GM Crop Database

Database Product Description

DBT418 (DKB-89614-9)
Host Organism
Zea mays (Maize)
Trade Name
Bt Xtra™
Trait
Resistance to European corn borer (Ostrinia nubilalis); 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

Country Food Feed Environment Notes
Argentina View
Australia 2002
Canada 1997 1997 1997
European Union View
Japan 2001 2007
Korea 2004
Philippines 2003 2003
Taiwan 2003 View
United States 1997 1997 1997

Introduction Expand

The maize line DBT418 (tradename Bt Xtra™) was developed through a specific genetic modification to be resistant to attack by European corn borer (ECB; Ostrinia nubilalis), a major insect pest of maize in agriculture. These novel plants produce a truncated version of the insecticidal protein, Cry1Ac, derived from Bacillus thuringiensis subp. kurstaki strain HD-73. Delta-endotoxins, such as the Cry1Ac protein expressed in DBT418, act by selectively binding to specific sites localized on the brush border midgut epithelium of susceptible insect species. Following binding, cation-specific pores are formed that disrupt midgut ion flow and thereby cause paralysis and death. Cry1Ac is insecticidal only to lepidopteran insects, and its specificity of action is directly attributable to the presence of specific binding sites in the target insects. There are no binding sites for delta-endotoxins of B. thuringiensis on the surface of mammalian intestinal cells, therefore, livestock animals and humans are not susceptible to these proteins.

DBT418 was also genetically modified to express the bar gene cloned from the soil bacterium Streptomyces hygroscopicus, which encodes a phosphinothricin-N-acetyltransferase (PAT) enzyme. 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, the active ingredient in the herbicides Basta, Rely, Finale, and Liberty). PAT catalyses the acetylation of phosphinothricin and thus detoxifies phosphinothricin into an inactive compound, eliminating its herbicidal activity.

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 2 1 copy rearranged
bla beta lactamase SM bacterial promoter 4 Not expressed in plant tissues
cry1Ac Cry1Ac delta-endotoxin IR CaMV 35S, octopine synthase double enhancers ; maize alcohol dehydrogenase (adh1) gene introns I and VI (promote transcript stability) potato pinII termination and poly(A) signal 2
pinII protease inhibitor IR CaMV 35S ; maize alcohol dehydrogenase (adh1) gene introns I and VI (promote transcript stability) potato pinII and Tr7 termination and poly(A) signal 1 Incomplete, non-functional

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.
Bacillus thuringiensis subsp. kurstaki cry1Ac

Although target insects are susceptible to oral doses of Bt proteins, there is no evidence of toxic effects in laboratory mammals or bird given up to 10 µg protein / g body wt. There are no significant mammalian toxins or allergens associated with the host organism.

Modification Method Expand

DBT418 maize was produced by biolistic transformation of the inbred line DBT418 with DNA from three different plasmid vectors, respectively containing the cry1Ac gene from B. thuringiensis, the bar gene from S. hygroscopicus,and the protease inhibitor gene (pinII) from potato.

Constitutive expression of the cry1Ac gene was under the control of a chimeric promotor consisting of the 35S promoter from cauliflower mosaic virus (CaMV) and two copies of the octopine synthase (OCS) enhancer from Agrobacterium tumefaciens. The OCS enhancer promotes the expression of genes in most vegetative tissues. An intron from a maize alcohol dehydrogenase gene (adhI intron VI) was positioned downstream from the promoter to enhance gene expression. The 3'-terminal sequences from the potato pinII gene were used as a termination signal for the cry1Ac gene.

Expression of the bar herbicide resistance gene was regulated using the CaMV 35S promoter and termination sequences from the Ttr7 transcript 7 polyadenylation region of the A. tumefaciens T-DNA. The third gene, pinII was under the control of the CaMV 35S promoter and adhI intron I but was not completely incorporated and therefore not expressed.

