Infection of Barley Protoplasts with Barley Stripe Mosaic Virus Detected and Assayed by Immunoperoxidase

The kinetics of infection of barley protoplasts with barley stripe mosaic virus (BSMV) was followed by enzyme-linked immunosorbent assay (ELISA) using the double-sandwich procedure. Infected protoplasts were identified by an immunoperoxidase method taking precautions to inhibit endogenous peroxidase. About 70% of the protoplasts were infected with BSMV when the inoculum contained 50 µg/ml purified BSMV in 10 mM-potassium citrate buffer pH 4.7 containing 0.65 M-mannitol and 5 µg/ml poly-L-ornithine. It was estimated by ELISA that about 1400 to 1600 virus particles were adsorbed per protoplast. Following an apparent latent period, virus increased linearly between about 14 and 48 h after inoculation. After 48 h the virus concentration reached between 1.62 x 10⁶ and 2.0 x 10⁶ particles per protoplast.