Protoplast Isolation and Cultivation from Embryogenic Suspension Cultures and Leaves of Grapevines (Vitis L.)

Abstract

Establishment of a protoplast-to-plant system in grapevines requires high quality protoplast isolation, capability of cell division and re- generation to plantlets. In this study, protoplasts were obtained from three somatic embryogenic suspension cultures and leaf tissues of four grapevines with a protoplast isolation mix of 1% cellulase R-10, 0.2% pectolyase Y 23, 1% macerozyme R-10 with 24 mM CaCl2.2H2O, 0.092 mM NaH2PO4, 0.7 M mannitol, and 6.15 mM 2-(N-morpholino) ethane sulfonic acid, plus 0.1% polyvinylpyrroli- done (PVP). The viability of the protoplasts was confirmed by fluo- rescein diacetate (FDA) staining, and subsequent cell wall formation, cell division, and callus development. Knowledge gained and meth- odology developed in this study are very helpful for the selection of parent tissue sources in the subsequent somatic hybridization.