USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 1 of 214

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Petition for Determination of Nonregulated Status for

Insect-Resistant DAS-81419-2 Soybean

OECD Unique Identifier: DAS-81419-2

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

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

should not be regulated under 7 CFR 340.

Submitting Company:

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

Submitted by:

_________________________________

Lei Han, Ph.D.

Regulatory Manager

Regulatory Sciences & Government Affairs

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

Phone: (317) 337-3398

Fax: (317) 337-4523

Email: lhan@dow.com

September 26, 2012

Revised November 2, 2012

Revised December 19, 2012
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Release of Information

Dow AgroSciences LLC (DAS) is submitting the information in this petition for deregulation to 

USDA APHIS as part of the regulatory process. By submitting this information, DAS does not 

authorize release of this information to any third party except to the extent the information is 

requested under the Freedom of Information Act (FOIA), 5 U.S.C., Section 522. In the event 

that USDA receives a FOIA request covering all or some of the information in this submission, 

DAS expects that, in advance of the release of the document(s), USDA will provide DAS with a 

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

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

competitive concerns. DAS expects that no information that has been identified as CBI 

(confidential business information) will be provided to any third party. DAS understands that a 

CBI-deleted copy of this information may be made available to the public as part of the public 

comment period. Except in accordance with the foregoing, DAS does not authorize the release, 

publication or other distribution of this information (including website posting) without prior 

notice and consent from DAS.

© 2012 Dow AgroSciences LLC. All Rights Reserved.

This document is property of Dow AgroSciences LLC and is for use by the regulatory authority 

to which it has been submitted, and only in support of actions requested by Dow AgroSciences. 

All data and information herein must not be used, reproduced or disclosed to third parties for any 

other purpose without the written consent of Dow AgroSciences.
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Certification

The undersigned certifies that, to the best knowledge and belief of the undersigned, this petition 

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

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

Lei Han, Ph.D.

Regulatory Manager

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, Indiana 46268

Phone: (317) 337-3398

Fax: (317) 337-4523

Email: lhan@dow.com
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Summary

Dow AgroSciences LLC (herein referred to as “DAS”) is submitting a Petition for Determination 

of Nonregulated Status for DAS-81419-2 soybean. DAS requests a determination from USDA 

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

DAS-81419-2 and any soybean lines derived from crosses between DAS-81419-2 soybean and 

conventional soybean or biotechnology-derived soybean granted non-regulated status by APHIS 

no longer be considered regulated articles under 7 CFR Part 340.

DAS-81419-2 Soybean

DAS-81419-2 soybean is a transgenic soybean that expresses the insecticidal proteins Cry1Ac 

and Cry1F originally from the naturally-occurring soil bacterium, Bacillus thuringiensis. Cry1Ac 

and Cry1F provide protection against several lepidopteran pests of soybean, including soybean 

looper (Chrysodeixis includens, formerly Pseudoplusia includens), velvetbean caterpillar 

(Anticarsia gemmatalis), fall armyworm (Spodoptera frugiperda) and tobacco budworm 

(Heliothis virescens). In addition, DAS-81419-2 soybean expresses the phosphinothricin 

acetyltransferase (PAT) protein from the soil bacterium Streptomyces viridochromogenes. The 

PAT protein provides tolerance to the herbicide glufosinate and was used as a selectable marker 

during the development of DAS-81419-2 soybean. The transgenes for Cry1Ac, Cry1F and PAT 

expression were introduced into soybean via Agrobacterium-mediated transformation to create 

DAS-81419-2 soybean.

DAS-81419-2 soybean provides an effective pest management tool for lepidopteran pests that 

could reduce or replace current insecticide applications in regions where these insect pests cause 

significant yield loss. In the U.S., the majority of soybeans are grown in the north central states 

and the remaining in the southeastern and Mississippi delta region. Insect pressure is generally 

greatest in the southeast, where climate allows the major soybean insect pests to overwinter and 

facilitates multiple generations per year.

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and 

Rodenticide Act (FIFRA), are subject to regulation by the U.S. Environmental Protection 

Agency (EPA). Pesticides produced in planta, known as plant-incorporated protectants (PIPs), 

are also subject to regulation by the EPA under FIFRA. DAS has submitted an application for a 

FIFRA Section 3 seed increase registration with EPA to support future breeding and seed 

increase activities in the U.S. Under a Section 3 seed increase registration, commercial sale of 

DAS-81419-2 soybean in the U.S. is prohibited. At present, DAS does not plan to 

commercialize DAS-81419-2 soybean in the U.S. due to the limited number of acres that 

consistently experience sufficient lepidopteran insect pressure. DAS will apply for a FIFRA 

Section 3 commercial use registration if DAS decides to commercialize DAS-81419-2 soybean 

in the U.S.

Evaluation of Plant Pest Potential of DAS-81419-2 Soybean

The data and information presented in this petition demonstrate that DAS-81419-2 soybean is no 

more likely to become a plant pest than conventional soybean or to have an adverse 

environmental impact. This conclusion is based on the outcome of extensive evaluations 

including i) detailed molecular characterization of the inserted DNA, ii) detailed biochemical 

characterization of the introduced proteins, iii) compositional assessment of forage and grain, iv) 
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 5 of 214

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agronomic and phenotypic characteristics and ecological interactions assessments, and v) 

assessment on the potential impact on non-target organisms and threatened and endangered 

species.

DAS-81419-2 soybean was genetically modified to express two insecticidal proteins, Cry1Ac 

and Cry1F, and phosphinothricin acetyltransferase (PAT) protein. Cry1Ac consists of 1156 

amino acids from three components: the N-terminal toxin core from Cry1Ac1 originating from 

B. thuringiensis subsp. kurstaki, followed by a very small portion of Cry1Ca3 originating from 

B. thuringiensis subsp. aizawai, and the C-terminal sequence from Cry1Ab1 originating from B. 

thuringiensis subsp. Berliner. Cry1F consists of 1148 amino acids from three components: the 

N-terminal toxin core from Cry1Fa2 following by a very small portion of Cry1Ca3 originating 

from B. thuringiensis subsp. aizawai, and the C-terminal sequence from Cry1Ab1 originating 

from B. thuringiensis subsp. Berliner. Cry1Ac and Cry1F expressed in DAS-81419-2 soybean 

are 100% identical in amino acid sequence to Cry1Ac and Cry1F expressed in the deregulated 

events comprising WideStrikecotton; DAS-21023-5 (also described as 3006-210-23 expressing 

Cry1Ac) and DAS-24236-5 (also described as 281-24-236 expressing Cry1F). The PAT 

enzyme, originating from Streptomyces viridochromogenes, acetylates the primary amino group 

of phosphinothricin rendering it inactive. The PAT enzyme expressed in DAS-81419-2 soybean 

is 100% identical in amino acid sequence to PAT expressed in a number of deregulated events, 

including LibertyLinksoybean ACS-GMØØ5-3 (also described as A2704-12) (USDA, 1996), 

HerculexI corn DAS-01507-1 (also described as TC1507) (USDA, 2001), DAS-21023-5 (also 

described as 3006-210-23) and DAS-24236-5 (also described as 281-24-236) comprising 

WideStrikecotton (USDA, 2004), and Herculex RW DAS-59122-7 (USDA, 2005).

The transgenes cry1Ac(synpro), cry1Fv3 and pat expressing Cry1Ac, Cry1F and PAT proteins 

were introduced into DAS-81419-2 soybean using Agrobacterium-mediated transformation. 

Molecular characterization by Southern blot analyses of DAS-81419-2 soybean confirmed that a 

single, intact DNA insert containing the cry1Ac(synpro), cry1Fv3, and pat gene expression 

cassettes was integrated into the soybean genome and the intact DNA insert was stably inherited 

in the five breeding generations tested. Southern blot analyses confirmed the absence of the 

plasmid backbone DNA in DAS-81419-2 soybean. Analyses of the segregating generations 

confirmed that segregation of the DNA insert followed the predicted Mendelian inheritance 

pattern. These data confirmed the stability of DAS-81419-2 soybean during traditional breeding 

procedures.

The expression levels of Cry1Ac, Cry1F and PAT proteins in DAS-81419-2 soybean were 

determined by protein-specific enzyme linked immunosorbent assays (ELISA). Protein 

expression was analyzed in leaf, root, whole-plant and grain tissues collected throughout the 

growing season from DAS-81419-2 soybean plants grown in the major soybean production 

regions in the U.S. Mean protein expression levels across sites ranged from 0.39 ng/mg dry 

weight (dwt) in root to 25.44 ng/mg dwt in V5 leaf tissue for Cry1Ac, 5.23 ng/mg dwt in root to 

56.75 ng/mg dwt in V5 leaf tissue for Cry1F, and 0.63 ng/mg dwt in root to 5.60 ng/mg dwt in 

V10-12 leaf tissue for PAT. Expression of the Cry1Ac and Cry1F proteins peaked during the 

 

 WideStrike and Herculex are registered trademarks of Dow AgroSciences LLC. 

 LibertyLink is a registered trademark of Bayer.
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vegetative growth stage, providing protection against target lepidopteran insect pests and 

potential yield loss as a result of plant damage. The overall low levels of Cry1Ac, Cry1F and 

PAT proteins in DAS-81419-2 soybean indicate a low exposure to humans and animals.

Cry1Ac and Cry1F have a long history of safe use. The proteins originate from the naturally 

occurring soil bacterium B. thuringiensis. The safety of the proteins has been demonstrated in 

sprayable Bt formulations for pest control in agriculture for over half a century (Mendelsohn et 

al., 2003; EPA, 2011; Sanahuja et al., 2011). Both proteins are expressed in WideStrikecotton 

which is authorized for cultivation in the U.S. and Brazil and for food and feed use in Australia, 

Brazil, Canada, European Union, Japan, Korea, Mexico, New Zealand and U.S. 

(www.biotradestatus.com). Bt corn and Bt cotton expressing variations of Cry1Ac or Cry1F 

have been cultivated for commercial use in the U.S. and other countries for more than a decade. 

In 1997, EPA established an exemption from the requirement of a tolerance for the plantincorporated protectant Cry1Ac in all plants (40 CFR §174.510). Later, EPA established an 

exemption from the requirement of a tolerance for the plant-incorporated protectant Cry1F in 

cotton (40 CFR §174.504) and in corn (40 CFR §174.520). The exemptions were based on 

safety assessments of the proteins including digestibility in simulated gastric fluid, lack of 

homology to known allergens and protein toxins, and lack of mammalian toxicity as 

demonstrated by acute oral mouse gavage studies. DAS has filed a petition with EPA for an 

exemption from the requirement of a tolerance for Cry1F as expressed in soybean in 2012.

An extensive set of biochemical evaluations confirmed the identity of the Cry1Ac and Cry1F 

proteins produced in DAS-81419-2 soybean. Moreover DAS-81419-2 soybean-derived Cry1Ac 

and Cry1F were determined to be biochemically equivalent to the corresponding proteins 

purified from a microbial expression host organism Pseudomonas fluorescens. The Cry1Ac and 

Cry1F purified from P. fluorescens had been extensively assessed to establish the safety of the 

proteins. The assessments included acute oral toxicity in mice and protein digestability in 

simulated gastric fluid. The proteins have a very low acute toxicity potential and are rapidly

degraded in simulated gastric fluid. Updated bioinformatic analyses showed that neither Cry1Ac 

nor Cry1F share meaningful amino acid sequence similarities with known allergens. No 

significant homology was identified when either protein sequence was compared with known 

allergen using the search criteria of either a match of eight or more contiguous identical amino 

acids, or greater than 35% identity over 80 amino acid residues. Likewise, neither protein share 

meaningful amino acid sequence similarities with known protein toxins.

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

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

protein. Updated bioinformatic analyses revealed no meaningful homologies to known or 

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

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

5000 mg/kg body weight of PAT protein. The low level expression of the PAT protein presents 

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

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

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

canola, corn, cotton, rice, soybeans, and sugar beets. 
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Nutrient compositional analyses of forage and grain were conducted to compare the composition 

of DAS-81419-2 soybean with that of a non-transgenic control. Compositional analyses were 

used to evaluate any changes in the levels of key nutrients and anti-nutrients in DAS-81419-2 

soybean. A total of 88 analytes were evaluated, nine in forage and the remaining 79 in seed 

including protein, fat, ash, moisture, carbohydrate, mineral, amino acids, fatty acids, vitamins, 

and bioactives. Seventeen analytes were excluded from statistical analysis because more than 

50% of the samples were below the limit of quantitation. Of the remaining 71 analytes, the 

results indicate that there were no statistical differences between DAS-81419-2 and the nontransgenic control for 61 analytes based on overall treatment effects and pair-wise comparisons. 

The statistical differences observed for the remaining ten analytes based on unadjusted P-values 

were non-existent after adjustment for multiplicity using the false discovery rate method. The 

numerical differences in mean values observed for the ten analytes between DAS-81419-2 and 

non-transgenic control were small relative to natural variation. The mean values of 

DAS-81419-2 soybean were within the literature ranges and/or within the range of the reference 

varieties included in the study. Nutrient compositional analyses indicate that DAS-81419-2 

soybean is substantially equivalent to conventional soybean and will not exhibit unexpected or 

unintended effects with respect to plant pest risk.

DAS-81419-2 soybean has been field tested in the major soybean growing regions in the U.S. 

All field tests were conducted under USDA APHIS notifications. Agronomic performance and 

phenotypic assessments were conducted on DAS-81419-2 soybean in multi-site field studies to 

measure characteristics such as emergence, seedling vigor, days to flowering, days to maturity, 

lodging, plant height, plant morphology, final population, shattering, and yield. All field trials 

were also observed for opportunistic disease or insect stressors as well as normal phenotypic 

characteristics. Laboratory studies were carried out to evaluate DAS-81419-2 soybean for seed 

dormancy and germination characteristics and to compare to that of the non-transgenic control. 

Agronomic, phenotypic and ecological evaluations indicate that DAS-81419-2 soybean is 

substantially equivalent to conventional soybean and does not possess a selective advantage that 

would result in increased plant pest risk compared to conventional soybean.

A review of potential environmental impact indicates that DAS-81419-2 soybean is unlikely to 

have adverse effects on non-target organisms including threatened or endangered species under 

normal agricultural practices. This conclusion is based on several lines of evidence including 

Cry protein specificity and long history of safe use, Cry protein content in DAS-81419-2 plant 

tissues and estimated likelihood for exposure, environmental fate, hazard characterization for 

representative non-target organisms including vertebrate and invertebrate species, significant 

margins of exposure, and non-target arthropod field trials comparing DAS-81419-2 soybean with 

non-transgenic soybean.

Because DAS-81419-2 soybean is agronomically and compositionally similar to conventional 

soybean, and Cry1Ac, Cry1F and PAT proteins have a history of safe use, no significant impact 

is expected on current crop production practices, non-target or endangered species, crop rotation, 

volunteer management, or commodity food and feed soybean products. The availability of 

DAS-81419-2 soybean is expected to have a beneficial impact on insect pest management by 

providing another tool to address insect control needs.
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Information collected during field trials and laboratory analyses presented herein demonstrate 

that DAS-81419-2 soybean exhibits no plant pathogenic properties or weediness characteristics. 

DAS-81419-2 soybean is no more likely to become a plant pest than conventional soybean, and 

the Cry1Ac, Cry1F and PAT proteins are unlikely to increase the weediness potential of any 

other cultivated plant or wild species. 

DAS hereby requests a determination from APHIS that insect-resistant DAS-81419-2 soybean 

and all progeny derived from crosses between DAS-81419-2 soybean and conventional soybean 

or biotechnology-derived soybean granted non-regulated status by APHIS no longer be 

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

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

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

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

Table of Contents.......................................................................................................................... 9

List of Figures.............................................................................................................................. 12

List of Tables............................................................................................................................... 15

Acronyms and Scientific Terms................................................................................................. 16

1. Rationale for the Development of DAS-81419-2 .......................................................... 18

1.1. Basis for the Request for Nonregulated Status..................................................... 18

1.2. Rationale for the Development of DAS-81419-2 Soybean and Benefits............. 18

1.3. Submission to Other Regulatory Agencies........................................................... 20

2. The Biology of Soybean .................................................................................................. 21

2.1. Overview of Soybean Biology.............................................................................. 21

2.2. Characterization of the Recipient Soybean Cultivar............................................. 21

3. Development of DAS-81419-2 Soybean......................................................................... 22

3.1. Description of the Transformation System........................................................... 22

3.2. Selection of Comparators for DAS-81419-2 Soybean.......................................... 26

4. Donor Genes and Regulatory Sequences...................................................................... 27

4.1. Identity and Source of Genetic Material in pDAB9582 ....................................... 27

5. Genetic Characterization ............................................................................................... 31

5.1. Overview of Genetic Characterization.................................................................. 31

5.2. Analysis of the Insert and Its Genetic Elements................................................... 41

5.2.1. Number of Insertion Sites............................................................................... 41

5.2.2. Structure of the Insert and Genetic Elements ................................................. 42

5.2.3. Absence of Plasmid Backbone Sequences...................................................... 44

5.2.4. Stability of the Insert Across Generations...................................................... 44

5.2.5. Conclusions..................................................................................................... 44

5.3. Segregation Analysis ............................................................................................ 66

5.3.1. Analysis of a Segregating Generation............................................................. 66

5.3.2. Segregation Analysis of Breeding Generations.............................................. 66

5.4. Summary of the Genetic Characterization............................................................ 67

6. Characterization of the Introduced Proteins ............................................................... 68

6.1. Cry1Ac and Cry1F................................................................................................ 68

6.1.1. Cry1 Protein Mode of Action ......................................................................... 68

6.1.2. Identity of the Cry1Ac Protein........................................................................ 68

6.1.3. Biochemical Characterization of the Cry1Ac Protein .................................... 69

6.1.4. Expression of the Cry1Ac Protein in Plant Tissues........................................ 70

6.1.5. Identity of the Cry1F Protein .......................................................................... 71

6.1.6. Biochemical Characterization of the Cry1F Protein....................................... 72

6.1.7. Expression of the Cry1F Protein in Plant Tissues .......................................... 73

6.1.8. Food and Feed Safety Assessment of Cry1Ac and Cry1F.............................. 74

6.1.9. Summary of Cry1Ac and Cry1F Characterization.......................................... 76

6.2. PAT....................................................................................................................... 78

6.2.1. Mode of Action of the PAT Protein................................................................ 78

6.2.2. Identity of the PAT Protein............................................................................. 78
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6.2.3. Biochemical Characterization of the PAT Protein.......................................... 78

6.2.4. Expression of the PAT Protein in Plant Tissues............................................. 79

6.2.5. Food and Feed Safety Assessment for PAT Protein....................................... 80

6.2.6. Summary of PAT Protein Characterization .................................................... 80

7. Grain and Forage Composition ..................................................................................... 81

7.1. Field Study Design and Data Analysis ................................................................. 81

7.2. Composition Analysis Results.............................................................................. 83

7.2.1. Proximate, Fiber, and Mineral Analysis of Forage......................................... 83

7.2.2. Proximate and Fiber Analysis of Seed............................................................ 86

7.2.3. Mineral Analysis of Seed................................................................................ 89

7.2.4. Amino Acid Analysis of Seed......................................................................... 93

7.2.5. Fatty Acid Analysis of Seed ........................................................................... 99

7.2.6. Vitamin Analysis of Seed ............................................................................. 104

7.2.7. Bioactive Analysis of Seed ........................................................................... 109

7.3. Summary of Composition Analysis.................................................................... 112

8. Agronomic Characteristics .......................................................................................... 113

8.1. Agronomic Study................................................................................................ 113

8.2. Ecological Evaluations........................................................................................ 119

8.3. Germination and Dormancy Evaluations............................................................ 121

8.4. Summary of Agronomic Characteristics............................................................. 122

9. Impact on Environment and Agronomic Practices ................................................... 123

9.1. Potential Impact on Environment ....................................................................... 123

9.1.1. Efficacy against Target Pests........................................................................ 123

9.1.2. Potential Impact on Non-Target Organisms................................................. 127

9.1.3. Potential Impact on Threatened and Endangered Species............................ 129

9.1.4. Environmental Fate of Introduced Proteins and Impact on Soil-dwelling 

Organisms..................................................................................................... 130

9.1.5. Weediness Potential...................................................................................... 130

9.1.6. Gene Flow Assessment................................................................................. 131

9.2. Current Agronomic Practices for Soybean and Potential Impact ....................... 131

9.2.1. Soybean Production ...................................................................................... 131

9.2.2. Weeds in Soybean and Weed Management.................................................. 133

9.2.3. Crop Rotation and Tillage Practices............................................................. 134

9.2.4. Management of Insect Pests.......................................................................... 135

9.2.5. Insect Resistance Management..................................................................... 138

9.3. Summary of Impact on Environment and Agronomic Practices ........................ 140

10. Adverse Consequences of Introduction ...................................................................... 141

11. Appendices..................................................................................................................... 142

Appendix 1. Materials and Methods for Molecular Characterization of DAS-81419-2 

Soybean............................................................................................................... 143

Appendix 2. Materials, Methods and Results for Characterization of Cry1Ac Protein......... 145

Appendix 3. Materials, Methods and Results for Characterization of Cry1F Protein ........... 164

Appendix 4. Materials, Methods and Results for Characterization of PAT Protein.............. 183

Appendix 5. Materials and Methods for Cry1Ac, Cry1F and PAT Protein Expression 

Analysis............................................................................................................... 188

Appendix 6. Analytical Methods for Compositional Analysis.............................................. 191
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Appendix 7. Literature Ranges for Compositional Analysis ................................................. 198

Appendix 8. Non-Target Arthropod Field Study ................................................................... 201

Appendix 9. USDA Notifications for DAS-81419-2 Soybean .............................................. 202

Appendix 10. Stewardship of Insect-Resistant DAS-81419-2 Soybean................................ 203

Appendix 11. References....................................................................................................... 204
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List of Figures

Figure 1. Plasmid map of pDAB9582........................................................................................... 23

Figure 2. Diagram of intended T-DNA insert from plasmid pDAB9582..................................... 23

Figure 3. Event sorting and selection process for DAS-81419-2 soybean. .................................. 24

Figure 4. Breeding diagram of DAS-81419-2 soybean. ............................................................... 25

Figure 5. Location of the probes on pDAB9582 used in Southern blot analysis of 

DAS-81419-2 soybean...................................................................................................... 35

Figure 6. Plasmid map of pDAB9582 with selected restriction enzyme sites used for 

Southern analysis. ............................................................................................................. 40

Figure 7. Actual T-DNA insert in DAS-81419-2 soybean with restriction enzymes used 

for DNA digestion and sizes of the expected hybridization bands................................... 41

Figure 8. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); AtUbi10 promoter probe................................................................................... 45

Figure 9. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); cry1F-5′ probe................................................................................................... 46

Figure 10. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); cry1F-3′A probe................................................................................................ 47

Figure 11. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); cry1F-3′ B probe. .............................................................................................. 48

Figure 12. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); AtuORF23-A 3′UTR probe............................................................................... 49

Figure 13. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); CsVMV promoter probe. .................................................................................. 50

Figure 14. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); cry1Ac-5′ probe. ................................................................................................ 51

Figure 15. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); cry1Ac-3′A probe. ............................................................................................. 52

Figure 16. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); cry1Ac-3′B probe. ............................................................................................. 53

Figure 17. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); AtuORF23-B 3′UTR probe............................................................................... 54

Figure 18. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); pat probe............................................................................................................ 55

Figure 19. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and 

SphI (B); AtuORF1 3′ UTR probe.................................................................................... 56

Figure 20. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A) 

AtUbi10 promoter probe, (B) cry1F-5′ probe................................................................... 57

Figure 21. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A) 

cry1F-3′A probe, (B) cry1F-3′B probe............................................................................. 58

Figure 22. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A) 

AtuORF23-A 3′UTR probe, (B) CsVMV promoter probe............................................... 59

Figure 23. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A)

cry1Ac-5′ probe, (B) AtuORF23-B 3′UTR probe............................................................. 60

Figure 24. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; ............... 61

Figure 25. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; ............... 62
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Figure 26. Southern blot analysis of DAS-81419-2 soybean digested with (A) SphI; Ori 

probe, (B) NcoI; SpecR probe........................................................................................... 63

Figure 27. Southern blot analysis of DAS-81419-2 soybean digested with (A) NcoI; 

Backbone 1 probe, (B) SphI; Backbone 2 probe............................................................... 64

Figure 28. Southern blot analysis of DAS-81419-2 soybean digested with NcoI; 

Backbone 3 and 4 probes. ................................................................................................. 65

Figure 29. Amino acid sequence of Cry1Ac protein. ................................................................... 69

Figure 30. Amino acid sequence of Cry1F protein....................................................................... 72

Figure 31. Mode of action of the PAT protein.............................................................................. 78

Figure 32. Amino acid sequence of the PAT protein.................................................................... 78

Figure 33. Proximates, fiber and minerals in control, DAS-81419-2 and reference variety 

soybean forage. ................................................................................................................. 85

Figure 34. Proximates and fiber in control, DAS-81419-2, and reference variety soybean 

seed. .................................................................................................................................. 88

Figure 35. Minerals in control, DAS-81419-2, and reference variety soybean seed.................... 91

Figure 36. Amino acids in control, DAS-81419-2, and reference variety soybean seed (% 

total amino acid). .............................................................................................................. 96

Figure 37. Fatty acids in control, DAS-81419-2, and reference variety soybean seed (% 

total fatty acid). ............................................................................................................... 103

Figure 38. Vitamins in control, DAS-81419-2, and reference variety soybean seed (mg/kg 

dry weight)...................................................................................................................... 107

Figure 39. Bioactives in control, DAS-81419-2, and reference variety soybean seed. .............. 111

Figure 40. Lateral flow strip assay for the Cry1Ac protein expression in Event 

DAS-81419-2 and Maverick grain extracts. ................................................................... 149

Figure 41. Western blot analysis of microbe- and soybean-derived Cry1Ac proteins. .............. 150

Figure 42. SDS-PAGE analysis of immunoaffinity-purified soybean-derived Cry1Ac............. 151

Figure 43. SDS-PAGE analysis of microbe- and soybean-derived Cry1Ac protein stained 

with GelCode Blue total protein and glycoprotein stains. .............................................. 152

Figure 44. Theoretical cleavage of the Cry1Ac protein with trypsin generated in silico

using Protein Analysis Worksheet (PAWS) from Proteometrics LLC........................... 153

Figure 45. Theoretical cleavage of the Cry1Ac protein with chymotrypsin generated in 

silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC. ................ 154

Figure 46. Tryptic digest sequence coverage map for Cry1Ac (DAS-81419-2) by 

MALDI-TOF MS and MALDI TOF/TOF. Cys residues were alkylated with 

iodoacetamide. Sequence coverage was 67.9% with PMF data and 67.9% by 

tandem MS...................................................................................................................... 156

Figure 47. Chymotryptic digest sequence coverage map for Cry1Ac (DAS-81419-2) by 

MALDI-TOF MS and MALDI TOF/TOF. Cys residues were alkylated with 

iodoacetamide. Sequence coverage was 53.5% with PMF data and 49.0% by 

tandem MS...................................................................................................................... 158

Figure 48. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1Ac 

(DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF. Overall sequence 

coverage was 86.1% with PMF data and 83.2% by tandem MS. ................................... 160

Figure 49. Lateral flow strip assay for the Cry1F protein expression in Event 

DAS-81419-2 and Maverick grain extracts. ................................................................... 168

Figure 50. Western blot analysis of microbe- and soybean-derived Cry1F proteins.................. 169
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Figure 51. SDS-PAGE analysis of immunoaffinity-purified soybean-derived Cry1F. .............. 170

Figure 52. SDS-PAGE analysis of microbe- and soybean-derived Cry1F protein stained 

with GelCode Blue total protein and glycoprotein stains. .............................................. 171

Figure 53. Theoretical cleavage of the Cry1F protein with trypsin generated in silico 

using Protein Analysis Worksheet (PAWS) from Proteometrics LLC........................... 172

Figure 54. Theoretical cleavage of the Cry1F protein with chymotrypsin generated in 

silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC................. 173

Figure 55. Tryptic digest sequence coverage map for Cry1F (DAS-81419-2) by MALDITOF MS and MALDI-TOF/TOF. Cys residues were alkylated with 

iodoacetamide. Sequence coverage was 65.1% with PMF data and 65.1% by 

tandem MS...................................................................................................................... 175

Figure 56. Chymotryptic digest sequence coverage map for Cry1F (DAS-81419-2) by 

MALDI-TOF MS and MALDI TOF/TOF. Cys residues were alkylated with 

iodoacetamide. Sequence coverage was 53.8% with PMF data and 52.4% by 

tandem MS...................................................................................................................... 177

Figure 57. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1F 

(DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF. Overall sequence 

coverage was 81.7% with PMF data and 81.7% by tandem MS. ................................... 179

Figure 58. Lateral flow strip assay for PAT protein expression in Event DAS-81419-2 

and Maverick leaf extracts.............................................................................................. 185

Figure 59. SDS-PAGE and Western blot analysis of microbe-derived PAT, transgenic 

soybean Event DAS-81419-2, and non-transgenic Maverick extracts. .......................... 186
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List of Tables

Table 1. Genetic elements from plasmid pDAB9582................................................................... 29

Table 2. Genetic elements in DAS-81419-2 soybean................................................................... 32

Table 3. List of probes and their positions in plasmid pDAB9582. ............................................. 34

Table 4. Predicted and observed sizes of hybridizing fragments in Southern blot analyses 

of DAS-81419-2 soybean. ................................................................................................ 36

Table 5. Results of F2 individual plants tested with LFS and event-specific PCR. ..................... 66

Table 6. Results of the individual plants from DAS-81419-2 soybean tested with eventspecific PCR within segregating BC1F2 generations....................................................... 67

Table 7. Expression of Cry1Ac in DAS-81419-2 soybean........................................................... 71

Table 8. Expression of Cry1F in DAS-81419-2 soybean. ............................................................ 74

Table 9. Expression of PAT in DAS-81419-2 soybean................................................................ 79

Table 10. Composition analytes.................................................................................................... 82

Table 11. Summary of the proximate, fiber, and mineral analysis of soybean forage from 

all sites and literature range. ............................................................................................. 84

Table 12. Summary of the proximate and fiber analysis of soybean seed from all sites and

literature range. ................................................................................................................. 87

Table 13. Summary of the mineral analysis of soybean seed from all sites and literature 

range.................................................................................................................................. 90

Table 14. Summary of the amino acid analysis of soybean seed from all sites and 

literature range. ................................................................................................................. 94

Table 15. Summary of the fatty acid analysis of soybean seed from all sites and literature 

range................................................................................................................................ 100

Table 16. Summary of the vitamin analysis of soybean seed from all sites and literature 

range................................................................................................................................ 105

Table 17. Summary of the bioactive analysis of soybean seed from all sites and literature 

range................................................................................................................................ 110

Table 18. Agronomic characteristics. ......................................................................................... 114

Table 19. Summary of agronomic characteristics for the combined site analysis...................... 117

Table 20. Insect and disease present by site. .............................................................................. 119

Table 21. Disease and insect incidence....................................................................................... 120

Table 22. Germination percentage for DAS-81419-2 and non-transgenic control soybean....... 122

Table 23. Efficacy of DAS-81419-2 soybean compared to ‘Maverick’..................................... 126

Table 24. Estimated margins of exposure (MOE) to non-target organisms based on highend exposure estimates (HEEE) for Cry1Ac and Cry1F proteins produced in 

DAS-81419-2.................................................................................................................. 128

Table 25. Soybean-derived PAT extraction buffer composition. ............................................... 185
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Acronyms and Scientific Terms

ADF Acid detergent fiber

ANOVA Analysis of variance

APHIS Animal and Plant Health Inspection Service, USDA

AtUbi10 Ubiquitin promoter from Arabidopsis thaliana

AtuORF1 Open reading frame 1 of Agrobacterium tumefaciens pTi15955

AtuORF23 Open reading frame 23 of Agrobacterium tumefaciens pTi15955

bp Base pair

Bt Bacillus thuringiensis

CFR US Code of Federal Regulations

Cry1 Insecticidal proteins from Bacillus thuringiensis

Cry1Ac Cry1Ac insecticidal protein derived from Bacillus thuringiensis 

subsp. kurstaki

cry1Ac(synpro) Coding sequence for Cry1Ac protein

Cry1F Cry1F insecticidal protein derived from Bacillus thuringiensis 

subsp. aizawai

cry1Fv3 Coding sequence for Cry1F protein

CsVMV Promoter from cassava vein mosaic virus

DAS Dow AgroSciences LLC

DAS-21023-5 OECD identifier for the cotton event expressing the Cry1Ac and 

PAT proteins. DAS-21023-5 is also described as 3006-210-23

DAS-24236-5 OECD identifier for the cotton event expressing the Cry1F and PAT 

proteins. DAS-24236-5 is also described as 281-24-236

DAS-81419-2 OECD identifier for the soybean event expressing the Cry1Ac, 

Cry1F, and PAT proteins

DNA Deoxyribonucleic acid

dwt Dry weight of tissue

EEC Estimated environmental concentration

ELISA Enzyme-linked immunosorbent assay

EPA US Environmental Protection Agency

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

FDA US Food and Drug Administration

FDR False Discovery Rate

FESTF FIFRA Endangered Species Task Force

FFDCA Federal Food, Drug, and Cosmetic Act

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

HAFT Highest average field trial

HEEE High-end exposure estimate

ILSI International Life Sciences Institute

IPM Integrated Pest Management

IRM Insect Resistance Management

Kb Kilobase pair

kDa Kilodalton

L Liter

LOD Limit of Detection

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

spectrometry

Maverick Publicly available soybean line used in transformation to produce 

DAS-81419-2 soybean

MOE Margin of exposure

NDF Neutral detergent fiber

NOEC No observed effect concentration

NTO Non-target organism

OECD Organisation for Economic Co-operation and Development

ORF Open reading frame

Ori Origin of replication

pat Gene from Streptomyces viridochromogenes which encodes the 

PAT protein

PAT Phosphinothricin N-acetyltransferase

PBN US FDA Pre-market Biotechnology Notice

PCR Polymerase chain reaction

pDAB9582 DNA vector carrying the cry1Ac(synpro), cry1Fv3 and pat 

expression cassettes

PIP Plant-incorporated protectant

PPT Phosphinothricin

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

sequences

RCB Randomized complete block

SCN Soybean cyst nematode

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE Standard error

SGF Simulated gastric fluid

Std. Dev. Standard deviation

STMR Supervised trials mean residue

subsp subspecies

T-DNA Transfer DNA

USDA United States Department of Agriculture

USDA ARS United States Department of Agriculture Agricultural Research 

Service

USDA ERS United States Department of Agriculture Economic Research 

Service

USDA NASS United States Department of Agriculture National Agricultural 

Statistics Service

UTR Untranslated region

w/v Weight per volume
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1. Rationale for the Development of DAS-81419-2

1.1. Basis for the Request for Nonregulated Status

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

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

introduction or dissemination of plant pests into or within the United States. The APHIS regulations

at 7 CFR 340.6 provide that an applicant may petition APHIS to evaluate submitted data on the 

genetically engineered crop to determine that a regulated article does not present a plant pest risk and 

therefore should no longer be regulated.

Dow AgroSciences LLC (herein referred to as ”DAS”) is submitting data for genetically engineered 

insect-resistant DAS-81419-2 soybean and requests a determination from APHIS that DAS-81419-2

soybean and crosses of DAS-81419-2 with nonregulated soybean lines no longer be considered 

regulated articles under 7 CFR 340.

1.2. Rationale for the Development of DAS-81419-2 Soybean and Benefits

Large scale soybean production began in the U.S. in the 1920s. Since then, the acres planted to 

soybeans have expanded rapidly due to ideal planting conditions, increased crop rotation, rising 

yield, and lower production cost (USDA ERS, 2012). With the exception of 2007, over the last 

15 years soybeans have become the second most planted field crop in the U.S. after corn with 

more than 70 million acres (USDA NASS, 2012). Soybeans are the dominant oilseed in the U.S. 

accounting for about 90 percent of U.S. oilseed production. In 2011 soybeans were planted on 75 

million acres producing 3.056 billion bushels of soybeans with a farm value of more than $35.7 

billion. About 1.615 billion bushels were crushed to yield soybean meal and soybean oil for 

domestic use and 1.275 billion bushels were processed for export worldwide. These sales 

accounted for 37% of the world’s soybean trade valued at $21.5 billion (USDA ERS, 2012; 

USDA NASS, 2012).

In the U.S. the majority of soybeans are grown in the north central states with the remaining 

primarily grown in the southeastern and Mississippi delta region. Insect injury has not 

traditionally resulted in substantial economic losses in most North American growing regions. 

However, in recent years, losses have been severe in some southern regions of the U.S. (Catchot, 

2011). Higher market prices for soybeans have lowered economic thresholds for mitigating 

damage caused by soybean pests, resulting in expansion of the area and number of insecticide 

treatments applied to soybeans (USDA ERS, 2011). Recent increases in pest insect populations 

in soybean have been observed; increases are attributed by some to global warming or insect 

adaptation to the soybean plant (Way, 1994). Lower economic thresholds coupled with higher 

insect populations favor more aggressive pest management strategies (Pedigo, 1996).

Insect pressure on soybeans in the southern U.S. states is derived primarily from four 

lepidopteran insect pests. Of these four, velvetbean caterpillar and soybean looper are the most 

damaging defoliating insects in the south (Way, 1994; Gianessi, 2009). On average about onethird of the soybean acres in Georgia, Louisiana, and North Carolina have been sprayed regularly 

with insecticides since 1991. Insecticides are used on approximately 50% of the soybean 

acreage in Georgia for lepidopteran pests with velvetbean caterpillar being the most targeted pest 

(Gianessi et al., 2002). Approximately 40% of the soybean acreage in Louisiana is treated with 
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insecticides for lepidopteran pests, with soybean looper being the main target (Gianessi et al., 

2002). 

Chemical insecticides can have limited efficacy in controlling lepidopteran infestations in 

soybean. Narrow application windows, the emergence of insecticide resistance, and public 

pressure for reduced pesticide use limit the desirability of this approach to pest management 

(Thomas and Boethel, 1994). Soybean looper has developed extensive insecticide resistance 

(Thomas and Boethel, 1994); resistance to pyrethroids is widespread across the southern U.S. 

(Felland et al., 1990; Leonard et al., 1990). Insecticides remain effective against velvetbean 

caterpillar. However, infestations can quickly reach damaging levels and cause economic loss if 

insecticides are not applied promptly.

Bt crops expressing one or more insecticidal proteins of Bacillus thuringiensis are effective in 

targeting specific insect pests. Increasing adoption rates of Bt corn and cotton since 1996 have 

coincided with increased yields (USDA ERS, 2006). Pesticide use has also decreased since the 

introduction of these insect-resistant Bt crops with one study showing an 8% reduction in 

insecticide use for adopters of Bt corn as compared to non-adopters (USDA ERS, 2006). 

Importantly, Bt crops have also been credited with increasing yields, even when economic 

thresholds have not been reached in either Bt- or non-Bt-fields due to regional insect population 

reductions (Hutchison et al., 2010).

DAS developed a transgenic soybean that is resistant to target lepidopteran pests. The unique 

identifier for the transgenic soybean, in accordance with the Organization for Economic Cooperation and Development’s (OECD) “Guidance for the Designation of a Unique Identifier for 

Transgenic Plants” (OECD, 2004), is DAS-81419-2.

DAS-81419-2 soybean expresses two insecticidal proteins, Cry1Ac and Cry1F, originally from 

the naturally-occurring soil bacterium, Bacillus thuringiensis. Cry1Ac and Cry1F provide plant 

protection against several target lepidopteran pests. In addition, DAS-81419-2 soybean 

expresses the phosphinothricin acetyltransferase (PAT) protein from the soil bacterium 

Streptomyces viridochromogenes. The PAT protein provides tolerance to the herbicide 

glufosinate and was used as a selectable marker during the development of DAS-81419-2 

soybean. The transgenes for Cry1Ac, Cry1F and PAT expression were introduced into soybean 

via Agrobacterium-mediated transformation to create DAS-81419-2 soybean. Cry1Ac and 

Cry1F are expressed in the leaf tissues of DAS-81419-2 soybean plants throughout the growing 

season, providing excellent control of target lepidopteran pests of soybeans. DAS-81419-2 

soybean offers several potential benefits, in particular:

 Efficient and environmentally sound alternative to chemical insecticides. DAS-81419-2 

soybean offers effective control of multiple lepidopteran pests of soybean. The 

availability of DAS-81419-2 soybean could reduce the need for insect scouting, preserve 

beneficial insect populations, and provide increased convenience and greater performance 

consistency. 

 Greater durability and improved insect resistance management. DAS-81419-2 soybean 

expresses two insecticidal proteins, Cry1Ac and Cry1F, whose modes of action differ 

with respect to receptor binding. For example, Cry1Ac and Cry1F have been shown to 

bind to different receptors in the midgut of the target soybean insect pest tobacco 
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budworm (H. virescens) (Jurat-Fuentes and Adang, 2001). Cry1Ac binds to at least three 

sets of receptors while Cry1F binds to at least two, only one of which also binds Cry1Ac. 

The major receptor for Cry1Ac is not recognized by Cry1F (Jurat-Fuentes and Adang, 

2006). Such incomplete shared binding is expected to lead to incomplete cross-resistance 

when resistance is mediated by receptor changes. Bt gene pyramiding offered by 

DAS-81419-2 soybean offers greater durability than Bt crops carrying a single Bt trait 

and provides protection against the development of insect resistance. 

Due to narrow commercial applicability in the U.S., DAS submitted an application for a FIFRA 

Section 3 seed increase registration with EPA to support future breeding and seed increase 

activities in the U.S. Under a Section 3 seed increase registration, commercial sale of 

DAS-81419-2 soybean in the U.S. is prohibited. If DAS chooses to market DAS-81419-2 

soybean for commercial purposes in the U.S. in the future, DAS would seek a FIFRA Section 3 

commercial use registration from EPA. DAS-81419-2 soybean is targeted for commercialization 

in Brazil and Argentina where insect pressure is heavy, resulting in significant yield loss. DAS 

expects to make submissions beginning 2012 to the regulatory authorities of trade partners for 

import clearance and cultivation approval. Deregulation of DAS-81419-2 soybean by APHIS is a 

prerequisite in achieving regulatory approvals in many of these countries. 

1.3. Submission to Other Regulatory Agencies

DAS-81419-2 soybean falls within the scope of the FDA policy statement, published in the 

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

varieties, including those developed via biotechnology. DAS intends to submit a pre-market 

biotechnology notification (PBN) to FDA in 2012.

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and 

Rodenticide Act (FIFRA) [7 U.S.C. §136(u)], are subject to regulation by the Environmental 

Protection Agency (EPA). Pesticides produced in planta, referred to as plant-incorporated 

protectants (PIPs), are also subject to regulation by the EPA under FIFRA. DAS submitted an 

application for a FIFRA Section 3 seed increase registration of DAS-81419-2 soybean on July 

27

th, 2012. Concurrent with the Section 3 application, DAS also petitioned the EPA for a 

permanent exemption from the requirement of a tolerance for Cry1F and the genetic material 

required for its production in soybean, under Federal Food, Drug and Cosmetic Act (FFDCA) 

Section 408. An exemption from the requirement of tolerance for Cry1Ac in all crops has 

already been established by the EPA and is published in the Code of Federal Regulations (40 

CFR §174.510). Under seed increase registration, commercial sale of DAS-81419-2 soybean in 

the U.S. is prohibited by law. If DAS chooses to cultivate DAS-81419-2 soybean for 

commercial purposes in the U.S. in the future, DAS will apply for a FIFRA Section 3 

commercial use registration with EPA and will develop a specific IRM program in connection 

with the commercial use registration.

DAS intends to submit dossiers beginning in 2012 to the regulatory authorities of trade partners 

for import clearance and production approval which may include Canada, Japan, Korea, Taiwan, 

European Union, Australia/New Zealand, South Africa, Brazil, Argentina and Mexico.
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2. The Biology of Soybean

2.1. Overview of Soybean Biology

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

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

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

plant

 agronomic practices

 centers of origin 

 reproductive biology

 cultivated Glycine max as a volunteer weed

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

other organisms

 summary of the ecology of Glycine max

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

nomenclature (Pedersen, 2004; Gaska, 2006):

Vegetative Stages Reproductive Stages

VE Emergence R1 Beginning bloom

VC Unrolled unifoliate leaves R2 Full bloom

V1 First-trifoliate R3 Beginning pod

V2 Second-trifoliate R4 Full pod

V3 Third-trifoliate R5 Beginning seed

V(n) n

th

-trifoliate R6 Full seed

R7 Beginning maturity

R8 Full maturity

2.2. Characterization of the Recipient Soybean Cultivar

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

DAS-81419-2 soybean.

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

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

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

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

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

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

phytophthora rot but is susceptible to brown stem rot and sudden death syndrome. 
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3. Development of DAS-81419-2 Soybean

3.1. Description of the Transformation System

Transgenic soybean (Glycine max) was generated through Agrobacterium-mediated 

transformation of soybean cotyledonary node explants. The disarmed Agrobacterium

tumefaciens strain EHA101 (Hood et al., 1986), carrying the binary vector, pDAB9582, with the 

selectable marker (pat) and the genes of interest (cry1Ac(synpro) and cry1Fv3) within the TDNA region, was used to initiate transformation.

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

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

cotyledonary nodes were isolated and infected with Agrobacterium EHA101 carrying 

pDAB9582. After infection with Agrobacterium, cotyledonary nodes were cultured on the 

cocultivation medium for 5 days before transferring to shoot initiation medium. All media, 

including shoot initiation, shoot elongation, and rooting media were supplemented with 

cefotaxime, timentin and vancomycin to inhibit the growth of Agrobacterium. Glufosinate 

selection (3-8 mg/L) was also employed in those media to inhibit the growth of non-transformed 

shoots. Selected shoots were transferred to rooting medium for root development and then 

transferred to soil mix for acclimatization of plantlets.

Terminal leaflets of regenerated plantlets were painted with glufosinate (0.05% - 2% w/v) to 

screen for putative transformants. Those plantlets exhibiting tolerance were transferred to the 

greenhouse, allowed to acclimate and then leaf painted again with glufosinate (0.05% - 2% w/v) 

to reconfirm herbicide tolerance. The glufosinate tolerant plants were sampled and analyzed at 

the molecular level to confirm the presence of the selectable marker gene and/or the genes of 

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

spectinomycin sequence as well as the presence of the cry1Ac(synpro) and cry1Fv3 genes. 

Invaderassay (Kwiatkowski et al., 1999) was carried out for copy number detection for pat, 

cry1Ac(synpro), and cry1Fv3 genes. Selected T0 plants were allowed to self-fertilize in the 

greenhouse to give rise to T1 seed. For T1 plants, Invader assay and Southern blot analyses were 

performed to determine copy number, integration number, and PTU integrity.

Figure 1 shows a plasmid map of pDAB9582.

Figure 2 shows a diagram of the intended T-DNA insert in plasmid pDAB9582.

Figure 3 outlines the development of DAS-81419-2 soybean.

Figure 4 shows a breeding diagram for DAS-81419-2 soybean including identification of the 

generations used in various safety assessment studies. 
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Figure 1. Plasmid map of pDAB9582.

Figure 2. Diagram of intended T-DNA insert from plasmid pDAB9582.

pDAB9582

18143 bp

trfA

pat

specR

cry1Ac(synpro)

cry1F v3

AtUbi10 promoter

CsVMV promoter

CsVMV promoter

ori

AtuORF23 3' UTR

AtuORF1 3' UTR

AtuORF23 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

Intended T-DNA Insert from pDAB9582 (12783 b p)

pat

cry1Ac(synpro)

cry1F v3

AtUbi10 promoter

CsVMV promoter

CsVMV promoter

AtuORF1 3' UTR

AtuORF23 3' UTR

AtuORF23 3' UTR

T-DNA Border B T-DNA Border A

T-DNA Border A

T-DNA Border A

1 12783 
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Figure 3. Event sorting and selection process for DAS-81419-2 soybean.

Assembled Agrobacterium binary vector pDAB9582 carrying the cry1Ac(synpro), 

cry1Fv3 and pat gene cassettes.

Introduced the cry1Ac(synpro), cry1Fv3 and pat gene cassettes into the soybean line 

Maverick via Agrobacterium tumefaciens-mediated transformation. 

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

Selected T0 soybean plants for regeneration. 

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

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

pollination to produce T1.

Continued evaluation of T1 and subsequent generations resulting from self 

pollination. Evaluated transgenic plants via molecular characterization, protein 

expression, agronomic performance and resistance to lepidopteran pests.

