow Fallow Fallow
CNPAF Clay sand Conventional Soybean Soybean Fallow Fallow
CNPH Clay Conventional Soybean Fallow Fallow Fallow

Rainfall and temperature were recorded at each location for the period from planting to harvest (Table F-3). While irrigation was required to supplement rainfall at ea ch location, the quantity of water and frequency of application was dependen t on the quantity and dist ribution of rainfall. In general, the amount of precipitation was be low average at EEA, near average at CNPH, CNPAF, SNT, CTTP and CNPSo, and above aver age at CNPMS. Temperatures were near average at all locations, except at SNT and CTTP where they were below and above average, respectively.

Table F-3. Rainfall Amount (R) and Average Monthly Temperature (T) at Each of the Soybean Field Trial Locations During the Conduct of the Trials, 2006/2007.

October

2006 November

2006 December

2006 January

2007 February

2007 March

2007 April

2007

R T R T R T R T R T R T R T Location

mm  ̊ C mm  ̊ C mm  ̊ C mm  ̊ C mm  ̊ C mm  ̊ C mm  ̊ C

SNT 91 18.1 172 22.1 155 24.1 210 21.0 207 20.9 228 21.4 86 18.

7

CNPSo 61 22.8 130 22.1 188 22.8 300 22.2 161 22.6 196 22.7 - -

EEA 41 22.7 125 22.4 201 24.0 241 22.3 81 24.4 89 24.1 - -

CTTP - - 178 24.5 178 24.5 413 25.5 198 25.8 105 25.9 - -

CNPMS - - 202 22.2 337 24.1 390 24.2 491 22.7 92 24.6 - -

CNPAF - - 203 22.7 295 22.4 211 22.7 196 22.2 49 24.1 - -

CNPH - - 161 22.6 192 22.7 309 22.8 203 22.4 132 25.0 - -CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 280 of 375 Field, and Plot Design

. In all field locations, fi ve treatments (T1-T5, Table F-6) were replicated four times in a randomized complete block desi gn. Each plot consisted of six 8 m-long rows with 0.5 m spacing between rows of the same pl ot and 1.0 m spacing betw een rows of adjacent plots. Soil, plant, and insect samples were co llected from (or between) the outer (rows 1 and 6) and inner (rows 2 and 5) border rows of each plot, while phenotypic, phenologic, and agronomic measurements were determined from plants within the center two rows (rows 3 and 4).

Land and Crop Management . The land and crop management practices at each location, including the management of pests, pathogens , and weeds, were as recommended for each specific region in which the field trials were located.

Pre-plant : All land used for the trials has a long history of use for the production of crops. Fields were prepared for planting by addi ng fertilizer (nitrogen, phosphorous , and/or potassium) based on soil analyses and local recommendations, and disc-tilled to ensure a uniform seedbed.

At-planting : Seeds were planted by hand at a rate of 13-15 seeds/m (depending on local recommendations) or approximately 260,000 seeds per ha between 10 October and 21 November 2006 (Table F-4). Heavy rain following the initial pl anting at Ponta Grossa negativel y impacted the germination of all entries and prompted the need to replant at this location. Th e replanted seed of the test and

isoline control were from an F 3 family that is related to, but not genetically identical to, the F 3 family used in all other test locations.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 281 of 375 Table F-4. Planting and Harvest Dates for Each Field Trial Location in Brazil, 2006/2007.

Location Site code Planting date (2006) Harvest date (2007)

Santo Antônio de Posse, SP EEA Oct. 25 Mar. 16

Oct. 10 Poor germination, replanted Ponta Grossa, PR SNT Oct. 31 † Destroyed prior to harvest, Apr. 20

Londrina, PR CNPSO Oct. 18 Mar. 14

Uberaba, MG CTTP Nov. 21 Mar. 29

Brasília, DF CNPH Nov. 16 Mar. 29

Santo Antônio de Goiás, GO CNPAF Nov. 7 Mar. 15

Sete Lagoas, MG CNPMS Nov. 9 Mar. 22

† Poor germination of initial pl anting on 10 October prompted need to replant on 31 October.

Post-plant To ensure the successful completi on of growth and development of soybean plants at each field trial, insecticides, fungicides, and herbicides were applied at each location according to local recommendations and as needed to protect the plan ts from insect, fungal, and weed infestations (Table F-5). Insecticidal chemicals applie d included fipronil, teflubenzuron, permethrin, cypermethrin, thiamethoxan, imidaclopride, ch lorpyrifos, methamidophos, and avermectin. Chemicals applied for the c ontrol of fungi, primarily Phakopsora pachyrhizi , included epoxiconazole, pyraclostrobin, fl utriafol, and tebuconazole. For treatment T1, weeds were controlled in plots containing CV127 soybean with a single application of imazapyr (70g ai/ha) and controlled with bentazon and acifluorfen (referred to as Volt ® herbicide) in all other plots (treatments T2 through T5). Th e herbicide tepraloxydim was applie d for grass weed control in plots of treatments T2-T5 on an as-needed basis. In most locations, more than one application and formulation of insecticide, fungicide , and herbicide was used during the season.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 282 of 375 Table F-5. Insecticides, Herbicides, and F ungicides Applied to Plots after Planting,

2006/2007.

Rate Date of application

Location Product L/ha kg/ha 2006 2007 Use

Fastac ® 0.12 15-Nov Insecticide

Lorsban ® 1 15-Nov Insecticide

Fastac ® 0.12 19-Nov Insecticide

Lorsban ® 1 19-Nov Insecticide

Arsenal ® 0.15 22-Nov Herbicide

Volt ® 1 22-Nov Herbicide

Aramo ® 0.5 24-Nov Herbicide
Opera ® 0.5 2-Dec Fungicide

Sevin ® 2 2-Dec Insecticide

Nomolt ® 0.08 2-Dec Insecticide

Connect ® 1 16-Dec Insecticide

Nomolt ® 0.08 16-Dec Insecticide
Opera ® 0.6 29-Dec Fungicide
Opera ® 0.6 11-Jan Fungicide
Engeo ® Pleno 0.25 11-Jan Insecticide
Opera ® 0.6 20-Jan Fungicide
Impact ® 0.6 3-Feb Fungicide

Connect ® 1 3-Feb Insecticide

Folicur ® 0.5 19-Feb Fungicide Santo

Antônio de

Posse

Engeo ® Pleno 0.25 19-Feb Insecticide

Arsenal ® 0.07 29-Nov Herbicide

Volt ® 0.57 29-Nov Herbicide

Aramo ® 0.1 14-Dec Herbicide
Opera ® 0.09 21-Dec Fungicide
Opera ® 0.09 11-Jan Fungicide
Opera ® 0.09 24-Jan Fungicide
Engeo ® Pleno 0.066 6-Feb Insecticide Sete Lagoas

RivalTM 0.1 16-Feb Fungicide

Arsenal ® 0.07 28-Nov Herbicide

Volt ® 0.57 28-Nov Herbicide

Talcord ® 0.1 6-Dec Insecticide

Aramo ® 0.1 7-Dec Herbicide
Opera ® 0.5 4-Jan Fungicide
Opera ® 0.5 15-Jan Fungicide

Folicur ® 0.75 20-Jan Fungicide Santo

Antônio de

Goiás

Abametrina 0.3 28-Jan Insecticide

Arsenal ® 0.07 13-Dec Herbicide

Volt ® 0.57 13-Dec Herbicide

Aramo ® 0.1 18-Dec Herbicide
Opera ® 0.09 17-Jan Fungicide
Opera ® 0.09 31-Jan Fungicide
Engeo ® Pleno 0.066 31-Jan Insecticide Uberaba

RivalTM 0.1 14-Feb FungicideCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 283 of 375 Table F-5 continued.

Rate Date of application

Location Product L/ha kg/ha 2006 2007 Use

Vexter ® 1 31-Oct Insecticide

Vexter ® 1 1-Nov Insecticide Glyphosate 2 3-Nov Herbicide

Vexter ® 1 3-Nov Insecticide

Vexter ® 1 21-Nov Insecticide

Arsenal ® 0.15 28-Nov Herbicide

Volt ® 1 28-Nov Herbicide

Aramo ® 0.5 8-Dec Herbicide
Opera ® 0.5 9-Jan Fungicide

Metafos ® 0.5 9-Jan Insecticide

Fastac ® 0.2 9-Jan Insecticide
Opera ® 0.5 31-Jan Fungicide
Engeo ® Pleno 0.15 31-Jan Insecticide

Folicur ® 0.5 23-Feb Fungicide

Metafos ® 0.5 23-Feb Insecticide

Abametrina 0.4 1-Mar Insecticide Ponta Grossa

Engeo ® Pleno 0.18 9-Mar Insecticide

Arsenal ® 0.15 15-Nov Herbicide

Volt ® 1 15-Nov Herbicide

Tamaron ® 0.5 24-Nov Insecticide

Klap ® 0.15 2-Dec Insecticide

Aramo ® 0.5 7-Dec Herbicide
Opera ® 0.5 26-Dec Fungicide

Fastac ® 0.2 28-Dec Insecticide

Nomolt ® 0.08 28-Dec Insecticide
Opera ® 0.5 12-Jan Fungicide

Tamaron ® 0.5 12-Jan Insecticide

Nomolt ® 0.08 13-Jan Insecticide
Opera ® 0.6 30-Jan Fungicide
Engeo ® Pleno 0.25 30-Jan Insecticide RivalTM 0.5 7-Feb Fungicide Londrina

Engeo ® Pleno 0.2 15-Feb Insecticide

Vexter ® 1 24-Nov Insecticide

Vexter ® 1 1-Dec Insecticide

Fastac ® 0.2 8-Dec Insecticide

Arsenal ® 0.07 5-Dec Herbicide

Volt ® 0.57 5-Dec Herbicide
Opera ® 0.5 2-Jan Fungicide

Folicur ® 0.75 13-Jan Fungicide

Folicur ® 0.75 21-Jan Fungicide

Engeo ® Pleno 0.3 23-Jan Insecticide

Folicur ® 0.75 3-Feb Fungicide Brasília

Folicur ® 0.75 15-Feb FungicideCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 284 of 375 Harvest Plants were manually cut below the lowest pod and removed from the middle 6 m of the two center rows of each plot and thresh ed with a stationary bulk plant thresher. Seed were dried to uniform moisture and weighed. Harvest was ini tiated on 14 March in Londrina and completed

on 29 March in Bras í lia and Uberaba (Table F-4). A late, bu t necessary replanti ng of the trial at

Ponta Grossa resulted in unchara cteristic plant growth and late maturity of all CV127, isoline control, and conventional soybean va rieties. Thus, all plants at Ponta Grossa were plowed into the soil and destroyed prior to harvest.

Treatments. The five treatments (Table F-6) included in these studies represent a combination of the specific soybean genotypes (test, isoline, and reference) and herbicide formulations (imidazolinone and non-imidazolinone herbicides) required to generate data appropriate for meeting the objectives of each study.

Test (CV127 soybean) and control soybeans. The imidazolinone-tolerant soybean, CV 127, utilized in the study was the F 5 generation of CV127, line 127 (Figure III-1), and the control was the corresponding isoline or null segregant.

Conventional reference varieties. Two reference soybean varieties were used in these fi eld trials: Monsoy 8001 (M8001) and Coodetec 217 (CD 217). Both of these vari eties are common nontransgenic Brazilian commercial varieties with a similar maturity classification as CV127 and the isoline control soybeans.

Two herbicide treatments were used in thes e evaluations: 1) Imazapyr, an imidazolinone herbicide, sprayed at a rate of 70 g ai/ha, and 2) Volt ® , a combination of bentazon (400 g ai/ha) and acifluorfen (170 g ai/ha), sprayed at a rate of 570 g ai/ha (Table F-6).

Table F-6. Experiment Treatments (Entries ) Included in Each Soybean Field Trial,

2006/2007.

Treatment Genotype Herbicide
Treatment 1 (T1) CV127 Imazapyr (70 g ai/ha)

Treatment 2 (T2) CV127 Volt ® (570 g ai/ha)

Treatment 3 (T3) Isoline control Volt ® (570 g ai/ha)

Treatment 4 (T4) Monsoy 8001 Volt ® (570 g ai/ha)

Treatment 5 (T5) Coodetec 217 (CD 217) Volt ® (570 g ai/ha)

Data Collection and Statistical Analyses. The phenotypic, phenologic, and agronomic similarities of the CV127, isolin e control, and conventional soyb ean varieties were determined by recording various charac teristics routinely used to describe the phenotype and behavior of a soybean genotype. The characteristics were reco rded for all plots at all locations (unless otherwise noted).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 285 of 375 Tukey’s test (Steel and Torrie, 1980) was used to compare means of sources of variation determined by ANOVA to have a significant effect on each of the measured traits.

Traits Evaluated A large number of characteristic agronomic tra its were evaluated duri ng the different growth stages of soybean development. The different growth stages of soybea n, including vegetative and reproductive stages of soyb ean plant growth and developmen t are defined below in Table F-7. A description of the evaluati on of each measured trait follows.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 286 of 375 Table F-7. Description of the Different Soybean Growth Stages

Stage

number Stage name Stage definition

VE Emergence Hypocotyl elongated an d hooked; cotyledons at soil surface

VC Cotyledonary Hypocotyl straighten ed; cotyledons unfolded; unifoliates

unfolded

V1 First node First trifoliate leaves unf olded on 50%+ of plants in the plot

V2 Second node Second trifoliate leaves unfolded on 50%+ of plants in the plot

V3 Third node Third trifoliate leaves unfolded on 50%+ of plants in the plot

Vn n th node N = no. of nodes on 50%+ of plants in the plot with an

unfolded trifoliate

R1 Begin flower 50%+ of plants in the plot with a flower on any node

R2 Full flower 50%+ of plants in th e plot with a flower on upper two nodes

R3 Begin pod 50%+ of plants in the pl ot with a 5 mm long pod (upper four nodes)

R4 Full pod 50%+ of plants in the plot with a 2 cm long pod (upper four nodes)

R5 Begin seed 50%+ of plants in the plot with 3 mm long seed (upper four nodes)

R6 Full seed 50%+ of plants in the pl ot with a full-size seed (upper four nodes)

R7 Begin maturity 50%+ of plants in the plot have a pod with mature (tan/brown)

color

R8 Full maturity All plants in the plot have pods with mature (tan/brown) colorCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 287 of 375 Seed germination : the percentage of emerge d plant seedlings at the V2 stage relative to the quantity of seed sown per plot.

Seedling vigor : the overall appearance and rate of plant deve lopment of plants in a plot at the V2 stage according to the index in Table F-8.

Table F-8. Scale Used to Evaluate the Aver age Vigor of Seedlings on a Plot Basis.

Initial and final plant stand : the number of plants per plot at the V2 and R8 stages of plant development.

Green stem : the percentage of plants in a plot that were green at the R8 stage relative to the total number of plants in that plot.

Plant height (cm) : the average height of five plants in a plot measured from soil level to the top of the plant at the R8 stage.

Shatter: the percentage of plants in a plot that show premature dehiscence of pods (shatter) resulting in the loss of seed at maturity (R8). Shatter was not identified in any plot at any

location and is not considered further here.

Lodging : the average standability (lodgi ng) of plants in a plot at maturity (R8) according to the

index in Table F-9.

Table F-9. Scale Used to Evaluate the Av erage Lodging of Plants on a Plot Basis

. Lod g in g Descri p tio n 1 All p lants are standin g erect ( 80-90 de g ree an g le )

. 2 Most p lants have a sli g ht lean ( 60-70 de g ree an g le )

. 3 Most p lants have a moderate lean ( 40-50 de g ree an g le )

. 4 Most p lants have a severe lean ( 20-30 de g ree an g le )

. 5 All p lants are p rostrate ( 0 to 10 de g ree an g le )

.

Vegetative life cycle (days to flowering) : the number of days between planting and full flower (R2).

Total life cycle (days to maturity) : the number of days between pl anting and full plant maturity (R8). Vigor Description 1 Plant emerged; no trifoliate leaves 2 One trifoliate leaf; unhealthy plant 3 One trifoliate leaf; healthy plant 4 Two trifoliate leaves; unhealthy plant 5 Two trifoliate leaves; healthy plantCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 288 of 375 Seed size : the weight (g) of 100 seeds.

Grain yield : the total weight (g) of all grain harveste d (adjusted to 12% mo isture) converted to kg/ha.

Results and Discussion

Based on analyses of variance (Table F-10):

1) location had a significant effect on all measur ed traits, including pe rcent germination (G), final plant stand (FS), seedling vigor (V), days to full flower (DF), days to full maturity (DM), percent plants with green stem (GS), plant height (PH), grain yi eld (Yield), seed size (100SW), and lodging (L),

2) treatment had a significant effect on seedling vi gor, days to full matur ity, plant height, seed size, and lodging, and

3) the combination of location and treatment had a significant effect on all dependent variables except germination, final plant stand, and seed size.

Table F-10. Agronomic and Phenotypic Evaluations 2006/2007: Statistically Significant and Nonsignificant Sources of Variation in th e Model Used to Analyze Data Collected for

Each Trait.

Trait † Source of

variation G FS V DF DM GS PH Yield 100SW L Location (L) 432.3** 3767.0** 1.8** 142.7** 3857.7** 104.1* 7249.7** 65.32** 38.7** 9.66** Treatment (T) 58.8 90.3 0.11** 5.1 151.6** 61.1 538.1** 4.78 213.3** 3.98**

L x T 25.9 33.2 0.11** 13.3** 23.1** 35.8** 62.4** 2.64** 1.22 0.87**

Error 17.9 30.3 2.38 a 0.12 0.05 5.1 13.3 1.11 0.77 0.18

Mean 86.6 92.1 4.89 45.2 124.1 2.0 80.4 3619.8 16.4 1.67

† G = germination; FS = final plant stand; V = seedling vigor; DF = days to full flower (vegetative cycle); DM = days to full maturity (total cycle); GS = green stem; PH = plan t height; Yield = grain yield; 100SW = seed size (weight of 100 seeds); and L = degree of lodging. *,** = significant at P<0.05 and P<0.01, respectively.

a the error value for vigor is 2.38 x 10 -18

Due to the naturally different environmental char acteristics (temperature, rainfall, humidity, day length, etc.) among trial locations, location had a sign ificant effect on all assessed traits. At Ponta Grossa, the most southerly location (latitude 25 o S) and the location for which the Conquista soybean variety is least adapted, plants were taller, green stem s were more frequent, lodging was most severe, and number of days to flower and to maturity were higher than at all other locations (Table F-11). As noted previously, yield was not reco rded from this site due to a late replanting of the study and subsequent unchara cteristic plant growth and late maturity of the CV127, isoline control, and conventional soybean vari eties. Seed size and yield were highest at Londrina (Table F-11). In fact, yield was above the Brazilian average yield at all locations except Brasília. At Brasília, seed ling vigor was slightly less than at other locations (rating of 4.2CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 289 of 375 versus 5.0 for all other locations) and yield appeared to be more adversely affected by a high and

late incidence of Asian rust than other locations similarly infected. Due to a high Coefficient of Variation at Brasília for yield, li kely due to the Asian rust infec tion, yield data from Brasília was

not included in the across-l ocation analysis for yield.

Table F-11. Agronomic and Phenotypic E valuations 2006/2007: Location Means and Standard Deviations (Shown in Parentheses ) across Treatments for Evaluated Traits.

Trait † Location G FS V PH GS L DF DM 100SW Yield

89.8 a ‡ 106.8 a 5.0 a 56.1 d 0.0 a 1.0 b 43 b 118 c 16.8 b 3224 c Sto. Antônio

de Goiás (4.9) (5.8) (0) (2.2) (0) (0) (0.6) (2.1) (3.1) (385.9)

80.4 c 94.5 b 4.2 b 57.5 d 0.0 a 1.0 b 43 b 124 b 15.3 c 1873 d Brasília (4.9) (5.8) (0.4) (1.4) (0) (0) (1.3) (3.3) (2.6) (623.4)

88.0 ab 102.8 a 5.0 a 86.6 b 1.5 a 2.4 a 44 b 111 d 16.0 bc 3967 b Sete Lagoas (3.7) (5.9) (0) (5.5) (2.5) (1.1) (1.6) (1.6) (3.0) (221.0)

91.5 a 89.0 b 5.0 a 94.8 a 3.3 a 1.8 ab 45 b 128 b 19.0 a 4459 a Londrina (3.2) (3.6) (0) (7.8) (2.3) (0.8) (1.2) (2.2) (2.9) (709.0)

91.1 a 102.2 a 5.0 a 70.0 c 0.1 a 1.0 b 45 b 110 d 15.3 c 3304 c Uberaba (4.0) (5.7) (0) (9.2) (0.3) (0) (1.2) (1.4) (2.9) (217.8)

83.8 bc 81.7 c 5.0 a 98.2 a 3.3 a 1.9 ab 44 b 126 b 16.0 bc 3144 c Sto. Antônio

de Posse (7.1) (7.8) (0) (6.1) (1.8) (0.7) (2.7) (3.2) (2.8) (508.9)

81.5 c 68.1 d 5.0 a 100.1 a 6.0 a 2.7 a 51 a 151 a - - Ponta Grossa (3.8) (4.7) (0) (7.6) (8.5) (0.9) (1.8) (5.2)

† G = initial germination (%); FS = final plant stand; V = vigor; PH = plant height (cm); GS = green stem (%); L = degree of lodging; DF = days to full flower (vegetative cycle); DM = days to full matu rity (total cycle); 100SW = seed size [weight of 10 0 seeds (g)]; and Yield = grain yield (kg/ha).

‡ Means followed by the same letter do not differ statistically significantly by the Tukey test at 5% probability.

As an average across locations, the performance of CV 127 treated with imaza pyr (T1) or with

Volt ® (T2) and the performance of the isoline control treated with Volt ® (T3) were not significantly different for germination, final stan d, green stem, lodging, days to flower, days to maturity, or yield (Table F-12). Only for vigor, plant height a nd seed size did T1 significantly differ from T3 and only for seed si ze did T2 significantly differ from T3. Only for seed size, was

the difference between both conve ntional soybean vari eties (T4 and T5) si gnificantly different from Treatments 1, 2, and 3.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 290 of 375 Table F-12. Agronomic and Phenotypic E valuations 2006/2007: Treatment Means and Standard Deviations (Shown in Parenth eses) Over Location for Each Trait.

Trait † Treatment ‡

G FS V PH GS L DF DM 100SW Yield

T1 85.0 90.7 5.0 a § 79.0 bc 4.5 1.5 ab 45 126 a 19.7 a 3748
(7.1) (13.4) (0) (16.8) (7.6) (0.6) (2.2) (14.8) (1.5) (1089.2)

T2 85.8 90.5 4.8 b 83.4 ab 2.0 1.6 ab 45 126 a 18.8 a 3816
(5.7) (13.3) (0.3) (19.6) (2.4) (0.8) (2.2) (13.8) (1.7) (1165.5)

T3 88.3 94.6 4.8 b 86.0 a 1.1 1.9 a 45 125 ab 16.9 b 3463
(6.3) (15.5) (0.4) (20.4) (2.1) (1.1) (2.7) (13.8) (1.6) (1047.7)

T4 87.9 93.5 4.8 b 79.0 bc 1.6 1.1 b 45 122 c 13.2 c 3502
(6.1) (14.8) (0.4) (16.6) (2.1) (0.4) (3.8) (12.2) (1.4) (530.0)

T5 85.9 91.4 4.8 b 74.7 c 0.9 2.1 a 46 122 bc 13.5 c 3570
(6.0) (14.8) (0.4) (16.6) (1.7) (1.2) (3.6) (11.4) (1.8) (718.7)

† G = initial germination (%); FS = final plant stand; V = seedling vigor; PH = plant height (cm); GS = green stem (%); L = degree of lodging; DF = days to full flower (veg etative cycle); DM = days to full maturity (total cycle); 100SW = seed size [weight of 100 seeds (g)]; and Yield = grain yield (kg/ha).

‡ T1 = CV127 treated w ith imazapyr, T2 = CV12 7 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. § Means followed by the same letter do not differ statistically significantly by the Tukey test at 5% probability.

