Fusion of Protoplasts from Wild Type of Bangia atropurpurea and Green Type of Porphyra tenera

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Three kinds of enzymes, beta-1, 4-mannanase, beta-1, 3-xylanase, and agarase, required for isolation of protoplats from a red alga, Bangia atropurpurea, were prepared from bacterial culture fluids of Vibrio sp. MA-138, Alcaligenes sp. XY-234, and Vibrio sp. P0-303, isolated from sea environment. The optimal pHs of all the crude enzymes were around 7.5. The suitable condition for protoplast isolation was examined. The treatment of the fronds with papain solution (20 mM MES buffer, pH 7.5, containing 2 % papain and 0.5 M mannitol) have contributed to isolatating protoplasts. When razorcut fragments of the fronds (about 200 mg in weight) dipped in the cell wall-digestive enzyme solution (1 unit each of 1, 4-beta-mannanase, 1, 3-beta-xylanase, and agarase, and 0.5 M mannitol in 20 mM MES buffer, pH 7.5) were incubated at 17 ﾟC for 90 min with gentle agitation, 7.1 x 10^6 of protoplasts were released from them.For carrying out cell fusion polyethylen glycol (PEG) was added into the suspension of protoplasts from the wild type of B.atropurpurea and the green varient of Porphyra tenera. Homo- and hetero-plasmic adhesion of protoplasts were observed. The PEG-treated protoplasts were cultured in artificial light of about 48 muEm^<-2>s^<-1> on a 9 h light/15 h dark cycle at 17 ﾟC.They grew into calli and plantrets of brown or green color after 6-week culture. A pair of protoplast which parts of their menbrans adhesed also developed into plantlets.