E

Determination of Plant 

Pest Risk for InsectResistant and Glufosinate 

Ammonium-Tolerant 

(TwinLinkTM) Cotton, 

Gossypium hirsutum, 

events T304-40 x GHB119

Petition 08-340-01p

Draft 

Plant Pest Risk Assessment

April 2011

United States

Department of 

Agriculture

Marketing and 

Regulatory 

Programs

Animal and 

Plant Health 

Inspection 

Service
2

Table of Contents

Table of Contents.............................................................................................. 2

Plant Pest Risk Assessment of Bayer CropScience TwinLinkTM Cotton ......... 3

A. Introduction..................................................................................... 3

B. History of Development of TwinLinkTM Cotton ............................ 3

1. Description of the modifications..................................................... 4

C. Plant Pest Risk Assessment ............................................................ 5

1. Potential impacts of altered disease and pest susceptibilities......... 6

2. Potential impacts from outcrossing of the BCS TwinLinkTM cotton 

to wild relatives............................................................................... 7

3. Potential impacts based on the relative weediness of BCS 

TwinLinkTM cotton.......................................................................... 9

4. Potential impacts on target and nontarget organisms, including 

beneficial organisms. .................................................................... 10

5. Potential impacts from transferring genetic information from BCS 

TwinLinkTM cotton to organisms with which it cannot interbreed.

11

D. Conclusion .................................................................................... 12

E. References..................................................................................... 12
Plant Pest Risk Assessment of Bayer CropScience 

TwinLinkTM Cotton

A. Introduction

Bayer CropScience (BCS) has petitioned APHIS for a determination that TwinLinkTM cotton is 

unlikely to pose a plant pest risk and, therefore, is no longer a regulated article under regulations 

at 7 CFR part 340. Bayer CropScience (BCS) has developed cotton (Gossypium hirsutum) plants 

that express two insecticidal proteins, Cry1Ab and Cry2Ae, from a common soil bacterium, 

Bacillus thuringiensis (Bt) and also expresses the bar gene that confers resistance to glufosinate 

ammonium herbicide from Streptomyces hygroscopicus. 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. In addition to the Cry1Ab and Cry2Ae proteins, TwinLinkTM

cotton also contains the PAT (phosphinothricin-acetyl-transferase) enzyme, encoded by the bar 

gene. This is the same protein that is expressed in Bayer CropScience Liberty Link cotton® 

(LLCotton25) that confers to the plant tolerance to the herbicide glufosinate ammonium.

B. History of Development of TwinLinkTM Cotton

In 2002, APHIS deregulated the first phosphinothricin (glufosinate)-tolerant cotton, event 

GHB119; Aventis CropScience cotton designated Liberty Link cotton® (LLcotton25). This 

event involved cotton that was transformed with a binary plasmid vector carry the bar gene 

construct within a disarmed transfer DNA (T-DNA) from Agrobacterium tumefaciens. Aventis 

CropScience (now Bayer CropScience) completed its food safety consultation with the U.S. 

Food and Drug Administration (FDA) on Liberty Link cotton® in 2003 

(http://www.accessdata.fda.gov; BNF 000086). Tolerance exemptions have been granted for the 

plant-incorporated protectant in Liberty Link cotton® (event LLcotton25) and the genetic 

material necessary for its production. On April 11 1997, the Environmental Protection Agency 

(EPA) granted exemptions from the requirement of a tolerance for residues of the plant-pesticide

phosphinothricin-acetyl-transferase protein in or on food and feed commodities of cotton (62 FR 

17719; 40 CFR 180.473). The Australia and New Zealand Food Authority (ANZFA) also 

completed a Toxicological Review and Risk Assessment, and a Technical Report was published 

in 2005 (ANZFA Application A533, 2005; http://www.foodstandards.gov.au). ANZFA 

concluded that Liberty Link cotton® was comparable to non-genetically engineered (non-GE) 

cotton in terms of their safety and nutritional adequacy. In 2004, the Plant Biosafety Office 

(PBO) of the Canadian Food Inspection Agency (CFIA) concluded that Bayer CropScience’s 

Liberty Link cotton® was substantially equivalent to currently grown cotton, in terms of their 

potential environmental impact and livestock feed safety and the novel traits would not have any 

substantial negative effect on the environment (CFIA Decision Document DD2004-49; 

http://www.inspection.gc.ca).

