GM Crop Database

Database Product Description
 
MON809
Host Organism
Zea mays L. L. (Maize)
 
Trait
Resistance to European corn borer (Ostrinia nubilalis); glyphosate herbicide tolerance.
 
Trait Introduction
Microparticle bombardment of plant cells or tissue
 
Proposed Use
Production of Z. mays for human consumption (wet mill or dry mill or seed oil), and meal and silage for livestock feed. These materials will not be grown outside the normal production area for corn.
 
Company Information
Pioneer Hi-Bred International Inc.
7100 NW 62nd Avenue
PO Box 1150
Johnston
IA  USA
 
 
Summary of Regulatory Approvals
 
Country Environment Food and/or Feed Food Feed Marketing
Canada 1996 1996 1996  
Japan 1997 1998  
United States 1996 1996  
Click on the country name for country-specific contact and regulatory information.
Introduction
 
Maize line MON809 was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. The novel variety produced the insecticidal protein, Cry1Ab, derived from Bacillus thuringiensis subsp. kurstaki (B.t.k.) HD-1 strain. Delta-endotoxins, such as the Cry1Ab protein expressed in MON809, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ab is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

General Description
 
1. Resistance to the European Corn Borer (ECB)
Bacillus thuringiensis var. kurstaki HD-1 (B.t.k.) is a common gram-positive soil-borne bacterium. In its spore forming stage, it produces several insecticidal protein crystals, including the -endotoxin Cry1A(b), which is active against certain lepidopteran insects, such as ECB. This protein has been shown to be non-toxic to humans, other vertebrates and beneficial insects. B.t.k. based foliar insecticides have been registered for over 30 years in Canada and have a long history of safe use.

A synthetic cryIA(b) gene was developed for maximum expression in corn, and introduced into line MON809. The gene codes for a protein similar to the Bacillus thuringiensis var. kurstaki HD-1 insecticidal crystal protein. The protein is insecticidal to lepidopteran larvae after cleavage to a bio-active, trypsin resistant core. Insecticidal activity is believed to depend on the binding of the active fragment to specific receptors present in susceptible insects on midgut epithelial cells, forming pores which disrupt osmotic balance and eventually results in cell lysis. Specific Lepidopteran pests of corn sensitive to the protein are ECB and corn earworm.

The cry1A(b) gene is linked to a strong constitutive promoter. Average protein expression evaluated at six locations was 1.63 g/g (f.w.) in leaves, 0.55 g/g (f.w.) in seeds and 1.23 g/g (f.w.)in the whole plant. Cry 1A(b) protein was not detected in pollen. Protein expression ranged from 0.88-2.37 g/g (f.w.) in leaves, from 0.28-0.73 g/g (f.w.) in grain and from 0.73-1.73 g/g (f.w.) in the whole plant. Protein expression declined over the growing season as indicated by the Cry1A(b) levels present in leaves assayed over the growing season.

The Cry1A(b) protein was shown to degrade readily in the environment. The plant expressed protein had DT50 and DT90 values (time to degrade to 50% and 90 % of the original bioactivity) of 2 and 15 days respectively.

Protein allergens are normally resistant to digestion unlike the Cry1A(b) protein which was shown to degrade readily in simulated gastric fluid. Unlike many known allergens the insecticidal protein is not glycosylated. A search for amino acid sequence similarity between the Cry1A(b) protein and known allergens, using a database assembled from the public domain databases GenBank, EMBL, Pir and SwissProt, revealed no significant amino acid sequence homologies. A search of a similarily constructed database of known toxins indicated no amino acid sequence homologies between known toxins and the Cry1A(b) protein, with the exception of homologies to other B.t. insecticidal proteins.

Corn products are an important alternative to wheat flour for individuals afflicted with coeliac disease, an immune mediated food intolerance for which wheat gliadins have been implicated as the causal agent. In light of the importance of corn products to these individuals, a sequence similarity search was conducted and no amino acid sequence homologies between the Cry1A(b) protein and gliadins were detected.

The full nucleotide sequence and corresponding amino acid sequence were provided.

