Plant Pest Risk Assessment for DAS-40278-9 Corn

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

09-233-01p) for a determination that genetically engineered (GE) corn (Zea mays) event DAS40278-9 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. The earlier 2010 version of 

the petition was revised on April 12, 2011 to update the summary on page 4 and was received by

APHIS on June 22, 2011. This version of the petition is referenced hereafter as DAS 2011. 

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-Tolerant

2 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 2011, 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 2011, pages 103-105).

1 Section 403 (14) of the Plant Protection Act (7 USC 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.

1
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

2011, 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. In addition, DAS has indicated 

that DAS-40278-9 may be stacked with glyphosate and other herbicide resistance traits to 

generate commercial hybrids with resistance to multiple herbicides (pg. 4, DAS 2011).

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 another tool to address 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, the petitioner submitted DAS-40278-9 corn for a Consultation of Bioengineered

Foods to the FDA on October1, 2009 under the FDA’s ‘Statement of Policy: Foods Derived 

from New Plant Varieties’ (FDA 1992). FDA evaluated DAS’ submission to determine 

whether DAS-40278-9 corn raises any safety or regulatory issues with respect to the intended 

modification or with respect to the food itself. Based on the information provided by the 

company and other information available to the agency, FDA did not identify any issues under 

the Federal Food, Drug and Cosmetic Act that would require further evaluation at this time

(BNF No. 000120, April 8, 2011: 

http://www.fda.gov/Food/FoodScienceResearch/Biotechnology/Submissions/ucm254647.htm.)

The U.S. Environmental Protection Agency (EPA) regulates the distribution, sale, use and 

testing of herbicides under the Federal Insecticide, Fungicide and Rodenticide Act (FIFRA) (7 

U.S.C. 136 et seq). EPA also sets tolerance limits for residues of 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) (21 U.S.C. Chapter 9). Prior to registration for a new 

use for a new or previously registered herbicide, EPA must determine through submitted test 

data that the herbicide does not cause unreasonable adverse effects on the environment and nontarget species when used in accordance with label instructions and will result in a reasonable 

certainty of no harm to humans. EPA must also approve the language used on the herbicide label 

in accordance with 40 CFR part 158. DAS has submitted information in support of label 

amendments to the EPA for the use of 2,4-D and quizalofop herbicides for weed management in 

fields with DAS-40278-9 corn (DAS 2011, pp 113-115). These actions are currently under 

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review by EPA. 

In 2009, the percent of corn planted in the United States that possessed either resistance to an 

herbicide or stacked resistance to both herbicides and insects, both of which were conferred 

through biotechnology, was approximately 22% and 46%, respectively, and 68% combined

(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 resistant biotypes 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 diversify and adapt their weed management strategies

to adopt best management practices (Norsworthy et al. 2012). If DAS-40278-9 corn 

becomes available to them, corn farmers who choose to grow it will have added options for

weed control if requested herbicide label changes mentioned above are approved by the EPA. 

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 proposed labeling changes to 

allow 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 

proposed label changes to allow over-the-top post-emergent use of quizalofop (DAS 2011, 

pages 108 and 113). The 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 if properly 

implemented within a diversified weed management cropping system (DAS 2011, pages 16-

18 and Appendix 6; Duke and Powles 2008, 2009; Dill et al. 2008; Owen 2008; Norsworthy 

et al. 2012).

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

reduced- till 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 

3
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). According to the USDA National 

Agricultural Statistics Service Chemical Use Database, the average percentage of all corn 

acreage treated with 2,4-D remained relatively stable from 1990 to 2003 typically ranging from 

about 8 to 12 percent, with slightly higher peaks in 1990, 1994 and 1995, and slightly lower 

usage in 2002 and 2003 for program states included in the survey (data accessed via 

http://www.pestmanagement.info/nass/index.html on 12/19/2013). These data demonstrate that 

there is a history of successful and effective use of 2,4-D as an herbicide, both generally and 

specifically for corn crops, as one of many options to manage weed species. Because corn is not

a broadleaf plant, it already has some tolerance to 2,4-D; and the addition of a trait for increased 

resistance to 2,4-D in 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, if 

approved by EPA (DAS 2011, 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 2011, 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

pre-emergent stage or later post emergent stages, and thus will allow for more robust weed 

control options. As stated earlier the label change request submitted by DAS to EPA is under 

review. 

