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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 1 of 375 

BASF Plant Science, L.P.

Petition for the Determination of Nonregulated Status for 

Imidazolinone-tolerant Soybean BPS-CV127-9 

The purpose of this petition is to request a determination that the article should not 

be regulated under 7 CFR Part 340 

Submitting Company

BASF Plant Science, L.P. 

26 Davis Drive 

Research Triangle Park, NC 27709 

Representing 

BASF Agrochemical Products B.V. 

Groningensingel 1, NL-6835 EA Arnhem, The Netherlands 

Submitted by 

James M. Ligon, Ph.D. 

BASF Plant Science, L.P. 

26 Davis Drive 

Research Triangle Park, NC 27709 

Prepared by

James M. Ligon, Ph.D., Andrew Reed, Ph.D., Bruce Luzzi, Ph.D. 

OECD Unique Identifier BPS-CV127-9 

January 13, 2009 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 2 of 375 

Release of Information 

As part of the regulatory requirement in the United States, BASF Plant Science, L.P. is 

submitting the information in this petition for review by the USDA. The submission of this 

information by BASF Plant Science, L.P. does not constitute an authorization to release any 

information contained within it to any third party. In the event the USDA receives a Freedom of 

Information Act request, pursuant to 5 U.S.C., § 552, and 7 CFR Part 1, covering all or some of 

this information, BASF Plant Science, L.P. expects that USDA will provide BASF Plant Science, 

L.P. with a copy of the material proposed to be released and the opportunity to object to the 

release of any information based on appropriate legal grounds, e.g. responsiveness, 

confidentiality, and/or competitive concerns in advance of the release of the document(s). BASF 

Plant Science, L.P. authorizes the posting of the petition on the USDA Biotechnology Regulatory 

Services website when the Plant Pest Risk Assessment and the Environmental Assessment are 

published in the Federal Register for public comment. BASF Plant Science, L.P. understands 

that a copy of this information may be made available to the public by individual request, as part 

of a public comment period. Except in accordance with the foregoing, BASF Plant Science, L.P. 

does not authorize the release, publication or other distribution of this information (including 

website posting) without prior notice and consent by BASF Plant Science, L.P. 

© 2009 BASF Plant Science, L.P. All Rights Reserved. 

This document is protected under copyright law. This document is for use only by the regulatory 

authority to which it has been submitted by BASF Plant Science, L.P. and only in support of 

actions requested by BASF Plant Science, L.P. Any other use or citation of this material, 

without prior written consent of BASF Plant Science, L.P., is strictly prohibited. By submitting 

this document, BASF Plant Science, L.P. does not grant any party or entity any right to license or 

to use the information or intellectual property described in this document.

Clearfield, Cultivance, OnDuty, Aramo, Arsenal, Clarity, Distinct, Fastac, Klap, Nomolt, Opera, 

Pirate, Pursuit, Raptor, Scepter, and Talcord are trademarks or registered trademarks of BASF or 

its affiliates. STS is a trademark of DuPont or its affiliates. Plenish is a trademark of Pioneer 

Hi-Bred. Roundup Ready is a registered trademark of Monsanto Technology L.L.C. Engeo and 

NK are registered trademarks and AAtrex is a trademark of Syngenta Group Company. Hornet, 

Lorsban, Vexter, and Widematch are registered trademarks of Dow AgroSciences LLC. 

Bronate, Connect, Folicur, Sevin, and Tamaron are registered trademarks and Bronate Advanced 

is a trademark of Bayer. Sumithion is a registered trademark of Sumitomo Chemical Co. Impact 

is a registered trademark of Cheminova. Metafos is a registered trademark of Milenia 

Agrociencias. Folisuper is a registered trademark and Rival is a trademark of NuFarm. 
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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, this petition includes all 

information and views on which to base a determination, and that it includes all relevant data and 

information known to the petitioner that are unfavorable to the petition. 

_________________________________ 

James M. Ligon, Ph.D. 

Regulatory Affairs Manager 

Address:

BASF Plant Science, L.P. 

26 Davis Drive 

Research Triangle Park, NC 27709 

Tel: (919)-547-2169 

Fax: (919)-547-2463 

Email: james.ligon@basf.com
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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 Determination 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 TRANSFORMATION 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 Elements of pAC321 Outside of the Transformation Fragment 41

V. GENETIC ANALYSIS OF THE INSERTION IN CV127 SOYBEAN 42

A. Copy Number and Insert Integrity 44

B. Analysis for Plasmid pAC321 Backbone 48

C. Stability of the Insert Across Multiple Generations 48

D. Organization of the Genetic Elements in the Insert of CV127 51

E. A. thaliana SEC61γ Subunit Gene 55

F. Inheritance of the Imidazolinone-Tolerance Trait in CV127 59

G. Conclusions of the Molecular Characterization 60
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Page

VI. CHARACTERIZATION OF PROTEINS EXPRESSED IN CV127 

SOYBEAN 

62

A. Biochemistry of the AtAHAS Protein and the Imidazolinone Herbicide 

Tolerance

63

B. Characterization of the 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 AtSEC61γ 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, Phenotypic and Ecological Evaluations of 

CV127 Soybeans 

133

IX. ENVIRONMENTAL ASSESSMENT OF CV127 SOYBEANS 134

A. Safety of the Imidazolinone-tolerance Trait 134

B. Ecological Characteristics of CV127 Soybean 136

B.1. Weediness Potential of CV127 Soybean 136

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

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 162
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Page

G. Summary of the Agronomic and Environmental Assessments of CV127 

Soybean

163

X. ADVERSE CONSEQUENCES OF INTRODUCTION 165

XI. REFERENCES 166

XII. APPENDICES 184

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

B. Characterization of AtAHAS Protein Produced in Imidazolinone-tolerant 

CV127 Soybean and Comparison with AtAHAS Protein Expressed in 

Recombinant Escherichia coli

195

C. Methods for Determining Levels of the AHAS and SEC61γ 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 Germination, Dormancy and Seed Quality 

Studies of CV127 Soybean

269

F. Materials and Methods and Results from the Agronomic, Phenotypic, and 

Ecological Interaction Evaluations of CV127 Soybean

277

G. Best Management Practice (BMP) Program for the Imidazolinone-tolerant 

CV127 Soybean Production System

347

H. Digestive Fate of the AtAHAS Produced in CV127 Soybean and in the 

Recombinant E. coli Expression System

364
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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 pAC321 and the Linear Map of the DNA 

Fragment Derived from the Plasmid Used for Plant Transformation 

37

IV-2 The Deduced Amino Acid Sequence of the AtAHAS Protein in 

CV127 Soybean Including the Predicted Chloroplast Transit Peptide 

(Underlined) 

40

V-1 Alignment of pAC321 Transformation Fragment with the Transgene 

Insert in CV127 Soybean 

43

V-2 Southern Blot Analysis of Insert 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 Demonstrating Intergenerational Stability of 

the Insert in CV127 Soybean 

50

V-5 Diagram of Overlapping PCR Amplicons 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 DNA from CV127 Soybean Confirming 

the Presence of ORF 501 

54

V-8 RT-PCR Analysis of Transcription of the AtSEC61 γ Subunit Gene in 

CV127 Soybean 

57

V-9 RLM-5’-RACE Secondary PCR Analysis of the AtSEC61 γ Subunit 

Gene in CV127 Soybean 

58

VI-1 SEC61γ Subunit Western Blot Analysis of Leaf and Grain 

Microsomal Membrane Protein Preparations 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 SEC61γ

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 Continental United States and Southern 

Canada where Various Soybean Maturity Groups (MG) Are Adapted 

for Full-Season Growth 

146
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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 Southern Blots of CV127 

Soybean Genomic DNA 

47

V-2 Chi-Square Analysis Demonstrating Single-Gene Mendelian Inheritance 

(1:2:1 Segregation) of the csr1-2 Gene in Two F3 Families, Each 

Derived from an Individual Hemizygous F2 Plant, Based on a 

Quantitative PCR Assay 

60

V-3 Chi-Square Analysis Demonstrating Single-Gene Mendelian Inheritance 

(3:1 Segregation) of the csr1-2 Phenotype in Two F4 Progeny Derived 

from Hemizygous Plants from Two F3 Families Based on a Herbicide 

Tolerance Assay 

60

VI-1 The Calculated Limit of Detection (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 Locations 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 115

 
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Table Page

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 Evaluated 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 Location 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 Location 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 through 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 Monthly 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 Soybean in the U.S. and South America 

(from Tolin and Lacy, 2004) 

154

IX-7 Causal Organisms of Commonly-Occurring 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) 

155
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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 Launch and the CFIA Decision Document 

Number for Imidazolinone-tolerant Clearfield® Crops 

161
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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

AtSEC61γ 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 

GmSEC61γ SEC 61 gamma subunit protein from Glycine max

ha Hectare 

HC Health Canada 

ILSI International Life Sciences Institute 

kb Kilobase pairs 

LD50 Dose that is lethal to 50% of treated animals 

LOD Limit of detection 

M Molar 

MAFF Ministry of Agriculture, Forestry and Fisheries of Japan 

MHLW Ministry of Health, Labour 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 Development 
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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 relative to the amino 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 

T0 generation resulting from regenerated transformed cells 

T4 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 volume 
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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 conventional breeding practices with other soybeans, 

including conventional varieties and genetically modified varieties 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 Plant 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 petition) are derived from a single transformation 

event and were produced by introduction of the imidazolinone-tolerant acetohydroxyacid 

synthase large subunit (AHAS) gene csr1-21 with its native promoter from Arabidopsis thaliana

into the soybean plant genome via biolistics transformation technology. The csr1-2 gene from A. 

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

to imidazolinone herbicides due to a point mutation that results in a single amino acid 

substitution in which the serine residue at position 653 is replaced by asparagine (S653N). The 

AtAHAS catalytic subunit encoded by the csr1-2 gene has altered herbicide binding properties 

such that imidazolinone herbicides 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, and isoleucine. Typically, inhibition of the AHAS 

enzyme by imidazolinone herbicides leads to a deficiency 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 allow growers to treat the 

soybean crop with imidazolinone herbicides for weed control without causing injury to the 

soybean plant at normal field application rates. Soybean CV127 was developed for cultivation 

primarily in Brazil and Argentina, and the introduction 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 herbicides 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 prevalent weeds 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 and the product concept of an imidazolinone-tolerant soybean fits well 

with the commercial needs for weed control in South 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 amino acid numbering of plant AHAS enzymes are cross-referenced to the corresponding 

amino acid numbers of the AHAS from Arabidopsis, including the residues comprising the chloroplast transit peptide. 
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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 benefit from reduced weed control costs. During the 

12-year period from 1996 through 2008, biotechnology-derived herbicide-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 herbicide use is also expected to benefit the 

environment. 

Several AHAS genes encoding AHAS enzymes that are tolerant to imidazolinone herbicides have 

been discovered in plants as naturally occurring 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 mutagenesis, selection, and conventional 

breeding technologies and have been commercialized under the Clearfield® brand name since 

1992, 2003, 2002, and 1996, respectively. Therefore, there 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 safety of CV127 soybeans was confirmed based on 

results of a series of inter-related safety assessment studies. The detailed molecular 

characterization of CV127 soybean confirmed that CV127 contains a single functional csr1-2

gene cassette integrated in the soybean genome. Biochemical characterization of the 

imidazolinone-tolerant AtAHAS protein expressed in CV127 soybean showed that the AtAHAS 

protein is typical of other AHAS proteins 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 potential environmental impact 

than cultivation of conventional soybeans. Finally, the composition and nutritional equivalence 

of CV127 soybean compared to conventional soybeans was demonstrated by analysis of key 

nutrients and antinutrients in both grain and forage, and nutritional equivalence 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 release as well as 

for food and feed uses. 

Molecular characterization of CV127 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 demonstrated 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 genome of CV127 soybeans. In the insert, there is also a 376 basepair (bp) duplication of a portion of the csr1-2 coding sequence directly before the 3’ integration 

point. This duplicated 376 bp segment creates a 501 bp open reading frame (ORF) that extends 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 15 of 375 

into the 3’ flanking sequence. Reverse transcription-polymerase chain reaction (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 region 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 transformation. This protein is part 

of a multi-subunit secretory complex that is ubiquitous in all eukaryotes. The AtSEC61γ 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 AtSEC61γ 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 transgene 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-tolerance trait over multiple breeding generations 

of CV127 soybean. Results of this study demonstrated that the trait is stably inherited according 

to classical Mendelian genetics, and results were consistent with the presence of a single 

dominant imidazolinone-tolerance gene in the soybean genome. 

The environmental safety of CV127 was shown to be comparable to an isoline control and other 

conventional soybean varieties through evaluation of phenotypic, agronomic and ecological 

interaction characteristics of CV127 and control comparator plants. Field trials with CV127 

soybeans were conducted at seven different locations in Brazil during 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 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 trial sites were representative of the different regions within Brazil 

where soybean is commercially cultivated 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 developmental stages, plant height, disease 

and insect susceptibility, and grain yield were evaluated. In addition, the impact of cultivating 

CV127 soybeans on factors relevant to the nitrogen fixation symbiosis with Bradyrhizobium was 

assessed and compared between CV127, the isoline 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 these comparative studies demonstrated that other 

than tolerance to imidazolinone herbicides, there are no biologically meaningful differences 

between CV127 and the isoline control and/or the other conventional soybean varieties with 

respect to the parameters measured as described above. 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 conventional 

soybean varieties. 
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The environmental as well as food and feed safety of the AtAHAS protein expressed in CV127 

soybeans was demonstrated in studies that confirmed that the AtAHAS protein 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 soybean varieties at different 

growth stages at two field locations in the Brazilian field trials during two growing seasons. In 

addition, leaf samples and grain samples were collected from all trial locations from young 

plants at the vegetative growth stage V2 and at harvest, respectively. These samples were 

analyzed by enzyme-linked immunosorbent assay (ELISA) to determine the quantity of AHAS 

enzyme in the tissues. As expected, the levels 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 endogenous soybean AHAS proteins. Generally, the 

level of AHAS was higher in young, actively-growing 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 tissues of CV127 are relatively 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 plant species, including crop plants with a history of safe use. 

The AtAHAS protein was readily degraded in simulated 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 showed no acute toxicity when administered to 

mice. Results of these studies show that the AtAHAS protein expressed in CV127 soybean 

tissues does not possess any attributes of known protein food allergens, 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 control as well as other conventional 

soybeans. Samples of grain or forage were harvested from CV127, the isoline control, and two 

conventional varieties from multi-location replicated field trials conducted in Brazil. Grain 

samples were collected from trials conducted in two separate growing seasons and analyzed for a 

comprehensive range of important nutrients and antinutrients of soybean. Forage samples were 

collected from trials in one growing season and were analyzed for proximates and fiber content. 

Statistical analysis of composition data from grain and forage samples demonstrated that the 

composition of grain and forage from CV127 soybeans is comparable to that of the isoline 

control and conventional soybean varieties. Furthermore, the nutritional equivalence of CV127 

soybean to the isoline control and conventional soybean varieties was confirmed in a poultry 

feeding study. A 42-day feeding 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 containing soybean meal from CV127 soybean was comparable to 

those fed a feed containing soybean meal from the grain of the isoline control and two 

conventional soybean varieties. Collectively, these results demonstrate that the grain and forage 
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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. 

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 conducted. 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 these studies demonstrated that the agronomic and environmental 

qualities of CV127 soybeans are comparable to those of conventional soybeans that have a long 

history of safe cultivation and consumption. 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 studies demonstrate that, other than tolerance to 

imidazolinone herbicides, CV127 soybeans possess the same agricultural and nutritional 

characteristics as conventional soybeans that have a long history of cultivation 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 during the growing season, including average 

temperatures and rainfall, are comparable between U.S. and Brazilian soybean production. 

Details of these similarities are presented in section IX-C.4 of this petition. Therefore, in the 

event that CV127 soybean were to be introduced into the U.S. environment, the data generated 

from field studies 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 

assessment of CV127 in the U.S. environment. 
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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 (APHIS) of the U.S. Department of Agriculture 

(USDA) is responsible under the Plant Protection 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 evaluate submitted data to determine that a 

particular regulated article does not present a plant pest risk and 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 referred 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 soybean varieties be considered nonregulated 

articles under 7 CFR 340. 

B. Rationale for the Development of Imidazolinone-tolerant Soybean CV127 

Soybean is the leading oilseed crop produced and 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 the world’s total oilseed production (Soy Stats, 2011). Soybean 

has been the dominant oilseed crop produced in the world since the 1960s (Smith and 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 (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 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 are 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 recommended weed control and other crop management 

practices and by using soybean varieties that have enhanced yield potential. 

For many years, genetically modified (GM) soybean varieties have constituted the largest 

percentage of genetically modified 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, virtually the entire area 

planted to soybeans in 2010, equal to 19.5 million hectares, was planted with herbicide-tolerant 
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GM soybeans (James, 2010). Cultivation of herbicide-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 herbicide-tolerant soybeans by growers worldwide 

has been due to increased yields, the reduced cost of effective weed control, 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 today are tolerant to the herbicide glyphosate. 

Glyphosate-tolerant soybeans were first grown commercially 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 glyphosate (Heap, 2011; Nandula et al., 

2005). It should be noted that most of the cases of glyphosate-resistant weeds occurred in nontransgenic crops. Exposure to glyphosate started well before the introduction of glyphosatetolerant crops and thus attributing 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 been developed by conventional 

mutagenesis procedures (Sebastian et al., 1989). The STSTM, or sulfonylurea-tolerant soybean, 

technology does not provide cross-tolerance to imidazolinone herbicides and it is currently 

marketed in the United States as a stacked trait together with glyphosate tolerance by NK®, 

among others. Use of STSTM soybeans allows effective weed control by sulfonylurea herbicides 

in soybean production areas where glyphosate-tolerant weeds are present. 

The introduction of imidazolinone herbicide-tolerant soybeans will provide the grower with the 

opportunity to cultivate soybeans that are tolerant to a different herbicide with a different mode 

of action than 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 classes in combination 

with the application of the corresponding herbicides is an effective strategy for managing the 

development of herbicide tolerance in weed populations. 

Although soybeans are naturally tolerant 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 readily 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 herbicides containing these active ingredients 

for effective weed control. Imidazolinone herbicide tolerance has been developed in several 

different crops by either conventional seed mutagenesis procedures or by natural mutation. For 

example, imidazolinone-tolerant bread wheat, canola, rice, lentils, and sunflower are marketed 

by BASF under the Clearfield® brand name. However, multiple attempts by BASF to introduce 

commercial levels of tolerance to postemergence applications of imidazolinone herbicides 

containing imazapic by seed mutagenesis methods were unsuccessful in soybeans (unpublished 

data). Therefore, BASF used plant genetic engineering technology to introduce the 

imidazolinone herbicide-tolerant gene csr1-2 from Arabidopsis thaliana into soybeans to 
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develop the imidazolinone-tolerance trait. BASF is developing the imidazolinone herbicidetolerant soybean CV127 for cultivation primarily 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 attractive proposition for soybean 

cultivation in South America. Therefore, regulatory approvals for this product are sought in 

Brazil and Argentina for production as well as for food and feed uses, 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 isoline control and two conventional soybean 

varieties with respect to phenotypic and agronomic characteristics as well as specific ecological 

parameters in multi-location field trials in Brazil 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 environmental impact than conventional soybean 

varieties. 

The introduction of CV127 soybeans in Argentina and Brazil will offer growers excellent weed 

control options which in turn is expected to provide 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 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 Argentina 

and Brazil has led to a shift in the species of prevalent weeds 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. Imidazolinone herbicides 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 and Senseman, 1998). For example, in Brazil, the 

recommended application rate and frequency that are proposed for imidazolinone herbicides 

used with CV127 soybean is one application per 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 tissue (Shaner and Singh, 

1998); they are among the most potent herbicides on the market, with effective weed control at 

application rates of 32 to 125 g ai/ha (Shaner and Singh, 1998). As a reference, the application 

rate of other conventional herbicides 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 imidazolinones 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 toxicity. Subchronic and 

chronic toxicity tests with rats showed no adverse effects 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 (LD50>2,150 mg/kg), fish (LD50>100 mg/liter), or honey bees (LD50>100 

μg/bee) (Gagne et al., 1991). Imidazolinone herbicides are readily degraded in the soil to non-
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toxic, naturally occurring compounds by the activity of soil microbes (Basham and Lavy, 1987; 

Flint and Witt, 1997). Furthermore, due to the relatively mild climate during the winters in 

Brazil and Argentina, there is increased microbial activity in the soils that leads to a shorter halflife and lower carry-over activity from the application of imidazolinones (Ulbrich et al., 2005). Due to the characteristics of the imidazolinone herbicides described above, it is anticipated that 

the use of imidazolinone herbicides with imidazolinone-tolerant CV127 soybeans will provide a 

safe and environmentally beneficial system of weed control for soybean growers. 

Grower adoption of imidazolinone-tolerant CV127 soybeans is expected to have other benefits to 

the environment. The adoption of herbicide-tolerant soybeans has resulted in a significant 

reduction in the adverse environmental impacts of intensive agricultural practices (Fawcett and 

Towery, 2002; Brookes and Barfoot, 2008; Brookes 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 weeds 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 soil 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 their soybean fields since the adoption of herbicide-tolerant 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 reduced tillage costs (Duke and Cerdeira, 2005). 

In Argentina there has also been a documented increase in the use of no-tillage agriculture in 

soybeans concomitant with the cultivation of herbicide-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 fields cultivated in herbicide-tolerant soybean 

varieties using reduced tillage practices (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 practices by soybean growers. 

The use of herbicide tolerant crops, including 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 reduced 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 herbicide-tolerant soybeans 

alone accounted for a 50.45 million kg decrease in the amount of active ingredient applied over 

this same time period (Brookes and Barfoot, 2010). 
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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, respectively, relative to conventional 

practices (Brookes and Barfoot, 2010). Carbon dioxide 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 release into the atmosphere 

(Holland, 2004). Brookes and Barfoot (2010) estimated that the increase in soil carbon 

sequestration in the United States due to reduced tillage soybean production prevented the 

atmospheric release of a maximum of 38,057 million 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 increased soil carbon storage was approximately 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 herbicide-tolerant soybeans is expected to promote 

reductions in fuel use and tillage with concomitant 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 statement addressing the regulation of products derived from new 

plant varieties, including those developed through biotechnology (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. Also, an import tolerance petition and supporting 

residue data were submitted to the U.S. Environmental Protection Agency (EPA) on September 

29, 2010 for the use of imazapyr and imazapic on imported CV127 soybeans (Federal Register 

volume 76, number 24; Federal 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 cultivation of CV127 is anticipated. Regulatory 

approval for CV127 was obtained in Brazil in December 2009. Regulatory submissions for the 

importation of CV127 soybeans and processed soybean products for food and feed uses have 

been or will be made in countries that import 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 number of governmental regulatory 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 Australia New Zealand (FSANZ), and 

the Taiwanese Department of Health (DOH). Submissions have also been completed to the 

Rural Development Administration (RDA) of the Republic of Korea, for determination of 

environmental and feed safety of CV127 soybean, and to the other Korean Regulatory 

Authorities including the Korea Food and Drug Administration (KFDA), the National Institute of 

Environmental Research (NIER), the National Fisheries Research and Development Institute 

(NFRDI), and the Korean Center for Disease Control and Prevention (KCDC). The Bureau of 

Plant Industry, Republic of the Philippines, granted regulatory approval for food, feed, and 
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processing uses of CV127 soybeans in October 2010. The Comisión Federal para la Protección 

contra Riesgos Sanitarios (COFEPRIS), Mexico, granted approval for CV127 import for food 

and feed uses as well as processing in May 2011. 
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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 crops (Wilcox, 2004; Soy Stats, 2011). 

In 2011, 258.4 million metric tons (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 and Huyser, 1987) and in the past twenty 

years soybean production has increased by 116 MMT (FAOSTAT, 2011). Soybean is grown as 

a commercial crop in over 35 different countries (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 soybeans, respectively, in 2010 (Soy Stats, 

2011). The fourth and fifth largest soybean producers 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 vegetable oils consumed in the world today with 2010 

production of 48.7 and 42.1 MMT, respectively, representing 33 and 29 percent of the world’s 

vegetable oil production (Soy Stats, 2011). Soybeans also dominate the 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 include 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, including trypsin inhibitors and lectins. However, 

heat treatment inactivates the anti-nutrient factors and allows the use of soybeans to produce soy 

sprouts, roasted soybeans and the traditional soy foods, for example miso, soy milk, soy sauce, 

and tofu (OECD, 2001). It is estimated that soybean oil accounts for 94% of soybean ingredients 

in the human diet (OECD, 2001). Examples of the use of soybean oil in food products include 

purified oil for use in margarines, shortenings, and cooking and salad oils (USDA-ERS, 2006a). 

Soybean oil is the second largest source of vegetable 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 valuable 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, including poultry, swine, and dairy and beef cattle, and is also used in 
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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 lysine, 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 Cahoon (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 China (Hymowitz, 1970; OECD, 2000). Historic and 

archeological evidence suggests that the soybean [Glycine max (L.) Merr.] first emerged as a 

domesticate during the Zhou dynasty in northeastern China (Hymowitz, 2004). Soybean 

domestication most likely occurred during the Shang dynasty (ca. 1500-1100 B.C.) or earlier. It 

is thought that by the first century A.D., the soybean 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 consolidation of territories and the collapse of the 

Chinese dynasties (Ho, 1969; Hymowitz, 1970). 

Due to the development of land races, the cultivation of soybeans spread to other regions in East 

Asia, including Japan, Indonesia, the Philippines, Vietnam, Thailand, Malaysia, Burma, Nepal, 

and north India from the first century A.D. to the 15th to 16th 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 16th century and during 

the 17th 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 employed 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 Savannah, 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. into 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 20th century, 

soybeans were grown at 30°S in Rio Grande do Sul, the most southerly state in Brazil, with 

climatic conditions similar to those in the southern U.S. Commercial 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 similar 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 26 of 375 

farm machinery and infrastructure, this policy also led to increased cultivation 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 Pantanal regions due to environmental protection 

restrictions and in the eastern areas due to the mountainous terrain (Guidelines for GAP, 2002). 

The primary centers of soybean cultivation in Brazil range from southern Brazil to the savannas 

(Cerrado) of central Brazil. Soybeans are typically planted from mid-October to mid-December 

with harvest from mid-March to late April, depending on latitude. Today 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 belongs to the family Fabaceae 

and is further classified taxonomically as follows: 

Kingdom Plantae -- Plants 

 Subkingdom Tracheobionta -- vascular plants 

 Division Magnoliophyta -- angiosperms, 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, China, 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 annual relatives from Asia, G. soja Sieb. 

and Zucc. are classified in the subgenus Soja. Glycine soja is an annual that grows in the wild in 

fields, hedgerows, roadsides, and riverbanks in many countries of East Asia (OECD, 2000). This 

plant has a slender build with narrow trifoliate leaves. The flowers are purple, or on rare 

instances white, and are found on short, slender racemes. The pods are short and tawny with 

hirsute pubescence and contain oval-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 only 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 species of the subgenus Soja are capable of 

cross-pollination and the hybrid seed that is produced can germinate normally and produce plants 
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with fertile pollen and seed (Singh and Hymowitz, 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 containing 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 ancestor 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 species 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 annual (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). 
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Table II-1. List of Species in the Genus Glycine Willd.

Includes 2n Chromosome Number, Genome Symbol, and Distribution (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 carry 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). 
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D. Morphology of Cultivated Soybean 

The following section dealing with the morphology 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 trifoliate and alternate, and compound 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 purple, 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 halves of a single carpel 

which are joined by a dorsal and ventral suture (OECD, 2000). The shape of the seed, usually 

oval, can vary amongst cultivars from almost spherical 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 indeterminate (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 activity 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 flowering period (OECD, 2000). 

E. Reproductive Biology and Hybridization with Cultivated Soybean 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 after flowering (OECD, 2000). The anthers, 

following maturation in the bud, directly pollinate the stigma within the same flower (OECD, 

2000). Pollination typically occurs 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 level of cross-pollination (below one percent) and is primarily selfpollinating (Caviness, 1966; Yoshimura et al., 2006). 
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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; Beard 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 close proximity to each other (15 cm) were found 

to have average outcrossing rates of 1.8%, while plants separated 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 are consistent with the low outcrossing rate 

recognized for soybean. These regulations place no restriction on the separation distance 

between different cultivars in the field provided that the distance is sufficient to prevent 

mechanical mixing during harvest. 

Due to the low level of genomic similarity 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 for 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 (OECD, 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 growing season to the next is very 

low. Soybean seeds normally germinate quickly under the appropriate environmental conditions 

that include adequate moisture and moderate temperatures and thus, could potentially grow as a 

volunteer. However, any volunteers that grow after harvest would be destroyed by the low and 

freezing temperatures encountered during the following winter. In the event that volunteers were 

to become established, they would not compete well with succeeding crops and they could be 

controlled by either mechanical or chemical means (OECD, 2000). 
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G. Characteristics of the Recipient Soybean Cultivar 

The soybean variety that was used as a recipient in transformation experiments to create the 

imidazolinone-tolerant CV127 soybean was the commercial variety ‘Conquista’. Conquista 

(also known as MG BR 46) is a highly productive, conventional Brazilian soybean variety in 

maturity group VIII. It is well adapted to cultivation in regions of less than 25 degrees latitude 

and was developed by Empresa Brasileira de Pesquisa Agropecuaria (EMBRAPA) with release 

for commercial cultivation in Brazil in 1995 (Pardey et al., 2004). A partial pedigree of 

Conquista (Figure II-1) shows that a significant portion of its genetic background is derived from 

varieties developed in the U.S. From the 1950s to the 1970s, a substantial amount of breeding 

was conducted by USDA researchers using soybean varieties 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 experiments conducted to support the safety assessments of CV127 presented 

in this petition, appropriate control soybean varieties were used as comparators. In field and 

laboratory studies, near-isogenic null segregants or, in some instances, the parental control 

variety Conquista were used as comparator 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 those studies that used the AtAHAS protein expressed in CV127 

soybean, this protein was derived either from CV127 soybean tissues or from protein produced 

and purified from an E. coli fermentation process. In the field studies, two other conventional 

varieties of soybean that are also typical of commercial varieties cultivated in Brazil were also 

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

cultivated in Brazil. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 32 of 375 

Figure II-1. Partial Pedigree for Soybean Variety Conquista (largely derived from Pardey 

et al., 2004). Where available, information on the name of the breeding 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

Philippines
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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 commercial 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 regeneration. 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 documented 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 were 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 continue their flight and eventual 

penetration and incorporation into the soybean plant 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 only those cells transformed with the csr1-2 gene 

continued to grow. From this process a tolerant T0 generation plant was identified and named 

Soybean Event 127 (CV127). 

The T0 generation plants resulting from the transformation of Conquista were advanced to the 

fourth transgenic generation (T4) via four cycles of self-pollination (Figure III-1). Some T4

plants were then backcrossed to Conquista to reduce the number of copies of the transgenic 

insert to a single locus (Figure V-4). The resulting progeny, the first filial (F1) generation, were 

advanced to the eighth filial (F8) generation by successive rounds of self-pollination and 

selection. The homozygous F8 line developed from this process 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, Conquista3

 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 parental line in the Conquista3

 x BRI98-641 pedigree, Conquista, 

is the commercial variety used in the original transformation to develop Line 603. As indicated 

by the superscript (3) in the pedigree, breeding line Conquista3

 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 tolerance trait from BRI98-641 into the high-performing Conquista variety. 

Thus, the genetic background of breeding line Conquista3

 x BRI98-641 is approximately 94% 

Conquista. The cross of Line 603 with this breeding line, Conquista3 x BRI98-641, produced a 

line identified as CV127 line 127 (Line 127) whose genetic background is approximately 97% 

Conquista (Figure III-1). 

A schematic diagram depicting the development of CV127 soybeans is shown in Figure III-2. 
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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 T0 generation and the fourth selfpollinated generation as T4. Key molecular and inheritance data was collected at the points in 

the history as indicated in the legend. Two elite lines are shown. CV127 line 603 was the 

product of a cross between the T4 generation and the conventional soybean variety Conquista. 

CV127 line 127 is the product of a cross between CV127 line 603 and a backcross-derived 

conventional soybean line known as ‘Conquista3

 x BRI98-641’. 

x

Conquista

x

(Conquista3 x

BRI98-641)

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

Conquista

x

(Conquista3 x

BRI98-641)

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 603
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Figure III-2. Schematic Diagram Depicting the 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 T4 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 T4

generation to Conquista followed by selfing to the F8 generation and crossing to a second 

conventional soybean variety (Conquista3

xBRI98-641) followed by selfing to the F7

generation. 

Laboratory and field studies were conducted to assess the food, feed and environmental 

safety of CV127 imidazolinone-tolerant soybeans. 
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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 plasmid 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 restriction endonuclease PvuII and was 

purified prior to use in transformation. 

Plasmid pAC321 consists primarily of the E. coli cloning plasmid pBluescript 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 its 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 Sections 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(-). 
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Figure IV-1. Circular Map of Plasmid pAC321 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 the PvuII fragment of pAC321 

containing the AtAHAS promoter and 5’ UTR, csr1-2 coding sequence and AtAHAS 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 stability 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

NcoI (2483)

PvuII (5916)

PvuII (8429) Xba I (2)

XbaI (5713)

SpeI (5342)

SpeI (5622)

SpeI (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: AtAHAS

Southern probe: 3' UTR

AtAHAS 5'UTR & putative promoter

AtAHAS 3' UTR

At3g48570 3' UTR

At3g48570 5' UTR 

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

Xba I (5713)

Spe I (5342)

Spe I (5622) Spe I (5719)

II (5916)

Arabidopsis gDNA, unannotated
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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 region1

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 origin 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 βgalactosidase (lacZ’). 

lacZ’ CDS, 

interrupted 

8590-8669, 

5718-5994 

E. coli β-galactosidase alpha fragment coding sequence, interrupted by 

Arabidopsis genomic DNA in pAC321; allows blue-white screening 

for 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(-) sequence in pAC321 differs from accession number X52324 at nucleotide 7751 of pAC321. 

Nucleotide 7751 of pAC321 is T (in agreement with the pUC19 origin of replication 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). 
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B. The csr1-2 Gene Coding Sequence and the AtAHAS Protein 

The mechanism of action of imidazolinone herbicides on weeds and non-tolerant plants is by 

inhibition of the AHAS enzyme and the resulting inhibition of branched-chain amino acid 

biosynthesis. AHAS catalyses the first common step in branched-chain amino acid biosynthesis 

that is specific to plants and microorganisms (Stidham and Singh, 1991). The enzyme catalyzes 

the condensation of two molecules of pyruvate to form acetolactate, the precursor of valine and 

leucine, or the condensation 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 biosynthetic pathway and is regulated by feedback inhibition by the end-product amino acids, 

valine, leucine and isoleucine. Imidazolinone herbicides readily inhibit the activity of AHAS by 

binding to the active site of the protein resulting in plant death (Duggleby and Pang, 2000; Tan et 

al., 2005). Studies have shown that specific 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 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 (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 herbicide 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 (S653N). This amino acid change in plant AHAS 

proteins is known to prevent the binding of imidazolinone herbicides 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 AHAS enzyme or its herbicide tolerance 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 this study it was demonstrated that both 

AtAHAS enzymes had equivalent levels of catalytic activity and tolerance to imidazolinone 

herbicides. Feedback regulation of AHAS activity 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 with the endogenous soybean 

AHAS small subunit protein to achieve 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, predicted to consist of 85 amino acids (Mazur et al., 1987). 

During transport into the chloroplast, it is predicted that the chloroplast transit peptide is 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 40 of 375 

removed to produce the mature AtAHAS enzyme that is approximately 64 kD 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 acid full-length protein containing the chloroplast 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 Sequence of the AtAHAS Protein in CV127 

Soybean Including the Predicted Chloroplast Transit Peptide (Underlined). 

The lysine (R272K) and asparagine 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 PTKPETFISR 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 MKLPGYMSRM 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 IPNGGTFNDV 

 670 

ITEGDGRIKY 
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C. The Arabidopsis thaliana SEC61 Gamma Subunit Protein 

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

csr1-2 coding region that was originally annotated as the AHAS promoter and 5’ untranslated 

region (UTR). More-recent sequence analysis has revealed that this segment also contains a 

previously unannotated Arabidopsis gene encoding the gamma subunit of SEC61 (AtSEC61γ), a 

multimeric transport protein of the endoplasmic reticulum that is ubiquitous in all plants and 

other eukaryotes (Hartmann et al., 1994). The CV127 insert contains the majority of the 

AtSEC61γ subunit gene including the complete coding sequence. The AtSEC61γ 5’ UTR, as 

annotated by The Arabidopsis Information Resource, 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 AtSEC61γ 

gene. The possible production of the Arabidopsis AtSEC61γ subunit protein in CV127 was 

evaluated and results showed that no AtSEC61γ subunit protein was detected in tissues of 

CV127 soybean above the levels of detection of the assay. (No AtSEC61γ 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 regulation of the csr1-2 gene in CV127 is under the 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 AtSEC61γ gene. Termination of transcription of the csr1-2 gene is also effected by the 

native A. thaliana csr1-2 gene transcription termination sequences 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(-) (Stratagene, La Jolla, CA). This plasmid contains the ColE1 

origin of replication for maintenance 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 encodes 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 βgalactosidase enzyme. The lacZ alpha fragment gene in plasmid 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 PvuII fragment containing the csr1-2 gene 

cassette was used in the transformation, very few 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). 
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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 region 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 detected 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 

AtAHAS 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 R272 to K272 does not affect the desired herbicide tolerance phenotype conferred 

by the csr1-2 gene. Southern blot analysis and sequence verification of the point mutation 

indicate that the insert is stably integrated in the soybean genome across the nine breeding 

generations studied. Also, trait inheritance studies across breeding generations confirmed the 

expected trait segregation ratios, further demonstrating that imidazolinone herbicide 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 base-pair (bp) duplication of a portion of the csr1-2

coding sequence directly before the 3’ integration point of the insert. This duplicated 376 bp 

segment creates a 501 bp open reading frame (ORF) that 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 from BPS-CV127-9 soybean showed the absence of 

a detectable ORF 501-specific PCR product, suggesting that this 501 bp ORF is not expressed. 

The insert also contains the majority of the Arabidopsis SEC61γ subunit gene locus (refer to 

Table IV-1 for genetic element At3g48570), which is a component of the DNA fragment used for 

transformation. This gene is discussed in more detail in section V.E. A more detailed 

description of these analyses and the results follows. 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 integrated into plasmid pAC321 are listed in Table IV-1 beginning 

with nucleotide 1 and continuing to nucleotide 5717. The hybridization probes used in the 

Southern blot analyses and the 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 that are present in the soybean CV127 insert in 

Figure V-1. 
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Figure V-1. Alignment of pAC321 Transformation Fragment with the Transgene Insert in CV127 Soybea1 The PvuII fragment from plasmid pAC321 that was used to transform soybean is shown in the upper portion of the figure. Parts of 

contained within the transgene insert in CV127 soybean are indicated by boxes filled with diagonal stripes. Characteristics of thflanking genomic soybean DNA in CV127 soybean are shown in the lower portion of the figure. The DNA between the vertical dothe maps of the transformation fragment and the transgene insertion region) is common to DNA from both sources. Restriction sitblot analysis are indicated. The numbering system of the PvuII transformation fragment corresponds to that of the pAC321 plasmid numbering system for the CV127 soybean DNA insert region begins with the first nucleotide at the 5’ end of the soybean genomic flsequences indicated by gray boxes). 

pAC321 PvuII fragment used for transformation

csr1-2 CDS

At3g48570 CDS Arabidopsis gDNA, unannotated

Arabidopsis gDNA, unannotated

AtAHAS 5'UTR & putative promoter

AtAHAS 3' UTR

PvuII NcoI (2483) PvuII (5916)

(8429)

XbaI (2) XbaI (5713)

SpeI (5622)

SpeI (5719)

SpeI

(1268)

SpeI(5342)

csr1-2 CDS

At3g48570 CDS

Arabidopsis gDNA, unannotated

Arabidopsis gDNA, unannotated

376 bp from csr1-2 CDS

5' flanking sequence 3' flanking sequence

AtAHAS 5'UTR & putative promoter

AtAHAS 3' UTR

XbaI NcoI (2761)

 (410)

XbaI 

SpeI (1268) SpeI (5620) SpeI (6505)

CV127 Soybean
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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 F8 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 AtAHAS 5’ UTR, the csr1-2 coding sequence for the AtAHAS protein, and 

the AtAHAS 3’ UTR) that spanned the entire DNA fragment 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 equivalents of pAC321 

plasmid DNA was included in the Southern blots as controls. 