Bayer CropScience has now developed the transgenic cotton, TwinLinkTM, by crossing cotton 

event GHB119 that expresses both the Cry2Ae protein and phosphinothricin-acetyl-transferase
4

(found in LiberyLink® cotton) proteins now with a newly developed cotton line (event 304-40) 

that expresses both Cry1Ab protein and phosphinothricin-acetyl-transferase (found in 

LiberyLink® cotton) proteins. TwinLinkTM cotton has been developed by BCS as an alternative 

insect resistant and herbicide tolerant cotton product.

TwinLinkTM cotton has been field tested under APHIS regulations since 2005. Data were 

provided in the petition for field trials completed prior to the petition submission. Field test 

reports can be found in the BCS TwinLinkTM cotton petition in Appendix 1, p.102.

On December 22 2008, BCS submitted a request that the Environmental Protection Agency 

(EPA) grant an exemption from the requirement of a tolerance for residues of the plantincorporated protectant (PIP), Bacillus thuringiensis Cry2Ae and Cry1Ab insect control proteins

and the genetic material necessary for its production, in or on all food commodities. BCS has

also submitted a food safety summary to FDA indicating that food and feed derived from 

TwinLinkTM cotton are as safe and nutritious as food and feed derived from conventional cotton. 

1. Description of the modifications

TwinLinkTM 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” (pg 2, BCS 

petition).

The two parent lines that were conventionally crossed to produce TwinLinkTM cotton contain the 

following genetic components (also described in BCS petition on page 21):

Cry2Ae + PAT (event GHB119) has been genetically engineered to contain the following 

transgene fragments:

Left border (LB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988).

3 ́nos: sequence including the 3’ untranslated region of the nopaline synthase gene from 

the T-DNA of pTiT37 in counter clockwise direction (Depicker, Stachel et al. 1982)

bar; the coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces 

hygroscopicus in counter clockwise direction (De Block, Botterman et al. 1987)

Pcsvmv XYZ sequence including the promoter region of the Cassava Vein Mosaic Virus 

in counter clockwise direction (Verdaguer, De Kochko et al. 1996)

P35S2 sequence including the promoter region from Cauliflower Mosaic Virus 35S 

transcript in clockwise direction (Odell, Nagy et al. 1985)

5'cab22L sequence including the leader sequence of the chlorophyl a/b binding protein 

gene from Petunia hybrida in clockwise direction (Harpster, Townsend et al. 1988)

TPssuAt coding sequence of the transit peptide of the ribulose-1,5-biphosphate 

carboxylase small subunit gene ats1A of Arabidopsis thaliana in clockwise direction 

(De Almeida, Gosselé et al. 1989)

Cry2Ae the coding sequence of an insecticidal protein gene of Bacillus thuringiensis, 

adapted to cotton codon usage in clockwise direction
5

3'35S sequence including the 3' untranslated region of the 35S transcript of Cauliflower 

Mosaic Virus in clockwise direction (Sanfaçon, Brodman et al. 1991)

Right border (RB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988)

Cry1Ab + PAT (event 304-40) has been genetically engineered to contain the following 

transgene fragments:

Right border (RB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988)

3’ me1 sequence that includes the 3’ untranslated region of the NADP-malic enzyme 

gene of Flaveria bidentis (yellowtop) in counter clockwise direction (Marshall, 

Stubbs et al. 1996)

Cry1Ab sequence from B. thuringiensis berliner 1715 in counter clockwise direction 

(Höfte, de Greve et al. 1986)

5’e1 sequence that includes the leader sequence of the tapetum specific E1 gene (GE1) of 

Oryza sativa (rice) in counter clockwise direction (Michiels F. 1992)

Ps7s7 sequence including the duplicated promoter region derived from subterranean 

clover stunt virus genome segment 7 in counter clockwise direction (Boevink, Chu et 

al. 1995)

P35S3 sequence including the promoter region from Cauliflower Mosaic Virus 35S 

transcript in clockwise direction (Odell, Nagy et al. 1985)

bar; the coding sequence of the phosphinothricin acetyltransferase gene of Streptomyces 

hygroscopicus in clockwise direction (De Block, Botterman et al. 1987)

3 ́nos sequence including the 3’ untranslated region of the nopaline synthase gene from 

the T-DNA of pTiT37 in clockwise direction (Depicker, Stachel et al. 1982)

Left border (LB) repeat from the T-DNA of A. tumefaciens (Zambryski 1988)

C. Plant Pest Risk Assessment

This plant pest risk assessment is to determine whether BCS TwinLinkTM cotton is unlikely to 

pose a plant pest risk. If APHIS determines that a GE organism is unlikely to pose a plant pest 

risk, APHIS then has no regulatory authority over that organism under 7 CFR part 340. 

