GM Crop Database

Database Product Description

DP-305423 (DP-3Ø5423-1)
Host Organism
Glycine max (Soybean)
Trait
Modified seed fatty acid content, specifically high oleic acid, low linolenic acid content.
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 2011 2011
European Union 2015 2015
Japan 2010 2010 2010
Korea 2010 2010
Mexico 2008 2008
Philippines 2013 2013
Singapore 2012 2012
South Africa 2011 2011
Taiwan 2010
United States 2009 2009 2010

Introduction Expand

The intended effect of the modification in 305423 soybean is to produce soybean seeds with increased levels of monounsaturated fatty acid (oleic) and decreased levels of polyunsaturated fatty acids (linoleic and linolenic). To accomplish this objective, Pioneer inserted a fragment of the soybean microsomal omega-6 desaturase gene (FAD2-1) into soybean. The fragment of the FAD2-1 gene does not code for a protein. Transcription of the gene fragment under the control of a seed-preferred KTi3 promoter acts to silence the expression of the endogenous soybean omega-6 desaturase, which results in an increased level of oleic acid and decreased levels of linoleic and linolenic acids in the soybean seed. A gene (gm-hra) encoding a modified version of the soybean acetolactate synthase (als gene) that confers tolerance to a sulfonylurea herbicide was used as a selectable marker for the transformation. The 305423 soybean will be commercialized for the production of TREUS™ brand high oleic soybean oil. The food service industry and food processors will benefit from the fact that high oleic soybean oil is a highly stable vegetable oil that is suitable for frying applications without the need for hydrogenation. In the industrial sector, high oleic soybean oil will offer an ingredient that is stable to oxidation for the formulation of cost effective, renewable, environmentally friendly industrial fluids.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
gm-fad2-1 delta(12)-fatty acid dehydrogenase FA Kti3 (Glycine max Kunitz trypsin inhibitor gene promoter) 3' untranslated region of the KTi3 gene 7 copies 597 bp fragment of FAD2-1 gene
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.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Glycine max gm-fad2-1

There is no published evidence that soybean is a human or animal pathogen.

Glycine max gm-fad2-1

There is no published evidence that soybean is a human or animal pathogen.

Modification Method Expand

Pioneer used microprojectile bombardment to co-transform secondary plant cell embryos with two purified linear DNA fragments: a 2924 base pair fragment (PHP19340A fragment) containing the gm-fad2-1 cassette, and the 4512 base pair fragment (PHP17752A fragment) containing the gm-hra cassette. The gm-fad2-1 cassette includes the promoter region from the soybean Kunitz trypsin inhibitor gene (KTi3), a fragment of the FAD2-1 gene that corresponds to approximately 40% of the middle portion of the FAD2-1 gene (597 bp), and the 3' untranslated region of the KTi3 gene (KTi3 terminator).

The gm-hra cassette contains the gm-hra gene (1971 bp), which is an optimized form of the endogenous als gene from soybean, with transcription regulated by the S-adenosyl-L-methionine synthetase (SAMS) constitutive promoter (1.3 kb) from soybean and with transcription terminated by the endogenous als gene terminator (0.6 kb) from soybean.

Characteristics of the Modification Expand

The Introduced DNA

Molecular characterization of 305423 soybean to evaluate insert copy number, insert integrity and presence of plasmid backbone sequences was conducted by Southern blot analysis in combination with sequencing of the inserted DNA in 305423 soybean.

Extensive Southern analyses demonstrate that multiple intact and truncated copies of fragment PHP19340A have been inserted into 305423 soybean that contain, in total, eight copies of the KTi3 promoter, seven copies of the gm-fad2-1 gene fragment and five copies of the KTi3 terminator. A single intact copy of fragment PHP17752A has been inserted into 305423 soybean. In addition, Southern blot analyses confirm the absence of all functional elements from the plasmid backbone in 305423 soybean, despite the presence of a small nonfunctional fragment of the plasmid backbone DNA.

Sequence characterization of the inserted DNA in 305423 soybean confirms that 305423 soybean contains four insertions that comprise:

  • Insertion 1: one truncated PHP19340A fragment with a truncated KTi3 terminator and intact gm-fad2-1 gene fragment and intact KTi3 promoter, one intact PHP19340A fragment, one intact PHP17752A fragment, one truncated PHP19340A fragment with an intact KTi3 promoter and a truncated gm-fad2-1 gene fragment, and one truncated PHP19340A fragment with a truncated KTi3 promoter and truncated gm-fad2-1 gene fragment.
  • Insertion 2: one truncated PHP19340A fragment with a truncated KTi3 promoter and with intact gm-fad2-1 gene fragment and intact KTi3 terminator.
  • Insertion 3: one truncated copy of the KTi3 promoter with a nonfunctional 495 bp fragment of the plasmid backbone; and
  • Insertion 4: two truncated PHP19340A fragments in an inverted repeat configuration, both with a truncated KTi3 promoter and intact gmfad2-1 gene fragment and KTi3 terminator.

The genetic material that is inserted in 305423 soybean is genetically linked and segregates following a typical pattern of Mendelian inheritance expected for a single, genetically-linked insertion locus.

