Database Product Description

B16 (DLL25) (DKB-8979Ø-5)

Host Organism

Zea mays (Maize)

Trait

Phosphinothricin (PPT) herbicide tolerance, specifically glufosinate ammonium.

Trait Introduction

Microparticle bombardment of plant cells or tissue

Proposed Use

Production for human consumption and livestock feed.

Product Developer

Dekalb Genetics Corporation

Summary of Regulatory Approvals

Introduction Expand

Maize line DLL25 (synonym B16) 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 bar gene, conferring tolerance to glufosinate ammonium, was isolated from the common aerobic soil actinomycete, Streptomyces hygroscopicus, and encodes the enzyme phosphinothricin-N-acetyltransferase (PAT). The PAT enzyme occurs naturally in the soil and acetyltransferases are ubiquitous in nature.

The PAT enzyme was used as a selectable marker enabling identification of transformed plant cells as well as a source of field tolerance to phosphinothricin containing herbicides. Glufosinate ammounium acts by inhibiting the plant enzyme glutamine synthetase, a key enzyme that detoxifies ammonia by incorporating it into glutamine. Inhibition of this enzyme leads to an accumulation of ammonia in the plant tissues, which kills the plant within hours of application. PAT catalyses the acetylation of phosphinothricin detoxifying it 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
bar	phosphinothricin N-acetyltransferase	HT	CaMV 35S	T-DNA transcript number 7 (Tr7) termination signal	1	Modified
bla	beta lactamase	SM	bacterial promoter			Not expressed in plant tissues

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 hygroscopicus	bar	S. hygroscopicus is ubiquitous in the soil and there have been no reports of adverse affects on humans, animals, or plants.

Modification Method Expand

Maize line DLL25 was produced by biolistic transformation of maize gentoype A188xB73, a cross between two publicly available inbred lines. The original transformant was then crossed with elite inbred lines. The constitutive expression of the bar gene was regulated using the cauliflower mosaic virus (CaMV) 35S promoter. The bar gene introduced in the transformant DLL25 was modified to optimize its expression in plants without altering the amino acid sequence it encodes.

The plasmid used for transformation contained the beta-lactamase encoding bla gene, which was included as a selectable marker to screen for bacterial colonies that had been transformed with the recombinant plasmid. The bacterial expression of beta-lactamase confers resistance to some beta-lactam antibiotics, including the moderate-spectrum penicillin and ampicillin. The bla gene was driven by a bacterial promoter and was therefore not functional in plants.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from DLL25 demonstrated that the bar gene was integrated at a single site of insertion into maize line DLL25. Further analysis demonstrated that DLL25 contained a partially integrated antiobiotic resistant marker (bla gene), which was not expressed.

Genetic Stability of the Introduced Trait

Segregation analysis over twelve generations indicated that the introduced bar gene was stably inherited and that it segregated as a single locus, consistent with the Southern blot data demonstrating a single site of insertion. Stability of expression was demonstrated to be consistent across several hybrids derived from DLL25, across several generations.

Expressed Material

The only newly expressed protein in DLL25 is PAT protein, which was expressed in leaves, roots, stalk, tassel, cob, husk, kernels, silk and pollen. Highest levels were found in leaves (1.0 - 4.6 µg/g fresh weight tissue, corresponding to 0.1 - 0.46% of total soluble protein), and no PAT protein was detected in either silks, pollen or mature seeds.

Environmental Safety Considerations Expand

Field Testing

The maize line DLL25 was field tested in the major maize growing regions of the United States starting in 1991, in Canada since 1994, and in Argentina. DLL25 has been evaluated extensively in the laboratory, greenhouse, and field experiments. The transgenic maize was compared to non-transgenic counterparts for agronomic characteristics including test weight, dropped ears, stalk lodging, barrenness, stay green, seedling vigour, plant height, ear height, growing day units to 50% pollen shed, growing degree day units to 50% silk emergence, silk delay, green snap, maturity, yield, grain moisture, disease and insect susceptibilities. Field data determined that vegetative vigour, flowering period, time to maturity, seed production, and disease and insect susceptibilities 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 line DLL25 had no potential to pose a plant pest risk.

Outcrossing

Since pollen production and viability were unchanged by the genetic modification resulting in maize line DLL25, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between maize line DLL25 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 and Canada, 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.

Weediness Potential

No competitive advantage was conferred to maize line DLL25 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 line DLL25 did not have a significant adverse impact on organisms beneficial to plants or agriculture, or nontarget organisms, and was not expected to impact on threatened or endangered species. The PAT enzyme responsible for glufosinate ammonium tolerance does not possess mammalian toxicity. The PAT enzyme has a very specific enzymatic activity, does not possess proteolytic or heat stability typical of toxic compounds, and does not affect the metabolism of the plant.