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. Quizalofop is currently under registration review 

(http://www.epa.gov/oppsrrd1/registration_review/) by the EPA (72 FR 71893-71896).

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

will be reviewed by the EPA, for the use of 2,4-D and “fop”-type herbicides (specifically

4
quizalofop) in DAS-40278-9 corn fields (DAS 2011, 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). 

When the petition was submitted, relatively few weed species had developed resistance to 

synthetic auxins or ACCase inhibitor herbicides compared to two 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. According to the ISHRW(http://www.weedscience.org, accessed 04/2010), there were

28 broadleaf weeds (8 in the U.S.) with selected 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”). At that time, within the U.S., none of the broadleaf weeds 

resistant to synthetic auxins had been found in corn fields, 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 were 108 weeds found 

world- wide 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 2011, pages 4, 18, 114-115, and 151) to use as part of a weed 

control strategy for growers. When the petition was submitted, within the U.S. there were not 

any weeds that had shown resistance to either glyphosate or atrazine and either a synthetic auxin

or a “fop” herbicide, nor were there any resistant to both 2,4-D and another class of herbicide, 

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

that there were 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, could 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. Herbicide resistant weeds continue 

to evolve, and herbicide use patterns and the impacts of DAS-40278-9 corn on herbicide 

resistant weed management are addressed in the Environmental Impact Statement (EIS) 

prepared for this petition (see 

http://www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml).

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 2011, page 21). 

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

as described in the petition (DAS 2011, 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

2011 pages 58-63 and 132-140).

5
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 2011, 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 2011, pages 30-57).

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

2011, 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

DAS-40278-9 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

2011, 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).

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) (see 

http://www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml ) 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

2011, Appendix 2, pages 132-140).

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

6
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 DAS40278-9 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 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 2011, 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

7
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) and subsequent EIS that have been prepared to 

analyze potential impacts of a determination of nonregulated status of DAS-40278-9 corn by

APHIS (see http://www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml.)

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.

None of the inserted genetic sequences contained in DAS-40278-9 corn 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 2011, pages 64-

75). There were no statistically significant differences in agronomic characteristics such as 

vigor, lodging or yield that might be indicative of altered sensitivity to pests and diseases

between DAS-40278-9 corn, derived hybrids and their control counterparts under standard 

corn management practices (DAS 2011, page 70) or in response to specific herbicide 

treatments (DAS 2011, 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 2011, 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 2011, pages 64-75).

The data presented in the petition (DAS 2011, 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 

8
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 2011, page 60), the composition of DAS-40278-9 corn is not biologically 

different than conventional corn. Data in the petition (DAS 2011, 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 altered 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

soil- column 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 DAS40278-9 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

described in the petition.

Potential Impacts Based on the Relative Weediness of DAS-40278-9 Corn

Corn (Zea mays) is not a plant pest in the United States (USDA-APHIS 2000). 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 DAS-40278-9 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

9
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 2011, 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 2011, 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

2011, 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 certain 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 DAS40278-9 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 (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

10
the susceptibility of DAS-40278-9 corn to both “dim” and ALS inhibitor herbicides at 

labeled-use levels (DAS 2011, 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 DAS-40278-9, 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

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). As previously analyzed by

USDA APHIS (2013), despite accounts of some taxa of teosinte as having been cultivated in the 

United States as fodder (Taba 1995) or as research specimens, it does not appear to be present in 

the United States other than as an occasional botanical garden or research specimen. There are 

11
no known 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, 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 2011,

pages 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, DAS40278-9 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 expected to 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.

12
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 2011, 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 2011, 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 2011, 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 and/or any new herbicide metabolites produced as a result of the 

genetic modification will be evaluated by the EPA. This is addressed in the EIS prepared 

for this DAS petition.

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

Organisms with which it cannot Interbreed

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 

13
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 DAS40278-9 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

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

14
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. APHIS 

concludes that DAS-40278-9 corn is unlikely to pose a plant pest risk for the following reasons:

• There is no plant pest risk from the inserted genetic material.