 

Non-transgenic Conquista DNA digested with all three 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 SEC61γ subunit genes are detected at the Southern 

blot stringency conditions used (Figure V-2, lanes 1, 5 and 9). The pAC321 DNA, spiked at 

one- and two-genome copy equivalents into non-transgenic 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 employed was sensitive enough to detect a singlecopy insert in the transgenic event. DNA samples from the CV127 soybean F8 generation treated 

with the different restriction enzyme and probe combinations all gave single hybridizing bands 

(Figure V-2, lanes 4, 8 and 12) except for 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 fragment 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.) thereby 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 equivalent control. The quantification of plasmid 

DNA versus genomic DNA coupled with the large 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 not 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 digested with NcoI and probed with AtAHAS 5’ 

UTR produced a hybridizing band approximately 4.5 kb in size (Figure V-2). The size of this 

band is consistent with the production of a single DNA fragment defined by the NcoI site within 

the insert (nt 2761, Figure V-2 panel D) and an NcoI site approximately 4.5 kb upstream in the 5’ 

genomic soybean flanking DNA sequence. The same digest probed with either the csr1-2 coding 

sequence or AtAHAS 3’UTR produced a hybridizing band approximately 9.0 kb in size. The size 

of this band is consistent with a single DNA fragment defined by the NcoI site in the insert (nt 

2761, Figure V-2 panel D) and an NcoI site approximately 9.0 kb downstream in the 3’ soybean 

genomic flanking DNA sequence. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 45 of 375 

Digestion of CV127 genomic DNA with SpeI and probed with AtAHAS 5’ UTR produced a 

hybridizing band of approximate size 4.4 kb. This is consistent with the production of a single 

DNA fragment from a SpeI site in the insert (nt 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 AtAHAS 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’ flanking DNA sequence junction. 

This hybridizing fragment was produced from the SpeI site in the DNA insert and a SpeI site 885 

bp downstream in the soybean genome (nt 5620 – 6505, Figure V-2, panel D). The 885 bp 

hybridizing band was not detected by the AtAHAS 3’ UTR probe, indicating that AtAHAS 3’ 

UTR DNA was not included in the 885 bp fragment, and 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 linear 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 analysis 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 digested with XbaI and probed with AtAHAS 5’ UTR 

shows a single hybridizing band of approximate size 10 kb. Based on the positions of the XbaI 

restriction sites in the linear DNA used for transformation (Figure V-1), a hybridizing band of 

approximately 5.7 kb was expected to be produced from within the DNA insert in the CV127 

soybean genome. However, DNA sequence analysis 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 was produced from XbaI restriction sites within 

the 5’ and 3’ DNA sequences flanking the insert (nt 410 and 10652, Figure V-2, panel D). 

Accordingly, the same digest probed with either the csr1-2 coding sequence or AtAHAS 3’ UTR 

produced the same 10 kb hybridizing 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 coding sequence for the 

AtSEC61γ subunit protein on the 5’ end of the csr1-2 gene, and a single DNA fragment 

containing a 376 bp duplicated segment of the csr1-2 gene at the 3’ end of the transgene insert. 
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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 F8

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

NcoI SpeI XbaI

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

NcoI SpeI XbaI

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

NcoI SpeI XbaI

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

Xba I (410) Nco I (2761) Xba I (10652)

Spe I (1268) Spe I (5620) Spe I (6505)

Regions of homology 

to hybridization probes
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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 5711b

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 5711b

2958 

~5500 

~3000 

NcoI none none 8669 ~9000 

V-3A VP1 SpeI none none 8292 ~8500 

XbaI none none 2958 ~3000 

NcoI none none 8669 ~9000 

V-3B VP2 SpeI none none 8292 ~8500 

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 therefore 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’ transgene integration site, confirming the identity of the 885 bp band observed in 

these Southern blots. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 48 of 375 

B. Analysis for Plasmid pAC321 Backbone 

Although the transformation was carried out with the PvuII restriction 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 above (Figure V-2) was hybridized with two vector 

backbone-specific probes designated VP-1 and VP-2 (Figure V-3, panel C). As expected, no 

hybridizing bands were detected in lanes containing non-transgenic Conquista genomic DNA. 

Non-transgenic Conquista genomic DNA spiked with one- or two-genome copy equivalents of 

transformation plasmid pAC321 showed hybridizing bands of the expected sizes (Figure V-3 

panels A and B, and Table V-1). No hybridizing bands were detected in CV127 soybean DNA, 

clearly indicating that no vector backbone DNA was integrated into the genome of CV127 

soybean. 

C. Stability of the Insert Across Multiple Generations 

In order to determine the stability of the insert in CV127, DNA samples from four different 

generations, T4, F4, F8 and F9, were subjected to Southern blot analysis. For reference, the 

breeding history of CV127 is presented in Figure III-1. Genomic DNA samples were digested 

with NcoI and SpeI and probed with either the AtAHAS 5’ UTR, csr1-2 coding sequence or 

AtAHAS 3’ UTR probes spanning the entire DNA fragment used for transformation (Figure IV1). The combination of these restriction enzymes and probes provides a unique fingerprint for 

the DNA insert in CV127 (Figure V-2). Non-transgenic Conquista genomic DNA was used as a 

negative control and Conquista spiked with one- and two-genome copy equivalents of pAC321 

was used as a positive control. Multiple bands from CV127 T4 generation DNA digested with 

either NcoI or SpeI were detected with all three probes, indicating that the T4 generation contains 

multiple copies of the csr1-2 gene cassette (Figure V-4). However, DNA from the F4, F8 and F9

generations all showed the same Southern pattern (Figure V-4) previously observed in the insert 

and copy number analyses (Figure V-2). This result clearly indicates that the multiple copies of 

the insert in the T4 generation segregated in the progeny of the cross between T4 and Conquista 

and that only a single copy is retained in the segregant selected. Moreover, these results 

demonstrate that this single copy is stably integrated in the soybean genome and is inherited 

across subsequent breeding generations. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 49 of 375 

Figure V-3. Southern Blot Analysis for the Absence of Vector Backbone Sequence 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 from the F8 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

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

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

 Nco I Spe I Xba I 

Fragment of pAC321 with probes

bla CDS

Southern probe: VP1

Southern probe: VP2

lacZ promoter phage f1 (-) ori

PvuII (5916) ColE1 ori PvuII (8429)
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 50 of 375 

23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

6.6

4.4

2.3

2.0

 b

9.4

6.6

4.4

2.3

2.0

M 1 2 3 4 5 6 7 8 9 10 11 12 13 14 M

Nc Sp

23.1 kb

9.4

6.6

4.4

2.3

2.0

23.1 kb

6.6

4.4

2.3

2.0

 b

9.4

6.6

4.4

2.3

2.0

Sp

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

NcoI SpeI

A Probe 5’UTR

B Probe AHAS

C Probe 3’UTR

Figure V-4. Southern Blot Analysis Demonstrating 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 (lanes 2 and 9), or 2-copy 

equivalents of pAC321 (lane 3 and 10); and 

genomic DNA of CV127 of either the T4 (lanes 

4 and 11), F4 (lanes 5 and 12), F8 (lanes 6 and 

13) or F9 (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

Xba I (410) NcoI (2761) Xba I (10652)

Spe I (1268) Spe I (5620) Spe I (6505)

Regions of homology 

to hybridization probes
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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 and sequencing of the insert was 

performed to confirm insert integrity. The complete 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 R272 to K272. This is a conservative amino acid 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 mutation, 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 PvuII linear DNA fragment of pAC321 (Figure IV-1) 

used to produce CV127 soybean, except for the 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. The possible transcription of this ORF was also 

investigated by RT-PCR analysis. 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 product was observed using total RNA from leaf, root, leaf bud 

and seed tissue as a template, suggesting that the ORF is not expressed in CV127 soybean 

(Figure V-6). 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Figure V-5. Diagram of Overlapping PCR Amplicons Used to Demonstrate the Integrity of the Insert in C

The insert and flanking sequences are displayed. Six amplicons used for sequencing of the insert that span th

from the 5’ to 3’ flanking regions are also indicated. 

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 sequ

PCR1 PCR2

PCR3

PCR4

PCR5

PCR6

AtAHASL 5'UTR & putative promoter

At3g48570 3' UTR

AtAHASL 3' UTR

At3g48570 5' UTR

Xba Nco I (2761) I (410) 

Spe I (1268) Spe I (5620) Spe I (6505)
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 53 of 375 

Figure 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. 

Total RNA was extracted from leaf, root, leaf bud and 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 specific for the ORF 501 (anticipated amplicon 

435 bp). Primers specific for the soybean endogenous cyclophilin gene, that is constitutively 

expressed, were used in positive control reactions (anticipated amplicon 315 bp). Panel A: 

Seed. Panel B: Leaves. Panel C: Leaf buds. Panel D: Roots. Lanes 1, 3, 5 and 7, ORF 501 

reactions; lanes 2, 4, 6, and 8, cyclophilin reactions; 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 μl reaction was loaded on the gel. Molecular 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

200
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 54 of 375 

Figure V-7. PCR Analysis of Genomic DNA from CV127 Soybean 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 conditions. PCR primers used were identical to those applied in 

the RT-PCR experiments in Figure V-6. Panel A: lanes 1 – 4, seed 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; lanes 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 μl 

reaction was loaded on the gel. Molecular weight 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

200
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E. A. thaliana SEC61γ Subunit Gene 

The linear DNA used to produce soybean CV127 contains 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 contains a previously unannotated Arabidopsis gene 

encoding the gamma subunit of SEC61 (AtSEC61γ), a small (69 amino acid) multimeric 

transport protein of the endoplasmic reticulum that is ubiquitous in all plants and other 

eukaryotes (Hartmann et al., 1994). The CV127 soybean insert sequence contains the majority 

of the AtSEC61γ subunit gene including the complete coding sequence. The AtSEC61γ 5’ UTR, 

as annotated by The Arabidopsis Information Resource, 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 

AtSEC61γ subunit gene. 

The possible transcription of the Arabidopsis AtSEC61γ 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 F7 generation of CV127 soybean as a template. Primers specific to 

two endogenous soybean genes, Iota and GmSEC61γ, were used as positive controls to confirm 

the quality of the template RNA. Total RNA from Arabidopsis leaf and root tissues without 

DNase treatment was also used as a positive control. Results showed that a very low amount of 

mRNA specific to the AtSEC61γ subunit gene in the CV127 F7 generation was present, 

indicating that the gene is only weakly transcribed (Figure V-8; for more details on the methods 

used see Appendix A). The amplified 393 bp AtSEC61γ subunit DNA band amplified by RTPCR is the same size as that amplified from Arabidopsis 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 AtSEC61γ subunit gene transcript, the 5’ end of 

the Arabidopsis SEC61γ subunit transcript in CV127 soybean was determined by RNA-ligase 

mediated rapid amplification of 5’ complementary DNA ends (RLM-5’-RACE). (For more 

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

shows the AtSec61γ RLM-5’-RACE secondary PCR result for CV127. Secondary PCR 

amplifications using soybean Sec61γ 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 cDNA sequence, it was difficult to calculate an 

expected size for the endogenous soybean Sec61γ 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 Sec61γ 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 GeneRacer RNA Oligo. The soybean positive control lanes in both 

conventional cultivar Conquista and CV127 show PCR products with similar intensity at the 

estimated size for the endogenous soybean Sec61γ subunit (Figure V-9). This proportional 

amplification of the positive control indicates that the band observed with Arabidopsis SEC61γspecific primers in CV127, but not in conventional Conquista, is not due to non-specific 

background amplification. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 56 of 375 

CV127 RLM-5’-RACE secondary PCR products amplified with Arabidopsis SEC61γ subunitspecific primer RS42 are not a single distinct band, indicating multiple transcript sizes (Figure 

V-9). The presence of PCR products in the corresponding “minus tobacco acid pyrophosphatase 

(TAP)” control lane indicates that some of these PCR products are derived from partial 

transcripts. However, because the largest PCR product in this “minus TAP” control lane is 

clearly smaller than the largest PCR products observed in the corresponding “plus TAP” lane, it 

is expected that the largest CV127 secondary PCR products amplified with Arabidopsis SEC61γ

subunit-specific primers represent full-length transcripts. The largest CV127 RLM-5’-RACE 

secondary PCR product amplified with Arabidopsis SEC61γ subunit−specific primers is clearly 

larger than the PCR product amplified from Arabidopsis ecotype Col-0 with Arabidopsis 

SEC61γ subunit-specific primers suggesting a transcriptional fusion in CV127 (Figure V-9). 

The resulting RLM-5’-RACE PCR product was sequenced and compared with the predicted 

mRNA sequence of the AtSEC61γ subunit gene. The sequence data obtained indicated that the 

Arabidopsis SEC61γ-subunit transcript in CV127 soybean is a transcriptional fusion with a short 

segment (89 nucleotides) of adjacent soybean flanking sequence. However, the first “ATG” start 

codon in the sequence coincides with the predicted translation initiation site of the native 

Arabidopsis SEC61γ-subunit gene. As there are no changes at the nucleotide level, the predicted 

protein sequence of the Arabidopsis SEC61γ-subunit protein in soybean CV127 does not differ 

from the native SEC61γ-subunit protein in Arabidopsis. Further experiments were conducted to 

determine if detectable amounts of the AtSEC61γ-subunit protein are produced in CV127 tissues. 

These experiments demonstrated that there are no detectable amounts of the SEC61γ-subunit 

protein present in CV127 tissues (LOD = 30 ng/g fresh weight for leaf tissue and 110 ng/g grain 

tissue). Further details of these experiments are presented in Section VI.E. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 57 of 375 

Figure V-8. RT-PCR Analysis of Transcription of the AtSEC61 γ Subunit Gene in CV127 

Soybean. 

Total RNA from CV127 F7 generation plant leaf tissue was extracted 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 SEC61γ subunit gene (GmSEC61γ) 

are used in positive control reactions. Reactions without template RNA using AtSEC61 

γ subunit-specific primers and GmSEC61γ subunit-specific primers are used as negative controls. 

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

AtSEC61γ subunit gene fragment amplified from soybean CV127. 

298

506, 517

At-L M IOTA Gmsec61 Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298 344

298

506, 517

At-L M IOTA Gmsec61 Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298 298

506, 517

At-L M IOTA Gmsec61 Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298

506, 517

At-L M IOTA Gmsec61 Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298

At-L M IOTA Gmsec61 Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298

At-L M IOTA Gmsec61 Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298

M

IOTA

Gmsec61 

Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298

M

IOTA

Gmsec61 

Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

M

IOTA

Gmsec61 

Atsec61

Atsec61

Gmsec61 

Atsec61

Event 127 F7 At-R

M

Atsec61

1018 bp

396

1018 bp

506, 517

396

344

298 344
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 58 of 375 

Figure V-9. RLM-5’-RACE Secondary PCR Analysis of the AtSEC61 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’-RACE. 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 Sec61γ (“Gm” in figure below) or a primer expected to 

specifically amplify Arabidopsis SEC61γ (“At” in figure below). Lanes 7 through 12 are 

identical to lanes 1 to 6, respectively, with the exception that lanes 7 through 12 were not treated 

with tobacco acid pyrophosphatase (TAP) during the RLM-5’-RACE procedure. Samples in 

lanes 7 through 12 serve as controls: any products observed in these 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 Results 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 Sec61γ, soybean ~199 bpa yes 

2 Conquista leaf SEC61γ, Arabidopsis none yes 

3 CV127 leaf Sec61γ, soybean ~199 bpa yes 

4 CV127 leaf SEC61γ, Arabidopsis ? smeared band 

5 Arabidopsis leaf Sec61γ, soybean none yes 

6 Arabidopsis leaf SEC61γ, Arabidopsis 342 bpb yes a

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

are derived from soybean Sec61γ. b

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

Arabidopsis SEC61γ. 

 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) 
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F. Inheritance of the Imidazolinone-Tolerance Trait in CV127. 

The inheritance of the imidazolinone herbicide tolerance trait in CV127 soybeans was 

determined by evaluating the F3 and F4 progeny from the cross of CV127 line CV603 (F8

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 F1 seed, hemizygous for 

the csr1-2 gene, were planted in a greenhouse and the plants were self-pollinated to produce 

individual populations of F2 seed segregating for the csr1-2 gene (one F2 population per each F1

plant). Seed from one F2 population were planted in the greenhouse and each plant was selfpollinated to produce individual F3 families (one F3 family per each F2 plant). 

Seed from each F2-derived F3 family (F2:3) were planted in a greenhouse and six to eight plants 

within each family were sprayed with imazapyr herbicide (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 families, ‘family 12’ and ‘family 13’, segregating for the csr1-2 gene 

was determined by a quantitative PCR assay. The 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 segregation pattern expected for a single 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 imidazolinone herbicide is expressed by a single gene that is inherited 

according to the principles of Mendelian genetics. 

Each F3 plant within families 12 and 13 which was identified as hemizygous based on the PCR 

assay, was self-pollinated. The F4 seed from each hemizygous F3 plant were bulked together, by 

family, and planted in a greenhouse. After spraying each F4 plant with imazapyr (100 g ai/ha), 

each plant was classified ‘resistant’ (no foliar symptoms) or ‘sensitive’ (foliar symptoms and/or 

plant death) to the herbicide. The proportion of individuals in families 12 and 13 that were 

resistant (homozygous or hemizygous) or sensitive (null) to the imidazolinone herbicide and the 

results of the chi-square test for the 3:1 segregation 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 are consistent with the 

conclusions of the molecular characterization studies conducted with CV127 soybean which 

showed that a single functional csr1-2 gene is integrated in the genome of CV127. 
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Table V-2. Chi-Square Analysis Demonstrating Single-Gene Mendelian Inheritance (1:2:1 

Segregation) of the csr1-2 Gene in Two F3 Families, Each Derived from an Individual 

Hemizygous F2 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 

X2

 = 3.00 

P = 0.22 

Total 57 57 

Family 13 

Hemizygous 23 26 

Homozygous (positive) 14 13 

Homozygous (negative) 15 13 

X2

 = 0.73 

P = 0.69 

Total 52 52 

Table V-3. Chi-Square Analysis Demonstrating Single-Gene Mendelian Inheritance (3:1 

Segregation) of the csr1-2 Phenotype in Two F4 Progeny Derived from Hemizygous Plants 

from Two F3 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 

X2

 = 1.89 

P = 0.17 

Total 678 678 

Family 13 

Tolerant (homozygous or 

hemizygous) 330 340 

Sensitive (null) 124 113 

X2

 = 1.30 

P = 0.26 

Total 454 454 

G. Conclusions of the Molecular Characterization 

Molecular analyses were performed to characterize 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 absence of plasmid backbone elements in the genome. The stability of the 

DNA insert across multiple generations 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 genetic elements within the insert. 
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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 unlinked 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 confirmed the expected trait segregation ratios, further confirming 

that the imidazolinone herbicide tolerance trait in CV127 soybean 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. 
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VI. Characterization of Proteins 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 protein that is tolerant to imidazolinone herbicides due to a 

single nucleotide mutation that results in a single amino acid substitution in which the serine 

residue at position 653 of the protein is replaced by asparagine (S653N). This amino acid 

change in plant AHAS proteins is known to prevent the binding of imidazolinone herbicides and 

thereby to result in tolerance to these herbicides (Tan et al., 2005). 

As part of the food, feed and environmental safety assessment of CV127 soybean, studies were 

conducted to confirm that the AtAHAS protein produced in CV127 soybeans is equivalent to 

other AHAS proteins that are found ubiquitously among 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 gavage study and a digestive fate study conducted 

with purified AtAHAS protein. Because the AtAHAS 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 quantities 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 AtAHAS protein were purified from 

the Escherichia coli over-expression system for the protein safety assessment studies. Key 

biochemical and functional parameters of the CV127-produced and Escherichia coli-produced 

AtAHAS proteins were characterized to demonstrate that the plant-produced protein is 

equivalent to the microbially-produced AtAHAS protein, and therefore justify the use of the 

microbially-produced protein for safety assessment studies of the AtAHAS protein produced in 

CV127 soybean. 

This section of the petition presents the following characterization studies of the AtAHAS 

protein expressed in CV127 soybeans: 

A. Biochemistry of the AtAHAS protein and imidazolinone herbicide tolerance; 

B. Characterization of the AtAHAS 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 environmental safety of the AtAHAS protein; 

D. Expression levels of the AtAHAS protein in CV127 soybean tissues. 

In addition, studies are presented in this section of the petition that address whether the 

AtSEC61γ subunit protein is expressed in CV127 soybean tissues, and a summary of the food, 

feed and environmental safety of the AtSEC61γ subunit protein is included. 
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A. Biochemistry of the AtAHAS Protein and Imidazolinone Herbicide Tolerance 

The mechanism of action of imidazolinone herbicides on weeds and non-tolerant plants is by 

inhibition of the AHAS enzyme and branched-chain amino acid biosynthesis. The AHAS 

enzyme is ubiquitous in plants and microbes and catalyzes the first step in the biosynthesis of the 

essential branched-chain amino acids valine, leucine, and isoleucine (Stidham and Singh, 1991). 

The AHAS enzyme of eukaryotes is composed of a large catalytic subunit (AHAS) and a small 

regulatory subunit. The enzyme catalyzes the condensation of two molecules of pyruvate to 

form acetolactate, the precursor of valine and leucine, or the condensation 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 acids, valine, leucine 

and isoleucine. Regulation is mediated through binding of these amino acids to the AHAS small 

(regulatory) subunit. 

Imidazolinone herbicides readily inhibit the activity 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 suggests that the herbicide binding pocket is at 

the entry site for the substrate of the AHAS enzyme, 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 herbicides on the mutant AHAS enzymes (Tan et al., 2005). 

Imidazolinone herbicide-tolerant amino acid substitutions are located in all three domains of the 

AHAS protein, but protein folding places all the amino acid substitutions that confer herbicide 

tolerance at the proposed herbicide-binding site of the protein. While these amino acid 

substitutions confer herbicide tolerance to the AHAS protein, they 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 amino 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 that the chloroplast transit peptide is removed to 

produce the mature AtAHAS enzyme that is approximately 64,000 molecular 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 acid 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 polyadenylation 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 herbicides. The mutation in the 

csr1-2 gene responsible for imidazolinone herbicide tolerance is a single nucleotide change of 
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guanine to adenine, which results in a codon change from AGT to AAT and a single amino acid 

substitution of serine to asparagine at position 653 of the AtAHAS protein. This amino acid 

change in plant AHAS proteins is known to confer imidazolinone herbicide tolerance, but have 

no effect on feedback regulation 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 replaced by lysine, does not impact the enzymatic 

function of the AHAS enzyme or herbicide tolerance properties. 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 tolerance to imidazolinone herbicides. Feedback regulation of AHAS 

activity by the branched-chain amino acids is effected 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 endogenous 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 compositional 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), indicating 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. These 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 S653N 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. 
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B. Characterization of the AtAHAS Protein Produced in CV127 Soybean and Equivalence 

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

As described above in this section of the petition, it was not technically feasible to purify gram 

quantities of the AtAHAS protein from CV127 soybean to conduct 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 study. 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 safety 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 peptide (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 present in the AtAHAS protein 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 from 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 herbicide 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 protein preparations. The results from these 

evaluations show that the CV127 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 and the reference AtAHAS produced in the Escherichia 

coli over-expression system. The microbially-produced AtAHAS and the plant-produced AHAS 

proteins demonstrated the expected enzymatic activity with the substrate pyruvate, confirming 

the same functional activity of the AHAS proteins from both microbial and plant sources. The 

specific activity of the AHAS produced in CV127 soybean was very similar to the endogenous 

soybean AHAS and was higher than the AHAS in AtAHAS produced in Escherichia coli. 

AHAS is a very unstable protein and readily loses activity upon purification (Chang and 

Duggleby, 1997). Therefore, it was not unexpected that the AHAS activity in the relatively 

impure state purified from plant tissues was higher than that retained in the much more pure 

material produced in Escherichia coli. Both CV127-produced and microbially-produced 

AtAHAS proteins showed tolerance to inhibition by the imidazolinone herbicide imazethapyr 
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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, leucine and valine, was very similar for both the 

AHAS proteins in CV127 and the nontransgenic isoline 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, 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). This result also confirms 

that the endogenous soybean AHASS must interact with the AtAHAS in CV127 soybean to 

effect feedback regulation by the branched-chain amino acids. There was no evidence of 

glycosylation associated with either the AtAHAS in CV127 soybean leaves or the Escherichia 

coli-produced protein. 

The amino acid sequence of the 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 protein 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 entire predicted AtAHAS amino acid sequence of 

the CV127-produced protein. All amino acid sequence data obtained from the AtAHAS of 

CV127 soybeans was identical to the corresponding amino acid sequence of the Escherichia 

coli-produced AtAHAS with the exception of a region near the N-terminus that 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 acid sequence of the mature AtAHAS has not been 

determined and the precise location of the cleavage site for the CTP is unknown. Amino acid 

sequence analysis of the AtAHAS protein produced in CV127 showed a peptide fragment 

corresponding to amino acid residues 52 to 73. Therefore, the CTP cleavage site of the AtAHAS 

protein produced in CV127 is most likely not at the predicted 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 mature protein (585 amino acids) plus additional amino acids in the 

CTP predicted by Mazur et al. (1987), the microbially-produced AtAHAS protein is considered 

equivalent to the CV127 protein for the following reasons. The plant-produced and E. coliproduced proteins should show structural, functional, and biochemical equivalence (Codex, 

2003). The CV127-produced AtAHAS and E. coli-produced AtAHAS demonstrated the same 

expected enzymatic activity with the substrate pyruvate, thus confirming that AtAHAS from 
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both sources possessed the same functional activity. Both are resistant to inhibition by the 

imidazolinone herbicide imazethapyr (Figure B-2), a characteristic produced 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 extracts 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 feedback inhibition by leucine and valine. This 

observation is not surprising given that the feedback inhibition is mediated by the regulatory 

(small) subunit. In planta, it is believed that the endogenous Glycine max acetohydroxyacid 

synthase regulatory (small) subunit is capable of interacting with the AtAHAS large (catalytic) 

subunit to impart the branched-chain amino acid feedback inhibition characteristic 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 specific 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 evidence of glycosylation. Also, both proteins shared the 

same amino acid sequence of the predicted mature protein of 585 amino acids, based upon amino 

acid sequence analysis of overlapping peptide fragments. Finally, in vitro digestibility and 

lability studies also supported the equivalence of the CV127-produced AtAHAS and E. coliproduced 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 pepsin in 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 allergenic proteins and was equivalent to 

the E. coli-produced AtAHAS in this respect. 

Furthermore, other studies confirm the food, feed 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 microbially-produced AtAHAS sequences do differ at 

the N-terminal end, numerous other dicotyledon AHAS sequences also show significant 

sequence divergence at the N-terminal end in the 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 AHAS from commonly-consumed crop species: 90% 

identity to Brassica napus AHASL1A (accession 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). Moreover, the model plant Arabidopsis thaliana is 
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closely related to Brassica oleracea, a species which includes the widely consumed cultivars 

broccoli, cabbage, brussel sprouts, cauliflower, collard greens, and kohlrabi (Zhang and Wessler, 

2004). Thus, while Arabidopsis thaliana itself does not have a history of safe use, it shares a 

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

The AtAHAS (R272K, S653N) protein expressed in CV127 soybean differs from the AtAHAS 

(P197S) protein expressed in sulfonylurea herbicide-tolerant Linum usitatissimum L. (flax) CDC 

Triffid (FP967) by only three amino 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 (University 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, was 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 deduced amino acid sequence did not 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 expressed 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 promoter which expresses low levels of AtAHAS, 

consistent with typical native AHAS expression patterns. The resulting low level of dietary 

exposure is a key consideration since acute toxicity is related to dosage. 

The level of exposure to a given protein is also a factor in potential allergenicity since the 

majority of major food allergens are expressed at a high level, greater 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 not quantifiable, in enzyme-linked immunosorbent 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 conditions present during standard soybean grain 

processing, further reducing the already-low levels of AtAHAS in CV127 in human food and 

animal feed. 

Additional safety assessment 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 demonstrated that there was no evidence of a significant difference in 

the performance of the broilers fed a feed containing soybean meal from CV127 soybean 
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compared to those fed a feed containing soybean meal from the grain of conventional soybeans. 

Thus, the nutritional equivalence of CV127 and its isoline control 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-produced 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 Environmental 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 reported in other sections of this petition. A 

comprehensive food and feed safety and nutritional 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 animal 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 structurally and biologically closely 

related to the AHAS proteins in all plants. For example, there is 79% amino acid identity 

between the AtAHAS and endogenous soybean AHASL2 (JGI locus Glyma13g31470.1) 

proteins. Similar levels of amino acid identity 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 AHAS proteins present in these crops with a 

history of safe production and use in food and feed products. 

3. Several imidazolinone-tolerant crops that produce 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 without 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 protein/kg body weight, the highest dose level 

tested. 

5. Bioinformatic analyses showed no biologically relevant amino acid sequence homology 

between the AtAHAS protein expressed in CV127 soybean and known toxins. In addition, 

analyses were conducted to confirm that the AtAHAS amino acid sequence did not share 

potentially immunologically relevant amino acid sequence segments or structure with known 

allergens. Results of these analyses showed that the amino acid sequence of the AtAHAS 
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protein expressed in CV127 soybean did not share either 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 AtAHAS protein is rapidly degraded in simulated 

digestive fluids and is rapidly inactivated by temperatures above 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 extremely 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 expression 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 protein 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 dietary safety factor of greater than 1010 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 structure 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 enzymes is well known and there is no scientific 

basis to suggest that its activity would result in toxicity. 

Therefore, results of these studies show that the AtAHAS protein 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 safety of the AtAHAS protein expressed in CV127 

soybean. 
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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 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 growing season were 

conducted in geographically distinct locations that were representative of commercial soybean 

production areas 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 

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 were sprayed with an imidazolinone 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 grain 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 different developmental stages, including the 

V2 (plants 15-20 cm tall with three nodes and two unfolded leaflets), R2 (plants in full bloom), 

and R8 (full maturity) stages. Three whole plants were maintained as such, and the remaining 

three whole plants were dissected into plant parts, including leaves, stems, roots, flowers and 

pods, depending on the stage of plant development. Because of a late planting date coupled with 

the southerly location of the Ponta Grossa field site in the 2006/2007 growing season, plants of 

all treatments failed to reach full 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 this field location. 

Expression levels of AHAS enzyme in different tissue samples were determined by enzymelinked immunosorbent assay (ELISA) using AHAS-specific antibodies. Due to the high level of 
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amino acid homology between the AtAHAS and the endogenous soybean AHAS enzymes, the 

AHAS-specific antibodies used in the ELISA assay 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 that only expresses the 

endogenous soybean AHAS proteins. The difference 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 each tissue type in each experiment and these are listed in Table 

VI-1. The limit of detection (LOD) is defined 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 least 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 quantified 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 concentrations of the AHAS protein. The assay is accurate 

only in the linear portion of this curve. Values which are above zero, but below the linear 

portion of the standard response curve are considered 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 assay detects both the AtAHAS protein encoded by the 

csr1-2 gene and the endogenous soybean AHAS enzymes. 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 (Stidham and Singh, 1991). The 

AHAS protein expression results from both trial seasons 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 (Tables VI-2 and VI-3). AHAS levels of 18 to 59 

ng AHAS/g fresh tissue and <80 to 254 ng AHAS/g dry 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 fresh weight basis at all 

sites ranged from below the limit of quantification (LOQ = 13 ng/g) to 16 ng/g during the 

2006/2007 season and were equal to or below the LOQ (14 ng/g) at all locations during the 2007 

season (Tables VI-2 and VI-3). 

Expression levels of the AHAS protein decreased with age of the 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-weight 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 during the 2006/2007 season had low levels of AHAS 
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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 levels 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 plant growth and development, these levels were sufficient to confer 

herbicide tolerance to CV127 soybean plants.
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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 field trials conducted in 

Brazil during the 2006/2007 (Season 1) and 2007 (Season 2) growing seasons. 

LOQ Season 1 Season 2 

(ng AHAS/g Fresh Wt.) 13 14 

Tissue Growth LOQ (ng AHAS/g Dry Wt.) LOD (ng AHAS/g Dry Wt.) 

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 

Leaves V2 70 76 16 16 

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 3 

 
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Table VI-2. AHAS Protein Levels (± Std. Dev.) on a Fresh- and Dry-Weight Basis in Different Tissues a

CV127 Soybean and the Isoline Control. Plants were cultivated at Londrina (Lond), Santo Antônio de Posse (SAP), Brasília (Bras), and Santo Antônio d

the 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 W

Tissue 

Season 1 Season 2 Season 1 

Growth 

Stage 

Soybean 

Line 

Location Lond SAP Bras SAG Lond SAP 

Whole Plant 6 ± 10 40 ± 6 <14 <14 314 ± 59 214 ± 37 Leaves 103 ± 24 128 ± 47 27 ± 9 18 ± 4 511 ± 116 714 ± 284 1Roots <132 <13 <14 <14 <41 <41 

CV127 

1st Trifoliate 55 ± 5 60 ± 12 22 ± 8 21 ± 4 278 ± 27 300 ± 47 1Whole Plant <13 <13 <14 <14 <68 <68 Leaves 15 ± 3 14 ± 1 <14 <14 78 ± 17 73 ± 8 Roots <13 <13 <14 <14 <41 <41 

V2 

Isoline 

Control 

1st Trifoliate <13 <13 <14 <14 <70 <70 Whole Plant ND3 34 ± 6 <14 <14 ND 160 ± 33 Leaves <13 24 ± 4 <14 <14 <62 106 ± 19 Roots <13 17 ± 4 <14 <14 <38 50 ± 13 

CV127 

Flowers <13 22 ± 9 <14 -4 <77 125 ± 45 Whole Plant ND <13 <14 <14 ND <65 Leaves ND <13 <14 <14 ND <62 Roots <13 <13 <14 <14 <38 <38 

R2 

Isoline 

Control 

Flowers <13 <13 <14 - <77 <77 
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Table VI-2. continued. 

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

Tissue Season 1 Season 2 Season 1 

Growth Stage Soybean Line Location Lond SAP Bras SAG Lond SAP Br

Whole Plant <13 <13 <14 <14 <15 <15 <Pods 24 ± 1 26 ± 2 <14 <14 26 ± 1 30 ± 2 <Roots <13 <13 15 ± 5 17 ± 8 <17 <17 42 ± 

CV127 

Grain <13 <13 <14 <14 <15 <15 <Whole Plant <13 <13 <14 <14 <15 <15 <Pods <13 <13 <14 <14 <15 <15 <Roots ND ND 15 ± 3 <14 ND ND 41 

R8 

Isoline 

Control 

Grain <13 <13 <14 <14 <15 <15 <

1The limit of quantification (LOQ) on a dry-weight basis varied for each tissue type and growth stage based on the moisture con

Values below the LOQ are reported as less than the LOQ for that tissue and growth stage. LOQ values for different tissues are show

2Sample had detectable but non-quantifiable amounts of AHAS. That is, value was below the limit of quantification (13 or 14 ng AH

2006/2007 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 

different tissues are shown in Table VI-1. 

4Not tested due to insufficient amount of flower tissues at this site. 
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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 during 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 <131

 <14 <70 <762

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

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 - <804

Teresina Isoline - <14 - <76 

CV127 - 39 ± 10 - 207 ± 56 

V2 / 

Leaves 

Vilhena Isoline - 14 - <834

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

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

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

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 LOQ 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. 
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E. Levels of the AtSEC61γ Subunit Protein in CV127 Soybean

As described in section V.E of this petition, the complete coding sequence of the A. thaliana

gene encoding the Arabidopsis SEC61γ (AtSEC61γ) subunit protein resides in the insert of 

CV127 soybean near the 5’ junction with genomic soybean DNA. Although the AtSEC61γ gene 

in CV127 soybean appears to lack the majority of its native promoter, experiments were 

performed to evaluate its possible expression. These demonstrated that a very low amount of 

mRNA is produced from this gene that, if translated, would encode the native 

AtSEC61γ−subunit protein. Since the transcript was highly amplified by RT-PCR to maximize 

detection capability, and only a faint band corresponding to the AtSEC61γ subunit transcript was 

detected, it was presumed that either no AtSEC61γ subunit protein is produced in CV127, or if 

produced the protein would be present at extremely low levels in the plant, acknowledging that 

transcript and protein levels may not be correlated. 

To determine if AtSEC61γ subunit protein is expressed in CV127 soybean tissues, western blot 

analysis of microsomal membrane protein fractions prepared from leaf and grain samples of 

CV127 was conducted (methods for these studies are presented in Appendix C of this petition). 

Identity of the microsomal membrane fraction 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 harvested from greenhouse-grown plants. 

The AtSEC61γ 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 VI-1). The rabbit anti-AtSEC61γ

polyclonal antibody can detect approximately 0.4 ng of SEC61γ subunit protein by the western 

blot development method utilized. 

The E. coli-produced SEC61γ subunit protein was readily detected by western blot analysis in 

the standard protein lanes at a molecular weight of approximately 7000 (Figure VI-1). 

Furthermore, expression of the endogenous AtSEC61γ subunit protein in Arabidopsis was 

detected in microsomal membrane protein preparations from seed but was not detected in 

Arabidopsis leaf tissue (Figure VI-1). This result showed that the AtSEC61γ subunit protein can 

be detected in tissues of the plant species which served as the donor of the AtSEC61γ 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, western blot analysis detected no 

AtSEC61γ subunit protein in leaf or grain microsomal membrane protein preparations of CV127 

(Figure VI-1, panels A and B). Because of the high amino acid sequence homology of SEC61γ

subunit proteins across species (e.g., 86% homology between soybean and Arabidopsis) it was 

expected that the antibody would detect the endogenous soybean SEC61γ subunit protein; 

however, no protein band corresponding to the SEC61γ subunit protein was detected in tissue 

extracts of the control soybean Conquista. Therefore, AtSEC61γ subunit protein was not 

detectable at levels greater than 30 ng/g leaf issue or 110 ng/g grain tissue or greater than 30 and 

110 ppb for leaf and grain of CV127, respectively. This result is not unexpected given the 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 79 of 375 

extremely low levels of the transcript corresponding to the AtSEC61γ subunit gene detected in 

CV127. 

These results demonstrate that no detectable amounts of the AtSEC61γ subunit protein are 

produced in either leaf tissue or grain of CV127 soybean. However, in the unlikely event that 

the AtSEC61γ subunit protein is present in tissues of CV127 at extremely low levels, below the 

limit of detection of the assay, the following summary confirms the food, feed and environmental 

safety of the AtSEC61γ subunit protein. 

1. The source organism of the AtSEC61γ subunit protein, Arabidopsis thaliana, is not a known 

human or animal pathogen and is not known to cause allergic reactions in humans. 

Similarly, A. thaliana is not known to produce toxic substances. 

2. The SEC61γ subunit is part of the protein translocation complex associated 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%) between the soybean SEC61γ subunit 

and that from Arabidopsis (see Figure VI-3 for the amino acid alignment). The amino acid 

sequence of the Arabidopsis SEC61γ subunit protein is 86% identical and 93% similar to the 

corresponding protein in soybean. Therefore the AtSEC61γ 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 biologically relevant amino acid sequence homology 

between the AtSEC61γ subunit protein and known toxins. In addition, bioinformatic 

analyses were conducted to confirm that the AtSEC61γ subunit protein amino acid sequence 

did not share potentially immunologically relevant amino acid sequence segments or 

structure with known allergens. Results of these analyses showed that the amino acid 

sequence of the AtSEC61γ subunit protein did not share either 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 AtSEC61γ subunit protein is rapidly degraded in 

simulated digestive fluids which is a characteristic typical of other dietary proteins with a 

history of safe use in food and feed products. 

Therefore, results of these studies show that the AtSEC61γ subunit protein does not possess the 

attributes of known food allergens, is not homologous to known protein toxins, and supports the 

environmental as well as food and feed safety of the AtSEC61γ subunit protein. 
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 Figure VI-1. SEC61γ Subunit Western Blot Analysis of Leaf and Grain Microsomal 

Membrane Protein Preparations from CV127 and Control (Conquista) Soybeans 

Microsomal membrane proteins were prepared from leaf and grain samples and subjected to 

electrophoresis on an 8 – 16% polyacrylamide gel. Western blot analysis was conducted using 

rabbit anti-AtSEC61γ subunit polyclonal antibodies and developed using a chemiluminescent 

substrate. The molecular weight of AtSEC61γ subunit protein is approximately 7000. All 

samples except for the AtSEC61γ subunit protein standard are from microsomal membrane 

protein preparations. Panel A. Lanes 1, and 5, purified SEC 61γ subunit protein standard, 2.4 

and 1.2 μg, respectively; lane 2, 20 μg protein from Arabidopsis leaf microsomal membrane 

protein preparation; lane 3, 20 μg protein from Conquista soybean leaf microsomal membrane 

protein preparation; lane 4, 20 μg protein from CV127 leaf microsomal membrane protein 

preparation; lane 6, 15 μg protein from Arabidopsis seed microsomal membrane protein 

preparation; lane 7, 15 μg protein from Conquista soybean seed microsomal membrane protein 

preparation and lane 8, 15 μg from CV127 soybean seed microsomal membrane protein 

preparation. Panel B. Longer exposure of same samples (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 SEC61γ 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 SEC61γ subunit

1 2 3 4

A. B.
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Figure VI-2. Cytochrome c Reductase Activity 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 centrifugation, 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

Fraction

Cyt. c red. activity (

ΔA547/min)

CV127

Crude Extract 100K Super 100K pellet

0.0

2.5

5.0

7.5

10.0

Fraction

Cyt. c Red. Activity (

ΔA547/min)
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Figure VI-3. Alignment of the Deduced Amino Acid Sequences of the SEC61γ Subunit 

Proteins from Soybean (Gmax) and Arabidopsis thaliana (Athal). 

The amino acid sequence for soybean SEC61γ (G. max gene Glyma02g42660) was obtained 

from the Joint Genome Institute Glyma 1.0 annotation available through Phytozome (release 

version 7.0), http://www.phytozome.net/soybean. The amino acid sequence for A. thaliana

SEC61γ was deduced from the sequence of the gene encoding the AtSEC61γ subunit protein in 

CV127 soybeans. Identical amino acids are shaded in grey, conservative replacements are 

presented as white text in a black background, and non-conservative amino acid differences are 

unshaded. 

