GM Crop Database

Database Product Description

MIR604 (SYN-IR6Ø4-5)
Host Organism
Zea mays (Maize)
Trait
Resistance to corn root worm (Coleopteran, Diabrotica sp.)
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Syngenta Seeds, Inc.

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Argentina 2012 2012 2012
Australia 2006
Brazil 2014 2014 2014
Canada 2007 2007 2007
China 2008 2008 View
Colombia 2012 2012
European Union 2009 2009
Indonesia 2011
Japan 2007 2007 2007
Korea 2007 2008
Malaysia 2016 2016
Mexico 2007 2007
New Zealand 2006
Philippines 2007 2007
Russia 2007 2008
South Africa 2011 2011
Taiwan 2007
Turkey 2015
United States 2007 2007 2007
Vietnam 2016 2016

Introduction Expand

Corn line MIR604 has been genetically modified to be resistant to western corn rootworm (Diabrotica vigifera vigifera), northern corn rootworm (D. berberi), and Mexican corn rootworm (D. vigifera zeae). These species are serious insect pests of dent corn in the major corn-producing states of the north-central United States and Canada. Protection is conferred by the expression in the plant of the bacterially derived protein toxin mCry3A, encoded by the mcry3A gene in the corn plants. A selectable marker gene, pmi, encodes phosphomannose isomerase and allows transformed cells to utilise carbon from phosphomannose media.

Commercial corn lines containing the cry genes from Bacillus thuringiensis can provide growers with effective methods for controlling corn rootworm. Formulations with insecticidal toxins from cultures of B. thuringiensis are widely used as biopesticides on a variety of cereal and vegetable crops grown organically or under conventional agricultural conditions.

Corn, together with rice and wheat, is one of the most important cereal crops in the world. The majority of grain and forage derived from maize is used in animal feed. Maize grain is also used in industrial products, such as ethanol by fermentation and highly refined starch by wet milling. Corn-based products, largely as high-fructose corn syrup, are processed into breakfast cereals, baking products, extruded confectionery and corn chips. Other corn products such as cornstarch are also used by the food industry for the manufacture of dessert mixes and canned foods. 

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
mcry3A Cry3A delta-endotoxin IR promoter derived from the metallo-thionein-like gene from Zea mays A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1 Modified to enhance expression in maize
pmi mannose-6-phosphate isomerase SM ZmUbiInt (Zea mays poly-ubiquitin gene promoter and first intron) A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1

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.

Modification Method Expand

MIR604 maize was produced by Agrobacterium-mediated transformation of the inbred maize line A188. The T-DNA segment of the vector plasmid pZM26 contained a modified cry3A (mcry3A) gene encoding the mCry3A insecticidal protein and the pmi (manA) gene from Escherichia coli encoding the enzyme phosphomannose isomerase as a selectable marker.

The maize-optimised modified mcry3A gene encodes a protein of 598 amino acids. The native cry3A gene was modified to incorporate a cathepsin-G serine protease recognition site within the expressed protein. The original N-terminal region of this protein has been removed and the mCry3A protein commences at a methionine residue in position 48 of the native protein. The entire coding region of the mcry3A gene was synthesised using codons that are preferred in maize. The amino acid sequence of the synthetic version of Cry3A is the same as the native protein, except for the modified serine-protease recognition site. Transcription of the mcry3A gene was directed by the MTL promoter derived from the Zea mays metallothionein-like gene, which demonstrates root-preferential expression. Terminator and polyadenylation sequences were derived from the nopaline synthase gene from Agrobacterium tumefaciens.

The pmi gene represents the E.coli manA gene and encodes the enzyme phosphomannose isomerase (PMI). It was used as a selectable marker gene during the transformation process. Mannose, a hexose sugar, is taken up by plants and converted to mannose-6-phosphate by hexokinase. This product cannot be further utilised in plants as they lack the PMI enzyme. The accumulation of mannose-6-phosphate inhibits phosphoglucose isomerase, causing a block in glycolysis. It also depletes cells of orthophosphate required for the production of ATP. Therefore, while mannose has no direct toxicity on plant cells, it causes growth. This does not occur in plants transformed with the pmi gene as they can utilise mannose as a source of carbon. Expression of the pmi gene was driven by the promoter and first intron from the Z. mays polyubiquitin gene which provides constitutive gene expression in monocots. The nopaline synthase gene from Agrobacterium tumefaciens was again used for termination and polyadenylation sequences. The left border sequence (from octopine Ti plasmid pTi15955) and right border (from nopaline Ti plasmid pTiT37) contained non-coding sequences essential for the transfer of the T-DNA segment.
 

