# Monsanto Petition (12-185-01p) for Determination of Nonregulated Status of Dicamba and Glufosinate

Resistant MON 88701 Cotton

OECD Unique Identifier:

MON-887Ø1-3



## Plant Pest Risk Assessment

April 2014

Agency Contact Cindy Eck Biotechnology Regulatory Services

4700 River Road USDA, APHIS Riverdale, MD 20737 Fax: (301) 734-8669

The U.S. Department of Agriculture (U SDA) prohibits discrimination in all its programs and activities on the basis of race, color, national origin, sex, religion, age, disability, political beliefs, sexual or ientation, or marital or family statu s. (Not all prohibited bases apply to all programs.) Persons with disabilitie s who require alternative means for communication of program information (Braille, large print, audiotape, etc.) should contact USDA’S TARGET Center at (202) 720–2600 (voice and TDD).

To file a complaint of discrimination, write USDA, Direct or, Office of Civil Rights, Ro om 326–W, Whitten Building, 1400 Independence Avenue, SW, Washington, DC 20250–9410 or call (202) 720–5964 (voice and TDD). USDA is an equal opportunity provider and employer.

Mention of companies or commercial products in this re port does not imply recommendation or endorsement by the U.S. Department of Agriculture over others not mentioned. USDA neither guarantees nor warrants the standard of any product mentioned. Product names are mentioned solely to re port factually on available data and to provide specific

information.

This publication reports research involving pesticides. All uses of pesticides must be regi stered by appropriate State and/or Federal agencies before they can be recommended.

### TABLE OF CONTENTS PAGE

i A. Introduction ................................................................................................................. 1

B. Development of MON 88701 Cotton ......................................................................... 3

C. Expression of the Gene Product and Changes to Plant Metabolism ........................... 6

D. Potential Impacts on Disease and Pest Susceptibilities ............................................ 10

E. Potential Impacts on Nontarget Organi sms Beneficial to Agriculture ..................... 14

F. Potential for Enhanced Weediness of MON 88701 .................................................. 16

G. Potential Impacts on the Weediness of Any Other Plants with which It Can

Interbreed ................................................................................................................. 18

H. Potential Changes to Agriculture or Cultivation Practices ....................................... 22

I. Potential Impacts from Transfer of Genetic Information to Organism with

which MON 87701 Cannot Interbreed .................................................................... 24

J. Conclusion ................................................................................................................ 26

K. References ................................................................................................................. 261 

### A. Introduction

Monsanto Company (referred hereafter to as Monsanto) has petitioned the Animal and Plant Health Inspection Servi ce (APHIS) of the United Stat es Department Agriculture (USDA) for a determination that the ge netically engineered (GE) dicamba and glusosinate herbicide-resistant 1 cotton event MON 88701 (hereafter referred to as MON 88701) is unlikely to pose a plant pest risk a nd, therefore, should no longer be a regulated

article under the APHIS’ 7 Code of Federa l Regulations (CFR) part 340. This petition was assigned the number 12-185-101p, and is he reafter referenced as Monsanto 2012. APHIS administers 7 CFR part 340 under the authority of the plant pest provisions of the Plant Protection Act (PPA) of 2000 (7 U.S.C. 7701 et seq. ) 2 . This plant pest risk assessment was conducted to determine if MON 88701 is unlikely to pose a plant pest

risk.

APHIS regulations in 7 CFR part 340 regulate the introduction (importation, interstate movement, or release into the environment) of certain GE organisms and products. A GE organism is no longer subject to the plant pest provisions of the PPA or to the regulatory requirements of 7 CFR part 340 when APHIS determines that it is unlikely to pose a plant pest risk. A GE organism is considered a regulated article under 7 CFR part 340 if the donor organism, recipient organism, or vector, or vector agent used in engineering the organism belongs any genera or taxa designated in 7 CFR § 340.2 and is also considered a plant pest. A GE organism is also regulated under 7 CFR part 340 when APHIS has reason to believe that the GE organism may be a plant pest or APHIS does not have sufficient information to determine if the GE organism is unlikely to pose a plant pest risk. MON 88701 was produced by Agrobacterium -mediated transformation and includes introduced genetic sequences derived from plant pest organi sms listed in 7 CFR § 340.2 (Monsanto 2012).

Monsanto has conducted introductions of MON 88701 as a regulated article under

APHIS-authorized notifications since 2007 (Table A-1, pages 264-265 in Monsanto 2012), in part, to gather information to support th at it is unlikely to pos e a plant pest risk.

Potential impacts in this Plant Pest Risk A ssessment are those that pertain to plant pest risk associated with MON 88701 and its pr ogeny and their use in the absence of

confinement. APHIS utilizes data and inform ation submitted by the applicant, in addition to current literature, to determine if MON 88701 is unlikely to pose a plant pest risk. APHIS regulations in 7 CFR § 340.6(c) specify the information needed for consideration

1 Monsanto has described the phenotype of MON 88701 as “herbicide to lerant” and historically APHIS has also referred to GE plants with reduced herbicide sensitivity as herb icide tolerant. However, the phenotype w ould fall under the Weed Science Society of

America’s definition of “herbicide resistance” since MON 88701 has an “inherited ability to survive and reproduce following exposure to a dose of herbicide normally leth al to the wild type” (WSSA, 1998). By the WSSA definition, “resistance [to an herbicide] may be naturally occurring or induced by such techni ques as genetic engineering or se lection of variants produced by tissue culture or mutagenesis.” Herbicide tolera nce, by the WSSA definition, only applies to plant species with an “inherent ability to survive and reproduce after herbicide treatment. This implies that there was no selection or gene tic manipulation to make the plant tolerant; it is naturally tolerant."

2 The Plant Protection Act in 7 U.S.C. 7702 § 403(14) defines plant pe st as: “Plant Pest - 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 p roduct: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A fungus. (F) A vi rus or viroid. (G) An infecti ous agent or other pathogen. (H) Any article si milar to or allied with an y of the articles specified in the preceding subparagraphs .”2 in a petition for nonregulated status. APHI S will evaluate information submitted by the applicant related to plant pest risk characte ristics, expression of the gene product, new enzymes, or changes to plant metabolism, w eediness of the regulated article, impact on the weediness of any other plan t with which it can interbreed, changes to agricultural or cultivation practices that may impact diseases and pests of plants, effects of the regulated article on nontarget organisms, indirect plant pest effects on other agricultural products, and transfer of genetic info rmation to organisms with which it cannot interbreed.

APHIS may also consider information releva nt to reviews conduc ted by other agencies that are part of the ‘Coordinated Framewor k for the Regulation of Biotechnology’(51 FR 23302, June 26, 1986). Under the Coordinated Framework, the oversight of biotechnology-derived plants rests with th e APHIS, the Food and Drug Administration (FDA), and the Office of Pesticide Programs of the U.S. Environmental Protection Agency (EPA). Depending on its characteri stics, certain biotec hnology-derived products are subjected to review by one or more of these agencies.

The EPA under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), regulates the distribution, sale, use and testing of pesticidal substances produced in plants and microbes, including those pesticides that are produced by an organism through techniques of modern biotechnology. The EPA also sets tolerance limits for residues of pesticides on and in food and animal feed, or establishes an exemption from the requirement for a tolerance, under the Federal Food, Drug a nd Cosmetic Act (FFDCA). Dicamba is currently labeled for use on cotton only as a preplant application, due to its ability to injure cotton (BASF 2010; U.S. EPA 2009). According to Monsanto, it is currently used on approximately 8% of cotton acreage, and is a lead ing herbicide for use against glyphosate-resistant marestail (hor seweed) in the Midsouth region (McClelland et
al 2006). Monsanto will submit an applicati on to amend EPA Registration Number 524- 582 to allow preemergence and in-crop poste mergence application of dicamba on MON 88701, an increase in the dicamba residue tolerance for cottonseed from 0.2 ppm to 3 ppm, the establishment of a tolerance of 70 ppm for cotton gin by-products, and the inclusion of the herbicidally inactive dicam ba metabolite 3,5-dichlorosalicylic acid (DCSA) in the residue definitions for both cottonseed and gin by-products (page 36 in Monsanto 2012). Glufosinate is currently labeled for use on glufosinate-tolerant cotton from emergence through the early bloom gr owth stage (Bayer CropScience 2011; U.S. EPA 2003; page 37 in Monsanto 2012). It is currently undergoing reregistration at EPA with the Reregistration Eligibility Deci sion expected by the end of 2013 (U.S. EPA 2008). According to Monsanto, it is curre ntly used on approximately 8% of cotton acreage (Table VIII-9, page 193, in Monsan to 2012). The use pa ttern and rate of glufosinate on MON 88701 will follow the ex iting glufosinate-tolerant cotton uses outlined on the glufosinate herbicide labe l, and glufosinate residues in MON 88701 treated with glufosinate are below the EPA- established residue to lerances (page 37 in Monsanto 2012). Therefore, Monsanto will not seek changes in existing glufosinate

labels or the established tolerances fo r its use on MON 88701 (page 37 in Monsanto 2012).3 The FDA under the FFDCA is responsible for ensuring the safety a nd proper labeling of all plant-derived foods and feeds, in cluding those developed through modern biotechnology. To help sponsor s of foods and feeds derived from genetically engineered crops comply with their obligations, the F DA encourages them to participate in its voluntary consultation process. Monsanto submitted a food and feed safety and compositional assessment to FDA (BNF No. 135, April 2012) (page 36 in Monsanto 2012).



### B. Development of MON 88701 Cotton

Cotton belongs to the genus Gossypium , which consists of approximately 50 species, four of which are generally cultivated in tropica l and subtropical regions around the world (Fryxell 1984; OECD 2008; Percival et al 1999) The most commonly cultivated species in the United States, G. hirsutum (Upland cotton), comprises 98% of the cotton crop (USDA-NASS 2012a) and is the subject of this risk assessment. Over the last decade, it has been grown on an average of 12.2 million acres annually in 17 states from Virginia

southward and westward to California - an area often referred to as the Cotton Belt (Figure 1). Smaller amounts of G. barbadense (Pima or Egyptian cotton) are cultivated in Arizona, California, New Mexico, and Texas ( USDA-NASS 2012a; Pleasants and Wendel 2005). Cotton is a perennia l plant cultivated as an annual, and is more limited geographically than other majo r crops in the United States because it can be grown only in regions with more than 180 frost-free da ys per year. Two other cultivated species, G.

arboretum and G. herbaceum , are not grown in the U.S. or its territories.

Weed control in cotton is essential to ma ximize both cotton fiber yield and quality. Historically, mechanical tillage and hand hoe ing were the primary means of controlling weeds in cotton. Herbicide use began to develop in the 1940s and 1950s, and rapidly

accelerated in the 1960s as a series of more se lective herbicides were introduced into the market. By the mid-1980s, over 30 herbicides and herbicide combinations were being used in cotton (Buchanan 1992; pages 187-188 in Monsanto 2012). With the commercial introduction of glyphosate resistant cotton in 1997, glyphosate used increased while the

use of other herbicides decr eased (Webster and Nichols, 2012). According to Monsanto, glyphosate is now used on approximately 90 % of cotton acres grown (Table VIII-9, pages 192-193 in Monsanto 2012). Although gluf osinate resistant cotton was introduced in 2003, as of 2010 it was planted on only 3% of cotton acres (USDA-ERS-FAS 2010; page 189 in Monsanto 2012).

Repeated and intensive use of herbicides with the same mechanisms of action can rapidly select for tolerant, difficult-to-control weeds and for herbicide-resist ant weeds, especially

in the absence of the concurre nt use of herbicides with different mechanisms of action

and/or use of different mechanical or cu ltural practices for weed control (Vencill et al 2012). Fourteen confirmed glyphosate-resistan t weeds are now known in the U.S. (Heap 2012), including Palmer amaranth and wate rhemp, two of the most troublesome weed species in cotton (Webster and Nichols 2012).4 Figure 1. Cotton production areas in the U.S. (USDA-NASS 2012b)

MON 88701 is an Upland cotton variety that has been genetically engineered to be

resistant to the herbicides dicamba a nd glufosinate. Dicamba (3,6-dichloro-2- methoxybenzoic acid) is a selec tive pre- and post-emergent he rbicide used to control a wide spectrum of broadleaf weeds and w oody plants, while glufosinate is a broadspectrum pre- and post-emergent contact herb icide that provides nonselective control of a wide range of broadleaf and grass weeds (BASF 2010; Bayer CropScience 2011; page 34 in Monsanto 2012). According to Monsan to, MON 88701 was developed to provide a wider window of application for dicamba be yond current pre-plant cotton uses and to enable improved control of a broad spectrum of weed species in cotton crops, including glyphosate resistant weeds and weeds that ar e difficult to control using glyphosate or other herbicides, in a manner that will impede and delay the emergence of further herbicide resistance in weeds (pages 4- 5, 34-35, and 206-207 in Monsanto 2012). MON 88701 will allow in-crop applications of di camba from preemergence to seven days preharvest. It will also allow in-crop applica tions of glufosinate from emergence through the early bloom growth stage, as is done w ith currently marketed glufosinate resistant

cotton. Monsanto will use traditional br eeding methods to combine MON 88701 with deregulated, glyphosate-resistant cotton events to allow the us e of herbicides with three distinct modes of action in an integrated weed management program (pages 35, 209, and 212 in Monsanto 2012).5 MON 88701 was generated via transforma tion of hypocotyl segments from the conventional variety Coker 130 (Bowman et al 2006) using a disarmed strain of Agrobacterium tumefaciens , to express the dicamba monooxygenase (DMO) and phosphinothricin acetyltransferase (PAT) proteins. Following transformation, the hypocotyl segments were grown on medium containing glufosinate to eliminate

untransformed cells and antibiotics to eliminat e the bacterial vector, and were regenerated into whole plants (pages 40-41 in Monsanto 2012). The DMO protein, encoded by the

dmo gene from Stenotrophomonas maltophilia , confers resistance to dicamba-containing herbicides by demethylating dicamba to th e herbicidally inactive metabolites DCSA and formaldehyde (pages 77-78 in Monsanto 2012). The PAT protein, encoded by the bar (bialaphos resistance) gene of Steptomyces hygroscopicus , confers resistance to glufosinate-containing herbicides by acetylat ing the free amino group of glufosinate to produce non-herbicidal N-acetyl glufosinate (pages 81-82 in Monsanto 2012).

Description of the ge netic modifications

As described in the petition (pages 40- 48 in Monsanto 2012), MON 88701 was produced by Agrobacterium -mediated transformation of Coker 130 with the binary plasmid vector PV-GHHT6997. The plasmid generates a T-DNA that contains two gene expression cassettes flanked by left and right border regions and non-co ding sequences used in DNA cloning, and separated by a short (12 base pair) intervening sequence.

