Isolation and Culture of Mesophyll Protoplasts of Phalaenopsis

蝴蝶兰叶肉原生质体的分离与培养

• 批准号: 63560029
• 负责人: TANAKA Michio
• 金额: $ 1.22万
• 依托单位: Kagawa University
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1988
• 资助国家: 日本
• 起止时间: 1988 至 1989
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-63560029/
• 关键词: Phalaenopsis; Protoplast isolation; Protoplast culture

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.

以蝴蝶兰（Phalaenopsis）乔治莫勒（George Moler）为材料，研究了蝴蝶兰花茎扦插苗叶肉原生质体的分离和培养方法。约1克（F。W.）最年轻的叶子使用刀片用平行的1 mm切口刻划（3- 4cm长），并将其置于预处理溶液中（400 ppmPVP在含有0.4M蔗糖的无蔗糖B_5培养基（1968）中）处理30分钟。然后用10 ml过滤灭菌的酶溶液处理这些叶条（0.1%果胶酶Y-23、2%纤维素酶YC和400 ppm PVP，在含0.4M蔗糖的无糖B_5培养基中，pH5.5）。在100 ml Erlemeyer烧瓶中于25 ℃下在黑暗中孵育3小时后，不振荡，将原生质体通过80 μ m尼龙网过滤以除去未染色的细胞和碎片，然后以90 x g离心5 min。收集漂浮的原生质体，并通过以90 x g离心10 min用洗涤培养基（与预处理溶液相同）洗涤2次。叶肉原生质体的产量约为1.1-1.2 × 10^6 /g F。W.洗完后。在含有2 ml结冷胶的30 x 15 mm塑料培养皿中，约0.01%的原生质体培养细胞密度为2.5 x 10^4 /ml（0.18%）-固化B_5培养基（pH5.5），补充0.1mg/1 NAA、5 mg/1BA、5 mg/1腺嘌呤、10 mg/1 L-盐酸鸟苷酸、1g/1 L-谷氨酰胺、400 ppm PVP、0.5M D-甘露醇和2%蔗糖，25 ° C，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。本文还尝试了从叶段培养的体细胞胚（PLB）中分离原生质体.在上述培养基（2 mg/l NAA，1 mg/l BA）中培养这些原球茎原生质体时，观察到细胞分裂和菌落形成的不均匀性。但是，该菌落没有发育成植株。