Please use this identifier to cite or link to this item: http://hdl.handle.net/11455/36870
標題: 建立高氏柴胡轉殖毛狀根系統之研究
Establishment on hairy root transformation system of Bupleurum kaoi Liu, Choa et Chuang
作者: 陳菀茹
Chen, Wan-Ju
關鍵字: Bupleurum kaoi Liu, Choa et Chuang;高氏柴胡;hairy root;毛狀根
出版社: 農藝學系所
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摘要: 
高氏柴胡(Bupleurum kaoi Liu, Choa et Chuang)為繖形花科(Umbelliferae)柴胡屬(Bupleurum)之草本植物,是保肝常用之重要藥材,利用組織培養技術,再配合農桿根群菌轉入發根基因於植物染色體中,藉以誘導培植體形成毛狀根之基因轉殖方法,已廣泛應用於中草藥植物生產二次代謝物之研究中。本研究主要探討農桿根群菌(Agrobacterium rhizogenes)感染高氏柴胡產生毛狀根之各種影響因子以及影響毛狀根生長之各種因子,以建立毛狀根之轉殖系統與毛狀根之最適生長條件。
以培養於含有0.25 mg/L BA之1/2 MS培養基之高氏柴胡瓶苗為材料,切取葉基部作為培植體於不含荷爾蒙之B5培養基預培養5天,使用農桿根群菌系R1601,於菌液濃度OD600介於0.8-1.2之間時,以含有200 μM乙酰丁香酮(acetosyringone, AS)之B5培養液等體積比例置換,作為感染培植體之菌液,感染時間為5分鐘,經過共培養三天後以含500 mg/L cefotaxime之B5培養基除菌,培植體可誘導毛狀根生成。毛狀根培養於B5液體培養基中,置於水平迴旋式震盪器中以85-90 rpm轉速,暗培養10週後,可得最大生物產量。高氏柴胡毛狀根與其不定根相較,在不含auxin的培養基中毛狀根有較佳的生物產量,未來將就提高轉殖毛狀根效率以及利用高氏柴胡毛狀根生產柴胡皂苷的可行性,進行更深入之探討。

Bupleurum kaoi Liu, Choa et Chuang (Umbelliferae) is an important traditional Chinese medicinal plant for liver protection. Hairy root produced by Agrobacterium rhizogenes mediated transformation through rol genes inserting to genomic DNA of target explants have been established using tissue culture process. There are many reports for secondary metabolite production using genetic transformed hairy root of medicinal plants. In this study, we reported that A. rhizogenes-mediated transformation of B. kaoi were investigated factors effecting to ultimate hairy root transformation efficiency and its production.
In vitro cultured B. kaoi maintaining on a half-strength Murashige and Skoog medium (1/2 MS) containing 0.25 mg/L BA was used in transformation experiments. Explants of leaf base were pre-cultivated on B5 medium for 5 days before inoculation. Bacterial solution of A. rhizogenes strain R1601 (OD600 0.8-1.2 ) in full strength with 200 μM acetosyringone was used for 5 min inoculation. After 3 days co-culturing, leaf base explants were subcultured into a B5 medium containg 500 mg/L cefotoxime for disinfection. Hairy root formation was observed after this stage.
The maximum biomass production of hairy root achieved at 10 weeks after culturing on a B5 liquid medium with 85-90 rpm shaking speed by a rotary shaker in dark condition. A comparison of the biomass of the hairy root and adventitious root on a auxin-free B5 liquid medium was conducted. The result showed hairy root superior to adventitious root on biomass production. Further investigation to improve transformation efficiency and production of saponin by hairy root of B. kaoi will be continued.
URI: http://hdl.handle.net/11455/36870
其他識別: U0005-2808200714134000
Appears in Collections:農藝學系

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