GM Crop Database

Database Product Description

DP-356043 (DP-356Ø43-5)
Host Organism
Glycine max (Soybean)
Trait
Tolerance to glyphosate and ALS-inhibiting herbicides
Trait Introduction
Microparticle bombardment of plant cells or tissue
Proposed Use

Production for human consumption and livestock feed.

Product Developer
DuPont Pioneer

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2010
Canada 2009 2009 2009
China 2010 2010
Colombia 2010 2010
European Union 2012 2012
Japan 2009 2009 2009
Korea 2010 2009
Mexico 2008 2008
Philippines 2009 2009
Singapore 2011 2011
South Africa 2011 2011
Taiwan 2009 View
United States 2007 2007 2008

Introduction Expand

Soybean (Glycine max L. Merr.), 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. 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. The major producers of soybeans are the United States, Brazil, Argentina, China, India, Paraguay and Canada.

DP356043 has been transformed to express two novel genes, providing tolerance to two different classes of herbicide. The line contains two genes; gat4601 which provides tolerance to glyphosate by detoxifying the compound, while gm-hra encodes for a modified acetolactate synthase (ALS) enzyme which is not affected by the imidazolinone class of ALS inhibiting herbicides. The gat4601 gene is based on the sequences of three gat genes from the common soil bacterium Bacillus licheniformis. The GAT4601 protein is 84% homologous to each of the three native GAT proteins from which it was derived. In DP356043, the expression of the gat4601 gene is driven by the constitutive synthetic core promoter SCP1. The GM-HRA protein in DP356043 is a modified version of the soybean GM-ALS protein. GM-ALS is involved in branched chain amino acid (leucine, isoleucine and valine) biosynthesis in the plastid. The herbicide tolerant gm-hra gene was made by isolating the herbicide sensitive soybean gm-als gene and introducing two specific amino acid changes, known to confer Imidazolinone tolerance. The GM-HRA protein is >99% homologous to the native GM-ALS protein from which it was derived. In DP356043, the expression of gm-hra gene is driven by the soybean constitutive S-adenosyl-L-methionine synthetase (SAMS) promoter.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
gat Glyphosate N-acetyltransferase HT

SCP1 - constitutive synthetic core promoter

TMV omega 5'-UTR

Solanum tuberosum proteinase inhibitor II (PINII)

1
gm-hra Acetolactate synthase HT

S-adenosyl-L-methionine synthetase (SAMS)

native soybean acetolactate synthase terminator

1

Characteristics of Glycine max (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.

Raw soybeans contain trypsin inhibitors, which are toxin when eaten.

Soy allergies are common, and eating soy products can cause rashes and swelling of the skin in sensitive individuals.

Modification Method Expand

DP356043 was produced by microprojectile-mediated bombardment of embryogenic somatic cell cultures, derived from explants from small, immature soybean seeds of the cultivar, Jack. The DNA was derived from the plasmid PHP20163, which contained two expression cassettes and an antibiotic (hygromycin) resistance marker gene. From this plasmid, a linear fragment, (PHP20163A transformation fragment), was excised, and adsorbed to gold microparticles. The PHP20163A transformation fragment contained two expression cassettes, the gat4601 and the gm-hra gene cassettes, but does not contain the antibiotic resistance marker gene.

The gat4601 gene cassette contains the coding sequence of the gat4601 gene, which encodes the GAT4601 protein. It is a synthetic gene derived from three gat genes from Bacillus licheniformis. In planta expression of the gat4601 gene is under the control of two regulatory elements, the SCP1 promoter and the TMV omega 5'-UTR. The SCP1 promoter is a synthetic constitutive promoter containing a portion of the CaMV 35S promoter and the Rsyn7-Syn II Core synthetic consensus promoter. The TMV omega 5'-UTR is the omega 5' untranslated leader of the Tobacco Mosaic Virus. The SCP1 promoter drives the transcription of the gat4601 gene. The TMV omega 5'-UTR, which is located downstream from the SCP1 promoter, enhances translation. Termination of transcription of the gat4601 gene is under the control of the pinII terminator, the 3' terminator sequence from the proteinase inhibitor II gene of Solanum tuberosum.

