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頁籤選單縮合
題名 | Establishment of Hairy Root Cultures of Nothapodytes nimmoniana to Produce Camptothecin=建立青脆枝毛狀根培養以生產喜樹鹼 |
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作者姓名(中文) | 張淑華; 陳芬蕙; 蔡錦瑩; 陳媶; 黃芷雲; 呂汶玲; 何政坤; | 書刊名 | 臺灣林業科學 |
卷期 | 29:3 2014.09[民103.09] |
頁次 | 頁193-204 |
分類號 | 371.8 |
關鍵詞 | 青脆枝; 農桿叢根菌; 毛狀根; 喜樹鹼; Nothapodytes nimmoniana; Agrobacterium rhizogenes; Hairy roots; Camptothecin; |
語文 | 英文(English) |
中文摘要 | 本研究利用農桿叢根菌AR281、AR1600與ATCC15834建立青脆枝的毛狀根培養系統。農桿菌的轉殖率在不同菌種與單株間有很大的差異,以ATCC15834轉殖率最高,在接種的10個營養系中,有7個營養系可成功誘導毛狀根,以H2營養系的莖段轉殖率在3個菌種都最高,將毛狀根培養於不含有植物生長調節劑的MS培養基中生長快速且有很高的分枝性,具有毛狀根的特性。選3個來自G1-4或H2接種ATCC15834產生之生長快速毛狀根進行鑑定與培養,以PCR與南方雜交法確認農桿叢根菌的rol B基因已轉移至毛狀根。將3個毛狀根在MS固體培養基培養40天,growth index為2.6~3.0,喜樹鹼含量為0.0537~0.1555%(乾重)。將毛狀根移入液體培養基中培養,其生長比固體培養增加1.8倍,培養30天後,喜樹鹼產量為9.8 mg/L,其中93.9%(9.2 mg/L)會從毛狀根系釋出於培養液中,因此可不斷收穫培養液提煉喜樹鹼,對未來利用生物反應器培養是一項有利的生產模式。 |
英文摘要 | "Nothapodytes nimmoniana" hairy roots were induced by infecting stem and leaf segments with 3 wild-type "Agrobacterium rhizogenes" strains, AR281, AR1600, and ATCC15834. Results showed great variations in the transformation efficiencies among different "A. rhizogenes" strains and "N. nimmoniana" clones in vitro. Strain ATCC15834 had the highest transformation ability. Among all 10 clones, 7 clones were infected, and hairy roots were successfully induced. These hairy roots grew vigorously and were highly branched on phytohormone-free solid medium. Three fast-growing hairy root lines, for which integration of the "rol" B gene into the root genome was verified using PCR and Southern blotting, were used for the growth and alkaloid production tests. After being cultured for 40 d, the growth index of hairy roots was 2.6~3 and camptothecin (CPT) contents were 0.0537~0.1555% dry weight (DW). When cultured in liquid medium, hairy roots grew up to 1.8-times faster than they did in solid medium. After 30 d in liquid culture, the CPT concentration was 9.8 mg/L. Approximately 93.9% (9.2 mg/L) of the total CPT produced was excreted into the culture medium. With continual harvesting of CPT from the liquid medium, this system will benefit the development of large-scale bioreactor culture in the future. |
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