The dmo gene expression cassette for producti on of the DMO protein contains the following genetic elements:

 Promoter: PC1SV from the full-length tr anscript of peanut chlorotic streak caulimovirus (Maiti a nd Shepherd 1998)

 Intervening sequence: Short segment (6 base pairs), used in DNA cloning

 Leader: 5’ untranslated region (UTR) leader sequence from the RNA of tobacco etch virus (Niepel and Gallie 1999)

 Intervening sequence: One base pair, used in DNA cloning

 Targeting sequence: coding sequence for the chloroplast transit peptide CTP2

derived from the CTP2 target sequence of the shkG gene from Arabidopsis

thaliana (Herrmann 1995; Klee et al 1987)

 Gene: dmo codon-optimized coding sequence for the DMO protein of S. maltophilia (Herman et al 2005; Wang et al 1997), with an insertion of a single codon (encoding the amino acid leucine at position 2 of the protein) (page 79 in Monsanto 2012)

 Intervening sequence: short segment (54 base pairs), used in DNA cloning

 Terminator: 3’ UTR sequence from the E6 gene of G. barbadense (John 1996)

The bar expression cassette for production of the PAT protein contains the following genetic elements:

 Promoter: e35S from the 35S RNA of cauliflower mosaic virus containing a duplicated enhancer region (Kay et al 1987; Odell et al 1985)

 Intervening sequence: short segment (3 base pairs), used in DNA cloning6  Leader: 5’ UTR of the DnaK gene from Petunia hybrid a (Rensing and Maier 1997; Winter et al 1988)

 Intervening sequence: short segment (6 base pairs), used in DNA cloning

 Gene: bar coding sequence for the PAT protein of S. hygroscopicus (Thompson et
al 1987)

 Intervening sequence: short segment (19 base pairs), used in DNA cloning

 Terminator: 3’ UTR sequence of the nos gene from A. tumefaciens pTi (Bevan et
al 1983; Fraley et al 1983)

Monsanto provided evidence demonstrating that,

 the DNA inserted into the MON 88701 genom e is present at a single locus and contains one functional copy each of the dmo and bar genes (Figure IV-1, page 53, Table IV-1, page 54, & Figures IV-2 – IV-4, pages 60-62 in Monsanto 2012);

 the right border sequence and a portion of the right side non-coding sequence, as well as a portion of the left border se quence, are deleted in MON 88701 while all other genetic elements in the insert ar e intact as in the plasmid PV-GHHT6997 (page 51 and Figure IV-6, page 66 in Monsanto 2012);

 the final product does not contain any of the backbone sequences from PVGHHT6997 (Figure IV-5, page 64 in Monsanto 2012);

 the inserted DNA was stably inherited acro ss five generations (Figures IV-8 and IV-9, pages 70-71 in Monsanto 2012); plants that cont ain the inserted DNA also express the glufosinate herbicide resi stance phenotype (as determined through glufosinate herbicide applic ation), and the inserted DNA and resistance trait both segregate according to Mende l’s laws of segregation co nsistent with the finding of a single insertion locus (Figure IV- 10 and Tables IV-3 and IV-4, pages 74-75, in Monsanto 2012).

In addition, Monsanto stated that there was a small, 123 ba se pair deletion from the conventional genomic DNA at the T-DNA inse rtion site (page 67 in Monsanto 2012).

Minor deletions and/or inserti ons of DNA are not uncommon during Agrobacterium

mediated transformation (Salomon and Puchta 1998).



### C. Expression of the Gene Product and Changes to Plant Metabolism

USDA-APHIS assessed whether changes inplant metabolism or composition in MON 88701 is likely to alter its plant pest risk. The assessment encompasse s a consideration of the expressed proteins and their effect on pl ant metabolism and an evaluation of whether

the nutrients and anti-nutri ent levels in harvested seed derived from MON 88701 are comparable to those in the conventional co tton control Coker 130 or to other reference cotton cultivars considered for the composition analysis. Forage is not considered, as harvested vegetative cotton biomass is neve r used as forage. Host plant quality (including such components as carbon, nitrog en, amino acid sources, trace elements, and defensive metabolites) is known to affect herbivore performance and fecundity; and higher-trophic level interactions, such as th e performance of predat ors and parasitoids, may also be affected (reviewed by Awmack a nd Leather 2002). Similarly a wide array of secondary metabolites in plants is known to provide defense against microbes (Dixon7 2001). Thus changes in host plant quality may ha ve the potential to a ffect the ability of MON 88701 to withstand attack by pests a nd diseases or have impacts non-target organisms including beneficial organisms.

The DMO protein expressed in MON 88701 is a variant of the DMO protein encoded by

the dmo gene of S. maltophilia . The dmo gene used was isolated from a stormwater retention pond outside a dicamba manufacturing plant (Krueger et al 1989). S. maltophilia is a gram negative bacterium that is ubiquitous in the environment and found associated with the rhizosphere of plan ts (Echemendia 2010; Pa lleroni and Bradbury 1993). It has been used as a biocontrol agen t for fungal plant pathoge ns and is not known to cause disease in plants (Berg et al 1999). The DMO protein expressed in MON 88701 is 349 amino acids in length and has an appa rent molecular weight of 39.5 kDa (Figure C-2, page 285, and Table C-4, page 291 in Monsanto 2012). It differs from the S. maltophilia DMO protein in that it includes an ad ditional leucine at position two and an

additional nine amino acids from CTP2 at its N-terminus that result from alternative processing of the transfer peptide (pages 79 and 275-276 in Monsan to 2012). Alternative processing of the CTP sequence has b een previously observed (Behrens et al 2007; Clark and Lamppa 1992), and these differences shoul d not have an effect on the structure, activity, or specificity of the MON 88701 DM O protein because the N-terminus and residue two are sterically distant from th e catalytic site of the enzyme (D’Ordine et al 2009; Dumitru et al 2009). Functional assays demonstrated that the MON 88701 DMO protein was active and protected MON 88701 from damage following dicamba application (Tables C-5 and C-7, pages 297 and 299, in Monsanto 2012). The MON 88701 DMO protein was found to be expressed throughout the life-cycle and tissues of the plant, including roots and s eed, with the highest level of expression in leaves (Table V-2, page 85 in Monsanto 2012). This is expected since expression of the dmo gene in MON 88701 is driven by the constitutive PC1SV promoter from peanut chlorotic streak caulimovirus (Maiti a nd Shepherd 1998).

DMO is an enzyme that confers herbicide resistance by catalyzing the demethylation of dicamba to the non-herbicidal compounds DCSA and formaldehyde (pages 77-78 in Monsanto 2012; Chakraborty et al 2005). In S. maltophilia

, DMO is the terminal component of a three component system comp rised of a reductase, a ferredoxin, and an oxygenase (DMO). These enzymes work t ogether to transport electrons from nictoninamide adenine dinucleotide (NADH) to oxygen and catalyze the demethylation of an electron acceptor substrate. In M ON 88701, the DMO protein is targeted to chloroplasts via the CTP2 sequence for co -localization with th e endogenous reductase and ferredoxin enzymes that supply electr ons for the DMO demethylation reaction (Behrens et al 2007). The DMO protein expressed in MON 88701 is highly specific for

dicamba. In vitro assays using plant produced and E. coli produced MON 88701 DMO demonstrated that five endogenous metabolites and the herbicide 2,4-D, all of which are structurally similar to dicamba, were not metabolized by the enzyme (pages 79-80 and 301-306 in Monsanto 2012). In addition, in vivo assays demonstrated that, of 10 different herbicides applied, MON 88701 and control Coke r 130 cotton exhibited similar levels of injury for all but dicamba, indicating that th e other nine herbicides do not serve as a substrate for MON 88701 DMO (pages 80-81 and 297-299 in Monsanto 2012).8 Therefore, because of the apparent high subs trate specificity, the DM O protein is unlikely to catalyze the conversion of other endogenous substrates and thereby affecting the metabolic system of MON 88701.

DCSA is a known metabolite of dicamba whose safety in soybean, soil, and livestock has been evaluated by the EPA (U.S. EPA 2009; 40 CFR part 180.227 [7-1-11 Edition]) and

whose structure is similar to salicylic acid (2- hydroxybenzoic acid), an endogenous plant benzoic acid (Frear 1976; p. 273 in Monsanto 2012). Because endogenous salicylic acid compounds are known to be involved in plant re sponses to stress, in cluding to pests and pathogens (Bi et al 1997; Colson-Hanks et al 2000; Inbar et al 2001; Martinez et al 2000;

Thaler et al 2010; Thaler et al 2012; Vlot et al 2009; Zarate et al 2007; Zhang et al 2011), the possible effect of DCSA produc tion on host defense in MON 88701 will be

further discussed in the later section on Potential Impacts of Genetic Modifications on Pest and Disease Susceptibilities . Formaldehyde has also been associated with plant stress responses, although its role if a ny in such responses is unclear (Sardi et al 1996; Szende and Tyihak 2010). In MON 88701, dicamba -derived formaldehyde is expected to be produced in small amounts; the maximum theo retical amount is estimated to be 6.3 – 33 mg/kg based on an assumption that the en tire amount of dicamba applied to MON 88701 is intercepted and instantaneously metabolized by DMO (page 274 in Monsanto 2012). This is well within the range of formalde hyde measured in a variety of dicot plants (up to several hundred mg/kg) (Adrian-Romero et al 1999) and agricultural commodities (WHO-IPCS 1989). Moreover, any additional fo rmaldehyde that would be produced in MON 88701 by dicamba treatment would be quick ly metabolized and incorporated into the 1-carbon pool of the pl ant through known pathways (Hanson and Roje 2001; Kalasz 2003). Thus, the incremental increase in form aldehyde over and above the levels already presumed to be present in the cotton plan t would likely be sma ll and transient.

The PAT protein expressed in MON 88701 is encoded by the bar gene of S. hygroscopicus (Thompson et al 1987), a saprophytic bacterium that is widespread in the environment and is not pathogenic to plants (Kämpfer 2006; Kutzne r 1981; Locci 1989). A homologous protei n is encoded by the bar gene of S. viridochromogenes (Wohlleben et
al 1988). The PAT protein expressed in MON 88701 is 183 amino acids in length and has an apparent molecular weight of 24.1 kD a (Figure C-10, page 315, and Table C-13, page 321, in Monsanto 2012). It differs from the S. hygroscopicus DMO protein by the deletion of one or two N-terminal amino acids (Table C-10, page 311, in Monsanto 2012). These differences should not affect the structure, activ ity, or specificity of the MON 88701 PAT protein because these amino acids are sterically distant from the catalytic site of the enzyme, based on the structure of a related acetyltransferase from Pseudomonas aeruginosa (Davies et al 2007). Functional assays demonstrated that the MON 88701 PAT protein was active and pr otected MON 88701 from damage following glufosinate application (Table C-14, page 327, and Tables IV-3 and IV-4, page 75, in Monsanto 2012). The MON 88701 PAT protein was found to be expressed throughout the life-cycle and tissues of the plant, includ ing roots and seed, with the highest level of expression in seed (Table V-3, page 87, in Monsanto 2012). This is expected since expression of the bar gene in MON 88701 is driven by the constitutive e35S promoter from cauliflower mosaic virus (Kay et al 1987; Odell et al 1985).9 PAT is an enzyme that confers herbicide resistance by catalyzing the acetylation of Lphosphinothricin, the active component in gl ufosinate, to the non-herbicidal compound N-acetyl L-phosphinothricin (Thompson et al 1987; Wehrmann et al 1996; pages 81-82 in Monsanto 2012). PAT proteins are highl y specific for L-phosphinothricin. Other Lamino acids, including the L-phosphinothricin analogue L-glutamate, are unable to be acetylated by PAT and do not inhibit acetyl ation of L-phosphinothricin in competition assays (Wehrmann et al 1996). Therefore, the PAT protein is unlikely to affect the metabolic system of MON 88701. Numerous gl ufosinate resistant crops containing the PAT protein encoded by the bar gene have undergone regulat ory review and approval in

the United States and several other countries (see list in ILSI-CERA 2011; OECD 1999, 2002a). The safety of the protein has been establ ished in the scientific literature (Herouet et al, 2005).

Monsanto carried out a compositional asse ssment comparing MON 88701 seed to seed

from conventional control variet ies using the principles outlined in the OECD consensus document on compositional considerations for cotton (OECD 2009). The samples for compositional assessment were collected fr om eight locations in 2010, chosen to represent typical cotton grow ing regions of the United States (page 99 in Monsanto 2012). Analytes were assessed quantitatively and included 47 nutrients (proximates, fiber, amino acids, fatty acids, minerals, and vitamin E) and five anti-nutrients (fatty acids, gossypol). Analyte levels in un treated MON 88701 and MON 88701 treated with both glufosinate (0.5 lbs acid eq uivalent/acre at the 3-5 leaf stage) and dicamba (0.5 lbs acid equivalent/acre at the 6 – 10 leaf stage) were compared to corresponding levels in the parental Coker 130 control ( untreated) and to statistical tolerance intervals generated from nine non-GE commercial varieties grown concurrently at the same field sites (four commercial varieties per site ) (page 99 and Table E-1, page 337, in Monsanto 2012). The latter group was included in the analysis to establish a spectrum of normal variation for the measured analytes in cotton. Statistical an alysis to detect significant differences in analyte levels was performed using data colle cted from individual field sites as well as

the combined set of data collected across all field sites.

Statistically significant (p <0.05) differences in the combined-site nutrient levels were

observed for 16 nutrients in both herbicid e treated and untreated MON 88701 compared to the Coker 130 control: the proximates ash, calories, carbohydrates , moisture, and total fat; acid detergent fiber, neutral detergent fiber, total dietary fiber; the amino acid arginine; the fatty acid 14:0 myristic acid; the minerals calcium, magnesium, manganese, potassium, and zinc; and vitamin E. Three additional nutrients were significantly different in herbicide treated MON 88701 only (t he amino acids methionine and proline, and the fatty acid 18:2 linoleic acid), while one additional nutrient was significantly different in untreated MON 88701 only (cru de fiber) (Table VI-1, pages 107-122, and Table E-19, pages 426-439, in Monsanto 2012). No statistically significant differences were observed for 27 nutrients (Table V I-2, pages 123-131, and Table E-20, pages 440- 448, in Monsanto 2012). With the exception of calcium (increased 14% and 15% relative

to control in treated and unt reated MON 88701 respectively), all differences in nutrient

levels were 10% or less. In addition, most of the statistica lly significant differences in10 nutrient levels were not cons istently observed across locations, with the exception of calcium (statistically significant differences observed in seven of eight locations), ash (statistically significant differences observe d at six (treated) a nd four (untreated) locations), 18:0 stearic acid (statistically significant differenc es observed at five locations), and manganese (stati stically significant differences observed at five locations for untreated MON 88701 only). However, in al l of these cases, the mean levels of all

nutrients in MON 88701 were w ithin the 99% tolerance inte rval established from the

conventional commercial reference varieties. While the mean level of methionine in herbicide treated (but not untreated) MON 88701 were slightly outside of the 99% tolerance interval, the mean increase in me thionine was less than 5%. Moreover, the increase was not consistently observed acro ss locations (a statistically significant difference was observed at only one of the eight locations), and the mean level of

methionine was within the natural varia tion observed for commerc ial cotton varieties (ILSI, 2010).