The Location x Treatment interaction was signifi cant for seedling vigor, da ys to full flower and maturity, green stem, plant height, yield, and lodgi ng (Table F-10). For all other traits, the mean

of the treatments over locations wa s a reliable indicator of the e ffect of each treatment on that particular trait. With the exception of plant height, percent green stem, lodging, and days to maturity at Ponta Grossa and seedling vigor at Brasília, the performance of CV127 treated with imazapyr (T1) and CV127 treated with Volt ® (T2) was not significantly different (Table F-13). Further, the performance of T2 and T3 were no t significantly different for germination, final plant stand, plant height, percent green stem, lodging, seed size or yi eld at any of the locations. T2 and T3 only differed significan tly for days to flowering and da ys to maturity at Londrina and Santo Antônio de Posse, and for days to floweri ng at Ponta Grossa (Table F-13). Similarly, the few instances where the performance of T1 and T3 differed significantly included seedling vigor at Brasília, as well as plant he ight, percent green stem , lodging, days to flow ering and maturity at Ponta Grossa, days to flowering and maturity at Londrina and Santo Antônio de Posse, and percent green stem at Sete Lagoas (Table F-13).

Although statistically si gnificant differences did exist betw een CV127 soybean and the isoline control for specific traits at specific locations, none of the di fferences are of any biological significance and all trait values are within the range for soybeans that are commercially cultivated in Brazil and elsewhere, including the conventional soybean varieties (T4 and T5)

included in these studies.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 291 of 375 Table F-13. Agronomic and Phenotypic Evaluations 2006/2007: Means for Each

Combination of Treatment and Location for All Traits and Standard Deviations (in Parentheses).

Trait †

Location Trt ‡

G FS V PH GS L DF DM 100SW Yield

T1 84.5 101.9 5.0 a § 56.2 ab 0.0 a 1.0 a 43 b 119 a 20.6 3313 a

(2.4) (5.9) (0.0) (1.4) (0.0) (0.0) (0.0) (0.0) (0.4) (332.2)

T2 86.8 103.1 5.0 a 56.9 ab 0.0 a 1.0 a 43 b 119 a 19.1 3275 a

(4.0) (4.7) (0.0) (0.5) (0.0) (0.0) (0.0) (0.0) (0.5) (151.9)

T3 92.5 109.4 5.0 a 57.9 a 0.0 a 1.0 a 43 b 119 a 17.1 3131 a

(3.7) (4.3) (0.0) (1.8) (0.0) (0.0) (0.0) (0.0) (0.7) (85.3)

T4 94.3 111.1 5.0 a 56.5 ab 0.0 a 1.0 a 43 b 114 b 14.2 3397 a

(2.7) (3.1) (0.0) (1.5) (0.0) (0.0) (0.0) (0.0) (0.9) (598.1)

T5 91.1 108.4 5.0 a 53.0 b 0.0 a 1.0 a 44 a 119 a 13.1 3007 a Santo

Antônio

de Goiás

(4.8) (6.1) (0.0) (2.4) (0.0) (0.0) (0.5) (0.0) (2.0) (562.4)

T1 78.6 92.5 5.0 a 56.6 a 0.0 a 1.0 a 44 a 126 a 18.0 1411 b

(4.7) (5.6) (0.0) (0.9) (0.0) (0.0) (0.8) (0.0) (0.5) (392.8)

T2 80.1 94.1 4.0 b 57.4 a 0.0 a 1.0 a 44 a 126 a 18.0 1553 b

(3.7) (4.4) (0.0) (0.7) (0.0) (0.0) (1.3) (0.0) (0.7) (386.4)

T3 81.9 96.3 4.0 b 580 a 0.0 a 1.0 a 44 a 126 a 15.6 1435 b

(4.6) (5.2) (0.0) (1.8) (0.0) (0.0) (0.5) (0.0) (1.4) (443.0)

T4 79.0 92.9 4.0 b 58.1 a 0.0 a 1.0 a 42 b 118 b 12.8 2622 a

(8.8) (10.6) (0.0) (1.6) (0.0) (0.0) (0.6) (0.0) (0.9) (321.8)

T5 82.3 96.9 4.0 b 57.3 a 0.0 a 1.0 a 43 ab 126 a 12.3 2347 ab Brasília

(1.7) (1.7) (0.0) (2.0) (0.0) (0.0) (1.0) (0.0) (1.1) (367.8)

T1 86.7 102.8 5.0 a 88.9 ab 5.0 a 2.3 ab 43 c 112 a 19.6 4114 a

(2.6) (3.4) (0.0) (2.2) (3.6) (0.5) (0.0) (0.0) (0.7) (155.2)

T2 89.6 99.8 5.0 a 87.3 ab 2.0 ab 2.3 ab 43 c 112 a 18.3 4028 a

(2.2) (9.1) (0.0) (3.8) (0.8) (1.0) (0.0) (0.0) (0.2) (66.2)

T3 90.6 108.0 5.0 a 91.9 a 0.0 b 3.5 a 43 c 112 a 16.8 3829 a

(4.1) (4.5) (0.0) (1.3) (0.0) (1.0) (0.0) (0.0) (0.4) (213.1)

T4 85.5 101.5 5.0 a 84.2 ab 0.0 b 1.0 b 45 b 112 a 12.2 3830 a

(4.4) (4.8) (0.0) (3.2) (0.0) (0.0) (0.0) (0.0) (0.4) (217.9)

T5 87.6 101.9 5.0 a 80.7 b 0.3 b 3.0 a 47 a 108 b 12.9 4033 a Sete Lagoas

(3.7) (5.6) (0.0) (7.7) (0.5) (0.8) (0.0) (0.0) (0.9) (312.7)

Table F-13 is continued on the next page.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 292 of 375

Table F-13 continued.

Trait †

Location Trt ‡

G FS V PH GS L DF DM 100SW Yield

T1 94.2 90.3 5.0 a 96.9 ab 2.8 a 2.0 a 46 b 129 b 21.5 4721 ab

(2.8) (4.3) (0.0) (3.8) (1.5) (0.8) (0.0) (0.0) (1.6) (572.2)

T2 91.5 89.0 5.0 a 100.3 a 3.5 a 1.8 a 46 b 129 b 22.1 5171 a

(3.6) (4.5) (0.0) (1.6) (2.1) (0.5) (0.0) (0.0) (0.3) (735.0)

T3 92.1 90.3 5.0 a 101.9 a 3.0 a 1.8 a 47 a 130 a 19.5 4463 ab

(3.4) (4.1) (0.0) (3.5) (3.5) (1.0) (0.0) (0.0) (0.6) (676.7)

T4 89.6 86.4 5.0 a 91.3 b 4.6 a 1.0 a 44 c 125 c 15.0 3799 b

(2.8) (2.2) (0.0) (6.8) (1.9) (0.0) (0.0) (0.0) (1.2) (358.3)

T5 90.1 89.0 5.0 a 83.5 c 2.8 a 2.3 a 44 c 125 c 16.9 4143 ab Londrina

(2.5) (2.9) (0.0) (2.9) (3.0) (1.0) (0.0) (0.0) (0.5) (498.9)

T1 89.7 100.9 5.0 a 70.5 ab 0.3 a 1.0 a 44 b 110 a 18.9 3432 a

(1.5) (2.1) (0.0) (2.0) (0.5) (0.0) (0.0) (0.5) (1.0) (185.0)

T2 89.9 101.0 5.0 a 75.9 a 0.0 a 1.0 a 44 b 111 a 17.2 3404 a

(3.1) (4.2) (0.0) (4.2) (0.0) (0.0) (0.0) (0.5) (0.4) (177.7)

T3 92.4 105.0 5.0 a 79.5 a 0.3 a 1.0 a 44 b 111 a 16.1 3107 a

(6.3) (9.4) (0.0) (6.7) (0.5) (0.0) (0.0) (0.6) (0.8) (252.8)

T4 92.5 104.3 5.0 a 61.6 b 0.0 a 1.0 a 44 b 111 a 11.9 3303 a

(2.7) (3.3) (0.0) (7.3) (0.0) (0.0) (0.0) (1.0) (0.6) (84.4)

T5 90.9 99.6 5.0 a 62.4 b 0.0 a 1.0 a 47 a 108 b 12.4 3276 a Uberaba

(5.6) (7.4) (0.0) (9.0) (0.0) (0.0) (0.0) (1.0) (0.3) (274.5)

T1 80.8 77.8 5.0 a 94.0 a 4.0 a 1.8 ab 47 a 130 a 19.5 3159 a

(11.8) (12.1) (0.0) (4.5) (0.8) (0.5) (0.0) (0.0) (1.6) (305.4)

T2 82.4 79.5 5.0 a 102.3 a 4.3 a 1.8 ab 47 a 130 a 18.1 3202 a

(6.4) (7.0) (0.0) (2.1) (2.9) (0.5) (0.0) (0.0) (1.2) (589.9)

T3 88.4 88.4 5.0 a 105.2 a 1.0 a 2.3 a 45 b 126 b 16.1 2788 a

(5.2) (6.2) (0.0) (1.0) (0.8) (0.5) (0.0) (0.0) (1.3) (799.3)

T4 87.5 84.9 5.0 a 98.2 a 4.0 a 1.0 b 41 c 123 c 13.0 3182 a

(2.7) (4.0) (0.0) (5.0) (0.8) (0.0) (0.0) (0.0) (1.1) (427.7)

T5 79.8 78.0 5.0 a 91.2 a 3.0 a 2.8 a 41 c 123 c 13.4 3392 a Santo

Antônio

de Posse

(4.4) (4.4) (0.0) (3.4) (1.2) (0.5) (0.5) (0.0) (0.7) (346.0)

Table F-13 is continued on the next page.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 293 of 375

Table F-13 continued.

Trait †

Location Trt ‡

G FS V PH GS L DF DM 100SW Yield

T1 80.7 69.1 5.0 a 90.1 c 19.8 a 1.8 c 49 c 157 a - -

(3.6) (3.7) (0.0) (4.3) (10.8) (0.5) (0.0) (0.0)

T2 80.1 67.1 5.0 a 104.0 ab 4.5 b 2.8 b 49 c 154 b - -

(2.9) (2.7) (0.0) (6.3) (2.1) (0.5) (0.0) (0.0)

T3 80.5 64.6 5.0 a 107.8 a 3.5 b 2.8 b 51 b 154 b - -

(4.0) (3.1) (0.0) (1.7) (2.6) (0.5) (0.0) (0.0)

T4 86.8 73.6 5.0 a 103.5 ab 2.3 b 2.0 bc 53 a 148 c - -

(0.6) (2.3) (0.0) (4.1) (1.3) (0.0) (0.0) (0.0)

T5 79.3 66.0 5.0 a 94.9 bc 0.0 b 4.0 a 53 a 143 d - - Ponta

Grossa

(2.2) (6.3) (0.0) (2.8) (0.0) (0.0) (0.0) (0.0)

† G = initial germination (%); FS = final plant stand; V = seedling vigor; PH = plant height (cm); GS = green stem (%); L = degree of lodging; DF = days to full flower (veg etative cycle); DM = days to full maturity (total cycle); 100SW = seed size [weight of 100 seeds (g)]; and Yield = grain yield (kg/ha).

‡ Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control treated with

Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. § Means followed by the same letter do not differ statistically significantly by the Tukey test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 294 of 375 b) 2007 growing season.

Since the methodology used in the agronomic evaluations was the same in the 2006/2007 and 2007 growing seasons, only important differences from the description of the methodology for

the 2006/2007 season are noted below.

Field Locations . The experiments were planted dur ing the short seas on in 2007 at six experimental stations in Brazil. The stations ar e located in regions that are representative of areas of commercial soybean production and areas in which the soybean trial entries are adapted.

Details of the stations are listed in Table F-14.

Table F-14. Experimental Station Lo cations with Location Code, 2007.

Research Station City, State Location (Experiment Code)

Embrapa / EPAMIG Uberaba, MG CTTP (001)
Embrapa Corn and Sorghum Sete Lagoas, MG CNPMS (002)
Embrapa Rice and Beans Santo Ant ônio de Goiás, GO CNPAF (003)
Embrapa Horticulture Brasília, DF CNPH (005)
Embrapa Soybean in Vilhena Vilhena, RO VILH (006)
Embrapa Mid-North Teresina, PI EMN (007)

The predominant soil type, the tillage system (c onventional disc tillage or no tillage), and the cropping history varied by location (Table F-15). All locations but one (EMN) had been used for crop production within the previous two years.

Table F-15. A Description of the Soybean Field Trial Locations, 2007

.

Cropping history

Location Soil type Tillage system Short

Season 2005 Summer

2005/06 Short

Season 2006 Summer

2006/07 CTTP Sandy No till Fallow Soybean Fallow Fallow
CNPMS Clay Conventional Fallow Fallow Fallow Pearl Millet
CNPAF Clay sand Conventional Fallow Soybean Fallow Rice
CNPH Clay Conventional Soybean Fallow Potato Fallow
VILH Clay Conventional Fallow Soybean Fallow Soybean
EMN Clay Conventional Fallow Fallow Fallow Fallow

Rainfall and temperature was recorded at each location for the period from planting to harvest (Table F-16). While irrigation was required to supplement rainfall at ea ch location, the quantity of water and frequency of application was dependen t on the quantity and dist ribution of rainfall. In general, precipitation was greatest during Ma rch and April, reaching means of 126 and 106 mm, respectively. Irrigation was necessary duri ng May and June, when the mean rainfall was only 30 and 0.3 mm, respectively. The mean temperature was 23.7°C and, in general,CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 295 of 375 temperatures were greater in March and April and decreased from May to July (winter season), except at EMN, where temperatures increased in June and July. The EMN location had precipitation and temperatures above the general mean, while the remaining locations experienced precipitation and temperatures near the mean. Excessive rainfall after planting caused the loss of three plots at EMN.

Table F-16. Rainfall Amount and Average Mo nthly Temperature at Each of the Soybean Field Trial Locations, 2007.

March April May June July

R* T R T R T R T R T Location

mm  ̊ C mm  ̊ C mm  ̊ C mm  ̊ C mm  ̊ C

CTTP 104.9 25.9 109.6 24.2 17.3 21.2 0.0 19.8 15.7 19.4

CNPMS 91.8 24.6 119.5 23.5 8.5 20.7 1.9 19.7 0.0 20.0

CNPAF 49.3 24.1 92.6 23.1 6.1 22.1 0.0 21.4 9.3 22.4

CNPH 132.2 25.0 63.7 24.3 6.0 23.1 0.0 22.5 0.0 23.1

VILH 76.0 23.5 90.0 23.6 22.5 21.8 0.0 22.2 19.0 22.2

EMN 300.4 27.2 161.2 27.2 118.6 27.4 0.1 33.2 4.9 34.0
*R=Rainfall (mm) and T=average monthly temperature (  ̊ C).

Field and Plot Design

. As described for the 2006/2007 field trial season.

Land and Crop Management . The land and crop management practices at each location, including the management of pests, pathogens , and weeds, were as recommended for each specific region in which the field trials were located.

At-planting Seeds were planted by hand at a rate of 13-15 seeds/m (depending on local recommendations) or approximately 260,000 seeds per ha between 27 Fe bruary and 14 March 2007 (Table F-17).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 296 of 375

Table F-17. Planting and Harvest Dates for Each Field Trial Location in Brazil, 2007.

Location Site Code Planting date (2007) Harvest date (2007)

Uberaba, MG CTTP March 02 July 03

Sete Lagoas, MG CNPMS March 06 July 11

Santo Antônio de Goiás, GO CNPAF February 27 July 08

Brasília, DF CNPH March 01 July 09

Vilhena, RO VILH March 12 July 10

Teresina, PI EMN March 14 July 04

Post-planting To ensure the successful completion of each trial, insecticides, fungicides, and herbicides were applied at each location according to local recomme ndations and as needed to protect the plants from insect, fungal, and weed infestations (Table F-18).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 297 of 375 Table F-18. Insecticides, Herbicides, and Fung icides Applied to Plots After Planting, 2007.

Location Product Rate

l/ha Active ingredient

concentration Date of application

2007 Use

Vexter ® 0.8 Chlorpyrifos 480g/l 12 Mar Insecticide

Arsenal ® 0.15 Imazapyr 480g/l 1 Apr Herbicide

Volt ® 1 Bentazon 400g/l + Acifluorfen 170g/l 1 Apr Herbicide

Nomolt ® 0.08 Teflubenzuron 150g/l 15 Apr Insecticide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 25 Apr Fungicide

Folicur ® 0.75 Tebuconazole 200g/l 4 May Fungicide

Folicur ® 0.75 Tebuconazole 200g/l 20 May Fungicide
EngeoTM Pleno 0.3 Cypermethrin 220g/l + Thiamethoxam 110g/l 21 May Insecticide
EngeoTM Pleno 0.3 Cypermethrin 220g/l + Thiamethoxam 110g/l 2 Jun Insecticide CNPH

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 27 May Fungicide

Arsenal ® 0.15 Imazapyr 480g/l 1 Apr Herbicide

Volt ® 1 Bentazon 400g/l + Acifluorfen 170g/l 1 Apr Herbicide

Aramo ® 0.5 Tepraloxydim 200g/l 7 Apr Herbicide

Fastac ® 0.2 Alpha-cypermethrin 100g/l 31 Mar Insecticide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 17 Apr Fungicide

Folicur ® 0.75 Tebuconazole 200g/l 26 Apr Fungicide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 1 May Fungicide

Folicur ® 0.75 Tebuconazole 200g/l 19 May Fungicide CTTP

Engeo ® Pleno 0.3 Cypermethrin 220g/l + Thiamethoxam 110g/l 19 May Insecticide

Arsenal ® 0.15 Imazapyr 480g/l 18 Apr Herbicide

Volt ® 1 Bentazon 400g/l + Acifluorfen 170g/l 18 Apr Herbicide

Aramo ® 0.5 Tepraloxydim 200g/l 24 Apr Herbicide

Fastac ® 0.2 Alpha-cypermethrin 100g/l 24 Apr Insecticide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 17 May Fungicide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 25 Apr Fungicide

Engeo ® Pleno 0.3 Cypermethrin 220g/l + Thiamethoxam 110g/l 17 May Insecticide CNPMS

Folicur ® 0.75 Tebuconazole 200g/l 6 Jun Fungicide

Arsenal ® 0.15 Imazapyr 480g/l 27 Mar Herbicide

Volt ® 1 Bentazon 400g/l + Acifluorfen 170g/l 27 Mar Herbicide

Vexter ® 0.8 Chlorpyrifos 480g/l 5 Apr Insecticide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 15 Apr Fungicide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 25 Apr Fungicide

Folicur ® 0.75 Tebuconazole 200g/l 4 May Fungicide

Engeo ® Pleno 0.3 Cypermethrin 220g/l + Thiamethoxam 110g/l 20 May Insecticide

Folicur ® 0.75 Tebuconazole 200g/l 21 May Fungicide CNPAF

Engeo ® Pleno 0.3 Cypermethrin 220g/l + Thiamethoxan 110g/l 3 Jun InsecticideCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 298 of 375 Table F-18. continued

Location Product Rate

l/ha Active ingredient

concentration Date of application

2007 Use

Vexter ® 0.8 Chlorpyrifos 480g/l 10 Apr Insecticide

Arsenal ® 0.15 Imazapyr 480g/l 11 Apr Herbicide

Volt ® 1 Bentazon 400g/l + Acifluorfen 170g/l 11 Apr Herbicide EMN

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 2 May Fungicide Folisuper ®

600BR 0.7 Parathion-methyl 600g/l

19 Mar Insecticide Folisuper ®

600BR 0.7 Parathion-methyl 600g/l

26 Mar Insecticide

Arsenal ® 0.15 Imazapyr 480g/l 3 Apr Herbicide

Volt ® 1 Bentazon 400g/l + Acifluorfen 170g/l 3 Apr Herbicide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 9 Apr Fungicide

Sumithion ®

500CE 1.2 Fenitrothion 500g/l 9 Apr Insecticide

Opera ® 0.5 Epoxiconazole 50g/l + Pyraclostrobin 133g/l 23 Apr Fungicide

Sumithion ®

500CE 1.2 Fenitrothion 500g/l 23 Apr Insecticide

Pirate ® 0.7 Chlorfenapyr 240g/l 3 May Insecticide

RivalTM 0.4 Tebuconazole 200g/l 11 May Fungicide

Engeo ® Pleno 0.17 Cypermethrin 220g/l + Thiamethoxan 110g/l 11 May Insecticide

Vexter ® 0.55 Chlorpyrifos 480g/l 11 May Insecticide

Engeo ® Pleno 0.17 Cypermethrin 220g/l + Thiamethoxan 110g/l 23 May Insecticide

Vexter ® 0.55 Chlorpyrifos 480g/l 23 May Insecticide VIL

RivalTM 0.5 Tebuconazole 200g/l 28 May Fungicide

Treatments. The same five treatments as described for the 2006/2007 field trial season were

included in the 2007 field trial season (Table F-6). The only difference was that the F 6 generation of CV127, line 127 (Figure III-1), a nd the corresponding generation of the isoline control (null segregant) were used in the 2007 studies.

Results and Discussion

Based on analyses of variance (Table F-19):

1) location had a significant effect on all measur ed traits, including pe rcent germination (G), seedling vigor (V), initial (IS) and final (FS) plan t stand, days to full flower (DF), days to full maturity (DM), percent plants with green stem (G S), plant height (PH), gr ain yield (Yield), seed size (100SW), and lodging (L),

2) no significant effect was observed fo r treatment for any of the traits,

3) the combination of location and treatment had a significant effect on plant height (PH), percent plants with green stem (GS), seed size (1 00SW), grain yield (Yield), lodging (L), days to full flower (DF) and days to full maturity (DM).CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 299 of 375Table F-19. Agronomic and Phenotypic Evaluations 2007: Statistically Significant and Non-Significant Sources of Variationin the Model Used to Analyze Data for Each Trait.1G = initial germination (%); V = seedling vigor; IS = initial plantstand; FS = final plant stand; PH = plant height (cm); GS =green stem (%); 100SW = seed size[weight of 100 seeds (g)]; Yield = grain yield (kg/ha); L = degreeof lodging; DF = days to fullflower (vegetative cycle) andDM = days to full maturity (totalcycle).*,** = significant at P<0.05 and P<0.01, respectively.Trait1Source ofVariationGVISFSPHGS100SWYieldLDFDMLocation (L)3678.37** 2.41** 4863.69** 5539.36** 3078.27** 8343.29** 16.86** 3723651.19** 20.83** 2367.07** 2675.40**Treatment (T)268.35 0.05 369.06 399.81 664.50 6801.09 85.14 423247.06 0.23 9.72 32.52L x T136.96 0.05 190.39 189.34 50.32** 1827.25** 2.61** 306005.05** 0.23** 59.70** 10.80**Error103.16 0.17 146.72 152.24 6.26 28.57 0.85 105353.70 0.05 0.15 0.54Mean80.04 4.86 93.72 89.59 57.57 24.66 17.83 2324.72 1.42 43.93 114.75CV%12.70 8.51 12.92 13.77 4.35 21.68 5.17 13.96 16.00 0.87 0.64CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 300 of 375 Due to the naturally different environmental char acteristics (temperature, rainfall, humidity, day length, etc.) among trial locations, location had a si gnificant effect on all assessed traits. The plant density was nearly ideal at all locations , except Teresina, where the final stand was the lowest due to low seed germination (Table F- 20) caused by excessive rainfall shortly after planting. As expected for short-season growi ng conditions, plant height was normally low, except at Sete Lagoas, where plants reached a me an stature of 82 cm and the lodging reached the higher index of 3.5. Percentage of green stem wa s higher at Uberaba, Sete Lagoas and Vilhena. An error in the preparation of the herbicide imaza pyr for application of treatment T1 at Uberaba and Sete Lagoas resulted in the application of more than three times the intended application rate (70 g a.i./ha) and caused high phytotoxicity, loss of pods a nd 100% of plants showing green stem over all the plots with treatment T1. Th ese plots were not harvested and no data was recorded for seed size (100SW), days to full matu rity (DM) and yield at these two sites (Table F22). The higher green stem percentage observe d at Vilhena was likel y caused by stink bug

attack.

The location mean for seed size ranged from 16.4 grams at Uberaba to 19 grams at Vilhena (Table F-20). Flowering occurred earlier at V ilhena and Teresina as a consequence of the lower latitudes of these sites. Similarly, the total cycle was shorter (less than 115 days) in these two sites and also in Uberaba and Sete Lagoas. The yield was relatively high at Bras í lia and Sete Lagoas, medium at Teresina and Uberaba and low at Santo Antônio de Go iás and Vilhena (Table F-20). The wide-ranging performa nce obtained for grai n yield among the different sites was expected and considered normal for short-seas on conditions. However, the coefficients of

variation for all traits were considered low and acceptable for the purposes of this work (Table F19).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 301 of 375 Table F-20. Agronomic and Phenotypic Evaluations 2007: Location Means Across Treatments and Standard Deviations (i n Parentheses) for Evaluated Traits.

