E

Draft Plant Pest Risk

Assessment for Double 

Herbicide-Tolerant Soybean

(Glycine max) event FG72

Petition 09-328-01p

September 2011

United States

Department of 

Agriculture

Marketing and 

Regulatory

Programs

Animal and 

Plant Health 

Inspection 

Service
Table of Contents

Table of Contents...................................................................................................................... 2

Plant Pest Risk Assessment of Bayer CropScience Event FG72 Soybean............................... 3

A. Introduction............................................................................................................. 3

B. History of Development of Event FG72 Soybean .................................................. 4

C. Description of the Modifications............................................................................ 6

D. Plant Pest Risk Assessment .................................................................................... 7

E. Conclusion ............................................................................................................ 14

F. References............................................................................................................. 14
Plant Pest Risk Assessment of Bayer CropScience 

Event FG72 Soybean

A. Introduction

Bayer CropScience (BCS) has petitioned APHIS for a determination that event FG72 double 

herbicide-tolerant soybean is unlikely to pose a plant pest risk (BCS, 2009) 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 20011. This plant pest risk assessment was conducted to determine if 

FG72 soybean is unlikely to pose a plant pest risk. The event FG72 soybean (Glycine max

cultivar Jack) expresses two added proteins which impart herbicide tolerance, 2mEPSPS from 

corn (Zea mays) and 4-hydroxyphenylpyruvate dioxygenase (HPPD), from Pseudomonas 

fluorescens (strain A32). The 2mEPSPS and HPPD proteins expressed in the soybean protect the 

plant from the application of the herbicides glyphosate and isoxaflutole (IFT), respectively.

FG72 soybean was produced by transformation of soybean tissue using direct gene transfer

(biolistics). Because two of the regulatory sequences used to facilitate expression of the herbicide 

tolerance genes in FG72 soybean (Tobacco Etch Virus promoter and A. tumefaciens nos

terminator) were derived from plant pests, the FG72 soybean has been considered a regulated 

article under APHIS regulations at 7 CFR part 340.

Potential impacts to be addressed in this risk assessment are those that pertain to the use of FG72 

soybean and its progeny in the absence of confinement. APHIS uses data and information 

submitted by the applicant, in addition to current literature, to determine if FG72 soybean is 

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

plant pest risk, then APHIS has no regulatory authority over that organism.

Potential impacts in this Plant Pest Risk Assessment are those that pertain to plant pest risk 

characteristics. APHIS regulation 7 CFR 340.6(c) specifies the information needed for 

consideration in a petition for nonregulated status. APHIS will evaluate information submitted 

by the applicant related to plant pest risk characteristics, disease and pest susceptibilities, 

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

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

and transfer of genetic information to organisms with which it cannot interbreed. 

An analysis of agricultural or cultivation practices associated with FG72 soybean and impacts on 

the environment are considered in the Environmental Assessment (EA) for FG72 soybean. A 

thorough assessment of the effects of the determination on nontarget organisms, beneficial 

organisms and threatened and endangered species will also be considered in the EA.

 

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.” 
4

B. History of Development of Event FG72 Soybean

The first glyphosate tolerant soybean that was given a determination of nonregulated status in 

May, 1994 by APHIS was Roundup Ready® soybean 40-3-2 (OECD Unique Identifier MON04032-6; submitted by Monsanto in petition number 93-258-01p). This event involved soybean

that contained the DNA as follows: an enhanced 35S promoter from cauliflower mosaic virus, a 

chloroplast transit peptide from Petunia hybrida fused to the 5-enolpyruvylshikimate-3-

phosphate synthase (EPSPS) gene from Agrobacterium spp. strain CP4, and the nopaline 

synthase 3’ terminator from A. tumefaciens. Monsanto completed its food safety consultation 

with the U.S. Food and Drug Administration (FDA) on soybean Event 40-3-2 on January 27, 

1995 (http://www.accessdata.fda.gov; BNF 00001). In September 1993, the Environmental 

Protection Agency (EPA) completed its Reregistration Eligibility Decision (RED) on the 

pesticide active ingredient glyphosate (EPA-738-F-93-011). In 1995, the Plant Biosafety Office 

