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Section G Proposed experimental program (Revised) 

1. Summary of participants 

The Participants and Cooperators who will supervise the experimental work conducted under this 

EUP are listed in the Confidential Attachment A. 

{CBI Cross Reference Number 1} 

2. Total Acreage by Protocol by State 

State Insect 

Efficacy 

Regulatory 

Sample 

Production 

Total Acreage 

by State 

AL 26.25 - 26.25 

AR 26.25 15.25 41.5 

AZ 94 82 176 

CA 53 31 84 

FL 8.75 3.75 12.5 

GA 41.5 10 51.5 

LA 64 17.5 81.5 

MS 141.25 113.5 254.75 

NC 101.75 20 121.75 

PR 51 10 61 

SC 184.25 - 184.25 

TN 55.25 10 65.25 

TX 267 174.75 441.75 

TOTAL 1114.25 487.75 1602 
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3. Acreage, seed and AI quantities by state 

State Protocol* Cry1Ab 

Cotton 

Acres 

Non-PIP 

Acres 

Total 

Acreage by 

State 

Max. lb of 

Cry1Ab 

Cotton 

Seed 

Max. Amt. of 

Cry1Ab Protein 

in Seed Planted 

(g) 

AL E 3.75 22.5 26.25 45 0.35 

AR E, R 8.5 33 41.5 102 0.79 

AZ E, R 18 158 176 216 1.67 

CA E, R 10 74 84 120 0.93 

FL E, R 1.5 11 12.5 18 0.14 

GA E, R 10.5 41 51.5 126 0.97 

LA E, R 8.5 73 81.5 102 0.79 

MS E, R 17.25 237.5 254.75 207 1.60 

NC E, R 12.25 109.5 121.75 147 1.13 

PR E, R 5 56 61 60 0.46 

SC E 9.75 174.5 184.25 117 0.90 

TN E, R 6.75 58.5 65.25 81 0.63 

TX E, R 40.25 401.5 441.75 483 3.73 

Total 152 1450 1602 1824 14.08 

1

 Protocols are abbreviated as follows: (E) Insect Efficacy, (R) Regulatory Sample Production 2

 Calculated as: Cry1Ab acres x 12 lb of seed/acre 

3

 Calculated as: max. lb of Cry1Ab seed x 0.007718g/lb of Cry1Ab seed (based on the label 

maximum concentration of 0.0017%) 
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4. Program overview 

Bayer CropScience (BCS) has developed cotton [Gossypium hirsutum] plants that express an 

insecticidal protein, Cry1Ab, from a common soil bacterium, Bacillus thuringiensis subsp. berliner

(B.t. berliner). The Cry1Ab protein is effective in controlling lepidopteran larvae, such as cotton 

bollworm (CBW, Helicoverpa zea) and tobacco budworm (TBW, Heliothis virenscens) larvae, 

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 Cry1Ab 

protein, conducted under notifications granted by the U.S. Department of Agriculture's Animal and 

Plant Health Inspection Service (APHIS), have shown the plant’s ability to protect itself against 

these pests. 

Transgenic cotton plants expressing Cry1Ab protein 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 Agency, 

but is has not been used in commercial cotton as a Plant Incorporated Protectant (PIP). 

BCS is applying for an extension of this EUP (264-EUP-140) to allow further evaluation of these 

cotton plant lines in a larger range of environmental conditions. All cotton plants to be evaluated 

under the EUP have been derived from transformation event T303-3 and T304-40. Several 

different experiments are planned, and they can be grouped into: insect efficacy trials (including 

a tobacco budworm high-dose verification study), herbicide efficacy evaluation, agronomic 

performance evaluation, breeding (line evaluations). 

In addition to these experimental plans, seed blocks may be grown as part of this program to 

evaluate effects on seed production and dormancy, and seed may be harvested for later 

plantings of experimental and regulatory field trials. 

The primary goal of this experimental research program is to evaluate the Cry1Ab proteincontaining cotton plants for their efficacy against insect pests of cotton, as well as to provide 

material for agronomic performance evaluations. These plants also contain an herbicide tolerant 

ingredient as a selectable marker, the phosphinothricin acetyltransferase (PAT) protein that 

confers tolerance to glufosinate-ammonium herbicides. 