Statistically significant (p < 0.05) increases in combined-sit e anti-nutrient levels were

observed for two anti-nutrients in both he rbicide treated and untreated MON 88701: the cyclopropenoid dihydrosterculic acid and to tal gossypol. In addition, a statistically significant increase in free gossypol was obs erved in treated MON 88701. The increase was less than 10% in all cases except di hydrosterculic acid in untreated MON 88701, which increased 12.6%. Moreover, the increases were not consistently observed across

locations and the anti-nutrient levels were all within the 99% tolerance interval

established from the conventional commerc ial reference varieties. There were no statistically significant differences in the cy cloproprenoids malvalic acid and sterculic acid (Tables VI-1, pages 107-122, VI-2, pa ges 123-131, E-19, pages 426-439, and E-20, pages 440-448, in Monsanto 2012).

The significant changes observed for the above -mentioned nutrients and anti-nutrients are unlikely to make MON 88701 more susceptible to pests and diseases, or to cause MON 88701 to have a greater impact on non-target organisms, than existing cotton varieties. As discussed further below, the disease, inse ct pest, arthropod abundance, and agronomic data presented for MON 88701 did not indi cate any significant difference for the

aforementioned observations.

Based on all the above noted data and cons iderations, APHIS concludes that MON 88701 poses no more of a plant pest risk from ne w gene products, changes to plant metabolism or composition than conven tional cotton varieties.



### D. Potential Impacts on Disease and Pest Susceptibilities

USDA-APHIS assessed whether MON 88701 is li kely to have significantly increased disease and pest susceptibility because of the introduced dmo and bar genes compared to the control cotton variety. This assessment encompasses a thorough consideration of the introduced traits, their impact on agronomic tr aits (discussed later in the document) and plant composition (discussed ear lier), and quantitative and/or observational data on pest and disease responses. Important changes in clude those which would (1) affect not only11 the new GE crop, but that would also result in significant introduction or spread of a damaging pest or disease to other plants; or (2) result in the introduction, spread, and/or creation of a new disease or (3) result in a sign ificant exacerbation of a pest or disease for which APHIS has a control program. Impor tant changes do not include changes in susceptibility to diseases a nd pests that are within the acceptable range of currently

cultivated varieties.

Plant Protection and Quarantine (PPQ) is an APHIS program that safeguards agriculture and natural resources from the entry, establishm ent, and spread of animal and plant pests

and noxious weeds into the United States of Am erica; and supports trade and exports of U.S. agricultural products. PPQ responds to many new introductions of plant pests to eradicate, suppress, or cont ain them through various programs in cooperation with state departments of agriculture a nd other government agencies. These may be emergency or longer term domestic programs that target a specific pest. A vari ety of insect, plant disease, mollusk, or weed programs exist (see http://www.aphis.usda.gov/plant_health/ plant_pest_info/index.shtml ). Among these, PPQ’s cotton pest programs specifically target boll weevil ( Anthonomus grandis) and pink bollworm ( Pectinophora gossypiella )

.

A number of other insects also feed on cotton. Between 2007 and 2011, the most important in terms of cotton yield loss were the bollworm/budworm (Heliothine) complex ( Helicoverpa zea and Heliothis virescens ), stink bugs (Pentatomidae), thrips (Thripidae), and Lygus species (Williams 2007, 2008, 2009, 2010, 2011). Of the various diseases affecting cotton, the most troublesome in recent years have been Phymatotrichum root rot ( P. omnivorum ), the boll rots, and the fungal seedling diseases (primarily Rhizoctonia solani, Pythium spp, and Fusarium spp ) (Blasingame 2010; National Cotton Council 2011).

Cotton is not itself a plant pest in the U. S. (USDA-NRCS 2012a; 7 CFR part 340.2). The A. tumefaciens transformed plants used in the generation of MON 88701 were treated

with antibiotics to kill A. tumefaciens cells (page 40 in Monsanto 2012). The description of the introduced genetic elements, expression of the gene products and their functions in

MON 88701 has been summarized above. That inserted genetic material which was derived from plant pests does not result in th e production of infectious agents or disease symptoms in plants.

Monsanto collected data relevant to cott on diseases and insect pests from field experiments conducted in two st udies at a total of 15 (Study 1) and 11 (Study 2) locations across the United States during the 2010 gr owing season (Table VII-1, pages 138-140, and VII-4, page 151, in Monsanto 2012). Th ese locations cover a diverse range of environmental conditions representative of most commercial cotton production areas and locations where MON 88701 is expected to be grown. All locations in Study 2 were also used in Study 1. The agronomic practices used to prepare and maintain each field site

were characteristic of eac h respective region (page 533 in Monsanto 2012). In both studies, plant mapping data, disease and pe st damage ratings, and arthropod abundance

data were collected for control Coke r 130 cotton and MON 88701. Data were also collected for 11 (Study 1) and eight (Study 2) commercial reference va rieties, with four12 varieties grown per site, to establish a range of natural variability for responses to the assessed stressors (Tables G-1 and G- 2, pages 534-537 in Monsanto 2012).

Plant mapping is a process commonly used by agronomists and breeders to quantify cotton growth and development parame ters such as boll retention (Kerby et al 2010).

Final boll retention and distri bution, as reflected in the pl ant mapping data, can provide

an indication of the effect of abiotic and bi otic on a cotton plant, as squares and early bolls tend to abort if the plant experiences stress (Guinn, 1982; Kerby et al 2010; University of California 1996). Within a st udy location and based on the proximity of plots within a location, it can be concluded that all plots would be subjected to similar stressors. Thus, if plant mapping results are similar between two cotton lines this generally indicates that the two lines had si milar responses to the overall set of abiotic and biotic stresses experienced. In Study 2, plant mapping data was collected for MON 88701 treated with the herbicides glufosinate (0.5 lbs a.i./acre at the 3 – 5 leaf stage) and dicamba (0.5 lbs acid equivalent (a.e.)/acre at the 6-10 leaf stage) to allow for assessment of MON 88701 under the agronomic system in whic h it is expected to be used (pages 99 and 149 in Monsanto 2012). This data is impor tant for this plant pest risk assessment since the DMO reaction product DCSA, which is formed in MON 88701 in the presence of dicamba, is an analog of salicylic aci d. Salicylic acid and it s endogenous derivatives are known to be involved in plant responses to stress, including to pests and pathogens, although their precise roles have not been fully elucidated (Bi et al 1997; Colson-Hanks

et al 2000; Inbar et al 2001; Martinez et al 2000; Thaler et al 2010; Thaler et al 2012;

Vlot et al 2009; Zarate et al 2007; Zhang et al 2011). It is plausi ble that DCSA could play a similar role as salicylic acid in plants or conversely, could in terfere with salicylic acid mediated responses (Park et al 2009; Silverman et al 2005).

The researchers at each field site were e xpected to be familiar with the growth, production, and evaluation of cotton characteristics, and to use well-established qualitative and/or quantitative techniques to observe and evaluate environmental interactions. For plant responses to abiotic stress, disease damage and arthropod-related damage, at least three abioti c stressors, three diseases a nd three arthropod pests were evaluated four times at all sites, starting about 30 days after plan ting and continuing at approximately 30 days intervals. The researcher at each field site chose abiotic stressors, diseases and arthropod pests that were activ ely causing injury. When fewer than three stressors were present, the researcher c hose additional stressors based on knowledge of those likely to occur in cotton during th e given observation period. Therefore, the stressors typically varied betw een observations at a site or among sites, as did the number of observations for each stressor (Table s G-21 and G-22, pages 580-581, in Monsanto 2012). Qualitative damage ratings were collect ed from each plot using a continuous 0 – 9 scale of increasing severity and then grouped in to four categories: non e (0), slight (1-3), moderate (4-6), and severe (7-9). These qualita tive categorical data were not statistically analyzed; they were considered different on a particular observation date at a site if the range of injury severity to MON 88701 did not overlap with the range of injury severity

to the control across all four replications at each site (p ages 540-541 in Monsanto 2012).13 In addition to these qualitative assessment s, in Study 1, quantitative damage assessments were performed at five sites for thrips (0 – 5 rating scale at 1 4, 21, and 28 days after planting) and heliothines (number of larvae and number of total and damaged fruiting bodies at 45, 60, 75, and 90 days after planti ng) (pages 540-541 in Monsanto 2012). At these same sites, arthropods were collected and quantified four times, starting about 30 days after planting and continuing at a pproximately 30 day intervals (page 542 in Monsanto, 2012). In both studies the total number of bolls per plant and the percent retention of first-pos ition bolls were among the plant mapping characteristics assessed (Table VII-1, page 140, in Monsanto 2012). Qu antitative numerical data for arthropod damage, arthropod abundance, and plant mappi ng characteristics und erwent statistical analysis (page 543 in Monsanto 2012).

Neither the introduced traits nor dicamba and glufosinate herbicide treatment altered the

assessed environmental interactions of MON 88701 compared to the conventional control. In Study 1, 498 out of 498 observa tions between untreated MON 88701 and the conventional control showed no meaningful differences in damage from 8 abiotic stressors, 14 diseases, and 14 types of ar thropod pests (Tables VII-8, page 160, and G-20 – G-22, pages 579-581, in Monsanto 2012). Similarly, in Study 2, 385 out of 385 observations showed no meaningful differences in damage from these stressors (Tables VII-11, page 163, and G-27 – G-29, pages 591-59 3, in Monsanto 2012). Combined site quantitative analyses showed no differe nce between MON 88701 and the conventional control in damage caused by thrips or heliot hine pests, or in the number of heliothine larvae in Study 1 (Tables VII-9 and VII-10, pages 160-161, in Monsanto 2012). The same results were seen at each individual site, except that there was less thrips and heliothine damage in MON 88701, and more live heliothine larvae, for a single time point

at one site for each of these characteris tics (Tables G-23 and G-25, pages 582-583, in Monsanto 2012). There were also no differe nces seen in Study 1 in pest abundance

between MON 88701 and the conventional cont rol for 87 out of 89 observations across five sites at four time points for 10 diffe rent pests (page 161 and Table G-25, pages 584- 587 in Monsanto 2012). Fewer stinkbugs and tarnished plant bugs were observed in MON 88701 at one site and one time point were observed in Study 1 for 87 out of 89

observations.

No differences were observed in comb ined-site analysis of 6 plant mapping characteristics, including the total number of bolls per plant and th e percent retention of first position bolls, between MON 88701 and conve ntional control. There was a small but statistically significant increase in the aver age number of first position bolls in MON 88701 relative to control. However, this valu e was within the refere nce range established using eight reference varieties and would not indicate an increase susceptibility of MON 88701 to stress (Table VII-7, page 157, in Monsanto 2012). The same results were seen

when MON 88701 was treated with dicamba a nd glufosinate (Table G-18, page 576, in Monsanto, 2012), indicating that herbicide treatment did not significantly affect the overall response of MON 88701 to the set of abiotic and biotic stresses experienced during the growing season.14 Finally, as discussed earlier, there we re no significant changes in MON 88701 compositions that would render MON 88701 more susceptible to pests and diseases over

its control or reference cotton varieties. As presented later in this document, the observed agronomic traits also did not reveal any signi ficant changes that w ould indirectly indicate that MON 88701 is or could be relatively mo re susceptible to pests and diseases over

control or reference cotton va rieties. Thus MON 88701 is exp ected to be susceptible to the same plant pathogens and insect pests as conventional cotton. The introduced genes did not significantly alter the observed insect pest infestation and disease occurrence or resulting damage on MON 88701 over the conventi onal control. For this reason, there is also unlikely to be any indirect plant pe st effects on other ag ricultural products.



### E. Potential Impacts on Nontarget Or ganisms Beneficial to Agriculture

MON 88701 is not engineered for pest resistan ce, thus there are no ‘target’ species, and thus no ‘nontarget’ species either. Howe ver, APHIS assessed whether exposure or consumption of herbicide-resistant MON 88701 containing the DMO and PAT proteins

would have an adverse effect on beneficial species or wildlif e associated with cotton.

Monsanto provided the following informa tion justifying the safety of MON 88701 (Sections V.D-V.F, pages 88-97, in Monsanto 2012):

 The donor organisms, S. maltophilia and S. hygroscopicus

, are not known for human or animal pathogenicity. S. maltophilia is an aerobic, ubiquitous, environmental, gram-negative bacterium which can be found in healthy individuals without causing harm to human health. Its incidental presence on foods and crops without any adverse repor ts establishes the safety of the donor organism (page 90 in Monsanto 2012). It ha s been previously reviewed as part of safety and nutritional assessments of ot her genetically engineered crop products (US-FDA 2011). S. hygroscopicus is a saprophytic, gram-positive, soil-borne bacterium with no known safety issues. Streptomyces species are widespread in the environment and present no known alle rgenic or toxicity issues (Kämpfer 2006; Kutzner 1981) though human exposur e is quite common (Goodfellow and Williams, 1983) (pages 92-93 in Monstanto 2012).

 The DMO enzyme present in MON 88701 has sequence similarity and many catalytic and domain structural similari ties with a wide variety of oxygenases found in numerous species of microorganism s widely distributed and prevalent in the environment (Chakraborty et al 2012). It also has similarity with oxygenases such as pheophorbide A oxygenase which are found in plants such as rice, maize, canola and pea (Rodoni et al 1997; Yang et al 2004) that are consumed in a variety of food and feed sources which ha ve a history of safe human consumption. Plants, animals and humans are extensivel y exposed to these types of enzymes (pages 90-91 in Monsanto, 2012).

 The PAT enzyme present in MON 88701 is identical to the wild-type protein produced in S. hygroscopicus and is analogous to the PAT proteins in commercially available glufosinate-tolera nt products in several crops including cotton, corn, soybean, and canola. OECD recognizes PAT proteins produced from15 different genes to be equivalent with regard to function and safety (OECD 1999). PAT proteins are structurally similar onl y to other acetyltransferases known to not

cause adverse effects af ter consumption (Herouet et al 2005). In 1997, a tolerance exemption was issued for PAT proteins by the U.S. EPA (U.S. EPA 1997).