The gm-hra gene cassette contains the coding sequence of the gm-hra gene, which encodes the GM-HRA protein. It is a modified version of the endogenous soybean acetolactate synthase gene (gm-als), which encodes the GM-ALS I protein. Compared to the GM-ALS I protein, the GMHRA protein sequence contains two amino acid substitutions important for tolerance to the ALS inhibiting class of herbicides, and five additional N-terminal amino acids derived from the translation of 15 nucleotides of the gm-als 5' untranslated region. In planta expression of the gm-hra gene is controlled by the promoter derived from the S-adenosyl-L-methionine synthetase (SAMS) gene from soybean, and an intron that interrupts the SAMS 5' untranslated region (5'-UTR). Termination of transcription of the gm-hra gene is under the control of the native soybean acetolactate synthase terminator (gm-als terminator).

Characteristics of the Modification Expand

The Introduced DNA

DNA blot (Southern) analyses were used to characterize the introduced DNA in DP356043 soybean confirming that DP356043 contains one insert with a single, intact copy of the transformation fragment PHP20163A containing the gat4601 and gm-hra gene cassettes. Southern blot analysis using a plasmid backbone-specific probe confirmed that plasmid backbone sequences, such as those coding for the hygromycin antibiotic resistance gene, were not present in DP356043.

Genetic Stability of the Introduced Trait

The inheritance of the new traits present in DP356043 was studied in five generations (T1, F2, F3, BC1F2, and C2F2). From the Chi-square analysis of the trait inheritance data, it was concluded that the inheritance of the integrated insert with the gat4601 and the gm-hra genes is consistent with the Mendelian inheritance pattern of a single locus. The stability of the insert in three generations was assessed using Southern analyses of genomic DNA digested with the restriction enzyme BglII. There is a single BglII site present in the PHP20163A fragment, and this generates a unique, event-specific, DNA hybridization pattern. No changes were detected in the hybridization pattern among the tested generations of the DP356043 soybean (the selfed T4 and T5 generations and the traditionally-bred F3 generation). Given the absence of pattern changes, it was concluded that the insertion of the PHP20163A DNA fragment in 356043 soybean was stable.

Expressed Material

The levels of the GAT4601 and GM-HRA proteins were assessed in the grain, forage, and root tissue of samples collected from plants grown at six field locations in North America. Three replicated samples per tissue per location were collected for DP356043 and one sample per tissue per location was collected for control soybean. Mean protein levels across six locations for the GAT4601 protein in forage, root, and grain were 1.6, 1.6, and 0.24 nanograms per milligram (ng/mg) of tissue (dry weight), respectively. The mean protein levels for the GM-HRA protein in forage, root, and grain were 27, 3.2, and 0.91 ng/mg of tissue (dry weight), respectively. Neither the GAT4601 nor the GM-HRA proteins were detected in non-transgenic Jack cultivar (control soybean) tissues sampled from the six locations. From this data, it was concluded that expression of the GAT4601 and GM-HRA proteins in 356043 soybean is constitutive. Levels of GAT4601 and GM-HRA proteins in toasted soybean meal and hulls were found to be below the limit of quantitation (LOQ).

Environmental Safety Considerations Expand

Field Testing

Soybean event 356043 has been field-tested in the United States, Canada, and Japan. During the field tests, several phenotypic characteristics were evaluated, including early population, seedling vigor, days to maturity, plant height, lodging, pod shattering, final population, and yield. The statistical analysis of these observations showed no biologically meaningful differences between soybean 356043 and the control plant and supports a conclusion of phenotypic equivalence to currently commercialized soybean lines. The seed dormancy and germination of soybean event 356043 were compared with the control, and no significant differences were detected in percent germinated seed, percent dead seed, and percent viable firm, swollen seed, and viable hard seed. Viable hard seed was not observed in seed germination and seed viability tests of soybean event 356043. Data on the susceptibility to a range of insect pests and diseases, and response to abiotic stressors were also recorded. No increase or decrease outside of the reference range was observed in any insect or abiotic stressor in soybean event 356043.

Outcrossing

Pollen-mediated gene flow and introgression from transformed soybean line 356043 is 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 356043 soybean.