The entire program will be done on a “Crop Destruct” basis; no cottonseed will enter commerce. 

Some of the plant material will be retained for scientific research and/or planting purposes. All 

other plant materials will be destroyed. There shall be no unintentional exposure to humans or 

domestic animals since the program will be conducted using containment precautions and in a 

crop destruct fashion. Isolation will be maintained in order to prevent any inadvertent outcrossing 

(pollination) from transgenic plants to non-transgenic cotton plants. No environmental impact 

issues related to the testing of these transgenic cotton plants have been identified. The Cry1Ab 

protein has a specific range of toxicity to the target lepidopteran pests without having effects on 

the non-target beneficial insects. 

In total, the program will be carried out across 13 states/territories on a total of 1602 acres, of 

which 152 acres will be planted to Cry1Ab cotton. The Cry1Ab-containing cotton will be planted at 

a maximum rate of 12 lbs/acre. The level of Cry1Ab protein is approximately 0.908 – 7.718 mg/lb 

of seed based on the label range of 0.0002 – 0.0017 %; therefore the planting of these Cry1Abcontaining seeds represents an application rate of approximately 10.8 – 92.6 mg of Cry1Ab 

protein per acre. Our proposed experimental research program would total 1.64 - 14.08 g of 

Cry1Ab protein (or 0.004 - 0.031 pounds of Cry1Ab protein) for 152 acres planted to Cry1Ab-
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containing cotton. The level of Cry1Ab protein in the different plant materials is only an 

estimation based on our current level of information. 

Competent Bayer CropScience employees will supervise the program which will be conducted by 

them, in addition to public and private cooperators. All responsible researchers listed are 

professionally qualified to accomplish their stated duties. 
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5. Program details 

Experimental Protocol: 

EFFICACY TESTING OF INSECT RESISTANT TRANSGENIC COTTON 

Objectives

The purpose of these trials is to compare plant growth, morphology, and agronomic performance 

among insect resistant transgenic cotton lines in different genetic backgrounds and their 

respective non-transgenic counterparts when infested with (either naturally or artificially) or 

devoid of the target insect. Another objective of these trials is to evaluate insect resistance 

transgene efficacy and subsequently determine if transgene expression and resulting insect 

resistance affect plant growth, morphology, or any facet of agronomic performance including fiber 

characteristics. 

Specific Objectives

 Breeding Line Evaluations - This is primarily for the purpose of transferring the Cry1Ab 

event into elite cotton lines, in order to evaluate performance of the gene in elite varieties. 

This will include backcrossing programs for initial evaluation of lines, and for production 

of hand-pollinated seed to conduct preliminary yield evaluations. 

 Insect Efficacy, Natural Infestations/ Herbicide Efficacy - These trials will be conducted to 

evaluate effectiveness of events T303-3 and T304-40 in controlling the primary target 

pests CBW and TBW. The trials will include sites where the infestations occur only as 

natural infestations. We expect large differences in the level of severity of the infestation 

and damage that can be seen across locations and environments; and for this reason, 

the evaluation of efficacy across a broad spectrum of environmental and geographical 

conditions is needed to accurately evaluate efficacy of the Cry1Ab events against the 

targeted pests under field conditions. Additionally, evaluation by independent experts, 

such as in trials conducted by university researchers and other cooperators, provides 

independently generated performance data. In addition, the tolerance to the herbicide 

glufosinate-ammonium provided by the marker gene will be evaluated. 

 Insect Efficacy TBW, Artificial Infestations – These trials will be conducted to provide 

high-dose verification as part of BCS IRM strategy and in response to a deficiency 

identified in a letter from the Agency dated May 27, 2010. 