 10 20 30 40 50 

 .........|.........|.........|.........|.........| 

Gmax MDAIDSVFDPLREFAKDSVRLVKRCHKPDRKEFSKVAVRTAIGFVVMGFV 

Athal MEAIDSAIDPLRDFAKSSVRLVQRCHKPDRKEFTKVAVRTAIGFVVMGFV 

60 70 

 .........|.........| 

Gmax GFFVKIIFIPINNIIVGSG 

Athal GFFVKLVFIPINNIIVGSS 

VII. Compositional and Nutritional Assessments of CV127 Soybean 

Soybean has many uses in human and animal nutrition. The grain is typically processed into two 

commodity products, oil and meal. The major food uses for soybean include the production of 

soybean oil that is utilized in margarines, shortenings, and salad and cooking oils. Soybeans are 

also used in various food products that include tofu, soya sauce, 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, including the forage, are also fed to a limited 

extent to animals, primarily to cattle. Therefore, the purpose of this section of the petition was to 

demonstrate that the grain and forage 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 nontransgenic, isoline control soybean 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 cultivated 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. Furthermore, results were compared to analytical 

data for soybean grain in the International Life Sciences Institute Crop Composition Database 

(ILSI, 2008), with comparisons to globally-derived values as well as data produced in Brazil, 

where available. A summary of the results of these comparisons are presented in this section of 

the petition, and detailed results for grain and forage analyses at each field trial location are 
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presented in Appendix D. Results showed that CV127 soybean is compositionally and 

nutritionally equivalent to conventional soybean varieties that are currently cultivated in Brazil 

as well as globally. 

Grain samples of CV127 soybean, the isoline control, and the two conventional soybean varieties 

were harvested from soybeans grown in four replicated plots at each of six field trial locations in 

Brazil during the 2006/2007 season and from four field trial locations in the 2007 short season. 

Forage tissues were harvested at the R2 growth stage from four replicated plots at each of six 

field trial locations in Brazil during the 2007/2008 growing season (all field trial locations for the 

production of grain and forage for compositional analyses are presented 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 soybeans 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 commercial 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 field trial location are presented in 

Appendix D. The samples that were obtained from the isoline control and the two conventional 

soybeans varieties were derived from plants that were treated with bentazon and acifluorfensodium (commercial name Volt®) at the rate of 1.0 liters/ha for the 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 where soybeans are cultivated commercially. The 

cultivation practices and a description of the protocol for the field trials that produced the grain 

used in this composition study are presented in Appendix F. 

Components analyzed for grain included proximates (protein, fat, ash, carbohydrates, calories, 

total dietary fiber [TDF], and moisture), crude fiber, acid detergent fiber (ADF), neutral 

detergent fiber (NDF), amino acids, fatty acids (C14-C24), minerals (calcium, iron, phosphorus, 

magnesium, and potassium), vitamins (α-, β-, γ-, δ-, and total tocopherols, vitamins E and B1, 

and folic acid), isoflavones (daidzin, malonyl-daidzin, daidzein, glycitin, malonyl glycitin, 

glycitein, genistin, malonyl genistin, and genistein), phospholipids (phosphatidyl ethanolamine, 

phosphatidic acid, phosphatidyl inositol, and phosphatidyl choline), and antinutrients (phytic 

acid, raffinose and stachyose, lectins, urease and trypsin inhibitor). Components analyzed in 

forage samples included proximates (protein, fat, ash, moisture, carbohydrates and calories), 

crude fiber, ADF and NDF. Compositional analyses were conducted at the Instituto de 

Tecnologia de Alimentos (ITAL), Campinas, Brazil on grain and forage samples, and complete 

methods of analyses are presented in Appendix D. 

In addition to the grain and forage compositional analyses, the nutritional equivalence of CV127 

soybean to the isoline control and conventional soybean varieties was confirmed in a poultry 

feeding study. A 42-day feeding 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 performance of the animals fed a feed containing soybean meal from 

CV127 soybean compared to those fed a feed containing soybean meal from the grain of 
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conventional soybeans, thereby showing that meal derived from CV127 grain is as wholesome as 

meal derived from grain of the isoline control or conventional soybean varieties 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 Identity 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 compositional analyses were conducted under standard 

agronomic practices in a complete randomized block design with four replicate blocks per 

location. For 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 conventional 

soybean varieties, the same grain harvest and separation procedures 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 trial 

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 variability for each analyte for soybeans 

grown in Brazil. 

Analysis of variance was carried out on the grain composition data using SAS Version 9.1 (SAS 

Institute Inc., Cary, North Carolina) following two procedures, the General Linear Model and the 

Mixed Model. With the exception of moisture content, all data were expressed on a dry-weight 
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basis for statistical analyses. Differences were assessed across location and by location. The 

model for across-location analyses is the following: 

y = variety + location + variety x location + block(location) + e 

Random effects: location, variety 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 were considered statistically 

significantly different at the p < 0.05 level. 

Proximates. Grain samples from the 2006/2007 (season 1) and 2007 (season 2) field trial 

seasons were analyzed for moisture, ash, protein, crude fat, total dietary 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 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-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 soybeans. In addition, in season 2 there was no significant difference for 

calories or crude fat between grain from the isoline control and CV127 soybeans. Slight grain 

moisture differences were observed between CV127 and the isoline control in both seasons, but 

the differences were inconsistent, since CV127 grain 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. Furthermore, 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 soybeans produced in Brazil. In season 1, grain 

from CV127 soybean was statistically significantly higher for crude fat and caloric content and 

statistically significantly lower for 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 protein content of grain from CV127 soybean was 

statistically significantly higher than that for grain 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 conventional soybean varieties cultivated in the same 

trials as well as to the range reported for soybeans 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. 
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Fiber composition. Grain samples were analyzed for crude fiber (CF), acid detergent fiber 

(ADF) and neutral detergent fiber (NDF). The means and ranges of values for CF, ADF and 

NDF in CV127 soybean, the isoline control and two conventional soybean control treatments 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-2. In 

seasons 1 and 2, there were no statistically significant differences in crude fiber values between 

the isoline control and CV127 soybeans, and no statistically significant differences 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 statistically 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 grain fiber content of the isoline control and comparable to and in 

the same range as the grain fiber content of other conventional soybean varieties with a history of 

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

Amino Acids. Grain samples from the two field trial seasons were analyzed for amino acid 

content. The means and ranges of values for all amino acids in CV127 soybean, the isoline 

control and the two conventional soybean varieties 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 control soybeans, except for small but statistically significant differences 

in values for cysteine and methionine between the two treatments. In the grain from season 2, 

there were no statistically significant differences in amino acid content between CV127 and the 

isoline control soybeans, except for alanine, cysteine, histidine, methionine, and tyrosine where 

statistically significant differences were observed between the two treatments. The means and 

ranges of values for the amino acid content of CV127 grain from both seasons 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 Brazil. These results demonstrate that the amino acid content of CV127 soybean grain is 

equivalent to the content of the isoline control grain and is comparable to, and in the same range 

as, grain amino acid content of conventional soybean 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 also 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 differences in levels of these amino acids between grain of CV127 and 

the isoline control. Therefore, these data confirm that the mutation in the AtAHAS enzyme has 
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no effect on the feedback regulation of the enzyme by the branched-chain amino acids or the 

biosynthetic function of the enzyme. 

Fatty Acids. Grain samples from the two field trial seasons 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, and behenic acids. In addition, low levels of 

eicosenoic acid were detected in grain of the different soybean treatments from season 1, but 

were not detected in the grain from season 2. The means and ranges of values for fatty acids 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 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 season 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 fatty acids (palmitic and oleic) were statistically 

significantly higher and two others (linoleic and linolenic) were statistically significantly lower 

in grain from CV127 soybean compared to the isoline control soybean in season 1. In grain from 

season 2, there were no statistically significant differences in the levels of myristic, palmitic, 

arachidic and behenic acids between the grain from CV127 and the isoline control soybeans 

(Table VII-4). Statistically significant differences in levels of stearic, oleic, linoleic, and 

linolenic fatty acids were observed between grain of CV127 and the isoline control in season 2. 

However, for all fatty acids, the means and ranges 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 Brazil (Table VII-4). These results demonstrate that the fatty acid 

content of CV127 grain is equivalent to the content in isoline control grain and is comparable to 

and in the same range as the grain fatty acid content 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 the animal feed diet (Baker, 2000). Soybeans are also a source of 

phosphorus and calcium in the animal feed diet; however, the bioavailability of these minerals is 

typically limited by chelation by phytate. Therefore, these five minerals (calcium, iron, 

magnesium, phosphorous and potassium) were quantified 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 and two conventional soybean varieties in two 

field trial seasons and across all locations in comparison 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 different from those of the isoline control, while 

levels of iron (lower), magnesium (higher), and potassium (lower) were statistically significantly 

different from the isoline control. All mineral mean values determined for grain from CV127 

soybean in season 2 were statistically 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 soybeans 
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(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 use 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 vitamins 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 ranges 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 statistically 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 isoline control soybeans from season 1 were not 

statistically significantly different. 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 CV127 soybean grain were either within or 

comparable to the range of values of the conventional soybean varieties cultivated in the same 

field trials. Global ranges for typical vitamin values in soybean grain are only available for folic 

acid, vitamin B1 and α-tocopherol. The mean values 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 higher 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 results demonstrate that vitamin levels in grain 

produced by CV127 soybean are comparable to and in the same range 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 analyzed for their isoflavone 

composition, including daidzin, malonyl daidzin, daidzein, glycitin, malonyl glycitin, glycitein, 

genistin, malonyl genistin, and genistein. The mean and range of values for the total isoflavones 

(total daidzein, total genistein, and total glycitein) 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 mean values for total glycitein between the grain 

from CV127 and the isoline control soybeans in season 1, but levels of glycitein were 

statistically significantly lower in grain of CV127 compared to levels in the grain of the isoline 

control in season 2. Levels of total daidzein and genistein were statistically significantly lower 

in grain of CV127 compared to levels in the isoline control in both field trial seasons. However, 

for each of the isoflavones, the mean and range of isoflavone values for CV127 soybean grain 
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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 reported globally for soybeans 

and for soybeans produced in Brazil (Table VII-7). These results demonstrate that the isoflavone 

content in grain produced from CV127 soybean is comparable to, and in the same range as, the 

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

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

Phospholipids. Phospholipids are common contaminants during soybean oil processing, often 

referred to as gums in the solvent-extracted oil. If not removed, the gum material typically 

settles out and can cause significant losses in oil refining. Therefore, phospholipid levels were 

analyzed in grain samples and the mean and range of values for phosphatidyl ethanolamine, 

phosphatidic acid, phosphatidyl inositol and phosphatidyl choline in grain from CV127, the 

isoline control soybeans and two conventional soybean 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, the 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 isoline control. Conversely, in season 2, there were 

no statistically significant 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 the ILSI Crop Composition Database, comparison of these values to 

this database was not possible. However, comparisons 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 that are commonly cultivated in Brazil and that have a long 

history of safe food and feed use and safety to the environment. 

Antinutrients. Grain samples were analyzed for antinutrient content, and the mean and range of 

values for phytic acid, raffinose, stachyose, lectins, urease and trypsin inhibitor 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 values for antinutrients in soybean 

grain published in the ILSI Crop Composition Database (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 soybeans in either field trial season, and the mean and range of 

phytic acid values for CV127 grain were either within or comparable 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 isoline control and the conventional soybean varieties values 

were lower than the range that is typical for soybeans cultivated globally (Table VII-9), and this 

most likely reflects a germplasm characteristic of soybean varieties adapted for cultivation in 

Brazil. 
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In the case of raffinose, the mean value for grain 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. Therefore the difference in grain raffinose levels 

observed between the two treatments in season 1 was not considered biologically meaningful 

since it was not observed consistently. Although 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 conventional soybean varieties included in the field trials 

(Table VII-9). Therefore the difference in raffinose 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 grain of CV127 soybeans in both field trial seasons 

were slightly, but statistically significantly lower 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 than 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 compared to levels recorded 

for soybeans cultivated globally, is a characteristic of soybean varieties adapted for cultivation in 

Brazil. 

The mean values for lectins in season 1, and urease and trypsin inhibitor in both seasons for 

CV127 were not statistically significantly different from levels of the same analytes in the isoline 

control soybeans. The mean value for lectin levels in grain from 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 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 reported globally for soybeans and 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 conventional soybean varieties, but 

values were below the range reported 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 has been a reliable method because 

trypsin inhibitor is readily reduced by heat as is urease activity. Urease is not harmful to nonruminant animals but can cause ammonia toxicity in cattle fed urea-supplemented diets (Dr. G. 

Weigel, personal communication). The means and ranges of urease values in grain of both 

CV127 and the isoline control soybeans 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 isoline control and the conventional Brazilian 
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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 varieties 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 demonstrate that the introduction of the csr1-2 gene into the 

soybean genome does not impact the nutritional 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 typically cultivated in Brazil. Therefore, grain derived from 

CV127 soybean is appropriate for use in human food and animal feed products. Minor 

differences in amounts of individual nutritional constituents that were detected between the grain 

of CV127 soybean and that of the isoline control 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 characteristics of soybean germplasm bred and 

developed for production under tropical growing conditions in Brazil. The complete results of 

these composition analyses are presented in Appendix D.
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Table VII-1. Grain Proximate Composition. Comparison of the proximate composition of grain derived from CV127 soybean with the isoline control soybean varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were c

single mean value (Conv Stds) for each analyte. 

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte (unit) Isoline CV127 Conv Stds Isoline CV127 Conv Stds Glo

N = 24 N = 12 N = 15 N = 8 N = 80 

Mean (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

(4.7 – 

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.

(3.89 

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

(33.19 

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

(8.10 – 

Total Dietary 

Fiber 

(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) N

Carbohydrate1

(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

(29.6 

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) N

†Values in the same season and row followed by the same letter are not significantly different at P<0.05. 

*NA = not available 

1Carbohydrates including total dietary fiber 
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Table VII-2. Grain Fiber Composition. Comparison of the fiber composition of grain derived from CV127 soybean with the isoline control and two varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILSI Crop Compomean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were combined to provid(Conv Stds) for each analyte.

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte Isoline CV127 Conv Stds Isoline CV127 Conv Stds Gl

(g/100 g 

DW) N = 24 N = 12 N = 15 N = 8 N = 8

Mean (range) 

Crude Fiber 8.5 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

(4.12 

ADF 11.39 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) 1

(7.81 

NDF 14.98 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) 1

(8.53 

†Values in the same season and row followed by the same letter are not significantly different at P<0.05. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-3. Grain Amino Acid Composition. Comparison of the amino acid composition of grain derived from CV127 soybean with the isoline control soybean varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were c

single mean value (Conv Stds) for each analyte. 

Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte Isoline CV127 Conv Stds Isoline CV127 Conv Stds Glob

(g/100 g 

DW) N = 24 N = 12 N = 15 N = 8 N = 80 

Mean (range) 

Alanine 1.64 a† (1.45 – 1.92 1.63 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) 1.7

(1.51 – 

Arginine 3.03 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) 2.8

(2.29 – 

Aspartate 4.64 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) 4.4

(3.81 – 

Cysteine 0.54 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) 0.5

(0.37 – 

Glutamate 7.61 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) 7.0

(5.84 – 

Glycine 1.65 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) 1.6

(1.46 – 

Histidine 0.87 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) 1.0

(0.88 – 

Isoleucine 1.61 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) 1.8

(1.54 – 

Leucine 2.89 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) 3.0

(2.59 – 

Lysine 2.48 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) 2.5

(2.29 – 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-3. continued.  Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte Isoline CV127 Conv Stds Isoline CV127 Conv Stds Glo

(g/100 g DW) N = 24 N = 12 N = 15 N = 8 N = 80 

 Mean (range) Methionine 0.63 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) 

0.5

(0.43 – 

Phenylalanine 1.99 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) 

1.9

(1.63 – 

Proline 1.98 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) 

2.0

(1.69 – 

Serine 2.09 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) 

2.0

(1.11 – 

Threonine 1.56 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) 

1.4

(1.14 – 

Tryptophan 0.73 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) 

0.4

(0.36 – 

Tyrosine 1.34 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) 

1.3

(1.02 – 

Valine 1.66 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) 

1.9

(1.60 – 

†Values in the same season and row followed by the same letter are not statistically significantly different at P<0.05. 

*N = 7 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-4. Grain Fatty Acid Content. Comparison of the fatty acid composition of grain derived from CV127 soybean with the isoline control 

soybean varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were csingle mean value (Conv Stds) for each analyte. 

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte (unit) Isoline CV127 Conv Stds Isoline CV127 Conv Stds Gl

(%Total FA) N = 24 N = 12 N = 15 N = 8 N = 8

Mean (range) 

Myristic 14:0 <0.09 a† (nd‡ – 0.10) <0.09 a (nd – 0.10) 0.10 (0.9-0.11) N=7 0.11 a N=5 (0.09 – 0.12) 0.11 a N=7 (0.10 – 0.11) 0.09 N=4 (0.06 – 0.12) NPalmitic 16:0 9.77 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) 1

(9.55 

Stearic 18:0 3.39 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

(2.70 

Oleic 18:1 20.07 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) 2

(14.3 

Linoleic 18:2 45.87 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) 5

(42.3 

Linolenic 18:3 5.65 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

(3.00 

Arachidic 20:0 0.37 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

(0.16 

Eicosenoic 20:1 0.13 a (0.09 – 0.19) 0.13 a (0.08 – 0.18) 0.14 (0.09-0.20) nd nd nd 0

(0.14 

Behenic 22:0 0.46 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

(0.28 

†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 available 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-5. Grain Mineral Composition. Comparison of the mineral composition of grain derived from CV127 soybean with the isoline control and two varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILSI Crop Compomean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were combined to provid(Conv Stds) for each analyte.

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte (unit) Isoline CV127 Conv Stds Isoline CV127 Conv Stds 

(mg/100 g DW) N = 24 N = 12 N = 15 N = 8 

Mean (range) 

Calcium 268 a† (221 – 330) 266 a (214 – 318) 254 (205-313) 246 b (172 – 359) 286 a (198 – 484) 234 (167-329) Iron 8.50 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) 

(

Magnesium 246 b (204 – 266) 266 a (227 – 304) 269 (225-308) 238 b (211 – 266) 287 a (268 – 309) 255 (223-293) Phosphorus 687 a (541 – 834) 667 a (546 – 760) 670 (527-875) 768 a (655 – 845) 732 b (580 – 871) 745 (614-808) Potassium 1928 a (1782 – 2071) 1881 b (1703 – 2069) 1961 (1772-2103) 1744 b (1593 – 2061) 1864 a (1599 – 2021) 1758 (1595-2033) 

(

†Values in the same season and row followed by the same letter are not significantly different at P<0.05. 

*NA = not available 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-6. Grain Vitamin Composition. Comparison of the vitamin composition of grain derived from CV127 soybean with the isoline control and two varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILSI Crop Compomean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were combined to provid(Conv Stds) for each analyte.

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte (unit) Isoline CV127 Conv Stds Isoline CV127 Conv Stds G

N = 24 N = 12 N = 15 N = 8 N = 

Mean (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) 

(24

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) 

(0.1

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) 

γ-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) 

δ-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) 

Total 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) 

Vitamin 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) 

(0.1

Vitamin 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) 

†Values in the same season and row followed by the same letter are not significantly different at P<0.05. 

*NA = not available 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-7. Grain Isoflavone Composition. Comparison of the isoflavone composition of grain derived from CV127 soybean with the isoline control soybean varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were c

single mean value (Conv Stds) for each analyte.

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte Isoline CV127 Conv Stds Isoline CV127 Conv Stds Glo

(mg/100 g 

DW) N = 24 N = 12 N = 15 N = 8 N = 80 

 Means (range) 

Total Daidzein 

72.2 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

(6.0 – 

Total Genistein 

101.7 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

(14.4 – 

Total Glycitein 

22.3 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

(1.5 – 

†Values in the same season and row followed by the same letter are not significantly different at P<0.05.
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-8. Grain Phospholipid Composition. Comparison of the phospholipid composition of grain derived from CV127 soybean with the isoline control soybean varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were c

single mean value (Conv Stds) for each analyte.

 Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte Isoline CV127 Conv Stds Isoline CV127 

(mg/g fat) N = 24 N = 12 N = 15 

 Means (range) 

Phosphatidyl ethanolamine 

101.9 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) 

Phosphatidic acid 4.0 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) Phosphatidyl inositol 11.8 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) Phosphatidyl choline 29.3 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) 

†Values in the same season and row followed by the same letter are not significantly different at P<0.05. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VII-9. Grain Antinutrient Composition. Comparison of the antinutrient composition of grain derived from CV127 soybean with the isoline control soybean varieties grown in two seasons as well as with global and Brazilian ranges of values from the ILDatabase. The mean values for the conventional soybean varieties, Monsoy 8001 and Coodetec 217, were c

single mean value (Conv Stds) for each analyte.

Season 1 

(2006/2007) 

Season 2 

(2007) 

Analyte (unit) Isoline CV127 Conv Stds Isoline CV127 Conv Stds Globa

N = 24 N = 15 N = 8 N = 80 – 

 Means (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

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

Stachyose (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

Lectins HU‡/mg DW 

2.24 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

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 

Trypsin 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 – 11

†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 available 
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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 the 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 treatment 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 Appendix D). The components analyzed included: 

proximates (moisture, fat, ash, protein, carbohydrates and calories) and fiber (crude fiber, acid 

detergent fiber and neutral detergent fiber). The results of analyses for CV127 were compared to 

the isoline control and to two conventional soybean varieties, 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 processed and analyzed for proximate and fiber composition. The three 

plants from each replicate plot were pooled together prior to analyses, and analyses were 

conducted for each replicate treatment at each field site. The methods used for this study are 

presented in Appendix D. Also, the forage composition data from each individual field site are 

presented in Appendix D of this petition. Results are reported on a dry-weight basis. Statistical 

analysis was conducted using the dry-weight data. In this study, statistical analyses of the 

compositional data included all treatments: CV127 + imi, isoline control, and the two 

conventional soybean varieties. Statistical analysis methods were as described for compositional 

analyses of the grain, except that for forage composition, contrasts were carried out to compare 

the isoline control as well as the conventional soybean varieties to CV127 soybean. Values were 

considered statistically significantly different at the P < 0.05 level. 

The proximate and fiber composition results calculated 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 isoline control and the two conventional varieties 

treated with conventional herbicide. Proximate analyses included 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 statistically significant differences in 

levels of moisture, ash, fat, protein, carbohydrates, calories, crude fiber, ADF or NDF in forage 

samples produced from CV127 soybean compared to levels of these analytes in the isoline 

control soybean. In addition, comparison of the forage composition of CV127 soybean and the 

isoline control with that of the two conventional soybean varieties demonstrated that there were 

no statistically significant differences among these treatments for fat, calories, ADF or NDF. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 103 of 375 

Where small differences in analyte values were 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 conventional soybean varieties that 

are currently cultivated. Therefore, forage derived from CV127 soybeans is appropriate for use 

in animal feed. These results further support 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. 
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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 varieties (Conv Std 1 and Conv Std 2) across 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 statistically significantly different at P < 0.05. 

**N=17 for Conv Std 1 and Conv Std 2 for ADF and NDF
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C. Poultry Feeding Study 

Soybeans are used primarily to produce oil and high protein soybean meal. Soybean meal is the 

predominant protein component of animal feeds with 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 soybeans and to compare it with that of soybean 

meals from conventional soybean varieties, a 42-day feeding 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 wholesomeness of feed 

ingredients (ILSI, 2003). 

In order to produce the grain for this feeding study, CV127 soybeans and three conventional 

soybean varieties, including Conquista, Monsoy 8001, and Coodetec 217, were grown at a field 

trial location near Santo Antônio de Posse, Brazil during the 2006/2007 growing season. 

Conquista is the conventional variety that was originally transformed with the csr1-2 gene 

encoding an imidazolinone-tolerant AtAHAS enzyme and it is closely related genetically to 

CV127 soybean. The soybeans were cultivated according to standard agricultural practices for 

soybean production in Brazil, and cultivation practices 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 F9 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 conventional 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 such 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 batch-type extractor, using indirect steam to heat 

the n-hexane solvent to 45-50oC. In the production 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) was applied for 30 minutes through an orifice plate 

with a 2.5 mm aperture. The residual solvent was removed by treating with direct steam for 30 

minutes under vacuum (250 mm Hg). To finish the process, the meal was subjected to a pressure 

of 0.2 kg/cm2

 for 10 minutes. The meal moisture was reduced 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 control soybean grain used to produce the meal as well as the soybean 

meal was confirmed using an event-specific 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 demonstrating that there was no cross-contamination 

of the conventional soybean grain or meal with grain or meal from CV127 soybean. In addition, 

the grain samples and the corresponding soybean meal samples were tested for the presence of 
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important mycotoxins, including aflatoxins B1, B2, G1, and G2, zearalenone, and ochratoxin A 

by the Instituto de Tecnologia de Alimentos (ITAL). None of these mycotoxins 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 facilities in Concórdia, Brazil. Preliminary 

experiments were conducted with feeds containing the soybean meals from CV127 soybean and 

the three conventional soybean varieties to determine the apparent metabolizable energy 

corrected for nitrogen and the true digestibility values of the amino acids. The results of these 

experiments were used to produce nutritionally-balanced feeds for the feeding study intended to 

assess the wholesomeness of soybean meal from CV127 soybeans. Five hundred and seventysix broiler chickens of the lineage AgRoss 508, half male and half female, were used in the study. 

The birds were organized randomly in blocks by body weight with 12 replications with 12 

broilers (six males and six females) per replication for a total of 144 broilers per treatment. 

Feeds were formulated for each of the four different treatments that included the soybean meal 

and all feeds were formulated to be isoenergetic and isoproteic. The experiment 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 animals. The feeds contained varying amounts of 

soybean meal that were approximately 40% of the 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 results 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 statistical analysis was conducted without 

segregation of the sexes. The results demonstrated that there were no statistically significant 

differences (P>0.05) in body weight, weight gain, feed intake or feed conversion between 

animals fed feed containing soybean meal from CV127 soybeans and those fed feeds containing 

soybean meal from the conventional varieties 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 from CV127 grain is appropriate for use in animal 

feed. 
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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, and as nutritious as, grain and forage produced by the isoline 

control and other conventional soybean varieties with a history of safe use in food and feed 

products, as well as safety to the environment. 
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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 standard errors for broiler chickens during the 

periods studied. 

Treatments 

Performance T1 T2 T3 T4 

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. 
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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 varieties through the evaluation of various agronomic, phenotypic, 

phenologic, and ecological interaction characteristics 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 greenhouse conditions. Field trials with CV127 

soybeans were conducted at seven different locations in Brazil during 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 soybean is commercially cultivated 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 practices used in soybean production between the 

United States and Brazil, including Maturity Groups of soybean cultivars as well as weed, insect 

and disease control practices. There are some similarities in the climatic conditions between the 

regions in the southeastern United States where soybeans of maturity 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, including 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. Therefore, in the event that CV127 soybeans were to be introduced into 

the United States environment, the data generated 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 assessment of CV127 for the United States 

environment. 

CV127 soybean was compared to the isoline control and two other conventional soybean 

varieties with respect to key vegetative and reproductive development 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 stages, plant height, and 

grain yield, as well as susceptibility to and interactions 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 conventional soybean varieties. Furthermore, 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 germination studies, the two other conventional soybean 

varieties. 
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Results of these studies demonstrated that, except for herbicide tolerance, soybean CV127 is 

agronomically, phenotypically, and phenologically equivalent to the isoline control and other 

conventional soybean varieties. Further, these studies showed no different biological effect of 

CV127 compared to the isoline control soybeans with respect 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 conventional soybean varieties. 

The treatments included in the seed germination, dormancy and quality studies, as well as in the 

field trials used 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) 

The methods as well as results for all treatments are included in Appendices E and F to this 

petition. However, because the objectives of the evaluations presented in this section of the 

petition were to compare the intrinsic agronomic, phenotypic and ecological characteristics of 

CV127 to those of the isoline control 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 used in these studies are shown below. 

Comparative Analyses Performed, Generations 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 

F7 (CV127 line 127) 

Isoline Control (F7 null) 

Monsoy 8001 

Coodetec 217 

Pollen Number and Germination: 

Greenhouse-Grown, Londrina, 

Brazil, 2007. 

F5 (CV127 line 127) Isoline Control (F5 null) 

Agronomic, Phenotypic and 

Ecological Evaluation: 

Field Trials 2006/07 

F5 (CV127 line 127) 

Isoline Control (F5 null) 

Monsoy 8001 

Coodetec 217 

Agronomic, Phenotypic and 

Ecological Evaluation: 

Field Trials 2007 

F6 (CV127 line 127) 

Isoline Control (F6 null) 

Monsoy 8001 

Coodetec 217 
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A. Seed Germination, Dormancy and Quality Characteristics 

Assessments of seed germination, dormancy and quality were compared between CV127 

soybean, the isoline control and two conventional 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 (Anderson, 1996; Lingenfelter and Hartwig, 2003). An 

assessment of seed dormancy is often used to assess the weediness potential of different plant 

species (Baker, 1974). Although soybeans do not typically display seed dormancy 

characteristics (OECD, 2000), the potential for seed dormancy in CV127 soybean was assessed 

and compared to that of the isoline control as well as two conventional soybean varieties 

(Monsoy 8001, Coodetec 217 [CD 217]). 

Data are presented in this section for comparisons 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 control and the two conventional soybean varieties produced with the 

same conventional herbicide application. Seed samples 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 experimental unit (four sub-samples of 50 seed derived 

from each replicate treatment at each field trial 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 initiation of the tests, the 

identities of the test (CV127), control (isoline 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 documentation 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 remained 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 soybean. Any seed that did not germinate and did not fall into the 

“abnormal”, “hard” or “firm” seed coat categories were designated as “dead” seed. Hard seed 

was determined at the final reading of the germination test and the percentage of hard seed was 

added to the germination percentage, 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 towel at 25oC for 16 h in an environment with a 

relative humidity of 100%. After conditioning, the seed were placed in plastic cups, and covered 

with 0.075% tetrazolium solution. The cups were incubated in an oven at 40oC for 150 minutes. 
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After staining, the seed were rinsed with tap water several times to stop the staining reaction. 

Seed were kept submerged in water to avoid dehydration. Individual seed were examined with a 

magnifying lens (6X) under fluorescent light. Seed were dissected longitudinally 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 outer surface of the cotyledons. 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 evaluation of the radiclehypocotyl axis and the vascular region on the cotyledons, which are structures of major 

importance for the viability of the seed. After the 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 tetrazolium-staining tests were statistically analyzed 

by Tukey’s test (Steel and Torrie, 1980) to compare 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 the 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 description 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 and 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 significant differences between 

the treatments across field trial locations 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 germination within each treatment were comparable, 

with the lowest levels associated with conventional 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 results demonstrate that seed germination, dormancy 

and other seed quality parameters of CV127 soybean are not different from those of conventional 

soybean varieties. Furthermore, these results support the conclusion that CV127 soybean does 

not pose any greater weediness potential or any different potential environmental impact than 

conventional soybean varieties. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table VIII-1. Means per Treatment Across Locations for Seed Quality, Germination and Dormancy TIsoline Control and Two Conventional Soybean Varieties. 

 Traits Assessed 1

Tetrazolium Test2 Germination Test 

Dmec Dweath Dstink Viab Germ Abnor Dead Hard 

(6-8) (6-8) (6-8) 

CV127 3.17 ab3 2.65 a 3.09 a 91.00 a 88.48 a 8.22 a 3.30 a 0.04 a 

Isoline 4.91 a 2.48 a 1.83 a 90.78 a 87.43 a 9.13 a 3.43 a 0.72 a 

Monsoy 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 

CD 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 

1Dmec: mechanical damage (%); Dweath: weathering damage (%); Dstink: damage by stink bugs (%); Viab: seed viability (%); Ge

Abnor: abnormal seedling (%); Dead: dead seed (%); Firm: firm seed (%); Hard: hard seed (%). 

2 Seed receiving a rating of 6 to 8 in the assessment are considered nonviable (Franca Neto et al., 1998). The cause of the nonviability 

investigated 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). 
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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 stigma 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 pollinate the stigma of the same flower and as a result, soybeans 

exhibit a high degree of self-fertilization and cross-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 isoline 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 conditions to stimulate germination and pollen 

germination was assessed. The results of this study demonstrate that there is no difference in 

pollen number or pollen germination between the imidazolinone-tolerant CV127 soybean or its 

isogenic, non-transgenic counterpart (isoline control). 

Plants of CV127 soybean (F5 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 flowers from each of five different plants one day 

before anthesis from both CV127 and isoline control plants to determine the number of pollen 

grains produced per flower. The anthers were collected 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 Eppendorf 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 sucrose solution (0.45 M) and shaken slightly. The 

solution containing the pollen grains from each plant was allowed to stand for approximately 3 

hours. A grain of pollen was considered germinated when the germination tube length was equal 

to the grain diameter. Germinated pollen grains were counted in a Neubauer chamber and the 

number of pollen grains that produced a pollen tube and the mean number of germinated pollen 

grains were determined (Figure VIII-1). In addition, 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. Statistical analysis of the data was by the t-test 

(Steel and Torrie, 1980). 
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Figure VIII-1. Counting the Pollen Grains (A) and Germinated 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 soybean 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 P2 

 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 plants, and results are presented in Table VIII-3. Statistical 

analysis of the data was by the t-test (Steel and Torrie, 1980). Results showed 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 pollen tube was measured for 20 pollen tubes of both CV127 soybean and the 

isoline control and results are presented in Table VIII-4. Results showed no statistically 

significant difference at the 5% level between pollen tube length of CV127 soybean compared to 

the isoline control. 

A B 
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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 pollen 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 P2 

 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.f1 P2 

 0.4104 8 0.6849 

1

d.f.=Degrees of freedom 

2

P=Probability 
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C. Agronomic and Phenotypic Evaluations in Replicated Field Trials 

Plant growth, development and yield characteristics were assessed under field conditions for 

CV127 soybean compared to the isoline control and two conventional 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 northern regions of Brazil that have a 

longer growing season. A similar cultivation practice 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 planted to soybeans in the U.S. followed this 

practice (CTIC, 2008). The locations and planting 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. 101 

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 F5 and F6 generations used in the 2006/2007 and 2007 

seasons, respectively; refer to breeding diagram 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. 
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 An isoline control that is a null segregant of CV127 soybean and lacks the csr1-2 gene 

cassette but is from the same stage of breeding as the CV127 soybean was included in the 

trial. Weed control was accomplished using the conventional soybean herbicide Volt® as 

described above. 

 Two conventional soybean varieties were used in these field trials: Monsoy 8001 and 

Coodetec 217 (CD 217). Both of these varieties are common nontransgenic Brazilian 

commercial varieties with a similar maturity classification as CV127 soybean (Maturity 

Groups VIII-IX). Weed control was by application 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 control was included in the study and the associated statistical analysis, and 

phenotypic and agronomic evaluations of this treatment are presented in Appendix F. However, 

to compare only the treatments cultivated in a similar manner, only the data for those treatments 

treated with the conventional 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 vigor, plant height, green 

stem, degree of shattering and lodging, days to full flower, days to full maturity, seed size and 

grain yield. Statistical analysis of the resulting data from all treatments was conducted by 

Tukey’s test (Steel and Torrie, 1980) to compare means of sources of variation determined by 

ANOVA to have a significant effect on each measured trait. 
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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 appearance 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 planting to full plant maturity 

Plant Height R8 Average height of five 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 (lodging) of plants in a plot 

according to the scale: 

 1=All plants standing 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 grain harvested (adjusted to 12% 

moisture) 

100-Seed 

Weight 

R8 Weight (g) of 100 seeds 
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Due to the naturally different environmental characteristics (temperature, rainfall, humidity, day 

length, etc.) among the trial locations, location had a significant effect 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 southerly location (latitude 25o S) and the location for which 

the Conquista soybean variety is least adapted, plants were taller and the number of days to 

flower and to maturity were higher than at all other locations during the 2006/2007 season (Table 

VIII-7). Yield was not recorded from this site due to a necessary late replanting of the study at 

this site and subsequent uncharacteristic plant growth and late maturity of the CV127, the isoline 

control, and the conventional soybean 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 other 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, seedling 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 location 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 control were observed for plant height, lodging, days 

to full flower, days to maturity and yield. Due to the different genetic backgrounds of CV127 

soybean and the isoline control compared to the two conventional soybean varieties, these 

differences were not unexpected. 

In the trials of the 2007 season, plant density was nearly ideal at all locations, 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, except at Sete Lagoas, where plants reached a 

mean stature of 82 cm and the lodging reached the 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 planting 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 relatively short (less than 115 days) in these two sites and also in 

Uberaba and Sete Lagoas. The yield was relatively high at Brasilia and Sete Lagoas, medium at 

Teresina and Uberaba and lower at Santo Antonio de Goiás and Vilhena (Table VIII-8). The 

wide-ranging performance obtained for grain yield 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 and within an acceptable range. 

In general, within locations, the performance of CV127 soybean treated with Volt® and the 

isoline control in the 2007 trials was not significantly different for germination, final stand, 

seedling vigor, lodging, days to flower, and days to maturity (Table VIII-8). CV127 soybean 
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treated with Volt® and the isoline control plants only differed 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 maturity. The conventional soybean varieties matured one to four 

days earlier in some locations and 100-seed weights were generally less in relation to CV127 

soybean and the isoline control. Due to the different genetic backgrounds of CV127 soybean and 

the isoline control compared to the two conventional soybean varieties, these differences were 

not unexpected. 

Although some statistically significant differences did exist between CV127 soybean and the 

isoline control for specific phenotypic and agronomic traits at specific locations in the trials 

conducted during the 2006/2007 and 2007 growing seasons, none of the trait differences were 

consistent across all locations and years, and therefore were not considered biologically 

significant. Furthermore, all trait values for CV127 soybeans and the isoline control were within 

the range for soybeans commercially cultivated in Brazil and elsewhere, including the 

conventional soybean varieties included 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 conventional soybean varieties. 
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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. 
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Table VIII-8. Mean Values for Phenotypic and Agronomic Characteristics of Each Combination of Trefor all Trials Conducted in the 2007 Season in Brazil. 

Trait†

Location Variety 

G IS FS V PH GS L DF DM 

CV127 70.25 84.00 76.50 5.00 a 48.80 ab 47.50 b 1.00 a 45.00 b 109.00 a 

Isoline 73.75 88.75 77.50 5.00 a 54.05 a 25.00 c 1.00 a 45.00 b 109.00 a 

M8001 78.00 93.50 88.00 5.00 a 46.90 b 22.50 c 1.00 a 46.00 a 106.00 b 

Uberaba 

CD217 76.50 92.00 79.25 5.00 a 38.50 c 1.00 d 1.00 a 46.00 a 106.25 b CV127 77.75 93.25 86.00 5.00 a 81.85 bc 35.00 bc 4.00 a 48.50 ab 114.00 a 

Isoline 77.25 93.00 85.75 5.00 a 91.75 a 23.75 c 4.00 a 49.00 ab 114.00 a 

M8001 78.00 93.50 90.75 5.00 a 85.55 ab 42.50 b 2.75 a 49.25 ab 112.25 b 

Sete Lagoas 

CD217 78.75 94.75 90.25 5.00 a 74.35 d 5.50 d 3.00 a 49.75 a 112.00 b CV127 94.50 113.25 113.13 5.00 a 58.00 a 10.00 a 1.00 a 44.00 a 131.00 a 

Isoline 93.25 111.63 110.38 5.00 a 58.00 a 10.00 a 1.00 a 44.00 a 130.50 a 

M8001 92.50 110.88 109.50 5.00 a 48.50 b 10.00 a 1.00 a 44.00 a 129.25 b 

Sto. Ant. de Goiás 

CD217 93.00 111.38 109.88 5.00 a 48.00 b 10.00 a 1.00 a 44.00 a 129.00 b CV127 90.00 108.00 107.63 5.00 a 59.00 a 5.00 a 1.00 a 49.00 a 128.00 a 

Isoline 87.25 104.88 103.50 5.00 a 58.75 a 5.00 a 1.00 a 49.00 a 127.25 a 

M8001 87.75 105.50 104.25 5.00 a 45.75 b 0.00 b 1.00 a 49.00 a 125.00 b 

Brasília 

CD217 91.50 109.75 108.38 5.00 a 45.00 b 0.00 b 1.00 a 49.00 a 124.75 b CV127 83.25 86.50 77.00 5.00 a 60.00 a 60.00 b 1.00 a 37.50 ab 99.50 bc 

Isoline 88.00 92.00 86.25 5.00 a 58.25 a 21.25 c 1.00 a 38.00 ab 98.00 bc 

M8001 96.50 100.50 93.25 5.00 a 54.25 a 90.00 a 1.00 a 35.25 c 104.75 a 

Vilhena 

CD217 89.25 93.25 87.25 5.00 a 41.25 b 3.25 c 1.00 a 39.00 a 97.00 c CV127 41.50 49.88 49.50 3.75 a 56.50 a 10.00 a 1.00 a 40.00 a 111.00 a 

Isoline 48.75 58.25 56.88 4.25 a 55.00 a 10.00 a 1.00 a 40.00 a 111.00 a 

M8001 70.25 84.25 82.88 4.25 a 49.75 b 0.00 b 1.00 a 38.00 b 107.00 b 

Teresina 

CD217 70.50 84.50 81.00 4.00 a 50.25 b 0.00 b 1.00 a 38.00 b 107.00 b 

†G = initial germination (%); IS= initial stand; FS = final plant stand; V = seedling vigor; PH = plant height (cm); GS = green stem (%

DF = days to full flower (vegetative cycle); DM = days to full maturity (total cycle); 100SW = seed size (weight of 100 seed, grams); Y

‡ Means followed by the same letter do not differ significantly by the Tukey test at 5% probability. 
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D. Ecological Evaluations 

Assessments of interactions between CV127 soybeans and the environment 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 diseases, insect pests and characteristics 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 isoline 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 diseases 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 were observed at the V4 stage with the exception 

of powdery mildew in Santo Antônio de Goiás in 2007. Due to the potential in Brazil for fungal 

diseases 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. 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 significant, Tukey’s procedure (Steel and Torrie, 

1980) was used to make pair-wise comparisons of treatment means. 

