GM Crop Database

Database Product Description

OXY-235 (ACS-BNØ11-5)
Host Organism
Brassica napus (Argentine Canola)
Trade Name
Westar OXY-235
Trait

Herbicide tolerant, oxynil.

Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption and livestock feed.

Product Developer
Aventis CropScience (formerly Rhône Poulenc Inc.)

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Australia 2002
Canada 1997 1997 1997
China 2004 2004
Japan 1999 1999 1998
United States 1999

Introduction Expand

Canola (Brassica napus) line Oxy-235 (ACS-BNØ11-5) was developed to allow the use of oxynil herbicides as weed control options in canola. Bromoxynil is very effective on broadleaf weeds that are common in canola fields and the deployment of transgenic bromoxynil-tolerant canola will allow the post-emergence control of these weeds without crop injury.

The herbicides ioxynil (3,5-di-iodo-4-hydroxybenzonitrile) and bromoxynil (3,5-dibromo-4-hydorxybenzonitrile) act on conventional dicotyledenous plants by blocking electron flow during the light reaction of photosynthesis (photosystem II). This causes super oxide production resulting in the destruction of cell membranes and an inhibition of chlorophyll formation, resulting in plant death.

Tolerance to oxynil herbicides in Oxy-235 was achieved by introduction of the bxn gene isolated from the bacterium Klebsiella pneumoniae (sub. sp ozaenae), which encodes a nitrilase enzyme that hydrolyzes oxynil herbicides to non-phytotoxic compounds.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
bxn nitrilase HT CaMV 35S Enhancer: non-translated leader of RuBisCO small subunit gene derived from maize A. tumefaciens nopaline synthase (nos) 3'-untranslated region 1 Native

Characteristics of Brassica napus (Argentine Canola) Expand

Center of Origin Reproduction Toxins Allergenicity

The species is native to India.

Canola flowers can self-pollinate, and they can also be cross-pollinated by insects and by wind.­

Brassica species can contain erucic acid and various glucosinolates, which can be toxic. However, commercial canola varieties have been bred to reduce the levels of these substances. Canola may contain elevated levels of tannins, which reduce the digestibility of seed protein, and sinapine, which is a bitter substance that can reduce the palatability of feeds made from canola meal.

­

Occupational exposure to pollen and seed flour have been associated with allergic reactions in humans. There are no known allergic reactions to canola oil.

­

Donor Organism Characteristics Expand

Latin Name Gene Pathogenicity
Klebsiella ozaenae bxn Member of the Enterobacteriaceae, a group of facultative gramnegative bacteria, classified as a Class 2 microorganism. This class contains microorganisms that could potentially cause disease in humans, however no known pathogenicity exists for the subspecies ozaenae. Klebsiella bacteria are widely distributed in nature, occurring naturally in the soil, water and in grain and are normal inhabitants of the intestinal tract

Modification Method Expand

Oxy-235 was produced by Agrobacterium-mediated transformation of the B. napus cultivar 'Westar' with plasmid pRPA-BL-150a using the EHA 101 strain of A. tumefaciens. Transformed plants were regenerated in tissue culture using bromoxynil as the sole selective agent. The original transformationevent --Westar-Oxy-235-- was used as a parental line in the production of commercial canola varieties marketed under the Navigator™ name.
Plasmid pRPA-BL-150a was a double border binary Ti vector containing one copy of the bxn gene (1150 bp) under the control of the CaMV 35S promoter and an enhancer region from the non-translated portion of the RuBisCo SSU gene from maize. The construct also contained the nopaline synthase 3' non-coding regions from A. tumefaciens (plasmid pTi37).

Characteristics of the Modification Expand

The Introduced DNA

Southern blot analysis of genomic DNA from Oxy-235 indicated that one complete copy of the bxn gene was integrated into the host genome at a single insertion site. The insert number was determined following digestion of genomic DNA with EcoRI or HindIII, which gave rise to a single hybridizing band in each case. No hybridizing bands were observed with restriction enzyme digested genomic DNA from non-transgenic control plants, and when Oxy-235 DNA was double-digested with EcoRI and HindIII a single hybridizing fragment of 1150 bp was detected. Additional Southern analyses using probes specific for the ori-322 region of pBR322 and polymerase chain reaction (PCR) amplification analysis for gentamycin encoding sequences demonstrated that these additional plasmid-derived sequences (located outside of the T-DNA region) were not incorporated into the host genome.

Genetic Stability of the Introduced Trait

Segregation analysis was consistent with a single site of insertion of the bxn gene into the Oxy-235 genome, as shown by a 3:1 ratio of susceptible to tolerant plants. Comparative analysis of third-generation progeny material, using Southern blots and PCR analysis, determined that the introduced DNA was stably inserted and stably inherited.

