GM Crop Database

Database Product Description
 
MON-8934-3 (MON89034)
Host Organism
Zea mays L. L. (Maize)
 
Trait
Resistance to lepidopteran pests.
 
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
 
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
Monsanto Company
Chesterfield Village Research Center (MO)
700 Chesterfield Parkway North
St. Louis
MO  USA
 
 
Summary of Regulatory Approvals
 
Country Environment Food and/or Feed Food Feed Marketing
Argentina 2010 2010  
Australia 2008  
Brazil 2009 2009  
Canada 2008 2008 2008  
Colombia 2010  
European Union 2009  
Japan 2008 2007 2008  
Korea 2009 2009  
Philippines 2009 2009  
Taiwan 2008  
United States 2008 2007  
Click on the country name for country-specific contact and regulatory information.
Introduction
 
Maize (Zea mays L), otherwise known as corn, is the world?s third leading cereal crop, behind wheat and rice, and is grown in over 25 countries worldwide. The majority of grain and forage derived from maize is used as animal feed, however maize also has a long history of safe use as food for human consumption. The grain can be processed into industrial products such as ethanol (by fermentation), and highly refined starch (by wetmilling) and sweetener products. In addition to milling, the maize germ can be processed to obtain corn oil and numerous other products. The majority of maize is grown for animal feed production and human food, however an increasing percentage is being used for the production of ethanol as a biofuel alternative to petroleum products from fossil sources.

MON 89034 was developed as a second generation insect-resistant maize product to provide enhanced benefits for the control of lepidopteran insect pests. MON 89034 produces the Cry1A.105 and Cry2Ab2 proteins derived from Bacillus thuringiensis, which are active against lepidopteran insect pests. MON 89034 is intended to serve farmers' needs for controlling a wider spectrum of lepidopteran pests and help assure the durability of insect-resistant maize. MON 89034 provides control of Ostrinia species such as European corn borer (ECB) and Asian corn borer, and Diatraea species such as southwestern corn borer (SWCB) and sugarcane borer. MON 89034 also provides a high level control of fall armyworm (FAW) throughout the season and improved protection from damage caused by corn earworm (CEW). In addition to the wider spectrum of insect control, the combination of the Cry1A.105 and Cry2Ab2 insecticidal proteins in a single plant provides a much more effective insect resistance management (IRM) tool. The results of mathematical modeling indicate that biotechnology-derived plants expressing two Cry proteins will have significantly greater durability than plants producing either of the single proteins if the cross-resistance between the Cry proteins is low and the mortality of susceptible insects caused by each of the individual proteins is at least 90%. Comparative biophysical studies indicate that the Cry1A.105 and Cry2Ab2 proteins have important differences in their mode of action, specifically in the way in which they bind to the lepidopteran midgut. Therefore, the probability of cross-resistance between these two proteins is low. Furthermore, in vitro and in planta studies with Cry1A.105 and Cry2Ab2 demonstrate that both proteins are highly active against the primary lepidopteran pests of corn (ECB, SWCB, CEW, and FAW), particularly ECB, achieving close to or greater than the critical 95% level of control in all cases.

Summary of Introduced Genetic Elements
 
Code Name Type Promoter, other Terminator Copies Form
cry1A.105 chimeric cry1 delta-endotoxin  (Bacillus thuringiensis) IR CaMV 35S
5' untranslated leader from wheat chlorophyll a/b-binding protein Rice actin gene intron
3' untranslated region of wheat heat shock protein 17.3 1  
cry2Ab cry2Ab delta-endotoxin  (Bacillus thuringiensis) IR FMV-35S - promoter from Figwort Mosaic Virus
Hsp70 intron from maize heat shock protein gene. Transit peptide from maize RBC-small subunit.
A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1  

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 cry2Ab2 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.