1 G = initial germination (%); V = seedling vigor; IS = initial plant stand; FS = final plant stand; PH = plant height (cm); GS = green stem (%); 100SW = seed size [weight of 1 00 seeds (g)]; Yield = grain yield (kg/ha); L = degree of lodging; DF = days to full flower (vegetative cycle) and DM = days to full maturity (total cycle).

* Means followed by the same letter do not differ statistically significantly by the Tukey test at 5% probability.

As an average across locations for any given tr ait, the means of all treatments showed no evidence of a significant difference (Table F-21). However, the Location x Treatment interaction was statistically si gnificant for plant height (PH), green stem (GS), seed size (100SW), yield, lodging (L), days to flowering (DF) and days to maturity (DM) (Table F-19). For all other traits, the means of the treatments over locations were a reliable indicator of the effect of each treatment on that particular trai t. With the exception of percent green stem at Uberaba and Sete Lagoas (caused by the applica tion of a higher concentration of imazapyr, as reported previously) and days to flowering at Uberaba the performance of CV127 treated with imazapyr (T1) and CV127 treated with Volt ® (T2) were not significantly different (Table F-22). Further, the performance of T2 (CV127 treated with Volt ® ) and T3 (isoline control treated with

Volt ® ) were not statistically significantly different for germ ination, seedling vigor, initial and final plant stand, lodging, days to fl owering or days to maturity at any of the field trial locations. T2 and T3 only differed statisti cally significantly for plant height at Sete Lagoas, percent green stem at Uberaba and Vilhena, seed size at Ub eraba, Sete Lagoas and Vilhena and yield at Vilhena (Table F-22). Similarly, the instances where the performance of T1 and T3 differed significantly included plant height at Sete La goas, green stem at Uberaba, Sete Lagoas and Vilhena, seed size and yield at Vilhena, and days to flowering at Uberaba (Table F-22). Trait 1 Location G V IS FS PH GS 100SW Yield L DF DM

73.95 c* 5.00 a 88.70 c 79.95 b 47.69 b 39.20 ab 16.44 c 2142.00 bc 1.00 b 45.20 b 107.56 d Uberaba (7.0) (0.0) (8.5) (9.6) (6.0) (35.0) (2.4) (509.8) (0.0) (0.8) (1.5)

79.75 bc 5.00 a 95.70 bc 90.60 b 82.37 a 41.35 ab 17.37 abc 2659.38 ab 3.50 a 48.90 a 113.06 c Sete Lagoas (5.5) (0.0) (6.4) (8.1) (6.6) (33.3) (2.2) (368.6) (0.8) (0.8) (1.0)

92.95 a 5.00 a 111.43 a 110.45 a 54.30 b 10.00 ab 18.00 abc 1948.60 c 1.00 b 44.00 b 130.15 a Santo

Antônio

de Goiás (2.5) (0.0) (3.0) (3.1) (5.3) (0.0) (1.9) (362.2) (0.0) (0.0) (1.0)

89.25 ab 5.00 a 107.18 ab 106.23 a 53.20 b 3. 00 b 18.60 ab 3054.55 a 1.00 b 49.00 a 126.60 b Brasília (3.3) (0.0) (3.9) (4.0) (7.1) (2.5) (1.6) (356.3) (0.0) (0.0) (1.6)

88.10 ab 5.00 a 91.85 c 84.15 b 54.45 b 48.40 a 19.02 a 1975.90 c 1.00 b 37.30 d 99.95 e Vilhena (5.9) (0.0) (6.2) (7.8) (7.6) (33.7) (2.6) (427.5) (0.0) (1.4) (3.1)

56.25 d 4.15 a 67.48 d 66.15 с 53.40 b 6.00 ab 17.11 bc 2175. 94 bc 1.00 b 39.20 c 109.40 d Teresina (22.7) (1.0) 27.3) (26.8) (3.4) (5.0) (2.0) (443.4) (0.0) (1.0) (2.0)CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 302 of 375 Table F-21. Agronomic and Phenotypic E valuations 2007: Treatment Means and Standard Deviations (in Parentheses) Over Location for Each Trait.

Trait 1

Trt 2 G V IS FS PH GS 100SW Yield L DF DM

T1 78.88 4.92 92.42 88.83 59.84 48.75 20.14 2256.27 1.46 43.63 117.63

(18.2) (0.3) (21.8) (22.5) (9.3) (43.1) (1.8) (787.0) (1.1) (4.4) (12.9)

T2 76.21 4.79 89.15 84.96 60.69 27.92 19.92 2284.04 1.50 44.00 115.42

(21.6) (0.6) (25.7) (26.2) (10.5) (22.2) (1.3) (614.6) (1.1) (4.3) (11.2)

T3 78.04 4.88 91.42 86.71 62.63 15.83 18.21 2510.04 1.50 44.17 114.96

(17.2) (0.4) (19.9) (20.8) (13.6) (9.4) (0.9) (466.3) (1.2) (4.2) (11.3)

T4 83.83 4.88 98.02 94.77 55.12 27.50 16.42 2158.75 1.29 43.58 114.04

(10.6) (0.6) (18.5) (11.2) (14.4) (32.7) (1.3) (508.8) (0.7) (5.4) (9.9)

T5 83.25 4.83 97.60 92.67 49.56 3.29 15.45 2394.83 1.33 44.29 112.67

(9.1) (0.6) (10.5) (13.3) (12.3) (4.0) (1.3) (538.8) (0.8) (4.6) (11.3)

1 G = initial germination (%); V = seedling vigor; IS = initial plant stand; FS = final plant stand; PH = plant height (cm); GS = green stem (%); 100SW = seed size [weight of 1 00 seeds (g)]; Yield = grain yield (kg/ha); L = degree of lodging; DF = days to full flower (vegetative cycle) and DM = days to full maturity (total cycle).

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control treated with

Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 303 of 375Table F-22. Agronomic and Phenotypic Evaluations 2007: Means and Standard Deviations(in Parentheses) for EachCombination of Treatment and Location for All Traits.Trait1LocationTrt2GVISFSPHGS100SWYieldLDFDMT1 71.25 5.00 85.25 78.50 50.20 ab* 100.00 a ND3ND 1.00 a 44.00 c ND(9.5) (0.0) (11.7) (12.1) (1.5) (0.0) (0.0) (0.0)T2 70.25 5.00 84.00 76.50 48.80 ab 47.50 b 19.53 a 1863.50 a 1.00 a 45.00 b 109.00 a(10.8) (0.0) (13.0) (11.5) (1.8) (5.0) (0.7) (97.5) (0.0) (0.0) (0.0)T3 73.75 5.00 88.75 77.50 54.05 a 25.00 c 17.60 b 2593.50 a 1.00 a 45.00 b 109.00 a(2.1) (0.0) (2.8) (11.2) (4.2) (10.0) (0.7) (811.4) (0.0) (0.0) (0.0)T4 78.00 5.00 93.50 88.00 46.90 b 22.50 c 14.28 c 2019.25 a 1.00 a 46.00 a 106.00 b(4.1) (0.0) (4.7) (4.2) (3.1) (5.0) (0.6) (316.4) (0.0) (0.0) (0.0)T5 76.50 5.00 92.00 79.25 38.50 c 1.00 d 14.35 c 2091.75 a 1.00 a 46.00 a 106.25 bUberaba(4.5) (0.0) (5.7) (7.0) (4.4) (1.4) (0.3) (364.4) (0.0) (0.0) (0.5)T1 87.00 5.00 104.00 100.25 78.35 cd 100.00 a ND ND 3.75 a 48.00 b ND(4.5) (0.0) (5.4) (5.3) (2.9) (0.0) (0.5) (0.0)T2 77.75 5.00 93.25 86.00 81.85 bc 35.00 bc 20.23 a 2446.00 a 4.00 a 48.50 ab 114.00 a(6.1) (0.0) (7.4) (8.1) (2.5) (5.8) (0.9) (102.8) (0.0) (0.6) (0.0)T3 77.25 5.00 93.00 85.75 91.75 a 23.75 c 18.15 b 2742.50 a 4.00 a 49.00 ab 114.00 a(3.7) (0.0) (4.1) (8.3) (3.2) (7.5) (0.3) (349.9) (0.8) (0.8) (0.0)T4 78.00 5.00 93.50 90.75 85.55 ab 42.50 b 16.10 c 2509.50 a 2.75 a 49.25 ab 112.25 b(4.1) (0.0) (4.7) (5.6) (2.9) (12.6) (1.0) (401.2) (0.5) (0.5) (0.5)T5 78.75 5.00 94.75 90.25 74.35 d 5.50 d 14.95 c 2939.50 a 3.00 a 49.75 a 112.00 bSeteLagoas(3.6) (0.0) (4.3) (6.4) (2.9) (3.3) (0.4) (425.8) (0.8) (0.5) (0.0)Table F-22 is continued on the next page.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 304 of 375Table F-22 continued.Trait1LocationTrt2GVISFSPHGS100SWYieldLDFDMT1 91.50 5.00 110.00 109.38 59.00 a 10.00 a 20.13 a 2017.75 a 1.00 a 44.00 a 131.00 a(4.3) (0.0) (5.2) (5.4) (1.8) (0.0) (1.0) (238.0) (0.0) (0.0) (0.0)T2 94.50 5.00 113.25 113.13 58.00 a 10.00 a 19.18 a 1928.75 a 1.00 a 44.00 a 131.00 a(1.8) (0.0) (2.2) (2.0) (1.4) (0.0) (1.3) (326.9) (0.0) (0.0) (0.0)T3 93.25 5.00 111.63 110.38 58.00 a 10.00 a 18.53 a 2179.50 a 1.00 a 44.00 a 130.50 a(2.0) (0.0) (2.4) (2.2) (2.0) (0.0) (1.0) (347.5) (0.0) (0.0) (1.0)T4 92.50 5.00 110.88 109.50 48.50 b 10.00 a 16.28 b 1985.50 a 1.00 a 44.00a 129.25 b(2.1) (0.0) (2.6) (2.7) (1.0) (0.0) (0.5) (287.9) (0.0) (0.0) (0.5)T5 93.00 5.00 111.38 109.88 48.00 b 10.00 a 15.90 b 1631.50 a 1.00 a 44.00 a 129.00 bSantoAntônio deGoiás(1.7) (0.0) (2.1) (1.7) (1.6) (0.0) (0.3) (499.5) (0.0) (0.0) (0.0)T1 89.75 5.00 107.75 107.3 57.50 a 5.00 a 20.20 a 3343.25 ab 1.00 a 49.00 a 128.00 a(2.2) (0.0) (2.6) (2.3) (4.5) (0.0) (1.2) (332.8) (0.0) (0.0) (0.0)T2 90.00 5.00 108.00 107.63 59.00 a 5.00 a 19.70 ab 3385.50 a 1.00 a 49.00 a 128.00 a(4.8) (0.0) (5.8) (5.8) (2.2) (0.0) (0.9) (241.4) (0.0) (0.0) (0.0)T3 87.25 5.00 104.88 103.50 58.75 a 5.00 a 18.88 ab 2898.25 ab 1.00 a 49.00 a 127.25 a(1.6) (0.0) (1.9) (2.1) (1.5) (0.0) (0.6) (260.4) (0.0) (0.0) (1.5)T4 87.75 5.00 105.50 104.25 45.75 b 0.00 b 16.60 c 2874.00 ab 1.00 a 49.00 a 125.00 b(4.3) (0.0) (5.1) (5.0) (1.5) (0.0) (0.9) (305.5) (0.0) (0.0) (0.0)T5 91.50 5.00 109.75 108.38 45.00 b 0.00 b 17.63 bc 2771.75 b 1.00 a 49.00 a 124.75 bBrasília(1.9) (0.00 (2.3) (2.5) (4.1) (0.0) (1.4) (185.4) (0.0) (0.0) (0.5)Table F-22 is continued on the next page.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 305 of 375Table F-22 continued.Trait1LocationTrt2GVISFSPHGS100SWYieldLDFDMT1 83.50 5.00 87.00 77.00 58.50 a 67.50 b 21.50 a 1701.5 b 1.00 a 36.75 bc 100.50 b(5.0) (0.0) (5.2) (4.0) (2.6) (15.0) (1.9) (206.6) (0.0) (0.5) (1.9)T2 83.25 5.00 86.50 77.00 60.00 a 60.00 b 21.58 a 1800.50 b 1.00 a 37.50 ab 99.50 bc(1.4) (0.0) (1.5) (4.1) (2.3) (14.1) (1.4) (187.8) (0.0) (1.3) (1.0)T3 88.00 5.00 92.00 86.25 58.25 a 21.25 c 18.58 b 2371.25 a 1.00 a 38.00 ab 98.00 bc(5.4) (0.0) (5.6) (5.3) (1.9) (6.3) (0.9) (128.6) (0.0) (0.0) (1.2)T4 96.50 5.00 100.50 93.25 54.25 a 90.00 a 18.20 b 1518.75 b 1.00 a 35.25 c 104.75 a(2.4) (0.0) (2.5) (1.3) (4.9) (7.1) (0.3) (128.3) (0.0) (0.5) (2.5)T5 89.25 5.00 93.25 87.25 41.25 b 3.25 c 15.25 c 2487.75 a 1.00 a 39.00 a 97.00 cVilhena(1.2) (0.0) (1.2) (6.9) (2.4) (2.4) (0.7) (261.1) (0.0) (0.0) (0.0)T1 50.25 4.50 60.50 60.50 55.50a 10.00 a 18.27 a 1865.00 a 1.00 a 40.00 a 111.00 a(26.9) (0.6) (32.3) (32.3) (2.5) (0.0) (2.3) (837.0) (0.0) (0.0) (0.0)T2 41.50 3.75 49.88 49.50 56.50 a 10.00 a 19.10 a 2278.67 a 1.00 a 40.00 a 111.00 a(31.3) (1.0) (37.6) (37.6) (1.7) (0.0) (1.7) (520.5) (0.0) (0.0) (0.0)T3 48.75 4.25 58.25 56.88 55.00 a 10.00 a 17.33 ab 2197.00 a 1.00 a 40.00 a 111.00 a(22.0) (1.0) (26.4) (26.0) (2.2) (0.0) (1.1) (292.2) (0.0 (0.0) (0.0)T4 70.25 4.25 84.25 82.88 49.75 b 0.00 b 17.05 ab 2045.50 a 1.00 a 38.00 b 107.00 b(11.6) (1.5) (13.9) (14.0) (1.7) (0.0) (0.5) (236.6) (0.0) (0.0) (0.0)T5 70.50 4.00 84.50 81.00 50.25 b 0.00 b 14.63 b 2446.75 a 1.00 a 38.00 b 107.00 bTeresina(4.1) (1.2) (4.9) (4.4) (1.3) (0.0) (1.0) (245.3) (0.0) (0.0) (0.0)1G = initial germination (%); V = seedling vigor; IS = initial plantstand; FS = final plant stand; PH = plant height (cm); GS =green stem (%); 100SW = seed size[weight of 100 seeds (g)]; Yield = grain yield (kg/ha); L = degreeof lodging; DF = days to fullflower (vegetative cycle) andDM = days to full maturity (totalcycle).2Treatments: T1 = CV127 treated withimazapyr, T2 = CV127treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monsoy 8001 treated with Volt®,T5 = Coodetec 217 treated with Volt®.3ND = Not determined*Means followed by the same letter do not differ statistically significantly by the Tukey test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 306 of 375 Conclusions

The above studies compared the agronomic, phenotypic, and phenologic characteristics of CV127 soybean relative to the isoline control and to other conventional soybean varieties. Although statistically significant differences did exist betw een CV127 and the isoline control for specific traits at specific locati ons, the only trait for which there was a trend toward a consistent

difference between CV127 and the isoline control wa s seed size. For all other traits, including seedling vigor, yield, germination, plant height, green stem, lodging, and days to flowering and maturity, there were no consistent significant differences between CV127 and the isoline control. The lack of any bi ologically sign ificant differences betw een CV127 and the isoline control for any of the evaluated traits demonstr ates that CV127 is agronomically, phenotypically, and phenologically equivalent to the isoline control as well as conventional soybean varieties under standard soybean production practices.

2.0. Disease susceptibility in the a) 2006/2007 and b) 2007 growing seasons

Introduction

Plant diseases can cause serious losses to soybean yield in commercial production in Brazil. Since 2001, Asian soybean rust, caused by the fungus Phakopsora pachyrhizi , has become one of the most important diseases in Brazilian so ybean agriculture due to yield losses and the expense of control measures. In addition to rust, downy mildew ( Peronospora manshurica ) can cause yield losses in soybean, and other leaf di seases known as end-of-cycle diseases (DC) caused mainly by Septoria glycines (brown spot or Septoriose) and Cercospora kikuchii (soybean leaf spot), can reduce the yield by more than 20%. According to Sinclair and Hartman (1999), powdery mildew can cause a 10 to 35% reductio n in the yield of susceptible soybean cultivars and can occur at any phase of the pl ant’s growth. These pa thogens are currently disseminated throughout the soyb ean-producing regions in Brazil, and the use of resistant cultivars and fungicide treatment are the main control methods.

The purpose of the current study wa s to evaluate the di sease susceptibility characteristics of CV127 soybean relative to the isol ine control and to tw o conventional soybean varieties. Field studies were conducted during the 2006/2007 and 2007 growing season s at the thirteen field

locations in Brazil described a bove in section 1.0 of Appendix F. Disease susceptibility of CV127 soybean relative to the iso line control and conven tional soybean varieties was determined at different stages of plant growth and development throughout the growing season.

Materials and Methods

Field design, management and treatments. The field experimental design and management practices at each field trial location, and treatmen ts in each study have been described above in section 1.0 of Appendix F (“Agronom ic and Phenotypic Evaluations”).

Disease evaluations . The incidence of all diseases pres ent at the time of assessment was recorded for each plot as the pe rcent of affected leaf area. The dominant diseases causing

infection were identified at each assessment. Assessments were made at or near vegetative stageCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 307 of 375 V4 and reproductive stages R1, R5, and R7. Exact stages in which assessments were made are indicated in the data tables. Due to the potential in Brazil for fungal di seases to devastate the soybean crop and thereby compromise the goals of this study, all plots were uniformly treated with fungicides in order to maintain plant health . Evaluations of disease incidence in each plot were typically made prior to disease control treatments.

Statistical Analyses. Data were analyzed by ANOVA on a pe r location, per disease, and per

time-of-assessment basis. In t hose cases where differences among treatments were significant, Tukey’s procedure (Steel and Torrie, 1980) wa s used to make pai r-wise comparisons of

treatment means.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 308 of 375 Results and Discussion

a) 2006/2007 growing season

Despite multiple applications of fungicide, Asian rust ( Phakopsora pachyrhizi ) was present at all locations. In addition, powdery mildew ( Erysiphe diffusa ), downy mildew ( Peronospora

manshurica ), and late-season diseases, brown spot ( Septoria glycines ) and leaf spot ( Cercospora

kikuchii ), were present at most loca tions (Tables F-23 to F-29). In the 2006/2007 field trials, no diseases were evident prior to the V4 stage of de velopment. In the data presented in Tables F-23 to F-29, where any of the four diseases listed above were not observe d on plants grown at a particular field location and stage of plant development, then no data is presented.

No significant differences in disease incide nce were observed among CV127 treated with imazapyr (T1), CV127 treated with Volt ® (T2), and the isoline control (T3), (Tables F-23 to F29). Significant differences in affected leaf area due to Asian rust between the treatments containing the Conquista genetic background (T1, T2 and T3) an d the conventional reference varieties, Monsoy 8001 (T4) and CD 217 (T5), were detected at one or more of the assessment stages (R1, R5, or R7) in Ponta Grossa (Table F-23), Londrina (Table F-24), Santo Antônio de Posse (Table F-25), and Brasília (Table F-28). Th ese differences in disease susceptibility were attributed to genetic differences between th e Conquista germplasm and the germplasm of the conventional reference soybean varieties.

Other than a high incidence of Asian rust at seve ral locations, the incidenc e of other diseases was generally low. Although a few isolated differences in disease susceptib ility were noted among the treatments, these were not consistent across fi eld trial locations and in general there were no differences in susceptibility between CV127 and the isoline c ontrol. Where susceptibility differences were observed, they were generally between varieties with the Conquista genetic background (CV127 and the isolin e control) and the two conven tional soybean varieties with different genetic backgrounds.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 309 of 375 Table F-23. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Ponta Grossa Field Site in 2006/2007.

Growth Stage at Assessment

V4 R1 R5 R7

Diseases ‡ Treatment †

PM DM DC AR DC AR

T1 ND § 6 a ¶ 2 5 8.4 a 5 48 a

T2 ND 6 a 2 5 6.0 ab 5 43 ab

T3 ND 7 a 2 5 6.0 ab 5 40 ab

T4 ND 16 b 2 5 5.3 b 5 30 b

T5 ND 8 a 2 5 4.8 b 5 36 ab

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ PM = powdery mildew ( Erysiphe diffusa ); DM = downy mildew ( Peronospora manshurica ); DC = end-of-cycle diseases ( Septoria glycines, Cercospora kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

¶ Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.

Table F-24. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Londrina Field Site in 2006/2007.

Growth Stage at Assessment

V4 R1 R5 R7

Diseases ‡ Treatment †

DM AR PM AR DC AR

T1 ND § 1.8 2.0 c ¶ 2 50 a 5 60 a

T2 ND 1.5 2.0 c 2 51 a 5 60 a

T3 ND 1.8 2.0 c 2 51 a 5 60 a

T4 ND 1.8 2.8 b 2 44 b 5 51 b

T5 ND 1.8 4.0 a 2 30 c 5 40 c

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ PM = powdery mildew ( Erysiphe diffusa ); DM = downy mildew ( Peronospora manshurica ); DC = end-of-cycle diseases ( Septoria glycines, Cercospora kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

¶ Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 310 of 375 Table F-25. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Santo Antônio de Posse Field Site in 2006/2007.

Growth Stage at Assessment

V4 R1 R5 R7

Diseases ‡ Treatment †

AR DM AR DC AR

T1 ND § 7.3 2.3 48 a ¶ 5 58 a

T2 ND 6.8 2.0 45 a 5 58 a

T3 ND 7.3 1.5 50 a 5 60 a

T4 ND 7.5 1.8 30 b 5 40 b

T5 ND 6.9 2.8 43 a 5 52 a

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ DM = downy mildew ( Peronospora manshurica ); DC = end-of-cycle diseases ( Septoria glycines, Cercospora

kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

¶ Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.

Table F-26. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Uberaba Field Site in 2006/2007.

Growth Stage at Assessment

V4 R1 R5 R7

Diseases ‡ Treatment †

PM AR AR

T1 ND § ND 1.5 2.0 2.5

T2 ND ND 1.5 2.0 2.5

T3 ND ND 1.5 2.0 2.5

T4 ND ND 1.5 2.0 2.5

T5 ND ND 1.5 2.2 2.5

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ PM = powdery mildew ( Erysiphe diffusa ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 311 of 375 Table F-27. Disease Assessment Presented as Mean Perce nt Affected Leaf Area at the Sete Lagoas Field Site in 2006/2007.

Growth Stage at Assessment

V4 R1 R5 R7

Diseases ‡ Treatment †

AR AR AR

T1 ND § 1.5 2.5 2.5

T2 ND 1.5 2.5 2.5

T3 ND 1.5 2.5 2.5

T4 ND 1.5 2.5 2.5

T5 ND 1.5 2.5 2.5

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

Table F-28. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Brasília Field Site in 2006/2007.

Growth Stage at Assessment

V4 R1 R5 R7

Diseases Treatment †

AR DC AR DC AR

T1 ND § 1.6 0.4 ab ¶ 39 a 4.4 45 a

T2 ND 2.0 0.6 ab 42 a 4.1 55 a

T3 ND 1.7 0.4 ab 40 a 4.4 45 a

T4 ND 1.9 1.1 a 19 b 5.0 23 b

T5 ND 1.8 0.0 b 19 b 4.7 23 b

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ DC = end-of-cycle diseases ( Septoria glycines, Cercospora kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

¶ Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 5% probability.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 312 of 375 Table F-29. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Santo Antônio de Goiás Field Site in 2006/2007.

Growth Stage at Assessment

V6 R1 R5 R7

Diseases ‡ Treatment †

DC AR DC AR DC AR

T1 ND § 0.8 0.9 18 2.8 24

T2 ND 1.0 0.5 17 2.9 22

T3 ND 0.9 0.1 19 2.4 22

T4 ND 0.6 0.3 18 3.6 21

T5 ND 0.8 0.6 18 3.1 22

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ DC = end-of-cycle diseases ( Septoria glycines, Cercospora kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

b) 2007 growing season

The short-season in Brazil typically has a low disease incidence of Asian rust ( Phakopsora pachyrhizi ) and this was reflected in the data collected. Asian rust was present in five of the six field site locations in 2007, but with very low severity. In addition, powdery mildew ( Erysiphe diffusa ) and late-season dise ases, brown spot ( Septoria glycines ) and leaf spot ( Cercospora

kikuchii ), were also present at some locations (Table s F-30 to F-34) but with low severity. No diseases were evident prior to the V4 stage of development, with the exception of powdery mildew in Santo Antônio de Goiás (Table F-32). In Tables F-30 to F-35, in cases where any of the four diseases listed above were not present at a specific field loca tion or stage of plant development, then no data is presented.

