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標題: | 青脆枝組織培養再生與轉殖之研究 Studies on Tissue Culture Regeneration and Genetic Transformation of Nothapodytes foetida |
作者: | Hung-Yu Yang 楊閎宇 |
指導教授: | 黃鵬林(Pung-Ling Huang) |
關鍵字: | 青脆枝,木本植物,體胚發生,不定芽誘導,瓶內發根,基因轉殖,農桿菌, Nothapodytes foetida,woody plant,somatic embryogenesis,adventitious shoot induction,in vitro rooting,transformation,Agrobacterium, |
出版年 : | 2011 |
學位: | 碩士 |
摘要: | 青脆枝 (Nothapodytes foetida) 含有重要的抗癌藥物成分喜樹鹼 (camptothecin, CPT),但於植株中含量低,故擬建立青脆枝癒傷組織再生及轉殖系統,以供未來進行喜樹鹼生合成相關基因轉殖有效且穩定的技術平台,以提高喜樹鹼含量。
再生系統之建立,係以青脆枝花藥誘導的癒傷組織為材料,將其培養於含1 mg/L TDZ之MS培養基,約80天可使部分癒傷組織形成體胚,將體胚移至含0.1 mg/L BA之MS培養基,可誘導根延長,再移至添加2 mg/L BA及1 mg/L GA的MS培養基,可使芽體抽長、葉片展開,再生成完整植株。另以青脆枝莖段進行不定芽誘導,0.5 mg/L TDZ處理可使莖段產生多芽體,再將多芽體置於含0.1 mg/L BA培養基抽長生長,將單一莖芽切下,扦插於添加 0.1 mg/L NAA及1 mg/L IBA之蛭石可得到根系較健全的植株,且可成功出瓶。 轉殖系統建立方面,以農桿菌媒介法將含3個報導基因Ds Red2、mGFP、gusA的質體轉殖至癒傷組織,並試驗菌種、菌液濃度、感染時間、界面活性劑、添加物等因子對於轉殖效率之影響,結果顯示青脆枝以A281或EHA105品系農桿菌菌液,稀釋至OD600 = 0.25,並添加0.25 mM Acetosyringone,感染6小時,共培養48小時後,以含500 mg/L cefotaxime、200 mg/L Timentin之無菌水淋洗,去除農桿菌,再將癒傷組織移到含300 mg/L cefotaxime、200 mg/L Timentin之X12固態培養基培養,以細胞分群配合低濃度 hygromycin進行篩選,60天後GUS活性組織化學染色的結果顯示A281及EHA105品系分別有約5% 及3% 的轉殖率,持續篩選可得到均質的轉殖癒傷組織。 Nothapodytes foetida contains camptothecin (CPT), an important anti-cancer component, but the content in plant is quite low. The CPT content can be augmented through genetic engineering of the CPT biosynthetic pathway. As a prerequisite we require an established regeneration and transformation system in N.foetida. Both of these objectives have been achieved in this present study. Regeneration was attempted and successfully optimized for calli induced from anthers. Media for various phases in the regeneration were optimized after detailed experiments with different plant growth regulators at different concentrations. Calli cultured on MS medium supplemented with 1 mg/L TDZ can induce somatic embryos within 80 days. For the development of somatic embryos into complete plantlets, MS medium supplemented with 0.1 mg/L BA enhances root elongation, whereas MS medium supplemented with 2 mg/L BA and 1 mg/L GA promotes shoot elongation and leaf expansion. Further the adventitious shoot pagation system was established using stem segments. MS medium supplemented with 0.5 mg/L TDZ was found to be optimal for inducing adventitious shoots, and MS medium with 0.1 mg/L BA was the best for shoot development. The shoot cuttages in sterile vermiculite containing 0.5 mg/L NAA and 1 mg/L IBA induced normal roots in vitro. An effective Agrobacterium-mediated transformation system was developed by studying the interaction effects of the Agrobacterium strains, bacterial concentration, infection time, surfactant concentration and the effectiveness of additives. The developed transformation system was validated by using a plasmid containing three reporter genes, DsRed2, mGFP, and gusA. Putative transformed cells were selected by using a cell separation method and with Hygromycin. The optimal transformation rates of A281 and EHA105 were 3% and 5%, respectively, based on GUS assay 60 days after infection. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/31051 |
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顯示於系所單位: | 園藝暨景觀學系 |
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