Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

Monsanto Company (referred to hereafter as Monsanto) has petitioned APHIS (APHIS 

number 09-082-01p) for a determination that genetically engineered (GE) soybean

(Glycine max) event MON 87701 is unlikely to pose a plant pest risk 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 if MON 87701 is unlikely to pose a plant pest risk. 

MON 87701 was produced by using Agrobacterium-mediated transformation (Monsanto, 

2010). Because Agrobacterium tumefaciens is a plant pest, this 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 MON 87701 and its progeny in the absence of confinement. APHIS utilizes data and 

information submitted by the applicant, in addition to current literature, to determine if 

MON 87701 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. 

The Environmental Assessment (EA) for this petition considered whether agricultural or 

cultivation practices for MON 87701 may result in impacts on the environment. A 

thorough assessment of the effects of the determination on non-target and beneficial 

organisms, and threatened and endangered species is included in the EA.

Development of Lepidopteran Resistant Soybean Event MON 87701

MON 87701 soybean expresses an insecticidal protein, Cry1Ac, and was developed for 

the South American soybean market (Monsanto, 2010). In this region, the lepidopteran 

pest, Epinotia aporema, causes severe economic damage through eating of soybean 

plants (Higley and Boethel, 1994). Because it bores into the stem, larvae are protected 

from insecticidal sprays. Control of these insects requires high levels of systemic 

 

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.”
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

2

insecticide treatment. To be effective, the application of these insecticides needs to be 

carefully timed (Higley and Boethel, 1994). 

Cry1Ac is a lepidopteran-specific (e.g., E. aporema) insecticide derived from the soil 

bacterium, Bacillus thuringiensis (Bt). This protein does not affect other orders of insects 

or animals (van Frankenhuyzen, 2009). Although initially developed for the South 

American soybean market, United States (U.S.) growers may eventually adopt MON 

87701 for commercial production if Monsanto obtains appropriate registrations from the 

EPA (Monsanto, 2010). Currently, MON 87701 has only received U.S. Environmental 

Protection Agency (EPA) approval for breeding and seed multiplication activities for a 

total of 15,000 acres in Georgia, South Carolina, North Carolina, Virginia, and Maryland 

with no more than 1,000 acres per county per year. This type of EPA registration 

precludes commercial sale of MON 87701 in the U.S. (US-EPA, 2010).

Soybean growers in the U.S., the world’s largest exporter of this commodity, suffer

0.001% yield loss due to insect pests (Heatherly et al., 2009). Insect pests in the U.S. that 

could be controlled by MON87701 include: soybean looper (Pseudoplusia includens), 

corn earworm / bollworm (Helicoverpa zea), fall armyworm (Spodoptera frugiperda), 

green cloverworm (Plathypena scabra), velvetbean caterpillar (Anticarsia gemmatalis),

lesser cornstalk borer (Elasmopalpus lignosellus), beet armyworm (Spodoptera exigua), 

and yellow stripe armyworm (S. ornithogalli) (Way, 1994)

Crystal (Cry) proteins derived from Bacillus thuringiensis (Bt) have a long history of safe 

use without adverse human health or environmental effects, and provide an option for the 

control of lepidopteran insect pests (Ferré and Van Rie, 2002). Bt-expressing soybean is 

a new commodity not commercially registered in the U.S. However, Bt-cotton and Btcorn, that also express Cry proteins, have a long history of safe use without adverse 

human health or environmental effects and provide an option for the control of 

lepidopteran insect pests (Glare and O'Callaghan, 2000; NAS, 2004; Romeis et al., 2008).

As additional information, tolerance exemptions and conditional pesticide registrations 

have been granted for the plant-incorporated protectant Cry1Ac in Bollgard® and 

Bollgard II® cottons, GE crops that express 100% amino acid sequence identity as MON 

87701 (Monsanto, 2010). MON 87701 is within the scope of the U.S. Food and Drug 

Administration (FDA) policy statement concerning regulation of products derived from 

new plant varieties, including those produced through genetic engineering. The 

Monsanto Company initiated the consultation process with FDA for the commercial 

distribution of MON 87701 and submitted a safety and nutritional assessment of food and 

feed derived from MON 87701 to the FDA on May 28, 2009 (BNF No. 000119) (FDA, 

2010a). FDA evaluated the submission and responded to the developer by letter on 

August 18, 2010 (FDA, 2010b). Based on the information the Monsanto Company 

submitted, and as of August 5, 2010, FDA has no further questions regarding MON 

87701 soybean. 
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

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Description of the Modification

MON 87701 was developed through Agrobacterium-mediated transformation of soybean 

meristem tissue utilizing transformation vector, PV-GMIR9 (Monsanto, 2010 page 38) of 

soybean variety A5547, a type V maturity group soybean (Monsanto, 2010 page 37). 

The PV-GMIR9 vector used for the transformation of soybean to produce MON 87701 is 

approximately 15.5 kb and contains two T-DNAs. Each of the two T-DNAs contains a 

single expression cassette. The first TDNA (designated as T-DNA I) contains the cry1Ac 

expression cassette, which results in the expression of Cry1Ac protein. The cry1Ac 

expression cassette contains the cry1Ac coding sequence under the regulation of the 

RbcS4 promoter and leader, CTP1 chloroplast targeting sequence, and the 7S' 3' nontranslated sequence. The second TDNA (designated as T-DNA II) contains the cp4 epsps 

gene expression cassette. The cp4epsps expression cassette contains the cp4 epsps coding 

sequence under the regulation of the FMV promoter, the shkG leader, the CTP2 

chloroplast targeting sequence and the E9 3' non-translated sequence. 

The backbone region outside of the T-DNAs contains two origins of replication for 

maintenance of plasmid in bacteria (ori V, ori-pBR322), a bacterial selectable marker 

gene (aadA), and a coding sequence for repressor of primer protein for maintenance of 

plasmid copy number in E. coli (rop). A description of the genetic elements in PVGMIR9 is provided in Table IV-1 of the petition.

Transformed plants were self-pollinated. The unlinked insertions of T-DNA I (cry1Ac 

gene expression cassette) and T-DNA II (cp4 epsps gene expression cassette) segregated

in the resulting progeny. A non-lethal dose of glyphosate herbicide was applied to these 

plants. The plants with minor injury were selected for further analyses, whereas plants 

showing no injury, i.e., containing T-DNA II (cp4 epsps gene expression cassette), were 

eliminated from subsequent development. Subsequently, plants containing only a single 

T-DNA I (cry1Ac gene cassette) were identified and selected by a combination of 

analytical techniques, including ELISA and TaqMan PCR analysis. MON 87701 was 

selected from these plants. (See section III of Monsanto, 2010 for a more detailed 

description.)

MON 87701 has been genetically engineered to contain one transgene construct2

: a

RbcS4 promoter, a full-length cry1Ac gene, a CTP1 chloroplast targeting sequence, and a 

7S α’ 3’ non-translated region.

 

2 The cp4 epsps gene from Arabidopsis thaliana (Monsanto, 2010, page 45), encoding for the selectable 

marker glyphosate tolerance, was also incorporated as part of the initial transformation process. However, 

the transformation process resulted in the introduction of the cry1Ac gene and the cp4 epsps gene into two 

different loci. The presence of the cp4 epsps gene allowed for selection of transformed cells containing 

cry1Ac gene in the presence of glyphosate. As part of the breeding process to develop MON 87701, the cp4

epsps gene was bred out, and is not present in MON 87701 (Monsanto, 2010 Figure IV-1 and table IV-1). 
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

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• The RbcS4 promoter and 5’ non-translated region from the ribulose 1,5-

bisphosphate carboxylase small subunit 1A transit peptide of Arabidopsis 

thaliana (Krebbers et al., 1988).

• CTP1 targeting sequence encoding the transit peptide of the Arabidopsis RbcS4 

encoding small subunit 1A transit peptide, from Arabidopsis thaliana, present to 

direct the cry1Ac protein to the chloroplast (Krebbers et al., 1988).

• cry1Ac, a codon-modified coding sequence of the Cry1Ac protein of Bacillus 

thuringiensis (Fischhoff and Perlak, 1996) 3

. The Cry1Ac protein is toxic to 

certain lepidopteran species. 

