# Genective Petition (11-342-01p) for Determination of Non-regulated Status of Event VCO-Ø1981-5 Corn

OECD Unique Identifier: VCO-Ø1981-5



## Plant Pest Risk Assessment

May 2013

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

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

B. Development of Event VCO-Ø1981-5 Corn .............................................................. 1

C. Potential for Event VCO-Ø1981-5 Corn to have Altered Disease and Pest Susceptibilities ........................................................................................................ 4

D. Potential for Enhanced Weediness of Event VCO-Ø1981-5 Corn ............................. 8

E. Potential to Impacts the Weediness of Other Plants with which It Can
Interbreed .............................................................................................................. 10

F. Potential Impacts on Non-target Organi sms, Including Those Beneficial to Agriculture ............................................................................................................ 11

G. Potential Impacts from Transfer of Genetic Information to Organism with which Event VCO-Ø1981-5 Corn Cannot Interbreed .......................................... 13

H. Potential Changes to Agriculture or Cultivation Practices ....................................... 14

I. Conclusion ................................................................................................................ 15

J. References ................................................................................................................. 151 

### A. Introduction

Genective SA in collaboration with Athenix Co rp., now an affiliate of Bayer CropScience LP, has developed herbicide-tolerant Even t VCO-Ø1981-5 corn. Gen ective SA (referred to as “Genective” hereafter) has petitione d APHIS (USDA-APHIS Petition Number #11- 342-01p) for a determination that gene tically engineered (GE) corn ( Zea mays ) Event VCO-Ø1981-5 is unlikely to pose a plant pest risk and, therefore, is no longer a regulated article under regulat ions at 7 CFR part 340. APHI S administers 7 CFR part 340 under the authority of the pl ant pest provisions of the Plant Protection Act of 2001 1 . The Genective petition request for Event VCO- Ø1981-5 corn was originally received on December 8, 2011. A revised version of th e petition, received March 28, 2012, was deemed technically complete and is the ve rsion of the petition referenced in this document. This plant pest risk assessmen t was conducted to determine whether Event VCO-Ø1981-5 corn is likely to pose a plant pest risk.



### B. Development of Event VCO-Ø1981-5 Corn

Corn ( Zea mays ) is among the most important crops in the United States, with more than 96 million acres cultivated for all purposes in 2012(USDA-NASS 2012). The United States is the larges t producer of corn in the worl d (USDA-ERS 2010). Corn has been used by U.S. farmers as the primary feed gr ain, accounting for more than 90 percent of the total value of feed grains (USDA-ERS 2010). Corn is also considered a major biofuel crop in the U.S. Recent strong demand for et hanol production has resulted in increased corn demand and higher corn prices and has prov ided incentives to increase corn acreage. USDA has projected that U.S. corn cultiva tion may continue to increase in the coming year (USDA-NASS 2012). Most re cently in the U.S., farmers planted 88 percent of their acreage with biotechno logy-derived varieties. Biotec hnology-derived traits in these genetically engineered (GE) varieties consist of: 21% herb icide tolerance, 15% insect
resistance and 52% stacked varieties contai ning both insect and herbicide resistance (USDA-NASS 2012).

Weed competition can be a major limiting factor in corn production leading to significant yield and quality reductions by direct compe tition for light, water and nutrients in the
soil. Late-season weed infestations do not reduce corn yield nearly as much as earlyseason weed competition; however, late-season w eeds can harbor destructive insect pests (UC-IPM 2009). In 2011, approximately 72% of corn planted in the U.S. possessed
tolerance to an herbicide that was co nferred through biotech nology (USDA-ERS 2011). The primary herbicide tolera nce trait in use has been glyphosate tolerance, and the adoption of this trait in soybeans is even higher (94%). The wide-spread adoption of herbicide –tolerant crops, es pecially GE-derived glyphosate- tolerant crops, has changed the approach that farmers take to avoid yi eld losses from weeds (Duke and Powles, 2009; Gianessi, 2008). Most transgenic herbicide tolerant corn has b een developed by the

1 Section 403 (14) of the Plant Protection Act (7USC Sec 7702(14) defi nes plant pest as: “Plant Pest - The term “plant pest” mean s any living stage of any of the fo llowing that can directly or i ndirectly injure, cause damage to , or cause disease in any plant or plant product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic pl ant. (D) A bacterium. (E) A fungus. (F) A virus or viroid. ( G) An infectious agent or other pathogen. (H) Any article similar to or allied with any of the ar ticles specified in the preceding subparagraphs.”2 insertion of genes that transfer toleran ce for glyphosate, whic h expands the control options for annual and perennial grass and broadleaf weeds.

Glyphosate ( N -(phosphonomethyl) glycine) is a broad-spectrum sy stemic herbicide used to kill weeds. It is regist ered with the Environmental Protection Agency (EPA) for nonselective weed control for both non-food use and food use plants. Glyphosate's mode of action is to inhibit the enzyme 5-enol pyruvylshikimate-3-phosphate synthase (EPSPS) involved in the synthesis of the aromatic amino acids: tyro sine, tryptophan and phenylalanine. Glyphosate functions due to its resemblance to the structure of the substrate for EPSPS enzyme and thereby competing with this substrate for the enzyme’s active site, thus preventing the synthesis of ar omatic amino acids and killing the plant.

The first corn line containing a glyphosate to lerance trait produced through the use of biotechnology was granted nonregulated status in 1995 and since that time several other corn lines containing glyphosat e tolerance have also been granted nonregulated status by APHIS-USDA. APHIS BRS completed Plant Pest Risk Assessments (PPRA) and Environmental Assessments (EAs) for glyphosate to lerant corn in a num ber of petitions ( http://www.aphis.usda. gov/biotechnology/not_reg.html ). The EAs fully addressed all environmental areas of potential concern. In these petitions, APHIS concluded on the basis of the EA that the impacts from maki ng a determination of nonregulated status would not be significant. The agency issued Findings of No Significant Impact (FONSI) and made determinations of nonregulated st atus for each. The 5-enolpyruvylshikimate-3- phosphate synthase (EPSPS) proteins fr om various GE crops have completed consultations procedures by Office of Premar ket Approval, Center for Food Safety and Applied Nutrition, Food and Drug Administ ration (FDA) since 1994 (US-FDA 2013). To date, regulatory authorities in twelve countries have approved the environmental (commercial) release of at least 30 GE plan t lines that contain non-plant derived EPSPS proteins. These are represented in a total of seven plant species: Beta vulgaris L. (sugar beet), Brassica napus L. and Brassica rapa L. (oilseed rape and turnip rape, respectively, although both can be referred to as canola) Glycine max L. (soybean), Gossypium
hirsutum L. (cotton), Medicago sativa L. (alfalfa) and Zea mays L. (maize) (ILSI 2010).

Genective has developed corn Event VCO- Ø1981-5 which is de signed to provide growers with weed control through over-t he-top glyphosate application. Event VCOØ1981-5 corn expresses a version of 5-e nolpyruvylshikimate-3-phosphate synthase protein derived from the common soil bacterium Arthrobacter globiformis (Genective 2011, p. 34) that is resistant to glyphosate. Ge nective conducted conf ined field tests of Event VCO-Ø1981-5 corn in the United States and its territories since 2007 and in Europe and Canada since 2009. The agronom ic evaluations have been conducted by Genective scientists and data in the petiti on are from those confined field trials.

Description of the Modification—Genetic material inserted and protein produced

Genective corn Event VCO-Ø1981-5 was produced using Agrobacterium tumefaciensmediated transformation of the hybrid corn line Hi-II from Mai ze Genetics Cooperation Stock Center (Genective 2011, page 19). The A. tumefaciens strain LBA4404 that was used to develop Event VCO-Ø1981-5 wa s made non-pathogenic by removing tumor3 inducing (Ti) DNA sequences normally present in A. tumefaciens (Koncz and Schell 1986). The disarmed A. tumefaciens carried a binary plasmid vector pAG3541containing
the epsps greg23ace5 gene (Genective 2011, page 19). In vitro selection of
transformation events was based on tolera nce to the herbicide glyphosate which is rendered non-toxic to plant tissues by the pres ence of the EPSPS protein. The size of the T-DNA was 3730 base pairs (bp) and contained a single T- DNA delineated by left and right border regions in which there was one expression cassette: epsps grg23ace5

.

The epsps grg23ace5 expression cassette consisted of the following genetic elements (Genective 2011, pp. 23-25):

 Border sequences: Left and right border sequences contain 25 base pairs of DNA
from A. tumefaciens to facilitate T-DNA transfer (Depicker et al. 1982; Barker et al. 1983)

 ScUbi4 Promoter: Promoter regi on of the ubiquitin-4 gene from Saccharum officinarum L. (sugarcane) (Alber t and Wei 2003) that dire cts transcription in plant cells.

 ScUbi4 5’ UTR: 5’untranslated region of the ubiquitin-4 gene from Saccharum officinarum L. (sugarcane) (Alb ert and Wei 2003) that helps regulate gene expression

 ScUbi4 intron: Intron region of the ubiquitin-4 gene from Saccharum officinarum L. (sugarcane) (Albert an d Wei 2003) mAHAS chloropl ast transit peptide: Nterminal chloroplast transit pe ptide sequence derived from the Zea mays L. acetohydroxyacid synthase ( ahas ) gene (Fang et al . 1992) that direct s transport of the expressed protein to the chloroplast.

 epsps grg23ace5 coding sequence: Codon-optimized version of the epsps grg23ace5 gene from Arthrobacter globiformis encoding EPSPS ACE5 protein (Schouten et al . 2010)

 35S terminator: Terminator region of the 35S transcript of the Cauliflower Mosaic Virus, which terminates mRNA transc ription and induces polyadenylation (Gardner et al . 1981)

In addition to the above-mentioned genetic el ements, the inserted T-DNA also contains short noncoding intervening DNA sequences . The intervening sequences contain restriction enzyme recognition sites and are used for cloning purposes.

Data provided from Genective and reviewed by APHIS demonstrated:

 The T-DNA inserted into the corn genome is present at a single locus and contains a single copy of the inserted transgene (Genective 2011, Appendix 3);

 No region from the genetic elements out side of the T-DNA borders from the
transformation vector was inse rted (Genective 2011, Appendix3);

 A short deletion (21bp) in the Even t VCO-Ø1981-5 genome was created by insertion of the transgene. Genectiv e has identified 12 pot ential open reading frames (ORFs) created in Event VCO- Ø1981-5. From Bioinformatics analysis, no homologies with known allergen sequen ces were identified and no significant homologies with known t oxins or other harmful proteins were found;4  Short deletions at the extremities of th e T-DNA borders (22 bp on the right and 16 bp on the left borders) resulted in the insert size being 3692 bp;

 All inserted regulatory sequences and the organization of the inserted gene in Event VCO-Ø1981-5 are intact and identical to their original a rrangements in the donor plasmid pAG3541;

 The stability of the introduced genes was determined by event-specific and genespecific endpoint PCR analyses for four generations (Genective 2011, pp.26-29). The stability integration was further c onfirmed by the Mende lian inheritance of the T-DNA in Event VCO-Ø1981-5 corn over four generations (Genective 2011, p. 29)



### C. Potential for Event VCO-Ø1981-5 Corn to have Altered Disease and Pest Susceptibilities

USDA-APHIS assessed whether Event VCO-Ø1981- 5 corn is likely to have significantly increased disease and pest susceptibility. This asse ssment encompasses a thorough
consideration of the tr ansformation process, introduced genes and their genetic elements, and their expression products to cause interactions with pests and diseases.

Transformation Process

APHIS considered the potential for the transformation process to cause or aggravate disease symptoms in Event VCO-Ø1981-5 corn or other plants or to cause the production of plant pathogens. Wild type Agrobacterium tumefaciens carries a tumor-inducing (Ti) plasmid that can be transferred to broadl eaf plants and cause crown gall disease. Agrobacterium tumefaciens strain LBA4404 contains a disarmed Ti plasmid that is incapable of inducing tumor formation due to the deletion of the phytohormone genes originally present in the Agrobacterium plasmid (Koncz and Schell 1986). Event VCOØ1981-5 corn was produced by Agrobacterium tumefaciens -mediated transformation with plasmid pAG3541 (Genective 2011, p.19). This transformation process did not lead to crown gall disease in Event VCO-Ø 1981-5. Instead, in the T-DNA region a maizeoptimized epsps grg23ace5 gene and regulatory component s necessary for its expression in the corn genome were introduced. Agrobacterium -mediated plant transformation has been used widely for decades, has not been implicated in causing plant disease, and is highly unlikely to pose a plant pest risk.

Introduced genes and their genetic elements

APHIS reviewed Southern blot analysis data that demonstrates that Event VCO-Ø1981-5 corn regenerated from the transformation ev ent contains a single copy of the gene cassette (Genective 2011, Appendix 3, pp. 134- 135). No region from the backbone of plasmid pAG3541 was inserted (Genective 2011, Appendix 3, pp. 140-142). The stability
of the inserted DNA was also evaluated ove r four generations by using molecular and phenotypic methodologies (Genective 2011, Appe ndix 3, p. 139). Plant phenotypes were evaluated to ensure stability of the traits during the plant breeding process by spraying plants with the herbicide glyphosate. Mendeli an segregation of the inserted genes was analyzed using Chi-square analysis over 4 generations (Genective 2011, p. 28-29). Each5 generation of plants was treate d with glyphosate to eliminate those plants that were not herbicide-tolerant, reflecting a lack of inher itance of the epsps gre23ace5 gene. Table 5 (p. 29) of the Petition show s the expected and observed segregation for the four generations tested. There was no significant deviation from the expected 1:1 ratio.

