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Petition for Determination of Nonregulated Status for

Herbicide Tolerant DAS-68416-4 Soybean

OECD Unique Identifier: DAS-68416-4

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

Submitted by:

__________________________________

Mark S. Krieger, Ph.D.

Regulatory Leader

Regulatory Sciences & Government Affairs

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, IN 46268

Telephone: (317) 337-3458

FAX: (317) 337-4649

mkrieger2@dow.com

December 8, 2009

Revised July 30, 2010

Revised November 15, 2010
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Release of Information

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

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

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

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

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

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

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

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

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

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

CBI-deleted copy of this information may be made available to the public in a reading room and

by individual request, as part of the public comment period. Except in accordance with the

foregoing, DAS does not authorize the release, publication or other distribution of this

information (including website posting) without DAS’ prior notice and consent.
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Certification

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

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

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

Mark S. Krieger, Ph.D.

Regulatory Leader

Dow AgroSciences LLC

9330 Zionsville Road

Indianapolis, Indiana 46268

Telephone: (317) 337-3458

Fax: (317) 337-4649

mkrieger2@dow.com
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Summary

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

Determination of Nonregulated Status for Herbicide Tolerant DAS-68416-4 Soybean. DAS

requests a determination from USDA Animal and Plant Health Inspection Service (APHIS) that

soybean transformation event DAS-68416-4 and any soybean lines derived from crosses with

DAS-68416-4 soybeans no longer be considered regulated articles under 7 CFR Part 340.

DAS-68416-4 soybean is a transgenic soybean product that provides tolerance to the herbicides

2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate. This herbicide-tolerant soybean 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 increased

incidence of glyphosate resistant weeds.

DAS-68416-4 soybean plants have been genetically modified to express the aryloxyalkanoate

dioxygenase-12 (AAD-12) and phosphinothricin acetyltransferase (PAT) proteins. The AAD-12

protein 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. The PAT enzyme acetylates the primary amino group of phosphinothricin rendering

it inactive. The pat gene expressing the PAT protein was derived from Streptomyces

viridochromogenes.

The aad-12 and pat genes were introduced into DAS-68416-4 soybean using

Agrobacterium-mediated transformation. Molecular characterization by Southern analyses of the

DAS-68416-4 event confirmed that a single, intact insert of the aad-12 and pat genes were stably

integrated into the soybean genome. A single copy of each of the genetic elements of the aad-12

expression cassette is present and the integrity of the inserted DNA fragment was demonstrated

in three different breeding generations, confirming the stability during traditional breeding

procedures. Southern analyses also confirmed the absence of unwanted DNA such as the

plasmid backbone DNA in DAS-68416-4 soybean. Segregation data for breeding generations

confirmed the predicted inheritance of the aad-12 and pat genes.

The AAD-12 and PAT proteins in DAS-68416-4 soybean were characterized biochemically and

measured using AAD-12 and PAT specific enzyme linked immunosorbent assays (ELISA).

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

the growing season from DAS-68416-4 plants treated with 2,4-D, glufosinate, both 2,4-D and

glufosinate, or not treated with either herbicide. The results showed a low level of expression of

the AAD-12 and PAT proteins across herbicide treatments and environments, indicating a low

exposure to humans and animals.

The AAD-12 protein was assessed for any potential adverse effects to humans or animals

resulting from the environmental release of crops containing the AAD-12 protein. 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 with known or

putative allergens or toxins for the AAD-12 amino acid sequence. The AAD-12 protein
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hydrolyzes rapidly in simulated gastric fluid and there was no evidence of acute toxicity in mice

at a dose of 2000 mg/kg body weight of AAD-12 protein. Glycosylation analysis of the plantand microbe-derived AAD-12 proteins revealed no detectable covalently linked carbohydrates.

Results of the overall safety assessment of the AAD-12 protein indicate that it is unlikely to

cause allergenic or toxic effects in humans or animals. The safety of the PAT protein has been

assessed previously and it has been approved for use in canola, corn, cotton, rice, soybeans, and

sugar beets.

DAS-68416-4 soybean has been field tested in the major soybean growing regions of the

continental United States as well as Puerto Rico. All field tests were conducted under field

permits granted by USDA APHIS. Agronomic performance assessments were conducted on

DAS-68416-4 soybean in multi-site field studies to measure characteristics such as emergence,

seedling vigor, plant height, lodging, and yield. All field trials were also observed for

opportunistic disease or insect stressors as well as normal phenotypic characteristics. There were

no meaningful differences observed between DAS-68416-4 soybean and the non-transgenic

control for plant pest characteristics and no indication of a selective advantage that would result

in increased weediness potential of DAS-68416-4 soybean.

Nutrient composition analyses of forage and grain was conducted to compare the composition of

DAS-68416-4 soybean with the composition of a non-transgenic control. Compositional

analyses were used to evaluate any changes in the levels of key nutrients and anti-nutrients in

DAS-68416-4 soybean which was sprayed with either 2,4-D, glufosinate, both 2,4-D and

glufosinate, or which was not sprayed with either herbicide. Along with the agronomic data, the

compositional analyses indicate that DAS-68416-4 soybean is substantially equivalent to

conventional soybean and will not exhibit unexpected or unintended effects with respect to plant

pest risk.

Since DAS-68416-4 soybean is agronomically and nutritionally similar to conventional soybean,

and the safety of the AAD-12 and PAT proteins has been demonstrated, no significant impact is

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

volunteer management, or commodity food and feed soybean products. The availability of DAS68416-4 soybean is expected to have a beneficial impact on weed control practices by providing

growers with another tool to address their weed control needs. The use of DAS-68416-4

soybean will allow growers to proactively manage weed populations while avoiding adverse

population shifts of troublesome weeds or the development of resistance, particularly glyphosateresistance in weeds.

Information collected during field trials and laboratory analyses presented herein demonstrate

that DAS-68416-4 soybean exhibits no plant pathogenic properties or weediness characteristics.

DAS-68416-4 soybean is no more likely to become a plant pest than conventional soybean, and

the AAD-12 and PAT proteins are unlikely to increase the weediness potential of any other

cultivated plant or wild species.

DAS hereby requests a determination from APHIS that herbicide-tolerant DAS-68416-4 soybean

and all progeny derived from the conventional breeding of this line no longer be considered

regulated articles under 7 CFR Part 340.
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Table of Contents

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

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

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

Table of Contents.......................................................................................................................... 6

List of Figures................................................................................................................................ 9

List of Tables............................................................................................................................... 11

Acronyms and Scientific Terms................................................................................................. 13

I. Rationale for the Development of DAS-68416-4 Soybean........................................... 15

I-A. Basis for the Request for Nonregulated Status..................................................... 15

I-B. Benefits of DAS-68416-4 Soybean ...................................................................... 15

I-C. Submissions to Other Regulatory Agencies ......................................................... 17

I-D. References............................................................................................................. 17

II. The Biology of Soybean .................................................................................................. 18

II-A. Overview of Soybean Biology.............................................................................. 18

II-B. Characterization of the Recipient Soybean Cultivar............................................. 18

II-C. References............................................................................................................. 19

III. Development of DAS-68416-4 Soybean......................................................................... 20

III-A. Description of the Transformation System........................................................... 20

III-B. Selection of Comparators for DAS-68416-4 Soybean.......................................... 24

III-C. References............................................................................................................. 24

IV. Donor Genes and Regulatory Sequences...................................................................... 25

IV-A. Identity and Source of Genetic Material in pDAB4468 ....................................... 25

IV-B. References............................................................................................................. 27

V. Genetic Characterization ............................................................................................... 30

V-A. Overview of Molecular Analysis.......................................................................... 30

V-B. Analysis of the Insert and Its Genetic Elements................................................... 36

V-B.1. Analysis of the aad-12 Gene........................................................................... 36

V-B.2. Analysis of the AtUbi10 Promoter ................................................................. 36

V-B.3. Analysis of the AtuORF23 3’UTR ................................................................. 36

V-B.4. Analysis of the pat Gene................................................................................. 37

V-B.5. Analysis of the CsVMV Promoter.................................................................. 37

V-B.6. Analysis of the AtuORF1 3’UTR ................................................................... 37

V-B.7. Analysis of the RB7 MAR.............................................................................. 37

V-C. Absence of Vector Backbone DNA...................................................................... 38

V-D. Stability of the Insert across Generations ............................................................. 38

V-E. Segregation Analysis ............................................................................................ 64

V-E.1. Genetic and Molecular Analysis of a Segregating Generation....................... 64

V-E.2. Segregation Analysis of Breeding Generations.............................................. 68

V-F. Summary of the Genetic Characterization............................................................ 68

VI. Characterization of the Introduced Proteins ............................................................... 69

VI-A. AAD-12................................................................................................................. 69

VI-A.1. Identity of the AAD-12 Protein ...................................................................... 69

VI-A.2. Biochemical Characterization of the AAD-12 Protein ................................... 69

VI-A.3. Expression of the AAD-12 Protein in Plant Tissues....................................... 70

VI-A.4. Food and Feed Safety Assessment for AAD-12 Protein ................................ 72
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VI-A.5. Summary of AAD-12 Protein Characterization.............................................. 73

VI-B. PAT....................................................................................................................... 73

VI-B.1. Identity of the PAT Protein............................................................................. 73

VI-B.2. Biochemical Characterization of the PAT Protein.......................................... 73

VI-B.3. Expression of the PAT Protein in Plant Tissues............................................. 74

VI-B.1. Food and Feed Safety Assessment for PAT Protein....................................... 75

VI-B.2. Summary of PAT Protein Characterization .................................................... 76

VI-C. References............................................................................................................. 76

VII. Agronomic Performance ................................................................................................ 79

VII-A. Phenotypic and Agronomic Characteristics.......................................................... 79

VII-A.1. Experiment 1................................................................................................... 79

VII-A.2. Experiment 2................................................................................................... 83

VII-A.3. Conclusions..................................................................................................... 85

VII-B. Ecological Evaluations.......................................................................................... 86

VII-C. Germination and Dormancy Evaluations.............................................................. 87

VII-D. Summary of Agronomic, Disease, and Pest Characteristics................................. 88

VII-E. References............................................................................................................. 88

VIII. Grain and Forage Composition..................................................................................... 89

VIII-A. Compositional Analysis of Soybean Forage......................................................... 90

VIII-B. Compositional Analysis of Soybean Grain........................................................... 93

VIII-B.1. Proximates and Fiber ...................................................................................... 93

VIII-B.2. Minerals .......................................................................................................... 96

VIII-B.3. Amino Acids................................................................................................... 99

VIII-B.4. Fatty Acids.................................................................................................... 103

VIII-B.5. Vitamins........................................................................................................ 107

VIII-B.6. Isoflavones.................................................................................................... 110

VIII-B.7. Antinutrients ................................................................................................. 113

VIII-C. Summary of Grain and Forage Composition...................................................... 115

VIII-D. References........................................................................................................... 115

IX. Environmental Consequences and Impact on Agronomic Practices....................... 116

IX-A. Mode of Action of the AAD-12 Protein ............................................................. 116

IX-A.1. Field Efficacy................................................................................................ 116

IX-B. Weediness Potential............................................................................................ 119

IX-C. Gene Flow Assessment....................................................................................... 120

IX-C.1. Vertical Gene Flow Assessment ................................................................... 120

IX-C.2. Horizontal Gene Flow Assessment............................................................... 120

IX-D. Current US Agronomic Practices for Soybeans.................................................. 120

IX-D.1. Soybean Production ...................................................................................... 120

IX-D.2. Weeds in Soybean......................................................................................... 121

IX-D.3. Weed Management in Soybean .................................................................... 123

IX-D.4. Crop Rotation and Tillage Practices............................................................. 124

IX-E. Potential Impact on Agronomic Practices........................................................... 124

IX-E.1. Potential Impact on Cultivation and Management Practices........................ 124

IX-E.2. Potential Impact on Weed Control Practices................................................ 125

IX-E.3. Potential Impact on Volunteer Management ................................................ 127

IX-E.4. Potential Impact on Non-Target Organisms and Endangered Species......... 128
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IX-F. Herbicide Resistance Management..................................................................... 129

IX-F.1. Herbicide Resistance..................................................................................... 129

IX-F.2. Factors Impacting Development of Resistance............................................. 131

IX-F.3. Herbicide Resistance Management............................................................... 132

IX-G. Summary of Environmental Assessment............................................................ 133

IX-H. References........................................................................................................... 134

X. Adverse Consequences of Introduction ...................................................................... 138

XI. Appendices..................................................................................................................... 139

Appendix 1. Methods for Molecular Characterization of DAS-68416-4 Soybean................ 140

Appendix 2. Methods and Results for Characterization of AAD-12 Protein ........................ 143

Appendix 3. Methods and Results for Characterization of PAT Protein............................... 155

Appendix 4. Methods for AAD-12 and PAT Protein Expression Analysis........................... 158

Appendix 5. USDA Notifications for DAS-68416-4 Soybean .............................................. 160

Appendix 6. Literature Ranges for Compositional Analysis................................................. 161

Appendix 7. Glyphosate, 2,4-D, Glufosinate and Herbicide Resistant Weeds...................... 165

Appendix 8. Stewardship of Herbicide Tolerant DAS-68416-4 Soybeans ........................... 178
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List of Figures

Figure 1. Plasmid map of pDAB4468........................................................................................... 21

Figure 2. Diagram of T-DNA insert in plasmid pDAB4468. ....................................................... 21

Figure 3. Development of DAS-68416-4 soybean. ...................................................................... 22

Figure 4. Breeding diagram of DAS-68416-4 soybean. ............................................................... 23

Figure 5. Location of probes on pDAB4468 used in Southern blot analysis of

DAS-68416-4 soybean...................................................................................................... 31

Figure 6. Plasmid map of pDAB4468 with restriction enzyme sites used for Southern blot

analysis.............................................................................................................................. 34

Figure 7. pDAB4468 T-DNA insert, restriction enzymes used in DNA digestion and

expected hybridization bands............................................................................................ 35

Figure 8. Southern blot analysis of Nco I digest with aad-12 probe. ........................................... 39

Figure 9. Southern blot analysis of Sph I/Xho I digest with aad-12 probe................................... 40

Figure 10. Southern blot analysis of Nhe I/Xho I digest with aad-12 probe. ............................... 41

Figure 11. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generation with aad-12 probe. .......................................................................................... 42

Figure 12. Southern blot analysis of Pst I digest with aad-12 probe............................................ 43

Figure 13. Southern blot analysis of Pst I digest of T5 generation with aad-12, AtUbi10,

and AtuORF23 probes. ..................................................................................................... 44

Figure 14. Southern blot analysis of Pst I digest with AtUbi10 probe......................................... 45

Figure 15. Southern blot analysis of Pst I digest with AtuORF23 probe. .................................... 46

Figure 16. Southern blot analysis of Nco I digest with pat probe................................................. 47

Figure 17. Southern blot analysis of Sph I/Xho I digest with pat probe. ...................................... 48

Figure 18. Southern blot analysis of Nhe I/Xho I digest with pat probe....................................... 49

Figure 19. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generation with pat probe. ................................................................................................ 50

Figure 20. Southern blot analysis of Pst I/Xho I digest with pat probe........................................ 51

Figure 21. Southern blot analysis of Pst I/Xho I digest of T5 generation with pat,

CsVMV, and AtuORF1 probes......................................................................................... 52

Figure 22. Southern blot analysis of Pst I/Xho I digest with CsVMV probe. .............................. 53

Figure 23. Southern blot analysis of Pst I/Xho I digest with AtuORF1 probe. ............................ 54

Figure 24. Southern blot analysis of BamH I/Nco I digest with RB7 probe................................. 55

Figure 25. Southern blot analysis of BamH I/Nco I digest with AtUbi10 probe.......................... 56

Figure 26. Southern blot analysis of BamH I/Nco I digest of T5 generation with RB7 and

AtUbi10 probes................................................................................................................. 57

Figure 27. Southern blot analysis of Nco I digest with backbone probe set 1 from plasmid

pDAB4468 vector backbone............................................................................................. 58

Figure 28. Southern blot analysis of Nco I digest with backbone probe set 2 from plasmid

pDAB4468 vector backbone............................................................................................. 59

Figure 29. Southern blot analysis of Sph I/Xho I digest with backbone probe set 1 from

plasmid pDAB4468 vector backbone. .............................................................................. 60

Figure 30. Southern blot analysis of Sph I/Xho I digest with backbone probe set 2 plasmid

pDAB4468 vector backbone............................................................................................. 61

Figure 31. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generations with the backbone probe set 1 from pDAB4468 vector backbone................ 62
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Figure 32. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generation with the backbone probe set 2 from pDAB4468 vector backbone. ................ 63

Figure 33. Southern blot analysis of Nco I digest with aad-12 probe. ......................................... 66

Figure 34. Southern blot analysis of Nco I digest with pat probe................................................. 67

Figure 35. Amino acid sequence of the AAD-12 protein. ............................................................ 69

Figure 36. Amino acid sequence of the PAT protein.................................................................... 73

Figure 37. Summary of the proximate, fiber and mineral analysis of soybean forage (%

dry weight)........................................................................................................................ 92

Figure 38. Summary of the proximate and fiber analysis of soybean grain. ................................ 95

Figure 39. Summary of the mineral analysis of soybean grain (mg/100 g dry weight). .............. 98

Figure 40. Summary of the amino acid analysis of soybean grain (% dry weight).................... 101

Figure 41. Summary of the fatty acid analysis of soybean grain (% total fatty acids). .............. 106

Figure 42. Summary of vitamin analysis of soybean grain (mg/kg dry weight). ....................... 109

Figure 43. Summary of isoflavone analysis of soybean grain (μg/g). ........................................ 112

Figure 44. Summary of anti-nutrient analysis of soybean grain (% dry weight)........................ 114

Figure 45. Degradation reaction of 2,4-D catalyzed by AAD-12............................................... 116

Figure 46. Adoption of genetically engineered crops in the U.S................................................ 125

Figure 47. 2,4-D herbicide application timing and rates for conventional and

DAS-68416-4 soybeans. ................................................................................................. 126

Figure 48. Glyphosate application rates in U.S. corn and soybeans from 1990 thru 2007......... 127

Figure 49. Resistant weed biotypes per herbicide mode of action. ............................................ 131

Figure 50. SDS-PAGE of soybean- and microbe-derived AAD-12. .......................................... 145

Figure 51. SDS-PAGE and western blot of soybean- and microbe-derived AAD-12

protein extracts................................................................................................................ 146

Figure 52. Glycosylation analysis of soybean- and microbe-derived AAD-12 proteins............ 147

Figure 53. Theoretical trypsin (top panel) and Asp-N (bottom panel) cleavage of the

AAD-12 protein. ............................................................................................................. 149

Figure 54. Sequence coverage in the tryptic and Asp-N peptide mapping analysis of

plant-derived AAD-12 protein with MALDI-TOF and ESI/LC MS.............................. 150

Figure 55. Sequence coverage in the peptide mapping analysis of microbe-derived AAD12 protein with MALDI-TOF and ESI/LC MS. ............................................................. 151

Figure 56. SDS-PAGE and western blots of DAS-68416-4 and non-transgenic Maverick

soybean. .......................................................................................................................... 156

Figure 57. U.S. adoption rates of glyphosate-tolerant soybean, cotton and corn. ...................... 166

Figure 58. Number of glyphosate-resistant weeds reported globally by year from 1996 to

2008................................................................................................................................. 171

Figure 59. Example weed resistance management statement on DAS glyphosate brand

labels. .............................................................................................................................. 179
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List of Tables

Table 1. Genetic elements of the T-DNA insert from plasmid pDAB4468. ................................ 25

Table 2. List of probes and their positions in plasmid pDAB4468. ............................................. 31

Table 3. Predicted and observed sizes of hybridizing fragments in Southern blot analyses

of DAS-68416-4 soybean. ................................................................................................ 32

Table 4. Results of F2 individual plants tested for of AAD-12 expression within a single

segregating generation. ..................................................................................................... 65

Table 5. Predicted and observed hybridizing fragments in Southern blot analysis of F2

population. ........................................................................................................................ 65

Table 6. Results of F2 individual plants analyzed by Southern blot with aad-12 and pat

probes within a single segregating generation.................................................................. 65

Table 7. Results of F2 individual plants tested for aad-12 within a single segregating

generation.......................................................................................................................... 68

Table 8. Summary of AAD-12 protein levels in tissues collected from DAS-68416-4

produced in the U.S. and Canada during 2008. ................................................................ 71

Table 9. Summary of PAT protein levels in tissues collected from DAS-68416-4

produced in the U.S. and Canada during 2008. ................................................................ 75

Table 10. Agronomic parameters evaluated in Experiment 1....................................................... 80

Table 11. Analysis of agronomic characteristics from Experiment 1........................................... 82

Table 12. Locations for Experiment 2 agronomic and yield trials, 2009. .................................... 83

Table 13. Data collected in Experiment 2 agronomic and yield trials, 2009................................ 84

Table 14. Agronomic parameters for sprayed (2,4-D) and unsprayed DAS-68416-4

soybean. ............................................................................................................................ 84

Table 15. Agronomic parameters for DAS-68416-4 and Maverick soybean. .............................. 85

Table 16. Analysis of disease incidence and insect damage from Experiment 1. ........................ 86

Table 17. Disease and insect stressors observed in trials of DAS-68416-4 and

conventional soybean........................................................................................................ 87

Table 18. Germination of DAS-68416-4 seeds under warm and cold conditions........................ 88

Table 19. Summary of the proximate, fiber and mineral analysis of soybean forage

(% dry weight). ................................................................................................................. 91

Table 20. Summary of the proximate and fiber analysis of soybean grain (% dry weight). ........ 94

Table 21. Summary of the mineral analysis of soybean grain (mg/100g dry weight).................. 97

Table 22. Summary of the amino acid analysis of soybean grain (% dry weight). .................... 100

Table 23. Summary of the fatty acid analysis of soybean grain (% total fatty acids). ............... 105

Table 24. Summary of vitamin analysis of soybean grain (mg/kg dry weight).......................... 108

Table 25. Summary of isoflavone analysis of soybean grain (μg/g). ......................................... 111

Table 26. Summary of anti-nutrient analysis of soybean grain (% dry weight). ........................ 114

Table 27. DAS-68416-4 soybean tolerance to preemergence applications of 2,4-D, 2008........ 117

Table 28. DAS-68416-4 soybean tolerance to post-emergence applications of 2,4-D,

2008................................................................................................................................. 118

Table 29. DAS-68416-4 soybean tolerance to postemergence applications of glufosinate,

2008................................................................................................................................. 119

Table 30. Common troublesome weeds in soybeans in 2006-2008............................................ 122

Table 31. Yield reduction from specific weed species in soybeans. .......................................... 123

Table 32. Number of herbicide resistant weeds globally by herbicide group and mode of

action............................................................................................................................... 130
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Table 33. Assessment of resistance risk by evaluation of cropping systems.............................. 132

Table 34. Glyphosate- and ALS-resistant weeds controlled by 2,4-D. ...................................... 133

Table 35. Summary of N-terminal sequence data of AAD-12 soybean- and microbederived proteins............................................................................................................... 152

Table 36. Summary of C-terminal sequence data of AAD-12 soybean- and microbederived proteins............................................................................................................... 153

Table 37. Summary of literature values for proximates in soybean forage. ............................... 161

Table 38. Summary of literature values for proximates in soybean grain.................................. 161

Table 39. Summary of literature values for amino acids in soybean grain................................. 162

Table 40. Summary of literature values for isoflavones in soybean grain.................................. 162

Table 41. Summary of literature values for fatty acids in soybean grain. .................................. 163

Table 42. Summary of literature values for vitamins in soybean grain. ..................................... 163

Table 43. Summary of literature values for minerals in soybean grain. ..................................... 164

Table 44. Summary of literature values for anti-nutrients in soybean grain. ............................. 164

Table 45. Summary of literature values for tocopherols in soybean grain. ................................ 164

Table 46. Herbicide-tolerant crops available to farmers in North America in 2005. ................. 165

Table 47. Weed species with reported glyphosate-resistant biotypes......................................... 170

Table 48. Global reports of glyphosate-resistant weed biotypes with resistance to other

herbicide modes of action. .............................................................................................. 171

Table 49. Potential weed shifts with use of glyphosate in U.S. corn and soybeans. .................. 172

Table 50. Weed species with reported 2,4-D-resistant biotypes................................................. 173
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Acronyms and Scientific Terms

2,4-D 2,4-Dichlorophenoxyacetic acid

A Acre

aad-12 Gene from Delftia acidovorans which encodes the AAD-12 protein

AAD-12 Aryloxyalkanoate Dioxygenase-12 protein

ACCase Acetyl CoA carboxylase

ADF Acid detergent fiber

ae Acid equivalent

ae/A Acid equivalent per acre

ae/ha Acid equivalent per hectare

ai Active ingredient

ai/A Active ingredient per acre

ai/ha Active ingredient per hectare

ALS Acetolactate synthase

ANOVA Analysis of variance

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

bp Base pair

bu Bushel

CFIA Canadian Food Inspection Agency

CFSAN Center for Food Safety and Nutrition, US FDA

CsVMV Promoter from cassava vein mosaic virus

DAS Dow AgroSciences LLC

DAS-68416-4 Soybean line containing event DAS-68416-4

DCP 2,4-Dichlorophenol

DMA Dimethylamine

DNA Deoxyribonucleic acid

ELISA Enzyme-linked immunosorbent assay

EPA Environmental Protection Agency (US)

EPSPS 5-enolpyruvylshikimate-3-phosphate synthase

ESA Endangered Species Act

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

Event DAS-68416-4 OECD identifier for the soybean event expressing the AAD-12

protein

FDA Food and Drug Administration (US)

FDR False Discovery Rate

FIFRA Federal Insecticide, Fungicide and Rodenticide Act

FWS Fish and Wildlife Service

GS Glutamine synthetase

ha Hectare

HRAC Herbicide Resistance Action Committee

IAA Indole acetic acid

ILSI International Life Sciences Institute

IWM Integrated weed management
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Kb Kilobase pair

kDa Kilodalton

L Liter

LOD Limit of Detection

LOQ Limit of Quantitation

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

spectrometry

Maverick Publicly available soybean line used in transformation to produce

event DAS-68416-4

MCPA 4-chloro-2-methylphenoxyacetic acid

MOA Mode of action

NDF Neutral detergent fiber

OECD Organisation for Economic Co-operation and Development

pat Gene from Streptomyces viridochromogenes which encodes the

PAT protein

PAT Phosphinothricin N-acetyl transferase protein

PBN US FDA Pre-market Biotechnology Notice

pDAB4468 DNA vector carrying the transgenes (aad-12 and pat) for insertion

into the plant genome

Pf Pseudomonas fluorescens

PPO Protoporphyrinogen oxidase

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

sequences

RB7 MAR Matrix attachment region (MAR) from Nicotiana tabacum

RCB Randomized complete block

SCN Soybean cyst nematode

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SGF Simulated gastric fluid

spp species

subsp subspecies

T-DNA Transfer DNA

USDA United States Department of Agriculture

WSSA Weed Science Society of America
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I. Rationale for the Development of DAS-68416-4 Soybean

I-A. 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) and the Plant

Quarantine Act (7 U.S.C. 151-167), to prevent the introduction or dissemination of plant pests

into or within the United States. Part 340 regulates introduction of organisms altered or

produced through genetic engineering which are plant pests or for which there is a reason to

believe are plant pests. The APHIS regulations at 7 CFR 430.6 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 is submitting data for genetically engineered herbicide-tolerant

DAS-68416-4 soybean and requests a determination from APHIS that event DAS-68416-4 and

crosses of this event with nonregulated soybean lines no longer be considered regulated articles

under 7 CFR 340.

I-B. Benefits of DAS-68416-4 Soybean

Dow AgroSciences LLC (herein referred to as “DAS”) has developed transgenic soybean plants

that are tolerant to the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and glufosinate.

DAS-68416-4 is the unique identifier of 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).

DAS-68416-4 soybean was developed using Agrobacterium-mediated transformation to stably

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

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

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

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

phosphinothricin acetyl transferase that inactivates glufosinate. The availability of DAS-68416-4

soybean is expected to have a beneficial impact on weed control practices by providing growers

with another tool to address their weed control needs. The availability of DAS-68416-4 soybean

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.

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, sugar beets, and canola are commercially

available in the United States and elsewhere in the Western Hemisphere. More glyphosatetolerant crops (e.g., wheat, rice, turf, etc.) are poised for introduction pending global market
USDA Petition for Nonregulated Status of DAS-68416-4 Soybean Page 16 of 181

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acceptance. Many other glyphosate-tolerant species are in experimental or development stages

(e.g., alfalfa, 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) (USDA APHIS, 2009). 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 crops

systems.

Extensive use of glyphosate-only weed control programs is resulting in the selection of

glyphosate-resistant weeds, and is selecting for the propagation of weed species that are

inherently more tolerant to glyphosate than most target species (i.e., weed shifts) (Heap, 2009).

Although glyphosate has been widely used globally for more than 30 years, only a handful of

weeds have been reported to have developed resistance to glyphosate; however, most of these

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, which now comprise >90% of U.S. soybean acres and

>60% of U.S. corn and cotton acres (USDA ERS 2009). 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 difficult-tocontrol 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 60 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 soybeans provides an excellent option for controlling broadleaf, glyphosateresistant (or highly tolerant and shifted) weed species for in-crop applications, allowing the

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

window without the need for specialized sprayer equipment. Combining the 2,4-D-tolerance

trait and a glyphosate-tolerance trait through conventional breeding (“stacking” traits) would

give growers the ability to use tank mixes of glyphosate/2,4-D over-the-top of the tolerant plants

to control the glyphosate-resistant broadleaf species.

DAS-68416-4 soybeans also provide tolerance to glufosinate herbicides. Glufosinate is a

non-selective, contact herbicide that controls a broad spectrum of annual and perennial grasses

and broadleaf weeds. The tolerance to glufosinate allows use of an additional mode of action as

part of effective herbicide resistance management strategies. Glufosinate herbicides can also be

used as selection agents in breeding nurseries to select herbicide-tolerant plants to maintain seed

trait purity.
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The commercial introduction of transgenic soybean exhibiting tolerance to 2,4-D and glufosinate

will bring new weed control alternatives for growers. This new weed management tool will

allow for the improved control of key broadleaf weeds which can affect the vigor and yield of

the crop, allow an increased herbicide application window for effective weed control, and

provide an effective resistance management/prevention solution to the increased incidence of

glyphosate- and acetolactate synthase (ALS)-resistant weeds.

I-C. Submissions to Other Regulatory Agencies

AAD-12 soybean event DAS-68416-4 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 premarket biotechnology notification (PBN) to FDA in December 2009.

As per EPA’s authority over the use of pesticidal substances under the Federal Insecticide,

Fungicide and Rodenticide Act (FIFRA), plant metabolism and residue data for 2,4-D on

DAS-68416-4 soybeans as well as proposed labeling for the use of 2,4-D on DAS-68416-4

soybeans will be submitted to EPA in Q1 2011.

DAS intends to submit dossiers in 2010 to the regulatory authorities of trade partners for import

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

Union, Australia/New Zealand, South Africa, Brazil, Argentina and Mexico.

I-D. References

Heap, I. (2009) The International Survey of Herbicide Resistant Weeds.

(http://www.weedscience.com)

OECD (2004) OECD Guidance for the Designation of a Unique Identifier for Transgenic Plants.

Organisation for Economic Co-operation and Development. Series on Harmonization of

Regulatory Oversight in Biotechnology, No. 23. ENV/JM/MONO(2002)7.

(http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/env-jm-mono(2002)7)

USDA APHIS (2009) Field Test Release Applications in the U.S. Accessed November 1, 2009.

http://www.isb.vt.edu/cfdocs/fieldtests1.cfm

USDA ERS (2009) USDA Economic Research Service Data Sets – Adoption of Genetically

Engineered Crops in the U.S.

(http://www.ers.usda.gov/Data/BiotechCrops/)
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II. The Biology of Soybean

II-A. Overview of Soybean Biology

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

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

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

plant

 agronomic practices

 centers of origin

 reproductive biology

 cultivated Glycine max as a volunteer weed

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

other organisms

 summary of the ecology of Glycine max

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

nomenclature (Pedersen, 2004; Gaska, 2006):

Vegetative Stages Reproductive Stages

VE Emergence R1 Beginning bloom

VC Unrolled unifoliate leaves R2 Full bloom

V1 First-trifoliate R3 Beginning pod

V2 Second-trifoliate R4 Full pod

V3 Third-trifoliate R5 Beginning seed

V(n) n

th

-trifoliate R6 Full seed

R7 Beginning maturity

R8 Full maturity

II-B. Characterization of the Recipient Soybean Cultivar

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

DAS-68416-4 soybean.

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

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

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

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

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

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

phytophthora rot but is susceptible to brown stem rot and sudden death syndrome.
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II-C. References

Gaska, J. (2006) Soybean Growth and Development

(http://soybean.uwex.edu/library/soybean/morphology/documents/04_soy_growth_development.

pdf)

OECD (2000) Consensus Document on the Biology of Glycine max (L.) Merr. (Soybean).

Organisation for Economic Co-operation and Development, Paris, France. Series on

Harmonization of Regulatory Oversight in Biotechnology No. 15. ENV/JM/MONO(2000)9.

(http:/www.olis.oecd.org/olis/2000doc.nsf/LinkTo/env-jm-mono(2000)9)

Pedersen, P. (2004) Soybean Growth and Development, Soybean Extension and Research

Program, Department of Agronomy, Iowa State University

(http://extension.agron.iastate.edu/soybean/production_growthstages.html)

Sleper, D.A., Nickell, C.D.; Noel, G.R., Cary, T.R., Thomas, D.J., Clark, K.M., Rao Arelli, A.P.

(1998) Registration of ‘Maverick’ Soybean. Crop Science 38:549.
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III.Development of DAS-68416-4 Soybean

III-A. Description of the Transformation System

Transgenic soybean (Glycine max) DAS-68416-4 was generated through Agrobacteriummediated transformation of soybean cotyledonary node explants. The disarmed Agrobacterium

tumefaciens strain EHA101 (Hood et al., 1986), carrying the binary vector pDAB4468 was used

to initiate transformation.

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

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

cotyledonary nodes were isolated and infected with Agrobacterium. Shoot initiation, shoot

elongation, and rooting media were supplemented with cefotaxime, timentin and vancomycin for

removal of Agrobacterium. Glufosinate selection was employed to inhibit the growth of nontransformed shoots. Selected shoots were transferred to rooting medium for root development

and then transferred to soil mix for acclimatization of plantlets.

Terminal leaflets of selected plantlets were leaf painted with glufosinate to screen for putative

transformants. The screened plantlets were transferred to the greenhouse, allowed to acclimate

and then leaf-painted with glufosinate to reconfirm tolerance. Surviving plantlets were deemed

to be putative transformants. The screened plants were sampled and molecular analyses for the

confirmation of the selectable marker gene and/or the gene of interest were carried out. T0 plants

were allowed to self fertilize in the greenhouse to give rise to T1 seed.

Figure 1 shows a plasmid map of pDAB4468 with all genetic elements identified.

Figure 2 shows a diagram of the T-DNA insert in plasmid pDAB4468.

Figure 3 shows the steps used to develop DAS-68416-4 soybean.

Figure 4 shows a breeding diagram for DAS-68416-4 soybean including identification of the

generations used in the various safety assessment studies.
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Figure 1. Plasmid map of pDAB4468.

Figure 2. Diagram of T-DNA insert in plasmid pDAB4468.

pDAB4468

12154 bp AAD-12

trfA

SpecR

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

Ori Rep

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR

pDAB4468

12154 bp AAD-12

trfA

SpecR

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

Ori Rep

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR

pDAB4468 T-DNA Insert

6794 bp

AAD-12

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR

pDAB4468 T-DNA Insert

6794 bp

AAD-12

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR
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Figure 3. Development of DAS-68416-4 soybean.

The aad-12 gene sequence that encodes the AAD-12 protein from Delftia acidovorans

was synthesized to produce an aad-12 gene sequence for expression in soybean.

A plasmid containing aad-12 gene and pat gene cassettes was assembled and inserted

into a plant expression cassette to make transformation plasmid pDAB4468.

The aad-12 gene was introduced into the soybean line Maverick via Agrobacterium

tumefaciens-mediated transformation.

Transformed plants were selected based on in vitro tolerance to glufosinate herbicide

and regeneration of T0 soybean plants.

T0 plants were screened for absence of vector backbone, presence of gene of interest,

copy number, and tolerance to 2,4-D and glufosinate herbicides. T0 plants that passed

the screen were self-pollinated.

Continued self pollination, molecular characterization, and evaluation of agronomic

performance and herbicide efficacy were performed.

Selection of homozygous plants, backcrossing and crossing for product development.

Selection of DAS-68416-4 event as the lead commercial candidate.
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Figure 4. Breeding diagram of DAS-68416-4 soybean.

Analysis Petition Section(s)

DAS-68416-4 Soybean

Generation Used Control

Molecular Analysis V-A, V-B, V-C, V-D T3, T4, T5 Maverick

Segregation Analysis V-E F2 Maverick

Protein Characterization VI-A.2, VI-B.2 T5 Maverick

Protein Expression VI-A.3, VI-B.3 T4 Maverick

Agronomics VII T4, T6 Maverick

Germination/Dormancy VII-C T6 Maverick

Composition VIII T4 Maverick

Efficacy IX-A T4 Maverick

T0

T1

T2

T3

T4

T5 F1

T6 F2 BC1F1

T7 F3 BC1F2 BC2F1

Self

Self

Self

Self

Self

Self

Self

Self

Self Self

Backcross to recurrent parents

Backcross to recurrent parents

Cross to elite lines

T0

T1

T2

T3

T4

T5 F1

T6 F2 BC1F1

T7 F3 BC1F2 BC2F1

Self

Self

Self

Self

Self

Self

Self

Self

Self Self

Backcross to recurrent parents

Backcross to recurrent parents

Cross to elite lines
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III-B. Selection of Comparators for DAS-68416-4 Soybean

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

of DAS-68416-4 soybean, a proper selection of comparator plants is important.

The control plants should have a genetic background similar to that of DAS-68416-4 soybean,

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

control. Maverick is the recipient variety that was transformed to generate DAS-68416-4

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

the transgenic insert DNA.

III-C. References

Hood, E.E.; Helmer, G.L.; Fraley, R.T.; Chilton, M.-D. The Hypervirulence of Agrobacterium

tumefaciens A281 Is Encoded in a Region of pTiBo542 Outside of T-DNA, Journal of

Bacteriology, 1986 168(3):1291-1301.

