TwinLink Cotton USDA Petition 

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Petition for Determination of Nonregulated Status for 

Insect-Resistant and Glufosinate Ammonium-Tolerant cotton: 

TwinLinkTM cotton (events T304-40 x GHB119) 

OECD Unique Identifier BCS-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, 2008 
TwinLink Cotton USDA Petition 

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SUMMARY 

Bayer CropScience LP (BCS) is submitting a Petition for Determination of Nonregulated Status 

to the Animal and Plant Health Inspection Service (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 TwinLink cotton, its parents, and any progeny derived 

from crosses of TwinLink cotton 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 (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 cry1Ab, cry2Ae 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 cry1Ab and cry2Ae 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 

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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 cotton 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 

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CERTIFICATION 

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

includes all information and views on which to base a determination, and that it includes 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: 919-549-3929 
TwinLink Cotton USDA Petition 

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ACRONYMS AND SCIENTIFIC 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 PCR 
TwinLink Cotton USDA Petition 

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TABLE OF CONTENTS 

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

CERTIFICATION................................................................................................................ 4

ACRONYMS AND SCIENTIFIC TERMS............................................................................ 5

TABLE OF CONTENTS ..................................................................................................... 6

LIST OF FIGURES........................................................................................................... 10

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

I. RATIONALE.......................................................................................................... 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 TRANSFORMATION SYSTEM ..................................................................... 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................................................................................... 27
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IV. CHARACTERIZATION OF TWINLINK COTTON (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 PROTEINS ....................................................... 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 INSERTED SEQUENCES ............................................... 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 THE MODIFIED ORGANISM.......................... 64

VIII. AGRONOMIC AND PHENOTYPIC EVALUATION ............................................... 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 ..................................................................... 69
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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 ..............................................................................................................................78

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 GROUNDS UNFAVORABLE ................................................... 94

XI. REFERENCES ...................................................................................................... 95

Appendix 1. FIELD TRIALS TERMINATION REPORTS 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 METHODS - PRODUCT CHARACTERIZATION ..... 131
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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. CHARACTERIZATION OF TWINLINK COTTON................................... 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 potentially disrupted genes at the integration 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 potentially disrupted genes at the integration site...............................................198

c. Identification of potentially unintended newly-created genes.......................................................199

Appendix 4. REGIONAL AGRONOMIC DATA........................................................... 201

 
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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 .......................52
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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.........92
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I. RATIONALE 

A. Basis for the Request for Determination 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 development 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 pest 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 and 
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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 crossing 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 naturally 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 proteins 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). Bt genes 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 Biopesticides 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 Cry1Ab 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 organism 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 derived from new plant varieties, including those 
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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. 
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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 primary 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, hollyhock, 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). 
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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 throughout 

the 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 particularly 

well adapted to the arid environmental conditions. The markets for ELS cotton are mainly highvalue products such as sewing thread and expensive apparel. 
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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 the 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 fluorescent 
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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 the 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 of 

herbicide 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 any 
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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 that 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 TwinLink 
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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 there 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 system 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. 
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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 T0 plant was crossed with 

conventional cotton in order to obtain homozygous and stable lines (BC3F3), 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. 
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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). 
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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. 
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pTEM12

12266 bp

2000

4000

6000

8000

10000

12000

LB 3'nos bar

Pcsvmv XYZ

P35S2

5'cab22L

TPssuAt

cry2Ae

3'35S

RB

nptI- fragment

ORI ColE1

ORI pVS1

aadA

Figure 2 Vector map of pTEM12 
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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 sequence 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 phosphinothricin acetyltransferase gene of 

Streptomyces hygroscopicus as described by Thompson et al. (1987). 

14096-14393 1-

12 

Clockwise 3 ́nos: sequence including the 3’ untranslated 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 phosphotransferase coding sequence of the nptI 

gene from transposon Tn903 (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 replication 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) 
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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 phosphinothricin 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 phosphotransferase coding sequence of the nptI

gene from transposon Tn903 (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 origin 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) 
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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. dakota
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IV. CHARACTERIZATION OF TWINLINK COTTON (EVENTS T304-40 x 

GHB119) 

A. Description, history and Mendelian inheritance 

T1 seed of transformation events T304-40 and GHB119 was harvested from self-pollinated T0

plants surviving a glufosinate-ammonium herbicide greenhouse screen. T1 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 T3 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 T3 homozygous 

seed (no segregation for tolerance) was used to produce homozygous T4 seed and was the 

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

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

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 (2005). Selfed T6 and T7 seed were used for the agronomic studies in 2007 and 2008, 

respectively. The TwinLink planting seed, individual F2 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 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. 

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 susceptible (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 F2

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

line. At the BC2F1 generation, the two events were combined and the progeny of this F1 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). 
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Cry1Ab/T0

Cry1Ab/T1

Cry1Ab/T6

Cry1Ab/T2

Cry1Ab/T7

Cry1Ab/T3 BC2F1

Cry1Ab/T4

Cry1Ab/T5

F1

BC1F1

Conventional 

Line (A)

X

9

9

9

9

9

9

9

Conventional 

Line

Conventional 

Line

X

X

Cry1Ab Breeding Diagram

Conventional 

Line

X

Cry2Ae/T0

Cry2Ae/T1

Cry2Ae/T6

Cry2Ae/T2

Cry2Ae/T7

Cry2Ae/T4

Cry2Ae/T5

9

9

9

9

9

9

9

Cry2Ae Breeding Diagram

BC2F1 Cry2Ae/T3

F1

BC1F1

Conventional 

Line (A)

X

X

X

Conventional 

Line

Conventional 

Line

Conventional 

Line

X

Cross of Cry1Ab and Cry2Ae

BC3F1 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

F1

TwinLink

9

2007

2008

TwinLink

Individual F2 plants 

selected and bulked

Figure 6 Breeding tree for the development of TwinLink cotton 
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Table 4 Segregation Analysis of the Independent Events in TwinLink cotton. 

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

At the BC2F1 generation, the two events were combined and the progeny of this F1 were analyzed by 

homozygosity PCR developed to identify the zygosity of each F2 plant. The individual plants were scored 

for presence or absence of the events. Four possible genotypes were identified; WT (wild type)/WT, or 

T304-40/WT, GHB119/WT and T304-40/GHB119. 

Parents of the F1 and zygosity 

of the TwinLink events Generation Ratio Observed a Expected 

χ2 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 F1 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. 
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B. Verification of the inserts and number 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 GHB119 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-cry2Ae

gene 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 EcoRV, which has two recognition sites in the integrated DNA 

fragment. Probing EcoRV 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). 
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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 assessed by analyzing the progeny of plants 

cultivated at 6 different field locations. The isolated DNA was digested with the restriction 

enzyme EcoRV, 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 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. 