Each plasmid also included a beta-lactamase (bla) gene which encodes resistance to the antibiotic ampicillin. The beta-lactamase gene was derived from E. coli and expression was under the control of its own bacterial promoter. This gene was included as a selectable marker to identify bacteria transformed with recombinant plasmid DNAs, but is not functional in plants.

Characteristics of the Modification Expand

Introduced DNA

Analysis of the DNA introduced into the DBT418 genome indicated integration of two intact copies of the cry1Ac gene, one intact copy of the bar gene and one rearranged copy of the bar gene. The incorporation of the pinII gene was incomplete due to rearrangements during or following the transformation process. There are four intact and one partial copy of the beta-lactamase (bla) gene and four intact copies of the ColE1 origin of replication. The beta lactamase gene was not expressed and the pinII gene was non-functional and therefore not expressed.

The synthetic cry1Ac gene introduced into line DBT418 encoded the N-terminal 613 amino acids of the native Cry1Ac protein and was modified to reflect plant preferred codon frequencies in order to maximize translation in maize cells. The gene codes for a 66 kDa protein of similar molecular weight to the protein tryptic core fragment of the Bacillus thuringiensis var. kurstaki HD-73 insecticidal crystal Cry1Ac protein.

Genetic Stability of the Introduced Trait

The expression of Cry1Ac and PAT proteins was demonstrated to be consistent across several generations in several different genetic backgrounds.

Expressed Material

Expression of the Cry1Ac protein and PAT occured in most, but not all, tissues of the maize plant with levels highest in leaves, and lower levels in roots, prop roots, stalk, tassel, cob, husk, and kernels. No Cry1Ac or PAT proteins were detected in silk or pollen.

Average protein expression of Cry1Ac in various tissues were as follows: harvested leaves ranged from 459.6 to 1194.4 ng Cry1Ac per gram tissue (dry weight); harvested kernels ranged from 36.0 to 42.8 ng Cry1Ac per gram tissue (d.w); harvested roots ranged from 58.0 to 125.4 ng Cry1Ac per gram tissue (d.w.); harvested stalks ranged from 40.9 to 123.6 ng Cry1Ac per gram tissue (d.w.).

The expressed PAT enzyme was compared to the bacterial protein: molecular weights were similar, indicating that the protein had not been glycosylated nor had it undergone post transcriptional modifications. The amino terminal sequence of PAT derived from DBT418 was determined to be the same as the native protein.

Average protein expression of PAT in various tissues, sampled at the pollen stage (except kernels), were as follows: leaves ranged from 501.8 to 1099.4 µg PAT per g tissue (d.w.); stalks ranged from 66 to 77 µg PAT per g tissue (d.w.),roots ranged from 27.5 to 69.5 µg PAT per g tissue (d.w.), and kernels ranged from 3.1 to 6.0 µg PAT per g tissue (d.w.).

Environmental Safety Considerations Expand

Field Testing

DBT418 maize has been field tested since 1993 in the major maize growing regions of the United States as well as in Argentina, Canada, France, and Italy. A number of traits (e.g., yield, grain moisture, final stand count, seedling vigor, plant height) were evaluated to determine how the genetic modifications altered agronomic performance. Very small differences were detected between the non-transformed control and DBT418 maize for grain moisture, seedling vigor, silk growing degree units, stay-green (a senescence rating) and intactness (primarily a measure of intactness of tassels and leaves above the ear). These differences were within the normal range of variation for maize plants and would not be expected to increase weediness. The transgenic line DBT418 provided significant protection from feeding damage from the European corn borer (Ostrinia nubilalis) and Southwestern corn borer (Diatraea grandiosella). Growth inhibition of corn earworm (Heliothis zea) was also observed as a result of silk and ear feeding on DBT418 plants. No significant differences were observed in resistance to a number of other significant insect pests or diseases compared to nontransgenic controls. Field data reports and data on agronomic traits confirmed that DBT418 exhibited the desired agronomic characteristics and did not pose a plant pest risk.

