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

<p style="text-align: justify;">The inserted DNA was characterized using a combination of restriction enzyme digestion and Southern blot labelling to confirm the number of sites of insertion of the pYTY32 T-DNA, and polymerase chain reaction (PCR) amplification using specific primer pairs to confirm gene organization and integrity. PCR amplification was also used to test for the incorporation of plasmid-derived sequences outside of the T-DNA region.</p>

<p style="text-align: justify;">The presence of an endogenous NtQPT1 gene in tobacco necessitated the use of a probe for the marker gene <i>nptII</i> as opposed to a probe for the NtQPTII antisense transgene. Southern Blot analysis confirmed the presence of two T-DNA insertions segregating as a single locus. The transgene conferring kanamycin resistance was found to segregate as a single locus and be co-segregated with the low nicotine phenotype. DNA blot analysis data demonstrated that Vector 21-41 T1 progeny carried 2 copies of the transgene.</p>

<p style="text-align: justify;">Between 2.1 kbp and 2.4 kbp of backbone pYTY32 sequences that flanked the T-DNA left border were also transferred to Vector 21-41, however these did not include any complete coding regions for any genes.</p>

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

<p style="text-align: justify;">Segregation and stability data indicated two copies of the T-DNA region of pYTY32 at a single locus. The stability of the insertion was demonstrated through multiple generations derived through self-pollination. Vector 21-41 was derived from the T1 generation and stable insertion of the insert was demonstrated through Southern blot analysis of T1 progeny as well as low nicotine expression in subsequent generations.</p>

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

<p style="text-align: justify;">The production of nicotine in Vector 21-41 was down-regulated in the roots of the plant by an antisense strategy. In this case, no new polypeptide was expressed, aside from the NPTII enzyme. QPTase activities are thus decreased in Vector 21-41 as compared to Burley 21LA.</p>