Other than a high incidence of Asian rust at several locations, the incidence of other diseases was 

generally low during both growing seasons (Tables VIII-9 and VIII-10). Although a few isolated 

differences in disease susceptibility 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 susceptibility differences were observed, they were 

generally between varieties with the Conquista genetic background (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 demonstrated 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 assessment methodology is included in Appendix F. 
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Table VIII-9. Disease Assessments for CV127 Soybean, the Isoline Control, and Conventional Soybea8001 and CD217) during the 2006/2007 Field Season. Evaluations were conducted at seven field trial lo

presented for three growth stages1 (R1, R5 and R7). Values are mean percent affected leaf area.

Location Treat- R1 R5 R

ment PM† DM DC AR PM DM DC AR PM DM 

CV127 6 a‡ 2 -§ - - - 5 6.0 ab - - 

Isoline 7 a 2 - - - - 5 6.0 ab - - 

M8001 16 b 2 - - - - 5 5.3 b - - 

Ponta Grossa 

CD217 8 a 2 - - - - 5 4.8 b - - CV127 - 1.5 - 2.0 c 2 - - 51 a - - 

Isoline - 1.8 - 2.0 c 2 - - 51 a - - 

M8001 - 1.8 - 2.8 b 2 - - 44 b - - 

Londrina 

CD217 - 1.8 - 4.0 a 2 - - 30 c - - CV127 - - - 6.8 - 2.0 - 45 a - - 

Isoline - - - 7.3 - 1.5 - 50 a - - 

M8001 - - - 7.5 - 1.8 - 30 b - - 

Sto. Ant. de Posse 

CD217 - - - 6.9 - 2.8 - 43 a - - CV127 - - - - 1.5 - - 2.0 - - 

Isoline - - - - 1.5 - - 2.0 - - 

M8001 - - - - 1.5 - - 2.0 - - 

Uberaba 

CD217 - - - - 1.5 - - 2.2 - - CV127 - - - 1.5 - - - 2.5 - - 

Isoline - - - 1.5 - - - 2.5 - - 

M8001 - - - 1.5 - - - 2.5 - - 

Sete Lagoas 

CD217 - - - 1.5 - - - 2.5 - - CV127 - - - 2.0 - - 0.6 ab 42 a - - 

Isoline - - - 1.7 - - 0.4 ab 40 a - - 

M8001 - - - 1.9 - - 1.1 a 19 b - - 

Brasilia 

CD217 - - - 1.8 - - 0.0 b 19 b - - 

CV127 - - - 1.0 - - 0.5 17 - - 

Isoline - - - 0.9 - - 0.1 19 - - 

M8001 - - - 0.6 - - 0.3 18 - - 

Sto. Ant. de Goiás 

CD217 - - - 0.8 - - 0.6 18 - - 

1Disease assessments were also conducted at the V4 growth stage but no diseases were observed at any location. † Diseases: PM = Powdery mildew (Erysiphe diffusa); DM = Downy mildew (Peronospora manshurica); DC = end of cycle di

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. 
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Table VIII-10. Disease Assessments for CV127 soybean, the Isoline Control, and Conventional Soybea8001 and CD217) during the 2007 Field Season. Evaluations were conducted at six field trial locations in Br

growth stages V4, R1, R5 and R7. Values are mean percent affected leaf area.

Location Treat- V4 R1 R5 

ment PM† PM DM DC AR PM DM DC AR PM D

CV127 -§ - - - 3.7 - - - 8.6 - 

Isoline - - - - 3.6 - - - 7.6 - 

M8001 - - - - 3.6 - - - 8.0 - 

Vilhena 

CD217 - - - - 2.1 - - - 5.2 - CV127 - - - - - - - - - - 

Isoline - - - - - - - - - - 

M8001 - - - - - - - - - - 

Teresina 

CD217 - - - - - - - - - - CV127 - - - - 1.5 - - 1.1 ab 0.7 - 

Isoline - - - - 1.5 - - 0.9 ab 0.6 - 

M8001 - - - - 1.5 - - 1.2 a 1.0 - 

Uberaba 

CD217 - - - - 1.5 - - 0.8 ab 0.8 - CV127 - 0.6 0.8 b‡ - - 0.8 - 

Isoline - 0.6 0.7 b - - 1.0 - 

M8001 - 0.6 1.7 a - - 0.7 - 

Sete Lagoas 

CD217 - 0.5 0.6 b - - 0.6 - CV127 - 1.6 - - - - - - 0.9 - 

Isoline - 1.3 - - - - - - 0.7 - 

M8001 - 1.6 - - - - - - 0.9 - 

Brasilia 

CD217 - 1.6 - - - - - - 1.4 - CV127 5 7.1 - - - - - - 2.2 - 

Isoline 5 6.6 - - - - - - 2.5 - 

M8001 5 6.3 - - - - - - 2.1 - 

Sto. Ant. de Goiás 

CD217 5 6.4 - - - - - - 3.1 - 

† Diseases: PM = Powdery mildew (Erysiphe diffusa); DM = Downy mildew (Peronospora manshurica); DC = end of cycle d

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. 
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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 these 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 seasons in Brazil. Insects on the plants 

were collected in the field by placing a shake cloth on the ground between the center two rows of 

each plot. The plants in the two adjacent rows were bent over the cloth and were shaken 

vigorously to remove all insects within the canopy. All insects thus collected were counted and 

identified by order. In addition, insect feeding damage on the leaves, stems and pods was 

evaluated at different stages of plant development according to the following rating scale. Insect 

populations as well as insect feeding damage were assessed on all plots at each field site at the 

V1, R1, R5 and R7 stages of plant development. Data were analyzed by ANOVA on a per 

location, per insect order, and per time of assessment basis. In those cases where differences 

among treatments were significant, Tukey’s procedure (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 different from numbers detected on plants 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 (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). 

Leaf, stem, or pod damage from insect feeding was below 2% for all soybean varieties across all 

treatments and locations during both growing seasons. 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 these 
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evaluations, in-field insect populations and resistance to insect damage of CV127 soybeans was 

found to be no different from that of the isoline control soybean or the conventional soybean 

varieties. 
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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 locations and growth stages. 

Growth Stage 

V1 R1 R5 R7 

Location Insect Order 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 0.25 
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Table 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. No significant differences were determined 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, where numbers followed by different letters are 

statistically significantly different 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 0.71 
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D.3. Nitrogen Fixation Characteristics 

An important agronomic characteristic of soybeans 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 creates an environment within the nodule that is 

conducive for nitrogen fixation by the endosymbiont. Through this symbiosis, B. japonicum is 

able to convert atmospheric nitrogen to fixed forms of nitrogen and reduce the soybean host’s 

dependency on nitrogen fertilizer. It is a common practice in Brazil and Argentina for farmers to 

inoculate soybean seed with B. japonicum prior to planting to ensure the development of 

effective root nodulation and strong 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 isoline control and conventional soybean varieties, including 

measurements of soil populations of B. japonicum, plant growth and nodulation and total 

nitrogen and ureide nitrogen accumulation 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 Brazil which are representative of areas of commercial soybean 

production and to which the soybean varieties included in the study are adapted. Identical 

evaluations of the B. japonicum nitrogen fixing symbiosis were also made at six trial locations in 

the 2007 short growing season in Brazil. The field trial locations are the same as described in 

section VIII.C. (“Agronomic and Phenotypic Evaluations 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 Significant Difference (LSD) statistical procedures, 

and across locations using the Tukey's test (Steel and Torrie, 1980) 

The results of these studies showed that CV127 soybean is equivalent to the isoline control with 

respect to biological nitrogen fixation, including nodule number, 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-ureide 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 differences in nitrogen fixation parameters, which 

were attributed to basic genetic differences between these soybean varieties in biological 

nitrogen fixation capabilities. A more detailed description of the methodology used in the study 

and the results obtained are presented in Appendix F. 
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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 performed in the summer of 2006/20071

. 

Treatment Parameter 

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 replicates, 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 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 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 locations in the 2007 short growing season1. 

Treatment Parameters 

NN NDW SDW CNS TNS %NU TNU 

CV127 29.4 150.0 a1

 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 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 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 N-ureide content in the 

above-ground portion of the plant or shoot (TNU) for treatment comparison purposes. 
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E. Conclusions from Agronomic, Phenotypic and Ecological Evaluations of CV127 

Soybeans 

The purpose of the studies summarized in this section was to: 

1) Evaluate the agronomic, phenotypic, and phenologic characteristics of CV127 soybean 

relative to an isoline control and to other conventional soybean varieties; 

2) Determine the ecological impact (interaction with diseases, insects and factors relevant to 

nitrogen fixation) of cultivating CV127 soybean relative 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 two other conventional soybean 

varieties with respect to key 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 interactions 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 conventional 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 interactions, seed germination, pollen number and 

pollen germination characteristics, or ability to support a nitrogen fixing symbiosis with 

Bradyrhizobium japonicum than do the isoline control 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. Further, where seed size 

differences were observed between CV127 and the isoline control, this did not confer a selective 

advantage with respect to germination, 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 antinutrients of the grain and forage derived from 

CV127 soybean and the isoline control and conventional soybean varieties (section VII). Results 

of these studies demonstrated that grain and 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 greater environmental or plant pest risk than 

does the cultivation of conventional soybeans. 
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IX. Environmental Assessment of CV127 Soybeans 

The potential for CV127 soybean to exhibit plant pest or weediness characteristics as well as an 

evaluation of interactions of CV127 soybean with both pest and non-pest organisms in the 

environment are discussed in this section. The potential for gene flow from CV127 and the 

impact of the cultivation of CV127 soybeans in the environment are also addressed. 

CV127 soybean was developed for cultivation primarily 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 attractive proposition for 

soybean cultivation in South America. Therefore, regulatory approvals for this product are 

sought in Brazil and Argentina for production as well as for food and feed uses, and in the U.S. 

and other countries for importation 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 introduced in to the U.S. environment, CV127 soybeans would 

present no different environmental impact compared to conventional soybean varieties with 

regard to cultivation practices, interactions with pest and non-pest organisms, and weed control 

measures, other than the ability to use imidazolinone herbicides for weed control in the CV127 

crop. 

A. Safety of the Imidazolinone-tolerance Trait 

The imidazolinone herbicides inhibit plant growth due to their inhibition of a plant enzyme, 

acetohydroxyacid synthase (AHAS), which has an important 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 mutations 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 asparagine 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-tolerance to other AHAS inhibitors such as the sulfonylurea herbicides 

(Lee et al., 1999). To date, imidazolinone-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 developed through mutagenesis 

or introgression from natural sources, selection, 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 present in CV127 soybean. These crops have been cultivated for many years 

without adverse environmental impact and without the development of weediness or other plant 

pest characteristics (Tan et al., 2005). Furthermore, the grain and other food or feed components 
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produced from the Clearfield® crops described above have been demonstrated to be as nutritious 

and as safe as the same food or feed components 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, CV127 soybean was compared to the 

isoline control and two other conventional soybean varieties with respect to key vegetative and 

reproductive development characteristics associated with the competitiveness and survival of 

plant species (section VIII of this petition). Also, CV127 was assessed for disease susceptibility 

and interactions with different orders of insects, as well as the impact of cultivating CV127 

soybeans on factors relevant to the nitrogen fixing symbiosis with Bradyrhizobium japonicum. 

Data from these studies demonstrated that CV127 soybean has no different agronomic or 

phenotypic characteristics, disease or insect interactions or ability to support a nitrogen fixing 

symbiosis with Bradyrhizobium japonicum than do conventional soybean varieties. In 

combination, all of these studies 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 varieties. Also, extensive analyses of the nutrient and antinutrient content 

of the grain and forage produced from CV127 soybeans 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 protein 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 herbicides. The AtAHAS protein expressed in CV127 

soybean is structurally and biologically closely related to the AHAS proteins found in all plants. 

For example, there is 79% amino acid identity between the AtAHAS and endogenous soybean 

AHASL2 (JGI locus Glyma13g31470.1) proteins. Also, the AtAHAS protein expressed in 

CV127 soybean does not share any biologically relevant amino acid sequence homology or 

biological characteristics with known toxins or allergens. Finally, the source organism of the 

csr1-2 gene that encodes the AtAHAS protein in CV127 soybean, Arabidopsis thaliana, is not a 

known human or animal pathogen and is not known to cause allergic reactions in humans. 

Similarly, A. thaliana is not known to produce toxic substances. Therefore, results of these 

studies show that neither the AtAHAS protein nor CV127 soybeans are likely to pose any 

increased safety risk to other plants and organisms in the environment compared to other plantproduced AHAS proteins or other conventional soybean plants. 
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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 disperse, 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 characteristics and only under certain environmental 

conditions will they grow as a volunteer in the year following cultivation (Raper and Kramer, 

1987; CFIA, 1996; OECD, 2000). In managed 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 soybeans 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 crop such as winter wheat is planted after soybean harvest, it is 

possible for soybean seed to be incorporated into the soil and to germinate that fall. These 

volunteer plants will be controlled 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 available to control 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 active ingredients applied to corn in the United 

States in 2010 are listed in Appendix G, Table G-1; there were 58 active ingredients reported as 

used on corn. Herbicides from group 4 (auxinic 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 volunteers were to become established, they would not compete well 

with succeeding crops and they could be controlled by either mechanical means or by herbicides 

other than the group two herbicides that target the AHAS enzyme. 

In Roundup Ready® (RR) corn, volunteer conventional or imidazolinone-tolerant soybeans will 

be controlled by any of the glyphosate 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 

soybeans. 
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The agronomic evaluations that were conducted with CV127 soybean in two growing seasons 

suggest no different characteristics compared to conventional soybeans that would impart the 

ability to be invasive or weedy. CV127 soybean was equivalent to the isoline control and to two 

conventional soybean varieties with respect to seed germination, quality and dormancy, as well 

as pollen number and pollen germination and key phenotypic characteristics including seedling 

vigor, days to flowering and to maturity and seed production. Also, no differences in the 

frequency of volunteers between CV127 soybean, the 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 support for the conclusion that CV127 soybean has no different 

survivability or persistence characteristics than conventional soybean varieties. Also, since wild 

species of Glycine are not indigenous to, or known to exist 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 species. Based upon the results of these studies it 

was concluded that CV127 soybean has no increased 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, viruses, 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 

symbiosis with Bradyrhizobium japonicum, a plant-associated nitrogen-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 interactions 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 soybean 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. Therefore, these results further confirmed that 

cultivation of CV127 soybeans poses no different environmental impact than the cultivation of 

conventional soybean varieties. 
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B.3. Potential for Gene Flow in Soybean 

Soybean is considered to be a self-pollinating species that is propagated commercially by seed. 

Commercial cultivars are developed through breeding by artificial hybridization. The longevity 

of soybean pollen is generally two to four hours at most (Andersson and 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 (Carlson and Lersten, 1987; Carlson and Lersten, 2004). Typically 

the anthers mature in the bud and directly pollinate the stigma of the same flower. 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 morphological characteristics that encourage the spread of the 

seed by animals. 

For a trait to become incorporated into 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 Yangtze river valley in northeast China and areas near the border 

with Russia. G. gracilis has been observed growing in northeast China (Skvortzow, 1927). 

Interspecific fertile 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 perennial 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 Islands, China, Papua New Guinea, the Philippines, 

and Taiwan (Hermann, 1962; Newell and Hymowitz, 1978; Hymowitz and Newell, 1981; Grant, 

1984; Tindale, 1984 and 1986). Hybridization 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, intersubgeneric 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 hybridization 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 these intersubgeneric hybrids were obtained with difficulty and only 

with human intervention and all resulting progeny were sterile. Soybean is not able to cross with 

other plants outside of the Glycine subgenus Soja and so intergeneric hybridization does not 

occur. 
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The potential for the genetic transfer of the imidazolinone-tolerance 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 South America. Consequently, the possibility of natural 

gene transfer between CV127 soybeans and compatible 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, the acquisition of the imidazolinone-tolerance trait would not be 

expected to enhance the weediness of the recipient plants. Such hypothetical imidazolinonetolerant weedy plants could be controlled by many other herbicides other than those of the 

imidazolinone class or by cultivation. 

There is no evidence that genetic information can transfer from soybean to other organisms that 

cannot sexually interbreed with soybean (horizontal gene transfer). Therefore, genetic 

information in CV127 soybean will not be transferred by horizontal gene flow to other organisms 

in the environment. 

C. Cultivation of Soybean 

Soybean is the leading oilseed crop produced and 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 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 and 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 producer of soybeans and in 

2010 it produced 90.6 MMT, 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 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 support 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 and other crops. Studies have demonstrated that 

the rotation of soybean with 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 

reduction of pest and weed cycles. 

For many years, genetically modified (GM) soybean 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 planted to soybeans (James, 2010). The vast majority 

of this acreage was planted in herbicide-tolerant soybean varieties. In 2010, 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 rapid and widespread adoption of 

herbicide-tolerant soybeans by growers worldwide has been due to increased yields, the reduced 

cost of effective weed control, and a simplified, more flexible weed control program (Reddy, 

2001; Gianessi, 2005) that is offered by the cultivation of herbicide-tolerant crops. Perhaps more 
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importantly to the grower, the benefits from the 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 herbicide-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 today are tolerant to the herbicide glyphosate. 

Glyphosate-tolerant soybeans were first grown commercially 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 glyphosate (Heap, 2011; Nandula et al., 

2005). It should be noted that most of the cases of glyphosate-resistant weeds occurred in nontransgenic crops. Exposure to glyphosate started well before the introduction of glyphosatetolerant crops and thus, attributing the development 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 grower with the opportunity to cultivate 

soybeans that are tolerant to a herbicide with a different mode 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 classes 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 world behind the U.S. The first soybean varieties 

produced in Brazil were varieties that were bred for production and commercialization 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 all major agricultural regions of Brazil. Today, 

many of the most productive Brazilian 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 February or early March with harvest in July. The 

cultivation of soybean plants in Brazil is similar to the cultivation of soybean plants in the U.S. 

as the plant’s requirements from the environment are not different. However, the input 

requirements to produce a crop in Brazil may be different from those required to produce a crop 

in the U.S. due to environmental differences that may exist between the two countries. Soybeans 

are commonly grown in rotations with grain crops such as maize or winter wheat. The use of 

reduced tillage that has been facilitated by the introduction of herbicide-tolerant varieties of 
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soybean and the correction of soil acidity and improved seed quality 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 worldwide (Soy Stats, 2011). There are many 

similarities in soybean production in Argentina and Brazil, including the fact 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 cultivated 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 grown as a full-season crop with the remaining 35% 

being double cropped with wheat. Soybean is also 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 largest producer of soybeans. 

As in the other major soybean producing countries of Brazil and Argentina, production has 

increased dramatically in the U.S. in recent years. 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 primarily in the eastern half of 

the U.S. from the Gulf of Mexico to the Canadian 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 crop that is seeded in the spring, but a small percentage of the 

crop is planted mid-June to early July as a second crop following winter wheat, rice or winter 

canola in the more southerly regions of the U.S. 

Soybeans with the non-recombinant DNA-derived STSTM trait are tolerant to sulfonylurea 

herbicides. The STSTM trait does not provide cross-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-tolerant weeds are present. [ 

 ] 

All of the GM soybeans commercially planted to date in the U.S. contain herbicide tolerance 

traits (Table IX-1, Table IX-2). In 2011 approximately 94% of the U.S. soybean crop was 

genetically modified for herbicide tolerance (USDA-ERS, 2011a). Since 2007 over 90% of the 

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soybean cultivated in the U.S. in recent years contains one or more biotechnology-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-derived soybeans planted in the U.S. 

As in other regions of the world where herbicide-tolerant soybeans 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 cultivation practices have lead to reduced: i) soil 

erosion, ii) contamination of surface waters with agricultural fertilizers and chemicals, iii) fuel 

consumption, and iv) release of greenhouse 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 

20111?

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 no2

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 soybeans is anticipated in 2012, upon full regulatory 

approval. Source: http://www.plenish.com/about plenish.aspx, accessed September 3, 2011.
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Table IX-2. Planted Acres of Soybeans and of Biotechnology-Derived Soybeans in the 

United States in 2001 through 2011 (USDA-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-herbicide 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 soybeans that are cultivated currently in the U.S. are tolerant to 

glyphosate, this herbicide is the leading 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 and 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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C.4. Comparison of the Cultivation of Soybean in Brazil and the U.S.

The following section reviews the similarities between soybean genotype, environment, and 

agronomic practices used in soybean production in Brazil and the U.S. This comparison 

demonstrates that the data generated in field 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 assessments 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 earliest maturity group (000 to II) are adapted to the northernmost soybean 

production regions of North America and the southernmost production regions of South America. 

Varieties in the latest MG (VII-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 developed 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, using classical methodologies, 

routinely recombine genes at the E locus to modify time to maturity 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 indeterminate 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 maximize production in a specific region. For example, 

in a study which included later-maturing determinate and indeterminate varieties adapted to the 

southern U.S., it was concluded that the indeterminate growth 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 variety 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 conditions across the soybean production regions supports the 

production of varieties in MG V to X (Alliprandini et al., 2009). In general, the soybean 
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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 original soybean varieties 

cultivated in Brazil during the early 20th century were varieties developed for cultivation in the 

southern U.S (refer to Figure II-1 as an example 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 programs 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 described in this petition are 

MG VIII. These lines are adapted to the northern soybean production regions of Brazil and the 

southern soybean production regions of the U.S. However, using conventional 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 varieties with determinate or indeterminate 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 Continental United States and Southern Canada 

where Various Soybean Maturity Groups (MG) Are Adapted for Full-Season Growth. 

(Willis, 1989). 
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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 season 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. The average monthly temperatures 

during the soybean growing season in Brazil are less variable across the season compared to 

those in the soybean growing regions of the southeastern 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 cultivated and cities closest to the individual field 

trial locations in Brazil where CV127 soybean was evaluated are shown. 

US Locations1 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 Locations2 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/united-states/weather/, accessed September 21, 2011. 

2

Source: http://www.weatherreports.com/ /Brazil, accessed September 22, 2011. Average 

monthly temperature over five to 36 years, depending on location, is reported. 

There are differences in the total amount of annual rainfall and in the distribution of rainfall 

throughout the year between the regions in Brazil where soybeans are cultivated and the 

southeastern U.S. where maturity group VIII soybeans 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 rainfall in this area ranges from 26 to 60 inches 
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per year. In the savannah, rainfall is highest during the growing season (November through 

March) and is much lower during the winter months (May through September). As rainfall is 

plentiful during the growing season 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 periods to insure that the crop 

receives adequate water for growth. A similar comparison of the monthly average rainfall during 

the growing season in the southeastern U.S. with locations near the individual Brazilian CV127 

field trial locations is shown in Table IX-5. In general, rainfall 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 soybean cultivation in the southeastern U.S. where 

irrigation is not available, soybean yield can be reduced during periods of inadequate rainfall. 

Table IX-5. Average Monthly and Total Rainfall (Inches) during the Soybean Growing 

Season. Different locations in the southeastern United States where soybeans in maturity group 

VIII are cultivated and cities closest to the individual field trial locations in Brazil where CV127 

soybean was evaluated are shown. 

U.S. Locations1 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 Locations2 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/united-states/weather/, accessed September 21, 2011. 

2

Source: http://weather.uk.msn.com/region.aspx?wealocations=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 regions of the U.S., and where differences between 

environments in Brazil and the US were observed, these were generally not different from the 

range of environments encountered across the soybean production regions within the southern 
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US. In any environment, the soybean plant’s requirements for resources from the environment 

are not significantly different. However, in those environments in which certain resources are 

limiting, the input requirements to produce 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 only hydrated oxides of iron and aluminum, are highly weathered 

with low pH and low in fertility and organic matter. The low pH of the soils often limits the 

availability of phosphorus to the soybean plant. 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 other 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 application is not necessary nor 

does it usually result in increased yield (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 optimum planting rate and row-spacing for soybean 

cultivation in Brazil varies depending on the climate of the various soybean cultivating regions. 

A planting rate of 220,000 to 320,000 seed/ha with row spacing of 0.4 to 0.5 m is typical for 

soybean cultivation in Brazil (Guidelines for GAP, 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 temperatures 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 soybeans in MG VII to VIII are 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 inches) 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 diseases (Aref and Pike, 1998). Since weeds 

compete with soybeans for light, nutrients and moisture, weed control is an important factor in 

optimizing yield. The main factors affecting yield 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 crop for the entire growing season, yield losses can 

be as high as 75% of the crop (Dalley et al., 2001). In addition, the impact of different weed 
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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 very competitive with weeds. However, soybeans 

are very sensitive to weed competition in the first weeks after emergence from the soil. It is 

recommended by weed specialists that weeds be controlled in soybeans within three to five 

weeks after emergence in order to prevent irreversible yield losses 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. However, 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 broad-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 adoption of no-till or reduced-tillage cultivation 

systems, the weed spectrum has shifted to favor the winter annual, biennial, and 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 lands in Brazil do not have as long a history of 

farming as those in the U.S. However, the practice of no-till farming in Brazil is common, just 

as it is in the U.S., and weed pressure is expected to increase over time in Brazil. For example, 

soybean farmers in the southern regions of Brazil 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 production in Brazil is that temperatures 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 herbicides 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 the 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 herbicides 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 that were not active on soybeans and their use 

increased dramatically until recently. The use 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 reductions in tractor passes, 

fuel consumption, soil erosion and the release of greenhouse gases. The more recent advent of 

genetically-modified (GM) soybeans that are tolerant to post-emergent treatment with broadspectrum herbicide has further facilitated the adoption of reduced- or no-tillage practices 

(Gianessi, 2005). 

The cultivation of glyphosate-tolerant soybeans treated with glyphosate has been widely adopted 

in the U.S., Argentina, and Brazil. In 2010, 60% of the United States treated acres were treated 

with glyphosate (Table IX-3). Similarly, nearly 100% of the soybean crop in Argentina is 

glyphosate-tolerant and the percentage of cultivation of glyphosate-tolerant soybeans cultivated 

in Brazil is rapidly increasing since regulatory approval in 2005 (approximately 75% in Brazil in 
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2010, [James, 2010]). Therefore, the uses of glyphosate herbicide to control weeds in soybeans 

is the most common practice in both the U.S. and Brazil. These trends 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 warmer 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-resistant 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 populations. Some weeds 

commonly found in soybeans have developed resistance 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 Brazil has led to a shift in the species of 

prevalent weeds 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. An 

effective method of managing and reducing the development 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 insecticides is second 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 three 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 soybeans 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 defoliator, 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 from 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 insect pest since its discovery in the U.S. in 

2000. 
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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 southern U.S., include the 

velvetbean caterpillar and the green stink bug (Turnipseed and 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 Mexico, to southern Brazil and Argentina where stand 

reductions frequently necessitate replanting in late-planted soybean in sandy soils, particularly 

under drier conditions (Turnipseed and Kogan, 1976). While the similarities 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 shown to be no different in its 

agronomic characteristics or environmental interactions compared to conventional soybeans. 

The susceptibility of CV127 to insects was found to be no different in CV127 soybean compared 

to conventional soybean varieties. Protease inhibitors 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 isoline 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 soybean varieties through breeding. 

The strong genetic similarity between soybeans cultivated 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 above considerations and on the 

extensive experience with many different imidazolinone-tolerant Clearfield® crops that have not 

demonstrated different sensitivity to insects compared to their corresponding imidazolinonesensitive conventional counterparts and on the extensive knowledge of the biological mode of 

action of the AHAS enzyme, it is not expected that CV127 soybean would demonstrate a 

different response to insects were it to be cultivated 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 found in both the U.S. and Argentina, Brazil, or 

South America (Tolin and Lacy, 2004). Of the others, five are present in the U.S. but not in 

South America and only two are present 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 reductions (Tolin and Lacy, 2004). 
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Over 40 fungal pathogens of soybean are reported to cause significant disease problems 

worldwide. The environment and crop management practices have a large impact on the 

prevalence and severity of these diseases (Yang and Feng, 2001). A key component in the 

management of soybean diseases is the incorporation of genetic resistance to important 

pathogens through breeding. However, resistance genes are not known for some pathogens and 

for these proper management practices are important. Due to economic considerations, the 

widespread application of foliar fungicides on soybeans in the U.S. is not common, but 

fungicides are commonly applied as seed treatments. In Brazil where Asian soybean rust 

(Phakopsora pachyrhizi) is especially problematic, foliar fungicides are commonly used to treat 

this pathogen. The major soybean pathogens in the top ten soybean-producing 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, effective 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. 
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Table IX-6. Important Viral Diseases of Soybean 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 resistance 
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Table IX-7. Causal Organisms of Commonly-Occurring 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). 

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 major pathogen in recent years. 

Differences in environmental conditions, whether between the U.S. and Brazil or between 

different regions within Brazil or the U.S., may result in differences 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 are 
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applied. In cases of heavy insect infestations, insecticides may be applied. Whether soybean is 

cultivated in the U.S. or in Brazil, it has the same general needs for growth and development. In 

some environments these needs are 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 between the southern U.S. and Brazil, including temperatures and 

rainfall, the maturity groups of soybean cultivated, 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 soybean varieties cultivated in the U.S. and Brazil as well as similarities 

in environmental conditions and agronomic practices that exist between the U.S.and Brazil, 

CV127 soybean would not be expected to present any new environmental risks relative to the 

cultivation of soybean varieties that are currently grown in the U.S. The long history of 

cultivation and safe use of Clearfield® crops also supports the conclusion that the introduction of 

the imidazolinone-tolerance trait into CV127 soybean will have no impact on its interaction with 

disease pathogens or the environment. Based on these considerations, the data generated in field 

studies 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 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 and are currently commercialized under the Clearfield®

brand name. Clearfield® crops are developed by seed mutagenesis 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 previously noted, 

soybean is naturally tolerant to some imidazolinone 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 other crops - field peas, snow peas, dry beans, snap 

beans, adzuki beans, fenugreek, seedling and established 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 Management 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 for use in the control of weeds on pastures and 

peanuts, while imazapyr is registered for use on pastures and corn. A list of the crops in the U.S. 

for which the use of different imidazolinone herbicides is registered and the total area treated is 

presented in Table IX-8. 

[CBI] 

deleted
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BASF developed CV127 soybean for commercial cultivation 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 will confer an incentive to grow the crop outside of 

its normal geographic range or habitat (e.g. cultivated agricultural lands). Furthermore, based on 

the use of imidazolinone herbicides for weed control on the different crops listed in Table IX-8, 

introduction of CV127 soybean in the U.S. is unlikely to have any significant effect on the total 

use of imidazolinone herbicides for weed control in U.S. agriculture. 
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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 excellent weed control options in addition to the 

environmental benefits that result from the cultivation of imidazolinone herbicide-tolerant 

[CBI] 

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soybeans. The imidazolinone herbicides possess 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 Argentina and Brazil has led to a shift in the species of prevalent 

weeds 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, so introduction of 

CV127 soybean will provide growers an effective means 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 cultivate soybeans that are tolerant to a herbicide 

with a different mode 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 

herbicide tolerant. The introduction of CV127 soybean varieties is not only expected to offer 

soybean growers an additional tool for controlling 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 weediness characteristics. CV127 soybean was 

compared to conventional soybean varieties with respect to important agronomic characteristics 

including seedling vigor, days to reach key developmental stages, plant height, disease and insect 

susceptibility, and grain yield. In addition, the impact of cultivating CV127 soybeans on factors 

relevant to the nitrogen 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 comparisons of these characteristics made between 

CV127 soybeans and the isoline control and, for the seed germination studies, two other 

conventional soybean varieties. The results of these comparative 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. 

Although it is not anticipated that 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-sensitive, conventional or glyphosate-tolerant soybean 

varieties. As noted above and elsewhere in this 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, the Brazilian soybean variety, Conquista, that was transformed with 

the csr1-2 gene cassette to produce CV127 soybean is closely 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 would interact with the environment in a similar 

manner to commercial soybeans that are currently cultivated in the U.S. 
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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 considered novel, regardless of the methods by 

which they are developed. These Plants with Novel Traits require review 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 required 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 compared to the herbicide-sensitive crop from which 

they were developed. A list of the Clearfield® crops that have been commercialized 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 tolerance to imidazolinone herbicides, none of these 

crops have demonstrated any different environmental characteristics compared to their 

imidazolinone-sensitive counterparts. Similarly, there is no reason to believe that the 

imidazolinone-tolerant CV127 soybean will present an altered risk to the environment compared 

to conventional soybeans. 

The agronomic and environmental assessments and the results and data from other studies of 

CV127 soybean that are described in this petition 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 imidazolinone-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 shorter growing seasons. Because 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 cultivated in the midwestern 

U.S. In addition, the experience with the safe cultivation of the many different Clearfield® crops 

in the U.S. and other countries supports the conclusion that CV127 soybeans that are adapted for 

cultivation in the midwestern U.S. would not present any different environmental risks compared 

to soybean varieties currently cultivated in this region. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 161 of 375 

Table IX-9. Identity, Year of Market Launch and the CFIA Decision Document Number 

for Imidazolinone-tolerant Clearfield® Crops. 

Crop 

First Market 

Introduction for 

Crop Line Name 

Decision 

Document1

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/english/plaveg/bio/dde.shtml. 

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: 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 162 of 375 

a) CV127 soybean will offer growers an additional tool for weed control as well as 

herbicide resistance management in weeds as part of an integrated weed management 

program, and is not expected to change weed control practices currently followed by 

soybean growers. 

b) CV127 soybean exhibited no different disease susceptibility or insect interactions 

compared to the conventional soybean varieties, so no change in disease and insect 

control practices in each different 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-tolerant crops currently commercialized under the 

Clearfield® brand name have been cultivated in North America since 1992 (Table 

IX-9). Each of these has been evaluated and reviewed by the Canadian Food 

Inspection Agency (CFIA) and has been determined to present no different 

environmental risk compared to their conventional, imidazolinone-sensitive 

counterparts. The collective 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 compared 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 environmental conditions, crop rotation, the numerous 

herbicide control options available, and the fact that soybeans do not compete well 

with the succeeding crop. However, the current practices used for management of 

volunteer soybeans are equally applicable to CV127 soybean. In the event that 

CV127 soybean volunteers were to become established, 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 imidazolinone herbicides in CV127 soybean crops. 

As detailed in Appendix G, grower compliance with this program is essential to delay 

the onset of imidazolinone herbicide tolerance in weeds 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 integrated weed management programs and not 

have any negative impact on integrated weed management programs that growers 

currently follow that include the use of a wide range of herbicides, cultural practices 

and crop rotations to manage weed populations. 

F. Herbicide Resistance Management for Imidazolinone-tolerant CV127 Soybean 

The objectives of herbicide resistance management are to achieve weed control while preserving 

the value of each herbicide and each herbicide group for the longer term. An integrated 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 163 of 375 

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 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, environmentally-sound products for many years. The 

key to the performance of the Imidazolinone-tolerant Soybean Production System is effective 

weed resistance management. BASF is committed to delivering sustainable cropping systems 

that incorporate best practice principles. The Imidazolinone-tolerant Soybean Production 

System provides alternative options for growers within a well-managed rotation. As part of its 

weed resistance management program, BASF has developed a detailed weed resistance 

management plan for implementation with imidazolinone-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-tolerant CV127 Soybean Resistance Management 

Recommendations: 

• DO NOT exceed a maximum of two exclusive AHAS-inhibiting herbicides on any one field, 

in any four-year period. 

• ALWAYS follow an Integrated Weed Management (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 crops that are uncontrolled by herbicides. 

• FOLLOW the Best Management Practices outlined in the Imidazolinone-tolerant Soybean 

Production System Stewardship Guide (presented in Appendix G). 

G. Summary of the Agronomic and Environmental Assessments for CV127 Soybean 

The agronomic characteristics and environmental interactions of CV127 soybean were 

thoroughly evaluated in Brazil during two different growing seasons. In addition, the molecular 

insert in CV127 soybean was completely characterized, 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 potential or environmental risk compared to that 

of conventional soybean varieties. This conclusion is further supported by the long history of the 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 164 of 375 

cultivation of other imidazolinone-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 environmental 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 food safety of the imidazolinone-tolerant AtAHAS 

introduced into CV127 soybean is further supported by the long history of safe consumption of 

food and feed products derived from the many different Clearfield® crops that contain the 

S653N AHAS mutation and by the grain composition studies of CV127 soybean that 

demonstrate that the grain from CV127 soybean is as nutritious as grain from conventional 

soybeans. 

The environmental safety of CV127 was shown to be comparable to conventional soybean 

varieties which do not exhibit plant pest or weediness characteristics. CV127 soybean was 

compared to conventional soybean varieties with respect to important agronomic characteristics 

including seedling vigor, days to reach key developmental stages, plant height, disease and insect 

susceptibility, and grain yield. In addition, the impact of cultivating CV127 soybeans on factors 

relevant to the nitrogen fixing symbiosis with Bradyrhizobium were assessed. Furthermore, 

laboratory- and greenhouse-based studies on seed germination, pollen number and pollen 

germination were conducted and comparisons of these characteristics made between CV127 

soybeans and the isoline control and, for the seed germination studies, two other conventional 

soybean varieties The results of these comparative 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 imidazolinone herbicides for weed control in CV127 

soybeans, the commercial introduction of CV127 soybeans is not expected to change the typical 

agronomic practices used for soybean production, will have no different environmental 

interactions compared to other commercially-produced 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 introduction of imidazolinone-tolerant CV127 soybeans is 

expected to expand the tools available to growers for the management of herbicide resistance in 

weed populations in soybean cultivation. 
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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 different weediness or plant pest potential and no 

different environmental impact than conventional soybean varieties. There are no adverse 

environmental consequences anticipated with the introduction of CV127 soybean. 

The environmental as well as food and feed safety of CV127 soybeans was confirmed based on 

the results of a series of interrelated safety assessment studies. The detailed molecular 

characterization of CV127 soybean confirmed that CV127 contains a single functional csr1-2

gene cassette integrated in the soybean genome. Biochemical characterization of the 

imidazolinone-tolerant AtAHAS protein expressed in CV127 soybean showed that the AtAHAS 

protein is typical of other AHAS proteins in this ubiquitous 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 potential 

environmental impact than cultivation of conventional soybean varieties. Finally, the 

composition and nutritional equivalence of CV127 soybean compared to conventional soybean 

varieties was demonstrated by analysis of key nutrients and antinutrients in both grain and 

forage, and nutritional equivalence 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 release as well as for food and feed uses. 

CV127 soybean was developed for cultivation primarily in Brazil and Argentina. Introduction of 

CV127 soybean will allow growers to treat the soybean crop with imidazolinone herbicides for 

weed control without causing injury to the soybean plant at normal field application rates. 

Introduction of CV127 soybean varieties is expected to offer soybean growers an additional tool 

for controlling weeds, as well as an important option for weed resistance management. 

Furthermore, it is expected that growers planting 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. 
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XII. Appendices 

Appendix A: Materials and Methods for the 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 Determining Levels of the AHAS and SEC61γ 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 Results 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 System 
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Appendix A

Materials and Methods for the Molecular 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 was isolated from leaf tissue of the non-transgenic soybean 

variety Conquista that is closely related to CV127 soybean. All plants were grown under 

greenhouse conditions in Londrina, Brazil, and young, fully-expanded 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 was 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 ethylenediamine tetraacetic acid (EDTA), pH 9.5 (5 

ml/60 mg dried leaf tissue) and β-mercaptoethanol (10 μl/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) added to a final concentration of about 500 ng/μl. 

The isolated DNA was quantified with 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 application. 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 AtAHAS promoter region, probe 2 the csr1-2

coding sequence (CDS), probe 3 the AtAHAS 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 using plasmid pAC321 as a template (Table 

A-1). The probes (25 – 50 ng each) were radiolabeled with 50 μCi of (α32P)-dCTP (3000 

Ci/mmol) (MP Biomedicals; Irvine, CA) using the RediprimeTM II DNA Labeling System 

(Amersham; Piscataway, NJ) according to the manufacturer’s instructions. The labeled 

probes were purified with a Spin-X® Centrifuge Tube Filter (Corning Costar Corporation; 

Acton, MA). 
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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 

AtAHAS CDS Reverse AGCAGGCAGATCAACAAC 4604-4621 

Probe 3 Forward GAACATGTGTTGCCGATGAT 4416-4435 

3' UTR Reverse CGCAACTGTTGGGAAGGG 5949-5966 

Probe 4 Forward GTTTTACAACGTCGTGACTG 5839-5858 

VP1 Reverse CGGTTAGCTCCTTCGGTC 6997-7014 

Probe 5 Forward CACTGCGGCCAACTTACT 6962-6979 

VP2 Reverse CTTGGCGTAATCATGGTC 8592-8609 

Restriction digestions and Southern blot analyses. Southern blot analyses 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 enzymes 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 fragments 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 restriction sites downstream of the AtAHAS 3’ UTR 

in CV127. The XbaI restriction sites flank the complete csr1-2 expression cassette. The 

number and sizes of the DNA fragments expected to be detected by Southern hybridization 

are listed in Table V-1 of the petition. 