• 7Sα’, the 3' region of the Sphas1 gene of Glycine max encoding the 7S α' seed 

storage protein, b-conglycinin, including 35 nucleotides of the carboxyl terminal 

b-conglycinin coding region with the termination codon and the polyadenylation 

sequence (Schuler et al., 1982). The element functions to terminate transcription 

and direct polyadenylation of the mRNA. 

• The right and left border regions of T-DNA from Agrobacterium tumefaciens

nopaline Ti-plasmid.

• Intervening sequence used for cloning.

Data from Southern analyses demonstrate that MON 87701 plants contain: (1) a single 

copy of cry1Ac (Monsanto, 2010, Figures V-2 to V-7, pages 58-65); (2) no sequences 

from the transformation plasmid (PV-GMIR9) (Monsanto, 2010) (i.e.‘backbone 

sequences’); (3) no sequences from T-DNA II. (Monsanto, 2010, Figure V-7). Analysis 

of DNA sequences of MON 87701 confirmed that the overall integrity of the intended 

insert and the contiguousness of the functional elements have been maintained 

(Monsanto, 2010, Response Appendix 1). Statistical analyses over multiple generations 

confirm that the cry1Ac gene is stably integrated and is inherited over generations in the 

expected fashion (Monsanto, 2010, Figures V-8 and V-9).

Potential of MON 87701 to Become Invasive and/or a Weed

Soybeans are not considered a weed in the U.S. (Holm, 1977; Holm, 1997; Muenscher, 

1980.; Reed and Hughes, 1977), and are not listed as a Federal noxious weed (7 CFR part 

3604

). APHIS assessed whether MON 87701 is any more likely to become a weed than 

the nontransgenic comparator, or other cultivated soybeans. The assessment encompassed

a thorough consideration of the basic biology of soybeans and an evaluation of the unique 

 

3 MON 87701 expresses the Cry1Ac protein, an insecticidal protein from Bacillus thuringiensis subsp. 

kurstaki, which provides resistance to certain lepidopteran pests. The Cry1Ac protein expressed in MON 

87701 shares >99% amino acid identity with Cry1Ac from B. thuringiensis (Bt) subsp. kurstaki and 100% 

amino acid sequence identity with the Cry1Ac protein present in Bollgard cotton, with the exception of four

additional amino acids at the N-terminus of the MON 87701-produced Cry1Ac protein. These four amino 

acids are derived from the chloroplast targeting sequence.

4 http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist2006.pdf
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

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characteristics of MON 87701. APHIS analyzed the field test reports and other data 

included in the petition, as well as data from scientific literature on the establishment, 

reproduction, and dispersal processes of soybeans as measures of invasiveness and 

weediness and the effectiveness of Cry1Ac-expressing soybeans. 

Monsanto provided measures of plant growth (Monsanto, 2010, pages 92-108) including; 

plant growth and development characteristics, seed germination parameters, pollen 

characteristics, and observations for plant-insect and plant-disease interactions and plant 

responses to abiotic stressors (Monsanto, 2010, Tables VIII-3, VIII-5, VIII-6, and VIII-7

and Appendix F, G, H, and I). The only significant difference between MON 87701 and 

the non-transformed soybean was found between the percentage of hard viable seed at 

20oC (Monsanto, 2010, Table VIII-3). The observed variation in germination was small 

(0.0% for MON 87701 and 0.5% for A5547), not found under any other temperature 

regime that ranged between 10 to 30 oC (Monsanto, 2010 Page 99).

A measure of the reproductive capacity of plants that are propagated by seed, such as 

soybeans, is the number of seeds that are produced and the germination and viability of 

those seeds. Overall, MON 87701 produced similar or slightly higher (not significant) 

percentages of viable seed when compared to A5547 or the null isoline (Monsanto, 2010,

Table VIII-3). Interestingly, the tests conducted in Mississippi had a consistent and 

significant lower germination than the other locations (Monsanto, 2010 Table F-2).

These results on growth characteristics and seed production and germination, indicate 

that the MON 87701 is not significantly different than its comparators. There is no 

indication that MON 87701 possesses a selective advantage that would result in increased 

weediness. Therefore, MON 87701 lacks the ability to persist as a troublesome weed, and 

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

cultivation.

Potential for Gene Flow and Gene Introgression from MON 87701 into its SexuallyCompatible Relatives 

Glycine max is cultivated as an annual crop in the U.S. in 43 states(USDA NASS 

(National Agricultural Statistics Service), 2007). Glycine max is generally 

self-pollinating, (Caviness, 1966; Free, 1970; Monsanto, 2010; Ray et al., 2003). In 

assessing the risk of gene introgression from MON 87701 to its sexually compatible 

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

introgression, and 2) the potential impact of introgression. Due to the fact that G. max

lacks wild and cultivated plants with which freely interbreeds, the USDA has determined 

that any adverse consequences of gene flow from MON 87701 to wild or weedy species 

in the U.S. are highly unlikely.

Potential for Transfer of Genetic Information to Organisms with which MON 87701

Cannot Interbreed

APHIS assessed whether horizontal gene transfer might occur between MON 87701 and 

inserted genes with other organisms. However, such transfer and expression of DNA 
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

6

from a plant species to bacteria is unlikely to occur (Keese, 2008). First, many bacteria 

(or parts thereof) that are closely associated with plants have been sequenced, including 

Agrobacterium and Rhizobium (Kaneko et al., 2002; Kaneko et al., 2000; Wood et al., 

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

Second, 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 et al., 2001). Third, transgene DNA promoters 

and coding sequences are optimized for plant expression, not bacterial expression. Thus, 

even if horizontal gene transfer occurred, proteins corresponding to the transgenes are not 

likely to be produced. Therefore, APHIS concludes that horizontal gene transfer is 

unlikely to occur and thus poses no significant environmental or plant pest risk.

Potential for MON 87701 to have Altered Disease and Pest Susceptibilities

APHIS assessed whether MON 87701 is likely to have significantly altered disease and 

pest susceptibility. This assessment encompassed a thorough consideration of the 

introduced trait and disease and pest susceptibility data from MON 87701 field trials. 

The applicant on Table G-6 (Monsanto, 2010) provided a summary of disease 

susceptibility evaluated in 131 occasions in 11 different sites, finding no differences 

between MON 87701 and the control line. Data submitted by Monsanto indicate that 

there are no significant differences for disease susceptibility between MON 87701 and its

comparators (Monsanto, 2010 Field trials summary, Table G-6). However, seed diseases 

caused by Cercospora kikuchii and Phomopsis spp. were observed in seeds produced in 

Mississippi and North Carolina trials (Monsanto, 2010, pages 96 and 104).

Susceptibility to arthropods was also evaluated in 11 sites in 133 occasions finding no 

differences in pest susceptibility of 20 taxa, except for four species, three of them in the 

order Lepidoptera (Plathypena scabra, Pseudoplusia includes and Anticarsia 

gemmatalis), which is commonly affected by Cry1A proteins, and one species that should 

not be affected by Cry1 toxins (Cerotoma trifurcate, Coleoptera: Chrysomelidae)

(Monsanto, 2010, page 295).

MON 87701 was grown during 2007 in 17 locations encompassing the range of 

geographical and environmental conditions (Monsanto, 2010) where it is anticipated that 

it will be grown for the intended purpose in the U.S. These field trials included two 

soybean comparators: MON 87701 and A5547, the comparator. MON 87701 plants were 

examined for changes in the types of plant pests that were observed and the relative pest 

damage that those plants incurred (field test summaries submitted for MON 87701

(Monsanto, 2010)) and published information (McPherson et al., 2001; McPherson and 

MacRae, 2009; Miklos et al., 2007; Walker et al., 2000). When expressed in MON 

87701, the Cry1Ac protein provides greater protection from the feeding damage incurred 

by several important lepidopteran pests of soybeans.
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

7

Soybean is not a plant pest in the United States5, and the introduced DNA in MON 87701

is unlikely to pose a plant pest risk. Based on the analysis of genetic modifications and 

their functions and field testing data submitted by petitioner, APHIS concludes that there 

are no significant differences between MON 87701 soybeans and the non-transgenic 

counterparts relative to pest and disease susceptibility. 

