TwinLink Cotton USDA Petition

Page 1 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## Petition for Determination of Nonregulated Status for

Insect-Resistant and Glufosinate Am monium-Tolerant cotton:

TwinLinkTM cotton (even ts T304-40 x GHB119)
OECD Unique Identifier BC S-GHØØ4-7 x BCS-GHØØ5-8

The undersigned submits this petition under 7 CFR 340.6 to request that the Administrator, Animal and Plant Health Inspection Service, makes a determination that the article should not be regulated under 7 CFR 340.

Submitted by:

Alejandra L. Scott, Ph.D. Regulatory Affairs Manager Region Americas Regulatory Affairs – BioScience

BayerCropScience LP

2 T.W. Alexander Drive

P.O. Box 12014 Research Triangle Park, NC 27709 Telephone: 919-549-2159

FAX: 919-549-3929

Contributors:

Dean Bushey Martine Freyssinet

Martha Poe

Mark Rinehardt

December 4, 2008TwinLink Cotton USDA Petition

Page 2 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## SUMMARY

Bayer CropScience LP (BCS) is submitting a Petition for Determination of Nonregulated Status to the Animal and Plant Health Inspection Serv ice (APHIS) for TwinLinkTM cotton. TwinLink cotton was obtained by the traditional breeding cross of events T304-40 and GHB119. BCS requests a determination from APHIS that Twi nLink cotton, its parents, and any progeny derived

from crosses of TwinLink cotton with traditi onal cotton varieties, and any progeny derived from crosses of TwinLink cotton with transgenic cotton varieties that have also received a determination of nonregulated status, no longer be considered regulated articles under 7 CFR Part 340. TwinLink cotton (events T304-40 x GHB119) is considered a regulated article because it contains sequences from the plant pests, Agrobacterium tumefaciens , Subterranean Clover Stunt Virus, Cauliflower Mosaic Virus (CaMV) and Cassava Vein Mosaic Virus (CsVMV).

TwinLink cotton contains the stably integrated genes cry 1Ab, cry 2Ae and bar , which encode respectively the Cry1Ab, Cry2Ae and PAT proteins. The genes were introduced by Agrobacterium -mediated gene transfer. Southern blot analyses show TwinLink cotton contains one complete copy of the cry 1Ab and cry 2Ae genes, and 2 copies of the bar gene.

Bayer CropScience (BCS) has developed cotton ( Gossypium hirsutum ) plants that express two insecticidal proteins, Cry1Ab and Cry2Ae, from a common soil bacterium, Bacillus thuringiensis (B.t.) . The Cry1Ab and Cry2Ae protein are effective in controlling lepidopteran larvae such as bollworm (CBW, Helicoverpa zea ), tobacco budworm (TBW, Heliothis virenscens ) larvae and fall armyworm (FAW, Spodoptera frugiperda ) which are common pests of cotton. These pests cause severe economic damage to the cotton crop if not controlled. If controlled by chemical pesticides, there is the need for large input annually to control these pests. Small scale field trial experiments of cotton expressing the Cry1Ab and Cry2Ae proteins, conducted under notifications and/or permits granted by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS), US EPA, Argentina and Spain, have shown the plant’s ability to protect itself against these pests.

Transgenic cotton plants expressing the Cry1Ab and Cry2Ae proteins will provide an excellent addition to growers' options for insect control that reduces or eliminates the need for other insecticide inputs and fits well within an integrated pest management program. Cry1Ab is a protein familiar to the Agencies, but is has not been used in commercial cotton. Cry2Ae is a B.t. insecticidal protein with many of the common characteristics of this group that is familiar to the agency, but has not been used in commercial cotton.

In addition to the Cry1Ab and Cry2Ae proteins, TwinLink cotton also contains the PAT (phosphinothricin-acetyl-transferase) enzyme, encoded by the bar gene. This is the same protein that is in Bayer CropScience LLCotton25 and confers to the plant tolerance to the

herbicide Glufosinate Ammonium.

TwinLink cotton has been developed by BCS as an alternative insect resistant and herbicide tolerant cotton product.TwinLink Cotton USDA Petition

Page 3 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### TwinLink cotton has been field tested by BCS beginning in 2005 in adapted growing regions of the United States and winter nursery. These tests have occurred at 66 sites under field release authorizations granted by USDA APHIS (USDA authorizations: 05-035-12n; 07-044-102n; 07- 044-103n; 07-044-104n; 07-059-101n; 07-059-104n; 07-065-117n; 07-065-118n; 07-065-119n; 08-022-101n; 08-036-127n) Data collected from these field trials and laboratory analyses presented herein demonstrate that TwinLink cotton: 1) exhibits no plant pathogenic properties; 2) is no more likely to become a weed than non-modified cotton; 3) is unlikely to increase the weediness potential of any other cultivated plant or native wild species; 4) does not cause damage to processed agricultural commodities; and 5) is unlikely to harm other organisms that are beneficial to agriculture.

Therefore, BCS requests a determination from USDA APHIS that TwinLink cotton, and any progeny derived from crosses of TwinLink cott on with traditional cotton varieties, and any progeny derived from crosses of TwinLink cotton with transgenic cotton varieties that have also received a determination of nonregulated status, no longer be considered regulated articles

under 7 CFR Part 340.TwinLink Cotton USDA Petition

Page 4 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## 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 relevant data and information known to the petitioner which is unfavorable to the petition.

Alejandra L. Scott, PhD Regulatory Affairs Manager Region Americas Regulatory Affairs – BioScience

Bayer CropScience LP

2 T.W. Alexander Drive

P.O. Box 12014 Research Triangle Park, NC 27709 Telephone: 919-549-2159

FAX: 919-549-3929TwinLink Cotton USDA Petition

Page 5 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## ACRONYMS AND SC IENTIFIC TERMS

A acre a.i. active ingredient AMS Agricultural Marketing Services ANOVA ANalysis Of VAriance APHIS Animal and Plant Health Inspection Service

bar Phosphinothricin acetyltransferase gene

BBMV Brush Border Membrane Vesicles BCS Bayer CropScience BLASTn Basic Local Alignment Search Tool comparing nucleotide sequences BLASTx BLAST search of protein databases using a translated nucleotide query bp base pairs Bt Bacillus thuringiensis BXN Bromoxynil-tolerant

CaMV Cauliflower Mosaic Virus

CBW Cotton Bollworm COA Certificate of Analysis

CsVMV Cassava Vein Mosaic Virus

cm centimeter dm dry matter DMPT Demethylphosphinothricin DNA Deoxyribonucleic Acid EDTA Ethylenediaminetetraacetic acid ELISA Enzyme Linked Immunosorbent Assay ELS Extra Long Staple EPA Environmental Protection Agency ESA Endngered Species Act EU European Union FAW Fall Armyworm FDA Food and Drug Administration FGENESH Find GENES using Hidden markov model FIFRA Federal Insecticide Fungicide and

Rodenticide Act FW fresh weight

FWS Fish and Wildlife Service

g gram GetORF EMBOSS database for ORFs HPLC High Pressure Liquid Chromatography

HRP Horseradish Peroxidase

ID identification

Kb Kilobases

kDa kiloDalton kg kilogram

L liter

LB Left Border lb pound (1 pound = 0,454 kg) LC/MS Liquid Chromatography/Mass Spectroscopy LOD Limit of Detection

LOQ Limit of Quantification L-PPT Phosphinothricin

m meter
M million mg milligram

mL milliter

mm millimeter

mM millimolar μL microliter μM micromolar MS Mass Spectrometry

MSMA Monosodium acid methanearsoate μg microgram NA Not Applicable

ng nanogram ND Not Detectable: Below the limit of detection

nm nanometer OD Optical Density OECD Organization for Economic Cooperation and Development ORF Open Reading Frame PAT Phosphinothricin-acetyl-transferase

PBW Pink bollworm PCR Polymerase Chain Reaction pg picogram ppb parts per billion RAC Raw Agricultural Product RB Right Border RBS Ribosome Binding Site RR Roundup Ready

SD Standard Deviation SDS Sodium Dodecyl Sulfate SDS-PAGE Sodium Dodecyl Sulfate-Polyacrylamide gel electrophoresis SIM Selected Ion Monitoring

TAE Tris Acetate EDTA T1, T2, etc generations after T0 (transformation)

TBW Tobacco Budworm T-DNA transfer DNA from Agrobacterium

TEP Total Extractable Protein

TEP Total Extractable Protein

TM Trademark TwinLink insect resistant and glufosinate -

ammonium tolerant cotton
US/USA United States of America USDA United States Department of Agriculture WT Wild type zPCR zygosity PCRTwinLink Cotton USDA Petition

Page 6 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## TABLE OF CONTENTS

SUMMARY ........................................................................................................................ . 2

CERTIFIC ATION................................................................................................................ 4

ACRONYMS AND SCIENT IFIC TERMS............................................................................ 5

TABLE OF CO NTENTS ..................................................................................................... 6

LIST OF FI GURES ........................................................................................................... 10

LIST OF TABLES............................................................................................................. 10

I. RATIONAL E.......................................................................................................... 12

A. Basis for the Request for Determination of Non-regulated status............................ 12

B. Rationale for the development of TwinLink cotton..................................................... 12

C. Adoption of Bt cotton .................................................................................................... 12

D. Benefits of Bt cotton...................................................................................................... 13

E. Submissions to other regulatory agencies ................................................................. 13



### II. THE COTTON FAMILY ......................................................................................... 15

A. Cotton as a crop............................................................................................................. 15

B. The taxonomy of cotton ................................................................................................ 16

C. The genetics of cotton................................................................................................... 17

D. Pollination of cotton ...................................................................................................... 17

E. Weediness potential of cotton...................................................................................... 18

F. Potential for outcrossing/gene escape in cotton........................................................ 18

G. Characteristics of the recipient plant........................................................................... 20

H. Cotton as a test system in this petition ....................................................................... 20



### III. THE TRANSFORMA TION SYST EM ..................................................................... 21

A. Description of the transformation system................................................................... 21

B. Parent lines................................................................................................................... .. 21

C. Construction of the plasmids used for transformation.............................................. 21

1. Plasmid pTDL008 ..........................................................................................................................21

2. Plasmid pTEM12 ...........................................................................................................................23



### D. Open reading frames and associated regulatory regions.......................................... 25

1. Plasmid pTDL008 ..........................................................................................................................25

2. Plasmid pTEM2 .............................................................................................................................26



### E. Deduced amino acid sequences................................................................................... 27TwinLink Cotton USDA Petition

Page 7 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### IV. CHARACTERIZATION OF TWINLINK CO TTON (EVENTS T304-40 x GHB119) 28

A. Description, history and Mendelian inheritance ......................................................... 28

B. Verification of the inserts and number of copies of the inserted sequences .......... 31

1. T304-40

............................................................................................................................... ............31

2. GHB119

............................................................................................................................... ...........31



### C. Stability of the inserted DNA sequence....................................................................... 31

1. T304-40

............................................................................................................................... ............31

2. GHB119

............................................................................................................................... ...........32

3. TwinLink cotton ............................................................................................................................32



### D. Presence of marker genes and origin of replication in the vector ............................ 32

E. Absence of remaining parts of the vectors ................................................................. 33

F. The flanking regions of the inserted sequence(s) ...................................................... 33

1. T304-40

............................................................................................................................... ............33

2. GHB119

............................................................................................................................... ...........34



### V. THE Cry1Ab, Cry2Ae AND PAT PROT EINS ....................................................... 36

A. History and background................................................................................................ 36

1. Cry proteins

............................................................................................................................... ....36

2. The PAT protein ............................................................................................................................37



### B. Characterization of the Cry1Ab, Cry2Ae and PAT proteins....................................... 38

1. Biochemistry and mode of action ...............................................................................................38

2. Protein safety

............................................................................................................................... .39



### VI. EXPRESSION OF THE IN SERTED SEQUE NCES ............................................... 41

A. Protein amounts in fuzzy seed of TwinLink cotton .................................................... 41

B. Cry1Ab, Cry2Ae, and PAT protein content in plant parts and during the life cycle 42

C. Expression of other parts of the insert........................................................................ 42

D. Verification of the biochemical and functional equivalence of Cry1ab, Cry2Ae and PAT/bar proteins produced in TwinLink cotton and in E. coli .............................................. 44

1. Molecular weight (SDS-PAGE) ....................................................................................................44

2. Immuno-reactivity (Western Blot) ...............................................................................................47

3. N-terminal sequencing .................................................................................................................48

4. Peptide profile (LC-MS) ................................................................................................................49

5. Biological activity .........................................................................................................................58

6. Expected and unexpected post-translational modifications ...................................................58



### VII. DETECTION TECHNIQUES FOR TH E MODIFIED ORGANISM.......................... 64

VIII. AGRONOMIC AND PHENOT YPIC EVAL UATION ............................................... 66

A. Agronomic performance and evaluation ..................................................................... 66

B. History of field activities................................................................................................ 67

C. Agronomic performance of TwinLink cotton (events T304-40 x GHB119) ............... 67

D. Biotic and abiotic stress characteristics ..................................................................... 69TwinLink Cotton USDA Petition

Page 8 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### E. Maturity and plant phenotype characteristics............................................................. 70

F. Yield and fiber quality.................................................................................................... 72

G. Equivalence between Coker-derived and commercial varieties................................ 72

H. Composition analysis.................................................................................................... 73

I. Conclusions for agronomic evaluation of TwinLink cotton....................................... 76



### IX. ENVIRONMENTAL SAFETY/IMPACT OF NON CONTAINED USE OF

TWINLINK COTTON ........................................................................................................ 78

A. Potential for gene transfer / out crossing.................................................................... 78

1. Bio-geography ..............................................................................................................................7 8

2. Vertical gene flow .........................................................................................................................78

3. Potential of horizontal gene flow from TwinLink cotton ..........................................................79



### B. Weediness potential of TwinLink cotton ..................................................................... 79

C. Effects on non-target organisms.................................................................................. 79

1. Cry1Ab and Cry2Ae cotton ..........................................................................................................80

2. Protein degradation in soils ........................................................................................................81

3. TwinLink cotton ............................................................................................................................81

4. Summary of assessment of effect on non-target organisms ...................................................84



### D. Endangered species considerations............................................................................ 85

E. Effects on current agricultural practices in cotton..................................................... 85

1. Insect resistant cotton .................................................................................................................87

2. Glufosinate-ammonium tolerant cotton .....................................................................................88



### F. Summary of Environmental Safety / Impact on non-contained use of TwinLink

cotton......................................................................................................................... ................ 92

G. Insect Resistance Management (IRM) Plan ................................................................. 93



### X. STATEMENT OF GRO UNDS UNFAVO RABLE ................................................... 94

XI. REFERENC ES ...................................................................................................... 95

Appendix 1. FIELD TRIALS TERMINATION R EPORTS 2005- 2007.......................... 102

Termination Report 1 05-035-12n ....................................................................................... 103

Termination Report 2 07-059-104n ..................................................................................... 105

Termination Report 3 07-044-102n ..................................................................................... 109

Termination Report 4 07-044-103n ..................................................................................... 114

Termination Report 5 07-044-104n ..................................................................................... 118

Termination Report 6 07-065-117n ..................................................................................... 121

Termination Report 7 07-065-118n ..................................................................................... 123

Termination Report 8 07-065-119n ..................................................................................... 125

Termination Report 9 07-059-101n ..................................................................................... 127



### Appendix 2. MATERIALS AND METH ODS - PRODUCT CHARACTERIZATION ..... 131TwinLink Cotton USDA Petition

Page 9 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Breeding diagram for each parent of TwinLink cotton used in agronomic evaluation.... 132

Materials and methods for molecular characterization – DNA tests ................................. 133

Materials and methods for protein characterization tests.................................................. 135

Materials and methods for protein levels in seeds.............................................................. 138

Materials and methods for protein levels in plant parts and during the life cycle ........... 150

Materials and methods for agronomic equivalence studies............................................... 153

Materials and methods for efficacy studies ......................................................................... 155

Method for agronomic efficacy studies................................................................................ 157

Materials and methods for composition analysis................................................................ 162



### Appendix 3. CHARACTERIZAT ION OF TWINLI NK COTTO N................................... 164

1. Verification of the insert in Cry1Ab cotton event T304-40 ....................................... 165

2. Verification of the insert in Cry2Ae cotton event GHB119....................................... 170

3. Genetic stability of the inserted DNA in Cry1Ab cotton event T304-40 .................. 175

4. Genetic stability of the inserted DNA in Cry2Ae cotton event GHB119.................. 180

6. Absence of vector backbone in Cry1Ab cotton event T304-40 ............................... 187

7. Absence of vector backbone in Cry2Ae cotton event GHB119 ............................... 191

8. Cry1Ab cotton event T304-40 pre-insertion locus .................................................... 195

a. The flanking sequences......................................................................................................... .......195

b. Identification of potentia lly disrupted genes at t he integrati on site ...............................................196

c. Identification of potentially unintended newly-created genes in the transformation event ...........196



### 9 Cry2Ae cotton event GHB119 pre-insertion locus.................................................... 198

a. The flanking sequences......................................................................................................... .......198

b. Identification of potentia lly disrupted genes at t he integrati on site ...............................................198

c. Identification of potentially unintended newly- created gene s.......................................................199



### Appendix 4. REGIONAL AGRONOMI C DATA ........................................................... 201TwinLink Cotton USDA Petition

Page 10 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## LIST OF FIGURES

Figure 1 Vector map of pTDL008 .....................................................................................22 Figure 2 Vector map of pTEM12 ......................................................................................24 Figure 3 Deduced amino acid sequence of the PAT protein encoded from the bar

gene .....................................................................................................................27 Figure 4 Deduced amino acid sequence of the Cry1Ab protein from Bacillus thuringiensis subsp . berliner ............................................................................27 Figure 5 Deduced amino acid sequence of the Cry2Ae protein from Bacillus thuringiensis subsp . dakota .............................................................................27 Figure 6 Breeding tree for the development of TwinLink cotton .................................29 Figure 7 Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein isolated from leaves of transgenic TwinLink cotton. .....................................45 Figure 8 Comparison of the Cry2Ae protein from Bt. to the Cry2Ae protein isolated from leaves of transgenic TwinLink cotton. ....................................................46 Figure 9 Comparison of the PAT/ bar protein from E. coli to the PAT/ bar protein isolated from leaves of transgenic TwinLink cotton. .....................................47 Figure 10 GlycoStain of the Cry1Ab protein from E. coli and the Cry1Ab protein isolated from leaves of transgenic TwinLink Cotton. .....................................59 Figure 11 GlycoStain of the Cry2Ae and PAT/ bar proteins from Bt. and E. coli and the Cry2Ae and PAT/ bar proteins isolated from leaves of transgenic TwinLink
Cotton. ................................................................................................................60 Figure 12 Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein isolated from ground seed of TwinLink cotton. ..............................................61 Figure 13 Comparison of the Cry2Ae protein from Bt. to the Cry2Ae protein isolated from ground seed of TwinLink cotton. ............................................................62 Figure 14 Comparison of the PAT/ bar protein from E. coli to the PAT/ bar proteins isolated from ground seed of TwinLink cotton. ..............................................63



## LIST OF TABLES

Table 1 Cotton: Production in specified countries and the world ..............................16

Table 2 Genetic elements of vector pTDL008 ...............................................................25

Table 3 Genetic elements of vector pTEM12 ................................................................26

Table 4 Segregation Analysis of the Independent Events in TwinLink cotton. .........30

Table 5 Fresh Weight average quantities of Cry1Ab, Cry2Ae and PAT proteins in raw agricultural commodities of TwinLink cotton as detected by ELISA ....43

Table 6 Dry Weight average quantities of Cry1Ab, Cry2Ae and PAT proteins in raw agricultural commodities of TwinLink cotton as detected by ELISA ............43

Table 7 Cry1Ab isolated from TwinLink cotton trypsin digest peptide map using Electrospray LC/MS ...........................................................................................50

Table 8 Amino acid coverage of Cry1Ab from E. coli and TwinLink Cotton Leaves 51

Table 9 Selected Ion Monitoring mass spectrometry results of the Cry1Ab sequence residues with the Asparagine-X-Serine/Threonine sequence .......................52TwinLink Cotton USDA Petition

Page 11 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 10 Cry2Ae isolated from TwinLink cotton trypsin digest peptide map using Electrospray LC/MS ...........................................................................................54

Table 11 Amino acid Coverage of Cry2Ae from Bt. and TwinLink Cotton Leaves ......55

Table 12 Selected Ion Monitoring mass spectrometry results of the Cry2Ae sequence residues with the Asparagine-X-Serine/Threonine sequence .......................56

Table 13 PAT/ bar isolated from TwinLink cotton trypsin digest peptide map using Electrospray LC/MS ...........................................................................................57

Table 14 Amino acid coverage of PAT/ bar from E. coli and TwinLink cotton leaves .57

Table 15 Bt cotton production in the United States by Region ....................................66

Table 16 Summary of field activities under USDA permits for TwinLink events ........67

Table 17 Description of Agronomic Parameters Evaluated ..........................................68

Table 18 Plant stand count data individual locations in 2008. ......................................69

Table 19 Lodging data by individual location in 2007 ...................................................70

Table 20 Plant part morphology data in 2008 .................................................................70

Table 21 Maturity data from individual locations in the Southeast and Texas in 2007

.............................................................................................................................71

Table 22 Maturity data from individual locations in the Mid-south in 2007 .................71

Table 23 Maturity data from individual locations in 2008. .............................................71

Table 24 Fiber Analysis from Sellers, SC location in 2008 ............................................72

Table 25 Proximate and fibre compounds in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges) .............................................................................74

Table 26 Minerals and Vitamin E in fuzzy seed of TwinLink Cotton and the nontransgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges) ............................................................................................74

Table 27 Anti-nutrients in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges) ................................................................................................................75

Table 28 Total Amino Acids in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges) ................................................................................................................75

Table 29 Total Fatty Acids in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges) ................................................................................................................76

Table 30 Summary of Non-Target Organism Data of the Cry1Ab protein ....................80

Table 31 Summary of Non-Target Organism Data of the Cry2Ae protein ....................81

Table 32 Genetically engineered (GE) upland cotton varieties by State and United States, 2000-2008 ...............................................................................................87

Table 33 Agricultural Chemical Usage 2007 - Cotton Summary ...................................89

Table 34 Common weed Species in US Cotton Production ..........................................90

Table 35 Important Weeds Labeled for Control by Ignite® Herbicide in Cotton .........92TwinLink Cotton USDA Petition

Page 12 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## I. RATIONALE

A. Basis for the Request for Dete rmination of Non-regulated status

The United States Department of Agriculture (USDA) Animal and Plant Health Inspection Services (APHIS) is responsible for protection of the US agricultural infrastructure against noxious pests and weeds. Under the Plant Protection Act (7 USC § 7701-7772) APHIS considers plants altered or produced by genetic engineers as restricted article under 7 CFR 340 which cannot be released into the environment without appropriate approvals. APHIS provides that petitions may be filed under 7 CFR §340.6 to evaluate data to determine that a particular regulated article does not present a risk as a noxious pest or weed to the agricultural infrastructure. Should APHIS determine that the submitted article does not present a plant pest risk, the article may be deregulated and released without further restriction.



### B. Rationale for the devel opment of TwinLink cotton

Cotton is an important fiber crop globally and in the South Plains of USA is the largest contiguous cotton-growing region in the world. The USA, China and India rank as the top three cotton producing countries (USDA-FAS, 2008; Table 1). According to USDA-NASS data, cotton production in the US was 12.73M acres in 2006 and was substantially lower in 2007 and 2008, most likely due to the increased value of corn and interest in biofuel production (USDA-NASS - http://www.nass.usda.gov/QuickStats/index2.jsp) . In 2008, 7.78M acres of cotton was harvested as compared to a 20-year high of approximately 16M acres in 1995, yet the need for a high yielding crop of high value cotton fiber at the lowest production cost is ever more important. The cotton industry relies heavily on pes t control measures for successful cotton fiber production, both to control weeds and lepidopteran insects, which account for the largest cotton insect pest problem.

To that end, cotton growers in the USA have readily adopted genetically improved cotton, primarily due to reductions in pest management costs, greater yield capture, decreased pesticide costs and overall enhanced grower returns. Ninety-one percent of pesticide reduction was derived from grower adoption of herbicide tolerant crops, mainly corn, cotton and soybeans, with herbicide tolerant cotton accounting for 22% of the total pesticide reduction (Johnson et al., 2007). In 2006, 86% of all cotton in the USA expressed herbicide tolerant traits and 57% expressed insect control traits.



### C. Adoption of Bt cotton

Reduction in the levels of insecticide use due to the adoption of Bt cotton have been described in terms of lower numbers and total quantity of chemical insecticide sprays. In Arizona, 5.4 fewer insecticide applications per crop per season was reported by Carriere et al. (2001), while a total reduction of 8.7 million and 15 million sprays for the US due to the adoption of Bt cotton in the 1998 and 1999 seasons, respectively (Carpenter and Gianessi, 2001). More recently,

Johnson et al. (2007) reported a total reduction of 2.8M pounds of insecticide (~2.5% of the total US pesticide reduction) during the 2006 growing season due to insect resistant Bt cotton. On average, profitability levels increased by about $48/acre in 2006 for dual gene Bt cotton (Brookes and Barfoot, 2008) based on technology costs, reduction in insecticide use andTwinLink Cotton USDA Petition

Page 13 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### improved yield capture. The total net value for US growers by using Bt cotton in 2006 was $315.5M (Johnson et al., 2007).

Bayer CropScience (BCS) has developed a combined-trait cotton known as TwinLink cotton using conventional breeding techniques by cros sing BCS’ Cry1Ab Cotton (Event T304-40) with BCS’ Cry2Ae Cotton (Event GHB119) to assist growers in controlling lepidopteran pests of cotton. TwinLink cotton will also express a herbicide tolerant trait, based on LibertyLink technology, that can tolerate glufosinate ammonium containing herbicides. Field trial evaluations of small-scale plots infested both na turally and artificially; indicate minimal damage of TwinLink cotton with infestations of cotton bollworm (CBW, Helicoverpa zea ), tobacco budworm (TBW, Heliothis virenscens ), pink bollworm (PBW, Plutella gossypiella ), and fall armyworm (FAW, Spodoptera frugiperda ). TwinLink not only demonstrates expanded target pest control by the expression of two Cry protei ns unique to cotton, but also offers growers a superior resistance management tool for comprehensive product durability.



### D. Benefits of Bt cotton

Bacillus thuringiensis ( Bt ) is a bacterium that occurs naturally in the soil and on plants. Bt products have an excellent safety record and can be used on crops until close to the day of harvest (EPA, 2005). Btgenes have also been transferred to plants for the production of insectprotected crops, such as TwinLink cotton. Mammalian safety of pesticidal proteins incorporated into plants is described in the EPA Biopes ticides Registration Documents (2007 - http://www.epa.gov/pesticides/biopesticides/pips/ index.htm) and in various publications. The results of these studies support the lack of mammalian toxicity or allergenic potential for Cry1Ab and Cry2Ae. Taken together there will not be a significant risk of toxic or allergenic effects to humans or other animals if exposed to the Cry1 Ab or the Cry2Ae proteins as expressed in

TwinLink cotton.

In summary, based upon the data provided within this application or from previously cited data, TwinLink cotton will provide growers:

1 Improved insect efficacy for major pests of cotton, independent of weather conditions 2 Combined Bt trait product for excellent insect resistance management 3 Herbicide tolerance based on LibertyLink technology providing growers with additional weed control options 4 Excellent human, animal, non-target or ganism and environmental safety profile 5 Reduced pesticide use 6 Enhanced yield capture 7 Decreased pest management costs

Leading to an overall enhancement of grower financial returns.



### E. Submissions to other regulatory agencies

Food and Drug Administration TwinLink cotton (events T304-40 x GHB119) is within the scope of the 1992 FDA policy statement concerning regulation of products deri ved from new plant varieties, including thoseTwinLink Cotton USDA Petition

Page 14 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### developed through biotechnology (FDA, 1992). In compliance with this policy, BCS will submit to FDA a food and feed safety and nutritional assessment summary for TwinLink cotton.

Environmental Protection Agency The United States Environmental Protection Agency has authority over the use of pesticidal substances under the Federal Insecticide Fungicide and Rodenticide Act (FIFRA) as amended (7 USA §136 et. Seq.). A tolerance exemption is in place for Cry1Ab protein in all crops (40 CFR Part 180, §180.1173), as well as for the PAT protein (40 CFR Part 180, §180.1151). A temporary exemption from the requirement of a tolerance has been issued for Cry2Ae (40 CFR Part 174 §174.530). EPA Section 3 registration applications will be submitted for Cry1Ab cotton, Cry2Ae cotton and

TwinLink cotton.

Foreign Governments BCS intends to submit dossiers to the proper regulatory authorities of foreign governments request for the importation of US Cottonseed and to have regulatory processes in place. These may include submissions to the relevant Regulatory Authorities in Canada, Mexico, EU, Japan, among others. TwinLink cotton has been, or is currently, in field trials in cotton growing regions

around the world.TwinLink Cotton USDA Petition

Page 15 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## II. THE COTTON FAMILY

Cotton, Gossypium hirsutum , has been cultivated for millennia in many parts of the world. About 90 percent of the production of cotton is G. hirsutum . Cotton is primarily used worldwide for its lint. Lint is produced on the seed coat, and is spun into fine strong threads. Only the United States and a few other countries have developed major commercial uses for the seed. Raw unprocessed cottonseed may be fed to ruminants in the form of cottonseed meal and hulls or the seed can be processed for oil, the primar y component consumed by humans. Linters, the

short fibers that remain on the hulls after the removal of the lint have both edible and non-edible

use.

Cotton belongs to the genus Gossypium , which is in the Malvaceae or Mallow family. Other members of this family include okra, hollyho ck, rose of sharon, and even such plants as teaweed, spurred anoda, and velvetleaf that are weed pests in cotton. Only the genus Gossypium , and a few isolated species of the other genera, is characterized by the seed hairs or trichomes, which are outgrowths of the epidermis of the seed coat. There are 50 diverse species in the genus Gossypium , but only four of them produce commercial-type lint (Fryxell, 1992).

The tribe Gossypieae has two specific characters: the form of the embryo (which is more complex than in the balance of the Malvaceae) and the presence of distinctive punctae in various parts of the plant but especially in the cotyledons. These punctae are now known as "gossypol glands" and are distinctive in morphology and chemical contents. They are believed to be unique to the tribe (Fryxell, 1979).



### A. Cotton as a crop

Cotton, Gossypium spp. has been grown for its fiber for several thousand years. Its cultivation and manufacture into cloth developed independently in both the Eastern and Western Hemispheres. One of the oldest records of cotton textiles, dating back about 5,000 years, was found in the Indus River Valley in what is now Pakistan. Excavations in Peru and Mexico have uncovered cotton cloth identified as being 4,500 to 7,000 years old. Cotton fabrics have also been found in the remains of some of the ancient civilizations of Egypt and in the ruins of Indian pueblos of the Southwestern United States, dating back hundreds of years before Christ. Other products, such as cottonseed oil, cake, and cotton linters are by-products of fiber production.

Cottonseed, a raw agricultural product which was once largely wasted, is now converted into food for people, feed for livestock, fertilizer and mulch for plants, fiber for furniture padding and cellulose for a wide range of products from explosives to computer chip boards. Cotton is indeed nature’s food and fiber plant. Although lint is the most valuable product from a field of cotton, it is very important to keep in mind that this versatile plant is also an important vegetable oil source. From this point of view, cotton is a food crop.

Cotton, Gossypium hirsutum L., is mainly produced in China, US, India, Pakistan and Uzbekistan, with these five countries contributing to nearly 75% of world production (Table 1).TwinLink Cotton USDA Petition

Page 16 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 1 Cotton: Production in specified countries and the world

Country 1,000 Metric Tons

2004/05 2005/06 2006/07 2007/08 Est Nov
2008/09
China Peoples Republic of 6,597 6,183 7,729 8,056 7,947

India 4,137 4,148 4,746 5,356 5,443
United States 5,062 5,201 4,700 4,182 2,945

Pakistan 2,425 2,213 2,155 1,938 1,960

Brazil 1,285 1,023 1,524 1,602 1,361
Uzbekistan Republic of 1,132 1,208 1,165 1,197 1,110
Turkey 904 773 827 675 501
Other 4,898 4,632 3,713 3,238 3,307

Total 26,440 25,382 26,560 26,245 24,574 Source: USDA-Foreign Agriculture Service (http ://www.fas.usda.gov/cotton/circular/Current.asp ). 2008 estimates



### In the US for the 2007 production year, cotton was grown on 9.4 million acres, the major producing states being Texas (4.9 million acres), Georgia (1.0 million acres), Arkansas (0.9 million acres), Mississippi (0.7 million acres), North Carolina and Tennessee (0.5 million acres). The world total planted area in 2007-2008 was 33.2 million hectares, for a production of 121 million bales (USDA-FAS, 2008).

The total production of cotton as an oilseed is estimated to be 46.1 million metric tons in 2007/2008 out of a world total of 391.4 million tons. Cottonseed oil, with a production estimated at 5 million tons in 2007/08, accounts only for 3.9% of total world oil production. With 1.6 million tons for that same year, China is by far the most important producer (http://www.fas.usda.gov/oilseeds/circular/Current.asp - USDA-FAS, 2008).



### B. The taxonomy of cotton

Scientific name: Gossypium hirsutum L. Family: Malvaceae

Genus: Gossypium Species: hirsutum (2n=52, Upland cotton), barbadense (2n=52, Pima cotton), arboreum (2n=26), herbaceum (2n=26) Cultivar/breeding line: numerous varieties and breeding lines

Common name: Cotton

The predominant type of cotton grown in the United States is Gossypium hirsutum , known as American Upland. The Upland type, which usually has a staple length of 1 to 1 1/4 inches, accounts for about 97 percent of the annual US cotton crop. Upland cotton is grown throughoutthe US Cotton Belt as well as in most major cotton-producing countries. The balance of USgrown cotton is Gossypium barbadense , commonly referred to as American Pima or extra-long staple (ELS). ELS cotton, which has a staple length of 1 1/2 inches or longer, is produced predominantly in California, Arizona, New Mexico and southwest Texas, where it is particularlywell adapted to the arid environmental conditions. The markets for ELS cotton are mainly highvalue products such as sewing thread and expensive apparel.TwinLink Cotton USDA Petition

Page 17 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### C. The genetics of cotton

The genus Gossypium consists of 50 species, of which 4 to 5 are generally cultivated (Fryxell, 1992). The cultivated species are G. hirsutum, G. barbadense, G. arboreum L ., G. herbaceum

and G. lanceolatum Todaro.

At least seven genomes, designated A, B, C, D, E, F, and G, are found in the genus (Endrizzi, 1984). Diploid species (2n=26) are found on all continents, and a few are of some agricultural importance. The A genome is restricted in diploids to two species ( G. arboreum and G.

herbaceum ) of the Old World. The D genome is restricted in diploids to some species of the New World, such as G. thurberi

.

By far, the most important agricultural cottons are G. hirsutum and G. barbadense . These are both allotetraploids (2n=4x=52) of New World origin, and presumably of ancient cross between Old World A genomes and New World D genomes. How and when the original crosses occurred has been subject to much speculation. Euploids of these plants have 52 somatic chromosomes, and are frequently designated as AADD (they behave as disomic polyploids). Four additional New World allotetraploids occur in the genus, including G. tomentosum , a native of Hawaii. Due to the difference in ploidy level, G. hirsutum cannot cross with wild diploid

cottons. G. hirsutum is readily cross-compatible only with other tetraploid members of the tribe Gossypium, which includes G. tomentosum in Hawaii , G. darwinii in the Galapagos, G.

mustelinum in northeastern Brazil, G. hirsutum and G. lanceolatum in tropical/subtropical America, and G. barbadense in South America, as well as cultivated forms of G. hirsutum and

G. barbadense (Fryxell, 1979). Gossypium tomentosum has been crossed with G. hirsutum in breeding programs; however, no commercial cotton is produced in Hawaii (Jenkins, 1993).

The New World allotetraploids are peculiar in the genus, because the species, at least in their wild forms, grow near the ocean, as invaders in the constantly disturbed habitats of strand and associated environs. It is from these "weedy" or invader species that the cultivated cottons developed (Fryxell, 1979).



### D. Pollination of cotton

Gossypium hirsutum is generally considered to be a self-pollinating crop (Niles and Feaster, 1984). The morphology of cotton pollen, is heavy and somewhat sticky, does not lend itself to wind pollination. Cotton can, however, be pollinated by insects. Bees – wild bees, honeybees ( Apis mellifera ) and bumblebees ( Bombus spp.), are the primary insect pollinators. Berger et al

. (1988) have found that pollination by Bombus was more efficient than by Apis mellifera , which is consistent with the amount of pollen found in th e hexapod. Bees collect mainly the nectar from the plants, and rarely the pollen. In addition, physical isolation with plants attractive to the bees significantly reduces the potential for pollen movement, as cotton flowers have a nectar high in glucose and low in sucrose, which probably makes it slightly repellent for bees (Moffett et al

., 1976).

McGregor (1976) traced the movement of pollen from a cotton field surrounded by a large number of honeybee colonies. Movement of the pollen was traced by means of fluorescentTwinLink Cotton USDA Petition

Page 18 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### particles. McGregor found that at 150 to 200 feet away from the source plant, only 1.6 percent showed the presence of the fluorescent particles. By comparison, the isolation distances for Foundation, Registered and Certified seeds in 7 CFR Part 201 are 1320, 1320 and 660 feet, respectively.



### E. Weediness potential of cotton

In the United States, cotton ( G. hirsutum ) is not a weed pest and has no sexually compatible weedy relatives except perhaps G. tomentosum in Hawaii, Puerto Rico, the Virgin Islands and south of Tampa (Florida Route 60) in Florida. A number of references confirm the lack of weediness of cotton: Crockett, 1977, Holm et al. , 1977, Muenscher, 1980. Some feral cotton populations do exist in the US, but they are rare and found in areas hundreds of miles from commercial cotton production areas.

Cotton is a domesticated crop that requires human intervention to survive in non-cotton production area. Since cotton is an exotic species in the US and has not become a weed pest over many centuries, there is no expectation that a new cotton variety with a single gene introduction would enhance that risk by becoming weedy in non-cotton production areas.

Within cotton production areas, the addition of t he TwinLink traits (Cry1Ab, Cry2Ae and PAT proteins) into domesticated cotton will not cause it to become weedy. Traditional cotton breeding has provided new cotton varieties with resistance to disease, insects and herbicides, tolerance to various environmental conditions (heat, cold, drought, etc.) and enhanced phenotypic traits, such as faster germination and rapid seedling growth. Despite the many enhanced cotton varieties, none have shown any evidence of weediness. Crops modified by molecular techniques, which are highly specific, should present no different risks than those introduced by traditional, less controlled methods. Of specific concern may be the addition ofherbicide tolerance to produce TwinLink cotton, but experience with many other herbicidetolerant crops demonstrates no change in weediness potential. For example, rapeseed, cotton, corn, soybean, tobacco, tomato and other crops have been transformed or modified to resist herbicides such as glufosinate ammonium, glufosinate, bromoxynil, and sulfonylurea without any evidence of weediness. The primary concern is with the control of volunteer plants. Yet these plants can easily be controlled by pre- or post- emergence herbicides that are not tolerated by the modified crop. For example, TwinLink cotton volunteers could easily be controlled by using any number of targeted and broad-specturm herbicides used to control broadleaf weeds in agricultural systems. Potential volunteer cotton plants with the TwinLink traits can be controlled with products such as flumioxazin, metribuzin, and bentazon in soybeans, and atrazine, 2,4-D, and mesotrione in corn. Volunteer cotton with the TwinLink traits, which emerges within conventional or glufosinate susceptible cotton varieties can be controlled with products such as flumioxazin during pre-plant burndown, pendimethlin, and paraquat. Expression of the Bt proteins, Cry1Ab and Cry2Ae, within the TwinLink cotton product, raises no additional concerns with regards to weediness.



### F. Potential for outcrossing/gene escape in cotton

The potential for outcrossing can be defined as the ability of gene escape to wild cotton relatives. While gene flow could occur vegetatively, by seed or pollen, only pollen flow has anyTwinLink Cotton USDA Petition

Page 19 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### potential risk for cotton. Vegetative propagation is uncommon for cotton and seed dispersal (wind, birds, and animals) is rarely successful due to the properties of the boll structure. Cotton pollen is not transferred by wind due to its large, heavy and sticky nature (Niles and Feaster, 1984). Natural cross-pollination results from pollen being carried by insects, bees being the most important cotton pollinators (McGregor, 1976).

In Upland cotton, outcrossing studies suggest t hat pollen carryover decreases very rapidly as the distance to the closest marker pollen row increases, and that very little pollen is transferred beyond 12 meters. Vaissière (1990) prepared a report containing a literature review on cotton pollination and a summary of his study, "Pollen Dispersal and Carryover in Upland Cotton," conducted in Texas in 1983. The Texas study was conducted using a male sterile line surrounded by male fertile plants. Sixty honeybee colonies were supplied. Results showed that the pollen carryover in upland cotton decreased in proportion to the inverse of the distance to the closest pollinator row, and there was no significant pollen carryover past 12 meters.

Meredith and Bridge (1973) detected no outcrossing between adjacent plants in a study conducted in Stoneville, MS; the approximate limit of detection for the sample size and methods was approximately 0.046%.

Outcrossing data using bromoxynil-tolerant cotton is reported for seven locations (Kareiva et al

., 1994) and described in USDA petition for GlyTol cotton 06-332-01p. Seed samples were collected in the border rows of Calgene's winter nursery sites in Catamarca, Argentina and Pongola, Republic of South Africa, as well as in Stoneville, MS, USA. Sampling distance was one to 20 meters away from the bromoxynil-tolerant cotton. The frequency of outcrossing is determined by the crop and the pollinator. It is interesting to note that although the rate is higher for Argentina and South Africa (most likely due to the behavioral differences between European and African honeybees) the pattern of decline with distance is the same. Van Deynze et al

. (2005) measured pollen-mediated gene flow (PGF) in four directions over two years from commercial seed fields of bromoxynil-tolerant (BXN) and Roundup Ready (RR) cotton in the California cotton growing region, at various distances from non-transgenic cotton fields and their

results confirm -and refine- those of Kareiva et al . (1994), as larger distances were studied. In spite of variations due to the respective cardinal positions of the fields, the same decline with

distance is observed.

In the US, there are four cotton species, two that are cultivated commercially – G. hirsutum L.

and G. barbadense L. and two wild relatives – G. thurberi Todaro and G. tomentosum Nuttall ex Seemann (Fryxell, 1979). Of these four species, only three Gossypium species could be recipients for G. hirsutum - G. hirsutum itself, G. barbadense and G. tomentosum

. G. hirsutum grows feral only in the southern tip of Florida and in Hawaii, which is hundreds of miles from any

commercial cotton fields. G. barbadense is only found in very small commercial plots and is not found in wild environments in the US. Thus outcrossing to wild G. hirsutum or commercial plots

of G. barbadense is unlikely.

Outcrossing of the tetraploid G. hirsutum to the wild diploid G. thurberi , which occurs in Arizona, is extremely unlikely. Crosses between these species in breeding programs have been done, but the vigor of the hybrid seed is much reduced and the plants are usually infertile. In addition, native populations of G. thurberi reside in the higher altitudes and are thus isolated from commercial cotton production (Fryxell, 1979). Therefore, outcrossing of commercial TwinLinkTwinLink Cotton USDA Petition

Page 20 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### cotton to G. thurberi is not a concern.

Gossypium tomentosum is only found in the Hawaiian archipelago, occurring in dry coastal areas far removed from agricultural areas. The flowers of G. tomentosum are only receptive at night, rather than in the day as for G. hirsutum and moths, rather than bees generally pollinate them. Finally, outcrossing is unlikely since t here are no commercial cotton production areas on the islands and there would be no selective advantage since glufosinate ammonium is not used in natural non-agricultural areas.



### G. Characteristics of the recipient plant

TwinLink cotton (events T304-40 x GHB119) has its origin in the varieties Coker 315 and Coker 312. The variety Coker 315 (PVP 8000087) and the variety Coker 312 (PVP 7200100) are both US Protected Varieties of SEEDCO Corporation, Texas and were developed from a cross of Coker 100 X D&PL-15 and selected through successive generations of line selection.



### H. Cotton as a test syst em in this petition

During the development of TwinLink cotton, the events T304-40 and GHB119 were carried in their Coker genetic background for purposes of equivalence testing. At the same time, the Cry1Ab and Cry2Ae traits were introgressed into commercial and/or advanced breeding varieties to evaluate performance and equivalence with the corresponding counterpart. The individual events were then combined by a conventional breeding cross. Each trial/test in the development of this product used an appropriate control.TwinLink Cotton USDA Petition

Page 21 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## III. THE TRANSFORMATION SYSTEM

A. Description of the transformation system

T304-40 and GHB119 cotton were both transformed by Agrobacterium -mediated gene transfer of the T-DNAs from pTDL008 and pTEM12 respectively. Each T 0 

### plant was crossed with conventional cotton in order to obtain homozygous and stable lines (BC 3 

### F 3 

### ), and the resulting

lines were crossed to obtain TwinLink.

Agrobacterium -mediated gene transfer of a plasmid results in transfer to the plant genome of the DNA fragment between the T-DNA border repeats. The left and right border repeats of A.

tumefaciens , as described in Tables 2 and 3, are also inserted in the individual events. Even though some of the regulatory elements used in the transformation process were derived from

A. tumefaciens , a known plant pathogen, the genes that cause crown gall disease were removed, and therefore not incorporated into the recipient plant (Deblaere et al ., 1985).

The Cry1Ab and Cry2Ae proteins, from Bacillus thuringiensis subsp. berliner and B. thuringiensis subsp. dakota respectively, are effective in controlling lepidopteran larvae such as bollworm (CBW, Helicoverpa zea ) and tobacco budworm (TBW, Heliothis virenscens ) larvae, which are common pests of cotton.

The bar gene is a common genetic element used in several transformations of agricultural crops as a selectable marker, as a means to confer tolerance to the herbicide glufosinate ammonium.



### B. Parent lines

Coker 312 and Coker 315 are older commercial varieties of upland cotton ( Gossypium hirsutum ) which are no longer commercially cultivated. However Coker 312 and Coker 315 are well suited for transformation because of their capacity for regeneration from tissue culture.



### C. Construction of the plasmids used for transformation

1. Plasmid pTDL008

a. Nature and source of the vector

The vector pTDL008 is a derivative of the vector pGSV20 in which the bar gene cassette coding for the phosphinothricin acetyltransferase protein of Streptomyces hygroscopicus (described by Thompson et al ., 1987) was inserted together with the cry1Ab gene cassette encoding a fragment of the Cry1Ab crystal protein of Bacillus thuringiensis subsp. berliner (described by

Höfte et al ., 1986).

A map of plasmid pTDL008 is provided in Figure 1 and the genetic elements borne by this plasmid are described in Table 2.TwinLink Cotton USDA Petition

Page 22 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

# pTDL008

14393 bps 2000

4000

6000 8000 10000

12000 14000 LB

aadA

nptI fragment

ORI pVS1

ORI ColE1 RB 3'me1 cry1Ab 5'e1 Ps7s7 P35S3 bar 3'nos



### Figure 1 Vector map of pTDL008

b. The genetic elements intended to be inserted into the plant genome

The sequences comprised between base pairs 8767 and 37, also referred to as the transferDNA (T-DNA), are intended to be inserted into the plant genome upon transformation. The sequences comprised between base pairs 38 and 8766 compose the vector backbone and are not intended to be inserted into the plant genome. Their absence in T304-40 was experimentally confirmed (Sections IV.D and IV.E).TwinLink Cotton USDA Petition

Page 23 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 2. Plasmid pTEM12

a. Nature and source of the vector

The vector pTEM12 (Figure 2) is derived from pGSC1700 (Cornelissen and Vandewiele, 1989). The vector backbone contains the following genetic elements:

1 the plasmid core comprising the origin of replication from the plasmid pBR322 (Bolivar et

al. , 1977) for replication in Escherichia coli (ORI ColE1) and a restriction fragment comprising the origin of replication from the Pseudomonas plasmid pVS1 (Itoh et al. , 1984) for replication in Agrobacterium tumefaciens (ORIpVS1).