APHIS administers the regulations 7 CFR part 340 under the authority of the plant pest 

provisions of the Plant Protection Act of 2000 (PPA). 

The PPA states that: 

“The term ‘plant pest’ means any living stage of any of the following that can 

directly or indirectly injure, cause damage to, or cause disease in any plant or plant 

product: 

(A) A protozoan. 

(B) A nonhuman animal. 

(C) A parasitic plant. 

(D) A bacterium. 

(E) A fungus. 

(F) A virus or viroid. 
6

(G) An infectious agent or other pathogen. 

(H) Any article similar to or allied with any of the articles specified in the preceding 

subparagraphs.” 

The Animal and Plant Health Inspection Service of the United States Department of Agriculture 

(USDA-APHIS) has prepared a Plant Pest Risk Assessment in response to a petition (APHIS 

Number 08-340-01p) from BCS. APHIS regulation 7 CFR 340.6(c) stipulates the information 

needed for consideration in a petition for nonregulated status. APHIS evaluated information 

submitted by the applicant related to plant pest risk characteristics, disease and pest 

susceptibilities, expression of the gene product, new enzymes, or changes to plant metabolism, 

weediness of the regulated article, any impacts on the weediness of any other plant with which it 

can interbreed for BCS TwinLinkTM cotton. Issues related to agricultural or cultivation practices 

and the effects of the regulated article on non-target organisms are considered in the 

Environmental Assessment for BCS TwinLinkTM cotton. 

Based on information on the biology of cotton (OECD 2008) data presented by BCS (APHIS 

Number 08-340-01p) and scientific data relevant to a discussion of plant pest risk, APHIS 

concluded the following regarding BCS TwinLinkTM cotton: 

1. Potential impacts of altered disease and pest susceptibilities

USDA-APHIS assessed whether BCS TwinLinkTM cotton is likely to have significantly 

increased disease and pest susceptibility. The assessment encompasses a thorough consideration 

of introduced traits and interactions with pest and disease. 

Cotton (Gossypium hirsutum) is not a plant pest in United States, and the introduced DNA in 

BCS TwinLinkTM cotton is unlikely to pose a plant pest risk because there are no pathogenic 

DNA sequences present. The description of the genetic modifications, including genetic 

elements, expression of the gene product and their functions for BCS TwinLinkTM cotton has 

been summarized above. Because the goal of TwinLinkTM cotton was to decrease the pest 

susceptibility, the data submitted by BCS indicated significant differences between BCS 

TwinLinkTM cotton and the non-transgenic counterparts for insect stresses (as measured by 32

agronomic parameters evaluated found on page 68 of the petition). An increase in yield was 

attributed to the inserted gene expression in TwinLinkTM cotton. There were no increases in pest 

susceptibility or disease as measured by the agronomic properties found on page 68 of the 

petition.

The introduced genes (cry1Ab, cry2Ae and bar) encode for Cry1Ab and Cry2Ae and glufosinate 

synthase proteins, respectively. The cry1Ab and cry2Ae genes come from the soil bacteria 

Bacillus thuringiensis and the bar gene is from Streptomyces hygroscopicus. Such genes have 

been used in field trials previously and are not known to cause plant disease. There is no 

indication that inserting the cry1Ab, cry2Ae and bar genes will result in increased likelihood of 

introduction or dissemination of a plant pest. Cry proteins were isolated and used commercially 

as a pesticide in France in 1938, marketed under the name Sporine. Since 1961, EPA approved 

Bt proteins for the treatment against lepidopteran pests under the trade name, Thuricide (UCSD 

1999; Erlandson and Litowski 2005). APHIS has not identified any plant pest risk following the 

introduction of Cry proteins or phosphinothricin acetyltransferase in prior deregulated products, 
7

such as rice, corn, cotton, beet, rapeseed, or soybean. A list of petitions of nonregulated status 

granted can be found at http://www.aphis.usda.gov/brs/not_reg.html. The Plant Biosafety Office 

(PBO) of the Canadian Food Inspection Agency (CFIA) also concluded that the intended effects 

herbicide-resistant plants such as phosphinothricin acetyltransferase are not related to altered 

plant pest potential (http://www.inspection.gc.ca/english/sci/biotech/enviro/herbice.shtml. CFIA 

has also reviewed the plant pest risk, the environmental risk and safety of plants with novel 

genes containing Cry proteins and found these proteins are not related to altered plant pest 

potential. CFIA’s decision documents can be found at 

http://www.inspection.gc.ca/english/plaveg/bio/dde.shtml.