The individual component lines (Cry1Ab and Cry2Ae), a control line and TwinLink cotton 

will be planted and cages (8 ft X 20 ft X 6 ft) will be placed over two 20 ft row sections 

(one cage per line). Beginning at early squaring each cage will be infested with 

approximately 50 male and female tobacco budworms from a lab colony supplied by 

USDA-ARS in Stoneville, MS. Plants will be inspected for sufficient oviposition to insure 

a heavy population of neonate TBWs in each line. Approximately 7-10 days after 

infestation at least 20 plants per line will be inspected for surviving larvae. Larvae will be 

collected, categorized according to size and identified to insure that surviving larvae are 

TBW. Any larvae reaching third instar stage will be considered a "survivor". Second 

instar larvae will be collected from the plants and placed on artificial diet to determine if 

they survive to third instar. Cages/Lines will be infested at least three times during the 

season and mortality/survivorship will be determined, thus infestation times will serve as 

replications. The objective of the study will be to determine if each component line 

(Cry1Ab and Cry2Ae) will control the TBW population at a high level to justify each Cry 

gene component of TwinLink as a "high dose". 

The trials may include sites where the infestations occur as natural infestations, as well 

as artificially infested to enhance the opportunity to observe extreme CBW pressure. We 

expect large differences in the level of severity of the infestation and damage that can be 
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seen across locations and environments; and for this reason, the evaluation of efficacy 

across a broad spectrum of environmental and geographical conditions is needed to 

accurately evaluate efficacy of the Cry1Ab events against the targeted pests under field 

conditions. Additionally, evaluation by independent experts, such as in trials conducted 

by university researchers and other cooperators, provides independently generated 

performance data. 

 Insect Efficacy CBW, Artificial Infestations - These trials will be conducted to evaluate 

effectiveness of events T303-3 and T304-40 in controlling the primary target pest CBW. 

The trials may include sites where the infestations occur as natural infestations, as well 

as artificially infested to enhance the opportunity to observe extreme CBW pressure. We 

expect large differences in the level of severity of the infestation and damage that can be 

seen across locations and environments; and for this reason, the evaluation of efficacy 

across a broad spectrum of environmental and geographical conditions is needed to 

accurately evaluate efficacy of the Cry1Ab events against the targeted pests under field 

conditions. Additionally, evaluation by independent experts, such as in trials conducted 

by university researchers and other cooperators, provides independently generated 

performance data. 

 Insect Efficacy, other Leps - Though the primary target pest for the Cry1Ab events are 

TBW and CBW, the Cry1Ab protein has been shown to have activity against other 

lepidopteran pests of cotton, including Fall Armyworm and Pink Bollworm. These pests 

can be significant in certain seasons and in certain geographical regions, though the 

overall incidence and damage attributable to these pests is probably less significant than 

for the two main pests. Since these and other lepidopteran pests can cause significant 

economic damage in some environments, the opportunity for Cry1Ab to provide control of 

additional lepidopteran pests will be evaluated. The acreage available under this EUP 

will allow the flexibility to test for these additional insect pests in planned experiments. 

 Agronomic Evaluation - These trials will be used to document yield and agronomic 

performance of the events containing Cry1Ab. Some trials will be small plot evaluations 

to look at effects on flowering, maturity, seed size, herbicide sensitivity, and yield, for 

example. Other trials will use larger fields to evaluate whether having Cry1Ab provides 

agronomic and yield benefits for production fields that affect the economics of producing 

cotton seed. One could reasonably expect that use of Cry1Ab in cotton lines will 

significantly reduce the amount of insecticides that will need to be applied to production 

fields, and will also substantially increase seed yields in production fields affected by the 

targeted pests. Evaluation of these questions is not possible under small plot situations, 

and requires the acreage provided by this EUP. 

Cooperators and Participants

The Participants and Cooperators who will supervise the experimental work conducted under this 

EUP are listed in the Confidential Attachment A. 