Zeng, P.; Vadnais, D.A.; Zhang, Z.; Polacco, J.C. Refined glufosinate selection in

Agrobacterium-mediated transformation of soybean [Glycine max (L.) Merrill], Plant Cell

Reports 2004 22:478–482
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IV.Donor Genes and Regulatory Sequences

IV-A. Identity and Source of Genetic Material in pDAB4468

Soybean event DAS-68416-4 was generated by Agrobacterium-mediated transformation using

the plasmid pDAB4468 (Figure 1). The T-DNA insert in the plasmid contains a synthetic, plantoptimized sequence of the aad-12 gene from Delftia acidovorans and the pat gene from

Streptomyces viridochromogenes (Figure 2). A summary of the genetic elements is given in

Table 1.

Table 1. Genetic elements of the T-DNA insert from plasmid pDAB4468.

Location on TDNA insert of

pDAB44681 Genetic Element

Size

(base

pairs) Description

1−24 T-DNA Border B 24 Transferring DNA sequences

25−160 Intervening

sequence

136 Sequence from Ti plasmid pTi15955 (Barker et al.,

1983)

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

tabacum (Hall et al., 1991)

1327−1421 Intervening

sequence

95 Sequence from plasmid pENTR/D-TOPO (Invitrogen

Cat. No. A10465) and multiple cloning sites

1422−2743 AtUbi10 1322

Arabidopsis thaliana polyubiquitin UBQ10

comprising the promoter, 5' untranslated region and

intron (Norris et al., 1993)

2744−2751 Intervening

sequence

8 Sequence used for DNA cloning

2752−3633 aad-12 882

Synthetic, plant-optimized version of an

aryloxyalkanoate dioxygenase gene from Delftia

acidovorans (Wright et al., 2007)

3634−3735 Intervening

sequence

102 Sequence used for DNA cloning

3736−4192 AtuORF23 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)

4193−4306 Intervening

sequence

114 Sequence from plasmid pENTR/D-TOPO (Invitrogen

Cat. No. A10465) and multiple cloning sites

4307−4823 CsVMV 517

Promoter and 5' untranslated region derived from the

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

4824−4830 Intervening

sequence

7 Sequence used for DNA cloning

4831−5382 pat 552

Synthetic, plant-optimized version of

phosphinothricin N-acetyl transferase (PAT) gene,

isolated from Streptomyces viridochromogenes

(Wohlleben et al., 1988)

5383−5484 Intervening

sequence

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

K205001) and multiple cloning sites
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Table 1 (cont.). Genetic elements of the T-DNA insert from plasmid pDAB4468.

Location on TDNA insert of

pDAB44681 Genetic Element

Size

(base

pairs) Description

5485−6188 AtuORF1 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)

6189−6416 Intervening

sequence

228 Sequence from Ti plasmid C58 (Zambryski et al.,

1982; Wood et al., 2001)

6417−6440 T-DNA border A 24 Transferring DNA sequence

6441−6459 Intervening

sequence

19 Sequence from Ti plasmid C58 (Zambryski et al.,

1982; Wood et al., 2001)

6460−6483 T-DNA border A 24 Transferring DNA sequence

6484−6770 Intervening

sequence

287 Sequence from Ti plasmid pTi15955 (Barker et al.,

1983)

6771−6794 T-DNA border A 24 Transferring DNA sequence

1 Base pair position.

Two gene expression cassettes were present in the pDAB4468 vector for insertion into soybeans.

The aad-12 expression cassette contained in the T-DNA insert of pDAB4468 is designed to

express the plant-optimized aryloxyalkanoate dioxygenase (aad-12) gene that encodes the

AAD-12 protein. The aad-12 gene was isolated from Delftia acidovorans and the synthetic

version of the gene was optimized to modify the G+C codon bias to a level more typical for plant

expression. The native and plant-optimized DNA sequences of aad-12 are 79.7% identical. The

aad-12 gene encodes a protein of 293 amino acids that has a molecular weight of approximately

32 kDa. The insertion of aad-12 into soybean plants confers tolerance to herbicides such as

2,4-D by production of the aryloxyalkanoate dioxygenase-12 enzyme (AAD-12).

Delftia acidovorans, which has previously been identified as Pseudomonas acidovorans and

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

present in soil, fresh water, activated sludge, and clinical specimens (von Gravenitz 1985,

Tamaoka et al. 1987, Wen et al., 1999). D. acidovorans can be used to transform ferulic acid

into vanillin and related flavor metabolites (Toms and Wood, 1970; Ramachandra Rao and

Ravishankar, 2000; Shetty et al., 2006). This utility has led to a history of safe use for

D. acidovorans in the food processing industry. For example, US Patent 5,128,253

“Bioconversion process for the production of vanillin” was issued on July 7, 1992 to Kraft

General Foods (Labuda et al., 1992).

Expression of aad-12 in the T-DNA insert of pDAB4468 is controlled by the AtUbi10 promoter

from Arabidopsis thaliana and AtuORF23 3’ UTR sequence from Agrobacterium tumefaciens

plasmid pTi15955. The AtUbi10 promoter is known to drive constitutive expression of the

genes it controls (Norris et al., 1993). The function of AtuORF23 (GenBank Accession:

CAA25184) in pTi15955 (Genbank Accession: X00493) was not identified (Barker et al., 1983),

however a search of its translated amino acid sequence returned no significant similarity with

known functional proteins.
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A matrix attachment region (MAR) of RB7 from Nicotiana tabacum was included at the 5’ end

of the aad-12 PTU (plant transcriptional unit, includes promoter, gene, and terminator

sequences) to potentially facilitate expression of the aad-12 gene in the plant. 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 (Abranches et al., 2005; Han et al., 1997; Verma et al., 2005). It is

hypothesized that MARs may act to buffer effects from neighboring chromosomal sequences that

could destabilize the expression of genes (Allen et al., 2000). A MAR was included at the 5’ end

of aad-12 PTU to potentially increase the consistency of aad-12 expression in transgenic plants.

The pat expression cassette contained in the T-DNA insert of pDAB4468 is designed to express

the plant-optimized phosphinothricin N-acetyl transferase (pat) gene that encodes the PAT

protein. The pat gene was isolated from Streptomyces viridochromogenes and the synthetic

version of the gene was optimized to modify the G+C codon bias to a level more typical for plant

expression. The insertion of the pat gene into soybean genome confers tolerance to glufosinate

and was used as a selectable marker during the soybean transformation. The pat gene encodes a

protein of 183 amino acids that has a molecular weight of approximately 21 kDa. The pat gene

has been widely used both as a selectable marker and herbicide tolerance trait in previously

deregulated products (e.g., USDA 1996, USDA 2001, USDA 2004, USDA 2005)

Expression of the pat gene in the T-DNA insert of pDAB4468 is controlled by the CsVMV

promoter from cassava vein mosaic virus and AtuORF1 3’ UTR sequence from Agrobacterium

tumefaciens plasmid pTi15955. The cassava vein mosaic virus is a double stranded DNA virus

which infects cassava plants (Manihot esculenta Crantz) and has been characterized as a plant

pararetrovirus belonging to the caulimovirus subgroup. The CsVMV promoter is known to drive

constitutive expression of the genes it controls (Verdaguer et al., 1996). The function of

AtuORF1 (GenBank Accession: CAA25163) in pTi15955 (GenBank Accession: X00493) was

not identified (Barker et al., 1983), but its translated amino acid sequence has a significant

similarity with an indole-3-lactate synthase (GenBank Accession: AAK90967) from

Agrobacterium tumefaciens str. C58.

IV-B. References

Abranches, R., Shultz, R. W., Thompson, W. F., Allen, G. C. (2005) Matrix attachment regions

and regulated transcription increase and stabilize transgene expression. Plant Biotechnology

Journal 3:535-543.

Allen, G.C., Spiker, S., Thompson, W.F. (2000) Use of matrix attachment regions (MARs) to

minimize transgene silencing. Plant Molecular Biology 43:361-376.

Barker, R.F., Idler, K.B., Thompson, D.V. and Kemp, J.D. (1983) Nucleotide sequence of the TDNA region from the Agrobacterium tumefaciens octopine Ti plasmid pTi15955. Plant Mol.

Biol. 2:335-350.
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Hall, G., Allen, G.C., Loer, D.S., Thompson, W.F., Spiker, S. (1991) Nuclear scaffolds and

scaffold-attachment regions in higher plants. Proceedings of the National Academy of Sciences

of the United States of America 88(20):9320-9324.

Han, K.-H., Caiping, M., Strauss, S.H. (1997) Matrix attachment regions (MARs) enhance

transformation frequency and transgene expression in poplar. Transgenic Research 6:415-420.

Labuda, I.M., Goers, S.K., Keon, K.A. (1992) Bioconversion Process for the Production of

Vanillin. US Patent #5,128,253.

Norris, S.R., Meyer, S.E., Callis, J. (1993) The intron of Arabidopsis thaliana polyubiquitin

genes is conserved in location and is a quantitative determinant of chimeric gene expression.

Plant Molecular Biology 21(5):895-906.

Ramachachandra Rao, S. and Ravishankar, G.A. (2000) Vanilla Flavor: Production by

Conventional and Biotechnological Routes. Journal of the Science of Food and Agriculture

80:289-304.

Shetty, K., Paliyath, G., Pometto, A., Levin, R.E. (2006) Food Biotechnology, CRC Press, ISBN

0824753291, pp. 1672-1673.

Tamaoka, J., Ha, D.M., Komagata, K. (1987) Reclassification of Pseudomonas acidovorans den

Dooren de Jong 1926 and Pseudomonas testosteroni Marcus and Talalay 1956 as Comamonas

acidovorans comb. nov. and Comamonas testosteroni comb. nov., with an emended description

of the genus Comamonas. International Journal of Systematic Bacteriology 37:52-59.

Toms, A., Wood, J.M. (1970) The Degradation of trans-Ferulic Acid by Pseudomonas

acidovorans. Biochemistry 9:337-343.

USDA (1996) Availability of Determination of Nonregulatated status for Soybeans Genetically

Engineered for Glufosinate Herbicide Tolerance. Federal Register Volume 61, Number

160:42581-42582.

USDA (2001) Availability of Determination of Non-regulatated status for Genetically

Engineered Corn for Insect Resistance and Glufosinate Herbicide Tolerance (Corn line 1507).

Federal Register Volume 66, Number 157:42624-42625.

USDA (2004) Availability of Determination of Nonregulated Status for Cotton Lines Genetically

Engineered for Insect Resistance. Federal Register Volume 69, Number 156:50154-50155.

USDA (2005) Availability of Determination of Non-regulated Status for Genetically Engineered

Corn (Corn line DAS-59122-7). Federal Register Volume 70, Number 194:58663-58664.

Verdaguer, B., Kochko de, A., Beachy, R. N. and Fauquet, C. (1996) Isolation and expression in

transgenic tobacco and rice plants, of the cassava vein mosaic virus (CVMV) promoter. Plant

Molecular Biology 31:1129-1139.
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Verma, D., Verma, M., Dey, M., Jain, R.K., Wu, R. (2005) Molecular dissection of the tobacco

Rb7 matrix attachment region (MAR): Effect of 5’ half on gene expression in rice. Plant Science

169:704-711.

Von Gravenitz, A, (1985) Ecology, clinical significance, and antimicrobial susceptibility

encountered glucose-non-fermenting gram-negative rods, p. 181-232. In G. L. Gilardi (ed.),

Nonfermentative gram-negative rods, laboratory identification and clinical aspects. Marcel

Dekker, Inc., New York.

Wen, A., Fegan, M., Hayward, C., Chakraborty, S., Sly, L.I. (1999) Phylogenetic relationships

among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren

de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov. International Journal of

Systematic Bacteriology 49:567-576.

Wohlleben, W,, Arnold, W., Broer, I., Hillemann, D., Strauch, E. and Pühler, A. (1988)

Nucleotide sequences of the phosphinothricin N-acetyltransferase gene from Streptomyces

viridochromogenes Tü494 and its expression in Nicotiana tabacum. Gene 70: 25-37.

Wood, D.W.; Setubal, J.C.; Kaul, R.; Monks, D.E.; Kitajima, J.P.; Okura, V.K.; Zhou, Y.;

Chen, L.; Wood, G.E.; Almeida, N.F. Jr.; Woo, L.; Chen, Y.; Paulsen, I.T.; Eisen, J.A.; Karp,

P.D.; Bovee, D. Sr.; Chapman, P.; Clendenning, J.; Deatherage, G.; Gillet, W.; Grant, C.;

Kutyavin, T.; Levy, R.; Li, M.J.; McClelland, E.; Palmieri, A.; Raymond, C.; Rouse, G.;

Saenphimmachak, C.; Wu, Z.; Romero, P.; Gordon, D.; Zhang, S.; Yoo, H.; Tao, Y.; Biddle, P.;

Jung, M.; Krespan, W.; Perry, M.; Gordon-Kamm, B.; Liao, L.; Kim, S.; Hendrick, C.; Zhao,

Z.Y.; Dolan, M.; Chumley, F.; Tingey, S.V.; Tomb, J.F.; Gordon, M.P.; Olson, M.V.; Nester,

E.W. (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58.

Science 294(5550):2317-2323.

Wright, T.R.; Lira, J.M.; Walsh, T.; Merlo, D.J., Jayakumar, P., Lin, G. (2007) Novel Herbicide

Resistance Genes. World Intellectual Property Organization International Publication Number

WO 2007/053482 A2

Zambryski, P.; Depicker, A.; Kruger,K.; Goodman,H.M. (1982) Tumor induction by

Agrobacterium tumefaciens: analysis of the boundaries of T-DNA. Journal of Molecular and

Applied Genetics 1(4):361-370.
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V. Genetic Characterization

V-A. Overview of Molecular Analysis

Molecular characterization of event DAS-68416-4 was conducted by Southern blot analysis.

The results demonstrate that the transgene insert in soybean event DAS-68416-4 occurred as a

simple integration of the T-DNA insert from plasmid pDAB4468, including a single, intact copy

of the aad-12 and pat expression cassettes. The event is stably integrated and inherited across

and within breeding generations, and no plasmid backbone sequences are present in

DAS-68416-4 soybean.

Detailed Southern blot analysis was conducted using probes specific to the gene coding

sequences, promoters, terminators, and other regulatory elements contained in the pDAB4468

transformation plasmid. The locations of each probe on the pDAB4468 plasmid are described in

Table 2 and shown in Figure 5. The expected and observed fragment sizes with specific digest

and probe combinations, based on the known restriction enzyme sites of the pDAB4468 plasmid

are shown in Table 3, Figure 6 and Figure 7, respectively. The Southern blot analyses described

here made use of two types of restriction fragments: a) internal fragments in which known

enzyme restriction sites are completely contained within the T-DNA insert of pDAB4468 and

b) border fragments in which a known enzyme site is located within the T-DNA insert and a

second site is located in the soybean genome flanking the insert. Border fragment sizes vary by

event because they rely on the DNA sequence of flanking genomic region. Since integration

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

DNA insertions and to specifically identify the event.

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

DAS-68416-4 soybean plants from three distinct breeding generations. Genomic DNA from

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

pDAB4468 added to genomic DNA from the conventional control served as the positive control

for the Southern blot analysis. Materials and methods used for the Southern analyses are further

described in Appendix 1.

The expected restriction fragments of the inserted DNA are shown in Figure 7. Southern blot

analysis showed that event DAS-68416-4 contains a single intact copy of the aad-12 and pat

expression cassettes integrated at a single locus (Section V-B). No vector backbone sequences

were detected in event DAS-68416-4 (Section V-C). The hybridization patterns across four

sample sets representing three generations of DAS-68416-4 soybean (T3, T4, and T5) were

identical, indicating that the insertion is stably integrated in the soybean genome (Section V-D).

The inheritance of DAS-68416-4 soybean in segregating generations was investigated using

Southern blot analysis, detection of the AAD-12 protein, and detection of the aad-12 gene; all

results confirmed the predicted inheritance of the transgene in a single locus (Section V-E).
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Table 2. List of probes and their positions in plasmid pDAB4468.

Probe Name Size (bp) Location in pDAB4468

Flanking B 303 11894 – 42

RB7 1010 306 – 1315

AtUbi10 771 1411 – 2181

aad-12 882 2752 – 3633

AtuORF23 413 3762 – 4174

CsVMV 478 4332 – 4809

pat 552 4820 – 5371

AtuORF1 684 5474 – 6157

Flanking A 339 6793 – 7131

Ori Rep 1068 7111 – 8178

Backbone 2 1728 8157 – 9884

Backbone 1 1310 9854 – 11163

Spec R 789 11092 – 11880

Figure 5. Location of probes on pDAB4468 used in Southern blot analysis of DAS-68416-4

soybean.

pDAB4468

12154 bp

AAD-12

trfA

SpecR

PAT

SpecR Probe

Backbone 1 Probe

Backbone 2 Probe

Ori Rep Probe

Flanking A Probe

AtuOrf1 Probe

pat Probe

aad-12 Probe

AtuOrf23 Probe

CsVMV Probe

AtUbi10 Probe

RB7 Probe

Flanking B Probe

T-DNA Border A

T-DNA Border A T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

Ori Rep AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR

pDAB4468

12154 bp

AAD-12

trfA

SpecR

PAT

SpecR Probe

Backbone 1 Probe

Backbone 2 Probe

Ori Rep Probe

Flanking A Probe

AtuOrf1 Probe

pat Probe

aad-12 Probe

AtuOrf23 Probe

CsVMV Probe

AtUbi10 Probe

RB7 Probe

Flanking B Probe

T-DNA Border A

T-DNA Border A T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

Ori Rep AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR
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Table 3. Predicted and observed sizes of hybridizing fragments in Southern blot analyses of

DAS-68416-4 soybean.

Probe Restriction

Enzyme Sample Southern Blot Figure Fragment Size (bp)

Expected Observed

aad-12

Nco I

Plasmid pDAB4468 Figure 8, Figure 11 7957 7957

DAS-68416-4 Figure 8, Figure 11 > 4043* ~5500*

Control (Maverick) Figure 8, Figure 11 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 9, Figure 11 12146 12146

DAS-68416-4 Figure 9, Figure 11 > 6229* ~8500*

Control (Maverick) Figure 9, Figure 11 none none

Nhe I/Xho I

Plasmid pDAB4468 Figure 10, Figure 11 12148 12148

DAS-68416-4 Figure 10, Figure 11 > 6229* ~7200*

Control (Maverick) Figure 10, Figure 11 none none

pat

Nco I

Plasmid pDAB4468 Figure 16, Figure 19 7957 7957

DAS-68416-4 Figure 16, Figure 19 > 4043* ~5500*

Control (Maverick) Figure 16, Figure 19 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 17, Figure 19 12146 12146

DAS-68416-4 Figure 17, Figure 19 > 6229* ~8500*

Control (Maverick) Figure 17, Figure 19 none none

Nhe I/Xho I

Plasmid pDAB4468 Figure 18, Figure 19 12148 12148

DAS-68416-4 Figure 18, Figure 19 > 6229* ~7200*

Control (Maverick) Figure 18, Figure 19 none none

aad-12

Pst I

(Release

PTU)

Plasmid pDAB4468 Figure 12, Figure 13 2868 2868

DAS-68416-4 Figure 12, Figure 13 2868 2868

Control (Maverick) Figure 12, Figure 13 none none

AtUbi10

Plasmid pDAB4468 Figure 14, Figure 13 2868 2868

DAS-68416-4 Figure 14, Figure 13 2868 2868

Control (Maverick) Figure 14, Figure 13 none none

AtuORF23

Plasmid pDAB4468 Figure 15, Figure 13 2868 2868

DAS-68416-4 Figure 15, Figure 13 2868 2868

Control (Maverick) Figure 15, Figure 13 none none

pat

Pst I/Xho I

(Release

PTU)

Plasmid pDAB4468 Figure 20, Figure 21 1928 1928

DAS-68416-4 Figure 20, Figure 21 1928 1928

Control (Maverick) Figure 20, Figure 21 none none

CsVMV

Plasmid pDAB4468 Figure 22, Figure 21 1928 1928

DAS-68416-4 Figure 22, Figure 21 1928 1928

Control (Maverick) Figure 22, Figure 21 none none

AtuORF1

Plasmid pDAB4468 Figure 23, Figure 21 1928 1928

DAS-68416-4 Figure 23, Figure 21 1928 1928

Control (Maverick) Figure 23, Figure 21 none none

RB7

BamH I/Nco I

Plasmid pDAB4468 Figure 24, Figure 26 2617 2617

DAS-68416-4 Figure 24, Figure 26 2617 2617

Control (Maverick) Figure 24, Figure 26 none none

AtUbi10

Plasmid pDAB4468 Figure 25, Figure 26 2617 2617

DAS-68416-4 Figure 25, Figure 26 2617 2617

Control (Maverick) Figure 25, Figure 26 none none
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Table 3 (cont.). Predicted and observed sizes of hybridizing fragments in Southern blot

analyses of DAS-68416-4 soybean.

Probe Restriction

Enzyme Sample Southern Blot Figure Fragment Size (bp)

Expected Observed

Flanking A

Nco I

Plasmid pDAB4468 Figure 27, Figure 31 7957 7957

DAS-68416-4 Figure 27, Figure 31 none none

Control (Maverick) Figure 27, Figure 31 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 29, Figure 31 12146 12146

DAS-68416-4 Figure 29, Figure 31 none none

Control (Maverick) Figure 29, Figure 31 none none

Backbone1

Nco I

Plasmid pDAB4468 Figure 27, Figure 31 4197, 7957 4197, 7957

DAS-68416-4 Figure 27, Figure 31 none none

Control (Maverick) Figure 27, Figure 31 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 29, Figure 31 12146 12146

DAS-68416-4 Figure 29, Figure 31 none none

Control (Maverick) Figure 29, Figure 31 none none

SpecR

Nco I

Plasmid pDAB4468 Figure 27, Figure 31 4197 4197

DAS-68416-4 Figure 27, Figure 31 none none

Control (Maverick) Figure 27, Figure 31 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 29, Figure 31 12146 12146

DAS-68416-4 Figure 29, Figure 31 none none

Control (Maverick) Figure 29, Figure 31 none none

Flanking B

Nco I

Plasmid pDAB4468 Figure 28, Figure 32 4197 4197

DAS-68416-4 Figure 28, Figure 32 none none

Control (Maverick) Figure 28, Figure 32 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 30, Figure 32 12146 12146

DAS-68416-4 Figure 30, Figure 32 none none

Control (Maverick) Figure 30, Figure 32 none none

Backbone2

Nco I

Plasmid pDAB4468 Figure 28, Figure 32 7957 7957

DAS-68416-4 Figure 28, Figure 32 none none

Control (Maverick) Figure 28, Figure 32 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 30, Figure 32 12146 12146

DAS-68416-4 Figure 30, Figure 32 none none

Control (Maverick) Figure 30, Figure 32 none none

Ori-Rep

Nco I

Plasmid pDAB4468 Figure 28, Figure 32 7957 7957

DAS-68416-4 Figure 28, Figure 32 none none

Control (Maverick) Figure 28, Figure 32 none none

Sph I/Xho I

Plasmid pDAB4468 Figure 30, Figure 32 12146 12146

DAS-68416-4 Figure 30, Figure 32 none none

Control (Maverick) Figure 30, Figure 32 none none

Note: * These bands include border region of soybean genome;

1. Expected fragment sizes are based on the plasmid map of the pDAB4468 and its T-DNA

insert as shown in Figure 6 and Figure 7,

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 II 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 6. Plasmid map of pDAB4468 with restriction enzyme sites used for Southern blot

analysis.

pDAB4468

12154 bp AAD-12

trfA

SpecR

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

Ori Rep

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR

SpeI (5433)

NheI (6235)

SphI (6245)

XhoI (6229)

PstI (1433)

PstI (4301)

NcoI (2751)

BamHI (134)

BamHI (5138)

BamHI (5427)

BamHI (6216)

Nco I (10708)

pDAB4468

12154 bp AAD-12

trfA

SpecR

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

Ori Rep

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR

SpeI (5433)

NheI (6235)

SphI (6245)

XhoI (6229)

PstI (1433)

PstI (4301)

NcoI (2751)

BamHI (134)

BamHI (5138)

BamHI (5427)

BamHI (6216)

Nco I (10708)
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Figure 7. pDAB4468 T-DNA insert, restriction enzymes used in DNA digestion and

expected hybridization bands.

Nco I

Nhe I/Xho I

Sph I/Xho I

Pst I

Pst I/Xho I

BamH I/Nco I

> 4043 bp

> 6229 bp

> 6229 bp

2868 bp

2868 bp 1928 bp

> 2493 bp

> 2751 bp

> 1433 bp

2617 bp 2387 bp

> 1433 bp

> 559 bp

> 549bp

> 565 bp

289 bp 789 bp > 578 bp

pDAB4468 T-DNA Insert

6794 bp

AAD-12

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR NcoI (2751)

NheI (6235)

SphI (6245)

XhoI (6229)

PstI (1433)

PstI (4301)

BamHI (134)

BamHI (5138)

BamHI (5427)

BamHI (6216)

Nco I

Nhe I/Xho I

Sph I/Xho I

Pst I

Pst I/Xho I

BamH I/Nco I

> 4043 bp

> 6229 bp

> 6229 bp

2868 bp

2868 bp 1928 bp

> 2493 bp

> 2751 bp

> 1433 bp

2617 bp 2387 bp

> 1433 bp

> 559 bp

> 549bp

> 565 bp

789 bp > 578 bp

Nco I

Nhe I/Xho I

Sph I/Xho I

Pst I

Pst I/Xho I

BamH I/Nco I

> 4043 bp

> 6229 bp

> 6229 bp

2868 bp

2868 bp 1928 bp

> 2493 bp

> 2751 bp

> 1433 bp

2617 bp 2387 bp

> 1433 bp

> 559 bp

> 549bp

> 565 bp

289 bp 789 bp > 578 bp

pDAB4468 T-DNA Insert

6794 bp

AAD-12

PAT

T-DNA Border A

T-DNA Border A

T-DNA Border A

T-DNA Border B

AtUbi10 promoter

CsVMV promoter

AtuORF23 3'UTR

AtuORF1 3' UTR

RB7 MAR NcoI (2751)

NheI (6235)

SphI (6245)

XhoI (6229)

PstI (1433)

PstI (4301)

BamHI (134)

BamHI (5138)

BamHI (5427)

BamHI (6216)
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V-B. Analysis of the Insert and Its Genetic Elements

V-B.1. Analysis of the aad-12 Gene

To characterize the aad-12 gene insert in event DAS-68416-4, restriction enzymes Nco I,

Sph I/Xho I and Nhe I/Xho I were used. These enzymes possess unique restriction sites in the

pDAB4468 T-DNA insert. Border fragments of >4043 bp, >6229 bp, >6229 bp were predicted

to hybridize with the aad-12 gene probe following digestion with Nco I, Sph I/Xho I and Nhe

I/Xho I enzymes respectively (Table 3). The results showed single hybridization bands of ~5500

bp, ~8500 bp and ~7200 bp respectively when Nco I, Sph I/Xho I and Nhe I/Xho I enzymes

were used, indicating a single insertion site of aad-12 in the soybean genome of event DAS68416-4 (Figure 8, Figure 9, Figure 10, Figure 11). An enzyme digestion with Pst I was

conducted to release a PTU (plant transcription unit) fragment of 2868 bp which contains the

AtUbi10 promoter, aad-12 gene, and AtuORF23 terminator sequences. The predicted 2868 bp

fragment was observed following the Pst I digestion and hybridization with aad-12 probe

(Figure 12, Figure 13A). Results obtained from the individual and double enzyme digestions

indicated that a single copy of an intact aad-12 expression cassette from pDAB4468 was inserted

into the soybean genome of event DAS-68416-4 as shown in the restriction map in Figure 7.

V-B.2. Analysis of the AtUbi10 Promoter

Restriction enzyme Pst I was used to characterize the AtUbi10 promoter region for aad-12 in

event DAS-68416-4. Pst I digestion was expected to release a PTU (plant transcription unit)

fragment of 2868 bp which contains the AtUbi10 promoter, aad-12 gene, and AtuORF23

terminator sequences. The predicted 2868 bp fragment was observed following the Pst I

digestion and hybridization with AtUbi10 promoter probe (Figure 13B, Figure 14). The AtUbi10

promoter was further characterized with a double digestion of BamH I and Nco I which releases

a fragment of 2617bp containing AtUbi10 promoter and RB7 MAR element. The predicted

2617 bp fragment was detected following the enzyme digestion and hybridization with AtUbi10

promoter probe (Figure 25, Figure 26B). Results obtained with Pst I or BamH I/Nco I digestion

of the DAS-68416-4 sample followed by AtUbi10 promoter probe hybridization further

confirmed that a single copy of an intact aad-12 PTU from plasmid pDAB4468, along with a

RB7 MAR element at its 5’ end, was inserted into the soybean genome of event DAS-68416-4.

V-B.3. Analysis of the AtuORF23 3’UTR

The terminator sequence, AtuORF23, for aad-12 in event DAS-68416-4 was characterized using

Pst I digestion, followed by hybridization of AtuORF23 probe. Pst I was expected to release a

PTU (plant transcription unit) fragment of 2868 bp which contains the AtUbi10 promoter, aad12 gene, and AtuORF23 terminator sequences. The predicted 2868 bp fragment was observed

following the enzyme digestion and hybridization with AtuORF23 probe (Figure 13C, Figure

15). Results obtained with Pst I digestion of the DAS-68416-4 sample followed by AtuORF23

probe hybridization further confirmed that a single copy of an intact aad-12 PTU from plasmid

pDAB4468 was inserted into the soybean genome of event DAS-68416-4.
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V-B.4. Analysis of the pat Gene

To characterize the pat gene insert in event DAS-68416-4, restriction enzymes Nco I, Sph I/Xho I

and Nhe I/Xho I were used. These enzymes possessed unique restriction sites in the pDAB4468

T-DNA insert. Border fragments of >4043 bp, >6229 bp, >6229 bp were predicted to hybridize

with the pat gene probe following digestion with Nco I, Sph I/Xho I and Nhe I/Xho I enzymes

respectively (Table 3). The results showed single hybridization bands of ~5500 bp, ~8500 bp

and ~7200 bp respectively when Nco I, Sph I/Xho and Nhe I/Xho I enzymes were used,

indicating a single site of pat gene insertion in the soybean genome of event DAS-68416-4

(Figure 16, Figure 17, Figure 18, Figure 19). An enzyme digestion with Pst I/Xho I was

conducted to release a PTU (plant transcription unit) fragment of 1928 bp which contained the

CsVMV promoter, pat gene, and AtuORF1 terminator sequences. The predicted 1928 bp

fragment was observed following the enzyme digestion and hybridization with pat probe (Figure

20, Figure 21A). Results obtained from the individual and double enzyme digestions indicated

that a single copy of an intact pat expression cassette from pDAB4468 was inserted into the

soybean genome of event DAS-68416-4 as shown in the restriction map in Figure 7.

V-B.5. Analysis of the CsVMV Promoter

Restriction enzyme combination of Pst I/Xho I was used to characterize the CsVMV promoter

region for pat in event DAS-68416-4. Pst I/Xho I digestion was expected to release a PTU (plant

transcription unit) fragment of 1928 bp which contains the CsVMV promoter, pat gene, and

AtuORF1 terminator sequences. The predicted 1928 bp fragment was observed following the

enzyme digestion and hybridization with CsVMV promoter probe (Figure 21B, Figure 22).

Results obtained with Pst I/Xho I digestion of the DAS-68416-4 sample followed by CsVMV

promoter probe hybridization further confirmed that a single copy of an intact pat PTU from

plasmid pDAB4468 was inserted into the soybean genome of event DAS-68416-4.

V-B.6. Analysis of the AtuORF1 3’UTR

The terminator sequence, AtuORF1, for pat in event DAS-68416-4 was characterized using Pst

I/Xho I double digestion, followed by hybridization of AtuORF1 probe. The double digestion of

Pst I/Xho I was expected to release a PTU (plant transcription unit) fragment of 1928 bp which

contained the CsVMV promoter, pat gene, and AtuORF1 terminator sequences. The predicted

1928 bp fragment was observed following the enzyme digestion and hybridization with

AtuORF1 probe (Figure 21C, Figure 23). Results obtained with Pst I/Xho I double digestion of

the DAS-68416-4 sample followed by AtuORF1 probe hybridization further confirmed that a

single copy of an intact pat PTU from plasmid pDAB4468 was inserted into the soybean genome

of event DAS-68416-4.

V-B.7. Analysis of the RB7 MAR

Restriction enzyme combination of BamH I and Nco I was selected to characterize the RB7

MAR elements from the T-DNA insert in pDAB4468 (Table 3). A double digestion with BamH

I and Nco I was expected to release a fragment of 2617 bp containing the RB7 MAR and

AtUbi10 promoter. The predicted 2617 bp fragment was observed following the double enzyme

digestion and hybridization with RB7 MAR and Atubi10 probe, respectively (Figure 24, Figure
USDA Petition for Nonregulated Status of DAS-68416-4 Soybean Page 38 of 181

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26A, Figure 26B). Results obtained with BamH I/Nco I double digestion of the DAS-68416-4

sample followed by hybridization with RB7 MAR (Figure 25, Figure 26B) further confirmed that

a single copy of an intact RB7 MAR, along with an intact aad-12 PTU from plasmid pDAB4468,

was inserted into the soybean genome of event DAS-68416-4.

V-C. Absence of Vector Backbone DNA

To verify that no plasmid vector backbone sequences exist in event DAS-68416-4, six probes

covering the whole backbone region of pDAB4468 were used to hybridize the blots from

digestions with Nco I and Sph I/Xho I (Table 2, Figure 5). For the T5 generation, a blot from

digestion with Nhe I/Xho I was also hybridized with backbone probes. The probes were grouped

into 2 sets by mixing them with equal ratio for hybridization purposes. Probe Set 1 included

backbone1, flanking A, and SpecR, and Probe Set 2 included backbone 2, flanking B, and OriRep (Figure 5, Table 2). The blots were hybridized with Probe Set 1, and then followed by

Probe Set 2 after complete removal of previously deployed probes. No hybridization signals

were detected in any sample across the T3 to T5 generations (Table 3, Figure 27, Figure 28,

Figure 29, Figure 30, Figure 31, Figure 32) except for the positive controls, indicating no

backbone sequences from pDAB4468 were incorporated into event DAS-68416-4.

V-D. Stability of the Insert across Generations

Southern blot hybridizations were conducted with four sample sets across three distinct

generations (T3, T4, and T5) of event DAS-68416-4. Prior to initiation of Southern blot

analysis, all plants were tested for AAD-12 protein expression using a lateral flow strip test kit to

allow confirmation of AAD-12 expression positive plants. All of the genetic element probes:

aad-12 gene, AtUbi10 promoter, AtuORF23 terminator, CsVMV promoter, pat gene, AtuORF1

terminator, and RB7 MAR, and the backbone of plasmid pDAB4468, were hybridized with the

DAS-68416-4 soybean samples. Results across all DAS-68416-4 samples in three generations

were as expected (Table 3, Figure 8 − Figure 32), indicating stable integration and inheritance of

the intact, single copy insert across multiple generations of DAS-68416-4 soybean.
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Figure 8. Southern blot analysis of Nco I digest with aad-12 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Nco I and hybridized with aad-12 probe. Nine (9) g of DNA

was digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of nontransgenic DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively

strong signal in Lane 15 was due to a larger amount of DNA recovered after digestion. Panel A and B were from

the same blot and hybridized in the same container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 9. Southern blot analysis of Sph I/Xho I digest with aad-12 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Sph I/Xho I and hybridized with aad-12 probe. Nine (9) g of

DNA was digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of

non-transgenic DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The

relatively strong signals in Lane 10 and 15 were due to a larger amount of DNA recovered after digestion. Panel A

and B were from the same blot and hybridized in the same container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4 #3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 10. Southern blot analysis of Nhe I/Xho I digest with aad-12 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Nhe I/Xho I and hybridized with aad-12 probe. Nine (9) g of DNA was digested and

loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a

ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively strong signal in Lane

9 was due to a larger amount of DNA recovered after digestion).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 11. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generation with aad-12 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 and the non-transgenic control

was digested with Nco I, Sph I/Xho I, and Nhe I/Xho I and hybridized with aad-12 probe. Nine (9) g of DNA was

digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic

DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively strong

signal in Lane 19 was due to a larger amount of DNA recovered after digestion).

Lane Description Enzyme Lane Description Enzyme

1 DNA molecular marker (bp) 12 T5 #1

Sph I/

Xho I

2 pDAB4468 +

control (Maverick) #2

Nco I

13 T5 #4

3 control (Maverick) #2 14 T5 #6

4 control (Maverick) #3 15 T5 #8

5 T5 #1 16 pDAB4468 +

control (Maverick) #2

Nhe I/

Xho I

6 T5 #4 17 control (Maverick) #2

7 T5 #6 18 control (Maverick) #3

8 T5 #8 19 T5 #1

9 pDAB4468 +

control (Maverick) #4 Sph I/

Xho I

20 T5 #4

10 control (Maverick) #4 21 T5 #6

11 control (Maverick) #5 22 T5 #8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

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Figure 12. Southern blot analysis of Pst I digest with aad-12 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Pst I and hybridized with aad-12 probe. Nine (9) g of DNA was digested and loaded

per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio

approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively weak signal in Lane 7 was

due to a lesser amount of DNA recovered after digestion).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5 #7

9 T4 #3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 5 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 1 5 16 17 18

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Figure 13. Southern blot analysis of Pst I digest of T5 generation with aad-12, AtUbi10, and

AtuORF23 probes.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 generation and the nontransgenic control were digested with Pst I and hybridized with aad-12 (Panel A), AtUbi10 (Panel B), and ORF23

probes (Panel C). Nine (9) g of DNA was digested and loaded per lane. The plasmid control contained plasmid

pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1 transgene copy per

soybean genome.

Lane Description

1 DNA molecular marker (bp)

2 pDAB4468 + control (Maverick) #2

3 control (Maverick) #2

4 control (Maverick) #3

5 T5 #1

6 T5 #4

7 T5 #6

8 T5 #8

A B C

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

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A B C

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

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Figure 14. Southern blot analysis of Pst I digest with AtUbi10 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Pst I and hybridized with AtUbi10 probe. Nine (9) g of DNA was digested and loaded

per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio

approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively weak signal in Lane 7 was

due to a lesser amount of DNA recovered after digestion).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 15. Southern blot analysis of Pst I digest with AtuORF23 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5and the non-transgenic

control were digested with Pst I and hybridized with AtuORF23 probe. Nine (9) g of DNA was digested and

loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a

ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively weak signal in Lane 7

was due to a lesser amount of DNA recovered after digestion).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 16. Southern blot analysis of Nco I digest with pat probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Nco I and hybridized with pat probe. Nine (9) g of DNA was

digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic

DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively strong

signals in Lane 12 and 15 were due to the greater amount of DNA recovered after digestion. Panel A and B were

from the same blot and hybridized in the same container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 17. Southern blot analysis of Sph I/Xho I digest with pat probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Sph I/Xho I and hybridized with pat probe. Nine (9) g of DNA

was digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of nontransgenic DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively

strong signals in Lane 10 and 15 were due to the greater amount of DNA recovered after digestion. Panel A and B

were from the same blot and hybridized in the same container.)