2 a selectable marker gene conferring resistance to streptomycin and spectinomycin ( aadA ) for propagation and selection of the plasmid in Escherichia coli and Agrobacterium

tumefaciens

. 3 a DNA region consisting of a fragment of the neomycin phosphotransferase coding sequence of the nptI gene from transposon Tn903 (Oka et al ., 1981).

These elements are outside the T-DNA borders and are not expected to be transferred into the cotton genome. Their absence in event GHB119 was experimentally confirmed (Sections IV.D and IV.E).

The genetic elements of plasmid pTEM12 are represented in Figure 2 and their description is provided in Table 3.

b. The genetic elements intended to be inserted into the plant genome

The sequence from bp 1 to bp 4345 of the pTEM12 plasmid is intended to be inserted into the plant genome. The genetic elements in this region are described in Table 3.TwinLink Cotton USDA Petition

Page 24 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

# pTEM12

12266 bp 2000

4000

6000 8000 10000 12000 LB 3'nos bar

Pcsvmv XYZ

P35S2

5'cab22L
TPssuAt

cry 2Ae

3'35S

RB

nptI - fragment

ORI ColE1 ORI pVS1 aadA



### Figure 2 Vector map of pTEM12TwinLink Cotton USDA Petition

Page 25 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### D. Open reading frames and associated regulatory regions

1. Plasmid pTDL008

Only the sequence from bp 8767 to bp 37 of the pTDL008 plasmid is intended to be inserted into the plant genome. Please refer to Table 2 below.

Table 2 Genetic elements of vector pTDL008

Nt Positions Orientation Origin

8767-8791 RB: right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)

8792-9728 Counter

clockwise 3 ́me1: sequence including the 3’ untranslated region of the NADP-malic enzyme gene of Flaveria bidentis (yellowtop) (Marshall et al ., 1996).

9729-11582 Counter

clockwise cry1Ab : a sequence encoding the Cry1Ab crystal protein of Bacillus thuringiensis berliner 1715 (Höfte et al ., 1986).

11583-11643 Counter

clockwise 5’e1: sequence including the leader sequenc e of the tapetum specific E1 gene ( GE1 ) of Oryza sativa (rice) (Michiels et al ., 1992).

11644-12685 Counter

clockwise Ps7s7: sequence including the duplicated promoter region derived from subterranean clover stunt virus genome segment 7 (Boevink et al ., 1995).

12686-13543 Clockwise P35S3: sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al ., 1985)

13544-14095 Clockwise bar : the coding sequence of the phosphinot hricin acetyltransferase gene of Streptomyces hygroscopicus as described by Thompson et al . (1987).

14096-14393 1-

12 Clockwise 3 ́nos: sequence including the 3’ untranslat ed region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al ., 1982)

13-37 LB: left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)

38-342 Residual plasmid sequences of pTiAch5 flanking the left border repeat (Zhu et

al ., 2000)

343-1965 Counter

clockwise aadA : fragment including the aminoglycoside adenyltransferase gene of

Escherichia coli as described by Fling et al . (1985)

1966-3486 Counter

clockwise Fragment of the neomycine phosphotra nsferase coding sequence of the nptI gene from transposon Tn 903 (Oka et al ., 1981)
3487-3632 Fragment including the residual sequences upstream of the aminoglycoside adenyltransferase gene of Escherichia coli as described by Fling et al . (1985)

3633-7403 ORI pVS1: fragment including the origin of replication from the Pseudomonas plasmid pVS1 for replication in Agrobacterium tumefaciens (Hajdukiewicz et al

., 1994)

7404-8576 ORI ColE1: fragment including the origin of r eplication from the plasmid pBR322 for replication in Escherichia coli (Bolivar et al ., 1977)

8577-8766 Residual plasmid sequences of pTiAch5 flanking the right border repeat (Zhu et

al ., 2000)TwinLink Cotton USDA Petition

Page 26 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 2. Plasmid pTEM2

The sequence from bp 1 to bp 4345 of the pTEM12 plasmid is intended to be inserted into the plant genome. Please refer to Table 3 below.

Table 3 Genetic elements of vector pTEM12

Nt Positions Orientation Origin

1-25 LB: left border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)

26-335 Counter

clockwise 3'nos: sequence including the 3' untranslated region of the nopaline synthase gene from the T-DNA of pTiT37 (Depicker et al. , 1982)

336-887 Counter

clockwise bar: the coding sequence of the phosphin othricin acetyltransferase gene of Streptomyces hygroscopicus as described by Thompson et al. (1987)

888-1423 Counter

clockwise Pcsvmv XYZ: sequence including the promoter region of the Cassava Vein Mosaic Virus (Verdaguer et al. , 1996)

1424-1920 Clockwise P35S2: sequence including the promoter region of the Cauliflower Mosaic Virus 35S transcript (Odell et al. , 1985)

1921-1990 Clockwise 5'cab22L: sequence including the leader sequence of the chlorophyl a/b binding protein gene from Petunia hybrida (Harpster et al. , 1988)

1991-2155 Clockwise TPssuAt: coding sequence of the transit peptide of the ribulose-1,5- biphosphate carboxylase small subunit gene ats1A of Arabidopsis thaliana , as described by De Almeida et al. (1989)

2156-4051 Clockwise cry2Ae : the coding sequence of an insecticidal protein gene of Bacillus thuringiensis , adapted to cotton codon usage

4052-4320 Clockwise 3'35S: sequence including the 3' untranslated region of the 35S transcript of the Cauliflower Mosaic Virus (Sanfaçon et al. ,1991)

4321-4345 RB: right border repeat from the T-DNA of Agrobacterium tumefaciens (Zambryski, 1988)

4346-4537 Residual plasmid sequences of pTiAch5 flanking the right border repeat (Zhu

et al. , 2000)

4538-5248 Fragment of the neomycine phosphotra nsferase coding sequence of the nptI gene from transposon Tn 903 (Oka et al. , 1981)

5249-6421 ORI ColE1: fragment including the origin of replication from the plasmid pBR322 for replication in Escherichia coli (Bolivar et al. ,1977)

6422-10192 ORI pVS1: fragment including the orig in of replication from the Pseudomonas plasmid pVS1 for replication in Agrobacterium tumefaciens (Hajdukiewicz et

al. , 1994)

10193-11961 Clockwise aadA : fragment including the aminoglycoside adenyltransferase gene of

Escherichia coli as described by Fling et al . (1985)

11962-12266 Residual plasmid sequences of pTiAch5 flanking the left border repeat (Zhu et

al. ,2000)TwinLink Cotton USDA Petition

Page 27 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### E. Deduced amino acid sequences

1 MDPERRPADI RRATEADMPA VCTIVNHYIE TSTVNFRTEP QEPQEWTDDL VRLRERYPWL
61 VAEVDGEVAG IAYAGPWKAR NAYDWTAEST VYVSPRHQRT GLGSTLYTHL LKSLEAQGFK
121 SVVAVIGLPN DPSVRMHEAL GYAPRGMLRA AGFKHGNWHD VGFWQLDFSL PVPPRPVLPV

181 TEI



### Figure 3 Deduced amino acid sequence of the PAT protein encoded from the bar

gene

1 MADNNPNINE CIPYNCLSNP EVEVLGGERI ETGYTPIDIS LSLTQFLLSE FVPGAGFVLG
61 LVDIIWGIFG PSQWDAFLVQ IEQLINQRIE EFARNQAISR LEGLSNLYQI YAESFREWEA
121 DPTNPALREE MRIQFNDMNS ALTTAIPLFA VQNYQVPLLS VYVQAANLHL SVLRDVSVFG 181 QRWGFDAATI NSRYNDLTRL IGNYTDHAVR WYNTGLERVW GPDSRDWIRY NQFRRELTLT
241 VLDIVSLFPN YDSRTYPIRT VSQLTREIYT NPVLENFDGS FRGSAQGIEG SIRSPHLMDI
301 LNSITIYTDA HRGEYYWSGH QIMASPVGFS GPEFTFPLYG TMGNAAPQQR IVAQLGQGVY
361 RTLSSTLYRR PFNIGINNQQ LSVLDGTEFA YGTSSNLPSA VYRKSGTVDS LDEIPPQNNN
421 VPPRQGFSHR LSHVSMFRSG FSNSSVSIIR APMFSWIHRS AEFNNIIPSS QITQIPLTKS
481 TNLGSGTSVV KGPGFTGGDI LRRTSPGQIS TLRVNITAPL SQRYRVRIRY ASTTNLQFHT
541 SIDGRPINQG NFSATMSSGS NLQSGSFRTV GFTTPFNFSN GSSVFTLSAH VFNSGNEVYI

601 DRIEFVPAEV TFEAEYD 617



### Figure 4 Deduced amino acid sequence of the Cry1Ab protein from Bacillus thuringiensis subsp . berliner

1 NNVLNNGRTT ICDAYNVVAH DPFSFEHKSL DTIRKEWMEW KRTDHSLYVA PIVGTVSSFL
61 LKKVGSLIGK RILSELWGLI FPSGSTNLMQ DILRETEQFL NQRLNTDTLA RVNAELEGLQ
121 ANIREFNQQV DNFLNPTQNP VPLSITSSVN TMQQLFLNRL PQFRVQGYQL LLLPLFAQAA
181 NMHLSFIRDV VLNADEWGIS AATLRTYQNY LKNYTTEYSN YCINTYQTAF RGLNTRLHDM
241 LEFRTYMFLN VFEYVSIWSL FKYQSLLVSS GANLYASGSG PQQTQSFTSQ DWPFLYSLFQ
301 VNSNYVLNGF SGARLTQTFP NIGGLPGTTT THALLAARVN YSGGVSSGDI GAVFNQNFSC
361 STFLPPLLTP FVRSWLDSGS DRGGVNTVTN WQTESFESTL GLRCGAFTAR GNSNYFPDYF

421 IRNISGVPLV VRNEDLRRPL HYNEIRNIES PSGTPGGLRA YMVSVHNRKN NIYAVHENGT
481 MIHLAPEDYT GFTISPIHAT QVNNQTRTFI SEKFGNQGDS LRFEQSNTTA RYTLRGNGNS

541 YNLYLRVSSL GNSTIRVTIN GRVYTASNVN TTTNNDGVND NGARFLDINM GNVVASDNTN
601 VPLDINVTFN SGTQFELMNI MFVPTNLPPI Y 631



### Figure 5 Deduced amino acid sequence of the Cry2Ae protein from Bacillus thuringiensis subsp . dakotaTwinLink Cotton USDA Petition

Page 28 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## IV. CHARACTERIZATION OF TWIN LINK COTTON (EVENTS T304-40 x GHB119)

A. Description, history and Mendelian inheritance

T 1 

### seed of transformation events T304-40 and GHB119 was harvested from self-pollinated T 0 

### plants surviving a glufosinate-ammonium herbicide greenhouse screen. T 1 

### plants were selected for survival following glufosinate-ammonium herbicide application, and at each generation, plants were sprayed with glufosinate-ammonium to eliminate those not expressing the bar gene. Homozygous T 3 

### plants were identified by planting 25 seed and spraying with glufosinateammonium to identify segregating seed lots. Homozygosity PCR based analysis was also performed as a secondary means of identifying homozygous plants. Selfed T 3 

### homozygous seed (no segregation for tolerance) was used to produce homozygous T 4 

### seed and was the

source of the lines that were used in ear ly event agronomic and stability studies.

The source of planting seed for the field studies are indicated in Figure 6.

Selfed T 3 

### homozygous seed (no segregation for resistance) was used to produce homozygous

T 4 

### seed and was the source of the lines that were used in early event agronomic and stability studies (2005). Selfed T 6 

### and T 7 

### seed were used for the agronomic studies in 2007 and 2008, respectively. The TwinLink planting seed, individual F 2 

### plants, identified by PCR to carry both events were identified, carried to maturity and seed harvested (2007) and selfed to produce the planting seed for field studies in 2008.

The source of the samples for the DNA analysis and stability studies are indicated in Figure 6. Generation BC2F4 (homozygous) was used for detailed insert characterization and protein expression levels. Generations T3, T4, T5, T6 and BC 2 

### F 2 

### were used for molecular stability analyses. Generation T5 was used for seed composition analysis. Generations T5 and BC 2 

### F 3 

### were used for replicated agronomic field tests. Generation T7 was used for analyses on absence/presence of vector backbone sequences.

During the development of TwinLink cotton (events T304-40 x GHB119), each locus was bred into selected cotton varieties. Herbicide tolerance screenings were conducted in the greenhouse using glufosinate ammonium at the recommended rate for field use. Plants were scored as tolerant (alive, no damage) or susc eptible (damaged severely and dead or dying) 5-7 days post-glufosinate ammonium application. Polymerase chain reaction (PCR) testing was used to verify the transformation events.

To demonstrate the independent inheritance of the two events in TwinLink, the results of an F 2 

### progeny screen are described. The two events were back-crossed into a conventional breeding

line. At the BC 2 

### F 1 

### generation, the two events were combined and the progeny of this F 1 

### were analyzed by PCR designed to identity the presence of two events. The plants identified as containing both events (genotype T304-40/GHB119) were carried to maturity and seed harvested for further study. The expected ratio for two independently inhereted loci was achieved in this population of 80 plants. (Table 4) These results complement the DNA analysis characterizing the individual events and demonstrating insert stability (Section IV.C).TwinLink Cotton USDA Petition

Page 29 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Cry1Ab/T 0

Cry1Ab/T 1

Cry1Ab/T 6 Cry1Ab/T 2

Cry1Ab/T 7 BC 2 F 1 Cry1Ab/T 3

Cry1Ab/T 4

Cry1Ab/T 5 F 1

BC 1 F 1 Conventional Line (A) X

9

9

9

9

9

9

9 Conventional

Line

Conventional

Line X

X Cry1Ab Breeding Diagram

Conventional

Line X Cry2Ae/T 0

Cry2Ae/T 1

Cry2Ae/T 6 Cry2Ae/T 2

Cry2Ae/T 7 Cry2Ae/T 4

Cry2Ae/T 5 9

9

9

9

9

9

9 Cry2Ae Breeding Diagram

Cry2Ae/T 3 BC 2 F 1

F 1

BC 1 F 1 Conventional Line (A)

X

X

X Conventional

Line

Conventional

Line

Conventional

Line X

Cross of Cry1Ab and Cry2Ae

BC 3 F 1 transgenic lines

Notes

- At each generation, plants were sprayed with glufosinate to eliminate those not expressing the bar gene

- 9 = self-cross - Homozygous T3 plants were identified by planting 25 seed, spraying with glufosinate to identify segregating seed lots. Homozygosity analysis was also performed to identify homozygous plants. - Selfed T3 homozygous seed (no segregation for resistance) was used to produce homozygous T4 seed and was the source of the lines that were used in early event agronomic and stability studies. - Generation BC2F4 (homozygous) was used for detailed insert characterization and protein expression levels. - Generations T3, T4, T5, T6 and BC2F2 were used for molecular stability analyses. - Generation T5 was used for seed composition analysis. - Generations T5 and BC2F3 were used for replicated agronomic field tests. - Generation T7 was used for analyses on absence/presence of vector backbone sequences. Origin of planting seed for agronomic/efficacy studies

2005 2005

2007

2008 2007

2008 TwinLink

F 1

TwinLink

9

2007

2008 TwinLink Individual F 2 plants

selected and bulked



### Figure 6 Breeding tree for the development of TwinLink cottonTwinLink Cotton USDA Petition

Page 30 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 4 Segregation Analysis of the Independent Events in TwinLink cotton.

Events T304-40 and GHB119 were independently back-c rossed into a conventional BCS breeding line.

At the BC 2 F 1 generation, the two events were combined and the progeny of this F 1 were analyzed by homozygosity PCR developed to identify the zygosity of each F 2 plant. The individual plants were scored for presence or absence of the events. Four possib le genotypes were identified; WT (wild type)/WT, or T304-40/WT, GHB119/WT and T304-40/GHB119.

Parents of the F 1 and zygosity

of the TwinLink events Generation Ratio Observed a Expected χ 2 b

calculated

WT/WT:

T304-40/WT:

GHB119/WT: T304-

40/GHB119 Hemizygous T304-40 ( cry1Ab/bar ) plant crossed with Hemizygous GHB119

( cry2Ae/bar ) plant F 1 1:1:1:1 19:15:25:21 20:20:20:20 2.6

a Tested by homozygosity PCR

b Assumes a one locus model for each gene. There was no significant difference (p=0.05) for the χ square goodness-of-fit test for the hypothesis of one locus for each gene. To reject the null hypothesis, the χ square value must be greater than 7.815, with three degrees of freedom.TwinLink Cotton USDA Petition

Page 31 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### B. Verification of the inserts and num ber of copies of the inserted sequences

1. T304-40

Genomic DNA isolated from cotton event T304-40 and control FiberMax 966 plants was subjected to Southern blot analysis using the different components of the transgene cassette (5’e1-Ps7s7 promoter, cry1Ab gene, 3’me1 terminator, P35S3 promoter, bar gene and 3’nos terminator) as well as the complete T-DNA fragment, as probes. The resulting DNA fragments were separated by agarose gel electrophoresis, transferred to a membrane and sequentially hybridized with six different probes, each representing a fragment of the transforming gene cassette, or the complete T-DNA probe.

The expected and observed hybridization fragments, as well as the hybridization strategy, are shown in Appendix 3. Results of this analysis (see Figure 3.2, Appendix 3) demonstrated that the inserted transgenic sequence in the cotton event T304-40 consists of one nearly complete copy of the T-DNA flanked by an inverted incomplete copy of the cry1Ab gene cassette and one

additional 3’me1 terminator.

2. GHB119

Genomic DNA isolated from cotton event GH B119 and control FiberMax 966 plants was subjected to Southern blot analysis using the different components of the transgene cassette (3’nos terminator, bar gene, PCsVMV promoter, P35S2-5'cab22L promoter

, 

### TPssuAt- cry2Aegene plus transit peptide and 3’35S-RB terminator plus right border) as well as the complete TDNA fragment, as probes. The resulting DNA fragments were separated by agarose gel electrophoresis, transferred to a membrane and sequentially hybridized with six different probes, each representing a fragment of the transforming gene cassette, or the complete T-DNA probe.

The expected and observed hybridization fragments, as well as the hybridization strategy, are shown in Appendix 3. Results of this analysis (see Figure 3.4, Appendix 3) demonstrated that a single copy of the T-DNA is integrated in cotton event GHB119 and that the transferred DNA in the plant corresponds to the DNA configuration as originally designed in the transformation

vector.



### C. Stability of the inserted DNA sequence

1. T304-40

To demonstrate the structural stability of cotton event T304-40, genomic DNA was prepared from several individual plants of different generations, different genetic backgrounds and from plants grown from seeds harvested at three different locations. The isolated DNA was digested with the restriction enzyme Eco RV, which has two recognition sites in the integrated DNA fragment. Probing Eco RV digested genomic T304-40 DNA with the cry1Ab probe showed all three expected fragments in all tested samples. Two of these fragments represent the junctions between the transgenic sequences and the plant DNA sequences and one represents an internal fragment. The hybridization results of the stability analysis over four different genetic backgrounds are presented in Figure 3.5 (Appendix 3), over four different generations in Figure 3.6 (Appendix 3) and over different environmental conditions in Figure 3.7 (Appendix 3).TwinLink Cotton USDA Petition

Page 32 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### The obtained results demonstrate the stability of the cotton event T304-40 at the genomic level over different generations, in different backgrounds and in plants grown from seeds harvested in different environments. Segregation data further confirm the stability of the insert, and show that it segregates as one dominant Mendelian locus (see Section IV.A).

2. GHB119

The structural stability of cotton event GHB119 over different generations and in different backgrounds was tested by means of Southern blot analysis. Genomic DNA was prepared from several individual plants of three consecutive generations and two different genetic backgrounds. The impact of environment was a ssessed by analyzing the progeny of plants cultivated at 6 different field locations. The isolated DNA was digested with the restriction

enzyme Eco RV, which has two recognition sites in the transforming DNA. Hybridization of these samples with the T-DNA probe revealed the expected profile in all tested samples (Figure 3.8 and Figure 3.9, Appendix 3). This demonstrates the stability of cotton event GHB119 at the genomic level in different generations, genetic backgrounds and growing environments. Segregation data further confirm the stability of the insert, and show that it segregates as one dominant Mendelian locus (see Section IV.A).

3. TwinLink cotton

The genetic stability of the inserted DNAs in TwinLink cotton was equally tested. Genomic DNA was isolated from 20 individual TwinLink plants and digested with the restriction enzyme Eco RV.

Eco RV recognizes the inserted DNA of each event, thus generating restriction profiles that are characteristic for each parent (T304-40 and GHB119) and TwinLink.

Probing DNA digests of the parents and the TwinLink cross with labeled DNA probes containing

the cry1Ab and cry2Ae genes, respectively, revealed the expected restriction fragments in all tested samples (Figures 3.10 and 3.11, Appendix 3). This demonstrates that the conventional breeding cross of events T304-40 and GHB119 has not affected the stability of the inserts, or created a rearrangement in the DNA structure.



### D. Presence of marker genes and or igin of replication in the vector

The bar gene was used as the selectable marker for both events, T304-40 and GHB119, therefore the same gene of interest acts as a marker. No other marker genes were present.

Cotton event T304-40 contains no vector back bone sequences as evidenced by using seven overlapping probes covering the complete pTDL008 vector backbone sequences (including

aadA , ORI pSV1 and ORI ColE1). See Figure 3.13, Appendix 3.

Southern blot analysis using five overlapping vector backbone probes covering the complete vector backbone sequence of the pTEM12 transformation vector confirmed the absence of vector backbone sequences as well as of the bacterial origin of replication, in the genome of cotton event GHB119. See Figure 3.15, Appendix 3.

No bacterial origin of replication was transferred with the Agrobacterium mediated transformation system. The inserted DNAs within cotton events T304-40 and GHB119 do notTwinLink Cotton USDA Petition

Page 33 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### add a bacterial origin of replication to the wild type cotton genome as a result of the

transformation.



### E. Absence of remaining parts of the vectors

The Southern blot analyses using overlapping probes covering the complete pTDL008 or pTEM12 vector backbone sequences demonstrate the absence of vector backbone sequences

in cotton events T304-40 and GHB119. Please refer to Section IV.D above.



### F. The flanking regions of the inserted sequence(s)

1. T304-40

Right and left border integration fragment Southern blot analysis demonstrated that the inserted transgenic sequence in cotton event T304-40 consists of one nearly complete copy of the T-DNA flanked by an inverted incomplete copy of the cry1Ab gene cassette and one additional 3’me1 terminator. The sequence of the transgenic locus and the sequence of the pre insertion locus were determined. 5’ flanking sequences and 3-prime flanking sequences were reported.

A PCR fragment of the pre-insertion locus of cotton event T304-40 was generated using genomic DNA from wild type cotton plants as template and a pair of primers specific for the 5’ and 3’ flanking sequence. 837 bp were determined at the pre-integration locus, including 480 bp of 5-prime flanking sequence, 325 bp of 3-prime flanking sequence and a target site deletion of 32 bp.

Between the sequences annotated as 5’ and 3’ flanking sequences at the pre-insertion locus, 32 base pairs can be detected which are present in the pre-insertion locus, but not at the transgenic locus of cotton event T304-40, and were deleted during transformation. A schematic overview of the T304-40 transgenic locus and pre-insertion locus is presented in Appendix 3, Figure 3.16.

BLASTn similarity search, and open reading frame search Cotton plants transformed using Agrobacterium -mediated transformation inserting the T-DNA from vector pTDL008 into the cotton genome generated the cotton event T304-40. Due to the

insertion of the cry1Ab - bar gene cassette in cotton, a 5-prime and a 3-prime junction, where cotton genomic DNA and inserted T-DNA are fused, were created. The junction regions were analyzed to confirm that no important cotton genes were interrupted and that no chimeric proteins would get expressed due to this insertion.

Open reading frame (ORF) and gene search tools were applied to predict the presence of potential newly created coding sequences in the 5-prime flanking genomic/insert DNA junction region and in the 3-prime flanking insert/genomic DNA junction region. No ORF was found, spanning the 5-prime junction or the 3-prime junc tion. One putative promoter was found in the 5’ flanking sequence. As there are no genes fo und further downstream, this predicted promoter is most probably not biologically active.

It can be concluded that, using the current databases and tools, no known genes wereTwinLink Cotton USDA Petition

Page 34 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### interrupted or influenced due to the insertion of transgenic DNA in the pre-insertion locus of

T304-40.

A bioinformatic analysis was performed to detect the possible presence of expression due to the formation of newly created open reading frames (ORF s) in the 5’, 3’and internal junction regions of cotton event T304-40. Two newly created chimer ic ORFs were identified; however as these ORFs lack the necessary regulatory elements, the probability of expression of newly created proteins due to the insertion of DNA containing the cry1Ab-bar gene construct is highly unlikely.

2. GHB119

Right and left border integration fragment Southern blot analysis demonstrated that a single copy of the T-DNA is integrated in cotton event GHB119 and that the transferred DNA in the plant corresponds to the DNA configuration as originally designed in the transformation vector.

A PCR fragment of the pre-insertion locus of cotton event GHB119 was generated using genomic DNA from wild type cotton plants as template and a pair of primers specific for the 5’ and 3’ flanking sequences. Analysis of the se quence obtained from this fragment shows that 684 bp of the pre-insertion locus were determined, including 367 bp 5’ flanking sequences, 309bp 3’ flanking sequences and 8 bp of the target site deletion. Bioinformatic analyses of the preinsertion locus sequence demonstrate that the characterized 5’ and 3’ flanking sequences are of

cotton plant origin.

The DNA of the pre-insertion locus of cotton event GHB119 was sequenced and subjected to bioinformatic tools in order to identify endogenous cotton genes and/or regulatory elements that may be influenced by the insertion of the transgenic DNA fragment. Homology was found with cotton genomic DNA but no function could be assigned to these sequences. Homology was also found with repetitive sequences indicating that the insertion of the GHB119 T-DNA took place in a region containing repetitive elements. In order to identify the presence of known functional genes in the pre-insertion locus of event GHB119, a BLASTx similarity search was performed, but no known proteins were found.

1 Using the prediction tools GetORF and FGENESH, no genes or ORFs were predicted that could be disrupted by the introduction of the transgenic DNA in the genome.

2 The surrounding sequence of the pre-insertion locus was subjected to a homology analysis comparing it with regulatory element s involved in transcription. Two promoter sequences were predicted (Figure 3.19, Appendix 3). Since no known endogenous genes were found or predicted, it is highly unlikely that these predicted promoters are biologically active. These bioinformatic analyses on the pre-insertion locus of cotton event GHB119, using the current databases and bioinformatic tools, allow us to conclude that the probability that a functional gene was interrupted is highly unlikely.

BLASTn similarity search, and open reading frame search A bioinformatic analysis was performed to detect the possible presence of cryptic expression due to the formation of newly created open reading frames (ORFs) in the 5’ or 3’ junction regions in event GHB119. These junctions are formed by the insertion of a cry2Ae - bar gene construct into the cotton genome.TwinLink Cotton USDA Petition

Page 35 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Several in silico tools were used to look for ORFs and regulatory elements taking into account the current scientific knowledge of gene expression. Three newly created chimeric ORFs were identified. The surrounding nucleotides of the three ORFs were also subjected to a homology analysis, comparing them with regulatory elements important for transcription and translation. Three newly created chimeric ORFs were identified. As not all regulatory elements were present in the DNA sequence at the 5’ or 3’ end of the newly created ORFs, the probability of expression of newly created proteins due to the insertion of DNA containing the cry2Ae - bar gene construct, is highly unlikely.TwinLink Cotton USDA Petition

Page 36 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## V. THE Cry1Ab, Cry2Ae AND PAT PROTEINS

A. History and background

1. Cry proteins

i. Bacillus thuringiensis history of safe use

Bacillus thuringiensis ( Bt ) is a Gram-positive, spore forming, rod-shaped bacterium present in soils, grain, on leaf surfaces, and in water. Not only is it found in various natural environments, but also in many animals including wild mammals ( e.g. voles, deers, rodents, insectivore mammals) and probably humans, as well as in food ( e.g. pasta, bread and processed food that contains flour) (OECD, 2007).

Bt

strains are generally classified as non-pathogenic bacteria in several national classifications for microorganisms. It has been rarely classified as an opportunistic pathogen.

Due to its insecticidal properties, microbial preparations of Bt isolates have been used as biopesticides since 1961 in commercial agriculture, forestry and mosquito control (OECD, 2007).

More than 100 microbial Bt products have been registered so far (USEPA, 2007). Bt sprayed crops that have been studied thus far are not toxic to humans or mammals (Betz et al ., 2000). Instead, their insecticidal activities are very specific to the groups of pest insects of field and vegetable crops.

The insecticidal effect of Btbacteria is largely attributed to proteins called crystal (Cry) deltaendotoxins. Since cloning of the first cry gene in 1981 (Schnepf and Whiteley, 1981), a total of

314 different cry genes had been identified by 2005 (OECD, 2007).

Results from numerous animal and human epidemiol ogical studies together, over the years, indicate that biopesticides pose minimal risks to humans and animals, and certainly there is no

evidence that Bt bacterial biopesticides caused any kind of significant infection or outbreaks of food poisoning (McClintock et al ., 1995; Betz et al ., 2000; Rosenquist et al ., 2005).

In conclusion, Bt bacterial biopesticides have a long history of safe use.

ii. Cry1Ab protein familiarity

Over the past 50 years, current use of Bt pesticides, including those expressing Cry1Ab, is estimated to be several tons annually. Moreover, Cry1Ab protein is expressed in a number of genetically modified crops that have been approved since 1995 and are currently commercialized. No records of allergenicity in humans and mammals were found associated

with Bt bacteria (OECD, 2007). In addition, microbial Bt biopesticides, including those containing the Cry1Ab protein, have shown no toxic effects in several mammalian toxicity studies (Betz et al ., 2000). Therefore, Cry1Ab has had a long history of safe use for 50 years.

More specifically, the safety profile of the Cry1Ab protein is based on a number of studies that have been submitted to regulatory agencies to support product registration or published in peerreviewed journals (summarized by Delaney et al ., 2008). All these studies concluded that the Cry1Ab protein exhibited no adverse potentials for allergenicity or toxicity in mammals. Mainly,TwinLink Cotton USDA Petition

Page 37 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Cry1Ab does not possess characteristics typical of known toxins and allergens. This includes that Cry1Ab originates from a non-pathogenic and non-allergenic organism. It has a known mode of action and has no protein sequence homology with known toxins and allergens. It is rapidly degraded by acid and/or enzymatic hydrol ysis when exposed to simulated human gastric fluids. The lack of allergenicity was further confirmed by skin prick tests in two sensitive groups, children with food and inhalant allergy and individuals with asthma-rhinitis (Batista et al ., 2005). The purified Cry1Ab protein is also devoid of toxic effects at high doses (up to 4000 mg/kg body weight).

In addition, a number of studies with Cry1Ab-incorporated GM crops support the notion that the Cry1Ab protein and the GM crops are non-toxic. Cry1Ab-incorporated GM crops were as safe and nutritious as their non-GM counterparts in rat or chicken feeding studies and no deleterious effects were observed (Wang et al ., 2002; Brake et al. , 2003; Hammond et al. , 2006; Schroder

et al. , 2007).

In conclusion, Cry1Ab has been safely consum ed by humans and animals for 50 years, without any records of safety issues.

iii. Cry2Ae protein familiarity

As summarized in the previous sections, Cry pr oteins have a good safety profile, as evidenced by the extensive use of Bt biopesticides, including those expressing the Cry2Ae protein, with excellent safety records.

Among the Cry proteins, Cry2Ae is a protein fr om the Cry2-type family. Although a number of data support the safety of the Bt organisms expressing Cry2Ae, no data have been published on safety assessment of the Cry2Ae protein itself. However, Cry2Ae has 86% identity with Cry2Ab, which has a safe profile.

A number of studies with the Cry2Ab protein have been submitted to regulatory agencies to support product registration. Assessment of those studies by various agencies concluded that the Cry2Ab protein exhibited no adverse potentials of allergenicity or toxicity (US-EPA, 2002; US-FDA, 2002; CFIA, 2004; OECD, 2007).

In addition, studies performed using Cry2Ab expressing GM crops support their safety profile. Short-term feeding studies on poultry, pigs, calves, catfish, quail and cattle demonstrated that no deleterious effects are associated with Cry2Ab expressing corn and that this GM crop is as nutritious and safe as its parental crop control (Hamilton et al. , 2004).

2. The PAT protein

PAT protein and PAT expressing cotton LLCotton25 have been approved in the US in 2003 (BNF 00086, US-FDA, 2003).

i. Streptomyces hygroscopicus history of safe use

S. hygroscopicus history of safe use data are included in the peer-reviewed scientific journal published by Herouet et al. (2005). This organism is a common soil saprophytic bacterial species of Streptomyces . Human exposure to Streptomyces is not new and these bacteria are widespread in nature worldwide (Kutzner, 1981). Further more, it is very likely that this exposure is frequent through the consumption of roots and vegetables. Nevertheless, there is noTwinLink Cotton USDA Petition

Page 38 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### evidence of adverse effects related to the exposure to this bacterial species.

ii. PAT protein familiarity

Safety data focused on PAT proteins have been ex tensively reviewed by international scientific peer-reviewed journals (Herouet et al. , 2005). PAT protein is expressed in a number of GM crops that have been approved by regulator y agencies and are consumed by humans and animals since 1995, with excellent safety records.

B. Characterization of the Cry1Ab, Cry2Ae and PAT proteins

1. Biochemistry and mode of action

i. Cry1Ab and Cry2Ae proteins

The insecticidal mode of action for Cry proteins has been studied extensively over the last 20 years. Prior to this, it was known that Cry proteins were not contact poisons, but rather insecticidal proteins that act on the midgut and must be ingested to be cleaved by midgut proteases to be effective against pest insects. It is now well-understood that the insecticidal mode of action is a multi-complex process, which includes a series of critical steps such as crystal solubilization, protoxin proteolysis, peritrophic membrane transport, brush border membrane binding, and pore formation (Aronson and Shai, 2001; Whalon and Wingerd, 2003).

The midgut environment of a lepidopteran larva is both alkaline (pH 8-10) and probably reductive, enabling the Cry proteins to be dissolved (Tojo and Aizawa, 1983; Ogiwara et al.

, 1992). Once dissolved, the specific larval midgut proteases begin to cleave the inactive protoxin to a biologically active toxin. The requirement of alkaline pH and specific larval gut proteases for activation is an important feature in insect-specific Btactivity. Since mammals and other nontarget pests (including most other insects) are unable to dissolve Bt crystals, their passage through the digestive system of non-target organs occurs most likely in the unaltered, and therefore non-toxic, form.

The toxic fragment of the protein then interacts with specific high affinity receptors on the microvilli of the target’s larval midgut (stomach) epithelium, in particular brush border membrane vesicles (BBMV). The surface glycosylation on these receptors is very important for interaction with toxic the fragment of Cry proteins. Invertebrates, but not vertebrates, were shown to have these glycosylations (Griffitts et al. , 2005).

Following insertion of at least part of the Cry proteins into the cell membrane, and subsequent oligomerization, a cation-selective channel is formed in the columnar cells of the midgut epithelium (Chen et al. , 1995). These pore formation and subsequent destruction of the cell lead to disruption of the integrity of the gut epithelium, starvation, and insect death (Knowles and Dow, 1993).

This overview of the mode of action accounts for the specificity of the Cry protein toxicity, as Cry proteins must be ingested to have an active inse cticidal effect, they must be dissolved under alkaline conditions specific to insect gut, they must be cleaved by specific larval midgut proteases to be active, and they must bind to glycoprotein or glycolipid receptors that are not present in the gut of vertebrate species.TwinLink Cotton USDA Petition

Page 39 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### In conclusion, there is no mechanistic evidence that Cry proteins pose risks for humans or

mammals.

Cry1Ab protein Cry1Ab protein as expressed in cotton has 617 amino acids. The deduced molecular weight is 69 kDa. The binding of the Cry1Ab toxin to brush border membranes of Manduca sexta was tested in buffers of varying pH (Van Rie et al. , 1989). No gross changes in affinity or binding site concentration occurred in the pH range 7.4-10.0, though the binding was somewhat reduced at pH 10.0. Heat sensitivity of the Cry1Ab protein was tested by incubating the protein at 45°C and 60°C and assaying the residual activity at various time intervals. The Cry1Ab protein appears to be deactivated by higher temperature. At 45°C the protein loses little activity. However, the Cry1Ab protein is deactivated after 120 minutes at 60°C.

Cry2Ae protein Cry2Ae protein as expressed in cotton has 631 amino acids. The deduced molecular weight is 71 kDa. Heat sensitivity of the Cry2Ae protein was tested by incubating the protein at 45°C and 60°C and assaying the residual activity at various time intervals. The Cry2Ae protein loses insecticidal potential when exposed to higher temperature. At 45°C the protein lost little activity. However, Cry2Ae is deactivated after 240 minutes at 60°C.

ii. PAT protein

PAT protein biochemistry and mode of action are included in the peer-reviewed scientific journal published by Herouet et al . (2005) and in US-FDA BNF 000086 (US-FDA, 2003). Briefly, phosphinothricin (L-PPT) and demethylphosphinothric in (DMPT) are inhibitors of glutamine synthetase. This inhibition results in the accumulation of toxic ammonium ions and a decrease in the amount of glutamine, an essential amino acid used in many anabolic processes. The PAT enzyme is an acetyltransferase that specifically catalyses the acetylation of both L-PPT and DMPT. Enzymatic properties of the PAT protein are well-characterized, in particular, pH and temperature dependancy are well-described and understood. From the perspective of safety, this characterization demonstrates that metabolic effects of the expression of the PAT protein are limited to conferring tolerance to the herbicide glufosinate ammonium.

2. Protein safety

In order to assess any potential adverse effects to humans or animals resulting from environmental release of the crops containing the Cry1Ab, Cry2Ae and PAT proteins, Bayer CropScience (BCS) has conducted a detailed safe ty evaluation based on Codex Alimentarius Commission (Codex; Alinorm 03/34A). As a basi s, BCS performed a series of safety studies with these proteins, including homology searches of the amino acid sequences with comparison to all known allergens and toxins from large public databases, an in vitro digestibility assay of the proteins, and an acute toxicity test in the mouse. Moreover, publicly available review documents issued by regulatory authorities, indica ting that similar protein family members are safe, have been used for supporting this safety assessment. The results of studies conducted by BCS are consistent with the published information, confirming that the crops containing these proteins can be safely used as food or feed.

Lack of allergenic potential

• The Cry1Ab, Cry2Ae and PAT proteins have no amino acid sequence similarity to knownTwinLink Cotton USDA Petition

Page 40 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### allergens, as demonstrated by overall am ino acid and epitope homology searches;

• As expected, the Cry1Ab, Cry2Ae and PAT protei ns only have similarities with other Cry or PAT proteins;

• The Cry1Ab, Cry2Ae and PAT proteins do not share epitopes with known allergens;

• The Cry1Ab, Cry2Ae and PAT proteins are not glycosylated;

• The Cry1Ab, Cry2Ae and PAT proteins are not heat stable;

• The Cry1Ab, Cry2Ae and PAT proteins are degraded by human simulated gastric and

intestinal fluids. This minimizes the likelihood th at these proteins could survive in the human digestive tract and be absorbed.

Lack of toxic potential

• The Cry1Ab, Cry2Ae and PAT proteins have no amino acid sequence similarity to known allergens, as demonstrated by overa ll amino acid homology searches;

• The Cry1Ab, Cry2Ae and PAT proteins are degraded by human simulated gastric and

intestinal fluids. This minimizes the likelihood th at these proteins could survive in the human digestive tract and be absorbed; - There were no mortalities, clinical signs or treatment-related effects in female OF1 mice after an acute oral administration by gavage of the Cry1Ab, Cry2Ae or PAT proteins at 2,000 mg protein/kg body weight.

In conclusion, it is considered that the cry 1Ab

, cry2Ae and bar genes as well as the Cry1Ab, Cry2Ae and PAT proteins are not toxic for mammals and do not possess any of the characteristics associated with food allergens. Therefore, no effects on animal and human health are to be expected by consumption of the cry1Ab

, cry2Ae or bar genes and the Cry1Ab, Cry2Ae or PAT proteins.TwinLink Cotton USDA Petition

Page 41 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## VI. EXPRESSION OF TH E INSERTED SEQUENCES

Several studies were performed to quantify the Cry1Ab, Cry2Ae, and PAT/ bar proteins in tissues of TwinLink cotton. The levels of the proteins in TwinLink cotton were determined by validated enzyme-linked immunosorbent assays (ELISA’s).



### A. Protein amounts in fuzz y seed of TwinLink cotton

Seven trials were conducted in 2007; the plants were grown under conditions typical ofproduction practices (see Appendix 2). There were six transgenic plots and three nontransgenic plots at each test site. Three of the transgenic plots were sprayed two times with Ignite® 280 SL glufosinate-ammonium herbicide at 0.53 lb ai/A, and the other plots were untreated. Samples of ginned cottonseed (fuzzy seed) were collected and shipped frozen to Bayer CropScience for ELISA determination of the amounts of Cry1Ab, Cry2Ae, and PAT/ bar proteins in the raw agricultural commodity. Results are summarized in Table 5 and Table 6 and provided in detail in Appendix 2. The subject proteins were detected in all kernel and lint coat fractions from transgenic TwinLink cotton. As expected, the kernel fraction contained most of the analyte.

The amount of each protein in fuzzy seed was calculated based on the amount of analyte present in the lint coat and kernel fractions. The amount of analyte measured in lint coat and kernel, then calculated in fuzzy seed, and the average as a percent of total crude protein on a fresh weight basis are given in Appendix 2, Tables 2.5, 2.7 and 2.9. The measured and calculated amounts of analyte on a dry weight basis are given in Appendix 2, Tables 2.6, 2.8 and 2.10. Ranges in values are also provided for fuzzy seed and the amounts of analyte as a percent of the total crude protein.

On a fresh weight basis, the average Cry1Ab protein content from all test sites ranged from 0.226 μg/g to 1.64 μg /g fresh weight in unsprayed fuzzy seed with an average value of 1.16 μg/g ± 0.482 μg/g. The amount of Cry1Ab determined to be in the sprayed transgenic fuzzy seed ranged from 0.962 to 1.77 μg/g with an average value of 1.38 ± 0.301 μg/g. The Cry1Ab protein comprised an average of 0.000561 ± 0.000206 % of the total crude protein in unsprayed fuzzy seed. For sprayed fuzzy seed, the Cry1Ab protein comprised an average of 0.000694 ± 0.000153 % of the total crude protein.

On a dry weight basis, the average Cry1Ab protein content from all test sites ranged from 0.258 μg/g to 1.81 μg/g dry weight in unsprayed fuzzy seed with an average value of 1.29 ± 0.520 μg/g. The amount of Cry1Ab determined to be in the sprayed transgenic fuzzy seed ranged from 1.09 μg/g to 1.96 μg/g with an average value of 1.54 ± 0.318 μg/g. The Cry1Ab protein comprised an average of 0.000561 ± 0.000206 % of the total crude protein in unsprayed fuzzy seed. For sprayed fuzzy seed, the Cry1Ab protein comprised an average of 0.000694 ± 0.000153% of the total crude protein.

On a fresh weight basis, the average Cry2Ae protein content from all test sites ranged from 4.69 μg/g to 11.7 μg/g fresh weight in unsprayed fuzzy seed with an average value of 8.34 ± 2.55 μg/g. The amount of Cry2Ae determined to be in the sprayed transgenic fuzzy seed ranged from 6.01 μg/g to 16.2 μg/g with an average value of 9.51 ± 3.56 μg/g. The Cry2Ae protein comprised an average of 0.00420 ± 0.00129 % of the total crude protein in unsprayed fuzzyTwinLink Cotton USDA Petition

Page 42 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### seed. For sprayed fuzzy seed, the Cry2Ae protein comprised an average of 0.00485 ± 0.00204% of the total crude protein.

On a dry weight basis, the average Cry2Ae protein content from all test sites ranged from 5.34 μg/g to 13.4 μg/g dry weight in unsprayed fuzzy seed with an average value of 9.35 ± 2.93 μg/g. The amount of Cry2Ae determined to be in the sprayed transgenic fuzzy seed ranged from 6.67 μg/g to 18.5 μg/g with an average value of 10.7 ± 4.17 μg/g. The Cry2Ae protein comprised an average of 0.00420 ± 0.00129 % of the total crude protein in unsprayed fuzzy seed. For sprayed fuzzy seed, the Cry2Ae protein comprised an average of 0.00485 ± 0.00204% of the total crude protein.

On a fresh weight basis, the average PAT/ bar protein content from all test sites ranged from 115 μg/g to 188 μg/g fresh weight in unsprayed fuzzy seed with an average value of 145 ± 28.0 μg/g.

The amount of PAT/ bar determined to be in the sprayed transgenic fuzzy seed ranged from 118 μg/g to 172 μg/g with an average value of 145 ± 22.2 μg/g. The PAT/ bar protein comprised an average of 0.0728 ± 0.0121 % of the total crude protein in unsprayed fuzzy seed. For sprayed fuzzy seed, the PAT/ bar protein comprised an average of 0.0736 ± 0.0151% of the total crude protein.

On a dry weight basis, the average PAT/ bar protein content from all test sites ranged from 125 μg/g to 213 μg/g dry weight in unsprayed fuzzy seed with an average value of 163 ± 32.4 μg/g.

The amount of PAT/ bar determined to be in the sprayed transgenic fuzzy seed ranged from 129 μg/g to 196 μg/g with an average value of 163 ± 27.4 μg/g. The PAT/ bar protein comprised an average of 0.0728 ± 0.0121 % of the total crude protein in unsprayed fuzzy seed. For sprayed fuzzy seed, the PAT/ bar protein comprised an average of 0.0736 ± 0.0151% of the total crude protein.



### B. Cry1Ab, Cry2Ae, and PAT protein cont ent in plant parts and during the life cycle

Additional data on protein content on plant parts and during the life cycle of the plant were analyzed and submitted as part of the Section 3 Registration package to USEPA. This information may be found in the Cry1Ab cotton, the Cry2Ae cotton and the TwinLink cotton

Confidential Statement of Formula documents.



