Plant Pest Risk Assessment for DAS-444Ø6-6 Soybean

Table of Contents

A. Introduction............................................................................................................................... 2

B. Development of DAS-444Ø6-6 herbicide tolerant soybean ..................................................... 3

C. Description of the modification................................................................................................. 6

D. Potential for DAS-444Ø6-6 to have altered disease and pest susceptibilities.......................... 8

E. Potential for effect on non-target organisms, including those beneficial to agriculture ......... 10

F. Potential for enhanced weediness or invasiveness .................................................................. 10

G. Potential of DAS-444Ø6-6 to impact the weediness of other plants with which it can interbreed

................................................................................................................................................ 11

H. Potential changes to agricultural or cultivation practices ....................................................... 12

I. Potential impacts from transfer of genetic information to organisms with which DAS-444Ø6-6 

cannot interbreed.................................................................................................................... 12

J. Conclusion................................................................................................................................ 13

K. References............................................................................................................................... 13
Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

A. Introduction

Dow AgroSciences LLC (DAS) and M.S. Technologies LLC (MS Tech) have petitioned 

APHIS (APHIS number 11-234-01p) for a determination that genetically engineered (GE) 

soybean (Glycine max) event DAS-444Ø6-6 is unlikely to pose a plant pest risk (DAS and 

MS Tech 2011) and, therefore, should no longer be a regulated article under APHIS’ 7 Code 

of Federal Regulations (CFR) part 340. APHIS administers 7 CFR part 340 under the 

authority of the plant pest provisions of the Plant Protection Act of 20001

. This plant pest 

risk assessment was conducted to determine whether DAS-444Ø6-6 is unlikely to pose a 

plant pest risk. 

Event DAS-444Ø6-6 was produced by transformation of cotyledonary node explants of soybean 

(Glycine max cv Maverick) with Agrobacterium tumefaciens. Because A. tumefaciens is a plant 

pest and some of the regulatory sequences (promoter from cassava vein mosaic virus and 

terminator from A. tumefaciens) used to facilitate expression of the genes in soybean were 

derived from plant pests, this soybean has been considered a regulated article under APHIS 

regulations at 7 CFR part 340.

Potential impacts considered in this Plant Pest Risk Assessment are those that pertain to the 

use of DAS-444Ø6-6 and its progeny in the absence of confinement. APHIS regulation 7 

CFR 340.6(c) specifies the information needed for consideration in a petition for 

nonregulated status. APHIS will evaluate information submitted by the applicant, in addition 

to current literature, related to plant pest risk characteristics, disease and pest susceptibilities, 

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

the regulated article, any impacts on the weediness of any other plant with which it can 

interbreed, potential changes to agricultural or cultivation practices, potential effects to nontarget organisms, and transfer of genetic information to organisms with which it cannot 

interbreed, to determine if DAS-444Ø6-6 is unlikely to pose a plant pest risk. If APHIS 

determines that a GE organism is not a plant pest risk, then APHIS has no regulatory 

authority over that organism. 

1 Section 403 (14) of the Plant Protection Act (7USC Sec 7702(14) defines plant pest as: “Plant Pest - The term “plant pest” means any 

living stage of any of the following that can directly or indirectly injure, cause damage to, or cause disease in any plant or plant 

product: (A) A protozoan. (B) A nonhuman animal. (C) A parasitic plant. (D) A bacterium. (E) A fungus. (F) A virus or viroid. (G) An 

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

subparagraphs.”

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

B. Development of DAS-444Ø6-6 herbicide tolerant2 soybean

In the U.S. soybean was grown on 75.0 million acres in 2011 (Figure 1, USDA NASS 

2011a) with a value of $29.6 billion in 2008/2009 (USDA ERS 2011a). Growers select 

soybean lines adapted to the different environmental and climatic features, operator’s 

education, weed and disease pressures, cost of seed and other inputs, technology fees, human 

safety, ease and flexibility of the productions system and marketing reasons (USDA ERS 

2002; Brookes 2011).

Figure 1. Soybean production areas in the U.S. (USDA NASS 2011b).