Selected homozygous plants and carried out crossing and backcrossing for further 

characterization.

Selected DAS-81419-2 as the lead commercial candidate. 
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Analysis Petition Section(s) DAS-81419-2 

Soybean Generations

Control

Genetic Characterization 5.2 T1, T2, T3, T4, F2 Non-transgenic soybean Maverick

Segregation Analysis 5.3 F2, BC1F2 None

Protein Characterization 6.1.3, 6.1.6, 6.2.3 T4, T5 Non-transgenic soybean Maverick

Protein Expression 6.1.4, 6.1.7, 6.2.4 T4 Non-transgenic soybean Maverick

Agronomics 8 T4 Non-transgenic soybean Maverick

Germination/Dormancy 8.3 T4 Non-transgenic soybean Maverick

Composition 7 T4 Non-transgenic soybean Maverick

NTO 9.1.2 T4 Non-transgenic soybean Maverick

Efficacy 9.1.1 T4 Non-transgenic soybean Maverick

Figure 4. Breeding diagram of DAS-81419-2 soybean.

T0

Self

T1

Self

T2

Self

T3

F1

Self

Self

T4

F2

Self

T5

Self

T6

Backcross to recurrent parent

Cross to elite line 

BC1F1

Self

BC1F2
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3.2. Selection of Comparators for DAS-81419-2 Soybean

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

of DAS-81419-2 soybean, a proper selection of comparator plants is important. 

The control plants should have a genetic background similar to that of DAS-81419-2 soybean but 

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

control. Maverick is the recipient variety that was transformed to generate DAS-81419-2 

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

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

4.1. Identity and Source of Genetic Material in pDAB9582

DAS-81419-2 soybean was generated by Agrobacterium-mediated transformation using plasmid 

pDAB9582 (Figure 1). The T-DNA insert in the plasmid contains two synthetic genes from 

Bacillus thuringiensis, cry1Ac(synpro) and cry1Fv3, as well as a pat gene from Streptomyces 

viridochromogenes (Figure 2). A summary of the genetic elements in pDAB9582 is provided in

Table 1.

Three gene expression cassettes are present in the T-DNA region of plasmid pDAB9582 for 

insertion into soybean. The cry1Fv3 expression cassette is designed to express a synthetic 

version of the Cry1F protein. The cry1Fv3 gene is comprised of three parts; at the 5′ end, a toxin 

core that was optimized from the native cry1Fa2 gene originally isolated from Bacillus 

thuringiensis subsp. aizawai strain PS811; in the middle, a very small portion of cry1Ca3 which 

was originally isolated from B. thuringiensis subsp. aizawai strain PS811; and at the 3′ end, a tail 

that was optimized from the native cry1Ab1 tail originally isolated from B. thuringiensis subsp. 

Berliner 1715. The cry1Fv3 gene encodes the Cry1F protein that is comprised of 1148 amino 

acids and has a molecular weight of ~130.2 kDa. The amino acid sequence of the Cry1F protein 

is identical to that expressed in cotton event DAS-24236-5 (also described as 281-24-236, 

expressing Cry1F) which was deregulated by USDA APHIS in 2004 (USDA, 2004).

Expression of cry1Fv3 is controlled by the AtUbi10 promoter from Arabidopsis thaliana and the 

AtuORF23 3′UTR sequence from Agrobacterium tumefaciens plasmid pTi15955. The AtUbi10 

promoter is known to drive constitutive expression of the genes it controls (Norris et al., 1993). 

The function of AtuORF23 (GenBank Accession: CAA25184) in pTi15955 (GenBank 

Accession: X00493) was not identified (Barker et al., 1983).

The cry1Ac(synpro) expression cassette is designed to express a synthetic version of the Cry1Ac

protein. The cry1Ac(synpro) gene is comprised of three parts; at the 5′ end, a toxin core that was 

optimized from the native cry1Ac1 gene originally isolated from B. thuringiensis subsp. kurstaki

strain HD73; in the middle, a very small portion of cry1Ca3 which was originally isolated from 

B. thuringiensis subsp. aizawai strain PS811; and at the 3′ end, a tail that was optimized from the 

native cry1Ab1 tail originally isolated from B. thuringiensis subsp. Berliner 1715. The

cry1Ac(synpro) gene encodes the Cry1Ac protein that is comprised of 1156 amino acids and has 

a molecular weight of ~130.7 kDa. The cry1Ac(synpro) gene sequence and the corresponding 

Cry1Ac amino acid sequence are identical to that expressed in cotton event DAS-21023-5 (also 

described as 3006-210-23, expressing Cry1Ac) which was deregulated by USDA APHIS in 2004 

(USDA, 2004).

Expression of cry1Ac(synpro) is controlled by the CsVMV promoter from Cassava Vein Mosaic 

virus and the AtuORF23 3′UTR sequence from Agrobacterium tumefaciens plasmid pTi15955. 

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

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

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

genes it controls (Verdaguer et al., 1996). The function of AtuORF23 (GenBank Accession: 

CAA25184) in pTi15955 (GenBank Accession: X00493) was not identified (Barker et al., 1983).
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The pat expression cassette is designed to express the phosphinothricin N-acetyltransferase 

(PAT) protein. The pat gene originates from the common soil bacterium Streptomyces 

viridochromogenes (Wohlleben et al., 1988). Expression of PAT protein in soybean plants 

confers tolerance to glufosinate and was used as a selectable marker during DAS-81419-2

soybean development. The pat gene encodes a protein of 183 amino acids that has a molecular 

weight of approximately 20.6 kDa. The pat gene has been widely used both as a selectable 

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

2004, 2005).

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

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

CsVMV promoter driving pat expression is the same as that driving expression of 

cry1Ac(synpro). The function of AtuORF1 (GenBank Accession: CAA25163) in pTi15955 

(GenBank Accession: X00493) was not identified (Barker et al., 1983), but its translated amino 

acid sequence has a significant similarity with an indole-3-lactate synthase (GenBank Accession: 

AAK90967) from Agrobacterium tumefaciens str. C58.
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Table 1. Genetic elements from plasmid pDAB9582.

Feature Name Feature 

Start

Feature 

Stop

Feature 

Length

Description

T-DNA Border B 1 24 24

T-DNA Border B sequence required for transfer of 

DNA from Agrobacterium tumefaciens into plant 

cells (Barker et al., 1983)

Intervening 

sequence

25 295 271 Non-specific DNA sequences necessary for cloning

AtUbi10 

promoter 296 1617 1322

AtUbi10 promoter along with the 5' untranslated 

region and intron from Arabidopsis thaliana 

polyubiquitin 10 (UBQ10) gene (Norris et al., 1993)

Intervening 

sequence

1618 1625 8 Non-specific DNA sequences necessary for cloning

cry1F v3 1626 5072 3447

cry1F v3 (synthetic version of the cry1F gene from 

Bacillus thuringiensis subsp. aizawai strain PS811) 

(Cardineau et al., 2001; Gao et al., 2006)

Intervening 

sequence

5073 5174 102 Non-specific DNA sequences necessary for cloning

AtuORF23 

3' UTR 5175 5631 457

AtuORF23 3' UTR (3' untranslated region (UTR) 

comprising the transcriptional terminator and 

polyadenylation site of open reading frame 23 

(ORF23) of Agrobacterium tumefaciens pTi15955) 

(Barker et al., 1983)

Intervening 

sequence

5632 5694 63 Non-specific DNA sequences necessary for cloning

CsVMV 

promoter 5695 6211 517

CsVMV promoter along with the 5' untranslated 

region derived from Cassava Vein Mosaic virus

(Verdaguer et al., 1996) 

Intervening 

sequence

6212 6220 9 Non-specific DNA sequences necessary for cloning

cry1Ac(synpro) 6221 9691 3471

cry1Ac (synpro) (synthetic version of the cry1Ac gene 

from Bacillus thuringiensis subsp. kurstaki strain 

HD73 ) (Adang et al., 1985; Gilroy and Wilcox, 

1992; Cardineau et al., 2001)

Intervening 

sequence

9692 9724 33 Non-specific DNA sequences necessary for cloning

AtuORF23 

3' UTR 9725 10181 457

AtuORF23 3' UTR (3' untranslated region (UTR) 

comprising the transcriptional terminator and 

polyadenylation site of open reading frame 23 

(ORF23) of Agrobacterium tumefaciens pTi15955)

(Barker et al., 1983)

Intervening 

sequence

10182 10295 114 Non-specific DNA sequences necessary for cloning

CsVMV 

promoter 10296 10812 517

CsVMV promoter along with the 5' untranslated 

region derived from Cassava Vein Mosaic virus 

(Verdaguer et al., 1996)

Intervening 

sequence

10813 10819 7 Non-specific DNA sequences necessary for cloning
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Feature Name Feature 

Start

Feature 

Stop

Feature 

Length

Description

pat 10820 11371 552

pat (synthetic version of the phosphinothricin acetyl 

transferase gene from Streptomyces 

viridochromogenes) (Wohlleben et al., 1988)

Intervening 

sequence

11372 11473 102 Non-specific DNA sequences necessary for cloning

AtuORF1 

3' UTR 11474 12177 704

AtuORF1 3' UTR (3' untranslated region (UTR) 

comprising the transcriptional terminator and 

polyadenylation site of open reading frame 1 (ORF1) 

of Agrobacterium tumefaciens pTi15955) (Barker et 

al., 1983)

Intervening 

sequence

12178 12405 228 Non-specific DNA sequences necessary for cloning

T-DNA Border 

A

12406 12429 24

T-DNA Border A sequence required for transfer of TDNA insert from Agrobacterium tumefaciens into 

plant cells (Barker et al., 1983)

Intervening 

sequence

12430 12448 19 Non-specific DNA sequences necessary for cloning

T-DNA Border 

A

12449 12472 24

T-DNA Border A sequence required for transfer of TDNA insert from Agrobacterium tumefaciens into 

plant cells (Barker et al., 1983)

Intervening 

sequence

12473 12759 287 Non-specific DNA sequences necessary for cloning

T-DNA Border 

A

12760 12783 24

T-DNA Border A sequence required for transfer of TDNA insert from Agrobacterium tumefaciens into 

plant cells (Barker et al., 1983)

Intervening 

sequence

12784 13162 379

Plasmid backbone sequences from gram-negative 

bacteria broad host-range RK2 plasmid (Stalker et al., 

1981)

ori 13163 14182 1020

ori (Replication origin sequences from gram-negative 

bacteria broad host-range RK2 plasmid) (Stalker et 

al., 1981)

Intervening 

sequence

14183 14727 545

Plasmid backbone sequences from gram-negative 

bacteria broad host-range RK2 plasmid (Stalker et al., 

1981)

trfA 14728 15876 1149

trfA (Plasmid replication sequences for Trf A protein 

from gram-negative bacteria broad host-range RK2 

plasmid) (Stalker et al., 1981)

Intervening 

sequence

15877 17080 1204

Plasmid backbone sequences from gram-negative 

bacteria broad host-range RK2 plasmid (Stalker et al., 

1981)

SpecR 17081 17869 789 SpecR (spectinomycin resistance gene from 

Escherichia coli Tn7 transposon ) (Fling et al., 1985)

Intervening 

sequence

17870 18143 274 Plasmid backbone sequences for cloning
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5. Genetic Characterization

5.1. Overview of Genetic Characterization

Molecular characterization of DAS-81419-2 soybean was conducted by Southern blot analyses

and DNA sequencing. The results demonstrated that the transgene insert in DAS-81419-2 

soybean occurred as a single integration of the T-DNA insert from plasmid pDAB9582, 

including a single, intact copy of each of the plant transcription units (PTUs) for the cry1Fv3, 

cry1Ac(synpro), and pat genes. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro)

gene was identified at the 5′ end of the T-DNA insert in a complementary orientation. The 

sequence of the insert in DAS-81419-2 soybean was confirmed and the genetic elements 

identified in DAS-81419-2 soybean are provided in Table 2. The full insert was stably integrated 

and inherited across breeding generations, and no plasmid backbone sequences are present in 

DAS-81419-2 soybean.

Detailed Southern blot analyses were conducted using probes specific to the genes, promoters, 

terminators, and other regulatory elements contained in the transformation plasmid pDAB9582. 

Locations of each probe on plasmid pDAB9582 are described in Table 3 and Figure 5. The 

expected and observed fragment sizes with specific digest and probe combinations, based on the 

known restriction enzyme sites of plasmid pDAB9582 and the actual T-DNA insert in 

DAS-81419-2 soybean are shown in Figure 6 and Figure 7, respectively. The Southern blot 

analyses described here made use of two types of restriction fragments: a) internal fragments 

generated by known restriction enzyme recognition sites contained within the T-DNA insert of 

pDAB9582, and b) border fragments generated by one known restriction enzyme recognition site 

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

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

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

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

transgene insertions and to specifically identify the event.

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

DAS-81419-2 soybean plants from five distinct breeding generations. Genomic DNA from 

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

pDAB9582 added to genomic DNA from the non-transgenic variety Maverick served as the 

positive control for Southern blot analyses. Materials and methods used for Southern analyses 

are further described in Appendix 1.

The expected restriction fragments of the inserted DNA are shown in Table 4 and Figure 7. 

Southern blot analysis results are shown in Figure 8 to Figure 28. Southern blot analyses showed 

that DAS-81419-2 soybean contains a single intact copy of each of the PTUs for the cry1Fv3, 

cry1Ac(synpro), and pat genes from pDAB9582 (Section 5.2.1 and Section 5.2.2). In addition, a 

minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified at the 5′ end of the T-DNA 

insert. No plasmid backbone sequences were detected in DAS-81419-2 soybean (Section 5.2.3). 

The hybridization patterns across five generations of DAS-81419-2 soybean (T1, T2, T3, T4, and 

F2) were identical, indicating that the insert is stably integrated in the soybean genome (Section 

5.2.4). The inheritance of DAS-81419-2 soybean insert in segregating generations was 

investigated using event-specific PCR and detection of the PAT protein expression. All results 

confirmed the predicted inheritance of the transgene in a single locus (Section 5.3).
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Table 2. Genetic elements in DAS-81419-2 soybean.

Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

5' Flanking 

border 1 1297 1297 Soybean genomic DNA flanking the 5' end of the insert in DAS-81419-2 soybean

Re-arranged 

sequence

1298 1321 24 Re-arranged DNA fragment at the 5' end of the insert

cry1Ac(synpro)

partial fragment 1322 1419 98

Complementary cry1Ac(synpro) partial fragment at 

the 5' end of the insert that is 99% identical to 1990 -

2087 bp of the full-length cry1Ac(synpro) gene

Re-arranged 

sequence

1420 1432 13 Re-arranged DNA fragment at the 5' end of the insert

Partial T-DNA 

Border B 1433 1433 1

Last nucleotide from T-DNA Border B which is 

required for transfer of DNA from Agrobacterium 

tumefaciens into plant cells (Barker et al., 1983)

Intervening 

sequence

1434 1704 271 Non-specific DNA sequences necessary for cloning

AtUbi10 

promoter 1705 3026 1322

AtUbi10 promoter along with the 5' untranslated 

region and intron from Arabidopsis thaliana 

polyubiquitin 10 (UBQ10) gene (Norris et al., 1993)

Intervening 

sequence

3027 3034 8 Non-specific DNA sequences necessary for cloning

cry1F v3 3035 6481 3447

cry1F v3 (synthetic version of the cry1F gene from 

Bacillus thuringiensis subsp. aizawai strain PS811) 

(Cardineau et al., 2001; Gao et al., 2006)

Intervening 

sequence

6482 6583 102 Non-specific DNA sequences necessary for cloning

AtuORF23 

3' UTR 6584 7040 457

AtuORF23 3' UTR (3' untranslated region (UTR) 

comprising the transcriptional terminator and 

polyadenylation site of open reading frame 23 

(ORF23) of Agrobacterium tumefaciens pTi15955 

(Barker et al., 1983)

Intervening 

sequence

7041 7103 63 Non-specific DNA sequences necessary for cloning

CsVMV 

promoter 7104 7620 517

CsVMV promoter along with the 5' untranslated 

region derived from Cassava Vein Mosaic virus

(Verdaguer et al., 1996) 

Intervening 

sequence

7621 7629 9 Non-specific DNA sequences necessary for cloning

cry1Ac(synpro) 7630 11100 3471

cry1Ac (synpro) (synthetic version of the cry1Ac gene 

from Bacillus thuringiensis subsp. kurstaki strain 

HD73 ) (Adang et al., 1985; Gilroy and Wilcox, 

1992; Cardineau et al., 2001)

Intervening 

sequence

11101 11133 33 Non-specific DNA sequences necessary for cloning
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Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

AtuORF23 

3' UTR 11134 11590 457

AtuORF23 3' UTR (3' untranslated region (UTR) 

comprising the transcriptional terminator and 

polyadenylation site of open reading frame 23 

(ORF23) of Agrobacterium tumefaciens pTi15955)

(Barker et al., 1983)

Intervening 

sequence

11591 11704 114 Non-specific DNA sequences necessary for cloning

CsVMV 

promoter 11705 12221 517

CsVMV promoter along with the 5' untranslated 

region derived from Cassava Vein Mosaic virus 

(Verdaguer et al., 1996)

Intervening 

sequence

12222 12228 7 Non-specific DNA sequences necessary for cloning

pat 12229 12780 552

pat (synthetic version of phosphinothricin acetyl 

transferase gene from Streptomyces 

viridochromogenes) (Wohlleben et al., 1988)

Intervening 

sequence

12781 12882 102 Non-specific DNA sequences necessary for cloning

AtuORF1 

3' UTR 12883 13586 704

AtuORF1 3' UTR (3' untranslated region (UTR) 

comprising the transcriptional terminator and 

polyadenylation site of open reading frame 1 (ORF1) 

of Agrobacterium tumefaciens pTi15955) (Barker et 

al., 1983)

Intervening 

sequence

13587 13784 198 Non-specific DNA sequences necessary for cloning

Re-arranged 

sequence

13785 13793 9 Re-arranged DNA fragment at the 3' end of the insert

3' Flanking 

border 13794 15172 1379

Soybean genomic DNA flanking the 3' end of the 

insert in DAS-81419-2 soybean
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Table 3. List of probes and their positions in plasmid pDAB9582.

Probe

Name

Location in

pDAB9582

Size 

(bp)

AtUbi10 Promoter

cry1F-5′

29-1620

1631-3376

1592

1746

cry1F-3′A 3354-3874 521

cry1F-3′B 3851-5081 1231

AtuORF23-A 3′ UTR 5080-5630 551

CsVMV Promoter 5664-6250 587

cry1Ac-5′ 6221-7940 1720

cry1Ac-3′A 7918-8352 435

cry1Ac-3′B 8329-9778 1450

AtuORF23-B 3′ UTR

CsVMV Promoter

9714-10180

10292-10817

467

526

pat 10820-11371 552

AtuORF1 3′ UTR 11342-12412 1071

Backbone 3 12389-13178 790

Ori 12804-14182 1379

Backbone 2 14149-15876 1728

Backbone 1 15846-17079 1234

SpecR 17081-18116 1036

Backbone 4 17426-18143 718
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Figure 5. Location of the probes on pDAB9582 used in Southern blot analysis of 

DAS-81419-2 soybean.

Note: * Since the CsVMV promoter element in the cry1Ac(synpro) and pat PTUs are 100% 

identical based on the nucleotide sequences, CsVMV Promoter probe generated from 

cry1Ac(synpro) PTU was used to hybridize CsVMV promoter in pat PTU.

pDAB9582

18143 bp

trfA

pat

SpecR

cry1Ac(synpro)

cry1Fv3

SpecR Probe

Ori Probe

cry1Ac-5' Probe

cry1Ac-3'A Probe

cry1Ac-3'B Probe

cry1F-3'A Probe

cry1F-3'B Probe

Backbone 3 Probe

Backbone 4 Probe

CsVMV Promoter Probe

AtuORF23-A 3' UTR Probe

AtuORF23-B 3' UTR Probe

AtuORF1 3' UTR Probe

AtUbi10 promoter

CsVMV promoter

CsVMV Promoter

Ori Rep

AtuORF23 3' UTR

AtuORF1 3' UTR

AtuORF23 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

Backbone 1 Probe

Backbone 2 Probe

cry1F-5' Probe

pat Probe

AtUbi10 Promoter Probe

CsVMV Promoter Probe*

SphI (12234)

NcoI (1625)

Nco I (16169)

Nco I (16697) Not I (241)

Not I (5634)

NotI (10184)

NotI (10224)

NotI (10264)

NotI (13687)
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Table 4. Predicted and observed sizes of hybridizing fragments in Southern blot analyses of 

DAS-81419-2 soybean.

Probe Restriction 

Enzyme

Sample Lane

Expected 

Fragment 

Sizes (bp)1

Observed 

Fragment 

Sizes (bp)2

Figure 

Number

AtUbi10 

promoter

NcoI

pDAB9582 2 3071 ~3100

Figure

8A

Maverick 3 none none

DAS-81419-2 4-18 >1737 ~7500

SphI

pDAB9582 2 18143 ~18100

Figure

8B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

cry1F-5′

NcoI

pDAB9582 2 14544 ~14500

Figure

9A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure

9B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

cry1F-3′A

NcoI

pDAB9582 2 14544 ~14500

Figure

10A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure

10B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

cry1F-3′B

NcoI

pDAB9582 2 14544 ~14500

Figure

11A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure

11B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

AtuORF23

-A 3′ UTR

NcoI

pDAB9582 2 14544 ~14500

Figure

12A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure

12B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100
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Probe Restriction 

Enzyme

Sample Lane

Expected 

Fragment 

Sizes (bp)1

Observed 

Fragment 

Sizes (bp)2

Figure 

Number

CsVMV 

Promoter

NcoI

pDAB9582 2 14544 ~14500

Figure

13A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure 

13B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

cry1Ac-5′

NcoI

pDAB9582 2 14544 ~14500

Figure

14A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure

14B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

cry1Ac-3′A

NcoI

pDAB9582 2 14544 ~14500

Figure

15A

Maverick 3 none none

DAS-81419-2 4-18 >10759, >1737 ~14500, ~7500

SphI

pDAB9582 2 18143 ~18100

Figure

15B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

cry1Ac-3′B

NcoI

pDAB9582 2 14544 ~14500

Figure

16A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure 

16B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

AtuORF23

-B 3′ UTR

NcoI

pDAB9582 2 14544 ~14500

Figure

17A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure 

17B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100
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Probe Restriction 

Enzyme

Sample Lane

Expected 

Fragment 

Sizes (bp)1

Observed 

Fragment 

Sizes (bp)2

Figure 

Number

pat

NcoI

pDAB9582 2 14544 ~14500

Figure 

18A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100

Figure 

18B

Maverick 3 none none

DAS-81419-2 4-18 >12346 ~18100

AtuORF1 

3′ UTR

NcoI

pDAB9582 2 14544 ~14500

Figure 

19A

Maverick 3 none none

DAS-81419-2 4-18 >10759 ~14500

SphI

pDAB9582 2 18143 ~18100 

Figure

19B

Maverick 3 none none

DAS-81419-2 4-18 >12346, >150 ~18100, ~4100

AtUbi10 

promoter

NotI/SphI 

(Release

cry1Fv3 

PTU)

pDAB9582 2 5393, 4697 ~5400, ~4700

Figure

20A

Maverick 3 none none

DAS-81419-2 4-18 5393, >353 ~5400, ~6500

cry1F-5′

pDAB9582 2 5393 ~5400

Figure 

20B

Maverick 3 none none

DAS-81419-2 4-18 5393 ~5400

cry1F-3′A

pDAB9582 2 5393, 4550 ~5400, ~4600

Figure 

21A

Maverick 3 none none

DAS-81419-2 4-18 5393, 4550 ~5400, ~4600

cry1F-3′B

pDAB9582 2 5393, 4550 ~5400, ~4600

Figure 

21B

Maverick 3 none none

DAS-81419-2 4-18 5393, 4550 ~5400, ~4600

AtuORF23

-A 3′ UTR

pDAB9582 2 5393, 4550 ~5400, ~4600

Figure

22A

Maverick 3 none none

DAS-81419-2 4-18 5393, 4550 ~5400, ~4600
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Probe Restriction 

Enzyme

Sample Lane

Expected 

Fragment 

Sizes (bp)1

Observed 

Fragment 

Sizes (bp)2

Figure 

Number

CsVMV 

Promoter

NotI/SphI 

(Release

cry1Ac(syn

pro) PTU)

pDAB9582 2 4550, 1970 ~4600, ~2000

Figure

22B

Maverick 3 none none

DAS-81419-2 4-18 4550, 1970 ~4600, ~2000

cry1Ac-5′

pDAB9582 2 4550 ~4600

Figure

23A

Maverick 3 none none

DAS-81419-2 4-18 4550 ~4600

cry1Ac-3′A

pDAB9582 2 5393, 4550 ~5400, ~4600

Figure 

24A

Maverick 3 none none

DAS-81419-2 4-18 >353, 4550, 

5393

~6500, ~4600, 

~5400

cry1Ac-3′B

pDAB9582 2 5393, 4550 ~5400, ~4600

Figure

24B

Maverick 3 none none

DAS-81419-2 4-18 5393, 4550 ~5400, ~4600

AtuORF23

-B 3′ UTR

pDAB9582 2 5393, 4550 ~5400, ~4600

Figure 

23B

Maverick 3 none none

DAS-81419-2 4-18 5393, 4550 ~5400, ~4600

pat

NotI/SphI 

(Release 

pat PTU)

pDAB9582 2 1970 ~2000

Figure

25A

Maverick 3 none none

DAS-81419-2 4-18 1970 ~2000

AtuORF1 

3′ UTR

pDAB9582 2 1453, 1970, ~1500, ~2000

Figure

25B

Maverick 3 none none

DAS-81419-2 4-18 >150, 1970 ~4000, ~2000

Ori SphI

pDAB9582 2 18143 ~18100

Figure

26A

Maverick 3 none none

DAS-81419-2 4-18 none none

SpecR NcoI

pDAB9582 2 3071 ~3100

Figure

26B

Maverick 3 none none

DAS-81419-2 4-18 none none

Backbone 

1

NcoI

pDAB9582 2

14544, 3071, 

528

~14500, ~3100, 

~500 Figure

Maverick 3 none none 27A

DAS-81419-2 4-18 none none
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Probe Restriction 

Enzyme

Sample Lane

Expected 

Fragment 

Sizes (bp)1

Observed 

Fragment 

Sizes (bp)2

Figure 

Number

Backbone 

2

SphI

pDAB9582 2 18143 ~18100

Figure

27B

Maverick 3 none none

DAS-81419-2 4-18 none none

Backbone 

3 + 4

NcoI

pDAB9582 2 14544, 3071 ~14500, ~3100

Figure

28 Maverick 3 none none

DAS-81419-2 4-18 none none

1Expected fragment sizes are based on the plasmid map of pDAB9582 (Figure 6) and the T-DNA insert in 

DAS-81419-2 soybean (Figure 7).

2Observed fragment sizes are considered approximately from these analyses and are based on the indicated sizes of 

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

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

weight.

Figure 6. Plasmid map of pDAB9582 with selected restriction enzyme sites used for 

Southern analysis.

pDAB9582

18143 bp

trfA

pat

specR

cry1Ac( synpro )

cry1Fv3

AtUbi10 promoter

CsVMV promoter

CsVMV promoter

Ori Rep

AtuORF23 3' UTR

AtuORF1 3' UTR

AtuORF23 3' UTR

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

Sph I (12234)

Nco I (1625)

Nco I (16169)

Nco I (16697)

NotI (241)

NotI (5634)

Not I (10184)

Not I (10224)

Not I (10264)

Not I (13687)
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Figure 7. Actual T-DNA insert in DAS-81419-2 soybean with restriction enzymes used for 

DNA digestion and sizes of the expected hybridization bands.

5.2. Analysis of the Insert and Its Genetic Elements

5.2.1. Number of Insertion Sites

The restriction enzymes NcoI and SphI were chosen to determine the number of insertions in 

DAS-81419-2 soybean (Figure 7). Probes derived from the DNA sequences of AtUbi10

Promoter, cry1Fv3 (split into three overlapping regions cry1F-5′, cry1F-3′A, and cry1F-3′B), 

AtuORF23 3′UTR (probes A and B contain small pieces of different intervening sequences at the 

5′ and 3′ ends of the AtuORF23 3′UTR sequence), CsVMV promoter, cry1Ac(synpro) (split into

three overlapping regions cry1Ac-5′, cry1Ac-3′A, and cry1Ac-3′B), pat, and AtuORF1 3′UTR 

were then hybridized to the digested genomic DNA to determine the number of insertion sites in 

DAS-81419-2 soybean.

When digested with restriction enzyme NcoI and independently hybridized with the cry1F-5′, 

cry1F-3′A, cry1F-3′B, AtuORF23-A 3′ UTR, CsVMV Promoter, cry1Ac-5′, cry1Ac-3′A, 

cry1Ac-3′B, AtuORF23-B 3′ UTR, pat, and AtuORF1 3′ UTR probes, a single hybridization 

band of ~14500 bp was detected in DAS-81419-2 soybean samples, consistent with the predicted 

size of >10759 bp for the NcoI fragment as shown in Table 4 and Figure 9A - Figure 19A. When 

NcoI digested samples were hybridized with AtUbi10 Promoter, a single band of ~7500 bp was 

detected in DAS-81419-2 soybean samples, consistent with the expected result of >1737 bp 

(Figure 8A).

When digested with restriction enzyme SphI and probed with the AtUbi10 Promoter, cry1F-5′, 

cry1F-3′A, cry1F-3′B, AtuORF23-A 3′ UTR, CsVMV Promoter, cry1Ac-5′, cry1Ac-3′A, 

pat

cry1Ac(synpro)

cry1Fv3

Partial cry1Ac(synpro) fragment

AtUbi10 Promoter

CsVMV Promoter

CsVMV Promoter

AtuORF23 3' UTR

AtuORF1 3' UTR

AtuORF23 3' UTR

Partial T-DNA Border B

NcoI (1737)

Sph I (12346)

Not I (353)

NotI (5746)

Not I (10296)

Not I (10336)

Not I (10376)

1

5 ’ 3 ’

DAS-81419-2 T-DNA Insert (12496 bp) 12496 

NcoI >1737 bp >10759 bp

NotI/SphI >353 bp 4550 bp 5393 bp 1970 bp

SphI >12346 bp >150bp

>150bp
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cry1Ac-3′B, AtuORF23-B 3′ UTR, and pat probes individually, a single band of ~18100 bp was 

detected in DAS-81419-2 soybean samples, consistent with the expected result of >12346 bp 

(Table 4, Figure 8B - Figure 18B). When SphI digested samples were hybridized with AtuORF1 

3′ UTR probe, two fragments of ~18100 bp and ~4100 bp were detected in DAS-81419-2 

soybean samples, consistent with the expected result of >12346 bp and >150 bp (Figure 19B).

Specific hybridization bands were detected in all of the positive samples at the expected sizes, 

while no specific hybridization bands were detected in any of the negative control samples. All 

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

elements from pDAB9582 in DAS-81419-2 soybean genome.

5.2.2. Structure of the Insert and Genetic Elements

According to the plasmid and T-DNA restriction maps shown in Figure 6 and Figure 7, the plant 

transcription units (PTUs) for cry1Fv3, cry1Ac(synpro), and pat could be released by restriction 

digestion with NotI/SphI. These digestions were performed to characterize the structure of the 

insert in DAS-81419-2 soybean.

When digested with NotI/SphI and hybridized with AtUbi10 Promoter probe, two bands of 

~5400 bp and ~6500 bp were detected in all DAS-81419-2 soybean and the positive control 

samples, consistent with the expected sizes of 5393 bp and > 353 bp for the cry1Fv3 PTU and 

the border fragment that the AtUbi10 promoter probe extended into, respectively (Figure 20A). 

When digested with NotI/SphI and hybridized with cry1F-5′ probe, a single band of ~5400 bp

was detected in DAS-81419-2 soybean and the positive control samples, consistent with the 

expected size of 5393 bp for the cry1Fv3 PTU (Figure 20B).

Since both cry1Fv3 and cry1Ac(synpro) genes contain synthetic cry1Ab sequence with 89% 

identity at nucleotide level, it is expected that the probes derived from the 3′ region of the cry1F

gene (cry1F-3′A, cry1F-3′ B) would cross-hybridize with the respective DNA fragments 

containing the 3′ region of the cry1Ac gene. When digested with NotI/SphI and hybridized with 

cry1F-3′A and cry1F-3′B probes individually, two bands of ~5400 bp and ~4600 bp which are 

consistent with the expected bands with sizes of 5393 bp for cry1Fv3 PTU and 4550 bp for 

cry1Ac(synpro) PTU (Figure 21), were detected in DAS-81419-2 soybean and the positive 

control samples. Since the AtuORF23 3′ UTR element in the cry1Fv3 and cry1Ac(synpro) PTUs 

are 100% identical based on the nucleotide sequences (Figure 5), it is expected that probes 

derived from AtuORF23 3′ UTR element (AtuORF23-A 3′ UTR and AtuORF23-B 3′ UTR) 

would cross-hybridize with the respective DNA fragments containing the AtuORF23 3′ UTR. 

When digested with NotI/SphI and hybridized with AtuORF23-A 3′ UTR probe, two 

hybridization bands of 5393 bp and 4550 bp are expected for DAS-81419-2 soybean and positive 

control samples. As shown in Figure 22A, two bands of ~5400 bp and ~4600 bp were detected 

in DAS-81419-2 soybean and the positive control samples, consistent with the predicted sizes for 

cry1Fv3 and cry1Ac(synpro) PTUs. These data suggest that an intact cry1Fv3 PTU is present in 

all generations tested.

Since the CsVMV promoter element in the cry1Ac(synpro) and pat PTUs are 100% identical 

based on the nucleotide sequences (Figure 5), it is expected that probe derived from CsVMV 

promoter (CsVMV Promoter probe) would cross-hybridize with the respective DNA fragments 

containing the CsVMV promoter element. When digested with NotI/SphI and hybridized with 
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CsVMV Promoter probe, two hybridization bands of 4550 bp and 1970 bp are expected for 

DAS-81419-2 soybean and the positive control samples. As shown in Figure 22B, two bands of 

~4600 bp and ~2000 bp were detected in DAS-81419-2 soybean and the positive control 

samples, consistent with the predicted sizes for cry1Ac(synpro) and pat PTUs. When digested 

with NotI/SphI and hybridized with cry1Ac-5′ probe, a single band of ~4600 bp was detected in 

DAS-81419-2 soybean and the positive control samples, consistent with the expected size of 

4550 bp for the cry1Ac(synpro) PTU (Figure 23A).

According to the sequence information, a 98-bp partial cry1Ac(synpro) fragment was also 

identified at the 5′end of the T-DNA insert in DAS-81419-2 soybean (Figure 7). This 98-bp 

partial cry1Ac(synpro) fragment is 99% identical to 1990 - 2087 bp region of the cry1Ac(synpro)

gene in a complementary orientation. As a consequence, when digested with NotI/SphI and 

hybridized with the cry1Ac-3′A probe, two hybridization bands, one of ~4600 bp which is 

consistent with the expected size of 4550 bp for full length cry1Ac(synpro) PTU and the other 

one of ~ 6500 bp which is consistent with the expected result of >353 bp for the DNA fragment 

containing the partial cry1Ac(synpro), were detected in DAS-81419-2 soybean samples In 

addition, a very faint band of ~5400 bp was also detected in DAS-81419-2 soybean and the 

positive control samples, consistent with the expected size of 5393 bp for cry1Fv3 PTU which is 

expected to be cross-hybridized by the cry1Ac-3′A probe (Figure 24A).

When digested with NotI/SphI and hybridized with cry1Ac-3′ B probe, two hybridization bands 

of 5393 bp and 4550 bp are expected for DAS-81419-2 soybean samples along with positive 

control sample. As shown in Figure 24B, two bands of ~5400 bp and ~4600 bp were detected in 

DAS-81419-2 soybean and positive control samples, consistent with the predicted sizes for 

cry1Ac(synpro) and cry1Fv3 PTUs. When digested with NotI/SphI and hybridized with 

AtuORF23-B 3′ UTR probe, two bands of 5393 bp and 4550 bp are expected for DAS-81419-2 

soybean and positive control samples. As shown in Figure 23B, two bands of ~5400 bp and 

~4600 bp were detected in DAS-81419-2 soybean and positive control samples, consistent with 

the predicted sizes for cry1Fv3 and cry1Ac(synpro) PTUs. These data suggest that an intact 

cry1Ac(synpro) PTU and a small partial cry1Ac(synpro) fragment is present in all generations 

tested.

When digested with NotI/SphI and hybridized with pat probe, a single band of ~2000 bp was 

detected in all DAS-81419-2 soybean and the positive control samples, consistent with the 

predicted size of 1970 bp for the pat PTU (Figure 25A). When digested with NotI/SphI and 

hybridized with AtuORF1 3′ UTR probe, two bands of ~4000 bp and ~2000 bp were detected in 

all DAS-81419-2 soybean and the positive control samples, consistent with expected bands of 

>150 bp and 1970 bp (Figure 25B). These data suggest that an intact pat PTU is present in all 

generations tested.

Hybridization bands of the expected sizes were detected in all of the positive samples, while no 

specific hybridization band was detected in the non-transgenic soybean negative control samples 

as expected. The hybridization pattern is consistent across all generations with all the tested 

restriction enzyme and probe combinations.
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Taken together, the Southern blot analyses reveal that the single insert in DAS-81419-2 soybean 

contains an intact PTU for each of the cry1Fv3, cry1Ac(synpro), and pat genes along with a 

partial cry1Ac(synpro) fragment at the 5′ end of the insert.

5.2.3. Absence of Plasmid Backbone Sequences

To verify that no plasmid vector backbone sequences exist in DAS-81419-2 soybean, six probes 

covering the entire region outside of the T-DNA in pDAB9582 were hybridized with NcoI or 

SphI digested samples (Table 3, Figure 5). The hybridization results demonstrated that no 

specific hybridization signals were detected in any DAS-81419-2 samples or the negative control 

samples, while hybridization fragments of the expected size were detected in the respective 

positive controls (Figure 26 - Figure 28). These data indicate that no backbone sequences from 

pDAB9582 have been integrated into DAS-81419-2 soybean.

5.2.4. Stability of the Insert Across Generations

Southern blot analyses were conducted with samples across five distinct generations (T1, T2, T3, 

T4, and F2) of DAS-81419-2 soybean. Prior to initiation of Southern blot analyses, all plants 

were tested for PAT protein expression using a lateral flow strip assay to identify PAT

expression-positive plants. All of the genetic element probes: AtUbi10 Promoter, cry1F-5′, 

cry1F-3′A, cry1F-3′B, AtuORF23-A 3′ UTR, CsVMV Promoter, cry1Ac-5′, cry1Ac-3′A, 

cry1Ac-3′B, AtuORF23-B 3′ UTR, pat, and AtuORF1 3′ UTR, as well as the probes covering the 

entire backbone regions of plasmid pDAB9582, were hybridized with the DAS-81419-2 soybean

samples. Results across all DAS-81419-2 soybean samples in the five generations were 

consistent with what were expected (Table 4), indicating stable integration and inheritance of the 

intact, single copy transgene insert across multiple generations of DAS-81419-2 soybean.

5.2.5. Conclusions

The Southern blot analyses confirm that DAS-81419-2 soybean contains a single insertion of the 

T-DNA from plasmid pDAB9582 with each of the intact PTUs for cry1Fv3, cry1Ac(synpro), and 

pat genes. In addition, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified at

the 5′ end of the T-DNA insert. Identical hybridization patterns were observed across five 

distinct generations of DAS-81419-2 soybean, indicating stable inheritance of the transgene 

insert across multiple generations. Moreover, the absence of the backbone sequences from 

plasmid pDAB9582 in DAS-81419-2 soybean was confirmed using probes covering the entire 

region flanking the T-DNA insert.
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Figure 8. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); AtUbi10 promoter probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lanes 7-9 of panel B may not be 

clearly visible on printed report.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

492

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

B

492
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Figure 9. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); cry1F-5′ probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

492

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564

359
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Figure 10. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); cry1F-3′A probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

492

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564

359
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Figure 11. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); cry1F-3′ B probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

492

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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Figure 12. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); AtuORF23-A 3′UTR probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

492

564
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 50 of 214

Dow AgroSciences LLC

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Figure 13. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); CsVMV promoter probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

564
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 51 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 14. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); cry1Ac-5′ probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 52 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 15. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); cry1Ac-3′A probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Fuzzy smear bands at ~18000 bp in lanes 4-18 

of panel A may be a result of DNA degradation and/or incomplete digestion.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710 564 492

564 492 359

359
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 53 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 16. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); cry1Ac-3′B probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lane 8 of panel A may not be clearly 

visible on printed report.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564 

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

bp

A

1164

992

718/710

492
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 54 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 17. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); AtuORF23-B 3′UTR probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

564

564
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 55 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 18. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); pat probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lane 4 of panel A may not be clearly 

visible on printed report.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

492

359

564 492

359
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 56 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 19. Southern blot analysis of DAS-81419-2 soybean digested with NcoI (A) and SphI 

(B); AtuORF1 3′ UTR probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Bands in lanes 7-8 of panel B may not be 

clearly visible on printed report.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

492

359

492

359

564
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 57 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 20. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A) 

AtUbi10 promoter probe, (B) cry1F-5′ probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

492

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

492

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 58 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 21. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A) 

cry1F-3′A probe, (B) cry1F-3′B probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Faint bands of ~ 4600 bp in positive control 

and DAS-81419-2 soybean samples observed in panel A are the expected cross-hybridization results of the cry1F3’A probe binding to the cry1Ac(synpro) PTU.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

492

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

492

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564

359

564
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 59 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 22. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A) 

AtuORF23-A 3′UTR probe, (B) CsVMV promoter probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

492

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

492

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564

359

564

359
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 60 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 23. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; (A)

cry1Ac-5′ probe, (B) AtuORF23-B 3′UTR probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

564 492

359
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 61 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 24. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; 

(A) cry1Ac-3′A probe, (B) cry1Ac-3′B probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. Note: Faint bands of ~ 5400 bp in positive control 

and DAS-81419-2 soybean samples observed in panel A are the expected cross-hybridization bands of the cry1Ac3’A probe binding to the cry1Fv3 PTU. Faint bands at ~9500 bp observed in lanes 2 and 4 in panel A are most 

likely a result of incomplete digestion.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

564 492

564

492

359
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 62 of 214

Dow AgroSciences LLC

Contains No Confidential Business Information

Figure 25. Southern blot analysis of DAS-81419-2 soybean digested with NotI/SphI; 

(A) pat probe, (B) AtuORF1 3′UTR probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

564 492

359

564

492

359
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Figure 26. Southern blot analysis of DAS-81419-2 soybean digested with (A) SphI; Ori 

probe, (B) NcoI; SpecR probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

492

359

564

492

359
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Figure 27. Southern blot analysis of DAS-81419-2 soybean digested with (A) NcoI; 

Backbone 1 probe, (B) SphI; Backbone 2 probe.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

A

718/710

492

359

564

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

718/710

564

B
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Figure 28. Southern blot analysis of DAS-81419-2 soybean digested with NcoI; Backbone 3 

and 4 probes.

Approximately 10 g of genomic DNA was used in the digestion. The positive control included plasmid pDAB9582 

at approximately one transgene copy per soybean genome and 10 g genomic DNA from non-transgenic soybean 

Maverick. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight Marker II and DIG-labeled DNA 

Molecular Weight Marker VII are indicated adjacent to image.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-81419-2-T2-2

2 Maverick + pDAB9582 12 DAS-81419-2-T2-3

3 Maverick 13 DAS-81419-2-T3-1

4 DAS-81419-2-F2-1 14 DAS-81419-2-T3-2

5 DAS-81419-2-F2-2 15 DAS-81419-2-T3-3

6 DAS-81419-2-F2-3 16 DAS-81419-2-T4-1

7 DAS-81419-2-T1-1 17 DAS-81419-2-T4-2

8 DAS-81419-2-T1-2 18 DAS-81419-2-T4-3

9 DAS-81419-2-T1-3 19 DIG Molecular Weight Marker VII

10 DAS-81419-2-T2-1

23130

9416

6557

4361

2322

2027

8576

7427

6106

4899

3639

2799

1953

1882

1515

1482

1164

992

718/710

bp

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

564

492

359
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5.3. Segregation Analysis

5.3.1. Analysis of a Segregating Generation

The inheritance pattern of the transgene insert within a segregating generation was demonstrated 

with a lateral flow strip assay and event-specific PCR analyses of individual plants within a F2 

generation of DAS-81419-2 soybean. The F1 generation was generated by crossing homozygous 

T3 plants of DAS-81419-2 soybean with a non-transgenic soybean line. The F1 plants were self 

pollinated to produce the F2 seeds.

A total of 123 plants from the F2 generation of DAS-81419-2 soybean were tested for PAT 

protein expression by a lateral flow strip assay. Of the 123 F2 plants tested, 91 plants were 

positive and 32 were negative (segregated nulls) for PAT protein expression. One plant died 

before sufficient tissue sample was harvested for genomic DNA extraction and thus was not 

subjected to event-specific PCR analysis. As a consequence, a total of 122 F2 plants were tested 

by event-specific PCR to determine the presence or absence of the DAS-81419-2 transgene 

insert. Of the 122 plants tested, 90 plants were positive for the presence of DAS-81419-2 

transgene insert, and the remaining 32 plants were negative (segregated null). All plants that 

tested positive for PAT protein expression by a lateral flow strip assay were also positive for the 

DAS-81419-2 transgene insert by event-specific PCR analysis. Similarly, all plants that tested

negative for PAT protein expression were also negative for the presence of the DAS-81419-2 

transgene insert by event-specific PCR (Table 5). This result confirmed that the phenotypic 

segregation matched the genotypic makeup of the tested F2 generation. Statistical analysis using 

a chi-square goodness of fit test indicated the ratio of 91 positive to 32 null segregants for the 

LFS test, and the ratio of 90 positive to 32 null segregants for the event-specific PCR analysis, 

did not significantly differ from the expected Mendelian 3:1 segregation pattern for a single 

independent locus.

Table 5. Results of F2 individual plants tested with LFS and event-specific PCR.

Tested 

Method

Total plants 

tested

Positive Negative Expected 

ratio

P-valuea

LFS 123 91 32 3:1 0.7946

EventSpecific PCR 122 90 32 3:1 0.7538

a Based on a chi-squared goodness of fit test

5.3.2. Segregation Analysis of Breeding Generations

Chi-square goodness of fit analyses of trait inheritance data from three populations of the BC1F2

breeding generation was conducted to determine the Mendelian inheritance of the transgene 

insert in DAS-81419-2 soybean. The presence or absence of the transgene insert was determined 

using an event-specific PCR assay for DAS-81419-2 soybean. The expected segregation ratio of 

3:1 for plants containing the transgene insert versus plants that do not contain the transgene 

insert (segregated nulls) was observed (Table 6).
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Table 6. Results of the individual plants from DAS-81419-2 soybean tested with eventspecific PCR within segregating BC1F2 generations.

Generation Total 

plants 

tested

Transgene

insert positive 

(with eventspecific PCR)

Transgene

insert negative 

(with eventspecific PCR)

Expected 

ratio

P-valuea

BC1F2 48 34 14 3:1 0.5050

BC1F2 30 22 8 3:1 0.8330

BC1F2 294 220 74 3:1 0.9463

a Based on a chi-square goodness of fit test

5.4. Summary of the Genetic Characterization

Molecular characterization of DAS-81419-2 soybean by Southern blot analyses confirmed that a 

single T-DNA insert containing each of the intact PTUs for the cry1Fv3, cry1Ac(synpro), and pat

genes from plasmid pDAB9582, were integrated into DAS-81419-2 soybean. In addition to the 

full-length insert, a minor (<100 bp) fragment of the cry1Ac(synpro) gene was identified on the 

5′ end of the T-DNA insert. The inserted DNA was stably inherited across the five generations 

(T1, T2, T3, T4, and F2) evaluated. No transformation plasmid backbone sequences were

identified in DAS-81419-2 soybean as demonstrated by Southern blot analyses using probes 

covering the entire region of the plasmid flanking the T-DNA insert. Moreover, the T-DNA 

insert displayed the expected Mendelian inheritance pattern for a single independent insert/locus 

in segregating generations (F2 and BC1F2). 
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6. Characterization of the Introduced Proteins

6.1. Cry1Ac and Cry1F

6.1.1. Cry1 Protein Mode of Action

Cry1 insecticidal crystal proteins from Bacillus thuringiensis are a class of structurally related 

delta endotoxins having three distinct structural domains. These proteins are generally toxic to a 

subset of lepidopteran larvae, and this class of Cry proteins has been investigated most 

thoroughly with regard to their mode of action. The most widely accepted hypothesis for Cry1 

mode of action is the two-receptor model proposed by (Bravo et al., 2004). In this model, upon 

ingestion, the protein is solubilized in the midgut of the insect and is processed to an active core 

structure by proteases located within the midgut. The processed core then binds to a cadherin 

protein through the interaction of specific residues on domains II and III of the protein. 