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 herbicide tolerance traits 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

Soybean (G. max) is not considered a weed and it does not persist in unmanaged ecosystems. In the United States, soybean is not listed as a weed in the major weed references and has been grown throughout the world without any report that it is a serious weed. Agronomic and phenotypic data collected from 16 field trial locations in North America over the 2004 and 2005 growing seasons indicated that 356043 was not different in any fitness characteristics from its parent that might cause 356043 to become invasive. There were no data indicating that the presence of the gat4601 or gm-hra genes improved the ability of this soybean line to survive without human intervention, nor is there any foreseeable reason to conclude that these genes would affect this line’s survival in the wild. The combination of phenotypic data and ecological observations supported a conclusion of agronomic and phenotypic comparability of 356043 soybean to conventional soybean varieties with respect to the lack of increased weed or plant pest potential.

Secondary and Non-Target Adverse Effects

Considering that GAT4601 and GM-HRA proteins lack the properties of known toxins or allergens, there should be no significant impacts to the environment or to non-target organisms, including threatened and endangered species, from the introduction of 356043 soybean.

Impact on Biodiversity

Analysis of available information demonstrates that 356043 does not exhibit any traits that should cause increased weediness, and that its unconfined cultivation should not lead to increased weediness of other sexually compatible relatives, of which there are none in North America. Soybean event 356043 has no effect on non-target organisms common to the agricultural ecosystem. Glyphosate use and crop production practices are not expected to change significantly, therefore, there should be no indirect or cumulative effect on biodiversity related to these practices. Use of ALS-inhibitor herbicides will likely increase in order to manage glyphosate-tolerant weed populations, but the use of glyphosate and ALS-inhibitors on 356043 soybean according to product labels is not expected to cause significant impacts on biodiversity outside the agroecosystem, based on the chemical and toxicological properties of these products.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Although commercialization of soybean 356043 could potentially increase dietary exposure to the acetylated amino acids NAGlu and NAAsp, acetylated amino acids are readily metabolized in humans and raise no safety issues. In addition, neither NAGlu nor NAAsp are detectable in soybean oil, which accounts for 94% of soybean consumption.

Nutritional Data

Compositional analyses were done to establish the nutritional adequacy of soybean event 356043, and to compare it to a non-transgenic conventional soybean under typical cultivation conditions. The components analyzed were protein, fat, carbohydrate, amino acids, fatty acids, vitamins, minerals, isoflavones, and the anti-nutrients stachyose, raffinose, lectins, phytic acid, and trypsin inhibitor.

The compositional analyses of key components in soybean 356043 indicate that, for the majority of components, there are no compositional differences of biological significance in forage or grain from transgenic soybean 356043, compared to the non-GM control. Several minor differences in key nutrients and other constituents were noted, but the mean levels observed were within the range of values observed for the non transgenic comparator and within the range of natural variation.

Increased levels of two fatty acids, heptadecanoic acid (C17:0) and heptadecenoic acid (C17:1) were observed. C17:0 and C17:1 in soybean 356043 together constitute approximately 0.5% of the total fatty acid content, compared to 0.2% in the conventional counterpart. C17:0 and C17:1 are present in vegetable oils and other commonly consumed foods. As these fatty acids are typical constituents of the human diet and readily metabolized, the increased levels raise no safety or nutritional concerns.

The introduction of dual herbicide-tolerant soybean 356043 into the food supply would be expected to have negligible nutritional impact. This was supported by the results of a feeding study in animals, where no differences in health and growth performance were found between broiler chickens fed diets containing soybean 356043 meal and those fed conventional soybean meal diets.

No potential public health and safety concerns have been identified in the assessment of dual herbicide-tolerant soybean 356043. On the basis of the data provided, and other available information, food derived from soybean 356043 is considered as safe for human consumption as food derived from conventional soybean varieties.

To date, all approved GM plants with modified agronomic production traits (e.g., herbicide tolerance) have been shown to be compositionally equivalent to their conventional counterparts. In the case of soybean 356043, there are unintended changes in the levels of two fatty acids (C17.0 and C17.1) and two-acetylated amino acids (NAGlu and NAAsp), that are outside the range of natural variation for soybean. All of these components are typical constituents of the human diet, and are readily metabolizable, and thus raise no safety issues resulting from their presence in food derived from soybean 356043.

Potential Toxicity

Bioinformatics studies with the GAT4601 and GM-HRA proteins confirmed the absence of any biologically significant amino acid sequence similarity to known protein toxins or allergens, and digestibility studies demonstrated that both proteins would be rapidly degraded following ingestion, similarly to other dietary proteins. Acute oral toxicity studies in mice with both proteins also confirmed the absence of toxicity. Taken together, the evidence indicated that neither protein is toxic or likely to be allergenic in humans.

