GM Crop Database

Database Product Description

Event 98140 (DP-Ø9814Ø-6)
Host Organism
Zea mays (Maize)
Trade Name
Optimum™ GAT™
Trait
Tolerance to glyphosate and ALS-inhibiting herbicides
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
DuPont Pioneer

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Argentina 2011 2011 2011
Australia 2010
Canada 2009 2009 2009
Korea 2010 2010
Mexico 2008 2008
United States 2008 2008 2009

Introduction Expand

Corn line 98140 was genetically engineered to express the GAT4621 (glyphosate acetyltransferase) and ZM-HRA (modified version of a maize acetolactate synthase) proteins. The GAT4621 protein, encoded by the gat4621 gene, confers tolerance to glyphosate-containing herbicides by acetylating glyphosate and thereby rendering it non-phytotoxic. The ZM-HRA protein, encoded by the zm-hra gene, confers tolerance to the ALS-inhibiting class of herbicides.

The gat4621 gene is based on the sequences of three gat genes from the common soil bacterium Bacillus licheniformis. B. licheniformis is widespread in the environment; therefore, animals and humans are regularly exposed without adverse consequences to this organism and its components, such as the glyphosate acetyltransferase (GAT) protein. GAT proteins are members of the GCN 5-related family of N-acetyltransferases (also known as the GNAT family). The GNAT superfamily is one of the largest enzyme superfamilies recognized to date with over 10,000 representatives from plants, animals and microbes. The GAT4621 protein is 75-78% identical and 90-91% similar at the amino acid level to each of the three native GAT enzymes from which it was derived. In 98140 corn, the expression of the gat4621 gene is driven by the maize ubiquitin promoter.

The ZM-HRA protein is a modified version of the maize acetolactate synthase (ALS) enzyme, which is involved in branched chain amino acid (leucine, isoleucine and valine) biosynthesis in the plastid. The herbicide tolerant zm-hra gene was made by isolating the herbicide sensitive maize als gene and introducing two specific amino acid changes known to confer herbicide tolerance to tobacco ALS. In 98140 corn, the expression of the zm-hra gene is driven by the maize ALS promoter.

The dual herbicide tolerance of 98140 corn will enable growers to choose an optimal combination of these herbicides to best manage their individual weed populations. The availability of 98140 corn will enable growers to proactively manage weed populations while delaying adverse population shifts of troublesome weeds or the development of resistance. Herbicide tolerant 98140 corn will be marketed in the U.S. under the brand name Optimum™ GAT™.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
gm-hra Acetolactate synthase HT

als (Z. mays)

CaMV 35S enhancer element

Solanum tuberosum proteinase inhibitor II (PINII)

1
gat Glyphosate N-acetyltransferase HT

ZmUbiInt (Zea mays polyubiquitin gene promoter and first intron).

CaMV 35S enhancer element

Solanum tuberosum proteinase inhibitor II (PINII)

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

Event 98140 was produced via Agrobacterium-mediated transformation of immature maize embryos using the plasmid vector PHP24279 (containing the gat4621 and zm-hra gene cassettes). Immature embryos of maize were aseptically removed from the developing caryopsis 9 - 10 days after pollination and infected with Agrobacterium tumefaciens strain LBA4404 containing plasmid PHP24279, essentially as described in Zhao et al., (2001). After 6 days of embryo and Agrobacterium co-cultivation on solid culture medium with no selection, the embryos were transferred to selective medium that was stimulatory to maize somatic embryogenesis containing glyphosate for selection of cells expressing the gat4621 transgene. The medium also contained carbenicillin to kill any remaining Agrobacterium.

After two weeks on the selective medium, healthy, growing calli that demonstrated resistance to glyphosate were identified. The putative transgenic calli were continually transferred to fresh selection medium for further growth until the start of the regeneration process. The presence of both transgenes was confirmed using PCR analysis, and regenerated whole transgenic plants (T0) were transferred to the greenhouse where they were evaluated for glyphosate and ALS-inhibitor herbicide tolerance.

Characteristics of the Modification Expand

The Introduced DNA

The inserted DNA in event 98140 was characterized via Southern hybridization analysis using hybridization probes derived from plasmid PHP24279. The T-DNA from plasmid PHP24279 contains two expression cassettes: the gat4621 cassette comprised of the ubiZM1 promoter, ubiZM1 intron, the gat4621 gene, and the pinII terminator. The zm-hra cassette is comprised of the maize als promoter, zm-hra gene, and pinII terminator. Located between the two cassettes are three copies of the CaMV 35S enhancer element, providing transcription enhancement to both cassettes. Individual plants of the T0S3 generation were analyzed to determine the copy number of each of the genetic elements inserted into event 98140 and to verify that the integrity of the PHP24279 T-DNA was maintained upon integration. The analysis confirmed that event 98140 contained a single, intact, copy of the PHP24279 T-DNA. Southern blot analysis was also used to confirm the absence of plasmid backbone sequences within the event 98140 genome.

Genetic Stability of the Trait

The stability of the gat4621 and zm-hra genes across four generations of event 98140 maize (T0S3, BC0S2, BC1, and BC1S1) was determined from Southern hybridization analysis of EcoRV digests of genomic DNA.

Chi-square analysis of trait inheritance data from four different generations (BC0S1, BC1S1, BC2 and BC3) was also performed to determine the heritability and stability of the gat4621 and zm-hra genes in 98140 maize. In order to confirm the expected segregation ratios, polymerase chain reaction (PCR) analysis was performed on leaf punches from seedlings. Qualitative PCR analysis for the gat4621 and zm-hra genes was conducted on all plants. The results of this analysis were consistent with the finding of a single locus of insertion of the gat4621 and zm-hra genes that segregates in 98140 progeny according to Mendel’s laws of genetics. In every case, plants that were positive for the gat4621 gene were also positive for the zm-hra gene and vice versa, confirming co-segregation of the two genes as expected.

Expressed Material

Concentrations of the GAT4621 and ZM-HRA proteins in event 98140 were measured in tissue samples collected from a replicated field study grown at six field locations in North America in 2006. Tissue samples were collected at various developmental growth stages, including V9 (the stage just after glyphosate application), R1 (pollen shed), R4 (forage harvest stage), and R6 (grain harvest stage). Samples of leaf tissue (V9, R1, R4 and R6), root tissue (R1, R4, and R6), pollen (R1), forage (R4), grain (R6), and whole-plant samples (R1 and R6), were tested for GAT4621 and ZM-HRA proteins using quantitative enzyme linked immunosorbent assay (ELISA).

Mean concentrations of GAT4621 protein in various tissues of 98140 maize throughout the growing season ranged from 2.6 ng/mg tissue on a dry weight basis (root at the R6 stage) to 51 ng/mg (leaf at the R1 stage). Mean concentrations of ZM-HRA protein in various tissues of 98140 corn throughout the growing season ranged from below the limit of quantification (pollen at the R1 stage) to 6.7 ng/mg on a dry weight basis (leaf at the V9 stage). As expected, all GAT4621 and ZM-HRA protein concentrations were below the limit of quantification in control corn tissues.

Environmental Safety Considerations Expand

Field Trial Evaluations

Agronomic data were collected in a series of three experiments (A, B, and C) from confined field trials of 98140 and non-transgenic control maize conducted at up to 15 locations in 2006. The trial locations provided a range of environmental and agronomic conditions representative of the major corn growing regions in the U.S. and Canada, where commercial production of event 98140 is expected. Agronomic practices used to prepare and maintain each field site were characteristic of each respective region.

Experiment A: Comprised seven locations in the major corn growing regions of the Midwest during the 2006 growing season and measured the following parameters: time to silking, time to pollen shed, plant height, ear height, final population, percent moisture, test weight and yield. For all characteristics measured, no statistical differences in mean values were seen between 98140 and control maize across locations (adjusted P-value > 0.05).

