Tobacco callus culture as a propagating medium for Cucumber mosaic cucumovirus

Abstract

This study was performed on Cucumber mosaic cucumovirus (CMV) as a model. The virus was isolated from naturally infected cucumber plants depending on polyclonal antibodies specific for the CMV (subgroup I) and local lesions produced on Chenopodium amaranticolor were used as a source of single lesion isolation. CMV isolate was maintained in Nicotiana tabacum cv. White Burley. The presence of viral spherical particles was confirmed by electron microscopic examination. Infected callus tissues were prepared by syringe injection with infectious clarified tobacco sap with the aid of Millipore® filter. CMV was purified from both infected tobacco leaves and calli tissues. The purification method was modified to omit the sap clarification step when using calli as a purification source. After spectrophotometry and electron microscopic evaluation purified preparations from both sources were used for rabbit immunization to produce polyclonal antibodies. IgGs were purified and evaluated by determination of their dilution end points using indirect enzyme linked immunosorbant assay (I-ELISA) and their reaction against healthy tobacco sap as a control. The CMV coat protein gene (cp) was isolated and amplified from both kinds of infected tissues using immunocapture reverse transcription polymerase chain reaction (IC-RT-PCR). PCR products were analyzed by agarose gel electrophoresis. © 2010 Academic Journals Inc.