Event VCO-Ø1981-5 corn expresses a modi fied 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). The native epsps gre23ace5 gene was isolated from Arthrobacter globiformis, a common soil-inhabiting bacterium. The native EPSPS GRG23 protein was modified for optimal expression in corn, in part, by changing ten amino acids to produce
EPSPS ACE5. EPSPS ACE5 is 97.6% identical and 98.5% homologous at the amino level to the originally isolated EPSPS GRG23 protein. EPSPS ACE5 encodes a 44.3 kDa (kilo Daltons) protein consisting of a single polypeptide of 402 am ino acids. The family of EPSPS proteins is ubiquitous in plants a nd microorganisms. EPSP synthase is a critical enzyme in the shikimate pathway mediating the biosynthesis of aromatic compounds in plants, bacteria and fungi. This pathway is absent in mammals, fis h, birds, reptiles and insects (Alibhai and Stallings 2001). EPSPS proteins have been extensively studied (ILSI 2010) and not found to cause disease or the prod uction of infectious agents in plants. A safety assessment of EPSPS ACE5 protein was submitted by Athenix to FDA in 2009 for the Early Food Safety Evaluation. FDA complete d its review with no further questions in 2010 (US-FDA 2010).

The terminator for the epsps gre23ace5 gene is derived from cauliflower mosaic virus (CaMV) which is a plant viral pathogen (which causes dise ase primarily in cruciferous plants). The CaMV 35S terminator seque nce is non-coding, contains regulatory sequences of known function and does not cause disease symptoms in plants nor encode for an infectious agent.

Genective performed sequencing analysis around the flanking regions of the insertion in event VCO-Ø1981-5 and found:

 A 21 base pairs (bp) deletion in the co rn genome was identified. Bioinformatics analysis identified 12 potential open reading frames (ORFs). Regarding allergenicity potential, no ORFs share any amino acid sequence similarities or homology compared with known allergen s. These ORF proteins were also analyzed against publically available databases and none showed significant homology with proteins known to be toxi ns. In addition, the sequencing analysis found no “start” codons upstream. It is highl y unlikely for these ORFs to generate any translatable mRNA. Therefore, the poten tial for any of the expressed proteins to be food allergens or toxins is minimal.

 The insertion of the T-DNA was integr ated at maize chromosome1 and has
occurred at the 5’ untrans lated region of endogenous Acanthoscurrin and could potentially code for an Acanthoscurrin- homolog protein. It has been noted in the
literature that Acanthoscurrin is a linear cationic antimicrobial glycine-rich protein (GRP) (Lorenzini et al. 2003). GRPs with a variety of functions have been found in different organisms (Remuzgo et al. 2005). Acanthoscurrins isolated
from Acanthoscurria gomesiana (tarantula spider) ha ve been noted in the6 literature when tested against microorganis ms to display activity against the gramnegative bacteria E. coli and against the fungus Candida albicans (Lorenzini et al. 2003). However, Acanthoscurrins isolated from Acanthoscurria gomesiana (tarantula spider) have no structural si milarities with already known glycine-rich antimicrobial peptides from animals and pl ants (Lorenzini et al. 2003). There are
no data or evidence that Acanthoscurr ins in the Event VCO-Ø1981-5 corn are

involved in resistance to b acterial or fungal disease.

Event VCO-Ø1981-5 corn was field tested in 17 U.S. field sites in 2009 to collect agronomic data. Genective evaluated whether pl ant-disease or plant- insect interactions, or plant response to abiotic stressors of event VCO-Ø1981-5 corn were altered compared to the negative segregant controls. Across all s ites, a total of 9 insect categories, 6 disease categories and 2 abiotic stressors were evaluated (Genective 2011, pp.51-52). There were no qualitative differences observed in Event VCO-Ø1981-5 corn compared to its negative segregants. Since no significant differences we re observed across sites, it was concluded that there is no apparent potential fo r significant impact on disease and pest susceptibilities.

Compositional Analysis

Compositional analyses were conducted to a ssess whether the composition and nutrient levels in grain and forage derived from Event VCO-Ø1981-5 corn were comparable to the null segregant, which has background ge netics very similar to Event VCO-Ø1981-5

corn but without the epsps grg23ace5 gene cassette. Compositional comparisons between Event VCO-Ø1981-5 corn and negative segreg ant controls were performed using the principles and analytes outlined in the OECD consensus documents for composition (OECD, 2002) of new varieties of corn. Three additional conventional corn varieties selected by Genective, which were commonl y planted commercially in their adapted geographic region and were currently in the marketplace, were also included in the analysis to establish a range of natura l variability for each analyte. For each compositional experiment, a 99% tolerance inte rval was calculated. This interval is expected to contain, with 95% confidence, 99% of the values obtained from the population of commercial reference varietie s. By comparison to the 99% tolerance interval, any statistically significant differences between Event VCO-Ø1981-5 corn and the comparators may be put into perspective, and can be assessed fo r biological relevance in the context of the natural variability in corn. In addition, for the compositional assessment, publicly available information was obtained by Genective from International Life Science Institute (ILSI) Crop Composition Database (ILSI 2006) for corn as “Published Range.” Event VCO-Ø1981-5 corn analyte ranges that fell within the tolerance interval and/or published range for that analyte were consider ed to be within the range of normal variability of commercial corn hybrids.

The Genective compositional analyses of Ev ent VCO-Ø1981-5 corn and the genetically very similar negative segregants were base d on forage and grain harvested from 2009 at
five field locations in thr ee states (Genective 2011, p. 53) . Compositional analyses of grain samples included moisture, protein, fat, acid detergent fibe r, neutral detergent fiber, crude fiber, total dietary fi ber, starch, ash, and carbohydrat es. Compositional analyses of7 forage samples included protein, fat, acid detergent fiber, ne utral detergent fiber, crude fiber, ash, calcium, and phosphorus and carbohydrates.

Genective also measured the concentrations of the expressed protein-EPSPS ACE5 in various growth stages (Genective 2011, Tabl e 8 and 9, p. 36-37). EPSPS ACE5 protein is homologous to EPSPS proteins naturally presen t in plants (e.g., s oybean and corn) and fungal and microbial sources, a ll of which have a history of safe human consumption (Harrison et al., 1996). The EPSPS ACE5 prot ein expressed in Ev ent VCO-Ø1981-5 corn is 97.6% identical and 98.5% homologous at the am ino acid level to its originally isolated native enzyme EPSPS GRG23 from Arthrobacter globiformis

. Arthrobacter species are found ubiquitously in soil. Athenix Corporat ion provided the FDA with information on the identity, function, and characterization of the gene for Event VCO-Ø1981-5 corn, including expression of the gene products in an Early Food Safe ty Evaluation of new proteins in new plant varieties. The FDA co mpleted its review of Athenix’ submittal and published a response with “no further que stions” in October 2010 (US-FDA 2010).

Overall, a comprehensive evaluation of Ev ent VCO-Ø1981-5 corn and the controls showed no biologically meaningful differen ces for grain and forage compositions for either major nutrients (Genective 2011, pp.53-65) or key anti-nutrients in corn grain (Genective 2011, p.64). The few detected differences were either exceedingly small in magnitude or the mean component values of Event VCO-Ø1981-5 corn and the control
were within the 99% tolerance interval. Therefore, based on the data presented by Genective on forage and grain, it is reasona ble to conclude that the foods and feeds derived from Event VCO-Ø1981-5 corn can be considered compositionally and nutritionally equivalent to those de rived from conventional corn.