(PBO) of the Canadian Food Inspection Agency (CFIA) concluded that Monsanto’s Event 40-3-

1 soybean was substantially equivalent to currently grown soybean, in terms of their potential 

environmental impact and livestock feed safety and the novel traits would not have any 

substantial negative effect on the environment (CFIA Decision Document DD95-05; 

http://www.inspection.gc.ca). In 2006, Monsanto developed a second generation glyphosatetolerant soybean called RReady2YieldTM, Event MON 89788. APHIS made a determination of 

nonregulated status for Event 89788 (RReady2YieldTM) on July 23, 2007. EPA and FDA did not 

re-evaluate MON 89788 because the gene of interest was identical to Event 40-3-2. MON 89788 

is very similar to MON-04032-6. Both plants were genetically engineered to be glyphosate 

tolerant by inserting a gene (from Agrobacterium spp. strain CP4) coding for epsps into the 

soybean genome. The three major differences between MON 89788 and MON-04032-6 are: the 

use of a different promoter for the cp4 epsps gene, a different transformation method, and the use 

of a different recipient variety. The Food Standards Australia New Zealand (FSANZ) approved 

MON 89788 soybean in a pre-market safety assessment on August 8, 2007 (Application A592) 

for import (but not for cultivation). 

There are no historical genetically engineered events for the insertion of the hppd gene into 

soybean or any other crop species. Page 33 of the BCS submitted petition describes the action of 

the protein made by the hppd gene along with diagram for the two metabolic pathways in plants; 

the biosynthesis of plastoquinone and biosynthesis of tocopherol:

“The biochemical pathways in which HPPD is involved differ between plants and 

nonphotosynthetic organisms. In bacteria and animals, it merely serves catabolic 

purposes by catalyzing the first committed step in tyrosine degradation that in the end 

yields energetically exploitable glucogenic and ketogenic products (Brownlee, JohnsonWinters et al., 2004). In plants, however, it is also involved in several anabolic pathways; 

its reaction product homogentisate (2,5-dihydroxyohenylacetate) being the aromatic 

precursor of tocopherol and plastoquinone, which are essential to the photosynthetic 

transport chain and antioxidative systems (Fritze, 2004).”

Figure 1. Biochemical pathways of HPPD proteins
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a) catabolism of tyrosine; b) biosynthesis of plastoquinone (plants); c) biosynthesis of 

tocopherol (plants)

The first herbicide developed in this family was isoxaflutole by Rhone Poulenc (now Aventis) 

and was registered for use in corn in 1998 by the EPA (FR Vol. 63, No.104 50773). 

From the EPA Isoxaflutole Fact Sheet (EPA, 1998):

“Isoxaflutole is a pigment inhibitor. It works by preventing the biosynthesis of 

carotenoid pigments, which protect chlorophyll from decomposition by

sunlight. Without carotenoid pigments, chlorophyll pigments are photo-oxidized 

and chloroplasts break down. Without the energy collecting action of the 

chlorophyll, the whole plant eventually dies.”

Once in the plant, isoxaflutole is rapidly converted into diketonitrile (DKN) which works by 

inhibiting the production of 4-hydroxyphenyl-pyruvate dioxygenase (HPPD). Inhibition of 

HPPD stops the biosynthesis of plastoquinone (Figure 1). Plastoquinone is a carotenoid pigment 

that is needed for electron transport process of photosynthesis 

(http://www.uniprot.org/keywords/618). 

HPPD controls a broad spectrum of grass and broadleaf weeds and is effective on difficult to 

control weeds such as velvetleaf and wooly cupgrass (Zollinger, 2009). HPPD is called a 

proherbicide because it degrades rapidly to the active molecule diketonitrile. The primary
6

breakdown product is mobile and breaks down more slowly than isoxaflutole itself (Zollinger, 

2009). DKN is reactivated by rainfall events, providing control of small weeds that have 

emerged. The combined effects of desorption (release of the compound from the soil) and 

degradation resupply the soil solution with a bioactive product. Because of its adsorption 

characteristics, the application rate of isoxaflutole is adjusted for soil texture and organic matter. 

Depending on the concentration of organic matter, the half-life of HPPD in soil can be up to 30

days (Inoue, Oliveira et al., 2009). Because of the potential for leaching, especially on 

permeable soils, isoxaflutole is a restricted-use pesticide. The label prohibits use on sandy soils 

with less than 2% organic matter where the water table is less than 25 feet from the surface. 

Isoxaflutole has a bleaching effect as chlorophyll is broken down in sunlight but is not replaced. 

The symptoms first appear on leaf edges and tips because this is the site of new carotenoid 

synthesis (Zollinger, 2009).

Since the FG72 soybean is tolerant to isoxaflutole, this herbicide can be applied prior to planting 

(pre-plant) without any significant damage to the plant while being toxic to weeds not managed 

by glyphosate.

Bayer CropScience has now developed the transgenic soybean, Event FG72, which expresses

both 2mEPSPS protein and HPPD proteins. Event FG72 soybean has been developed by BCS as

an alternative soybean product that could help manage herbicide resistant weed populations.

Event FG72 soybean has been field tested under APHIS regulations since 2001. Data were 

provided in the petition for field trials completed prior to the petition submission. Field test 

reports can be found in the BCS Event FG72 soybean petition (BCS, 2009, pp. 93).

BCS will make a request to the Environmental Protection Agency (EPA) to allow the use of an 

isoxaflutole formulation to be used on Event FG72 soybean. BCS initiated the consultation 

process with FDA for the commercial distribution of FG72 soybean and submitted a safety and 

nutritional assessment of food and feed derived from FG72 soybean to the FDA on December 3, 

2009. FDA is currently evaluating the submission, and as of March 16, 2012, has not completed 

the consultation. 

C. Description of the Modifications

Event FG72 soybean contains the stably integrated genes 2mepsps and hppdPfW336, which 

encode respectively the 2mEPSPS and HPPD proteins. The 2mepsps and the hppdPfW336 genes 

were introduced into the soybean genome by means of direct gene transfer (biolistics). “Event

FG72 contains a single insert of two consecutive complete copies of the linear fragment.” (BCS, 

2009, pp.25).

BCS’ double herbicide-tolerant soybean (Event FG72) has been genetically engineered to 

contain the following transgene fragments:

The 2mepsps gene expression cassette borne by pSF10 is represented by the following string:

“Ph4a748-intron1 h3At-TPotpC::2mepsps::3’histonAt” (BCS, 2009, pp.22):
7

Ph4a748 promoter and intron 1 h3At: The Ph4a748 promoter sequence is derived from 

the histone H4 gene of Arabidopsis thaliana (Chabouté, Clement et al., 1987) and 

controls expression of the 2mepsps gene. The Ph4a748 promoter, combined with the 

first intron of gene II of the histone H3.III variant of Arabidopsis thaliana (Chaubet, 

Clement et al., 1992) directs high level constitutive expression, especially in rapidly

growing plant tissues.

TPotp C transit peptide: The optimized transit peptide, which contains sequences from 

the RuBisCO small subunit genes of corn and sunflower, targets the mature protein to 

the plastids, which is where the wildtype protein would be located (Lebrun, Leroux et 

al., 1996).

2mepsps coding sequence: The wildtype epsps gene isolated from maize (Zea mays) was 

mutated using site-directed mutagenesis. Two point mutations resulted in the double 

mutant 2mepsps gene (Lebrun, Sailland et al., 1997). A methionine codon is added to 

the N-terminal of the 2mEPSPS protein sequence in order to restore the cleavage site 

of the optimized plastid transit peptide. The 2mepsps gene encodes a 47 kDa protein 

consisting of 445 amino acids.

3’histonAt terminator: The 3’ untranslated region of the histone from Arabidopsis 

thaliana (Chabouté, Clement et al., 1987).

The hppdPfW336 gene expression cassette is represented by the following string: “Ph4a748

ABBC-5’tev -TPotpY::hppdPfW336::3’nos” (p.23, BCS Event FG72 petition: 

Ph4a748 ABBC promoter and 5’tev enhancer: The same Ph4a748 promoter was used 

to drive the expression of the hppdPfW336 gene, but an internal portion of the 

promoter sequence (referred to as “B”) was duplicated to increase the promoter 

activity in plant cells. In combination with the leader sequence of the tobacco etch

virus (5' tev; Carrington and Freed, 1990), this promoter brings the level of 

expression of hppdPfW336 gene to an appropriate level that enables tolerance at 

agronomic doses of IFT.

