USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 1 of 214

Dow AgroSciences LLC

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Petition for Determination of Nonregulated Status for

Herbicide Tolerant DAS-8191Ø-7 Cotton

OECD Unique Identifier: DAS-8191Ø-7

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:

Gary Rudgers, Ph.D.

U.S. Regulatory Manager

Regulatory Sciences & Government Affairs

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

Telephone: (317) 337-3581

FAX: (317) 337-4649

gwrudgers@dow.com

September 17, 2013

Revised November 25, 2013
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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 its release to any third party. If USDA receives a Freedom of Information Act request 

pursuant to 5 U.S.C., § 552 and 7 CFR Part 1 regarding this information, DAS expects that 

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 on appropriate legal grounds (e.g., 

responsiveness, confidentiality, and/or competitive concerns). DAS understands that a copy of 

this information may be made available to the public in a reading room and upon individual 

request as part of a 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.

© 2013 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, unfavorable as well as favorable, 

associated with DAS-8191Ø-7 cotton.

Gary Rudgers, Ph.D.

U.S. Regulatory Manager

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, Indiana 46268

Telephone: (317) 337-3581

Fax: (317) 337-4649

gwrudgers@dow.com
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 4 of 214

Dow AgroSciences LLC

Summary

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

Determination of Nonregulated Status for DAS-8191Ø-7 cotton. DAS requests from USDA 

Animal and Plant Health Inspection Service (APHIS) that cotton transformation event DAS8191Ø-7 and any cotton lines derived from crosses between DAS-8191Ø-7 cotton and 

conventional cotton or biotechnology-derived cotton granted nonregulated status by APHIS,

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

DAS has developed transgenic cotton plants containing aad-12 and pat, which confer

tolerance to the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate. DAS8191Ø-7 cotton will provide growers with greater flexibility in selection of herbicides for the 

improved control of economically important weeds; allow an increased application window 

for effective weed control; and provide an effective weed resistance management solution to 

the growing incidence of glyphosate resistant weeds.

DAS-8191Ø-7 cotton plants have been genetically modified to express aryloxyalkanoate 

dioxygenase-12 (AAD-12). AAD-12 is an enzyme with an alpha ketoglutarate-dependent 

dioxygenase activity which results in metabolic inactivation of the herbicides of the 

aryloxyalkanoate family. The aad-12 gene, which expresses the AAD-12 protein, was 

derived from Delftia acidovorans, a gram-negative soil bacterium.

In addition to AAD-12, DAS-8191Ø-7 cotton plants have been genetically modified to 

express the phosphinothricin acetyltransferase (PAT) protein. The PAT enzyme acetylates 

the primary amino group of the herbicide phosphinothricin, rendering the herbicide inactive. 

The pat gene expressing the PAT protein was derived from Streptomyces viridochromogenes

and provides tolerance to the herbicide glufosinate in DAS-8191Ø-7 cotton plants. The pat

gene has been extensively reviewed by regulatory authorities in over eleven countries and 

has a long history of safe use, being used in over eight crop species representing over twentytwo biotechnology plant events. 

The aad-12 and pat expression cassettes introduced into DAS-8191Ø-7 cotton are the same 

as those introduced into DAS-68416-4 soybean, currently under review by USDA-APHIS

(USDA petition number 09-349-01p). In addition, the aad-12 and pat expression cassettes 

are the same as those introduced into DAS-444Ø6-6 soybean, also currently under USDAAPHIS review (USDA petition number 11-234-01p).

Plant Pest Assessment of DAS-8191Ø-7 Cotton

Data and information presented in this petition to USDA-APHIS confirm the lack of plant 

pest potential of DAS-8191Ø-7 cotton compared to non-transgenic cotton. The data 

demonstrate that DAS-8191Ø-7 cotton is agronomically, phenotypically, and compositionally 

comparable to non-transgenic cotton, with the exception of the presence of the aad-12 and pat 

genes. DAS-8191Ø-7 cotton is unlikely to pose an increased plant pest risk, weediness

potential or an adverse environmental impact compared to non-transgenic cotton. These 

conclusions are based on the outcome of extensive data and evaluation including:
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 Cotton is a familiar crop that does not possess any of the attributes commonly

associated with weeds and has a long history of safe use.

 Detailed molecular characterization of the inserted DNA demonstrated a single, intact 

copy of the T-DNA insert within the cotton genome.

 Detailed biochemical characterization of the expressed AAD-12 and PAT proteins

confirmed they are unlikely to be allergens or toxins. In addition, PAT has been 

commercially available in crops including cotton, corn, rice, canola, soybean and several 

other crops previously reviewed by USDA. 

 Compositional assessment of cottonseed confirmed the DAS-8191Ø-7 cotton is 

compositionally equivalent to non-transgenic cotton.

 Phenotypic, agronomic and ecological characterization assessments of DAS-8191Ø-7 

cotton demonstrated no increased plant pest or weediness potential compared to nontransgenic cotton.

 Assessment on the potential impact on non-target and endangered species indicated that 

DAS-8191Ø-7 cotton is unlikely to have adverse effects on these organisms compared to 

non-transgenic cotton.

 Current agronomic management practices for cotton conclude that DAS-8191Ø-7 

cotton is not likely to impact cotton agronomic practices or land use, with the exception 

of 2,4-D application on DAS-8191Ø-7 cotton varieties.

Cotton is a Familiar Crop

U.S. cotton is grown across 17 states in the southern region of the country. Cotton is the 

world’s most widely grown fiber crop, accounting for over 35% of the total world fiber use. 

The United States is the top exporter of raw cotton, and third in world production behind 

China and India. In 2012, 9.4 million acres of cotton were harvested in the US; with 

production value estimated at just under $6 billion.

Cotton is a slow-growing plant that competes poorly with weeds. The commercial cotton 

species grown in the U.S. do not exhibit weedy characteristics, do not outcross to weedy 

relatives and are not invasive. Cotton is not listed as a weed and is not listed on the list of 

noxious weeds by USDA (7 CFR Part 360). Volunteers that may appear in subsequent 

rotational crops are easily controlled through traditional tillage practices or the use of 

appropriate herbicides.

Although feral populations of cultivated variants of cotton exist in the U.S., these populations 

do not occur in cotton growing areas. In addition, DAS-8191Ø-7 cotton would not be 

expected to confer a selective advantage or result in a plant pest or weediness potential if 

crossing with feral populations were to occur. Such unlikely outcomes could be controlled by 

current agronomic practices. 
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Molecular Characterization of DAS-8191Ø-7 Cotton

The aad-12 and pat genes were introduced into DAS-8191Ø-7 cotton using Agrobacterium

mediated transformation. Molecular characterization by Southern blot analyses of DAS8191Ø-7 cotton confirmed that a single, intact DNA insert containing the aad-12 and pat

gene expression cassettes was stably integrated into the cotton genome. Southern blot 

analyses also confirmed the absence of the plasmid backbone DNA in DAS-8191Ø-7 cotton. 

The integrity of the inserted DNA was demonstrated in five different breeding generations. 

Data from segregating generations confirmed the predicted Mendelian inheritance pattern. 

These data confirmed the stability of DAS-8191Ø-7 cotton during traditional breeding 

procedures.

Biochemical Characterization of DAS-8191Ø-7 Cotton

Microbial-derived AAD-12 and PAT have been extensively assessed to establish the safety 

of the protein. DAS-8191Ø-7 cotton-derived AAD-12 and PAT proteins were determined to 

be biochemically equivalent to the corresponding proteins from microbial expression host 

organisms. A step-wise, weight-of-evidence approach was used to assess the potential for 

toxic or allergenic effects from the AAD-12 and PAT proteins. Bioinformatic analyses 

revealed no meaningful homologies with known or putative allergens or toxins for the AAD12 or PAT amino acid sequences. Both proteins hydrolyzed rapidly in simulated gastric fluid. 

There was no evidence of acute toxicity in mice at a dose of 2000 mg/kg body weight of 

AAD-12 protein and 5000 mg/kg body weight of PAT protein. 

AAD-12 and PAT expression levels in DAS-8191Ø-7 cotton were measured using a proteinspecific enzyme-linked immunosorbent assay (ELISA). Protein expression was analyzed in 

multiple tissues collected throughout the growing season from DAS-8191Ø-7 cotton plants 

both non-sprayed and sprayed with 2,4-D plus glufosinate. Glycosylation analysis revealed 

no detectable covalently linked carbohydrates in either AAD-12 or PAT proteins expressed 

in DAS-8191Ø-7 cotton plants. The low level expression of these proteins presents a low 

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

AAD-12 and PAT indicate that DAS-8191Ø-7 cotton is unlikely to cause allergenic or toxic

effects in humans or animals.

Compositional Assessment of DAS-8191Ø-7 Cotton

A compositional assessment was conducted in which levels of key nutrients and antinutrients of DAS-8191Ø-7 cottonseed were compared with the appropriate non-transgenic 

near isogenic control and non-transgenic reference lines. Samples were analyzed for 

proximates, fiber, minerals, amino acids, fatty acids, vitamins, and anti-nutrients. Fifty-nine 

cotton analytes were assayed and the analyses conclude that DAS-8191Ø-7 cotton is 

compositionally equivalent to non-transgenic cotton.

Phenotypic, Agronomic and Ecological Characterization of DAS-8191Ø-7 Cotton

Evaluations of nine phenotypic, agronomic and ecological characteristics (early population, 

seedling vigor, flower initiation, nodes above white flower, plant height, percent open bolls, 

lint yield, disease incidence and insect damage) were conducted to investigate the 

equivalency of DAS-8191Ø-7 (non-sprayed and sprayed with 2,4-D plus glufosinate) cotton 

to non-transgenic cotton. Phenotypic, agronomic and ecological analysis of DAS-8191Ø-7 
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cotton revealed no statistically significant differences between non-sprayed DAS-8191Ø-7 

cotton and the isoline (control). It was also determined that the phenotypic, agronomic and 

ecological characteristics of DAS-8191Ø-7 cotton sprayed with 2,4-D plus glufosinate are 

equivalent to non-sprayed DAS-8191Ø-7 cotton. In addition, germination and dormancy 

studies conducted under warm and cool conditions did not differ significantly from the nontransgenic cotton, near isogenic comparator. 

The data support the conclusion that DAS-8191Ø-7 cotton is substantially equivalent to nontransgenic cotton with the exception of the introduced and expression of aad-12 and pat 

genes and therefore no more likely to pose a plant pest risk or have a significant 

environmental impact compared to non-transgenic cotton.

Assessment on the Potential Impact on Non-Target and Endangered Species 

A review of potential environmental impacts indicates that DAS-8191Ø-7 cotton 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.

 The aad-12 gene and expressed protein are present in nature in the soil bacterium Delftia 

acidovorans. The pat gene and the expressed protein are present in other crops grown in the 

United States with no effects on non-target organisms or endangered species. 

 AAD-12 and PAT are not potential food allergens or toxins.

 DAS-8191Ø-7 cotton has been shown to be substantially equivalent to non-transgenic cotton 

based on the compositional analysis of cottonseed.

 Observations made during field testing of DAS-8191Ø-7 cotton revealed no effects on 

invertebrate populations.

 Agronomic characteristics were found to be equivalent to non-transgenic cotton. 

Impact on Current Agronomic Management Practices

Because DAS-8191Ø-7 cotton is agronomically and compositionally similar to conventional 

cotton, no significant impact is expected on current crop production practices, non-target or 

endangered species, crop rotation, volunteer management, or commodity food and feed 

cotton products. The availability of DAS-8191Ø-7 cotton is expected to have a beneficial 

impact on weed control practices by providing growers with an advanced tool to address their 

weed control needs. The availability of DAS-8191Ø-7 cotton will allow growers to 

proactively manage weed populations while avoiding adverse population shifts of 

troublesome weeds or the development of resistance, particularly glyphosate resistance in 

weeds.

Conclusion

Information collected during field trials and laboratory analyses presented herein conclude 

that DAS-8191Ø-7 cotton is not likely to be a plant pest or result in weediness potential. 

DAS therefore requests a determination from USDA-APHIS that herbicide tolerant DAS8191Ø-7 cotton and all progeny derived from crosses between DAS-8191Ø-7 cotton and 

non-transgenic cotton or biotechnology derived cotton be granted nonregulated status under 7 

CFR Part 340.
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Table of Contents

RELEASE OF INFORMATION ...................................................................................................................................2

CERTIFICATION ......................................................................................................................................................3

SUMMARY .............................................................................................................................................................4

TABLE OF CONTENTS..............................................................................................................................................8

LIST OF FIGURES................................................................................................................................................... 11

LIST OF TABLES .................................................................................................................................................... 13

LIST OF ACRONYMS AND SCIENTIFIC TERMS........................................................................................................ 14

1. RATIONALE FOR THE DEVELOPMENT OF DAS-8191Ø-7 COTTON................................................................. 17

1.1. BASIS FOR THE REQUEST FOR NONREGULATED STATUS .........................................................................................17

1.2. BENEFITS OF DAS-8191Ø-7 COTTON ..............................................................................................................17

1.3. SUBMISSION TO OTHER REGULATORY AGENCIES..................................................................................................19

2. THE BIOLOGY OF COTTON AND DAS-8191Ø-7 RECIPIENT COTTON CULTIVAR............................................. 20

2.1. OVERVIEW OF COTTON BIOLOGY ......................................................................................................................20

2.2. CHARACTERIZATION OF THE RECIPIENT COTTON CULTIVAR.....................................................................................20

3. METHOD OF DEVELOPMENT OF DAS-8191Ø-7 COTTON.............................................................................. 21

3.1. PDAB4468 .................................................................................................................................................21

3.2. DESCRIPTION OF THE TRANSFORMATION SYSTEM.................................................................................................21

3.3. SELECTION OF THE COMPARATORS FOR DAS-8191Ø-7 COTTON ...........................................................................25

3.4. DONOR GENES AND REGULATORY SEQUENCES IN DAS-8191Ø-7 COTTON..............................................................25

3.4.1. aad-12 expression cassette..................................................................................................................25

3.4.2. pat expression cassette........................................................................................................................25

3.4.3. RB7 MAR ..............................................................................................................................................28

4. GENETIC CHARACTERIZATION OF DAS-8191Ø-7 COTTON............................................................................ 29

4.1. OVERVIEW OF GENETIC CHARACTERIZATION .......................................................................................................29

4.1.1. Analysis of the Insert and Its Genetic Elements ...................................................................................37

4.1.2. Absence of Plasmid Backbone Sequences............................................................................................38

4.2. STABILITY OF THE INSERT ACROSS GENERATIONS .................................................................................................39

4.3. SOUTHERN BLOT ANALYSIS CONCLUSIONS..........................................................................................................39

4.4. SEGREGATION ANALYSIS .................................................................................................................................67

4.4.1. Genetic and Molecular Analysis of a Segregating Generation ............................................................67

4.4.2. Segregation Analysis of Breeding Generations....................................................................................67

4.5. SUMMARY OF THE GENETIC CHARACTERIZATION..................................................................................................68

5. CHARACTERIZATION OF THE DAS-8191Ø-7 AAD-12 AND PAT PROTEINS..................................................... 69

5.1. AAD-12......................................................................................................................................................69

5.1.1. Identity of the AAD-12 Protein.............................................................................................................69

5.1.2. Mode of Action of the AAD-12 Protein ................................................................................................70

5.1.3. Biochemical Characterization of the AAD-12 Protein ..........................................................................71

5.1.4. Expression of the AAD-12 Protein in Plant Tissues...............................................................................72

5.1.5. Food and Feed Safety Assessment of AAD-12......................................................................................73

5.1.6. Summary of AAD-12 Protein Characterization ....................................................................................77

5.2. PAT............................................................................................................................................................78

5.2.1. Identity of the PAT Protein...................................................................................................................78

5.2.2. Mode of Action of the PAT Protein ......................................................................................................78
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5.2.3. Biochemical Characterization of the PAT Protein ................................................................................79

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

5.2.5. Food and Feed Safety Assessment of PAT............................................................................................81

5.2.6. Summary of PAT Protein Characterization...........................................................................................82

6. COMPOSITION ASSESSMENT OF DAS-8191Ø-7 COTTON............................................................................. 83

6.1. DAS-8191Ø-7 COTTONSEED COMPOSITION .....................................................................................................83

6.1.1. Field Study Design................................................................................................................................83

6.1.2. Compositional Analysis........................................................................................................................84

6.2. STATISTICAL ANALYSIS ....................................................................................................................................84

6.3. COMPOSITION ANALYSIS RESULTS.....................................................................................................................86

6.3.1. Sub-experiment One - Proximate and Fiber Analysis of Seed ..............................................................86

6.3.2. Sub-experiment One - Mineral Analysis of Seed ..................................................................................90

6.3.3. Sub-experiment One - Amino Acid Analysis of Seed.............................................................................94

6.3.4. Sub-experiment One - Fatty Acid Analysis of Seed.............................................................................100

6.3.5. Sub-Experiment One - Vitamin Analysis of Seed ................................................................................105

6.3.6. Sub-Experiment One - Anti-Nutrient Analysis of Seed .......................................................................107

6.3.7. Sub-Experiment Two - Composition Results.......................................................................................110

6.4. COMPOSITION SUMMARY .............................................................................................................................110

6.5. CONCLUSIONS.............................................................................................................................................111

7. PHENOTYPIC, AGRONOMIC AND ECOLOGICAL CHARACTERISTICS ............................................................ 112

7.1. STUDY DESIGN............................................................................................................................................112

7.2. DATA COLLECTION AND STATISTICAL ANALYSIS..................................................................................................113

7.2.1. Phenotypic and Agronomic Results....................................................................................................115

7.2.2. Ecological Evaluations........................................................................................................................116

7.2.3. Cottonseed Germination and Dormancy Evaluation .........................................................................117

7.3. SUMMARY OF PHENOTYPIC, AGRONOMIC AND ECOLOGIC CHARACTERISTICS ...........................................................119

7.4. FIELD EFFICACY ...........................................................................................................................................119

8. IMPACT ON ENVIRONMENTAL AND AGRONOMIC PRACTICES .................................................................. 121

8.1. CURRENT U.S. AGRONOMIC PRACTICES FOR COTTON.........................................................................................121

8.1.1. Cotton Production..............................................................................................................................121

8.1.2. Cotton Seed Production .....................................................................................................................124

8.1.3. Planting and Cotton Development.....................................................................................................125

8.1.4. Weeds in Cotton.................................................................................................................................126

8.1.5. Weed Management in Cotton ...........................................................................................................127

8.1.6. Crop Rotation and Tillage Practices...................................................................................................128

8.2. POTENTIAL IMPACTS OF DAS-8191Ø-7..........................................................................................................128

8.2.1. Environment – Weediness Potential, Gene Flow and Non-Target / Endangered Species..................129

8.2.2. Agronomic Practices – cultivation/management, weed control and volunteer management..........133

8.3. HERBICIDE RESISTANCE MANAGEMENT............................................................................................................135

8.3.1. Herbicide Resistance ..........................................................................................................................135

8.3.2. Factors Impacting Herbicide Resistance ............................................................................................136

8.3.3. Herbicide Resistance Management ...................................................................................................138

8.4. SUMMARY OF ENVIRONMENTAL CONSEQUENCES AND IMPACT ON AGRONOMIC PRACTICES.......................................140

9. ADVERSE CONSEQUENCES OF INTRODUCTION ......................................................................................... 141

10. APPENDICES ............................................................................................................................................. 141

APPENDIX 1. METHODS FOR MOLECULAR CHARACTERIZATION OF DAS-8191Ø-7 COTTON ....................................................142

APPENDIX 2. METHODS AND RESULTS FOR CHARACTERIZATION OF AAD-12 PROTEIN ............................................................144

APPENDIX 3. METHODS AND RESULTS FOR CHARACTERIZATION OF PAT PROTEIN ..................................................................159
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APPENDIX 4. METHODS FOR AAD-12 & PAT PROTEIN EXPRESSION ANALYSIS......................................................................173

APPENDIX 5. METHODS FOR COMPOSITIONAL ANALYSIS ...................................................................................................177

APPENDIX 6. COMPOSITIONAL LITERATURE RANGES FOR NON-TRANSGENIC COTTONSEED.......................................................185

APPENDIX 7. EVOLUTION OF HERBICIDE RESISTANT WEEDS IN TRANSGENIC CROPS ................................................................187

APPENDIX 8. STEWARDSHIP OF HERBICIDE TOLERANT TRAIT TECHNOLOGY FOR DAS-8191Ø-7 COTTON...................................196

APPENDIX 9. USDA NOTIFICATIONS FOR DAS-8191Ø-7 COTTON.....................................................................................202

APPENDIX 10. REFERENCES .........................................................................................................................................203
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List of Figures

FIGURE 1. PLASMID MAP OF PDAB4468.....................................................................................................................22

FIGURE 2. DIAGRAM OF T-DNA REGION IN PLASMID PDAB4468...............................................................................22

FIGURE 3. SCHEMATIC OF THE DEVELOPMENT OF DAS-8191Ø-7 COTTON ...............................................................23

FIGURE 4. BREEDING DIAGRAM OF DAS-8191Ø-7 COTTON.......................................................................................24

FIGURE 5. DIAGRAM OF SEQUENCED TRANSGENE INSERT IN DAS-8191Ø-7 COTTON ..............................................29

FIGURE 6. PROBE LOCATIONS ON PDAB4468 USED IN SOUTHERN BLOT ANALYSIS ..................................................32

FIGURE 7. PDAB4468 SHOWING LOCATION OF RESTRICTION ENZYMES USED FOR SOUTHERN ANALYSIS ...............36

FIGURE 8. T-DNA INSERT RESTRICTION ENZYME MAP OF DAS-8191Ø-7 COTTON.....................................................36

FIGURE 9. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH NCOI; AAD-12 PROBE ........................40

FIGURE 10. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; AAD-12 PROBE ........................41

FIGURE 11. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH NCOI; PAT PROBE .............................42

FIGURE 12. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; PAT PROBE ..............................43

FIGURE 13. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH MSCI; ATUBI10 PROBE......................44

FIGURE 14. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; ATUBI10 PROBE ......................45

FIGURE 15. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH NCOI; ATUORF23 3′ UTR PROBE.......46

FIGURE 16. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; ATUORF23 3′ UTR PROBE........47

FIGURE 17. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH NCOI; CSVMV PROBE........................48

FIGURE 18. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; CSVMV PROBE.........................49

FIGURE 19. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH NCOI; ATUORF1 3′ UTR PROBE.........50

FIGURE 20. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; ATUORF1 3′ UTR PROBE..........51

FIGURE 21. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI TO RELEASE AAD-12 EXPRESSION 

CASSETTE; AAD-12 PROBE..................................................................................................................................52

FIGURE22. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI TO RELEASE AAD-12 EXPRESSION 

CASSETTE; ATUBI10 PROBE................................................................................................................................53

FIGURE 23. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI TO RELEASE AAD-12 EXPRESSION 

CASSETTE; ATUORF23 3′ UTR PROBE .................................................................................................................54

FIGURE 24. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI/XHOI TO RELEASE PAT 

EXPRESSION CASSETTE; PAT PROBE...................................................................................................................55

FIGURE 25. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI/XHOI TO RELEASE PAT 

EXPRESSION CASSETTE; CSVMV PROBE .............................................................................................................56

FIGURE 26. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI/XHOI TO RELEASE PAT 

EXPRESSION CASSETTE; ATUORF1 3′ UTR PROBE ..............................................................................................57

FIGURE 27. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH SPHI; RB7 PROBE ..............................58

FIGURE 28. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH MSCI; ORI PROBE..............................59

FIGURE 29. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI; ORI PROBE ...............................60

FIGURE 30. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH MSCI; BACKBONE2 PROBE................61

FIGURE 31. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI; BACKBONE2 PROBE .................62

FIGURE 32. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH MSCI; BACKBONE1 PROBE................63

FIGURE 33. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI; BACKBONE1 PROBE .................64

FIGURE 34. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH MSCI; SPECR PROBE .........................65

FIGURE 35. SOUTHERN ANALYSIS OF DAS-8191Ø-7 COTTON DIGESTED WITH PSTI; SPECR PROBE ...........................66

FIGURE 36. AMINO ACID SEQUENCE OF EXPRESSED AAD-12 IN DAS-8191Ø-7 ..........................................................69

FIGURE 37. DEGRADATION REACTION OF 2,4-D CATALYZED BY AAD-12 ....................................................................70

FIGURE 38. GENERAL REACTION CATALYZED BY AAD-12 (R=H OR CH3)......................................................................71

FIGURE 39. AMINO ACID SEQUENCE OF PAT...............................................................................................................78

FIGURE 40. MODE OF ACTION OF THE PAT PROTEIN..................................................................................................79

FIGURE 41. PROXIMATES & FIBER IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY COTTONSEED.....................88

FIGURE 42. MINERALS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY COTTONSEED.......................................92

FIGURE 43. PERCENT TOTAL AMINO ACIDS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY COTTONSEED ......97

FIGURE 44. PERCENT TOTAL FATTY ACIDS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY COTTONSEED ......103
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FIGURE 45. VITAMINS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY COTTONSEED.....................................106

FIGURE 46. ANTI-NUTRIENTS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY COTTONSEED .........................109

FIGURE 47. PLANTED UPLAND COTTON ACRES BY COUNTY IN THE U.S. IN 2012 (USDA NASS, 2012B) ..................123

FIGURE 48. PLANTED PIMA COTTON ACRES BY COUNTY IN THE U.S. IN 2012.........................................................124

FIGURE 49. 2,4-D USE IN DAS-8191Ø-7 COTTON......................................................................................................134

FIGURE 50. ADOPTION OF GE CROPS IN THE U.S. ....................................................................................................139

FIGURE 51. SDS-PAGE AND WESTERN BLOT ANALYSIS OF MICROBE- & DAS-8191Ø-7 COTTON-DERIVED AAD-12 

PROTEINS .........................................................................................................................................................150

FIGURE 52. SDS-PAGE ANALYSIS OF IMMUNOAFFINITY PURIFIED DAS-8191Ø-7 COTTON DERIVED AAD-12 .........151

FIGURE 53. GLYCOPROTEIN ANALYSIS OF PURIFIED DAS-8191Ø-7 COTTON AAD-12 ..............................................152

FIGURE 54. IN SILICO TRYPSIN CLEAVAGE OF DAS-8191Ø-7 COTTON AAD-12.........................................................153

FIGURE 55. IN SILICO CHYMOTRYPSIN CLEAVAGE OF DAS-8191Ø-7 COTTON AAD-12 ............................................154

FIGURE 56. IN SILICO ASP-N CLEAVAGE OF DAS-8191Ø-7 COTTON AAD-12 ............................................................154

FIGURE 57. COMPREHENSIVE MS AND MS/MS SEQUENCE COVERAGE MAP FOR TRYPSIN, CHYMOTRYPSIN AND 

ASP-N DIGEST OF DAS-8191Ø-7 COTTON-DERIVED AAD-12............................................................................155

FIGURE 58. KINETIC ANALYSIS OF MICROBE AND DAS-8191Ø-7 COTTON DERIVED AAD-12 PROTEINS WITH SDICHLOROPROP SUBSTRATE............................................................................................................................158

FIGURE 59. SDS-PAGE AND WESTERN BLOT ANALYSIS OF MICROBE- AND DAS-8191Ø-7 COTTON-DERIVED PAT 

PROTEINS .........................................................................................................................................................166

FIGURE 60. SDS-PAGE ANALYSIS OF THE IMMUNOAFFINITY PURIFIED DAS-8191Ø-7 COTTON DERIVED PAT ........167

FIGURE 61. GLYCOPROTEIN ANALYSIS OF IMMUNOPURIFIED DAS-8191Ø-7 DERIVED PAT ....................................168

FIGURE 62. IN SILICO TRYPSIN CLEAVAGE OF DAS-8191Ø-7 COTTON PAT...............................................................169

FIGURE 63. IN SILICO ASP-N CLEAVAGE OF DAS-8191Ø-7 COTTON PAT ..................................................................170

FIGURE 64. COMPREHENSIVE MS AND MS/MS SEQUENCE COVERAGE MAP FOR TRYPSIN AND ASP-N DIGESTS OF 

DAS-8191Ø-7 COTTON-DERIVED PAT ..............................................................................................................171

FIGURE 65. KINETIC ANALYSIS OF MICROBE- AND DAS-8191Ø-7 COTTON-DERIVED PAT PROTEINS ......................172

FIGURE 66. ADOPTION OF GE COTTON IN THE U.S. (USDA ERS, 2013B)..................................................................188
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List of Tables

TABLE 1. GENETIC ELEMENTS FROM PLASMID PDAB4468..........................................................................................26

TABLE 2. GENETIC ELEMENTS IN DAS-8191Ø-7 COTTON ............................................................................................30

TABLE 3. LIST OF PROBES AND THEIR POSITIONS IN PLASMID PDAB4468..................................................................31

TABLE 4. PREDICTED & OBSERVED SIZES OF HYBRIDIZING FRAGMENTS IN SOUTHERN BLOT ANALYSES ..................33

TABLE 5. SEGREGATING GENERATION RESULTS OF BC1F2 INDIVIDUAL PLANTS..........................................................67

TABLE 6. SEGREGATING BREEDING GENERATION RESULTS OF DAS-8191Ø-7 COTTON PLANTS ................................68

TABLE 7. DAS-8191Ø-7 COTTON AAD-12 EXPRESSION ANALYSIS TISSUE SAMPLES....................................................72

TABLE 8. SUMMARY OF AAD-12 PROTEIN EXPRESSION..............................................................................................73

TABLE 9. DAS-8191Ø-7 COTTON PAT EXPRESSION ANALYSIS TISSUE SAMPLES..........................................................80

TABLE 10. SUMMARY OF PAT PROTEIN EXPRESSION IN DAS-8191Ø-7 COTTON ........................................................80

TABLE 11. COTTONSEED COMPOSITION ANALYTES ....................................................................................................85

TABLE 12. COMBINED SITE ANALYSIS RESULTS FOR PROXIMATES & FIBER IN ISOLINE, DAS-8191Ø-7 AND 

REFERENCE VARIETY COTTONSEED AND LITERATURE RANGES.........................................................................87

TABLE 13. COMBINED SITE ANALYSIS RESULTS FOR MINERALS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY 

COTTONSEED AND LITERATURE RANGES...........................................................................................................91

TABLE 14. COMBINED SITE ANALYSIS RESULTS FOR AMINO ACIDS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE 

VARIETY COTTONSEED AND LITERATURE RANGES ............................................................................................95

TABLE 15. COMBINED SITE ANALYSIS RESULTS FOR FATTY ACIDS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE 

VARIETY COTTONSEED AND LITERATURE RANGES ..........................................................................................101

TABLE 16. COMBINED SITE ANALYSIS RESULTS FOR VITAMINS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE VARIETY 

COTTONSEED AND LITERATURE RANGES.........................................................................................................105

TABLE 17. COMBINED SITE ANALYSIS RESULTS FOR ANTI-NUTRIENTS IN ISOLINE, DAS-8191Ø-7 AND REFERENCE 

VARIETY COTTONSEED AND LITERATURE RANGES ..........................................................................................108

TABLE 18. MEASURED AGRONOMIC CHARACTERISTICS AND SAMPLE COLLECTION DETAILS .................................114

TABLE 19. COMBINED SITE ANALYSIS RESULTS FOR AGRONOMIC CHARACTERISTICS.............................................115

TABLE 20. INSECTS AND DISEASES PRESENT AT FIELD TESTING SITES......................................................................117

TABLE 21. GERMINATION PERCENTAGES FOR DAS-8191Ø-7 AND CONTROL COTTONSEED ...................................119

TABLE 22. DAS-8191Ø-7 COTTON TOLERANCE TO APPLICATION OF HERBICIDES ...................................................120

TABLE 23. TOTAL U.S. COTTON GROWING REGIONS, STATES AND 2010 – 2012 HARVEST, YIELD & PRODUCTION122

TABLE 24. COTTON PRODUCTION IN THE U.S., 2002 - 20131,2.................................................................................123

TABLE 25. MAJOR WEEDS TREATED IN COTTON BY REGION IN 2011 (DESCENDING ORDER)1................................127

TABLE 26. NUMBER OF HERBICIDE RESISTANT WEEDS REPORTED GLOBALLY BY HERBICIDE GROUP AND MODE OF 

ACTION1............................................................................................................................................................137

TABLE 27. ASSESSMENT OF RESISTANCE RISK BY EVALUATION OF CROPPING SYSTEMS1.......................................138

TABLE 28. GLYPHOSATE AND ALS RESISTANT WEEDS CONTROLLED BY 2,4-D

1

........................................................139

TABLE 29. TEST MATERIAL FOR AAD-12 CHARACTERIZATION..................................................................................144

TABLE 30. COMMERCIALLY AVAILABLE REFERENCE SUBSTANCES FOR AAD-12 CHARACTERIZATION.....................145

TABLE 31. SUMMARY OF N-TERMINAL SEQUENCES OF AAD-12 DERIVED PROTEINS ..............................................156

TABLE 32. SUMMARY OF C-TERMINAL SEQUENCES OF AAD-12 DERIVED PROTEINS ..............................................157

TABLE 33. TEST MATERIAL FOR PAT CHARACTERIZATION........................................................................................159

TABLE 34. COMMERCIALLY AVAILABLE REFERENCE SUBSTANCES FOR PAT CHARACTERIZATION...........................160

TABLE 35. SUMMARY OF N-TERMINAL SEQUENCES OF PAT DERIVED PROTEINS ....................................................170

TABLE 36. SUMMARY OF C-TERMINAL SEQUENCES OF PAT DERIVED PROTEINS ....................................................170

TABLE 37. WEED SPECIES WITH REPORTED GLYPHOSATE RESISTANT BIOTYPES1....................................................189

TABLE 38. GLOBAL REPORTS OF GLYPHOSATE RESISTANT WEED BIOTYPES WITH RESISTANCE TO OTHER HERBICIDE 

MODES OF ACTION5.........................................................................................................................................190

TABLE 39. POTENTIAL WEED SHIFTS WITH USE OF GLYPHOSATE ............................................................................191

TABLE 40. WEED SPECIES WITH REPORTED 2,4-D RESISTANT BIOTYPES..................................................................193

TABLE 41. U.S. GLYPHOSATE RESISTANT WEEDS IN COTTON GROWING STATES1...................................................194
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List of Acronyms and Scientific Terms

Symbol or 

Abbreviation

Definition

°C Degree Celsius

2,4-D 2,4-Dichlorophenoxyacetic acid

AA Amino Acid

aad-12 Gene encoding the AAD-12 protein

AAD-12 Aryloxyalkanoate dioxygenase-12 protein

ACCase Acetyl CoA carboxylase

ADF Acid detergent fiber

Adj. P False Discovery Rate Adjusted P-value

ae Acid equivalent

ae/ha Acid equivalent per hectare

ai Active ingredient

ai/ha Active ingredient per hectare

AL Alabama

ALS Acetolactate synthase

ANOVA Analysis of variance

AOSA Association of Official Seed Analysis

APHIS Animal and Plant Health Inspection Service, USDA

AtUbi10 Ubiquitin promoter from Arabidopsis thaliana

AtuORF1 3′ untranslated region from Agrobacterium tumefaciens

AtuORF23 3′ untranslated region from Agrobacterium tumefaciens

BLASTp Basic Local Alignment Search Tool protein

bp Base pair

BSA Bovine Serum Albumin

CFR Code of Federal Regulations

CFSAN Center for Food Safety and Nutrition, U.S. FDA

cm centimeter

CsVMV Promoter from cassava vein mosaic virus

DAS Dow AgroSciences LLC

DAS-8191Ø-7 OECD identifier for the cotton event expressing the AAD-12 and PAT 

proteins

DCP 2,4-Dichlorophenol

DIG Digoxigenin

DNA Deoxyribonucleic acid

ELISA Enzyme-linked immunosorbent assay

EPA Environmental Protection Agency (US)

ESA Endangered Species Act

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

Abbreviation

Definition

FAO Food and Agriculture Organization of the United Nations

FARRP Food Allergy Research and Resource Program

FDA Food and Drug Administration (US)

FDR False Discovery Rate

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

ft feet

FWS U.S Fish & Wildlife Service

g gram

GA Georgia

GS Glutamine synthetase

ha Hectare

IAA Indole acetic acid

ILSI International Life Sciences Institute

Kb Kilobase pair

kDa Kilodalton

kPa Kilopascals

L Liter

LA Louisiana

LFS Lateral Flow Strip

LOD Limit of Detection

LOQ Limit of Quantitation

m meter

M Molar

MALDI-TOF 

MS

Matrix assisted laser desorption/ionization time-of-flight mass spectrometry

MARC Metabolism Assessment Review Committee - U.S. EPA

MCPA ((4-chlro-2-methyphenoxy) acetic acid)

MO Missouri

MOA Mode of Action

MS Mississippi

μg microgram

μL microliter

μM micromolar

mg Milligrams

min minute

mL Milliliter

mM millimolar

MOE Margin of exposure

MW Molecular Weight
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Symbol or 

Abbreviation

Definition

NA Not Applicable

NC North Caroline

ng/mg Nanogram / milligram

NOAEL No observed adverse effect level

NOEL No observed effect level

OD Optical Density

OECD Organisation for Economic Co-operation and Development

Ori Rep replication Origin Sequence

pat Gene from Streptomyces viridochromogenes which encodes the PAT protein

PAT Phosphinothricin N-acetyltransferase protein

PBN US FDA Pre-market Biotechnology Notice

PCR Polymerase chain reaction

pDAB4468 DNA vector carrying the aad-12 and pat expression cassettes

ppb Parts Per Billion

ppm Parts Per Million

PPO Protoporphyrinogen oxidase

PPT Phosphinothricin

PSI Pounds per Square Inch

RB7 MAR Matrix attachment region (MAR) from Nicotiana tabacum

RCB Randomized complete block

SDS Sodium dodecyl sulfate

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SE Standard Error

sec second

SGF Simulated gastric fluid

SpecR Spectinomycin Resistance

STI Short Term Intake

T-DNA Transfer DNA

TX Texas

USDA United States Department of Agriculture

V volt

w/v weight / volume

w/w weight / weight

WHO World Health Organization
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1. Rationale for the Development of DAS-8191Ø-7 Cotton

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 herbicide 

tolerant DAS-8191Ø-7 cotton and requests a determination from APHIS that DAS-8191Ø-7 

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

1.2. Benefits of DAS-8191Ø-7 Cotton 

DAS has developed transgenic cotton plants containing aad-12 and pat, which confers

tolerance to the herbicides 2,4-D and glufosinate. The unique identifier for these plants, in 

accordance with the Organisation for Economic Co-operation and Development’s (OECD) 

“Guidance for the Designation of a Unique Identifier for Transgenic Plants” (OECD, 2004), 

is DAS-8191Ø-7.

DAS-8191Ø-7 cotton was developed using Agrobacterium-mediated transformation to stably 

incorporate the aad-12 gene from Delftia acidovorans and the pat gene from Streptomyces 

viridochromogenes into cotton. The aad-12 gene encodes the aryloxyalkanoate 

dioxygenase-12 (AAD-12) enzyme which, when expressed in plants, degrades 2,4-D to 

herbicidally-inactive 2,4-dichlorophenol (DCP). The pat gene encodes the enzyme 

phosphinothricin acetyltransferase that inactivates glufosinate. 

With the introduction of genetically engineered, glyphosate tolerant crops in the mid-1990’s, 

growers were enabled with a simple, convenient, flexible, and inexpensive tool for 

controlling a wide spectrum of broadleaf and grass weeds that was unparalleled in 

agriculture. Consequently, producers were quick to adopt glyphosate tolerant crops, and in 

many instances, abandon many of the accepted best agronomic practices such as crop 

rotation, herbicide mode of action rotation, tank mixing, and incorporation of mechanical 

with chemical and cultural weed control. Currently glyphosate tolerant soybean, cotton, corn, 

alfalfa, sugar beets, and canola are commercially available in the United States and elsewhere 

in the Western Hemisphere. More glyphosate tolerant crops (e.g., wheat, rice, turf, etc.) are 

poised for introduction pending global market acceptance. Many other glyphosate tolerant

species are in experimental or development stages (e.g., sugar cane, sunflower, beets, peas, 

carrot, cucumber, lettuce, onion, strawberry, tomato, and tobacco; forestry species like poplar 

and sweetgum; and horticultural species like marigold, petunia, and begonias) (Information 

Systems for Biotechnology, 2011). Additionally, the cost of glyphosate has dropped 

dramatically in recent years to the point that few conventional weed control programs can 

effectively compete on price and performance with glyphosate tolerant crop systems.
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Extensive use of glyphosate-only weed control programs has resulted in the selection of 

glyphosate resistant weeds, and continues to select for the propagation of weed species that 

are inherently more tolerant to glyphosate than most target species (i.e., weed shifts). 

Although glyphosate has been widely used globally for more than 30 years, the vast majority 

of resistant weeds have been identified in the past 5-8 years. 

Resistant weeds in the U.S. include both grass and broadleaf species—Lolium rigidum (Rigid 

ryegrass), Lolium multiflorum (Italian ryegrass), Sorghum halepense (Johnsongrass),

Amaranthus palmeri (Palmer amaranth), Amaranthus rudis (Common waterhemp), Ambrosia 

artemisiifolia (Common ragweed), Ambrosia trifida (Giant ragweed), Conyza canadensis 

(Horseweed), and Conyza bonariensis (Hairy fleabane). Additionally, weeds that had 

previously not been an agronomic problem prior to the wide use of glyphosate tolerant crops 

are now becoming more prevalent and difficult to control in the context of glyphosate

tolerant crops. According to a 2012 survey across 31 states in the U.S., 49% of growers 

reported the presence of glyphosate resistant weed on their farms (Pucci, 2013). In 2011, the 

number was 34%. The problem is more pronounced in the South, with 92% of growers 

reporting glyphosate resistant weeds. These weed shifts are occurring predominantly, but not 

exclusively, with difficult-to-control broadleaf weeds. Some examples include Ipomoea, 

Amaranthus, Chenopodium, Taraxacum, and Commelina species. 

In areas where growers are faced with glyphosate resistant weeds or a shift to more difficultto-control weed species, growers can compensate by tank mixing or alternating with other 

herbicides that will control the surviving weeds. One popular and efficacious tank mix active 

ingredient for controlling broadleaf escapes has been 2,4-diclorophenoxyacetic acid (2,4-D). 

2,4-D has been used agronomically and in non-crop situations for broad spectrum, broadleaf 

weed control for more than 70 years. Individual cases of more tolerant weed species have 

been reported, but 2,4-D remains one of the most widely used herbicides globally. The 

development of 2,4-D-tolerant cotton provides an excellent option for controlling glyphosate

resistant (or highly tolerant and shifted) broadleaf weed species for in-crop applications, 

allowing the grower to focus applications at the critical weed control stages and extending 

the application window.

The availability of DAS-8191Ø-7 cotton is expected to have a beneficial impact on weed 

control practices by providing growers with an advanced tool to address their weed control 

needs. The availability of DAS-8191Ø-7 cotton will allow growers to proactively manage 

weed populations while avoiding adverse population shifts of troublesome weeds or the 

development of resistance, particularly glyphosate resistance in weeds.

DAS-8191Ø-7 cotton will be commercialized in combination with other herbicide tolerant 

(e.g. glyphosate) and insect resistance cotton events. Stacked varieties provide growers with 

built-in, sustainable resistance management tools to address glyphosate resistant and hard to 

control weeds, as well as incorporate the recognized environmental benefits of insect

resistant traits. The combination of herbicide tolerance traits will allow the use of multiple 

herbicides in an integrated weed management program to control a broad spectrum of grass 

and broadleaf weed species in cotton. These herbicides will provide distinct modes of actions 

for use in conjunction with other herbicide active ingredients and modes of action for an 
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effective weed management program in cotton. 2,4-D will provide improved in-crop postemergence control of hard to control glyphosate resistant broad-leaf weeds, such as pigweed, 

waterhemp, horseweed, and morning glory.

1.3. Submission to Other Regulatory Agencies 

DAS-8191Ø-7 cotton 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 submitted a pre-market

biotechnology notification (PBN) to FDA in June 2013, BNF 000142 with anticipated review 

completed in late 2014.

The regulation and use of herbicides on DAS-8191Ø-7 cotton is governed by the U.S. EPA 

under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), which prohibits the 

sale and distribution of any pesticide that is not registered by EPA. In order to register a 

pesticide or a new use of a previously registered pesticide EPA must find that it will not 

pose an unreasonable risk to humans or the environment. Once registered, it is a violation 

of FIFRA to use a pesticide in a manner inconsistent with its EPA-approved label. The use 

of herbicides on DAS-8191Ø-7 cotton will be consistent with either currently authorized 

herbicide uses on cotton or uses that are currently in review by U.S. EPA.

Pesticides produced in planta, known as plant-incorporated protectants (PIPs), are also 

subject to regulation by the U.S. EPA under FIFRA (e.g. Cry proteins). Since AAD-12 and 

PAT are neither a herbicide or PIP, AAD-12 and PAT fall outside the scope of EPA 

regulations.

DAS intends to submit dossiers beginning in 2013 to the regulatory authorities of trade 

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

Korea, European Union, Australia / New Zealand, South Africa, Brazil and Mexico.
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2. The Biology of Cotton and DAS-8191Ø-7 Recipient Cotton Cultivar

2.1. Overview of Cotton Biology

Refer to The OECD Consensus Document on the Biology of Cotton (Gossypium, spp.)

(OECD, 2008), for information related to the following aspects of cotton biology: 

 General description and morphology of cotton (p 11 – 12)

 Uses of cotton as a crop plant (p 13)

 Taxonomy (p 14 – 15)

 Centers of origin, diversity and domestication (p 15 – 18)

 Agronomic practices (p 19 – 23)

o Biotic Environment (pest, diseases, weeds) (p 21 – 23)

o Harvest, processing and crop rotation (p23)

 Reproductive biology (p 24 – 27)

o Seed dormancy and germination (p 26)

o Weediness and naturalization (p 26-27)

 Genetics & Hybridization (p 28 – 29)

o Ability to cross intra and inter-species/genus (p 29)

 Toxin and Allergen potential (p 34– 35)

o Gossypol (p 34-35)

o Cyclopropenoid fatty acids (p 35)

o Allergens (p 35)

2.2. Characterization of the Recipient Cotton Cultivar

The publically available cotton variety Coker 310 (G. hirsutum) was used as the recipient 

lines for the generation of event DAS-8191Ø-7. The variety Coker 310 was developed by the 

cotton division of Coker’s Pedigreed Seed Company and is an older commercial variety of 

upland cotton generated from a cross of Coker 100 Staple and Deltapine 15 and selected 

through successive generations of line selection (Smith et al., 1999; Bowman et al., 2006).
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3. Method of Development of DAS-8191Ø-7 Cotton

DAS-8191Ø-7 cotton was generated by Agrobacterium mediated transformation of cotton 

tissues from Coker 310 variety using plasmid pDAB4468 (Figure 1). This section describes 

the plasmid vector (pDAB4468), the transformation method, the donor genes (aad-12 and

pat), and the regulatory elements used in the development of DAS-8191Ø-7 cotton. The 

transfer DNA (T-DNA) refers to DNA that is transferred to the plant during transformation. 

The T-DNA insert in pDAB4468 (Figure 2) contains two synthetic genes, aad-12 from 

Delftia acidovorans and pat from Streptomyces viridochromogenes. 

3.1. pDAB4468

The plasmid vector, pDAB4468, was used in the transformation of cotton to generate DAS8191Ø-7. A vector map and the summary of the genetic elements in pDAB4468 are provided 

in Figure 1 and Table 1, respectively. pDAB4468 is approximately 12-kb and contains one TDNA that is delineated by T-DNA borders B and A (Figure 2). The T-DNA contains the aad12 and pat expression cassettes along with the RB7-MAR sequence. A gene expression 

cassette is comprised of sequences to be transcribed (the gene coding sequence) and the 

regulatory elements necessary for the expression of those sequences (e.g. promoter, 

terminator). Details of the expression cassettes and RB7-MAR in pDAB4468 are described 

below in sections 3.4.1, 3.4.2, 3.4.3 and in Table 1.

The backbone region of pDAB4468, located outside the T-DNA region, contains two origins 

of replication (OriRep and Trf A) for the maintenance of the plasmid vector in bacteria and a 

bacterial selectable marker gene (SpecR). Details of the genetic elements are in Table 1.

3.2. Description of the Transformation System

Cotton (Gossypium hirsutum L.) event DAS-8191Ø-7 was developed through 

Agrobacterium-mediated transformation of pDAB4468. The disarmed A. tumefaciens strain 

LBA4404 (Ooms et al., 1982), carrying the binary vector, pDAB4468, was used to initiate 

transformation of cotton hypocotyl segments. 

Agrobacterium-mediated transformation was carried out using a modified procedure based 

on Umbeck et al. (Umbeck, 1991). Briefly, cotton seeds (cv. Coker 310) were germinated on 

basal media and hypocotyl segments were isolated and infected with Agrobacterium strain 

LBA4404 carrying pDAB4468. Following infection, the hypocotyl segments were cultured 

on a sequence of media containing carbenicillin and glufosinate to inhibit the growth of 

Agrobacterium and untransformed cells, respectively. 

Gene-specific PCR analyses were performed on embryogenic callus to identify transgenic 

lines containing the target genes (aad-12 and pat). Selected calli were transferred to culture 

medium containing plant growth regulators to stimulate root regeneration. Rooted plants (T0) 

were transferred to soil mixtures under high humidity in growth chambers for 2-4 weeks. The 

hardened plants were then transferred to greenhouse facilities. 

