Maize line NK603 was developed to allow the use of glyphosate containing herbicides as a weed control option for maize crops. The gene encoding a glyphosate tolerant form of the enzyme 5-enolpyruvlyshikimate-3-phosphate synthase (EPSPS) was isolated from the soil bacterium Agrobacterium tumefaciens strain CP4 and introduced into maize using recombinant DNA techniques. Glyphosate specifically binds to and inactivates EPSPS, which is involved in the biosynthesis of the aromatic amino acids tyrosine, phenylalanine and tryptophan. This enzyme is present in all plants, bacteria and fungi, but not in animals, which do not synthesize their own aromatic amino acids. Thus, EPSPS is normally present in food derived from plant and microbial sources.

For environmental release in the United States, another glyphosate tolerant maize line, GA21, was designated as the antecedent organism for NK603. Maize line NK603 and the antecedent organism GA21 were genetically engineered using the same transformation method and contain a functionally equivalent enzyme that makes the plants tolerant to the herbicide glyphosate.

Maize line NK603 was produced by biolistic transformation of the inbred maize line LH82xB73 with a 6706 bp DNA fragment containing two adjacent EPSPS expression cassettes. Each cassette contained a single copy of the CP4 EPSPS gene and respective regulatory sequences. In the first cassette, expression of the CP4 EPSPS gene was regulated using the rice actin 1 promoter and associated intron, and the 3’ non-translated region of the nopaline synthase gene from Agrobacterium tumefaciens (3’ NOS). Expression of the CP4 EPSPS gene within the second cassette was controlled by the duplicated, enhanced, 35S promoter from cauliflower mosaic virus (E35S) and the heat shock protein 70 intron from maize (ZmHSP70), and the 3’ NOS transcriptional termination signal. In both cases, post-translational translocation of the CP4 EPSPS protein to the chloroplast was directed by inclusion of the chloroplast transit peptide (CTP2; isolated from Arabidopsis thaliana EPSPS) signal sequence at the 5’ terminus of the CP4 EPSPS coding sequence.

The DNA segment used for transformation (6706 bp) was isolated as a single band following agarose gel electrophoresis of restriction enzyme digested PV-ZMGT32 plasmid DNA. The purified DNA fragment did not contain antibiotic resistance marker genes, bacterial origin of replication sequences, or any plasmid derived sequences other than those described above. Transformed plant cells were selected and regenerated in tissue culture in the presence of glyphosate.

The Introduced DNA
The incorporated DNA was characterized using a combination of Southern blot analyses, polymerase chain reaction (PCR) amplification of specific sequences, and nucleotide sequencing of the entire inserted fragment, including flanking sequences from the host genome. In addition, bioinformatics analyses were conducted on the inserted sequence, including host genome junction regions, to demonstrate the lack of any unforeseen, or chimeric, open reading frames (ORFs) that could potentially result in the expression of unanticipated novel proteins.

These analyses demonstrated the introduction of single copy of the transforming DNA at a single insertion site within the host genome. Some anomalies were, however, observed:

1. In addition to a complete copy of the introduced DNA, the insert also included a 217 bp fragment (50 bp of polylinker sequence + 167 bp of the enhancer region of the rice actin promoter) inversely linked to the 3’ terminus of the introduced DNA.
2. Nucleotide sequencing indicated that the sequence of the CP4 EPSPS gene within the second (3’ proximal) cassette differed by two nucleotides from the inserted sequence. This gave rise to a single amino acid substitution at position 214 of the expressed protein (leucine --> proline; variant protein referred to as: CP4 EPSPS L214P).
3. Analyses of specific PCR products from the 3’ terminus of the inserted DNA revealed that an additional segment comprising 305 bp of chloroplast DNA had been co-integrated. Bioinformatics analysis indicated that this sequence corresponded to a portion of the maize DNA-directed RNA polymerase alpha-subunit and ribosomal S11 protein. The source of this DNA was believed to be the chloroplast of the transformed cell.

Genetic Stability of the Introduced Trait
Southern blot analyses of genomic DNA isolated from plants over six generations of crossing and three generations of self-pollination were used to confirm that the introduced DNA was stably inherited and segregated as a single locus according to Mendelian genetics. Multigenerational stable expression of the glyphosate tolerant trait was demonstrated using bioassay (tolerance to glyphosate application) and enzyme linked immunosorbent assay (ELISA) to measure CP4 EPSPS protein concentration.

Expressed Material
Expression of full-length (approx. 47 kDa) CP4 EPSPS protein was confirmed by Western immunoblot analysis and protein concentrations were estimated using ELISA. Plant samples from line NK603 and the non-transformed parental control line were collected from six non-replicated and two replicated field sites during the 1998 growing season and assayed using a double antibody sandwich (DAS)-ELISA employing monoclonal anti-CP4 EPSPS antibody as the capture antibody and a horseradish peroxidase-conjugated anti-CP4 EPSPS polyclonal antibody as the detection reagent. Mean CP4 EPSPS protein levels (across all sites) were 25.6 µg/g (fwt; range 18.0 – 31.2) and 10.9 µg/g (fwt; range 6.9 – 15.6) for forage and grain tissue, respectively.

