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

• Published in: Plant cell reports 1988-12, Vol.7 (7), p.531-534
• Main Authors: Lee, N, Wetzstein, H.Y
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Biological and medical sciences; Biotechnology; callus; culture media; Eukaryotic cell cultures; Fundamental and applied biological sciences. Psychology; General agronomy. Plant production; isolation; leaves; Methods. Procedures. Technologies; Plant cells and fungal cells; Protoplast preparation, cell fusion and regeneration; protoplasts; tissue culture; Vegetative propagation. Micropropagation; Vegetative propagation. Sowing and planting. Harvesting; Vitis rotundifolia; Vitis vinifera
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/bf00272749

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 μM 6-benzyladenine, 4.5 μM 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.