1 

## Plant Pes t Risk Assessment for Event 5307 Corn
Syngenta Biotechnology, Inc. has petitioned APHIS (APHIS number 10

- 336

- 01p ) for a determination that genetically engineered (GE) corn ( Zea mays L. subsp. mays ) Event 5307 is unlikely to pose a plant pest risk (Syngenta 2011) and, therefore, should no longer be a regulated article under APHIS’ 7 Code of Federal Regulations (CFR) part 340. APHIS administers 7 CFR part 340 under the authority of the plant pest provisions of t he Plant Protection Act of 2000 1

. This plant pest risk assessment was conducted to determine whether corn Event 5307 is unlikely to pose a plant pest risk under full deregulation. Event 5307 was produced by transformation of an immature corn embryo of li ne NP222 with Agrobacterium tumefaciens. Because A. tumefaciens is a plant pest and some of the regulatory sequences ( promoter from Cestrum yell ow leaf curling virus and terminator from A. tumefaciens ) used to facilitate expression of the genes in corn wer e derived from plants pests, this corn has been considered a regulated article under APHIS regulations at 7 CFR part 340. Potential impacts to be addressed in this risk assessment are those that pertain to the use of Event 5307 and its progeny in the absence of confinement. APHIS utilizes data and information submitted by the applicant, in addition to current literature, to determine if Event 5307 is unlikely to pose a plant pest risk. If APHIS determines that a GE organism is n ot a plant pest risk , then APHIS has no regulatory authority over that organis m. Potential impacts in this Plant Pest Risk Assessment are those that pertain to plant pest risk characteristics. APHIS regulation 7 CFR 340.6(c) specifies the information need ed for consideration in a petition for nonregulated status. APHIS will evaluate information submitted by the applicant related to plant pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new enzymes, or changes to plant metabolism, weediness of the regulated article, any impacts on the weediness of any other plant with which it can interbreed, and transfer of genetic information to organisms with which it cannot interbreed. The Environmental Assessment (EA) for thi s petition considers whether agricultural or cultivation practices for Event 5307 may result in impacts on the environment. A thorough assessment of the effects of the determination on non

- target and beneficial organisms, and threatened and endangered spec ies is included in the EA

. Development of Event 5307 Corn Corn is the most widely produced feed grain in the U.S., accounting for more than 90

1 Section 403 (14) of the Plant Protection Act (7USC Sec 7702(14) defines plant pest 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 product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant

. (D) A bacterium. (E) A fungus. (F) A virus or viroid. (G) An infectious ag ent or other pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding subparagraphs.”2 percent of the total value and production of feed grain (USDA

- ERS 2011). There is a need to provide growers with a cost effective method to control insects, while taking into account the potential haz ardous nature of insecticides. GE corn expressing Cry proteins derived from Bacillus thuringiensis ( Bt ), commonly referred to as Bt corn , have a long history of safe use without adverse human health or environmental effects and provide an option for the control of coleopteran insect pests ( NAS 2004). The Bt proteins used in this product called eCry3.1Ab controls corn rootworm, which is a highly destructive pest respons ible for the single largest use o f conventional insecticides in the United States (EPA 2007 ). Corn rootworms have long been the major economic pests of corn in the U.S. (Calvin 2003 ). Syngenta h as genetically engineered corn to express a n eCry3.1Ab protein for use in the control of coleo pteran pests. Event 5307 deters feeding by several insects known to cause significant damage to corn in the U.S. These include: western corn rootworm ( Diabrotica vergifera virgifera Le Conte), northern corn rootworm ( D. long icornis barberi Smi th and Lawrence ) and Mexican corn rootworm ( D. virgifera zeae Krysan and Smith) (Peairs and Pilcher 2010)

. GE corn containing insect

- resistant traits, first appro ved in the U.S. in 1996, has been wide ly adopted by U.S. growers (Figure1)

. Adoption of stacked varieties (those containing both herbicide and insect tolerance traits) has accelerated in recent years. Planting of stacked corn made up 49 percent of corn acres in 2011 (Figure 2).