Genomic DNA (7 μg) from the F8 generation of CV127 line 603 (Figure III-1 of the petition) 

and from the non-transgenic control Conquista was digested overnight in a volume of 40 μl 

with the restriction enzymes listed above (8 units/μg DNA) under the conditions specified by 

the enzyme manufacturers (New England Biolabs; Ipswich, MA; or Amersham). Two 

additional non-transgenic Conquista genomic DNA samples were spiked with one- and twogenome copy equivalents2

 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 109

 bp (Arumuganathan and Earle, 1991). 

• Plasmid pAC321 is 8669 bp. 

Since the insert in Cultivance® Event 127 is homozygous and 7 μg 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 μg genomic DNA (1.115 X 109

 bp genomic DNA) x 2 

mass of pAC321 DNA = 27 pg 
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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/cm2

) of 

hybridization buffer (2x SSC, 0.6% SDS, 50 mM Na2HPO4, 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/cm2) at room temperature for 15 min, 2x SSC, 0.1% SDS (4 ml/cm2) at 65°C for 

30 min, and finally with 0.1x SSC, 0.1% SDS (4 ml/cm2

) at 65°C for 15 min. After washing, 

the membranes were wrapped in plastic wrap and exposed to HyperfilmTM MP film 

(Amersham) for 2-5 days, depending on the radioactive 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 T4, F4, F8 and F9 generations of CV127 

line 603 (Figure III-1). Genomic DNA from these samples was digested with NcoI and SpeI 

(as described above) and Southern blot analysis 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 the junctions with the adjacent soybean genomic sequences (Figure 

V-5). The primer pairs used to generate these six amplicons are described in Table A-2. The 

complete sequence of the inserted DNA was obtained by PCR amplification of these six 

overlapping fragments followed by DNA sequence analysis. PCR amplicons containing 

sequence discrepancies relative to the sequence 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 2229-2247 
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Reverse transcription-polymerase chain reaction (RT-PCR) analysis of the AtSEC61γ

subunit transcript. Young leaf tissue of silica gel-desiccated young leaves derived from F7

generation plants of CV127 soybean and from leaves of the non-transgenic parental soybean 

variety Conquista was provided to DNA Landmarks, Inc. (Montreal, Canada), by BASF SA 

for RNA isolation and characterization. All plants were grown under greenhouse conditions 

in Londrina, Brazil, and young, fully-expanded trifoliate leaves were harvested for molecular 

analyses. 

Total RNA was extracted from silica gel-desiccated young leaves with the Qiagen RNeasy 

Mini Kit (Qiagen; Valencia, CA). About 25 mg of silica gel-desiccated leaf tissue was frozen 

with liquid nitrogen and ground with an Autogrinder. The total RNA isolation procedure was 

carried out according to the manufacturer’s directions. 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 AtSEC61γ subunit gene present in 

CV127 was transcribed. The primers used for this RT-PCR analysis are 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 AtSEC61γ 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 Plant Science (Research Triangle Park, NC). 

The Arabidopsis total RNA samples were prepared with TRIzol reagent (Invitrogen) without 

DNase treatment. The RT-PCR reactions contained 1x Qiagen OneStep RT-PCR Buffer, 400 

μM of each dNTP, 0.6 μM of each primer, 2 μl of Qiagen OneStep RT-PCR Enzyme mix, 

and 500 ng or 125 ng of total RNA in a total volume of 50 μl. The RT-PCR was conducted 

using the GeneAmp PCR System 9700. Following a 30 min reverse transcription step at 

50°C, PCR amplification was carried out under 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 SEC61γ subunit (GmSEC61) 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 AtSEC61γ subunit transcript 

Purpose Direction Primer Sequence Position in pAC321 

RT-PCR Forward ACGAACCTGCTGAAACCCTAAT 1338-1359 

AtSEC61 Reverse TAAGAATGGAGAATTTGGCTACA 2280-2302 

RT-PCR Forward TGAAGCAGCAGCTGAGTTTCGC N/A* 

Iota Reverse GGCAGTCTGAACCGTCTCCTC N/A 

RT-PCR Forward GCTTGGGAGACAGAGAAAGAGA N/A* 

GmSEC61 Reverse CCTTTTGCTTGACAACCTGAAT N/A 

RT-PCR Forward TTGGAATGCATGGGACTGT 3807-3825, 5733-5751 

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 transcript unrelated to the CV127 insert. 
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RNA ligase-mediated rapid amplification of 5’ complementary DNA ends (RLM-5’-RACE) 

of the AtSEC61γ 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 (F8) generation and its 

comparator non-transgenic line, Conquista, 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 Arabidopsis and soybean leaf tissue using the 

FastTrack MAG Maxi mRNA Isolation Kit (Invitrogen GmbH; Karlsruhe, Germany). 

Approximately 200 mg of leaf tissue was ground in liquid nitrogen using a mortar and pestle. 

Pre-mixed Lysis Buffer L4/Protein Degrader (510 μl) was added to each sample and samples 

were suspended by vortexing. Samples were centrifuged for five minutes at approximately 

16,100 x g at room temperature. Supernatants were transferred to new tubes and incubated 

for 15 minutes at 45 ̊C. FastTrack MAG Beads (200 μl per sample) were pre-washed and 

bound to each sample in accordance with the manufacturer’s instructions. The recommended 

wash procedure was followed with the inclusion of an optional DNase I digestion. Two 

additional washes (beyond the standard protocol) with Wash Buffer W7 were performed 

immediately before elution of the mRNA. Messenger RNA was eluted under the 

recommended conditions in 20 μl RNase-free water followed by a second elution in 10 μl 

RNase-free water. The two mRNA eluates were combined and stored at –80 ̊C. 

Messenger RNA was diluted in 1 M Tris-HCl, pH 7.5 and quantitated by measuring the 

absorbance at 260 nm using a BioPhotometer (Eppendorf AG; Hamburg, Germany). 

RNA ligase-mediated rapid amplification of 5’ cDNA ends (RLM-5’-RACE) was conducted 

using the GeneRacer Kit (Invitrogen) in accordance with the manufacturer’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 preparation. 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 steps 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 mRNA. 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 the RLM-5’-RACE experiment is only amplifying 

capped full-length message, then no PCR products would be evident in these “minus TAP” 

controls. 

TAP treatment generates a free 5’ phosphate which 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 reverse transcribed into first-strand cDNA using 

SuperScript III RT (Invitrogen) and the GeneRacer Oligo dT Primer in a 75 minute reaction. 

The region of the first-strand cDNA corresponding to the GeneRacer RNA Oligo contains 

binding sites for the GeneRacer 5’ Primer and the GeneRacer 5’ Nested Primer which will be 

used in the primary and secondary PCR amplifications, respectively. 
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Primary PCR amplification was performed using 2 μl of each reverse transcription reaction as 

a template for amplification by Platinum Taq DNA Polymerase High Fidelity (Invitrogen) 

under the reaction conditions specified in the GeneRacer Kit instructions. 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 SEC61γ subunit transcripts. Due to the lack of 

a published full-length cDNA sequence, soybean SEC61γ 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, was expected to amplify the 5’ end of cDNAs 

derived from Arabidopsis SEC61γ subunit (locus At3g48570) transcripts. Although there are 

two additional putative SEC61γ subunit genes in Arabidopsis, primer RS46 was designed 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-4. Gene-Specific Primers Used in RLM-5’-RACE Experiment: Primary PCR 

Name Primer Sequence and Description 

RS41 CATCCTAACCAGATCCGACGATGATGTTG 

SEC61γ subunit, soybean. Reverse primer 

RS46 CAAAGGGCTGATAATGTCGTTTGGTTCGTTCTTC 

SEC61γ subunit, Arabidopsis. Reverse primer 

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 PCR amplification was performed 

using nested primers. The secondary PCR utilized 1 μl of each primary amplification 

reaction as a template for amplification in 50 μl 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 μM of each primer, and 0.2 mM of 
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each dNTP (Invitrogen). In this secondary PCR, the GeneRacer 5’ Nested Primer was paired 

with either nested gene-specific primer RS43 (Integrated DNA Technologies) or nested genespecific primer RS42 (Integrated DNA Technologies). 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 SEC61γ 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 SEC61γ subunit 

(locus At3g48570) transcripts. Although there are two additional putative SEC61γ subunit 

genes in Arabidopsis, primer RS42 was designed 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 

SEC61γ subunit, Arabidopsis. Reverse primer, nested. Spans intron. 

RS43 CAGTACGGACGGCAACCTTGGAGAAT 

SEC61γ subunit, soybean. Reverse primer, 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 μl) were separated by electrophoresis at 10 V/cm in a 1% (w/v) 

agarose gel containing 0.2 μg/ml ethidium bromide in TAE (40 mM Tris-acetate, 1 mM 

EDTA). 

The CV127 RLM-5’-RACE secondary PCR products generated with Arabidopsis SEC61γ

subunit-specific primers were cloned using the TOPO TA Cloning Kit for Sequencing 

(Invitrogen). TOPO vector pCR4-TOPO (1 μl) was incubated with an aliquot of the 

secondary PCR (4 μl) and 1 μl 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 μl) 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 μg/ml 

kanamycin. 

Twelve transformants were screened for positive clones by colony PCR. Each colony was 

suspended in 4 μl of sterile water. One microliter of this suspension was added to a 25 μl 

reaction containing final concentrations of 1X Eppendorf MasterMix (Eppendorf AG; 
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Hamburg, Germany) and 0.2 μM of each primer. Due to the bidirectional nature of the 

cloning, a gene-specific primer (RS42) was paired with either vector-specific primer RS7 

(Integrated DNA Technologies) or vector-specific primer RS8 (Integrated DNA 

Technologies) to determine the 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 SEC61γ subunit, Arabidopsis. 

Reverse primer, nested. Spans 

intron. 

PCR amplification was carried out on a PrimusHT 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 μl) were separated by electrophoresis at 10 V/cm in a 1% (w/v) 

agarose gel containing 0.2 μg/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-Bertani broth containing 50 μg/ml kanamycin. Plasmids were purified from 

these cultures using the QIAprep Spin Miniprep Kit (Qiagen GmbH; Hilden, Germany) in 

accordance with manufacturer’s instructions. 

Double-stranded sequencing of selected clones was performed at the BASF AG Sequencing 

Facility (Ludwigshafen, Germany). Sequencing data was assembled using Sequencher, 

version 4.5 (Gene Codes Corporation; Ann Arbor, Michigan). 

RT-PCR analysis of the 501 bp open reading frame (ORF). RT-PCR was conducted to 

determine if the 376 bp duplication of a portion of the csr1-2 coding sequence present in 

CV127 was transcribed. Tissues (roots, leaves, 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 and seed tissue using 

the NucleoSpin® Plant II kit (Macherey-Nagel; Bethlehem, PA) in accordance with the 

manufacturer’s protocol with the following modifications. Powdered tissue sample (100 mg) 

was mixed with 400 μl Buffer PL1. Following vortexing, 10 μl RNase A was added and after 
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mixing, the sample was incubated at 65oC for 30 minutes. The lysate of each sample was 

loaded onto a single NucleoSpin Plant II Column and centrifuged for 1 minute at 12,000 rpm 

at room temperature. The flow-through was mixed with 450 μl 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 the column was washed consecutively with 400 μl 

PW1, 700 μl PW2 and 200 μl PW2. Finally, the DNA was eluted with 50 μl Elution Buffer 

PE. All genomic DNA samples were stored at 4oC. 

Genomic DNA was quantitated using the Quant-iT PicoGreen dsDNA Assay Kit (Invitrogen; 

Carlsbad, CA) in accordance with the manufacturer’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 Digital Filter Fluorometer (Barnstead International; 

Dubuque, IA), λex= 490 nm, λem= 515 nm. All genomic DNA samples were quantitated in 

duplicate. 

RNA was isolated from 100 mg of powdered tissue using the Qiagen RNeasy Plant Mini Kit 

(Qiagen Inc., Valencia, CA) and treated 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 was mixed with 0.5 ml ethanol, loaded onto the 

RNeasy mini spin column, centrifuged at 11,000 rpm for 1 min and washed with 350 μl 

Buffer RW1. DNase I was added directly onto the column and incubated at room temperature 

for 15 min. The column was washed with 350 μl Buffer RW1 followed by two washes with 

500 μl Buffer RPE. RNA was eluted in 35 μl RNase free water and quantitated using 1.5 μl 

of the RNA sample on the NanoDrop 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 transcript 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 reverse transcriptase was omitted were included to 

confirm that amplification was not occurring from contaminating genomic DNA. 

Table A-10. Primers Used for RT-PCR Analysis of the 501 bp ORF 

Purpose Direction Primer Sequence (5’ → 3’) 

Position in 

pAC321 

Forward TTGGAATGCATGGGACTGT 3807 – 3825 

ORF 501 5733 - 5751 

Reverse TGTCTTCCCTCATTGGACTG 6148 - 6167 

Forward AGCTCTATGCCGACGTGACTCC N/A* Cyclophilin Reverse ACTCCGTCTTCGTCGTGCAGAT N/A 

*N/A – not applicable. Cyclophilin was used as a positive control; the primer set is expected 

to amplify cDNA derived from an endogenous soybean transcript unrelated to the CV127 

insert. 

Total RNA (500 ng) was used as a template for RT-PCR using the Superscript First Strand 

Synthesis System for RT-PCR (Invitrogen; Carlsbad, 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 and 4oC for 5 minutes. Each 20 μl RT reaction contained final 
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concentrations of 1.25 ng/μl random hexamers, 0.5 mM dNTP mix, 1x RT Buffer, 5 mM 

magnesium chloride, 10 μM DTT, 2 U/μl RNaseOUT, and 10 U/μl Superscript III Reverse 

Transcriptase. The reaction was incubated at 25oC for 10 minutes, 50oC for 50 minutes, 85oC 

for 5 minutes and then stored at 4oC to cool down followed by addition of 1 μl of RNase H 

and incubation at 37oC for 20 minutes. The second step is the PCR amplification using 1 μl of 

the cDNA template generated in the first step. Each 25 μl 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 μM of each primer. All 

primers were synthesized by Integrated DNA Technologies (Coralville, IA). Following a 30 

minute reverse transcription step at 50oC, PCR amplification was carried out under the 

following cycling conditions: 95oC for 10 minutes, 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 cyclophilin primers described above. 
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Appendix B

Characterization of AtAHAS Protein Produced 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 AtAHAS protein. In addition, a second amino acid 

substitution of an arginine replaced by lysine was identified at position 272 of the AtAHAS 

protein; this amino acid substitution does not impact the enzymatic 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 chloroplast, 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 protein is equivalent to other AHAS proteins 

that are found ubiquitously among plant species and 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 conducted with purified AtAHAS protein. The 

AtAHAS protein is expressed 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 protein from soybean tissues for the safety 

assessment studies of the protein. The same AtAHAS coding sequence introduced in the 

CV127 soybean genome was introduced into Escherichia coli for over-expression of the 

AtAHAS protein. The AtAHAS protein, as encoded in this expression system, lacks the 

predicted 85-amino acid N-terminal CTP sequence that targets the protein in planta to the 

chloroplast. The microbially-produced AtAHAS protein contained the same S653N and 

R272K amino acid substitutions as found in the plant-produced protein. 

Microbial fermentation provided sufficient starting material for purification of gram 

quantities of the AtAHAS protein required for the safety assessment studies. The purpose of 

the following study was to characterize the AtAHAS protein produced in the transgenic 

imidazolinone-tolerant CV127 soybean to evaluate key biochemical and functional 

parameters and to demonstrate that it is substantially 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 extracts of young leaves of fieldgrown CV127 plants. In some studies, immuno-purified AHAS was used, and in others, 

ammonium sulfate precipitated AHAS-enriched extract 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 leaves 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 protein is identified as 

AtAHAS-0107. Soybean-expressed AHAS protein was evaluated for its molecular weight, 

immunoreactivity, enzymatic activity, sensitivity to branched-chain amino acid feedback 
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inhibition and imidazolinone herbicide inhibition. In addition, the soybean-expressed AHAS 

protein was evaluated for post-translational modifications and amino acid sequence 

confirmation. 

Western blot analyses using antibodies specific 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 produced in CV127 and the microbially-produced 

protein was demonstrated by monitoring the enzymatic activity, its lack of sensitivity to 

inhibition by imidazolinone herbicide, and feedback inhibition by branched-chain amino 

acids. While both the plant-produced and microbially-produced proteins demonstrated the 

expected enzymatic activity with the substrate pyruvate, the specific activity of the AHAS 

protein in CV127 was found to be higher compared to the microbially-produced protein, most 

likely a result of enzyme activity losses incurred during extensive purification of the 

microbially-produced protein. Both CV127-produced AHAS and microbially-produced 

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 retained in the 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 analysis 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 functionality and was substantially similar to the AtAHAS 

produced in a microbial over-expression system for use in safety assessment studies. 

Therefore, this study demonstrates the functional 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. 
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Preparation of test substance. AHAS expressed in CV127 used in these experiments was 

either from an ammonium sulfate precipitated AHAS-enriched fraction or it was immunopurified from young leaves (collected from the F6 generation of CV127 line 127, grown in 

regulatory field trials in Santo Antônio de Goiás, GO, Brazil in 2007). Crude extracts were 

prepared by homogenizing frozen powdered leaves (1:3 w:v) in extraction buffer [50 mM 

potassium phosphate, pH 7.2, 100 mM sodium pyruvate, 5 mM MgCl2, with HALTTM 

protease inhibitor cocktail (Pierce Biotechnology, Inc., Rockford, IL)] using a PolytronTM 

(Brinkmann, Westbury, NY). Extracts were filtered through miracloth and subjected to 

centrifugation for 15 min at approx. 10,000 x g. The resulting supernatant was considered to 

be the crude extract. The ammonium sulfate precipitated AHAS-enriched fraction was 

obtained by adding an equal volume of saturated ammonium sulfate to the 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 purification was achieved by using goat anti-AtAHAS 

antibody (which had been purified using Protein 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 ammonium sulfate precipitated AHAS-enriched 

fraction prepared from young leaves [collected from the isoline control soybean (F6 null) 

corresponding to the F6 generation of CV127 line 127, and grown 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 ammonium sulfate pellet to the 

Regulatory Science Laboratory at BASF Plant Science, 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 Emulsiflex cell disruptor (Avestin, Ontario, 

Canada). Insoluble material was removed by centrifugation. The His-tagged AtAHAS 

present in the supernatant was purified by chromatography using Talon® cobalt resin 

(Clontech, Inc., Mountain View, CA). Once eluted, 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 sequence 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. 
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mM ammonium bicarbonate buffer, pH 7.9, containing 20 μM FAD. The dialyzed material 

was then lyophilized and designated AtAHAS-0107. This reference substance was 

characterized (Privalle, 2007a), and results are summarized in Table B-2. 

Table B-2. Characterization 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 reference substance and ammonium sulfatetreated test and control AHAS-enriched fractions from plant tissues was quantified by the 

BCATM procedure (bicinchoninic acid procedure; Pierce) in accordance with the 

manufacturer’s instructions, using 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 μl) were loaded onto a multiwell plate in triplicate, reacted with 

200 μl of a 1:50 (B:A) mixture of BCA reagent, incubated at 37oC for 30 min and allowed to 

cool at room temperature for 10 min. The absorbance at 550 nm was measured using a 

Multiskan Ascent V1.24 multiwell plate reader (Therma Labsystems, Helsinki, Finland). The 

results were analyzed using DeltaSoft PC software (Version 1.71.4, Biometallics, Inc.; 

Princeton, NJ) using the linear regression curve fit. 

AHAS quantification. Samples were quantitatively analyzed for AHAS protein by a 

sandwich enzyme-linked immunosorbent assay (ELISA) [Tijssen, 1985] using 

immunoaffinity-purified polyclonal rabbit anti-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 incubated 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 37oC. 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 washed 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 peroxidase (HRP) was added. After incubation at 

37oC for 1 hr, the plates were washed three times and HRP substrate was added (1 Step Ultra 

TMB; Pierce). After 20 min at room temperature 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; Biometallics, Inc.; Princeton, NJ). The fourparameters algorithm was used to generate a curve. The AHAS component of the samples 

was quantified from the standard concentration curve generated 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-PAGE, SOP BPS 510.02), using an 8 – 16% 

polyacrylamide gradient Tris-glycine gel (Invitrogen) followed by electroblotting onto PVDF 

membrane (Invitrogen; SOP BPS 510.03). Samples of the ammonium sulfate AHAS-
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enriched preparations and AtAHAS-0107 preparations were loaded onto the gel. After 

electroblotting the membrane was probed with rabbit anti-AtAHAS peptide 2 polyclonal 

antibody. Donkey anti-rabbit IgG linked to horseradish 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 antibody and was visualized by development with 

ECL Plus Western Blotting Detection Reagents (Amersham, GE Healthcare).

Western blot analysis, using rabbit anti-AtAHAS peptide 2 polyclonal antibodies, of extracts 

prepared from both CV127 and the isoline control soybean contained immuno-reactive bands 

at a molecular weight of approximately 64,000 (Figure B-1). This was the same apparent 

molecular weight as the microbially-produced AtAHAS reference substance, AtAHAS-0107, 

indicating that the AHAS produced in the CV127 and isoline control soybeans was of the 

same anticipated size and of similar immunoreactivity to the microbially-produced protein. 

Both plant extracts included a smaller immunoreactive band at approximately 47,000 

molecular weight, which is most likely a degradation product of the mature AtAHAS protein. 

The molecular weights of the microbially-produced AtAHAS and plant-produced AHAS 

proteins were also shown to be similar using SDS-PAGE and Coomassie blue staining. The 

approximate molecular weights of the immuno-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, the microbially-produced 

AtAHAS protein consisted of the predicted mature AtAHAS (585 amino acids) plus an Nterminal addition of 38 amino acid to facilitate protein purification, folding and solubility. 

Therefore, it was expected that the molecular weight of the microbially-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 molecular weights of the plant-produced 

AHAS and microbially-produced AtAHAS 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 residues 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 soybeans compared to the predicted cleavage site in 

Arabidopsis. This conclusion is supported by data presented in the later 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 the synthesis of acetolactate (an acetohydroxy 

acid) by the condensation of two molecules of pyruvate. The acetolactate produced by 

AHAS in this assay is converted to acetoin in the presence of acid and acetoin is detected 

colorimetrically (A530 nm) after interaction with creatine and naphthol. One unit of AHAS 

activity is defined as 1 μmole acetoin produced per minute. The ammonium sulfate AHASenriched preparations of CV127 and the isoline control soybean as well as AtAHAS-0107, 

dissolved in 50 mM Tris-HCl, pH ∼7.0 and diluted to the desired concentration, were mixed 

with an equal volume of 2x assay buffer [100 mM potassium phosphate, pH 7.2, 10 mM 

MgCl2, 200 mM sodium pyruvate, with 20 μM FAD and 2 mM thiamine pyrophosphate 

(TPP)] and incubated at 37oC for 90 minutes prior to the addition of 20 μl 5% H2SO4

followed by a 15 min incubation at 60oC. Background absorbance was determined by 
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immediately quenching the reactions 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, final concentration) for 15 min at 60°C and the 

absorbance was measured at 550 nm. Absorbance values were compared to an acetoin 

standard curve and corrected for background absorbance. Several dilutions of a single 

dissolved sample of AtAHAS were prepared and each dilution was assayed in triplicate; the 

mean corrected value is reported. 

Feedback inhibition of AHAS enzymatic activity by leucine and valine was confirmed by 

including leucine and valine in the assay mix. Leucine and valine solutions were prepared in 

2x assay buffer at twice the desired final assay concentration (1 mM). Fifty μl of 20-fold 

diluted AtAHAS-0107, the ammonium sulfate AHAS-enriched preparation of CV127 and the 

ammonium sulfate AHAS-enriched preparation of isoline control soybean were incubated 

with 50 μl 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 two times the desired final assay concentration, 

where the final concentration of herbicide ranged from 0 to approx. 500 μM. Fifty μl of the 

sample was incubated with 50 μl 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 absorbances 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 plant-produced AHAS proteins demonstrated the 

expected enzymatic activity with the substrate pyruvate, confirming the same functional 

activity of the AHAS proteins from both microbial and plant sources. However, the specific 

activity of ammonium sulfate AHAS-enriched plant extracts was considerably higher than the 

highly purified microbially-produced 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 was 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 extensively for the protein safety studies, 

whereas the plant AHAS used in these enzymatic 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 extracts (Table B-3). Activity was very 

similar between CV127 soybean and the isoline 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 imazethapyr compared to the isoline control plantproduced protein (Figure B-2). An intermediate level of inhibition of the CV127-produced 
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AtAHAS protein was observed, and this was as expected due to the mixture of sensitive 

(endogenous) and tolerant (transgenic) AHAS present 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 lower than anticipated but may reflect the tolerance that soybean 

naturally has to this particular imidazolinone herbicide, imazethapyr. 

Feedback inhibition by the branched-chain amino acids leucine and valine was very similar 

for both the CV127 soybean AHAS and the isoline control soybean AHAS (Figure B-3). No 

feedback inhibition was observed for the AtAHAS present in the microbially-produced 

reference substance as expected. 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). 

Glycosylation analysis. The DIG Glycan Detection kit (Roche Diagnostics GmbH, 

Mannheim, Germany) was used in accordance with the manufacturer’s instructions (method 

B, described below) to monitor for glycosylation 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 treatment. The spaced-linked steroid hapten 

digoxigenin (DIG) is then covalently attached to these aldehydes via a hydrazide group. 

Digoxigenin-labeled glycoconjugates are then subsequently detected by western blot analysis 

using a digoxigenin-specific antibody 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 metaperiodate (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 with 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 can 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 would not be present in the mature protein. 

There are, however, numerous potential O-glycosylation 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 glycosylation, but rather it is destined to be localized in the

chloroplast where proteins are not typically glycosylated. Likewise, the AtAHAS produced 

in the microbial expression system should not be glycosylated as glycosylation does not 
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occur in prokaryotes. The results shown here demonstrate that no glycosylation was detected 

above the limit of detection (2 – 4 molecules of glucose equivalents/molecule of AtAHAS). 

Amino acid sequence analysis. To further confirm that the AtAHAS protein produced in 

CV127 soybean was the protein expected, amino acid sequence was determined. The 

attempts to obtain N-terminal amino acid sequence 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 bands were excised and transferred into 0.5 ml 

Eppendorf cups. The gel pieces were washed consecutively with 100 μl of acetonitrile/ water 

(1:1, v:v), acetonitrile and acetonitrile/water (1:1). For each of the wash steps, the samples 

were shaken at 800 rpm for 5 min at 30°C, subjected 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 μl of 100 mM ammonium bicarbonate, the gel pieces were shaken for 5 

minutes at 30°C. Then 50 μl of the trypsin solution [0.76 μg trypsin, (Roche Mannheim), 

0.12% CaCl2, 8% acetonitrile, 100 mM ammonium bicarbonate] were added and the samples 

were incubated at 37°C for 16 hours with shaking (300 rpm). After stopping the reaction by 

addition of 90 μl 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 μl 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 peptides 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, Germany. MS-analysis of the trypsin-digested 

protein samples was carried out on a quadrupole 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 unit (Fa. SunChrom GmbH), allowing separation 

of the peptide mix prior to the MS-analysis. 

The peptides were separated on the μHPLC unit 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 particle size. A gradient of 5 – 50% solvent 

A and B (A: 95% H2O, 5% ACN, 0.08% HCOOH; B: 95% ACN, 5% H2O, 0.08% HCOOH) 

in 60 min was selected for separation of the trypsin-digested peptides. The 10 μl/min flow 

from an Eldex HPLC pump (Fa. SunChrome) was 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 alternating 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 selected for MS/MS-analyses. In an MS/MSexperiment a specific ion is selected (by its m/z value) and subjected 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, dissociation occurs mainly 

 

[4] C18 material, i.e. covalently-bonded octadecylsilane, is used as bonded phase in the column. 
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at the peptide bonds, with an ideally statistical distribution. Two series of ions are formed: 

b-ions, where the charge is retained on the N-terminal fragment, and 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 sequences 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, accurately 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 highly specific for the originating protein. In summary, the MSexperiments provide the molecular masses of the proteolytic peptides and either full-length 

sequences or specific sequence tags. 

In order to identify the originating 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 with close homology can be identified. The MASCOT search was carried 

out without specifying a digesting enzyme. All MASCOT results 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 for 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 the major protein band at approximately 64,000 

molecular weight in CV127 soybean is AtAHAS since the amino acid sequences obtained 

from peptide fragments derived from it were identical 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 fragments derived from the microbially-produced 

AtAHAS were identical to regions of the deduced 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 fragments overlapped between the plant-produced 

and microbially-produced sources, the amino acid sequences were the same (data not 

presented). Since the same DNA coding sequence 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 sequences 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 sequence of AtAHAS with the S653N 

and R272K amino acid substitutions. 

Two of the four potential N-glycosylation sites were included in the amino acid sequence 

obtained, providing confirmatory support for the lack 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 mature AtAHAS protein produced in CV127 soybean. The csr1-2 gene from Arabidopsis 

thaliana encodes a single polypeptide of 670 amino acids that includes a chloroplast transit 
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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 sequences of known AHAS proteins and looking 

for the first conserved residue (i.e. threonine 86). An identical CTP cleavage site was 

experimentally determined for the AHAS enzyme in maize (B. K. Singh, personal 

communication). During transport into the chloroplast, 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 expected to be present in CV127 soybean. The 

microbially-produced AtAHAS protein was designed to represent the mature form of the 

AtAHAS protein and lacks the 85-amino acid N-terminal sequence corresponding to the 

predicted chloroplast transit peptide sequence. The microbial protein does contain an 

additional 38 amino acids (see footnote on page 197) to aid in bacterial expression and 

purification. The molecular 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-terminal 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 apparently only 47 amino acids in length (Rood et al., 2006). 

Conclusions. Key biochemical and functional parameters were evaluated to demonstrate that 

the AtAHAS protein produced in CV127 soybean is substantially similar 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 AtAHAS. 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 material. 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 retained in the plant extracts but not in the 

microbially-produced material as expected. 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 was found associated with the AtAHAS in CV127 leaves or the 

microbially-produced AtAHAS. 

To compare the amino acid sequence of the AtAHAS protein produced in CV127 soybean 

with that produced in the E. coli expression system, the amino 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 plant tissues, coupled with the repetitive amino 

acid content of the putative CTP. The high content of serine, threonine and proline within the 

first 85 amino acids of the full-length AtAHAS (the putative CTP segment of the protein) 
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contributes to the difficulties in obtaining unambiguous results. Serines and threonines 

negatively impact the yield of amino acid sequence and have high carryover into subsequent 

chromatograms, leading to ambiguous interpretation of results of the amino acid sequence 

analysis. Proline is not efficiently cleaved (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-terminal 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 performed. 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 amino 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 peptide. Based on a sequence comparison of the 

deduced amino acid sequence of plant and microbial AHAS proteins, Mazur et al. (1987) 

predicted that the chloroplast transit peptide of AtAHAS is cleaved at the C-terminal side of 

residue 85. Amino acid sequence analysis of the AtAHAS protein produced in CV127 

soybean showed a peptide fragment corresponding to amino acid residues 52 to 73. 

Therefore, the CTP cleavage site of the AtAHAS protein produced in CV127 soybean is most 

likely not at the predicted site at the C-terminal side of residue 85, but is located at least at the 

C-terminal side of residue 51. This conclusion was further confirmed because the apparent 

molecular weights of the CV127-produced AtAHAS protein and the microbially-produced 

AtAHAS protein were equivalent, yet the microbially-produced 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 study suggest that the mature AtAHAS protein 

produced in CV127 soybean consists of the predicted mature protein (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 considered equivalent to the CV127 protein for 

the following reasons. The microbially-produced AtAHAS protein was functionally active 

and had the expected enzymatic activity with the substrate pyruvate. Furthermore, the 

microbially-produced protein lacked sensitivity 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-produced AtAHAS protein and CV127-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. Finally, both proteins had 

the same amino acid sequence of the predicted 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 produced in CV127 soybean in safety assessment 

studies of the AtAHAS protein. 
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Standard Operating Procedures 

BPS 510.02 SDS-Polyacrylamide Gel Electrophoresis 

BPS 510.03 Western Blot Analysis 

BPS 510.04 Protein Determination Using the BCA Procedure 

BPS 510.09 Enzymatic Assay for Acetohydroxyacid Synthase (AHAS) 
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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/μg 

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 0.005 
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Figure B-1. Western Blot Analysis of AHAS Protein in CV127 Soybean, the Isoline 

Control Soybean and Reference Substance AtAHAS-0107. 

The immunoreactivity and integrity 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 μg protein from the ammonium sulfate AHASenriched fraction from CV127 soybean; and lane 3, 81 μg protein from the ammonium sulfate 

AHAS-enriched fraction of the isoline control soybean. Blots were probed 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

98

64

50

36

22

16

1 2 3

AHAS

148

98

64

50

36

22

16
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Figure B-2. Sensitivity of the Enzymatic Activity of AHAS from CV127 Soybean, the 

Isoline Control Soybean and Reference Substance 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 extracts enriched by ammonium sulfate 

precipitation, prepared from CV127 soybean leaves (-▲-) and isoline control soybean leaves 

(-■-), as well as in the reference AHAS microbially-produced test substance, AtAHAS-0107 

(-▼-).
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Figure B-3. Sensitivity of the Enzymatic Activity 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 valine 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 (-▼-).
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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 membrane was divided so that lane 1 and part of 

the marker lane (M) were developed by western blot analysis using goat anti-AtAHAS, Lanes 

2 – 4 were developed in the absence of oxidation, 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 (negative control). The AtAHAS position is 

indicated by arrows. 

148

98

64

50

36

22

16

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

148

98

64

50

36

22

16

148

98

64

50

36

22

16

1 M 2 3 4 M 5 6 7 8 9 10
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Figure B-5. AtAHAS Amino Acid Sequence. 

Panel A. SDS-PAGE with Coomassie blue staining of immuno-purified 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 the event designated CV127. The amino 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-glycosylation 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 (K272, N653) 

A. 

B. 

1 MAAATTTTTT SSSISFSTKP SPSSSKSPLP ISRFSLPFSL NPNKSSSSSR 

51 RRGIKSSSPS SISAVLNTTT NVTTTPSPTK PTKPETFISR FAPDQPRKGA 

101 DILVEALERQ GVETVFAYPG GASMEIHQAL TRSSSIRNVL PRHEQGGVFA

151 AEGYARSSGK PGICIATSGP GATNLVSGLA DALLDSVPLV AITGQVPRRM

201 IGTDAFQETP IVEVTRSITK HNYLVMDVED IPRIIEEAFF LATSGRPGPV 

251 LVDVPKDIQQ QLAIPNWEQA MKLPGYMSRM PKPPEDSHLE QIVRLISESK 

301 KPVLYVGGGC LNSSDELGKF VELTGIPVAS TLMGLGSYPC DDELSLHMLG

351 MHGTVYANYA VEHSDLLLAF 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 IPNGGTFNDV ITEGDGRIKY 

148

98

64

50

36

22

16

1 2 3 4 5

AHAS

6

148

98

64

50

36

22

16

1 2 3 4 5

AHAS

6
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Appendix C

Methods for Determining Levels of the AHAS and SEC61γ Subunit Proteins in Soybean 

Tissues 

A. Determining the level of AHAS protein in soybean tissues 

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 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 areas 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 rate of 1.0 liters/ha; treatment T3) as 

described in Section VIII.C and Appendix F. Leaf 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 grain was harvested from each plot at all of the 

field test locations in the 2006/2007 and 2007 growing seasons, except for Ponta Grossa in 

the 2006/2007 growing season. In addition, six whole plants per plot, including 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 season (Brasília and Sto. 

Antônio de Goiás ) at three different developmental stages, including the V2 (plants 15-20 

cm tall with three nodes and two unfolded leaflets), R2 (plants in full bloom), and R8 (full 

maturity) stages. Each whole plant was placed in a separate, labeled plastic bag and was 

shipped on ice within 24 hr to the test facility: 
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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 and pods, if present. The plant parts from each 

plant were maintained as a single sample except 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. The whole plant and leaf tissue samples were 

shipped on wet ice within 24 hrs to the test facility 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 analysis. 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 KH2PO4, 35.2 mM K2HPO4, 100 mM Na-Pyruvate, 5 mM MgCl2) 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 through miracloth and was centrifuged again at 

14,000 rpm at 5°C for 15 min. The liquid phase was recovered and stored at 5°C prior to 

analysis by ELISA. 

Sample analysis by ELISA. The amount of AHAS enzyme in the different plant tissues was 

analyzed using an Enzyme-Linked Immunosorbent Assay (ELISA) protocol 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 distinguishing between these 

enzymes but rather measures the total AHAS protein 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 spectrophotometer with a microplate reader at 450 nm. A 

dilution series of purified AtAHAS 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-weight tissue. The laboratory portion of this 

study was conducted under Good Laboratory Practices (GLP). 

B. Determining the level of SEC61γ subunit protein in soybean tissues

Source of plant materials. Leaf tissue was harvested from greenhouse-grown plants of 

CV127 soybean (grown from F9 generation seed) and the conventional soybean variety 

Conquista for microsomal membrane protein isolation. The soybean seeds used to produce 

the plants from which the leaf material was harvested were used as the grain samples. 
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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 tissues and grain derived from CV127 soybean. Microsomal 

membranes include the endoplasmic reticulum and were selected to enrich the SEC61γ

subunit in the samples to be analyzed. 

Control material. The control materials for this study were microsomal membrane protein 

preparations from leaf tissues and grain derived 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. 

AtSEC61γ protein standard. The At3g48570 gene, encoding the AtSEC61γ 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 expression cassette encodes a glutathione S-transferase 

(GST)–AtSEC61γ subunit fusion protein (mol. wt. ca. 35,000). The GST-AtSEC61γ subunit 

fusion protein was purified from E. coli using an immobilized glutathione column according 

to the manufacturer’s instructions (GE Healthcare). On-column digestion using PreScission 

ProteaseTM (GE Healthcare) was performed to release the AtSEC61γ subunit protein (mol. wt. 

ca. 7000). This was the AtSEC61γ subunit protein that was used as a standard on the western 

blots. 

Antibody generation and sensitivity. Rabbit polyclonal antibodies used in western blot 

analysis were generated against two synthetic peptides which together spanned the entire 69-

amino acid AtSEC61γ 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 routinely diluted 1:3000 in blocking buffer for use 

in western blot analysis as described below. 

Microsomal membrane protein preparations. Arabidopsis and greenhouse-grown soybean 

leaf extracts were prepared by homogenizing 6 g frozen powdered leaves in 18 ml extraction 

buffer [8 mM sodium phosphate, 2 mM potassium phosphate, 0.14 M sodium chloride, 10 

mM potassium chloride, pH 7.4 (PBS) with protease inhibitor cocktail (Thermo Scientific, 

Inc., Rockford, IL) and 2 mM MgCl2]. The homogenates were filtered through miracloth and 

centrifuged at 12,000 x g for 15 minutes. The supernatant was then centrifuged at 100,000 x 

g for 1.5 hours. The resulting microsomal membrane pellet was resuspended in 250 μl 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 miracloth and microsomal 

membranes were isolated as described above for leaf extracts. The pellets were resuspended 

in 250 μl PBS. 

Cytochrome c reductase assay. The activity of cytochrome c reductase, a marker enzyme of 

the endoplasmic reticulum (microsomal membrane fraction), was measured in the crude 

extract, 100,000 x g supernatant and pellet (microsomal membrane fraction) by monitoring 

the rate of reduction of cytochrome c (the change in absorbance at 547 nm with time) to 

provide assurance that the fraction being analyzed for the presence of the SEC61 gamma 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 216 of 375 

subunit protein was indeed the microsomal fraction 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 μl and contained 300 mM potassium 

phosphate, pH 7.8, 0.1 mM EDTA, 85 μg NADPH, and 36 μM cytochrome c. The reaction 

was initiated by the addition of NADPH and the 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, BioMetallics, Inc., Princeton, NJ) using the kinetic 

mode to calculate the rate. 

Western blot analysis. Aliquots of the microsomal membrane preparations were subjected to 

SDS-polyacrylamide gel electrophoresis on an 8 – 16% polyacrylamide gradient gel followed 

by electroblotting onto a PVDF membrane (Invitrogen; SOPs BPS 510.02 and 510.03). The 

membrane was probed with rabbit anti-AtSEC61γ polyclonal antibody which can detect 

approximately 0.4 ng SEC61γ subunit protein. Donkey anti-rabbit IgG linked to horseradish 

peroxidase (Jackson ImmunoResearch Laboratories, 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 antibody and was visualized by 

development with the SuperSignal® West Femto Maximum Sensitivity Substrate (Thermo 

Fisher). The limit of detection of this development method with this antibody was found to 

be approximately 0.4 ng SEC61γ subunit protein. 
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Appendix D

Methods Used and Results of the Grain 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 isoline 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 varieties commonly grown in Brazil (Monsoy 8001 and Coodetec 

217 presented as “Std1” and “Std2”, respectively, 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 treatment 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. However, because this treatment will not be used 

in the commercial production of CV127 soybean, this treatment was not included in the mean 

and range analyte data determined across field trial 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 standard agronomic practices in a complete 

randomized block design with four replicate blocks per location. For 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 conventional soybean varieties (“Std1” and “Std2”), 

the same grain harvest and separation procedures 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 variability for each analyte for soybeans grown in 

Brazil. All compositional analyses 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 results are reported on both 

a fresh- and dry-weight basis; all other analytes are reported 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 following tables of this Appendix (D-3 to D-22). Statistical 

analysis was conducted using the dry-weight data. 

b) Forage Composition Analyses 

Imidazolinone-treated CV127 plants together with the isoline control, and two other 

conventional soybean varieties [Monsoy 8001 (“Std 1”) and Coodetec 217 (“Std 2”)] were 

grown at six locations in Brazil during the 2007/2008 growing season. The field trial 

locations are presented in Table D-1. The plants were grown under standard agronomic 

practices in a complete randomized block design with three replicate blocks per location. 
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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 plants at the full-flowering stage of 

development in each replicate plot were harvested 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 compositional analysis. At ITAL, the pooled samples 

consisting of three plants from each replicate plot were chopped and mixed to produce a 

composite sample. After partial drying and weighing, the samples were reduced to a fine 

powder using a Waring blender. Aliquots (25 g each) of each sample were removed to 

separate air-tight containers and were shipped on dry ice to Eurofins (Des Moines, IA) for 

analysis of cysteine and methionine. Samples were stored in air-tight glass bottles until 

analyzed. Results were recorded on a fresh-weight (FW) basis and adjusted for moisture 

content to calculate the corresponding dry-weight (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 Brazil 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 √
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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 general description of the methods used for measurement of each 

analyte. 

