<p style="text-align: justify;">The ZW-20 squash line was produced using an <i>Agrobacterium</i>-meditated transformation protocol in which the transfer-DNA (T-DNA) region of the bacterial tumour inducing (Ti) plasmid was modified using recombinant DNA techniques to contain the coat protein (CP) encoding sequences from ZYMV and WMV2. During transformation, the T-DNA portion of the plasmid was transferred into the plant cells and stably integrated into the plant&#39;s genome, and there was no incorporation of backbone plasmid sequences outside of the T-DNA region.</p>

<p style="text-align: justify;">The constitutive expression of the CP genes was regulated by the 35S promoter and termination sequences from cauliflower mosaic virus (CaMV). In addition, WMV2 CP gene was fused to the 5&#39; intergenic region and the first 48 nucleotides (N-terminus) of the CMV coat protein gene to enhance translation of the transgene mRNA.</p>

<p style="text-align: justify;">The plasmid vector used for transformation also contained sequences encoding the enzyme neomycin phosphotransferase II (NPTII), which conferred the trait of resistance to aminoglycoside antibiotics that was used as a selection mechanism during plant tissue culture regeneration and propagation. During varietal development, only plants lacking this gene were selected for commercialization and the NPTII encoding sequences were, therefore, not present in the ZW-20 genome.</p>