 Bioinformatics analyses demonstrated that the DMO and PA T proteins in MON 88701 do not share amino acid sequence sim ilarities with known allergens, gliadins, glutenins, or prot eins toxins which could have adverse effects to human or animal health (pages 88 – 89 in Monsanto 2012).

 The DMO and PAT proteins in MON 88701 ar ea readily digestible in simulated gastric and intestinal fluids, making it highly unlikely that it would be absorbed in the small intestine and have any adverse e ffects on human or animal health (pages 94-95 in Monsanto 2012).

 Acute oral toxicity studies have indicat ed that the DMO and PAT proteins have

no adverse effects in mice at the highest dose tested (Herouet et al 2005; page 84 in Monsanto 2010). By extrapolation, ther e is no meaningful risk to human or animal health from dietary exposure to MON 88701.

As indicated earlier in this plant pest risk assessment, the petitioner’s characterization of

MON 88701 showed that it is similar in nut ritional and compositional analysis to the unmodified control variety Coker 130 (Sect ion C. Expression of the gene product, enzymes or changes to plant metabolism). Gossypol and the cycl opropenoid sterculic acid play a role in defense of cotton agains t insect pests (Chan et al 1978; Kong et al 2010; Rani and Rajasekharreddy 2012), suggest ing that increases in these compounds could have negative effects on non-target or ganisms. Although statistically significant increases in combined-site levels of gossypol and the related cycl opropenoid fatty acid dihydrosterculic acid were observed in bot h herbicide treated and untreated MON 88701, the increases were not consistently observed across locations and the levels of these compounds were all within the 99% tolerance interval establ ished from the conventional commercial reference varieties (page 105 and Tables VI-1, pages 109-110, and E-19, page 428, in Monsanto 2012).

As discussed in the previous section, M onsanto conducted field experiments in two

studies at locations across th e United States during the 2010 growing season. In one of these studies, arthropods were collected and quantified four times at five locations, starting about 30 days after planting and c ontinuing at approximately 30 day intervals (page 542 in Monsanto 2012). No differences were seen in the abundance of seven

different beneficial arthr opods between MON 88701 and the conventional control for 86 out of 89 observations (page 161 and Table G-26, pages 588 – 590, in Monsanto 2012). More damselbugs were seen at one location for one of four time points, while fewer

Orius species (which include predators of thrips ) were observed at one location for two of three time points. Although the mean abundance values for Orius species at this site were outside the reference range established by four commercial cotton vari eties also grown at the site, differences in Orius abundance were not consistent ly detected across sites.

Based on APHIS’s analyses of the data provid ed by Monsanto (as described here and in section C. Expression of the gene product, enzymes or changes to plant metabolism)16 MON 88701 is unlikely to cause any significant adverse e ffects on nontarget organisms (including beneficial species or wildlife associated with cotton) compared to other commercial cotton varieties. Any effects on non-target organisms that could potentially result from proposed changes in herbicide labels will be evaluated by the U.S. EPA. APHIS has concluded that adverse impacts to non-target organisms exposed to MON 88701 are unlikely.



### F. Potential for Enhanced Weediness of MON 88701

Upland cotton ( G. hirsutum ) possesses few of the characte ristics common to plants that are successful weeds (Baker 1965; Keeler 1989) and is not cons idered to be a serious or

common weed in the United States. It is not listed as a weed in the major weed references (Crocket 1977; Holm et al 1979; Muenscher 1980), nor is it present on Federal or State lists of noxious weed species (USDA- APHIS 2012; USDA-NRCS 2012b). Modern Upland cotton is a domesticated perennial grow n as an annual crop that is not generally persistent in unmanaged or undisturbed environments without human intervention.

Modern cultivars are not frost tolerant a nd do not survive freezi ng winter conditions, do not produce abundant or long-live d seeds that can persist or lie dormant in soil, do not exhibit vegetative propagation or rapid vege tative growth, and do not compete effectively with other cultivated plants (OECD 2008). In areas where winter temperatures are mild and freezing does not occur, cotton plants can occur as volunteers in the following growing season. However, these volunteers can be easily controlled by herbicides or mechanical means. Cotton can become locally feral or naturalized in suitable areas, such as southern Florida, Hawaii, and Puerto Rico (Coile and Garland 2003; Fyxell 1979; USDA-NRCS 2012a; Wunderlin and Hansen 2008).

Monsanto collected seed germination data (as an indicator of seed dormancy) for seed grown at three field locations and tested under controlled laboratory conditions at six different temperature regimes (10 °C, 20 °C, 30°C and three diurnal combinations of these temperatures). Germination data were collected for MON 88701 seed, control Coker 130 seed, and nine commercial refere nce varieties (pages 145-147 in Monsanto 2012). In a combined-site analysis, no statis tically significant diffe rences (p < 0.05) in seed germination rates, dead seed, or viab le but ungerminated seed were observed for five of the temperature regimes. At 30 °C, MON 88701 seed germinated at a slightly higher rate than Coker 130 seed (96.7% ve rsus 94.4%) and had a correspondingly lower amount of dead seed. However, these slight differences were well within the range of

rates observed for the commercial referen ce varieties. Moreover, at no temperature regime was viable hard seed observed. Theref ore, APHIS concludes that there were not biologically meaningful differences in the seed germination characteristics between MON 88701 and its parental control.

Monsanto also collected agronomic data releva nt to weedy traits su ch as plant vigor and height and seed yield from field experiments conducted in two studies at a total of 15 (Study 1) and 11 (Study 2) lo cations across the United Stat es during the 2010 growing season (Tables VII-1, pages 138-40, and V II-4, page 151, in Monsanto 2012). All locations in Study 2 were also included in Study 1. Data were collect ed for control Coker 130 cotton and MON 88701, as well as for MON 88701 treated with the herbicides17 glufosinate (0.5 lbs a.i./a cre at the 3 – 5 leaf stage) and dicamba (0.5 lbs a.e./acre at the 6- 10 leaf stage) to allow for assessment of MON 88701 under the agronomic system that it is expected to be used (pages 99 and 149 in Monsanto 2012). Data were also collected for 11 (Study 1) and eight (Study 2) commercial re ference varieties (four varieties grown per site) to establish statistical tolerance interv als for the various trai ts assessed (Tables G-1 and G-2, pages 534-537 in Monsanto 2012).

Plant vigor was assessed qualita tively. No differences in vigor were observed between

MON 88701 and the Coker 130 control at 14 a nd 30 days after planting for 73 out of 74 comparisons across all sites and treatments. At one site, MON 88701 plants were slightly less vigorous than Coker 130 at 30 days after planting, but were within the range of vigor ratings of the commercial reference varieties.

Other agronomic data were assessed quantit atively. In the combined-site analysis, no statistically significant (p < 0.05) differe nces were observed between MON 88701 and

the Coker 130 control for stand count at 14 and 30 days after plan ting, final stand count, seedcotton yield, number of immature seed pe r boll, boll weight, or a variety of fiber characteristics. In contrast, in both studies and whether untreated or herbicide treated, MON 88701 plants were shorte r than Coker 130 control plan ts, took slightly longer to mature (as indicated by an increased number of nodes a bove white flower), produced more but smaller seed, and ha d a slightly increa sed fiber strength (Tables VII-5, page 153, VII-6, page 155, and G-13, page 567, in Monsanto 2012). These differences were all

small and in all cases the mean values for these characteristics were with the range

observed for the commercial reference varietie s. Finally, as discussed above, changes in disease or insect pest susceptibility or in response to abiotic stre ss were not observed in

MON 88701 relative to control.

Given these data, the herbicide-re sistance traits conferred by the dmo and bar genes are very unlikely to provide MON 88701 with a selective advantage in unmanaged ecosystems. However, the herbicide-resistance traits could complicat e efforts to control volunteer cotton in settings where dicamba and/ or glufosinate are be ing applied for weed control, such as in subsequent cotton (p lanted on over 50% of cotton acreage grown in 2010, see Table VIII-20, page 217 in Monsan to 2012) or rotation crops (Roberts et al 2002; Fannin 2010; Ledbetter 2011). In particular , dicamba was used for weed control on 20% of sorghum acres in 2011 (sorghum was planted as a rotation crop on 8% of cotton acreage in 2010) and in 11% of winter wh eat acres in 2009 (wheat was planted as a rotation crop on 9% of cotton acreage in 2010), as well as in 7% of cotton acres in 2007 (Table VIII-20, page 217, in Monsanto 2012) . Although cotton volunteers typically do not reduce crop yield, they can act as rese rvoirs for insect pests of cotton (York et al 2004). However, both mechanical means (til lage) and a variety of other herbicide

treatments are available for control of vol unteer cotton in such circumstances (Keeling et
al 2009; Morgan et al 2011; Thompson and Steckel 2009; Fannin 2010; Ledbetter 2011;

York et al 2004; see also pages 228-230, including Table VIII-25, in Monsanto 2012). For instance, volunteer MON 88701 in subseque nt corn (planted as a rotation crop on 16% of cotton acreage, Table VIII-20, page 217, in Monsanto 2012), sorghum, or wheat crops can be well controlled at various time points with the herbicides Sharpen, atrazine,18 2,4-D, Status (a mix of diflufenzopyr and dicamba), or Starane (Morgan et al 2011; Fannin 2010; Table VII-25, page 230, in Mons anto 2012). The specificity of the DMO and PAT proteins for dicamba and glufosin ate respectively is hi gh and other common herbicides are not metabolized by these en zymes (see Section C above). Therefore, excepting dicamba and glufosinate, MON 88701 is expected to be sensitive to the same

herbicides as othe r cotton varieties.

Therefore, based on this characterization, APHIS concludes that MON 88701 is no more likely to establish weedy populat ions than existing cotton cult ivars, and such populations can be controlled using curre nt weed control practices.



### G. Potential Impacts on the Weedine ss of Any Other Plants with which

It Can Interbreed

Gene introgression is the movement of a gene or genes from one population of organisms into the gene pool of another, genetically distinct, population via sexual crossing. The process begins with the pollination of one species by the other, followed by the establishment of one or more hybrid offspr ing and maintenance of introgressed genes during repeated backcrossing of the hybrid to on e of the parental spec ies or to a different species. Gene introgression is a natural biol ogical process with si gnificant evolutionary importance. A number of angiosperm taxa are believed to be derived from hybridization or introgression between closel y related taxa (Hegde et al . 2006; Rieseberg 1997; Soltis and Soltis 1993), and even in the existing floras, the occurrence of hybridization or introgression is reported to be widespread (Peterson et al 2002; Rieseberg and Wendel 1993; Stace 1987). It has been a common pr actice by plant breeders to artificially introgress traits from wild relatives into crop plants to develop new cultivars. However, gene introgression from crops to wild relativ es is also thought to have a potential to

enhance the weediness of wild relatives, as observed in rice, sorghum, sunflower and few other crops (see Table 1 in Ellstrand et al 1999).

APHIS considers two primary issues when a ssessing weediness of sexually compatible plants because of transgene flow: 1) the poten tial for transgene flow and introgression to sexually compatible cultivated, wild, or free-living re latives and, 2) if transgene flow and introgression are determined to be biologically meaningf ul, the potential impact of introgression with respect to weediness of the recipient.

Potential for hybridization and gene introgression

Two cultivated and two wild species of co tton grow in the United States and its

territories. G. hirsutum (Upland cotton) is the most widely cultivated species, comprising 97% of the U.S. cotton crop (USDA-NASS 2012a). The vast majority of Upland cotton is cultivated in the Cotton Belt, which stretc hes across the southern United States from Virginia to California (USD A-NASS 2012a). Small amounts are also grown in Puerto Rico for breeding and seed production pur poses (Bayer CropScience 2006; Monsanto19 Company 2004). In addition to cultivat ed varieties, naturalized or native 3 populations of

G. hirsutum grow in Florida, Puerto Rico, and the Virgin Islands while naturalized populations grow in some of the Hawaiian Islands (Coile and Garland 2003; Fryxell 1984; USDA-NRCS 2012a; Wunderlin and Hans en 2008). The second cultivated species,

G. barbadense (Pima or Egyptian cotton), is grown in Arizona, California, New Mexico, and Texas (Pleasants and Wendel 2005; USDA-NASS 2012a). Naturalized populations

of G. barbadense grow in Puerto Rico, the Virgin Islands and most of the major Hawaiian Islands (Bates 1990; Fryxell 1984; USDA-NRCS 2012d). Two wild species of cotton are native to the United States, G. thurberi and G. tomentosum , and grow in Arizona and Hawaii respectively (Fr yxell 1984; USDA-NRCS 2012e; USDA-NRCS 2012f).

G. hirsutum is tetraploid and thus effectively incompatible with diploid species such as

G. thurberi. Plants from these two groups do not normally hybridize spontaneously and produce fertile offspring, and experiment al crosses are difficult (OECD 2008). In

contrast, G. hirsutum is sexually compatible with the tetraploids G. barbadense and G.

tomentosum and can form viable and fertile progeny with both species (Brubaker et al 1993; OECD 2008; Saha et al 2006). Thus, unassisted outc rossing and gene introgression could potentially occur in areas wh ere these species are co-located.

Wind dispersal of cotton pollen is negligible because of its large si ze and self-adherent properties (Hutmacher et al 2006; Jenkins 1993; OECD 2008). However, crosspollination between cotton species can occur through the activity of pollinating insects (Llewellyn et al 2007; McGregor 1976; OE CD 2008; Van Deynze et al 2005). For transgene introgression from MON 88701 to occur there would have to be spatial proximity between MON 88701 and the recipient variety or species, overlap in their flowering phenology, and overlap in their pol linators (Pleasants and Wendel 2005). In general, the extent of transgene intr ogression will depend on the species pool, preferences, and abundance of pollinators, wh ich can vary according to region, location, season, time of day, and use of insecticides . In addition, transgene introgression will decrease with increasing geographic di stance between the source and receiver populations and physical barriers and intermedia te pollinator-attractive plants can reduce the potential for pollen movement (Green and Jones 1953; Llewellyn et al 2007; McGregor 1976; OECD 2008; Umbeck et al 1991; Van Deynze et al 2005; Zhang et al 2005). Additional information on the biology of cotton can be found within the OECD cotton consensus document (OECD 2008).

Although naturalized populations of G. hirsutum grow in Southern and Central Florida, their northernmost reported location (Gilchrest County, FL )) is separated by over 120

miles from the nearest commercial cotton production areas in the Florida panhandle (Wunderlin and Hansen 2008; USDA-NA SS 2012b). Thus, outcrossing from MON

88701 to naturalized G. hirsutum in Florida is highly unlikely.

