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Plant Pest 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 the Plant Protection Act of 20001. 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 line NP222 

with Agrobacterium tumefaciens. Because A. tumefaciens is a plant pest and some of the 

regulatory sequences (promoter from Cestrum yellow leaf curling virus and terminator 

from A. tumefaciens) used to facilitate expression of the genes in corn were 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 not a plant pest risk, then APHIS has no regulatory authority over that organism.

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 needed 

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 this 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 species 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 agent or other pathogen. (H) Any article similar to or allied with any of the articles specified in the preceding 

subparagraphs.”
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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 hazardous 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 

responsible for the single largest use of 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 has genetically engineered corn to express an eCry3.1Ab protein for use 

in the control of coleopteran 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. longicornis 

barberi Smith and Lawrence) and Mexican corn rootworm (D. virgifera zeae Krysan and 

Smith) (Peairs and Pilcher 2010). 

GE corn containing insect-resistant traits, first approved in the U.S. in 1996, has been 

widely 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 engineered crops in the U.S., 1996-2011 

(USDA ERS 2011).
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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 protectant 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, Figures III-1, III-2). Immature embryos of Syngenta inbred 

corn line NP2222 (Plant Variety Protection Certificate 200200071; USDA-AMS 2004) 

were infected with A. tumefaciens strain LBA4404 containing plasmid SYN12274. Plants 

containing the introduced DNA were selected based on survival on a mannose substrate 

due to the expression of phosphomannose 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).

eCry3.1Ab – expression cassette consists of three genetic elements (Syngenta 2011, 

Figure III-1, Table IV-1). 
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• The Cestrum Yellow Leaf Curling Virus (CmYLCV, Caulimoviridae dsDNA 

plant virus2) genomic full-length transcript promoter (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 III) 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, 

Figures V-4, V-5, Table V-2); (2) a single copy of the ecry3.1Ab gene (Syngenta 2011, 

Figures V-2, V-3, Table V-1); (3) two copies of the NOS terminator (Syngenta 2011, 

Figures V-10, V-11, Table V-5); and (4) a single copy of the pmi gene (Syngenta 2011, 

Figures V-6, V-7, Table V-3). Southern blot analysis demonstrated the absence of the 

plasmid backbone elements (Syngenta 2011, Figures V-12, V-13, Table V-6). Data from 

Southern analysis was provided and reviewed by APHIS that demonstrates stable

integration and inheritance of the 5307 corn insert over four generation (Syngenta 2011, 

Figures V-14, V-15, V-16, Table V-7).

Potential of 5307 to Become Invasive and/or a Weed

Corn possesses 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 3603

). 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 previous 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 the 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.pdf
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APHIS assessed whether Event 5307 is any more likely to become a weed than a nearisogenic non-transgenic corn line. The assessment encompasses a thorough consideration 

of the basic biology of corn and an evaluation of the unique characteristics of Event 5307 

(Syngenta 2011 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 Tables 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 phenotypic endpoints 

assessed in these field trials or in greenhouse or laboratory experiments (Syngenta 2011, 

Tables 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, dormancy, plant 

emergence, plant height, ear height, stalk lodging, barren plants, timing of pollen shed or 

silking, pollen viability and morphology, and yield (Syngenta 2011, Tables VII-7, VII-8, 

VII-9, VII-10, VI-12, Figure VII-1). None of the measure attributes 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, Event 5307 lacks the ability to persist as a troublesome weed, 

and there would be no significant impact on current weed management practices for corn 

cultivation. 

Potential for Gene Flow and Gene Introgression from Event 5307 into SexuallyCompatible 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 from 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 reported to be 

widespread (Stace 1987; Rieseberg 1997). It has been a common 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 relatives and considered whether such introgression would 

result in increased 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 (OECD 2003). The closest wild relatives of corn, 

various Zea species referred to as teosinte, are normally confined to the tropical and 
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subtropical regions of Mexico, Guatemala, and Nicaragua. In the U.S. a fairly rare, 

sparsely dispersed feral population of teosinte has been reported in Florida (USDA 

PLANTS Database4

, 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 

typically withstand even the mildest winters (Galinat 1988; Ellstrand 2007). Firstgeneration hybrids are generally less fit for survival and dissemination in the wild and 

show substantially reduced reproductive capacity, which thus acts as a significant 

constraint 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 

phosphomannose 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 material 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 ingredient in all plants.

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 2000; OMRI 2011; Shelton 

2002). Because Cry protein receptors are not present 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=ZEME
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affect non-target invertebrate and vertebrate organisms (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 invertebrate community structure where Bt crops are grown. The use of 

transgenic crops producing Cry proteins has been shown to reduce the use of broad 

spectrum insecticides5 without significant impacts on non-target organisms (Cattaneo 

2006; Dively 2005; Marvier, 2007; Mendelsohn 2003; Naranjo, 2005; OECD 2007; 

Romeis 2006; Torres and Ruberson, 2005; Torres and Ruberson, 2007; Yu 2011, 

Whitehouse 2005). Specifically, a meta-analysis6 of the data collected from 42 field 

studies indicated that non-target invertebrates 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 include 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 species at exposure levels in excess of that 

anticipated in the environment (Syngenta 2010, pp. 134-152). Data was submitted for two 

above-ground arthropods (insidious flower bug (Orius insidious)) and spotted ladybird 

beetle (Coleomegilla maculata)), two soil 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 chicken (Gallus gallus domesticus)), a 

mammal (mouse (Mus 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 Meta-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.
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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 transfer 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 with 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 transfer occurred, these events are inferred to occur on an 

evolutionary time scale on the order of 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 microorganisms 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 Altered Disease and Pest Susceptibilities

APHIS assessed whether Event 5307 is likely to have significantly 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 through 2009 (Syngenta 

2011, Appendix A). Except for differences in rootworm damage, no observations of 

differences were observed in pest damage or in disease susceptibility (Syngenta 2011, 

Table III-1, Figures III-3, VIII-1,VIII-2, p. 101). In field trials conducted 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 cells (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 plant 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 or 
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pests. The data presented in the petition indicate no difference in compositional and 

nutritional quality of Event 5307 compared to conventional corn, apart from the presence 

of the eCry3.1Ab 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, Tables VII-13, VII-14, VII-15, VII16, VII-17, VII-18, VII-19, VII-20, VII-21). Therefore, the composition of Event 5307 is 

not biologically different than conventional corn (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 pest 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 to disease or plant pests in the field, effects on non-targets 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.

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