The presence of weeds in soybean fields can cause greater production losses than either 

insects or diseases (Gibson 2005; Oerke 2006). Before the development of effective 

herbicides for the selective control of weeds in soybeans in the early 1960’s, cultural 

practices including tillage, use of weed free seed, row spacing and crop rotation were the 

only ways to control weeds (Wax 1973). By 1987, over 30 herbicides were being used on 

soybeans (Jordan 1987). With the 1996 commercial introduction and rapid adoption of 

glyphosate tolerant soybeans, a major change in herbicide usage occurred with an increasing 

use of glyphosate concurrent with the increased planting of glyphosate tolerant soybeans and 

a decrease in use of other soybean herbicides (Figure 2; NRC 2010; Young 2006). 

2

The applicant has described DAS-444Ø6-6 soybean 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 (WSSA) definition of “herbicide resistance” since DAS-444Ø6-6 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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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

Consequently, the diversity of herbicides used for weed management has declined in soybean 

(Table 1; Young 2006) resulting in weed species shifts (Johnson 2009). Deregulation of 

DAS-444Ø6-6 would provide soybean growers with additional options for the post-emergent 

control of both broadleaf and grass weeds. The integration of other herbicides with different 

modes of action with glyphosate has been encouraged to improve the duration of weed 

control, to enhance control of glyphosate tolerant weeds, to reduce the risk of developing 

glyphosate resistant weeds and to control glyphosate-resistant weeds (WSSA 2010). This 

soybean product would also provide a potential remedy to the increased incidence of weed 

species that are more tolerant to glyphosate (DAS and MS Tech 2011; NRC 2010; WSSA 

2010).

Figure 2. Percent acreage of genetically engineered crops in the U.S. (USDA ERS 2011b).

Herbicide 1990 1995 2001 2006

2,4 D 3 10 4

2,4-D Dimethlyl salt 3

Acetic acid (2,4 D) 7

2,4-DB 1

Acifluorfen 3

Alachlor 13 4

Bentazon 16 12 1

Chloramben 1

Chlorimuron, ethyl 20 16 5 4

Clethodim 5 4 3

Clomazone 7 4

Cloransulam, methyl 5 1

Dimethenamid 1

Ethalfluralin 5 1

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

Fenoxaprop 6 3

Fluazifop, P, butyl 6 10 3 1

Flumetsulam 2

Flumiclorac, pentyl 1

Fomesafen 2 4 7 2

Glyphosate 5 21 73 92

Imazamox 5

Imazaquin 16 15 2 1

Imazethapyr 11 44 9 3

Lactofen 1 5 1

Linuron 6 2

Metolachlor 10 7

Metribuzin 19 11 2 2

Paraquat 2 2 1

Pendimethalin 14 26 10 3

Quizalofop 3 6

S-Metolachlor 1

Sethoxydim 4 7 1

Sulfentrazone 5 1

Sulfosate 3 1

Thifensulfuron 4 12 2 1

Tribenuron, methyl 1

Trifluralin 37 20 7 2

Table 1. Percent of U.S. Soybean Acres Treated with Herbicides in 1990, 1995, 2001 

and 2006 (USDA NASS 2010c)

DAS-444Ø6-6 is a GE soybean line that has been developed to increase tolerance to the 

herbicides 2,4-D, glufosinate and glyphosate. The introduced genetic material results in the 

production of aryloxyalkanoate dioxygenase-12 (AAD-12) that degrades the herbicide 2,4-D 

into herbicidally-inactive 2,4-dichlorophenol (Müller 1999; Westendorf 2002 and 2003; 

Wright 2010a), phosphinothricin acetyltransferase (PAT) that metabolizes the L-isomer3 of 

glufosinate into non-phytotoxic N-acetyl-L-glufosinate (2-acetamido-4-methylphosphinicobutanoic acid) (OECD 2002), and modified 5-enolpyruvylshikimate-3-phosphate synthase 

(EPSPS) that is insensitive to glyphosate (Lebrun 1996; Lebrun 2003). If given nonregulated status, DAS-444Ø6-6 soybean would be the first soybean variety with increased

tolerance to 2,4-D, glufosinate, and glyphosate.

3 The herbicide glufosinate, which is utilized as an ammonium salt in commercial formulation, consists of a racemic mixture of D and L 

enantiomers (Liu 2009). D-glufosinate in the racemic D,L mixture is not acetylated by PAT and is not phytotoxic to plants (Beriault

1999).

