GM Crop Database

Database Product Description

MON-Ø4Ø32-6 (GTS 40-3-2)
Host Organism
Glycine max L. (Soybean)
Trait
Glyphosate herbicide tolerance.
Trait Introduction
Microparticle bombardment of plant cells or tissue
Proposed Use
Production of soybeans for animal feed (mostly defatted toasted meal and flakes) and human consumption (mostly oil, protein fractions, and dietary fibre).
Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Argentina 1996 1996 1996
Australia 2000
Brazil 1998 1998 1998 View
Canada 1996 1995 1995
China 2004 2004
Colombia 2005 2005
Czech Republic 2001 2001
European Union 2005 2005 View
Japan 1996 1996 1996
Korea 2000 2004
Mexico 1998 1998 1998 View
Paraguay 2004 2004 2004
Philippines 2003 2003
Russia 1999 View
South Africa 2001 2001 2001
Switzerland 1996 1996
Taiwan 2002
United Kingdom 1996 1996
United States 1994 1994 1994
Uruguay 1997 1997 1997 View

Introduction Expand

The soybean line GTS 40-3-2 was developed to allow for the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option for soybean. This genetically engineered soybean variety contains a glyphosate tolerant form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) isolated from the common soil bacterium, Agrobacterium tumefaciens strain CP4 (CP4 EPSPS).

The EPSPS enzyme is part of the shikimate pathway that is involved in the production of aromatic amino acids and other aromatic compounds in plants (Steinrucken & Amrhein, 1980). When conventional plants are treated with glyphosate, the plants cannot produce the aromatic amino acids needed to survive. This enzyme is present in all plants, bacteria, fungi, but not in animals, which do not synthesize their own aromatic amino acids. Because the aromatic amino acid biosynthetic pathway is not present in mammalian, avian or aquatic life forms, glyphosate has little if any toxicity for these organisms (U.S. EPA, 1993; WHO, 1994; Williams et al. 2000). The EPSPS enzyme is normally present in food derived from plant and microbial sources.

GTS 40-3-2 was developed by introducing the CP4 EPSPS coding sequence into the soybean variety A5403, a commercial soybean variety of Asgrow Seed Company, using particle-acceleration (biolistic) transformation. A5403 is a maturity group V cultivar that combines consistently high yield potential with resistance to races 3 and 4 of the soybean cyst nematode (SCN). It also possesses good standability, excellent emergence and tolerance to many leaf and stem diseases. The glyphosate tolerance trait has since been transferred into more than one thousand commercial soybean varieties by traditional breeding techniques. Globally, glyphosate-tolerant soybeans were planted on more than 50 million hectares in 2007. One of the reasons growers have rapidly adopted the glyphosate-tolerant soybean is the simplicity it offers in weed control. Since glyphosate containing herbicides are highly effective against the vast majority of annual and perennial grasses and broadleaf weeds, growers planting glyphosate tolerant soybeans are able to reduce the number of herbicides used to control the economically destructive weeds that grow in their fields and thereby realize a savings in weed control costs. This reduction in herbicides used has benefited the environment by reducing the number of herbicide applications and also allows growers to implement integrated weed management practices in their fields - practices that are generally not possible when pre-plant or pre-emergent herbicides are used.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase HT enhanced CaMV 35S chloroplast transit peptide from Petunia hybrida A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1* Native; Also 2 partial gene sequences (250 bp; 72 bp)

Characteristics of Glycine max L. (Soybean) Expand

Center of Origin Reproduction Toxins Allergenicity

Southeast Asia; wild soybean species endemic in China, Korea, Japan, Taiwan

self-polinated; rarely displays any dormancy characteristics; does not compete well with other cultivated plants

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Agrobacterium tumefaciens strain CP4 CP4 epsps

A. tumefaciens is a common soil bacterium that is responsible for causing crown gall disease in susceptible plants. There have been no reports of adverse affects on humans or animals.