• DAS-40278-9 corn was not observed to display atypical responses to disease or plant 

pests in the field.

• DAS-40278-9 corn does not have phenotypic characteristics that would contribute to 

enhanced weediness; and despite its greater herbicide resistance, volunteers of DAS40278-9 can still be controlled with some other currently available herbicides or other 

control methods in situations where they are considered weeds. As with other herbicide 

resistant corn varieties available on the market, management strategies can be used to 

minimize the potential for stacked herbicide resistant corn volunteers to become a weed 

management problem.

• DAS-40278-9 corn is not expected to increase weediness of other sexually-compatible 

wild relatives with which it can interbreed.

• DAS-40278-9 corn is not expected to have adverse effects on non-targets or beneficial 

organisms in the agro-ecosystem. 

• Horizontal gene transfer from DAS-40278-9 to other species with which it cannot 

interbreed is unlikely to occur and thus poses no plant pest risk. 

References

Ainley, M., K. Armstrong, S. Belmar, O. Folkerts, N. Hopkins, M. A. Menke, D. Pareddy, J. F. Petolino, 

K. Smith, and A. Woosley. 2002. Regulatory Sequences from Transgenic Plants (U.S. Patent 

US6384207)

Allen, G. C., S. Spiker, and W. F. Thompson. 2000. Use of matrix attachment regions (MARs) to 

minimize transgene silencing. Plant Molecular Biology 43:361.

Baker, H. B. 1965. Characteristics and modes of origin of weeds.in H.G. Baker and G.L. Stebbins (eds). 

The genetics of colonizing species. Academic Press, London, U.K.

15
Balkwill, D., J. Fredrickson, and M. Romine. 2006. Chapter 6.10: Sphingomonas and related genera. The

Prokaryotes 7:605-629.

Beckett, T. H. and E. W. Stoller. 1988. Volunteer corn (Zea mays) interference in soybeans (Glycine 

max). Weed Science 36:159-166.

Beckie, H. J. and M. D. K. Owen. 2007. Herbicide-resistant crops as weeds in North America. CAB

reviews: PAVSNNR 2:1-22.

Brown, J. R. 2003. Ancient horizontal gene transfer. Nature Reviews Genetics 4:121-132.

Christensen, A. H., R. A. Sharrock, and P. H. Quail. 1992. Maize polyubiquitin genes: structure, thermal 

perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts 

by electroporation. Plant Molecular Biology 18:675-689.

Christensen, A.H., Quail, P.H. 1996. Ubiquitin promoter-based vectors for high-level expression of 

selectable and/or screenable marker genes in monocotyledonous plants.Transgenic Research 5: 13-218.

Clewis, S., B., W. Thomas, E., W. Everman, J., and J. Wilcut, W. 2008. Glufosinate-resistant corn 

interference in glufosinate-resistant cotton. Weed Technology 22:211-216.

Crockett, L. 1977. Wildly Successful Plants: North American Weeds. University of Hawaii Press, 

Honolulu.

DAS 2011. Petition for Determination of Nonregulated Status for Herbicide Tolerant DAS-40278-9 

Corn. Submitted by L. Tagliani. Dow Agrosciences LLC, Indianapolis, IN (USDA APHIS BRS Petition 

09-233-01p, see http://www.aphis.usda.gov/brs/aphisdocs/09_23301p.pdf

Deen, W., A. Hamill, C. Shropshire, N. Soltani, and P. H. Sikkema. 2006. Control of Volunteer 

Glyphosate-Resistant Corn (Zea mays) in Glyphosate-Resistant Soybean (Glycine max). Weed 

Technology 20:261-266.

Devine, M. D. 1997. Mechanisms of resistance to acetyl-coenzyme A carboxylase inhibitors: a review. 

Pesticide Science 51:259-264.

Dill, G. M., C. A. Cajacob, and S. R. Padgette. 2008. Glyphosate-resistant crops: adoption, use and 

future considerations. Pest Management Science 64:326-331.

