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Plant Pest Risk Assessment for DAS-40278-9 Corn

Dow AgroSciences LLC (referred to hereafter as DAS) has petitioned APHIS (APHIS number 

09-233-01p, referenced as DAS 2010) for a determination that genetically engineered (GE) corn 

(Zea mays) event DAS-40278-9 is unlikely to pose a plant pest risk and, therefore, should no 

longer be a regulated article under regulations at 7 Code of 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 

event DAS-40278-9 corn is unlikely to pose a plant pest risk. 

History of Development of DAS-40278-9 Herbicide-Tolerant2 Corn 

DAS-40278-9 corn is a GE corn line that has been provided increased tolerance to treatment with 

phenoxy auxin herbicides and resistance to aryloxyphenoxypropionate (AOPP) acetyl coenzyme 

A carboxylase (ACCase) inhibitor (“fop”) herbicides. The introduced genetic material (DNA)

results in the production of an aryloxyalkanoate dioxygenase (AAD-1) enzyme that inactivates 

herbicides of the aryloxyalkanoate family, including phenoxy auxins and AOPP ACCase

inhibitors. If a determination of nonregulated status is reached, DAS-40278-9 corn would be the 

first commercially available corn variety with both improved tolerance to phenoxy auxin 

herbicides and resistance to “fop” herbicides.

The most well-known and widely-used phenoxy auxin herbicide is 2,4-dichlorophenoxyacetic 

acid (2,4-D) which has been used for many decades as a pre-plant or post-emergent herbicide to 

control broadleaf (dicot) weeds in corn fields. 2,4-D is also labeled for use for many other weed 

control applications including with other crops, on pastures and rangeland, and by both

commercial and residential entities for weed control in turf and lawns. The mode of action and 

the relative biochemical selectivity of 2,4-D for broadleaf plants is unclear, but it is believed to 

function as a plant growth regulator (auxin hormone) mimic which causes abnormal cell division 

and growth leading to plant injury and death (DAS 2010, pages 154-155; EPA 2005). Though it 

is not a broadleaf plant, corn can suffer injury from 2,4-D depending on the growth stage and 

method of application (DAS 2010, pages 103-105). 

 

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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.” 

2

The applicant has described DAS-40278-9 corn as “herbicide tolerant” and historically APHIS has also referred to 

GE plants with diminished herbicide sensitivity as “herbicide tolerant”. However, the phenotype would fall under 

the Weed Science Society of America’s definition of “herbicide resistance” since DAS-40278-9 has an inherited 

ability to survive and reproduce following exposure to a dose of herbicide normally lethal to the wild type variety 

(WSSA 1998). By the WSSA definition, “resistance [to an herbicide] may be naturally occurring or induced by 

such techniques as genetic engineering or selection of variants produced by tissue culture or mutagenesis.” 

Herbicide tolerance, by the WSSA definition, only applies to plant species with an “inherent ability” to survive and 

reproduce after herbicide treatment. 
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AOPP ACCase inhibitors, or “fop” herbicides, are post-emergent graminicides, meaning that the 

herbicides selectively control emerged grass (Poaceae) weeds. The selectivity of the “fops” is 

based on an increased sensitivity of grass species to ACCases compared to other plant species

(Devine 1997; Konishi and Sasaki 1994). The consequence of treatment with an ACCase 

inhibitor herbicide is to block production of fatty acid biosynthesis in sensitive plants. Corn, a 

plant in the Poaceae family, along with other related cereal plant varieties, is conventionally 

sensitive to treatment with “fop” herbicides, and these herbicides have not been traditionally 

labeled for weed control in corn fields (WSSA 2007). While some cereal crop plants have 

developed some tolerance to “fop” treatment due to improved herbicide detoxification (DAS 

2010, pages 155-156; Devine 1997), and corn varieties with resistance to “fop” treatment have 

been isolated using mutation screening (Marshall et al. 1992), if a determination of nonregulated 

status is reached, DAS-40278-9 corn may be the first corn variety with resistance to these 

herbicides to become widely commercially available.