Characteristics of the Modification Expand

The Introduced DNA

Data from Southern hybridisation analysis and DNA sequencing demonstrate that single copies of both the mcry3A and pmi genes are present in MIR604. Additionally, MIR604 does not contain any of the backbone sequences from the transformation plasmid pZM26. Sequence analysis of the entire T-DNA insert present in MIR604 confirms that the overall integrity of the intended insert and of the functional elements have been maintained. A 43 base-pair truncation at the right border junction of the T-DNA insert and 44 base-pair truncation at the left border junction of the T-DNA insert were identified. Three single nucleotide changes were also identified relative to the intended DNA sequence. One of these changes occurred within a promoter, a regulatory region that does not encode a protein. The remaining two changes occurred within the pmi coding sequence and resulted in the substitution of (1) alanine in place of valine-61 and (2) histidine in place of glutamine-210 in the amino acid sequence of the PMI protein. These substitutions have not resulted in any apparent functional change in PMI as expressed in MIR604.

Genetic Stability of the Introduced Trait

The hybridization pattern over three generations of MIR604 (BC4, BC5 and BC6) was identical, which demonstrates that the TDNA insert from pZM26 incorporated into MIR604 is stable over several generations. Furthermore, levels of mCry3A and PMI proteins were determined to be stable in MIR604 plants over four successive backcross generations.

Homozygous plants from the T3 generation were crossed to a non-transgenic inbred to yield the T4 generation, which were selfed in the field and seed was collected in bulk, creating the T5 seed. Individual T5 plants were assayed for the presence of the trait by immuno-detection (ELISA) of mCry3A, as well as by TaqMan® PCR for both mcry3A and pmi genes. The expected Mendelian inheritance ratio of positive and negative plants for a hemizygous trait in these populations is 3:1 and statistical analysis confirmed the expected Mendelian inheritance ratio for both genes.

Expressed Material

To characterize the range of expression of the mCry3A and PMI proteins in maize (corn) plants derived from MIR604, the concentrations of mCry3A protein and PMI were determined by ELISA in several plant tissues and whole plants at four growth stages (whorl, anthesis, seed maturity and senescence) in two field maize hybrids and one maize inbred.

Quantifiable levels of mCry3A protein were detected in all MIR604-derived plant tissues analyzed except pollen. Across all growth stages, mean mCry3A levels measured in leaves, roots and whole plants ranged from ca. 3 - 23 µg/g fresh wt, ca. 2 - 14 µg/g fresh wt and ca. 0.9 - 11 µg/g fresh wt, respectively. Mean mCry3A levels measured in kernels from the MIR604 hybrids at senescence (corresponding to the stage closest to grain harvest) were ca. 0.7 µg/g fresh wt. The levels of mCry3A were generally similar between hybrids for each tissue type at each time point. For the inbred line, mCry3A expression was generally higher than in the hybrids in leaves, roots and whole plants at whorl and anthesis stages and in roots at seed maturity.

PMI protein was detected in most of the MIR604-derived plant tissues analyzed, albeit at low levels. Across all plant stages, mean PMI levels measured in leaves, roots and whole plants ranged from not detectable (ND) to ca. 0.4 µg/g fresh wt., below the LOQ (ca. 0.2 µg/g fresh wt. and below the LOQ (ca 0.3 µg/g fresh wt, respectively. Mean PMI levels measured in kernels from the MIR604 hybrids at senescence (corresponding to the stage closest to grain harvest) were ca. 1.9 – 2.6 µg/g fresh wt. The levels of PMI were generally similar among the inbred and hybrid genotypes for each tissue type at each time point. PMI was not detectable in silage at all three sampling times (day 15, 29 and 75). By comparison, the level of PMI measured in the chopped plant material prior to ensiling was ca. 0.3 µg/g fresh wt.
 

