GM Crop Database

The spring wheat variety MON 71800 (Roundup Ready® wheat) was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option in spring wheat. This genetically engineered wheat variety contains a glyphosate-tolerant form of the plant enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), isolated from the soil bacterium Agrobacterium tumefaciens strain CP4. The novel form of this enzyme is termed hereafter 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 and Amrhein, 1980). When conventional plants are treated with glyphosate, the herbicide binds to EPSPS, thereby preventing the synthesis of aromatic amino acids needed for plant growth. The CP4 EPSPS enzyme in MON 71800 spring wheat has a reduced affinity for glyphosate; its enzymatic activity is therefore not hindered by the herbicide.

EPSPS 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.

MON 71800 was developed by introducing the CP4 EPSPS coding sequences into the spring wheat variety ‘Bobwhite’ using Agrobacterium-mediated transformation.

The wheat line MON 71800 was produced by Agrobacterium-mediated transformation of plant cells from ‘Bobwhite’ spring wheat. The plasmid vector PV-TXGT10 used for the transformation contained two cp4 epsps gene cassettes coding for glyphosate tolerance. Each gene cassette consisted of chloroplast transit peptide coding sequences from the Arabidopsis thaliana epsps gene (ArabTP) associated with the sequences of the cp4 epsps gene. Two different promoters were used to regulate the expression of each cp4 epsps gene: 1) the enhanced 35S promoter from the cauliflower mosaic virus and 2), the promoter, transcription start site, and first intron of the 5’ region of the rice actin1 gene. Terminator sequences in each gene casette consisted of the 3’ non-translated region of the nopaline synthase gene (nos 3’). The PV-TXGT10 vector backbone contained the origin of replication sequences ori-V and ori-322/rop. The vector backbone also contained the aad gene, which codes for streptomycin adenyltransferase, to allow the selection of bacteria containing the PV-TXGT10 vector.

The Introduced DNA
Southern blot analysis and Polymerase Chain Reaction (PCR) amplification of the genomic DNA of MON 71800 wheat demonstrated one site of integration of a single copy of the T-DNA insert of PV-TXGT10. Southern blot analysis also confirmed the insertion of one intact copy of each cp4 epsps gene cassette, including the promoter, terminator and chlorophyll transit peptide sequences. None of the vector backbone sequences were integrated into the genome of MON 71800 wheat.

Genetic Stability of the Introduced Trait
The stability of the inserted DNA was evaluated, across several generations of wheat plants, using Southern blot analysis. The plants tested were progeny from several generations of self-fertilization, as well as from crosses with commercial varieties. The results of the genomic DNA blot analysis confirmed the stable inheritance of the inserted cp4 epsps gene cassettes. The stability of the introduced trait was also demonstrated after 18 generations of selfing of the original homozygous glyphosate-resistant plants. These generations showed no decrease in tolerance to glyphosate.

Mendelian segregation studies for the inheritance of the glyphosate tolerant trait were conducted with heterozygous first generation plants: these were selfed and the progeny were sprayed with glyphosate. The resulting 3:1 ratio of tolerant to sensitive plants was statistically significant and confirmed the inheritance of a single insertion site of the glyphosate tolerant trait.

Expressed material
In plant cells, the EPSPS enzyme is transported to the chloroplast by a transit peptide, which then cleaves from the enzyme. The introduced gene sequences in MON 71800 included a gene for a chloroplast transit peptide from Arabidopsis thaliana. Thorough analyses were conducted to investigate whether the same mechanism of binding, transporting, and cleaving of the chloroplast transit peptide to the CP4 EPSPS enzyme exists in MON 71800. The analyses revealed two forms of CP4 EPSPS expressed in MON 71800: one in which the chloroplast transit peptide has completely cleaved from the full length enzyme, and the other in which only part of the transit peptide is bound to the enzyme. Further analysis of Western blots showed that of the total amount of CP4 EPSPS expressed in MON 71800, 80% is in the form where the transit peptide is fully cleaved and 20%, where part of the transit peptide is still bound to the enzyme.

An enzyme-linked immunosorbent assay (ELISA) analysis was used to quantify the levels of the CP4 EPSPS proteins in forage and grain from MON 71800. The mean levels of both CP4 EPSPS proteins, on a fresh weight basis, were 106 μg/g in forage and 13 μg/g in grain. Assuming 14.5% moisture in stored grain, the concentration of both CP4 EPSPS proteins in the grain is approximately 0.0015%, on a dry matter basis.