Figure 1. Percent of acreage of genetically engi neered crops in the U.S., 1996

- 2011 (USDA ERS 2011 ).3 Figure 2. Percent of planted acres of corn in the U.S. by trait, 2000

- 2011 (USDA

- ERS 2011). The eCry3.1Ab protein is a chimeric Bacillus thuringiensis protein, composed of portions of Cry1Ab and mCry3A proteins both of which are EPA

- registered Plant Incorporated Protectants (75 FR 34040

- 34045). Syngenta completed the consultation process with FDA for Event 5307 corn on February 29, 2012. EPA has granted an exemption from food and feed tolerance for the phosphomannose isomerase (PMI) protein on April 25, 2007 and the eCry3.1Ab protein on August 8, 2012. On July 31, 2012, EPA announced the approval of a conditional registration as a plan

- incorporated protec tant for the eCry3.1Ab protein in Event 5307

. Description of the Modification Event 5307 corn was produced by transformation using disarmed A. tumefaciens ( Syngenta 2011, pp. 26

- 28, Figure s II I

- 1, III

- 2). Immature embryos of Syngenta inbred corn line NP 2222 (Plant Variety Protection C ertificate 2 00200071; US DA

- AMS 2004) were infected with A. tumefaciens strain LBA440 4 containing plasmid SYN12274. Plants containing the introduced DNA were selected based on survival on a mannose substrate d ue to the expr ession of phospho mannose isomerase (Syngenta 2011, p
. 27, Figure III

- 2). The plasmid SYN12274 contained two gene expression cassettes flanked by the right and left border of T

- DNA from the A. tumefaciens Ti

- plasmid ( Syngenta 2011, pp. 28, 35

- 36, Figure III

- 1, Table IV

- 1). eCry3.1 Ab

– expression cassette consists of three genetic elements (Syngenta 2011, Figure III

- 1, Table IV

- 1).4 • The Cestrum Yellow Leaf Curling Virus (CmYLCV, Caulimoviridae dsDNA plant virus 2 ) genomic full

- length transcript pr omoter (Hohn 2007; Stavone 2003 ).

• The ecry3.1Ab gene consists of a fusion between the 5’ end (Domain I, Domain II and 15 amino acids of Domain I II) of a modified mcry3A gene from B. thuringiensis subsp. tenebrionis and the 3’end (Domain III and variable region 6 ) of a cry1Ab (Syngenta 2011, Table IV

- 1, Figure IV

- 1 ; Walters 2010 ).

• The nopaline synthase (NOS) terminator from A. tumefaciens (Depicker 1982). Phosphomannose isomerase

– expression cassette consists of three genetic elements ( Syngenta 2011, Figure III

- 1, Table IV

- 1).

• The polyubiquitin promoter from Z. mays (Christensen 1992).

• The pmi gene from Escherichia coli strain K

- 12 (Negrotto 2000).

• The NOS terminator from A

. tumefaciens (Depicker 1982). Data from Southern blot analysis was provided and reviewed by APHIS that showed that Event 5307 contains: (1) a single copy of the CmYLCV promoter (Syngenta 2011, Figure s V

- 4, V

- 5, Table V

- 2); (2 ) a single copy of the ecry3.1Ab gene (Syngenta 2011, Figure s V

- 2, V

- 3, Table V

- 1); (3) two copies of the NOS t erminator ( Syngenta 2011, Figure s V

- 10, V

- 11, Table V

- 5); and (4) a single copy of the pmi gene (Syngenta 2011, Figure s V

- 6, V

- 7, Table V

- 3). Southern blot analysis demonstrated the absence of the plasmid backbone elements ( Syngenta 2011, Figure s V

- 12, V

- 1 3, Table V

- 6). Data from Southern analysis was provided and revie wed by APHIS that demonstrates st able integration and inheritance of the 5307 corn insert over four generation (Syngen ta 2011, Figure s V

- 14, V

- 15, V

- 16, Table V

- 7).
Potential of 5307 to Become Invasive and/or a Weed Corn possess es few of the characteristics of those plants that are notably successful as weeds (Baker 1965; Keeler 1989). In the U.S., corn is not listed as a weed (Crockett 1977; Holm 1991 ; Muenscher 1980), nor is it present in the Federal noxious weed list (7 CFR part 360 3 ). Furthermore, corn is grown throughout the world without any report that it is a serious weed or that it forms persistent feral populations (Gould 1968). Like many domesticated crops, corn seed from a prev ious year’s crop can overwinter and germinate the following year. For instance, the appearance of corn seedlings in soybean fields following a corn crop is a common occurrence. Manual or chemical measures are often applied to remove these volunteers, but t he plants that are not removed do not typically result in feral populations in following years.

