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

从海洋环境中分离的弧菌MA-138、产碱菌XY-234和弧菌P0-303的培养液中制备了分离红球藻原生质体所需的β-1，4-甘露聚糖酶、β-1，3-木聚糖酶和琼胶酶。所有粗酶的最适pH均在7.5左右。对原生质体分离的适宜条件进行了研究。用木瓜蛋白酶溶液（20 mM MES缓冲液，pH 7.5，含有2%木瓜蛋白酶和0.5 M甘露醇）处理叶有助于分离原生质体。当刀片切割的叶子碎片（重量约200毫克）浸在细胞壁-消化酶溶液中（1单位的1，4-β-甘露聚糖酶、1，3-β-木聚糖酶和琼胶酶，以及0.5M甘露醇，在20 mM MES缓冲液中，pH 7.5）在17 ℃下温育90分钟，同时轻轻搅拌，为了进行细胞融合，将聚乙二醇（PEG）加入到来自野生型黑紫B.atropurpurea和细紫菜绿色变体的原生质体悬浮液中。观察到原生质体的同质和异质粘附。将PEG处理的原生质体在约48 μ Em·s<-2>·L<-1>-1的人工光下在17 ℃下以9 h光/15 h暗循环培养。培养6周后，它们生长成褐色或绿色的愈伤组织和小植株。一对原生质体与其部分膜粘连，也发育成小植株。

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.