Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun ( The pressure of 200 psi and the...)

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

The pressure of 200 psi and the distance of 0 cm were optimal for A6, differing significantly (p< 0.01) from the other pressures used. The higher pressures of 250 and 300 psi ripped the leaves and are thus not recommended for use. Although bombardment at a 2 cm distance from the target tissue resulted in spreading of the micro carriers (and subsequently the necrotic lesions) over a larger area on the leaf, the total number of necrotic lesions was no greater than after bombardment at a distance of 0 cm. No differences were observed between bombardment using PVA RNA or cDNA (data not shown).

PVP Concentration and Micro c a rrier Size. PVP serves as an adhesive during the coating of DNA/(or RNA)/micro carrier suspension to the walls of the Goldcoat tubing. With both DNA and RNA of PVA, the optimal concentration was shown to be 0.05 mg PVP/ml 99.5% ethanol. This resulted in significantly more lesions (p< 0.01) in A6 leaves than at PVP concentrations of 0 or 0.1 mg/ml (Figure 5).

Three sizes of micro carriers were used: 0.6, 1.0 and 1.6 m. The 0.6 m carriers were significantly more efficient in initiation of infection (p< 0.01) than the other two sizes used (Figure 6).

Ratios of Microcarrier and DNA Amounts. Various ratios of microcarriers and viral cDNA (Table 1) were tested by bombarding A6 leaves. The numbers of infection sites observed with different ratios of microcarriers and DNA were not significantly different statistically. As little as 0.1 g viral cDNA per bombardment was sufficient to trigger development of necrotic lesions in A6.

Discussion
The Helios gene gun is being used in several laboratories f
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( The pressure of 200 psi and the...)Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun