Abstract
The potato plantlets singly infected by PVA, PLRV, PVS, PVX and PVY and mix-infected by PVM, PVS and PVY were cultured on MS medium with different concentration of ribavirin. The effects of ribavirin on growth of the plantlets and efficiency of virus elimination were investigated. Results showed that the plant height and fresh weight obviously decreased with increase of ribavirin concentration from 0 mg/L to 150 mg/L, and most of the plantlets could not survive when the concentration reached 200 mg/L. According to the ELISA tests, ribavirin was more efficient for eradicating PVA, PVM, PVS and PVX than PVY and PLRV, and healthy plantlets could be obtained with high frequency (up to 100 %) by culturing with 75 ~ 150 mg/L ribavirin after 2 ~ 3 subcultures. Whereas, only 33 ~ 66 % PVY and PLRV infected plantlets were found to be virus-free after 3 subcultures with 75 ~ 150 mg/L ribavirin. The results of quantitative RT-PCR (qPCR) indicated that ribavirin could obviously reduce virus content in the plantlets. Except PLRV was detected positive after 3 subcultures with ribavirin, the healthy seedlings were obtained from infected stocks at the first or end of propagation and no viruses could be detected at the post-eradication stage. No apparent difference of genetic variation resulted from ribavirin treatment was found by SSR analysis between the control and the treated plantlets. All of these results above proved that ribavirin treatment in vitro was an effective method to eliminate viruses in the propagation of potato.
Resumen
Las plántulas de papa infectadas individualmente con los virus PVA, PLRV, PVS, PVX y PVY e infectadas con la mezcla de PVM, PVS y PVY, se cultivaron en un medio MS con diferentes concentraciones de ribavirin. Se investigaron los efectos de ribavirin en el crecimiento de las plántulas y la eficiencia en la eliminación de virus. Los resultados demostraron que la altura de la planta y el peso fresco disminuyeron obviamente con el aumento en la concentración de ribavirin de 0 mg/L a 150 mg/L, y la mayoría de las plántulas no sobrevivieron cuando la concentración alcanzó 200 mg/L. De acuerdo con las pruebas de ELISA, ribavirin fue más eficiente en la erradicación de PVA, PVM, PVS y PVX, que en la de PVY y PLRV, y se pudieron obtener plántulas sanas con alta frecuencia (hasta de 100 %) mediante el cultivo con 75 ~ 150 mg/L de ribavirin después de 2 ~ 3 subcultivos, mientras que solo 33 ~ 66 % de plántulas infectadas con PVY y PLRV se encontraron libres de virus después de tres subcultivos con 75 ~ 150 mg/L de ribavirin. Los resultados de RT-PCR (qPCR) cuantitativos indicaron que ribavirin pudo reducir obviamente el contenido de virus en las plántulas. Excepto por el PLRV que fue detectado después de tres subcultivos con ribavirin, las plántulas sanas se obtuvieron de lotes infectados al principio o al final de la propagación y no se pudieron detectar virus en la etapa de post-erradicación. No hubo diferencia aparente en variación genética del tratamiento con ribavirin mediante análisis SSR entre las plántulas testigo y las tratadas. Estos resultados prueban que el tratamiento in vitro con ribavirin fue un método efectivo para eliminar virus en la propagación in vitro de papa.
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Acknowledgments
We thank Prof. Conghua Xie and Dr. Xianzhou Nie for their valuable discussions and language editing and Dr. Xun Liu for figures modifying. This research was supported by Earmarked Fund for Modern Agro-industry Technology Research System (CARS-10-P08), National Science Foundation of China (31101191) and Fundamental Research Funds for the Central Universities (2011PY145).
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Yang, L., Nie, B., Liu, J. et al. A Reexamination of the Effectiveness of Ribavirin on Eradication of Viruses in Potato Plantlets in vitro Using ELISA and Quantitative RT-PCR. Am. J. Potato Res. 91, 304–311 (2014). https://doi.org/10.1007/s12230-013-9350-z
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DOI: https://doi.org/10.1007/s12230-013-9350-z