Journal of Taiwan Agricultural Research

Author:Chi-Ni Hsia, Chin-Chih Chen, Nan-Hsin Lin, and Wei-Ting Tsai*

Abstract:

    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.

Key words:Orchid, Thin cell layer culture, Protocorm-like body

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