Doebley, J. 1990a. Molecular evidence for gene flow among Zea species. BioScience 40:443-448. 

Doebley, J. 1990b. Molecular systematics of Zea (Gramineae). Maydica 35:143-150.

Dröge, M., A. Puhler, and W. Selbitschka. 1998. Horizontal gene transfer as a biosafety issue: a natural 

phenomenon of public concern. Journal of Biotechnology 64:75-90.

Duke, S. and S. Powles. 2008. Glyphosate: a once-in-a-century herbicide. Pest Management Science

64:319-325.

Duke, S. and S. Powles. 2009. Glyphosate-resistant crops and weeds: now and in the future. 

AgBioForum

12:346-357.

16
Ellstrand, N. C., L. C. Garner, S. Hegde, R. Guadagnuolo, and L. Blancas. 2007. Spontaneous 

hybridization between maize and teosinte. Journal of Heredity 98:183-187

Ellstrand, N. C., H. C. Prentice, and J. F. Hancock. 1999. Gene flow and introgression from domesticated 

plants into their wild relatives. Annual Review of Ecology and Systematics 30:539-563.

EPA (Environmental Protection Agency). 2005. Reregistration Eligibility Decision for 2,4-D. 

http://www.epa.gov/oppsrrd1/REDs/24d_red.pdf (Last updated 8/8/2005)

FDA (Food and Drug Administration). 1992. Statement of Policy - Foods Derived from New Plant 

Varieties. FDA Federal Register 57: 22984.

http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/biotechnology/u

cm096095.htm (Page last updated 8/15/2013) 

FDA (Food and Drug Administration). 1998. Use of antibiotic resistance marker genes in transgenic 

plants. Guidance for Industry 

http://www.fda.gov/food/guidanceregulation/guidancedocumentsregulatoryinformation/biotechnology/u

cm096135.htm (Page last updated: 8/15/2013)

Fukunaga, K., J. Hill, Y. Vigouroux, Y. Matsuoka, J. S. Sanchez, K. Liu, E.S. Buckler, and J. Doebley.

2005. Genetic diversity and population structure of teosinte. Genetics 169: 2241–2255.

Galinat, W. 1988. The origin of corn. Pp 1-31 in Sprague, GF and J.W. Dudley (eds.). Corn and Corn 

Improvement. American Society of Agronomy, Crop Science Society of America, and Soil Science 

Society of America, Madison, WI.

Gianessi, L. P. 2008. Economic impacts of glyphosate-resistant crops. Pest Management Science 64:346-

352.

Gianessi, L. P. and N. Reigner. 2006. Pesticide Use in U.S. Crop Production: 2002 with Comparison to

1992 and 1997. Fungicides & Herbicides. CropLife Foundation. Accessible at:

http://www.croplifefoundation.org/Documents/PUD/NPUD%202002/Fung%20&%20Herb%202002%2

0Data%20Report.pdf

Grant, V. 1981. Plant speciation. Columbia University Press, New York.

Guadagnuolo, R., J. Clegg, and N. C. Ellstrand. 2006. Relative fitness of transgenic vs. non-transgenic 

maize x teosinte hybrids: a field evaluation. Ecological Applications 16: 1967-1974.

Hall, G., G. C. Allen, D. S. Loer, W. F. Thompson, and S. Spiker. 1991. Nuclear scaffolds and scaffoldattachment regions in higher plants. Proceedings of the National Academy of Sciences (USA) 88:9320-

9324.

Hegde, S. G., J. D. Nason, J. M. Clegg, and N. C. Ellstrand. 2006. The evolution of California's wild 

radish has resulted in the extinction of its progenitors. Evolution 60:1187-1197.

Holm, L., J. V. Pancho, J. P. Herbarger, and D. L. Plucknett. 1979. A Geographical Atlas of World

Weeds. John Wiley and Sons, New York.

Horvath, M., G. Ditzelmüller, M. Loidl, and F. Streichsbier. 1990. Isolation and characterization of a 2-

(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium. Applied Microbiology and 

17
Biotechnology 33:213-216.

ILSI (International Life Sciences Institute). 2006. Crop Composition Database, version 3.0. Accessible at

http://www.cropcomposition.org

Kaneko, T., Y. Nakamura, S. Sato, E. Asamizu, T. Kato, S. Sasamoto, A. Watanabe, K. Idesawa, A. 