Experiment B: Comprised nine locations in the major corn growing region of North America during the 2006 growing season and measured the following parameters: early population, final population, seedling vigor, time to silking, time to pollen shed, stalk lodging, root lodging, stay green, disease incidence, insect damage, yield, plant height and ear height. For all characteristics measured, no statistical differences in mean values were seen between 98140 and control maize across locations (adjusted P-value > 0.05).

Experiment C: Comprised six locations in the major corn growing regions of North America during the 2006 growing season and measured the following parameters: early population, final population, seedling vigor, time to silking, time to pollen shed, stalk lodging, root lodging, stay green, disease incidence, insect damage, plant height, ear height and pollen viability (shape & color) over time. For all characteristics measured, no statistical differences in mean values were seen between 98140 and control maize across locations (adjusted P-value > 0.05).

Ecological Observations

Ecological observations (plant interactions with insects and diseases) were recorded for all USDA-APHIS permitted field trials of 98140 maize during the 2005 and 2006 growing seasons. Plant breeders and field staff familiar with plant pathology and entomology observed event 98140 and control lines at least every four weeks for insect and disease pressure and recorded the severity of any stressor seen. In every case, the severity of insect or disease stress on event 98140 was not qualitatively different from various control lines growing at the same location. These results support the conclusion that the ecological interactions for event 98140 were comparable to control maize lines with similar genetics or to conventional maize lines.

Weediness Potential

Commercial maize varieties are not effective in invading established ecosystems (CFIA, 1994). Maize hybrids have been domesticated for such a long period of time that the seeds cannot be disseminated without human intervention, nor can corn seed readily survive in the U.S. and Canadian environments from one growing season to the next because of the poor dormancy. Any volunteer corn plants are easily identified and controlled through manual or chemical means.

There is little probability that event 98140 maize could become a problem weed. Various characteristics that might impart weediness potential were evaluated for 98140 and control maize in comparative studies, and no differences were seen in characteristics such as seed germination, emergence, seedling vigor, yield, and disease/insect susceptibility. Assessment of these data detected no biologically significant differences between event 98140 and control maize indicative of a selective advantage that would result in increased weediness potential. Furthermore, postharvest monitoring of field trial plots containing 98140 maize have shown no differences in survivability or persistence of event 98140 as compared to conventional maize.

Outcrossing

Maize is an open pollinated monoecious plant that produces abundant pollen. Maize pollen is among the largest and heaviest of the wind-dispersed pollen grains, thus limiting the distance it can travel. The potential transfer of traits in pollen to other corn plants is sometimes called pollen-mediated gene flow. There are many factors that affect pollen-mediated gene flow in maize including isolation distance between pollen source and recipient field, size of pollen source and recipient field, shape and orientation of pollen source and recipient field, wind direction and velocity, rain, temperature and humidity, pollen viability, silk receptivity, synchrony of flowering between source and recipient fields, pollen competition and physical barriers. Event 98140 is unchanged with respect to pollen characteristics.

Maize 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 or Canada, 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.

Potential Impact on Farming Practices

Cultivation and Management Practices: No negative impact is expected from the introduction of 98140 maize on current cultivation and management practices for maize. Event 98140 has been shown to be comparable to conventional maize in phenotypic, ecological and compositional characteristics. Event 98140 maize is expected to be similar in its agronomic characteristics and have the same levels of resistance to insects and diseases as other commercially available maize.

Weed Control: The commercialization of herbicide tolerant 98140 maize is expected to have a beneficial impact on weed control practices, as growers will have more herbicide options available to address their regional weed problems. Event 98140 maize will enable growers to choose an optimal combination of glyphosate, ALS-inhibiting herbicides, and other complementary herbicides to best manage their individual weed populations. Growers value the glyphosate-resistant crop trait and the utility of glyphosate. The availability of event 98140 maize will enable growers to proactively manage weed populations while delaying population shifts to troublesome weeds or the evolution of resistant weeds. Alternating herbicides with different modes of actions to control weeds generally is recommended to help delay the evolution of herbicide-resistant weeds. Therefore, incorporating tolerance to two herbicides with different modes of action in maize, as in event 98140, will be useful.

