Establishment, purification, maintenance and serological diagnosis of Sunflower necrosis virus in callus

The calli induction from different Sunflower necrosis virus (SNV)-infected explants of sunflower was initiated on Murashige & Skoog medium amended with benzyladenine at 1 mg l -1 and indole acetic acid at 0.5 mg l -1. The virus was purified from the induced calli by sucrose density gradient centrifugation and the viral particles were confirmed by transmission electron microscopy. The SNV infection in uninfected calli was established by a soak prick method and the viral development was confirmed by a plant infectivity test. Serological analyses such as Ouchterlony double immnuodiffusion test, enzyme-linked immunosorbent assay and Western blot using anti-SNV antibodies yielded positive reactions with the purified SNV. These investigations have demonstrated that the SNV could be cultured in calli and maintained in vitro in pure form for a long period of time.