No statistically significant differences in diseas e incidence occurred between the isoline control and CV127 treatments in any of the locations in the 2007 field trials, except in Uberaba where CV127 treated with imazapyr had a hi gher level of end-of-cycle dis eases than the isoline at the R7 stage of development (Table F-30).

Conclusions

Although a few isolated differences in disease susceptibility were noted among the treatments across both the 2006/2007 and 2007 growing seasons, these differences were not consistent across field trial locations and in general ther e were no differences in susceptibility between CV127 and the isoline control. Where susceptib ility differences were observed, they were generally between treatments with the Conquista genetic b ackground (CV127 and the isolineCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 313 of 375 control) and the two conventional soybean va rieties with different genetic backgrounds. Therefore disease susceptibility of CV127 was found to be no different from that of the isoline control plants. This study demons trates that the insertion of the csr1-2 gene into the genome of

CV127 did not affect the dis ease susceptibility of CV127.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 314 of 375 Table F-30. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Uberaba Field Site in 2007.

Growth Stage at Assessment

V4 R1 R5.1 R7

Diseases ‡ Treatment †

AR DC AR DC AR

T1 ND § 1.5 0.7 b ¶ 0.8 1.5 a 0.6

T2 ND 1.5 1.1 ab 0.7 1.4 ab 0.6

T3 ND 1.5 0.9 ab 0.6 1.3 b 0.5

T4 ND 1.5 1.2 a 1.0 1.5 a 0.6

T5 ND 1.5 0.8 ab 0.8 1.5 a 0.5

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ DC = end-of-cycle diseases ( Septoria glycines, Cercospora kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

¶ Means followed by the same letter do not differ statistical ly significantly by the Tukey’s test at 1% probability.

Table F-31. Disease Assessment Presented as Mean Perce nt Affected Leaf Area at the Sete Lagoas Field Site in 2007.

Growth Stage at Assessment

V4 R1 R5.1 R7

Diseases ‡ Treatment †

AR AR PM AR

T1 ND § 0.5 0.7 1.0 b ¶ 1.0

T2 ND 0.6 0.8 0.8 b 1.1

T3 ND 0.6 1.0 0.7 b 1.0

T4 ND 0.6 0.7 1.7 a 1.1

T5 ND 0.5 0.6 0.6 b 1.0

† T1 = CV127 treated with imazapyr, T2 = CV 127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ AR = Asian rust ( Phakopsora pachyrhizi ); PM = powdery mildew ( Erysiphe diffusa ).

§ ND = no diseases were observed.

¶ Means followed by the same letter do not differ statistical ly significantly by the Tukey’s test at 1% probability.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 315 of 375 Table F-32. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Santo Antônio de Goiás Field Site in 2007.

Growth Stage at Assessment

V4 R1 R5.1 R7

Diseases ‡ Treatment †

PM PM AR AR

T1 5 6.1 3.4 1.3

T2 5 7.1 2.2 1.9

T3 5 6.6 2.5 0.6

T4 5 6.3 2.1 1.3

T5 5 6.4 3.1 0.9

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ PM = powdery mildew ( Erysiphe diffusa ); AR = Asian rust ( Phakopsora pachyrhizi ).

Table F-33. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Brasília Field Site in 2007.

Growth Stage at Assessment

V4 R1 R5.1 R7

Diseases ‡ Treatment †

PM AR AR

T1 ND § 1.4 1.4 1.6

T2 ND 1.6 0.9 1.6

T3 ND 1.3 0.7 1.6

T4 ND 1.6 0.9 1.3

T5 ND 1.6 1.4 1.6

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ PM = powdery mildew ( Erysiphe diffusa ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 316 of 375 Table F-34. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Vilhena Field Site in 2007.

Growth Stage at Assessment

V4 R1 R5.1 R7

Diseases ‡ Treatment †

AR AR DC AR

T1 ND § 3.7 8.4 5.0 22.0

T2 ND 3.7 8.6 5.0 20.6

T3 ND 3.6 7.6 4.4 20.2

T4 ND 3.6 8.0 5.0 19.8

T5 ND 2.1 5.2 5.0 10.5

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. ‡ DC = end-of-cycle diseases ( Septoria glycines, Cercospora kikuchii ); AR = Asian rust ( Phakopsora pachyrhizi ).

§ ND = no diseases were observed.

Table F-35. Disease Assessment Presented as Mean Percent Affected Leaf Area at the

Teresina Field Site in 2007.

Growth Stage at Assessment

V4 R1 R5.1 R7 Treatment †

Diseases

T1 ND § ND ND ND

T2 ND ND ND ND

T3 ND ND ND ND

T4 ND ND ND ND

T5 ND ND ND ND

† T1 = CV127 treated with imazapyr , T2 = CV127 treated with Volt ® , T3 = Isoline control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = CD 217 treated with Volt ®

. § ND = no diseases were observed.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 317 of 375 3.0. Insect interactions in the a) 2006/2007 and b) 2007 growing seasons

Introduction

Populations of several different insect orders are common in commercial soybean production fields in Brazil and include the Coleopterans ( Diabrotica and Aracanthus spp.), Lepidopterans ( Anticarsia

, Agrotis

, Pseudoplusia , and Spodoptera spp.), and Hemipterans ( Euschistus

, Piezodorus , and Nezara spp.). Insect pests in these orders can cause serious losses to soybean yield in commercial production in Brazil. The purpose of the current study was to evaluate resistance to insect damage and the effect on in-f ield insect populations of imidazolinone-tolerant CV127 soybean relative to the iso line control and the two conventional soyb ean varieties. Field studies were conducted during the 2006/2007 and 2007 growing season at the thirteen field

locations in Brazil described above in section 1.0 of Appendix F. Resistance to insect damage

and effects on in-field insect populations of CV127 soybean relati ve to the isoline control and conventional soybean varieties we re determined at different stages of plant growth and development throughout each growing season.

Materials and Methods

Field design, management and treatments. The field experimental design and management practices at each field trial location, and treatmen ts in each study have been described above in section 1.0 of Appendix F (“Agronom ic and Phenotypic Evaluations”).

Insect interaction evaluations

. Insects were collected by placing a shake cloth (a 1.0 m x 0.8 m tarp supported on either side by a wooden rod) on the ground betw een the center two rows of each plot. The plants in the two rows adjacent to the shake cloth were bent over the cloth and shaken vigorously to remove a ll insects within the canopy. All insects that fell on the cloth were counted and identified by order. This method was initially conducted at or near the V4 stage and repeated at or near the R1, R5, and R7 stages . Leaf, stem, and/or pod damage caused by feeding of each insect type was also evaluated at each sampling date using a damage rating scale (Table F-36). However, insect feeding damage acr oss all treatments and across locations was

considered minimal and no differences were di stinguished among the treatments. Therefore these data are not presented.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 318 of 375 Table F-36. Rating Scale Used to Evaluate Each Plot for Damage Caused by Insect Feeding.

Insect damage rating Percent of leaves with insect damage

0 0

1 1 – 10

2 11 – 20

3 21 – 30

4 31 – 40

5 41 – 50

6 51 – 60

7 61 – 70

8 71 – 80

9 81 – 90

10 91+

Statistical Analysis. Data were analyzed by ANOVA on a per location, per insect order, and per

time-of-assessment basis. In t hose cases where differences among treatments were significant, Tukey’s procedure (Steel and Torrie, 1980) wa s used to make pai r-wise comparisons of

treatment means.

Results and Discussion

a) 2006/2007 growing season

Insect orders that were common across most or all locations included the Coleopterans ( Diabrotica and Aracanthus spp.), Lepidopterans ( Anticarsia

, Agrotis

, Pseudoplusia , and Spodoptera spp.), and Hemipterans ( Euschistus, Piezodorus , and Nezara spp.). The Coleopterans were the most comm on order at Ponta Grossa, Londrin a, Santo Antônio de Goiás,

and Bras í lia, while the Lepidopterans were most co mmon at Santo Antônio de Posse, Uberaba, and Sete Lagoas (Table F-37). The location w ith the largest insect populations was Ponta Grossa, followed by Santo Antônio de Posse and Londrina. Relatively few insects were found at Sete Lagoas, Santo Antônio de Goiás, Uberaba, and Bras í lia. The largest populations of insects were detected at the R5 and R7 sampling dates.

A statistical analysis of the data detected no evidence of a significant difference among

treatments for the number of individual insect species detected at a ny location in the 2006/2007

season.

Regardless of the size of each insect population at each sampling date, at no time were populations high enough to compromise plant healt h. In fact, leaf, stem, or pod damage from insect feeding was below 2% for all treatments and was therefore not statistically analyzed nor is it presented herein.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 319 of 375Table F-37. The Mean Number of Coleopteran, Lepidopteran,and Hemipteran Insects atEach Field Site in 2006/2007.Growth StageV1R1R5R7OrderLocationTrt†ColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraT1 0.25 0.25 0 2.5 1.50 6.75 3.25 2.5 2.5 0 1T2 0.25 0.25 0 2 2 0 5.75 3 2 2.25 0 0.75T3 0.75 0.25 0 2.25 1.25 05.5 2.75 2.5 2.75 0 0.75T4 0.25 0.25 0 3.75 2.250 4.75 3.25 2.75 2 0 1.25T5 0.5 0.25 0 3.25 2.25 0 6 3 2.25 2.25 0 1PontaGrossaMean 0.4 0.25 0 2.75 1.850 5.75 3.05 2.4 2.35 0 0.95T1 0.5 0 0.5 1.75 2.5 01.5 0.5 0.75 0.25 0 1.75T2 1 0 0.25 2.25 2.25 0 1 2.25 1.25 0 0.25 2.25T3 1 0 1 1.75 1.75 0 1.25 1.25 0.25 0.75 0.25 1.5T4 1 0 0.25 2.25 2.25 0 0.75 2.25 1.25 0.25 0.75 1.25T5 1.25 0 0.5 1.75 1.75 01.25 1 2.25 0.25 0.25 2.75LondrinaMean 0.95 0 0.5 1.95 2.1 01.15 1.45 1.15 0.3 0.3 1.9T1 0.5 0.5 0 0 0 0 2 4 4 0 3.75 2.75T2 0.25 0 0.25 0 0 0 2.25 3.75 4 0 4.25 3T3 0.5 0.5 0.25 0 0 0.5 1.75 4 3.75 0 3.25 2.75T4 0.5 0.5 0.25 0 0 0.5 2.25 4 3.75 0 4.25 3.25T5 0.5 0.5 0.25 0 0 0.252.75 4.5 4.25 0 4.25 3.25SantoAntôniode PosseMean 0.45 0.4 0.2 0 0 0.25 2.2 4.05 3.95 0 3.95 3T1 0.5 0 0 0 0.25 0 0 0.5 0 0 0.5 0.25T2 0.25 0 0 0 0 0 0 0.5 0 0 0 0.25T3 0 0 0 0 0 0 0 0.25 0 0 0.25 0.5T4 0.75 0 0 0 0.25 0 0 0 0.25 0 0 0T5 0.5 0 0 0 0.25 0.25 0 0.75 0.25 0 0.75 0.25UberabaMean 0.4 0 0 0 0.15 0.05 0 0.4 0.1 0 0.3 0.25†Treatment: T1 = CV127 treated withimazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monsoy 8001 treated with Volt®,T5 = Coodetec 217 treated with Volt®.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 320 of 375Table F-37 continued.Growth StageV1R1R5R7OrderLocationTrt†ColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraT1 0 0 0 0 0 0 0.25 0 0 1 0.5 0T2 0.25 0 0 0 0.25 0.25 0 0.25 0 0.5 1 0T3 0.5 0 0 0 0.25 0 0 0.25 0 0 1.25 0.5T4 0.5 0 0 0 0.5 0 0.25 0 0 0.25 0.75 0.25T5 0.75 0 0 0 0.5 0 0.75 0.25 0 0.75 0.5 0SeteLagoasMean 0.4 0 0 0 0.3 0.050.25 0.15 0 0.5 0.8 0.15T1 1.5 0 0 0.5 0.25 0 0 0 0 0 0 0.25T2 0.25 0 0 0 0 0 0 0 0.5 0 0 0.5T3 0.5 0 0 1 0 0 0 0 0.25 0 0 0.25T4 1.25 0 0 0.75 1.25 0 0 0 00 0 0T5 0.25 0 0 0 0.5 0 0 0 0.25 0 0 0.25SantoAntôniode GoiásMean 0.75 0 0 0.45 0.4 0 0 0 0.2 0 0 0.25T1 0.5 0.25 0 0 0 0 0 0 0.25 0 0 0T2 0 0.25 0 0.5 0 0 0 0 0 0 0 0T3 0.25 0 0 0.25 0.25 0 0 0.25 0.5 0 0 0T4 0.25 0.25 0 0 1 0 0 0 0.5 0 0 0.25T5 0.5 0 0 0.25 0 0 0 0 0 0 0 0.25BrasíliaMean 0.3 0.15 0 0.2 0.25 0 0 0.05 0.25 0 0 0.1†Treatment: T1 = CV127 treated withimazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monsoy 8001 treated with Volt®,T5 = Coodetec 217 treated with Volt®.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 321 of 375 b) 2007 growing season

Insect orders that were common across most or all locations in the tria ls conducted in the 2007 short season included the Coleopterans ( Diabrotica and Aracanthus spp.), Lepidopterans ( Anticarsia

, Agrotis

, Pseudoplusia , and Spodoptera spp.), and Hemipterans ( Euschistus

, Piezodorus , and Nezara spp.). The Coleopterans and Hemipterans were the most common orders in Vilhena, while Lepidopterans were co mmon at Teresina (Table F-38). The location with the largest insect populations was Vilh ena, followed by Sete Lagoas and Uberaba. Relatively few insects were f ound at Santo Antônio de Goiás and Brasília. The largest populations of insects of the orders Coleoptera a nd Lepidoptera were detected at the V4 growth stage, while insects of the order Hemiptera were greatest at the R5 and R7 growth stages.

A statistical analysis of the data did not detect any statistically significant differences among

treatments for the number of i ndividual insect species detected at any location, except for one instance at the Teresina field site in 2007 for Coleopterans at the R5 growth stage, where levels on the conventional soybean variety Monsoy 8001 were statistically significantly higher compared to the other treatments.

Regardless of the size of each insect population at each sampling date, at no time were populations high enough to compromise plant healt h. In fact, leaf, stem, or pod damage from insect feeding was below 2% of the total leaf area for all treatments and was therefore not statistically analyzed nor is it presented herein.CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 322 of 375Table F-38.The Mean Number of Coleopteran, Lepidopteran, and Hemipteran Insects at EachField Trial Site in 2007.†Treatment: T1 = CV127 treated withimazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monsoy 8001 treated with Volt®,T5 = Coodetec 217 treated with Volt®.Growth StageV4R1R5R7OrderLocationTrt1ColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraT11.39 0.71 0.71 1.06 1.10 0.710.71 1.10 0.71 0.71 0.71 0.84T21.56 0.71 0.71 0.97 0.84 0.710.71 0.93 0.84 0.71 0.84 0.71T31.48 0.71 0.71 1.06 0.84 0.710.71 1.06 0.84 0.71 0.84 0.71T41.56 0.71 0.71 0.84 0.71 0.710.71 0.84 0.84 0.71 0.71 0.71T51.40 0.71 0.71 0.97 0.97 0.710.71 0.97 0.71 0.71 0.71 0.71UberabaMean1.48 0.71 0.71 0.98 0.89 0.710.71 0.98 0.78 0.71 0.76 0.73T11.22 0.71 0.71 0.84 0.84 0.840.71 1.40 0.84 0.71 0.71 0.84T21.06 0.71 0.71 0.84 1.06 0.710.71 1.31 0.84 0.71 0.84 0.97T31.18 0.71 0.71 1.06 1.06 0.710.71 1.10 0.71 0.71 0.84 0.71T41.22 0.71 0.71 0.97 1.18 0.710.71 0.84 0.97 0.71 0.97 0.71T51.10 0.71 0.71 0.97 0.97 0.710.71 1.06 0.97 0.71 1.06 0.71SeteLagoasMean1.16 0.71 0.71 0.93 1.02 0.730.71 1.14 0.86 0.71 0.88 0.78T11.36 0.71 0.71 0.84 0.84 0.840.71 0.71 0.84 0.71 0.71 0.84T21.06 0.84 0.71 0.84 0.71 0.710.71 0.71 0.84 0.71 0.71 0.71T31.06 0.71 0.71 0.84 0.84 0.710.71 0.71 0.84 0.71 0.71 0.84T40.93 0.84 0.71 0.84 0.71 0.710.71 0.71 0.71 0.71 0.71 0.71T50.93 0.71 0.71 0.71 0.84 0.710.71 0.71 0.84 0.71 0.71 0.71SantoAntônio deGoiásMean1.06 0.76 0.71 0.81 0.78 0.730.71 0.71 0.81 0.71 0.71 0.76T10.71 0.71 0.71 0.71 0.84 0.710.71 0.84 0.84 0.71 0.71 0.71T20.71 0.84 0.71 0.71 0.84 0.710.71 0.71 0.71 0.71 0.71 0.84T30.84 0.71 0.71 0.84 0.71 0.710.71 0.84 0.97 0.71 0.71 0.84T40.84 0.71 0.71 0.71 0.71 0.710.71 0.71 0.71 0.71 0.71 0.71T50.97 0.71 0.71 0.71 0.71 0.710.71 0.71 0.71 0.71 0.71 0.71BrasíliaMean0.81 0.73 0.71 0.73 0.76 0.710.71 0.76 0.78 0.71 0.71 0.76CONFIDENTIAL BUSINESS INFORMATION DELETEDBASF Plant ScienceImidazolinone-tolerant Soybean BPS-CV127-9Page 323 of 375Table F-38 continued.Growth StageV4R1R5R7OrderLocationTrt1ColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraColeopteraLepidopteraHemipteraT1 0.97 0.71 0.71 1.49 0.71 0.71 1.40 1.10 1.18 0.71 0.71 1.10T2 0.97 0.71 0.71 1.49 0.71 0.71 1.61 0.84 1.27 0.71 0.71 1.22T3 1.10 0.71 0.71 1.65 0.71 0.71 1.31 1.10 1.40 0.71 0.71 1.10T4 1.06 0.71 0.71 1.49 0.71 0.71 1.79 1.13 1.44 0.71 0.71 1.18T5 0.97 0.71 0.71 1.40 0.71 0.71 1.59 0.84 1.49 0.71 0.71 1.06VilhenaMean 1.01 0.71 0.71 1.51 0.71 0.71 1.54 1.00 1.36 0.71 0.71 1.13T1 0.71 1.78 0.71 0.71 0.71 0.710.71 b* 0.71 0.84 0.71 0.71 0.97T2 0.71 2.04 0.71 0.71 0.84 0.710.71 b 0.71 0.84 0.71 0.71 0.71T3 0.71 1.90 0.71 0.71 0.97 0.710.71 b 0.71 0.71 0.71 0.71 0.71T4 0.71 1.83 0.71 0.71 0.71 0.711.79 a 1.13 0.71 0.71 0.71 0.84T5 0.71 1.63 0.71 0.71 0.71 0.710.71 b 0.71 0.71 0.71 0.71 0.71TeresinaMean 0.71 1.84 0.71 0.71 0.78 0.71 0.92 0.79 0.76 0.71 0.71 0.78†Treatment:T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monsoy 8001 treated with Volt®,T5 = Coodetec 217 treated with Volt®.*Means followed by the same letter, in the same column, are not statistically different at the 5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 324 of 375 Conclusions

The numbers of Coleopteran, Lepidopteran, and Hemipteran insects detected on CV127 soybean plants were not statistically signi ficantly different from numbers detected on plants of the isoline control at any of four sampling dates during both the 2006/2007 and 2007 growing seasons. Furthermore, insect feeding damage in both growing seasons

across all treatments and across locations wa s considered minimal and no biologically significant differences were distinguished among the treatments. Theref ore, resistance to insect damage and effect on in-field ins ect populations of CV127 soybean was found to

be no different from that of the isoline contro l plants or conventional soybean varieties. These results further support the conclusion th at the cultivation of CV127 soybeans poses no different environmental impact than the cu ltivation of conventional soybean varieties that have a long history of safe cultivation and food and feed use.

4.0. Nitrogen fixation characteristics in the a) 2006/2007 and b) 2007 growing

seasons

Introduction

An important agronomic characteristic of soybeans is the ability to support a nitrogenfixing symbiosis with the soil bacterium Bradyrhizobium japonicum

. B. japonicum cells in the soil stimulate the development of root nodules on the soybean. The B. japonicum cells populate the interior of the nodule and th e plant creates an environment within the

nodule that is conducive to nitr ogen fixation. Through this symbiosis, soybeans are able to convert atmospheric nitrogen to fixed forms of nitrogen and reduce their need for chemical fertilizer. It is a common pract ice in Argentina and Brazil for farmers to inoculate soybean seed with B. japonicum at planting to ensure the development of plentiful root nodulation and st rong nitrogen fixation activity. Studies have demonstrated that the rotation of soybean w ith other crops generally leads to increased yields for the soybean and subsequent crops (Johnson, 1987; Wesley, 1999). Other benefits for the farmer from rotation with soybeans include reduced nitrogen input in the following crop, increased residue cover, and the redu ction of pest and weed cycles.

The purpose of the current study was to confirm that CV127 soybean is equivalent to the isoline control soybean and comp arable to conventional soybea n varieties with respect to nitrogen fixation and plant N accumulation. Characteristics of nitrogen fixation in CV127 soybean were evaluated and compared with the characteris tics of the isoline control and conventional soybean varieties, including measurements of soil populations

of B. japonicum , plant growth and nodulation and to tal nitrogen and ureide nitrogen accumulation in the plants. These determina tions were made on plants grown in field studies during the 2006/2007 growing season and the 2007 short growing season at

thirteen field locations in Brazil described above in sect ion 1.0 of Appendix F; these locations are representative of areas of commercial soyb ean production and the soybean varieties included in the study ar e adapted to th ese areas.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 325 of 375 Materials and Methods

Field design, management and treatments. The field experimental design and management practices at each field trial location, and treatments in each study have been

described above in section 1.0 of Appendix F (“Agronomic and Phenotypic Evaluations”).

Evaluation of biological nitrogen fixation . Evaluation of biologi cal nitrogen fixation was performed following the protocols of RELARE (Rede de Laboratórios para Recomendação de Estirpes de Rizóbio e I noculantes Microbianos), a network of Brazilian microbiologists offi cially recognized by the Ministry of Agriculture.

Population of soybean Bradyrhizobium japonicum

. The Bradyrhizobium japonicum population was estimated with soil samples colle cted at the pre-sowing stage, using the most probable number (MPN) te chnique (Vincent, 1970) and the statis tical tables of Andrade and Hamakawa (1994). Evalua tion of the nodulation capacity of the Bradyrhizobium japonicum population was performed using soybean plants of cultivar Conquista, grown under greenhouse conditions, that were inoculated with soil dilutions. Each replicate was analyzed in triplicate.

Plant growth, nodulation, total N and N- ureides accumulated in plant tissues. At the R2 stage, ten plants were randomly collected per replicate (avoiding areas established for harvesting grains) for evaluation of nodulatio n and plant growth. In the laboratory, shoots were separated from roots and the la tter were carefully washed and placed in a forced-air dryer at 65oC until constant weight was obtained (approximately 72 h).

Nodules were removed from the roots and dried again. Nodulati on (nodule number and dry weight), shoot dry weight and shoot total N (Kjeldahl di gestion and determination of N concentration using a Tecator automatic N analyzer) were determined (Hungria et al

., 2006). In soybeans, nitrogen is fixed in th e nodules and converted and exported to the plant principally in the form of N-ureides. Therefore, biological nitrogen fixation by nodules associated with the soybean plant was estimated using the N-ureide technique in dry tissues of plants harvested at th e R2 stage, as described by Hungria et al . (2006). Analysis was performed with stems includ ing the petioles according to Herridge and Peoples (1990). Dry stems (generated by incubation at 65oC until a constant weight) of ten plants per replicate we re ground (20 mesh), extract ed with 0.1 M phosphate buffer (pH 7.0) and 2.5 mL of ethanol (2:1, v/ v, buffer:ethanol), and heated at 80 ° C for 5 min. Samples were filtered in cheesecloth, centr ifuged at 10,000 g for 5 min. and stored at -5°C. Concentrations of ureides (allantoin and allantoic acid) were determined based on the method of Vogels and Van der Drift (1970), as described byHungria (1994).

