Plant Pest Risk Assessment for HCEM485 Corn

APHIS’ authority to regulate genetically engineered (GE) organisms under the Plant Protection 

Act (PPA) (7 U.S.C. Sec 7701 et seq.) is limited to those GE organisms that are plant pests as 

defined under Section 403(14) of the PPA (7 U.SC. 7702(14)

1

). APHIS regulations at 7 CFR part 

340, which were promulgated in 1987 under the authority of the Federal Plant Pest Act and Plant 

Quarantine Act, provided APHIS with the mechanism to regulate articles altered or produced 

through genetic engineering as plant pests and that the Administer considers a plant pest based 

upon the definition of a regulated article (7 CFR part 340.1). In 1993, 7 CFR part 340 was 

revised to include a process whereby a person may petition the agency to make a determination 

that a regulated article should not be regulated (7 CFR part 340.6). Required data and 

information (as per 7 CFR part 340.6(c)) is submitted to the agency to demonstrate that the 

article that is the subject of the petition does not pose a greater plant pest risk than the organism 

from which it was derived and that it should no longer be subject to the regulations of 7 CFR part

340. If APHIS determines that a GE organism is unlikely to pose a plant pest risk, APHIS then 

has no regulatory authority under 7 CFR Part 340 or the plant pest provisions of the Plant 

Protection Act to continue to regulate that organism. Additionally, under 7 CFR part 340.6(e), a 

person may request that APHIS extend a determination of nonregulated status, based on 

similarity of a regulated article to an antecedent organism which has previously been determined 

to be unlikely to pose a plant pest risk and is no longer subject to the plant pest provisions of the 

Plant Protection Act and 7 CFR part 340.

Stine Seed Farm, Inc. (hereafter referred to as Stine Seed), has submitted to APHIS an extension 

request for a determination of nonregulated status of GE corn (Zea mays) event HCEM485, 

based on similarity to the corn event GA21 for which a petition (97-099-01p) for nonregulated 

status was approved in 1997 (62 FR 64350). The Stine Seed extension request for HCEM485 

corn was originally received on March 4, 2009. A revised version (09-063-01p_a3) received on 

April 1, 2011 (Stine, 2011) was deemed technically complete and is the version of the petition 

referenced in this document. Both HCEM485 corn and GA21 corn are similarly genetically 

engineered for resistance to the herbicide glyphosate. This plant pest risk assessment was 

conducted to determine whether, as with the antecedent GA21 corn, event HCEM485 corn is 

unlikely to pose a greater plant pest risk than the unmodified organism from which it was 

derived. If the submitted extension request provides evidence of sufficient similarity to the 

antecedent GA21, allowing APHIS to make a determination that HCEM485 is unlikely to pose a

1 The term “plant pest” is defined in the PPA as 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) protozoan.

(B) A nonhuman animal.

(C) A parasitic plant.

(D) bacterium.

(E) A fungus.

(F) 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.

1
plant pest risk, HCEM485 and its progeny will no longer be considered regulated articles under

regulations at 7 CFR part 340.

Potential impacts to be addressed in this risk assessment are those that pertain to the use of 

HCEM485 corn and its progeny in the absence of confinement. APHIS examined information 

submitted by the petitioner (Stine, 2011) as well as information contained in our determination 

of nonregulated status and the supporting documents for the antecedent organism GA21 (as per 7

CFR part 340.6(c)(4)) related to plant pest risk characteristics, including disease and pest 

susceptibilities, expression of the gene product, new enzymes or changes to plant metabolism, 

weediness (including control options) of the regulated article, and any impacts on the weediness 

of any other plant with which it can interbreed, effects of the regulated article on non-target 

beneficial organisms, changes to agricultural and cultivation practices that might impact plant 

pest control measures, and potential for plant pest effects from horizontal gene transfer. APHIS 

also examined information submitted by the petitioner (as per 7 CFR part 340.6(e)(2)) related to 

the similarity of the regulated article to the antecedent organism GA21. Other issues related to 

agricultural or cultivation practices (including but not limited to those related to changes in 

herbicides used to control weeds in corn) and other impacts to the environment are examined in 

the separately drafted Environmental Assessment for HCEM485 corn.

APHIS may also consider information relevant to reviews conducted by other agencies that are 

part of the ‘Coordinated Framework for the Regulation of Biotechnology’ (51 FR 23302, June 

26, 1986). Under the Coordinated Framework, the oversight of biotechnology-derived plants 

rests with the APHIS, the Food and Drug Administration (FDA), and the Office of Pesticide 

Programs of the U.S. Environmental Protection Agency (EPA). Depending on its characteristics, 

certain biotechnology-derived products are subjected to review by one or more of these agencies. 

The EPA under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA), regulates the 

distribution, sale, use and testing of pesticidal substances produced in plants and microbes, 

including those pesticides that are produced by an organism through techniques of modern 

biotechnology. The EPA also sets tolerance limits for residues of pesticides, including 

herbicides, on and in food and animal feed, or establishes an exemption from the requirement for 

a tolerance, under the Federal Food, Drug and Cosmetic Act (FFDCA). HCEM485, as well as

the antecedent organism GA21, are not engineered to express pesticidal substances, however 

tolerance levels for residues of glyphosate have already been established for GA21 corn. 

Supplemental labels are required to allow over-the-top applications of glyphosate herbicide on 

crops genetically modified for glyphosate resistance and have already been approved for GA21 

corn. The FDA under the FFDCA is responsible for ensuring the safety and proper labeling of all 

plant-derived foods and feeds, including those developed through modern biotechnology. To 

help sponsors of foods and feeds derived from genetically engineered crops comply with their 

obligations, the FDA encourages them to participate in its voluntary consultation process (57 FR 

22984). Stine Seed has submitted a food and feed safety and nutritional assessment for events 

HCEM485 on Dec. 27, 2010, and received a completed consultation letter from the FDA in July 

2012 (BNF 106). Likewise,, a consultation process has been completed for GA21 (BFN 51) and

both letters are available for public review via the FDA Completed Consultations on

Bioengineered Foods page at www.fda.gov/bioconinventory.

History of Development of HCEM485 Glyphosate Herbicide-Resistant Corn

HCEM485 corn is a GE corn line that has been provided resistance to treatment with the 

herbicide glyphosate. The particular genetic element conferring this trait is an altered sequence 

of DNA derived from the genome of corn. Specifically, Stine Seed removed a 6 kilobase (six 
thousand nucleotides) section of DNA from an inbred corn line, modified the 5-

enolpyruvylshikimate-3-phosphate synthase (epsps) gene contained within that DNA section, 

and then reintroduced the DNA into corn. The modified genetic sequence of the epsps gene 

encodes a double mutated 2mEPSPS enzyme product identical to that produced in the nonregulated GE corn line, GA21, that is also resistant to glyphosate (Funke et al. 2009), although 

the regulatory sequences controlling expression of the gene do differ between HCEM485 and 

GA21 (see “Description of the Inserted Genetic Material...” below).

A petition request for a determination of nonregulated status of the first corn line containing a 

glyphosate resistance trait produced through the use of biotechnology was approved by APHIS in 

1995, and since that time several other corn lines containing glyphosate resistance have also been 

determined by APHIS to be no longer subject to the regulatory requirements of 7 CFR part 340 

or the plant pest provisions of the Plant Protection Act. In 2011, approximately 72% of corn 

planted in the United States possessed resistance to an herbicide that was conferred through 

biotechnology (USDA-ERS, 2011). The primary herbicide resistance trait in use is glyphosate 

resistance, and the adoption of this trait in other major crops, such as cotton and soybean, is even 

higher. Weed resistance to herbicides is a concern in agricultural production, and the widespread adoption of herbicide resistant crops, especially GE-derived glyphosate-resistant crops, 

has dramatically changed the approach that farmers take to avoid yield losses from weeds 

(Vencill et al., 2012).

Glyphosate-resistant crops have become widely adopted since their introduction in the mid-late 

1990s for several reasons. Glyphosate works non-selectively on a wide range of plant species, is 

a relatively low-cost herbicide, facilitates ‘no-till’ farming practices, and has minimal animal 

toxicological and environmental impact (Gianessi, 2008; Duke and Powles, 2009)

Stine Seed conducted confined field tests of HCEM485 corn in the United States beginning in 

2005 under requested notifications acknowledged by APHIS (Stine 2011, page 50 and Appendix 

2), and has provided data in the petition from several of those confined field trials.

If a determination of nonregulated status is extended to HCEM485, this cultivar is expected to 

add to the options that growers have when deciding on a glyphosate-resistant corn line. It is not 

expected that the availability of HCEM485 will significantly impact the adoption rate of 

glyphosate resistant corn crops, nor will it greatly impact current or future trends of glyphosate 

resistance among weed species. These specific issues are discussed in greater detail in the 

Environmental Assessment that is written for HCEM485.

Description of the Inserted Genetic Material in HCEM485 and the Antecedent

The similarity of the transformation process and inserted genetic elements and gene products 

expressed in HCEM485 corn compared to the antecedent are summarized here and inform the

plant pest risk assessment.

HCEM485 corn was produced using an aerosol beam injector (Held, 2004) to introduce a 6.0 

kilobase corn genomic fragment, originally isolated from a bacterial artificial chromosome 

(BAC) corn DNA library. The method of direct DNA introduction is a similar method to the 

particle bombardment, or “biolistics”, approach used to generate the antecedent event GA21. 

