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

pacer, polyvinylpyrrolidone (PVP) concentration, viral DNA and RNA amounts, and the size and number of gold particles (micro carriers ) .

PVA belongs to the family Potyviridae, the largest and economically the most important group of plant viruses (Bartels 1971). PVA is widely distributed in potato-growing areas and decreases the yield of infected potato plants by up to 40% (Brunt 1992). A full-length cDNA clone of the RNA genome of PVA has been constructed under the control of a T7 promoter (PVA - T 7 ), f rom which infectious transcripts can be produced in vitro for inoculation (Puurand et al. 1996). The same construct is also available under the control of a cauliflower mosaic virus (CMV) 35S promoter (PVA-35S) that can be used directly for inoculation (. Puurand pers. comm., Rajamaki and Valkonen 1999).


Methods
Plant Material. Tobacco (Nicotiana tabacum L. cv. Samsun) is susceptible to PVA, whereas the test potato clone A6 reacts with the development of necrotic lesions (hypersensitive response) at the sites of infection (Valkonen et al. 1995 ). Tobacco and potato plants were grown in a growth chamber under an 18 hr photoperiod (250 E s-1 m- 2) at 19/17 C (day/night) and a relative humidity of 40%. Tobacco plants were inoculated by particle bombardment when the first true leaf was fully expanded. Potato plants propagated by taking cuttings were grown in the growth chamber for four weeks before bombardment.

Preparation of DNA or RNA for Bombardment. The PVA-T7 construct was linearized with AgeI prior to in vitro synthesis of the 5'-capped transcripts according to the manufacturers instructions (RiboMax, Promega). Trans
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