### C. Expression of other parts of the insert

There is no expression of other genes (coding sequences) of the inserts since the inserted sequences consist only of the cry1Ab - bar cassette in T304-40 and cry2Ae - bar cassette in GHB119. The absence of any additional DNA from the vectors used for the transformation has

been documented in Section IV.E.TwinLink Cotton USDA Petition

Page 43 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 5 Fresh Weight average quantities of Cry1Ab, Cry2Ae and PAT proteins in raw agricultural commodities of TwinLink cotton as detected by ELISA

Average protein contents (μg/g sample) ± SD Average protein content (as % of total crude protein) ± SD Protein *

No Glufosinate Glufosinate Treated No Glufosinate Glufosinate Treated

Cry1Ab

Range in values 0.226 – 1.64 0.962 – 1.77 0.000147 – 0.000751 0.000455 – 0.000871

Average ± SD 1.16 ± 0.482 1.38 ± 0.301 0.00056 ± 0.000206 0.000694 ± 0.000153

Cry2Ae

Range in values 4.69 – 11.7 6.01 – 16.2 0.00248 – 0.00601 0.00283 – 0.00870

Average ± SD 8.34 ± 2.55 9.51 ± 3.56 0.00420 ± 0.00129 0.00485 ± 0.00204

PAT/ bar

Range in values 115 – 188 118 – 172 0.0505 – 0.0896 0.0512 – 0.0923

Average ± SD 145 ± 28.0 145 ± 22.2 0.0728 ± 0.0121 0.0736 ± 0.0151

* Data based on 7 sites and 12 measurements (2 sample extracts assayed in duplicate from three

replicate plots).



### Table 6 Dry Weight average quantities of Cry1Ab, Cry2Ae and PAT proteins in raw agricultural commodities of TwinLink cotton as detected by ELISA

Average protein contents (μg/g sample) ± SD Average protein content (as % of total crude protein) ± SD Protein *

No Glufosinate Glufosinate Treated No Glufosinate Glufosinate Treated

Cry1Ab

Range in values 0.258 – 1.81 1.09 – 1.96 0.000147 – 0.000751 0.000455 – 0.000871

Average ± SD 1.29 ± 0.520 1.54 ± 0.318 0.000561 ± 0.000206 0.000694 ± 0.000153

Cry2Ae

Range in values 5.34 – 13.4 6.67 – 18.5 0.00248 – 0.00601 0.00283 – 0.00870

Average ± SD 9.35 ± 2.93 10.7 ± 4.17 0.00420 ± 0.00129 0.00485 ± 0.00204

PAT/ bar

Range in values 125 – 213 129 – 196 0.0505 – 0.0896 0.0512 – 0.0923

Average ± SD 163 ± 32.4 163 ± 27.4 0.0728 ± 0.0121 0.0736 ± 0.0151

* Data based on 7 sites and 12 measurements (2 sample extracts assayed in duplicate from three

replicate plots).TwinLink Cotton USDA Petition

Page 44 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### D. Verification of the biochemical and functional equivalence of Cry1ab, Cry2Ae and PAT/bar proteins produced in TwinLink cotton and in E. coli

Studies on potential toxicology and allergenici ty for food, feed and the environment were conducted using Cry1Ab, Cry2Ae and PAT/bar proteins expressed in E. coli . The results of these experiments are used to show safety of the same protein produced in TwinLink cotton. In order to utilize the safety data of the protein produced in a microorganism for the safety assessment of the same protein produced in a genetically modified plant, it is important to confirm that the protein produced in a microorganism is representative of the protein produced in the modified plant. The Cry1Ab, Cry2Ae and PAT/ bar proteins isolated from E. coli were compared to the Cry1Ab, Cry2Ae and PAT/ bar proteins isolated from TwinLink cotton leaves, using the 6 following criteria and associated methods.

1. Molecular weight (SDS-PAGE)

The Cry1Ab, Cry2Ae and PAT/ bar proteins from E. coli and Bt. and the Cry1Ab, Cry2Ae and

PAT/ bar proteins isolated from TwinLink cotton leaves were analyzed by SDS-PAGE. The proteins from the plant and the corresponding protein from E. coli and Bt. were denatured and analyzed by electrophoresis on a denaturing polyacrylamide gel where mobility can be correlated to molecular weight. Molecular weight markers on the gel are comprised of a series of recombinant proteins of known molecular weight. The gel was then stained with Pierce Imperial Stain to visualize the protein bands.

Figure 7, Panel A shows the Pierce Imperial stained gel of Cry1Ab. The electrophoretic mobilities of the Cry1Ab protein produced in E. coli and isolated from TwinLink cotton leaves are indistinguishable. The Cry1Ab from TwinLink has other lower molecular weight bands that are attributed to degradation from the isolation procedure. The Cry1Ab proteins from E. coli and TwinLink cotton have measured electrophoretic mobilities of 24.5 mm. The electrophoretic mobility of each standard protein was plotted versus its respective molecular weight and an approximate molecular weight of 66 kDa was determined for Cry1Ab. The theoretical molecular weight calculated from the amino acid sequence deduced from the DNA sequence is approximately 69.5 kDa.

Figure 8, Panel A shows the Pierce Imperial stained gel of Cry2Ae. The electrophoretic mobilities of the Cry2Ae proteins produced in Bt. or isolated from TwinLink cotton leaves are indistinguishable. The Cry2Ae protein isolated from TwinLink has other higher molecular weight bands in the gel, in which one is believed to be a protein dimer and the other is not related to the protein. The Cry2Ae proteins from Bt . and TwinLink have measured electrophoretic mobilities of 33.5 mm. The electrophoretic mobility of each standard protein was plotted versus its respective molecular weight and an approximate molecular weight of 65 kDa was determined for Cry2Ae. The theoretical molecular weight ca lculated from the amino acid sequence deduced from the DNA sequence is approximately 70.9 kDa.

Figure 9, Panel A shows the Pierce Imperial stained gel of PAT/ bar . The electrophoretic

mobilities of the PAT/ bar proteins produced in E. coli. or isolated from TwinLink cotton leaves are indistinguishable. The PAT/ bar proteins from E. coli and TwinLink have measured electrophoretic mobilities of 76.5 mm. The electrophoretic mobility of each standard protein wasTwinLink Cotton USDA Petition

Page 45 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### plotted versus its respective molecular weight and an approximate molecular weight of 21.9 kDa

was determined for PAT/ bar . The theoretical molecular weight calculated from the amino acid sequence deduced from the DNA sequence is approximately 21 kDa.

Panel A Panel B Cry1Ab SDS-PAGE Cry1Ab Western Blot

A B C kDa

250-

100-

50-

37-



### Lanes: A- Kaleidoscope Marker B- Cry1Ab Isolated from TwinLink Cotton Leaves C- Cry1Ab Thrombin Standard A B C D kDa

220-

120-

70-

50-

40-

30-

20-



### Lanes:

A- BenchMark Protein Ladder B- Cry1Ab Thrombin Standard C- Cry1Ab Isolated from TwinLink

D- BenchMark Protein Ladder A B C kDa

250-

100-

50-

37-



### Lanes: A- Kaleidoscope Marker B- Cry1Ab Isolated from TwinLink Cotton Leaves C- Cry1Ab Thrombin Standard

A B C kDa

250-

100-

50-

37-



### Lanes: A- Kaleidoscope Marker B- Cry1Ab Isolated from TwinLink Cotton Leaves C- Cry1Ab Thrombin Standard A B C D kDa

220-

120-

70-

50-

40-

30-

20- 

### A B C D kDa

220-

120-

70-

50-

40-

30-

20-



### Lanes:

A- BenchMark Protein Ladder B- Cry1Ab Thrombin Standard C- Cry1Ab Isolated from TwinLink

D- BenchMark Protein Ladder



### Figure 7 Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein isolated from leaves of transgenic TwinLink cotton.

Panel A shows the SDS-PAGE gel stained with Pierce Imperial Protein Stain. Lanes A and D contain molecular weight markers of 220 , 160, 120 , 100, 90, 80, 70 , 60, 50 , 40 , 30

, 25, 20 , 15 and 10 kDa. Underlined molecular weights are shown on the gel. Lane B contains the Cry1Ab Thrombin protein standard produced in E. coli. Lane C contains the Cry1Ab protein isolated from TwinLink cotton leaves. Panel B shows the western blot. Lane A contains the molecular weight markers of 250 , 150, 100 , 75, 50 , 37 , 25, 20, 15 and 10 kDa. Underlined molecular weights are shown on the western blot. Lane B contains the Cry1Ab protein isolated from TwinLink cotton leaves. Lane C, contains the Cry1Ab Thrombin protein standard from E. coli.TwinLink Cotton USDA Petition

Page 46 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION



### Panel A Panel B Cry2Ae SDS-PAGE Cry2Ae Western Blot

A B C D

220-

120-



### Lanes:
A- Benchmark Protein Standard B- Cry2Ae from TwinLink (pooled) C- Cry2Ae Standard

D- Benchmark Protein Standard kDa

60-

50- 80-

40-

30-

20- 

### A B C kDa

250-
150-
100-

50-

37-

20-

15-



### Lanes: A- Kaleidoscope Marker B- Cry2Ae from TwinLink C- Cry2Ae Standard A B C D

220-

120-



### Lanes:
A- Benchmark Protein Standard B- Cry2Ae from TwinLink (pooled) C- Cry2Ae Standard

D- Benchmark Protein Standard kDa

60-

50- 80-

40-

30-

20- 

### A B C kDa

250-
150-
100-

50-

37-

20-

15-



### Lanes: A- Kaleidoscope Marker B- Cry2Ae from TwinLink C- Cry2Ae Standard

A B C kDa

250-
150-
100-

50-

37-

20-

15-



### Lanes: A- Kaleidoscope Marker B- Cry2Ae from TwinLink C- Cry2Ae Standard



### Figure 8 Comparison of the Cry2Ae protein from Bt. to the Cry2Ae protein isolated from leaves of transgenic TwinLink cotton.

Panel A shows the SDS-PAGE gel stained with Pi erce Imperial Protein Stain. Lane A and D contains molecular weight markers of 220 , 160, 120 , 100, 90, 80, 70 , 60 , 50 , 40 , 30 , 25, 20 , 15 and 10 kDa. Underlined molecular weights are shown on the gel. Lane B contains the Cry2Ae protein

isolated from TwinLink cotton leaves. Lane C contains the Cry2Ae protein standard from Bt . Panel B shows the western blot for Cry2Ae. Lane A contains the molecular weight markers of 250 , 150

, 100 , 75, 50 , 37 , 25, 20, 15 and 10 kDa. Underlined molecula r weights are shown on the western blot. Lane B contains the Cry2Ae protein isolated from TwinLink cotton leaves. Lane C, contains the Cry2Ae protein standard from E. coli.TwinLink Cotton USDA Petition

Page 47 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Panel A Panel B

PAT/ bar SDS-PAGE PAT/ bar Western Blot

A B C kDa

250-

100-

50-

37-

20-

10-

Lanes: A- Kaleidoscope Marker
B- PAT/ bar isolated from TwinLink Cotton Leaves

C- PAT/ bar Standard A B C kDa

-220

-120

-70

-50

-30

-25 -20

Lanes:

A- PAT/ bar isolated from TwinLink

B- PAT/ bar Standard

C- BenchMark Protein Ladder A B C kDa

250-

100-

50-

37-

20-

10- A B C kDa

250-

100-

50-

37-

20-

10-

Lanes: A- Kaleidoscope Marker
B- PAT/ bar isolated from TwinLink Cotton Leaves

C- PAT/ bar Standard A B C kDa

-220

-120

-70

-50

-30

-25 -20

Lanes:

A- PAT/ bar isolated from TwinLink

B- PAT/ bar Standard

C- BenchMark Protein Ladder



### Figure 9 Comparison of the PAT/ bar protein from E. coli to the PAT/ bar protein isolated from leaves of transgenic TwinLink cotton.

Panel A shows the SDS-PAGE gel stained with Pierce Imperial Protein Stain. Lane A contains the

PAT/ bar protein isolated from TwinLink cotto n leaves. Lane B contains the PAT/ bar protein from E.

coli. Lane C contains molecular weight markers of 220 , 160, 120 , 100, 90, 80, 70 , 60, 50 , 40, 30

, 25 , and 20 kDa. Underlined molecular weights are sh own on the gel. Panel B shows the western blot. Lane A contains the molecular weight markers of 250 , 150, 100 , 75, 50 , 37 , 25, 20, 15 and 10 kDa. Underlined molecular weights are shown on the western blot. Lane B contains the PAT/ bar protein isolated from TwinLink cott on leaves. Lane C contains the PAT/ bar protein standard from E.

coli.



### 2. Immuno-reactivity (Western Blot)

The Cry1Ab western blot results are shown in Figure 7, Panel B. These results show that the electrophoretic mobilities of the Cry1Ab proteins produced from E. coli or isolated from TwinLink cotton leaves are indistinguishable and both proteins are immuno-reactive. The Cry1Ab protein isolated from TwinLink cotton appears to be partially degraded based on the multiple low molecular weight bands beneath the Cry1Ab band in the western. The Cry1Ab protein degradation is believed to come from the isolation process.

The Cry2Ae western blot results are shown in Figure 8, Panel B. These results show that the electrophoretic mobilities of the Cry2Ae proteins produced from Bt. or isolated from TwinLink cotton leaves are indistinguishable and both proteins are immuno-reactive. A higher molecular weight band is seen in the western blot for Cry2Ae isolated from TwinLink cotton leaves and aTwinLink Cotton USDA Petition

Page 48 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### faint band can be seen for the Cry2Ae protein from Bt. This higher molecular weight band is believed to be a dimer of the Cry2Ae protein. Some lower molecular weight bands were also seen for the Cry2Ae protein isolated from TwinLink cotton. These lower molecular weight bands are believed to be degraded Cry2Ae formed as a result of the protein isolation procedure.

The PAT/ bar western blot results are shown in Figure 9, Panel B. These results show that the electrophoretic mobilities of the PAT/ bar proteins produced from E. coli. or isolated from TwinLink cotton leaves are indistinguishable and both proteins are immuno-reactive. An additional band in the western, possibly caused by a PAT/ bar protein dimer, was detected in the

PAT/ bar protein sample isolated from TwinLink cotton leaves.

3. N-terminal sequencing

The theoretical N-terminal sequence of the first five amino acids of the Cry1Ab protein deduced from the DNA sequence of the gene in E. coli and TwinLink cotton leaves is: methionine, aspartic acid, asparagine, asparagine and proline. An N-terminal sequence was not determined by Edman Degradation because the concentration of the Cry1Ab protein isolated from TwinLink cotton leaves was too low. However, the N-terminal peptide for the Cry1Ab protein isolated from

TwinLink was detected in the selected ion mass s pectrometry analysis of the protein, indicating that the protein is not modified.

The theoretical N-terminal sequence of the first five amino acids of the Cry2Ae protein deduced from the DNA sequence of the gene in Bt. and TwinLink cotton leaves is: asparagine, asparagine, valine, leucine and asparagine. The Cry2Ae protein isolated from TwinLink cotton leaves gave the following sequence for the firs t five amino acids by Edman Degradation: ?- asparagine-valine-leucine-asparagine. The first amino acid which is asparagine was not detected or could not be distinguished from the second amino acid which is also asparagine. Since the selected ion monitoring mass spectrometry analysis of the Cry2Ae protein isolated from TwinLink cotton was detected, the peptide is not blocked or modified.

The following N-terminal sequence was obtained by Edman Degradation for the Cry2Ae protein

from Bt. : methionine, asparagine, asparagine, valine and leucine. The asparagine to leucine sequence matches the N-terminal peptide sequence for Cry2Ae. Since the full scan and selected ion monitoring mass spectrometry analysis of the Cry2Ae bacterial N-terminal peptide was detected, indicating that the peptide is not modified and does not contain a methionine amino acid, the identification of a methionine on the N-terminal sequence by Edman Degradation may be caused by contamination in the N-terminal sequencing analysis.

The theoretical N-terminal sequence of the first five amino acids of the PAT/ bar protein deduced from the DNA sequence of the gene in E. coli and TwinLink cotton leaves is: methionine, aspartic acid, proline, glutamic acid and arginine. The following N-terminal sequence was

obtained for the PAT/ bar protein from E. coli. : methionine, aspartic acid, proline, glutamic acid and arginine. This sequence is an exact match to the sequence deduced from the DNA sequence of the PAT/ bar gene for residues 1-5. The PAT/ bar protein isolated from TwinLinkcotton leaves gave a sequence by Edman Degradation of: ?-aspartic-methionine-prolineglutamic acid. Since the PAT/ bar N-terminal peptide was not detected by selected ion monitoring mass spectrometry indicating that the peptide is modified, not detecting the first amino acid and having an incorrect assignment for the third by Edman Degradation confirmsTwinLink Cotton USDA Petition

Page 49 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### that the peptide is modified. Post-translational modifications, such as removal of a methionine are often found in proteins from both prokaryotic and eukaryotic organisms (Bradshaw et al.

, 1998).

4. Peptide profile (LC-MS)

i. Cry1Ab Protein

Peptides from a trypsin digest of the Cry1Ab protein from E. coli and the Cry1Ab protein isolated from TwinLink cotton were analyzed by LC/MS using the selected ion monitoring method developed for the analysis. The method was developed based on the protein sequence of the Cry1Ab protein from E. coli. The expected peptides from the trypsin digest of the Cry1Ab protein from E. coli were identified from their full scan spectra. The most abundant ion for each peptide was chosen for selected ion monitoring. The peptides at positions 116 to 127, EWEADPTNPALR, 128 to 131, EEMR, 450 to 458, APMFSWIHR and 459 to 478, SAEFNNIIPSSQITQIPLTK could not be detected by fu ll scan mass spectrometry, which resulted in a peak for that peptide not being assigned and not detected by selected ion monitoring, Table 7. The peptide not being detected by full scan mass spectrometry could be caused by a missed cleavage.

A 91% sequence coverage of the Cry1Ab protein from E. coli was obtained by full scan and selected ion monitoring combined, and 80% sequence coverage was obtained by the selected ion monitoring method, Tables 8 and 9. The analysis of the tryptic digest of the Cry1Ab protein isolated from TwinLink cotton leaves by the selected ion monitoring method obtained 74% sequence coverage of the protein, Tables 8 and 9. The N-terminal peptide, MDNNPNINECIPYNCLSNPEVEVLGGER, of the Cry1Ab protein isolated from TwinLink cotton leaves was detected demonstrating that the N-terminal is not modified. The peptides at positions 100 to 115, LEGLSNLYQIYAESFR, a nd 602 to 620, IEFVPAEVTFEAEYDLVPR were not detected in the Cry1Ab protein isolated from TwinLink cotton leaves; but were detected in the Cry1Ab protein from E. coli. These peptides were not detected possibly because of a missed cleavage, modification, or the sample concentration was too low. The selected ion monitoring results for the Cry1Ab protein demonstrate that 81% of the Cry1Ab protein isolated from TwinLink cotton leaves is identical to the Cry1Ab protein from E. coli., Table 8.

The Cry1Ab protein has sites with a potential for N-glycosylation. N-glycosylated proteins are glycosylated on the asparagine residue and have a tripeptide asparagine-X-serine/threonine sequence, where X is any amino acid except proline. However, the presence of this sequence does not indicate that the protein will be glycosylated. The Cry1Ab protein sequence has 5 tryptic peptides with the N-X-S/T sequence. The selected ion monitoring analysis results of these 5 tryptic peptides from the bacterial standard Cry1Ab protein and the Cry1Ab protein isolated from TwinLink cotton leaves is shown in Table 9. All the Cry1Ab tryptic peptide residues containing the N-X-S/T sequence were detected in both the bacterial standard and Cry1Ab protein isolated from TwinLink cotton. The glycostain analysis results of the Cry1Ab protein isolated from TwinLink and bacterial st andard also indicates that the protein is not glycosylated.TwinLink Cotton USDA Petition

Page 50 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 7 Cry1Ab isolated from TwinLink cotton trypsin digest peptide map using Electrospray LC/MS

91%cov. (full scan and SIM) 80 % cov, SIM 74% cov, SIM Cry1Ab Theoretical mass Cry1Ab Std Cry1Ab Mol. Wt. by residue number [M+H] TwinLink Mass Spec.

______________________________________________________________________________________________________

1 to 28 3135 1568 [M+2H] 1568 [M+2H] 3134 29 to 87 6569 1643 [M+4H] 1643 [M+4H] 6568

88 to 93 765 766 a [M+H] NA* 765 94 to 99 689 689 [M+H] 689 [M+H] 688 100 to 115 1904 952 [M+2H] ND 951

116 to 127 1400 ND NA ND

128 to 131 565 ND NA ND 132 to 173 4675 1559 [M+3H] 1559 [M+3H] 4674

174 to 181 908 908 [M+H] NA* 907 182 to 192 1238 1238 [M+H] 1238 [M+H] 1237

193 to 198 782 782 [M+H] NA* 781 199 to 209 1259 1259 [M+H] 1259 [M+H] 1258

210 to 217 1039 1039 [M+H] NA* 1038

218 to 224 817 817 [M+H] NA* 816

225 to 228 590 589 a [M+H] NA* 588

229 to 233 728 728 [M+H] NA* 727

234 to 234 175 NA NA NA 235 to 253 2197 1099 [M+2H] 1099 [M+2H] 2196

254 to 258 650 649 a [M+H] NA* 648

259 to 265 805 805 [M+H] NA* 804 266 to 281 1902 1902 [M+H] 1902 [M+H] 1901 282 to 292 1075 1075 [M+H] 1075 [M+H] 1074 293 to 311 2199 2199 [M+H] 2199 [M+H] 2198 312 to 349 4182 2091 [M+2H] 2091 [M+2H] 4180 350 to 360 1204 1204 [M+H] 1204 [M+H] 1203

361 to 368 941 941 [M+H] NA* 940 369 to 402 3731 1866 [M+2H] 1866 [M+2H] 3730

403 to 403 147 NA NA NA 404 to 423 2150 2150 [M+H] 2150 [M+H] 2149 424 to 429 732 732 [M+H] 732 [M+H] 731

430 to 437 977 977 [M+H] NA* 976 438 to 449 1254 1254 [M+H] 1254 [M+H] 1253

450 to 458 1145 ND NA ND

459 to 478 2202 ND NA ND 479 to 490 1150 1150 [M+H] 1150 [M+H] 1149 491 to 501 1090 1090 [M+H] 1090 [M+H] 1089

502 to 502 175 NA NA NA 503 to 512 1060 1060 [M+H] 1060 [M+H] 1059 513 to 522 1099 1099 [M+H] 1099 [M+H] 1098

523 to 524 338 NA NA NA

525 to 526 274 NA NA NA

527 to 528 288 NA NA NA 529 to 567 4181 1394 [M+3H] 1394 [M+3H] 4179 568 to 601 3714 1239 [M+3H] 1239 [M+3H] 3714 602 to 620 2226 1113 [M+2H] ND 2224
= Mass Spectrometer is a unit resolution inst rument (uncertainty is ± 1 amu) resulting in some ions being off by 1 amu in the full scan spectrum.

NA* = Not analyzed due to software and sample limitations. NA = Not analyzed. ND = Not Detected Data obtained from Cry1Ab protein is olated from TwinLink on 3/12/2008.TwinLink Cotton USDA Petition

Page 51 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 8 Amino acid coverage of Cry1Ab from E. coli and TwinLink Cotton Leaves

Number of amino acids not detected or analyzed Calculation of % Amino Acid Coverage E. coli Cry1Ab TwinLink Cry1Ab Residue Number

0 6 88-93

0 16 100-115

12 12 116-127

4 4 128-131

0 8 174-181

0 6 193-198

0 8 210-217

0 7 218-224

0 4 225-228

0 5 229-233

1 1 234-234

0 5 254-258

0 7 259-265

0 8 361-368

1 1 403-403

0 8 430-437

9 9 450-458

20 20 459-478

1 1 502-502

2 2 523-524

2 2 525-526

2 2 527-528

0 19 602-620

Total 54 161

Total number of Amino Acids 620 620 % Amino Acid Not Detected or Analyzed 8.7 26 % Amino Acid Sequence Coverage 91 74 % Amino Acid Coverage of Cry1Ab from E. coli. 100 81 NA a

a NA = Not ApplicableTwinLink Cotton USDA Petition

Page 52 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 9 Selected Ion Monitoring mass spectrometry results of the Cry1Ab sequence residues with the Asparagine-X-Serine/Threonine sequence

Cry1Ab Theoretical mass Cry1Ab Std Cry1Ab residue number [M+H] TwinLink

199 to 209 1259 1259 [M+H] 1259 [M+H] 438 to 449 1254 1254 [M+H] 1254 [M+H] 513 to 522 1099 1099 [M+H] 1099 [M+H] 529 to 567 4181 1394 [M+3H] 1394 [M+3H] 568 to 601 3714 1239 [M+3H] 1239 [M+3H]



### ii. Cry2Ae Protein

Peptides from a trypsin digest of the Cry2Ae protein from Bt. and the Cry2Ae protein isolated from TwinLink cotton were analyzed by LC/MS using the selected ion monitoring method developed for the analysis. The method was developed based on the protein sequence of the Cry2Ae protein from Bt. The expected peptides from the trypsin digest of the Cry2Ae protein

from Bt. were identified from their full scan spectra. The most abundant ion for each peptide was chosen for selected ion monitoring. The peptides at positions

315 to 338 = LTQTFPNIGGLPGTTTTHALLAAR

433 to 437 = NEDLR

438 to 446 = RPLHYNEIR

447 to 459 = NIESPSGTPGGLR

460 to 468 = AYMVSVHNR

508 to 513 = TFISEK
514 to 522 = FGNQGDSLR

532 to 535 = YTLR

536 to 546 = GNGNSYNLYLR

557 to 562 = VTINGR

could not be detected by full scan mass spectrometry which resulted in a peak for that peptide not being assigned and not detected by selected ion monitoring, Table 10. The peptides not being detected by full scan mass spectrometry could be caused by a missed cleavage or poor ionization under the conditions of analysis.

A 80% sequence coverage of the Cry2Ae protein from Bt. was obtained by full scan and selected ion monitoring combined and 68% sequence coverage was obtained by the selected ion monitoring method, Tables 11 and 12. The analysis of the tryptic digest of the Cry2Ae protein isolated from TwinLink cotton leaves by the selected ion monitoring method obtained 46% sequence coverage of the protein, Tables 11 and 12. The N-terminal peptide, NNVLNNGR, of the Cry2Ae protein isolated from TwinLink cotton leaves was detected demonstrating that the N-terminal is not modi fied. The N-terminal sequencing analysis also confirmed the NVLN portion of the N-terminal peptide. The peptides at positionsTwinLink Cotton USDA Petition

Page 53 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 339 to 373 = V NYS GGVSSGDIGAVFNQ NFS CSTFLPPLLTPFVR

374 to 382 = SWLDSGSDR
383 to 403 = GGVNTVTNWQTESFESTLGLR

563 to 584 = VYTASNV NTT TNNDGVNDNGAR

585 to 631 = FLDINMGNVVASDNTNVPLDI NVT FNSGTQFELMNIMFVPTNLPPIY

were not detected in the Cry2Ae protein isolated from TwinLink cotton leaves; but were detected in the Cry2Ae protein from Bt . These peptides were not detected possibly because of a missed cleavage, non-glycosylated modifications, or the sample concentration was too low. The selected ion monitoring results for the Cry2Ae protein demonstrate that 58% of the Cry2Ae protein isolated from TwinLink cotton leaves is identical to the Cry2Ae protein from Bt., Table 11

.

The Cry2Ae protein also has sites with a potent ial for N-glycosylation. The Cry2Ae protein sequence has 9 tryptic peptides with the N-X-S/T sequence. The selected ion monitoring analysis results of these 9 tryptic peptides from the bacterial standard Cry2Ae protein and the Cry2Ae protein isolated from TwinLink cotton leaves is shown in Table 12. Cry2Ae tryptic peptide residues 339 to 373, 563 to 584, and 585 to 631, shown above with the N-X-S/T sequence in bold, were not detected by selected ion monitoring. Since a glycostain analysis of the Cry2Ae protein isolated from TwinLink indicates that the protein is not glycosylated, not detecting a peak for these residues can be attributed to a non-glycosylated modification, or low sample concentration.TwinLink Cotton USDA Petition

Page 54 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 10 Cry2Ae isolated from TwinLink cotton trypsin digest peptide map using Electrospray LC/MS

80%cov. (full scan and SIM) 68 % cov, SIM 46% cov, SIM Cry2Ae Theoretical mass Cry2Ae Std Cry2Ae Mol. Wt. by residue number [M+H] TwinLink Mass Spec.
1 to 8 901 901 [M+H] 901 [M+H] 900 9 to 28 2296 1149 [M+2H] 1149 [M+2H] 2296

29 to 34 705 704 a [M+H] NA* 703

35 to 35 147 NA NA NA

36 to 41 909 908 b [M+H] 908 b [M+H] 907

42 to 42 175 NA NA NA

43 to 62 2149 1075 [M+2H ] NA* 2148

63 to 63 147 NA NA NA

64 to 70 674 674 [M+H] NA* 673

71 to 71 175 NA NA NA

72 to 94 2605 1302 a [M+2H] 1302 a [M+2H] 2602

95 to 103 1165 1166 a [M+H] NA* 1165 104 to 111 904 904 [M+H] 904[M+H] 903

112 to 124 1428 1429 a [M+H] NA* 1428

125 to 159 4037 1346 b [M+3H] 1346 b [M+3H] 4035 160 to 164 661 661 [M+H] 661 [M+H] 660

165 to 188 2745 916 [M+3H] NA* 2745

189 to 205 1831 NA NA NA

206 to 212 930 929 a [M+H] 929 a [M+H] 928 213 to 231 2354 1177 [M+2H] 1177 [M+2H] 2352

232 to 236 561 560 a [M+H] 560 a [M+H] 559

237 to 244 1061 1061 [M+H] NA* 1060

245 to 262 2289 1144 a [M+2H] 1144 a [M+2H] 2286 263 to 314 5685 1422 [M+4H] 1422 [M+4H] 5684

315 to 338 2453 ND NA ND 339 to 373 3693 1848 [M+2H] ND 3694

374 to 382 1023 1022 a [M+H] ND 1021

383 to 403 2297 1149 a [M+2H] ND 2296 404 to 410 726 726 [M+H] 726 [M+H] 725 411 to 422 1494 1493 [M+H] 1493 [M+H] 1492 423 to 432 1054 1054 [M+H] 1054 [M+H] 1053

433 to 437 647 ND NA ND

438 to 446 1198 ND NA ND

447 to 459 1258 ND NA ND

460 to 468 1077 ND NA ND

469 to 469 147 NA NA NA 470 to 507 4270 856 [M+5H] 856 [M+5H] 4275

508 to 513 725 ND NA ND

514 to 522 994 ND NA ND 523 to 531 1054 1054[M+H] 1054[M+H] 1053

532 to 535 553 ND NA ND

536 to 546 1271 ND NA ND

547 to 556 1034 1034 b [M+H] 1034 b [M+H] 1033

557 to 562 660 ND NA ND

563 to 584 2298 1151 a [M+2H] ND 2300 585 to 631 5233 1309 [M+4H] ND 5232

a = Mass Spectrometer is a unit resolution in strument (uncertainty is ± 1 amu) resulting in some ions being off by 1 amu in the full scan spectrum.

b = Selected ion monitoring (SIM) mass is 1 amu off from full scan spectrum because SIM method was developed from an earlier ful l scan spectrum. NA* = Not analyzed due to software and sample limitations. NA = Not analyzed. ND = Not DetectedTwinLink Cotton USDA Petition

Page 55 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 11 Amino acid Coverage of Cry2Ae from Bt. and TwinLink Cotton Leaves

Number of amino acids not detected or analyzed Calculation of % Amino Acid Coverage Bt. Cry2Ae TwinLink Cry2Ae Residue number

0 6 29-34

1 1 35-35

1 1 42-42

0 20 43-62

1 1 63-63

0 7 64-70

1 1 71-71

0 9 95-103

0 13 112-124

0 24 165-188

17 17 189-205

8 8 237-244

24 24 315-338

0 35 339-373

0 9 374-382

0 21 383-403

5 5 433-437

9 9 438-446

13 13 447-459

9 9 460-468

1 1 469-469

6 6 508-513

9 9 514-522

4 4 532-535

11 11 536-546

6 6 557-562

0 22 563-584

0 47 585-631

Total 126 339

Total number of Amino Acids 631 631 % Amino Acid Not Detected or Analyzed 20 54 % Amino Acid Sequence Coverage 80 46 % Amino Acid Coverage of Cry2Ae from Bt. 100 58 NA a

a NA = Not ApplicableTwinLink Cotton USDA Petition

Page 56 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 12 Selected Ion Monitoring mass spectrometry results of the Cry2Ae sequence residues with the Asparagine-X-Serine/Threonine sequence

Cry2Ae Theoretical mass Cry2Ae Std Cry2Ae residue number [M+H] TwinLink

125 to 159 4037 1346 b [M+3H] 1346 b [M+3H] 213 to 231 2354 1177 [M+2H] 1177 [M+2H] 339 to 373 3693 1848 [M+2H] ND 423 to 432 1054 1054 [M+H] 1054 [M+H] 470 to 507 4270 856 [M+5H] 856 [M+5H] 523 to 531 1054 1054[M+H] 1054 [M+H]

547 to 556 1034 1034 b [M+H] 103 b [M+H]

563 to 584 2298 1151 a [M+2H] ND 585 to 631 5233 1309 [M+4H] ND

a = Mass Spectrometer is a unit resolution instrument (uncerta inty is ± 1 amu) resulting in some ions being off by 1 amu in the full scan spectrum.

b = Selected ion monitoring (SIM) mass is 1 amu off fr om full scan spectrum because SIM method was developed from an earlier full scan spectrum. ND = Not Detected due to low sample concentration or a non-glycosylated modification because glycostain analysis indicated that the protei n was not glycosylated.



### iii. PAT/ bar Protein

Peptides from a trypsin digest of the PAT/ bar protein from E. coli and the PAT/ bar protein isolated from TwinLink cotton were analyzed by LC/MS using the selected ion monitoring method previously developed for the analysis of cotton. The method previously developed was based on the protein sequence of the PAT/ bar protein from E. coli. The expected peptides from the trypsin digest of the PAT/ bar protein from E. coli were identified from their full scan spectra. The most abundant ion for each peptide was chosen for selected ion monitoring.

A 96% sequence coverage of the PAT/ bar protein from E. coli. was obtained by selected ion monitoring, Tables 13 and 14. The analysis of the peptides from the tryptic digest of the

PAT/ bar protein isolated from TwinLink cotton l eaves by the selected ion monitoring method obtained 81% sequence coverage of the protein, Tables 13 and 14. The N-terminal peptide, MDPER, of the PAT/ bar protein isolated from TwinLink cotton leaves was not detected indicating that the N-terminal peptide may be modified. Post-translational modifications, such as removal of a methionine are often found in proteins from both prokaryotic and eukaryotic organisms (Bradshaw et al. , 1998). The peptides at positions 97 to 99, HQR, 136 to 145, MHEALGYAPR, 146 to 149, GMLR and 150 to 154, AAGFK were not detected in the PAT/ bar protein isolated from TwinLink cotton leaves; but were detected in the PAT/ bar protein from E.

coli. These peptides were not detected possibly because of a missed cleavage, modification, or the sample concentration was too low. The selected ion monitoring results for the PAT/ bar protein demonstrate that 84% of the PAT/ bar protein isolated from TwinLink cotton leaves is

identical to the PAT/ bar protein from E. coli., Table 14.TwinLink Cotton USDA Petition

Page 57 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 13 PAT/ bar isolated from TwinLink cotton trypsin digest peptide map using Electrospray LC/MS

96 % cov, SIM 81 % cov, SIM

PAT/bar Theoretical mass PAT/ bar Std PAT/bar Mol. Wt. by residue number [M+H] TwinLink Mass Spec.

1 to 5 647 648 [M+H] ND 647 6 to 11 727 728 [M+H] 728 [M+H] 727

12 to 12 175 NA* NA* NA 13 to 37 2782 930 [M+3H] 930 [M+3H] 2787 38 to 52 1842 923 [M+2H] 923 [M+2H] 1844

53 to 54 288 NA* NA* NA

55 to 56 304 NA* NA* NA 57 to 78 2391 1198 [M+2H] 1198 [M+2H] 2394

79 to 80 246 NA* NA* NA 81 to 96 1859 931 [M+2H] 931 [M+2H] 1860 97 to 99 440 222 [M+2H] ND 442 100 to 112 1404 703 [M+2H] 703 [M+2H] 1404 113 to 120 879 880 [M+H] 880 [M+H] 879 121 to 135 1523 763 [M+2H] 763 [M+2H] 1524 136 to 145 1145 1146 [M+H] ND 1145 146 to 149 476 477 [M+H] ND 476 150 to 154 493 494 [M+H] ND 493 155 to 183 3353 843** [M+4H] 843** [M+4H] 3368

NA* = Two amino acid masses were not analyzed because it was not indicative of only the PAT/bar protein.

ND = Not Detected ** The peptide is oxidized, possibly on a tryptophan residue.



### Table 14 Amino acid coverage of PAT/ bar from E. coli and TwinLink cotton leaves

Number of amino acids not detected or analyzed Calculation of % Amino Acid Coverage

E. coli PAT/bar TwinLink

PAT/bar Residue number

0 5 1-5

1 1 12-12

2 2 53-54

2 2 55-56

2 2 79-80

0 3 97-99

0 10 136-145

0 4 146-149

0 5 150-154

Total 7 34

Total number of Amino Acids 183 183 % Amino Acid Not Detected or Analyzed 4 19 % Amino Acid Sequence Coverage 96 81 % Amino Acid Coverage of PAT/ bar from E. coli. 100 84 NA a

a NA = Not ApplicableTwinLink Cotton USDA Petition

Page 58 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 5. Biological activity

PAT/ bar proteins from E. coli or isolated from TwinLink cotton leaves, was shown to generate free Coenzyme A sulfhydryl groups during the transfer of the acetyl group to phosphinothricin, resulting in an increase of the absorption at 412 nm by more than 10%. This increase in absorption indicated that both the PAT/ bar proteins from E. coli and TwinLink cotton leaves are biologically active.

The activity of the Cry2Ae protein isolated from TwinLink cotton was determined by feeding the isolated Cry2Ae protein to Helicoverpa zea larvae, at the first larvae stage. The Cry2Ae protein standard was also analyzed for comparison. The insects fed the isolated Cry2Ae protein from TwinLink cotton lost approximately the same amount of weight as the insects fed the Cry2Ae protein standard. Therefore, the Cry2Ae protein isolated from TwinLink cotton leaves has the same activity as the Cry2Ae protein standard from Bt.

Activity determination could not be done with Cry1 Ab protein isolated from TwinLink cotton because the protein concentration was too low.

6. Expected and unexpected post-translational modifications

i. Cry1Ab protein

The glyco-staining results for the Cry1Ab protein are shown in Figure 10. The N-glycosylated proteins for Avidin and alpha acidic glycoprotein in lane C are seen as bright bands in the gel. The Cry1Ab protein from E. coli. and the Cry1Ab protein isolated from TwinLink cotton leaves in lanes B and D are not bright bands. This indicates that the Cry1Ab proteins are not glycosylated. The mass spectrometry data also confirmed that the Cry1Ab proteins are not glycosylated by detecting the peptides with a potential site for glycosylation.

ii. Cry2Ae and PAT/bar proteins

The glycol-staining results for the Cry2Ae and PAT/ barproteins are shown in Figure 11. The Nglycosylated proteins for Avidin and alpha acidic glycoprotein in lane D are seen as bright bands in the gel. The Cry2Ae protein from Bt. and the Cry2Ae protein isolated from TwinLink cotton leaves in lanes B and C are not detected as bright bands indicating that the proteins are not glycosylated. The PAT/ bar protein from E. coli. and the PAT/ bar protein isolated from TwinLink cotton leaves in lanes F and E are not seen as bright bands indicating also that the proteins are not glycosylated.TwinLink Cotton USDA Petition

Page 59 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION A B C D E

220-

120-

80-

70-

60-

50-

25-

20-

Lanes:

A- BenchMark Protein Ladder B- Cry1Ab Thrombin Protein Standard C- BTS4 N-Glycosylated Protein Standard D- Cry1Ab Isolated from TwinLink Cotton Leaves

E- BenchMark Protein Ladder

A B C D E

220-

120-

80-

70-

60-

50-

25-

20-

Lanes:

A- BenchMark Protein Ladder B- Cry1Ab Thrombin Protein Standard C- BTS4 N-Glycosylated Protein Standard D- Cry1Ab Isolated from TwinLink Cotton Leaves

E- BenchMark Protein Ladder



### Figure 10 GlycoStain of the Cry1Ab protein from E. coli and the Cry1Ab protein isolated from leaves of transgenic TwinLink Cotton.

GlycoprofileTM III Fluorescent Glycoprotein analy sis of the Cry1Ab protein from E. coli. and the Cry1Ab protein isolated from TwinLink cotton leav es. Lanes A and E contain molecular weight

markers of 220 , 160, 120 , 100, 90, 80 , 70 , 60 , 50 , 40, 30, 25 , 20 , 15 and 10 kDa. Underlined molecular weights are shown on the stained gel. Lane B contains the Cry1Ab protein from E. coli. Lane C contains the BTS#4 Glycoprotein Standar d mix of Phosphorylase B, alpha-Acidic glycoprotein, Carbonic anhydrase and Avidin. Lane D contains Cry1Ab isolated from TwinLink

cotton leaves.TwinLink Cotton USDA Petition

Page 60 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION A B C D E F G kD

220-

120-

80-

70- 60-

50-

25-

20- 40-

30-

Lanes:

A- BenchMark Protein Ladder B- Cry2Ae Protein Standard C- Cry2Ae Isolated from TwinLink Cotton Leaves D- BTS4 N-Glycosylated Protein Marker

E- PAT/bar Isolated from TwinLink Cotton Leaves

F- PAT/bar Protein Standard

G- BenchMark Protein Ladder A B C D E F G kD

220-

120-

80-

70- 60-

50-

25-

20- 40-

30-

Lanes:

A- BenchMark Protein Ladder B- Cry2Ae Protein Standard C- Cry2Ae Isolated from TwinLink Cotton Leaves D- BTS4 N-Glycosylated Protein Marker

E- PAT/bar Isolated from TwinLink Cotton Leaves

F- PAT/bar Protein Standard

G- BenchMark Protein Ladder



### Figure 11 GlycoStain of the Cry2Ae and PAT/ bar proteins from Bt. and E. coli and the Cry2Ae and PAT/ bar proteins isolated from leaves of transgenic TwinLink
Cotton.

GlycoprofileTM III Fluorescent Glycoprotein analysis of the Cry2Ae protein from Bt. and the PAT/ bar protein from E. coli. and the Cry2Ae and PAT/ bar proteins isolated from TwinLink cotton leaves. Lanes A and G contain molecular weight markers of 220 , 160, 120 , 100, 90, 80 , 70 , 60 , 50 , 40 , 30

, 25 , 20 , 15 and 10 kDa. Underlined molecular weights are shown on the stained gel. Lane B contains the Cry2Ae protein from Bt.. Lane C contains the Cry2Ae pr otein isolated from TwinLink cotton leaves. Lane D contains the BTS#4 Glyc oprotein Standard mix of Phosphorylase B, alphaAcidic glycoprotein, Carbonic anhydrase and Avidin. Lane E contains the PAT/ bar protein isolated

from TwinLink cotton leaves. Lane F contains the PAT/ bar protein from E. coli.



### Conclusion

The results from the six analytical tests indicate that the Cry1Ab, Cry2Ae and PAT/ bar proteins

from E. coli and Bt. are equivalent to the Cry1Ab, Cry2Ae and PAT/ bar proteins isolated from TwinLink cotton leaves. The molecular weights, mobilities, immuno-reactivities, activities for Cry2Ae and PAT/ bar , as well as 81% of Cry1Ab, 58% of Cry2Ae and 84% of PAT/ bar protein sequence coverages, are identical. These data demonstrate that the safety data obtained for the Cry1Ab, Cry2Ae and PAT/ bar proteins produced in E. coli and Bt. can be used to support the safety of the Cry1Ab, Cry2Ae and PAT/ bar proteins produced in TwinLink cotton.TwinLink Cotton USDA Petition

Page 61 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Equivalence of Proteins Expressed in Seed

To complement the equivalence evaluation of proteins produced in cotton leaves, an immunoreactivity study (Western blot) was completed for TwinLink cotton seed. The Western blots indicate that the proteins have the same molecular weights, mobilities and immuno-reactivities. These Western blot studies indicate that the Cry1Ab, Cry2Ae and PAT/ bar proteins produced in the seed of individual events (T304-40 and GHB119) and TwinLink cotton are the same as the Cry1Ab, Cry2Ae and PAT/ bar proteins produced from Escherichia coli and Bacillus thuringiensis.

The results of the Cry1Ab western blot show that the molecular weights, electrophoretic mobilities and immuno-reactivities of the Cry1Ab proteins produced in E. coli or isolated from event T304-40 and TwinLink cotton seed are indistinguishable (Figure 12).

A B C D kDa

250-

100-

75-

50-

37-

20-

15-

Lanes: A- Kaleidoscope Protein Standard B- Cry1Ab isolated from TwinLink cotton seed C- Cry1Ab Thrombin Standard D- Cry1Ab isolated from T304-40 cotton seed A B C D kDa

250-

100-

75-

50-

37-

20-

15-

Lanes: A- Kaleidoscope Protein Standard B- Cry1Ab isolated from TwinLink cotton seed C- Cry1Ab Thrombin Standard D- Cry1Ab isolated from T304-40 cotton seed



### Figure 12 Comparison of the Cry1Ab protein from E. coli to the Cry1Ab protein isolated from ground seed of TwinLink cotton.

Lane A contains molecular weight markers of 250 , 150, 100 , 75 , 50 , 37 , 20 , 15 and 10 kDa. Underlined molecular weights are shown on the gel. Lane B contains the Cry1Ab protein isolated

from TwinLink cotton seed

. Lane C contains the Cry1Ab Thrombin protein standard form E. coli. Lane D contains the Cry1Ab protein is olated from event T304-40 cotton seed.TwinLink Cotton USDA Petition

Page 62 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION A B C D E kD

250-

150- 100-

75-

50-

37-

Lanes: A- Kaleidoscope Protein Std B- Cry2Ae from TwinLink seed C- Cry2Ae Standard D- Cry2Ae from GHB119 seed E- Kaleidoscope Protein Std A B C D E kD

250-

150- 100-

75-

50-

37-

Lanes: A- Kaleidoscope Protein Std B- Cry2Ae from TwinLink seed C- Cry2Ae Standard D- Cry2Ae from GHB119 seed E- Kaleidoscope Protein Std



### Figure 13 Comparison of the Cry2Ae protein from Bt. to the Cry2Ae protein isolated from ground seed of TwinLink cotton.

Lanes A and E contain molecular weight markers of 250, 150, 100, 75, 50, 37, 20, 15 and 10 kDa. Underlined molecular weights are shown on the gel. Lane B contains the Cry2Ae protein isolated

from TwinLink cotton seed. Lane C cont ains the Cry2Ae protein standard from Bt . Lane D contains the Cry2Ae protein isolated from event GHB119 cotton seed.



### The results of the Cry2Ae western blot show that the molecular weights, electrophoretic mobilities and immuno-reactivities of the Cry2Ae proteins produced from Bt. or isolated from event GHB119 and TwinLink cotton seed are indistinguishable. The western blot result for the Cry2Ae protein produced from Bt. in lane C had a higher molecular weight band believed to be a Cry2Ae protein dimer. Some lower molecular weight bands were also seen for the Cry2Ae protein standard indicative of protein degradation (Figure 13).