The original DNA from the BAC library was modified to incorporate the desired change to 

EPSPS functionality, however all genetic material inserted by aerosol beam injection was 

otherwise entirely derived from corn DNA. All promoter, intron, and terminator sequences that 
inserted into HCEM485 are the same as those already associated with the native EPSPSencoding gene in corn. No DNA from plant pests, nor any transformation method or vector 

involving a plant pest, was used to produce HCEM485 corn. The only modification to the corn 

DNA sequence is the introduction of two single-nucleotide alterations, which results in two 

changes to the amino acids (Threonine-102→Isoleucine and Proline-106→Serine) of the 

expressed EPSPS protein. Corn plants containing the inserted genetic material were initially 

isolated by growing them on selective media containing glyphosate. Stine Seed used a Southern 

blot analysis to confirm insertion and to estimate the number of copies inserted into the recipient 

corn product, HCEM485 (Stine 2011, pages 14-18). Evidence from the submitted petition 

establishes that multiple copies of the modified epsps gene were inserted at a single locus (also 

similar to the antecedent) and that there does not appear to be any truncated or abnormally sized 

protein products resulting from the inserted genetic material (as determined by western blot 

analysis) (Stine 2011, pages 20-22).

The notable difference between HCEM485 and GA21 is the inserted non-coding DNA 

regulatory sequences before and after the sequences encoding the modified EPSPS protein. In 

HCEM485 all regulatory sequences up- and down-stream of the modified epsps are derived 

directly from native corn DNA, whereas in GA21 those sequences are derived from various 

DNA isolated from other organisms, including rice (Oryza sativa), sunflower (Helianthus annus) 

and the bacterium Agrobacterium tumefaciens that is listed as a plant pest in 7 CFR part 340.2 

(see Stine 2011, Table 1). As APHIS has already approved the petition request for a 

determination of nonregulated status of GA21and therefore GA21 is not considered to present a

plant pest risk, these differences in the introduced DNA do not provide any increase in the plant 

pest risk potential for HCEM485 compared to GA21.

Expression of the Gene Product, Enzymes, or Changes to Plant Metabolism

APHIS assessed whether changes in plant metabolism or composition in HCEM485 are likely to 

alter plant pest risk. The assessment encompasses a consideration of the expressed modified 

epsps gene and its effect on plant metabolism, and an evaluation of whether HCEM485 is 

nutritionally equivalent to other corn, as has been determined for GA21. The modified epsps 

gene inserted does not replace the native, unmodified epsps gene or EPSPS protein; both 

modified and unmodified versions of the gene and protein are present within HCEM485. The 

introduced gene responsible for conferring the herbicide resistance encodes a form of EPSPS that 

is insensitive to the herbicide glyphosate, similar to the antecedent GA21. This gene encodes a 

protein that differs from the native corn EPSPS protein by two key amino acids, which is the

4
same modification as introduced into GA21 (Funke, 2009) making the EPSPS enzyme 

significantly less sensitive to the presence of glyphosate compared to the native form (see Stine 

2011, Section IV for further details of the inserted genetic material and resulting protein 

product). The engineered change does not impact the essential cellular functions of the EPSPS 

enzyme, and there is no evidence that the protein contributes to any changes to the agronomic 

properties, disease susceptibility, or composition of HCEM485 corn compared to other 

commercially available corn (see “Potential Impacts of Genetic Modifications on Altered 

Disease and Pest Susceptibilities” below). The only expected difference is the desired change in 

sensitivity to the herbicide glyphosate. GA21 and its progeny, containing a similar form of 

modified EPSPS, have been available as nonregulated corn plant varieties since 1997, alongside 

other varieties of glyphosate resistant corn that also express glyphosate resistant versions of the 

EPSPS enzyme (e.g. NK603, MON 88017, and MON 802). The wide and successful adoption of 

these very similar corn varieties establishes that extending a determination of nonregulated status 

to glyphosate resistant corn line HCEM485 is unlikely to present new plant pest risk issues.

The data presented in the submitted request also indicate no substantive difference in 

compositional and nutritional or anti-nutritional quality of HCEM485 corn treated with 

glyphosate at the normal commercial application rate compared to other commercially available 

corn (Stine 2011, Tables 12 -20). None of the values for the forage and grain composition 

characteristics were outside the range of natural variability of conventional corn reported by the 

International Life Sciences Institute Crop Composition Database (ILSI, 2006) and/or in the 

OECD consensus document on corn composition (OECD, 2002). Stine Seed also evaluated 

levels of phytosterols, a common component of plants, and did not find significant differences in 

mean levels of total phytosterols or individual phytosterols (cholesterol, campesterol, 

stigmasterol, β-sitosterol, stigmastanol) between grain samples from HCEM485 and the 

combined samples of the control hybrids. Although host plant quality and composition is known 

to affect herbivore performance and fecundity (Awmack and Leather, 2002) and secondary 

metabolites in plants can affect defense against microbes (Dixon, 2001), the similar 

compositional profiles between HCEM485 and other commercial corn cultivars would suggest 

that pest populations or infestations would not be expected to be altered if a determination of 

nonregulated status is extended to HCEM485. The collective data and the similarity of 

HCEM485 to other commercially produced corn including GA21, support the petitioner’s claim 

that HCEM485 corn does not have increased susceptibility to any insects, plant pathogens, or 

other plant pests.