TPotp Y transit peptide: The optimized transit peptide, which contains sequences from 

the RuBisCO small subunit genes of corn and sunflower, targets the mature protein to 

the plastids, which is where the wildtype protein would be located (Lebrun, Leroux et 

al., 1996).

hppdPfW336 gene coding sequence: The wildtype hppd gene isolated from 

Pseudomonas fluorescens was mutated using site directed mutagenesis. A point 

mutation resulted in the hppdPfW336 gene (Boudec, Rodgers et al., 2001). The 

hppdPfW336 gene encodes a 40 kDa protein consisting of 358 amino acids.

3' nos terminator: The 3’ untranslated region of the nopaline synthase from 

Agrobacterium tumefaciens is a polyadenylation signal (Depicker, Stachel et al., 

1982).

D. Plant Pest Risk Assessment
8

This plant pest risk assessment is to determine whether BCS Event FG72 is unlikely to pose a 

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

APHIS then has no regulatory authority over that organism under 7 CFR part 340. 

APHIS administers the regulations 7 CFR part 340 under the authority of the Plant Protection 

Act of 2000 (PPA). 

The Animal and Plant Health Inspection Service of the United States Department of Agriculture 

(USDA-APHIS) has prepared a Plant Pest Risk Assessment in response to a petition (APHIS 

Number 09-328-01p) from BCS. APHIS regulation 7 CFR 340.6(c) stipulates the information 

needed for consideration in a petition for nonregulated status. APHIS evaluated information 

submitted by the applicant related to plant pest risk characteristics, disease and pest 

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

potential impacts on non-target organisms, weediness of the regulated article, and any impacts 

on the weediness of any other plant with which it can interbreed. Issues related to agricultural or 

cultivation practices are considered in the Environmental Assessment for BCS Event FG72 

soybean. 

Based on information on the biology of soybean (OECD, 2000), data presented by BCS (BCS, 

2009, Appendix I, pp. 93-114) and scientific data relevant to a discussion of plant pest risk, 

APHIS concluded the following regarding BCS double herbicide-tolerant soybean Event FG72:

Potential Impacts of Genetic Modifications on Disease and Pest Susceptibilities 

USDA-APHIS assessed whether BCS Event FG72 soybean is likely to have significantly

increased disease and pest susceptibility. The assessment encompasses a thorough consideration 

of introduced traits, their impact on agronomic traits and plant composition, and interactions with 

pests and diseases.

BCS observed field trials for damage due to diseases (Alternaria leaf spot, Anthracnose, Asian 

rust, Bacterial blight, Bacterial pustule, Brown spot, Brown stem rot, Cercospora leaf blight, 

Charcoal rot, Downy mildew, Frogeye leaf spot, Fusarium, Phytophthora, Powdery mildew, 

Pythium, Rhizoctonia, Sclerotinia, Septoria, Soybean mosaic virus, Soybean rust, Stem canker, 

Sudden death, White mold) and insect pests (aphid, bean leaf beetle, blister beetle, corn 

rootworm, flea beetle, grasshopper, green cloverworm, Japanese beetle, leafhopper, leafroller, 

Mexican bean beetle, spidermite, seed corn maggot, soybean stem borer, stink bug, tarnished 

plant bug, thistle caterpillar, thrips, velvetbean caterpillar, white fly, wireworm, and woolybear 

caterpillar) for four consecutive years conducted across different agroecological conditions (from 

2001-2004) and then for three years (2007-2009). Data were collected on pest and disease 

damage across 15 sites from 2001-2003 and 18 sites from 2007-2009. The observations

submitted by BCS indicated no meaningful differences between Event FG72 soybean and the 

non-transgenic counterparts for diseases or insect pests (BCS, 2009, Section VII.D, Table 22). 

