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Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun

cript quality and concentration were determined by electrophoresis in an agarose gel. RNA was precipitated on gold particles by addition of 1/10 volume of 3 M NaAc (pH 7.4) and 3 volumes of 99.5% ethanol. The suspension was mixed by tapping the tube and placing it at -20 C for 30 min. After a quick centrifugation (~5 sec), the supernatant was discarded and the gold pellet washed three times with 1 ml 99.5% ethanol. The gold particles were then resuspended in 200 l of ethanol containing the appropriate concentration of PVP. The suspension was transferred to a 15 ml polypropylene centrifuge tube, and 2.8 ml ethanol/PVP solution was added. The suspension was immediately used for preparation of cartridges.

P VA-35S plasmid was isolated from an E. coli XL1-Blue culture using the QIAGEN Plasmid Maxi Kit (Q IAGEN Ltd., UK) according to the manufacture rs instructions. PVA-35S was linearized prior to precipitation of the DNA on gold particles , as described above for RNA. Cartridges were prepared as described in the Helios gene gun instruction manuals (catalog # 165-2431 and 165-2432, Bio-Rad).

Inoculation. For each tobacco plant, the first full-grown true leaf was inoculated with one bombardment. For the potato clone A6, one or two full-grown leaves were bombarded either one or multiple times on different sides of the midvein. The latter arrangement permitted comparison of two or more different bombardment parameters on the same A6 leaf (Figure 1). Before bombardment, plants were kept in the dark for at least five hr. After bombardment, plants were sprayed with water and placed in a transparent box in dim light overnight . The next day, the plants were returned to the growth chamber, and the ex
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