Agronomic Properties

Genective conducted agronomic evalua tions on Event VCO-Ø1981-5 corn and

conventional and /or control corn lines. Thes e evaluations are used to determine whether Event VCO-Ø1981-5 corn is agronomically comp arable to conventional corn. They also provide reasonable scientific measures as to whether Event VCO-Ø1981-5 corn has plant pest potential. These agronomic assessments included seven (7) parameters of vegetative characteristics (plant height, ear height, final stand count, stalk lodging, root lodging, grain moisture and grain weight), 5 paramete rs of reproductive char acteristics (days to 50% pollen shed, days to 50% silking, dr opped ears, barrenness and yield), seed emergence and dormancy, and several biotic an d abiotic stress characteristics (disease incidence and insect damage) (Genective 2011, pp. 46-53). The agronomic data showed no significant differences between Event VCO-Ø1981-5 corn and control corn lines. These data support Genective’s claim that Event VCO-Ø1981-5 corn is agronomically comparable to conventional corn except fo r the intended traits and that Event VCOØ1981-5 corn does not possess characteristics that constitute plant pest risk compared to
conventional corn.

Event VCO-Ø1981-5 corn and its control lines ha ve been field tested in the U.S. and its
territories since 2007 and outside of the U.S. (Spain, Czech Republic, Romania, Slovak Republic and Canada) since 2009 (Genective 2011, pp. 42-43). The principal investigator8 of the trials surveyed the fields for natu rally occurring insects, diseases and any unexpected differences between Event VC O-Ø1981-5 corn and control lines are
summarized in Table 19-21 of the Petiti on. The data of phenotypic and ecological assessments of Event VCO-Ø1981-5 corn pr ovided by Genective collectively support the petitioner’s claim that Event VCO-Ø1981-5 corn does not exhibit any meaningful differences when compared to its non-tran sgenic negative segregant counterparts in regards to naturally-occurring in sect or disease infestations.

The data (transformation method, introduced genes and their genetic elements, and compositional analysis and agronomic proper ties) cited by Genectiv e and reviewed by APHIS indicate that Event VCO-Ø1981-5 corn is not biologically different from nontransgenic conventional corn lines (with the exceptions of intended introduced genetic constructs and trait) and Event VCO-Ø1981-5 corn is no more susceptible to pests and diseases compared to conventional corn. Th e introduced genetic c onstructs and trait (tolerance to glyphosate herb icide) are not expected to alter disease and pest susceptibilities.



### D. Potential for Enhanced Weedin ess of Event VCO-Ø1981-5 Corn

In the U.S., corn is not listed as a weed (Crockett 1977; Muenscher 1980), nor is it present on the Federal Noxious Weed Li st (7 CFR part 360.200). Corn is grown throughout the world without any repo rt that it is a serious weed or that it forms persistent feral populations (Gould 1968). Like many domesticated crops, corn seed from a previous year’s crop can overwinter and germinate the following year. Manual or chemical measures are often applied to remove these volunteers, but the plants that are not removed do not typically result in feral popul ations in following years because corn is incapable of sustained reproduction outside of domestic cultivation and corn is noninvasive in natural habitats (Gould 1968). Co rn possesses few of the characteristics of those plants that are notably successful as weeds (Baker 1965; Keeler 1989). Compared to other corn varieties, Ev ent VCO-Ø1981-5 corn has improve d fitness in the presence of glyphosate herbicide. But, there are many av ailable options for the control of Event VCO-Ø1981-5 corn if unwanted plants might be growing in the field.

APHIS assessed whether Event VCO-Ø1981-5 corn is any more likely to become a weed than the non-transgenic comparator corn li ne or other corn va rieties currently under cultivation. The assessment encompasses a thor ough consideration of the basic biology of corn and an evaluation of the unique char acteristics of Event VCO-Ø1981-5 corn. Genective has been conducting agronomic ev aluations in both la boratory experiments
and field trials.

The germination and dormancy evaluations were conducted under la boratory conditions. Seed dormancy is an important ch aracteristic that is often asso ciated with plants that are considered weeds (Anderson 1996). Although dorma ncy is not associated with modern corn cultivars, corn seed dormancy tests can be used to determine whether Event VCOØ1981-5 corn is agronomically comparable to conventional corn and determine whether

2 http://www.aphis.usda.gov/plant_health/plant_p est_info/weeds/downloads/weedlist-2010doc.pdf9 Event VCO-Ø1981-5 corn is more likely to pos e a plant pest risk when compared to
conventional corn.

Standardized germination assays of the Asso ciation of Official Seed Analysts (AOSA 2007) are often used as a baseline to meas ure seed germination potential. Under warm germination, Event VCO-Ø1981-5 corn and its non-transgenic controls have an equivalent germination rate of 99.6%. Under cold germination, Event VCO-Ø1981-5 corn has a 97% germination rate vs. 93% for non-transgenic cont rols (Genective 2011, pp. 52-53). The results (Genective 2011, Tabl e 22, p.53) support th e conclusion that Event VCO-Ø1981-5 corn is comparable to c onventional corn with minor differences and Event VCO-Ø1981-5 corn does not exhibit char acteristics that would cause it to be weedier than the parental line s or conventiona l varieties.

A total of 13 different agronomic phenotypic characteristics, as we ll as observations for plant responses to plant-diseas e and plant-insect interactions were evaluated by Genective (Genective 2011, Tables16-21, pp.48-52) at 17 U.S. locations, 2 Canadian locations and more than 20 locations in Europe fr om 2007 through 2011 (Genective 2011, Tables12-13, p.42-43). At these locations, the range of values for agronomic parameters is within the range of values expected for non-GE commercial corn hybrids. Agronomic characteristics evaluated (Genective 2011, Table 16-18, pp.48-50) include: emergence, plant height, final stand count , stalk lodging, root lodging, gr ain moisture, grain weight, days to 50% pollen shed, days to 50% silk ing, dropped ears, barrenn ess, yield, disease incidence and insect damage. No differences in undesirable phenotypic characteristics, including weediness traits, were identified. Th e mean values of these agronomic traits observed for Event VCO-Ø1981-5 corn fall with in the range of values observed for the
commercial corn varieties. Field trial data (Genective 2011, Appendix 1, pp. 85-114) indicates that Event VCO-Ø1981-5 corn does no t exhibit characteristics that would cause
it to be weedier than the contro ls and non-GE conventional corn.