Cadherins are a superfamily of proteins made up of a series of 5 to 34 calcium binding repeat 

elements. The first report of a cadherin interacting with a Cry protein was in Manduca sexta L.

(Lepidoptera: Sphingidae) (Vadlamudi et al., 1993). The cadherin was shown to bind Cry1Ac, 

Cry1Aa and Cry1Ab. Cry1Aa and Cry1Ac competed with Cry1Ab binding, suggesting a 

common epitope within the cadherin protein. Subsequently, cadherins have been implicated in 

Cry1 susceptibility in a number of different insect species (Gahan et al., 2001; Morin et al., 

2003; Coates et al., 2005; Yang et al., 2011). Upon binding to the cadherin receptor, structural 

changes in the Cry1 active core allow for further processing of the N-terminus enabling it to 

oligomerize to a tetramer. In addition to cadherins, receptors in the aminophosphatase-N family 

have been identified in several lepidopteran species while alkaline phosphatases are also believed 

to be involved. Thus the mode of action of Cry1 protein corresponds to proteolytic processing of

the protein and sequential multiple receptor binding interactions leading to pore formation in the 

insect gut.

6.1.2. Identity of the Cry1Ac Protein

The Cry1Ac protein expressed in DAS-81419-2 soybean is a synthetic version of Cry1Ac1, from 

B. thuringiensis subsp. kurstaki strain HD73. In this synthetic version, the first 612 amino acids 

are comprised of the insect-active portion of Cry1Ac1; the remaining portion consists of 

C-terminal sequences from Cry1Ca3 (B. thuringiensis subsp. aizawai PS81I), and Cry1Ab1 (B.

thuringiensis subsp. Berliner 1715). Together, the portions of Cry1Ca3 and Cry1Ab1 that 

comprise the chimeric C-terminal domain are approximately those removed by alkaline proteases 

in the lepidopteran mid-gut during formation of the active Cry1Ac core toxin. The synthetic 

Cry1Ac protein is comprised of 1156 amino acids and has a molecular weight of ~130.7 kDa

(Figure 29). The amino acid sequence of the Cry1Ac protein expressed in DAS-81419-2 

soybean is identical to that expressed in WideStrikecotton event DAS-21023-5 (also described 

as 3006-210-23 expressing Cry1Ac) (Dow AgroSciences, 2003a).
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 1 MDNNPNINECIPYNCLSNPEVEVLGGERIE 30

 31 TGYTPIDISLSLTQFLLSEFVPGAGFVLGL 60

 61 VDIIWGIFGPSQWDAFLVQIEQLINQRIEE 90

 91 FARNQAISRLEGLSNLYQIYAESFREWEAD 120

121 PTNPALREEMRIQFNDMNSALTTAIPLFAV 150

151 QNYQVPLLSVYVQAANLHLSVLRDVSVFGQ 180

181 RWGFDAATINSRYNDLTRLIGNYTDYAVRW 210

211 YNTGLERVWGPDSRDWVRYNQFRRELTLTV 240

241 LDIVALFPNYDSRRYPIRTVSQLTREIYTN 270

271 PVLENFDGSFRGSAQGIERSIRSPHLMDIL 300

301 NSITIYTDAHRGYYYWSGHQIMASPVGFSG 330

331 PEFTFPLYGTMGNAAPQQRIVAQLGQGVYR 360

361 TLSSTLYRRPFNIGINNQQLSVLDGTEFAY 390

391 GTSSNLPSAVYRKSGTVDSLDEIPPQNNNV 420

421 PPRQGFSHRLSHVSMFRSGFSNSSVSIIRA 450

451 PMFSWIHRSAEFNNIIASDSITQIPAVKGN 480

481 FLFNGSVISGPGFTGGDLVRLNSSGNNIQN 510

511 RGYIEVPIHFPSTSTRYRVRVRYASVTPIH 540

541 LNVNWGNSSIFSNTVPATATSLDNLQSSDF 570

571 GYFESANAFTSSLGNIVGVRNFSGTAGVII 600

601 DRFEFIPVTATLEAESDLERAQKAVNALFT 630

631 SSNQIGLKTDVTDYHIDRVSNLVECLSDEF 660

661 CLDEKKELSEKVKHAKRLSDERNLLQDPNF 690

691 RGINRQLDRGWRGSTDITIQGGDDVFKENY 720

721 VTLLGTFDECYPTYLYQKIDESKLKAYTRY 750

751 QLRGYIEDSQDLEIYLIRYNAKHETVNVPG 780

781 TGSLWPLSAPSPIGKCAHHSHHFSLDIDVG 810

811 CTDLNEDLGVWVIFKIKTQDGHARLGNLEF 840

841 LEEKPLVGEALARVKRAEKKWRDKREKLEW 870

871 ETNIVYKEAKESVDALFVNSQYDRLQADTN 900

901 IAMIHAADKRVHSIREAYLPELSVIPGVNA 930

931 AIFEELEGRIFTAFSLYDARNVIKNGDFNN 960

961 GLSCWNVKGHVDVEEQNNHRSVLVVPEWEA 990

991 EVSQEVRVCPGRGYILRVTAYKEGYGEGCV 1020

1021 TIHEIENNTDELKFSNCVEEEVYPNNTVTC 1050

1051 NDYTATQEEYEGTYTSRNRGYDGAYESNSS 1080

1081 VPADYASAYEEKAYTDGRRDNPCESNRGYG 1110

1111 DYTPLPAGYVTKELEYFPETDKVWIEIGET 1140

1141 EGTFIVDSVELLLMEE 1156

Figure 29. Amino acid sequence of Cry1Ac protein.

6.1.3. Biochemical Characterization of the Cry1Ac Protein

Large quantities of purified Cry1Ac protein are required to perform safety assessment studies. 

As it is technically infeasible to extract and purify sufficient amounts of recombinant protein 

from transgenic plants (Evans, 2004), the Cry1Ac protein was produced in Pseudomonas 

fluorescens. Characterization studies were performed to confirm the equivalency of the Cry1Ac

protein expressed in DAS-81419-2 soybean with the P. fluorescens-derived Cry1Ac protein.
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Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, 

glycoprotein detection, and protein sequence analysis by matrix assisted laser 

desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF 

MS/MS were used to characterize the biochemical properties of the proteins. Using these 

methods, the Cry1Ac protein from P. fluorescens and DAS-81419-2 soybean were shown to be 

biochemically equivalent, thereby supporting the use of the microbe-derived protein in safety 

assessment studies.

The methods and results of the biochemical characterization of DAS-81419-2 soybean- and 

microbe-derived Cry1Ac proteins are described in detail in Appendix 2. Briefly, both the plant 

and P. fluorescens-derived Cry1Ac proteins showed the expected molecular weight of ~130 kDa 

by SDS-PAGE and were immunoreactive to Cry1Ac protein-specific polyclonal antibodies by 

Western blot analysis. There was no evidence of any post-translational modifications (i.e. 

glycosylation) of the DAS-81419-2 soybean-derived Cry1Ac protein. The amino acid sequence 

was confirmed by enzymatic peptide mass fingerprinting using MALDI-TOF MS and MALDITOF MS/MS and was shown to be as expected and was identical to the protein expressed in P. 

fluorescens. The result is consistent with those for the Cry1Ac protein expressed in WideStrike

cotton event DAS-21023-5 (also described as 3006-210-23) (Dow AgroSciences, 2003a).

6.1.4. Expression of the Cry1Ac Protein in Plant Tissues

A field expression study was conducted at ten locations in U.S. during 2011 (Appendix 5). The 

test sites represented regions of diverse agronomic practices and environmental conditions for 

soybean in North America. Plant tissues sampled included leaf, grain, root, and forage. Leaf 

tissues were collected at V5 and V10-12 stages, and root and forage were collected at the R3 

stage of development. The grain was collected at the R8 stage of development (Gaska, 2006). 

The soluble, extractable Cry1Ac protein was measured using a validated enzyme-linked 

immunosorbent assay (ELISA). Cry1Ac protein levels for all tissue types were calculated on 

ng/mg dry weight basis. Methods used for tissue sampling and quantification of protein 

expression by ELISA are detailed in Appendix 5.

A summary of the Cry1Ac protein concentrations (averaged across sites) in the various soybean 

matrices is shown in Table 7. Average expression values ranged from 0.39 ng/mg dry weight in 

R3 stage root to 25.44 ng/mg dry weight in V5 stage leaf tissue. No Cry1Ac protein was 

detected in the control (Maverick) tissues across the ten locations.
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Table 7. Expression of Cry1Ac in DAS-81419-2 soybean.

Cry1Ac ng/mg Tissue Dry Weight

Matrix Description Overall 

Mean

Std. Dev. 

(n=10)

Min/Max 

Range STMRa HAFTb

V5 Leaf DAS-81419-2 25.44 6.61 12.10 - 40.20 26.90 35.25

V10-12 Leaf DAS-81419-2 23.16 6.17 10.70 - 37.45 22.15 34.61

Forage DAS-81419-2 5.54 2.54 1.38 - 11.83 5.44 10.28

Root DAS-81419-2 0.39 0.24 [0.12]* - 1.12 0.32 0.97

Grain DAS-81419-2 1.04 0.10 0.79 - 1.40 1.04 1.20

 a

Supervised Trials Mean Residue is calculated as the median of all the individual results across different sites. 

b Highest Average Field Trial is the maximum value of all the means across different sites.

* Expression level below LOQ

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the Cry1Ac ELISA in the 

tissue matrices were as follows:

Cry1Ac (ng/mg

sample dry weight)

Matrix LOD LOQ

Leaf V5 0.1 0.2

Leaf V10-12 0.1 0.2

Root 0.1 0.2

Forage 0.1 0.2

Grain 0.1 0.2

6.1.5. Identity of the Cry1F Protein

The Cry1F protein expressed in DAS-81419-2 soybean is a synthetic version of Cry1F from 

Bacillus thuringiensis subsp. aizawai strain PS81I. In this synthetic version, the first 604 amino 

acids are comprised of the insect-active portion of Cry1F; the remaining portion consists of 

C-terminal sequences from Cry1Ca3 (B. thuringiensis subsp. aizawai PS81I), and Cry1Ab1 (B.

thuringiensis subsp. Berliner 1715). Together, the portions of Cry1Ca3 and Cry1Ab1 that 

comprise the chimeric C-terminal domain are approximately those removed by alkaline proteases 

in the lepidopteran mid-gut during the formation of the active Cry1F core toxin. The synthetic 

Cry1F protein is comprised of 1148 amino acids and has a molecular weight of ~130.2 kDa

(Figure 30). The amino acid sequence of the Cry1F protein expressed in DAS-81419-2 soybean 

is identical to that expressed in WideStrikecotton event DAS-24236-5 (also described as 281-

24-236) (Dow AgroSciences, 2003b).
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 1 MENNIQNQCVPYNCLNNPEVEILNEERSTG 30

 31 RLPLDISLSLTRFLLSEFVPGVGVAFGLFD 60

 61 LIWGFITPSDWSLFLLQIEQLIEQRIETLE 90

 91 RNRAITTLRGLADSYEIYIEALREWEANPN 120

121 NAQLREDVRIRFANTDDALITAINNFTLTS 150

151 FEIPLLSVYVQAANLHLSLLRDAVSFGQGW 180

181 GLDIATVNNHYNRLINLIHRYTKHCLDTYN 210

211 QGLENLRGTNTRQWARFNQFRRDLTLTVLD 240

241 IVALFPNYDVRTYPIQTSSQLTREIYTSSV 270

271 IEDSPVSANIPNGFNRAEFGVRPPHLMDFM 300

301 NSLFVTAETVRSQTVWGGHLVSSRNTAGNR 330

331 INFPSYGVFNPGGAIWIADEDPRPFYRTLS 360

361 DPVFVRGGFGNPHYVLGLRGVAFQQTGTNH 390

391 TRTFRNSGTIDSLDEIPPQDNSGAPWNDYS 420

421 HVLNHVTFVRWPGEISGSDSWRAPMFSWTH 450

451 RSATPTNTIDPERITQIPLVKAHTLQSGTT 480

481 VVRGPGFTGGDILRRTSGGPFAYTIVNING 510

511 QLPQRYRARIRYASTTNLRIYVTVAGERIF 540

541 AGQFNKTMDTGDPLTFQSFSYATINTAFTF 570

571 PMSQSSFTVGADTFSSGNEVYIDRFELIPV 600

601 TATLEAESDLERAQKAVNALFTSSNQIGLK 630

631 TDVTDYHIDRVSNLVECLSDEFCLDEKKEL 660

661 SEKVKHAKRLSDERNLLQDPNFRGINRQLD 690

691 RGWRGSTDITIQGGDDVFKENYVTLLGTFD 720

721 ECYPTYLYQKIDESKLKAYTRYQLRGYIED 750

751 SQDLEIYLIRYNAKHETVNVPGTGSLWPLS 780

781 APSPIGKCAHHSHHFSLDIDVGCTDLNEDL 810

811 GVWVIFKIKTQDGHARLGNLEFLEEKPLVG 840

841 EALARVKRAEKKWRDKREKLEWETNIVYKE 870

871 AKESVDALFVNSQYDRLQADTNIAMIHAAD 900

901 KRVHSIREAYLPELSVIPGVNAAIFEELEG 930

931 RIFTAFSLYDARNVIKNGDFNNGLSCWNVK 960

961 GHVDVEEQNNHRSVLVVPEWEAEVSQEVRV 990

991 CPGRGYILRVTAYKEGYGEGCVTIHEIENN 1020

1021 TDELKFSNCVEEEVYPNNTVTCNDYTATQE 1050

1051 EYEGTYTSRNRGYDGAYESNSSVPADYASA 1080

1081 YEEKAYTDGRRDNPCESNRGYGDYTPLPAG 1110

1111 YVTKELEYFPETDKVWIEIGETEGTFIVDS 1140

1141 VELLLMEE

Figure 30. Amino acid sequence of Cry1F protein.

6.1.6. Biochemical Characterization of the Cry1F Protein

Large quantities of purified Cry1F protein are required to perform safety assessment studies. As

it is technically infeasible to extract and purify sufficient amounts of recombinant protein from 

transgenic plants (Evans, 2004), the Cry1F protein was produced in Pseudomonas fluorescens. 

Characterization studies were performed to confirm the equivalency of the Cry1F protein 

expressed in DAS-81419-2 soybean with the P. fluorescens-derived Cry1F protein. Sodium 

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot, glycoprotein 

detection, and protein sequence analysis by matrix assisted laser desorption/ionization time-of-
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flight mass spectrometry (MALDI-TOF MS) and MALDI-TOF MS/MS were used to 

characterize the biochemical properties of the proteins (Gao et al., 2006). Using these methods, 

the Cry1F protein from P. fluorescens and DAS-81419-2 soybean were shown to be 

biochemically equivalent, thereby supporting the use of the microbe-derived protein in safety 

assessment studies.

The methods and results of the biochemical characterization of DAS-81419-2 soybean- and 

microbe-derived Cry1F proteins are described in detail in Appendix 3. Briefly, both the plant and 

P. fluorescens-derived Cry1F proteins showed the expected molecular weight of ~130 kDa by 

SDS-PAGE and were immunoreactive to Cry1F protein-specific polyclonal antibodies by 

Western blot analysis. There was no evidence of any post-translational modifications (i.e. 

glycosylation) of the DAS-81419-2 soybean-derived Cry1F protein. The amino acid sequence 

was confirmed by enzymatic peptide mass fingerprinting using MALDI-TOF MS and MALDITOF MS/MS and was shown to be as expected and was identical to the protein expressed in P. 

fluorescens. The result is consistent with those for the Cry1F protein expressed in WideStrike

cotton event DAS-24236-5 (also described as 281-24-236) (Dow AgroSciences, 2000; Gao et al., 

2006).

6.1.7. Expression of the Cry1F Protein in Plant Tissues

A field expression study was conducted at ten locations in U.S. during 2011 (Appendix 5). The 

test sites represented regions of diverse agronomic practices and environmental conditions for 

soybean in North America. Plant tissues sampled included leaf, grain, root, and forage. Leaf 

tissues were collected at V5 and V10-12 stages, and root and forage were collected at the R3 

stage of development. The grain was collected at the R8 stage of development (Gaska, 2006). 

The soluble, extractable Cry1F protein was measured using a validated enzyme-linked 

immunosorbent assay (ELISA). Cry1F protein levels for all tissue types were calculated on 

ng/mg dry weight basis. Methods used for tissue sampling and quantification of protein 

expression by ELISA are detailed in Appendix 5.

A summary of the Cry1F protein concentrations (averaged across sites) in the various soybean 

matrices is shown in Table 8. Average expression values ranged from 5.23 ng/mg dry weight in 

R3 stage root to 56.75 ng/mg dry weight in V5 stage leaf tissue. No Cry1F protein was detected 

in the control (Maverick) tissues across the ten locations.
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 Table 8. Expression of Cry1F in DAS-81419-2 soybean.

Cry1F ng/mg Tissue Dry Weight

Matrix Description Overall 

Mean

Std. Dev. 

(n=10)

Min/Max 

Range STMRa HAFTb

V5 Leaf DAS-81419-2 56.75 15.03 24.60 - 99.50 56.30 76.05

V10-12 Leaf DAS-81419-2 39.07 16.60 12.75 - 76.71 38.70 59.98

Forage DAS-81419-2 20.28 11.29 5.34 - 44.62 20.64 40.23

Root DAS-81419-2 5.23 3.74 1.09 - 16.08 4.12 14.21

Grain DAS-81419-2 13.80 1.24 10.41 - 16.95 13.71 16.21

 a

Supervised Trials Mean Residue is calculated as the median of all the individual results across different sites. 

b Highest Average Field Trial is the maximum value of all the means across different sites.

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the Cry1F ELISA in the 

tissue matrices were as follows:

Cry1F (ng/mg

sample dry weight)

Matrix LOD LOQ

Leaf V5 0.25 0.5

Leaf V10-12 0.25 0.5

Root 0.15 0.3

Forage 0.05 0.1

Grain 0.2 0.4

6.1.8. Food and Feed Safety Assessment of Cry1Ac and Cry1F

The food and feed safety assessment of the Cry1Ac and Cry1F proteins expressed in 

DAS-81419-2 soybean considers several factors including safety of the donor organism, history 

of safe use, allergenic potential, toxicity potential, and dietary risk assessment based on 

consumption patterns. Results of the overall safety assessments of Cry1Ac and Cry1F indicate 

that each protein is unlikely to cause an allergenic reaction in humans or be a toxin to humans or 

animals. 

The donor organism, Bacillus thuringiensis, is a naturally occurring microorganism commonly 

found in soil. B. thuringiensis was first registered as a pesticide in the U.S. in 1961. Today there 

are more than 100 registered pesticide products containing B. thuringiensis as an active 

ingredient (EPA, 2011). An exemption from the requirement of tolerance for residues of B. 

thuringiensis was first established by FDA in 1960 and was republished and then amended after 

EPA was established.
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Cry1Ac and Cry1F have a long history of safe use. The safety of the proteins has been 

demonstrated in sprayable Bt formulations for pest control in agriculture for over half a century 

(Mendelsohn et al., 2003; EPA, 2011; Sanahuja et al., 2011). Both proteins are expressed in

events comprising Dow AgroSciences’ WideStrikecotton authorized for cultivation in the U.S. 

and Brazil and for food and feed use in Australia, Brazil, Canada, European Union, Japan, 

Korea, Mexico, New Zealand and U.S. (www.biotradestatus.com). Bt corn and Bt cotton 

expressing variations of either Cry1Ac or Cry1F have been cultivated for commercial use in the 

U.S. and other countries for more than a decade. In 1997, EPA established an exemption from 

the requirement of a tolerance for the plant-incorporated protectant Cry1Ac in all plants (40 CFR 

§174.510). Later EPA established an exemption from the requirement of a tolerance for the 

plant-incorporated protectant Cry1F in cotton (40 CFR §174.504) and in corn (40 CFR 

§174.520). The exemptions were based on safety assessments of the proteins including 

digestibility in simulated gastric fluid, lack of homology to known allergens and protein toxins, 

and lack of mammalian toxicity as demonstrated by acute oral mouse gavage studies. DAS has

filed a petition with EPA for an exemption of a tolerance for Cry1F as expressed in soybean in 

2012.

Allergenicity assessments of Cry1Ac and Cry1F found no evidence of allergenic potential for 

either protein. Both Cry1Ac and Cry1F were rapidly digested in simulated gastric fluids in less 

than one minute indicating that the proteins are unlikely to elicit allergenic reactions when 

consumed. Glycosylation analysis revealed no detectable covalently linked carbohydrates in the 

Cry1Ac and Cry1F proteins expressed in DAS-81419-2 soybean. Bioinformatics analysis of 

Cry1Ac and Cry1F amino acid sequences using an updated allergen database (Food Allergy 

Research and Resource Program (FARRP), www.allergenonline.org, Allergen Database Version 

12, Released February, 2012) demonstrated that the proteins do not share any amino acid 

sequence similarities with known allergens. No significant homology was identified when either 

protein sequence was compared with known allergen using the search criteria of either a match of 

eight or more contiguous identical amino acids, or greater than 35% identity over 80 amino acid 

residues.

Toxicity assessments identified no potential for toxic effects in humans or animals for Cry1Ac or 

Cry1F. Mouse acute oral toxicity studies demonstrated that neither protein was acutely toxic or

caused any adverse effects with doses up to 700 mg/kg body weight and 600 mg/kg body weight

for Cry1Ac and Cry1F, respectively. BLASTp search of Cry1Ac and Cry1F amino acid 

sequences against an up-to-date GenBank non-redundant protein sequences (nr) database 

(www.ncbi.nlm.nih.gov/genbank; updated to May 18, 2012) demonstrated that neither Cry1Ac 

nor Cry1F shared any amino acid sequence similarities with known protein toxins.

In 2005, EPA concluded, based upon the human health assessment of Cry1Ac and Cry1F 

expressed in WideStrikecotton events DAS-21023-5 (also described as 3006-210-23 

expressing Cry1Ac) and DAS-24236-5 (also described as 281-24-236 expressing Cry1F), that 

“there is a reasonable certainty that no harm will result from exposure to Cry1F and Cry1Ac”

and “data provided by Dow AgroSciences support issuance of a tolerance exemption for the 

Bacillus thuringiensis Cry1F protein and the genetic material necessary for its productin in or on 

cotton” (EPA, 2005). Cry1Ac and Cry1F expressed in DAS-81419-2 soybean are identical in 

amino acid sequence to Cry1Ac and Cry1F expressed in WideStrikecotton events DAS-21023-

5 and DAS-24236-5 (Sections 6.1.2 and 6.1.5). The Cry proteins expressed in DAS-81419-2 
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soybean are biochemically equivalent to Cry1Ac and Cry1F (Sections 6.1.3 and 6.1.6) that had 

been previously assessed in terms of allergenic and toxicity potential.

Potential dietary risk to humans and livestock from the consumption of the Cry1Ac and Cry1F 

proteins in food and feed derived from DAS-81419-2 soybean were evaluated by determining 

margins of exposure (MOE). MOE is the ratio of the No Observable Effect Level (NOEL) of 

Cry1Ac or Cry1F determined by the mouse acute oral toxicity study (700 mg/kg body weight for 

Cry1Ac, 600 mg/kg body weight for Cry1F) to estimated dietary intake of the respective Cry 

protein. To evaluate dietary risk for human food consumption, MOEs were determined for the 

overall U.S. population and for several subpopulations based on mean protein expression levels 

in the seed. The dietary risk assessments revealed that the MOEs for the overall U.S. population 

were greater than 1067073 and 68918 for Cry1Ac and Cry1F, respectively. The MOEs for 

infants (an age group with highest soybean intake) were greater than 268817 and 17366 for 

Cry1Ac and Cry1F, respectively. It is a common regulatory practice that MOE values greater 

than 100 are considered protective when using animal data to protect human health. Large MOE 

values for the overall U.S. population and for each subpopulation indicate there is no concern for 

Cry1Ac and Cry1F from acute dietary exposure through DAS-81419-2 soybean. The U.S. 

livestock dietary assessments considered several soybean commodity forms as potential animal 

feeds including seed, meal, hulls, aspirated grain fractions and optional forage. For the typical 

diet without soybean forage, the highest exposed animal in the U.S. is beef cow. For the worstcase diet with soybean forage, the highest exposed animal in the U.S. is dairy cow. Using the 

worst-case diet for assessments the MOEs were greater than 2941 and 596 for Cry1Ac and 

Cry1F, respectively, indicating an adequate margin of safety for livestock.

6.1.9. Summary of Cry1Ac and Cry1F Characterization

The Cry1Ac protein expressed in DAS-81419-2 soybean is a synthetic version of Cry1Ac1, from 

B. thuringiensis subsp. kurstaki strain HD73. The Cry1Ac protein is comprised of 1156 amino 

acids and has a molecular weight of ~130.7 kDa. Detailed biochemical characterization of the 

Cry1Ac protein derived from DAS-81419-2 soybean confirmed the identity of the protein. 

Moreover, biochemical characterization demonstrated that the DAS-81419-2 soybean-derived 

and Pseudomonas-derived Cry1Ac proteins are equivalent thereby supporting the use of 

Pseudomonas-derived Cry1Ac protein for safety assessment. Additionally, characterization of 

Cry1Ac protein expression in DAS-81419-2 soybean over the growing season was determined 

by analyzing leaf, root, whole plant, and grain tissues.

The Cry1F protein expressed in DAS-81419-2 soybean is a synthetic version of Cry1F from B.

thuringiensis subsp. aizawai strain PS81I. The Cry1F protein is comprised of 1148 amino acids 

and has a molecular weight of ~130.2 kDa. Detailed biochemical characterization of the Cry1F

protein derived from DAS-81419-2 soybean confirmed the identity of the protein. Moreover, 

biochemical characterization demonstrated that the DAS-81419-2 soybean-derived and 

Pseudomonas-derived Cry1F proteins are equivalent, thereby supporting the use of 

Pseudomonas-derived Cry1F protein for safety assessment. Additionally, characterization of 

Cry1F protein expression in DAS-81419-2 soybean over the growing season was determined by 

analyzing leaf, root, whole plant, and grain tissues.

A step-wise, weight-of-evidence approach was used to assess the potential for toxic or allergenic 

effects from the Cry1Ac and Cry1F proteins. Bioinformatic analyses revealed no meaningful 
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homologies to known or putative allergens or toxins for the Cry1Ac and Cry1F amino acid

sequence. Both Cry1Ac and Cry1F hydrolyzed rapidly in simulated gastric fluid and 

glycosylation analysis revealed no detectable covalently linked carbohydrates in Cry1Ac and 

Cry1F. Additionaly, neither protein caused adverse effects in mouse acute oral toxicity studies. 

Therefore, the low level Cry1Ac and Cry1F content in DAS-81419-2 soybean relative to total 

plant proteins presents a low exposure risk to humans and animals, and the results of the overall 

safety assessment of the Cry1Ac and Cry1F proteins indicate that the proteins are unlikely to 

cause allergenic or toxic effects in humans or animals.
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6.2. PAT

6.2.1. Mode of Action of the PAT Protein

The phosphinothricin acetyltransferase (PAT) has been expressed in a variety of crops to provide 

tolerance to the herbicide glufosinate, which contains the active ingredient phosphinothricin 

(PPT). The L-isomer of PPT is a potent inhibitor of glutamine synthetase (GS) in plants and is 

used as a non-selective herbicide (OECD, 1999). Inhibition of GS by PPT causes rapid 

accumulation of intracellular ammonia which leads to cessation of photorespiration and results in 

the death of the plant cell (Duan et al., 2009). The pat gene which encodes phosphinothricin 

acetyltransferase (PAT) acetylates the free NH2 group of PPT (in the presence of acetyl 

coenzyme A) and thereby prevents autotoxicity in the producing organism (Figure 31, (Duke, 

1996)).

Figure 31. Mode of action of the PAT protein.

6.2.2. Identity of the PAT Protein

The phosphinothricin acetyltransferase (PAT) protein was derived from Streptomyces 

viridochromogenes, a gram-positive soil bacterium (Strauch et al., 1988; OECD, 1999). The pat

transgene in DAS-81419-2 encodes a protein sequence that is identical to the native PAT protein 

(UniProt Accession Number: Q57146). PAT is comprised of 183 amino acids and has a 

molecular weight of ~20.6 kDa (Figure 32).

 1 MSPERRPVEIRPATAADMAAVCDIVNHYIE 30

 31 TSTVNFRTEPQTPQEWIDDLERLQDRYPWL 60

 61 VAEVEGVVAGIAYAGPWKARNAYDWTVEST 90

 91 VYVSHRHQRLGLGSTLYTHLLKSMEAQGFK 120

121 SVVAVIGLPNDPSVRLHEALGYTARGTLRA 150

151 AGYKHGGWHDVGFWQRDFELPAPPRPVRPV 180

181 TQI

 Figure 32. Amino acid sequence of the PAT protein.

6.2.3. Biochemical Characterization of the PAT Protein

Characterization of the biochemical properties of the DAS-81419-2 soybean-derived PAT 

protein was accomplished through the use of sodium dodecyl sulfate polyacrylamide gel 
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electrophoresis (SDS-PAGE), Western blot analysis, protein sequence alignment, and a lateral 

flow strip assay. The methods and results are described in Appendix 4. Using these methods, 

the PAT protein produced in DAS-81419-2 soybean was shown to be substantially equivalent to 

that produced in Escherichia coli. Furthermore, the sequence of the PAT protein is identical to 

the PAT protein expressed in other deregulated transgenic crops (USDA, 1996, 2001, 2004, 

2005).

6.2.4. Expression of the PAT Protein in Plant Tissues

A field expression study was conducted at ten locations in U.S. during 2011 (Appendix 5). The 

test sites represented regions of diverse agronomic practices and environmental conditions for 

soybean in North America. Plant tissues sampled included leaf, grain, root, and forage. Leaf 

tissues were collected at V5 and V10-12 stages, and root and forage were collected at the R3 

stage of development. The grain was collected at the R8 stage of development (Gaska, 2006). 

The soluble, extractable PAT protein was measured using a validated enzyme-linked 

immunosorbent assay (ELISA) method. PAT protein levels for all tissue types were calculated 

on ng/mg dry weight basis. Methods used for tissue sampling and quantification of protein 

expression by ELISA are detailed in Appendix 5.

A summary of the PAT protein concentrations (averaged across sites) in the various soybean 

matrices is shown in Table 9. Average expression values ranged from 0.63 ng/mg dry weight in 

R3 stage root to 5.60 ng/mg dry weight in V10-12 stage leaf tissue. No PAT protein was 

detected in the control (Maverick) tissues across the ten locations.

 Table 9. Expression of PAT in DAS-81419-2 soybean.

PAT ng/mg Tissue Dry Weight

Matrix Description Overall 

Mean Std. Dev. Min/Max Range STMRa HAFTb

V5 Leaf DAS-81419-2 5.23 0.88 3.25 - 7.35 5.30 6.93

V10-12 Leaf DAS-81419-2 5.60 1.14 2.55 - 7.56 5.76 7.32

Forage DAS-81419-2 4.06 1.30 1.24 - 6.12 4.02 5.69

Root DAS-81419-2 0.63 0.12 0.44 - 1.05 0.63 0.85

Grain DAS-81419-2 0.86 0.13 0.63 - 1.12 0.83 1.06

 a

Supervised Trials Mean Residue is calculated as the median of all the individual results across different sites. 

b Highest Average Field Trial is the maximum value of all the means across different sites.

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the PAT ELISA in the tissue 

matrices were as follows:

PAT (ng/mg

sample dry weight)

Matrix LOD LOQ

Leaf V5 0.06 0.12

Leaf V10-12 0.06 0.12
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PAT (ng/mg

sample dry weight)

Matrix LOD LOQ

Root 0.06 0.12

Forage 0.06 0.12

Grain 0.06 0.12

6.2.5. Food and Feed Safety Assessment for PAT Protein

The PAT protein expressed in DAS-81419-2 soybean originates from a common soil bacterium 

Streptomyces viridochromogenes (Wohlleben et al., 1988). The protein is 100% identical in 

amino acid sequence to PAT expressed in other transgenic crops that have been previously 

deregulated by USDA (USDA, 1996, 2001, 2004, 2005). The food and feed safety of PAT was 

assessed in these products and in published findings (OECD, 1999; Herouet et al., 2005) and 

shown to present no significant food or feed safety risk. The PAT protein is hydrolyzed rapidly 

in simulated gastric fluid and there was no evidence of acute toxicity in mice at a dose of 5000 

mg/kg body weight of PAT protein (OECD, 1999). Additionally, updated bioinformatic 

analyses revealed no meaningful homologies to known or putative allergens or toxins for the 

PAT amino acid sequence.

Additionally, the US EPA has concluded, after reviewing data on the acute toxicity and 

digestibility of the PAT protein, that there is a reasonable certainty that no harm will result from 

aggregate exposure to the U.S. population, including infants and children, to the PAT protein and 

the genetic material necessary for its production (EPA, 1997). The US EPA has consequently 

established an exemption from tolerance requirements pursuant to FFDCA section 408(j)(3) for 

PAT and the genetic material necessary for its production in all plants.

The dietary risk assessments showed large margins of exposure for the overall U.S. populations 

as well as for infants (age group with the highest soybean consumption). The analyses also 

revealed large margins of exposure for poultry and livestock. Large margins of exposure 

indicate lack of meaningful risks to humans and animals from dietary exposure.

6.2.6. Summary of PAT Protein Characterization

The phosphinothricin acetyltransferase (PAT) protein was derived from Streptomyces 

viridochromogenes, a gram-positive soil bacterium. PAT is comprised of 183 amino acids and 

has a molecular weight of ~20.6 kDa. The PAT protein, as expressed in DAS-81419-2, has the 

same amino acid sequence as the PAT expressed in several other transgenic events. Western blot 

analysis and lateral flow strip assays demonstrated that the PAT protein expressed in 

DAS-81419-2 soybean had the expected molecular weight and immunoreactivity. 

Characterization of PAT protein expression in DAS-81419-2 soybean was determined by 

analyzing leaf, root, whole plant, and grain tissues over the growing season. The PAT protein 

has a long history of safe use and the food and feed safety of PAT has previously been assessed 

in other products and in published findings. The low level expression of the PAT protein in 

DAS-81419-2 soybean presents a low exposure risk to humans and animals, and the results of 

the overall safety assessment of the PAT protein indicate that it is unlikely to cause allergenic or 

toxic effects in humans or animals.
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7. Grain and Forage Composition

Field trials with DAS-81419-2 soybean, the non-transgenic control (Maverick), and reference 

lines were conducted in 2011 at ten sites located in the U.S. This study used the same plots that 

were used for protein expression (Section 6) and agronomic characterization (Section 8) studies. 

No biologically meaningful unintended compositional differences were observed between the 

non-transgenic near-isogenic control (Maverick) and DAS-81419-2 soybean. Results from this 

study demonstrate compositional equivalence between event DAS-81419-2 soybean and nontransgenic soybean.

7.1. Field Study Design and Data Analysis

A crop composition study with DAS-81419-2 soybean, a near-isogenic non-transgenic control 

(Maverick) and six non-transgenic reference lines (IL 3503, Porter 75148, DSR 75213-72, 

Pioneer 93M62, HiSOY 38C60, Williams 82) was conducted in 2011 at ten sites located in

Richland, Iowa; Atlantic, Iowa; Carlyle, Illinois; Wyoming, Illinois; Frankfort, Indiana; Fisk, 

Missouri; La Plata, Missouri; York, Nebraska; Brunswick, Nebraska; and Germansville, 

Pennsylvania. 

Each trial site included event DAS-81419-2, the non-transgenic near-isogenic control

(Maverick), and three non-transgenic reference lines. At each of the ten sites, all entries were 

arranged in a randomized complete block design with four blocks. Across all sites, each control 

(Maverick) and DAS-81419-2 entry was represented by a total of 40 plots (10 sites, 4 replicate 

plots per entry at each site). Three of the six reference lines were included at each site by 

randomizing across sites in a balanced incomplete-block design. Each of the six reference lines 

was assigned to five sites; therefore, each reference line was represented by a total of 20 plots 

across sites (5 sites per reference line, 4 replicate plots per entry at each site). 

At each site, four replicate plots of each entry were established, with each plot consisting of four 

25 ft (7.62 m) rows. Soybean seeds were planted at a seeding rate of approximately 125 seeds 

per row with seed spacing within each row of approximately 2.4 inches (6 cm). Each soybean 

plot was bordered by two rows of a non-transgenic soybean cultivar of similar maturity. The 

entire trial site was surrounded by a minimum of four rows (10 ft or 3.0 m) of a non-transgenic 

soybean cultivar of similar maturity. Appropriate insect, weed, and disease control practices 

were applied to produce an agronomically acceptable crop. Soybean forage samples used for 

composition analysis were composed of the above-ground portion of three whole plants that 

collected from the center two rows of each four row plot at the R3 growth stage. Soybean seed 

samples used for composition analysis were composed of a representative 500 g sample of the 

soybean seed harvested from the center two rows of each four row plot.

Composition Analysis

Samples of soybean forage and seed were analyzed for nutrient and antinutrient content 

(Appendix 6). The analytes examined are presented in Table 10.
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Table 10. Composition analytes.

Matrix Category Analyte Matrix Category Analyte

Forage Proximate Protein Grain Amino Acid Valine

Forage Proximate Fat Grain Fatty Acid 8:0 Caprylic

Forage Proximate Ash Grain Fatty Acid 10:0 Capric

Forage Proximate Moisture Grain Fatty Acid 12:0 Lauric

Forage Proximate Carbohydrates Grain Fatty Acid 14:0 Myristic

Forage Fiber ADF Grain Fatty Acid 14:1 Myristoleic

Forage Fiber NDF Grain Fatty Acid 15:0 Pentadecanoic

Forage Mineral Calcium Grain Fatty Acid 15:1 Pentadecenoic

Forage Mineral Phosphorus Grain Fatty Acid 16:0 Palmitic

Grain Proximate Protein Grain Fatty Acid 16:1 Palmitoleic

Grain Proximate Fat Grain Fatty Acid 17:0 Heptadecanoic

Grain Proximate Ash Grain Fatty Acid 17:1 Heptadecenoic

Grain Proximate Moisture Grain Fatty Acid 18:0 Stearic

Grain Proximate Carbohydrates Grain Fatty Acid 18:1 Oleic

Grain Fiber ADF Grain Fatty Acid 18:2 Linoleic

Grain Fiber NDF Grain Fatty Acid 18:3 Linolenic

Grain Fiber Total Dietary Fiber Grain Fatty Acid 18:3 γ-Linolenic

Grain Mineral Calcium Grain Fatty Acid 20:0 Arachidic

Grain Mineral Copper Grain Fatty Acid 20:1 Eicosenoic

Grain Mineral Iron Grain Fatty Acid 20:2 Eicosadienoic

Grain Mineral Magnesium Grain Fatty Acid 20:3 Eicosatrienoic

Grain Mineral Manganese Grain Fatty Acid 20:4 Arachidonic

Grain Mineral Phosphorus Grain Fatty Acid 22:0 Behenic

Grain Mineral Potassium Grain Vitamin Vitamin A (Beta Carotene)

Grain Mineral Selenium Grain Vitamin Vitamin B1 (Thiamine HCl)

Grain Mineral Sodium Grain Vitamin Vitamin B2 (Riboflavin)

Grain Mineral Zinc Grain Vitamin Vitamin B3 (Niacin)

Grain Amino Acid Alanine Grain Vitamin Vitamin B5 (Pantothenic Acid)

Grain Amino Acid Arginine Grain Vitamin Vitamin B6 (Pyridoxine HCl)

Grain Amino Acid Aspartic Acid Grain Vitamin Vitamin B9 (Folic Acid)

Grain Amino Acid Cystine Grain Vitamin Vitamin C (Ascorbic Acid)

Grain Amino Acid Glutamic Acid Grain Vitamin α-tocopherol (Vitamin E)

Grain Amino Acid Glycine Grain Vitamin β-tocopherol

Grain Amino Acid Histidine Grain Vitamin γ-tocopherol

Grain Amino Acid Isoleucine Grain Vitamin δ-tocopherol

Grain Amino Acid Leucine Grain Vitamin Total tocopherol

Grain Amino Acid Lysine Grain Bioactive Lectin

Grain Amino Acid Methionine Grain Bioactive Phytic Acid

Grain Amino Acid Phenylalanine Grain Bioactive Raffinose

Grain Amino Acid Proline Grain Bioactive Stachyose

Grain Amino Acid Serine Grain Bioactive Trypsin Inhibitor

Grain Amino Acid Threonine Grain Bioactive Total Daidzein Equivalent

Grain Amino Acid Tryptophan Grain Bioactive Total Genistein Equivalent

Grain Amino Acid Tyrosine Grain Bioactive Total Glycitein Equivalent

The results of the compositional analysis for soybean forage and seed were compared with 

values reported in the literature (Kakade et al., 1972; Iskander, 1987; Hartwig and Kilen, 1991; 

Padgette et al., 1996; Taylor et al., 1999; OECD, 2001; McCann et al., 2005; Harrigan et al., 

2007; Bilyeu et al., 2008; Lundry et al., 2008; Berman et al., 2009, 2010; ILSI, 2010; Berman et 

al., 2011; Zhou et al., 2011). A summary of the compositional data from the literature can be 

found in Appendix 7.
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Statistical Analysis

Analysis of variance was conducted across field sites (combined-site analysis) for composition 

data using a mixed model (SAS Institute Inc., 2009). Entry was considered a fixed effect, and

location, block within location, and location-by-entry, were designated as random effects. 

Significant differences were declared at the 95% confidence level. Data were not rounded off for 

statistical analysis. The significance of an overall treatment effect was estimated using an F-test. 

Comparisons were made between DAS-81419-2 and the control entry using t-tests.

Due to the large number of comparisons made in this study, multiplicity was an issue. 

Multiplicity is an issue when a large number of comparisons are made in a single study to look 

for unexpected effects. Under these conditions, the probability of falsely declaring differences 

based on comparison-wise P-values is very high (1-0.95number of comparisons). In this study there 

were 71 analytes analyzed for composition; therefore, 71 comparisons were made in the 

combined-site composition analysis. As a result, the probability of declaring one or more false 

differences based on unadjusted P-values was 97.38% (1-0.9571).

One method to account for multiplicity is to adjust P-values to control the experiment-wise error 

rate, but when many comparisons are made in a study, the power for detecting specific effects 

can be reduced significantly. An alternative with much greater power is to adjust P-values to 

control the probability that each declared difference is significant (Curran-Everett, 2000). This 

can be accomplished using a False Discovery Rate (FDR) control procedure (Benjamini and 

Hochberg, 1995); FDR methods are commonly applied in studies examining transgenic crops 

(Herman et al., 2007; Coll et al., 2008; Huls et al., 2008; Jacobs et al., 2008; Stein et al., 2009; 

Herman et al., 2010). Therefore, the P-values from the composition contrasts were each adjusted 

using the FDR method to improve discrimination of true differences among treatments from 

random effects (false positives). Differences were considered significant if the FDR-adjusted Pvalue was less than 0.05.

7.2. Composition Analysis Results

An across-site summary and statistical analysis of composition data from the non-transgenic 

near-isogenic control (Maverick) and DAS-81419-2 soybean is found in Table 11 to Table 17

and Figure 33 to Figure 39. For each analyte and entry, the least-square mean, standard error, 

and minimum and maximum sample value are reported. Also for comparison, the minimum and 

maximum values for the six reference lines and literature range are reported (values are for 

individual plot results except where noted). Arithmetic means from each field site are plotted in 

figures and literature ranges are shaded (literature ranges reported as not detected or <LOD are 

plotted as zeros).

7.2.1. Proximate, Fiber, and Mineral Analysis of Forage

Soybean forage samples from the non-transgenic control (Maverick), DAS-81419-2, and 

reference variety entries were analyzed for proximates, fiber, and minerals (nine analytes). A 

summary of the results across all locations is presented in Table 11 and Figure 33. No statistical 

differences were observed in the combined-site analysis between the control and DAS-81419-2 

entries for all proximate, fiber, and mineral analytes tested. Additionally, all mean values were 

within literature ranges (when available) and within ranges for reference varieties included in the 

study. 
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Table 11. Summary of the proximate, fiber, and mineral analysis of soybean forage from 

all sites and literature range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Proximate

Protein

0.786

20.4 ± 0.8 20.6 ± 0.8

13.9 - 29.8 15.8 - 25.8 14.0 - 35.5 11.2 - 24.71

(% DW) (0.786, 0.828)

Fat

0.274

2.49 ± 0.19 2.70 ± 0.20

0.898 - 3.89 0.857 - 4.32 0.685 - 5.32 1.01 - 9.87

(% DW) (0.274, 0.589)

Ash

0.926

8.96 ± 0.31 8.99 ± 0.32

7.01 - 16.9 7.51 - 10.9 6.80 - 15.2 4.68 - 10.782

(% DW) (0.926, 0.931)

Moisture

0.578

79.7 ± 0.8 79.4 ± 0.8

75.8 - 84.3 75.5 - 83.6 75.3 - 86.6 32.05 - 84.60

(% FW) (0.578, 0.719)

Carbohydratese

0.510

68.2 ± 1.1 67.7 ± 1.1

57.2 - 76.4 60.3 - 75.7 50.1 - 75.6 59.8 - 80.18

(% DW) (0.510, 0.696)

Fiber

Acid Detergent

0.310

34.7 ± 2.1 33.3 ± 2.1

Fiber (ADF) 22.4 - 56.7 22.2 - 45.4 19.4 - 64.1 22.72 - 59.03

(% DW) (0.310, 0.595)

Neutral Detergent

0.300

41.6 ± 2.6 39.9 ± 2.6

Fiber (NDF) 27.8 - 70.9 27.2 - 59.3 25.2 - 82.0 19.61 - 73.05

(% DW) (0.300, 0.592)

Mineral

Calcium

0.333

1378 ± 64 1401 ± 64

940 - 1840 908 - 1740 874 - 2000 NR

(mg/100g dry wt.) (0.333, 0.615)

Phosphorus

0.793

266 ± 7 268 ± 7

206 - 327 201 - 342 187 - 381 NR

(mg/100g dry wt.) (0.793, 0.828)

Abbreviations: NR = Not Reported.

a Unit of measure was not converted prior to analysis, except for calcium and phosphorus. Unit of measure

 for calcium and phosphorus was converted from % DW to mg/100g dry wt. prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR) procedure; 

P-values < 0.05 were considered significant.

d Combined range from Appendix 7.

e % Carbohydrates = 100 % - (% Protein + % Fat + % Ash + % Moisture)
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Figure 33. Proximates, fiber and minerals in control, DAS-81419-2 and reference variety 

soybean forage. 

moisture = % fresh weight, calcium and phosphorous = mg/100g dry wt., all others = % dry weight

Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 33 (Cont). Proximates, fiber and minerals in control, DAS-81419-2 and reference 

variety soybean forage. 

moisture = % fresh weight, calcium and phosphorous = mg/100g dry wt., all others = % dry weight

Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.

7.2.2. Proximate and Fiber Analysis of Seed

Soybean seed samples from the control (Maverick), DAS-81419-2, and reference variety entries 

were analyzed for proximates and fiber (eight analytes). A summary of the results across all 

locations is presented in Table 12 and Figure 34. All mean results were within literature ranges

(when available) and within ranges for reference varieties included in the study. Statistically 

significant overall treatment effects were found for fat, ash, and moisture, and pair-wise 

comparisons between the control and DAS-81419-2 were also significant for those analytes 

based upon unadjusted P-values (but not after adjustment for multiplicity using FDR methods). 

