Database Product Description

T14 (ACS-ZMØØ2-1)

Host Organism

Zea mays (Maize)

Trade Name

Liberty-Link™

Trait

Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.

Trait Introduction

Chemically mediated introduction into protoplasts and regeneration.

Proposed Use

Production for livestock feed.

Product Developer

Bayer CropScience (Aventis CropScience(AgrEvo))

Summary of Regulatory Approvals

Introduction Expand

Maize line T14 was developed through a specific genetic modification to allow the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta, Rely, Finale, and Liberty), as a weed control option in maize crops. The pat gene, conferring tolerance to glufosinate ammonium, was cloned from the common aerobic soil actinomycete, Streptomyces viridochromogenes, and encodes the enzyme phosphinothricin-N-acetyltransferase (PAT).

The PAT enzyme was used as a selectable marker enabling identification of transformed plant cells as well as a source of resistance to the herbicide phosphinothricin (also known as glufosinate ammonium). Phosphinothricin containing herbicides, such as glufosinate ammonium, act by inhibiting glutamine synthetase resulting in the accumulation of toxic levels of ammonia that kill the plant within hours of application. The PAT enzyme detoxifies phosphinothricin by acetylation into an inactive compound. The modified corn permits farmers to use phosphinothricin-containing herbicides for weed control in the cultivation of corn.

Summary of Introduced Genetic Elements Expand

Code	Name	Type	Promoter, other	Terminator	Copies	Form
bla	beta lactamase	SM	bacterial promoter			Truncated beta-lactamase gene missing about 25 % of the gene from the 5' end; not expressed
pat	phosphinothricin N-acetyltransferase	HT	CaMV 35S	CaMV 35S poly(A) signal	3 or 1*	Modified for enhanced expression; PEG mediated uptake into plant protoplasts; 3 copies in T14, 1 copy in T25

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.

Donor Organism Characteristics Expand

Latin Name	Gene	Pathogenicity
Streptomyces viridochromogenes	pat	S. viridochromogenes is ubiquitous in the soil. It exhibits very slight antimicrobial activity, is inhibited by streptomycin, and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

The T14 lines were derived by chemically mediated transformation of cultured protoplasts obtained from a yellow dent corn line with purified DNA containing the pat gene isolated from S. viridochromogenes. Transformed cell colonies were selected and regenerated on medium containing glufosinate ammonium. The primary transformants, T14 and T25, were then backcrossed with both commercial public inbred lines and proprietary inbred lines of the yellow dent corn type.

The pat gene introduced in the transformants T14 was modified to optimize its expression in plants without altering the amino acid sequence of the PAT enzyme.

The transformed line T14 also contained the beta lactamase (bla) gene included as a selectable marker to identify transformed bacterial cell colonies during the initial stages of cloning the recombinant pat gene. The bla gene codes for a beta-lactamase enzyme that confers resistance to some beta-lactam antibiotics, including the moderate-spectrum penicillin and ampicillin antibiotics. The bla gene is not functional in the modified maize lines, as its promoter is only active in bacteria.

Characteristics of the Modification Expand

The Introduced DNA

Molecular analyses showed that maize line T14 contained 3 copies of the pat gene. Further analyses demonstrated that T14 did not possess a complete sequence of the antibiotic resistance encoding bla gene. Northern blot analyses showed that maize lines derived from T14 did not produce mRNA from the disrupted bla gene, nor did they produce beta-lactamase, the enzyme encoded by the bla gene. Additionally, the bla gene would not have been expected to be functional in the modified maize lines, as it did not have the necessary regulatory sequences for expression in plants.

Genetic Stability of the Introduced Trait

The integration of the transferred DNA into event T14 was demonstrated to be stable by segregation analysis. The data provided demonstrated the integration of three copies of the pat gene in transformation event T14. Commercial maize lines containing the transformation events are several generations removed from the two original transformation events, further demonstrating the stable inheritance of the herbicide resistant trait.

Expressed Material

The only newly expressed protein in T14-derived plant was the PAT protein. The pat gene was linked to a constitutive promoter, and protein expression was detected in plant tissues, grain and silage. Expression of the pat gene in T14 resulted in the production of PAT protein in maize tissues at low levels (< 0.003% (w/w) of total crude protein). The PAT enzyme was not detected in protein extracts from the pollen.

Environmental Safety Considerations Expand

Field Testing

The maize event T14 has been field tested in the major maize growing regions of the United States since 1992 and in Canada since 1993. T14 has been evaluated extensively in the laboratory, greenhouse, and field experiments. Field reports on T14 compared to non-transgenic counterparts, determined that agronomic characteristics such as yield, plant height, over-wintering capacity, flowering period, and disease susceptibility were within the normal range of expression currently displayed by commercial maize hybrids. Overall the field data reports and data on agronomic traits showed that maize event T14 and lines derived from these events, have no potential to pose a plant pest risk.

Outcrossing

Since pollen production and viability were unchanged by the genetic modification resulting in maize line T14, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between maize line T14 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 appears remote. Cultivated maize, or 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.