Ash. The sample was placed in an electric furnace 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. The 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 the following equation: % carbohydrates = 100% 

- (% protein + % fat + % moisture + % ash). Calories 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 quantitation 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 CuSO4 + K2SO4 + 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 glutamine), glycine, histidine, isoleucine, leucine, 

lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and 

valine. This was accomplished for all amino acids except cysteine, methionine, and 

tryptophan through direct acid hydrolysis with 6N hydrochloric acid. Once hydrolyzed, 

the individual amino acids were then quantified using an automated amino acid analyzer 

and detected at 520 nm. The limit of quantitation for this study was 0.1 mg/g. The 

reference standards were 2.5 μmol/mL per amino acid with the exception of cystine 

which was 1.25 μmol/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 this method is 0.01% on an “as is” basis. 

Tryptophan levels were determined by measuring the absorbance at 590 nm following 

direct enzymatic hydrolysis with pronase at 40o C for 24 hours. The reference standard 
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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 fritted 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 quantitated 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 amyloglucosidase to remove protein and starch. 

Four volumes of 95% ethyl alcohol were added to precipitate soluble dietary fiber. The 

total residues were filtered and washed consecutively with 78% ethyl alcohol, 95% ethyl 

alcohol, and acetone. After drying, the residue was weighed. One duplicate was then 

analyzed for protein, and the other was incinerated 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 saponified with 0.5 N sodium hydroxide in 

methanol. The mixture was methylated with a solution of NH4Cl and H2SO4 in methanol 

based on Hartman and Lago (1973). The resulting methyl esters were extracted with 

hexane. The methyl esters of the fatty acids were analyzed by gas chromatography using 

area normalization for quantitation. The 37-Component FAME mix from Supelco 

(Sigma, Saint Louis, MO) was used for reference 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 was centrifuged and filtered. The sample was 

analyzed on a high-performance liquid chromatography (HPLC) system with a diode 

array detector. Quantification was conducted against an external standard curve of 

known standards. The limit of quantification 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 lecithins 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) suspension was added to an equal volume of 
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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 last well to show visible agglutination was 

considered the point of equivalence. One hemagglutinating unit (HU) was defined as the 

reciprocal of dilution at the point of equivalence, the specific activity being given as one 

hemagglutinating unit per μg or mg of protein (HU) in the undiluted sample. The 

minimum dose was defined as the minimum concentration necessary to show visible 

agglutination. These assays are only semiquantitative 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 limit 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). Proteins and lipids which co-extracted were eliminated by 

precipitation, followed by filtration. Raffinose and stachyose were separated and 

quantified by HPLC with a refractive index detector. 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 cleavage of benzoyl-DL-arginine-p-nitroanalide 

hydrochloride. The sample was ground and/or defatted with petroleum ether, if 

necessary. A sample of matrix was extracted 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-nitroanalide 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 filtered 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 meal. 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 which uses microplates and the Lactobacillus casei subspecies 

rhamnosus – ATCC 7469. The antioxidants 2-mercaptoethanol and ascorbic acid were 

used in all preparation stages of the extract. The samples were hydrolyzed in an autoclave 

with potassium dihydrogen phosphate 0.1 M pH 6.8, followed by consecutive enzymatic 

hydrolysis steps. Each enzyme was inactivated 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 necessary dilutions, the extracts were applied 

to the microplates. 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 was 
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measured at 630 nm in a microplate reader. Quantitation was by comparison to a 

standard curve. The limit of quantitation for this study was 0.1 μg/g FW sample. 

Vitamin E (Tocopherols). The samples were saponified at 90-95o C with reflux using 

nitrogen forced through the condenser, with potassium 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 Lichrospher 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 extraction of niacin, the samples 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 recommended by AOAC (2005) method 961.14. The 

extracts were passed over an ion-exchange resin prior to HPLC on a Lichrospher 100 RP18 (250 x 4 mm) column (Merck, Germany). The mobile phase consisted of 

heptanesulfonic acid sodium salt, triethylamine, potassium dihydrogen phosphate, and 

methanol in a gradient system. Niacin levels were monitored at 265 nm. The limit of 

quantification for this study 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 hydrochloric acid in an autoclave for 15 minutes; 

the pH was adjusted to 4.5 with sodium acetate. 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 monitor 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 vitamin 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. Differences were assessed across location and by 

location. The model for across-location analysis was the following: 

y = variety + location + variety x location + block(location) + e 

Random effects: location, variety 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 error 
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Contrasts were carried out to compare each of the sprayed and unsprayed CV127 treatments 

with the isoline control. Differences were considered statistically significant at the 0.05 

confidence level. 
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Table D-2. Methods for Compositional Analyses 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 X 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-3. Proximate Composition of Grain on a Fresh-Weight Basis of CV127 Soybean TreatmenIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in B

Location Treatment N Moisture

(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) 

Mean ± Standard Deviation (range) 

Isoline 4 10.2 ± 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) 

Santo Antônio de Goias CV127 4 9.1 ± 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) 

 CV127 

+ imi 

4 9.6 ± 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) 

 Std 1 1 10.5 23.29 35.4 18.1 4.4  Std 2 1 9.9 22.84 33.5 21.2 4.2 Uberaba Isoline 4 9.9 ± 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) 

 CV127 4 8.9 ± 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) 

 CV127 

+ imi 

4 9.0 ± 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) 

 Std 1 1 9.5 22.55 34.9 19.1 4.1  Std 2 1 9.6 24.19 35.0 21.8 4.1 Sete Lagoas 

Isoline 4 10.6 ± 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) 

 CV127 4 9.7 ± 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) 

 CV127 

+ imi 

4 9.8 ± 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) 

 Std 1 1 9.8 23.31 34.0 19.4 4.4  Std 2 1 9.8 22.80 33.7 21.7 4.3 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-3. continued.

*Statistically significantly different from the isoline control at P < 0.05. 

Location Treatment N Moisture

(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

Mean ± Standard Deviation (range) 

Londrina Isoline 4 9.4 ± 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) 

 CV127 4 9.1 ± 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) 

 CV127 

+ imi 

4 9.4 ± 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) 

 Std 1 1 9.9 21.30 32.8 20.5 4.7  Std 2 1 9.4 24.81 34.1 22.5 4.6 Brasília Isoline 4 10.1 ± 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) 

 CV127 4 9.1 ± 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) 

 CV127 

+ imi 

4 9.0 ± 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) 

 Std 1 1 9.5 22.63 33.7 19.9 4.5  Std 2 1 9.5 22.30 32.9 21.5 4.6 

Isoline 4 10.3 ± 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) 

Santo Antônio de Posse CV127 4 10.1 ± 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) 

 CV127 

+ imi 

4 9.6 ± 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) 

 Std 1 1 10.1 20.04 33.2 19.6 4.5  Std 2 1 9.7 25.31 33.5 21.6 4.8 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-4. Proximate Composition of Grain on a Fresh-Weight Basis of CV127 Soybean TreatmenIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in 

Location Treatment N Moisture 

(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

Mean ± Standard Deviation (range) 

Isoline 4 7.7 ± 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) 

Santo Antônio de Goiás CV127 4 8.1 ± 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) 

 CV127 

+ imi 

4 8.1 ± 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) 

 Std 1 1 7.9 20.16 38.1 15.6 4.8  Std 2 1 7.7 21.35 37.6 17.8 4.8 Teresina Isoline 3 7.4 ± 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) 

 CV127 3 7.6 ± 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) 

 CV127 

+ imi 

3 7.8 ± 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) 

 Std 1 1 7.5 22.02 36.9 20.7 5.1  Std 2 1 7.0 25.36 34.7 23.3 5.1 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-4. continued.

Location Treatment N Moisture 

(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

Mean ± Standard Deviation (range) 

Vilhena Isoline 4 7.4 ± 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) 

 CV127 4 7.8 ± 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) 

 CV127 

+ imi 

4 7.8 ± 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) 

 Std 1 1 7.8 22.33 38.7 16.8 4.8  Std 2 1 7.4 24.60 35.6 20.8 4.6 Brasília Isoline 4 8.1 ± 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) 

 CV127 4 7.9 ± 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) 

 CV127 

+ imi 

4 7.9 ± 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) 

 Std 1 1 8.2 23.90 33.8 18.1 4.5  Std 2 1 8.0 24.70 34.3 18.7 4.7 

*Statistically significantly different from the isoline control at P < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-5. Proximate Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatment

Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in B

Location Treatment N Total Dietary Fiber 

(g/100 g DW) 

Protein 

(g/100 g DW) 

Fat 

(g/100 g DW) 

Ash 

(g/100 g DW) 

Carbohydr

(g/100 g D

Mean ± Standard Deviation (range) 

Isoline 4 24.65 ± 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.

Santo Antônio de Goiás CV127 4 25.00 ± 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.

(6.0 – 8.7

 CV127 

+ imi 

4 26.08 ± 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

 Std 1 1 26.02 39.6 20.2 4.9 9.3  Std 2 1 25.34 37.2 23.5 4.7 9.3 Uberaba Isoline 4 23.79 ± 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.

(6.6 – 10.

 CV127 4 23.66 ± 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

(8.0 – 12.

 CV127 

+ imi 

4 24.20 ± 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.

(6.6 – 11.

 Std 1 1 24.91 38.6 21.1 4.5 10.9  Std 2 1 26.75 38.7 24.1 4.5 5.9 Sete Lagoas 

Isoline 4 24.02 ± 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.

(5.6 – 9.9

 CV127 4 23.87 ± 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.

(7.5 – 10.

 CV127 

+ imi 

4 24.57 ± 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

 Std 1 1 25.84 37.7 21.5 4.9 10.1  Std 2 1 25.28 37.4 24.1 4.8 8.5 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-5. continued. 

*Statistically significantly different from the isoline control at P < 0.05. 

Location Treatment N Total Dietary Fiber 

(g/100 g DW) 

Protein 

(g/100 g DW) 

Fat 

(g/100 g DW) 

Ash 

(g/100 g DW) 

Carbohydra

(g/100 g D

Mean ± Standard Deviation (range) 

Londrina Isoline 4 26.53 ± 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

 CV127 4 25.37 ± 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.

 CV127 

+ imi 

4 24.85 ± 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

 Std 1 1 23.63 36.4 22.8 5.2 12.0  Std 2 1 27.38 37.6 24.8 5.1 5.1 Brasília Isoline 4 24.51 ± 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.

 CV127 4 23.92 ± 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.

 CV127 

+ imi 

4 22.80 ± 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.

(9.4 – 12.

 Std 1 1 25.00 37.2 22.0 5.0 10.8  Std 2 1 24.64 36.4 23.8 5.1 10.2 

Isoline 4 24.04 ± 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.

Santo Antônio de Posse CV127 4 25.46 ± 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

 CV127 

+ imi 

4 25.72 ± 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

 Std 1 1 22.29 36.9 21.8 5.0 14.0  Std 2 1 28.03 37.1 23.9 5.3 5.6 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-6. Proximate Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatment

Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in 

Location Treatment N Total Dietary Fiber (g/100 g DW) Protein (g/100 g DW) Fat (g/100 g DW) Ash (g/100 g DW

Mean ± Standard Deviation (range

Isoline 4 21.33 ± 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) 

Santo Antônio de Goiás CV127 4 24.14 ± 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) 

 CV127 

+ imi 

4 25.27 ± 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) 

 Std 1 1 21.88 41.4 16.9 5.2  Std 2 1 23.13 40.7 19.3 5.2 Teresina Isoline 3 24.34 ± 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) 

 CV127 3 24.86 ± 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) 

 CV127 

+ imi 

3 26.12 ± 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) 

 Std 1 1 23.80 39.9 22.4 5.5  Std 2 1 27.26 37.3 25.1 5.5 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-6. continued.

Location Treatment N Total Dietary Fiber 

(g/100 g DW) 

Protein 

(g/100 g DW) 

Fat 

(g/100 g DW) 

Ash 

(g/100 g DW) 

Mean ± Standard Deviation (range

Vilhena Isoline 4 26.21 ± 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) 

 CV127 4 24.61 ± 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) 

 CV127 

+ imi 

4 22.76 ± 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) 

 Std 1 1 24.22 42.0 18.2 5.2  Std 2 1 26.57 38.4 22.5 5.0 Brasília Isoline 4 26.23 ± 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) 

 CV127 4 25.34 ± 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) 

 CV127 

+ imi 

4 24.86 ± 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) 

 Std 1 1 26.03 36.8 19.7 4.9  Std 2 1 26.84 37.3 20.3 5.1 

*Statistically significantly different from the isoline control at P < 0.05.
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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 Two Conventional 

Soybean Varieties (Std 1 and Std 2) Grown 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. 
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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) Grown 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.
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-9. Amino Acid Composition of Grain on a Dry-Weight Basis of CV127 Soybean TreatmenIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in B

Location/ Treatment Asp Thr Ser Glu Pro Gly ASanto Antônio de Goiás 

Mean ± Standard Deviation (range) 

g/100 g dry weight

Isoline 4.53 ± 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 ± 

(1.57 

CV127 4.50 ± 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 ± 

(1.52 

CV127 + imi 

4.85 ± 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 ± 

(1.54 

Std 1 4.72 1.55 2.16 7.68 1.99 1.71 1.Std 2 4.13 1.40 1.90 6.58 1.78 1.46 1.UberabaIsoline 5.02 ± 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 ± 

(1.68 

CV127 4.36 ± 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 ± 

(1.36 

CV127 + imi 

4.28 ± 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 ± 

(1.37 

Std 1 4.44 1.50 2.05 7.24 1.87 1.66 1.Std 2 4.64 1.52 2.14 7.52 1.97 1.68 1.Sete LagoasIsoline 4.75 ± 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 ± 

(1.45 

CV127 4.44 ± 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 ± 

(1.48 

CV127 + imi 

4.86 ± 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 ± 

(1.67 

Std 1 4.33 1.51 1.96 7.05 1.84 1.56 1.Std 2 4.14 1.43 1.90 6.83 1.77 1.50 1.
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-9. continued.

Location/ Treatment Asp Thr Ser Glu Pro Gly ALondrina Mean ± Standard Deviation (range) g/100 g dry weightIsoline 4.42 ± 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 ± 

(1.52 – 

CV127 4.23 ± 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 ± 

(1.32 – 

CV127 + imi 

4.50 ± 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 ± 

(1.61 – 

Std 1 4.27 1.52 1.92 6.98 1.76 1.56 1.5Std 2 4.43 1.51 2.00 7.25 1.86 1.58 1.5BrasíliaIsoline 4.61 ± 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 ± 

(1.56 – 

CV127 4.51 ± 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 ± 

(1.56 – 

CV127 + imi 

4.64 ± 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 ± 

(1.57 – 

Std 1 4.26 1.49 1.88 6.93 1.76 1.55 1.5Std 2 3.94 1.36 1.78 6.16 1.58 1.41 1.3Santo Antônio de PosseIsoline 4.54 ± 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 ± 

(1.54 – 

CV127 4.74 ± 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 ± 

(1.48 – 

CV127 + imi 

4.54 ± 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 ± 

(1.27 – 

Std 1 4.17 1.46 1.88 6.66 1.64 1.50 1.5Std 2 4.32 1.51 1.94 6.93 2.05 1.51 1.5*Statistically significantly different from the isoline control at P < 0.05.
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-9. continued. 

Location/ Treatment Met Ile Leu Tyr Phe Lys HSanto Antônio de Goiás 

Mean ± Standard Deviation (range) 

g/100 g dry weight

Isoline 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 

(0.79 

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 

(0.86 

CV127 + imi 

0.61 ± 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 

(0.78 

Std 1 0.61 1.64 2.97 1.39 2.04 2.54 0Std 2 0.55 1.50 3.65 1.20 1.83 2.22 0UberabaIsoline 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 

(0.92 

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 ± 

(0.74 

CV127 + imi 

0.62 ± 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 ± 

(0.75 

Std 1 0.60 1.58 2.83 1.35 1.96 2.46 0Std 2 0.55 1.71 3.03 1.38 2.09 2.54 0Sete 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 

(0.79 

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 

(0.79 

CV127 + imi 

0.63 ± 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 

(0.86 

Std 1 0.65 1.60 2.75 1.32 1.92 2.38 0Std 2 0.59 1.55 2.73 1.20 1.88 2.31 0
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-9. continued.

Location/ Treatment Met Ile Leu Tyr Phe Lys HiLondrina

Mean ± Standard Deviation (range) 

g/100 g dry weight

Isoline 0.64 ± 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 ± 

(0.79 – 

CV127 0.62 ± 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 ± 

(0.69 – 

CV127 + imi 

0.62 ± 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 ± 

(0.83 – 

Std 1 0.63 1.48 2.59 1.27 1.78 2.31 0.7Std 2 0.60 1.53 2.76 1.27 1.88 2.36 0.7BrasíliaIsoline 0.64 ± 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 ± 

(0.78 – 

CV127 0.64 ± 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 ± 

(0.75 – 

CV127 + imi 

0.63 ± 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 ± 

(0.83 – 

Std 1 0.63 1.49 2.60 1.22 1.77 2.30 0.8Std 2 0.60 1.38 2.45 1.12 1.64 2.08 0.7Santo Antônio de PosseIsoline 0.62 ± 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 ± 

(0.83 – 

CV127 0.61 ± 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 ± 

(0.77 – 

CV127 + imi 

0.60 ± 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 ± 

(0.67 – 

Std 1 0.60 1.39 2.51 1.22 1.69 2.18 0.7Std 2 0.56 1.61 2.76 1.32 1.97 2.31 0.7

*Statistically significantly different from the isoline control at P < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-10. Amino Acid Composition of Grain on a Dry-Weight Basis of CV127 Soybean TreatmenIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in 

Location/ Treatment Asp Thr Ser Glu Pro Gly 

Mean ± Standard Deviation (range) 

g/100 g dry weight 

Santo Antônio de GoiásIsoline 4.50 ± 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 

(1.6

CV127 4.48 ± 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 

(1.5

CV127 + imi 

4.50 ± 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 

(1.5

Std 1 4.77 1.51 2.05 7.54 1.87 1.72 Std 2 4.93 1.55 2.21 8.02 2.05 1.72 TeresinaIsoline 4.43 ± 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 

(1.4

CV127 4.05 ± 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 

(1.3

CV127 + imi 

4.38 ± 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 

(1.4

Std 1 5.06 1.34 1.91 8.55 2.05 1.60 Std 2 4.03 1.31 1.78 6.47 1.69 1.41 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-10. continued.

Location/ Treatment Asp Thr Ser Glu Pro Gly 

Mean ± Standard Deviation (range) 

g/100 g dry weight 

VilhenaIsoline 4.20 ± 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 

(1.43 

CV127 4.12 ± 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 

(1.58 

CV127 + imi 

4.27 ± 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 

(1.58 

Std 1 4.66 1.47 2.00 7.51 2.03 1.61 1Std 2 4.32 1.42 1.90 6.86 1.81 1.55 1BrasíliaIsoline 3.63 ± 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 

(1.29 

CV127 3.93 ± 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 

(1.33 

CV127 + imi 

4.03 ± 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 

(1.38 

Std 1 4.11 1.38 1.84 6.57 1.75 1.53 1Std 2 3.70 1.23 1.67 5.90 1.60 1.35 1
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-10. continued

Location/ Treatment Met Ile Leu Tyr Phe Lys H

Mean ± Standard Deviation (range) 

g/100 g dry weight 

Santo Antônio de Goiás Isoline 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 ± 

(1.27 – 

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 ± 

(0.99 – 

CV127 + imi 

0.66 ± 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 ± 

(0.91 – 

Std 1 0.62 1.57 2.92 1.39 1.96 2.45 1.Std 2 0.60 1.65 3.15 1.45 2.12 2.58 1.TeresinaIsoline 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 ± 

(0.80 – 

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 ± 

(1.18 – 

CV127 + imi 

0.58 ± 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 ± 

(1.10 – 

Std 1 0.56 1.35 2.85 1.24 1.90 2.29 0.Std 2 0.53 1.47 2.39 1.16 1.71 2.09 0.
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-10. continued.

Location/ Treatment Met Ile Leu Tyr Phe Lys H

Mean ± Standard Deviation (range) 

g/100 g dry weight 

VilhenaIsoline 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 ± 

(1.07 – 

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 ± 

(1.40 – 

CV127 + imi 

0.64 ± 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 ± 

(1.17 – 

Std 1 0.69 1.57 2.77 1.18 1.93 2.51 1.Std 2 0.62 1.53 2.72 1.19 1.90 2.36 1.BrasíliaIsoline 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 ± 

(1.04 – 

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 ± 

(0.93 – 

CV127 + imi 

0.58 ± 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 ± 

(0.96 – 

Std 1 0.59 1.53 2.63 1.25 1.79 2.28 1.Std 2 0.57 1.37 2.43 1.14 1.64 2.03 1.

*Statistically significantly different from the isoline control at P < 0.05.
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-11. Fatty Acid Composition of Grain on a Dry-Weight Basis of CV127 Soybean TreatmenIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in B

Location/ Treatment

C14:0 

Myristic

C16:0 

Palmitic

C18:0 

Stearic

C18:1 

Oleic

C18:2 

Linoleic 

C18:3 

Linolenic 

C

Ara

Santo Antônio de Goiás

Mean ± Standard Deviation (range) 

g/100 g DW 

Isoline <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 

(0.08 

CV127 0.02 ± 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 

(0.07 

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 

(0.07 

Std 1 0.02 2.08 0.66 4.06 10.75 1.56 0Std 2 0.02 2.21 0.70 4.94 12.71 1.65 0UberabaIsoline 0.02 ± 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 

(0.08 

CV127 0.02 ± 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) 

0.0

(

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) 

0.0

(

Std 1 0.02 2.18 0.76 3.99 11.35 1.65 0Std 2 nd 2.19 0.76 5.07 13.09 1.68 0Sete 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 

(0.08 

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) 

0.0

(

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) 

0.0

(

Std 1 nd 2.28 0.84 4.28 11.39 1.54 0Std 2 nd 2.34 0.84 5.14 12.94 1.52 0
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-11. continued. 

Location/ Treatment

C14:0 

Myristic

C16:0 

Palmitic

C18:0 

Stearic

C18:1 

Oleic

C18:2 

Linoleic 

C18:3 

Linolenic 

C

Ara

Londrina Mean ± Standard Deviation (range) 

g/100 g DW 

Isoline <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) 

0.0

(

CV127 0.02 ± 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) 

0.0

(

CV127 + imi 

0.02 ± 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) 

0.0

(

Std 1 nd 2.49 0.79 4.22 12.21 1.85 0Std 2 nd 2.42 0.81 5.41 13.22 1.63 0BrasíliaIsoline 0.02 ± 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 

(0.09 

CV127 0.02 ± 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 

(0.08 

CV127 + imi 

0.02 ± 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 

(0.08 

Std 1 0.02 2.45 0.85 4.20 11.51 1.69 0Std 2 0.02 2.42 0.86 4.82 12.63 1.69 0Santo Antônio de PosseIsoline 0.02 ± 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 

(0.09 

CV127 0.02 ± 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 

(0.09 

CV127 + imi 

0.02 ± 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 

(0.09 

Std 1 0.02 2.38 0.78 4.05 11.72 1.65 0Std 2 0.02 2.34 0.80 4.83 13.01 1.61 0

*Statistically significantly different from the isoline control at P < 0.05. ^ nd 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-12. Fatty Acid Composition of Grain on a Dry-Weight Basis of CV127 Soybean TreatmenIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in 

Location/ Treatment 

C14:0 

Myristic 

C16:0 

Palmitic 

C18:0 

Stearic 

C18:1 

Oleic 

C18:2 

Linoleic 

C18:3 

Linolenic 

C20:0 

Arachid

Mean ± Standard Deviation (range) 

g/100 g DW 

Santo Antônio de GoiásIsoline 0.02 ± 0 

(0.02) N=2 

1.83 ± 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

(0.06 – 0.

CV127 0.02 ± 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

(0.08 – 0.

CV127 + imi 

0.02 ± 0 

(0.02) N=3 

1.96 ± 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

(0.08 – 0.

Std 1 0.01 1.80 0.83 3.87 8.10 1.37 0.07 Std 2 NA 1.77 0.89 4.82 9.44 1.27 0.08 TeresinaIsoline 0.02 ± 0 

(0.02) N=2 

2.37 ± 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

(0.08 – 0.

CV127 0.02 ± 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

(0.10 – 0.

CV127 + imi 

NA 2.33 ± 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

(0.10 – 0.

Std 1 NA 2.14 0.70 5.48 11.49 1.26 0.06 Std 2 0.02 2.09 0.76 7.04 12.44 1.19 0.06 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-12. continued.

Location/ Treatment 

C14:0 

Myristic 

C16:0 

Palmitic 

C18:0 

Stearic 

C18:1 

Oleic 

C18:2 

Linoleic 

C18:3 

Linolenic 

C20:0 

Arachid

Mean ± Standard Deviation (range) 

g/100 g DW 

VilhenaIsoline NA 2.24 ± 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

(0.08 – 0

CV127 0.02 ± 0 

(0.02) N=2 

2.18 ± 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 ± 

(0.09) 

CV127 + imi 

0.02 

N=1 

2.12 ± 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

(0.08 – 0

Std 1 0.02 1.88 0.91 4.52 8.51 1.33 0.09 Std 2 NA 2.21 0.81 4.70 11.88 1.64 0.06 BrasíliaIsoline 0.02 

N=1

1.97 ± 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

(0.07 – 0

CV127 0.02 ± 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 ± 

(0.08) 

CV127 + imi 

0.02 ± 0 

(0.02) N=3 

2.00 ± 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

(0.05 – 0

Std 1 0.02 2.10 0.78 3.97 10.10 1.68 0.05 Std 2 NA 1.97 0.74 4.24 10.61 1.67 0.05 

NA=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) Grown 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 1933 
CONFIDENTIAL 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 DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-14. Mineral Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (CVControl and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in Brazil 

Location Treatment N Calcium Iron Phosphorus M

Mean ± Standard Deviation (range) 

mg/100 g dry weight 

Santo Antônio de Goiás 

Isoline 4 235 ± 14 

(223 – 252) 

8.67 ± 0.44 

(8.24 – 9.24) 

831 ± 14 

(817 – 845) 

(2

 CV127 4 267 ± 13* 

(255 – 279) 

9.63 ± 1.08 

(8.64 – 10.93) 

739 ± 21* 

(719 – 769) 

(2

 CV127 

+ imi 

4 256 ± 9* 

(244 – 264) 

9.03 ± 0.25 

(8.70 – 9.26) 

706 ± 41* 

(654 – 743) 

2

(2

 Std 1 1 192 8.68 793  Std 2 1 213 9.98 808 Teresina Isoline 3 354 ± 8 

(345 – 359) 

8.44 ± 0.41 

(7.98 – 8.77) 

758 ± 20 

(735 – 771) 

(2

 CV127 3 377 ± 48 

(332 – 427) 

8.44 ± 0.32 

(8.23 – 8.81) 

711 ± 63 

(672 – 784) 

(2

 CV127 

+ imi 

3 448 ± 61* 

(377 – 484) 

8.45 ± 0.49 

(7.90 – 8.81) 

797 ± 70 

(733 – 871) 

2

(2

 Std 1 1 329 7.93 734  Std 2 1 323 8.27 703 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-14. continued.

Location Treatment N Calcium Iron Phosphorus M

Mean ± Standard Deviation (range) 

mg/100 g dry weight 

Vilhena Isoline 4 234 ± 5 

(230 – 242) 

9.90 ± 0.84 

(8.96 – 10.90) 

704 ± 52 

(655 – 752) 

(2

 CV127 4 273 ± 13* 

(257 – 287) 

12.10 ± 0.99* 

(10.70 – 13.00) 

708 ± 35 

(679 – 759) 

2

(2

 CV127 

+ imi 

4 275 ± 17* 

(260 – 297) 

10.33 ± 0.99 

(9.00 – 11.40) 

753 ± 14 

(741 – 770) 

(2

 Std 1 1 231 14.13 790  Std 2 1 224 12.23 614 Brasília Isoline 4 188 ± 11 

(172 – 194) 

9.47 ± 0.26 

(9.23 – 9.80) 

778 ± 47 

(708 – 809) 

(2

 CV127 4 205 ± 17 

(190 – 228) 

9.01 ± 0.49 

(8.60 – 9.70) 

707 ± 38 

(654 – 736) 

(

 CV127 

+ imi 

4 206 ± 11 

(198 – 223) 

8.63 ± 0.60* 

(7.80 – 9.20) 

690 ± 90 

(580 – 799) 

(2

 Std 1 1 167 9.43 754  Std 2 1 195 10.60 755 *Statistically significantly different from the isoline control at p < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-15. Selected Vitamin Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatthe Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations 

Location/ Treatment 

α-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)

V

(mg

Santo Antônio de Goiás Mean ± Standard Deviation (range) Isoline 2.87 ± 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.

(5

CV127 3.05 ± 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.

(5

CV127 + imi 

2.81 ± 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.

(4

Std 1 3.29 0.85 14.22 6.31 24.66 6 Std 2 2.45 0.12 20.88 7.42 30.86 6 UberabaIsoline 2.82 ± 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.

(4

CV127 3.18 ± 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) 

5.6

(5

CV127 + imi 

3.30 ± 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) 

5.8

(5

Std 1 2.98 0.71 12.68 5.31 21.68 5 Std 2 2.53 0.55 17.89 6.06 27.04 6 Sete LagoasIsoline 2.53 ± 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.

(4

CV127 3.04 ± 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.

(5

CV127 + imi 

2.95 ± 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.

(5

Std 1 3.27 0.47 14.50 6.04 24.28 6 Std 2 2.48 0.49 17.83 6.47 27.05 6 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-15. continued.

Location/ Treatment 

α-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)

V

(mg/

Londrina Mean ± Standard Deviation (range) Isoline 3.42 ± 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.

(5

CV127 3.68 ± 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) 

6.2

(5

CV127 + imi 

3.91 ± 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) 

6.6

(6

Std 1 3.89 0.99 13.99 6.26 25.15 7 Std 2 3.39 0.73 20.02 6.48 31.03 7 BrasíliaIsoline 2.93 ± 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.

(4

CV127 3.24 ± 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.

(5

CV127 + imi 

3.47 ± 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) 

5.6

(5

Std 1 3.21 0.91 13.44 5.65 23.20 6 Std 2 2.74 0.67 16.81 5.51 25.73 6 Santo Antônio de PosseIsoline 3.65 ± 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.

(5

CV127 4.48 ± 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) 

6.9

(6

CV127 + imi 

4.48 ± 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.

(6

Std 1 4.61 1.08 15.31 6.03 27.00 8 Std 2 3.80 0.80 17.77 5.76 28.14 7 

*Statistically significantly different from the isoline control at P < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-16. Selected Vitamin Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatthe Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Location

Location/ Treatment 

α-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)

Vit

(mg/1

Mean ± Standard Deviation (range) 

Santo Antônio de GoiásIsoline 2.50 ± 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) 

4.7

(4.4

CV127 2.81 ± 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 

(4.9

CV127 + imi 

2.53 ± 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) 

4.7

(4.5

Std 1 2.67 1.24 11.65 7.51 23.07 5 Std 2 2.53 0.66 15.44 7.18 25.81 5 TeresinaIsoline 6.61 ± 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) 

9.4

(8.1

CV127 6.85 ± 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) 

9.8

(9.5

CV127 + imi 

7.84 ± 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) 

10.2

(9.2

Std 1 7.51 1.34 14.62 5.61 29.09 11 Std 2 5.23 0.53 22.27 5.16 33.19 9 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-16. continued.

Location/ Treatment 

α-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) 

Vitam

(mg/100 

Mean ± Standard Deviation (range)

VilhenaIsoline 2.77 ± 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 

(4.93 

CV127 3.21 ± 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 

(5.48 

CV127 + imi 

3.17 ± 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 

(5.16 

Std 1 3.43 1.04 12.63 7.04 24.14 6 5Std 2 2.69 0.66 20.93 8.94 33.23 6 5BrasíliaIsoline 1.80 ± 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 

(3.94 

CV127 2.09 ± 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 

(4.35 

CV127 + imi 

2.20 ± 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 ± 

(4.35 

Std 1 2.07 0.65 11.98 6.86 21.56 4 3Std 2 2.16 0.89 15.94 7.63 26.61 5 4*Statistically significantly different from the isoline control at P < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-17. Isoflavone Composition of Grain on a Dry-Weight Basis of CV127 Soybean TreatmentIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Locations in B

Location/ Treatment Daidzin Malonyl- Daidzin Daidzein Glycitin Malonyl- glycitin Glycitein GenistSanto Antônio de Goiás

Mean ± Standard Deviation (range) 

mg/100 g dry weight

Isoline 13.2 ± 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

(11.7 – 18

CV127 11.7 ± 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

(14.5 – 16

CV127 + imi 

11.2 ± 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

(14.8 – 15

Std 1 14.2 60.1 1.3 9.4 26.3 0.6 18.1 Std 2 11.5 55.5 1.7 12.7 36.2 1.6 15.1 UberabaIsoline 12.1 ± 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

(11.2 – 16

CV127 10.9 ± 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

(11.6 – 14

CV127 + imi 

11.4 ± 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

(13.2 – 14

Std 1 12.2 70.5 1.7 10.3 34.5 1.4 15.6 Std 2 12.6 65.0 0.8 12.6 34.6 1.5 16.2 Sete LagoasIsoline 15.7 ± 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

(16.0 – 19

CV127 11.7 ± 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

(11.9 – 18

CV127 + imi 

12.5 ± 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

(14.3 – 17

Std 1 15.3 57.0 2.8 12.1 31.9 1.1 21.0 Std 2 15.2 53.9 2.7 15.3 39.7 1.8 24.3 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-17. continued. 

Location/ Treatment Daidzin Malonyl- Daidzin Daidzein Glycitin Malonyl-glycitin Glycitein GeLondrina Mean ± Standard Deviation (range) 

mg/100 g dry weight 

Isoline 19.6 ± 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 

(21.8 

CV127 13.1 ± 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 

(17.8 

CV127 + imi 

12.6 ± 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 

(16.0 

Std 1 18.3 72.7 2.7 10.4 29.5 1.3 2Std 2 19.3 70.8 2.9 11.5 28.5 1.5 3BrasíliaIsoline 14.2 ± 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 

(11.7 

CV127 10.8 ± 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 

(9.5 

CV127 + imi 

11.7 ± 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 

(10.3 

Std 1 14.4 67.3 1.7 11.7 33.8 1.4 2Std 2 15.1 70.7 1.2 12.8 34.8 0.6 2Santo Antônio de PosseIsoline 12.5 ± 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 

(9.2 

CV127 11.2 ± 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 

(11.1 

CV127 + imi 

11.1 ± 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 

(12.3 

Std 1 15.2 54.9 2.4 14.0 38.8 0.7 2Std 2 20.6 68.1 2.9 22.9 50.7 1.8 2

*Statistically significantly different from the isoline control at P < 0.05.^nd = not detected 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-18. Isoflavone Composition of Grain on a Dry-Weight Basis of CV127 Soybean TreatmentIsoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locations in 

Location/ Treatment Daidzin Malonyl- Daidzin Daidzein Glycitin Malonyl-glycitin Glycitein Genistin 

Mean ± Standard Deviation (range) 

Santo Antônio mg/100 g dry weight de Goiás Isoline 14.2 ± 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

CV127 9.2 ± 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) 

CV127 + imi 

9.3 ± 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) 

nd 11.8 ± 0.6

(11.2 – 12.4

Std 1 13.9 94.1 0.7 7.5 26.9 0.7 17.7 Std 2 10.4 71.5 0.8 6.6 23.4 1.8 16.0 TeresinaIsoline 6.2 ± 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) 

CV127 6.9 ± 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) 

CV127 + imi 

5.7 ± 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) 

Std 1 9.4 28.0 1.8 9.6 20.6 1.4 15.9 Std 2 8.0 22.1 1.0 10.8 23.4 1.4 10.2 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-18. continued.

Location/ Treatment Daidzin Malonyl- Daidzin Daidzein Glycitin Malonyl- glycitin Glycitein Genistin 

Mean ± Standard Deviation (range) 

mg/100 g dry weight

VilhenaIsoline 14.5 ± 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

CV127 9.7 ± 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

CV127 + imi 

9.8 ± 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

Std 1 15.7 73.9 1.2 7.5 22.2 0.8 14.9 Std 2 15.5 63.4 2.0 11.0 26.2 1.5 20.4 BrasíliaIsoline 29.6 ± 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

CV127 19.6 ± 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

CV127 + imi 

20.6 ± 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

Std 1 26.9 157.5 1.4 8.0 28.0 1.2 29.0 Std 2 25.1 156.5 1.0 10.5 30.2 1.4 35.7 

*Statistically significantly different from the isoline control at P < 0.05. 

^nd = not detected 
CONFIDENTIAL 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.
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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 DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-21. Antinutrient Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (imi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Six Loca2006/07 Season

Location Treatment N Rafffinose 

(g/100 g dry wt.) 

Stachyose 

(g/100 g dry wt.) 

Trypsin Inhib. 

(TIU/mg dry wt.) 

Urease 

(ΔpH)

Lectin 

(HU/mg dry 

Mean ± Standard Deviation (range) 

Santo Antônio de Goiás 

Isoline 4 1.1 ± 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.

(1.36 – 2.7

CV127 4 1.1 ± 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.

(1.54 – 3.0

CV127 + imi 

4 1.1 ± 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.

(1.57 – 3.4

Std 1 1 1.2 4.1 6.11 2.05 3.53 Std 2 1 1.0 3.7 9.59 2.10 1.52 

Uberaba Isoline 4 1.1 ± 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.

(1.75 – 3.3

CV127 4 1.2 ± 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 ± 0.4

(1.46 – 2.5

CV127 + imi 

4 1.2 ± 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 ± 0.1

(1.27 – 1.5

Std 1 1 1.1 4.6 5.40 2.07 1.38 Std 2 1 1.0 3.7 5.03 2.06 1.58 

Sete Lagoas 

Isoline 4 1.4 ± 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.

(2.20 – 3.1

 CV127 4 1.7 ± 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.

(1.68 – 3.7

 CV127 

+ imi 

4 1.5 ± 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.

(2.42 – 3.2

 Std 1 1 1.6 3.7 10.73 1.43 2.13  Std 2 1 1.0 3.1 11.06 1.65 1.68 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-21. continued. 

Location Treatment N Rafffinose (g/100 g dry wt.) Stachyose (g/100 g dry wt.) Trypsin Inhib. (TIU/ mg dry wt.) Urease (ΔpH) Lectin (HU/mg dry 

Mean ± Standard Deviation (range) 

Londrina Isoline 4 1.1 ± 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.

(1.52 – 2.1

CV127 4 1.3 ± 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.

(31.86 – 65

CV127 + imi 

4 1.2 ± 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.

(1.49 – 3.2

Std 1 1 1.2 4.1 13.43 1.78 1.85 Std 2 1 0.8 3.6 17.40 1.83 2.17 

Brasília Isoline 4 1.0 ± 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.

(1.35 – 3.2

CV127 4 1.2 ± 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.

(2.59 – 3.4

CV127 + imi 

4 1.2 ± 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.

(1.44 – 2.8

Std 1 1 1.0 4.2 14.54 2.15 1.60 Std 2 1 0.9 3.8 19.64 2.16 2.48 

Santo Antônio de Posse 

Isoline 4 1.2 ± 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.

(1.60 – 3.1

 CV127 4 1.7 ± 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 ± 0.0

(1.43 – 1.5

 CV127 

+ imi 

4 1.5 ± 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 ± 0.8

(1.36 – 3.1

 Std 1 1 1.3 4.1 10.88 1.96 2.35  Std 2 1 1.0 3.3 13.58 1.94 2.13 

*Statistically significantly different from the isoline control at P < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-22. Antinutrient Composition of Grain on a Dry-Weight Basis of CV127 Soybean Treatments (Cimi), the Isoline Control and Two Conventional Soybean Varieties (Std 1 and Std 2) Grown at Four Locati2007 Season

Location Treatment N Raffinose (g/100 g dry wt.) Stachyose (g/100 g dry wt.) Trypsin Inhib. (TIU/mg dry wt.) Urease (ΔpH) Lectin (HU/mg dry w

Mean ± Standard Deviation 

(range) 

Isoline 4 1.4 ± 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

Santo Antônio de Goiás CV127 4 1.2 ± 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

 CV127 

+ imi 

4 1.2 ± 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

 Std 1 1 1.4 4.8 13.62 1.92 0.67  Std 2 1 1.2 4.0 16.76 1.74 1.66 Teresina Isoline 3 1.4 ± 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

 CV127 3 1.3 ± 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) 

 CV127 

+ imi 

3 1.5 ± 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

 Std 1 1 1.5 3.8 9.84 1.98 1.34  Std 2 1 1.4 3.1 12.56 2.02 2.67 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-22. continued. 