Modification Method Expand

Soybean line GTS 40-3-2 was produced by biolistic transformation of plant cells from soybean cultivar A5403 with DNA-coated gold particles. The plasmid PV-GMGT04 used for transformation contained the genes coding for glyphosate tolerance and for production of ß-glucuronidase (gus gene), a selectable marker. Expression of the CP4 EPSPS gene was regulated by an enhanced 35S promoter (E35S) from cauliflower mosaic virus (CaMV), a chloroplast transit peptide (CTP4) coding sequence from Petunia hybrida, and a nopaline synthase (nos 3') transcriptional termination element from Agrobacterium tumefaciens. The transit peptide facilitated the translocation of newly translated EPSP synthase into chloroplasts, the site of aromatic amino acid biosynthesis and glyphosate site of action.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from GTS 40-3-2 demonstrated that there were two sites of integration, one site containing a functional copy of CP4 EPSPS gene and a second site containing a non-functional segment of the CP4 EPSPS gene. The gus gene was not integrated into the host genome and there were no antibiotic resistance marker genes introduced into GTS 40-3-2.

Southern blots, chromosome walking and DNA sequencing were used to analyze the insertion sites. The largest insert, containing the function CP4 EPSPS gene, contained a deletion in the enhancer region of the E35S promoter and the remainder of the E35S promoter was functional. A single copy of the CP4 EPSPS gene (1365 bp) was integrated and analysis of the 3' terminus revealed a complete nos 3' transcriptional termination element, rather than a portion of nos as previously reported. Also, an additional 250 bp sequence corresponding to a portion of the CP4 EPSPS gene was recently detected adjacent to the 3' end of the nos 3' element, which was not detected in earlier studies (Additional information submitted by Monsanto to regulatory authorities in the spring of 2000).

Analysis of the second insert site showed the presence of a 72 bp sequence corresponding to a portion of the CP4 EPSPS gene. Sequence analysis, western and northern blotting confirmed that neither the additional 250 bp sequence identified in the first insertion site nor the 72 bp fragment inserted at a second site were functional. Pedigree analyses confirmed that these segments of CP4 EPSPS were present in GTS 40-3-2 soybeans evaluated during all safety assessment studies and are present in commercial varieties.

Genetic Stability of the Introduced Trait

DNA analyses over six generations demonstrated that the CP4 EPSPS gene was stably inserted. Observations over multiple generations showed the CP4 EPSPS gene was no longer segregating, demonstrating that soybean line GTS 40-3-was homozygous for the herbicide tolerance trait. These multi-generation studies on GTS 40-3-2 also demonstrated that the 72 bp CP4 EPSPS segment co-segregated with the primary insert, indicating that the two sites of integration are close linked and behave as a single locus.

Expressed Material

Expression of the full-length CP4 EPSPS gene, which encodes a 456 amino acid polypeptide (46 kDa), was confirmed by Western immunoblot analysis and the levels of expression were quantitated using enzyme linked immunosorbent assay (ELISA). The concentrations of CP4 EPSPS averaged 239 µg/g fresh weight tissue in seeds and 495 µg/g f.w.t. in leaves.

Soybean line GTS 40-3-2 was found to express only the full length mRNA and complete CP4 EPSPS protein. The additional DNA segments, the 72 bp CP4 EPSPS segment that comprises the second insert or the 250 bp of the CP4 EPSPS element adjacent to the 3' end of NOS on the functional insert, were both non-functional and did not result in the expression of any mRNA or protein. These results were expected as neither segment contained a promoter or terminator sequence, and only the full-length mRNA and full-length CP4 EPSPS protein were detected.

Environmental Safety Considerations Expand

Field Testing

GTS 40-3-2 soybean has been field tested in the United States (1991-1993), Canada (1992), Puerto Rico (1993), Argentina, and Costa Rica. Agronomic studies on seed yield, and visual observations on germination characteristics of seeds, final stands, disease and insect susceptibility supported the conclusion that soybean line GTS 40-3-2 was as safe to grow as other soybean varieties and had no potential to pose a plant pest risk.

