GM Crop Database

Database Product Description

BW255-2, BW238-3
Host Organism
Triticum aestivum (Wheat)
Imidazolinone herbicide tolerance.
Trait Introduction
Chemically induced seed mutagenesis
Proposed Use

Production for human consumption and livestock feed.

Product Developer

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 2006 2006 2006

Introduction Expand

BW255-2 and BW238-3 (Clearfield™) bread wheat was developed to allow the use of imidazolinone herbicides as a weed control option in spring wheat. This trait was developed using chemically induced seed mutagenesis and whole plant selection procedures. This wheat line expresses a mutated form of the acetohydroxyacid synthase (AHAS) enzyme, which renders the plant tolerant to levels of imidazolinone herbicides used in weed control.

AHAS catalyses the first step in the biosynthesis of the branched-chain amino acids isoleucine, leucine, and valine, and is active in the glycolytic pathway of plant metabolism. When conventional plants (i.e., weeds) are treated with imidazolinone herbicides, the herbicide binds to a specific site on the enzyme, thereby inhibiting its activity. The result of this enzyme inhibition is a decrease in the synthesis of branched-chain amino acids and an accumulation of toxic levels of ?-ketoglutarate, which results in the eventual death of the plant.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
als acetolactate synthase MUT Native AHAS Selected following chemical mutagenesis

Characteristics of Triticum aestivum (Wheat) Expand

Center of Origin Reproduction Toxins Allergenicity
Asia Minor, Tigris-Euphrates drainage basin of the Middle East, as well as the regions of southern Caucasus and Crimea. Primarily self-pollinated (autogamous). Some outcrossing by wind-pollination of less than 10%. Seed does not display dormancy. Phytic acid, trypsin inhibitor, lectins. Gliadins responsible for celiac enteropathy. Glutenins and gliadins (e.g., the IgE-inducing alpha-gliadin).

Modification Method Expand

The wheat line BW255-2 and BW238-3 was isolated from populations derived by chemical-induced mutagenesis of wheat varieties BW255 and BW238 respectively, utilizing sodium azide as the mutagenizing chemical. The selection of herbicide tolerance was made on whole plants. The designation BW255-2 and BW238-3 was given to one herbicide tolerant mutant selected from each population.

Characteristics of the Modification Expand

The Introduced DNA:

Since BW255-2 and BW238-3 wheat are products of mutagenesis and conventional seed increase techniques there was no introduction or incorporation of heterologous DNA into the plant genome. The tolerance to imidazolinone herbicides is due to a point mutation of a single nucleotide and the amino acid sequence of the mutated enzyme differs by one amino acid from that of the unmodified enzyme. This single change in the amino acid sequence alters the binding site of AHAS to imidazolinone herbicides, such that the herbicide cannot inhibit the enzyme’s activity. The mutation of the AHAS gene was identified from information on the gene sequence.

Genetic Stability of the Introduced Trait:

Segregation analysis in generation three BW255-2 and BW238-3 populations was shown to be consistent with the inheritance of a single gene.

Expressed material:

The modified AHAS gene, conferring tolerance to imidazolinone herbicides, is under control of the native AHAS promoter and is believed to be constitutively expressed. Whole plant tolerance to this herbicide was expressed in BW255-2 and BW238-3.

In the unmodified plant, the levels of valine, leucine and isoleucine are regulated by feedback inhibition of AHAS. A mutation in the AHAS enzyme could affect the regulation of the biosynthesis of these amino acids. Data were submitted to show that the modified AHAS was feedback inhibited by valine and leucine, similar to the unmodified enzyme. Levels of valine, leucine and isoleucine in BW255-2 and BW238-3 were comparable to those in the unmodified parental lines.