However, these differences were small relative to natural variation and not biologically 

meaningful, as all results were within literature ranges and within the ranges of the reference 

varieties included in this study.
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Table 12. Summary of the proximate and fiber analysis of soybean seed from all sites and 

literature range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Proximate

Protein

0.442

37.9 ± 0.6 38.1 ± 0.6

34.3 - 41.9 32.9 - 42.8 34.4 - 46.0 32 - 48.4

(% DW) (0.442, 0.662)

Fat

0.008

18.2 ± 0.5 17.7 ± 0.5

15.4 - 21.5 14.2 - 21.0 14.1 - 22.7 8.104 - 24.7

(% DW) (0.008, 0.102)

Ash

0.026

5.06 ± 0.07 5.18 ± 0.07

4.62 - 5.68 4.57 - 6.05 3.79 - 6.79 3.885 - 6.994

(% DW) (0.026, 0.233)

Moisture

0.019

12.3 ± 0.9 11.7 ± 0.9

8.29 - 19.2 7.56 - 17.9 7.91 - 22.7 4.7 - 34.4

(% FW) (0.019, 0.196)

Carbohydratese

0.372

38.8 ± 0.7 39.0 ± 0.7

33.6 - 41.2 33.3 - 43.3 29.9 - 40.8 29.3 - 50.2

(% DW) (0.372, 0.643)

Fiber

Acid Detergent

0.813

15.3 ± 0.7 15.2 ± 0.7

Fiber (ADF) 10.6 - 22.8 10.5 - 22.5 10.2 - 21.0 7.81 - 26.26

(% DW) (0.813, 0.837)

Neutral Detergent

0.576

17.5 ± 0.8 17.7 ± 0.8

Fiber (NDF) 11.7 - 24.2 11.6 - 25.5 10.6 - 22.6 8.53 - 23.90

(% DW) (0.576, 0.719)

Total Dietary

0.689

23.8 ± 0.9 24 .0 ± 0.9

Fiber 17.6 - 29.1 17.4 - 31.3 16.1 - 29.5 NR

(% DW) (0.689, 0.765)

Abbreviations: NR = Not Reported.

a Unit of measure was not converted prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.

e % Carbohydrates = 100 % - (% Protein + % Fat + % Ash + % Moisture)
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Figure 34. Proximates and fiber in control, DAS-81419-2, and reference variety soybean 

seed. 

moisture = % fresh weight, all others = % dry weight

Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 34 (Cont). Proximates and fiber in control, DAS-81419-2, and reference variety 

soybean seed. 

moisture = % fresh weight, all others = % dry weight

Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.

7.2.3. Mineral Analysis of Seed

Soybean seed samples from the control (Maverick), DAS-81419-2, and reference variety entries 

were analyzed for mineral content (10 analytes). A summary of the results across all locations is 

presented in Table 13 and Figure 35. For sodium, statistical analysis was not performed because 

more than 50% of the samples were found to be below the LOQ. All mean results were within 

literature ranges (when available) and/or within ranges for reference varieties included in the 

study. Additionally, no statistical differences were observed in the combined-site analysis 

between the control and DAS-81419-2 entries for all mineral analytes.
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Table 13. Summary of the mineral analysis of soybean seed from all sites and literature 

range.

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Calcium

0.137

270 ± 8 267 ± 8

233 - 335 205 - 328 181 - 339 116.55 - 510

(mg/100 g DW) (0.137, 0.456)

Copper

0.655

1.32 ± 0.06 1.33 ± 0.06

0.922 - 1.64 0.894 - 1.72 0.693 - 1.86 0.632 - 1.092

(mg/100 g DW) (0.655, 0.747)

Iron

0.500

9.56 ± 1.40 10.26 ± 1.40

6.61 - 27.3 6.21 - 42.9 6.33 - 151 3.734 - 10.954

(mg/100 g DW) (0.500, 0.696)

Magnesium

0.663

233 ± 4 232 ± 4

204 - 256 197 - 257 205 - 278 219.40 - 312.84

(mg/100 g DW) (0.663, 0.747)

Manganese

0.548

2.64 ± 0.09 2.67 ± 0.09

2.13 - 3.09 2.01 - 3.96 2.22 - 7.18 2.52 - 3.876

(mg/100 g DW) (0.548, 0.719)

Phosphorus

0.118

607 ± 14 619 ± 14

536 - 704 494 - 708 471 - 759 506.74 - 935.24

(mg/100 g DW) (0.118, 0.456)

Potassium

0.168

1799 ± 21 1819 ± 21

1660 - 1940 1490 - 1980 1650 - 2050 1868.01 - 2510

(mg/100 g DW) (0.168, 0.459)

Selenium

0.401

468 ± 188 507 ± 187

<LOQ - 2370 <LOQ - 2560 <LOQ - 3060 NR

(ppb DW) (0.401, 0.647)

Sodium

NA

NA NA

<LOQ <LOQ <LOQ - 22.2 4.05 - 30

(mg/100 g DW)

Zinc

0.066

4.53 ± 0.15 4.63 ± 0.15

3.66 - 5.73 3.66 - 5.83 3.12 - 6.33 4.98 - 7.578

(mg/100 g DW) (0.066, 0.381)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation);

NR = Not Reported

a Unit of measure was not converted prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Figure 35. Minerals in control, DAS-81419-2, and reference variety soybean seed.

selenium = ppb dry weight, all others = mg/100 g dry weight

Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 35 (Cont). Minerals in control, DAS-81419-2, and reference variety soybean seed.

selenium = ppb dry weight, all others = mg/100 g dry weight

Symbols for each location shown: open circle = IA1,  = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.
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7.2.4. Amino Acid Analysis of Seed

Soybean seed samples from the control (Maverick), DAS-81419-2, and reference variety entries 

were analyzed for the content of 18 amino acids. A summary of the results across all locations is 

presented in Table 14 and Figure 36. All mean results were within literature ranges (when 

available) and/or within ranges for reference varieties included in the study. With the exception 

of phenylalanine, no statistical differences were observed in the combined-site analysis between 

the control and DAS-81419-2 entries for amino acid content. The overall treatment effect and 

pair-wise comparison between the control and DAS-81419-2 were significant for phenylalanine 

based on unadjusted P-values (but not after adjustment for multiplicity using FDR methods). 

However, these differences were small relative to natural variation and not biologically 

meaningful, as all results were within literature ranges and within the range of the reference 

varieties included in this study.
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Table 14. Summary of the amino acid analysis of soybean seed from all sites and literature 

range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Alanine

0.212

4.57 ± 0.03 4.59 ± 0.03

4.31 - 4.76 4.30 - 4.82 4.28 - 4.75 4.16 - 4.74

(% total amino acid) (0.212, 0.520)

Arginine

0.111

7.49 ± 0.05 7.44 ± 0.05

7.04 - 7.79 7.05 - 7.74 7.22 - 8.20 6.41 - 8.41

(% total amino acid) (0.111, 0.456)

Aspartic Acid

0.099

11.51 ± 0.01 11.48 ± 0.01

11.29 - 11.64 11.30 - 11.76 9.99 - 11.74 11.37 - 12.68

(% total amino acid) (0.099, 0.440)

Cystine

0.283

1.610 ± 0.028 1.629 ± 0.028

1.432 - 1.946 1.448 - 1.861 1.240 - 1.792 1.02 - 1.87

(% total amino acid) (0.283, 0.591)

Glutamic Acid

0.161

17.32 ± 0.11 17.23 ± 0.11

16.29 - 18.01 16.28 - 17.98 17.04 - 18.56 17.71 - 20.48

(% total amino acid) (0.161, 0.456)

Glycine

0.745

4.50 ± 0.02 4.50 ± 0.02

4.34 - 4.67 4.30 - 4.66 4.14 - 4.54 4.19 - 4.62

(% total amino acid) (0.745, 0.802)

Histidine

0.567

2.714 ± 0.016 2.703 ± 0.016

2.583 - 2.818 2.165 - 2.836 2.434 - 2.776 2.49 - 2.89

(% total amino acid) (0.567, 0.719)

Isoleucine

0.931

4.80 ± 0.02 4.80 ± 0.02

4.65 - 4.96 4.52 - 4.99 4.61 - 4.99 4.13 - 5.11

(% total amino acid) (0.931, 0.931)

Leucine

0.605

7.65 ± 0.01 7.64 ± 0.01

7.51 - 7.83 7.49 - 7.75 7.45 - 7.98 7.46 - 8.29

(% total amino acid) (0.605, 0.728)

a Unit of measure was converted from % DW to % total amino acid prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate 

(FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Table 14 (Cont). Summary of the amino acid analysis of soybean seed from all sites and 

literature range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Lysine

0.151

6.32 ± 0.08 6.44 ± 0.08

5.90 - 7.51 5.92 - 7.41 5.61 - 7.29 6.23 - 7.38

(% total amino acid) (0.151, 0.456)

Methionine

0.211

1.419 ± 0.011 1.435 ± 0.011

1.305 - 1.608 1.325 - 1.607 1.223 - 1.618 1.18 - 1.71

(% total amino acid) (0.211, 0.520)

Phenylalanine

0.009

5.15 ± 0.01 5.11 ± 0.01

4.99 - 5.25 4.92 - 5.22 4.88 - 5.37 4.91 - 5.44

(% total amino acid) (0.009, 0.102)

Proline

0.240

5.14 ± 0.04 5.20 ± 0.04

4.86 - 5.97 4.91 - 5.73 4.80 - 6.02 4.75 - 5.62

(% total amino acid) (0.240, 0.550)

Serine

0.466

5.13 ± 0.02 5.16 ± 0.02

4.74 - 5.37 4.85 - 5.38 4.81 - 5.53 3.25 - 6.04

(% total amino acid) (0.466, 0.674)

Threonine

0.381

4.19 ± 0.03 4.20 ± 0.03

3.99 - 4.38 3.96 - 4.44 3.86 - 4.25 3.15 - 4.24

(% total amino acid) (0.381, 0.643)

Tryptophan

0.650

1.519 ± 0.016 1.524 ± 0.016

1.423 - 1.640 1.401 - 1.653 1.271 - 1.686 0.95 - 1.49

(% total amino acid) (0.650, 0.747)

Tyrosine

0.418

3.97 ± 0.01 3.96 ± 0.01

3.88 - 4.10 3.83 - 4.06 3.82 - 4.16 2.62 - 3.72

(% total amino acid) (0.418, 0.659)

Valine

0.532

4.98 ± 0.02 4.96 ± 0.02

4.74 - 5.22 4.70 - 5.32 4.59 - 5.18 4.28 - 5.57

(% total amino acid) (0.532, 0.712)

a Unit of measure was converted from % DW to % total amino acid prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate 

(FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Figure 36. Amino acids in control, DAS-81419-2, and reference variety soybean seed (% 

total amino acid). 

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 36 (Cont). Amino acids in control, DAS-81419-2, and reference variety soybean seed 

(% total amino acid). 

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 36 (Cont). Amino acids in control, DAS-81419-2, and reference variety soybean seed 

(% total amino acid). 

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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7.2.5. Fatty Acid Analysis of Seed

Soybean seed samples from the control (Maverick), DAS-81419-2, and reference variety entries 

were analyzed for the content of 22 fatty acids. A summary of the results across all locations is 

presented in Table 15 and Figure 37. Statistical analysis was not performed on the following 

fatty acids because greater than 50% of the samples were found to be below the LOQ of 0.02% 

fresh weight: 8:0 caprylic, 10:0 capric, 12:0 lauric, 14:0 myristic, 14:1 myristoleic, 15:0 

pentadecanoic, 15:1 pentadecenoic, 16:1 palmitoleic, 17:0 heptadecanoic, 17:1 heptadecenoic, 

18:3 γ-linolenic, 20:2 eicosadienoic, 20:3 eicosatrienoic, 20:4 arachidonic. The mean values of

the remaining fatty acids were within literature ranges (when available) and/or within ranges for 

reference varieties included in the study. Overall treatment effects and pair-wise comparisons 

between the control and DAS-81419-2 were significant for 16:0 palmitic, 18:3 linolenic, and 

20:1 eicosenoic. However, these differences were small relative to natural variation and not 

biologically meaningful, as all results were within literature ranges and/or within the range of the

reference varieties included in this study.
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Table 15. Summary of the fatty acid analysis of soybean seed from all sites and literature 

range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

8:0 Caprylic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.148

(% total fatty acid)

10:0 Capric

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.27

(% total fatty acid)

12:0 Lauric

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.132

(% total fatty acid)

14:0 Myristic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.238

(% total fatty acid)

14:1 Myristoleic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.125

(% total fatty acid)

15:0 Pentadecanoic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - <LOQ

(% total fatty acid)

15:1 Pentadecenoic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - <LOQ

(% total fatty acid)

16:0 Palmitic

<0.001

11.12 ± 0.08 11.56 ± 0.08

10.04 - 11.59 10.88 - 12.27 9.12 - 11.53 1.40 - 15.77

(% total fatty acid) (<0.001, <0.001)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation);

NR = Not Reported.

a Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Table 15 (Cont). Summary of the fatty acid analysis of soybean seed from all sites and 

literature range.

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

16:1 Palmitoleic

NA

NA NA

<LOQ <LOQ - 0.236 <LOQ <LOQ - 0.194

(% total fatty acid)

17:0 Heptadecanoic

NA

NA NA

<LOQ - 0.128 <LOQ - 0.132 <LOQ - 0.133 <LOQ - 0.146

(% total fatty acid)

17:1 Heptadecenoic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.087

(% total fatty acid)

18:0 Stearic

0.18

4.40 ± 0.12 4.46 ± 0.12

3.67 - 5.27 3.62 - 5.22 3.19 - 5.07 0.50 - 5.88

(% total fatty acid) (0.180, 0.474)

18:1 Oleic

0.08

21.6 ± 0.4 21.2 ± 0.4

19.6 - 25.2 19.8 - 25.5 18.8 - 24.6 2.60 - 45.68

(% total fatty acid) (0.080, 0.404)

18:2 Linoleic

0.124

54.1 ± 0.4 53.8 ± 0.4

51.2 - 55.8 50.4 - 55.7 53.6 - 57.5 7.58 - 58.8

(% total fatty acid) (0.124, 0.456)

18:3 Linolenic

0.04

7.97 ± 0.15 8.17 ± 0.15

6.91 - 8.76 7.32 - 8.94 6.58 - 9.88 1.27 - 12.52

(% total fatty acid) (0.040, 0.314)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation);

a Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Table 15 (Cont). Summary of the fatty acid analysis of soybean seed from all sites and 

literature range.

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

18:3 γ-Linolenic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - <LOQ

(% total fatty acid)

20:0 Arachidic

0.068

0.319 ± 0.007 0.325 ± 0.007

0.276 - 0.370 0.282 - 0.372 0.254 - 0.383 0.038 - 0.57

(% total fatty acid) (0.068, 0.381)

20:1 Eicosenoic

0.048

0.157 ± 0.004 0.153 ± 0.004

<LOQ - 0.189 <LOQ - 0.171 <LOQ - 0.196 0.024 - 0.35

(% total fatty acid) (0.048, 0.339)

20:2 Eicosadienoic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - 0.245

(% total fatty acid)

20:3 Eicosatrienoic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - <LOQ

(% total fatty acid)

20:4 Arachidonic

NA

NA NA

<LOQ <LOQ <LOQ <LOQ - <LOQ

(% total fatty acid)

22:0 Behenic

0.159

0.317 ± 0.003 0.321 ± 0.003

0.283 - 0.347 0.263 - 0.345 0.277 - 0.390 0.043 - 0.65

(% total fatty acid) (0.159, 0.456)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation);

NR = Not Reported.

a Unit of measure was converted from % dry wt. to % total fatty acid prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Figure 37. Fatty acids in control, DAS-81419-2, and reference variety soybean seed (% total 

fatty acid).

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 37 (Cont). Fatty acids in control, DAS-81419-2, and reference variety soybean seed 

(% total fatty acid).

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.

7.2.6. Vitamin Analysis of Seed

Soybean seed samples from the control (Maverick), DAS-81419-2, and reference variety entries 

were analyzed for the content of 13 vitamins. A summary of the results across all locations is 

presented in Table 16 and Figure 38. Statistical analysis was not performed on Vitamin A 

because greater than 50% of the samples were found to be below the LOQ. All mean results 

were within literature ranges (when available) and/or within ranges for reference varieties

included in the study. With the exception of γ-tocopherol and Vitamin B5, no statistical 

differences were observed in the combined-site analysis between the control and DAS-81419-2 

entries for vitamin content. The overall treatment effect and pair-wise comparison between the 

control and DAS-81419-2 were significant for γ-tocopherol and Vitamin B5 based on unadjusted 

P-values (but not after adjustment for multiplicity using FDR methods). However, these 

differences were small relative to natural variation and not biologically meaningful, as all results 

were within the range of the reference varieties included in this study.
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Table 16. Summary of the vitamin analysis of soybean seed from all sites and literature 

range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Vitamin A

NA

NA NA

(β-Carotene) <LOQ <LOQ <LOQ - 0.244 NR

(mg/kg DW)

Vitamin B1

0.447

3.51 ± 0.24 3.43 ± 0.24

(Thiamine) 2.35 - 5.44 2.20 - 5.16 1.82 - 4.92 1.01 - 2.54

(mg/kg DW) (0.447, 0.662)

Vitamin B2

0.146

3.40 ± 0.08 3.51 ± 0.08

(Riboflavin) 2.63 - 4.65 2.58 - 4.64 2.42 - 5.00 1.90 - 3.21

(mg/kg DW) (0.146, 0.456)

Vitamin B3

0.07

25.0 ± 0.7 25.6 ± 0.7

(Niacin) 20.2 - 30.5 20.3 - 32.1 20.5 - 29.0 NR

(mg/kg DW) (0.070, 0.381)

Vitamin B5

0.004

14.8 ± 0.5 14 ± 0.5

(Pantothenic Acid) 12.3 - 19.5 11.8 - 16.8 8.97 - 18.0 NR

(mg/kg DW) (0.004, 0.074)

Vitamin B6

0.721

5.23 ± 0.11 5.18 ± 0.11

(Pyridoxine) 4.42 - 6.43 4.53 - 6 3.01 - 6.36 NR

(mg/kg DW) (0.721, 0.787)

Vitamin B9

0.599

4.21 ± 0.20 4.15 ± 0.20

(Folic Acid) 3.05 - 5.62 2.75 - 5.51 2.94 - 5.59 2.386 - 4.709

(mg/kg DW) (0.599, 0.728)

Vitamin C

0.135

141 ± 13 133 ± 13

(Ascorbic Acid) 75.2 - 231 74 - 230 49.2 - 210 NR

(mg/kg DW) (0.135, 0.456)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation);

NR = Not Reported.

a Unit of measure was not converted prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Table 16 (Cont). Summary of the vitamin analysis of soybean seed from all sites and 

literature range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

α-Tocopherol

0.267

14.3 ± 1.3 13.6 ± 1.3

8.56 - 29.3 9.62 - 26.6 6.51 - 25.0 1.934 - 84.9

(mg/kg DW) (0.267, 0.589)

β-Tocopherol

NA

NA NA

<LOQ <LOQ <LOQ NR

(mg/kg DW)

γ-Tocopherol

0.004

69.4 ± 2.8 74.7 ± 2.8

41.3 - 83.1 45.4 - 90.8 49.7 - 104.0 NR

(mg/kg DW) (0.004, 0.074)

δ-Tocopherol

0.338

168 ± 8 172 ± 8

97 - 220 118 - 219 77.5 - 240 NR

(mg/kg DW) (0.338, 0.615)

Total Tocopherole

0.096

237.5 ± 6.9 247.1 ± 6.9

159.5 - 280.5 194.7 - 282.3 150.5 - 299.9 NR

(mg/kg DW) (0.096, 0.440)

Abbreviations: NA (Not Available) = analysis not performed, majority of data was < LOQ (Limit of Quantitation);

NR = Not Reported.

a Unit of measure was not converted prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.

eTotal Tocopherol = α-Tocopherol + β-Tocopherol + γ-Tocopherol + δ-Tocopherol 
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Figure 38. Vitamins in control, DAS-81419-2, and reference variety soybean seed (mg/kg 

dry weight).

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.
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Figure 38 (Cont). Vitamins in control, DAS-81419-2, and reference variety soybean seed 

(mg/kg dry weight).

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range (when available) is shaded for each analyte.
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7.2.7. Bioactive Analysis of Seed

Soybean seed samples from the control (Maverick), DAS-81419-2, and reference variety entries 

were analyzed for the content of eight bioactives. A summary of the results across all locations 

is presented in Table 17 and Figure 39. All mean results were within literature ranges and/or

within ranges for reference varieties included in this study. With the exception of total glycitein 

equivalent, no statistical differences were observed in the combined-site analysis between the 

control and DAS-81419-2 entries for bioactive content. The overall treatment effect and pairwise comparison between the control and DAS-81419-2 were significant for total glycitein 

equivalent based on unadjusted P-values (but not after adjustment for multiplicity using FDR 

methods). However, these differences were small relative to natural variation and not 

biologically meaningful, as all results were within the literature range and the range of the 

reference varieties included in this study.
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Table 17. Summary of the bioactive analysis of soybean seed from all sites and literature 

range. 

Analytical 

Component 

(Units)a

Overall

Treatment

Effect

(Pr > F)b

Control

(Maverick) DAS-81419-2

Reference Variety

Range

Combined 

Literature Ranged

Mean ± SE Mean ± SE

Min - Max Min - Max Min - Max Min - Max

(P-value, Adj.P)c

Lectin

0.401

30.8 ± 2.1 32.2 ± 2.1

(H.U./mg protein 13.9 - 50.1 12.4 - 52.6 7.89 - 45.2 37 - 323

DW) (0.401, 0.647)

Phytic Acid

0.447

1.22 ± 0.06 1.24 ± 0.06

0.857 - 2.02 0.911 - 1.52 0.678 - 1.71 0.41 - 2.68

(% DW) (0.447, 0.662)

Raffinose

0.475

0.750 ± 0.038 0.766 ± 0.038

0.505 - 1.02 0.475 - 0.977 0.570 - 1.16 0.212 - 1.85

(% DW) (0.475, 0.674)

Stachyose

0.648

3.68 ± 0.08 3.69 ± 0.08

3.19 - 4.14 3.15 - 4.29 3.01 - 5.28 1.21 - 6.65

(% DW) (0.648, 0.747)

Trypsin Inhibitor

0.226

29.1 ± 1.2 30.2 ± 1.2

22.2 - 46.5 21.3 - 49.9 19.5 - 53.8 18.14 - 118.68

(TIU/mg) (0.226, 0.536)

Total Daidzein

0.299

950 ± 48 932 ± 48

Equivalent 462 - 1200 504 - 1190 585 - 1460 25 - 2453.5

(mcg/g DW) (0.299, 0.592)

Total Genistein

0.362

1296 ± 63 1276 ± 63

Equivalent 808 - 1680 922 - 1620 753 - 1950 28 - 2837.2

(mcg/g DW) (0.362, 0.643)

Total Glycitein

0.002

197 ± 6 180 ± 6

Equivalent 156 - 266 140 - 237 40.3 - 259 15.3 - 349.19

(mcg/g DW) (0.002, 0.074)

a Unit of measure was not converted prior to analysis.

b Overall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

d Combined range from Appendix 7.
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Figure 39. Bioactives in control, DAS-81419-2, and reference variety soybean seed.

Lectin = H.U./mg protein dry weight, Trypsin inhibitor = TIU/mg, total daidzein equivalent, total genistein equivalent, and total glycitein 

equivalent = mcg/gram dry weight, all others = % dry weight

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.
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Figure 39 (Cont). Bioactives in control, DAS-81419-2, and reference variety soybean seed.

Lectin = H.U./mg protein dry weight, Trypsin inhibitor = TIU/mg, total daidzein equivalent, total genistein equivalent, and total glycitein 

equivalent = mcg/gram dry weight, all others = % dry weight

Symbols for each location shown: open circle = IA1, × = IA2, + = IL1, open triangle = IL2, open square = IN, open diamond = MO1, filled circle 

= MO2, filled triangle = NE1, filled square = NE2, filled diamond = PA. Literature range is shaded for each analyte.

7.3. Summary of Composition Analysis

All overall mean values for the control (Maverick) and DAS-81419-2 were within literature 

ranges (when available) for soybean and/or within ranges for non-transgenic soybean reference

varieties included in the study. A limited number of statistically significant differences were 

observed between DAS-81419-2 and the control; however, these differences were not 

biologically meaningful because the differences were small relative to natural variation and the 

results were within ranges found for non-transgenic soybean. In conclusion, forage and grain 

derived from DAS-81419-2 soybean are compositionally equivalent to those of non-transgenic 

soybean.
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8. Agronomic Characteristics

Field trials with DAS-81419-2 soybean, a non-transgenic control (Maverick), and reference 

varieties were conducted in 2011 at ten sites located in Iowa (2 sites), Illinois (2 sites), Indiana, 

Missouri (2 sites), Nebraska (2 sites), and Pennsylvania. This study used the same plots that 

were used for protein expression (Section 6) and nutrient composition studies (Section 7). No

biologically relevant differences, other than resistance to target insect pests, were identified

between DAS-81419-2 soybean and non-transgenic soybean. Agronomic characteristics of 

DAS-81419-2 were statistically indistinguishable from the non-transgenic control Maverick or 

were within the reference variety ranges for non-transgenic soybean. Ecological evaluations 

indicated that there were no statistically significant differences between DAS-81419-2 soybean 

and the non-transgenic control Maverick in susceptibility to and interactions with diseases and 

non-target insects. The germination and dormancy evaluation indicated that seed dormancy 

characteristics have not been altered in DAS-81419-2 soybean. Taken together, results from the 

field and laboratory studies demonstrate agronomic equivalence between DAS-81419-2 soybean 

and non-transgenic soybean.

8.1. Agronomic Study

An agronomic study with DAS-81419-2 soybean, a non-transgenic near-isogenic control 

(Maverick) and six non-transgenic reference lines (IL 3503, Porter 75148, DSR 75213-72, 

Pioneer 93M62, HiSOY 38C60, Williams 82) was conducted in 2011 at ten sites located in

Richland, Iowa; Atlantic, Iowa; Carlyle, Illinois; Wyoming, Illinois; Frankfort, Indiana; Fisk, 

Missouri; La Plata, Missouri; York, Nebraska; Brunswick, Nebraska; and Germansville, 

Pennsylvania. 

Each trial site included event DAS-81419-2, the non-transgenic near-isogenic control

(Maverick), and three non-transgenic reference lines. At each of the ten sites, all entries were 

arranged in a randomized complete block design with four blocks. Across all sites, each control

(Maverick) and DAS-81419-2 was represented by a total of 40 plots (10 sites, 4 replicate plots 

per entry at each site). Three of the six reference lines were included at each site by randomizing 

across sites in a balanced incomplete-block design. Each of the six reference lines was assigned 

to five sites; therefore, each reference line was represented by a total of 20 plots across sites (5 

sites per reference line, 4 replicate plots per entry at each site). 

At each site, four replicate plots of each entry were established, with each plot consisting of four 

25 ft (7.62 m) rows. Observations of agronomic characteristics were conducted on the center 

two rows of each plot; row number one and four were included as additional border rows. 

Soybean seeds were planted at a seeding rate of approximately 125 seeds per row with seed 

spacing within each row of approximately 2.4 inches (6 cm). Each soybean plot was bordered by 

two rows of a non-transgenic soybean cultivar of similar maturity. The entire trial site was 

surrounded by a minimum of four rows (10 ft or 3.0 m) of a non-transgenic soybean cultivar of 

similar maturity. Appropriate insect, weed, and disease control practices were applied to 

produce an agronomically acceptable crop.

Agronomic Data Collection

The following agronomic characteristics were measured and recorded for all test entries at each 

location on a per plot basis (Table 18).
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Table 18. Agronomic characteristics.

Trait Evaluation 

Timing

Description Scale

Early Population 

(Stand Count)

V2 Number of plants in a 

representative 1 meter section 

of one row per plot

Number of emerged plants in 1 meter

Seedling Vigor V2 Visual estimate of average 

vigor of plants in each plot

0-100% rating scale (e.g., 0 = all plants are 

short with small, thin leaves; 100 = all plants 

are tall with robust leaves). Not based on 

growth of the control entries; Did not consider 

germination/ emergence (stand count)

Days to 50% 

Flowering

R1/R2 Date at which 50% of plants 

are flowering

Recorded the date when 50% of the plants in 

each plot were flowering. Days since planting 

calculated. 

Disease Incidence R6 Visual estimate of disease 

incidence

0-100%; Estimated % plant tissue/leaf area 

diseased over all plants in plot; did not record % 

of plants in plot that had detectable disease; 

100% = all plant tissues in plot were diseased; 

recorded type of disease

Insect Damage R6 Visual estimate of insect 

damage

0-100%; Estimated % plant tissue/leaf area 

damaged over all plants in plot; Did not record 

% of plants in plot that had detectable damage; 

100% = all plant tissues had feeding damage; 

Recorded type of damage, e.g. chewing, 

stippling, distortion; Recorded type of insect(s) 

if present 

Days to Maturity R8 Date at which 95% of plants 

had reached physiological 

maturity/dry down color

Recorded the date when 95% of the plants in 

each plot reached physiological maturity/dry 

down color; Days since planting calculated

Lodging R8 Visual estimate of incidence 

of lodging severity

0-100%; Estimated % of plants lodged in plot; 

100% = all plants in plot were lodged

Plant Height R8 Average plant height: from 

soil surface to growing tip (at 

senescence / after leaf shed)

Recorded the average height of all plants in plot 

(stand) in centimeters (cm); One value for each 

plot; If plot was lodged, a representative group 

of plants was held up to obtain a measurement

Final Population 

(Stand Count)

R8 Number of plants in a 

representative 1 meter section 

of one row per plot 

Number of plants in 1 meter; Did not sample a 

section where plants were removed during 

previous sampling

Plant 

Morphology(number 

of seeds and pods)

R8 (prior to 

harvest)

Number of pods and seeds 

from 5 plants collected from 

each plot

Recorded the number of pods and seeds present 

on 5 representative plants collected from each 

plot
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Trait Evaluation 

Timing

Description Scale

Shattering R8 (prior to 

harvest)

Visual estimate of pod 

shattering 

0-100%; Estimated % of shattered pods for each 

plot; 100% = all pods shattered

Yield R8 Weight of grain harvested 

from each plot

Recorded the weight in grams of grain 

harvested from each plot

100 seed weight R8 Weight of 100 representative 

seeds from bulk yield sample

in grams

Recorded the weight in grams for 100 

representative seeds taken from the bulk yield 

sample

Analysis of variance was conducted across field sites (combined-site analysis) for agronomic 

data using a mixed model (SAS Institute Inc., 2009). Entry was considered a fixed effect and 

location, block within location, and location-by-entry were designated as random effects. 

Significant differences were declared at the 95% confidence level. The significance of an overall 

treatment effect was estimated using an F-test. Comparisons were made between DAS-81419-2 

soybean and the control (Maverick) using t-tests.

Due to the large number of comparisons made in this study, multiplicity was an issue. 

Multiplicity is an issue when a large number of comparisons are made in a single study to look 

for unexpected effects. Under these conditions, the probability of falsely declaring differences 

based on comparison-wise P-values is very high (1-0.95number of comparisons). In this study, there 

was one comparison per endpoint (14 analyzed observation types for agronomics), resulting in 

14 comparisons made in the combined-site agronomic analysis. Therefore, the probability of 

declaring one or more false differences based on unadjusted P-values was >51% for agronomics 

(1-0.9514).

One method to account for multiplicity is to adjust P-values to control the experiment-wise error 

rate, but when many comparisons are made in a study, the power for detecting specific effects

can be reduced significantly. An alternative with much greater power is to adjust P-values to 

control the probability that each declared difference is significant. This can be accomplished 

using False Discovery Rate (FDR) control procedures (Benjamini and Hochberg, 1995); FDR

methods are commonly applied in studies examining transgenic crops (Herman et al., 2007; Coll

et al., 2008; Huls et al., 2008; Jacobs et al., 2008; Stein et al., 2009; Herman et al., 2010). 

Therefore, the P-values from the agronomics evaluations were each adjusted using the FDR 

method to improve discrimination of true differences among treatments from random effects 

(false positives). Differences were considered significant if the FDR-adjusted P-value was less 

than 0.05.

Agronomic Results

A statistical analysis of the agronomic data collected from the control (Maverick) and 

DAS-81419-2 entries was conducted. For each agronomic characteristic and entry, the least 

squares mean, standard error, and minimum and maximum sample values are reported. 
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Additionally, the minimum and maximum values from reference varieties across all sites 

(reference variety ranges) for each agronomic characteristic are provided for comparison. Each 

minimum and maximum value is an individual data point reported for a single test plot.

For the combined-site analysis (Table 19), no statistically significant differences were observed 

following False Discovery Rate (FDR) adjustment of P-values for all of the agronomic 

characteristics evaluated: early population, seedling vigor, days to flowering, disease incidence, 

insect damage, days to maturity, lodging, plant height, final population, number of pods per five 

plants, number of seeds per five plants (plant morphology), shattering, yield, and 100 seed 

weight. Unadjusted P-values were significant at the 0.05 level for paired t-tests for days to 

flowering and number of pods per five plants (plant morphology). For each significant 

unadjusted P-value, differences in mean values between DAS-81419-2 and control (Maverick) 

entries were negligible and the mean values of DAS-81419-2 were within the range observed for 

reference varieties included in the study. Results from this study demonstrate agronomic 

equivalence between event DAS-81419-2 and non-transgenic soybean.
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Table 19. Summary of agronomic characteristics for the combined site analysis.

Overall 

Control

(Maverick) DAS-81419-2 Reference Variety 

Agronomic 

Component 

(Units)a

Treatment Mean ± SE Mean ± SE Range

Effect Min - Max Min - Max Min - Max

(Pr > F)b(P-value, Adj.P)c

Early Population - V2

0.267

17 ± 0 16 ± 0

(number of plants in a 13 - 21 11 - 21 7 - 22

1 m section of row) (0.267, 0.386)

Seedling Vigor - V2

0.074

86 ± 5 82 ± 5

(0-100% scale, 0% = all plants are 60 - 100 50 - 100 60 - 100

short with small thin leaves; 100% = 

all plants are tall with robust leaves) (0.074, 0.137)

Days to Flowering - R1/R2

0.008

42 ± 2 43 ± 2

(days since planting) 32 - 58 33 - 58 32 - 58

(0.008, 0.053)

Disease Incidence - R6

0.343

4 ± 3 5 ± 3

(0-100% scale, 0% = no disease 0 - 30 0 - 30 0 - 50

100% = all plants diseased) (0.343, 0.446)

Insect Damage - R6

0.504

7 ± 3 6 ± 3

(0-100% scale, 0% = no damage 0 - 30 0 - 30 0 - 30

100% = all plants damaged) (0.504, 0.596)

Days to Maturity - R8

0.957

114 ± 3 114 ± 3

(days since planting) 91 - 130 93 - 133 96 - 133

(0.957, 0.957)

Lodging - R8

0.053

19 ± 6 7 ± 6

(0-100% scale, 0% = no lodging 0 - 90 0 - 30 0 - 35

100% = all plants lodged) (0.053, 0.137)

Plant Height - R8

0.066

108 ± 4 103 ± 4

(Representative plant height in cm) 80 - 135 71 - 126 63 - 129

(0.066, 0.137)

Final Population - R8

0.742

14 ± 1 14 ± 1

(number of plants in a 7 - 22 8 - 22 7 - 17

1 m section of row) (0.742, 0.804)

Number of Pods - R8

0.006

331 ± 10 304 ± 10

(number of pods on 5 plants) 203 - 476 172 - 408 153 - 808

(0.006, 0.053)

Number of Seeds - R8

0.059

783 ± 38 728 ± 38

(number of seeds from 5 plants) 491 - 1154 318 - 1031 313 - 1244

(0.059, 0.137)

a Unit of measure was not converted prior to analysis.

bOverall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.
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Table 19 (Cont). Summary of agronomic characteristics for the combined site analysis.

Overall 

Control

(Maverick) DAS-81419-2 Reference Variety 

Agronomic 

Component 

(Units)a

Treatment Mean ± SE Mean ± SE Range

Effect Min - Max Min - Max Min - Max

(Pr > F)b(P-value, Adj.P)c

Shattering - R8

NA

NA NA

(0-100% scale, 0% = no shattering 0 - 0 0 - 0 0 - 0

100% = all pods shattered)

Yield - R8

0.092

37 ± 4 35 ± 4

(bushels per acre) 5 - 55 6 - 54 7 - 62

(0.092, 0.149)

100 Seed Weight - R8

0.051

14 ± 1 13 ± 1

(grams) 9.76 - 17.61 10.83 - 16.65 11.69 - 25.82

(0.051, 0.137)

Abbreviations: NA (Not Available) = analysis not performed, insufficent variation in response variable to

enable ANOVA.

a Unit of measure was not converted prior to analysis except for yield. Unit of measure for yield was converted

 from grams per plot to bushels per acre prior to analysis using the formula:

 (X g/125 ft2) × (43560 ft2/A) × (1 kg/1000 g) × (1 bu./27.2155 kg) , where X is the individual sample value.

bOverall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR)

 procedure; P-values < 0.05 were considered significant.

Conclusions

Agronomic characteristics of DAS-81419-2 soybean were evaluated in field trials in 2011. 

DAS-81419-2 agronomic characteristics were statistically indistinguishable from the control or 

were within the reference variety ranges for non-transgenic soybean. No unintended agronomic 

effects were observed for DAS-81419-2 soybean. Results from this study demonstrate 

agronomic equivalence between event DAS-81419-2 and non-transgenic soybean.
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8.2. Ecological Evaluations

The DAS-81419-2 soybean field trials were monitored and observed by personnel familiar with 

soybean cultivation practices (breeders, field station managers, field agronomists, field 

associates). The personnel conducting the field tests visually monitored the incidence of plant 

disease and pests on DAS-81419-2 soybeans compared with the conventional soybean varieties, 

including Maverick and six reference lines, in the same trials. Disease and insect damage was 

rated on a numerical scale of 0-100%, with 0% representing no damage due to disease incidence 

or insect pests. The insect and disease pressure observed in these trials was typical of the 

growing locations. Table 20 summarizes the insects and diseases that caused the insect damage 

and disease incidence at each field site. In all cases, the insects and diseases observed were found 

on both test and control plots. Table 21 summarizes the results obtained from the field trials 

conducted in 2011 at ten sites. There were no statistically significant differences between 

DAS-81419-2 soybean and the non-transgenic control Maverick in susceptibility to and 

interactions with diseases and insects. It should be noted that efficacy evaluation of 

DAS-81419-2 soybean was not the objective of this study (refer to Section 9.1.1 for efficacy 

evaluation of DAS-81419-2 soybean). All plots were uniformly treated with pest control 

measures at each location based on pressure in the non-transgenic plots such that low incidence 

of these pests was expected. This design mimics commercial practice and allows comparison to 

typical cultivation conditions for non-transgenic soybean. The results do not indicate an 

ecological impact of DAS-81419-2 soybean on non-target insects or disease.

Table 20. Insect and disease present by site.

Site Insects Present Diseases Present

110000IA1 Not Recordeda None Present

110000IA2 Grasshoppers Bacterial Pustule

110000IL1 Green Cloverworms, Bean Leaf Beetles, 

Grasshoppers

None Present

110000IL2 Not Recordeda Not Recordeda

110000IN Bean Leaf Beetles None Present

110000MO1 None Present Not Recordeda

110000MO2 None Present None Present

110000NE1 Wooly Bear Caterpillars Not Recordeda

110000NE2 Caterpillars Mold, Mildew, Blight

110000PA Loopers, Skippers None Present

a

Insects or Diseases were not recorded due to their presence on less than 30% of the site.
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Table 21. Disease and insect incidence. 

Overall Control DAS-81419-2 Reference Variety 

Agronomic 

Component 

(Units)a

Treatment Mean ± SE Mean ± SE Range

Effect Min - Max Min - Max Min - Max

(Pr > F)b(P-value, Adj.P)c

Disease Incidence* - R6

0.343

4 ± 3 5 ± 3

0 - 30 0 - 30 0 - 50

(0.343, 0.446)

Insect Damage** - R6

0.504

7 ± 3 6 ± 3

0 - 30 0 - 30 0 - 30

(0.504, 0.596)

a Unit of measure was not converted prior to analysis.

bOverall treatment effect estimated using an F-test. 

c

P-value = Comparison to the control using t-tests; Adj. P = P-values adjusted using False Discovery Rate (FDR) procedure; P-values 

< 0.05 were considered significant.

*Disease incidence 0 – 100% scale. 0% = no disease; 100% = all plants diseased.

**Insect damage 0 – 100% scale. 0% = no damage; 100% = all plants damaged.
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8.3. Germination and Dormancy Evaluations

The germination of DAS-81419-2 soybean seed compared with the non-transgenic control 

(Maverick) under warm and cold conditions was evaluated to determine any impact on seed 

dormancy characteristics. The cold and warm germination tests consisted of four replications of 

each entry with 100 seeds per replication in a completely randomized design.

Cold Germination Methods: The seeds were covered with approximately 1/8 inch of a soil 

mixture and placed in an incubator set at 10°C for 7 days; the temperature was then raised to 

25°C for an additional 7 days. After the seven-day incubation period at 25°C, the number of 

seedlings germinated was determined in accordance with the AOSA Seedling Evaluation 

guidelines (AOSA, 2010b) and documented. This method is adapted from the AOSA Seed 

Vigor Testing Handbook (AOSA, 2009).

Warm Germination Methods: Twenty-five soybean seeds per petri dish were saturated with 

water and positioned with four dishes placed closely together in a square to make one replication. 

The dishes were placed in an incubator set at 25°C and on day five, the number of seeds 

germinated in each replication was recorded. On day 8, all remaining seeds were scored as 

germinated (normal seedlings), abnormal, or hard seeds. This method is adapted from the AOSA 

Rules for Testing Seeds (AOSA, 2010a). Seedlings were evaluated in accordance with the 

AOSA Seedling Evaluation Guidelines (AOSA, 2010b).

Data were transformed using the arcsine of the square root of the decimal fraction of seeds 

germinated per replicate and then subjected to analysis of variance based on a completely 

randomized design (SAS Institute Inc., 2009).

Germination data and P-values for the significance of the effect of entry on germination are 

provided in Table 22. Germination of DAS-81419-2 soybean did not differ significantly (α = 

0.05) from that of the non-transgenic, near-isogenic control Maverick under cold (P = 0.5372) 

and warm (P = 0.6129) conditions. The results indicate that seed dormancy characteristics have 

not been changed in DAS-81419-2 soybean.
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Table 22. Germination percentage for DAS-81419-2 and non-transgenic control soybean.

Soybean Germination (%)

Temperature Entry Rep 1 Rep 2 Rep 3 Rep 4 Mean (±SE) P

Cold DAS-81419-2 88 93 90 91 90.5 (±1.0)

0.5372

Cold Control 90 91 90 88 89.8 (±0.6)

Warm DAS-81419-2 99 98*100 99 99.0 (±0.4)

0.6129

Warm Control 98*100 100 99 99.3 (±0.5)

*One hard seed was found in these replicates.

8.4. Summary of Agronomic Characteristics

The agronomic study evaluating plant growth characteristics throughout the growing season 

demonstrated the equivalence of DAS-81419-2 soybean with conventional non-transgenic 

soybean. Agronomic and phenotypic characteristics, including response to ecological stressors 

as indicated by susceptibility to disease and insect pressure, were not different between 

DAS-81419-2 soybean and conventional soybean grown across diverse environments. In 

addition, no differences were detected in germination and dormancy of seed from DAS-81419-2 

compared with control soybean. The data support the conclusion that agronomic, disease, and 

pest characteristics of DAS-81419-2 soybean are not significantly different from that of 

conventional soybean, and there is no indication that DAS-81419-2 soybean will pose an 

increased plant pest risk.
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9. Impact on Environment and Agronomic Practices

This section describes the potential impact of DAS-81419-2 soybean on non-target organisms 

including threatened and endangered species and an assessment on the impact of introducing 

DAS-81419-2 soybean on the current agronomic practices for soybean in the U.S. and insect 

resistance management. This section also provides an evaluation of weediness potential and 

gene flow.

9.1. Potential Impact on Environment

9.1.1. Efficacy against Target Pests

The efficacy of DAS-81419-2 soybean against lepidopteran pests was evaluated under field 

conditions and through laboratory bioassay of field collected foliage at the Dow AgroSciences 

Southern U.S. Research Center located near Greenville, Mississippi. DAS-81419-2 soybean was

compared directly with the non-transgenic soybean control Maverick. T4 seed (four generations 

post-transformation) bearing DAS-81419-2 were planted for field evaluation. Bioassay of 

excised leaves with soybean looper (Chrysodeixis includens, formerly Pseudoplusia includens),

velvetbean caterpillar (Anticarsia gemmatalis), fall armyworm (Spodoptera frugiperda), and

tobacco budword (Heliothis virescens) was used to measure the efficacy. In addition, the 

number of natural C. includens larvae and associated levels of leaf damage were observed in the 

DAS-81419-2 and ‘Maverick’ plots. Excellent efficacy against soybean looper, velvetbean 

caterpillar, fall armyworm, and tobacco budworm was demonstrated.

Trial Design: Plots were planted in randomized complete block with 4 replicates of each entry 

replicated one time per block. Each replicate plot consisted of 4 rows, 20 ft (6.1 m) in length, 

spaced 40 inches (1.02 m) apart. There were 160 seeds planted per row, spaced 1.5 inches (3.8

cm) apart within the row. Two additional rows of ‘Maverick’ were planted between the 

experimental plots and four rows of ‘Maverick’ were planted adjacent to the outermost 

experimental plots to attract natural insect pest populations.

Dates of Efficacy Evaluations: Leaves for the first bioassay were collected 15 July 2011, when 

the soybean plants were in the R2 growth stage. Leaves for the second bioassay were collected 

from the same plants on 12 August 2011, when the soybean plants were in the R5 growth stage. 

Field populations of C. includens were sampled on 8, 15, 22, and 29 August 2011.

Bioassay of Excised Leaves at Growth Stage R2: Fully-expanded, mainstem trifoliate leaves, 

showing no signs of damage or discoloration and located four nodes below the meristem, were 

excised for the feeding bioassay. A single trifoliate leaf was excised from each of 15 plants per 

replicate; 4 trifoliates per replicate were used for the bioassay of C. includens (formerly P. 

includens), 4 trifoliates per replicate were used for the bioassay of S. frugiperda, 4 trifoliates per 

replicate were used for the bioassay of H. virescens, and 3 trifoliates per replicate were used for 

the bioassay of A. gemmatalis.

The two side leaflets of each trifoliate were excised and placed in separate, labeled petri plates 

containing a thin layer of agar. Two second instars of C. includens, A. gemmatalis, S. frugiperda

or H. virescens were placed on each leaflet. For C. includens, S. frugiperda, and H. virescens, 16 

larvae per replicate (64 larvae in total) were exposed to leaf tissue from event DAS-81419-2 and 

‘Maverick’. For A. gemmatalis, 12 larvae per replicate (48 larvae in total) were exposed to leaf 
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tissue from event DAS-81419-2 and ‘Maverick’. All insect larvae were provided by the Dow 

AgroSciences Insectary in Indianapolis, Indiana. The petri plates holding the infested leaflets 

were held at 25 C and 40% RH. After 2 days, larvae were determined to be dead (no movement 

when stimulated with a sharp probe), moribund (larva responds to stimulus but unable to right 

itself if placed on side), stunted (smaller in size than larvae held on ‘Maverick’ leaves), or alive 

(normal in size and response to stimulus). Also, the percentage of each leaflet consumed was 

estimated visually. If no feeding damage was evident, 0% consumption was recorded. If only 

“pinholes” in the leaf tissue were evident, 0.5% consumption was recorded. For greater amounts 

of consumption, a photographic guide was used to aid in estimating the percentage consumed

(Rice, 2002). Larval condition and leaf consumption were observed again 4 days after larvae 

had been placed on leaflets.

Bioassay of Excised Leaves at Growth Stage R5: The feeding bioassay procedure at R5 was the 

same as the procedure used at R2 with the exception that for S. frugiperda, and H. virescens, all 

three leaflets were excised from each trifoliate and a single, second instar was placed on each 

leaflet. This resulted in 12 larvae per replicate (48 larvae in total) of S. frugiperda and H. 

virescens being exposed to leaflets from DAS-81419-2 and ‘Maverick’.

Field Sampling of Natural Chrysodeixis includens Infestations: Insect counts were initiated when 

the natural C. includens (formerly P. includens) population began to cause damage in ‘Maverick’ 

plots, and continued weekly while C. includens larvae were present. The center two rows of 

each plot were sampled. A 3 ft × 3 ft (91 cm × 91 cm) white cloth was placed at a randomly 

selected location between the center two rows. The plants in the section of row adjacent to one 

edge of the sheet were bent over the cloth and shaken 15 times to dislodge any insects present. 