Outcrossing

Gene introgression from transformed soybean line GTS 40-3-2 was extremely unlikely as there are no relatives of cultivated soybean in the continental United States and Canada, and soybean plants are almost completely self-pollinated. Furthermore, the reproductive characteristics such as pollen production and viability were unchanged by the genetic modification resulting in GTS 40-3-2.

Cultivated soybean, Glycine max, naturally hybridizes with the wild annual species G. soja. However, G. soja is only found naturally occurring in China, Korea, Japan, Taiwan and the former USSR, and is not naturalized in North America, although it may possibly be grown in research plots. It was concluded that the potential for transfer of the glyphosate tolerance trait from the transgenic line to soybean relatives through gene flow was negligible in managed ecosystems, and that there was no potential for transfer to wild species in Canada and the continental United States

Weediness Potential

No competitive advantage was conferred to GTS 40-3-2, other than that conferred by resistance to glyphosate herbicide. Resistance to glyphosate-containing herbicides will not, in itself, render soybean weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified. In the unlikely event of the formation of a herbicide tolerant hybrid, there would be no competitive advantage conferred on any hybrid progeny in the absence of sustained glyphosate use. The glyphosate-tolerant plant could easily be controlled by mechanical means or by using herbicides that are not based on glyphosate. Cultivated soybean does not exhibit any weedy characteristics in the United States and Canada, although related species are reported as weeds in Japan and China. It was concluded that soybean line GTS 40-3-2 had no altered weed of invasiveness potential compared to commercial soybean varieties.

Secondary and Non-Target Adverse Effects

Field observations of line GTS 40-3-2 revealed no negative effects on nontarget organisms, suggesting that the relatively higher levels of the protein in the transgenic plant tissues were not toxic to beneficial organisms. The novel protein CP4 EPSPS did not result in altered toxicity or allergenicity properties as demonstrated from studies including the acute oral mouse gavage study, the digestive fate study, and the fact that homologous EPSPS proteins are ubiquitous in nature and common in plants, fungi and microbes. Furthermore, the high specificity of the enzyme for its substrates makes it unlikely that the introduced enzyme would metabolize endogenous substrates to produce compounds toxic to beneficial organisms. It was determined that the genetically modified soybean line GTS 40-3-2 did not have a significant adverse impact on organisms beneficial to plants or agriculture, or on nontarget organisms, and was not expected to impact on threatened or endangered species.

Impact on Biodiversity

GTS 40-3-2 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of soybean production. Since there are no wild relatives of soybean in Canada and continental United States and since soybean is not an invasive species, the novel trait will not be transferred to unmanaged environments.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The genetic modification of GTS 40-3-2 soybean will not result in any change in the consumption pattern of soybean products. Consequently, the dietary exposure of consumers in the United States and Canada to GTS 40-3-2 soybean products was anticipated to be the same as for other lines of commercially available soybean. Dietary exposure to EPSPS is not novel in that all plants, bacteria and fungi produce this enzyme and the CP4 EPSPS will be ingested as inactive denatured protein since all soybean-derived human food products are heated prior to consumption.

Nutritional Data

The analysis of nutritional composition of transgenic GTS 40-3-2 soybean and non-transgenic soybean did not reveal any significant differences in the levels of protein, fat, fibre and starch. A number of these parameters were also measured in glyphosate-treated soybeans with the same results. Comparisons of the amino acid composition of the raw soybeans and the fatty acid profiles of extracted oil from transgenic and control plants did not reveal any significant differences. The anti-nutritive activity in raw soybean meal and flour is caused by soybean trypsin inhibitor, which acts to inhibit normal protein digestion in humans and animals. There was no significant difference in the trypsin inhibitor activity between transgenic GTS 40-3-2 soybeans and non-transgenic control soybeans. Likewise, there were no significant differences in the levels of plant lectins, as determined by hemagglutination assay, or isoflavone glucosides between transgenic and control soybeans. These latter substances, which include genistin and daidzin, can exhibit estrogenic and hypocholesterolemic activities. It was determined that the consumption of refined soybean oil from GTS 40-3-2 would have no significant impact on the nutritional quality of the food supply in the United States and Canada.