Ishikawa, K. Kawashima, T. Kimura, Y. Kishida, C. Kiyokawa, M. Kohara, M. Matsumoto, A. 

Matsuno,Y. Mochizuki, S. Nakayama, N. Nakazaki, S. Shimpo, M. Sugimoto, C. Takeuchi, M. Yamada, 

and S. Tabata. 2000. Complete genome structure of the nitrogen-fixing symbiotic bacterium

Mesorhizobium loti. DNA Research 7:331-338.

Kaneko, T., Y. Nakamura, S. Sato, K. Minamisawa, T. Uchiumi, S. Sasamoto, A. Watanabe, K. Idesawa, 

M. Iriguchi, K. Kawashima, M. Kohara, M. Matsumoto, S. Shimpo, H. Tsuruoka, T. Wada, M. Yamada, 

and S. Tabata. 2002. Complete genomic sequence of nitrogen-fixing symbiotic bacterium 

Bradyrhizobium japonicum. DNA Research 9:189-197.

Keeler, K. 1989. Can genetically engineered crops become weeds? Bio/Technology

7:1134-1139.

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

123-149.

Kiely, T., D. Donaldson, and A. Grube. 2004. Pesticides Industry Sales and Usage. 2000 and 2001

Market Estimates. Pg 48. Environmental Protection Agency, Biological and Economic Analysis Division

- Office of Pesticide Programs - Office of Prevention, Pesticides, and Toxic Substances. Accessible at:

http://www.epa.gov/pesticides/pestsales/01pestsales/market_estimates2001.pdf

Knobloch, I. 1972. Intergeneric hybridization in flowering plants. Taxon 21:97-103.

Kohler, H. P. E. 1999. Sphingomonas herbicidovorans MH: a versatile phenoxyalkanoic acid herbicide 

degrader. Journal of Industrial Microbiology and Biotechnology 23:336-340.

Konishi, T. and Y. Sasaki. 1994. Compartmentalization of two forms of acetyl-CoA caroxylase in plants 

and the origin of their tolerance toward herbicides. Proceedings of the National Academy of Sciences 

(USA) 91: 3598-3601.

Koonin, E. V., K. S. Makarova, and L. Aravind. 2001. Horizontal gene transfer in prokaryotes:

quantification and classification. Annual Review of Microbiology 55:709-742.

Mankin, S. L., G. C. Allen, T. Phelan, S. Spiker, and W. F. Thompson. 2003. Elevation of transgene 

expression level by flanking matrix attachment regions (MAR) is promoter dependent: a study of the 

interactions of six promoters with the RB7 3' MAR. Transgenic Research 12:3-12.

Marshall, L. C., D. A. Somers, P. D. Dotray, B. G. Gengenbach, D. L. Wyse, and J. W. Gronwald. 1992. 

Allelic mutations in acetyl-coenzyme A carboxylase confer herbicide tolerance in maize. Theoretical and 

Applied Genetics 83: 435-442.

Martin, J.R. 2008. Strategies for controlling volunteer corn and wheat. Kentucky Pest News. Number

1176. University of Kentucky, College of Agriculture. Available at 

http://www.uky.edu/Ag/kpn/kpn_08/pn080825.htm,. Accessed on 7/24/10.

Miki, B. and S. McHugh. 2004. Selectable marker genes in transgenic plants: applications, alternatives 

18
and biosafety. Journal of Biotechnology 107:193-232.

Muenscher, W. C. 1980. Weeds. 2nd edition. Cornell University Press, New York.

Müller, R. H., S. Kleinsteuber, and W. Babel. 2001. Physiological and genetic characteristics of two 

bacterial strains utilizing phenoxypropionate and phenoxyacetate herbicides. Microbiological Research

156:121-131.

Müller, T. A., S. M. Byrde, C. Werlen, J. R. van der Meer, and H.P. E. Kohler. 2004. Genetic analysis of 

phenoxyalkanoic acid degradation in Sphingomonas herbicidovorans MH. Applied Environmental 

Microbiology. 70:6066-6075.

Norsworthy, J. K., S. M. Ward, D. R. Shaw, R. S. Llewellyn, R. L. Nichols, T. M. Webster, K. W. 

Bradley, G. Frisvold, S. B. Powles, N. R. Burgos, W. W. Witt, and M. Barrett. 2012. Reducing the risks 

of herbicide resistance: best management practices and recommendations. Weed Science (Special Issue): 

31-62.

OECD (Organization for Economic Co-operation and Development). 2002. Consensus document on 

compositional considerations for new varieties of maize (Zea mays): Key food and feed nutrients, antinutrients and secondary plant metabolites. Series on the Safety of Novel Foods and Feeds, No. 6. OECD, 

Paris.

OECD (Organization for Economic Co-operation and Development). 2003. Consensus document on the 

biology of Zea mays subsp. mays (maize). Series on Harmonisation of Regulatory Oversight in 

Biotechnology, No. 27, OECD, Paris.

Owen, M. D. K. 2008. Weed species shifts in glyphosate-resistant crops. Pest Management Science

64:377-387.

Peterson, C. D., D. A. Pearman, and T. D. Dines. 2002. New Atlas of the British flora, London, U.K. 

Petolino, J., F. and N. Arnold, L. . 2009. Whiskers-mediated maize transformation. Methods in

Molecular Biology 526:59-67.

Petolino, J., F., M. Welter, and C. Q. Cai. 2003. Whiskers-mediated transformation of maize. Molecular

Methods of Plant Analysis 23:147-158.

Ridley, W. P., R. D. Shillito, I. Coats, H. Y. Steiner, M. Shawgo, A. Phillips, P. Dussold, and L. Kurtyka.

2004. Development of the International Life Sciences Institute Crop Composition Database. Journal of

Food Composition and Analysis 17:423-438.

Rieseberg, L. H. 1997. Hybrid origins of plant species. Annual Review of Ecology and Systematics

28:359-389.

Rieseberg, L. H. and J. F. Wendel. 1993. Introgression and its consequences in plants. Pp. 70-109 in R. 

G. Harrison (ed.). Hybrid Zones and the Evolutionary Process. Oxford University Press, Oxford, U.K.

Sánchez, G. J. J. and C. J. A. Ruiz. 1997. Teosinte distribution in Mexico. Pp. 18-39 in J. A. Serratos, M. 

C. Willcox, and F. Castillo-Gonzalez (eds.). Gene Flow among Maize Landraces, Improved Maize 

Varieties, and Teosinte: Implications for Transgenic Maize. CIMMYT, Mexico, D.F.

19
Soltani, N., C. Shropshire, and P. Sikkema, H. 2006. Control of volunteer glyphosate-tolerant maize (Zea 

mays) in glyphosate-tolerant soybean (Glycine max). Crop Protection 25:178-181.

Soltis, D. E. and P. S. Soltis. 1993. Molecular data and the dynamic nature of polyploidy. Critical

Reviews in Plant Sciences 12:243 - 243.

Stace, C. A. 1987. Hybridization and the plant species. Pp. 115-127 in K. M. Urbanska (ed.). 

Differentiation Patterns in Higher Plants. Academic Press, New York.

Taba, S. 1995. Teosinte: Geographic variations and conservations. Pp. 59-73 in S. Taba (ed.). Maize

Genetic Resources. CIMMYT, Mexico, D.F.Thomas, W., E. , W. Everman, J. , S. Clewis, B. , and J. 

Wilcut, W. 2007. Glyphosate-resistant corn interference in glyphosate-resistant cotton. Weed

Technology 21:372-377.

USDA-APHIS (U.S. Department of Agriculture, Animal and Plant Health Inspection Service). 2000. 