This process was repeated for the row on the opposite edge of the cloth. Larvae were counted by 

species and size: larvae < 0.25 in (< 6 mm) in length were counted as small larvae and larvae ≥

0.25 in (≥ 6 mm) in length were counted as large larvae. All insects were removed from the 

cloth before taking the next measurement. The cloth was moved to a second randomly selected 

location between the two center rows and the sampling process was repeated, resulting in two 

subsamples per plot at each sampling date. On the last sample date, percent defoliation for 20 

plants per replicate was estimated visually on the trifoliate at the fifth mainstem node below the 

meristem with the aid of a photographic guide (Rice, 2002).

Data Analysis: Given the binomial nature of the insect mortality data (alive and healthy vs. dead, 

moribund or stunted), the data were arranged as a 2×2 contingency table (as shown below) and 

subjected to Pearson’s chi-square test. The calculated chi-square values and significance levels 

(P values) of these chi-square values are presented in Table 23.
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DAS-81419-2 ‘Maverick’

Total number of live, 

healthy larvae a b Row 1 total

Total number of dead, 

moribund, or stunted 

larvae 

c d Row 2 total

Column 1 

total

Column 2 

total Grand total

All other results were subjected to analysis of variance to determine if significant treatment or 

replicate effects existed. There were no significant replication effects for any variable measured. 

Bartlett’s test was used to test for the equality of treatment variances. Where variances were 

unequal, the ln(x+1) transformation was applied. If the ln(x+1) transformation did not equalize 

treatment variances, the square root(x+0.5) transformation was applied. If the square root(x+0.5) 

transformation did not equalize treatment variances, the arcsine square root transformation was 

applied. The calculated F values and significance levels (P values) of these F values are 

presented in Table 23.

Results

Bioassays of field-grown foliage of soybean event DAS-81419-2 demonstrated excellent control 

of C. includens, A. gemmatalis, S. frugiperda, and H. virescens at both the R2 and R5 plant 

development stages (Table 23). Likewise, field evaluations indicated excellent control of C. 

includens.
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Table 23. Efficacy of DAS-81419-2 soybean compared to ‘Maverick’.

1

DAS81419-2

‘Maverick’ Test χ

2

or F

value P

Excised leaf bioassay at R2, 15 July 2011

P. includens, total number of live 2nd instars, 4d* 0 64 χ

2

128.0 <0.0001

P. includens, average percent leaf consumption, 4d 0.50 46.28 F 32.91 0.0105

A. gemmatalis, total number of live 2nd instars, 4d** 0 48 χ

2

96.00 <0.0001

A. gemmatalis, average percent leaf consumption, 4d† 0.60 26.66 F 278.5 0.0005

S. frugiperda, total number of live 2nd instars, 4d* 0 64 χ

2

128.0 <0.0001

S. frugiperda, average percent leaf consumption, 4d† 1.55 27.68 F 164.0 0.0010

H. virescens, total number of live 2nd instars, 4d* 0 64 χ

2

128.0 <0.0001

H. virescens, average percent leaf consumption, 4d‡ 0.52 31.73 -- -- --

Excised leaf bioassay at R5, 12 August 2011

P. includens, total number of live 2nd instars, 4d* 0 63 χ

2

127.0 <0.0001

P. includens, average percent leaf consumption, 4d 0.50 32.20 F 600.5 0.0001

A. gemmatalis, total number of live 2nd instars, 4d** 0 44 χ

2

81.23 <0.0001

A. gemmatalis, average percent leaf consumption, 4d 0.50 20.00 F 466.6 0.0002

S. frugiperda, total number of live 2nd instars, 4d** 0 47 χ

2

92.08 <0.0001

S. frugiperda, average percent leaf consumption, 4d† 1.00 11.72 F 1519 <0.0001

H. virescens, total number of live 2nd instars, 4d** 0 45 χ

2

84.71 <0.0001

H. virescens, average percent leaf consumption, 4d† 0.57 15.98 F 534.5 0.0002

Sampling of natural infestation

P. includens, average number of small larvae, 8 Aug 0.00 0.00 F 0.000 1.0000

P. includens, average number of large larvae, 8 Aug 0.00 2.25 F 4.765 0.1170

P. includens, average number of small larvae, 15 Aug 0.00 2.75 F 5.418 0.1024

P. includens, average number of large larvae, 15 Aug 0.00 3.50 F 29.40 0.0123

P. includens, average number of small larvae, 22 Aug 0.25 5.25 F 75.00 0.0032

P. includens, average number of large larvae, 22 Aug 0.00 17.50 F 193.4 0.0008

P. includens, average number of small larvae, 29 Aug 0.00 0.00 F 0.000 1.0000

P. includens, average number of large larvae, 29 Aug† 0.25 4.00 F 7.784 0.0684

Average percent defoliation, 29 Aug 0.00 2.50 F 75.00 0.0320

* 16 larvae tested per replicate, 64 larvae tested in total.

** 12 larvae tested per replicate, 48 larvae tested in total.

† Data were ln(x+1) transformed to equalize treatment variances prior to Analysis of Variance, untransformed 

means are presented in the table.

‡ Data transformations were unable to equalize treatment variances.

 C. includens (formerly P. includens).

In conclusion, laboratory bioassay of field grown event DAS-81419-2 soybean foliage, and 

evaluation of field infestations, demonstrate the efficacy of this event against four lepidopteran 

pests of soybean.
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9.1.2. Potential Impact on Non-Target Organisms

A risk assessment was conducted to characterize the potential for adverse effects of cultivating 

DAS-81419-2 soybean, expressing Cry1Ac and Cry1F proteins, on non-target organisms 

(NTOs). The assessment focused on non-target arthropods associated with soybean 

agroecosystems with special emphasis on beneficial taxa and threatened or endangered species. 

Assessment factors included: established safety data for Cry proteins including data supporting 

previous deregulations for corn and cotton products expressing the Cry1Ac and Cry1F proteins, 

the expression levels of the Cry1Ac and Cry1F proteins in DAS-81419-2 soybean (Section 6), 

the environmental fate of the Cry1Ac and Cry1F proteins (Section 9.1.4), feeding tests of 

Cry1Ac and Cry1F proteins to representative NTOs, results from a field survey of non-target 

arthropods associated with DAS-81419-2, and information from key databases for threatened and 

endangered species. Results from the assessment indicate that DAS-81419-2 is unlikely to have 

adverse effects on non-target organisms, including threatened or endangered species, under 

normal agricultural practices.

Regulatory guidelines for risk assessment of genetically modified crops expressing plantincorporated protectants (PIPs) were developed by the U.S. EPA (EPA, 1998), and followed by 

Scientific Advisory Panel (SAP) meetings (EPA, 2001c, 2001b, 2004), and publications that 

provide refinements applicable to Bt PIPs (EPA, 2007; Wolt et al., 2010; Carstens et al., 2011). 

The assessment for DAS-81419-2 focused on non-target arthropods associated with soybean 

agroecosystems using a step-wise process that included problem formulation, hazard 

identification and characterization, exposure assessment, and risk characterization. Problem 

formulation accounted for the familiarity of mode of action for Cry proteins (Aronson and Shai, 

2001; Rang et al., 2004; Lee et al., 2006; Gouffon et al., 2011; Liu et al., 2011), the narrow 

spectrum of activity for Cry proteins (McClintock et al., 1995; Romeis et al., 2006; Marvier et 

al., 2007; Duan et al., 2008; Wolfenbarger et al., 2008; Duan et al., 2009), the demonstrated 

history of safe use for Bt cops (Naranjo, 2005; Sanvido et al., 2007) and the agronomic 

equivalence of DAS-81419-2 soybean to conventional soybean (Section 8). The hazard 

characterization was informed by existing safety data for the Cry1Ac and Cry1F proteins, 

spectrum of activity data that were produced to support the deregulation and registration of 

Cry1Ac- and Cry1F-expressing WideStrike®cotton events DAS-21023-5 (also described as 

3006-210-23) and DAS-24236-5 (also described as 281-24-236) (EPA, 2005), and lack of 

synergy between these two proteins (EPA, 2005). The exposure characterization was based on 

the expression analyses for Cry1Ac and Cry1F in DAS-81419-2 soybean (Section 6) and 

environmental fate of Cry proteins (Section 9.1.4). Special consideration for threatened or 

endangered species is addressed in Section 9.1.3.

Laboratory studies used Cry1Ac and Cry1F proteins that have been demonstrated to be 

equivalent with the Cry1Ac and Cry1F proteins produced by DAS-81419-2 (Section 6). 

Laboratory dose levels were based on Cry1Ac and Cry1F protein expression levels in 

WideStrikecotton events DAS-21023-5 and DAS-24236-5, and therefore for the present 

assessment, the margins of exposure (MOE) for representative NTOs were recalculated based on 

high-end exposure estimates (HEEEs) (EPA, 2009) derived from Cry1Ac and Cry1F protein 

expression in DAS-81419-2. Based on the level of concern (LOC) of 50% mortality at 10× the 

estimated environmental concentration (EEC) (EPA, 2007), and HEEEs for Cry1Ac and Cry1F 

in DAS-81419-2 cropping systems, the resulting MOE values support the conclusion of 
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negligible risk to NTOs representing diverse taxonomic groups and exposure scenarios (Table 

24).

Table 24. Estimated margins of exposure (MOE) to non-target organisms based on highend exposure estimates (HEEE) for Cry1Ac and Cry1F proteins produced in DAS-81419-2.

Organism Protein1 HEEE2 NOEC3 MOE4

Mouse Cry1Ac 28.33 ng/mg > 700 mg/kg > 100

(CD-1) Cry1F 63.32 ng/mg > 600 mg/kg > 100

Earthworm5 Cry1Ac 0.0910 mg/kg soil 107 mg/kg soil > 100

(Eisenia fetida) Cry1F 0.345 mg/kg soil 247 mg/kg soil > 100

Collembola5 Cry1Ac 0.0910 mg/kg soil > 22.6 mg/kg diet > 100

(Folsomia candida) Cry1F 0.345 mg/kg soil > 702 mg/kg diet > 100

Lady beetle Cry1Ac 4.76 ng/mg fw > 20.5 ng/mg fw > 4

(Hippodamia convergens) Cry1F 10.6 ng/mg fw > 273 ng/mg fw > 25

1 Microbe-derived Cry1Ac and Cry1F proteins used in laboratory studies have been demonstrated to be equivalent 

with the Cry1Ac and Cry1F proteins expressed in DAS-81419-2 (Section 6).

2 High-End Exposure Estimate (HEEE) represents the 90% upper bound of the reported expression; HEEE = mean 

expression concentration + (t0.1, upper tail, n-1 × std. dev.) / n1/2; HEEE values based on Cry1Ac and Cry1F expression 

level determined for DAS-81419-2 plant tissue relevant to the potential exposure route for the organism of 

interest; values for earthworm and Collembola are expressed as estimated environmental concentrations for soil 

(soil EECs) based on the HEEEs for forage tissue at the R3 growth stage; values for mice and lady beetles were 

based on leaf expression at the V5 growth stage.

3 No Observed Effect Concentration (NOEC).

4 Margin of Exposure (MOE) was calculated based on the ratio of the NOEC to HEEE.

5 The MOE values for earthworm and Collembola were calculated using the following parameter assumptions: 

175,000 soybean plants/acre, soybean plant dry weight of 71.2 g/plant, soil bulk density of 1500 kg/cubic meter, 

soil volume in a one-acre area with a depth of 0.15 m, and Cry1Ac and Cry1F soil EEC values of 6.65 and 25.22 

ng/mg dw, respectively, for forage tissue at the R3 growth stage.

The estimated MOEs ranged from approximately 4 to greater than 100. Where the MOE for lady 

beetles (>4×) in the Cry1Ac test was not demonstrated to be greater than 10× NOEC, the 

potential risk was examined in the context of the known spectrum of activity of Cry1Ac being 

restricted to the order Lepidoptera, an additional high-dose laboratory study of the Cry1Ac 

protein against lady beetles (Li et al., 2011) as well as data for coccinellids monitored in 

DAS-81419-2 field trials that demonstrated no adverse effects on lady beetle performance and 

abundance, respectively. Given no detrimental effects were observed in independent laboratory 

and field studies, risk to lady beetles was concluded to be negligible.

A lack of adverse effects following exposure to Cry1Ac and/or Cry1F proteins has also been 

shown for collembolans (Folsomia candida and Xenylla grisea) (Sims and Martin, 1997), the 

minute pirate bug (Orius albidipennis) (González-Zamora et al., 2007), green lacewing 

(Chrysopa carnea) (Rodrigo-Simón et al., 2006), hymenopteran parasitoids (Nasonia 

vitripennis), honeybees (Apis mellifera), and bobwhite quail. Agreement between laboratory and 

field observations for transgenic Bt proteins (Duan et al., 2009) has been supported through 
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meta-analyses of NTO data at the order- (Naranjo, 2009) and species-level (Duan et al., 2008). 

The meta-analysis for honeybees conducted by Duan et al. (2008) included six studies 

incorporating Cry1Ac or Cry1F (truncated or full length protein), suggesting that negative effects 

on pollinators are unlikely given exposure to the Cry1Ac and Cry1F proteins expressed in 

DAS-81419-2.

To confirm previous laboratory findings and to address any uncertainties in the risk assessment 

focused on taxonomic groups that were most likely to be exposed to the Cry proteins produced 

by DAS-81419-2 soybean in field settings, the potential for adverse effects of DAS-81419-2 on 

non-target arthropod populations was assessed in a US field study. Field study details are 

provided in Appendix 8. Assessment of population abundance and composition was conducted 

for several taxonomic groups, including: Oribatida (soil mites), Araneae (foraging and web 

spiders), Collembola (springtails), Hemiptera (e.g. Orius spp.), Coleoptera (e.g. ground beetles, 

lady beetles), Neuroptera (e.g. Chrysopa spp.), Lepidoptera, Diptera, and Hymenoptera (e.g. 

parasitoid wasps). Consistent across study site locations and sampling methods, arthropod 

populations associated with DAS-81419-2 were similar to those of non-transgenic soybean. 

Field monitoring confirmed the results of laboratory studies examining direct exposure to 

Cry1Ac and Cry1F proteins, and demonstrated no adverse effects of DAS-81419-2 cultivation on 

non-target arthropod communities. Field trial results for DAS-81419-2 contribute to a series of 

studies demonstrating no adverse effects of Bt crops on NTOs across a diversity of receiving

environments (Pilcher et al., 1997; Lozzia et al., 1998; Bhatti et al., 2005; Bitzer et al., 2005; 

Daly and Buntin, 2005; Dively, 2005; Head et al., 2005; Lopez et al., 2005; Naranjo, 2005; 

Pilcher et al., 2005; Torres and Ruberson, 2005; Whitehouse et al., 2005). 

The Cry1Ac and Cry1F proteins expressed in DAS-81419-2 have been previously demonstrated 

to have no adverse effects on fish (rainbow trout) or aquatic invertebrates (e.g. Daphnia magna). 

Risk posed to aquatic invertebrates (including the aquatic Lepidoptera) through DAS-81419-2 

cultivation was concluded to be negligible due to a low likelihood of exposure (Carstens et al., 

2011) and no verified instances of sensitive species. Therefore, the existing studies in Daphnia

and rainbow trout were considered to be adequate in supporting the conclusion that 

DAS-81419-2 deployment poses little risk to aquatic organisms.

9.1.3. Potential Impact on Threatened and Endangered Species

Given the narrow spectrum of activity observed for Cry proteins (McClintock et al., 1995; 

Romeis et al., 2006; Marvier et al., 2007; Wolfenbarger et al., 2008; Duan et al., 2009)

consideration for threatened and endangered (T/E) species in the assessment for DAS-81419-2 

focused on taxa within the order Lepidoptera.

Proximity relationships between potential growing sites where DAS-81419-2 could be planted 

and locations of T/E lepidopteran species were determined from the information contained in the 

FIFRA Endangered Species Task Force (FESTF) databases. Ecology and life history 

information for species identified as being present in soybean-growing counties was accessed 

from the US Fish and Wildlife Service and NatureServe Explorer databases 

(http://www.natureserve.org/explorer/).

Four lepidopteran butterfly species (Karner blue, Lycaeides melissa samuelis; Mitchell's satyr, 

Neonympha mitchellii mitchellii; Saint Francis' satyr, Neonympha mitchellii francisci; and 
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Uncompahgre fritillary, Boloria acrocnema) were indicated to co-occur with soybean use sites in

66 counties located in 13 states. Ecology and life history information demonstrated that habitat 

requirements for larvae and adults of three of the identified species (Mitchell's satyr, Saint 

Francis' satyr, and Uncompahgre fritillary) do not overlap with commercial soybean acreage, and 

are therefore not expected to be impacted by DAS-81419-2 cultivation. There is a possible 

overlap between the geographic range for Karner blue butterfly and soybean use sites; however, 

the potential for direct exposure to the Cry proteins produced by DAS-81419-2 soybean is 

negligible. Karner blue larvae feed only on lupines (Lupinus spp.) and soybean is neither 

identified as a larval food source, nor is soybean open-pollinated so DAS-81419-2 soybean 

pollen is highly unlikely to be deposited on lupine leaves. Furthermore, soybean is not identified 

as a nectaring source for Karner blue adults. Therefore, the risk posed to the Karner blue 

butterfly was considered to be negligible. In agreement with previous T/E species assessments 

for Cry1-containing corn (Herculex®) and cotton (WideStrike®) products, the assessment for

DAS-81419-2 supports a conclusion of negligible risk to T/E species.

9.1.4. Environmental Fate of Introduced Proteins and Impact on Soil-dwelling 

Organisms

Proteins are not known to persist in the environment due to the ubiquitous nature of proteases in 

microbes. The Cry1Ac protein has been used in sprayable Bt formulations for over half a 

century and is present in both WideStrikecotton event DAS-21023-5 (also described as 3006-

210-23) and Bollgardcotton (event MON 531, as well as a discontinued corn event: 

DBT418Cry1Ac corn) (Mendelsohn et al., 2003; Sanahuja et al., 2011). The Cry1F protein has 

also been used in sprayable Bt formulations and is present in WideStrike



cotton event 

DAS-24236-5 (also described as 281-24-236), as well as, in Herculexand SmartStaxcorn

(event TC1507) (Mendelsohn et al., 2003; Sanahuja et al., 2011). Laboratory experiments have 

shown that both Cry1Ac and Cry1F are quickly inactivated, and field studies have shown no 

accumulation of the proteins as a result of continuous planting of crops containing these proteins

(Head et al., 2002; Herman et al., 2002; Shan et al., 2008). It has further been suggested that the 

Cry proteins in general are unlikely to represent a significant risk in the soil environment (Icoz 

and Stotzky, 2008).

9.1.5. Weediness Potential

Commercial soybean varieties in the U.S. are not considered weeds and are not effective at

invading established ecosystems.

Cultivated soybean seed rarely displays any dormancy characteristics and only under certain 

environmental conditions grows as a volunteer in the year following cultivation. If this should 

occur, volunteers do not compete well with the succeeding crop, and can easily be controlled 

mechanically or chemically. The soybean plant is not weedy in character. In North America, 

Glycine max is not found outside of cultivation. In managed ecosystems, soybean does not 

effectively compete with other cultivated plants or primary colonizers (OECD, 2000).

 

 Bollgard and SmartStax are registered trademarks of Monsanto Technology LLC.
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The introduction of insecticidal proteins Cry1Ac and Cry1F into soybean does not alter the 

weediness characteristics of soybean. Agronomic properties of DAS-81419-2 soybean related to 

weediness, such as emergence (Section 8.1), seedling vigor (Section 8.1), response to 

environmental stressors (Section 8.2), and germination (Section 8.3), have been shown to be 

equivalent to conventional soybean. These findings support the conclusion that DAS-81419-2 

soybean does not have increased weediness potential compared to conventional soybean. 

9.1.6. Gene Flow Assessment

Vertical Gene Flow

Soybean is considered to be a self-pollinated species, although natural crossing can occur at low 

rates (OECD, 2000). The level of cross pollination can vary as a function of growing conditions, 

spatial arrangement of plants, and genotype. Cross pollination levels ranging from 0.09% up to 

3.6% in adjacent rows have been reported (Beard and Knowles, 1971; Ahrent and Caviness, 

1994), but outcrossing frequency rapidly declines with distance. Cross pollination rates have 

been reported to be <0.1% at distances of >5 m (Caviness, 1966; Ray et al., 2003). Cultivated 

soybeans can cross only with members of its subgenus Soja. Wild soybean species of the 

subgenus Soja are native to Korea, Japan, Taiwan, northeastern China, and areas of the former 

USSR, but do not exist naturally in the United States (OECD, 2000). Therefore, there is no 

potential for gene flow from DAS-81419-2 soybeans to wild soybean relatives in the United 

States.

Horizontal Gene Flow

There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to 

microbes (Conner et al., 2003). Even if such a transfer were to take place, transfer of the cry1Ac 

(synpro), cry1Fv3 and pat genes from DAS-81419-2 soybean would not present a human health 

or plant pest risk, based on the safety data presented in this petition.

9.2. Current Agronomic Practices for Soybean and Potential Impact

9.2.1. Soybean Production

Large scale soybean production in the U.S. began in the early 1920s (USDA ERS, 2012). 

According to USDA NASS (2012), approximately 1.5 million acres were planted to soybean in 

1924. Over the following two decades soybean acreage expanded rapidly reaching 15 million 

acres. Since 1997 more than 70 million acres were planted to soybean every year with an 

exception in 2007 when an increase in corn acreage limited soybean cropland. This increase in 

soybean acreage is largely due to ideal planting conditions, increased crop rotation, rising yield, 

and lower production costs (USDA ERS, 2012). Average soybean yield per acre varied from 

33.9 bushels to 44 bushels between 1997 and 2011 with the lowest yield recorded in 2003 and 

the highest in 2009. Total annual soybean production during the same time period ranged from 

approximately 2.45 billion bushels in 2003 to 3.36 billion bushels in 2009. Farm value of U.S. 

soybean production ranged from $12.21 billion in 1999 to $37.55 billion in 2010 (USDA NASS, 

2012). An average of 1.6 billion bushels of soybean were crushed annually in the last ten years 

to produce soybean meal for livestock and soybean oil for consumption and industrial products 

(Soy Stats, 2012).
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In the U.S. the majority of soybeans are grown in the north central states with the remaining crop 

primarily grown in the southeastern and Mississippi-delta regions. About 80% of the planted 

soybean acres and 80% of the harvested soybean acres are concentrated in 11 states, including 

Iowa, Illinois, Minnesota, Indiana, Missouri, Nebraska, Ohio, South Dakota, North Dakota, 

Kansas and Arkansas, where growers achieve higher yields at lower per-acre production cost 

(USDA ERS, 2012). Soybean yield in the north central region in 2011 ranged from 27 bushels 

per acre in Kansas to 53.5 bushels per acre in Nebraska (USDA NASS, 2012). Soybean yield is 

largely dependent on variety selection, environmental conditions and agronomic decisions. 

Variety selection is critical in achieving high yield while environmental conditions and 

agronomic decisions such as weeds and disease management and fertilizer application have a 

significant impact on genetic yield potential. In 2011, seed, chemicals and fertilizers contributed 

to 70% of the operating cost (USDA ERS, 2012). In the southern U.S. states higher fertilizer and 

chemical insecticide applications per acre soybean planted contributed to higher operating cost 

(Soy Stats, 2012).

U.S. is the world’s leading soybean exporter. In 2011, U.S. exported 1.275 billion bushels of 

soybean which accounted for 42% of the U.S. production and 37% of the world’s soybean trade 

(Soy Stats, 2012; USDA ERS, 2012). U.S. soybean and soybean product exports exceeded $21.5 

billion in 2011 with soybean accounting for 82%, soybean meal 13% and soybean oil 6% (Soy 

Stats, 2012). China was the largest export market for U.S. soybean with purchases exceeding 

$10.4 billion. Mexico was the second largest market for U.S. soybean with purchases of nearly 

$1.6 billion. Other significant export markets for U.S. soybean are Japan, Indonesia and Taiwan. 

Canada was the largest export market for U.S. soybean meal in 2011 with purchases reaching 

$375 million, Mexico was second with $369 million followed by Venezuela at $260 million. 

The top three export markets for U.S. soybean oil were Morocco, Mexico and China with 

combined purchases exceeding $659 million (Soy Stats, 2012).

Soybean seed for commercial planting is produced throughout most of the U.S. soybean growing 

regions. Soybean varieties are developed and adapted to certain geographical zones and are 

separated into ten maturity groups (OECD, 2000). Seed production for these maturity groups is 

grown in respective geographical zone for each maturity group.

Seed quality, including genetic purity, vigor, and presence of weed seed, seed-borne diseases, 

and inert materials, is a major factor affecting crop yield. Genetic purity in commercial seed 

production is generally regulated through a system of seed certification which is intended to 

ensure that the desired traits in the seed are maintained throughout all stages in cultivation.

The U.S. Federal Seed Act, enacted in 1939, recognizes seed certification and official certifying 

agencies. Implementing regulations further recognize land history, field isolation, and varietal 

purity standards for seed. States have developed laws to regulate the quality of seed available to

farmers (Bradford, 2006). Most of the laws are similar in nature and have general guidelines for

providing information on the label for the following: 1) Commonly accepted name of 

agricultural seed, 2) Approximate total percentage by weight of purity, 3) Approximate total 

percentage of weight of weed seeds, 4) Name and approximate number per pound of each kind 

of noxious weed seeds, 5) Approximate percentage of germination of the seed, and 6) Month and 

year the seed was tested.
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The Association of Official Seed Certifying Agencies (AOSCA, 2012) defines the classes of 

seed as follows: 1) breeder seed, 2) foundation seed, 3) registered seed, and 4) certified seed. 

Breeder seed is seed directly controlled by the originating or sponsoring plant breeding 

organization. Foundation seed is the progeny of Breeder seed or Foundation seed that is handled 

to maintain specific genetic identity and purity. Registered seed is the progeny of Breeder or 

Foundation seed that is so handled as to maintain satisfactory genetic identity and purity.

Certified seed is the progeny of Breeder, Foundation, or Registered seed that is handled to 

maintain satisfactory genetic identity and purity.

Production of all classes of certified seed requires that 1) each certifying agency shall determine 

that genetic purity and identity are maintained at all stages of certification including seeding, 

harvesting, processing, and labeling of the seed; 2) the unit of certification shall be a clearly 

defined field or fields; 3) one or more field inspection shall be made prior to harvest and when 

genetic purity and identity can best be determined; and 4) a certification sample shall be drawn in 

a manner approved by the certifying agency from each cleaned lot of seed eligible for 

certification (7 CFR §201.72). Federal regulation 7 CFR §201.76 specifies minimum land, 

isolation, field, and seed standards required for soybean Foundation, Registered and Certified 

seed. To produce all classes of certified seed, the land requirement is that the crop shall not be 

grown on land where soybeans were grown the previous year unless the preceding soybean crop 

was planted with a class of certified seed of the same variety or unless the preceding soybean 

crop and the variety being planted have an identifiable character difference. Isolation 

requirement varies somewhat among states and seed producers. Some states require an isolation 

distance of five feet while other states and seed producers require 10 feet of isolation distance. 

To qualify as Certified seed, only one plant of another variety in 200 soybean plants and 0.5% of 

seed of other varieties or off-types are permitted (7 CFR §201.76). To qualify as Registered 

seed, only one plant of another variety in 500 soybean plants and 0.2% of seed of other variety or 

off-types are permitted. To be certified as Foundation seed, only one plant of another variety in 

1,000 soybean plants and 0.1% of seed of other variety or off-types are permitted.

DAS-81419-2 does not differ from conventional soybean in agronomic characteristics (Section 

8) and thus is not expected to impact U.S. soybean grain or seed production.

9.2.2. Weeds in Soybean and Weed Management

Weeds in soybean compete with the crop for light, nutrients and soil moisture. When weeds are 

uncontrolled for the entire season, yield losses can exceed 68 to 75% (Krausz et al., 2001; Dalley

et al., 2002). Hartzler and Pringnitz (2005) define the critical period of competition in soybeans 

as the point of time when weeds that emerge with the crop begin to impact yields. Most studies 

have found that soybean yields are protected if weeds are controlled before they reach a height of 

6 to 8 inches. The critical period varies widely, depending upon weed species and densities, 

environmental conditions, and cultural practices.

To maximize soybean yields, weeds must be removed or controlled when they are no more than 

6 inches tall (Loux et al., 2006). Field trials conducted in Ohio in 2001 found that applying 

glyphosate when weeds are already 9 to 12 inches tall resulted in a 6-10% yield loss due to weed 

competition prior to control of the weeds (Loux et al., 2006). Dalley et al. (2002) studied the 

effect of row width and weed competition on soybean yield in 1998-2001. They found that weed 
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competition could reduce soybean yields by the time weeds reached 6, 6, and >12 inches in 

height in 7.5, 15, and 30 inch rows, respectively.

The most common weed problems in soybeans are the annual grass and broadleaf weeds. Some 

fields are also infested with perennial weeds (quackgrass, Johnsongrass, field bindweed, Canada 

thistle, others) which are more difficult to control since they can reproduce from seed or 

underground root buds or rhizomes.

In 2006, USDA NASS surveyed 19 states (Arkansas, Illinois, Indiana, Iowa, Kansas, Kentucky, 

Louisiana, Michigan, Minnesota, Mississippi, Missouri, Nebraska, North Carolina, North 

Dakota, Ohio, South Dakota, Tennessee, Virginia, and Wisconsin; about 96% of the total U.S. 

soybean acreage) and found that 98% of the planted soybean acreage was treated with 

herbicides, an indication of the intensive weed management that is used in U.S. soybeans (USDA 

NASS, 2007). In 2006, 97.5% of the planted soybean acres received an average of 2.2 herbicide 

applications to control weeds (USDA ERS ARMS, 2006). Many herbicides are registered for 

pre-plant, pre-emergent and/or post-emergent application to selectively control most weed 

species commonly found in soybeans. In general, soybeans sometimes receive a soil applied or 

burndown herbicide prior to planting or at plant, but almost always receive a post-emergence 

herbicide application. In 2006, the most widely used herbicide in soybeans was glyphosate, 

driven by the high adoption of glyphosate tolerant soybeans (USDA NASS, 2007). Soybeans 

received an average of 1.7 applications of glyphosate on 96% of the acres planted in 2006. 

Herbicides such as 2,4-D (10% of acres), chlorimuron (4% of acres), and trifluralin (2% of acres) 

were applied on significantly fewer acres. All other herbicides applied in soybeans that year 

comprised 3% or less of the total acres planted. Integrated Weed Management (IWM) programs 

advocate the use of a combination of preventive, cultural, mechanical and chemical tools to keep 

weed pressure below threshold levels that reduce yield and profits (Knezevic, 2010).

DAS-81419-2 soybean expresses the phosphinothricin acetyltransferase (PAT) protein and 

therefore it is tolerant to the herbicide glufosinate. With the exception of glufosinate tolerance, 

no impact is expected from the deregulation of DAS-81419-2 soybean on current weed 

management practices.

DAS-81419-2 soybean expresses the phosphinothricin acetyltransferase (PAT) protein and 

therefore it is tolerant to the herbicide glufosinate. As glufosinate tolerance in soybeans is 

currently available today to U.S. growers, no impact is expected from the deregulation of 

DAS-81419-2 soybean on current weed management practices.

9.2.3. Crop Rotation and Tillage Practices

In 2006, 71% of U.S. soybean acres were grown in rotation after corn, 13% were grown after 

soybeans, and 16% were grown after small grains, cotton, fallow or other crops (USDA ERS 

ARMS). Crop rotation is a widespread management practice that has been recognized and 

exploited for centuries to increase crop yields (Lauer, 2007). In the Midwestern U.S., a cornsoybean rotation produces at least 10% greater yields in both crops, and sometimes as much as 

19% higher corn yields. The exact mechanism for the rotation effect is unknown, but may be 

influenced by increases in organic matter and soil fertility, as well as management of diseases, 

insects, and weeds.
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The introduction of numerous herbicides in the decades following the launch of 2,4-D in the 

mid-1940s allowed reduced and conservation tillage systems to become more feasible and 

popular (Givens et al., 2009). The introduction of glyphosate-resistant (GR) crops in 1996 

brought a technology that enabled many producers to adopt reduced-tillage production systems. 

By 2002, only 17% of U.S. soybeans were produced under conventional tillage (<15% residue) 

systems (USDA ERS ARMS). Some form of conservation tillage was being used on the other 

83% of U.S. soybean acres, as follows: 18% reduced tillage (15-30% residue), 32% mulch till, 1-

2% ridge till, and 31% no-till. Increased yields coupled with decreases in erosion and water 

runoff and lower fuel use and fuel cost strongly support the premise that soybean produced with 

conservation tillage in any production system is more economically and environmentally 

sustainable (CAST, 2009).

Given the data presented in this petition demonstrating no difference between DAS-81419-2 and 

conventional soybean in agronomic characteristics (Section 8), no impact is expected from the 

deregulation of DAS-81419-2 soybean on current crop rotation and tillage practices.

9.2.4. Management of Insect Pests

Agricultural production of soybean is a relatively recent development; soybean did not become a 

major export crop in North America until the 1970s (Smith, 1994). Insect injury has not 

traditionally resulted in economic losses in most North American growing regions. However, in 

recent years, losses have been severe in some southern regions of the United States (Catchot, 

2011). Economic thresholds, which are the level of insect pressure where the benefits of 

treatment cover the cost of that treatment, have traditionally been based on $4-6 per bushel 

soybeans. However, recent prices of $12-15 per bushel have significantly lowered economic 

thresholds associated with many soybean pests, expanding the area and number of insecticide 

treatments applied to soybeans (USDA ERS, 2011). Increases in pest insect populations in 

soybean have also been observed; increases are attributed by some to global warming or insect 

adaptation to the soybean plant (Way, 1994). Lower economic thresholds coupled with higher 

insect populations favor more aggressive pest management strategies (Pedigo, 1996).

Insect injury can negatively impact yield, plant maturity, and seed quality. Insect injury is most 

often defined by the plant parts that are injured: roots, stems, foliage, or pods. Root- and stemfeeding insect groups typically remain undetected until damage has occurred. Although leaffeeding insects are the most diverse group, pod-feeding insects are generally considered to be 

proportionately more important, because high-value soybean reproductive parts are affected. 

Many soybean foliar feeders are caterpillars (Lepidoptera larvae) in the family Noctuidae. These 

include the soybean looper, Chrysodeixis includens formerly Pseudoplusia includens Walker,

velvetbean caterpillar, Anticarsia gemmatalis Hübner and green cloverworm, Hypena scabra

(Fab.). These species commonly occur sympatrically and procedures are being developed for 

making management decisions that acknowledge combined effects of these pests.

A major effect of foliar feeding is reduced light interception by the soybean canopy; which

ultimately decreases yield (Higley, 1994). Soybean plants can withstand up to 35% leaf loss prebloom without lowering yield (de Freitas Bueno et al., 2011). However, defoliation during the 

flowering and pod-filling stages poses a greater threat to yield because the plant has less time to 

compensate for injury. From pod-set to maturity, the plant can tolerate only a 20% defoliation 
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level before yield is affected (de Freitas Bueno et al., 2011). The tolerance of soybeans for 

defoliation varies somewhat depending on cultivar (de Freitas Bueno et al., 2011).

In addition to the plant tissue attacked, soybean responses to injury are influenced by the mode 

of feeding. For example, yield loss incurred from pod-feeding stink bugs, such as the southern 

green stinkbug Nezara viridula (L.) (Hemiptera: Pentatomidae), is linear in relation to the 

amount of plant tissue damage caused by feeding. In contrast, for seedcorn maggot, Delia 

platura (Meigen) (Diptera: Anthomyiidae), larval feeding on roots reduces yield only when 

injury levels are relatively high (Higley, 1994). Environmental factors also impact the response 

of soybean to insect injury; yield reductions from insect injury are more severe under conditions 

associated with water stress.

Although soybean is attacked by many pests, only a few pose an economic threat. Damage is 

concentrated in certain production regions (Way, 1994). Eight insect species are responsible for 

most insect damage in U.S. soybean production: velvetbean caterpillar, soybean looper, green 

cloverworm, Mexican bean beetle, Epilachna varivestis Mulsant (Coleoptera: Coccinellidae), 

southern green stinkbug, bean leaf beetle, Cerotoma trifurcata Forster (Coleoptera: 

Coccinellidae), southern green podworm/corn earworm, Helicoverpa zea (Boddie) (formerly 

Heliothis zea) (Lepidoptera: Noctuidae), and green stink bug (Higley and Boethel, 1994). 

Soybean insect pest populations and extent of soybean damage varies annually and regionally 

due to differences in climatic and weather conditions, species distributions and environmental 

tolerances and production practices (Way, 1994).

Generally, soybean insect pest problems are less severe in the Midwest states of the U.S. than in 

other soybean producing areas (Way, 1994). Green cloverworm is the only known lepidopteran 

pest that occurs frequently in the Midwest; cutworms, Agrostis ipsilon (Hufnagel) and

Peridroma saucia (Hübner) (Noctuidae) and Painted Lady, Vanessa cardui L. (Nymphalidae) 

have also been reported but are less commonly economically important. Economic insect 

problems increased in the 1990s (Hammond, 2012) with greater increases in the north-central 

U.S. around 2000 (McWilliams et al., 1999). Generally, insect challenges still remain below 

economic threshold levels for the U.S. Midwest. 

Several insect pests have been reported in the northeastern United States and Southern Canada in 

numbers high enough to cause economic losses in soybean (Penn State Extension, 2012). 

Defoliators include: green cloverworm, Japanese beetle, Popillia japonica Newman (Coleoptera: 

Scarabaeidae), Mexican bean beetle, bean leaf beetle, and grasshoppers, Melanoplus spp.

(Orthoptera: Acrididae). Sap-sucking insects include: soybean aphid, Aphis glycines Matsumura

(Hemiptera: Aphididae), potato leafhopper, Empoasca fabae (Harris) (Hemiptera: Cicadellidae)

and stink bugs. Stink bugs in particular will feed on both foliage and pods. Seedcorn maggots 

feed on seeds.

Insect pressure is generally greatest in the southern U.S. states bordering the Gulf of Mexico and 

the Atlantic Ocean. Four of the major soybean insect pests in the Southeast region are 

lepidopteran insect pests. Velvetbean caterpillar and soybean looper infestations are greatest in 

the southeastern states because of their close proximity to the tropics. The warm climate allows 

these pests to overwinter and facilitates multiple generations per year.
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Stink bugs (Nezara viridula, Acrosternum hilare, Euschistus servus) are responsible for most 

infestations and greatest economic losses in the southeastern United States (McPherson et al., 

1997; Guillebeau et al., 2008). Lepidopteran insects, primarily soybean looper, velvetbean 

caterpillar, corn earworm (soybean podworm), and lesser cornstalk borer, Elasmopalpus 

lignosellus (Zeller) (Lepidoptera: Pyralidae) are responsible for most of the remaining damage 

(McPherson et al., 1997; Guillebeau et al., 2008). Velvetbean caterpillar and soybean looper are 

considered the most damaging defoliating insects in the South (Way, 1994; Hammond, 2012). 

Insecticides are used on approximately 50% of the soybean acreage in Georgia for lepidopteran 

pests with velvetbean caterpillar being the most targeted pest (Gianessi et al., 2002). 

Approximately 40% of the soybean acreage in Louisiana is treated with insecticides for 

lepidopteran pests, with soybean looper being the main target (Gianessi et al., 2002).

Although insecticides provide effective and economical control or suppression of soybean insect 

pests that reach economic thresholds, Integrated Pest Management (IPM) programs that 

incorporate preventive pest management with insecticidal control have proven to be highly 

effective in managing pest problems (Pedigo, 1996; Kogan, 1998). Effective management of 

insects in soybean requires consideration of agronomic, economic and biological factors. For 

example, preventive pest management practices are most important where pest problems are 

anticipated. Agricultural practices can be adjusted to adversely affect pest species or aid 

beneficial species. Similarly, variety selection, crop rotation, tillage, planting dates, and adjacent 

crops all impact the potential for pest outbreaks and the success of natural enemies in 

suppressing pest populations.

As it is not possible to eliminate all economic losses due to insects, IPM programs are 

implemented to minimize these losses. IPM practice includes monitoring fields for insect growth 

stage, insect development and population density, and occasionally natural enemy development 

and population density. Management decisions for insect populations in individual fields are 

based on economic injury level, which is defined as the lowest population density of each insect 

likely to cause economic damage. The economic injury level usually changes during the 

growing season, with later stages of development warranting action at a lower level of 

defoliation. IPM programs integrate chemical control and biological control, cultural control and 

plant resistance to minimize insecticide resistance and reduce dependence on insecticides 

(Pedigo, 1996). Effective IPM requires intense insect scouting and is justified in high value 

crops or where insect pressure is common. However, the majority of soybean fields are not 

scouted because damage is infrequent. Failure to scout risks allowing damage that greatly 

exceeds the economic threshold to occur unnoticed; discovery may occur too late for subsequent 

insecticide applications to be effective.

The growing problem of soybean insect pest pressure is evidenced by increasing insecticide 

treatments in recent years (USDA NASS, 2007). According to USDA-NASS statistics, about 

16% of the U.S. soybean acreage in 2006 received an insecticide treatment, with chlorpyrifos 

(6%), esfenvalerate (5%), and lambda-cyhalothrin (3%); these accounted for nearly all treated 

acres (USDA NASS, 2007). Each of these insecticides controls a similar broad spectrum of 

insect pests including lepidopteran and non-lepidopteran pests. USDA-NASS (2007) also 

reported that approximately 15% of the soybean acreage in the Midwest region and 

approximately 25% of the soybean acreage in the Southeast and Eastern Coastal regions of the 
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U.S. received an insecticide treatment in 2006 with some states in the Southeast requiring 

treatments on up to 75% of their acreage (USDA NASS, 2007)

Chemical insecticides can have limited efficacy in controlling lepidopteran infestations in 

soybean. Narrow application windows, the emergence of insecticide resistance, and public 

pressure for reduced pesticide use limit the desirability of this approach to pest management 

(Thomas and Boethel, 1994). Soybean looper has developed extensive insecticide resistance 

(Thomas and Boethel, 1994); resistance to pyrethroids is widespread across the southern U.S. 

(Felland et al., 1990; Leonard et al., 1990). Insecticides remain effective against velvetbean 

caterpillar. However, infestations can quickly reach damaging levels and cause economic loss if 

insecticides are not applied promptly.

Bt crops (expressing at least one of several insecticidal proteins isolated from Bacillus 

thuringiensis) are effective in targeting specific insect pests, easy to adopt, and cost effective; 

Increasing adoption rates of Bt corn and cotton since 1996 have coincided with increased yields 

(USDA ERS, 2006). Pesticide use has also decreased since the introduction of these insectresistant crops, with one study showing an 8% reduction in insecticide use for adopters of Bt

corn than non-adopters (USDA ERS, 2006). Importantly, Bt crops have also be credited with 

increasing yields, even when economic thresholds have not been reached in either Bt- or non-Btfields due to regional insect population reductions (Hutchison et al., 2010).

Like Bt corn and cotton, DAS-81419-2 soybean offers an efficient and environmentally sound 

alternative to chemical insecticides for controlling lepidopteran soybean pests. Genetically 

modified crops are now an important component of many IPM programs, providing effective 

management of lepidopteran pests with reduced insecticide use (Perlak et al., 2001; Pilcher and 

Rice, 2003). Additional benefits from DAS-81419-2 soybean include a reduced need for 

scouting, preservation of beneficial insect populations, increased convenience and greater 

performance consistency.

9.2.5. Insect Resistance Management

DAS-81419-2 soybean expresses two insecticidal proteins, Cry1Ac and Cry1F, originally from 

the naturally occurring soil bacterium Bacillus thuringiensis. Data presented in this petition 

demonstrate that Cry1Ac and Cry1F are expressed in the tissues of the field-grown 

DAS-81419-2 soybean throughout the growing season. DAS-81419-2 soybean provides an 

effective pest management tool while reducing or replacing current insecticide applications to 

control lepidopteran pests in regions where these insect pests cause significant plant damage and 

yield loss.

Substances that are pesticides, as defined under the Federal Insecticide, Fungicide and 

Rodenticide Act (FIFRA), are subject to regulation by the U.S. Environmental Protection 

Agency (EPA). Pesticides produced in planta, known as plant-incorporated protectants (PIPs), 

are also subject to regulation by the EPA under FIFRA. To prevent or delay the onset of insect 

resistance to Bt proteins expressed in transgenic crops, EPA has mandated specific insect 

resistance management (IRM) programs to protect the benefits of Bt crops and sustainable longterm use of Bt crops (EPA, 2001a). EPA requires that an IRM plan is based on the 

characteristics of the crop, the biology and sensitivity of the target insect pests, and the cropping 

systems in which the product will be deployed. A core element of an IRM program is the use of 
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refuges, which are non-Bt host plants of the target insects that support susceptible target insects 

and reduce selection pressure for resistance. Susceptible insects produced from the refuge may 

mate with any resistant insects that survive in the Bt crop producing heterozygote insects that 

may be susceptible to the Bt crop (EPA, 2001a). Given that the registration DAS is currently 

seeking from EPA for planting of DAS-81419-2 soybean in the U.S. will be limited in acreage 

for breeding and seed increase purposes to support product development, DAS has proposed that 

planting of a refuge is not necessary and an IRM program for DAS-81419-2 soybean is not 

needed. The proposal is based on previous registration decisions, impact on the natural refuge for 

key target pests of Bt cotton, durability of DAS-81419-2 soybean, and target pest biology. A 

summary of the analyses is presented below. Detailed analyses will be provided to EPA in 

connection with the FIFRA Section 3 seed increase registration application.

In previous registration decisions, EPA indicated that cultivation of crops containing PIPs for 

seed production on up to 20,000 acres per county and 250,000 acres total does not require 

planting of a refuge (for example, HerculexI corn, EPA registration number 68467-2 as 

amended on September 29th, 2010). Similarly, on July 27th 2012, EPA issued registrations under 

FIFRA Section 3 that permit cultivation of each Bt cotton, Cry1Ac Insect Protected cotton seed 

(EPA registration number 68467-17) or Cry1F Insect Protected cotton seed (EPA registration 

number 68467-18), on up to a total of 20,000 acres per county and up to a combined total of 

30,000 acres per year without planting of a refuge for breeding, agronomic testing and seed 

production purposes. 

In the U.S., the majority of the soybeans are grown in the north central states and the remaining 

in the southeastern and Mississippi delta regions. In the southern and southeastern U.S., soybean 

is a component of the Helicoverpa zea (bollworm) natural refuge for Bt cotton (Jackson et al., 

2008) and DAS-81419-2 soybean is expected to cause reduced survival of H. zea larvae feeding 

on it relative to non-Bt soybean. EPA has oversight of insect resistance management for Bt 

crops in the U.S. Through the terms of registration for DAS-81419-2 soybean, EPA will impose 

resistance management requirements to promote the durability of Bt traits in cotton, corn, and 

soybean.

DAS-81419-2 soybean expresses two insecticidal proteins whose modes of action differ with 

respect to receptor binding. For example Cry1Ac and Cry1F have been shown to bind to 

different receptors in the midgut of the target soybean insect pest tobacco budworm H. virescens

(Jurat-Fuentes and Adang, 2001). Cry1Ac binds to at least three sets of receptors while Cry1F 

binds to at least two, only one of which also binds Cry1Ac. The major receptor for Cry1Ac is 

not recognized by Cry1F (Jurat-Fuentes and Adang, 2006). Such incomplete shared binding is 

expected to lead to incomplete cross-resistance when resistance is mediated by receptor changes. 

Bt gene pyramiding offered by DAS-81419-2 soybean provides additional protection against the 

development of insect resistance and offers greater durability than Bt crops carrying a single Bt 

trait.

In addition to H. zea, other lepidopteran pests of soybean that are sensitive to the Cry1F and/or 

Cry1Ac Bt proteins, such as soybean looper (Chrysodeixis includens, formerly Pseudoplusia 

includens), velvetbean caterpillar (Anticarsia gemmatalis), and fall armyworm (Spodoptera 

frugiperda), are polyphagous, feeding on numerous wild and cultivated plants (Sparks, 1979; 

Waters and Barfield, 1989; Jost and Pitre, 2002). With other Bt crops grown for breeding and 
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seed increase purposes, plantings of up to 20,000 acres per county are permitted by EPA without 

a refuge because with these limits the selection pressure for resistance in sensitive insect species 

is negligible. The natural refuge of non-Bt crops and wild hosts for these polyphagous species 

that are pests of soybean further reduces selection pressure for resistance.

The information presented here indicates that planting of DAS-81419-2 soybean under limited 

acreage for breeding and seed increase purposes in the U.S. will have very little effect on 

resistance development in H. zea to Bt cotton. Furthermore, the level of selection pressure on 

other lepidopteran pests of soybean will be negligible under the proposed acreage limits. We 

therefore anticipate that EPA will not require a structured refuge for DAS-81419-2 soybean 

under the conditions of use. If DAS chooses to cultivate DAS-81419-2 soybean for commercial 

purposes in the U.S. in the future, DAS will apply for a FIFRA Section 3 commercial use 

registration with EPA and will develop a specific IRM program in connection with the 

commercial use registration.

Under the terms of the EPA’s registrations for commercial cultivation of Bt cotton and Bt corn, 

DAS is required to monitor for resistance in several lepidopteran pests of cotton that are also 

pests of soybean. This monitoring will continue after the introduction of DAS-81419-2 soybean.

9.3. Summary of Impact on Environment and Agronomic Practices

The efficacy study demonstrates that Cry1Ac and Cry1F expressed in DAS-81419-2 soybean 

provide protection against target lepidopteran pests. There are no new phenotypic characteristics 

in DAS-81419-2 soybean to indicate it is any different from conventional soybean in weediness 

potential, and like conventional soybean, the risk of gene flow from DAS-81419-2 soybean to 

wild relatives in the U.S. is negligible.

No significant impact is expected on current crop management practices, non-target or 

endangered species, or crop rotation from the introduction of DAS-81419-2 soybean. 

DAS-81419-2 offers an efficient and environmentally sound alternative to chemical insecticides. 

In addition, DAS-81419-2 offers greater durability and therefore improved insect resistance 

management. The availability of DAS-81419-2 is expected to reduce the need for scouting, 

preserve beneficial insect population, and provide increased convenience and greater 

performance consistency.
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10. Adverse Consequences of Introduction

Field and laboratory testing of DAS-81419-2 soybean demonstrated that the transgenic soybean 

is substantially equivalent to non-transgenic conventional soybean apart from the intended 

change of insect resistance. DAS knows of no study results or other observations associated with 

DAS-81419-2 soybean that indicate there would be anticipated to be adverse consequences from 

introduction. 
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11. Appendices

Appendix 1. Materials and Methods for Molecular Characterization of DAS-81419-2 Soybean

Appendix 2. Materials, Methods and Results for Characterization of Cry1Ac Protein

Appendix 3. Materials, Methods and Results for Characterization of Cry1F Protein

Appendix 4. Materials, Methods and Results for Characterization of PAT Protein

Appendix 5. Materials and Methods for Cry1Ac, Cry1F and PAT Protein Expression Analysis

Appendix 6. Analytical Methods for Compositional Analysis

Appendix 7. Literature Ranges for Compositional Analysis

Appendix 8. Non-Target Arthropod Field Study

Appendix 9. USDA Notifications for DAS-81419-2 Soybean

Appendix 10. Stewardship of Insect-Resistant DAS-81419-2 Soybean

Appendix 11. References
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Appendix 1. Materials and Methods for Molecular Characterization of DAS-81419-2 

Soybean

DAS-81419-2 Soybean Material

Transgenic soybean seeds from five distinct generations of soybean containing event DAS81419-2 were planted in the greenhouse. After at least one week of growth for emerged 

seedlings, leaf punches were taken from each plant and were tested for PAT protein expression 

using a lateral flow strip assay according to the manufacturer’s instructions (Envirologix Inc.). 

Each plant was given a “+” or “-” for the presence or absence of the PAT protein. 

Control Soybean Material

Seeds from the non-transgenic soybean line Maverick were planted in the greenhouse. The 

Maverick seeds had a genetic background representative of the transgenic seeds but did not 

contain the cry1Ac(synpro), cry1Fv3, or pat genes.

Reference Materials

DNA of the plasmid pDAB9582 was added to samples of the non-transgenic control genomic 

DNA at a ratio approximately equivalent to 1 copy of the transgene per soybean genome with a 

soybean genome size of ~1.1 × 109bp (Arumuganathan and Earle, 1991) and used as the positive 

control for the Southern hybridization. DIG-labeled DNA Molecular Weight Marker II and 

DIG-labeled DNA Molecular Weight Marker VII (Roche Diagnostics), each containing a 

mixture of DNA fragments with different sizes, served as size standards for agarose gel 

electrophoresis and Southern blot analysis. 

DNA Probe Preparation

DNA probes were generated by a PCR-based incorporation of a digoxigenin (DIG) labeled 

nucleotide, [DIG]-dUTP, into fragments generated by primers specific to genetic elements and 

backbone regions from plasmid pDAB9582. Generation of DNA probes by PCR synthesis was 

carried out using a PCR DIG Probe Synthesis Kit (Roche Diagnostics). Labeled probes were 

purified from agarose gels and were quantified by PicoGreen reagent (Invitrogen).

Sample Collection and DNA Extraction

Leaf samples were collected from greenhouse-grown plants for genomic DNA extraction. 

Genomic DNA was extracted following a modified CTAB method. Briefly, leaf samples were 

individually ground in liquid nitrogen followed by addition of CTAB extraction buffer (~5:1 

ratio milliliter CTAB extraction buffer: gram leaf tissue) and RNase-A (>10 μL) (Qiagen). After 

approximately 1 hour of incubation at ~65°C with gentle shaking, samples were spun down and 

the supernatants were extracted with equal volumes of chloroform:octanol = 24:1 (Sigma). DNA 

was precipitated by mixing the supernatants with equal volumes of CTAB precipitation buffer. 

The precipitated DNA was dissolved in high salt TE buffer followed by precipitation with 

isopropyl alcohol. The precipitated DNA was rinsed with 70% ethanol, air-dried, and dissolved 

in appropriate volume of 1 × TE buffer (pH 8.0). The DNA was quantified with PicoGreen 

reagent (Invitrogen), and was visualized on an agarose gel to check for genomic DNA quality.
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DNA Digestion and Electrophoretic Separation of the DNA Fragments

Genomic DNA extracted from the soybean leaf tissue was digested with restriction enzymes by 

combining approximately 10 g of genomic DNA with approximately 5-10 units of the selected 

restriction enzyme per g of DNA in the corresponding reaction buffer. Each sample was 

incubated at 37oC overnight for digestion. The digested DNA samples were precipitated with 

Quick-Precip (Edge BioSystems) and re-suspended to achieve the desired volume for gel 

loading. The DNA samples and molecular size markers were then electrophoresed through 0.8% 

agarose gels with 1 TBE buffer at 35-65 V for 18-22 hr to achieve fragment separation. The 

gels were stained with ethidium bromide and the DNA was visualized under UV light. A

photographic record was made for each stained gel.

Southern Transfer

DNA fragments in the agarose gels were depurinated, denatured, neutralized in situ, and 

transferred to nylon membranes in 10 SSC buffer using a wicking system. After transfer to the 

membrane, the DNA was fixed to the membrane by crosslinking through UV treatment.

Hybridization

Labeled probes were hybridized to the target DNA on the nylon membranes using the DIG Easy 

Hyb Solution according to manufacturer’s instructions (Roche Diagnostics). DIG-labeled DNA 

molecular weight marker II and VII were used to determine the hybridizing fragment size on the 

Southern blots.

Detection

DIG-labeled probes bound to the nylon membranes after stringent wash were incubated with 

Alkaline Phosphatase (AP)-conjugated anti-Digoxigenin antibody for ~1 hr at room temperature. 

The anti-DIG antibody specifically bound to the probes was then visualized using CDP-Star 

Chemiluminescent Nucleic Acid Detection System (Roche Diagnostics). Blots were exposed to 

chemiluminescent film to detect the hybridizing fragments and to visualize the molecular weight 

markers. The images were then scanned and stored. The number and size of all the detected 

band were documented for each digest and probe combination.

Once the data were recorded, membranes were rinsed with milli-Q water and then stripped of the 

bound probe in a solution containing 0.2 M NaOH and 1.0% SDS. The alkali-based stripping 

procedure successfully removes the labeled probes from the membranes, allowing them to be rehybridized with a different DNA probe.
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Appendix 2. Materials, Methods and Results for Characterization of Cry1Ac Protein

Materials and Methods

Test Substance/Test System

The test substance was the Cry1Ac protein expressed in and extracted from grain of transgenic

soybean event DAS-81419-2: Source ID: YX10MX500029.0001 (T4 generation). The seeds

were planted, grown, and harvested under DAS protocol number 110000. After harvest, the 

grain was frozen, lyophilized, ground, and stored at -20C. The presence of Cry1Ac protein in 

the soybean grain was confirmed by a commercially available lateral flow assay kit from

EnviroLogix Inc., as described below.

Control Substances

The control substance used in this study was a non-transgenic soybean grain grown from seed 

(Glycine max cv Maverick). Grain of the Maverick soybean line (Source ID:

YX10MX030001.0002 – T4 generation) were planted, grown, harvested, and processed under 

the same conditions as the transgenic plants described above. The absence of Cry1Ac protein in

the non-transgenic soybean tissue was confirmed by a commercially available lateral flow assay 

kit as described below.

Recombinant Cry1Ac microbial protein, (TSN102591, Lot #: 1757-66), has a molecular weight 

of ~130 kDa and a concentration of 144 g/mg. The microbial preparation was produced and 

purified from recombinant Pseudomonas fluorescens at Dow AgroSciences and an aliquot of the 

purified sample was sent to the Test Substance Coordinator located in Indianapolis, IN for 

dispensation. 

Reference Substances

The commercially available (non-GLP) reference substances used in this study are listed in the 

following table:

Reference Substance Product Name Lot Number Assay Reference

Mass Spectrometry 

Mass Standards Kit

Mass Standards Kit for 

Calibration of AB 

SCIEX TOF/TOF 

Instruments

A1068 Protein 

sequence 

analysis

AB SCIEX

Soybean Trypsin 

Inhibitor (STI)

A component of the 

GelCode glycoprotein 

staining kit

MH161385 Glycosylation 

assay

ThermoFisher

Horseradish 

Peroxidase (HRP)

A component of the 

GelCode glycoprotein 

staining kit

ND171686 Glycosylation 

assay

ThermoFisher

Bovine Serum 

Albumin (BSA)

Pre-diluted BSA 

protein assay standard 

set

NA165380 & 

NE170914

SDS-PAGE & 

glycosylation 

assay

ThermoFisher

Unstained Molecular 

Weight Markers

Novex Sharp 

unstained protein 

standards

1143231 SDS-PAGE Invitrogen: Molecular 

Weight Markers of 260, 

160, 110, 80, 60, 50, 40, 

30, 20, 10, and 3.5 kDa
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Reference Substance Product Name Lot Number Assay Reference

Prestained Molecular 

Weight Markers

Novex Sharp 

prestained protein 

standards

1095889, 

1141762, &

1022458

SDS-PAGE, 

western blot & 

glycosylation 

assay

Invitrogen: Molecular 

Weight Markers of 260, 

160, 110, 80, 60, 50, 40, 

30, 20, 10, and 3.5 kDa

Lateral Flow Strip Assay

The soybean grain tissues of the transgenic event DAS-81419-2 and non-transgenic Maverick 

were planted, grown, and harvested under protocol 110000 in 2011. After harvest, the samples 

were shipped frozen, ground, and stored at –20C until use. To confirm the presence/absence of 

the Cry1Ac protein in the ground grain, approximately 250-mg samples of the grain were 

weighed out in 2-mL microfuge tubes and tested by the lateral flow strip assay as described by 

Envirologix Inc. Briefly, the soluble proteins were extracted by adding 1.5 mL of EB2 

extraction buffer and grinding in a Geno-Grinder for 3 minutes at 500 strokes per minute. The 

test strips were added to the tubes and incubated at room temperature in the samples for 10 

minutes to develop. After the assay was complete, the strips were removed and allowed to air 

dry and the results were recorded.

SDS-PAGE and Western Blot

SDS-PAGE and western blot analysis of the crude protein extracts prepared from DAS-81419-2 

soybean grain and non-transgenic Maverick soybean grain were performed with Bio-Rad 

Criterion gels fitted in a Criterion Gel chamber with XT MES running buffer (Bio-Rad). 

Extracts were prepared by Geno-Grinding ~15 mg of the ground soybean grain in 1.5 mL 

PBS/Triton/ascorbic acid buffer for 3 minutes in a 2 mL micro-centrifuge tube. The supernatants 

were clarified by briefly centrifuging the samples at >20,000×g (4°C), and 250 L of each 

extract was mixed with 250 L of Laemmli sample buffer (Bio-Rad) containing freshly added -

mercaptoethanol (Bio-Rad) and heated for 5 minutes at ~95C. After a brief centrifugation (2 

min at 20,000 ×g), 40 μL of each supernatant was loaded directly on the gel. The reference 

standard, microbe-derived Cry1Ac (TSN102591), and control standard, BSA (ThermoScientific), 

were diluted to an appropriate concentration and combined with Laemmli sample buffer 

containing -mercaptoethanol. The electrophoresis was conducted at a constant voltage of 150 

V for ~60 minutes. After separation, the gel was cut in half and one half was stained with 

ThermoScientific GelCode Blue protein stain and scanned with a densitometer (GE Healthcare) 

to obtain a permanent record of the gel. The remaining half of the gel was electro-blotted to a 

nitrocellulose membrane (Bio-Rad) with a Criterion transfer cell (Bio-Rad) for 60 minutes under 

a constant voltage of 100V. The transfer buffer contained 20% methanol and Tris/glycine buffer 

from Bio-Rad. After transfer, the membrane was probed with a Cry1Ac specific polyclonal 

rabbit antibody (α-Cry1Ac PAb, NB1434-63, 1.46 mg/mL) for 60 minutes (1:2500 dilution) at 

room temperature. A 1:5000 dilution of conjugated goat anti-rabbit IgG (H+L) with horseradish 

peroxidase (ThermoScientific) was used as the secondary antibody. GE Healthcare ECL 

chemiluminescent substrate was used for development and visualization of the immunoreactive 

protein bands. The membranes were exposed to detection film (ThermoScientific) for various 

time points and subsequently developed with an All-Pro 100 Plus film developer.

Purification of Soybean-Derived Cry1Ac from Event DAS-81419-2

The Cry1Ac protein was extracted from the ground soybean grain with a modified RIPA buffer 

(phosphate buffered saline containing an additional 0.1 M NaCl, 12.5 mM EDTA, 1% Tween-20, 
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0.1% SDS, and 0.5% sodium deoxycholate) by weighing ~25 grams of ground grain into a 

chilled Waring blender cup and adding ~175 mL of buffer and 150 μL of protease inhibitor 

cocktail mix. The tissue was blended on high speed four times in 20 second pulses and the 

solution was filtered through 2 layers of pre-wetted miracloth (Calbiochem) and clarified by 

centrifugation at 38,000×g for 30 minutes. The supernatant was removed and filtered through 2 

layers of miracloth and another 150 μL of protease inhibitor cocktail was added to the extract 

and held on ice.

The Cry1Ac protein was purified from the supernatant by immuno-precipitation using 

monoclonal antibodies (cell line #158E6, lot #: 200.687-3-5 (6 mg/mL) or cell line #158E7 (5.8 

mg/ml)) cross-linked to Thermo Scientific’s Protein A/G Agarose resin at 1.0 μg of antibody per 

mL of resin. For each 15 mL of clarified supernatant, 100 μL (100 g of antibody) of coupled 

resin was added and allowed to incubate on a rotating mixer for 30 minutes at 4°C. The resin 

was recovered by centrifugation at 500×g for 5 minutes at 4°C and then resuspended in an 

additional 15 mL of extract for a total of 30 mL of extract applied to each resin preparation. The 

resin was then washed with 15 mL of extraction buffer for 15 minutes at 4oC, followed by two 

washes with 15 mL of wash buffer (PBS, 0.7 M NaCl, 25 mM EDTA, 25 mM galactose, 1% 

Tween 20). After the final wash, spin, and decant, the resin was transferred to a 1.5 mL 

Eppendorf tube with 1 mL of wash buffer and centrifuged for 2 minutes at 500×g at 4°C to pellet 

the resin. The supernatant was carefully aspirated and the pellet was washed an additional two 

times with 1 mL of PBS, 5 mM EDTA. The pelleted resin was resuspended in 1 mL of PBS, 5 

mM EDTA and 500 μL of the slurry was removed and centrifuged at 21000×g for 1 minute to 

pellet the resin. After the supernatant was removed, the resin was stored at -80°C for later use. 

From the remaining 500 μL of slurry, the resin was pelleted as described above and the bound 

proteins were eluted by incubating the resin for 5 minutes in 80 μL of 2% SDS at room 

temperature. The resin was pelleted with a brief centrifugation and 75 μL of supernatant was 

removed and the elution procedure was repeated for a total of 150 μL of eluted protein. The 

extracted resin, eluted protein, and other fractions were all stored at -80°C for future analysis.

Detection of Post-Translational Glycosylation

The immunoaffinity-purified, soybean-derived Cry1Ac protein was mixed with Laemmli sample 

buffer (37.5 μL + 12.5 μL of 4x LSB) and heated at 95°C for 10 minutes. The microbe-derived 

Cry1Ac, soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were 

diluted with 2x LSB to the approximate concentration of the purified soybean-derived Cry1Ac 

protein. After mixing the proteins with Laemmli sample buffer, the proteins were heated at 

~95C for 10 minutes and centrifuged at 20,000×g for 2 minutes to obtain a clarified supernatant. 

The resulting supernatants were applied directly to a Bio-Rad Mini-Protean TGX gel and 

electrophoresed at 100V for ~90 minutes. After electrophoresis, the gel was cut in half and one 

half was stained with GelCode Blue stain for total protein according to the manufacturers’ 

protocol. After the staining was complete, the gel was scanned with a densitometer to obtain a 

permanent visual record of the gel. The remaining half of the gel was stained with a GelCode 

Glycoprotein Staining Kit (ThermoScientific) according to the manufacturers’ protocol to 

visualize the glycoproteins. The procedure for glycoprotein staining is briefly described as 

follows: After electrophoresis, the gel was fixed in 50% methanol for 30 minutes and rinsed 

with 3% acetic acid. This was followed by an incubation period with the oxidation solution from 

the staining kit for 15 minutes. The gel was once again rinsed with 3% acetic acid and incubated 

with GelCode glycoprotein staining reagent for 15 minutes. Finally, the gel was immersed in the 
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reduction solution for 5 minutes, and rinsed with 3% acetic acid. The glycoproteins (with a 

detection limit as low as 0.625 ng per band) were visualized as magenta bands on a light pink 

background. After the glycoprotein staining was complete, the gel was scanned with a GE 

Healthcare densitometer to obtain a permanent visual record of the gel.

MALDI-TOF MS and LC/MS Peptide Mass Fingerprinting and Sequence Analysis of Soybeanand Microbe-Derived Cry1Ac

Sample preparation and deposition

Tryptic (in-gel digest), and Chymotryptic (in-gel digest) peptides were purified using Millipore 

ZipTip C18 as per manufacturer’s procedure. Purified peptides were eluted sequentially with 

aqueous 10%, 25%, 50%, and 100% ACN (supplemented with 0.1% TFA). The ZipTip C18 

fractions were mixed with 4 μL of CHCA matrix (10 mg/mL CHCA in 50% ACN supplemented 

with 0.1% TFA), and 1 μL of the sample-matrix mixture was deposited on the MALDI target and 

allowed to air dry.

MALDI-TOF MS

The sample preparations were analyzed directly by MALDI-TOF mass spectrometry. All mass 

spectra were acquired on an AB Sciex 4800 MALDI-TOF/TOF mass spectrometer (S/N 

AK011030605H). Mass calibration was performed with a mass standards kit for calibration of 

AB SCIEX TOF/TOF instruments. The plate wide calibration model was used for MS 

calibration.

MALDI-TOF MS, Electrospray Ionization-Liquid Chromatography Mass Spectrometry (ESILC/MS), glycoprotein analysis, and N-terminal and internal protein sequence analysis of the 

microbe-derived Cry1Ac (TSN102591) was conducted previously at Dow AgroSciences.

Results

Lateral Flow Strip Assay

The presence of the Cry1Ac protein in the ground grain of DAS-81419-2 soybean was confirmed 

using commercially prepared lateral flow strips from EnviroLogix Inc. The strips, capable of 

detecting one transgenic seed in 400, easily discriminated between transgenic and non-transgenic 

tissue. The extracts prepared from DAS-81419-2 soybean tested positive for Cry1Ac protein and 

the non-transgenic extracts of Maverick did not contain detectable immunoreactive protein 

(Figure 40). This result was also confirmed by the western blot analysis using polyclonal 

antibodies specific to the Cry1Ac protein (Figure 41).
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Figure 40. Lateral flow strip assay for the Cry1Ac protein expression in Event DAS-81419-

2 and Maverick grain extracts.

Western Blot Analysis of Transgenic Grain Extracts

The microbe-derived Cry1Ac protein showed a positive signal of the expected size (the fulllength Cry1Ac protein is ~130 kDa) by polyclonal antibody western blot analysis (Figure 41). 

This was also observed in the DAS-81419-2 transgenic soybean grain extract whereas the nontransgenic Maverick extract did not contain any immunoreactive proteins. In the Cry1Ac 

western blot analysis, some immunoreactive proteins of truncated Cry1Ac were observed in the 

microbe-derived standard; however no alternate size Cry1Ac proteins (aggregates or degradation 

products) were observed in the transgenic samples (Figure 41). These results add to the evidence 

that the protein expressed in soybean is not glycosylated or post-translationally modified which 

would add to or subtract from the overall protein molecular weight.

Event

DAS-81419-2 Maverick

Control Line

Test Line
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Figure 41. Western blot analysis of microbe- and soybean-derived Cry1Ac proteins.

Lane Sample Amount Loaded

M Novex unstained MW markers 10 L

 1 (gel) Bovine serum albumin 1.3 μg 

 1 (blot) Bovine serum albumin 2.5 ng

 2 (gel) Cry1Ac protein standard (TSN102591) 0.72 g

 2 (blot) Cry1Ac protein standard (TSN102591) 1.9 ng

 3 Maverick crude (non-transgenic) grain extract 40 L

 4 DAS-81419-2 crude (transgenic) grain extract 40 L

Purification of the Cry1Ac Protein from Transgenic Event DAS-81419-2 Grain Extracts

Immunoaffinity precipitation was conducted on an aqueous extract of ~25 grams of ground 

DAS-81419-2 soybean grain. The protein that bound to the monoclonal antibody columns was 

eluted from the column and examined by SDS-PAGE which demonstrated that the final 

concentrated fractions contained the intact Cry1Ac protein at an approximate molecular weight 

of ~130 kDa and fragments that contain the active core toxin (Figure 42). Once isolated, the 

soybean-derived Cry1Ac was compared with the microbe-derived protein.

SDS-PAGE Analysis of the Immuno-purified Cry1Ac

In the toxicology-lot preparation of P. fluorescens-produced Cry1Ac (TSN102591), the major 

protein bands, as visualized on Coomassie stained SDS-PAGE gels, were approximately 130, 

110, and 90 kDa. As expected, the corresponding soybean-derived Cry1Ac protein was identical 

in size to the full-length microbe-expressed protein (Figure 42). Predictably, the plant purified 

fractions contained a minor amount of impurities in addition to the full-length and truncated 

Cry1Ac core protein. The co-eluted proteins were likely retained on the column by weak 

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

Coomassie Stained Ge l Western Blot Film

M 1 2 3 4 1 2 3 4

Cry1Ac

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

- 3.5

kDa
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interactions with the column matrix or antibody leaching off of the column under the elution 

conditions. Other researchers have also reported the non-specific adsorption of proteins, 

peptides, and amino acids on activated agarose immuno-adsorbents (Holroyde et al., 1976; 

Kennedy and Barnes, 1983; Williams et al., 2006) as well as antibody leaching from the column 

(Goldberg et al., 1991).

Figure 42. SDS-PAGE analysis of immunoaffinity-purified soybean-derived Cry1Ac.

Lane Sample Amount Loaded

M Novex prestained MW markers 10 L

 1 Bovine serum albumin ~1 μg 

 2 DAS-81419-2 crude grain extract 3 L

 3 Cry1Ac protein standard (TSN102591) ~1 g

 4 Soybean-derived Cry1Ac (DAS-81419-2) 20 L

 5 Soybean-derived Cry1Ac (DAS-81419-2) 20 L

 6 Soybean-derived Cry1Ac (DAS-81419-2) 20 L

 7 Soybean-derived Cry1Ac (DAS-81419-2) 20 L

Detection of Glycosylation

Potential protein glycosylation of soybean-derived Cry1Ac was assessed with a GelCode 

Glycoprotein Staining Kit from ThermoScientific. The immunoaffinity-purified Cry1Ac protein 

was electrophoresed simultaneously with a set of control and reference protein standards. A 

glycoprotein, horseradish peroxidase, was loaded as a positive indicator for glycosylation, and 

non-glycoproteins, microbe-derived Cry1Ac, soybean trypsin inhibitor, and bovine serum 

albumin, were employed as negative controls. The results showed that both the soybean- and 

microbe-derived Cry1Ac proteins had no detectable covalently linked carbohydrates (Figure 43).

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

Coomassie Stained Gel

M 1 2 3 4 5 6 7

Cry1Ac

Cry1Ac
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Figure 43. SDS-PAGE analysis of microbe- and soybean-derived Cry1Ac protein stained 

with GelCode Blue total protein and glycoprotein stains.

Lane Sample Amount Loaded

M Novex prestained MW markers 10 L

 1 Cry1Ac protein standard (TSN102591) ~1 μg 

 2 Soybean-derived Cry1Ac (DAS-81419-2) 20 L

 3 Bovine serum albumin (- control) 0.5 g

 4 Horseradish peroxidase (+ control) 0.5 g

 5 Soybean trypsin inhibitor (- control) 0.5 g

MALDI-TOF MS and MS/MS Peptide Mass Fingerprinting and Sequence Analysis

The Cry1Ac protein derived from DAS-81419-2 soybean tissue was separated by SDS-PAGE 

(Figure 42) and the respective Cry1Ac bands were excised and subjected to in-gel digestion by 

trypsin and chymotrypsin. The resulting peptide mixture was analyzed by MALDI-TOF MS and 

sequence verified by MS/MS to determine the peptide sequences. The masses of the detected 

peptides were compared with expected masses based on trypsin or chymotrypsin cleavage sites

within the sequence of the soybean-derived Cry1Ac protein. Figure 44 and Figure 45 illustrates 

the theoretical cleavage which was generated in silico using Protein Analysis Worksheet 

(PAWS) freeware from Proteometrics LLC. The Cry1Ac protein, once denatured, is readily 

digested by endoproteases to yield numerous peptides that are able to be detected using mass 

spectrometry.

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

Coomassie Stained Ge l Glycoprotein Stained Gel

M 1 2 3 4 5 1 2 3 4 5

HRP
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Figure 44. Theoretical cleavage of the Cry1Ac protein with trypsin generated in silico using 

Protein Analysis Worksheet (PAWS) from Proteometrics LLC.
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Figure 45. Theoretical cleavage of the Cry1Ac protein with chymotrypsin generated in 

silico using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.
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In the endoprotease digests of the transgenic-soybean-derived Cry1Ac protein, the peptide 

sequence coverage from peptide mass fingerprint (PMF) data was extensive at 86.1%. Of the 

86.1% sequence coverage from PMF data, 83.2% was confirmed by tandem mass spectrometry 

sequencing. The detected peptide fragments covered nearly the entire protein sequence with 

only a few peptide fragments undetected (Figure 46 - Figure 48). This analysis confirmed the 

soybean-derived protein amino acid sequence matched that of the microbe-derived Cry1Ac 

protein near the N- and C-terminus as well as a major portion of the internal sequence. In the 

MS spectra, there were unidentified peptides detected in the enzyme digest preparations (data not 

shown). Many factors contribute to the formation of these unidentified peptides, such as over 

digestion (which results in non-specific cleavage), self-digestion products of trypsin and 

chymotrypsin, as well as random breakage of peptides during ionization. Unidentified peptides 

do not indicate the protein is different from the predicted amino acid sequence. The results of 

these analyses indicate that the amino acid sequence of the soybean-derived Cry1Ac protein was 

equivalent to the P. fluorescens-expressed protein previously characterized.
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Figure 46. Tryptic digest sequence coverage map for Cry1Ac (DAS-81419-2) by MALDITOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. 

Sequence coverage was 67.9% with PMF data and 67.9% by tandem MS. 

Cry1Ac peptide mapping - trypsin Protocol 110840

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E 30

Trypsin 15 15

31 T G Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L 60

Trypsin 0 0

61 V D I I W G I F G P S Q W D A F L V Q I E Q L I N Q R I E E 90

Trypsin 3 3

91 F A R N Q A I S R L E G L S N L Y Q I Y A E S F R E W E A D 120

Trypsin 30 30

121 P T N P A L R E E M R I Q F N D M N S A L T T A I P L F A V 150

Trypsin 7 7

151 Q N Y Q V P L L S V Y V Q A A N L H L S V L R D V S V F G Q 180

Trypsin 7 7

181 R W G F D A A T I N S R Y N D L T R L I G N Y T D Y A V R W 210

Trypsin 30 30

211 Y N T G L E R V W G P D S R D W V R Y N Q F R R E L T L T V 240

Trypsin 30 30

241 L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N 270

Trypsin 30 30

271 P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L 300

Trypsin 27 27

301 N S I T I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G 330

Trypsin 11 11

331 P E F T F P L Y G T M G N A A P Q Q R I V A Q L G Q G V Y R 360

Trypsin 11 11

361 T L S S T L Y R R P F N I G I N N Q Q L S V L D G T E F A Y 390

Trypsin 30 30

391 G T S S N L P S A V Y R K S G T V D S L D E I P P Q N N N V 420

Trypsin 29 29

421 P P R Q G F S H R L S H V S M F R S G F S N S S V S I I R A 450

Trypsin 30 30

451 P M F S W I H R S A E F N N I I A S D S I T Q I P A V K G N 480

Trypsin 30 30

481 F L F N G S V I S G P G F T G G D L V R L N S S G N N I Q N 510

Trypsin 30 30

511 R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H 540

Trypsin 16 16

541 L N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F 570

Trypsin 0 0

571 G Y F E S A N A F T S S L G N I V G V R N F S G T A G V I I 600

Trypsin 10 10

601 D R F E F I P V T A T L E A E S D L E R A Q K A V N A L F T 630

Trypsin 27 27

631 S S N Q I G L K T D V T D Y H I D R V S N L V E C L S D E F 660

Trypsin 30 30

661 C L D E K K E L S E K V K H A K R L S D E R N L L Q D P N F 690

Trypsin 19 19

691 R G I N R Q L D R G W R G S T D I T I Q G G D D V F K E N Y 720

Trypsin 16 16

721 V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y T R Y 750

Trypsin 1 1

751 Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G 780

Trypsin 26 26

781 T G S L W P L S A P S P I G K C A H H S H H F S L D I D V G 810

Trypsin 15 15

811 C T D L N E D L G V W V I F K I K T Q D G H A R L G N L E F 840

Trypsin 6 6

841 L E E K P L V G E A L A R V K R A E K K W R D K R E K L E W 870

Trypsin 18 18

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
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Figure 46 (Cont). Tryptic digest sequence coverage map for Cry1Ac (DAS-81419-2) by 

MALDI-TOF MS and MALDI TOF/TOF.

511 R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H 540

Trypsin 16 16

541 L N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F 570

Trypsin 0 0

571 G Y F E S A N A F T S S L G N I V G V R N F S G T A G V I I 600

Trypsin 10 10

601 D R F E F I P V T A T L E A E S D L E R A Q K A V N A L F T 630

Trypsin 27 27

631 S S N Q I G L K T D V T D Y H I D R V S N L V E C L S D E F 660

Trypsin 30 30

661 C L D E K K E L S E K V K H A K R L S D E R N L L Q D P N F 690

Trypsin 19 19

691 R G I N R Q L D R G W R G S T D I T I Q G G D D V F K E N Y 720

Trypsin 16 16

721 V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y T R Y 750

Trypsin 1 1

751 Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G 780

Trypsin 26 26

781 T G S L W P L S A P S P I G K C A H H S H H F S L D I D V G 810

Trypsin 15 15

811 C T D L N E D L G V W V I F K I K T Q D G H A R L G N L E F 840

Trypsin 6 6

841 L E E K P L V G E A L A R V K R A E K K W R D K R E K L E W 870

Trypsin 18 18

871 E T N I V Y K E A K E S V D A L F V N S Q Y D R L Q A D T N 900

Trypsin 27 27

901 I A M I H A A D K R V H S I R E A Y L P E L S V I P G V N A 930

Trypsin 29 29

931 A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N N 960

Trypsin 30 30

961 G L S C W N V K G H V D V E E Q N N H R S V L V V P E W E A 990

Trypsin 30 30

991 E V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V 1020

Trypsin 25 25

1021 T I H E I E N N T D E L K F S N C V E E E V Y P N N T V T C 1050

Trypsin 30 30

1051 N D Y T A T Q E E Y E G T Y T S R N R G Y D G A Y E S N S S 1080

Trypsin 28 28

1081 V P A D Y A S A Y E E K A Y T D G R R D N P C E S N R G Y G 1110

Trypsin 30 30

1111 D Y T P L P A G Y V T K E L E Y F P E T D K V W I E I G E T 1140

Trypsin 22 22

1141 E G T F I V D S V E L L L M E E 1156

Trypsin 0 0

Identified in MS Analysis Total Residues Observed 785 785

Identified in MS and MS/MS Analysis Trypsin Percent Coverage 67.9% 67.9%
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Figure 47. Chymotryptic digest sequence coverage map for Cry1Ac (DAS-81419-2) by 

MALDI-TOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. 

Sequence coverage was 53.5% with PMF data and 49.0% by tandem MS.

Cry1Ac peptide mapping - chymotrypsin Protocol 110840

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E 30

Chymotrypsin 0 0

31 T G Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L 60

Chymotrypsin 15 15

61 V D I I W G I F G P S Q W D A F L V Q I E Q L I N Q R I E E 90

Chymotrypsin 27 27

91 F A R N Q A I S R L E G L S N L Y Q I Y A E S F R E W E A D 120

Chymotrypsin 8 8

121 P T N P A L R E E M R I Q F N D M N S A L T T A I P L F A V 150

Chymotrypsin 16 0

151 Q N Y Q V P L L S V Y V Q A A N L H L S V L R D V S V F G Q 180

Chymotrypsin 30 25

181 R W G F D A A T I N S R Y N D L T R L I G N Y T D Y A V R W 210

Chymotrypsin 9 0

211 Y N T G L E R V W G P D S R D W V R Y N Q F R R E L T L T V 240

Chymotrypsin 16 16

241 L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N 270

Chymotrypsin 20 20

271 P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L 300

Chymotrypsin 10 10

301 N S I T I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G 330

Chymotrypsin 22 21

331 P E F T F P L Y G T M G N A A P Q Q R I V A Q L G Q G V Y R 360

Chymotrypsin 8 8

361 T L S S T L Y R R P F N I G I N N Q Q L S V L D G T E F A Y 390

Chymotrypsin 23 23

391 G T S S N L P S A V Y R K S G T V D S L D E I P P Q N N N V 420

Chymotrypsin 30 30

421 P P R Q G F S H R L S H V S M F R S G F S N S S V S I I R A 450

Chymotrypsin 26 16

451 P M F S W I H R S A E F N N I I A S D S I T Q I P A V K G N 480

Chymotrypsin 30 25

481 F L F N G S V I S G P G F T G G D L V R L N S S G N N I Q N 510

Chymotrypsin 1 1

511 R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H 540

Chymotrypsin 27 27

541 L N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F 570

Chymotrypsin 5 5

571 G Y F E S A N A F T S S L G N I V G V R N F S G T A G V I I 600

Chymotrypsin 21 21

601 D R F E F I P V T A T L E A E S D L E R A Q K A V N A L F T 630

Chymotrypsin 30 30

631 S S N Q I G L K T D V T D Y H I D R V S N L V E C L S D E F 660

Chymotrypsin 30 30

661 C L D E K K E L S E K V K H A K R L S D E R N L L Q D P N F 690

Chymotrypsin 0 0

691 R G I N R Q L D R G W R G S T D I T I Q G G D D V F K E N Y 720

Chymotrypsin 15 15

721 V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y T R Y 750

Chymotrypsin 13 13

751 Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G 780

Chymotrypsin 14 10

781 T G S L W P L S A P S P I G K C A H H S H H F S L D I D V G 810

Chymotrypsin 7 7

811 C T D L N E D L G V W V I F K I K T Q D G H A R L G N L E F 840

Chymotrypsin 14 14

841 L E E K P L V G E A L A R V K R A E K K W R D K R E K L E W 870

Chymotrypsin 9 9

871 E T N I V Y K E A K E S V D A L F V N S Q Y D R L Q A D T N 900

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
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Figure 47 (Cont). Chymotryptic digest sequence coverage map for Cry1Ac (DAS-81419-2) 

by MALDI-TOF MS and MALDI TOF/TOF.

Chymotrypsin 27 27

541 L N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F 570

Chymotrypsin 5 5

571 G Y F E S A N A F T S S L G N I V G V R N F S G T A G V I I 600

Chymotrypsin 21 21

601 D R F E F I P V T A T L E A E S D L E R A Q K A V N A L F T 630

Chymotrypsin 30 30

631 S S N Q I G L K T D V T D Y H I D R V S N L V E C L S D E F 660

Chymotrypsin 30 30

661 C L D E K K E L S E K V K H A K R L S D E R N L L Q D P N F 690

Chymotrypsin 0 0

691 R G I N R Q L D R G W R G S T D I T I Q G G D D V F K E N Y 720

Chymotrypsin 15 15

721 V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y T R Y 750

Chymotrypsin 13 13

751 Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G 780

Chymotrypsin 14 10

781 T G S L W P L S A P S P I G K C A H H S H H F S L D I D V G 810

Chymotrypsin 7 7

811 C T D L N E D L G V W V I F K I K T Q D G H A R L G N L E F 840

Chymotrypsin 14 14

841 L E E K P L V G E A L A R V K R A E K K W R D K R E K L E W 870

Chymotrypsin 9 9

871 E T N I V Y K E A K E S V D A L F V N S Q Y D R L Q A D T N 900

Chymotrypsin 11 11

901 I A M I H A A D K R V H S I R E A Y L P E L S V I P G V N A 930

Chymotrypsin 12 12

931 A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N N 960

Chymotrypsin 16 13

961 G L S C W N V K G H V D V E E Q N N H R S V L V V P E W E A 990

Chymotrypsin 5 5

991 E V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V 1020

Chymotrypsin 7 7

1021 T I H E I E N N T D E L K F S N C V E E E V Y P N N T V T C 1050

Chymotrypsin 0 0

1051 N D Y T A T Q E E Y E G T Y T S R N R G Y D G A Y E S N S S 1080

Chymotrypsin 23 23

1081 V P A D Y A S A Y E E K A Y T D G R R D N P C E S N R G Y G 1110

Chymotrypsin 30 30

1111 D Y T P L P A G Y V T K E L E Y F P E T D K V W I E I G E T 1140

Chymotrypsin 23 23

1141 E G T F I V D S V E L L L M E E 1156

Chymotrypsin 16 16

Identified in MS Analysis 619 566

Identified in MS and MS/MS Analysis Chymotrypsin Percent Coverage 53.5% 49.0%
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Figure 48. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1Ac 

(DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF. Overall sequence coverage 

was 86.1% with PMF data and 83.2% by tandem MS.

Cry1Ac peptide mapping - overall Protocol 110840

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M D N N P N I N E C I P Y N C L S N P E V E V L G G E R I E 30

Trypsin 15 15

Chymotrypsin 0 0

Overall 15 15

31 T G Y T P I D I S L S L T Q F L L S E F V P G A G F V L G L 60

Trypsin 0 0

Chymotrypsin 15 15

Overall 15 15

61 V D I I W G I F G P S Q W D A F L V Q I E Q L I N Q R I E E 90

Trypsin 3 3

Chymotrypsin 27 27

Overall 27 27

91 F A R N Q A I S R L E G L S N L Y Q I Y A E S F R E W E A D 120

Trypsin 30 30

Chymotrypsin 8 8

Overall 30 30

121 P T N P A L R E E M R I Q F N D M N S A L T T A I P L F A V 150

Trypsin 7 7

Chymotrypsin 16 0

Overall 23 7

151 Q N Y Q V P L L S V Y V Q A A N L H L S V L R D V S V F G Q 180

Trypsin 7 7

Chymotrypsin 30 25

Overall 30 27

181 R W G F D A A T I N S R Y N D L T R L I G N Y T D Y A V R W 210

Trypsin 30 30

Chymotrypsin 9 0

Overall 30 30

211 Y N T G L E R V W G P D S R D W V R Y N Q F R R E L T L T V 240

Trypsin 30 30

Chymotrypsin 16 16

Overall 30 30

241 L D I V A L F P N Y D S R R Y P I R T V S Q L T R E I Y T N 270

Trypsin 30 30

Chymotrypsin 20 20

Overall 30 30

271 P V L E N F D G S F R G S A Q G I E R S I R S P H L M D I L 300

Trypsin 27 27

Chymotrypsin 10 10

Overall 27 27

301 N S I T I Y T D A H R G Y Y Y W S G H Q I M A S P V G F S G 330

Trypsin 11 11

Chymotrypsin 22 21

Overall 28 27

331 P E F T F P L Y G T M G N A A P Q Q R I V A Q L G Q G V Y R 360

Trypsin 11 11

Chymotrypsin 8 8

Overall 19 19

361 T L S S T L Y R R P F N I G I N N Q Q L S V L D G T E F A Y 390

Trypsin 30 30

Chymotrypsin 23 23

Overall 30 30

391 G T S S N L P S A V Y R K S G T V D S L D E I P P Q N N N V 420

Trypsin 29 29

Chymotrypsin 30 30

Overall 30 30

421 P P R Q G F S H R L S H V S M F R S G F S N S S V S I I R A 450

Trypsin 30 30

Chymotrypsin 26 16

Overall 30 30

451 P M F S W I H R S A E F N N I I A S D S I T Q I P A V K G N 480

Trypsin 30 30

Chymotrypsin 30 25

Overall 30 30

481 F L F N G S V I S G P G F T G G D L V R L N S S G N N I Q N 510

Trypsin 30 30

Chymotrypsin 1 1

Overall 30 30

511 R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H 540

Trypsin 16 16

Chymotrypsin 27 27

Overall 30 30

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
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Figure 48 (Cont). Overall sequence coverage of trypsin and chymotrypsin digests for 

Cry1Ac (DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF.

361 T L S S T L Y R R P F N I G I N N Q Q L S V L D G T E F A Y 390

Trypsin 30 30

Chymotrypsin 23 23

Overall 30 30

391 G T S S N L P S A V Y R K S G T V D S L D E I P P Q N N N V 420

Trypsin 29 29

Chymotrypsin 30 30

Overall 30 30

421 P P R Q G F S H R L S H V S M F R S G F S N S S V S I I R A 450

Trypsin 30 30

Chymotrypsin 26 16

Overall 30 30

451 P M F S W I H R S A E F N N I I A S D S I T Q I P A V K G N 480

Trypsin 30 30

Chymotrypsin 30 25

Overall 30 30

481 F L F N G S V I S G P G F T G G D L V R L N S S G N N I Q N 510

Trypsin 30 30

Chymotrypsin 1 1

Overall 30 30

511 R G Y I E V P I H F P S T S T R Y R V R V R Y A S V T P I H 540

Trypsin 16 16

Chymotrypsin 27 27

Overall 30 30

541 L N V N W G N S S I F S N T V P A T A T S L D N L Q S S D F 570

Trypsin 0 0

Chymotrypsin 5 5

Overall 5 5

571 G Y F E S A N A F T S S L G N I V G V R N F S G T A G V I I 600

Trypsin 10 10

Chymotrypsin 21 21

Overall 21 21

601 D R F E F I P V T A T L E A E S D L E R A Q K A V N A L F T 630

Trypsin 27 27

Chymotrypsin 30 30

Overall 30 30

631 S S N Q I G L K T D V T D Y H I D R V S N L V E C L S D E F 660

Trypsin 30 30

Chymotrypsin 30 30

Overall 30 30

661 C L D E K K E L S E K V K H A K R L S D E R N L L Q D P N F 690

Trypsin 19 19

Chymotrypsin 0 0

Overall 19 19

691 R G I N R Q L D R G W R G S T D I T I Q G G D D V F K E N Y 720

Trypsin 16 16

Chymotrypsin 15 15

Overall 17 17

721 V T L L G T F D E C Y P T Y L Y Q K I D E S K L K A Y T R Y 750

Trypsin 1 1

Chymotrypsin 13 13

Overall 14 14

751 Q L R G Y I E D S Q D L E I Y L I R Y N A K H E T V N V P G 780

Trypsin 26 26

Chymotrypsin 14 10

Overall 27 26

781 T G S L W P L S A P S P I G K C A H H S H H F S L D I D V G 810

Trypsin 15 15

Chymotrypsin 7 7

Overall 22 22

811 C T D L N E D L G V W V I F K I K T Q D G H A R L G N L E F 840

Trypsin 6 6

Chymotrypsin 14 14

Overall 20 20

841 L E E K P L V G E A L A R V K R A E K K W R D K R E K L E W 870

Trypsin 18 18

Chymotrypsin 9 9

Overall 22 22

871 E T N I V Y K E A K E S V D A L F V N S Q Y D R L Q A D T N 900

Trypsin 27 27

Chymotrypsin 11 11

Overall 30 30

901 I A M I H A A D K R V H S I R E A Y L P E L S V I P G V N A 930

Trypsin 29 29

Chymotrypsin 12 12

Overall 29 29

931 A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N N 960

Trypsin 30 30

Chymotrypsin 16 13

Overall 30 30

961 G L S C W N V K G H V D V E E Q N N H R S V L V V P E W E A 990

Trypsin 30 30
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Figure 48 (Cont). Overall sequence coverage of trypsin and chymotrypsin digests for 

Cry1Ac (DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF.

Trypsin 6 6

Chymotrypsin 14 14

Overall 20 20

841 L E E K P L V G E A L A R V K R A E K K W R D K R E K L E W 870

Trypsin 18 18

Chymotrypsin 9 9

Overall 22 22

871 E T N I V Y K E A K E S V D A L F V N S Q Y D R L Q A D T N 900

Trypsin 27 27

Chymotrypsin 11 11

Overall 30 30

901 I A M I H A A D K R V H S I R E A Y L P E L S V I P G V N A 930

Trypsin 29 29

Chymotrypsin 12 12

Overall 29 29

931 A I F E E L E G R I F T A F S L Y D A R N V I K N G D F N N 960

Trypsin 30 30

Chymotrypsin 16 13

Overall 30 30

961 G L S C W N V K G H V D V E E Q N N H R S V L V V P E W E A 990

Trypsin 30 30

Chymotrypsin 5 5

Overall 30 30

991 E V S Q E V R V C P G R G Y I L R V T A Y K E G Y G E G C V 1020

Trypsin 25 25

Chymotrypsin 7 7

Overall 29 29

1021 T I H E I E N N T D E L K F S N C V E E E V Y P N N T V T C 1050

Trypsin 30 30

Chymotrypsin 0 0

Overall 30 30

1051 N D Y T A T Q E E Y E G T Y T S R N R G Y D G A Y E S N S S 1080

Trypsin 28 28

Chymotrypsin 23 23

Overall 30 30

1081 V P A D Y A S A Y E E K A Y T D G R R D N P C E S N R G Y G 1110

Trypsin 30 30

Chymotrypsin 30 30

Overall 30 30

1111 D Y T P L P A G Y V T K E L E Y F P E T D K V W I E I G E T 1140

Trypsin 22 22

Chymotrypsin 23 23

Overall 30 30

1141 E G T F I V D S V E L L L M E E 1156

Trypsin 0 0

Chymotrypsin 16 4

Overall 16 4

Identified in Trypsin MS Analysis Total Residues Observed 995 962

Identified in Trypsin MS and MS/MS Analysis

Overall Percent Coverage 86.1% 83.2%

Identified in Chymo MS Analysis

Identified in Chymo MS and MS/MS Analysis

Identified in Overall MS Analysis

Identified in Overall MS and MS/MS Analysis
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Conclusions

It was demonstrated that the biochemical identity of P. fluorescens-produced Cry1Ac protein 

was equivalent to the protein purified from grain of soybean event DAS-81419-2. Both the 

soybean- and microbe-derived Cry1Ac proteins had an apparent molecular weight of ~130 kDa

and were immunoreactive to Cry1Ac protein-specific antibodies in both lateral flow strip and 

Western blot assays. The amino acid sequences were confirmed by enzymatic peptide mass 

fingerprinting by MALDI-TOF MS and verified by MS/MS. In addition, the lack of 

glycosylation of the soybean-derived Cry1Ac protein provided additional evidence that the

Cry1Ac protein produced by P. fluorescens and transgenic soybean were essentially equivalent 

molecules.
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Appendix 3. Materials, Methods and Results for Characterization of Cry1F Protein

Materials and Methods

Test Substance/Test System

The test substance was the Cry1F protein expressed and extracted from grain of transgenic 

soybean event DAS-81419-2: Source ID: YX10MX500029.0001 (T4 generation). The seeds

were planted, grown, and harvested under DAS protocol number 110000. After harvest, the 

grain was frozen, lyophilized, ground, and stored at -20C. The presence of Cry1F protein in the 

soybean grain was confirmed by a commercially available lateral flow assay kit from

EnviroLogix Inc., as described below.