Potential Impacts of MON 87701 on Target and Non-target Organisms, Including 

Beneficial Organisms

For decades, microbial products containing Bacillus thuringiensis (the organism that 

produces the Cry1A protein) have been used to control insect pests on a commercial scale 

and for home garden applications (Glare and O'Callaghan, 2000; OMRI, 2009; Shelton et 

al., 2002). Plants that are genetically engineered to express the Cry proteins have a 

history of safe use in the U.S. Since the mid-1990s, corn and cotton lines have been 

commercialized without substantiated reports of significant deleterious impacts on nontarget organisms (Mendelsohn et al., 2003; OECD, 2007; US-EPA, 2008). The use of 

transgenic crops producing Cry1A proteins has been shown to reduce the use broad 

spectrum insecticides

6 without significant impacts on diversity of non-target insects 

(Cattaneo et al., 2006; Dively, 2005; Marvier, 2007; Naranjo, 2005; Romeis et al., 2006; 

Torres and Ruberson, 2005; Torres and Ruberson, 2007; Whitehouse et al., 2005). MON 

87701 is expected to be similar with respect to the low potential harm to the environment 

(Monsanto, 2010). Because Cry receptors are not present in non-target birds and

mammals (Glare and O'Callaghan, 2000; Hofmann et al., 1988a; Hofmann et al., 1988b; 

Shimada et al., 2006a; Shimada et al., 2006b; Van Rie et al., 1989; Van Rie et al., 1990), 

these insecticidal proteins are not expected to adversely affect non-target invertebrate and 

vertebrate organisms (Glare and O'Callaghan, 2000; US-EPA, 2008). 

In addition to the 21 references that the petitioner provided (Monsanto, 2010), other 

information has been reviewed by APHIS (Carrière et al., 2009; Chen et al., 2009; de 

Vaufleury et al., 2007; Ferry et al., 2007; Ferry et al., 2006; Ludy and Lang, 2006; Obrist 

et al., 2006; Rauschen et al., 2009; Schoenly et al., 2003; Sharma and Pampapathy, 2006; 

Torres and Ruberson, 2008; Vercesi et al., 2006; Zeilinger et al., 2010; Zwahlen and 

Andow, 2005) that describes the non-effects of Bt crops or toxins in non-target 

 

5 http://www.aphis.usda.gov/plant_health/plant_pest_info/weeds/downloads/weedlist2006.pdf

6 Broad spectrum insecticides are chemical insecticides which kill insects that are causing injury to plants 

and also kill other insects that are not causing injury to the plant. Insects that are inadvertently killed by the 

application of insecticide are called “non-target” insects. Because Cry proteins are specific for a narrow 

range of insects, use of Cry1Ac to control plant pests is recognized as being beneficial to the survival of 

non-target insects US-EPA. (2008) Biopesticides Registration Action Document Bacillus thuringiensis 

modified Cry1Ab (SYN-IR67B-1) and Vip3Aa19 (SYN-IR102-7) insecticidal proteins and the genetic 

material necessary for their production in COT102 XCOT67B cotton, U.S. Environmental Protection 

Agency, Office of Pesticide Programs, Biopesticides and Pollution Prevention Division, Washington, D.C. 

pp. 135.. 
Plant Pest Risk Assessment for Monsanto MON 87701 Soybeans

8

organisms, except minor and unclear results found by Zwahlen et al.(2007) using Bt corn 

(Cry1Ab) as a model.

Based on the above information, APHIS believes that a determination of nonregulated 

status for MON 87701 will have no adverse effects on non-target organisms. Another 

consideration by APHIS was the potential that additional 15,000 acres of Cry1Acexpressing crops may have on the development of Bt resistance in important lepidopteran 

pests. 

Conclusion

APHIS has prepared this plant pest risk assessment in order to determine if event MON 

87701 is unlikely to pose a plant pest risk. Based on the information provided by the 

applicant and the lack of plant pest risk from the inserted genetic material, weedy 

characteristics, atypical responses to disease or plant pests in the field, effects on nontargets or beneficial organisms in the agro-ecosystem, and horizontal gene transfer,

APHIS has concluded that event MON 87701 is unlikely to pose a plant pest risk. 

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