2 http://ictvdb.cumc.columbia.edu/servlet/Virus?id=944 3 http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist.pdf5 APHIS assessed whether Event 5307 is any more l ikely to become a weed than a near

is ogenic non

- transgenic corn line. The assessment encompasses a thorough consi deration of the basic biology of corn and an evaluation of the unique characteristics of Event 5307 (Syngenta 201 1 Table VII

- 1 , Appendix A Table A

- 1 ). Syngenta has been conducting agronomic field trials of Event 5307 since 2005 at locations that provide a range of environmental conditions representative of where Event 5307 is expected to be grown (Syngenta 2011, Appendix A, Table A

- 1)

. Field trial data indicated that Event 5307 does not exhibit characteristics that would cause it to be weedier than the parental corn line (Syngenta 2011 Table s VII

- 5, VII

- 6, VII

- 7, VII

- 8, VII

- 9, VII

- 10) . No differences in phenotypic characteristics that might contribute to enhanced weediness were observed between Event 5307 and control lines for the wide range of phenotypi c endpoints assessed in these field trials or in greenhouse or laboratory exper iments (Syngenta 2011 , Table s VII

- 7, VII

- 8, VII

- 9, VII

- 10, VI

- 12, Figure VII

- 1 ). There was no increase in weediness potential as measured by differences in seed germination, dor mancy, plant emergence, plant height, ear height, stalk lodging, barren plants, timing of pollen shed or silking, pollen viability and morp hology, and yield (Syngenta 2011 , Table s VII

- 7, VII

- 8, VII

- 9, VII

- 10, VI

- 12, Figure VII

- 1). None of the measure attri butes showed any differences relative to its comparator, suggesting that Event 5307 is not weedier than current corn cultivars. These results on growth characteristics, seed production and germination indicate that Event 5307 is not significantly different from its comparators. There is no indication that Event 5307 possesses a selective advantage that would result in increased weediness. As with other corn varieties, E vent 5307 lacks the ability to persist as a troublesome weed, and there would be no signi ficant impact on current weed management practices for corn cultivation. Potential for Gene Flow and Gene Introgression from Event 5307 into Sexually
Compatible Relatives Gene flow from crops to wild relatives may have the potential to enhance the weediness of wild relatives , as observed in rice, sorghum, sunflower, and a few other crops (Ellstrand 1999). Gene flow is a natural biological process with significant evolutionary importance. A number of angiosperm taxa are believed to be derived fr om hybridization or introgression between closely related taxa (Soltis and Soltis 1993; Rieseberg 1997) and even in existing floras, the occurrence of hybridization or introgression is report ed to be widespread (Stace 1987; Rieseberg 1997 ) . It has been a c ommon practice by plant breeders to artificially introgress traits from wild relatives into crop plants to develop new cultivars. APHIS evaluated the potential for gene introgression to occur from Event 5307 to sexually compatible wild relative s and consid ered whether such introgression would result in increase d weediness (Syngenta 2011, Figure III

- 3, Table III

- 1) . Cultivated corn is sexually compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum (OEC D 2003). The closest wild relatives of corn, various Zea species referred to as teosinte, are normally confined to the tropical and6 subtropical regions of Mexico, Guatemala, and Nicaragua. In the U.S. a fairly rare, sparsely dispersed feral population of t eosinte has been reported in Florida (USDA PLANTS D atabase 4 , accessed 8/23/2011 ).
The genus Tripsacum contains up to 16 recognized species, most of which are native to Mexico, Central and South America. Three of these species ( T. dactyloides, T. floridatum , and T. lanceolatum ) exist as wild and/or cultivated species in the continental U.S.; and two taxa ( T. faciculatum and T. latifolium ) also occur in Puerto Rico (USDA PLANTS Database, accessed 8/23/2011). Though many of these species occur where corn might be grown , gene introgression from Event 5307 under natural conditions is highly unlikely. Hybrids of Tripsacum species with Zea are difficult to obtain outside of a laboratory and are often sterile or have greatly reduced fertility and none of them typica lly withstand even the mildest winters (Galinat 1988; Ellstrand 2007). First

generation hybrids are generally less fit for survival and dissemination in the wild and show substantially reduced reproductive capacity, which thus acts as a significa nt constra int to introgression. Therefore, USDA has concluded that adverse consequences of gene flow from Event 5307 to wild or weedy species in the U.S. are highly unlikely. Potential Impacts on Target and Non
- target Organisms, Including Beneficial Organisms Based on the data provided by the applicant and existing literature, APHIS evaluated the potential for plant pest

