- 期刊
- OpenAccess
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
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。
並列摘要
＂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.
延伸閱讀
- Lai, H. C. (2005). 青脆枝(Nothapodytes foetida)組織培養,植株再生及喜樹鹼含量分析 [master's thesis, Tatung University]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0081-0607200917234453
- 何政坤、張淑華（2007）。利用青脆枝優良種苗枝葉生產喜樹鹼。林業研究專訊，14(1)，35-35。https://doi.org/10.29953/FRN.200702.0013
- 蔡杏枚、廖宇賡（2011）。篩選適合落花生毛狀根生長之簡化培養基。作物、環境與生物資訊，8(4)，242-253。https://doi.org/10.30061/CEB.201112.0004
- 呂芳舉、李國彰、王均琍（2014）。接種叢枝菌根菌促進孤挺花（Hippeastrum hybridum Hort.）植株生長及地下鱗莖之發育。植物種苗，16(2)，35-47。https://www.airitilibrary.com/Article/Detail?DocID=15622746-201406-201504200024-201504200024-35-47
- 陳國峰、何政坤、郭幸榮（2003）。農桿根群菌促進台灣紅豆杉成熟樹插穗發根。臺灣林業科學，18(3)，213-223。https://doi.org/10.7075/TJFS.200309.0213