Environmental Safety Considerations Expand

Field Testing

Filed trials were performed at a total of 32 locations in the United Stated during 2002 and 2003 with a number of different hybrids all containing the MIR604 event. As controls, non-transgenic segregants with the same genetic background were used at each site, as well as a range of conventional maize hybrids. For the majority of the traits assessed, there were no statistically significant differences and for the few traits where there was a significant difference, these differences were not consistent between sites over the two years of trials. The range of values for the agronomic characters, even when statistically significantly different from the controls, was within the range of values expected for maize hybrids and was thus not considered biologically significant.

MIR604 hybrids and their non-segregant controls were exposed to various corn pathogens and susceptibility was measured revealing no significant differences in disease susceptibility. Insect efficacy trials were conducted in field plots that were infested with naturally occurring populations of corn rootworm, with populations enhanced by use of trap crops the preceding season, or artificially infested with rootworm eggs.

The outcome of these studies indicated that, except for reduced damage to roots caused by corn rootworm, the agronomic performance of MIR604-derived hybrids was similar, and for most traits, equivalent to their nontransformed near isogenic counterparts. In contrast to the similarity in most agronomic parameters, the yield performance of MIR604-derived hybrids in the presence of corn rootworm pressure was significantly increased relative to corn hybrids lacking this trait.

Outcrossing

Since pollen production and viability were unchanged by the genetic modification resulting in MIR604, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between MIR604 maize and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In the United States, where there are no plant species closely-related to maize in the wild, the risk of gene flow to other species appears remote. Feral species in the United States related to corn cannot be pollinated due to differences in chromosome number, phenology (periodicity or timing of events within an organism’s life cycle as related to climate, e.g., flowering time) and habitat.

Maize (Zea mays ssp. mays) freely hybridizes with annual teosinte (Zea mays ssp. mexicana) when in close proximity. These wild maize relatives are native to Central America and are not present in the United States, except for special plantings. Tripsacum, another genus related to Zea, contains sixteen species, of which twelve are native to Mexico and Guatemala. Three species of Tripsacum have been reported in the continental United States: T. dactyloides, T. floridanum and T. lanceolatum. Of these, T. dactyloides, Eastern Gama Grass, is the only species of widespread occurrence and of any agricultural importance. It is commonly grown as a forage grass and has been the subject of some agronomic improvement (i.e., selection and classical breeding). T. floridanum is known from southern Florida and T. lanceolatum is present in the Mule Mountains of Arizona and possibly southern New Mexico. Even though some Tripsacum species occur in areas where maize is cultivated, gene introgression from maize under natural conditions is highly unlikely, if not impossible. Hybrids of Tripsacum species with Zea mays are difficult to obtain outside of the controlled conditions of laboratory and greenhouse. Seed obtained from such crosses are often sterile or progeny have greatly reduced fertility.

Weediness Potential

The introduced traits in MIR604 are not expected to cause MIR604 maize to become a weed. No competitive advantage was conferred to MIR604 that would render maize weedy or invasive of natural habitats, since none of the reproductive or growth characteristics were modified and no significant differences in these traits were noted in the field trials. Cultivated maize is unlikely to establish in non-cropped habitats and there have been no reports of maize surviving as a weed. Zea mays is not invasive and is a weak competitor with very limited seed dispersal. Volunteers can occur in fields, the year following cultivation, when maize is grown in rotation with other crops. Although maize volunteers are a minor weed problem, these can cause harvesting problems.

Secondary and Non-Target Adverse Effects

Three sources of data were used to evaluate the environmental safety of the modified Cry3A (mCry3A) Bacillus thuringiensis-related insect control protein expressed in MIR604. First, experimental data was used to establish how the spectrum of activity of mCry3A differs from native Cry3A. These data suggest that the toxicity of mCry3A differs from native Cry3A only in the intended increase in activity against certain pest species in the genus Diabrotica (Coleoptera: Chrysomelidae). Secondly, data on the spectrum of activity of native Cry3A was reviewed to provide evidence about the likely spectrum of activity (i.e., the hazard) of mCry3A. As the previous studies showed that mCry3A and native Cry3A differ only in the intended increase of activity of mCry3A to certain Diabrotica species, the toxicity of native Cry3A is a good predictor of the hazard of mCry3A to non-target organisms (NTOs).