Environmental Safety Considerations Expand


Wheat (Triticum aestivum) is primarily a self-pollinating plant. Gene transfer can occur by wind-mediated pollination to other T. aestivum plants, however outcrossing rates are usually low (The potential for introgression of the novel trait into weedy relatives of wheat in Canada and the United States is also very low. Wild species closely related to T. aestivum in the continental United States and Canada are Aegilops cylindria Host (jointed goat grass) and Agropyron repens (L.) Beauv. (quackgrass). Both of these are introduced species, native to Eurasia, and are considered weedy and invasive in Canada and the United States. Jointed goat grass is an annual, found in most of the United States, but not in Canada. It is considered a noxious weed in British Columbia due to the proximity of populations in Washington, Idaho and Montana, but is also found in other states contiguous to Canada, i.e., North Dakota, Michigan, Ohio and New York. It would not be expected to become established in Canada except in southwestern British Columbia and southwestern Ontario. Hybridization of jointed goat grass with wheat can occur under field conditions and has been reported in Oregon. The hybridization was successful to the first backgross generation with jointed goat grass as the female plant. This has raised concerns of possible introgression of wheat into jointed goat grass populations as introgression of an herbicide tolerant trait from wheat into jointed goatgrass would confer an additional advantage to this weedy species. Quackgrass is a perennial species found extensively in Canada and the United States. Reports of hybridization between wheat and quackgrass have been reported; however, these have not been successfully reproduced by manual pollination. Due to the doubtful nature of any hybridizations between wheat and quackgrass, gene introgression into quackgrass populations is not expected to occur.

Weediness potential:

Cultivated wheat is not considered a weed of agriculture. Wheat plants can volunteer in fields following a wheat crop but these can be controlled by cultivation or herbicides. No competitive advantage was conferred to BW255-2 or BW238-3, other than resistance to imidazolinone herbicides. Results from agronomic performance field trials and compositional data show that the physiology of BW255-2 and BW238-3 was not affected by the mutation of the AHAS gene. BW255-2 and BW238-3 are not expected to become weedy or invasive of natural habitats since neither vegetative growth or reproductive characteristics were altered. Wheat volunteers expressing the modified AHAS gene are therefore not expected to become weedier than conventional wheat volunteers. These will not be controlled by imidazolinone herbicides, thereby providing less flexibility in herbicide use.

Secondary and Non-Target Effects:

The characterization of the modified AHAS gene containing a single base pair change, and the resulting modified enzyme, led to the conclusion that the expression of the modified enzyme does not result in altered toxic or allergenic properties. The modified AHAS enzyme was found to be substantially equivalent to AHAS in nonmodified plants. The AHAS enzyme is not a known toxin, does not confer resistance to agricultural pests, and is naturally present in plants and micro-organisms. It is similar to conventional wheat in terms of vegetative growth and seed production. Based on this information, BW255-2 and BW238-3 would not result in altered impacts on non-target organisms, compared to conventional wheat varieties.

Impact on Biodiversity:

BW255-2 and BW238-3 do not possess novel phenotypic characteristics that would extend cultivation beyond the current geographic range of spring wheat. Neither do they possess characteristics that would allow them to grow and compete under soil and climatic conditions for which they are not adapted. The herbicide-tolerant trait, in itself, will not confer to any potential hybrids any competitive advantage other than tolerance to imidazolinone herbicides. BW255-2 and BW238-3 have also been found safe to non-target organisms and do not display altered plant pest potential since agronomic and biological characteristics were found to be within the range of conventional wheat varieties.

Food and/or Feed Safety Considerations Expand

Dietary exposure:

The modification to the AHAS enzyme in BW255-2 and BW238-3 will not result in any change in the consumption or use pattern of wheat and wheat-based products. The availability of many wheat cultivars for cultivation, the diversity of wheat in phenotypic traits, and the normal variation in wheat composition due to differences in growing conditions all result in a wide variation in the composition of conventional wheat grain. Since the modification to the AHAS enzyme would not be expected to change the gluten content in BW255-2 and BW238-3, the cultivation and use of this wheat would not result in any new or additional concern for individuals with celiac disease. Thus, the cultivation of BW255-2 and BW238-3 would not be expected to change the dietary exposure in Canada and the United States, any more than commercially available wheat cultivars.