Outcrossing
Pollen production and viability were unchanged in line NK603 and, therefore, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between NK603 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. None of the sexually compatible relatives of maize in Canada or the United States are considered to be weeds in these countries and it is therefore unlikely that introgression of the CP4 EPSPS gene would provide a selective advantage to these populations as they would not be routinely subject to herbicide treatments.

Weediness Potential
No competitive advantage was conferred to NK603, other than that conferred by resistance to glyphosate herbicide. Resistance to glyphosate containing herbicides 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 that are not based on glyphosate as appropriate. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects
No environmentally toxic components were detected in NK603. CP4 EPSPS protein is not a known toxin and analogous proteins are found in all plants and microorganisms. There are no anticipated adverse effects of NK603 on non-target organisms that would be different from conventional maize varieties.

Impact on Biodiversity
Maize line NK603 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 NK603 to species in unmanaged environments was not a significant concern.

Other Considerations
Consideration was made as to whether the introduction of crops tolerant to glyphosate would result in a significant increase in the use of the herbicide, and lead to the evolution of glyphosate resistant weeds. It was determined that the risk of increasing the selection of glyphosate tolerant weeds was low and could be mitigated through the use of other approved herbicides with a mode of action dissimilar to glyphosate.

Nutritional Data
Compositional analyses were performed on grain and forage samples of NK603 (treated with glyphosate) and the non-transformed parental control line together with a number of other commercial maize hybrids planted at trial sites in the U.S. and Europe. Analyses of grain samples included measurements of proximates (protein, fat, ash, moisture), acid detergent fibre (ADF), neutral detergent fibre (NDF), amino acids, fatty acids, vitamin E, minerals (calcium, copper, iron, magnesium, manganese, phosphorus, potassium, sodium, and zinc), and the antinutritional components, phytic acid and trypsin inhibitor.

In all of these tests, small statistical differences between NK603 and control lines were observed only in: six amino acids (alanine, arginine, glutamic acid, histidine, lysine, and methionine) as measured in grain from European trials (no differences were observed in material from U.S. trials); and stearic (C18:0) acid levels. Overall, these differences were not consistent across all trial sites and they were considered to reflect random variation. All compositional results were within the ranges observed for commercial non-transformed lines.

The nutritional quality of NK603 grain was assessed in feeding trials with broiler chickens, finisher swine, and laboratory rats. These studies showed that there were no differences between the transformed and non-transformed maize.

Toxicity
The CP4 EPSPS gene encodes a single polypeptide of 455 amino acids (47.6 kDa) which exhibits about 50% amino acid sequence similarity with the analogous plant EPSPS enzyme. The family of bacterial and plant EPSPS proteins are not known to display any toxic or allergenic properties. The potential toxicity of the CP4 EPSPS protein was assessed by comparing its amino acid sequence against a database of 4,677 protein sequences (not all unique) that have been associated with toxicity, and in an acute oral toxicity study in mice. The CP4 EPSPS protein did not display any sequence homology with known protein toxins and did not result in any adverse effects on test animals (50 males, 50 females) receiving doses up to 400 mg/kg of bacterially derived CP4 EPSPS protein. The single amino acid substitution within the CP4 EPSPS L214P protein did not alter the sequence comparison results.

Allergenicity
The CP4 EPSPS encoding gene was not derived from an organism known to cause allergic reactions and the allergenic potential of this protein was further evaluated by comparing its amino acid sequence against a database of known allergens, and by assessing its stability to digestion in the presence of simulated gastric fluids. There was no sequence homology between CP4 EPSPS and known allergens when checked against a database of 567 protein sequences using an 8-amino acid length window. As assessed by Western immunoblot analysis, the CP4 EPSPS was rapidly degraded (T50 < 15 sec) upon exposure to pepsin-containing simulated gastric fluid or trypsin-containing simulated intestinal fluid (T50 <= 10 min). Similar results were obtained with the variant CP4 EPSPS L214P protein.

Final risk assessment report: glyphosate-tolerant corn line NK603

Decision Document DD2002-35 Determination of the Safety of Monsanto Canada Inc.’s Roundup ReadyTM Corn (Zea mays L.) Line 603

Risk Assessment of Herbicide Tolerant Maize (NK603)

COMMISSION DECISION of 3 March 2005 authorising the placing on the market of foods and food ingredients derived from genetically modified maize line NK 603 as novel foods or novel food ingredients under Regulation (EC) No 258/97 of the European Parliament and of the Council

Notification of the placing on the Community Register of MON-ØØ6Ø3-6.

Opinion of the Scientific Panel on Genetically Modified Organisms on a request from the Commission related to the safety of foods and food ingredients derived from herbicide-tolerant genetically modified maize NK603, for which a request for placing on the market was submitted under Article 4 of the Novel Food Regulation (EC) No 258/97 by Monsanto (QUESTION NO EFSA-Q-2003-002). Opinion adopted on 25 November 2003

Novel food decision summary for glyphosate-tolerant maize line NK603

Outline of the biological diversity risk assessment report: Type 1 use approval for NK603

Product safety description

Determination of the Safety of Monsanto’s Corn NK 603 (Glyphosate-Tolerant Corn) for Direct Use as Food, Feed and for Processing and for Propagation

Monsanto Co. Request for Extension of Determination of Nonregulated Status to the Additional Regulated Article: Roundup Ready Corn Line NK603

Letter to Monsanto regarding NK603

Memorandum to file concerning glyphosate-tolerant maize link NK603.