Regeneration of protoplasts into complete thalli in the marine green alga Ulva pertusa

Protoplasts could be isolated from thalli of Ulva pertusa using a mixed enzyme solution of Cellulase Onozuka R-10, Macerozyme R-10, and Driselase. The isolated protoplasts were spherical and 10-40 μm in diameter. The number of protoplasts was 4.8×106 from 1g of fresh weight of the thalli. The purification and fractionation of heterogeneous protoplasts were carried out by a Percoll/mannitol discontinuous density gradient centrifugation, successively, affording a homogeneous population of protoplasts. The percentage of viable protoplasts were more than 90%. The isolated protoplasts developed to the thalli through regeneration of new cell walls, repeating cell divisions, and forming cell colonies. The regenerated plants were obtained on a preparative scale by using an aeration culture of the thalli through an axenic filter in Provasoli's enriched sea water medium (PES) containing antibiotics at 14-18°C under white fluorescent light (14:10h L:D). The present method of the preparative scale-culture might be a large supply of plants for production of valuable substances.