- related impacts from Event 5307 on non

- target or beneficial organisms. The genes introduced into Event 5307 result in the production of phosph omannose isomerase (PMI) and eCry3.1Ab proteins.
The pmi gene, which was introduced along with the ecry3.1Ab gene via the same pSYN12274 transformation vector, was employed as a selectable marker during the process of regenerating plant ma terial following transformation (Syngenta 2011, p. 40, Figure V

- 1). The pmi gene was isolated from E. coli , which is a common intestinal bacterium and is considered a non

- allergenic source of proteins (FAO/WHO 2001). The PMI protein is widely distributed in nature and has been found to be expressed in enteric bacteria, fungi, insects, some species of plants, and mammals (69 FR 26770 , Slein 1950). Additionally, on April 25, 2007 EPA granted an exemption from the requirement of a tolerance for the PMI protein as an inert ing redient in all pl ants

. The ecry3.1Ab gene from Bacillus thuringiensis expressed in Event 5307 is a chimeric gene composed of portions of the N

- terminus of mcry3A gene and the C

- terminus of cry1Ab gene (Syngenta 2010, Figures IV

- 1A, IV

- 1B). For decades, microbial products containing B. thuringiensis have been used to control insect pests on a commercial scale and for home garden applications (Glare and O'Callaghan 2 000; OMRI 2011; Shelton 2002). Because Cry protein receptors are not pre sent in non

- target organisms (Glare and O'Callaghan 2000; Hofmann 1988a; Hofmann 1988b; Shimada 2006a; Shimada 2006b; Van Rie 1989; Van Rie 1990), these insecticidal proteins are not expected to adversely

4 http://plants.usda.gov/java/county?state_name=Florida&statefips=12&symbol=ZEME7 affect non

- target invertebrate and vertebrate organ isms (Glare and O'Callaghan, 2000; EPA 2007 ; EPA 2008).
Since the commercialization of Bt crops, a large number of field studies published in the scientific literature have addressed the issue of long range effects of cultivation of Cry proteins on the inv ertebrate community structure where Bt crops are grown. The use of transgenic crops pro ducing Cry proteins has been shown to reduce the use of broad spectrum insecticides 5 without significant impacts on non

- target organisms ( Cattan eo 2006; Dively 2005; Mar vier, 2007; Mendelsohn 2003; Naranjo, 2005; OECD 2007; Romeis 2006; Torres and Ruberson, 2005; Torres and Ruberson, 2007; Yu 2011, Whitehouse 2005). Specifically, a meta

- analysis 6 of the data collected from 42 field studies indicated that non

- target invert ebrates are generally more abundant in Bt cotton and Bt corn fields than in non

- transgenic fields managed with insecticides (Marvier 2007). In addition, a comprehensive review of short and long term field studies on the effects of invertebrate populations in Bt corn and cotton fields indicated that no unreasonable adverse effects are taking place as a result of wide scale Bt crop cultivation (Sanvido 2007). Environmental exposure of non
- target aquatic organisms is generally dependent upon the input of crop debris into streams and waterways. Because of the short

- term exposure and rapid degradation of Cry proteins, there is limited opportunity for exposure 2 weeks after a Cry protein is introduced into the environment by crop debris or pollen shed (Jensen 2010 ; Wolt 2010). Therefore, the level of Cry proteins found in any surrounding aquatic ecosystem is expected to be below that at which biological activity would be observed (Jensen 2010, Syngenta 2010). Assessment of insecticidal transgenic crops should inclu de laboratory tests with indicator species to determine potential toxicity at doses of the toxin higher than would be anticipated under field conditions (Rose 2007). Syngenta submitted data from laboratory and field studies on non

- target representative spe cies at exposure levels in excess of that anticipated in the environment (Syngenta 2010, pp. 134

- 152). Data was submitted for two abo ve
- ground arthropods (insidious flower bug ( Orius insidious )) and spotted ladybird beetle ( Coleomegilla maculata )), two soi l dwelling arthropods (rove beetle ( Aleochara bilineata ) and ground or carabid beetle ( Poecilus cupreus )), a soil dwelling annelid (earthworm ( Eisenia fetida )), a pollinator (honeybee ( Apis mellifera )), two birds (Bobwhite quail ( Colinus virginianus ) and c hicken ( Gallus gallus domesticus )), a mammal (mouse ( M us musculus )), an aquatic arthropod ( fresh water shrimp ( Gammarus fasciatus )), and a fish (catfish ( Ictalurus punctatus )). The data submitted in the petition indicate that no significant adverse effects were observed at the maximum test dose for any of the tested species.

