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

tent of mechanical damage on bombarded areas of the leaves was observed. Symptoms of PVA infection in A6 (necrotic lesions) were observed four days after bombardment. In tobacco, PVA infection was detected twelve days after bombardment of upper uninoculated leaves.

The assay was a double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA), using monoclonal antibodies and alkaline phosphatase-conjugated monoclonal antibodies to PVA (MAb 58/0; Rajamaki et al. 1998) obtained from Adgen, UK.

Optimization of Bombardment Parameters. We tested various bombardment conditions in order to identify those that resulted in the largest number of necrotic lesions on A6 leaves and in infection of all inoculated tobacco plants. First, different helium pressures (80, 100, 150, 200, 250 and 300 psi) were tested using 2 g PVA-35S or PVA-T7 per mg gold (1.0 m), 0.05 mg PVP per ml 99.5% ethanol, and 0.5 mg gold per bombardment as recommended for starting conditions in the Helios gene gun instruction manual. Bombardments were done with 0 or 2 cm distance between the spacer and the target. The first experiments showed that bombardment at 250 and 300 psi damaged leaves extensively, so these pressures were not included in further experiments.

Next, PVP concentrations were varied (0, 0.05 and 0.1 mg per ml 99.5% ethanol). Third, using the previously determined optimal helium pressure and PVP concentration, different sizes of microcarriers (0.6, 1.0 and 1.6 m) were tested. In the last experiment, various amounts of microcarriers per shot (the microcarrier loading quantity, or MLQ) and various amounts of PVA cDNA coated on microcarriers (the DNA loading ratio, or DLR) were tested
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