The 131 kDa protein expressed in plants and the resulting proteolytic fragments were compared to the bacterial proteins and shown to be of similar molecular weight, amino acid sequence, immunological reactivity and trypsin resistance. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein.

2. Glyphosate Tolerance
Two genes have been introduced into MON809 to provide tolerance to glyphosate, the active ingredient in Roundup herbicide. The tolerance, although not sufficient to provide field tolerance to the herbicide, was used to select transformed plants.

The CP4 EPSPS gene expresses a bacterial version from Agrobacterium tumefaciens strain CP4, of 5-enolpyruvlshikimate-3-phosphate synthase, a plant enzyme involved in the shikimate biochemical pathway for the production of aromatic amino acids. Glyphosate inhibits the native plant EPSPS, thus blocking the shikimate pathway and halting the production of these necessary amino acids. The CP4-EPSPS is a glyphosate tolerant version of the enzyme with high catalytic activity. Under glyphosate selection, the addtion of the bacterial, glyphosate tolerant enzyme will permit the normal production of aromatic amino acids.

The second gene, gox, is derived from Achromobacter sp., a ubiquitous soil bacteria species which expresses an enzyme that degrades glyphosate by conversion to aminomethlyphosphonic acid (AMPA) and glyoxylate. In line MON809 only a partial copy of the gene is inserted and no gene expression was detected.

A plant-derived coding sequence expressing a chloroplast transit peptide was co-introduced with each of the glyphosate resistance genes. The chloroplast transit peptides facilitates the import of the protein products from the glyphosate resistance genes into the chloroplast, which is the location of the shikimate pathway and the site of action for glyphosate. Expression of the CP4 EPSPS gene averaged 21.68 g/g (f.w.) in leaves, 9.41 g/g (f.w.) in grain and 1.6 g/g (f.w.) in the whole plant.

The plant expressed enzyme showed no significant homology with any known toxins or allergens. It is common in nature and no toxic or allergenic effects would be expected to occur. Data was presented which showed that the CP4 EPSPS enzyme is rapidly inactivated by heat and by digestion in simulated mammalian gastric fluid.

3. Development Method
MON809 was co-transformed with two vectors, one carrying a synthetic cry1A(b) gene and the second bearing the two herbicide resistance genes. The two plasmid vectors were introduced by microprojectile bombardment into cultured plant cells. Glyphosate tolerant transformed cells were selected, then cultured in tissue culture medium for plant regeneration.

4. Stable Integration into the Plant's Genome
Southern analysis indicated one integrated DNA segment which included a complete and a partial copy of the cryIA(b) gene, two complete copies of the CP4 EPSPS genes and a partial gox gene.

Segregation data indicate a stable insertion, with the trait segregating according to Mendelian genetics, through five generations of crossing.

5. Nutrition
The nutritional quality of grain from MON 809 is similar to that of grain from an equivalent non-transgenic
line and to grain that is currently in commerce. Grain was analysed for proximate (i.e., ash, moisture content, crude protein, crude fat), amino acid and fatty acid composition. Some minor differences were observed between MON 809 and an equivalent non-transgenic line, but these components were within the range of variation normally observed in the commercial grain supply and are attributed to variations in the plant genotypes rather than to the presence of the introduced genes.

6. Safety
The CryIA(b) and CP4 EPSPS proteins should pose no risks for consumption of products produced from insect resistant maize. The introduced proteins were compared to databases of known protein toxins and neither shows any significant amino acid sequence similarity to known protein toxins. Both proteins are rapidly broken down and their enzymatic activity lost under conditions that simulate mammalian digestion. Neither protein was toxic when fed to mice.
Reference: Canadian Food Inspection Agency, Plant Biotechnology Office; Office of Food Biotechnology, Health Canada

