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regime.and and protectionpublishing data and/orcontents property its AG therefore or regulatory may Bayer a of intellectual reproduction document as parties.under document fall this property such third of this owner. the may distribution,of and use its is rights affiliates. of to and its owner of owner document rights the publication, the documentany subject of of this the be any This exploitation and/ormay rights violate It copy permissionand Furthermore, commercialthe Consequently, any withoutprohibited be regime.and and protectionpublishing data and/orcontents property its AG therefore or regulatory may Bayer a of intellectual reproduction document as parties.under document fall this property such third of this owner. the may distribution,of and use its is rights affiliates. of to and its owner of owner document rights the publication, the documentany subject of of this the be any This exploitation and/ormay rights violate It copy permissionand Furthermore, commercialthe Consequently, any withoutprohibited be regime.and and protectionpublishing data and/orcontents property its AG therefore or regulatory may Bayer a of intellectual reproduction document as parties.under document fall this property such third of this owner. the may distribution,of and use its is rights affiliates. of to and its owner of owner document rights the publication, the documentany subject of of this the be any This exploitation and/ormay rights violate It copy permissionand Furthermore, commercialthe Consequently, any withoutprohibited be Petition for the Determination of Nonregulated Status for ® Roundup Ready Flex Cotton MON 88913 Summary The Animal and Plant Health Inspection Service (APHIS) of the United States Department of Agriculture (USDA) has responsibility, under the Plant Protection Act (7 CFR U.S.C. 7701-7772), to prevent the introduction and/or dissemination of plant pests into the United States or interstate introduction and/or dissemination. The APHIS regulations, (7 CFR § 340.6) provide that an applicant may petition APHISregime. to evaluateand submitted data to determine that a particular regulated article does not present a plant pest risk and should no longer be regulated. If APHIS determines that the regulated article does not present a plant pest risk, the petition is granted, thereby allowing unrestricted introduction of the article. and protectionpublishing Since the commercialization of the first cotton varieties withdata biotechnology traits in the contents mid-1990s, one of the most successful in terms of farmer adoptionand/or has been Roundup ® property its AG therefore Ready cotton event 1445 (hereinafter referred to as Roundup Readyor cotton), which is tolerant to glyphosate, the active ingredient in Roundup® agricultural herbicides. Roundup Ready cotton, the first-generation herbicide-tolerantregulatory cottonmay product from Bayer a Monsanto, was commercialized in the U.S. in 1997. Roundup Ready cotton has been of intellectual reproduction widely adopted by cotton farmers and has made up adocument significant portion of the U.S. cotton production. Cotton varietiesas parties. contunderaining the Roundup Ready trait are currently document cultivated on more than 7.8 million fallacres annuallythis within the U.S. (USDA-NASS, property such third of 2003a). this owner. the may distribution,of and use its is rights Monsantoaffiliates. Company has now developed a second-generationof glyphosate-tolerant cotton product, Roundupto Ready Flex cottonand MON 88913, that provides increased tolerance to its owner glyphosateof duringowner the critical reproductive phases of growth compared to Roundup document rights Ready cotton.the Use of MONpublication, 88913 will the enable the application of a Roundup agricultural documentany subject of herbicide overof thethis top of the cotton cropthe at later stages of development than is possible be any This with the current product.exploitation This will allow for effective weed control during crop production, because Roundup agricultural herbicides are highly effective against the and/ormay rights violate It majority of annual and perennial weeds that can be problematic during the later stages of copycrop development, withpermission minimaand l risk of crop injury. The increased level of glyphosate toleranceFurthermore, in MON 88913 is achieved through the use of improved promoter sequences commercialthe thatConsequently, regulate the expression of the cp4 epsps coding sequence. any MON 88913without wasprohibited developed using the same cp4 epsps coding sequence and chloroplast targeting sequencebe and produces the same CP4 EPSPS protein (5-enolpyruvylshikimate- 3-phosphate synthase) as Roundup Ready cotton. The transformation methodology used to