Weediness Potential

No competitive advantage was conferred to maize line T14, other than that conferred by resistance to glufosinate ammonium herbicides. Resistance to glufosinate ammonium 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, other than glufosinate ammonium. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects

It was determined that genetically modified maize event T14 did not have a significant adverse impact on organisms beneficial to plants or agriculture, nontarget organisms. These events were not expected to impact on threatened or endangered species. The PAT enzyme responsible for glufosinate ammonium tolerance has very specific enzymatic activity, does not possess proteolytic or heat stability typical of toxic compounds, and does not affect the metabolism of the plant. Other crops such as wheat, barley, lentils, peas, flax and alfalfa, have been modified by recombinant DNA techniques to express the PAT enzyme with no apparent effect on the agronomic performance of succeeding crops. Expression levels of PAT in T14 were comparable to other transformed species and therefore it was concluded that no significant residual effects from T14 were expected.

Impact on Biodiversity

T14 has no novel phenotypic characteristics that would extend their 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 T14 maize lines to species in unmanaged environments was insignificant. It was determined that the overall relative impact on plant biodiversity was neutral, as was the impact on animal and microbe biodiversity since the introduced PAT enzyme was not expected to alter the plant's metabolism and as such, novel compounds would not be produced.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Maize line T14 was intended mainly for use in animal feed. The major human food uses for maize are extensively processed starch and oil fractions prepared by wet or dry milling procedures and include products such as corn syrup and corn oil, bran, grits, meal and flour. There was no PAT protein detected in starch or oil. Neither was PAT protein detected in wet milled corn grain fractions with the exception of the bran. Wet milled bran and all of the dry milled corn fractions contained very low levels of the PAT protein (< 0.00035% of crude protein). Overall, the dietary exposure of North Americans to grain from maize from line T14 was anticipated to be the same as for other lines of commercially available field corn.

Nutritional Data

Compositional analyses of maize grain from T14-derived lines and current commercial maize varieties were compared for compositional and nutritional parameters including moisture, crude fat, crude protein, crude fiber, ash, carbohydrates, mineral content (calcium and phosphorous), amino acid profile, phytates and oil composition. Overall protein content in the grain and whole plant was significantly higher in the transgenic lines compared to the unmodified controls. The statistical differences were explained by the fact that the transformed plant lines were not complete genetic isolines of the control plants and thus the variation arose from genetic differences unrelated to the transformation event. In all cases protein content was within the normal published range for maize. Similarly, an evaluation of the nutrient values determined that they were well within the range of nutrient values reported for corn grain. The use of corn grain from T14-derived maize lines was determined not to have a significant impact on the nutritional quality of the North American food supply.

Toxicity

The low potential for toxicity of the PAT protein expressed in the transgenic maize line T14 was demonstrated by examining the amino aid sequence homology, chemical characteristics of the protein and by acute oral toxicity testing in rats. The nucleotide sequence of the pat gene and the deduced amino acid sequence of the PAT protein were compared with sequences available for known toxins in the GenBank database and showed no significant homology with any known toxins or allergens. PAT protein expressed in bacterial systems was used as the test protein for mammalian feeding trials and studies were performed to demonstrate equivalence with the plant-expressed protein. The molecular weight and immunoreactivity of the PAT enzyme from the plant derived sources were similar to the PAT derived from a bacterial expression system. These similarities indicated that the PAT protein was unlikely to have been glycosylated or undergone other post translational modifications. An acute oral toxicity study demonstrated no evidence of toxicity for PAT protein when administered to rats at dietary concentrations up to 50,000 ppm for 14 days, which represents a concentration 6 orders of magnitude greater than that in grain from T14-derived maize lines.

Allergenicity

The PAT enzyme expressed in T14-derived maize lines does not possess characteristics typical of known protein allergens and is extremely unlikely to be allergenic. There were no regions of homology when the sequences of the introduced protein was compared to the amino acid sequences of known protein allergens. There was no evidence found of post-translational modifications such as acetylation, glycosylation or phosphorylation of the PAT protein. Unlike known protein allergens, the PAT protein was rapidly degraded by acid and/or enzymatic hydrolysis when exposed to simulated gastric fluids. In vitro digestibility studies, under simulated mammalian gastric conditions, demonstrated that the PAT enzyme was inactivated within one minute and was rapidly degraded. No adverse effects have been reported to be associated with this enzyme.

Abstract Collapse

Maize (Zea mays L.), or corn, is grown primarily for its kernel, which is largely refined into products used in a wide range of food, medical, and industrial goods.

Only a small amount of whole maize kernel is consumed by humans. Maize oil is extracted from the germ of the maize kernel and maize is also a raw material in the manufacture of starch. A complex refining process converts the majority of this starch into sweeteners, syrups and fermentation products, including ethanol. Refined maize products, sweeteners, starch, and oil are abundant in processed foods such as breakfast cereals, dairy goods, and chewing gum.
In the United States and Canada maize is typically used as animal feed, with roughly 70% of the crop fed to livestock, although an increasing amount is being used for the production of ethanol. 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 makes up 10-12% of the annual corn acreage, and 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, pharmaceuticals and car parts.
The maize line T14 was genetically engineered to express tolerance to glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Finale®, and Liberty®). Glufosinate chemically resembles the amino acid glutamate and acts to inhibit an enzyme, called glutamine synthetase, which is involved in the synthesis of glutamine. Essentially, glufosinate acts enough like glutamate, the molecule used by glutamine synthetase to make glutamine, that it blocks the enzyme's usual activity. Glutamine synthetase is also involved in ammonia detoxification. The action of glufosinate results in reduced glutamine levels and a corresponding increase in concentrations of ammonia in plant tissues, leading to cell membrane disruption and cessation of photosynthesis resulting in plant withering and death.

