Protoplast isolation and callus production from leaves of tissue-cultured Vitis spp.

Protoplasts were isolated from mesophyll of axenic cultures of grape, Vitis rotundifolia cv. Summit and V. vinifera cv. Cabernet Sauvignon. Enzymes effective for protoplast isolation were Macerozyme R-10 (0.5% and 0.1%) and Cellulase Onozuka R-10 (1.0% and 0.5%) for V. rotundifolia and V. vinifera, respectively. Polyvinylpyrrolidone and 2-(N-morpholino)ethanesulfonic acid were essential in the isolation media. Protoplasts were purified using flotation/centrifugation. The protoplasts of V. rotundifolia cultured in Gamborg's B5 basal medium with 2.2 M 6-benzyladenine, 4.5 M 2,4-dichlorophenoxyacetic acid and 0.4% agarose gave the best plating efficiency of conditions tried in this study. Cell division occurred within 5 to 6 days and visible microcalli developed within one month. After 6 weeks in culture, microcalli transferred to liquid medium exhibited active callus growth. Protoplasts of V. vinifera cultured under these conditions had similar results.Abbreviations MES 2-(N-morpholino)ethanesulfonic acid monohydrate - PVP polyvinylpyrrolidone - PDS potassium dextran sulfate - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate