Production of Potato Spindle Tuber Viroid (PSTV) by Large Scale Fermentation of PSTY–Infected Potato Cell Suspension Cultures

Abstract

Berlin, J., Wray, V., Forche, E., Reng, H.–G , Sch€ler, H, Luckinger, R. and M€hlbach, H.–P. 1985. Production of potato spindle tuber viroid (PSTV) by large scale fermentation of PSTV–infected potato cell suspension cultures.—J. exp. Bot 36: 1985–1995. Cell suspension cultures of Solatium demissum, infected with the potato spindle tuber viroid (PSTV), were scaled up to volumes of up to 800 dm3 to provide sufficient and uniform plant material for subsequent studies on viroid biosynthesis. Here we describe the technological aspects of producing the required amounts of biomass and viroid. The cells, which had been maintained on a medium containing expensive coconut milk, were first adapted to rapid growth on the less expensive B5–medium. The physiological state of the cells was monitored by in vivo31P–NMR spectroscopy Under the chosen conditions the scale–up from 10 dm3 inoculum from shake flasks to the harvest of the 800 dm3 stirred fermenter lasted 38 d and provided 112 kg biomass. Growth characteristics and viroid production in shake flasks and large bioreactors were rather similar. Gel electrophoretic analysis of isolated nucleic acids using silver staining and Northern blot hybridization revealed a PSTV–content of approximately 700 μg PSTV per kg fresh mass of cultured cells.