GM Crop Database

Database Product Description

MON-89Ø34-3 (MON89034)
Host Organism
Zea mays 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.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Argentina 2010 2010 2010
Australia 2008
Brazil 2009 2009 2009
Canada 2008 2008 2008
Colombia 2010
European Union 2009 2009
Japan 2007 2008 2008
Korea 2009 2009
Philippines 2009 2009
Taiwan 2008
United States 2007 2007 2008

Introduction Expand

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 Expand

Code Name Type Promoter, other Terminator Copies Form
cry1A.105 chimeric cry1 delta-endotoxin 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 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) 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
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 µg protein/g body weight.

Modification Method Expand

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 Expand

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 Expand

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.

Abstract Collapse

Maize, or corn (Zea mays L.) is grown commercially in over 100 countries with a combined harvest of nearly 700 million metric tonnes in 2006. The top five producers of maize in 2005 were the United States, China, Brazil, Argentina, and Mexico, accounting for 70% of world production. Maize is grown primarily for its kernel (grain), the majority of which is used for animal feed, but with significant amounts refined into products used in a wide range of food, medical, and industrial goods.

In the United States maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, although an increasing share is now being used to produce ethanol for fuel. 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 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, car parts.

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.

MON 89034 was produced by Agrobacterium-mediated transformation of corn with PVZMIR245, which is a binary vector containing 2 T-DNAs. The first T-DNA, designated as T-DNA I, contained the cry1A.105 and the cry2Ab2 expression cassettes. The second T-DNA, designated as T-DNA II, contained the nptII (neomycin phosphotransferase II) expression cassette. During transformation, both T-DNAs were inserted into the genome. The nptII gene was used as the selectable marker which was needed for selection of the transformed cells. Once the transgenic cells were identified, the selectable marker gene was no longer needed and traditional breeding was used to isolate plants that only contained the cry1A.105 and cry2Ab2 expression cassettes (T-DNA I). Molecular characterization of MON 89034 by Southern blot analyses demonstrated that the DNA inserted into the corn genome is present at a single locus and contains one functional copy of the cry1A.105 and the cry2Ab2 expression cassettes. All genetic elements are present in the inserted DNA as expected with the exception that the e35S promoter, which 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 MON 89034. No backbone plasmid DNA or nptII sequences were detected.

As the selectable marker gene employed in the transformation process was removed during development of the final event, the two insecticidal proteins are the only new proteins expressed in this plant. This was confirmed by a molecular analysis that identified only the genes coding for the insecticidal proteins as being present in the final event, with no evidence for presence of the selecable marker gene. A review of the potential toxicity and allergenicity of these two proteins was made, in addition to a comparison of the composition of the grain and forage from this transgenic event with non-transgenic maize. Agronomic characters were evaluated in field trials, also in comparison to non-transgenic maize. No evidence was found to suggest that this product would pose a greater risk to human or animal health, or to the environment.

The food and livestock feed safety of MON89034 maize grain and forage was established based on several standard criteria. As part of the safety assessment, the nutritional composition of MON89034 grain was found to be equivalent to conventional maize as shown by the analyses of key nutrients including proximates (i.e., moisture, crude protein, crude fat, ash and carbohydrates), acid detergent fibre, neutral detergent fibre, total dietary fibre, amino acid composition, fatty acid profiles, minerals (e.g., calcium, phosphorus, magnesium), and vitamins (i.e., folic acid, niacin, vitamin E), as well as antinutrient compounds. Similar compositional analyses were conducted on MON89034 forage harvested at the late dough to early dent stages.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office Comissão Técnica Nacional de Biossegurança - CTNBio (Brazil) European Food Safety Authority Food Standards Australia New Zealand Japanese Biosafety Clearing House, Ministry of Environment U.S. Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Friday, March 26, 2010