Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun ( . In each optimization step ...)

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

In each optimization step, three cartridges of PVA-T7 transcript (RNA) or PVA-35S (cDNA) were bombarded onto tobacco and potato plants. Each test was carried out at least three times. After each optimization step, the optimal parameter was used in further experiments, as explained above. Significance of observed differences was tested using analysis of variance and calculation of the least significant differences (Steel and Torrie 1981).

Results
Pressure and Distance. In tobacco, 100% infection was achieved using a helium pressure of 150 or 200 psi at a distance of 0 cm, but extensive death of the cells in the middle of the bombarded area was observed (Figure 2). At the higher pressure of 250 or 300 psi, leaves tended to be ripped. No infection was detected in tobacco plants bombarded at the low pressures of 80 and 100 psi, and at these pressures the bombarded areas usually remained undamaged. Results were similar for PVA-T7 (RNA) and PVA-35S (cDNA).

In the potato clone A6, infection initiation sites could be visually identified by the development of necrotic lesions as a result of the hypersensitive response to PVA. A few necrotic lesions characteristic of PVA infection (Valkonen et al. 1995) were observed at 80 psi, and their numbers increased with increased pressure (Figure 3). At lower pressures (80 and 100 psi), the bombarded area of leaf was usually undamaged and just a few necrotic lesions were observed. Bombardment with higher pressures (150 and 200 psi) led to cell death in the middle part of the bombarded tissue, but numerous necrotic lesions were still observed in the surrounding surviving tissue (Figure 4).

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( . In each optimization step ...)Inoculation of Viral RNA and cDNA to Potato and Tobacco Plants Using the Helios Gene Gun