2. Potential impacts from outcrossing of the BCS TwinLinkTM cotton to wild 

relatives

In assessing the risk of gene introgression from BCS’ TwinLinkTM cotton into its sexually 

compatible relatives, APHIS considers two primary issues: 1) the potential for gene flow and 

introgression via pollen movement and horizontal gene transfer1

a) Gene Flow via Pollen Movement

; and 2) the potential plant pest 

risk of introgression.

Movement of genetic material by pollen is possible only to those plants with the proper 

chromosomal type. In the United States, this would only include G. hirsutum, G. barbadense, 

and G. tomentosum. Native G. barbadense is only found in Hawaii, Virgin Islands and Puerto 

Rico, while G. tomentosum is only found in Hawaii (Fryxell 1979). G. hirsutum is generally 

self-pollinating but some cross-pollination can occur, albeit at relatively low incidence through 

activity of pollinating insects (Fryxell 1979). Gene movement between G. hirsutum and G. 

barbadense is possible if suitable insect pollinators are present, and if there is a short distance 

from host plants to recipient plants (Fryxell 1979). Physical barriers, intermediate pollinatorattractive plants, and other temporal or biological impediments (geography or absence of 

pollinators) reduce the potential for pollen movement (Fryxell 1979). Table 1 outlines the 

compatibility of all species on an international level. Concentration of suitable pollinators varies 

from location to location and by season, and is considerably suppressed by insecticide use.

Table 1. Cotton Species

Species Common Name Native 

location Comments

G. hirsutum Upland cotton

Central 

America, 

Mexico, 

Caribbean 

and 

southern 

Florida. 

Commercial Species, Grown in U.S.A. and 

comprises 97% of U.S.A cotton crop. 

Sexually compatible with G. barbadense

and G. tomentosum.

G. barbadense

Pima, Creole, 

Egyptian or Sea 

Island cotton

S. America 

Commercial species, grown in U.S.A. 

Grown in Hawaii, Virgin Islands and Puerto 

Rico. Sexually compatible with G. 

hirsutum and G. tomentosum.

 1 Horizontal gene transfer is any process in which an organism transfers genetic material to another cell that is not its 

offspring.
8

G. tomentosum Ma’o or Hawaiian 

cotton Hawaii

Non-commercial species. Thought to be 

only pollinated by moths when the flowers 

open at night, but more recent evidence 

suggests it can also be pollinated in early 

morning by bees. Only found in Hawaii. 

Sexually compatible with G. hirsutum and 

G. barbadense.

G. arboreum Asiatic tree or tree 

cotton

Pakistan, 

India

Commercial species, grown in Europe, 

Africa and eastern countries. Sexually 

compatible with G. herbaceum.

G. herbaceum Levant cotton Africa, 

Arabia

Commercial species, grown in Europe, 

Africa and eastern countries. Sexually 

compatible with G. arboreum.

G. thurberi

Thurber’s, Desert or 

Arizona desert 

cotton

Mexico, 

Arizona 

Non-commercial species. Sexually 

compatible with G. arboreum and G. 

herbaceum.

Historically, it was reported that cross-pollination between G. tomentosum and G. hirsutum was

unlikely because they use different insect pollinators and are receptive to pollination at different 

times of the day (McGregor 1976). Field and laboratory studies demonstrated that the historic 

literature is incorrect, and that these three species share common pollinators, and further that 

differences in flower structure and flowering habits do not serve as barriers to cross pollination 

(Pleasants and Wendel 2010). However, DNA marker analyses have not found evidence of genes 

from G hirsutum occurring in native populations of G. tomentosum (DeJoode and Wendel 1992). 

It is possible that the lack of evidence of movement of G. hirsutum genes into G. tomentosum is 

the result of lack of opportunity because cotton has not been grown commercially in Hawaii for 

at least the last 45 years (USDA-APHIS 2010).

In farm scale studies using traditional Upland cotton in California, it was found that the outcrossing distance was strongly dependent on the presence of bee colonies. When only native 

pollinators were present in the field, 1% out-crossing was detectable over a distance of 1 meter 

(approximately 3 ft) and 9 m (29.5 ft) when there was high pollinator activity (Van Deynze, 

Sunderstrom et al. 2005). Out-crossing declined exponentially with increasing distance from the 

source plot (Van Deynze, Sunderstrom et al. 2005). Current cultivation practices to prevent outcrossing (distance being primarily used) have been deemed sufficient to prevent unwanted gene 

flow. For Upland cotton, the Association of Official Seed Certifying Agencies (AOSCA) 

mandates an isolation distance being a nature barrier or crop boundary with a minimal isolation 

distance of 100 ft “if the contaminating source differs by easily observed morphological 

characteristics from the field to be inspected”. For Pima or Egyptian type cotton “the isolation 

shall be 1320 feet from any other type of cotton for Foundation and Registered and 660 feet for 

Certified seed”2

. Since TwinLinkTM cotton is not morphologically distinguishable from 

traditional Upland cotton much like Pima or Egyptian type cotton, cultivation practices using 

AOSCA standards of 1320 ft for Foundation and Registered and 660 ft for Certified seed are 

used.

 2 From AOSCA “Yellow Books” 2003 OPERATIONAL PROCEDURES, CROP STANDARDS AND SERVICE 

PROGRAMS PUBLICATION (Genetic and Crop Standards), pg 194.
9

Wind is rarely seen as a means for cross-pollination of cotton pollen because of its adherent 

properties and large size (mean diameter of 53-56 μm). The pollen of cultivated Gossypium

species is described as being sticky and having pronounced spines, with a marked tendency for 

groups of pollen grains to clump together (Hutmacher and Wright 2006).

b) Gene Flow via Horizontal Gene Transfer3

Transfer and expression of DNA from TwinLinkTM cotton to soil bacteria is unlikely to occur. 

Gebhard and Smalla (Gebhard and Smalla 1999) and Schlüter et al. (Schlüter, Fütterer et al. 

1995) have studied transgenic DNA movement to bacteria, and although theoretically possible, 

determined mathematically it would occur at extremely low rates (approximately 1 in 10-14

Kaneko, Nakamura et al. 2000

). 

Many genomes (or parts thereof) have been sequenced from bacteria that are closely associated 

with plants including Agrobacterium and Rhizobium ( ) and there is 

no evidence for recent horizontal transfer. Koonin et al. (Koonin, Makarova et al. 2001) and 

Brown (Brown 2003) presented reviews based on sequencing data that revealed horizontal gene 

transfer occurs occasionally on an evolutionary time scale of millions of years. Even in the 

unlikely event transfer were to occur, the gene would be poorly expressed at best because 

transgene promoters and coding sequences are optimized for plant expression and function 

poorly in prokaryotic cells. 

Based on the above considerations, TwinLinkTM cotton will not adversely impact sexually 

compatible wild relatives or their weediness characters.

3. Potential impacts based on the relative weediness of BCS TwinLinkTM 

cotton.

In the United States, cotton is not listed as a weed in the major weed references

(Crockett 1977; Holm, Pancho et al. 1979; Muenscher 1980), nor is it present on the list of 

noxious weed species (7 CFR 360) distributed by the Federal Government (USDA-APHIS 

2006). Furthermore, cotton has been grown throughout the world without any report that it is a 

serious weed. Cotton is unlikely to become a weed. It is not persistent in undisturbed 

environments without human intervention. In the year following cultivation, cotton may grow as 

a volunteer only under specific conditions (disturbed or cultivated soil that had cotton grown in 

the last growing season) and can be easily controlled by herbicides or mechanical means. It does 

not compete effectively with cultivated plants or primary colonizers because it is such a slow 

grower, especially in the cooler soils in the northern cotton belt (OECD 2004).

APHIS assessed whether TwinLinkTM cotton is any more likely to become a weed than the 

isogenic nontransgenic cotton line, or other cotton varieties currently under cultivation. The 

assessment encompasses a thorough consideration of the basic biology of cotton and an 

evaluation of the unique characteristics of TwinLinkTM cotton evaluated under field conditions.