5 Broad spectrum insecticides are chemical insecticides which kill insects that are causing injury to plants and also kill other insects that are not causing injury to the plant. Insects that are inadvertently killed by the application of insecticide are called “non

- target” insects. 6 M eta

- analysis combines the results of several studies that address a set of related research hypotheses. The general aim of a meta

- analysis is to more powerfully estimate the true "effect size" as opposed to a smaller "effect size" derived in a single study under a given single set of assumptions and conditions.8 Event 5307 is expected to be similar with respect to the low potential harm to the environment (Syngenta 2010). Based on the above information, APHIS has concluded that adverse impacts to non

- target organisms exposed to Event 5307 corn are unlikely.
Potential for Transfer of Genetic Information with which Event 5307 Cannot Interbreed Horizontal gene transfe r and expression of DNA from a plant species to bacteria is unlikely to occur (Keese 2008). First, many bacteria (or parts thereof) that are closely associated w ith plants have been sequenced, including Agrobacterium and Rhizobium (Kaneko 2000; Kaneko 2002 ; Wood 2001). There is no evidence that these organisms contain genes derived from plants. Second, in cases where review of sequence data implied that horizontal gene tran sfer occurred, these events are inferred to occur on an evolutionary time scale on th e order o f millions of years (Brown 2003; Koonin 2001). Third, FDA has evaluated horizontal gene transfer from the use of antibiotic resistance genes, and concluded that the likelihood of transfer of antibiotic resistance genes from plant genomes to microo rganisms in the gastrointestinal tract of humans or animals, or in the environment, is remote (FDA 1998). Therefore, APHIS concludes that horizontal gene transfer is unlikely to occur and thus poses no plant pest risk.
Potential for Event 5307 to have Alte red Disease and Pest Susceptibilities
APHIS assess ed whether Event 5307 is likely to have significant ly altered disease and pest susceptibility. This assessment encompassed a thorough consideration of the introduced trait and disease and pest susceptibility data from Event 5307 field trials. Syngenta collected field trial data on Event 5307 from 2005 throug h 2009 (Syngenta 2011, Appendix A). Except for differences in rootworm damage, no observatio ns of differences were observed in pest damage or in disease susceptibility (Syngenta 2011, Table III

- 1, Figure s III

- 3, VIII

- 1, VIII

- 2, p. 101)

. In f ield trials cond ucted in 2007 where gray leaf spot and northern corn leaf blight was present, Event 5307 did not show increased susceptibility to gray leaf spot or northern corn leaf blight diseases (Syngenta 2011 , Table VII

- 8)

. In 2009, no significant differences were observed in the disease ratings for eyespot, southern corn leaf blight and gray leaf spot (Syngenta 2011, Table VII

- 11).
The Agrobacterium transformed plants used in the generation of Event 5307 were treated with an antibiotic to kill the Agrobacterium c ells (Syngenta 2011, p. 26). Furthermore, DNA sequences derived from plant pests that were incorporated in Event 5307 do not result in the production of infectious agents or disease symptoms in plants, and so it is unlikely that Event 5307 could pose a pla nt pest risk. The description of the genetic modifications, including genetic elements, expression of the gene product and their functions for Event 5307 has been summarized above

. APHIS considered whether corn constituents that differed between Event 5307 corn and other corn varieties might be sufficient to lead to increased susceptibility to pathogens or9 pests. The data presented in the petition indicate no difference in compositional and nutritional quality of Event 5307 compared to conventional corn, ap art from the presence of the eCry3.1A b and PMI proteins (Syngenta 2011). None of the values for seed and forage composition characteristics were outside the range of natural variability of conventional corn seed and forage (Syngenta 2011, Table s VII

- 13, VI I

- 14, VII

- 15, VII

16, VII

- 17, VII

- 18, VII

- 19, VII

- 20, VII

- 21). Therefore, the composition of Event 5307 is not biologically different than conventional cor n (with the exception of the eCry3.1Ab and PMI proteins). Based on the known functions and mechanism of actions of these proteins (summarized in Syngenta 2011), none of these proteins are expected to directly alter susceptibility to plant pests. Thus, Event 5307 is expected to be susceptible to the same pests as conventional corn except for a reduction in rootworm damage. Corn is not a plant pest in the U.S., and the introduced DNA in Event 5307 is unlikely to pose a plant pest risk. Based on the analysis of genetic modifications and their functions and field testing data submitted by the petitioner, APHIS concludes that with the exception of resistance to corn rootworm there are no significant differences between Event 5307 corn and the non