Regulated Plant Pest List. http://www.invasivespecies.org/RegulatedPestList.pdf. (Last modified:

6/16/2000)

USDA-APHIS. 2013. Plant Pest Risk Assessment for HCEM485 corn. (Prepared in response to Stine 

Seed requests for an extension of nonregulated status (09-063-01p).) Riverdale, MD: US Department 

of Agriculture, Animal and Plant Health Inspection Service, Biotechnology Regulatory Services. 

http://www.aphis.usda.gov/biotechnology/petitions_table_pending.shtml

USDA-NASS (U.S. Department of Agriculture, National Agricultural Statistics Service). 2006. 

Agricultural Chemical Usage 2005 Field Crops Summary. 

http://usda.mannlib.cornell.edu/usda/nass/AgriChemUsFC//2000s/2006/AgriChemUsFC-05-17-2006.pdf

(Last Modified 5/17/2006)

USDA-NASS (U.S. Department of Agriculture, National Agricultural Statistics Service). 2009. Acreage. 

http://usda.mannlib.cornell.edu/usda/nass/Acre/2000s/2009/Acre-06-30-2009.pdf

USDA, NRCS. 2010. The PLANTS Database (http://plants.usda.gov, 26 July 2010). National Plant Data

Center, Baton Rouge, LA 70874-4490 USA.

Vangessel, M.J., Q. Johnson, and Issacs M. 1997. Response of sethoxydim-resistant corn (Zea mays)

hybrids to postemergence graminicides. Weed Technology 11:598-601.

Wen, A., M. Fegan, C. Hayward, S. Chakraborty and L. I. Sly. 1999. Phylogenetic relationships among 

members of the Comamonadaceae, and description of Delftia acidovorans (den Dooren de Jong 1926 

and Tamaoka et al. 1987) gen. nov., comb. nov. International Journal of Systematic Bacteriology 49:

567–576

Wilkes, H. G. 1967. Teosinte: The closest relative of maize. The Bussey Institute, Harvard University, 

Cambridge, MA.

Wood, D. W., J. C. Setubal, R. Kaul, D. E. Monks, J. P. Kitajima, V. K. Okura, Y. Zhou, L. Chen, G. E. 

Wood, N. F. Almeida, Jr., L. Woo, Y. Chen, I. T. Paulsen, J. A. Eisen, P. D. Karp, D. Bovee, Sr., P. 

Chapman, J. Clendenning, G. Deatherage, W. Gillet, C. Grant, T. Kutyavin, R. Levy, M.-J. Li, E. 

McClelland, A. Palmieri, C. Raymond, G. Rouse, C. Saenphimmachak, Z. Wu, P. Romero, D. Gordon, 

S. Zhang, H. Yoo, Y. Tao, P. Biddle, M. Jung, W. Krespan, M. Perry, B. Gordon-Kamm, L. Liao, S. 

Kim, C. Hendrick, Z.-Y. Zhao, M. Dolan, F. Chumley, S. V. Tingey, J.-F. Tomb, M. P. Gordon, M. V. 

Olson, and E. W. Nester. 2001. The Genome of the Natural Genetic Engineer Agrobacterium

20
tumefaciens C58. Science 294:2317-2323.

Wright, T. R., J. M. Lira, D. J. Merlo, and N. Hopkins. 2009. Novel Herbicide Resistance Genes. (U.S. 

Patent # 2009/0093366)

WSSA (Weed Science Society of America). 2007. Herbicide Handbook. 9th Edition. Weed Science

Society of America, Lawrence.

WSSA (Weed Science Society of America). 1998. “Herbicide Resistance” and “Herbicide Tolerance”

defined. Weed Technology. 12: 789

Yoshida, S., Maruyama, S., Nozaki, H., and K. Shirasu. 2010. Horizontal gene transfer by the parasitic 

plant Striga hermonthica. Science 328:1128.

Young, B.G., and S.E. Hart. 1997. Control of volunteer sethoxydim-resistant corn (Zea mays) in soybean

(Glycine max). Weed Technology 11:649-655.

Zipper, C., K. Nickel, W. Angst, and H. P. Kohler. 1996. Complete microbial degradation of both 

enantiomers of the chiral herbicide mecoprop [(RS)-2-(4-chloro-2-methylphenoxy)propionic acid] in an 

enantioselective manner by Sphingomonas herbicidovorans sp. nov. Applied Environmental 

Microbiology 62:4318-4322.

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