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 origin 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 backbone 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 not 
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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 junction. 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 biologically active. 

It can be concluded that, using the current databases and tools, no known genes were 
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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 (ORFs) in the 5’, 3’and internal junction regions 

of cotton event T304-40. Two newly created chimeric 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 sequence obtained from this fragment shows that 

684 bp of the pre-insertion locus were determined, including 367 bp 5’ flanking sequences, 309 

bp 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 elements 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. 
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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. 
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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 Bt bacteria 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 epidemiological 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, 
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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 hydrolysis 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 consumed by humans and animals for 50 years, without 

any records of safety issues. 

iii. Cry2Ae protein familiarity 

As summarized in the previous sections, Cry proteins 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 from 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). Furthermore, it is very likely that this exposure 

is frequent through the consumption of roots and vegetables. Nevertheless, there is no 
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evidence of adverse effects related to the exposure to this bacterial species. 

ii. PAT protein familiarity

Safety data focused on PAT proteins have been extensively 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 regulatory 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 Bt activity. 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 insecticidal 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. 
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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 demethylphosphinothricin (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 safety evaluation based on Codex Alimentarius 

Commission (Codex; Alinorm 03/34A). As a basis, 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, indicating 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 known 
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allergens, as demonstrated by overall amino acid and epitope homology searches; 

• As expected, the Cry1Ab, Cry2Ae and PAT proteins 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 that 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 overall amino acid homology searches; 

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

intestinal fluids. This minimizes the likelihood that 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 cry1Ab, 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. 
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VI. EXPRESSION OF THE 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 fuzzy seed of TwinLink cotton 

Seven trials were conducted in 2007; the plants were grown under conditions typical of 

production 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 fuzzy 
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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 content 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. 
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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 

Protein * (as % of total crude protein) ± SD 

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 

Protein * (as % of total crude protein) ± SD 

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). 
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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 allergenicity 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 calculated 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 was 
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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 

kDa A B C

250-

100-

50-

37-

Lanes:

A- Kaleidoscope Marker

B- Cry1Ab Isolated from TwinLink Cotton Leaves

C- Cry1Ab Thrombin Standard

kDa A B C D

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

kDa A B C

250-

100-

50-

37-

Lanes:

A- Kaleidoscope Marker

B- Cry1Ab Isolated from TwinLink Cotton Leaves

C- Cry1Ab Thrombin Standard

kDa A B C

250-

100-

50-

37-

Lanes:

A- Kaleidoscope Marker

B- Cry1Ab Isolated from TwinLink Cotton Leaves

C- Cry1Ab Thrombin Standard

kDa A B C D

220-

120-

70-

50-

40-

30-

20-

kDa A B C D

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. 
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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-

kDa A B C

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-

kDa A B C

250-

150-

100-

50-

37-

20-

15-

Lanes:

A- Kaleidoscope Marker

B- Cry2Ae from TwinLink

C- Cry2Ae Standard

kDa A B C

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 Pierce 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 molecular 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. 
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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 cotton 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 shown 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 cotton 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 a 
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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 spectrometry 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 first 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 TwinLink 

cotton 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 confirms 
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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 full 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, and 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 standard also indicates that the protein is not 

glycosylated. 
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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 766a

 [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 instrument (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 isolated from TwinLink on 3/12/2008. 
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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 

NAa

 

a

 NA = Not Applicable 
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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 modified. The N-terminal sequencing analysis also 

confirmed the NVLN portion of the N-terminal peptide. The peptides at positions 
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339 to 373 = VNYSGGVSSGDIGAVFNQNFSCSTFLPPLLTPFVR 

374 to 382 = SWLDSGSDR 

383 to 403 = GGVNTVTNWQTESFESTLGLR 

563 to 584 = VYTASNVNTTTNNDGVNDNGAR 

585 to 631 = FLDINMGNVVASDNTNVPLDINVTFNSGTQFELMNIMFVPTNLPPIY 

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 potential 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. 
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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 704a

 [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 instrument (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 full 

scan spectrum. NA* = Not analyzed due to software and sample limitations. NA = Not analyzed. ND = Not Detected 
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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 

NAa

a

 NA = Not Applicable 
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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] 1346b

 [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] 103b [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 (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 full scan spectrum. 

 ND = Not Detected due to low sample concentration or a non-glycosylated modification because glycostain analysis 

indicated that the protein 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 leaves 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. 
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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 

NAa

a

 NA = Not Applicable 
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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 Cry1Ab 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/bar proteins 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. 
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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 analysis of the Cry1Ab protein from E. coli. and the 

Cry1Ab protein isolated from TwinLink cotton leaves. 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 Standard mix of Phosphorylase B, alpha-Acidic 

glycoprotein, Carbonic anhydrase and Avidin. Lane D contains Cry1Ab isolated from TwinLink 

cotton leaves. 
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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 protein isolated from TwinLink 

cotton leaves. Lane D contains the BTS#4 Glycoprotein 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. 
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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 isolated from event T304-40 cotton seed. 
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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 contains 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). 
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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

kDa A B C D E F

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

kDa A B C D E F

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 the PAT/bar protein isolated from event GHB119. 

Lane D contains the PAT/bar protein 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. 
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VII. DETECTION TECHNIQUES FOR 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 important 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 negatively 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 peroxidase 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 two 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 the 
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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 through 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. 
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VIII. AGRONOMIC AND PHENOTYPIC 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. 
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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, T3 seed harvested from T2 plants was 

imported for planting in the first field evaluation in Mississippi. T3 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.
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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 retained 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 bundle 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 the 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 cotyledonary 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 germination 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 selective insecticides for lepidopteran pests 

Strain Uniformity Used to evaluate the uniformity of the event on a 1 to 9 scale. (1 = uniform, 9 

= highly variable) 

Total number of nodes Number of reproductive nodes present on the main stem of the plant 

Treated plots TwinLink plots sprayed with glufosinate ammonium herbicide 

Unsprayed plots Plots unsprayed for lepidopteran pests 

Yield: Lbs. lint per acre Productivity expressed 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 of 
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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 populations 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 selective conventional insecticides to control lepidopteran pests. 
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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 plots 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 insecticide 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 selective 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 fruit 
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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 

1st boll 

opening 

Days to 

1st flower 

Days to 

1st boll 

opening 

Days to 

1st flower 

Days to 1st

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 selective conventional insecticides to control lepidopteran pests. 
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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 1.25ab 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 selective 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 transformed 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 