Nutritional Data:

Nutritional components of BW255-2 and BW238-3 wheat were measured analytically and compared to those of the parental lines. These components included: moisture, crude protein, crude fat, crude fibre, and the amino acids valine, isoleucine, leucine, threonine, cystine, lysine and methionine. Also measured were fatty acids, B vitamins (thiamine, niacin, pantothenic acid, pyridoxine), vitamin E, and minerals (phosphorus, zinc, magnesium and iron). Statistically significant differences were observed in the levels of protein between BW255-2 and BW238-3 and their parental controls. Significant differences were also observed in the concentration of branched-chain amino acids and Vitamin B1 between BW238-3 and its parental control and in Mg, Fe and panthothenic acid between BW255-2 and its parental control. However, the levels of all of these nutritional components fell within the range of conventional wheat varieties supporting the conclusion that the nutritional composition of BW255-2 and BW238-3 is equivalent to conventional bread wheat varieties.

The anti-nutrients phytic acid and trypsin inhibitor were measured in BW255-2 and BW238-3 grain samples. Phytic acid occurs naturally in wheat and other cereals. It is indigestible by humans and non-ruminant livestock, and inhibits the absorption of iron and other minerals. Trypsin inhibitor interferes with protein digestion. Phytic acid levels in BW255-2 and BW238-3 were not significantly different from those of the parental controls and trypsin inhibitor was not detected in events BW255-2 and BW238-3 or their parental controls.

Toxicity and Allergenicity:

The potential for toxicity and allergenicity of BW255-2 and BW238-3 wheat were determined by examining the characteristics of the modified AHAS protein and the amino acid sequence homology between the modified protein and known toxins and allergens. The unmodified form of AHAS is heat sensitive and susceptible to trypsin degradation. Data from studies on the heat sensitivity and trypsin degradability of the modified AHAS in BW255-2 and BW238-3 showed similar sensitivity to heat and trypsin degradability compared to unmodified AHAS. The unmodified form of AHAS shows no amino acid similarity to known toxins and allergens. The modified AHAS is substantially equivalent to the unmodified enzyme in that it differs by only one amino acid. Evidence was also provided to show that the protein components of BW255-2 and BW238-3 were not altered compared to those of unmodified parental controls. Results from HPLC on protein extracts demonstrated that no new major proteins or increased protein expression occurred as a result of the mutation to the AHAS enzyme. From these results it was concluded that BW255-2 and BW23803 wheat did not demonstrate any potential for toxicity and allergenicity compared to conventional unmodified wheat.

Abstract Collapse

BW255-2 and BW238-3 (Clearfield™) bread wheat was not subject to regulation in any jurisdiction except Canada since the development of this herbicide-tolerant line did not employ recombinant DNA technologies. In Canada, regulatory approval is required for use in human food and livestock feed, and for environmental release.

Commercial wheat is comprised mainly of two species: common, or bread wheat (T. aestivum L.) and durum wheat (T. durum Desf.). Bread wheat is classified into several types, based on the spring and winter forms of growth habit, and the hardness of the kernels. Winter wheat requires vernalisation to produce flowers, whereas spring wheat does not have this requirement. The hard types of bread wheat are high in protein, especially gliadins and glutenins. The high levels of these protein fractions in the flour impart elasticity to bread dough and allow it to expand during leavening and baking. Soft wheats are low in protein, and have low levels of gliadin and glutenin; these qualities are desirable in products such as cakes and pastries, and in unleavened breads. Durum wheat produces very hard, almost vitreous kernels due to its high protein content. This wheat is milled into semolina for the production of pasta and couscous.
Harvested wheat consists of a naked kernel, unlike other cereals such as rice, barley or oats that retain their hull (i.e., the palea and lemma). The wheat kernel is loosely enclosed within the palea and lemma of each spikelet; these are eliminated as chaff during threshing. The wheat kernel is milled into white flour by removing the bran, aleurone layers and the germ prior to grinding; whole-wheat flour retains these fractions. By-products of wheat milling include: bran, germ, shorts and middlings. Some of these by-products are used as human food (i.e., bran, germ), and others, as livestock feed. Grain that does not meet the grade for food use can be used as animal feed, mainly for poultry and swine, but also for cattle. Wheat can also be fed as forage, either as pasture prior to stem elongation, or as ensilage. Wheat is also used in the brewing and distilling industries.