Following the transfer to the greenhouse, young leaves of T0 plants were painted with 

glufosinate (1.5% w/v) to screen for putative transformants. Those glufosinate tolerant plants 

were sampled and analyzed at a molecular level to confirm the presence of the target genes 
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and the absence of the vector backbone. Specifically, for T0 plants, PCR analyses were 

performed to verify the absence of the bacterial selectable marker sequence, spectinomycin, 

in the vector backbone of pDAB4468 as well as the presence of the aad-12 and pat genes. 

PCR and Invader assays (Kwiatkowski et al., 1999), were carried out to determine the copy 

number of the pat and aad-12 genes. T0 plants conferring the desirable copy number were 

self-pollinated to produce T1 seed. For T1 plants, Invader assay and Southern blot analyses 

were performed to identify plants containing a single pat and aad-12 gene insertion. DAS8191Ø-7 cotton was selected as the lead event based on molecular and phenotypic 

characteristics. Genetic characterization studies on DAS-8191Ø-7 cotton were initiated to 

further characterize the transgenic insert and the expressed proteins (Section 4). The major 

steps described in the development of DAS-8191Ø-7 cotton are described in Figure 3 with 

the breeding diagram for DAS-8191Ø-7 cotton in Figure 4.

Figure 1. Plasmid map of pDAB4468

Figure 2. Diagram of T-DNA Region in Plasmid pDAB4468

pDAB4468

12154 bp

pat

aad-12

SpecR

TrfA

T-DNA Border A

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR

CsVMV promoter

AtUbi10 promoter

Ori Rep AtuORF23 3'UTR

AtuORF1 3' UTR pDAB4468 T-DNA Insert (6794bp)

pat

aad-12 T-DNA Border A

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR

CsVMV promoter

AtUbi10 promoter AtuORF23 3'UTR AtuORF1 3' UTR

1 6794
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Figure 3. Schematic of the Development of DAS-8191Ø-7 Cotton 

Assembled Agrobacterium binary vector pDAB4468 and transformed into disarmed 

Agrobacterium tumefaciens strain LBA4404

Transformed Coker 310 hypocotyl segments with pDAB4468 using Agrobacterium 

tumefaciens-mediated transformation

Regenerated calli on media containing glufosinate and carbenicillin to select for 

transformed calli expressing PAT and to inhibit Agrobacterium growth, respectively

Performed gene specific PCR analysis (aad-12, pat) on selected transformants 

Generated rooted shoots from transformed tissues positive for both genes

Performed preliminary evaluation on transformed T0 plants for the absence of vector 

backbone and copy number of the genes of interest. Allowed self pollination to 

produce T1 plants

Evaluated T1 plants and subsequent generations resulting from self pollination

Evaluated transgenic plants via molecular characterization, protein expression, 

agronomic performance, and herbicide resistance

Selected homozygous plants and carried out crossing and backcrossing into DAS 

proprietary cotton strain 98M-2983 to identify the lead candidate, DAS-8191Ø-7. 

Further evaluated progeny to evaluate and assess insert integrity, copy number, 

herbicide tolerance and agronomic performance
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Analysis Petition 

Section(s)

DAS-8191Ø-7

Cotton Generations Control

Molecular Analysis 4, 4.2 T2, T3, T4, T5, BC1F2Coker 310 

Segregation Analysis

(single generation) 4.4.1 BC1F2

None

Segregation Analysis

(breeding generations) 4.4.2 T1, BC1F2

None

Protein Characterization 5.1.3, 5.2.3 T3 Coker 310 

Protein Expression 5.1.4, 5.2.4 BC1F3 98M-2983 

Composition 6.1 BC1F398M-2983 

Agronomics 7.1 BC1F398M-2983 

Germination & Dormancy 7.2.3 BC1F498M-2983 X Coker 310 

Figure 4. Breeding Diagram of DAS-8191Ø-7 Cotton
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3.3. Selection of the Comparators for DAS-8191Ø-7 Cotton

Proper selection of comparator (control) plants is important to ensure the accurate assessment 

of the impact of transgene insertion on various characteristics of DAS-8191Ø-7 cotton. The 

control plants should have a genetic background similar to that of DAS-8191Ø-7 cotton but 

lack the transgenic insert. In all cases, a genotypically similar non-transgenic near isogenic

control was used as a comparator (see Figure 4).

3.4. Donor Genes and Regulatory Sequences in DAS-8191Ø-7 Cotton

The transgenic insert in DAS-8191Ø-7 cotton contains two expression cassettes, aad-12 and 

pat in addition to a matrix attachment region (MAR) of RB7 from Nicotiana tobacum (Figure 

2). This section describes the details of each expression cassette including the source 

organism, history of safe use, the expressed traits and the genetic organization of each 

expression cassette. In addition, details of the RB7-MAR sequence are described.

3.4.1. aad-12 expression cassette

The aad-12 expression cassette consists of the AtUbi10 promoter, aad-12 gene and 

AtuORF23 3′ UTR terminator (Figure 2, Table 1) and is identical to the aad-12 expression 

cassette in DAS-68416-4 soybean and DAS-444Ø6-6 soybean (currently under USDAAPHIS review, petition numbers 09-349-01p, 11-234-01p respectively). The aad-12 gene 

was isolated from Delftia acidovorans and the synthetic version of the gene was optimized 

for plant expression by modifying the G+C content bias to the plant system. The native and 

plant-optimized DNA sequences of aad-12 are 80% identical. The aad-12 gene is designed to 

express the Aryloxyalkanoate Dioxygenase-12 (AAD-12) protein, which consists of 293 

amino acids with a molecular weight of approximately 32 kDa. Expression of AAD-12 

protein in plants confers tolerance to herbicides such as 2,4-D. 

D. acidovorans, which was previously described as Pseudomonas acidovorans and 

Comamonas acidovorans, is a non glucose-fermenting, gram-negative, non spore-forming 

rod-shaped bacterium present in soil, fresh water, activated sludge, and clinical specimens 

(Von Graevenitz, 1985; Tamaoka et al., 1987; Wen et al., 1999). Strains of D. acidovorans

can be used to transform ferulic acid into vanillin and related flavor metabolites (Toms and 

Wood, 1970; Labuda et al., 1992; Rao and Ravishankar, 2000; Shetty et al., 2006).

aad-12 expression is controlled by the AtUbi10 promoter from Arabidopsis thaliana, which 

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

The terminator sequence, AtuORF23 3′ UTR, is derived from Agrobacterium tumefaciens 

plasmid pTi15955 (Barker et al., 1983).

3.4.2. pat expression cassette

The pat expression cassette consists of the CsVMV promoter, pat gene and AtuORF1 3′ 

UTR terminator (Figure 2) and is identical to the pat expression cassette in DAS-68416-4 

soybean and DAS-444Ø6-6 soybean (currently under USDA-APHIS review, petition 

numbers 09-349-01p, 11-234-01p respectively). The pat expression cassette is designed to 

express the PAT protein. The pat gene was isolated from the common soil bacterium

Streptomyces viridochromogenes (Wohlleben et al., 1988) and the synthetic version of the 

gene was optimized for plant expression by modifying the G+C content bias to the plant 

system.
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Table 1. Genetic Elements from Plasmid pDAB4468

Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Size

Description

T-DNA Region

T-DNA 

Border B 1 24 24 Transferring DNA sequences

Intervening 

sequence

25 160 136 Sequence from Ti plasmid pTi15955 (Barker et al., 1983)

RB7-MAR 161 1326 1166 Matrix attachment region (MAR) from Nicotiana tobacum

(Hall et al., 1991)

Intervening 

sequence

1327 1421 95 Sequence from plasmid pENTR/D-TOPO (Invitrogen Cat. 

No. A10465) and multiple cloning sites

AtUbi10 1422 2743 1322

Arabidopsis thaliana polyubiquitin UBQ10 gene 

comprising the promoter, 5′ untranslated region and intron 

(Norris et al., 1993)

Intervening 

sequence

2744 2751 8

Sequence used for DNA cloning

aad-12 2752 3633 882

Synthetic, plant-optimized version of an aryloxyalkanoate 

dioxygenase from Delftia acidovorans (Wright et al., 

2009)

Intervening 

sequence

3634 3735 102 Sequence used for DNA cloning

AtuORF23 3736 4192 457

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

4193 4306 114 Sequence from plasmid pENTR/D-TOPO (Invitrogen Cat. 

No. A10465) and multiple cloning sites

CsVMV 4307 4823 517 Promoter and 5′ untranslated region derived from the 

cassava vein mosaic virus (Verdaguer et al., 1996)

Intervening 

sequence

4824 4830 7

Sequence used for DNA cloning

pat 4831 5382 552

Selectable marker. Synthetic, plant-optimized version of 

phosphinothricin N-acetyl transferase gene, isolated from 

Streptomyces viridochromogenes (Wohlleben et al., 1988)

Intervening 

sequence

5383 5484 102

Sequence from plasmid pCRI2.1(Invitrogen Cat. No. 

K205001) and multiple cloning sites

AtuORF1 5485 6188 704

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

6189 6416 228 Sequence from Ti plasmid C58 (Zambryski et al., 1982; 

Wood et al., 2001)
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The presence of PAT protein in plants confers tolerance to glufosinate. The pat gene encodes 

a protein of 183 amino acids that has a molecular weight of approximately 20 kDa. The pat

gene was used both as a selectable marker and herbicide tolerance trait in previously 

deregulated products (USDA, 1996; FDA, 1998, 2001; USDA, 2001; FDA, 2003, 2004a, 

2004b, 2004c; USDA, 2004, 2005; FDA, 2011). 

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

and the AtuORF1 3′ UTR sequence from A. tumefaciens plasmid pTi15955. The CsVMV 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 that it controls 

(Verdaguer et al., 1996). 

Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Size Description

T-DNA 

border A 6417 6440 24

Transferring DNA sequences

intervening 

sequence

6441 6459 19

Sequence from Ti plasmid C58 (Zambryski et al., 1982; 

Wood et al., 2001)

T-DNA 

border A 6460 6483 24

Transferring DNA sequences

intervening 

sequence

6484 6770 287 Sequence from Ti plasmid pTi15955 (Barker et al., 1983)

T-DNA 

border A 6771 6794 24 Transferring DNA sequences

Plasmid Backbone Region

Plasmid 

backbone 

sequences

6795 7173 379

Plasmid backbone sequences from RK2 plasmid (Stalker

et al., 1981)

Ori Rep 7174 8193 1020 Replication origin sequences from RK2 plasmid (Stalker

et al., 1981)

Plasmid 

backbone 

sequences

8194 8738 545

Plasmid backbone sequences from RK2 plasmid (Stalker

et al., 1981)

Trf A 8739 9887 1149 Plasmid replication sequences for Trf A protein from RK2 

plasmid (Stalker et al., 1981)

Plasmid 

backbone 

sequences

9888 11091 1204

Plasmid backbone sequences from RK2 plasmid (Stalker

et al., 1981)

Spec R 11092 11880 789 Sequences for Spectinomycin resistance gene (Fling et al., 

1985)

Plasmid 

backbone 

sequences

11881 12154 274

Plasmid backbone sequences for cloning
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3.4.3. RB7 MAR

In addition to the two expression cassettes, a matrix attachment region (MAR) of RB7 from 

Nicotiana tabacum was included at the 5′ end of the T-DNA. Matrix attachments regions are 

natural and abundant regions found in genomic DNA that are thought to attach to the matrix 

or scaffold of the nucleus. When positioned on the flanking ends of gene cassettes, some 

MARs have been shown to increase expression of transgenes and to reduce the incidence of 

gene silencing (Han et al., 1997; Abranches et al., 2005; Verma et al., 2005). It is 

hypothesized that MARs may act as a buffer to protect transgenes from neighboring 

chromosomal sequences that could destabilize their expression (Allen et al., 1993; Allen et 

al., 2000).
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4. Genetic Characterization of DAS-8191Ø-7 Cotton 

4.1. Overview of Genetic Characterization

Characterization of DAS-8191Ø-7 cotton was conducted by Southern blot and DNA 

sequence analyses. The sequence of the insert in DAS-8191Ø-7 cotton was confirmed 

(Figure 5) and the genetic elements identified in DAS-8191Ø-7 cotton are provided in Table 

2. Results demonstrate that the transgene insert in DAS-8191Ø-7 cotton occurred as a single

integration of the respective T-DNA regions from plasmid pDAB4468, including a single, 

intact copy of each of the aad-12 and pat gene expression cassettes along with a RB7-MAR 

element. The transgene insert is stably integrated and inherited across breeding generations, 

and no plasmid backbone sequences are present in DAS-8191Ø-7 cotton. 

Figure 5. Diagram of Sequenced Transgene Insert in DAS-8191Ø-7 Cotton

Southern blot analyses were used to determine the copy and insertion number of the 

integrated DNA as well as the presence or absence of plasmid vector backbone sequences. 

The Southern analysis was designed to ensure that all potential transgenic segments from

pDAB4468 would be identified in DAS-8191Ø-7 cotton. Locations of each probe on plasmid 

pDAB4468 are described in Figure 6 and Table 3 respectively. 

Restriction enzymes were specifically chosen to fully characterize the transgene insert and 

detect any potential fragments of the T-DNA and backbone sequences in DAS-8191Ø-7 

cotton. The expected and observed fragment sizes for DAS-8191Ø-7 cotton genomic DNA, 

generated by specific restriction enzyme and probe combinations, based on the known

restriction enzyme sites of plasmid pDAB4468 and the intended T-DNA insert from 

pDAB4468, are shown in Figure 7 and Figure 8, respectively.

The Southern blot analyses described here consist of two types of DNA restriction fragments: 

a) internal fragments generated by known restriction enzyme recognition sites located

within the T-DNA insert of pDAB4468, and b) border fragments generated by one known 

restriction enzyme recognition site located within the T-DNA insert and another site located 

in the cotton genome flanking the insert (Figure 8). Border fragment sizes vary by event 

because they rely on the location of the restriction enzyme recognition sites within the DNA 

sequence flanking the transgene insert. 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.
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Table 2. Genetic Elements in DAS-8191Ø-7 Cotton

Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

5′ Flanking 

border 1 1373 1373

Cotton genomic DNA flanking the 5′ end of the 

transgene insert in DAS-8191Ø-7 cotton

Transgene Insert

Partial TDNA Border 

B

1374 1375 2

Partial sequence from T-DNA Border Bs

Intervening 

sequence

1376 1511 136

Sequence from Ti plasmid pTi15955 (Barker et al., 1983)

RB7-MAR 1512 2677 1166

Matrix attachment region (MAR) from Nicotiana 

tobacum (Hall et al., 1991)

Intervening 

sequence

2678 2772 95

Sequence from plasmid pENTR/D-TOPO (Invitrogen 

Cat. No. A10465) and multiple cloning sites

AtUbi10 2773 4094 1322

Arabidopsis thaliana polyubiquitin UBQ10 gene 

comprising the promoter, 5′ untranslated region and 

intron (Norris et al., 1993)

Intervening 

sequence

4095 4102 8

Sequence used for DNA cloning

aad-12 4103 4984 882

Synthetic, plant-optimized version of an 

aryloxyalkanoate dioxygenase from Delftia acidovorans

(Wright et al., 2010)

Intervening 

sequence

4985 5086 102

Sequence used for DNA cloning

AtuORF23 5087 5543 457

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

5544 5657 114

Sequence from plasmid pENTR/D-TOPO (Invitrogen 

Cat. No. A10465) and multiple cloning sites

CsVMV 5658 6174 517 Promoter and 5′ untranslated region derived from the 

cassava vein mosaic virus (Verdaguer et al., 1996)

Intervening 

sequence

6175 6181 7

Sequence used for DNA cloning

pat 6182 6733 552

Synthetic, plant-optimized version of phosphinothricin 

N-acetyl transferase (PAT) gene, isolated from 

Streptomyces viridochromogenes (Wohlleben et al., 

1988)

Intervening 

sequence

6734 6835 102 Sequence from plasmid pCRI2.1 (Invitrogen Cat. 

No. K205001) and multiple cloning sites
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Genomic DNA for Southern blot analysis was prepared from leaf material of individual 

DAS-8191Ø-7 cotton plants from five distinct breeding generations (Figure 4). Genomic 

DNA from leaves of non-transgenic variety Coker 310 was used as a control material. 

Plasmid DNA of pDAB4468 added to genomic DNA from the non-transgenic variety Coker

310 served as the positive control for Southern blot analysis. 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 8. 

Southern blot analysis results are shown in Figure 9 through Figure 35. 

Table 3. List of Probes and their Positions in Plasmid pDAB4468

Probe Name Position in pDAB4468 Length (bp)

RB7 25-1432 1408

AtUbi10 1433-2750 1318

aad-12 2752-3633 882

AtuORF23 3′ UTR 3607-4300 694

CsVMV 4301-4871 571

pat 4831-5382 552

AtuORF1 3′ UTR 5361-6411 1051

Ori 6412-8193 1782

Backbone2 8160-9887 1728

Backbone1 9857-11110 1254

SpecR 11092-24 1087

Feature 

Name

Feature 

Start

Feature 

Stop

Feature 

Length

Description

AtuORF1 6836 7539 704

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

7540 7763 224

Sequence from Ti plasmid C58 (Zambryski et al., 1982; 

Wood et al., 2001)

Cotton Genomic DNA

3′ Flanking 

border 7764 8834 1071 Cotton genomic DNA flanking the 3′ end of the 

transgene insert in DAS-8191Ø-7 cotton
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Figure 6. Probe Locations on pDAB4468 used in Southern Blot Analysis

The eleven probes described in Table 3 are shown as hashed boxes in the inner circle of the 

pDAB4468 plasmid map (outer circle).

pDAB4468

12154 bp

pat

aad-12

SpecR

TrfA

pat Probe

aad-12 Probe

ORF1 3' UTR Probe

RB7 Probe

Backbone 1 Probe

Backbone 2 Probe

Ori Probe

SpecR Probe

AtuORF23 3' UTR Probe

AtUbi10 Probe

T-DNA Border A CsVMV Probe

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR

CsVMV promoter

AtUbi10 promoter

Ori Rep AtuORF23 3'UTR

AtuORF1 3' UTR

SphI (6245)

XhoI (6229)

MscI (1330)

MscI (7555)

PstI (1433)

PstI (4301)

NcoI (2751)

NcoI (10180)

NcoI (10708)
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Table 4. Predicted & Observed Sizes of Hybridizing Fragments in Southern Blot Analyses 

Probe Restriction 

enzyme

Sample Lane

Expected 

fragment sizes 

(bp)1

Observed 

fragment sizes 

(bp)2

Figure

aad-12

NcoI

pDAB4468 2 7429 ~7400

Coker 310 3 none none Figure 9

DAS-8191Ø-7 4-18 >4043 ~9500

SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 10

DAS-8191Ø-7 4-18 >6245 ~7000

pat

NcoI

pDAB4468 2 7429 ~7400

Coker 310 3 none none Figure 11

DAS-8191Ø-7 4-18 >4043 ~9500

SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 12

DAS-8191Ø-7 4-18 >6245 ~7000

AtUbi10

MscI

pDAB4468 2 6225 ~6200

Coker 310 3 none none Figure 13

DAS-8191Ø-7 4-18 >5464 ~15000

SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 14

DAS-8191Ø-7 4-18 >6245 ~7000

AtuORF23 

3′UTR

NcoI

pDAB4468 2 7429 ~7400

Coker 310 3 none none Figure 15

DAS-8191Ø-7 4-18 >4043 ~9500

SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 16

DAS-8191Ø-7 4-18 >6245 ~7000

CsVMV

NcoI

pDAB4468 2 7429 ~7400

Coker 310 3 none none Figure 17

DAS-8191Ø-7 4-18 >4043 ~9500

SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 18

DAS-8191Ø-7 4-18 >6245 ~7000

AtuORF1 

3′UTR NcoI

pDAB4468 2 7429 ~7400

Coker 310 3 none none Figure 19

DAS-8191Ø-7 4-18 >4043 ~9500
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Probe Restriction 

enzyme

Sample Lane

Expected 

fragment sizes 

(bp)1

Observed 

fragment sizes 

(bp)2

Figure

SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 20

DAS-8191Ø-7 4-18 >6245 ~7000

aad-12

PstI

Release aad-12

expression cassette

pDAB4468 2 2868 ~2900

Coker 310 3 none none Figure 21

DAS-8191Ø-7 4-18 2868 ~2900

AtUbi10

pDAB4468 2 2868 ~2900

Coker 310 3 none none Figure22

DAS-8191Ø-7 4-18 2868 ~2900

AtORF23 

3′UTR

pDAB4468 2 2868 ~2900

Coker 310 3 none none Figure 23

DAS-8191Ø-7 4-18 2868 ~2900

pat

PstI/XhoI 

Release pat

expression cassette

pDAB4468 2 1928 ~1900

Coker 310 3 none none Figure 24

DAS-8191Ø-7 4-18 1928 ~1900

CsVMV

pDAB4468 2 1928 ~1900

Coker 310 3 none none Figure 25

DAS-8191Ø-7 4-18 1928 ~1900

AtuORF1 

3′UTR

pDAB4468 2 1928 ~1900

Coker 310 3 none none Figure 26

DAS-8191Ø-7 4-18 1928 ~1900

RB7 SphI

pDAB4468 2 12154 ~12200

Coker 310 3 none none Figure 27

DAS-8191Ø-7 4-18 >6245 ~7000

Ori

MscI

pDAB4468 2 5929, 6225 ~5900, ~6200

Coker 310 3 none none Figure 28

DAS-8191Ø-7 4-18 none none

PstI

pDAB4468 2 9286 ~9300

Coker 310 3 none none Figure 29

DAS-8191Ø-7 4-18 none none

Backbone 2 MscI

pDAB4468 2 5929 ~5900

Figure 30

Coker 310 3 none none

DAS-8191Ø-7 

4-18 none none
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Probe Restriction 

enzyme

Sample Lane

Expected 

fragment sizes 

(bp)1

Observed 

fragment sizes 

(bp)2

Figure

Backbone 2 PstI

pDAB4468 2 9286 ~9300

Coker 310 3 none none Figure 31

DAS-8191Ø-7 4-18 none none

Backbone 1

MscI

pDAB4468 2 5929 ~5900

Figure 32

Coker 310 3 none none

DAS-8191Ø-7 4-18 none none

PstI

pDAB4468 2 9286 ~9300

Figure 33

Coker 310 3 none none

DAS-8191Ø-7 4-18 none none

SpecR

MscI

pDAB4468 2 5929 ~5900

Coker 310 3 none none Figure 34

DAS-8191Ø-7 4-18 none none

PstI

pDAB4468 2 9286 ~9300

Figure 35 Coker 310 3 none none

DAS-8191Ø-7 4-18 none none

1

Expected fragment sizes are based on the plasmid map of pDAB4468 (Figure 7) and the intended T-DNA insert in DAS-8191Ø-7 cotton 

(Figure 8).

2 Observed 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.
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Figure 7. pDAB4468 Showing Location of Restriction Enzymes used for Southern Analysis

Figure 8. T-DNA Insert Restriction Enzyme Map of DAS-8191Ø-7 Cotton

Top: Intended T-DNA insert map of DAS-8191Ø-7 cotton showing restriction enzymes used for DNA digestion. 

Dashed blue lines on either end of insert represent cotton genomic DNA flanking T-DNA insert. Bottom: table of 

restriction enzymes (left) showing expected Southern blot hybridization band sizes for each restriction enzyme 

(right). A greater than symbol (>) denotes border fragments in which the hybridization band size is expected to be 

greater than the indicted size shown.

pDAB4468

12154 bp

pat

aad-12

SpecR

TrfA

T-DNA Border A

T-DNA Border A

T-DNA Border B

T-DNA Border A

RB7 MAR

CsVMV promoter

AtUbi10 promoter

Ori Rep AtuORF23 3'UTR

AtuORF1 3' UTR

Sph I (6245)

XhoI (6229)

MscI (1330)

MscI (7555)

Pst I (1433)

Pst I (4301)

NcoI (2751)

NcoI (10180)

NcoI (10708)

NcoI > 2751 bp > 4043 bp

SphI > 6245 bp > 549 bp

PstI > 1433 bp 2868 bp > 2493 bp

PstI/XhoI > 1433 bp 2868 bp 1928 bp >565 bp

MscI > 1330 bp > 5464 bp

pDAB4468 T-DNA Insert (6794bp)

pat

aad-12 T-DNA Border A

T-DNA Border A

T-DNA Border B

RB7 MAR T-DNA Border A

CsVMV promoter

AtUbi10 promoter AtuORF23 3'UTR

AtuORF1 3' UTR

MscI (1330)

NcoI (2751)

SphI (6245)

XhoI (6229)

PstI (1433) PstI (4301)
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4.1.1. Analysis of the Insert and Its Genetic Elements

4.1.1.1. Number of Transgenic Insertion Sites & Copy Number 

To determine the number of pDAB4468 transgenic insertion sites as well as the copy 

numbers of the transgenes in DAS-8191Ø-7 cotton, detailed Southern blot analysis was 

conducted on genomic DNA from five distinct breeding generations of DAS-8191Ø-7 cotton

(Figure 4). Restriction enzymes NcoI, SphI, and MscI were chosen to determine the number 

of inserts in DAS-8191Ø-7 cotton since each restriction enzyme cuts only once in the TDNA insert (Figure 8) and at an undefined location in the cotton genome to generate a border 

fragment. Since integration sites are unique for each event, border fragments provide a means 

to determine the insertion and copy numbers to specifically identify the event.

To determine insertion and copy number, probes specific to the T-DNA insert (aad-12, pat, 

AtUbi10 promoter, AtuORF23 3 UTR, CsVMV promoter, AtuORF1 3UTR, and RB7 

MAR) were used to screen Southern blots containing digested DAS-8191Ø-7 cotton genomic 

DNA. 

When genomic DNA from DAS-8191Ø-7 cotton was digested with NcoI and hybridized with 

the aad-12, pat, ORF23, CsVMV, and AtuORF1 3 UTR probes a single hybridization band 

of >4043 bp was expected (Figure 8). As anticipated, all DAS-8191Ø-7 cotton samples 

displayed a single band of ~9500 bp, consistent with the expected result of >4043 bp (Figure 

8, Figure 9, Figure 11, Figure 15, Figure 17, Figure 19).

When digested with MscI and hybridized with the AtUbi10 promoter probe, all DAS-8191Ø7 cotton samples displayed a single band of ~15000 bp, consistent with the expected result of 

>5464 bp (Figure 8, Figure 13).

When digested with SphI and hybridized with aad-12, pat, AtUbi10, ORF23 3 UTR, 

CsVMV, and AtuORF1 3 UTR and RB7probes, all DAS-8191Ø-7 cotton samples displayed 

a single band of ~7000 bp, consistent with the expected result of >6245 bp (Figure 8, Figure 

10, Figure 12, Figure 14, Figure 16, Figure 18, Figure 20 and Figure 27).

In addition to DAS-8191Ø-7 cotton DNA, genomic DNA from leaves of non-transgenic 

variety Coker 310 was used as a negative control material (Appendix 1). Plasmid DNA of 

pDAB4468 added to genomic DNA from the non-transgenic variety Coker 310 served as the 

positive control for Southern blot analysis. As expected, specific hybridization bands were 

detected in all of the positive control samples at the expected sizes for each restriction 

enzyme and probe combinations tested (Figure 9 - Figure 20 and Figure 27). For negative 

control plants, in which genomic DNA from non-transgenic variety Coker 310 was used, no 

hybridization bands were detected, as expected (Figure 9 - Figure 20 and Figure 27).

These data confirm that DAS-8191Ø-7 cotton contain a single integration and a single copy 

of the respective T-DNA sequence from pDAB4468 plasmid (including the AtUbi10 

promoter, add-12, AtuORF23 3′ UTR, CsVMV promoter, pat, AtuORF1 3′ UTR along with 

the RB7 MAR element).
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 38 of 214

Dow AgroSciences LLC

4.1.1.2. Structure of the Insert and Genetic Elements

To further characterize the structure of transgene insert and confirm that DAS-8191Ø-7

cotton contains a single intact copy of the aad-12 expression cassette (AtUbi10 promoter, 

add-12, AtuORF23 3′ UTR) and pat expression cassette (CsVMV promoter, pat, AtuORF1 

3′ UTR), DAS-8191Ø-7 cotton genomic DNA was digested with PstI and PstI/XhoI

restriction enzymes and hybridized with the cassette element-based probes. As shown in

Figure 7 and Figure 8, PstI allows for the release of the full length aad-12 expression 

cassette, while PstI/XhoI allows for the release of the full length pat expression cassette.

Southern blot analysis was conducted on genomic DNA from five distinct breeding 

generations of DAS-8191Ø-7 cotton (Figure 4). When digested with PstI and separately 

hybridized with the aad-12, AtUbi10, and AtuORF23 3′ UTR probes, all DAS-8191Ø-7

cotton samples along with the positive control displayed a single band of ~2900 bp, 

consistent with the predicted size of 2868 bp for the aad-12 expression cassette (Figure 8, 

Figure 21, Figure22, Figure 23). These data indicate that an intact aad-12 expression cassette

is present in all tested generations of DAS-8191Ø-7 cotton. 

When digested with PstI/XhoI and separately hybridized with the pat probe, CsVMV, and 

AtuORF1 3 UTR probes, all DAS-8191Ø-7 cotton samples along with the positive control 

displayed a single band of ~1900 bp, consistent with the predicted size of 1928 bp for pat 

expression cassette (Figure 8, Figure 24, Figure 25, Figure 26). These data indicate that an 

intact pat expression cassette is present in all tested generations of DAS-8191Ø-7 cotton. 

Hybridization bands of the expected sizes were detected in all positive samples, while no 

specific hybridization band was detected in the non-transgenic cotton samples; as expected

(Figure 21 - Figure 26). The hybridization pattern is consistent across all generations with all 

the tested restriction enzyme and probe combinations. 

Taken together, the Southern blot analyses reveal that DAS-8191Ø-7 cotton contains a single 

intact insert of aad-12 expression cassette and the pat expression cassette.

4.1.2. Absence of Plasmid Backbone Sequences 

To verify that no plasmid vector backbone sequences were inserted in DAS-8191Ø-7 cotton, 

four probes (Backbone 1, Backbone 2, Ori, and SpecR) covering the entire backbone region 

of pDAB4468 plasmid DNA were generated and hybridized to MscI and PstI digested DAS8191Ø-7 cotton DNA samples.

When digested with MscI and independently hybridized with Backbone 1, Backbone 2, and 

SpecR probes, it would be anticipated that the positive control samples would have a band of 

~5900 bp, while in the absence of plasmid backbone, DAS-8191Ø-7 cotton samples and the 

negative control would be expected to have no hybridization bands (Figure 8).

When digested with MscI and hybridized with Backbone 1, Backbone 2, and SpecR probes, 

no specific hybridization bands were detected in any DAS-8191Ø-7 cotton samples, except 

for the positive controls. A single band of ~5900 bp was detected in the positive control 
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 39 of 214

Dow AgroSciences LLC

sample, which was consistent with the predicted size of 5929 bp (Figure 30, Figure 32 and 

Figure 34).

When blots containing the same digested genomic DNA and hybridized with Ori probe, two 

expected bands at ~5900 and ~6200 bp were detected only in the positive control sample and 

not in the DAS-8191Ø-7 cotton samples (Figure 28). This is consistent with the expected

fragment sizes of 5929 and 6225 bp, since the Ori probe binds to DNA sequences on both 

sides of MscI digested pDAB4468 plasmid DNA (Figure 6). 

When digested with PstI and hybridized with Backbone 1, Backbone 2, Ori, and SpecR 

probes, no specific hybridization bands were detected in any DAS-8191Ø-7 cotton samples, 

except for the positive controls. A single band of ~9300 bp was detected in positive control 

sample, consistent with the predicted size of 9286 bp (Figure 6, Figure 29, Figure 31, Figure 

33 and Figure 35). 

These Southern analysis data, along with the positive and negative control results confirm 

that no backbone sequences from pDAB4468 are incorporated into DAS-8191Ø-7 cotton.

4.2. Stability of the Insert Across Generations

All DAS-8191Ø-7 cotton Southern hybridization samples, across all five generations (T2, 

T3, T4, T5, and BC1F2 see Figure 4) revealed an intact, single copy aad-12 expression 

cassette, pat expression cassette and RB7 MAR insertion. These data clearly show stable 

integration and inheritance of the intact, single copy transgene insert across multiple 

generations of DAS-8191Ø-7 cotton.

4.3. Southern Blot Analysis Conclusions

Southern blot analysis confirms that DAS-8191Ø-7 cotton contains a single copy of the 

transgene insert from pDAB4468, gene expression cassettes aad-12 and pat, and a RB7-

MAR element (Section 4.1.1). No plasmid backbone sequences were detected in DAS8191Ø-7 cotton (Section 4.1.2). The hybridization patterns across five generations of DAS8191Ø-7 cotton (T2, T3, T4, T5, and BC1F2) were identical, indicating that the insert is 

stably integrated and inherited in the DAS-8191Ø-7 cotton genome (Section 4.2). 
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 40 of 214

Dow AgroSciences LLC

Figure 9. Southern Analysis of DAS-8191Ø-7 Cotton Digested with NcoI; aad-12 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-03

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-03

6 DAS-8191Ø-7-T2-03 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-06

9 DAS-8191Ø-7-T3-03 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 41 of 214

Dow AgroSciences LLC

Figure 10. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; aad-12 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

b p

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

b p

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 42 of 214

Dow AgroSciences LLC

Figure 11. Southern Analysis of DAS-8191Ø-7 Cotton Digested with NcoI; pat Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-03

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-03

6 DAS-8191Ø-7-T2-03 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-06

9 DAS-8191Ø-7-T3-03 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

b p

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

b p

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 43 of 214

Dow AgroSciences LLC

Figure 12. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; pat Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs.

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 44 of 214

Dow AgroSciences LLC

Figure 13. Southern Analysis of DAS-8191Ø-7 Cotton Digested with MscI; AtUbi10 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 45 of 214

Dow AgroSciences LLC

Figure 14. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; AtUbi10 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 46 of 214

Dow AgroSciences LLC

Figure 15. Southern Analysis of DAS-8191Ø-7 Cotton Digested with NcoI; AtuORF23 3′

UTR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. (Note: Differential migration of hybridization bands in lanes 10 and 12 are attributable to 

minor impurities in DNA samples.)

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-03

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-03

6 DAS-8191Ø-7-T2-03 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-06

9 DAS-8191Ø-7-T3-03 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 47 of 214

Dow AgroSciences LLC

Figure 16. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; AtuORF23 3′

UTR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. (Note: Differential migration of hybridization bands in lanes 10 and 12 are attributable to 

minor impurities in DNA samples) 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-06

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 48 of 214

Dow AgroSciences LLC

Figure 17. Southern Analysis of DAS-8191Ø-7 Cotton Digested with NcoI; CsVMV Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. (Note: Differential migration of hybridization bands in lane 10 and 12 are attributable to 

minor impurities in DNA samples.)

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-03 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576 7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 49 of 214

Dow AgroSciences LLC

Figure 18. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; CsVMV Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

b p

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 50 of 214

Dow AgroSciences LLC

Figure 19. Southern Analysis of DAS-8191Ø-7 Cotton Digested with NcoI; AtuORF1 3′

UTR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-03

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-03

6 DAS-8191Ø-7-T2-03 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-06

9 DAS-8191Ø-7-T3-03 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

b p

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 51 of 214

Dow AgroSciences LLC

Figure 20. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; AtuORF1 3′

UTR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7 BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 52 of 214

Dow AgroSciences LLC

Figure 21. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI to Release aad-12 

Expression cassette; aad-12 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7-BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 53 of 214

Dow AgroSciences LLC

Figure22. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI to Release aad-12 

Expression Cassette; AtUbi10 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7-BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 54 of 214

Dow AgroSciences LLC

Figure 23. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI to Release aad-12

Expression Cassette; AtuORF23 3′ UTR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 55 of 214

Dow AgroSciences LLC

Figure 24. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI/XhoI to Release

pat Expression Cassette; pat Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7-BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 56 of 214

Dow AgroSciences LLC

Figure 25. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI/XhoI to Release

pat Expression Cassette; CsVMV Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7- BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 57 of 214

Dow AgroSciences LLC

Figure 26. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI/XhoI to Release

pat Expression Cassette; AtuORF1 3′ UTR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. (Note: Differential migration of hybridization bands in lanes 10 and 12 are attributable to 

minor impurities in DNA samples.)

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7- BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7 -T3-02 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 58 of 214

Dow AgroSciences LLC

Figure 27. Southern Analysis of DAS-8191Ø-7 Cotton Digested with SphI; RB7 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7- BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 59 of 214

Dow AgroSciences LLC

 

Figure 28. Southern Analysis of DAS-8191Ø-7 Cotton Digested with MscI; Ori Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7- BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 60 of 214

Dow AgroSciences LLC

Figure 29. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI; Ori Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7- BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 61 of 214

Dow AgroSciences LLC

Figure 30. Southern Analysis of DAS-8191Ø-7 Cotton Digested with MscI; Backbone2 

Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 62 of 214

Dow AgroSciences LLC

Figure 31. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI; Backbone2 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 63 of 214

Dow AgroSciences LLC

Figure 32. Southern Analysis of DAS-8191Ø-7 Cotton Digested with MscI; Backbone1 

Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

b p

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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Figure 33. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI; Backbone1 Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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Figure 34. Southern Analysis of DAS-8191Ø-7 Cotton Digested with MscI; SpecR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-03

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-03

5 DAS-8191Ø-7-T2-03 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-05

7 DAS-8191Ø-7-T3-02 17 DAS-8191Ø-7- BC1F2-06

8 DAS-8191Ø-7-T3-03 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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Figure 35. Southern Analysis of DAS-8191Ø-7 Cotton Digested with PstI; SpecR Probe

Approximately 10 g of genomic DNA was digested and loaded per lane. The positive control included plasmid 

pDAB4468 at approximately one copy of plasmid pDAB4468 per cotton genome in 10 g of genomic DNA 

extracted from non-transgenic control Coker 310. Fragment sizes in bp of the DIG-labeled DNA Molecular Weight 

Marker II and VII were indicated adjacent to membrane image. The last two digits in Sample Description indicate 

individual sample IDs. 

Lane Sample Lane Sample

1 DIG Molecular Weight Marker II 11 DAS-8191Ø-7-T4-02

2 Coker 310 + pDAB4468 12 DAS-8191Ø-7-T4-05

3 Coker 310 13 DAS-8191Ø-7-T5-01

4 DAS-8191Ø-7-T2-01 14 DAS-8191Ø-7-T5-02

5 DAS-8191Ø-7-T2-02 15 DAS-8191Ø-7-T5-04

6 DAS-8191Ø-7-T2-04 16 DAS-8191Ø-7-BC1F2-04

7 DAS-8191Ø-7-T3-01 17 DAS-8191Ø-7- BC1F2-05

8 DAS-8191Ø-7-T3-02 18 DAS-8191Ø-7- BC1F2-07

9 DAS-8191Ø-7-T3-04 19 DIG Molecular Weight Marker VII

10 DAS-8191Ø-7-T4-01

bp

6557

4361

2027

2322

564

9416

23130

1482

8576

7427

6106

4899

3639

2799

1882

1515

1164

992

718/710

492

359

bp

1953

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19
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4.4. Segregation Analysis

4.4.1. Genetic and Molecular Analysis of a Segregating Generation

DAS-8191Ø-7 cotton transgene insert resides at a single locus within the cotton genome and 

is inherited according to Mendelian inheritance principles. Chi-square goodness of fit 

analyses of trait inheritance data within a segregating generation was conducted to confirm 

the Mendelian inheritance of the transgene insert in DAS-8191Ø-7 cotton. 

The inheritance pattern of the transgene insert within a segregating generation was 

demonstrated with Lateral Flow Strip (LFS) and event specific PCR analyses of individual 

plants within a BC1F2 generation of DAS-8191Ø-7 cotton (Figure 4). 

A total of 136 plants from the BC1F2 generation of DAS-8191Ø-7 cotton were tested for 

AAD-12 protein expression by LFS assay. Of the 136 BC1F2 plants tested, 104 plants were 

positive and 32 were negative (segregated nulls) for AAD-12 protein expression. Genomic 

DNA was extracted from all these samples and followed by event-specific PCR analysis to 

determine the presence or absence of the DAS-8191Ø-7 transgene insert. Similarly, of the 

136 plants tested by event-specific PCR, 104 plants were positive for the presence of DAS8191Ø-7 transgene insert and the remaining 32 plants were negative (segregated null). All 

plants that tested positive for AAD-12 protein expression by LFS were also positive for the 

DAS-8191Ø-7 transgene insert by event-specific PCR analysis, and all plants that tested

negative for AAD-12 protein expression were also negative for the presence of the DAS8191Ø-7 transgene insert by event-specific PCR (Table 5). 

This result confirmed that the phenotypic segregation matched the genotypic makeup of the 

tested plants in the BC1F2 generation. Statistical analysis using a chi-square goodness of fit 

test indicated that the ratio of 104 positive to 32 null segregants did not significantly differ 

from the expected Mendelian 3:1 segregation pattern for a single independent locus. 

Table 5. Segregating Generation Results of BC1F2 Individual Plants 

Tested Method Total plants 

tested

Positive Negative Expected 

ratio

P-value 1

AAD-12 LFS 136 104 32 3:1 0.6921

Event-Specific PCR 136 104 32 3:1 0.6921

1 Based on a chi-squared goodness of fit test

4.4.2. Segregation Analysis of Breeding Generations

Chi-square goodness of fit analyses of trait inheritance data from a population of T1 and 

BC1F2 breeding generations was also conducted to determine the Mendelian inheritance of 

the transgene insert in DAS-8191Ø-7 cotton (Figure 4). The presence or absence of the 

transgene insert was determined using a PAT protein assay, a pat gene zygosity assay, or an 

aad-12 gene zygosity assay for DAS-8191Ø-7 cotton. Gene zygosity assays were conducted 

using TaqMan real time PCR with primers and probes specific to either pat or aad-12

sequence to determine gene copy number of each assayed sample. DNA samples were 

extracted from 2571 BC1F2 and 196 T1 plants and assayed for aad-12 and pat, respectively. 

Quantitative analysis of the PCR results revealed the expected segregation ratio of 3:1 for 

plants containing the transgene insert versus plants that do not contain the transgene insert 

(segregated nulls) (Table 6). The observed segregation ratio does not significantly differ from 
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the expected Mendelian 3:1 segregation pattern for a single independent locus. These results 

support the conclusion that DAS-8191Ø-7 transgene insert resides at a single locus within the 

cotton genome and is inherited according to Mendelian principles. 

 

Table 6. Segregating Breeding Generation Results of DAS-8191Ø-7 Cotton Plants 

Generation Total 

Plants 

Tested

Tested Method Positive Null Expected 

Ratio

P-value1

T1 191 PAT LFS 146 45 3:1 0.65

T1 196 pat gene zygosity 153 43 3:1 0.32

BC1F2 2571 aad-12 gene zygosity 1916 655 3:1 0.58

1 Based on a chi-square goodness of fit test

4.5. Summary of the Genetic Characterization

Molecular characterization of DAS-8191Ø-7 cotton by Southern blot analysis confirmed that 

a single transgene insert containing each of the intact expression cassettes for aad-12 and pat, 

along with the RB7 MAR element at the 5 end, were integrated into DAS-8191Ø-7 cotton. 

No transformation plasmid backbone sequence was found in DAS-8191Ø-7 cotton as

demonstrated by Southern blot analysis using probes covering the entire region of the 

plasmid flanking the T-DNA insert. 

Southern blot analysis of five generations (T2, T3, T4, T5, and BC1F2) demonstrated the 

inserted DNA was stably inherited through multiple generations. Moreover, the transgene 

insert displayed the expected Mendelian inheritance pattern for a single independent 

insert/locus in segregating generations (T1 and BC1F2), confirming that the transgene insert in 

DAS-8191Ø-7 cotton occurs at a single chromosomal locus with expected inheritance 

patterns. 
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5. Characterization of the DAS-8191Ø-7 AAD-12 and PAT Proteins

As described in Section 3.4, DAS-8191Ø-7 cotton contains the aad-12 and pat expression 

cassettes that, when transcribed and translated, result in the expression of the AAD-12 and 

PAT proteins, respectively. This section summarizes: 1) the identity and mode of action of 

the AAD-12 and PAT proteins expressed in DAS-8191Ø-7 cotton; 2) demonstration of 

equivalence between the plant-produced and microbially-produced AAD-12 and PAT 

proteins, which were used in various protein safety studies; 3) the expression levels of AAD12 and PAT proteins in DAS-8191Ø-7 plant tissues; 4) the assessment of the potential 

allergenicity and toxicity of AAD-12 and PAT proteins; 5) the food and feed safety 

assessment of AAD-12 and PAT proteins. Based on several lines of evidence described 

below, the data support a conclusion that these two proteins produced in DAS-8191Ø-7

cotton are safe for the environment and safe for human and animal consumption. These data 

were supplied to FDA for their evaluation in consultation BNF No. 000142 on the food and 

feed safety and compositional assessment of DAS-8191Ø-7 cotton.

5.1. AAD-12

5.1.1. Identity of the AAD-12 Protein

The expressed DAS-8191Ø-7 cotton AAD-12 protein is comprised of 293 amino acids and 

has a molecular weight of 32 kDa (Figure 36). The wild-type aryloxyalkanoate dioxygenase 

(AAD-12) protein coding sequence was derived from Delftia acidovorans, a gram-negative 

soil bacterium (Wright et al., 2010). The wild-type AAD-12 amino acid sequence and the 

DAS-8191Ø-7 cotton AAD-12 amino acid sequence (encoded by the plant-optimized aad-1

gene (see section 3.4.1)) are greater than 99% identical, differing only by a single amino acid 

addition, alanine (A), at position number two (Figure 36). The additional alanine codon 

encodes part of an NcoI restriction enzyme recognition site (CCATGG) spanning the ATG 

translational start codon of aad-12. This additional codon serves the dual purpose of 

facilitating subsequent cloning operations and improving the sequence context surrounding 

the ATG start codon to optimize translation initiation. The AAD-12 protein produced in 

DAS-8191Ø-7 cotton is expressed from the identical genetic aad-12 sequence present in 

DAS-68416-4 soybean and DAS-444Ø6-6 soybean (currently under USDA-APHIS review, 

petition numbers 09-349-01p, 11-234-01p respectively).

001 MAQTTLQITPTGATLGATVTGVHLATLDDAGFAALHAAWLQHALLIFPGQ

051 HLSNDQQITFAKRFGAIERIGGGDIVAISNVKADGTVRQHSPAEWDDMMK

101 VIVGNMAWHADSTYMPVMAQGAVFSAEVVPAVGGRTCFADMRAAYDALDE

151 ATRALVHQRSARHSLVYSQSKLGHVQQAGSAYIGYGMDTTATPLRPLVKV

201 HPETGRPSLLIGRHAHAIPGMDAAESERFLEGLVDWACQAPRVHAHQWAA

251 GDVVVWDNRCLLHRAEPWDFKLPRVMWHSRLAGRPETEGAALV

Figure 36. Amino Acid Sequence of Expressed AAD-12 in DAS-8191Ø-7

DAS-8191Ø-7 cotton AAD-12 amino acid sequence was designed to have a single amino acid addition, alanine, at 

position number two (underlined in figure above) as compared to the native D. acidovorans AAD-12 amino acid 

sequence. See text for details.
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5.1.2. Mode of Action of the AAD-12 Protein

Expression of the AAD-12 protein in transgenic crops has been shown to provide tolerance to 

the herbicide 2,4-D by catalyzing the conversion of 2,4-D to 2,4-dichlorophenol (DCP), a 

herbicidally inactive compound, through an Fe(II)/α-keto acid-dependent dioxygenase 

reaction (Figure 37 and Figure 38) (Müller et al., 1999; Westendorf et al., 2002; Westendorf

et al., 2003; Wright et al., 2009; Wright et al., 2010). 

O

O

OH

Cl Cl

2,4-D

OH

Cl Cl AAD-12

DCP

Figure 37. Degradation Reaction of 2,4-D Catalyzed by AAD-12

5.1.2.1. DCP

DCP (2,4-dichlorophenol) is a known primary degradate of 2,4-D in plants (Roberts, 1998).

DCP has been observed as a degradate of 2,4-D in environmental matrices and is also 

observed in animal metabolism studies (Roberts, 1998; Barnekow et al., 2001).

The U.S. EPA tolerance expression for 2,4-D does not include DCP in the plant residue 

definition, but DCP was at one point included in the livestock meat and milk tolerance 

expression. In 2004 the U.S. EPA’s Health Effects Division, Metabolism Assessment Review 

Committee (MARC) recommended that DCP be deleted from the livestock tolerance 

expression for 2,4-D. The MARC committee stated DCP is “not of concern for either the 

tolerance expression or for risk assessment at the levels expected in livestock tissues and 

considering the likely lower toxicity of 2,4-DCP compared to 2,4-D” (EPA, 2003). This 

decision was included in the 2005 Registration Eligibility Decision document (EPA, 2005)

and posted in the 2007 Federal Register (EPA, 2007). This action harmonizes U.S. tolerances 

with Codex, Japanese and European residue definitions which do not include DCP in any 

tolerance expression.

DAS will be submitting information to the U.S. EPA on the Nature of Residue and 

Magnitude of Residue resulting from the use of 2,4-D on cotton that expresses the AAD-12 

protein. The U.S. EPA will evaluate this information as a component of their review of the 

use of 2,4-D on cotton that expresses the AAD-12 protein.