BCS conducted agronomic field trials during the 2005, 2007 and 2008 growing seasons across 8 

states and 23 locations representative of the major cotton-growing areas of the cotton belt (field 

test reports can be found in the BCS TwinLinkTM cotton petition in Appendix 1, p.102). The data 

 3 Horizontal gene transfer is any process in which an organism transfers genetic material to another cell that is not its 

offspring.
10

submitted by BCS indicated no significant differences between BCS TwinLinkTM cotton and the 

non-transgenic counterparts (as measured by 32 agronomic parameters evaluated found on page 

68 of the petition).

Based on the agronomic field data and literature survey about cotton weediness potential, 

TwinLinkTM cotton lacks ability to persist as troublesome weed, and there would be no direct 

impact on current weed management practices for cotton cultivation.

4. Potential impacts on target and nontarget organisms, including 

beneficial organisms.

The Cry1Ab and Cry2Ae proteins expressed in TwinLinkTM cotton are effective in controlling 

specific 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 Cry proteins are not expected to adversely affect non-target 

invertebrates such as bees, ladybugs, lacewings, spiders or other arthropods and vertebrate 

organisms, including birds, mammals and humans, because they do not contain the receptor 

found in the midgut of target insects (OECD 2007).

Cry1Ab and Cry2Ae are crystal delta-endotoxins from Bacillus thuringiensis. A total of 314 

different cry genes have been identified by 2005 (OECD 2007). The insecticidal activity of 

Cry1Ab and Cry2Ae proteins occurs when the C-terminal half of these inactive protoxins are 

enzymatically cleaved within the midgut of susceptible insect larvae by trypsin-like proteases to 

the active toxin, which consists of the N-terminal portion of the molecule (Federici 1993; Brar, 

Verma et al. 2007). The susceptibility of the insect depends on what trypsin-like protease is 

expressed in the specific insect’s gut (OECD 2007).

For many decades microbial products containing Bacillus thuringiensis (the organism that 

produces Cry proteins) have been used to control insect pests on a commercial scale and for 

home garden applications (Glare and O’Callaghan 2000; Shelton, Zhao et al. 2002). Plants that 

were genetically engineered to express the Cry1A protein have a history of safe use in the U.S. 

Since the mid-1990s, corn and cotton lines have been commercialized without substantiated 

reports of significant deleterious impacts on non-target organisms (OECD 2007; USEPA 2008). 

As stated on pp.36 of the submitted petition (08-340-01p) “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).”

Cry proteins are not expected to adversely affect non-target invertebrates, such as bees, and 

vertebrate organisms, including birds, mammals and humans, because they do not contain the 

receptor found in the midgut of target insects. Data provided in the petition (summarized in the 

Table 30 and 31, pg. 80-81 and data found on pg. 82-83) confirmed that in the mammal, bird, 

honey bee, above ground arthropod, and soil dwelling invertebrate studies, no observable 
11

adverse effects or differences in survival were noted at for both Cry proteins. The amount of the 

Cry proteins tested were well above those expected from exposure to the Cry proteins from 

TwinLinkTM cotton planted in the field. The nontarget above-ground arthropods and soildwelling invertebrates studied (ladybugs, green lacewings, springtail, daphnia, honeybees, 

collembola and earthworms) were considered to be representative of the cotton agro-ecosystem. 

Although not an endangered or threatened species, Danaus plexippus (monarch butterfly) is a 

species of high conservation interest, and there has been concern that it may be harmed by 

consuming pollen from transgenic insect-protected cotton and corn. The monarch is susceptible 

to Cry1Ab (Hellmich, Siegfried et al. 2001) the most common insecticidal protein in transgenic 

maize and cotton. However, the distribution of the monarch’s food plant (Asclepias syriaca -

common milkweed), the monarch’s pattern of migration (Sears, Hellmich et al. 2001), and the 

lack of cotton pollen movement and adherence to available milkweed (cotton pollen is heavy and 

sticky) means that very few monarchs are exposed to harmful concentrations of Cry1Ab. 

TwinLinkTM cotton, therefore, poses low risk to monarchs because of minimal hazard of Cry1Ab 

and Cry2ac and low exposure to Cry protein-containing pollen. TwinLinkTM cotton is expected 

to have no harmful effects on any endangered or threatened species in the U.S.

5. Potential impacts from transferring genetic information from BCS 

TwinLinkTM cotton to organisms with which it cannot interbreed.