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 18. Southern blot analysis of Nhe I/Xho I digest with pat probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Nhe I/Xho I and hybridized with pat probe. Nine (9) g of DNA was digested and loaded

per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio

approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively strong signal in Lane 9

was due to the greater amount of DNA recovered after digestion).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 19. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generation with pat probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 and the non-transgenic control

was digested with Nco I, Sph I/Xho I and Nhe I/Xho I and hybridized with pat probe. Nine (9) g of DNA was

digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic

DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively strong

signal in Lane 19 was due to the greater amount of DNA recovered after digestion. The faint band in Lane 16 is

probably degraded plasmid DNA).

Lane Description Enzyme Lane Description Enzyme

1, 23 DNA molecular marker (bp) 12 T5 #1

Sph I/

Xho I

2 pDAB4468 +

control (Maverick)l #2

Nco I

13 T5 #4

3 control (Maverick) #2 14 T5 #6

4 control (Maverick) #3 15 T5 #8

5 T5 #1 16 pDAB4468 +

control (Maverick) #2

Nhe I/

Xho I

6 T5 #4 17 control (Maverick) #2

7 T5 #6 18 control (Maverick) #3

8 T5 #8 19 T5 #1

9 pDAB4468 +

control (Maverick) #4 Sph I/

Xho I

20 T5 #4

10 control (Maverick) #4 21 T5 #6

11 control (Maverick) #5 22 T5 #8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

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Figure 20. Southern blot analysis of Pst I/Xho I digest with pat probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Pst I/Xho I and hybridized with pat probe. Nine (9) g of DNA was digested and loaded

per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio

approximately equivalent to 1 transgene copy per soybean genome.

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #3 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

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Figure 21. Southern blot analysis of Pst I/Xho I digest of T5 generation with pat, CsVMV,

and AtuORF1 probes.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 generation and the nontransgenic control were digested with Pst I/Xho I and hybridized with pat (Panel A), CsVMV (Panel B), and

AtuORF1 probes (Panel C). Nine (9) g of DNA was digested and loaded per lane. The plasmid control contained

plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1 transgene

copy per soybean genome.

Lane Description

1 DNA molecular marker (bp)

2 pDAB4468 + control (Maverick) #2

3 control (Maverick) #2

4 control (Maverick) #3

5 T5 #1

6 T5 #4

7 T5 #6

8 T5 #8

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

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A B C

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

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Figure 22. Southern blot analysis of Pst I/Xho I digest with CsVMV probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Pst I/Xho I and hybridized with CsVMV probe. Nine (9) g of DNA was digested and

loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a

ratio approximately equivalent to 1 transgene copy per soybean genome.

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #3 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130
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Figure 23. Southern blot analysis of Pst I/Xho I digest with AtuORF1 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with Pst I/Xho I and hybridized with AtuORF1 probe. Nine (9) g of DNA was digested and

loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a

ratio approximately equivalent to 1 transgene copy per soybean genome.

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #3 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130
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Figure 24. Southern blot analysis of BamH I/Nco I digest with RB7 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with BamH I/Nco I and hybridized with RB7 probe. Nine (9) g of DNA was digested and

loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a

ratio approximately equivalent to 1 transgene copy per soybean genome. (Note: The relatively weak signals in Lane

6 and 7 were due to the less amount of DNA recovered after digestion).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130
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Figure 25. Southern blot analysis of BamH I/Nco I digest with AtUbi10 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3, T4, T5 and the non-transgenic

control were digested with BamH I/Nco I and hybridized with AtUbi10 probe. Nine (9) g of DNA was digested

and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at

a ratio approximately equivalent to 1 transgene copy per soybean genome.

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130
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Figure 26. Southern blot analysis of BamH I/Nco I digest of T5 generation with RB7 and

AtUbi10 probes.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 generation and the nontransgenic control were digested with BamH I/Nco I and hybridized with RB7 (Panel A), AtUbi10 (Panel B). Nine

(9) g of DNA was digested and loaded per lane. The plasmid control contained plasmid pDAB4468 mixed with 9

g of non-transgenic DNA at a ratio approximately equivalent to 1 transgene copy per soybean genome.

Lane Description

1 DNA molecular marker (bp)

2 pDAB4468 + control (Maverick) #2

3 control (Maverick) #2

4 control (Maverick) #3

5 T5 #1

6 T5 #4

7 T5 #6

8 T5 #8

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

564

4361

6557

2322

2029

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23130

A B

1 2 3 4 5 6 7 8 1 2 3 4 5 6 7 8

564

4361

6557

2322

2029

9416

23130

A B
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Figure 27. Southern blot analysis of Nco I digest with backbone probe set 1 from plasmid

pDAB4468 vector backbone.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Nco I and hybridized with Backbone Probe Set 1 (Backbone1,

Flanking A, and SpecR). Nine (9) g of DNA was digested and loaded per lane. The plasmid control contained

plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1 transgene

copy per soybean genome. (Note: Panel A and B were from the same blot and hybridized in the same container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130

A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

564

4361

6557

2322

2029

9416

23130

A B
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Figure 28. Southern blot analysis of Nco I digest with backbone probe set 2 from plasmid

pDAB4468 vector backbone.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Nco I and hybridized with Backbone Probe Set 2 (Backbone2,

Flanking B, and Ori-Rep) probes. Nine (9) g of DNA was digested and loaded per lane. The plasmid control

contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1

transgene copy per soybean genome. (Note: Panel A and B were from the same blot and hybridized in the same

container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

4361

6557

2322

2029

9416

A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

4361

6557

2322

2029

9416

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

4361

6557

2322

2029

9416

A B
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Figure 29. Southern blot analysis of Sph I/Xho I digest with backbone probe set 1 from

plasmid pDAB4468 vector backbone.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Sph I/Xho I and hybridized with Backbone Probe Set 1

(Backbone1, Flanking A, and SpecR). Nine (9) g of DNA was digested and loaded per lane. The plasmid control

contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1

transgene copy per soybean genome. (Note: Panel A and B were from the same blot and hybridized in the same

container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

4361

6557

2322

2029

9416

A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

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6557

2322

2029

9416

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

4361

6557

2322

2029

9416

A B
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Figure 30. Southern blot analysis of Sph I/Xho I digest with backbone probe set 2 plasmid

pDAB4468 vector backbone.

DNA isolated from individual plants of soybean event DAS-68416-4 generations T3 (Panel A), T4, T5 (Panel B)

and the non-transgenic control were digested with Sph I/Xho I and hybridized with Backbone Probe Set 2

(Backbone2, Flanking B, and Ori-Rep). Nine (9) g of DNA was digested and loaded per lane. The plasmid control

contained plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1

transgene copy per soybean genome. (Note: Panel A and B were from the same blot and hybridized in the same

container).

Lane Description Lane Description

1 DNA molecular marker (bp) 10 T4#6

2 pDAB4468 + control (Maverick) #2 11 T4#9

3 control (Maverick) #3 12 T4#10

4 control (Maverick) #2 13 DNA molecular marker (bp)

5 T3#2 14 T5#2

6 T3#7 15 T5#4

7 T3#8 16 T5#5

8 DNA molecular marker (bp) 17 T5#7

9 T4#3 18 DNA molecular marker (bp)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

4361

6557

2322

2029

9416

A B

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

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2322

2029

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18

23130

564

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6557

2322

2029

9416

A B
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Figure 31. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generations with the backbone probe set 1 from pDAB4468 vector backbone.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 and the non-transgenic control

was digested with Nco I, Sph I/Xho I, and Nhe I/Xho I and hybridized with Backbone Probe Set 1 (Backbone1,

Flanking A, and SpecR). Nine (9) g of DNA was digested and loaded per lane. The plasmid control contained

plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1 transgene

copy per soybean genome. (Note: the faint bands in lane 16 may be a result of hybridization to the degraded plasmid

DNA).

Lane Description Enzyme Lane Description Enzyme

1, 23 DNA molecular marker (bp) 12 T5 #1

Sph I/Xho

I

2 pDAB4468 +

control (Maverick) #2

Nco I

13 T5 #4

3 control (Maverick) #2 14 T5 #6

4 control (Maverick) #3 15 T5 #8

5 T5 #1 16 pDAB4468 +

control (Maverick) #2

Nhe

I/Xho I

6 T5 #4 17 control (Maverick) #2

7 T5 #6 18 control (Maverick) #3

8 T5 #8 19 T5 #1

9 pDAB4468 +

control (Maverick) #4 Sph

I/Xho I

20 T5 #4

10 control (Maverick) #4 21 T5 #6

11 control (Maverick) #5 22 T5 #8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

23130

564

4361

6557

2322

2029

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

23130

564

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6557

2322

2029

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Figure 32. Southern blot analysis of Nco I, Sph I/Xho I, and Nhe I/Xho I digests of T5

generation with the backbone probe set 2 from pDAB4468 vector backbone.

DNA isolated from individual plants of soybean event DAS-68416-4 generation T5 and the non-transgenic control

was digested with Nco I, Sph I/Xho I and Nhe I/Xho I and hybridized with Backbone Probe Set 2 (Backbone2,

Flanking B, and Ori-Rep). Nine (9) g of DNA was digested and loaded per lane. The plasmid controls contained

plasmid pDAB4468 mixed with 9 g of non-transgenic DNA at a ratio approximately equivalent to 1 transgene

copy per soybean genome. (Note: The faint bands in lane 16 may be a result of hybridization to the degraded

plasmid DNA).

Lane Description Enzyme Lane Description Enzyme

1, 23 DNA molecular marker (bp) 12 T5 #1

Sph I/Xho I

2 pDAB4468 +

control (Maverick) #2

Nco I

13 T5 #4

3 control (Maverick) #2 14 T5 #6

4 control (Maverick) #3 15 T5 #8

5 T5 #1 16 pDAB4468 +

control (Maverick) #2

Nhe I/Xho I

6 T5 #4 17 control (Maverick) #2

7 T5 #6 18 control (Maverick) #3

8 T5 #8 19 T5 #1

9 pDAB4468 +

control (Maverick) #4 Sph I/Xho I

20 T5 #4

10 control (Maverick) #4 21 T5 #6

11 control (Maverick) #5 22 T5 #8

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

23130

564

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2322

2029

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23

23130

564

4361

6557

2322

2029

9416
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V-E. Segregation Analysis

V-E.1. Genetic and Molecular Analysis of a Segregating Generation

The inheritance pattern of the transgene insert within a segregating generation was demonstrated

with protein expression detection and Southern analysis of individual plants from a F2

population of DAS-68416-4 soybean. The F2 generation was generated by crossing T4 plants of

DAS-68416-4 soybean with a conventional soybean line. The F1 plants were self pollinated to

produce the F2 seeds.

A total of 147 F2 seedlings were leaf tested for the presence or absence of the AAD-12 protein

using an AAD-12 specific lateral flow strip test kit. Of the 147 plants tested, 102 plants were

positive for AAD-12 protein expression, and 45 plants were negative (segregated null) (Table 4).

Statistical analysis using a 2 goodness of fit test indicated the phenotypic segregation ratio of

the plants with positive AAD-12 protein expression versus negative is consistent with the 3:1

segregation ratio characteristic of the Mendelian inheritance pattern of a single dominant trait.

Similarly, Southern blot analysis was used to determine the genetic equivalence of the inserted

DNA among the same F2 individual plants. Among 147 emerged plants, four plants (2 positive

and 2 negative for AAD-12 protein expression) died prior to proceeding with DNA extraction.

To further confirm if the phenotypic segregation matched the genotypic makeup of the tested F2

population, genomic DNA samples from each of the remaining 143 plants, along with DNA

samples from the non-transgenic control, were analyzed by Southern blot using Nco I restriction

enzyme digestion followed by hybridization with aad-12 and pat probes. All the DNA samples

from AAD-12 expression positive plants displayed a ~5500 bp expected single band of the 3’

border of the transgene insert when digested by Nco I and hybridized with either the aad-12 or

pat probes (Table 5). Two representative Southern blots are presented in Figure 33 and Figure

34. The hybridization patterns across all the individual plants that tested positive for AAD-12

protein expression were identical, which indicated that all individual plants contained the same

insert and were equivalent to one another. None of the DNA samples from AAD-12 protein

expression negative plants and non-transgenic control showed any hybridization bands. The

Southern blot analysis data matches what was observed in the AAD-12 protein expression

testing, i.e., individual plants which tested positive for AAD-12 expression displayed the

expected hybridization bands, while plants negative for AAD-12 protein expression (segregated

nulls) did not have any hybridization signals. As observed in the protein expression testing, the

ratio of aad-12 or pat hybridization positive versus negative plants in the F2 population also fit

the expected 3:1 segregation ratio characteristic of the Mendelian inheritance pattern of a single

gene (Table 6).
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Table 4. Results of F2 individual plants tested for of AAD-12 expression within a single

segregating generation.

Generation

Total plants

tested

AAD-12 protein

positive

AAD-12 protein

negative

Expected

ratio P-valuea

F2 147 102 45 3:1 0.116

a Based on a chi-squared goodness of fit test

Table 5. Predicted and observed hybridizing fragments in Southern blot analysis of F2

population.

Restriction

Enzymes

DNA

Probe Sample Source

Southern

Blot

Figure

Expected

Fragment

Sizes (bp) a

Observed

Fragment

Size (bp)b

Nco I

aad-12

pDAB4468 Figure 33 7429 7429

Control (Maverick) Figure 33 none none

AAD-12 positive plants in F2 Figure 33 >4043(border) ~5500

AAD-12 negative plants in

F2 (null segregants)

Figure 33 none none

pat

pDAB4468 Figure 34 7429 7429

Control (Maverick) Figure 34 none none

AAD-12 positive plants in F2 Figure 34 >4043(border) ~5500

AAD-12 negative plants in

F2 (null segregants)

Figure 34 none none

a. Expected fragment sizes are based on the plasmid map of the pDAB4468 as shown in Figure 6.

b. Observed fragment sizes are considered approximate from these analyses and are based on the

indicated sizes of the DIG-labeled DNA Molecular Weight Marker II 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.

Table 6. Results of F2 individual plants analyzed by Southern blot with aad-12 and pat

probes within a single segregating generation.

Generation

Total plants

analyzed

Southern

hybridization

positive

Southern

hybridization

negative

Expected

ratio P-valuea

F2 143 100 43 3:1 0.162

a Based on a chi-squared goodness of fit test
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Figure 33. Southern blot analysis of Nco I digest with aad-12 probe.

DNA isolated from individual plants of soybean event DAS-68416-4 F2 population and non-transgenic Maverick was digested with Nco I and hybridized with

aad-12 probe. Nine (9) μg of DNA was digested and loaded per lane. The plasmid controls contained plasmid pDAB4468 mixed with 9 μg of non-transgenic

DNA at a ratio approximately equivalent to 1 transgenic copy per soybean genome.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1, 12, 22 Molecular Marker 11 416-72 1, 12, 22 Molecular Marker 11 416-90

2 pDAB4468+ Maverick C6 13 416-73 2 pDAB4468+ Maverick C2 13 416-91

3 Maverick C6 14 416-74 3 Maverick C2 14 416-92

4 Maverick C10 15 416-75 4 Maverick C3 15 416-93

5 416-66 16 416-76 5 416-83 16 416-94

6 416-67 17 416-77 6 416-85 17 416-95

7 416-68 18 416-78 7 416-86 18 416-96

8 416-69 19 416-79 8 416-87 19 416-98

9 416-70 20 416-80 9 416-88 20 416-99

10 416-71 21 416-82 10 416-89 21 416-100

564-

4361-

6557-

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23130-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

- 564

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- 6557

- 2322

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- 23130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

B

A

B

564-

4361-

6557-

2322-

2029-

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23130-

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

- 564

- 4361

- 6557

- 2322

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- 23130

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

B

A

B
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Figure 34. Southern blot analysis of Nco I digest with pat probe.

DNA isolated from individual plants of soybean event DAS-68416-4 F2 population and non-transgenic Maverick was digested with Nco I and hybridized

with pat probe. Nine (9) μg of DNA was digested and loaded per lane. The plasmid controls contained plasmid pDAB4468 mixed with 9 μg of nontransgenic DNA at a ratio approximately equivalent to 1 transgenic copy per soybean genome.

Panel A Panel B

Lane Sample Lane Sample Lane Sample Lane Sample

1, 12, 22 Molecular Marker 11 416-40 1, 12, 22 Molecular Marker 11 416-56

2 pDAB4468+ Maverick C2 13 416-41 2 pDAB4468+ Maverick C2 13 416-57

3 Maverick C2 14 416-42 3 Maverick C2 14 416-58

4 Maverick C3 15 416-43 4 Maverick C3 15 416-59

5 416-34 16 416-44 5 416-50 16 416-60

6 416-35 17 416-45 6 416-51 17 416-61

7 416-36 18 416-46 7 416-52 18 416-62

8 416-37 19 416-47 8 416-53 19 416-63

9 416-38 20 416-48 9 416-54 20 416-64

10 416-39 21 416-49 10 416-55 21 416-65

564-

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2322-

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

- 564

- 4361

- 6557

- 2322

- 2029

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- 23130

A B

564-

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1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

- 564

- 4361

- 6557

- 2322

- 2029

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- 23130

A B
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V-E.2. Segregation Analysis of Breeding Generations

Chi-square analysis of trait inheritance data from a F2 breeding generation was conducted to

determine the Mendelian inheritance of aad-12 in DAS-68416-4 soybeans. The presence or

absence of aad-12 was determined using a gene-specific fluorescence-based detection method

specific for aad-12. The expected segregation ratio of 3:1 for plants containing aad-12

(homozygous + hemizygous) versus plants not containing aad-12 was observed (Table 7).

Table 7. Results of F2 individual plants tested for aad-12 within a single segregating

generation.

Generation

Total plants

tested

aad-12 gene

positive

aad-12 gene

negative

Expected

ratio P-valuea

F2 6774 5056 1718 3:1 0.492

a Based on a chi-squared goodness of fit test

V-F. Summary of the Genetic Characterization

AAD-12 soybean event DAS-68416-4 was produced using Agrobacterium-mediated

transformation with the plasmid pDAB4468. The T-DNA insert of pDAB4468 consists of the

aad-12 gene, controlled by the AtUbi10 promoter and AtuORF23 3’ UTR regulatory sequences,

the pat gene, controlled by the CsVMV promoter and AtuORF1 3’ UTR regulatory sequences,

and a RB7 MAR element at the 5’ of the AtUbi10 promoter. Various breeding generations were

developed and used to examine the integrity, stability, and inheritance of the aad-12 and pat

transgenic insert in soybean event DAS-68416-4.

Molecular characterization of soybean event DAS-68416-4 by Southern blot analysis confirmed

the insertion of a single intact copy of the aad-12 and pat expression cassettes from the T-DNA

insert of pDAB4468. No additional DNA fragments from the aad-12 and pat expression

cassettes were identified in DAS-68416-4 and no plasmid backbone sequences were present.

DAS-68416-4 was also shown to be stably integrated across three breeding generations (T3, T4,

and T5) and displayed the expected inheritance pattern of a single insert/locus in a generation

(F2) that was segregating for the DAS-68416-4 event.
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VI.Characterization of the Introduced Proteins

VI-A. AAD-12

VI-A.1. Identity of the AAD-12 Protein

The arylalkanoate dioxygenase (AAD-12) protein was derived from Delftia acidovorans, a gramnegative soil bacterium. The amino acid sequence is identical to the native enzyme sequence

except for the addition of an alanine at position number 2. The additional alanine codon encodes

part of an Nco I restriction enzyme recognition site (CCATGG) spanning the ATG translational

start codon. 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 proteins encoded by the native and plant-optimized coding regions are

99.3% identical, differing only at amino acid number 2. The AAD-12 protein is comprised of

293 amino acids and has a molecular weight of ~32 kDa (Figure 35).

Figure 35. Amino acid sequence of the AAD-12 protein.

001 MAQTTLQITPTGATLGATVTGVHLATLDDAGFAALHAAWLQHALLIFPGQ

051 HLSNDQQITFAKRFGAIERIGGGDIVAISNVKADGTVRQHSPAEWDDMMK

101 VIVGNMAWHADSTYMPVMAQGAVFSAEVVPAVGGRTCFADMRAAYDALDE

151 ATRALVHQRSARHSLVYSQSKLGHVQQAGSAYIGYGMDTTATPLRPLVKV

201 HPETGRPSLLIGRHAHAIPGMDAAESERFLEGLVDWACQAPRVHAHQWAA

251 GDVVVWDNRCLLHRAEPWDFKLPRVMWHSRLAGRPETEGAALV

VI-A.2. 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), the AAD-12 protein was microbially-produced

using Pseudomonas fluorescens (Pf). Characterization studies were performed to confirm the

equivalency of the AAD-12 protein expressed in planta in soybean line DAS-68416-4 with the

Pf microbe-derived AAD-12 protein. Sodium dodecyl sulfate polyacrylamide gel

electrophoresis (SDS-PAGE), western blot, glycoprotein detection, and protein sequence

analysis by matrix assisted laser desorption/ionization time-of-flight mass spectrometry

(MALDI-TOF MS) and electrospray ionization-liquid chromatography mass spectrometry (ESILC/MS) were used to characterize the biochemical properties of the protein. Using these

methods, the AAD-12 protein from Pf and the transgenic soybean event DAS-68416-4 were

shown to be biochemically equivalent, thereby supporting the use of the microbially-produced

protein in safety assessment studies.

The methods and results of the biochemical characterization of the DAS-68416-4 soybean- and

microbe-derived AAD-12 proteins are described in detail in Appendix 2. Briefly, both the plant

and Pf-derived AAD-12 proteins showed 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 the DAS-68416-4 soybean-derived AAD-12

protein. Amino acid sequence was confirmed by enzymatic peptide mass fingerprinting using

MALDI-TOF MS and ESI-LC/MS. The N-terminal methionine was found to be cleaved from
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both protein sources and the N-terminal peptide of the plant AAD-12 was determined to be

acetylated after the N-terminal methionine was cleaved. These two co-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).

VI-A.3. Expression of the AAD-12 Protein in Plant Tissues

A field expression study was conducted at six locations in U.S. and Canada during 2008. Six

sites (Iowa, Illinois, Indiana, Nebraska and Ontario, Canada (2 sites)) were planted with

DAS-68416-4 soybean and the conventional control (Maverick). The test sites represented

regions of diverse agronomic practices and environmental conditions for soybean in North

America. Four treatments of the DAS-68416-4 soybean (unsprayed, sprayed with 2,4-D, sprayed

with glufosinate, or sprayed with both 2,4-D and glufosinate) were tested (see Appendix 4 for

application rates and timings). Plant tissues sampled included leaf, grain, root, and forage. Leaf

tissues were collected at V5 and V10 stage, and root and forage were collected at the R3 stage of

development. The grain was collected at the R8 stage of development (Gaska, 2006). The

soluble, extractable AAD-1 protein was measured using a validated enzyme-linked

immunosorbent assay (ELISA) method. AAD-12 protein levels for all tissue types were

calculated on ng/mg dry weight basis. Methods used for tissue sampling and quantification of

protein expression by ELISA are detailed in Appendix 4.

A summary of the AAD-12 protein concentrations (averaged across sites) in the various soybean

matrices is shown in Table 8. Average expression values ranged from 15.48 ng/mg dry weight in

R3 stage root to 66.08 ng/mg dry weight in V5 stage leaf tissue. Expression values were similar

for the all sprayed treatments as well as for the plots sprayed and unsprayed with 2,4-D and

glufosinate herbicides. No AAD-12 protein was detected in the control tissues across the six

locations.
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Table 8. Summary of AAD-12 protein levels in tissues collected from DAS-68416-4

produced in the U.S. and Canada during 2008.

AAD-12 ng/mg Tissue Dry Weight

Tissue Treatment Mean Std. Dev. Range

V5 Leaf DAS-68416-4 Unsprayed 51.42 25.22 26.37 - 97.66

DAS-68416-4 + Glufosinate 50.63 23.69 28.03 - 94.00

DAS-68416-4 + 2,4-D 51.68 25.41 27.16 - 100.79

DAS-68416-4 + Glufosinate and 2,4-D 66.08 37.82 25.14 - 164.58

V10 Leaf DAS-68416-4 Unsprayed 53.95 20.85 29.83 - 90.89

DAS-68416-4 + Glufosinate 56.06 21.95 25.06 - 91.95

DAS-68416-4 + 2,4-D 55.24 20.62 30.84 - 91.80

DAS-68416-4 + Glufosinate and 2,4-D 57.07 22.97 32.02 - 95.16

Root DAS-68416-4 Unsprayed 17.10 5.68 8.80 - 27.62

DAS-68416-4 + Glufosinate 15.48 4.58 6.30 - 23.08

DAS-68416-4 + 2,4-D 16.01 6.64 3.16 - 27.91

DAS-68416-4 + Glufosinate and 2,4-D 16.66 6.81 1.84 - 26.50

Forage DAS-68416-4 Unsprayed 41.11 25.72 5.70 - 91.17

DAS-68416-4 + Glufosinate 39.35 24.47 5.49 - 87.96

DAS-68416-4 + 2,4-D 40.56 25.58 5.02 - 88.02

DAS-68416-4 + Glufosinate and 2,4-D 39.65 22.41 4.96 - 69.62

Grain DAS-68416-4 Unsprayed 16.47 3.55 9.40 - 21.86

DAS-68416-4 + Glufosinate 16.94 3.15 11.9 - 22.74

DAS-68416-4 + 2,4-D 16.47 3.78 9.71 - 21.95

DAS-68416-4 + Glufosinate and 2,4-D 16.21 3.62 9.91 - 23.40

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the AAD-12 ELISA in the

tissue matrices were as follows:

Tissue LOD

(ng/mg DW)

LOQ

(ng/mg DW)

Leaf (V5) 0.50 1.00

Leaf (V10) 0.50 1.00

Root 0.50 1.00

Forage 0.50 1.00

Grain 0.50 1.00
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VI-A.4. Food and Feed Safety Assessment for AAD-12 Protein

Results of the overall safety assessment of the AAD-12 protein indicate that it is unlikely to

cause an allergic reaction in humans or be a toxin in humans or animals.

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 present in soil, fresh water, activated sludge, and clinical specimens (von

Gravenitz 1985, Tamaoka et al. 1987, Wen et al., 1999).

 Delftia acidovorans can be used to transform ferulic acid into vanillin and related flavor

metabolites (Toms and Wood, 1970; Ramachandra Rao and Ravishankar, 2000; Shetty et al.,

2006). This utility has led to a history of safe use for Delftia acidovorans in the food

processing industry. For example, see US Patent 5,128,253 “Bioconversion process for the

production of vanillin” issued on July 7, 1992 to Kraft General Foods (Labuda et al., 1992).

Lack of allergenic potential

 The step-wise, weight-of-evidence approach (Codex, 2003) 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 homology was identified when the AAD-12 protein sequence was

compared with known allergens in the FARRP (Food Allergy Research and Resource

Program) version 9.00 allergen database, using the search criteria of either a match of eight

or more contiguous identical amino acids, or >35% identity over 80 amino acid residues.

 The AAD-12 protein is rapidly degraded below the level of detection in simulated gastric

fluid (SGF). The AAD-12 protein was readily digested, i.e., not detectable after 30 seconds,

under in vitro SGF conditions (0.32% pepsin, pH 1.2; 37 oC) as demonstrated by both

SDS-PAGE and western blot analyses.

 The AAD-12 protein is not present in a glycosylated state. No glycosylation of the AAD-12

protein was detected using SDS-PAGE and a glycosylation detection system. (see Appendix

2)

Lack of toxic potential

 The AAD-12 protein does not share meaningful amino acid sequence similarities with known

toxins. Amino acid homologies were evaluated using a global sequence similarity search

against the GenBank non-redundant protein dataset. The search identified 618 similar

proteins, which can be broken down into a few major subclasses. The largest subclass,

containing 474 proteins, was identified as tauD or taurine dioxygenases. These are proteins

involved in the degradation of taurine (Eichorn et al., 2007). The next largest class, with 138

members, was clavaminic acid synthetases or “CAS-like”(Zhang et al., 2000). There were 2

TolC proteins which are known efflux pumps (Koronakis et al., 2000). The last four proteins

were: 1) a (S)-2-(2,4-dichlorophenoxy)propionate, 2-oxoglutarate dioxygenase (Schleinitz et

al., 2004); 2) a pvcB protein which is a known CAS-like protein (see accession page of

NP_968348); 3) an inosine-uridine preferring nucleoside hydrolase (Gopaul et al., 1996); and
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4) a hypothetical protein with no functional annotation. None of these protein classes are

known toxins.

 In acute mouse toxicity testing, there were no mortalities or clinical signs in CD-1 mice after

oral administration by gavage of AAD-12 protein at 2000 mg protein/kg body weight.

VI-A.5. Summary of AAD-12 Protein Characterization

The arylalkanoate dioxygenase (AAD-12) protein was derived from Delftia acidovorans, a

gram-negative soil bacterium. AAD-12 is comprised of 293 amino acids and has a molecular

weight of ~32 kDa. Detailed biochemical characterization of the AAD-12 protein derived from

plant and microbial sources was conducted. Additionally, characterization of AAD-12 protein

expression in DAS-68416-4 plants over the growing season was determined by analyzing leaf,

root, whole plant, and grain tissues from DAS-68416-4 plants sprayed with 2,4-D, glufosinate,

both 2,4-D and glufosinate, and non-sprayed.

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.

The AAD-12 protein hydrolyzes 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. Glycosylation

analysis revealed no detectable covalently linked carbohydrates in AAD-12 protein expressed

in DAS-68416-4 soybean plants. Therefore, the low level expression of the AAD-12 protein

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

assessment of the AAD-12 protein indicate that it is unlikely to cause allergenic or toxic effects

in humans or animals.

VI-B. PAT

VI-B.1. Identity of the PAT Protein

The phosphinothricin acetyltransferase (PAT) protein was derived from Streptomyces

viridochromogenes, a gram-positive soil bacterium. The pat transgene in DAS-68416-4 encodes

a protein sequence that is identical to the native PAT protein (Accession number: Q57146).

PAT is comprised of 183 amino acids and has a molecular weight of ~21 kDa (Figure 36).

Figure 36. Amino acid sequence of the PAT protein.

001 MSPERRPVEIRPATAADMAAVCDIVNHYIETSTVNFRTEPQTPQEWIDDL

051 ERLQDRYPWLVAEVEGVVAGIAYAGPWKARNAYDWTVESTVYVSHRHQRL

101 GLGSTLYTHLLKSMEAQGFKSVVAVIGLPNDPSVRLHEALGYTARGTLRA

151 AGYKHGGWHDVGFWQRDFELPAPPRPVRPVTQI

VI-B.2. Biochemical Characterization of the PAT Protein

Characterization of the biochemical properties of the plant-derived PAT protein was

accomplished through the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE), lateral flow strips and western blot analysis. The methods and results are

described in detail in Appendix 3. Using these methods the PAT protein produced in DAS-
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68416-4 soybean was shown to be equivalent to that produced in other transgenic crops (USDA

1996, USDA 2001, USDA 2004, USDA 2005).

VI-B.3. Expression of the PAT Protein in Plant Tissues

A field expression study was conducted at six locations in U.S. and Canada during 2008. Six

sites (Iowa, Illinois, Indiana, Nebraska and Ontario, Canada (2 sites)) were planted with

DAS-68416-4 soybean and the conventional control (Maverick). The test sites represented

regions of diverse agronomic practices and environmental conditions for soybean in North

America. Four treatments of the DAS-68416-4 soybean (unsprayed, sprayed with 2,4-D, sprayed

with glufosinate, or sprayed with both 2,4-D and glufosinate) were tested (see Appendix 4 for

application rates and timings). Plant tissues sampled included leaf, grain, root, and forage. Leaf

tissues were collected at V5 and V10 stage, and root and forage were collected at the R3 stage of

development. The grain was collected at the R8 stage of development (Gaska, 2006). The

soluble, extractable PAT protein was measured using a validated enzyme-linked immunosorbent

assay (ELISA) method. PAT protein levels for all tissue types were calculated on ng/mg dry

weight basis. Methods used for tissue sampling and quantification of protein expression by

ELISA are detailed in Appendix 4.

A summary of the PAT protein concentrations (averaged across sites) in the various soybean

matrices is shown in Table 9. Average expression values ranged from 1.73 ng/mg dry weight in

R3 stage root to 11.76 ng/mg dry weight in V10 stage leaf tissue. Expression values were

similar for the all sprayed treatments as well as for the plots sprayed and unsprayed with 2,4-D

and glufosinate herbicides. No PAT protein was detected in the control tissues across the six

locations.
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Table 9. Summary of PAT protein levels in tissues collected from DAS-68416-4 produced in

the U.S. and Canada during 2008.

PAT ng/mg Tissue Dry Weight

Tissue Treatment Mean Std. Dev. Range

V5 Leaf DAS-68416-4 Unsprayed 9.17 2.99 4.33 - 13.75

DAS-68416-4 + Glufosinate 9.83 2.66 3.67 - 13.78

DAS-68416-4 + 2,4-D 9.01 3.03 4.87 - 13.92

DAS-68416-4 + Glufosinate and 2,4-D 10.05 3.76 3.00 - 15.03

V10 Leaf DAS-68416-4 Unsprayed 10.94 1.31 8.43 - 13.35

DAS-68416-4 + Glufosinate 11.51 1.69 9.08 - 14.44

DAS-68416-4 + 2,4-D 11.76 2.02 7.49 - 14.81

DAS-68416-4 + Glufosinate and 2,4-D 11.58 1.45 9.26 - 14.15

Root DAS-68416-4 Unsprayed 1.73 0.51 0.47 - 2.84

DAS-68416-4 + Glufosinate 1.92 0.45 1.01 - 2.67

DAS-68416-4 + 2,4-D 1.73 0.68 0.42 - 2.83

DAS-68416-4 + Glufosinate and 2,4-D 1.93 0.55 0.36 - 2.68

Forage DAS-68416-4 Unsprayed 3.63 2.88 0.06 - 12.54

DAS-68416-4 + Glufosinate 4.81 3.75 0.40 - 12.10

DAS-68416-4 + 2,4-D 5.28 4.20 0.12 - 12.13

DAS-68416-4 + Glufosinate and 2,4-D 4.73 3.63 0.45 - 12.35

Grain DAS-68416-4 Unsprayed 2.73 0.34 1.96 - 3.37

DAS-68416-4 + Glufosinate 2.74 0.28 2.29 - 3.39

DAS-68416-4 + 2,4-D 2.79 0.26 2.21 - 3.13

DAS-68416-4 + Glufosinate and 2,4-D 2.82 0.23 2.43 - 3.25

The Limit of Detection (LOD) and Limit of Quantitation (LOQ) of the PAT ELISA in the tissue

matrices were as follows:

Tissue LOD

(ng/mg DW)

LOQ

(ng/mg DW)

Leaf (V5) 0.06 0.12

Leaf (V10) 0.06 0.12

Root 0.06 0.12

Forage 0.06 0.12

Grain 0.06 0.12

VI-B.1. Food and Feed Safety Assessment for PAT Protein

The PAT protein produced in DAS-68416-4 soybean was shown to be equivalent to that

produced in other transgenic crops that have been previously deregulated by USDA (USDA

1996, USDA 2001, USDA 2004, USDA 2005). The food and feed safety of PAT was assessed

in these products and shown to present to significant food or feed safety risk.
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Additionally, the US EPA has concluded, after reviewing data on the acute toxicity and

digestibility of the PAT protein, that there is a reasonable certainty that no harm will result from

aggregate exposure to the U.S. population, including infants and children, to the PAT protein and

the genetic material necessary for its introduction (US EPA, 1997). US EPA has consequently

established an exemption from tolerance requirements pursuant to FFDCA section 408(j)(3) for

PAT and the genetic material necessary for its production in all plants.

VI-B.2. Summary of PAT Protein Characterization

The phosphinothricin acetyltransferase (PAT) protein was derived from Streptomyces

viridochromogenes, a gram-positive soil bacterium. PAT is comprised of 183 amino acids and

has a molecular weight of ~21 kDa. Detailed biochemical characterization of the PAT protein

derived from DAS-68416-4 soybean was conducted. Western blot analysis techniques were used

to determine that the PAT protein expressed in soybean was the same molecular weight and

immunoreactivity as the native protein. Polyclonal and monoclonal antibodies were used that

recognize antigenic epitopes on the protein. Any protein that was smaller (a degradation

fragment) or larger (fusion protein or post-translationally modified version) in size would have

been detected as a band of different molecular weight.

Characterization of PAT protein expression in DAS-68416-4 plants over the growing season was

determined by analyzing leaf, root, whole plant, and grain tissues from DAS-68416-4 plants

sprayed with 2,4-D, glufosinate, both 2,4-D and glufosinate, and non-sprayed.

VI-C. References

Codex Alimentarius Commission (2003) Guideline for the conduct of food safety assessment of

foods derived from recombinant-DNA plants, Annex 1: Assessment of Possible Allergenicity,

CAC/GL 45-2003, pg 9-11.

(www.codexalimentarius.net/download/standards/10021/CXG_045e.pdf)

Eichhorn, E., Van Der Ploeg, J.R., Kertesz, M.A., Leisinger, T. (1997) Characterization of alphaketoglutarate-dependent taurine dioxygenase from Escherichia coli. Journal of Biological

Chemistry 272:23031-23036.

Evans, S. L. (2004) Producing Proteins from Genetically Modified Organisms for Toxicology

and Environmental Fate Assessment of Biopesticides. In: Parekh, S. R. (Ed), The GMO

Handbook: Genetically Modified Animals, Microbes, and Plants in Biotechnology, Humana

Press Inc., Totowa, New Jersey, pp. 53-84.

Gaska, J. (2006) Soybean growth and development

(http://soybean.uwex.edu/library/soybean/morphology/documents/04_soy_growth_development.

pdf)

Gopaul, D.N., Meyer, S.L., Degano, M., Sacchettini, J.C., Schramm, V.L. (1996) Inosine-uridine

nucleoside hydrolase from Crithidia fasciculata. Genetic characterization, crystallization, and

identification of histidine 241 as a catalytic site residue. Biochemistry 35:5963-5970.
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Koronakis, V., Sharff, A., Koronakis, E., Luisi, B., Hughes, C. (2000) Crystal structure of the

bacterial membrane protein TolC central to multidrug efflux and protein export. Nature

405:914-9.

Labuda, I.M., Goers, S.K., Keon, K.A. (1992) Bioconversion Process for the Production of

Vanillin. US Patent #5,128,253.

Polevoda, B. and Sherman, F. (2003) N-terminal Acetyltransferases and Sequence Requirements

for N-terminal Acetylation of Eukaryotic Proteins. Journal of. Molecular Biology. 325:595-

622.

Ramachachandra Rao, S. and Ravishankar, G.A. (2000) Vanilla Flavor: Production by

Conventional and Biotechnological Routes. Journal of the Science of Food and Agriculture

80:289-304.

Schleinitz, K.M., Kleinsteuber, S., Vallaeys,T., Babel, W. (2004) Localization and

Characterization of Two Novel Genes Encoding Stereospecific Dioxygenases Catalyzing 2(2,4-

Dichlorophenoxy)propionate Cleavage in Delftia acidovorans MC1. Applied and Environmental

Microbiology 70:5357-65.

Shetty, K., Paliyath, G., Pometto, A., Levin, R.E. (2006) Food Biotechnology, CRC Press, ISBN

0824753291, pp. 1672-1673.

Tamaoka, J., Ha, D.M., Komagata, K. (1987) Reclassification of Pseudomonas acidovorans den

Dooren de Jong 1926 and Pseudomonas testosteroni Marcus and Talalay 1956 as Comamonas

acidovorans comb. nov. and Comamonas testosteroni comb. nov., with an emended description

of the genus Comamonas. International Journal of Systematic Bacteriology 37:52-59.

Toms, A., Wood, J.M. (1970) The Degradation of trans-Ferulic Acid by Pseudomonas

acidovorans. Biochemistry 9:337-343.

US EPA (1997) Phosphinothricin Acetyltransferase and the Genetic Material Necessary for Its

Production in All Plants; Exemption From the Requirement of a Tolerance On All Raw

Agricultural Commodities. Federal Register Volume 62, Number 70:17717-17720.

USDA (1996) Availability of Determination of Nonregulatated status for Soybeans Genetically

Engineered for Glufosinate Herbicide Tolerance. Federal Register Volume 61, Number

160:42581-42582.

USDA (2001) Availability of Determination of Non-regulatated status for Genetically

Engineered Corn for Insect Resistance and Glufosinate Herbicide Tolerance (Corn line 1507).

Federal Register Volume 66, Number 157:42624-42625.

USDA (2004) Availability of Determination of Nonregulated Statusfor Cotton Lines Genetically

Engineered for Insect Resistance. Federal Register Volume 69, Number 156:50154-50155.

USDA (2005) Availability of Determination of Non-regulated Status for Genetically Engineered

Corn (Corn line DAS-59122-7). Federal Register Volume 70, Number 194:58663-58664.
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Von Gravenitz, A, (1985) Ecology, clinical significance, and antimicrobial susceptibility

encountered glucose-non-fermenting gram-negative rods, p. 181-232. In G. L. Gilardi (ed.),

Nonfermentative gram-negative rods, laboratory identification and clinical aspects. Marcel

Dekker, Inc., New York. Winkelmann G, Drechsel H (1997).

Wen, A., Fegan, M., Hayward, C., Chakraborty, S., Sly, L.I. (1999) Phylogenetic relationships

among members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren

de Jong 1926 and Tamaoka et al. 1987) gen. nov., comb. nov. International Journal of

Systematic Bacteriology 49:567-576.

Zhang, Z., Ren, J.-S., Stammers, D.K., Baldwin, J.E., Harlos, K., Schofield, C.J. (2000)

Structural origins of the selectivity of the trifunctional oxygenase clavaminic acid synthase.

Nature Structural and Molecular Biology 7:127-133.
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VII. Agronomic Performance

VII-A. Phenotypic and Agronomic Characteristics

Agronomic trials were conducted with DAS-68416-4 soybeans in 2008 at 6 locations in the U.S.

and Canada (Experiment 1) and in 2009 at 21 locations in the U.S (Experiment 2). These studies

compared DAS-68416-4 soybeans with the non-transgenic control (Maverick). Experiment 1

used the same plots that were used for the protein expression (Sections VI-A.3 and VI-B.3) and

nutrient composition (Section VIII) studies. In Experiment 1, DAS-68416-4 soybeans were

evaluated with and without 2,4-D and glufosinate herbicide spray treatments. Experiment 2 was

conducted at 21 locations to allow for testing in a broader range of environmental conditions.

The results of both experiments showed that the DAS-68416-4 soybean was equivalent to nontransgenic soybean.

VII-A.1. Experiment 1

An agronomic study with DAS-68416-4 soybean and a non-transgenic control (Maverick) was

conducted in 2008 at six sites located in Iowa (Keokuk county), Illinois (Clinton county), Indiana

(Parke county), Nebraska (York county) and Ontario, Canada (2 sites). Agronomic

determinants, including stand count, emergence, seedling vigor, plant height, lodging, days to

flowering, and final population were evaluated to investigate the equivalency of the DAS-68416-

4 transgenic soybeans (with and without herbicide treatments) to the control (Table 10).

The test and control soybean seed were planted at a seeding rate of approximately 112 seeds per

25 ft row with a row spacing of approximately 30 inches. At each site, 3 replicate plots of each

treatment were established, with each plot consisting of 2-25 ft rows. Plots were arranged in a

randomized complete block (RCB) design, with a unique randomization at each site. Each

soybean plot was bordered by 2 rows of a non-transgenic soybean of similar maturity. The entire

trial site was surrounded by a minimum of 10 ft of a non-transgenic soybean of similar relative

maturity. Appropriate insect, weed, and disease control practices were applied to produce an

agronomically acceptable crop.

Herbicide treatments were applied with a spray volume of approximately 20 gallons per acre

(187 L/ha). These applications were designed to replicate maximum label rate for commercial

practices. 2,4-D was applied as 3 broadcast over-the-top applications for a seasonal total of

3 lb ae/A. Individual applications of 1.0 lb ae/A (1120 g ae/ha) were made at pre-emergence and

approximately V4 and R2 growth stages. Glufosinate was applied as 2 broadcast over-the-top

applications for a seasonal total of 0.74 lb ai/A (828 g ai/ha). Individual applications of

0.33 lb ai/A and 0.41 lb ai/A (374 and 454 g ai/ha) were made at approximately V6 and R1

growth stages.
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Table 10. Agronomic parameters evaluated in Experiment 1.

Parameter Evaluation Timing Description Scale or Units

Stand count VC-V2 Number of plants

emerged in rows of

each plot

Actual count per plot

Emergence VC-V2 Stand count divided

by the number of

seeds planted

%

Seedling vigor VC-V2 Visual estimate of

average vigor of

emerged plants per

plot

1-10 scaled based on growth

of the non-transformed

soybeans

10 = Growth equivalent to

non-transformed

9 = Plant health is 90% as

compared to nontransformed, etc.

Plant vigor / injury After post-emergent

herbicide applications

Injury from herbicide

applications

1-10 scale based on growth of

the non-transformed soybeans

10 = Growth equivalent to

non-transformed

9 = Plant health is 90% as

compared to nontransformed, etc.

Days to flower R1 Number of days from

planting to when 50%

of plants reached R1

Days

Plant height Approximately R6 Height from soil

surface to the tip of

the highest leaf when

extended by hand

Height in cm

(average of 10 plants per plot)

Lodging Approximately R8 Visual estimate of

lodging severity

Visual estimate on 0-100%

scale based on the number of

plants lodged

Final population Approximately R8 The number of plants

remaining in rows of

each plot

Actual count per plot,

including plants removed

during previous sampling

Analysis of variance was conducted across the field sites for the agronomic data using a mixed

model (SAS Version 8; SAS Institute 1999). Entry was considered a fixed effect, and location,

block within location, location-by-entry, and entry-by-block within location were designated as

random effects. The significance of an overall treatment effect was estimated using an F-test.

Paired contrasts were made between DAS-68416-4 (unsprayed AAD-12), DAS-68416-4 sprayed

with glufosinate (AAD-12 + glufosinate), DAS-68416-4 sprayed with 2,4-D (AAD-12 + 2,4-D),

and DAS-68416-4 sprayed with both glufosinate and 2,4-D (AAD-12 + both herbicides), and the

control entry using t-tests.

Multiplicity is an issue when a large number of comparisons are made in a single study to look

for unexpected effects. Under these conditions, the probability of falsely declaring differences

based on comparison-wise p-values is very high (1-0.95number of comparisons). In this study there
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were four comparisons per analyte and 12 quantitated observation types (including disease

incidence and insect damage, Section VII-B), resulting in 48 comparisons made in the across-site

analysis. Therefore, the probability of declaring one or more false differences based on

unadjusted p-values was >91% (1-0.9548).

One method to account for multiplicity is to adjust p-values to control the experiment-wise error

rate (probability that all declared differences are significant), but when many comparisons are

made in a study, the power for detecting specific effects can be reduced significantly. An

alternative with much greater power is to adjust p-values to control the probability that each

declared difference is significant. This can be accomplished using False Discovery Rate (FDR)

procedures (Benjamini and Hochberg, 1995). Therefore the p-values were adjusted using FDR

to improve discrimination of true differences among treatments from random effects (false

positives).

Experiment 1 Results

An analysis of the agronomic data collected from the control, AAD-12 unsprayed, AAD-12 +

2,4-D, AAD-12 + glufosinate, and AAD-12 + both herbicides was conducted. No statistically

significant differences were observed for stand count, emergence, seedling vigor, days to flower,

lodging, or final population (Table 11Table 11. Analysis of agronomic characteristics from

Experiment 1.). For plant height, a significant paired t-test was observed between the control

and the AAD-12 + 2,4-D spray. However, no significant overall treatment effect was observed,

differences were very small between the AAD-12 treatment and the control, and differences were

not shared among the different AAD-12 treatments. Based on these results, DAS-68416-4

soybean was agronomically equivalent to the non-transgenic control.
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Table 11. Analysis of agronomic characteristics from Experiment 1.

P-value for Treatment Means

Overall (P-valueb

, Adj. Pc)

Parameter

Treatment

Effecta Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Stand Count 0.774 170 172 175 173 175

(0.709,0.824) (0.311,0.575) (0.476,0.672) (0.269,0.575)

Emergence 0.714 76.7 77.4 79.1 79.0 79.4

(0.738,0.824) (0.301,0.575) (0.327,0.575) (0.256,0.575)

Seedling Vigor 0.547 9.72 9.39 9.50 9.44 9.39

(0.146,0.575) (0.326,0.575) (0.222,0.575) (0.146,0.575)

Vigor/Injury 0.511 10.0 9.86 9.89 9.83 9.67

App. 2d

(0.461,0.671) (0.555,0.718) (0.378,0.611) (0.087,0.575)

Vigor/Injury 0.462 10.0 10.0 9.89 9.83 9.89

App. 3e

(1.000,1.000) (0.320,0.575) (0.141,0.575) (0.320,0.575)

Vigor/Injury 0.431 9.94 9.89 9.78 9.67 9.78

App. 5f

(0.721,0.824) (0.289,0.575) (0.085,0.575) (0.289,0.575)

Days to Flower 0.452 49.0 49.5 49.4 48.7 49.2

(0.261,0.575) (0.395,0.611) (0.568,0.718) (0.668,0.801)

Plant Height 0.144 101 98.1 99.2 96.1 97.2

(0.145,0.575) (0.390,0.611) (0.020,0.575) (0.062,0.575)

Lodging (%) 0.948 17.2 18.2 21.3 20.7 21.7

(0.885,0.904) (0.551,0.718) (0.606,0.746) (0.511,0.700)

Final Population 0.268 156 154 161 155 163

(0.770,0.840) (0.335,0.575) (0.817,0.853) (0.127,0.575)

a Overall treatment effect estimated using an F-test.

b Comparison of the sprayed and unsprayed treatments to the control using a t-test.

c

P-values adjusted using a False Discovery Rate (FDR) procedure.

d Evaluated after application of 2,4-D at the V4 growth stage.

e Evaluated after application of 2,4-D at the R2 growth stage.

f Evaluated after application of glufosinate at the R1 growth stage.

Bolded P-values are significant (<0.05).
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VII-A.2. Experiment 2

An agronomic study with DAS-68416-4 soybean and a non-transgenic control (Maverick) was

conducted in 2009 at 21 sites in the U.S. (Table 12). Ten locations had both a 2,4-D sprayed

and unsprayed component, while the remaining eleven locations had no 2,4-D applied.

Table 12. Locations for Experiment 2 agronomic and yield trials, 2009.

County State County State

Sprayed and non-sprayed Non-sprayed only

Keokuk IA Madison IA

Clinton IL Story IA

Benton IN Ford IL

LaGrange IN Saline IL

Parke IN Stark IL

Butler MO Benton IN

Henry MO Newton IN

Seward NE Queen Anne MD

York NE Adair MO

Fulton OH Jasper MO

Polk NE

Agronomic determinants, including emergence, seedling vigor, days to flower, days to maturity,

plant height, lodging, shattering, and yield were evaluated to investigate the equivalency between

the non-sprayed and sprayed DAS-68416-4 soybeans, and between non-sprayed DAS-68416-4

soybeans and the control (Table 13).

Each plot consisted of 2 rows planted 0.762 meters apart. Plots were 5.3 or 3.8 meters in length

depending on the location of the trial. Planting density was 27.5 seeds per meter. A preemergent herbicide typical of soybean production was applied at most locations. Hand weeding

or conventional post-emergent herbicides were used for weeds that were not controlled by the

pre-emergent herbicide or by 2,4-D application.

A randomized-complete-block design was used for the 21 experiments that did not have a 2,4-D

spray component. Each location of the trial consisted of 4 replications of each treatment.

A modified split-plot design with 4 replications was used for the 10 trials that included the 2,4-D

spray component. Whole plots were herbicide treatments and subplots were test lines.

Treatments consisted of a non-2,4-D-sprayed treatment and 2,4-D applied at the V3 and R2

growth stages. For both applications, 2,4-D was applied at a rate of 2 lbs ae/A (2240 g ae/ha,

equivalent to twice the maximum proposed use rate). Whole-plots which received the 2,4-D

treatment were grouped together for ease of application. The non-transgenic control (Maverick)

was forced to a set position within the herbicide treated whole-plots. This was done to reduce

any chance of border effect which would have occurred had the control been placed beside a 2,4-

D-tolerant entry. The control plots were expected to die after herbicide application and so would

have left bare ground adjacent to resistant plots had they been randomized among the other

entries. Both experiments were anayzed using a mixed model with entry designated as a fixed

effect and location, block within location, and location-by-entry designated as random effects.

Differences were considered statistically significant at alpha < 0.05.
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Table 13. Data collected in Experiment 2 agronomic and yield trials, 2009.

Parameter

Evaluation

Timing Description Scale or Units

Emergence VC - V2 Stand count in 1 meter section of row divided

by number of seeds planted per meter

%

Seedling vigor V1 - V3 General seedling vigor 1 (low) to 10 (high)

Days to Flower R1 Number of days from planting to when 50%

of plants are at R1

days

Stand count R1 Number of plants in one meter section of row

Disease incidence ~R6 Opportunistic note on any disease that

occurred at a location

%

Insect damage ~R6 Opportunistic note on any insect damage that

occurred at a location

%

Plant Height R8 Final height of plot at R8 cm

Days to Maturity R8 Number of days from planting to when 95%

of plants in plot have reached their mature

color

days

Lodging R8 Degree of lodging in a plot 1 (none) - 5 (flat)

Shattering R8 Percent of pods shattered %

Yield R8 Quantity of seed produced by the plot bu/acre

100 seed weight R8 Weight of 100 random seeds from the

harvested plot

g

Experiment 2 Results – Comparison of Sprayed vs. Non-Sprayed DAS-68416-4 Soybean

All agronomic observations for the 2,4-D sprayed and non-sprayed DAS-68416-4 soybeans were

statistically indistinguishable across the ten field locations, indicating phenotypic equivalence

even when sprayed with 2,4-D at twice the maximum proposed use rate (Table 14). Insect

damage ratings were taken at seven of the ten locations and were identical for sprayed and nonsprayed plots, and all values were ≤5% damage.

Table 14. Agronomic parameters for sprayed (2,4-D) and unsprayed DAS-68416-4 soybean.

Mean

Parameter Unit Sprayed Unsprayed SE1 Pr>F

Emergence % 78.4 79.8 3.6 0.5820

Seedling vigor 1-10 scale 8.8 8.9 0.4 0.4757

Days to flower days 40.6 39.8 3.8 0.3822

Stand count No./m 21.1 21.4 1.0 0.6601

Disease incidence % 8.7 8.7 3.0 1.0000

Plant height at senescence cm 79.8 83.1 8.7 0.0888

Days to maturity days 133.1 132.8 5.3 0.2847

Lodging 1-5 scale 1.5 1.8 0.3 0.1866

Shattering % 1.2 1.2 0.6 0.3434

Yield bu/acre 48.2 51.3 6.6 0.2818

100 seed weight g 15.1 15.3 0.4 0.6912

1

SE = pooled standard error of the mean.

Bolded P-values are significant (<0.05).
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Experiment 2 Results - Comparison of DAS-68416-4 and Control (Maverick) Soybean

All agronomic observations for non-sprayed control (Maverick) and DAS-68416-4 soybeans

were statistically indistinguishable across the 21 locations except for days to maturity and 100

seed weight (Table 15). On average, Maverick took less than one additional day to mature

compared with event DAS-68416-4 soybean. This difference in maturity is not biologically

significant. The weight per 100 seeds was statistically lower for event DAS-68416-4 soybean

compared with Maverick soybean, but the difference was less than 3.5% and was not

accompanied by reduced yield. Therefore this difference is not considered to be biologically

significant. Overall, event DAS-68416-4 soybean was agronomically equivalent to the nontransgenic control (Maverick) soybean.

Table 15. Agronomic parameters for DAS-68416-4 and Maverick soybean.

Mean

Parameter Unit Maverick DAS-68416-4 SE1 Pr>F

Emergence % 79.4 77.6 2.7 0.3154

Seedling vigor 1-10 scale 8.7 8.7 0.2 1.0000

Days to flower days 42.3 42.3 2.1 0.7961

Stand count No./m 21.8 21.7 0.8 0.7482

Disease incidence % 5.9 6.0 1.8 0.6703

Insect damage % 2.4 2.4 0.7 0.3364

Plant height cm 92.7 91.6 5.5 0.1389

Days to maturity days 129.3 128.4 3.3 0.0017

Lodging 1-5 scale 2.0 2.1 0.2 0.3100

Shattering % 0.8 0.8 0.3 0.6787

Yield bu/acre 53.9 54.1 3.7 0.8043

100 seed weight g 15.8 15.3 0.3 0.0014

1

SE = pooled standard error of the mean.

Bolded P-values are significant (<0.05).

VII-A.3. Conclusions

Results from experiments in 2008 and 2009 indicate that DAS-68416-4 soybean is

agronomically equivalent to non-transgenic control (Maverick) soybean. Treatments of 2,4-D

and/or glufosinate did not significantly change the agronomic performance of DAS-68416-4

soybean.
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VII-B. Ecological Evaluations

The DAS-68416-4 soybean field trials were monitored and observed by personnel familiar with

soybean cultivation practices (breeders, field station managers, field agronomists, field

associates). The personnel conducting the field tests visually monitored the incidence of plant

disease and pests on DAS-68416-4 soybeans compared to the conventional soybean varieties in

the same trials. As part of Experiment 1 described in Section VII-A.1, disease and insect damage

was rated on a numerical scale of 0-100%, with 0% representing no damage due to disease

incidence or insect resistance. Table 16 shows results across the 6 sites described in Experiment

1.

Table 16. Analysis of disease incidence and insect damage from Experiment 1.

P-value for Treatment Means

Overall (P-valueb

, Adj. Pc)

Parameter

Treatment

Effecta Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Disease 0.422 13.1 12.6 11.8 11.1 10.1

Incidence (%)d

(0.803,0.853) (0.456,0.671) (0.251,0.575) (0.091,0.575)

Insect Damaged0.332 24.1 21.8 22.1 22.3 20.9

(0.140,0.575) (0.204,0.575) (0.236,0.575) (0.044,0.575)

a Overall treatment effect estimated using an F-test.

b Comparison of the sprayed and unsprayed treatments to the control using a t-test.

c

P-values adjusted using a False Discovery Rate (FDR) procedure.

d Visual estimate on 0-100% scale; 0% = no damage.

No statistically significant differences were observed for disease incidence. For insect damage, a

significant paired t-test was observed between the control and the AAD-12 + both herbicides.

However, no significant overall treatment effect was observed, the difference between the

AAD-12 treatment and the control was small, and differences were not shared among the

different AAD-12 treatments.

Insect damage and disease incidence was also measured at 21 field sites in Experiment 2

(Section VII-A.2). No significant difference was observed between DAS-68416-4 soybeans that

had been sprayed with 2,4-D and non-sprayed. No significant differences were observed

between DAS-68416-4 soybeans and the non-transgenic control.

Ecological observations were also made from all USDA APHIS notified field trials conducted in

2006-2009 (Appendix 5). Incidence of disease and insect presence in trials of DAS-68416-4

soybeans were recorded and differences in incidence or response of DAS-68416-4 soybeans

compared with the conventional control were examined. In all cases, no differences were seen in

any of the trials of DAS-68416-4 soybeans compared with the conventional controls. The

disease and insect stressors observed in trials of DAS-68416-4 and conventional soybeans are

summarized in Table 17. These observations support the conclusion that the response of

DAS-68416-4 soybean to ecological stressors does not differ from that of conventional soybean.
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Table 17. Disease and insect stressors observed in trials of DAS-68416-4 and conventional

soybean.

Year

USDA

Notification

Number State (County) Diseases Insects

2007 06-292-105n IN (Benton) none aphids, bean leaf

beetles, corn

rootworm beetles,

Japanese beetles,

lady bugs, leaf

hoppers

2007-

2008

07-242-107n PR (Santa Isabel) Carla virus spider mites,

whiteflies

2008 08-071-107n CA (Tulare), IL

(Clinton), IN (Benton,

Parke), IA (Jefferson,

Story), MN (Dakota),

MS (Washington), NE

(York)

brown spot,

Cercospora leaf blight,

rust,

Septoria leaf spot

aphids, bean leaf

beetles, grasshoppers,

Japanese beetles,

lady bugs, stink bugs,

thrips, yellow-striped

armyworms

2008 08-121-102n IL (Schuyler, Stark), IN

(LaGrange), MO (Adair,

Henry), NE (Polk,

Seward, York), OH

(Fulton, Lucas)

Septoria brown aphids, grasshoppers,

lady bugs, thrips

2008 08-121-103n IA (Boone) none none

VII-C. Germination and Dormancy Evaluations

Changes in seed dormancy characteristics were evaluated by looking at the germination of

DAS-68416-4 seed compared with the non-transgenic control (Maverick) under warm and cold

conditions.

For the warm germination test, event DAS-68416-4 and control soybean seeds were placed at

25 seeds/plate into Petri dishes containing germination pads saturated with water and excess

water drained. The plates were placed at 25 oC and held under these conditions for 5 days.

Sixteen plates (400 seeds) were prepared per line. After five days, the number of nongerminated seeds was recorded.

For the cold germination test, seeds were planted at 100 seeds per half-flat filled with potting

soil. Flats were sub-watered and held at 10 oC for 7 days followed by exposure to 25 oC for 5

days, after which the number of non-germinated seed was recorded.

Data from each test were analyzed by ANOVA using a completely randomized design with four

replicates of 100 seeds per replicate. Data were transformed using the arcsine of the square root

of the number of germinated seeds divided by 100 for statistical analysis. Percent germination is

summarized in Table 18. Differences were considered statistically significant at alpha < 0.05.
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Table 18. Germination of DAS-68416-4 seeds under warm and cold conditions.

Replicate

Test Line 1 2 3 4 Mean

Warm DAS-68416-4 97 100 99 100 99.0

Warm Control 100 100 99 97 99.0

Cold DAS-68416-4 92 92 92 89 91.3

Cold Control 98 88 98 96 95.0

There were no significant differences in germination between DAS-68416-4 and control

soybeans in either the warm or cold germination experiments (Pr > F = 1.0 and 0.13,

respectively). These results indicate that the seed dormancy characteristics have not been

changed in DAS-68416-4 soybeans.

VII-D. Summary of Agronomic, Disease, and Pest Characteristics

Agronomic data evaluating plant growth characteristics throughout the growing season

demonstrate the equivalence of DAS-68416-4 soybean with conventional non-transgenic

soybean. Plant growth and phenotypic characteristics, response to ecological stressors as

indicated by susceptibility to disease and insect pressure, and germination and dormancy

characteristics were unchanged between DAS-68416-4 soybeans and conventional soybeans

across diverse environments. Therefore, these data support the conclusion that agronomic,

disease and pest characteristics of DAS-68416-4 soybean are not significantly different from that

of conventional soybeans, and there is no indication that DAS-68416-4 soybeans will pose an

increased plant pest risk.

VII-E. References

Benjamini, Y., Hochberg, Y. (1995) Controlling the false discovery rate: A practical and

powerful approach to multiple testing. J. Royal Statistical Soc. B, 57:289-300.
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VIII. Grain and Forage Composition

Compositional analysis was performed on soybean forage and grain to investigate the

equivalency between DAS-68416-4 soybean (sprayed with 2,4-D, glufosinate, 2,4-D +

glufosinate, or not sprayed with 2,4-D or glufosinate) and conventional soybean. Trials were

conducted at six test sites located within the major soybean-producing regions of the U.S and

Canada; with one site each in Iowa (Keokuk county), Illinois (Clinton county), Indiana (Parke

county), Nebraska (York county) and two sites in Ontario, Canada.

The herbicide treatments were identical to those used in studies for protein expression analysis

(Sections VI-A.3, VI-B.3) and agronomic Experiment 1 (Section VII-A.1). Herbicide treatments

were applied with a spray volume of approximately 20 gallons per acre (187 L/ha). These

applications were designed to replicate maximum label rate for commercial practices. 2,4-D was

applied as 3 broadcast over-the-top applications for a seasonal total of 3 lb ae/A. Individual

applications of 1.0 lb ae/A (1120 g ae/ha) were made at pre-emergence and approximately V4

and R2 growth stages. Glufosinate was applied as 2 broadcast over-the-top applications for a

seasonal total of 0.74 lb ai/A (828 g ai/ha). Individual applications of 0.33 lb ai/A and 0.41 lb

ai/A (374 and 454 g ai/ha) were made at approximately V6 and R1 growth stages.

Samples of soybean forage and grain were analyzed for nutrient content with a variety of tests

(OECD 2001). The analyses performed for forage included protein, fat, ash, moisture,

carbohydrate, acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium and

phosphorus. The analyses performed for grain included proximates (ash, total fat, moisture,

protein, cholesterol, carbohydrate), fiber, minerals, amino acids, fatty acid, vitamins, antinutrients.

The results of the nutritional analysis for soybean forage and grain were compared with values

reported in literature. A summary of the compositional data used for comparison can be found in

Appendix 6. Analysis of variance was also conducted across the field sites using a mixed model.

Entry was considered a fixed effect, and location, block within location, and location-by-entry

were designated as random effects. The significance of an overall treatment effect was estimated

using an F-test. Paired contrasts were made between DAS-68416-4 (unsprayed AAD-12), DAS68416-4 sprayed with glufosinate (AAD-12 + glufosinate), DAS-68416-4 sprayed with 2,4-D

(AAD-12 + 2,4-D), and DAS-68416-4 sprayed with both glufosinate and 2,4-D (AAD-12 + both

herbicides), and the control entry using t-tests.

Multiplicity is an issue when a large number of comparisons are made in a single study to look

for unexpected effects. Under these conditions, the probability of falsely declaring differences

based on comparison-wise p-values is very high (1-0.95number of comparisons). In this study there

were four comparisons per analyte (75 quantitated analytes), resulting in 300 comparisons made

in the across-site composition analysis. Therefore, the probability of declaring one or more false

differences based on unadjusted p-values was >99.99%.

One method to account for multiplicity is to adjust p-values to control the experiment-wise error

rate (probability that all declared differences are significant), but when many comparisons are

made in a study, the power for detecting specific effects can be reduced significantly. An

alternative with much greater power is to adjust p-values to control the probability that each

declared difference is significant. This can be accomplished using False Discovery Rate (FDR)
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procedures (Benjamini and Hochberg, 1995). Therefore the p-values were adjusted using FDR

to improve discrimination of true differences among treatments from random effects (false

positives).

VIII-A. Compositional Analysis of Soybean Forage

An analysis of the protein, fat, ash, moisture, carbohydrate, acid detergent fiber (ADF), neutral

detergent fiber (NDF), calcium and phosphorus in soybean forage samples from the control,

unsprayed AAD-12, AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides

was performed. A summary of the results across all locations is shown in Table 19 and Figure

37.

No statistical differences were observed in the across-site analysis between the control and

transgenic entries for protein, fat, ash, moisture, carbohydrates, ADF, NDF, calcium or

phosphorus.

Mean ash values across sites for AAD-12 + glufosinate and AAD-12 + both herbicides was

outside of the literature range as was the NDF value for AAD-12 + glufosinate and AAD-12 +

2,4-D. ADF values for all treatments including the non-transgenic control were also outside of

the literature values. Mean values were not significantly different between the non-transgenic

control and any transgenic entry for any proximate, fiber type, or mineral in forage.

Based on these compositional constituents, the forage from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 19. Summary of the proximate, fiber and mineral analysis of soybean forage (% dry

weight).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Proximate

Protein 11.2-24.7 0.805 19.1 19.0 19.4 18.9 18.6

(0.881,0.930) (0.666,0.819) (0.744,0.860) (0.441,0.634)

Fat 1.30-5.1 0.046 4.11 4.46 3.66 4.17 3.74

(0.216,0.403) (0.107,0.254) (0.844,0.908) (0.186,0.360)

Ash 6.7-10.8 0.092 10.6 10.1 11.1 10.2 12.3

(0.567,0.767) (0.546,0.741) (0.672,0.819) (0.051,0.151)

Moisture 73.5-81.6 0.569 77.8 78.5 78.4 77.8 77.8

(% fresh weight) (0.255,0.444) (0.330,0.539) (0.960,0.970) (0.976,0.979)

Carbohydrates 59.8-74.7 0.675 66.2 66.5 65.9 66.7 65.3

(0.830,0.902) (0.739,0.860) (0.641,0.808) (0.366,0.564)

Fiber

Acid Detergent 32.0-38.0 0.967 30.2 30.4 30.6 29.7 30.7

Fiber (ADF) (0.904,0.936) (0.797,0.875) (0.746,0.860) (0.740,0.860)

Neutral Detergent 34.0-40.0 0.375 34.4 34.7 33.1 32.0 34.5

Fiber (NDF) (0.877,0.930) (0.397,0.596) (0.135,0.297) (0.948,0.962)

Minerals

Calcium NR 0.246 1.39 1.36 1.40 1.38 1.43

(0.361,0.560) (0.664,0.819) (0.842,0.908) (0.178,0.352)

Phosphorus NR 0.957 0.263 0.266 0.269 0.266 0.265

(0.671,0.819) (0.442,0.634) (0.696,0.831) (0.754,0.860)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

NR = not reported

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 37. Summary of the proximate, fiber and mineral analysis of soybean forage (% dry

weight).

Percent dry-weight for all analytes, except moisture which was percent fresh-weight.

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded.

ADF

20

25

30

35

40

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Protein

5

10

15

20

25

30

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Total Fat

0.2

1.2

2.2

3.2

4.2

5.2

6.2

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Ash

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Carbohydrates

50

55

60

65

70

75

80

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Calcium

0.0

0.5

1.0

1.5

2.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Phosphorus

0.0

0.1

0.2

0.3

0.4

0.5

0.6

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

NDF

20

25

30

35

40

45

50

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Moisture

65

70

75

80

85

90

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B. Compositional Analysis of Soybean Grain

VIII-B.1. Proximates and Fiber

An analysis of the protein, fat, ash, moisture, cholesterol, carbohydrate, ADF, NDF and total

dietary fiber in soybean grain samples from the control, unsprayed AAD-12, AAD-12 +

glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides was performed. A summary of the

results across all locations is shown in Table 20 and Figure 38.

No statistical differences were observed in the across-site analysis between the control and

transgenic entries for the fat, ADF or total dietary fiber. However, ADF was slightly higher than

the literature range for the AAD-12 + 2,4-D entry.

Protein levels were significantly different in the across-site analysis based on the unadjusted

p-value for the unsprayed, AAD-12 + 2,4-D, and AAD-12 + both herbicides compared with the

control. However, after FDR adjustment, only the p-value for the AAD-12 + 2,4-D was

significant, and overall mean protein values for all treatments were within the reported literature

values, indicating that the differences were not biologically meaningful.

A significant unadjusted p-value was observed in the across site analysis of ash between the

control and the 2,4-D sprayed AAD-12 treatment, but no overall treatment effect or adjusted

p-value was observed. Ash values were also within the reported literature values, indicating that

the differences were not biologically meaningful.

Moisture levels were significantly different in the across-site analysis based on the unadjusted

p-value for the unsprayed, AAD-12 + 2,4-D, and AAD-12 + both herbicides compared with the

control. However, the overall treatment effect was not significant for moisture, only the

AAD-12 + 2,4-D treatment had a significant FDR-adjusted p-value, and the mean moisture

levels for all treatments were within the literature ranges. This indicated that the differences

were not biologically meaningful.

Cholesterol values were all <LOQ and no literature values were reported.

Carbohydrate levels were significantly different in the across-site analysis based on the

unadjusted p-value for the unsprayed, AAD-12 + glufosinate, and AAD-12 + 2,4-D compared

with the control. However, only the AAD-12 + 2,4-D treatment was significantly different from

the control based on the FDR adjusted p-value and all treatment means were within the reported

literature values, indicating equivalence to non-transgenic soybean.

NDF levels were significantly different in the across-site analysis based on the unadjusted pvalue for AAD-12 + glufosinate compared with the control, but this was not accompanied by a

significant adjusted p-value or an overall treatment effect. NDF across-site values were slightly

higher than the reported literature values for the AAD-12 + glufosinate and AAD-12 + 2,4-D

entries, but the differences were <9% compared with the non-transgenic control.

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 20. Summary of the proximate and fiber analysis of soybean grain (% dry weight).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Proximate

Protein 32.0-45.5 0.004 39.2 38.3 38.8 37.8 38.5

(0.009,0.051) (0.186,0.360) (0.0003,0.009) (0.035,0.122)

Fat 8.10-24.7 0.105 17.1 17.1 16.6 16.7 17.2

(0.877,0.930) (0.059,0.169) (0.142,0.305) (0.674,0.819)

Ash 3.89-6.99 0.315 4.92 5.04 5.04 5.10 5.07

(0.176,0.351) (0.175,0.351) (0.048,0.145) (0.099,0.240)

Moisture 4.70-34.4 0.066 14.9 14.1 14.3 13.7 14.0

% fresh weight (0.047,0.143) (0.122,0.276) (0.006,0.043) (0.037,0.124)

Cholesterol NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

Carbohydrate 29.6-50.2 0.010 38.8 39.6 39.6 40.3 39.3

(0.046,0.143) (0.044,0.138) (0.001,0.011) (0.241,0.432)

Fiber

Acid Detergent 7.81-18.6 0.561 17.8 17.6 18.0 18.8 18.1

Fiber (ADF) (0.772,0.868) (0.772,0.868) (0.190,0.362) (0.685,0.825)

Neutral Detergent 8.53-21.3 0.184 20.1 20.8 21.9 21.6 20.3

Fiber (NDF) (0.386,0.585) (0.042,0.134) (0.090,0.225) (0.754,0.860)

Total Dietary NR 0.770 31.6 31.7 31.7 32.1 32.5

Fiber (0.899,0.936) (0.897,0.936) (0.466,0.653) (0.286,0.482)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

NA = statistical analysis was not performed since a majority of the data was < LOQ.

NR = not reported.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 38. Summary of the proximate and fiber analysis of soybean grain.

Percent dry-weight for all analytes, except moisture which was percent fresh-weight.

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded. Grain was also analyzed for

cholesterol, but results were less than the limit of quantitation.

ADF

5.0

10.0

15.0

20.0

25.0

30.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Protein

25.0

30.0

35.0

40.0

45.0

50.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Total Fat

5.0

10.0

15.0

20.0

25.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Ash

2.0

4.0

6.0

8.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Moisture

0.0

5.0

10.0

15.0

20.0

25.0

30.0

35.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Carbohydrate

20.0

25.0

30.0

35.0

40.0

45.0

50.0

55.0

60.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Total Fiber

20.0

25.0

30.0

35.0

40.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

NDF

5.0

10.0

15.0

20.0

25.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B.2. Minerals

The analysis of the calcium, chromium, copper, iodine, iron, magnesium, manganese,

molybdenum, phosphorus, potassium, selenium, sodium and zinc in soybean grain samples from

the control, unsprayed AAD-12, AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both

herbicides was performed. A summary of the results across all locations is shown in Table 21

and Figure 39.

No statistical differences were observed in the across-site analysis between the control and

transgenic entries based on the unadjusted p-value for the chromium, copper, iodine, iron,

manganese, molybdenum, phosphorus, selenium and sodium (not detected).

Calcium had a significant difference in the across-site analysis based on the unadjusted p-value

for the AAD-12 + 2,4-D, but this was not associated with a significant FDR adjusted p-value or

overall treatment effect, and all treatment means fell within the literature range, indicating that

the difference was not biologically meaningful.

Magnesium levels were significantly different in the across-site analysis for the AAD12 + both

herbicides and AAD-12 + glufosinate compared with the control based on the unadjusted pvalues, but this was not associated with a significant FDR adjusted p-value or overall treatment

effect. Magnesium across site mean values were slightly lower than the reported literature

values, but the differences were small (<3%) in comparison to the control and all AAD-12

entries were closer to literature values compared with the control. The mean value for the

control is further from the literature range than any of the transgenic treatments, therefore by

including the control values as part of the range seen for non-transgenic varieties, all the

transgenic treatments fall within the range.

All AAD-12 entries had significantly higher potassium values compared with the control in the

across-site analysis. However, differences were small (<5%) in comparison to the control, and

all AAD-12 entries were closer to the literature range compared with the control. The mean

value for the control is further from the literature range than any of the transgenic treatments,

therefore by including the control values as part of the range seen for non-transgenic varieties, all

the transgenic treatments fall within the range.

A difference in zinc levels was significant in the across-site analysis based on the unadjusted

p-value for AAD-12 + both herbicides, however this was not accompanied by a significant

FDR-adjusted p-value or overall treatment effect, and the difference was small (<4%).

