<p style="text-align: justify;">The 23-198 and 23-18 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 &ldquo;bay leaf&rdquo; 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 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. 1. Production of High Levels of Laurate in Seed Oil A gene from the California bay (Umbellularia californica) was introduced into the B. napus cultivar 212/86. This gene codes for a thioesterase (Bay TE), an enzyme active in the fatty acid biosynthetic pathway of the developing seed. The gene is linked to a seed specific promoter, active only in developing seed embryos. As a consequence, expression occurs only in maturing seeds. Dry seeds and any other plant parts have immunologically undetectable levels of the enzyme. Gene expression results in the production of 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. This enzyme accounts for 0.015% of the total extractable protein at mid-maturation of the developing seed. Studies showed that the introduced Bay TE enzyme is not heat stable and is 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. Thioesterases are ubiquitous in plants and animals and Bay TE is not expected to be toxic or allergenic to non-target organisms. When subjected to comparative analyses with genetic sequence databases, no significant homology was found with any known sequenced toxins or allergens, whereas significant homology was found with thioesterases from edible plant species. The full nucleotide sequence of the gene was provided. Activity of the Bay TE results in the accumulation of the saturated fatty acid laurate (12:0), and to a lesser extent, of the saturated fatty acid myristate (14:0), in triacylglycerol molecules of seed oil, at the expense of oleic acid and linoleic acid. 2. Neomycin phosphotransferase II (NPTII) Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell. The kanamycin-resistance gene (neo), isolated from the bacterium Escherichia coli, codes for an enzyme that prevents kanamycin from binding to ribosomes, through phosphorylation, thereby rendering the cells resistant. It is very specific and is not known to affect any plant metabolic pathway. The gene is linked to a strong constitutive promoter. Dry seeds have immunologically undetectable levels of the enzyme. The enzyme is ubiquitous in the environment. It degrades rapidly in simulated mammalian gastric and intestinal fluids. The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database. Toxic properties are not expected since: (1) the enzyme catalyses only a very specific reaction using aminoglycosidic antibiotics as a substrate, (2) despite wide usage, there have been no reports of toxicity of the protein in the literature and (3) enzyme levels are less than 0.0008% of total protein in seeds and leaves, and undetected in meal. The full nucleotide sequence of the gene was provided. The presence of the kanamycin resistance gene in livestock feed should 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, and was not different as compared to the control line. 3. Development Method The 23-198 and 23-18 lines were created by Agrobacterium-mediated transformation 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). The seed specific expression of this gene was regulated by the inclusion of sequences from the promoter and 3&rsquo;-polyadenylation signal regions of the napin gene from B. rapa. In addition, the T-DNA contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII) from the Tn5 transposon of Escherichia coli, strain K12. The expression of NPTII activity 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. The data from several generations of backcrossing demonstrated that the ACP thioesterase encoding gene was stably inherited. The original transformation event 23 was estimated to have 15 copies of the genes, at five independent genetic loci, as shown by Southern and segregation analyses. Lines 23-198 and 23-18-17 are several generations removed from the original transformant. Mendelian inheritance, and Southern and PCR analyses of third-generation material show the stability of the introduced genes as the bands did not change position. Some of the copies are segregating out, as expected, so that these lines may not be homogeneous for the number of copies present.</p>

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