If a determination of nonregulated status is reached, DAS-40278-9 would provide corn growers 

with additional options for the post-emergent control of both broadleaf and grass weeds. This 

corn product would also provide a potential remedy to the increased incidence of glyphosate and 

acetolactate synthase (ALS) inhibitor resistant weeds. The petitioner has studied various 

agronomic and compositional components of DAS-40278-9 corn since 2006 through confined 

field tests, and, in accordance with 7 CFR part 340.6(c), has presented data in the submitted 

petition for a determination that DAS-40278-9 corn should no longer be regulated under 7 CFR 

part 340. Additionally, per correspondence with the petitioner, APHIS is aware that DAS40278-9 corn was submitted for a Consultation of Bioengineered Foods to the FDA on October 

1, 2009 under the FDA’s Statement of Policy: Foods Derived from New Plant Varieties (Federal 

Register Volume 57 No. 104, 1992, page 22984). The petitioner also communicated (DAS 2010, 

page 18, and personal communication to APHIS on July 11 and 22, 2010) that label change 

requests would be made by the fourth quarter of 2010 to the EPA for the use of 2,4-D and 

quizalofop herbicides for weed management in fields with DAS-40278-9 corn. 

In 2009, approximately 68% of corn planted in the United States possessed resistance to an 

herbicide that was conferred through biotechnology (USDA NASS 2009). The primary 

herbicide resistance trait in use has been 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 wide-spread 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 (Duke and Powles 2009; Gianessi 2008). 

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, allows ‘no-till’ farming practices, and has minimal animal 

toxicological and environmental impact. However, increased selection pressure resulting from 

the wide-spread adoption of glyphosate-resistant crops, along with the reductions in the use of 

other herbicides and weed management practices, has resulted in both weed population shifts and 

growing numbers of glyphosate resistance among some weed populations (Duke and Powles 

2009; Owen 2008). In order to combat this trend, and to avoid decreased crop yields that result 

from weed competition, growers must continue to adapt their weed management strategies. If 
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DAS-40278-9 corn becomes available to them, corn farmers who choose to grow it will have 

added options for weed control. The use of DAS-40278-9 corn could allow for improved corn 

crop yields when it is grown in the vicinity of weeds resistant to glyphosate, particularly with 

anticipated labeling changes allowing the expanded application of 2,4-D at a critical time period 

for controlling weeds that compete with corn during the first 3-5 weeks before the corn reaches 

6-8 inches in height and label changes allowing over-the-top post-emergent use of quizalofop 

(DAS 2010, pages 108 and 113). This practice of using herbicides with alternative modes of 

action could also potentially diminish the populations of those glyphosate-resistant weeds and

reduce the likelihood of the development of new herbicide-resistant weed populations (DAS 

2010, pages 16-18 and Appendix 6; Duke and Powles 2008, 2009; Dill et al. 2008; Owen 2008). 

The herbicide 2,4-D was introduced more than 60 years ago and is used throughout the world for 

the treatment of broadleaf weeds. In 2002, 2,4-D was ranked as the third most used herbicide by 

active ingredient in the U.S. for all purposes (~ 40 million pounds), behind glyphosate (~102 

million pounds) and atrazine (~77 million pounds) (Gianessi and Reigner 2006). That same 

report found that the use of 2,4-D remained relatively steady from 1992 to 2002, whereas 

glyphosate usage increased more than 5-fold over the same time period. This increase in 

glyphosate use is attributable to the introduction and wide-spread adoption of GE glyphosate 

resistant crop species (e.g., soybeans, corn, cotton, canola) and rising adoption of no- or reducedtill farming practices that typically accompanies the use of glyphosate. The report also indicated

that overall herbicide use on corn decreased during that same 10 year time frame (from ~213 

million pounds in 1992 to ~159 million pounds in 2002), as farmers increasingly favored the use 

of GE crops which allowed for fewer types and a smaller overall amount of herbicide to be used.

The EPA did an analysis of herbicide usage in corn fields for 2004 that also showed that the use 

of glyphosate increased dramatically from 1987 to 2001 whereas 2,4-D usage remained 

essentially unchanged during that time (Kiely 2004). In 2005, herbicides were applied to 97 

percent of corn acreage planted in 19 states representing 93 percent of all corn planted in the 

United States. The herbicide most widely used on corn was atrazine (66 percent, ~57 million 

pounds applied), glyphosate was second (31 percent, ~23 million pounds), followed by smetolachlor and acetochlor (both at 23 percent, <24 million pounds and <30 million pounds, 

respectively). By comparison, 2,4-D was applied on less than 8 percent of 2005 corn acreage (~2 

million pounds applied) (USDA NASS 2006). The highest recorded use of 2,4-D is 14 percent of 

U.S. corn acres in 1994 (http://www.pestmanagement.info/nass/index.html, Accessed 3/2010).

These data demonstrate that there is a history of successful and effective use of 2,4-D as an 

herbicide, both generally and specifically on corn crops, to eliminate weed species. Because 

corn is not a broadleaf plant, it already has tolerance to 2,4-D, and the addition of a trait for 

increased tolerance to 2,4-D is not anticipated by the petitioner to greatly change the historical 

levels of herbicide use at the field level. Instead, DAS-40278-9 corn will give growers the 

option to apply 2,4-D during different growing windows, using different application methods and 

herbicide mixes (DAS 2010, pages 113-115). The expectation from the petitioner is that this 

added trait will help to minimize the emergence of herbicide resistant weeds because based on 

field efficacy studies (DAS 2010, pages 103-105) the DAS-40278-9 corn plants are less 

susceptible to injury associated with proposed application rates of 2,4-D applied either at the preemergent stage or later post emergent stages, and thus will allow for more robust weed control 

options. As per the Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), the petitioner 
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will submit to the Environmental Protection Agency (EPA) metabolism and residue data, along 

with proposed labeling changes, for the use of 2,4-D on DAS-40278-9 corn (DAS 2010, pages

18 and 113). Under FIFRA, without an experimental use permit issued by the EPA, it is unlawful

to use an herbicide “in a manner inconsistent with its labeling” (7 U.S.C. 136j). 

The “fop” herbicides (AAOP ACCase inhibitors) have been registered for crop use for over 20 

years. They have not traditionally been used to control weed species in corn fields because, as a 

grass (Poaceae family) species, corn is damaged by AOPP ACCase inhibitor activity. The use of 

“fop” herbicides has been primarily on broadleaf crops, such as soybean, to control grass weed 

species, although certain cereal plant varieties have a level of tolerance to some “fops.”

According to the USDA National Agricultural Statistics Service (NASS) Agricultural Chemical 

Use Database, “fop” type herbicides were used for weed control on at least 23 food crop species 

between 1990 and 2006, totaling over 16 million pounds of active ingredient 

(http://www.pestmanagement.info/nass/index.html, Accessed 3/2010). If a determination of 

nonregulated status is reached, DAS-40278-9 corn would be the first GE corn variety with 

nonregulated status that also has resistance to the “fop” class of herbicides. The petitioner has 

indicated that “fop” herbicides could be used to maintain seed purity in DAS-40278-9 corn 

breeding nurseries, hybrid production fields, and generally for the control of grass weeds in corn. 

As required under FIFRA, metabolism and residue data, along with proposed labeling changes

will be submitted to the EPA, for the use of “fop”-type herbicides (specifically quizalofop) in

DAS-40278-9 corn fields (DAS 2010, page 18). Under FIFRA, it is unlawful to use an herbicide 

“in a manner inconsistent with its labeling” without an experimental use permit issued (7 U.S.C. 

136j). Quizalofop is currently under registration review 

(http://www.epa.gov/oppsrrd1/registration_review/) by the EPA with a Final Decision expected 

sometime in 2013 (EPA-HQ-OPP-2007-1089 at http://www.regulations.gov, accessed 3/2010). 

Relatively few weed species have developed resistance to synthetic auxins or ACCase inhibitor 

herbicides compared to several other classes of herbicides. The International Survey of 

Herbicide Resistant Weeds (ISHRW) regularly updates a website with lists of “field selected, 

genetically inherited resistant weed biotypes that survive a rate of herbicide to which the 

indigenous population was controlled” that are discovered and verified globally

(http://www.weedscience.org, accessed 04/2010). According to the ISHRW, there are 28

broadleaf weeds (8 in the U.S.) that have been selected for resistance to synthetic auxins

(including phenoxy auxins such as 2,4-D), and 38 grass weeds (15 in the U.S.) with resistance to 

ACCase inhibitors (including “fops”). Within the U.S., none of the broadleaf weeds resistant to 

synthetic auxins have been found in corn fields to date, and grass weeds resistant to “fops” have 

also not been a problem for corn growers as ACCase inhibitors have not traditionally been used 

to control weeds in corn fields after planting. By comparison, there are 108 weeds found worldwide that have developed resistance to ALS inhibitor-type herbicides, 68 that have developed 

resistance to Photosystem II inhibitors (such as atrazine), 18 that have been found to be resistant 

to glyphosate, and, according to the ISHRW, many of these resistant weeds were found in U.S. 

corn fields. The petitioner has also indicated that the herbicide resistance traits of DAS-40278-9 

corn might be combined (or “stacked”) with other herbicide resistance traits (i.e. glyphosate and 

glufosinate) (DAS 2010, pages 18, 115, and 151) to use as part of a weed control strategy for 

growers. Within the U.S. there have not been any weeds that have shown resistance to either

glyphosate or atrazine and either a synthetic auxin or a “fop” herbicide. Additionally, there are 
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no weeds identified within the U.S. that are resistant to both 2,4-D and another class of herbicide, 

though instances have been identified in other countries. The ISHRW has indicated however, 

that there are five instances of weeds within the U.S. showing resistance to both ACCase 

inhibitors and another herbicide mode of action class, though none of these were located within 

corn fields. The use of DAS-40278-9 corn, regardless of the stacking with other herbicide 

resistance traits that may or may not occur, will provide growers with another weed control 

option, and should enable the continued use of no- or reduced-tillage in corn crops in areas 

where glyphosate herbicide resistant weeds have appeared. 

