1. Glufosinate Ammonium Tolerance<br>Phosphinothricin (L-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 DLL25 codes for PPT- acetyltransferase (PAT). This enzyme detoxifies phosphinothricin by acetylation into an inactive compound. It has extremely high substrate specificity. <br>The PAT gene was originally isolated from Streptomyces hygroscopicus, an aerobic soil actinomycete. The gene was slightly modified to conform with plant codon usage, which did not result in any modification to the expressed protein. The PAT enzyme is naturally occurring in the soil. More generally, acetyltransferases are ubiquitous in nature. <br>The gene is linked to a well characterized constitutive promoter. Expression levels were quantified in leaves, roots, stalk, tassel, cob, husk, kernels, silk and pollen. Highest levels were found in leaves (1.0 - 4.6 mg/g of proteins (f.w.), corresponding to 0.1 - 0.46% of total soluble protein), and no PAT was detected in either silks, pollen or mature seeds. <br>Studies showed that the enzyme was heat and pH labile, and was inactivated within one minute when subjected to typical mammalian stomach and intestinal conditions. <br>The gene nucleotide sequence and the enzyme amino acid sequence were provided. The nucleotide sequence showed no significant homology to toxins or allergens entered in the GenBank DNA database.<br>2. Development Method<br>The Zea mays genotype A188xB73, a cross between two publicly available inbred lines, was transformed through microprojectile bombardment of embryogenic cell cultures. <br>The original transformant was then crossed with Dekalb's elite inbred lines.<br>3. Stable Integration into the Plant's Genome<br>The provided data showed that gene integration occurred at only one insertion site. The insertion site was very well characterized. <br>An antibiotic resistance marker, driven by a bacterial promoter, was used for selection in bacteria. Although this gene was partially integrated into the plant's genome, it is not expressed and the plant is not resistant to the antibiotic. <br>The novel gene was stably inherited over twelve generations.<br>4. Nutrition<br>Forage and grain from DLL25 maize hybrids were analyzed for nutritional composition and compared to the nutritional composition of non-transgenic versions of the same maize hybrids. Proximate and amino acid analyses were performed. Small differences between DLL25 plants and their non-transgenic counterparts were occasionally observed. However, the nutrient composition of DLL25 maize falls within the range of variability for the relevant nutrients reported for maize. The use of corn products derived from DLL25 would therefore have no significant impact on the nutritional quality of the Canadian food supply.