Location Treatment N Raffinose 

(g/100 g dry wt.) 

Stachyose 

(g/100 g dry wt.) 

Trypsin Inhib. 

(TIU/mg dry wt.) 

Urease 

(ΔpH) 

Lectin 

(HU/mg dry 

Mean ± Standard Deviation 

(range) 

Vilhena Isoline 4 1.3 ± 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 ± 0.4

(0.85 – 1.7

 CV127 4 1.2 ± 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) 

 CV127 

+ imi 

4 1.2 ± 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 – 0.1

 Std 1 1 1.2 4.4 14.61 1.16 1.71  Std 2 1 1.4 3.7 16.66 0.27 1.71 Brasília Isoline 4 1.1 ± 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 ± 1.1

(1.29 – 3.4

 CV127 4 1.0 ± 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 – 1.3

 CV127 

+ imi 

4 1.0 ± 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 – 2.0

 Std 1 1 1.1 4.8 14.91 1.98 1.72  Std 2 1 1.3 4.3 13.18 2.02 1.72 *Statistically significantly different from the isoline control at P < 0.05. 
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-23. Proximate Composition of Forage of CV127 Soybean (CV127 + Imi), the Isoline Control anSoybean Varieties (Std 1 and Std 2), Grown at Six Locations in Brazil in the 2007/2008 Season

Location Treatment N 

Moisture 

(g/100 g FW)

Ash 

(g/100 g DW)

Fat 

(g/100 g DW)

Protein 

(g/100 g DW)

Carbohydrates 

(g/100 g DW)

Mean ± Standard Deviation 

(range) 

Santo Antônio de Goiás 

Isoline 3 82.0 ± 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)

CV127 + imi 3 81.8 ± 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)

Std 1 3 82.9 ± 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)

Std 2 

3 

82.0 ± 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)

Uberaba Isoline 3 79.8 ± 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)

CV127 + imi 3 79.1 ± 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)

Std 1 3 80.0 ± 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)

Std 2 3 80.1 ± 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)

Sete Lagoas Isoline 3 80.8 ± 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)

CV127 + imi 3 79.4 ± 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)

Std 1 3 81.7 ± 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)

Std 2 3 77.9 ± 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)
CONFIDENTIAL BUSINESS INFORMATION DELETED 

BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table D-23. continued

Location Treatment N 

Moisture 

(g/100 g FW)

Ash 

(g/100 g DW)

Fat 

(g/100 g DW)

Protein 

(g/100 g DW)

Carbohydrates 

(g/100 g DW)

Mean ± Standard Deviation 

(range) 

Londrina Isoline 3 80.7 ± 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)

CV127 + imi 3 81.2 ± 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)

Std 1 3 81.8 ± 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)

Std 2 3 82.4 ± 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)

Brasilia Isoline 3 81.9 ± 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)

CV127 + imi 3 81.4 ± 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)

Std 1 3 82.1 ± 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)

Std 2 3 81.9 ± 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)

Santo Antônio de Posse 

Isoline 3 83.3 ± 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)

CV127 + imi 3 83.6 ± 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)

Std 1 3 85.0 ± 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)

Std 2 3 84.0 ± 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)

*Statistically significantly different from CV127 + imi at P < 0.05. 
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Table D-24. Fiber Composition of Forage of CV127 Soybean (CV127 + imi), the Isoline 

Control and Two Conventional Soybean Varieties (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)
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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 - 38
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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 developed that are tolerant to the 

imidazolinone class of agricultural herbicides and are referred to as CV127 soybean. Seed 

quality and germination parameters are important characteristics used in comparisons of 

agronomic performance among different varieties of a crop species and seed dormancy is an 

important biological characteristic used to assess 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 treatment for weed control) were compared to 

the isoline control and two conventional soybean varieties to confirm that CV127 soybean is 

equivalent to the isoline control 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 during the 2007 short growing season. The results 

of seed quality, germination and dormancy tests demonstrated 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 isoline 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. Furthermore, these results support the conclusion that 

CV127 soybean does not pose any greater weediness potential or any different environmental 

impact than conventional soybean varieties. 

Introduction

Assessments of seed germination, dormancy and quality were compared between CV127 

soybean, the isoline control and two conventional 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 (Anderson, 1996; Lingenfelter and 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 soybean varieties [Monsoy 

8001 and Coodetec 217 (CD 217)] to confirm that CV127 soybean is equivalent to conventional 

soybean varieties with respect to these biological characteristics. Also, even though soybeans do 

not typically display seed dormancy characteristics (OECD, 2000), the 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 characteristics. These include tetrazolium staining for 

seed quality and the standard germination test (SGT). Seed dormancy was assessed by hard seed 
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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 trial 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 petition, 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 regulatory field trials in 

Brazil in the 2007 short-season. The test seed was harvested from two different 

treatments, T1 and T2. T1 seed was harvested from CV127 plants treated with an 

imidazolinone herbicide (imazapyr, 70 g ai/ha), whereas T2 seed was harvested from 

CV127 plants treated with conventional herbicide (bentazon + acifluorfen-sodium, 

commercial name Volt®, 570 ai g/ha). Grain production 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) Control Seed. (Treatment T3) 

The isoline control seed was harvested from plants grown in the regulatory field trials 

in Brazil in the 2007 short-season. The isoline control seed was harvested from the 

near-isogenic (null segregant) soybean that is genetically similar to, and derived from, 

imidazolinone-tolerant CV127 soybean by breeding, 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 two different conventional soybean varieties 

that are typical commercial varieties grown in Brazil and these were cultivated and 

harvested in the same 2007 field trials as the CV127 seed. The reference soybean 

varieties were Monsoy 8001 (T4) and Coodetec 217 (T5). These reference seeds will 

provide a range of background values for dormancy and germination characteristics 

that are common to soybean. T4 and T5 plants 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 with 
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dehydrogenase enzymes in living tissues to form a red pigment, thereby distinguishing living 

from nonliving tissues. Two sub-samples of 50 seed each were tested for all treatments (T1, T2, 

T3, T4, and T5) harvested from each of the six field 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 were placed in plastic cups, and 

covered with a 0.075% tetrazolium solution. The cups were incubated in an oven at 40oC for 150 

minutes. After staining, the seeds were rinsed with tap water several times to stop the staining 

reaction. Seeds were kept submerged in water to avoid dehydration. Individual seeds were 

examined with a magnifying lens (6X) under fluorescent light. Seed were dissected 

longitudinally through the midsection of the embryonic axis with a single-edge razor blade. 

After the seed was sectioned, the seed halves were 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 revealed by the staining procedure and attributed to 

either mechanical, weathering, or stink bug damage for each sample. Special care was applied 

during the evaluation of the radicle-hypocotyl axis and the vascular region on the cotyledons, 

which are structures of major importance for the 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 cotyledons, and viability was measured 

accordingly. 

Seeds were rated for each type of damage (mechanical, 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, and T5) harvested from each of six field trial 

locations] were tested for germination, following 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 incubated 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. Seeds 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 water and were swollen but did not germinate during 

the test period. The hard or firm seed coat characteristic 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 the 
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germination test and the percentage 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 were 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 ANOVA 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 significant (P<0.01) effect on all the traits except for 

firm seed (which was zero for all treatments in all locations). Given the vast geographical and 

environmental differences among the trial locations, significant differences among locations 

were expected. In contrast to location, and despite genetic differences between the CV127 lines 

and the commercial varieties, there was no statistically significant treatment (“Treatment” in 

Table E-1) effect for any of the traits. 

In general, seed damage (due to mechanical, weathering, and stink bugs), seed viability, and seed 

germination levels were very good for seed of all treatments produced at all 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 weathering and stink bugs) and the highest percent 

abnormal and dead seed. In addition, seed produced in Teresina had the lowest percent seed 

viability and germination. It is likely that the quality of seed 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 interaction 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 seeds at Vilhena; 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). Differences among T1, T2, and T3, were significant for mechanical 

damage at Santo Antônio de Goiás, 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 treatments 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 differences 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 either 
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treatments T2 or T3 and at Teresina, where the T3 treatment (isoline) was higher than the T1 or 

T2 (CV127) treatments. In either 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 germination (percent germination, 

abnormal, dead, hard, or firm seeds), and dormancy tests (percent hard seeds) demonstrated that 

there were statistically significant differences among location means for all measured traits 

(“Location” in Table E-1), but there were no statistically significant differences among treatment 

means for any of the measured traits (“Treatment” in Table E-1). The difference among 

locations was expected given the environmental differences among the locations. However, lack 

of significant treatment differences demonstrates that the seed quality, germination and 

dormancy characteristics of CV127 soybean are not different from that of the isoline control or 

other conventional soybean varieties. These results support the conclusion that CV127 does not 

pose any greater weediness potential or any different environmental impact than conventional 

soybean varieties. 
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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 Test2 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 Test2 Germination Test 

Dmec3 Dweath Dstink Viab Germ Abnor Dead Hard3 Firm 

Locations 

(6-8) (6-8) (6-8) (1-5) 

Uberaba 3.13 a4

 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 the 5% level (Tukey’s test). 
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Table E-3. Mean of Each Treatment Across Locations for the Traits Assessed in the 

Tetrazolium and Germination Tests. 

Traits Assessed 1

Tetrazolium Test2 Germination Test 

Dmec Dweath Dstink Viab Germ Abnor Dead Hard Firm 

Trt 

(6-8) (6-8) (6-8) (1-5) 

T1 2.403

 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 considered nonviable. The cause of the nonviability of these seeds was further 

assessed and the results are reported here. 
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Table E-4. Means of Each Combination of Location and Treatment for the Traits Assessed 

in the Tetrazolium and Germination Tests. 

Traits Assessed1, 4

Tetrazolium Test2 Germination Test 

Dmec Dweath Dstink Viab Germ Abnor Dead Hard Firm 

Location Trt 

(6-8) (6-8) (6-8) (1-5) 

T1 *3 * * * * * * * * 

T2 3.25 a4

 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 

Uberaba 

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 

Lagoas 

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 

Antônio 

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 

Brasília 

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 

Vilhena 

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 field sites because of a misapplication of imazapyr herbicide. 4 Means in a column followed by the same letter do not differ statistically significantly at the 5% level (Tukey’s 

test). 
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Appendix F

Materials and Methods and Results from the Agronomic, Phenotypic, and Ecological 

Interaction Evaluations of CV127 Soybean 

Introduction

Evaluations of the agronomic, phenotypic and ecological interactions of CV127 soybeans were 

conducted at a total of thirteen field trial locations 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 conventional soybean varieties commonly 

cultivated in Brazil. The field trial sites were representative of the different regions within Brazil 

where soybean is commercially cultivated and each trial location consisted of four replications of 

the two CV127 treatments, isoline control and two 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 germination 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, the 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 used 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, except 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 isoline control soybeans with respect 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 conventional soybean varieties. 

The methods and results presented in this Appendix are organized as follows: 
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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 characteristics 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 during the 2006/2007 growing season at seven 

experimental stations in Brazil. One station is owned and operated by BASF S.A. and six by 

Embrapa. The stations are located in regions that are representative of areas of commercial 

soybean production and areas in which the soybean trial entries are adapted. All locations had a 

Certificate of Quality in Biosafety (CQB), an established infrastructure, laboratory facilities and 

field equipment, and personnel experienced in agricultural research and trained in biosafety 

(Table F-1). 

Table F-1. Experimental Station Locations 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 Antônio de Goiás, GO CNPAF (014) 

Embrapa Corn and Sorghum Sete Lagoas, MG CNPMS (013) 

The predominant soil type, the tillage system (conventional disc tillage or no tillage), and the 

cropping history varied by location (Table F-2). All but one location (CNPMS) had been used 

for crop production within the previous two years. 
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Table F-2. A Description of the Soybean 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 each location, the quantity 

of water and frequency of application was dependent on the quantity and distribution of rainfall. 

In general, the amount of precipitation was below average at EEA, near average at CNPH, 

CNPAF, SNT, CTTP and CNPSo, and above average 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 - - 
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Field, and Plot Design. In all field locations, five treatments (T1-T5, Table F-6) were replicated 

four times in a randomized complete block design. Each plot consisted of six 8 m-long rows 

with 0.5 m spacing between rows of the same plot and 1.0 m spacing between rows of adjacent 

plots. Soil, plant, and insect samples were collected 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 adding 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 planting at Ponta Grossa negatively impacted the germination of 

all entries and prompted the need to replant at this location. The replanted seed of the test and 

isoline control were from an F3 family that is related to, but not genetically identical to, the F3

family used in all other test locations. 
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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 planting on 10 October prompted need to replant on 31 October. 

Post-plant

To ensure the successful completion 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 plants from insect, fungal, and weed infestations 

(Table F-5). Insecticidal chemicals applied included fipronil, teflubenzuron, permethrin, 

cypermethrin, thiamethoxan, imidaclopride, chlorpyrifos, methamidophos, and avermectin. 

Chemicals applied for the control of fungi, primarily Phakopsora pachyrhizi, included 

epoxiconazole, pyraclostrobin, flutriafol, 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). The herbicide tepraloxydim was applied 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. 
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Table F-5. Insecticides, Herbicides, and Fungicides 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 Fungicide 
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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 Fungicide 
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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 threshed with a stationary bulk plant thresher. Seed were dried to 

uniform moisture and weighed. Harvest was initiated on 14 March in Londrina and completed 

on 29 March in Brasília and Uberaba (Table F-4). A late, but necessary replanting of the trial at 

Ponta Grossa resulted in uncharacteristic plant growth and late maturity of all CV127, isoline 

control, and conventional soybean varieties. 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, CV127, utilized in the study was the F5 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 field trials: Monsoy 8001 (M8001) and 

Coodetec 217 (CD 217). Both of these varieties are common nontransgenic Brazilian 

commercial varieties with a similar maturity classification as CV127 and the isoline control 

soybeans. 

Two herbicide treatments were used in these 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, isoline control, and conventional soybean varieties were determined 

by recording various characteristics routinely used to describe the phenotype and behavior of a 

soybean genotype. The characteristics were recorded for all plots at all locations (unless 

otherwise noted). 
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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 traits were evaluated during the different growth 

stages of soybean development. The different growth stages of soybean, including vegetative 

and reproductive stages of soybean plant growth and development are defined below in Table 

F-7. A description of the evaluation of each measured trait follows. 
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Table F-7. Description of the Different Soybean Growth Stages 

Stage 

number 

Stage name Stage definition 

VE Emergence Hypocotyl elongated and hooked; cotyledons at soil surface 

VC Cotyledonary Hypocotyl straightened; cotyledons unfolded; unifoliates 

unfolded 

V1 First node First trifoliate leaves unfolded 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 nth 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 the plot with a flower on upper two nodes 

R3 Begin pod 50%+ of plants in the plot 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 plot 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) color 
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Seed germination: the percentage of emerged plant seedlings at the V2 stage relative to the 

quantity of seed sown per plot. 

Seedling vigor: the overall appearance and rate of plant development 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 Average 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 (lodging) of plants in a plot at maturity (R8) according to the 

index in Table F-9. 

Table F-9. Scale Used to Evaluate the Average Lodging of Plants on a Plot Basis. 

Lodging Description

1 All plants are standing erect (80-90 degree angle). 

2 Most plants have a slight lean (60-70 degree angle).

3 Most plants have a moderate lean (40-50 degree angle).

4 Most plants have a severe lean (20-30 degree angle).

5 All plants are prostrate (0 to 10 degree angle).

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 planting 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 plant 
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Seed size: the weight (g) of 100 seeds. 

Grain yield: the total weight (g) of all grain harvested (adjusted to 12% moisture) converted to 

kg/ha. 

Results and Discussion

Based on analyses of variance (Table F-10): 

1) location had a significant effect on all measured traits, including percent 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 yield (Yield), seed size (100SW), 

and lodging (L), 

2) treatment had a significant effect on seedling vigor, days to full maturity, 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 the 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 2.38a 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 = plant 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 characteristics (temperature, rainfall, humidity, day 

length, etc.) among trial locations, location had a significant effect on all assessed traits. At 

Ponta Grossa, the most southerly location (latitude 25o S) and the location for which the 

Conquista soybean variety is least adapted, plants were taller, green stems 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 recorded from this site due to a 

late replanting of the study and subsequent uncharacteristic plant growth and late maturity of the 

CV127, isoline control, and conventional soybean varieties. 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, seedling vigor was slightly less than at other locations (rating of 4.2 
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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, likely due to the Asian rust infection, yield data from Brasília was 

not included in the across-location analysis for yield. 

Table F-11. Agronomic and Phenotypic Evaluations 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‡ Sto. Antônio 106.8 a 5.0 a 56.1 d 0.0 a 1.0 b 43 b 118 c 16.8 b 3224 c

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)

Sto. Antônio 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

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 maturity (total cycle); 100SW = 

seed size [weight of 100 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 CV127 treated with imazapyr (T1) or with 

Volt® (T2) and the performance of the isoline control treated with Volt® (T3) were not 

significantly different for germination, final stand, green stem, lodging, days to flower, days to 

maturity, or yield (Table F-12). Only for vigor, plant height and seed size did T1 significantly 

differ from T3 and only for seed size did T2 significantly differ from T3. Only for seed size, was 

the difference between both conventional soybean varieties (T4 and T5) significantly different 

from Treatments 1, 2, and 3. 
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Table F-12. Agronomic and Phenotypic Evaluations 2006/2007: Treatment Means and 

Standard Deviations (Shown in Parentheses) 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 (vegetative 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 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. 

The Location x Treatment interaction was significant for seedling vigor, days to full flower and 

maturity, green stem, plant height, yield, and lodging (Table F-10). For all other traits, the mean 

of the treatments over locations was a reliable indicator of the effect 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 not significantly different for germination, final 

plant stand, plant height, percent green stem, lodging, seed size or yield at any of the locations. 

T2 and T3 only differed significantly for days to flowering and days to maturity at Londrina and 

Santo Antônio de Posse, and for days to flowering 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 height, percent green stem, lodging, days to flowering 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 significant differences did exist between CV127 soybean and the isoline 

control for specific traits at specific locations, none of the differences 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.
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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. 
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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.
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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 (vegetative 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. 
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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 during the short season in 2007 at six 

experimental stations in Brazil. The stations are 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 Locations 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 Antônio 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 (conventional 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 each location, the quantity 

of water and frequency of application was dependent on the quantity and distribution of rainfall. 

In general, precipitation was greatest during March and April, reaching means of 126 and 106 

mm, respectively. Irrigation was necessary during 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, 
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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 Monthly Temperature at Each of the Soybean 

Field Trial Locations, 2007. 

March April May June July 

Location R* T R T R T R T R T 

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 February and 14 March 2007 (Table F-17). 
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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 recommendations and as needed to protect the plants 

from insect, fungal, and weed infestations (Table F-18). 
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Table F-18. Insecticides, Herbicides, and Fungicides 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 Insecticide 
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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 F6

generation of CV127, line 127 (Figure III-1), and 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 measured traits, including percent germination (G), 

seedling vigor (V), initial (IS) and final (FS) plant stand, days to full flower (DF), days to full 

maturity (DM), percent plants with green stem (GS), plant height (PH), grain yield (Yield), seed 

size (100SW), and lodging (L), 

2) no significant effect was observed for 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 (100SW), grain yield (Yield), lodging (L), days to 

full flower (DF) and days to full maturity (DM). 
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Table F-19. Agronomic and Phenotypic Evaluations 2007: Statistically Significant and Non-Significant 

in the Model Used to Analyze Data for Each Trait.

1G = initial germination (%); V = seedling vigor; IS = initial plant stand; FS = final plant stand; PH = plant height (cm); GS = green ste

[weight of 100 seeds (g)]; Yield = grain yield (kg/ha); L = degree of lodging; DF = days to full flower (vegetative cycle) and DM = 

cycle). *,** = significant at P<0.05 and P<0.01, respectively. 

Trait1 Source of 

Variation G V IS FS PH GS 100SW Yield L Location (L) 3678.37** 2.41** 4863.69** 5539.36** 3078.27** 8343.29** 16.86** 3723651.19** 20.83** 2Treatment (T) 268.35 0.05 369.06 399.81 664.50 6801.09 85.14 423247.06 0.23 L x T 136.96 0.05 190.39 189.34 50.32** 1827.25** 2.61** 306005.05** 0.23** Error 103.16 0.17 146.72 152.24 6.26 28.57 0.85 105353.70 0.05 Mean 80.04 4.86 93.72 89.59 57.57 24.66 17.83 2324.72 1.42 CV% 12.70 8.51 12.92 13.77 4.35 21.68 5.17 13.96 16.00 
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Due to the naturally different environmental characteristics (temperature, rainfall, humidity, day 

length, etc.) among trial locations, location had a significant 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 growing conditions, plant height was normally low, 

except at Sete Lagoas, where plants reached a mean stature of 82 cm and the lodging reached the 

higher index of 3.5. Percentage of green stem was higher at Uberaba, Sete Lagoas and Vilhena. 

An error in the preparation of the herbicide imazapyr 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 and 100% of plants showing green 

stem over all the plots with treatment T1. These plots were not harvested and no data was 

recorded for seed size (100SW), days to full maturity (DM) and yield at these two sites (Table F22). The higher green stem percentage observed at Vilhena was likely 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 Vilhena 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 Brasília and Sete 

Lagoas, medium at Teresina and Uberaba and low at Santo Antônio de Goiás and Vilhena (Table 

F-20). The wide-ranging performance obtained for grain yield among the different sites was 

expected and considered normal for short-season conditions. However, the coefficients of 

variation for all traits were considered low and acceptable for the purposes of this work (Table F19). 
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Table F-20. Agronomic and Phenotypic Evaluations 2007: Location Means Across 

Treatments and Standard Deviations (in 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 100 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 trait, the means of all treatments showed no 

evidence of a significant difference (Table F-21). However, the Location x Treatment 

interaction was statistically significant 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 trait. With the exception of percent green stem at 

Uberaba and Sete Lagoas (caused by the application 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 germination, seedling vigor, initial and 

final plant stand, lodging, days to flowering or days to maturity at any of the field trial locations. 

T2 and T3 only differed statistically 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 F-22). Similarly, the instances where the performance of T1 and T3 differed 

significantly included plant height at Sete Lagoas, green stem at Uberaba, Sete Lagoas and 

Vilhena, seed size and yield at Vilhena, and days to flowering at Uberaba (Table F-22). 

Trait1

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) 

Sete 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

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) 
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Table F-21. Agronomic and Phenotypic Evaluations 2007: Treatment Means and 

Standard Deviations (in Parentheses) Over Location for Each Trait. 

Trait1

Trt2

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 100 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®. 
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 Table F-22. Agronomic and Phenotypic Evaluations 2007: Means and Standard Deviations (in PaCombination of Treatment and Location for All Traits. 

Trait1 Location Trt2

G V IS FS PH GS 100SW Yield L 

T1 71.25 5.00 85.25 78.50 50.20 ab* 100.00 a ND3 ND 1.00 a 

 (9.5) (0.0) (11.7) (12.1) (1.5) (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 

 (10.8) (0.0) (13.0) (11.5) (1.8) (5.0) (0.7) (97.5) (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 

 (2.1) (0.0) (2.8) (11.2) (4.2) (10.0) (0.7) (811.4) (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 

 (4.1) (0.0) (4.7) (4.2) (3.1) (5.0) (0.6) (316.4) (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 

Uberaba 

 (4.5) (0.0) (5.7) (7.0) (4.4) (1.4) (0.3) (364.4) (0.0) 

T1 87.00 5.00 104.00 100.25 78.35 cd 100.00 a ND ND 3.75 a 

 (4.5) (0.0) (5.4) (5.3) (2.9) (0.0) (0.5) 

T2 77.75 5.00 93.25 86.00 81.85 bc 35.00 bc 20.23 a 2446.00 a 4.00 a 

 (6.1) (0.0) (7.4) (8.1) (2.5) (5.8) (0.9) (102.8) (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 

 (3.7) (0.0) (4.1) (8.3) (3.2) (7.5) (0.3) (349.9) (0.8) 

T4 78.00 5.00 93.50 90.75 85.55 ab 42.50 b 16.10 c 2509.50 a 2.75 a 

 (4.1) (0.0) (4.7) (5.6) (2.9) (12.6) (1.0) (401.2) (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 

Sete 

Lagoas 

 (3.6) (0.0) (4.3) (6.4) (2.9) (3.3) (0.4) (425.8) (0.8) 

Table F-22 is continued on the next page. 
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Table F-22 continued. 

Trait1 Location Trt2

G V IS FS PH GS 100SW Yield L 

T1 91.50 5.00 110.00 109.38 59.00 a 10.00 a 20.13 a 2017.75 a 1.00 a  (4.3) (0.0) (5.2) (5.4) (1.8) (0.0) (1.0) (238.0) (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  (1.8) (0.0) (2.2) (2.0) (1.4) (0.0) (1.3) (326.9) (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  (2.0) (0.0) (2.4) (2.2) (2.0) (0.0) (1.0) (347.5) (0.0) T4 92.50 5.00 110.88 109.50 48.50 b 10.00 a 16.28 b 1985.50 a 1.00 a  (2.1) (0.0) (2.6) (2.7) (1.0) (0.0) (0.5) (287.9) (0.0) T5 93.00 5.00 111.38 109.88 48.00 b 10.00 a 15.90 b 1631.50 a 1.00 a 

Santo 

Antônio de 

Goiás 

 (1.7) (0.0) (2.1) (1.7) (1.6) (0.0) (0.3) (499.5) (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  (2.2) (0.0) (2.6) (2.3) (4.5) (0.0) (1.2) (332.8) (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  (4.8) (0.0) (5.8) (5.8) (2.2) (0.0) (0.9) (241.4) (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  (1.6) (0.0) (1.9) (2.1) (1.5) (0.0) (0.6) (260.4) (0.0) T4 87.75 5.00 105.50 104.25 45.75 b 0.00 b 16.60 c 2874.00 ab 1.00 a  (4.3) (0.0) (5.1) (5.0) (1.5) (0.0) (0.9) (305.5) (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 

Brasília 

 (1.9) (0.00 (2.3) (2.5) (4.1) (0.0) (1.4) (185.4) (0.0) 

Table F-22 is continued on the next page. 
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Table F-22 continued. 

Trait1 Location Trt2 G V IS FS PH GS 100SW Yield L 

T1 83.50 5.00 87.00 77.00 58.50 a 67.50 b 21.50 a 1701.5 b 1.00 a 

 (5.0) (0.0) (5.2) (4.0) (2.6) (15.0) (1.9) (206.6) (0.0) 

T2 83.25 5.00 86.50 77.00 60.00 a 60.00 b 21.58 a 1800.50 b 1.00 a 

 (1.4) (0.0) (1.5) (4.1) (2.3) (14.1) (1.4) (187.8) (0.0) 

T3 88.00 5.00 92.00 86.25 58.25 a 21.25 c 18.58 b 2371.25 a 1.00 a 

 (5.4) (0.0) (5.6) (5.3) (1.9) (6.3) (0.9) (128.6) (0.0) 

T4 96.50 5.00 100.50 93.25 54.25 a 90.00 a 18.20 b 1518.75 b 1.00 a 

 (2.4) (0.0) (2.5) (1.3) (4.9) (7.1) (0.3) (128.3) (0.0) 

T5 89.25 5.00 93.25 87.25 41.25 b 3.25 c 15.25 c 2487.75 a 1.00 a 

Vilhena 

 (1.2) (0.0) (1.2) (6.9) (2.4) (2.4) (0.7) (261.1) (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 

 (26.9) (0.6) (32.3) (32.3) (2.5) (0.0) (2.3) (837.0) (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 

 (31.3) (1.0) (37.6) (37.6) (1.7) (0.0) (1.7) (520.5) (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 

 (22.0) (1.0) (26.4) (26.0) (2.2) (0.0) (1.1) (292.2) (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 

 (11.6) (1.5) (13.9) (14.0) (1.7) (0.0) (0.5) (236.6) (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 

Teresina 

 (4.1) (1.2) (4.9) (4.4) (1.3) (0.0) (1.0) (245.3) (0.0) 

1G = initial germination (%); V = seedling vigor; IS = initial plant stand; FS = final plant stand; PH = plant height (cm); GS = green ste

[weight of 100 seeds (g)]; Yield = grain yield (kg/ha); L = degree of lodging; DF = days to full flower (vegetative cycle) and DM = 

cycle). 

2Treatments: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monso

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. 
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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 between CV127 and the isoline control for 

specific traits at specific locations, the only trait for which there was a trend toward a consistent 

difference between CV127 and the isoline control was 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 biologically significant differences between CV127 and the isoline 

control for any of the evaluated traits demonstrates 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 soybean 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 diseases 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% reduction in the yield of susceptible soybean 

cultivars and can occur at any phase of the plant’s growth. These pathogens are currently 

disseminated throughout the soybean-producing regions in Brazil, and the use of resistant 

cultivars and fungicide treatment are the main control methods. 

The purpose of the current study was to evaluate the disease susceptibility characteristics of 

CV127 soybean relative to the isoline control and to two conventional soybean varieties. Field 

studies were conducted during the 2006/2007 and 2007 growing seasons at the thirteen field 

locations in Brazil described above in section 1.0 of Appendix F. Disease susceptibility of 

CV127 soybean relative to the isoline control and conventional 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 treatments in each study have been described above in 

section 1.0 of Appendix F (“Agronomic and Phenotypic Evaluations”). 

Disease evaluations. 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 diseases causing 

infection were identified at each assessment. Assessments were made at or near vegetative stage 
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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 diseases 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 per location, per disease, and per 

time-of-assessment basis. In those cases where differences among treatments were significant, 

Tukey’s procedure (Steel and Torrie, 1980) was used to make pair-wise comparisons of 

treatment means. 
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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 locations (Tables F-23 to F-29). In the 2006/2007 field trials, no 

diseases were evident prior to the V4 stage of development. In the data presented in Tables F-23 

to F-29, where any of the four diseases listed above were not observed on plants grown at a 

particular field location and stage of plant development, then no data is presented. 

No significant differences in disease incidence 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) and 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). These differences in disease susceptibility were 

attributed to genetic differences between the Conquista germplasm and the germplasm of the 

conventional reference soybean varieties. 

Other than a high incidence of Asian rust at several locations, the incidence of other diseases was 

generally low. Although a few isolated differences in disease susceptibility were noted among 

the treatments, these were not consistent across field trial locations and in general there were no 

differences in susceptibility between CV127 and the isoline control. Where susceptibility 

differences were observed, they were generally between varieties with the Conquista genetic 

background (CV127 and the isoline control) and the two conventional soybean varieties with 

different genetic backgrounds. 
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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. 
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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. 
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Table F-27. Disease Assessment Presented as Mean Percent 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. 
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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 diseases, brown spot (Septoria glycines) and leaf spot (Cercospora 

kikuchii), were also present at some locations (Tables 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 location or stage of plant 

development, then no data is presented. 

No statistically significant differences in disease 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 higher level of end-of-cycle diseases 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 there were no differences in susceptibility between 

CV127 and the isoline control. Where susceptibility differences were observed, they were 

generally between treatments with the Conquista genetic background (CV127 and the isoline 
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control) and the two conventional soybean varieties with different genetic backgrounds. 

Therefore disease susceptibility of CV127 was found to be no different from that of the isoline 

control plants. This study demonstrates that the insertion of the csr1-2 gene into the genome of 

CV127 did not affect the disease susceptibility of CV127. 
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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 statistically significantly by the Tukey’s test at 1% probability. 

Table F-31. Disease Assessment Presented as Mean Percent 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 = 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); PM = powdery mildew (Erysiphe diffusa). 

§

ND = no diseases were observed. 

¶

Means followed by the same letter do not differ statistically significantly by the Tukey’s test at 1% probability. 
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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. 
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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 

Treatment V4 R1 R5.1 R7 †

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. 
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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-field insect populations of imidazolinone-tolerant 

CV127 soybean relative to the isoline control and the two conventional soybean 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 relative to the isoline control and 

conventional soybean varieties were 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 treatments in each study have been described above in 

section 1.0 of Appendix F (“Agronomic 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 between 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 all 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 across all treatments and across locations was 

considered minimal and no differences were distinguished among the treatments. Therefore 

these data are not presented. 
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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 those cases where differences among treatments were significant, 

Tukey’s procedure (Steel and Torrie, 1980) was used to make pair-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 common order at Ponta Grossa, Londrina, Santo Antônio de Goiás, 

and Brasília, while the Lepidopterans were most common at Santo Antônio de Posse, Uberaba, 

and Sete Lagoas (Table F-37). The location with 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 Brasília. 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 any 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 health. 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. 
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Table F-37. The Mean Number of Coleopteran, Lepidopteran, and Hemipteran Insects at Each Field Site 

Growth Stage 

V1 R1 R5 

Order 

Location Trt†

Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleopter

T1 0.25 0.25 0 2.5 1.5 0 6.75 3.25 2.5 2.5 T2 0.25 0.25 0 2 2 0 5.75 3 2 2.25 T3 0.75 0.25 0 2.25 1.25 0 5.5 2.75 2.5 2.75 T4 0.25 0.25 0 3.75 2.25 0 4.75 3.25 2.75 2 T5 0.5 0.25 0 3.25 2.25 0 6 3 2.25 2.25 

Ponta 

Grossa 

Mean 0.4 0.25 0 2.75 1.85 0 5.75 3.05 2.4 2.35 

T1 0.5 0 0.5 1.75 2.5 0 1.5 0.5 0.75 0.25 T2 1 0 0.25 2.25 2.25 0 1 2.25 1.25 0 T3 1 0 1 1.75 1.75 0 1.25 1.25 0.25 0.75 T4 1 0 0.25 2.25 2.25 0 0.75 2.25 1.25 0.25 T5 1.25 0 0.5 1.75 1.75 0 1.25 1 2.25 0.25 

Londrina 

Mean 0.95 0 0.5 1.95 2.1 0 1.15 1.45 1.15 0.3 

T1 0.5 0.5 0 0 0 0 2 4 4 0 T2 0.25 0 0.25 0 0 0 2.25 3.75 4 0 T3 0.5 0.5 0.25 0 0 0.5 1.75 4 3.75 0 T4 0.5 0.5 0.25 0 0 0.5 2.25 4 3.75 0 T5 0.5 0.5 0.25 0 0 0.25 2.75 4.5 4.25 0 

Santo 

Antônio 

de Posse 

Mean 0.45 0.4 0.2 0 0 0.25 2.2 4.05 3.95 0 

T1 0.5 0 0 0 0.25 0 0 0.5 0 0 T2 0.25 0 0 0 0 0 0 0.5 0 0 T3 0 0 0 0 0 0 0 0.25 0 0 T4 0.75 0 0 0 0.25 0 0 0 0.25 0 T5 0.5 0 0 0 0.25 0.25 0 0.75 0.25 0 

Uberaba 

Mean 0.4 0 0 0 0.15 0.05 0 0.4 0.1 0 

† Treatment: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monso

T5 = Coodetec 217 treated with Volt®. 
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Table F-37 continued. 

Growth Stage 

V1 R1 R5 

Order 

Location Trt†

Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleopte

T1 0 0 0 0 0 0 0.25 0 0 1 T2 0.25 0 0 0 0.25 0.25 0 0.25 0 0.5 T3 0.5 0 0 0 0.25 0 0 0.25 0 0 T4 0.5 0 0 0 0.5 0 0.25 0 0 0.25 T5 0.75 0 0 0 0.5 0 0.75 0.25 0 0.75 

Sete 

Lagoas 

Mean 0.4 0 0 0 0.3 0.05 0.25 0.15 0 0.5 

T1 1.5 0 0 0.5 0.25 0 0 0 0 0 T2 0.25 0 0 0 0 0 0 0 0.5 0 T3 0.5 0 0 1 0 0 0 0 0.25 0 T4 1.25 0 0 0.75 1.25 0 0 0 0 0 T5 0.25 0 0 0 0.5 0 0 0 0.25 0 

Santo 

Antônio 

de Goiás 

Mean 0.75 0 0 0.45 0.4 0 0 0 0.2 0 

T1 0.5 0.25 0 0 0 0 0 0 0.25 0 T2 0 0.25 0 0.5 0 0 0 0 0 0 T3 0.25 0 0 0.25 0.25 0 0 0.25 0.5 0 T4 0.25 0.25 0 0 1 0 0 0 0.5 0 T5 0.5 0 0 0.25 0 0 0 0 0 0 

Brasília 

Mean 0.3 0.15 0 0.2 0.25 0 0 0.05 0.25 0 

† Treatment: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monso

T5 = Coodetec 217 treated with Volt®. 
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b) 2007 growing season 

Insect orders that were common across most or all locations in the trials 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 common at Teresina (Table F-38). The location 

with the largest insect populations was Vilhena, followed by Sete Lagoas and Uberaba. 

Relatively few insects were found at Santo Antônio de Goiás and Brasília. The largest 

populations of insects of the orders Coleoptera and 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 individual 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 health. 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. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 

Table F-38. The Mean Number of Coleopteran, Lepidopteran, and Hemipteran Insects at Each Field Tria† Treatment: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monso

T5 = Coodetec 217 treated with Volt®. 

Growth Stage 

V4 R1 R5 

Order 

Location Trt1

Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleopte

T1 1.39 0.71 0.71 1.06 1.10 0.71 0.71 1.10 0.71 0.71 T2 1.56 0.71 0.71 0.97 0.84 0.71 0.71 0.93 0.84 0.71 T3 1.48 0.71 0.71 1.06 0.84 0.71 0.71 1.06 0.84 0.71 T4 1.56 0.71 0.71 0.84 0.71 0.71 0.71 0.84 0.84 0.71 T5 1.40 0.71 0.71 0.97 0.97 0.71 0.71 0.97 0.71 0.71 

Uberaba 

Mean 1.48 0.71 0.71 0.98 0.89 0.71 0.71 0.98 0.78 0.71 T1 1.22 0.71 0.71 0.84 0.84 0.84 0.71 1.40 0.84 0.71 T2 1.06 0.71 0.71 0.84 1.06 0.71 0.71 1.31 0.84 0.71 T3 1.18 0.71 0.71 1.06 1.06 0.71 0.71 1.10 0.71 0.71 T4 1.22 0.71 0.71 0.97 1.18 0.71 0.71 0.84 0.97 0.71 T5 1.10 0.71 0.71 0.97 0.97 0.71 0.71 1.06 0.97 0.71 

Sete 

Lagoas 

Mean 1.16 0.71 0.71 0.93 1.02 0.73 0.71 1.14 0.86 0.71 T1 1.36 0.71 0.71 0.84 0.84 0.84 0.71 0.71 0.84 0.71 T2 1.06 0.84 0.71 0.84 0.71 0.71 0.71 0.71 0.84 0.71 T3 1.06 0.71 0.71 0.84 0.84 0.71 0.71 0.71 0.84 0.71 T4 0.93 0.84 0.71 0.84 0.71 0.71 0.71 0.71 0.71 0.71 T5 0.93 0.71 0.71 0.71 0.84 0.71 0.71 0.71 0.84 0.71 

Santo 

Antônio de 

Goiás 

Mean 1.06 0.76 0.71 0.81 0.78 0.73 0.71 0.71 0.81 0.71 T1 0.71 0.71 0.71 0.71 0.84 0.71 0.71 0.84 0.84 0.71 T2 0.71 0.84 0.71 0.71 0.84 0.71 0.71 0.71 0.71 0.71 T3 0.84 0.71 0.71 0.84 0.71 0.71 0.71 0.84 0.97 0.71 T4 0.84 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 T5 0.97 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 0.71 

Brasília 

Mean 0.81 0.73 0.71 0.73 0.76 0.71 0.71 0.76 0.78 0.71 
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Table F-38 continued.

Growth Stage 

V4 R1 R5 

Order 

Location Trt1

Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleoptera Lepidoptera Hemiptera Coleopte

T1 0.97 0.71 0.71 1.49 0.71 0.71 1.40 1.10 1.18 0.71 T2 0.97 0.71 0.71 1.49 0.71 0.71 1.61 0.84 1.27 0.71 T3 1.10 0.71 0.71 1.65 0.71 0.71 1.31 1.10 1.40 0.71 T4 1.06 0.71 0.71 1.49 0.71 0.71 1.79 1.13 1.44 0.71 T5 0.97 0.71 0.71 1.40 0.71 0.71 1.59 0.84 1.49 0.71 

Vilhena 

Mean 1.01 0.71 0.71 1.51 0.71 0.71 1.54 1.00 1.36 0.71 

T1 0.71 1.78 0.71 0.71 0.71 0.71 0.71 b* 0.71 0.84 0.71 T2 0.71 2.04 0.71 0.71 0.84 0.71 0.71 b 0.71 0.84 0.71 T3 0.71 1.90 0.71 0.71 0.97 0.71 0.71 b 0.71 0.71 0.71 T4 0.71 1.83 0.71 0.71 0.71 0.71 1.79 a 1.13 0.71 0.71 T5 0.71 1.63 0.71 0.71 0.71 0.71 0.71 b 0.71 0.71 0.71 

Teresina 

Mean 0.71 1.84 0.71 0.71 0.78 0.71 0.92 0.79 0.76 0.71 

† Treatment: T1 = CV127 treated with imazapyr, T2 = CV127 treated with Volt®, T3 = Isoline control treated with Volt®, T4 = Monso

T5 = Coodetec 217 treated with Volt®. 

