GM Crop Database

Database Product Description

PWC16
Host Organism
Oryza sativa (Rice)
Trait
Imidazolinone herbicide tolerance, specifically imazethapyr.
Trait Introduction
Chemically induced seed mutagenesis
Proposed Use

Production for human food, livestock feed and industrial uses.

Product Developer
BASF Inc.

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 2003 2002

Introduction Expand

The rice line PWC16 was developed to allow the use of imazethapyr, an imidazolinone herbicide, as a weed control option in rice production. This trait was developed using chemically induced seed mutagenesis and whole plant selection procedures. This rice line expresses a mutated form of the acetohydroxyacid synthase (AHAS) enzyme, which renders the plant tolerant to levels of imazethapyr 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 are treated imazethapyr, 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 these amino acids, and an accumulation of toxic levels of alpha-ketoglutarate, all of 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 Whole plant selection of imidazolinone tolerant mutants, followed by introgression of HT trait into commercial varieties through cross-breeding.

Characteristics of Oryza sativa (Rice) Expand

Center of Origin Reproduction Toxins Allergenicity
Northern India and Southeast Asia, and southern China are believed to be the centre of origin of Asian rice (O. sativa). Wild progenitors of African cultivated rice (O. glaberrima) are grasses endemic to West Africa. Basically an autogamous plant propagating through seeds produced by self-pollination. Cross pollination between wild species and O. sativa cultivars has been reported to occur in natural habitats. Antinutrients including phytic acid, trypsin inhibitor, hemagglutinins (lectins) are present in the bran fraction. Allergenic proteins, 14-15 kDa range, present in the albumin plus globulin protein fraction from rice endosperm. Major 16 kDa allergenic protein is a member of the alpha-amylase/trypsin inhibitor family of proteins.

Modification Method Expand

The rice line PWC16 was isolated from a population derived from seed of the variety ‘Cypress’ which had been treated with ethyl methanesulfonate (EMS), a substance know to induce point mutations within the genome of organisms. The selection of herbicide tolerant plants was made from whole plants treated with imazethapyr. The designation of PWC16 was given to the imazethapyr-tolerant plant selected from the population. Seed increase was achieved through self-pollination or natural (wind) pollination occurring among plants of this line.

Characteristics of the Modification Expand

The Introduced DNA

Since PWC16 is a product of mutagenesis and conventional seed increase techniques there was no introduction or incorporation of heterologous DNA into the plant genome. The tolerance to imazethapyr is due to a single point mutation in the AHAS encoding gene such that 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 on AHAS to imazethapyr, such that the herbicide cannot inhibit the enzyme’s activity. This mutation of the AHAS gene was identified by sequencing the gene, which showed the single nucleotide change in the coding sequence for AHAS.

Genetic Stability of the Trait

Segregation analysis of crosses with PWC16 demonstrated that a single semi-dominant gene was inherited. All F1 progeny are resistant to imazethapyr, while F2 progeny segregate in a 1:2:1 ratio (herbicide resistant: intermediate resistant: susceptible to herbicide).

Expressed Material

The modified AHAS gene, conferring tolerance to imazethapyr, is under control of the native AHAS promoter and is believed to be constitutively expressed. Whole plant tolerance to this herbicide was expressed in PWC16 rice.

In the unmodified plant, the levels of valine, leucine and isoleucine are regulated by feedback inhibition. A mutation in the AHAS enzyme could affect the regulation of the biosynthesis of these amino acids. Data were submitted to show that the activity of the modified AHAS was not altered and did not affect feedback inhibition and therefore, the regulation and levels of these amino acids. The amino acid composition of PWC16 was compared to that of commercial cultivars and found to be equivalent in terms of the levels of valine, leucine and isoleucine.

Environmental Safety Considerations Expand

Outcrossing

Species sexually compatible with rice occur in most rice producing areas, especially those within the centre of origin of the cultivated species. In the United the only species known to be compatible with cultivated rice are O. rufipogon, which has been found in a single location in the Everglades of Florida, and red rice (O. sativa ssp.). Due to the relative isolation of O. rufipogon, it is unlikely to hybridize with cultivated rice. Cross-pollination may, however, occur with red rice, under suitable conditions, and form imazethapyr-tolerant hybrids.