- transgenic counterparts relative to pest and disease susceptibility.
Conclusion APHIS has prepared this plant pe st risk assessment in order to determine if Event 5307 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 inserted genetic material, weedy characteristics, atypical responses t o disease or plant pests in the field, effects on non

- target s or beneficial organisms in the agro

- ecosystem, and horizontal gene transfer, APHIS has concluded that Event 5307 is unlikely to pose a plant pest risk.
References Baker, H.G. (1965) Characterist ics and Modes of Origin of Weeds. In : The Genetics of Colonizing Species. H.G. Baker and G.L. Stebbins (eds.). pp. 147

- 172. Academic Press, New York and London. Brown, J.R. (2003) Ancient horizontal gene transfer. Nature Reviews Genetics 4:121

- 132.
Calvi n , D

. (2003) Western and Northern corn rootworm management in Pennsylvania

. Pennslyvania State University Fact Sheet. < http://ento.psu.edu/extension/factsheets/corn

- rootworm > Accessed July 19, 20 11

. Cattaneo, M.G., Yafuso, C., Schmidt, C., Huang, C.

- Y., Rahman, M., Olson, C., Ellers

Kirk, C., Orr, B.J., Marsh, S.E., Antilla, L., Dutilleul, P., Carrière, Y. (2006) Farm

scale evaluation of the impacts of transgenic. Proceedings of the Nat ional Academy of Sciences of the United States of America 10:7571

– 7576.10 Crockett , L

. (1977) Wildly Successful Plants: North American Weeds. University of Hawaii Press, Hawaii. Depicker, A., Stach el, S., Dhaese, P., Zambryski, P., Goodman , H.M. (1982) No paline synthase: transcript mapping and DNA sequence. Journal of Molecular and Applied Genetics 1:561

- 573. Dively, G.P. (2005) Impact of transgenic VIP3A x Cry1Ab lepidopteran

- resistant field corn on the non

- target arthropod community. Environmental Ento mology 34:1267

- 1291.
Ellstrand , N

. C

. , Prentice , H

. C

. , Hancock , J

. F

. (1999) Gene flow and introgression from domesticated plants into their wild relatives. Annual Review of Ecology, Evolution, and Systematics 30:539

- 563. Ellstrand, N.C., Garner, L. C. , Hegde, S., G uadagnuolo, R., Blancas, L. (2007) Spontaneous hybridization between maize and teosinte. Journal of Heredity 98:183

- 187. EPA (2007 ) U.S. Environmental Protection Agency. Biopesticide Registration Action Document. Modified Cry3A Protein and the Genetic Material Necessary for its Production (Via Elements of pZM26) in Event MIR604 Corn SYN

- IR604

- 8. U.S. Environmental Protection Agency, Office of Pesticide Programs, Biopesticides and Pollution Prevention Division, Washington, D.C. pp.106.
EPA ( 2008) U.S. Environmental Protection Agency. Biopesticide Registration Action Document

. Bacillus thuringiensis modified Cry1Ab (SYN

- IR67B

- 1) and Vip3Aa19 (SYN

- IR102

- 7) insecticidal proteins and the genetic material necessary for their production in COT102 XCOT67B cotton, U.S. Environmental Protection Agency, Office of Pesticide Programs, Biopesticides and Pollutio n Prevention Division, Washington, D.C. pp. 135. FAO/WHO (2001) (Food and Agriculture Organization of the United Nations/World Health Organization) Evaluation of Allergenicity of Genetically Modified Foods. Report of a Join FAO/WHO Expert Consultation on Allergenicity of Foods Derived from Biotechnology. Rome, Italy. < ftp://ftp.fao.org/es/esn/food/allergygm.pdf > Accessed September 15, 2011. FDA ( 1998) Guidance for Industry: Use of Antibiotic Res istance Marker Gen es in Transgenic Plants. < http://www.fda.gov/Food/GuidanceComplianceRegulatoryInformation/GuidanceDo cuments/Biotec hnology/default.htm > Accessed August 24, 2011

. Galinat, W .C. (1988) The origin of corn. In : Corn and corn improvement. G.F. Sprague and J.W. Dudley (eds.). pp. 1

- 31. American Society of Agronomy, Crop Science Society of America, and Soil Science Society of America, Madison, WI.11 Glare, T. R ., O’Callaghan , M. (2000) Bacillus thuringiensis Biology, Ecology and Safety. John Wiley & Sons, Chichester. 350pp.
Gould FW (1968) Grass Systematics. McGraw

- Hill, New York. Hofmann, C., Luthy, P., Hutter, R., Pliska , V. (1988a) Binding of the delta endotoxins from Bacillus thuringiensis to brush