3 A “native” plant is one that has grown in a particular region or ecosystem for hundreds or thousands of years without intentional or accidental human help. A “n aturalized” plant is a non- native plant that does not need human help to reproduce and maintain itself over time in an area where it is not native. (USDA-NRCS 2012c).20 In contrast, G. hirsutum is cultivated in many areas where G. barbadense is also grown (USDA-NASS 2012b). In addition, as noted abov e, native and/or na turalized populations of both species are present in Hawaii, Pu erto Rico, and the Virgin Islands. Althoughcultivated varieties of both species are largely self-pollinated, insect-mediated crosspollination can occur both within and between the species (Brubaker et al 1993; Llewellyn et al 2007; OECD 2008; Van Deynze et al 2005; Van Deynze et al 2011). Bumble bees ( Bombus spp.), Melissodes and Halictus bees, honey bees ( Apis mellifera ),

and Scolia wasps are the primary pollinators (McGregor 1976).

Published studies report that there has been relatively l ittle gene introgression from G.

hirsutum into native or naturalized G. barbadense in Mesoamerica and the Caribbean, despite the fact that G. barbadense has been grown in the presence of the predominant G.

hirsutum since prehistoric times. In contrast, introgression from G. barbadense to native

or naturalized G. hirsutum in these areas has been relatively common (Brubaker et al 1993; Wendel et al 1992). Various mechanisms have b een suggested to account for this asymmetry (Brubaker et al 1993; Jiang et al 2000; OGTR 2008; Percy and Wendel 1990). While none of these mechanisms leads to complete isolation between the two species, the reported asymmetry in gene fl ow suggests that gene introgression from

cultivated G. hirsutum varieties such as MON 88701 to native or naturalized G.

barbadense should be rare.

However, gene introgression from cultivated G. hirsutum to cultivated G. barbadense may be more likely, since gene flow between cultivated varieties of these species appears to occur with the opposite asymmetry from that observed between native or naturalized varieties (CDFA 2009; Van Deynze et al 2011). The mechanism underlying this reversal in the directionality of gene flow, which ha s also been reported in Caribbean accessions (Wendel, Brubaker et al. 1992; Brubaker, Jas on et al. 1993), is not known. Nonetheless, outcrossing rates from M ON 88701 to cultivated G. barbadense are still likely to be low. For instance, Van Deynze et al. (2005) repor ted that Upland/Pima hybrid plants have

been observed at a rate of 0.01% in fields so wn with seeds of cult ivated varieties that were obtained from production fields separated by at least 800 meters.

With regard to G. tomentosum

, natural populations of this species are found on all Hawaiian Islands except Kauai and Hawaii; the species is dominant on Kohoolawe and several sizable populations are found on Oahu and Maui. Populations are located on the drier, leeward coastal plains of the islands at low elevations, which are also the areas that are primarily used for agricultu re (Pleasants and Wendel 2005). G. hirsutum has not been grown as an agricultural commodity in Hawa ii for decades, and to the best of APHIS’ knowledge, seed companies no longer use th e Hawaiian Islands as a winter nursery (Grace, personal communication, 2012).

The flowering period for G. tomentosum corresponds to the end of the rainy season; it may begin as early as January, with peak fl owering occurring in April and May, and may extend through August in a very wet year (Pleasants and Wendel 2010). Thus, any

cultivated cotton that blooms between January and August c ould potentially overlap with

G. tomentosum . Previously, it was thought that peak anthesis and receptivity in G.21 tomentosum occurs at dusk, whereas in G. hirsutum the flowers open in the morning and whither by evening (OECD 2008). However, Pleasants and Wendel (2010) found that G.

tomentosum flowers also open in the morning, dehi sce rapidly, and begin to senesce by late afternoon. These results suggest that there is substantial overlap in flowering phenology between G. hirsutum and G. tomentosum

.

Spontaneous self-pollin ation is rare in G. tomentosum , perhaps due to th e structure of its flowers. Instead, the species appears to re ly on the action of pollinators (Münster and Wieczorek 2007; Pleasants and Wendel 2005). It was previously thought that moths were the only insects that pollinated G. tomentosum , and thus that there wa s little overlap with pollinators of G. hirsutum (OECD 2008; Pleasants and Wendel 2005). However, more

recent studies have shown that G. tomentosum is pollinated by honeybees and carpenter bees, which are among the species that also pollinate commercially grown G. hirsutum

. In addition, both of these polli nators are long-distance fora gers; for instance, honeybees may forage up to 6 – 10 miles from their nest (Pleasants and Wendel 2010). Thus, in addition to overlap in flowering phenology, there is overlap in pollinators between G.

tomentosum and G. hirsutum . However, no hybrids between G. hirsutum and G.

tomentosum have been identified to date, alt hough only a relatively small number of

accessions and marker loci have been examined (DeJoode and Wendel 1992).

Expression of the DMO and PAT proteins do es not cause any major changes in the phenotype of cotton plants other than to conf er resistance to the herbicides dicamba and glufosinate. No significant di fferences in viability, size, or morphology were observed in pollen obtained from MON 88701 compared to pollen from the conventional control (pages. 164-166 in Monsanto 2012). Thus, the in troduced genetic material is unlikely to

cause an increased rate of out crossing of MON 88701 relative to non-transgenic varieties. Should outcrossing from MON 88701 to G. barbadense or G. tomentosum occur, transgene introgression would still require the establis hment of hybrid progeny followed by persistence of the transgen e through self-crossing or back-c rossing into the recipient species in subsequent generations.

The low level of introgression from G. hirsutum to native or naturalized G. barbadense observed in the Caribbean and the phe nomenon of hybrid breakdown suggests that transgene introgression from MON 88701 to native or naturalized G. barbadense can occur but is likely to be rare (Jiang et al 2000; OGTR 2008). In the absence of herbicide treatment, the transgenic material in M ON 88701 is unlikely to confer a selective advantage on any hybrid progeny that may re sult from outcrossing. Thus, the transgenes present in MON 88701 are unlikely to increa se the rate of successful transgene introgression from MON 88701 in to native or naturalized G. barbadense relative to the rate of gene intr ogression from conventional cultivars.

Transgene introgression from MON 88701 to cultivated G. barbadense can also occur but is also likely to be rare since cultivated G. barbadense is regularly harvested. While the likelihood of transgene movements to G. barbadense is likely greater with cultivated

varieties than with native or naturalized G. barbadense , such movements would tend to involve plants producing seeds intended for processing rather than planting because seed22 production fields are isolated from commercial fields. Seed production isolation standards will help ensure that any movement of transgenes into seed production fields will remain at very low levels (AOSCA 2003; Van Deynze et al 2005). The transgenes present in MON 88701 unlikely to increase the rate of successful transgene introgression

from MON 88701 into cultivated G. barbadense relative to the rate of gene introgression

from conventional cultivars.

Finally, introgression into G. tomentosum in Hawaii is also likely to be rare, both because

of barriers to intr ogression (Brubaker et al 1993; Jiang et al 2000; OGTR 2008; Percy and Wendel 1990), and because there is no commercial cotton production on these islands (Grace, personal communication, 2012) . If any Upland cotton is grown in the Hawaiian Islands, it is grown at a ve ry small scale and outcrossing to G. tomentosum is unlikely to occur. Should outcrossing nonethel ess occur, transfer of the transgenes present in MON 88701 would not be expected to confer a selective advantage on the hybrid progeny or to reduce hybrid breakdown, which would be expected to eliminate introgressed genes from the G. tomentosum population. Thus, the transgenes present in MON 88701 are unlikely to increase the rate of successful transgene introgression from

MON 88701 to G. tomentosum

.

In summary the available evidence indicates th at there is a low potential for introgression of transgenic material from MON 88701cotton to G. tomentosum or to native or

naturalized G. barbadense . There is no evidence that any of the genetic elements used in

MON 88701 would increase the rate of outcrossing or gene introgression of MON 88701

relative to non-tr ansformed cotton.

Relative fitness/weediness of recipients after introgression

As discussed in the previous section, th e genetic material introduced into MON 88701 does not confer or enhance weedy characteri stics of cultivated Upland cotton. There is no

reason to believe that it would do so in naturalized or native G. hirsutum , in G.

tomentosum , or in cultivated, naturalized, or native G. barbadense . Thus, in the unlikely event that transgene introgression from MON 88701 to one of these other types of cotton were to occur, the herbicide resistance tra its would provide a sele ctive advantage only when the resulting hybrids were in contact with the herbicide (i.e., in an agricultural field or treated rights of way). However, APHI S could find no reports that any of these potential recipient populations are actively controlled by herbic ides. Therefore, transgene introgression from MON 88701 would not be e xpected to adversely impact recipient plants or increase their fitness or weedin ess any more than would gene flow from cultivated non-transgenic Upland cotton. Nor would it affect efforts to remove wild populations, as no such efforts exist.



### H. Potential Changes to Agricul ture or Cultivat ion Practices

APHIS considered whether there are likely to be significant changes to agricultural practices associated with cu ltivation of MON 88701, and if so, are they likely to significantly exacerbate plant diseases or pest s, especially those for which APHIS has a control program. Relative to currently cultivat ed cotton varieties, th e only agricultural or23 cultivation practices that are expected to change if MON 88701 is no longer subject to regulation are those related to weed management : in particular, choice of herbicide(s) or herbicide combinations, times of application.

The current and proposed uses of dicamba in cotton are described in the petition (Section VIII.G, pages 206- 214, in Monsanto 2012). Di camba is currently labeled only for early preplant applications in cotton. In addition, due to insufficient crop tolerance of cotton to applications of dicamba, preplant restric tions are required to avoid cotton injury: a maximum application rate of 0.25 lbs a.e. per acre and a minimum of 21 days and one inch of rainfall or overhead irrigation betw een preplant application and planting of cotton. Due to insufficient crop tolerance, di camba currently also cannot be used for incrop postemergence applications. If EPA a pproves Monsanto's submitted application to amend Registration Number 524-582 for a DGA salt formulation of dicamba, growers would be authorized to apply dicamba alone or in mixtures with glufosinate, glyphosate (when MON 88701 is stacked with glyphosate resi stant cotton) or ot her herbicides for preplant applications without restrictions a nd for in-crop postemergence applications on MON 88701. Non-aerial applications of dicam ba would be authorized preemergence up to crop emergence as a single application or sp lit applications up to a total of 1.0 lb a.e. per acre, while up to two postemergence appli cations of up to 0.5 lb a.e. per acre each through seven days prior to harvest. The tota l maximum annual application rate would be 2.0 lb dicamba a.e. per acre. The use of dicamba on MON 88701 is not expected to impact dicamba-sensitive crops from drift (spray or vola tility) because Monsanto is registering a low volatility (DGA salt) di camba formulation for use on MON 88701 and aerial applications of dicamba would not be al lowed. Issues related to herbicide drift and

volatilization will be further addressed in the NEPA document for this petition.

Upon integration of MON 88701 into glyphosate resistant cotton systems, aside from the anticipated dicamba label changes requested, Monsanto expects that growers will have the ability to continue to use established cotton production practices including the crop rotation, tillage systems, labeled herbicid es, and planting and harvesting machinery currently being utilized (Section VIII.G, pages 206 – 212, in Monsanto 2012). The anticipated label changes would facilitate a wider window of application for dicamba in cotton, which is expected to provide a tool for improved control of broadleaf weeds (including some with resistance to other he rbicides such as glyphosate, ALS, and PPO chemistries) that can be integrated into weed management programs using no-till or reduced tillage or conventiona l tillage. Monsanto's anticipated weed management

recommendations for MON 88701 combined with glyphosate resistant cotton also include a preemergence (burndown at planting) application of a residual herbicide either alone or combined with gl yphosate and dicamba depending on location and the type of tillage employed (Tables VIII-16 and VIII- 17, pages 208-209, in Monsanto 2012). The impacts of this system for reducing or ma naging weeds and the evolution of herbicideresistant weeds will be examined as part of the NEPA analysis. Greater weed control could potentially reduce disease and pest pressure in cotton if the diseases and pests of

the weeds also use cotton as a host.24 Crop rotation practices are not expected to be adversely impacted by the use of dicamba on fields planted to MON 88701. Crop rotation pr actices in cotton were analyzed in the petition (Section VIII.H, pages 215-230, in M onsanto 2012). The primary crops planted after cotton are cotton (54%), corn (16%), wheat (9%), soybean (8%), sorghum (8%), and peanut (4%). Dicamba can be absorbed through leaves and roots and translocated, but is considered only moderately persistent in soil , with a half-life of six days for dicamba acid

under aerobic soil conditions with formation of the non-persistent degradate DCSA, and a half-life of 141 days under anaerobic soil conditions (U.S. EPA 2009). Crop rotation restrictions range from 30 to approximat ely 180 days, depending on the rate applied, inches of rainfall and the following crop, acco rding to the Clarity® label (BASF 2010), and these should be adequate for rotation to other crops th e spring following harvest of

cotton.

As described above (see Potential Impacts of Genetic M odifications on Disease and Pest Susceptibilities ), field studies on MON 87701 cotton demonstrated that neither the

herbicide resistance traits nor the herbicide treatments appear to alter the response of MON 87701 cotton to abiotic stress, diseases, or arthropod pests under natural levels of these stressors, nor were pest arthrop ods more abundant around MON 88701 plots. Agronomic practices used to prepare and mainta in each study site were characteristic of those used in each respective geographic re gion and all maintenance operations were performed uniformly over the entire trial area (page 533 in Monsan to 2012). Although pest and disease susceptibility data was not presented for MON 88701 stacked with the glyphosate-resistant trait, a recent review indi cates that nether the glyphosate resistance trait nor glyphosate use in glyphosate resi stant crops increases crop disease (Duke et al 2012), and there is no evidence that either in crease susceptibility to insect pests. Therefore, changes in agricu ltural practices related to weed control in MON 88701 or MON 88701 stacked with the glyphosate resistan ce trait are unlikely to adversely impact pest and disease control practices or any ot her cultivation and management practices in

cotton.

In conclusion, as discussed throughout this document, MON 88701 is similar to conventional cotton in its agronomic, phenot ypic, environmental, and compositional

characteristics and has levels of toleran ce to insects and diseases comparable to conventional cotton. Therefore, no significant adverse impacts on curre nt agricultural or cultivation practices are expected following the intr oduction of MON 88701.