{CBI Cross Reference Number 1} 

Locations

The total maximum acreage by state planted with seed containing Cry1Ab protein for efficacy 

testing is listed below. 
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Efficacy Testing Locations and Acreage

State Non-PIP 

Acres 

Cry1Ab 

Cotton 

Acres 

Total 

Acreage by 

State 

Max. lb of 

Cry1Ab 

Cotton Seed 

AL 22.5 3.75 26.25 45 

AR 22.5 3.75 26.25 45 

AZ 83 11 94 132 

CA 46 7 53 84 

FL 7.5 1.25 8.75 15 

GA 33 8.5 41.5 102 

LA 58 6 64 72 

MS 132.5 8.75 141.25 105 

NC 93.5 8.25 101.75 99 

PR 48 3 51 36 

SC 174.5 9.75 184.25 117 

TN 50.5 4.75 55.25 57 

TX 243 24 267 288 

Total 1014.5 99.75 1114.25 1197 

1

 Calculated as: Cry1Ab acres x 12 lb of seed/acre 

Genotypes and vectors

Transgenic cotton plants expressing the cry1ab gene (event T304-40 and T303-3) 

Experimental lines with different backgrounds 

Non transgenic plants Coker 315, Coker 312 or equivalents 

Transgenic commercial control 

Non transgenic commercial control 

Trial Design

The preferred statistical design is a split-plot where level of insect infestation (sprayed vs. nonsprayed) is the main plot and line/transgene is the subplot. A split-strip design may also be used 

which facilitates insecticide treatments. A randomized complete block design (RCBD) would 

suffice, but would not provide maximal precision. 

Agronomic Treatments

Typical agronomic inputs for conventionally grown cotton for the area, 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 insecticide applications for non-target and/or target insects only 

(see test treatments below) 

 Growth regulator application (this should be done sparingly if at all) 

 Additional hand weeding as necessary 

 Chemical defoliation without boll-opening desiccants 

Test Treatments
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Test treatments involve natural infestations of target insects, chemical control of target insects, 

and chemical control of non-target insects. Treatments include but are not limited to: 

 Complete insect control, both target and non-target in sprayed plots 

 Insect control of non-target insects that allows or encourages natural infestation of target 

insect, in non sprayed plots. 

Border rows

The EUP test plants or the trial will be surrounded by one or more border rows of cotton. 

Schedule

Planting dates: February - June 

Harvest dates: July - November 

Activities and Agronomic Practices 

Plots will be harvested by hand or mechanically. If by hand, the bolls will be placed in cloth or 

paper bags of such construction to avoid loss of seed outside of the bags. If by machine, seed 

cotton will be harvested, transported and processed under conditions appropriate for the handling 

of regulated material. This includes separate, redundant labeled packaging of all regulated 

material leaving the location. 

Use of GPS coordinates, stakes, markers or other methods will be used to identify the area where 

the transgenic plants are grown, and such an area will be subsequently monitored for volunteers 

for an appropriate period of time. Volunteer plants will be terminated by hand weeding, disking, 

herbicide spraying or other method. 

Containment

EUP test plants will be isolated in accordance with USDA-APHIS Performance Standards for 

regulated cotton trials. Isolation methods will include one or more of the following: (1) EUP test 

plants will be located at least 660 feet from other parties’ sexually receptive cotton; (2) a 40 footwide perimeter of non-transgenic cotton will surround the transgenic plants planted at a density 

within 20% (greater or less than) the trial density to act as pollen sink for insect pollinators (the 

perimeter cotton would be disposed of by harvesting, disking and monitoring); (3) temporal 

isolation, where the flowering period for the EUP plants will not coincide with the presence of 

other parties’ sexually receptive cotton within 660 feet of the EUP test plants. Open flowering 

EUP test plants may be located within 660 feet of sexually receptive cotton provided such other 

cotton is used only for experimental purposes and/or destroyed. 

Following the trial completion, all remaining plant debris will be destroyed by incorporation in the 

soil. All equipment used during crop destruction practices will be inspected and cleaned before 

leaving the field. Seed cotton not destined for further experimentation will be destroyed by 

incineration or deep burial. 

If harvested material is to be ginned, seed cotton will securely be transported to a gin. 

Processing of harvested seed cotton will consist of either hand ginning or research/commercial 

scale ginning. Hand ginning will be on small, table-top gins. Machine ginning will occur at the 

ginning facility on-site on a limited number of research/commercial scale gins. All packaging and 

waste will be destroyed by devitalization. No seed cotton or ginning by-products will be used for 

food or feed. Ginned seed will be stored under containment practices for regulated materials. 

Agronomic Data Collection

If available, the following data may be collected from the plots, using a 1-9 scale, where 

applicable: 

 Strain uniformity: 1=uniform, 9=highly variable 

 Leaf pubescence: 1=highly pubescent, 5=semi-smooth, 9=glabrous 
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 Disease reaction (verticillium wilt, bacterial blight, bronze wilt, etc. IF applicable): 1=no 

symptoms, 5=some symptoms apparent, 9=severe 

 Stalk lodging: 1=upright, 9=severely lodged 

 % open bolls as a visual average when uninfested recurrent parent is 40-60% open 

 Yield in lb lint per acre 

 % lint 

 # seed per boll 

 Boll size 

 Seed index 

 Fiber properties: length, length uniformity, strength, micronaire, elongation 

 Plant mapping: plant map 10 plants per plot, each of 3 reps at maturity shortly before 

defoliation. Data will include plant height, number of nodes, and boll position. Boll 

damage ratings may be a part of the mapping data. Information collected will reflect 

overall plant architecture and maturity. 

Target Insect Evaluation Data Collection

Data will be taken on insect damage for 6-8 weeks to measure resistance to the infested insect. 

Insect infestation and damage data are collected by examining the terminal and one square or 

boll on 10 plants per row on each of the two center rows in each plot. Data collected may 

include: 

 Number of live larvae in squares 

 Number of live larvae in bolls 

 Number of squares damaged by larvae 

 Number of bolls damaged by larvae 

 Number of damaged white flowers 

 Number of live larvae in white flowers 

Quality Control Sample Collection

Leaf samples are to be taken from two individual plants per plot for QC purposes. 
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 Experimental Program:

PRODUCTION OF SAMPLE MATERIAL FOR USE IN REGULATORY STUDIES 

Objectives

The purpose of these trials is to produce sample materials for regulatory trials, as part of the 

requirement of various regulatory agencies for the submission of registration packages in different 

countries. 

Cooperators and participants

The Participants and Cooperators who will supervise the experimental work conducted under this 

EUP are listed in the Confidential Attachment A. 

{CBI Cross Reference Number 1} 

Locations 

The total maximum acreage by state planted with seed containing Cry1Ab protein for production 

of regulatory samples is listed below. 

Regulatory Study Sample Material Generation Locations and Acreage

State Non-PIP 

Acres 

Cry1Ab 

Cotton 

Acres 

Total 

Acreage 

by State 

Max. lb of 

Cry1Ab Cotton 

Seed 

AL - - - N/A2

AR 10.5 4.75 15.25 57 

AZ 75 7 82 84 

CA 28 3 31 36 

FL 3.5 0.25 3.75 3 

GA 8 2 10 24 

LA 15 2.5 17.5 30 

MS 105 8.5 113.5 102 

NC 16 4 20 48 

PR 8 2 10 24 

SC - - - - 

TN 8 2 10 24 

TX 158.5 16.25 174.75 195 

TOTAL 435.5 52.25 487.75 627 

1

 Calculated as: Cry1Ab acres x 12 lb of seed/acre 

2

 Not Applicable 

Genotypes and vectors

Transgenic cotton plants expressing the cry1ab gene (event T304-40 and T303-3) 

Experimental lines with different backgrounds 

Non transgenic plants Coker 315, Coker 312 or equivalents 

Transgenic commercial control 

Non transgenic commercial control 
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Trial Design

For seed increased to plant future regulatory studies, a block consisting of transgenic seed will be 

planted with rows of non transgenic border rows of cotton planted on all four sides of the 

perimeter of the seed block. The spacing between each row is approximately 38 inches. 

Agronomic Treatments

Typical agronomic inputs for conventionally grown cotton for the area, 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 insecticide applications 

 Growth regulator application (this should be done sparingly if at all) 

 Additional hand weeding as necessary 

 Chemical defoliation without boll-opening desiccants as this will mask maturity 

Border rows

The EUP test plants or the trial will be surrounded by one or more border rows of cotton. 