APHIS examined the potential for the new genetic material inserted into TwinLinkTM cotton to 

be horizontally transferred to other organisms without sexual reproduction and whether such an 

event could lead directly or indirectly to disease, damage, injury or harm to plants, including the 

creation of more virulent pathogens. The horizontal gene transfer between unrelated 

organisms is one of the most intensively studied fields in the biosciences since 1940, and the 

issue gained extra attention with the release of transgenic plants into the environment (Dröge, 

Puhler et al. 1998). Potential risks from stable horizontal gene transfer (HGT) from genetically 

engineered organisms to another organism without reproduction or human intervention were

recently reviewed (Keese 2008). Mechanisms of HGT include conjugation, transformation and 

transduction, and other diverse mechanisms of DNA and RNA uptake and recombination and 

rearrangement, most notably through viruses and mobile genetic elements. HGT has been a 

major contributor to the spread of antibiotic resistance amongst pathogenic bacteria and the 

emergence of increased virulence in bacteria, eukaryotes and viruses and in the long run has 

contributed to major transitions in evolution. 

a) Potential for Horizontal Gene Transfer to Bacteria or Fungi 

TwinLinkTM cotton has three bacteria genes. Horizontal gene transfer and expression of DNA 

from a plant species to other bacterial species is unlikely to occur based on the following 

observations. Although there are many opportunities for plants to directly interact with fungi and 

bacteria (e.g. as commensals, symbionts, parasites, pathogens, decomposers, or in the guts of 

herbivores), there are almost no evolutionary examples of HGT to bacteria from eukaryotes or 

from plants to fungi (Keese 2008). The only genes likely to be transferred successfully from 

genetically engineered plants to bacteria are other bacterial genes. Horizontal transfer from and 

expression in bacteria of the foreign DNA inserted into the nuclear genome of TwinLinkTM 
12

cotton is unlikely to occur. First, many genomes (or parts thereof) have been sequenced from 

bacteria that are closely associated with plants including Agrobacterium and Rhizobium 

(Kaneko, Nakamura et al. 2000; Wood, Setubal et al. 2001). There is no evidence that these 

organisms contain genes derived from plants. Second, in cases where review of sequence data 

implied that horizontal gene transfer occurred, these events are inferred to occur on an 

evolutionary time scale on the order of millions of years (Koonin, Makarova et al. 2001; Brown 

2003). Third, transgene DNA promoters and coding sequences are optimized for plant 

expression, not prokaryotic bacterial expression. Thus even if horizontal gene transfer occurred, 

proteins corresponding to the transgenes are not likely to be produced. Fourth, the FDA has 

evaluated horizontal gene transfer from the use of antibiotic resistance marker genes and 

concluded that the likelihood of transfer of antibiotic resistance genes from plant genomes to 

microorganisms in the gastrointestinal tract of humans or animals, or in the environment, is 

remote (http://vm.cfsan.fda.gov/~dms/opa-armg.html). 

b) Potential for Horizontal Gene Transfer to Viruses 

APHIS also considered whether horizontal transfer of DNA from TwinLinkTM cotton to plant 

viruses was likely to occur and would lead to the creation or selection of a more virulent plant 

pathogen through recombination with other plant viruses. This issue has been considered before 

by other science review panels and government regulatory bodies (Keese 2008). The only virus 

sequences contained within TwinLinkTM cotton encode regulatory elements: the cauliflower 

mosaic virus, the cassava vein mosaic virus and the subterranean clover stunt virus. Regulatory 

elements such as promoters and terminators have not been implicated in viral recombination. 

Therefore, APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses no 

significant environmental or plant pest risk. Finally, under natural conditions; no transfer of an 

intact functional gene has been demonstrated to date (Miki and McHugh 2004). Therefore 

APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses no significant 

environmental or plant pest risk.

D. Conclusion

APHIS has reviewed and conducted a plant pest risk assessment on BCS TwinLinkTM cotton. 

Due to the lack of plant pest risk from the inserted genetic material, the lack of weediness 

characteristics of BCS TwinLinkTM cotton, the lack of atypical responses to disease or plant pests 

in the field, the lack of deleterious effects on non-targets or beneficial organisms in the agroecosystem, and the lack of horizontal gene transfer, APHIS concludes that BCS TwinLinkTM 

cotton is unlikely to pose a plant pest risk.

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