GM Crop Database

Database Product Description

23-18-17, 23-198 (CGN-89465-2)
Host Organism
Brassica napus (Argentine Canola)
Trade Name
Laurical™
Trait
Modified seed fatty acid content, specifically high laurate levels and myristic acid production.
Trait Introduction
Agrobacterium tumefaciens-mediated plant transformation.
Proposed Use

Production for human consumption, livestock feed, industrial applications.

Product Developer
Monsanto Company

Summary of Regulatory Approvals

Country Food Feed Environment Notes
Canada 1996 1996 1996
United States 1994 1994 1994

Introduction Expand

The 23-198 and 23-18-17 lines of canola (Brassica napus) were developed through a specific genetic modification to produce oil that contains significant levels of lauric acid. The novel varieties were developed from the 212/86 canola variety by introduction of a gene from the California bay tree, the leaves of which are an alternative source of the spice “bay leaf” which is commercially harvested from Laurus nobilis. The introduced gene encodes a thioesterase enzyme that is active in the fatty acid biosynthetic pathway of the developing seed resulting in the accumulation of triacylglycerides containing esterified lauric acid (12:0) and, to a lesser extent, myristic acid (14:0). The processed oil derived from these novel varieties has a level of lauric acid similar to that of coconut ant palm kernel oil.

Summary of Introduced Genetic Elements Expand

Code Name Type Promoter, other Terminator Copies Form
Bay TE thioesterase FA

seed-specific promoter

15 copies at 5 loci (original transformant)

Native

nptII neomycin phosphotransferase II SM CaMV 35S tml3' from Agrobacterium tumefaciens 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.

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

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Modification Method Expand

The 23-198 and 23-18-17 lines were created by Agrobacterium-mediated transformation of line 212/86 in which the transfer-DNA (T-DNA) contained the gene encoding the enzyme 12:0 ACP thioesterase (bay TE) from the California bay tree (Umbellularia californica). Expression of the bay TE gene was under the regulation of a seed specific plant promoter. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII). The expression of NPTII activity, regulated by the CaMV 35S promoter, was used as a selectable trait to screen transformed plants for the presence of the bay TE gene. No other translatable DNA sequences were incorporated into the plant genome.

Characteristics of the Modification Expand

The Introduced DNA

Southern blot and segregation analysis of the genomic DNA from the original transformation event 23 indicated the presence of at least 15 copies of the bay TE gene, inserted at five independent loci.

Genetic Stability of the Introduced Trait

Lines 23-198 and 23-18-17 were several generations removed from the original transformant 23 and comparisons of the original transformant with third-generation material showed that the introduced genes were stably inserted and stably inherited. It was anticipated that due to the high copy number of the bay TE gene, some copies would segregate out and that the lines may not be homogeneous for the number of copies present.

Expressed Material

The bay TE gene is linked to a seed specific promoter limiting expression to developing seed embryos. The bay TE gene encodes a 382 amino acid long preprotein that is transported to the stroma of plastids where a 60 amino acid transit peptide is removed to produce the mature Bay TE enzyme. Using Western blot analysis, the expression of Bay TE was monitored over the seed maturation process and was detectable only in seeds samples at mid-maturity at a level of 0.015% of the total protein. Dry seeds and any other plant parts have immunologically undetectable levels of the enzyme. The NTPII encoding gene was linked to a strong constitutive promoter and expression of the protein was detected in seeds and other tissues of transgenic canola. Its presence was judged to be insignificant with respect to any human health risk.

Environmental Safety Considerations Expand

Field Testing

The canola lines 23-198 and 23-18-17 were field tested in the United States (1991-1992) and Canada (1993 to 1995). Agronomic and adaptation characteristics such as seed germination, seed yield, seedling growth, flowering and maturity dates, were within the normal range of expression of characteristics in unmodified counterparts. The laurate canola lines were generally taller, later maturing and lower yielding, reflecting characteristics of the parent line 212/86. Disease incidence (i.e. stem rot, blackleg, black spot, black rot, powdery mildew, gray stem, damping off, turnip mosaic, cauliflower mosaic, crown gall) and insect susceptibilities (flea beetles and aphids) were monitored during field trials and in greenhouses and were shown to the same as those of unmodified counterparts. The only significant consistent difference between high laurate canola and the parent variety was the increase in laurate content from less than 0.1% to greater than 10%. The high laurate B. napus lines are grown under contract to preserve their identity and prevent mixing with conventional canola. Overall the field data reports demonstrated that canola lines 23-198 and 23-18-17 had no potential to pose a plant pest risk.

