沙巴蛇草之再生與轉殖系统之建立

Abstract

沙巴蛇草 (Clinacanthus nutans L.) 為馬來西亞民俗藥用植物,具有預防癌症之潛力。本研究所建立之沙巴蛇草組織培養再生及轉殖系統，將有助於大量繁殖及代謝體學研究。以含 1 mg/L BA 及1 mg/L Kinetin 之MS培養基，誘導莖頂組織生成不定芽，可獲得最高增殖率。將不定芽移至僅含0.5 mg/L之MS培養基可順利抽長並發根。以各種生長素及細胞分裂素誘導葉圓片生成癒傷組織，並依形態進行分類，經選取黃色且緊密結實之癒傷組織，培養於含2,4-二氯苯氧乙酸 (2,4-dichlorophenoxyacetic acid) 之MS培養基，可生成具有胚性之細胞並分化為體胚。癒傷組織及芽體以濃度為OD600 0.5之含有pCRFG質體的農桿菌 LBA4404浸染1小時後，共培養72小時，經1 mg/L Hygomycin篩選，獲得轉殖之癒傷組織及芽體；將癒傷組織以45℃進行熱休克處理後，再與膿桿菌共培養，可獲最高效率GUS陽性反應。以超音波震盪前處理節間，所誘導之芽體伴隨農桿菌菌液再進行轉殖，可檢測到報導基因GFP表現，經聚合酶連鎖反應分析，結果顯示超音波處理5秒，可達90%轉殖率。

In Acanthaceae family, Clinacanthus nutans L. is a popular folk medicine and has potential in cancer prevention. Therefore, establishing in vitro regeneration and genetic transformation systems of C. nutans is important for mass production and metabolomics studies of this medicinal plant. Successful regeneration system from difference explant types of C. nutans was developed. The adventitious shoots induced from tissue mass formed from shoot tips gave high proliferation rate on MS medium containing 1 mg/L BA and 1mg/L kinetin. MS medium containing 0.5 mg/L kinetin promoted shoot elongation and rooting. For callus initiation, various types of auxins and cytokinins were tested on leaf laminas and types of callus were classified. Callus induced from leaf explants on MS medium containing 2,4-dichlorophenoxyacetic acid (2,4-D) produced competent embryogenic cells. In addition, MS supplemented with 2 mg/L 2,4-D was found to be optimal in callus maintenance while 0.3 mg/L 2,4-D was optimal in the development of somatic embryos. Furthermore, Agrobacterium-mediated transformation system of C. nutans was established. The effects of bacterial concentration, infection time, co-cultivation, sonication period, heat shock and centrifugation were tested. Transgenic calli and shoots were obtained through selection with 1 mg/L hygromycin after transformation with Agrobacterium tumefaciens strain LBA4404 harboring plasmid pCRFG. The bacterial concentration of A600=0.5 with co-cultivation periods of 72 hours was optimal conditions for transformation. Callus treated with heat shock showed highest frequency of GUS positive tissues. GFP expression was observed in shoots induced from nodal explants with sonication treatment. PCR was carried out on hygromycin-resistant plants to identify possible transformants. Results showed optimal transformation efficiency for shoots were explants treated with 5 s of sonication.