Modification Method
 
MON89034 was produced by Agrobacterium-mediated transformation of corn with PVZMIR245, which is a binary vector containing 2 separate T-DNA regions. The first T-DNA, designated as T-DNA I, contained the cry1A.105 and the cry2Ab2 expression cassettes which express the insecticidal proteins Cry1A.105 and Cry2Ab2. The cry1A.105 gene, is a chimeric gene comprising of 4 domains from other cry genes previously used in transgenic plants. The amino acid sequences of Domains I and II are identical with the respective domains from Cry1Ab and Cry1Ac proteins, Domain III is almost identical to the Cry1F protein, and the C-terminal Domain is identical to Cry1Ac protein. Overall, the amino acid sequence homology of Cry1A.105 is 93.6%, 90.0%, and 76.7% respectively with the Cry1Ac, Cry1Ab and Cry1F proteins. The expression cassette for the coding sequence of the Cry1A.105 protein consists of the promoter (P-e35S) and leader for the cauliflower mosaic virus (CaMV) 35S RNA with a duplicated enhancer region. The cassette also contains the 5' untranslated leader of the wheat chlorophyll a/b/ binding protein (L-Cab), the intron from the rice actin gene (I-Ract1), the cry1A.105 coding sequence that was optimized for expression in monocots, and the 3? nontranslated region of the coding sequence for wheat heat shock protein 17.3 (T-Hsp17), which terminates transcription and provides the signal for mRNA polyadenylation

The Cry2Ab2 protein is a member of the Cry2Ab class of proteins that share >95% amino sequence homology. Like the Cry2Ab2 protein produced in the transgenic cotton event 15985, the Cry2Ab2 protein produced in MON89034 is a slight variant of the wild-type Cry2Ab2 protein isolated from Bacillus thuringiensis subsp. kurstaki. The Cry2Ab2 produced in MON 89034 is identical to the Cry2Ab2 produced in 15985 cotton, and they differ from the wild-type Cry2Ab2 of Bt by only one amino acid. The promoter region of the cry2Ab2 gene expression cassette consists of the 35S promoter from figwort mosaic virus (P-FMV) and the first intron from the corn heat shock protein 70 gene (I-Hsp 70). The cassette also contains a cry2Ab2 coding sequence with a modified codon usage fused to a chloroplast transit peptide region of corn ribulose 1,5-biphosphate carboxylase small subunit including the first intron (TSSSU-CTP). The 3? nontranslated region of the nopaline synthase (T-nos) coding region from Agrobacterium tumefaciens T-DNA terminates transcriptionand directs polyadenylation.

The second T-DNA, designated as T-DNA II, contains the nptII (neomycin phosphotransferase II) expression cassette. The nptII gene cassette that produces the NPTII protein consists of the promoter (P-e35S) from the cauliflower mosaic virus (CaMV) 35S RNA. The cassette also contains the coding sequence for the NPTII protein, followed by the 3? nontranslated region of the nopaline synthase (T-nos) sequence from Agrobacterium tumefaciens which terminates the transcription and directs polyadenylation.

During transformation, both T-DNAs were inserted into the genome. The nptII gene confers resistance to Kanamycin and similar antibiotics and was used as the selectable marker for isolation of transformed cells and regeneration of transgenic plants. Once transgenic plants had been regenerated, the selectable marker gene was no longer needed and conventional breeding was used to isolate plants that only contain the cry1A.105 and the cry2Ab2 expression cassettes (T-DNA I) and did not contain the nptII expression cassette (T-DNA II), thereby, producing marker-free transgenic lines, one of which was selected and designated as MON89034.


Characteristics of the Modification
 
The Introduced DNA
Molecular characterization of MON89034 by Southern blot analyses demonstrated that the DNA inserted into the genome is present at a single locus and contains one functional copy of the cry1A.105 and the cry2Ab2 expression cassettes. Furthermore, no backbone plasmid DNA or nptII sequences were detected in these analyses. The organization of the elements within the insert in MON89034 was confirmed by DNA sequencing analyses. PCR primers were designed to amplify seven overlapping regions of DNA that span the entire length of the insert (9317 bp), and the amplified DNA fragments were subjected to DNA sequencing analyses. The results confirmed that the sequence of the DNA insert in MON89034 matched the designed, corresponding sequences in PV-ZMIR245 with one exception.

This exception is that the e35S promoter that regulates expression of the cry1A.105 gene has been modified and that the Right Border sequence present in PV-ZMIR245 was replaced by a Left Border sequence in MON89034. This molecular rearrangement can be explained by a recombination event which occurred, either prior to or during the process of T-DNA transfer to the plant cell, between the DNA sequences near the 35S promoters in T-DNA I and T-DNA II. Due to this recombination event, the reconstituted e35S promoter in MON89034 no longer has the duplicated enhancer elements compared to the original e35S promoter in PV-ZMIR245. Despite the deletion of the enhance elements, the Cry1A.105 protein expression levels in MON89034 are still sufficiently high under the regulation of the modified e35S promoter to deliver the required efficacy against target insect pests.