Description of Inserted Genetic Material 

DAS-40278-9 corn was produced by transformation of embryogenic suspension cultures of the 

recipient corn line Hi-II using silicon carbide whisker fibers (Petolino et al. 2003; Petolino and 

Arnold 2009) to introduce a 6236 base pair restriction fragment expression cassette containing

the genetic material that enables the production of the AAD-1 enzyme (DAS 2010, page 21). 

The petitioner has established that DAS-40278-9 corn contains only this expression cassette as

described in the petition (DAS 2010, pages 26-57) and that this inserted genetic material results 

in the production of the AAD-1 protein within the DAS-40278-9 corn plants (DAS 2010, pages 

58-63).

Corn plants containing the inserted genetic material were initially isolated by growing

transformed plant tissue on medium containing R-haloxyfop, an AOPP ACCase inhibitor (DAS 

2010, page 21). Whole plants were generated from the surviving tissue and selected plants were 

bred with desirable corn lines to generate elite corn varieties containing the genetic insertion. 

DAS confirmed via Southern blot analyses that the final corn product (DAS-40278-9) does not 

contain any plasmid sequences outside of the intended 6236 base pairs and contains only a single 

intact insert in the corn genome with no evidence of duplicated or missing DNA from that 

insertion. Inheritance patterns of the inserted genetic material also demonstrate the intactness 

and functional stability of the inserted DNA over several breeding generations. (DAS 2010, 

pages 30-57).

A detailed description of the inserted genetic material is provided in the petition (DAS 2010, 

pages 26-27), and is summarized below:

The introduced gene responsible for conferring the herbicide resistance encodes an

aryloxyalkanoate dioxygenase (AAD-1) enzyme. The gene, aad-1, was originally obtained from 

the soil bacterium Sphingobium herbicidovorans, and was subsequently altered to be plantoptimized (the original nucleic acid sequence was changed because plants favor different codons 

for optimal DNA-protein translation). The final protein product (AAD-1) produced in DAS40278-9 corn is nearly identical (99.3% identity) to the protein produced by S. herbicidovorans

differing only by the addition of one amino acid, alanine, at position 2 (DAS, page 58; Wright et 

al. 2009). DAS provided evidence that this single amino acid change did not impact the function 

of the enzyme nor was there any evidence that the protein contributed to any changes to the 

agronomic properties, disease susceptibility, or composition of DAS-40278-9 corn compared to 

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

Disease and Pest Susceptibilities” below). 
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The aad-1 gene is flanked on either side by regulatory (non-coding) sequences that have been 

derived from the corn genome. In front of aad-1 (on the 5’ end) is the Z. mays ubiquitin 1 

(ZmUbi1) promoter which is a sequence of DNA that promotes the constitutive expression of 

genetic material, particularly in monocots (Christensen et al. 1992, Christiansen and Quail 1996). 

This particular promoter sequence was previously used in a plant product that has been

previously determined to have nonregulated status (APHIS BRS Petition 03-181-01p) and the 

use of that sequence in DAS-40278-9 corn does not raise any new issues. On the downstream

side of the aad-1 gene is the Z. mays peroxidase 3’ untranslated region (ZmPer5 3’ UTR), which 

is a regulatory sequence of corn DNA that promotes the efficient transcription and translation of 

a corn gene (Ainley et al. 2002). Both the ZmUbi1 promoter and the ZmPer5 3’ UTR genetic 

sequences serve only as a regulatory role, and are not translated with the AAD-1 protein (DAS 

2010, Appendix 2, pages 129-131). 

On either side of the above genetic material, DAS has also included the RB7 Matrix attachment 

region (MAR). This genetic material, derived from tobacco (Nicotiana tabacum) has been 

demonstrated to improve the stability of expression of inserted genetic material by binding to 

nuclear matrices within a plant cell (Hall et al. 1991; Allen et al. 2000; Mankin et al. 2003). 

MAR sequences do not encode for a protein product and are also considered solely as regulatory 

elements. Also part of the 6236 base pair introduced fragment are short intervening DNA 

sequences that were part of the original cloning plasmid. These sequences do not contain any 

coding sequence but represent the linking portions of the plasmid that lie between the DNA 

sequence insertion points for the regulatory regions and coding region of the expression cassette. 