Control Substances

The control substance used in this study was a non-transgenic soybean grain grown from seed 

(Glycine max cv Maverick). Grain of the Maverick soybean line (Source ID: 

YX10MX030001.0002) were planted, grown, harvested, and processed under the same 

conditions as the transgenic plants described above. The absence of Cry1F protein in the nontransgenic soybean tissue was confirmed by a commercially available lateral flow assay kit as 

described below.

Recombinant Cry1F microbial protein, (TSN103748, Lot #: 020404), has a molecular weight of 

~130 kDa and a concentration of 233 g/mg. The microbial preparation was produced and 

purified from recombinant Pseudomonas fluorescens at Dow AgroSciences and the purified 

Cry1F protein was sent to the Test Substance Coordinator located in Indianapolis.

Reference Substances

The commercially available (non-GLP) reference substances used in this study are listed in the 

following table:

Reference Substance Product Name Lot Number Assay Reference

Mass Spectrometry 

Mass Standards Kit

Mass Standards Kit 

for Calibration of AB 

SCIEX TOF/TOF 

Instruments

A1068 Protein 

sequence 

analysis

AB SCIEX

Soybean Trypsin 

Inhibitor (STI)

A component of the 

GelCode glycoprotein 

staining kit

MH161385 Glycosylation 

assay

ThermoFisher

Horseradish 

Peroxidase (HRP)

A component of the 

GelCode glycoprotein 

staining kit

ND171686 Glycosylation 

assay

ThermoFisher

Bovine Serum 

Albumin (BSA)

Pre-diluted BSA 

protein assay standard 

set

NA165380 

and 

NE170914

SDS-PAGE 

& 

glycosylation 

assay

ThermoFisher

Unstained Molecular 

Weight Markers

Novex Sharp 

unstained protein 

standards

1143231 SDS-PAGE Invitrogen: Molecular 

Weight Markers of 260, 160, 

110, 80, 60, 50, 40, 30, 20, 

10, and 3.5 kDa
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Reference Substance Product Name Lot Number Assay Reference

Prestained Molecular 

Weight Markers

Novex Sharp 

prestained protein 

standards

1095889 & 

1022458

SDS-PAGE, 

western blot 

& 

glycosylation 

assay

Invitrogen: Molecular 

Weight Markers of 260, 160, 

110, 80, 60, 50, 40, 30, 20, 

10, and 3.5 kDa

Lateral Flow Test Strip Assay

The soybean grain tissues of the transgenic event and non-transgenic Maverick were planted, 

grown and harvested under protocol 110000 in 2011. The samples after harvest were shipped

frozen, ground, and stored at -20C until use. To confirm the presence/absence of the Cry1F 

protein in the ground grain, approximately 250-mg samples of the grain were weighed out in 2-

mL micro-centrifuge tubes and tested by the lateral flow test strip assay as described by 

Envirologix Inc. Briefly, the soluble proteins were extracted by adding 1.5 mL of EB2 

extraction buffer and grinding in a Geno-Grinder for 3 minutes at 500 strokes per minute at room 

temperature. The test strips were added to the tubes and developed for 10 minutes. After the 

assay was complete, the strips were removed and allowed to air dry and the results were 

recorded.

SDS-PAGE and Western Blot

SDS-PAGE and western blot analysis of the crude protein extracts prepared from the transgenic 

DAS-81419-2 soybean grain and non-transgenic Maverick soybean grain were performed with 

Bio-Rad Criterion gels fitted in a Criterion Gel chamber with XT MES running buffer (Bio-Rad). 

Extracts were prepared by geno-grinding ~15 mg of the ground soybean grain in 1.5 mL 

PBS/Triton/ascorbic acid buffer for 3 minutes in a 2 mL micro-centrifuge tube. The supernatants 

were clarified by briefly centrifuging the samples at >20,000×g (4°C), and 250 L of each 

extract was mixed with 250 L of Laemmli sample buffer (Bio-Rad) containing freshly added -

mercaptoethanol (Bio-Rad) and heated for 5 minutes at ~95C. After a brief centrifugation (2 

min at 20,000 ×g), 40 μL of each supernatant was loaded directly on the gel. The reference 

standard, microbe-derived Cry1F (TSN103748), and control standard, BSA (ThermoScientific), 

were diluted to an appropriate concentration and combined with Laemmli sample buffer 

containing -mercaptoethanol. The electrophoresis was conducted at a constant voltage of 150 

V for ~60 minutes. After separation, the gel was cut in half and one half was stained with 

ThermoScientific GelCode Blue protein stain and scanned with a densitometer (GE Healthcare) 

to obtain a permanent record of the gel. The remaining half of the gel was electro-blotted to a 

nitrocellulose membrane (Bio-Rad) with a Criterion transfer cell (Bio-Rad) for 60 minutes under 

a constant voltage of 100 volts. The transfer buffer contained 20% methanol and Tris/glycine 

buffer from Bio-Rad. After transfer, the membrane was probed with a Cry1F specific polyclonal 

rabbit antibody (α-Cry1F PAb, Lot#: 200.310-4-63, 6.48 mg/mL) for 60 minutes (1:5000 

dilution) at room temperature. A 1:5000 dilution of conjugated goat anti-rabbit IgG (H+L) with 

horseradish peroxidase (ThermoScientific) was used as the secondary antibody. GE Healthcare 

ECL chemiluminescent substrate was used for development and visualization of the 

immunoreactive protein bands. The membranes were exposed to detection film 

(ThermoScientific) for various time points and subsequently developed with an All-Pro 100 Plus 

film developer.
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Purification of Soybean-Derived Cry1F from Event DAS-81419-2

The Cry1F protein was extracted from the ground soybean grain with a modified RIPA buffer 

[phosphate buffered saline containing an additional 0.1 M NaCl, 12.5 mM EDTA, 1% Tween-20, 

0.1% SDS, and 0.5% sodium deoxycholate] by weighing ~25 grams of ground grain into a 

chilled Waring blender cup and adding ~175 mL of buffer and 150 μL of a broad spectrum 

protease inhibitor cocktail mix. The tissue was blended on high speed four times in 20 second 

pulses and the solution was filtered through 2 layers of pre-wetted miracloth (Calbiochem) and 

clarified by centrifugation at 38,000×g for 30 minutes. The supernatant was removed and 

filtered through 2 layers of miracloth and another 150 μL of protease inhibitor cocktail was 

added to the extract and held on ice.

The Cry1F protein was purified from the supernatant by immuno-precipitation using monoclonal 

antibodies (cell lines #96A22.2 and 96A19) cross-linked to Thermo Scientific’s Protein A/G 

Agarose resin at 1.0 μg of antibody per mL of resin. For each 15 mL of clarified supernatant, 

100 μL (100 g of antibody) of coupled resin was added and allowed to incubate on a rotating 

mixer for 30 minutes at 4°C. The resin was recovered by centrifugation at 500×g for 5 minutes 

at 4°C and then resuspended in an additional 15 mL of extract for a total of 30 mL of extract 

applied to each resin preparation. The resin was then washed with 15 mL of extraction buffer for 

15 minutes at 4°C, followed by two washes with 15 mL of wash buffer (PBS, 0.7 M NaCl, 25 

mM EDTA, 25 mM galactose, 1% Tween 20). After the final wash, spin, and decant, the resin 

was transferred to a 1.5 mL Eppendorf tube with 1 mL of wash buffer and centrifuged for 2 

minutes at 500×g at 4°C to pellet the resin. The supernatant was carefully aspirated and the 

pellet was washed an additional two times with 1 mL of PBS, 5 mM EDTA. The pelleted resin 

was resuspended in 1 mL of PBS, 5 mM EDTA and 500 μL of the slurry was removed and 

subjected to centrifugation at 21,000×g for 1 minute to pellet the resin. After the supernatant 

was removed the resin was stored at -80°C. From the remaining 500 μL of slurry, the resin was 

pelleted as described above and the bound proteins were eluted by incubating the resin for 5 

minutes in 80 μL of 2% SDS at room temperature. The resin was pelleted with a brief 

centrifugation and 75 μL of supernatant was removed and the elution procedure was repeated for 

a total of 150 μL of eluted protein. The extracted resin, eluted protein and other fractions were 

all stored at -80°C.

Detection of Post-Translational Glycosylation

The immunoaffinity-purified, soybean-derived Cry1F protein was mixed with Laemmli sample 

buffer (37.5 μL + 12.5 μL of 4x LSB) and heated at 95 °C for 10 minutes. The microbe-derived 

Cry1F, soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were 

diluted with 2x LSB to the approximate concentration of the purified soybean-derived Cry1F 

protein. After mixing the proteins with Laemmli sample buffer, the proteins were heated at ~95 

C for 10 minutes and centrifuged at 20000×g for 2 minutes to obtain a clarified supernatant. 

The resulting supernatants were applied directly to a Bio-Rad Mini-Protean TGX gel and 

electrophoresed at 100V for ~90 minutes. After electrophoresis, the gel was cut in half and one 

half was stained with GelCode Blue stain for total protein according to the manufacturers’ 

protocol. After the staining was complete, the gel was scanned with a densitometer to obtain a 

permanent visual record of the gel. The remaining half of the gel was stained with a GelCode 

Glycoprotein Staining Kit (ThermoScientific) according to the manufacturers’ protocol to 

visualize the glycoproteins. The procedure for glycoprotein staining is briefly described as 

follows: After electrophoresis, the gel was fixed in 50% methanol for 30 minutes and rinsed 
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with 3% acetic acid. This was followed by an incubation period with the oxidation solution from 

the staining kit for 15 minutes. The gel was once again rinsed with 3% acetic acid and incubated 

with GelCode glycoprotein staining reagent for 15 minutes. Finally, the gel was immersed in the 

reduction solution for 5 minutes, and rinsed with 3% acetic acid. The glycoproteins (with a 

detection limit as low as 0.625 ng per band) were visualized as magenta bands on a light pink 

background. After the glycoprotein staining was complete, the gel was scanned with a GE 

Healthcare densitometer to obtain a permanent visual record of the gel.

MALDI-TOF MS and LC/MS Peptide Mass Fingerprinting and Sequence Analysis of Soybeanand Microbe-Derived Cry1F

Sample preparation and deposition

Tryptic (in-gel digest), and Chymotryptic (in-gel digest) peptides were purified using Millipore 

ZipTip C18 as per manufacturer’s procedure. Purified peptides were eluted sequentially with 

aqueous 10%, 25%, 50%, and 100% ACN (supplemented with 0.1% TFA). The ZipTip C18 

fractions were mixed with 4 μL of CHCA matrix (10 mg/mL CHCA in 50% ACN supplemented 

with 0.1% TFA), and 1 μL of the sample-matrix mixture was deposited on the MALDI target and 

allowed to air dry.

MALDI-TOF MS

The sample preparations were analyzed directly by MALDI-TOF mass spectrometry. All mass 

spectra were acquired on an AB Sciex 4800 MALDI-TOF/TOF mass spectrometer (S/N 

AK011030605H). Mass calibration was performed with a mass standards kit for calibration of 

AB SCIEX TOF/TOF instruments. The plate wide calibration model was used for MS 

calibration.

MALDI-TOF MS, Electrospray Ionization-Liquid Chromatography Mass Spectrometry (ESILC/MS), glycoprotein analysis, and N-terminal and internal protein sequence analysis of the 

microbe-derived Cry1F (TSN103748) was conducted previously by (Gao et al., 2006).

Results

Lateral Flow Test Strip Assay

The presence of the Cry1F protein in the ground grain of event DAS-81419-2 was confirmed 

using commercially prepared lateral flow test strips from EnviroLogix Inc. The strips, capable of 

detecting one transgenic seed in 400, easily discriminated between transgenic and non-transgenic 

tissue. The transgenic extracts were positive for Cry1F protein and the non-transgenic extracts 

of Maverick did not contain detectable immunoreactive protein (Figure 49). This result was also 

confirmed by the western blot analysis using polyclonal antibodies specific to the Cry1F protein 

(Figure 50).
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Figure 49. Lateral flow strip assay for the Cry1F protein expression in Event DAS-81419-2 

and Maverick grain extracts.

Western Blot Analysis of Transgenic Grain Extracts

The microbe-derived Cry1F protein showed a positive signal of the expected size (the full-length 

Cry1F protein is ~130 kDa) by polyclonal antibody western blot analysis (Figure 50). This was 

also observed in the DAS-81419-2 transgenic soybean grain extract whereas the non-transgenic 

Maverick extracts did not contain any immunoreactive proteins. In the Cry1F western blot 

analysis, some immunoreactive proteins of truncated Cry1F were observed in the microbederived standard; however no alternate size Cry1F proteins (aggregates or degradation products) 

were observed in the transgenic samples (Figure 50). These results add to the evidence that the 

protein expressed in soybean is not glycosylated or post-translationally modified which would 

add to or subtract from the overall protein molecular weight.

Event

DAS-81419-2 Maverick

Control Line

Test Line
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Figure 50. Western blot analysis of microbe- and soybean-derived Cry1F proteins.

Lane Sample Amount Loaded

M Novex unstained MW markers 10 L

 1 (gel) Bovine serum albumin 1.3 μg

 1 (blot) Bovine serum albumin 2.5 ng

 2 (gel) Cry1F protein standard (TSN103748) 0.47 g

 2 (blot) Cry1F protein standard (TSN103748) 1.2 ng

 3 Maverick crude (non-transgenic) grain extract 40 L

 4 DAS-81419-2 crude (transgenic) grain extract 40 L

Purification of the Cry1F Protein from Transgenic Event DAS-81419-2 Grain Extracts

Immunoaffinity precipitation was conducted on an aqueous extract of ~25 grams of ground 

DAS-81419-2 transgenic grain. The protein that bound to the monoclonal antibody columns was 

examined by SDS-PAGE which demonstrated that the final concentrated fractions contained the 

intact Cry1F protein at an approximate molecular weight of ~130 kDa and fragments that contain 

the active core toxin (Figure 51). Once isolated, the soybean-derived Cry1F was then compared 

with the microbe-derived protein.

SDS-PAGE Analysis of the Immuno-purified Cry1F

In the toxicology-lot preparation of P. fluorescens-produced Cry1F (TSN103748), the major 

protein bands, as visualized on Coomassie stained SDS-PAGE gels, were approximately 130, 

110, and 90 kDa. As expected, the corresponding soybean-derived Cry1F protein was identical 

in size to the full-length microbe-expressed proteins (Figure 51). Predictably, the plant purified 

fractions contained a minor amount of impurities in addition to the full-length and truncated 

Cry1F core protein. The co-eluted proteins were likely retained on the column by weak 

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

Coomassie Stained Ge l Western Blot Film

M 1 2 3 4 1 2 3 4

Cry1F

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

- 3.5

kDa
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interactions with the column matrix or antibody leaching off of the column under the elution 

conditions. Other researchers have also reported the non-specific adsorption of proteins, 

peptides, and amino acids on activated agarose immunoadsorbents (Holroyde et al., 1976; 

Kennedy and Barnes, 1983; Williams et al., 2006) as well as antibody leaching from the column 

(Goldberg et al., 1991).

Figure 51. SDS-PAGE analysis of immunoaffinity-purified soybean-derived Cry1F.

Lane Sample Amount Loaded

M Novex prestained MW markers 10 L

 1 Bovine serum albumin ~1 μg 

 2 DAS-81419-2 crude grain extract 3 L

 3 Cry1F protein standard (TSN103748) 0.25 g

 4 Soybean-derived Cry1F (DAS-81419-2) 20 L

 5 Soybean-derived Cry1F (DAS-81419-2) 20 L

 6 Soybean-derived Cry1F (DAS-81419-2) 20 L

 7 Soybean-derived Cry1F (DAS-81419-2) 20 L

Detection of Glycosylation

Potential protein glycosylation of soybean-derived Cry1F was assessed with a GelCode 

Glycoprotein Staining Kit from ThermoScientific. The immunoaffinity-purified Cry1F protein 

was electrophoresed simultaneously with a set of control and reference protein standards. A 

glycoprotein, horseradish peroxidase, was loaded as a positive indicator for glycosylation, and 

non-glycoproteins, microbe-derived Cry1F, soybean trypsin inhibitor, and bovine serum 

albumin, were employed as negative controls. The results showed that both the soybean- and 

microbe-derived Cry1F proteins had no detectable covalently linked carbohydrates (Figure 52).

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

Coomassie Stained Gel

M 1 2 3 4 5 6 7

Cry1F

Cry1F
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Figure 52. SDS-PAGE analysis of microbe- and soybean-derived Cry1F protein stained 

with GelCode Blue total protein and glycoprotein stains.

Lane Sample Amount Loaded

M Novex prestained MW markers 10 L

 1 Cry1F protein standard (TSN103748) ~1 μg 

 2 Soybean-derived Cry1F (DAS-81419-2) 20 L

 3 Bovine serum albumin (- control) 0.5 g

 4 Horseradish peroxidase (+ control) 0.5 g

 5 Soybean trypsin inhibitor (- control) 0.5 g

MALDI-TOF MS and MS/MS Peptide Mass Fingerprinting and Sequence Analysis

The Cry1F protein derived from DAS-81419-2 soybean tissue was separated by SDS-PAGE 

(Figure 51) and the respective bands were excised and subjected to in-gel digestion by trypsin 

and chymotrypsin. The resulting peptide mixture was analyzed by MALDI-TOF MS and 

sequence verified by MS/MS to determine the peptide sequences. The masses of the detected 

peptides were compared with the expected masses based on trypsin or chymotrypsin cleavage 

sites in the sequence of the soybean-derived Cry1F protein. Figure 53 and Figure 54 illustrate

the theoretical cleavage of the Cry1F protein which was generated in silico using Protein 

Analysis Worksheet (PAWS) freeware from Proteometrics LLC. The Cry1F protein, once 

denatured, is readily digested by endoproteases to yield numerous peptides that are able to be 

detected using mass spectrometry.

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

Coomassie Stained Ge l Glycoprotein Stained Gel

M 1 2 3 4 5 1 2 3 4 5

HRP
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Figure 53. Theoretical cleavage of the Cry1F protein with trypsin generated in silico using 

Protein Analysis Worksheet (PAWS) from Proteometrics LLC.
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Figure 54. Theoretical cleavage of the Cry1F protein with chymotrypsin generated in silico 

using Protein Analysis Worksheet (PAWS) from Proteometrics LLC.
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In the endoprotease digests of the transgenic-soybean-derived Cry1F protein, the peptide 

sequence coverage from peptide mass fingerprint (PMF) data was extensive at 81.7%. Of the 

81.7% sequence coverage from PMF data, all peptide sequences were confirmed by tandem mass 

spectrometry sequencing. The detected peptide fragments covered nearly the entire protein 

sequence with only a few peptide fragments undetected (Figure 55, Figure 56 and Figure 57). 

This analysis confirmed the soybean-derived protein amino acid sequence matched that of the 

microbe-derived Cry1F protein near the N- and C-terminus as well as a major portion of the 

internal sequence (Gao et al., 2006). In the MS spectra, there were unidentified peptides 

detected in the enzyme digest preparations (data not shown). Many factors contribute to the 

formation of these unidentified peptides, such as over digestion (which results in non-specific 

cleavage), self-digestion products of trypsin and chymotrypsin, as well as random breakage of 

peptides during ionization. Unidentified peptides do not indicate the protein is different from the 

predicted amino acid sequence. The results of these analyses indicate that the amino acid 

sequence of the soybean-derived Cry1F protein was equivalent to the P. fluorescens-expressed

protein previously characterized (Gao et al., 2006).
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 175 of 214

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Figure 55. Tryptic digest sequence coverage map for Cry1F (DAS-81419-2) by MALDITOF MS and MALDI-TOF/TOF. Cys residues were alkylated with iodoacetamide. 

Sequence coverage was 65.1% with PMF data and 65.1% by tandem MS.

Cry1F peptide mapping - Trypsin Protocol 110841

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M E N N I Q N Q C V P Y N C L N N P E V E I L N E E R S T G 30

Trypsin 15 15

31 R L P L D I S L S L T R F L L S E F V P G V G V A F G L F D 60

Trypsin 11 11

61 L I W G F I T P S D W S L F L L Q I E Q L I E Q R I E T L E 90

Trypsin 5 5

91 R N R A I T T L R G L A D S Y E I Y I E A L R E W E A N P N 120

Trypsin 22 22

121 N A Q L R E D V R I R F A N T D D A L I T A I N N F T L T S 150

Trypsin 5 5

151 F E I P L L S V Y V Q A A N L H L S L L R D A V S F G Q G W 180

Trypsin 9 9

181 G L D I A T V N N H Y N R L I N L I H R Y T K H C L D T Y N 210

Trypsin 27 27

211 Q G L E N L R G T N T R Q W A R F N Q F R R D L T L T V L D 240

Trypsin 24 24

241 I V A L F P N Y D V R T Y P I Q T S S Q L T R E I Y T S S V 270

Trypsin 30 30

271 I E D S P V S A N I P N G F N R A E F G V R P P H L M D F M 300

Trypsin 30 30

301 N S L F V T A E T V R S Q T V W G G H L V S S R N T A G N R 330

Trypsin 24 24

331 I N F P S Y G V F N P G G A I W I A D E D P R P F Y R T L S 360

Trypsin 3 3

361 D P V F V R G G F G N P H Y V L G L R G V A F Q Q T G T N H 390

Trypsin 30 30

391 T R T F R N S G T I D S L D E I P P Q D N S G A P W N D Y S 420

Trypsin 27 27

421 H V L N H V T F V R W P G E I S G S D S W R A P M F S W T H 450

Trypsin 30 30

451 R S A T P T N T I D P E R I T Q I P L V K A H T L Q S G T T 480

Trypsin 30 30

481 V V R G P G F T G G D I L R R T S G G P F A Y T I V N I N G 510

Trypsin 15 15

511 Q L P Q R Y R A R I R Y A S T T N L R I Y V T V A G E R I F 540

Trypsin 19 19

541 A G Q F N K T M D T G D P L T F Q S F S Y A T I N T A F T F 570

Trypsin 6 6

571 P M S Q S S F T V G A D T F S S G N E V Y I D R F E L I P V 600

Trypsin 6 6

601 T A T L E A E S D L E R A Q K A V N A L F T S S N Q I G L K 630

Trypsin 27 27

631 T D V T D Y H I D R V S N L V E C L S D E F C L D E K K E L 660

Trypsin 28 28

661 S E K V K H A K R L S D E R N L L Q D P N F R G I N R Q L D 690

Trypsin 9 9

691 R G W R G S T D I T I Q G G D D V F K E N Y V T L L G T F D 720

Trypsin 26 26

721 E C Y P T Y L Y Q K I D E S K L K A Y T R Y Q L R G Y I E D 750

Trypsin 19 19

751 S Q D L E I Y L I R Y N A K H E T V N V P G T G S L W P L S 780

Trypsin 26 26

781 A P S P I G K C A H H S H H F S L D I D V G C T D L N E D L 810

Trypsin 7 7

811 G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G 840

Trypsin 14 14

841 E A L A R V K R A E K K W R D K R E K L E W E T N I V Y K E 870

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 176 of 214

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Figure 55 (Cont). Tryptic digest sequence coverage map for Cry1F (DAS-81419-2) by 

MALDI-TOF MS and MALDI-TOF/TOF. 

511 Q L P Q R Y R A R I R Y A S T T N L R I Y V T V A G E R I F 540

Trypsin 19 19

541 A G Q F N K T M D T G D P L T F Q S F S Y A T I N T A F T F 570

Trypsin 6 6

571 P M S Q S S F T V G A D T F S S G N E V Y I D R F E L I P V 600

Trypsin 6 6

601 T A T L E A E S D L E R A Q K A V N A L F T S S N Q I G L K 630

Trypsin 27 27

631 T D V T D Y H I D R V S N L V E C L S D E F C L D E K K E L 660

Trypsin 28 28

661 S E K V K H A K R L S D E R N L L Q D P N F R G I N R Q L D 690

Trypsin 9 9

691 R G W R G S T D I T I Q G G D D V F K E N Y V T L L G T F D 720

Trypsin 26 26

721 E C Y P T Y L Y Q K I D E S K L K A Y T R Y Q L R G Y I E D 750

Trypsin 19 19

751 S Q D L E I Y L I R Y N A K H E T V N V P G T G S L W P L S 780

Trypsin 26 26

781 A P S P I G K C A H H S H H F S L D I D V G C T D L N E D L 810

Trypsin 7 7

811 G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G 840

Trypsin 14 14

841 E A L A R V K R A E K K W R D K R E K L E W E T N I V Y K E 870

Trypsin 17 17

871 A K E S V D A L F V N S Q Y D R L Q A D T N I A M I H A A D 900

Trypsin 28 28

901 K R V H S I R E A Y L P E L S V I P G V N A A I F E E L E G 930

Trypsin 25 25

931 R I F T A F S L Y D A R N V I K N G D F N N G L S C W N V K 960

Trypsin 26 26

961 G H V D V E E Q N N H R S V L V V P E W E A E V S Q E V R V 990

Trypsin 30 30

991 C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N N 1020

Trypsin 9 9

1021 T D E L K F S N C V E E E V Y P N N T V T C N D Y T A T Q E 1050

Trypsin 25 25

1051 E Y E G T Y T S R N R G Y D G A Y E S N S S V P A D Y A S A 1080

Trypsin 28 28

1081 Y E E K A Y T D G R R D N P C E S N R G Y G D Y T P L P A G 1110

Trypsin 21 21

1111 Y V T K E L E Y F P E T D K V W I E I G E T E G T F I V D S 1140

Trypsin 14 14

1141 V E L L L M E E 1148

Trypsin 0 0

Identified in MS Analysis Total Residues Observed 747 747

Identified in MS and MS/MS Analysis Trypsin Percent Coverage 65.1% 65.1%
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 177 of 214

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Figure 56. Chymotryptic digest sequence coverage map for Cry1F (DAS-81419-2) by 

MALDI-TOF MS and MALDI TOF/TOF. Cys residues were alkylated with iodoacetamide. 

Sequence coverage was 53.8% with PMF data and 52.4% by tandem MS.

Cry1F peptide mapping - Chymotrypsin Protocol 110841

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M E N N I Q N Q C V P Y N C L N N P E V E I L N E E R S T G 30

Chymotrypsin 0 0

31 R L P L D I S L S L T R F L L S E F V P G V G V A F G L F D 60

Chymotrypsin 0 0

61 L I W G F I T P S D W S L F L L Q I E Q L I E Q R I E T L E 90

Chymotrypsin 8 8

91 R N R A I T T L R G L A D S Y E I Y I E A L R E W E A N P N 120

Chymotrypsin 7 7

121 N A Q L R E D V R I R F A N T D D A L I T A I N N F T L T S 150

Chymotrypsin 14 14

151 F E I P L L S V Y V Q A A N L H L S L L R D A V S F G Q G W 180

Chymotrypsin 0 0

181 G L D I A T V N N H Y N R L I N L I H R Y T K H C L D T Y N 210

Chymotrypsin 30 30

211 Q G L E N L R G T N T R Q W A R F N Q F R R D L T L T V L D 240

Chymotrypsin 24 24

241 I V A L F P N Y D V R T Y P I Q T S S Q L T R E I Y T S S V 270

Chymotrypsin 8 8

271 I E D S P V S A N I P N G F N R A E F G V R P P H L M D F M 300

Chymotrypsin 16 16

301 N S L F V T A E T V R S Q T V W G G H L V S S R N T A G N R 330

Chymotrypsin 16 16

331 I N F P S Y G V F N P G G A I W I A D E D P R P F Y R T L S 360

Chymotrypsin 21 21

361 D P V F V R G G F G N P H Y V L G L R G V A F Q Q T G T N H 390

Chymotrypsin 20 20

391 T R T F R N S G T I D S L D E I P P Q D N S G A P W N D Y S 420

Chymotrypsin 30 30

421 H V L N H V T F V R W P G E I S G S D S W R A P M F S W T H 450

Chymotrypsin 28 28

451 R S A T P T N T I D P E R I T Q I P L V K A H T L Q S G T T 480

Chymotrypsin 0 0

481 V V R G P G F T G G D I L R R T S G G P F A Y T I V N I N G 510

Chymotrypsin 21 21

511 Q L P Q R Y R A R I R Y A S T T N L R I Y V T V A G E R I F 540

Chymotrypsin 24 24

541 A G Q F N K T M D T G D P L T F Q S F S Y A T I N T A F T F 570

Chymotrypsin 15 15

571 P M S Q S S F T V G A D T F S S G N E V Y I D R F E L I P V 600

Chymotrypsin 23 23

601 T A T L E A E S D L E R A Q K A V N A L F T S S N Q I G L K 630

Chymotrypsin 30 30

631 T D V T D Y H I D R V S N L V E C L S D E F C L D E K K E L 660

Chymotrypsin 22 22

661 S E K V K H A K R L S D E R N L L Q D P N F R G I N R Q L D 690

Chymotrypsin 8 8

691 R G W R G S T D I T I Q G G D D V F K E N Y V T L L G T F D 720

Chymotrypsin 30 29

721 E C Y P T Y L Y Q K I D E S K L K A Y T R Y Q L R G Y I E D 750

Chymotrypsin 20 14

751 S Q D L E I Y L I R Y N A K H E T V N V P G T G S L W P L S 780

Chymotrypsin 11 11

781 A P S P I G K C A H H S H H F S L D I D V G C T D L N E D L 810

Chymotrypsin 15 15

811 G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G 840

Chymotrypsin 22 22

841 E A L A R V K R A E K K W R D K R E K L E W E T N I V Y K E 870

Chymotrypsin 11 11

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 178 of 214

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Figure 56 (Cont). Chymotryptic digest sequence coverage map for Cry1F (DAS-81419-2) 

by MALDI-TOF MS and MALDI TOF/TOF.

511 Q L P Q R Y R A R I R Y A S T T N L R I Y V T V A G E R I F 540

Chymotrypsin 24 24

541 A G Q F N K T M D T G D P L T F Q S F S Y A T I N T A F T F 570

Chymotrypsin 15 15

571 P M S Q S S F T V G A D T F S S G N E V Y I D R F E L I P V 600

Chymotrypsin 23 23

601 T A T L E A E S D L E R A Q K A V N A L F T S S N Q I G L K 630

Chymotrypsin 30 30

631 T D V T D Y H I D R V S N L V E C L S D E F C L D E K K E L 660

Chymotrypsin 22 22

661 S E K V K H A K R L S D E R N L L Q D P N F R G I N R Q L D 690

Chymotrypsin 8 8

691 R G W R G S T D I T I Q G G D D V F K E N Y V T L L G T F D 720

Chymotrypsin 30 29

721 E C Y P T Y L Y Q K I D E S K L K A Y T R Y Q L R G Y I E D 750

Chymotrypsin 20 14

751 S Q D L E I Y L I R Y N A K H E T V N V P G T G S L W P L S 780

Chymotrypsin 11 11

781 A P S P I G K C A H H S H H F S L D I D V G C T D L N E D L 810

Chymotrypsin 15 15

811 G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G 840

Chymotrypsin 22 22

841 E A L A R V K R A E K K W R D K R E K L E W E T N I V Y K E 870

Chymotrypsin 11 11

871 A K E S V D A L F V N S Q Y D R L Q A D T N I A M I H A A D 900

Chymotrypsin 9 9

901 K R V H S I R E A Y L P E L S V I P G V N A A I F E E L E G 930

Chymotrypsin 20 20

931 R I F T A F S L Y D A R N V I K N G D F N N G L S C W N V K 960

Chymotrypsin 21 21

961 G H V D V E E Q N N H R S V L V V P E W E A E V S Q E V R V 990

Chymotrypsin 0 0

991 C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N N 1020

Chymotrypsin 7 7

1021 T D E L K F S N C V E E E V Y P N N T V T C N D Y T A T Q E 1050

Chymotrypsin 5 5

1051 E Y E G T Y T S R N R G Y D G A Y E S N S S V P A D Y A S A 1080

Chymotrypsin 26 16

1081 Y E E K A Y T D G R R D N P C E S N R G Y G D Y T P L P A G 1110

Chymotrypsin 30 30

1111 Y V T K E L E Y F P E T D K V W I E I G E T E G T F I V D S 1140

Chymotrypsin 26 26

1141 V E L L L M E E 1148

Chymotrypsin 0 0

Identified in MS Analysis Total Residues Observed 618 601

Identified in MS and MS/MS Analysis Chymotrypsin Percent Coverage 53.8% 52.4%
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 179 of 214

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Figure 57. Overall sequence coverage of trypsin and chymotrypsin digests for Cry1F 

(DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF. Overall sequence coverage 

was 81.7% with PMF data and 81.7% by tandem MS.

Cry1F peptide mapping - overall Protocol 110841

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30

1 M E N N I Q N Q C V P Y N C L N N P E V E I L N E E R S T G 30

Trypsin 15 15

Chymotrypsin 0 0

Overall 15 15

31 R L P L D I S L S L T R F L L S E F V P G V G V A F G L F D 60

Trypsin 11 11

Chymotrypsin 0 0

Overall 11 11

61 L I W G F I T P S D W S L F L L Q I E Q L I E Q R I E T L E 90

Trypsin 5 5

Chymotrypsin 8 8

Overall 13 13

91 R N R A I T T L R G L A D S Y E I Y I E A L R E W E A N P N 120

Trypsin 22 22

Chymotrypsin 7 7

Overall 22 22

121 N A Q L R E D V R I R F A N T D D A L I T A I N N F T L T S 150

Trypsin 5 5

Chymotrypsin 14 14

Overall 19 19

151 F E I P L L S V Y V Q A A N L H L S L L R D A V S F G Q G W 180

Trypsin 9 9

Chymotrypsin 0 0

Overall 9 9

181 G L D I A T V N N H Y N R L I N L I H R Y T K H C L D T Y N 210

Trypsin 27 27

Chymotrypsin 30 30

Overall 30 30

211 Q G L E N L R G T N T R Q W A R F N Q F R R D L T L T V L D 240

Trypsin 24 24

Chymotrypsin 24 24

Overall 30 30

241 I V A L F P N Y D V R T Y P I Q T S S Q L T R E I Y T S S V 270

Trypsin 30 30

Chymotrypsin 8 8

Overall 30 30

271 I E D S P V S A N I P N G F N R A E F G V R P P H L M D F M 300

Trypsin 30 30

Chymotrypsin 16 16

Overall 30 30

301 N S L F V T A E T V R S Q T V W G G H L V S S R N T A G N R 330

Trypsin 24 24

Chymotrypsin 16 16

Overall 24 24

331 I N F P S Y G V F N P G G A I W I A D E D P R P F Y R T L S 360

Trypsin 3 3

Chymotrypsin 21 21

Overall 21 21

361 D P V F V R G G F G N P H Y V L G L R G V A F Q Q T G T N H 390

Trypsin 30 30

Chymotrypsin 20 20

Overall 30 30

391 T R T F R N S G T I D S L D E I P P Q D N S G A P W N D Y S 420

Trypsin 27 27

Chymotrypsin 30 30

Overall 30 30

421 H V L N H V T F V R W P G E I S G S D S W R A P M F S W T H 450

Trypsin 30 30

Chymotrypsin 28 28

Overall 30 30

451 R S A T P T N T I D P E R I T Q I P L V K A H T L Q S G T T 480

Trypsin 30 30

Chymotrypsin 0 0

Overall 30 30

481 V V R G P G F T G G D I L R R T S G G P F A Y T I V N I N G 510

Trypsin 15 15

Chymotrypsin 21 21

Overall 28 28

511 Q L P Q R Y R A R I R Y A S T T N L R I Y V T V A G E R I F 540

Trypsin 19 19

Chymotrypsin 24 24

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
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Figure 57 (Cont). Overall sequence coverage of trypsin and chymotrypsin digests for 

Cry1F (DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF.

Trypsin 3 3

Chymotrypsin 21 21

Overall 21 21

361 D P V F V R G G F G N P H Y V L G L R G V A F Q Q T G T N H 390

Trypsin 30 30

Chymotrypsin 20 20

Overall 30 30

391 T R T F R N S G T I D S L D E I P P Q D N S G A P W N D Y S 420

Trypsin 27 27

Chymotrypsin 30 30

Overall 30 30

421 H V L N H V T F V R W P G E I S G S D S W R A P M F S W T H 450

Trypsin 30 30

Chymotrypsin 28 28

Overall 30 30

451 R S A T P T N T I D P E R I T Q I P L V K A H T L Q S G T T 480

Trypsin 30 30

Chymotrypsin 0 0

Overall 30 30

481 V V R G P G F T G G D I L R R T S G G P F A Y T I V N I N G 510

Trypsin 15 15

Chymotrypsin 21 21

Overall 28 28

511 Q L P Q R Y R A R I R Y A S T T N L R I Y V T V A G E R I F 540

Trypsin 19 19

Chymotrypsin 24 24

Overall 25 25

541 A G Q F N K T M D T G D P L T F Q S F S Y A T I N T A F T F 570

Trypsin 6 6

Chymotrypsin 15 15

Overall 17 17

571 P M S Q S S F T V G A D T F S S G N E V Y I D R F E L I P V 600

Trypsin 6 6

Chymotrypsin 23 23

Overall 23 23

601 T A T L E A E S D L E R A Q K A V N A L F T S S N Q I G L K 630

Trypsin 27 27

Chymotrypsin 30 30

Overall 30 30

631 T D V T D Y H I D R V S N L V E C L S D E F C L D E K K E L 660

Trypsin 28 28

Chymotrypsin 22 22

Overall 28 28

661 S E K V K H A K R L S D E R N L L Q D P N F R G I N R Q L D 690

Trypsin 9 9

Chymotrypsin 8 8

Overall 16 16

691 R G W R G S T D I T I Q G G D D V F K E N Y V T L L G T F D 720

Trypsin 26 26

Chymotrypsin 30 29

Overall 30 30

721 E C Y P T Y L Y Q K I D E S K L K A Y T R Y Q L R G Y I E D 750

Trypsin 19 19

Chymotrypsin 20 14

Overall 28 28

751 S Q D L E I Y L I R Y N A K H E T V N V P G T G S L W P L S 780

Trypsin 26 26

Chymotrypsin 11 11

Overall 26 26

781 A P S P I G K C A H H S H H F S L D I D V G C T D L N E D L 810

Trypsin 7 7

Chymotrypsin 15 15

Overall 22 22

811 G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G 840

Trypsin 14 14

Chymotrypsin 22 22

Overall 30 30

841 E A L A R V K R A E K K W R D K R E K L E W E T N I V Y K E 870

Trypsin 17 17

Chymotrypsin 11 11

Overall 22 22

871 A K E S V D A L F V N S Q Y D R L Q A D T N I A M I H A A D 900

Trypsin 28 28

Chymotrypsin 9 9

Overall 30 30

901 K R V H S I R E A Y L P E L S V I P G V N A A I F E E L E G 930

Trypsin 25 25

Chymotrypsin 20 20

Overall 25 25

931 R I F T A F S L Y D A R N V I K N G D F N N G L S C W N V K 960
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Figure 57 (Cont). Overall sequence coverage of trypsin and chymotrypsin digests for 

Cry1F (DAS-81419-2) by MALDI-TOF MS and MALDI TOF/TOF.

781 A P S P I G K C A H H S H H F S L D I D V G C T D L N E D L 810

Trypsin 7 7

Chymotrypsin 15 15

Overall 22 22

811 G V W V I F K I K T Q D G H A R L G N L E F L E E K P L V G 840

Trypsin 14 14

Chymotrypsin 22 22

Overall 30 30

841 E A L A R V K R A E K K W R D K R E K L E W E T N I V Y K E 870

Trypsin 17 17

Chymotrypsin 11 11

Overall 22 22

871 A K E S V D A L F V N S Q Y D R L Q A D T N I A M I H A A D 900

Trypsin 28 28

Chymotrypsin 9 9

Overall 30 30

901 K R V H S I R E A Y L P E L S V I P G V N A A I F E E L E G 930

Trypsin 25 25

Chymotrypsin 20 20

Overall 25 25

931 R I F T A F S L Y D A R N V I K N G D F N N G L S C W N V K 960

Trypsin 26 26

Chymotrypsin 21 21

Overall 30 30

961 G H V D V E E Q N N H R S V L V V P E W E A E V S Q E V R V 990

Trypsin 30 30

Chymotrypsin 0 0

Overall 30 30

991 C P G R G Y I L R V T A Y K E G Y G E G C V T I H E I E N N 1020

Trypsin 9 9

Chymotrypsin 7 7

Overall 13 13

1021 T D E L K F S N C V E E E V Y P N N T V T C N D Y T A T Q E 1050

Trypsin 25 25

Chymotrypsin 5 5

Overall 25 25

1051 E Y E G T Y T S R N R G Y D G A Y E S N S S V P A D Y A S A 1080

Trypsin 28 28

Chymotrypsin 26 16

Overall 30 30

1081 Y E E K A Y T D G R R D N P C E S N R G Y G D Y T P L P A G 1110

Trypsin 21 21

Chymotrypsin 30 30

Overall 30 30

1111 Y V T K E L E Y F P E T D K V W I E I G E T E G T F I V D S 1140

Trypsin 14 14

Chymotrypsin 26 26

Overall 26 26

1141 V E L L L M E E 1148

Trypsin 0 0

Chymotrypsin 0 0

Overall 0 0

Identified in Trypsin MS Analysis Total Residues Observed 938 938

Identified in Trypsin MS and MS/MS Analysis

Overall Percent Coverage 81.7% 81.7%

Identified in Chymo MS Analysis

Identified in Chymo MS and MS/MS Analysis

Identified in Overall MS Analysis

Identified in Overall MS and MS/MS Analysis
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Conclusions

It was demonstrated that the biochemical identity of P. fluorescens-produced Cry1F protein was 

equivalent to the protein purified from grain of soybean event DAS-81419-2. Both the soybeanand microbe-derived Cry1F proteins had an apparent molecular weight of ~130 kDa and were 

immunoreactive to Cry1F protein-specific antibodies in both lateral flow strip and western blot 

assays. The amino acid sequences were confirmed by enzymatic peptide mass fingerprinting by 

MALDI-TOF MS and verified by MS/MS. In addition, the lack of glycosylation of the soybeanderived Cry1F protein provided additional evidence that the Cry1F protein produced by P. 

fluorescens and transgenic soybean were essentially equivalent.
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Appendix 4. Materials, Methods and Results for Characterization of PAT Protein

Materials and Methods

Test Substance/Test System

The test substance was the PAT protein expressed and extracted from tissues grown from the T5 

seeds of transgenic soybean event DAS-81419-2: Source ID: YTR11ETR760037 (TSN303074). 

The seeds were planted in the greenhouise and 3 to 7 week old leaf tissue was harvested, frozen, 

lyophilized, ground, stored at -20°C to vent the dry ice, and stored at -80C. The presence of 

PAT protein in the soybean tissue was confirmed by a commercially available lateral flow assay 

kit from EnviroLogix Inc., as described below.

Control Substances

The control substance used in this study was a non-transgenic soybean plant extract (Glycine 

max cv Maverick). Seeds of the Maverick soybean line (Source ID: YX11AX090001) were 

planted, grown, harvested and processed under the same conditions as the transgenic plants 

described above. The absence of PAT protein in the non-transgenic soybean tissue was 

confirmed by a commercially available lateral flow assay kit as described below.

Recombinant PAT microbial protein, (TSN031116-0001, Lot #: 55238-1A), has a molecular 

weight of ~21 kDa and a concentration of 810 g/mL. An enzymatically active microbial 

preparation was produced in recombinant E. coli and based on confirmation of the gene sequence 

in the E. coli expression system (Madduri and Snodderley, 2007), the E. coli-derived PAT 

protein sequence is identical to the proteins expressed in deregulated transgenic corn, cotton, 

soybean, canola, event DAS-81419-2, and the native organism S. viridochromogenes (Wohlleben

et al., 1988; AgrEvo, 1994; Northrup King, 1995; AgrEvo, 1996, 1997; Dow AgroSciences, 

2003c, 2003b, 2003a). The DAS-81419-2 and E. coli-derived PAT protein sequences are also 

identical to the PAT protein sequence described in the OECD consensus document (OECD, 

1999) and in other publications (Herouet et al., 2005).

Reference Substances

The commercially available (non-GLP) reference substances used in this study are listed in the 

following table:

Reference Substance Product Name Lot Number Assay Reference

Prestained Molecular 

Weight Markers

Novex Sharp 

prestained protein 

standards

1095889 SDS-PAGE 

and Western

blot 

Invitrogen: Molecular 

Weight Markers of 260, 160, 

110, 80, 60, 50, 40, 30, 20, 

15, 10 and 3.5 kDa

Unstained Molecular 

Weight Markers

Novex Sharp protein 

standards

1030454 SDS-PAGE Invitrogen: Molecular 

Weight Markers of 260, 160, 

110, 80, 60, 50, 40, 30, 20, 

15, 10 and 3.5 kDa
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Lateral Flow Strip Assay

The soybean leaf tissues of the transgenic and non-transgenic events were harvested fresh as 

described above and were frozen, lyophilized, ground, and stored at approximately –80C until 

use. To confirm the presence/absence of the PAT protein in the pooled tissues, approximately 16

mg of the lyophilized tissues (Event DAS-81419-2 and Maverick) were weighed into 2.0-mL 

microfuge tubes and tested as described by EnviroLogix Inc. Briefly, the soluble proteins were 

extracted by adding 2 metal beads to the extraction buffer (0.25 mL EB2 buffer supplied by the 

manufacturer) and grinding in a Spex Geno-Grinder for 3 minutes at 500 strokes per minute. 

The samples were subjected to centrifugation for 5 minutes at 20,000×g and the resulting 

supernatants were transferred to a fresh tube and the strips were developed according to the 

manufacturer’s instructions.

SDS-PAGE and Western Blot

SDS-PAGE analysis of the transgenic (DAS-81419-2) and non-transgenic Maverick soybean 

extracts was performed with Bio-Rad Criterion gels fitted in a Criterion Cell gel module with 

MES running buffer. Extracts were prepared by grinding ~43 mg of tissue for 3 minutes in a 

Geno-Grinder with steel ball bearings in ~ 1 mL of PBST based buffer (Table 25). The 

supernatants were clarified by centrifuging for 5 minutes at 20,000×g, and then 120 L of each 

extract was mixed with 30 L of 5x Laemmli sample buffer (LSB, 2% SDS, 50 mM Tris pH 6.8, 

0.2 mg/mL bromophenol blue, 50% (w/w) glycerol containing 10% freshly added 2-

mercaptoethanol). Samples were heated for 5 minutes at ~95C, and after a brief centrifugation, 

20 μL of the supernatant was loaded directly on the gel. The reference standard, microbederived PAT (TSN031116-0001) was diluted with Bio-Rad 2x LSB containing 5% βmercaptoethanol and processed as described earlier. The electrophoresis was conducted at a 

constant voltage of 150 V for ~60 minutes using MES running buffer from Bio-Rad. After 

separation, the gel was cut in half and one half was stained with Thermo Scientific GelCode Blue 

protein stain and scanned with a densitometer to obtain a permanent record of the image. The 

remaining half of the gel was electro-blotted to a nitrocellulose membrane from Bio-Rad with a 

Criterion trans-blot electrophoretic transfer cell for ~60 minutes under a constant voltage of 100 

volts. The transfer buffer contained 20% methanol and Tris/glycine buffer from Bio-Rad. After 

transfer, the membrane was probed with a PAT specific polyclonal rabbit antibody (Lot #: 

D2976-27, 1.1 mg/mL). A conjugate of goat anti-rabbit IgG (H+L) and horseradish peroxidase 

from Pierce was used as the secondary (detection) antibody. Pierce ECL chemiluminescent 

substrate was used for development and visualization of the immunoreactive protein bands. The 

membranes were exposed to Thermo Scientific CL-XPosure detection film for various time 

points and subsequently developed with a Radiation Services film developer.
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Table 25. Soybean-derived PAT extraction buffer composition.

