Isolation and Culture of Mesophyll Protoplasts of Phalaenopsis

为建立蝴蝶兰切花培养茎段叶肉原生质体的分离和培养方法，以在叶片切段培养中表现出较高再生能力的蝴蝶兰为材料。大约一克(F.W.)取3-4 cm长的幼叶，用刀片平行切开1 mm的叶片，放入预处理液(400ppm PVP，含0.4M蔗糖的无激素B5(1968)培养基中)处理30min。然后用10ml过滤灭菌的酶溶液(0.1%果胶酶Y-23，2%纤维素酶YC，400ppm PVP，在含有0.4M蔗糖的无激素B_5培养基中，pH 5.5)处理这些叶片。在25ﾟC的100mlErlmeyer瓶中黑暗培养3小时后，原生质体通过80um尼龙网过滤，去除波动的细胞和碎片，然后在90xg的条件下离心5min。收集漂浮的原生质体，90xg离心10min，洗涤2次(与前处理液相同)。洗涤后叶肉原生质体得率约为1.1-1.2×10~(-6)/g鲜重。在30×15 mm的塑料培养皿中，细胞密度约为2.5×10~(-4)/ml，其中含有2ml结冷胶(0.18%)固化的B_5培养基(pH 5.5)，附加0.1 mg/1NAA，5 mg/1BA，5 mg/l腺嘌呤，10 mg/l L-鸟氨酸，1g/l L-谷氨酰胺，400ppm PVP，0.5M D-甘露醇和2%蔗糖，在25ﾟ℃的黑暗中，细胞分裂不均匀。较小的子细胞继续分裂，产生不相等的子细胞。从叶段培养获得的体细胞胚胎中分离出原生质体的尝试也已取得。在上述培养基中(NAA 2 mg/l，BA 1 mg/l)培养这些原生质体，细胞分裂和集落形成不均匀。然而，这个菌落并没有发育成植株。

To develop a procedure for isolation and culture of mesophyll protoplasts from the leaves of shoots derived from flower-stalk cutting culture of Phalaenopsis, Phalaenopsis George Moler which shows higher regenerative capability in the in vitro culture of leaf segments was used as a material. About one gram (F. W.) of the youngest leaves (3-4 cm long) was scored with parallel 1 mm cuts using blade and placed the in pretreatment solution (400 ppm PVP in hormone-free B_5 medium (1968) containing 0.4M sucrose) for 30 min. These leaf strips were then treated with 10 ml of a filtered-sterilized enzyme solution (0.1% Pectolyase Y-23, 2% Cellulase YC, and 400 ppm PVP, in hormone-free B_5 medium containing 0.4M sucrose, pH 5.5). After 3 hours of incubation in a 100ml Erlemeyer flask at 25ﾟC in the dark without shaking, protoplasts were filtered through 80 um nylon mesh to remove undsted cells and debris then centrifugated at 90 x g for 5 min. The protoplasts floated were collected and washed 2 times by centrifugation at 90 x g for 10 min with washing medium (the same as the pretreatment solution). Yields of mesophyll protoplasts were appoximately 1.1-1.2 x 10^6 /g F. W. after washing. In approximately 0.01% of protoplasts cultured a cell density of 2.5x 10^4 /ml in 30 x 15 mm plastic petridishes containing 2 ml of Gellan Gum (0.18%)-solidified B_5 medium (pH 5.5) supplemented with 0.1mg/1NAA, 5mg/1BA, 5mg/1 adenine, 10mg/1 L-ornithine HCl, 1g/1 L-glutamine, 400ppm PVP, 0.5M D-mannitol and 2% sucrose at 25ﾟC in the dark, an unequal cell division occurred. The smaller daughter cell continued to divide to give unequal daughter cells. Attempts to isolate protoplasts from PLB (somaticembryo) derived from the leaf segment culture have also made. An unequal cell division and colony formation were observed, when culturing these PLB protoplasts in above described culture medium (2mg/1 NAA, 1mg/1 BA). However, this colony did not develop into plantlet.