(BC2F3) 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. Commercial 
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germplasm was backcrossed with the T0 generation of the respective Coker transformant and 

then progressed to the BC2F3 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 Georgia, 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-transgenic 

samples 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 nutritional factors proximate, minerals, vitamins, 

amino acids, and fatty acids (cottonseed oil). Antinutritional factors of cotton include the 

gossypols, phytic acid, and the cyclic fatty acids. 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. 
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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 100% - (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 

rangesb 

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, 2001 
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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. 
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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 - 100.5 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. 
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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. 
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IX. ENVIRONMENTAL SAFETY/IMPACT 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 offspring. 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. 
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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 are 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, glufosinate-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 comparative 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 assessment, 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 are 
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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 NOEC1 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/g3

Springtail 

(Folsomia candida) 

larva Mortality, reproduction NOEC 4.5 μg/g2

Daphnia immature Mortality, development, reproduction NOEC 48 μg/g3

1

NOEC = No Effect Concentration

2

This is a conservative estimate based on ELISA analysis of diet samples. Actual exposure was probably significantly 

higher. 

3

 Preliminary results 
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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 NOEC1 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/g3

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/g3

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 DT50 (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 DT50 was 

determined from the soil protein concentration first-order exponential regression curve. An 

average DT50 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. 
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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 material 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 emergence. 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 survival and development, since if larvae 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. 
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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 (DT50 for Cry1Ab is 3.6 days, DT50 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. 
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4. Summary of assessment of effect on non-target organisms 

Insects are the organisms most likely to have significant 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 representative 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 environmentally 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 extremely 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. 
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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 TwinLink 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 that 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 positive 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 herbicide), this problem is less likely to occur 

in GM HT crops; 
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• 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 farm workers (from reduced handling and use of 

insecticides); 

Genetically modified herbicide tolerant cotton was first grown commercially in the US in 1997 

and 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. 
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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 Statistics 

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. 
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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-regulated 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 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 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). 
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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 important 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 collected 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 Agricultural Statistics Service (NASS), Agricultural Statistics Board, U.S. Department of 

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. 
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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 methods 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 ammonium) 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 be 
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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, such as glyphosate (RoundUp®) and flumioxazin 

(Valor®), can be used for burndown in no-till planting systems common in herbicide tolerant 

cropping systems. 

In conventional cultivation systems, post-directed 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 tolerant 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. Glufosinate-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 class of herbicide in an international survey of 

herbicide resistant weeds (Heap, 2002). Glufosinate ammonium is registered by EPA for use 

in cotton. 
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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 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. 

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, composition analysis, and agronomic performance 

evaluation. These assessments of TwinLink cotton and the Cry1Ab, Cry2Ae and PAT proteins 

were conducted across a wide variety of environmental 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. 
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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 TwinLink cotton will have long-term durability in the 

marketplace. A plan for monitoring of insect susceptibility is also provided. 
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X. STATEMENT OF GROUNDS 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. 
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XI. REFERENCES 

Amann M. M. 1999. Identification of Nutrients and Anti-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 Content 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., Stipanovic R., Harvey P., Patzer S., Fuchs, R.L. 1996. 

The composition of insect-protected cottonseed is equivalent 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. Regul. 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 for the quantitation of microgram 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. Trends Biochem. Sci. 23: 263-267. 

Brake J., Faust M.A., and Stein J. 2003. Evaluation of transgenic event Bt11 hybrid corn in broiler chickens. 

Poult. Sci. 82:551-559. 

Brookes G, Barfoot P. 2008. GM crops: global socio-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. Decision 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 Policy. http://www.ncfap.org/pesticid1.htm 

Carrière, Y. and T.J. Dennehy, B. Pedersen, S. Haller, C. Ellers-Kirk, 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 sustained? 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 thuringiensis deltaendotoxin 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 transcriptional activity of the tobacco plasmid psbA promoter. 

Nucleic Acids Research. 17 (1) 19-29. 

Crockett L. 1977. Wildly Successful Plants: North American 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 genes under the control of an Arabidopsis rbcs 

promoter: sequence encoding the Rubisco transit peptide 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 Agrobacterium 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 Meta-Analysis of Effects of Bt Crops 

on Honey Bees (Hymenoptera: Apidae). PLoSONE 3(1): e1415. 

http://www.plosone.org/article/info:doi/10.1371/journal.pone.0001415 

Endrizzi, J.E., Turcotte, E.I. and Kohel, R.J. 1984. cotton, 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 

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CONTAINS NO CONFIDENTIAL BUSINESS INFORMATION

EPA- see United States Environmental Protection Agency. 

Fawcett, R., Towery, D. 2004. Conservation Tillage and Plant Biotechnology: 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. Nucleotide sequence of the transposon Tn7 gene encoding an 

aminoglycoside-modifying enzyme, 3"(9)-O-nucleotidyltransferase. 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 Agriculture. 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., Robinson E., Gallagher S.P., Sorbet R., Chen Y. 2004. 

Bollgard II cotton: compositional analysis and feeding 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-protected 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 synthase promoters in transformed tobacco, sugarbeet 

and oilseed rape callus tissue. Molec. Gen Genet. 212:182-190. 

Herouet C., Esdaile D.J., Mallyon B.A., Debruyne E., Schulz A., Currier T., Hendrickx K., van der Klis R.J., 

Rouan D. 2005. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the 

pat and bar sequences that confer tolerance to glufosinate-ammonium herbicide in transgenic plants. 

Regul Toxicol Pharmacol. 41:134-149. 

Höfte H., de Greve H., Seurinck J., Jansens S., Mahillon J., Ampe C., Vandekerckhove J., Vanderbruggen 

H., van Montagu M., Zabeau M.,Vaeck M. 1986. Structural 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., Van Rie, J., Jansens, S., and Van Mellaert, H. 1988a. 

Specificity of Bacillus thuringiensis delta-endotoxins is correlated with the presence of high-affinity 

binding sites in the brush border membrane of target 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. Quantification 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 genes 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. Pesticide 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., Stipanovic 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. Identification of DNA sequences required 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 juices of various insect larvae. J. Invertebr. 

Pathol. 60:121-126. 

Oka A., Sugisaki H., Takanami M. 1981. Nucleotide sequence of the kanamycin resistance transposon 

Tn903. Journal of Molecular Biology, 147, 217-226. 