From the native Cry3A data it was predicted that mCry3A is unlikely to be hazardous except to certain species in 3 families of Coleoptera: the Chrysomelidae (leaf beetles, flea beetles and rootworms), the Curculionidae (weevils and snout beetles) and the Tenebrionidae (darkling beetles). The third source of data which was then used to test this hypothesis, was single-species laboratory studies exposing representative NTOs to concentrations of mCry3A in excess of the expected environmental concentrations of mCry3A resulting from the proposed cultivation of MIR604. Test species were chosen to represent taxa that might be exposed to mCry3A from tissues of MIR604, or taxa related to the western and northern corn rootworm. In addition, test species were selected to include functional groups found in agricultural fields and other habitats into which mCry3A might spread: birds, freshwater fish, predators and parasitoids of crop pests, soil invertebrates and pollinators.

Impact on Biodiversity

Analysis concluded that MIR604 maize exhibited no traits that would cause increased weediness and that cultivation would not lead to increased weediness of other maize or sexually compatible species, nor would cultivation be likely to harm non-target organisms. Based on this conclusion, there was no apparent potential for a significant impact on biodiversity.
 

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 MIR604 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.

Equivalence of Bacterial Expressed Proteins

As it is very difficult to extract and purify sufficient quantities of the subject protein from transgenic corn plants for laboratory studies, it has become standard practice to instead use equivalent proteins that have been produced using bacterial expression systems.  mCry3A and PMI were produced in recombinant E. coli, purified and used in laboratory toxicity and allergenicity studies.

Prior to use in these laboratory studies, the bacterially produced proteins are compared to the proteins produced in planta in order to establish their equivalence. The molecular identity and biochemical characteristics of the proteins expressed in planta and in the bacterial-expression systems were examined by analysis of biochemical and functional parameters, including molecular weight determination, immunoreactivity, glycosylation analysis and biological activity.  SDS-PAGE and Western blotting confirmed the expected molecular weight of mCry3A to be approximately 67,700 Daltons. In the plant derived protein sample, a second band was present and was thought to represent mCry3A that had been degraded in the plant cell.  PMI was confirmed to be approximately 45,000 Daltons.  No glycosylation was observed for either protein.

The insecticidal activity of plant-derived mCry3A was similar to the bacterially produced protein and the enzyme activity of plant-derived PMI was equivalent to that of the bacterially produced enzyme.  The two amino acid changes in the plant expressed PMI do not affect the function of this enzyme.

These studies established that bacterially produced mCry3A and PMI are equivalent to those proteins produced in corn line MIR604, thus support the use of the bacterial proteins in the toxicity testing.

Toxicity

The mCry3A protein extracted and purified from the recombinant E. coli was used as the test material for an acute oral toxicity study with mice.  The mice received a single dose of 2377 mg/kg bw mCry3A and were observed for two weeks.  Parameters evaluated included body weights, food consumption and detailed clinical observations. At the end of the study all animals were killed and examined post mortem.  Brain, liver, kidneys and spleen were weighed and selected tissues were taken for histopathological examination.

One female mouse was killed on day 2 of the study due to clinical signs consistent with a dosing injury and not related to the test substance.  No test substance-related mortalities occurred.  There were no test substance-related effects on body weight, food consumption, organ weights or macroscopic and microscopic pathology.  Therefore, under the conditions of this study, the acute oral LD50 of mcry3A in mice is greater than 2377 mg/kg bw, the highest dose tested

For PMI, the mice received a single dose of 3080 mg/kg bw of the bacterially expressed protein and were observed for two weeks.  Parameters evaluated included body weights and detailed clinical observations. At the end of the study all animals were killed and examined post mortem.  Brain, liver, kidneys and spleen were weighed.

One male in the control group and two in the test group died shortly after dosing or were in distress after dosing and subsequently died.  Necropsy revealed perforated oesophagi in these animals, a sign of gavage error and not test-substance related.  One replacement animal was available for each group and dosed in the same manner on day 0.  There was no test article related mortality during the study.  No clinical signs of toxicity were observed in either group.  There were no test-substance related effects on body weight, organ weights or gross pathology.  Under the conditions of this study, the acute oral LD50 of the PMI protein in mice is greater than 3080 mg /kg bw.

To determine whether mCry3A has any significant homology with known protein toxins, its amino acid sequence was systematically compared to the latest posting of the National Centre for Biotechnology Information Entrez Protein Database containing all the publicly available protein sequences.  The appropriate cut-off expectation (E) value was determined to be 0.38 and amino acid sequences with E values lower than this were considered to be significant.