*Means followed by the same letter, in the same column, are not statistically different at the 5% level. 
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Conclusions

The numbers of Coleopteran, Lepidopteran, and Hemipteran insects detected on CV127 

soybean plants were not statistically significantly 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 was considered minimal and no biologically 

significant differences were distinguished among the treatments. Therefore, resistance to 

insect damage and effect on in-field insect populations of CV127 soybean was found to 

be no different from that of the isoline control plants or conventional soybean varieties. 

These results further support the conclusion that the cultivation of CV127 soybeans poses 

no different environmental impact than the cultivation 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 the plant creates an environment within the 

nodule that is conducive to nitrogen 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 practice in Argentina and Brazil for farmers to 

inoculate soybean seed with B. japonicum at planting to ensure the development of 

plentiful root nodulation and strong nitrogen fixation activity. Studies have demonstrated 

that the rotation of soybean with 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 reduction 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 comparable to conventional soybean varieties with respect to 

nitrogen fixation and plant N accumulation. Characteristics of nitrogen fixation in 

CV127 soybean were evaluated and compared with the characteristics of the isoline 

control and conventional soybean varieties, including measurements of soil populations 

of B. japonicum, plant growth and nodulation and total nitrogen and ureide nitrogen 

accumulation in the plants. These determinations 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 section 1.0 of Appendix F; these 

locations are representative of areas of commercial soybean production and the soybean 

varieties included in the study are adapted to these areas. 
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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 biological nitrogen fixation 

was performed following the protocols of RELARE (Rede de Laboratórios para 

Recomendação de Estirpes de Rizóbio e Inoculantes Microbianos), a network of 

Brazilian microbiologists officially recognized by the Ministry of Agriculture. 

Population of soybean Bradyrhizobium japonicum. The Bradyrhizobium japonicum

population was estimated with soil samples collected at the pre-sowing stage, using the 

most probable number (MPN) technique (Vincent, 1970) and the statistical tables of 

Andrade and Hamakawa (1994). Evaluation 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 nodulation and plant growth. In the laboratory, 

shoots were separated from roots and the latter 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. Nodulation (nodule number and 

dry weight), shoot dry weight and shoot total N (Kjeldahl digestion and determination of 

N concentration using a Tecator automatic N analyzer) were determined (Hungria et al., 

2006). In soybeans, nitrogen is fixed in the 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 the R2 stage, as described by Hungria et al. (2006). 

Analysis was performed with stems including the petioles according to Herridge and 

Peoples (1990). Dry stems (generated by incubation at 65oC until a constant weight) of 

ten plants per replicate were ground (20 mesh), extracted 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, centrifuged 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 by Hungria (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 described 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 plant and provide an estimate of the contribution of nitrogen 

fixation to total N accumulated by the plant. The N-ureide concentration in the stem and 
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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 conducted by analysis of variance 

(ANOVA) on a within- and across-location basis. Mean comparisons across treatments 

at each site (within-location) were conducted by the Least Significant Difference (LSD) 

statistical procedure and across sites (location 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 usually 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 specific to any one treatment. 

Locations Number of Cells1

Santo Antônio de Posse 2.75x103 

Ponta Grossa 3.00x104

Londrina 4.83x105

Uberaba 3.98x103

Sete Lagoas 1.60x103

Santo Antônio de Goiás 1.39x104

Brasília 0.95x103

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 trial sites the coefficients of variance (CV) for the 

nodule number values were acceptable and were 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 nodulation was observed in all field trials and no 

statistical differences were observed for nodule 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 CV127
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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 the capacity for nodulation 

in CV127 soybean is equivalent to that of the isoline control. 

In the analysis of nodule number across treatments for each field trial location, higher 

nodule number at the R2 stage occurred in Santo Antônio de Goiás, Uberaba, Ponta 

Grossa, and Londrina (Table F-47). In the analysis of nodule number for each treatment 

across field trial locations (the treatment effect), highest nodulation was demonstrated for 

CV127 and the isoline control 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) (Table 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 treatments, these were generally between 

CV127 together with the isoline control compared to the conventional soybean varieties, 

and this was most likely associated with 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 Season1

. 

Locations 

Treatment2 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. 
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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, Londrina, Uberaba, and Sete Lagoas), 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 three out of the seven sites, Ponta Grossa, 

Londrina, and Brasília (Table F-41). Lower nodule dry weights at those sites were 

observed in the conventional 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 weights 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 all field trial locations (Table F-47). There 

were no statistically significant differences in nodule dry weights between CV127 

soybean and the isoline control or the conventional soybean varieties 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 conventional 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 Season1. 

Location 

Trt2 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 generally 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 treatment at each field location, no statistical 
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differences in plant dry weight were detected among treatments 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 significant differences in shoot dry weight values between 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 treatments, there were no statistically significant 

differences in shoot dry weights between CV127 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 dry 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 Season1

. 

Location 

Treatment2 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 the analysis across treatments for each field 

trial location and most likely reflects differences in nodulation and soil fertility between 

field trial locations. However, the within-location analyses demonstrated no statistical 

differences among the three treatments including CV127 and the isoline control 

(treatments T1, T2, and T3). Some statistical differences were observed between these 

treatments and the conventional soybean varieties T4 and T5 (Table F-43). Total N 

accumulated in the shoot (column “TNS” of Table F-48) was statistically significantly 
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lower in the conventional soybean varieties (T4 and T5 treatments) compared to the 

CV127 and isoline control treatments (T1-T3), but total N concentration in the soybean 

shoot tissue (column “CNS” of Table F-48) was 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 statistically 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 

Season1

. 

Location 

Treatment2 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 expressed as total N in the shoot per plant (Tables 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 Antônio de Posse, Ponta Grossa, and Brasília, 

and the lowest value was observed at Uberaba (Table F-47). In the analysis of total shoot 

N for each treatment at each field location, at five of the sites 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. Therefore, there was no consistent trend of any 
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differences between these treatments with respect to shoot N content. This was further 

confirmed in the across-location analysis for each treatment, where there were no 

statistical differences in the total N content in the shoot per plant between the treatments 

with CV127 (T1 and T2) and the isoline control (expressed as total N in the shoots per 

plant in Table F-48). Overall, these results demonstrate that there is no significant 

difference in either N concentration or total N in the shoot between 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 Season1, 4. 

Location 

Treatment2 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 field 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-ureides plus nitrate) in the stem and petiole 

tissues, the % N-ureide values measured ranged from 67.36% in the trial at Uberaba to 

76.28% in the trial at Sete Lagoas, with no evidence of statistical 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 statistical differences between these treatments and 

the conventional soybean varieties T4 and T5 at four of the seven field sites (Table F-45). 

However, in three out of the seven sites, Santo Antônio de Posse, Santo Antônio de Goiás 

and Brasília, there was a statistically lower contribution of biological nitrogen fixation in 

the conventional soybean varieties, Monsoy 8001 and CD 217, compared to CV127 and 

the isoline control (Table F-45), and this difference in shoot ureide concentration is to be 

expected between different soybean varieties grown at different locations. 
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When all data were analyzed for treatments across locations (across-location analysis), 

there were no differences in N-ureide concentration between CV127, treated with either 

imazapyr herbicide or with Volt® herbicide, and the isoline control, but lower values 

were present in the conventional soybean varieties Monsoy 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 Season1. 

Location 

Trt2 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 biological nitrogen fixation was 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 content 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 observed 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 analysis 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 varieties Monsoy 8001 and CD 217 at two field trial 

locations (Santo Antônio de Posse and Santo Antônio de Goiás) compared to CV127 and 

the isoline control (Table F-46). Among the CV127 and isoline control 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 with 
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imazapyr (T1) had a statistically significantly 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 analysis for each treatment for total N-ureide shoot content per 

plant, there was no evidence of a statistically significant difference between the CV127 

soybean treatments or the isoline control (Table F-48). These results demonstrate that 

there is no significant difference in either 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. 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 

Season1

. 

Location 

Treatment2 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 treatment of shoot N and N-ureides has shown that 

there was no evidence of statistical differences between CV127 soybean and the isoline 

control in the total N or N-ureide contents (Table F-48). In general, the values of total N 

and N-ureide observed in CV127 soybean and the isoline control were higher than those 

obtained with conventional soybean varieties Monsoy 8001 and CD 217 (Table F-48). 
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Table F-47. Effect of Location on Nitrogen Fixation Parameters in the 2006/2007 

Growing Season. 

Parameter1

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 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 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. 
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Table F-48. Effect of the Different Treatments on Nitrogen Fixation Parameters in 

the 2006/2007 Growing Season. 

Parameter1

Treatment2

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 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 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. 
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b) 2007 growing season 

Population of soybean Bradyrhizobium 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 evaluating nodulation in soils 

cropped with soybean that have previously received rhizobial inoculants. 

Table F-49. Population of Bradyrhizobium japonicum in Soils Cropped with 

Soybean in the Six Trials Performed 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 1.89x103

Vilhena, RO, ER 2.41x103

Uberaba, MG, CTTP 1.58x104

Sete Lagoas, MG, CNPMS 2.60x104

Santo Antônio de Goiás, GO, CNPAF 2.05x104

Brasília, DF, CNPH 1.48x103

*

Means of four replicates with analyses performed in triplicate. 

Soybean nodulation. In the trials conducted during the 2007 short season, 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 high 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 within-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 log10, promoting a significant reduction in the 

coefficients of variation to values between 8.2% and 12.8% in Santo Antônio de Goiás 

and Sete Lagoas, respectively (data not shown). Despite the high coefficients of 

variation, good nodulation was observed in Teresina, Brasília, 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 (Table F-50). The analysis 

across treatments for each field location showed 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).
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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 Season1. 

Location 

Treatment2

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 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. 

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 (Table 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 treatments T1 and T2 (Table F-51). No 

statistically significant differences in nodulation 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 nodulation parameters, with poor nodulation at Uberaba, Sete 

Lagoas, Santo Antônio de Goiás and Teresina (Table F-57). The excellent nodulation 

potential of CV127 soybean and the isoline control soybean, which were superior to the 

conventional soybean varieties Monsoy 8001 and CD217, was also observed when the 

treatments were compared across all field trial locations (across-location analysis) with 

respect to nodulation parameters (Table F-58). These results confirmed that there is no 

evidence of a statistically significant difference in root nodule formation capacity 

between CV127 compared to the isoline control. Even though CV127 soybean and the 

isoline control had slightly higher nodule numbers and nodule dry weights compared to 

the conventional soybean varieties, the only statistically significant difference was 

between CD217 and the other treatments with respect to nodule dry weight (Table F-58). 
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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 Season1. 

Location 

Treatment2

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 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. 

Shoot biomass production. Analysis of shoot dry weight values across treatments for 

each field location showed a high level of variability 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 within-location analysis for each treatment at 

each location showed there were no significant differences in shoot dry weights between 

the CV127 and isoline control soybeans except at Uberaba (Table F-52). Furthermore, 

there were no significant differences in this parameter between CV127 soybean and the 

two conventional soybean varieties at any location except for a statistically significant 

difference observed with CD217 (T5) at Santo Antônio de Goiás (Table F-52). It is 

concluded that shoot dry matter accumulation in CV127 soybean is not different from 

that of the isoline control or other conventional soybean varieties. Comparison of shoot 

dry weight accumulation for each treatment across locations confirmed that CV127 

soybeans were not statistically significantly different for shoot dry matter accumulation 

compared to the isoline control or the conventional soybean varieties (Table F-58). 
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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 Season1. 

Location 

Treatment2

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 treatments 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).
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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 Season1. 

Location 

Treatment2

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 accumulated 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 locations for total N accumulated in shoots per 

plant (Table F-57), as expected, since this value 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 significant differences in total shoot N accumulation among the 

treatments (Table F-58). Overall, these results demonstrate that there is no evidence of 

statistically significant differences in either N concentration or total N in the shoot 

between CV127, treated with either imazapyr herbicide or with Volt® herbicide, and the 

isoline control and conventional soybean varieties (Table F-58). 
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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 Season1, 4. 

Location 

Treatment2

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 field 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-ureides plus nitrate) in the stem and petiole 

tissues, the % N-ureide values measured ranged from 58.0% in the trial at Teresina to 

82.2% in the trial at Sete Lagoas, with statistically significant differences in % N-ureide 

among the six locations (Table F-57). These 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 treatments within a location, there was no 

evidence of a statistically significant difference 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 lower value for the conventional soybean variety 

CD 217 compared to the isoline control treatment at Sete Lagoas. Across locations, there 

were no differences in % N-ureide values between CV127, treated with either imazapyr 

herbicide or with Volt® herbicide, and the isoline control as well as the Monsoy 8001 

conventional soybean variety. There was a statistically 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 nitrogen 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 for each field location showed that total Nureide varied between field sites, as expected, since this parameter is the product of both 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 342 of 375

plant growth as well as N-ureide accumulation (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 differences between any of the five treatments 

at four of the six field trial 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 treated with imazapyr herbicide (T1) compared to the 

conventional soybean variety CD 217. Levels of total N-ureide per plant were equivalent 

between all other treatments at these two field trial locations. 

When total N-ureide content per plant was analyzed for each treatment across all 

locations, there was no evidence of statistically significant difference between the CV127 

soybean treatments and the isoline control as well as the conventional soybean variety 

Monsoy 8001 (Table F-58). However, levels of total N-ureide content per plant were 

statistically significantly lower in conventional soybean variety CD 217 (T5) compared to 

the other four treatments in the study. These results demonstrate that there is no evidence 

of a significant difference in either 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-mentioned difference at Uberaba. These results are 

consistent with results reported for the same studies conducted in the 2006/2007 growing 

season. 

The analysis of shoot N and N-ureides across all 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 soybean and the isoline control were higher than, or 

comparable to, those obtained with conventional soybean varieties Monsoy 8001 and CD 

217 (Table F-58). 
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Table F-55. Percent N-Ureides (%NU) of Total N (N-ureides Plus Nitrate)§

 in the 

Stem and Petiole Tissues of Soybean at the R2 Stage in Field Trials Conducted in 

Brazil During the 2007 Growing Season1. 

Location 

Treatment2

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. 
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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 Season1. 

Location 

Treatment2

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 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 

§ 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. 
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Table F-57. Effect of Location on Nitrogen Fixation Parameters in the 2007 

Growing Season. 

Parameter1 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 trials 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 letter, in the same column, are not statistically 

different at the 5% level. 
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Table F-58. Effect of the Different Treatments on Nitrogen Fixation Parameters in 

the 2007 Growing Season. 

Parameters1

Treatment2

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 letter, in the same column, are not statistically 

different at the 5% level. 

Conclusions

Studies on nitrogen fixation characteristics of CV127 soybean compared to the isoline 

control and two conventional soybean varieties were conducted in both the 2006/2007 

and the 2007 growing seasons in Brazil. The results across both growing seasons were 

consistent and showed that CV127 soybean, irrespective of herbicide treatment, is 

equivalent to the isoline control soybean with respect to biological nitrogen fixation, 

including nodule number and dry weight, shoot 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 isoline control as a group 

to the conventional soybean varieties showed some differences in nitrogen fixation 

parameters, which were attributed to basic genetic differences between soybean varieties 

in biological nitrogen fixation capacities. Therefore, these results support the conclusion 

that CV127 soybean is no different in its interactions with the symbiotic nitrogen-fixing 

Bradyrhizobium japonicum or in nitrogen fixation capacity than conventional soybean 

varieties. 
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Appendix G

Best Management Practice (BMP) Program for the Imidazolinone-tolerant CV127 

Soybean Production System 
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The Imidazolinone-tolerant Soybean Production System 

Introduction

The Production System for imidazolinone-tolerant soybean is an innovative cropping 

system that offers broad-spectrum weed control. It creates a number of new opportunities 

for soybean producers: 

• Superior one-pass, broad-spectrum control of grass and annual 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 adopting 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 developed by BASF Plant Science, L.P and 

EMBRAPA (Empresa Brasileira de Pesquisa Agropecuaria, Brasil) that are tolerant to the 

imidazolinone class of agricultural herbicides. The herbicide-tolerant CV127 soybean 

plants (also referred to as CV127) are derived from a single transformation event and 

were produced by introduction of the 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 imidazolinone-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 commercial product being developed for this 

production system. These imidazolinone-tolerant 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, feed, and processing 

uses. [ 

 ] 

[CBI] 

deleted 
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The introduction of CV127 soybeans in Argentina and Brazil is expected 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 herbicides possess 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 Argentina and Brazil has 

led to a shift in the species of prevalent weeds, 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. Imidazolinone herbicides are effective at low 

application rates (Shaner and Singh, 1998), possess residual activity in the soil and are 

absorbed by roots and translocated throughout the plant, resulting in fewer herbicide 

applications. For example, in Brazil, the recommended application rate and frequency 

that are proposed for imidazolinones used with CV127 soybean is one application per 

season at a rate of approximately 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 the AHAS enzyme that is the target 

for imidazolinones, imidazolinone herbicides have very low mammalian toxicity. Based 

on the known safety profile of imidazolinone herbicides, it is anticipated that the 

Imidazolinone-tolerant Soybean Production 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 system and must address the following key 

issues: 

• Herbicide resistance management using an integrated approach. 

• Control of herbicide-tolerant crop volunteers. 

• Managing out-crossing to other crops and weedy relatives. 

These key sustainability issues form the basis 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: 
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Imidazolinone-tolerant Soybean Production System – GUIDING 

PRINCIPLES 

There are a number of GUIDELINES 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 Group 2 herbicides on any one field, in 

any 4-year period. 

• ALWAYS follow an Integrated Weed Management (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 crops that are uncontrolled by herbicides. 

• FOLLOW the Best Management Practices outlined in the Imidazolinone-tolerant 

Soybean Stewardship Guide. 

Resistance Management in the Imidazolinone-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 (acetolactate synthase)/AHAS (acetohydroxyacid 

synthase) inhibitors. BASF markets 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, high-performance, environmentally sound products for many 

years. The key to the performance of the Imidazolinone-tolerant Soybean Production 

System is effective weed resistance management. BASF is committed to delivering 

sustainable cropping systems that incorporate best practice principles. The 

Imidazolinone-tolerant Soybean Production System provides alternative options for 

growers within a well-managed crop rotation system. 
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Herbicide Resistance Management Using an Integrated Weed Management (IWM) 

Approach 

The objectives of herbicide resistance management 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 number of useful tools available to growers. 

Development of Resistance: 

The Herbicide Resistance Action Committee (HRAC) is an industry initiative which 

fosters co-operation between plant protection manufacturers, government researchers, 

advisors and farmers. The objective of the working group is to facilitate the effective 

management of herbicide resistance. HRAC has identified a number of factors to 

consider when evaluating herbicide resistance risk. The most important factors 

influencing a plant's potential to develop 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 susceptible to an herbicide or that 

are prolific seed producers and have a large amount of genetic variation within the 

species may have a greater potential to become resistant to an herbicide. The initial 

frequency of naturally-occurring resistant biotypes in a weed population influences a 

weed population's potential to develop resistance. Also, the relative fitness or vigor 

of resistant weed biotypes affects resistance development. 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 years, without tank mixing or sequentially 

applying herbicides with other modes-of-action, may increase the risk for resistant 

populations to develop. The greater the selection 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 control, exert less selection pressure. Label 

herbicide rates are a reflection of efficacy trials that indicated the best control and 

crop yield responses. Some herbicides, including Group 2 herbicides, have a greater 

ability to select for resistant weeds than others 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 practices, with herbicide applications may 

increase the potential for resistant populations to develop. Crop rotation practices 
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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 are 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. The longer a herbicide persists, the longer it 

exerts selection pressure on a weed population, particularly if there 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 dormancy 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 germinate 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 exist, 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, and depends on many factors, including the 

following: 

• Selection pressure exerted by the herbicide; 

• Herbicide rotation patterns; 

• Seed germination dynamics; 

• Use of herbicide combinations with different modes of action; 

• Initial frequency of naturally occurring resistant individuals in the weed 

population; and 

• The relative vigor of resistant 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 these indications are an essential input to the development 

of resistance management strategies and practices. 

Identifying Weed Resistance: 

It is important to avoid confusing herbicide 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 considering the possibility of resistance. These 

include application error and poor environmental conditions at the time of herbicide 

application. Shifts in weed populations from susceptible species to species that are less 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 353 of 375

sensitive can also cause weed control problems. 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 application error or adverse weather conditions are ruled 

out. 

• Irregular-shaped patches of a weed develop in the field and are not controlled 

with the particular herbicide or herbicide group. 

• Weed control records for the field indicate repeated use of a particular 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 weed populations under the Imidazolinone-tolerant Soybean 

Production System. The guidelines for managing the development of weed resistance 

presented here are consistent with recommendations put forward in crop protection 

guides and the Weed Resistance Education and Action Program (WREAP). 

Development of herbicide-resistant weeds can be avoided or delayed through good 

management practices. The recommendations listed 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 onset of weed resistance. Three key areas of 

integrated weed management are chemical 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 herbicide classification by product group is 

necessary to develop an effective weed management strategy. 

• Keep records of herbicide application. 

Herbicide application records and field mapping are necessary to effectively rotate 

herbicide groups. 

• Always read and follow herbicide label recommendations. 
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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 resistance. Use the minimum number of 

applications of any one herbicide or herbicide group per season. 

• Utilize non-selective herbicides. 

Non-selective herbicides applied pre-emergence 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 practices 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 integrated 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 spectrum. It also makes it easier to rotate 

between herbicide groups. 

• Plant competitive crops. 

Crops vary in their ability to compete with weeds. Crops such as barley that establish 

a heavy stand are much more competitive with weeds than crops such as lentils or 

flax. In most instances, crop competition will increase with higher seeding rates and 

by planting varieties with a tall stature. A competitive crop can reduce weed 

pressure. 
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• Seed early. 

Early seeding generally leads 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 with herbicide treatments, if practical. 

Direct seeding has been shown to reduce the number of annual weeds over time. 

However, if the crop production system includes spring cultivation, seed as soon as 

possible after working the ground to give the crop a head start over the weeds. 

An effective weed management strategy is comprised of multiple weed control options. 

Herbicide-tolerant cropping systems provide yet another mechanism for effective weed 

control and should be considered as one of the tools for managing the development of 

weed resistance. 

Integrated Weed Management (IWM) in the Imidazolinone-tolerant Soybean 

Production System

Imidazolinone-tolerant soybean is tolerant to the imidazolinones, which are Group 2 

herbicides. Group 2 herbicides work by inhibiting ALS/AHAS, an enzyme that is 

required for the production of the amino acids leucine, isoleucine and valine in plants. 

Group 2 herbicides are known as ‘ALS inhibitors’. 

Continuous use of Group 2 herbicides may result 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 management strategies outlined below, should be 

followed when using the Imidazolinone-tolerant 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. 
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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 four years will slow 

the development of resistance. Consideration should be given to less frequent use to 

delay the onset of resistance development. This includes the use of Group 2 

herbicides both within the Imidazolinone-tolerant Soybean Production System and in 

conventional crops. 

• If a Group 2 herbicide has been applied as a pre-emergence application, DO NOT 

apply further Group 2 herbicides to that crop. Make any further post-emergence 

applications with herbicides from 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, effective weed control 

has been achieved with methods other than 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 within 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 consideration the history of the field well as 

the future use options. 

• Resistance management guidelines for other herbicide Groups, especially Group 1, 

should be taken into account when developing 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, approximately 80% of the soybeans were grown in 

rotation with corn (USDA-ERS, 2006b). The area 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). 
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Figure G-1. Area Planted to Soybeans 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 volunteer 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-tolerant 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 stages of sowing, harvesting, storage and 

transport are important in the effective control of volunteers. 

• Best Management Practice is to make volunteer control part of weed, pest and disease 

management strategies for the farm. 
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Important reasons for control of volunteers

• Volunteer plants act as competitive weeds in subsequent crops or pastures. 

• Volunteers may be important in the build-up and spread of major diseases. 

• Volunteers increase the risk of herbicide tolerance spreading from cross-pollination of 

volunteer plants and conventional 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 harvest 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 harvest and during transport of grain. Secure loads to 

prevent losses during transport. 

• Practice good stubble management after harvest 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 control weeds and volunteers that emerge 

prior to seeding. 

• Control volunteers along field edges and borders. 

• Control ALL volunteers in subsequent crops with the proper selection of in-crop 

herbicides or other agronomic practices such as tillage where appropriate. 

• Keep good field records of herbicides used in previous crops and herbicide-tolerant 

varieties in neighboring fields to develop effective plans for controlling volunteers. 

• Soybeans should not follow an imidazolinone-tolerant soybean crop, as controlling 

volunteers within the same crop species is difficult. Generally speaking, this would 

also not be a good agronomic practice for disease and weed management. 

Management of volunteer Imidazolinone-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 management 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 rotational crop such as winter wheat is 
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planted after soybean harvest, it is possible for soybean seed to be incorporated into the 

soil and to germinate that fall. These volunteer plants will be controlled by freezing 

temperatures in the fall and do not become a competitive consideration. Normally 

volunteer soybean is not a target for weed control measures in winter wheat. 

When corn is the rotational crop, the first option available to control 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 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 ingredient reported as used on corn (29% of 

the total) in 2010 are members of one of these herbicide groups. Therefore, there are 

numerous tools available to control volunteer soybeans in corn. 

 

 

 

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In Roundup Ready® (RR) corn, volunteer conventional or imidazolinone-tolerant 

soybeans will be controlled by any of the glyphosate 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 soybeans. 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 

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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 number of products available for 

volunteer soybean management. Products such as Bronate® AdvancedTM (bromoxynil), 

Clarity® (dicamba) and Widematch® (clopyralid + fluroxypyr) will control all types of 

volunteer soybean (Zollinger, 2011). 

Managing Out-Crossing to Imidazolinone-sensitive 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 their subsequent incorporation in the gene pool 

of the new locality (Slatkin, 1987). Gene flow is a natural biological process and in 

plants it primarily occurs via pollen or seed dispersal (Levin and Kerster, 1974). The 

relative importance of gene flow to a population’s genetic structure depends on the 

distance between donor and recipient populations, population size, how long the process 

has been in effect, and whether the new gene confers any fitness 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 generally 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-mediated 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 receptivity, pollen production, pollen dispersal, 

pollen viability and environmental factors. The likelihood of out-crossing varies greatly 

from species to species and variety to variety. Successful out-crossing may result in the 

offspring displaying characteristics 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 volunteers and managing herbicide-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 habit can be found amongst soybean cultivars: 

determinate, semi-determinate and indeterminate (Bernard and Weiss, 1973). Generally 

determinate types are grown in the southern United States and indeterminate types are 

grown in central and northern regions of North America. In Canada the majority of the 

soybean cultivars have an indeterminate growth habit. 
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The herbicide-tolerant CV127 soybean plants are derived from a single transformation 

event and were produced by introduction of the imidazolinone-tolerance conferring 

acetohydroxyacid synthase large subunit gene csr1-2 with its native promoter from 

Arabidopsis thaliana into the soybean plant genome via biolistics transformation 

technology. 

Potential for out-crossing of Imidazolinone-tolerant Soybean

Soybean is considered to be a self-pollinating species. Based on the reproductive 

morphology of soybean, the anthers mature in the bud and directly pollinate the stigma of 

the same flower (OECD, 2000). Natural out-crossing levels range from less than 0.5% to 

about 1.0% (Carlson and Lersten, 1987). Therefore the potential for gene transfer from 

an Imidazolinone-tolerant Soybean variety to another commercial variety 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, China 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 are 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 artificially with great 

difficulty, and the progeny of such hybrids were sterile (as reviewed 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 potential 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 herbicides 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 corresponding conventional 

varieties of the equivalent maturity group. Therefore, there would be no change to 

agronomic practice due to the occurrence of Imidazolinone-tolerant Soybean. 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 363 of 375

Always read and follow label directions. 

Clearfield, OnDuty, Clarity, and Distinct are registered trademarks of BASF. AAtrex is a 

trademark of Syngenta Group Company. Hornet and Widematch are registered 

trademarks of Dow AgroSciences LLC. Roundup Ready is a registered trademark of 

Monsanto Technology L.L.C. Bronate and Bronate Advanced are trademarks or 

registered trademarks of Bayer. 
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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 System 
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INTRODUCTION 

The purpose of this study was to determine the susceptibility of 1) a test substance (Lot 

number AtAHAS-0107) containing AtAHAS protein encoded by the imidazolinonetolerant acetohydroxyacid synthase large subunit gene isolated from Arabidopsis thaliana 

(AtAHAS, containing R272K and S653N mutations) and 2) AHAS protein present in 

extracts of leaf and grain from CV127 plants, to digestion 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 ubiquitously in plants. The mutation in the 

Arabidopsis acetohydroxyacid synthase large subunit gene responsible for imidazolinone 

herbicide tolerance is a single nucleotide change of guanine to adenine, which results in a 

codon change from AGT to AAT and a single amino acid substitution of serine to 

asparagine at position 653 of the AtAHAS protein. In addition, a second mutation was 

identified after this gene was introduced into the soybean genome, resulting in the 

transformation event designated as CV127. The second mutation, in which arginine at 

position 272 was replaced by lysine (R272K), does 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, inhibition of the AHAS enzyme by imidazolinone 

herbicides leads to a deficiency in branched-chain amino acids and other compounds 

derived from this pathway that are needed 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, concentration, and solubility (Privalle, 2007a) and has been found to 

be substantially equivalent to that produced by CV127 plants (Privalle, 2007b). The 

purpose of Phase A of this study was to confirm that the microbially-produced AtAHAS 

protein is rapidly digested, in a similar manner as typical dietary proteins. The purpose 

of also examining the digestion of the AtAHAS protein as expressed in leaf and grain of 

CV127 (Phase B of this study) was to determine whether the plant matrix has an effect on 

the digestibility and to confirm that the plant-produced AtAHAS protein had a similar 

digestive fate to the E. coli-produced AtAHAS protein as well as the endogenous soybean 

AHAS protein. After incubation for various times in the appropriate simulated digestive 
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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 predicted 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 amino acids which include 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 two aforementioned point mutations. The replacement of serine with asparagine at 

amino acid residue 653 of the AtAHAS protein results in decreased binding of 

imidazolinone herbicide to AtAHAS, and imidazolinone tolerance. The point mutation 

that results in replacement of arginine with lysine at amino acid residue 272 has no 

apparent impact on AHAS functionality. 

AtAHAS protein was produced and purified by Invitrogen, Inc. (Madison, WI) and 

transferred as an ammonium sulfate pellet to the Regulatory Science Laboratory at BASF 

Plant Science, Research Triangle Park, NC where it was received on February 27, 2007. 

The AtAHAS protein was purified from 2,400 g of cell paste after lysis in 20 L buffer (50 

mM sodium phosphate, 300 mM NaCl, 10 mM β-mercaptoethanol, 30 mM imidazole) 

using an Emulsiflex cell disruptor (Avestin, Ontario, Canada). Insoluble material was 

removed by centrifugation. The His-tagged AtAHAS present in the supernatant was 

purified by chromatography using Talon® cobalt resin (Clontech, Inc., Mountain View, 

CA). Once eluted, 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 against 20 mM ammonium 

bicarbonate buffer, pH 7.9, containing 20 μM 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 below 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 chloride, pH 7.4, and used on the day of 

preparation. 

Control substances. An extract of nontransgenic soybean 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 nontransgenic soybean grain was prepared similarly 

except that two grams of material was extracted with nine ml of buffer. Extracts were 
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centrifuged 15 min at approx. 13,000 x g. The protein concentration was determined in 

the supernatants after centrifugation and the supernatants were used directly in the 

digestive reactions. Digestion reactions were 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 procedure; Pierce Biotechnology, Inc., Rockford, 

IL) using bovine serum albumin as the standard. Dilutions of the extract (final volume 

100 μl) 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 allowed to cool at room temperature for 10 

min. The absorbance at 562 nm was measured 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 with 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 centrifuged 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 concentration 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 procedure; Pierce Biotechnology, Inc., Rockford, 

IL) using bovine serum albumin as the standard. Dilutions of the extract were prepared 

such that the expected concentration of protein would be within the standard curve. 

Samples (25 μl) were loaded into a multiwell plate in triplicate, reacted with 200 μl of a 

1:50 (B:A) mixture of BCA reagent, incubated at 37oC for 30 min and allowed to cool at 

room temperature for 10 min. The absorbance 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 mammalian 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 μl of SGF (2X) with 

350 μl 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 μl 1X SGF with 35 μl extract. The reaction mixtures were immediately 

placed in a 37°C water bath and 100 μl 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 35 
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μl of 200 mM NaHCO3, pH 11, and 35 μl 5X Laemmli buffer (40% glycerol, 5% βmercaptoethanol, 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 addition, reactions that 

served as controls for pepsin auto-digestion and test protein stability were prepared 

containing SGF without test protein and SGF with test protein but without pepsin 

(“G-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. 

Simulated mammalian intestinal fluid reactions. Simulated mammalian intestinal 

fluid (SIF) containing phosphate (8.2 mg KH2PO4/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 using azoalbumin as a substrate (SOP BPS 

510.01). A single tube containing enough reaction mix for all time points was prepared 

by mixing 280 μl of SIF with 70 μl of AtAHAS-0107 solution or CV127 leaf or grain 

extract. The reaction mixtures were immediately placed in a 37°C water bath and 50 μl 

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 μl 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 μl 2X Laemmli buffer 

and reheating for 10 min at > 75°C. In addition, reactions that served as controls for 

pancreatin auto-digestion and test protein stability 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-polyacrylamide gel electrophoresis. To monitor the integrity 

of AtAHAS (ca. 64,000 mol. wt.) in sample AtAHAS-0107 after incubation in the 

various digestion mixtures, aliquots of the quenched and heated 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 (Invitrogen). Aliquots 

of the samples that contained ca. 2.5 μg of protein based on determinations made prior to 

digestion were loaded onto the gel. Mark 12TM molecular weight markers (Invitrogen) 

were used to establish the approximate molecular weight of proteins. The protein bands 

were stained with 0.1% Coomassie Brilliant Blue R (Sigma Chemical, Saint Louis, MO). 

Western blot analysis. To monitor the integrity (intactness) 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 protein 
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loaded were 24 μg for leaves and grain, and 8 and 30 μg for CV127 and conventional 

soybean leaves, respectively. For the SIF samples, the amounts loaded onto the gel were 

84 and 67 μg for CV127 and conventional soybean grain, respectively. For western blot 

analysis of the AtAHAS-0107 SIF reactions, 0.2 μg 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 horseradish 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 and was visualized by 

development with the chemiluminescent ECL kit from Amersham (Buckinghamshire, 

UK) according to the manufacturer’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). AtAHAS (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 after 0.5 min of incubation. Some degradation of the AtAHAS is 

observed in the 60 min “I-con” sample indicating protein sensitivity to incubation at 60 

min in simulated intestinal fluid in the absence of pancreatin. Pancreatin is comprised of 

multiple proteases and lipases and this accounts 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 using western blot analysis (Figure H-3 panel A) confirm 

that no immunoreactive degradation products of AtAHAS were obscured by bands 

associated with the pancreatin, 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 leaves 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 acid 
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sequence similarity of the endogenous AHAS and AtAHAS proteins, they are 

immunologically indistinguishable. Both soybean AHAS and AtAHAS proteins are 

rapidly digested in SGF and, although the AHAS protein band is visible at time 0, no 

AHAS protein band is detectable within two minutes of incubation (Figure H-1 panel B). 

An additional immunoreactive band at approximately 50,000 molecular weight is 

observed in leaf extracts (Figure H-1 panel B). It is stable in the absence of pepsin (“Gcon” lanes) but is not visible during the digestion time course. A different 

immunoreactive band at approximately 36,000 molecular weight is observed in the grain 

extracts, and it is also rapidly digested by pepsin within 0.5 min (Figure H-1 panel C). 

These bands may represent proteins that cross-react with the AHAS-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 rapidly digested and is no longer visible within 

0.5 min in SIF (Figure H-2 panel B). Similar to the results observed with the E. coliproduced AtAHAS protein, the leaf AHAS protein is not stable in SIF without pancreatin 

(“I-con”). Also, a lower molecular weight protein of approximately 50,000 molecular 

weight appears to increase in intensity with time of digestion. Furthermore, an 

immunoreactive band of approximately 16,000 molecular weight appears to be associated 

with the pancreatin since the band was not visible in the absence of SIF (“I-con” lanes) or 

at the zero time point where the SIF was inactivated by heating prior 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 degradation product of pancreatin. Full-length AHAS 

protein is just barely detected by western blot analysis in the leaf extract of conventional 

soybean and the lower molecular weight protein at approximately 50,000 molecular 

weight (observed in the leaf extract of CV127 soybean) is apparently below the detection 

limit for this method as the 50,000 molecular weight band was not observed in the 

conventional soybean leaf extract 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 approximately 600 ng AHAS/g dry weight 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 digestion of leaf extracts of CV127, were again 

observed in SIF digestion of the leaf extract of conventional soybean (Figure H-3 panel 

B), supporting the conclusion that these bands are associated with pancreatin 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 expression levels, as reported 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 with digestion of the leaf 

extract of CV127, where a lower molecular weight (approx. 50,000) protein band was 
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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 immunoreactive 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 (Figures 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 bands corresponding to approximately 50,000 and 

36,000 molecular weights, respectively, appeared to increase in intensity 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 (Figure H-3 panel A), it is unlikely 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 from 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 intestinal fluid. Degradation of the AHAS and 

AtAHAS proteins in plant extracts was slightly slower compared to the purified E. coliproduced protein in both SGF and SIF, showing 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 range of proteins are consumed in human diets each day, 

rarely do any of these tens of thousands of proteins elicit an allergenic response (Taylor, 

1992). There are no predictive bioassays available to assess the allergenic potential of 

proteins in humans; however, physicochemical and human exposure profiles of the 

protein provide a basis for assessing potential allergenicity relative to known protein 

allergens. Results of the current study show 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 proteases of the mammalian digestive tract. 

Furthermore, the transgenic AtAHAS protein expressed in CV127 soybean was 

immunologically indistinguishable from the endogenous soybean AHAS protein in the 

current studies, and results suggest that the digestive 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, which has a history of safe consumption in 

food and feed products. 
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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 Laboratory 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 soybean. A protocol change was made to add digestion 

reactions with conventional soybean grain and leaf preparations. This was judged to have 

no adverse impact on the study and allowed better interpretation of background protein 

bands. 

Standard Operating Procedures

SOP BPS 510.01 Simulated Mammalian In vitro Digestibility Method 

SOP BPS 510.02 SDS-Polyacrylamide Gel Electrophoresis 

SOP BPS 510.03 Western Blot Analysis 
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BASF Plant Science Imidazolinone-tolerant Soybean BPS-CV127-9 Page 373 of 375

Figure H-1. Susceptibility of AtAHAS to Digestion in Simulated Mammalian 

Gastric Fluid (SGF). 

Incubation time at 37oC is indicated in minutes. “SGF” is simulated mammalian gastric 

fluid containing pepsin; “G-con” is SGF without pepsin. “SGF only” indicates no 

AtAHAS sample was included. Panel A. Coomassie blue stained 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 digestion reactions carried 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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50

36

22

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0 60 0 0.5 2 5 10 30 60 0 60 min

G-con SGF + Ext. SGF only

+ Ext.

6

97.4

14.4

21.5

31.0

36.5

55.4

66.3

116.3

6

M 0 60 0 0.5 2 5 10 30 60 0 60 min

G-con + AHAS SGF + AHAS SGF only

AtAHAS

Pepsin

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50

36

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6

 

 

 

A 

B C

AHAS 

AHAS 
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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 indicated in minutes. “SIF” is simulated mammalian intestinal 

fluid containing pancreatin; “I-con” is SIF without pancreatin. “SIF only” indicates no 

AtAHAS sample was included. Panel A. Coomassie blue stained 4-20% polyacrylamide 

gel containing digestion reactions carried out with the sample AtAHAS-0107. Panel B. 

Western blot analysis of digestion reactions carried out with leaf extract from CV127 

soybean. Panel C. Western blot analysis of digestion reactions carried out with grain 

extract from CV127 soybean. AtAHAS is ca. 64,000 mol wt. and molecular weight (x 

10-3) markers are indicated. 

148

98

64

50

36

22

16

0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

97.4

14.4

21.5

31.0

36.5

55.4

66 3

116 3

6

M 0 60 0 0.5 2 5 10 30 60 0 60 min

I-con + AHAS SIF + AHAS SIF only

AtAHAS

148

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50

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

A 

B C

AHAS 

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

Figure H-3. Western Blot Analysis of Simulated Mammalian Intestinal Fluid (SIF) 

Reactions for AtAHAS-0107, Conventional Soybean Leaf and Conventional 

Soybean Grain Protein Extracts. 

Incubation time at 37oC is indicated in minutes. “SIF” is simulated mammalian intestinal 

fluid containing pancreatin; “I-con” is SIF without pancreatin. “SIF only” indicates no 

AtAHAS sample was included. Panel A. Western blot analysis using a 4-20% 

polyacrylamide gel containing the same digestion 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. 

148

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50

36

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

AtAHAS

148

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50

36

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

AtAHAS

B C

148

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36

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

AtAHAS

148

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

AtAHAS

A 

148

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

AtAHAS

148

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0 60 0 0.5 2 5 10 30 60 0 60 min

I-con SIF + Ext. SIF only

+ Ext.

6

AHAS AtAHAS

AHAS