Potential Impacts of Genetic Modifications on Altered Disease and Pest 

Susceptibilities

USDA-APHIS assessed whether HCEM485 corn is likely to have significantly increased disease 

and pest characteristics or susceptibility compared to both nonregulated GE corn line GA21 and 

non-GE control corn.

Corn is not a plant pest in the United States (USDA-APHIS, 2000). Furthermore, none of the 

inserted genetic sequences are derived from any plant pests listed in 7 CFR part 340.2. The

description of the genetic modification, including the introduced genetic elements and the

resulting expression and function of the modified EPSPS enzyme in HCEM485 corn, has been 

summarized above. Stine Seed conducted agronomic field trials during the 2007 growing

season across 15 locations in 9 states, representative of the major corn-growing areas of the

U.S., to evaluate hybrids of HCEM485 in comparison to three control hybrids derived from the

same inbred lines used to generate the HCEM485 hybrids. (Stine 2011, pages 23-43). Stine
Seed routinely monitored their corn field trials for corn diseases such as Southern rust disease,

gray leaf spot, Northern maize leaf blight, common rust and smut (Stine 2011, page 30). There

were no meaningful differences in agronomic characteristics that might be indicative of altered 

sensitivity to pests and diseases between HCEM485 glyphosate resistant corn derived hybrids, 

and the control counterparts lacking the glyphosate resistant trait under standard corn 

management practices and without glyphosate treatment. Comparison of disease ratings in one

or more of four sites (one each in Iowa, Nebraska, Minnesota, and Illinois) where the

individual disease incidence was sufficiently high to warrant assessment did not show any 

remarkable differences between the HCEM485 and control hybrids. The data submitted by

Stine Seed confirms that the inserted genetic material did not confer any altered disease or pest 

susceptibility to HCEM485 corn.

In summary, all evidence reviewed by APHIS indicate that as with GA21 corn, HCEM485 corn 

is no more susceptible to plant pathogens and other plant pest species than other commercially 

available corn, and that there is no added plant pest risk that results from the insertion and 

expression of the genetic material described in the Petition. It therefore follows that relative to 

other commercially available corn, including GA21, there should be no indirect plant pest effects 

on other agricultural products.

Potential Impacts Based on the Relative Weediness of HCEM485 Corn

Corn is not typically considered as a weed (Crockett, 1977; Holm et al., 1979; Muenscher, 1980) 

and is not listed on the Federal noxious weed list (7 CFR part 360). Corn possesses few of the 

characteristics of notably successful weeds and is grown as a crop throughout the world without 

any report that it is a serious weed or that it forms persistent feral populations (Baker, 1965; 

Keeler, 1989). However, as is the case with other crop cultivation, after harvest any seeds left in 

the field can germinate and become volunteers during subsequent cropping seasons. A common 

example of this is the appearance of corn seedlings in soybean fields following a corn crop, 

associated with reduced soybean yields (Beckett and Stoller, 1988). Compared to other corn

varieties, HCEM485 has improved fitness (reduced injury) in the presence of glyphosate

herbicides, which translates into fewer options for the removal of volunteer plants. Nonetheless, 

similar to the antecedent GA21 and other glyphosate tolerant corn varieties, there are many

available options for the control of HCEM485 plants so that growers can avoid losses in fields

where unwanted HCEM485 might be growing.

APHIS assessed whether HCEM485 corn is any more likely to become a weed than any other 

corn varieties currently under cultivation. This assessment encompasses a thorough 

consideration of the basic biology of corn and an evaluation of the unique characteristics of 

HCEM485 corn evaluated under field conditions. Stine Seed conducted agronomic field trials 

during the 2007 growing season across 15 locations representative of the major corn-growing 

areas of the U.S. (Stine 2011, pages 25-30 and Annex 1). Though not all traits were assessed at 

all locations, up to 17 different agronomic characteristics were assessed, including the percent of 

barren plants, percent dropped ears, early growth rating, early stand count, seedling vigor, ear 

height, early root lodging, grain moisture percent, final stand count, heat units to 50% pollen 

shed, heat units to 50% silking, leaf color ratings, plant height, root lodging rating, stalk lodging 

rating, grain weight, and grain yield. No differences in phenotypic characteristics that would 

contribute to enhanced weediness were observed between HCEM485 corn and control lines for 

the range of phenotypic endpoints assessed. There were some statistically significant differences 

between from HCEM485 corn and control counterparts. The significant differences that were

seen in ear height, plant height, heat units to 50% silking, heat units to 50% pollen shed, grain
yield, and final stand count are actually representative of very small differences, compared to the

overall range of corn, and these differences did not appear both genotype specific and consistent 

across tested field locations (Stine 2011, pages 28-30 and Annex 1).

