Surface Sterilization of Grape Phylloxera Eggs in Preparation for In Vitro Culture with Vitis Species

¹ Department of Viticulture and Enology, University of California, Davis, CA 95616-8749.

The development of an in vitro dual culture system with grape phylloxera (Daktulosphaira vitifoliae Fitch) and Vitis species is dependent upon effective surface sterilization of the eggs to prevent microbial contamination of the system. A range of commercial bleach (5.25% sodium hypochlorite) and ethanol solutions were tested for their effect on phylloxera eggs. Treatments were judged by how they affected egg hatch and nymph survival and how well they prevented microbial contamination in sterile culture. About 80% of the eggs survived a seven-minute treatment in either 50% ethanol or 50% bleach with no surface contamination detected in any of the replicates after seven days. However, all of the bleach treatments required rinsing with sterile water to avoid greatly reduced egg hatch. Fifty percent ethanol for seven minutes had the advantage of not needing a sterile water rinse after treatment, thus simplifying the procedure and reducing further risk of contamination. This sterilization procedure allows in vitro dual culture studies of phylloxera biology, phylloxera resistance and susceptibility within Vitis, and development of a rapid screening tool for grape rootstock breeding.

Key words: grape, phylloxera, tissue culture, dual culture, ethanol, sodium hypochlorite

Submitted on August 31, 1995