- border membrane vesicles of the cabbage butterfly ( Pieris brassicae ). European Journal of Biochemistry 173:85

- 91. Hofmann, C., Vanderbruggen, H., Höfte, H., Van Rie, J., Jan sens, S., Van Mellaert, H. (1988b) Specificity of Bacillus thuringiensis δ

- endotoxins is correlated with the presence of high

- affinity binding sites in the brush border membrane of target insect midguts. Proceedings of the National Academy of Sciences of t he United States of America 85:7844

- 7848. Holm, L.G., Pancho, J.V., Herberger, J.P., Plucknett, D.L. (1991) A Geographical Atlas of World Weeds. Krieger Publishing Company, Malabar, Florida. Hohn, T., Stavolone, L., DeHann, P

. , Ligon, H., Kon onova, M. (200 7) Cestrum yellow leaf curling virus promoters. U.S. Patent No. 7,166,770. Washington, D.C.: U.S. Patent Office.
Honee , G

. , Convents , D

. , Van Rie , J

. , Jansens , S

. , Peferoen , M

. , Visser , B. (1991) The C

terminal domain of the toxic f ragment of a Bacillus thuringiensis crystal protein determines receptor b inding. Molecular Microbiology 5:2799

- 2806. Jensen, P.D., Dively, G.P, Swan, C.M, Lamp, W.O. (2010) Exposure and nontarget effects of transgenic Bt corn debris in streams. Environmental Entomology 39:707

- 7 14. Kaneko, T. et al. (2000) Complete genome structure of the nitrogen

- fixing symbiotic bacterium Mesorhizobium loti . DNA Research 7:331

- 338. Kaneko, T. et al. (2002) Complete genomic sequence of nitrogen

- fixing symbiotic bacterium Bradyrhizobium japonicum USDA110. DNA Research 9:189

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

- 1139

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

- 149. Koonin, E.V., Makarova, K.S., Aravind, L. (2001) Horizontal gene transfer in prokaryotes: quantification and classification . Annual Review of Microbiology 55:709

- 742.12 Marvier, M., McCreedy, C., Regetz, J. Kareiva, P. (2007) A meta

- analysis of effects of Bt cotton and maize on nontarget invertebrates. Science 316: 1475

– 1477. Mendelsohn, M., Kough, J., Vaituzis, Z., Matthews, K. (2003) Are Bt crops safe? Nature Biotechnology 21:1003

- 1009.
Muenscher , W

. C

. (1980) Weeds. 2nd edition. Cornell University Press, Ithaca and London. Naranjo, S.E. (2005) Long

- term assessment of the effects of transgenic Bt cotton on the function of the natural enemy community. Environmental Entomology 34:1211

- 1223.
NAS (2004) National Academy of Sciences. Safety of genetically engineered foods: Approaches to Assessing Unintended Health E ffects. National Academy Press, Washington, D.C. < http://www.nap.edu/openbook.php?record_id=10977&page=147 > Accessed August 22, 2011

. Negrotto, D., Jolley, M., Beer, S., Wenck, A.R., Hansen, G. (2000) The use of phosphomannose

- isomerase as a selec Table marker to recover transgenic maize plants ( Zea mays L.) via Agrobacterium transformation. Plant Cell Reports 19:798

- 803.
OECD (2003) Organisation for Economic Co

- operation and Development Consensus

. Document on the Biology of Zea mays subsp. mays (Maize). p p
. 1

- 49. OECD. (2007) Organisation for Economic Co

- operation and Development. Consensus Document of Safety Information on Transgenic Plants E xpressing Bacillus thuringiensis Derived Insect Control P roteins. pp 1

- 108. OMRI (2009) Organic Materials Review Institute Products List < http://www.omri.org/OMRI_products_list.html> Accessed September 15 , 2011.
Peairs , F

. B

. , Pilcher , S

. D

. (2010) Western Corn Rootworm. Insect Series No. 5.570. Colorado State University. < http://www.ext.colostate.edu/pubs/insect/05570.pdf > Accessed July 19, 2011. Rieseberg, L.H., Wendel, J.F. (1993) Introgression and its Consequences in Plants. In : Hybrid Zones and the Evolut ionary Process. R.G. Harrison (ed.). pp. 70

- 109. Oxford University Press, Oxford. Rose, R. (ed.) (2007) White paper on tier

- based testing for the effects of proteinaceous insec ticidal plant

- incorporated protectants on non

- target arthropods or regulatory risk assessments. Available at: http://www.aphis.usda.gov/brs/pdf/NTO_White_Paper_1.pdf13 Romeis, J., Meiss le M., Bigler, F. (2006) Transgenic crops expressing Bacillus thuringiensis toxins and biological control. Nature Biotechnology 24:63

- 71. Sanvido,O., Romeis, J., Bigler, F. (2007). Ecological Impacts of Genetically Modified Crops: Ten Years of Field Resea rch and Commercial Cultivation. Adv Biochem Engin/Biotechnol 107: 235

– 278. Shelton, A.M., Zhao, J.

