Infection of Protoplasts from Soybean Cell Culture with Southern Bean Mosaic and Cowpea Mosaic Viruses

Abstract

SUMMARY Soybean protoplasts, isolated from liquid suspension culture, were successfully infected with cowpea mosaic virus (CPMV) and with southern bean mosaic virus (SBMV). Poly-l-ornithine (PLO) was required for infection with either virus. As detected by fluorescent antibody, 70 to 90% of the protoplasts were infected by CPMV when the inoculation medium contained 0.4 m-sorbitol, 0.5 µg virus/ml, 1.5 µg PLO/ml, 10 mm-potassium phosphate buffer, pH 6.3, and 0.5 mm-CaCl2, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaCl2 was omitted. Inoculation at temperatures below 10 °C also decreased infection substantially. With SBMV a maximum of 30 to 35% of the protoplasts were infected when 0.4 m-sorbitol, 2 to 2.5 µg virus/ml, 2 µg PLO/ml, 10 mm-tris-HCl buffer, pH 8.0, 1 mm-MgSO4 and 0.5 mm-CaCl2 were present in the inoculum. Less than 5% of the protoplasts were infected when magnesium and calcium salts were omitted. The major advantage of the use of soybean cell suspension culture for plant virus studies is that it can provide a reliable and continuous source of cells for protoplast isolation. These soybean protoplasts allow synchronous infection and replication under sterile conditions without antibiotics and also a high degree of reproducibility.