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

以海洋环境中分离的弧菌sp. MA-138、Alcaligenes sp. XY-234和Vibrio sp. P0-303为培养基，制备了分离红藻原生植物所需的3种酶——β - 1,4 -甘露聚糖酶、β - 1,3 -木聚糖酶和琼脂酶。所有粗酶的最适ph值均在7.5左右。考察了原生质体分离的适宜条件。用木瓜蛋白酶溶液（20 mM MES缓冲液，pH 7.5，含2%木瓜蛋白酶和0.5 M甘露醇）处理叶片有助于原生质体的分离。将刮切的叶片片段（重约200mg）浸于细胞壁消化酶溶液（1,4 - β -甘露聚糖酶、1,3 - β -木聚糖酶和琼脂酶各1单位，0.5 M甘露醇在20 mM MES缓冲液中，pH为7.5）中，在17℃下温和搅拌90 min，释放原生质体7.1 × 10^6个。为了进行细胞融合，将聚乙二醇（PEG）添加到野生型B.atropurpurea和绿色品种Porphyra tenera原生质体的悬浮液中。观察原生质体的同质和异质粘附。经peg处理的原生质体在约48 μ m^<-2 bbb s^<-1>的人工光照条件下，在17‰C的光照/暗循环条件下培养9 h。培养6周后形成愈伤组织和褐色或绿色植株。被膜部分粘附的一对原生质体也发育成植株。

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.