Progress in leaf protoplast isolation and culture from virus-free axenic shoot cultures of Vitis vinifera L.

Abstract

Axenic shoot cultures of virus-free Vitis vinifera L. cv. Soultanina were a highly efficient source for isolation of viable protoplasts. Optimum results were obtained with leaves of 50–100 mg fresh weight, leaf discs of 0.7 cm in diameter, 100 and 15 U ml-1 Cellulase R-10 and Macerozyme R-10, respectively, and 18 h reaction time in either light or in darkness. Protoplast yield was approx. 25×106 viable protoplasts per g fresh weight and their size ranged from 12 to 44 μm. During a 20-day culture period, the maximum survival rate obtained was approx. 40%. A plating density of 10×105 protoplasts per ml resulted in increased survival rates. Various growth regulators and glutamine did not significantly improve survival rates of protoplasts, whereas extract from coconut added to the culture medium caused an increase in the survival rates of protoplasts. Cell elongation at a significant rate and divisions were observed. [14C]glucose uptake was studied as an index of cell membrane integrity and functioning. Uptake rate of glucose by protoplasts was linear for up to 60 min, fully inhibited by NaN3, with an optimum pH of 4.8. Protoplasts 24 h old exhibited significantly lower rates of glucose uptake.