The results of the PAT/ bar western blot demonstrate that the molecular weights and electrophoretic mobilities of the PAT/ bar proteins produced from E. coli. or isolated from events T304-40, GHB119 and TwinLink cotton seed are indistinguishable and all four proteins are immunoreactive. Faint bands of protein degradation from the protein isolation process can be

seen for the PAT/ bar proteins isolated from TwinLink, GHB119 and T304-40 (Figure 14).TwinLink Cotton USDA Petition

Page 63 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Lanes: A- Kaleidoscope Marker
B- PAT/bar TwinLink seed

C- PAT/bar GHB119 seed

D- PAT/bar T304-40 seed

E- PAT/bar standard F- Kaleidoscope Marker A B C D E F kDa

100-

50-

37-

20-

15- 250-



### Lanes: A- Kaleidoscope Marker
B- PAT/bar TwinLink seed

C- PAT/bar GHB119 seed

D- PAT/bar T304-40 seed

E- PAT/bar standard F- Kaleidoscope Marker A B C D E F kDa

100-

50-

37-

20-

15- 250-



### Figure 14 Comparison of the PAT/ bar protein from E. coli to the PAT/ bar proteins isolated from ground seed of TwinLink cotton.

Lanes A and F contain molecular weight markers of 250, 150, 100, 75, 50, 37, 20, 15 and 10 kDa. Underlined molecular weights are shown on the gel. Lane B contains the PAT /bar protein isolated

from TwinLink cotton seed. Lane C contains t he PAT/bar protein isolated from event GHB119. Lane D contains the PAT/bar protei n isolated from event T304-40 cotton seed. Lane E contains the

PAT/ bar protein from E. coli.



### Conclusion

The results from the western blot analyses indicate that the Cry1Ab, Cry2Ae and PAT/ bar proteins from E. coli. and Bt. are equivalent to the Cry1Ab, Cry2Ae and PAT/ bar proteins isolated from seed of the individual events (T304-40 and GHB119) and from TwinLink cotton. The molecular weights, mobilities and immuno-reactivities are identical. These data demonstrate that the safety data obtained for the Cry1Ab, Cry2Ae and PAT/ bar proteins produced in E. coli and Bt. can be used to support the safety of the Cry1Ab, Cry2Ae and

PAT/ bar proteins produced in TwinLink cotton seed.TwinLink Cotton USDA Petition

Page 64 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## VII. DETECTION TECHNIQUES FO R THE MODIFIED ORGANISM

The trait could be detected either on molecular genetic level or on protein biochemical level.

The molecular genetic detection can be performed with a PCR based method to confirm the presence of the introduced material in cotton plant material.

The polymerase chain reaction (PCR) is a technique used to amplify a small quantity of target DNA in order to make it detectable. Most of the PCR reactions can be carried out as multiplex reactions, which means they involve more than one PCR reaction, therefore more than one target. One targets a DNA sequence endogenous to the plant; another pair targets a DNA sequence specific to the inserted transgene. The endogenous reaction acts as a control in order to determine whether plant DNA is present and that reaction conditions are sufficient to allow amplification. The transgene reaction will only amplify a product from the inserted DNA, making it possible to distinguish between non-transgenic and transgenic samples.

There are specific protocols for each transgene within each type of plant. An individual protocol usually requires optimization to account for differences between laboratories, matrices, or reagents. This optimization is especially import ant when performing multiplex reactions. Some loci are more efficiently amplified than others due to base composition, length of product, and secondary structure. In multiplex reactions, the more efficiently amplified loci compete better for the available reaction components, and will negativ ely influence the yield of product from the less efficient loci, making them less visible or undetectable. It is important to obtain reaction conditions that amplify equimolar quantities of both the enodogenous and transgenic sequences in a known transgenic DNA sample.

The detection tools for the protein level are based on immunoassays. These assays are a Sandwich Enzyme Linked Immunosorbent Assay (ELISA) based on the specific interaction between antibody and antigen. The wells of the solid phase are coated with affinity-purified polyclonal antibodies (capture antibodies) specifically recognizing the protein of interest from the inserted gene. The protein from the introduced gene present in the samples is bound to the capture antibody. The immobilized protein can be detected by sequential incubation with monoclonal or polyclonal antibodies (detection antibody or second antibody) recognizing the protein to be tested, and a horseradish peroxidase-conjugated polyclonal antibody (antibody conjugate) against the second antibody. A peroxi dase substrate, tetramethylbenzidine, is added and converted by the peroxidase to a blue product in proportion to the amount of tested protein present in the sample. Upon the addition of the stop solution, the blue product turns yellow. The optical density of the yellow product at 450 nm reflects proportionally the amount of protein present in the sample.

Another protein detection method is the lateral flow strip (LFS). This method allows qualitative detection of the introduced protein, and can be performed under field and/or laboratory

conditions.

The method uses a double antibody sandwich format to detect the introduced protein. Antibodies specific for the protein are present in tw o places in the strip. One antibody is fixed to the strip in the area where the band is expected, and its purpose is to capture the protein (if theTwinLink Cotton USDA Petition

Page 65 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### protein is present) while the extract flows up the strip. The other antibody is found in the pad that is located near the bottom of the strip, and its purpose is to report the presence of the protein by binding to it. This detection antibody is conjugated to gold particles. When the lateral flow strip is placed in an extract from plant tissue that contains the protein of interest, the extract flows through the pad where the reporting antibody binds to the protein, if present. The extract, reporting antibody and any inserted protein flow thr ough the strip until they come in contact with the capture antibody. A sandwich is formed between the capture antibody, the protein of interest and with some, but not all the reporting antibody that is coupled to the gold. A second band of antibodies to the reporting antibodies capture any remaining antibody to develop the control band. The bands display as a reddish color when the gold-conjugated antibodies are captured in the specific zones on the membrane. The presence of only one band (control band) on the membrane indicates a negative sample and the presence of two bands indicates a positive sample.

Reference material (specific PCR primers, genomic DNA, seeds) of TwinLink cotton can be provided upon request, and upon agreement with BCS.TwinLink Cotton USDA Petition

Page 66 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## VIII. AGRONOMIC AND PH ENOTYPIC EVALUATION

A. Agronomic performance and evaluation

TwinLink cotton, comprised of transformation events T304-40 and GHB119, was derived by transformation of upland Coker cotton varieties to express the Cry1Ab and Cry2Ae proteins,

which are derived from the soil bacterium Bacillus thuringiensis . These Cry protein events were selected based on demonstrated production of protein toxins selective to lepidopteran pests ( i.e. Cotton Bollworm, Tobacco Budworm, Armyworm and Pink Bollworm). Bayer CropScience plans

to commercialize both events as a combined insect-resistant trait under the trade name TwinLink. Cry proteins are widely used as plant incorporated insecticides in US cotton. In 2008, 69% of US cotton acreage was planted in Bt cotton. Today, Bt cotton is grown in every cotton production region of the US (Table 15) and in the Southwest, represents more than half the area in production. TwinLink cotton and its parents also produce the PAT protein that confers tolerance to the herbicide glufosinate ammonium.

Table 15 Bt cotton production in the United States by Region

Region Total acres per region % of Bt cotton planted

Southeast (AL, GA, NC, SC, VA, FL) 1,947,000 21
Mid-South (MS, LA, MO, AR, TN) 1,900,000 20
Southwest (TX, OK, KS) 5,121,000 54
West (CA, NM, AZ) 446,000 4
Source: USDA Agricultural Marketing Service (http://www.ams.usda.gov/), 2008



### TwinLink cotton was evaluated by comparison to its non-transgenic parent though-out the US cotton belt. Agronomic performance trials were conducted in a manner representative of standard agricultural practices within the respective testing regions. Conventional insecticide applications were made in accordance with university extension recommendations to determine trait effectiveness. Thus, comparisons of agronomic performance can be made with conventional and transgenic production practices.

The findings of these tests show:

1. The agronomic performance of TwinLink cotton was equal to or better than that of its nontransgenic counterpart both when sprayed and unsprayed for lepidopteran pests with

conventional insecticides.

2. No significant differences were observed in leaf, flower, plant, or boll morphology between TwinLink and the conventional parental pedigree.

3. Laboratory and field experimentation show significant reduction in lepidopteran plant damage and larval survival due to expression of Cry proteins from the component events

T304-40 and GHB119 and TwinLink.TwinLink Cotton USDA Petition

Page 67 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 4. The agronomic performance of TwinLink cotton treated with the herbicide glufosinate ammonium was equal to or better than that of its unsprayed TwinLink cotton and its unsprayed non-transgenic counterpart.

5. Evaluation of the agronomic performance of component events T304-40 and GHB119, and TwinLink cotton has identified neither safety nor environmental concerns.

B. History of field activities

TwinLink cotton events T304-40 and GHB119 were regenerated via tissue culture after transformation of the individual plant cells. In 2005, T 3 

### seed harvested from T 2 

### plants was imported for planting in the first field evaluation in Mississippi. T 3 

### plants were evaluated for agronomic equivalence and line selection. Selected lines of the two events were increased in 2006. Breeding activities to move the events into FiberMax germplasm and to combine the events for TwinLink progressed in the greenhouse. Sufficient seed was produced to conduct the

2007 field trials. Seed harvested from US seed increases in 2007 was used in 2008 field trials. Fields were routinely sprayed with insecticide to prevent possible pollen transfer by insects. Table 16 presents a summary of the field trials and associated authorization permits.

Table 16 Summary of field activities under USDA permits for TwinLink events

Notification

Number Year Number of

Locations Locations Termination Report

05-035-12n 2005 1 MS 1 07-059-104n 2007 10 LA(2), MS(2), TX(6) 2

07-065-119n 2007 1 NC 8

07-065-118n 2007 1 NC 7

07-065-117n 2007 1 NC 6 07-044-104n 2007 6 LA(1), AZ(1). TX(2), MS(2) 5 07-044-103n 2007 7 LA(1), AZ(1), TX(2), MS(3) 4 07-044-102n 2007 11 LA(2), AZ(1),TX(4), MS(2), NC(1), SC(1) 3 07-059-101n 2007 9 AR(3), GA(2), MS(1), TX(3) 9 08-022-101n 2008 10 TX(2),CA(1),LA(1 ),MS(3),NC(1),SC(1),AZ(1) * 08-036-127n 2008 9 TX(2),GA(2),AR(3),AZ(1),MS(1) *
*Copies of the termination reports for these field trials are provided in Appendix I. Termination reports for the 2008 field trials will be provided to the USDA according to the 6 month post-termination reporting requirement.



### C. Agronomic performance of TwinLink cotton (events T304-40 x GHB119)

Replicated field trials were conducted in 2007 and 2008 to compare agronomic performance of the TwinLink cotton events T304-40 and GHB119 with the non-transformed Coker counterpart (see Table 16 above). Agronomic performance was measured with cotton plant mapping methods and observation of defined growth parameters. Samples of seed and lint were harvested to evaluate the fiber quality characteristics. The agronomic parameters used to evaluate the transgenic and non-transgenic lines are defined in Table 17.TwinLink Cotton USDA Petition

Page 68 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 17 Description of Agronomic Parameters Evaluated

Agronomic Characteristic Description

Boll Type Visual rating of boll type; 1= loose, 5=intermediate, 9=stormproof.

Boll retention (p1, p2) Percent bolls reta ined on plant at first and second position

Days to bloom The number of days from planting to first bloom

Days to first open boll The number of days from planting to first open boll

Disease reaction Visual rating of disease pathogen; 1 = no symptoms 5 = some symptoms apparent, 9 = severe
Fiber elongation % Measure of the % change in length based on original fiber length

Fiber Length Average length of the longer one-half of cotton fibers

Fiber uniformity % Ratio between the mean length and upper half mean length of the fibers expressed as a percentage

Fiber Micronaire A measure of fiber fineness and maturity as indicated by specific surface area

Fiber strength The force in grams required to break a bund le of fibers one tex unit in size (1 tex = weight in grams of 1,000 meters of fiber)
Height to node ratio Plant height divided by total number of nodes.

Leaf Uniformity Consistency of leaf type

Lint Percent Lint weight divided by seed cotton weight, expressed as a percentage.

Lodging Visual rating of plant stature; 1=fully upright, no leaning, 5=Leaning 45 degrees from ground, 9=laying on soil surface
Number first position bolls Total number of bolls per plant set on the first position of fruiting branches
Number second position bolls Total number of bolls per plant set on the second position of fruiting branches
Number third position bolls Total number of bolls per plant set on t he third position of fruiting branches

Number of seeds per boll The number of ovules that are fertilized and develop into mature seed is an indication of pollination efficiency, most usually affected by heat.

Number of seeds per plant An expression of yield component combining numbers of seed per boll and average boll retention.
Number of open Bolls Total number of bolls mature and open (ready for picking) at a given time point.

Number of Total Bolls Total number of bolls on an individual cotton plant at a given time point.

Percent open bolls Differences in percent open bolls at a given time are an indication of differences in crop maturity.
Plant height Average plant height from cotyle donary node to terminal, expressed in inches
Plant morphology rating (leaf, flower, bolls) A scale rating of leaf, flower and boll type.

Plant Stand Evaluation of the ger mination rates and plant population

Seed index Average weight in grams of 100 seed, an indication of seed size and maturity.

Sprayed plots Plots sprayed with selectiv e insecticides for lepidopteran pests

Strain Uniformity Used to evaluate the uniformity of the ev ent on a 1 to 9 scale. (1 = uniform, 9 = highly variable)
Total number of nodes Number of reproductive no des present on the main stem of the plant

Treated plots TwinLink plots sprayed wi th glufosinate ammonium herbicide

Unsprayed plots Plots unsprayed for lepidopteran pests

Yield: Lbs. lint per acre Productivity expre ssed as pounds of lint produced per acre



### Data from replicated field trials was recorded from 23 locations in 8 states (North Carolina, South Carolina, Georgia, Arkansas, Mississippi, Louisiana, Texas, and Arizona over the 2007 and 2008 growing seasons. Studies were conducted in geographic regions representative ofTwinLink Cotton USDA Petition

Page 69 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 80% and 87% of the total upland cotton production in the US in 2007 and 2008, respectively. Trials were conducted across the cotton belt to capture the various environmental stresses that upland cotton varieties undergo during the course of a normal production year. These areas represent a large majority of the Bt cotton acreage and target market area for TwinLink cotton.

Findings across locations show that TwinLink cotton is similar for maturity and yield to its conventional variety counterpart. Limited phenotypic differences were recorded between TwinLink and its conventional variety counterpart. These differences can be attributed to premature fruit shed due to lepidopteran damage in the conventional pedigree line. The majority of phenotypic differences recorded can be considered as positive attributes of the TwinLink technology: earlier maturity, increased fruit retention and increased lint yield, due to lepidopteran

control.

D. Biotic and abiotic stress characteristics

Field trial managers noted no performance difference of T304-40, GHB119, TwinLink and their non-transgenic parent lines to abiotic and biotic stress. Weed management in all trials was performed in accordance with local University Extension recommendations: weeds were not evaluated in these trials, as all weed populatio ns were controlled using appropriate herbicide applications to eliminate this variable. Summary data (across locations) for the Agronomic Performance evaluations can be found in Appendix 4.

Seedling vigor and plant stand counts were similar for conventional cotton and lines containing the T304-40 and GHB119 event transformations for all the trials planted in optimal conditions and where irrigation was available. Reduced germination was observed in all test plots under less than optimal conditions (marginal moisture, deeper than optimal planting depth, inconsistent soil type).

Data for plant stand were recorded at 13 of the 22 locations over the 2007 and 2008 growing seasons (Table 18). TwinLink stand counts were significantly increased at 9 of the 13 locations compared with its conventional counterpart. Plant stands were within commercially acceptable

limits for all trial locations. [In order to avoid confusion the term “sprayed” refers to insecticide treatments, and “treated” refers to herbicide treatment]

Table 18 Plant stand count data individual locations in 2008.

Southeast Mid-South Texas Arizona Martin, NC Sellers, SC Leland, MS Franklin, LA Lubbock,

TX Pinal, AZ

Coker not sprayed 2.73b 2.60a 2.83a 2.31b 1.57a 3.90ab Coker sprayed 3.53a 2. 60a 3.00a 2.88a 1.59a 3.30b TwinLink not sprayed 2. 83b 2.27a 2.75a 2. 31b 1.32a 4.49a TwinLink sprayed 3.68a 2.36a 2.85a 3.03a 1.86a 3.99ab LSD (0.05) 0.419 0. 428 0.644 0.466 0.701 0.906

CV 8.22 10.91 14. 1 11.08 27.7 14.45 * Sprayed plots indicate use of selectiv e conventional insecticides to control lepidopteran pests.TwinLink Cotton USDA Petition

Page 70 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Disease and lodging ratings were made using a scale of 1 to 9 (1 = normal, 9 = severe) to rate plant pathogen susceptibility or lodging problems. Disease ratings across locations showed no significant differences between transformed pl ots and their non-transformed counterparts (Table 19).

Table 19 Lodging data by individual location in 2007

Southeast Mid-south Southwest
Chula, GA (#1) Chula, GA (#2) Tate, MS Jackson,

AR Crittenden AR (#1) Crittenden AR (#2) Hockley,

TX

Coker 1 1 1 2 1 1 1

TwinLink * 1 1 1 2 1 1 1

TwinLink treated 1 1 1 2 1 1 1

LSD (0.05) 0 0 0 0 0 0 0

CV 0 0 0 0 0 0 0
* TwinLink plots were either treated or not treated with glufosinate ammonium herbicide to eliminate wild type plants from transformed seed lots.



### Leaf, flower and boll morphology data was recorded at three locations in 2008 with no significant difference between TwinLink and the conventional parent (Table 20). Ratings are 1=normal and

2=abnormal.

Table 20 Plant part morphology data in 2008

Comparing conventional insectic ide and Bt cotton treatments. Sellers, SC Leland, MS Lubbock, TX

Leaf Flower Boll Leaf Flower Boll Leaf Flower Boll Coker unsprayed 1 1 1 1 1 1 1 1 1 Coker sprayed 1 1 1 1 1 1 1 1 1 TwinLink unsprayed 1 1 1 1 1 1 1 1 1 TwinLink sprayed 1 1 1 1 1 1 1 1 1 LSD (0.05) 0 0 0 0 0 0 0 0 0

CV 0 0 0 0 0 0 0 0 0 * Sprayed plots indicate use of selectiv e conventional insecticides to control lepidopteran pests.



### E. Maturity and plant phenotype characteristics

Plant maturity data were collected in seven locations in 2007 and four locations in 2008. No significant differences were recorded in plant maturity at three of the seven locations, while four of the seven locations in 2007 recorded significant differences in maturity (Table 21, Table 22 and Table 23). Where significant differences in maturity were recorded, there is a clear trend towards increased earliness (shorter period to first flower and greater % open bolls) in the

TwinLink plots compared with the Coker plots which were not sprayed with insecticide (Table 23). This would be expected as a result of control of lepidopteran pests and higher fruitTwinLink Cotton USDA Petition

Page 71 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### retention in the TwinLink plots relative to Coker. In contrast, no difference was observed in maturity between TwinLink plots treated with glufosinate ammonium herbicide and untreated TwinLink plots (Tables 21 and 22).

Boll type visual assessments were taken at seven locations in 2007 (1-9, 1=loose, 5=intermediate, 9=storm-proof). No significant differences were observed in boll type across the

seven locations.

Table 21 Maturity data from individual locations in the Southeast and Texas in 2007

Chula, GA (Loc #1) Chula, GA (Loc #2) Texas Days to 1st

flower % open

bolls Days to 1st

flower % open

bolls Days to 1st

flower % open

bolls

Coker 55.7a 43b 54.7a 33.3b 73.0a 50b

TwinLink 53.0b 70a 52.7b 69.3a 63.0b 75a

TwinLink treated * 54. 0ab 59ab 52.0b 72.0a 63.0b 75a LSD (0.05) 2.20 22.1 2.00 21.15 0 0

CV 1.79 16.97 1.66 16.03 0 0 * TwinLink plots were either treated or not treated with glufosinate ammonium herbicide to eliminate wild type plants from transformed seed lots.



### Table 22 Maturity data from individual locations in the Mid-south in 2007

Tate, MS Jackson, AR Crittenden, AR (#1) Crittenden, AR (#2) Days to

1st

flower %

open bolls Days to

1st

flower %

open bolls Days to

1st

flower % open

bolls Days to

1st

flower %

open bolls

Coker 55.0a 50.0a 51. 0a 58a 45.0a 50a 45.0a 50a

TwinLink 55.0a 50.0a 49.0b 55a 45.0a 50a 45.0a 50a

TwinLink treated * 55.0a 50.0a 49.0b 57a 45.0a 50a 45.0a 50a LSD (0.05) 0 0 0 12.2 0 0 0 0

CV 0 0 0 9.53 0 0 0 0 * TwinLink plots were either treated or not treated with glufosinate ammonium herbicide to eliminate wild type plants from transformed seed lots.



### Table 23 Maturity data from individual locations in 2008.

Sellers, SC Leland, MS Lubbock, TX Pinal, AZ Days to

1st flower Days to

1 st

boll opening Days to

1st flower Days to

1 st

boll opening Days to

1st flower Days to 1 st

boll opening % open

bolls

Coker unsprayed 63.0a 121a 58b 102b 67.0a 128a 55a Coker sprayed 63.0a 120a 60a 103ab 67.0a 128a 50a TwinLink unsprayed 63.0a 120a 59ab 103ab 67.0a 128a 58a TwinLink sprayed 63.0a 120a 58b 104a 67.0a 128a 50a LSD (0.05) 0 0.8 2.0 2.1 0 0 15.6

CV 0 0.42 2.12 1.27 0 0 18.36

* Sprayed plots indicate use of selectiv e conventional insecticides to control lepidopteran pests.TwinLink Cotton USDA Petition

Page 72 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### F. Yield and fiber quality

Yield was recorded at all of the 23 locations over the 2007 and 2008 growing seasons (Appendix 4, Tables 4.2, 4.4, 4.6 and 4.10). Yields were within expectations for the Coker varietal background, but significantly lower than would be expected for commercial varieties. TwinLink had significantly greater yield than its conventional counterpart at multiple locations (Appendix 4, Tables 4.2, 4.4, 4.6, 4.8 and 4.10) and at the Sellers location (Table 24). Increases in yield would be expected as a result control of lepidopteran pests and higher fruit retention in the TwinLink plots relative to the Coker plots.

Table 24 Fiber Analysis from Sellers, SC location in 2008

Coker TwinLink Significance Unsprayed Sprayed* Unsprayed Sprayed LSD** CV (0.05) SIG

Yield 1227.1b 1326.9ab 1334. 6ab 1434.0a 201.81 9.48 ab Fiber Length 1.27a 1.25a b 1.22b 1.22b 0.031 1.57 ab Fiber strength 36.0a 35.8ab 33.8c 34.2bc 1.66 2.97 abc Fiber uniformity % 86.1a 85.3ab 84.8b 84. 8b 1.17 0.86 ab

Micronaire 4.9 4.9 4. 7 4.7 0.29 3.77 N.S.

% lint 40.8b 41.7ab 41. 9ab 42.4a 1.50 2.25 ab

* Sprayed plots indicate use of selectiv e conventional insecticides to control lepidopteran pests.
** LSD = 0.05



### Laboratory tests were conducted to analyze commercially important fiber qualities of harvested lint from test plots compared to the non-transformed plots. Significant differences were found in the 2007 season for fiber length when analyzed across seven locations (Appendix 4, Table 4.2 and Table 4.4, and at the Sellers location in Table 24). No significant differences were observed in 2007 for length uniformity, lint percent, fiber strength, and micronaire.

Fiber quality data is available for only two locations for 2008 at time of dossier preparation. Significant differences were observed at the Sellers, SC location (Table 24), but trends were not

consistent across locations.

During equivalence evaluation of the transform ed trait (see Section VIII.G) no significant differences were recorded in fiber characteristics between the T304-40, GHB119, and TwinLink lines compared to their conventional counterpart. Backcrossing reduces the influence of the Coker background in which the transformation took place. Therefore, it was determined that the

differences in fiber characteristics recorded in the 2007 and 2008 trials were associated with the somaclonal variation in the Coker background, and the relative early stage of backcrossing (BC 2 

### F 3 

### ) rather than the T304-40 or GHB119 traits per se . These differences would be expected to be overcome with further backcrossing in elite germplasm backgrounds.

G. Equivalence between Coker-derived and commercial varieties

Somaclonal variation can occur when transformed plant cells are stressed by the surrounding environment, which can result in differences between a regenerated Coker plant and the original plant from which the tissue was taken. Somaclonal variation can lead to variances in the plant which are not linked to the transgene event and are removed by backcrossing. CommercialTwinLink Cotton USDA Petition

Page 73 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### germplasm was backcrossed with the T 0 

### generation of the respective Coker transformant and then progressed to the BC 2 

### F 3 

### generation for evaluation along with the Coker variety (see Breeding diagram in Appendix 2).

H. Composition analysis

A study was conducted to obtain composition analysis data on RAC (cottonseed) samples of TwinLink cotton and its non-transgenic counterpart. The nutritional components of cottonseed were compared for the TwinLink cotton and the non-transgenic counterpart Coker 315.

Cotton plants were grown in the field by Bayer CropScience in 2007. Seven field trials were conducted in EPA Regions II, IV, and VIII in Geor gia, Arkansas, Mississippi and Texas, all important cotton growing regions of the Southern United States. The plants in this study were grown under conditions typical of production practices. There were six TwinLink cotton transgenic plots and three non-transgenic plots at each test site. Three of the TwinLink cotton transgenic plots were sprayed two times with glufosinate-ammonium herbicide at a target rate of 0.53 lb ai/A, and three transgenic plots were non-treated. Three replicate non-transgenicsamples and six replicate transgenic samples (3 non-treated and 3 treated with glufosinateammonium herbicide) of fuzzy seed were collected from each of the field test sites and shipped frozen to Bayer CropScience BioAnalytics laboratory, Research Triangle Park, North Carolina. The fuzzy seeds were sub-sampled and shipped frozen to the analytical facility, Covance Laboratories, Inc., Madison, Wisconsin for composition analyses.

Composition data were obtained for 63 samples (9 samples from each of 7 field trials) of ginned cottonseed (also known as fuzzy seed). There were 21 samples from each of three groups: 1. Non-transgenic, non-tolerant Coker 315 cotton 2. TwinLink cotton from combined-trait transgenic events T304-40 and GHB119 that was not treated with glufosinate herbicide 3. TwinLink cotton from combined-trait transgenic events T304-40 and GHB119 that was treated two times with glufosinate herbicide.

Chemical analysis of the samples included the nutri tional factors proximate, minerals, vitamins, amino acids, and fatty acids (cottonseed oil). Antinutritional factors of cotton include the gossypols, phytic acid, and the cyclic fatty acid s. The mean values and the literature standards are provided in Tables 25-29.

Data were obtained from the transgenic non-treated, transgenic treated and non-transgenic samples and are presented on a fresh weight basis. The data are also presented on a dry weight basis by correcting the fresh weight values for the moisture content determined for each sample. Mean values and standard deviations were calculated for the dry matter data. Fatty acid data are presented on a fresh weight basis and additionally, together with the cyclopropenoid fatty acid data, as relative quantities of the total sum of fatty acids.TwinLink Cotton USDA Petition

Page 74 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

### Table 25 Proximate and fibre compounds in fuzzy seed of TwinLink Cotton and the nontransgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges)

NonTransgenic Transgenic

Non-treated Transgenic

Treated a Parameter

Mean ± SD Mean ± SD Mean ± SD Reference

ranges c

Moisture % fw 11.13 ± 2.17 10.70 ± 2.32 10.72 ± 2.43 4.0 - 15.9

Fat % dm 23.30 ± 2.29 22.38 ± 2.40 22.38 ± 2.29 11.8 - 36.3

Protein % dm 18.40 ± 2.40 18.53 ± 2.38 18.76 ± 1.83 11.7 - 34.2

Ash % dm 3.94 ± 0.33 4.01 ± 0.22 4.04 ± 0.30 3.2 - 5.0

Total carb. % dm b 54.38 ± 2.37 55.10 ± 2.63 54.82 ± 2.27 36.4 - 74.4

ADF % dm 39.64 ± 2.64 39.53 ± 2.89 38.92 ± 2.60 29.0 - 66.9

NDF % dm 46.05 ± 3.77 46.44 ± 3.75 47.24 ± 3.05 38.1 - 71.4

a Treated with glufosinate ammonium

b Total carbohydrates calculated as 10 0% - (protein %dm + fat %dm + ash %dm)

c References: Amann, 1999. Berberich et al ., 1996. Bertrand et al ., 2005. Calhoun et al ., 1995.

ILSI, 2007. Lundquist, 1995. Nida et al ., 1996. OECD, 2004. USCA, 1982.



### Table 26 Minerals and Vitamin E in fuzzy seed of TwinLink Cotton and the nontransgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges)

NonTransgenic Transgenic

Non-treated Transgenic

Treated a Parameter

Mean ± SD Mean ± SD Mean ± SD Reference

ranges b

Calcium (% dm) 0.126 ± 0.014 0.136 ± 0.023 0.133 ± 0.021 0.09 - 0.33

Phosphor (% dm) 0.569 ± 0.100 0.574 ± 0.075 0.578 ± 0.071 0.31 - 0.86

Potassium (% dm) 1.12 ± 0.06 1.14 ± 0.06 1.14 ± 0.07 0.96 - 1.42

Magnesium (% dm) 0.349 ± 0.036 0.365 ± 0.031 0.364 ± 0.028 0.27 - 0.49

Iron (mg/kg dm) 45.2 ± 11.2 43.8 ± 9.7 43.3 ± 7.5 23.2 - 160

Zinc (mg/kg dm) 33.7 ± 4.3 34.7 ± 4.8 35.0 ± 4.3 17.8 - 63.0

Alpha Tocopherol (mg/kg dm) 122 ± 14 130 ± 13 132 ± 9 16 - 245

a Treated with glufosinate ammonium

b References: Calhoun et al., 1995. ILSI, 2007. Lundquist, 1995. OECD, 2004. USCA, 1982.

FAO/WHO, 2001TwinLink Cotton USDA Petition

Page 75 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 27 Anti-nutrients in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges)

Parameter NonTransgenic Transgenic

Non-treated Transgenic

Treated a

Mean ± SD Mean ± SD Mean ± SD Reference

ranges b

Free gossypol (% dm) 0.709 ± 0.128 0.754 ± 0.139 0.715 ± 0.146 0.23 - 1.40

Total gossypol (% dm) 0.799 ± 0.103 0.835 ± 0.114 0.801 ± 0.096 0.46 - 1.99

Phytic acid (% dm) 1.62 ± 0.47 1.60 ± 0.31 1.55 ± 0.37 0.85 - 2.57

Malvalic acid (% rel.) 0.467 ± 0. 075 0.473 ± 0.106 0.474 ± 0.094 0.17 - 1.5

Sterculic acid (% rel.) 0.308 ± 0. 063 < 0.10 - 0.389 0.314 ± 0.080 0.12 - 0.92

Dihydrosterculic acid (% rel.) 0.145 ± 0.015 < 0.10 - 0.155 0. 135 ± 0.012 0.11 - 0.50

a Treated with glufosinate ammonium

b References: Berberich et al . 1996. Calhoun et al ., 1995. ILSI, 2007. Nida et al ., 1996. OECD, 2004. Phelps et al ., 1965. Wozenski and Woodburn, 1975.



### Table 28 Total Amino Acids in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges)

% dry matter

NonTransgenic Transgenic

Non-treated Transgenic

Treated a Parameter

Mean ± SD Mean ± SD Mean ± SD Reference

ranges b

Alanine 0.79 ± 0.09 0.76 ± 0.07 0.77 ± 0.10 0.42 - 1.51

Arginine 2.40 ± 0.31 2.28 ± 0.26 2.32 ± 0.34 1.05 - 4.40

Aspartic acid 1.92 ± 0.24 1.84 ± 0.19 1.86 ± 0.27 1.00 - 3.55

Cysteine 0.36 ± 0.04 0.35 ± 0.04 0.34 ± 0.05 0.16 - 0.86

Glutamic acid 3.96 ± 0.51 3.74 ± 0.43 3.82 ± 0.57 1.96 - 8.16

Glycine 0.85 ± 0.10 0.82 ± 0.07 0.83 ± 0.11 0.44 - 1.58

Histidine 0.57 ± 0.07 0.54 ± 0.06 0.55 ± 0.08 0.31 - 1.03

Isoleucine 0.66 ± 0.08 0.63 ± 0.06 0.65 ± 0.09 0.35 - 1.17

Leucine 1.19 ± 0.15 1.14 ± 0.11 1.16 ± 0.16 0.63 - 2.23

Lysine 0.94 ± 0.10 0.90 ± 0.07 0.91 ± 0.11 0.52 - 1.65

Methionine 0.32 ± 0.04 0.31 ± 0.03 0.30 ± 0.04 0.15 - 0.54

Phenylalanine 1.11 ± 0.14 1.06 ± 0.11 1.08 ± 0.15 0.54 – 2.03

Proline 0.74 ± 0.09 0.70 ± 0.07 0.71 ± 0.10 0.41 - 1.39

Serine 0.88 ± 0.12 0.84 ± 0.09 0.86 ± 0.12 0.50 - 1.63

Threonine 0.65 ± 0.08 0.63 ± 0.07 0.65 ± 0.08 0.34 - 1.21

Tryptophan 0.20 ± 0.02 0.19 ± 0.02 0.19 ± 0.03 0.10 - 0.49

Tyrosine 0.55 ± 0.07 0.53 ± 0.06 0.53 ± 0.07 0.32 - 1.17

Valine 0.93 ± 0.11 0.89 ± 0.08 0.91 ± 0.12 0.45 - 1.67

a Treated with glufosinate ammonium

b References: Bertrand. et al . 2005. ILSI, 2007. Lawhon. et al ., 1977. OECD, 2004.TwinLink Cotton USDA Petition

Page 76 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 29 Total Fatty Acids in fuzzy seed of TwinLink Cotton and the non-transgenic counterpart Coker 315 compared to commercial cotton varieties (Reference ranges)

% relative

NonTransgenic Transgenic

Non-treated Transgenic

Treated a Parameter

Mean ± SD Mean ± SD Mean ± SD Reference

ranges b

Saturated

C12:0 (lauric acid) < 0.10 < 0.10 - 0.21 < 0.10 0 - 0.20

C14:0 (myristic) 0.66 ± 0.11 0.62 ± 0.10 0.63 ± 0.10 0.53 - 1.17

C16:0 (palmitic) 23.19 ± 0.77 23.27 ± 0.56 23.32 ± 0.62 21.1 - 29.9

C17:0 (margaric acid) < 0.10 - 0.13 < 0.10 - 0.11 < 0.10 - 0.11 ND

C18:0 (stearic) 2.44 ± 0.08 2.52 ± 0.13 2.52 ± 0.13 2.15 - 3.4

C20:0 (arachidic) 0.27 ± 0.02 0.26 ± 0.01 0.26 ± 0.02 0 - 0.48

C22:0 (behenic) < 0.10 - 0.22 < 0.10 - 0. 21 < 0.10 - 0.17 0 - 0.27

C24:0 (lignoceric) < 0.10 - 0.18 < 0.10 - 0.15 < 0.10 0 - 0.30

Sum Saturated 26.56 - 27.09 26.67 - 27.35 26.73 - 27.01 23.78 - 35.52

Mono-unsaturated

C16:1 (palmitoleic) 0.46 ± 0.03 0.47 ± 0.02 0.47 ± 0.03 0.46 - 0.86

C18:1 (oleic) 14.70 ± 0.78 15.95 ± 0.40 15.95 ± 0.45 13.4 - 22.0

Sum Mono-unsaturated 15.16 16.42 16.42 13.86 - 22.86

Poly-unsaturated

C18:2 (linoleic) 57.90 ± 1.45 56.53 ± 0.86 56.52 ± 1.01 36.3 - 64.0

C18:3 (alpha linolenic) 0.20 ± 0.03 0.20 ± 0.02 0. 19 ± 0.03 < 0.10 - 0.62

Sum Poly-unsaturated 58.10 56.73 56.71 36.3 - 64.62

Sum of total fatty acids 99.82 - 100.4 99.82 - 10 0.5 99.86 - 100.1 -

ND No data available

a Treated with glufosinate ammonium

b References: Berberich et al. 1996 (values for Coker 312). Bertrand et al. 2005. ILSI, 2007.

Nida et al ., 1996. OECD, 2004.



### I. Conclusions for agronomic evaluation of TwinLink cotton

Results across locations show that TwinLink and the conventional parent lines are similar for yield, plant morphology, and fiber quality.

Seedling vigor and plant stand counts were similar for conventional cotton and lines containing the T304-40 and GHB119 event transformations for all the trials planted in optimal conditions and where irrigation was available. Reduced germination was observed in all test plots under less than optimal conditions.TwinLink Cotton USDA Petition

Page 77 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### No significant differences were found when evaluating plant responses to biotic and abiotic stresses in 2007. In a minority of trials TwinLink cotton plants demonstrate a potential for increased lodging due to heavy fruit load. Differences in lodging seen in the data appear not to be a deleterious result of the transformation, but rather an indirect effect of higher fruit retention as a result of reduced lepidopteran damage.TwinLink Cotton USDA Petition

Page 78 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## IX. ENVIRONMENTAL SAFETY/IMPAC T OF NON CONTAINED USE OF
TWINLINK COTTON

A. Potential for gene transfer / out crossing

1. Bio-geography

As discussed in Section II of this petition, only two wild Gossypium species are present in the

US: G. thurberi Todaro found in mountain regions of Arizona at altitudes of 2500 to 5000 feet

and G. tomentosum which is found in Hawaii. Only G. tomentosum is capable of crossing with domesticated cotton that will produce fertile offs pring. There is no expected selective advantage conferred by the transfer of the TwinLink trait if that cross would occur.

2. Vertical gene flow

Cotton pollination Gossypium hirsutum is considered to be a self-pollinating crop. Cotton pollen is heavy and sticky thus cross pollination by wind is unlikely. Cotton can, however, be pollinated by insects. Honeybees ( Apis mellifera ) and bumblebees ( Bombus spp.) are the primary insect pollinators.

As previously discussed in Section II, McGregor (1976) traced the movement of pollen from a cotton field surrounded by a large number of honeybee colonies. Movement of the pollen was traced by means of fluorescent particles. McGregor found that at 150 to 200 feet away from the source plant, only 1.6 percent showed the presence of the fluorescent particles. By comparison, the isolation distances for Foundation, Registered and Certified seeds in 7 CFR Part 201 are 1320, 1320 and 660 feet, respectively. The trend for cross pollination to decrease as the distance from the source increased has been established by several research groups over the years. (Kareiva et al ., 1994; Van Deynze et al., 2005).

Outcrossing potential to wild/weedy relatives The potential for outcrossing can be defined as the ability of gene escape to wild cotton relatives. Previously the USDA stated in the environmental assessment document of LLCotton25 cotton that “gene flow from genetically engineered cotton into wild cotton relatives is not likely, and if it occurs, would not lead to increased weediness” (USDA, 2003). G. tomentosum, found only in Hawaii, is the only species capable of crossing with domesticated cotton that will produce fertile offspring. Outcrossing to G. tomentosum is unlikely as there is neither selective advantage nor cotton production in Hawaii other than winter nursery breeding activities where isolation practices are employed, and therefore the potential for gene flow to

these wild relatives is low. There are other wild relatives known to exist in Southern Florida and Puerto Rico that are capable of crossing with cultivated cotton. However, these wild relatives are found hundreds of miles from where cotton production occurs.

Outcrossing potential to feral or cultivated cotton No feral cotton populations (domesticated plants capable of surviving outside of cultivation) of G.

barbadense have been found in the US Cotton production fields (production of planting seed) are required to be isolated from other cotton fields to prevent cross pollination. Therefore if any cross pollination were to occur to either G. barbadense or G hirsutum it would be from a lint production field where seed is crushed and not propagated.TwinLink Cotton USDA Petition

Page 79 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 3. Potential of horizontal gene flow from TwinLink cotton

Bayer CropScience is not aware of any reports of incidents of naturally occurring transgene movement from transgenic crops to sexually incompatible species.

B. Weediness potential of TwinLink cotton

In the United States, cotton ( G. hirsutum ) is not a weed pest and has no sexually compatible weedy relatives except perhaps G. tomentosum in Hawaii where there is no commercial cotton production. A number of references confirm the lack of weediness of cotton: Crockett, 1977,

Holm et al. , 1977, Muenscher, 1980. The USDA has previously determined that “cotton is not considered to be a serious, principal or common weed pest in the US” (USDA, 2003). Previous findings by the USDA of similar herbicide-tolerant cotton during environmental assessment expected no change in weediness potential, and an example is glufosinate-tolerant cotton (LibertyLink), commercially sold today. The largest concern is that of volunteer plants that could become weedy in subsequent years. Volunteers ar e also limited by the geography in which they may exist as cotton does not survive as a perennial where freezing temperatures are reached during the winter. Volunteers can easily be controlled by crop rotation, tillage and/or pre- or post-emergence herbicides. For example, glufosi nate-tolerant cotton volunteers could easily be controlled by using the herbicide glyphosate.

There is limited probability that TwinLink cotton or any Gossypium species containing TwinLink cotton would become a weed problem. In the co mparative studies presented in this petition there were no consistent significant differences in phenotypic or plant morphological characteristics between the transgenic TwinLink cotton and the conventional counterpart line Coker that would impact plant pest or noxious weed potential. Based on these data there was no evidence to suggest that TwinLink cotton has a higher likelihood to become a weed than conventional cotton. There were no instances in which volunteer monitoring after harvest revealed any differences in survival or persistence relative to other cotton varieties.

C. Effects on non-target organisms

An assessment of the risk to non-target species has been performed for Twinlink cotton and for the Cry1Ab protein and Cry2Ae protein produced by TwinLink cotton. Each assessment comprised: hazard identification, exposure as sessment, testing of species potentially exposed and, if necessary, dose-response evaluation for affected species.

The mode of action of Cry proteins in target organisms is well understood and is mediated by binding proteins in the gut, which have considerable inter-species variability. Bacillus thuringiensis (Bt) Cry proteins (including several Cry1 and Cry2 proteins) have been extensively studied in laboratory assays and field tests, and cotton and corn expressing these proteins have been grown on significant acreage in the US for several years with no reports of significant adverse effects. All these studies indicate that Cry proteins pose no unacceptable risk for any organisms except the narrowly-targeted pest species and very close relatives (USEPA, 2001). BCS has generated additional data on the Gossypium hirsutum transformation events T304-40 and GHB119 and the Cry1Ab and Cry2Ae proteins encoded in these events and the results areTwinLink Cotton USDA Petition

Page 80 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### summarized in this chapter. There is much detailed information about the specificity of Cry1Ab and Cry2 proteins to lepidopteran species ( e.g. Hoffmann et al. 1988). Cry1Ab and Cry2Ae are both narrowly targeted to lepidopteran insects. Whilst there is no evidence of toxicity to other non-target organisms, it is prudent to identify those organisms most likely to be exposed to cotton plants or plant material, and assess their sensitivity to the Cry proteins produced by

TwinLink cotton.

The main routes of exposure to non-target organisms are:

• Grazing by herbivorous insects or other animals

• Predation on insects that have fed on Bt cotton

• Consumption of cotton seeds (birds, mammals)

• Pollen transfer

• Leaf fall

• Plowing in of senescent plants

1. Cry1Ab and Cry2Ae cotton

For most species, testing was carried out using purified protein expressed in the bacterium E.

coli . This purified protein could be incorporated into the appropriate diet for the non-target organism, and in this way, the organisms could be treated with a dose significantly higher than the expected environmental concentration (EEC). The basis for concluding ‘no risk’ to a target species is the presence of evidence that there are no significant effects at the maximum expected environmental concentration (EEC).

Table 30 Summary of Non-Target Organism Data of the Cry1Ab protein

Organism Lifestage Endpoint(s) Result

Mammal ( Mus musculus) Young adult Mortality, body weight, clinical signs NOEC 1 2000 mg/kg

Honeybee larva Mortality, development, Adult emergence, behavior NOEC 20 μg/g

Ladybug ( Coleomegilla maculata ) larva Mortality, development, Adult emergence, behavior NOEC 10 μg/g

Lacewing ( Chrysoperla carnea) larva mortality NOEC 29 μg/g 3

Springtail ( Folsomia candida ) larva Mortality, reproduction NOEC 4.5 μg/g 2

Daphnia immature Mortality, development, reproduction NOEC 48 μg/g 3

1 NOEC = No Effect Concentration

2 This is a conservative estimate based on ELISA analysis of di et samples. Actual exposure was probably significantly higher.

3 Preliminary resultsTwinLink Cotton USDA Petition

Page 81 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 31 Summary of Non-Target Organism Data of the Cry2Ae protein

Organism Lifestage Endpoint(s) Result

Mammal ( Mus musculus) Young adult Mortality, body weight, clinical signs NOEC 1 2000 mg/kg

Honeybee larva Mortality, development, Adult emergence, behavior NOEC 50 μg/g

Ladybug ( Coleomegilla maculata ) larva Mortality, development, Adult emergence, behavior NOEC 64 μg/g

Lacewing ( Chrysoperla carnea) larva mortality NOEC 27 μg/g 3

Springtail ( Folsomia candida ) larva Mortality, reproduction No mortality at 44 μg/g.

Earthworm adult Mortality NOEC 100 mg/kg soil Daphnia immature Mortality, development, reproduction NOEC 48 μg/g 3

1 NOEC = No Effect Concentration

3 Preliminary results



### The conclusion from the risk assessments for the two individual proteins is that Cry1Ab and Cry2Ae pose minimal risk to any species except the target Lepidoptera and very close relatives. There was no indication of risk to birds, mammalian wildlife, aquatic organisms, soil-dwelling organisms or several species of non-target insects.

2. Protein degradation in soils

An aerobic soil degradation study using the most sensitive target insect, Heliothis virescens , to

determine the DT 50 

### (time for 50% of initial concentration of bioactive material to dissipate) was conducted to ascertain the environmental risk assessment of the individual Cry1Ab and Cry2Ae proteins produced from Escherichia coli and Bacillus thuringiensis respectively. A 30 day aerobic soil degradation study was performed with the proteins produced from bacteria in three US soils at a concentration of a) Cry1Ab 15 μg/g, which represents fifteen times the 1 μg/g concentration expressed in cotton leaves and b) Cry2Ae 50 μg/g, which represents five times the 10 μg/g concentration expressed in cotton leaves. The three US soils from Proctor (Arkansas), Senatobia (Mississippi) and East Bernard (Texas) selected for the study are from areas where transgenic cotton is grown. The insect bioassay monitored growth inhibition of

larval Heliothis virescens . A dose response curve run concurrently with the insect bioassay was used to determine the Cry1Ab and Cry2Ae protein concentration in the soil. The DT 50 

### was determined from the soil protein concentration fi rst-order exponential regression curve. An average DT 50 

### of 3.6 days for Cry1Ab and 3.4 days for Cry2Ae for all three soils was obtained from the insect assay data.

3. TwinLink cotton

A B. thuringiensis strain expressing more than one type of crystal protein might possibly be expected to have synergistic or additive effects on the intended target pest insect. Binding studies demonstrate that Cry1Ab and Cry2Ae bind to distinct receptor sites in the insect gut. Comparison of insect efficacy data on Cry1Ab cotton (event T304-40), Cry2Ae cotton (event GHB119), and TwinLink cotton (event T304-40 x event GHB119) indicates an additive effect of the two insect control proteins and not any synergy of those two proteins.TwinLink Cotton USDA Petition

Page 82 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Since no significant effects were seen with either protein on non-target organisms, an effect from a combination of the two proteins is unlikely. However, in order to investigate whether there was an unexpected interaction, three species were selected for testing with TwinLink plant material. This approach has previously been used in the assessment of non-target effects of cotton expressing more than one insecticidal protein (USEPA 2005, 2008)

1) Birds The possibility of intrinsic toxicity of the cotton plant to birds was addressed via a broiler feeding study using meal prepared from plant materi al from the TwinLinkTM cotton plant, which expresses both Cry1Ab and Cry2Ae protein. The birds were fed with a diet containing 10%TwinLink cotton seed, and the two control groups were fed on diet containing either nontransgenic "near isogenic" cottonseed, or commercial cottonseed. Birds were 1 day old at start of study, and were housed 10 per pen (replicate), 14 replicates per group.

