A small-scale procedure for extracting nucleic acids from grapevine dormant cuttings infected with GFLV
2004
Buzkan, N. (University of Kahramanmaras Sutcu Imam (Turkey). Dept. of Plant Protection) | Walker, A.
Detection of Grapevine fanleaf virus (GFLV) in grapevine samples is difficult due to seasonal effect, low viral titer and uneven distribution of the viral particles within the plant and presence of phenolic compounds. To circumvent this problem in grape tissue, a nucleic acid purification (viral RNA) procedure is aimed to improve the extraction methods to remove the inhibitory components of the tissue. In this work, RNA from hybrid grapevines of five different populations (9621, 9623, 9630, 9631 and 9635) from the breeding program of the Department of Viticulture and Etiology, University of California, Davis (CA) on which nematodes (Xiphinema index) carrying GFLV was extracted with silica particle suspension and phenol/chloroform methods. Extracted RNAs were submitted to cDNA synthesis and amplification procedure with primers specific to GFLV. The silica method could detect the virus infection from 43 cuttings although viral nucleic acid of the same samples from phenol/chloroform extraction were not amplified in PCR reaction The success of the silica methods was because phenolic compounds were bound to silica particles, eliminated by buffer solution with ethanol. The protocol does not require ultracentrifugation and is possible to complete in few hours which is much shorter than phenol/chloroform application. It also appears to be widely applicable to particularly difficult plant tissues.
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