5.1.2.2. Enzyme Specificity

AAD-12 is able to degrade related achiral phenoxyacetate herbicides such as MCPA 

((4-chloro-2-methylphenoxy) acetic acid) and pyridyloxyacetate herbicides such as triclopyr 

and fluroxypyr to their corresponding inactive phenols and pyridinols, respectively (Figure 

38). AAD-12 has enantiomeric selectivity for the (S)-enantiomers of the chiral phenoxy acid 

herbicides (e.g., dichlorprop and mecoprop), but does not catalyze degradation of the (R)-

enantiomers. It is the R-enantiomers in this class of chemistry that are herbicidally active;
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therefore AAD-12 does not provide tolerance to commercially-available chiral phenoxy acid 

herbicides (Wright et al., 2010).

Figure 38. General Reaction Catalyzed by AAD-12 (R=H or CH3)

The AAD-12 enzyme was screened for the ability to utilize endogenous plant substrates 

using a sensitive coupled in vitro enzyme assay (Luo et al., 2006). Potential plant substrates 

were determined based on chemical structure, similar physiological function to known AAD12 substrates, and abundance within primary/secondary metabolic pathways of plants. The 

substrates tested were separated into three groups; natural plant hormones (indole acetic acid, 

abscisic acid, gibberellin, and aminocyclopropane-1-carboxylate), phenylpropanoid 

intermediates (cinnamate, coumarate, and sinapate), and L-amino acids. 2,4-

dichlorphenoxyacetic acid (2,4-D), the positive control substrate, showed a high level of 

activity in the enzyme assay. Under the same reaction conditions, the plant compounds

identified and tested were not oxidized upon incubation with AAD-12, resulting in values at 

or below the background limit of detection (<3% positive control rate). Based on this survey 

of potential substrates, there is no indication that AAD-12 has activity on endogenous plant 

substrates (Griffin et al., 2013).

5.1.3. Biochemical Characterization of the AAD-12 Protein

Large quantities of purified AAD-12 protein are required to perform safety assessment 

studies. Because it is technically infeasible to extract and purify sufficient amounts of 

recombinant protein from transgenic plants (Evans, 2004; Raybould et al., 2012), large 

quantities of AAD-12 protein was produced in Pseudomonas fluorescens. Characterization 

studies were performed to confirm the equivalency of the AAD-12 protein produced in P. 

fluorescens with the AAD-12 protein produced in planta in DAS-8191Ø-7 cotton. Sodium 

dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), western blot, glycoprotein 

detection, enzymatic assay, and protein sequence analysis by matrix assisted laser 

desorption/ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF 

MS/MS) were used to characterize the biochemical properties of the proteins. Using these 

methods, the AAD-12 protein isolated from P. fluorescens and DAS-8191Ø-7 cotton were 

shown to be biochemically and biologically equivalent, thereby supporting the use of the 

microbe-derived protein in safety assessment studies.

The methods and results of the biochemical characterization of the DAS-8191Ø-7 cotton and 

microbe-derived AAD-12 proteins are described in detail in Appendix 2. Both the plant and 
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P. fluorescens-derived AAD-12 proteins were observed at the expected molecular weight of 

32 kDa by SDS-PAGE and were immunoreactive to AAD-12 protein-specific antibodies by 

western blot analysis. There was no evidence of glycosylation of either the DAS-8191Ø-7

cotton or P. fluorescens-derived AAD-12 proteins. Cotton- and P. fluorescens-derived AAD12 were equally active using S-dichloroprop as a substrate and displayed similar kinetic 

parameters, indicating that the proteins are enzymatically equivalent. In addition, greater than 

88% of the cotton-derived protein amino acid sequence was confirmed by either enzymatic 

peptide mass fingerprinting or MS/MS sequence analysis by MALDI-TOF MS/MS. The Nterminal methionine was found to be cleaved from both protein sources and the N-terminal 

peptide of the plant-derived AAD-12 was determined to be acetylated after the N-terminal 

methionine was cleaved. These two post-translational processes, cleavage of the N-terminal 

methionine residue and N-terminal acetylation, are common modifications that have been 

found to occur on the vast majority (~85%) of eukaryotic proteins (Polevoda and Sherman, 

2003). The C-terminal peptides from DAS-8191Ø-7 cotton and P. fluorescens were intact 

and empirically determined to be identical.

5.1.4. Expression of the AAD-12 Protein in Plant Tissues

A field expression study was conducted in the U.S. during 2012. Six sites (Alabama, 

Georgia, Louisiana, Missouri, North Carolina, and Texas) were planted with DAS-8191Ø-7

cotton and the non-transgenic control (98M-2983). The test sites represented regions of 

diverse agronomic practices and environmental conditions for cotton in North America. Two

treatments of DAS-8191Ø-7 cotton (non-sprayed or sprayed with 2,4-D and glufosinate) were 

tested and plant tissue was collected from leaf, squares, bolls, pollen, flower, whole plant, 

root, and seed (Table 7).

Table 7. DAS-8191Ø-7 Cotton AAD-12 Expression Analysis Tissue Samples

Expression

Tissue

Growth

Stage1

Sample

Size

No. of Samples per 

Entry/Site

Bolls Peak Bloom 10-14 Bolls 4

Flower Peak Bloom 14-18 Flowers 4

Leaf 4-Leaf 10-14 Leaves 4

Leaf 1

st White Bloom 10-14 Leaves 4

Leaf 1

st Open Boll 10-14 Leaves 4

Pollen Early Bloom 0.2-0.5 mL 4

Root Maturity 1-2 Plants 4

Seed Maturity 175-250 grams 4

Squares 1

st White Bloom 10-14 Squares 4

Whole Plant Maturity 1-2 Plants 4

1 Approximate growth stage

AAD-12 protein was extracted from DAS-8191Ø-7 cotton tissue and the soluble, extractable 

AAD-12 protein from each tissue was measured using a draft enzyme-linked immunosorbent 

assay (ELISA) method. AAD-12 protein levels for all tissue types were calculated on ng/mg 

dry weight basis. The details of the materials and methods are described in Appendix 4.
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A summary of the AAD-12 protein concentrations (averaged across sites) in the various 

cotton matrices is shown in Table 8. The mean AAD-12 protein levels were highest in the 

leaf at 71.17 ng/mg followed by pollen at 70.71 ng/mg, squares at 38.33 ng/mg, flower at 

30.63 ng/mg, seed at 18.75 ng/mg, bolls at 17.17 ng/mg, whole plant at 16.42 ng/mg, and 

root at 10.74 ng/mg. AAD-12 expression levels were comparable for both sprayed and nonsprayed treatments. No AAD-12 protein was detected in the control tissues.

Table 8. Summary of AAD-12 Protein Expression

Expression 

Tissue

Growth 

Stage

AAD-12 ng/mg Tissue Dry Weight1,2

Overall 

Mean

Std. Dev.3

(n = 48)

Min/Max Range4

Bolls Peak Bloom 17.17 7.91 4.36-33.39

Flower Peak Bloom 30.63 8.36 18.76-52.28

Leaf 4-Leaf 71.17 46.63 7.78-180.72

Leaf 1

st white Bloom 17.53 8.6 1.45-40.56

Leaf 1

st Open Boll 51.26 19.63 17.38-89.23

Pollen Early Bloom 70.71 19.58 35.15-107.1

Root Maturity 10.74 5.27 NDe-22.89

Seed Maturity 18.75 4.81 6.75-27.77

Squares 1

st White Bloom 38.33 12.21 16.42-66.72

Whole Plant Maturity 16.42 12.18 ND5-46.98

1Calculated from AAD-12 expression raw data.

2Table represents overall results for non-sprayed and sprayed cotton tissue across all sites.

3

Standard deviation of individual cotton samples across all sites.

4Represents the min and max for individual cotton samples across all sites.

5ND = Not Detected, expression level below LOD (Limit of Detection). A zero value was used for ND results for mean calculation.

5.1.5. Food and Feed Safety Assessment of AAD-12

The primary food source from cotton is refined cottonseed oil, which contains undetectable 

amounts of protein (Reeves III and Weihrauch, 1979). Therefore, assessing the allergenicity 

and toxicity to humans of the expressed proteins in cotton is less relevant compared with

other crops such as soybean, since little to no protein is present in consumed cotton products. 

Regardless, a detailed safety assessment of AAD-12 was conducted to assess any potential 

adverse effects to humans or animals resulting from the environmental release of crops 

containing AAD-12 (Codex Alimentarius Commission, 2009). The food and feed safety 

assessment of AAD-12 expressed in DAS-8191Ø-7 cotton 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. The conclusion from the assessment 

is that AAD-12 is unlikely to cause allergic reaction in humans or be a toxin to humans or 

animals. 

5.1.5.1. Safety of Donor Organism and history of safe use

The donor organism, Delftia acidovorans (formerly designated as Pseudomonas acidovorans

and Comamonas acidovorans) is a non glucose-fermenting, gram-negative, non sporeforming rod, bacterium present in soil, fresh water, activated sludge, and clinical specimens 

(Von Graevenitz, 1985; Tamaoka et al., 1987; Wen et al., 1999).
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Strains of Delftia acidovorans can be used to transform ferulic acid into vanillin and related 

flavor metabolites (Toms and Wood, 1970; Labuda et al., 1992; Rao and Ravishankar, 2000; 

Shetty et al., 2006).

5.1.5.2. Assessment of Allergenicity Potential

Studies were conducted to ascertain the potential allergenicity of AAD-12 and conclusions 

were as follows:

1) DAS-8191Ø-7 cotton AAD-12 originates from Delftia acidovorans, an organism that 

has not been reported as a source of allergens (5.1.5.1).

2) Bioinformatics analyses demonstrated that DAS-8191Ø-7 cotton AAD-12 does not

share amino acid sequence similarities with known allergens and is highly unlikely to 

contain immunologically cross-reactive allergenic epitopes.

3) In vitro digestive fate experiments conducted with AAD-12 demonstrate that the 

protein is rapidly digested in simulated gastric fluid (SGF).

4) AAD-12 is heat labile, in which enzyme activity is rapidly eliminated at temperatures 

as low as 50 °C in less than thirty minutes. Due to the harsh processing conditions of 

cottonseed oil and the lack of detectable proteins in processed cottonseed oil, AAD12 is most likely absent in the oil and linters used in food products.

Taken together, these data conclude that DAS-8191Ø-7 cotton AAD-12 does not pose a 

significant allergenic risk. 

Amino Acid Sequence Comparison to Known Allergens

The step-wise, weight-of-evidence approach (Codex Alimentarius Commission, 2009) was 

used to assess the allergenic potential of the AAD-12 protein. The AAD-12 protein does not 

share meaningful amino acid sequence similarities with known allergens. No significant 

sequence homology was identified when the AAD-12 protein sequence was compared with 

known allergens in the FARRP (Food Allergy Research and Resource Program) version 

13.00 allergen database (Released in February, 2013), using the search criteria of either a 

match of eight or more contiguous identical amino acids, or >35% identity over 80 or longer 

amino acid residues.

Lability in Simulated Gastric Fluid

The digestibility of the P. fluorescens-derived AAD-12 protein was tested in vitro using 

simulated gastric fluid (SGF). The AAD-12 protein was incubated in SGF (0.32% w/v pepsin 

at pH 1.2; (The United States Pharmacopeia, 1995) for various periods of time. The samples 

were then analyzed via SDS-PAGE and western blot analysis using a polyclonal antibody 

specific to AAD-12. The results demonstrated the AAD-12 protein was readily digested in 

less than thirty seconds in SGF, indicating that the proteins are unlikely to elicit allergenic

reactions when consumed.
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Heat Lability

The thermal stability of the P. fluorescens-derived AAD-12 protein was evaluated by heating 

protein solutions for 30 min at 50, 70 and 95 °C; and 20 min in an autoclave (120 °C at ~117 

kPa (~17 PSI)) in a phosphate-based buffer. The AAD-12 protein activity was measured by a 

modified enzyme assay based on the procedure described in Fukumori and Hausinger 

(Fukumori and Hausinger, 1993). In the presence of Fe(II), the AAD-12 protein catalyzes the 

conversion of dichlorphenoxyacetate to 2,4-dichlorophenol and glyoxylate concomitant with 

the decomposition of α-ketoglutarate to form succinate and carbon dioxide. The resulting 

phenol is measured with an AAPPC assay or the Emerson reaction (Emerson, 1943). All

heating conditions eliminated the enzymatic activity of the AAD-12 protein. 

5.1.5.3. Assessment of Toxicity Potential 

Amino Acid Sequence Comparison to Known Toxins

The AAD-12 protein does not share meaningful amino acid sequence similarities with known 

toxins. Sequence homologies between the AAD-12 protein and known protein toxins were 

evaluated using BLASTp search algorithm against the GenBank non-redundant protein 

sequences (updated February 18, 2012). By their annotations, the proteins returned by 

BLASTp search can be grouped into the following 10 categories: 2,4-D/alpha-ketoglutarate 

dioxygenase, putative alkylsulfatase, alpha-ketoglutarate (dependent) dioxygenase, alphaketoglutarate-dependent sulfonate dioxygenase, taurine catabolism dioxygenase, taurine 

dioxygenase, dioxygenase, oxidoreductase, pyoverdine biosynthesis protein, and hypothetical 

(putative) or unnamed proteins. AAD-12 (aryloxyalkanoate dioxygenase-12) itself is an 

alpha-ketoglutarate dependent dioxygenase. Hypothetical and unnamed proteins are derived 

from conceptual translation of DNA sequences generated from massive genome sequencing 

projects of various fungi and bacteria. Those proteins have functional annotations such as 

“probable taurine catabolism dioxygenase”, “clavaminic acid synthetase-like”, and “putative 

alpha-ketoglutarate dependent dioxygenase”. None of those proteins are associated with 

protein toxins that are harmful to humans or animals.

Acute Oral Toxicity

An acute oral toxicity study with the P. fluorescens-derived AAD-12 protein was conducted 

in mice administered 2000 mg AAD-12/kg body weight after adjustment for purity (5666 

mg/kg of test substance at 35.3% purity). The study was conducted following OECD 

guideline 423 and used a total of 10 mice (5 male and 5 female) (OECD, 2001). There were 

no treatment-related gross pathological observations. Therefore, the acute oral LD50 and no 

observed effect level (NOEL) of AAD-12 in mice was greater than 2000 mg/kg based on fact 

that no mortality was observed and there were no observable effects (adverse or non-adverse 

effects) with the AAD-12-treated animals. AAD-12 protein displays very low acute toxicity 

potential.

5.1.5.4. Human Dietary Risk Assessment for AAD-12

For the human diet, only the cottonseed of cotton plants is useful for food. Cottonseed is not 

directly consumed by humans because the majority of commercial cotton varieties contain 

the anti-nutrients gossypol and cyclopropenoid fatty acids. For the human diet, cottonseed is 

used in food applications in which the seeds are mainly used to obtain refined edible oil, 
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which contains undetectable amounts of protein (Reeves III and Weihrauch, 1979; OECD, 

2009); therefore, oil produced from DAS-8191Ø-7 will contain extremely low levels to no 

detectable levels of AAD-12 protein.

This assessment assumes that 100% of consumed cotton products are derived from 

DAS-8191Ø-7 cottonseed. This is a highly conservative estimate for exposure to the AAD12 protein from DAS-8191Ø-7 cotton. The actual dietary exposure of the protein from 

DAS-8191Ø-7 cotton will be lower because: 1) there may be protein degradation during 

transport and storage; 2) cottonseed containing AAD-12 will be mixed with non-AAD-12

cottonseed; 3) human consumption of cotton products is primarily in food forms which are 

cooked and heat is known to denature the AAD-12 protein and 4) the majority of consumer 

dietary exposure to cotton is via edible cottonseed oil, which according to USDA National 

Nutrient Database for Standard Reference, Release 25 (USDA ARS, 2012) does not contain 

any protein. Therefore, no human dietary exposure to the AAD-12 protein is expected from 

consumption of oils derived from DAS-8191Ø-7 cotton based on USDA Nutrient Database. 

Potential dietary risk to humans and livestock from the consumption of AAD-12 protein in food 

and feed derived from DAS-8191Ø-7 cotton was evaluated by determining margins of exposure 

(MOE). MOE is the ratio of the No Observable Effect Level (NOEL) of AAD-12 determined by 

a mouse acute oral toxicity study (2000 mg/kg body weight) to estimated dietary intake of AAD12. To evaluate dietary risk for human food consumption, MOEs were determined for the U.S. 

population based on mean protein expression levels in cottonseed. For a U.S. assessment, the 

MOE values were calculated using the DEEM dietary exposure model program, DEEMFCID version 3.15 (EPA, 2013). The only cotton commodity included in the dietary model in 

DEEM is cottonseed oil. There is no protein in cottonseed oil; therefore, estimated exposure 

to AAD-12 based on this model is 0 mg/kg bw/day.

The U.S. assessment is incongruent with an assessment performed based on the WHO 

maximum global consumption data for acute exposures, which indicated that the U.S. was 

the top global consumer for both cottonseed and cottonseed oil. The 97.5thpercentile 

exposure estimates from WHO for cottonseed were 0.05 g/kg bw/day, indicating very low 

consumption levels. When the WHO “SO 691 Cottonseed” acute consumption information is 

coupled to the protein average field expression level for cottonseed (Table 8), the dietary risk 

assessments revealed that the calculated MOE values for the AAD-12 protein in cottonseed 

for both adults and infants are very large with both great than 2,000,000. For chemicals, 

MOEs greater than 100 are typically considered acceptable. Based on the available safety 

threshold information, all MOEs for AAD-12 protein indicate negligible concern for adverse 

effects from DAS-8191Ø-7 cotton. Accordingly, there is negligible risk to human health 

when DAS-8191Ø-7 AAD-12 is present in the human diet.

5.1.5.5. Livestock Dietary Risk Assessment for AAD-12

Expression levels of the AAD-12 protein in DAS-8191Ø-7 cotton were used with 

conservative (i.e. protective) livestock dietary assumptions for cotton commodities to 

estimate livestock dietary exposure. In addition, the relevance of the exposure estimate is 

placed into context based on the known mammalian toxicity information. A dietary exposure 

assessment reveals large margin of exposure (MOE) values for the AAD-12 protein in 
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DAS-8191Ø-7 cotton, indicating negligible concern for adverse effects from acute dietary 

exposure.

AAD-12 Livestock Dietary Risk Assessment Analysis

The cotton commodity forms that are considered potential animal feeds are derived primarily 

from the cottonseed, which can be fed whole or processed into meal and hulls; cotton gin 

byproducts are also sometimes used as a supplemental cattle feed. Cotton gin byproducts 

(also called cotton gin trash) consist of the plant residues remaining from ginning cotton and 

can contain several parts of the cotton plant including stems, leaves, seed, burr, and lint. Gin 

byproducts are not a high value feed commodity and so are not processed for removal of the 

anti-nutrient, gossypol; as such, they are only a relevant feed commodity for ruminants.

This assessment has assumed that 100% of cotton consumed is derived from DAS-8191Ø-7

cotton. Actual dietary exposure to AAD-12 protein will be lower because: 1) cotton is a 

blended commodity; cotton-derived food and feed will contain cotton from a mixture of 

sources, 2) degradation of the protein will occur during transport and storage, and 3) heat 

applied during preparation of cotton derived foods and feeds may lessen exposure to AAD12, as AAD-12 is functionally unstable when heated (Section 5.1.5.2).

A U.S. livestock assessment was conducted based on the Maximum Reasonably Balanced 

Diet (MRBD) animal burden procedures (EPA, 2008). This U.S. assessment includes several 

cotton commodity forms as potential animal feeds: seed, meal, hulls and gin byproducts. The

U.S. MRBD guidance is used to construct a maximum cotton feed contribution for swine, 

poultry and cattle. No guidance on sheep diets is offered in the MRBD guidance, so all 

possible commodities were included as a conservative approach. 

The highest estimated exposed U.S. animal is the sheep with 0.27 mg AAD-12/kg-bw. Lower 

estimates for beef cattle, dairy cattle, swine and poultry were <0.23 mg AAD-12/kg-bw. 

When these values are compared to the acute mammalian NOAEL of >2000 mg/kg-bw (see 

section 5.1.5.3), there is a high margin of safety for livestock because the MOEs are large 

(>7200). This dietary exposure assessment for AAD-12 in DAS-8191Ø-7 cotton supports the 

conclusion that there is negligible risk to animal health when AAD-12 is present in their 

diets.

5.1.6. Summary of AAD-12 Protein Characterization

Detailed biochemical characterization of the AAD-12 protein derived from DAS-8191Ø-7 

cotton was conducted. SDS-PAGE, western blot, glycoprotein detection, enzymatic assay, 

and protein sequence analysis by MALDI-TOF MS/MS were used to characterize the 

biochemical properties of the proteins. Using these methods, the AAD-12 protein isolated 

from P. fluorescens and DAS-8191Ø-7 cotton were shown to be biochemically and 

biologically equivalent.

A step-wise, weight-of-evidence approach was used to assess the potential for toxic or 

allergenic effects from the AAD-12 protein. Bioinformatic analyses revealed no meaningful 

homologies to known or putative allergens or toxins for the AAD-12 amino acid sequence. 

AAD-12 protein is rapidly degraded in simulated gastric fluid and is readily denatured by 
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heat. There is no evidence of acute toxicity of the AAD-12 protein in mice at a dose of 2000 

mg/kg body weight of AAD-12. These results indicate that AAD-12 is unlikely to cause 

allergenic or adverse effects in humans or animals.

AAD-12 protein expression analysis was conducted using DAS-8191Ø-7 cotton tissue 

samples collected over the growing season. The low level expression of the AAD-12 protein

in the various assayed tissue samples in DAS-8191Ø-7 cotton presents a low exposure risk to 

humans and animals. The overall safety assessment of the AAD-12 protein supports the 

conclusion that DAS-8191Ø-7 cotton is as safe as non-transgenic cotton currently on the 

market and unlikely to cause allergenic or toxic effects to humans or animals.

5.2. PAT

5.2.1. Identity of the PAT Protein

The expressed DAS-8191Ø-7 cotton PAT protein is comprised of 183 amino acids and has a 

molecular weight of ~20 kDa (Figure 39). The PAT protein was derived from Streptomyces 

viridochromogenes, a gram-positive soil bacterium (Strauch et al., 1988; OECD, 1999). The 

pat transgene in DAS-8191Ø-7 encodes a protein sequence that is identical to the native S.

viridochromogenes PAT protein (UniProt Accession Number: Q57146) and is identical to 

that produced in DAS-68416-4 soybean and DAS-444Ø6-6 soybean (currently under USDAAPHIS review, petition numbers 09-349-01p, 11-234-01p respectively).

001 MSPERRPVEIRPATAADMAAVCDIVNHYIETSTVNFRTEPQTPQEWIDDL

051 ERLQDRYPWLVAEVEGVVAGIAYAGPWKARNAYDWTVESTVYVSHRHQRL

101 GLGSTLYTHLLKSMEAQGFKSVVAVIGLPNDPSVRLHEALGYTARGTLRA

151 AGYKHGGWHDVGFWQRDFELPAPPRPVRPVTQI

Figure 39. Amino Acid Sequence of PAT

5.2.2. Mode of Action of the PAT Protein

The L-isomer of phosphinothricin (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). PAT acetylates 

the free NH2 (amine) group of PPT (in the presence of acetyl coenzyme A). The acetylated 

NH2 does not bind GS, thus preventing autotoxicity (Figure 40, (Duke, 1996)).
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Figure 40. Mode of Action of the PAT Protein

5.2.3. Biochemical Characterization of the PAT Protein

Large quantities of purified PAT protein are required to perform safety assessment studies. 

Because it is technically infeasible to extract and purify sufficient amounts of recombinant 

protein from transgenic plants (Evans, 2004; Raybould et al., 2012), the PAT protein was 

heterologously-expressed in Pseudomonas fluorescens. Characterization studies were 

performed to confirm the equivalency of the PAT protein produced in P. fluorescens with the 

PAT protein produced in planta in DAS-8191Ø-7 cotton. Sodium dodecyl sulfate 

polyacrylamide gel electrophoresis (SDS-PAGE), western blot, glycoprotein detection, 

enzymatic assay, and protein sequence analysis by matrix assisted laser desorption /

ionization time-of-flight mass spectrometry/mass spectrometry (MALDI-TOF MS/MS) were 

used to characterize the biochemical properties of the proteins. Using these methods, the 

PAT protein isolated from P. fluorescens and DAS-8191Ø-7 cotton were shown to be 

biochemically and biologically equivalent, thereby supporting the use of the microbe-derived

protein in safety assessment studies.

The methods and results of the biochemical characterization of the DAS-8191Ø-7 cottonand microbe-derived PAT proteins are described in detail in Appendix 3. Both the DAS8191Ø-7 cotton and P. fluorescens-derived PAT proteins were observed at the expected 

molecular weight of ~20 kDa by SDS-PAGE and were immunoreactive to PAT proteinspecific antibodies by western blot analysis. There was no evidence of glycosylation of either 

the DAS-8191Ø-7 cotton or P. fluorescens-derived PAT proteins. Cotton and P. fluorescensderived PAT were equally active using glufosinate as a substrate and displayed similar 

kinetic parameters, indicating that the proteins are enzymatically equivalent. In addition, 

greater than 90% of the cotton-derived protein amino acid sequence was confirmed by 

enzymatic peptide mass fingerprinting and MS/MS sequence analysis by MALDI-TOF 

MS/MS. The N-terminal methionine was found to be cleaved from both protein sources

which is a common modification that has been found to occur on many proteins (Li and 

Chang, 1995). The C-terminal peptides from DAS-8191Ø-7 cotton and P. fluorescens were 

intact and empirically determined to be identical.

5.2.4. Expression of the PAT Protein in Plant Tissues

A field expression study was conducted in the U.S. during 2012. Six sites (Alabama, 

Georgia, Louisiana, Missouri, North Carolina, and Texas) were planted with DAS-8191Ø-7

cotton and the non-transgenic control. The test sites represented regions of diverse agronomic 

practices and environmental conditions for cotton in North America. Two treatments of the 
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DAS-8191Ø-7 cotton (non-sprayed or sprayed with 2,4-D and glufosinate) were tested with 

PAT protein levels being determined in nine tissue types including leaf, squares, bolls, 

pollen, flower, whole plant, root, and seed (Table 9). 

Table 9. DAS-8191Ø-7 Cotton PAT Expression Analysis Tissue Samples

Expression

Tissue

Growth

Stage1

Sample

Size

No. of Samples per 

Entry/Site

Bolls Peak Bloom 10-14 Bolls 4

Flower Peak Bloom 14-18 Flowers 4

Leaf 4-Leaf 10-14 Leaves 4

Leaf 1

st White Bloom 10-14 Leaves 4

Leaf 1

st Open Boll 10-14 Leaves 4

Pollen Early Bloom 0.2-0.5 mL 4

Root Maturity 1-2 Plants 4

Seed Maturity 175-250 grams 4

Squares 1

st White Bloom 10-14 Squares 4

Whole Plant Maturity 1-2 Plants 4

1 Approximate growth stage

PAT protein was extracted from DAS-8191Ø-7 cotton tissue and the soluble, extractable PAT 

protein was measured using an enzyme-linked immunosorbent assay (ELISA) method. PAT

protein levels for all tissue types were calculated on ng/mg dry weight basis. The details of 

the materials and methods are described in Appendix 4.

Table 10. Summary of PAT Protein Expression in DAS-8191Ø-7 Cotton

Expression 

Tissue

Growth 

Stage

PAT ng/mg Tissue Dry Weight1,2

Overall 

Mean

Std. Dev3

(n = 48) Min/Max Range4

Bolls Peak Bloom 3.16 1.11 1.62-6.27

Flower Peak Bloom 5.3 1.09 2.92-8.20

Leaf 4-Leaf 13.29 4.76 1.11-20.06

Leaf 1

st White Bloom 8.18 2.57 1.64-14.34

Leaf 1

st Open Boll 9.14 3.92 4.11-18.56

Pollen Early Bloom 0.11 0.22 NDe-0.99

Root Maturity 1.63 0.7 NDe-3.11

Seed Maturity 3.85 0.79 2.37-5.71

Squares 1

st White Bloom 7.91 2.39 4.38-14.48

Whole Plant Maturity 0.97 1.02 ND5-3.97

1Calculated from PAT expression raw data.

2 Table represents overall results for non-sprayed and sprayed cotton tissue across all sites.

3

Standard deviation of individual cotton samples across all sites.

4 Represents the min and max for individual cotton samples across all sites.

5 ND = Not Detected, expression level below LOD (Limit of Detection). A zero value was used for ND results for mean calculation.

A summary of the PAT protein concentrations (averaged across sites) in the various cotton

matrices is shown in Table 10. The mean PAT protein levels were highest in the leaf at 13.29 

ng/mg, followed by squares at 7.91 ng/mg, flower at 5.30 ng/mg, seed at 3.85 ng/mg, bolls at 
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3.16 ng/mg, root at 1.67 ng/mg, whole plant at 0.97 ng/mg and pollen at 0.11 ng/mg. PAT 

expression levels were comparable for both sprayed and non-sprayed treatments. No PAT 

protein was detected in the control tissue above the limit of quantitation (0.06 ng/mg) with 

the exception of a single seed sample. This was most likely attributed to sampling error 

and/or contamination.

5.2.5. Food and Feed Safety Assessment of PAT 

Safety evaluation of the PAT protein was conducted to assess any potential adverse effects to 

humans or animals resulting from the environmental release of crops containing the PAT 

protein (Codex Alimentarius Commission, 2009). The conclusion from the assessment is that 

the PAT protein is unlikely to cause allergic reactions in humans or to be toxic to humans or 

animals.

The safety of phosphinothricin acetyltransferase (PAT) in biotech crops has been extensively 

studied and environmental release of biotech crops containing PAT have been issued by 

regulatory agencies in eleven different countries involving over thirty eight events in eight 

plant species including cotton, maize and soybean, among other crops (CERA - ILSI 

Research Foundation, 2011). Numerous transgenic crops expressing PAT have been 

reviewed by USDA and FDA with no concerns identified (USDA, 1996; FDA, 1998, 2001; 

USDA, 2001; FDA, 2004a, 2004b, 2004c; USDA, 2004, 2005)

Amino Acid Sequence Comparison to Known Allergens

The PAT protein has no biologically meaningful sequence similarities to known allergens 

using a sequence evaluation program based on that formulated by the joint FAO/WHO 

Expert Consultation (2001) and by the Codex Alimentarius (Codex Alimentarius 

Commission, 2001). This search looks for a match of at least eight contiguous amino acids or 

greater than 35% identity over 80 or longer amino-acid stretches using an allergen database 

(FARRP, 2013) and no such matches were found.

Amino Acid Sequence Comparison to Known Toxins

The PAT protein does not share any amino acid sequence similarities with known protein 

toxins that would present any safety concerns. Amino acid sequence similarities with known 

proteins were evaluated using BLASTp search algorithm against the GenBank non-redundant 

protein sequences (updated February 23, 2012). By their annotations, the majority proteins 

returned by BLASTp with statistically significant alignments are phosphinothricin 

acetyltransferase, other acetyltransferases, and hypothetical proteins without assigned 

function. Furthermore, most of the non-significant alignments are related with 

phosphinothricin N-acetyltransferase or GCN5 related N-acetyltransferase. None of these 

proteins are associated with known protein toxins that are harmful to humans or animals.

Lability in Simulated Gastric Fluid and Heat Lability

The PAT protein is rapidly degraded in simulated gastric fluid (EPA, 1997; OECD, 1999; 

Herouet et al., 2005) and is readily denatured by heat (EPA, 1997; OECD, 1999). Numerous 

glufosinate tolerant products including those in canola, soybean, corn and cotton have been 

reviewed by USDA with no concerns identified.
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There is no evidence available indicating that the PAT protein is toxic to either humans or 

animals. In acute toxicity studies mice gavaged with high levels of PAT protein showed no 

treatment-related significant toxic effects (EPA, 1997; OECD, 1999). The U.S. 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 introduction (EPA, 1997). U.S. 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, which would include DAS8191Ø-7 cotton (40 CFR §174.522).

5.2.6. Summary of PAT Protein Characterization

Detailed biochemical characterization of the PAT protein derived from DAS-8191Ø-7 cotton 

was conducted. SDS-PAGE, western blot, glycoprotein detection, enzymatic assay, and 

protein sequence analysis by MALDI-TOF MS/MS were used to characterize the 

biochemical properties of the proteins. Using these methods, the PAT protein isolated from 

P. fluorescens and DAS-8191Ø-7 cotton were shown to be biochemically and biologically 

equivalent with no glycosylation.

A step-wise, weight-of-evidence approach was used to assess the potential for toxic or 

allergenic effects from the PAT protein. Bioinformatic analyses revealed no meaningful 

homologies to known or putative allergens or toxins for the PAT amino acid sequence. PAT 

protein is rapidly degraded in simulated gastric fluid and is readily denatured by heat. There 

is no evidence of acute toxicity of the PAT protein in mice. 

PAT protein expression analysis was conducted using DAS-8191Ø-7 cotton tissue samples 

collected over the growing season. The low level expression of the PAT protein in the 

various assayed tissue samples in DAS-8191Ø-7 cotton 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 adverse effects in humans or animals.

The overall safety assessment of PAT supports the conclusion that food and feed products 

containing DAS-8191Ø-7 cotton or derived from DAS-8191Ø-7 cotton are as safe as nontransgenic cotton currently on the market for human and animal consumption.
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6. Composition Assessment of DAS-8191Ø-7 Cotton

6.1. DAS-8191Ø-7 Cottonseed Composition

Composition assessment of DAS-8191Ø-7 cotton was conducted to examine key nutrient and 

anti-nutrient levels. In one experiment, non-sprayed DAS-8191Ø-7 cottonseed were 

compared with the appropriate non-transgenic near isogenic isoline (control) (Figure 4) and 

non-transgenic reference lines. In another experiment, non-sprayed DAS-8191Ø-7 cottonseed 

were compared with herbicide-treated DAS-8191Ø-7 cottonseed and non-sprayed DAS8191Ø-7 cottonseed.

The assessment was conducted using the principles and analytes in the OECD consensus 

document for cotton composition (OECD, 2009). Samples were analyzed for proximates, 

fiber, minerals, amino acids, fatty acids, vitamins, and anti-nutrients. Levels of the analytes 

in DAS-8191Ø-7 cottonseed were compared with: 1) corresponding levels in cottonseed 

from a non-transgenic, near isogenic control (Figure 4), grown concurrently, under the same 

field conditions; 2) natural ranges generated from an evaluation of six commercial nontransgenic cotton reference lines (ALL-TEX 1203, ALL-TEX A102, ALL-TEX LA122, 

Paymaster HS 200, Raider 271, and Speed) grown concurrently, under the same field 

conditions; and 3) data published in the scientific literature for non-transgenic cotton. 

Comparison with data published in the literature places any potential differences between the 

assessed crop and its comparators in the context of the documented variations in the 

concentrations of crop nutrients and anti-nutrients. 

Of the 73 cotton analytes assayed, 59 analytes had sufficient quantitative data for inclusion in 

the combined site statistical analysis. Although a limited number of statistically significant 

differences occurred between DAS-8191Ø-7 cotton and the control, these differences were 

not biologically relevant because the results were within ranges found for non-transgenic 

cotton reference lines included in this study and/or within available literature ranges for nontransgenic cotton. Based on these results, it is concluded that DAS-8191Ø-7 cotton is 

compositionally equivalent to non-transgenic cotton.

6.1.1. Field Study Design

Field trials were conducted at eight U.S. sites (one site each in Tallassee, Alabama;

Sycamore, Georgia; Washington, Louisiana; Stoneville, Mississippi; Fisk, Missouri; Mebane,

North Carolina; Groom, Texas; and East Bernard, Texas) in 2012 to produce cottonseed 

samples of DAS-8191Ø-7 cotton (non-sprayed and sprayed with 2,4-D plus glufosinate), 

near isogenic non-transgenic isoline (control) and non-transgenic cotton varieties, referred to 

as reference lines, for nutrient composition analysis. For the reference lines, six unique 

reference lines were included across all sites of the field production with three varieties per 

site to provide data on natural variability of each composition analyte analyzed. 

Because non-transgenic cotton plants (e.g. isoline and reference lines) are sensitive to 2,4-D, 

two sub-experiments were conducted at each site to spatially separate the non-transgenic 

entries from the entries where 2,4-D was applied. Sub-experiment one was not sprayed and 

contained the isoline, reference varieties and one entry of DAS-8191Ø-7 cotton. Subexperiment two contained two entries of only DAS-8191Ø-7 cotton; one not sprayed and one 

sprayed with 2,4-D plus glufosinate. 
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The entries were arranged in a randomized complete block design within each subexperiment. The sub-experiments were separated by 100 ft (30 m) to prevent isoline and 

reference entries in sub-experiment one from potential injury by 2,4-D drift from applications 

in sub-experiment two. Both sub-experiment one and two were present at all field testing 

sites. Randomization of entries within blocks was unique at each field testing site. Test, 

isoline, and reference variety cotton was planted at a seeding rate of approximately 100 seeds 

per 25 ft (7.62 m) of row (one seed per 3 in or 7.6 cm). Four replicate plots of each entry

were established at each site for each sub-experiment, with each plot consisting of four rows 

that were 25 ft (7.62 m) long with a row spacing of approximately 30 in (76 cm). Each four 

row plot was bordered by two rows of a non-transgenic cotton cultivar.

Herbicides were applied in a spray volume of approximately 20 gallons per acre (187 L/ha), 

and all herbicide applications included 2% v/v ammonium sulfate. In sub-experiment two, 

2,4-D (GF-2654) and glufosinate (Ignite 280 SL) were applied in a tank mixture as two 

broadcast applications to one entry of DAS-8191Ø-7 cotton. Application timings were at the 

3 node and 6 node growth stages. The target application rates for both application timings 

were 1120 g ae/ha 2,4-D and 596 g ai/ha glufosinate. Actual application rates ranged from 

1108 – 1142 g ae/ha 2,4-D and 589 – 622 g ai/ha glufosinate for the 3 node application and 

1061 – 1141 g ae/ha 2,4-D and 564 – 621 g ai/ha glufosinate for the 6 node application. 

6.1.2. Compositional Analysis

Samples of acid delinted cottonseed were analyzed at Covance Laboratories Inc. for 73 

cotton analytes (Table 11). Methods for compositional analysis are described in Appendix 5. 

Data from the composition analysis was statistically analyzed for each sub-experiment 

comparing 1) non-sprayed DAS-8191Ø-7 cotton in sub-experiment one to the appropriate 

non-sprayed non-transgenic near isogenic isoline and non-transgenic reference lines in subexperiment one and 2) non-sprayed DAS-8191Ø-7 cotton in sub-experiment one to the nonsprayed DAS-8191Ø-7 cotton and to herbicide-treated DAS-8191Ø-7 cotton in subexperiment two. The results were then placed into context with reported scientific literature 

values.

6.2. Statistical Analysis

Analysis of variance was conducted for each sub-experiment separately across field testing 

sites for composition data using a mixed model (SAS Institute Inc., 2009). Entry was 

considered a fixed effect; location, block within location, and location-by-entry were 

designated as random effects. Paired contrasts were conducted using t-tests, and the 

significance of overall treatment effects was estimated using an F-test. Significant differences 

were declared at the 95% confidence level (α = 0.05). 

Of the 73 analytes evaluated in this study, 59 produced sufficient data (>50% of data points 

above the limited of quantitation (LOQ)) for inclusion in the combined site statistical 

analysis. Therefore, 59 comparisons were made in each sub-experiment. Inclusion of analytes 

in the statistical analysis where a predominance of the data are less than the LOQ violates the 

assumptions of ANOVA due to non-normal data distributions and artificially reduced 

variance estimates.
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In the 59 comparisons in the statistical analysis, the probability of declaring one or more false 

differences based on unadjusted P-values was very high due to a multiplicity effect in each 

sub-experiment. Multiplicity occurs when a large number of comparisons are made in a 

single study to look for unexpected effects. As a result, the probability of falsely declaring 

differences based on comparison-wise P-values is very high at 95.151% (1-0.9559) (1-

0.95number of comparisons).

Table 11. Cottonseed Composition Analytes

Proximates and Fiber (9)

Protein Moisture Neutral Detergent Fiber (NDF)

Fat Carbohydrates Total Dietary Fiber

Ash Acid Detergent Fiber (ADF) Crude Fiber

Minerals (12)

Calcium Manganese2Selenium

Copper Molybdenum Sodium

Iron Phosphorus Sulfur

Magnesium Potassium Zinc

Amino Acids (18)

Alanine Histidine Proline

Arginine Isoleucine Serine

Aspartic Acid Leucine Threonine

Cystine Lysine Tryptophan

Glutamic Acid Methionine Tyrosine

Glycine Phenylalanine Valine

Fatty Acids (22)

8:0 Caprylic116:1 Palmitoleic220:0 Arachidic

10:0 Capric117:0 Heptadecanoic120:1 Eicosenoic1

12:0 Lauric117:1 Heptadecenoic120:2 Eicosadienoic1

14:0 Myristic218:0 Stearic 20:3 Eicosatrienoic1

14:1 Myristoleic118:1 Oleic220:4 Arachidonic1

15:0 Pentadecanoic118:2 Linoleic222:0 Behenic

15:1 Pentadecenoic118:3 Linolenic

16:0 Palmitic 18:3 γ-Linolenic1

Vitamins (7)

Vitamin A (Beta Carotene) 1 Vitamin B3 (Niacin) α-tocopherol (Vitamin E)

Vitamin B1 (Thiamine HCl) Vitamin B6 (Pyridoxine HCl)

Vitamin B2 (Riboflavin) Vitamin B9 (Folic Acid)

Anti-Nutrients (5)

Dihydrosterculic Acid Sterculic Acid Total Gossypol2

Malvalic Acid2Free Gossypol

1

Analytes excluded from combined site and analysis due to more than 50% of samples < LOQ

2

Analytes with statistically significant adjusted FDR differences in the combined site analysis.

One method that has been used to handle multiplicity is to adjust P-values to control the 

experiment-wise error rate. However, the power for detecting specific effects can be reduced 

significantly when many comparisons are made. An alternative approach 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), which is a commonly used 

approach 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).
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Therefore, the P-values from the composition analysis were each adjusted using the FDR 

method to improve discrimination of true differences among treatments from random effects 

(false positives). In this study differences were considered significant if the FDR-adjusted Pvalue was less than 0.05.

6.3. Composition Analysis Results

Combined summary and statistical analysis of composition data from the non-transgenic,

near isogenic isoline (control) and DAS-8191Ø-7 cotton is found in Table 12 through Table

17 and Figure 41 through Figure 46 for both sub-experiment one and two. For each analyte 

and entry, the least-square mean, standard error, and minimum and maximum sample values

are reported. Also, for comparison, the minimum and maximum values for the reference lines 

and literature ranges are reported. Arithmetic means from each field site are plotted in figures 

and literature ranges are shaded. Literature ranges reported as not detectable or <LOD are 

plotted as zeros.

The following sections discuss results first for sub-experiment one (non-sprayed DAS8191Ø-7, isoline, and reference lines), followed by a discussion of results for sub-experiment 

two (sprayed and non-sprayed DAS-8191Ø-7). Sub-experiment one was used to evaluate if 

the composition of non-sprayed DAS-8191Ø-7 cotton is equivalent to reference and isoline

lines, and sub-experiment two was used to evaluate if applying 2,4-D plus glufosinate 

impacts the composition of DAS-8191Ø-7 cotton.

6.3.1. Sub-experiment One - Proximate and Fiber Analysis of Seed 

Results from the combined site analysis of the proximate and fiber composition for the nontransgenic isoline (control) and DAS-8191Ø-7 cottonseed are provided in Table 12 and 

Figure 41. Nine analytes were analyzed including ash, carbohydrates, crude fat, protein, 

moisture, acid detergent fiber (ADF), crude fiber, neutral detergent fiber (NDF) and dietary 

fiber. No significant FDR-adjusted P-values were observed for DAS-8191Ø-7 non-sprayed 

for all nine analytes. In addition, mean results for all nine DAS-8191Ø-7 non-sprayed 

proximate and fiber analytes fell within the reference variety ranges and literature ranges. 

Statistical analyses found no differences between the levels of nutrient components in 

cottonseed from DAS-8191Ø-7 and the non-transgenic isoline, supporting the findings of 

composition equivalence of DAS-8191Ø-7 to non-transgenic cotton. 
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Table 12. Combined Site Analysis Results for Proximates & Fiber in Isoline, DAS-8191Ø-7 and 

Reference Variety Cottonseed and Literature Ranges

Analytical 

Component

(Units)

Isoline 

(control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7

Sprayed1

Literature 

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

Ash

(% Dry Weight)

4.29 ± 0.11

3.67 - 5.14

4.17 ± 0.11

3.62 - 4.76

(0.047, 0.185)

3.53 - 5.21 4.31 ± 0.13

3.69 - 5.08

4.28 ± 0.13

3.79 - 5.02

(0.411, 0.945)

3.7 - 5.342

Carbohydrates

(% Dry Weight)

47.5 ± 0.9

44.5 - 53.6

49.1 ± 0.9

44.7 - 57.9

(0.007, 0.050)

42.3 - 54.2 50.2 ± 1.1

46.9 - 56.6

50.5 ± 1.1

45.7 - 58.6

(0.485, 0.945)

39.0 - 53.62

Crude Fat

(% Dry Weight)

21.7 ± 0.7

18.3 - 25.2

21.3 ± 0.7

15.6 - 26.2

(0.368, 0.642)

15.8 - 27.9 20.9 ± 0.9

17.3 - 25.9

20.5 ± 0.9

15.1 - 26.6

(0.319, 0.945)

14.4 - 27.292

Protein

(% Dry Weight)

26.4 ± 0.9

22.3 - 31.5

25.4 ± 0.9

22.2 - 31.1

(0.008, 0.055)

21.5 - 32.3 24.6 ± 1.0

19.2 - 29.7

24.7 ± 1.0

19.2 - 29.7

(0.669, 0.945)

12 - 32.97

Moisture

(% Fresh Weight)

8.2 ± 0.2

6.64 - 9.74

8.0 ± 0.2

6.56 - 9.39

(0.663, 0.815)

6.37 - 10.2 8.2 ± 0.2

6.95 - 9.20

8.2 ± 0.2

6.92 - 9.13

(0.985, 0.998)

2.25 - 15.9

ADF3

(% Dry Weight)

25.9 ± 0.5

23.0 - 28.6

25.3 ± 0.5

21.3 - 28.6

(0.381, 0.642)

20.4 - 29.4 26.0 ± 0.5

22.1 - 29.8

25.7 ± 0.5

22.4 - 30.0

(0.405, 0.945)

19.74 - 40.5

Crude Fiber

(% Dry Weight)

18.1 ± 0.3

16.0 - 21.3

17.9 ± 0.3

14.8 - 22.7

(0.677, 0.815)

15.1 - 23.5 18.6 ± 0.3

13.7 - 23.6

17.8 ± 0.3

15.6 - 19.8

(0.060, 0.945)

13.45 - 23.10

NDF4

(% Dry Weight)

34.0 ± 0.6

30.7 - 40.3

33.8 ± 0.6

28.3 - 39.7

(0.667, 0.815)

27.2 - 38.2 35.2 ± 0.6

30.6 - 39.3

34.8 ± 0.6

31.3 - 39.3

(0.557, 0.945)

25.56 - 53.6

Dietary Fiber

(% Dry Weight)

44.8 ± 1.0

40.3 - 53.5

45.7 ± 1.0

40.7 - 56.5

(0.283, 0.591)

37.6 - 51.3 46.6 ± 1.1

40.0 - 54.0

47.2 ± 1.1

41.7 - 54.1

(0.309, 0.945)

33.69 - 47.55

1 Sprayed with 2,4-D and glufosinate 

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values

3 Acid detergent fiber

4 Neutral detergent fiber
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Figure 41. Proximates & Fiber in Isoline, DAS-8191Ø-7 and Reference Variety Cottonseed

Y axis: Moisture = % fresh weight, all others = % dry weight. Reference Variety cottonseed: All-TEX 1203, ALLTEX A102, ALL-TEX LA122, Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 

unsprayed in sub-experiment 1, 191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed 

= DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open 

circle = AL,  = GA, + = LA, open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, 

filled triangle = TX2. The shaded band represents the literature range.
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Figure 41. Proximates & Fiber in Isoline, DAS-8191Ø-7 and Reference Variety Cottonseed

(continued)

Y axis: Moisture = % fresh weight, all others = % dry weight. Reference Variety cottonseed: All-TEX 1203, ALLTEX A102, ALL-TEX LA122, Paymaster HS 200, Raider 271 and Speed.191Ø Unsprayed 1 = DAS-8191Ø-7 

unsprayed in sub-experiment 1, 191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed 

= DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open 

circle = AL,  = GA, + = LA, open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, 

filled triangle = TX2. The shaded band represents the literature range.
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6.3.2. Sub-experiment One - Mineral Analysis of Seed

Results from the combined site analysis of the mineral composition for the non-transgenic 

isoline (control) and DAS-8191Ø-7 cottonseed are provided in Table 13 and Figure 42. 

Twelve analytes were measured including calcium, copper, iron, magnesium, manganese, 

molybdenum, phosphorus, potassium, selenium, sodium, sulfur and zinc. Mean results for all 

minerals tested in DAS-8191Ø-7 non-sprayed fell within the reference variety ranges and 

literature ranges where available (no values were reported in the literature for selenium and 

sulfur). 