Nitrate was evaluated in the same extracts by the salicylic acid technique (Cataldo et al

., 1975) that was adapted for smaller sample sizes as describe d by Hungria (1994). Therefore N-ureides were expressed as a percent of total N (N-ureides plus nitrate) in the stem and petiole tissues. N- ureides are representative of the major forms of nitrogen translocated in the soybean pl ant and provide an estimate of the contribution of nitrogen fixation to total N accumulated by the plant. The N-ureide concentration in the stem andCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 326 of 375 petiole plant tissues was transformed to total N-ureide content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes.

Statistical Analyses. Statistical analyses were conduc ted by analysis of variance (ANOVA) on a within- and across-location ba sis. Mean comparisons across treatments at each site (within-location) were conducted by the Least Significant Difference (LSD) statistical procedure and across sites (loc ation across treatment and treatment across location) by the Tukey's test (Steel and Torrie, 1980).

Results and Discussion

a) 2006/2007 growing season

Population of Bradyrhizobium japonicum in the soils. The soil collected from all of the experimental areas prior to planting in the 2006/2007 growing season had high populations of Bradyrhizobium japonicum capable of nodulating soybean (Table F-39). The values are in agreement with those usua lly obtained in studies of nodulation in soils cropped with soybeans at the Embrapa Soja field trial site that have previously received

rhizobial inoculants.

Table F-39. Population of Bradyrhizobium Measured in Soils Prior to Planting the CV127 Soybean Field Trials at the Seven Trial Locations Conducted in the 2006/2007 Growing Season. The values are expressed in number of B. japonicum cells

g -1 dry soil and were obtained using the most probable number (MPN) method (Vincent, 1970). These data are not spec ific to any one treatment.

Locations Number of Cells 1

Santo Antônio de Posse 2.75x10 3

Ponta Grossa 3.00x10 4

Londrina 4.83x10 5

Uberaba 3.98x10 3

Sete Lagoas 1.60x10 3

Santo Antônio de Goiás 1.39x10 4

Brasília 0.95x10 3

1 Means of four replicates performed in triplicate.

Soybean nodulation

. Nodulation was evaluated in each of seven trials at the R2 growth stage in ten plants per replicate. At all tria l sites the coefficients of variance (CV) for the nodule number values were acceptable and we re in the range of the mean values (between 12.03 and 37.73%) proposed by Souza et al . (2008), (Table F-40).

In the within-location analysis, good nodulatio n was observed in all field trials and no

statistical differences were observed for nodul e number among the five treatments in the field trials performed at Ponta Grossa, Sete Lagoas, Santo Antônio de Goiás and Brasília (Table F-40). At Santo Antônio de Posse higher nodulation was observed in CV127CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 327 of 375 soybean and the isoline control compared to the other treatments. However, at Santo Antônio de Posse, Londrina, and Uberaba, there was no evidence of statistically significant differences among the CV127 soybean and isoline control treatments in terms of nodule number. At these three sites, nodulation of soybean cultivars Monsoy 8001 and CD 217 was lower than the nodulation exhibited by the CV127 and isoline control soybean plants (Table F-40). These results demonstrate that th e capacity for nodulation

in CV127 soybean is equivalent to that of the isoline control.

In the analysis of nodule number across trea tments for each field trial location, higher nodule number at the R2 stage occurred in Sa nto Antônio de Goiás, Uberaba, Ponta Grossa, and Londrina (Table F-47). In th e analysis of nodule number for each treatment across field trial locations (the treatment eff ect), highest nodulation was demonstrated for

CV127 and the isoline cont rol treated with Volt ® herbicide (treatments T2 and T3, respectively) compared to CV127 treated with imazapyr (T1) and the conventional varieties Monsoy 8001 (T4) and CD 217 (T5) (T able F-48). However, there were no statistically significant differences between nodule number for CV127 treated with either imazapyr or Volt ® herbicide compared to the isoline control (Table F-48). Furthermore, there were no statistically significant differences in nodule number between CV127 treated with either imazapyr herbicide or with Volt ® herbicide or the isoline control within any field trial location (Table F-40). These results confirm that there is no difference in root nodule formation capacity between CV127 and the isoline control and show that CV127 is equivalent to other conventional soybean varieties. Where nodulation differences were observed among tr eatments, these were generally between CV127 together with the isolin e control compared to the c onventional soybean varieties, and this was most likely associated w ith differences in genetic backgrounds.

Table F-40. Nodule Number (number plant -1 ) in Soybeans at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing Season 1

.

Locations

Treatment 2 Santo

Antônio

de Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio

de Goiás Brasília

T1 28.9 ab* 32.8 44.4 ab 50.7 ab 27.2 30.8 28.5

T2 42.3 a 54.3 58.4 a 56.0 ab 43.2 70.1 43.8

T3 42.9 a 45.4 43.5 ab 72.3 a 29.8 56.0 33.3

T4 22.0 b 57.8 25.6 b 35.1 b 33.5 48.3 34.8

T5 19.2 b 41.8 32.8 b 37.6 ab 32.0 51.9 21.3 CV(%) 27.5 24.9 21.3 32.4 36.0 36.9 31.4

Pr>F 0.004 0.063 0.002 0.044 0.425 0.126 0.084

LSD 19.2 n.s. 3 19.6 36.7 n.s. n.s. n.s.

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 328 of 375 For the within-location analysis of nodule dry weights, the CV values were within the medium variability range (between 8.84 and 32.60%), as proposed by Souza et al . (2008), in four sites (Ponta Grossa, L ondrina, Uberaba, and Sete Lago as), but higher values were observed at Santo Antônio de Posse (41.1 %), Santo Antônio de Goiás (49.1%) and Brasília (36.7%), (Table F-41). Significant statistical differences in nodule dry weight were detected among the treatments at thre e out of the seven si tes, Ponta Grossa, Londrina, and Brasília (Table F-41). Lo wer nodule dry weights at those sites were

observed in the conventiona l soybean varieties Monsoy 8001 (T4) and CD 217 (T5), except for Monsoy 8001 in Ponta Grossa. There was no evidence of a statistical difference in the nodule dry we ights among CV127 soybean and the isoline control at any of the locations (Table F-41).

In the analysis of nodule dry weight across treatments for each field trial location, the

results confirm normal levels of nodulation at a ll field trial locations (Table F-47). There were no statistically signi ficant differences in nodule dry weights between CV127 soybean and the isoline control or the conve ntional soybean varie ties when treatments were analyzed across locations (Table F-48). These results confirm that there is no difference in root nodule formation capacity between CV127 soybean and the isoline

control or other conventi onal soybean varieties.

Table F-41. Nodule Dry Weight (mg plant -1 ) of Soybean at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing Season 1

.

Location

Trt 2 Santo

Antônio

de Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio

de Goiás Brasília

T1 138.1 94.9 b* 104.0 ab 167.8 117.4 79.9 187.0 ab

T2 199.8 143.6 ab 123.2 a 160.8 189.5 154.2 207.8 a

T3 184.1 138.0 ab 111.0 ab 236.3 149.7 162.9 163.2 ab

T4 106.5 194.4 a 75.0 b 171.3 238.3 204.1 122.8 ab

T5 89.7 106.6 b 70.1 b 176.9 193.7 158.4 74.1 b CV(%) 41.1 26.6 19.4 25.4 32.4 49.1 36.7

Pr>F 0.089 0.017 0.007 0.207 0.093 0.276 0.035

LSD n.s. 3 81.2 42.3 n.s. n.s. n.s. 124.9

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

Shoot biomass production. The analysis of shoot dry weight values across treatments for each field trial location indicated that a ge nerally higher plant dry weight production was

obtained at Santo Antônio de Posse, Ponta Grossa and Brasília (Table F-47). In the analysis of shoot dry weight for each treat ment at each field location, no statisticalCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 329 of 375 differences in plant dry wei ght were detected among treatme nts in five of the seven locations, including Santo Antônio de Posse, Ponta Grossa, Londrina, Sete Lagoas, and Uberaba, while at two sites, Santo Antônio de Goiás and Brasília, there was no evidence of statistically signif icant differences in shoot dry we ight values betw een CV127 (T1 and T2 treatments) and the isoline control (T3) , but lower values were determined for Monsoy 8001 (T4) and CD 217 (T5), (Table F- 42), which most likely reflects varietal differences in growth characteristics.

In the across-location analysis of treatmen ts, there were no statistically significant differences in shoot dry weights between CV 127 treated with either imazapyr or Volt ®
herbicide compared to the isoline control but values for the conventional soybean varieties were significantly lower compared to the other treatments (Table F-48). These

results demonstrate that there is no evidence of a significant difference in the production of shoot biomass, as indicated by shoot dr y weights, between CV127, treated with either imazapyr herbicide or with Volt ® herbicide, and the isoline control.

Table F-42. Shoot Dry Weight (g plant -1 ) of Soybean at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing Season 1

.

Location

Treatment 2 Santo

Antônio

de Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio

de Goiás Brasília

T1 23.25 35.07 15.43 11.25 15.70 19.30 a* 27.48 ab

T2 24.05 37.40 19.94 10.25 13.40 15.28 ab 25.08 ab

T3 26.69 35.90 20.23 10.70 14.33 14.54 abc 30.45 a

T4 19.49 27.33 17.28 7.88 12.03 12.66 bc 21.98 b

T5 20.44 33.40 16.17 7.24 12.86 9.43 c 20.57 b CV(%) 14.8 20.8 15.4 31.0 24.8 18.1 14.5

Pr>F 0.066 0.348 0.096 0.266 0.610 0.002 0.014

LSD n.s. 3 n.s. n.s. n.s. n.s. 5.80 8.20

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

Total N and N as ureides in the soybean shoot. The total N concentration in soybean shoot tissues at the R2 stage was higher in the trials at Santo Antônio de Posse, Ponta Grossa, and Brasília (Table F-47) based on th e analysis across treatments for each field trial location and most likely reflects differences in nodulat ion and soil fertility between field trial locations. However, the within-l ocation analyses demonstrated no statistical differences among the three treatments including CV127 and the isoline control (treatments T1, T2, and T3). Some statisti cal differences were observed between these treatments and the conventional soybean vari eties T4 and T5 (Table F-43). Total N accumulated in the shoot (column “TNS” of Ta ble F-48) was statistically significantlyCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 330 of 375 lower in the conventional soyb ean varieties (T4 and T5 tr eatments) compared to the

CV127 and isoline control treatm ents (T1-T3), but total N concentration in the soybean shoot tissue (column “CNS” of Table F-48) wa s similar across all treatments with the exception of treatment T1 (CV127 treated with imazapyr) and treatment T4 (Monsoy 8001) which were statistically different due to the higher concentration of N in the shoots of Monsoy 8001. Therefore, the differences observed for total N accumulated in the shoot (column “TNS” of Table F-48) between treatments T1-T3 and the conventional soybean varieties were attributed to the stat istically significantly lower shoot dry weight values of treatments T4 and T5 compared to treatments T1-T3 (column “SDW” of Table F-48).

Table F-43. Total N Concentration in Shoot (g N kg -1 plant -1 ) of Soybean at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing

Season 1

.

Location

Treatment 2 Santo

Antônio de

Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio

de Goiás Brasília

T1 56.65 ab* 55.70 47.05 36.20 41.18 44.62 ab 53.55 ab

T2 54.90 ab 51.82 48.00 42.35 44.70 42.65 ab 52.50 b

T3 56.95 ab 57.18 54.50 42.65 40.95 47.98 a 54.45 ab

T4 59.55 a 59.90 54.18 43.48 52.82 43.42 ab 56.42 a

T5 50.78 b 58.18 51.28 40.95 49.35 36.30 b 54.65 ab CV(%) 5.6 7.2 9.5 8.0 11.7 8.7 2.84

Pr>F 0.020 0.129 0.156 0.059 0.053 0.012 0.039

LSD 6.97 n.s. 3 n.s. n.s. n.s. 8.42 3.84

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

These data were also expresse d as total N in th e shoot per plant (T ables F-44, F-47 and F48). In the analysis across treatments for each field location, considerable differences were detected among locations for total N accumulated in shoots per plant (Table F-47) as expected, since this value is a combination of shoot dry weight and N accumulation. Higher total N values were found at Santo Antô nio de Posse, Ponta Grossa, and Brasília,

and the lowest value was observe d at Uberaba (Table F-47). In the analysis of total shoot

N for each treatment at each fiel d location, at five of the s ites there was no evidence of

statistical differences between CV127 treated with either imazapyr herbicide or with

Volt ® herbicide and the isoline control treatment (Table F-44). At the Londrina field site,

shoot N accumulation in CV127 treated with imazapyr herbicide (T1) was statistically significantly lower than CV127 treated with Volt ® herbicide (T2) and the isoline control (T3). In contrast, at the Brasília field site, total shoot N in treatment T2 was statistically significantly lower than treatment T3. Theref ore, there was no cons istent trend of anyCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 331 of 375 differences between these treatments with resp ect to shoot N content. This was further confirmed in the across-location analysis for each treatment, wh ere there were no

statistical differences in the total N content in the shoot pe r plant between the treatments with CV127 (T1 and T2) and the isoline cont rol (expressed as total N in the shoots per plant in Table F-48). Overall, these resu lts demonstrate that there is no significant

difference in either N concentration or tota l N in the shoot betw een CV127, treated with either imazapyr herbicide or with Volt ® herbicide, and the isoline control.

Table F-44. Total N in Shoot (mg N plant -1 ) of Soybean at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing Season 1, 4

.

Location

Treatment 2 Santo

Antônio

de Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio

de Goiás Brasília

T1 1317.1ab* 1953.4 ab 725.5 c 407.3 646.5 861.2 1471.5 ab

T2 1320.3 ab 1938.1 ab 957.1 ab 434.1 599.0 651.7 1316.7 bc

T3 1520.0 a 2052.7 a 1102.5 a 456.4 586.8 697.6 1658.0 a

T4 1160.6 b 1637.1 b 936.2 ab 342.6 635.4 549.7 1240.1 bc

T5 1037.9 b 1943.2 a 829.2 bc 296.5 634.6 342.3 1124.2 c CV(%) 22.3 19.2 21.1 27.7 33.6 32.1 25.2

Pr>F 0.005 0.003 0.004 0.368 0.414 0.556 0.003

LSD 295.0 350.1 200.2 n.s. 3 n.s. n.s 340.5

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences

4 Total shoot N was calculated from the shoot N concentration and the total shoot dry weight. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

In the analysis across treatments for each fiel d location of the contribution of biological nitrogen fixation to the total N accumulated by the soybean crop, determined as percent N-ureides of total translocated nitrogen (N-ure ides plus nitrate) in the stem and petiole tissues, the % N-ureide values measured ra nged from 67.36% in the trial at Uberaba to

76.28% in the trial at Sete La goas, with no evidence of sta tistical differences among the seven sites (Table F-47). When % N-ureide values were presented for the different treatments at each field site (within-location analysis), there was no evidence of statistical differences among the three treatments including CV127 and the isoline control (treatments T1, T2, and T3), and no statisti cal differences between these treatments and the conventional soybean varieties T4 and T5 at four of the seven fiel d sites (Table F-45). However, in three out of the seven sites, Sa nto Antônio de Posse, Santo Antônio de Goiás and Brasília, there was a statistically lower contribution of biologica l nitrogen fixation in the conventional soybean varieties, Mons oy 8001 and CD 217, compared to CV127 and the isoline control (Table F-45), and this differe nce in shoot ureide c oncentration is to be expected between different soybean va rieties grown at different locations.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 332 of 375 When all data were analyzed for treatments across locations (across-location analysis),

there were no differences in N-ureide con centration between CV127, treated with either imazapyr herbicide or with Volt ® herbicide, and the isoline control, but lower values were present in the conven tional soybean varieties M onsoy 8001 and CD 217 (Table F48).

Table F-45. N-ureides as Percent of Total N (N-ureides plus nitrate) § in the Stem and Petiole Tissues of Soybean at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing Season 1

.

Location

Trt 2 Santo

Antônio

de Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio de

Goiás Brasília

T1 72.1 a* 76.8 67.9 68.9 78.2 80.2 a 74.2 a

T2 73.3 a 75.0 73.1 73.3 77.1 77.3 a 76.2 a

T3 76.1 a 79.9 74.4 72.8 76.7 76.9 a 80.1 a

T4 63.2 b 69.1 68.8 61.1 75.0 70.2 b 65.0 b

T5 60.0 b 73.9 67.2 60.7 74.4 65.5 b 62.1 b CV(%) 8.2 7.7 8.1 6.6 9.2 9.0 8.2

Pr>F 0.003 0.111 0.092 0.066 0.071 0.004 0.004

LSD 8.2 n.s. 3 n.s. n.s. n.s. 6.5 7.2

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences

§ N-ureides were measured in stem and petiole tissues only and expressed as a percent of total N (Nureides plus nitrate) in these tissues. N-ureides are an estimate of the contribution of nitrogen fixation to total N accumulated by the plant. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

The final parameter used to evaluate biolog ical nitrogen fixation wa s the total N-ureide content on a per-plant basis. The N-ureide concentration in the stem and petiole plant

tissues was transformed to total N-ureide c ontent in the above-ground portion of the plant or shoot for treatment comparison purposes. Higher levels of total N-ureide shoot content per plant were observe d in the trials performed in Ponta Grossa, Brasília and Santo Antônio de Posse, with lower values in Londrina, Uberaba, Santo Antônio de Goiás and Sete Lagoas according to the anal ysis across treatments for each field location (Table F-47).

In the within-location analysis of each treatment, there was statistically lower N-ureide content in the conventional soybean varie ties Monsoy 8001 and CD 217 at two field trial locations (Santo Antônio de Posse and Sant o Antônio de Goiás) compared to CV127 and the isoline control (Table F-46). Among th e CV127 and isoline cont rol treatments (T1, T2, T3), there was no evidence of a significant difference in N-ureide content at five of the seven sites, including Santo Antônio de Posse, Ponta Grossa, Uberaba, Sete Lagoas, and Brasília (Table F-46). At the Santo Antônio de Goiás site, the CV127 treated withCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 333 of 375 imazapyr (T1) had a statistically significantl y higher N-ureide content compared to the

CV127 treated with Volt ® herbicide and the isoline control, while at the Londrina site, the reverse was noted (Table F-46).

In the across-location analys is for each treatment for tota l N-ureide shoot content per plant, there was no evidence of a statistica lly significant difference between the CV127 soybean treatments or the isoline control (T able F-48). These results demonstrate that there is no significant difference in either N-ur eide concentration or total N-ureide in the shoot between CV127, treated with eith er imazapyr herbicide or with Volt ® herbicide,

and the isoline control. These results are consistent with previous results showing no

differences in nodule number or dry weight associated with CV127 compared to the

isoline control.

Table F-46. Total N-Ureide Content (mg N-ureide plant -1 ) § in Soybean at the R2 Stage, in the Field Trials Performed in Brazil During the 2006/2007 Growing

Season 1

.

Location

Treatment 2 Santo

Antônio

de Posse Ponta

Grossa Londrina Uberaba Sete Lagoas Santo

Antônio

de Goiás Brasília

T1 949.6 a* 1500.2 a 492.6 d 280.6 505.6 690.7 a 1091.9 ab

T2 967.8 a 1453.6 a 699.6 ab 318.2 461.8 503.8 b 1003.3 abc

T3 1156.7 a 1640.2 a 820.3 a 332.2 450.1 536.5 b 1328.1 a

T4 733.5 b 1131.2 b 644.1 bc 209.3 476.6 385.9 c 806.1 bc

T5 622.7 b 1436.0 a 557.2 cd 180.0 472.2 224.2 d 698.1 c CV(%) 20.2 23.1 17.7 32.3 27.7 11.9 16.6

Pr>F 0.005 0.005 0.005 0.411 0.334 0.005 0.004

LSD 212.7 223.0 128.6 n.s. 3 n.s. 126.4 236.5

1 The data represent the mean of four replicates, with ten plants per replicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences

§ The N-ureide concentration in the stem and petiole plant tissues was transformed to total N-ureide

content in the above-ground portion of the plant or shoot for treatment comparison purposes. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

The across-location analysis for each treatmen t of shoot N and N-ureides has shown that

there was no evidence of statistical diffe rences between CV127 soybean and the isoline control in the total N or N-ureide contents (Tab le F-48). In general, the values of total N and N-ureide observed in CV127 soybean and th e isoline control were higher than those obtained with conventional soybean vari eties Monsoy 8001 and CD 217 (Table F-48).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 334 of 375 Table F-47. Effect of Location on Ni trogen Fixation Parameters in the 2006/2007 Growing Season.

Parameter 1

Location NN NDW SDW CNS TNS %NU TNU

Santo Antônio

de Posse 31.0 c* 143.7 ab 22.78 b 55.77 a 1271.2 b 68.94 886.1 b

Ponta Grossa 46.4 abc 135.5 ab 33.82 a 56.56 a 1904.9 a 74.94 1432.2 a

Londrina 41.0 abc 96.7 b 17.81 c 51.00 ab 910.1 c 70.28 642.8 c

Uberaba 50.3 ab 182.6 a 9.46 d 41.12 c 387.4 e 67.36 264.1 d

Sete Legoas 33.1 bc 177.7 a 13.66 c 45.80 bc 620.5 d 76.28 473.2 c

Santo Antônio

de Goiás 51.4 a 151.9 ab 14.25 c 43.00 c 620.5 d 74.02 468.2 cd

Brasília 32.3 c 151.0 ab 25.11 b 54.32 a 1362.1 b 71.52 985.5 b

1 Nodule number (NN, number plant -1 ) and dry weight (NDW, mg plant -1 ), shoot dry weight (SDW, g

plant -1 ), concentration of N in shoots (CNS, g N kg -1 plant -1 ), total N accumulated in shoots (TNS, mg N plant -1 ), %N as N-ureide (%NU) 2 and total N as N-ureide (TNU, mg N-ureide plant -1 ) 3 in soybean harvested at the R2 stage, in the seven trials performed in Brazil during the 2006/2007 growing season.

2 N-ureides were measured in stem and petiole tissues only and expressed as a percentage (%NU) of total

N (N-ureides plus nitrate) in thes e tissues. N-ureides are an estima te of the contribution of nitrogen fixation to total N accumulated by the plant.

3 The N-ureide concentration in the stem and petiole plant tissues was transformed to total N-ureide

content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 335 of 375 Table F-48. Effect of the Different Treatm ents on Nitrogen Fixation Parameters in the 2006/2007 Growing Season.

Parameter 1

Treatment 2 NN NDW SDW CNS TNS %NU TNU

1 34.8 b* 127.0 21.07 a 47.85 b 1054.6 a 74.04 a 787.3 a

2 52.6 a 168.4 20.78 a 48.13 ab 1031.0 a 75.04 a 772.6 a

3 46.2 ab 163.6 21.84 a 50.66 ab 1153.4 a 76.70 a 894.9 a

4 36.7 b 158.9 16.95 b 52.82 a 928.8 b 67.49 b 626.7 b

5 33.8 b 124.2 17.16 b 48.78 ab 886.8 b 66.26 b 598.6 b

1 Nodule number (NN, number plant -1 ) and dry weight (NDW, mg plant -1 ), shoot dry weight (SDW, g

plant -1 ), concentration of N in shoots (CNS, g N kg -1 plant -1 ), total N accumulated in shoots (TNS, mg N plant -1 ), %N as N-ureide (%NU) 3 and total N as N-ureide (TNU, mg N-ureide plant -1 ) 4 in soybeans harvested at the R2 stage, in the seven trials performed in Brazil during the 2006/2007 growing season.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 N-ureides were measured in stem and petiole tissues only and expressed as a percentage (%NU) of total

N (N-ureides plus nitrate) in thes e tissues. N-ureides are an estima te of the contribution of nitrogen fixation to total N accumulated by the plant.

4 The N-ureide concentration in the stem and petiole plant tissues was transformed to total N-ureide

content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 336 of 375 b) 2007 growing season

Population of soybean Bradyrhizob ium japonicum in the soils. The soil collected from all of the experimental field areas had high populations of B. japonicum capable of nodulating soybean (Table F-49). The values are in agreement with those usually obtained at the Embrapa Soja field trial site in studies evaluati ng nodulation in soils cropped with soybean that have previ ously received rhizobial inoculants.