Potential Impact on Biodiversity

Event 98140 maize does not have an increased weediness potential, and unconfined cultivation of 98140 maize hybrids should not lead to increased weediness of other sexually compatible relatives, as non cultivated Zea mays species are not found in the United States or Canada. Therefore, it is unlikely to have effects on non-target organisms common to the agricultural ecosystem or threatened or endangered species, and there is no apparent potential for significant impact to biodiversity.

Food and/or Feed Safety Considerations Expand

Food and Feed Use

Maize grain and its processed fractions are consumed as human food and animal feed. The majority of maize is used as animal feed, and a small percentage is harvested as forage and made into silage and fed to ruminants. The remainder of maize is exported, processed into food products, or converted to ethanol. Maize can be processed by wet and dry milling processes to convert the grain into food, feed, and fuel products.

Compositional Analysis

The composition of forage and grain from event 98140 maize and a near isogenic, nontransgenic control maize was analyzed to assess whether there had been any unintentional compositional changes as a result of the genetic modification. Forage samples were analyzed for proximates (protein, fat, and ash), amino acids, free amino acids, acetylated amino acids [N-acetylaspartate (NAA) and N-acetylglutamate (NAG)], acid detergent fiber (ADF), neutral detergent fiber (NDF), calcium and phosphorus. Grain samples were analyzed for proximates, ADF, NDF, fatty acids, total amino acids, acetylated amino acids [NAA and NAG, N-acetylthreonine (NAThr), N-acetylserine (NASer), N-acetylglycine (NAGly)], free amino acids, minerals, vitamins, antinutrients, and secondary plant metabolites.

Forage Analysis: No statistically significant differences between event 98140 and the nontransgenic control samples were observed in the mean levels of proximates, minerals, carbohydrates, ADF, NDF, total and free amino acids. The mean levels were also within the 99% tolerance intervals and combined literature ranges. Literature values and statistical tolerance intervals were not available for the amino acid content of maize forage, however, most of the means for the specific amino acids fell within or close to the literature ranges for maize silage published by OECD (2002). As expected, the mean levels for NAA and NAG were statistically significantly higher (p < 0.05) for the 98140 samples compared to the non-transgenic control samples. No literature data were found regarding the level of the two acetylated amino acids in maize forage. In order to address the biological significance of the elevated levels of the two acetylated amino acids, their effect on the free amino acid pool was investigated. The free amino acids in maize forage were measured in both samples of nontransgenic control maize and samples of event 98140 maize, and no significant differences were detected in the mean levels of any of the free amino acids. NAG and NAA make up less than 1.2 % of the total amino acids in event 98140 forage samples and the protein levels and free amino acid pool were comparable to the non-transgenic control samples. The low levels of acetylation of aspartate and glutamate in the forage of event 98140 did not affect amino acid incorporation into proteins or the level of the free amino acid pool.

Grain Analysis: No statistically significant differences between event 98140 and the non- transgenic control maize grain samples were observed in the mean levels of protein, ash, NDF, ADF, minerals, vitamins, antinutrients (raffinose, phytic acid, and trypsin inhibitor), secondary plant metabolites (furfural, p-coumaric acid, and ferulic acid), and fatty acids. All mean levels were within the 99% tolerance intervals and the combined literature ranges. No statistically significant differences between the 98140 and the non-transgenic control samples were observed in the mean levels of total amino acids in the grain, with the exception of the mean level of tryptophan, which was slightly higher in 98140 samples compared to control samples but still within the 99% tolerance interval and/or literature range.