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

2,4-D (2,4-dichlorophenoxyacetic acid) is an herbicide in the phenoxy or phenoxyacetic acid 

family used for selective control of broadleaf weeds since the mid-1940s in over 600 

products in agricultural and residential applications (USEPA 2005). The mode of action of 

2,4-D for broadleaf plants is unclear, but it is believed to function as a plant growth 

regulator4 with synthetic auxin hormone-like properties. When applied as an herbicide 2,4-D

causes abnormal cell division and growth leading to plant injury and death. 

Glufosinate (phosphinothricin; DL-homoalanin-4-yl(methyl)phosphinic acid) is a nonselective foliar herbicide used for pre-plant and post-emergent control of broadleaf plants 

and annual and perennial grasses (OECD 2002; USEPA 2008). Glufosinate acts by 

inhibiting the enzyme glutamine synthetase, which leads to poisoning in plants because of 

the overproduction of ammonia. Glufosinate was first registered by EPA for use in 2000 as a 

non-selective foliar herbicide that is used for pre-plant and post-emergent control of 

broadleaf weeds (USEPA 2008). EPA registration authorizes use on many crops including; 

apples, berries, canola, corn, cotton, currants, grapes, grass grown for seed, potatoes, rice, 

soybeans, sugar beets, and tree nuts and in non-crop areas including lawns and residential 

areas (USEPA 2008). The first soybean line containing a glufosinate resistance trait 

produced through the use of biotechnology was granted nonregulated status in 1996 (USDA 

APHIS 2011). 

Glyphosate (N-(phosphonomethyl) glycine) is a phosphonomethyl derivative of the amino 

acid glycine that inhibits the enzyme 5-enolpyruvylshikamate-3-phosphate synthase (EPSPS) 

resulting in the disruption of the pathway to synthesize aromatic amino acids (Amrhein 1980; 

Steinrucken 1980). Glyphosate is widely used to control weeds in agricultural crops and 

non-agricultural sites and is registered for use on a variety of fruit, vegetable, and field crops 

as well as for aquatic and terrestrial uses (USEPA 2009). Glyphosate is also registered for 

use on glyphosate-resistant (transgenic) crop varieties such as canola, corn, cotton, soybeans, 

alfalfa, and sugar beets. Labeled uses of glyphosate include over 100 terrestrial food crops 

as well as other non-food sites including forestry, greenhouse, non-crop, and residential. The 

first soybean line containing a glyphosate resistance trait produced through the use of 

biotechnology was granted nonregulated status in 1994 (USDA APHIS 2011). 

C. Description of the modification

Soybean DAS-444Ø6-6 was produced by transformation using disarmed Agrobacterium

tumefaciens (DAS and MS Tech 2011, p. 21). Soybean (cultivar Maverick) cotyledonary 

nodes were infected with Agrobacterium strain EHA101 (Hood 1986) containing plasmid 

4 Plant Growth Regulators are synthetic plant hormones that regulate cellular processes, plant growth and development. Auxin compounds 

represent a class of hormones that along with other plant hormones determine patterns of plant development.

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

pDAB8264. Plants containing the introduced DNA were selected based on growth in the 

present of glufosinate.

The plasmid pDAB8264 contained three gene expression cassettes flanked by T-DNA border 

sequences from the Agrobacterium Ti-plasmid (DAS and MS Tech 2011, pp. 22 and 26-28).

Feature Name Length Description 

T-DNA Border B 24 Required for transfer of T-DNA insert from Agrobacterium tumefaciens into 

plant cells (Barker 1983) 

Intervening 

sequence 

136 Non-specific DNA sequences necessary for cloning 

RB7 MAR 1166 Matrix attachment region from the Nicotiana tabacum rb-7-5A gene (Hall 

1991) 

Intervening 

sequence 

39 Non-specific DNA sequences necessary for cloning 

Histone H4A748 

3′ UTR 

661 3′ untranslated region (UTR) comprising the transcriptional terminator and 

polyadenylation site of the histone H4A748 gene from Arabidopsis thaliana

(Chaboute 1987) 