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 21. Summary of the mineral analysis of soybean grain (mg/100g dry weight).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Calcium 117-307 0.102 256 265 264 274 269

(0.174,0.351) (0.237,0.432) (0.010,0.057) (0.050,0.148)

Chromium NR 0.775 145 149 175 126 137

(ppb) (0.912,0.941) (0.468,0.653) (0.613,0.796) (0.855,0.916)

Copper NR 0.887 1.31 1.28 1.30 1.27 1.28

(0.534,0.728) (0.788,0.873) (0.367,0.564) (0.461,0.649)

Iodine NR 0.285 0.027 0.023 0.021 0.032 0.023

(0.430,0.632) (0.182,0.358) (0.348,0.551) (0.348,0.551)

Iron 5.54-11.0 0.917 8.15 8.46 8.95 8.53 8.59

(0.719,0.853) (0.353,0.552) (0.656,0.819) (0.608,0.796)

Magnesium 219-313 0.082 210 212 215 213 215

(0.437,0.634) (0.020,0.087) (0.143,0.305) (0.021,0.088)

Manganese NR 0.984 2.56 2.60 2.60 2.58 2.59

(0.608,0.796) (0.618,0.799) (0.781,0.873) (0.698,0.831)

Molybdenum NR 0.845 2165 2557 2462 2563 2284

(ppb) (0.353,0.552) (0.479,0.665) (0.346,0.551) (0.722,0.853)

Phosphorus 507-935 0.675 583 589 599 596 594

(0.630,0.804) (0.191,0.363) (0.272,0.469) (0.349,0.551)

Potassium 1868-2316 0.0005 1801 1876 1882 1883 1864

(0.0003,0.009) (0.0001,0.006) (0.0001,0.006) (0.001,0.019)

Selenium NR 0.490 490 523 520 511 418

(ppb) (0.626,0.802) (0.659,0.819) (0.758,0.861) (0.280,0.475)

Sodium NR NA <LOQ < LOQ <LOQ <LOQ <LOQ

Zinc NR 0.096 5.06 5.07 5.19 5.21 5.25

(0.868,0.926) (0.117,0.268) (0.074,0.197) (0.027,0.105)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

NR = not reported.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 39. Summary of the mineral analysis of soybean grain (mg/100 g dry weight).

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded. Grain was also analyzed for

sodium, but results were less than the limit of quantitation.

Iodine

0.00

0.01

0.02

0.03

0.04

0.05

0.06

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Iron

0.0

2.0

4.0

6.0

8.0

10.0

12.0

14.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Magnesium

100

150

200

250

300

350

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Manganese

0.0

1.0

2.0

3.0

4.0

5.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Phosphorus

450

550

650

750

850

950

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Molybdenum

500

1500

2500

3500

4500

5500

6500

7500

8500

9500

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Chromium

0

100

200

300

400

500

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Copper

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Calcium

0

100

200

300

400

500

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Zinc

2.0

4.0

6.0

8.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Selenium

100

300

500

700

900

1100

1300

1500

1700

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Potassium

1500

1600

1700

1800

1900

2000

2100

2200

2300

2400

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B.3. Amino Acids

An analysis of the following amino acids: alanine, arginine, aspartic acid, cysteine, glutamic acid

glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine,

threonine, tryptophan, tyrosine, and valine; in soybean grain samples from the control, unsprayed

AAD-12, AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides was

performed. A summary of the results across all locations is shown in Table 22 and Figure

40Figure 40. Summary of the amino acid analysis of soybean grain (% dry weight).

No statistical differences were observed between the control and transgenic entries for cysteine,

methionine, proline, tyrosine or tryptophan. The isoleucine level for AAD-12 + 2,4-D was

significantly different from the control based on the unadjusted p-value, but this was not

accompanied by a significant FDR-adjusted p-value or a significant overall treatment effect. The

levels of the remaining 12 amino acids were slightly lower (<7%) for two or more of the

AAD-12 entries compared with the control, but all fell within the literature range for nontransgenic soybean. All amino acids for all entries were within the literature ranges, indicating

that the differences were not biologically meaningful.

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 22. Summary of the amino acid analysis of soybean grain (% dry weight).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Alanine 1.51-2.10 0.003 1.74 1.70 1.70 1.69 1.71

(0.001,0.017) (0.004,0.033) (0.0003,0.009) (0.014,0.067)

Arginine 2.29-3.40 0.007 3.15 2.97 3.00 2.94 2.96

(0.004,0.033) (0.012,0.066) (0.001,0.015) (0.003,0.026)

Aspartic Acid 3.81-5.12 0.007 4.52 4.41 4.44 4.38 4.43

(0.004,0.033) (0.037,0.124) (0.0005,0.010) (0.014,0.067)

Cysteine 0.37-0.81 0.254 0.60 0.60 0.60 0.61 0.61

(0.637,0.808) (0.787,0.873) (0.900,0.936) (0.110,0.260)

Glutamic Acid 5.84-8.20 0.002 6.98 6.76 6.83 6.70 6.80

(0.001,0.015) (0.019,0.086) (0.0001,0.006) (0.006,0.043)

Glycine 1.46-2.00 0.001 1.74 1.69 1.70 1.69 1.70

(0.0004,0.009) (0.002,0.023) (0.0001,0.006) (0.001,0.017)

Histidine 0.88-1.22 0.003 1.09 1.06 1.07 1.05 1.07

(0.002,0.023) (0.014,0.067) (0.0002,0.007) (0.013,0.067)

Isoleucine 1.54-2.08 0.232 1.87 1.83 1.85 1.82 1.85

(0.100,0.241) (0.450,0.642) (0.042,0.134) (0.514,0.708)

Leucine 2.20-4.00 0.010 3.06 3.00 3.02 2.98 3.01

(0.007,0.046) (0.068,0.186) (0.001,0.011) (0.037,0.124)

Lysine 2.29-2.84 0.005 2.56 2.51 2.52 2.49 2.52

(0.004,0.034) (0.028,0.105) (0.0003,0.009) (0.022,0.093)

Methionine 0.43-0.68 0.433 0.56 0.55 0.55 0.55 0.55

(0.377,0.575) (0.245,0.438) (0.089,0.225) (0.742,0.860)

Phenylalanine 1.60-2.35 0.008 2.02 1.97 1.98 1.94 1.97

(0.014,0.067) (0.044,0.138) (0.0004,0.009) (0.027,0.105)

Proline 1.69-2.28 0.374 1.91 1.85 1.88 1.87 1.87

(0.059,0.169) (0.400,0.597) (0.155,0.324) (0.240,0.432)

Serine 1.11-2.48 0.063 1.99 1.95 1.95 1.91 1.93

(0.082,0.210) (0.115,0.268) (0.006,0.043) (0.021,0.088)

Threonine 1.14-1.89 0.001 1.62 1.57 1.58 1.55 1.57

(0.002,0.020) (0.008,0.048)(<0.0001,0.006) (0.002,0.022)

Tryptophan 0.36-0.67 0.330 0.43 0.43 0.43 0.43 0.42

(0.593,0.787) (0.981,0.981) (0.904,0.936) (0.095,0.235)

Tyrosine 1.02-1.61 0.449 1.36 1.34 1.35 1.33 1.33

(0.275,0.471) (0.517,0.708) (0.096,0.235) (0.153,0.321)

Valine 1.50-2.44 0.159 1.97 1.92 1.94 1.92 1.95

(0.032,0.116) (0.279,0.475) (0.038,0.124) (0.346,0.551)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 40. Summary of the amino acid analysis of soybean grain (% dry weight).

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded.

Aspartic Acid

3.0

3.5

4.0

4.5

5.0

5.5

6.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Arginine

2.0

2.2

2.4

2.6

2.8

3.0

3.2

3.4

3.6

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Histidine

0.7

0.8

0.9

1.0

1.1

1.2

1.3

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Leucine

1.5

2.0

2.5

3.0

3.5

4.0

4.5

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Alanine

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Cystine

0.2

0.3

0.4

0.5

0.6

0.7

0.8

0.9

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Glutamic Acid

5.0

6.0

7.0

8.0

9.0

10.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Glycine

1.2

1.4

1.6

1.8

2.0

2.2

2.4

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Isoleucine

1.0

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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Figure 40 (cont.) Summary of the amino acid analysis of soybean grain (% dry weight).

Methionine

0.2

0.3

0.4

0.5

0.6

0.7

0.8

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Valine

1.0

1.5

2.0

2.5

3.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Proline

1.0

1.5

2.0

2.5

3.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Serine

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Threonine

0.75

0.95

1.15

1.35

1.55

1.75

1.95

2.15

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Tryptophan

0.20

0.30

0.40

0.50

0.60

0.70

0.80

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Tyrosine

0.5

0.7

0.9

1.1

1.3

1.5

1.7

1.9

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Phenylanlanine

1.2

1.4

1.6

1.8

2.0

2.2

2.4

2.6

2.8

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Lysine

2.0

2.2

2.4

2.6

2.8

3.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B.4. Fatty Acids

An analysis of 22 fatty acids in soybean grain samples from the control, unsprayed AAD-12,

AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides was performed. A

summary of the results across all locations is shown in Table 23 and Figure 41.

The fatty acids 10:0 capric, 15:0 pentadecanoic, 15:1 pentadecenoic, 20:3 eicosatrienoic, 20:4

arachidonic, 8:0 caprylic, 12:0 lauric, 14:0 myristic, 14:1 myristoleic, 17:1 heptadecenoic, 18:3

gamma linolenic, and 20:2 eicosadienoic acids were analyzed and the results were <LOQ. The

fatty acids 16:0 palmitic, 17:0 heptadecanoic, and 20:1 eicosenoic were not significantly

different between the control and the AAD-12 entries, although 20:1 eicosenoic values were

lower than the reported literature values for AAD-12 + glufosinate and AAD-12 + both

herbicides. However, the differences were small (<5%) in comparison to the control.

The level of 16:1 palmitoleic was significantly different between the control and the unsprayed,

AAD-12 + glufosinate, AAD-12 + 2,4-D, and AAD-12 + both herbicides based on unadjusted pvalues. However, only the unsprayed AAD-12 entry had a FDR-adjusted p-value that was

significant for 16:1 palmitoleic. The 16:1 palmitoleic across-site value was lower for this

treatment compared with the reported literature values, but the difference was small (<13%) in

comparison to the non-transgenic control.

The level of 18:0 stearic was significantly different between the control and the unsprayed and

AAD-12 + glufosinate, based on unadjusted p-values. However, no significant differences were

observed based on the adjusted p-values or the overall treatment effect, and all entries were

within the reported literature values, indicating equivalence to non-transgenic soybean.

The level of 18:1 oleic was significantly different between the control and the unsprayed, AAD12 + glufosinate, AAD-12 + 2,4-D, and AAD-12 + both herbicides. However, 18:1 oleic levels

were within the reported literature values for all treatments, indicating equivalence to nontransgenic soybean.

The level of 18:2 linoleic was significantly different between the control and the unsprayed and

AAD-12 + 2,4-D, based on unadjusted p-values. However, no significant differences were

observed in the adjusted p-values or the overall treatment effect, and 18:2 linoleic levels were

within the reported literature values for all treatments, indicating equivalence to non-transgenic

soybean.

Levels of 18:3 linolenic were significantly different between each of the AAD-12 entries and the

control based on unadjusted p-values, and the adjusted p-values were also significant between

the unsprayed AAD-12 and AAD-12 + both herbicide treatment compared with the control.

Differences between the AAD-12 and control treatment were small (<6%) and all fell within the

literature range.

The level of 20:0 arachidic was significantly different between the control and the unsprayed,

AAD-12 + glufosinate, AAD-12 + 2,4-D, and AAD-12 + both herbicides based on unadjusted pvalues, and 20:0 arachidic also had significant differences in the across-site analysis in the

adjusted p-value for the unsprayed and AAD-12 + glufosinate treatments. However, 20:0
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arachidic levels were within the reported literature values for all treatments, indicating

equivalence to non-transgenic soybean.

The level of 22:0 behenic was significantly different between the control and the unsprayed,

AAD-12 + glufosinate, AAD-12 + 2,4-D, and AAD-12 + both herbicides based on unadjusted pvalues, and the level of 22:0 behenic also had a significant difference in the across-site analysis

in the adjusted p-value for the AAD-12 + glufosinate. However, there were no significant

overall treatment effect, and 22:0 behenic levels were within the reported literature values for all

treatments, indicating equivalence to non-transgenic soybean.

Of the 22 fatty acids investigated, all four AAD-12 entries were either statistically

indistinguishable from the control or within literature values for 21 of the fatty acids. In one case

(unsprayed AAD-12; 16:1 palmitoleic), the value was slightly under the minimum literature

values and statistically different from the control (<13% lower), however, all three sprayed

treatments were within the literature range.

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 23. Summary of the fatty acid analysis of soybean grain (% total fatty acids).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

8:0 Caprylic 0.15 NA < LOQ < LOQ < LOQ < LOQ < LOQ

10:0 Capric NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

12:0 Lauric 0.08-0.13 NA < LOQ < LOQ < LOQ < LOQ < LOQ

14:0 Myristic 0.07-0.24 NA < LOQ < LOQ < LOQ < LOQ < LOQ

14:1 Myristoleic 0.12-0.13 NA < LOQ < LOQ < LOQ < LOQ < LOQ

15:0 Pentadecanoic NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

15:1 Pentadecenoic NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

16:0 Palmitic 9.55-15.77 0.607 10.1 10.0 9.78 9.94 9.85

(0.625,0.802) (0.148,0.313) (0.455,0.644) (0.249,0.441)

16:1 Palmitoleic 0.09-0.19 0.029 0.097 0.085 0.088 0.087 0.089

(0.003,0.028) (0.038,0.124) (0.027,0.105) (0.029,0.109)

17:0 Heptadecanoic 0.09-0.15 0.640 0.111 0.114 0.113 0.114 0.113

(0.162,0.336) (0.331,0.539) (0.239,0.432) (0.296,0.493)

17:1 Heptadecenoic 0.07-0.09 NA < LOQ < LOQ < LOQ < LOQ < LOQ

18:0 Stearic 2.70-5.88 0.136 4.28 4.03 3.98 4.05 4.06

(0.048,0.145) (0.018,0.081) (0.060,0.169) (0.073,0.196)

18:1 Oleic 14.3-32.2 0.010 21.8 19.8 19.5 19.9 19.9

(0.004,0.033) (0.001,0.017) (0.006,0.043) (0.006,0.043)

18:2 Linoleic 42.3-58.8 0.145 50.3 52.5 51.9 52.6 52.0

(0.030,0.109) (0.116,0.268) (0.024,0.095) (0.087,0.222)

18:3 γ-Linolenic NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

18:3 Linolenic 3.00-12.52 0.022 7.83 8.23 8.15 8.10 8.21

(0.003,0.031) (0.016,0.073) (0.034,0.119) (0.004,0.034)

20:0 Arachidic 0.16-0.48 0.023 0.307 0.284 0.282 0.285 0.287

(0.007,0.045) (0.004,0.033) (0.009,0.052) (0.014,0.067)

20:1 Eicosenoic 0.14-0.35 0.683 0.143 0.140 0.136 0.141 0.138

(0.582,0.779) (0.201,0.380) (0.794,0.875) (0.327,0.538)

20:2 Eicosadienoic 0.08-0.25 NA < LOQ < LOQ < LOQ < LOQ < LOQ

20:3 Eicosatrienoic NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

20:4 Arachidonic NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

22:0 Behenic 0.28-0.60 0.053 0.305 0.288 0.285 0.288 0.288

(0.023,0.095) (0.008,0.048) (0.020,0.087) (0.020,0.087)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

NA = statistical analysis was not performed since a majority of the data was < LOQ.

NR = not reported.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 41. Summary of the fatty acid analysis of soybean grain (% total fatty acids).

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded. Grain was also analyzed for

8:0 Caprylic, 10:0 Capric, 12:0 Lauric, 14:0 Myristic, 14:1 Myristoleic, 15:0 Pentadecanoic,

15:1 Pentadecenoic, 17:1 Heptadecenoic, 18:3 gamma-Linolenic, 20:2 Eicosadienoic,

20:4 Arachidonic, 20:3 Eicosatrienoic, but levels were below the limit of quantitation.

16:0 Palmitic

8.0

12.0

16.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

16:1 Palmitoleic

0.05

0.10

0.15

0.20

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

17:0 Heptadecanoic

0.05

0.07

0.09

0.11

0.13

0.15

0.17

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

18:0 Stearic

0.0

1.0

2.0

3.0

4.0

5.0

6.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

18:1 Oleic

3.0

8.0

13.0

18.0

23.0

28.0

33.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

18:2 Linoleic

30.0

40.0

50.0

60.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

22:0 Behenic

0.20

0.25

0.30

0.35

0.40

0.45

0.50

0.55

0.60

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

18:3 Linolenic

0.0

3.0

6.0

9.0

12.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

20:0 Arachidic

0.00

0.50

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

20:1 Eicosenoic

0.0

0.1

0.2

0.3

0.4

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B.5. Vitamins

An analysis of vitamins in soybean grain samples from the control, unsprayed AAD-12, AAD-12

+ glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides was performed. A summary of

the results across all locations is shown in Table 24 and Figure 42.

No literature values were found for beta-tocopherol, delta-tocopherol, gamma-tocopherol,

Vitamin A, Vitamin B5, Vitamin B6, Vitamin B12, Vitamin C, Vitamin D and niacin in soybean

grain. Beta tocopherol, Vitamin A, Vitamin B12 and Vitamin D were all <LOQ. No differences

were observed between the control, unsprayed AAD-12 and the treated AAD-12 for Vitamin B1,

Vitamin B2, Vitamin B6, Vitamin C, Vitamin E or niacin. Of those vitamins with available

literature ranges, all treatments fell within these ranges with the exception of vitamin B2 where

values exceeded the range for all treatments including the non-transgenic control.

Delta-tocopherol levels were significantly different between the control and the AAD-12 +

glufosinate and AAD-12 + 2,4-D entries based on unadjusted p-values. However this was not

accompanied by a significant adjusted p-value or overall treatment effect. Gamma-tocopherol

was significantly different between the control and the unsprayed and AAD-12 + 2,4-D entries

based on unadjusted and adjusted p-values. However, gamma tocopherol was <11% higher for

the AAD-12 treatments compared with the non-transgenic control.

Vitamin B5 levels were significantly different between the control and the AAD-12 + glufosinate

entry based on the adjusted p-value. However this was not accompanied by a significant overall

treatment effect.

Folic acid was significantly different between the control and the unsprayed, AAD-12 + 2,4-D

and AAD-12 + both herbicides based on unadjusted p-values. Folic acid also had significant

differences in the adjusted p-values for two of the AAD-12 entries compared with the control.

However, folic acid levels were within the reported literature values for all treatments and the

AAD-12 entries differed from the non-transgenic control by <9%, indicating equivalence to nontransgenic soybean.

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 24. Summary of vitamin analysis of soybean grain (mg/kg dry weight).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Beta Carotene

(Vitamin A) NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

Vitamin B1 1.01-2.54 0.560 2.10 2.14 1.94 1.97 2.14

(Thiamin) (0.809,0.886) (0.312,0.517) (0.414,0.615) (0.787,0.873)

Vitamin B2 1.90-3.21 0.994 4.49 4.52 4.60 4.52 4.55

(Riboflavin) (0.933,0.952) (0.677,0.819) (0.922,0.948) (0.817,0.891)

Vitamin B3 NR 0.211 27.4 25.3 25.4 26.9 26.7

(Niacin) (0.060,0.169) (0.076,0.201) (0.698,0.831) (0.513,0.708)

Vitamin B5 NR 0.183 15.1 14.9 14.2 14.5 14.3

(Pantothenic acid) (0.601,0.794) (0.041,0.134) (0.170,0.350) (0.065,0.178)

Vitamin B6 NR 0.788 5.50 5.51 5.40 5.40 5.39

(Pyridoxine) (0.929,0.951) (0.439,0.634) (0.451,0.642) (0.420,0.620)

Vitamin B12 NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

Vitamin C NR 0.338 84.1 79.6 85.4 82.5 83.5

(0.126,0.281) (0.639,0.808) (0.580,0.779) (0.838,0.907)

Vitamin D NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

Vitamin E 1.90-61.7 0.182 14.8 15.1 14.5 15.9 14.3

(Alpha-tocopherol) (0.762,0.863) (0.611,0.796) (0.137,0.301) (0.439,0.634)

Beta-tocopherol NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

Delta-tocopherol NR 0.095 92.6 95.1 96.5 97.1 94.5

(0.142,0.305) (0.030,0.109) (0.013,0.067) (0.257,0.446)

Gamma-tocopherol NR 0.0004 153 164 158 169 157

(0.002,0.021) (0.117,0.268) (0.0005,0.006) (0.174,0.351)

Folic Acid 2.39-4.71 0.006 3.70 3.49 3.56 3.38 3.48

(0.011,0.060) (0.078,0.203) (0.0004,0.009) (0.008,0.048)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

NR = not reported.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 42. Summary of vitamin analysis of soybean grain (mg/kg dry weight).

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded. Grain was also analyzed for

Beta-Tocopherol, Vitamin A, Vitamin B12, and Vitamin D, but results were less than the limit of

quantitation.

Delta Tocopherol

0

20

40

60

80

100

120

140

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Thiamine Hydrochloride/Vitamin B1

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Panthotenic acid/Vitamin B5

0.0

5.0

10.0

15.0

20.0

25.0

30.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Riboflavin/Vitamin B2

1.0

2.0

3.0

4.0

5.0

6.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Gamma Tocopherol

100

150

200

Control No spray Glufosinate 2,4-D 2,4-D +Glufos

Pyridoxine HCl/Vitamin B6

2.0

3.0

4.0

5.0

6.0

7.0

8.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Vitamin C

0

25

50

75

100

125

150

175

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Alpha Tocopherol/Vitamin E

0.0

10.0

20.0

30.0

40.0

50.0

60.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Niacin/Vitamin B3

15

20

25

30

35

40

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Folic Acid

1.0

2.0

3.0

4.0

5.0

6.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B.6. Isoflavones

The analysis of isoflavones in soybean grain samples from the control, unsprayed AAD-12,

AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides was performed. A

summary of the results across all locations is shown in Table 25 and Figure 43.

The genistein and glycitein results were below the LOQ for the treated samples. Diadzin levels

were significantly different between the control and the AAD-12 + both herbicides entries based

on unadjusted and adjusted p-values. However, the overall treatment effect was not significant.

Although there are no reported literature values, the AAD-12 + both herbicides treatment was

<9% different from the non-transgenic control. Genistin levels were significantly different

between the control and the AAD-12 + both herbicides entries based on unadjusted and adjusted

p-values. However, the overall treatment effect was not significant. Although there are no

reported literature values for genistin, the AAD-12 treatments were <9% different compared with

the non-transgenic control. Glycitin values were significantly different between the control and

the AAD-12 + both herbicides based on unadjusted and adjusted p-values. While there were no

reported literature values for glycitin, all AAD-12 entries were <13% different compared with

the non-transgenic entry. In addition, all total isoflavone aglycone equivalents were within

reported literature ranges.

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 25. Summary of isoflavone analysis of soybean grain (μg/g).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Daidzein NR NA <LOQ <LOQ <LOQ <LOQ < LOQ

Daidzine60.0-2454f0.068 1085 1103 1112 1128 1179

(0.584,0.779) (0.391,0.589) (0.187,0.360) (0.007,0.045)

Genistein NR NA <LOQ < LOQ < LOQ < LOQ < LOQ

Genistine144-2837f0.069 1282 1321 1327 1357 1389

(0.292,0.490) (0.220,0.408) (0.052,0.152) (0.007,0.044)

Glycitein NR NA < LOQ < LOQ < LOQ < LOQ < LOQ

Glycitine15.3-310f

0.032 253 267 270 268 285

(0.142,0.305) (0.076,0.201) (0.121,0.274) (0.002,0.021)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

e Expressed as aglycone equivalent.

f Ranges represent total aglycone equivalents.

NA= statistical analysis was not performed since a majority of the data was < LOQ.

NR = not reported.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).
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Figure 43. Summary of isoflavone analysis of soybean grain (μg/g).

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded. Grain was also analyzed for

genistein and glycitein, but results were less than the limit of quantitation.

Total Daidzein

0

500

1000

1500

2000

2500

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Total Genisetin

0

500

1000

1500

2000

2500

3000

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Daidzin

0

500

1000

1500

2000

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Genistin

0

500

1000

1500

2000

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Glycitin

0

100

200

300

400

500

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Total Glycitein

0

200

400

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-B.7. Antinutrients

An analysis of anti-nutrients in soybean grain samples from the control, unsprayed AAD-12,

AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides was performed. A

summary of the results across all locations is shown in Table 26 and Figure 44.

No statistical differences were observed between the control and transgenic entries for lectin,

phytic acid, or trypsin inhibitor. These three anti-nutrients were also all within the literature

ranges, indicating equivalence to non-transgenic soybean.

Raffinose was significantly lower (<10%) for the AAD-12 + glufosinate treatment compared

with the control based on unadjusted p-values. Raffinose was not significantly different in the

across-site analysis based on the adjusted p-value or the overall treatment effect. Raffinose

levels were also within the reported literature values for all treatments, indicating equivalence to

non-transgenic soybean.

Stachyose was significantly different between the control and the AAD-12 + glufosinate entry

based on the unadjusted p-value. Stachyose levels were not significant different in the acrosssite analysis based on the adjusted p-value or the overall treatment effect. Stachyose levels were

also within the reported literature values for all treatments, indicating equivalence to nontransgenic soybean.

Anti-nutrient analysis for lectin, phytic acid, raffinose, stachyose and trypsin inhibitor were all

within the reported literature values, and the two significant differences based on unadjusted

p-values had lower levels of anti-nutrients for the AAD-12 treatments compared with the control.

Based on these compositional constituents, the grain from DAS-68416-4 soybean was

substantially equivalent to that of non-transgenic soybean.
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Table 26. Summary of anti-nutrient analysis of soybean grain (% dry weight).

P-value for Treatment Means

Overall (P-value,c Adj. Pd

)

Analyte

Literature

Valuesa

Treatment

Effectb Control Unsprayed

Sprayed

Glufosinate

Sprayed

2,4-D

Sprayed

Both

Lectin 0.11-9.04 0.552 2.18 2.74 2.84 2.98 3.09

(H.U./mg) (0.333,0.540) (0.254,0.444) (0.176,0.351) (0.124,0.277)

Phytic Acid 0.63-2.74 0.725 1.20 1.20 1.22 1.21 1.25

(0.949,0.962) (0.673,0.819) (0.896,0.936) (0.253,0.444)

Raffinose 0.212-0.661 0.111 0.344 0.339 0.310 0.317 0.315

(0.753,0.860) (0.033,0.118) (0.082,0.210) (0.062,0.173)

Stachyose 1.2-3.5 0.217 2.42 2.34 2.23 2.28 2.32

(0.378,0.575) (0.027,0.105) (0.105,0.253) (0.231,0.425)

Trypsin Inhibitor 19.6-184 0.435 25.3 27.2 24.7 24.9 25.3

(TIU/mg) (0.204,0.383) (0.657,0.819) (0.748,0.860) (0.973,0.979)

a Combined range from Appendix 6.

b Overall treatment effect estimated using an F-test.

c Comparison of the transgenic treatments to the control using t-tests.

d

P-values adjusted using a False Discovery Rate (FDR) procedure.

Bolded mean values are outside of the reported literature range.

Bolded P-values are significant (<0.05).

Figure 44. Summary of anti-nutrient analysis of soybean grain (% dry weight).

Values at each location shown: diamond = IA, square = IL, triangle = IN, X = NE, star = ON1,

and circle = ON2. Literature ranges (Appendix 6) are shaded.

Phytic Acid

0.0

0.5

1.0

1.5

2.0

2.5

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Lectin

0.0

2.0

4.0

6.0

8.0

10.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Stachyose

1.0

2.0

3.0

4.0

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Trypsin Inhibitor

15

65

115

165

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos

Raffinose

0.0

0.2

0.4

0.6

0.8

Control No spray Glufosinate 2,4-D 2,4-D +

Glufos
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VIII-C. Summary of Grain and Forage Composition

The composition of DAS-68416-4 soybean was either statistically indistinguishable from the

non-transgenic control, <13% different from the non-transgenic control, or within the literature

range for non-transgenic soybean. Plots of the composition results do not indicate any

biologically meaningful treatment-related compositional differences among unsprayed AAD-12,

AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both herbicides soybean and the control

soybean line.

In conclusion, unsprayed AAD-12, AAD-12 + glufosinate, AAD-12 + 2,4-D and AAD-12 + both

herbicides composition results confirm the substantial equivalence of DAS-68416-4 soybean and

conventional soybean.

VIII-D. References

Benjamini, Y., Hochberg, Y. (1995) Controlling the false discovery rate: A practical and

powerful approach to multiple testing. J. Royal Statistical Soc. B, 57:289-300.

OECD (2001) Consensus Document on Compositional Considerations for New Varieties of

Soybean: Key Food and Fee Nutrients and Anti-Nutrients.
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IX.Environmental Consequences and Impact on Agronomic Practices

IX-A. Mode of Action of the AAD-12 Protein

Expression of the AAD-12 protein in transgenic crops provides tolerance to the herbicide 2,4-

dichlorophenoxyacetic acid (2,4-D) by catalyzing the conversion of 2,4-D to 2,4-dichlorophenol

(DCP) (Müller et al., 1999; Westendorf et al., 2002 and 2003; Wright et al., 2007), a herbicidally

inactive compound.

AAD-12 is also 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.

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,

therefore AAD-12 does not provide tolerance to commercially-available chiral phenoxy acid

herbicides.

Figure 45. Degradation reaction of 2,4-D catalyzed by AAD-12.

O

O

OH

Cl Cl

2,4-D

OH

Cl Cl AAD-12

DCP

IX-A.1. Field Efficacy

The efficacy of DAS-68416-4 soybean for the detoxification of 2,4-D and subsequent protection

from injury caused by this compound has been characterized in field studies conducted in 2007

and 2008. Trials were designed to evaluate the preemergence (2008 only) and postemergence

tolerance to 2,4-D. The preemergence trials were conducted as a randomized complete block

design and postemergence trials were a split plot design with application timing as the main plots

and herbicide rates as the sub plots. Experimental units consisted of single-row plots for

postemergence trials and two-row plots for preemergence trials, approximately 3 m in length and

all trials contained three replications. Postemergence trials received an application of glufosinate

at 0.41 lb ae/A at the V1 growth stage on all DAS-68416-4 plots to remove unintended nulls

prior to application of herbicide treatments. All herbicide applications were made with gaspressurized small-plot spray equipment delivering approximately 140 – 190 l/ha of spray

volume. The formulation of 2,4-D used was a 456 g ae/liter dimethylammonium salt (DMA).

Visual injury ratings were taken approximately 7 days after crop emergence in the case of

preemergence trials and 7 days after application of herbicide treatments in the case of

postemergence trials. Ratings were taken on a 0 to 100 scale which reflects a visual composite
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of all injury symptoms observed across all plants in a plot, with 0 = no injury as compared to

untreated plots and 100 = death of all plants.

Tolerance to 2,4-D

In 2007, data from a field study in Indiana indicated excellent tolerance of DAS-68416-4

soybean to 2,4-D at rates ranging from 0.5 to 4.0 lb ae/A applied at either the V4 or R2 growth

stages. At 7 days after the V4 application mean injury to DAS-68416-4 soybeans ranged from 0

to 8% as the 2,4-D rate increased from 0.5 to 4.0 lb ae/A. Injury to the non-transgenic Maverick

soybean from the same treatments ranged from 58 to 85% injury. Similar evaluations after the

R2 application produced mean injury ranging from 0 to 5% for DAS-68416-4 soybeans and 32 to

72% for non-transgenic Maverick. Injury symptoms to the non-transgenic Maverick soybean

consisted mainly of stem and leaf malformation, growth inhibition, and tissue necrosis.

Further field evaluations of 2,4-D tolerance were conducted in 2008. Studies examining the

response of DAS-68416-4 soybeans and non-transgenic Maverick soybeans to applications of

2,4-D DMA preemergence and at the V2-V3, V6 and R2 growth stages were conducted at

locations in Mississippi, Indiana, and Minnesota.

Averaged across the three locations, preemergence application of 2,4-D DMA to DAS-68416-4

resulted in ≤2% injury regardless of application rate (Table 27). The same treatments caused 34

to 60% injury to non-transgenic Maverick. Injury from pre-emergence applications of 2,4-D

consisted of stand loss and reduction in growth.

Table 27. DAS-68416-4 soybean tolerance to preemergence applications of 2,4-D, 2008.

Herbicide Ratea

Application

Stageb

Percent Plant Injuryc

DAS-68416-4 Maverick

2,4-D amine 1.0 lb ae/A Pre-emergence 2 ns 34 b

2,4-D amine 2.0 lb ae/A Pre-emergence 2 ns 58 b

2,4-D amine 4.0 lb ae/A Pre-emergence 0 ns 63 a

a

ae/A = acid equivalent/acre

b Application stage in terms of soybean plant growth development.

c Ratings were taken approximately 7 days after application. Means within each column followed by the

same letter are not significantly different as determined by restricted maximum likelihood methods for

mixed models and Tukey, or for unbalanced data, Tukey-Kramer HSD test (0.05). ns indicates no

significant differences.

The most evident symptoms from post-emergence 2,4-D treatments to non-transgenic Maverick

were stem twisting, leaf malformation, and necrosis at this evaluation timing, with overall injury

ranging from 60 to 93% depending on application rate and timing. Injury to DAS-68416-4

soybeans (Table 28) was <5% and consisted almost entirely of small necrotic patches on the

leaves observed at the 2.0 to 4.0 lb ae/A rates and was confined to the leaves near the top of the

canopy a the time of application. No injury was observed on newly emerging leaves. Sequential

applications of 2,4-D at 1.0 lb ae/A followed by a second application of 1.0 lb ae/A caused no

injury to DAS-68416-4 soybeans, but caused 89% injury to non-transgenic Maverick when

applied at V2 followed by V6, and 95% injury when applied at V6 followed by R2.
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Table 28. DAS-68416-4 soybean tolerance to post-emergence applications of 2,4-D, 2008.

Herbicide Ratea

Application

Stageb

Percent Plant Injuryc

DAS-68416-4 Maverick

2,4-D amine 1.0 lbs ae/A V2-V3 0 ns 81 b

2,4-D amine 2.0 lbs ae/A V2-V3 1 ns 90 ab

2,4-D amine 4.0 lbs ae/A V2-V3 5 ns 93 a

2,4-D amine 1.0 lbs ae/A V6 0 b 67 cd

2,4-D amine 2.0 lbs ae/A V6 0 b 81 abc

2,4-D amine 4.0 lbs ae/A V6 4 a 86 ab

2,4-D amine 1.0 lbs ae/A R2 0 b 60 a

2,4-D amine 2.0 lbs ae/A R2 1 ns 69 c

2,4-D amine 4.0 lbs ae/A R2 2 ns 74 bc

2,4-D amine 1.0 f/b 1.0 lbs ae/A V2 f/b V6 0 ns 89 b

2,4-D amine 1.0 f/b 1.0 lbs ae/A V6 f/b R2 0 ns 95 a

a

ae/A = acid equivalent/acre

b Application stage in terms of soybean plant growth development.

c Ratings were taken approximately 7 days after application. Means within each columns followed by the

same letter are not significantly different as determined by restricted maximum likelihood methods for

mixed models and Tukey, or for unbalanced data, Tukey-Kramer HSD test (0.05). ns indicates no

significant differences.

Tolerance to Glufosinate

Since DAS-68416-4 soybeans also contain the pat gene, the post-emergence tolerance to

glufosinate as the 183 g ae/liter ammonium salt was also evaluated in trials during 2007 and

2008 at the same locations as the 2,4-D tolerance studies. Trial methods and evaluations were

similar to those utilized for 2,4-D studies.

In 2007, field testing of applications of glufosinate to DAS-68416-4 soybeans at the V4 stage of

development resulted in 3, 10, and 22% injury 7 days after application of 0.375, 0.75, and

1.5 lbs ae/A (420, 840, and 1680 g ae/ha), respectively. The same applications to nontransformed Maverick resulted in 78, 90, and 98% injury. Injury symptoms were mainly of

chlorosis, necrosis, and growth inhibition, typical of injury normally observed from glufosinate.

Field trials with glufosinate in 2008 demonstrated results consistent with the 2007 data (Table

29). A similar dose response of DAS-68416-4 soybean to glufosinate was noted, regardless of

soybean growth stage at application. Glufosinate injury to non-transformed Maverick ranged

from 54 to 98% depending on application rate and timing.
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Table 29. DAS-68416-4 soybean tolerance to postemergence applications of glufosinate,

2008.

Application

Stageb

Percent Plant Injuryc

Herbicide Ratea DAS-68416-4 Maverick

glufosinate 0.366 lbs ae/A V2-V3 2 b 91 ns

glufosinate 0.73 lbs ae/A V2-V3 9 b 97 ns

glufosinate 1.46 lbs ae/A V2-V3 19 a 98 ns

glufosinate 0.366 lbs ae/A V6 3 b 78 b

glufosinate 0.73 lbs ae/A V6 9 b 88 ab

glufosinate 1.46 lbs ae/A V6 16 a 92 a

glufosinate 0.366 lbs ae/A R2 2 b 54 a

glufosinate 0.73 lbs ae/A R2 3 b 77 b

glufosinate 1.46 lbs ae/A R2 18 b 90 ab

glufosinate 0.366 f/b 0.366 lbs ae/ha V2 f/b V6 5 a 98 a

glufosinate 0.366 f/b 0.366 lbs ae/ha V6 f/b R2 1 b 87 a

a

ae/A= acid equivalent/acre

b Application stage in terms of soybean plant growth development.

c Ratings were taken approximately 7 days after application. Means within each column followed by the

same letter are not significantly different as determined by restricted maximum likelihood methods for

mixed models and Tukey, or for unbalanced data, Tukey-Kramer HSD test (0.05). ns indicates no

significant differences.

Summary of Herbicide Tolerance

For DAS-68416-4 soybeans, the proposed maximum single application rate of 2,4-D will be

1.0 lb ae/A (see section IX-E.2). Results of field testing indicate that DAS-68416-4 soybeans

provide acceptable tolerance to 2,4-D at rates of at least two times this proposed maximum use

rate.

The maximum use rate of glufosinate on herbicide-tolerant soybeans is 2 applications at

0.366 lb ae/A. Results of field testing indicate that DAS-68416-4 soybeans provide acceptable

tolerance to glufosinate at rates of at least two times this rate.

IX-B. Weediness Potential

Commercial soybean varieties in the United States are not considered weeds and are not effective

in invading established ecosystems.

Cultivated soybean seed rarely displays any dormancy characteristics and only under certain

environmental conditions grows as a volunteer in the year following cultivation. If this should

occur, volunteers do not compete well with the succeeding crop, and can easily be controlled

mechanically or chemically. The soybean plant is not weedy in character. In North America,

Glycine max is not found outside of cultivation. In managed ecosystems, soybean does not

effectively compete with other cultivated plants or primary colonisers (OECD, 2000).