A series of animal feeding studies were completed using diets incorporating seed or processed fractions from soybean line GTS 40-3-2. These studies addressed the nutritional equivalence of transgenic soybean when used as animal feed, the safety of any expressed protein or peptide (or any other newly produced constituent), and the potential of any pleiotropic effect caused by the insertion process or site of insertion. The animal feeding studies included two independent four week studies in rats (one with unprocessed and one with processed soybeans), a four week dairy cow study, a six week chicken study, a ten week catfish study and a five day quail study. Animals were fed either unprocessed or processed soybeans (dehulled, defatted, toasted). Included in these studies were control groups fed a non-commercial glyphosate-tolerant soybean (61-67-1) and the non-modified parental soybean line (A5403) from which both glyphosate tolerant events were derived. Results from all groups were compared using conventional statistical methods to detect differences between groups in measured parameters. All three soybean samples tested provided similar growth and feed efficiency for rats, chickens, catfish and quail. The nutritional value or wholesomeness of GTS 40-3-2, even when fed to animals at levels much higher than humans would encounter in the diet, was the same as conventional varieties of soybeans.

Toxicity

The low potential for toxicity of transgenic soybean line GTS 40-3-2 was demonstrated by examining the amino acid sequence homology, acute oral toxicity studies on mice, and the characteristics of the proteins. The amino acid sequence of CP4 EPSPS was determined to be closely related to the sequence of the endogenous soybean EPSPS enzyme. An analysis of the amino acid sequence of the inserted CP4 EPSPS enzyme did not show homologies with known mammalian protein toxins and was not judged to have any potential for human toxicity. Additionally, acute oral toxicity studies with purified CP4 EPSPS did not reveal any deleterious effects when mice were administered a dose of 572 mg/kg body weight, which was approximately 1300-fold greater than the highest anticipated potential consumption of CP4 EPSPS from soybean. Furthermore, EPSPS is an enzyme that is ubiquitous in nature, present in plants, fungi and micro-organisms and therefore would not be expected to be toxic or allergenic.

Allergenicity

The CP4 EPSPS protein is extremely unlikely to be an allergen. A search for amino acid sequence similarity between the CP4 EPSPS protein and known allergens revealed no significant amino acid sequence homologies. In addition, the potential for allergenicity was assessed based upon the characteristics of known food allergens (stability to digestion, stability to processing). Unlike known protein allergens, CP4 EPSPS was rapidly degraded by acid and/or enzymatic hydrolysis when exposed to simulated gastric or intestinal fluids. Overall, the CP4 EPSPS does not possess characteristics typical of known protein allergens.

Abstract Collapse

Soybean (Glycine max) is grown primarily for its seed, which has many uses in the food and industrial sectors, and represents one of the major sources of edible vegetable oil and of proteins for livestock feed use. The major producers of soybeans were the United States, Brazil, Argentina, China, India, Paraguay and Canada.
A major food use of soybean in North America and Europe is as purified oil, used in margarines, shortenings, and cooking and salad oils. It is also a major ingredient in food products such as tofu, tempeh, soya sauce, simulated milk and meat products, and is a minor ingredient in many processed foods. Soybean meal is used as a supplement in feed rations for livestock.

Weeds are a major production problem in soybean cultivation. Typically, weeds are managed using a combination of cultural (e.g. seed bed preparation, using clean seed, variety selection, and planting date) and chemical controls. Depending on the production area and the prevalent weed species, herbicides may be applied before planting (e.g. pendimethalin, trifluralin, metribuzin), after planting but before emergence (e.g. pendimethalin, linuron, imazethapyr), and/or after emergence (e.g. bentazon, acifluorfen, fomesafen). Commonly, several different herbicides are required to adequately control weeds in soybean fields.

The soybean line GTS 40-3-2 was developed to allow for the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option. This genetically engineered soybean line contains a form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that allows GTS 40-3-2 to survive the otherwise lethal application of glyphosate. The EPSPS gene put into GTS 40-3-2 was isolated from a strain of the common soil bacterium Agrobacterium tumefaciens called CP4 and the form of EPSPS enzyme produced by this gene is tolerant to glyphosate.