Ingredient Amount

Phosphate Buffered Saline with 

0.5% Tweena20, pH 7.4

5.924 mL

0.5 M EDTA 60 L

Protease inhibitor cocktail 6.0 L

-mercaptoethanol 10 L

Note: the extraction buffer was prepared immediately before use.

Results

Lateral Flow Strip Assay

The presence of the PAT protein in the pooled T5 leaf tissue of DAS-81419-2 was confirmed 

using a commercially prepared lateral flow test strip from EnviroLogix Inc. The assay easily 

discriminated between transgenic and non-transgenic plants as the non-transgenic extracts of 

Maverick did not contain detectable amounts of immunoreactive protein (Figure 58).

Figure 58. Lateral flow strip assay for PAT protein expression in Event DAS-81419-2 and 

Maverick leaf extracts.

* Note: Event DAS-81419-2 is positive for the PAT protein

SDS-PAGE and Western Blot Analysis

In the toxicology-lot preparation of E. coli-produced PAT protein (TSN031116-0001), the major 

protein band, as visualized on Coomassie stained SDS-PAGE gels, was approximately 21 kDa 

(Figure 59). As expected, the corresponding soybean-derived PAT protein was visualized by 

immunospecific polyclonal antibodies at an identical size to the microbe-expressed proteins. In 

the PAT Western blot analysis, no immunoreactive proteins, consistent with the PAT protein, 

Event

DAS-81419-2 Maverick

Control Line

Test Line
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were observed in the control Maverick extract; however a non-specific band was detected at ~40 

kDa and was detected in both the transgenic and non-transgenic extracts. In addition the 

polyclonal antibody did detect a small amount of a protein dimer in the microbe-derived PAT 

protein preparation. These results add to the evidence that the PAT protein expressed in soybean 

is not post-translationally modified (the PAT enzyme it does not contain N-glycosylation sites 

(Herouet et al., 2005) or processed which would have added to or subtracted from the overall 

protein molecular weight.

Figure 59. SDS-PAGE and Western blot analysis of microbe-derived PAT, transgenic 

soybean Event DAS-81419-2, and non-transgenic Maverick extracts.

Lane Sample Amount Loaded

M Novex unstained MW Markers 10 L

 1 PAT Protein Standard (TSN031116-0001) ~1 μg 

 2 Transgenic Soybean - Event DAS-81419-2 20 L

 3 Non-transgenic Soybean – Maverick 20 L

 4 PAT Protein Standard (TSN031116-0001) ~5 ng

 5 Transgenic Soybean - Event DAS-81419-2 20 L

 6 Non-transgenic Soybean – Maverick 20 L

M Novex Prestained MW Markers* 10 L

 *Note: The molecular weight markers were manually transferred to the film after development.

SDS-PAGE gel Western blot

M 1 2 3 4 5 6 M

- 260

- 160

- 110

- 8 0

- 6 0

- 5 0

- 4 0

- 3 0

- 2 0

- 1 5

- 1 0

- 3.5

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

kDa

non-specific

binding

PAT protein

PAT dimer
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Conclusion

The results of this study demonstrated that both the transgenic soybean-plant extract and the 

microbe-derived PAT toxicological lot contained the intact, full-length PAT protein. This was 

confirmed by SDS-PAGE molecular-weight approximation, Western blot analysis, and 

immunoreactivity using a commercially available lateral flow strip assay. Together, these 

biochemical tests indicate that the plant- and microbe-derived PAT proteins are substantially 

equivalent and consistent with the published data on PAT.
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Appendix 5. Materials and Methods for Cry1Ac, Cry1F and PAT Protein Expression 

Analysis

Experimental Design

This study used the same plots that were used for agronomic performance and nutrient 

composition studies. The experimental design included ten field sites located in Iowa (2 sites), 

Illinois (2 sites), Indiana, Missouri (2 sites), Nebraska (2 sites), and Pennsylvania. Each trial site 

included DAS-81419-2 soybean, the non-transgenic near-isogenic control (Maverick), and three 

non-transgenic reference lines. At each of the ten sites, all entries were arranged in a randomized 

complete block design with four blocks. Across all sites, each control and DAS-81419-2 was 

represented by a total of 40 plots (10 sites, 4 replicate plots per entry at each site). Three of the 

six reference lines were included at each site by randomizing across sites in a balanced 

incomplete-block design. Each of the six reference lines was assigned to five sites; therefore, 

each reference line was represented by a total of 20 plots across sites (5 sites per reference line, 4 

replicate plots per entry at each site). At each site, four replicate plots of each entry were 

established, with each plot consisting of four 25 ft (7.62 m) rows. Soybean seeds were planted at 

a seeding rate of approximately 125 seeds per row with seed spacing within each row of 

approximately 2.4 inches (6 cm). Each soybean plot was bordered by two rows of a nontransgenic soybean cultivar of similar maturity. The entire trial site was surrounded by a 

minimum of four rows (10 ft or 3.0 m) of a non-transgenic soybean cultivar of similar maturity.

Appropriate insect, weed, and disease control practices were applied to produce an 

agronomically acceptable crop.

Sample Collection

Plant tissue samples were collected as described below and shipped to Dow AgroSciences 

Regulatory Sciences and Government Affairs laboratories where they were maintained frozen 

until use. Samples of soybean tissues were prepared for expression analysis by coarse grinding, 

lyophilizing and/or fine-grinding with a Geno/Grinder (Certiprep, Metuchen, New Jersey).

Leaf (V5 and V10-12)

One leaf sample per plot, each sample containing 8 trifoliate sets of leaves collected from 

separate plants, was collected for each test and control entry. Each leaf sample was the youngest 

set of fully expanded trifoliate leaves.

Root (R3)

One root sample (representing 3 plants) per plot was collected for each test and control entry at 

the R3 stage by cutting a circle around the base of the plant. The root ball was removed and 

cleaned.

Forage (R3)

One forage sample (representing 3 plants) per plot each consisting of the aerial portion (no roots) 

of 3 whole plants was collected from each test and control entry.

Grain (R8 – Maturity)

One individual sample was collected from each plot of each test and control entry. Each sample 

contained approximately 500-grams of grain.
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Determination of Cry1Ac Protein Concentration

Soybean tissue samples were analyzed using Dow AgroSciences method 110674. In this 

method, the soluble, extractable Cry1Ac protein was quantified using an enzyme-linked 

immunosorbent assay (ELISA) kit from Romer Labs, Inc.

The Cry1Ac protein was extracted from lyophilized, ground soybean tissues with a phosphate 

buffered saline solution containing 0.05% Triton X-100 with 1 mg/mL sodium ascorbate 

(PBS/Triton/AA). The extract was centrifuged, and then the aqueous supernatant was collected 

and subjected to trypsin digestion to convert full length Cry1Ac to the truncated Cry1Ac core 

toxin. After truncation by trypsin, the reaction was stopped by the protease inhibitor 

phenylmethanesulfonyl fluoride (PMSF). The digested extract was diluted and assayed using a 

specific Cry1Ac ELISA kit. A sequential sandwich ELISA format was applied in this assay. An 

aliquot of the diluted sample was incubated in the wells of a plate pre-coated with an 

immobilized monoclonal anti-Cry1Ac antibody, and then the unbound samples are removed 

from the plate by washing with phosphate buffered saline solution containing 0.05% Tween 20 

(PBST). An excess amount of enzyme-conjugated monoclonal antibody specific to the Cry1Ac 

protein was then added to the wells for incubation. These antibodies bind with Cry1Ac protein 

in the wells and form a “sandwich” with Cry1Ac protein bound between the soluble and 

immobilized antibodies. At the end of an incubation period, the unbound reagents are removed 

from the plate by washing with PBST. The presence of Cry1Ac was detected by incubating the 

antibody-bound enzyme conjugate with an enzyme substrate, generating a colored product. 

Since the Cry1Ac is bound in the antibody sandwich, the level of color development is 

proportional to the concentration of Cry1Ac in the sample (i.e., lower protein concentrations 

result in lower color development). The absorbance at 450 nm was measured either using a 

Grifols Triturus Automated Immunoassay Analyzer with a 620 nm background subtraction or 

using a microplate reader with a 650 nm background subtraction. A calibration curve was 

estimated from 7 standard concentrations using a quadratic regression equation with a coefficient 

of determination > 0.990.

Determination of Cry1F Protein in Soybean Tissue Samples

Soybean tissue samples were analyzed using Dow AgroSciences method 110675. In this 

method, the soluble, extractable Cry1F protein was quantified using an enzyme-linked 

immunosorbent assay (ELISA) kit from Romer Labs, Inc.

The Cry1F protein was extracted from lyophilized, ground soybean tissues with a phosphate 

buffered saline solution containing 0.05% Triton X-100 with 1 mg/mL sodium ascorbate 

(PBS/Triton/AA). The extract was centrifuged; the aqueous supernatant was collected, diluted, 

and assayed using a specific Cry1F ELISA kit. A sandwich ELISA format was applied in this 

assay. An aliquot of the diluted sample was incubated in the wells of a plate pre-coated with an 

immobilized monoclonal anti-Cry1F antibody coated plate along with an enzyme-conjugated 

monoclonal antibody specific to the Cry1F protein. These antibodies bind with Cry1F protein in 

the wells and form a “sandwich” with Cry1F protein bound between the soluble and immobilized 

antibodies. At the end of an incubation period, the unbound reagents are removed from the plate 

by washing with PBST. The presence of Cry1F was detected by incubating the antibody-bound 

enzyme conjugate with an enzyme substrate, generating a colored product. Since the Cry1F is 
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bound in the antibody sandwich, the level of color development is proportional to the 

concentration of Cry1F in the sample (i.e., lower protein concentrations result in lower color 

development). The absorbance at 450 nm was measured either using a Grifols Triturus 

Automated Immunoassay Analyzer with a 620 nm background subtraction or using a microplate 

reader with a 650 nm background subtraction. A calibration curve was estimated from 7 

standard concentrations using a quadratic regression equation.

Determination of PAT Protein in Soybean Tissue Samples

Soybean tissue samples were analyzed using Dow AgroSciences method GRM 08.05. In this 

method, the soluble, extractable PAT protein was quantified using an enzyme-linked 

immunosorbent assay (ELISA) kit from EnviroLogix, Inc.

The PAT protein was extracted from lyophilized, ground soybean samples with a phosphate 

buffered saline solution containing 0.05% Tween 20 and 1% polyvinylpyrrolidone (PBST/PVP). 

The extract was centrifuged; the aqueous supernatant was collected, diluted and assayed using a 

specific PAT ELISA kit. An aliquot of the diluted sample was incubated with enzymeconjugated anti-PAT protein monoclonal antibody in the wells of an anti-PAT polyclonal 

antibody coated plate in a sandwich ELISA format. Both antibodies in the sandwich pair capture 

the PAT protein in the sample. At the end of the incubation period, the unbound reagents were

removed from the plate by washing with PBST. The presence of PAT was detected by 

incubating the antibody-bound enzyme conjugate with an enzyme substrate, generating a colored 

product. Since the PAT is bound in the antibody sandwich, the level of color development is 

proportional to the concentration of PAT in the sample (i.e., lower protein concentrations result 

in lower color development). The absorbance at 450 nm was measured either using a Grifols 

Triturus Automated Immunoassay Analyzer with a 620 nm background subtraction or using a 

microplate reader with a 650 nm background subtraction. A calibration curve was estimated 

from the 7 standard concentrations using a quadratic regression equation.
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Appendix 6. Analytical Methods for Compositional Analysis

Acid Detergent Fiber (ADFA)

The ANKOM2000 Fiber Analyzer automated the process of removal of proteins, carbohydrates, 

and ash. Fats and pigments were removed with an acetone wash prior to analysis. The fibrous 

residue that was primarily cellulose and lignin and insoluble protein complexes remained in the 

Ankom filter bag, and was determined gravimetrically.

Amino Acid Composition (TALC/TPLC)

Total aspartic acid (including asparagine)

Total threonine

Total serine

Total glutamic acid (including glutamine)

Total proline

Total glycine

Total alanine

Total valine

Total isoleucine

Total leucine

Total tyrosine

Total phenylalanine

Total histidine

Total lysine

Total arginine

Total tryptophan

Sulfur-containing amino acids: Total methionine

 Total cystine (including cysteine)

The samples were hydrolyzed in 6N hydrochloric acid for approximately 24 hours at 

approximately 106-118oC. Phenol was added to the 6N hydrochloric acid to prevent halogenation 

of tyrosine. Cystine and cysteine are converted to S-2-carboxyethylthiocysteine by the addition 

of dithiodipropionic acid. Tryptophan was hydrolyzed from proteins by heating at approximately 

110oC in 4.2N sodium hydroxide for approximately 20 hours.

The samples were analyzed by HPLC after pre-injection derivatization. The primary amino acids 

were derivatized with o-phthalaldehyde (OPA) and the secondary amino acids are derivatized 

with fluorenylmethyl chloroformate (FMOC) before injection.

Reference Standards:

Component Manufacture Lot No. Purity (%)

L-Alanine Sigma-Aldrich 1440397 99.9

L-Arginine Monohydrochloride Sigma-Aldrich 1361811 100.0

L-Aspartic Acid Sigma-Aldrich BCBB9274 100.6

L-Cystine Sigma-Aldrich 1418036

1451329

99.9

100

L-Glutamic Acid Sigma-Aldrich 1423805 100.2

Glycine Sigma-Aldrich 1119375 100.0

L-Histidine Monohydrochloride Monohydrate Sigma-Aldrich BCBB1348 99.9

L-Isoleucine Sigma-Aldrich 1423806 100.0
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Component Manufacture Lot No. Purity (%)

L-Leucine Sigma-Aldrich BCBB1733 98.6

L-Lysine Monohydrochloride Sigma-Aldrich 1362380 100.2

L-Methionine Sigma-Aldrich 1423807 99.9

L-Phenylalanine Sigma-Aldrich BCBB9200 100

L-Profile Sigma-Aldrich 1414414 99.7

L-Serine Sigma-Aldrich 1336081 99.9

L-Threonine Sigma-Aldrich 1402329 100.0

L-Tryptophan Sigma-Aldrich BCBB1284 99.8

L-Tyrosine Sigma-Aldrich BCBB5393 99.5

L-Valine Sigma-Aldrich 1352709 100.0

Ash (ASHM)

All organic matter was driven off when the samples were ignited at approximately 550oC in a 

muffle furnace for at least 5 hours. The remaining inorganic material was determined

gravimetrically and referred to as ash.

Beta Carotene (BCLC)

The samples were saponified and extracted with hexane. The samples were then injected on a 

reverse phase high-performance liquid chromatography system with ultraviolet light detection. 

Quantitation was achieved with a linear regression analysis.

Reference Standard:

Sigma-Aldrich, Beta Carotene, 99.0%, Lot No. 021M1304V

Carbohydrate (CHO)

The total carbohydrate level was calculated by difference using the fresh weight-derived data and 

the following equation:

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

Fat by Acid Hydrolysis (FAAH)

The samples were hydrolyzed with hydrochloric acid. The fat was extracted using ether and 

hexane. The extracts were dried down and filtered through a sodium sulfate column. 

The remaining extracts were then evaporated, dried, and weighed.

Fat by Soxhlet Extraction (FSOX)

The samples were weighed into a cellulose thimble containing sodium sulfate and dried to 

remove excess moisture. Pentane was dripped through the samples to remove the fat.

The extract was then evaporated, dried, and weighed.

Fatty Acids (FAPM)

The lipid was extracted and saponified with 0.5N sodium hydroxide in methanol. The 

saponification mixture was methylated with 14% boron trifluoride in methanol. The resulting 

methyl esters were extracted with heptane containing an internal standard. The methyl esters of 

the fatty acids were analyzed by gas chromatography using external standards for quantitation.
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 Reference Standards:

Manufacturer Lot No. Component Weight

(%)

Purity 

(%)

Nu-Chek Prep

GLC Reference

Standard

Covance 1

Covance 2

S27-V

S28-V

S27-V S28-V

Methyl Octanoate 3.0 1.25 99.7

Methyl Decanoate 3.25 1.25 99.6

Methyl Laurate 3.25 1.25 99.8

Methyl Myristate 3.25 1.25 99.8

Methyl Myristoleate 1.0 1.25 99.5

Methyl Pentadecanoate 1.0 1.25 99.6

Methyl Pentadecenoate 1.0 1.25 99.4

Methyl Palmitate 10.0 15.0 99.8

Methyl Palmitoleate 3.0 1.25 99.7

Methyl Heptadecanoate 1.0 1.25 99.6

Methyl 10-Heptadecenoate 1.0 1.25 99.5

Methyl Stearate 7.0 11.0 99.8

Methyl Oleate 10.0 15.0 99.8

Methyl Linoleate 10.0 15.0 99.8

Methyl Gamma Linolenate 1.0 1.25 99.4

Methyl Linolenate 3.0 1.25 99.5

Methyl Arachidate 2.0 1.25 99.8

Methyl 11-Eicosenoate 2.0 1.25 99.6

Methyl 11-14 Eicosadienoate 1.0 1.25 99.5

Methyl 11-14-17 

Eicosatrienoate

1.0 1.25 99.5

Methyl Arachidonate 1.0 1.25 99.4

Methyl Behenate 1.0 1.5 99.8

Folic acid (FOAN)

The samples were hydrolyzed in a potassium phosphate buffer with the addition of ascorbic acid 

to protect the folic acid during autoclaving. Following hydrolysis by autoclaving, the samples 

were treated with a chicken-pancreas enzyme and incubated approximately 18 hours to liberate 

the bound folic acid. The amount of folic acid was determined by comparing the growth response 

of the samples, using the bacteria Lactobacillus casei, with the growth response of a folic acid 

standard. This response was measured turbidimetrically.

Reference Standard:

USP, Folic acid, 98.9%, Lot No. Q0G151

ICP Emission Spectrometry (ICPS)

The sample was dried, precharred, and ashed overnight in a muffle set to maintain 500C. The 

ashed sample was re-ashed with nitric acid, treated with hydrochloric acid, taken to dryness, and 

put into a solution of 5% hydrochloric acid. The amount of each element was determined at 

appropriate wavelengths by comparing the emission of the unknown sample, measured on the 

inductively coupled plasma spectrometer, with the emission of the standard solutions.

 Inorganic Ventures Reference Standards and Limits of Quantitation:
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Mineral Lot No. Concentration (μg/mL)

Calcium E2-MEB393070MCA, E2-MEB393072 200, 1000

Copper E2-MEB393070MCA, E2-MEB393071MCA 2.00, 10.00

Iron E2-MEB393070MCA, E2-MEB393073 10.0, 50.0

Magnesium E2-MEB393070MCA, E2-MEB393071MCA 50.0, 250

Manganese E2-MEB393070MCA, E2-MEB393071MCA 2.0, 10.0

Phosphorus E2-MEB393070MCA, E2-MEB393072 200, 1000

Potassium E2-MEB393070MCA, E2-MEB393072 200, 1000

Sodium E2-MEB393070MCA, E2-MEB393072 200, 1000

Zinc E2-MEB393070MCA, E2-MEB393071MCA 10.0, 50.0

Isoflavones (ASOF)

The samples were extracted at approximately 65°C with a 80/20 methanol:water solution and the 

extract was saponified with dilute NaOH solution. The extract was then acidified, filtered, and 

diluted. The samples were analyzed on a high-performance liquid chromatography system with 

ultraviolet spectrophotometric detection and was compared against an external standard curve.

Reference Standards:

Component Manufacturer Lot No. Purity (%)

Daidzein LC Labs DA-121 99.7

Glycitein LC Labs ARH-114 99.8

Genistein LC Labs CH-148 99.7

Daidzin LC Labs ARF-114 99.7

Glycitin LC Labs ARG-113 99.6

Genistin LC Labs ARE-109 99.4

Lectins (LCTN)

The determination of lectins was based on the ability of lectin (a hemagglutinin) to bind to 

specific sugars present on the surface of red blood cells (RBCs) of different animal species 

resulting in the agglutination of RBCs. Samples were defatted and extracted with a saline 

solution. Agglutination of trypsinized rabbit RBCs was measured with a spectrophotometer at a 

wavelength of 620 nm.

Moisture (M100)

The samples were dried in a vacuum oven at approximately 100°C. The moisture weight loss 

was determined and converted to percent moisture. 

Neutral Detergent Fiber (NDFA)

The ANKOM2000 Fiber Analyzer automated the process of the removal of protein, 

carbohydrate, and ash. Fats and pigments were removed with an acetone wash prior to analysis. 

Hemicellulose, cellulose, lignin and insoluble protein fraction were left in the filter bag and 

determined gravimetrically.

Niacin (NIAP)

The samples were hydrolyzed with sulfuric acid and the pH was adjusted to remove

interferences. The amount of niacin was determined by comparing the growth response of the 
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samples, using the bacteria Lactobacillus plantarum, with the growth response of a niacin 

standard. This response was measured turbidimetrically.

Reference Standard:

USP, Niacin, 99.8%, Lot No. I0E295

Pantothenic Acid (PANN)

The samples were diluted with water or treated with an enzyme mixture to liberate the 

pantothenic acid from coenzyme A and the pH was adjusted to remove interferences. The 

amount of pantothenic acid was determined by comparing the growth response of the samples, 

using the bacteria Lactobacillus plantarum, with the growth response of a calcium pantothenate 

standard. This growth response was measured turbidimetrically.

Reference Standard:

USP, Calcium Pantothenate, 99.0%, Lot No. O1H081

Phytic Acid (PHYT)

The samples were extracted using hydrochloric acid and sonication, purified using a silica based 

anion exchange column, concentrated and injected onto a high-performance liquid 

chromatography (HPLC) system with a refractive index detector.

Reference Standard:

Sigma-Aldrich, Phytic Acid Sodium Salt Hydrate, 93.5%, Lot No. BCBF5728V

Protein (PGEN)

The BUCHI Automated Digester was used to automate the following process. The protein and 

other organic nitrogen in the samples were converted to ammonia by digesting the samples with 

sulfuric acid containing a catalyst mixture. The acid digest was made alkaline. The ammonia was 

distilled and then titrated with a previously standardized acid. The percent nitrogen was alculated 

and converted to equivalent protein using the factor 6.25.

Selenium by Inductively Coupled Plasma-Mass Spectrometry (SEMS)

The samples were closed-vessel microwave digested with nitric acid (HNO3) and water. After 

digestion, the solution was brought to a final volume with water. To normalize the organic 

contribution between samples and standards, a dilution was prepared for analysis that contained 

methanol. The selenium concentration was determined with Se78 using an inductively coupled 

plasma-mass spectrometer (ICP-MS) with a dynamic reaction cell (DRC) by comparing the 

counts generated by standard solutions.

Reference Standard:

SPEX, Selenium, 1000 mg/L, Lot No. 16-177SE

Sugar Profile (SUGT)

Sugars in the samples were extracted with a 50:50 water:methanol solution. Aliquots were taken, 

dried under inert gas, and then reconstituted with a hydroxylamine hydrochloride solution in 

pyridine containing phenyl-β-D-glucopyranoside as the internal standard. The resulting oximes 

were converted to silyl derivatives by treatment with hexamethyldisilazane and trifluoracetic 

acid treatment, and then analyzed by gas chromatography using a flame ionization detector.
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Reference Standards:

Component Manufacturer Lot No. Purity (%)

D-(+)-Raffinose pentahydrate Sigma-Aldrich 019K1156 99.6

Stachyose hydrate Sigma-Aldrich 049K3800 98

Total Tocopherols (TTLC)

The samples were saponified to break down any fat and release vitamin E. The saponified 

mixtures were extracted with an organic solvent, dried down and brought to a suitable volume in 

hexane. The samples were then quantitated by high-performance liquid chromatography using a 

silica column.

Reference Standards:

Component Manufacturer Lot No. Purity (%)

Alpha Tocopherol USP N0F068 98.9

rac-beta-Tocopherol Matreya LLC 23260 >99

Gamma Tocopherol ACROS A0083534 99.3

(+)-δ-Tocopherol (Delta) Sigma-Aldrich 090M1916V 92

Total Dietary Fiber (TDF)

Duplicate samples were gelatinized with α-amylase and digested with enzymes to break down 

starch and protein. Ethanol was added to each of the samples to precipitate the soluble fiber. The 

samples were filtered, and the residue was rinsed with ethanol and acetone to remove starch and 

protein degradation products and moisture. Protein content was determined for one of the 

duplicates; ash content was determined for the other. The total dietary fiber in the samples were 

calculated using protein and ash values and the weighed residue fractions.

Trypsin Inhibitor (TRIP)

The samples were ground and defatted with petroleum ether. A sample of matrix was extracted 

with 0.01N sodium hydroxide. Varying aliquots of the sample suspensions were exposed to a 

known amount of trypsin and benzoy1-DL-arginine~p~nitroanilide hydrochloride. The samples 

were allowed to react for 10 minutes at 37°C. After 10 minutes, the reaction was halted by the 

addition of acetic acid. The absorbance was determined at 410 nm against a sample blank. 

Trypsin inhibitor activity was determined by photometrically measuring the inhibition of 

trypsin’s reaction with benzoyl-DLarginine~p~nitroanilide hydrochloride.

Thiamine Hydrochloride (BIDE)

The samples were autoclaved under weak acid conditions to extract the thiamine. The resulting 

solutions were incubated with a buffered enzyme solution to release any bound thiamine. The 

solutions were purified on a cation-exchange column. Aliquots were reacted with potassium 

ferricyanide to convert thiamine to thiochrome. The thiochrome was extracted into isobutyl 

alcohol, measured on a fluorometer, and quantitated by comparison to a known standard.

Reference Standard:

USP, Thiamine Hydrochloride, 99.8%, Lot No. O1F236

Vitamin B2 (Riboflavin) (B2FV)
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The samples were hydrolyzed with dilute hydrochloric acid and the pH was adjusted to remove 

interferences. The amount of riboflavin was determined by comparing the growth response of the 

samples, using the bacteria Lactobacillus rhamnosus, with the growth response of multipoint 

riboflavin standards. The growth response was measured turbidimetrically.

Reference Standard:

USP, Riboflavin, 99.7%, Lot No. N1J079

Pyridoxine Hydrochloride

The samples were hydrolyzed with dilute sulfuric acid in the autoclave and the pH was adjusted 

to remove interferences. The amount of pyridoxine was determined by comparing the growth 

response of the samples, using the yeast Saccharomyces cerevisiae, with the growth response of 

a pyridoxine standard. The response was measured turbidimetrically. Results were reported as 

pyridoxine hydrochloride. 

Reference Standard:

USP, Pyridoxine hydrochloride, 99.8%, Lot No. Q0G409

Vitamin C (VCF)

The vitamin C in the samples were extracted, oxidized, and mixed with o-phenylenediamine to 

produce a fluorophor having an activation maximum at approximately 350 nm and a 

fluorescence maximum at 430 nm. Fluorescence was proportional to concentration. Development 

of the fluorescence compound with the vitamin was prevented by forming a boric aciddehydroascorbic acid complex prior to addition of the o-phenylenediamine solution. Any 

remaining fluorescence was due to extraneous material and served as the blank.

Reference Standard:

USP, Ascorbic Acid, 99.9%, Lot No. Q1G135
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Appendix 7. Literature Ranges for Compositional Analysis

Matrix Category Analyte Units OECD1

ILSI2Literature Literature Citations

Min Max Min Max Min Max Min Max

Forage Fiber ADF % DW 32 38 NR NR 22.72 59.03 Harrigan et al. 2007 Berman et al. 2010

Forage Fiber NDF % DW 34 40 NR NR 19.61 73.05 Lundry et al. 2008 Berman et al. 2010

Forage Proximate Ash % DW 8.8 10.5 6.718 10.782 4.68 9.24 Harrigan et al. 2007 Lundry et al. 2008

Forage Proximate Carbohydrates % DW NR NR 59.8 74.7 60.61 80.18 Berman et al. 2009 Berman et al. 2010

Forage Proximate Moisture % FW 74 79 73.5 81.6 32.05 84.60 Berman et al. 2009 Berman et al. 2010

Forage Proximate Crude Protein % DW 11.2 17.3 14.38 24.71 11.77 24.29 Berman et al. 2010 Lundry et al. 2008

Forage Proximate Total Fat % DW NR NR 1.302 5.132 1.01 9.87 Berman et al. 2010 Lundry et al. 2008

Forage Mineral Calcium mg/100g dry wt. NR NR NR NR NR NR NR NR

Forage Mineral Phosphorus mg/100g dry wt. NR NR NR NR NR NR NR NR

Grain Fiber ADF % DW 9.0 11.1 7.81 18.61 9.22 26.26 Lundry et al. 2008 Lundry et al. 2008

Grain Fiber NDF % DW 10.0 14.9 8.53 21.25 10.79 23.90 Lundry et al. 2008 Lundry et al. 2008

Grain Proximate Ash % DW 4.5 6.4 3.885 6.994 4.29 6.44 Padgette et al. 1996 Harrigan et al. 2007

Grain Proximate Carbohydrates % DW 31.7 31.8 29.6 50.2 29.3 44.35 Padgette et al. 1996 Harrigan et al. 2007

Grain Proximate Moisture % FW NR NR 4.7 34.4 4.71 14.30 Harrigan et al. 2007 Taylor et al. 1999

Grain Proximate Crude Protein % DW 32 43.6 33.19 45.48 32.29 48.4 Berman et al. 2011 Hartwig and Kilen 1991

Grain Proximate Total Fat % DW 15.5 24.7 8.104 23.562 14.10 23.67 Padgette et al. 1996 Berman et al. 2010

Grain Mineral Calcium mg/100g dry wt. NR NR 116.55 307.10 258 510 Iskander 1987 Bilyeu et al. 2008

Grain Mineral Copper mg/100g dry wt. NR NR NR NR 0.632 1.092 Bilyeu et al. 2008 Bilyeu et al. 2008

Grain Mineral Iron mg/100g dry wt. NR NR 5.536 10.954 3.734 6.624 Bilyeu et al. 2008 Bilyeu et al. 2008

Grain Mineral Magnesium mg/100g dry wt. NR NR 219.40 312.84 261 280 Iskander 1987 Bilyeu et al. 2008

Grain Mineral Manganese mg/100g dry wt. NR NR NR NR 2.52 3.876 Iskander 1987 Bilyeu et al. 2008

Grain Mineral Phosphorus mg/100g dry wt. NR NR 506.74 935.24 770 790 Bilyeu et al. 2008 Bilyeu et al. 2008

Grain Mineral Potassium mg/100g dry wt. NR NR 1868.01 2316.14 1910 2510 Iskander 1987 Bilyeu et al. 2008

Grain Mineral Selenium ppb DW NR NR NR NR NR NR NR NR

Grain Mineral Sodium mg/100g dry wt. NR NR NR NR 4.05 30 Iskander 1987 Bilyeu et al. 2008

Grain Mineral Zinc mg/100g dry wt. NR NR NR NR 4.98 7.578 Iskander 1987 Bilyeu et al. 2008

Grain Amino Acid Alanine % total amino acid NR NR 4.23 4.74 4.16 4.54 Berman et al. 2011 Harrigan et al. 2007

Grain Amino Acid Arginine % total amino acid NR NR 6.70 8.41 6.41 7.60 Berman et al. 2010 Lundry et al. 2008

Grain Amino Acid Aspartic Acid % total amino acid NR NR 11.39 12.04 11.37 12.68 Taylor et al. 1999 Berman et al. 2009

Grain Amino Acid Cystine % total amino acid NR NR 1.02 1.87 1.40 1.75 Berman et al. 2010 Berman et al. 2011

Grain Amino Acid Glutamic Acid % total amino acid NR NR 17.71 19.24 18.25 20.48 Harrigan et al. 2007 Berman et al. 2010

Grain Amino Acid Glycine % total amino acid NR NR 4.19 4.61 4.24 4.62 Berman et al. 2010 Berman et al. 2011

Grain Amino Acid Histidine % total amino acid NR NR 2.49 2.85 2.65 2.89 Lundry et al. 2008 Berman et al. 2011

Grain Amino Acid Isoleucine % total amino acid NR NR 4.13 5.11 4.51 4.62 Berman et al. 2009 Taylor et al. 1999

Grain Amino Acid Leucine % total amino acid NR NR 7.62 8.29 7.46 7.88 Berman et al. 2010 Lundry et al. 2008
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Matrix Category Analyte Units OECD1

ILSI2Literature Literature Citations

Min Max Min Max Min Max Min Max

Grain Amino Acid Lysine % total amino acid NR NR 6.29 7.16 6.23 7.38 Berman et al. 2011 Berman et al. 2010

Grain Amino Acid Methionine % total amino acid NR NR 1.18 1.71 1.31 1.47 Lundry et al. 2008 Lundry et al. 2008

Grain Amino Acid Phenylalanine % total amino acid NR NR 4.91 5.44 4.95 5.44 Berman et al. 2010 Berman et al. 2011

Grain Amino Acid Proline % total amino acid NR NR 4.75 5.62 4.96 5.23 Berman et al. 2010 Taylor et al. 1999

Grain Amino Acid Serine % total amino acid NR NR 3.25 6.04 5.03 5.47 Berman et al. 2010 Lundry et al. 2008

Grain Amino Acid Threonine % total amino acid NR NR 3.15 4.24 3.92 4.08 Berman et al. 2009 Berman et al. 2011

Grain Amino Acid Tryptophan % total amino acid NR NR 0.95 1.29 0.97 1.49 Lundry et al. 2008 Padgette et al. 1996

Grain Amino Acid Tyrosine % total amino acid NR NR 2.83 3.72 2.62 3.67 Berman et al. 2010 Padgette et al. 1996

Grain Amino Acid Valine % total amino acid NR NR 4.28 5.57 4.69 4.93 Padgette et al. 1996 Berman et al. 2011

Grain Fatty Acid 8:0 Caprylic % total fatty acid NR NR 0.148 0.148 ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 10:0 Capric % total fatty acid NR NR ND ND ND 0.27 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 12:0 Lauric % total fatty acid NR NR 0.082 0.132 ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 14:0 Myristic % total fatty acid NR NR 0.071 0.238 ND 0.11 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 14:1 Myristoleic % total fatty acid NR NR 0.121 0.125 ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 15:0 Pentadecanoic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 15:1 Pentadecenoic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 16:0 Palmitic % total fatty acid NR NR 9.55 15.77 1.40 12.63 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 16:1 Palmitoleic % total fatty acid NR NR 0.086 0.194 ND 0.14 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 17:0 Heptadecanoic % total fatty acid NR NR 0.085 0.146 ND 0.13 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 17:1 Heptadecenoic % total fatty acid NR NR 0.073 0.087 ND 0.064 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 18:0 Stearic % total fatty acid NR NR 2.70 5.88 0.50 5.63 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 18:1 Oleic % total fatty acid NR NR 14.3 32.2 2.60 45.68 Harrigan et al. 2007 Berman et al. 2010

Grain Fatty Acid 18:2 Linoleic % total fatty acid NR NR 42.3 58.8 7.58 57.72 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 18:3 Linolenic % total fatty acid NR NR 3.00 12.52 1.27 9.90 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 18:3 gamma Linolenic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 20:0 Arachidic % total fatty acid NR NR 0.163 0.482 0.038 0.57 Harrigan et al. 2007 Berman et al. 2009

Grain Fatty Acid 20:1 Eicosenoic % total fatty acid NR NR 0.140 0.350 0.024 0.35 Harrigan et al. 2007 Berman et al. 2010

Grain Fatty Acid 20:2 Eicosadienoic % total fatty acid NR NR 0.077 0.245 ND 0.065 Harrigan et al. 2007 Berman et al. 2010

Grain Fatty Acid 20:3 Eicosatrienoic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 20:4 Arachidonic % total fatty acid NR NR ND ND ND ND Harrigan et al. 2007 Harrigan et al. 2007

Grain Fatty Acid 22:0 Behenic % total fatty acid NR NR 0.277 0.595 0.043 0.65 Harrigan et al. 2007 Berman et al. 2009

Grain Vitamin Alpha Tocopherol (Vitamin E) mg/kg DW NR NR 1.934 61.693 10.8 84.9 Berman et al. 2010 Zhou et al. 2011

Grain Vitamin Beta Tocopherol mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Delta Tocopherol mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Gamma Tocopherol mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Total Tocopherol mg/kg DW NR NR NR NR NR NR NR NR
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Matrix Category Analyte Units OECD1

ILSI2Literature Literature Citations

Min Max Min Max Min Max Min Max

Grain Vitamin Vitamin A (Beta Carotene) mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Vitamin B1 (Thiamine HCl) mg/kg DW NR NR 1.01 2.54 NR NR NR NR

Grain Vitamin Vitamin B2 (Riboflavin) mg/kg DW NR NR 1.90 3.21 NR NR NR NR

Grain Vitamin Vitamin B3 (Niacin) mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Vitamin B5 (Pantothenic Acid) mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Vitamin B6 (Pyridoxine HCl) mg/kg DW NR NR NR NR NR NR NR NR

Grain Vitamin Vitamin B9 (Folic Acid) mg/kg DW NR NR 2.386 4.709 NR NR NR NR

Grain Vitamin Vitamin C (Ascorbic acid) mg/kg DW NR NR NR NR NR NR NR NR

Grain Bioactive Total Daidzein Equivalent mcg/g DW NR NR 60.0 2453.5 25 2100.00 McCann et al. 2005 Berman et al. 2011

Grain Bioactive Total Genistein Equivalent mcg/g DW NR NR 144.3 2837.2 28 2600.70 McCann et al. 2005 Harrigan et al. 2007

Grain Bioactive Total Glycitein Equivalent mcg/g DW NR NR 15.3 310.0 32 349.19 Berman et al. 2011 Harrigan et al. 2007

Grain Bioactive Lectin HU/mg Protein DW 37 323 NR NR 37 323 Kakade et al. 1972 Kakade et al. 1972

Grain Bioactive Phytic Acid % DW NR NR 0.634 1.960 0.41 2.68 Lundry et al. 2008 Berman et al. 2010

Grain Bioactive Raffinose % DW NR NR 0.212 0.661 0.22 1.85 Harrigan et al. 2007 Berman et al. 2011

Grain Bioactive Stachyose % DW NR NR 1.21 3.50 1.52 6.65 Harrigan et al. 2007 Berman et al. 2011

Grain Bioactive Trypsin Inhibitor TIU/mg DW NR NR 19.59 118.68 18.14 75.5 Berman et al. 2009 McCann et al. 2005

ND = Not Detected, NR = Not Reported

1

(OECD, 2001) Consensus document on compositional considerations for new varieties of soybean: Key food and feed nutrients and anti-nutrients. 

ENV/JM/MONO(2001)15. 30. www.oecd.org/dataoecd/15/60/46815135.pdf.

2

International Life Sciences Institute, Crop Composition Database v4.2 (accessed January 10 and May 16, 2012)
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 201 of 214

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Appendix 8. Non-Target Arthropod Field Study

A non-target arthropod field study with DAS-81419-2 soybean, a near-isogenic non-transgenic 

control (Maverick) and four non-transgenic reference lines (DSR 3590; IL 3505; Porter 75148; 

Williams 82) was conducted in 2011 at two sites in the U.S. located in Richland, Iowa and York, 

Nebraska. Each trial site included six entries, including one entry of DAS-81419-2, one control 

entry (Maverick), and four entries of non-transgenic reference varieties. At each site, entries 

were arranged in a randomized complete block design with four blocks. Across sites, each 

control, reference, and DAS-81419-2 entry was represented by a total of eight plots (2 sites, 4 

replicate plots per entry at each site). Each study plot (observational unit) measured 

approximately 335 m2(3600 ft2; 24 rows wide, 60 ft length). Soybean seeds were planted at a 

seeding rate of approximately 300 seeds per row with seed spacing within each row of 

approximately 2.4 inches (6 cm; 5 seeds per row foot). Study plots were separated by four rows 

of a non-transgenic soybean cultivar of similar maturity. The entire trial site was surrounded by 

a minimum of four rows (3.0 m; 10 ft) of a non-transgenic soybean cultivar of similar maturity. 

Therefore, the entire trial area formed a continuous field of soybean. Appropriate weed and 

disease control practices were applied to produce an agronomically acceptable crop. Arthropod 

pest management, including sprayed insecticides and miticides, was excluded from study.

Arthropod abundance was monitored using three sampling methods: pitfall trapping, sticky card 

trapping, and vertical beat sheet sampling. Sample timings included vegetative and reproductive 

soybean stages. Arthropods collected during the study included representatives from multiple 

orders and diverse ecological roles, including phytophagous, predatory, parasitic, and 

saprophagous modes of feeding. Assessment was conducted for several taxonomic groups, 

including: Oribatida (soil mites), Araneae (foraging and web spiders), Collembola (springtails), 

Hemiptera (e.g. Orius spp.), Coleoptera (e.g. ground beetles, lady beetles), Neuroptera (e.g. 

Chrysopa spp.), Lepidoptera, Diptera, and Hymenoptera (e.g. parasitoid wasps). A step-wise 

approach to statistical analysis was used, where community-level data were first analyzed using 

redundancy analysis (RDA) and Monte Carlo simulations (Dively, 2005) to test for differences 

between arthropod populations associated with DAS-81419-2 and the near-isogenic control. 

Redundancy analysis results were used to construct principal response curves (PRCs) to visualize 

population patterns. When defined criteria were met, a second step assessment included 

univariate analysis of variance (ANOVA) and application of the false discovery rate (FDR) 

procedure (Benjamini and Hochberg, 1995; Curran-Everett, 2000). Results of both the PRC and 

ANOVA analyses confirmed no unintended effects of DAS-81419-2 soybean on any of the 

arthropod groups monitored. Consistent across locations and sampling methods, arthropod 

populations associated with DAS-81419-2 soybean were similar to those of non-transgenic 

soybean. Field monitoring confirmed the results of established laboratory studies examining the 

Cry1Ac and Cry1F proteins, and demonstrated no adverse effects of DAS-81419-2 cultivation on 

non-target arthropod communities.
USDA Petition for Nonregulated Status of DAS-81419-2 Soybean Page 202 of 214

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Appendix 9. USDA Notifications for DAS-81419-2 Soybean

USDA 

Notification 

Number

Notification 

Authorization

Date

Notification 

Expiration 

Date

States Number 

of Trials

Trial 

Status1

12-088-113n 4/23/2012 4/23/2013

AL, AR, GA, IA, 

IL, IN, MD, MO, 

NE

24 pending

12-072-105n 4/2/2012 4/2/2013 IA, IL, MO, NE, 

PA, IN 8 pending

12-060-107n 4/1/2012 4/1/2013 IA, IL, MO, NE, 

PA, IN 12 pending

11-305-101n 11/30/2011 11/30/2012 PR 1 pending

11-216-106n 9/9/2011 9/9/2012 PR 8 pending

11-088-112n 5/6/2011 5/6/2012 IA, IN, MO 4 pending

11-088-101n 5/9/2011 5/9/2012 IA, NE 2 pending

11-087-115n 4/21/2011 4/21/2012

AR, GA, IA, IL, 

IN, MD, MI, MO, 

MS, NE, PR

26 pending

11-087-113n 4/15/2011 4/15/2012 IA, IL, IN, MO, 

NE, PA 10 pending

10-243-105n 9/30/2010 9/30/2011 PR 3 submitted

10-077-106n 4/14/2010 4/14/2011 IN 1 submitted

09-259-107n 9/30/2009 9/30/2010 PR 1 submitted

1

Pending reports as of July 28, 2012 to be submitted within six months of the notification expiration date.
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Appendix 10. Stewardship of Insect-Resistant DAS-81419-2 Soybean

Dow AgroSciences is committed to promoting the responsible use and stewardship of its 

products throughout the product life cycle. DAS has a long-standing commitment to product 

stewardship and is a founding member of Excellence through Stewardship. In addition, DAS has 

voluntarily established a Biotechnology Quality Management System (BQMS) which has been 

verified by third-party audits as conforming to the USDA APHIS BQMS Program Audit Standard.

Consistent with its existing stewardship programs and quality management systems, DAS will 

implement comprehensive stewardship measures for insect-resistant DAS-81419-2 soybean. 

These programs and practices include rigorous field compliance, quality assurance, quality 

control, and auditing. 

DAS has submitted an application for a FIFRA Section 3 seed increase registration with EPA to 

support future breeding and seed increase activities in the United States. Under a Section 3 seed 

increase registration, commercial sale of DAS-81419-2 soybean in the U.S. is prohibited. If DAS 

chooses to cultivate DAS-81419-2 soybean for commercial purposes in the U.S. in the future, 

DAS will apply for a FIFRA Section 3 commercial use registration with EPA. 

As with all products, DAS is committed to the rigorous product stewardship of DAS-81419-2 

soybean. This includes having processes in place to restrict and steward the use of DAS-81419-2 

soybean based on the registered uses in the U.S. All breeding or seed multiplication work will 

be done directly by DAS or its licensees under a contract. As long as DAS-81419-2 soybean is 

cultivated in the U.S. under the Section 3 seed increase registration, the limitations on the EPA 

registration, combined with contractual production of the seed, will serve to effectively limit use 

of DAS-81419-2 soybean and product stewardship requirements will be implemented 

accordingly. In the future, should DAS choose to register DAS-81419-2 soybean for 

commercial production in the U.S., product stewardship measures would be expanded to reflect 

commercial grower use and insect resistance management requirements.

Before commercially launching DAS-81419-2 soybean in any country, DAS will obtain 

regulatory approval from key soybean import countries with functioning regulatory systems to 

assure regulatory compliance, promote product launch stewardship, and support the flow of 

international trade. DAS is in the process of making submissions to the regulatory authorities of 

trade partners for import approval and/or cultivation approval. DAS also commits to industry 

practices to maintain plant product integrity and seed purity of DAS-81419-2 soybean. A 

detection method will be made available to soybean producers, processors and buyers before 

commercializing DAS-81419-2 soybean in any country.
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Appendix 11. References

Adang, M.J., Staver, M.J., Rocheleau, T.A., Leighton, J., Barker, R.F., Thompson, D.V., 1985. 

Characterized full-length and truncated plasmid clones of the crystal protein of Bacillus 

thuringiensis subsp. kurstaki HD-73 and their toxicity to Manduca sexta. Gene 36, 289-

300.

AgrEvo, 1994. Petition for determination of nonregulated status: Glufosinate resistant corn 

transformation events T14 and T25. 

http://www.aphis.usda.gov/brs/aphisdocs/94_35701p.pdf

AgrEvo, 1996. Petition for determination of nonregulated status: Glufosinate resistant soybean 

transformation events. http://www.aphis.usda.gov/brs/aphisdocs/96_06801p.pdf

AgrEvo, 1997. Petition for determination of nonregulated status: Glufosinate tolerant canola 

transformation event T45. http://www.aphis.usda.gov/brs/aphisdocs/97_20501p.pdf.

Ahrent, D.K., Caviness, C.E., 1994. Natural cross-pollination of twelve soybean cultivars in 

Arkansas. Crop Science 34, 376-378.

AOSA, 2009. Seed vigor testing handbook contribution no. 32. In: Baalbaki, R.J., Marcos-Filho, 

J., Elias, S.G., McDonald, M. (Eds.).

AOSA, 2010a. AOSA rules for testing seeds volume 1. Principles and procedures. Association of 

Official Seed Analysts Inc.

AOSA, 2010b. AOSA rules for testing seeds volume 4. Seeding evaluation. Association of 

Official Seed Analysts Inc.

AOSCA, 2012. Seed certification. http://www.aosca.org/seed%20certification.htm.

Aronson, A.I., Shai, Y., 2001. Why Bacillus thuringiensis insecticidal toxins are so effective: 

Unique features of their mode of action. FEMS Microbiology Letters 195, 1-8.

Arumuganathan, K., Earle, E., 1991. Nuclear DNA content of some important plant species. 

Plant Molecular Biology Reporter 9, 208-218.

Barker, R.F., Idler, K.B., Thompson, D.V., Kemp, J.D., 1983. Nucleotide-sequence of the TDNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant 

Molecular Biology 2, 335-350.

Beard, B.H., Knowles, P.F., 1971. Frequency of cross-pollination of soybeans after seed 

irradiation. Crop Science 11, 489-492.

Benjamini, Y., Hochberg, Y., 1995. Controlling the false discovery rate: A practical and 

powerful approach to multiple testing. Journal of the Royal Statistical Society. Series B 

(Methodological) 57, 289-300.

Berman, K.H., Harrigan, G.G., Nemeth, M.A., Oliveira, W.S., Berger, G.U., Tagliaferro, F.S., 

2011. Compositional equivalence of insect-protected glyphosate-tolerant soybean MON 

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