In addition, agronomic field observation data for Event VC O-Ø1981-5 corn and the nontransgenic counterparts shows no significant differences in di sease incidence and insect damage. The introduced EPSPS protein provi des tolerance to th e herbicide glyphosate for Event VCO-Ø1981-5 corn and this trait occurs in a total of seven commercially

grown plant species given non-regulated status by USDA-APHIS since 1994 (e.g., Petition 93-258-01p, Petition 95-045-01p, Petition 97-099-01p, Petition 98-173-01p, Petition 98-216-01p, etc. found here: http://www.aphis.usda.gov/biotechnology/ not_reg.html ). The plant species that have been engineered to expr ess the protein EPSPS

are: Beta vulgaris L. (sugarbeet), Brassica napus L., Brassica rapa L., Glycine max L. (soybean), Gossypium hirsutum L. (cotton), Medicago sativa L. (alfalfa) and Zea mays L. (maize). These GE plant species also have some potential to “volunteer” as weeds in following growing seasons (OECD 2009; OECD 2003). The available data indicate there is no linkage between expression of EPSPS pr oteins and any increased survival or overwintering capacity that would alter the pr evalence of volunteer plants in subsequent growing seasons

. These changes will not necessita te a major departure from wellestablished and broadly used ag ricultural protocols. The firs t glyphosate tolerant corn was given nonregulated status in 1997 and was on the market soon after. Based on 2008 survey data, glyphosate tolerant corn, soyb ean and cotton accounted for approximately10 three quarter of the total commercially grown corn, cotton and soybean planted in the US (Nandula 2011). After many years of commerc ialization of glyphosate tolerant corn, soybean and cotton, these plants have not e xhibited characteristics that would indicate
increased weediness. There is no reason to expect that Event VC O-Ø1981-5 corn would result in increased weediness, since Even t VCO-Ø1981-5 corn contains a version of EPSPS protein that provides similar glyphosat e tolerance as thes e other varieties. Based on agronomic and compositional data showi ng that EPSPS does not have a significant impact on agronomic or compositional traits (including those that are related to weediness), there is no evidence to date that expression of EPSPS pr otein has resulted in any altered potential for weediness in these GE plants. The above considerations, together
with the fact that the novel trait has no intended effect on weediness, leads USDA-APHIS to conclude that Event VCO-Ø1981-5 corn ha s no altered weedine ss potential compared to current commercialized glyphos ate tolerant varieties. Add itionally, field evaluation of phenotypic and agronomic characteristics s howed no differences relative to its comparators and supports the conclusion that Event VCO-Ø1981-5 corn is not likely to have increased weediness compared to conventional corn varieties.



### E. Potential to Impacts the Weedine ss of Other Plants with which It
Can Interbreed

APHIS evaluated the potential for gene intr ogression to occur from Event VCO-Ø1981-5 corn to sexually compatible wild relatives and considered whether such introgression
would result in increased weediness. Intr ogression is a proc ess whereby gene(s) successfully incorporate into the ge nome of a recipient plant.

Corn belongs to the grass family, Poaceae . The genus Zea has five species: diploperennis HH, perennis, luxurians

, mays , and nicaraguensis (OGTR 2008). Zea mays is further divided into four subspecies: huehuetenangensis

, mexicana

, parviglumis , and mays . The first three subspecies are teosintes. Z. mays ssp mays is the only cultivated species and has been domesticated for its current use by selection of key agronomic characters, such as a non-shattering rachis, grain yield, and resistance to pests (Wozniak 2002). Zea mays

ssp mays occurs only where it is cultivated in the U.S. Occasionally it is found in
abandoned fields or on roadsides. The closest wild relatives of corn are the teosintes (wild Zea spp.) (Ellstrand et al. 2007) wh ich are sexually compatible with Zea mays . All
teosinte members can be crossed with cultivat ed corn to produce fertile first generation hybrids (Doebley1990; Wilkes 1967). However co rn teosinte hybrids exhibit low fitness and have little impact on gene introgressi on in subsequent gene rations (Galinat 1988). Additionally, teosintes are normally confined to the tropical and s ubtropical regions of Mexico, Guatemala, and Nicaragua. A fairly rare, sparsely disper sed feral population of teosinte has been reported in Florida (USDA PLANTS Database 3 , accessed 4/18/2012).

Tripsacum is a genus of grass in the Po aceae family. Although it is difficult, Tripsacum can be successfully hand crossed with corn to form hybrids. However these hybrids have a high degree of sterility (Doebley 1990; Wilkes 1967) and are generally unstable
because of differences in chromosome numbe r and lack of pairing between chromosomes

3 http://plants.usda.gov/java/county?state_name=Florida&statefips=12&symbol=ZEME11 (Eubanks 1997). First generation hybrids are mu ch less fit for survival and dissemination in the wild and typically show reduced re productive capacity. Furthermore, gene flow
from corn to Tripsacum is virtually impossible because of several factors including distribution, genetic incompatibil ity, temporal separation of fl owering time, etc. (Galinat 1988). These distinctions between related species directly aff ect the ability of cultivated
corn to interbreed with wild relatives. Mode rn corn is highly domesticated and requires significant human intervention to grow and repr oduce. As with all domesticated corn, the likelihood that Event VCO-Ø1981-5 corn w ould reproduce and sustain populations outside of cultivation is extremely small.

Corn is predominantly an outcrossing plant species. The rate of se lf-pollination is 5% (Sleper and Poehlman, 2006). The short viability period of polle n grains limits significant outcrossing. Since Event VCO-Ø 1981-5 corn does not exhibit characteristics that can cause it to be any weedier than other cultiv ated corn, its potential for gene introgression into teosinte is not expected to be any di fferent from that of other cultivated corn
varieties.

None of the sexually compatible relatives of co rn in the U.S. are considered to be weeds in the U.S. (Holm et al. 1979); and, as di scussed in the sectio n above, the unlikely acquisition of an herbicide tole rance gene would not be expect ed to transform them into weeds. Therefore, USDA has concluded that adverse consequences of gene flow from Event VCO-Ø1981-5 corn to wild or weedy sp ecies in the U.S. ar e highly unlikely.



### F. Potential Impacts on Non-target Organisms, Including Those Beneficial to Agriculture

APHIS evaluated the potential for Event VC O-Ø1981-5 corn to have damaging or toxic effects directly or indire ctly on non-target organisms. The non-target organisms considered were representatives of the expos ed species in the agricultural environment. As discussed earlier, Event VCO-Ø1981-5 corn is similar (by compositional analysis) to unmodified control corn varieties except for the intended changes in herbicide resistance, associated with the production of EPSPS ACE5 protein in the plant.

Event VCO-Ø1981-5 corn expresses the EPSPS ACE5 protein which provides resistance to the herbicide glyphosate. It was derived from EPSPS GRG23 pr otein isolated from
common soil bacteria Arthrobacter globiformis. The EPSPS ACE5 protein was optimized for incorporation into corn by changing te n amino acids from the EPSPS GRG23 protein (Schouten etal. 2011; Genective 2011, page 31). This codon modification makes EPSPS ACE 5 closely match the native maize EPSPS and did not alter its enzymatic profile (Genective 2011, page 33-34). The EPSPS ACE5 is still 97.6% identical to the originally
isolated native EPSPS GRG23.

For EPSPS ACE5 protein, a bioinformatics an alysis was performed by Athenix which determined that the amino acid sequence of the EPSPS ACE5 protein exhibits no significant homology to known or suspected allergens and toxins (Athenix 2009). Further, the resistance to enzymatic peps in digestion of EPSPS ACE5 protein was analyzed (Thomas et al. 2004). The results indi cate that EPSPS ACE5 is readily digested12 in mammalian digestive systems and rapidl y degraded (Atheni x, 1999; Genective 2011, p.40). Rapid degradation of the full-length EPSPS ACE5 protein makes it highly unlikely that EPSPS protein would be absorbed by epith elial cells of the sm all intestine in a biologically-active form. Acute oral toxicity studies in mice show that EPSPS ACE5 has no adverse effect on acutely gavaged mice (Athenix 1999). Doses of 1783 mg/kg bodyweight were administered to mice, and there were no signs of toxicity in any of the groups of mice tested over a period of 14 days . Acute toxicity studies in mice show the EPSPS ACE5 protein has no toxicity even at doses much higher than mice could encounter due to exposure to GE plants expressing the EPSPS protein. Based on this study, EPSPS ACE5 protein did not s how any evidence of toxicity.

