<p style="text-align: justify;"><b>The Introduced DNA</b></p>

<p style="text-align: justify;">Southern blot analysis of genomic DNA from the original transformant (event 260-05) identified inserts at three loci which were designated as locus A, B and C. At one locus (locus A), the GmFad2-1 construct was causing silencing of the endogenous GmFad2-1 gene, resulting in seeds like G168 with a high oleic acid content only. Locus A was characterized using Southern blotting and shown to contain two copies of the GmFad2-1 expression cassette as indicated by two hybridizing bands on the Southern blot. The second locus (locus B) contained a copy of GmFad2-1 that was over-expressing, thus decreasing the oleic acid levels to around 4% (G175). This locus also contained a functioning <i>dapA</i> gene as evidenced by an increase in the seed lysine levels. Locus B contained only a single copy of the GmFad2-1 expression cassette as indicated by a single hybridizing band on the Southern blot. In seeds with both locus A and locus B (G94), lysine levels were increased and oleic acid levels were increased but not as high as in those with locus A alone.</p>

<p style="text-align: justify;">Lines G94 and G168 were selected for further characterization as they contained the silencing locus A with the high oleic acid phenotype. As G94 plants contained both locus A and locus B, an additional round of selection was used on the segregating R2 plants to isolate plants containing locus A and not locus B. Southern blot analysis on R2 leaf tissue grown from G94 R2 seed identified two sub lines, G94-1 and G94-19, that contained locus A without locus B which had been removed through segregation. Locus B was not further characterized for the purposes of this application.</p>

<p style="text-align: justify;">The three sub lines, G94-1, G94-19 and G168, identified as containing the GmFad2-1 silencing locus A, were selected as the high oleic acid soybeans for subsequent analyses. G94-1, G94-19 and G168 containing only locus A and locus C and were advanced to produce high oleic soybeans. The presence of the high oleic phenotype was the primary screen for the advancement of sublines since G94-1, G94-19 and G168 were GUS negative. These sublines were all homozygous for the GmFad2-1 encoding sequences arranged in the sense orientation.</p>

<p style="text-align: justify;"><b>Genetic Stability of the Introduced Trait</b></p>

<p style="text-align: justify;">All three sublines G94-1, G94-19 and G168 were kept separate for six generations and all maintained an identical Southern pattern indicating the stability of the introduced trait for high levels of oleic acid. Multiple years of field testing at several locations determined that the fatty acid phenotype for these plants was stable. In contrast, a high oleic mutant produced by traditional breeding techniques shows a very broad range of oleic levels depending on the environmental conditions.</p>

<p style="text-align: justify;"><b>Expressed Material</b></p>

<p style="text-align: justify;">There were no new proteins expressed in the sublines G94-1, G94-19 and G168. Biochemical and molecular tests confirmed that the beta-lactamase gene was not active in plants, and thus no protein (enzyme) was produced by this gene. The GUS gene was also silent and beta-glucuronidase was not expressed. Similarly no protein affecting the lysine levels was produced in any of the sublines.</p>

<p style="text-align: justify;">Suppression of the GmFad2-1 genes in soybean sublines G94-1, G94-19 and G168 resulted in the expression of levels of oleic acid in soybean oil exceeding 80%, compared to 23% found in a typical conventional soybean oil.</p>