Thus Event FG72 is expected to be susceptible to the same plant pathogens and insect pests as 

conventional soybean with the noted exceptions listed in the variety registration for Jack. From 

the BCS petition (pp. 65) “The variety registration of Jack, claims resistance to soybean cyst 

nematode (SCN) (Races 3 and 4) Heterodera glycines Ichinohe) and susceptibility to 
9

Phytophthora rot (Races 1, 4, and 7) caused by Phytophthora megasperma (Drechs.) f. sp. 

glycinea T. Kuan & D.C. Erwin. Neither of these phytopathologies was observed consistently in 

the trials, so it was not possible to confirm xpression of these variety traits. At the Perry location, 

one plant of event FG72 was presumed to have died of Phytophthora root rot.”

BCS’ aforementioned data indicate that Event FG72 soybean is not biologically different from 

conventional soybean (with the exception of the HPPD and 2mEPSPS proteins) and the 

herbicide resistant phenotypes did not alter the pest and disease incidences on Event FG72 

soybean; therefore, Event FG72 soybean is no more susceptible to pests and diseases compared 

to conventional soybean cultivars. 

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

The HPPD and 2mEPSPS proteins expressed in Event FG72 soybean are nearly identical to the 

native proteins produced by Pseudomonas fluorescens and corn, respectively. The HPPD protein 

was modified by the replacement of the amino acid glycine with a tryptophan and the 2mEPSPS

protein has two point mutations along with a methionine codon added to the N-terminal end. 

The functional activities of the HPPD and 2mEPSPS proteins were confirmed in vivo from fieldgrown Event FG72 soybean plants (BCS, 2009, pp. 38). BCS collected samples from grain, 

seeds, leaf samples from three different growth stages (V4, V6, V8), stem and root at V4 and V8 

stages to quantify HPPD and 2mEPSPS protein expression in Event FG72 soybean (BCS, 2009, 

Appendix 2). Both proteins were expressed at varying levels during all stages of the plant life 

cycle (BCS, 2009, Table 7 and 8).

USDA-APHIS assessed whether changes in plant metabolism or composition in Event FG72 

soybean is likely to alter plant pest risk. The assessment encompasses a consideration of the 

expressed added proteins or enzymes and their effect on plant metabolism and an evaluation of 

whether the nutrients and anti-nutrient levels in harvested seed and forage derived from Event 

FG72 soybean are comparable to those in the non-transgenic parent line, Jack variety as well as 

three commercial soybean varieties, Stine 2686-6, 2788 and 3000-0. (BCS, 2009, Tables 25-29, 

pp. 71-74).

Detailed compositional and nutritional comparisons of Event FG72 soybean and the 

conventional soybean control, Jack, were conducted on samples collected from ten sites across 

the U.S. in 2008 and compared against reference ranges calculated from three commercial 

soybean varieties listed in the above paragraph. The analysis included moisture, protein, fat, ash, 

carbohydrates, amino acids, fatty acids, anti-nutrients and isoflavones, consistent with OECD 

guidelines (OECD, 2001). 

Appendix 2.G (BCS, 2009, pp. 134) describes the materials and methods used for the 

compositional analysis. The overall data set was examined for evidence of biologically relevant 

changes. All Event FG72 soybean compositional analysis values were similar to the two test and 

control entries and fell within the commercial variety and literature reference ranges (BCS, 2009, 

Tables 25-29, pp. 71-74). The compositional analyses confirmed that Event FG72 soybean is 

compositionally equivalent to the three conventional soybean lines (Stine 2686-6, 2788 and 
10

3000-0) with no single data entry outside any reference ranges (commercial varieties or literature 

references). Collectively, the compositional data support the conclusion that Event FG72 

soybean does not have biologically meaningful differences from conventional soybean from a 

food/feed safety and/or nutritional perspective.

Based on all the above noted considerations, APHIS concludes that Event FG72 soybean poses 

no more of a plant pest risk from new gene products, changes to plant metabolism or 

composition (with the exception of intended changes in herbicide resistance) than conventional 

soybean.

Weediness of Event FG72 Soybean

Soybean is a highly domesticated legume species, and cultivated varieties of soybean in the US

do not exhibit weedy characteristics, nor is soybean listed as a weed in any major weed 

references (Muenscher, 1952; Crockett, 1977; Holm, Pancho et al., 1979). Likewise, soybean is 

not identified as a noxious weed in the Federal Noxious Weed List (7 CFR § 360; 

http://plants.usda.gov/java/noxious?rptType=Federal). Moreover, 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. 