Table F-49. Population of Bradyrhizobium japonicum in Soils Cropped with Soybean in the Six Trials Perf ormed in the 2007 Growing Season. The values are expressed in number of Bradyrhizobium japonicum cells g -1 dry soil and were obtained using the most probable number (MPN) method (Vincent, 1970).

Locations No. cells g -1 soil*

Teresina, PI, EMN 1.89x10 3

Vilhena, RO, ER 2.41x10 3

Uberaba, MG, CTTP 1.58x10 4

Sete Lagoas, MG, CNPMS 2.60x10 4

Santo Antônio de Goiás, GO, CNPAF 2.05x10 4

Brasília, DF, CNPH 1.48x10 3

* Means of four replicates with an alyses performed in triplicate.

Soybean nodulation. In the trials conducted during th e 2007 short seas on, nodulation at the R2 stage was evaluated at six locations on ten plants per replicate. Field nodulation by legumes, especially nodule number (NN) per plant, can present hi gh variability, and the maximum acceptable value of coefficient of variation (CV) established by Souza et
al. (2008) was 37.7% (medium). The observed variability for nodule number was very high at all locations, as evident by the w ithin-location analysis, and medium CV was observed only in Vilhena (Table F-50). In this case, as recommended by Souza et al

. (2008), values were transformed into log 10 , promoting a significant reduction in the

coefficients of variation to values betw een 8.2% and 12.8% in Santo Antônio de Goiás and Sete Lagoas, respectively (data not show n). Despite the high coefficients of variation, good nodulation was ob served in Teresina, Bras ília, Vilhena, and Santo

Antônio de Goiás. Due to adverse climatic conditions, nodulation was lower in Uberaba and Sete Lagoas (Table F-50).

The within-location analysis demonstrated that significant differences among treatments

for nodule number were observed in Vilhena and Uberaba, with the highest values observed in treatment T3, the isoline control treated with conventional herbicide, and the lowest in T4, conventional soybean variety Monsoy 8001 (Tab le F-50). The analysis

across treatments for each field location s howed that a lower number of nodules were present on plants at the Uberaba and Sete Lagoas sites (Table F-57). However, no significant differences were observed among the five treatments in the across-location analysis (Table F-58).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 337 of 375 Table F-50. Number of Nodules (number plant -1 ) in Soybeans at the R2 Stage in the Field Trials Conducted in Brazil During the 2007 Growing Season 1

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio

de Goiás

T1 30.8 45.0 48.4 ab* 12.8 b 23.0 32.0

T2 27.0 39.6 53.6 a 15.7 b 17.2 23.2

T3 25.6 36.1 62.9 a 26.1 a 20.0 36.7

T4 33.4 32.2 25.2 c 5.7 b 11.5 20.3

T5 26.8 30.7 30.2 bc 8.6 b 8.0 26.6 CV(%) 45.6 38.6 27.8 48.8 64.3 47.9

Pr>F 0.891 0.0016 0.009 0.032 0.513 0.657

LSD n.s. 3 n.s. n.s. n.s.

1 Data are means of four replicates w ith analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

In the observations for nodule dry weight, CV values of up to 32.6% were within the range of medium variability proposed by Souza et al . (2008) at two locations, Vilhena and Santo Antônio de Goiás (T able F-51). Based on the within-location analysis, significant differences for nodule dry matter were observed among treatments at three locations, Brasília, Vilhena and Uberaba. At Uberaba the isoline control soybean (T3) had higher nodule mass compared to the CV127 tr eatments T1 and T2 (Table F-51). No statistically significant differences in nodul ation parameters were observed between CV127 treated with either imazapyr or Volt ® herbicide in the 2007 season (Tables F-50 and F-51).

The data analysis across treatments for each field location showed differences among locations with respect to nodul ation parameters, with poor nodulation at Uberaba, Sete Lagoas, Santo Antônio de Goiá s and Teresina (Table F-57) . The excellent nodulation potential of CV127 soybean and the isoline control soybean, wh ich were superior to the conventional soybean varieties Monsoy 8001 and CD217, was also observed when the treatments were compared across all field tr ial locations (across-location analysis) with respect to nodulation parameters (Table F-58). These results confirmed that there is no evidence of a statistically significant di fference in root nodule formation capacity between CV127 compared to the isoline cont rol. Even though CV127 soybean and the isoline control had slightly higher nodule numbers and nodule dry weights compared to the conventional soybean varieties, the onl y statistically signi ficant difference was

between CD217 and the other treatm ents with respect to nodule dry weight (Table F-58).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 338 of 375 Table F-51. Nodule Dry Weight (mg plant -1 ) of Soybeans at the R2 Stage in the Field Trials Conducted in Brazil During the 2007 Growing Season 1

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio

de Goiás

T1 121.3 319.1 a* 154.0 ab 51.8 b 147.8 125.1

T2 94.3 313.9 a 196.7 ab 58.0 b 99.6 137.7

T3 99.2 253.2 ab 209.0 a 106.8 a 131.1 193.3

T4 155.6 187.7 b 147.6 ab 21.6 b 82.2 126.5

T5 117.6 180.9 b 131.4 b 31.4 b 66.0 149.9 CV(%) 61.0 38.4 25.6 49.2 59.0 29.2

Pr>F 0.912 0.0072 0.065 0.018 0.621 0.303

LSD n.s. 3 n.s. n.s. n.s.

1 Data are means of four replicates w ith analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

Shoot biomass production. Analysis of shoot dry weight values across treatments for each field location showed a high level of va riability among locations, with the highest

values observed at Santo Antônio de Goiás and Brasília and the lowest values at Uberaba (Table F-57). The variation in shoot dry weight values most likely reflects environmental differences between field locations with, for example, higher rainfall at the Uberaba site compared to Santo Antônio de Goiás. The wi thin-location analysis for each treatment at each location showed there were no significant differences in shoot dry weights between

the CV127 and isoline control s oybeans except at Uberaba (Tab le F-52). Furthermore, there were no significant differences in this parameter between CV127 soybean and the two conventional soybean varieties at any lo cation except for a statistically significant difference observed with CD217 (T5) at Sant o Antônio de Goiás (Table F-52). It is concluded that shoot dry matter accumulati on in CV127 soybean is not different from

that of the isoline control or other conventi onal soybean varieties. Comparison of shoot dry weight accumulation for each treatment across locations confirmed that CV127 soybeans were not statistically significantl y different for shoot dry matter accumulation compared to the isoline control or the c onventional soybean varieties (Table F-58).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 339 of 375 Table F-52. Shoot Dry Weight (g plant -1 ) of Soybean at the R2 Stage in Field Trials Conducted in Brazil During the 2007 Growing Season 1

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio de

Goiás

T1 9.82 14.27 6.86 2.63 b* 8.23 14.96 a

T2 9.24 14.92 7.36 1.67 b 7.21 14.01 a

T3 11.05 14.83 7.44 4.46 a 8.55 13.99 a

T4 10.79 14.53 7.00 3.22 ab 7.25 12.39 ab

T5 9.46 12.32 7.51 3.26 ab 5.84 9.71 b CV(%) 29.5 24.5 16.0 45.5 26.4 13.95

Pr>F 0.891 0.444 0.924 0.103 0.406 0.023

LSD n.s. 3 n.s. n.s. n.s.

1 Data are means of four replicates with analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

Total N and N as ureides in the soybean shoot. The total N concentration in soybean shoot tissues at the R2 stage across treatm ents for each field location was comparable among all locations (Table F-57), and small differences observed most likely reflect differences in nodulation and soil fertility between field locations. For each treatment at each field location, there was no evidence of statistically significant differences among treatments for total shoot N concentration at any of the field trial locations (Table F-53), and this was confirmed in data analysis comparing treatments across field trial locations (Table F-58).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 340 of 375 Table F-53. Shoot N Concentration (g N kg -1 plant -1 ) of Soybean at the R2 Stage in Field Trials Conducted in Brazil During the 2007 Growing Season 1

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio

de Goiás

T1 30.5 49.9 44.0 39.4 42.9 47.0

T2 37.6 42.9 42.7 40.5 42.4 47.8

T3 28.0 44.2 41.1 43.1 45.8 46.6

T4 33.1 46.6 41.7 39.8 41.4 41.1

T5 32.5 43.0 42.0 39.8 40.0 37.3 CV(%) 11.77 19.89 8.36 10.88 15.73 17.21

Pr>F 0.131 0.916 0.370 0.943 0.824 0.246

LSD n.s. 3 n.s. n.s. n.s. n.s. n.s.

1 Data are means of four replicates with analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

The values of total N accumu lated in the shoots of the soybean plants were also determined (Table F-54). The data analysis across treatments for each location detected statistically significant differences among locat ions for total N accumulated in shoots per plant (Table F-57), as expected, since this va lue is a combination of shoot dry weight and N accumulation. In the analysis of total shoot N within a location for each treatment, at four of the locations there was no evidence of statistically significant differences for total shoot N among any of the treatments. Only at the Uberaba site was a statistically significant difference observed between CV127 treated with Volt ® herbicide and the isoline control, and at the Santo Antônio de Goiás field site the total shoot N content of the conventional soybean variety CD 217 was statistically significantly less than the CV127 and isoline control treatments (Table F-54). Across locations, there was no evidence of statistically si gnificant differences in total shoot N accumulation among the treatments (Table F-58). Overall, these resu lts demonstrate that there is no evidence of statistically significant differe nces in either N concentra tion or total N in the shoot between CV127, treated with either imazapyr herbicide or with Volt ® herbicide, and the

isoline control and conventional s oybean varieties (Table F-58).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 341 of 375 Table F-54. Total N in the Shoot (mg N plant -1 ) of Soybean at the R2 Stage in Field Trials Conducted in Brazil During the 2007 Growing Season 1, 4

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio

de Goiás

T1 302 720 304 103 ab* 354 699 a

T2 351 660 314 69 b 306 666 a

T3 306 648 304 191 a 395 652 a

T4 382 718 292 123 ab 300 517 ab

T5 308 529 314 132 ab 230 363 b CV(%) 29.5 38.83 14.47 42.56 28.56 19.10

Pr>F 0.940 0.822 0.948 0.068 0.1779 0.0056

LSD n.s. 3 n.s. n.s. n.s.

1 Data are means of four replicates with analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences

4 Total shoot N was calculated from the shoot N concentration and the total shoot dry weight. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.

In the analysis across treatments for each fiel d location of the contribution of biological nitrogen fixation to the total N accumulated by the soybean crop determined as percent N-ureides of total translocated nitrogen (N-ure ides plus nitrate) in the stem and petiole tissues, the % N-ureide values measured range d from 58.0% in the trial at Teresina to 82.2% in the trial at Sete Lagoa s, with statistically significan t differences in % N-ureide among the six locations (Table F-57). Thes e differences among locations were most likely related to soil fertility and plant nodulation levels between sites. When % N-ureide values were presented for the different tr eatments within a location, there was no evidence of a statistically significant differen ce in this parameter among the five different treatments at five of the field trial sites (Table F-55). The only statistically significant

difference for % N-ureide content was a lowe r value for the conventional soybean variety CD 217 compared to the isoline control treatment at Sete Lagoas. Ac ross locations, there

were no differences in % N-ureide values between CV127, treated with either imazapyr

herbicide or with Volt ® herbicide, and the isoline co ntrol as well as the Monsoy 8001 conventional soybean variety. There was a stat istically significantly lower value in the conventional soybean variety CD 217 compared to the isoline control and Monsoy 8001 (Table F-58).

Finally, total N-ureide content on a per-plant basis was measured to evaluate biological nitrogen fixation contribution to overall nitroge n accumulation in the plant. The N-ureide concentration in the stem and petiole plant tissues was transformed to total N-ureide content in the above-ground portion of the plant or shoot for treatment comparison purposes. An analysis across treatments fo r each field location showed that total Nureide varied between field sites, as expect ed, since this parameter is the product of bothCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 342 of 375 plant growth as well as N-ureide accumula tion (Table F-57). When total N-ureide content per plant was presented for the different treatments within each field site, there was no evidence of statistically significant diffe rences between any of the five treatments

at four of the six field tria l locations (Table F-56). At the Uberaba field site, total Nureide content was statistically significantly higher for the isoline control soybean (T3) compared to CV127 soybean treated with Volt ® herbicide (T2). Also, at the Santo Antônio de Goiás field site, levels of total N- ureide content were statistically significantly higher in CV127 soybean trea ted with imazapyr herbicide (T1) compared to the conventional soybean variety CD 217. Levels of total N-ureide per pl ant were equivalent

between all other treatments at th ese two field trial locations.

When total N-ureide content per plant wa s analyzed for each treatment across all locations, there was no evidence of statistically significant difference between the CV127 soybean treatments and the is oline control as we ll as the conventional soybean variety Monsoy 8001 (Table F-58). However, levels of total N-ureide content per plant were statistically significantly lower in conventiona l soybean variety CD 217 (T5) compared to the other four treatments in the study. Thes e results demonstrate that there is no evidence of a significant difference in ei ther N-ureide concentration or total N-ureide in the shoot between CV127, treated with either imazapyr herbicide or with Volt ® herbicide, and the isoline control, aside from the above-mentione d difference at Uberaba. These results are consistent with results reported for the sa me studies conducted in the 2006/2007 growing

season.

The analysis of shoot N and N-ureides across a ll field trial locations has shown that there was no evidence of statistical differences between CV127 soybean and the isoline control in the total N or N-ureide content (Table F-58 ). In general, the values of total N and Nureide observed in CV127 s oybean and the isoline cont rol were higher than, or comparable to, those obtained with conven tional soybean varieties Monsoy 8001 and CD 217 (Table F-58).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 343 of 375 Table F-55. Percent N-Ureides (%NU) of Total N (N-ureides Plus Nitrate) § in the Stem and Petiole Tissues of Soybean at th e R2 Stage in Field Trials Conducted in Brazil During the 2007 Growing Season 1

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio

de Goiás

T1 56.3 76.2 60.8 68.5 85.2 ab* 86.2

T2 58.3 73.8 60.2 60.0 81.2 ab 78.2

T3 57.0 74.8 59.0 77.2 89.0 a 84.5

T4 60.0 73.2 59.0 68.8 81.0 ab 81.0

T5 58.3 63.5 59.5 72.5 74.8 b 71.0 CV(%) 11.75 20.06 6.77 9.17 5.84 12.05

Pr>F 0.981 0.745 0.960 0.173 0.0141 0.2516

LSD n.s. 3 n.s. n.s. n.s. n.s.

1 Data are means of four replicates with analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences

§ N-ureides were measured in stem and petiole tissues only and expressed as a percent of total N (Nureides plus nitrate) in these tissues. N-ureides are an estimate of the contribution of nitrogen fixation to total N accumulated by the plant. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 344 of 375 Table F-56. Total N-Ureide (mg N-ureide plant -1 ) § in Soybean at the R2 Stage in Field Trials Conducted in Brazil During the 2007 Growing Season 1

.

Location

Treatment 2 Teresina Brasília Vilhena Uberaba Sete Lagoas Santo

Antônio

de Goiás

T1 174 567 186 71 ab* 303 605 a

T2 210 516 190 43 b 251 526 ab

T3 173 496 179 150 a 354 552 ab

T4 245 582 173 90 ab 245 431 ab

T5 180 345 187 100 ab 176 276 b CV(%) 50.58 55.85 19.47 52.19 31.72 27.5

Pr>F 0.8388 0.768 0.954 0.073 0.096 0.0302

LSD n.s. 3 n.s. n.s. n.s.

1 Data are means of four replicates w ith analyses performed in duplicate.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline control

treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated with Volt ®

. 3 n.s. = no significant differences

§ The N-ureide concentration in the stem and petiole plant tissues wa s transformed to total N-ureide

content in the above-ground portion of the plant or shoot for treatment comparison purposes. * Means followed by no letter or by the same letter, in the same column, are not statistically different at the

5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 345 of 375 Table F-57. Effect of Location on Nitrogen Fixation Parameters in the 2007 Growing Season.

Parameter 1

Location

NN NDW SDW CNS TNS %NU TNU

Teresina 28.7 ab 117.6 bc 10.07 b 32.4 b 330 b 58.0 d 196 bc

Brasilia 36.7 a 251.0 a 14.18 a 45.3 a 655 a 72.3 bc 501 a

Vilhena 44.1 a 167.7 ab 7.23 c 42.3 a 305 b 59.7 d 184 bc

Uberaba 13.8 b 53.9 c 3.05 d 40.5 a 124 c 69.4 c 91 c Sete Lagoas 15.9 b 105.3 bc 7.42 c 42.5 a 317 b 82.2 a 266 b

Santo Antônio

de Goiás 27.8 ab 146.5 bc 14.18 a 44.0 a 580 a 80.2 ab 487 a

1 Number (NN, number plant -1 ) and dry weight (NDW, mg plant -1 ) of nodules, shoot dry weight

(SDW, g plant -1 ), concentration of N in shoots (CNS, g N kg -1 plant -1 ), total N accumulated in the shoots (TNS, mg N plant -1 ), %N as N-ureide (%NU) 2 and total N as ureides (TNU, mg ureide-N plant -1 ) 3 in soybean at the R2 stage in field tr ials conducted at six locations in Brazil during the 2007 growing season.

2 N-ureides were measured in stem and petiole tissues only and expressed as a percent (%NU) of

total N (N-ureides plus nitrate) in these tissues. N-ureides are an estimate of the contribution of nitrogen fixation to total N accumulated by the plant.

3 The N-ureide concentration in the stem and petiole plant tissues was transformed to total Nureide content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes. * Means followed by no letter or by the same le tter, in the same column, are not statistically

different at the 5% level.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 346 of 375 Table F-58. Effect of the Different Treatments on Nitrogen Fixation Parameters in the 2007 Growing Season.

Parameters 1

Treatment 2 NN NDW SDW CNS TNS %NU TNU

T1 32.0 153.2 a* 9.45 ab 42.8 418 72.9 ab 324 a

T2 29.4 150.0 a 9.06 ab 42.5 396 69.1 ab 293 ab

T3 34.6 165.4 a 10.01 a 42.0 421 74.3 a 324 a

T4 21.4 120.2 ab 9.12 ab 40.9 388 71.0 a 297 ab

T5 21.8 112.9 b 7.96 b 39.4 313 67.0 b 212 b

1 Number (NN, number plant -1 ) and dry weight (NDW, mg plant -1 ) of nodules, shoot dry weight

(SDW, g plant -1 ), concentration of N in shoots (CNS, g N kg -1 plant -1 ), total N accumulated in the shoots (TNS, mg N plant -1 ), %N as N-ureide (%NU) 3 and total N as ureides (TNU, mg ureide-N plant -1 ) 4 in soybean at the R2 stage in field trials conducted in Brazil at six locations in the 2007 growing season.

2 Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt ® , T3 = Isoline

control treated with Volt ® , T4 = Monsoy 8001 treated with Volt ® , T5 = Coodetec 217 treated

with Volt ®

. 3 N-ureides were measured in stem and petiole tissues only and expressed as a percentage (%NU)

of total N (N-ureides plus nitrate) in these tissues. N-ureides are an estimate of the contribution of nitrogen fixation to total N accumulated by the plant.

4 The N-ureide concentration in the stem and petiole plant tissues was transformed to total Nureide content in the above-ground portion of the plant or shoot (TNU) for treatment comparison purposes. * Means followed by no letter or by the same le tter, in the same column, are not statistically

different at the 5% level.

Conclusions

Studies on nitrogen fixation ch aracteristics of CV127 soybean compared to the isoline

control and two conventional soybean varieties were condu cted in both the 2006/2007 and the 2007 growing seasons in Brazil. Th e results across both growing seasons were consistent and showed that CV127 soybean, irrespective of herbic ide treatment, is equivalent to the isoline c ontrol soybean with respect to biological nitrogen fixation, including nodule number and dry weight, shoo t dry weight, total N content and the contribution of nitrogen fixation to the total N accumulated in tissues as evaluated by the N-ureide content of the shoot. Comparison of CV127 and the isolin e control as a group to the conventional soybean va rieties showed some differ ences in nitrogen fixation parameters, which were attribut ed to basic genetic differenc es between soybean varieties in biological nitrogen fixation capacities. Therefore, thes e results support the conclusion that CV127 soybean is no different in its inte ractions with the symb iotic nitrogen-fixing Bradyrhizobium japonicum or in nitrogen fixation capac ity than conven tional soybean

varieties.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 347 of 375 Appendix G

Best Management Practice (BMP) Program for the Imidazolin one-tolerant CV127 Soybean Production SystemCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 348 of 375 The Imidazolinone-tolerant Soybean Production System

Introduction

The Production System for imidazolinone-toler ant soybean is an innovative cropping system that offers broad-spectrum weed contro l. It creates a numb er of new opportunities for soybean producers:

• Superior one-pass, broad-spect rum control of grass and an nual broadleaf weeds with

imidazolinone herbicides.

• Superior in-crop weed control of a range of difficult-to-control weeds, including glyphosate-resistant weeds. It is expected that growers adopti ng this technology will

be able to reduce the number of herbicide applications and benefit from an overall

reduction in weed control costs.

• An additional weed management tool for weed control and resistance management.

• Allows the maintenance of glyphosate as an effective pre-seed tool.

Soybean ( Glycine max L.) plants have been develope d by BASF Plant Science, L.P and EMBRAPA (Empresa Brasileira de Pesquisa Agr opecuaria, Brasil) that are tolerant to the imidazolinone class of agricu ltural herbicides. The herb icide-tolerant CV127 soybean plants (also referred to as CV127) are de rived from a single transformation event and were produced by introduction of th e imidazolinone-tolerance conferring acetohydroxyacid synthase large subunit ( AHAS ) gene csr1-2 with its native promoter

from Arabidopsis thaliana into the soybean plant genome via biolistics transformation technology.

BASF Plant Science is developing im idazolinone-tolerant CV127 soybeans for cultivation primarily in Brazil and Argentina. The herbicide OnDuty ® (525 grams ai/L imazapic + 175 grams ai/L imazapyr) is the co mmercial product being developed for this production system. These imidazolinone-toler ant CV127 soybeans are distinct from the imidazolinone-tolerant crops sold under the Clearfield ® brand name; Clearfield ® -brand crops are not derived from recombinant DNA technology.

Regulatory approval will be sought in the United States and Canada, as well as other countries, for importation of soybean grain from CV127 for food, f eed, and processing uses. [

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 349 of 375 The introduction of CV127 soybeans in Argentina and Brazil is ex pected to provide enhanced soybean yield potential and offer growers excellent weed control options in

addition to the environmental benefits that result from the cultivation of herbicidetolerant soybeans. The imidazolinone herb icides possess several environmentally beneficial characteristics compared to other herbic ide classes (Tan et al ., 2005). Imidazolinone herbicides control a wide spect rum of grass and broadleaf weeds. The growing use of glyphosate with glyphosate-tole rant soybeans in Arge ntina and Brazil has led to a shift in the species of prevalent w eeds, with those that are more tolerant to glyphosate predominating. The most common weeds in this category include Benghal dayflower ( Commelina benghalensis L.)

, morning glory ( Ipomoea spp.), Brazil pusley ( Richardia brasiliensis ), and winged false buttonweed ( Spermacoce alata ). These weeds

are sensitive to imidazolinone herbicides. Im idazolinone herbicides are effective at low application rates (Shaner and Singh, 1998), pos sess residual activity in the soil and are absorbed by roots and translocated throughout the plant, resulting in fewer herbicide applications. For example, in Brazil, th e recommended application rate and frequency that are proposed for imidazolinones used with CV127 soybean is one application per season at a rate of approx imately 70 g ai/hectare.

Imidazolinone herbicides have a very favorable toxicology profile. Due to the fact that imidazolinones are rapidly excreted before they can accumulate in blood or tissues (Gagne et al ., 1991) and since animals do not possess th e AHAS enzyme that is the target for imidazolinones, imidazolinone herbicides ha ve very low mammalian toxicity. Based on the known safety profile of imidazolinone herbicides, it is anticipated that the Imidazolinone-tolerant Soybean Producti on System will provide a safe and environmentally beneficial system of weed control for soybean growers.

Key Sustainability Issues

The users, developers and marketers of herbicide-tolerant cropping systems are responsible for sustaining the production sy stem and must address the following key

issues:

• Herbicide resistance management using an integrated approach.

• Control of herbicide-tolerant crop volunteers.

• Managing out-crossing to othe r crops and weedy relatives.

These key sustainability issues form the ba sis for our stewardship plans for crops grown using the Imidazolinone-tolerant Soybean Production System. The Imidazolinonetolerant Soybean Stewardship Plan can be summarized by the following guiding principles:CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 350 of 375 

### Imidazolinone-tolerant Soybean Production System – GUIDING

PRINCIPLES

There are a number of GU IDELINES that must be understood and followed by Agronomists and Growers when using the Imidazolinone-tolerant Soybean Production System.