Two hundred and twenty three entries in GenBank returned E values below 0.38.  Most of these (216) were identified as known or putative delta-endotoxins  with a similar function to mCry3A.  None of other entries were identified as known or putative toxins other than delta-endotoxins.

To determine whether the PMI protein sequence has any significant homology with known protein toxins, it was compared to known protein sequences in a similar manner.  The sequence used in this study did not take into account the two amino acid changes present in PMI in MIR604.  The appropriate cut-off expectation (E) value was determined to be 0.17 and amino acid sequences with E values lower than this were considered to be significant.

One hundred and thirty three protein entries in GenBank returned E values below 0.17. One hundred and fourteen of these were identified as known or putative PMI proteins.  Sixteen entries were hypothetical proteins and 3 were unnamed proteins.  No association with known or putative toxins was described for these entries.

Allergenicity

Current scientific knowledge indicates that many food allergens tend to be abundant proteins within the food product, are resistant to proteolytic digestion and heat inactivation and are often glycosylated.  The mCry3A and PMI proteins were assessed for allergenicity by examining their digestibility, glycosylation profile, heat stability and amino acid sequence homology to known allergens.

The results of an in vitro digestive fate study using simulated gastric fluid (SGF) indicated mCry3A protein is rapidly degraded by gastric fluid. In a solution of simulated gastric fluid, 1 mg/mL mCry3A test protein mixed with simulated gastric fluid (pH 1.2, containing 2 mg/mL NaCl, 14 mL 6 N HCl, and 2.7 mg/mL pepsin) resulting in 10 pepsin activity units/ mg test protein (complies with 2000 US Pharmacopoeia recommendations), complete degradation of detectable mCry3A protein occurred within 2 minutes, determined by both SDS-PAGE and western blot analysis.

PMI was digested in simulated gastric fluid containing pepsin and in simulated intestinal fluid containing pancreatin.  Samples were examined by SDS-PAGE.  PMI was degraded rapidly by pepsin: no PMI was detected by SDS-PAGE upon immediate sampling of the reaction mix (0 seconds).  When the pepsin was diluted to 0.0001X of the standard concentration, no PMI remained after 10 minutes of incubation.  Similarly, no PMI enzymatic activity was detectable after 10 minutes under these conditions.  PMI was degraded by pancreatin in simulated intestinal fluid after two minutes.  In the unlikely event that PMI survived digestion by pepsin, it would be digested in the mammalian intestinal environment by pancreatin.  This indicates that PMI is not stable to digestion and is unlikely to be a food allergen

mCry3A was assessed for glycosylation by DIG Glycan analysis.  The limit of detection was 2.5 ng. mCry3A samples of 1000 ng were tested and neither the E. coli-expressed mCry3A nor the corn-expressed mCry3A were found to be glycosylated.  The PMI amino acid sequence contains no consensus sequences for N-glycosylation, although O-glycosylation could theoretically occur.  Mass spectrometric analysis of human PMI indicates that this protein is not post-translationally modified

The effect of temperature on mCry3A was determined by incubation for 30 minutes at a range of temperatures (4ºC, 25ºC, 37ºC, 65ºC, and 95ºC) followed by a bioassay against Western corn rootworm larvae.  At 95ºC mcry3A was completely inactivated.  Some reduction in activity was observed after incubation at 65ºC and temperatures of 4ºC, 25ºC and 37ºC had no effect on mCry3A bioactivity.

The heat stability of PMI was evaluated in a similar manner.  Loss of enzyme activity was used to determine the instability of the protein after exposure to various temperatures (25, 37, 55, 65 and 95ºC) for 30 minutes.  Incubation at ambient temperature (25ºC), 37ºC or 55ºC for 30 minutes had little effect on enzyme activity.  Incubation at 65ºC and 95ºC essentially inactivated the protein.

To determine whether mCry3A has any significant homology with allergenic proteins, the protein sequence was systematically compared to a database of allergen sequences.  This database was compiled from entries identified as allergens or putative allergens in public protein databases, and was supplemented with additional amino acid sequences identified from the scientific literature.  Overall similarity was examined by comparing sequential 80-amino acid sequences covering the entire mcry3A protein sequence (such that each 80-amino acid window was offset from the previous one by one residue and overlapped by 79 residues) to the allergen sequences using the FASTA search algorithm. Any 80-amino acid peptide having greater than 35% amino acid identity was defined as having significant similarity to the allergen sequence.  The mCry3A sequence was also screened for matches of eight or more contiguous amino acids.  The purpose of this is to identify any short local regions of identity that might indicate the presence of common IgE binding epitopes.