APHIS considered the similarity in struct ure and function of the CP4 EPSPS found in a number of Roundup Ready® crops with nonreg ulated status to EPSPS ACE5, and other EPSPS proteins endogenous to plants and pr esent throughout the environment. The EPSPS ACE5 protein in Event VCO-Ø1981-5 corn is highly homologous to the family of EPSPS proteins and the CP4 EPSP S protein that has a long hist ory of safe use in these plants. In 2011, glyphosate-resist ant varieties were grown on approximately 93 percent of soybean acres, 78 percent of upland cotton ac res, and 70 percent of corn acres in the United States (USDA-ERS 2011). The CP4 EPSPS proteins have been commercialized since 1994 and are present in crops which ar e grown on millions of acres in the U.S. every year. At present, APHIS is not aware of any identified significant adverse effects of EPSPS proteins on the abundance of non-target organisms in the field. The family of EPSPS proteins present in a va riety of plants has been subject to extensive human consumption without eviden ce of health concerns.

The Environmental Protection Agency (EPA) also has previously reviewed the safety of the EPSPS protein and has established a tole rance exemption for the protein (US-EPA 1997). The exemption was based on a safety assessment. CP4 EPSPS and other versions of EPSPS proteins were evaluated for sa fety by FDA concluding that “no further questions” were needed. Th e food and feed products containing EPSPS proteins are apparently as safe as corn currently on the market for human and animal consumption. In 2009, Athenix submitted EPSPS ACE5 protein for the Early Food Safety Evaluation. FDA completed the review with no further questions in 2010 (US-FDA 2010). To assess unintended effects, APHIS analyzed data subm itted by the developer to determine if there were changes to phenotype, germination, vege tative growth, reproduc tive parameters and response to biotic stressors (insect and disease stress) associated with Event VCOØ1981-5 corn in comparison to the various co ntrol lines (nontransge nic). Data presented in the Petition (Genect ive 2011, page 43-55) indicates that the ecological interactions between Event VCO-Ø1981-5 corn and the control lines were similar.

Considering all the data noted from field obser vations, the lack of t oxicity in an acute mouse feeding study, lack of ho mology to toxins and allergen s, widespread presence in the environment and known safety informa tion on EPSPS proteins, APHIS concludes that Event VCO-Ø1981-5 corn is unlikely to have any adverse impacts to nontarget organisms and beneficial organisms.13 

### G. Potential Impacts from Transfer of Genetic Information to Organism with which Event VCO-Ø 1981-5 Corn Cannot Interbreed

APHIS examined the potential for the new ge netic material inserted into Event VCOØ1981-5 corn to be horizontally transfe rred to other organisms without sexual reproduction and whether such an event could le ad directly or indirectly to disease, damage, injury or harm to plants. Horizontal gene transfer and expression of DNA from a plant species to other species is highly unlikely to occur ba sed on the following reasons.

The horizontal gene transfer (HGT) between unrelated organisms is one of the most intensively studied fields of science. Hori zontal gene transfer and expression of DNA from a plant species to bacteria or animal speci es is unlikely to occur. A number of points support this conclusion:

 Many genomes (or parts thereof) from bacter ia that are closely associated with plants have been sequenced including Agrobacterium and Rhizobium (Kaneko et al. 2000; Wood et al. 2001; Kaneko et al. 2002). There is no evidence that these organisms contain genes de rived from plants. Also, Agrobacterium tumefaciens

or Arthrobacter globiformis species are generally co mmon in soil and therefore
various epsps genes have been available for long periods of time for horizontal
transfer from Agrobacterium tumefaciens or Arthrobacter globiformis to plants or other soil microorganisms and decaying plant material. Therefore the likelihood of any impact or new horizontal gene tr ansfer that is not already capable of taking place in the soil is extremely unlikely.

 No evidence has been identified for a ny mechanism by which maize genes could be transferred to humans or animals, or any evidence that such gene transfer has occurred for any plant species during e volutionary history, despite animals and humans eating large quantities of plant DNA. In cases where review of sequence data implied that horizontal gene transfer occurred, these events are inferred to occur on an evolutionary time scale on the order of millions of years (Koonin et al. 2001; Brown 2003).

 Transgene DNA promoters and coding se quences are optimized for plant expression, not prokaryotic bacterial expr ession. Thus, even if horizontal gene transfer occurred, proteins corresponding to the transgenes are not likely to be produced.

 FDA has evaluated horizontal gene transfer from the use of antibiotic resistance marker genes, and concluded that th e likelihood of transfer of antibiotic resistance genes from plant genomes to microorganisms in the gastrointestinal tract of humans or animals, or in the environment, is extremely unlikely (USFDA 1998).

 APHIS also considered whether horiz ontal transfer of DNA from Event VCOØ1981-5 corn to plant viruses was likely to occur and would l ead to the creation14 or selection of a more vi rulent plant pathogen through recombination with other plant viruses. This issue has been cons idered before by other science review panels and government regulatory bodi es (Keese 2008). Alth ough sequences of
the Cauliflower Mosaic Vi rus are contained within Event VCO-Ø1981-5 corn, those sequences are limited to the regulator y elements. Regulatory elements such as promoters and terminators have not been implicated in viral recombination.

Finally, under natural conditions ; no transfer of an intact functional gene has been demonstrated to date (Miki and McH ugh, 2004).Therefore, APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses no plant pest risk.



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

APHIS considered potential impacts associated with cultivation of Event VCO-Ø1981-5 corn on current agricultural practices in corn and other crops. Genective has provided data which indicate that Event VCO-Ø1981-5 corn is comparable to conventional corn in phenotypic, ecological and compositional char acteristics (Genective 2011). Event VCOØ1981-5 corn, a glyphosate tolerant variety, is expected to replace some presently available commercial glyphosate tolerant corn varieties without aff ecting the overall total corn acreage or glyphosate tolerant corn acr eage. The availability of more herbicide tolerant corn products will likely incr ease grower choice and price competition.

Compared to currently-available glyphosate tolerant corn products containing EPSPS, no
increased use of herbicides from the use of Event VCO-Ø1981-5 corn is expected. In 2010, glyphosate-resistant varieties were grow n on approximately 93 percent of soybean acres, 78 percent of upland cotton acres, and 70 percent of corn acres in the United States. As these varieties were adopted, farm ers generally used glyphosate as an herbicide and weed-management tactic. In general, gl yphosate is less toxic to humans than other common herbicides and not as likely to pe rsist in the environment as many of the herbicides it replaces (IPCS 1994; US DA-ERS 2006). APHIS doe s not foresee any increased glyphosate use by the addition of Event VCO-Ø1981-5 corn to the market.

