1. Tolerance to Soil Residues of Triasulfuron and Metsulfuron-methyl<br>Sulfonylurea herbicides, such as triasulfuron and metsulfuron-methyl, target and bind to the enzyme acetolactate synthase (ALS) thereby inhibiting the biosynthesis of the branched chain amino acids valine, leucine and isoleucine and resulting in the accumulation of toxic levels of alpha-ketoglutarate. <br>In addition to its native ALS gene, CDC Triffid contains an als gene from a chlorsulfuron tolerant line of A. thaliana. This variant als gene differs from the wild type A. thaliana gene by one nucleotide and the resulting ALS enzyme differs by one amino acid from the wild type ALS enzyme. The inserted als gene is linked to its native promoter and terminator. <br>Enzyme extracts from CDC Triffid exhibited a slightly higher ALS activity compared to its non-modified counterpart cv. Norlin. Whereas the statistical significance of this higher activity could not be verified, it may be expected due to the presence of at least two additional copies of the als gene in CDC Triffid.<br>2. Kanamycin Resistance<br>Kanamycin is an aminoglycosidic antibiotic that binds to bacterial ribosomes thus disrupting normal protein synthesis and killing the bacterial cell. <br>The kanamycin-resistance gene, neo, isolated from the bacterium Echerichia coli, codes for an enzyme, neomycin phosphotransferase II (NPTII) that phosphorylates kanamycin. This prevents kanamycin from binding to ribosomes and renders the cells resistant to the antibiotic. Plant cells that have been co-cultured with A. tumefaciens are plated onto a growth medium containing kanamycin. Those plant cells that have been successfully transformed and thus contain the kanamycin-resistance gene, are able to grow on this medium. <br>The nptII gene is linked to a constitutive promoter. The CDC has demonstrated, using PCR analysis, that the inserted nptII gene was unaltered when compared to the native E. coli gene. The gene product, NPTII, was detected in seeds, cotyledons and leaves of CDC Triffid. <br>NPTII is ubiquitous in the environment. It degrades rapidly in vitro in simulated mammalian gastric and intestinal fluids.<br>3. Nopaline Synthase<br>Nopaline synthase is an enzyme which catalyses the synthesis of nopaline, an opine which is formed as the result of the condensation of the amino acid arginine and alpha-ketoglutaric acid. When wild-type A. tumefaciens infects a host plant, the opine synthase gene present on the T-DNA region of the Ti plasmid of the bacterium directs infected host cells to synthesize an opine, such as nopaline. The type of opine produced is specific to the particular strain of A. tumefaciens. Opines are metabolized as a source of carbon and nitrogen only by a bacterium possessing a Ti-plasmid and the gene specific for catabolism of the particular opine. <br>The nopaline synthase gene, nos, is linked to its native promoter which is known to be constitutive with a slightly stronger expression in roots. <br>Levels of nopaline were quantified at two growth stages in CDC Triffid: seedling and pre-flowering, and was detected in roots, stems and leaves. Expression in roots ranged from 0.05 to 0.63 mg of nopaline/g tissue (fresh weight) at the seedling stage, and 0.07 to 0.47 mg/g (f.w.) at pre-flowering; in stems, at 0.01 to 0.33 mg/g (f.w.) at the seedling stage, and 0.02 to 0.12 mg/g (f.w.) at pre-flowering; and leaves, at 0.05 to 2.4 mg/g (f.w.) at the seedling stage, and 0.23 to 0.72 mg/g (f.w.) at pre- flowering. Nopaline was not detected in seeds. <br>A sequence homology search for nopaline synthase revealed similarity only to other opine synthases and no similarity was found to known sequenced toxins or allergens. <br>This trait was introduced to permit the identification of transformed plant embryos. Nopaline was detected in successfully transformed plant embryos.<br>4. Other novel DNA sequences<br>Genes conferring resistance to the antibiotics ampicillin, carbenicillin and spectinomycin were present in an E. coli vector used as an intermediary during the cloning of the target sequences to be transferred to flax. These three antibiotic resistance genes were under the control of a bacterial promoter which is active only in bacteria and not active in plants. These genes are therefore not expected to be expressed in CDC Triffid. Portions of Hind III fragments 10 and 23 (Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M., and Schell, J. 1983. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO 2:2143-2150.) from the nopaline Ti plasmid pTiC58 were also incorporated. <br>5. Development Method<br>The flax cv. Norlin was transformed using the disarmed A. tumefaciens Ti-plasmid vector pGV3850. The vector contained the T-DNA region of an A. tumefaciens plasmid from which oncogenic regions had been deleted and replaced with the mutant als gene and the kanamycin resistance gene. <br>Successful transformants were selected in vitro, on the basis of expression of kanamycin resistance and presence of nopaline. These were subsequently grown on medium containing chlorsulfuron to confirm the expression of the inserted chlorsulfuron-tolerant als gene. <br>6. Stability of Insertion of the Traits<br>Data were obtained from the literature (Mc Sheffrey, S. A., Mc Hughen, A., and Devine, M. D. 1992. Characterization of transgenic sulfonylurea-resistant flax (Linum usitatissimum). Theoretical and Applied Genetics 84:480-486.) showing that the genes inserted into CDC Triffid were integrated in at least two unlinked loci, with a possible third locus exhibiting partial linkage to one of the other two. The expression of the genes remained stable after eight generations of selfing CDC Triffid plants.