Dietary exposure
The genetic modification of MON 71800 spring wheat will not result in any change in the consumption pattern of wheat and wheat-based products. MON 71800 is expected to be used in similar applications as other spring wheat cultivars by the food industry. MON 71800 did not express any novel compositional characteristics, as confirmed by the similarity in composition of the modified line to its parental counterpart, and other conventional spring wheat varieties. Furthermore, the availability of many spring wheat cultivars for cultivation, and the normal variation in wheat composition due to differences in grade and growing conditions, result in a wide variation in the composition of conventional wheat grain. Consequently, the dietary exposure of consumers in the United States to MON 71800 is anticipated to be the same as for other varieties of commercially available spring wheat.

Nutritional and Compositional Data
The nutritional components of MON 71800 grain and forage were determined analytically and compared to those of the parental line ‘Bobwhite’ and several commercial varieties grown at five locations in the United States and Canada. For the grain, these components included proximates (crude protein, crude fat, ash, moisture, total carbohydrates), total dietary fibre, sugars, starch, amino acids, fatty acids, B vitamins, vitamin E and minerals. Forage samples were analyzed for proximates, acid detergent fibre, neutral detergent fibre, calcium and phosphorus. At some of the locations, for both the grain and the forage, there were statistically significant differences in the levels of certain components between MON 71800 and its parental line. These differences within locations could be attributed to environmental effects rather than to any unintended effect of the genetic modification in MON 71800. However, the combined data from all test locations demonstrated that the nutritional composition of MON 71800 grain and forage was comparable to that of the parental line ‘Bobwhite,’ and other commercial spring wheat varieties.

Phytic acid occurs naturally in wheat and other cereals. It is indigestible by humans and non-ruminant livestock, and inhibits the absorption of iron and other minerals. Grain samples of MON 71800, the parental line ‘Bobwhite,’ other commercial wheat varieties were analyzed to determine levels of phytic acid. The concentration of phytic acid in MON 71800 was comparable to that in the parental line, and was within the range of values determined for the commercial varieties and those found in the literature.

Toxicity and Allergenicity
The potential for toxicity and allergenicity of MON 71800 wheat was investigated using the following data and information: results from the determination of amino acid sequence similarity between the CP4 EPSPS proteins and known toxins and allergens; analysis for possible glycosylation of the CP4 EPSPS proteins; analysis of the stability the novel proteins in simulated gastric fluids; results from an acute oral toxicity study in mice using the CP4 EPSPS proteins; and, information on the safety of the cp4 epsps gene donor, A. tumefaciens strain CP4. The potential for increased allergenicity of the grain from MON 71800 was investigated, specifically with regard to endogenous wheat allergens that induce an IgE reaction in susceptible humans. Various immunilogical assays, using sera from humans with an IgE-mediated wheat allergy, were performed with extracts from MON 71800, the parental line, and several other commercial wheat varieties. The possibility that the genetic modification would have also altered the levels of gliadin, proteins that cause celiac enteropathy in susceptible persons, was also investigated. Gliadin levels were measured, and gluten levels calculated in MON 71800, its parental line, and other commercial varieties.

The CP4 EPSPS proteins in MON 71800 showed no amino acid sequence similarity with known toxins and allergens and neither of the two forms of the protein are glycosylated. Both forms of the CP4 EPSPS protein were rapidly digested under simulated gastric fluid conditions, and the enzyme activity of each protein was substantially diminished within the same period as for the digestion. The results of the acute oral toxicity study on mice, at the highest administered dose, showed no adverse effects of the CP4 EPSPS proteins. Gluten levels were not significantly altered by the genetic modification of MON 71800, neither were the endogenous allergens, as demonstrated by the results of the immunological studies.

Both forms of the CP4 EPSPS protein were expressed at very low levels in MON 71800 grain and forage. This fact, along with negative results of the various safety studies, sequence homology investigations, and immunological assays led to the conclusion that MON 71800 spring wheat did not demonstrate any potential for toxicity and novel allergenicity, nor any altered endogenous allergenicity, compared to conventional spring wheat varieties.