Schedule

Planting dates: February - June 

Harvest dates: July - November 

PR – Planting dates: October –November 

 Harvest dates: April-May 

Activities and Agronomic Practices 

After planting, transgenic and control seeds that were not planted will be collected. The 

transgenic seeds will be returned to the attention of the Study Director. Any non-transgenic 

seeds that are not planted will be disposed of at the discretion of the Cooperator. The disposition 

of the seeds will be documented. 

Plots will be harvested by hand or mechanically. The harvested seed cotton will be ginned at or 

near the field location to obtain required fuzzy seed samples. Lint obtained from the ginning will 

be disposed of. Seed samples will be packed, placed in frozen storage and ultimately shipped, 

frozen, to the Study Director and/or to a designated analytical laboratory for compositional 

analyses. 

After the field phase of the study is complete, the remaining transgenic and non-transgenic cotton 

plants will be destroyed by plowing the crop into the ground. 

Containment

EUP test plants will be isolated in accordance with USDA-APHIS Performance Standards for 

regulated cotton trials. Isolation methods will include one or more of the following: (1) EUP test 

plants will be located at least 660 feet from other parties’ sexually receptive cotton; (2) a 40 footwide perimeter of non-transgenic cotton will surround the transgenic plants planted at a density 

within 20% (greater or less than) the trial density to act as pollen sink for insect pollinators (the 

perimeter cotton would be disposed of by harvesting, disking and monitoring); (3) temporal 

isolation, where the flowering period for the EUP plants will not coincide with the presence of 

other parties’ sexually receptive cotton within 660 feet of the EUP test plants. Open flowering 

EUP test plants may be located within 660 feet of sexually receptive cotton provided such other 

cotton is used only for experimental purposes and/or destroyed. 

Following the trial completion, all remaining plant debris will be destroyed by incorporation in the 

soil. All equipment used during crop destruction practices will be inspected and cleaned before 
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leaving the field. Seed cotton not destined for further experimentation will be destroyed by 

incineration or deep burial. 

After completion of sampling, all perimeter cotton, if perimeters are planted, will be disposed of by 

mowing, followed by disking into the soil. The transgenic plots and any perimeter cotton fields will 

be monitored the following season for volunteers. Volunteer plants will be terminated by hand 

weeding, disking, herbicide spraying or other method. 

If harvested material is to be ginned, seed cotton will securely be transported to a gin. All 

packaging and waste will be destroyed by devitalization. Ginned seed will be stored under 

containment practices for regulated materials. 

Data Requirement

Depending upon the study, one or two treatments of Cry1Ab cotton seed will be produced. If two 

treatments are produced, one treatment will be from plants that were treated with glufosinate 

ammonium and one treatment will be from plants that were not treated with glufosinate 

ammonium. In addition to the Cry1Ab Cotton material, a non-genetically modified line of the 

same genetic background will also be produced to provide material for comparative treatments. 

Cotton seed will be harvested from each plot, including edge rows, at normal maturity. Material 

from the control non-transgenic plots will be harvested before harvesting the transgenic plots, if 

they were established in the same field. The harvested seed cotton will be ginned to obtain the 

required seed samples to prepare meal for feeding studies. Ginning will be done at or near the 

field site. If the seed cotton is transported to the gin, the samples will be contained in an 

enclosed or covered vehicle or module. Ginning may be performed on a commercial gin, under 

the supervision of the Cooperator. 

The ginned seed samples will be stored for transport at ambient temperature in suitable 

containers such as large bulk bags. Sub-samples will be sent to BCS. The bulk samples will be 

shipped to a processing facility for processing into meal. Transgenic cotton lint may be retained 

for BCS after ginning for immediate storage by BCS and possible future commercial use after 

registration. Alternatively, it will be disposed of. Any “waste” transgenic lint (e.g., spillage or 

clean-out waste) will be disposed of. 

After samples are collected, remaining transgenic crop (left in the field after harvesting) will be 

destroyed. 

Future Plans

Transgenic seed harvested from trial may be used for regulatory trials at a later date.