Organization for Economic Co-operation and Development (OECD). 2004. Consensus Document on 

Compositional Considerations for New Varieties of cotton (Gossypium hirsutum and Gossypium 

barbadense): Key Food and Feed Nutrients and Anti-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 thuringiensis-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., Wilcks A. 2005. Occurrence and significance of 

Bacillus cereus and Bacillus thuringiensis in ready-to-eat food. 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 dissection of the Cauliflower Mosaic Virus polyadenylation 

signal. Genes and Development. 5:141-149. 

Sankula S., Marmon G., Blumenthal E. 2005. Biotechnology-Derived Crops Planted in 2004 - Impacts on 

US Agriculture. National Center for Food and Agricultural 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, sensitive and versatile assay for protein using Coomassic 

brilliant blue G250. Anal. Biochem. 79, 544. 

Thompson C., Van Montagu M., Leemans J. 1987. Engineering 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. United States Department of Agriculture. Foreign 

Agricultural Service. Circular Series FOP 9-06. September 2006. 26 pages 

USDA-NASS. http://www.nass.usda.gov/QuickStats/index2.jsp#top. 

United States Environmental Protection Agency (USEPA,) (2001). Biopesticides Registration Action 

Document (BRAD) – Bacillus thuringiensis Plant-Incorporated Protectants, 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 (USEPA). 2005. Bt Cry1F/Cry1Ac WideStrike® Cotton 

Registration Action Document. 

http://www.epa.gov/oppbppd1/biopesticides/ingredients/tech_docs/brad_006512-006513.pdf 

United States Environmental Protection Agency (USEPA). 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 proteins and the genetic material necessary for 

their production in COT102 X COT67B cotton 

http://www.epa.gov/pesticides/biopesticides/ingredients/tech_docs/brad_006529.pdf 

United States Food and Drug Administration (US-FDA). 1992. Statement of Policy: Foods Derived From 

New Plant Varieties. http://www.cfsan.fda.gov/~lrd/biotechm.html#reg 

United States Food and Drug Administration (US-FDA). 2002. Biotechnology Consultation: BNF No. 

000074. Insect-Protected Bollgard II Cotton 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 Administration (US-FDA). 2003. Biotechnology Consultation: Note to the File 

BFN No. 000086. Glufosinate Tolerant Cotton Transformation Event LLCotton25. June 05, 2003. 

http://www.cfsan.fda.gov/~rdb/bnfm086.html (Accessed 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 mosaic virus (CVMV) promoter. Plant Mol. Biol. 

31:1129-1139. 

Wang Z.-H., Wang Y., Cui H.-R., Xia 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, presented at the 2008 Beltwide Cotton Conferences. 

January 8-11, 2008. Nashville, TN 

Wozenski J., Woodburn, M. 1975. Phytic acid (myoinositol 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., Banks 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 Agrobacterium-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. 
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Appendix 1. FIELD TRIALS TERMINATION REPORTS 2005-2007 
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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 were 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 trial 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. 
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Weather Synopsis: Cooperator reported that the growing season had above average moisture, with 

above average temperatures on average. 

Containment Measures: Plot was contained utilizing a 40 ft. isolation border which encompassed this field 

site. 
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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 Damage 
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Section III. Summarized results from the 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. 
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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. 
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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. 
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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 Characteristics 
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f Weather Synopsis 

g Plot Damage 

Section III. Summarized results from the 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. 
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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. 
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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 yield 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. 
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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. 
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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 the BCS regulatory In-Season Observation forms:
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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. 
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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: 
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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. 
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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 the BCS regulatory In-Season Observation forms:

1. Franklin Co., LA 
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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. 
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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 

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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 the 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. 
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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. 
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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 the 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. 
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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 

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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 the 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. 
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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 

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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 the BCS regulatory In-Season Observation forms:
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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. 
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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. 
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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. 
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Appendix 2. MATERIALS AND METHODS - PRODUCT CHARACTERIZATION 
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Breeding diagram for each parent of TwinLink cotton used in agronomic 

evaluation 
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Materials and methods for molecular 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 equivalent) 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 PstI and/or EcoRI and/or

HindIII 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 

[α32P] 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: 4.5x109

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-
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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 PstI and/or EcoRI and/or HindIII 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. 
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Materials and methods for protein characterization tests 

Studies on potential toxicology and allergenicity 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/bar proteins 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 proteins 

by 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. 
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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 trypsin 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 leaves 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 at 
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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. 
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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, 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 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 field 

were 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. 
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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 appropriate 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. 
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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 expressed as the average of 10 data points from 

duplicate extracts of 5 samples at each fortification 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 expressed as the average of 10 data points from 

duplicate extracts of 5 samples at each fortification level using non-transgenic matrix. 
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Table 2.3 Validation of Sample Extraction for the PAT/bar ELISA 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 expressed as the average of 10 data points from 

duplicate extracts of 5 samples at each fortification 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 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. 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. 
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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 for 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, Version 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 the 
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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: 

% Crude Protein FW/ g matrix 

Average μg Target Protein/ g sample AverageTarget Protein as % Crude Protein = 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: 

Averagemg TEP / g sample 

Average μg analyte / g sample AverageAnalyte as % Total Extractable Protein = x 10-1

Protein content (Bradford) μg

SoftMax ProTM software (Molecular Devices, Version 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 tables 
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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 ± 0.844 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 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 Cry1Ab protein present in kernel and lint coat fractions taking into account 

their respective weights. b Average Cry1Ab as % of crude protein is not applicable (NA) because crude protein determinations 

were not made on the individual lint coat and kernel; but only on the entire fuzzy seed samples. 
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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 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 Cry1Ab protein present in kernel and lint coat fractions taking into 

account their respective weights b Average Cry1Ab as % of crude protein is not applicable (NA) because crude protein determinations 

were not made on the individual lint coat and kernel; but only on the entire fuzzy seed samples. 
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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 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 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 Cry2Ae protein present in kernel and lint coat fractions taking into 

account their respective weight. b Average Cry2Ae as % of crude protein is not applicable (NA) because crude protein determinations 

were not made on the individual lint coat and kernel; but only on the entire fuzzy seed samples. 
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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 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 Cry2Ae 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 were made only on the entire fuzzy seed samples 

and not the individual lint coat and kernel. b Average Cry2Ae as % of crude protein is not applicable (NA) because crude protein determinations 

were not made on the individual lint coat and kernel; but only on the entire fuzzy seed samples. 
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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 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 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 protein 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 kernel; but only on the entire fuzzy seed samples. 
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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 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 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 were 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 kernel; but only on the entire fuzzy seed samples. 
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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 total 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. 
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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 LOQa