No significant sequence homology was found between any of the sequential mCry3A 80-amino acid peptides and any entries in the database.  No alignments of eight or more contiguous identical amino acids between mcry3A and any of the proteins in the database were identified.

The PMI protein sequence was compared to allergenic proteins in a similar manner and there was no significant similarity between any of the sequential PMI 80-amino acid peptides and any entries in the database.  There was one region of eight identical amino acids between PMI and the known allergen Α-parvalbumin from Rana species CH2001.  One case of severe food-induced anaphylaxis in a single individual who consumed Indonesian frogs legs has been shown to be due to the protein Α-parvalbumin from Rana species.  To determine if the IgE antibodies in this patient’s serum recognized PMI, a PMI protein sample was tested for cross-reactivity.  

The PMI sample was bacterially derived and did not contain the two amino acid changes present in MIR604 PMI, however, the region of identity between PMI and Α-parvalbumin was not affected by this change and therefore the use of bacterially derived PMI would not affect the outcome of this test.  No cross reactivity between the human serum IgE and PMI occurred.  This indicates that the allergic patient’s serum IgE does not recognise any portion of the PMI protein as an allergenic epitope.  Therefore, the similarity between PMI and Rana species Α-parvalbumin is not considered to be biologically relevant.

In summary, no significant homology was found between the novel proteins and known protein allergens.  In vitro digestibility studies indicate that the novel proteins are quickly digested.  Glycosylation and thermolability studies did not indicate a cause for concern.  It is unlikely that either mCry3A or PMI have any allergenic potential.

Nutritional and Compositional Data
Grain and forage from transgenic event MIR604 derived plants and their near-isogenic, non-transgenic control were grown at 13 different locations over a two year period, with three replicate plots of each genotype planted in randomized complete blocks. Compositional data were statistically analyzed using a randomized block design with locations serving as the blocks. Statistical significance was assigned at p<0.05 indicating that the difference between the treatments was statistically different at the 5% customary level. The treatment-location interaction was also assessed.  Values for each analyte from published literature  were provided to assess whether statistically significant differences in the composition of the test and control maize are biologically meaningful.

Forage samples were analyzed for proximates and the minerals calcium and phosphorus.  Of all the analytes measured, only the moisture level was different in MIR604 when compared with its near-isogenic non-transgenic control. However, moisture is a function of maturity and conditions at harvest and this difference was not considered meaningful. All other values measured for the analytes fell within published literature ranges.

The levels of various nutritional components were determined from mature grain, including: proximates (crude protein, crude fat, moisture, ash, and carbohydrate, acid detergent fibre, neutral detergent fibre, total dietary fibre); minerals (calcium, phosphorus, magnesium, copper, iron, manganese, potassium, sodium, selelnium, chromium and zinc); amino acids; fatty acids; vitamins (beta carotene, cryptoxanthin, folic acid, B1, B2, B3, B5, B6, C and tocopherols); anti-nutrients (phytic acid, trypsin inhibitor); and secondary metabolites (raffinose, furfural, ferulic acid, p-coumaric acid and inositol).  

The MIR604 hybrids had statistically significantly higher levels of protein and total fat, and statistically significantly lower levels of starch and total dietary fibre than the near-isogenic non-transgenic control corn. MIR604 hybrids also had statistically significantly higher levels of the amino acids asparagine, threonine, serine, glutamate, alanine, valine, methionine, isoleucine, leucine, tyrosine, and phenylalanine than did the near-isogenic non-transgenic control corn, whereas, the control corn showed statistically higher cystine levels than did the MIR604 corn. However, mean values for all proximates and amino acids, measured for grain from MIR604 and non-isogenic non-transgenic controls were within published literature ranges and it was concluded that the differences in composition between MIR604 and control lines are not biologically significant.