Based on the agronomic field data and literature survey about corn weediness potential, 

HCEM485 corn lacks the ability to persist as a troublesome or invasive weed. Still, because 

HCEM485 corn might grow in areas where it is undesired, the GE corn, similar to other corn 

varieties, would be considered a weed (Beckett and Stoller, 1988). Because the genetic elements 

inserted into HCEM485 corn confers greater resistance to glyphosate herbicides the measures 

that can be used to remove volunteer HCEM485 corn are more limited compared to other 

glyphosate-sensitive corn varieties. Nonetheless, as discussed in the next paragraph, most of the 

currently used control measures for removing volunteer corn are also available for the removal of 

HCEM485 corn. Furthermore, similar glyphosate resistant corn plants, such as GA21 and its 

progeny, are already widely distributed for agricultural production and growers are familiar with 

procedures for removing any unwanted corn that is glyphosate resistant.

Corn is commonly grown in rotation with other crop varieties including, but not necessarily 

limited to, oats, forage crops, peanut, wheat, rye, cotton, and soybean. Corn seed left in a field 

after harvest is common, and that seed can germinate, or “volunteer,” during the cultivation of

the following rotational crop. If volunteer corn is not destroyed or removed, the subsequent crop 

planting may have lower yields and reduced value due to competition and mixing with the

volunteers (Beckett and Stoller, 1988; Beckie and Owen, 2007). The advent and wide adoption of

herbicide resistant crops have already changed the approaches growers can adopt to reduce crop

yield losses

7
resulting from volunteer corn. For example, glyphosate-resistant soybean plants, grown after 

glyphosate-resistant corn, can have reductions in soybean yield, due to competition with 

volunteer corn, if glyphosate is the only weed control method used (Soltani et al., 2006). Similar 

studies have also been done for corn-cotton rotations (Thomas et al., 2007; Clewis et al., 2008) 

and corn-wheat rotations (Martin, 2008). Similar to other corn varieties, including the GA21 

antecedent, HCEM485 plants or seed left in a field after harvest may be considered a weed when 

present during the subsequent planting of other crops, including rotational crops such as soybean. 

When this occurs, farmers have the option to use other herbicides to control the volunteer plants 

(Deen et al., 2006; Soltani et al., 2006; Thomas et al., 2007; WSSA, 2007; Clewis et al., 2008). 

Use of any herbicide for the control of volunteer HCEM485 corn is subject to any labeling 

restrictions established by the EPA, as per FIFRA. Additionally, there are also many nonchemical methods of weed control (tilling, mechanical removal, etc.) that are effective for 

removing any unwanted HCEM485 corn.

In summary, APHIS found that HCEM485 is no more likely than the antecedent GA21 corn or the 

control to become a troublesome weed or present weed management issues.

Potential Impacts from Outcrossing (Gene Flow) to Sexually-compatible Wild 

Relatives

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 (Grant, 1981; Soltis and Soltis, 1993; Rieseberg, 1997; Hegde et al., 2006). Even in 

existing floras, the occurrence of hybridization or introgression is reported to be widespread 

(Knobloch, 1972; Stace, 1987; Rieseberg and Wendel, 1993). It has been a common practice by 

plant breeders to artificially introgress traits from wild relatives into crop plants to develop new 

cultivars. However, gene flow from crops to wild relatives is also thought of as having a 

potential to enhance the weediness of wild relatives, as observed in rice, sorghum, sunflower, 

and other crops (Ellstrand et al., 1999).

Corn is a monoecious species with separate male and female inflorescences that enable cross 

pollination. Corn is predominantly a wind pollinated outcrosser with occasional bee visitation for 

pollen. Bees rarely visit female inflorescences (silk). Researchers recognize that: (i) the percent 

gene flow will vary by population, hybrid or inbred, (ii) the level of gene flow decreases with 

greater distance between the source and recipient plants; (iii) environmental factors affect the

level of gene flow, (iv) corn pollen is viable for a relatively short period of time under field 

conditions, (v) corn produces ample pollen over an extended period of time, and (vi) corn is not 

pollinated by insects (pollinating insects, especially bees, are occasional visitors to the tassels but 

rarely visit silks of corn) (Luna, 2001; Jemison and Vayda, 2002).

APHIS evaluated the potential for gene introgression to occur from HCEM485 corn to sexually 

compatible wild relatives and considered whether such introgression would result in increased 

weediness. Cultivated corn, or maize, Zea mays L. subsp. mays, is sexually compatible with 

other members of the genus Zea with a few exceptions (Kermicle, 1997), and to a much lesser

degree with members of the genus Tripsacum (OECD, 2003). Wild diploid and tetraploid 

members of Zea, collectively referred to

8
as teosinte, are normally confined to the tropical and subtropical regions of Mexico, Guatemala, 

and Nicaragua (Wilkes, 1967; Fukunaga, 2005). Teosinte does not appear to be present in the

U.S. other than as an occasional botanical garden or research specimen. Although the Plants 

Profile for Z. mexicana (http://plants.usda.gov/java/profile?symbol=ZEME ) in the PLANTS 

Database (USDA-NRCS, 2012) shows distributions in South Carolina, Alabama, Maryland, and 