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 approximately 0.1 g matrix per 6 mL extraction buffer for 

leaves and squares and 0.1 g matrix per 2 mL extraction 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 LOQa

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 approximately 0.1 g matrix per 6 mL extraction buffer for 

leaves and squares and 0.1 g matrix per 2 mL extraction 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 LOQa

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 approximately 0.1 g matrix per 6 mL extraction buffer for 

leaves and squares and 0.1 g matrix per 2 mL extraction 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, Version 4.0) was used to derive the concentration 

of protein from the Bradford assay (Bradford, 1976). The optical density was converted to the 
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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, Version 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 obtained from analysis at Covance Laboratories. The complete 

set of data is given in Appendix 3. 
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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 measured 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. 
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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). 
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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 supplemental 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 supplemental 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 desiccants 
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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, white flowers, and bolls at multiple time frames 

through fruiting period (n=2-8). An additional 22 agronomic 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). 
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Figure 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 example 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 slightly 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 5th and 6th 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. 
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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 5th and 6th 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 5th and 6th 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 insecticides should be applied to ‘Sprayed’ or 

‘Treated’ plots to minimize lepidopteran damage. Allow 48 hours after an infestation before 

applying any insecticide sprays. 
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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 material 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 separate 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 lepidopteran pests – see protocols. 
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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. 
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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. Fiber 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%. 
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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 SL 

herbicide 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 were 

indeed 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. 
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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 AOAC 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. 
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Appendix 3. CHARACTERIZATION OF TWINLINK COTTON 
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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: ApaI, DraIII, EcoRI, EcoRV, NdeI, BgII, SwaI, SspI, SacI and 

XbaI. Wild type genomic DNA digested with XbaI was used as negative control; wild type 

genomic DNA digested with XbaI and supplemented with an equimolar amount of pTDL008 

plasmid DNA digested with XbaI 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 hybridization 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 cry1Ab 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 cry1Ab 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 bp 
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Figure 3.1 Schematic overview of Southern blot strategy for Cry1Ab cotton event 

T304-40 
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Table 3.2 Expected and obtained hybridization fragments – Cry1Ab cotton event T304-40 
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Figure 3.2 Cry1Ab cotton event T304-40 Insert verification – Southern blot results 
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Figure 3.2 (Continued) Cry1Ab cotton event T304-40 Insert verification – Southern blot 

results 
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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: DraI, EcoRV, HindIII, NcoI, BamHI, EcoRI, PstI, AvaI, NdeI, XhoI, 

EcoRI/PstI and EcoRI/NdeI. 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-cry2Ae probe 2100 bp 

PT047 3’35S-RB probe 264 bp 
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NcoI BamHI NcoI

NdeI

HindIII

NdeI NcoI

BamHI

EcoRI

HindIII EcoRV

HindIII

PstI

LB 3'nos bar PCsVMV P35S2

5'cab22L

TPssuAt cry2Ae 3'35S RB

PT024

DraI > 4345 bp

EcoRV X X > 1829 bp 1373 bp > 1143 bp

HindIII XX X

> 1427 bp 1280 bp 1352 bp > 286 bp

NcoI X X X

854 bp 1105 bp > 2356 bp > 30 bp

BamHI X X

> 316 bp 2051 bp > 1978 bp

EcoRI X > 2528 bp > 1817 bp

PstI X

> 4303 bp > 42 bp

NdeI X X

> 1318 bp 131 bp > 2896 bp

PT023 PT044 PT045 PT046 PT047

PT026

EcoRV

EcoRI/PstI X> 42 bp > 2528 bp 1775 bp

X

EcoRI/NdeI > 1318 bp > 1817 bp X X X

131 bp 1079 bp

Figure 3.3 Schematic overview of Southern blot strategy for Cry2Ae cotton event 

GHB119 
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Table 3.4 Expected and obtained hybridization fragments – event GHB119 

Yes

No

YesYes

YesYes

YesYes

YesYes

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

10893

1373

10893

1373

Positive controlPositive control

WT genomic 

DNA + pTEM12 

- EcoRV

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 

No fragment. No fragment. Negative control WT genomic DNA - EcoRV

NoNoNo

Yes

NoNo

YesYes

NoNo

YesYes

NoNo

Yes

No

NoNo

Yes

No

Yes

NoNoNo

Yes

No *

NoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Ye 

NoNoNo

3960

No hybr.

10792450

>1318

131

1079

>1817

5' integration fragm.internal fragm.internal fragm.

3' integration fragm.

EcoRI/NdeI

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

Yes

NoNo

Yes

No *

No

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

4200

No hybr.

5380

>1318

131

>2896

5' integration fragm.internal fragm.3' integration fragm.

NdeI

No

Yes

YesYes

YesYes

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

52001775

>2528

1775

5' integration fragm.internal fragm. EcoRI/PstI

PstI 5' integration fragm. >4303 7800 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 

No

Yes

YesYes

YesYes

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

51802480

>2528>1817

5' integration fragm.3' integration fragm. EcoRI

NoNo

Yes

No

YesYes

No

YesYes

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

Yes

NoNo

Yes

NoNo

3120 20517070

>316

2051

>1978

5' integration fragm.internal fragm.3' integration fragm.

BamHI

NoNo

Yes

NoNo

Yes

NoNo

Yes

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

854

11055940

854

1105

>2356

5' integration fragm.internal fragm.3' integration fragm.

NcoI

NoNoNo

Yes

No

YesYes

No

No

YesYes

No

No

Yes

NoNo

No

Yes

NoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

5150128013528830

>1427

12801352

>286

5' integration fragm.internal fragm.internal fragm.3' integration fragm.

HindIII

NoNo

Yes

No

YesYes

No

YesYes

YesYes

No

YesYes

No

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

338013738890

>1829

1373

>1143

5' integration fragm.internal fragm.3' integration fragm.

EcoRV

DraI Complete insert >4345 4430 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 

Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. 

PT04

3'35S

PT046:

TPssuATcry2Ae

PT045:

P35S2-

5'cab22L

PT044:

Pcsvmv

PT023:

bar

PT024:

LB-3'nos

Obtained 

fragment 

sizes (bp)

Expected 

fragment 

sizes (bp)

Digest Description

Yes

No

YesYes

YesYes

YesYes

YesYes

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

10893

1373

10893

1373

Positive controlPositive control

WT genomic 

DNA + pTEM12 

- EcoRV

Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 

No fragment. No fragment. Negative control WT genomic DNA - EcoRV

NoNoNo

Yes

NoNo

YesYes

NoNo

YesYes

NoNo

Yes

No

NoNo

Yes

No

Yes

NoNoNo

Yes

No *

NoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Ye 

NoNoNo

3960

No hybr.