No significant FDR-adjusted P-values were observed for DAS-8191Ø-7 non-sprayed except 

for manganese. Manganese showed no significant differences for FDR-adjusted P-values 

among the eight individual field sites. Significant FDR-adjusted P-values were only 

identified in the combined site analysis. The relative magnitude of the difference for 

manganese between the mean values for DAS-8191Ø-7 non-sprayed and the non-transgenic 

isoline for the combined site analysis was a decrease of 15.0%, which was less than the 

variability observed for the control sample for manganese (range 1.17 – 2.36, a relative 

difference of 101.7%). The observed differences in manganese between DAS-8191Ø-7 and 

the non-transgenic isoline were not considered to be meaningful from a food, nutritional or 

safety perspective because they were small, less than the variability seen in the isoline, 

statistically insignificant when compared at each individual field site and the mean DAS891Ø-7 value for manganese was within both reference variety and literature ranges.

For the remaining mineral analytes, mean results for DAS-8191Ø-7 non-sprayed fell within 

the reference variety ranges and literature ranges. Statistical analyses found no differences 

between the levels of nutrient components in cottonseed from DAS-8191Ø-7 and the nontransgenic isoline, supporting the findings of composition equivalence of DAS-8191Ø-7 to 

non-transgenic cotton. 
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Table 13. Combined Site Analysis Results for Minerals in Isoline, DAS-8191Ø-7 and Reference 

Variety Cottonseed and Literature Ranges

Analytical 

Component

(mg/100 g Dry 

Weight)

Isoline 

(Control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature 

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

Calcium 124 ± 7

88.1 - 164

133 ± 7

98.0 - 172

(0.011, 0.059)

78.9 - 204 136 ± 7

113 - 178

136 ± 7

114 - 178

(0.960, 0.998)

100 - 330

Copper 0.90 ± 0.07

0.525 - 1.37

0.86 ± 0.07

0.532 - 1.30

(0.021, 0.096)

0.466 - 1.44 0.86 ± 0.08

0.446 - 1.32

0.86 ± 0.08

0.426 - 1.41

(0.944, 0.998)

0.313 - 2.457

Iron 4.20 ± 0.24

3.24 - 6.26

4.25 ± 0.24

3.45 - 5.76

(0.546, 0.747)

3.43 - 6.45 4.25 ± 0.21

3.28 - 5.71

4.30 ± 0.21

3.43 - 5.57

(0.353, 0.945)

3.671 - 31.838

Magnesium 387 ± 18

291 - 487

384 ± 18

282 - 488

(0.420, 0.679)

285 - 470 391 ± 19

250 - 494

388 ± 19

283 - 498

(0.278, 0.945)

340 - 493.12

Manganese 1.59 ± 0.10

1.17 - 2.36

1.35 ± 0.10

0.968 - 2.20

(0.002, 0.021)

0.983 - 2.28 1.31 ± 0.08

0.985 - 1.93

1.31 ± 0.08

1.01 - 2.04

(0.789, 0.990)

1.069 - 2.216

Molybdenum 0.039 ± 0.012

0.00412 -

0.107

0.036 ± 0.012

0.00427 - 0.106

(0.460, 0.702)

0.00326 -

0.122

0.036 ± 0.011

0.00557 - 0.0980

0.034 ± 0.011

0.00539 - 0.0999

(0.331, 0.945)

NR

Phosphorus 652 ± 42

491 - 924

633 ± 42

469 - 921

(0.079, 0.259)

460 - 901 655 ± 45

412 - 936

649 ± 45

472 - 933

(0.378, 0.945)

482.54 - 991.57

Potassium 1078 ± 23

958 - 1230

1055 ± 23

937 - 1190

(0.106, 0.331)

938 - 1290 1074 ± 23

939 - 1200

1070 ± 23

934 - 1190

(0.613, 0.945)

960 - 1448.35

Selenium

(ppb Dry Weight)

110 ± 30

<LOQ - 382

122 ± 30

<LOQ - 378

(0.379, 0.642)

<LOQ - 676 194 ± 95

<LOQ - 2010

180 ± 95

<LOQ - 1070

(0.670, 0.945)

NR

Sodium 123 ± 8

77.5 - 178

111 ± 8

64.7 - 188

(0.249, 0.577)

73.9 - 192 128 ± 9

78.7 - 207

136 ± 9

75.3 - 192

(0.187, 0.945)

5.4 - 740

Sulfur 495 ± 67

305 - 952

535 ± 67

311 - 1850

(0.541, 0.747)

331 - 847 520 ± 74

279 - 1430

499 ± 74

303 - 1180

(0.734, 0.945)

NR

Zinc 3.36 ± 0.13

2.63 - 3.93

3.47 ± 0.13

2.47 - 5.29

(0.290, 0.591)

2.77 - 4.26 3.51 ± 0.21

2.33 - 5.53

3.48 ± 0.21

2.23 - 5.94

(0.821, 0.998)

2.70 - 5.95

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
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Figure 42. Minerals in Isoline, DAS-8191Ø-7 and Reference Variety CottonseedY axis: 

selenium = ppb dry weight, all others = mg/100 g dry weight. Reference Variety cottonseed: All-TEX 1203, ALLTEX A102, ALL-TEX LA122, Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 

unsprayed in sub-experiment 1, 191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed 

= DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open 

circle = AL,  = GA, + = LA, open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, 

filled triangle = TX2. The shaded band represents the literature range (where available).
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Figure 42. Minerals in Isoline, DAS-8191Ø-7, and Reference Variety Cottonseed (continued)

Y axis: selenium = ppb dry weight, all others = mg/100 g dry weight. Reference Variety cottonseed: All-TEX 1203, 

ALL-TEX A102, ALL-TEX LA122, Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 

unsprayed in sub-experiment 1, 191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed 

= DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open 

circle = AL,  = GA, + = LA, open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, 

filled triangle = TX2. The shaded band represents the literature range (where available).
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6.3.3. Sub-experiment One - Amino Acid Analysis of Seed

Results from the combined site analysis of the amino acid composition for non-transgenic 

isoline (control) and DAS-8191Ø-7 cottonseed are provided in Table 14 and Figure 43.

Eighteen analytes were measured including alanine, arginine, aspartic acid, cystine, glutamic 

acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, 

threonine, tryptophan, tyrosine and valine. Mean results for all eighteen of the amino acids 

tested in DAS-8191Ø-7 non-sprayed fell within the reference variety ranges and/or literature 

ranges. 

For all amino acid analytes measured, no significant FDR-adjusted P-values were observed 

for DAS-8191Ø-7 non-sprayed. In addition, mean results for all eighteen DAS-8191Ø-7 nonsprayed analytes fell within the reference variety ranges and literature ranges. Based on these 

data, statistical analyses found no differences between the levels of nutrient components in 

cottonseed from DAS-8191Ø-7 and the non-transgenic isoline, supporting the findings of 

composition equivalence of DAS-8191Ø-7 cotton to non-transgenic cotton. 
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Table 14. Combined Site Analysis Results for Amino Acids in Isoline, DAS-8191Ø-7 and 

Reference Variety Cottonseed and Literature Ranges

Analytical 

Component

(% Total Amino 

Acids)

Isoline

(Control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

Alanine 4.44 ± 0.03

4.29 - 4.598

4.45 ± 0.03

4.22 - 4.741

(0.705, 0.815)

4.18 - 4.656 4.48 ± 0.05

4.265 - 4.734

4.49 ± 0.05

4.25 - 4.746

(0.549, 0.945)

4.08 - 5.30

Arginine 12.64 ± 0.16

11.87 - 13.73

12.49 ± 0.16

11.50 - 13.67

(0.020, 0.096)

11.57 - 13.67 12.41 ± 0.20

11.27 - 13.47

12.39 ± 0.20

11.31 - 13.77

(0.677, 0.945)

10.83 - 15.18

Aspartic 

Acid 10.09 ± 0.15

9.67 - 11.03

10.25 ± 0.15

9.75 - 12.03

(0.349, 0.642)

9.59 - 11.42 10.32 ± 0.22

9.69 - 11.74

10.56 ± 0.22

9.70 - 12.33

(0.182, 0.945)

9.00 - 12.37

Cystine 1.785 ± 0.040

1.563 - 2.126

1.833 ± 0.040

1.529 - 2.214

(0.145, 0.427)

1.593 - 2.339 1.799 ± 0.029

1.559 - 2.235

1.786 ± 0.029

1.542 - 2.165

(0.701, 0.945)

1.53 - 2.35

Glutamic 

Acid 20.11 ± 0.13

19.17 - 20.73

20.07 ± 0.13

18.58 - 20.72

(0.701, 0.815)

19.44 - 21.23 19.92 ± 0.20

18.91 - 20.69

19.83 ± 0.20

18.33 - 20.65

(0.437, 0.945)

20.24 - 22.90

Glycine 4.42 ± 0.04

4.14 - 4.61

4.41 ± 0.04

4.081 - 4.546

(0.949, 0.969)

4.10 - 4.594 4.45 ± 0.05

4.053 - 4.633

4.44 ± 0.05

4.070 - 4.738

(0.382, 0.945)

4.29 - 5.72

Histidine 2.841 ± 0.016

2.695 - 3.048

2.868 ± 0.016

2.604 - 3.001

(0.267, 0.583)

2.651 - 3.077 2.840 ± 0.019

2.685 - 2.999

2.829 ± 0.019

2.606 - 2.978

(0.652, 0.945)

2.91 - 3.88

Isoleucine 3.62 ± 0.02

3.284 - 3.790

3.61 ± 0.02

3.391 - 3.799

(0.746, 0.847)

3.180 - 3.817 3.63 ± 0.03

3.268 - 3.880

3.62 ± 0.03

3.329 - 3.876

(0.737, 0.945)

3.10 - 4.46

Leucine 6.30 ± 0.02

6.15 - 6.49

6.31 ± 0.02

6.18 - 6.45

(0.702, 0.815)

6.04 - 6.54 6.34 ± 0.04

6.15 - 6.60

6.32 ± 0.04

6.14 - 6.60

(0.153, 0.945)

6.03 - 8.11

Lysine 4.69 ± 0.06

4.36 - 5.09

4.73 ± 0.06

4.44 - 5.107

(0.254, 0.577)

4.27 - 5.03 4.72 ± 0.07

4.40 - 5.209

4.74 ± 0.07

4.39 - 5.149

(0.727, 0.945)

4.62 - 6.60

Methionine 1.640 ± 0.023

1.406 - 1.801

1.625 ± 0.023

1.460 - 1.774

(0.464, 0.702)

1.339 - 1.785 1.651 ± 0.033

1.434 - 1.988

1.645 ± 0.033

1.449 - 1.908

(0.728, 0.945)

1.27 - 2.28

Phenylalanine 5.70 ± 0.06

5.49 - 6.12

5.68 ± 0.06

5.201 - 6.01

(0.504, 0.726)

5.44 - 6.02 5.68 ± 0.06

5.35 - 5.88

5.63 ± 0.06

5.223 - 5.97

(0.137, 0.945)

5.44 - 7.23

Proline 4.04 ± 0.01

3.908 - 4.116

4.04 ± 0.01

3.895 - 4.187

(0.983, 0.983)

3.780 - 4.192 4.04 ± 0.02

3.903 - 4.200

4.05 ± 0.02

3.92 - 4.160

(0.897, 0.998)

3.81 - 5.30

Serine 4.63 ± 0.04

4.352 - 4.88

4.63 ± 0.04

4.32 - 4.875

(0.878, 0.925)

4.26 - 5.05 4.63 ± 0.03

4.354 - 4.915

4.63 ± 0.03

4.291 - 4.928

(0.897, 0.998)

4.15 - 5.87

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
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Table 14. Combined Site Analysis Results for Amino Acids in Isoline, DAS-8191Ø-7 and 

Reference Variety Cottonseed and Literature Ranges (continued)

Analytical 

Component

(% Total 

Amino Acids)

Isoline

(Control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

Threonine 3.54 ± 0.03

3.312 - 3.705

3.54 ± 0.03

3.291 - 3.684

(0.849, 0.911)

3.186 - 3.756 3.57 ± 0.04

3.261 - 3.800

3.58 ± 0.04

3.338 - 3.799

(0.705, 0.945)

2.67 - 4.26

Tryptophan 1.433 ± 0.019

1.240 - 1.592

1.422 ± 0.019

1.267 - 1.651

(0.557, 0.747)

1.295 - 1.677 1.422 ± 0.018

1.254 - 1.595

1.420 ± 0.018

1.199 - 1.652

(0.927, 0.998)

0.91 - 1.40

Tyrosine 3.335 ± 0.015

3.201 - 3.457

3.313 ± 0.015

3.180 - 3.422

(0.155, 0.436)

3.190 - 3.459 3.331 ± 0.020

3.192 - 3.490

3.320 ± 0.020

3.157 - 3.468

(0.507, 0.945)

2.63 - 3.46

Valine 4.75 ± 0.02

4.43 - 4.96

4.74 ± 0.02

4.50 - 4.92

(0.426, 0.679)

4.36 - 5.02 4.76 ± 0.04

4.36 - 5.023

4.74 ± 0.04

4.45 - 5.059

(0.610, 0.945)

4.49 - 6.24

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 97 of 214

Dow AgroSciences LLC

Figure 43. Percent Total Amino Acids in Isoline, DAS-8191Ø-7 and Reference Variety 

Cottonseed Y axis: % Total Amino Acid. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALLTEX LA122, Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in subexperiment 1, 191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 

sprayed with 2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = 

GA, + = LA, open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = 

TX2. The shaded band represents the literature range.
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 98 of 214

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Figure 43. Percent Total Amino Acids in Isoline, DAS-8191Ø-7 and Reference Variety 

Cottonseed (continued)

Y axis: % Total Amino Acid. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALL-TEX LA122, 

Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in sub-experiment 1, 

191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 sprayed with 

2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = GA, + = LA, 

open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = TX2. The shaded 

band represents the literature range
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 99 of 214

Dow AgroSciences LLC

Figure 43. Percent Total Amino Acids in Isoline, DAS-8191Ø-7 and Reference Variety 

Cottonseed (continued)

Y axis: % Total Amino Acid. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALL-TEX LA122, 

Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in sub-experiment 1, 

191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 sprayed with 

2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = GA, + = LA, 

open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = TX2. The shaded 

band represents the literature range

F

F
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6.3.4. Sub-experiment One - Fatty Acid Analysis of Seed

Results from the combined site analysis of the fatty acid composition (22 analytes) for nontransgenic isoline (control) and DAS-8191Ø-7 cottonseed are provided in Table 15 and

Figure 44. Of the 22 fatty acids analyzed (Table 15), thirteen were excluded from the 

analysis because the majority of the results were less than the LOQ (Table 11 and Table 15). 

Mean results in DAS-8191Ø-7 non-sprayed for the remaining nine fatty acids including 14:0 

myristic, 16:0 palmitic, 16:1 palmitoleic, 18:1 oleic, 18:2 linoleic, 18:0 stearic, 18:3 

linolenic, 20:0 arachidic, and 22:0 behenic, fell within the reference variety ranges and/or 

literature ranges. 

The FDR-adjusted P-values for 14:0 myristic, 16:1 palmitoleic, 18:1 oleic, and 18:2 linoleic 

were significant. Statistical differences for 16:1 palmitoleic and 18:1 oleic were not 

consistently observed among individual sites, in which 16:1 palmitoleic decreased at one of 

the eight sites by a mean difference of -2.3%, while 18:1 oleic acid decreased at one of the 

eight sites by a mean difference of -5.3%. In addition, the relative magnitudes of the 

difference between the mean values for non-sprayed DAS-8191Ø-7 and the non-transgenic 

control for the combined site analysis were a decrease of 6.9% for 16:1 palmitoleic and a 

decrease of 5.9% for 18:1 oleic acid. This was less than the variability observed for the 

control sample for 16:1 palmitoleic (range 0.4105 – 0.639, a relative difference of 55.7%) 

and 18:1 oleic (range 13.74 – 17.16 a relative difference of 24.9%). The observed differences 

in 16:1 palmitoleic and 18:1 oleic between DAS-8191Ø-7 and the non-transgenic isoline

were not considered to be meaningful from a food, nutritional or safety perspective because 

they were small, not consistently reproducible across the individual sites and the mean DAS8191Ø-7 values for 16:1 palmitoleic and 18:1 oleic were within both reference variety and 

literature ranges. 

For 14:0 myristic and 18:2 linoleic, no significant differences for FDR-adjusted P-values 

were observed among the individual sites. Significant FDR-adjusted P-values were only 

identified in the combined site analysis. The relative magnitudes of the differences between 

the mean values for DAS-8191Ø-7 non-sprayed and the non-transgenic isoline for the 

combined site analysis were a decrease of 8.75% for 14:0 myristic and an increase of 2.05% 

for 18:2 linoleic. This was less than the variability observed for the control sample for 14:0 

myristic (range 0.545 – 0.907, a relative difference of 66.4%) and 18:2 linoleic (range 54.4 –

61.1, a relative difference of 12.3%). The observed differences in 14:0 myristic and 18:2 

linoleic between DAS-8191Ø-7 and the non-transgenic isoline were not considered to be 

meaningful from a food, nutritional or safety perspective because they were small and the 

mean DAS-891Ø-7 values for 14:0 myristic and 18:2 linoleic were within both reference 

variety and literature ranges.

For 16:0 palmitic, 18:0 stearic, 18:3 linolenic, 20:0 arachidic, and 22:0 behenic, mean results 

for DAS-8191Ø-7 non-sprayed fell within the reference variety ranges and literature ranges. 

Statistical analyses found no FDR-adjusted differences between the levels of these nutrient 

components in cottonseed from DAS-8191Ø-7 and the non-transgenic isoline, supporting the 

findings of composition equivalence of DAS-8191Ø-7 to non-transgenic cotton. 
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 101 of 214

Dow AgroSciences LLC

Table 15. Combined Site Analysis Results for Fatty Acids in Isoline, DAS-8191Ø-7 and 

Reference Variety Cottonseed and Literature Ranges

Analytical 

Component

(% Total Fatty 

Acid)

Isoline 

(Control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature 

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

8:0 Caprylic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

10:0 Capric NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

12:0 Lauric NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

14:0 Myristic 0.720 ± 0.041

0.545 - 0.907

0.657 ± 0.041

0.489 - 0.872

(<0.001, 0.002)

0.4324 - 1.046 0.651 ± 0.041

0.508 - 0.871

0.648 ± 0.041

0.506 - 0.866

(0.541, 0.945)

0.455 - 2.40

14:1 

Myristoleic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

15:0 

Pentadecanoic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ 0.050 - 0.481

15:1 

Pentadecenoic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

16:0 Palmitic 22.55 ± 0.58

20.37 - 25.67

22.26 ± 0.58

20.04 - 26.12

(0.027, 0.114)

18.76 - 26.07 22.26 ± 0.66

19.71 - 26.14

22.16 ± 0.66

19.53 - 25.99

(0.216, 0.945)

15.11 - 28.10

16:1 

Palmitoleic

0.494 ± 0.022

0.4105 - 0.639

0.460 ± 0.022

0.3917 - 0.588

(<0.001, 0.003)

0.3787 - 0.636 0.454 ± 0.023

0.3776 - 0.587

0.451 ± 0.023

0.3728 - 0.592

(0.203, 0.945)

0.464 - 1.190

17:0 

Heptadecanoic

NA

<LOQ - 0.0990

NA

<LOQ - 0.1046 <LOQ - 0.1085 NA<LOQ - 0.1044

NA

<LOQ -

0.1030

0.092 - 0.119

17:1 

Heptadecenoic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

18:0 Stearic 2.311 ± 0.068

1.951 - 2.685

2.334 ± 0.068

1.943 - 2.645

(0.373, 0.642)

1.801 - 2.962 2.341 ± 0.073

1.959 - 2.628

2.313 ± 0.073

1.875 - 2.612

(0.124, 0.945)

0.20 - 3.11

18:1 Oleic 14.84 ± 0.35

13.74 - 17.16

13.95 ± 0.35

12.76 - 16.50

(<0.001, 0.001)

12.93 - 17.09 13.82 ± 0.34

12.54 - 16.50

13.80 ± 0.34

12.64 - 16.08

(0.698, 0.945)

12.8 - 25.3

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 102 of 214

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Table 15. Combined Site Analysis Results for Fatty Acids in Isoline, DAS-8191Ø-7 and 

Reference Variety Cottonseed and Literature Ranges (continued)

Analytical 

Component

(% Total Fatty 

Acid)

Isoline 

(Control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature 

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

18:2 Linoleic 58.5 ± 0.8

54.4 - 61.1

59.7 ± 0.8

54.9 - 62.5

(<0.001, 0.003)

52.36 - 63.9 59.9 ± 0.9

54.45 - 62.9

60.0 ± 0.9

54.7 - 62.91

(0.247, 0.945)

46.00 - 59.4

18:3 Linolenic 0.2032 ± 0.0083

0.1733 - 0.2412

0.2117 ± 0.0083

0.1782 - 0.2991

(0.249, 0.577)

0.1460 -

0.2567

0.2126 ± 

0.0090

0.1829 -

0.2761

0.2150 ± 0.0090

0.1828 - 0.2688

(0.617, 0.945)

0.11 - 0.35

18:3 γLinolenic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ 0.097 - 0.232

20:0 

Arachidic 0.2509 ± 0.0105

0.2088 - 0.2949

0.2492 ± 0.0105

0.2029 - 0.3063

(0.489, 0.722)

0.1855 -

0.3242

0.2530 ± 

0.0111

0.2010 -

0.3113

0.2521 ± 0.0111

0.2067 - 0.2991

(0.660, 0.945)

0.186 - 0.414

20:1 

Eicosenoic NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ 0.095 - 0.098

20:2 

Eicosadienoic

NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

20:3 

Eicosatrienoic

NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

20:4 

Arachidonic

NA

<LOQ

NA

<LOQ <LOQ NA<LOQ

NA

<LOQ NR

22:0 Behenic 0.1373 ± 0.0060

0.1119 - 0.1692

0.1341 ± 0.0060

0.1057 - 0.1693

(0.181, 0.464)

0.1035 -

0.1749

0.1358 ± 

0.0071

0.1101 -

0.1757

0.1386 ± 0.0071

0.1092 - 0.1726

(0.373, 0.945)

0.104 - 0.295

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 103 of 214

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Figure 44. Percent Total Fatty Acids in Isoline, DAS-8191Ø-7 and Reference Variety 

CottonseedY axis: % Total Fatty Acid. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALL-TEX 

LA122, Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in subexperiment 1, 191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 

sprayed with 2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = 

GA, + = LA, open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = 

TX2. The shaded band represents the literature range.
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 104 of 214

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Figure 44. Percent Total Fatty Acids in Isoline, DAS-8191Ø-7 and Reference Variety 

Cottonseed (continued)

Y axis: % Total Fatty Acid. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALL-TEX LA122, 

Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in sub-experiment 1, 

191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 sprayed with 

2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = GA, + = LA, 

open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = TX2. The shaded 

band represents the literature range.
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 105 of 214

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6.3.5. Sub-Experiment One - Vitamin Analysis of Seed

Results from the combined site analysis of the vitamin composition from non-transgenic 

isoline (control) and DAS-8191Ø-7 cottonseed are provided in Table 16 and Figure 45.

Seven vitamin analytes were measured including vitamin E, A, B1, B2, B3, B6, and B9 

(Table 16). Vitamin A (beta carotene) was excluded from the analysis because the majority 

of the results were less than the LOQ. Mean results in DAS-8191Ø-7 non-sprayed for the 

remaining six vitamins fell within the reference variety ranges and the literature ranges (only 

available for vitamin E). No significant FDR-adjusted P-values were observed for 

DAS-8191Ø-7 non-sprayed in the six vitamins that were included in the statistical analysis, 

supporting the findings of composition equivalence of DAS-8191Ø-7 to non-transgenic 

cotton. 

Table 16. Combined Site Analysis Results for Vitamins in Isoline, DAS-8191Ø-7 and Reference 

Variety Cottonseed and Literature Ranges

Analytical 

Component

(mg/kg Dry 

Weight)

Isoline 

(Control)

DAS-8191Ø-7 

non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature 

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

Alpha 

Tocopherol

(Vitamin E)

90 ± 11

30.3 - 134

88 ± 11

26.7 - 136

(0.612, 0.803)

31.1 - 151 90 ± 10

20.4 - 135

89 ± 10

27.6 - 129

(0.360, 0.945)

70.825 - 197.243

Vitamin A

(Beta 

Carotene)

NA

<LOQ -

0.264

NA

<LOQ - 0.323

<LOQ -

0.267

NA

<LOQ - 0.249

NA

<LOQ - 0.238 NR

Vitamin B1 

(Thiamine 

HCl)

10.3 ± 0.4

7.52 - 12.6

10.3 ± 0.4

8.28 - 13.2

(0.953, 0.969)

5.54 - 14.7 10.4 ± 0.5

6.14 - 13.4

10.4 ± 0.5

7.42 - 15.4

(0.912, 0.998)

NR

Vitamin B2

(Riboflavin) 6.2 ± 0.5

3.52 - 9.95

6.3 ± 0.5

3.82 - 9.82

(0.813, 0.888)

3.44 - 9.52 6.6 ± 0.5

3.69 - 12.5

6.6 ± 0.5

3.97 - 10.0

(0.998, 0.998)

NR

Vitamin B3

(Niacin) 27.8 ± 1.2

21.7 - 35.6

28.5 ± 1.2

22.7 - 35.3

(0.315, 0.620)

20.4 - 36.8 28.2 ± 1.1

21.3 - 37.7

28.1 ± 1.1

23.5 - 34.3

(0.905, 0.998)

NR

Vitamin B6

(Pyridoxine 

HCl)

3.83 ± 0.09

3.15 - 4.49

3.72 ± 0.09

3.10 - 4.54

(0.176, 0.464)

2.84 - 5.12 3.70 ± 0.13

3.18 - 4.72

3.77 ± 0.13

3.08 - 5.28

(0.535, 0.945)

NR

Vitamin B9

(Folic Acid) 1.66 ± 0.07

1.14 - 2.38

1.67 ± 0.07

1.17 - 2.26

(0.806, 0.888)

1.10 - 2.40 1.56 ± 0.09

0.872 - 2.31

1.64 ± 0.09

1.15 - 2.29

(0.160, 0.945)

NR

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 106 of 214

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Figure 45. Vitamins in Isoline, DAS-8191Ø-7 and Reference Variety Cottonseed

Y axis: mg/kg Dry Weight. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALL-TEX LA122, 

Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in sub-experiment 1, 

191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 sprayed with 

2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = GA, + = LA, 

open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = TX2. The shaded 

band represents the literature range.
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 107 of 214

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6.3.6. Sub-Experiment One - Anti-Nutrient Analysis of Seed

Results from the combined site analysis of the five anti-nutrient analytes including 

dihydrosterculic acid, malvalic acid, sterculic acid, free gossypol and total gossypol from 

non-transgenic isoline (control) and DAS-8191Ø-7 cottonseed are provided in Table 17 and

Figure 46. Mean results in DAS-8191Ø-7 non-sprayed for the five anti-nutrients analyzed 

fell within the reference variety ranges and the literature ranges. 

The FDR-adjusted P-values were significant in DAS-8191Ø-7 non-sprayed for malvalic acid 

and total gossypol. For both analytes, no significant differences for FDR-adjusted P-values 

were observed among the individual sites. Significant FDR-adjusted P-values were only 

identified in the combined site analysis. The relative magnitudes of the differences between 

the mean values for DAS-8191Ø-7 non-sprayed and the non-transgenic isoline for the 

combined site analysis were an increase of 10.1% for malvalic acid and a decrease of 12% 

for total gossypol. This was less than the variability observed for the control sample for both

malvalic acid (range 0.403 – .645, a relative difference of 60.0%) and total gossypol (range 

0.829 – 1.44, a relative difference of 73.7%). The observed differences in malvalic acid and 

total gossypol between DAS-8191Ø-7 and the non-transgenic isoline were not considered to 

be meaningful from a food, nutritional or safety perspective: differences were 1) small, 2) 

less than the variability seen in the isoline, and 3) the mean DAS-891Ø-7 values for malvalic 

acid and total gossypol were within both reference variety and literature ranges.

For dihydrosterculic acid, sterculic acid and free gossypol mean results for DAS-8191Ø-7 

non-sprayed fell within the reference variety ranges and literature ranges. Statistical analyses 

found no FDR-adjusted differences between the levels of nutrient components in cottonseed 

from DAS-8191Ø-7 and the non-transgenic isoline, supporting the findings of composition 

equivalence of DAS-8191Ø-7 to non-transgenic cotton. 
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Table 17. Combined Site Analysis Results for Anti-Nutrients in Isoline, DAS-8191Ø-7 and Reference 

Variety Cottonseed and Literature Ranges

Analytical 

Component

(Units)

Isoline 

(Control)

DAS-8191Ø-7 

Non-sprayed

Reference 

Variety 

Range

DAS-8191Ø-7 

Non-sprayed

DAS-8191Ø-7 

Sprayed1

Literature 

Range

Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max Mean ± SE

Min - Max

Mean ± SE

Min - Max

(P-Val., Adj. P)2

Min - Max

Sub-Experiment 1 

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)

Dihydrosterculic 

Acid

(% Total Fatty Acid)

0.205 ± 0.007

0.150 - 0.247

0.214 ± 0.007

0.174 - 0.261

(0.074, 0.259)

0.153 -

0.325

0.216 ± 0.006

0.175 - 0.269

0.214 ± 0.006

0.181 - 0.254

(0.578, 0.945)

0.075 - 0.310

Malvalic Acid

(% Total Fatty Acid)

0.524 ± 0.023

0.403 - 0.645

0.577 ± 0.023

0.426 - 0.762

(0.003, 0.029)

0.402 -

0.854

0.591 ± 0.026

0.474 - 0.722

0.612 ± 0.026

0.450 - 0.806

(0.174, 0.945)

0.17 - 0.759

Sterculic Acid

(% Total Fatty Acid)

0.275 ± 0.011

0.173 - 0.447

0.297 ± 0.011

0.209 - 0.358

(0.077, 0.259)

0.196 -

0.440

0.301 ± 0.011

0.215 - 0.366

0.301 ± 0.011

0.196 - 0.391

(0.964, 0.998)

0.13 - 0.56

Free Gossypol

(% Dry Weight)

0.96 ± 0.06

0.593 - 1.36

0.83 ± 0.06

0.556 - 1.17

(0.010, 0.059)

0.492 - 1.28 0.82 ± 0.06

0.479 - 1.20

0.81 ± 0.06

0.498 - 1.13

(0.624, 0.945)

0.454 - 1.399

Total Gossypol

(% Dry Weight)

1.08 ± 0.05

0.829 - 1.44

0.95 ± 0.05

0.719 - 1.19

(0.001, 0.010)

0.551 - 1.41 0.92 ± 0.05

0.519 - 1.31

0.93 ± 0.05

0.624 - 1.32

(0.867, 0.998)

0.547 - 1.522

1 Sprayed with 2,4-D and glufosinate

2

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values
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Figure 46. Anti-Nutrients in Isoline, DAS-8191Ø-7 and Reference Variety CottonseedY axis: 

see Table 17 for specific values. Reference Variety cottonseed: All-TEX 1203, ALL-TEX A102, ALL-TEX LA122, 

Paymaster HS 200, Raider 271 and Speed. 191Ø Unsprayed 1 = DAS-8191Ø-7 unsprayed in sub-experiment 1, 

191Ø Unsprayed 2 = DAS-8191Ø-7 unsprayed in sub-experiment 2, 191Ø Sprayed = DAS-8191Ø-7 sprayed with 

2,4-D plus glufosinate in sub-experiment 2. Symbols for each location shown: open circle = AL,  = GA, + = LA, 

open triangle = MO, open square = MS, open diamond = NC, filled circle = TX1, filled triangle = TX2. The shaded 

band represents the literature range.
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6.3.7. Sub-Experiment Two - Composition Results

The comparisons between sprayed and non-sprayed entries of DAS-8191Ø-7 cotton in subexperiment two indicated no statistically significant differences for all 59 analytes included 

in the combined site analysis (Table 12 - Table 17 and Figure 41 - Figure 46). In addition, 

comparisons of non-sprayed DAS-8191Ø-7 cotton in sub-experiment two with non-sprayed

DAS-8191Ø-7 cotton in sub-experiment one indicated no statistically significant differences 

for all 59 analytes. Based on these comparisons, sprayed DAS-8191Ø-7 cotton in subexperiment two is compositionally equivalent to non-sprayed DAS-8191Ø-7 cotton in subexperiment one and indicates that spraying DAS-8191Ø-7 with 2,4-D plus glufosinate has no 

significant effect on cottonseed composition. Based on these results, it is concluded that 

DAS-8191Ø-7 (non-sprayed or sprayed with 2,4-D plus glufosinate) cottonseed is 

compositionally equivalent to non-transgenic cottonseed.

6.4.Composition Summary

Field trials were conducted at eight U.S. field sites in 2012 to produce cottonseed samples of 

DAS-8191Ø-7 cotton (non-sprayed and sprayed with 2,4-D plus glufosinate), non-transgenic 

isoline (control) and non-transgenic cotton lines for nutrient composition analysis. 

Cottonseed samples were analyzed for 73 analytes including proximates, fiber, minerals, 

amino acids, fatty acids, vitamins, and anti-nutrients, which included analytes that are 

deemed important for the assessment of new cotton varieties for use in food and feed 

(OECD, 2009). Of the 73 analytes tested, 14 were excluded from the combined site analysis 

because more than 50% of the results for those analytes were less than the limit of 

quantitation (<LOQ) (Table 11).

Statistical analyses found no FDR-adjusted P-value differences in cottonseed from 

DAS-8191Ø-7 and the non-transgenic control for proximate, fiber, amino acid or vitamin 

analytes. In addition, mean values for these analytes all fell within reference variety ranges 

and/or literature ranges supporting the findings of composition equivalence of DAS-8191Ø-7 

to non-transgenic cotton.

For mineral, fatty acid and anti-nutrient composition analysis, all but seven analytes showed 

no statistical differences in FDR-adjusted P-values in combined site analysis of DAS-8191Ø7 non-sprayed cotton and the non-transgenic isoline. The mean values for mineral, fatty acid 

and anti-nutrient analytes with no statistical difference also fell within reference variety 

ranges and/or literature ranges.

Seven analytes including manganese, 14:0 myristic, 16:1 palmitoleic, 18:1 oleic, 18:2 

linoleic, malvalic acid and total gossypol all showed statistical differences in FDR-adjusted 

P-values between the isoline and DAS-8191Ø-7 non-sprayed and the non-transgenic control.

However, the observed differences between the seven analytes in DAS-8191Ø-7 and the nontransgenic isoline were not considered to be meaningful from a food, nutritional or safety

perspective because the differences between DAS-8191Ø-7 and the non-transgenic isoline

were small, were less than the variability seen in the non-transgenic isoline and the mean 

values for the seven analytes were all within reference variety and/or literature ranges.

Additionally, no statistically significant differences were detected between DAS-8191Ø-7 

non-sprayed and DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate, which indicates that 
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spraying DAS-8191Ø-7 with 2,4-D plus glufosinate has no significant effect on cottonseed 

composition. Based on these results, it is concluded that DAS-8191Ø-7 (non-sprayed or 

sprayed with 2,4-D plus glufosinate) cotton is compositionally equivalent to non-transgenic 

cotton.

6.5. Conclusions

Overall, for DAS-8191Ø-7, mean analyte values observed to be significantly different from

those of the non-transgenic isoline (control) were generally shown to be of small relative 

magnitudes. All combined-site mean values and individual site mean values of 

DAS-8191Ø-7 for all analytes measured were within the context of the natural variability of 

reference cotton line composition data and/or published in the scientific literature.

Additionally, no statistically significant differences were detected between DAS-8191Ø-7 

non-sprayed and DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate, which indicates that 

spraying DAS-8191Ø-7 with 2,4-D plus glufosinate has no significant effect on cottonseed 

composition. Based on the results of this composition analysis, it is concluded that 

cottonseed from DAS-8191Ø-7 is compositionally equivalent to non-transgenic cottonseed

and therefore the food and feed safety and nutritional quality of this product is comparable to 

that of commercially cultivated cotton.
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7. Phenotypic, Agronomic and Ecological Characteristics

This section provides a comparative assessment of the phenotypic, agronomic and ecological

characteristics of DAS-8191Ø-7 cotton compared to non-transgenic isoline (control). As part 

of 7 CFR § 340.6, a detailed description of the regulated article phenotype is requested as 

part of the petition for determination of nonregulated status to substantiate that the regulated

article is unlikely to pose a greater plant pest risk than the unmodified organism from which 

it was derived. A list of all DAS-8191Ø-7 cotton field evaluations conducted under USDA 

permit or notification is provided in Appendix 9.

The characterization of DAS-8191Ø-7 cotton encompasses 1) vegetative growth, 2) 

ecological (insect and disease) and 3) seed germination, dormancy, and emergence. The data 

were evaluated by individuals familiar with the production and evaluation of cotton. In each 

assessment, DAS-8191Ø-7 cotton was compared to an appropriate, near isogenic isoline 

(control) that does not contain the add-12 or pat genes. The data collected here support a 

conclusion that DAS-8191Ø-7 cotton is substantially equivalent to non-transgenic cotton 

with the exception of the introduced and expression of aad-12 and pat genes and therefore no 

more likely to pose a plant pest risk or have a significant environmental impact compared to 

non-transgenic cotton.

7.1. Study Design

Agronomic studies used the same field studies and plots as those for composition assessment 

of DAS-8191Ø-7 cotton, as described in detail in section 6.1.1. Briefly, field trials were 

conducted at eight U.S. sites in 2012 to evaluate phenotypic, agronomic and ecological

characteristics of DAS-8191Ø-7 cotton (non-sprayed and sprayed with 2,4-D plus 

glufosinate), near isogenic non-transgenic isoline (control) (Figure 4) and reference variety 

cotton plants. For the reference lines, six unique reference lines (ALL-TEX 1203, ALL-TEX 

A102, ALL-TEX LA122, Paymaster HS 200, Raider 271, and Speed) were included across 

all sites of the field production with three varieties per site. 

Because non-transgenic cotton plants (e.g., isoline and reference lines) are sensitive to 2,4-D, 

two sub-experiments were conducted at each site to spatially separate the non-transgenic 

entries from the entries in which 2,4-D was applied. Sub-experiment one was not sprayed and 

contained the isoline, reference varieties and one entry of DAS-8191Ø-7 cotton. Subexperiment two contained two entries of only DAS-8191Ø-7 cotton; one not sprayed and one 

sprayed with 2,4-D plus glufosinate. The objective of sub-experiment one was to determine if 

non-sprayed DAS-8191Ø-7 cotton is agronomically equivalent to non-transgenic cotton, and 

the objective of sub-experiment two was to determine if applying 2,4-D plus glufosinate 

impacts the agronomic characteristics of DAS-8191Ø-7 cotton.

The entries were arranged in a randomized complete block design within each subexperiment. The sub-experiments were separated by 100 ft (30 m) to prevent isoline and 

reference entries in sub-experiment one from potential injury by 2,4-D drift from applications 

in sub-experiment two. Both sub-experiments were present at all field testing sites. 

Randomization of entries within blocks was unique at each field testing site. Test, isoline, 

and reference variety cotton was planted at a seeding rate of approximately 100 seeds per 25 

ft (7.62 m) of row (one seed per 3 in or 7.6 cm). Four replicate plots of each entry were 
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established at each site for each sub-experiment, with each plot consisting of four rows that 

were 25 ft (7.62 m) long with a row spacing of approximately 30 in (76 cm). Each four row 

plot was bordered by two rows cotton.

Herbicides were applied in a spray volume of approximately 20 gallons per acre (187 L/ha), 

and all herbicide applications included 2% v/v ammonium sulfate. 2,4-D (GF-2654) and 

glufosinate (Ignite 280 SL) were applied in a tank mixture as two broadcast applications to 

one entry of DAS-8191Ø-7 cotton in sub-experiment two. Application timings were at the 3 

node and 6 node growth stages. The target application rates for both application timings were 

1120 g ae/ha 2,4-D and 596 g ai/ha glufosinate. Actual application rates ranged from 1108 –

1142 g ae/ha 2,4-D and 589 – 622 g ai/ha glufosinate for the 3 node application and 1061 –

1141 g ae/ha 2,4-D and 564 – 621 g ai/ha glufosinate for the 6 node application. 

7.2. Data Collection and Statistical Analysis

Evaluations of nine phenotypic, agronomic and ecological characteristics (early population, 

seedling vigor, flower initiation, nodes above white flower, plant height, percent open bolls, 

lint yield, disease incidence and insect damage) were conducted to investigate the 

equivalency of DAS-8191Ø-7 (non-sprayed and sprayed with 2,4-D plus glufosinate) cotton 

to non-transgenic cotton. In all cases, the agronomic data was collected and evaluated by 

individuals familiar with the production and evaluation of cotton. Details regarding the 

agronomic data collection of the nine agronomic characteristics measured are described in 

Table 18.

Analysis of variance was conducted across field testing sites for agronomic data using a 

mixed model (SAS Institute Inc., 2011). Entry was considered a fixed effect; location, block 

within location, and location-by-entry were designated as random effects. Paired contrasts 

were conducted using t-tests, and the significance of overall treatment effects was estimated 

using an F-test. Significant differences were declared at the 95% confidence level (α = 0.05).

Multiplicity occurs when a 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 can be high (1-0.95number of comparisons). There were nine 

analytes included in the combined site statistical analysis; therefore, nine comparisons were 

made in each sub-experiment in this study (DAS-8191Ø-7 non-sprayed compared with the 

control in sub-experiment one and DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate 

compared with DAS-8191Ø-7 non-sprayed in sub-experiment two). As a result, the 

probability of declaring one or more false differences based on unadjusted P-values was 

36.975% (1-0.959) due to multiplicity in each sub-experiment (or >60% across both subexperiments).

One method to account for multiplicity is to adjust P-values to control the experiment-wise 

error rate; however, the power for detecting specific effects can be reduced significantly 

when many comparisons are made in a study. An alternative with much greater power is to 

adjust P-values to control the probability that each declared difference is significant (CurranEverett, 2000). This can be accomplished using a False Discovery Rate (FDR) control 

procedure (Benjamini and Hochberg, 1995), which is a commonly used approach in studies 

examining transgenic crops (Herman et al., 2007; Coll et al., 2008; Huls et al., 2008; Jacobs
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et al., 2008; Stein et al., 2009; Herman et al., 2010). Therefore, the P-values from the 

contrasts were each adjusted using the FDR method to improve discrimination of true 

differences among treatments from random effects (false positives). Differences were 

considered significant if the FDR-adjusted P-value was less than 0.05.

Table 18. Measured Agronomic Characteristics and Sample Collection Details

Evaluation

Category

Evaluation 

Timing Description of Data Evaluation Description 

(scale/measurements)

Early 

Population 

(Stand Count)

28 days after 

planting

Total number of emerged plants in 

the center two rows of each plot

Plants were considered emerged when the cotyledons had

assumed an erect posture and were completely unrolled.

Seedling Vigor 28 days after 

planting

Visual estimate of the plant vigor 

in each plot

0-100% rating scale; 0 = all plants were dead, 10 = short 

plants with small, thin leaves, 100 = all plants were tall 

with robust leaves. Evaluation was not based on growth of 

control entries and did not consider germination/ 

emergence (stand count).

Flower 

Initiation

First white 

flower

1) Date when approximately 50% 

of plants in plot produced at least 

one white flower

2) Number of days from planting 

and the date when 50% of plants in 

plot have reached first white flower

Visual approximation of when 50% of the plants in each 

plot had produced one open white flower.

Nodes Above 

First White 

Flower

28 days after 

first white 

flower

Number of nodes above the 

uppermost node containing a firstposition white flower on six plants

per plot.

Recorded the number of the uppermost node and the total 

number of nodes on six cotton plants that contained a firstposition white flower. The number of nodes above first 

white flower were calculated by subtracting the number of 

the uppermost node that contained a first-position white 

flower from the total number of nodes.

Plant Height

28 days after 

first white 

flower

Plant height from soil surface to 

terminal bud on six plants per plot Recorded the height each plant in centimeters.

Percent Open 

Bolls

Maturity but 

prior to 

defoliation

1. Total number of bolls and the 

number of open bolls on six plants 

from each plot.

2. Calculated percent open bolls

1. Recorded the total number of bolls on six representative 

plants from each plot. Recorded the number of open bolls 

(bolls greater than 1 in.) on the same six plants. Cracked 

bolls were considered to be open. 

2. Calculated percent open bolls: (Number of open bolls /

total number of bolls)*100.

Lint Yield Maturity Weight of cotton lint from the 

center two rows of each plot.

Seedcotton from rows 2 and 3 of each plot was harvested 

and a sub-sample of the seedcotton yield was ginned.

Gin turnout (lint/seedcotton) was multiplied by the 

seedcotton yield of each plot to determine lint yield per plot.

Disease 

Incidence Peak bloom 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 Peak bloom 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 and type of insects if 

present.
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7.2.1. Phenotypic and Agronomic Results

Seven agronomic characteristics were evaluated in this study: early population, seedling 

vigor, flower initiation, nodes above first white flower, plant height, percent open bolls and 

lint yield. A statistical analysis of agronomic data from the control and DAS-8191Ø-7 cotton

entries across field testing sites was conducted. For each agronomic characteristic and entry, 

the least squares mean, standard error, and minimum and maximum values were reported

(Table 19). 

Table 19. Combined Site Analysis Results for Agronomic Characteristics

Agronomic 

Characteristic

(Units)

Isoline

(Control)

DAS-8191Ø-7 

Non-Sprayed

Reference 

Range

DAS-8191Ø-7 

Non-Sprayed

DAS-8191Ø-7

Sprayed1

Mean ± SE

Min - Max2

Mean ± SE

Min – Max2

(P-Val., Adj. P)3

Min – Max2

Mean ± SE

Min – Max2

(P-Val., Adj. P) 3

Mean ± SE

Min – Max2

(P-Val., Adj. P) 3

Sub-Experiment 1

(No Herbicide)

Sub-Experiment 2 

(Herbicide Applied)1

Early Population

(Emerged Plants per Plot)

142 ± 10

91 – 194

129 ± 10

62 - 179

(0.050, 0.450)

4 - 209 137 ± 10

87 - 185

137 ± 10

84 - 179

(0.856, 0.963)

Seedling Vigor

(0 - 100% scale: 0% = short, thin 

plants, 100% = tall, robust plants)

85 ± 3

40 – 100

82 ± 3

62 - 100

(0.288, 0.555)

10 - 100 87 ± 3

60 - 100

84 ± 3

40 - 100

(0.037, 0.335)

Flower Initiation

(Days from Planting to First White 

Bloom)

62 ± 2

51 – 69

62 ± 2

51 - 76

(0.915, 0.995)

51 - 70 62 ± 2

51 - 70

62 ± 2

51 - 69

(0.642, 0.931)

Nodes Above First 

White Flower

(Number of Nodes)

8 ± 2

0 – 18

7 ± 2

0 - 17

(0.161, 0.555)

0 - 19 8 ± 2

0 - 17

8 ± 2

1 - 19

(0.625, 0.931)

Plant Height

(cm)

99 ± 9

48 – 158

99 ± 9

49 - 147

(0.774, 0.995)

45 - 164 103 ± 9

55 - 161

102 ± 9

60 - 163

(0.624, 0.931)

Percent Open Bolls

(Percent of Total Bolls)

65 ± 8

0 – 100

68 ± 8

17 - 100

(0.199, 0.555)

11 - 100 68 ± 8

15 - 100

68 ± 8

14 - 100

(0.989, 0.989)

Lint Yield

(kilograms per hectare)

762 ± 191

146 – 1991

762 ± 191

78 - 1891

(0.995, 0.995)

74 - 2225 745 ± 193

208 - 2187

687 ± 193

144 - 1585

(0.423, 0.931)

Ecological Eval.

Disease Incidence4

(0 - 100%: 0% = no damage, 

100% = all plants diseased)

0 ± 0

0 – 7

0 ± 0

0 - 5

(0.855, 0.995)

0 - 7

0 ± 0

0 - 3

0 ± 0

0 - 3

(0.724, 0.931)

Insect Damage4

(0 - 100%: 0% = no damage, 

100% = all plants damaged)

1 ± 1

0 – 10

1 ± 1

0 - 5

(0.308, 0.555)

0 - 10 1 ± 1

0 - 5

1 ± 1

0 - 10

(0.270, 0.931)

1

Sprayed with 2,4-D and glufosinate

2 Min, Max value is an individual data point reported for a single test plot

3

P-Val – unadjusted P-value, Adj. P – FDR Adjusted P-values

4 Evaluation conducted at peak bloom

7.2.1.1. Comparison of the Isoline (Control) and Non-Sprayed DAS-8191Ø-7 

For the assessment of the nine phenotypic, agronomic and environmental characteristics

measured there were no statistically significant differences (Adj. P<0.05) observed between 

the control and non-sprayed DAS-8191Ø-7 entries (sub experiment 1); moreover, mean 
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results for non-sprayed DAS-8191Ø-7 entries fell within the reference variety ranges (Table 

19). Therefore, non-sprayed DAS-8191Ø-7 was found to be agronomically equivalent to the 

isoline (control).