Following 42 days of daily exposure to TwinLink cottonseed meal at 10% in diet, there were no negative effects detected in feed consumption, feed conversion ratio, survival, body weight gain, or in weight of chilled carcass, leg, thigh, wing or breast between ROSS #308 broiler chickens fed with the genetically modified cottonseed, and the two control groups.

This study indicates that TwinLink cotton poses minimal risk to birds.

2) Bees Cotton is wind pollinated, and bees are not therefore brought in to cotton for pollination purposes. However, honey bees and other wild pollinators will visit cotton on occasion, and may consume cotton nectar. Some pollen may also be brought back to the hive, where it may be fed to developing larvae. A recent meta-analysis of bee studies (Duan et al. 2008) indicated that Bt proteins pose minimal risk to bees. However, bee populations are the subject of intense scrutiny at the moment, due to significant unexplained declines, and the intrinsic toxicity of Cry1Ab protein and Cry2Ae protein have therefore been investigated. Neither showed any effect on bee larval development or adult emer gence. The honey bee larval assay provides a useful test system for investigating any possible harmful effect of TwinLink pollen.

Pollen was collected from field-grown TwinLink cotton and the non-transgenic near-isoline, Coker 315 and shipped on dry ice to the laboratory in California for testing. Honeybee larvae were treated with 2mg of pollen and 10 μL 30% sucrose solution. Honey bee larvae were treated in brood frames selected from outdoor beehives and brought into the laboratory just prior

to treatment. The test treatment consisted of 4 replicates of 20 larvae treated with TwinLink pollen, and the control treatment consisted of 4 replicates of 20 larvae treated with Coker 315 pollen. A fourth set of 4 replicates were treated with a reference substance, potassium arsenate (2,000 μg/mL) which is known to act as a stomach poison. This treatment was included to confirm exposure of the larvae.

Treated frames were returned to the hives, and checked at Day 6 for capping. This gives an

overall measure of larval surv ival and development, since if la rvae do not develop normally, the cells will not be capped by the nurse bees. At Day 13, frames were brought into the laboratory and the test area was covered with an adult emergence cage. Frames were kept in a growth chamber at 29.9 to 32.3 degrees centigrade and 56 to 74 percent for relative humidity.TwinLink Cotton USDA Petition

Page 83 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Data recorded Day 6 revealed the percent survival from dosing to capping was 88.8% for larvae treated with the TwinLink pollen at 2 mg/cell and 86.3% for larvae treated with Coker 315 pollen at 2 mg/cell. Comparatively, the potassium arsenate treatments at 2,000 μg/ml had a reduced survival rate of 0.0%, none of the larvae survived to capping.

Frames were checked for adult emergence twice daily until Day 15. The percent larval survival from dosing to adult emergence was identical to the survival from dosing to capping (88.8% for TwinLink, 86.3% for Coker 315). There were no obvious effects on behavior of morphology of the emerged adults.

This data indicates that TwinLink cotton poses minimal risk to honeybees.

c) Collembola Soil dwelling organisms could be exposed to Cry1Ab or Cry2Ae from TwinLink cotton through falling leaves or incorporation of senescent plant material. Exposure levels will be rather low, however, as both proteins break down rapidly in soil (DT 50 

### for Cry1Ab is 3.6 days, DT 50 

### for Cry2Ae is 3.4 days).

This study was conducted using leaf material from greenhouse-grown TwinLink cotton and a near-isoline, Coker 315. The leaves were collected and rapidly lyophilized.

The study was started with 12 day old larvae which were maintained in 4 ounce glass arenas containing a substrate of plaster of Paris and activated charcoal mixed in a ratio of 8:1 by weight. Collembola were fed a diet prepared by mixing dry Brewer's yeast (human consumption quality) with the lyophilized plant material. TwinLink cotton leaf material was added to the diet at 5, 20 and 50% of the total diet. There were four replicate arenas for the test and four control arenas treated with diet mixed with Coker 315 leaf material. Aliquots of diet were kept in the fridge, and fresh diet was added to the arenas every four days.

Percent mortality of adult organisms and the number of offspring produced during the exposure period were determined at test termination (day 28).

Results There was no significant difference in mortality or reproduction between the larvae treated with

TwinLink cotton and those treated with Coker 315 cotton.

Conclusion TwinLink cotton leaf material had no effect on Collembola even when incorporated into the diet at 50%. This represents a much higher exposure than would normally be found in a cotton field, and indicates that TwinLink cotton does not pose any risk to Collembola.TwinLink Cotton USDA Petition

Page 84 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 4. Summary of assessment of effect on non-target organisms

Insects are the organisms most likely to have si gnificant exposure to Twinlink cotton either by direct feeding on plants or pollen, or by feeding on other insects which have fed on the cotton plants. Cry proteins have been extensively studied for many years on many species of insects. Additional data was generated by Bayer CropScience on the effects of Cry1Ab, Cry2Ae and TwinLink cotton on sensitive insect species repres entative of insects found in cotton plants. No effects were observed on the predaceous insect Coleomegilla maculata (ladybug), or on the honey bee, which might be exposed to pollen from TwinLink plants.

There is also some possibility of exposure to soil-dwelling organisms to plant material. This exposure will be limited, as the Cry proteins have a very short half-life in soil. The half-lives for both Cry1Ab and Cry2Ae protein were measured in soils typical of cotton-growing regions, and the mean half-life was approximately three days for both proteins. Both proteins were, however, tested against the springtail, Folsomia candida, since springtails play an important role in the breakdown of plant material in the soil. Neither protein had a significant effect on this collembolan (springtail) species at environmenta lly relevant concentrations. An additional study using leaf material from TwinLink cotton also indicated no significant effect. Data had previously been generated indicating that Cry1Ab poses minimal risk to earthworm, and data for Cry2Ae was generated by Bayer CropScience to confirm that it too poses minimal risk.

Some exposure to birds and wildlife in cotton fields might also occur, but studies indicate that Cry1Ab or Cry2Ae are not intrinsically toxic to birds or mammals.

The risk of exposure to aquatic organisms is extr emely small. There is a slight chance of exposure via pollen drifting to streams or rivers, but cotton pollen is heavy and sticky and will not drift far from the cotton field. Expression levels of the Cry proteins in pollen are low and any exposure will be minimal.

With reference to the herbicide tolerance of TwinLink cotton, the shift in agronomic practices as a result of herbicide tolerance technology could potentially impact the habitat for non-target organisms. Herbicide tolerant cotton has made practices such as no-till planting more viable, resulting in an “ecosystem” in the cultivated field that is less disturbed due to the lack of cultivation and reduced need to enter the field to maintain the crop. Increased cotton canopy in the field during the growing season results in increased habitat for birds, insects, and other animals to thrive (Fawcett and Towery, 2004). In addition to the increased use of practices such as no-till agriculture, reductions in soil erosion, chemical use, fuel consumption, and other reduced inputs all have a direct positive impact on the well being of species found in agricultural settings. Since weed populations are currently controlled by both cultivation and chemical applications, use of herbicide tolerant crops would at the very least, add no additional burdens on non-target organisms.

It can be concluded that the TwinLink proteins pose minimal risk to non-target organisms.TwinLink Cotton USDA Petition

Page 85 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### D. Endangered species considerations

The US Fish & Wildlife Services (FWS) is responsible under the Endangered Species Act (ESA) (16 USC §1531). Section 6 of the ESA 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 the use of TwinLink cotton will impact any current listed species of concern. Of the total 747 plants listed as endangered, fewer than half (355) reside in states which commercially produce cotton (US FWS, 2006). Species of concern that may inhabit areas close to commercial cotton operations would not be impacted by the use of TwinLink cotton. Commercial agriculture routinely disturbs the ground in which crops are currently planted. As a result, perennial vegetative species would not grow in these areas. Additionally, because horizontal gene flow to sexually incompatible species is not an issue, there is negligible potential for exposure to the transgene contained in Twin Link cotton through sexual reproduction.

There are no reports of threatened or endangered species feeding on cotton plants; therefore, such species would not be exposed to cotton tissue containing these proteins. Cry1Ab and Cry2Ae protein is highly specific to Lepidoptera, so the only organisms which might be considered to be ‘at risk’ from TwinLink cotton would be endangered lepidopteran insects. While there are endangered lepidopteran species in cotton growing counties ( e.g. Kern Primrose Sphinx moth, Saint Francis' Satyr butterfly) the larvae are highly unlikely to be exposed to Cry2Ae proteins because their habitats do not overlap with cotton fields and their larvae do not feed on cotton and will not be exposed to Cry1Ab or Cry2Ae protein in pollen. The amount of pollen that would drift from these cotton plants onto plants fed upon by endangered/threatened species, would be very small, and the expression level of both Cry1Ab and Cry2Ae in pollen is very low. It is also therefore highly unlikely th at TwinLink could outcross to any wild or weedy

relatives of cotton.

For these reasons, it is not believed that the use of TwinLink cotton in commercial cotton production will adversely impact endangered species of concern.

E. Effects on current agricultural practices in cotton

TwinLink cotton is an alternative cotton product that contains insect resistant and herbicide tolerant traits. Agricultural practices that are today used for both products are expected to be maintained when incorporating TwinLink to the farmer’s choice of cotton products. Adoption and use of genetically modified cotton can provide posi tive impacts on agricultural practices. These positive impacts have been detailed in a study by Brookes and Barfoot (Brookes and Barfoot 2006) and include:

Herbicide tolerant crops • Increased management flexibility that comes from a combination of the ease of use associated with broad-spectrum, post-emergent herbicides; • Compared to conventional crops, where post-emergent herbicide application may result in ‘knock-back’ (some risk of crop damage from the herbi cide), this problem is less likely to occur in GM HT crops;TwinLink Cotton USDA Petition

Page 86 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### • Facilitation of adoption of no/reduced tillage practices with resultant savings in time and equipment usage; • Improved weed control has reduced harvesting costs – cleaner crops have resulted in reduced times for harvesting; • Elimination of potential damage caused by soil-incorporated residual herbicides in follow-on

crops.

Insect resistant crops • Production risk management/insurance purposes – taking away the worry of significant pest damage occurring; • A ‘convenience’ benefit (less time spent on crop walking and/or applying insecticides); • Savings in energy use – mainly associated with less spraying; • Savings in machinery use (for spraying and possibly reduced harvesting times); • Improved health and safety for farmers and fa rm workers (from reduced handling and use of insecticides);

Genetically modified herbicide tolerant cotton was first grown commercially in the US in 1997and by 2006, was planted on 65% of the total cotton plantings. Genetically modified insectresistant cotton has been grown commercially in the US since 1996 and by 2006, was used in 57% (3.5 million ha) of total cotton plantings (Brookes and Barfoot 2006). Table 32 describes percentages of the total cotton planted and their composition based on trait in 2007.TwinLink Cotton USDA Petition

Page 87 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 32 Genetically engineered (GE) upland cotton varieties by State and United States, 2000-2008

Insect-resistant (Bt) only Herbicide-tolerant only

State 2000 2001 2002 2003 2004 2005 2006 2007 2008 2000 2001 2002 2003 2004 2005 2006 2007 2008

Percent of all upland cotton planted Percent of all upland cotton planted

AL 1/ 10 10 10 18 28 25 25 15

AR 33 21 27 24 34 42 28 32 30 23 29 37 25 15 12 21 16 4

CA 3 11 6 9 6 8 9 4 7 17 27 26 27 39 40 40 51 45

GA 18 13 8 14 13 29 19 17 19 32 43 55 32 23 11 13 10 5

LA 37 30 27 30 26 21 13 17 19 13 14 9 15 7 10 13 11 6

MS 29 10 19 15 16 14 7 16 19 13 15 22 16 23 23 22 19 13

MO 1/ 20 32 13 12 59 40 63 68

NC 11 9 14 16 18 17 19 13 19 29 37 27 29 27 24 19 16 14

TN 1/ 13 16 10 10 8 10 17 14

TX 7 8 7 8 10 14 18 16 16 33 35 40 39 40 35 34 36 31

Other 17 18 19 18 22 18 21 27 22 21 33 35 32 24 26 24 20 20

U.S. 15 13 13 14 16 18 18 17 18 26 32 36 32 30 27 26 28 23

Stacked gene varieties All GE varieties

State 2000 2001 2002 2003 2004 2005 2006 2007 2008 2000 2001 2002 2003 2004 2005 2006 2007 2008

Percent of all upland cotton planted Percent of all upland cotton planted

AL 1/ 54 60 60 65 92 95 95 98

AR 14 28 26 46 45 42 45 47 64 70 78 90 95 94 96 94 95 98

CA 4 2 1 3 7 5 8 6 8 24 40 33 39 52 53 57 61 60

GA 32 29 30 47 58 55 64 68 73 82 85 93 93 94 95 96 95 97

LA 30 47 49 46 60 64 68 68 73 80 91 85 91 93 95 94 96 98

MS 36 61 47 61 58 59 69 62 66 78 86 88 92 97 96 98 97 98

MO 1/ 16 25 23 19 95 97 99 99

NC 36 38 45 48 46 54 60 64 62 76 84 86 93 91 95 98 93 95

TN 1/ 75 67 71 73 96 93 98 97

TX 6 6 4 6 8 14 18 28 31 46 49 51 53 58 63 70 80 78

Other 36 33 32 38 45 46 45 42 48 74 84 86 88 91 88 90 89 90

U.S. 20 24 22 27 30 34 39 42 45 61 69 71 73 76 79 83 87 86
1/ Estimates published individually beginning in 2005. Source : 2007-2008: U.S. Dept. of Agriculture, National Agricultural Sta tistics Service (NASS), Acreage. June 30, 2008.



### 1. Insect resistant cotton

As described in Section I of this petition, TwinLink cotton is an alternative for control of common cotton lepidopteran pests. Heliothine pests (budworm/bollworm) infested about 83 percent of the U.S. cotton crop in 2005, second only to thrips, which were found in 92 percent of the U.S. crop. Heliothine damage to the crop resulted in the loss of 520,000 bales of cotton in the United

States.TwinLink Cotton USDA Petition

Page 88 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Oklahoma reported the largest loss to insects, 13.3 percent, representing 63,900 bales of cotton. Arizona was second with 11.98 percent loss, followed by South Carolina, 8 percent, Alabama, 7.2 percent, North Carolina, 6.8 percent, California, 6.6 percent and Virginia, 5.6 percent. All other states reported a less than 5 percent loss.

The largest number of bales lost by state was in Texas, 392,800 bales, followed by Mississippi, 168,000 bales, California, 160,000 bales, and Arkansas, 132,000 bales. The biggest cost plus loss state was not surprising, Texas at $222 million, followed by Arkansas, $165 million, Mississippi, $162 million, and Georgia, $102 million.

The bollworm/budworm complex retained its position as the nation’s No. 1 pest in cotton production in 2007, reducing yields by 0.91 percent, a slight increase over 2006, according to Williams, 2008. Total losses from all insect pests in 2007 were 3.61 percent. According to the report, losses below 5 percent “continue to reflect the outstanding contribution technology has made to managing pest complexes which long have plagued cotton producers.”

Bollworms were the dominant species (92 percent) of the heliothine complex in 2007. Heliothine damages resulted in the loss of 229,000 bales of cotton. South Carolina (4.5 percent) and Alabama (2.88 percent) reported the highest loss to heliothines, with Georgia (1.58 percent), Florida (1.28 percent) and North Carolina (1.04 percent) rounding out the top five. Williams (2008) reported that Texas, which lost 78,000 bales, was the only state to report losses greater than 50,000 bales.

Bt cotton acreage decreased in 2007 to 7.1 million acres, although this was more reflective of the overall decrease in cotton acres, according to the report. Bt cotton comprised 65 percent of total acreage. Heliothine were sprayed with insecticide on 2.21 million Bt cotton acres in 2007. Foliar insecticide applications represent 47 percent of the cost of insect management in cotton.

2. Glufosinate-ammonium tolerant cotton

A detailed evaluation of glufosinate ammonium tolerant cotton was submitted in the LLCotton25 USDA Petition for determination of non-regulat ed status. The same arguments are applicable for the evaluation of TwinLink cotton. USDA responded in their Environmental Assessment and Finding of No Significant Impact (2003).

Glufosinate-ammonium may positively impact cu rrent agronomic practices in cotton by 1) offering broad spectrum, post-emergence weed control with a wide application window, which allows treatment only when weeds reach economical thresholds; 2) providing the opportunity to continue to move away from pre-emergent and residually active compounds; 3) providing a new herbicidal mode of action that allows for improved weed shift and resistance management; 4) decreasing cultivation needs; 5) allowing the application of less total pounds of active ingredient than used presently, and, 6) providing a more profitable and sustainable cotton system for cotton growers.

Weeds are a significant challenge for US cotton producers which must be managed in order to successfully produce an economically viable cotton crop (Bryson, 1999). It is estimated that without weed control, crop yields in cotton would be reduced by 77% (Gianessi et al., 2002).TwinLink Cotton USDA Petition

Page 89 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Weeds compete with cultivated cotton for nutrients and water in the soil, and if large enough can compete for sunlight required for photosynthesis in the plant. Because cotton prefers warmer climates, early weed control is especially impor tant to establishing a solid stand shortly after planting in the spring when temperatures are not consistently high. It is only when soil temperatures are consistently at 75°F that cotton becomes competitive with weed species (Chandler, 1984). Weeds also can host a variety of insect pests, and can interfere with the harvesting process, and can impact fiber quality by staining cotton lint during harvest. Weeds also contribute to the amount of gin trash coll ected during ginning, and can negatively impact equipment.

The total acreage planted with upland cotton in the US is shown in Table 33, 97% of these acres received herbicide application for weed control (USDA, NASS 2008). Many acres are often treated multiple times using herbicide tolerant cotton during pre-plant burndown and at least one application post-emergence.

Table 33 Agricultural Chemical Usage 2007 - Cotton Summary

State Planted Acreage Herbicide Herbicide Insecticide 1/ Insecticide 1/

1000 acres Percent 1000 lbs. Percent 1000 lbs.

AL 400 98 941 55 88

AR 860 97 2399 92 1092

CA 455 90 565 90 506

GA 1030 100 3163 85 956

LA 335 98 992 99 562

MS 660 100 2132 97 1231

MO 380 100 995 83 270

NC 500 100 1479 79 300

SC 180 100 535 92 85

TN 515 100 1482 94 228

TX 4925 96 11532 43 2624

Total 10240 97 26214 66 7943
Released May 21, 2008, by the National Agricult ural Statistics Service (NASS), Agricultur al Statistics Board, U.S. Department o f Agriculture 1/ Total Applied excludes Bt's ( Bacillus thuringiensis ) and other biologicals.



### The main weed species across all cotton include redroot pigweed ( Amaranthus retroflexus ) and other amaranth species, morning glories ( Ipomoea spp), cocklebur ( Xanthium strrumarium ), Johnsongrass ( Sorghum halepense ), crabgrass ( Digitaria spp), barnyardgrass and watergrass ( Echinochloa spp), sicklepod ( Cassia obtusifolia ), and Texas panicum ( Panicum texanum ). Cotton is grown across the southern United States in 4 distinct regions (southeast, mid-south, southwest and west). Weed species infestations change across these regions and weed control methods are adjusted accordingly.TwinLink Cotton USDA Petition

Page 90 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 34 Common weed Species in US Cotton Production

Common Name Scientific Name Region Morning glory Ipomoea spp SE, MS, SW, W Prickly sida Sida spinosa SE, MS, SW Sicklepod Senna obtusifolia SE, MS, SW Pigweed spp Amaranthus spp SE, MS, SW Nutsedge spp Cyperus spp SE, MS, SW, W

Velvet leaf Abutilon theophrasti SE, MS Smartweed spp Polygonum spp SE, MS, SW Tropic croton Croton glandulosus var. septentrionalis SE, MS, SW Hemp sesbania Sesbania herbacea SE, MS, SW

Redvine Brunnichia ovata MS Johnsongrass Sorghum halep SE, MS, SW

Common Cockleburr Xanthium strumarium SE, MS, SW Nightshade spp Solanum spp SE, MS, SW, W Lambsquarter spp Chenopodium spp SE, MS, W

Field blindweed Convolvulus arvensis SW, W Grass spp Various species SE, MS, SW, W Texas panicum Panicum texanum SW, W

SE = Southeast MS = Mid-south MW = Midwest W = West Source: 2001 proceedings, SWSS vol. 54; NCSU Crop profiles 2006



### Prior to the development of herbicide tolerant crops, control of these diverse species required the use of multiple herbicide families and multiple applications. Development of crops which are tolerant to broad spectrum herbicides has changed agricultural tillage, weed control, and ecological practices. The volume of herbicide sprayed has been reduced greatly using herbicide tolerant cotton varieties (Sankula et al ., 2005). Additionally, cultivation of herbicide tolerant cotton has provided multiple benefits in the form of reduced inputs to manage cultivated crops, and reduced losses of those inputs from the field due to erosion, run-off, and waste (USDA-ERS, 2002).

Conventional methods of weed control Successful weed control utilizing conventional met hods is achieved by a combination of crop rotation, cultivation, and herbicides.

Crop rotation allows for the use of complimentary chemical and agricultural practices. Certain weeds do not grow well in other crops, therefore reducing the weed seed bank of the seed so in subsequent years there is no build-up of weed populations from recurrent cotton plantings.

Herbicide use is the most effective and direct form of weed control. Herbicides are used in pre-plant burndown applications where established weed populations are removed prior to planting. Herbicide formulations are also available for broadcast and directed application post-emergence to help establish the stand of the cotton to provide competitive advantage over weed species (Ferrell, 2006). Many herbicides used in herbicide tolerant cotton production systems (including glufosinate ammoni um) have no residual soil activity, which contributes to their more favorable environmental profile. Herbicides used in conventional systems often have residual soil activity to increase the duration of the herbicidal effect, and to reduce the number of herbicide applications made to a field. Late into the season, hooded spray applications of herbicides, which would normally be harmful to cotton crops, may beTwinLink Cotton USDA Petition

Page 91 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### applied between cotton rows to help reduce the population of weed species. Should herbicide application fail to control weed populations, mechanical cultivation can be used to remove weed species from between cotton rows.

Other weed management programs have been attempted over the course of cotton production which have been effective in some cases, but the methods mentioned above are by far the most commonly used methods of weed control used in conventional cotton production systems.

Volunteer management TwinLink cotton is sensitive to many other chemicals registered for pre-plant burndown and post-emergence in cotton. All cotton varieties are sensitive to many herbicides, such as 2,4- D, used for weed management in monocotyledonous crops such as corn in rotational systems. Additionally, other herbicides, su ch as glyphosate (RoundUp®) and flumioxazin (Valor®), can be used for burndown in no-till plant ing systems common in herbicide tolerant cropping systems.

In conventional cultivation systems, post-dire cted sprays of herbicides such as MSMA in combination with traditional cultivation would be successful in removing volunteer cotton plants.

In rotational situations with other glufosinate to lerant crops such as corn or soybeans, many herbicides, ( e.g. 2,4-D) are used for broadleaf control in monocotyledon crops ( i.e. corn). Soybean crops can use soil incorporated, pre-plant, and post-emergence herbicides to control a broad spectrum of broadleaf plants, such as cotton. Products such as Lexon® and Lorox® and others are available should cotton volunteers emerge.

There are no reported glufosinate-tolerant weeds.

The Herbicide Glufosinate-ammonium and Current Uses Glufosinate ammonium is currently registered under various trade names for control of weeds in cotton and other crops, and as Ignite®/Liberty® for use on LibertyLink® corn, cotton, soy, rice and canola varieties. It acts by inhibiting the enzyme glutamine synthase which causes a toxic buildup of ammonia within the weed. Glufosinate-ammonium is a nonselective herbicide for both non-crop and crop uses. It is highly biodegradable, has no residual activity, and has very low toxicity for humans. Gluf osinate-ammonium is an ecologically sound herbicide that degrades rapidly in microbially active soils and also readily binds to soil particles. Glufosinate-ammonium poses less risk of adverse effects of drift to non-target

areas than current market standards. Glufosinate-ammonium and its short-lived metabolites have not been found to accumulate in the environment. Soil microorganisms, bees, earthworms, birds and mammals are unaffected by glufosinate-ammonium. This herbicide has a different mode of action than the other major herbicides used in cotton that it is intended to replace or supplement, and unlike the other herbicides, there are no weed biotypes with resistance reported to this clas s of herbicide in an international survey of herbicide resistant weeds (Heap, 2002). Glufosinate ammonium is registered by EPA for use

in cotton.TwinLink Cotton USDA Petition

Page 92 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 35 Important Weeds Labeled for Control by Ignite® Herbicide in Cotton

Provided are the common and scientific names.

Grass weeds Barnyardgrass Echinochloa crus-galli Crabgrass, large Digitaria sangulinalis Johnsongrass Sorghum halepense

Texas Panicum Panicum texanum Watergrass Echinochloa oryzoides

Broadleaf weeds Pigweed species Amaranthus spp. Sicklepod Cassia obtusifolia Cocklebur, common Xanthium strumarium Prickly Sida Sida spinosa Black nightshade Solanum nigrum Morning glory species Ipomoea spp.
Sunflower Helanthus annuus



### TwinLink Cotton and Glufosinate-ammonium may positively impact current agronomic practices in cotton by 1) offering broad spectrum, post-emergence weed control with a wide application window, which allows treatment only when weeds reach economical thresholds; 2) providing the opportunity to continue to move away from pre-emergent and residually active compounds; 3) providing an effective herbicidal mode of acti on that allows for improved weed shift and resistance management; 4) decreasing cultivation needs; 5) allowing the application of less total pounds of active ingredient than used presently, and, 6) providing a more profitable and sustainable cotton system for cotton growers.

F. Summary of Environmental Safety / Impact on non-contained use of TwinLink cotton

TwinLink cotton was evaluated for agronomic impacts during seed germination and dormancy studies, protein safety assessment, compos ition analysis, and agronomic performance evaluation. These assessments of TwinLink cotton and the Cry1Ab, Cry2Ae and PAT proteins were conducted across a wide variety of envi ronmental and climatic conditions which are representative of the majority of upland cotton acres produced in the United States. These assessments demonstrate that TwinLink cotton does not pose a greater plant pest potential than conventional cotton produced in the United States.

The environmental impacts of pollen transfer to other cotton varieties is not considered to be an issue with the production of TwinLink cotton. The limited range of movement of cotton pollen described in Section II coupled with the low acute oral toxicity of the three expressed proteins demonstrates that the opportunities for exposure and the impacts of this exposure are minimal. Additionally, the opportunities for outcrossing with sexually compatible cotton species is highly unlikely due to the limited number of species, and their isolation from cotton production regions in the United States. Therefore the agronomic consequences of introduction of TwinLink cotton are also expected to be minimal due to the wide range of methods of control of transgenic cotton, and cotton’s inability to establish itself as a major weed species.TwinLink Cotton USDA Petition

Page 93 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### The resulting conclusion is that TwinLink cotton is not expected to have an adverse impact on non-target organisms found in and around agricultural production systems, or to the environment around these regions.

G. Insect Resistance Management (IRM) Plan

A comprehensive insect resistance management program is described in the EPA Section 3 Registration application for TwinLink Cotton. The IRM program provides a full characterization of the TwinLink product, demonstrating that it expresses a combination of two Bt proteins, Cry1Ab and Cry2Ae, at a high dose level for all major target pests. Binding data confirm the literature results that these two proteins bind to unique binding sites in the insect midgut. Computer modeling results show that TwinLi nk cotton will have long-term durability in the marketplace. A plan for monitoring of insect susceptibility is also provided.TwinLink Cotton USDA Petition

Page 94 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## X. STATEMENT OF GR OUNDS UNFAVORABLE

Data generated from agronomic tests and molecular characterization indicate that no unfavorable grounds are associated with TwinLink cotton.

Therefore Bayer CropScience requests that TwinLink cotton no longer be considered a regulated article under 7 CFR 340.TwinLink Cotton USDA Petition

Page 95 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

## XI. REFERENCES

Amann M. M. 1999. Identification of Nutrients and An ti-nutrients to Analyze in Cotton-derived Foods for Human and Animal Consumption. Environ Arlington, VA, USA.

Aronson, A.I. and Shai, Y. 2001. Why Bacillus thuringiensis insecticidal toxins are so effective: unique

features of their mode of action. FEMS Microbiol. Lett. 195:1-8.

Arumuganathan K., Earle E.D. 1991. Nuclear DNA C ontent of Some Important Plant Species. Plant Molecular Biology Reporter, 9 (3), 208-218.

Batista R., Nunes B., Carmo M., Cardoso C., Jose H.S., de Almeida A.B., Manique A., Bento L., Ricardo C.P., Oliveira M.M. 2005. Lack of detectable allergenicity of transgenic maize and soya samples. J. Allergy Clin. Immunol. 116:403-410.

Berberich S.A., Ream J.E, Jackson T.L., Wood R., Stipanov ic R., Harvey P., Patzer S., Fuchs, R.L. 1996. The composition of insect-protected cottonseed is equi valent to that of conventional cottonseed. J. Agric. Food Chem. 44. 365-371.

Berger L.A., Vaissière B.E., Moffett J.O., Merritt, S. J. 1988. Bombus spp. (Hymenoptera: Apidae) as pollinators of male-sterile Upland cotton on the Texas High Plains. Environ. Entomol. 17. 789-794.

Bertrand, J.A., Sudduth T.Q., Condon A., Jenkins T.C ., Calhoun C. 2005. Nutrient Content of Whole cottonseed. J. Dairy Sci. 88:1470-1477.

Betz F.S., Hammond B.G., Fuchs R.L. 2000. Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. R egul. Toxicol. Pharmacol. 32:156-173. Boevink P., Chu P.W.G., Keese P. (1995). Sequence of subterranean clover stunt virus DNA: Affinities with the geminiviruses. Virology 207: 354-361.

Bolivar, F, Rodrigues, R.L, Greene, P.J, Betlach, M.C., Haymeker, H.L., Boyer, H.W., Crosa J., and Talkow, S.

1977. Construction and Characterization of New Cloning Vehicles. II. A Multipurpose Cloning System.

Gene 2:95-113.

Bradford, M.M. 1976. A refined and sensitive method fo r the quantitation of microg ram quantities of protein utilizing the principle of protein-dye binding. Anal. Biochem., 72, 248.

Bradshaw R.A., Brickey W.W., Walker K.W. 1998. N- terminal processing: the methionine aminopeptidase and N-acetyl transferase families. Tr ends Biochem. Sci. 23: 263-267.

Brake J., Faust M.A., and Stein J. 2003. Evaluation of transgenic event Bt11 hybrid co rn in broiler chickens.

Poult. Sci

. 82:551-559.

Brookes G, Barfoot P. 2008. GM crops: global soci o-economic and environmental impacts 1996-2006. PG Economics Ltd, UK. 118 pages.

Bryson, C.T., Salisbury, C., and McCloskey, W.B. 1999. Weeds and their control. In Cotton: Origin, History, Technology, and Production. C.W. Smith and J. Cothern, (eds.). John Wiley & Sons, Inc.,

New York: 617-658.TwinLink Cotton USDA Petition

Page 96 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Calhoun M.C., Kuhlmann S.W., Baldwin B.C. 1995. cotton feed composition and gossypol availability and toxicity. In Proc. 2nd National Alternative Feeds Symp., Wooster, OH, Sept. 24-26, 1995. 125-145.

Canadian Food Inspection Agency (CFIA). 2004. Deci sion Document DD2003-45 - Determination of the safety of Monsanto’s insect resistant Bollgard IITM cotton ( Gossypium hirsutum L.). CFIA, Plant Products Directorate, Plant Biosafety Office. June 6, 2004.

Carpenter, J.E., and L.P. Gianessi. 2001. Agricultural Biotechnology: Updated benefit estimates. National Center for Food and Agricultural Poli cy. http://www.ncfap. org/pesticid1.htm

Carrière, Y. and T.J. Dennehy , B. Pedersen, S. Haller, C. Ellers-Kir k, L. Antilla, Y.B. Liu, E. Willott, B. Tabashnik. 2001. Large-scale management of insect resistance to transgenic cotton in Arizona: can transgenic insecticidal crops be su stained? Journal of Economic Entomology 94(2): 315-325.

Chen X.J., Curtiss A., Alcantara E., Dean D.H. 1995. Mutations in domain I of Bacillus thuringiensisdeltaendotoxin Cry1Ab reduce the irreversible binding of toxin to Manduca sexta brush border membrane
vesicles. J. Biol. Chem

. 270:6412-6419.

Cornelissen M., Vandewiele M. 1989. Nuclear transcriptio nal activity of the tobacco plasmid psbA promoter.

Nucleic Acids Research . 17 (1) 19-29.

Crockett L. 1977. Wildly Successful Plants: North Am erican Weeds. University of Hawaii Press, Honolulu,

Hawaii.

De Almeida E.R.P., Gosselé V., Muller C.G., Dockx J. , Reynaerts A., Botterman J, Krebbers E., Timko M.P. 1989. Transgenic expression of two marker gene s under the control of an Arabidopsis rbcs promoter: sequence encoding the Rubisco transit pept ide increase expression levels. Molec. Gen.

Genet. 218:78-86.

Deblaere R., Bytebier B., De Greve H., Deboeck F., Schell J., Van Montagu M., Leemans J. 1985. Efficient octopine Ti-plasmid derived vectors for Agroba cterium mediated gene transfer to plants. Nucleic

Acid Research . 13. 4777-4788.

Delaney B., Astwood J.D., Cunny H ., Conn R.E., Herouet-Guicheney C., Macintosh S., Meyer L.S., Privalle L., Gao Y., Mattsson J., Levine, M. 2008. Evaluation of protein safety in the context of agricultural biotechnology. Food Chem. Toxicol. 46 Suppl 2:S71-97.

Depicker A., Stachel S., Dhaese P., Zambryski P., Goodman H.M. 1982. Nopaline Synthase: transcript mapping and DNA sequence. J. Molec. Appl. Genet. 1:561-573.

Devore, J., Peck, R. 1986. Statistics. West Publishing Company, St. Paul.

D'Halluin, K., De Block, M., Denecke, J., Janssens, J ., Leemans, J., Reynaerts, A., Botterman, J. 1992. The

bar Gene as Selectable and Screenable Marker in Plant Engineering. Methods in Enzymology, 216,

415-426.

Duan, J.J., Marvier, M., Huesing, J ., Dively, G., Huang, Z.Y. 2008. A Me ta-Analysis of Effects of Bt Crops on Honey Bees (Hymenoptera: Apidae). PLoSONE 3(1): e1415. http://www.plosone.org/article/in fo:doi/10.1371/journal.pone.0001415

Endrizzi, J.E., Turcotte, E.I. and Kohel, R.J. 1984. cott on, Agronomy No. 24, p 82-129, Soil Science Society of America, Inc. (Kohel, R.J. and C.F. Lewis, eds) Wisconsin, USA.TwinLink Cotton USDA Petition

Page 97 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION EPA- see United States Envir onmental Protection Agency.

Fawcett, R., Towery, D. 2004. Co nservation Tillage and Plant Biotech nology: How New Technologies Can Improve the Environment By Reducing the Need to Plow. Conservation Technology Information Center. 1220 Potter Drive, Suite 170, West Lafayette, IN 47906. www.ctic.purdue.edu.

FAO/WHO Food Standards. 2001. Named Vegetable Oils . Codex Stan 210 from Codex Alimentarius vol. 8. http://codexalimentarius.net/standard -list accessed December 5, 2002.

Ferrell, J.A., G.E. MacDonald, and B.J. Brecke. 2006. Weed management in cotton. Available at http://edis.ifas.ufl.edu/WG003. Univ. of Florida IFAS, Gainesville.

Fling M.E., Kopf J., Richards C. 1985. Nucl eotide sequence of the transposon Tn7 gene encoding an aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyl transferase. Nucleic Acids Research. 13(19):

7095-7106.

Fryxell P.A. 1979. The natural history of the cotton tribe ( Malvaceae , tribe Gossypieae ), Texas A&M University Press. College Station and London.

Fryxell PA. 1992. A revised taxonomic interpretation of Gossypium L. (Malvaceae). Rheedea 2: 108-165.

Gianessi L.P., Silvers C.S., Sankula S., Carpenter J.E. 2002. Plant Biotechnology: Current and Potential Impact For Improving Pest Management In US Agri culture. An Analysis of 40 Case Studies.

Herbicide Tolerant Cotton. National Center for Food and Agricultural Policy. 1616 P Street, NW. Washington, DC 20036. 61 pages.

Griffitts J.S., Haslam S.M., Yang T., Garczynski S.F ., Mulloy B., Morris H., Cremer P.S., Dell A., Adang M.J., Aroian R.V. 2005. Glycolipids as receptors for Bacillus thuringiensis crystal toxin. Science.

307:922-925.

Hajdukiewicz P., Svab Z., Maliga P. 1994. The small, versatile pPZP family of Agrobacteium binary vectors for plant transformation. Plant Molecular Biology. 25:989-994.

Hamilton K.A., Pyla P.D., Breeze M., Olson T., Li M., R obinson E., Gallagher S.P., Sorbet R., Chen Y. 2004. Bollgard II cotton: compositional analysis and feedi ng studies of cottonseed from insect-protected cotton ( Gossypium hirsutum L .) producing the Cry1Ac and Cry2Ab2 proteins. J. Agric. Food Chem

. 52:6969-6976.

Hammond B.G., Dudek R., Lemen J.K. and Nemeth M.A. 2006. Results of a 90-day safety assurance study with rats fed grain from corn borer-protec ted corn. Food Chem. Toxicol. 44:1092-1099.

Harpster M.H., Townsend J.A., Jones J.D.G., Bedbrook J., Dunsmuir P. 1988. Relative strengths of the 35S Cauliflower Mosaic Virus, 1’, 2’ and nopaline syntha se promoters in transf ormed tobacco, sugarbeet and oilseed rape callus tissue. Molec. Gen Genet. 212:182-190.

Herouet C., Esdaile D.J., Mallyon B.A., Debruyne E., Sc hulz A., Currier T., Hendrickx K., van der Klis R.J., Rouan D. 2005. Safety evaluation of the phosphinot hricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to gl ufosinate-ammonium herbicide in transgenic plants. Regul Toxicol Pharmacol. 41:134-149.

Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandek erckhove J., Vanderbruggen H., van Montagu M., Zabeau M.,Vaeck M. 1986. Stru ctural and functional analysis of a cloned delta endotoxin of Bacillus thuringiensis berliner 1715. Eur. J. Biochem. 161:273-280.TwinLink Cotton USDA Petition

Page 98 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Hofmann, C., Vanderbruggen, H. V., Höfte, H., V an Rie, J., Jansens, S., and Van Mellaert, H. 1988a. Specificity of Bacillus thuringiensis delta-endotoxins is correlated wi th the presence of high-affinity binding sites in the brush border membrane of ta rget insect midguts. Proc. Natl. Acad. Sci. USA

85:7844-7848

Holm L., Pancho J.V., Herbarger J.P., Plucknett D.L. 1979. A geographical Atlas of World Weeds. John Wiley and Sons, NY. 391 pp.

ILSI. 2007. ILSI Crop Composition database. Accessed March 13, 2007. http://www.cropcomposition.org/cgi-perl/search_ora.cgi.

Itoh Y., Watson J.M., Haas D., Leisinger T. 1984. Genetic and molecular characterization of the Pseudomonas plasmid pVS1. Plasmid, 11, 206.

Jenkins J.N. 1993. Cotton. In Traditional crop breeding practices: an historical review to serve as a baseline for assessing the role of modern biotechnology. OECD.

Jenkins N., Parekh R.B., James D.C. 1996. Getting the glycosylation right: implications for the biotechnology industry. Nat. Biotechnol

. 14:975-981.

Johnson, S.R., Strom, S., Grillo, K. 2007. Quantificati on of the Impacts on US Agriculture of BiotechnologyDerived Crops Planted in 2006. National Center for Food and Agricultural Policy, accessed at: http://ncfap.org/index.html

Kareiva P., Morris W., Jacobi C. 1994. Studying and managing the risk of cross-fertilization between transgenic crops and their wild relatives. Molec. Ecology 3. 15-21.

Knowles B.H., Dow J.A.T. 1993. The crystal ∂ -endotoxins of Bacillus thuringiensis : Models for their mechanism of action on the insect gut. BioEssays. 469-476.

Kützner H.J. 1981. The family Streptomycetaceae (Chapter 156). In : The prokaryotes: a handbook on habitats, isolation and identification of bacteria. Starr M.P., Stolp H., Trüper H.G., Ballows A., Schlegel H.G., Eds. Pages 2028-2090.

Lawhon J.T., Cater C.M., Mattil K.F. 1977. Evaluation of the food use potential of sixteen varieties of

cottonseed. J. Am. Oil Chem. Soc. 54. 75-80. Abstract.

Lundquist R. 1995. Current Uses of Traditional Co -Products. Wooster, OH: September 24-26, Proceedings of the 2nd National Alternative Feeds Symposium. 95-104.

Marshall J.S., Stubbs,J.D., Taylor W.C. 1996. Two g enes encode highly similar choroplastic NADP-malic enzymes in Flaveria. Plant Physiol. 111:1251-1261.

McClintock J.T., Schaffer C.R., Sjoblad R.D. 1995. A comparative review of the mammalian toxicity of Bacillus thuringiensis -based pesticides. Pestic ide Science. 45:95-105.

McGregor, S.E. 1976. Insect Pollination of Cultivated Crop Plants, Agricultural Handbook No. 496, Unites States Department of Agriculture Research Service, Washington, DC.

Meredith W.R., Bridge R.R. 1973. Natural crossing in cotton Gossypium hirsutum L. in the delta of Mississippi. Crop Sci. 13. 551-552.TwinLink Cotton USDA Petition

Page 99 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Michiels F., Morioka S., Scheirlinck T., Komari T. 1992. Stamen-specific promoters from rice. Patent Application WO92/13956A1 (20AUG-1992). PLANT GENETIC SYSTEMS N.V. (BE).

Moffett J.O., Stith L.S., Burkhart C.C., Shipman, C.W. 1976. Honey bee visits to cotton flowers. Environ.

Entomol. 4. 203-206.

Muenscher W.C. 1980. Weeds. Second Edition. Cornell University Press, Ithaca and London.

Nida D.L., Patzer S., Harvey P., Sti panovic R., Wood R., Fuchs, R.L. 1996. Glyphosate-tolerant cotton: the composition of the cottonseed is equivalent to that of conventional cottonseed. J. Agric. Food Chem.

44. 1967-1974.

Niles, G.A. and Feaster, C.V. 1984. Cotton, Agronomy No. 24, p 205, Soil Science Society of America, Inc., (Kohel, R.J. and C.F. Lewis, eds) Wisconsin, USA

Odell J.T., Nagy F., Chua N.-H. 1985. Identificat ion of DNA sequences requi red for activity of the Cauliflower Mosaic Virus 35S promoter. Nature. 313:810-812.

Ogiwara K., Indrasith L.S., Asano S., Hori H. 1992. Processing of delta-endotoxin from Bacillus thuringiensis subsp. kurstaki HD-1 and HD-73 by gut juic es of various insect larvae. J. Invertebr.

Pathol. 60:121-126.

Oka A., Sugisaki H., Takanami M. 1981. Nucleoti de sequence of the kanamyci n resistance transposon Tn903. Journal of Molecular Biology, 147, 217-226.

Organization for Economic Co-operation and Deve lopment (OECD). 2004. Consensus Document on Compositional Considerations for New Varietie s of cotton (Gossypium hirsutum and Gossypium barbadense): Key Food and Feed Nutrients and An ti-Nutrients. ENV/JM/FOOD(2004)16. 18-Aug2004.

Organization for Economic Co-operation and Development (OECD). 2007. Consensus document on safety information on transgenic plants expressing Bacillus thur ingiensis -derived insect control protein. 109

pages.

Phelps R.A., Shenstone F.S., Kemmerer R.J., Evans, R. J. 1965. A review of cyclopropenoid compounds: biological effects of some derivatives. Poult. Sci. 44. 358-394.

Rosenquist H., Smidt L., Andersen S.R., Jensen G.B., W ilcks A. 2005. Occurrence and significance of

Bacillus cereus and Bacill us thuringiensis in ready-to-eat f ood. FEMS Microbiol. Lett. 250:129-136.

Sambrook, J., Fritsch, E.F. and Maniatis, T. 1989. Molecular cloning, a laboratory manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

Sanfaçon H., Brodman P., Hohn T. 1991. A dissect ion of the Cauliflower Mosaic Virus polyadenylation signal. Genes and Development. 5:141-149.

Sankula S., Marmon G., Blumenthal E. 2005. Biot echnology-Derived Crops Planted in 2004 - Impacts on US Agriculture. National Center for Food and Agri cultural Policy. 1616 P-street, NW. Washington, DC 20036. www.ncfap.org.

Schnepf H.E., Whiteley H.R. 1981. Cloning and expression of the Bacillus thuringiensis crystal protein gene

in Escherichia coli . Proc. Natl. Acad. Sci. U.S.A. 78:2893-2897.TwinLink Cotton USDA Petition

Page 100 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Schroder M., Poulsen M., Wilcks A., Kroghsbo S., Miller A., Frenzel T., Danier J., Rychlik M., Emami K., Gatehouse A., Shu Q., Engel K.H., Altosaar I., Knudsen I. 2007. A 90-day safety study of genetically modified rice expressing Cry1Ab protein ( Bacillus thuringiensis toxin) in Wistar rats. Food

Chem. Toxicol. 45:339-349.

Sedmak J.J., Grossberg S. E. 1977. A rapid, sens itive and versatile assay fo r protein using Coomassic brilliant blue G250. Anal. Biochem. 79, 544.

Thompson C., Van Montagu M., Leemans J. 1987. Engi neering herbicide resistance in plants by expression of a detoxifying enzyme. EMBO Journal. 6(9). 2513-2518.

Tojo A., Aizawa K. 1983. Dissolution and degradation of Bacillus thuringiensis delta-endotoxin by gut juice protease of the silkworm Bombyx mori. Appl. Environ. Microbiol. 45:576-580.

USCA. 1982. United States – Canadian Tables of Feed Composition. 1982. 3rd Revision. National Academy Press, Washington D.C.

USDA. 2003. Aventis CropScience USA LP Petition 02-042-01p to USDA/APHIS Seeking a Determination of Non-regulated Status for Glufosinate-ammonium Herbicide-tolerant Cotton Transformation Event LLCotton25. Environmental Assessment and Finding of No Significant Impact.

USDA-FAS. 2006. Cotton: world markets and trade. Un ited States Department of Agriculture. Foreign Agricultural Service. Circular Seri es FOP 9-06. September 2006. 26 pages

USDA-NASS. http://www.nass.us da.gov/QuickStats/index2.jsp#top.

United States Environmental Protection Agency (USEPA,) (2001). Biopesticides Registration Action Document (BRAD) – Bacillus thuringiensis Plant-Incorporated Protect ants, October 15 2001. U.S. Environmental Protection Agency, Washington, D.C. http://www.epa.gov/pesticides/biopesticides/pips/bt_brad.htm

United States Environmental Protection Agency (USEPA). 2002. Biopesticides Registration Action

Document: Bacillus thuringiensis Cry2Ab2 protein and its genetic material necessary for its production in cotton. 74 pages.

United States Environmental Protection Agency (USE PA). 2005. Bt Cry1F/Cry1Ac WideStrike® Cotton Registration Action Document. http://www.epa.gov/oppbppd1/biopesticides/in gredients/tech_docs/brad_006512-006513.pdf

United States Environmental Protection Agency (U SEPA). 2007. Biopesticide active ingredients and products containing them. 39 pages.

United States Environmental Protection Agency (USEPA) 2008 Bacillus thuringiensis modified Cry1Ab (SYNIR67B-1) and Vip3Aa19 (SYN-IR102-7) insecticidal pr oteins and the genetic material necessary for their production in COT102 X COT67B cotton http://www.epa.gov/pesticides/biopesticide s/ingredients/tech_docs/brad_006529.pdf

United States Food and Drug Administra tion (US-FDA). 1992. Statement of Policy: Foods Derived From New Plant Varieties. http://www.c fsan.fda.gov/~lrd/bi otechm.html#reg

United States Food and Drug Administ ration (US-FDA). 2002. Biotechnology Consultation: BNF No. 000074. Insect-Protected Bollgard II Cott on Line 15985. July 16, 2002. 7 pages.TwinLink Cotton USDA Petition

Page 101 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION United States Food and Drug Administrati on (US-FDA). 2003. Biotechnology Consultation: Note to the File

BFN No. 000086. Glufosinate Tolerant Cotton Tr ansformation Event LLCotton25. June 05, 2003. http://www.cfsan.fda.gov/~rdb/bnfm086.html (A ccessed on November 11, 2008). 6 pages.

Vaissière B. 1990. Pollen dispersal and carryover in Upland cotton, Gossypium hirsutum L. Study report for the Department of Entomology, Texas A & M University, College Station, TX. USA.

Van Deynze A.E., Sundstrom F.J., Bradford K.J. 2005. Pollen-mediated gene flow in California cotton depends on pollinator activity. Crop Science, 45: 1565-1570.

Van Rie J., Jansens S., Hofte H., Degheele D ., Van Mellaert H. 1989. Specificity of Bacillus thuringiensis delta-endotoxins. Importance of specific receptors on the brush border membrane of the mid-gut of target insects. Eur. J. Biochem. 186:239-247.

Verdaguer B., De Kochko A., Beachy R.N., Fauquet C. 1996. Isolation and expression in transgenic tobacco and rice plants, of the cassava vein mosa ic virus (CVMV) promoter. Plant Mol. Biol.

31:1129-1139.

Wang Z.-H., Wang Y., Cui H.-R., Xi a Y.-W., Altosaar I., and Shu Q.-Y. 2002. Toxicological evaluation of transgenic rice flour with a synthetic cry1Ab gene from Bacillus thuringiensis . J. Science Food Agric.

82:738-744.

Whalon M.E., Wingerd B.A. 2003. Bt: mode of action and use. Arch. Insect Biochem. Physiol. 54:200-211.

Williams, M.R. 2008. Cotton Loss Estimates, 2007, pr esented at the 2008 Beltwide Cotton Conferences. January 8-11, 2008. Nashville, TN

Wozenski J., Woodburn, M. 1975. Phytic acid (myoin ositol hexaphosphate) and phytase activity in four cottonseed protein products. Cereal Chemistry 52. 665-669.

Xin Z.Y., Brettell R.I.S., Cheng Z.M ., Waterhause P.M., Appels R., Bank s P.M., Zhou G.H., Chen X., Larkin P.J. 1988. Characterization of a potential source of barley yellow dwarf virus resistance for wheat.

Genome. 30: 250-257.

Zambryski P. 1988. Basic processes underlying Agrobac terium-mediated DNA transfer to plant cells. Ann.

Rev. Genet. 22: 1-30.

Zhu J., Oger P.M., Schrammeijer B., Hooykaas P.J. J., Farrand S.K., Winans S.C. 2000. The Bases of Crown Gall Tumorigenesis. Journal of Bacteriology. 182 (14):3885-3895.TwinLink Cotton USDA Petition

Page 102 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 1. FIELD TRIALS TERMINATION REPORTS 2005-2007TwinLink Cotton USDA Petition

Page 103 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 1 05-035-12n



## USDA Field Termination Report

USDA Notification Number: 05-035-12n

Applicant Internal Number: BT-5C-Cotton-MR

Applicant: Bayer CropScience LP Research Triangle Park, NC 27709 (919) 549-2655

Regulated Article: Gossypium hirsutum; plant incorporated protectant (PIP); tolerant to glufosinateammonium herbicide

Site Release Information : Trials utilizing this trait we re conducted at one location:

County / State Acreage

Planted Date

Planted Date

Terminated Isolation Method

Oktibbeha Co., MS 0.98 6/16/05 11/2/05 40 ft. isolation buffer

Purpose of Release : This trial was established to evaluate the performance of Plant Incorporated

Protectant traits in various cotton varieties.

Observations : Cooperator did not note any differences between transgenic and non-transgenic varieties other than effects of the PIP on bollworm populations. No other differences in beneficial insect populations, plant diseases, phenotypical or weediness characteristics were reported.

Plant Disposition : Plants were harvested on 11/2/05 to evaluate yield and were then destroyed in field. No seed was retained after the completion of the trial, and cooperator indicated that all seed provided was utilized in the creation of the plots.

Volunteer Monitoring : Volunteer monitoring has been discontinued due to the replant of regulated

cotton in the same tria l area on 5/19/06.

Weediness Characteristics : There was no indication of increased weediness characteristics in either the transgenic or non-transgenic varieties.

Non-target Organisms : There was no indication of any adverse effects to non-target insect populations or

beneficial insects. Various beneficial insect populations were present with lacewings, assassin bugs, lady beetles, and various coleopteran species

observed.TwinLink Cotton USDA Petition

Page 104 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Weather Synopsis : Cooperator reported that the growing season had above average moisture, with above average temperatures on average.

Containment Measures : Plot was contained ut ilizing a 40 ft. isolat ion border whic h encompassed this field

site.TwinLink Cotton USDA Petition

Page 105 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 2 07-059-104n



## USDA Field Termination Report

USDA Notification Number: 07-059-104n

Applicant Internal Number : IR-7J-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Lines Planted : GEM1

Regulated Article : Gossypium hirsutum ; cotton

Section I. Site Release Information:

Trials utilizing this trait were conducted at ten (10) locations:

County / State Acreage

Planted Date

Planted Date

Terminated 1 St. Landry / LA (1) 1.28 6/24/07 11/13/2007 2 St. Landry / LA (2) 1.28 6/24/07 11/13/2007 3 Tate / MS (1) 0.72 5/25/07 10/9/2007 4 Tate / MS (2) 0.80 5/16/07 11/02/2007 5 Hockley / TX (1) 0.96 6/4/07 11/15/2007 6 Hockley / TX (2) 0.96 6/5/07 11/29/2007 7 Uvalde / TX (1) 0.81 5/7/07 10/25/2007 8 Uvalde / TX (2) 2.40 5/7/07 10/26/2007 9 Wharton / TX (1) 1.10 6/5/07 11/1/2007 10 Wharton / TX (2) 3.47 6/8/07 11/16/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot DamageTwinLink Cotton USDA Petition

Page 106 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Section III. Summarized results from th e BCS regulatory In-Season Observation forms:

1. St. Landry Co., LA (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

2. St. Landry Co., LA (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

3. Tate Co., MS (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

4. Tate Co., MS (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment.TwinLink Cotton USDA Petition

Page 107 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

5. Hockley Co., TX (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

6. Hockley Co., TX (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

7. Uvalde Co., TX (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

8. Uvalde Co., TX (2) : a) Cooperator noted no unusual plant growth characteristics.TwinLink Cotton USDA Petition

Page 108 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

9. Wharton Co., TX (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

10. Wharton Co., TX (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects or the

environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 109 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 3 07-044-102n



## USDA Field Termination Report

USDA Notification Number : 07-044-102n

Applicant Internal Number : IR-7D-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Regulated Article : Gossypium hirsutum ; cotton

Lines: GEM1

Section I. Site Release Information:

Trials were conducted at eleven (11) locations:

County / State Acreage

Planted Date

Planted Date

Terminated

1 Franklin / LA 0.293 6/7/2007 11/16/2007

2 Pinal / AZ 0.363 5/31/2007 12/19/2007 3 Hidalgo / TX 0..333 4/17/2007 8/30/2007

4 Oktibbeha / MS 0.263 5/23/2007 11/5/2007

5 Madison / LA 1.50 5/12/07 11/5/2007

6 Washington / MS 2.10 5/17/07 &
5/29/07 11/5/2007

7 Halifax / NC 0.67 5/20/07 &
6/14/07 6/12/07 &
11/15/07

8 Dillon / SC 0.67 5/22/07 10/8/2007

9 Lubbock / TX 0.49 5/26/07 11/13/2007

10 Uvalde / TX 0.95 5/7/07 10/25/2007

11 Wharton / TX 1.02 5/16/07 11/2/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness CharacteristicsTwinLink Cotton USDA Petition

Page 110 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION



### f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:

1. Franklin Co., LA a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

2. Pinal Co., AZ : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

3. Hidalgo Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

4. Oktibbeeha Co., MS : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease.TwinLink Cotton USDA Petition

Page 111 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

5. Madison Co., LA : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

6. Washington Co., MS : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

7. Halifax Co., NC replant : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location.TwinLink Cotton USDA Petition

Page 112 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### g) This test was destroyed by an overspray of roundup on 6/12/2007 and replanted on 6/14/2007 the subsequent test was planted to late for reliable yi eld data so it was terminated without harvesting on 11/15/2007. Trial was successfully conducted without any environmental releases.

8. Dillon Co., SC : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

9. Lubbock Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

10. Uvalde Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

11. Wharton Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations.TwinLink Cotton USDA Petition

Page 113 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 114 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 4 07-044-103n



## USDA Field Termination Report

USDA Notification Number: 07-044-103n

Applicant Internal Number : IR-7E-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Regulated Article: Gossypium hirsutum; cotton

Lines: GEM2

Section I. Site Release Information:

Trials were conducted at seven (7) locations:

County / State Acreage

Planted Date

Planted Date

Terminated

1 Franklin / LA 0.293 6/7/2007 11/16/2007

2 Pinal / AZ 0.363 5/31/2007 12/19/2007 3 Hidalgo / TX 0.333 4/17/2007 8/30/2007

4 Oktibbeha / MS 0.263 5/23/2007 11/5/2007

5 Washington / MS (1) 0.10 5/17/07 &
5/29/07 11/5/2007

6 Washington / MS (2) 0.94 6/15/2007 11/01/2007

7 Lubbock / TX 0.17 5/26/07 11/13/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:TwinLink Cotton USDA Petition

Page 115 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 1. Franklin Co., LA a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

2. Pinal Co., AZ : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

3. Hidalgo Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

4. Oktibbeeha Co., MS : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots.TwinLink Cotton USDA Petition

Page 116 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

5. Washington Co., MS 1 : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

6. Washington Co., MS 2: a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

7. Lubbock Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

Section IV. Plant Disposition :TwinLink Cotton USDA Petition

Page 117 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 118 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 5 07-044-104n



## USDA Field Termination Report

USDA Notification Number: 07-044-104n

Applicant Internal Number : IR-7F-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Regulated Article: Gossypium hirsutum ; cotton

Lines: TwinLink

Section I. Site Release Information:

Trials were conducted at six (6) locations:

County / State Acreage

Planted Date

Planted Date

Terminated

1 Franklin / LA 0.293 6/7/2007 11/16/2007

2 Pinal / AZ 0.363 5/31/2007 12/19/2007 3 Hidalgo / TX 0.333 4/17/2007 8/30/2007

4 Oktibbeha / MS 0.263 5/23/2007 11/5/2007

5 Washington / MS 0.10 5/17/07 &
5/29/07 11/5/2007

6 Lubbock / TX 0.17 5/26/07 11/13/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:

1. Franklin Co., LATwinLink Cotton USDA Petition

Page 119 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

2. Pinal Co., AZ : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

3. Hidalgo Co., TX : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

4. Oktibbeeha Co., MS : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location.TwinLink Cotton USDA Petition

Page 120 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

5. Washington Co., MS : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

6. Lubbock Co., TX : h) Cooperator noted no unusual plant growth characteristics. i) No observed differences in plant disease. j) No unusual differences in insect pest or beneficial insect’s populations. k) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. l) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. m) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. n) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 121 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 6 07-065-117n



## USDA Field Termination Report

USDA Notification Number: 07-065-117n

Applicant Internal Number : IR-7K-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Lines Planted : GEM1 (T304-40)

Regulated Article: Gossypium hirsutum ; cotton

Section I. Site Release Information:

Trials utilizing this trait were conducted at one (1) location:

County / State Acreage

Planted Date

Planted Date

Terminated

1 Martin / NC 0.137 5/24/07 10/17/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:

1. Martin Co., NC : a) No observed differences between transgenic and non-transgenic agronomic growth. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment.TwinLink Cotton USDA Petition

Page 122 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) Early season Deer feeding controlled by electric fence (USDA letter 7/24/2007). Trial was successfully conducted without any unauthorized environmental releases or compliance incidents.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 123 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 7 07-065-118n



## USDA Field Termination Report

USDA Notification Number: 07-065-118n

Applicant Internal Number : IR-7L-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Lines Planted : GEM2 (GHB119)

Regulated Article: Gossypium hirsutum ; cotton

Section I. Site Release Information:

Trials utilizing this trait were conducted at one (1) location:

County / State Acreage

Planted Date

Planted Date

Terminated

1 Martin / NC 0.137 5/24/07 10/17/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:

1. Martin Co., NC : a) No observed differences between transgenic and non-transgenic agronomic growth. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment.TwinLink Cotton USDA Petition

Page 124 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) Early season Deer feeding controlled by electric fence (USDA letter 7/24/2007). Trial was successfully conducted without any unauthorized environmental releases or compliance incidents.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 125 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 8 07-065-119n



## USDA Field Termination Report

USDA Notification Number: 07-065-119n

Applicant Internal Number : IR-7M-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Lines Planted: TwinLink (GHB119/T304-40)

Regulated Article: Gossypium hirsutum; cotton

Section I. Site Release Information:

Trials utilizing this trait were conducted at one (1) location:

County / State Acreage

Planted Date

Planted Date

Terminated

1 Martin / NC 0.137 5/24/07 10/17/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:

1. Martin Co., NC : a) No observed differences between transgenic and non-transgenic agronomic growth. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment.TwinLink Cotton USDA Petition

Page 126 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) Early season Deer feeding controlled by electric fence (USDA letter 7/24/2007). Trial was successfully conducted without any unauthorized environmental releases or compliance incidents.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 127 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Termination Report 9 07-059-101n



## USDA Field Termination Report

USDA Notification Number: 07-059-101n

Applicant Internal Number : IR-7I-GH-MR

Applicant : Bayer CropScience LP Research Triangle Park, NC 27709

Lines Planted: TwinLink

Regulated Article: Gossypium hirsutum ; cotton

Section I. Site Release Information:

Trials utilizing this trait were conducted at nine (9) locations:

County / State Acreage

Planted Date

Planted Date

Terminated 1 Crittenden / AR (1) 0.29 6/4/07 10/20/2007 2 Crittenden / AR (2) 0.29 6/4/07 10/20/2007

3 Jackson / AR 0..29 6/5/07 11/14/2007 4 Tift / GA (1) 0.29 6/13/07 11/30/2007 5 Tift / GA (2) 0.28 6/15/07 11/29/2007

6 Tate / MS 0.20 6/5/07 10/21/2007 7 Hockley / TX (1) 2.02 6/9/07 12/7/2007 8 Hockley / TX (2) 0.27 6/4/07 11/16/2007

9 Wharton / TX 0.29 6/11/07 8/14/2007

Section II. List of characteristic observations between the transgenic & non-transgenic plants, insects and

environment:

a Plant Growth Characteristics

b Disease Differences c Insect Populations d Adverse Effects on Non-Target Insects or Environment

e Weediness Characteristics f Weather Synopsis g Plot Damage

Section III. Summarized results from th e BCS regulatory In-Season Observation forms:TwinLink Cotton USDA Petition

Page 128 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 1. Crittenden Co., AR (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

2. Crittenden Co., AR (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

3. Jackson Co., AR : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

4. Tift Co., GA (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots.TwinLink Cotton USDA Petition

Page 129 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

5. Tift Co., GA (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

6. Tate Co., MS : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

7. Hockley Co., TX (1) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations. d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

8. Hockley Co., TX (2) : a) Cooperator noted no unusual plant growth characteristics. b) No observed differences in plant disease. c) No unusual differences in insect pest or beneficial insect’s populations.TwinLink Cotton USDA Petition

Page 130 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### d) There was no indication of any adverse effects to non-target insect populations, beneficial insects

or the environment. e) There were no indications of increased weediness characteristics in either the transgenic or nontransgenic plots. f) Weather was reported as normal, with expected variations in rainfall and temperatures during the growing season for this location. g) No plot damage occurred. Trial was successfully conducted without any environmental releases or compliance incidents.

9. Wharton Co., TX : This test was terminated early, August 14, 2007 following flood damage. The potential compliance incident was reported to USDA/BRS and resolved. Volunteer monitoring is scheduled to end September 2008.

Section IV. Plant Disposition :

All plot locations were harvested and all remaining harvested material that was not sent to a Bayer CropScience Facility or other Laboratories for analysis were destroyed. Unplanted seed which was not utilized by the cooperator was either returned to Bayer for storage or destroyed by deep burial within the plot.

Section V. Volunteer Monitoring :

Volunteer monitoring is ongoing and is scheduled for completion one year from harvest.TwinLink Cotton USDA Petition

Page 131 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 2. MATERIALS AND ME THODS - PRODUCT CHARACTERIZATIONTwinLink Cotton USDA Petition

Page 132 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Breeding diagram for each parent of TwinLink cotton used in agronomic

evaluationTwinLink Cotton USDA Petition

Page 133 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for molecu lar characterization – DNA tests

Materials DNA for the analyses was isolated from leaves of T304-40, GHB119, TwinLink, and the conventional control produced in the greenhouse. The references included the plasmids (pTDL008 for T304-40 and pTEM12 for GHB119) that were used in their respective transformations. For Southern blot analysis of cotton genomic DNA, digested plasmid DNA (approximately 0.1, 1, 10 genomic copies equ ivalent) was mixed with genomic DNA of the conventional control and separated by electrophoresis on agarose gels. Standard DNA molecular weight markers, such as phage Lambda DNA digested with Pst I and/or Eco RI and/or

Hind III were used for size estimation of the DNA fragments.

Identity of the materials The identity of the greenhouse produced material was confirmed by PCR analysis prior to use, to confirm the presence or absence of T304-40 or GHB119, as appropriate. In the case of segregating seedlots, the zygosity of the harvested plants was determined by means of zPCR. The integrity of the isolated DNA was verified in each Southern analysis by observation of the DNA samples on an ethidium bromide stained agarose gel. The identity of the materials used in generational stability analyses was confirmed by chain-of-custody documents and by PCR analysis.

DNA preparation for Southern blot and PCR analyses Harvested plant tissues were directly transferred and frozen in liquid nitrogen, then stored in an ultrafreezer until DNA preparation. Leaf material, stored in the ultrafreezer, is stable for at least 10 years. Genomic DNA was extracted following standard procedures, and stored at 4°C. Plasmid DNA was prepared from an E.coli cell strain containing plasmid pTDL008 or pTEM12, as appropriate. Concentration of the different DNA preparations was determined by spectrophotometry using a Nanodrop ND-8000, or by fluorescence using the Quant-iTTM PicoGreen® dsDNA Reagent.

Approximately 10 μg of genomic DNA was digested with restriction enzymes (see Chapter IV) following the procedure indicated by the manufacturer. Digestions took place in a total reaction volume of 50 μl, and the digests were incubated overnight at 37°C.

Probe template DNA was prepared either by means of PCR amplification using the Taq DNA polymerase or the Expand enzyme, following standard procedures. Probe templates were [ α

32 

### P] labeled.

Southern blot analysis of genomic DNA. Digested genomic DNA samples were loaded on 1% TAE agarose gels and separated based on size, following standard procedures (Sambrook et al ., 1989).

An appropriate dilution of the restriction enzyme digested plasmid DNA was prepared. With a single copy integration of the transgene into the Gossypium hirsutum genome, ten μg of genomic heterozygous DNA would correspond to ca. 32 pg of pTDL008, and ca. 27 pg of pTEM12 plasmid DNA [ Gossypium hirsutum genome size: 4.5x10 9 

### bp (Arumuganathan and Earle, 1991); pTDL008 = 14393 bp; pTEM12 = 12266 bp]. The amount representing approximately 0.1, 1, 10 plasmid copies per genome was added to 10 μg of digested non-TwinLink Cotton USDA Petition

Page 134 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### transgenic DNA. This reconstitution sample served as a positive control and was used to show that the hybridizations were performed under conditions allowing hybridization of the probe with target sequences. Standard DNA molecular weight markers, such as phage Lambda DNA digested with Pst I and/or Eco RI and/or Hind III were used for size estimation of the DNA fragments.

PCR preparation of DNA probes The DNA templates used for probe preparation were synthesized by means of polymerase chain reaction (PCR) amplification, using the ExpandTM High Fidelity PCR system (Boehringer Mannheim). Five hundred pg of target DNA were mixed with 10 pmoles of each primer, 200 μM of each deoxyribonucleoside triphosphate, 5 μl expand high fidelity Buffer 2 and 2.6 Units Expand High Fidelity polymerase enzyme in a 50 μl PCR reaction. The amplification of the different products was performed under the following conditions: 95°C for 4 minutes, 5 cycles at 94°C for 1 minute, 57°C for 1 minute, 72°C for 2 minutes, 25 cycles at 94°C for 15 seconds, 60°C for 45 seconds, 72°C for 2 minutes, and 1 cycle at 72°C for 10 minutes. Aliquots of each product were separated on 1% (w/v) agarose gel in 1X TAE buffer and visualized by ethidium bromide staining to verify that the amplified fragments were of the expected size.

The DNA templates were labeled using the ‘Ready-to-go DNA labeling system’ from GE Healthcare. Unincorporated nucleotides were removed by separation on a micro Bio-Spin-30

column from Bio-Rad.TwinLink Cotton USDA Petition

Page 135 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for pr otein characterization tests

Studies on potential toxicology and allergeni city for food, feed and the environment are conducted with Cry1Ab, Cry2Ae and PAT/ bar proteins expressed in Bacillus thuringiensis (Bt.)

and E. coli . In order to utilize the safety data of the protein produced in a microorganism for the safety assessment of the same protein produced in a genetically modified plant, it is important to confirm that the protein produced in a microorganism is representative of the protein produced in the modified plant.. Six analytical tests show that the proteins produced in Bt . and E. coli are representative of Cry1Ab, Cry2Ae and PAT/ bar proteins produced in TwinLink cotton.

Materials The plant-produced Cry1Ab, Cry2Ae and PAT/ barproteins were isolated from greenhousegrown TwinLink plants. The identity of the plants was confirmed by PCR. Leaf extract was purified on an antibody affinity column, and the purified protein solution was stored at -10 C or lower until further analyses were performed. The antibody affinity columns used for purification were purchased from Pierce (Rockford, IL, product number 44894), and prepared using a covalently attached monoclonal antibodies specific for each protein.

The Cry1Ab (BCS reference standard, Batch No. MIN1443, purity 98.59%) and PAT/ bar (BCS reference standard, Batch N° NB010905P44P2, purity 65.82%) protein reference standards were produced in E. coli . The Cry2Ae protein reference standard (BCS reference standard, Batch No. NB170907, purity 94.37%) was produced in Bacillus thuringiensis . The protein

solutions are stored in an Ultrafreezer.

Analysis by N-terminal sequencing The affinity purified Cry2Ae and PAT/ bar proteins isolated from TwinLink cotton leaves and the bacterial proteins were loaded onto PVDF membranes and sent to Eurosequence bv (Groningen, the Netherlands) for analysis of the N-terminal amino acid sequence of the proteinsby Edman degradation. Prior to analysis, the samples were reduced and alkylated using Nisopropyliodoacetamide.

For an N-terminal sequence the concentration of the Cry1Ab protein isolated from TwinLink cotton leaves was too low for N-terminal sequencing. However, the N-terminal peptide for the Cry1Ab protein isolated from TwinLink was detected in the selected ion mass spectrometry analysis of the protein, indicating that the protein is not modified.

Analysis by SDS-PAGE The Cry1Ab, Cry2Ae and PAT/ bar proteins from E. coli and Bt. and the Cry1Ab, Cry2Ae and

PAT/ bar proteins isolated from TwinLink cotton leaves were analyzed by SDS-PAGE. The proteins from the plant and the corresponding protein from E. coli and Bt. were denatured and analyzed by electrophoresis on a denaturing polyacrylamide gel where mobility can be correlated to molecular weight. SDS-PAGE was performed using an Invitrogen Bis-Tris 10% polyacrylamide gel (InVitrogen, CA, product number NP0301) and a MOPS SDS running buffer according to the manufacturer’s instructions. Molecular weight markers on the gel are comprised of a series of recombinant proteins of known molecular weight. The gel was then stained with Pierce Imperial Stain to visualize the protein bands.TwinLink Cotton USDA Petition

Page 136 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Analysis by western blotting Western blotting was performed in the same electrophoresis system as used for SDS-PAGE and the gel was blotted to PVDF membranes (New England Nuclear, MA, product number NEF1001) according to the instructions provided by InVitrogen. The proteins in the gel were transferred out of the gel perpendicular to the direction of the first electrophoresis. They were adsorbed to the membrane giving an exact replica of the positions of all the proteins in the gel. The membrane was then exposed to a monoclonal antibody to the subject protein, and through a series of additional steps a luminescent tag was attached to the bound antibody to reveal the position of the protein of interest. The second antibody was a horseradish peroxidase (HRP) linked anti-mouse antibody. All reagents except the monoclonal primary antibodies used for western blotting were obtained from GE Healthcare (NJ) as an ECL Plus luminescent detection kit (product number RPN 2108). Kaleidoscope molecular weight markers from GE Healthcare

were used.

Analysis by HPLC/Electrospray Mass Spectrometry The Cry1Ab, Cry2Ae and PAT/ bar proteins from Bt. and E. coli and the Cry1Ab, Cry2Ae and

PAT/ bar proteins isolated from TwinLink cotton leaves were denatured for 1 hour at 37 °C in Rapid Gest (Waters Corporation, 1mg/mL) containing 10 mM dithiothreitol and digested with trypsin for 1 hour at 37°C. A selected ion monitoring method was developed for Cry1Ab and Cry2Ae using the peptides generated from the tryp sin digest of the Cry1Ab and Cry2Ae proteins

from E. coli. and Bt. A method for PAT/bar was developed earlier and this was used for the analysis of the PAT/bar protein. A full scan mass spectrum was generated for the Cry1Ab and Cry2Ae peptides that were detected in the trypsin digest of the Cry1Ab and Cry2Ae proteins

from E. coli. and Bt. The most abundant ions indicative of each peptide were used for selected ion monitoring (SIM) of the peptides in the sample. The peptides from the Cry1Ab E. coli.

, Cry2Ae Bt. and PAT/ bar E. coli standard trypsin digest and peptides from the the trypsin digest of the Cry1Ab, Cry2Ae and PAT/ bar proteins isolated from TwinLink cotton leaves were analyzed using the selected ion methods. The presence of a peak for the peptide in the selected ion monitoring chromatogram of the sample at a retention time detected for a peak of the same peptide in the trypsin digest of the Cry1Ab from E. coli., Cry2Ae from Bt. and PAT/ bar

from E. coli standards confirms the presence of that peptide in the trypsin digest sample of the Cry1Ab, Cry2Ae and PAT/ bar proteins isolated from TwinLink cotton. The limit of detection of a peak is defined as approximately three times signal to noise.

Glycoprotein staining analysis The Cry1Ab, Cry2Ae and PAT/ bar proteins from E. coli and Bt. and the Cry1Ab, Cry2Ae and

PAT/ bar proteins isolated from TwinLink cotton l eaves were analyzed by SDS-PAGE. A set of glycoprotein molecular weight standards was included on the gel. This set of marker proteins forms an alternating ladder of glycosylated and non-glycosylated proteins. Glyco-Staining was performed using a Sigma GlycoProfileTM III Fluorescent Glycoprotein Detection Kit (Sigma, cat # PP0300) according to the manufacturers instructions.

Analysis of enzymatic activity The enzymatic activity of the PAT/ bar protein isolated from TwinLink cotton leaves was determined according to D’Halluin et al . (1995). The method measures the activity of PAT/ bar by the generation of free Coenzyme A sulphydryl groups during the transfer of the acetyl group of Acetyl Coenzyme A to phosphinothricin. The reaction of the reduced Coenzyme A with 5,5- dithiobis(2-nitrobenzoic acid) produces 5-thio-2-nitrobenzoic acid which has an absorption atTwinLink Cotton USDA Petition

Page 137 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 412 nm. The detection of an absorbance at 412 nm indicates that the PAT/ bar protein is active.

The activity of the Cry2Ae protein was determined from an insect assay. The Cry2Ae protein isolated from TwinLink cotton and the Cry2Ae protein from Bt. were added to Lepidoptera diet

and fed to Helicoverpa zea larvae, at the first larvae stage. The Cry1Ab protein could not be isolated from TwinLink cotton leaves in enough quantity to perform an insect assay.TwinLink Cotton USDA Petition

Page 138 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for protein levels in seeds

Field design TwinLink cotton plants containing events T304-40 and GHB119, along with cotton plants representing the non-transgenic (non-transformed) counterpart Coker 315 were field tested by Bayer CropScience in 2007 under USDA APHIS Notification 07-059-101n. Field trials were established in EPA Regions II, IV, and VIII in the following locations: Trial number 02-01-Tift County, Georgia; Trial number 02-02-Tift County, Georgia; Trial 04-03-Jackson County Arkansas; Trial number 04-04-Crittenden County, Arkansas; Trial number 04-06-Tate County, Mississippi; Trial number 08-08-Hockley County, Tex as. These areas are typical cotton growing regions of the southeastern United States, and the plants in this study were grown underconditions typical of production practices. There were six transgenic plots and three nontransgenic plots at each test site. The test plots were randomized at each field location. Seed for planting was supplied for each field trial by Bayer CropScience.

Three of the transgenic event plots in each field trial were sprayed two times with Ignite® 280 SL glufosinate-ammonium herbicide, and the other plots were untreated. The Ignite herbicide contained a labeled concentration of 2.34 pounds per U.S. gallon of glufosinate-ammonium active ingredient (280 grams of active ingredient per liter). Each application of Ignite herbicide was made at a target rate of 29 fluid ounces of product per acre (2.1 liters per hectare), equivalent to 0.53 lb ai/acre (0.60 kg ai/hectare).

Two replicate samples of ginned cottonseed (fuzzy seed) were obtained from each test plot. The samples were shipped frozen to the BioAnalytics laboratories of Bayer CropScience. Shipping and storage of the regulated seed was carried out under applicable USDA regulations and Bayer CropScience guidelines.

Certificates of Analysis (COA) were produced by Bayer CropScience for the two seed lots used for planting in this study. Identity and purity of the TwinLink cottonseed and the corresponding non-transgenic (Coker 315) cottonseed were confirmed to be acceptable for use.

Sample preparation A CoA to determine identity of the samples harvested from the seven field sites was prepared by Bayer CropScience. The data certified that the transgenic samples harvested from the fieldwere indeed TwinLink cotton, and that the non-transgenic samples were harvested from nontransgenic plots.

Fuzzy seed was difficult to grind to homogeneity with dry ice due to the residual lint adhering to the seed. Gently grinding the fuzzy seed in a mortar and pestle in the presence of liquid nitrogen produced a relatively clean seed fraction (designated as a kernel sample) and a sample of short strands of cotton lint and seed coat material, identified as lint coat sample. The weights of the kernel and lint coat fractions were recorded for each sample of fuzzy seed that was analyzed in order to reconstruct the amount of protein analyte in the fuzzy seed as it was

received from the field.

Each of the frozen kernel samples was ground in a Waring Laboratory Blender prechilled with dry ice, adding dry ice as necessary to ensure the samples remained frozen during preparation.TwinLink Cotton USDA Petition

Page 139 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### In between samples, the blender was washed with soapy water using a brush, rinsed twice with hot water and twice with deionized water, and dried with an air stream. The ground samples were stored in a freezer at approximately -20oC for overnight or longer to allow the dry ice to dissipate before extraction. The lint coat samples were not ground in dry ice. They were used directly in all the assays.

Protein extraction The Cry1Ab, Cry2Ae, and PAT/bar proteins were extracted from raw agricultural products of cotton using a buffer described by Xin et al ., (1988). A representative portion (approximately 0.1 g) of ground sample was mixed with an appropri ate amount of extraction buffer (approximately 20 mL) in a 50 mL polypropylene centrifuge tube, shaken for 15 minutes at ~ 4°C on a shaker (IKA-SCHÜTTLER MTS 4) at 250 rpm and then centrifuged at approximately 4100 x g for 5 minutes at ~ 4°C. The supernatant was transferred to a clean centrifuge tube for another cycle of centrifugation at approximately 18000 x g for 5 minutes at ~ 4°C. The clear supernatant was then used for the protein and TEP analyses. Duplicate extracts were prepared for each sample.

Bioassay All quantitative determinations of the Cry1Ab, Cry2Ae, and PAT/ bar proteins were conducted at Bayer CropScience, Research Triangle Park, NC. A commercial ELISA (EnviroLogix, MA) specific for each protein was used to measure the amounts of Cry1Ab, Cry2Ae, and PAT/bar. Each ELISA was validated in kernel and lint coat matrices for the each protein.

Validation A validation was performed for Cry1Ab, Cry2Ae, and PAT/ bar proteins separately, using non-transgenic kernel and lint coat samples fortified at the concentrations listed in Tables 2.1 through 2.4. The standards were added to the extraction buffer at the indicated concentrations prior to extraction in 5 replicates. Each replicate was analyzed using duplicate wells. A summary of the validation data is shown for kernel and lint coat matrices in Table 2.1.TwinLink Cotton USDA Petition

Page 140 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

### Table 2.1 Validation of Sample Extraction for the Cry1Ab ELISA with Fortified Nontransgenic Controls of Kernel and Lint Coat

Sample

ID Validation of Cotton Seed Kernel
for Cry1Ab
(Field Sample ID: 02-01; BTID: 1243A) Validation of Lint Coat
for Cry1Ab
(Field Sample ID: 02-01; BTID: 1243A)

Cry1Ab

Fortified (ng/mL) Cry1Ab

Detected
(ng/mL) a

Mean ± SD % Cry1Ab Recovery

Mean ± SD Cry1Ab Recovery

% CV Cry1Ab

Detected
(ng/mL)

Mean ± SD % Cry1Ab
Recovery

Mean ± SD Cry1Ab
Recovery

% CV

100 121 ± 17.6 121 ± 17.6 14.6 79. 5 ± 5.52 79.5 ± 5.52 6.94

60 50.1 ± 3.47 83.5 ± 5.78 6.92 57.3 ± 3. 79 95.4 ± 6.32 6.63

5 5.19 ± 0.630 104 ± 12.6 12.2 4.42 ± 0.169 88. 4 ± 3.37 3.81

2.5 2.86 ± 0.381 114 ± 15.2 13.3 1.80 ± 0. 069 72.1 ± 2.76 3.82

1.25 1.54 ± 0.126 123 ± 10.1 8.23 1.15 ± 0. 038 92.3 ± 3.08 3.34

0.645 0.771 ± 0.086 120 ± 13.4 11.2 0.556 ± 0. 121 88.9 ± 19.3 21.8

a The Cry1Ab protein detected and its recovery are ex pressed as the average of 10 data points from

duplicate extracts of 5 samples at each fortif ication level using non-transgenic matrix.



### Table 2.2 Validation of Sample Extraction for the Cry2Ae ELISA with Fortified Nontransgenic Controls of Kernel and Lint Coat

Sample

ID Validation of Cotton Seed Kernel
for Cry2Ae
(Field Sample ID: 02-01; BTID: 1243A) Validation of Lint Coat
for Cry2Ae
(Field Sample ID: 02-01; BTID: 1243A)

Cry2Ae

Fortified (ng/mL) Cry2Ae

Detected
(ng/mL) a

Mean ± SD % Cry2Ae Recovery

Mean ± SD Cry2Ae
Recovery

% CV Cry2Ae

Detected
(ng/mL) a

Mean ± SD % Cry2Ae Recovery

Mean ± SD Cry2Ae
Recovery

% CV

100 65.0 ± 5.47 65.0 ± 5.47 8.41 99.2 ± 2. 45 99.2 ± 2.45 2.47

30 23.8 ± 3.86 79.3 ± 12.9 16.2 27.6 ± 0. 47 92.0 ± 1.56 1.69

15 15.9 ± 1.60 106 ± 10.6 10.0 12.3 ± 0.69 81.9 ± 4.61 5.63

10 10.2 ± 0.560 102 ± 5.63 5.53 8.00 ± 0.55 80.0 ± 5.47 6.83

7.5 6.66 ± 0.190 88.7 ± 2.56 2.88 6.74 ± 0. 33 89.9 ± 4.34 4.83

3.75 3.30 ± 0.160 88.0 ± 4.26 4.84 3.58 ± 0. 35 95.4 ± 9.43 9.88

1.875 1.85 ± 0.120 98.7 ± 6.21 6.29 1.97 ± 0.17 105 ± 9.07 8.65

0.938 0.980 ± 0.250 105 ± 26.9 25. 7 1.02 ± 0.06 109 ±6.87 6.32

0.469 0.700 ± 0.120 149 ± 25.4 17. 0 0.82 ± 0.26 175 ± 55.7 31.8

a The Cry2Ae protein detected and its recovery are ex pressed as the average of 10 data points from

duplicate extracts of 5 samples at each fortif ication level using non-transgenic matrix.TwinLink Cotton USDA Petition

Page 141 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.3 Validation of Sample Extraction for the PAT/ barELISA with Fortified Nontransgenic Controls of Kernel and Lint Coat

Sample

ID Validation of Cotton Seed Kernel

for PAT/bar
(Field Sample ID: 02-01; BTID: 1243A) Validation of Lint Coat

for PAT/bar
(Field Sample ID: 02-01; BTID: 1243A)

PAT/bar

Fortified (ng/mL) PAT/bar

Detected (ng/mL) a

Mean ± SD % PAT/bar Recovery

Mean ± SD PAT/bar
Recovery

% CV PAT/bar

Detected
(ng/mL)

Mean ± SD % PAT/bar
Recovery

Mean ± SD PAT/bar Recovery

% CV

1000 65.0 ± 5.47 65.0 ± 5.47 8. 41 99.2 ± 2.45 99.2 ± 2.45 2.47

7.5 23.8 ± 3.86 79.3 ± 12.9 16.2 27.6 ± 0. 47 92.0 ± 1.56 1.69

3.75 15.9 ± 1.60 106 ± 10.6 10.0 12.3 ± 0. 69 81.9 ± 4.61 5.63

1.875 10.2 ± 0.560 102 ± 5.63 5.53 8.00 ± 0. 55 80.0 ± 5.47 6.83

0.938 6.66 ± 0.190 88.7 ± 2.56 2.88 6.74 ± 0. 33 89.9 ± 4.34 4.83

0.469 0.700 ± 0.120 149 ± 25.4 17. 0 0.82 ± 0.26 175 ± 55.7 31.8

a The PAT/ bar protein detected and its recovery are expr essed as the average of 10 data points from duplicate extracts of 5 samples at each fortif ication level using non-transgenic matrix.



### Limit of detection and limit of quantification The limit of detection (LOD) is determined for each matrix using the average standard curve and the concentration derived from the background optical density (OD) of the negative control samples. The LOD is the concentration corresponding to an OD value three standard deviations above the mean background OD.

The limit of detection is expressed in the unit of concentration (ng/mL) and the unit of weight ratio (ng/g matrix, i.e. ppb) calculated based on the extraction of an amount of the matrix with a known volume of extraction buffer, e.g. , 1 g of matrix/20ml extraction buffer. The data are summarized in Table 2.4. An absorbance reading giving rise to a protein analyte concentration above this limit of detection level is assumed to be greater than the zero dose reading.

The limit of quantification (LOQ) is defined as the lowest concentration of the standard that meets the validity criteria for the LOQ. Validity criteria are a) analyte recoveries from fortified matrix samples are > 60 % and < 130 % and b) the coefficient of variance (relative standard deviation) is less than 25%. When a lower recove ry is caused by the nature of a specific matrix or the effect of a process, the lowest concentration of the standard that gives a coefficient of variance equal to or less than 25% is used as the LOQ. Values below the LOD are reported as ND (Non-detectable) and values below the LOQ but above the LOD are reported as ‘<LOQ’.
The calculated LOQs are summarized in Table 2.4 below.TwinLink Cotton USDA Petition

Page 142 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.4 Limits of Detection and Quantitation of Cry1Ab, Cry2Ae and PAT/ bar Protein in Raw Agricultural Commodities of TwinLink Cotton as Detected by ELISA.

LOD LOQ

(ng/mL) ng/g a Sample (ng/mL) ng/g a Sample

Cry1Ab Kernel 0.0990 19.8 0.645 129

Cry1Ab Lint Coat 0.0660 13.2 0.645 129

Cry2Ae Kernel 0.463 92.6 1.88 375

Cry2Ae Lint Coat 0.503 101 0.938 188

PAT/ bar Kernel 0.148 29.6 0.938 188

PAT/ bar Lint Coat 0.220 44.0 1.88 375

a Calculated based on the extraction of 0.1 g matrix per 20 mL of extraction buffer fo r kernel and lint coat.



### Protein determination Protein determinations were made in order to confirm that protein was extracted from the samples. The Bradford assay (Bradford, 1976; Sedmak, 1977) was used to determine the concentration of total extractable protein (TEP). The assay relies on the binding of the dye Coomassie blue G250 to protein. The anionic form of the dye, which binds to protein, has a maximum absorption at 595 nm. The amount of absorption at 595 nm produced is therefore correlated to the protein concentration. Bovine Serum Albumin (BSA) was used as protein standard at 0.05, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 mg/mL in the assay.

The total extractable protein was determined for each sample extract. Duplicate 10μL aliquots of the sample extract were placed in wells of a 96-well plate (Costar No. 3590) and 200 μL of Bradford Reagent (Sigma-Aldrich Chemical Company, Product number: B-6916) was added. After 12 ± 3 minutes of incubation on a shaker (IKA-SCHÜTTLER MTS 4) at 700 rpm at room temperature, the optical density (OD) was measured in a microplate reader (Molecular Devices THERMOmax) at 595 nm.

Protein analyte content SoftMax ProTM software (Molecular Devices, Vers ion 4.0) was used to derive the concentration of the Cry1Ab, Cry2Ae, and PAT/ bar proteins. Absorbance units were adjusted for the buffer blank and then any background due to the matrix was subtracted, using values from wells containing non-transgenic extracts, assayed on the same plate. This correction was used for all samples except the transgenic seed as discussed in the following paragraph. The absorbance readings corrected for both buffer blank and non-transgenic background were converted to the protein concentration using the standard curve.

A set of wells containing samples of the corresponding non-transgenic matrix was always included on a plate for background subtraction. The appropriate background corrections for the transgenic kernel samples were obtained from background values of a non-transgenic kernel sample (BTID 1243A), which was diluted on the same plate and to the same extent as the transgenic kernel sample. Thus the dilution of the non-transgenic sample used for background subtraction was the same as the dilution of the transgenic sample that was required to place theTwinLink Cotton USDA Petition

Page 143 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### OD reading in the center portion of the standard curve.

The absorbance assays give results in units of ng of analyte per milliliter of extract that are then converted into ng or μg of analyte per gram of fresh sample. As different tissues have different protein and water contents, the results are also expressed as percent of Crude Protein and as percent of Total Extractable Protein (TEP). Samples were analyzed for crude protein and moisture at Eurofins and for TEP at the BioAnalytics Laboratories, BCS, NC. The calculations

and conversions were done as described below.

The values of percent crude protein on both a fresh weight and dry weight basis were used to calculate the Cry1Ab, Cry2Ae, and PAT/ bar protein concentration as percent of crude protein. The following is the conversion formula used:



### matrix g FW/ Protein Crude % sample g Protein/ Target μg Average Protein Crude % as Protein Target Average = x 10 -2



### The values of analyte protein as percent of TEP were calculated using the values generated by ELISA assay [Average analyte protein (μg/g sample)] and the values generated by the Bradford Assay [Average TEP (mg/g sample)]. The following is the conversion formula used:



### sample g / TEP mg Average sample g / analyte μg Average Protein e Extractabl Total % as Analyte Average = x 10 -1



### Protein content (Bradford) μg SoftMax ProTM software (Molecular Devices, Vers ion 4.0) was used to derive the concentration of protein from the Bradford assay (Bradford, 1976; Sedmak, 1977). The optical density was converted to the TEP concentration using the standard curve. The data point for the dilution falling nearest the center of the standard curve was used. If two points were near the center of the curve, the data for the least diluted sample was used.

Statistical analysis Descriptive statistics (mean, standard deviation, and coefficient of variance) were calculated for each sample matrix and treatment (Devore and Peck, 1986). An analysis of variance (ANOVA) was performed on the data for each subject protein content in kernel and lint coat fractions at a significance level of 0.05 ( α = 5%). Independent variables evaluated were the site, treatment, extract and assay. The null hypothesis states that there are no differences between the values of analyte protein content (dependent variable) due to the independent variables. A small probability (p-value) means that an observed difference is unlikely to occur by chance, so the null hypothesis should be rejected. A low p-value (< 0.05) suggests that there is a significant difference caused by the effect analyzed. StatView® 5 (SAS Institute, Cary, NC) was used for ANOVA. BCS conducted all statistical analyses. All statistical analyses were done on data with full precision. Results may be rounded to two or three significant numbers.

