E



# Determination of Plant Pest Risk for Insect

Resistant and Glufosinate Ammonium

- Tolerant ( TwinLink TM) C otton, **Gossypium hirsutum** , event s T304

- 40 x GHB119

Petition 08

- 340

- 01 p

Draft Plant Pest Risk Assessment April 2011 United States Department of Agriculture

Marketing and Regulatory Programs

Animal and Plant Health Inspection Service2 

## 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 p art 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, TwinLink TM cotton also contains the PAT (phosphinothricin

- acetyl

- transferase) enzyme, encoded by the bar gene. This is the same protein t hat is expressed in Bayer CropScience Liberty Link cotton® ( LLCotton25 ) that confers to the plant tolerance to the herbicide g lufosinate a mmonium.



### B. History of Development of TwinLinkTM Cotton

In 2002, APHIS deregulated the first phosphinothricin (glufosin ate)

- 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) fr om 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, t he Environmental Protection Agency (EPA) granted exemption s fr om the require ment 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 complete d a Toxicological Review and Risk Assessment, and a Technical Report was published in 200 5 (ANZFA Application A533 , 200 5; h ttp://www.foodstandards.gov.au ). ANZFA concluded that Liberty Link cotton® was comparable to non

- genetically engineered (non

- GE)
cott on in terms of their safety and nutritional adequacy. In 2004, t he 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

- transferase4 (found in LiberyLink® cotton) proteins now with a newly developed cotton line (event 304

- 40) that express es both Cry1Ab protein and phosphinothricin

- acetyl

- transferase (found in LiberyLink® cotton) proteins

. TwinLink TM 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 cott on 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 requirem ent of a tolerance for residues of the plant

incorporated protectant (PIP), Bacillus thuringi ensis Cry2Ae and Cry1Ab insect control protein s 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 modification s**  TwinLink TM cotton contains the stably integrated genes cry 1Ab, cry 2Ae and bar , which encode respectively the Cry1Ab, Cry2Ae and PAT proteins. “ T he genes were introduced by Agrobacterium

- mediated gene transfer. Southern blot analyses show TwinLink cotton contains one complete copy of the cry 1Ab and cry 2Ae genes, and 2 copies of the bar gene ” (pg 2 , BCS petition)

. The two parent lines that were c onventionally crossed to produce TwinLink TM 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 ) P35S 2 sequence including the promoter region from Cauliflower Mosaic Virus 35S transcript in clockwise dir ection ( Odell, Nagy et al. 1985 ) 5'cab22L sequence including the le ader 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

c arboxylase small subunit gene ats1A of Arabidopsis thaliana in clockwis e direction ( De Almeida, Gosselé et al. 1989 ) C ry2Ae the coding sequence of an insecticidal protein gene of Bacillus thuringiensis

, adapted to cotton codon usage in clockwise direction5 3'35S sequence including the 3' untranslated region of the 35S transcr ipt of Cauliflower Mosaic Virus in clockwise direction ( Sanfa çon, 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 direct ion ( 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 Strepto myces hygroscopicus in clockwise direction ( De Block, Botterman et al. 1987 ) 3 ́n os sequence including the 3’ untranslated region of the nopaline synthase gene from the T

- DNA of pTiT37 in clockwise direction ( Depicker, S tachel 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 TwinLink TM 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 th e information needed for consideration in a petition for nonregulated status. APHIS evaluate d 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 TwinLink TM 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 TwinLink TM cotton

. Based on information on the biology of cotton ( OECD 2008 ) da ta 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 TwinLink TM 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 o f 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 cotto n 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 ( cry 1Ab

, cry 2Ae 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 cry 1Ab

, cry 2Ae and bar gene s will result in increased likelihood of introduction or dissemination of a plant pest. Cr y 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 product s,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 effect s 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 revi ewed 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 transfer 1



### 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 I slands 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 suitab le insect pollinators are present, and if there is a short distance from host plants to recipient plants ( Fryxell 1979 ) . Physical barriers, intermediate pollinator

attractive plants, and other temporal or biological impediments (geography or absence of pollinators) reduce the potential for pollen movement ( Fryxell 1979 ) . Tabl e 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 N ative 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 proc ess 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 p ollinated 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. barbad ense

.

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 c ross

- 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. hirs utum 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

- A PHIS 2010 )

. In farm scale studies using traditional Upland cotton in California, it was found that the out

crossing 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 practi ces to prevent out

crossing (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 o ther 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 sta ndards 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 **μ** m). 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 Transfer 3
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 transg enic 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

w ith 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 milli ons 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 c onsiderations, 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 t he 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 interven tion. 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 ( O ECD 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 f ield 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 (f ield test reports can be found in the BCS TwinLinkTM cotton petition in Appendix 1

, p.102 ). The dat a

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, i ncluding**  **beneficial organisms.** The Cry1Ab and Cry2Ae protein s 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 bir ds, 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

- end otoxins from Bacillus thuringiensis . A total of 314 different cry genes have been identified by 2005 ( OECD 2007 )

. The insecticidal activity of Cry1Ab and C ry2Ae 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 o rganism that produces Cry p rotein s ) have been used to control insect pests on a commercial scale and for home garden applications ( Glare and O’Callagha n 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 observable11 adverse e ffects 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 protein s from TwinLink TM cotton planted in the field. The nontarget above

- ground arthropods and soil

dwell ing 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 pl exippus (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 mo narch 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 h armful concentrations of Cry1Ab

. TwinLink TM cotton , therefore, poses low risk to monarchs becaus e of minimal hazard of Cry1Ab and Cry2ac and low exposure to Cry protein

- containing pollen. TwinLink TM cotton is expected to have no harmful effects on any endangered or threatened species in the U.S.



### 5. **P otential 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 t ransfer between unrelated organisms is one of the most intens ively studied fields in the bio sciences 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) P otential 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 b acteria (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 geneti cally 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 TwinLinkTM12 cotton is unlikely to occur. First, many genomes (or parts thereof) have bee n 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 contai n 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 evaluat ed 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 e nvironment, 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 th e 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
sequence s contained within TwinLinkTM cotton enco de 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 Twi nLinkTM 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 n on

- targets or beneficial organisms in the agro

ecosystem, and the lack of horizontal gene transfer, APHIS concludes that BCS TwinLinkTM cotton is unlikely to pose a plant pest risk.



### E. Refer e nces

Boevink, P., P. W. G. Chu, et al. (1995). "S equence of subterranean clover stunt virus DNA: Affinities with the geminiviruses." Virology Brar, S. K., M. Verma, et al. (2007). "Bacillus thuringiensis proteases: Production and role in growth, sporulation and synergism." 207 : 354

- 361.

Process Biochemis try Brown, J. R. (2003). "Ancient horizontal gene transfer." 42 (5): 773

- 790. Genetics Crockett, L. (1977). Wildly successful plants: North American weeds. Honolulu, Hawaii, University of Hawaii. 4 : 121

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