Summary of Introduced Genetic Elements
 
Code Name Type Promoter, other Terminator Copies Form
goxv247 glyphosate oxidoreductase  (Ochrobactrum anthropi) HT NULL
chloroplast transit peptide from A. thaliana SSU1A gene (CTP1)
NULL   partial copy, no expression detected
cry1Ab Cry1Ab delta-endotoxin (Btk HD-1)  (Bacillus thuringiensis subsp. kurstaki (Btk)) IR enhanced CaMV 35S
NULL
A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1 Truncated, modified; second partial copy present
CP4 epsps 5-enolpyruvyl shikimate-3-phosphate synthase  (Agrobacterium tumefaciens CP4) SM enhanced CaMV 35S
chloroplast transit peptide from A. thaliana EPSPS gene (CTP2)
NULL 2  

Characteristics of Zea mays L. (Maize)
 
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
 
Latin Name Gene Pathogenicity
Bacillus thuringiensis subsp. kurstaki cry1Ab While target insects are susceptible to oral doses of Bt proteins, no evidence of toxic effects in laboratory mammals or birds given up to 10 g protein/g body weight.
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
 
Maize line MON809 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. Plasmid PV-ZMBK07 also contained sequences from lacZ operon (a partial E. coli lacI coding sequence, the promoter plac and a partial coding sequence for beta-galactosidase from pUC119), ori-pUC (replication origin for pUC plasmids), and the neo gene, which encodes neomycin phosphotransferase II (NPT II) that imparts resistance to the antibiotic kanamycin.

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. The expression of these genes was used as a selectable marker to identify transformed plants on medium containing glyphosate but was not sufficient to provide field level tolerance to glyphosate containing herbicides. 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
 
The Introduced DNA
Southern blot analysis of MON809 genomic DNA indicated one integrated DNA segment, of approximately 23Kb DNA, which included a complete and a partial copy of the cry1Ab gene, two complete copies of the CP4 EPSPS encoding gene, a partial gox gene and the NPT II encoding antibiotic resistance marker gene.

Additional Southern blot analyses were performed to demonstrate that additional plasmid backbone DNA sequences from PV-ZMBK07 were not integrated into the host genome.

Genetic Stability of the Introduced Trait
Segregation analysis over five generations of crossing indicated that the cry1Ab gene was stably integrated and segregated according to Mendelian rules of inheritance.

Expressed Material
Western blot analysis of protein extracts from MON809 demonstrated the presence of an immunoreactive protein of the predicted size. Lower molecular weight species corresponding to the predicted size of the truncated cry1Ab gene were not detected, indicating that this partial gene sequence is not expressed. The expression of Cry1Ab protein was evaluated using enzyme linked immunosorbent assay (ELISA) of tissue samples obtained from plants grown at 6 locations. The average levels of expressed Cry1Ab protein were: 1.63 0.75 ?/g fresh weight (fwt) in leaves, 0.55 0.23 ?/g fwt in grain and 1.23 0.5 ?/g fwt in the whole plant. Cry1Ab protein was not detected in pollen and the levels expressed in leaves declined over the growing season.

Similarly, CP4 EPSPS gene activity, used as a selectable marker conferring tolerance to glyphosate, was quantified by testing for EPSPS protein levels and averaged results were as follows: 21.68 ?/g fwt in leaves, 9.41 ?/g fwt in grain and 1.6 ?/g fwt in the whole plant. Data were provided showing that the level of expression of CP4 EPSPS was too low to confer significant glyphosate resistance in the field. Expression of the second gyphosate tolerance gene, gox, which encodes the enzyme glyphosate oxidase, could not be detected by Western blot analysis.

The Cry1Ab protein was shown to degrade readily in the environment. The plant expressed protein had DT50 and DT90 values (time to degrade to 50% and 90 % of the original bioactivity) of 2 and 15 days respectively.

Environmental Safety Considerations
 
Field Testing
MON809 maize was field tested under confined conditions in Canada from 1994-1996. Field reports on MON809 and corn hybrids derived from MON809 determined that early stand establishment, vegetative vigour, time to maturity and seed production were within the normal range of expression of these traits in commercial corn hybrids. Hybrids derived from MON809 produced similar yields in the absence of ECB, but under heavy infestations of ECB, MON809 hybrids out-yielded their non-transgenic counterparts by 10-15%. Hybrids derived from MON809 were not tolerant to the concentrations of glyphosate herbicide that are typically used for weed control. No significant differences were observed in resistance to a number of other significant insect pests or diseases compared to non-transgenic controls. Overall the field data reports and data on agronomic traits showed that MON809 lines have no potential to pose a plant pest risk.

