GM Crop Database

Database Product Description

MON832
Host Organism
Zea mays (Maize)
Trait
Glyphosate herbicide tolerance.
Trait Introduction
Microparticle bombardment of plant cells or tissue
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1997 View
United States 1996

Introduction Expand

Maize line MON832 was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option for maize crops. Two novel genes were introduced into maize line MON832, which in combination provide field level tolerance to glyphosate. The novel plants express a glyphosate tolerant version of the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) encoding gene isolated from Agrobacterium tumefaciens strain CP4 (CP4 EPSPS). Glyphosate specifically binds to and inactivates EPSPS, which is involved in the biosythesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan. The EPSPS enzyme is present in all plants, bacteria and fungi, but not in animals, which do not synthesize their own aromatic amino acids. The modified enzyme (CP4 EPSPS) has a reduced binding affinity for glyphosate and allows the plant to function normally in the presence of the herbicide.

The second gene codes for glyphosate oxidase (gox gene), a bacterial enzyme from Ochrobactrum anthropi and is ubiquitous in nature. Glyphosate oxidase (GOX) accelerates the normal degradation of glyphosate into aminomethylphosphonic acid (AMPA) and glyoxylate. AMPA is the principal metabolite of glyphosate and is degraded by several microorganisms while glyoxylate is commonly found in plant cells and is broken down by the glyoxylic pathway for lipid metabolism.

These two enzymes, CP4 EPSPS and glyphosate oxidase, allow MON832 maize to be protected against herbicide damage when glyphosate is used for weed control in the cultivation of maize.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
goxv247 glyphosate oxidoreductase HT CaMV 35S chloroplast transit peptide from A. thaliana SSU1A gene (CTP1) 3
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase HT enhanced CaMV 35S, maize HSP70 intron chloroplast transit peptide from A. thaliana EPSPS gene (CTP2) A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1
nptII neomycin phosphotransferase II SM bacterial promoter 1 complete, 1 rearranged Not expressed in plant tissues because of bacterial promoter.

Characteristics of Zea mays (Maize) Expand

Center of Origin Reproduction Toxins Allergenicity

Mesoamerican region, now Mexico and Central America

Cross-pollination via wind-borne pollen is limited, pollen viability is about 30 minutes. Hybridization reported with teosinte species and rarely with members of the genus Tripsacum.

No endogenous toxins or significant levels of antinutritional factors.

Although some reported cases of maize allergy, protein(s) responsible have not been identified.

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Agrobacterium tumefaciens strain CP4 CP4 epsps

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

Modification Method Expand

Maize line MON832 was produced by biolistic transformation of embryogenic maize cells with a mixture of DNA from two plasmids, PV-ZMBK07 and PV-ZMGT10. Plasmid PV-ZMBK07 contained the synthetic cr1Ab gene regulated by the enhanced duplicated cauliflower mosaic virus 35S promoter (CaMV E35S) and the maize hsp70 heat shock protein intron. The polyadenylation signal was from the Agrobacterium tumefaciens nopaline synthase (nos) gene.

The second plasmid, PV-AMGT10, contained genes encoding the CP4 EPSPS enzyme from the common soil bacterium, A. tumefaciens sp. CP4, and glyphosate oxidoreductase (goxv247) from Ochrobactrum anthropi. Constitutive expression of these genes in plant cells was under the control of the CaMV E35S promoter, the hsp70 intron, and nos 3' terminator. Post-translational targeting of the CP4 EPSPS and glyphosate oxidoreductase enzymes to the chloroplast was accomplished by fusion of the 5'-terminal coding sequences with the chloroplast transit peptide DNA sequences from the Arabidopsis thaliana EPSPS gene (CTP2) and from A. thaliana SSU1A gene (CTP1), respectively. Plasmid PV-ZMGT10 also contained sequences from the lacZ operon, ori-pUC and the neo gene. The neo gene on both plasmids was included as a selectable marker to identify bacteria transformed with recombinant plasmid DNAs. Expression of the neo gene was regulated by a bacterial promoter and therefore is not functional in plants.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from MON832 confirmed that sequences from plasmid PV-ZMBK07, containing the cry1Ab gene, were not incorporated but that the CP4 EPSPS encoding sequences along with three copies of the gox gene and two NPTII/ori-pUC sequences (one complete and one rearranged) were integrated as a single 16 kb insert.

Genetic Stability of the Introduced Trait

Segregation and stability data were consistent with a single site of insertion into the genomic DNA of line MON832.

Expressed Material

The CP4 EPSPS and GOX enzymes were constitutively expressed in tissues throughout the plant and at all plant growth stages. Enzyme linked immunosorben assay (ELISA) was used to quantitate the expression levels of CP4 EPSPS and GOX in the leaves, forage, and kernels of plants grown in field trials. Average amounts were as follows: in leaves 49.61 EPSPS µg/g fresh weight (fwt) and 4.92 GOX µg/g fwt; in forage 21.32 EPSPS µg/g fwt and 3.32 GOX µg/g fwt; and in kernels 5.83 EPSPS µg/g fwt and 1.97 GOX µg/g fwt.

Western immunoblot analysis was used to confirm that NPT II was not detectable in MON832, which was predicted since expression of the neo gene was regulated by a bacterial promoter known not to be active in plants.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Grain from maize line MON832 was intended mainly for use in animal feed. The major human food uses for maize are extensively processed starch and oil fractions prepared by wet or dry milling procedures and products include corn syrup and corn oil, neither containing protein. Human exposure to the modified protein from whole grain corn in the diet was considered to be very low due both to its low abundance in the protein fraction of the grain and to the proportionately low percentage of protein in the kernel, compared with the major starch component. Overall, the dietary exposure of consumers in the United States and Canada to grain from MON832 maize was anticipated to be the same as for other lines of commercially available field corn.