Expressed Material

Levels of nitrilase enzyme were quantified in samples of transgenic leaf (1000 ng/mg total protein) and seed (<10 ng/mg total protein) tissue by Western immunoblot analysis (detection of single 37 kDa species). No nitrilase was detectable in samples of refined oil (detection limit 20 ppb).

Environmental Safety Considerations Expand

Field Testing

The Oxy-235 line was field tested in Canada from 1992 to 1996 and assessed for a number of agronomic characteristics, including seed yield, days to maturity, silique shattering, and overwintering capacity. In comparison with non-transgenic commercial canola varieties, all of these parameters were within the normal range of expression. Stress adaptation was evaluated, including resistance to major B. napus pests such as the fungal pathogen Leptosphaeria maculans (blackleg) and the susceptibility of Oxy-235 was within the ranges currently displayed by commercial varieties. The only significant difference between Oxy-235 canola and the parental non-transformed variety was that Oxy-235 exhibited field tolerance to ioxynil and bromoxynil.

Outcrossing

B. napus plants are known to outcross up to 30% with other plants of the same species, and potentially with plants of related species B. rapa, B. juncea, B. carinata, B. nigra, Diplotaxis muralis, Raphanus raphanistrum, and Erucastrum gallicum. Previous studies have demonstrated that gene flow is most likely to occur with B. rapa.
The bxn gene is not expected to confer an ecological advantage to potential hybrid offspring. If tolerant individuals arose through interspecific or intergeneric hybridization, the novel trait would confer no competitive advantage to these plants unless these populations were routinely subject to herbicide treatments. This may occur in managed ecosystems where oxynil herbicides are applied for broad spectrum weed control, or when used to control weeds in crop plants which exhibit herbicide tolerance such as cereals, or plant varieties genetically engineered for tolerance. In the event that a tolerant plant survived, the herbicide-tolerant individual would be easily controlled using mechanical and other available chemical means. This can be avoided by the use of sound crop management practices including both crop and herbicide rotations.

Weediness Potential

No competitive advantage was conferred to Oxy-235 canola, other than that conferred by resistance to ioxynil and bromoxynil herbicides. Resistance to these herbicides did not confer any pest resistance, alter reproductive biology or change any physiology related to survival. In the event that a tolerant plant survived, the herbicide-tolerant individual would be easily controlled using mechanical and/or alternative herbicides with different modes of action such as 2,4-D and MCPA. It was concluded that Oxy-235 canola had no altered weed or invasiveness potential compared to currently commercialized B. napus varieties.

Secondary and Non-Target Adverse Effects

It was determined that Oxy-235 did not have a significant adverse impact on interacting organisms including humans. The nitrilase protein expressed in Oxy-235 did not result in altered toxicity or allergenicity properties as determined from studies using simulated digestive fluids, nutritional composition and amino acid sequence homology studies.

Impact on Biodiversity

Oxy-235 has no novel phenotypic characteristics that would extend its use beyond the current geographic range of canola/rapeseed production. Since most related species are only found in agricultural ecosystems, the transfer of novel traits would not have an impact on unmanaged environments. It was determined that the relative impact on biodiversity from Oxy-235 was equivalent to that of currently commercialized canola lines.

Food and/or Feed Safety Considerations Expand

Dietary Exposure

The human consumption of canola products is limited to the refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortening, salad oil and cooking oils. Refined edible canola oil consists of purified triglycerides (96-97%) and does not contain any detectable protein. As the nitrilase protein was not found in the refined oil produced from transgenic canola Oxy-235, it can be concluded that there will be no human exposure to these proteins. The genetic modification of Oxy-235 canola will not result in any change in the consumption pattern for this product.

Nutritional Data

The nutritional composition of Oxy-235 was shown to be equivalent to non-transgenic canola. There were no significant differences in the fatty acid composition of oil from Oxy-235 and unmodified counterparts and all values were within the range of quality standards for canola. It was also found that the modification did not change the levels of sterols, tocopherols, and unsaponifiable matter in crude oil.
An analysis of the nutrient value of canola meal from Oxy-235 and non-transgenic canola did not reveal any significant differences in the levels of dry matter, crude fibre, neutral detergent fibre (NDF), acid detergent fibre and lignin (ADF and ADL), crude protein, total fat, soluble sugars, amino acid content as well as gross energy level in seeds and meal. Seeds of Oxy-235 were shown to have the same levels of glucosinolates and erucic acid as commercial canola varieties.