Phenotypic and agronomic characteristics of Event FG72 soybean were evaluated in a 

comparative manner to assess plant pest potential (OECD, 1993). These assessments included 

17 plant growth and development characteristics: early stand count, plant vigor, days to 

flowering, flower color, leaf shape, health rating at stage V4-5, health rating at stage R1, health 

rating of mature plants, pubescence color, pod color, hilum color, canopy, days to maturity, yield 

in bu/ac, lodging, final stand count and pod shatter (BCS, 2009, Table 57, pp. 181). BCS

presented the following results to show that Event FG72 soybean is phenotypically and 

agronomically similar to conventional control reference varieties:

 Seed dormancy is one of the potential traits effecting volunteerism and weediness. No 

statistically or biologically significant differences were detected between Event FG72 

soybean and the conventional control (BCS, 2009, Table 24, pp. 64). Seed tests were 

completed by Iowa State Seed Lab using standard testing (BCS, 2009, pp. 133). No 

dormant seeds were identified. There was a slight difference (94% vs. 96%) in 

germination at day 6 (BCS, 2009, Table 24, pp. 64) between Event FG72 and the parent 

line Jack, but when germination was extended to 13 days, the percent viable seed of the 

samples (95% vs. 96%) were statistically similar. The germination values of Event FG72 

soybean were not different from the conventional control and were within the range of 

accepted germination values for certified soybean seed. Although soybean seeds can 

potentially grow as volunteer plants in a subsequent crop rotation, volunteer plants would

most likely be killed by frost in the soybean growing regions during autumn or winter of 

the year they were produced. Even if soybean volunteers get established, there are 

effective weed management strategies to control such volunteers (OECD, 2000; York, 

Beam et al., 2005).
11

 The results of the 17 plant growth and development characteristics showed the introduced 

trait did not unexpectedly alter the phenotypic or agronomic characteristics of Event 

FG72 soybean compared to conventional soybean. The early plant density and plant 

vigor ratings were lower in unsprayed Event FG72 plots than in commercial variety plots 

which carried through to late season possibly due to seed lot quality. The days to 50% 

flowering were shorter for Event FG72 compared to commercial varieties, but for days to 

95% flowering, there were no differences between the Event FG72 soybean and 

commercial varieties. There are indicative of no increased weediness of Event FG72 

soybean (BCS, 2009, pp. 53-68).

Results of these evaluations indicate that there is no fundamental difference between Event FG72 

soybean and the conventional control for traits associated with weediness. Collectively, these 

findings support the conclusion that Event FG72 soybean is no more likely to be a weed 

compared to conventional soybean. 

Impacts on the Weediness of Any Other Plant with which Event FG72 Soybean

can Interbreed

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, 1991; Rieseberg and Wendel, 1993; Soltis and Soltis, 1993; Hegde, Nason et 

al., 2006), and even in the existing floras, the occurrence of hybridization or introgression is 

reported to be widespread (Stace, 1987; Rieseberg and Wendel, 1993; Peterson, Pearman 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 few other crops (see Table 1 in Ellstrand, Prentice et 

al., 1999).

Soybean is predominantly a self-pollinated species (OECD, 2000), yet a small amount of 

outcrossing does occur. Soybean typically exhibits a level of cross-pollination below one 

percent. Adjacent rows are measured to have between 0.03 and 3.62% outcrossing, but plants 

more than 4.5 meters apart cross at less than 0.02% (Caviness, 1966; Yoshimura, Matsuo et al., 

2006). While most sources agree that insects do not greatly increase outcrossing rate in domestic 

soybean (Erickson, 1984), there is some evidence that some insects (notably honeybees) can 

increase hybridization rates (Free, 1970; McGregor, 1976). Pollen is only viable for 2-4 hours (it 

desiccates quickly) with anthesis (pollen shed) normally occurring in the late morning (Caviness, 

1966). Current cultivation practices to prevent out-crossing have been deemed sufficient to 

prevent unwanted gene flow. For soybean, the Association of Official Seed Certifying Agencies 

(AOSCA) mandates a zero isolation distance where “Fields of soybeans shall be separated from 

any other variety or uncertified seed of the same variety by a distance adequate to prevent 

mechanical mixture”.