Outcrossing

Since pollen production and viability were unchanged by the genetic modification resulting in DBT418, pollen dispersal by wind and outcrossing frequency should be no different than for other maize varieties. Gene exchange between DBT418 and other cultivated maize varieties will be similar to that which occurs naturally between cultivated maize varieties at the present time. In North America, where there are few wild plant species closely-related to maize, the risk of gene flow to other species is remote. Furthermore, none of the sexually compatible relatives of maize in the U.S. are considered to be weeds in the U.S., therefore it is unlikely that introgression of the bar 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 DBT418, other than that conferred by resistance to European corn borer and herbicide tolerance to glufosinate. Maize is not a weed, and resistance to European corn borer or herbicide tolerance will not 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 other non-glufonsinate containing herbicides as appropriate. Zea mays is not invasive and is a weak competitor with very limited seed dispersal.

Secondary and Non-Target Adverse Effects

The history of use and literature suggest that the bacterial Bt protein is not toxic to humans, other vertebrates, and beneficial insects. This protein is active only against specific lepidopteran insects and no lepidopteran species are listed as threatened or endangered species in North America.

Other invertebrates and all vertebrate organisms, including non- target birds, mammals and humans would not be effected by Cry1Ac as these organisms do not have the receptor protein found in the midgut of target insects. Experimental studies demonstrated that soil-dwelling organims (collembola, earthworms) and birds (quail) were not adversely affected by exposure to pollen from DBT418 corn, or as a result of exposure to DBT418 leaf material or Cry1Ac on detritus.

Impact on Biodiversity

DBT418 had 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 North America is remote, it was determined that the risk of transferring genetic traits from DBT418 to species in unmanaged environments was insignificant.

Other Considerations

In order to prolong the effectiveness of plant-expressed Bt toxins, and the microbial spray formulations of these same toxins, regulatory authorities in Canada and U.S. have required developers to implement specific Insect Resistant Management (IRM) Programs. These programs are mandatory for all transgenic Bt-expressing plants, including DBT418, and require that growers plant a certain percentage of their acreage to non-transgenic varieties in order to reduce the potential for selecting Bt-resistant insect populations. Details on the specific design and requirements of individual IRM programs are published by the relevant regulatory authority.

DBT418 maize plants are not likely to eliminate the use of chemical insecticides which are traditionally applied to about 25 to 35% of the total maize acreage planted, since the primary target for most of these applications has been the coleopteran, corn rootworm. DBT418 maize may positively impact current agricultural practices used for insect control by 1) offering an alternative method for control of European corn borer (and potentially other Cry1Ac-susceptible pests of maize); 2) reducing the use of insecticides to control European corn borer and the resulting potential adverse effects of such insecticides on beneficial insects, farm worker safety, and ground water contamination; and 3) offering a new tool for managing insects that have become resistant to other insecticides currently used or expressed in maize, including other Bt-based insecticides.

DBT418 maize, along with glufosinate ammonium herbicides, is expected to positively impact current agricultural practices used for weed control by 1) offering growers a broad spectrum, post-emergent weed control system; 2) providing the opportunity to continue to move away from pre-emergent and residually active herbicides; 3) providing a new herbicidal mode of action in maize that allows for improved management of weeds which have developed resistance to herbicides with different modes of action; and 4) decreasing cultivation needs and increasing the amount of no-till acres. Volunteers of DBT418 can be easily controlled by selective mechanical or manual weed removal or by the use of herbicides with active ingredients other than glufosinate ammonium.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

Grain from DBT418 is intended primarily for animal feeding. However, such field maize may be dry- or wet-milled into various processed maize products for human food use. The human food uses of grain from DBT418 plants are not expected to be different from the uses of non-transgenic field maize varieties. As such, the dietary exposure of humans to grain from DBT418 will not be different from that for other commercially available field maize varieties.