Intervening 

sequence 

23 Non-specific DNA sequences necessary for cloning 

2mepsps 1338 Native 5-enolpyruvylshikimate-3-phosphate synthase gene from Zea mays with 

two mutations providing glyphosate tolerance (Lebrun 1996, Lebrun 2003) 

TPotp C 372 Optimized chloroplast transit peptide derived from maize and sunflower 

RuBisCO (Lebrun 1996, Lebrun 2003) 

Intervening 

sequence 

4 Non-specific DNA sequences necessary for cloning 

Histone H4A748 

promoter 

1430 Promoter along with the 5′ untranslated region of the Histone H4A748 gene 

from Arabidopsis thaliana including an intron from the Histone 3 gene from 

Arabidopsis thaliana (Chaboute 1987) 

Intervening 

sequence 

92 Non-specific DNA sequences necessary for cloning 

AtUbi10 promoter 1322 Promoter along with the 5' untranslated region and intron from the Arabidopsis 

thaliana polyubiquitin 10 (UBQ10) gene (Norris 1993) 

Intervening 

sequence 

8 Non-specific DNA sequences necessary for cloning 

aad-12 882 Plant-optimized version of an aryloxyalkanoate dioxygenase gene from Delftia 

acidovorans encoding an enzyme with an alpha ketoglutarate-dependent 

dioxygenase activity which results in metabolic inactivation of the herbicide(s) 

(Wright 2009; Wright 2010) 

Intervening 

sequence 

102 Non-specific DNA sequences necessary for cloning 

AtuORF23 3′ 

UTR 

457 3′ untranslated region (UTR) comprising the transcriptional terminator and 

polyadenylation site of open reading frame 23 (ORF23) of plasmid pTi15955 

from Agrobacterium tumefaciens (Barker 1983) 

Intervening 

sequence 

114 Non-specific DNA sequences necessary for cloning 

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

CsVMV promoter 517 Promoter along with the 5′ untranslated region derived from the Cassava Vein 

Mosaic virus (Verdaguer 1996) 

Intervening 

sequence 

7 Non-specific DNA sequences necessary for cloning 

pat 552 Plant-optimized version of phosphinothricin acetyltransferase (PAT) gene, 

isolated from Streptomyces viridochromogenes, encoding a protein that confers 

tolerance to glufosinate (Wohlleben 1988) 

Intervening 

sequence 

102 Non-specific DNA sequences necessary for cloning 

AtuORF1 3′ UTR 704 3′ untranslated region (UTR) comprising the transcriptional terminator and 

polyadenylation site of open reading frame 1 (ORF1) of plasmid pTi15955 

from Agrobacterium tumefaciens (Barker 1983) 

Intervening 

sequence 

228 Sequence from Ti plasmid C58 (Zambryski 1982; Wood 2001) 

T-DNA Border A 24 Required for transfer of T-DNA insert from Agrobacterium tumefaciens into 

plant cells (Barker et al., 1983) 

Intervening 

sequence 

19 Sequence from Ti plasmid C58 (Zambryski 1982, Wood 2001) 

T-DNA Border A 24 Required for transfer of T-DNA insert from Agrobacterium tumefaciens into 

plant cells, aiming to prevent vector DNA being transferred into plant genome 

(Barker 1983) 

Intervening 

sequence 

287 Sequence from Ti plasmid pTi15955 (Barker 1983) 

T-DNA Border A 24 Required for transfer of T-DNA insert from Agrobacterium tumefaciens into 

plant cells, aiming to prevent vector DNA being transferred into plant genome 

(Barker 1983) 

Data from Southern blot analysis (DAS and MS Tech 2011, Table 3, Figures 6 & 7) 

demonstrate that DAS-444Ø6-6 contains a single copy of : (1) RB7 (Figures 8A & 8B); (2) 

histone H4748 promoter (Figures 10A, 10B & 18A), 2mepsps gene (Figures 15A, 15B, 15C 

& 18B) and histone H4A748 terminator (Figures 9A, 9B & 18C); (3), polyubiquitin promoter 

(Figures 11A, 11B & 19A), aad-12 gene (Figures 16A, 16B, 16C & 19B) and ORF 23 

terminator (Figures 12A, 12B & 19C); (4) CsVMV promoter (Figures 13A, 13B & 20A), pat

gene (Figures 17A, 17B, 17C & 20B) and ORF 1 terminator (Figures 14A, 14B & 20C). No 

plasmid backbone sequences were detected (Figures 21A, 21B, 22A, 22B, 23A, 23B, 24A, 

24B, 25A and 25B). 