7.2.1.2. Comparison of Non-Sprayed and Sprayed DAS-8191Ø-7 Cotton

Comparisons between DAS-8191Ø-7 sprayed with 2,4-D plus glufosinate and non-sprayed 

DAS-8191Ø-7 cotton (sub experiment 2) were not statistically significant (Adj. P<0.05) for 

the nine agronomic characteristics included in the combined site analysis based on FDRadjusted P-values (Table 18). 

The unadjusted P-value for seedling vigor in DAS-8191Ø-7 sprayed with 2,4-D plus 

glufosinate was significant. Significant P-values for seedling vigor were identified at only 

one of the eight field locations. The relative magnitudes of the differences between the mean 

values for non-sprayed DAS-8191Ø-7 cotton vs. sprayed DAS-8191Ø-7 cotton for the 

combined site analysis were small (-3.45%). Mean results for DAS-8191Ø-7 sprayed with 

2,4-D plus glufosinate fell within the reference variety ranges, and the FDR-adjusted P-value 

was insignificant; therefore, the statistically significant difference for seedling vigor based on 

the unadjusted P-value is not considered biologically meaningful. The agronomic 

characteristics of DAS-8191Ø-7 cotton sprayed with 2,4-D plus glufosinate are equivalent to

non-sprayed DAS-8191Ø-7. 

7.2.1.3. Phenotypic and Agronomics Conclusions

Phenotypic and agronomic analysis of DAS-8191Ø-7 cotton revealed no statistically 

significant differences between non-sprayed DAS-8191Ø-7 cotton and the isoline (control). 

In addition, all mean results from the isoline and DAS-8191Ø-7 entries were within the 

ranges observed in the non-transgenic cotton reference varieties included in the study. It was 

also determined that the agronomic characteristics of DAS-8191Ø-7 cotton sprayed with 2,4-

D plus glufosinate are equivalent to non-sprayed DAS-8191Ø-7 cotton. 

The data collected here support a conclusion that DAS-8191Ø-7 cotton is substantially 

equivalent to non-transgenic cotton with the exception of the introduced and expression of

aad-12 and pat genes and therefore no more likely to pose a plant pest risk, increase in 

weediness, or have an altered environmental impact compared to non-transgenic cotton.

7.2.2. Ecological Evaluations

As part of the plant characterization for DAS-8191Ø-7 cotton, evaluations of disease 

incidence and insect damage were conducted to determine the potential for increase in plant 

pest characteristics compared to non-transgenic cotton. The same field trials and plots as 

described for the phenotypic and agronomic analyses for DAS-8191Ø-7 cotton were 

monitored for both insect damage and disease incidence at peak bloom. Monitoring was 

performed and observed by personnel familiar with cotton cultivation practices (field station 

managers, field agronomists, field associates). 

The personnel conducting the field tests visually monitored the incidence of plant disease and 

pests on DAS-8191Ø-7 cotton and non-transgenic cotton varieties included in the same trials. 

Disease and insect damage was rated at peak bloom on a numerical scale of 0-100%, with 

0% representing no damage due to disease incidence or insect pests (Table 19). Table 20

summarizes the insects and diseases that caused the insect damage and disease incidence at 

each field site. 
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Insect damage or disease was limited, and where present, was found in both test and control 

plots (Table 19). The insect and disease pressure observed in these trials was typical of the 

growing locations. There were no statistically significant differences between non-sprayed 

DAS-8191Ø-7 cotton and the isoline (control) in susceptibility to and interactions with 

diseases and insects (Table 19). Likewise, spraying 2,4-D and glufosinate on DAS-8191Ø-7 

cotton did not statistically significantly affect the occurrence of insect damage or disease 

incidence. 

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 cotton. 

These data support the conclusion that DAS-8191Ø-7 cotton is substantially equivalent to 

non-transgenic cotton and is no more likely to be susceptible or resistant to insects or 

diseases typical to U.S. cotton growing regions. The data collected here support a conclusion 

that, with the exception of the introduced and expression of aad-12 and pat genes DAS8191Ø-7 cotton is no more likely to pose a plant pest risk, increase in weediness or have an 

altered environmental impact compared to non-transgenic cotton.

Table 20. Insects and Diseases Present at Field Testing Sites 

Site1

Insects Present Type of Insect 

Damage

Diseases 

Present

AL Whitefly Stippling Leaf spot

GA Bollworm/Tobacco Budworm Chewing None

LA None None None

MO None None None

MS Plant bug Square Feeding None

NC None None None

TX1 None None None

TX2 None None Alternaria

1

Site location listed by U.S. state abbreviation with two locations shown for Texas (TX1, TX2)

7.2.3. Cottonseed Germination and Dormancy Evaluation

Seed dormancy is a characteristic that is often associated with plants that are considered 

weeds. Cotton, however, has had this characteristic removed through selection and breeding. 

Since USDA-APHIS considers weediness as a factor in the plant pest determination, an 

assessment of seed dormancy and germination was conducted on DAS-8191Ø-7 cottonseed. 

The germination and dormancy of DAS-8191Ø-7 cottonseed compared with non-transgenic 

isoline (control) (98M-2983XCoker 310) cottonseed under warm and cool conditions was 

evaluated to determine any impact of AAD-12 and PAT expression on seed germination and 

dormancy characteristics. 

7.2.3.1. Study Design

The warm and cool germination tests consisted of four replications of each cotton entry with 

100 seeds per replication in a completely randomized design.
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Warm Germination Methods: One hundred acid delinted cotton seeds were evenly 

distributed onto two water saturated sheets of non-toxic, germination paper towels. A third 

water saturated towel was used to cover the seeds and all three towels were loosely rolled and 

placed upright in a plastic container. This process was repeated for each of four replications. 

The rolled towels were placed in an incubator set at 30° C. Sufficient water was added 

throughout the experiment to maintain a moist substrate while ensuring that a film of excess 

water did not accumulate around the seeds. On day four and day eight, seeds were observed 

for germination. 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). 

Cool Germination Methods: One hundred acid delinted cotton seeds were evenly distributed

onto two water saturated sheets of germination non-toxic paper towels. A third water 

saturated towel was used to cover the seeds and all three towels were loosely rolled and 

placed upright in a plastic container. Prior to use all towels had been kept at 18° C for at least 

16 hours. Each container was covered to retain moisture and then kept at 18° C for 7 days in 

the dark. On day seven, the germination was recorded. For cool germination, normal 

seedlings were defined as “normal seedlings having a combined hypocotyl and root length of 

4 cm (1 and 10/16 in) or longer (AOSA, 2009).” All other germinated seedlings that did not 

meet these criteria were classified as abnormal. This method is adapted from the AOSA Seed 

Vigor Testing Handbook (AOSA, 2009). 

Data were transformed using the arcsine of the square root of the decimal fraction of cotton 

seeds normally germinated per replicate and then subjected to analysis of variance based on a 

completely randomized design (SAS Institute Inc., 2011).

7.2.3.2. Germination and Dormancy Results

Percentages of normally germinated cotton seedlings and P-values for the significance of the 

effect of entry on germination are provided in Table 21. Germination of event DAS-8191Ø-7

cotton did not differ significantly (α = 0.05) from that of the non-transgenic cotton 

comparator under warm (P = 0.1189) or cool (P = 0.0717) conditions, indicating that the 

presence of the transgenic insert in DAS-8191Ø-7 cotton did not alter cottonseed 

germination. These data support the conclusion that DAS-8191Ø-7 cottonseed is 

substantially equivalent to non-transgenic cottonseed in its germination characteristics. The 

data support a conclusion that with the exception of the introduced and expression of aad-12

and pat genes DAS-8191Ø-7 cottonseed is no more likely to pose a plant pest risk or increase 

in weediness compared to non-transgenic cotton.
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Table 21. Germination Percentages for DAS-8191Ø-7 and Control Cottonseed

Temperature Entry

Cotton Germination (%)1

PValue2

Rep 1 Rep 2 Rep 3 Rep 4 Mean (±SE)

Warm

30 °C DAS-8191Ø-7 93 95 95 99 95.5 (±1.3)

0.1189

30 °C Control 89 93 91 95 92.0 (±1.3)

Cool

18 °C DAS-8191Ø-7 60 73 48 50 57.8 (±5.7)

0.0717

18 °C Control 85 81 76 59 75.3 (±5.7)

1

Four replications of each cotton sample at the indicated temperature with 100 seeds per replication in a completely randomized design

2 Differences were considered significant if the P-value was less than 0.05.

7.3. Summary of Phenotypic, Agronomic and Ecologic Characteristics

A comparative assessment of the phenotypic, agronomic and ecological characteristics of 

DAS-8191Ø-7 cotton compared to non-transgenic isoline (control) was conducted. The 

characterization of DAS-8191Ø-7 cotton encompassed 1) vegetative growth, 2) ecological

(insect and disease) and 3) seed germination and dormancy. The data were evaluated by 

individuals familiar with the production and evaluation of cotton. 

In each assessment, DAS-8191Ø-7 cotton was compared to an appropriate, near isogenic 

isoline (control) that does not contain the add-12 or pat genes (Figure 4). Phenotypic, 

agronomic and ecological analysis of DAS-8191Ø-7 cotton revealed no statistically 

significant differences between non-sprayed DAS-8191Ø-7 cotton and the isoline (control). 

It was also determined that the phenotypic, agronomic and ecological characteristics of 

DAS-8191Ø-7 cotton sprayed with 2,4-D plus glufosinate are equivalent to non-sprayed 

DAS-8191Ø-7 cotton. In addition, studies conducted under warm and cool conditions 

revealed that the germination and dormancy of DAS-8191Ø-7 cotton did not differ 

significantly from its non-transgenic cotton, near isogenic comparator. 

The data collected here support a conclusion that DAS-8191Ø-7 cotton is substantially 

equivalent to non-transgenic cotton with the exception of the introduced and expression of

aad-12 and pat genes and therefore no more likely to pose a plant pest risk or have a 

significant environmental impact compared to non-transgenic cotton.

7.4. Field Efficacy

As part of the phenotypic, agronomic and ecologic field studies, herbicide injury ratings 

were also collected from DAS-8191Ø-7 cotton entries (non sprayed and sprayed with 2,4-D 

plus glufosinate). Field trial designs and herbicide application ratings are described in 

section 7.1. Herbicide applications were applied at the 3 node and 6 node stage and plants 

were evaluated 14 days after application on a scale of 0 – 100% where 0 represents no injury

and 100 represents complete plant death. 

Results of the herbicide injury data are shown in Table 22. DAS-8191Ø-7 cotton displayed 

minimal herbicide injury for applications at both the 3 node and 6 node stages. These data 

support the excellent tolerance of DAS-8191Ø-7 cotton at the proposed maximum herbicide 

use rate.
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Table 22. DAS-8191Ø-7 Cotton Tolerance to Application of Herbicides

Herbicide1 Application Rate2 Application 

Stage

Percent Plant Injury3

(Mean ± SE)

DAS-8191Ø-7

No Herbicide

DAS-8191Ø-7

Sprayed

2,4-D plus 

glufosinate

1120 g ae/ha (2,4-D)

596 g ai/ha (glufosinate)

3 node 0 ± 0 4 ± 2

6 node 0 ± 0 4 ± 2

1

2,4-D and glufosinate were applied in combination as a tank mixture

2

g ae/ha = grams acid equivalent per hectare, g ai/ha = grams active ingredient per hectare

3 Means for no herbicide treatment (No Herbicide) and sprayed were not statistically compared since the data contained 

insufficient variability to enable ANOVA. Data from the Louisiana field testing site were excluded from this analysis since data 

was not collected on non sprayed plants.
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8. Impact on Environmental and Agronomic Practices

As part of the plant pest assessment required by 7 CFR § 340.6(c)(4), impacts to agricultural 

and cultivation practices must be considered. This section provides a summary of current 

agronomic practices in the U.S. for producing cotton and describes potential impacts

regarding the introduction of DAS-8191Ø-7 cotton into the environment, including gene flow 

and on non-target organisms and endangered species. In addition, this section provides an 

evaluation of the potential impact of DAS-8191Ø-7 cotton on current agronomic practices 

including cultivation, weed control, volunteer management and herbicide resistance 

management. 

With the exception of the presence of the AAD-12 and PAT proteins, DAS-8191Ø-7 cotton 

is phenotypically equivalent to commercially cultivated cotton and is no more susceptible to 

diseases or pests than commercially cultivated cotton. Therefore, there are no likely impacts 

of DAS-8191Ø-7 cotton to the majority of the agronomic practices currently employed in 

cotton production and based on substrate specificity of AAD-12 and PAT, no effect on nontarget organisms or endangered species is anticipated for DAS-8191Ø-7 cotton. 

8.1. Current U.S. Agronomic Practices for Cotton

8.1.1. Cotton Production

Only the cotton boll (consisting of fibers and seeds) is useful for fiber, food and feed with the 

primary use of cotton in the U.S. being fibers which are mainly used in the manufacturing of 

a large number of textiles. Approximately 85% of cotton value is derived from the fiber. The 

boll is processed to remove the fiber for textile use; the remaining fuzzy seed is processed 

into four major food and feed products: oil, meal, hulls and linters. Cottonseed currently 

results in approximately 10% of the world’s oilseed production.

Cotton is the world’s most widely grown fiber crop, accounting for over 35% of the total 

world fiber use (USDA ERS, 2013a). The United States is the top exporter of raw cotton, and 

third in world production behind China and India (USDA ERS, 2013a). U.S. cotton 

production alone accounts for more than $25 billion in products and services annually and 

generates approximately 200,000 U.S. jobs (USDA ERS, 2013a).

Upland cotton, Gossypium hirsutum, accounts for approximately 97% of U.S. annual cotton 

crop production, with Pima (Egyptian) cotton, G. barbadense, accounting for the remaining 

3% of cotton acreage (USDA NASS, 2013c, p.42). In the U.S., cotton is grown across the 

southern states in four major cotton growing regions which include the Southeast, Midsouth, 

Southwest and West regions (Table 23, Figure 47 & Figure 48). Over the past 10 years, 

harvested cotton acres have varied from around 7.5 to 13.8 million acres harvested with the 

lowest harvested acres recorded in 2009 and the highest in 2005 (Table 24).

Texas devotes more area to cotton production and produces more cotton than any other state, 

producing over five million bales in 2012 accounting for nearly 30% of all cotton production 
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in the U.S. (Table 23). Other states producing over one million bales in 2012 included 

Georgia, North Carolina, Arkansas, and California (Table 23).

In 2012, 9.4 million acres of cotton were harvested in the US; with production value 

estimated at just under $6 billion (USDA NASS, 2013a, 2013b). Current estimates of all 

cotton planted in the U.S. for 2013 are 10.2 million acres, with 10.0 million acres estimated 

for upland cotton and the remaining U.S. acreage dedicated to pima cotton which is mainly 

grown and limited to the Southwestern U.S. due to the longer required growing season 

compared to upland cotton (Figure 48).

Table 23. Total U.S. Cotton Growing Regions, States and 2010 – 2012 Harvest, Yield & Production
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Table 24. Cotton Production in the U.S., 2002 - 20131,2 

Figure 47. Planted Upland Cotton Acres by County in the U.S. in 2012

(USDA NASS, 2012b)
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Figure 48. Planted Pima Cotton Acres by County in the U.S. in 2012

(USDA NASS, 2012a)

8.1.2. Cotton Seed Production

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.

The Association of Official Seed Certifying Agencies (AOSCA, 2012) defines the classes of 

cottonseed 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 handled to maintain satisfactory genetic identity and 
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purity. Certified seed is the progeny of Breeder, Foundation, or Registered seed that is 

handled to maintain satisfactory genetic identity and purity. Commercial cotton seed sold and 

planted for typical cotton production is produced predominately to meet or exceed certified 

seed standards.

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 (USDA, 2012a). 

Federal regulation 7 CFR §201.76 specifies minimum land, isolation, field, and seed 

standards required for cotton Foundation, Registered and Certified seed. To qualify as 

Certified seed, only one plant of another variety in 1000 cotton plants or 0.1% of seed of 

other varieties or off-types are permitted (USDA, 2012b). To qualify as Registered seed, only 

one plant of another variety in 5000 cotton plants and 0.05% of seed of other variety or offtypes are permitted. To be certified as Foundation seed, only one plant of another variety in 

10,000 cotton plants and 0.03% of seed of other variety or off-types are permitted.

Agronomic practices for producing cottonseed are similar to commercial production with 

enhanced management practices to ensure high quality, genetically pure seeds. Commercial 

certified cotton seed must meet state and federal seed standards and labeling requirements. 

State seed certification standards vary from state to state, but can be more restrictive than 

standards set forth by AOSCA.

DAS-8191Ø-7 cotton does not differ from conventional cotton in agronomic characteristics 

(Section 7) and thus is not expected to impact U.S. cotton or seed production. Once 

deregulated, DAS-8191Ø-7 cottonseed will be produced in the same manner as commercially 

certified cottonseed, such that it will meet all state and federal seed standards and labeling 

requirements.

8.1.3. Planting and Cotton Development

Soil temperatures have a strong influence on the rate of germination and seedling 

development in cotton, which responds most favorably to soil temperatures around 64°C or 

greater at the appropriate seedling planting depth (Smith et al., 1999). Planting cotton in soil 

conditions with temperatures less than 55°C will generally result in poor vigor and seedling 

disease problems. Under favorable conditions, germination generally occurs with five to 

fifteen days after planting.

Optimal cotton yield is highly dependent on achieving a satisfactory cotton stand which is 

dependent on location, soil conditions, environment conditions, row-spacing and the cultivar. 

Soil conditions can be optimized and cotton has the ability to compensate in response to row 

spacing and plant populations. However, cotton plants in thick stands tend to grow taller, 
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develop more vegetative growth and often cause delays in fruiting; this may alter the 

reproductive/vegetative balance of the plant (Smith et al., 1999). Low plant populations can 

contribute to weed problems and produce inadequate fruit and impact overall yield.

The first cotton squares become visible normally five to eight weeks after planting, 

depending on growing conditions. The majority of cotton bolls harvested tend to come from 

squares which set during the first month of squaring, emphasizing the importance of proper 

crop management during this period including water, weed, nutrient and pest management 

which all have the potential to dramatically decrease crop yield if not managed properly.

Once cotton develops into the later stages of the season, management of the crop is typically 

orientated to harvest. Effective defoliation is an essential step in the overall process of 

harvesting high quality lint. Quick, efficient defoliation is accomplished by chemical means 

and is dependent on the plant-water status, nitrogen fertility status, insect damage, weather 

conditions and the chemical defoliant (Smith et al., 1999). Once defoliated, the cotton is 

harvested mechanically.

8.1.4. Weeds in Cotton

Cotton is a slow-growing plant early in the season and competes poorly with weeds. This is 

especially true in the northern cotton-growing areas, when cotton is planted into cooler soils. 

Studies show that control of weeds during the first four to eight weeks after planting is 

critical to prevent weeds for competing for water, light, nutrients and other resources 

essential for cotton germination and growth (Smith et al., 1999).

Weeds that are most common are not necessarily the most troublesome. The degree of 

importance depends on the expense of control, interference to cotton growth and yield, and 

reduction in lint quality. The type, severity, and impact of weeds in cotton vary between 

geographical regions. In addition, weed populations do not remain static and change over 

time due to reproductive ability, control methods, herbicide regimes, herbicide resistance, 

climate changes and other environmental factors (Smith et al., 1999). It is important for 

proper identification of invading weed species to help develop an effective weed 

management program.

Major weeds in cotton include broadleaf species such as pigweeds, horseweed, and common 

cocklebur, as well as grass species such as Johnsongrass (sorghum halepense) and 

goosegrass (Heap, 2013b). Palmer amaranth, morning glory spp., and nutsedge species are 

reported as both common and troublesome (Webster and Sosnoskie, 2010). Table 25

provides a list of top weeds in cotton as reported by growers in specific cotton-growing 

regions, with glyphosate resistant biotypes being the most pervasive and problematic in the 

Southeast and parts of the Midsouth.
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Table 25. Major Weeds Treated in Cotton by Region in 2011 (Descending Order)1

8.1.5. Weed Management in Cotton

Weed control in cotton uses a combination of cultural, mechanical and chemical methods 

(University of California Agriculture & Natural Resources, 2013c). With herbicide tolerant 

cotton planted on 82% of U.S. acres (USDA NASS, 2013c), herbicides are the primary basis 

of weed management programs. These regimens incorporate herbicides pre-emergence, 

during planting, post-emergence, or a combination (Burgos et al., 2006). Mechanical 

cultivation remains common, particularly in areas of herbicide resistant weeds (University of 

Western Australia, 2010).

A 2010 USDA survey reports that herbicides were applied to 99% of upland cotton, with 

glyphosate being the most widely and heavily used (USDA NASS, 2011). Other commonly 

used herbicides include diuron, pendimethalin, metalochlor, pyrithriobac, and trifluralin 

(University of California Agriculture & Natural Resources, 2013c).

The widespread adoption of glyphosate tolerant crops and subsequent glyphosate use on a 

large portion of cropland has created strong selection pressure for glyphosate resistant weeds. 

In 1996, no weed species were known to be resistant to glyphosate; currently there are 24 

confirmed glyphosate resistant weed species globally. Fourteen are confirmed in the U.S., 

and all occur in cotton growing states (Hurley et al., 2009; Heap, 2013d). For more details 

see Appendix 7.
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Heavy reliance on glyphosate and the resulting emergence of persistent, resistant weeds has 

resulted in increasing emphasis on integrated management approaches. These approaches 

include tillage, hand-weeding, crop rotation, and alternate herbicidal chemistries (WSSA, 

2013).

Few herbicides have been developed for use on cotton as compared to corn and soybean 

(Kendig et al., 1994). DAS-8191Ø-7 cotton and the herbicide systems it enables, provide 

growers needed tools for use in an integrated weed management strategy. 

Weed management decisions are difficult for growers because a single tool cannot effectively 

control all possible weed problems. Effective weed control requires grower implementation 

of management practices that limit the introduction and spread of weeds, help the crop to 

compete with weeds, and prevent weeds from adapting. The combination of weed control 

practices that a grower chooses depends upon the weed spectrum, level of infestation, soil 

type, cropping system, weather, and time and labor available for the treatment option.

8.1.6. Crop Rotation and Tillage Practices

Cotton is commonly rotated with other crops, including corn, soybeans, sorghum, peanut and 

wheat (USDA APHIS, 2011). Market dynamics, geography and grower needs influence the 

choice of rotation crop; crop rotation is used to manage weeds, pests, diseases, erosion, and 

improve soil health (University of California Agriculture & Natural Resources, 2013b). 

While some of the cotton belt still operates under continuous cotton monoculture practice, 

there is evidence that rotation practice is becoming more common due to the benefits it 

provides (Cotton Incorporated, 2013). 

Types of tillage currently used in cotton include conventional, reduced, and conservation 

tillage (Albers and Reinbott, 1994). Data demonstrates that new technologies, such as 

herbicides to control early season weeds, allowed growers to shift away from conventional 

tillage (University of California Agriculture & Natural Resources, 2013c). A Cotton 

Incorporated grower survey found a “significant increase” in conservation tillage from 1990-

2004 (Smith, 2009). However, high weed pressure in recent years has resulted in a reduction 

in conservation tillage practice (Shaw et al., 2012; Robinson, 2013).

8.2. Potential Impacts of DAS-8191Ø-7

Dow AgroSciences has developed DAS-8191Ø-7 cotton, tolerant to the herbicides 2,4-D and 

glufosinate. DAS-8191Ø-7 cotton will provide growers with greater flexibility in selection of 

herbicides for the improved control of economically important weeds; allow an increased 

application window for effective weed control; and provide an effective weed resistance 

management solution to glyphosate resistant weeds.

DAS-8191Ø-7 cotton will be combined with glyphosate tolerant cotton utilizing traditional 

breeding techniques. The combination of herbicide tolerant traits will allow the use of 

multiple herbicides in an integrated weed management program to control a broad spectrum 

of grass and broadleaf weed species in cotton. These herbicides will provide distinct modes 

of actions for use in conjunction with other herbicide active ingredients and modes of action 
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for an effective weed management program in cotton. 2,4-D will provide improved in-crop 

post-emergence control of hard to control glyphosate resistant broadleaf weeds, such as 

pigweed, waterhemp, horseweed, and morningglory. 

8.2.1. Environment – Weediness Potential, Gene Flow and Non-Target /

Endangered Species

8.2.1.1. Weediness Potential

Cotton is not considered to have weedy characteristics in the U.S. and USDA-APHIS has 

determined that cotton (Gossypium hirsutum) is not a plant pest in the United States (USDA 

APHIS, 2011). In addition, cotton is not present on the lists of noxious weed species 

distributed by the federal government (7 CFR Part 360) nor does cotton possess attributes 

commonly associated with weeds; it is relatively slow-growing and does not compete 

effectively with other cultivated plants or primary colonizers (OECD, 2010). In addition, 

commercial varieties of cotton in the U.S. are not effective in invading established 

ecosystems. Cultivated cotton rarely displays any dormancy characteristics and may grow as 

a volunteer only under specific conditions. Volunteers are easily controlled by herbicides or 

mechanical means (Morgan et al., 2011a; Morgan et al., 2011b).

The introduction of 2,4-D, glufosinate, and glyphosate tolerance into cotton will not alter its 

weediness characteristics. Agronomic properties of DAS-8191Ø-7 cotton related to 

weediness, such as germination, emergence, seedling vigor and response to environmental 

stressors have been shown to be identical to conventional cotton (Section 7). If individual 

DAS-8191Ø-7 cotton plants were to overwinter, they can still be effectively controlled 

mechanically or with other herbicide modes of action such as paraquat in burndown, atrazine 

to control 2,4-D cotton volunteers in corn fields, and flumioxazin to control 2,4-D tolerant 

cotton volunteers in soybean fields (Morgan et al., 2011a). Collectively, these findings 

support the conclusion that DAS-8191Ø-7 cotton has no increased weediness compared to 

commercially cultivated cotton. 

8.2.1.2. Gene Flow Assessment 

8.2.1.2.1. Vertical Gene Flow 

Cotton is considered self-pollinating; pollen grains are large, heavy and somewhat sticky; 

thus dissemination by wind is absent or negligible. Pollen-mediated gene flow declines 

steeply, typically below 1% beyond 10 meters from the source (Van Deynze et al., 2005).

Thus, the potential for gene flow from DAS-8191Ø-7 cotton to cultivated, wild or feral 

cotton is highly unlikely. 

Cotton is generally self-pollinating but some cross-pollination can occur, albeit at relatively 

low incidence through activity of pollinating insects (Hutmacher et al., 2006). In the presence 

of pollinators, cross pollination may occur at low levels, with decreasing frequency as 

distance from the source increases (OECD, 2010). In farm scale studies using traditional 

Upland cotton in California, it was found that the outcrossing distance was strongly 

dependent on the presence of bee colonies. When only native pollinators were present in the 

field, 1% out-crossing was detectable over a distance of 1 meter (approximately 3 feet) and 9 
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meters (29.5 feet) when there was high pollinator activity (Van Deynze et al., 2005). Outcrossing declined exponentially with increasing distance from the source plot (Van Deynze et 

al., 2005). In addition, DAS-8191Ø-7 cotton would not be expected to confer a selective 

advantage to, or enhance the pest potential of, progeny resulting from such crosses if they 

were to occur. Therefore, the environmental consequences of gene flow from DAS-8191Ø-7 

cotton to other cotton species is considered to be negligible.

The potential for outcrossing and gene introgression from DAS-8191Ø-7 to cultivated cotton 

in the U.S. (G. hirsutum and G. barbadense) is low since cotton pollen movement by wind is 

limited due to its large and sticky nature. In addition, studies have shown that even in the 

presence of pollinator activity, cross-pollination is limited by distance (Van Deynze et al., 

2005). 

Only two ‘wild’ Gossypium species are known to persist in the U.S. including G. thurberi

found in Arizona and feral populations of G. hirsutum known to occur in South Florida and 

Puerto Rico. In addition, G. thurberi is a diploid (2x) cotton species while DAS-8191Ø-7 

cotton (present in G. hirsutum) is allotetraploid (4x) species, if pollen or gene exchange did 

occur, it would result in a triploid (3X) sterile plant. Such sterile plants have not been 

reported in the wild. Although DAS-8191Ø-7 cotton is capable of crossing with wild G. 

hirsutum, wild species of G. hirsutum are not commonly present in cotton growing areas of 

the U.S. 

G. tomentosum is endemic to the Hawaiian Islands. Cultivated cotton is not grown 

commercially in Hawaii (USDA NASS, 2012c), but may occur in counter-season breeding 

nurseries. In such cases of counter-season nurseries, appropriate isolation distances and 

practices are required for seed productions (Federal regulation 7 CFR §201.76 ) and growing 

regulated cotton (660 feet physical isolation from non-transgenic cotton fields; or minim of 

40 feet of continuous and uninterrupted non-regulated cotton border, plus 10 feet separation 

between the border and adjacent field; or destruction of material prior to flowering),

significantly limiting any potential for gene flow between domesticated cotton and G. 

tomentosum.

Overall, the potential for DAS-8191Ø-7 cotton for crossing to cultivated, wild or feral cotton 

populations is low due to cotton’s self-pollination nature, geographic isolation barriers and or 

genetic make-up. In addition, DAS-8191Ø-7 cotton would not be expected to confer a 

selective advantage to, or enhance the pest potential of, progeny resulting from such crosses 

if they were to occur. Therefore, the environmental consequences of gene flow from DAS8191Ø-7 cotton to other cotton species is considered to be negligible. 

8.2.1.2.2. Horizontal Gene Flow

DAS is unaware of any reports regarding the unintended transfer of genetic material from 

cotton to other sexually-incompatible species. In the unlikely event that such a transfer were 

to take place, the aad-12 and/or pat genes from DAS-8191Ø-7 cotton would not present a 

human health or plant pest risk; this conclusion is based on the safety data presented in this 

petition. Therefore, in the highly unlikely event that horizontal gene flow were to occur, the 
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presence of aad-12 and/or pat traits would not be expected to increase pest or weed potential 

in the recipient species.

8.2.1.3. Non-Target Organisms and Endangered Species

Based on substrate specificity of the AAD-12 enzyme activity, no effect on non-target 

organisms or endangered species is anticipated for DAS-8191Ø-7 cotton. The aad-12 gene 

and expressed protein are present in nature in the soil bacterium Delftia acidovorans. The pat 

gene and the expressed protein are present in other crops grown in the United States with no 

effects on non-target organisms or endangered species. AAD-12, and PAT are not potential 

food allergens or toxins (Section 5.1.5.2, 5.1.5.3 and 5.2.5) and DAS-8191Ø-7 cotton has 

been shown to be substantially equivalent to non-transgenic cotton based on the 

compositional analysis of cottonseed (Section 6). Observations made during field testing of 

DAS-8191Ø-7 cotton revealed no effects on invertebrate populations (Section 7.2.2) and 

agronomic characteristics were found to be equivalent to non-transgenic cotton (Section 7). 

The Endangered Species Act (ESA) (16 USC 1531) is administered by the U.S. Fish & 

Wildlife Service (FWS). Under ESA, Section 6 requires federal agencies who conduct 

activities which may affect listed species to consult with the FWS to ensure that listed 

species are protected should there be a potential impact. It is not anticipated that DAS8191Ø-7 cotton will impact any currently listed species of concern since it is not anticipated 

that DAS-8191Ø-7 cotton will cause new cotton acres to be planted in areas that are not 

already in agricultural use. Habitat disruption within DAS-8191Ø-7 cotton fields would be 

comparable to other herbicide tolerant cropping systems.

Cultivated cotton is not invasive or weedy, and these properties are not anticipated to be 

altered by the insertion of the genes conferring tolerance to herbicides. Based on the data, it 

is reasonable to conclude that DAS-8191Ø-7 cotton will not affect threatened or endangered 

species or adversely affect or change designated critical habitats as compared to current 

commercial agricultural practices.

2,4-D is not currently registered for uses specific to cotton production. The environmental 

fate and ecological effects on non-target organisms for the use of 2,4-D in cotton will be 

addressed by the EPA as part of its review process under FIFRA.

Glufosinate is currently registered for use in cotton production as a post-emergent herbicide 

treatment. The environmental fate and ecological effects of these herbicides on non-target 

organisms and endangered species have been addressed by the EPA as part of its review 

process. 

8.2.1.4. Potential Impacts on Boll Weevil Eradication Program

The National Boll Weevil Eradication Program (BWEP) was launched in the late 1970’s by 

USDA's Animal and Plant Health Inspection Service (APHIS) to eradicate the boll weevil in 

the cotton-growing areas of the U.S. Prior to implementation of the program, boll weevils 

were found throughout southern cotton growing states, including California, Arizona, and 

New Mexico. By 2009, eradication was completed in all U.S. cotton growing regions except 
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for a small region in Texas (National Cotton Council of America, 2013). In 2012, boll 

weevils were captured in only three of the sixteen Texas eradicated zones, with a 

substantially reduced number of boll weevils in two of the three zones (Texas Boll Weevil 

Eradication Foundation Inc., 2012). Eradication continues today for a small region of Texas.

The BWEP and the Texas Boll Weevil Eradication Foundation (TBWEF) have developed 

comprehensive eradication programs over the past several decades to monitor, trap and treat 

infected boll weevil zones in the U.S. and northern Mexico (Texas Boll Weevil Eradication 

Foundation Inc., 2012; National Cotton Council of America, 2013). Eradication and control 

of boll weevil is primary based on the use of the insecticide malathion. This product has been 

highly efficacious in the control and eradication of boll weevil across the southern US. In 

addition to chemical control, the boll weevil eradication programs rely on and promotes 

farmers to use cultural controls such as planting during the most advantageous periods of 

time, harvesting early and thoroughly, and destroying crop residues and failed plantings in a 

timely manner (Texas Boll Weevil Eradication Foundation Inc., 2012).

 

Because of the warm climate and rainfall in parts of south Texas, cotton stalks left in the field 

after harvest can regrow. This provides hostable cotton structure (squares and bolls) that boll 

weevils can use as both food and reproductive harbors, potentially increasing overwintering 

survivability. Methods available for the management of cotton stalks include mechanical 

destruction or removal (shredding, tillage, and stalk pullers), flood irrigation, and herbicide 

application. Applied herbicides may include 2,4-D, dicamba,thifensulfuron-methyl + 

tribenuronmethyl and others. Based on the cotton growing zones in Texas, The Texas 

Department of Agriculture has provided guidelines on appropriate cotton stalk destruction 

methods (Texas Department of Agriculture, 2013).

In order for destruction of cotton plants to be deemed complete, both plants re-growing from 

stalks and those growing from seed must be eliminated. To ensure compliance of cotton crop 

destruction, the BWEP has extensive quality control measures in place to monitor and 

enforce the destruction of cotton plants post-harvest (National Cotton Council of America, 

2013). Herbicides are typically the management option of choice, largely due to efficacy, 

convenience and cost. Among the herbicides labeled for the management of cotton stalks, 

sequential applications of 2,4-D are most commonly used.

Although methods other than 2,4-D are currently available for the management of cotton 

stalks, Dow AgroSciences recognizes the utility of 2,4-D in the BWEP and is committed to 

identifying other herbicides that can be used for cotton stalk control. Since 2010, Dow 

AgroSciences has been actively engaged in research focused on identifying alternative 

herbicides that meet the BWEP performance criteria of no hostable cotton structures during 

the prescribed period for the pest management zones of interest.

Effective treatments which result in no hostable structure have been identified including 

dicamba and dichlorprop and combinations of dicamba plus dichlorprop. Additional testing 

will be conducted over the next several years to further improve efficacy of these alternative 

treatments for controlling hostable structures. 
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The introduction of DAS-8191Ø-7 cotton in Texas is not anticipated to significantly impact 

the BWEP or TBWEF in the U.S or Mexico. With the exception of fields where 

DAS-8191Ø-7 cotton is planted, the standard management practices of the BWEP and 

TBWEF remain unaffected. In fields planted with DAS-8191Ø-7 cotton, identical, effective 

boll weevil control measures (mapping, trapping, insecticide control and framing practices) 

set forth by BWEP, TBWEF and Texas Department of Agriculture will remain effective 

measures for control and eradication of boll weevil populations. The only potential impact 

could be the need to use a herbicide stalk destruct method other than 2,4-D that meets the 

BWEP performance criteria. Collaborative research by Dow AgroSciences and public-sector 

scientists have identified herbicides other than 2,4-D that effectively meet the BWEP 

performance criteria, to mitigate this potential impact. Dow AgroSciences is committed to 

the long-term success of BWEP, and will continue its efforts to identify effective herbicide 

alternatives and management plans for cultivation of cotton in boll weevil regions of the US.

8.2.2. Agronomic Practices – cultivation/management, weed control and 

volunteer management

8.2.2.1. Potential Impacts on Cultivation and Management Practices

Transgenic varieties of cotton have been rapidly adopted. USDA estimates 10.3 million acres 

for all U.S. cotton planted in 2013, with 10 million of these acres being upland cotton 

(USDA NASS, 2013c). Of those acres, 90% have been planted to transgenic varieties: 8% 

are insect resistant varieties only, 15% are herbicide tolerant varieties only, and 67% are

stacked varieties (herbicide resistance plus insect resistance) (USDA NASS, 2013c). 

Current cotton cultivation and management practices incorporate the management of 

herbicide tolerant traits. DAS-8191Ø-7 cotton is phenotypically and agronomically 

comparable to conventional cotton (Section 7). Ecological observations during field testing 

have shown no changes in insect susceptibility (Section 7.2.2); therefore no impacts are 

expected on insect control practices for DAS-8191Ø-7 cotton. It is anticipated that the same 

management practices for today for herbicide tolerant cotton will also be appropriate for 

DAS-8191Ø-7 cotton.

8.2.2.2. Potential Impact on Weed Control Practices

DAS-8191Ø-7 cotton will provide expanded weed management options in cotton. Postemergence applications of 2,4-D control a broad spectrum of broadleaf weeds. 2,4-D also has 

some short-lived soil residual activity (approximately three weeks) which provides limited 

residual control of later germinating broadleaf weeds (Lajeunesse, 1997). Thus, 

DAS-8191Ø-7 cotton will provide greater flexibility to farmers compared to glyphosate 

alone in weed management systems. Inclusion of 2,4-D would contribute to the control of 

glyphosate resistant and hard to control broadleaf weeds, plus slow down the selection for 

more glyphosate resistant broadleaf weeds (Powles, 2008a).

2,4-D is not currently registered for use on cotton; however, 2,4-D is used for weed control in 

burndown programs in no-till fields, due to 2,4-D’s efficacy against broadleaf weeds, 

including glyphosate resistant biotypes (University of Delaware College of Agriculture & 

Natural Resources, 2008). 2,4-D has approximately three weeks of residual soil activity. For 
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growers who choose to use 2,4-D in a burndown regimen, a 30 day waiting interval is 

typically required prior to planting. This time period may fluctuate slightly depending on the 

individual label.

In DAS-8191Ø-7 cotton, the proposed use pattern will allow application of 2,4-D at 

burndown or pre-emergence (1 lb ae/A or 1120 g ae/ha) without plant back restrictions, 

followed by one or two post-emergence (0.5 – 1.0 lb ae/A or 560-1120 g ae/ha) applications 

at least 12 days apart, over-the-top of the cotton, up to the mid-bloom stage of development 

(Figure 49). Thus the proposed maximum seasonal rate of 2,4-D on cotton is estimated to be 

3.0 lbs ae/ha. DAS-8191Ø-7 cotton will allow growers to apply 2,4-D from burndown or preemergence up through the mid-bloom stage without risk of crop injury. This will provide 

new options for improved weed control during the cotton development period when weeds 

have the greatest potential yield impact. 

Figure 49. 2,4-D Use in DAS-8191Ø-7 Cotton

While 2,4-D is currently registered as a pesticide, supporting information on proposed label 

changes for its use with DAS-8191Ø-7 cotton is being provided by Dow AgroSciences to 

U.S. EPA for review. Application for Dow AgroSciences’ 2,4-D formulation (Enlist Duo) 

registration on DAS-8191Ø-7 cotton and tolerance petition is scheduled for submission to 

U.S. EPA in early 2014 with anticipated approval in mid-2015 based on set PRIA timelines. 

Dow AgroSciences is also developing an extensive stewardship program that will include 

technological advancements in application to reduce potential for off-target movement, as 

well as utilizing several media venues to educate and facilitate adoption of the technology 

and decision management tools to ensure the proper use and stewardship of both the trait and 

the associated chemical technologies. DAS’ stewardship program is detailed further in 

Appendix 8.
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When combined with additional herbicide mode of actions through traditional breeding 

methods, DAS-8191Ø-7 cotton varieties will improve weed control by allowing use of 

herbicide combinations or mixtures which can provide more consistent performance in postemergence weed control programs. They counteract glyphosate rate-creep (steady increase in 

the amount of glyphosate herbicide rates to effectively control weeds) needed over time to 

obtain effective weed control on hard to control weeds (Jaehnig, 2005). DAS-8191Ø-7 cotton

will allow use of a low cost, high performance solution to reduce the escalation of glyphosate 

and ALS resistance in weed populations.

8.2.2.3. Volunteer Management

Commercial varieties of cotton in the U.S. are not considered weeds and are not effective in 

invading established ecosystems (USDA APHIS, 2011). Cultivated cotton rarely displays any 

dormancy characteristics and may grow as a volunteer only under specific conditions. Any 

volunteers are easily controlled by herbicides or mechanical means (Morgan et al., 2011a; 

Morgan et al., 2011b). Cotton is a slow grower, and does not compete effectively with other 

cultivated plants or primary colonizers (OECD, 2010).

Cultivated cotton is considered self-pollinating (OECD, 2010) and is not classified as a weed 

either in the scientific literature or by USDA (USDA APHIS, 2011). If DAS-8191Ø-7 cotton 

were to cross with cotton varieties expressing tolerance to herbicides with different modes of 

action and produce cotton volunteers with multiple herbicide tolerance, volunteers can still 

be effectively controlled mechanically or with other herbicide modes of action. Additionally, 

agronomic practices such as appropriate variety selections, crop rotation, and rotation of 

herbicides with different modes of action can be used to avoid or manage volunteer cotton

tolerance to one or a few herbicides.

8.3. Herbicide Resistance Management

8.3.1. Herbicide Resistance

Herbicides have revolutionized weed control in most countries around the world. Herbicides 

are often the most reliable and least expensive method of weed control available, but reliance 

upon herbicides as the primary method of weed control can have unintended consequences

(Appendix 7). The widespread use of herbicides can lead to weed populations that are no 

longer susceptible to the herbicide being used. The Weed Science Society of America defines 

herbicide resistance as "the inherited ability of a plant to survive and reproduce following 

exposure to a dose of herbicide normally lethal to the wild type” (WSSA, 2013a). Target 

plants with genes conferring resistance to a given herbicide can occur naturally within a 

population, but in extremely small numbers. Such plants remain reproductively compatible 

with the wild-type and can pass resistance genes on to their progeny (Mallory-Smith, 2010). 

Repeated use of the herbicide may allow these resistant plants to survive and reproduce. The 

number of resistant plants then increases in the population until the herbicide no longer 

effectively controls the weed. Thus, this is an evolutionary process (Moss, 2002), whereby a 

population changes from being susceptible to being resistant. Individual plants do not change 

from being susceptible to being resistant; rather, the proportion of resistant individuals within 

the population increases over time. 
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Herbicide resistant weeds have been a problem for growers for decades. During the 1970s, up 

to 30 different weed species were reported to be resistant to the triazine herbicides (Bandeen

et al., 1982). Today, there are 403 unique cases of herbicide resistant weeds globally, with 

217 species: 129 dicots and 88 monocots (Heap, 2013c). A weed biotype is a sub-type or 

sub-population of a weed species; in this case one that has developed resistance to one or 

more herbicides. Report of a resistant biotype for a given weed species does not mean that 

weed resistance is common, widespread, or persistent in that species. There are generally 

many other options available to control these resistant biotypes. 

Table 26 shows a tabular summary of the total number of resistant species for each herbicide 

mode of action as of August 2013. Two different classification systems have been developed 

independently by Herbicide Resistance Action Committee (HRAC) and the Weed Science 

Society of America (WSSA) to communicate the mode of action of herbicides; both are 

included in Table 26. Weeds which have developed resistance to one herbicide may also be 

resistant to other herbicides which have the same mode of action. Additional information on 

glyphosate, the evolution of herbicide resistant weeds, and 2,4-D can be found in Appendix 7.

8.3.2. Factors Impacting Herbicide Resistance

There are several factors to consider when assessing the risk for herbicide resistance in a 

weed species. Some of these relate to the biology of the weed species in question, others 

relate to particular farming practices. The key factors influencing a plant’s potential to 

develop resistance have been outlined by the HRAC, an industry initiative that fosters 

cooperation between plant protection manufacturers, government, researchers, advisors and 

farmers. These key factors include the number or density of weeds, natural frequency of 

resistant plants in the population, seed soil dormancy potential, frequent use of herbicides 

with a similar mode of action, cropping rotations with reliance primarily on herbicides for 

weed control, and lack of non-chemical weed control practices (HRAC, 1998).

A matrix that can be used to evaluate the risk of selection for herbicide resistant weeds based 

on cropping system practices in shown in Table 27. This table assesses the risk of herbicide 

resistance development for each management practice as either “low”, “medium”, or “high”. 

There is a high risk of resistance when limited management tools are used – for example, 

sole reliance on a chemical, such as glyphosate. When additional methods are used, the risk 

of weed resistance decreases. Cultural controls are an effective tool in battling weed 

resistance; these include practices such as delayed planting, cultivation or plowing prior to 

sowing, using certified crop seed free of weeds, and others (HRAC, 2013b). 
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Table 26. Number of Herbicide Resistant Weeds Reported Globally by Herbicide Group 

and Mode of Action1

Herbicide Group Mode of Action

HRAC 

GroupWSSA Group

Example 

Herbicide

Total 

Count

ALS Inhibitors Inhibition of acetolactate synthase ALS 

(acetohydroxyacid synthase AHAS) B 2 Chlorsulfuron 132

Photosystem II 

Inhibitors Inhibition of photosynthesis at photosystem II C1 5 Atrazine 71

ACCase Inhibitors Inhibition of acetyl CoA carboxylase 

(ACCase) A 1 Diclofop-methyl 43

Synthetic Auxins Synthetic auxins (action like indoleacetic 

acid) O 4 2,4-D 30

Bipyridiliums Photosystem-I-electron diversion D 24 Paraquat 28

Ureas and Amides Inhibition of photosynthesis at photosystem II C2 7 Chlorotoluron 23

Glycines Inhibition of EPSP synthase G 9 Glyphosate 24

Dinitroanilines and 

others Microtubule assembly inhibition K1 10 Trifluralin 11

Thiocarbamates and 

others

Inhibition of lipid synthesis – not ACCase 

inhibition N 8 Triallate 8

Chloroacetamides and 

others

Inhibition of cell division (inhibition of very 

long chain fatty acids) K3 15 Butachlor 4

Triazoles, Ureas, 

Isoxazolidiones

Bleaching: Inhibition of carotenoids 

biosynthesis (unknown target) F3 11 Amitrole 5

4-HPPD Inhibitors Bleaching: Inhibition of 4-hydroxyphenylpyruvate-dioxygenase (4-HPPD) F2 27 Isoxaflutole 2

PPO Inhibitors Inhibition of protoporphyrinogen oxidase 

(PPO)

E

14 Oxyfluorfen 6

Glutamine Synthase 

Inhibitors Inhibition of glutamine synthetase H 10 Glufosinateammonium 2

Nitriles and others Inhibition of photosynthesis at photosystem II C3 6 Bromoxynil 4

Carotenoid 

Biosynthesis Inhibitors

Bleaching: Inhibition of carotenoids 

biosynthesis at the phytoene desaturase step F1 12 Flurtamone 3

Arylaminopropionic 

Acids Unknown Z 25 Flamprop-methyl 2

Unknown Unknown Z 26 (chloro) – flurenol 2

Mitosis Inhibitors Inhibition of mitosis / microtubule 

polymerization inhibitor K2 23 Propham 1

Cellulose Inhibitors Inhibition of cell wall (cellulose) synthesis L 27 Dichlobenil 1

Organoarsenicals Unknown Z 17 MSMA 1

Total herbicide resistant weeds 403

1

(Heap, 2013a)
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Table 27. Assessment of Resistance Risk by Evaluation of Cropping Systems1

Management Option Risk of Resistance

Low Moderate High

Herbicide mix or rotation in 

cropping system

>2 modes of action 2 modes of action 1 mode of action

Weed control in cropping system Cultural, mechanical and 

chemical

Cultural and chemical Chemical only

Use of same MOA per season Once More than once Many times

Cropping system Full rotation Limited rotation No rotation

Resistance status to MOA Unknown Limited Common

Weed infestation Low Moderate High

Control in last 3 years Good Declining Poor

1

(Nevill et al., 1998; HRAC, 2013a)

Development of herbicide resistance is often thought of as a problem caused by the herbicide 

itself. However, it is well documented that resistance results from management practices that 

have relied too heavily on a particular herbicide as the sole method of weed control. Under 

these conditions, the risk of weeds developing herbicide resistance is greatest: the best 

defense is diversity in weed management practices.

8.3.3. Herbicide Resistance Management

Although no cases of glyphosate resistant weeds were documented for 20 years after the 

launch of glyphosate (Dyer, 1994), glyphosate resistant biotypes of several weed species 

have now been reported in the United States (Powles, 2008b). This may be attributed to 

increased reliance on glyphosate for weed control after the launch of glyphosate tolerant

soybeans (1996), cotton (1997), and corn (1998). The adoption of genetically engineered 

(GE) technology from 1996 onwards, primarily glyphosate tolerant crops, is illustrated in 

Figure 50: herbicide tolerant (HT) cotton is shown in dark blue; insect resistant (Bt) cotton is 

shown in green (USDA ERS, 2013b). The resulting evolution of glyphosate resistant weed 

populations threatens the ongoing sustainability of glyphosate and its contributions to world 

food production (Duke and Powles, 2008a).

