<p style="text-align: justify;">The transgenic potato lines RBMT15-101, SEMT15-02 and SEMT15-15 were produced by <i>Agrobacterium</i>-mediated transformation of the potato cultivars &#39;Russet Burbank&#39; and &#39;Shepody&#39;, respectively. The transfer-DNA (T-DNA) region of a &quot;disarmed&quot; form of the bacterial tumour inducing (Ti) plasmid was modified to contain genes encoding Cry3A delta-endotoxin, PVY-O coat protein, and NPTII. This plasmid was designated PV-STMT15. As a matter of course, Ti plasmids used for plant transformation are &quot;disarmed&quot; such that they do not contain the virulence genes (<i>vir</i>A) that are normally associated with the pathogenicity and disease-causing properties of <i>A. tumefaciens</i>.</p>

<p style="text-align: justify;">A 5&#39;-truncated copy of the <i>cry3A</i> gene, obtained from <i>B. thuringiensis</i>, ssp. tenebrionis (Btt) strain BI 256-82, was modified by site-directed mutagenesis to enhance expression by generating plant-preferred codons. These modifications in nucleotide sequence did not alter the expected amino acid sequence of the Cry3A protein. Constitutive expression of the <i>cry3A</i> gene was controlled by the promoter region of the ribulose-1,5-bisphosphate carboxylase small subunit gene from <i>Arabidopsis thaliana</i> and the terminator sequence from the nontranslated 3&#39; region of the nopaline synthase (<i>nos</i>) gene from <i>A. tumefaciens</i>.</p>

<p style="text-align: justify;">During normal virus replication, the PVY-O CP is derived from a large poly-protein precursor by proteolytic cleavage. In order to express the PVY-O CP transgene in plant cells, the cloned complementary-DNA sequence corresponding to the CP gene was modified to contain a translation start codon (ATG). Expression of the PVY-O CP gene was under the control of the 35S promoter region of figwort mosaic virus, the leader sequence from the <i>Glycine max</i> (soybean) heat-shock protein, Hsp 17.9, and the terminator sequence from the nontranslated 3&#39; region of the pea (<i>Pisum sativum</i>) ribulose-1,5-bisphosphate carboxylase small subunit gene E9 3&#39;.</p>

<p style="text-align: justify;">Expression of NPTII, encoded by the <i>neo</i> gene, was regulated by promoter and termination sequences from the nopaline synthase (<i>nos</i>) gene.</p>

<p style="text-align: justify;">In addition to the plant expressible genes described above, plasmid PV-STMT15 also contained the following non-expressed elements: (1) <i>oriV</i> , which is the origin of replication from plasmid RK2 isolated from Agrobacterium strain ABI; (2) ori-322/rop , which is a segment of plasmid pBR322 which provides the origin of replication for maintenance of PV-STMT15 in <i>Escherichia coli</i> [rop] and the bom site, which enables conjugal transfer into Agrobacterium; (3) the <i>aad</i> gene from transposon Tn7 of <i>E. coli</i>, which encodes the enzyme 3&quot;(9)-O-aminoglycoside adenylyltransferase and confers resistance to the antibiotics streptomycin and spectinomycin; and (4) the left (LB) and right (RB) sequences, which facilitate integration of the T-DNA into the plant host genome. Inclusion of the selectable marker <i>aad</i> gene, which was only expressed in bacterial cells, facilitated the isolation of bacterial colonies that had been transformed with recombinant plasmid DNA.</p>
