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

Tuija Kekarainen and Jari P. T. Valkonen, Department of Plant Biology, Swedish University of Agricultural Sciences (SLU), P.O. Box 7080, SE-75007 Uppsala, Sweden


Introduction
Particle bombardment is a physical method widely used for gene transfer into plants, mammals, fungi and bacteria (Armaleo et al. 1990, Klein et al. 1992, Shark et al. 1991, Williams et al. 1991, Zelenin et al. 1989). The hand-held Helios gene gun provides wider possibilities for gene transfer to plant tissue both in vivo and in vitro, because it is not limited by the large size of the target, and bombardment does not need to be carried out under vacuum.

The particle bombardment technique has been used to infect plants with DNA viruses, RNA viruses and cloned RNA viruses (Gal-On et al. 1995, Gilbertson et al. 1991, Klein et al. 1992 ) . The PDS-1000/He system is efficient for inoculation of detached leaves, but the Helios gene gun enables inoculation of an intact plant. In this study we have used the Helios gene gun to inoculate whole tobacco and potato plants with the cDNA of an RNA virus, potato virus A (PVA; Puurand et al. 1996). We wanted to optimize bombardment parameters to achieve 100% efficiency of PVA infection in susceptible tobacco plants. In the case of potato plants, we used potato clone A6 (Solanum demissum Lindl. x S . tuberosumcv. Aquila), which develops necrotic lesions at the site of PVA infection (Valkonen et al. 1995 ), and thus enables counting of the number of initial infection sites. The number of lesions can be used as a readily observed measure for optimal inoculation conditions. The bombardment parameters examined in this study were helium pressure, distance of target tissue from the s
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