Glufosinate tolerance in T14 maize is the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, Streptomyces viridochromogenes, the same organism from which glufosinate was originally isolated. The PAT enzyme catalyzes the acetylation of phosphinothricin, detoxifying it into an inactive compound. The PAT enzyme is not known to have any toxic properties.

Cultured protoplasts obtained from a yellow dent corn line were transformed using a chemically mediated direct DNA introduction method. Transformed cell colonies were selected for the presence of the pat gene by regeneration on medium containing glufosinate ammonium. The primary transformants, T14 and T25, were then backcrossed with parental lines of the yellow dent corn type. The resulting lines displayed field tolerance to phosphinothricin-containing herbicides, thereby permitting farmers to use this herbicide for weed control in maize cultivation.

The maize event T14 has been field tested in the major maize growing regions of the United States since 1992 and in Canada since 1993. Field reports on T14 were compared to those of their non-transgenic counterparts, and it was determined that agronomic characteristics such as yield, plant height, over-wintering capacity, flowering period, and disease susceptibility were within the normal range of expression currently displayed by commercial maize hybrids. Overall the field data reports indicated that the transformed maize lines did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms, and were not expected to impact on threatened or endangered species.

Maize does not have any closely related species growing in the wild in the continental United States and Canada. Cultivated maize can naturally cross with annual teosinte (Zea mays ssp. mexicana) when grown in close proximity, however, these wild maize relatives are native to Central America and are not naturalized in North America. Additionally, reproductive and growth characteristics were unchanged in T14. Gene exchange between the modified lines and maize relatives was determined to be negligible in managed ecosystems, with no potential for transfer to wild species in Canada and the United States.

Compositional analyses of T14 maize grain and of current commercial maize varieties were compared for a number of parameters, including moisture, crude fat, crude protein, crude fibre, ash, carbohydrates, mineral content (calcium and phosphorous), amino acid profile, phytates and oil composition. Overall protein content in the grain and whole plant was significantly higher in the transgenic lines than in the unmodified controls. It was determined that this variation arose from genetic differences unrelated to the transformation event. In all cases protein content and nutrient values were within the normal published range for maize. The use of grain from T14-derived maize lines was determined not to have a significant impact on the nutritional quality of the North American food supply.

Potential toxicity and allergenicity of the PAT protein expressed in the transgenic maize lines T14 was investigated by searching for amino acid sequence homologies with known toxins and allergens, by examining its physiochemical properties, and by acute oral toxicity testing in mice. No homologies between the deduced amino acid sequence of the PAT protein and the sequences of known toxins or allergens were detected. The PAT enzyme does not possess the stability to digestive enzymes or heat stability characteristic of toxic compounds, and was readily degraded under conditions simulating mammalian digestion. In the acute mouse toxicity study mice were fed high doses of the PAT protein, with no observable adverse effects. Based on these properties, it was concluded that the PAT protein, and thus T14 maize, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office

• Decision Document 98-22: Determination of the Safety of AgrEvo Canada Inc.'s Glufosinate Ammonium Tolerant Corn (Zea mays) lines, T14 and T25
  [PDF Size: 149.05K bytes]
• Supplement to Decision Document DD96-11: Determination of Livestock Feed Safety of AgrEvo Canada Inc.'s Glufosinate Ammonium-Tolerant Canola Line HCN28
  [PDF Size: 51.46K bytes]

European Commission: Community Register of GM Food and Feed

• Notification of the placing on the Community Register of ACS-ZMØØ3-2.
  [PDF Size: 12.25K bytes]

Japanese Biosafety Clearing House, Ministry of Environment

• Outline of the biological diversity risk assessment report: Type 1 use approval for T14
  [PDF Size: 165.11K bytes]

Office of Food Biotechnology, Health Canada

• NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGYGLUFOSINATE AMMONIUM TOLERANT CORN (T14 AND T25)
  [PDF Size: 21.56K bytes]

U.S.Department of Agriculture, Animal and Plant Health Inspection Service

• AgrEvo Petition for Determination of Nonregulated Status for Glufosinate Resistant Corn Transformation Events T14 and T25
  [PDF Size: 792.23K bytes]

US Food and Drug Administration

• Memorandum to file concerning glufosinate herbicide tolerant maize lines T14, T25.
  [PDF Size: 544.29K bytes]

USDA-APHIS Environmental Assessment

• USDA/APHIS Petition 94-357-01 for Determination of NonregulatedStatusfor Glufosinate Resistant Corn Transformation Events T14 and T25
  [PDF Size: 45.79K bytes]

This record was last modified on Wednesday, January 13, 2016