In summary, the only portion of the introduced DNA fragment that is translated into a protein 

product or that could potentially alter the nutritional or fundamental component characteristics of 

the corn plants is the aad-1 gene. An analysis of the impacts of that insertion within DAS-40278-

9 corn as it pertains to plant pest risk is presented through the rest of this document. 

Plant Pest Risk Assessment 

APHIS is responsible for regulating GE organisms and plants under the plant pest provisions in 

the Plant Protection Act (PPA) of 2000, as amended (7 USC § 7701 et seq.) to ensure that they 

do not pose a plant pest risk to the environment. APHIS regulations at 7 Code of Federal 

Regulations (CFR) Part 340, which were promulgated pursuant to authority granted by the PPA, 

regulate the introduction (importation, interstate movement, or release into the environment) of 

certain GE organisms and products. A GE organism is no longer subject to the plant pest 

provisions of the PPA or to the regulatory requirements of Part 340 when APHIS determines that 

it is unlikely to pose a plant pest risk. A GE organism is considered a regulated article under Part

340 if the donor organism, recipient organism, vector, or vector agent used in engineering the 

organism belongs to one of the taxa listed in the regulation (7 CFR 340.2) and is also considered 

a plant pest. A GE organism is also regulated under Part 340 when APHIS has reason to believe 

that the GE organism may be a plant pest or APHIS does not have information to determine if 

the GE organism is unlikely to pose a plant pest risk. 

A person may petition the agency that a particular regulated article is unlikely to pose a plant 
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pest risk, and therefore, is no longer regulated under the plant pest provisions of the PPA or the 

regulations of Part 340. The petitioner is required to provide information under §340.6(c)(4)to 

demonstrate that the regulated article that is the subject of the petition does not pose a plant pest 

risk and that it should no longer be subject to the regulations of 7 CFR part 340. A GE organism 

is no longer subject to the regulatory requirements of Part 340 or the plant pest provisions of the 

PPA when APHIS determines that it is unlikely to pose a plant pest risk.

As described earlier in this document, the introduced genetic material in DAS-40278-9 corn

confers enhanced tolerance and resistance to treatment with select herbicides to the GE plants. 

No plant pest or plant pest-derived material was used to generate the DAS-40278-9 corn plants. 

DAS has planted DAS-40278-9 corn as a regulated article under APHIS BRS Notification of 

Environmental Release since 2006 (DAS 2010, page 144). 

Potential impacts to be addressed in this plant pest risk assessment are those that pertain to the 

use of DAS-40278-9 corn and its progeny in the absence of confinement as otherwise afforded 

under conditions of 7 CFR part 340 for the introduction of genetically engineered organisms. Of 

the information requested by APHIS for submission of a petition for nonregulated status, APHIS 

examined information submitted by the petitioner (as per 7 CFR part 340.6(c)(4)) related to plant 

pest risk characteristics, disease and pest susceptibilities, expression of the gene product, new 

enzymes or changes to plant metabolism, weediness (including control options) of the regulated 

article, the possibility of effects of the regulated article on non-target organisms, and any impacts 

on the weediness of any other plant with which it can interbreed. 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), non-target organisms, beneficial organisms, threatened 

and endangered species and other environmental impacts have been addressed in an

Environmental Assessment (EA) that has been prepared to analyze potential impacts of a 

determination of nonregulated status of DAS-40278-9 corn by APHIS. 

Potential Impacts of Genetic Modifications on Altered Disease and Pest 

Susceptibilities 

APHIS assessed whether DAS-40278-9 corn is likely to have significantly increased disease and 

pest susceptibility compared to conventional corn. This assessment encompasses a thorough 

consideration of introduced traits and interactions with pests and disease. 

Corn (Zea mays) 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 or pests listed elsewhere in 7 CFR parts 300-399. The description of the genetic 

modification, including the introduced genetic elements and the resulting expression and 

function of the AAD-1 protein product in DAS-40278-9 corn, has been summarized above. DAS 

routinely monitored their corn field trials for corn diseases such as ear rots, leaf blights, rusts, 

and leaf spots along with insect damage from corn pests such as leaf hoppers, mites, thrips, 

aphids, beetles, grasshoppers, and corn earworms. DAS conducted agronomic field trials during 

the 2008 growing season across 27 locations representative of the major corn-growing areas of 

the U.S. (DAS 2010, pages 64-75). There were no statistically significant differences in 

agronomic characteristics such as vigor, lodging or yield that might be indicative of altered 
8

sensitivity to pests and diseases between DAS-40278-9 corn, derived hybrids and their control 

counterparts under standard corn management practices (DAS 2010, page 70) or in response to 

specific herbicide treatments (DAS 2010, page. 67). In one experiment, disease and insect 

damage ratings were taken for treatments that included DAS-40278-9 hybrid corn sprayed with 