### I. Potential Impacts from Transfer of Genetic Information to Organism with which MON 87701 Cannot Interbreed

APHIS examined the potential for the new ge netic material inserted into MON 88701 to be horizontally transferred without sexual re production to other or ganisms (horizontal gene transfer (HGT)) and whether such an ev ent could lead directly or indirectly to disease, damage, injury or harm to plants, including the creation of new or more virulent pests or pathogens. HGT between unrelated or ganisms has been intensively studied in recent years, and the issue gained extra attenti on with the release of tr ansgenic plants into the environment (Dröge et al 1998). Potential risks from HGT from genetically25 engineered organisms to another organism without reproduction or human intervention were recently reviewed (Keese 2008). M echanisms of HGT include conjugation, transformation and transduction, and other di verse mechanisms of DNA and RNA uptake and recombination and rearrangement, most not ably through viruses and mobile genetic elements. HGT has been a major contributor to the spread of antibiotic resistance amongst pathogenic bacteria and the emergen ce of increased virulence in bacteria, eukaryotes and viruses and in the long r un has contributed to major transitions in

evolution.

Potential for horizontal gene tr ansfer to bacteria or fungi

MON 88701 has two bacterial genes. HGT a nd 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 inter act with fungi and bacteria (e.g. as commensals, symbionts, pa rasites, pathogens, decomposers, or in the guts of herbivores), there are almost no evolut ionary examples of HGT to bacteria from eukaryotes or from plants to fungi (Keese 2008). The only genes likel y to be transferred successfully from genetically e ngineered plants to bacteria are other bacterial genes. Horizontal transfer from and expression in ba cteria of the foreign DNA inserted into the nuclear genome of MON 88701 is unlikely to occur. First, many genomes (or parts thereof) have been sequenced from bacteria that are closely asso ciated with plants, including Agrobacterium and Rhizobium (Kaneko et al 2002; Wood et al 2001), and there is no evidence that these organisms contain genes derived from plants. Second, in cases where review of sequence data has implied that HGT has occurred, these events are inferred to have occured on an evolutionary time scale on the order of millions of years (Brown 2003; Koonin et al 2001). Third, under natural conditions; no transfer of an intact functional gene from a genetically engineer ed plant to bacteria or fungi has been demonstrated to date (Miki and McHugh 2004). Fourth, the transgene DNA promoters and coding sequences used in MON 88701 are optimized for plant expression, not prokaryotic bacterial expression. Thus even if horizontal gene transfer occurred, proteins corresponding to the transgen es are not likely to be pr oduced. Finally, 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 gastrointestin al tract of humans or animals, or in the environment, is remote (U.S. FDA 1998). Th erefore, APHIS concludes that horizontal gene transfer from MON 88701 to bacteria or fungi is unlikely to occur and thus poses no significant environmental or plant pest risk.

Potential for horizontal gene transfer to viruses

APHIS also considered whether horizontal transfer of DNA fro m MON 88701 to plant viruses was likely to occur and would lead to the creation or selecti on of a more virulent plant pathogen through recombination with ot her plant viruses. This issue has been considered before by other science review panels and government regulatory bodies (Keese 2008). The only virus se quences contained within MON 88701 encode regulatory elements from the peanut chlorotic streak virus, the tobacco etch virus, and the cauliflower mosaic virus. Regulatory elements such as promoters and terminators have26 not been implicated in viral recombination. Therefore, APHIS concl udes that horizontal

transfer of DNA from MON 88701 to viruses is unlikely to occur and thus poses no significant environmental or plant pest risk.

Potential for horizontal gene transfer to parasitic plants

Recently, Yoshida et al (2010) through a comparative genomics analysis implicated HGT for the incorporation of a specific genetic sequence in the parasitic plant purple witchweed ( Striga hermonthica ), which infests cereal fiel ds (monocots) including corn and sorghum ( Sorghum bicolor ). According to this study, in corporation of the specific genetic sequence (with an unknown function) occurred between sorghum and purple witchweed. However, this horizontal gene tr ansfer occurred before the speciation of purple witchweed ( S. hermonthica ) and related cowpea witchweed ( S. gesnerioides ), a parasitic plant of dicots, from their comm on ancestor. In other words, HGT between a parasitic plant and its host is an extremely rare event. Therefore, APHIS concludes that horizontal gene transfer from MON 88701 to para sitic plants is unlikely to occur and thus poses no significant environm ental or plant pest risk.



### J. Conclusion

APHIS prepared this plant pest risk asse ssment in order to determine if MON 88701 is unlikely to pose a plant pest risk. The inserted genetic material does not pose a plant pest

risk. MON 88701 is no weedier than the unmodif ied control or commercial varieties. There is low potential for introgression of transgenic material from MON 88701cotton to sexually compatible wild or naturalized rela tives, and should such introgression occur it would not be expected to increase the fitne ss or weediness of the recipient plants any more than would gene flow from cultiv ated non-transgenic Upland cotton. MON 88701 also exhibits no greater susceptibility to disease or plant pests than the unmodified control or commercial varieties, is unlikely to cause delete rious effects on non-target or beneficial organisms in the agro-ecosyst em, and is unlikely to adversely affect agricultural or cultivation practices. Horizontal gene transfer of the introduced material to non-sexually compatible organisms is extremel y unlikely to occur. Therefore, APHIS has

concluded that MON 88701 is unlikel y to pose a plant pest risk.



### K. References

7 Code of Federal Regulations, Part 340 [1 -1-12 Edition]. Introduction of organisms and products altered or produced through genetic engineering which are plant pests or which there is reason to believe are plant pests. Retrieved on August 4, 2012 from http://www.gpo.gov/fdsys/pkg/CFR-2012-titl e7-vol5/pdf/CFR-2012-title7-vol5- part340.pdf

7 United States Code, Sections 7701- 7772 [2011 Edition]. Plant Protection Act. Retrieved on August 4, 2012 from http://www.gpo.gov/fdsys/pkg/USCODE-2011- title7/pdf/USCODE-2011-title7-chap104.pdf27 40 Code of Federal Regulations, Part 180.227 [7 -1-11 Edition]. Dicamba; tolerances for residues. Retrieved on July 20, 2012 from http://www.gpo.gov/fdsys/pkg/CFR-2011- title40-vol24/pdf/CFR-2011- title40-vol24-sec180-227.pdf

51 Federal Register 23302 [June 26, 1986]. C oordinated Framework for Regulation of Biotechnology. Retrieved on August 9, 2012 from http://www1.usgs.gov/usbiotechreg/index.jsp

Adrian-Romero M, G Blunden, BG Carpente r and E Tyihák. 1999. HPLC quantification of formaldehyde, as formaldemethone, in plants and plant-like organisms. Chromatographia 50(3/4):160-166.

AOSCA. 2003. "Yellow Books" 2003: Operatio nal Procedures, Crop Standards and Service Programs Publication (Genetic and Crop Standards). Association of Official Seed Certifying Agencies Meridian, ID.

Awmack CS and SR Leather. 2002. Host plan t quality and fecundity in herbivorous insects. Annual Review of Entomology 47:817-844.

Baker HG. 1965. Characteristics and mode s of origin of weeds. pp. 147-168. In HG Baker and GL Stebbins (eds), The Genetic s of Colonizing Species. London: Academic

Press.

BASF. 2010. CLARITY® Herbicide Labe l, Soybean, pp. 18-19. BASF Corporation 007969-00137.20100927c.NVA 2010-04-065-0154, 22 pp. Retrieved on June 27, 2012

from http://agproducts.basf.us/app/ cdms?manuf=16&pd=229&ms=2274

Bates, DM. 1990. Malvaceae. Manual of the fl owering plants of Hawaii. WL Wagner, DR Herbst and SH Sohmer. Honolulu, Univ ersity of Hawaii Press, Bishop Museum

Press : 868-902.

Bayer CropScience. 2006. Petition for Determ ination of Nonregulated Status for Glyphosate-Tolerant cotton: GlyTol TM cotton Event GHB614. Submitted by Alejandra Scott, Bayer CropScience USA LP, Res earch Triangle Park, North Carolina, 173pp. Retrieved on November 26, 2012 from http://www.aphis.usda.gov/ biotechnology/not_reg.html

.

Bayer CropScience. 2011. Liberty herbicide label, Cotton, pp. 12-14. Bayer CropScience LP, Research Triangle Park, North Carolina , 21 pp. Retrieved on November 2, 2012 from http://fs1.agrian.com/pdfs/Liberty_280_SL_Label2.pdf

Behrens MR, N Mutlu, S Chakraborty, R Dum itru, WZ Jiang, BJ LaVallee, PL Herman,

TE Clemente and DP Weeks. 2007. Dicamba resistance: Enlarging and preserving biotechnology-based weed management strategies. Science 316:1185-1188.28 Berg, G, N Roskot and K Smalla. 1999. Genot ypic and phenotypic relationships between

clinical and environmental isolates of Stenotrophomonas maltophilia . Journal of Clinical Microbiology 37:3594-3600.

Bevan, M, WM Barnes and M-D Chilton. 1983. Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Research 11:369-385.

Bi, JL, JB Murphy and GW Felton. 1997. Does sali cylic acid act as a signal in cotton for

induced resistane to Helicoverpa zea ? Journal of Chemical Ecology 23(7):1805-1818.

Blasingame, D. 2010. Personal communication.

Bowman, DT, OA Gutierrez, RG Percy, DS Calhoun and OL May. 2006. Pedigrees of upland and pima cotton cultivars releas ed between 1970 and 2005. Mississippi State University, Mississippi State, Mississippi. Available at http://msucares.com/pubs/bulletins/b1155.pdf , accessed November 5, 2012.

Brown JR. 2003. Ancient horizontal gene transfer. Nature Re views Genetics 4:121-132.

Brubaker, CL, JA Koontz and JF Wendel. 1993. Bidirectional Cytoplasmic and Nuclear Introgression in the New Wo rld Cottons, Gossypium barbadense and G. hirsutum (Malvaceae). American Jour nal of Botany 80(10):1203-1208.

Buchanan, GA. 1992. Trends in weed control methods. Pages 47-72 in Weeds of Cotton:

Characterization and Control. CG McWhor ter and JR Abernathy (eds.). The Cotton Foundation, Memphis, Tennessee.

Chakraborty J, D Ghosal, A Dutta and TK Dutta. 2012. An insight into the origin and

functional evolution of bacterial aromatic ring-hydroxylating oxygenases. Journal of

Biomolecular Structure a nd Dynamics 30(1):1-19.

Chakraborty S, M Behrens, PL Herman, AF Arendsen, WR Hagen, DL Carlson, X-Z Wang and DP Weeks. 2005. A three-component dicamba O -demethylase from Pseudomonas maltophilia , strain DI-6: Purification and characterization. Archives of Biochemistry and Biophysics 437:20-28.

Chan, BG, AC Waiss Jr., RG Binder and CA Elliger. 1978. Inhibition of lepidopterous larval growth by cotton constituents. Entomo logia Experimentalis et Applicata 24(3):294-

300.

Clark, SE and GK Lamppa. 1992. Processing of the precursors for the light-harvesting chlorophyll binding proteins of photosystem II and photosystem I during import and in an organelle-free assay. Plant Physiology 98:595-601.29 Coile, NC and MA Garland. 2003. Notes on Flor ida's Endangered and Threatened Plants. Florida Department of Agriculture and Cons umer Services. Gainseville. Available at http://www.virtualherbarium.org/EPAC/Notes2003.pdf , accessed November 5, 2012.

Colson-Hanks, ES, SJ Allen and BJ Deverall . 2000. Effect of 2,6- dichloroisonicotinic acid or benzothiadiazole on Alternaria leaf spot, bacterial blight and VErticillium wilt in

cotton under field conditions. Austra lasian Plant Pathology 29:170-177.

Crockett LJ. 1977. Wildly successful plants : A handbook of North American weeds. Honolulu, Hawaii: University of Hawaii Press, 609 pp.

D'Ordine, RL, TJ Rydel, MJ Storek, EJ Sturma n, F Moshiri, RK Bartlett, GR Brown, RJ Eilers, C Dart, Y Qi, and others. 2009. Dicam ba monooxygenase: Structur al insights into a dynamic Rieske oxygenase that catalyzes an exocyclic monooxygenation. Journal of Molecular Biology

392:481-497.

Davies, AM, R Tata, RL Beavil, BJ Sutt on, PR Brown. 2007. L-Methionine sulfoximine, but not phosphinothricin, is a substrate for an acetyltransferase (gene PA 4866) from Pseudomonas aeruginosa : structural and functional st udies. Biochemistry 46:1829-1839.

DeJoode, DR and JF Wendel. 1992. Genetic Dive rsity and Origin of the Hawaiian Islands Cotton, Gossypium tomentosum. Ameri can Journal of Botany 79(11):1311-1319.

Dixon RA. 2001. Natural products and plant disease resistance . Nature 411: 843

- 847

.

Dröge M, A Pühler and W Selbitschka. 1998. Ho rizontal gene transfer as a biosafety issue: A natural phenomenon of public conc ern. Journal of Bi otechnology 64:75-90.

Duke SO, J Lydon, WC Koshinen, TB Moorma n, RL Chaney and R Hammerschmidt. 2012. Glyphosate effects on plant mineral nutr ition, crop rhizosphere microbiota, and plant disease in glyphosate-resi stant crops. Journal of Agri cultural and Food Chemistry

60:10375-10397.

Dumitru R, WZ Jiang, DP Weeks and MA Wilson. 2009. Crystal structure of dicamba monooxygenase: A Rieske nonheme oxygenase that catalyzes oxidative demethylation. Journal of Molecular Biology 392:498-510.

Echemendia, Y. 2010. Microor ganism of the month: Stenotrophomonas maltophilia

. Environmental Microbiology Laborator y, Inc., Cherry Hill, New Jersey. http://www.emlab.com/s/sampling/env-report-07-2007.html, accessed November 2, 2012.

Ellstrand NC, HC Prentice and JF Hancoc k. 1999. Gene flow and introgression from domesticated plants into their wild relative s. Annual Review of Ecology and Systematics

30:539-563.30 Fannin, B. 2010. Producers battling volunteer cott on in corn, sorghum fields. AgriLife Today, Texas A&M AgriLife, December 24, 2010. Available at http://today.agrilife.or g/2010/12/24/volunteer-cotton/ , accessed November 5, 2012.

Fraley, RT, SG Rogers, RB Horsch, PR Sande rs, JS Flick, SP Adams, ML Bittner, LA Brand, CL Fink, JS Fry and others. 1983. Expressi on of bacterial gene s in plant cells. Proceedings of the National Academy of Scie nces of the United States of America

80:4803-4807.

Frear DS. 1976. The benzoic acid herbicides: IV - Dicamba and Tricamba. pp. 563-579.

In PC Kearney and DD Kaufmann (eds), Herb icides: Chemistry, Degradation and Modeof-Action – Second Edition. New York: Marcel Dekker.

