Plant Regeneration from Embryogenic Suspension-Derived Protoplasts of Sandalwood (Santalum Album)
Protoplasts of sandalwood (Santalum album L.), an economically important tree in India and Southeast Asia, divided to produce embryogenic callus from which plants could be regenerated. Suspension cultures which provided a convenient source for protoplasts were established with callus originating from cultured shoot segments from 20-year-old trees. Suspensions maintained by subculture every 4 to 5 days in a modified Murashige and Skoog (MS) medium containing 0.5 or 1.0 mg/1 2,4-dichlorophenoxyacetic acid (2,4-D) produced embryoids when plated in stationary liquid media or on agar media with 0.1 to 2.0 mg/1 benzylaminopurine (BA). Plants could be obtained from secondary embryos. Cells grown in suspension afforded a good protoplast yield with an enzyme solution consisting of 1% Cellulase RS, 1% Macrozyme R-10, 0.5% Driselase, and 0.55 M mannitol. Protoplasts cultured in V47 medium [H. Binding (1974) Z. Pflanzenphysiol. 74:327] containing 3 mM 2-(N-morpholino)ethane-sulfonic acid, 0.5 mg/1 2,4-D, and 0.5 mg/1 BA, and adjusted with mannitol to 750 m0s/kg H2O, divided within 3 to 4 days. Multicellular colonies present after 2 weeks continued to develop when the osmalality was gradually reduced by dilution every 5 to 7 days.