Florida, the cited species account (Wunderlin et al., 1996; Wunderlin and Hansen, 2002) as 

updated (Wunderlin and Hansen, 2008b) only shows distribution of native or naturalized 

populations in Florida (Miami-Dade, Orange and Levy Counties). The citation for this 

distribution, however, links to a University of Florida Herbarium Collections Catalog accession 

number 191119 that lists the specimen as cultivated at a USDA Soil Conservation Service Plant 

Materials Center in Heranando County and collected in October 21, 1993. Contrary to the Plants 

Profile for Z. mexicana, it is not listed in the citation provided for distribution in Maryland 

(Brown and Brown, 1984), nor are any Zea species or subspecies other than Z. mays (corn) listed 

in Alabama (Kral et al., 2012). The species account in the PLANTS Database (USDA-NRCS,

2012) written by Hugh Iltis for Zea perennis 

(see http://herbarium.usu.edu/webmanual/info2.asp?name=Zea_perennis&type=treatment, 

accessed 6/4/2012) describes it as crossing infrequently with Z. mays subsp. mays, but the

hybrids, being triploid, are sterile. While it is described as having been cultivated at research 

stations in the United States for many years, and Hitchcock (Hitchcock, 1951) reported that it 

was established at James Island, South Carolina, it is not known if the population has persisted. 

There are no Zea species other than Zea mays (corn) found in the comprehensive online South 

Carolina Plant Atlas hosted by the University of South Carolina A.C. Moore Herbarium and the

South Carolina Heritage Trust (South Carolina Heritage Trust, 2011)(available

at http://herbarium.biol.sc.edu/scplantatlas.html, accessed June 11, 2012); the absence of Zea 

perennis was confirmed with herbarium curators (Nelson and Damrel, 2012). Isolated plants of 

teosinte identified as Zea mays ssp parviglumis var. parviglumis were collected in 1975 in

Miami-Dade County, Florida (FLAS accession numbers 121428-121430) (Wunderlin and

Hansen, 2008a) in an area that is now urban, and Z. luxurians has been reported as cultivated as a 

fodder in tropical Florida in the early 1900s (Taba, 1995). Experts familiar with the teosinte 

collections in the United States (T. Chochrane, University of Wisconsin-Madison Herbarium; M. 

Barkworth, Utah State University; and J. Doebley, and H. Iltis, University of Wisconsin, 

personal communication, June, 2012) (Barkworth, 2012; Cochrane, 2012) some of whom are 

currently involved with revision of the Manual of Grasses for North America, are not aware of 

any naturalized or native populations of teosinte currently growing in the United States.

The genus Tripsacum contains up to 16 recognized species, most of which are native to Mexico, 

Central and South America, but three (T. dactyloides, T. floridatum, and T. lanceolatum) exist as 

wild and/or cultivated species in the continental U.S. (OECD, 2003), and two taxa (T. 

fasciculatum and T. latifolium) also occur in Puerto Rico) (USDA-NRCS, 2011). Though many 

of these species occur where corn might be cultivated, gene introgression from HCEM485 corn 

under natural conditions is highly unlikely. In contrast with corn and teosinte, which may 

hybridize relatively easily under certain conditions, special techniques are required to hybridize 

corn and Tripsacum thereby making hybrids of Tripsacum species with Zea difficult to obtain 

outside of a laboratory; and offspring are often sterile or have greatly reduced fertility, and are 

unable to withstand even mild winter conditions (Mangelsdorf, 1974; Russell and Hallauer,

9
1980; Galinat, 1988). Furthermore, none of the sexually compatible relatives of corn in the U.S. 

are considered to be weeds in the U.S. (Holm et al., 1979).

Introgression of genes from corn into teosinte or Tripsacum species in the U.S. has not been 

described to occur in nature. While some teosinte may be considered weeds in certain instances, 

they are also used by some farmers for breeding improved corn (Sánchez and Ruiz, 1997). 

Teosinte is described as being susceptible to many of the same pests and diseases that attack 

cultivated corn. In the wild, introgressive hybridization from corn to teosinte is currently limited 

by several factors including geographic isolation, differing degrees of genetic incompatibility, 

differences in flowering time, developmental morphology, dissemination, and dormancy 

(Galinat, 1988; Doebley, 1990a; Doebley, 1990b; Ellstrand et al., 2007). First-generation hybrids 

are generally less fit for survival and dissemination in the wild, and show substantially reduced 

reproductive capacity, which serves as a significant constraint to introgression. Guadagnuolo et 

al. (2006) did report that hybrids between a glyphosate-resistant maize cultivar and “chalco 

teosinte” (Z. mays ssp. mexicana) showed greater vigor and produced more seeds than the wild 

parent. Nonetheless, in the absence of selective pressure from glyphosate there was no direct 

positive or negative impact of the transgene on the fitness or vigor of either the hybrids or the 

pure maize progeny. There have been no reports that the widespread cultivation of glyphosate 

resistant corn in the United States has resulted in the introgression of the engineered trait into 

related species in the field; no related species are listed as glyphosate resistant (Heap, 2012).