The metabolite residues generated by glyphosate-treated soybean 356043 plants are considered less toxic than glyphosate, which itself is considered of very low potential toxicity in animals. Hence, there is no increase in overall toxicity arising from the presence of glyphosate residues on soybean 356043, and the current acceptable daily intake (ADI) for glyphosate is considered to be protective of public health and safety.

Potential Allergenicity

Soybean naturally contains allergenic proteins and is one of a group of known allergens, including milk, eggs, fish, shellfish, wheat, peanuts, tree nuts, and sesame. This group of foods accounts for approximately 90% of all food allergies. The presence of allergenic proteins in the diets of hypersensitive individuals can cause severe adverse reactions. The allergenic effect of soybeans is attributed to the globulin fraction of soybean proteins that comprises about 85% of total protein. Soybean-allergic individuals will also be allergic to soybean 356043.

In order to assess whether soybean 356043 has altered endogenous allergenic potential, a study was conducted to determine binding levels of IgE antibody to protein extracts prepared from soybean 356043 and the parental soybean line “Jack.” The data indicate that soybean 356043 and control Jack had similar protein/allergen profiles when tested with soy-allergic material. Thus, soybean 356043 appears to be equivalent to the non-transgenic counterpart in terms of its endogenous allergenicity.

Abstract Collapse

Soybean (Glycine max L. Merr.), 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. 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. The major producers of soybeans are the United States, Brazil, Argentina, China, India, Paraguay and Canada.

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.

DP356043 has been transformed to express two novel genes, providing tolerance to two different classes of herbicide. The line contains two genes; gat4601 which provides tolerance to glyphosate by detoxifying the compound, while gm-hra encodes for a modified acetolactate synthase (ALS) enzyme which is not affected by the imidazolinone class of ALS inhibiting herbicides. The gat4601 gene is based on the sequences of three gat genes from the common soil bacterium Bacillus licheniformis. B. licheniformis is widespread in the environment; therefore, animals and humans are regularly exposed without adverse consequences to this organism and its components, such as the glyphosate N-acetyltransferase (GAT) protein. The GAT4601 protein is 84% homologous to each of the three native GAT proteins from which it was derived. In DP356043, the expression of the gat4601 gene is driven by the constitutive synthetic core promoter SCP1.

The GM-HRA protein in DP356043 is a modified version of the soybean GM-ALS protein. GM-ALS is involved in branched chain amino acid (leucine, isoleucine and valine) biosynthesis in the plastid. The herbicide tolerant gm-hra gene was made by isolating the herbicide sensitive soybean gm-als gene and introducing two specific amino acid changes, known to confer herbicide tolerance to tobacco ALS. The GM-HRA protein is >99% homologous to the native GM-ALS protein from which it was derived. In DP356043, the expression of the gm-hra gene is driven by the soybean constitutive S-adenosyl-L-methionine synthetase (SAMS) promoter.

There was no evidence of acute toxicity in mice for either GAT4601 or GM-HRA at doses of 1680 or 582 mg protein per kg of body weight, respectively. Based on the GAT4601 and GM-HRA protein levels in DP356043, exposure levels would be well below the tested doses. These data support the food and feed safety of the GAT4601 and GM-HRA proteins. The allergenic potential of the GAT4601 and GM-HRA proteins was assessed using a step-wise, weight-of-the evidence approach. Bioinformatic analyses revealed no identities to known or putative protein allergens or toxins for either the GAT4601 or GM-HRA amino acid sequences. Both the GAT4601 and GM-HRA proteins were non-glycosylated and heat labile. Both proteins hydrolyzed rapidly (within 30 seconds) in simulated gastric fluid. In simulated intestinal fluid the GAT4601 protein hydrolyzed within 2 minutes and the GMHRA protein hydrolyzed within 30 seconds.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office Columbian Ministry of Health and Social Protection European Food Safety Authority (EFSA) Food Standards Australia New Zealand Health Canada Japanese Biosafety Clearing House, Ministry of Environment Korean Products Safety Agency of Foods Mexican Health Secretary Federal Commission for the Protection against Sanitary Risk U.S. Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Monday, November 2, 2015