Outcrossing

B. napus plants, including lines 23-198 and 23-18-17, 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. Other studies concluded that transgenic canola was not any more aggressive than nontransgenic canola, was not invasive of undisturbed habitats, and did not persist in the environment into which it was introduced. It was concluded that gene flow from the transgenic lines 23-198 and 23-18-17 to canola relatives was possible, but would not result in increased weediness or invasiveness of these relatives.

Weediness Potential

The introduced trait, laurate content, was determined to be extremely unlikely to increase weediness of canola lines 23-198 and 23-18-17. It was determined that the increased levels of lauric acid did not confer a competitive advantage to canola lines 23-198 and 23-18-17 over non-transgenic varieties since the novel trait did not confer any pest resistance, alter reproductive biology or change any physiology related to survival. Other studies on fitness characteristics determined that there was no obvious increase in volunteers from seed, increase in seed dormancy, or other variations indicative of increased weediness.

Secondary and Non-Target Adverse Effects

It was determined that there was no reason to believe that deleterious effects or significant impacts on nontarget organisms, including beneficial organisms, would result from the cultivation of high laurate canola. Neither lauric acid, nor the gene that produces increased laurate, nor the gene for aminoglycoside resistance were found to have any toxic or allergenic properties. Both Bay TE and NPTII enzymes are ubiquitous in nature and no adverse effects have been reported to be associated with either enzyme.

Impact on Biodiversity

Canola lines 23-18-17 and 23-198 have no novel phenotypic characteristics that would extend their use beyond the current geographic range of canola production. Since outcross species are only found in disturbed habitats, transfer of novel traits would not impact unmanaged environments. It was concluded that the potential impact on biodiversity of high laurate canola lines 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. The increased levels of lauric acid in the oil allow its use as a replacement for other lauric acid oils such as coconut and palm kernel oil in products such as confectionery coatings and fillings, margarines, spreads, shortenings and commercial frying oils. The level and composition of laurate produced by lines 23-18-17 and 23-198 was no different from that in common food sources, such as coconuts and palm oil.

Nutritional Data

The modified fatty acid composition of the oil from transgenic canola lines 23-198 and 23-18-17 does not raise any nutritional concerns regarding the intended use of the subject oil. The analysis of levels of erucic acid and glucosinolates were within acceptable levels for canola quality. Similarly, the quality and quantity of seed protein were as expected. The fatty acid profile of high laurate canola was different from all control varieties, with increased levels of lauric acid (up to 40%) and myristic acid, and lower levels of oleic acid and linoleic acid, and less palmitic acid, less stearic, linoleic, arachidic, gadoleic, and slightly more behenic acid. Nonetheless, the overall lipid content of the seed remained unchanged. The consumption of products formulated with this oil would have no significant impact on the nutritional quality of the food supply in North America and Canada. No statistical differences in crude protein, crude fibre, gross energy content, and amino acids were noted between the processed meal of high laurate and control B. napus cultivars. Based on a maximum of 4% oil in the meal, the meal was expected to have 1.6% laurate and 0.16% myristate. As the use rate of meal in livestock rations ranges from 5-25%, the maximum laurate and myristate in meal would be 0.4% and 0.94% respectively. These levels would have no significant impacts on nutrition or carcass quality. In addition, the presence of the aminoglycoside resistance gene in livestock feed would not compromise the efficacy of added antibiotics. Neomycin activity when mixed with meal from high laurate canola remained stable for a period of 56 days responding in the same manner as the control line. It was determined that canola meal derived from lines 23-18-17 and 23-198 was equivalent to meal from traditional B. napus varieties in terms of nutritional composition and safety.

Toxicity and Allergenicity

The low potential for toxicity of the thioesterase (Bay TE) and NPTII enzymes expressed in the transgenic canola lines 23-18-17 and 23-198 was demonstrated by studies on the amino aid sequence homology and characteristics of the proteins. The amino acid sequences of the Bay TE and NPTII enzymes were compared with sequences available in GENBank databases and no significant homology was found with any known sequenced toxins or allergens. Significant homology was found with thioesterases from edible plant species. Studies showed that the introduced Bay TE enzyme was not heat stable and was fully degraded into inactive peptides and amino acids following protease digestion. Two potential glycosylation sites were identified but glycosylation of plastid proteins is not known to occur. Similarly, the NPTII enzyme was found to degrade rapidly in simulated mammalian gastric and intestinal fluids. The NPTII protein was not expected to have toxic properties since: the enzyme catalyses only a very specific reaction using aminoglycosidic antibiotics as a substrate; despite wide usage, there have been no reports of toxicity of the protein in the literature; and enzyme levels are less than 0.0008% of total protein in seeds and leaves, and undetected in meal. Both Bay TE and NPTII enzymes are ubitiquitous in nature and no toxic or allergenic effects have ever been reported.