Results

Please refer to tablesTwinLink Cotton USDA Petition

Page 144 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.5 Fresh Weight average quantities of Cry1Ab protein in raw agricultural commodities of TwinLink cotton as detected by ELISA

Sample Trial

Number a Average Cry1Ab content (μg/g sample) ± SD b Average Cry1Ab content (as % of total crude protein) ± SD

No Glufosinate Glufosinate

Treated No Glufosinate Glufosinate

Treated

Kernel B02-01 2.47 ± 0.326 2.49 ± 0.270 NA NA

B02-02 2.65 ± 0.396 3.33 ± 0.761 NA NA

B04-03 0.467 ± 0.0475 2.77 ± 0.346 NA NA

B04-04 1.87 ± 0.0357 1.91 ± 0.610 NA NA

B04-05 2.27 ± 0.317 2.22 ± 0.150 NA NA

B04-06 3.45 ± 0.0306 3.70 ± 0.878 NA NA

B08-08 3.09 ± 0.587 2.94 ± 0.995 NA NA

Average NA 2.32 ± 0.8 44 2.77 ± 1.71 NA NA

Lint Coat B02-01 0 0 NA NA

B02-02 0 0 NA NA

B04-03 0 0 NA NA

B04-04 0 0 NA NA

B04-05 0 0 NA NA

B04-06 0 0 NA NA

B08-08 0 0.0565 ± 0.0978 NA NA

Average NA 0 ± 0 0.00807 ± 0.0978 NA NA

Fuzzy Seed B02-01 1.19 1.22 0.000598 0.000609

B02-02 1.28 1.62 0.000676 0.000817

B04-03 0.226 1.38 0.000147 0.000871

B04-04 0.956 0.962 0.000455 0.000455

B04-05 1.17 1.09 0.000597 0.000591

B04-06 1.64 1.77 0.000751 0.000833

B08-08 1.64 1.60 0.000704 0.000681

Range in Values NA 0.226 – 1.64 0.962 – 1.77 0.00047 –

0.000751 0.000455 –

0.000871

Average ± SD NA 1.16 ± 0.482 1.38 ± 0.301 0.00056 ±

0.000206 0.000694 ±

0.000153

a Standard Deviation was not calculated for individual fuzzy seed data because the value is the weighted

numerical sum of the average kernel and average lint coat values. A standard deviation was calculated for the average Cry1Ab value of fuzzy seed. This is based only on the calculated average values (kernel + lint coat) obtained at t he seven sites. Standard deviations for the individual sites were based on 12 measurements (two sample extracts assayed in duplicate from three replicate plots). Standard deviations for the averages of kernel and lint c oat are based on 84 actual measurements (2 sample extracts assayed in duplicate from 3 replicate plots at seven sites). The data for the fuzzy seed were calculated from the amount of Cry1Ab protein present in kernel and lint coat fractions taking into account their respective weights.

b Average Cry1Ab as % of crude protein is not app licable (NA) because crude protein determinations

were not made on the individual lint coat and ke rnel; but only on the entire fuzzy seed samples.TwinLink Cotton USDA Petition

Page 145 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.6 Dry Weight average quantities of Cry1Ab protein in raw agricultural commodities of TwinLink cotton as detected by ELISA

Sample Trial

Number a Average Cry1Ab content (μg/g sample) ± SD b Average Cry1Ab content (as % of total crude protein) ± SD

No Glufosinate Glufosinate

Treated No Glufosinate Glufosinate

Treated

Kernel B02-01 NA NA NA NA

B02-02 NA NA NA NA

B04-03 NA NA NA NA

B04-04 NA NA NA NA

B04-05 NA NA NA NA

B04-06 NA NA NA NA

B08-08 NA NA NA NA

Average NA NA NA NA NA

Lint Coat B02-01 NA NA NA NA

B02-02 NA NA NA NA

B04-03 NA NA NA NA

B04-04 NA NA NA NA

B04-05 NA NA NA NA

B04-06 NA NA NA NA

B08-08 NA NA NA NA

Average NA NA NA NA NA

Fuzzy Seed B02-01 1.34 1.37 0.000598 0.000609

B02-02 1.46 1.84 0.000676 0.000817

B04-03 0.258 1.56 0.000147 0.000871

B04-04 1.08 1.09 0.000455 0.000455

B04-05 1.33 1.25 0.000597 0.000591

B04-06 1.81 1.96 0.000751 0.000833

B08-08 1.74 1.70 0.000704 0.000681

Range in Values NA 0.258 – 1.81 1.09 – 1.96 0.000147 –

0.000751 0.000455 –

0.000871

Average ± SD NA 1.29 ± 0.520 1.54 ± 0.318 0.000561 ±

0.000206 0.000694 ±

0.000153

a Standard Deviation was not calculated for individual fuzzy seed data because the value is the weighted

numerical sum of the average kernel and average lint coat values. A standard deviation was calculated for the average Cry1Ab value of fuzzy seed. This is based only on the calculated average values (kernel + lint coat) obtained at the seven sites. Standard deviations for the individual sites were based on 12 measurements (two sample extracts assayed in duplicate from three replicate plots). Standard deviations for the averages of kernel and lint c oat are based on 84 actual measurements (2 sample extracts assayed in duplicate from 3 replicate plots at seven sites). The data for the fuzzy seed were

calculated from the amount of Cr y1Ab protein present in kernel and lint coat fractions taking into account their respective weights

b Average Cry1Ab as % of crude pr otein is not applicable (NA) because crude protein determinations

were not made on the individual lint coat and ke rnel; but only on the entire fuzzy seed samples.TwinLink Cotton USDA Petition

Page 146 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.7 Fresh Weight average quantities of Cry2Ae protein in raw agricultural commodities of TwinLink cotton as detected by ELISA

Sample Trial

Number a Average Cry2Ae content (μg/g sample) ± SD b Average Cry2Ae content (as % of total crude protein) ± SD

No Glufosinate Glufosinate

Treated No Glufosinate Glufosinate

Treated

Kernel B02-01 21.6± 1.96 18.8 ± 2.60 NA NA

B02-02 8.37 ± 1.15 11.6 ± 1.78 NA NA

B04-03 12.2 ± 1.94 16.4 ± 0.302 NA NA

B04-04 17.1 ± 0.467 20.6 ± 6.51 NA NA

B04-05 21.6 ± 2.64 31.4 ± 10.0 NA NA

B04-06 15.0 ± 0.619 12.3 ± 1.78 NA NA

B08-08 12.8 ± 3.28 13.9 ± 5.35 NA NA

Average NA 15.5 ± 5.22 17.9 ± 13.6 NA NA

Lint Coat B02-01 1.13 ± 0.218 1.16 ± 0.628 NA NA

B02-02 1.22 ± 0.756 0.990 ± 0.802 NA NA

B04-03 1.21 ± 0.775 0.971 ± 0.168 NA NA

B04-04 1.98 ± 0.524 2.83 ± 1.76 NA NA

B04-05 1.31 ± 0.407 1.45 ± 0.341 NA NA

B04-06 0.464 ± 0.334 0.266 ± 0.240 NA NA

B08-08 1.11 ± 0.0350 1.12 ± 1.08 NA NA

Average NA 1.20 ± 1.33 1.26 ± 2.35 NA NA

Fuzzy Seed B02-01 11.0 9.81 0.00552 0.00490

B02-02 4.69 6.16 0.00248 0.00311

B04-03 6.52 8.68 0.00423 0.00546

B04-04 9.70 11.7 0.00462 0.00555

B04-05 11.7 16.2 0.00601 0.00870

B04-06 7.39 6.01 0.00339 0.00283

B08-08 7.35 7.99 0.00315 0.00339

Range in Values NA 4.69 – 11.7 6.01 – 16.2 0.00248 –

0.00601 0.00283 –

0.00870

Average ± SD NA 8.34 ± 2.55 9.51 ± 3.56 0.00420 ±

0.00129 0.00485 ±

0.00204

a Standard Deviation was not calculated for individual fu zzy seed data because the value is the weighted

numerical sum of the average kernel and average lint coat values. A standard deviation was calculated for the average Cry2Ae value of fuzzy seed. This is based only on the calculated average values (kernel + lint coat) obtained at the seven sites. Standard deviations for the individual sites were based on 12 measurements (two sample extracts assayed in duplicate from three replicate plots). Standard deviations for the averages of kernel and lint c oat are based on 84 actual measurements (2 sample extracts assayed in duplicate from 3 replicate plots at seven sites). The data for the fuzzy seed were

calculated from the amount of Cr y2Ae protein present in kernel and lint coat fractions taking into account their respec tive weight.

b Average Cry2Ae as % of crude pr otein is not applicable (NA) because crude protein determinations

were not made on the individual lint coat and ke rnel; but only on the entire fuzzy seed samples.TwinLink Cotton USDA Petition

Page 147 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.8 Dry Weight average quantities of Cry2Ae protein in raw agricultural commodities of TwinLink cotton as detected by ELISA

Sample Trial

Number a Average Cry2Ae content (μg/g sample) ± SD b Average Cry2Ae content (as % of total crude protein) ± SD

No Glufosinate Glufosinate

Treated No Glufosinate Glufosinate

Treated

Kernel B02-01 NA NA NA NA

B02-02 NA NA NA NA

B04-03 NA NA NA NA

B04-04 NA NA NA NA

B04-05 NA NA NA NA

B04-06 NA NA NA NA

B08-08 NA NA NA NA

Average NA NA NA NA NA

Lint Coat B02-01 NA NA NA NA

B02-02 NA NA NA NA

B04-03 NA NA NA NA

B04-04 NA NA NA NA

B04-05 NA NA NA NA

B04-06 NA NA NA NA

B08-08 NA NA NA NA

Average NA NA NA NA NA

Fuzzy Seed B02-01 12.3 11.0 0.00552 0.00490

B02-02 5.34 6.99 0.00248 0.00311

B04-03 7.43 9.84 0.00423 0.00546

B04-04 11.0 13.4 0.00462 0.00555

B04-05 13.4 18.5 0.00601 0.00870

B04-06 8.19 6.67 0.00339 0.00283

B08-08 7.79 8.48 0.00315 0.00339

Range in Values NA 5.34 – 13.4 6.67 – 18.5 0.00248 –

0.00601 0.00283 –

0.00870

Average ± SD NA 9.35 ± 2.93 10.7 ± 4.17 0.00420 ±

0.00129 0.00485 ±

0.00204

a Standard Deviation was not calculated for individual fuzzy seed data because the value is the weighted

numerical sum of the average kernel and average lint coat values. A standard deviation was calculated for the average Cry2Ae value of fuzzy seed. This is based only on the calculated average values (kernel + lint coat) obtained at the seven sites. Standard deviations for the individual sites were based on 12 measurements (two sample extracts assayed in duplicate from three replicate plots). Standard deviations for the averages of kernel and lint c oat are based on 84 actual measurements (2 sample extracts assayed in duplicate from 3 replicate plots at seven sites). The data for the fuzzy seed were

calculated from the amount of Cr y2Ae protein present in kernel and lint coat fractions taking into account their respective weights. Average dry weight content of Cry2Ae in kernel and lint coat is not applicable (NA) because moisture determinations we re made only on the entire fuzzy seed samples

and not the individual lint coat and kernel.

b Average Cry2Ae as % of crude pr otein is not applicable (NA) because crude protein determinations

were not made on the individual lint coat and ke rnel; but only on the entire fuzzy seed samples.TwinLink Cotton USDA Petition

Page 148 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.9 Fresh Weight average quantities of PAT/ bar protein in raw agricultural commodities of TwinLink cotton as detected by ELISA

Sample Trial

Number a Average PAT/bar content (μg/g sample) ± SD b Average PAT/bar content (as % of crude protein) ± SD

No Glufosinate Glufosinate

Treated No Glufosinate Glufosinate

Treated

Kernel B02-01 278 ± 24.5 238 ± 7.51 NA NA

B02-02 275 ± 40.3 276 ± 33.9 NA NA

B04-03 234 ± 19.1 280 ± 23.8 NA NA

B04-04 365 ± 42.6 338 ± 73.7 NA NA

B04-05 283 ± 15.2 342 ± 19.0 NA NA

B04-06 370 ± 22.4 329 ± 50.2 NA NA

B08-08 221 ± 11.9 223 ± 98.8 NA NA

Average NA 290 ± 72.7 289 ± 141 NA NA

Lint Coat B02-01 2.34 ± 0.544 2.67 ± 0.369 NA NA

B02-02 4.41 ± 3.17 3.39 ± 1.87 NA NA

B04-03 2.28 ± 2.34 2.02 ± 0.915 NA NA

B04-04 3.56 ± 1.94 4.71 ± 2.28 NA NA

B04-05 3.63 ± 1.47 4.66 ± 3.06 NA NA

B04-06 1.65 ± 1.15 2.77 ± 2.65 NA NA

B08-08 2.43 ± 0.552 4.09 ± 4.35 NA NA

Average NA 2.90 ± 4.83 3.47 ± 6.70 NA NA

Fuzzy Seed B02-01 135 118 0.0680 0.0590

B02-02 136 136 0.0711 0.0684

B04-03 115 140 0.0742 0.0885

B04-04 188 172 0.0896 0.0814

B04-05 147 170 0.0751 0.0923

B04-06 176 158 0.0809 0.0744

B08-08 118 121 0.0505 0.0512

Range in Values NA 115 – 188 118 – 172 0.0515 – 0.0896 0.0512 – 0.0923
Average ± SD NA 145 ± 28.0 145 ± 22.2 0.0728 ± 0.0121 0.0736 ± 0.0151

a Standard Deviation was not calculated for indivi dual fuzzy seed data because the value is the

weighted numerical sum of the average kernel a nd average lint coat values. A standard deviation was calculated for the average PAT/ bar value of fuzzy seed. This is based only on the calculated average values (kernel + lint coat) obtained at the seven sites. Standard deviations for the individual sites were based on 12 measurements (two sample extracts assayed in duplicate from three replicate plots). Standard deviations for the averages of kernel and lint coat are based on 84 actual measurements (2 sample extracts assayed in duplicate from 3 replicate plots at seven sites). The data for the fuzzy seed were calculated from the amount of PAT/bar prot ein present in kernel and lint coat fractions taking into account their respective weights.

b Average PAT/ bar as % of crude protein is not applicable (NA) because crude protein determinations

were not made on the individual lint coat and ke rnel; but only on the entire fuzzy seed samples.TwinLink Cotton USDA Petition

Page 149 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.10 Dry Weight average quantities of PAT/ bar protein in raw agricultural commodities of TwinLink cotton as detected by ELISA

Sample Trial

Number a Average PAT/bar content (μg/g sample) ± SD b Average PAT/bar content (as % of crude protein) ± SD

No Glufosinate Glufosinate

Treated No Glufosinate Glufosinate

Treated

Kernel B02-01 NA NA NA NA

B02-02 NA NA NA NA

B04-03 NA NA NA NA

B04-04 NA NA NA NA

B04-05 NA NA NA NA

B04-06 NA NA NA NA

B08-08 NA NA NA NA

Average NA NA NA NA NA

Lint Coat B02-01 NA NA NA NA

B02-02 NA NA NA NA

B04-03 NA NA NA NA

B04-04 NA NA NA NA

B04-05 NA NA NA NA

B04-06 NA NA NA NA

B08-08 NA NA NA NA

Average NA NA NA NA NA

Fuzzy Seed B02-01 152 132 0.0680 0.0590

B02-02 154 154 0.0711 0.0684

B04-03 131 159 0.0742 0.0885

B04-04 213 196 0.0896 0.0814

B04-05 168 195 0.0751 0.0923

B04-06 195 175 0.0809 0.0744

B08-08 125 129 0.0505 0.0512

Range in Values NA 125 – 213 129 – 196 0.0505 – 0.0896 0.0512 – 0.0923
Average ± SD NA 163 ± 32.4 163 ± 27.4 0.0728 ± 0.0121 0.0736 ± 0.0151

a Standard Deviation was not calculated for individual fuzzy seed data because the value is the weighted

numerical sum of the average kernel and average lint coat values. A standard deviation was calculated for the average PAT/ bar value of fuzzy seed. This is based only on the calculated average values (kernel + lint coat) obtained at the seven sites. Standard deviations for the individual sites were based on 12 measurements (two sample extracts assayed in duplicate from three replicate plots). Standard deviations for the averages of kernel and lint c oat are based on 84 actual measurements (2 sample extracts assayed in duplicate from 3 replicate plots at seven sites). The data for the fuzzy seed were

calculated from the amount of PAT/ bar protein present in kernel and lint coat fractions taking into account their respective weights. Average dry weight content of PAT/ bar in kernel and lint coat is not applicable (NA) because moisture determinations we re made only on the entire fuzzy seed samples

and not the individual lint coat and kernel.

b Average PAT/ bar as % of crude protein is not applicable (NA) because crude protein determinations

were not made on the individual lint coat and ke rnel; but only on the entire fuzzy seed samples.TwinLink Cotton USDA Petition

Page 150 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for protein levels in plant parts and during the life cycle

This study was carried out to determine the amount of PAT protein, Cry1Ab protein, and Cry2Ae protein in various TwinLink cotton plant matrices produced during the growth of the plant. The matrices included leaves, squares, and grain.

Materials Transgenic plants were grown under greenhouse conditions and received one spray application of Ignite® herbicide at 0.52 lb glufosinate ai/A. Coker 315 plants were grown as controls and did not receive glufosinate (Ignite herbicide) treatment. Five replicate samples of all matrices were collected for analysis. Samples for each treatment were frozen and analyzed for the PAT/ bar protein, Cry1Ab protein, and Cry2Ae protein content by ELISA at the BioAnalytics Laboratories, Bayer CropScience, NC.

Sample preparation and protein extraction The vegetative plant sample consisted of the to tal amount of each matrix from five plants. Samples for analysis were prepared either by grinding in a Waring Laboratory Blender prechilled with dry ice or by grinding in a mortar and pestle after freezing in liquid nitrogen. Each tissue was ground for 30 seconds in a Waring blender pre-cooled with dry ice. Frozen tissue specimens, together with dry ice, were ground until all crushed material was homogeneous, adding dry ice as necessary. Grinding with liquid nitrogen involved placing the sample in a mortar surrounded with dry ice. The sample was placed in the mortar and covered with liquid nitrogen. The pestle was then placed in the mortar for cooling. The sample was then ground to a fine powder immediately after the liquid nitrogen dissipated. The liquid nitrogen method was usually used when the amount of available sample was small. The ground samples were stored in a freezer at approximately -20°C for overnight or longer to allow the dry ice to dissipate before

extraction.

The extraction of vegetative samples was carried out using a buffer described by Xin et al

., 1988. A representative portion of ground sample was mixed with the extraction buffer in a 50 mL polypropylene centrifuge tube. Samples were extracted for 30 minutes at approximately 4°C on a shaker (IKA-SCHÜTTLER MTS 4) at 250 rpm and then centrifuged at approximately 18,000 x g for 10 minutes at approximately 4°C. The clear supernatant was then used for Cry1Ab, Cry2Ae, PAT/ bar, and TEP analyses. Duplicate extracts were prepared for each sample.

Limit of Detection (LOD) and Limit of Quantitation (LOQ) The limit of detection (LOD) is determined for each matrix using the average standard curve and the concentration derived from the background optical density (OD) of the negative control samples. The LOD is the concentration corresponding to an OD value three standard deviations above the mean background OD.

The limit of detection is expressed in the unit of concentration in the extract (ng/mL) and the unit weight (ng/g fresh weight) in the matrix. This relationship is based on the ratio of the weight of the matrix to the volume of the extraction buffer. An ELISA reading resulting in a PAT/ bar

, Cry1Ab, or Cry2Ae protein concentration at or above the limit of detection level is assumed to be greater than the zero dose reading.TwinLink Cotton USDA Petition

Page 151 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### The limit of quantitation is given by the lowest concentration of the standard that meets the following two validity criteria: a) analyte recoveries from fortified matrix samples are > 60 % and < 130 % and b) the coefficient of variance (relative standard deviation) is less than 25%. When a lower recovery is caused by the nature of a specific matrix or the effect of a process, the lowest concentration of the standard that gives a coefficient of variance equal to or less than
25% is used as the LOQ.

Table 2.11 Limit of Detection and Quantitation of PAT/bar Protein in Control Cotton Coker 315 Leaves, Squares, and Grain as Detected by ELISA

LOD LOQ a

Matrix ng/mL

Extract ng/g Sample ng/mL

Extract ng/g Sample

Leaves 0.002 0.12 0.469 28.1

Squares 0.096 5.76 0.469 28.1

Grain 0.013 0.26 0.469 9.38

a Calculated based on the extraction of approximat ely 0.1 g matrix per 6 mL extraction buffer for leaves and squares and 0.1 g matrix per 2 mL extr action buffer for grain. The data are expressed in ng/mL in the extract and ng/g in the sample.



### Table 2.12 Limit of Detection and Quantitation of Cry1Ab Protein in Control Cotton Coker 315 Leaves, Squares, and Grain as Detected by ELISA

LOD LOQ a

Matrix ng/mL

Extract ng/g Sample ng/mL

Extract ng/g Sample

Leaves 0.013 0.78 0.078 4.68

Squares 0.025 1.50 0.078 4.68

Grain 0.040 0.80 0.078 1.56

a Calculated based on the extraction of approximat ely 0.1 g matrix per 6 mL extraction buffer for leaves and squares and 0.1 g matrix per 2 mL extr action buffer for grain. The data are expressed in ng/mL in the extract and ng/g in the sample.



### Table 2.13 Limit of Detection and Quantitation of Cry2Ae Protein in Control Cotton Coker 315 Leaves, Squares, and Grain as Detected by ELISA

LOD LOQ a

Matrix ng/mL

Extract ng/g Sample ng/mL

Extract ng/g Sample

Leaves 0.211 12.7 0.938 56.3

Squares 0.203 12.2 0.469 28.1

Grain 0.183 3.66 0.469 9.38

a Calculated based on the extraction of approximat ely 0.1 g matrix per 6 mL extraction buffer for leaves and squares and 0.1 g matrix per 2 mL extr action buffer for grain. The data are expressed in ng/mL in the extract and ng/g in the sample.



### Determination of Total Extractable Protein content SoftMax ProTM software (Molecular Devices, Vers ion 4.0) was used to derive the concentration of protein from the Bradford assay (Bradford, 1976). The optical density was converted to theTwinLink Cotton USDA Petition

Page 152 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### TEP concentration using the standard curve. The data point for the dilution falling nearest the center of the standard curve was used. If two points were near the center of the curve, the

measurement with the lowest CV was used.

Protein analyte content

The amount of PAT/ bar, Cry1Ab, and Cry2Ae proteins in the extracts were determined by ELISA. The assays used were commercial ELISA kits (catalogue # AP014 for PAT/ bar protein, AP003 for Cry1Ab protein, and # AP005 for Cry2Ae protein) manufactured by EnviroLogix, Inc. (Portland, ME). All assays resulted in color development in the last step, which was measured in a microplate reader (Molecular Devices THERMOmax) at 450 nm.

Average concentration of Cry1Ab, Cry2Ae, and PAT/bar in leaves, squares, and grain on a fresh and dry weight basis are given in Table 2.14.

Calculations and conversions SoftMax ProTM software (Molecular Devices, Vers ion 4.0) was used to derive the concentration

of immunoreactive PAT/ bar , Cry1Ab, and Cry2Ae proteins in the ELISA. The optical density (OD) values were adjusted for the buffer blank and then any background due to the matrix was subtracted, using the average value from 2 wells containing non-transgenic extracts, assayed on the same plate. The optical density corrected for buffer blank and non-transgenic background was converted to the protein analyte concentration using the standard curve also from the same plate. The data point for the dilution falling nearest the center of the standard curve was used. If OD values from different dilutions were equally distant from the midpoint of the Standard Curve, the measurement with the lowest CV were used. To obtain the amount of protein analyte in the matrix, the amount of protein determined from the standard curve was multiplied by the ratio of volume of extraction buffer to grams matrix extracted.

Statistics Descriptive statistics (mean, standard deviation, and coefficient of variance) were calculated for each tissue and growth stage using Microsoft ® 

### Excel 2002. All statistical analyses were done on data with full precision. Results may be rounded to two or three significant numbers.

Table 2.14 Average Analyte Concentrations for Leaves, Squares and Grain on a Fresh Weight and Dry Weight Basis for TwinLink Transgenic Cotton

Matrix Average Cry1Ab

Protein

Content (ug/g fresh weight) ± SD Average Cry2Ae Protein

Content (ug/g fresh weight) ± SD Average PAT/bar

Protein Content

(ug/g fresh weight) ± SD Percent

moisture a Average Cry1Ab

Protein

Content (ug/g dry weight) ±

SD Average Cry2Ae

Protein

Content (ug/g dry weight) ±

SD Average PAT/bar

Protein Content (ug/g dry weight)

± SD

Leaves 0.42 0.26 7.18 3.83 77.08 38.6 82.6 ± 3.4 2.40 ± 1.51 41.3 ± 22 443 ± 222

Squares 0.27 0.09 1.07 0.32 2.08 5.02 78.1 ± 3.2 1.23 ± 0.41 4.89 ± 1.46 95.9 ± 22.9

Grain 0.39 0.21 0.21 0.19 1.53 0.66 8.56 ±0.46 0.43 ± 0.23 0.23±0.21 1.67 ± 0.72

a The values for percent moisture in the samples were obtai ned from analysis at Covance Laboratories. The complete set of data is given in Appendix 3.TwinLink Cotton USDA Petition

Page 153 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for agronomic equivalence studies

Materials Materials for agronomy evaluation were created at field sites in 2007 and 2008 in the southeastern, mid-southern, and mid-western regions of the United States. Seven locations in 2007, and two locations in 2008 (only data available at time of dossier preparation) in eight states were used to produce the reference material for fiber analysis and agronomic performance. Agronomic trials consisted of three treatment regimes; two of the transformed cotton and the other the corresponding non-transgenic Coker counterpart. Agronomic field studies utilized an experimental treatment regime which compared the transformed events T304-40 and GHB119 sprayed, and unsprayed with glufosinate ammonium compared to the non-transformed Coker counterpart. Agronomic data was also taken on efficacy trials in 2007 and 2008 (discussed in following section).

Characterization of the Materials Identity of the materials was preserved through chain of custody documentation. Chain of custody documentation was utilized to identify the materials shipped to their respective field sites for proper identification of the evaluated plots in the field. Harvested materials contained chain of custody documentation for samples sent from the field to analytical laboratories to preserve identity.

Performing Facility and Experimental Methods Trials in 2007 and 2008 were utilized to characterize and evaluate agronomic performance of the selected events, and develop materials for nutritional and compositional testing. Trials were conducted in three geographic regions of the United States (see xxx).

All trials received similar agronomic treatments for the care and upkeep of the plots. Field studies were managed in a manner representative of normal agricultural practices for inputs including, but not limited to:

• Conventional herbicide treatments, both pre- and post- planting

• Granular insecticide and/or fungicide application at planting

• Fertilizer applications

• Necessary in-season herbicide applications

• Growth regulator application

• Additional hand weeding as necessary

• Chemical defoliation without boll-opening desiccants

Trials were performed using a randomized complete block design using four row plots with four replications and either 3,6 or 8 treatments. A total of 22 agronomic parameters were used to measure the growth and development of the plant, and provide visual observations on the effect of any biotic and abiotic stressors upon the field plots across regions. Of the agronomic parameters observed, seven determined yield quality and quantity, 9 were conducted to evaluate growth habit and agronomics, and six measur ed impact of abiotic and biotic stress. These parameters were selected as key indicators of commercial and agronomic importance to commercial cotton growers, and the ability of the crop to perform under a variety of stresses from the different growing regions around the county.TwinLink Cotton USDA Petition

Page 154 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.15 Treatment schedule for agronomic field tests in 2007 and 2008

Entry Treatment Description Coker Not sprayed Non-transgenic parent unsprayed T304-40xGHB119 Sprayed with glufosinate ammonium Transgenic TwinLink sprayed T304-40xGHB119 Not sprayed Transgenic TwinLink sprayed



### Plant mapping was conducted on 5 consecutive plants in the plots which were representative of general field conditions. Plant height, number of nodes, first fruiting position, second fruiting position, third fruiting position (one location), and total boll count (including vegetative bolls) were taken as a measure of agronomic performance throughout the year. This data showed the development and potential reproductive success of the cotton plant as an indication of the yield of the plant. Visual ratings of 1-9 (1 = most favorable rating and 9 = least favorable rating) were taken for lodging and boll type. Visual ratings of 1-2 (1=normal and 2=abnormal) were taken for morphology characteristics.

Statistical Analysis Analysis of variance (ANOVA) between groups was calculated to analyze data for significant differences. All treatments were analyzed in comparison to their non-transgenic counterpart across regions, regionally, and locally. Data was reviewed using a confidence interval of 95%. Least significant difference (LSD) and coefficients of variance (CV) are presented in data tables 4.1 - 4.10).TwinLink Cotton USDA Petition

Page 155 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for efficacy studies

Materials Materials for efficacy evaluation were created at field sites in 2007 and 2008 in the southeastern, mid-southern, and mid-western regions of the United States. Six locations in 2007 and eight locations in 2008 in six states were used to produce the reference material for fiber analysis, agronomic performance, and lepidopteran control efficacy. External university and USDA trials consisted of 8 treatment regimes; 4 genotypes either treated or untreated for lepidopteran pests (Table 2.2). Internal locations had the same treatments in 2007, however, the treated T304-40 and GHB119 were dropped in 2008.

Table 2.16 Treatment schedule for external/university efficacy field tests in 2007 and

2008

Entry Treatment Description Coker Unsprayed No lepidopteran supplemental control Coker Sprayed Lepidopteran suppleme ntal control with conventional

insecticides T304-40 Unsprayed No lepidopteran supplemental control T304-40 Sprayed Lepidopteran supplemental control with conventional

insecticides GHB119 Unsprayed No lepidopteran supplemental control GHB119 Sprayed Lepidopteran supplemental control with conventional

insecticides T304-40xGHB119 Unsprayed No lepidopteran supplemental control T304-40xGHB119 Sprayed Lepidopteran supp lemental control with conventional

insecticides



### Characterization of the Materials Identity of the materials was preserved through chain of custody documentation. Chain of custody documentation was utilized to identify the materials shipped to their respective field sites for proper identification of the evaluated plots in the field. Harvested materials contained chain of custody documentation for samples sent from the field to analytical laboratories to preserve identity. Additionally, two leaf samples were taken from two individual plants per plot for PCR analysis to confirm identity of plots in the tests.

Performing Facility and Experimental Methods Field studies were managed in a manner representative of normal agricultural practices for inputs including, but not limited to:

• Conventional herbicide treatments, both pre- and post- planting

• Granular insecticide and/or fungicide application at planting

• Fertilizer applications

• Necessary in-season herbicide applications

• Growth regulator application

• Additional hand weeding as necessary

• Chemical defoliation without boll-opening desiccantsTwinLink Cotton USDA Petition

Page 156 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Trials were performed using a randomized complete block design using two row plots with four replications of four treatments. A total of 8 efficacy parameters were used to measure the amount of plant damage and lepidopteran larval survival. Damage assessments and larval survival were taken on squares, plant terminals, wh ite flowers, and bolls at multiple time frames through fruiting period (n=2-8). An additional 22 ag ronomic parameters were also evaluated.. Of the 22 potential agronomic parameters observed, six determined yield quality and quantity, 10 were conducted to evaluate growth habit and agronomics, and six were visual measures of plant morphology, biotic and abiotic stress (disease and lodging). These parameters were used to evaluate equivalence in crop development, fiber qualities, and environmental tolerance.

Plant mapping was conducted on 5 consecutive plants in the plots which were representative of general field conditions. Plant height, number of nodes, first fruiting position, second fruiting position, third fruiting position (one location), and total boll count (including vegetative bolls) were taken as a measure of agronomic performance throughout the year. This data showed the development and potential reproductive success of the cotton plant as an indication of the yield of the plant. Visual ratings of 1-9 (1 = most favorable rating and 9 = least favorable rating) were taken for lodging and boll type. Visual ratings of 1-2 (1=normal and 2=abnormal) were taken for morphology characteristics.

Statistical Analysis Analysis of variance (ANOVA) between groups was calculated to analyze data for significant differences. All treatments were analyzed in comparison to their non-transgenic counterpart across regions, regionally, and locally. Data was reviewed using a confidence interval of 95%. Least significant difference (LSD) and coefficients of variance (CV) are presented in data tables 4.1 - 4.10).TwinLink Cotton USDA Petition

Page 157 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Fi g ure 2.1 

### Method for agronomic efficacy studies

Artificial Infestations of Helicoverpa spp. (Cotton Bollworm) in Bt cotton

Objective: Artificial Infestations This method increases cotton bollworm (CBW) pressure in experimental field trial plots testing Bt cotton. Timing of infestations will vary depending on location, but should commence at early bloom. Weekly infestations will continue for 6 consecutive weeks.

Materials and Methods 1) The R&D specialist in your region will arrange for egg orders according to trial planting date and supply sufficient corn grits for entire season. Eggs should be kept in temperature controlled environment upon arrival. 2) Preparation of inoculants: a. Weigh 25g of corn grits per plot to be infested (ex. 2 untreated, infested plots with 4 replications would be 8 total plots, or 200g of corn grits) into a large Tupperware

container. b. Gently mix egg shipment, corn grits, and 25mL of water by rotating and swirling container. The total egg count in shipment will be determined prior to arrival at testing

location.

c. Obtain total number of application devices needed for all plots (For example above, 8 total containers). An exampl e of an application device is shown in Figure 2.X. Equally divide mixture into application devices. d. Incubate mixture 12-24 hours to allow for egg hatching. 3) Once neonates are observed in the mixture, infestation of experimental plots can proceed

. Place application devices into a cooler with cool packs for transport to the field. Place paper barrier between cool packs and application devices to minimize

condensation. 4) Only infest unsprayed plots. Evenly distribute corn grit and neonate mixture to the center two rows of all experimental plots. All infestations should be carried out during the early morning. Avoid infesting plants after 10am when higher temperatures will cause significant insect mortality. Leaves should be sl ightly damp prior to infestation. Moisture should be present with morning applications, but if not, use a spray bottle to dampen cotton terminals prior to infestation. For the 5 th 

### and 6 th 

### week infestation, dispense eggs and grits into white flowers.

Damage Assessments after Infestations Insect damage ratings and larval counts should be conducted prior to the first infestation. Weekly assessments will be conducted 5-7 days after infestations for the subsequent 5-6 weeks. Trial protocols with describe insect and damage assessments needed for trial.TwinLink Cotton USDA Petition

Page 158 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Assessing the plots. Ensure that you assess the plots prior to each infestation. For the first 4 infestations, concentrate sampling in the top third of the plants. For the 5 th 

### and 6 th 

### infestation, begin checking for insect and damage lower in the plants and commence boll assessments. Continue to assess the plots for 2 weeks after the final infestation.

5-7 days after each infestation, record larval survival in the unsprayed untreated control (UTC) ‘check’ plots of Coker conventional cotton. We are aiming for an average of 1 surviving larva per cotton plant. For the 5 th 

### and 6 th 

### infestation, dispense eggs and grits into white flowers.

Control of Secondary Insect Pests All plots should be sprayed with selective chemistry for non-lepidopteran pests as required by best local agronomic practices. Conventional in secticides should be applied to ‘Sprayed’ or ‘Treated’ plots to minimize lepidopteran damage. Allow 48 hours after an infestation before applying any insecticide sprays.TwinLink Cotton USDA Petition

Page 159 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Method for Terminal Leaf Bioassays of Helicoverpa spp. (Cotton Bollworm) in Bt cotton

Objective: Bioassays Leaf tissue bioassays will be performed in parallel with artificial infestations of CBW neonates. First in-star CBW larvae will be placed on leaf mate rial taken weekly from experimental plots for six consecutive weeks. Mortality will be compared between transgenic lines and their conventional counterparts to determine Bt efficacy against CBW.

Materials and Methods 1) Each shipment of CBW eggs for field infestations will also have neonates on artificial diet. Neonates on diet will be left overnight to reach the L1 stage. 2) At each sampling date 10-20 of the most recently expanded leaves will be sampled from each plot prior to infestation. Sampled leaves should measure approximately 5 cm across and be removed from the plant with as much petiole attached as possible. Leaves from each replicate are kept separat e in labeled paper sacks and placed in a small plastic cooler containing ‘blue ice’ for transport to the laboratory bioassay the same day. 3) Place individual leaves in a 50 x 9 mm Tight-Fit Lid sealing Petri dish (BD Falcon #351006, VWR International) and infest with a single day old fed L1 larva. Infested larvae should be of consistent size and movement for all leaf samples. 4) Bioassays are kept at a constant temperature (25-30 degrees centigrade) and >50% relative humidity prior to assessment. 5) Five days after exposure (DAE), larvae are prodded with a camel-hair brush and

considered alive if coordinated movement is observed. Larvae from each dish are scored according to the criteria in Table 2.2 and Figure 3 :

Sampling and Scoring

Table 2.17 Sampling schedule for bioassays

Sample number and timing

Protocol # Unsprayed* plots

evaluated 1

64

DAP 2

71

DAP 3

78

DAP 4

85

DAP 5

92

DAP 6

99

DAP

BD-08-NAR-HA8 Coker

TwinLink * Plots unsprayed for lepidopter an pests – see protocols.TwinLink Cotton USDA Petition

Page 160 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.18 Scoring criteria for bioassays

Condition Score

Dead 0

Alive L1 1

Alive L2 2

Alive L3 3

Analysis Percent survival and development of larvae is analyzed using SCOUT or REML-ANOVA, and means are separated according to Fisher’s Protected LSD (Littell et al. 1996; PROC MIXED, SAS Institute 2001).

Figure 2.2 Bioassay set up

Figure 2.3 Size comparison for bioassay.TwinLink Cotton USDA Petition

Page 161 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for fiber quantification studies

Materials Materials were generated from agronomic performance field trials in 2004 and 2005 (see table 9). Ginned cotton fiber from 25 boll samples were taken from each plot, and sent to be analyzed at various cotton fiber analytical laboratories (ITC, Star, USDA, etc.). Samples were analyzed for traditional cotton fiber quality parameters using high volume instrumentation (HVI).

Characterization of the Materials Identity of the materials was preserved through chain of custody documentation. Chain of custody documentation was utilized to identify the materials shipped to their respective field sites for proper identification of the evaluated plots in the field. Harvested materials contained chain of custody documentation for samples sent from the field to analytical laboratories to preserve identity.

Performing Facility and Experimental Methods Fiber was analyzed using HVI standard procedures at Star Labs in Knoxville, TN and the International Textile Center in Lubbock, TX. Fi ber was analyzed for fiber strength, elongation, % lint, micronaire, fiber uniformity and fiber length. These parameters are the standard classing parameters used by the United States Department of Agriculture. Measurements for color and trash were not taken because the samples were hand harvested. Samples harvested by hand do not have the same issues as fiber harvested with cotton picking equipment, therefore making these measurements irrelevant.

Statistical Analysis Analysis of variance (ANOVA) between groups was calculated to analyze data for significant differences. All treatments were analyzed in comparison to their non-transgenic counterpart across regions, regionally, and locally. Data was reviewed using a confidence interval of 95%.TwinLink Cotton USDA Petition

Page 162 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Materials and methods for composition analysis

Field design TwinLink cotton plants containing events T304-40 and GHB119, along with cotton plants representing the non-transgenic (non-transformed) counterpart Coker 315 were field tested by Bayer CropScience in 2007 under USDA APHIS Notification 07-059-101n. Field trials were established in EPA Regions II, IV, and VIII in the following locations: Trial number 02-01-Tift County, Georgia; Trial number 02-02-Tift County, Georgia; Trial 04-03-Jackson County Arkansas; Trial number 04-04-Crittenden County, Arkansas; Trial number 04-05-Crittenden County, Arkansas;Trial number 04-06-Tate County, Mississippi; Trial number 08-08-Hockley County, Texas. These areas are typical cotton growing regions of the southeastern United States, and the plants in this study were grown under conditions typical of production practices. There were six transgenic plots and three non-transgenic plots at each test site. The test plots were randomized at each field location. Seed for planting was supplied for each field trial by Bayer CropScience.

Three of the transgenic event plots in each field trial were sprayed two times with Ignite 280 SL glufosinate-ammonium herbicide, and the other plots were untreated. The Ignite 280 SLherbicide contained a labeled concentration of 2.34 pounds per U.S. gallon of glufosinateammonium active ingredient (280 grams of active ingredient per liter). Each application of Ignite herbicide was made at a target rate of 29 fluid ounces of product per acre (2.1 liters per hectare), equivalent to 0.53 lb ai/acre (0.60 kg ai/hectare).

Three replicate non-transgenic samples and six replicate transgenic samples (3 untreated and 3 treated with Ignite herbicide) of fuzzy seed were collected from each of the field test sites and shipped frozen to Bayer CropScience BioAnalytics lab, Research Triangle Park, North Carolina. The fuzzy seeds were sub-sampled and shipped frozen to the analytical facility, Covance Laboratories, Inc., Madison, Wisconsin for composition analyses. Shipping and storage of the regulated seed was carried out under applicable USDA regulations and Bayer CropScience guidelines.

Characterization of the material Certificates of Analysis (COA) were produced by Bayer CropScience for the two seed lots used for planting in this study. Identity and purity of the TwinLink cottonseed and the corresponding non-transgenic (Coker 315) cottonseed were confirmed to be acceptable for use. A COA to determine identity of the samples harvested from the seven field sites was prepared by Bayer CropScience. The data certified that the transgenic samples harvested from the field wereindeed TwinLink cotton, and that the non-transgenic samples were harvested from nontransgenic plots.

Analytical procedures The composition of the cottonseed was determined at Covance Laboratories, Inc. The analyses performed and the methods used are listed in Table 2.19.TwinLink Cotton USDA Petition

Page 163 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 2.19 Analyses Performed on TwinLink Cottonseed and Its Non-transgenic Counterpart

Parameter Covance

Mnemonic Covance Method Reference

Moisture M100 AOAC 926.08 and 925.09

Crude protein PGEN AO AC 955.04 and 979.09

Crude fat FAAH AOAC 922.06 and 954.02

Ash ASHM AOAC 923.03

Carbohydrate CHO Difference between 100 and the sum of moisture, crude protein, fat, and ash. Agric. Handbook No. 74

Acid Detergent Fiber ADF Agric. Handbook No. 379

Neutral Detergent Fiber NDFE AACC 32.20 + Agric. Handbook No. 379

Ca, K, Mg, Fe, Zn, P ICPS AOAC 984.27 and 985.01

Vitamin E and Tocopherols TTLC HPLC method is based on the references below

Amino Acids TAA5 AOAC 982.30

Fatty Acids FALC AOAC 996.06 and AOCS Ce 1-62

Phytic acid PHYT HPLC method is based on the references below

Gossypol (total) GOSS AOCS Ba 7-58 abd Ba 8-78

Gossypol (free) GOSF AOCS Ba 7-58 abd Ba 8-78

Amino Acids TAA5 AOAC 982.30

Cyclopropenoid Fatty Acids CPFA HPLC method is based on the references below



### Data were obtained from the transgenic unsprayed, transgenic sprayed and non-transgenic samples and presented on a fresh weight basis. The data were also presented on a dry weight basis by correcting the fresh weight values for the moisture content determined for each sample. Mean values and standard deviations were calculated for the dry matter data. Fatty acid data were presented on a fresh weight basis and additionally, together with the cyclopropenoid fatty acid data, as relative quantities of the total sum of fatty acids.TwinLink Cotton USDA Petition

Page 164 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 3. CHARACTERIZ ATION OF TWINLINK COTTONTwinLink Cotton USDA Petition

Page 165 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 1. Verification of the insert in Cry1Ab cotton event T304-40

Genomic DNA isolated from Cry1Ab cotton event T304-40 was digested with one of the following restriction enzymes: Apa I, Dra III, Eco RI, Eco RV, Nde I, BgI I, Swa I, Ssp I, Sac I and

Xba I. Wild type genomic DNA digested with Xba I was used as negative control; wild type genomic DNA digested with Xba I and supplemented with an equimolar amount of pTDL008 plasmid DNA digested with Xba I was used as positive control. The resulting DNA fragments were analyzed by Southern blot with six different probes, corresponding to the different genetic elements contained in the pTDL008 T-DNA. Probe information is presented in Table 3.1. A schematic overview of the Southern blot strategy is presented in Figure 3.1.

Table 3.2 summarizes the expected and obtained hybr idization fragments. An overview of the obtained Southern blot results is presented in Figure 3.2.

The results of the Southern blot analysis demonstrate that the inserted transgenic sequence in Cry1Ab cotton event T304-40 consists of one nearly complete copy of the T-DNA flanked by an inverted incomplete copy of the cry 1Ab gene cassette and one additional 3’me1 terminator.

Table 3.1 Event T304-40 Insert verification – probe information

Probe template ID Genetic element Size probe template

PT020-1 3’me1 probe 859 bp

PT021-1 cry 1Ab probe 1822 bp PT022-1 P35S3 probe 801 bp

PT023-1 bar probe 425 bp PT024-1 3’nos probe 214 bp PT040-1 5’e1-Ps7s7 probe 1144 bp PT041-3 T-DNA probe 5050 bpTwinLink Cotton USDA Petition

Page 166 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.1 Schematic overview of Southern blot strategy for Cry1Ab cotton event

T304-40TwinLink Cotton USDA PetitionPage 167 of 205CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 3.2 Expected and obtained hybridization fragments – Cry1Ab cotton event T304-40TwinLink Cotton USDA Petition

Page 168 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.2 Cry1Ab cotton event T304-40 Insert verification – Southern blot resultsTwinLink Cotton USDA Petition

Page 169 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.2 (Continued) Cry1Ab cotton event T304-40 Insert verification – Southern blot

resultsTwinLink Cotton USDA Petition

Page 170 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 2. Verification of the insert in Cry2Ae cotton event GHB119

Genomic DNA prepared from GHB119 plants was digested with one of the following enzymes or enzyme combinations: Dra I, Eco RV, Hind III, Nco I, Bam HI, Eco RI, Pst I, Ava I, Nde I, Xho I,

Eco RI/ Pst I and Eco RI/ Nde I. The resulting DNA fragments were analyzed by Southern blot with six different probes, corresponding to the different genetic elements contained in the pTEM12

T-DNA.

Probe information is given in Table 3.3, and a schematic of the Southern blot strategy is presented in Figure 3.3. Table 3.4 summarizes the expected and obtained hybridization fragments and an overview of the Southern Blot results is shown in Figure 3.4.

Hybridization results demonstrated that a single copy of the T-DNA is integrated in event GHB119 and that the configuration of the inserted DNA corresponds to that of the original

transformation vector.