Nutritional Data

Forage and grain from DBT418 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 DBT418 plants and their non-transgenic counterparts were occasionally observed. However, the nutrient composition of DBT418 maize falls within the range of variability for the relevant nutrients reported for maize. The use of maize products derived from DBT418 would therefore have no significant impact on the nutritional quality of the North American food supply

Toxicity

The amino acid sequences of the Cry1Ac and PAT proteins were compared to databases of known protein toxins and showed no homology with known mammalian protein toxins. An acute mouse toxicity study was performed using microbially-produced, purified Cry1Ac or PAT protein. The test protein was administered at doses up to 3325 mg/kg body weight and 2500 mg/kg body weight for Cry1Ac and PAT, respectively, without any observable adverse affects. Based on the results of these studies, the acute oral LD50 was estimated to be greater than 3325 mg of Cry1Ac or 2500 mg of PAT/kg body weight.

Allergenicity

The likelihood of the Cry1Ac and PAT proteins being allergens was judged to be remote. No homologies were found when the deduced amino acid sequences of the introduced proteins were compared to the sequences of known allergens. In addition, the potential for allergenicity was assessed based upon the physiochemical propterties of known food allergens, such as stability to acid and/or proteolytic digestion, heat stability, glycosylation, and molecular weight. Neither the Cry1Ac protein or the PAT protein possessed these characteristics normally associated with food allergens. For example, studies demonstrated that the PAT enzyme was completely degraded within five minutes when subjected to typical mammalian gastric conditions and was thus digested as a conventional dietary protein.

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 forthe 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, car parts and pharmaceuticals.
The European corn borer (ECB), Ostrinia nubilalis, is the most damaging insect pest of maize in the United States and Canada; losses resulting from ECB damage and control costs exceed $1 billion each year. An average of one ECB cavity per maize stalk across an entire field can reduce yield by as much as 5% when caused by first generation larvae, and 2.5% when caused by second generation larvae, with annual yield losses estimated at 5 to 10 %.

Despite consistent losses to ECB, chemical insecticides are utilized on a relatively small acreage (less than 20%). Historically, this reluctance stems from the difficulties in identifying and managing ECB in maize crops: ECB larval damage is hidden, heavy infestations are unpredictable, insecticides are costly, timing of insecticide application is difficult and multiple applications may be required to guarantee ECB control.

Weeds are also a major production problem in maize cultivation. Even a light infestation of weeds can reduce yields by 10 to 15%; severe infestations can reduce yields by 50% or more. Typically, weeds are managed using a combination of cultural (e.g., seed bed preparation, clean seed, variety selection) and chemical controls. Depending on the production area and the prevalent weed species, herbicides may be incorporated into the soil before planting (pre-plant), applied after planting but before emergence (pre-emergence), or applied after the maize plants emerge (post-emergence). Ideally, for maize production, weeds should be controlled for the full season. However, the most critical period for weed control is usually about six to eight weeks after crop emergence, during the 4th to 10th leaf stages. This critical period in the life cycle of maize must be kept weed free in order to prevent yield loss.

Maize line DBT418 was genetically modified to contain two novel genes, cry1Ac and bar, for insect and herbicide tolerance respectively. Both genes were introduced into a maize line by particle acceleration (biolistic) transformation.
The transgenic maize line DBT418 was developed to resist ECB by producing its own insecticide. This event was genetically engineered by introducing the cry1Ac gene, isolated from the common soil bacterium Bacillus thuringiensis (Bt), into the maize line AT824. The cry1Ac gene produces the insect control protein Cry1Ac, a delta-endotoxin. Cry proteins, of which Cry1Ac 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. Cry1Ac is lethal only when eaten by the larvae of lepidopteran insects (moths and butterflies), and its specificity of action is directly attributable to the presence of specific binding sites in the 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.