Genetic Stability of the Trait
Analysis of the stability of the integrated DNA demonstrated that the unique Southern blot fingerprint of MON89034 was maintained through seven generations of conventional breeding, thereby confirming the stability of the insert. Additionally, the Southern blo analysis of multiple generations in the MON89034 breeding history indicated that there were no detectable T-DNA II elements other than those which are common to T-DNA I, i.e., 35S promoter, nos 3 end sequence and the Left Border sequence. Furthermore, these generations were shown not to contain any detectable backbone sequence from plasmid PVZMIR245. The stability was further confirmed by the fact that the inheritance of the lepidopteran protection trait in MON89034 follows Mendelian segregation principle.

Expressed Material
The expression levels of the Cry1A.105 and Cry2Ab2 proteins in different tissues of MON89034 are relatively low. Therefore, it was necessary to produce the proteins in a high-expressing, recombinant microorganism in order to obtain sufficient quantities of the protein for safety studies. Recombinant Cry1A.105 and Cry2Ab2 proteins were produced in Escherichia coli, with the sequences engineered to match the Cry1A.105 and Cry2Ab2 proteins produced in MON89034. The equivalence of the physicochemical characteristics and functional activity between the MON89034-produced and E. coli-produced proteins was confirmed by a panel of analytical techniques, including sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Western blot analysis, matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), glycosylation analysis, and assays of biological activity.

The levels of the Cry1A.105 and Cry2Ab2 proteins in various tissues of MON89034 were determined using enzyme-linked immunosorbent assays (ELISA). To produce the tissues for analysis, MON89034 and conventional maize were planted at five field locations during the 2005 growing season. In tissues harvested throughout the growing season, mean Cry1A.105 protein levels across all sites varied from 72-520 ?g/g dwt in leaf, 11-79 ?g/g dwt in root, 5.9 ?g/g dwt in grain and 42-380 ?g/g dwt in whole plant. In tissues harvested throughout the growing season, mean Cry2Ab2 protein levels across all sites varied from 130-180 ?g/g dwt in leaf, 21-58 ?g/g dwt in root, 1.3 ?g/g dwt in grain and 38-130 ?g/g dwt in whole plant. In general, levels of both proteins declined in vegetative tissues over the growing season.

Food and/or Feed Safety Considerations
 
Dietary Exposure
Humans consume relatively little whole kernel or processed maize, compared to maize-based food ingredients. Maize is a raw material for the manufacture of starch, the majority of which is converted to a variety of sweetener and fermentation products, including high fructose syrup and ethanol. Maize oil is commercially processed from the germ. These materials are components of many foods including bakery and dairy goods, and the human food uses of grain from MON89034 are not expected to be different from the uses of non-transgenic field maize varieties. As such, the dietary exposure to humans of grain from insect resistant hybrids will not be different from that for other commercially available field maize varieties. Maize grain and by-products of wet and dry milling are used as animal feed. The whole maize plant can also be harvested at an appropriate stage and fed to animals or stored as silage. As for food use, the dietary exposure of animals to grain or forage from MON89034 will not be different to exposure from other commercially available maize.

Potential toxicity and allergenicity
Cry1A.105 is a chimeric Cry1A protein of Bacillus thuringiensis that is comprised of three N-terminal domains (I, II, and III) and the C-terminal region. The domains I and II of Cry1A.105 are 100% identical to the respective domains of Cry1Ab or Cry1Ac. The domain III of Cry1A.105 is 99% identical to the domain III of Cry1F. The C-terminal region of Cry1A.105 is 100% identical to that of Cry1Ac. The overall amino acid sequence identity of 93.6%, 90.0%, and 76.7% to Cry1Ac, Cry1Ab, and Cry1F proteins, respectively. Cry2Ab2 is a Bacillus thuringiensis (subsp. kurstaki) protein. Bacillus thuringiensis strains producing Cry1Ac, Cry1Ab, Cry1F, and Cry2A proteins have been used for decades as biopesticides, and pesticides that contain Cry1Ac/Cry1F chimeric protein have been used since 1997. Transgenic maize and cotton expressing Cry1Ac, Cry1Ab, Cry1F, or Cry2Ab2 proteins have been cultivated in large areas in the U.S. and other countries for up to a decade. There are no known reports of allergy or toxicity to Bt or to these Cry proteins..

