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

<p style="text-align: justify;"><u>Line RBMT21-129</u> - insertion of the T-DNA occurred at two sites. One insertion site contained the right border of the T-DNA as well as the PLRV replicase gene cassette, the <i>cry3A</i> gene and the NPTII coding region, terminating within the NOS promoter. This T-DNA insertion was not completely resolved at the left border, resulting in the partial deletion of the 5&#39; end of the NOS promoter used to express the NPTII encoding gene. The second insert consisted of the PLRV replicase gene and a partially deleted <i>cry3A</i> gene cassette. The ArabSSU1A promoter of the <i>cry3A</i> gene, as well as a portion of the 5&#39; coding region of the <i>cry3A</i> gene, were deleted upon integration into the plant genome. The partial <i>cry3A</i> gene was still associated with its <i>nos</i> 3&#39; terminator region. Northern and western blot analysis were unable to detect any protein or mRNA from the truncated <i>cry3A</i> gene. This T-DNA insertion was not completely resolved at the right border, resulting in the deletion of the FMV 35S promoter as well as a portion of the 5&#39; end of the PLRV replicase gene. Northern blot analysis confirmed that the partial PLRV replicase gene did not give rise to any detectable mRNA.</p>

<p style="text-align: justify;"><u>Line RBMT21-350</u> - insertion of the T-DNA occurred at two sites. At one site, intact copies of all three genes were inserted. At the second site, a less than full-length copy of the T-DNA was inserted resulting in a truncated copy of the PLRV replicase gene, lacking the FMV 35S promoter region. Northern blot analysis demonstrated that no detectable RNA transcript was produced from the truncated PLRV replicase gene. The remaining two genes were intact.</p>

<p style="text-align: justify;"><u>Line RBMT22-082</u> - insertion of the T-DNA occurred at three sites. All three copies of the T-DNA contained intact coding regions for the PLRV replicase gene and the <i>cry3A</i> gene. Two copies of the T-DNA contained an intact coding region of the CP4 EPSPS gene. At one site, however, a less than full-length copy of the CP4 EPSPS gene had been inserted. Northern analysis was not able to detect any mRNA being produced from the truncated CP4 EPSPS gene, suggesting that, as the promoter region for the truncated CP4 EPSPS gene is intact, mRNA did not accumulate to any detectable levels. At the third insertion site, DNA sequences beyond the T-DNA right border were also inserted. This DNA was adjoined to the right border of the T-DNA and contained the <i>aad</i> gene and the ori-322 region. This result conflicted with PCR analyses, which were unable to detect the <i>aad</i> gene. The failure to detect the <i>aad</i> gene by PCR suggested that the gene was probably not intact.</p>

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

<p style="text-align: justify;">Lines RBMT21-129 and RBMT21-350 expressed three novel proteins, Cry3A, the PLRV replicase, and NPTII. The Cry3A protein was expressed in tubers at levels ranging from 0.11 - 0.9 &micro;g protein/g fresh weight (equivalent to 0.0006 to 0.005% of the total tuber protein). The production of viral replicase protein was undetectable in both leaf and tuber tissue, although other evidence suggested that it was expressed. NPTII was expressed in the tuber, but at levels below the limit of detection (0.3 ng protein/g fresh weight). The levels of expression of Cry3A protein and PLRV replicase in RBMT22-082 were equivalent to the levels determined for lines RBMT21-129 and RBMT21-350. CP4 EPSPS was expressed in RBMT22-082 tubers at levels ranging from 0.21 - 0.78 &micro;g protein/g fresh weight (equivalent to 0.001 to 0.004% of the total tuber protein).</p>