Based upon these factors, it is unlikely that Event FG72 soybean will naturally outcross or 

hybridize to a significant extent with other soybean varieties in agricultural settings.
12

In assessing the risk of gene introgression from Event FG72 soybean into its sexually compatible 

relatives, APHIS considers two primary issues: 1) the potential for gene flow and introgression 

and, 2) the potential impact of introgression. 

The genus Glycine is divided into two subgenera, Glycine and Soja. 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, the cultivated soybean. These species grow wild or 

semi-wild in Asia. Fertile hybrids between G. max and G. soja (Nakayama and Yamaguchi, 

2002; Mizuguti, Ohigashi et al., 2010), and between G. max and G. gracilis (Karasawa, 

1952)occur. Glycine soja and G. gracilis grow naturally only in Asia, not in the United States 

(Lu, 2005). The subgenus Glycine consists of twelve wild perennial species. These species grow 

wild in Australia, South Pacific Islands and Asia (Newell and Hymowitz, 1978), and do not exist 

naturally in the US. Hybrids between perennial Glycine species are fertile. 

Glycine max is the only Glycine species located in the United States, thus there are no other plant 

species with which G. max can interbreed. Glycine max has never been found in the wild 

(Hymowitz and Singh, 1987) without human intervention. Therefore, it is highly unlikely that 

soybean plants in the United States will be found outside of an agricultural setting. It is also 

highly unlikely that gene flow and introgression will occur between Event FG72 soybean and 

soybean plants in a natural environment. USDA has therefore determined that any adverse 

consequences of gene flow from Event FG72 soybean to wild or weedy species in the United 

States are highly unlikely.

Effects of Event FG72 Soybean on Non-target Organisms

Event FG72 soybean is not engineered for insect pest resistance, thus there are no ‘target’ 

species, and thus no ‘nontarget’ species either. APHIS assessed whether exposure or 

consumption of Event FG72 soybeans would have an adverse effect on beneficial species or 

wildlife associated with soybeans. As discussed earlier, Event FG72 soybean is similar in 

nutritional and compositional analysis to unmodified control soybean variety except for the 

intended changes in herbicide resistance associated with the production of HPPD and 2mEPSPS 

proteins in the plant.

BCS also assessed the potential allergenicity and toxicity of introduced traits (HPPD and 

2mEPSPS proteins obtained from Pseudomonas fluorescens and corn, respectively) according to 

the recommendations of the Codex Alimentarius Commission (Codex, 2003). The donor 

organism of the 2mepsps gene (corn) is not toxic and consumed by humans and animals alike

and the donor organism of the hppd gene, Pseudomonas fluorescens, can be an opportunistic 

pathogen in immunocompromised patients, but is generally seen as having poor virulence due to 

its inability to multiply at body temperature and having to compete against host defenses (Liu, 

1964). 

BCS’s bioinformatic analyses of both proteins demonstrated that they do not share structurally or 

immunologically relevant amino acid sequence similarities with known allergens. Additionally, 

BCS conducted digestive fate experiments with the HPPD and 2mEPSPS proteins and found that 

the full-length proteins are rapidly digested in simulated gastric fluid (SGF), a characteristic 
13

shared among many proteins with a history of safe consumption. The transiently stable protein 

fragments in the SGF assay were quickly degraded during a short exposure to simulated 

intestinal fluid (SIF). Rapid digestion of the full-length proteins in SGF and SIF, together with 

rapid degradation of the transiently stable fragments from the SGF assay by SIF, indicates that it 

is highly unlikely that the HPPD and 2mEPSPS proteins and their fragments will reach 

absorptive cells of the intestinal mucosa. Finally, the HPPD and 2mEPSPS proteins are present at 

very low concentrations which average no more than 0.00023% and 0.041%, respectively, of the 

total protein in Event FG72 soybean mature seed.