D. Potential for DAS-444Ø6-6 to have altered disease and pest 

susceptibilities 

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

APHIS assessed whether DAS-444Ø6-6 is likely to have significantly altered disease and 

pest susceptibility. This assessment encompassed a consideration of the introduced trait and 

disease and pest susceptibility data from DAS-444Ø6-6 field trials.

DAS-444Ø6-6 was released in the U.S. from 2009 through 2011 in twenty five locations 

across a diverse range of environmental conditions representative of where DAS-444Ø6-6 is 

expected to be grown (DAS and MS Tech 2011, pp. 88 & 194). DAS and MS Tech used 

well-established qualitative and quantitative techniques to observe insect and disease damage

(DAS and MS Tech 2011, pp. 78-89). The following disease and insect pests were evaluated

in the field: brown spot, Carlavirus, frogeye leaf spot, rust, sudden death syndrome; aphids, 

bean leaf beetles, green cloverworm, grasshoppers, ground beetles, ladybugs, leaf hoppers, 

Lepidoptera spp., and stink bugs. In 2010 the combined-site analysis across ten locations 

found no statistically significant differences for seed germination and emergence, seedling 

vigor, days to flowering, disease incidence, insect damage, days to maturity, lodging, plant 

height, final population, number of pods, number seeds per plant, shattering, yield and seed 

weight (DAS and MS Tech 2011, pp. 83 - 84). Data were collected on pest and disease 

damage across twenty five release sites in 2009 and 2010 (DAS and MS Tech 2011, p. 78). 

In all cases, no significant differences between DAS-444Ø6-6 and the conventional parental 

line Maverick were observed. No qualitative or quantitative observations indicated any 

biologically meaningful differences from control lines or differences outside the range of 

different soybean varieties.

The description of the introduced genetic elements, expression of the gene products and their 

functions of DAS-444Ø6-6 have been summarized above. The A. tumefaciens transformed 

plants used in the generation of DAS-444Ø6-6 were treated with an antibiotic to kill the A. 

tumefaciens cells. DNA sequences derived from plant pests that were incorporated in DAS444Ø6-6 do not result in the production of infectious agents or disease symptoms in plants, 

and so it is unlikely that DAS-444Ø6-6 could pose a plant pest risk. The description of the 

introduced genetic elements, expression of the gene products and their functions in DAS444Ø6-6 has been summarized above. 

Given the interactions between the environment, the genetic backgrounds of the cultivars 

used and some inherent genetic variability within soybean varieties, APHIS concludes that 

these results do not indicate an increased pest risk. Expression of AAD-12, EPSPS and PAT 

in event DAS-444Ø6-6 soybean is not expected to cause plant disease or influence 

susceptibility of DAS-444Ø6-6 or its progeny to diseases or other pests. 

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

E. Potential for effect on non-target organisms, including those 

beneficial to agriculture

There is no reason to believe that deleterious effects or significant impacts on non-target 

organisms, including beneficial organisms, would result from the cultivation of DAS-444Ø6-

6. Field observations of DAS-444Ø6-6 (DAS and MS Tech 2011, section 7) revealed no 

negative effects on non-target organisms, suggesting that the production of the ADD-12, 

PAT and EPSPS proteins in the plant tissues are not toxic to organisms. The introduced 

genetic material does not result in the production of novel proteins, enzymes, or metabolites 

in the plant that are known to have toxic properties. The lack of known toxicity of ADD-12, 

PAT and EPSPS suggests no potential for deleterious effects on beneficial organisms such as 

bees and earthworms. The use of glyphosate, 2,4-D and glufosinate herbicides in the 

cultivation of DAS-444Ø6-6 or its offspring is regulated by EPA under its existing 

regulations for the registration of pesticide use. EPA considers the impacts on the 

environment, including effects on non-target organisms in establishing residue tolerances for 

glyphosate, 2,4-D and glufosinate tolerant lines (USEPA 1993; USEPA 2005; USEPA 

1997). APHIS has not identified any other potential mechanisms for deleterious effects on 

non-target organisms.