As the number of glyphosate resistant weed species increases, it becomes increasingly 

important for growers to introduce greater diversity into their weed management programs 

(Powles, 2008a). This diversity could be achieved with herbicide rotations/sequences, 

mixtures of robust herbicides with different modes of action, and use of non-herbicide weed 

control tools. Glyphosate is increasingly being mixed with effective doses of other herbicides 

to manage these hard to control and resistant weed species. New herbicide tolerant traits that 

allow selective use of additional herbicides with a wider weed control spectrum compared to 

conventional herbicides can be used to control glyphosate resistant weed populations and 

reduce selection pressure for additional glyphosate resistant weed species. Table 28 shows 

that several common weeds in U.S. agriculture which are resistant to or difficult to control 

with glyphosate or ALS herbicides can be effectively controlled with 2,4-D.
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Figure 50. Adoption of GE Crops in the U.S.

Introduction of DAS-8191Ø-7 cotton will give farmers one more tool for use in their weed 

management programs and help ensure the long term sustainability of weed management 

programs, including the use of glyphosate. DAS-8191Ø-7 cotton will be combined with 

glyphosate tolerant cotton utilizing traditional breeding techniques. The combination of 

herbicide tolerance traits will allow the use of multiple herbicides in an integrated weed 

management program to control a broad spectrum of grass and broadleaf weed species in 

cotton. 

DAS-8191Ø-7 cotton will allow use of 2,4-D to control glyphosate resistant weeds and 

significantly delay the selection for glyphosate resistance in other weed species. The same 

benefits of DAS-8191Ø-7 cotton can be extended to other herbicide tolerant cropping 

systems, such as those with tolerance to glufosinate or ALS-inhibiting herbicides.

Table 28. Glyphosate and ALS Resistant Weeds Controlled by 2,4-D

1

Weed Species Glyphosate (year)2 ALS Herbicides (year)22,4-D

Common lambsquarters Difficult: Suspected Resistant (2004) Resistant (2001) Susceptible

Common ragweed Confirmed Resistant (2004) Resistant (1998) Susceptible

Eastern black nightshade Difficult (2004) Resistant (1999) Susceptible

Giant ragweed Confirmed Resistant (2004) Resistant (1998) Susceptible

Marestail (horseweed) Confirmed Resistant (2000) Resistant (2000) Susceptible

Palmer amaranth Confirmed Resistant (2005) Resistant (1991) Susceptible

Waterhemp spp. Confirmed Resistant (2005) Resistant (1993) Susceptible

Prickly sida Difficult (2004) Resistant (1993) Susceptible

1

(Heap, 2013c)

2 Year first reported
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DAS-8191Ø-7 cotton will expand the range of herbicides that can be used in herbicide

tolerant cotton production systems, improving the ease and effectiveness of managing 

resistant and hard to control weeds and delaying the evolution of resistance to glyphosate and 

other herbicides.

8.4. Summary of Environmental Consequences and Impact on Agronomic Practices

Field testing results confirm that AAD-12 and PAT proteins expressed in DAS-8191Ø-7 

cotton provide robust tolerance to 2,4-D and glufosinate respectively. There are no new 

phenotypic characteristics in DAS-8191Ø-7 cotton to indicate it is any different from 

conventional cotton in weediness potential, and like conventional cotton, the risk of gene 

flow from DAS-8191Ø-7 cotton to wild relatives in the U.S. is negligible.

No significant impact is expected on current crop management practices, non-target or 

endangered species, crop rotation, or volunteer management from the introduction of DAS8191Ø-7 cotton. The availability of DAS-8191Ø-7 cotton will have a beneficial impact on 

weed control practices by providing growers with another tool to address their weed control 

needs. The availability of DAS-8191Ø-7 cotton will allow growers to proactively manage 

weed populations while avoiding adverse population shifts of troublesome weeds or the 

development of resistance.

With the exception of the presence of the AAD-12 and PAT proteins, DAS-8191Ø-7 cotton 

is phenotypically equivalent to commercially cultivated cotton and is no more susceptible to 

diseases or pests than commercially cultivated cotton. Based on the data presented 

throughout this petition, DAS submits that DAS-8191Ø-7 cotton is unlikely to pose a plant 

pest risk.
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9. Adverse Consequences of Introduction

Field and laboratory testing of DAS-8191Ø-7 cotton demonstrated that the transgenic cotton 

is substantially equivalent to non-transgenic cotton apart from the intended change of 

herbicide tolerance. DAS knows of no study results or other observations indicating that 

there would be adverse consequences from the introduction of DAS-8191Ø-7 cotton.

10. Appendices

Appendix 1. Methods for Molecular Characterization of DAS-8191Ø-7 Cotton

Appendix 2. Methods and Results for Characterization of AAD-12 Protein

Appendix 3. Methods and Results for Characterization of PAT Protein

Appendix 4. Methods for AAD-12 & PAT Protein Expression Analysis

Appendix 5. Methods for Compositional Analysis

Appendix 6. Compositional Literature Ranges for Non-Transgenic Cottonseed

Appendix 7. Evolution of Herbicide Resistant Weeds in Transgenic Crops

Appendix 8. Stewardship of Herbicide Tolerant Trait Technology for DAS-8191Ø-7 Cotton

Appendix 9. USDA Notifications for DAS-8191Ø-7 Cotton

Appendix 10. References
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Appendix 1. Methods for Molecular Characterization of DAS-8191Ø-7 Cotton

DAS-8191Ø-7 cotton Material

Transgenic cotton seeds from five distinct generations of cotton containing event DAS8191Ø-7 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 (LFS) test according to the manufacturer’s 

instructions (Envirologix Inc.). Each plant was given a “+” or “-” for the presence or 

absence of the PAT protein. 

Control Cotton Material

Seeds from the non-transgenic cotton variety Coker 310 were planted in the greenhouse. 

The Coker 310 seeds had a genetic background representative of the transgenic seeds but 

did not contain the aad-12 or pat genes.

Reference Materials

DNA of the plasmid pDAB4468 was added to samples of the non-transgenic control 

genomic DNA at a ratio approximately equivalent to 1 copy of the transgene per cotton

genome with a cotton genome size of ~2.2 × 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 pDAB4468. 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 a 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 ground individually in liquid nitrogen, and then CTAB extraction buffer (100 mM 

Tris-HCl, pH 8.0, 20 mM EDTA, 1.4 M NaCl, 2% CTAB, 2% PVP-10, 2% -ME) was 

added to samples at a ratio of about 5:1 plus over 10 μL each of RNase-A (Qiagen) and 

proteinase K (Qiagen). After approximately 2 hours of incubation at ~65 °C with gentle 

shaking, samples were centrifuged and the supernatants were extracted with equal volume 

of chloroform : octanol = 24:1 three times. DNA was precipitated by mixing the 

supernatants with approximately 0.7 volume of isopropanol. The precipitated DNA was 

rinsed with 70% ethanol, air-dried, then dissolved in appropriate volume of 1 TE buffer 

(pH 8.0). The resultant genomic DNA was further purified with a Genomic-tip 500/G 

following the manufacturer’s instructions (Qiagen). The DNA was quantified with a 
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PicoGreen reagent (Invitrogen), and was visualized on an agarose gel to check for genomic 

DNA quality.

DNA Digestion and Electrophoretic Separation of the DNA Fragments

Genomic DNA extracted from the cotton 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). DIGlabeled 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 re-hybridized with a different DNA probe. 
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Appendix 2. Methods and Results for Characterization of AAD-12 Protein

Materials & Methods for Characterization of AAD-12 Protein

DAS-8191Ø-7 transgenic cotton material

The AAD-12 protein was extracted from the leaf tissue of greenhouse grown transgenic 

cotton event DAS-8191Ø-7 (T3 generation see Figure 4). Prior to harvest, the AAD-12 

protein expression was verified by lateral flow strip testing and the leaf tissue from DAS8191Ø-7 cotton was harvested, frozen, lyophilized, ground, and stored at -80 C. Test 

material used for characterization of DAS-8191Ø-7 cotton AAD-12 are listed in Table 29.

Control cotton material

The control cotton line had a genetic background representative of DAS-8191Ø-7 cotton 

plants, but did not contain aad-12. Seeds from this isogenic non-transgenic cotton line were 

planted and the resulting leaf tissue was harvested and processed under the same conditions 

as DAS-8191Ø-7 cotton.

AAD-12 Reference material

Recombinant AAD-12 protein was produced and purified from the microbe Pseudomonas

fluorescens and had a molecular weight of 32 kDa. The commercially available reference 

substances used in this study are listed in Table 30. 

Table 29. Test Material for AAD-12 Characterization

Test Substance Source Lot Number Assay Reference

Cotton AAD-12 Cotton 

DAS-8191Ø-7 

TSN304178 SDS-PAGE, 

Glycosylation, 

MALDI-TOF, MALDITOF/TOF MS/MS, 

Activity Assay

NA

Control Cotton 

Line

isogenic / nontransgenic cottonTSN304177 SDS-PAGE, Western NA

Cotton crude 

leaf extract

Cotton 

DAS-8191Ø-7

NA SDS-PAGE, Western NA

Microbe-derived 

AAD-12

Pseudomonas 

fluorescens

TSN030732-003 

(466-028B)

SDS-PAGE, Western, 

Glycosylation, Activity 

Assay

Microbe-derived 

AAD-12

Pseudomonas 

fluorescens

TSN030732-002 

(466-028A)

MALDI-TOF, MALDITOF/TOF MS/MS or 

ESI-LC/MS
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Table 30. Commercially available reference substances for AAD-12 Characterization

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

NH175044 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

NH175569 SDS-PAGE 

& 

Glycosylation 

assay

ThermoFisher

Prestained Molecular 

Weight Markers

Novex Sharp 

prestained protein 

standards

1167391 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

SDS-PAGE and western blot analysis of crude cotton leaf extracts

SDS-PAGE and western blot analyses of the crude protein extracts prepared from the 

transgenic DAS-8191Ø-7 and non-transgenic-cotton leaf were performed with Bio-Rad 

Criterion gels fitted in a Criterion Gel chamber with XT MES running buffer (Bio-Rad). 

Extracts were prepared by bead-grinding (Geno-grinder) ~40 mg of the ground cotton leaf 

tissue for 3 minutes in a micro-centrifuge tube containing ~1.0 mL PBST supplemented 

with 5 mM EDTA, 23 mM β-mercaptoethanol, and protease inhibitors. Supernatants were 

isolated by subjecting the homogenate to centrifugation at ~20,000 × g (4 °C), and 100 L 

of each extract was mixed with 100 L of 2× Laemmli sample buffer (Bio-Rad) containing 

freshly added -mercaptoethanol (Bio-Rad) and heated for ~10 minutes at ~95 C. After a 

brief centrifugation (2 min at 20,000 × g), 30 μL of each supernatant was loaded directly on 

the gel. The reference standards, microbe-derived AAD-12 and BSA (ThermoScientific), 

were diluted to an appropriate concentration and combined with freshly prepared Laemmli 

sample buffer containing 5% -mercaptoethanol. The electrophoresis was conducted at a 

constant voltage of 150 V for ~60 minutes. 
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After separation, the gel was cut in half and one half was stained with ThermoScientific 

GelCode Blue protein stain and then 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 V. The transfer buffer contained 20% methanol and 

Tris/glycine buffer from Bio-Rad. After transfer, the membrane was probed with an AAD12 specific polyclonal rabbit antibody (α-AAD-12 PAb, DAS 1197-167-2, 4.3 mg/mL) for 

60 minutes (1:5000 dilution) at room temperature. A 1:10,000 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.

Protein purification of AAD-12 from DAS-8191Ø-7 cotton plant tissue for structural 

analyses

An AAD-12 immunoaffinity resin was prepared by mixing 50 μL of a slurry of Protein A/G 

resin (ThermoScientific) with 200 μg α-AAD-12 monoclonal antibody and incubating 

overnight at 4 °C. The resin was washed with PBST and the bound IgG irreversibly coupled 

to the resin by incubating in 200 μL PBST containing 1.8 mM DSS for 60 minutes at room 

temperature with mixing. Cross-linking was terminated by incubating for 5 minutes 

following the addition of 500 μL of 1.0 M Tris-HCl pH 8.0. The resin was then washed 

extensively with PBST and used immediately for immune-capture of the AAD-12 protein.

To generate a cotton leaf extract suitable for AAD-12 isolation, 5-10 grams of ground, 

lyophilized DAS-8191Ø-7 cotton leaf tissue was suspended by stirring in cold extraction 

buffer (50 mM HEPES pH 7.8, 300 mM NaCl, 10 mM EDTA) at a ratio of ~15 mL of 

buffer per gram of dry tissue. A protease inhibitor tablet (Roche) and 10% (w/w) PVPP 

were added to the suspended tissue. The mixture was stirred at 4 °C for 30 minutes to fully 

hydrate and extract soluble proteins. The mixture was then filtered through a single layer of 

pre-wetted miracloth (Calbiochem) and the extract then further clarified by centrifugation at 

38,000 × g for 30 minutes. The resulting primary supernatant was removed and mixed with 

an equal volume of extraction buffer supplemented with 2 M Urea and 0.2% Tween-20 for a 

final concentration of 1 M and 0.1% Tween-20 respectively. To isolate AAD-12, typically 

15 mL of the leaf extract was incubated with 50 μL of freshly prepared AAD-12 capture 

resin. Binding reactions were incubated in batch at 4 °C for 3 hours to overnight. The resin 

was collected by centrifugation and then successively washed twice with 1.0 mL extraction 

buffer containing 1 M urea + 0.1% Tween-20 and then washed one time each with 1.0 mL 

extraction buffer with 0.5 M Urea + 0.05% Tween-20 followed by 1.0 mL extraction buffer 

alone. Lastly, the resin was washed once with either1.0 mL of 50 mM HEPES pH 7.8, 0.1 

mM EDTA or 1.0 mL of PBST. The resin containing captured AAD-12 was either stored at 

-80 oC or analyzed immediately.
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Detection of post-translational glycosylation

Glycosylation analysis was used to determine whether DAS-8191Ø-7 cotton AAD-12 was 

post-translationally modified with covalently bound carbohydrate moieties. The resin 

containing the immunoaffinity-captured, cotton-derived AAD-12 protein was mixed with 50 

– 100 μL Laemmli sample buffer (Bio-Rad) lacking -mercaptoethanol and the sample was 

then incubated at ~95 °C for 10 minutes to solubilize AAD-12. The microbe-derived AAD12, soybean trypsin inhibitor, bovine serum albumin, and horseradish peroxidase were 

similarly diluted with 2X SDS-PAGE sample buffer to the approximate concentration of the 

purified cotton AAD-12 protein. As with AAD-12, all control proteins were also incubated 

for 10 minutes at ~95 C. All samples were then centrifuged at 20,000 × g for 2 minutes to 

obtain a clarified supernatant. The resulting supernatants were applied directly to duplicate 

Mini-Protean TGX gels (Bio-Rad) and electrophoresed at 150 V for ~50 minutes. 

After electrophoresis, one gel was stained with GelCode Blue stain (ThermoScientific) 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 

second 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 30 minutes. Finally, the gel was immersed in the reduction solution for 

5 minutes, and rinsed with 3% acetic acid. Glycoproteins 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.

Mass spectrometry peptide mass fingerprinting and sequence analysis of plant and microbederived AAD-12 protein

The immunoaffinity purified plant-derived AAD-12 protein was subjected to in-gel 

digestion by trypsin, chymotrypsin, and Asp-N followed by matrix assisted laser 

desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) and MALDITOF MS-MS. Prior to each enzyme digestion, purified AAD-12 protein was resolved on a

SDS-PAGE gel and bands corresponding to AAD-12 protein (one band was used per digest 

containing approximately 100 ng per lane) were excised with a sterile scalpel and processed 

as follows (a half lane section of the standard protein of the gel was processed alongside 

with the protein sample, in a separate tube, using identical procedure). Gel pieces were 

crushed with a sterile micro-pestle in a siliconized microcentrifuge tube, and destained as 

follows: 0.4 mL of 50% ACN and 0.4 mL of ammonium bicarbonate buffer were added, the 

tube was sealed and shaken at room temperature for 30 min in a Thermomixer R at 1100 

rpm; the tube was centrifuged to settle the gel pieces, and the supernatant was removed with 

a pipette tip and discarded; the destaining procedure was repeated 2 times.

The proteins were reduced and alkylated in-gel as follows: (1) 0.2 mL of DTT solution was 

added to gel pieces, and the tube was incubated at room temperature for 30 min in a 
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Thermomixer R; (2) the tube was centrifuged, and the DTT solution was removed by a 

pipette tip; (3) the gel pieces were washed with 0.5 mL of 25 mM ammonium bicarbonate 

buffer, the tube was centrifuged, and the buffer was removed; (4) 0.2 mL of IAA solution 

was added to the gel pieces, and the tube was incubated in darkness at ambient temperature 

for 1 hour; (5) the gel pieces were washed twice with 0.5 mL of 25 mM ammonium 

bicarbonate buffer (the tube was centrifuged, and the buffer was removed after each wash). 

After the destaining/ reduction/ alkylation procedures, the gel pieces were shrunk in neat 

acetonitrile and then dried in a centrifugal evaporator for 30 min.

The dried gel pieces were re-hydrated with a trypsin solution (25 g in 500 L of 25 mM 

ammonium bicarbonate buffer, pH 7.8; prepared fresh), chymotrypsin solution (25 g in 

500 L of 1 mM HCl; prepared fresh), or Asp-N solution (2 g in 50 L of 50 mM sodium 

phosphate buffer, pH 8.0; prepared fresh) and incubated in an incubator at 37 oC for 

approximately 16 hours (overnight). Afterwards, the peptides were extracted from the gel 

slices sequentially with 0.4 mL of 50% ACN/ 0.1% TFA, then 0.4 mL of 50% ACN/ 5% 

FA, and finally 0.4 mL of 75% ACN/ 5% FA (30 min per extraction in a Thermomixer R at 

room temperature, shaking at 1100 rpm). The extracts for each sample were combined and 

dried in a centrifugal evaporator.

Dried peptides were reconstituted in 30 L of 0.1% TFA in water and were purified for 

MALDI MS analysis using C18 Zip-Tips (Millipore), according to the manufacturer’s 

procedure. Purified peptides were eluted directly onto MALDI plate sequentially, with 

aqueous 10%, 25%, 50%, and 75% ACN (buffered 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. 

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. 

Mass calibration was performed with a Mass Standards Kit for Calibration of AB SCIEX 

TOF/TOF Instruments, consisting of the calibration mixture (theoretical monoisotopic 

[M+H]+ m/z values used): des-Arg1-Bradykinin, m/z 904.4681; Angiotensin I, m/z

1296.6853; Glu1-Fibrinopeptide B, m/z 1570.6774; ACTH (fragment 1-17), m/z 2093.0867; 

ACTH (fragment 18-39), m/z 2465.1989; ACTH (fragment 7-38) m/z 3657.9294. The plate 

wide calibration model was used for MS calibration.

The peptide fragments of the cotton-derived ADD-12 protein (including the N- and Ctermini) were analyzed and compared with the sequence of the microbe-derived protein.

Activity assay of the AAD-12 protein derived from DAS-8191Ø-7 cotton plant tissue

To prepare a cotton-derived AAD-12 fraction suitable for enzymatic analysis, a primary leaf 

extract was prepared as previously described. Following the initial centrifugation at 38,000 

× g, powdered ammonium sulfate was added to the primary supernatant to achieve a final 

concentration of 30%. The mixture was incubated with stirring at 4 °C for 1 hour. The 

sample was subjected to centrifugation at 38,000 × g for 30 minutes at 4 °C. The 30% 
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ammonium sulfate pellet was discarded and additional ammonium sulfate was added to the 

30% supernatant to achieve a final concentration of 55%. The sample was then incubated 

overnight at 4 °C with stirring. The sample was again subjected to centrifugation at 38,000 × 

g for 30 minutes at 4 °C. The resulting supernatant was discarded and the pellet containing 

the bulk of the AAD-12 was dissolved in 50 mM HEPES pH 7.8, 1 mM EDTA. The 30 –

50% ammonium sulfate protein fraction was divided into 850 μL aliquots, snap frozen in 

liquid nitrogen and stored at -80 °C. 

A colorimetric assay was used to assess the activity of both the cotton leaf- and microbederived enzymes. Assays were performed in 1.5 mL Eppendorf tubes at room temperature 

with a total assay volume of 150 μL as previously described (Fukumori and Hausinger, 

1993). Typical assays contained 100 mM HEPES pH 7.0, 1 mM Fe(II)(SO4)2(NH4)2, 1 mM 

sodium ascorbate, S-Dichloroprop (in DMSO), and 0.05 μM AAD-12. All reactions were 

initiated by addition of α-ketoglutarate to a final concentration of 2 mM. 

Prior to assay, the AAD-12 protein was serially diluted in 50 mM HEPES pH 7.0, 1 mM 

EDTA to match the concentration of the cotton-derived enzyme (0.0865 μM). After the 

appropriate incubation time, assays were terminated by addition of 10 μL 100 mM sodium 

EDTA followed by 15 μL of borate buffer pH ~10 (0.309 g boric acid + 0.373 g KCl + 4.4 

mL 1 N KOH). Phenol products were detected by the addition of 2 μL 2 % 4-

aminoantipyrine and 2 μL 8% potassium ferricyanide. Following centrifugation for 30 

seconds at 10,000 × g, 150 μL of the supernatant was transferred to a 96-well plate and the 

absorbance at 510 nm was recorded on a SpectraMax® M2 microplate reader. Authentic 

product standard curves were run in parallel with 2,4 dichlorophenol in the range of 0 – 125 

μM added in place of substrate. Control reactions contained all reagents except for 

substrates. Blanks lacking enzyme were analyzed to account for the contaminating phenols

in the substrate formulation which were found to be negligible (not shown). Kinetic 

experiments were performed as described with S-dichloroprop varied over a concentration 

range from 0 – 125 μM.

Results for Characterization of AAD-12 Protein

SDS-PAGE and western blot analysis of crude cotton leaf protein extracts

SDS-PAGE and western analysis was conducted to confirm the identity DAS-8191Ø-7 

cotton-derived AAD-12 and to show molecular weight and immunoreactive equivalence 

between the microbe-derived and DAS-8191Ø-7 cotton-derived AAD-12 proteins. Both the 

microbe-derived AAD-12 protein and the transgenic DAS-8191Ø-7 cotton leaf extract 

contained a positive signal at the expected molecular weight of 32 kDa by polyclonal 

antibody western blot analysis (Figure 51). Importantly, the non-transgenic cotton extracts 

and the BSA control samples did not contain any immunoreactive protein bands, as 

expected. The results demonstrated that the anti-AAD-12 antibodies recognized the DAS8191Ø-7 cotton AAD-12 protein and the identical migration pattern for both transgenic 

cotton and microbe-derived AAD-12 strongly suggests that DAS-8191Ø-7 cotton AAD-12

is not fragmented, glycosylated or otherwise post-translationally modified which would add 

or subtract from the overall protein molecular weight. Both SDS-PAGE and western 
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analysis results demonstrate the microbe-derived AAD-12 and DAS-8191Ø-7 cotton 

derived proteins to be equivalent in both molecular weight and immunoreactive. 

Purification results of DAS-8191Ø -7 cotton AAD-12

To conduct additional equivalency analysis of DAS-8191Ø-7 cotton AAD-12 to that of 

microbe-derived AAD-12, AAD-12 immunoaffinity precipitation was conducted on an 

aqueous extract of 5 – 10 grams of ground DAS-8191Ø-7 cotton lyophilized transgenic leaf 

tissue. Proteins bound to the anti-AAD-12 monoclonal antibody column were examined by 

SDS-PAGE along with the microbe-derived AAD-12. For the microbe-derived AAD-12, a 

single major protein band of 32 kDa was visualized following Coomassie staining of the 

SDS-PAGE gel and as expected, the corresponding DAS-8191Ø-7 cotton-derived AAD-12 

protein was identical in size to the microbe-derived AAD-12 protein (Figure 52).

Lane Sample Amount Loaded

 M Novex prestained MW markers 10.0 L

 1 DAS-8191Ø-7 cotton crude leaf extract 30.0 μL

 2 Microbe-derived AAD-12 1.00 g

 3 Control cotton (non-transgenic) leaf extract 30.0 L

 4 BSA protein standard 1.00 g

 5 DAS-8191Ø-7 cotton crude leaf extract 30.0 μL

 6 Microbe-derived AAD-12 standard 1.30 ng

 7 Blank -

 8 Control cotton (non-transgenic) leaf extract 30.0 L

 9 BSA protein standard 1.25 ng

Figure 51. SDS-PAGE and Western Blot Analysis of Microbe- & DAS-8191Ø-7 

Cotton-Derived AAD-12 Proteins

Coomassie Stained Gel Western Blot Film

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

3.5 -

M 1 2 3 4 5 6 7 8 9 

AAD-1 2
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Predictably, the plant purified fractions contained a minor amount of impurities in addition 

to the AAD-12 protein. The co-eluted proteins were likely retained on the resin by weak 

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).

Lane Sample Amount Loaded

1 Microbe-derived AAD-12 Standard 0.34 μg

2 Novex prestained MW markers 10 μL

3 DAS-8191Ø-7 purified AAD-12 25 μL

4 DAS-8191Ø-7 purified AAD-12 25 μL

5 DAS-8191Ø-7 purified AAD-12 25 μL

6 DAS-8191Ø-7 purified AAD-12 25 μL

Figure 52. SDS-PAGE Analysis of Immunoaffinity Purified DAS-8191Ø-7 

Cotton Derived AAD-12

Glycosylation detection of DAS-8191Ø-7 cotton AAD-12 protein

Detection of carbohydrates, possibly covalently linked to immunoaffinity-purified DAS8191Ø-7 cotton-derived AAD-12 protein, was assessed using the GelCode Glycoprotein 

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

Coomassie Stained Gel

1 2 3 4 5 6

AAD-12
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Staining Kit from ThermoScientific. The purified DAS-8191Ø-7 cotton AAD-12 was 

electrophoresed simultaneously with a set of control and reference protein standards. 

As seen in the Coomassie stained gel in Figure 53, the glycoprotein, horseradish peroxidase, 

was loaded as a positive control indicator for glycosylation and two non-glycoproteins, 

soybean trypsin inhibitor and bovine serum albumin, were employed as negative controls. 

As expected, the glycoprotein stained gel shows a positive signal from the glycosylated 

horseradish peroxidase positive control, while the non-glycosylated negative controls, 

soybean trypsin inhibitor and bovine serum albumin, show no signal. Importantly, as with 

the negative control samples, neither the microbe-derived or DAS-8191Ø-7 purified AAD12 exhibited detectable glycosylation following glycoprotein staining. These results confirm 

that microbe-derived and DAS-8191Ø-7 cotton derived AAD-12 contains no detectable 

covalently linked carbohydrates.

Lane Sample Amount Loaded

M Novex prestained MW markers 10 L

1 Horseradish peroxidase (+ control) 0.5 g

2 Horseradish peroxidase (+ control) 1.0 g

3 Soybean trypsin inhibitor (- control) 0.5 g

4 Soybean trypsin inhibitor (- control) 1.0 g

5 Bovine serum albumin (- control) 0.5 g

6 Bovine serum albumin (- control) 1.0 g

7 Microbe-derived AAD-12 0.5 g

8 Microbe-derived AAD-12 1.0 g

9 DAS-8191Ø-7 immunopurified AAD-12 20 μL

Figure 53. Glycoprotein Analysis of Purified DAS-8191Ø-7 Cotton AAD-12

Coomassie (left) and glycoprotein (right) stained SDS-PAGE gels containing control proteins and microbederived and cotton derived AAD-12 protein.

kDa

260 -

160 -

110 -

80 -

60 -

50 -

40 -

30 -

20 -

15 -

10 -

Coomassie Stained Gel

M 1 2 3 4 5 6 7 8 9

Glycoprotein Stained Gel

M 1 2 3 4 5 6 7 8 9

3.5 -
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Results of MALDI-TOF MS and MALDI-TOF MS/MS amino acid sequence analysis of DAS8191Ø-7 cotton-derived AAD-12 protein

To confirm the amino acid sequence of the purified DAS-8191Ø-7 cotton-derived AAD-12 

protein, the purified protein was resolved by SDS-PAGE (Figure 52) and the respective 

bands were excised and subjected to in-gel digestion with trypsin, chymotrypsin, or Asp-N. 

The masses of the digested DAS-8191Ø-7 cotton AAD-12 protein were compared with those 

deduced from expected masses of trypsin, chymotrypsin, and ASP-N cleavage sites in AAD12 using Protein Analysis Worksheet (PAWS) freeware from Proteometric LLC (Figure 54 -

Figure 56). The masses of the DAS-8191Ø-7 cotton detected peptides were compared with 

the expected in silico masses to confirm the identity of the purified AAD-12 protein.

Figure 54. In Silico Trypsin Cleavage of DAS-8191Ø-7 Cotton AAD-12 

Theoretical cleavage of the AAD-12 protein with trypsin generated in silico using Protein Analysis Worksheet 

(PAWS) from Proteometrics LLC. Theoretical trypsin digest peptides are indicated by the continuum of upper 

(black) or lower (red) case letters.

A A D - 1 2 

[ 1 - 2 9 3 ] m a s s = 3 1 7 2 9 . 1

C le a v a g e a t K R

S m a ll p o l a r : D ( 1 7 ) E ( 1 1 ) N ( 4 ) Q ( 1 4 )

L a r g e p o la r : K ( 6 ) R ( 1 8 ) H ( 1 6 )

S m a ll n o n - p o la r : S ( 1 3 ) T ( 1 8 ) A ( 4 4 ) G ( 2 5 )

L a r g e n o n -p o l a r : L ( 2 4 ) I ( 1 1 ) V ( 2 4 ) M ( 1 0 ) F ( 8 ) Y ( 5 ) W ( 8 )

S p e c ia l : C ( 3 ) P ( 1 4 )

1 M A Q T T L Q I T P T G A T L G A T V T G V H L A T L D D A 30 

31 G F A A L H A A W L Q H A L L I F P G Q H L S N D Q Q I T F 60 

61 A K r F G A I E R i g g g d i v a i s n v k A D G T V R q h 90 

91 s p a e w d d m m k V I V G N M A W H A D S T Y M P V M A Q 120 

121 G A V F S A E V V P A V G G R t c f a d m r A A Y D A L D E 150 

151 A T R a l v h q r S A R h s l v y s q s k L G H V Q Q A G S 180 

181 A Y I G Y G M D T T A T P L R P L V K v h p e t g r p s l l 210 

211 i g r H A H A I P G M D A A E S E R f l e g l v d w a c q a 240 

241 p r V H A H Q W A A G D V V V W D N R c l l h r A E P W D F 270 

271 K l p r V M W H S R l a g r p e t e g a a l v 293 

(1) [1-62] = 6496.4 (2) [63-63] = 174.2 (3) [64-69] = 691.8

(4) [70-82] = 1242.4 (5) [83-88] = 617.7 (6) [89-100] = 1474.6

(7) [101-135] = 3619.2 (8) [136-142] = 843.0 (9) [143-153] = 1195.2

(10) [154-159] = 722.8 (11) [160-162] = 332.4 (12) [163-171] = 1048.2

(13) [172-199] = 2945.4 (14) [200-213] = 1531.8 (15) [214-228] = 1591.7

(16) [229-242] = 1604.8 (17) [243-259] = 1960.1 (18) [260-264] = 640.8

(19) [265-271] = 892.0 (20) [272-274] = 384.5 (21) [275-280] = 815.0

(22) [281-293] = 1283.4
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Figure 55. In Silico Chymotrypsin Cleavage of DAS-8191Ø-7 Cotton AAD-12

Theoretical cleavage of the AAD-12 protein with chymotrypsin generated in silico using Protein Analysis 

Worksheet (PAWS) from Proteometrics LLC. Theoretical trypsin digest peptides are indicated by the continuum of 

upper (black) or lower (red) case letters.

Figure 56. In Silico Asp-N Cleavage of DAS-8191Ø-7 Cotton AAD-12

Theoretical cleavage of the AAD-12 protein with Asp-N generated in silico using Protein Analysis Worksheet 

(PAWS) from Proteometrics LLC. Theoretical trypsin digest peptides are indicated by the continuum of upper 

(black) or lower (red) case letters

Figure 57 is a comprehensive peptide coverage map illustrating the peptides identified and 

sequence verified for each endoproteinase (trypsin, chymotrypsin, and Asp-N) digest. 

Observed sequence coverage for trypsin, chymotrypsin and Asp-N was 61.4%, 49.1%, and 

36.5%, respectively. For DAS-8191Ø-7 cotton-derived AAD-12, overall sequence coverage 

analysis was excellent at 88.1%. Peptide sequence that was missed did not contain sequence 

motifs that are typically required for glycosylation (N-X-S or N-X-T where X is any amino 

acid) (Hamby and Hirst, 2008). 

S m a ll p o l a r : D ( 1 7 ) E ( 1 1 ) N ( 4 ) Q ( 1 4 )

L a r g e p o la r : K ( 6 ) R ( 1 8 ) H ( 1 6 )

S m a ll n o n - p o la r : S ( 1 3 ) T ( 1 8 ) A ( 4 4 ) G ( 2 5 )

L a r g e n o n -p o l a r : L ( 2 4 ) I ( 1 1 ) V ( 2 4 ) M ( 1 0 ) F ( 8 ) Y ( 5 ) W ( 8 )

S p e c ia l : C ( 3 ) P ( 1 4 )

1 M A Q T T L q i t p t g a t l G A T V T G V H L a t l D D A 30 

31 G F a a l H A A W l Q H A L l I F P G Q H L s n d q q i t f 60 

61 A K R F g a i e r i g g g d i v a i s n v k a d g t v r q h 90 

91 s p a e w D D M M K V I V G N M A W h a d s t y M P V M A Q 120 

121 G A V F s a e v v p a v g g r t c f A D M R A A Y d a l D E 150 

151 A T R A L v h q r s a r h s l V Y s q s k l G H V Q Q A G S 180 

181 A Y i g y G M D T T A T P L r p l V K V H P E T G R P S L l 210 

211 I G R H A H A I P G M D A A E S E R F l E G L v d w A C Q A 240 

241 P R V H A H Q W a a g d v v v w D N R C L l H R A E P W d f 270 

271 K L P R V M W h s r l A G R P E T E G A A L v 293 

(1) [1-6] = 663.8 (2) [7-15] = 901.0 (3) [16-24] = 854.0

(4) [25-27] = 303.4 (5) [28-32] = 523.5 (6) [33-35] = 273.3

(7) [36-39] = 483.5 (8) [40-40] = 131.2 (9) [41-44] = 467.5

(10) [45-45] = 131.2 (11) [46-52] = 810.9 (12) [53-60] = 952.0

(13) [61-64] = 520.6 (14) [65-95] = 3204.5 (15) [96-108] = 1509.8

(16) [109-114] = 692.7 (17) [115-124] = 1050.3 (18) [125-138] = 1392.6

(19) [139-145] = 796.9 (20) [146-148] = 317.3 (21) [149-155] = 774.8

(22) [156-165] = 1190.3 (23) [166-167] = 280.3 (24) [168-172] = 561.6

(25) [173-182] = 1017.1 (26) [183-185] = 351.4 (27) [186-194] = 906.0

(28) [195-197] = 384.5 (29) [198-209] = 1319.5 (30) [210-210] = 131.2

(31) [211-229] = 2065.3 (32) [230-230] = 131.2 (33) [231-233] = 317.3

(34) [234-236] = 418.4 (35) [237-248] = 1403.6 (36) [249-256] = 815.9

(37) [257-261] = 619.7 (38) [262-262] = 131.2 (39) [263-268] = 794.9

(40) [269-270] = 280.3 (41) [271-277] = 929.2 (42) [278-281] = 511.6

(43) [282-292] = 1071.2 (44) [293-293] = 117.1

A A D - 1 2 

[ 1 - 2 9 3 ] m a s s = 3 1 7 2 9 . 1

C le a v a g e a t D

S m a ll p o l a r : D ( 1 7 ) E ( 1 1 ) N ( 4 ) Q ( 1 4 )

L a r g e p o la r : K ( 6 ) R ( 1 8 ) H ( 1 6 )

S m a ll n o n - p o la r : S ( 1 3 ) T ( 1 8 ) A ( 4 4 ) G ( 2 5 )

L a r g e n o n -p o l a r : L ( 2 4 ) I ( 1 1 ) V ( 2 4 ) M ( 1 0 ) F ( 8 ) Y ( 5 ) W ( 8 )

S p e c ia l : C ( 3 ) P ( 1 4 )

1 M A Q T T L Q I T P T G A T L G A T V T G V H L A T L d D A 30 

31 G F A A L H A A W L Q H A L L I F P G Q H L S N d q q i t f 60 

61 a k r f g a i e r i g g g D I V A I S N V K A d g t v r q h 90 

91 s p a e w D d m m k v i v g n m a w h a D S T Y M P V M A Q 120 

121 G A V F S A E V V P A V G G R T C F A d m r a a y D A L d e 150 

151 a t r a l v h q r s a r h s l v y s q s k l g h v q q a g s 180 

181 a y i g y g m D T T A T P L R P L V K V H P E T G R P S L L 210 

211 I G R H A H A I P G M d a a e s e r f l e g l v D W A C Q A 240 

241 P R V H A H Q W A A G d v v v w D N R C L L H R A E P W d f 270 

271 k l p r v m w h s r l a g r p e t e g a a l v 293 

(1) [1-27] = 2668.1 (2) [28-28] = 133.1 (3) [29-54] = 2799.2

(4) [55-73] = 2064.3 (5) [74-83] = 1029.2 (6) [84-95] = 1382.5

(7) [96-96] = 133.1 (8) [97-110] = 1603.0 (9) [111-139] = 2962.4

(10) [140-145] = 725.8 (11) [146-148] = 317.3 (12) [149-187] = 4273.8

(13) [188-221] = 3640.3 (14) [222-234] = 1435.6 (15) [235-251] = 1904.1

(16) [252-256] = 616.7 (17) [257-268] = 1509.7 (18) [269-293] = 2837.3
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Figure 57. Comprehensive MS and MS/MS Sequence Coverage Map for Trypsin, 

Chymotrypsin and Asp-N Digest of DAS-8191Ø-7 Cotton-Derived AAD-12

AAD-12 peptide mapping - Consensus Protocol 110819

Event DAS-8191Ø-7 

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 A Q T T L Q I T P T G A T L G A T V T G V H L A T L D D A 30

Trypsin 0 0

Chymotrypsin 15 15

Asp-N 26 26

Consensus 29 29

31 G F A A L H A A W L Q H A L L I F P G Q H L S N D Q Q I T F 60

Trypsin 0 0

Chymotrypsin 18 18

Asp-N 6 6

Consensus 18 18

61 A K R F G A I E R I G G G D I V A I S N V K A D G T V R Q H 90

Trypsin 25 25

Chymotrypsin 4 4

Asp-N 30 30

Consensus 30 30

91 S P A E W D D M M K V I V G N M A W H A D S T Y M P V M A Q 120

Trypsin 0 0

Chymotrypsin 6 6

Asp-N 5 5

Consensus 11 11

121 G A V F S A E V V P A V G G R T C F A D M R A A Y D A L D E 150

Trypsin 15 15

Chymotrypsin 23 23

Asp-N 6 6

Consensus 30 30

151 A T R A L V H Q R S A R H S L V Y S Q S K L G H V Q Q A G S 180

Trypsin 27 27

Chymotrypsin 20 20

Asp-N 0 0

Consensus 27 27

181 A Y I G Y G M D T T A T P L R P L V K V H P E T G R P S L L 210

Trypsin 30 30

Chymotrypsin 30 30

Asp-N 0 0

Consensus 30 30

211 I G R H A H A I P G M D A A E S E R F L E G L V D W A C Q A 240

Trypsin 30 30

Chymotrypsin 0 0

Asp-N 6 6

Consensus 30 30

241 P R V H A H Q W A A G D V V V W D N R C L L H R A E P W D F 270

Trypsin 30 30

Chymotrypsin 12 12

Asp-N 28 28

Consensus 30 30

271 K L P R V M W H S R L A G R P E T E G A A L V 293

Trypsin 23 23

Chymotrypsin 16 16

Asp-N 0 0

Consensus 23 23

Identified in MS Analysis - Trypsin Trypsin Trypsin # of AA covered 180 180

Identified in MS and MS/MS Analysis - Trypsin Chymotrypsin Chymotrypsin # of AA covered 144 144

Identified in MS Analysis - Chymotrypsin Asp-N Asp-N # of AA covered 107 107

Identified in MS and MS/MS Analysis - Chymotrypsin Consensus Consensus # of AA covered 258 258

Identified in MS Analysis - Asp-N

Identified in MS and MS/MS Analysis - Asp-N Trypsin Percent Coverage 61.4 61.4

Identified in MS Analysis - Consensus Chymotrypsin Percent Coverage 49.1 49.1

Identified in MS and MS/MS Analysis - Consensus Asp-N Percent Coverage 36.5 36.5

Consensus Percent Coverage 88.1 88.1

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
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The amino acid residues at the N-and C-termini of the cotton-derived AAD-12 protein were 

measured and compared with the previously determined sequence of the microbe-derived 

protein. The protein sequences were measured by MALDI-TOF, MALDI-TOF/TOF 

MS/MS or ESI-LC/MS. Asp-N digestion revealed that both the microbe- and DAS-8191Ø-7 

cotton-derived AAD-12 N-termini were identical with the N-terminal amino acid of the 

cotton-derived AAD-12 containing an acetylated alanine (A) (Table 31).

Table 31. Summary of N-terminal Sequences of AAD-12 Derived Proteins

Expected M

1

A

2

Q T T L Q I T P T G A T L G...

DAS-8191Ø-7 N-acetyl- A

2

Q T T L Q I T P T G A T L G...

Microbe-derived A

2

Q T T L Q I T P T G A T L G...

Based on the aad-12 DNA sequence, the N-terminal DAS-8191Ø-7 cotton and microbederived AAD-12 protein sequences are expected to contain a methionine residue (Table 31). 

This result indicated that the N-terminal methionine of the microbe-derived and DAS8191Ø-7 cotton proteins had been removed. In addition, in DAS-8191Ø-7 the second amino 

acid alanine was acetylated. This result is encountered frequently with eukaryotic (plant) 

expressed proteins since approximately 80-90% of the N-terminal protein residues are 

modified in such a way (Wellner et al., 1990; Polevoda and Sherman, 2003). This result 

determined that during or after AAD-12 translation in cotton and P. fluorescens, the Nterminal methionine is cleaved by a methionine aminopeptidase (MAP). MAPs cleave 

methionyl residues rapidly when the second residue on the protein is small, such as Gly, Ala, 

Ser, Cys, Thr, Pro, and Val (Walsh, 2005). 

Also, it has been shown that proteins with serine and alanine at the N-termini are frequently 

acetylated (Polevoda and Sherman, 2003). The two cotranslational processes, cleavage of Nterminal methionine residue and N-terminal acetylation, are by far the most common 

modifications and occur on the vast majority (~85%) of eukaryotic proteins (Polevoda and 

Sherman, 2003). However, examples demonstrating that N-terminal protein acetylation result 

in biological significance are rare (Polevoda and Sherman, 2003).

The C-terminal sequence of the DAS-8191Ø-7 cotton- and microbe-derived AAD-12 

proteins were determined essentially as described above for AAD-12 N-terminal sequence. 

The resulting amino acid C-terminal sequences were compared to the expected translated 

aad-12 DNA sequence (Table 32). The results indicated that the measured DAS-8191Ø-7 

cotton- and microbe-derived AAD-12 C-terminal sequences were identical to the expected 

protein sequence, and both the cotton- and microbe-derived AAD-12 proteins were 

indistinguishable and unaltered at the C-terminus.
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Table 32. Summary of C-terminal Sequences of AAD-12 Derived Proteins

Expected R

284 P E T E G A A L V293

DAS-8191Ø-7 R

284 P E T E G A A L V293

Microbe-derived R

284 P E T E G A A L V293

 

Results of the activity assay of the AAD-12 protein derived from DAS-8191Ø-7 cotton plant 

tissue

To evaluate AAD-12 enzymatic activity, kinetic assays were performed with both microbederived and DAS-8191Ø-7 cotton-derived AAD-12. Assays were performed for 6 minutes at 

28 °C with 0.05 μM enzyme. AAD-12 isolated from both P. flourescens and DAS-8191Ø-7 

cotton displayed the expected hyperbolic activity profiles when evaluated over a range of Sdichloroprop substrate concentrations (Figure 58). Michaelis-Menten curve fitting revealed 

apparent Km values of 41.2 ± 4.8 μM and 29.8 ± 1.0 μM for microbe-derived and cottonderived AAD-12 respectively. For these assays, the apparent Vmax values for microbe-derived

and cotton-derived AAD-12 were also similar at 6.8 ± 0.3 and 5.0 ± 0.1 μM/min respectively. 

The relative agreement in the measured kinetic parameters for both microbe- and DAS8191Ø-7 cotton-derived AAD-12 indicates that the enzymes are functionally equivalent.

Conclusions

This study demonstrated that the biochemical identity and biological function of P. 

fluorescens-produced AAD-12 protein was equivalent to the protein purified from the leaf 

tissue of DAS-8191Ø-7 cotton. Both the DAS-8191Ø-7 cotton and microbe-derived AAD-12 

proteins had an apparent molecular weight of 32 kDa and were immunoreactive on western 

blots using an AAD-12 specific polyclonal antibody. The observation that DAS-8191Ø-7 

cotton and microbe-derived AAD-12 co-migrate on both SDS-PAGE and western blots 

strongly indicates a lack of posttranslational modifications apart from the N-terminal 

acetylation. Moreover, it was experimentally determined that both DAS-8191Ø-7 cotton and 

microbe-derived AAD-12 are not glycosylated as empirically determined by MALDI-MS 

and glycoprotein stained gels. DAS-8191Ø-7 cotton - and P. fluorescens-derived AAD-12 

were equally active using S-dichloroprop as a substrate and displayed similar kinetic 

parameters, indicating that the proteins are enzymatically equivalent. Finally, the amino acid 

sequence of cotton-derived AAD-12 was directly confirmed using enzymatic peptide mass 

fingerprinting by MALDI-TOF MS and verified by MS/MS analysis. Collectively, these data 

support the conclusion that the AAD-12 protein produced by P. fluorescens and transgenic 

cotton are biochemically and functionally equivalent.
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0

1

2

3

4

5

6

0 20 40 60 80 100 120 140

AAD-12 Activity Analysis

AAD-12 Cotton DAS-81910-7

AAD-12 TSN030732-0003

uM / min

S-Dichloroprop (uM)

AAD-12 Cotton DAS-81910-7

Value Error

Vmax 4.9598 0.062438

Km (uM) 29.836 0.98063

R 0.99978 NA

AAD-12 TSN030732-0003

Value Error

Vmax 6.7825 0.33204

Km (uM) 41.184 4.7848

R 0.99767 NA

Figure 58. Kinetic Analysis of Microbe and DAS-8191Ø-7 Cotton Derived AAD-12 

Proteins with S-Dichloroprop Substrate

The Michaelis-Menten plots of microbe-derived AAD-12 (black circles) and cotton-derived AAD-12 (blue 

squares) using S-Dichloroprop as a substrate for AAD-12. The average of three independent experiments is 

shown and error bars indicate the standard deviation of the analyses.
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Appendix 3. Methods and Results for Characterization of PAT Protein

MaterØials & Methods for Characterization of PAT Protein

DAS-8191Ø-7 transgenic Cotton material

The PAT protein was extracted from the leaf tissue of greenhouse grown transgenic cotton 

event DAS-8191Ø-7 (T3 generation see Figure 4). Prior to harvest, the PAT protein 

expression was verified by western blot and the leaf tissue from DAS-8191Ø-7 cotton was 

harvested, frozen, lyophilized, ground, and stored at -80 C. Test material used for 

characterization of DAS-8191Ø-7 cotton PAT are listed in Table 33.

Control Cotton material

The control cotton line had a genetic background representative of DAS-8191Ø-7 cotton 

plants, but did not contain the pat gene. Seeds of the non-transgenic cotton line were planted, 

grown, tested, harvested, and processed under the same conditions as DAS-8191Ø-7 cotton. 

Microbe-Derived PAT

Recombinant microbe-derived PAT protein was produced and purified from Pseudomonas

fluorescens and has an apparent molecular weight of ~20 kDa. 

Reference materials

The commercially available reference substances used in this study are listed in 

Table 34.