Continuous use of one herbicid e to control weeds will select for weed resistance. In general, weed problems in fields of GE glyphosate-resistant crops will become more common as weeds evolve resistance to glyphosat e or weeds less susceptible to glyphosate
become established in areas treated exclusiv ely with the same herbicide. A number of new genetically engineered, herbicide-resi stant corn varieties are currently under development and may provide growers with other herbicides and weed management options when fully commercialized. Growers need to consider other effective weedmanagement tools or use the alternative herbic ides with different modes of actions. Such practices should be encourag ed through collaborative effo rts by federal and state government agencies, private-sector technol ogy developers, universities, and farmer organizations to develop cost -effective resistant-management programs and practices that preserve effective weed control in herbicide-resistant crops (Ervin 2010).

Overall, APHIS concludes that agricultural or cultivation practices are not expected to change and thus no changes in plant pest ri sks are expected due to such practices.15 

### I. Conclusion

APHIS has prepared the plant pest risk assessment in order to determine if Event VCOØ1981-5 corn is unlikely to pose a plant pest risk. Based on the information provided by the applicant and the lack of plant pest risk from the inse rted genetic material, weedy characteristics, atypical respons es to disease, insects or plan t pests in the field, effects on non-targets or beneficial organisms in th e agro-ecosystem, cha nges to agricultural practices, and horizontal gene transfer, APHIS has concl uded that Event VCO-Ø1981-5 corn is highly unlikely to pose a plant pest risk.



### J. References

Albert, H., Wei, H. (2003) Promoter of the sugarcane UBI4 gene. United States Patent:
6638766.

Alibhai, M., and Stallings, W. (2001) Closing down on glyphos ate inhibition-with a new structure for drug discovery. Proc . Natl. Acad. Sci. USA 98:2944-2946.

AOSA (2007) Rules for testing seeds. Associat ion of Official Seed Analysts, Lincoln,

NE.

Anderson, W. (1996) Weed Ecology. Principles and applications. Third Edition. West Publishing Company. Page 27-38.

Athenix (2009) Early Food Safety Evaluation for EPSPS ACE5 protein http://www.fda.gov/downloads/Food/Bio technology/Submissions/UCM233624.pdf

Baker, H. (1965) Characteristics and Modes of Origin of Weeds. In : The Genetics of Colonizing Species. H.G. Baker and G.L. St ebbins (eds.). pp. 147-172. Academic Press,
New York and London.

Barker, R., Chibata, K., Thompson, D., and Ke mp, J. (1983) Nucleotide sequence of the T-DNA Region from the Agrobacterium tumefaciens Octopine Ti Plasmid pTi15955. Plant Molecular Biology 2: 335-350.

Brown, J. (2003) Ancient hor izontal gene transfer. Na t. Rev. Genet. 4: 121–132.

Crockett, L. (1977) Wildly Successful Plants : North American Weeds. University of Hawaii Press, Honolulu, Hawaii. 609 pp.

Depicker, A., Stachel, S., Dhaese, P., Za mbryski, P., and Goodman, H. M. (1982) Nopaline synthase:transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics 1:561-573.

Doebley, J. (1990) Molecular evidence and th e evolution of maize. Econ. Bot. 44 (3 supplement): 6-27.16 Duke , S., Powles , S. ( 2009) Glyphosate-Resistant Crops a nd Weeds: Now and in the Future. Agbioforum. 12(3&4):346-357.

Ellstrand, N., Garner, L., Hegde, S., Guad agnuolo, R., Blancas, L. Hered, J. (2007) Spontaneous hybridization between maize and teosinte. Mar-Apr; 98(2):183-7. Epub
2007 Mar 30.

Ervin, D (2010) Impact of genetically engi neered crops on farm sustainability in the
United States. The National Academies Press. http://www.nap.edu/openbook.php?record_id=12804&page=R1

Eubanks, M. (1997) Molecular Analysis of Crosses between Tripsacum dactyloides and

Zea diploperennis (Poaceae). Theoretical and Applied Genetics 94:707-712.

Fang, L., Gross, P., Chen, C. and Li llis, M. (1992) Sequence of two acetohydroxyacidsynthase genes from Zea mays. Plant. Mol. Biol. 18:1185-1187.

Galinat, W. (1988) The Origin of Corn. pp. 1-31. ( In Sprague, G. F., Dudley, and J. W., Editors). Corn and Corn Improvement, Thir d Edition. American Society of Agronomy, Crop Science Society of America, and So il Science Society of America, Madison, Wisconsin. 986 pp.

Gardner, R., Howarth, A., Hahn, P., Brow n-Luedi, M., Shepherd, R. and Messing, J.(1981) The complete nucleotide sequence of an infectious clone of cauliflower mosaic virus by M13mp7 shotgun sequencing. Nucleic Acids Res 9:2871-2888.

Genective (2011) Petition for Determination of Nonregulated Status for herbicidetolerant Event VCO-Ø1981-5 maize. Submitte d by Isabelle Coats, US Registration Manager Bayer CropScience LP for Genective SA 1rue Limagrain BP1 63720 CHAPPES, FRANCE (USDA APHIS BRS Petition 11-342-01p)

Gianessi, L. (2008). Economic impacts of gl yphosate-resistant crops. Pest Management Science, 64(4), 346-352. doi: 10.1002/ps.1490.

Gould, F (1968) Grass Systematic s. McGraw-Hill, New York.

Harrison, L., Bailey, M., Naylor, M., Ream, J., Hammond, B., Nida, D., Burnette, B., Nickson, T., Mitsky, T., Taylor, M., Fuchs, R ., Padgette, S. (1996) The expressed protein in glyphosate-tolerant soybeans, 5-enolpyr uvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4, is rapidly digested in vitro and is not toxic to acutely gavaged mice. J. Nutr. 126: 728-740.

Holm, L., Pancho, J., Herbarger, J., and Pl ucknett, D. (1979) A Geographical Atlas of World Weeds. John Wiley and Sons, New York. 391 pp.

ILSI (Center for Environmental Risk Assessment) (2006) ILSI Crop Composition
Database Version 3.0 Internationa l Life Sciences Institute. http://www.cropcomposition.org/17 ILSI (Center for Environmental Risk Assessm ent) (2010) A review of the Environmental Safety of the CP4 EPSPS protein. http://ceragmc.org/docs/cera_publications/pub_01_2010.pdf

IPCS (International Progra mme on Chemical Safety) (1994) Environmental Health Criteria for Glyphosate. http://www.inchem.org/documents/ehc/ehc/ehc159.htm

Kaneko, T., Nakamura, Y., Sato , S., Mina misawa, K., Uchiumi, T., Sasamoto, S., Watanabe, A., Idesawa, K., Iriguchi, M., Ka washima, K., Kohara, M., Matsumoto, M., Shimpo, S., Tsuruoka, H., Wada, T., Yamada, M ., Tabata, S. (2002) Complete genomic sequence of nitrogen-fix ing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Research 9(6):189-197.

Kaneko, T., Y. Nakamura, S. Sato, E. Asamizu, T. Kato, and S. Sasamoto. (2000) Complete genome structure of the nitrogen- fixing xymbiotic bacterium Mesorhizobium
loti. DNA Research. 7:331-338.

Keeler, K. (1989) Can genetically engin eered crops become weeds? Bio/Technology
7:1134-1139.

Keese, P. (2008). "Review Article: Risks from GMOs due to horizontal gene transfer." Environmental Biosafety Research 7 (123-149).

