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Studies on the protoplast of Phalaenopsisy hybrida HORT.

By Min-Chang Huang and 黃敏展

Abstract

使用蝴蝶蘭無菌播種的實生苗葉片以及嘉德麗亞蘭花瓣為材料,獲得較多原生質體的處理是以D-mannitol的濃度為0.7M,MES buffer 20mM,MgCl2 5mM,Ph5.6的基本液。添加酵素的配方,在蝴蝶蘭葉片為Cellulase "Onozuka" R-10 1.0%,Macerozyme R-10 0.5%,Pectolyase Y-23 0.1%。在嘉德麗蘭花瓣為Cellulase "Onozuka" R-10 2.0%,Macerozyme R-10 0.5%,Driselase 0.5%,處理時間為6-12小時。細胞融合採用電氣平行極融合方法,使用日本島津公司製造之SSH-1型融合器,經多次測試結果,原生質體融合條件為Frequency 1 MHz,形成原生質體連珠的磁場電壓VAC為30-50Vp.p,PW為30-60μs,pulse時的磁場強度Field strength 以1-1.5KV/cm為宜。Pulse的次數,經30-60秒,可按1-3次,視原生質體的狀況而定,用酵素處理期間的溫度為25-28℃,宜保持較高之外,精製過程和融合操作間,以保持4-5℃較低溫度為佳

Topics: protoplast, Phalaenopsisy hybrida, 原生質體, 蝴蝶蘭
Year: 2014
OAI identifier: oai:ir.lib.nchu.edu.tw:11455/81483
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