The introduction of aryloxyalkanoate herbicide-tolerance into soybean will not alter the

weediness characteristics of soybean. Agronomic properties of DAS-68416-4 soybean related to

weediness, such as germination, emergence, seedling vigor, and response to environmental
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stressors, have been shown to be identical to conventional soybean. These findings demonstrate

that DAS-68416-4 soybean do not have increased weediness potential relative to conventional

soybean.

IX-C. Gene Flow Assessment

IX-C.1. Vertical Gene Flow Assessment

Soybean is considered to be a self-pollinated species, although natural crossing can occur

(OECD, 2000) at low rates. The level of cross pollination can vary as a function of growing

conditions, spatial arrangement of plants, and genotype. Cross pollination levels ranging from

0.09% up to 3.6% in adjacent rows have been reported (Beard and Knowles, 1971; Ahrent and

Caviness, 1994), but outcrossing frequency rapidly declines with distance. Cross pollination

rates have been reported to be <0.1% at distances of >5 m (Caviness, 1966; Ray et al., 2003).

Cultivated soybeans can cross only with members of its subgenus Soja. Wild soybean species of

the subgenus Soja are native to Korea, Japan, Taiwan, northeastern China, and areas of the

former USSR, but do not exist naturally in the United States (OECD, 2000). Therefore there is

no potential for gene flow from DAS-68416-4 soybeans to wild soybean relatives in the United

States.

IX-C.2. Horizontal Gene Flow Assessment

There is no known mechanism for, or definitive demonstration of, DNA transfer from plants to

microbes (Connor et al., 2003). Even if such a transfer were to take place, transfer of the aad-12

gene from DAS-68416-4 soybean would not present a human health or plant pest risk, based on the

safety data presented in this petition. The gene encoding the AAD-12 protein is from a naturally

occurring soil bacterium, Delftia acidovorans, and is already present in nature. Transfer recipients

would, therefore, not pose a greater plant pest risk than the environmentally prevalent wild type

microbes from which the genes originated.

IX-D. Current US Agronomic Practices for Soybeans

IX-D.1. Soybean Production

Processed soybeans are the largest source of protein feed and the second largest source of

vegetable oil in the world. The United States is the world's leading soybean producer and

exporter. Farm value of U.S. soybean production in 2007/08 was $27 billion, the second-highest

value among U.S.-produced crops, trailing only corn. Soybean and soybean product exports

accounted for 43 percent of U.S. soybean production in 2007/08. Soybeans are about 90 percent

of U.S. total oilseed production, while other oilseeds—such as cottonseed, sunflower seed,

canola, and peanuts—account for the remainder (USDA ERS, 2008).

A total of 75.7 million acres were planted to soybeans in the United States in 2008 (USDA

NASS, 2008). Of these planted acres, about 74.6 million acres were harvested, valued at $27.4

billion. The majority of these soybeans were grown within the North Central states. About 80%

of the planted soybean acres and 80% of the harvested soybean acres were concentrated in 11
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states – Iowa, Illinois, Minnesota, Indiana, Missouri, Nebraska, Ohio, South Dakota, North

Dakota, Kansas, and Arkansas.

IX-D.2. Weeds in Soybean

Weeds in soybean compete with the crop for light, nutrients and soil moisture. When weeds are

uncontrolled for the entire season, yield losses can exceed 68 to 75% (Krausz et al. 2001; Dalley

et al. 2002). Hartzler and Pringnitz (2000) define the critical period of competition in soybeans

as the point of time when weeds that emerge with the crop begin to impact yields. Most studies

have found that soybean yields are protected if weeds are controlled before they reach a height of

6 to 8 inches. The critical period varies widely, depending upon weed species and densities,

environmental conditions, and cultural practices.

To maximize soybean yields, weeds must be removed or controlled when they are no more than

6 inches tall (Loux et al. 2006). Field trials conducted in Ohio in 2001 found that applying

glyphosate when weeds are already 9 to 12 inches tall resulted in a 6-10% yield loss due to weed

competition prior to control of the weeds (Loux et al. 2006). Dalley et al. (2002) studied the

effect of row width and weed competition on soybean yield in 1998-2001. They found that weed

competition could reduce soybean yields by the time weeds reached 6, 6, and >12 inches in

height in 7.5, 15, and 30 inch rows, respectively.

The most common weed problems in soybeans are the annual grass and broadleaf weeds (see

Table 30). Some fields are also infested with perennial weeds (quackgrass, Johnsongrass,

Canada thistle, others) which are more difficult to control since they can reproduce from seed or

underground rhizomes.
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Table 30. Common troublesome weeds in soybeans in 2006-2008.

(Data from DMR-Kynetec).

Weed Species

Total Soybean Acres Treated1

2006 2007 2008

Annual Broadleaf Weeds

Lambsquarters, Common 21,859,614 24,459,895 28,242,972

Velvetleaf 23,820,731 23,373,573 26,786,349

Pigweed, Redroot 21,093,224 21,788,121 26,715,150

Cocklebur, Common 23,657,980 22,389,376 23,962,063

Waterhemp, Common 18,399,609 15,970,794 21,364,980

Ragweed, Giant 13,369,296 14,684,000 16,565,209

Marestail 4,044,060 5,382,190 11,257,267

Morningglory Spp. 10,711,087 11,432,904 11,011,185

Ragweed, Common 9,417,252 9,438,871 9,518,051

Sunflower, Wild 5,558,526 5,759,216 5,709,292

Kochia 4,859,759 3,671,795 5,317,528

Smartweed Pennsylvania 2,366,851 1,835,825 3,529,114

Waterhemp, Tall 2,301,380 2,926,358 3,826,647

Horseweed 2,188,359 3,159,712 3,470,274

Mustard, Wild 2,019,346 1,975,291 2,688,590

Sicklepod 2,024,031 1,650,086 2,535,829

Sida, Prickly 1,639,261 1,567,275 2,432,701

Sunflower, Volunteer 1,089,460 1,007,691 1,913,860

Chickweed 1,652,712 1,259,096 1,823,638

Nightshade, Black 1,766,649 1,277,416 1,385,751

Buckwheat, Wild 1,167,746 855,879 1,331,675

Pigweed, Smooth 188,160 801,569 1,322,732

Annual Grass Weeds

Foxtail Spp. 24,409,043 18,489,746 18,446,420

Foxtail, Giant 11,817,612 17,513,493 17,804,622

Foxtail, Yellow 10,870,761 11,217,512 13,947,018

Foxtail, Green 5,629,880 7,109,316 7,610,855

Crabgrass 5,170,684 5,928,919 7,424,879

Barnyardgrass 4,189,156 3,967,425 3,805,391

Corn, Volunteer 2,292,705 2,088,371 3,704,330

Oat, Wild 1,792,389 1,478,890 2,886,300

Cupgrass, Woolly 1,765,244 2,470,437 2,108,135

Shattercane 2,408,592 2,715,388 1,879,416

Panicum, Fall 2,251,014 2,241,088 1,852,417

Perennial / Biennial Weeds

Johnsongrass 10,152,393 11,057,825 10,368,155

Thistle, Canada 4,123,437 3,584,676 4,840,383

Quackgrass 2,628,187 2,570,688 2,786,633

Dandelion 1,578,579 1,528,332 2,154,008

Thistle 1,479,038 647,315 1,513,566

1 Total soybean acres in 2006, 2007, and 2008 was 75.5, 64.7, and 75.7 million acres, respectively (USDA

NASS, 2008). However, the total soybean herbicide-treated acreage is much more, due to multiple sprays

on each acre.
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The economic threshold for weeds is the density of a weed population at which control is

economically justified due to the potential for yield reduction, quality loss, harvesting

difficulties, or other problems that weeds may cause (Penn State Agronomy Guide, 2009-2010).

Broadleaf and grass weeds compete at different levels of intensity depending upon the

competitiveness of the crop, tillage system, environmental conditions, and other weeds present.

Generally, broadleaf weeds are more damaging to a broadleaf crop, while grass weeds are more

competitive in a grass crop. Crop yield loss information is available for certain single weed

species growing with soybeans in the U.S. Midwest (Table 31).

Table 31. Yield reduction from specific weed species in soybeans.

(from Penn State Agronomy Guide, 2009-2010).

Weed Species

Percent soybean yield reduction1

1 2 4 6 8 10

Weeds per 100 feet of row

Cocklebur 1 2 4 6 8 10

Pigweed or Lambsquarters 2 4 6 10 15 20

Shattercane (5-8/clump) 2 5 8 11 14 17

Giant foxtail 15 25 80 300 400 600

Velvetleaf 1 3 6 10 13 16

Smartweed 2 4 6 10 15 20

Volunteer corn 1 2 3 4 5 6

1

Interference data are from Stoller et al., 1985, Reviews of Weed Science; E. L. Knake and

F. W. Slife, 1962, Weeds 10:26; and E. L. Werner and W. S. Curran, 1995, Proc. NEWSS

49:23.

IX-D.3. Weed Management in Soybean

In 2005, USDA NASS surveyed 17 states (AR, IL, IN, IA, KS, KY, LA, MI, MN, MS, MO, NE,

NC, OH, SD, TN, VA; about 90% of the total U.S. soybean acreage) and found that 98% of the

planted soybean acreage was treated with herbicides, an indication of the intensive weed

management that is used in U.S. soybeans (USDA NASS, 2006). In 2006, 95% of the planted

soybean acres received an average of 2.2 herbicide applications to control weeds (USDA ERS

ARMS, 2006). Many herbicides are registered for pre-plant, pre-emergent and/or post-emergent

application to selectively control most weed species commonly found in soybeans. In general,

soybeans sometimes receive a soil applied or burndown herbicide prior to planting or at plant,

but always receive a post-emergence herbicide application. In 2005, the most widely used

herbicide in soybeans was glyphosate, driven by the high adoption of glyphosate tolerant

soybeans (USDA NASS, 2006). Soybeans received an average of 1.5 applications of glyphosate

on 91% of the acres planted in 2005. Herbicides such as 2,4-D (6% of acres), chlorimuron (4%

of acres), and trifluralin (23% of acres) were applied on significantly fewer acres. All other

herbicides applied in soybeans that year comprised 3% or less of the total acres planted.

Integrated weed management (IWM) programs advocate the use of a combination of preventive,

cultural, mechanical and chemical tools to keep weed pressure below threshold levels that reduce

yield and profits (Knezevic, 2002). Herbicides are only one of several tools available for

growers to consider using in an IWM approach. Herbicide-tolerant soybeans represent another

tool that can be used in an IWM program.
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As with any technology, some concerns have been raised about using herbicides and herbicidetolerant crops as part of an IWM program (Knezevic, 2002). Some of these concerns include

selection for herbicide-resistant weeds, shifts in weed species, drift to sensitive non-target crops

or non-agricultural habitats, herbicide persistence in the soil resulting in carryover to the next

crop, herbicide misapplications, and over-reliance on herbicides to control weeds.

Weed management decisions are difficult decisions for growers since no one tool will effectively

control all possible weed problems. In general, growers need to implement management

practices that limit the introduction and spread of weeds, help the crop to compete with weeds,

and not allow weeds to adapt. The combination of weed control practices that a grower chooses

is dependent upon the weed spectrum, level of infestation, soil type, cropping system, weather,

and time and labor available for the treatment option.

IX-D.4. Crop Rotation and Tillage Practices

In 2006, 71% of U.S. soybean acres were grown in rotation after corn, 13% were grown after

soybeans, and 16% were grown after small grains, cotton, fallow or other crops (USDA ERS

ARMS, 2006). Crop rotation is a widespread management practice that has been recognized and

exploited for centuries to increase crop yields (Lauer, 2007). In the Midwestern U.S., a cornsoybean rotation produces at least 10% greater yields in both crops, and sometimes as much as

19% higher corn yields. The exact mechanism for the rotation effect is unknown, but may be

influenced by increases in organic matter and soil fertility, as well as management of diseases,

insects, and weeds.

The introduction of numerous herbicides in the decades following launch of 2,4-D in the

mid-1940s allowed reduced and conservation tillage systems to become more feasible and

popular (Givens et al., 2009). The introduction of glyphosate-resistant (GR) crops in 1996

brought a technology that enabled many producers to adopt reduced-tillage production systems.

By 2002, only 17% of U.S. soybeans were produced under conventional tillage (<15% residue)

systems (USDA ERS ARMS 2002). Some form of conservation tillage was being used on the

other 83% of U.S. soybean acres, as follows: 18% reduced tillage (15-30% residue), 32% mulch

till, 1-2% ridge till, and 31% no-till. Increased yields coupled with decreases in erosion and

water runoff and lower fuel use and fuel cost strongly support the premise that soybean produced

with conservation tillage in any production system is more economically and environmentally

sustainable (CAST, 2009). In fact, considering the generally accepted 30% minimum residue

cover and the 3 to 5 tons/acre/year tolerable soil loss for a sustainable soybean production system

(CAST 2009), conservation tillage is the only system that seems to support sustainable soybean

production from an environmental standpoint.

IX-E. Potential Impact on Agronomic Practices

IX-E.1. Potential Impact on Cultivation and Management Practices

Soybean lines that contain herbicide-tolerance traits (glyphosate) have been on the market since

1996 and have experienced broad adoption (Figure 46). The adoption track record of

biotechnology-derived soybean represents the most rapid case of technology adoption in the

history of agriculture (Sankula and Blumenthal, 2004). Based on USDA survey data,
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herbicide-tolerant soybeans (glyphosate-tolerant) went from 17 percent of U.S. soybean acreage

in 1997 to 68 percent in 2001 and 91 percent in 2009 (USDA ERS 2009a). Glyphosate has

provided simple, inexpensive, and highly effective weed control and has resulted in an increase

in no-till soybean production, a practice that is now accepted as improving soil health and

agricultural sustainability.

With 91% of soybean acres in the U.S. in 2009 planted to glyphosate-tolerant soybeans, typical

cultivation and management practices used by growers today already take into account the

management of herbicide-tolerant traits. DAS-68416-4 soybeans are comparable to conventional

soybeans phenotypically and agronomically (Section VII-D), and are not expected to alter the

geographic range or seasonality of soybean cultivation. Furthermore, ecological observations

during field testing have shown no changes in insect susceptibility of DAS-68416-4 soybeans

(Section VII-B) and therefore, no impacts are expected on insect control practices for

DAS-68416-4 soybeans. It is anticipated that the same management practices used today for

soybeans with the glyphosate-tolerance trait will also be appropriate for DAS-68416-4 soybeans.

Figure 46. Adoption of genetically engineered crops in the U.S.

From USDA ERS, 2009b

IX-E.2. Potential Impact on Weed Control Practices

DAS-68416-4 soybeans confers tolerance to the herbicides 2,4-D and glufosinate, both of which

will provide expanded weed management options in soybeans. Post-emergence applications of

2,4-D control a broad spectrum of broadleaf weeds. 2,4-D also has some short-lived soil residual

activity (4-10 day soil half life) which provides limited residual control of later germinating

broadleaf weeds. Post-emergence applications of glufosinate will control a broad spectrum of

grass and broadleaf weeds. Thus, DAS-68416-4 soybeans will provide alternatives to glyphosate

in weed management systems. 2,4-D and/or glufosinate would control the already glyphosate-
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resistant and hard to control broadleaf weeds, plus slow down the selection for more glyphosateresistant broadleaf weeds (Powles, 2008a).

Currently, for soybeans without the aad-12 gene, 2,4-D can be applied only as a burndown or

pre-emergence application at up to 1.0 lb ae/A (1120 g ae/ha). 2,4-D currently cannot be applied

at burndown or pre-emergence to conventional soybeans any later than 7-15 days

(0.5 - 1.0 lbs ae/A, or 560 - 1120 g ae/ha of ester formulations) or 15-30 days (0.5 – 1.0 lbs ae/A,

or 560 - 1120 g ae/ha of amine formulations) prior to planting, due to potential for crop injury.

In DAS-68416-4 soybeans, the proposed use pattern will be to 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 10

days apart over-the-top of the soybeans up to the R2 stage (full flower) of development (Figure

47). Thus, the proposed maximum seasonal rate of 2,4-D on soybeans will increase from 1.0 lb

ae/A (current) to 3.0 lbs ae/ha (DAS-68416-4 soybeans). DAS-68416-4 soybeans will allow

growers to apply 2,4-D from burndown or pre-emergence up through R2 stage soybeans without

risk of crop injury. This will provide new options for improved weed control during the soybean

development period when weeds have the greatest potential yield impact.

Figure 47. 2,4-D herbicide application timing and rates for conventional and DAS-68416-4

soybeans.

The use pattern for glufosinate on DAS-68416-4 soybeans will be consistent with the current use

pattern of glufosinate on other soybean products that contain the pat gene.

While 2,4-D is currently registered as a pesticide, supporting information on proposed label

changes for its use with DAS-68416-4 soybeans is being provided by Dow AgroSciences to U.S.

EPA for review. Dow AgroSciences is also developing an extensive stewardship program that

will include technological advancements in application and 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 chemical

technologies.

Post 0.5-1 lb

(up to 2 applications 10 days apart) Burndown/Pre 1 lb

Burndown

0.5-1 lb

R2

Full flower

DAS-68416-4 Soybean

Conventional Soybean

Post 0.5-1 lb

(up to 2 applications 10 days apart) Burndown/Pre 1 lb

Burndown

0.5-1 lb

R2

Full flower

DAS-68416-4 Soybean

Conventional Soybean
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In the future, DAS-68416-4 soybeans could be stacked with glyphosate-tolerance traits. Such

combined trait products (stacks) would have the potential to improve weed control by allowing

use of herbicide combinations or mixtures which can provide more consistent performance in

post-emergence weed control programs, and counteract glyphosate “rate-creep” (steady increase

in rates needed to obtain effective weed control; Figure 48) on hard-to-control weeds (Jaehnig,

2005). DAS-68416-4 soybeans, which will enable the use of 2,4-D, will allow use of a low cost,

high performance solution to reduce the escalation of glyphosate- and ALS-resistance in weed

populations.

Figure 48. Glyphosate application rates in U.S. corn and soybeans from 1990 thru 2007.

(Data from DMR-Kynetec)

0.55

0.60

0.65

0.70

0.75

0.80

0.85

19

9

0

19

91

19

9

2

19

9

3

19

9

4

19

9

5

19

9

6

19

9

7

19

9

8

19

9

9

2

0

0

0

2

0

01

2

0

0

2

2

0

0

3

2

0

0

4

2

0

0

5

2

0

0

6

2

0

0

7

Glyphosate Rate in Lbs ae/acre

IX-E.3. Potential Impact on Volunteer Management

Cultivated soybean seed rarely displays any dormancy characteristics and only under certain

environmental conditions grows as a volunteer in the year following cultivation (OECD 2000).

If this should occur, volunteers do not compete well with the succeeding crop, and can easily be

controlled mechanically or chemically. The soybean plant is not weedy in character. In North

America, Glycine max is not found outside of cultivation. In managed ecosystems, soybean does

not effectively compete with other cultivated plants or primary colonisers.

DAS-68416-4 soybeans are tolerant to 2,4-D and glufosinate. In the unlikely event that they

would grow as volunteers in the year following cultivation, they can still be effectively

controlled with herbicides (York et al., 2005).

Soybean is considered a self-pollinated species (OECD 2000). Crossing does not generally

occur. Soybean can only cross with other members of Glycine subgenus Soja. Approximately

91% of all soybeans planted in the U.S. in 2009 were glyphosate-tolerant soybeans (USDA ERS

2009c). If DAS-68416-4 soybeans cross with soybean varieties expressing tolerance to
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herbicides with different modes of action to produce soybean volunteers with multiple herbicidetolerance, they can still be effectively controlled mechanically or with other herbicide modes of

action; such as paraquat in burndown programs, fluometuron in cotton (Hayes, 2000), and

atrazine in corn. 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 soybeans tolerant to one or a few herbicides.

IX-E.4. Potential Impact on Non-Target Organisms and Endangered

Species

Based on substrate specificity of the aryloxyalkanoate dioxygenase-12 (AAD-12) enzyme

activity, no effect on non-target organisms or endangered species is anticipated for DAS-68416-4

soybean. The aad-12 gene and expressed protein are present in nature in the soil bacterium

Delftia acidovorans. AAD-12 is not a potential food allergen or toxin (Section VI-A.4) and

DAS-68416-4 soybean has been shown to be substantially equivalent to conventional soybean

based on the compositional analysis of grain and forage (Section VIII). Observations made

during field testing of DAS-68416-4 soybean revealed no effects on invertebrate populations

(Section VII-B) and agronomic characteristics equivalent to conventional soybean (Section VIIA).

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 DAS-68416-4 soybean will impact

any currently listed species of concern since it is not anticipated that DAS-68416-4 soybean will

cause new soybean acres to be planted in areas that are not already in agricultural use.

Commercial cropping systems routinely disturb the ground in which crops are grown. Habitat

disruption within DAS-68416-4 soybean fields would be comparable to other no-till or

herbicide-tolerant cropping systems.

Soybean is not an invasive or weedy species, there are no invasive or weedy sexually compatible

relatives of soybean in the US, and these properties are not anticipated to be altered by the

insertion of the aad-12 gene conferring tolerance to 2,4-D. It is therefore reasonable to conclude

that DAS-68416-4 soybean 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 currently registered for use in soybean production for pre-plant and burndown herbicide

treatment. The environmental fate and ecological effects on non-target organisms for the

proposed extension of the existing use pattern of 2,4-D in soybeans will be addressed by the EPA

as part of their review process.

Corn and soybean are typically planted in rotation in the US. In corn, 2,4-D is used both as a

pre-plant burn down prior to planting and post-emergence. The proposed new post-emergent and

seasonal use patterns in DAS-68416-4 soybeans are consistent with those currently approved for

use in conventional corn, and are consistent with those proposed for use in DAS-40278-9 corn

that also provides tolerance to 2,4-D. Therefore, no significant new geography will be treated

with 2,4-D beyond what is already available through pre-emergent applications in soybeans and
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pre- and post-emergent applications in corn. This consistency in the application rates, timings,

and the maximum seasonal rate with currently registered use patterns indicates that there should

be no change in the ecological risk assessments or endangered species assessments for 2,4-D

with DAS-68416-4 soybean.

IX-F. Herbicide Resistance Management

IX-F.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. 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, 1998). 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, 2008). 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.

Herbicide-resistant weeds have been a problem for growers for decades (Heap, 1997). The

earliest documented reports of herbicide-resistant weeds were resistance to 2,4-D in wild carrot

(Daucus carota) in 1952 and spreading dayflower (Commelina diffusa) in 1957 (Heap, 2009).

During the 1970s, up to 30 different weed species were reported to be resistant to the triazine

herbicides (Bandeen et al., 1982). Today, more than 330 weed biotypes around the world have

been reported to have some populations that are resistant to one or more herbicides (Heap, 2009).

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 32

shows a tabular summary of the total number of resistant species for each herbicide mode of

action as of November 2009. Figure 49 shows the number of resistant weed biotypes that have

been reported over time for each herbicide mode of action. Additional information on

glyphosate, 2,4-D, glufosinate, and the evolution of herbicide resistant weeds can be found in

Appendix 7.
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Table 32. Number of herbicide resistant weeds globally by herbicide group and mode of

action.

(Heap, 2009)

Herbicide Group Mode of Action

H

R

A

C

Gro

u

p

a

W

S

S

A

Gro

u

p

a

Example

Herbicide T

otal

C

o

u

nt

ALS Inhibitors

Inhibition of acetolactate synthase

ALS (acetohydroxyacid synthase

AHAS)

B 2 Chlorsulfuron 101

Photosystem II

Inhibitors

Inhibition of photosynthesis at

photosystem II C1 5 Atrazine 68

ACCase Inhibitors Inhibition of acetyl CoA carboxylase

(ACCase) A 1 Diclofop-methyl 36

Synthetic Auxins Synthetic auxins (action like

indoleacetic acid) O 4 2,4-D 27

Bipyridiliums Photosystem-I-electron diversion D 24 Paraquat 24

Ureas and Amides Inhibition of photosynthesis at

photosystem II C2 7 Chlorotoluron 21

Glycines Inhibition of EPSP synthase G 9 Glyphosate 16

Dinitroanilines and

others Microtubule assembly inhibition K1 10 Trifluralin 10

Thiocarbamates and

others

Inhibition of lipid synthesis – not

ACCase inhibition N 8 Triallate 8

Triazoles, Ureas,

Isoxazolidiones

Bleaching: Inhibition of carotenoids

biosynthesis (unknown target) F3 11 Amitrole 4

PPO Inhibitors Inhibition of protoporphyrinogen

oxidase (PPO) E 14 Oyxfluorfen 3

Chloroacetamides and

others

Inhibition of cell division (inhibition

of very long chain fatty acids) K3 15 Butachlor 3

Carotenoid

Biosynthesis Inhibitors

Bleaching: Inhibition of carotenoids

biosynthesis at the phytoene

desaturase

F1 12 Flurtamone 2

Arylaminopropionic

Acids Unknown Z 25 Flamprop-methyl 2

Nitriles and others Inhibition of photosynthesis at

photosystem II C3 6 Bromoxynil 1

Mitosis Inhibitors Inhibition of mitosis / microtubule

polymerization inhibitor K2 23 Propham 1

Cellulose Inhibitors Inhibition of cell wall (cellulose)

synthesis L 27 Dichlobenil 1

Unknown Unknown Z 8 (chloro) – flurenol 1

Unknown Unknown Z 17 MSMA 1

Total Number of

Unique Herbicide

Resistant Biotypes

330

a Two different classification systems have been developed independently by HRAC (alphabetical) and

the WSSA (numerical) to communicate the mode of action of herbicides. Weeds which have developed

resistance to one herbicide will also be resistant to all other herbicides which have the same mode of

action.
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Figure 49. Resistant weed biotypes per herbicide mode of action.

IX-F.2. Factors Impacting Development of 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 Herbicide Resistance Action Committee (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 33. This table assesses the risk of herbicideresistance development for each management practice as either “low”, “medium”, or “high”.

The greatest chance for resistance development occurs when several of these management

practices fall into the “high” category.
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Table 33. Assessment of resistance risk by evaluation of cropping systems.

(Nevill et al., 1998)

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

*Cultural control can be by using cultivation, stubble burning, competitive crops, stale seedbeds, etc. See

HRAC guidelines for more details.

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 and the best defense is

diversity in weed management practices.

IX-F.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). This evolution of glyphosate-resistant weed populations threatens the

ongoing sustainability of glyphosate and its contributions to world food production (Duke and

Powles, 2008).

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 34 shows that several common

weeds in U.S. corn and soybeans which are resistant to or difficult to control with glyphosate or

ALS herbicides and can be effectively controlled with 2,4-D.
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Introduction of DAS-68416-4 soybeans will give farmers one more tool for use in their weed

management programs which will help insure the long term sustainability of weed management

programs, including the use of glyphosate. DAS-68416-4 soybeans 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-68416-4 soybeans can be extended to other

herbicide-tolerant cropping systems, such as those with tolerance to glufosinate or ALSinhibiting herbicides. Furthermore, DAS-68416-4 soybeans will allow use of glufosinate

herbicides to significantly delay the selection for glyphosate-resistance in grass and broadleaf

weed species.

Table 34. Glyphosate- and ALS-resistant weeds controlled by 2,4-D.

(Heap, 2009)

Weed Species Glyphosate ALS Herbicides 2,4-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

DAS-68416-4 soybeans will expand the range of herbicides that can be used in herbicide-tolerant

soybean 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.

IX-G. Summary of Environmental Assessment

The AAD-12 protein is an enzyme with alpha-ketoglutarate-dependent dioxygenase activity

which results in metabolic inactivation of several herbicides of the aryloxyalkanoate family.

Field testing results confirm that AAD-12 protein expressed in DAS-68416-4 soybean provides

robust tolerance to 2,4-D. There are no new phenotypic characteristics in DAS-68416-4 soybean

to indicate it is any different from conventional soybean in weediness potential, and like

conventional soybean, the risk of gene flow from DAS-68416-4 soybean to wild relatives in the

U.S. is negligible.

No significant impact is expected on current crop management practices, non-target or

endangered species, crop rotation, or volunteer management from the introduction of

DAS-68416-4 soybean. The availability of DAS-68416-4 soybean 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-68416-4 soybean 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.
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IX-H. References

Ahrent, D.K., Caviness, C.E. (1994) Natural cross-pollination of twelve soybean cultivars in

Arkansas. Crop Science 34:376-378.

Bandeen, J.D., Stephenson, G.R., Cowett, E.R. (1982) Discovery and distribution of herbicideresistant weeds in North America. In Lebaron H.M. and Gressel J., eds., Herbicide Resistance in

Plants. John Wiley & Sons, Inc., New York, pp 9-30.

Beard, B.H., Knowles, P.F. (1971) Frequency of cross-pollination of soybeans after seed

irradiation. Crop Science 11:489-492.

CAST (2009) Sustainability of U.S. Soybean Production: Conventional, Transgenic, and Organic

Production Systems. Council for Agricultural Science and Technology - Special Publication No.

30, Ames, Iowa. June, 2009

Caviness, C.E. (1966) Estimates of natural cross-pollination in Jackson soybeans in Arkansas,

Crop Science 6:211-212.

Connor, A.J., Glare, T.R., Nap, J.P. (2003) The release of genetically modified crops into the

environment. Part II. Overview of ecological risk assessment. The Plant Journal 33:19-46.

Dalley, C.D., Renner, K.A, Kells, J.J. (2002) Weed Competition in Roundup Ready Soybeans

and Corn. Michigan State University, Integrated Pest Management Fact Sheet.

(www.ipm.msu.edu/CAT05_fld/pdf/5-26FactSheet.pdf)

Duke, S.O. and Powles, S.B. (2008) Editorial: Glyphosate-Resistant Weeds and Crops. Pest

Management Science 64:317-318.

Dyer, W.E. (1994) Resistance to glyphosate, in Herbicide Resistance in Plants: Biology and

Biochemistry, ed. by S.B. Powles and J.A.M. Holtum. CRC Lewis Publishers, New York, NY,

pp. 229-241.

Givens, W.A., Shaw, D.R., Kruger, G.R., Johnson, W.G., Weller, S.C., Young, B.G., Wilson,

R.G., Owen, M.D.K., Jordan, D. (2009) Survey of Tillage Trends Following the Adoption of

Glyphosate-Resistant Crops. Weed Technology 23:150-155.

Hartzler, B. and Pringnitz, B. (2000) Early Season Weed Competition - Integrated Crop

Management IC-484 (8), Iowa State University.

(http://www.ipm.iastate.edu/ipm/icm/2000/5-8-2000/earlyweed.html)

Hayes, R.M. (2000) Herbicide Resistant Crops and Weed Species Shifts. Proceedings: Third

Annual National Conservation Tillage Cotton and Rice Conference.

(http://www.btinternet.com/~nlpWESSEX/Documents/cottonrrproblems.htm)

Heap, I.M. (1997) The Occurrence of Herbicide-Resistant Weeds Worldwide. Pesticide Science

51:235-243.
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Heap, I. (2009) The International Survey of Herbicide Resistant Weeds.

(http://www.weedscience.com)

HRAC (1998) Herbicide Resistance Action Committee. Guidelines to the management of

herbicide resistance.

(http://www.hracglobal.com/Publications/ManagementofHerbicideResistance/tabid/225/Default.

aspx#guidelines)

Jaehnig, K.C. (2005) Farmers facing rise of herbicide resistant weeds. Southern Illinois

University Carbondale Country Column.

(http://news.siu.edu/news/April04/042704kj4032.jsp)

Knezevic, S. Z. (2002) Use of Herbicide Tolerant Crops as a Component of an Integrated Weed

Management Program, University of Nebraska NebGuide, p.1484.

(http://elkhorn.unl.edu/epublic/live/g1484/build/#concerns)

Krausz, R.F., B.G. Young, G. Kapusta, and J.L. Matthews. (2001) Influence of Weed

Competition and Herbicides on Glyphosate-Resistant Soybean (Glycine max). Weed

Technology 15:530-534.

Lauer, J. (2007) Continuous Corn or Rotate in 2008? The Principle of Crop Rotation. University

of Wisconsin Agronomy Department, Agronomy Advice, Field Crops 28.426-51.

(http://corn.agronomy.wisc.edu/AA/A051.aspx)

Loux, M., Stachler, J., Johnson, B, Nice, G. (2006) The Benefits of Preemergence Herbicides in

Roundup Ready Soybean. Fact Sheet from The Ohio State University and Purdue University.

(http://agcrops.osu.edu/weeds/documents/prefactfinal.pdf)

Mallory-Smith, C. (2008) Pacific Northwest Weed Management Handbook – Managing

Herbicide-resistant Weeds.

(http://weeds.ippc.orst.edu/pnw/weeds?06W_HRES01.dat)

Moss, S. (2002) Chapter 11 – Herbicide-Resistant Weeds. Weed Management Handbook, Ninth

Edition, Edited by Robert Naylor, Blackwell Publishing, British Crop Protection Council

Müller, R.H., Jorks, S., Kleinsteuber, S., Babel, W. (1999) Comamonas acidovorans strain MC1:

a new isolate capable of degrading the chiral herbicides dichlorprop and mecoprop and the

herbicides 2,4-D and MCPA. Microbiological Research 154:241-246.

Nevill, D., Cornes, D. and Howar, S. (1998) The Role of HRAC in the Management of Weed

Resistance.

(http://www.hracglobal.com/Publications/HRACManagementandWeedResistance/tabid/228/Def

ault.aspx)

OECD (2000) Consensus Document on the Biology of Glycine max (L.) Merr. (Soybean).

Organisation for Economic Co-operation and Development, Paris, France. Series on

Harmonization of Regulatory Oversight in Biotechnology No. 15. EVN/JM/MONO(2000)9.

(http:/www.olis.oecd.org/olis/2000doc.nsf/LinkTo/env-jm-mono(2000)9)
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Penn State Agronomy Guide. 2009-2010. Part 2, Section 1: Pest Management.

(http://agguide.agronomy.psu.edu/pm/sec1/sec12f.cfm)

Powles, S.B. (2008a) Evolution in action: glyphosate-resistant weeds threaten world crops.

Outlooks on Pest Management 19:256-259.

Powles, S.B. (2008b) Review: Evolved glyphosate-resistant weeds around the globe: lessons to

be learnt. Pest Management Science 64:360-365.

Ray, J.D., Kilen, T.C., Abel, C.A., Paris, R.L. (2003) Soybean natural cross-pollination rates

under field conditions. Environmental Biosafety Research 2:133-138.

Sankula, S., Blumenthal, E. (2004) Impacts on U.S. Agriculture of Biotechnology-Derived Crops

Planted in 2003– An Update of Eleven Case Studies. National Center for Food and Agricultural

Policy.

(http://www.ncfap.org/documents/2004finalreport.pdf.)

USDA ERS (2008) USDA Economic Research Service – Soybeans and Oil Crops Briefing

Room

(http://www.ers.usda.gov/Briefing/SoybeansOilCrops/)

USDA ERS (2009a) USDA Economic Research Service Adoption of Genetically Engineered

Crops in the U.S.: Extent of Adoption.

(http://www.ers.usda.gov/Data/BiotechCrops/adoption.htm)

USDA ERS(2009b) USDA Economic Research Service Data Sets – Adoption of Genetically

Engineered Crops in the U.S.

(http://www.ers.usda.gov/Data/BiotechCrops/)

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Engineered Crops in the U.S. – Soybean Varieties.

(http://www.ers.usda.gov/Data/BiotechCrops/ExtentofAdoptionTable3.htm)

USDA ERS ARMS (2002) USDA Economic Research Service – Agricultural Resource

Management Survey.

(http://www.ers.usda.gov/data/ARMS/app/Crop.aspx.)

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Management Survey.

(http://www.ers.usda.gov/data/ARMS/app/Crop.aspx.)

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Usage – 2005 Field Crops Summary.

(http://usda.mannlib.cornell.edu/usda/nass/AgriChemUsFC/2000s/2006/AgriChemUsFC-05-17-

2006.pdf)

USDA NASS (2008) USDA National Agricultural Statistics Service – Quick Stats.

(http://www.nass.usda.gov/Data_and_Statistics/Quick_Stats/)
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WSSA (1998) Resistance and Tolerance Definitions. Weed Science Society of America.

(http://www.wssa.net/Weeds/Resistance/definitions.htm)

Westendorf, A.; Benndorf, D.; Müller, R.; Babel, W. (2002) The two enantiospecific

dichlorprop/α-ketoglutarate-dioxygenases from Delftia acidovorans MC1-protein and sequence

data of RdpA and SdpA Microbiological Research 157:317-22.

Westendorf, A., Müller, R.H., Babel, W. (2003) Purification and characterization of the

enantiospecific dioxygenases from Delftia acidovorans MC1 initiating the degradation of

phenoxypropionates and phenoxyacetate herbicides. Acta Biotechnologica 23: 3-17.

Wright, T.R.; Lira, J.M.; Walsh, T.; Merlo, D.J., Jayakumar, P., Lin, G. (2007) Novel Herbicide

Resistance Genes. World Intellectual Property Organization International Publication Number

WO 2007/053482 A2

York, A.C., Beam, J.B., Culpepper, A.S. (2005) Control of Glyphosate-resistant Soybean in

Cotton. Journal of Cotton Science 9:102-109.
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X. Adverse Consequences of Introduction

Field and laboratory testing of DAS-68416-4 soybean has demonstrated that it has no significant

differences from non-transgenic conventional soybean apart from the intended change of

herbicide-tolerance. DAS knows of no study results or other observations associated with

DAS-68416-4 soybean that would be anticipated to result in adverse consequences from

introduction. Soybean varieties derived from DAS-68416-4 will be the first soybean varieties to

express tolerance to 2,4-D. As such, they will be an important tool to growers in areas where

weeds have become resistant to other broad spectrum herbicides, such as glyphosate.
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XI.Appendices
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Appendix 1. Methods for Molecular Characterization of DAS-68416-4 Soybean

1.1. DAS-68416-4 Soybean Material

Transgenic soybean seeds from three distinct generations of soybean containing event

DAS-68416-4 were planted in the greenhouse. After at least two weeks of growth, leaf punches

were taken from each plant and were tested for AAD-12 protein expression using a rapid lateral

flow test strip according to the manufacturer’s instructions. Each plant was given a “+” or “-“

for the presence or absence of the AAD-12 protein.

1.2. Control Soybean Material

Seeds from the unmodified Maverick were planted in the greenhouse. The Maverick seeds had a

genetic background representative of the transgenic seeds but did not contain the aad-12 gene.

1.3. Reference Materials

DNA of the plasmid pDAB4468 was added to samples of the Maverick control genomic DNA at

a ratio approximately equivalent to 1 copy of the transgene per soybean genome and used as the

positive control to verify probe hybridization and sizes of internal fragments.

