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

<p style="text-align: justify;">Southern blot analysis of genomic DNA from Oxy-235 indicated that one complete copy of the bxn gene was integrated into the host genome at a single insertion site. The insert number was determined following digestion of genomic DNA with EcoRI or HindIII, which gave rise to a single hybridizing band in each case. No hybridizing bands were observed with restriction enzyme digested genomic DNA from non-transgenic control plants, and when Oxy-235 DNA was double-digested with EcoRI and HindIII a single hybridizing fragment of 1150 bp was detected. Additional Southern analyses using probes specific for the ori-322 region of pBR322 and polymerase chain reaction (PCR) amplification analysis for gentamycin encoding sequences demonstrated that these additional plasmid-derived sequences (located outside of the T-DNA region) were not incorporated into the host genome.</p>

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

<p style="text-align: justify;">Segregation analysis was consistent with a single site of insertion of the bxn gene into the Oxy-235 genome, as shown by a 3:1 ratio of susceptible to tolerant plants. Comparative analysis of third-generation progeny material, using Southern blots and PCR analysis, determined that the introduced DNA was stably inserted and stably inherited.</p>

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

<p style="text-align: justify;">Levels of nitrilase enzyme were quantified in samples of transgenic leaf (1000 ng/mg total protein) and seed (&lt;10 ng/mg total protein) tissue by Western immunoblot analysis (detection of single 37 kDa species). No nitrilase was detectable in samples of refined oil (detection limit 20 ppb).</p>