Statistically significantly higher levels of palmitic, oleic, and linoleic acids and statistically significantly lower levels of linolenic acid in MIR604 compared to the near-isogenic non-transgenic control were noted. MIR604 also had statistically significantly higher levels of calcium and phosphorus than did their near-isogenic non-transgenic control corn. Once again, mean values for these were within published ranges and the differences in composition between MIR604 and control lines are not considered to be biologically significant.

Vitamin levels in both MIR604 hybrids and the near-isogenic non-transgenic controls were found to be within published literature ranges.  The values for phytic acid were at or below literature values for both MIR604 and the near-isogenic non-trangenic control corn. The values for the other anti-nutrient, trypsin-inhibitor, and all of the secondary metabolites for both the MIR604 hybrids and the near-isogenic non-trangenic control corn were within published literature ranges.

The results of these compositional analyses led to the conclusion that MIR604 forage and grain is 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. Refined maize products are also used in bioproducts such as antibiotics.

Corn rootworm (Diabrotica spp.) is considered one of the most damaging insects pests of maize. The species of corn rootworm most prevalent in the United States are the northern corn rootworm (Diabrotica barberi) and the western corn rootworm (D. virgifera). The larvae of these beetles (coleopterans) feed on the corn roots. Feeding damage to the roots impedes the absorption of water and nutrients. Corn rootworms also feed on the brace roots and cause plant lodging. Adults feed on the silks thus interfering with pollination and seed set. Crop rotation is a recommended practice to reduce the population of these insects; thus, corn should not follow corn in a rotation. The protection offered by insecticides is limited: these will protect the crop from rootworm damage, but will only reduce a small percentage of the beetles from emerging.

The transgenic maize line MIR604 was genetically engineered to resist the western corn rootworm (D. virgifera virgifera), northern corn rootworm (D. barberi) and the Mexican corn rootworm (D. virgifera zeae) by producing an insecticidal protein. MIR604 contains two novel genes, a modified cry3A (mcry3A) gene encoding the mCry3A insecticidal protein and the pmi (manA) gene from Escherichia coli encoding the enzyme phosphomannose isomerase as a selectable marker.

The mcry3A gene encodes for a protein with a similar amino acid sequence to the Cry3Aa2 protein from Bacillus thuringiensis (Bt) subspecies tenebrionsis, with the inclusion of a protease cleavage site which results in a greater toxicity to certain species of corn rootworm. Cry proteins, of which mCry3A is only one, act by selectively binding to specific sites localized on the lining of the midgut of susceptible insect species. Following binding, pores are formed that disrupt midgut ion flow, causing gut paralysis and eventual death due to bacterial sepsis. mCry3A is lethal only when eaten by the larvae of coleopteran insects (i.e. , beetles) and exhibits no toxicity to other corn pests or non-target insects. There are no binding sites for the delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins. The phosphomannose isomerase protein (encoded by the pmi gene) was used as a selectable marker as plants expressing this gene can utilise mannose as a primary carbon source, whereas cells lacking this gene fail to proliferate on mannose-based medium.

The food and livestock feed safety of MIR604 maize grain and forage was established based on several standard criteria, including the potential toxicity and allergenicity of the two proteins, mCry3A and phosphomannose isomerase, which was determined to be negligible. As part of the safety assessment, the nutritional composition of MIR604 grain and forage was found to be equivalent to conventional maize as shown by the analyses of key nutrients including proximates, fatty acid profiles, minerals and vitamins, as well as anti-nutrient compounds.

Field trials with MIR604 were conducted during 2002 and 2003 with few significant differences between MIR604 and non-transgenic controls. None of these differences were outside the range of values expected for maize hybrids. Disease susceptibility was not different to existing maize hybrids, neither was there any difference in resistance to pests, except corn rootworms. It was thus determined that there would be little impact on potential weediness of MIR604 and commercial cultivation of MIR604 is unlikely to harm non-target organisms.

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Canadian Food Inspection Agency, Plant Biotechnology Office European Food Safety Authority Food Standards Australia New Zealand Health Canada Novel Foods Instituto Colombiano Agropecuario (ICA) Japanese Biosafety Clearing House, Ministry of Environment U.S. Department of Agriculture, Animal and Plant Health Inspection Service U.S. Environmental Protection Agency, FIFRA Scientific Advisory Panel Report U.S. Environmental Protection Agency, Office of Pesticide Programs United States Food and Drug Administration

This record was last modified on Monday, August 7, 2017