A dietary safety assessment was performed to evaluate the risks to humans and animals from the Cry1A.105 and Cry2Ab2 proteins present in the foods and feeds derived from MON89034. Risks are quantified as a margin of exposure (MOE), which is defined as the ratio of the No Observable Effect Level (NOEL) from an acute mouse gavage study to estimates of the dietary intake of the respective Cry protein. Mice acute oral toxicity studies demonstrated that the two proteins are not acutely toxic and do not cause any adverse effects even at the higest dose levels tested, which are 2072 and 2198 mg/kg body weight for Cry1A.105 and Cry2Ab2 proteins, respectively. The dietary safety assessment showed that the MOEs for the overall U.S. population were greater than or equal to 199,000 and 981,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For children aged 3-5 years old, an age group with the highest corn consumption, the MOEs were greater than or equal to 79,400 and 390,000 for the Cry1A.105 and Cry2Ab2 proteins, respectively. For poultry and livestock, the MOEs ranged between 1,930 13,500 and 2,160 47,600 for the Cry1A.105 and Cry2Ab2 proteins, respectively.

Cry1A.105 and Cry2Ab2 proteins do not share any amino acid sequence similarities with known allergens, gliadins, glutenins, or protein toxins which have adverse effects to mammals. This has been shown by extensive assessments with bioinformatic tools, such as FASTA sequence alignment tool and an eight amino acid sliding window search. Furthermore, Cry1A.105 and Cry2Ab2 proteins are rapidly digestible in simulated gastric fluids. Greater than 95% to 99% of the proteins were digested in simulated gastric fluids in less than 30 seconds. Proteins that are rapidly digestible in mammalian gastrointestinal systems are unlikely to be allergens when consumed.

Nutritional and Compositional Data
The composition of forage and grain from MON89034 and a non-transgenic control was compared to assess whether the transgenic corn differs from non-transgenic corn in nutritional characteristics. The non-transgenic control has a genetic background similar to that of MON89034 but does not contain the cry1A.105 and cry2Ab2 genes. In addition, grain and forage from 15 different conventional maize hybrids produced in the same field trials with MON89034 and the non-transgenic control was evaluated, with three different hybrids planted at each of the five sites. Data derived from these 15 conventional hybrids as references to generate a 99% tolerance interval for each analyzed component.

The levels of proximates (ash, fat, protein and moisture), fiber (acid detergent fiber-ADF and neutral detergent fiber-NDF) and minerals (calcium and phosphorous) were compared in forage from MON89034 and the non-transgenic control. No statistically significant differences were found in the levels of all analyzed components between forage from MON89034 and forage from the conventional control, with the exception of phosphorus. MON89034 had statistically significantly higher levels of phosphorus than its control, but these levels were within the 99% tolerance intervals for the commercial varieties and within the ranges reported in the literature and the ILSI Crop Composition Database.

The levels of proximates, minerals, amino acids, fatty acids, anti-nutrients, secondary metabolites and vitamins were compared in mature grain from MON89034 and control maize. A statistical analysis of the analytical results combined from all field trials found no statistically significant differences between MON89034 and the conventional non-transgenic control, except for stearic acid and arachidic acid (calculated as a percentage of total fatty acids). However, differences in the levels of these fatty acids were small. The levels of all measured components were within the ranges reported in the literature and the ILSI Crop Composition Database and within the 99% tolerance interval established from the 15 control varieties. There were several statistically significant differences in the level of nutrients within the individual locations, but these differences were not consistent across all locations and it was concluded that these differences were not biologically significant.

The results of these compositional analyses led to the conclusion that MON89034 forage and grain are not different in nutritional and anti-nutritional composition compared to maize hybrids currently marketed, grown and consumed.

Links to Further Information
 
Canadian Food Inspection Agency, Plant Biotechnology Office[PDF Size: 88415 bytes]
Decision Document DD2008-74: Determination of the Safety of Monsanto Canada Inc.'s Corn (Zea mays L.) Event MON 89034
European Food Safety Authority[PDF Size: 235813 bytes]
Scientific Opinion: Application (Reference EFSA-GMO-NL-2007-37) for the placing on the market of the insect-resistant genetically modified maize MON89034, for food and feed uses, import and processing under Regulation (EC) No 1829/2003 from Monsanto
Food Standards Australia New Zealand[PDF Size: 462511 bytes]
Final Assessment Report - Application A595: Food Derived From Insect-Protected Corn Line MON 89034
Japanese Biosafety Clearing House, Ministry of Environment[PDF Size: 485858 bytes]
Outline of the biological diversity risk assessment report: Type 1 use approval for MON89034
U.S. Department of Agriculture, Animal and Plant Health Inspection Service[PDF Size: 558415 bytes]
Petition for Non-Regulated Status for Corn Event MON89034 (APHIS 06-289-01p): Finding of No Significant Impact
U.S. Department of Agriculture, Animal and Plant Health Inspection Service[PDF Size: 60152 bytes]
Determination of Nonregulated Status for Corn Genetically Engineered for Insect Resistance
U.S. Department of Agriculture, Animal and Plant Health Inspection Service[PDF Size: 5216014 bytes]
Petition for Non-Regulated Status for Corn Event MON89034 (APHIS 06-289-01p)
United States Food and Drug Administration[PDF Size: 41091 bytes]
Biotechnology Consultation Note to the File BNF No. 000107