To clarify whether low levels of acetylation of amino acids affected the composition of the free amino acid pool in event 98140 grain, levels of individual free amino acids were measured. Two non-amino acid compounds (ethanolamine and ammonia) were measured along with the amino acids because they are recognized as analytes by the method used. There were no statistically significant differences in the mean levels of the free amino acids between 98140 and the non-transgenic control samples. Furthermore, all mean levels were within the 99% tolerance intervals; hence the free amino acid pools in event 98140 grain and non-transgenic control grain samples were comparable.

Levels of acetylated amino acids were also compared between event 98140 and non-transgenic control grain samples. The mean levels of NAA and NAG in 98140 grain samples were 0.0403 % dw and 0.0079 % dw, respectively, compared to 0.00009 % dw and 0.00005 % dw, respectively, for control samples. It was calculated that NAA and NAG make up less than 0.05 % dw of maize grain and less than 0.5% of the total amino acids in event 98140 grain. Although the levels of NAThr, NASer, and NAGly were statistically significantly higher in 98140 grain samples than in the non-transgenic control samples, they were more than 100 fold lower than the levels of NAA and NAG. For example, the mean levels of NAThr, NASer, and NAGly in 98140 grain samples were less than 0.0003% on a dw basis.

In addition to being found in conventional corn grain and forage, NAA and NAG are components of commonly consumed food such as eggs, chicken, turkey, and beef. An exposure assessment for humans to compare the estimates of dietary intake of NAA and NAG with and without exposure from event 98140 maize was conducted and it was concluded that commercialization of event 98140 maize may increase the dietary exposure to NAA and NAG in the U.S. population above current levels of exposure. However, based on the relatively low level of dietary exposure as well as the presence of deacetylases (aminoacylases that deacetylate N-acetylated amino acids) in the human body, it was concluded that no safety issues are expected as a result of the estimated increased consumption of acetylated amino acids in the human diet due to commercialization of 98140 maize.

The biological effects of increased levels of NAA and NAG were also assessed in a 42-day broiler chicken feeding trial. No statistically significant differences were noted in mortality, weight gain, feed efficiency, and organ and carcass yield variables between broilers consuming diets produced with 98140 maize and those consuming diets produced with the non-transgenic control maize. No safety issues are expected as a result of the estimated increase in exposure to NAA and NAG in the broiler diet. Dietary exposure estimates for NAA and NAG in other livestock animal species were also calculated, and these were less than for the broiler chicken feeding trial.

Potential Allergenicity and Toxicity

The GAT4621 and ZM-HRA proteins were evaluated for potential allergenicity and toxicity using a weight of evidence approach that examined: amino acid sequence similarities with known allergens and toxins, digestibility in model pepsin (simulated gastric fluid) and pancreatin (simulated intestinal fluid) systems, post-translational modification, and characteristics of the donor organism. Neither protein displayed significant amino acid sequence similarity with known allergens or toxins, both proteins were rapidly and completely digested in SGF within 30 seconds, and neither protein is glycosylated.

The potential toxicity of GAT4621 protein was also assessed in an acute oral toxicity study in mice. A single dose of 1640 milligrams per kilogram of body weight (mg/kg bw) of E. coli-produced and purified GAT4621 protein was administered by oral gavage to five male and five female mice. No clinical symptoms of toxicity, body weight loss, gross organ lesions, or mortality were observed. It was concluded that GAT4621 protein is not acutely toxic. A similar study was conducted using the ZM-HRA protein with mice (5 male and 5 female) receiving a single oral dose of 1236 mg/kg bw of E. coli-produced and purified ZM-HRA protein. No clinical symptoms of toxicity, body weight loss, gross organ lesions, or mortality were observed, indicating that the ZM-HRA protein is not acutely toxic.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office European Food Safety Authority Food Standards Australia New Zealand Food Standards Australia New Zealand (FSANZ) Secretariat of Agriculture, Livestock, Fisheries and Food Argentina U.S. Department of Agriculture, Animal and Plant Health Inspection Service United States Food and Drug Administration

This record was last modified on Thursday, October 29, 2015