Table 33. Test Material for PAT Characterization

Test Substance Source Lot Number Assay Reference

Cotton PAT Cotton 

DAS-8191Ø-7 

TSN304178 SDS-PAGE, Western 

blot, Glycosylation, 

MALDI-TOF, MALDITOF/TOF MS/MS, 

Activity Assay

NA

Control Cotton 

Line

isogenic / nontransgenic 

cotton

TSN304177 SDS-PAGE, Western 

blot, Glycosylation, 

MALDI-TOF, MALDITOF/TOF MS/MS, 

Activity Assay

NA

Cotton crude leaf 

extract

Cotton 

DAS-8191Ø-7

NA SDS-PAGE, Western NA

Microbe-derived 

PAT

Pseudomonas 

fluorescens

TSN303589 (ENBK 

132436-001)

SDS-PAGE, Western, 

Glycosylation, MALDITOF, MALDI-TOF/TOF 

MS/MS, Activity Assay
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Table 34. Commercially Available Reference Substances for PAT Characterization

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

NH175044 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

NH175569 SDS-PAGE, 

Glycosylation 

assay

ThermoFisher

Prestained Molecular 

Weight Markers

Novex Sharp 

prestained protein 

standards

1167391 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

SDS-PAGE and western blot analysis of crude cotton leaf extracts

SDS-PAGE and western blot analysis of the crude protein extracts prepared from the 

transgenic DAS-8191Ø-7 cotton tissue and non-transgenic isoline tissue were performed with 

Bio-Rad Criterion gels fitted in a Criterion Gel chamber with XT MES running buffer (BioRad). Extracts were prepared by Geno-grinding the ground cotton tissue at 40 mg/mL in 

PBST buffer containing 5 mM EDTA, 0.16% (v/v) β-mercaptoethanol, and 1× protease 

inhibitor cocktail with 2 steel beads for 3 minutes at 1,500 strokes/min in a 2 mL microcentrifuge tube. The supernatants were clarified by centrifuging the samples at ~20,000 × g 

(4 °C) for 5 minutes, and 100 L of each extract was mixed with 100 L of 2× Laemmli 

sample buffer (Bio-Rad) containing freshly added β-mercaptoethanol (Bio-Rad) and heated 

for 5 minutes at ~95 C. After a brief centrifugation (2 min at 20,000 × g, 4 °C), 20 μL of 

each supernatant was loaded directly on the gel. The reference standards, microbe-derived 

PAT, and control standard, BSA (Thermo Scientific), were diluted to an appropriate 

concentration and combined with Laemmli sample buffer containing β-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 Thermo Scientific 

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) using a Criterion trans-blot electrophoretic transfer cell

(Bio-Rad) with transfer buffer containing 20% methanol, 10% Tris/glycine buffer under a 

constant voltage of 100 V for 60 minutes. After transfer, the membrane was probed with a 

PAT specific polyclonal rabbit antibody for 60 minutes (1:40,000 dilution) at room 
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temperature. A 1:80,000 dilution of conjugated goat anti-rabbit IgG (H+L) with horseradish 

peroxidase (Thermo) was used as the secondary antibody. ThermoPierce ECL 

chemiluminescent substrate was used for development and visualization of the 

immunoreactive protein bands. The membrane was exposed to detection film (Thermo 

Scientific) for various time points and subsequently developed with an All-Pro 100 Plus film 

developer.

Protein purification of PAT from DAS-8191Ø-7 cotton plant tissue for structural analyses

The PAT protein was extracted from the ground cotton tissue using 50 mM HEPES (pH 7.8), 

300 mM NaCl, 10 mM EDTA (Extraction buffer) by weighing and transferring ground leaf 

tissue into a chilled glass beaker and adding Extraction buffer to 20 mL per 1 gram of tissue. 

The tissue was mixed briefly and PVPP (insoluble) was added to the mixture at 10% (w/w). 

The mixture was stirred for 20 min and the solution was filtered through 1 layer of prewetted miracloth (Calbiochem) and clarified by centrifugation at ~30,000 × g, 4 °C for 30 

minutes. The supernatant was removed and combined with an equal volume of Extraction 

buffer supplemented with 2 M urea, 0.2% Tween-20 to yield a final extract containing 1 M 

urea and 0.1% Tween-20.

The PAT protein was purified from the supernatant by immuno-precipitation using 

polyclonal antibodies cross-linked to Protein A/G Agarose resin (Thermo Scientific) at 4 mg 

of antibody per milliliter of resin. For each 15 mL of clarified supernatant, 50 μL (200 g of 

antibody) of coupled resin was added and allowed to incubate on a rotating mixer for 3 hours 

at 4 °C. The resin was recovered by centrifugation at ~3,500 × g for 5 minutes at 4 °C and 

the resin was transferred to a 1.5 mL microcentrifuge tube using 1 mL of wash buffer. The 

resin was then washed sequentially for a total of 5 washes using different buffers (vide infra). 

Each wash was accomplished by adding buffer to the tube containing resin, rotating the tube 

on a mixer for ~5 min at 4 °C, centrifuging the sample at ~500 x g for 1 min, and discarding 

the supernatant. The resin was first washed twice with 0.9 mL of Extraction buffer 

supplemented with 1 M urea and 0.1% Tween-20, once with 0.9 mL of Extraction buffer 

supplemented with 0.5 M urea and 0.05% Tween-20, once with Extraction buffer, and once 

with 50 mM HEPES (pH 7.8), 0.1 mM EDTA. After the final wash, spin, and decant, the 

resin was centrifuged for 1 min at 500 × g at 4 °C to pellet the resin and the supernatant was 

discarded. Finally, the resin was centrifuged for 10 sec at 20,000 × g at 4 °C to pellet the 

resin and supernatant was again discarded. The immuno-purified protein was stored bound to 

the resin at -20 °C until used for SDS-PAGE followed by mass spectrometry analysis and 

SDS-PAGE followed by glycosylation analysis.

Detection of post-translational glycosylation

For detection of potential post-translational glycosylation, cotton-derived PAT protein was 

immuno-purified as described above. The resin with bound PAT protein from a single 

immuno-precipitation preparation was combined with 60 μL of 2× Laemmli sample buffer

(no β-mercaptoethanol added) and was heated at 95 °C for 5 minutes. Protein standards and 

controls including microbe-derived PAT, soybean trypsin inhibitor, bovine serum albumin, 

and horseradish peroxidase proteins were diluted with 2× Laemmli sample buffer (Bio-Rad) 

to the approximate concentration of the purified cotton-derived PAT protein and heated at 95 
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C for 5 minutes. All samples were centrifuged at 20,000 × g for 1 minute prior to loading on 

SDS-PAGE gel. 

The resulting sample supernatants were applied directly to a Bio-Rad Mini-Protean TGX gel 

and were electrophoresed at 150 V for ~50 minutes. Two identical gels were run in parallel

and after electrophoresis, one gel was stained with GelCode Blue stain (Thermo Scientific)

for detection of 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 second gel was stained with a GelCode Glycoprotein Staining Kit (Thermo 

Scientific) 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 30 minutes. Finally, the gel was immersed in the reduction 

solution for 5 minutes, and rinsed with 3% acetic acid. The glycoproteins 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.

Mass spectrometry peptide mass fingerprinting and sequence analysis of cotton event DAS8191Ø-7- and microbe-derived PAT proteins

Immunoaffinity purified plant-derived PAT protein was subjected to in-gel digestion by 

trypsin and Asp-N followed by matrix assisted laser desorption/ionization time-of-flight mass 

spectrometry (MALDI-TOF MS) and MALDI-TOF MS-MS. For each enzyme digestion,

bands resolved on a SDS-PAGE gel at ~20 kDa corresponding to PAT protein (two bands 

were used per digest containing approximately 100 ng per lane, resulting in 200 ng total 

protein per digest reaction) were excised with a sterile scalpel and processed as follows (a 

half lane section of the standard protein of the gel was processed alongside with the protein 

sample, in a separate tube, using identical procedure). Gel pieces were crushed with a sterile 

micro-pestle in a siliconized microcentrifuge tube, and destained as follows: 0.4 mL of 50% 

ACN and 0.4 mL of ammonium bicarbonate buffer were added, the tube was sealed and 

shaken at room temperature for 30 min in a Thermomixer R at 1100 rpm; the tube was 

centrifuged to settle the gel pieces, and the supernatant was removed with a pipette tip and 

discarded; the destaining procedure was repeated 2 times.

The proteins were reduced in-gel as follows: (1) 0.2 mL of DTT solution was added to the

gel pieces, and the tube was incubated at room temperature for 30 min in a Thermomixer R; 

(2) the tube was centrifuged, and the DTT solution was removed by a pipette tip; (3) the gel 

pieces were washed twice with 0.5 mL of 25 mM ammonium bicarbonate buffer, the tube 

was centrifuged, and the buffer was removed. After the destaining/ reduction procedures, the 

gel pieces were shrunk in neat acetonitrile and then dried in a centrifugal evaporator for 30 

min.
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The dried gel pieces were re-hydrated with a trypsin solution (25 g in 500 L of 25 mM 

ammonium bicarbonate buffer, pH 7.8; prepared fresh) or Asp-N solution (2 g in 50 L of 

50 mM sodium phosphate buffer, pH 8.0; prepared fresh) and incubated in an incubator at 37 

oC for approximately 16 hours (overnight). Afterwards, the peptides were extracted from the 

gel slices sequentially with 0.4 mL of 50% ACN/ 0.1% TFA, then 0.4 mL of 50% ACN/ 5% 

FA, and finally 0.4 mL of 75% ACN/ 5% FA (30 min per extraction in a Thermomixer R at 

room temperature, shaking at 1100 rpm). The extracts for each sample were combined and 

dried in a centrifugal evaporator.

Dried peptides were reconstituted in 30 L of 0.1% TFA in water and were purified for 

MALDI MS analysis using C18 Zip-Tips (Millipore), according to the manufacturer’s 

procedure. Purified peptides were eluted directly onto MALDI plate sequentially, with 

aqueous 10%, 25%, 50%, and 75% ACN (buffered 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. 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. Mass calibration was performed with an Mass 

Standards Kit for Calibration of AB SCIEX TOF/TOF Instruments, consisting of the 

calibration mixture (theoretical monoisotopic [M+H]+ m/z values used): des-Arg1-

Bradykinin, m/z 904.4681; Angiotensin I, m/z 1296.6853; Glu1-Fibrinopeptide B, m/z 

1570.6774; ACTH (fragment 1-17), m/z 2093.0867; ACTH (fragment 18-39), m/z

2465.1989; ACTH (fragment 7-38) m/z 3657.9294. The plate wide calibration model was 

used for MS calibration. The peptide fragments of the cotton-derived PAT protein (including 

the N- and C-termini) were analyzed and compared with the sequence of the microbe-derived 

protein.

Protein purification of PAT from DAS-8191Ø-7 cotton plant tissue for functional analyses

The PAT protein was extracted with 350 mL of 50 mM Tris (pH 7.5), 150 mM NaCl, 1 mM 

EDTA, 1 mM TCEP, supplemented with 1% PVPP (polyvinylpolypyrrolidone, insoluble) 

and a protease inhibitor cocktail by blending ground cotton leaf tissue (15 g) using a stick 

blender (~1 g of tissue per 20 mL of buffer). The blended material was strained through 2 

layers of pre-wetted Miracloth (Calbiochem), and clarified by centrifuging at ~35,000 × g, 4 

°C for 45 minutes to remove all particulates and PVPP. 

The supernatant was transferred to a pre-chilled beaker and solution was stirred at 4 °C. 

Ammonium sulfate was slowly added to the extract over a 1 hr time-course until 40% 

ammonium sulfate saturation was achieved. The solution was stirred for an additional 2 hrs 

following final addition of salt. The solution was centrifuged at 35,000 × g, 4 °C for 60 

minutes to remove precipitated proteins. 

The supernatant, containing PAT, was collected and returned to the chilled beaker at 4 °C 

with stirring. Ammonium sulfate was added to the extract over 1 hr until a concentration of 

70% saturation was reached. The solution was allowed to equilibrate overnight. The 
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precipitated protein material was pelleted by centrifuging the mixture at ~35,000 × g, 4 °C 

for 60 minutes and the supernatant was discarded. The pellet containing PAT was 

resuspended in 45 mL of chilled HIC Buffer A (HIC Buffer A = 50 mM Tris (pH 7.5), 5% 

glycerol with 1.25 M ammonium sulfate; HIC Buffer B = 50 mM Tris (pH 7.5), 5% 

glycerol). The resuspension was centrifuged at ~35,000 × g, 4 °C for 60 minutes to remove 

any non-dissolved particulates prior to chromatography.

The clarified supernatant was loaded onto a 10 mL Phenyl HP HiTrap column (two 5 mL 

columns connected in serial, GE Healthcare) equilibrated in HIC Buffer A. The column was 

washed with 15 column volumes (CV) of HIC Buffer A, followed by 5 CV of 5% HIC 

Buffer B. The bound proteins were eluted with a 50 CV linear elution gradient to 100% HIC 

Buffer B. Fractions (3.5 mL volume) were collected starting at 10% HIC Buffer B conditions 

and odd fractions were assayed for PAT content by Lateral Flow Strip Assay (Envirologix, 

Inc) and by the PAT Activity Assay. 

Fractions containing the PAT protein were pooled and concentrated using a 10 kDa MWCO 

filter device (Amicon) from 122.5 mL to 4.2 mL. The salt content of the concentrated sample 

was adjusted to match the initial chromatographic conditions (100% HIC Buffer A) and was 

loaded onto a 9.4 mL Phenyl HP HiScreen column (two 4.7 mL columns connected in serial, 

GE Healthcare) equilibrated in HIC Buffer A. The column was washed with 6 column 

volumes (CV) of HIC Buffer A, and the bound proteins were eluted with a 20 CV linear 

elution gradient to 100% HIC Buffer B. Fractions (2.0 mL volume) were collected starting at 

0% HIC Buffer B conditions and odd fractions were assayed for PAT content by Lateral 

Flow Strip Assay (Envirologix, Inc) and by the PAT activity assay. 

Fractions containing the PAT protein were pooled and concentrated using a 10 kDa MWCO 

filter device (Amicon) from 46 mL to 3.0 mL. The concentrated sample was then applied to a 

120 mL HiLoad 16/600 Superdex 200 column (GE Healthcare) equilibrated with 50 mM Tris 

(pH 7.5), 150 mM NaCl, and 5% glycerol. One-milliliter fractions were collected and 

assayed for PAT content by Lateral Flow Strip (Envirologix, Inc) and by the PAT Activity 

Assay. Fractions containing the PAT protein were pooled and concentrated using a 10 kDa 

MWCO filter device (Amicon) from 8 mL to 0.8 mL. The final purification product was 

aliquoted, flash frozen in liquid nitrogen, and stored at -80 °C.

Activity assay of the PAT protein derived from DAS-8191Ø-7 cotton plant tissue

Cotton-derived PAT was purified using traditional purification procedures as described 

above. The activity of both the cotton- and microbe-derived enzymes was verified using an 

established spectrophotometric assay with minor modifications (De Block et al., 1987; 

Mahan et al., 2006). 

Assays were performed in 96-well plates at room temperature in a total volume of 150 μL (a 

150 μL total volume results in a 0.446 cm path length). Assays contained 50 mM HEPES 

(pH 7.0), 1 mM EDTA, 1 mM 5,5'-dithiobis-2-nitrobenzoic acid (DTNB), 500 μM of acetylCoA, 25 nM PAT, and varied levels of DL-glufosinate (25 μM –2 mM). Reactions were 

initiated by the addition of acetyl-CoA and the absorbance at 412 nm was recorded every 30 
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seconds for a total of 5 minutes on a Molecular Devices SpectraMax® M2 plate reader. 

Separate positive control reactions were assayed using microbe-derived PAT at the same 

concentration as the plant-derived protein (25 nM). Negative control reactions were assayed 

in the absence of DL-glufosinate to monitor non-specific acetyl-CoA consumption. All 

reactions were run in triplicate.

PAT activity was quantified by measuring the liberation of the free CoA sulfhydryl group 

which forms concomitantly with transfer of the acetyl group to glufosinate. The reaction of 

the sulfhydryl group of free CoA with DTNB yields a molar equivalent of the chromophore 

5-thio-2-nitrobenzoic (ε = 14,150 M

-1

cm-1at 412 nm). The initial rates were determined 

from the raw data, and were corrected for non-specific acetyl-CoA consumption (based upon 

control reactions lacking glufosinate) and converted to μM/min. A racemic mixture of 

glufosinate was used for the assays; therefore the final values are presented as a function of 

L-glufosinate, the active enantiomer. These converted initial rates were plotted against Lglufosinate concentrations and subsequently fitted to the Michaelis–Menten equation 

allowing for the extrapolation of Km and Vmax. The data were analyzed using KaleidaGraph 

software (v.4.03). 

Results for Characterization of PAT Protein

SDS-PAGE and western blot analysis of cotton leaf protein extracts

SDS-PAGE and western analysis was conducted to confirm the identity DAS-8191Ø-7 

cotton-derived PAT and to show molecular weight and immunoreactive equivalence between 

the microbe-derived and DAS-8191Ø-7 cotton derived PAT proteins. For the microbederived PAT protein, the major protein band was approximately ~20 kDa (Figure 59) as 

visualized on Coomassie stained SDS-PAGE gels. As expected, the corresponding DAS8191Ø-7 cotton-derived PAT protein was visualized by immunospecific polyclonal 

antibodies at an identical size to the microbe-expressed protein. In the PAT western blot 

analysis, no immunoreactive proteins, consistent with the PAT protein, were observed in the 

control isoline extract or the BSA lanes (Figure 59). The results demonstrated that the antiPAT antibodies recognized the DAS-8191Ø-7 cotton PAT protein and the identical migration 

pattern for both transgenic cotton- and microbe-derived PAT strongly suggests that the PAT 

protein expressed in cotton is not post-translationally glycosylated (note: the PAT enzyme 

does not contain N-glycosylation sites (Herouet et al., 2005)) or processed in such a manner 

which would have added to or subtracted from the overall protein molecular weight. Both 

SDS-PAGE and western analysis results demonstrate the microbe-derived PAT and DAS8191Ø-7 cotton derived proteins to be equivalent in both molecular weight and 

immunoreactive. 

Purification results of DAS-8191Ø-7 cotton PAT for structural analysis

To conduct additional equivalency analysis of DAS-8191Ø-7 cotton-derived PAT to that of 

microbe-derived PAT, immuno-precipitation was conducted on an aqueous extract of ~6 

grams of ground DAS-8191Ø-7 cotton leaf tissue to further purify the DAS-8191Ø-7 cottonderived PAT protein. The total protein bound to the polyclonal antibody resin was examined 

by SDS-PAGE analysis which demonstrated that DAS-8191Ø-7 cotton-derived PAT protein 

from the tissue extract with an approximate molecular weight of ~20 kDa was captured 
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during the immuno-precipitation procedure (Figure 60). Following immuno-precipitation, the 

DAS-8191Ø-7 cotton-derived PAT was then compared with the microbe-derived protein. For 

the microbe-derived PAT protein, the major protein band was approximately 20 kDa (Figure 

60) as visualized on Coomassie stained SDS-PAGE gels. 

Lane Sample Amount Loaded

M Novex Sharp Pre-Stained MW marker 10.0 L

1 Bovine Serum Albumin (BSA) 1.00 μg

2 Control cotton (non-transgenic) leaf extract 20.0 L

3 Microbe-derived PAT 1.00 g

4 DAS-8191Ø-7 cotton extract 20.0 L

5 Bovine Serum Albumin 1.25 ng

6 Control cotton (non-transgenic) leaf extract 20.0 L

7 No sample ---

8 Microbe-derived PAT 1.30 ng

9 DAS-8191Ø-7 cotton crude leaf extract 20.0 L

Figure 59. SDS-PAGE and Western Blot Analysis of Microbe- and DAS-8191Ø-7 

Cotton-Derived PAT Proteins

M 1 2 3 4 5 6 7 8 9

Coomassie Stained Gel Western Blot Film

260

110

160

80

60

50

40

30

20

15

10

3.5

Lane

MW

(kDa)
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Lane Sample Loaded

1 Microbe-derived PAT 0.50 g

2 No sample ---

3 Novex Sharp Pre-Stained MW marker 10.0 L

4 No sample ---

5 DAS-8191Ø-7 immunopurified PAT 25.0 L

6 DAS-8191Ø-7 immunopurified PAT 25.0 L

7 DAS-8191Ø-7 immunopurified PAT 25.0 L

8 DAS-8191Ø-7 immunopurified PAT 25.0 L

Figure 60. SDS-PAGE Analysis of the Immunoaffinity Purified DAS-8191Ø-7 Cotton 

Derived PAT

As expected, the corresponding cotton-derived PAT protein was visualized by 

immunospecific polyclonal antibodies at an identical size to the microbe-expressed protein 

(Figure 60). The plant-derived immuno-precipitation samples contained a minor amount of 

impurities in addition to the full-length PAT protein. The co-bound proteins were likely 

retained to the resin by weak interactions with the matrix or antibody released during sample 

preparation for SDS-PAGE analysis. 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 resin (Goldberg et al., 1991).
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Results of detection of glycosylation of PAT protein

Detection of carbohydrates, possibly covalently linked to DAS-8191Ø-7 cotton-derived PAT 

protein, was assessed by resolving immuno-precipitated PAT using SDS-PAGE followed by 

visualization of the gel with a stain that specifically detects glycosylated proteins. The 

immunoaffinity-purified DAS-8191Ø-7 cotton PAT protein was electrophoresed 

simultaneously with a set of control and reference protein standards. A glycoprotein, 

horseradish peroxidase, was loaded as a positive control indicator for glycosylation, and nonglycoproteins including microbe-derived PAT, soybean trypsin inhibitor, and bovine serum 

albumin, were employed as negative controls.

Lane Sample Amount Loaded

M Novex Sharp Pre-Stained MW marker 10.0 L

1 Horseradish Peroxidase (+ control) 0.50 g

2 Horseradish Peroxidase (+ control) 1.00 g

3 Soybean Trypsin Inhibitor (- control) 0.50 g

4 Soybean Trypsin Inhibitor (- control) 1.00 g

5 Bovine Serum Albumin (- control) 0.50 g

6 Bovine Serum Albumin (- control) 1.00 g

7 Microbe-derived PAT 0.50 g

8 Microbe-derived PAT 1.00 g

9 DAS-8191Ø-7 immunopurified PAT 25.0 L

Figure 61. Glycoprotein Analysis of Immunopurified DAS-8191Ø-7 Derived PAT 

SDS-PAGE gels containing microbe- and cotton-derived PAT protein and standards visualized for total protein 

and glycoprotein stains.

M 1 2 3 4 5 6 7 8

Lane

MW

(kDa)

9 1 2 3 4 5 6 7 8 9

Coomassie Stained Gel Glycoprotein Stained Gel

260

110

160

80

60

50

40

30

20

15

10

3.5
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As expected, the glycoprotein stained gel shows a positive signal from the glycosylated 

horseradish peroxidase positive control, while the non-glycosylated negative controls, 

soybean trypsin inhibitor and bovine serum albumin, shows no signal. As with the negative 

control sample, both the microbial-derived and DAS-8191Ø-7 cotton purified PAT samples 

were not detected on the glycoprotein stained gel. This was anticipated as the PAT protein 

does not contain any sites predicted for N-glycosylation, the most common form of 

glycosylation found on proteins (Herouet et al., 2005). The results showed that both the

DAS-8191Ø-7 cotton and microbe-derived PAT proteins had no detectable covalently linked 

carbohydrates (Figure 61).

Results of MALDI-TOF MS and MALDI-TOF MS/MS tryptic and Asp-N peptide sequencing 

of DAS-8191Ø-7 cotton-derived PAT protein

The PAT protein derived from DAS-8191Ø-7 cotton tissue was separated by SDS-PAGE

(Figure 60) and the respective bands were excised and subjected to in-gel digestion by 

trypsin and Asp-N endoproteases. The resulting peptide mixture was analyzed by MALDITOF 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 Asp-N 

cleavage sites in the sequence of the cotton-derived PAT protein.

Figure 62 and Figure 63 illustrate the theoretical cleavage of the PAT protein generated in 

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

PAT protein, once denatured, is readily digested by endoproteases to yield numerous 

peptides that are able to be detected using mass spectrometry.

Figure 62. In Silico Trypsin Cleavage of DAS-8191Ø-7 Cotton PAT

Theoretical cleavage of the PAT protein with trypsin generated in silico using Protein Analysis Worksheet (PAWS) 

from Proteometrics LLC. Trypsin endoprotease specifically hydrolyzes protein and peptide bonds C-terminally of 

lysine and arginine. Theoretical trypsin digest peptides are indicated by the continuum of upper (black) or lower 

(red) case letters.

P A T 

[ 1 - 1 8 3 ] m a s s = 2 0 6 1 8 . 3

C le a v a g e a t K R

S m a ll p o l a r : D ( 9 ) E ( 1 2 ) N ( 4 ) Q ( 7 )

L a r g e p o la r : K ( 4 ) R ( 1 5 ) H ( 7 )

S m a ll n o n - p o la r : S ( 8 ) T ( 1 2 ) A ( 1 8 ) G ( 1 3 )

L a r g e n o n -p o l a r : L ( 1 3 ) I ( 7 ) V ( 1 8 ) M ( 3 ) F ( 4 ) Y ( 8 ) W ( 6 )

S p e c ia l : C ( 1 ) P ( 1 4 )

1 M S P E R r p v e i r p a t a a d m a a v c d i v n h y i e 30 

31 t s t v n f r T E P Q T P Q E W I D D L E R l q d r Y P W L 60 

61 V A E V E G V V A G I A Y A G P W K a r N A Y D W T V E S T 90 

91 V Y V S H R h q r L G L G S T L Y T H L L K s m e a q g f k 120 

121 S V V A V I G L P N D P S V R l h e a l g y t a r G T L R a 150 

151 a g y k H G G W H D V G F W Q R d f e l p a p p r p v r p v 180 

181 t q i 183 

(1) [1-5] = 618.7 (2) [6-37] = 3561.0 (3) [38-52] = 1857.0

(4) [53-56] = 530.6 (5) [57-78] = 2375.7 (6) [79-80] = 245.3

(7) [81-96] = 1927.1 (8) [97-99] = 439.5 (9) [100-112] = 1415.7

(10) [113-120] = 897.0 (11) [121-135] = 1522.8 (12) [136-145] = 1130.3

(13) [146-149] = 445.5 (14) [150-154] = 508.6 (15) [155-166] = 1481.6

(16) [167-183] = 1932.2
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Figure 63. In Silico Asp-N Cleavage of DAS-8191Ø-7 Cotton PAT

Theoretical cleavage of the PAT protein with Asp-N generated in silico using Protein Analysis Worksheet (PAWS) 

from Proteometrics LLC. Asp-N endoprotease specifically hydrolyzes protein and peptide bonds N-terminally of 

aspartic acid. Theoretical Asp-N digest peptides are indicated by the continuum of upper (black) or lower (red) case 

letters.

In the endoprotease digests of the transgenic cotton-derived PAT protein, the peptide 

sequence coverage from peptide mass fingerprint (PMF) data was extensive at 91.3%. Of the 

91.3% 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 64). This analysis 

confirmed the DAS-8191Ø-7 cotton-derived protein amino acid sequence matched that of the 

microbe-derived PAT protein at the N- and C-terminus as well as a major portion of the 

internal sequence.

Table 35. Summary of N-terminal Sequences of PAT Derived Proteins

Expected M

1

S

2

P E R R P V E I R P A T A A

DAS-8191Ø-7 S

2

P E R R P V E I R P A T A A

Microbe-derived S

2

P E R R P V E I R P A T A A

Table 36. Summary of C-Terminal Sequences of PAT Derived Proteins

Expected P

173 P R P V R P V T Q I183

DAS-8191Ø-7 P

173 P R P V R P V T Q I183

Microbe-derived P

173 P R P V R P V T Q I183

Based on the described results, one post-translational modification was observed for the 

DAS-8191Ø-7 cotton-derived PAT protein which included the removal of the N-terminal 

methionine. The excision of Met at position 1 is a common modification found in plantexpressed proteins (Li and Chang, 1995). No other post-translational modifications were 

observed. Collectively, the mass spectrometry data, glycosylation staining, and SDS-PAGE 

western blot analyses all provide evidence for the lack of glycosylation of both the DAS8191Ø-7 cotton-derived PAT and the microbe-derived PAT proteins. The results of these 

analyses indicate that the amino acid sequence of the DAS-8191Ø-7 cotton-derived PAT 

protein was equivalent to the P. fluorescens-expressed protein previously characterized.

S m a ll n o n - p o la r : S ( 8 ) T ( 1 2 ) A ( 1 8 ) G ( 1 3 )

L a r g e n o n -p o l a r : L ( 1 3 ) I ( 7 ) V ( 1 8 ) M ( 3 ) F ( 4 ) Y ( 8 ) W ( 6 )

S p e c ia l : C ( 1 ) P ( 1 4 )

1 M S P E R R P V E I R P A T A A d m a a v c D I V N H Y I E 30 

31 T S T V N F R T E P Q T P Q E W I d D L E R L Q d r y p w l 60 

61 v a e v e g v v a g i a y a g p w k a r n a y D W T V E S T 90 

91 V Y V S H R H Q R L G L G S T L Y T H L L K S M E A Q G F K 120 

121 S V V A V I G L P N d p s v r l h e a l g y t a r g t l r a 150 

151 a g y k h g g w h D V G F W Q R d f e l p a p p r p v r p v 180 

181 t q i 183 

(1) [1-16] = 1781.1 (2) [17-22] = 608.7 (3) [23-47] = 3018.3

(4) [48-48] = 133.1 (5) [49-54] = 772.9 (6) [55-83] = 3222.6

(7) [84-130] = 5227.0 (8) [131-159] = 3177.5 (9) [160-166] = 907.0

(10) [167-183] = 1932.2
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Figure 64. Comprehensive MS and MS/MS Sequence Coverage Map for Trypsin and 

Asp-N digests of DAS-8191Ø-7 Cotton-Derived PAT

Sequence coverage was 91.3% with PMF data and 91.3% by tandem MS.

Results of the enrichment and activity assay of the PAT protein derived from DAS-8191Ø-7 

cotton plant tissue

Microbe and DAS-8191Ø-7 cotton-derived PAT proteins were assayed using an established 

spectrophotometric assay with minor modifications (De Block et al., 1987; Mahan et al., 

2006). Both the DAS-8191Ø-7 cotton and microbe-derived PAT displayed hyperbolic kinetic 

plots when evaluated over a range of glufosinate concentrations (Figure 9). MichaelisMenten curve fitting revealed that microbe and DAS-8191Ø-7 cotton-derived PAT have 

similar Vmax values of 9.30 ± 0.10 and 7.19 ± 0.06 μM/min, respectively. The Km value for 

the microbe-derived PAT (150 ± 3 μM) was comparable to that of the cotton-derived enzyme 

(117 ± 4 μM). Both enzyme preparations also displayed similar kcat values of 6.20 s-1for 

microbe-derived and 4.80 s-1for cotton-derived PAT. The calculated catalytic efficiencies 

PAT peptide mapping - Consensus Protocol 120051

Event DAS 81910-7

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 S P E R R P V E I R P A T A A D M A A V C D I V N H Y I E 30

Trypsin 0 0

Asp-N 23 23

Consensus 23 23

31 T S T V N F R T E P Q T P Q E W I D D L E R L Q D R Y P W L 60

Trypsin 23 23

Asp-N 26 26

Consensus 30 30

61 V A E V E G V V A G I A Y A G P W K A R N A Y D W T V E S T 90

Trypsin 28 28

Asp-N 0 0

Consensus 28 28

91 V Y V S H R H Q R L G L G S T L Y T H L L K S M E A Q G F K 120

Trypsin 27 27

Asp-N 0 0

Consensus 27 27

121 S V V A V I G L P N D P S V R L H E A L G Y T A R G T L R A 150

Trypsin 30 30

Asp-N 0 0

Consensus 30 30

151 A G Y K H G G W H D V G F W Q R D F E L P A P P R P V R P V 180

Trypsin 26 26

Asp-N 21 21

Consensus 26 26

181 T Q I 183

Trypsin 3 3

Asp-N 3 3

Consensus 3 3

Identified in MS Analysis - Trypsin Trypsin Trypsin # of AA covered 137 137

Identified in MS and MS/MS Analysis - Trypsin Asp-N Asp-N # of AA covered 73 73

Identified in MS Analysis - Asp-N Consensus Consensus # of AA covered 167 167

Identified in MS and MS/MS Analysis - Asp-N

Identified in MS Analysis - Consensus Trypsin Percent Coverage 74.9 74.9

Identified in MS and MS/MS Analysis - Consensus Asp-N Percent Coverage 39.9 39.9

Consensus Percent Coverage 91.3 91.3

A.a. 

covered 

by MS

A.a. 

covered 

by MS-MS
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(kcat/Km) were nearly identical at 4.13 x 104and 4.11 x 104 M

-1

s

-1

for both the microbe- and 

DAS-8191Ø-7 cotton-derived enzymes, respectively. These results demonstrate that PAT 

derived from DAS-8191Ø-7 cotton leaf displays similar kinetic parameters to PAT purified 

from P. fluorescens and are therefore functionally equivalent.

Figure 65. Kinetic Analysis of Microbe- and DAS-8191Ø-7 Cotton-Derived PAT 

Proteins

The Michaelis-Menten plots of microbe-derived PAT (black circles) and cotton-derived PAT (blue squares)

using L-Glufosinate as a substrate for PAT. The average of three independent experiments is shown and error 

bars indicate the standard deviation of the analyses.

Conclusions

It was demonstrated that the biochemical identity and biological function of the microbederived PAT protein was equivalent to the protein purified from leaf tissue of cotton event 

DAS-8191Ø-7. Both the cotton- and microbe-derived PAT proteins had an apparent 

molecular weight of ~20 kDa and were immunoreactive to a PAT protein-specific polyclonal 

antibody in western blot assays. Greater than 90% of the amino acid sequences were 

confirmed by enzymatic peptide mass fingerprinting by MALDI-TOF MS and verified by 

MS/MS. The lack of glycosylation of the cotton-derived PAT protein provided additional 

support that the PAT protein produced by P. fluorescens and transgenic cotton were 

equivalent. Enzymatic activity assays using glufosinate as a substrate were performed to 

evaluate the kinetic parameters of both enzymes. These results demonstrate that PAT derived 

from DAS-8191Ø-7 cotton leaf displays similar kinetic parameters to PAT purified from 

Pseudomonas fluorescens. Collectively, these biochemical tests indicate that the plant- and 

microbe-derived PAT proteins are biochemically and biologically equivalent and therefore 

the microbe-derived protein is acceptable for use in regulatory studies.
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Appendix 4. Methods for AAD-12 & PAT Protein Expression Analysis 

Experimental Design 

Cotton samples were collected from a field study conducted in the U.S. in 2012 that included six 

(6) field sites; Alabama, Georgia, Louisiana, Missouri, North Carolina, and Texas. Each site 

consisted of one plot of each treatment per block, with 4 blocks per location. Plots were arranged 

in a randomized complete block (RCB) design, with a unique randomization at each site. Plot 

size was 4 rows by 25 feet with row spacing of approximately 30-40 inches. Each cotton plot 

was bordered by 2 rows of non-transgenic cotton of similar maturity. Blocks were separated 

from each other and outside border rows by an alley of at least 10 ft of bare soil or a non‐

sexually compatible non‐crop buffer (e.g., turf grass). At each location, all blocks were used for 

collection of samples for protein expression analysis. With the exception of seed, expression 

samples were collected from rows 1 and 4 of each four row plot. Seed expression samples were

collected from rows 2 and 3 (the center two rows) of each four row plot. Herbicide treatments 

were designed to replicate maximum label rates.

2,4-D plus glufosinate Treatment:

2,4-D (GF-2654) + Glufosinate (Ignite 280 SL) as a tank mixture was applied as two 

broadcast applications to DAS‐8191Ø‐7 cotton. Individual applications were at 

approximately 3 node and 6 node stages. Individual target application rates were 1.0 lb ae/A 

for GF-2654, or 1120 g ae/ha. Individual target application rates were 0.53 lb ae/A for Ignite 

280 SL, or 596 g ai/ha. 

Tissue Sampling and Processing

Tissue Sampling

A total of ten tissue samples were collected for AAD-12 and PAT protein expression 

analysis. Details of each tissue type are as described: 

a. Leaf (4-leaf, first white bloom, and first open boll growth stages) 

One leaf sample per plot (representing 10-14 leaves collected from separate plants) was 

collected for each test and control entry. Each leaf was collected from a different plant in the 

plot. Each leaf sample was the youngest set of fully expanded leaves. 

b. Squares (first white bloom growth stage)

One square sample per plot (representing 10-14 squares) was collected for each test and 

control entry. Each square was collected from a different plant in the plot consisting of a 

flower bud and bracts, but not the stem.

c. Pollen (early bloom growth stage)

One pollen sample per plot (representing a volume of 0.2-0.5 mL of pollen) was collected 

from each plot for each test and control entry. Each pollen sample was collected across the 

plants within each plot from white flowers just starting to open.
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d. Flower (peak bloom growth stage)

One flower sample per plot (representing 14-18 flowers) was collected for each test and 

control entry. Each flower consisted of white flowers from a different plant in the plot.

e. Bolls (peak bloom)

One boll sample per plot (representing 10-14 bolls) was collected for each test and control 

entry. Each boll was collected from a different plant in the plot. Boll samples were either 

open or closed on the first position of fruiting branches.

f. Root (Maturity) 

One root sample (representing 1-2 plants) per plot was collected for each test and control 

entry at the maturity stage by cutting a circle around the base of the plant. The root ball was 

removed and cleaned. 

g. Whole plant 

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. 

h. Seed (Maturity)

One individual sample was collected from each plot of each test and control entry. Each 

sample contained approximately 500 grams of grain. 

Tissue Processing

Samples were shipped to Dow AgroSciences laboratories and maintained frozen until use. 

Samples of cotton tissues were prepared for expression analysis by coarse grinding, 

lyophilizing and/or fine-grinding with a Geno/Grinder (Certiprep, Metuchen, NJ). 

Determination of AAD-12 Protein in Cotton Tissue Samples 

ELISA method DAS 120999 was used to determine AAD-12 protein concentration in cotton 

tissue samples.

AAD-12 Protein Extraction and Analysis

The AAD-12 protein was extracted from cotton tissues except grain and pollen with a 

phosphate buffered saline solution containing 0.05% (v/v) Tween-20 (PBST), 0.75% 

ovalbumin (OVA) and 1.0% polyvinylpyrrolidone (PVP). For grain and pollen, the protein 

was extracted with a phosphate buffered saline solution containing 0.30% (v/v) Tween-20 

(PBST), 0.75% OVA and 1.0% PVP. 

The plant tissue and grain extracts were centrifuged; the aqueous supernatant was collected, 

diluted with appropriate buffer if necessary, and analyzed using an AAD-12 ELISA kit. 

Briefly, an aliquot of the diluted sample and a horseradish peroxidase (HRP)/anti-AAD-12 

monoclonal antibody conjugate were incubated in the wells of a microtiter plate coated with 

an immobilized anti-AAD-12 polyclonal antibody. These antibodies bind with AAD-12 
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protein in the wells and form a "sandwich" with AAD-12 protein bound between soluble and 

the immobilized antibodies. The unbound samples and excess conjugate were then removed 

from the plate by washing with PBST. Subsequent addition of an enzyme substrate generated 

a colored product. The reaction was stopped by adding a dilute acid solution. 

Since the AAD-12 was bound in the antibody sandwich, the level of color development, 

determined by measuring the absorbance of the solution, was related to the concentration of 

AAD-12 in the sample (i.e., lower protein concentrations result in lower color development). 

The absorbance at 450 nm with a background subtraction at 650 nm was measured using a 

Molecular Devices Spectra Max M2 plate reader or a Grifols Triturus Automated 

Immunoassay Analyzer. A calibration curve was generated and the AAD-12 concentration in 

unknown samples was calculated from the polynomial regression equation using Soft-MAX 

ProTM or Triturus Version 4.01B software which was compatible with the plate reader. 

Samples were analyzed in duplicate wells with the average concentration of the duplicate 

wells being reported. 

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the AAD-12 ELISA in 

the tissue matrices were as follows:

Tissue AAD-12 (ng/mg)

LOD LOQ

Bolls

0.5 1

Flower

Leaf 

Pollen

Root

Seed

Squares

Whole Plant

Determination of PAT Protein in Cotton Tissue Samples 

ELISA method GRM07.26 was used to determine PAT protein concentration in cotton tissue 

samples.

PAT Protein Extraction and Analysis

The PAT protein was extracted from cotton tissues with a phosphate buffered saline solution 

with 0.05% Tween-20 (PBST) and 1.0% polyvinylpyrrolidone (PVP). The extract was 

centrifuged; the aqueous supernatant was collected, diluted with PBST/1.0% PVP if 

necessary, and analyzed using a PAT ELISA kit. Briefly, an aliquot of the diluted sample 

was incubated with enzyme-conjugated anti-PAT monoclonal antibody and anti-PAT 

polyclonal antibodies coated in the wells of a 96-well plate in a sandwich ELISA format. At 

the end of the incubation period, the unbound reagents were removed from the plate by 

washing. Subsequent addition of an enzyme substrate generated a colored product. The 

reaction was stopped by adding a dilute acid solution. 
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Since PAT was bound in the antibody sandwich, the level of color development, determined 

by measuring the absorbance of the solution, was related to the concentration of PAT in the 

sample (i.e., lower residue concentrations result in lower color development). The 

absorbance at 450 nm with a background subtraction at 650 nm was measured using a 

Molecular Devices Spectra Max M2 plate reader or a Grifols Triturus Automated 

Immunoassay Analyzer. A calibration curve was generated and the PAT concentration in 

unknown samples was calculated from the polynomial regression equation using Soft-MAX 

ProTM or Triturus Version 4.01B software which was compatible with the plate reader. 

Samples were analyzed in duplicate wells with the average concentration of the duplicate 

wells being reported.

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the PAT ELISA in the 

tissue matrices were as follows:

Tissue PAT (ng/mg)

LOD LOQ

Bolls

0.025 0.06

Flower

Leaf 

Pollen

Root

Seed

Squares

Whole Plant
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Appendix 5. Methods for Compositional Analysis

Samples of DAS-8191Ø-7 (non-sprayed and sprayed with 2,4-D + glufosinate), near isogenic 

non-transgenic control (98M-2983), and reference variety cottonseed were analyzed at 

Covance Laboratories Inc. for 73 composition analytes. The following methods were used by 

Covance Laboratories to determine Proximates & Fiber (9), Minerals (12), Amino Acids 

(18), Fatty Acids (22), Vitamins (7) and Anti-Nutrients (5). See section 6.1 for composition 

analysis study design and results.

Proximates and Fiber

 Protein

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. Instrumentation was used to automate the digestion, distillation and 

titration processes. The percent nitrogen was calculated and converted to equivalent 

protein using the factor 6.25.

 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.

 Ash 

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.

 Moisture

The samples were dried in a vacuum oven at approximately 100°C. The moisture weight 

loss was determined and converted to percent moisture.

 Carbohydrates

The total carbohydrate level was calculated by difference using the fresh weight-derived 

data and the following equation:

% carbohydrates = 100 % - (% protein + % fat + % moisture + % ash)

 Acid Detergent Fiber (ADF)

Sample aliquots were weighed into pre-weighed filter bags. The fats and pigments were then

removed by an acetone wash. Due to the high fat content of the samples, this was followed

by an additional 12-hour acetone soak with no agitation. The filter bags were placed in an

ANKOM2000 Fiber analyzer where the protein, carbohydrate, and ash content were
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dissolved by boiling acidic detergent solution. After drying, the bags were reweighed and

the acid detergent fiber was determined gravimetrically.

 Neutral Detergent Fiber (NDF)

Sample aliquots were weighed into pre-weighed filter bags. The fats and pigments were 

then removed by an acetone wash. Due to the high fat content of the samples, this was 

followed by an additional 12-hour acetone soak with no agitation. The filter bags were 

placed in an ANKOM2000 Fiber analyzer where the protein, carbohydrate, and ash 

content were dissolved by a boiling detergent solution at a neutral pH. Hemicellulose, 

cellulose, lignin and insoluble protein fraction were left in the filter bag and determined 

gravimetrically.

 Total Dietary Fiber

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 was calculated using protein and ash values and the 

weighed residue fractions.

 Crude Fiber

Crude fiber was quantitated as the loss on ignition of dried residue remaining after 

digestion of the samples with 1.25% sulfuric acid and 1.25% sodium hydroxide solutions 

under specific conditions.
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Minerals

Mineral Analysis (12 Total): ICP Emission Spectrometry

The samples were dried, precharred, and ashed overnight in a muffle furnace set to 

maintain 500°C. The ashed samples were 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 samples, measured on the inductively coupled plasma 

spectrometer, with the emission of the standard solutions.

Mineral Reference Standards:

Manufacturer Mineral Lot No. Concentration (μg/ml)

Inorganic Ventures Calcium F2-MEB453071MCA, 200, 1000

F2-MEB453073

Inorganic Ventures Copper F2-MEB453071MCA, 2.00, 10.0

F2-MEB453072MCA

Inorganic Ventures Iron F2-MEB453071MCA, 10.0, 50.0

F2-MEB453074

Inorganic Ventures Magnesium F2-MEB453071MCA, 50.0, 250

F2-MEB453072MCA

Inorganic Ventures Manganese F2-MEB453071MCA, 2.00, 10.0

F2-MEB453072MCA

Inorganic Ventures Phosphorus F2-MEB453071MCA, 200, 1000

F2-MEB453073

Inorganic Ventures Potassium F2-MEB453071MCA, 200, 1000

F2-MEB453073

Inorganic Ventures Sodium F2-MEB453071MCA, 200, 1000

F2-MEB453073

Inorganic Ventures Zinc F2-MEB453071MCA, 10.0, 50.0

F2-MEB453072MCA

Molybdenum & Sulfur: ICP-Mass Spectrometry

The samples were wet-ashed with nitric acid using microwave digestion. Using 

inductively coupled plasma mass spectrometry, the amount of each element was 

determined by comparing the counts generated by the unknowns to those generated by 

standard solutions of known concentrations.

Molybdenum and Sulfur Reference Standards:

Manufacturer Mineral Lot No. Concentration (μg/ml)

Inorganic Ventures Molybdenum F2-MEB421115MCA 1.0

Inorganic Ventures Sulfur E2-S01119 10000

Selenium by Inductively Coupled Plasma-Mass Spectrometry (SEICPMS)

The samples were closed-vessel microwave digested with nitric acid (HNO3) and water. 

After digestion, the solutions were 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 Se

78 using an

inductively coupled plasma-mass spectrometer (ICP-MS) with a dynamic reaction cell

(DRC) by comparing the counts generated by standard solutions.

Selenium Reference Standard:

Manufacturer Mineral Lot No. Concentration (mg/L)

SPEX CertiPrep. Selenium 18-29SE 10

00
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Amino Acid Composition (TAALC/TRPLC)

 Samples Analyzed (18 total)

Alanine (total) Lysine (total)

Arginine (total) Methionine (total)

Aspartic acid (including asparagines) (total) Phenylalanine (total)

Cystine (including cysteine) (total) Proline (total)

Glutamic acid (including glutamine) (total) Serine (total)

Glycine (total) Threonine (total)

Histidine (total) Tryptophan (total)

Isoleucine (total) Tyrosine

Leucine (total) Valine (total)

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 were 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 were 

derivatized with fluorenylmethyl chloroformate (FMOC) before injection.

Amino Acid Reference Standards:

Component Manufacturer Lot No. Purity (%)

L-Alanine Sigma-Aldrich BCBC5470 99.8

L-Arginine Monohydrochloride Sigma-Aldrich 1361811 100

L-Aspartic Acid Sigma-Aldrich BCBB9274 100.6

L-Cystine Sigma-Aldrich 1451329 100

L-Glutamic Acid Sigma-Aldrich 1423805 100.2

Glycine Sigma-Aldrich 1119375 100

L-Histidine Monohydrochloride Monohydrate Sigma-Aldrich BCBB1348 99.9

L-Isoleucine Sigma-Aldrich 1423806 100

L-Leucine Sigma-Aldrich BCBC6907 99.9

L-Lysine Monohydrochloride Sigma-Aldrich 1362380 100.2

L-Methionine Sigma-Aldrich 1423807 99.9

L-Phenylalanine Sigma-Aldrich BCBC5774 100

L-Proline Sigma-Aldrich 1414414 99.7

L-Serine Sigma-Aldrich 1336081 99.9

L-Threonine Sigma-Aldrich 1402329 100

L-Tryptophan Sigma-Aldrich BCBC1685 >99

BCBB1284 99.8

L-Tyrosine Sigma-Aldrich BCBC2417 100

L-Valine Sigma-Aldrich 1352709 100
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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. 

Fatty acid results were converted to their triglyceride equivalents.