2,4-D, quizalofop, 2,4-D and quizalofop, and neither herbicide, as well as the near-isoline control 

hybrid planted at six (6) field sites; Richland, IA; Carlyle, IL; Wyoming, IL; Rockville, IN; 

York, NE; and Branchton, Ontario, Canada (DAS 2010, page 71). The data submitted by DAS

confirmed that the inserted genetic material did not confer any altered disease or pest 

susceptibility to DAS-40278-9 corn. Specific disease and insect stressor observations were

collected from 2006 to 2008 across locations representing a broad range of U.S. corn growing 

regions (DAS 2010, pages 64-75). 

The data presented in the petition (DAS 2010, pages 76-101) indicate no material difference in 

compositional and nutritional quality of DAS-40278-9 corn compared to other commercially 

available corn apart from the presence of the AAD-1 protein. A few variables did show 

statistically significant differences between the compositional characteristics of DAS-40278-9

corn and control corn grown at the same locations. However, these differences were relatively 

small and 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 (Ridley et al. 2004; ILSI 2006) or in the OECD consensus 

document on corn composition (OECD 2002). Therefore, with the exception of the AAD-1 

protein which is at most 0.02% of corn tissue dry weight (range: 0.42-210 ng/mg depending on 

the tissue, see DAS 2010, page 20), the composition of DAS-40278-9 corn is not biologically 

different than conventional corn. Data in the petition (DAS 2010, pages 68-70) showed no 

significant difference in grain yield between a DAS-40278-9 corn hybrid and a near isogenic 

control line across 21 locations (6 states). These data collectively support the petitioner’s claim 

that DAS-40278-9 corn does not have increased susceptibility to any insects, plant pathogens, or 

other plant pests over its non-transgenic control. 

The AAD-1 protein is derived from a gram-negative soil bacterium Sphingobium

herbicidovorans that is not associated with plant disease (Balkwill et al. 2006; Kohler 1999; 

Zipper et al. 1996; Horvath et al. 1990). S. herbicidovorans was originally isolated using a soilcolumn enrichment followed by culture growth with phenoxy auxin herbicide as the sole carbon 

and energy sources (Horvath et al. 1990). The AAD-1 protein expressed by the DAS-40278-9

corn, with the exception of the second amino acid is identical to proteins found in other soil 

bacteria (Non-redundant Protein Basic Local Alignment Search Tool (BLAST), 

http://www.ncbi.nlm.nih.gov/, accessed 4/2010), including Rhodoferax sp P230 and Delftia 

acidovorans, which have also been isolated from herbicide-enriched substrate (Müller et al. 

2001) and are not considered plant pests (Wen et al. 1999). Using BLAST, the AAD-1 protein 

was classified as an R-2,4-dichlorophenoxypropionate dioxygenase, and it has also been named 

as an (R)-dichlorprop/α-ketoglutarate dioxygenase (RdpA) (Müller, et al. 2004; Wright et al.

2009). The enzyme has only been associated with the metabolism of herbicides and there is no 

indication that it has any other function. 

In summary, all evidence reviewed by APHIS indicate that DAS-40278-9 corn is just as 

susceptible to plant pathogens and other plant pest species as conventional corn and that there is 

no added plant pest risk that results from the insertion and expression of the genetic material 
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described in the petition.

Potential Impacts Based on the Relative Weediness of DAS-40278-9 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 corn 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, corn seed can germinate in undesired locations and would then 

be considered a weed. A common example of this is the appearance of corn seedlings in soybean 

fields following a corn crop which has been associated with reduced soybean yields (Beckett and 

Stoller 1988). Compared to other corn varieties, DAS-40278-9 has improved fitness (reduced 

injury) in the presence of certain herbicides, which translates into fewer options for the removal 

of volunteer plants. Nonetheless, there are many available options for the control of DAS40278-9 plants so that growers can avoid losses in fields where unwanted DAS-40278-9 might 

be growing.

APHIS assessed whether DAS-40278-9 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 

DAS-40278-9 corn evaluated under field conditions. DAS conducted agronomic field trials 

during the 2008 growing season across 27 locations representative of the major corn-growing 

areas of the U.S. (DAS 2010, pages 64-75). There were few statistically significant differences 

between DAS-40278-9 corn, derived hybrids and their control counterparts, regardless of 

herbicide treatment. The significant differences that were seen (DAS 2010, page 67), in ear 

height and a visual estimate of plant health (stay green), reflected very small differences in 

morphological features and were not accompanied by significant overall treatment effects (DAS 

2010, page 66). No differences in phenotypic characteristics that would contribute to enhanced 

weediness were observed between DAS-40278-9 corn and control lines for the range of 

phenotypic endpoints assessed. 

Based on the agronomic field data and literature survey about corn weediness potential, DAS40278-9 corn lacks the ability to persist as a troublesome or invasive weed. Still, because DAS40278-9 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 DAS-40278-9 corn confers greater tolerance or resistance to herbicides the

measures that can be used to remove volunteer DAS-40278-9 corn are more limited compared to 

other corn varieties. Nonetheless, as discussed in the next paragraphs, most of the currently used 

control measures for removing volunteer corn are also available for the removal of DAS-40278-9 

corn.

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 growth of the 

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

may have lower yields and reduced value due to competition and mixing with the volunteer corn
10

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

resistant crops has already changed the approaches growers can adopt to reduce crop yield losses 

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 (Clewis et al. 2008; Thomas et al. 2007)

and corn-wheat rotations (Martin 2008). 

One suggested chemical method for the control of volunteer corn among broadleaf crops is the 

use of graminicide ACCase inhibitors which include “fops” (Deen et al. 2006; Soltani et al. 

2006). However, the use of “fop” ACCase inhibitor herbicides would not be effective for the 

control of volunteer DAS-40278-9 corn due to the resistance that has been conferred by genetic 

engineering. Nonetheless, other ACCase inhibitor graminicides, such as cyclohexanedione 

(“dim”) herbicides (e.g. clethodim or sethoxydim) are also effective for the control of volunteer 

corn in broadleaf rotational crops (Clewis et al. 2008; Thomas et al. 2007; Deen et al. 2006; 

Soltani et al. 2006; WSSA 2007), although the level of control with sethoxydim has been

reported to be less effective at manufacturer recommended doses than the “fop” herbicides tested 

(Soltani et al, 2006). Another large class of herbicides that can be used for the elimination of 

volunteer corn includes the acetolactate synthesis (ALS) inhibitors, such as imazamox, 

imazaquin, and imazethapyr (WSSA 2007). The petitioner has confirmed the susceptibility of 

DAS-40278-9 corn to both “dim” and ALS inhibitor herbicides at labeled-use levels (DAS 2010, 

page 115 and April 29, 2010 correspondence with APHIS). ALS inhibitor herbicides as well as 

paraquat can also be used to control volunteer corn in corn to wheat rotations (Martin 2008).

Other commonly used methods for the control of volunteer corn are also still available for use on 

DAS-40278-9. For example, non-selective herbicides (e.g. glyphosate and glufosinate) are 

options for eliminating DAS-40278-9 corn and its progeny, assuming that there has been no 

breeding (or stacking) or cross-pollination with other corn lines that have resistance traits to 

those herbicides. Use of any herbicide for the control of volunteer DAS-40278-9 corn is subject 

to any labeling restrictions established by the EPA, as per FIFRA. Additionally, there are also 

many non-chemical methods of weed control (tilling, mechanical removal, etc.) that are effective 

for removing any unwanted DAS-40278-9 corn.

The possibility of herbicide-resistant volunteer corn competing with other crops is not confined 

to GE varieties. Other cultivar-development methods have generated corn varieties that have

reduced sensitivity to herbicides that could otherwise control for volunteer corn in crops such as 

soybean. For example, there are commercially available corn hybrid varieties with resistance to 

sethoxydim, a cyclohexanedione ACCase inhibitor (Vangessel et al. 1997). Similar to DAS40278-9, many options for control of the herbicide resistant corn are available (Young and Hart, 

1997). While farmers may have to change their management strategies due to DAS-40278-9, 

these changes will not necessitate a major departure from well-established and broadly used 

agricultural protocols. 

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

Relatives 
11

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; Peterson et al. 2002). 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). 

APHIS evaluated the potential for gene introgression to occur from DAS-40278-9 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, and to a much lesser degree with members of the genus

Tripsacum (OECD 2003). Wild diploid and tetraploid members of Zea, collectively referred to 

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

and Nicaragua (Wilkes 1967; Fukunaga et al. 2005). In the U.S. isolated populations of teosinte 

have been reported in Florida (http://www.florida.plantatlas.usf.edu/, Taba 1995). 