BCS also assessed both proteins for their potential toxicity. HPPD is ubiquitous in nature across 

all kingdoms including bacteria, fungi, plants and animals. HPPD has been characterized in 

organisms present in human food such as carrot, barley, pork and beef (BCS, 2009, pp. 38). The 

proteins lack structural similarity to known toxins or biologically active proteins known to have 

adverse effects to mammals. Both proteins occur at extremely low levels in the harvested seed

(see above paragraph for concentrations), in other words, they make up a negligible portion of 

the total protein present in food and feed derived from Event FG72 soybean. BCS provided data 

showing that feeding high rates of the HPPD and 2mEPSPS proteins purified from Event FG72 

soybean immature seed did not cause adverse effects on mice (BCS, 2009, pp. 47-52). 

Furthermore, BCS has submitted food and feed safety data to FDA as part of a voluntary

consultation process. Based on the food and feed safety data, lack of toxicity and allergenicity of 

introduced gene products, APHIS concludes that feeding of Event FG72 soybean plant or seed 

by mammals and other nontarget organisms is unlikely to cause any adverse impact on their 

survival and reproduction.

Transfer of Genetic Information to Organisms with which Event FG72 Soybean

cannot Interbreed

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

studied fields since 1940, and the issue gained extra attention with the release of transgenic 

plants into the environment (Dröge, Puhler et al., 1998). HGT has been implicated as a major 

contributor to the spread of antibiotic resistance amongst pathogenic bacteria and the emergence 

of increased virulence in bacteria, eukaryotes, and viruses; and has contributed to major 

transitions in evolution. Gene exchange has been documented for nearly all types of genes and 

between unrelated organisms (Gogarten, Doolittle et al., 2002). For example, recently, Yoshida 

and colleagues (Yoshida, Maruyama et al., 2010) through a comparative genomics analysis 

implicated HGT for the presence of a similar genetic sequence between the parasitic plant purple 

witchweed (Striga hermonthica), which infests cereal fields (monocots), and sorghum (Sorghum 

bicolor).

APHIS examined the potential for the new genetic material inserted into Event FG72 soybean 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. The Event 

FG72 soybean contains one coding sequence from bacteria, the modified hppdPfW336 gene from 

Pseudomonas fluorescens (as described by Boudec, Rodgers et al., 2001) and two non-coding 

regulatory sequences from the bacteria, Agrobacterium tumefaciens. Horizontal gene transfer 
14

and expression of DNA 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, bacteria, and 

parasitic plants (e.g. as commensals, symbionts, parasites, pathogens, decomposers, or in the guts 

of herbivores), so far there are no reports of significant horizontal gene transfer between sexually

incompatible or evolutionarily distant organisms (as reviewed in Keese, 2008). Accumulated 

evidence show that there are universal gene-transfer barriers, regardless of whether transfer 

occurs among closely or distantly related organisms (Kaneko, Nakamura et al., 2000; Koonin, 

Makarova et al., 2001; Wood, Setubal et al., 2001; Kaneko, Nakamura et al., 2002; Brown, 2003; 

Sorek, Zhu et al., 2007). Many genomes (or parts thereof) from bacteria that are closely 

associated with plants have been sequenced including Agrobacterium and Rhizobium (Kaneko, 

Nakamura et al., 2000; Wood, Setubal et al., 2001; Kaneko, Nakamura et al., 2002). There is no 

evidence that these organisms contain genes derived from plants. 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 (Koonin, Makarova et al., 2001; 

Brown, 2003). This is similar to the case in a recent report about of HGT between sorghum and 

purple witchweed. According to the authors (Yoshida, Maruyama et al., 2010), the incorporation 

of a specific genetic sequence 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 is 

an extremely rare event, and a majority of those rare events occur over millions of years. 

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

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

corresponding to the transgenes are not likely to be produced. 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 remote (FDA, 1998). 

Therefore APHIS concludes that horizontal gene transfer is unlikely to occur from Event FG72 

soybean to microorganisms and thus no significant plant pest risk is expected from horizontal 

gene transfer.

E. Conclusion

APHIS has reviewed and conducted a plant pest risk assessment on BCS Event FG72 soybean. 

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

characteristics of BCS Event FG72 soybean, the lack of atypical responses to disease or plant 

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

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

FG72 soybean is unlikely to pose a plant pest risk.

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