10792450

>1318

131

1079

>1817

5' integration fragm.internal fragm.internal fragm.

3' integration fragm.

EcoRI/NdeI

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

Yes

NoNo

Yes

No *

No

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

4200

No hybr.

5380

>1318

131

>2896

5' integration fragm.internal fragm.3' integration fragm.

NdeI

No

Yes

YesYes

YesYes

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

52001775

>2528

1775

5' integration fragm.internal fragm. EcoRI/PstI

PstI 5' integration fragm. >4303 7800 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 

No

Yes

YesYes

YesYes

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

Yes

No

51802480

>2528>1817

5' integration fragm.3' integration fragm. EcoRI

NoNo

Yes

No

YesYes

No

YesYes

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

Yes

NoNo

Yes

NoNo

3120 20517070

>316

2051

>1978

5' integration fragm.internal fragm.3' integration fragm.

BamHI

NoNo

Yes

NoNo

Yes

NoNo

Yes

No

Yes

No

No

Yes

No

No

Yes

No

No

Yes

No

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

854

11055940

854

1105

>2356

5' integration fragm.internal fragm.3' integration fragm.

NcoI

NoNoNo

Yes

No

YesYes

No

No

YesYes

No

No

Yes

NoNo

No

Yes

NoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

Yes

NoNoNo

5150128013528830

>1427

12801352

>286

5' integration fragm.internal fragm.internal fragm.3' integration fragm.

HindIII

NoNo

Yes

No

YesYes

No

YesYes

YesYes

No

YesYes

No

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

Yes

NoNo

338013738890

>1829

1373

>1143

5' integration fragm.internal fragm.3' integration fragm.

EcoRV

DraI Complete insert >4345 4430 Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes Yes 

Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. Obt. Exp. 

PT04

3'35S

PT046:

TPssuATcry2Ae

PT045:

P35S2-

5'cab22L

PT044:

Pcsvmv

PT023:

bar

PT024:

LB-3'nos

Obtained 

fragment 

sizes (bp)

Expected 

fragment 

sizes (bp)

Digest Description

(*) 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.
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Figure 3.4 Cry2Ae cotton event GHB119 Insert verification - Hybridization results 

Lane 1 : Phage Lambda - PstI digested 

Lane 2 : Phage Lambda - HindIII digested 

Lane 3 : GHB119 - DraI digested 

Lane 4 : GHB119 - EcoRV digested 

Lane 5 : GHB119 - HindIII digested 

Lane 6 : GHB119 - NcoI digested 

Lane 7 : GHB119 - BamHI digested 

Lane 8 : GHB119 - EcoRI digested 

Lane 9 : GHB119 - PstI digested 

Lane 10 : GHB119 - EcoRI/PstI digested 

Lane 11 : GHB119 - PstI digested 

Lane 12 : GHB119 - NdeI/EcoRI digested 

Lane 13 : Wild type variety FM966 - EcoRV digested 

Lane 14 : Wild type variety FM966 - EcoRV digested + an 

equimolar amount of pTEM12 - EcoRV digested 

Lane 15 : Empty lane 

Lane 16 : Phage Lambda - PstI digested 

Lane 17 : Phage Lambda - HindIII digested
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Figure 3.4 (Continued) Cry2Ae cotton event GHB119 Insert verification - Hybridization 

results 
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3. Genetic stability of the inserted 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 EcoRV, which has two recognition sites in the integrated 

DNA fragment. Probing EcoRV digested genomic T304-40 DNA with the cry1Ab 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. 
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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 F1 05GAGH000743 15 

Generation BC1F1 05GAGH000742 14 

Generation BC2F2 05GAGH000741 12 

Test item 

samples 

Generation BC2F2 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. 
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Figure 3.5 Hybridization results of the stability analysis of cotton event T304-40 in 

different genetic backgrounds 
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Figure 3.6 Hybridization results of the stability analysis of cotton event T304-40 over 

different generations 
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Figure 3.7 Hybridization results of the stability analysis of cotton event T304-40 over 

different environmental conditions 
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4. Genetic stability of the inserted DNA in Cry2Ae cotton event GHB119 

The structural stability of Cry2Ae cotton event GHB119 over different generations, in different 

backgrounds, and over different environmental conditions 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 assessed 

by analyzing the progeny of plants cultivated at six different field locations. The isolated DNA 

was digested with the restriction enzyme EcoRV, 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 demonstrates 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. 
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Table 3.6 GHB119 Structural stability over generations and genetic backgrounds - Expected and ob

fragments 

Samples Condition tested Seedlot N° of plants Expected fragment sizes (*) Expected fragments obtained ? Des

Background Coker 312 05GC03 22 Generation F1 05GC16 4 Generation BC1F1 05GC33 18 

Test item samples 

Generation BC2F1 05GC59 13 

ca. 3380 bp 1373 bp ca. 8890 bp 

Yes Yes Yes 

5’ininte3’in

DNA negative control Wild type variety FM966 (Seedlot 04GAGH003113) No hybridization Yes DNA positive control Wild type variety FM966 + an equimolar amount pTEM12 1373bp 10893 bp Yes Yes Pos

(*) Fragment sizes as determined in the detailed insert characterization of GHB119 Table 3.7 GHB119 Structural stability over environments - Expected and obtained hybridization frag

Samples Condition tested Seedlot N° of plants Expected fragment sizes (*) Expected fragments obtained ? Des

Location Chula, Georgia CY06B001-201-31 13 Location Newport, Arkansas CY06B001-402-32 12 Location Proctor, Arkansas CY06B001-403-33 10 Location Senatobia, Mississippi CY06B001-404-32 10 Location East Bernard, Texas CY06B001-605-33 14 

Test item samples 

Location Levelland, Texas CY06B001-806-33 14 

ca. 3380 bp 1373 bp ca. 8890 bp 

Yes Yes Yes 

5’ininte3’in

DNA negative control Wild type variety FM966 (Seedlot 04GAGH003113) No hybridization Yes DNA positive control Wild type variety FM966 + an equimolar amount pTEM12 1373bp 10893 bp Yes Yes Pos

(*) Fragment sizes as determined in the detailed insert characterization of GHB119 
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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 8 14 17 19 10 15 20 21 22 16 18

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 8 10 14 17 19 15 20 21 22 16 23 24 26 18 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

4 5 6 7 8 9 10 11 12 13 14 15 16 17

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 results 
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A: GHB119 samples; B: Negative control; C: Positive control 

Figure 3.9 Structural stability of event GHB119 over environments - Hybridization 

results
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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 TwinLink plants. Two separate aliquots of each 

plant DNA extract were digested with the restriction 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 positive control for GHB119 analysis was genomic 

DNA prepared from WT leaf material mixed with 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 that 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. 
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Figure 3.10 Demonstration of the stability of TwinLink cotton for the cry1Ab DNA 

insert. 