Nutritional Data

The major components of maize grain and forage from MON832 were analysed on plant material harvested from field trials. Compositional data for protein, fat, ash, carbohydrates, calories, moisture, amino acids, and fatty acids for line MON832 grain were comparable to the data from the control line and within published ranges for commercial hybrids. Similar promximate analysis, including acid detergent fibre and neutral detergent fibre analyses, were performed on forage for line MON832 and an appropriate control. Based on these compositional data, it was concluded that there were no significant differences between the forage from line MON832 and the control line.

Toxicity

The low potential for toxicity of transgenic maize line MON832 was demonstrated by examining the amino aid sequence homology, protein characterization, digestive fate studies in simulated gastric and intestinal fluids and acute oral toxicity in mice. Using sophisticated computerized search capabilities, the evidence showed that the CP4 EPSPS and GOX proteins did not show meaningful amino acid sequence homology when compared to known allergens or protein toxins. These studies support the safety of the proteins and are consistent with the history of safe presence and ubiquitous distribution of Agrobacterium and Achromobacter spp. in the soil. An acute mouse toxicity study further indicated that there were no toxic effects caused by CP4 EPSPS or GOX proteins administered by oral gavage, as measured by the absence of treatment related adverse effects in mice.

Allergenicity

The CP4 and GOX proteins are not likely to be toxic or allergenic. The amino acid sequences of both proteins were compared with known toxins and allergens did not show meaningful amino acid sequence homology. 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, studies have shown that the CP4 and GOX proteins are rapidly degraded and their enzymatic activity lost under conditions that simulate mammalian digestion.

Abstract Collapse

Maize (Zea mays L.), or corn, is grown primarily for its kernel, which is largely refined into products used in a wide range of food, medical, and industrial goods.

Only a small amount of whole maize kernel is consumed by humans. Maize oil is extracted from the germ of the maize kernel and maize is also a raw material in the manufacture of starch. A complex refining process converts the majority of this starch into sweeteners, syrups and fermentation products, including ethanol. Refined maize products, sweeteners, starch, and oil are abundant in processed foods such as breakfast cereals, dairy goods, and chewing gum.
In the United States and Canada maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, although an increasing amount is being used for ethanol production. The entire maize plant, the kernels, and several refined products such as glutens and steep liquor, are used in animal feeds. Silage made from the whole maize plant makes up 10-12% of the annual corn acreage, and is a major ruminant feedstuff. Livestock that feed on maize include cattle, pigs, poultry, sheep, goats, fish and companion animals.

Industrial uses for maize products include recycled paper, paints, cosmetics, pharmaceuticals and car parts.
The MON832 line of maize was developed to allow the use of glyphosate, the active ingredient in the herbicide Roundup®, as a weed control option. In order to obtain field tolerance to glyphosate herbicide, two novel genes, EPSPS and goxv247, were introduced maize by particle acceleration (biolistic) transformation.

The EPSPS gene codes for the enzyme 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) that is present in all plants, bacteria and fungi. The EPSPS gene put into MON832 was isolated from strain CP4 of the common soil bacterium Agrobacterium tumefaciens and is a glyphosate tolerant form of EPSPS. 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 maize plants are treated with glyphosate, they cannot produce the aromatic amino acids needed to grow and survive. EPSPS is not present in mammals, birds or aquatic life forms, which do not synthesize their own aromatic amino acids. For this reason, glyphosate has little toxicity to these organisms. The EPSPS enzyme is naturally present in foods derived from plant and microbial sources.

MON832 contains a second gene that codes for a modified version of glyphosate oxidase (GOX) enzyme that is ubiquitous in nature. The goxv247 gene inserted into MON832 was isolated from strain LBAA of the bacterium Ochrobactrum anthropi. The GOX enzyme accelerates the normal breakdown of the herbicide glyphosate into two non-toxic compounds, aminomethylphosphonic acid (AMPA) and glyoxylate. AMPA is the principal breakdown product of glyphosate and is degraded by several microorganisms, while glyoxylate is commonly found in plant cells and is broken down by the glyoxylic pathway for lipid metabolism.

The major components of grain and forage from MON832 maize were analyzed in order to determine their safety for use as food and as livestock feed. Proximate analysis determined that MON832 grain was comparable to that of the non-transgenic control line in its protein, fat, ash, carbohydrate, fibre, moisture and caloric content. Additionally, the amino acid and fatty acid composition of MON832 grain was comparable to that of a non-transgenic line. Based on this data, it was concluded that there was no significant difference between the forage from MON832 maize and the control line.

The low potential for toxicity of the transgenic maize line MON832 was demonstrated by examining the amino acid sequence homology and protein characterization of the line. The CP4 EPSPS and GOX proteins did not show meaningful amino acid sequence homology with known allergens or protein toxins. Further assessment of the allergenic potential of these proteins consisted of digestibility studies and an acute oral toxicity study involving mice. Results of the digestibility studies indicated that, unlike known protein allergens, the CP4 EPSPS and GOX proteins are rapidly degraded and their enzymatic activity lost under conditions that simulate mammalian digestion. The mouse toxicity study indicated that there were no toxic effects caused by consumption of the CP4 EPSPS or GOX proteins, as measured by the absence of treatment-related adverse effects in the mice. It was therefore concluded that the CP4 EPSPS and GOX proteins are not likely to pose any toxicity or allergenicity concerns.

Links to Further Information Expand

Office of Food Biotechnology, Health Canada US FDA

This record was last modified on Monday, March 28, 2016