produce MON 88913, Agrobacterium tumefaciens-mediated plant transformation, is ® Roundup and Roundup Ready are registered trademarks of Monsanto Technology LLC. 04-CT-112U Page 4 of 239 comparable to the method used in development of Roundup Ready cotton. MON 88913 utilizes the same cp4 epsps coding sequence in the same crop, and confers the same glyphosate-tolerant phenotype as the nonregulated commercial Roundup Ready cotton product. The data presented in this petition characterize the nature and stability of the genetic modification, characterize the CP4 EPSPS protein produced in MON 88913 and demonstrate that MON 88913 is not phenotypically different than the negative segregant control, MON 88913(-). As determined by Southern blot analysis, MON 88913 contains a single, intact DNA insert from the binary plasmid PV-GHGT35 at a single integration locus within the cotton genome. The DNA insert in MON 88913 contains two intact cp4 epsps gene expression cassettes containing identical cp4 epsps coding sequences. Polymerase chain regime.and reaction was performed to confirm the 5’ and 3’ insert-to-genomic DNA junctions and the organization of the elements within the insert in MON 88913. The DNA insert and the Roundup Ready trait are stable across multiple sexual generations. Phenotypic segregation data confirmed that the single insert locusand and Roundupprotection Readypublishing trait behave as a single dominant locus with the expected Mendelian segregation pattern across multiple generations. data contents and/or property its The production of CP4 EPSPSAG protein was confirmed by determining thereforethe level of CP4 or EPSPS in tissues produced under field conditions. The levels of CP4 EPSPS protein were determined by validated enzyme-linked immunosorbentregulatory assaymay methods in leaf tissue Bayer sampled throughout the growing season, as wella as in root, pollen and seed. The cotton of intellectual reproduction plants were tolerant to over-the -top applications of adocument Roundup agricultural herbicide at later stages of development thanas isparties. possiunderble with cotton varieties that contain the Roundup Ready 1445 event. this document property fall such third of this owner. The CP4the EPSPS protein producedmay in distribution, MON 88913of is targeted to the chloroplasts via an N- and use its terminalis fusion withrights the chloroplast transit peptide, CTP2, to form a CTP2-CP4 EPSPS affiliates. of precursor protein.to The precursor proteinand produced in the cytoplasm is processed to its owner removeof the transitowner peptide upon translocation into the plant chloroplast, resulting in the document rights mature CP4 EPSPSthe protein.publication, The identity the of the plant-produced protein was confirmed documentany subject of using dataof from this western blot analysis andthe N-terminal sequence analysis. On the basis of be any This western blot analysis, theexploitation electrophoretic mobility and immunoreactive properties of the plant-produced CP4 EPSPS protein were found to be equivalent to those of the E. coli- and/ormay rights violate It produced CP4 EPSPS reference standard protein. copy permissionand SafetyFurthermore, assessment of the CP4 EPSPS protein included protein characterization commercialthe demonstratingConsequently, the lack of similarity to known allergens and toxins, the long history of safe consumptionany of similar EPSPS proteins from a variety of food sources, CP4 EPSPS digestibilitywithout in vitroprohibited , and the lack of acute oral toxicity in mice. These data will be presented tobe the U.S. FDA for evaluation of food and feed safety for MON 88913 as part of the pre-market consultation process for products of modern biotechnology. The phenotypic evaluation of MON 88913 included the key agronomic characteristics of seed germination, plant growth and development under field conditions, crop productivity 04-CT-112U Page 5 of 239 and harvest quality. These agronomic characteristics were evaluated by replicated field observations at 14 locations in 11 states across the U.S. cottonbelt to evaluate whether the presence of the DNA insert or the presence of the CP4 EPSPS protein altered the phenotypic characteristics and/or ecological interactions of MON 88913. In these evaluations, MON 88913 was compared to MON 88913(-), a negative segregant of MON 88913 that contains similar background genetics but does not contain the DNA insert. No differences were detected for 439 of a total of 458 comparisons between MON 88913 and MON 88913(-) by field location at p≤0.05. The majority of the 19 differences that were observed occurred for a single characteristic at a single field location. When all data were pooled across locations, only a single statistically significant difference in the growth and development