PWC16 has not been modified for cultivation in areas outside of its adapted range. Thus, in areas such as Canada, any unintentional release would not be of concern. Species sexually compatible with cultivated rice do not occur in Canada and any seeds unintentionally released in the environment would not persist due to the unfavourable climate.

Weediness Potential

Cultivated rice can become a weed in certain areas where it is grown. PWC16 has not demonstrated characteristics that would confer any competitive advantage, other than tolerance to imazethapyr. Vegetative vigour, time to maturity and seed production of PWC16 were within the normal range of expression of these traits expressed by currently commercialized rice varieties. Tolerance to an herbicide does not, in itself, increase the potential for rice to become weedy or invasive of natural habitats. In the event of the formation of an herbicide tolerant hybrid, there would be no competitive advantage conferred on any hybrid progeny in the absence of sustained imazethapyr use. The imazethapyr-tolerant plant could be controlled by cultural or mechanical means, or by using herbicides other than imazethapyr. PWC16 would not be expected to express greater weediness characteristics than commercial rice varieties.

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 AHAS does not result in altered toxic or allergenic properties. The AHAS enzyme is not a known toxin, does not confer resistance to agricultural pests and is present in plants and micro-organisms with a history of safe use. Based on this information, it was determined that PWC16 will not result in altered impacts on non-target organisms, compared to conventional rice varieties.

Impact on Biodiversity

PWC16 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of rice production. The novel herbicide-tolerant trait could, however, be transferred to unmanaged areas in the United States where species sexually compatible with cultivated rice occur. The herbicide-tolerant trait, in itself, will not render any hybrids weedier than conventional rice-wild species hybrids. The line has also been found safe to non-target organisms and did not display altered plant pest potential since the agronomic and biological characteristics were within the normal range of conventional rice varieties. Furthermore, the novel herbicide-tolerant trait has not altered the ability of PWC16 to persist in areas outside of rice growing areas, such as Canada.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The modification to the AHAS enzyme in PWC16 will not result in any change in the consumption pattern of rice and rice-based products. The availability of many rice varieties for cultivation, the diversity of rice varieties in phenotypic traits, and the normal variation in rice composition due to differences in growing conditions, all result in a wide variation in the composition of conventional rice grain. Thus, the cultivation of PWC16 would not be expected to change the dietary exposure in the United States and Canada, any more than commercially available rice cultivars. Furthermore, PWC16 is expected to be used in similar applications as other rice cultivars by the food industry.

Nutritional and Compositional Data

Nutritional components of line PWC16 were measured analytically and compared to those of its parental line, Cypress. These components included crude fat, crude fibre, crude protein, moisture, amino acids, fatty acids, B vitamins, vitamin E, phosphorous, magnesium, iron and zinc. There were no differences between PWC16 and Cypress in the levels of these nutrients, and the nutritional profile was comparable to other commercial rice varieties.

The levels of the anti-nutritional factors phytic acid and trypsin inhibitor were determined in rice grain samples from PWC16 and Cypress. Phytic acid is indigestible by humans and non-ruminant livestock and inhibits the absorption of iron and other minerals. Trypsin inhibitor interferes with normal protein digestion. No differences were found between the modified and unmodified lines for either anti-nutritional factor.

Toxicity and Allergenicity

The potential for toxicity and allergenicity of PWC16 was investigated by examining the amino acid sequence homology and the characteristics of the altered AHAS protein. 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 AHAS in PWC16 were submitted and showed similar sensitivity to heat and degradability compared to unmodified AHAS. The unmodified form of AHAS showed no amino acid similarity to known allergens. The amino acid sequence of the mutated AHAS differs by only one amino acid compared to unmodified rice. Data were also submitted to show that levels of endogenous rice allergens in PWC16, such as trypsin inhibitor, were similar to those of conventional cultivated rice. Evidence was also provided to show that the protein components of PWC16 were not altered compared to the unmodified parent ‘Cypress.’ Results of SDS-PAGE electrophoresis and HPLC showed 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 PWC16 did not demonstrate any potential for toxicity and allergenicity compared to conventional rice varieties.