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 canola lines 23-18-17 and 23-198 were genetically engineered to express modified seed fatty acid content, specifically high levels of lauric acid and myristic acid. The increased levels of lauric acid in oil from the modified canola lines allow for its use as a replacement for other lauric acid oils, such as coconut and palm kernel oil, in products such as confectionery coatings and fillings, margarines, spreads, shortenings and commercial frying oils. The modified fatty acid content of the canola lines 23-18-17 and 23-198 is a result of the insertion of a thioesterase (TE) encoding gene from the California bay tree,Umbellularia californica, which is an alternative source of the spice “bay leaf” that is harvested commercially from Lauris nobilis. The introduced thioesterase enzyme is active in the fatty acid biosynthetic pathway of the developing seed and causes the accumulation of triacylglycerides containing esterified lauric acid and, to a lesser extent, myristic acid. The canola lines 23-198 and 23-18-17 were field tested in the United States from 1991 to 1992, and in Canada from 1993 to 1995. Agronomic and adaptation characteristics such as seed germination, seed yield, seedling growth, and flowering and maturity dates were within the normal range of expression found in conventional canola varieties. The laurate canola lines were generally taller, later maturing and lower yielding, reflecting characteristics of the parent line. Disease incidence and insect susceptibilities were shown to the same as those of unmodified counterparts. The only significant difference between high laurate canola and the parent variety was the modified laurate content. Overall the field data reports demonstrated that the transformed canola lines did not exhibit weedy characteristics, or negatively affect beneficial or non-target organisms. Canola lines 23-18-17 and 23-198 were not expected to impact on threatened or endangered species. Brassica napus may outcross (up to 30% of the time) with 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 cross breeding is most likely to occur with B. rapa. The ability of rapeseed to outcross with related plants makes the formation of high laurate content hybrids possible. However, the high laurate content trait is not expected to provide a competitive advantage to hybrid plants, and the high laurate B. napus lines are grown under contract to preserve their identity and prevent mixing with conventional canola. Any hybrids that do arise would be found in managed environments and could easily be controlled using mechanical and/or chemical means. The human consumption of canola products is limited to the refined oil. The level and composition of laurate produced by lines 23-18-17 and 23-198 was no different from that found in common food sources, such as coconut and palm oil. The modified fatty acid composition of the oil from transgenic canola lines 23-198 and 23-18-17 does not raise any nutritional concerns regarding its intended use. The analysis of levels of erucic acid and glucosinolates, as well as the quality and quantity of seed protein, were within acceptable levels for canola. The fatty acid profile of high laurate canola was different from all control varieties, with increased levels of lauric acid (up to 40%) and myristic acid, and lower levels of oleic acid and linoleic acid, as well as less palmitic, stearic, linoleic, arachidic, and gadoleic acids, and slightly more behenic acid. Nevertheless, the overall lipid content of the seed remained unchanged. It was concluded that the consumption of products formulated with this oil would have no significant impact on the nutritional quality of the food supply in North America and Canada. No statistical differences in crude protein, crude fibre, gross energy content, or amino acids were noted between the processed meal of high laurate lines and control B. napus cultivars. The levels of both laurate and myristate oils were within the accepted ranges for canola meal used in livestock feed. It was determined that canola meal derived from lines 23-18-17 and 23-198 was equivalent to meal from traditional B. napus varieties in terms of nutritional composition and safety. Potential toxicity and allergenicity of the Bay TE enzyme expressed in the transgenic canola lines 23-18-17 and 23-198 was investigated by searching for amino acid sequence homologies with known toxins and allergens, and by examining their physiochemical properties. No homologies between the amino acid sequences of the Bay TE enzyme and the sequences of known toxins or allergens were detected. Bay TE does not possess the proteolytic or heat stability characteristic of toxic compounds, and was readily digested under conditions simulating mammalian digestion. Thioesterase enzymes like Bay TE are widespread in nature and no toxic or allergenic effects have ever been reported. Based on these properties, it was concluded that Bay TE enzyme possessed little or no potential for allergenicity or toxicity.

Links to Further Information Expand

Canadian Food Inspection Agency, Plant Biotechnology Office Office of Food Biotechnology, Health Canada U.S.Department of Agriculture, Animal and Plant Health Inspection Service US Food and Drug Administration USDA-APHIS Biotechnology Regulatory Services USDA-APHIS Environmental Assessment

This record was last modified on Friday, March 27, 2015