Data included in the Petition (Stine 2011, page 31, Table 11 and Figure 12) demonstrated that 

there were no discernible differences between HCEM485 corn and control corn in the absence of 

glyphosate treatment in terms of mean pollen diameter and overall pollen morphology. The 

petitioner did find a statistical difference in pollen viability between HCEM485 and control 

plants, though the values for both corn varieties were still within the normal range of corn pollen 

reference samples and the out-crossing rate of HCEM485 corn will likewise not be different 

compared to other corn varieties. Based on the data presented in the Petition, HCEM485 corn 

does not exhibit characteristics that cause it to be any weedier than other cultivated corn; and the 

extremely limited potential for gene introgression from HCEM485 corn into teosinte or 

Tripsacum species is not expected to be any different than that of other cultivated corn varieties. 

Even in the unlikely event that gene flow were to occur between HCEM485 corn and wild 

relatives, there is no reason to expect that the possible expression of modified EPSPS would 

transform corn wild relatives into more weedy species, especially in the absence of herbicide 

selection pressure. Hybrids, if they occurred, could possibly have diminished susceptibility to 

glyphosate herbicides if the gene encoding the double mutated 2mEPSPS is inherited and 

expressed in the hybrid, but the hybrids would still be susceptible to many other herbicides, as is 

HCEM485, and as are the many other glyphosate resistant corn varieties that have been available 

for approximately 15 years.

In summary, HCEM485 corn, like the antecedent G21 or the control corn, will not adversely

impact sexually compatible wild relatives or the weediness attributes of those wild relatives.

10
Potential Impacts of HCEM485 on Non-target Organisms Considered

Beneficial to Agriculture

HCEM485 is agronomically and compositionally similar to GA21 and other corn varieties in 

commercial production and therefore will not adversely affect other organisms in any new or 

novel way. In particular, with respect to the secondary metabolites and antinutrients (ferulic 

acid, ρ-coumaric acid, furfural, inositol, phytic acid, raffinose and trypsin inhibitor) analyzed in 

grain samples from HCEM485 and control hybrids (Stine 2011, Table 19, page 41), the only 

significant difference noted was a small increase (ca. 3.4%) in trypsin protease inhibitor in 

HCEM485 grain samples, but this difference was not consistent across all growing locations 

and the level is well within the range reported in the literature for corn. APHIS has found no 

evidence or reason to believe that any differences between HCEM485 and GA21, or between 

HCEM485 and other corn varieties, in either the genetic material present or the proteins 

produced, would have any impact on other organisms, including organisms beneficial to 

agriculture (such as earthworms, honeybees, predators or parasites of corn pests). The EPSPS 

protein is expressed in a variety of plant tissues in corn, and expression of 2mEPSPS in 

HCEM485 corn is expected to be expressed in the same tissues since it is driven by the same 

regulatory sequences as the native glyphosate-sensitive EPSPS gene. Limited data from 

western blot analysis provided in the petition (Stine 2011, Figure 8, page 20) demonstrated that 

the level of expression of 2mEPSPS in leaf and seed of HCEM485 is less than that observed in 

the antecedent organism GA21. The inserted genetic material is not secreted, is not toxic, and 

does not produce any substance that is secreted or that would be considered toxic. The safety of 

modified EPSPS proteins from both plant and bacterial sources has been previously addressed 

in other petitions for deregulation (e.g. Monsanto submitted petitions 97-099-01p for GA21 

corn and 00-011-01p for NK603 corn) and by the OECD (1999)). The EPSPS protein, both 

modified and unmodified, does not share meaningful amino acid sequence similarities with 

known toxins and is already present in a large portion of corn that is present in the food supply

chain.

Potential Impacts of Associated Changes in Agricultural Practices on Plant 

Pests

APHIS evaluated whether the genetic modification causes changes in agricultural practices that 

affect the incidence, severity or control of plant pests. Considering that glyphosate resistant corn 

already has achieved a wide-adoption rate among U.S. growers, (approximately 66% of corn 

acres were treated with glyphosate in 2010, per http://quickstats.nass.usda.gov/, accessed June 

2011) and that HCEM485 will be grown in a similar manner to other glyphosate resistant corn, 

with the same glyphosate application rates, it can be concluded that there will not be any 

significant changes in agricultural practices if a determination of nonregulated status is extended 

to HCEM485. While growers may opt for different corn varieties to meet their anticipated needs, 

the availability of HCEM485 will not alter current or future methods that growers use to produce 

corn nor any of the methods used to minimize the damage caused by any plant pests. The 

methods applicable to production of GA21 corn are expected to be applicable to HCEM485 

corn.

11
Potential to Transfer Genetic Information from HCEM485 Corn

to Organisms with which it cannot Interbreed

APHIS examined the potential for the genetic material inserted into HCEM485 corn to be 

horizontally transferred to other organisms without sexual reproduction and whether such an 

event could lead directly or indirectly to disease, damage, injury or harm to plants, including

the creation of more virulent plant pests. The horizontal gene transfer (HGT) between unrelated 

organisms is one of the most intensively studied fields in the bio-sciences since 1940, and the 

issue gained extra attention with the release of transgenic plants into the environment (Dröge et

al., 1998). Potential risks from stable HGT from genetically engineered organisms to another 

organism without reproduction or human intervention was recently reviewed (Keese, 2008). 

