利用不同培植體配合利巴韋林處理對蝴蝶蘭去病毒之影響

Effect of Explant Type and Ribavirin Treatment on Virus Elimination of Phalaenoposis spp.

夏奇鈮(Chi-Ni Hsia) ； 陳金枝(Chin-Chih Chen) ； 林南欣(Nan-Hsin Lin) ； 蔡媦婷(Wei-Ting Tsai)

《台灣農業研究》 66卷2期 (2017/06) Pp. 146-157

https://doi.org/10.6156/JTAR/2017.06602.07

摘要

本研究利用切取自短縮莖及擬原球體 (protocorm-like body; PLB) 之薄片培植體配合利巴韋林 (ribavirin)不同濃度與處理時間進行蝴蝶蘭去病毒研究。試驗材料為確知感染蕙蘭嵌紋病毒 (Cymbidium mosaic virus; CymMV) 之蝴蝶蘭「台農1 號小精靈」組織培養瓶苗，以瓶苗去葉後之短縮莖切取厚約1 mm 薄片作為培植體，將薄片培植體置入含有不同濃度ribavirin 之液體培養基中震盪培養1-5 d 後，移至不含ribavirin 之固體培養基中培養；或將薄片培植體於含有較低濃度ribavirin 之固體培養基中持續培養一代或二代，培養期間除調查培植體的存活率外，當長出之小芽其葉片或根長度達1 cm 以上時，切取其葉片或根以間接式酵素連結免疫吸附反應法 (indirect enzyme-linked immunosorbent assay; Indirect-ELISA) 進行病毒檢測，其中ELISA 檢測呈陰性反應之芽體續以反轉錄核酸聚合酶鏈鎖反應 (reverse transcription polymerase chain reaction; RT-PCR) 進行檢測。試驗結果顯示，以250 mg L^(-1) ribavirin 液體培養1 d 以上即明顯降低培植體之成活率；ribavirin 濃度降至25-75 mg L^(-1) 之間處理1-5 d，則不具去病毒之效果；但若於50 mg L^(-1) ribavirin 液體培養基中浸泡2 d 後，再繼代於含有25 mg L^(-1) ribavirin 之固體培養基中繼續8-12 wk 培養，去病毒率為26.7%。此外，切取自短縮莖與切取自PLB 之薄片培植體於低濃度 (12.5-37.5 mg L^(-1)) ribavirin 固體培養基中進行二階段培養，短縮莖與PLB 培植體之去病毒率最高分別為29.2% 及22.2%，但PLB 薄片培植體之對照組 (未施用ribavirin) 去病毒率可高達100%。綜合本研究之結果，建議採用具有高再生能力之培植體，以對植物細胞毒傷害較小但具抑制病毒增生之低濃度ribavirin 配合繼代處理，具有提高去病毒之效果。

關鍵字

蘭科植物 ； 薄層細胞培養 ； 擬原球體

並列摘要

This study was conducted to determine thin section explants derived from stem or protocormlike body (PLB) in combination with ribavirin treatments on virus elimination of the Cymbidium mosaic virus (CymMV)-infected Phalaenopsis 'Tainung No. 1 Pixie'. Leafless in vitro shoots were cut into 1 mm thin sections as culture explants. Explants were soaked in liquid media containing various concentrations of ribavirin for less than 5 d culturing before culturing into the solid medium without ribavirin; or explants were cultured directly on solid media containing lower concentrations of ribavirin for one or consecutive culturing. Other than collecting survival rates of explants, leaves or roots which developed longer than 1 cm were cut out from regenerants for indirect enzyme-linked immunosorbent assay (ELISA) tests. Samples reacted in negative by ELISA were tested further using reverse transcription polymerase chain reaction (RT-PCR). Results showed that high concentration of ribavirin (250 mg L^(-1)) suppressed explant survival as well as shoot regeneration significantly. However, lower concentrations of ribavirin (25-75 mg L^(-1)) for short duration were found not effective on virus elimination. A virus elimination rate of 26.7% was obtained from explants soaked in the liquid medium containing 50 mg L^(-1) ribavirin for 2 d before subculturing on the solid medium containing 25 mg L^(-1) ribavirin for 12-wk of culturing. Thin section explants cut from in vitro shoot stem or PLB were both cultured on the solid media containing 12.5-37.5 mg L^(-1) ribavirin for two consecutive culturing. The highest virus elimination rate derived from stem or PLB thin section explants were 29.2% and 22.2%, respectively. However, a virus elimination rate of 100% was obtained from the control PLB explants which treated without any ribavirin. In conclusion, in order to obtain an efficient virus elimination system, highly regenerable explants cultured in the medium containing low concentration of ribavirin with least cytotoxic effect on regenerable cell growth but with suppression on virus proliferation for consecutive cultures would be recommended.

並列關鍵字

Orchid ； Thin cell layer culture ； Protocorm-like body