Fryxell, PA. 1984. Taxonomy and Germplasm Resources. In: RJ Kohel and CF Lewis (Eds) Cotton, Agronomy Monograph 24, ASA-CSSA-SSA, Madison, WI, p27-57.

Goodfellow, M. and S.T. Williams. 1983. Ecolog y of actinomycetes. Annual Review of Microbiology 37:189-216.

Green, JM and MD Jones. 1953. Isolation of co tton for seed increase. Agronomy Journal 45(8):366-368.

Guinn, G. 1982. Causes of square and boll shedding in cotton. Technical Bulletin Number 1672. United States Department of Agri culture, Agricultural Research Services, Washington, D.C.

Hanson AD and S Roje. 2001. One-carbon metabo lism in higher plants. Annual Review of Plant Physiology and Plant Molecular Biology 52: 119-137.

Heap I. 2012. International surv ey of herbicide resistant weeds. Retrieved on November 26, 2012 from http://www.weedscience.org/in.asp

Hegde SG, JD Nason, JM Clegg and NC Ells trand. 2006. The evolution of California’s

wild radish has resulted in the extinction of its progen itors. Evolution 60(6):1187-1197.

Herman PL, M Behrens, S Chakraborty, BM Chrastil, J Barycki and DP Weeks. 2005. A three-component dicamba O -demethylase from Pseudomonas maltophilia , strain DI-6: Gene isolation, characterization, and heterol ogous expression. The Journal of Biological Chemistry 280(26):24759-24767.

Hérouet, C, DJ Esdaile, BA Mallyon, E De bruyne, A Schulz, T Currier, K Hendrickx, RJ van der Klis and D Rouan. 2005. Safety evaluation of the phosphinothricin acetyltransferase proteins encoded by the pat and bar sequences that confer tolerance to glufosinate-ammonium herbic ide in transgenic plants. Regulatory Toxicology and Pharmacology 41:134-149.31 Herrmann KM. 1995. The shikimate pathway: Earl y steps in the biosynthesis of aromatic compounds. Plant Cell 7:907-919.

Holm LG, JV Pancho, JP Herberger, DL Plucknett. 1979. A Geographical Atlas of World Weeds. John Wiley and Sons, New York, 391 pp.

Hutmacher, RB, RN Vargas and SD Wright. 2006. Methods to Enable the Coexistence of Diverse Cotton Production Systems, University of California, Divisi on of Agriculture and

Natural Resources.

ILSI-CERA. 2011. A review of the envir onmental safety of the PAT protein. International Life Sciences Institute, Ce nter for Environmental Risk Assessment, Washington, D.C. Retrieved on November 2, 2012 from http://ceragmc.org/docs/protein_monographs/pat_en.pdf

.

ILSI. 2010. International Life Sciences In stitute, Crop Composition Database, Version 4.2. Retrieved on November 5, 2012 from www.cropcomposition.org

Inbar, M, H Doostdar, D Gerling and RT Mayer. 2001. Induction of systemic acquired resistance in cotton by BTH has a neg ligible effect on phytophagous insects. Entomologia Experimentalis et Applicata 99:65-70.

Jenkins, JN. 1993. Cotton. In Traditional Crop Br eeding Practices: An Historical Review to Serve as a Baseline for Assessing the Ro le of Modern Biotechnology. Paris, OECD (Organisation for Economic Co-Operation and Development) : 61-70.

Jiang, C-X, PW Chee, X Draye, PL Morrell, CW Smith and AH Paterson. 2000.

Multilocus Interactions Restrict Gene Intr ogression in Intersp ecific Populations of Polyploid Gossypium (Cotton). Evolution 54(3):798-814.

John, ME. 1996. Structural characterization of genes corresponding to cotton fiber mRNA, E6: Reduced E6 protein in transgenic plants by antisense gene. Plant Molecular Biology 30:297-306.

Kalasz H. 2003. Biological role of formalde hyde, and cycles related to methylation, demethylation and formaldehyde production. Mini Reviews in Medicinal Chemistry 3(3):

175-192.

Kämpfer, P. 2006. The family Streptomycetaceae , Part I: Taxonomy. Pages 538-604 in The Prokaryotes. A Handbook on the Biology of Bacteria: Archaea. Bacteria: Firmicutes, Actinomycetes. Volume 3. M Dworkin, S Fal kow, E Rosenberg, K-H Schleifer, and E Stackebrandt (eds.). Springer+ Busine ss Media, LLC., New York, New York.

Kaneko T, Y Nakamura, S Sato, K Minamisa wa, T Uchiumi, S Sasamoto, A Watanabe, K Idesawa, M Iriguchi, K Kawashima, M Kohara, M Matsumoto, S Shimpo, H Tsuruoka,32 T Wada, M Yamada and S Tabata. 2002. Comple te genomic sequence of nitrogen-fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Research 9: 189–197.

Kay, R, A Chan, M Daly and J McPh erson. 1987. Duplication of CaMV 35 S promoter sequences creates a strong enhancer for plant genes. Science 236:1299-1302.

Keeler, K. 1989. Can Genetically Engineer ed Crops Become Weeds? Bio/Technology

7 :1134-1139.

Keeling, JW., JD Reed, DM Petty and BA Brown. 2009. Control of volunteer Roundup Ready Flex cotton with postemergence herb icides. Page 1333 in 2009 Beltwide Cotton Conferences, San Antonio, Texas.

Keese P. 2008. Risks from GMOs due to horizontal gene transfer. Environmental Biosafety Research 7:123-149.

Kerby, TA, FM Bourland and KD Hake. 2010. Physiologyical rationales in plant monitoring and mapping. Pages 304-317 in Physiology of Cotton. JM Stewart, DM Oosterhuis, JJ Heitholt, and JR Mauney (eds.). Springe r Science+Business Media, Dordrecht,

Netherlands.

Klee HJ, YM Muskopf and CS Gasser. 1987. Cloning of an Arabidopsis thaliana gene encoding 5-enolpyruvylshikimate-3-phospha te synthase: Sequence analysis and manipulation to obtain glyphosate-tolerant plants. Molecular a nd General Genetics

210:437-442.

Kong, G, MK Daud and S Zhu. 2010. Effects of pigment glands and gossypol on growth, development and insecticide-re sistance of cotton bollworm ( Heliothis armigera (Hubner)). Crop Protection 29:813-819.

Koonin EV, KS Makarova and L Aravind. 2001. Ho rizontal gene transf er in prokaryotes: Quantification and classification. A nnual Review of Microbiology 55:709-742.

Krueger JP, RG Butz, YH Atallah and DJ Cork. 1989. Isolation and identification of microorganisms for the degradation of di camba. Journal of Agricultural and Food Chemistry 37:534-538.

Kutzner, HJ. 1981. The family streptomycetaceae. Pages 2028-2090 in The Prokaryotes: A Handbook on Habitats, Isolati on, and Identification of Bact eria. Volume 2. MP Starr, H Stolp, HG Trüper, A Balo ws, and HG Schlegel (eds.) . Springer-Verlag, Berlin, Germany.

Ledbetter, K. 2011. AgriLife extension specialis t: Cotton planting should be delayed in times of drought. AgriLife Today, Texas A&M AgriLife, May 3, 2011. Available at33 http://today.agrilife.org/2011/05/ 03/agrilife-extension-specia list-cotton-planting-shouldbe-delayed-in-times-of-drought/ , accessed November 5, 2012.

Llewellyn, D, C Tyson, G Constable, B D uggan, S Beale, and P Steel. 2007. Containment of regulated genetically modified cotton in the field. Agriculture, Ecosystems & Environment 121(4):419-429.

Locci, R. 1989. Streptomycetes and related genera. Pages 2451-2508 in Bergey's Manual of Systematic Bacteriology. Volume 4. ST Williams and ME Sharpe (eds.). Williams & Wilkins, Baltimore, Maryland.

Maiti IB and RJ Shepherd. 1998. Isolation and expression analysis of peanut chlorotic streak caulimovirus (PCISV) full-length transcri pt (FLt) promoter in transgenic plants. Biochemical and Biophysical Resear ch Communications 244:440-444.

Martinez, C, J-C Baccou, E Bresson, Y Baissac, J-F Daniel, A Jalloul, J-L Montillet, J-P Geiger, K Assigbetse and M Nicole. 2000. Salicy lic acid mediated by the oxidative burst is a key molecule in local and systemic re sponses of cotton challe nged by an avirulent

race of Xanthomonas campestris pv malvacearum . Plant Physiology 122:757-766.

McClelland, MR, KL Smith and JK Nors worthy. 2006. Managing glyphosate-resistant horseweed in conservation-tillage co tton production: Final summary and

recommendations. AAES Research Series 552. University of Arkansas Agricultural Experiment Station, Fa yetteville, Arkansas.

McGregor, SE. 1976. Insect Pollination of Cultivated Crop Plants. A. R. S. U.S. Department of Agriculture. Washingt on, DC, Government Printing Office.

Miki, B and S McHugh. 2004. Selectable marker ge nes in transgenic pl ants: applications, alternatives and biosafety. Journal of Biotechnology 107:193-232.

Monsanto Company. 2010. Petition for the Dete rmination of Nonregulated Status for Dicamba-Tolerant Soybean MON 87708. Submitted by R. Mannion, Monsanto Company, St. Louis, Missouri, 721 p p. Retrieved on November 26, 2012 from http://www.aphis.usda.gov/ biotechnology/not_reg.html

Monsanto Company. 2004. Petition for the Dete rmination of Nonregulated Status for MON 88017 Corn. Submitted by Ravinder Si nhu, Monsanto Company, St. Louis, Missouri, 278 pp. Retrieved on November 26, 2012 from http://www.aphis.usda.gov/ biotechnology/not_reg.html

.

Monsanto Company. 2012. Petition for the Dete rmination of Nonregulated Status for Dicamba and Glufosinate-Tolerant Cotton MON 88701. Submitted by M. Malven, Monsanto Company, St. Louis, Missouri, 620 pp. Retrieved on May 28, 2012 from http://www.aphis.usda.gov/ biotechnology/not_reg.html34 Morgan, GD, DA Fromme, PA Baumann, J Gric har, B Bean, ME Matocha and DA Mott. 2011. Managing volunteer cotton in grain crops (corn, sorghum, soybean, and wheat). Texas AgriLife Extension Service, Texas A&M University. Available at http://publications.tamu.edu/COTTON/PU B_cotton_Managing%20Volunteer%20Cotton %20in%20Grain%20Crops.pdf , accessed November 5, 2012.

Muenscher, WC. 1980. Weeds. Cornell University Press, New York and London.

Münster, P and AM Wieczorek. 2007. Potential gene flow from agricultural crops to native plant relatives in the Hawaiian Islands. Agriculture, Ecosystems & Environment 119(1-2):1-10.

National Cotton Council. 2011. National Cott on Council Disease Da tabase. Retrieved November 26, 2012 from http://www.cotton.org/tech/pest/

.

Niepel M and DR Gallie. 1999. Identification and characterization of the functional

elements within the tobacco etch virus 5' l eader required for cap-independent translation. Journal of Virology 73:9080-9088.

Odell, JT, F Nagy and N-H Chua. 1985. Iden tification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810-812.

OECD. 1999. Consensus document on general information concerning the genes and their enzymes that confer tolera nce to phosphinothricin herbicide. ENV/JM/MONO(99)13. Organisation for Econom ic Co-operation and Development, Paris, France, 26 pp. At http://www.oecd.org/scien ce/biosafety-biotrack/46815618.pdf

, accessed November 2, 2012.

OECD. 2002. Module II: Herbicid e biochemistry, herbicide metabolism and the residues in glufosinateammonium (Phosphinothric in)-tolentant transgenic plants. ENV/JM/MONO(2002)14. Organisation for Economic Co-operation a nd Development, Paris, France, 22 pp. At http://www.oecd.org/scien ce/biosafety-biotrack/46815748.pdf , accessed November 2,

2012.

OECD. 2008. Consensus document on the biology of cotton ( Gossypium spp.). ENV/JM/MONO(2008)33. Organisation for Econom ic Co-operation and Development, Paris, France, 64 pp. At http://www.oecd.org/scien ce/biosafety-biotrack/46815918.pdf

, accessed November 2, 2012.

OECD. 2009. Consensus document on the compositional considerations for new varieties of cotton ( Gossypium hirsutum and Gossypium barbadense ): Key food and feed nutrients and anti-nutrients. ENV/JM/MONO(2004)16. Or ganisation for Economic Co-operation and Development, Paris, France, 32 pp. At http://www.oecd.org/science/biosafetybiotrack/46815236.pdf , accessed November 2, 2012.35 OGTR. 2008. The Biology of Gossypium hirsutum L. and Gossypium barbadense L. (cotton), version 2. Department of Health and Ageing, Office of the Gene Technology Regulator, Government of Australia. Available at http://www.ogtr.gov.au/internet/ogt r/publishing.nsf/content/cotton3/$FILE/biologycotton08.pdf , accessed November 5, 2012.

Palleroni NJ and JF Bradbury. 1993. Stenotrophomonas , a new bacterial genus for Xanthomonas maltophilia (Hugh 1980) Swings et al. 1983. International Journal of Systematic Bacteriology 43(3):606-609.

Park S-W, P-P Liu, F Forouhar, AC Vlot , L Tong, L Tietjen and DF Klessig. 2009. Use of a salicylic acid analog to investigate the roles of methyl salicylate and its esterases in plant disease resistance. The Journa l of Biological Chemistry 284(11):7307-7317.

Percival, AE, JF Wendel and JM Stewar t. 1999. Taxonomy and germplasm resources. Pages 33-63 in Cotton: Origin, Histor y, Technology, and Production. W.C. Smith and J.T. Cothren (eds.). John Wiley & Sons, Inc., New York, New York.

Percy, RG and JF Wendel. 1990. Allozyme eviden ce for the origin and diversification of Gossypium barbadense L. Theoritical and Applied Genetics 79:529-542.

Peterson, C.D., Pearman, D. A., and Dines, T. D. 2002. New Atlas of the British flora. Oxford University Press, London, U.K.

Pleasants, JM and JF Wendel. 2005. Assessm ent of potential for gene flow between transgenic cotton and the endemic Hawa iian cotton - Final Report. EPA Grant R83032901.

Pleasants, JM and JF Wendel. 2010. Reproduc tive and Pollination Biology of the Endemic Hawaiian Cotton, Gossypium tomentosum (Malvaceae). Pacific Science 64(1):45-55.

Rani, PR and P Rajasekharreddy. 20 10. Insecticidal activity of (2 n -octyl cyclo prop-1- enyl)-octanoic acid (I) against three co leopteran stored product insects from Sterculia foetida (L.). Journal of Pest Science 83:273-279.

