Highly efficient system of plant regeneration from protoplasts of grapevine ( Vitis vinifera L.) through somatic embryogenesis by using embryogenic callus culture and activated charcoal

Abstract

A simple protocol is described for high frequency plant regeneration from protoplasts isolated from leaf-derived embryogenic calli of grapevine ( Vitis vinifera L. cv. Koshusanjaku). The protoplasts successfully divided to form somatic embryos by culturing in gellan gum disc-method in which protoplasts were embedded in 2 g/l gellan gum-solidified Nitsch's medium containing 2.0 mg/l NAA, 0.5 mg/l BA, 0.09 M sucrose and 0.3 M glucose at a density of 1 × 10 5 protoplasts/ml. For the continuous growth of the colonies without browning, it was essential to add 0.3% (w/v) AC in the liquid reservoir medium from the beginning of the culture. In this culture condition, protoplasts started to divide after 10 days of culture and grew into torpedo embryos 4 months after initiation of culture. The torpedo embryos thus obtained germinated normally by transferring onto 2 g/l gellan gum-solidified PGR-free Nitsch's medium containing 30 g/l sucrose. The regenerated plants were successfully transferred to the greenhouse and showed normal morphology.