F. Potential for enhanced weediness or invasiveness

APHIS assessed whether DAS-444Ø6-6 soybean is any more likely to become a weed than 

the non-transgenic recipient soybean line or other soybean lines currently cultivated. The 

assessment encompasses a consideration of the basic biology of soybean and an evaluation of 

unique characteristics of DAS-444Ø6-6 soybean.

Weediness for the purposes of this part of the plant pest risk assessment is an attribute, which 

causes a crop to act as a weed due to the addition of genes, in comparison to the non-GE 

comparator (parental line Maverick). If the fitness of DAS-444Ø6-6 soybean improves in 

natural or agricultural ecosystems due to the inserted DNA, the potential for weediness could 

increase. The following analysis of the inserted DNA is intended to document that DAS444Ø6-6 soybean has a negligible likelihood of increased weediness. 

In the U.S., soybean is not listed as a weed in the major weed references (Crockett 1977; 

Holm 1979; Muenscher 1980) nor is it designated as noxious weed by the federal 

government (USDA NRCS 2012a). Soybean does not possess any of the attributes 

commonly associated with weeds (Baker 1965), such as long persistence of seed in the soil, 

the ability to disperse, invade, and become a dominant species in new or diverse landscapes, 

or the ability to compete well with native vegetation. Furthermore, mature soybean seeds 

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

have no innate dormancy, are sensitive to cold, are not expected to survive in freezing winter 

conditions and do not reproduce vegetatively (Hermann 1962; OECD 2000; Padgette 1996; 

Raper Jr. & Kramer 1987). 

From 2009 through 2011, DAS and MS Tech conducted field trials to evaluate phenotypic 

characteristics comparing DAS-444Ø6-6 with the non-transgenic soybean parental variety 

Maverick (DAS and MS Tech 2011, pp. 88 & 194). Results on growth characteristics, seed 

production and germination indicate that DAS-444Ø6-6 is not significantly different from its 

comparators (DAS and MS Tech 2011, pp. 79 - 85). No biologically meaningful differences 

were observed across sites between DAS-444Ø6-6 and the parental variety Maverick. 

To increase weediness of the soybean plant there would have to be selection pressure on the 

line (Tiedge 1989). Because neither 2,4-D, glufosinate or glyphosate will affect the survival 

of DAS-444Ø6-6 and because soybean is not itself weedy, this type of selection pressure 

does not now and is unlikely to exist.

There is no indication that DAS-444Ø6-6 possesses a selective advantage that would result 

in increased weediness. DAS-444Ø6-6 lacks the ability to persist as a troublesome weed, 

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

cultivation.

G. Potential of DAS-444Ø6-6 to impact the weediness of other plants 

with which it can interbreed 

The genus Glycine, a member of the Fabaceae (= Leguminosae or pea family), consists of 

two subgenera, soja and glycine (OECD 2000; USDA NRCS 2012b). Perennial species in 

the subgenus glycine do not occur in the U.S. (USDA NRCS 2012b), except in the U.S. 

territories in the South Pacific (Hymowitz & Singh 1987). The subgenus soja consists of 

three annual species: G. soja Sieb. and Zucc., the wild form of soybean; G. gracilis Skvortz., 

the weedy form of soybean; and G. max (L.) Merr., the cultivated soybean. G. soja and G. 

max do not occur naturally in the U.S. (Hermann 1962; Hymowitz 1987; USDA NRCS 

2012b). Hybrids from crosses between the subspecies have generally been sterile, and further 

progeny have only been obtained with extreme difficulty (OECD 2000). 

Cultivated soybean is highly self-pollinating (Ahrent 1994). When soybean plants are grown 

directly adjacent to other soybean plans, the amount of natural cross pollination has generally 

been found to be 0.5 - 1 percent (Fehr 1980; OECD 2000) although higher values (2.5 

percent) occur in some varieties (Abud 2007). Outcrossing can be reduced to 0 – 0.01 

percent with a separation distance of 10 meters (Abud 2007).

