Preparation and regeneration of protoplasts from wild-type of Porphyra yezoensis and green variant of P. tenera

Porphyranase, β-1, 4-mannanase, and β-1, 3-xylanase, capable of degrading the cell wall of red algae, genus Porphyra, were prepared from the culture fluids of Vibrio sp. AP-2, Aeromonas sp. F-25, and Vibrio sp. AX-4, respectively, The thalli of P. yezoensis, after treatment with a 2% papain solution, were suspended in 5ml of MES buffer, pH 6.0, containing 0.7M mannitol and 1 unit each of porphyranase, β-1, 4-mannanase, and β-1, 3-xylanase. When a 100mg weight of the thalli was shaken in the enzyme solution at 22°C for 90 min, ca. 8.6×105 protoplasts were released. Protoplasts from P. yezoensis grew in a ASP12 (NTA) medium and developed into brown thalli of ca. 10cm in length after 3 months. A number of protoplasts were obtained from green variant of P. tenera, too. These green protoplasts developed into green thalli of ca. 10cm in length after 2 months. A lot of monospores released from regenerated thalli of P. yezoensis (wild type) and P. tenera (green type) developed into brown thalli and green thalli, respectively.