In addition to the cry1Ac gene, DBT418 was developed to allow for the use of glufosinate ammonium, the active ingredient in phosphinothricin herbicides (Basta®, Rely®, Liberty®, and Finale®), as a weed control option, and as a breeding tool for selecting plants containing the cry1Ac gene. DBT418 contains the bar gene isolated from a common soil actinomycete, Streptomyces hygroscopicus. This gene allows for the production of the enzyme phosphinothricin N-acetyltransferase (PAT) which confers tolerance to glufosinate.

The PAT enzyme in maize line DBT418 converts L-phosphinothricin (PPT), the active ingredient in glufosinate ammonium, to an inactive form, thereby conferring resistance to the herbicide. In the absence of PAT, application of glufosinate leads to reduced production of the amino acid glutamine and increased ammonia levels in the plant tissues, resulting in the death of the plant. The PAT enzyme is not known to have any toxic properties.
Expression of the Cry1Ac protein and PAT occurred in most, but not all, tissues of the maize plant with levels highest in leaves, and lower levels in roots, prop roots, stalk, tassel, cob, husk, and kernels. No Cry1Ac or PAT proteins were detected in silk or pollen.

Maize line DBT418 was tested in field trials in the United States, beginning in 1993, and in Canada, beginning in 1995. Data collected from these trials demonstrated that DBT418 was not different from conventional maize varieties. Agronomic characteristics such as seedling vigor and yield were within the expected range of expression reported for commercial maize hybrids. The transgenic line DBT418 provided significant protection from feeding damage caused by the European corn borer (Ostrinia nubilalis) and Southwestern corn borer (Diatraea grandiosella). Maize line DBT418 was comparable to conventional maize lines in all other respects and did not exhibit weedy characteristics, or negatively affect beneficial or nontarget organisms. DBT418 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 or 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 maize line DBT418. Gene exchange between DBT418 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.

Regulatory authorities in Canada and the United States have mandatory requirements for developers of Bt maize to implement specific Insect Resistant Management (IRM) Programs. The potential for ECB populations to develop tolerance or become resistant to the Bt toxin is expected to increase as more maize acreage is planted with Bt hybrids. These IRM programs are designed to reduce the potential development of Bt-resistant insect populations, as well as prolonging the effectiveness of plant-expressed Bt toxins, and the microbial Bt spray formulations of these same toxins.

The food and livestock feed safety of maize line DBT418 was established based on several standard criteria. As part of the safety assessment, the nutritional composition of the grain and forage of DBT418 were analyzed in order to determine protein, oil, fibre, ash, moisture, and starch content, and were found to be equivalent to conventional maize. The grain’s amino acid composition was also analyzed and found to be identical to that of conventional maize hybrids.

The potential for toxicity and allergenicity of the DBT418-expressed Cry1Ac and PAT proteins was demonstrated by examining their physiochemical characteristics and amino acid sequence homologies to known protein toxins and allergens. Stability to acid and/or proteolytic digestion, heat stability, glycosylation, and molecular weight were determined and digestibility and acute oral toxicity studies were also conducted. The Cry1Ac protein has a history of safe use, as demonstrated by its use in microbial Bt spray formulations in agriculture for more than 30 years with no evidence of adverse effects. Similarly, PAT has a very specific enzymatic activity and does not possess proteolytic or heat stability, and does not affect plant metabolism. Acetyltransferases such as PAT are common enzymes in bacteria, plants and animals and no toxic or allergic effects were expected. These facts, combined with the lack of amino acid sequence homologies between Cry1Ac and PAT proteins, and known allergens and toxins, and the rapid degradation of the novel proteins in acidic gastric fluids, was sufficient to provide with reasonable certainty a lack of toxicity and allergenic potential.

Links to Further Information Expand

Australia New Zealand Food Authority Canadian Food Inspection Agency, Plant Biotechnology Office Impact of Bt corn pollen on monarch butterfly populations: A risk assessment Japanese Biosafety Clearing House, Ministry of Environment Office of Food Biotechnology, Health Canada U.S.Department of Agriculture, Animal and Plant Health Inspection Service USDA-APHIS Environmental Assessment

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