A 28-day rat feeding study was conducted where rats were fed up to 10% canola meal in their food. There were no toxic effects from the study and results were the same regardless of whether the source of canola meal was Oxy-235 or non-transgenic canola.

Toxicity and Allergenicity

It was determined that there were no toxicity or allergenicity concerns with Oxy-235, since refined canola oil is the only product for human consumption and does not contain any detectable amount of protein. The absence of toxicity was further demonstrated by examining the amino aid sequence homology and the characteristics of the novel nitrilase enzyme.

The amino acid sequence of the nitrilase protein was compared to the amino acid sequences of known protein toxins and allergens and no significant similarities were found. Furthermore, the nitrilase protein did not share characteristics common to allergens.

Digestive fate studies demonstrated that the nitrilase protein was rapidly inactivated in mammalian stomach and intestinal fluids by enzymatic degradation and pH-mediated proteolysis. To confirm the absence of toxic effects from nitrilase protein, a 14-day sub-acute toxicity feeding study was conducted with mice feed bacterial nitrilase protein. There were no toxic effects observed.

Abstract Collapse

Argentine or oilseed rape (Brassica napus) was grown as a commercial crop in over 50 countries, with a combined harvest of 48.9 million metric tonnes in 2006. The major producers of rapeseed are China, Canada, India, Germany, France, the United Kingdom and Australia. Canola is a genetic variation of B. napus that was developed through conventional breeding to contain low levels of the natural rapeseed toxins, glucosinolate and erucic acid. Canola is grown for its seed, which represents a major source of edible vegetable oil and is also used in livestock feeds.
The only food use of canola is as a refined oil. Typically, canola oil is used by itself as a salad oil or cooking oil, or blended with other vegetable oils in the manufacture of margarine, shortenings, cooking and salad oils. Canola meal, a byproduct of the oil production process, is added to livestock feed rations. An increasing amount of oil is being used for biodiesel production, especially in Europe.

The removal of weeds early in the growing season is extremely important in canola production. Young canola seedlings are not very competitive and early weed pressure has detrimental effects on final yield. Precautions such as pre-plant tillage or herbicide application are common approaches for reducing weed competition. Once established, canola forms a dense canopy and is very competitive, making weed control less of a concern.

Line Oxy-235 (ACS-BNØ11-5) was developed to allow the use of oxynil herbicides, ioxynil and bromoxynil, as weed control options in canola. Bromoxynil is very effective on broadleaf weeds that are common in canola fields and the deployment of transgenic bromoxynil-tolerant canola will allow the post-emergence control of these weeds without crop injury.

Transgenic Oxy-235 contains a single copy of the bxn gene isolated from the bacterium Klebsiella pneumoniae (sub. sp ozaenae), which encodes a nitrilase enzyme that hydrolyzes oxynil herbicides to non-phytotoxic compounds. Constitutive expression of the bxn gene was regulated via the 35S promoter from cauliflower mosaic virus (CaMV) and levels of nitrilase enzyme were detected in samples of leaf (1000 ng/mg total protein) and seed (The Oxy-235 line was field tested in Canada from 1992 to 1996 and assessed for a number of agronomic characteristics, including seed yield, days to maturity, silique shattering, and overwintering capacity. In comparison with non-transgenic commercial canola varieties, all of these parameters were within the normal range of expression. Stress adaptation was evaluated, including resistance to major B. napus pests such as the fungal pathogen Leptosphaeria maculans (blackleg) and the susceptibility of Oxy-235 was within the ranges currently displayed by commercial varieties. The only significant difference between Oxy-235 canola and the parental non-transformed variety was that Oxy-235 exhibited field tolerance to ioxynil and bromoxynil.

As the nitrilase protein was not found in the refined oil produced from transgenic canola Oxy-235, it can be concluded that there will be no human exposure to this proteins. The genetic modification of Oxy-235 canola will not result in any change in the consumption pattern for this product.

Further studies were conducted to assess the potential toxicity of the introduced nitrilase protein. These included an acute oral toxicity study in mice, amino acid sequence comparisons with known protein toxins, an evaluation of the prior human history of ingestion of similar enzymes and the potential toxicity of bromoxynil metabolites. None of these studies indicated a potential for human toxicity. Similarly, studies on digestive fate (stability to pepsin digestion) and similarity to known allergens demonstrated that the nitrilase enzyme is rapidly degraded (<= 15 s upon exposure to simulated gastric fluid) and has no homology with known allergens.

Links to Further Information Expand

Australia New Zealand Food Authority Canadian Food Inspection Agency, Plant Biotechnology Office Office of Food Biotechnology, Health Canada U.S. Food and Drug Administration

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