Table 3.3 Cry2Ae cotton event GHB119 Insert verification – probe information

Probe template ID Description Size probe template

PT026 T-DNA probe 4325 bp PT024 3’ nos probe 214 bp

PT023 bar probe 425 bp PT044 PCsVMV probe 572 bp PT045 P35S2-5’cab22L probe 532 bp

PT046 TPssuAt- cry 2Ae probe 2100 bp PT047 3’35S-RB probe 264 bpTwinLink Cotton USDA Petition

Page 171 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION Nco I Bam HI Nco I Nde I

Hin dIII

Nde I Nco I Bam HI

Eco RI

Hind III Eco RV Hin dIII

Pst I

LB 3'nos bar PCsVMV P35S2

5'cab22L TPssuAt cry 2Ae 3'35S RB

PT024 Dra I > 4345 bp

Eco RV X X > 1143 bp 1373 bp > 1829 bp

Hin dIII XX X > 1427 bp 1280 bp 1352 bp > 286 bp

Nco I X X X 854 bp 1105 bp > 2356 bp > 30 bp

Bam HI X X > 316 bp 2051 bp > 1978 bp

Eco RI X > 1817 bp > 2528 bp

Pst I X > 4303 bp > 42 bp

Nde I X X > 1318 bp 131 bp > 2896 bp

PT023 PT044 PT045 PT046 PT047

PT026 Eco RV

Eco RI/ Pst I X > 42 bp 1775 bp > 2528 bp X

Ec o

RI/ Nde I > 1817 bp > 1318 bp X X X 131 bp 1079 bp



### Figure 3.3 Schematic overview of Southern blot strategy for Cry2Ae cotton event

GHB119TwinLink Cotton USDA PetitionPage 172 of 205CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 3.4 Expected and obtained hybridization fragments – event GHB119YesYesYesYesYesNoYesNoYesYesYesYesYesYesYesYesYesNoYesNoYesNoYesNoYesNoYesNo108931373108931373Positive controlPositive controlWT genomicDNA + pTEM12-EcoRVYesYesYesYesYesYesYesYesYesYesYesYesYesYesNofragment.Nofragment.Negative controlWT genomicDNA - EcoRVYesNoYesYesYesNo *YesYesNoNoNoYesNoNoNoYesNoNoYesYesNoNoYesYesNoNoYesNoNoNoYesNoYesNoNoNoYesNo *NoNoYesNoNoNoYesNoNoNoYesNoNoNoYeNoNoNo3960No hybr.10792450>13181311079>18175' integration fragm.internal fragm.internal fragm.3' integration fragm.EcoRI/NdeIYesNoYesYesNo *YesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesYesNoNoYesNo *NoYesNoNoYesNoNoYesNoNoYesNoNo4200No hybr.5380>1318131>28965' integration fragm.internal fragm.3' integration fragm.NdeIYesYesYesYesNoYesNoYesYesYesYesYesYesNoYesNoYesNoYesNoYesNoYesNoYesNoYesNo52001775>252817755' integration fragm.internal fragm.EcoRI/PstIYesYesYesYesYesYesYesYesYesYesYesYesYesYes7800>43035' integration fragm.PstIYesYesYesYesNoYesNoYesYesYesYesYesYesNoYesNoYesNoYesNoYesNoYesNoYesNoYesNo51802480>2528>18175' integration fragm.3' integration fragm.EcoRIYesYesYesYesYesYesNoNoYesNoNoYesNoYesYesNoYesYesNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoYesNoNoYesNoNo312020517070>3162051>19785' integration fragm.internal fragm.3' integration fragm.BamHIYesYesYesYesYesYesNoNoYesNoNoYesNoNoYesNoNoYesNoYesNoNoYesNoNoYesNoNoYesNoYesNoNoYesNoNoYesNoNoYesNoNo854110559408541105>23565' integration fragm.internal fragm.3' integration fragm.NcoIYesYesYesNo**YesYesYesYesNoNoNoYesNoNoNoYesNoYesYesNoNoYesYesNoNoYesNoNoNoYesNoNoYesNoNoNoYesNoNoNoYesNoNoNoYesNoNoNoYesNoNoNoYesNoNoNo5150128013528830>142712801352>2865' integration fragm.internal fragm.internal fragm.3' integration fragm.HindIIIYesYesYesYesYesYesNoNoYesNoNoYesNoYesYesNoYesYesYesYesNoYesYesNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNo338013738890>18291373>11435' integration fragm.internal fragm.3' integration fragm.EcoRVYesYesYesYesYesYesYesYesYesYesYesYesYesYes4430>4345Complete insertDraIObt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.PT026:T-DNA probePT047:3'35S-RBPT046:TPssuATcry2AePT045:P35S2-5'cab22LPT044:PcsvmvPT023:barPT024:LB-3'nosObtainedfragmentsizes (bp)Expectedfragmentsizes (bp)DescriptionDigestYesYesYesYesYesNoYesNoYesYesYesYesYesYesYesYesYesNoYesNoYesNoYesNoYesNoYesNo108931373108931373Positive controlPositive controlWT genomicDNA + pTEM12-EcoRVYesYesYesYesYesYesYesYesYesYesYesYesYesYesNofragment.Nofragment.Negative controlWT genomicDNA - EcoRVYesNoYesYesYesNo *YesYesNoNoNoYesNoNoNoYesNoNoYesYesNoNoYesYesNoNoYesNoNoNoYesNoYesNoNoNoYesNo *NoNoYesNoNoNoYesNoNoNoYesNoNoNoYeNoNoNo3960No hybr.10792450>13181311079>18175' integration fragm.internal fragm.internal fragm.3' integration fragm.EcoRI/NdeIYesNoYesYesNo *YesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesYesNoNoYesNo *NoYesNoNoYesNoNoYesNoNoYesNoNo4200No hybr.5380>1318131>28965' integration fragm.internal fragm.3' integration fragm.NdeIYesYesYesYesNoYesNoYesYesYesYesYesYesNoYesNoYesNoYesNoYesNoYesNoYesNoYesNo52001775>252817755' integration fragm.internal fragm.EcoRI/PstIYesYesYesYesYesYesYesYesYesYesYesYesYesYes7800>43035' integration fragm.PstIYesYesYesYesNoYesNoYesYesYesYesYesYesNoYesNoYesNoYesNoYesNoYesNoYesNoYesNo51802480>2528>18175' integration fragm.3' integration fragm.EcoRIYesYesYesYesYesYesNoNoYesNoNoYesNoYesYesNoYesYesNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoYesNoNoYesNoNo312020517070>3162051>19785' integration fragm.internal fragm.3' integration fragm.BamHIYesYesYesYesYesYesNoNoYesNoNoYesNoNoYesNoNoYesNoYesNoNoYesNoNoYesNoNoYesNoYesNoNoYesNoNoYesNoNoYesNoNo854110559408541105>23565' integration fragm.internal fragm.3' integration fragm.NcoIYesYesYesNo**YesYesYesYesNoNoNoYesNoNoNoYesNoYesYesNoNoYesYesNoNoYesNoNoNoYesNoNoYesNoNoNoYesNoNoNoYesNoNoNoYesNoNoNoYesNoNoNoYesNoNoNo5150128013528830>142712801352>2865' integration fragm.internal fragm.internal fragm.3' integration fragm.HindIIIYesYesYesYesYesYesNoNoYesNoNoYesNoYesYesNoYesYesYesYesNoYesYesNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNoYesNoNo338013738890>18291373>11435' integration fragm.internal fragm.3' integration fragm.EcoRVYesYesYesYesYesYesYesYesYesYesYesYesYesYes4430>4345Complete insertDraIObt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.Obt.Exp.PT026:T-DNA probePT047:3'35S-RBPT046:TPssuATcry2AePT045:P35S2-5'cab22LPT044:PcsvmvPT023:barPT024:LB-3'nosObtainedfragmentsizes (bp)Expectedfragmentsizes (bp)DescriptionDigest(*) The fragment is too small to be visible.(**) The fragment is very small as compared to the probe size. As a result, this fragment is not visualized.TwinLink Cotton USDA Petition

Page 173 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.4 Cry2Ae cotton event GHB119 Insert verification - Hybridization results

Lane 1 : Phage Lambda - Pst I digested Lane 2 : Phage Lambda - Hind III digested

Lane 3 : GHB119 - Dra I digested

Lane 4 : GHB119 - Eco RV digested

Lane 5 : GHB119 - Hind III digested

Lane 6 : GHB119 - Nco I digested

Lane 7 : GHB119 - Bam HI digested

Lane 8 : GHB119 - Eco RI digested

Lane 9 : GHB119 - Pst I digested Lane 10 : GHB119 - Eco RI/ Pst I digested

Lane 11 : GHB119 - Pst I digested

Lane 12 : GHB119 - Nde I/ Eco RI digested Lane 13 : Wild type variety FM966 - Eco RV digested Lane 14 : Wild type variety FM966 - Eco RV digested + an equimolar amount of pTEM12 - Eco RV digested Lane 15 : Empty lane Lane 16 : Phage Lambda - Pst I digested Lane 17 : Phage Lambda - Hind III digestedTwinLink Cotton USDA Petition

Page 174 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.4 (Continued) Cry2Ae cotton event GHB119 Insert verification - Hybridization

resultsTwinLink Cotton USDA Petition

Page 175 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 3. Genetic stability of the inserte d DNA in Cry1Ab cotton event T304-40

Southern blot analysis was performed to assess the impact of environmental conditions or different backgrounds on the structural stability of the transgenic sequence and to assess the structural stability over different generations.

To demonstrate the structural stability of cotton event T304-40, genomic DNA was prepared from several individual plants of different generations and different genetic backgrounds, and from plants grown from seeds harvested at three different locations. The isolated DNA was digested with the restriction enzyme Eco RV, which has two recognition sites in the integrated DNA fragment. Probing Eco RV digested genomic T304-40 DNA with the cry 1Ab probe (Figure 3.5) showed all three expected fragments in all samples tested. Two of these fragments represent the junctions between the transgenic sequences and the plant DNA sequences and one represents an internal fragment.

Table 3.5 summarizes the expected and obtained hybridization results. The hybridization results of the stability analysis over different genetic backgrounds are presented in Figure 3.5, over different generations in Figure 3.6, and over different environmental conditions in Figure

3.7.TwinLink Cotton USDA Petition

Page 176 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 3.5 Expected and obtained hybridization fragments – event T304-40

Samples Condition tested Seed lot N° of plants Expected fragment sizes (*) Expected

results

obtained

? Description

Background A 32CON0466 20

Background C315 32CON0467 20

Background B 32CON0468 20

Background C 32CON0469 20

Location Vinyols / 11

Location Camarles / 18

Location Santa Oliva / 18

Generation F 1 05GAGH000743 15

Generation BC 1 F 1 05GAGH000742 14

Generation BC 2 F 2 05GAGH000741 12 Test item samples

Generation BC 2 F 2 05GAGH000740 18 ca 3100 bp

3476 bp

ca 6800 bp Yes

Yes

Yes 5’ integration fragment

Internal fragment

3’ integration fragment

DNA negative

control Wild type variety FM966 (Seed lot 04GAGH003113) No hybridization Yes Negative

control

DNA positive

control Wild type variety FM966 + an equimolar amount pTDL008 14393bp Yes Positive

control fragment (*) Fragment sizes as determined in the detailed insert characterization of T304-40.TwinLink Cotton USDA Petition

Page 177 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.5 Hybridization results of the stability analysis of cotton event T304-40 in different genetic backgroundsTwinLink Cotton USDA Petition

Page 178 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.6 Hybridization results of the stability analysis of cotton event T304-40 over different generationsTwinLink Cotton USDA Petition

Page 179 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.7 Hybridization results of the stability analysis of cotton event T304-40 over

different environmental conditionsTwinLink Cotton USDA Petition

Page 180 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 4. Genetic stability of the inserte d DNA in Cry2Ae cotton event GHB119

The structural stability of Cry2Ae cotton event GHB119 over different generations, in different backgrounds, and over different environmental condi tions was tested by means of Southern blot analysis. Genomic DNA was prepared from severa l individual plants of three consecutive generations and two different genetic backgrounds. The impact of environment was assessed by analyzing the progeny of plants cultivated at six different field locations. The isolated DNA was digested with the restriction enzyme Eco RV, which has two recognition sites in the transforming DNA. Hybridization of these samples with the T-DNA probe revealed the expected profile in all tested samples. This demonstrat es the stability of cotton event GHB119 at the genomic level in different generations, different genetic backgrounds, and over different

environmental conditions.

Hybridization results are shown in Figures 3.8 and 3.9, and are summarized in Tables 3.6 and

3.7.TwinLink Cotton USDA PetitionPage 181 of 205CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 3.6 GHB119 Structural stability over generations and genetic backgrounds - Expected and obtained hybridizationfragmentsSamplesCondition testedSeedlotN° of plantsExpectedfragment sizes (*)Expectedfragmentsobtained ?DescriptionBackground Coker 312 05GC03 22Generation F105GC16 4Generation BC1F105GC33 18Test itemsamplesGeneration BC2F105GC59 13ca. 3380 bp1373 bpca. 8890 bpYesYesYes5’integration fragmentinternal fragment3’integration fragmentDNA negativecontrolWild type variety FM966 (Seedlot 04GAGH003113) No hybridization YesDNA positivecontrolWild type variety FM966 + an equimolar amount pTEM121373bp10893 bpYesYesPositive control fragments(*) Fragment sizes as determined in the detailed insert characterization of GHB119Table 3.7 GHB119 Structural stability over environments - Expected and obtained hybridization fragmentsSamplesCondition testedSeedlotN° ofplantsExpectedfragment sizes (*)Expectedfragmentsobtained ?DescriptionLocation Chula, Georgia CY06B001-201-31 13Location Newport, Arkansas CY06B001-402-32 12Location Proctor, Arkansas CY06B001-403-33 10Location Senatobia, Mississippi CY06B001-404-32 10Location East Bernard,Texas CY06B001-605-33 14Test itemsamplesLocation Levelland, Texas CY06B001-806-33 14ca. 3380 bp1373 bpca. 8890 bpYesYesYes5’integration fragmentinternal fragment3’integration fragmentDNA negativecontrolWild type variety FM966 (Seedlot 04GAGH003113) No hybridization YesDNA positivecontrolWild type variety FM966 + an equimolar amount pTEM121373bp10893 bpYesYesPositive control fragments(*) Fragment sizes as determined in the detailed insert characterization of GHB119TwinLink Cotton USDA Petition

Page 182 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 23130 bp
14057 bp 9416 bp

6557 bp

5077 bp 4749 bp 4507 bp

2838 bp

2459 - 2443 bp 2322 bp 2140 bp 1986 bp

1700 bp

1159 bp 1093 bp

805 bp 345678

2459-2443 bp

2140 bp 1986bp

1093 bp 23130 bp 14057 bp 11501 bp 9416 bp

6557 bp 5077 bp 4749 bp 4507 bp

2838 bp

2322 bp

1700 bp

1159 bp

805 bp 3 4 5 6 7 9 11 12 13 10 8 14 17 19 18 15 20 21 22

16 2459-2443 bp

2140 bp 1986bp

1093 bp 23130 bp 14057 bp 11501 bp 9416 bp

6557 bp

5077 bp 4749 bp 4507 bp

2838 bp

2322 bp

1700 bp

1159 bp

805 bp 3 4 5 6 7 9 11 12 13 10 8 14 17 19 18 15 20 21 22

16 23 24 26 25

3

6557 bp

4507 bp

2140 bp 1986 bp

1700 bp 14057 bp

9416 bp

4749 bp

2459-2443 bp 23130 bp

11501 bp

5077 bp

2838 bp

2322 bp

1159 bp 1093 bp 17 4 5 6 7 8 9 10 11 12 13 14 15 16 

### Background Coker 312 A B C

A B C A B C

A B C 

### Generation F1

Generation BC1.F1 Generation BC2.F1

A: GHB119 samples; B: Negative control; C: Positive control



### Figure 3.8 Structural stability of cotton event GHB119 over genetic backgrounds and generations - Hybridization resultsTwinLink Cotton USDA Petition

Page 183 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION A: GHB119 samples; B: Negative control; C: Positive control


### Figure 3.9 Structural stability of event GHB119 over environments - Hybridization

resultsTwinLink Cotton USDA Petition

Page 184 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 5. Genetic Stability of TwinLink cotton (events T304-40 x GHB119)

The genetic stability of the inserted DNA in Cry1Ab cotton event T304-40 and in Cry2Ae cotton event GHB119 cotton was demonstrated in plants grown in different genetic backgrounds, in a single genetic background grown for multiple generations, and in a single genetic background grown in different environments. Southern blot analysis was used to determine whether DNA rearrangements occurred during the production of TwinLink from a conventional breeding cross between T304-40 and GHB119 cotton. T304-40 contains the cry1Ab and bar genes on a single genetic insert. GHB119 contains the cry2Ae and bar genes on a single genetic insert.

Genomic DNA was isolated from 20 individual Twi nLink plants. Two separate aliquots of each plant DNA extract were digested with the rest riction enzyme EcoRV. EcoRV recognizes the inserted DNA of each event, thus generating restriction profiles that are characteristic for each parent (T304-40 and GHB119) and TwinLink.

DNA from non-transgenic cotton plants was used as a negative control. This DNA was also supplemented with vector DNA containing either the cry1Ab gene or the cry2Ae gene to serve as a positive control on their respective Southern blots. Each blot also contained the positive control DNA from the appropriate parent. The positive control for T304-40 analysis was genomic DNA prepared from WT leaf material mixed with an equimolar amount of pTDL008 plasmid DNA and digested with EcoRV. The posi tive control for GHB119 analysis was genomic DNA prepared from WT leaf material mixed wi th an equimolar amount of pTEM12 plasmid DNA and digested with EcoRV. Probing DNA digests of the parents and the cross with labeled DNA probes containing the cry1Ab and cry2Ae genes, respectively, revealed the expected restriction fragments in all tested samples.

The three expected fragments were observed in the DNA from all 20 TwinLink cotton plants digested with EcoRV and hybridized with the cry1Ab probe (Figure 3.10). The EcoRV restriction of TwinLink genomic DNA yields four fragments t hat can be detected by hybridization with the GHB119 T-DNA probe (Figure 3.11). Three of the fragments are produced by the GHB119 event DNA in TwinLink. A fourth fragment (6200 bp) results from the presence of the T304-40 event in TwinLink cotton. This fourth fragment is not detected in the GHB119 parent DNA (Lane 21). The four expected fragments were observed in the DNA from all 20 TwinLink cotton plants digested with EcoRV and hybridized with the GHB119 T-DNA probe.TwinLink Cotton USDA Petition

Page 185 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.10 Demonstration of the stability of TwinLink cotton for the cry1Ab DNA

insert.

Genomic DNA was isolated from TwinLink cotton pl ants and from the non-transgenic counterpart (Coker 315). Genomic DNAs were digested with Eco RV and probed with part of the T-DNA (1822 bp fragment of pTLD008).

Lane 1: TwinLink – plant 1 – Eco RV Lane 12: TwinLink – plant 12 – Eco RV Lane 2: TwinLink – plant 2 – Eco RV Lane 13: TwinLink – plant 13 – Eco RV Lane 3: TwinLink – plant 3 – Eco RV Lane 14: TwinLink – plant 14 – Eco RV Lane 4: TwinLink – plant 4 – Eco RV Lane 15: TwinLink – plant 15 – Eco RV Lane 5: TwinLink – plant 5 – Eco RV Lane 16: TwinLink – plant 16 – Eco RV Lane 6: TwinLink – plant 6 – Eco RV Lane 17: TwinLink – plant 17 – Eco RV Lane 7: TwinLink – plant 7 – Eco RV Lane 18: TwinLink – plant 18– Eco RV Lane 8: TwinLink – plant 8 – Eco RV Lane 19: TwinLink – plant 19 – Eco RV Lane 9: TwinLink – plant 9 – Eco RV Lane 20: TwinLink – plant 20 – Eco RV Lane 10: TwinLink – plant 10 – Eco RV Lane 21: T304-40 parent – Eco RV Lane 11: TwinLink – plant 11 – Eco RV Lane 22: WT variety Coker 315 plus 1 copy pTDL0008 – Eco RV Lane 23: WT variety Coker 315 – Eco RVTwinLink Cotton USDA Petition

Page 186 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.11 Demonstration of the stability of TwinLink cotton for the cry2Ae DNA

insert.

Genomic DNA was isolated from TwinLink cotton pl ants and from the non-transgenic counterpart (Coker 315). Genomic DNAs were digested with Eco RV and probed with part of the T-DNA (4346 bp fragment of pTEM12).

Lane 1: TwinLink – plant 1 – Eco RV Lane 12: TwinLink – plant 12 – Eco RV Lane 2: TwinLink – plant 2 – Eco RV Lane 13: TwinLink – plant 13 – Eco RV Lane 3: TwinLink – plant 3 – Eco RV Lane 14: TwinLink – plant 14 – Eco RV Lane 4: TwinLink – plant 4 – Eco RV Lane 15: TwinLink – plant 15 – Eco RV Lane 5: TwinLink – plant 5 – Eco RV Lane 16: TwinLink – plant 16 – Eco RV Lane 6: TwinLink – plant 6 – Eco RV Lane 17: TwinLink – plant 17 – Eco RV Lane 7: TwinLink – plant 7 – Eco RV Lane 18: TwinLink – plant 18– Eco RV Lane 8: TwinLink – plant 8 – Eco RV Lane 19: TwinLink – plant 19 – Eco RV Lane 9: TwinLink – plant 9 – Eco RV Lane 20: TwinLink – plant 20 – Eco RV Lane 10: TwinLink – plant 10 – Eco RV Lane 21: GHB119 parent – Eco RV Lane 11: TwinLink – plant 11 – Eco RV Lane 22: WT variety Coker 315 plus 1 copy pTDL0008 – Eco RV Lane 23: WT variety Coker 315 – Eco RVTwinLink Cotton USDA Petition

Page 187 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 6. Absence of vector backbone in Cry1Ab cotton event T304-40

Genomic DNA isolated from T304-40 plants was digested with either Eco RV or Not I restriction enzymes and analyzed by Southern blot. Sev en overlapping vector backbone probes covering the complete vector backbone sequence were assessed. A positive control hybridization was performed using a T-DNA probe.

A schematic representation of the restriction fragments and hybridization probes is given in Figure 3.12. An overview of the expected and obtained hybridization results is presented in Table 3.8. The obtained results are presented in Figure 3.13.

The absence of hybridization signal with the vector backbone probes demonstrates the absence of vector backbone sequences, while the positive control hybridization assures that the experimental conditions for this Southern blot analysis allowed detection of the integrated DNA fragments.

EcoRV

T-DNA

PT012 ca. 6300 bp > 2069 bp

T304-40- Eco RV

EcoRV Not I

PT012 ca. 3100 bp > 7467 bp

ca. 3500 bp T304-40- Not I



### Figure 3.12 Absence of vector backbone in cotton event T304-40 -- Schematic overview of the hybridization strategyTwinLink Cotton USDA PetitionPage 188 of 205CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 3.8 Absence of vector backbone in event T304-40 - Expected and obtained hybridization resultsT304-40 - EcoRVT304-40 - NotIWT (FM966) - NotIWT - NotI + 0.1; 1;or 10 copies pTDL008 - NotIObtained hybridization fragments(bp)Probe templateIDExpectedhybridizationfragments(bp)Obtainedhybridizationfragments(bp)Expectedhybridizationfragments(bp)Obtainedhybridizationfragments(bp)Expectedhybridizationfragments(bp)Obtainedhybridizationfragments(bp)Expectedhybridizationfragments(bp)0.1 copy1 copy10 copiesPT002 none none none none none none1290(1532)154041290540412901532540412901532540461672PT003 none none none none none none12901532129015321290153212901532PT005 none none none none none none 6167 6167 6167 6167PT007 none none none none none none 5404 5404 5404 5404PT008 none none none none none none1532616715326167153261671532540426167PT009 none none none none none none 6167 6167 6167 6167PT010 none none none none none none 6167 6167 6167 6167PT012ca. 6300ca. 3500ca. 30006300 ± 2003500 ± 1003100 ± 100> 7467 >14K none none 5404 5404 5404 54041Probably not visible due to limited overlap (85 bp) between PT002 and this vector fragment.2Background hybridizationTwinLink Cotton USDA Petition

Page 189 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.13 Absence of vector backbone in cotton event T304-40– Southern blot results

Lane 1: Phage Lambda – Pst I digested (not shown)

Lane 2: Cotton event T304-40– Eco RV digested

Lane 3: Cotton event T304-40– Not I digested Lane 4: Non-trangenic control – Not I digested Lane 5: Non-trangenic control + 0.1 copy pTDL008 – Not I digested Lane 6: Non-trangenic control + 1 copy pTDL008 – Not I digested Lane 7: Non-trangenic control + 10 copies pTDL008 – Not I digestedTwinLink Cotton USDA Petition

Page 190 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.13 (Continued) Absence of vector backbone in cotton event T304-40–
Southern blot resultsTwinLink Cotton USDA Petition

Page 191 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 7. Absence of vector backbone in Cry2Ae cotton event GHB119

Genomic DNA isolated from GHB119 plants was digested with either Eco RV or Dra I restriction enzymes and analyzed by Southern blot using pTEM12 vector backbone probes. Five overlapping vector backbone probes covering the complete vector backbone sequence were assessed. A positive control hybridization was performed using a T-DNA probe. The absence of hybridization signal with the vector backbone probes demonstrates the absence of vector backbone sequences, while the positive control hybridization assures that the experimental conditions for this Southern blot analysis allowed an effective detection of the integrated DNA fragments (Figure 3.15)

A schematic representation of the restriction fragments and hybridization probes is given in Figure 3.14. An overview of the expected and obtained hybridization results is presented in Table 3.9. The obtained results are presented in Figure 3.15.

Ec o RV

PT026 PT001

PT002

PT003

PT004

PT005 Eco RV

LB RB

npt I homology

region ORI ColE1 ORI pVS1 aadA pTEM12: X GHB119

- Eco RV

GHB119

- Dra I > 4345 bp

10893 bp pTEM12

- Eco RV > 1829 bp

X > 1143 bp 1373 bp

X X 1373 bp

3'nos bar

Pcsvmv P35S2

5'cab22L TPssuA t

cry2Ae 3'35S Ec o RV

PT026 PT001

PT002

PT003

PT004

PT005 Eco RV

LB RB

npt I homology

region ORI ColE1 ORI pVS1 pTEM12: X GHB119

- Eco RV

Dra I GHB119

- > 4345 bp

10893 bp pTEM12

- Eco RV > 1829 bp

X > 1143 bp 1373 bp

X X 1373 bp

3'nos bar

Pcsvmv P35S2

5'cab22L TPssuA t

cry2Ae



### Figure 3.14 Absence of vector backbone in cotton event GHB119 -- Schematic overview of the hybridization strategyTwinLink Cotton USDA PetitionPage 192 of 205CONTAINS NO CONFIDENTIAL BUSINESS INFORMATIONTable 3.9 Absence of vector backbone in event GHB119 -- Expected and obtained hybridization fragmentsGHB119 - EcoRVGHB119 - DraIWT (FM966) - EcoRVWT (FM966) - EcoRV +0.1 and 1 copy pTEM12 - EcoRVObtained hybridizationfragmentsProbe template IDExpectedhybridization fragmentsObtainedhybridization fragmentsExpectedhybridization fragmentsObtainedhybridization fragmentsExpectedhybridizationfragmentsObtainedhybridization fragmentsExpectedhybridizationfragments0.1 copy1 copyPT001-2 / / / / / / 10893 bp 10893 bp 10893 bpPT002-2 / / / / / / 10893 bp 10893 bp 10893 bpPT003-2 / / / / / / 10893 bp 10893 bp 10893 bpPT004-3 / / / / / / 10893 bp 10893 bp 10893 bpPT005-2 / / / / / / 10893 bp 10893 bp 10893 bp3380 bp1373 bp8890 bp3380 bp1373 bp8890 bp4430 bp 4430 bp / /10893 bp1373 bp10893 bp1373 bp310893 bp1373 bpPT026-23380 bp1373 bp8890 bpobtainedobtainedobtained4430 bp obtained / /10893 bp1373 bp10893 bpnot visible10893 bpobtained3Faint band, not always visible on all blotsTwinLink Cotton USDA petition

Page 193 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Figure 3.15 Southern blot analysis of GHB119 – Absence of vector backbone

Lane 1: Phage Lambda – Pst I digested

Lane 2: Cotton event GHB119 – Eco RV digested

Lane 3: Cotton event GHB119 – Dra I digested Lane 4: Wild type cotton variety FM966 – Eco RV digested Lane 5: Wild type cotton variety FM966 – Eco RV digested + 0.1 copy pTEM12 – Eco RV digested Lane 6: Wild type cotton variety FM966 – Eco RV digested + 1 copy pTEM12 – Eco RV digested Lane 7: Phage Lambda – Pst I digestedTwinLink Cotton USDA petition

Page 194 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

23456

1093 bp 14057 bp

4749 bp 5077 bp

4507 bp

1159 bp

805 bp 11497 bp

2838 bp

2140 bp

1700 bp 1986 bp 2459-2443 bp

1093 bp 14057 bp

4749 bp 5077 bp

4507 bp

1159 bp

805 bp 11497 bp

2838 bp

2140 bp

1700 bp 1986 bp 2459-2443 bp 23456

Probe: PT005-2 Probe: PT026-2 Lane 1: Phage Lambda – Pst I digested

Lane 2: Cotton event GHB119 – Eco RV digested
Lane 3: Cotton event GHB119 – Dra I digested Lane 4: Wild type cotton variety FM966

Eco RV digested Lane 5: Wild type cotton variety FM966

Eco RV digested + 0.1 copy pTEM12 –

Eco RV digested Lane 6: Wild type cotton variety FM966

Eco RV digested + 1 copy pTEM12 –

Eco RV digested Lane 7: Phage Lambda – Pst I digested



### Figure 3.15 (Continued) Southern blot analysis of cotton event GHB119 – Absence of

vector backboneTwinLink Cotton USDA petition

Page 195 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 8. Cry1Ab cotton event T304-40 pre-insertion locus

a. The flanking sequences

The flanking sequences of T304-40 were determined by PCR. Based on this sequence information, a PCR fragment was generated usi ng genomic DNA from wild type cotton plants as template and a pair of primers specific for the 5’ and 3’ flanking sequence. This fragment (828 bp) corresponds to the region of the original cotton genome where the insertion took place,referred to as the pre-insertion locus. Figure 3.16 gives a schematic representation of the preinsertion locus and the inserted DNA surrounded by the flanking sequences.

Figure 3.16 Schematic drawing of the T304-40 transgenic locus and pre-insertion locus 5' flanking

sequences (479 bp) RB

3'me1
(truncated)

Ps7s7
(truncated) 5'e1 cry1Ab 3'me1 3'me1
(truncated) cry1Ab 5'e1 Ps7s7 P35S3 bar 3'nos
(truncated)

3' flanking

sequences (320 bp) T304-40 - transgenic locus

T304-40 – pre-insertion locus 5' flanking

sequences (480 bp) 3' flanking

sequences (325 bp)

Target site Deletion (32 bp) RB

3'me1 cry1Ab 5'e1 Ps7s7 P35S3 bar 3'nos LB pTDL008 – T-DNA

Inserted transgenic sequences (9056 bp) RBTwinLink Cotton USDA petition

Page 196 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### b. Identification of potentially disrupted genes at the integration site

The sequence of the pre-insertion locus of cotton event T304-40 was subjected to a bioinformatic analysis to determine the possibility that endogenous genes are interrupted by the insertion of the transgenic sequences.

Homology was found between the cotton genomic DNA and small sequences from the cotton DNA database but no function was assigned to these sequences. Also homology was found with repetitive sequences indicating that the insertion of the T304-40 T-DNA took place in a region containing such elements.

In order to identify the presence of known func tional genes in the pre-insertion locus of event T304-40, a BLASTx similarity search was performed, but no known proteins were found.

Also no ORFs or genes were predicted. One putative promoter was found in the 5’ flanking sequence. As there are no genes found further downstream, this predicted promoter is most probably not biological active.

It can be concluded that, using the current databases and tools, no known genes were interrupted or influenced due to the insertion of transgenic DNA in the pre-insertion locus of

T304-40.

Figure 3.17 Schematic overview of the regulatory element search result on the preinsertion locus of T304-40

c. Identification of potentially unintended newly-created genes in the transformation

event

A bioinformatic analysis was performed to detect the possible presence of cryptic expression due to the formation of newly created open reading frames (ORFs) in the 5’, 3’and internal junction regions of cotton event T304-40. These junctions are formed by the T-DNA insertion from the pTDL008 vector, containing the cry 1Ab and bar gene cassettes into the cotton genome. Several

in silico tools were used to look for ORFs and regulatory elements taking into account the current scientific knowledge on gene expression.

- Two newly created chimeric ORFs were identified (GetORF analysis) 5’ flanking DNA Target

Site

Deletion 3’ flanking DNA 1 480 513 837

Putative promoter region 1 5’ flanking DNA Target

Site

Deletion 3’ flanking DNA 1 480 513 837

5’ flanking DNA Target

Site

Deletion 3’ flanking DNA 1 480 513 837

Putative promoter region 1TwinLink Cotton USDA petition

Page 197 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### - No putative newly created gene was predicted (FGENESH analysis) - Eleven promoters and promoter-like regions were identified (TSSP analysis) - None of the two ORFs will potentially be tran scribed by any of the predicted promoters - None of the two ORFs possesses a highly conserved RBS region, leading most probably

to absence of translation

Based on this analysis, the probability of newly created protein expression in the cotton event T304-40 is highly unlikely.TwinLink Cotton USDA petition

Page 198 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### 9 Cry2Ae cotton event GHB119 pre-insertion locus

a. The flanking sequences

The flanking sequences of GHB119 insert were determined by PCR. Based on this sequence information, a PCR fragment was generated from non-transgenic cotton using a pair of primers specific for the 5’ and 3’ flanking sequence. This fragment (684 basepairs) corresponds to theregion of the original cotton genome where the insertion took place, referred to as the preinsertion locus. Figure 3.18 shows a schematic representation of the pre-insertion locus and the inserted DNA surrounded by the flanking sequences.

Figure 3.18 Schematic overview of the transgenic and pre-insertion locus of the cotton

event GHB119

b. Identification of potentially disrupted genes at the integration site

The DNA of the pre-insertion locus of the cotton event GHB119 was sequenced and subjected to bioinformatic tools in order to identify endogenous cotton genes and/or regulatory elements that may be influenced by the insertion of the transgenic DNA fragment.

Homology was found with cotton genomic DNA but no function could be assigned to these sequences. Also homology was found with repetitiv e sequences indicating that the insertion of the GHB119 T-DNA took place in a region containing repetitive elements. In order to identify the presence of known functional genes in the pre-insertion locus of event GHB119, a BLASTx similarity search was performed, but no known proteins were found. 5' flanking seq. (358 bp)

LB

3' nos bar Pcsvmv

XYZ P35S2

5'cab22L TPssuAt cry2Ae RB 3' flanking

seq. (320 bp)

GHB119 – transgenic locus

Target Site Deletion (8 bp) 5' flanking seq. (367 bp) 3' flanking seq. (309 bp) GHB119 – pre insertion locus 3'35S Inserted transgenic sequences (4302 bp)TwinLink Cotton USDA petition

Page 199 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Using the prediction tools GetORF and FGENESH, no genes or ORFs were predicted that could be disrupted by the introduction of the transgenic DNA in the genome.

The surrounding sequence of the pre-insertion locus was subjected to a homology analysis comparing it with regulatory elements involved in transcription. Two promoter sequences were predicted (Figure 3.19). Since no known endogenous genes were found or predicted, it is highly unlikely that these predicted promoters are biologically active.

These bioinformatic analyses on the pre-insertion locus of cotton event GHB119, using the current databases and bioinformatic tools, allow us to conclude that the probability that a functional gene was interrupted is highly unlikely.

5’ flanking DNA Target

Site

Deletion 3’ flanking DNA 1 367 376 684

5’ flanking DNA Target

Site

Deletion 3’ flanking DNA 1 367 376 684

Putative promoter region 1

Putative promoter region 2



### Figure 3.19 Schematic overview of the regulatory element search results on the preinsertion locus of GHB119

c. Identification of potentially unintended newly-created genes

A bioinformatic analysis was performed to detect the possible presence of cryptic expression due to the formation of newly created open reading frames (ORFs) in the 5’ or 3’ junction region in event GHB119. These junctions are formed by the insertion of a cry 2Ae - bar gene construct into the cotton genome.

Several in silico tools were used to look for ORFs and regulatory elements taking into account the current scientific knowledge of gene expression. Three newly created chimeric ORFs were identified. The surrounding nucleotides of the three ORFs were also subjected to a homology analysis, comparing them with regulatory elements important for transcription and translation. Three newly created chimeric ORFs were identified. The surrounding nucleotides of the three ORFs were also subjected to a homology analys is, comparing them with regulatory elements important for transcription and translation.

- ORF-1 at the 3’ junction (sense strand): two promoters were predicted at the 5’ end of ORF-1, however more than 200 bp away from the ATG codon. No homology was found

with a RBS.TwinLink Cotton USDA petition

Page 200 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### - ORF-2 at the 3’ junction (sense strand): two promoters were predicted at the 5’ end of ORF-2 however more than 200 bp away from the ATG codon. No homology was found

with a RBS. - ORF-3 at the 5’ junction (antisense strand): no promoters were predicted at the 5’ end of ORF-3 and no homology was found with a RBS.

ORF-3 is very likely inactive since no regulatory elements that could lead to transcription or translation were predicted. Initiation of transcription of ORF-1 and ORF-2 cannot be excluded, but translation is very unlikely.

One promoter crossing the 5’ junction was predict ed but it is highly unlikely that this predicted promoter region would lead to a changed expression level of endogenous genes.

As not all regulatory elements were present in the DNA sequence at the 5’ or 3’ end of the newly created ORFs, the probability of an expression of newly created proteins due to the insertion of DNA containing the cry 2Ae - bar gene construct, is highly unlikely.TwinLink Cotton USDA petition

Page 201 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Appendix 4. REGIO NAL AGRONOMIC DATA

For ease of understanding: - “GA treated” means “treated with glufosinate ammonium” - “Sprayed” means “sprayed with conventional insecticides”TwinLink Cotton USDA petition

Page 202 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 4.1 Agronomic data across regions from BCS Regulatory trials in 2007

Growth Habit & Phenotype Significance Agronomic Parameter (locations)* Coker TwinLink

GA treated TwinLink

Non treated LSD CV Plant Stand (7) 2. 11b 2.73a 2.79a 0.15 1.84 Days to bloom (7) 54.19 a 51.81b 51.86b 2.11 1.24 % open bolls (7) 47.86b 59.86a 58.95b 9.73 5.40 Plant height in cm (6) 101.69a 96.87b 96. 59b 3.56 1.02 Total # of plant nodes (7) 17.89a 16.97b 16. 95b 0.76 1.36 Height to Node Ratio (6 ) 5.65a 5.63a 5.63a 0.21 1.04 # of 1st position bolls (7) 4.40a 4.70a 4.61a 1.10 7.40

% retention 1 st position (2) 100.00b 167.11a 171.28a 9.57 0.98 # of total bolls (7) 9.51a 9.05a 8. 89a 1.81 6.10 Strain Uniformity (7) 1.43a 1.43a 1.43a 0 0 Boll Type (7) 4.29a 4.29a 4.29a 0 0 Lodging (7) 1.14a 1.14a 1.14a 0 0 * Numbers in parentheses indicate the number of sites used to generate data for that parameter



### Table 4.2 Yield and Fiber Quality data across regions from BCS Regulatory trials in

2007

Yield and Fiber Quality Significance Agronomic Parameter (locations)* Coker TwinLink

GA treated TwinLink

Not treated LSD CV Yield (7) 608.28a 619. 12a 608.00a 78.59 3.96 Fiber Length (7) 1.21a 1.20b 1.19b 0.02 0.46 Fiber Strength (7) 31.73a 31.74a 31.82a 0.57 0.55 Fiber Uniformity % (7) 84.93 a 84.31a 84.37a 0.69 0.25 Micronaire (7) 4.26a 4. 50a 4.55a 0.31 2.13 % lint (7) 38.65a 39.02a 38.71a 0.64 0.51 * Numbers in parentheses indicate the number of sites used to generate data for that parameter



### Table 4.3 Agronomic data from BCS Development Efficacy trials in 2007

Growth Habit & Phenotype
Significance Agronomic parameter (locations)* Coker

Not sprayed Coker Sprayed TwinLink Not sprayed TwinLink Sprayed LSD CV

Plant Stand (1) 1.31cd 1. 26d 1.49bc 1.36c d 0.21 10.0 days to bloom (1) 60.00ab 61.00ab 61.33ab 62. 00a 2.69 2.52 # open bolls (1) 88.75 ab 70. 00 e 82.50 cd 90.00ab 5.40 4.33 Days to 1 st open bolls (1) 123.33a 123. 00a 123.33a 123.00a 1.33 0.62 Lodging (1) 8.00a 7.50a 5.00b 3.75bc 1.58 22.13 * Note: All data from this table from a single location, Sellers, South CarolinaTwinLink Cotton USDA petition

Page 203 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 4.4 Yield and Fiber Quality data across regions from BCS Development Efficacy

trials in 2007

Yield and Fiber Quality
Significance Agronomic

Parameter (locations)* Coker Not sprayed Coker Sprayed TwinLink Not sprayed TwinLink Sprayed LSD CV

Yield (5) 815.72e 1009.30c 860. 14de 968.66cd 118.60 2.95 Fiber Length (1) 1.23a 1.23a 0.04 1.85 Fiber strength (1) 31.50b 32.30b 2.23 4.21 Fiber uniformity % (1) 87.05b 86.98b 0.98 0.7 Micronaire (1) 4.65b 4.75b 0.37 4.78 % lint (3) 38.94cd 38.76d 39.51bc 39.45bc 0.61 0.31 * Numbers in parentheses indicate the number of sites used to generate data for that parameter Non-sprayed plots were analyzed from only one location



### Table 4.5 Agronomic data across regions from BCS Regulatory trials in 2008

Growth Habit & Phenotype Significance Agronomic Parameter (locations)* Coker TwinLink

GA treated TwinLink

Not treated LSD CV Plant stand (plants/ft) (2 ) 1.20b 1.55a 1.53a 0.02 0.25 Days to bloom (2) 45.0a 45.0a 45.0a 0 0 Days to 1 st open boll (2) 98.0a 98.0a 98.0a 0 0 % open bolls (2) 60.0a 60.0a 60.0a 0 0 Plant height in cm (2) 32.17a 32.33a 30. 33b 1.02 0.48 Total # of plant nodes (2) 18.83a 18.00b 17. 17c 0.62 0.52 Height to Node Ratio (2 ) 2.12a 1.71b 1.62b 0.35 2.89 # of 1st position bolls (2 ) 7.50c 8.83a 8.33b 0.41 0.74 # of total bolls (2) 15. 33b 20.17a 18.50a 2.85 2.37 Strain Uniformity (2) 5.00a 5.00a 5.00a 0 0 Leaf Uniformity (2) 1.00a 1.00a 1.00a 0 0 Boll Type (2) 5.00a 5.00a 5.00a 0 0 Disease reaction (2) 1.00a 1.00a 1.00a 0 0 Lodging (2) 3.00a 3.00a 3.00a 0 0 * Numbers in parentheses indicate the number of sites used to generate data for that parameterTwinLink Cotton USDA petition

Page 204 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 4.6 Yield and Fiber Quality data across regions from BCS (Regulatory) trials in

2008

Yield and Fiber Quality Significance Agronomic

Parameter (locations)* Coker TwinLink

GA treated TwinLink

Not treated LSD CV

Yield (2) 689.83a 805.3 3a 812.67a 188.00 3.66 Fiber Length (2) 1.29a 1. 30a 1.28a 0.02 0.22 Fiber strength (2) 35.58 34.23 a 34.77a 1.87 0.80 Fiber uniformity % (2) 87.32b 87.87a 86.78c 0.14 0.02 Fiber elongation (2) 5.30a 5.47a 5.65a 0.41 1.11 Micronaire (2) 3.92a 4.12a 3.77a 0.45 1.70 % lint (2) 36.78a 35.97b 35.97b 0.58 0.24 * Numbers in parentheses indicate the number of sites used to generate data for that parameter



### Table 4.7 Agronomic data across regions from University and USDA Efficacy trials in

2008

Growth Habit and Phenotype Significance Agronomic Parameter (locations)* Coker Not sprayed Coker Sprayed TwinLink Not sprayed TwinLink Sprayed LSD CV Plant Stand (3) 2.98b 3.21a b 3.23ab 3.57a 0.36 1.65 Plot uniformity (2) 1.00b 1.50ab 2.00a 1. 25ab 0.76 40.51 Plant Height in cm (2) 92.04bc 94.68ab 89.57c 94. 42ab 3.79 0.50 Total nodes (2) 22.73a 23.23a 20.70c 23.00a 0.60 0.39 Height-Node ratio (2) 2.27ab 2.25ab 2.45a 1.98c 0.25 1.38

1 st position bolls (2) 6.55bc 6. 75ab 6.65b 6.25bc 0.65 1.23

2 nd position bolls (2) 3.45ab 3.08a bc 3.53a 3.23abc 0.41 1.65

3 rd position bolls (1) 3. 50a 3.35a 3.05a 4. 00a 1.96 41.74 Total Bolls (2) 13.98a 12.95 ab 13.08ab 12.38b 1.25 1.21

% retention 1 st position (2) 58.93d 67.13b 62.84c 59.89d 2.40 0.47

% retention 2 st position (2) 29.65a 25.05ab 26.49ab 22.87ab 7.68 4.01

% retention 3rd position (1) 29.35a 24. 44a 24.49a 24.45a 18.81 48.81 Plant Uniformity (1) 1. 00b 1.50ab 2.00a 1.5b 0.50 29.18 Disease Reaction (1) 1.00a 1.00a 1.00a 1.00a 0 0 Lodging (1) 1.00a 1.00a 1.00a 1.00a 0 0 Leaf morphology (1) 1.00a 1.00a 1.00a 1.00a 0 0 Plant morphology (1) 1.00a 1.00a 1.00a 1.00a 0 0 Boll morphology (1) 1.00a 1.00a 1.00a 1.00a 0 0 * Numbers in parentheses indicate the number of sites used to generate data for that parameter NOTE: Data received from only one location was analyzed individually.TwinLink Cotton USDA petition

Page 205 of 205

CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION 

### Table 4.8 Yield and Fiber Quality data across regions from University and USDA Efficacy trials in 2008

Yield and Fiber Quality
Significance Agronomic Parameter (locations)* Coker Not sprayed Coker Sprayed TwinLink Not sprayed TwinLink Sprayed LSD CV Yield (2) 1078.66bc 1070.26bc 1030.46c 1018. 46c 58.57 0.69 Fiber Length (1) 1.11a 1.11a 1.11a 1. 11a 0.04 2.3 Fiber strength (1) 30.90bc 33. 60ab 30.05c 30.53 bc 3.53 7.46 Fiber uniformity % (1) 82.48 ab 83.15a 82.40ab 81.75b 1.07 0.88 Micronaire (1) 5.45a 5.10a bc 5.25ab 5.20abc 0.36 4.73 % lint (1) 44.08bcd 43.84cd 45.40b 44.83bcd 1.46 2.19 * Numbers in parentheses indicate the number of sites used to generate data for that parameter



### Table 4.9 Agronomic data across regions from BCS (Development) Efficacy trials in

2008

Growth Habit & Phenotype
Significance Agronomic Parameter (locations)* Coker Not sprayed Coker Sprayed TwinLink Not sprayed TwinLink Sprayed LSD CV Stand uniformity (3) 1.00a 1.00a 1.00a 1.00a 0 0 Plant uniformity (3) 1.00a 1.00a 1.00a 1.00a 0 0 Disease rating (3) 1.00a 1.00a 1.00a 1.00a 0 0 Leaf morphology (3) 1.00a 1.00a 1.00a 1.00a 0 0 Boll morphology (3) 1.00a 1.00a 1.00a 1.00a 0 0 Plant morphology (3) 1.00a 1.00a 1.00a 1.00a 0 0 Plant Height in cm (2) 91.18bc 88.15cd 90.71bc 92.32b 3.57 0.49 Total nodes (2) 17.73b 17.53b 17.63 b 18.15ab 0.83 0.58
Bolls P1 (2) 4.30d 4.58cd 4.80bc 4.60cd 0.46 1.21
Bolls P2 (2) 2.40bc 2.53abc 2.65a b 2.30cd 0.32 1.65
Total bolls (2) 10.45ab 10.55ab 9.48bc 9.58bc 1.40 1.77 Height Node Ratio (2) 3.62a b 3.75ab 3.74ab 3.51bc 0.28 0.98 Boll Retention P1 (2) 46.12c 50. 18bc 52.80ab 50. 86bc 6.07 1.47 Boll Retention P2 (2) 36.48b 38.14ab 40.78a 39. 53ab 4.14 1.33 * Numbers in parentheses indicate the number of sites used to generate data for that parameter



### Table 4.10 Yield and Fiber Quality data from BCS Development Efficacy trials in 2008

Significance Agronomic Parameter (locations)* Coker Not sprayed Coker Sprayed TwinLink Not sprayed TwinLink Sprayed LSD CV Yield (1) 1227.1c 1326.9bc 1334.6bc 1434.0ab 162.60 7.82 Fiber Length (1) 1.27a 1.25a b 1.22bc 1.22c 0.03 1.57 Fiber strength (1) 36.00b 35.80b 33.80c 34.20c 1.41 2.65 Fiber uniformity % (1) 86.10 a 85.30ab 84.80b 84.80b 1.07 0.84 Micronaire (1) 4.90ab 4. 90a 4.70b 4.70b 0.22 3.07 Lint % (1) 40.80cd 41.70bc 41.90bc 42.4b 1.29 2.04 * Numbers in parentheses indicate the number of sites used to generate data for that parameter Note: All data from this table from a single location in Sellers, South Carolina