Impact on Biodiversity

DLL25 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 the United States and Canada is remote, it was determined that the risk of transferring genetic traits from DLL25 maize lines to species in unmanaged environments was not a significant concern. 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 DLL25 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. Oral exposure of the introduced PAT protein, at very low levels, may occur from ingestion of processed corn products, however a lack of mammalian toxicity and the rapid digestibility of this protein has been well demonstrated. The human food uses of grain from DLL25 were not expected to be different from the uses of non-transgenic field corn varieties. As such, the dietary exposure of people to grain from DLL25 would not be different from that for other commercially available field corn varieties

Nutritional Data

Forage and grain from DLL25 maize hybrids were analyzed for nutritional composition and compared to the nutritional composition of non-transgenic versions of the same maize hybrids. Proximate and amino acid analyses were performed. Small differences between DLL25 plants and their non-transgenic counterparts were occasionally observed. However, the nutrient composition of DLL25 maize fell within the range of variability for the relevant nutrients reported for maize. The use of corn products derived from DLL25 was not anticipated to have any significant impact on the nutritional quality of the food supply.

Toxicity

The low potential for toxicity of the PAT protein expressed in the transgenic maize line DLL25 was investigated by searching for amino acid sequence homology with known toxins, examining its physiochemical properties and by acute oral toxicity testing in rats. A search of the GenBank database did not reveal any homologies between the deduced amino acid sequence of the PAT protein and the sequences of known protein toxins or allergens.
The PAT enzyme does not possess proteolytic or heat stability typical of toxic compounds, and does not affect the metabolism of the plant. The PAT enzyme has a very specific enzymatic activity that is highly specific for glufosinate and is not expected to have any effect on metabolic pathways in the plant other than detoxification of glufosinate herbicides.

An acute mouse toxicity study of bacterially-derived PAT protein was performed with no toxicity observed. Results indicated that the acute oral LD50 was estimated to be greater than 2500 mg of PAT/kg body weight. The acute oral toxicity data supported the prediction that the PAT protein was non-toxic to humans, by demonstrating a lack of mammalian toxicity

Allergenicity

The PAT protein does not possess characteristics typical of known protein allergens. In addition to a lack of sequence homology with known allergens, the PAT protein was rapidly degraded by simulated gastric fluids (< 1 min), unlike known protein allergens. Based on these properties, it was concluded that there was little or no potential for allergenicity.

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 od 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 DLL25 (synonym B16) 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 this maize line is the result of introducing a gene encoding the enzyme phosphinothricin-N-acetyltransferase (PAT) isolated from the common aerobic soil actinomycete, Streptomyces hygroscopicus, 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. The PAT encoding gene (bar) was introduced into the soybean genome by micro-particle acceleration (biolistic) transformation, and the resulting maize line displayed field tolerance to phosphinothricin-containing herbicides, thereby permitting farmers to use this herbicide for weed control in maize cultivation.

The maize line DLL25 was field tested in the major maize growing regions of the United States starting in 1991, in Canada beginning 1994, and in Argentina. DLL25 has been evaluated extensively in the laboratory and greenhouse, and in field experiments. The transgenic maize was compared to its non-transgenic counterparts, assessing agronomic characteristics such as vegetative vigour, flowering period, time to maturity, seed production, and disease and insect susceptibilities. Field data determined that the values observed for DLL25 maize 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 line DLL25 did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. DLL25 was 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 DLL25. Gene exchange between DLL25 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.

In an assessment of food and livestock feed safety forage and grain from DLL25 maize hybrids were analyzed for nutritional composition and compared to the nutritional composition of non-transgenic versions of the same maize hybrids. Proximate and amino acid analyses were performed. Small differences between DLL25 plants and their non-transgenic counterparts were occasionally observed. However, the nutrient composition of DLL25 maize fell within the range of variability for the relevant nutrients reported for maize. The use of maize products derived from DLL25 was not anticipated to have any significant impact on the nutritional quality of the food supply.

Potential toxicity and allergenicity of the PAT protein expressed in the transgenic maize line DLL25 was investigated by searching for amino acid sequence homology 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 proteolytic or heat stability characteristic of toxic compounds, and was readily digested 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 DLL25 maize, possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office

• Decision Document 96-15: Determination of Environmental Safety of Dekalb Canada Inc.'s Glufosinate Ammonium-Tolerant Corn Line DLL25
  [PDF Size: 139.61K bytes]
• Supplement to Decision Document DD96-15: Determination of Livestock Feed Safety of Dekalb Canada Inc.'s Glufosinate Ammonium-Tolerant Corn Line DLL25
  [PDF Size: 53.12K bytes]

Japanese Biosafety Clearing House, Ministry of Environment

• Outline of the biological diversity risk assessment report: Type 1 use approval for DLL25
  [PDF Size: 147.56K bytes]

Office of Food Biotechnology, Health Canada

• NOVEL FOOD INFORMATION - FOOD BIOTECHNOLOGYGLUFOSINATE RESISTANT MAIZE (CORN), DLL25
  [PDF Size: 19.06K bytes]

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

• Dekalb Genetics Corporation Petition for Determination of Nonregulated Status: Glufosinate Resistant Corn Line B16
  [PDF Size: 1.00M bytes]

US Food and Drug Administration

• Memorandum to file concerning glufosinate tolerant maize line DLL25.
  [PDF Size: 390.08K bytes]

USDA-APHIS Environmental Assessment

• Response to the Dekalb Genetics Corporation Petition for a Determination of Nonregulated Status for Glufosinate Resistant Corn Line B16
  [PDF Size: 45.66K bytes]

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