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

The cultivated soybean, G. max, lacks sexually compatible wild relatives in the U.S. and its 

territories. Consequently, there is no potential for gene flow from cultivated soybean plants 

to wild relatives in the U.S. Therefore, it is not likely that gene flow and introgression will 

occur between DAS-444Ø6-6 and other species of soybean. APHIS has determined that any

adverse consequences of gene flow from DAS-444Ø6-6 soybean to wild or weedy species in 

the United States are highly unlikely.

H. Potential changes to agricultural or cultivation practices

None of the management practices currently employed for soybean production is expected to 

change if DAS-444Ø6-6 is determined to be no longer subject to the regulatory requirements 

of 7 CFR part 340 or the plant pest provisions of the Plant Protection Act. DAS and MS 

Tech’s (2011) studies demonstrate that the agronomic characteristics and cultivation 

practices employed when growing DAS-444Ø6-6 soybean are essentially indistinguishable 

from practices used to grow other soybean varieties, including other herbicide-tolerant 

varieties (DAS and MS Tech 2011, Section 7.4). Although DAS-444Ø6-6 soybean might be 

expected to replace other varieties of soybean currently cultivated, additional acreage is not 

expected to be developed to accommodate the cultivation of DAS-444Ø6-6 (DAS and MS 

Tech 2011). DAS-444Ø6-6 is comparable to currently available soybean varieties in terms 

of resistance to insects and disease (DAS and MS Tech 2011, pp. 78-89). Therefore, no 

changes are expected for insect and disease control practices with DAS-444Ø6-6. Because 

agricultural and cultivation practices would not be significantly different than that of 

conventional soybean, APHIS does not foresee changes in on insects or diseases damage or 

control measures employed due to agricultural or cultivation practices with DAS-444Ø6-6.

I. Potential impacts from transfer of genetic information to organisms 

with which DAS-444Ø6-6 cannot interbreed

APHIS examined the potential for the new genetic material inserted into DAS-444Ø6-6 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. 

Horizontal gene transfer and expression of DNA from a plant species to other species is 

highly unlikely to occur based on the following reasons.

The horizontal gene transfer (HGT) between unrelated organisms is one of the most 

intensively studied fields of science. Horizontal gene transfer and expression of DNA from a 

plant species to bacteria or animal species is unlikely to occur (Keese 2008). 

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Plant Pest Risk Assessment for Herbicide-Tolerant DAS-444Ø6-6 Soybean

1. Many genomes (or parts thereof) from bacteria that are closely associated with plants have 

been sequenced, including Agrobacterium and Rhizobium (Kaneko 2000; Kaneko 2002; 

Wood 2001). There is no evidence that these organisms contain genes derived from plants. 

Therefore the likelihood of any impact or new horizontal gene transfer that is not already 

capable of taking place in the soil is extremely unlikely.

2. No evidence has been identified for any mechanism by which soybean genes could be 

transferred to humans or animals, or any evidence that such gene transfer has occurred for 

any plant species during evolutionary history, despite animals and humans eating large 

quantities of plant DNA. In cases where review of sequence data implied that horizontal gene 

transfer occurred, these events are inferred to occur on an evolutionary time scale on the 

order of millions of years (Brown 2003; Koonin 2001). 

3. Transgene DNA promoters and coding sequences are optimized for plant expression, not 

prokaryotic bacterial expression. Thus even if horizontal gene transfer occurred, proteins 

corresponding to the transgenes are not likely to be produced. 

4. FDA has evaluated horizontal gene transfer 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 extremely unlikely (FDA 1998). Therefore APHIS concludes that horizontal 

gene transfer is highly unlikely to occur and thus poses no significant plant pest risk.

J. Conclusion

APHIS has prepared this plant pest risk assessment in order to determine if event DAS444Ø6-6 is likely to pose a plant pest risk. Based on the information provided by the 

applicant and the lack of atypical responses to disease or plant pests in the field, weedy 

characteristics of the DAS-444Ø6-6 or other plants with which it can interbreed, changes to 

agricultural or cultivation practices, effects on non-targets or beneficial organisms in the 

agro-ecosystem, indirect effects on other agricultural products and the unlikelihood of 

horizontal gene transfer, APHIS has concluded that soybean event DAS-444Ø6-6 is highly 

unlikely to pose a plant pest risk. 

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