Genomic DNA was isolated from TwinLink cotton plants and from the non-transgenic counterpart (Coker 

315). Genomic DNAs were digested with EcoRV and probed with part of the T-DNA (1822 bp fragment of 

pTLD008). 

Lane 1: TwinLink – plant 1 – EcoRV Lane 12: TwinLink – plant 12 – EcoRV 

Lane 2: TwinLink – plant 2 – EcoRV Lane 13: TwinLink – plant 13 – EcoRV 

Lane 3: TwinLink – plant 3 – EcoRV Lane 14: TwinLink – plant 14 – EcoRV 

Lane 4: TwinLink – plant 4 – EcoRV Lane 15: TwinLink – plant 15 – EcoRV 

Lane 5: TwinLink – plant 5 – EcoRV Lane 16: TwinLink – plant 16 – EcoRV 

Lane 6: TwinLink – plant 6 – EcoRV Lane 17: TwinLink – plant 17 – EcoRV 

Lane 7: TwinLink – plant 7 – EcoRV Lane 18: TwinLink – plant 18– EcoRV 

Lane 8: TwinLink – plant 8 – EcoRV Lane 19: TwinLink – plant 19 – EcoRV 

Lane 9: TwinLink – plant 9 – EcoRV Lane 20: TwinLink – plant 20 – EcoRV 

Lane 10: TwinLink – plant 10 – EcoRV Lane 21: T304-40 parent – EcoRV 

Lane 11: TwinLink – plant 11 – EcoRV Lane 22: WT variety Coker 315 

 plus 1 copy pTDL0008 – EcoRV 

 Lane 23: WT variety Coker 315 – EcoRV 
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Figure 3.11 Demonstration of the stability of TwinLink cotton for the cry2Ae DNA 

insert. 

Genomic DNA was isolated from TwinLink cotton plants and from the non-transgenic counterpart (Coker 

315). Genomic DNAs were digested with EcoRV and probed with part of the T-DNA (4346 bp fragment of 

pTEM12). 

Lane 1: TwinLink – plant 1 – EcoRV Lane 12: TwinLink – plant 12 – EcoRV 

Lane 2: TwinLink – plant 2 – EcoRV Lane 13: TwinLink – plant 13 – EcoRV 

Lane 3: TwinLink – plant 3 – EcoRV Lane 14: TwinLink – plant 14 – EcoRV 

Lane 4: TwinLink – plant 4 – EcoRV Lane 15: TwinLink – plant 15 – EcoRV 

Lane 5: TwinLink – plant 5 – EcoRV Lane 16: TwinLink – plant 16 – EcoRV 

Lane 6: TwinLink – plant 6 – EcoRV Lane 17: TwinLink – plant 17 – EcoRV 

Lane 7: TwinLink – plant 7 – EcoRV Lane 18: TwinLink – plant 18– EcoRV 

Lane 8: TwinLink – plant 8 – EcoRV Lane 19: TwinLink – plant 19 – EcoRV 

Lane 9: TwinLink – plant 9 – EcoRV Lane 20: TwinLink – plant 20 – EcoRV 

Lane 10: TwinLink – plant 10 – EcoRV Lane 21: GHB119 parent – EcoRV 

Lane 11: TwinLink – plant 11 – EcoRV Lane 22: WT variety Coker 315 

 plus 1 copy pTDL0008 – EcoRV 

 Lane 23: WT variety Coker 315 – EcoRV 
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6. Absence of vector backbone in Cry1Ab cotton event T304-40 

Genomic DNA isolated from T304-40 plants was digested with either EcoRV or NotI restriction 

enzymes and analyzed by Southern blot. Seven 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-EcoRV

EcoRV NotI

PT012

ca. 3100 bp

> 7467 bp 

ca. 3500 bp

T304-40-NotI

Figure 3.12 Absence of vector backbone in cotton event T304-40 -- Schematic overview 

of the hybridization strategy 
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Table 3.8 Absence of vector backbone in event T304-40 - Expected and obtained hybridization resul

T304-40 - EcoRV T304-40 - NotI WT (FM966) - NotI WT 

or 10 cop

Obt

Probe template 

ID 

Expected 

hybridization 

fragments 

(bp) 

Obtained 

hybridization 

fragments 

(bp) 

Expected 

hybridization 

fragments 

(bp) 

Obtained 

hybridization 

fragments 

(bp) 

Expected 

hybridization 

fragments 

(bp) 

Obtained 

hybridization 

fragments 

(bp) 

Expected 

hybridization 

fragments 

(bp) 0.1 

PT002 none none none none none none 

1290 

 (1532)1

5404 

15

PT003 none none none none none none 1290 1532 1

1

PT005 none none none none none none 6167 6PT007 none none none none none none 5404 5PT008 none none none none none none 

1532 6167 

16

PT009 none none none none none none 6167 6PT010 none none none none none none 6167 6PT012 

ca. 6300 ca. 3500 ca. 3000 

6300 ± 200 3500 ± 100 3100 ± 100 

> 7467 >14K none none 5404 5

1 Probably not visible due to limited overlap (85 bp) between PT002 and this vector fragment. 2 Background hybridization 
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Figure 3.13 Absence of vector backbone in cotton event T304-40– Southern blot results 

Lane 1: Phage Lambda – PstI digested (not shown) 

Lane 2: Cotton event T304-40– EcoRV digested 

Lane 3: Cotton event T304-40– NotI digested 

Lane 4: Non-trangenic control – NotI digested 

Lane 5: Non-trangenic control + 0.1 copy pTDL008 – NotI digested 

Lane 6: Non-trangenic control + 1 copy pTDL008 – NotI digested 

Lane 7: Non-trangenic control + 10 copies pTDL008 – NotI digested 
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Figure 3.13 (Continued) Absence of vector backbone in cotton event T304-40– 

Southern blot results 
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7. Absence of vector backbone in Cry2Ae cotton event GHB119 

Genomic DNA isolated from GHB119 plants was digested with either EcoRV or DraI 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. 