References
 
Compositional Analysis
Drury, SM; Reynolds, TL; Ridley, WP; Bogdanova, N; Riordan, S; Nemeth, MA; Sorbet, R; Trujillo, WA; and Breeze, ML. (2008). Composition of forage and grain from second-generation insect-protected corn MON 89034 is equivalent to that of conventional corn (Zea mays L.). J. Agric. Food Chem. 56 (12): 4623?4630.
Reynolds, T.L., Drury, S.M., Nemeth, M.A., Trujillo, W.A. and Sorbet, R. (2006). Compositional analyses of corn forage and grain collected from MON 89034 grown in 2004 U.S. field trials. Unpublished study number: MSL-20403. Amended report for MSL-20097. Monsanto Company.
Development & Molecular-Genetic Characterization
Rice, J.F., Wolff, B.J., Groat, J.R., Scanlon, N.K., Jennings, J.C. and Masucci, J.D. (2006). Molecular analysis of corn MON 89034. Unpublished study number: 05-01-39-12; MSL-20311. Monsanto Company.
Efficacy
Wu, X; Rogers, LB; Zhu, YC; Abel, CA; Head, GP; and Huang, F. (2009). Susceptibility of Cry1Ab-resistant and -susceptible sugarcane borer (Lepidoptera: Crambidae) to four Bacillus thuringiensis toxins. J. Invertebr. Pathol. 100(1): 29-34.
Nutritional Equivalence
Taylor, M; Hartnell, G; Nemeth, M; Lucas, D; and Davis, D. (2007). Comparison of broiler performance when fed diets containing grain from second-generation insect-protected and glyphosate-tolerant, conventional control or commercial reference corn. Poultry Science 86: 1972?1979.
Patents
Anderson, H.M., Allen, J.R., Groat, J.R., Johnson, S.C., Kelly, R.A., Korte, J. and Rice, J.F. (2008). Corn plant and seed corresonding to transgenic event MON89034 and methods for detection and use thereof. United States Patent Application 2008/0260932 A1.
Protein Safety
Goley, M.E. and Thorp, J.J. (2005). Immunodetection of Cry2Ab2 and Cry1A.105 proteins in corn grain from MON 89034 following heat treatment. Unpublished study number: 05-01-39-27; MSL-19899. Monsanto Company.
Kapadia, S.A. and Rice, E.A. (2005). Assessment of the in vitro digestibility of the Cry1A.105 protein in simulated gastric fluid. Unpublished study number: 05-01-62-02; MSL-19929. Monsanto Company.
Kapadia, S.A. and Rice, E.A. (2006). Assessment of the in vitro digestibility of the Cry2Ab2 protein in simulated gastric fluid. Unpublished study number: 05-01-62-04; MSL-19931. Monsanto Company.
McClain, J.S. and Knupp, G. (2008). Updated bioinformatics evaluation of the Cry1A.105 protein utilizing the AD8 database. Unpublished study number: REG-08-133; MSL0021226. Monsanto Company.
McClain, J.S. and Knupp, G. (2008). Updated bioinformatics evaluation of the Cry2Ab2 protein utilizing the AD8 database. Unpublished study number: REG-08-134; MSL0021227. Monsanto Company.
McClain, J.S. and Silvanovich, A. (2006). Bioinformatics evaluation of the Cry1A.105 protein utilizing the AD6, TOXIN5, and ALLPEPTIDES databases. Unpublished report number: 06-01-62-04; MSL number: 20351. Monsanto Company.
McClain, J.S. and Silvanovich, A. (2006). Bioinformatics evaluation of the Cry2Ab2 protein utilizing the AD6, TOXIN5, and ALLPEPTIDES databases. Unpublished report number: 06-01-62-01; MSL number: 20307. Monsanto Company.


THIS RECORD WAS LAST MODIFIED ON SUNDAY, NOVEMBER 08, 2009
Query Page
 
> New Database query
Go to Event