ISOLATION AND REGENERATION OF HAPLOID PROTOPLASTS FROM BANGIA-ATROPURPUREA (RHODOPHYTA) WITH MARINE BACTERIAL ENZYMES

Three Kinds of enzymes, agarase, beta-1,4-mannanase, and beta-1,3-xylanase, required for isolation of protoplasts from the red alga Bangia atropurpurea (Roth) C. Ag. were prepared from bacterial culture fluids of Vibrio sp. PO-303, Vibrio sp. MA-138, and Alcaligenes sp. XY234, respectively, isolated from the sea environment. The optimal pH of all enzymes was around 7.5. Suitable conditions for protoplast isolation from B. atropurpurea were examined. The pretreatment of the fronds with papain solution (20 mM Mes buffer, pH 7.5, containing 2% papain and 0.5 M mannitol) contributed to successful protoblast isolation. When razor-cut fragments of the fronds (about 200 mg in fresh weight) immersed in 20 mM Mes buffer, 7.5, containing 0.5 M mannitol and one unit each of agarase, beta-1,4-mannanase, and beta-1,3-xylanase were incubated at 22 degrees C for 90 min with gentle agitation, 5.7 x 10(6) protoplasts were released from them. Many protoplasts regenerated into fronds of regular or irregular shape.