Expressed Material

The 305423 soybean has been obtained by introducing the gm-fad2-1 gene fragment and the gm-hra gene into the soybean genome. The gm-fad2-1 gene fragment is part of the coding region of the soybean omega-6 desaturase gene 1 (FAD2-1) and does not code for a functional protein. Transcription of the gm-fad2-1 gene fragment in 305423 soybean seeds acts to suppress transcription of endogenous omega-6 desaturase, resulting in the high oleic phenotype.

The gm-hra gene encodes the GM-HRA protein, an optimized version of the soybean acetolactate synthase (ALS). Expression of the GM-HRA protein in 305423 soybean, used as a selectable marker, confers tolerance to ALS-inhibiting herbicides.

Northern analyses demonstrate that transcription of the endogenous FAD2-1 gene is effectively silenced in 305423 soybean seed as intended by the genetic modification.

The GM-HRA protein levels in 305423 soybean were measured in replicated samples of leaf, root, forage and grain tissues using a quantitative enzyme-linked immunosorbent assay (ELISA). Pioneer reports that the mean GM-HRA protein concentrations in 305423 soybean leaf, root, forage, and grain were 4.0, 0.18, 5.7, and 2.5 nanograms per milligram (ng/mg) tissue dry weight respectively. Pioneer concluded that the above results confirm that the expression of the GM-HRA protein in 305423 soybean is constitutive.

Food and/or Feed Safety Considerations Expand

Food and Feed Use

The primary use of commodity soybeans is for soybean meal that is consumed by animals. Raw soybeans contain several antinutritional factors (trypsin inhibitors, urease, and hemagglutinins). Heat treatment is the most common processing method used to minimize the activity of such factors. Soybean oil is the major soybean fraction consumed by humans. Soybean oil accounts for 80% of total United States consumption of oils and fats.

Pioneer states that the 305423 soybean variety is intended to be used for the production of high oleic soybean oil. The oil is intended to be a highly stable vegetable oil suitable for frying applications without the need for hydrogenation which produces trans fatty acids and "hydrogenated flavor." Pioneer states that it is aware of no food or feed uses of current soybean varieties for which the 305423 soybean variety would not be equally suitable.

Compositional Analysis

Pioneer assessed the composition of grain and forage from the 305423 soybean and a null segregant (non-transgenic isoline) control. Pioneer states that the null segregant plants are an appropriate control because they are almost genetically identical to the corresponding 305423 soybean plants with the exception that they do not carry the transgenic DNA. Both the transgenic and control soybeans were grown at six field locations in soybean-growing areas of North America using a randomized complete block design with three replicates at each location. Pioneer also analyzed grain and forage from four different commercial soybean varieties.

Pioneer measured 52 components in grain and 5 in forage. Composition data obtained from the commercial varieties were used to calculate 99% tolerance intervals with 95% confidence for all measured components. To interpret the composition results for 305423 soybeans, Pioneer used the confidence intervals and established a combined literature range using data from published literature and databases on soybean composition. Grain samples were analyzed for proximates (protein, fat, and ash), acid detergent fiber (ADF), neutral detergent fiber (NDF), fatty acids, amino acids, isoflavones, and antinutrients. Compositional analysis of forage samples included proximates, ADF, and NDF.

Grain Analysis: Analysis of the levels of 14 fatty acids confirmed the expected high oleic acid phenotype as shown by a substantial increase in the mean level of oleic acid to 76.5% in 305423 soybean as compared to a mean level of 21% in the control soybean. As expected, the increase in the level of oleic acid was accompanied by a decrease in the level of linoleic acid. The mean level of linoleic acid in the control soybean was 52.5% and that in the 305423 soybean was 3.6%. The level of linolenic acid also decreased since linolenic acid is formed in soybeans directly from linoleic acid. Levels of two minor fatty acids, heptadecanoic and heptadecenoic, increased in the 305423 soybean to 0.8% and 1.2% of the total fatty acids, respectively. This increase in the levels of heptadecanoic and heptadecenoic fatty acids is not unexpected because the GM-HRA enzyme most likely results in the increased concentration of 2-ketobutyrate, which is the substrate in the biosynthesis of hepta fatty acids in soybeans.

Mean levels of protein and fiber in grain from the 305423 and control soybean were not statistically significantly different. While the mean level of fat was statistically significantly lower in the 305423 soybean than in the control using the unadjusted P-value, no statistical difference was detected using the false discovery rate (FDR)-adjusted P-value. The mean level of ash was statistically significantly lower in the 305423 soybean as compared to the control soybean. Mean levels of the proximates and fiber in grain from 305423 and control soybean lines were within the 99% tolerance intervals for the reference varieties and within the combined literature range.

There were no statistically significant differences observed in the mean levels of amino acids between 305423 and control soybean grain with the exception of threonine and glutamic acid. The mean levels of these two amino acids were statistically significantly increased in 305423 soybean when the unadjusted P-values were used, but not when the FDR-adjusted P-values were used. All these levels were within the 99% tolerance intervals.