Rensing SA and U-G Maier. 1994. Phylogenetic analysis of the stress-70 protein family.

Journal of Molecula r Evolution 39:80-86.

Rieseberg LH. 1997. Hybrid origins of plan t species. Annual Review of Ecology and Systematics 28:359-389.

Rieseberg LH and JF Wendel. 1993. Introgress ion and its consequences in plants. pp. 70-

109. In RG Harrison (ed), Hybrid Zones and the Evolutionary Process. Oxford, U.K.: Oxford University Press.36 Roberts, G, S Kerlin and M Hickman. 2002. C ontrolling volunteer cotton. Page F4 in WEEDpak – A Guide for Integrated Management of Weeds in Cotton. Cotton Catchment Communities CRC, Australian Cotton Research Institute, Narrabri, New South Wales.

Retrieved from http://www.cottoncrc.org.au/in dustry/Publications/Weeds/ WEEDpak/F1_Farm_Hygiene , November 5, 2012.

Rodoni S, W Mühlecker, M Anderl, B Kräutle r, D Moser, H Thomas, P Matile and S Hörtensteiner. 1997. Chlorophyll breakdown in senescent chloroplasts: Cleavage of pheophorbide α in two enzymic steps. Plant Physiology 115:669-676.

Saha, S, DA Raska and DM Stelly. 2006. Upland Cotton ( Gossypium hirsutum L.) x Hawaiian Cotton ( G. tomentosum Nutt. ex Seem.) F 1 Hybrid Hypoaneuploid

Chromosome Substitution Series. Journal of Cotton Science 10:263-272.

Salomon S and H Puchta. 1998. Capture of genomic and T-DNA sequences during double-strand break repair in somatic pl ant cells. The EM BO Journal 17:6086-6095.

Sardi E, J Velich and J Szarka. 1996. The pr esence of Selye’s stre ss-syndrome in the Snap Bean – Halo Blight relationship. Re ports of Bean Improvement Cooperative and National Dry Bean Council Research Conference, Annual Report 39:278-279.

Silverman FP, PD Petracek, DF Heima n, CM Fledderman and P Warrior. 2005. Salicylate activity. 3. Structure relationship to systemic acquired resistance. Journal of Agricultural and Food Chemistry 53(25):9775-9780.

Soltis DE and PS Soltis. 1993. Molecular data and the dynamic nature of polyploidy.

Critical Reviews in Plant Sciences 12:243-273.

Stace, C.A. 1987. Hybridization and the plant species. Pp. 115–127 in K.M. Urbanska (ed.) Differentiation Patterns in Higher Plants. Academic Press, New York.

Szende B and E Tyihak. 2010. Effect of formaldehyde on cell proliferation and death. Cell Biology Intern ational 34:1273-1282.

Thaler, JS, AA Agrawal and R Halitschke. 2010. Salicylate-mediated interactions between pathogens and herb ivores. Ecology 91(4):1075-1082.

Thaler, JS, PT Humphrey and NK White man. 2012. Evolution of jasmonate and salicylate signal crosstalk. Trends in Plant Sciences 17(5):260-270.

Thompson, A and L Steckel. 2010. Controlli ng volunteer cotton in soybeans. W168. University of Tennessee Extension, Me mphis, Tennessee. Retrieved from https://utextension.tennessee.e du/publications/Documents/W168.pdf , accessed November 5, 2012.37 Thompson, CJ, NR Movva, R Tizard, R Cr ameri, JE Davies, M Lauwereys and J Botterman. 1987. Characterization of the herbicide-resistance gene bar from Streptomyces hygroscopicus . EMBO Journal 6:2519-2523.

Umbeck, PF, KA Barton, EV Nordheim, JC Mc Carty, WL Parrott and JN Jenkins. 1991. Degree of pollen dispersal by insects from a fi eld test of genetically engineered cotton. Journal of Economic Entomology 84:1943-1950.

University of California. 1996. Development and growth requirements of the cotton plant. Pages 11-23 in Integrated Pest Mana gement for Cotton in the Western Region of

the United States. Publication 3305. Second Edit ion. University of California, Oakland,

California.

USDA-APHIS. 2012. United States Department of Agriculture, Animal Plant Health Inspection Service. Federal Noxious Weed Li st as of 2/1/2012. Retrieved November 5,

2012 from http://www.aphis.usda.gov/plan t_health/plant_pest_info/w eeds/downloads/weedlist.pdf

USDA-ERS-FAS. 2010. Outlook for U.S. agricultur al trade: FY 2010 exports expected to rise to $104.5 billion; Impor ts drop to $76.5 billion. U.S. Department of Agriculture, Economic Research Service and Foreign Ag ricultural Service, Washington, D.C. http://www.fas.usda.gov/cmp/outl ook/2010/May-10/May2010_fullreport.pdf , accessed November 5, 2012.

USDA-NASS. 2012a. United States Department of Agriculture, National Agricultural Statistics Service. Crop Production, pp. 14- 15. Retrieved on November 5, 2012 from http://usda01.library.cornell.edu/usda /nass/CropProd//2010s /2012/CropProd-05-10- 2012.pdf

.

USDA-NASS. 2012b. United States Department of Agriculture, National Agricultural

Statistics Service. Nationa l Statistics for Cotton. Cotton County Maps: Planted Acreage By County. Retrieved on November 26, 2012 from http://www.nass.usda.gov/Statistics_ by_Subject/result.php?6 AC9DE2A-0B22-34E4-

BC77- 4797B1AA5DAD§or=CROPS&group= FIELD%20CROPS&comm=COTTON

USDA-NRCS. 2012a. Plant profile for Gossypium hirsutum L. The PLANTS Database. National Plant Data Team, Greensboro, NC. At http://plants.usda.gov , accessed November 26, 2012.

USDA-NRCS. 2012b. United States Department of Agriculture, Natural Resources

Conservation Service. Invasi ve and Noxious Weeds. At http://plants.usda.gov/java /invasiveOne?startChar=G , accessed November 26, 2012.38 USDA-NRCS. 2012c. United States Departme nt of Agriculture, Natural Resources Conservation Service. Native, Invasive, and Other Plant-Related Definitions. At http://www.ct.nrcs.usda. gov/plant_definitions.html , accessed November 19, 2012.

USDA-NRCS. 2012d. Plant profile for Gossypium barbadense . The PLANTS Database. National Plant Data Team, Greensboro, NC. At http://plants.usda.gov , accessed November 26, 2012.

USDA-NRCS. 2012d. Plant profile for Gossypium thurberi . The PLANTS Database. National Plant Data Team, Greensboro, NC. At http://plants.usda.gov , accessed November 26, 2012.

USDA-NRCS. 2012f. Plant profile for Gossypium tomentosum . The PLANTS Database. National Plant Data Team, Greensboro, NC. At http://plants.usda.gov , accessed November 26, 2012.

U.S. EPA. 1997. Phosphinothricin acetyltransf erase and the genetic material necessary for its production in all plants; Exemption fr om the requirement of a tolerance on all raw agricultural commodities. Federal Register 62:17717-17720.

U.S. EPA. 2003. Memorandum: Glufosinate ammonium (PC code 128850). Section 3 registrations for transgenic cotton and cotton (ID# - 0F06140), tr ansgenic rice (ID# - 0F06210), and bushberry (ID# - 2E06404). Human h ealth risk assessment. DP barcode:

D290086. Case number: 293386. Submission: S635308. 40 CFR 180.473. U.S. Environmental Protection Agency, Office of Pevention, Pesticides, and Toxic Substances, Washington, D.C.

U.S. EPA. 2008. Glufosinate final work pl an registration revi ew: August 2008. Docket Number: EPA-HQ-OPP-2008-0190. U.S Environm ental Protection Agency, Washington,

D.C.

U.S. EPA. 2009. Environmental Protection Ag ency. Reregistration Eligibility Decision for Dicamba and Associated Salts (Amended). EPA, Office of Prevention, Pesticides and Toxic Substances, Washington, D.C. Retrieved on May 29, 2012 from http://www.regulations.gov/#!docum entDetail;D=EPA-HQ-OPP-2005-0479-0026

U.S. FDA. 1998. Guidance for industry: use of antibiotic resistance marker genes in transgenic plants. Draft guidance. US F ood and Drug Administration, Center for Food Safety and Applied Nutrition, Center fo r Veterinary Medicine. September 4, 1998.

Available at http://www.fda.gov/Food/GuidanceComplianc eRegulatoryInformation/GuidanceDocume nts/Biotechnology/ucm096135.htm , accessed November 5, 2012.

U.S. FDA. 2011. Food and Drug Administration, Office of Food Additive Safety, Center for Food Safety and Applied Nutrition. Co mpleted Consultations on Bioengineered Foods: BNF No. 125. Retrieved on May 29, 2012 from39 http://www.fda.gov/Food/Biotec hnology/Submissions/ucm282993.htm and http://www.fda.gov/Food/Biotec hnology/Submissions/ucm282994.htm

Van Deynze, AE, RB Hutmacher and KJ Bradford. 2011. Gene flow between Gossypium

hirsutum L. and Gossypium barbadense L. is asymmetric. Crop Science 51:298-305.

Van Deynze, AE, FJ Sundstrom, and KJ Br adford. 2005. Pollen-Mediated Gene Flow in California Cotton Depends on Pollinator Activity. Crop Science 45(4):1565-1570.

Vencill W, RL Nichols, TM Webster, J Soteres, C Mallory-Smith, NR Burgos, WG

Johnson and MR McClelland. 2012. Herbicide re sistance: Toward an understanding of resistance development and the impact of he rbicide-resistant crops. Weed Science 60 (Special Issue). Available at http://www.wssajournals.o rg/doi/pdf/10.1614/WS-D-11-

00206.1 , accessed May 29, 2012.

Vlot AC, DA Dempsey and DF Klessig. 2009. Salicylic acid, a multifaceted hormone to

combat disease. Annual Review of Phytopathology 47:177:206.

Wang X-Z, B Li, PL Herman and DP Week s. 1997. A three-component enzyme system catalyzes the O demethylation of the herbicide dicamba in Pseudomonas maltophilia DI6. Applied and Environmenta l Microbiology 63(4):1623-1626.

Webster, TM and RL Nichols. 2012. Changes in the prevalence of weed species in the major agronomic crops of the southern United States: 1994/1995 to 2008/2009. Weed Science. 60(2):145-157. Available at http://www.bioone.org/doi/full/10.1614/WS-D-11-

00092.1 , accessed November 2, 2012.

Wehrmann, A, AV Vliet, C Opsomer, J Botte rman and A Schulz. 1996. The similarities

of bar and pat gene products make them equally applicable for plant engineers. Nature Biotechnology 14:1274-1278.

Wendel, JF, CL Brubaker and AE Pe rcival. 1992. Genetic Diversity in Gossypium

hirsutum and the Origin of Upland Cotton. Am erican Journal of Botany 79(11):1291-

1310.

WHO-IPCS. 1989. World Health Organization, International Programme on Chemical Safety. Environmental health criteria 89: Formaldehyde. Retrieved on June 24, 2012 from http://www.inchem.org/documents/ehc/ehc/ehc89.htm

Williams, MR. 2007. Cotton Losses – 2007. Table 7 – Summary of all states. Mississippi State University Extension Service, Mississi ppi State University. Retrieved on November 20, 2012 from http://www.entomology.msstate.edu/ resources/tips/cotton-losses/data/

.

Williams, MR. 2008. Cotton Losses – 2008. Table 6 – Summary of all states. Mississippi State University Extension Service, Mississi ppi State University. Retrieved on November 20, 2012 from http://www.entomology.msstate.edu/ resources/tips/cotton-losses/data/

.40 Williams, MR. 2009. Cotton Losses – 2009. Table 8 – Summary of all states. Mississippi State University Extension Service, Mississi ppi State University. Retrieved on November 20, 2012 from http://www.entomology.msstate.edu/ resources/tips/cotton-losses/data/

.

Williams, MR. 2010. Cotton Losses – 2010. Table 8 – Summary of all states. Mississippi State University Extension Service, Mississi ppi State University. Retrieved on November 20, 2012 from http://www.entomology.msstate.edu/ resources/tips/cotton-losses/data/

.

Williams, MR. 2011. Cotton Losses – 2011. Table 8 – Summary of all states. Mississippi State University Extension Service, Mississi ppi State University. Retrieved on November 20, 2012 from http://www.entomology.msstate.edu/ resources/tips/cotton-losses/data/

.

Winter, J, R Wright, N Duck, C Gasser, R Fraley and D Shah. 1988. The inhibition of petunia hsp70 mRNA processing during CdCl 2 stress. Molecular and General Genetics

211:315-319.

Wood DW, JC Setubal, R Kaul, DE Monks, JP Kitajima et al. 2001. The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294:2317-2323.

WSSA. 1998. Weed Science Society of Ameri ca. Resistance and to lerance definitions. Weed Technology 12(4):789. Retr ieved on August 10, 2012 from http://www.wssa.net/Weeds/Resistance/definitions.htm

Wunderlin, RP and BF Hansen. 2008. Atlas of Florida Vascular Plants, [SM Landry and KN Campbell (application development), Fl orida Center for Co mmunity Design and Research.] Institute for Systematic Bota ny, University of South Florida, Tampa.

Yang M, E Wardzala, GS Johal and J Gray. 2004. The wound-inducible Lls1 gene from maize is an orthologue of the Arabidopsis Acd1 gene, and the LLS1 protein is present in non-photosynthetic tissues. Plant Molecular Biology 54: 175-191.

York, AC, AM Stewart, PR Vidrine and AS Culpepper. 2004. Control of volunteer glyphosate resistant cotton in glyphosate-r esistant soybean. Weed Technology 18:532-

539.

Yoshida S, S Maruyama, H Nozaki and K Shir asu. 2010. Horizontal gene transfer by the parasitic plant Striga hermonthica. Science 328:1128.

Zarate, SI, LA Kempema and LL Walling. 2007. S ilverleaf whitefly induces salicylic acid defense and suppresses effectual jasm onic acid defenses. Plant Physiology 143:866-

875.

Zhang, B-H, X-P Pan, T-L Guo, Q-L Wang and TA Anderson. 2005. Measuring gene flow in the cultivation of transgenic cotton ( Gossypium hirsutum L.). Molecular Biotechnology 31:11-20.41 Zhang, P, X Zhu, F Huang, Y Liu, J Zha ng, Y Lu and Y Ruan. 2011. Suppression of jasmonic acid-dependent defense in cotton plant by the mealybug Phenacoccus solenopsis . PLoS ONE 6(7): e22378. doi:10.1371/journal.pone.0022378.