- Z,, Roush, R.T. (2002) Economic, ecological, food safety, and social consequences of the deployment of Bt transgenic plants. Annual Review of Entomology 47 :845

- 881. Shimada, N., Murata, H., Mikami, O., Yoshioka, M. , Guruge , K

. S

. , Yamanaka , N

. , Nakajima , Y

. , Miyazaki , S

. (2006a) Effects of feeding calves genetically modified corn Bt11: A clinico

- biochemical study. Journal of Veterinary Medical Science 68:111 3

- 1115.
Shimada , N

. K

. , Miyamoto , K

. , Kanda , K

. , Murata , H

. (2006b) Bacillus thuringiensis insecticidal Cry1Ab toxin does not affect the membrane integrity of the mammalian intestinal epithelial cells: an in vitro study. In Vitro Cellular & Developmental Biology

- Animal 42:45

- 49. Slein, M.W. (1950) Phosphomannose isomerase. Journal of Biological Chemistry 186:753

- 761. Soltis, D.E., Soltis, P.S. ( 1993) Molecular data and the dynamic nature of polypoloidy. Critical Reviews in Plant Science s 12:243

- 273. Stace, C.A. (1987) Hybrid ization and the plant species. In : Differentiation Patterns in Higher Plants. K.M. Urbanska (ed.). pp. 115

- 127. Academic Press, New York. Stavolone, L., Kononova, M., Pauli, S., Ragozzino, A., de Hann , P., Milligan, S., Lawton, K., Hohn, T. (2003) Cestrum yellow leaf curling virus (CmYLCV) promoter: a new strong constitutive promoter for heterologous gene expression in a wide variety of crops. Plant Molecular Biology 53: 703

- 713. Syngenta (2011) Petit ion for Determination of Nonregulated Status for Rootworm

Resistant Event 5307 Corn. Submitted by D. Vlachos. Syngenta Biotechnology, Inc., Research Triangle Park, NC < http://www.aphis.us da.gov/biotechnology/not_reg.html >.
Torres , J.B, Ruberson, J. R. (2005) Canopy

- and ground

- dwelling predatory arthropods in commercial Bt and non

- Bt cotton fields: patterns and mechanisms. Environmental Entomology 34:1242

- 1256.14 Torres, J.B., Ruberson, J.R. (2007) Abundance and diversity of ground

- dwelling arthropods of pest management importance in commercial Bt and non

- Bt cotton fields. Annals of Applied Biology 150:27

- 39.
USDA

- AMS (2004 ) United States Department of Agriculture. Agricultural Marketing Service. Plant Variety Protection Office. < http://apps.ams.usda.gov/CMS/ >

. USDA

- ERS (2011) United States Department of Agriculture. Economic Research Service < http://w ww.ers.usda.gov/ >. Walters, F., deFontes , C.M., Hart, H., Warren, G.W., Chen, J.S. (2010) Lepidopteran

active variable

- region sequence imparts Coleopteran activity in eCry3.1Ab, an engineered Bacillus thuringiensis hybrid insecticidal protein. Applied and Environmental Microbiology 76:3082

- 3088. Whitehouse, M.E.A., Wilson, L.J., Fitt, G.P. (2005) A comparison of arthropod communities in transgenic Bt and conventional cotton in Australia. Environmental Entomology 34:1224

- 1241. Wolt, J.D., Peterson, R.K.D. (2010) Prospective formulation of environmental risk assessments: probalistic screening for Cry1A(b) maize risk to aquatic insects. Ecotoxicology and Environmental Safety 73:1182

- 1188.
Wood , D

. W

. , et al. (2001) The genome of the natural genetic engineer Agrobacterium tumefaciens C58. Science 294:2317

- 2323. Yu, H.

- L., Li, Y.

- H., Wu, K.

- M. (2011) Transgenic Bacillus thuringiensis crops on non
target organisms. Journal of Integrative Plant Biology 53:520

- 538.