Mean levels of nine isoflavones were quantified in grain samples. The mean levels for daidzin, malonylgenistin, and malonyldaidzin were statistically significantly increased in the 305423 versus the control soybean. Mean genestin levels were only shown to be statistically significantly increased when the unadjusted P-value was used. Mean values for all the measured isoflavones were within the 99% tolerance intervals. In both the control and 305423 soybean the mean levels of genistin, glycitin, malonylgenistin, malonylglycitin, and malonyldaidzin were above the combined literature range. Daidzin levels were only above the combined literature range in the 305423 soybean. No literature data were available for the level of glycitein. The values for the other two isoflavones (genistein and daidzein) were within the combined literature range.

Concentrations of several antinutrients were measured in soybean grain including non-digestible oligosaccharides stachyose and raffinose, lectins, phytic acid, and trypsin inhibitor. No statistically significant differences were observed between the 305423 and control soybean in mean values for raffinose, lectins, and phytic acid. The mean value for trypsin inhibitor was statistically significantly lower for 305423 soybean as compared to the control soybean. The reduction in the mean value of trypsin inhibitor in 305423 soybean was expected as Pioneer reported that the promoter used to drive expression of the FAD2-1 gene, the Kunitz trypsin inhibitor promoter, has been shown to silence the KTi3 gene which encodes the Kunitz trypsin inhibitor. The mean value for stachyose was statistically significantly increased when the unadjusted P-value was used, but not when the FDR-adjusted P-value was used. Mean values for all the measured antinutrients were within the 99% tolerance intervals and within the combined literature ranges.

Forage Analysis: Pioneer analyzed forage for protein, fat, ash, ADF, and NDF. No statistically significant differences were observed between the mean levels of these components in forage from the 305423 and control soybeans with the exception of the level of fat. The mean level of fat was statistically significantly decreased in 305423 using the unadjusted P-value, but not when using the FDR-adjusted P-value. All mean levels were within the 99% tolerance intervals and combined literature ranges, with the exception of NDF. The mean level of NDF in both the control and 305423 soybean was above the combined literature range.

Endogenous Allergens: A study was conducted to assess whether the transformation process may have increased the overall allergenicity of 305423 soybean compared to conventional soybean. Using sera from clinically reactive soy allergic patients, Pioneer conducted IgE immunoblot and ELISA studies using protein extracts from 305423 soybean and conventional soybean. The SDS-PAGE Coomassie blue-stained protein profiles for 305423 and control soybean extracts appeared to be the same; they are similar in their IgE binding profile, and showed the same IgE binding capacity for 305423 and control soy extracts. It was concluded that the levels of endogenous allergens in and the allergic potential of 305423 soybean are comparable to those in nontransgenic control soybean.

Nutritional Quality

A poultry feeding study over a period of 42 days has been carried out with diets containing 305423 soybean, non-GM control soybean with comparable genetic background and three commercial non-GM soybeans. The mortality, body weight gain, feed efficiency, organ yield, carcass yield, breast, thigh, wing and leg yield and abdominal fat of the chickens fed with 305423 soybean were compared to chickens fed non-GM control soybean diets. The results from this 42-day poultry study confirm the safety of 305423 soybean and that 305423 soybean is nutritionally equivalent to non-GM control soybean with a comparable genetic background and to commercial soybeans.

Potential Toxicity and Allergenicity

The only newly expressed protein in 305423 soybean is the GM-HRA protein. Based on amino acid sequence similarity searches against the National Center for Biotechnology Information (NCBI) Protein dataset, there were no significant similarities with known protein toxins. Pioneer conducted an acute oral toxicity study in mice in which a single dose of 582 mg per kilogram of body weight (mg/kg bw) of GM-HRA protein was administered by oral gavage to five male and five female mice. No clinical symptoms of toxicity, body weight loss, gross organ lesions or mortality were observed. The result of this study shows that the GM-HRA protein does not cause acute toxicity.

While soybean is one of the major food allergens, none of the identified allergens is a member of the ALS family and the ALS protein from soybean has not been characterized as a soy allergen. Comparisons of the amino acid sequence of the GM-HRA protein to the amino acid sequences of known allergens in the Food Allergy Research and Resource Program (FARRP, version 6.0) database using the FASTA34 sequence alignment program showed that none of the identified alignments met or exceeded the threshold of greater than or equal to 35% identity over 80 amino acids and no contiguous stretches of 8 or greater amino acids were shared between the GM-HRA protein and the proteins in the allergen database. Additionally, the GM-HRA protein is not glycosylated.

The GM-HRA protein is rapidly (< 30 seconds) hydrolyzed in simulated gastric fluid (SGF) to fragments of < 3 kDa; and when subjected to simulated intestinal fluid (SIF), the GM-HRA protein, including the low weight molecular fragments seen in SGF, is completely and rapidly (< 2 minutes) hydrolyzed.

Based on this weight of evidence assessment, it was concluded that the GM-HRA protein is unlikely to be a toxin and is not a potential allergen.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office European Commission European Food Safety Authority Food Standards Australia New Zealand (FSANZ) Health Canada Japan (MAFF) Republic of Korea, Ministry of Trade, Industry and Energy Republic of the Philippines Department of Agriculture 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