The EPSPS enzyme is part of an important biochemical pathway in plants called the shikimate pathway, that is involved in the production of aromatic amino acids and other aromatic compounds. When conventional plants are treated with glyphosate, the plants cannot produce the aromatic amino acids needed to grow and survive. EPSPS is present in all plants, bacteria, and fungi. It is not present in animals, which do not synthesize their own aromatic amino acids. Because the aromatic amino acid biosynthetic pathway is not present in mammals, birds or aquatic life forms, glyphosate has little if any toxicity for these organisms. The EPSPS enzyme is naturally present in foods derived from plant and microbial sources.

GTS 40-3-2 was developed by introducing the CP4 EPSPS gene into a commercial soybean variety using particle-acceleration (biolistic) transformation. The glyphosate tolerance trait expressed in GTS 40-3-2 has since been transferred into more than one thousand commercial soybean varieties by traditional breeding techniques.
GTS 40-3-2 has been tested in field trials in the United States, Central and South America, Europe, Central Europe and Canada since 1991. Data collected from over 150 field trials conducted over a three-year period prior to commercialization in the United States demonstrated that GTS 40-3-2 did not differ significantly from conventional soybeans in morphology, seed production (yield), agronomic characteristics (such as time to flowering and pod set, or vigor) and tendency to weediness. GTS 40-3-2 did not negatively affect beneficial or nontarget organisms, and was not expected to impact on threatened or endangered species.

Soybean does not have any weedy relatives with which it can crossbreed in the continental United States and Canada. Cultivated soybean can naturally cross with the wild annual species G. soja, however G. soja, which occurs naturally in China, Korea, Japan, Taiwan and the former USSR, is not naturalized in North America. Additionally, soybean plants are almost completely self-pollinated and reproductive characteristics such as pollen production and viability were unchanged by the genetic modification resulting in GTS 40-3-2. It was therefore concluded that the potential for transfer of the glyphosate tolerance trait from the transgenic line to soybean relatives through gene flow (outcrossing) was negligible in managed ecosystems, and that there was no potential for transfer to wild species in Canada and the continental United States.

The food and livestock feed safety of GTS 40-3-2 soybean was established based on: the evaluation of the similarity of the structure and function of CP4 EPSPS protein to this same enzyme naturally present in foods and livestock feeds; the fact that CP4 EPSPS protein constitutes a small amount of the protein in GTS-40-3-2 soybeans so there is little dietary exposure; the lack of toxicity or allergenicity of EPSPS proteins from plants, bacteria and fungi; and by direct laboratory studies of the CP4 EPSPS protein. The nutritional equivalence and wholesomeness of GTS 40-3-2 soybeans compared to conventional (non-GM) soybeans was demonstrated by the analysis of key nutrients, including proximates (e.g. protein, fat, fibre, ash, and carbohydrates), amino acid and fatty acid composition, as well as anti-nutrients. A number of these parameters were also measured in glyphosate-treated soybeans. The equivalence of GTS 40-3-2 soybeans to conventional soybeans was confirmed in numerous feeding studies with rats, fish, broiler chickens, quail, and cows.

Links to Further Information Expand

Brazillian National Technical Biosafety Commission (CTNBio) Canadian Food Inspection Agency, Plant Biotechnology Office European Commission: Community Register of GM Food and Feed Food Standards Australia New Zealand Japan Biosafety Clearing House Monsanto Company Office of Food Biotechnology, Health Canada Philippines Department of Agriculture, Bureau of Plant Industry Secretaria de Agricultura, Ganaderia, Pesca y Alimentos: Republica Argentina Swiss Federal Office of Public Health THE COMMISSION OF THE EUROPEAN COMMUNITIES U.S.Department of Agriculture, Animal and Plant Health Inspection Service US Food and Drug Administration USDA-APHIS Environmental Assessment

This record was last modified on Monday, January 13, 2014