Outcrossing
Since pollen production and viability were unchanged by the genetic modification resulting in maize line MON809, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between MON809 and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In Canada and the United States, where there are few plant species closely-related to maize in the wild, the risk of gene flow to other species is remote. Cultivated maize, Zea mays L. subsp. mays, is sexually compatible with other members of the genus Zea, and to a much lesser degree with members of the genus Tripsacum.

Weediness Potential
No competitive advantage was conferred to MON809, other than that conferred by resistance to ECB, which will not, in itself, render maize weedy or invasive of natural habitats since none of the reproductive or growth characteristics were modified.

Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. In agriculture, maize volunteers are not uncommon but are easily controlled by mechanical means or by using herbicides. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects
MON809 maize and the Cry1Ab protein, in particular, should not have a significant potential to harm organisms beneficial to agricultural ecosystems. The history of use and literature suggest that the bacterial Bt protein is not toxic to humans, other vertebrates, and beneficial insects. Most insecticidal protein toxins from B. thuringiensis subspecies kurstaki, including Cry1Ab, have been shown to be specifically toxic to Lepidopteran larvae on ingestion and appear non-toxic to other species of insects, either directly or through secondary ingestion (predation).

Laboratory studies of honeybee larvae and adults, predaceous insects (green lacewing larvae, ladybird beetles), and parasitic hymenoptera exposed to the Cry1Ab protein recorded no discernible effects at approximately 10 times the usual LC50 dose for a target insect. Field examination of beneficial arthropods (the Anthocorid Orius insidiosus and spiders) in genetically modified MON809 maize, concluded that any potential impact on non-target arthropods would be less than that from the use of conventional insecticides. Results from feeding studies of young quail fed with modified maize meal in their diet showed no adverse effects. Studies were conducted to evaluate the impact of residual MON809 maize on soil organisms and soil function. It was shown that the Bt endotoxin may become adsorbed to some soil fractions and that degradation was by microbial action. However, since very little of the crop material remains after harvest for incorporation it was concluded based on evidence available that the risks to organisms and soil function were very low.

In summary, it was determined that genetically modified maize MON809 did not present an increased risk to, or impact on interacting organisms, including humans, with the exception of specific lepidopteran insect species. Furthermore, MON809 will not impact on threatened or endangered lepidopteran species, as none are listed which feed on maize plants in Canada or the United States. In addition, these organisms are not expected to be affected by the expressed CP4 EPSPS.

Impact on Biodiversity
MON809 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of maize production. Since the risk of outcrossing with wild relatives in Canada and the United States is remote, it was determined that the risk of transferring genetic traits from MON809 maize to species in unmanaged environments was insignificant.

Other Considerations
In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in Canada and U.S. have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including MON809 maize, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

MON809 maize plants are not likely to eliminate the use of chemical insecticides, which are traditionally applied to about 25 to 35% of the total planted maize acreage. MON809 maize may positively impact current agricultural practices used for insect control by 1) offering an alternative method for control of European corn borer (and potentially other Cry1Ab-susceptible pests of maize); 2) reducing the use of insecticides to control European corn borer and the resulting potential adverse effects of such insecticides on beneficial insects, farm worker safety, and ground water contamination; and 3) offering a new tool for managing insects that have become resistant to other insecticides currently used or expressed in maize, including other Bt-based insecticides.

Food and/or Feed Safety Considerations
 
Dietary Exposure
MON809 maize 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. The potential 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 insect resistant hybrids of MON809 maize was anticipated to be the same as for other lines of commercially available field maize.

Nutritional Data
The nutritional quality of grain from MON809 maize was similar to that of grain from an equivalent non-transgenic line and to grain currently available in commerce. Grain was subjected to proximate analysis (e.g., ash, moisture content, crude protein, crude fat), amino acid and fatty acid composition. Some minor differences were observed between MON809 and an equivalent non-transgenic line, but the levels were within the range of variation normally observed in the commercial grain supply and were attributed to variations in the plant genotypes rather than to the presence of the introduced genes.

