Isolation and Culture of Mesophyll Protoplasts of Phalaenopsis

以在离体培养中具有较高再生能力的蝴蝶兰（Phalaenopsis George Moler）叶片为材料，建立了从蝴蝶兰（Phalaenopsis Phalaenopsis）花茎扦插培养的嫩枝叶片中分离培养叶肉原生质体的方法。将最年轻叶片（3-4 cm长）约1克（F. W.）用叶片平行切1 mm，置于预处理溶液（400 ppm PVP，含0.4M蔗糖的无激素B_5培养基（1968））中30 min。然后将这些叶片条用10 ml过滤灭菌的酶溶液（0.1%果胶酶Y-23, 2%纤维素酶YC， 400 ppm PVP，含0.4M蔗糖的无激素B_5培养基，pH 5.5）处理。在100ml Erlemeyer烧瓶中孵育3小时后，在黑暗中不摇晃，在25℃的温度下，原生质体通过80 um尼龙网过滤，去除未染色的细胞和碎片，然后在90 x g离心5分钟。收集漂浮的原生质体，用与预处理液相同的洗涤介质（90 x g）离心洗涤2次，洗涤10 min。洗涤后的叶肉原生质体产量约为1.1-1.2 × 10^6 /g f.w.。约0.01%的原生质体细胞密度为2.5 × 10^4 /ml，在30 × 15 mm的塑料培养皿中培养，培养皿中含有2 ml结冷胶（0.18%）固化的B_5培养基（pH 5.5），在黑暗中添加0.1mg/1NAA, 5mg/1BA， 5mg/1腺嘌呤，10mg/1 l -鸟氨酸HCl， 1g/1 l -谷氨酰胺，400ppm PVP， 0.5M d -甘尼醇和2%蔗糖（25‰），细胞密度为2.5 × 10^4 /ml。较小的子细胞继续分裂，产生不等的子细胞。从叶段培养的体胚（PLB）中分离原生质体也进行了尝试。在上述培养基（2mg/1 NAA, 1mg/1 BA）中培养这些PLB原生质体时，观察到细胞分裂和集落形成不均。然而，这个群体并没有发育成植株。

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.