1. Glufosinate Ammonium Tolerance<br>Phosphinothricin (PPT), the active ingredient of glufosinate ammonium, inhibits glutamine synthetase, which results in the accumulation of lethal levels of ammonia in susceptible plants within hours of application. <br>The phosphinothricin tolerance gene engineered into HCN92 codes for PPT-acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. It has extremely high substrate specificity; experimental data clearly showed that neither L-PPT's analog L-glutamic acid, D-PPT, nor any protein amino acid can be acetylated by the PAT enzyme. <br>The PAT gene was originally isolated from Streptomyces viridochromogenes, an aerobic soil actinomycete. The PAT enzyme is therefore naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature. <br>The gene is linked to a constitutive promoter, and protein expression was detected in roots, leaves, buds and seeds. However, it was not detected in stem tissue, protein extracts from the pollen, or unprocessed honey. Maximum expression was 0.001% of total plant protein. <br>The expressed PAT enzyme was compared to the bacterial protein: molecular weights were similar, indicating that the protein had not been glycosylated nor had it undergone post transcriptional modifications. Studies showed that the enzyme was inactivated within one minute when subjected to typical mammalian stomach conditions and was inactivated during processing of canola seed into feed ingredients. <br>The gene nucleotide sequence and the enzyme amino acid sequence were provided. The nucleotide sequence showed no significant homology the toxins or allergens entered in to GENEBANK DNA database.<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 codes for an enzyme that prevents kanamycin from binding to ribosomes, thereby rendering the cells resistant. The exact nature of the enzyme is considered Confidential Business Information by AgrEvo. The source of the gene was described, and the full nucleotide sequence was provided. <br>The gene is linked to a weak constitutive promoter; expression was consistently stronger in root tissue, but was also observed in buds, leaves, and crude seed samples. The enzyme was not detected in unprocessed honey or pollen samples and was inactivated during processing of canola seed into feed ingredients. <br>The expressed enzyme was compared to the bacterial protein: molecular weights were similar, indicating that the protein had not been glycosylated nor had it undergone post-transcriptional modifications. <br>The nucleotide sequence showed no significant homology with the toxins or allergens entered in the GENEBANK DNA database.<br>3. Development Method<br>Brassica napus cultivar Topas was transformed using a disarmed non-pathogenic Agrobacterium tumefaciens vector; the vector contained the T-DNA region of an Agrobacterium plasmid from which virulence and plant disease-causing genes were removed, and replaced with genes coding for glufosinate ammonium tolerance and kanamycin resistance. The T-DNA portion of the plasmid is known to insert randomly into the plant's genome and the insertion is usually stable, as was shown to be the case in HCN92. <br>The transformant was crossed with B. napus line ACSN3, then with AC Excel; HCN92 was derived from a bulk of single F3 plants selected from the cross. <br>4. Stable Integration into the Plant's Genome<br>The provided data showed that there was no incorporation of any coding region from outside the T-DNA borders and that gene integration occurred at only one insertion site. <br>HCN92 is several generations removed from the original transformant. Comparisons between the original transgenic plant and the HCN92 line show no difference in the presence and expression of both genes, nor in the insertion site.