Toxicity
Data were submitted that demonstrated that the active Cry1Ab protein product of the inserted cry1Ab gene was equivalent to the protein contained in microbial Bt spray formulations that have been safely used in agriculture for more than 40 years. Studies on the Cry1Ab protein expressed in plants and the resulting proteolytic fragments were compared to the bacterial proteins and shown to be of similar molecular weight, amino acid sequence, immunological reactivity and trypsin resistance. The protein was not glycosylated and showed similar bioactivity and host range specificity to the native protein.

The Cry1Ab and CP4 EPSPS proteins should pose no risks for consumption of products produced from insect resistant maize. The introduced proteins were compared to databases of known protein toxins and neither showed any significant amino acid sequence similarity to known protein toxins. Neither protein was toxic when fed to mice.

Allergenicity
The Cry1Ab and CP4 EPSPS proteins are extremely unlikely to be allergens. The deduced amino acid sequences of the introduced proteins were compared to sequences of known allergens contained in the public domain databases GenBank, EMBL, Pir and SwissProt. No significant sequence homologies were detected. Neither the Cry1Ab and CP4 EPSPS protein originated from an allergenic source, or were structurally similar to known allergens. EPSPS is ubiquitous in bacteria, plants and animals and no toxic or allergenic effects have ever been reported.

In addition, the potential for allergenicity was assessed based upon the characteristics of known food allergens (stability to digestion, stability to processing, abundance in foods).
Protein allergens are normally resistant to digestion unlike the Cry1Ab protein that was shown to degrade readily in simulated gastric fluid. Similarly, data were presented showing that the CP4 EPSPS enzyme was rapidly inactivated by heat and by digestion in simulated mammalian gastric fluid.

Maize products are an important alternative to wheat flour for individuals afflicted with coeliac disease, an immune mediated food intolerance for which wheat gliadins have been implicated as the causal agent. In light of the importance of corn products to these individuals, a sequence similarity search was conducted and no amino acid sequence homologies between the Cry1Ab protein and gliadins were detected.

Links to Further Information
 
Canadian Food Inspection Agency, Plant Biotechnology Office[PDF Size: 175811 bytes]
Decision Document 97-18: Determination of the Safety of Pioneer Hi-Bred International Inc.'s European Corn Borer (ECB) Resistant Corn (Zea mays L.) Line MON809
European Commission Scientific Committee on Plants[PDF Size: 146707 bytes]
Opinion of the Scientific Committee on Plants regarding the submission for placing on the market of genetically modified, insect-resistant maize lines notified by the pioneer genetique S.A.R.L. Company (notification No C/F/95/12-01/B) (Submitted by the Scientific Committee on Plants, 19 May 1998)
Impact of Bt corn pollen on monarch butterfly populations: A risk assessment[PDF Size: 166577 bytes]
Mark K. Sears, Richard L. Hellmich, Diane E. Stanley-Horn, Karen S. Oberhauser, John M. Pleasants, Heather R. Mattila, Blair D. Siegfried, and Galen P. Dively (2001). Proc. Natl. Acad. Sci. USA Early Edition
Office of Food Biotechnology, Health Canada[PDF Size: 12423 bytes]
Novel Food Information: Insect resistant maize (corn), MON809
U.S.Department of Agriculture, Animal and Plant Health Inspection Service[PDF Size: 1505444 bytes]
Monsanto Co. Petition for Determination of Non-regulated Status of Additional Yieldgard Corn Lines MON 809 and 810
UK Register C/F/95/12-01/B[PDF Size: 10778 bytes]
UK Department of the Environment,Transport and the Regions: Registry entry for MON809.

References
 
Betz, F.S., Hammond, B.G. & Fuchs, R.L. (2000). Safety and advantages of Bacillus thuringiensis-protected plants to control insect pests. Regulatory Toxicology 32, 156-173.


THIS RECORD WAS LAST MODIFIED ON THURSDAY, JULY 21, 2005
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