1.4. DNA Probe Preparation

DNA probes specific to the genetic elements in the T-DNA insert of pDAB4468 and the vector

backbone were produced via polymerase chain reaction (PCR) amplification using pDAB4468

plasmid DNA as a template, followed by purification.

1.5. Sample Collection and DNA Extraction

Labeled leaf samples were collected from green house for DNA extraction or being stored in -

80°C freezer for future use. Genomic DNA was extracted with 2 methods. Method 1 is based on

the method of Guillemant, 1992. Briefly, leaf samples were ground individually in liquid

nitrogen, and then extraction buffer was added to samples at a ratio of about 3:1 plus 10 μL of

RNase-A (Qiagen, Valencia, catalog # 1007885). After precipitation using isopropyl alcohol,

crude DNA samples were purified using PCI (phenol:chloroform:isoamyl alcohol = 25:24:1,

Sigma, St. Louis, MO, catalog #: P2069) and CI (chloroform:isoamyl alcohol = 24:1, Sigma, St.

Louis, MO, catalog # C0549) extraction. DNA was precipitated again by addition of 1/10

volume of 3 M NaOAc and equal volume of isopropyl alcohol. The precipitated DNA was

rinsed with 70% ethanol, then dissolved in appropriate volume of 0.1X TE buffer.

Method 2 is based on the modified CTAB method. Briefly, leaf samples were individually

ground in liquid nitrogen followed by the addition of extraction buffer (~5:1 ratio milliliter

CTAB extraction buffer: gram leaf tissue) and RNase-A (>10 μL) (Qiagen, Valencia, catalog #

1007885). After approximately 2 hours of incubation at ~65 °C with gentle shaking, samples

were spun down and the supernatants were extracted with equal volume of chloroform:octanol =

24:1 (chloroform, Sigma, Catalog # 366922-4L; octanol, Sigma, catalog # O4504-100mL).

DNA was precipitated by mixing the supernatants with equal volume of precipitation buffer (1%

CTAB, Sigma, Catalog # H6269-2506; 50 mM Tris-HCl, Invitrogen, Catalog # 15568-025; 10
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mM EDTA, AcruGene, Catalog # 51234). The precipitated DNA was dissolved in high salt TE

buffer (1X TE pH8.0, thermo, Catalog # 17890; 1.0M NaCl, AccuGene, Catalog # 51202)

followed by precipitation with isopropyl alcohol (Mallinckrot, Catalog # 3031-08). The

precipitated DNA was rinsed with 70% ethanol, air-dried, then dissolved in appropriate volume

of 1 X TE buffer (pH8.0).

To check the quality of the resultant genomic DNA, an aliquot of the DNA samples was

electrophoretically separated on a 1% agarose gel containing ethidium bromide (~1 μg/mL) with

1X TBE buffer (89 mM Tris-Borate, 20 mM EDTA, pH 8.3). The gel was visualized under

ultraviolet (UV) light to confirm that the DNA was not degraded and that the RNA had been

removed by the RNase-A. The concentration of DNA in solution was determined by a picogreen

kit (Invitrogen, Carlsbad, CA, catalog # P7589) in a fluorometer (Bio-TEK, FLX800).

1.6. DNA Digestion and Electrophoretic Separation of the DNA Fragments

Genomic DNA extracted from the soybean leaf tissue was digested with restriction enzymes by

combining approximately 9 g of genomic DNA with approximately 5-11 units of the selected

restriction enzyme per g of DNA in the corresponding reaction buffer. Each sample was

incubated at 37oC overnight. The positive control sample was prepared by combining

pDAB4468 plasmid DNA with genomic DNA from the Maverick control (at a ratio

approximately equivalent to 1 copy of the transgene per soybean genome) and was digested

using the same procedures and restriction enzymes as the transgenic DNA samples. DNA from

the Maverick control was digested using the same procedures and restriction enzymes as the test

samples to serve as the negative control.

The digested DNA samples were precipitated with Quick-Precip (Edge BioSystems) and resuspended 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 (89mM Tris,

89mM Boric acid, 2mM EDTA) at 55-65 V for 18-22 hours to achieve fragment separation. The

gels were stained with ethidium bromide and the DNA was visualized under UV light. A

photographic record was made of each stained gel.

1.7. Southern Transfer

The DNA fragments on the agarose gels were transferred to nylon membranes via Southern

transfer, essentially as described by Memelink et al., 1994. The agarose gels were depurinated,

denatured, neutralized in situ and transferred to a nylon membrane in 10 SSC buffer (3M NaCl,

0.3M Na citrate) using a wicking system. Following transfer to the membrane, the DNA was

bound to the membrane by crosslinking through UV treatment.

1.8. Probe Synthesis and Hybridization

The hybridization probes were generated using a PCR-based incorporation of a digoxigenin

(DIG) labeled nucleotide, [DIG-11]-dUTP, from DNA fragments generated by primers specific

to the gene elements and other regions from plasmid pDAB4468. The PCR synthesis of the

probes was performed using PCR DIG Probe Synthesis Kit (Roche Diagnostics) and following

the manufacturer’s recommended procedures.
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Labeled probes were hybridized to the target DNA on the nylon membranes using the DIG Easy

Hyb Solution according to manufacturer’s instructions (Roche Diagnostics). DIG-labeled DNA

molecular weight marker II was used to determine the hybridizing fragment size on the Southern

blots.

1.9. Detection

DIG-labeled probes bound to the nylon membranes after stringent washing were incubated with

AP (Alkaline Phosphatase)-conjugated anti-Digoxigenin antibody for ~1 hr in 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 for one or more time points to detect the hybridizing fragments and to

visualize the molecular weight standards. The images were then scanned and stored. The

number and size of each of the detected bands were documented for each digest and for each

probe.

Once the data was recorded, membranes were rinsed with milli-Q water and then stripped of the

probe in a solution of 0.2M NaOH and 1.0% SDS. The alkali-based stripping procedure

successfully removes the labeled probes from the membranes, allowing them to be re-probed

with a different gene probe. After stripping, the membranes were exposed to chemiluminescent

film to ensure all the previous DNA probes had been removed.

1.10. References

Guillemant, P. (1992) Isolation of Plant DNA: A Fast, Inexpensive, and Reliable Method. Plant

Molecular Biology Reporter 10(1): 60-65.

Memelink, J., Swords, K., Harry, J., Hoge, C. 1994. Southern, Northern, and Western Blot

Analysis. Plant Molecular Biology Manual F1:1-23.
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Appendix 2. Methods and Results for Characterization of AAD-12 Protein

2.1. DAS-68416-4 Transgenic Soybean Material

Greenhouse-grown DAS-68416-4 soybean plants (T5 generation) were used as the plant source

of the AAD-12 protein. Prior to use, individual plants were leaf tested to confirm expression of

the AAD-12 protein using a rapid lateral flow test strip according to the manufacturer’s

instructions. Leaves (and some stems) from AAD-12 expressing plants were harvested,

lyophilized, ground to a fine powder, and stored frozen until needed.

2.2. Control Soybean Material

Control soybean line Maverick had a genetic background representative of the DAS-68416-4

soybean plants, but did not contain the aad-12 gene. Absence of AAD-12 expression in the

control plants was confirmed by immunoassay using an AAD-12 specific rapid lateral flow test

strip. Leaves (and some stems) of control plants were harvested, lyophilized, ground and stored

under the same conditions as the DAS-68416-4 soybean.

2.3. Reference Material

Recombinant AAD-12 microbial protein was produced in Pseudomonas fluorescens (Pf) and

purified to a lyophilized powder. The microbe-derived AAD-12 protein preparation was stored

dry and resuspended in a buffer to maintain activity prior to use.

2.4. Protein Purification of AAD-12 from DAS-68416-4 Soybean Plant Tissue

The AAD-12 protein was extracted from lyophilized leaf tissue in a PBST (Phosphate Buffered

Saline with 0.05% Tween 20, pH 7.4) based buffer with added stabilizers, and the soluble

proteins were separated from the insoluble plant material by centrifugation. The supernatant was

filtered and the soluble proteins were allowed to bind to Phenyl Sepharose (PS) beads (GE

Healthcare). After an hour of incubation, the PS beads were washed with PBST and the bound

proteins were eluted with Milli-Q water. Sodium chloride was added to increase the

conductivity and the PS purified proteins were loaded onto an anti-AAD-12 immunoaffinity

column which had been conjugated with an AAD-12 specific polyclonal antibody. The nonbound proteins were collected from the column and the column was washed extensively with

pre-chilled PBS (phosphate buffered saline, pH 7.4). The bound proteins were eluted from the

column with a 3.5 M NaSCN, 50 mM Tris, pH 8.0 buffer and examined by SDS-PAGE (stained

with GelCode Blue total protein stain from Thermo-Pierce) and western blotting.

2.5. SDS-PAGE and Western Blot Analysis of Crude Extracts

Lyophilized leaf tissue from event DAS-68416-4 and Maverick was mixed with PBST buffer

containing ~2.0% protease inhibitor cocktail (Sigma) and the protein was extracted by grinding

with ball bearings in a Geno-Grinder. The samples were centrifuged and the supernatants were

mixed with Laemmli sample buffer, heated, and briefly centrifuged. The samples were loaded

directly on to a Bio-Rad Criterion SDS-PAGE gel. The positive reference standard, microbederived AAD-12, was also mixed with sample buffer and loaded on to the gel. Electrophoresis

was conducted with Tris/glycine/SDS buffer (Bio-Rad). Following electrophoresis, the gel was
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cut in half, with one half stained with Pierce GelCode Blue protein stain and the other gel half

was electro-blotted onto a nitrocellulose membrane. The nitrocellulose membrane was then

probed with AAD-12 specific polyclonal and monoclonal antibodies. A chemiluminescent

substrate was used to visualize the immunoreactive bands.

2.6. Detection of Post-Translational Glycosylation

The immunoaffinity-purified, plant-derived AAD-12 protein was analyzed for evidence of

glycosylation by electrophoresis with microbe-derived AAD-12 protein, soybean trypsin

inhibitor, bovine serum albumin, and horseradish peroxidase as controls. The control protein

samples were adjusted to concentrations approximately equal with the plant-derived AAD-12

protein and mixed with Laemmli buffer. The proteins were heated, centrifuged, and applied

directly to a Bio-Rad Criterion SDS-PAGE gel. Following electrophoresis, the gel was cut in

half. One gel half was stained with Pierce GelCode Blue stain for total protein. The remaining

half of the gel was stained with GelCode Glycoprotein Stain to visualize the glycoproteins. The

glycoproteins present on the gel were visualized as magenta bands on a light pink background.

2.7. Mass Spectrometry Peptide Mass Fingerprinting and Sequence Analysis of Plant- and

Microbe-Derived AAD-12 Protein

The immunoaffinity purified AAD-12 plant-derived protein was subjected to in-solution

digestion by trypsin and Asp-N followed by matrix assisted laser desorption/ionization time-offlight mass spectrometry (MALDI-TOF MS) and electrospray-ionization liquid

chromatography/mass spectrometry (ESI-LC/MS). The peptide fragments of the plant-derived

AAD-12 protein (including the N- and C-termini) were analyzed and compared with the

sequence of the microbe-derived protein.

2.8. Results of the SDS-PAGE and Western Blot Analysis

In the microbe-derived AAD-12, the major protein band, as visualized on the Coomassie stained

SDS-PAGE gel, was approximately 32 kDa (Figure 50). As expected, the corresponding plantderived AAD-12 protein was identical in size to the microbe-derived protein. Predictably, the

plant purified fractions contained a minor amount of non-immunoreactive impurities in addition

to the AAD-12 protein. The co-purified proteins were likely retained on the column by weak

interactions with the column matrix (Holroyde et al., 1976, Kennedy and Barnes, 1983 and

Williams et. al., 2006).

The microbe-derived AAD-12 and DAS-68416-4 plant tissue extract showed a positive signal of

the expected size on the western blot using the anti-AAD-12 polyclonal antibody (Figure 51). In

the AAD-12 western blot analysis, no immunoreactive proteins were observed in the control

Maverick extract and no alternate size proteins (aggregates or degradation products) were seen in

the samples from the transgenic plant. The monoclonal antibody did detect a small amount of

the AAD-12 dimer in the microbe-derived protein. These results add to the evidence that the

protein expressed in soybean is not glycosylated which would add to the overall protein

molecular weight.
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Figure 50. SDS-PAGE of soybean- and microbe-derived AAD-12.

Note: Affinity-purified soybean-derived AAD-12 and microbe-derived AAD-12 were separated

by SDS-PAGE. Following electrophoresis, the gel was stained with Thermo-Pierce GelCode

Blue stain for total protein according to the manufacturer’s protocol.

AAD-12

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

Soy Pf

MW AAD-12 AAD-12

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

Soy Soybean-Derived AAD-12 (DAS-68416-4) 500 L

Pf Microbe-Derived AAD-12 1000 ng

SDS-PAGE

AAD-12

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

Soy Pf

MW AAD-12 AAD-12

AAD-12

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

Soy Pf

MW AAD-12 AAD-12

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

Soy Soybean-Derived AAD-12 (DAS-68416-4) 500 L

Pf Microbe-Derived AAD-12 1000 ng

SDS-PAGE
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Figure 51. SDS-PAGE and western blot of soybean- and microbe-derived AAD-12 protein

extracts.

Note: Crude extracts from lyophilized leaf tissue of event DAS-68416-4 and Maverick were

separated by SDS-PAGE along with microbe-derived AAD-12 and bovine serum albumin.

Following electrophoresis the gel was cut in half, one half was stained with Pierce GelCode Blue

stain for total protein and the remaining half was electro-blotted onto a nitrocellulose membrane.

The nitrocellulose membrane was then probed with AAD-12 specific polyclonal and monoclonal

antibodies and detected with HRP-labeled antibodies. A chemiluminescent substrate was used to

visualize the immunoreactive bands.

Mav 416 12

- 15 -

- 20 -

- 30 -

- 40 -

- 50 -

- 60 -

- 80 -

- 110 -

- 160 -

- 260 -

kDa

- 10 -

- 3.5 -

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

M Mav 416 12

AAD

BSA P M Mav 416 12 P

AAD AAD

SDS-PAGE Western blot film

a) monoclonal b) polyclonal

AAD-12

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

Mav Nontransgenic Soybean Extract 32 L

416 Event DAS-68416-4 extract 32 L

AAD-12 Microbe-Derived AAD-12 ~785 ng

BSA Bovine Serum Albumin (BSA) ~785 ng

P Novex Prestained MW Markers 10 L

Mav 416 12

- 15 -

- 20 -

- 30 -

- 40 -

- 50 -

- 60 -

- 80 -

- 110 -

- 160 -

- 260 -

kDa

- 10 -

- 3.5 -

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

M Mav 416 12

AAD

BSA P M Mav 416 12 P

AAD AAD

SDS-PAGE Western blot film

a) monoclonal b) polyclonal

AAD-12

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

Mav Nontransgenic Soybean Extract 32 L

416 Event DAS-68416-4 extract 32 L

AAD-12 Microbe-Derived AAD-12 ~785 ng

BSA Bovine Serum Albumin (BSA) ~785 ng

P Novex Prestained MW Markers 10 L
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2.9. Results of Detection of Glycosylation of AAD-12 Protein

No covalently-linked carbohydrates were detectable on the plant- or microbe-derived AAD-12

proteins (Figure 52). Horseradish peroxidase, a glycoprotein, was used as a positive indicator for

glycosylation. Soybean trypsin inhibitor and bovine serum albumin, both non-glycoproteins,

served as negative controls.

Figure 52. Glycosylation analysis of soybean- and microbe-derived AAD-12 proteins.

Note: The affinity-purified soybean-derived AAD-12, microbe-derived AAD-12, soybean trypsin

inhibitor, bovine serum albumin, and horseradish peroxidase were diluted to a similar concentration prior

to separation by SDS-PAGE. After electrophoresis, the gel was cut in half and one half was stained with

GelCode Blue stain for total protein, and the other half of the gel was stained with a GelCode

Glycoprotein Staining Kit to visualize the glycoproteins.

- 15 -

- 20 -

- 30 -

- 40 -

- 50 -

- 60 -

- 80 -

- 110 -

- 160 -

- 260 -

kDa

- 10 -

- 3.5 -

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

M 1 2 3 4 5 P P 1 2 3 4 5 M

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

1 Soybean-Derived AAD-12 (Frac 3) 500 L

2 Microbe-Derived AAD-12 500 ng

3 Soybean Trypsin Inhibitor (STI) 500 ng

4 Horseradish Peroxidase (HRP) 500 ng

5 Bovine Serum Albumin (BSA) 500 ng

P Novex Prestained MW markers 10 L

Total protein stain Glycoprotein stain

- 15 -

- 20 -

- 30 -

- 40 -

- 50 -

- 60 -

- 80 -

- 110 -

- 160 -

- 260 -

kDa

- 10 -

- 3.5 -

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

M 1 2 3 4 5 P P 1 2 3 4 5 M

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

1 Soybean-Derived AAD-12 (Frac 3) 500 L

2 Microbe-Derived AAD-12 500 ng

3 Soybean Trypsin Inhibitor (STI) 500 ng

4 Horseradish Peroxidase (HRP) 500 ng

5 Bovine Serum Albumin (BSA) 500 ng

P Novex Prestained MW markers 10 L

- 15 -

- 20 -

- 30 -

- 40 -

- 50 -

- 60 -

- 80 -

- 110 -

- 160 -

- 260 -

kDa

- 10 -

- 3.5 -

6.0 -

14.4 -

21.5 -

31.0 -

36.5 -

55.4 -

66.3 -

97.4 -

116.3 -

200 -

kDa

3.5 -

2.5 -

M 1 2 3 4 5 P P 1 2 3 4 5 M

Lane Sample Amount

M Invitrogen Mark12 MW markers 10 L

1 Soybean-Derived AAD-12 (Frac 3) 500 L

2 Microbe-Derived AAD-12 500 ng

3 Soybean Trypsin Inhibitor (STI) 500 ng

4 Horseradish Peroxidase (HRP) 500 ng

5 Bovine Serum Albumin (BSA) 500 ng

P Novex Prestained MW markers 10 L

Total protein stain Glycoprotein stain
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2.10. Results of MALDI-TOF and ESI/LC- MS Tryptic and Asp-N Peptide Mass Fingerprints of

AAD-12 Proteins

Following digestion of the plant-derived AAD-12 protein by trypsin and Asp-N, the masses of

the detected peptides were compared with those deduced based on potential cleavage sites in the

sequence of the AAD-12 protein. Figure 53 illustrates the theoretical peptide cleavage which

was generated in silico using PAWs software (Proteometrics LLC).

The trypsin and Asp-N digestion of soybean-derived AAD-12 protein yielded high detection of

the expected peptides, resulting in 73.4% coverage of the AAD-12 protein sequence (Figure 54).

The analysis confirmed the plant-derived protein amino acid sequence matched that of the

microbe-derived AAD-12 protein and that of the predicted amino acid sequence. Results of

these analyses indicated that the amino acid sequence of the soybean-derived AAD-12 protein

was equivalent to the P. fluorescens-expressed protein.

2.11. Results of Tryptic and Asp-N Peptide N- and C-terminal Sequence Analysis of AAD-12

The N-terminal sequence of the first 27 residues of the plant-derived and all 292 residues of the

microbe-derived AAD-12 protein was obtained by mass spectrometry. The amino acid

sequences for N-terminus of both proteins was A2 H A A L S P L S Q I T P T G A T L G A T V

T G V H L A T L27, indicating the N-terminal methionine had been removed (Table 35 and

Figure 54). These results suggest that during or after translation in the plant and P. fluorescens,

the N-terminal methionine is cleaved by a methionine aminopeptidase. In addition to the

methionine being removed, the N-terminal peptide of the AAD-12 protein was shown to be

acetylated after the N-terminal methionine was cleaved. These two co-translational processes,

cleavage of N-terminal 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, 2000; Polevoda and Sherman, 2002).

The C-terminal sequences of the plant- and microbe-derived AAD-12 proteins were determined

to be identical to the expected sequences (Table 36 and Figure 54).
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Figure 53. Theoretical trypsin (top panel) and Asp-N (bottom panel) cleavage of the AAD12 protein.

Note: Alternating blocks of upper (black) and lower (red) case letters within the amino acid

sequence are used to differentiate the potential peptides after protease digestion. The numbers

on the left and right sides indicate the amino acid residue numbers.

Digestion at K (lysine) and R (arginine)

Digestion at D (aspartate)
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Figure 54. Sequence coverage in the tryptic and Asp-N peptide mapping analysis of plantderived AAD-12 protein with MALDI-TOF and ESI/LC MS.

Note: The numbers on the left and right sides of the protein sequence indicate the amino acid

residue numbers. Letters highlighted in gray represent tryptic peptide sequence detected by

MALDI-TOF MS and ESI-LC/MS. Underlined letters represent Asp-N peptide sequence

detected. The overall sequence coverage was 73.4%. The down arrow indicates the N-terminal

methionine was removed by an aminopeptidase and the N-terminal alanine was N-acetylated.

N-Ac

001 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

031 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

061 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

091 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

- Trypsin

- Asp N

Total number of amino acids covered: 215*

Total number of amino acids in protein: 293

Combined sequence coverage: 73.4%

*The N-terminal methionine was missing

N-Ac

001 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

031 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

061 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

091 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

- Trypsin

- Asp N

Total number of amino acids covered: 215*

Total number of amino acids in protein: 293

Combined sequence coverage: 73.4%

*The N-terminal methionine was missing

N-Ac

001 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

031 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

061 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

091 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

- Trypsin

- Asp N

Total number of amino acids covered: 215*

Total number of amino acids in protein: 293

Combined sequence coverage: 73.4%

*The N-terminal methionine was missing
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Figure 55. Sequence coverage in the peptide mapping analysis of microbe-derived AAD-12

protein with MALDI-TOF and ESI/LC MS.

Note: The numbers on the left and right sides of the protein sequence indicate the amino acid

residue numbers. Letters highlighted in gray represent tryptic peptide sequence detected by

MALDI-TOF MS and ESI-LC/MS. Letters in boxes indicates sequence coverage detected with

Arg-C digestion. Underlined letters indicates sequence coverage detected with Asp-N,

chymotrypsin and Glu-C digestions. The overall sequence coverage was 99.7%. The down

arrow indicates the N-terminal methionine was removed by an aminopeptidase.

Total number of amino acids covered: 292*

Total number of amino acids in protein: 293

Combined sequence coverage: 99.7%

*The N-terminal methionine was missing

Total number of amino acids covered: 292*

Total number of amino acids in protein: 293

Combined sequence coverage: 99.7%

*The N-terminal methionine was missing
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Table 35. Summary of N-terminal sequence data of AAD-12 soybean- and microbe-derived

proteins.

Source Expected N-terminal Sequence1

P. fluorescens M

1 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 D27

Soybean Event M

1 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 D27

DAS-68416-4

Source Detected N-terminal Sequence2

P. fluorescens A

2 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 D27

Soybean Event N-AcA

2 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 27

DAS-68416-4

3

1

Expected N-terminal sequence of the first 27 amino acid residues of P. fluorescens- and

soybean-derived AAD-12.

2Detected N-terminal sequences of P. fluorescens- and soybean-derived AAD-12.

3

The MALDI-TOF MS data for the N-terminal peptide revealed that the soybean-derived AAD12 protein was acetylated (N-Acetyl-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).

Notes:

Numbers in superscript (Rx) indicate amino acid residue numbers in the sequence. Amino

acid residue abbreviations:

A: alanine D: Aspartate G: glycine

H: histidine I: isoleucine L: leucine

M: methionine P: proline Q: glutamine

T: threonine V: valine
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Table 36. Summary of C-terminal sequence data of AAD-12 soybean- and microbe-derived

proteins.

Source Expected C-terminal Sequence1

P. fluorescens 281L A G R P E T E G A A L V293

Soybean Event

DAS-68416-4

281L A G R P E T E G A A L V293

Source Detected C-terminal Sequence2

P. fluorescens 281L A G R P E T E G A A L V293

Soybean Event 281L A G R P E T E G A A L V293

DAS-68416-4

1

Expected C-terminal sequence of the last 13 amino acid residues of P. fluorescens- and

soybean-derived AAD-12.

2Detected C-terminal sequences of P. fluorescens- and soybean-derived AAD-12.

Notes:

Numbers in superscript (Rx) indicate amino acid residue numbers in the sequence.

Amino acid residue abbreviations:

A: alanine E: glutamate G: glycine

L: leucine P: proline R: arginine

T: threonine V: valine
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2.12. Conclusions

The biochemical identity of microbe-derived AAD-12 protein was equivalent to the protein

purified from leaf tissue of event DAS-68416-4. The plant- and microbe-derived AAD-12

proteins showed the expected molecular weight of ~32 kDa by SDS-PAGE and were

immunoreactive to AAD-12 protein specific antibodies by western blot analysis. The amino acid

sequence of both proteins was confirmed by enzymatic peptide mass fingerprinting by MALDITOF MS and ESI-LC/MS. In addition, the lack of glycosylation of the plant-derived AAD-12

protein provided additional evidence that the AAD-12 protein produced by P. fluorescens and

DAS-68416-4 soybean are biochemically equivalent.

2.13. References

Holroyde, M. J., Chesher, J. M. E., Trayer, I. P., and Walker, D. G. (1976) Studies on the Use of

Sepharose –N-(6-Aminohexanoyl)-2-Amino-2-Deoxy-D-Glucopyranose for the Large-Scale

Purification of Hepatic Glucokinase. Journal of Biochemistry 153:351-361.

Kennedy J. F. and Barnes J. A. (1983) Immunochemical studies of the non-specific interactions

of cyanogen bromide-activated macroporous agarose-based immunoadsorbents. Journal of

Chromatography 281, 83-93.

Polevoda, B. and Sherman, F. (2000) N



-terminal Acetylation of Eukaryotic Proteins. Journal of

Biological Chemistry 275:36479–36482.

Polevoda, B. and Sherman, F. (2002) The Diversity of Acetylated Proteins. Genome Biology

3:0006.1–0006.6.

Williams, A. J. K., Norcross, A. J., Chandler, K. A. and Bingley, P. J. (2006) Non-specific

binding to protein A Sepharose and protein G Sepharose in insulin autoantibody assays may be

reduced by pre-treatment with glycine or ethanolamine. Journal of Immunological Methods 314,

170–173.
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Appendix 3. Methods and Results for Characterization of PAT Protein

3.1. DAS-68416-4 Transgenic Soybean Material

Greenhouse-grown DAS-68416-4 T5 plants were used as the plant source of the PAT protein.

Prior to use, individual plants were leaf tested to confirm expression of the PAT protein using a

rapid lateral flow test strip according to the manufacturer’s instructions. Leaves (and some

stems) from PAT expressing plants were harvested, lyophilized, ground to a fine powder, and

stored frozen until needed.

3.2. Control Soybean Material

Control soybean line Maverick had a genetic background representative of the DAS-68416-4

soybean plants, but did not contain the pat gene. Absence of PAT expression in the control

plants was confirmed by immunoassay using a PAT specific rapid lateral flow test strip. Leaves

(and some stems) of control plants were harvested, lyophilized, ground and stored under the

same conditions as the DAS-68416-4 soybean.

3.3. Reference Material

Recombinant PAT microbial protein was produced in Pseudomonas fluorescens (Pf) and purified

to homogeneity. The microbe-derived PAT protein preparation was aliquoted and stored at -80

oC to maintain activity.

3.4. SDS-PAGE and Western Blot Analysis of Crude Extracts

Lyophilized leaf tissue from event DAS-68416-4 and Maverick was mixed with PBST buffer

containing ~2.0% protease inhibitor cocktail (Sigma) and the protein was extracted by grinding

with ball bearings in a Geno-Grinder. The samples were centrifuged and the supernatants were

mixed with Laemmli sample buffer, heated and briefly centrifuged. The samples were loaded

directly on to a Bio-Rad Criterion SDS-PAGE gel. The positive reference standard, microbederived PAT, was also mixed with sample buffer and loaded on to the gel. Electrophoresis was

conducted with Tris/glycine/SDS buffer (Bio-Rad). Following electrophoresis, the gel was cut in

half, with one half stained with Pierce GelCode Blue protein stain and the other gel half was

electro-blotted onto a nitrocellulose membrane. The nitrocellulose membrane was then cut in

half with one probed with a PAT specific polyclonal rabbit antibody and the remaining half

probed with a PAT specific monoclonal antibody. A chemiluminescent substrate was used to

visualize the immunoreactive bands.

3.5. Results of the SDS-PAGE and Western Blot Analysis

The soybean-derived PAT protein was visualized by immunospecific polyclonal and monoclonal

antibodies and showed the expected band at approximately 21 kDa (Figure 56, Panel B and C).

In the PAT western blot analysis, no immunoreactive proteins were observed in the control

Maverick extract and no alternate size proteins (aggregates or degradation products) were seen in

the transgenic soybean extract. This result adds to the evidence that the protein expressed in

soybean is not post-translationally modified which would have added to the overall protein

molecular weight.
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Figure 56. SDS-PAGE and western blots of DAS-68416-4 and non-transgenic Maverick

soybean.

Note: Crude extracts from lyophilized leaf tissue of event DAS-68416-4 and Maverick were

separated by SDS-PAGE along with microbe-derived PAT and bovine serum albumin.

Following electrophoresis the gel was cut in half, one half was stained with Pierce GelCode Blue

stain for total protein and the remaining half was electro-blotted onto a nitrocellulose membrane.

The nitrocellulose membrane was then probed with PAT specific polyclonal and monoclonal

antibodies and detected with HRP-labeled antibodies. A chemiluminescent substrate was used to

visualize the immunoreactive bands

Lane Sample Amount

M Invitrogen Mark12 molecular weight markers 10 L

1 Non-transgenic (Maverick) soybean extract 40 L

2 Transgenic (Event DAS-68416-4) soybean extract 40 L

3 Microbe-derived PAT protein (TSN105742) 750 ng gel, 35 ng blot

4 Bovine serum albumin (BSA) 780 ng gel

P Novex Sharp prestained molecular weight markers 10 L

3.6. Conclusions

The PAT protein produced in DAS-68416-4 soybean was shown to be equivalent to that

produced in other transgenic crops (USDA 1996, USDA 2001, USDA 2004, USDA 2005).

3.7. References

USDA (1996) Availability of Determination of Nonregulatated status for Soybeans Genetically

Engineered for Glufosinate Herbicide Tolerance. Federal Register Volume 61, Number

160:42581-42582.

kDa

- 260 -

- 110 -

- 160 -

- 60 -

- 80 -

- 50 -

- 40 -

- 30 -

- 20 -

- 15 -

- 10 -

- 3.5 -

kDa

200 -

97.4 -

116.3 -

55.4 -

66.3 -

36.5 -

31.0 -

21.5 -

14.4 -

6.0 -

3.5 -

2.5 -

kDa

- 260

- 110

- 160

- 60

- 80

- 50

- 40

- 30

- 20

- 15

- 10

- 3.5

M 1 2 3 4 P P 1 2 3 P 1 2 3 P

Panel A: SDS-PAGE Panel B: Polyclonal Panel C: Monoclonal

antibody western blot antibody western blot

PAT dimer

PAT monomer

kDa

- 260 -

- 110 -

- 160 -

- 60 -

- 80 -

- 50 -

- 40 -

- 30 -

- 20 -

- 15 -

- 10 -

- 3.5 -

kDa

200 -

97.4 -

116.3 -

55.4 -

66.3 -

36.5 -

31.0 -

21.5 -

14.4 -

6.0 -

3.5 -

2.5 -

kDa

- 260

- 110

- 160

- 60

- 80

- 50

- 40

- 30

- 20

- 15

- 10

- 3.5

M 1 2 3 4 P P 1 2 3 P 1 2 3 P

Panel A: SDS-PAGE Panel B: Polyclonal Panel C: Monoclonal

antibody western blot antibody western blot

kDa

- 260 -

- 110 -

- 160 -

- 60 -

- 80 -

- 50 -

- 40 -

- 30 -

- 20 -

- 15 -

- 10 -

- 3.5 -

kDa

200 -

97.4 -

116.3 -

55.4 -

66.3 -

36.5 -

31.0 -

21.5 -

14.4 -

6.0 -

3.5 -

2.5 -

kDa

- 260

- 110

- 160

- 60

- 80

- 50

- 40

- 30

- 20

- 15

- 10

- 3.5

M 1 2 3 4 P P 1 2 3 P 1 2 3 P

Panel A: SDS-PAGE Panel B: Polyclonal Panel C: Monoclonal

antibody western blot antibody western blot

PAT dimer

PAT monomer
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USDA (2001) Availability of Determination of Non-regulatated status for Genetically

Engineered Corn for Insect Resistance and Glufosinate Herbicide Tolerance (Corn line 1507).

Federal Register Volume 66, Number 157:42624-42625.

USDA (2004) Availability of Determination of Nonregulated Statusfor Cotton Lines Genetically

Engineered for Insect Resistance. Federal Register Volume 69, Number 156:50154-50155.

USDA (2005) Availability of Determination of Non-regulated Status for Genetically Engineered

Corn (Corn line DAS-59122-7). Federal Register Volume 70, Number 194:58663-58664.
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Appendix 4. Methods for AAD-12 and PAT Protein Expression Analysis

4.1. Experimental Design

The experimental design included six (6) field sites; Iowa, Illinois, Indiana, Nebraska and

Ontario, Canada (2 sites) (referred to as IA, IL, IN, NE, ON1 and ON2). Each site consisted of

one plot of each treatment per block, with 3 blocks per location. Plot size was 2 rows by 25 feet.

Plots were arranged in a randomized complete block (RCB) design, with a unique randomization

at each site. Each soybean plot was bordered by 2 rows of a non-transgenic soybean of similar

maturity. The entire trial site was surrounded by a minimum of 20 feet of a non-regulated

soybean of similar relative maturity. At each location, all blocks were used for collection of

samples for expression and nutrient composition analysis.

Herbicide treatments were designed to replicate maximum label rate commercial practices.

2,4-D (Weedar 64) was applied as 3 broadcast over-the-top applications (seasonal total of

3 lb ae/A). Individual applications were at pre-emergence and approximately V4 and R2 stages.

Individual target application rates were 1.0 lb ae/A for Weedar 64 (1120 g ae/ha). Glufosinate

(Liberty) was applied as 2 broadcast over-the-top application. Application timing was at

approximately V6 and R1 growth stages. The target application rate was 0.33 lb ai/A and 0.41 lb

ai/A (374 and 454 g ai/ha).

4.2. Sample Collection

Samples were shipped to Dow AgroSciences Regulatory Science and Government Affairs

laboratories and maintained frozen until use. Samples of soybean tissues were prepared for

expression analysis by coarse grinding, lyophilizing and/or fine-grinding with a Geno/Grinder

(Certiprep, Metuchen, New Jersey).

Leaf (V5 and V10)

One leaf sample per plot, each sample containing 8 trifoliate set of leaves collected from separate

plants, were collected for each test and control entry. Each leaf sample was the youngest set of

fully expanded trifoliate leaves.

Root (R3)

One root sample (representing 3 plants) per plot were collected for each test and control entry at

the R3 stage by cutting a circle around the base of the plant. The root ball was removed and

cleaned.

Forage (R3)

One forage sample (representing 3 plants) per plot each consisting of the aerial portion (no roots)

of 3 whole plants were collected from each test and control entry.

Grain (R8 – Maturity)
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One individual sample was collected from each plot of each test and control entry. Each sample

contained approximately 500-gram of grain.

4.3. Determination of AAD-12 Protein Concentration

The AAD-12 protein was extracted from soybean tissues except grain with a phosphate buffered

saline solution with Tween-20 (PBST) and 0.75% ovalbumin (OVA). For grain, the protein was

extracted with a PBST buffer containing 0.1% Triton-100. 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 in a sandwich format. Briefly, an aliquot

of the diluted sample and a horseradish peroxidase (HRP)/anti-AAD-12 monoclonal antibody

conjugate are incubated in the wells of a microtiter plate coated with an immobilized antiAAD-12 polyclonal antibody. These antibodies bind with AAD-12 protein in the wells and form

a "sandwich" with AAD-12 protein bound between soluble and the immobilized antibodies. The

unbound samples and conjugate are 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 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 minus 650 nm was measured using a Molecular Devices Spectra Max 190 or Spectra

Max M2 plate reader. A calibration curve was generated and the AAD-12 concentration in

unknown samples was calculated from the polynomial regression equation using Soft-MAX

ProTM software which was compatible with the plate reader. Samples were analyzed in duplicate

wells with the average concentration of the duplicate wells being reported.

4.4. Determination of PAT Protein in Soybean Tissue Samples

The PAT protein was extracted from soybean tissues with a phosphate buffered saline solution

with Tween-20 (PBST) and 1% polyvinylpyrrolidone (PVP). The extract was centrifuged; the

aqueous supernatant was collected, diluted with PBST/1% PVP, and analyzed using a PAT

ELISA kit. Briefly, an aliquot of the diluted sample was incubated with enzyme-conjugated antiPAT antibody and anti-PAT 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. Since the PAT was bound in the

antibody sandwich, the level of color development was related to the concentration of PAT in the

sample (i.e., lower residue concentrations result in lower color development). The absorbance at

450 minus 650 nm was measured using a Molecular Devices Spectra Max 190 or Spectra max

M2 plate reader. A calibration curve was generated and the PAT concentration in unknown

samples was calculated from the polynomial regression equation using Soft-MAX ProTM

software which was compatible with the plate reader. Samples were analyzed in duplicate wells

with the average concentration of the duplicate wells being reported.
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Appendix 5. USDA Notifications for DAS-68416-4 Soybean

USDA

Notification

Number

Notification

Authorization

Date

Notification

Expiration

Date State(s)

Total

Number

of Trials

Planted1

Status of

Trial2

09-259-105n 9/25/2009 9/25/2010 PR TBD Pending

09-086-101n 5/30/2009 5/30/2010 IL, IN, IA, MN, MO,

NE, WI

10 Pending

09-084-110n 4/15/2009 4/15/2010 AL, AR, CA, GA, IL,

IN, IA, MI, MN, MO,

MS, NE, OH

45 Pending

09-075-105n 4/15/2009 4/15/2010 HI, IN, IA, PR 0 Pending

09-068-101n 4/13/2009 4/13/2010 AR, IL, IN, IA, MD,

MI, MO, ND, NE, OH,

PR, WI

77 Pending

09-061-104n 4/6/2009 4/6/2010 AR, IL, IN, IA, MN,

MS, NY, OH, TN

29 Pending

09-005-108n 1/1/2009 1/1/2010 HI 2 Pending

08-323-102n 12/3/2008 12/3/2009 PR 0 Pending

08-254-110n 9/26/2008 9/26/2009 PR 10 Pending

08-170-103n 6/26/2008 6/26/2009 MO 1 Pending

08-137-103n 6/5/2008 6/5/2009 MD 1 Pending

08-121-103n 5/14/2008 5/14/2009 IA 1 Submitted

08-121-102n 5/15/2008 5/15/2009 IL, IN, MO, NE, OH 11 Submitted

08-071-107n 4/14/2008 4/14/2009 CA, IL, IN, IA, MN,

MN, NE

22 Submitted

07-242-107n 9/30/2007 9/30/2008 PR 4 Submitted

06-292-105n 12/1/2006 12/1/2007 IN 2 Submitted

1Trials not yet planted as of October 15, 2009 are indicated as TBD (to be determined).