Fatty Acids Analyzed and Reference Standards (22 AA total)

Manufacturer Lot No. Component

Weight (%) Purity

JY-10 MA7-W (%)

Nu-Chek Prep GLC 

Reference Standard 

Covance 1

Covance 2

JY-10

MA7-W

Methyl Octanoate (8:0 Caprylic) 3.0 1.25 99.7

Methyl Decanoate (10:0 Capric) 3.25 1.25 99.6

Methyl Laurate (12:0 Lauric) 3.25 1.25 99.8

Methyl Myristate (14:0 Myristic) 3.25 1.25 99.8

Methyl Myristoleate (14:1 Myristoleic) 1.0 1.25 99.5

Methyl Pentadecanoate (15:0 Pentadecanoic) 1.0 1.25 99.6

Methyl Pentadecenoate (15:1 Pentadecenoic) 1.0 1.25 99.4

Methyl Palmitate (16:0 Palmitic) 10.0 15.75 99.8

Methyl Palmitoleate (16:1 Palmitoleic) 3.0 1.25 99.7

Methyl Heptadecanoate (17:0 Heptadecanoic)) 1.0 1.25 99.6

Methyl 10-Heptadecenoate (17:1 

Heptadecenoic) 1.0 1.25 99.5

Methyl Stearate (18:0 Stearic) 7.0 14.00 99.8

Methyl Oleate (18:1 Oleic) 10.0 15.75 99.8

Methyl Linoleate (18:2 Linoleic) 10.0 15.75 99.8

Methyl Gamma Linolenate (18:3 γ-Linolenic) 1.0 1.25 99.4

Methyl Linolenate (18:3 Linolenic) 3.0 1.25 99.5

Methyl Arachidate (20:0 Arachidic) 2.0 1.25 99.8

Methyl 11-Eicosenoate (20:1 Eicosenoic) 2.0 1.25 99.6

Methyl 11-14 Eicosadienoate

(20:2 Eicosadienoic) 1.0 1.25 99.5

Methyl 11-14-17 Eicosatrienoate

(20:3 Eicosatrienoic) 1.0 1.25 99.5

Methyl Arachidonate (20:4 Arachidonic) 1.0 1.25 99.4

Methyl Behenate (22:0 Behenic) 1.0 1.25 99.8
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Vitamins

 Vitamin A (Beta Carotene) 

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

Vitamin A Reference Standard:

Component Manufacturer Lot No. Purity (%)

Beta-Carotene Sigma-Aldrich 091M1417V 98.4 

(Lambda Maximum 450 to 451 nm in 

hexane)

 Vitamin B1 (Thiamine HCl)

The samples were subjected to acid hydrolysis to denature matrix and free bound

thiamine analogs. The treated sample was brought to volume, filtered and injected onto a 

reversed phase column using a high-performance liquid chromatography system with a 

post-column derivatization reaction coil and detected via a fluorescence detector. As 

thiamine monophosphate is not completely reacted, thiamine and thiamine monophosphate 

are both quantitated separately. Final results are the sum of the two components converted 

to thiamine hydrochloride form.

Vitamin B1 Reference Standards:

Component Manufacture

r

Lot No. Purity (%)

Thiamine monophosphate chloride dihydrate Sigma-Aldrich BCBF5554V 99.3

Thiamine hydrochloride USP P0K366 99.7

 Vitamin B2 (Riboflavin)

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.

Vitamin B2 Reference Standards:

Component Manufacturer Lot No. Purity (%)

Riboflavin USP N1J079 99.7
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 Vitamin B3 (Niacin)

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 samples, using the bacteria Lactobacillus plantarum, with the growth response of a 

niacin standard. This response was measured turbidimetrically.

Vitamin B3 Reference Standards:

Component Manufacturer Lot No. Purity (%)

Niacin USP J0J235 99.8

 Vitamin B6 (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.

Vitamin B6 Reference Standards:

Component Manufacturer Lot No. Purity (%)

Pyridoxine hydrochloride USP Q0G409 99.8

 Vitamin B9 (Folic Acid)

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.

Vitamin B9 Reference Standards:

Component Manufacturer Lot No. Purity (%)

Folic Acid USP Q0G151 98.9

 α-Tocopherol (Vitamin E)

The samples were saponified to break down any fat and release vitamin E. The 

saponified mixtures were extracted with ethyl ether and then quantitated by highperformance liquid chromatography using a silica column. Note: Alpha tocopherol is

part of a mixed standard which also includes beta, delta, and gamma isomers. The reference

standard material for those isomers may contain small amounts of alpha tocopherol. All reference

standards that contributed to the alpha tocopherol concentration are listed below.

α-Tocopherol Reference Standards:

Component Manufacturer Lot No. Purity (%)

Alpha Tocopherol USP O0K291 98.5

D-gamma-Tocopherol Acros Organics A0083534 99.3

(+)-δ-Tocopherol Sigma-Aldrich 090M1916V 92.0
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Anti-Nutrients

Sterculic Acid, Malvalic Acid, Dihydrosterculic Acid

The total lipid fraction was extracted from the samples using chloroform and methanol. A 

portion of the lipid fraction was then saponified with a mild alkaline hydrolysis. The free

fatty acids were extracted with ethyl ether and hexane. The free fatty acids were then

converted to their phenacyl derivatives with 2-bromoacetophenone. The derivatized 

extracts were injected on a high-performance liquid chromatography system equipped 

with an ultraviolet detector. The relative percent of total fatty acids for each peak was 

calculated from peak areas.

Sterculic, Malvalic and Dihydrosterculic Acid Reference Standards:

Manufacturer Lot No. Component Weight (%) Purity (%)

Nu-Chek Prep N-22A-A17-Q Behenic acid NA >99

Nu-Chek Prep N-24A-A28-Q Lignoceric acid NA >99

Matreya LLC 22990 Dihydrosterculic acid NA >98

Nu-Chek Prep 

GLC Reference 

Standard PSA 1

AU19-V

Methyl Myristate 2.2 99.6

Methyl Pentadecanoate 0.7 99.5

Methyl Palmitate 47.0 99.5

Methyl Palmitoleate 0.5 99.4

Methyl Heptadecanoate 2.0 99.6

Methyl Stearate 36.0 99.7

Methyl Oleate 9.0 99.5

Methyl Linoleate 1.0 99.4

Methyl Linolenate 0.5 99.5

Methyl Nonadecanoate 0.6 99.4

Methyl Arachidate 0.5 99.6

NA=Not applicable

Gossypol, Free

The samples were extracted with an aqueous acetone solution and filtered. Duplicate 

aliquots were made and the active aliquot was reacted with aniline with heat applied in a 

water bath. Active and inactive aliquots were brought to volume with an aqueous 

isopropyl alcohol solution and read on a spectrophotometer at 440 nm. The absorbance 

difference was then compared to a linear curve calculated from standards that were 

aliquoted, reacted, and read in the same fashion as the samples.

Gossypol, Free & Total Reference Standard:

Component Manufacturer Lot No. Purity (%)

Gossypol from cotton seeds Sigma-Aldrich 041M4117V 99.64

Gossypol, Total (GOSS)

Total gossypol defines gossypol and gossypol derivatives, both free and bound, in 

cottonseed products that are capable of reacting with 3-amino-1-propanol in 

dimethylformamide solution to form a diaminopropanol complex, which then reacts with 

aniline to form dianilinogossypol under the conditions of the method. Gossypol, gossypol 

analogs, and gossypol derivatives having an available aldehyde moiety were measured by 

the method (see references above).
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Appendix 6. Compositional Literature Ranges for Non-Transgenic Cottonseed

Analyte Units ILSI 2010 Literature Literature Citations

Min Max Min Max Min Max

Proximates

& Fiber

(9 Total)

Ash % DW 3.761 5.342 3.7 5.29 (Nida et al., 1996) (Hamilton et al., 2004)

Carbohydrates % DW 39.0 53.6 41.0 53.62 (Nida et al., 1996) (Hamilton et al., 2004)

Fat % DW 17.201 27.292 14.4 25.5 (Bertrand et al., 2005) (Nida et al., 1996)

Protein % DW 21.48 32.97 12 32 (Kohel et al., 1985) (Kohel et al., 1985)

Moisture % FW 2.3 9.9 2.25 15.9 (Hamilton et al., 2004) (Berberich et al., 1996)

ADF % DW 19.74 38.95 21.10 40.5 (Hamilton et al., 2004) (Bertrand et al., 2005)

Crude Fiber % DW 13.86 23.10 13.45 19.31 (Hamilton et al., 2004) (Hamilton et al., 2004)

NDF % DW 25.56 51.87 32.92 53.6 (Hamilton et al., 2004) (Bertrand et al., 2005)

Total Dietary Fiber % DW 33.69 47.55 NR NR NR NR

Minerals

(12 Total)

Calcium mg/100g dry wt. 103.23 325.81 100 330 (Hamilton et al., 2004) (Hamilton et al., 2004)

Copper mg/100g dry wt. 0.313 2.457 0.333 1.114 (Hamilton et al., 2004) (Hamilton et al., 2004)

Iron mg/100g dry wt. 3.671 31.838 3.927 7.215 (Hamilton et al., 2004) (Hamilton et al., 2004)

Magnesium mg/100g dry wt. 347.09 493.12 340 470 (Belyea et al., 1989) (Hamilton et al., 2004)

Manganese mg/100g dry wt. 1.069 2.196 1.106 2.216 (Hamilton et al., 2004) (Hamilton et al., 2004)

Molybdenum mg/100g dry wt. NR NR NR NR NR NR

Phosphorus mg/100g dry wt. 482.54 991.57 560 860 (Hamilton et al., 2004) (Hamilton et al., 2004)

Potassium mg/100g dry wt. 983.45 1448.35 960 1240 (Belyea et al., 1989) (Hamilton et al., 2004)

Selenium ppb_DW NR NR NR NR NR NR

Sodium mg/100g dry wt. 11.183 735.477 5.4 740 (Hamilton et al., 2004) (Hamilton et al., 2004)

Sulfur mg/100g dry wt. NR NR NR NR NR NR

Zinc mg/100g dry wt. 2.70 5.95 2.89 4.862 (Belyea et al., 1989) (Hamilton et al., 2004)

Amino Acids (AA)

(18 Total)

Alanine % total amino acid 4.08 4.51 4.15 5.30 (Hamilton et al., 2004) (Hamilton et al., 2004)

Arginine % total amino acid 10.85 12.77 10.83 15.18 (Hamilton et al., 2004) (Hamilton et al., 2004)

Aspartic Acid % total amino acid 9.00 10.60 9.63 12.37 (Hamilton et al., 2004) (Hamilton et al., 2004)

Cystine % total amino acid 1.53 2.35 1.60 2.32 (Hamilton et al., 2004) (Hamilton et al., 2004)

Glutamic Acid % total amino acid 20.61 22.90 20.24 21.61 (Hamilton et al., 2004) (Hamilton et al., 2004)

Glycine % total amino acid 4.29 4.68 4.44 5.72 (Hamilton et al., 2004) (Hamilton et al., 2004)

Histidine % total amino acid 2.91 3.22 3.00 3.88 (Hamilton et al., 2004) (Hamilton et al., 2004)

Isoleucine % total amino acid 3.10 3.71 3.10 4.46 (Hamilton et al., 2004) (Hamilton et al., 2004)

Leucine % total amino acid 6.03 6.65 6.27 8.11 (Hamilton et al., 2004) (Hamilton et al., 2004)

Lysine % total amino acid 4.62 5.46 4.85 6.60 (Hamilton et al., 2004) (Hamilton et al., 2004)

Methionine % total amino acid 1.27 2.16 1.46 2.28 (Hamilton et al., 2004) (Hamilton et al., 2004)

Phenylalanine % total amino acid 5.44 6.04 5.51 7.23 (Hamilton et al., 2004) (Hamilton et al., 2004)

Proline % total amino acid 3.81 4.49 3.93 5.30 (Hamilton et al., 2004) (Hamilton et al., 2004)

Serine % total amino acid 4.15 5.31 4.16 5.87 (Hamilton et al., 2004) (Hamilton et al., 2004)

Threonine % total amino acid 2.67 3.59 3.26 4.26 (Hamilton et al., 2004) (Hamilton et al., 2004)

Tryptophan % total amino acid 0.91 1.31 0.94 1.40 (Hamilton et al., 2004) (Hamilton et al., 2004)

Tyrosine % total amino acid 2.63 2.93 2.65 3.46 (Hamilton et al., 2004) (Hamilton et al., 2004)

Valine % total amino acid 4.49 5.31 4.72 6.24 (Hamilton et al., 2004) (Hamilton et al., 2004)
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Appendix 6. Compositional Literature Ranges for Non-Transgenic Cottonseed (continued)

Analyte Units ILSI 2010 Literature Literature Citations

Min Max Min Max Min Max

Fatty Acids

(22 Total)

8:0 Caprylic % total fatty acid ND ND NR NR NR NR

10:0 Capric % total fatty acid ND ND NR NR NR NR

12:0 Lauric % total fatty acid ND ND NR NR NR NR

14:0 Myristic % total fatty acid 0.455 2.400 0.55 2.40 (Hamilton et al., 2004) (Hamilton et al., 2004)

14:1 Myristoleic % total fatty acid ND ND NR NR NR NR

15:0 Pentadecanoic % total fatty acid 0.103 0.481 0.050 0.17 (Hamilton et al., 2004) (Hamilton et al., 2004)

15:1 Pentadecenoic % total fatty acid ND ND NR NR NR NR

16:0 Palmitic % total fatty acid 15.11 27.90 21.23 28.10 (Hamilton et al., 2004) (Hamilton et al., 2004)

16:1 Palmitoleic % total fatty acid 0.464 1.190 0.57 0.57 (Bertrand et al., 2005) (Bertrand et al., 2005)

17:0 Heptadecanoic % total fatty acid 0.092 0.119 NR NR NR NR

17:1 Heptadecenoic % total fatty acid ND ND NR NR NR NR

18:0 Stearic % total fatty acid 0.20 3.11 1.99 3.11 (Hamilton et al., 2004) (Hamilton et al., 2004)

18:1 Oleic % total fatty acid 12.8 25.3 12.90 20.10 (Hamilton et al., 2004) (Hamilton et al., 2004)

18:2 Linoleic % total fatty acid 46.0 59.4 46.00 57.10 (Hamilton et al., 2004) (Hamilton et al., 2004)

18:3 Linolenic % total fatty acid 0.11 0.35 0.18 0.18 (Bertrand et al., 2005) (Bertrand et al., 2005)

18:3 gamma 

Linolenic

% total fatty acid 0.097 0.232 NR NR NR NR

20:0 Arachidic % total fatty acid 0.186 0.414 0.24 0.34 (Hamilton et al., 2004) (Hamilton et al., 2004)

20:1 Eicosenoic % total fatty acid 0.095 0.098 NR NR NR NR

20:2 Eicosadienoic % total fatty acid ND ND NR NR NR NR

20:3 Eicosatrienoic % total fatty acid ND ND NR NR NR NR

20:4 Arachidonic % total fatty acid ND ND NR NR NR NR

22:0 Behenic % total fatty acid 0.104 0.295 0.12 0.24 (Hamilton et al., 2004) (Hamilton et al., 2004)

Vitamins

(7 Total)

Alpha Tocopherol 

(Vitamin E)

mg/kg DW 70.825 197.243 NR NR NR NR

Vitamin A 

(Beta Carotene)

mg/kg DW NR NR NR NR NR NR

Vitamin B1 

(Thiamine HCl)

mg/kg DW NR NR NR NR NR NR

Vitamin B2 

(Riboflavin)

mg/kg DW NR NR NR NR NR NR

Vitamin B3 

(Niacin)

mg/kg DW NR NR NR NR NR NR

Vitamin B6 

(Pyridoxine HCl)

mg/kg DW NR NR NR NR NR NR

Vitamin B9 

(Folic Acid)

mg/kg DW NR NR NR NR NR NR

Anti-Nutrients

(5 Total)

Dihydrosterculic 

Acid

% total fatty acid 0.075 0.310 0.12 0.24 (Hamilton et al., 2004) (Hamilton et al., 2004)

Malvalic Acid % total fatty acid 0.229 0.759 0.17 0.61 (Hamilton et al., 2004) (Hamilton et al., 2004)

Sterculic Acid % total fatty acid 0.190 0.556 0.13 0.56 (Hamilton et al., 2004) (Hamilton et al., 2004)

Free Gossypol mg/kg DW 0.454 1.399 0.53 1.20 (Hamilton et al., 2004) (Hamilton et al., 2004)

Total Gossypol % DW 0.547 1.522 0.55 1.46 (Bertrand et al., 2005) (Nida et al., 1996)
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Appendix 7. Evolution of Herbicide Resistant Weeds in Transgenic Crops 

Herbicide Tolerant Crops

Herbicide resistance in weeds has long driven the development of herbicide tolerant crops. 

Soon after the first weeds evolved resistance to herbicides, scientists began to consider 

altering crops to make them resistant to herbicides (Duke, 2005). Initially, non-transgenic 

methods were used until the early 1980s when the tools for producing transgenic crops were 

becoming available. The first transgenic herbicide tolerant crops included bromoxynil 

resistant cotton and canola. However, transgenic crops with resistance to broad-spectrum, 

non-selective herbicides were perceived as a better approach for weed management. This was 

soon realized with the development of glyphosate and glufosinate tolerant crops. Since the

transgenic crops would tolerate the application of those broad spectrum herbicides, they 

could survive and prosper while reducing the amount and number of applications of 

herbicides by the growers.

Herbicide tolerance (often called resistance) in plants employs one of two strategies (or a 

combination) to make the plant tolerant to the applied herbicide:

 the plant produces a new protein which detoxifies the herbicide (e.g., glufosinate 

tolerance, 2,4-D tolerance), or

 the protein in the plant which is normally the target of the herbicide’s action is replaced 

by a new protein which is unaffected by the herbicide (e.g., glyphosate tolerance).

Herbicide tolerant crops available to farmers since 1996 include corn, soybean, cotton, sugar 

beet, alfalfa and canola among others. Initially, transgenes were only used to confer tolerance 

to bromoxynil, glufosinate, and glyphosate. The bromoxynil-tolerant crops are no longer 

sold. Until recently, this only left glyphosate and glufosinate tolerant transgenic crops, and of 

those, glyphosate has had the strongest impact on weed management (Duke, 2005).

Growers choose glyphosate tolerant crops because they make weed control easier and more 

effective, increase profit, require less tillage, and do not restrict crop rotations (Green, 2009). 

Thus, glyphosate tolerant corn, soybeans and cotton have experienced an unprecedented 

rapid adoption rate by U.S. farmers (Figure 50). The planting of glyphosate tolerant crops has 

increased steadily since their introduction in 1996: herbicide tolerant cotton is currently 

planted to 82% of U.S. acres; the vast majority is glyphosate tolerant (University of 

California Agriculture & Natural Resources, 2013a; USDA NASS, 2013c). Figure 66

illustrates the breakdown between herbicide tolerant (HT), insect tolerant (Bt), and stacked 

varieties of transgenic cotton planted in the U.S.
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Evolution of Resistance to Glyphosate, 2,4-D, and Glufosinate

Glyphosate Resistance and Weed Shifts

Glyphosate Resistance

It was initially thought that evolution of glyphosate resistant weeds would be slow and the 

levels of resistance would be low (Bradshaw et al., 1997). This was based on the amount of 

glyphosate applied over many years, the repeated applications made to perennial crops, the 

high level of herbicidal activity that it had demonstrated, and the uniqueness of its metabolic 

activity in the plant. More than twenty years after the launch of glyphosate, rigid ryegrass in 

Australia was reported as the first glyphosate resistant weed in 1996 (Powles et al., 1998). 

About the same time, sales of glyphosate began to increase dramatically in the U.S. due to 

the launch of glyphosate tolerant transgenic soybeans (1996), cotton (1997), and corn (1998). 

Rapid adoption of this new technology drove dramatic increases in the use of glyphosateonly weed control, which resulted in increased selection pressure for glyphosate resistant

weeds.

Figure 66. Adoption of GE Cotton in the U.S. (USDA ERS, 2013b)

Table 37 shows a summary of the glyphosate resistant weed species that have been reported 

from 1996 to August 2013. The data clearly shows that glyphosate resistance in weeds is 

expanding around the globe. Two of these glyphosate resistant weed species have already 
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become a significant problem for farmers across a large geographic area: Conyza canadensis

(Horseweed) infests at least two million hectares of glyphosate tolerant crops in the U.S.; and

Amaranthus palmeri (Palmer amaranth) has heavily impacted cotton growers, particularly in 

the Southeast. 

Table 37. Weed Species with Reported Glyphosate Resistant Biotypes1

Common Name Species Name

First Confirmed 

Report

Later 

Confirmed 

Year Country Reports

Rigid ryegrass Lolium rigidum 1996 Australia USA, S. Africa, 

France, Spain

Goosegrass Eleusine indica 1997 Malaysia Colombia, USA

Horseweed/Marestail Conyza Canadensis 2000 USA

Brazil, China, 

Spain, Czech 

Republic

Italian ryegrass Lolium multiflorum 2001 Chile Brazil, USA, 

Spain, Argentina

Hairy fleabane Conyza bonariensis 2003 S. Africa Spain, Brazil, 

Colombia, USA

Buckhorn plantain Plantago lanceolata 2003 S. Africa

Common ragweed Ambrosia artemisiifolia 2004 USA

Giant ragweed Ambrosia trifida 2004 USA

Ragweed parthenium Parthenium hysterophorus 2004 Colombia

Palmer amaranth Amaranthus palmeri 2005 USA

Common waterhemp Amaranthus rudis 2005 USA Canada

Johnsongrass Sorghum halepenses 2005 Argentina USA

Sourgrass Digitaria insularis 2006 Paraguay Brazil

Junglerice Echinochloa colona 2007 Australia USA

Kochia Kochia scoparia 2007 USA

Liverseedgrass Urochloa panicoides 2008 Australia

Perennial ryegrass Lolium perenne 2008 Argentina

Gramilla mansa Cynodon hirsutus 2008 Argentina

Sumatran fleabane Coryza sumatrensis 2009 Spain

Australian fingergrass Chloris truncate 2010 Australia

Annual bluegrass Poa annua 2010 USA

Tropical Sprangletop Leptochloa virgate 2010 Mexico

Ripgut Brome Bromus diandrus 2011 Australia

Spiny Amaranth Amaranthus spinosus 2012 USA

1

(Heap, 2013d)
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Table 38. Global Reports of Glyphosate Resistant Weed Biotypes with Resistance to Other 

Herbicide Modes of Action5

Common Name Species Name Year – Country 

(State/Providence)

Multiple Resistance to 

Other Herbicide 

Mode of Actions

Kochia Kochia scoparia 2012 – CAN (Alberta) ALS1

Sumatran Fleabane Conyza sumatrensis 2011 – Brazil ALS

Palmer amaranth Amaranthus palmeri 2008 – U.S. (MS, GA) ALS

2009 – U.S. (TN)

Common waterhemp Amaranthus rudis

2005 – U.S. (MO) ALS, PPO2

2006 – U.S. (IL) ALS

2011 – U.S. (IA) ALS, HPPD3

Common ragweed Ambrosia artemisiifolia 2006 – U.S. (OH) ALS

2012 – CAN (Ontario)

Hairy fleabane Conyza bonariensis 2009 – U.S. (CA) Bipyridiliums

Horseweed Conyza canadensis

2003 – U.S. (OH) ALS

2007 – U.S. (MS) Bipyridiliums

2010 – U.S. (DE) ALS

2011 – CAN (Ontario) ALS

Giant ragweed Ambrosia trifida 2006 – U.S. (OH) ALS

2008 – U.S. (MN) ALS

Goosegrass Eleusine indica 1997 – Malaysia ACCase4

Italian ryegrass Lolium multiflorum

2002 – Chile ALS

2006 – Chile ACCase

2007 – Chile ACCase, ALS

2010 - Argentina ALS

2010 – Argentina ACCase

2010 – Brazil ACCase

2010 – U.S. (OR) Glutamine synthase

Rigid ryegrass Lolium rigidum

1999 - Australia ACCase, ALS, 

Dinitroanilines

2003 – South Africa ACCase, Bipyridiliums

2007 - Israel ACCase, ALS

2008 - Australia Triazoles

2010 - Australia Bipyridiliums

Acetolactase synthase inhibitors

2

Polyphenol oxidase inhibitors

3 Hydroxyphenyl-pyruvate-dioxygenase inhibitors

4 Acetyl CoA carboxylase inhibitors

5

(Heap, 2013b)

In addition, researchers in Virginia have been testing a biotype of common lambsquarters 

that survived 1.0 lb ae/acre (1120 g ae/ha) glyphosate, and thus appears to have low level 

resistance to glyphosate (Hite et al., 2008). Weed scientists in Ohio and Indiana have also 

identified a biotype of common lambsquarters in at least a dozen fields that appears to have 

low-level glyphosate resistance (Curran et al., 2007). The increased reports of glyphosate

resistant species, plus the geographic spread of their infestations, have caused some to raise 

concerns about the long term sustainability for glyphosate. Some researchers have stated that 
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applying glyphosate alone over wide areas on highly variable and prolific weeds made the 

evolution of resistant weeds inevitable (Owen, 2001; Thill and Lemerle, 2001).

Researchers have reported that eleven individual biotypes of the twenty-four glyphosate

resistant species are also resistant to herbicides with other modes of action (Table 38). In 

recent years, chemical costs have doubled, and many growers have returned to hand weeding, 

in-crop cultivation, tillage, and post-harvest deep-turning to control glyphosate resistant 

weeds (Sosnoskie and Culpepper, 2013). Although herbicide options to control these 

biotypes with multiple herbicide resistance will be more limited, 2,4-D remains a viable 

control option for the broadleaf weeds.

Table 39. Potential Weed Shifts with Use of Glyphosate 

Common Name Species Name

Asiatic dayflower Commelina communis

Brazil callalily Richardia brasiliensis

Broadleaf buttonweed Spermacoce latifolia

Common waterhemp Amaranthus rudis

Common lambsquarters Chenopodium album

Eastern black nightshade Solanum ptycanthum

Giant ragweed Ambrosia trifida

Hemp sesbania Sesbania exaltata

Kochia Kochia scoparia

Marestail / Horseweed Conyza canadensis

Morning glory spp. Ipomoea spp.

Nutsedge spp. Cyperus spp.

Prickly sida Sida spinosa

Russian thistle Salsola iberica

Tall waterhemp Amaranthus tuberculatus

Tridax daisy Tridax procumbens

Tropical spiderwort Commelina benghalensis

Velvetleaf Abutilon theophrasti

Palmer Amaranth Amaranthus Palmieri

Weed Shifts

When glyphosate tolerant crops are grown intensively with high reliance on glyphosate for 

weed control, species which possess some level of natural tolerance to glyphosate will 

become more prevalent. These “weed shifts” can occur more rapidly than selection for 

glyphosate resistance (Shaner, 2000). Some common hard to control weed species that could 

become “weed shifts” in U.S. cotton are listed in Table 39, with pigweed and morning glory 

most commonly reported by cotton growers (Duke and Powles, 2008b; Owen, 2008; Prince

et al., 2012). 
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2,4-D Resistance

2,4-D has been used continually and widely for over 70 years, and reports of resistance have 

been rare (Wright et al., 2010; Greene and Reid, 2012). The earliest documented reports of 

herbicide resistant weeds were for resistance to 2,4-D in wild carrot (Daucus carota) 

(observed in 1952 but not reported until 1957) and spreading dayflower (Commelina diffusa) 

in 1957 (Heap, 2011). Today, a total of seventeen weed species have documented reports of 

2,4-D resistant biotypes someplace around the globe (Table 40). Wild carrot, yellow burhead, wild radish, musk thistle, and corn poppy are the only 2,4-D resistant weeds that have 

reported infestations in more than 1,000 acres. Some of these 2,4-D resistant biotypes have 

documented cross resistance to other auxin herbicides or multiple resistance to some ALSinhibiting herbicides. Most of these resistant species do not appear to be spreading, as 

indicated by few reports of additional sites after the initial report.

Few of these auxin resistant weeds have had a significant economic impact due to the wide 

array of alternatives that successfully control these resistant weeds (Heap, 1997). The overall 

incidence of auxinic herbicide resistance after more than seventy years of use is low, 

particularly when compared with other herbicide families in use over shorter time periods:

ALS inhibitors (imidazolinones, sulfonylureas, and sulfonamides), triazines, and ACCase 

herbicides. 

It has been suggested by various researchers that the rarity of auxinic herbicide resistant

biotypes in the field is due to: a) a commonly held belief that these herbicides have multiple 

sites of action in the plant (Jasieniuk et al., 1996), b) redundancy in auxin receptors (AFBs) 

and other components of the auxin signal response (Walsh et al., 2006), c) moderate 

selection pressure and their use in mixtures with other herbicides (Kern et al., 2005), d) 

fitness penalties (Bourdot et al., 1996), and e) quantitative inheritance of the resistance trait

(Cranston et al., 2001).

The mechanism of resistance to auxinic herbicides has been investigated, in varying degrees 

for only a few of these resistant biotypes. Resistance mechanisms in these biotypes have 

proven to be difficult to elucidate. A lack of differences between biotypes in auxinic 

herbicide absorption, translocation, and metabolism has led to the hypothesis that auxinic 

herbicide resistance is most often likely due to differences at the target site or differences 

along the signal transduction pathway (Van Eerd et al., 2005).

Patterns and mechanisms of cross resistance in auxin herbicide resistant biotypes to other 

classes of auxin herbicides are not yet well understood. Further research is needed across a 

range of resistant species and biotypes to identify the potentially numerous gene mutations 

that cause resistance. It is also possible, but less likely, that a biotype might be resistant to all 

of these auxin herbicides. Due to the diversity of chemistry representing the synthetic auxin 

mode of action, it is unlikely plants will derive a single metabolic mechanism for tolerance to 

this class of herbicides.
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Table 40. Weed Species with Reported 2,4-D Resistant Biotypes

Common Name Species Name Herbicide(s) Year Country /

U.S. State

Wild carrot Daucus carota

2,4-D 1952 Ontario

2,4-D 1993 Michigan

2,4-D 1994 Ohio

Dayflower Commelina diffusa 2,4-D 1957 Hawaii

Field bindweed Convolvulus arvensis 2,4-D 1964 Kansas

Musk thistle Carduus nutans 2,4-D, MCPA 1981 New Zealand

Scentless chamomile Matricaria perforate

2,4-D 1975 France

2,4-D 1975 United 

Kingdom

Gooseweed Spenoclea zeylanica

2,4-D 1983 Philippines

2,4-D 1995 Malaysia

2,4-D 2000 Thailand

Canada thistle Cirsium arvense 2,4-D, MCPA 1985 Hungary

Globe fringerush Fimbristylis miliacea 2,4-D 1989 Malaysia

Wild mustard Sinapsi arvensis 2,4-D, most other auxins 1990 Manitoba

Corn poppy Papaver rhoeas 2,4-D, tribenuron 1993 Spain

Yellow bur-head Limnocharis flava 2,4-D 1995 Indonesia

2,4-D, bensulfuron-methyl 1998 Malaysia

Italian thistle Carduus pycnocephalus 2,4-D 1997 New Zealand

Wild radish Raphanus raphanistrum 2,4-D 1999 Australia

Marshweed Limnophila erecta 2,4-D, ALS 2002 Malaysia

Indian hedge 

mustard Sisymbrium orientale 2,4-D, metsulfuron-methyl 2005 Australia

Prickly lettuce Lactuca serriola 2,4-D, dicamba, MCPA 2007 Washington

Common 

Waterhemp Amaranthus tuberculatus 2,4-D 2009 Nebraska

1

(Heap, 2013e)

To summarize, selection for auxin resistant weed biotypes after more than 70 years of use has 

been slow. Auxin resistant weeds have not shown significant spread from initial sites, nor 

have they demonstrated significant economic importance. Thus far, there have been no 

reports of auxin resistant weed biotypes in cotton fields. Use of 2,4-D in DAS-8191Ø-7 

cotton should not result in 2,4-D resistant weeds becoming a significant issue in cotton. 

Additionally, 2,4-D will likely be used in a mixture with one or more other herbicides. Other 

alternative herbicides which are effective on the same weeds can be used to control any 2,4-

D resistant weeds that might occur.

Glufosinate Resistance

There are currently only two reported weed biotypes that have developed resistance to 

glufosinate, Goosegrass (Eleusine indica) in Malaysia and Italian ryegrass (Oregon) (Heap, 

2013a). Italian Ryegrass is also resistant to glyphosate. 
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Weed Resistance Management

Cotton growers in the U.S. are currently battling a number of glyphosate resistant weeds

(Table 41) due to years of exclusive use of glyphosate for weed control (Foresman and 

Glasgow, 2008; Gustafson, 2008). As described above, overreliance on this single method of 

control has greatly increased selection pressure for glyphosate resistant weeds. 

Table 41. U.S. Glyphosate Resistant Weeds in Cotton Growing States1

Number Species Common Name State

1 Amaranthus palmeri Palmer Amaranth

Georgia

North Carolina

Arkansas

Tennessee

New Mexico

Alabama

Georgia

Mississippi

Missouri

Tennessee

Louisiana

Kansas

Arizona

California

2 Amaranthus spinosus Spiny Amaranth Mississippi

3

Amaranthus tuberculatus 

(syn. rudis) Common Waterhemp

Missouri

Kansas

Mississippi

Oklahoma

Tennessee

4 Ambrosia artemisiifolia Common Ragweed

Arkansas

Missouri

Kansas

5 Ambrosia trifida Giant Ragweed

Arkansas

Kansas

Tennessee

Missouri

Mississippi

6 Conyza bonariensis Hairy Fleabane California

7 Conyza canadensis Horseweed

Kentucky

Tennessee

Missouri

Arkansas

Mississippi

North Carolina

California

Kansas

Mississippi

Oklahoma

8 Echinochloa colona Junglerice California

9 Eleusine indica Goosegrass Mississippi

Tennessee

10 Kochia scoparia Kochia Kansas

11 Lolium multiflorum Italian Ryegrass

Mississippi

Arkansas

California

12 Lolium rigidum Rigid Ryegrass California

13 Poa annua Annual Bluegrass Missouri

Tennessee

14 Sorghum halepense Johnsongrass

Arkansas

Mississippi

Louisiana

1

(Webster and Sosnoskie, 2010)
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The magnitude of current weed resistance challenges has prompted outreach efforts by 

industry and academics to educate growers on integrated weed management strategies. For 

example, the Weed Science Society of America has extensive resources on its website, 

including lesson modules for herbicide resistant weeds (WSSA, 2013b). These modules 

explain the current status of resistance, how herbicides work, what herbicide resistance is, 

and principles of managing herbicide resistance (WSSA, 2013c). Similarly, the Herbicide 

Resistance Action Committee, an international body founded by the agrochemical industry, 

provides herbicide resistance management strategies and principles to teach growers an 

integrated approach to weed resistance management (HRAC, 2013b).

One cornerstone of integrated weed resistance management is the use of herbicides with 

multiple modes or sites of action. DAS-8191Ø-7 cotton will be combined with glyphosate 

tolerant cotton utilizing traditional breeding techniques and fits into a recommended 

integrated weed resistance management program. Additionally, DAS-8191Ø-7 cotton is a 

needed tool to mitigate the current selection pressure for glyphosate resistant weed species.

Conclusions 

The adoption of herbicide tolerant crops, primarily glyphosate tolerant varieties, and 

resulting reliance on a single mode of action for weed control, has resulted in a rapidly 

growing population of resistant and hard to control weed species. Growers now understand 

the need for an integrated weed management approach; DAS-8191Ø-7 cotton offers an 

alternative chemistry for control of troublesome weeds, in accordance with recommended 

management practices.

DAS-8191Ø-7 cotton will be combined with glyphosate tolerant cotton utilizing traditional 

breeding techniques. The combination of herbicide tolerance traits will allow the use of 

multiple herbicides in an integrated weed management program to control a broad spectrum 

of grass and broadleaf weed species in cotton. These herbicides will provide distinct modes 

of actions for use in conjunction with other herbicide active ingredients and mode of action 

for an effective weed management program in cotton. 
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Appendix 8. Stewardship of Herbicide Tolerant Trait Technology for DAS-8191Ø-7 Cotton

Introduction

DAS-8191Ø-7 cotton is a transgenic cotton product that provides tolerance to 2,4-

dichlorophenoxyacetic acid (2,4-D) and glufosinate. This herbicide tolerant cotton will 

provide growers with greater flexibility in selection of herbicides for the improved control of 

economically important weeds; allow an increased application window for effective weed 

control; and provide an effective resistance management prevention solution to the increased 

incidence of hard to control and glyphosate resistant weeds1. 

Dow AgroSciences is committed to promoting the responsible use and stewardship of this 

new herbicide tolerant trait technology and will implement a comprehensive stewardship 

program for DAS-8191Ø-7 cotton and its associated herbicide technology containing a new

2,4-D choline salt. The stewardship program will focus on educating and training retailers, 

growers and applicators on the appropriate use of this new technology as part of an integrated 

weed management program. This will be accomplished by using a multi-faceted approach, 

including use of a variety of tools and delivery methods, and working with customers, 

stakeholders and industry organizations to promote responsible use of the technology. The 

stewardship program is designed to:

• Provide comprehensive guidance and education on responsible use 

• Promote compatibility with other crops and cropping systems 

• Minimize the potential for off-target movement 

• Promote Weed Resistance Management2

• Promote responsible use and worker safety 

• Support compliance with applicable regulatory requirements

Stewardship Program Components

The comprehensive stewardship program is based on Dow AgroSciences’ ongoing 

commitment to stewardship and input from retailers, growers, applicators, regulators, and 

stakeholders. Key components of the stewardship program include:

 Authorized use of DAS-8191Ø-7 cotton (and associated herbicide products) through a 

Technology Use Agreement (TUA)

 Herbicide technology advancements, including new 2,4-D choline based herbicide 

technologies 

 Comprehensive product use information, including mandatory use requirements and 

restrictions

 Education and training for distributors, retailers, growers, and applicators, and

 

1 As used in this document, a herbicide resistant weed is a member of a population within a species that has an 

inherited ability to survive and reproduce following exposure to a dose of a herbicide normally lethal to susceptible 

populations of the species. Through repeated herbicide selection, the resistant population becomes dominant in a 

given area. A hard-to-control weed is a weed species that is inherently able to withstand treatment with a herbicide 

as a result of natural factors not involving herbicide selection or genetic mutation.

2

Stewardship information on insect resistance management will also be included, as applicable.
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 Compliance monitoring.

Authorized Use of DAS-8191Ø-7 Cotton and Associated Herbicide Products Through a 

Technology Use Agreement 

Dow AgroSciences will authorize growers of DAS-8191Ø-7 cotton through a Technology 

Use Agreement that requires growers to use the cotton trait technology responsibly and 

prohibits the use of unauthorized herbicides. 

The Technology Use Agreement is a legal and contractual obligation that will require all 

growers of DAS-8191Ø-7 cotton to: 

 Use only EPA accepted and Dow AgroSciences’ authorized herbicide products for postplant applications to DAS-8191Ø-7 cotton; 

 Read and follow the Product Use Guide (PUG) requirements;

 Read and follow FIFRA pesticide product label directions, which will include 

requirements to use in a manner protective of the environment; 

 Read and follow seed packaging information; and 

 Provide access to information about use of the technology and allow on-farm 

assessments.

Herbicide Technology Advancements

Dow AgroSciences has discovered and developed a novel (non-ester or non-amine) 

2,4-D choline3. Exceptionally low volatility of DAS’ 2,4-D choline salt has been 

demonstrated in both laboratory and field studies. In a laboratory study, wheat plants treated 

with rates ten times higher than specified on the label of 2,4-D choline were placed in an 

enclosed chamber with grape and tomato plants (Ouse et al., 2010). After 24 hours of 

exposure in the chamber at 104°F, neither sensitive crop showed any symptoms of injury. 

The lack of any symptoms on the grape and tomato plants is a clear indication that there was 

no detectable volatility following application of 2,4-D choline salt. Field results are 

consistent with these laboratory findings (Hillger et al., 2010).

Comprehensive Product Use Information

Product Use Guides 

Dow AgroSciences will provide DAS-8191Ø-7 cotton growers a comprehensive Product Use 

Guide that is a material part of the Technology Use Agreement and details the requirements 

for responsible use. As a standard practice, Dow AgroSciences provides a Product Use Guide 

to users (e.g., retailers, dealers, growers, applicators) of its technology. The Product Use 

Guide provides growers comprehensive information including required and recommended 

management practices. The Product Use Guide for DAS-8191Ø-7 cotton will include 

detailed requirements for responsible use of the cotton and associated herbicide products. It 

will include information on weed resistance management, application and management of 

 

3 Applications for 2,4-D choline and associated herbicides for use with DAS-8191Ø-7 cotton will be submitted to 

EPA.
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 198 of 214

Dow AgroSciences LLC

off-target herbicide movement, as well as seed planting directions and cotton seed and byproduct stewardship. 

The Product Use Guide will require DAS-8191Ø-7 cotton growers to: 

 Use only EPA accepted and Dow AgroSciences’ authorized herbicide products with 

DAS-8191Ø-7 cotton

 Read and follow FIFRA pesticide product label directions 

 Follow Weed Resistance Management guidelines, recommending utilization of herbicide 

products with additional modes of action 

 Read and follow marketing guidelines 

 Provide management practices to promote compatibility with other crops, which may 

include setbacks, consulting sensitive crop registries, and communicating with neighbors.

The Product Use Guide will promote appropriate cultural practices including diversified 

weed control strategies (e.g. herbicide use or crop rotation) to minimize selection for 

herbicide resistant weed populations. Responsible use of the cotton seed and herbicides in an 

integrated farming system promotes compatibility with other crops.

Herbicide Application and Management of Off-Target Movement 

The new 2,4-D choline-containing herbicide products will minimize off target movement by 

reducing volatility and the number of driftable fines. To further minimize the potential for 

off-target movement with the new 2,4-D choline-containing herbicide products, the Product 

Use Guide will include recommendations on application equipment and methods such as 

following proper equipment maintenance, calibration and use. 

A variety of factors including weather conditions (e.g., wind direction, wind speed, 

temperature, and relative humidity) and method of application can influence pesticide drift. 

The Product Use Guide will require applicators to evaluate factors and make appropriate 

adjustments when applying 2,4-D choline salt for use with DAS-8191Ø-7 cotton consistent 

with the final product label approved by EPA. Dow AgroSciences is conducting significant 

research on nozzles for spray application. The Product Use Guide will include instructions on 

using specific nozzles that minimize driftable spray droplets and specified environmental 

conditions such as wind speed and field temperature that minimize the potential for off-target 

movement due to particle or vapor drift. 

Product Labels, Seed Bags and Tags 

Product Labels

Dow AgroSciences will submit a product label to EPA that includes comprehensive 

information and requirements for responsible use of 2,4-D choline with DAS-8191Ø-7 

cotton. That label language will include detailed use directions and application requirements 

designed to minimize the potential for weed resistance development, minimize the potential 

for off-target movement, and promote worker safety. For example, the submitted label will
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 199 of 214

Dow AgroSciences LLC

not allow herbicide application through any type of irrigation equipment and will prohibit

aerial application. 

Seed Bags and Tags 

Packages of DAS-8191Ø-7 cotton seed will have product stewardship information on the 

seed bags and seed tags, including use requirements and a customer information telephone 

number. Information will be included on seed packaging to communicate the presence of 

herbicide tolerant traits. This information will also be included in the Product Use Guide, 

training, and marketing materials.

Education and Training for Retailers, Growers and Applicators

Dow AgroSciences will provide comprehensive education and training in multiple formats to 

distributors, retailers, growers, and applicators to reinforce the requirements for proper 

stewardship and responsible use. Dow AgroSciences provides comprehensive training on its 

portfolio of products through a variety of formats. Education and training, reinforced through 

product profiles, technical bulletins, sales literature, direct mailing and websites will also be 

presented in multiple formats to enhance learning and mastery of core concepts related to the 

stewardship program. 

A variety of educational formats will be used to promote concept learning. Training will be 

provided through face-to-face meetings, retailer visits, field visits, and self-paced learning 

modules on: 

 stewardship requirements for responsible use of DAS-8191Ø-7 cotton and associated 

herbicides, including

 weed resistance management.

This training will reinforce the cotton grower’s obligation to adhere to product use 

guidelines, FIFRA Pesticide Product Labels, seed product information and all product 

stewardship requirements for responsible use and proper stewardship. Training is an ongoing 

process, with interactive learning incorporated, where applicable, to promote responsible use 

and stewardship. 

Multiple resources and tools will be available to the growers and applicators to promote and 

communicate the importance of stewardship and responsible technology use. In addition to 

the Technology Use Agreement, Product Use Guide, and training, additional resources will 

be available, including: 

 Customer Service 

 Technical Bulletins 

 AdvanceFarming.com 

 On-line training library 

 In-field experts 

 Trait stewardship website4

 

4 www.traitstewardship.com
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 200 of 214

Dow AgroSciences LLC

 Stewardship tools 

An extensive network of Dow AgroSciences sales representatives, field scientists, and 

agronomists will play an important role in educating and training retailers, growers and 

applicators on the proper use of the technology and application guidelines. Dow 

AgroSciences field personnel, in addition to product suppliers, will work with growers to 

select the seed and herbicide products appropriate to their needs, growing conditions, and 

proper application equipment for field conditions. 

Compliance Monitoring 

Dow AgroSciences will monitor compliance with the Technology Use Agreement and 

Product Use Guide through surveys and on-farm assessments. Upon request by Dow 

AgroSciences, DAS-8191Ø-7 cotton growers must provide information regarding the 

location of fields planted with DAS-8191Ø-7 cotton, and the herbicides applied to these 

fields. 

Failure to follow the stewardship requirements will result in: 

 Education and reinforcement of the stewardship requirements with growers, applicators 

and retailers, 

 Additional education and training, and/or 

 Loss of access to DAS-8191Ø-7 cotton.

Stakeholder Outreach and Industry Involvement

In addition to managing the technology in accordance with the applicable requirements of federal 

and state government agencies, Dow AgroSciences is a member of the American Chemistry 

Council's Responsible Care®initiative and Founding Member of the biotechnology industry’s 

Excellence Through Stewardship®organization which encourages effective and comprehensive 

stewardship programs and quality management systems throughout a trait product’s life cycle. 

Dow AgroSciences participates in several organizations and associations globally to promote the 

safe research and development, production, distribution, and responsible use of agricultural 

chemical and biotechnology products. 

Dow AgroSciences is a participant in the Herbicide Resistance Action Committee (HRAC)5, 

an industry-based group supported by CropLife International6. HRAC focuses on 

encouraging responsible herbicide usage, communicating herbicide resistance management 

strategies and supporting their implementation through practical guidelines. HRAC engages 

in active collaboration with public and private researchers, especially in the areas of problem 

identification and devising and implementing herbicide management strategies. 

Dow AgroSciences’ personnel interact with academic weed scientists in addressing weed 

resistance management7issues. Dow AgroSciences conducts joint trials at university sites as 

 

5 www.hracglobal.com

6 www.croplife.org/public/resistance_management

7 www.wssa.net/Weeds/Resistance
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 201 of 214

Dow AgroSciences LLC

well as seeks input from university researchers regarding weed management. Dow 

AgroSciences also participates in a wide range of professional organizations including 

agronomy societies, seed trade groups, weed science societies, and crop commodity groups.

Conclusion

Dow AgroSciences is committed to promoting the responsible use and stewardship of DAS8191Ø-7 cotton and associated herbicide products. The comprehensive stewardship program 

outlined here incorporates elements to:

 Promote responsible use of DAS-8191Ø-7 cotton and associated herbicide products, 

 Promote compatibility with other crops and cropping systems,

 Promote Weed Resistance Management, and 

 Support compliance with all relevant state and federal requirements.

The stewardship program, focusing on education and management practices for proper use of 

the cotton and herbicides, coupled with the physical attributes of DAS’ 2,4-D choline 

formulation (e.g., ultra-low volatility, minimized potential for drift, decreased odor and 

improved handling characteristics) enables coexistence with organic crops, crops 

traditionally sensitive to 2,4-D, and non-genetically engineered cotton. 

Education and training efforts are also an integral part of the stewardship effort, promoting 

awareness and understanding of proper use of the cotton and herbicides. The combination of 

EPA mandated herbicide label requirements, Dow AgroSciences’ stewardship program, and 

associated proprietary herbicide technology will provide the grower of DAS-8191Ø-7 cotton

with the tools needed to mitigate potential risk to human health and the environment without 

compromising farm productivity. 
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 202 of 214

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Appendix 9. USDA Notifications for DAS-8191Ø-7 Cotton

USDA 

Notification 

Number

Authorization 

Date

Expiration 

Date

State(s) Number 

of Trials

Trial Report 

Status1

13-063-109n 4/17/2013 4/17/2014 TX 12 Pending

13-053-106n 4/1/2013 4/1/2014 TX 3 Pending

13-063-110n 3/25/2013 3/25/2014 AR, CA, GA, 

LA, MS, NC, 

OK, SC

27 Pending

12-089-109n 4/26/2012 4/26/2013 LA 1 Pending

12-089-106n 4/26/2012 4/26/2013 TX 2 Pending

12-089-109n 4/26/2012 4/26/2013 AL, AR, CA, FL, 

GA, MO, MS, 

NC

9 Pending

12-089-101n 4/15/2012 4/15/2013 TX 13 Pending

13-066-104n 4/12/2012 4/12/2013 AL, AR, CA, 

GA, LA, MO, 

MS, NC

10 Pending

12-081-113n 4/6/2012 4/6/2013 AR, CA, GA, 

MO, MS, NC

13 Pending

12-062-102n 4/5/2012 4/5/2013 AR, CA, GA, 

LA, MO, MS, 

NC, OK, SC, TN

50 Pending

12-081-114n 4/4/2012 4/4/2013 TX 8 Pending

11-249-101n 11/1/2011 11/1/2012 PR 1 Submitted 

(Apr 2013)

11-147-101n 6/10/2011 6/10/2012 IN 1 Submitted 

(Nov 2012)

11-087-112n 4/29/2011 4/29/2012 AR, CA, GA, 

LA, MS, NC, 

SC, TN, TX

24 Submitted 

(Oct 2012)

10-076-102n 4/19/2010 4/19/2011 AL, AR, CA, 

GA, LA, MS, 

NC, SC, TN

29 Submitted 

(Oct 2011)

1

Pending reports to be submitted within six months of the notification expiration date
USDA-APHIS Petition for Nonregulated Status of DAS-8191Ø-7 Cotton Page 203 of 214

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