鐵砲百合花粉電穿孔基因轉殖法之研究

Study of gene transfer using pollen electrotransformation method in Easter lily(Lilium longiflorum Thunb.)

李光智(Lee Kung Chge)

指導教授 ： 張有明 ； 闕甫伈

亞洲大學/健康學院/生物科技學系碩士班/碩士(2006年)

摘要

鐵砲百合為世界重要花卉之一，可惜花色變化少，僅以白色系為主。本研究之目的在探討經由花粉電穿孔轉殖法進行鐵砲百合花色基因轉殖的可能性。在春季採集的花粉，以開花當日有較佳之活性，隨開花天數的增加，其發芽率隨之下降；花粉在含有蔗糖6%、硼酸40mg/L及氯化鈣40mg/L的液態培養基中以25~30℃的溫度培養180分鐘，其發芽率可達80%。開花當日至開花後五日內的柱頭活性佳，有較高的結實率；剪柱液態授粉則是以剪去3/4的處理效果較佳。
百合幼苗葉片插於50mg/L 的hygromycine液中經8天葉片全部白化，可做為抗hygromycine轉殖株篩選的標準。採上述條件，將dfr基因經電穿孔處理送入花粉中，再攜至田間授粉採種。採收的轉殖後代種子經播種育苗，採其葉片抽取DNA，經聚合酶連鎖反應(PCR)及其南方氏雜交法檢測，在轉殖後代均可測得含有外來的基因片段，證實以花粉電穿孔轉殖法，可將外源基因導入鐵砲百合中。

關鍵字

百合 ； 鐵砲百合 ； 百合花色 ； 百合花粉 ； 結實率 ； 可能性 ； 培養基 ； 花粉電穿孔 ； 花粉

並列摘要

Easter lily is one of the important floral crops in the world, but the pity is that of less variation in flower color except the white clor is noted. The purpose of this study was to evaluate the possibility of flower color gene transformation in Easter lily using pollen electrotransformation method. Pollen germination rate was the highest with the best activity at first blooming day, and decreased with following days after anthesis. The germination rate was up to 80% in special liquid medium supplemented with 6% sucrose, 40mg/L boric acid and 40mg/L CaCl2 at 25~30C. The stigma vigor has a better activity and with higher fruit setting at first blooming day to 5 days after flowering. Sitgma with 3/4 style cut-off was formol to have better effect in liquid pollination process. Young leaves were cultured on medium supplemented with 50mg/L hygromycine for selecting transformed plantlets. According to this pollen electrotransformation method, dfr gene was transferred into germinating pollen, then pollinated onto the normal flowers. After pollination, seeds were gathered and planted. Genomic DNA was extracted from the leaves of putative transformats. Results form PCR and DIG-labelled Southern assays indicated that dfr gene were transferred into transgenic progenies, this suggested that foreign gene could be transformed into Easter lily by means of pollen electrotransformation method.

並列關鍵字

lily ； pollen ； pollen electrotransformation

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