Weeds are a major production problem in wheat cultivation. Weeds compete for light, water and nutrients, and can also cause lodging and problems with harvesting. The seeds of several weed species are almost impossible to clean out of harvested wheat (e.g., Avena fatua L. wild oats), causing loss of quality and downgrading of the crop. Weeds can be managed using a combination of cultural practices (e.g., seed bed preparation, use of clean [certified] seed, narrow row spacing, fertilizer banding), integrated weed management (e.g., weed scouting, economic thresholds) and the use of herbicides. Depending on the weed species present, herbicides can be applied before the crop emerges (e.g., amitrole, glyphosate, trifluralin), or after (e.g., 2-4D, bromoxynil, dicamba, fenoxaprop-p-ethyl, MCPA, metsulfuron methyl). The build-up of weed populations can be stemmed by applying herbicides on summer-fallowed fields, and by practicing crop rotation, which allows the use of different herbicides. Rotating among herbicide groups also prevents the development of herbicide-resistant biotypes.

BW255-2 and BW238-3 (Clearfield™) bread wheat lines were developed to allow the use of imidazolinone herbicides, as a weed control option in spring wheat production. The mode of action of imidazolinone herbicides consists of inhibiting the activity of acetohydroxyacid synthase (AHAS), an enzyme in plants active in glycolysis and in the biosynthesis of the branched-chain amino acids isoleucine, leucine and valine. The result of the inhibition of AHAS activity is a decrease in protein synthesis and an accumulation of toxic levels of ?-ketoglutarate, which causes the eventual death of the plant. While unmodified wheat is not tolerant to imidazolinone herbicides, BW255-2 and BW238-3 have been modified to survive an otherwise lethal application. BW255-2 and BW238-3 were developed using chemically induced seed mutagenesis and whole plant selection procedures. The herbicide tolerance is due to a single point mutation in the AHAS gene, such that the resulting enzyme has a single amino acid substitution and is no longer affected by imidazolinone herbicides.

Events BW255-2 and BW238-3 have been field tested in multiple locations in Canada in 1999 and in the United States in 2003. Data collected from replicated field trials demonstrated that BW255-2 and BW238-3 did not differ significantly from the parental line in terms of vegetative vigour, time to maturity, seed production (yield), disease resistance, and tendency to weediness.

The potential for transfer of the herbicide tolerant trait from BW255-2 and BW238-3 wheat to other nonmodified wheat plants, or to wild relatives of wheat, has been investigated. Common wheat (T. aestivum) is primarily self-pollinating. While outcrossing can occur by wind-pollination, the rates are usually very low (< 10%). Under the most ideal conditions, outcrossing will occur to nearby plants, but does not usually occur 3 m beyond the source plant. Given the variability of outcrossing rates among genotypes, an isolation distance of 30 m is required for certified seed production.

The potential for introgression of the novel trait into weedy relatives of wheat in Canada and the United States is also very low. Two species closely related to wheat are Aegilops cylindria Host (jointed goat grass) and Agropyron repens (L.) Beauv. (quackgrass). Both are introduced species and are considered weedy and invasive. Recent research has shown a possibility of hybridization between wheat and jointed goatgrass. Introgression of an herbicide tolerant trait from wheat into jointed goatgrass would confer an additional advantage to this weedy species.

The food and livestock safety of BW255-2 and BW238-3 wheat was based on: the evaluation of the similarity of AHAS, in structure and function, to the enzyme naturally present in food and livestock feeds; the lack of toxicity or allergenicity of the modified AHAS. The nutritional equivalence and wholesomeness of BW255-2 and BW238-3 wheat compared to conventional wheat was demonstrated by the analysis of key nutrients in the grain including proximates (e.g., crude protein, crude fat, crude fibre, ash, moisture), branched chain amino acids (valine, leucine and isoleucine), essential amino acids (cystine, methionine, threonine and lysine) fatty acid composition, vitamins and minerals, as well the levels of anti-nutrients (phytic acid and trypsin inhibitor). Significant differences were observed between BW255-2 and BW238-3 and parental controls for some of the tested nutrients, but levels of all parameters measured were within the range of conventional bread wheat varieties.

Links to Further Information Expand

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