菊花利用農桿菌轉殖色素基因之研究

Abstract

利用基因轉殖技術導入單個或多個目標基因於植物染色體中，配合組織培養技術再生大量植株，有助於跨越遺傳鴻溝並提高育種效率。全世界的菊花栽培種種類繁多，但目前所建立的再生程序大都僅適用於特定的品種，並無法廣泛的應用在眾多的菊花品種之中。本論文主要以15個菊花栽培品種，首先建立其無菌瓶苗，再以瓶苗葉柄培植體為試驗材料，以不同生長調節劑之組合，測試各品種之再生反應，並以癒傷組織誘導再生的｀紅美人＇及可直接再生芽體之｀黃秀芳＇菊花栽培品種，應用於後續之轉殖試驗中。以農桿菌EHA105做為媒介進行轉殖試驗，轉殖過程中所使用之3種除菌抗生素（carbenicillin、cefotaxime及timentin）試驗中，發現參試之三種抗生素對於菊花芽體再生皆不會產生明顯的抑制影響，甚至200 mg/L timentin處理有助於芽體的產生。此外hygromycin濃度對篩選擬轉植株之影響，培植體在hygromycin濃度高於10 mg/L時即會抑制芽體再生。目前經hygromycin篩選之擬轉植株，經馴化後培養於溫室中觀察其園藝性狀之表現，25株｀紅美人＇栽培種之擬轉殖株中，其中20株可觀察到花色與對照組有明顯之差異，未來將就花色基因表現之部分進一步探討，並持續對農桿菌轉殖菊花系統加以改進。

Using gene transformation technology for inserting foreign target gene(s) into plant chromosome, following by tissue culture techniques for regeneration of transgenic plants, contributes to overcome the hereditary gap and to improve the efficiency of breeding. There are various chrysanthemum cultivars in the world, however the regeneration protocols were developed mostly only suitable for the specific cultivar(s) and unable to apply to the numerous varieties. In this study, regeneration systems using 15 cultivars of chrysanthemum were established. The factors of explant type, combinations of plant growth regulators were tested for regeneration, and a somatic embryogenesis system and a organogenesis regeneration system were developed for｀Hon-Mei-Zen＇and｀Huang-Shiou-Fan＇, respectively. Three kinds antibiotic (carbenicillin, cefotaxime and timentin ) were tested for their bacteriostatic effects and influences on regeneration. It was found that all three antibiotics had no obvious suppressing influence on shoots regeneration of the chrysanthemum. More over the 200 mg/L timentin treatment was found beneficial for the shoot regeneration in a A. tumefaciens EHA105 mediated transformation. A study of proper hygromycin concentration for screening putative transgenic plants was also conducted, the 10 mg/L hygromycin was found effective for shoot regeneration. The putative transgenic plants screened from hygromycin selection were potted in the greenhouse for their horticultural characteristics observation. A total 25 putative｀Hon-Mei-Zen＇transgenic plants for the flower color investigation, and 20 of them recorded with various levels of color expression. Further investigations for the pigment gene expression and improving of Agrobacterium-mediated transformation system for chrysanthemum are needed for the future work.