( or plant transformation and ...)Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun

or plant transformation and also virus inoculation. We have optimized bombardment parameters for a cloned RNA virus (PVA) using an experimental host widely used for many plant v i ruses (N. tabacum L. cv. Samsun) and also using a host that reacts with a hypersensitive response to virus infection (potato clone A6). The latter type of host is useful for an optimization experiment because the sites of infection can easily be identified visually after only a few days. Because the aim of our study was to inoculate a cloned virus (RNA transcripts or linearized viral cDNA), it was inferred that the optimal bombardment parameters could be different from those used for plant transformation. For this reason, optimization of parameters was needed.

For successful infection, it is important to optimize the condition of plants before and after inoculation. We noticed that shading the plants overnight before bombardment and keeping them under humid conditions in dim light after bombardment improved initiation of infection considerably. Such treatments are known to increase susceptibility of plants to mechanical inoculation (Matthews 1995).

This study showed that it is important to determine the optimal pressure, PVP concentration and micro carrier size, because differences in these parameters provided varying success in infection. These parameters may also vary for different host plants. In our study, the low pressure of 80 psi caused a few necrotic lesions in A6 leaves, whereas in tobacco no PVA infection was detected following inoculation at a pressure of 80 or 100 psi. In contrast, at a pressure of 150 or 200 psi, all tobacco plants became infected. PVP concentration of 0.05 mg/ml and micro carrier
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