Abstract Collapse

This product 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 was required for use in human food and livestock feed, but not for environmental release as these lines were not intended for cultivation in Canada.

Harvested rice, also known as rough rice or paddy rice, is encased by an inedible hull, which is removed prior to milling. Rice hulls are used as fuel, mulch, abrasives, and in animal feed products. Brown rice is what remains after the hulls are removed. The light brown colour of brown rice is due to the presence of bran layers and the rice germ surrounding the rice kernel. Brown rice is further milled to remove the bran and germ to yield white rice, or ‘polished rice.’ The bran and germ are high in protein and other nutrients, and are used in specialty foods, such as rice bran oil, and in livestock feed. Rough rice may be parboiled prior to milling: this involves soaking, steaming and drying the rice. Parboiling improves milling yield and also preserves some of the B vitamin content of the milled rice. Both brown and white rice kernels can be processed into rice flour for use in breakfast cereals, baby foods, desserts and other food products. Rice kernels are also used to produce beer and wine.

Weeds are a significant production problem in rice cultivation. Weeds of rice producing areas, such as the United States, include red rice (Oryza spp.) and barnyard grass (Echinochloa crus-galli (L.) Beauv.). These are typically managed using a combination of cultural practices (e.g., dry vs wet tillage, using clean seed, crop rotations) and chemical controls (herbicides such as molinate and propanil).

The rice line PWC16 was developed to allow the use of imazethapyr, an imidazolinone herbicide, as a weed control option in rice production. The mode of action of imazethapyr 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 in an accumulation of toxic levels of alpha-ketoglutarate, all of which causes the eventual death of the plant. While unmodified rice is not tolerant to imazethapyr, the line PWC16 has been modified to survive an otherwise lethal application of this herbicide. PWC16 was developed using chemically induced seed mutagenesis and whole plant selection procedures. The herbicide tolerance is due to a mutation in the AHAS gene, which codes for an alteration in the binding site for imazethalpyr in the AHAS enzyme.

Cultivated rice is primarily self-pollinating; however, it may cross-pollinate with other cultivated rice varieties, although the rates are less than one percent. Factors limiting cross-pollination include flower morphology (short style and stigma, short anthers), inability of the pollen to remain viable longer than a few minutes, and a lack of insect vectors for pollen spread. In the United States, the only wild species known to be compatible with cultivated rice are O. rufipogon, which has been found in a single location in the Everglades of Florida, and red rice (O. sativa ssp.). Due to the relative isolation of O. rufipogon, it is unlikely to hybridize with cultivated rice. Cross-pollination may, however, occur with red rice under suitable conditions, form imazethapyr-tolerant hybrids. Although PWC16 is not intended for cultivation in Canada, any unintentional release would not be of concern since there are no species that are sexually compatible with rice, and due to environmental conditions, the plants would not persist.

Data submitted on the vegetative vigour, time to maturity and seed production of PWC16 were found to be within the normal range of expression of these traits displayed by unmodified rice varieties. It was determined that the tolerance to imazethapyr in PWC16 will not render it weedy, invasive of natural habitats, or allow it to grow outside of its adapted environment, since none of the reproductive or growth characteristics were modified.

The food and livestock safety of PWC16 rice was established based on: the evaluation of the similarity of AHAS, in structure and function, to the enzyme naturally present in food and livestock feeds; the fact that the modified AHAS constitutes a small amount of the protein in PWC16 rice, so there is little dietary exposure; the lack of toxicity or allergenicity of AHAS from plants; and, studies on the function of this enzyme in PWC16 compared to its unmodified counterpart. The nutritional equivalence and wholesomeness of PWC16 was demonstrated by the analysis of key nutrients, including proximates (protein, crude fat, crude fibre, ash and carbohydrates), amino acid and fatty acid composition, vitamins, as well as anti-nutrients.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biosafety Office Health Canada, Novel Foods

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