EcoRV

PT026

PT001

PT002

PT003

PT004

PT005

EcoRV

LB RB

npt I

homology

region

ORI ColE1 ORI pVS1 aadA

pTEM12:

GHB119 - EcoRV X

GHB119 - DraI

> 4345 bp

10893 bp

pTEM12- EcoRV

> 1829 bp

X

1373 bp > 1143 bp

X X

1373 bp

3'nos bar

Pcsvmv

P35S2

5'cab22L

TPssuAt

cry2Ae 3'35S

EcoRV

PT026

PT001

PT002

PT003

PT004

PT005

EcoRV

LB RB

npt I

homology

region

ORI ColE1 ORI pVS1

pTEM12:

GHB119 - EcoRV X

GHB119 - DraI

> 4345 bp

10893 bp

pTEM12- EcoRV

> 1829 bp

X

1373 bp > 1143 bp

X X

1373 bp

3'nos bar

Pcsvmv

P35S2

5'cab22L

TPssuAt

cry2Ae

Figure 3.14 Absence of vector backbone in cotton event GHB119 -- Schematic overview 

of the hybridization strategy 
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Table 3.9 Absence of vector backbone in event GHB119 -- Expected and obtained hybridization frag

GHB119 - EcoRV GHB119 - DraI WT (FM966) - EcoRV WT (FM9

0.1 and 1 cop

Probe template ID Expected 

hybridizatio

n fragments 

Obtained 

hybridizatio

n fragments 

Expected 

hybridizatio

n fragments 

Obtained 

hybridizatio

n fragments 

Expected 

hybridization 

fragments 

Obtained 

hybridizatio

n fragments 

Expected 

hybridization 

fragments 

PT001-2 / / / / / / 10893 bp PT002-2 / / / / / / 10893 bp PT003-2 / / / / / / 10893 bp PT004-3 / / / / / / 10893 bp PT005-2 / / / / / / 10893 bp 

3380 bp 1373 bp 8890 bp 

3380 bp 1373 bp 8890 bp 

4430 bp 4430 bp / / 10893 bp 1373 bp 

PT026-2 

3380 bp 1373 bp 8890 bp 

obtained obtained obtained 

4430 bp obtained / / 10893 bp 1373 bp 

3 Faint band, not always visible on all blots 
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Figure 3.15 Southern blot analysis of GHB119 – Absence of vector backbone 

Lane 1: Phage Lambda – PstI digested 

Lane 2: Cotton event GHB119 – EcoRV digested 

Lane 3: Cotton event GHB119 – DraI digested 

Lane 4: Wild type cotton variety FM966 – EcoRV digested 

Lane 5: Wild type cotton variety FM966 – EcoRV digested + 0.1 copy pTEM12 – EcoRV digested 

Lane 6: Wild type cotton variety FM966 – EcoRV digested + 1 copy pTEM12 – EcoRV digested 

Lane 7: Phage Lambda – PstI digested 
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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 – PstI digested 

Lane 2: Cotton event GHB119 – EcoRV digested 

Lane 3: Cotton event GHB119 – DraI digested 

Lane 4: Wild type cotton variety FM966 

 EcoRV digested 

Lane 5: Wild type cotton variety FM966 

EcoRV digested + 0.1 copy pTEM12 – 

EcoRV digested 

Lane 6: Wild type cotton variety FM966 

EcoRV digested + 1 copy pTEM12 – 

EcoRV digested 

Lane 7: Phage Lambda – PstI digested 

Figure 3.15 (Continued) Southern blot analysis of cotton event GHB119 – Absence of 

vector backbone 
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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 using 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)

RB
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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 functional 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 cry1Ab 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 1
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- 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 transcribed 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. 
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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 the 

region 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 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. 

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)
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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 cry2Ae - 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 analysis, 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. 
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- 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 predicted 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 cry2Ae - bar gene construct, is highly unlikely. 
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Appendix 4. REGIONAL AGRONOMIC DATA 

For ease of understanding: 

- “GA treated” means “treated with glufosinate ammonium” 

- “Sprayed” means “sprayed with conventional insecticides” 
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Table 4.1 Agronomic data across regions from BCS Regulatory trials in 2007 

Growth Habit & Phenotype

Agronomic Parameter Significance 

(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) 54.19a 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 1st 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 

Agronomic Parameter Significance 

(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) 84.93a 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 

Agronomic parameter Significance 

(locations)* 

Coker 

Not 

sprayed 

Coker 

Sprayed 

TwinLink 

Not sprayed 

TwinLink 

Sprayed LSD CV 

Plant Stand (1) 1.31cd 1.26d 1.49bc 1.36cd 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 1st 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 Carolina 
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Table 4.4 Yield and Fiber Quality data across regions from BCS Development Efficacy 

trials in 2007 

Yield and Fiber Quality 

Agronomic Significance 

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

Agronomic Parameter Significance 

(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 1st 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 parameter 
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Table 4.6 Yield and Fiber Quality data across regions from BCS (Regulatory) trials in 

2008 

Yield and Fiber Quality 

Agronomic Significance 

Parameter 

(locations)* 

Coker TwinLink 

GA treated 

TwinLink 

Not treated LSD CV 

Yield (2) 689.83a 805.33a 812.67a 188.00 3.66 

Fiber Length (2) 1.29a 1.30a 1.28a 0.02 0.22 

Fiber strength (2) 35.58 34.23a 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 

Agronomic Parameter Significance 

 (locations)* 

Coker 

Not sprayed 

Coker 

Sprayed 

TwinLink 

Not sprayed 

TwinLink 

Sprayed LSD CV 

Plant Stand (3) 2.98b 3.21ab 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 

1st position bolls (2) 6.55bc 6.75ab 6.65b 6.25bc 0.65 1.23 

2nd position bolls (2) 3.45ab 3.08abc 3.53a 3.23abc 0.41 1.65 

3rd position bolls (1) 3.50a 3.35a 3.05a 4.00a 1.96 41.74 

Total Bolls (2) 13.98a 12.95ab 13.08ab 12.38b 1.25 1.21 

% retention 1st position (2) 58.93d 67.13b 62.84c 59.89d 2.40 0.47 

% retention 2st 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. 
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Table 4.8 Yield and Fiber Quality data across regions from University and USDA 

Efficacy trials in 2008 

Yield and Fiber Quality 

Agronomic Parameter Significance 

(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 30.53bc 3.53 7.46 

Fiber uniformity % (1) 82.48ab 83.15a 82.40ab 81.75b 1.07 0.88 

Micronaire (1) 5.45a 5.10abc 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 

Agronomic Parameter Significance

(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 17.63b 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 2.65ab 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) 3.62ab 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 

Agronomic Parameter Significance

(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 1.25ab 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) 86.10a 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