Koncz, C., Schell, J. (1986) The promoter of Tl-DNA gene 5 controls the tissue specific expression of chimeric genes carried by a nove l type of Agrobacterium binary vector.
Molecular and General Genetic s 204: 383-396.Weeds. John W iley and Sons, New York. 391 pp.

Koonin, E., Makarova, K., Aravind, L. (2001) Ho rizontal gene transfer in prokaryotes: Quantification and classification. Annu Rev Microbiol, 2001; 55:709-42.

Lorenzini, D., da Silva, P., Fogaca, A., Bule t, P., Daffre, S. (2003) Acanthoscurrin: a novel glycine-rich antimicrobial peptide constitu tively expressed in the hemocytes of the spider Acanthoscurria gomesiana. Deve lopmental & Comparative Immunology Volume 27, pp. 781-791.

Miki, B., S. McHugh (2004). "Selectable marker genes in transgenic plants: applications,
alternatives and biosafet y." Journal of Biotechnology 107 : 193-232.

Muenscher, W. (1980) Weeds. Second Edition. Cornell University Press, New York and London. 586 pp.

Nandula, V. (2010) Glyphosate resistance in crops and weeds: History, development and management. Wiley Publisher. http://books.google.com/books?id=Vwfe qZxBzcwC&pg=PA168&lpg=PA168&dq=glyp hosate+resistance+corn+planted+in+US &source=bl&ots=EHSqFGpM1w&sig=8HnSdT 3a_uT6Q0KIohCrOjW4uxM&hl=en&sa=X& ei=V3qRT8fVF4awiQKf7PzMAw&sqi=218 &ved=0CGYQ6AEwCQ#v=onepage&q=glyph osate%20resistance%20corn%20planted
%20in%20US&f=false

OECD (2002) Consensus Document on Com positional Considerations for New Varieties of Maize ( Zea mays ): key Food and Feed Nutrients, Anti-Nutrients and Secondary Plant Metabolites. Organization for Econo mic Co-operation and Development, ENV/JM/MONO (2002)25, http://www.oecd.org/da taoecd/15/63/46815196.pdf

OECD (2003) Consensus document on the biology of Zea mays subsp, mays (maize) http://www.oecd.org/officialdocuments/d isplaydocumentpdf/?cote=env/jm/mono(2003)1 1&doclanguage=en

OECD (2009) Module III: Drafe consensu s documen on general information concerning agronomic and environmental aspects of the cu ltivation of genetically modified herbiced resistant plants. http://www.oecd.org/officialdocuments/ publicdisplaydocumentpdf/?cote=ENV/JM/BIO( 2004)8/REV4&docLanguage=En

OGTR (2008). Australian Government, Departme nt of Health and Ageing, Office of the Gene Technology Regulator. The Biology of Zea mays L. ssp mays (maize or corn). Version 1: September 2008.

Remuzgo, C., Andrade, G, Temperini, M., Daffre, S., Miranda, M. (2005) The CTerminal fragment of Acanthoscurriin is a difficult sequence. American Peptide Society.

Schouten, L, Peters, C, Vande Berg, B. ( 2010) GRG23 EPSPS Synthases: Compositions
and methods of use. US Patent N o 7,834, 249 B2.

Sleper, D., Poehlman, J. (2006). Breeding Corn (Maize). Chapter 17. In: Breeding Field Crops, 5 th Edition. Blackwell Publishing pp 277-296. Blackwell Publishing.

Thomas, K., Aalbers, M., Bannon, G.A., Bartels, M ., Dearman, R.J., Esdaile, D.l., Fu, TJ.,Glatt, C.M., Hadfield, N., Hatzos, C ., Helfe, S.L., Heylings, J.R., Goodman , R.E., Henry, B.,

Herouet, C., Holsapple, M., Ladies, G.S., Land ry, T.D., MacIntosh, S.C., Rice, E.A., Privalle,

L.S., Steiner H.Y., Teshima, R., van Ree, R., Woolhiser, M., and Zawodny, J. (2004) A multilaboratory evaluation of a common in vitro pepsin digestion assay protocol used in assessing the safety of novel proteins, Regulatory Tox. Pharm. 39, 87-98.

UC-IPM (2009) University of California Pest Manage ment Guidelines: Corn.Agriculture and Natural Resources, UC Statewide Integr ated Pest Management Program, Oakland,
CA.

US-EPA (1997) Glyphosate; Pesticide Tolerances Final Rule.62 FR 1772319 USDA-ERS (2006) The first deca de of genetically engineered crops in the United States. http://www.ers.usda.gov/publ ications/eib11/eib11.pdf

USDA-ERS (2010) Briefing Rooms-Corn. ( http://www.ers.usda.gov/Briefing/Corn/ )

USDA-ERS (2011) Adoption of Genetic ally Engineered Crops in the
U.S. http://www.ers.usda.gov/Data/Biotech Crops/ExtentofAd optionTable1.htm

US-FDA (2013) Completed Consultations on Bioengineered Foods http://www.accessdata.fda.gov/scripts/fc n/fcnNavigation.cfm?rpt=bioListing

US-FDA (1998) Use of antibio tic resistance marker gene s in transgenic plants Draft Guidance: Use of Antibiotic Resistance Marker Genes in Transgenic Plants

US-FDA (2010) NPC 00012: Agency Respons e Letter CFSAN/Office of Food Additive Safety (EPSPS ACE5) http://www.fda.gov/Food/FoodScienceResear ch/Biotechnology/Submissions/ucm231052
.htm

USDA-NASS (2012) Acreage ISSN: 1949-1522 http://usda01.library.cornell.edu/ usda/current/Acre/Acre-06-29-2012.pdf

Wilkes, H. (1967) Teosinte: The closest relative of maize. Cambridge, MA: The Bussey Institute, Harvard University.

Wood, D. W., J. C. Setubal, R. Kaul, D. E. Monks, J. P. Kitajima, V. K. Okura, Y. Zhou, L. Chen, G. E. Wood, N. F. Almeida Jr., L. W oo, Y. Chen, I. T. Paulsen, J. A. Eisen, P. D. Karp, D. Bovee Sr., P. Chapman, J. Clende nning, G. Deatherage, W. Gillet, C. Grant, T. Kutyavin, R. Levy, M.-J. Li, E. McClelland, A. Palmieri, C. Raymond, G. Rouse, C. Saenphimmachak, Z. Wu, P. Romero, D. Gor don, S. Zhang, H. Yoo, Y. Tao, P. Biddle, M. Jung, W. Krespan, M. Perry, B. Gordon-Ka mm, L. Liao, S. Kim, C. Hendrick, Z.-Y. Zhao, M. Dolan, f. Chumley, S. V. Tingey, J. -F. Tomb, M. P. Gordon, M. V. Olson, and E. W. Nester. (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science. 294:2317-2323.

Wozniak, C. (2002). Gene Flow Assessment fo r Plant-Incorporated Protectants by the Biopesticide and Pollution Pr evention Division, U.S. EPA. Paper presented at the Scientific Methods Workshop: Ecological and Agronomic Consequences of Gene Flowfrom Transgenic Crops to Wild Relatives, Columbus, Ohio.