• DO NOT exceed a maximum of 2 exclusive Gr oup 2 herbicides on any one field, in any 4-year period.

• ALWAYS follow an Integrated Weed Mana gement (IWM) program that includes a wide range of herbicides, cultural practices and crop rotations in order to manage weed populations and minimize weed seed development.

• ALWAYS control volunteers in the seas on following an imidazolinone-tolerant soybean crop.

• USE practices which minimize the likelihood of out-crossing to similar crops or

related weeds.

• Scout fields for weeds or volunteer cr ops that are uncontro lled by herbicides.

• FOLLOW the Best Management Practices outlined in the Imidazolinone-tolerant Soybean Stewardship Guide.

Resistance Management in th e Im idazolinone-tolerant Soybean Production System

Herbicides have been grouped based on their mode of action. Herbicides that are in Group 2 are those classified as ALS (acet olactate synthase)/AHAS (acetohydroxyacid synthase) inhibitors. BASF ma rkets herbicides that are in the imidazolinone chemical family and they are members of Group 2. BASF is therefore a key stakeholder in resistance management of ALS/AHAS-resistant weeds.

BASF is committed to maintaining the efficacy of all of its herbicides in order to provide growers with effective, hi gh-performance, environmentally sound products for many years. The key to the perf ormance of the Imidazolinone-tolerant Soybean Production System is effective weed resistance ma nagement. BASF is committed to delivering sustainable cropping systems that incorp orate best practice principles. The Imidazolinone-tolerant Soybean Production Sy stem provides alternative options for growers within a well-manag ed crop rotation system.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 351 of 375 Herbicide Resistance Management Using an Integrated Weed Management (IWM) Approach

The objectives of herbicide resistance manage ment are to achieve weed control while preserving the value of each herbicide and each herbicide group for the longer term. An integrated approach to weed control is the Best Management Practice to delay the onset of weed resistance to herbicides. Integrated weed management involves the use of a range of methods available to the grower in order to provide effective weed control incrop. The use of herbicides is one of a num ber of useful tools av ailable to growers.

Development of Resistance:

The Herbicide Resistance Ac tion Committee (HRAC) is an industry ini tiative which fosters co-operation between plant protecti on manufacturers, govern ment researchers, advisors and farmers. The objective of the wo rking group is to facilitate the effective management of herbicide resistance. HRAC has identified a number of factors to consider when evaluating herbicide resist ance risk. The most important factors influencing a plant's potential to de velop resistance are the following:

• Biology and genetic make up of the weed species in question: Points of consideration include the following

. Weeds that are extremely suscepti ble to an herbicide or that are prolific seed producers and have a large amount of ge netic variation within the species may have a greater potential to become resistant to an herbicide. The initial frequency of naturally-occurri ng resistant biotypes in a weed population influences a weed population's potential to de velop resistance. Also, the relative fitness or vigor

of resistant weed bi otypes affects resistance devel opment. Generally speaking, for any particular weed species, the greater the initial frequency of resistance and the greater the fitness of the resistant biotype , the greater the potential for herbicide resistance to develop.

• History of herbicide use and mode of action : Continuous use of the same mode-ofaction herbicide for several consecutive y ears, without tank mi xing or sequentially applying herbicides with other modes-of-a ction, may increase the risk for resistant populations to develop. The greater the sele ction pressure exerted by an herbicide, the greater the potential for resistance. Although a higher rate of application or sequential applications results in a high level of weed control, it also represents an increased potential for the development of resistance. Likewise, lower herbicide rates, which provide less effective weed cont rol, exert less selection pressure. Label herbicide rates are a reflection of efficacy tr ials that indicated the best control and crop yield responses. Some herbicides, in cluding Group 2 herbicides, have a greater ability to select for resistant weeds than ot hers because of the frequency of resistance alleles in weedy populations.



### • Crop management practices : Weed control that relies solely on herbicide use and does not combine tillage, or other cultural pr actices, with herbicide applications may increase the potential for resistant populati ons to develop. Crop rotation practicesCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 352 of 375 that allow for non-chemical control options in addition to chemical group rotations are sound management procedures.



### • Environmental conditions : Environmental conditions that ar e not conducive to herbicide breakdown in the soil may increase the potential for resistant populations to develop. Continuous dry weather can slow the breakdown of many herbicides (e.g. imidazolinones). Also, high soil pH inhibits the breakdown of some herbicides, such as some of the sulfonylurea herbicides. Th e longer a herbicide persists, the longer it exerts selection pressure on a weed populati on, particularly if th ere are multiple weed flushes in one growing season.

• Weed seed bank/Seed soil dormancy : A high soil seed bank within an individual field increases the selection pressure, which in turn increases the likelihood of resistance developing. Seed soil dormancy will also have an impact on weed resistance development. Plants with longer soil dorma ncy will tend to exhibit slower resistance development since selection pressure is reduced

. Seeds that can survive for years in the soil may slow the onset of resistance. Weeds with a long seed life may create a large seed bank in the soil. This seed bank serves as a buffer against genetic changes in the weed population, since the seeds do not normally all germinat e within one year. Conversely, weed seed with a short seed life germinate within one or two years. This rapidly depletes the quantity of susceptible weed seed and gives any resistant seed a competitive advantage when a selection pressure is applied.

Many exceptions to these generalizations ex ist, and this makes it difficult to predict which species will develop a resistant population. The time required for a weed population to develop resistance will vary, a nd depends on many factors, including the following:



### • Selection pressure exer ted by the herbicide;



### • Herbicide rotation patterns;



### • Seed germination dynamics;



### • Use of herbicide combinations with different modes of action;



### • Initial frequency of natu rally occurring resistant individuals in the weed population; and



### • The relative vigor of resist ant biotypes of weeds.

Based on these factors, models have been developed to predict the development of resistance in a weed population. Current models provide an indication of the development of resistance and th ese indications are an essential input to the development of resistance management strategies and practices.

Identifying Weed Resistance:

It is important to avoid confusing herbic ide failure caused by weed resistance with herbicide failure caused by other factors. All other possible reasons for poor herbicide performance should be ruled out before consid ering the possibility of resistance. These include application error a nd poor environmental conditions at the time of herbicide application. Shifts in weed populations from susceptible species to species that are lessCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 353 of 375 sensitive can also cause weed control pr oblems. Herbicide resistance should be suspected under the following conditions:

• A weed species that is normally controlled by the herbicide now escapes treatment, while other weeds on the label are controlled.

• Other factors such as app lication error or adverse w eather conditions are ruled

out.

• Irregular-shaped patches of a weed develop in the field and are not controlled with the particular herb icide or herbicide group.

• Weed control records for the field indicate repeated use of a pa rticular herbicide, or herbicides from the same herbicide group.

General Recommendations to Minimize Development of Weed Resistance:

The following is a discussion and proposed strategies for managing ALS/AHAS

herbicide resistance in w eed populations under the Im idazolinone-tolerant Soybean Production System. The guidelin es for managing the development of weed resistance presented here are consistent with reco mmendations put forward in crop protection guides and the Weed Resistance Educati on and Action Program (WREAP).

Development of herbicide-resistant weed s can be avoided or delayed through good management practices. The recommendations lis ted below take into consideration many of the points discussed so far and are designed to provide an integrated approach to weed management in order to prevent or delay the ons et of weed resistance. Three key areas of integrated weed management are chemi cal control, cultural practices and crop

management.

Chemical Control



### • Know your herbicide groups.

Reliance on one product or on products within the same group may eventually lead to weed resistance. Understanding herb icide classification by product group is necessary to develop an effec tive weed management strategy.



### • Keep records of herbicide application.

Herbicide application record s and field mapping are necessa ry to effectively rotate herbicide groups.



### • Always read and follow herbicide label recommendations.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 354 of 375 The recommended rate of herbicide provides the most effective control over a wide range of environmental conditions. This will help to ensure weed seed is not added to the seed bank, while minimizing selection pressure.



### • Use tank mixes or sequential applications of herbicides that have different modes of

action.

Herbicide tank mixes allow weed control in more than one way by combining two or

more modes of herbicide action on the same weed. In order to be effective, both active ingredients need to provide control of the target weed. This minimizes selection pressure and delays the development of weed resistance.



### • Rotate among herbicide groups.

Rotate among herbicide groups for both grass and broadleaf weed control. Alternating products used according to mode -of-action is one of the most effective means of delaying development of weed re sistance. Use the minimum number of applications of any one herbicid e or herbicide group per season.



### • Utilize non-selective herbicides.

Non-selective herbicides applied pre-emerge nce are an effective means of controlling early flushes of weeds and/or weed escapes. They should be used in conjunction with an in-crop herbicide that has an alternative mode of action.

Cultural Control/Crop Management

Cultural (non-chemical) weed control practi ces do not exert any chemical selection pressure and can help to reduce the level of weeds in the soil seed bank. These practices are important components of an in tegrated weed control strategy.



### • Use crop rotations, notably rotations from broadleaf crops to grass crops, winterseeded and spring-seeded crops, and perennial and annual crops.

Different crops can help to alter the weed sp ectrum. It also makes it easier to rotate between herbicide groups.



### • Plant competitive crops.

Crops vary in their ability to compete with w eeds. Crops such as barley that establish a heavy stand are much more competitive w ith weeds than crops such as lentils or flax. In most instances, crop competition will increase with hi gher seeding rates and by planting varieties with a tall stature. A competitive crop can reduce weed

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 355 of 375 

### • Seed early.

Early seeding generally lead s to better crop establishment and increased crop competitive ability. Delayed seeding of most crops results in a potential yield loss

and is not recommended.



### • Plant quality seed/Increase the seeding rate.

Certified seed offers a number of benefits and helps to ensure a uniform stand that will compete more effectively with weeds. A more dense plant canopy helps to reduce weed competition.



### • Combine tillage and/or timely cultivation w ith herbicide treatments, if practical.

Direct seeding has been shown to reduce the number of annual weeds over time. However, if the crop production system incl udes spring cultivation, seed as soon as possible after working the gr ound to give the crop a h ead start over the weeds.

An effective weed management strategy is co mprised of multiple weed control options. Herbicide-tolerant cropping systems provide yet another mechanism for effective weed

control and should be consider ed as one of the tools for managing the development of

weed resistance.

Integrated Weed Management (IWM) in the Imidaz olinone-tolerant Soybean Production System

Imidazolinone-tolerant soybean is tolerant to the imidazolinones, which are Group 2 herbicides. Group 2 herbicides work by in hibiting ALS/AHAS, an enzyme that is required for the production of th e amino acids leucine, isoleu cine and valine in plants. Group 2 herbicides are known as ‘ALS inhibitors’.

Continuous use of Group 2 herbicides may resu lt in the selection of weed biotypes with a resistance to this Group of herbicides. Preservation of the effectiveness of this Group of herbicides is vital for efficient and cost-effective agricultural production in North America. Therefore effective management strategies for weed control delay or avoid the potential for the development of resistant weeds and are an important focus of the Imidazolinone-tolerant Soybean Production System.

In addition to the general chemical and cultural control recommendations outlined in the previous section, a number of specific manageme nt strategies outlined below, should be followed when using the Imidazolinone-toler ant Soybean Production System. Growers and agronomists should give consideration to each of the following points, in order to develop an integrated weed management system when using imidazolinone-tolerant soybeans as part of their production system.

• Make only one application of a Group 2 herbicide per season.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 356 of 375 • Apply no more than two (2) exclusive Group 2 herbicides in any four (4) year period

on the same field. The use of no more than two applications in f our years will slow the development of resistance. Considerati on should be given to less frequent use to delay the onset of resistance developmen t. This includes the use of Group 2

herbicides both within the Imidazolinone-t olerant Soybean Production System and in conventional crops.

• If a Group 2 herbicide has been applied as a pre-em ergence application, DO NOT apply further Group 2 herbicides to that crop. Make any further post-emergence applications with herbicides fr om a different mode-of-action group.

• Where it is possible, care should be taken to avoid applications of Group 2 herbicides in consecutive years unless in at least the two previous years, e ffective weed control

has been achieved with methods other th an the use of Group 2 herbicides.

• Farm practices, including herbicide and crop rotations, should be developed which

allow for the use of alternative mode-of-action herbicides.

• Where Group 2 resistance is suspected with in a weed population, testing of the relevant weeds should be carried out prior to the use of crops in the Imidazolinonetolerant Soybean Production System.

• Integrated Weed Management should be undertaken on a field-by-field basis. Specific field planning should take into consid eration the history of the field well as the future use options.

• Resistance management guidelines for othe r herbicide Groups, especially Group 1,

should be taken into account whendeveloping and planning rotations.

Crop Rotation and Imidazolinone-tolerant Soybeans

The most common rotational crops for soybeans in the United States are corn, other row crops, and small grains. In 2002, approximate ly 80% of the soybeans were grown in rotation with corn (USDA-ERS, 2006b). The ar ea planted to soybean in the U.S. over the past 20 years is presented in Figure G-1. For the years 2006-2011, the area planted to soybeans in the United States has ranged between 65 and 77 million acres (USDA-NASS, 2011; USDA-ERS, 2011b).CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 357 of 375 Figure G-1. Area Planted to S oybeans in the United States.

Controlling Volunteers from Imidazolinone-tolerant Soybeans

Objective: control of all volunteers from Imidazolinone -tolerant Soybeans before flowering.

Summary

• Best Management Practice is to control vol unteer plants in the year following the cultivation of imidazolinone-tolerant soybeans.

• Do NOT rely on Group 2 herbicides to control imidazolinone-tolerant soybean volunteers. Group 4 + 5 herbicides are the most commonly used management tool for

control of imidazolinone-toler ant soybean volunteers.

• Volunteers from imidazolinone-tolerant soybeans will be controlled by all herbicides currently registered for control of volunteer conventional soybeans

, except for

imidazolinone herbicides.

• Clean-up of farm equipment during all st ages of sowing, harvesting, storage and transport are important in the e ffective control of volunteers.

• Best Management Practice is to make voluntee r control part of w eed, pest and disease management strategies for the farm.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 358 of 375 Important reasons for control of volunteers

• Volunteer plants act as competitive weeds in subsequent crops or pastures.

• Volunteers may be importan t in the build-up and spr ead of major diseases.

• Volunteers increase the risk of herbicide tolerance spreading from cross-pollination of volunteer plants and conventi onal plants of the same crop species in neighboring

fields.

Cultural and chemical options for volunteer management

A number of cultural and chemical control options are available to control volunteers including the following:

• Minimize seed losses at harv est by the following means:

 Pay close attention to timeliness of harvesting.

 Make correct adjustments in the header.

 Seal all holes and cracks in harvesting equipment which allow spillage, even of small quantities of seed (especially in the table, front elevator and grain tank).

 Clean out harvest equipment before switching fields.

• Practice good hygiene at harves t and during transport of grain. Secure loads to prevent losses during transport.

• Practice good stubble management after harves t in light of crop rotation decisions. Leaving soybean seed on the surface will increase seed mortality and help to reduce the seed bank population in the soil.

• Use pre-seed glyphosate application to c ontrol weeds and volunteers that emerge prior to seeding.

• Control volunteers along fi eld edges and borders.

• Control ALL volunteers in subsequent crops with the proper se lection of in-crop herbicides or other agronomic practices such as tillage where appropriate.

• Keep good field records of herbicides used in previous crops a nd herbicide-tolerant varieties in neighboring fields to develop effective plans for controlling volunteers.

• Soybeans should not follow an imidazolinon e-tolerant soybean crop, as controlling volunteers within the same crop species is difficult. Generally speaking, this would also not be a good agronomic practice fo r disease and weed management.

Management of volunteer Im idazolin one-tolerant Soybean

Cultivated soybean seed rarely displays any dormancy characteristics, and only under certain environmental conditions will it grow as a volunteer (OECD, 2000). If soybean plants do grow as a volunteer, they do not compete well with the succeeding crop.

Volunteer soybean management is normally not a significant mana gement concern in

North America because of environmental conditions, crop rotation and the numerous herbicide control options available. When soybean seeds are left on the soil surface they are typically not viable after winter. When a rotationa l crop such as winter wheat isCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 359 of 375 planted after soybean harvest, it is possible fo r soybean seed to be incorporated into the soil and to germinate that fall. These volun teer plants will be controlled by freezing temperatures in the fall and do not beco me a competitive consideration. Normally volunteer soybean is no t a target for weed control measures in winter wheat.

When corn is the rotational crop, the first option available to c ontrol volunteer soybean the following spring is pre-plant tillage or the use of a pre-plant burn-off herbicide such as glyphosate. Table G-1 lists the herbicide active ingredients applied to corn in the

United States in 2010. Herbicides from Group 4 (auxinic herbicides), Group 5 (triazines), Group 10 (phosphorylated am ino acids) and Group 27 or Group 28 (4- hydroxyphenyl pyruvate dioxygenase or HPPD i nhibitors) all will control volunteer soybeans in corn. Seventeen of the 58 active in gredient reported as used on corn (29% of the total) in 2010 are members of one of th ese herbicide groups. Therefore, there are

numerous tools available to contro l volunteer soybeans in corn.

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 360 of 375 In Roundup Ready ® (RR) corn, volunteer conventi onal or imidazolinone-tolerant soybeans will be controlled by any of the gl yphosate products (Group 9) applied. In some cases the glyphosate is tank-mixed or used in sequence with another broadleaf herbicide. Any of the broadleaf herbicide products from Groups 4, 5 or 27 will also control volunteer soybeans. Therefore, virtually all the herbicide options available in Roundup Ready ® corn will control volunteer soybean s. The two most commonly-used herbicide active ingredients for use on corn, glyphosate and atrazine, [ ] will both control volunteer soybeans.

In the United States, volunteer soybean management is normally not a concern in common rotational crops such as corn and wheat. In addition to cultural control measures such as tillage, there are numerous herbicide options available in these crops to [CBI]

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 361 of 375 control volunteer soybeans. In corn, post- emergence application of products such as

AAtrex ® (atrazine), Clarity ® (dicamba), Distinct ® (diflufenzopyr + dicamba), Hornet ®
(flumetsulam + clopyralid) and Widematch ® (clopyralid + fluroxypyr) will control volunteer conventional, volunteer Roundup Ready ® or imidazolinone-tolerant soybean (Zollinger, 2011). Similarly, in wheat there are also a numbe r of products available for volunteer soybean management. Products such as Bronate ® AdvancedTM (bromoxynil), Clarity ® (dicamba) and Widematch ® (clopyralid + fluroxypyr) wi ll control all types of volunteer soybean (Zollinger, 2011).

Managing Out-Crossing to Imidazo linone-se nsitive Crops and Weeds

What is out-crossing and gene flow?

Gene flow is the movement of gametes, zygotes (seeds), individuals, or groups of individuals from one place to another and thei r subsequent incorporation in the gene pool of the new locality (Slatkin, 1987). Gene fl ow is a natural biological process and in plants it primarily occurs via pollen or seed dispersal (Levin a nd Kerster, 1974). The relative importance of gene flow to a popul ation’s genetic structure depends on the

distance between donor and re cipient populations, population size, how long the process has been in effect, and whether the new ge ne confers any fitne ss advantage to the recipient population (Waines and Hegde, 2003).

Out-crossing (or cross-pollination) is a type of mating in plants in which a male gamete

of one individual fertilizes a female gamete of another individual (Waines and Hegde, 2003). The term out-crossing gene rally refers to mating within a species and the term has been used synonymously with gene flow (Gleaves, 1973; Handel, 1983). However, as defined earlier, gene flow can occur through means other then cross-pollination. This discussion is restriced to pollen-me diated gene flow (out-crossing).

A wide range of factors influence the ability of a given plant to out-cross with others, including synchrony of flowering, stigma r eceptivity, pollen production, pollen dispersal, pollen viability and environmental factors. The likelihood of out-crossing varies greatly from species to species and variety to variet y. Successful out-crossi ng may result in the offspring displaying characteris tics of both parent plants.

The risk of out-crossing can vary among crop and weed species. The focus for management must be on the control of volunt eers and managing herbic ide-tolerant crops and related species.

Description of the Imidazolinone -tolerant Soybean technology

Cultivated soybean, Glycine max (L.) Merr., is a diploidized tetraploid (2n=40), in the family Fabaceae. Three types of growth ha bit can be found amongst soybean cultivars: determinate, semi-determinate and indeterm inate (Bernard and Weiss, 1973). Generally determinate types are grown in the southern United States and i ndeterminate types are grown in central and northern regions of North America. In Canada the majority of the soybean cultivars have an indeterminate growth habit.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 362 of 375 The herbicide-tolerant CV127 soybean plants are derived from a single transformation event and were produced by introduction of the imidazolinone-t olerance conferring acetohydroxyacid synthase large subunit gene csr1-2 with its native promoter from Arabidopsis thaliana into the soybean plant genom e via biolistics transformation technology.

Potential for out-crossing of Imidazolinone-tolerant Soybean

Soybean is considered to be a self-pol linating species. Based on the reproductive morphology of soybean, the anthers mature in the bud and directly pol linate the stigma of the same flower (OECD, 2000). Natural out-cr ossing levels range from less than 0.5% to about 1.0% (Carlson an d Lersten, 1987). Therefore the pot ential for gene transfer from an Imidazolinone-tolerant Soybean variety to another commercial variet y is very low.

Potential for out-crossing to species related to Imidazolinone-tolerant Soybean

Soybeans, Glycine max , belong to the subgenus Soja . This subgenus also includes G.
soja and G. gracilis which are wild and semi-wild soybean relatives from Asia. Interspecific, fertile hybrids between G. max and G. soja or between G. max and G. gracilis have been easily obtained (as reviewed in OECD, 2000). However, the potential for gene flow to wild soybean relatives is limited geographically. Wild soybean species are limited to Korea, Taiwan, Japan, Chin a and areas around the border of the former

USSR. These wild relatives do not exist naturally in North America.

The genus Glycine also contains the subgenus Glycine . This subgenus consists of 22 wild perennial species that ar e indigenous to Australia, the South Pacific Islands, China, Papua New Guinea, the Philippines and Taiwan. Intersubgeneric hybrids between the subgenus Soja and the subgenus Glycine have been created ar tificially with great difficulty, and the progeny of such hybrids we re sterile (as revi ewed in OECD, 2000).

The risk for introgression into other relatives is extremely low. Soybeans can only cross

with other members of Glycine subgenus Soja . Therefore, the pote ntial for outcrossing to other members of the subgenus Soja is limited by geographic isolation and the wild soybean species of the subgenus Soja are not naturalized in North America (OECD, 2000).

Conclusion

In summary, the accidental presence of Imidazolinone-tolerant Soybean in the North American environment would have no impact on current soybean control practices, since all of the conventional control methods (including all herbicid es currently registered for

control of volunteer conventional soybeans

, except for imidazolinone herbicides) would continue to control this plant. There are no significant differences between the growth and productivity of Imidazolinone-tolerant Soybean and the corre sponding conventional varieties of the equivalent maturity group. Therefore, th ere would be no change to agronomic practice due to the occurren ce of Imidazolinone- tolerant Soybean.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 363 of 375 Always read and follow label directions.

Clearfield, OnDuty, Clarit y, and Distinct are registered trad emarks of BASF. AAtrex is a trademark of Syngenta Group Company. Ho rnet and Widematch are registered trademarks of Dow AgroScie nces LLC. Roundup Ready is a registered trademark of Monsanto Technology L.L.C. Bronate an d Bronate Advanced are trademarks or registered trademarks of Bayer.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 364 of 375 Appendix H

Digestive Fate of the AtAHAS Produced in CV127 Soybean and in a Recombinant

E. coli Expression SystemCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 365 of 375 INTRODUCTION

The purpose of this study was to determine the susceptibility of 1) a test substance (Lot number AtAHAS-0107) containi ng AtAHAS protein encoded by the imidazolinonetolerant acetohydroxyacid synthase la rge subunit gene isolated from Arabidopsis thaliana ( AtAHAS, containing R272K an d S653N mutations) and 2) AHAS protein present in extracts of leaf and grain from CV127 plants, to diges tion in both simulated mammalian gastric fluid (SGF) and simulated mammalian intestinal fluid (SIF).

