ISHS Acta Horticulturae 164: VI International Symposium on Virus Diseases of Ornamental Plants
REPLICATION OF CHRYSANTHEMUM STUNT VIROID IN ISOLATED LEAF CELLS AND IN CELL CULTURES OF CHRYSANTHEMUM |
| Authors: | M. Monsion, P. Merel, M. Vitale, G. Macquaire, J. Dunez |
| Abstract:
Replication of Chrysanthemum stunt viroid (CSV) was investigated in cells isolated from Chrysanthemum fresh leaves and in Chrysanthemum cell suspensions cultured in vitro. Cells were extracted from leaves after enzymatic digestion with pectinase and cellulase : they differ from protoplasts because of the presence of uncompletely digested walls. Suspension cultures of infected and healthy cells derived from calli produced from leaf or stem tissues. Calli were grown on agar solidified Skoog medium supplemented with 2–4 D and kinetin. After 2–3 months, friable calli were selected and transferred to liquid medium and incubated on an orbital checker. Cells were transferred after filtration every two weeks. Calli and cell suspensions were cultured at 25°C under continuous light. Both cells isolated from fresh leaves and those cultured from calli were metabolically active. They incorporate radioactive precursors (3H Uridine) into TCA insoluble material. Infected cells sustain viroid replication. Persistence of CSV replication was observed after 18 months culture. Presence of the viroid was detected by gel electrophoresis and by infectivity. After transmission to Chrysanthemum plants typical symptoms develop. Despite the fact that viroids were reported to multiply preferentially at high temperatures, best results in this long term replication of CSV were obtained at about 25°C. Synthesis of new CSV molecules was demonstrated after incorporation of labelled precursors in viroid RNA by polyacrylamide gel electrophoresis and autoradiography. | |
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