Mechanisms of HGT include conjugation, transformation and transduction, and other diverse 

mechanisms of DNA and RNA uptake, recombination, and rearrangement, that occur most 

notably through viruses and mobile genetic elements. HGT has been implicated as a contributor 

to major transitions in evolution, including the spread of antibiotic resistance amongst 

pathogenic bacteria and the emergence of increased virulence in bacteria, eukaryotes, and 

viruses.

Potential for Horizontal Gene Transfer to Bacteria or Fungi

HGT from a plant species to other bacterial species is unlikely to occur based on the following 

observations. Although there are many opportunities for plants to directly interact with fungi 

and bacteria (e.g. as commensals, symbionts, parasites, pathogens, decomposers, or in the guts 

of herbivores), there are almost no evolutionary examples of HGT to bacteria from eukaryotes 

or from plants to fungi (as reviewed in Keese 2008). The only genes likely to be transferred 

successfully from genetically engineered plants to bacteria are other bacterial genes. Horizontal 

transfer of the gene construct from the nuclear genome of HCEM485 corn and subsequent 

expression in bacteria is unlikely to occur. First, many genomes (or parts thereof) have been 

sequenced from bacteria that are closely associated with plants including Agrobacterium and 

Rhizobium (Kaneko et al., 2000; Wood et al., 2001; Kaneko, 2002). There is no evidence that 

these organisms contain genes derived from plants. Second, in cases where review of sequence 

data implied that HGT 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). Third, the 6000-base pair 

insertion made to confer the herbicide resistance to HCEM485 corn is a small fraction of a 

percent (approximately 0.00024%) of the total DNA (approximately 2500 million base pairs) 

found in the corn genome, and all of the 6000 base pairs were derived directly from the corn 

genome. Forth, transgene DNA promoters and coding sequences are optimized for plant 

expression, not prokaryotic bacterial expression so even if HGT occurred, proteins 

corresponding to the transgenes are not likely to be produced. Finally, the FDA evaluated HGT 

from the use of antibiotic resistance marker genes and concluded that the likelihood of transfer

of antibiotic resistance genes from plant genomes to microorganisms in the gastrointestinal tract

of humans or animals, or in the environment, is remote (FDA, 1998).

Potential for Horizontal Gene Transfer to Viruses

APHIS also considered whether horizontal transfer of DNA from HCEM485 corn to plant 

viruses was likely to occur and would lead to the creation or selection of a more virulent plant 

12
pathogen through recombination with other plant viruses. This issue has been considered 

before by other science review panels and government regulatory bodies (for a general review

of the issue see (Keese 2008). Because there are no virus sequences used as part of the inserted

gene cassette and no sequences that have been implicated in viral recombination or

pathogenicity, there is no reason to think that viral recombination of the herbicide-resistance

conferring genetic material will occur, or that such theoretical recombination would have any

impact on plant pest risk.

Potential for Horizontal Gene Transfer to Parasitic Plants

Recently, Yoshida et al. (2010) through a comparative genomics analysis implicated HGT for

the incorporation of a specific genetic sequence in the parasitic plant purple witchweed (Striga 

hermonthica), which infests cereal fields (monocots) including corn and sorghum (Sorghum 

bicolor). According to this study, the incorporation of the specific genetic sequence (with an 

unknown function) occurred between sorghum and purple witchweed before speciation of

purple witchweed and related cowpea witchweed (S. gesnerioides), a parasitic plant of dicots, 

from their common ancestor. In other words, HGT between a parasitic plant and its host

is an extremely rare event, and furthermore, purple witchweed is not found in the U.S. and 

Asiatic witchweed (S. asiatica), another related parasite of cereal crops, is only present in North 

Carolina and South Carolina (USDA-NRCS, 2011).

For all the reasons listed above, APHIS concludes that as with GA21 corn or the control corn,

HGT from HCEM485 corn to other organisms is unlikely to occur and thus poses no 

significant plant pest risk.

Conclusion

APHIS has reviewed and conducted a plant pest risk assessment on glyphosate-resistant 

HCEM485 corn. Due to similarities to the antecedent glyphosate-resistant GA21corn and to 

the control corn, including the lack of plant pest risk from the inserted genetic material, the

lack of atypical responses of HCEM485 corn to disease or plant pests in the field, the lack of 

weediness characteristics of HCEM485 corn, the lack of gene flow potential to sexually 

compatible wild relatives to impact their weediness attributes, the lack of deleterious effects

on non-targets or beneficial organisms in the agro-ecosystem, the lack of changes in 

agricultural practices which would affect the incidence, severity or control of plant pests, and

the lack of concerns of horizontal gene transfer, APHIS concludes that HCEM485 corn is

unlikely to pose a plant pest risk.

13
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