香蕉基因組亞群華蕉系之原生質體培養

Culture and Early Embryogenesis of Suspension-cell-derived Protoplasts of Banana AAA Cavendish Subgroup cv. Robusta and Pei Chiou

香蕉華蕉系＇北蕉＇及'Robusta'兩參試品種，以幼雄花序衍生之懸浮細胞供當原生質體分離來源。混合酵素以cellulase 2.5%、macerozyme 1.5%、driselase 0.5%及pectolyase Y23 0. I%組合，分離效果最佳，抽取效率依細胞團之類型而定，以橢圓體及平頭體、平頭伸長體（早期）較佳，原生質體生產值可達9.3x 106個／每克鮮重，球狀體次之為4.3x 106·個／每克鮮重。原生質體接種密度以4x 105個／每毫升以上，有利其胞型再生及生長。培養基含不同無機鹽類組合（Kao , MS , SH）之CPIHI , MSB, SHI ,皆可促使分裂生長。起始接種及持續生長於液體內，早期細胞生長受限，包埋於agarose則有利其細胞增生。植物生長調節物質2,4-D I mg/I ,加或不加NAA 0.5 mg/I , Kinetin 0.01 mg/I ,均可以促使原生質體進一步分裂形成具有明顯胚柄及胚本體的原胚體，而大多數的原胚體可進一步分裂形成多胞體，少部分甚至可以發育至早期球狀體。植物生長調節劑NAA 0.2 mg/I , Kinetin 0.1 mg/I , Zeatin 0.05 mg/I , 2iP 0.2 mg/I ,則可以促使原生質體更快分裂在2個月內形成球狀胚。由'Robusta'懸浮細胞原生質體再生過程的觀察顯示，其顯然為原生質體直接發育成早期擬胚，而不必經過癒合組織的階段。至於目前之培養，至多只能達於早期球胚，其可能因持續Auxin（2,4-D）的存在有關。

Young male inflorescence-derived suspension cells of banana Cavendish subgroup cv. Pei Chiao and Robusta were the source for protoplast isolation material. The best enzyme composition was the mixture of cellulase 2. 5%, macerozyme 1.5%, diselase 0.5%, and pectolylase Y23 0.1 %. Digestion efficiency was varied with the growth types of cell clusters. Oblong bodies, flatten bodies, and the early stage of the flatten elongated bodies produced approximately 9.3x I 06 protoplasts /g(fresh weight).The globular bodies produced 4.3x I 06 protoplasts lg High plating density about 6x 106 cells/dish(3.5cmx 1.3 cm) was beneficial for the further growth and development of protoplasts. Culture media based on salt compositions of Kao , SH and _MS were all suitable for cell division. Initial inoculation in liquid medium limited the growth of protoplasts. Embedding in agarose (I%) improved cell proliferation. 2,4-0 (I mg/1), with or without NAA( 0.5 mg/1), kinetin (0.01 mg/1) promoted protoplast division , most treated protoplasts grew well to form proembryoid structures. A few early globular bodies were observed Medium supplemented with NAA 0.2 mg/1 , kinetin 0.1 mg/1 , zeatin 0.05 mg/1 ,2iP 0.2 mg/1 promote more division and eventually formed globular body-like structures within two months The regeneration in'Robusta'protoplasts developed into proembryo directly, without an intervening callus phase. As to the cultures at present just grow to the globular body structure, and couldn't further develop should pertain to the long exist of the 2,4-0.