2

Pending reports as of December 1, 2009 to be submitted within 6 months of the notification expiration

date.
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Appendix 6. Literature Ranges for Compositional Analysis

Literature ranges for compositional analysis of soybean grain and forage are from

ILSI (International Life Sciences Institute) (2006) ILSI Crop Composition Database.

Version 3.0

(http://www.cropcomposition.org/)

OECD (2001) Consensus Document on Compositional Considerations for New Varieties

of Soybean: Key Food and Fee Nutrients and Anti-Nutrients.

Table 37. Summary of literature values for proximates in soybean forage.

Tissue/Component OECD ILSI

Proximate (% DW)

Moisture (% FW) NA 73.5-81.6

Protein 11.2-17.3 14.38-24.71

Total Fat 3.1-5.1 1.302-5.132

Ash 8.8-10.5 6.718-10.782

Carbohydrates (calculated) NA 59.8-74.7

Fiber (% DW)

Neutral Detergent Fiber (%) 34-40 NA

Acid Detergent Fiber (%) 32-38 NA

Minerals (% DW)

Calcium NA NA

Phosphorus NA NA

NA – Literature Values Not Available

FW=Fresh Weight; DW=Dry Weight

Table 38. Summary of literature values for proximates in soybean grain.

NA – Literature Values Not Available

FW=Fresh Weight; DW=Dry Weight

Tissue/Component OECD ILSI

Proximate (% DW)

Moisture (% FW) NA 4.7-34.4

Protein 32-43.6 33.19-45.48

Total Fat 15.5-24.7 8.1-23.56

Ash 4.5-6.4 3.89-6.99

Carbohydrates (calculated) 31.7-31.8 29.6-50.2

Cholesterol NA NA

Fiber (% DW)

Neutral Detergent Fiber 10.0-14.9 8.53-21.25

Acid Detergent Fiber 9-11.1 7.81-18.61

Total Dietary Fiber NA NA
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Table 39. Summary of literature values for amino acids in soybean grain.

Amino Acids

OECD

(% DW)

ILSI

(% DW)

Aspartic Acid NA 3.81-5.12

Threonine 1.4-1.89 1.14-1.86

Serine NA 1.11-2.48

Glutamic Acid NA 5.84-8.20

Proline NA 1.69-2.28

Glycine NA 1.46-2.00

Alanine NA 1.51-2.10

Cysteine 0.45-0.67 0.370-0.808

Valine 1.5-2.44 1.60-2.20

Methionine 0.5-0.67 0.431-0.681

Isoleucine 1.76-1.98 1.54-2.08

Leucine 2.2-4.0 2.59-3.62

Tyrosine NA 1.02-1.61

Phenylalanine 1.6-2.08 1.63-2.35

Lysine 2.5-2.66 2.29-2.84

Histidine 1.0-1.22 0.88-1.18

Arginine 2.45-3.1 2.29-3.40

Tryptophan 0.51-0.67 0.356-0.502

NA – Literature Values Not Available

DW=Dry Weight

Table 40. Summary of literature values for isoflavones in soybean grain.

Isoflavones (μg/g) OECD ILSI

Daidzein NA 60-2453.5

Glycitein NA 15.3-310.4

Genistein NA 144.3-2837.2

Daidzin NA NA

Glycitin NA NA

Genistin NA NA

NA – Literature Values Not Available
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Table 41. Summary of literature values for fatty acids in soybean grain.

Fatty Acids

ILSI

(% Total FA)

8:0 Caprylic 0.148

10:0 Capric NA

12:0 Lauric 0.082-0.132

14:0 Myristic 0.071-0.238

14:1 Myristoleic 0.121-0.125

15:0 Pentadecanoic NA

15:1 Pentadecenoic NA

16:0 Palmitic 9.55-15.77

16:1 Palmitoleic 0.086-0.194

17:0 Heptadecanoic 0.085-0.146

17:1 Heptadecenoic 0.073-0.087

18:0 Stearic 2.70-5.88

18:1 Oleic 14.3-32.2

18:2 Linoleic 42.3-58.8

18:3 γ-Linolenic NA

18:3 Linolenic 3.00-12.52

20:0 Arachidic 0.163-0.482

20:1 Eicosenoic 0.140-0.350

20:2 Eicosadienoic 0.077-0.245

20:4 Arachidonic NA

20:3 Eicosatrienoic NA

22:0 Behenic 0.277-0.595

NA – Literature Values Not Available

FA – Fatty Acids

Table 42. Summary of literature values for vitamins in soybean grain.

Vitamins (mg/kg) ILSI

Thiamine Hydrochloride 1.01-2.54

Riboflavin/Vitamin B2 1.90-3.21

Niacin/Vitamin B3 NA

Pyridoxine HCl NA

Folic Acid 2.39-4.71

Pantothenic acid NA

Vitamin B12 NA

Vitamin D NA

Vitamin C NA

Vitamin A NA

NA – Literature Values Not Available
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Table 43. Summary of literature values for minerals in soybean grain.

Minerals (mg/100g) ILSI

Calcium 116.55-307.1

Copper NA

Iron 5.54-10.95

Magnesium 219.4-312.8

Manganese NA

Phosphorus 506.7-935.2

Potassium 1868.01-2316.14

Sodium NA

Zinc NA

Iodine NA

Minerals (ppb)

Chromium NA

Selenium NA

Molybdenum NA

NA – Literature Values Not Available

Table 44. Summary of literature values for anti-nutrients in soybean grain.

Anti-Nutrients OECD ILSI

Phytic Acid (% DW) 1.0-2.74 0.63-1.960

Raffinose (% DW) NA 0.212-0.661

Stachyose (% DW) NA 1.21-3.50

Lectin (H.U./mg)* NA 0.105-9.038

Trypsin Inhibitor (TIU/mg)** NA 19.59-118.68

NA – Literature Values Not Available

*H.U. - Hemagglutinating Unit

**TIU - Trypsin Inhibitor Unit

DW – dry weight

Table 45. Summary of literature values for tocopherols in soybean grain.

OECD ILSI

Alpha Tocopherol NA 1.9-61.7

Beta Tocopherol NA NA

Gamma Tocopherol NA NA

Delta Tocopherol NA NA

NA – Literature Values Not Available
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Appendix 7. Glyphosate, 2,4-D, Glufosinate and Herbicide Resistant Weeds

7.1. 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-resistant 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 and for capturing market share. This

was soon realized with development of glyphosate- and glufosinate-tolerant crops. Since these

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, 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.

Herbicide-tolerant crops which were available to farmers in 2005 are listed in Table 46 (Duke,

2005). Transgenes were only used to confer tolerance to bromoxynil, glufosinate, and

glyphosate. The bromoxynil-tolerant crops are no longer sold. This leaves only glyphosate- and

glufosinate-tolerant transgenic crops, and of those, glyphosate has had a strongest impact on

weed management (Duke, 2005).

Table 46. Herbicide-tolerant crops available to farmers in North America in 2005.

Herbicide Crop Year Available

Bromoxynil Cottonb

1995

Canolab2000

Cyclohexanediones (sethoxydim)ab Corn 1996

Glufosinate

Canola 1995

Corn 1997

Cotton 2004

Glyphosate

Soybean 1996

Canola 1996

Cotton 1997

Corn 1998

Imidazolinonesa

Corn 1993

Canola 1997

Wheat 2002

Rice 2002

SulfonylureasaSoybean 1994

Triazinesa Canola 1984

aNot transgenic, bNo longer available by 2005
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Growers choose glyphosate-tolerant crops because it makes weed control easier and more

effective, increases profit, requires less tillage, and does 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 57. U.S. adoption rates of glyphosate-tolerant soybean,

cotton and corn.). The planting of glyphosate-tolerant crops has increased steadily since their

introduction in 1996 (glyphosate-tolerant soybean plantings are currently >90% of all soybean

planting in the US).

Figure 57. U.S. adoption rates of glyphosate-tolerant soybean, cotton and corn.

Many growers now rely only on glyphosate for their weed control in these crops (Foresman and

Glasgow, 2008; Gustafson, 2008). This has significantly increased selection pressure for

glyphosate-resistant weeds. Thus, introduction of combined event products (“stacks”) which are

tolerant to glyphosate plus at least one other class of herbicides is needed as a tool to delay

selection for glyphosate-resistant weed species.

7.2. Characteristics of Glyphosate, 2,4-D, and Glufosinate Herbicides

Glyphosate

Glyphosate is a nonselective, foliar applied herbicide which is registered for use on over one

hundred food and feed crops, several non-food field crops (fallow, fencerows, woody

ornamentals, golf courses, etc.), forestry uses (conifer release and reforestation), and non-crop

areas where total vegetation control is desired (aquatics, rights of way, industrial sites, etc.) (US

EPA, 1993). When applied at lower rates, glyphosate also is a plant growth regulator. Pre-plant

or pre-emergence uses of glyphosate in food and feed crops include most of the major

agricultural crops around the globe, including alfalfa, barley, buckwheat, corn, dry beans, grass

forage/fodder/hay, lentils, millet, oats, pastures, rye, sorghum, soybeans, and wheat. In addition,

0

10

20

30

40

50

60

70

80

90

100

1996 1997 1998 1999 2000 2001 2002 2003 2004 2005 2006 2007 2008

%

of A

cre

s

RR Soybean RR Cotton RR Corn Source: DMRKynetec

Estimate for 1997 Cotton
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it can be foliar applied over-the-top of recent glyphosate-tolerant transgenic crops, including

corn, soybeans, cotton, canola, and sugarbeets.

Glyphosate is absorbed relatively rapidly through plant surfaces (Duke and Powles, 2008a).

Leaf uptake rates vary considerably between species, accounting for at least some of the

difference in glyphosate susceptibility between species. Once in the plant, glyphosate moves in

the phloem with sugar to the growing points. Foliar post-emergence applications of glyphosate

at 560 to 1120 g acid equivalent/hectare (ae/ha) control a broad spectrum of grass, broadleaf, and

sedge weeds in agronomic crops.

The mode of action for glyphosate is unique in that it is the only herbicide that is highly effective

at inhibiting an essential plant enzyme called EPSPS (5-enolpyruvylshikimate-3-phosphate

synthase), which produces EPSP from shikimate-3-phosphate and phosphoenolpyruvate in the

shikimic acid pathway (Vencill, 2002). Many assume that this EPSPS inhibition leads to

depletion of the aromatic amino acids tryptophan, tyrosine, and phenylalanine (Duke and

Powles, 2008a). However, others support the view that increased carbon flow to the shikimate

pathway by deregulation of the pathway by inhibiting EPSPS results in shortages of carbon for

other essential pathways (Siehl, 1997). The EPSPS of all higher plants appears to be inhibited by

glyphosate (Duke and Powles, 2008a), which makes it nonselective with activity on a wide range

of plant species.

In general, glyphosate is an environmentally benign molecule (Franz et al., 1997). It is degraded

microbially in soil and water. Glyphosate binds tightly to most types of soil, so it is not available

for uptake by roots of nearby plants (Monsanto, 2005). Therefore, even though glyphosate has a

typical field half life of 47 days, crops can be planted immediately after application due to its

strong adsorption to soil. Glyphosate is not appreciably metabolized in plants when applied at

normal use rates for weed control. It is slowly metabolized to amino methylphosphonic acid

(AMPA) (FAO, 1997; Vencill, 2002). Glyphosate is also one of the least toxic pesticides to

animals (Duke and Powles, 2008a). The enzyme, EPSP synthase, is not present in humans or

animals, which contributes to the low risk to human health when glyphosate is used according to

label directions (Monsanto, 2005).

2,4-Dichlorophenoxyacetic Acid (2,4-D)

2,4-Dichlorophenoxyacetic acid (2,4-D) was introduced in 1946 as the first selective herbicide

and rapidly became the most widely used herbicide in the world (Industry Task Force II, 2005).

Today, it is still the third most widely used herbicide in the U.S. and Canada, as well as the most

widely used herbicide globally. Approximately 46 million pounds of 2,4-D is used domestically

in the U.S. annually, with 30 million pounds (66%) used by agriculture and 16 million pounds

(34%) used in non-agriculture settings such as pasture/rangeland and lawn/garden (US EPA,

2006). 2,4-D is an ingredient in approximately 660 agricultural and home use products as a sole

active ingredient and in conjunction with other active ingredients. Agriculturally, it is used on a

variety of crops including corn, rice, sorghum, sugar cane, wheat, rangeland and pasture as well

as being used on rights-of-way, roadsides, non-crop areas, forestry, lawn and turf care and on

aquatic weeds (Industry Task Force II, 2005). A major use today of 2,4-D is in combination with

other herbicides because it economically enhances the weed control spectrum of many other

herbicides such as glyphosate, dicamba, mecoprop, ALS herbicides, etc (US EPA, 2006). 2,4-D
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controls many broadleaf weeds including carpetweed, dandelion, cocklebur, horseweed, morning

glory, pigweed sp., lambsquarters, ragweed spp., shepherd’s-purse and velvetleaf. It has little to

no activity on grasses (Industry Task Force II, 2005).

In over 60 years since its discovery, probably few other compounds have been as thoroughly and

extensively evaluated for health and safety as 2,4-D. There have been more than 40,000 research

studies conducted and more than 140 peer-reviewed published epidemiologic studies specific to

2,4-D. In August, 2005, the U.S. Environmental Protection Agency (EPA) completed its

reregistration assessment of 2,4-D. The EPA concluded that 2,4-D does not present risks of

concern to human health when users follow its product instructions (US EPA, 2006). 2,4-D data

has been reviewed by more than a dozen government and expert panels since 1986 and not one

regulatory agency has ever identified 2,4-D as a human carcinogen.

The mode of action of 2,4-D is described as an “auxin mimic”, meaning that it kills the target

weed by mimicking auxin plant growth hormones like IAA. Auxins and synthetic auxinic

herbicides all regulate virtually every aspect of plant growth and development (Mockaitis and

Estelle, 2008). At low doses, auxinic herbicides possess similar hormonal properties to natural

auxin (Kelley and Riechers, 2007). However, as rates increase, they can cause various growth

abnormalities in sensitive dicots. Observable plant responses to 2,4-D can include epinasty, root

growth inhibition, meristematic proliferation/callusing, leaf cupping/narrowing, stem cracking,

adventitious root formation, senescence, and chlorosis. This uncontrolled and disorganized plant

growth eventually leads to plant death when applied at effective doses (Tu et. al., 2001).

IAA and auxin herbicides work through stimulation of the ubiquination and degradation of the

Aux/IAA family of transcriptional regulators. Degradation of these Aux/IAA proteins results in

derepression of auxin-regulated genes that in turn leads to the physiological and morphological

events associated with auxin action (Mockaitis and Estelle, 2008; Walsh et al., 2006; Kelley and

Riechers, 2007). Stated another way, high concentrations of IAA or auxin herbicides (like

2,4-D) promote ubiquitin mediated degradation of Aux/IAA protein repressors, which permits

auxin response factor (ARF) dependent transcription of auxin-regulated genes. This results in

“uncontrolled” growth which leads to plant death in susceptible species.

In the environment, 2,4-D is mainly degraded by soil microorganisms. Once it contacts soils, all

2,4-D forms are rapidly converted to the acid form and thus, the rate of soil dissipation is often

the same as for the acid (Tu et al., 2001). 2,4-D has a relatively short soil half-life and no

significant carryover effects to subsequent crops are encountered, adding to 2,4-D’s herbicidal

utility. 2,4-D has different levels of selectivity on certain plants, i.e., dicots are more sensitive

than monocots. Differential metabolism of 2,4-D by different plants is one explanation for

varying levels of selectivity. In general, plants metabolize 2,4-D slowly, so varying plant

response to 2,4-D may be more likely explained by different activity at the target site(s). Plant

metabolism of 2,4-D typically occurs via a two-phase mechanism of hydroxylation followed by

conjugation with amino acids or glucose (Vencill, 2002).

Glufosinate

Glufosinate was first reported as a herbicide in 1981 (Vencill, 2002). Glufosinate

(phosphinothricin; DL-homoalanin-4-yl(methyl)phosphinic acid) is a racemic phosphinic amino
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acid (Vencill, 2002). Its ammonium salt (glufosinate-ammonium) is widely used as a nonselective herbicide and is the active ingredient of the commercial herbicide formulations, Liberty

and Ignite. The L-isomer of glufosinate is a structural analogue of glutamate and, therefore, is a

competitive inhibitor of the enzyme glutamine synthetase (GS) of bacteria and plants. The

D-isomer is not a GS inhibitor and is not herbicidally active.

Due to the inhibition of GS, non-tolerant plant cells accumulate large amounts of toxic ammonia

produced by nitrate assimilation and photorespiration and the level of available glutamine drops

(OECD, 2002). Damage of cell membranes and inhibition of photosynthesis are followed by

plant cell death.

In genetically modified glufosinate-tolerant plants (OECD, 2002), the L-isomer of glufosinate is

rapidly metabolized by the action of the enzyme phosphinothricin acetyltransferase (PAT) into

the non-phytotoxic stable metabolite N-acetyl-L-glufosinate (2-acetamido-4-methylphosphinicobutanoic acid). N-acetyl-L-glufosinate does not inhibit glutamine synthetase. Therefore, no

phytotoxic physiological effects are observed in genetically modified glufosinate-tolerant plants.

Glufosinate is a contact herbicide which is taken up by the plant primarily through the leaves.

There is no uptake from the soil through the roots, presumably because of the rapid degradation

of glufosinate by soil microorganisms. There is limited translocation of glufosinate within the

plant.

Glufosinate is a nonselective herbicide (Vencill, 2002). It controls a broad spectrum of annual

and perennial grasses and broadleaf weeds. Due to its limited systemic action, there is no

enduring effect on perennial weeds. Weeds which emerge after herbicide application are not

affected.

Glufosinate is rapidly broken down in soil due to microbial degradation (Vencill, 2002). At

20°C, the soil half life is less than 7 days. The end products of microbial degradation are CO2

and natural phosphorus compounds.

7.3. 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 very slow, and the

levels of resistance would be very low (Bradshaw et al., 1997). This was based on the amount of

glyphosate applied over many years, the repeated applications made to many 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 glyphosate-only weed

control, which resulted in increased selection pressure for glyphosate-resistant weeds.
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Table 47 shows a summary of the sixteen glyphosate-resistant weed species that have been

reported from 1996 to November 2009. These data clearly show that glyphosate-resistance in

weeds is expanding around the globe. Most notably, there have been reports of nine new weed

species with some biotypes resistant to glyphosate in the U.S. since 2000 (Figure 58). Two of

these glyphosate-resistant weed species have already become a significant problem for farmers

across a large geographic area. Conyza canadensis infests at least two million hectares of

glyphosate-tolerant crops in the U.S. (Main et al., 2004) and glyphosate-resistance in Palmer

amaranth has serious ramifications for future weed management in the Southeast U.S. due to its

rapid growth rate, extremely competitive nature, and resistance to other herbicide modes of

action (Culpepper et al., 2008). Researchers have also reported that individual biotypes of seven

of the 16 glyphosate-resistant species are also resistant to herbicides with other modes of action

(Table 48). The Palmer amaranth, common waterhemp, and Conyza canadensis biotypes were

reported in the corn and soybean growing states. Although herbicide options to control these

biotypes with multiple herbicide resistance will be more limited, 2,4-D is still a viable control

option for the broadleaf weeds.

Table 47. Weed species with reported glyphosate-resistant biotypes.

(Heap, 2009)

First Confirmed

Report

Later

Confirmed

Common Name Species Name Year Country** Reports

Rigid ryegrass Lolium rigidum 1996 Australia USA, S.Africa,

France, Spain

Goosegrass Eleusine indica 1997 Malaysia Colombia

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

Johnsongrass* Sorghum halepenses 2005 Argentina USA

Sourgrass Digitaria insularis 2006 Paraguay Brazil

Wild poinsettia* Euphorbia heterophylla 2006 Brazil

Junglerice Echinochloa colona 2007 Australia

Liverseedgrass Urochloa panicoides 2008 Australia

* Important weeds in US corn, soybean, and cotton production.

** Nine new species confirmed resistant in US since 2000.
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Figure 58. Number of glyphosate-resistant weeds reported globally by year from 1996 to

2008.

(Compiled from Heap, 2009)

0

2

4

6

8

10

12

14

16

18

1996199719981999200020012002200320042005200620072008

#

of Gly-R

es W

e

e

d

Species

Table 48. Global reports of glyphosate-resistant weed biotypes with resistance to other herbicide

modes of action.

(Heap, 2009)

Common Name Species Name Year – Country (State)

Multiple Resistance to

Other Herbicide

MOAs

Palmer amaranth Amaranthus palmeri 2008 – US (MS) ALS

Common waterhemp Amaranthus rudis 2005 – US (MO) ALS, PPO

2006 – US (IL) ALS

Horseweed Conyza Canadensis 2003 – US (OH) ALS

2007 – US (MS) Bipyridiliums

Goosegrass Eleusine indica 1997 – Malaysia ACCase

Wild poinsettia Euphorbia heterophylla 2006 – Brazil ALS

Italian ryegrass Lolium multiflorum 2002 – Chile ALS

Rigid ryegrass Lolium rigidum 1999 - Australia ACCase, ALS,

Dinitroanilines

2003 – S.Africa ACCase, Bipyridiliums

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., 2007). 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 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).
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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). Coble and Warren (1997) demonstrated that continuous use of glyphosate

caused an increase in the infestation of morningglory (Ipomoea spp.) species over a three year

period compared with other herbicide programs. Some common hard to control weed species

that could become “weed shifts” in U.S. corn and soybeans are listed below in Table 49.

Table 49. Potential weed shifts with use of glyphosate in U.S. corn and soybeans.

(Duke and Powles, 2008b; Owen, 2008)

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

Morningglory 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

2,4-D Resistance

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, 2009). Today, a total of sixteen weed species have

documented reports of 2,4-D resistant biotypes someplace around the globe (Table 50). Wild

carrot in soybeans and roadsides, field bindweed in cropland, and prickly lettuce in cereals are

the only ones reported on the U.S. mainland (Heap, 2009). Wild carrot, yellow bur-head, 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 ALS-inhibiting

herbicides. It is notable that most of these resistant species do not appear to be spreading, as

indicated by few reports of additional sites after the initial report.
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Table 50. Weed species with reported 2,4-D-resistant biotypes.

(Heap, 2009)

Common Name Species Name Herbicide(s) Year

Country

or 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, bensulfuronmethyl1998 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, metsulfuronmethyl2005 Australia

Prickly lettuce Lactuca serriola 2,4-D, dicamba,

MCPA

2007 Washington

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 60 years of use is low compared with

other herbicide families such as the ALS inhibitors (imidazolinones, sulfonylureas, and

sulfonamides), triazines, and ACCase herbicides in a much shorter period of use (Section IX-F.1,

Figure 49). Furthermore, there is no widespread resistance to auxinic 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
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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

broadly: 2,4-D (phenoxy auxins), fluroxypyr (pyridyloxy auxin), dicamba (benzoate structure) or

clopyralid (picolinate structure).

To summarize, selection for auxin resistant weed bioytpes after more than 60 years of use has

been slow, none show significant spread from initial sites, none are of significant economic

importance, and none have been found in corn fields to date. Use of 2,4-D in DAS-68416-4

soybeans should not result in 2,4-D resistant weeds becoming significant issue in soybeans.

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 no reports of weed biotypes which have developed resistance to glufosinate

(Heap, 2009). Thus, glufosinate is an excellent tool to include in a weed management program.

7.4. References

Bourdot, G.W., Saville, D.J., and Hurrell, G.A. (1996) Ecological fitness and the decline of

resistance to the herbicide MCPA in a population of Ranunculus acris. Journal of Applied

Ecology 33:151-160.

Bradshaw, L.D., Padgette, S.R., Kimball, S.L. and Wells, F.H. (1997) Perspectives on

Glyphosate Resistance. Weed Technology 11:189-198.

Coble, H.D. and Warren, L.S. (1997) Weed control investigations in corn, cotton, crop rotations,

soybean, small grain. Annual Report, Department of Crop Science, North Carolina State

University 28:103-113.

Cranston, H.J., Kern, A.J., Hackett, J.L., Miller, E.K., Maxwell, B.D., and Dyer, W.E. (2001)

Dicamba resistance in kochia. Weed Science 49:164-170.

Culpepper, A.S., Whitaker, J.R., MacRae, A.W. and York, A.C. (2008) Distribution of

Glyphosate-Resistant Palmer Amaranth (Amaranthus palmeri) in Georgia and North Carolina

during 2005 and 2006. Journal of Cotton Science 12:306-310.

Curran, B., Sprague, C., Stachler, J. and Loux, M. (2007) The Glyphosate, Weeds, and Crops

Series: Biology and management of common lambsquarters (GWC-11).

(http://www.glyphosateweedscrops.org/Info/GWC-11.pdf)
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Duke, S.O. (2005) Taking stock of herbicide-resistant crops ten years after introduction. Pest

Management Science 61:211-218.

Duke, S.O. and Powles, S.B. (2008a) Editorial: Glyphosate-resistant weeds and crops. Pest

Management Science 64:317-318.

Duke, S.O. and Powles, S.B. (2008b). Mini-review – Glyphosate: a once-in-a-century herbicide.

Pest Management Science 64:319-325.

FAO (1997) Pesticide Residues in Food – Glyphosate report.

(http://www.fao.org/docrep/W8141E/w8141e0u.htm)

Foresman, C. and Glasgow, L. (2008) Grower perception and experiences with glyphosateresistant weeds. Pest Management Science 64:388-391.

Franz J.E., Mao, M.K, Sikorski, J.A. (1997) Glyphosate: A Unique and Global Herbicide. ACS

Monograph No. 189. American Chemical Society, Washington, DC, 653 pp.

Green, J.M. (2009) Evolution of glyphosate-resistant crop technology. Weed Science 57:108-

117.

Gustafson, D.I. (2008) Sustainable use of glyphosate in North American cropping systems. Pest

Management Science 64:409-416.

Heap, I.M. (1997) The occurrence of herbicide-resistant weeds worldwide. Pesticide Science

51:235-243.

Heap, I. (2009) The International Survey of Herbicide Resistant Weeds.

(http://www.weedscience.com)

Hite, G.A., King, S.R., Hagood, E.S. (2007) Response of selected Virginia common

lambsquarters (Chenopodium album) biotypes to glyphosate. Weed Science Society of America

Abstracts 47:22.

Industry Task Force II on 2,4-D Research Data: Issue Backgrounder – What is 2,4-D (2005)

(http://www.24d.org/background/Backgrounder-What-is-24D-Dec-2005.pdf)

Jasieniuk, M., Brule-Babel, A.L. and Morrison, I.N. (1996) The evolution and genetics of

herbicide resistance in weeds. Weed Science 44:176-193.

Kelley, K.B. and Riechers, D.E. (2007) Recent developments in auxin biology and new

opportunities for auxinic herbicide research. Pesticide Biochemistry and Physiology 89:1-11.

Kern, A.J., Chaverra, M.E. and Dyer, W.E. (2005) Dicamba-responsive genes in herbicideresistant and susceptible bioytpes of kochia (Kochia scoparia). Weed Science 53:139-145.
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Main, C., Mueller, T.C., Hayes, R.M., and Wilkerson, J.B. (2004) Response of selected

horseweed (Conyza canadensis) populations to glyphosate. Journal of Agricultural and Food

Chemistry 52:879-883.

Mockaitis, K. and Estelle, M. (2008) Auxin receptors and plant development: A new signaling

paradigm. Annual Review of Cell Developmental Biology 24:55-80.

Monsanto (2005). Backgrounder: History of Monsanto’s Glyphosate Herbicides.

(http://www.monsanto.com/monsanto/content/products/productivity/roundup/back_history.pdf)

OECD (2002) Module II - Herbicide Biochemistry, Herbicide Metabolism and the Residues in

Glufosinate-Ammonium (Phosphinothricin)-Tolerant Transgenic Plants.

(http://www.olis.oecd.org/olis/2002doc.nsf/LinkTo/NT00002CCE/$FILE/JT00125605.PDF)

Owen, M.D.K. (2001) World maize/soybean and herbicide resistance. Pages 101-163 in S. B.

Powles and D. L. Shaner, eds. Herbicide Resistance and World Grains. New York: CRC.

Owen, M.D.K. (2008) Review: Weed species shifts in glyphosate-resistant crops. Pest

Management Science 64:377-387.

Powles, S.B., Lorraine-Colwill, D.F. , Dellow, J.J. and Preston, C. (1998) Evolved resistance to

glyphosate in rigid ryegrass (Lolium rigidum) in Australia. Weed Science 46:604-607.

Shaner, D.L. (2000) The impact of glyphosate-tolerant crops on the use of other herbicides and

on resistance management. Pest Management Science 56:320-326.

Siehl, D.L. (1997) Inhibitors of EPSP synthase, glutamine synthase and histidine synthesis, in

Herbicide Activity: Toxicology, Biochemistry and Molecular Biology, ed. by Roe R.M., Burton

J.D. and Kuhr R.J. IOS Press, Amsterdam, The Netherlands, pp. 37-67.

Thill, D.C. and Lemerle, D. (2001) World wheat and herbicide resistance. Pages 61-70 in S. B.

Powles and D. L. Shaner, eds. Herbicide Resistance and World Grains. New York: CRC.

Tu, M., Hurd, C. and Randall, J.M. (2001) Weed Control Methods Handbook, The Nature

Conservancy.

(http://www.invasive.org/gist/handbook.html)

US EPA (1993) Glyphosate Reregistration Eligibility Decision (RED). EPA-738-F-93-011.

(http://www.epa.gov/oppsrrd1/REDs/old_reds/glyphosate.pdf)

US EPA (2006) Pesticides Reregistration, 2,4-D RED Facts. 738-R-05-002

(http://www.epa.gov/oppsrrd1/REDs/factsheets/24d_fs.htm.)

Van Eerd, L.L., Stephenson, G.R., Kwiatkowski, J., Grossmann, K., and Hall, C. (2005)

Physiological and biochemical characterization of quinclorac resistance in a false cleavers

(Galium spurium L.) biotype. Journal of Agriculture and Food Chemistry 53:1144-1151.
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Vencill, W.K. (2002) Herbicide Handbook (8th edition) ed. Lawrence, KS: Weed Science Society

of America.

Walsh, T.A., Neal, R., Merlo, A.O., Honma, M., Hicks, G.R., Wolff, K., Matsumura, W. and

Davies, J.P. (2006) Mutations in an auxin receptor homolog AFB5 and in SGT1b confer

resistance to synthetic picolinate auxins and not to 2,4-Dichlorophenoxyacetic acid or indole-3-

acetic acid in Arabidopsis. Plant Physiology 142:542-552.
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Appendix 8. Stewardship of Herbicide Tolerant DAS-68416-4 Soybeans

Dow AgroSciences (DAS) takes product stewardship seriously and recognizes the importance of

ongoing stewardship regarding both our agricultural chemical and biotechnology trait products.

Dow AgroSciences will effectively steward DAS-68416-4 soybeans through both the agricultural

chemical and seed business units using a variety of means with our sales force, commercial

channels and grower customers.

8.1. Communication to Agricultural Chemical and Seed Customers

Technical Bulletins

Dow AgroSciences creates comprehensive technical bulletins on biotechnology and chemical

products including elements on resistance management strategies, and ensures those are available

to our channel and grower customers as well as available on our DAS customer websites. These

bulletins help communicate key elements of the technology and how the products need be used

effectively. The technical bulletins are also utilized with university cooperators, seed &

chemical agronomists, crop consultants and other technical professionals.

Direct Mail

Dow AgroSciences uses direct mail newsletters sent on a monthly basis through both our

chemical products business and seed brands. The Mycogen seed brand sends six “Let’s Talk

Agronomy” newsletters per year. These newsletters go directly to our farmer customers

highlighting agronomics and production practices, as well as information regarding traits and

other new technologies. The DAS chemical products newsletters contain product usage

guidelines, positioning and best use practices. DAS has highlighted weed resistant management

strategies and proper stewardship, and in the future will highlight proper stewardship of

herbicide programs compatible with DAS-68416-4 soybeans through both chemical products and

seed brands newsletters.

Sales Literature

Dow AgroSciences also uses sales literature to promote our products and position proper usage.

DAS uses this sales literature to properly position both trait and herbicide technologies properly

as well as highlight weed resistance management strategies for effective use of the technology.

Information on Websites

The Dow AgroSciences seed brands websites focus on agronomic and proper hybrid placement.

The websites also contain a specific section devoted to trait stewardship and following specific

guidelines in planting transgenic traits (see the Mycogen Seed website,

http://www.mycogen.com ). In the future, websites can also provide information on

DAS-68416-4 soybeans that will feature trait and herbicide stewardship programs. The websites

will also contain links to technical bulletin and herbicide resistance management strategies to

provide a comprehensive resource.
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Dow AgroSciences agricultural chemicals website (http://www.dowagro.com/usag/) features

commercial and technical information and today contains information on pest resistance

management such as rotating insecticides for effective stewardship. DAS can feature herbicide

resistance management and effective stewardship of herbicide information labeled for

DAS-68416-4 soybeans upon commercial launch. Industry and university links will be included

to expose the reader to a wider range of technical information.

Dow AgroSciences also has an internal Technology Transfer website where internal technical

training and materials are posted. This information is made widely available to employees

through links and kept up to date with regular information feeds.

8.2. Trait and Herbicide Field Testing

Dow AgroSciences uses a wide variety of field trial collaborators including universities,

consultants, other biotech commercial partners and internal resources in reviewing hundreds of

field trials every year, including DAS-68416-4 soybean trials. These field trials allow Dow

AgroSciences to precisely characterize, position, and recommend the proper herbicide approach

for best long term success and efficacy for the trait and herbicide usage. These also allow us to

monitor a consistent set of field trial data over a long period of time to observe any efficacy

trends and adjust our herbicide recommendations if necessary to mitigate resistance threats.

8.3. Agricultural Chemical Labeling

Key Dow AgroSciences herbicides carry a weed resistance management statement. Figure 59 is

an example of the weed resistance management statement printed on DAS glyphosate brand

labels. Dow AgroSciences can place a similar statement on all products labeled for use in

conjunction with DAS-68416-4 soybeans to help communicate proper weed resistant

management strategies.

Figure 59. Example weed resistance management statement on DAS glyphosate brand

labels.

Weed Resistance Management

Glyphosate, the active ingredient in this product, is a group 9 herbicide (inhibitor of EPSP synthase).

Some naturally occurring weed biotypes that are tolerant (resistant) to glyphosate may exist due to genetic

variability in a weed population. Where resistant biotypes exist, the repeated use of herbicides with the

same mode of action can lead to the selection for resistant weeds. Certain agronomic practices reduce the

likelihood that resistant weed populations will develop, and can be utilized to manage weed resistance

once it occurs.

To delay the selection for glyphosate resistant weeds, use the following practices:

Herbicide Selection:

 Rotate the use of glyphosate with non-glyphosate herbicides.

 Avoid using more than two applications of a glyphosate-based herbicide in a given field over a twoyear period. Utilize tank mixes or sequential applications of herbicides with alternative modes of

action if this is not possible.

 Use herbicides with alternative modes of action for burndown applications prior to planting Roundup

Ready® crops that are likely to require more than one over-the-top application of glyphosate.
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 Apply full rates of glyphosate at the specified time (correct weed size) to minimize escapes of tolerant

weeds.

Crop Selection and Cultural Practices:

 Rotate Roundup Ready crops with conventional crops and use non-glyphosate herbicides to manage

resistant volunteers.

 Use alternative weed control practices whenever possible, such as mechanical cultivation, delayed

planting and weed-free crop seeds.

 Do not allow weed escapes to produce seeds, roots or tubers.

 Thoroughly clean plant residues from equipment before leaving fields suspected to contain resistant

weeds.

 Scout fields after application to detect weed escapes or shifts in weed species.

 Report any incidence of repeated non-performance of this product against a particular weed species to

the local retailer, county extension agent, or Dow AgroSciences representative.

Specific Directions:

 In burndown programs, always tank mix glyphosate with 2,4-D and/or other non-glyphosate

herbicide. This product may be tank mixed with the products listed provided the product tank-mixed

is registered for use on this site.

 Use soil-applied herbicides at full or reduced rates on some or all of your Roundup Ready crop fields

to provide early season weed control, allow for optimal post-emergence applications of glyphosate,

and to interrupt or delay selection for glyphosate resistant weeds.

8.4. Training and Education of Sales Representatives and Agronomists

Dow AgroSciences provides significant agronomic and herbicide usage training. This training is

conducted for new sales representatives with updates provided to all sales representatives

regularly by DAS Technical staff. Particular focus is on new products prior to launch and

ensuring proper recommendations are communicated to our customer base for long-term product

efficacy. DAS also employs extensive on-line training and includes agronomic training as

needed. The Dow AgroSciences seed brand sales force and agronomists also engage in continual

training including biotechnology trait technical training and positioning in addition to yearly

product and agronomic training. The trait training includes specifics on stewardship and proper

management of the traits. We produce product usage guides and technical use guides for current

biotechnology traits and provide them to all growers planting biotechnology traits.

8.5. Tracking Customer Satisfaction and Managing Issues

Dow AgroSciences is directly involved with farmer and distributor/retail customers on a daily

basis throughout the U.S. Customer satisfaction is of ultimate importance to the continued

success of DAS, so continual positioning and follow up on the usage of the DAS-68416-4

soybeans and associated herbicides will be important in our launch and subsequent sales. As a

result of our continual follow up directly with customers, we are able to implement and monitor

the proper usage and stewardship of this technology.

DAS also electronically tracks any weed control non-performance issues and crop injury as

serviced directly by our sales representatives. All new DAS sales representatives receive
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extensive in-field training on weed control issues and handling customer complaints. This

complaint handling and data entry helps us track any emerging issues specific to products, pests

(including resistant weeds) or crops and address them on a broad scale.

8.6. Involvement in Industry Groups

Dow AgroSciences is a participant in the Herbicide Resistance Action Committee (HRAC), an

industry-based group supported by CropLife International. HRAC focuses on encouraging

responsible attitudes towards 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 also interact with academic weed scientists in tackling weed

resistance management issues. DAS conducts joint trials at university sites as well as seeking

input from universities regarding weed management. We also participate in a wide range of

professional organizations including agronomy societies, seed trade groups, weed science

societies, the American Soybean Association and their Biotech Working Group and many other

industry organizations.