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, NRC, 2010). Though many of 

these species occur where corn might be cultivated, gene introgression from DAS-40278-9 corn 

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 can withstand even the mildest winters. 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, and 

references therein). 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 in some cases, developmental morphology and 

timing of the reproductive structures, dissemination, and dormancy (Doebley 1990a and 1990b; 

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 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. 

Data included in the petition demonstrated that there were no significant differences in viability 

and morphology of pollen collected from DAS-40278-9 corn and control corn (DAS 2010, pages 
12

73-74) and therefore the out-crossing rate of DAS-40278-9 corn is not expected to be different

compared to other corn varieties. Based on the data presented in the petition, DAS-40278-9 corn 

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

extremely limited potential for gene introgression 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 DAS-40278-9 corn and wild relatives, there is no 

reason to expect that the possible expression of aad-1 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 some herbicides but would still be 

susceptible to many others, as is DAS 40278-9. 

In summary, DAS-40278-9 corn will not adversely impact sexually compatible wild relatives or 

the weediness attributes of those wild relatives.

Potential Impacts of DAS-40278-9 on Organisms Considered Beneficial to 

Agriculture 

The petitioner also assessed the potential for DAS-40278-9 corn to impact “non-target” 

organisms, including those considered beneficial to agriculture, and determined that there would 

be no effect (DAS 2010, page 116). DAS-40278-9 is agronomically and compositionally similar 

to other corn varieties and will not adversely affect other organisms compared to other corn 

varieties. Furthermore, APHIS has found no evidence or reason to believe that the presence of 

the aad-1 gene or the expressed AAD-1 protein in DAS-40278-9 corn would have any impact on 

other organisms, including organisms beneficial to agriculture (such as earthworms, honeybees, 

predators or parasites of corn pests). The AAD-1 protein is expressed in a variety of plant tissues 

in DAS-40278-9 corn with average expression values ranging from 2.87 ng/mg dry weight in R1 

stage root to 127 ng/mg in pollen tissue (DAS 2010, page 60) and expression values were similar 

for sprayed treatments as well as for plots sprayed and unsprayed with 2,4-D and quizalofop 

herbicides. 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 AAD-1 protein does not share 

meaningful amino acid sequence similarities with known toxins based on information reported 

by DAS (DAS 2010, page 62). Amino acid homologies were evaluated using a global sequence 

similarity search against the GenBank non-redundant protein dataset and the only significant 

homologies identified were with other alpha-ketoglutarate dependent dioxygenases, the same 

class of enzymes as AAD-1. DAS reported that none of the similar proteins returned by the 

search identified any safety concerns that might arise from the expression of AAD-1 protein in 

plants. The aad-1 gene, isolated from a soil bacterium, and the AAD-1 protein are both present 

in nature. The soil bacterium, Sphingobium herbicidovorans, is neither a plant pest nor a known 

pest of organisms beneficial to agriculture and the AAD-1 protein is similar to proteins found in 

other soil bacteria that are also not considered pests (Wright et al. 2009; Balkwill et al. 2006).

Any effects on non-target organisms that could potentially result from proposed changes in 

herbicide labels will be evaluated by the EPA.

Potential to Transfer Genetic Information from DAS-40278-9 Corn to 

Organisms with which it cannot Interbreed 
13

APHIS examined the potential for the genetic material inserted into DAS-40278-9 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 aad-1 gene construct from the nuclear genome of DAS-40278-9 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 et al. 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 6236 base pair insertion made to confer the herbicide tolerance to DAS-40278-9 corn

is a small fraction of a percent (approximately 0.00025%) of the total DNA (approximately 2500 

million base pairs) found in 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 DAS-40278-9 corn to plant 

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

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 
14

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 (S. hermonthica) 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, S. hermonthica is not found in the U.S. and S. 

asiatica, another related parasite of cereal crops, is only present in North Carolina and South 

Carolina (USDA, NRC, 2010). 

For all the reasons listed above, APHIS concludes that HGT is unlikely to occur and thus poses 

no plant pest risk. 

Conclusion

APHIS has reviewed and conducted a plant pest risk assessment on DAS-40278-9 corn. Due to 

the lack of plant pest risk from the inserted genetic material, the lack of weediness characteristics 

of DAS-40278-9 corn, the lack of atypical responses of DAS-40278-9 corn to disease or plant 

pests in the field, the lack of deleterious effects on non-targets or beneficial organisms in the 

agro-ecosystem, and the lack of concerns of horizontal gene transfer, APHIS concludes that 

DAS-40278-9 corn is unlikely to pose a plant pest risk. 

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