The introduction of an imidazolinone-tolerant acetohydroxyacid synthase large subunit gene from Arabidopsis thaliana into the plant genome allows growth and, hence, selection of the transformed cells in the presence of imidazolinone herbicides. The Arabidopsis thaliana AHAS large subunit (AtAHAS) protein is a member of the class of AHAS large subunit proteins found ubiquitous ly in plants. The mutation in the Arabidopsis acetohydroxyacid synthase large subunit gene res ponsible for imidazolinone herbicide tolerance is a single nucleotide change of guanine to adenin e, which results in a codon change from AGT to AAT and a singl e amino acid substitu tion of serine to asparagine at position 653 of the AtAHAS protein. In ad dition, a second mutation was identified after this gene was introduced into the soybean genome, resulting in the transformation event designated as CV127. Th e second mutation, in which arginine at position 272 was replaced by lysine (R272K), doe s not impact the enzymatic function of the AHAS enzyme (Stevenson-Paulik, 2011).

The AHAS enzyme catalyzes the first step in the biosynthesis of branched-chain amino acids in plants. In conventional plants, i nhibition of the AHAS en zyme by imidazolinone herbicides leads to a deficiency in bran ched-chain amino acids and other compounds derived from this pathway that are need ed for plant survival . The acetohydroxyacid synthase large subunit gene from Arabidopsis confers tolerance to imidazolinone herbicides by encoding an AtAHAS enzyme (large subunit) with altered herbicide binding properties. However, the enzyme has normal biosynthetic function in the transgenic plant.

The lot of test substance, AtAHAS-0107, was prepared from a recombinant Escherichia

coli over-expression system and was intended for use in an acute oral mouse toxicity study, as well as other studies to confirm the food, feed and environmental safety of the protein. The AtAHAS protein preparation was characterized to determine identity, purity, functionality, concentrati on, and solubility (Privalle, 2007a) and has been found to be substantially equivalent to that pr oduced by CV127 plants (Privalle, 2007b). The purpose of Phase A of this study was to c onfirm that the microbi ally-produced AtAHAS protein is rapidly digested, in a similar ma nner as typical dietary proteins. The purpose of also examining the digesti on of the AtAHAS protein as expr essed in leaf and grain of CV127 (Phase B of this study) wa s to determine whether the pl ant matrix has an effect on the digestibility and to conf irm that the plant-produced AtAHAS protein had a similar digestive fate to the E. coli -produced AtAHAS protein as we ll as the endogenous soybean AHAS protein. After incubation for various times in the appropriate simulated digestiveCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 366 of 375 fluids, the remaining protein was subjected to electrophoresis and visualized by either Coomassie blue staining or western blot analysis.

MATERIALS AND METHODS

PHASE A

. Preparation of test substance . The Arabidopsis thaliana acetohydroxyacid synthase large subunit gene ( ahas R272K, S653N ) was cloned into the inducible, over-expression vector pTrcHis A ® (Invitrogen, Carlsbad, CA) in E. coli strain BL21(DE3)pLysS. The AtAHAS protein encoded by this vector lacks the predicte d 85-amino acid N-terminal leader sequence that targets the protein in planta to the chloroplast. This leader sequence has been replaced in this vector with 38 am ino acids which include a 6x His tag to allow for ease in purification, a gene 10 leader sequence to enhanc e solubility, and an XpressTM tag for detection. The remainde r of the protein is identical in amino acid sequence to that produced by the native gene from Arabidopsis thaliana (Mazur et al ., 1987) except for the two aforementioned point mutations. The replacement of serine with asparagine at amino acid residue 653 of the AtAHAS prot ein results in decreased binding of imidazolinone herbicide to AtAHAS, and imidazolinone tolerance. The point mutation that results in repl acement of arginine with lysine at amino acid residue 272 has no apparent impact on AHAS functionality.

AtAHAS protein was produced and purified by Invitroge n, Inc. (Madison, WI) and

transferred as an ammonium su lfate pellet to the Regulatory Science Laboratory at BASF Plant Science, Research Triangl e Park, NC where it was receiv ed on February 27, 2007. The AtAHAS protein was purified from 2,400 g of cell paste after lysi s in 20 L buffer (50 mM sodium phosphate, 300 mM NaCl, 10 mM β -mercaptoethanol, 30 mM imidazole) using an Emulsiflex cell disr uptor (Avestin, Ontario, Canada ). Insoluble material was removed by centrifugation. The His-tagged AtAHAS present in th e supernatant was purified by chromatography using Talon ® cobalt resin (Clontech, Inc., Mountain View, CA). Once eluted, the fractions cont aining AtAHAS were pool ed and immediately precipitated by ammonium su lfate at 40% saturation

. The ammonium sulfate pellet was resuspended in 100 mM Tris-HCl, pH 7.0 a nd dialyzed against 20 mM ammonium
bicarbonate buffer, pH 7.9, containing 20 μ M FAD. The dialyzed material was then lyophilized and designated AtAHAS-0107. This material has been characterized (Privalle, 2007a). The test substance in this study is also the test system. The test solution used in the digestion reactions be low was prepared by resuspending 2.2 mg of

AtAHAS-0107 in 1.1 ml of 8 mM sodium phosphate, 2 mM potassium phosphate, 0.14 M sodium chloride, 10 mM potassium ch loride, pH 7.4, and used on the day of preparation.

Control substances . An extract of nontransgenic s oybean leaves was prepared by homogenizing 0.67 g of leaf material with two ml extraction buffer (8 mM sodium phosphate, 2 mM potassium phosphate, 0.14 M sodium chloride, 10 mM potassium chloride, pH 7.4). An extract of nontransge nic soybean grain was prepared similarly except that two grams of materi al was extracted with nine ml of buffer. Extracts wereCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 367 of 375 centrifuged 15 min at approx. 13,000 x g. The pr otein concentration was determined in the supernatants after centrifugation and the supernatants were used directly in the digestive reactions. Digestion reactions we re conducted the same day that the extracts were prepared.

Protein quantification . Total protein in the leaf and grain extracts was quantified by the BCATM procedure (bicinchoninic acid procedur e; Pierce Biotechnology, Inc., Rockford, IL) using bovine serum albumin as the standar d. Dilutions of the extract (final volume

100 μ l) were prepared such that the expected concentration of protein would be within the standard curve. Samples were reacted with two ml of a 1:50 (B:A) mixture of BCA reagent, incubated at 37oC for 30 min and a llowed to cool at room temperature for 10

min. The absorbance at 562 nm was m easured using a UV1600 Spectrophotometer (Shimadzu, Columbia, MD). The results were analyzed by the instrument’s software using the linear regression curve fit.

PHASE B

. Preparation of test substances . Extracts of CV127 leaves were prepared by homogenizing two grams of leaf material wi th six ml extraction buffer (8 mM sodium phosphate, 2 mM potassium phosphate, 0.14 M sodium chloride, 10 mM potassium chloride, pH 7.4). Extracts were centrifuge d 15 min at approx. 13,000 x g. An extract of CV127 grain was prepared similarly except that two grams of material was extracted with nine ml of buffer. The protein concentratio n was determined in the supernatants after centrifugation and the supernatants were used directly in the digestive reactions. Digestion reactions were conducted the same day extracts were prepared.

Protein quantification . Total protein in the leaf and grain extracts was quantified by the BCATM procedure (bicinchoninic acid procedur e; Pierce Biotechnology, Inc., Rockford, IL) using bovine serum albumin as the standard . Dilutions of the extract were prepared such that the expected concentration of prot ein would be within the standard curve. Samples (25 μ l) were loaded into a multiwell pl ate in triplicate, reacted with 200 μ l of a 1:50 (B:A) mixture of BCA reagen t, incubated at 37oC for 30 min and allowed to cool at room temperature for 10 min. The absorb ance at 550 nm was measured using a Multiskan Ascent V1.24 multiwell plate reader (Therma Labsystems, Helsinki, Finland) and concentrations were determined using a standard curve analyzed by linear regression

.

PHASE A and B. Simulated mammalian gastric fluid reactions . Simulated mammalia n gastric fluid [2X SGF; 0.016 N HCl, 0.75 mM NaCl, pH 1.2, and 3500 Units of pepsin (Sigma)] was prepared as described by Thomas et al. (2004) and proteolytic activity was confirmed using azoalbumin as a substrate (SOP BPS 510.01). A single tube containing sufficient reaction mix for all assay time points was prepared by mixing 350 μ l of SGF (2X) with

350 μ l of AtAHAS-0107 solution or CV127 leaf extract. For CV127 grain extract, a single tube containing sufficient reaction mix for all assay time points was prepared by mixing 665 μ l 1X SGF with 35 μ l extract. The reaction mixtures were immediately placed in a 37 ° C water bath and 100 μ l samples were removed at 0.5, 2, 5, 10, 30 and 60 min after initiation of the experiment. Each sample was quenched by the addition of 35CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 368 of 375 μ l of 200 mM NaHCO 3 , pH 11, and 35 μ l 5X Laemmli buffer (40% glycerol, 5% β

mercaptoethanol, 10% SDS, 0.33 M Tris, 0.05% bromophenol blue, pH 6.8). Quenched samples were then heated to >75 ° C for 10 min and stored at ca . –20 ° C until subjected to electrophoresis as described below. The “zero” time point for all reactions was prepared by first quenching SGF and then adding the test protein. In additi on, reactions that served as controls for pepsin auto-digesti on and test protein stab ility were prepared containing SGF without test protein and SGF with test protein but without pepsin (“G-con”), respectively. These control reacti ons were treated exactly as described above except samples were only taken at 0 a nd 60 minutes after the start of the 37 ° C incubation.

Simulated mammalian intestinal fluid reactions . Simulated mammalian intestinal fluid (SIF) containing phosphate (8.2 mg KH 2 PO 4 /ml), tap water and pancreatin (12 mg/ml), pH 7.5, was prepared as described in the United States Pharmacopoeia (2000) and was checked for proteolytic activity us ing azoalbumin as a substrate (SOP BPS 510.01). A single tube contai ning enough reaction mix for all time points was prepared by mixing 280 μ l of SIF with 70 μ l of AtAHAS-0107 solution or CV127 leaf or grain

extract. The reaction mixtures were immediately placed in a 37 ° C water bath and 50 μ l samples were removed at 0.5, 2, 5, 10, 30 and 60 min after initiation of the experiment. Each sample was quenched by addition of 50 μ l of 2X Laemmli buffer (20% glycerol,

2% β -mercaptoethanol, 4% SDS, 0.13 M Tris , 0.02% bromophenol blue, pH 6.8). Samples were then heated to >75 ° C for 10 min and stored at ca . –20 ° C until subjected to electrophoresis as described below. The “zero” time point was prepared by first heating

SIF for 10 min at > 75 ° C and then adding the test protein and 50 μ l 2X Laemmli buffer and reheating for 10 min at > 75 ° C. In addition, reactions th at served as controls for pancreatin auto-digestion and test protein st ability were prepared containing SIF without test protein and SIF with test protein but without pancreatin (“I-con”), respectively. These control reactions were treated exactly as described above except samples were only

taken at 0 and 60 minutes after the start of the 37 ° C incubation.

Sodium dodecyl sulfate-polya crylamide gel electrophoresis

. To monitor the integrity of AtAHAS ( ca . 64,000 mol. wt.) in sample At AHAS-0107 after incubation in the various digestion mixtures, al iquots of the quenched and heat ed mixes were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE, SOP BPS 510.02), using a 4 - 20% polyacrylamide gradient Tris-glycine gel (Inv itrogen). Aliquots of the samples that contained ca. 2.5 μ g of protein based on determinations made prior to digestion were loaded onto the gel. Mark 12 TM molecular weight markers (Invitrogen) were used to establish the approximate molecular weight of proteins. The protein bands

were stained with 0.1% Coomassie Brilliant Bl ue R (Sigma Chemical, Saint Louis, MO).

Western blot analysis

. To monitor the integrity (intact ness) of the AtAHAS protein in the leaf and grain extracts as well as in sample AtAHAS -0107 after incubation in the various digestion mixtures, western blot analysis was performed. Aliquots of the quenched and heated mixes were subjected to SDS-PAGE on a 4 - 20% polyacrylamide gradient gel followed by electroblotting onto PVDF membrane (Invitrogen; SOP BPS 510.03). The samples were loaded onto the gel to achieve maximum total protein based on determinations made prior to digestion. For the SGF samples, the amounts of proteinCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 369 of 375 loaded were 24 μ g for leaves and grain, and 8 and 30 μ g for CV127 and conventional soybean leaves, respectively. For the SIF sa mples, the amounts loaded onto the gel were

84 and 67 μ g for CV127 and conventional soybean grai n, respectively. For western blot analysis of the AtAHAS-0107 SIF reactions, 0.2 μ g of protein was loaded per lane. After electroblotting, the membranes were probed with rabbit anti-AHAS peptide 2 polyclonal antibody. Donkey anti-rabbit IgG linked to hor seradish peroxidase (Pierce), diluted 1:3000 in blocking buffer (3% nonfat dry milk in 0.1% Tween-20, 10 mM Tris-HCl, 150 mM NaCl, pH 7.5) was used to bind to the primary antibody a nd was visualized by development with the chemiluminescent EC L kit from Amersham (Buckinghamshire, UK) according to the manufact urer’s instructions.

RESULTS

PHASE A

. Sensitivity of E. coli -produced AtAHAS (Lot number AtAHAS-0107) to degradation

in SGF . The E. coli -produced AtAHAS protein in test material lot number AtAHAS0107 was rapidly degraded in SGF (Figure H-1 panel A). AtAHA S (molecular weight

ca . 64,000) is readily detected in the time 0 sample and in the “G-con” samples. However, no full-length protein is visible in the sample removed at 0.5 min after the

initiation of the reaction. Some low molecular weight, Coomassie blue-staining bands (<6000) were observed in the 0.5 min sample, but these were degraded after 2 min in the reaction. A low molecular weight band of approx. 6000 MW is apparent that is also present in the “SGF only” lane and hence must be derived from the pepsin enzyme preparation.

Sensitivity of E. coli -produced AtAHAS (Lot number AtAHAS-0107) to degradation

in SIF . The E. coli -produced AtAHAS protein in test material lot number AtAHAS0107 was also rapidly degraded in SIF (Figure H-2 panel A). Similar to the SGF results,

AtAHAS is detected in the time 0 and “I-con ” samples. However, no full-length protein is visible in the sample afte r 0.5 min of incubation. Some degradation of the AtAHAS is observed in the 60 min “I-con” sample indica ting protein sensitivity to incubation at 60

min in simulated intestinal fluid in the absen ce of pancreatin. Pancreatin is comprised of multiple proteases and lipases and this account s for the multiple staining bands in the SIF lanes. All of the lower molecular weight bands visible in the digestion time course correspond to those present in the “SIF only” sample (no AtAHAS-0107). The results of the AtAHAS digestion in SIF us ing western blot analysis (F igure H-3 panel A) confirm that no immunoreactive degradation products of AtAHAS were obscured by bands associated with the pancreati n, and confirm that the AtAHAS protein is rapidly degraded

in SIF within a 0.5 min incubation time.

PHASE B

. Sensitivity of CV127 soybean leaf and grain AtAHAS to degradation in SGF . The AHAS protein produced in CV127 soybean l eaves and grain is a mixture of the endogenously encoded soybean AHAS proteins and the AtAHAS protein encoded by the

introduced imidazolinone-tolerant ahas R272K, S653N gene. Due to the high amino acidCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 370 of 375 sequence similarity of the endogenous AHAS and AtAHAS proteins, they are immunologically indistinguishable. Both soybean AHAS and At AHAS proteins are rapidly digested in SGF and, although the AH AS protein band is vi sible at time 0, no AHAS protein band is detectable within two minutes of inc ubation (Figure H-1 panel B). An additional immunoreactive band at ap proximately 50,000 molecular weight is observed in leaf extracts (Figur e H-1 panel B). It is stable in the absence of pepsin (“Gcon” lanes) but is not vi sible during the di gestion time course. A different immunoreactive band at approximately 36,000 mol ecular weight is observed in the grain extracts, and it is also rapidl y digested by pepsin within 0.5 min (Figure H-1 panel C). These bands may represent proteins that cr oss-react with the AHA S-specific antibody or smaller fragments of the AHAS protein, but in either case, they are rapidly degraded.

Sensitivity of CV127 soybean leaf and grain AtAHAS to degradation in SIF . The full-length soybean leaf AHAS protein is rapi dly digested and is no longer visible within 0.5 min in SIF (Figure H-2 panel B). Si milar to the results observed with the E. coli

produced AtAHAS protein, the leaf AHAS protein is not stable in SIF w ithout pancreatin (“I-con”). Also, a lower molecular wei ght protein of approximately 50,000 molecular weight appears to increase in intensity w ith time of digestion. Furthermore, an immunoreactive band of approximately 16,000 mol ecular weight appears to be associated with the pancreatin since the ba nd was not visible in the absen ce of SIF (“I-con” lanes) or at the zero time point where the SIF was inactivated by heating prio r to addition of the

leaf extract. A smaller immunoreactive band of >6000 molecular weight is also observed and since it is also present in the “SIF only” incubations, it appears to be associated with pancreatin. This band may be a degradati on product of pancreatin. Full-length AHAS protein is just barely detected by western blot anal ysis in the leaf ex tract of conventional soybean and the lower molecular weight protein at approximately 50,000 molecular weight (observed in the leaf extract of CV127 soybean) is a pparently below the detection limit for this method as the 50,000 molecula r weight band was not observed in the conventional soybean leaf extr act subjected to digestion in SIF (Figure H-3 panel B). This result was not unexpected since the AHAS protein expression levels, as reported in da Silva (2007) were appr oximately 600 ng AHAS/g dry we ight in young CV127 leaves as compared to approx. 75 ng AHAS/g dry wt in young leaves of conventional soybean. The lower molecular weight bands at approximately 16,000 and >6000 molecular weights, previously observed in SIF digest ion of leaf extracts of CV127, were again observed in SIF digestion of the leaf extrac t of conventional soyb ean (Figure H-3 panel B), supporting the conclusion that these bands ar e associated with panc reatin and not with the AtAHAS transgenic protein encoded by the introduced imidazolinone-toleranceconferring ahas R272K, S653N gene.

In the grain extracts of CV127, full-length AHAS is rapidly degraded by pancreatin and is not observed at 0.5 min (Figure H-2 panel C). Full-length AHAS protein was not detected in the SIF digest of the conventional soybean grain sample (Figure H-3 panel C). This result was not unexpected since the expr ession levels, as reporte d in da Silva (2007) were below the level of quantification in the conventional soybean grain, and approximately 28 ng/g dry weight in CV127 grain. As observed w ith digestion of the leaf extract of CV127, where a lower molecula r weight (approx. 50,000) protein band wasCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 371 of 375 observed, a lower molecular weight immuno-reactive band at approximately 36,000 molecular weight appears to accumulate with digestion time in SIF digestions of both CV127 and the conventional soybean grain (Figures H-2 panel C and H-3 panel C respectively). Also, the same immunoreactiv e bands associated with pancreatin (at approximately 16,000 and >6000 molecular weights) , which appeared in the digest of the leaf extracts, are also observed in digestion of the grain extracts (F igures H-2 panel C and H-3 panel C). The grain AtAHAS appears more stable in the SIF without pancreatin (“Icon”) and perhaps this is due to the presence of other components in the grain extract that stabilize AHAS (Figure H-2 panel C).

In leaf and grain extracts, protein ba nds corresponding to approximately 50,000 and 36,000 molecular weights, respectively, appeared to increase in intens ity with increasing time of SIF digestion. Because no lower molecular weight protein bands at approximately 50,000 or 36,000 molecular weights were observed to accumulate in SIF digestion of AtAHAS-0107 (Figur e H-3 panel A), it is unlikel y that these protein bands are a degradation product of the full-length AHAS protein produced in leaf or grain tissue, and are more likely immunoreactive peptides generated fr om digestion of a different protein present in the leaf or grain extracts.

CONCLUSIONS

E. coli -produced AtAHAS, and both forms of AHAS produced in CV127 leaves and grain (endogenous soybean AHAS and AtAHAS), are rapidly degraded in simulated mammalian gastric fluid. Full-length AHAS, regardless of source, is also rapidly degraded in simulated mammalian intestin al fluid. Degradation of the AHAS and AtAHAS proteins in plant ex tracts was slightly slower compared to the purified E. coli

produced protein in both SGF and SIF, showi ng that the plant matrix has only a slight effect on protein digestion. Therefore, AtAHAS expressed in CV127 soybean is digested the same as conventional dietary protein, and the same as endogenous soybean AHAS has been digested in conventional plants to date.

Although large quantities of a ra nge of proteins are consumed in human diets each day, rarely do any of these tens of thousands of proteins elicit an allergenic re sponse (Taylor, 1992). There are no predictive bioassays availa ble to assess the alle rgenic potential of proteins in humans; however, physicochemi cal and human exposure profiles of the protein provide a basis for assessing potenti al allergenicity rela tive to known protein allergens. Results of the current study s how that the AtAHAS protein expressed in CV127 soybean is highly digestible under the simulated digestion conditions, which is typical of most proteins exposed to the pr oteases of the mammalia n digestive tract. Furthermore, the transgenic AtAHAS protein expressed in CV127 soybean was immunologically indistinguishable from th e endogenous soybean AHAS protein in the current studies, and results suggest that the di gestive fate of both proteins in SGF and SIF is identical. Therefore, the AtAHAS protein shares the same digestive fate properties with the endogenous soybean AHAS protein, whic h has a history of safe consumption in food and feed products.CONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 372 of 375 GLP COMPLIANCE AND PROTOCOL CHANGES: This study was conducted in compliance with the relevant provisions of Good Laborator y Practice Standards (40 CFR 160, Federal Register , 1989) pursuant to the Federal Insecticide, Fungicide and Rodenticide Act and subsequent revisions with the exception of Phase B, the digestibility of AtAHAS produced in CV127 s oybean. A protocol change was made to add digestion reactions with conventional soybean grain and le af preparations. This was judged to have no adverse impact on the study and allowed better interpre tation of background protein

bands.

Standard Operating Procedures

SOP BPS 510.01 Simulated Mammalian In vitro Digestibility Method

SOP BPS 510.02 SDS-Polyacryl amide Gel Electrophoresis

SOP BPS 510.03 Western Blot AnalysisCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 373 of 375 Figure H-1. Susceptibility of AtAHAS to Dige stion in Simulated Mammalian Gastric Fluid (SGF).

Incubation time at 37oC is i ndicated in minutes. “SGF” is simulated mammalian gastric fluid containing pepsin; “G-con” is SGF w ithout pepsin. “SGF only” indicates no AtAHAS sample was included. Panel A . Coomassie blue stai ned 4-20% polyacrylamide gel containing digestion reactions carried out with test substance AtAHAS-0107. Panel

B . Western blot analysis of digestion reactions carried out with leaf extract from CV127 soybean. Panel C . Western blot analysis of diges tion reactions carrie d out with grain extract from CV127 soybean. AtAHAS is ca. 64,000 mol wt.; pepsin is ca. 40,000 mol. wt. Molecular weight (x 10 -3 ) markers are indicated.

148

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14.4 21.5 31.0 36.5 55.4 66.3 116.3

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## A

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AHAS AHASCONFIDENTIAL BUSINESS INFORMATION DELETED

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 374 of 375 Figure H-2. Susceptibility of AtAHAS to Digestion by Simulated Mammalian Intestinal Fluid (SIF).

Incubation time at 37oC is indica ted in minutes. “SIF” is simulated mammalian intestinal fluid containing pancreatin; “I-con” is SIF wi thout pancreatin. “SIF only” indicates no AtAHAS sample was included. Panel A . Coomassie blue stai ned 4-20% polyacrylamide gel containing digestion reactions carri ed out with the sample AtAHAS-0107. Panel B

. Western blot analysis of dige stion reactions carried out wi th leaf extract from CV127 soybean. Panel C . Western blot analysis of diges tion reactions carrie d out with grain extract from CV127 soybean. AtAHAS is ca. 64,000 mol wt. and molecular weight (x

10 -3 ) markers are indicated.

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+ Ext.

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AtAHAS

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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 375 of 375 Figure H-3. Western Blot Analysis of Simulate d Mammalian Intestinal Fluid (SIF) Reactions for AtAHAS-0107, Conventional Soybean Leaf and Conventional Soybean Grain Protein Extracts.

Incubation time at 37oC is indica ted in minutes. “SIF” is simulated mammalian intestinal fluid containing pancreatin; “I-con” is SIF wi thout pancreatin. “SIF only” indicates no AtAHAS sample was included. Panel A . Western blot anal ysis using a 4-20% polyacrylamide gel containing the same diges tion as in Figure H-2 panel A for AtAHAS0107. Panel B . Western blot analysis of digestion reactions carried out with leaf extract from conventional soybean leaves. Panel C . Western blot analysis of digestion reactions carried out with grain extract from conventional soybeans. AtAHAS is ca. 64,000 mol wt. and molecular weight (x 10 -3 ) markers are indicated.

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## A

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