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

Abstract

SUMMARYSoybean 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 9o % of the protoplasts were infected by CPMV when the inoculation medium contained o'4 M-sorbitol, o'5/~g virus/ml, 2"5/zg PkO/ml, IO raM-potassium phosphate buffer, pH 6.3, and o'5 mM-CaCI~, and inoculation was performed at 23 °C. This maximum level of infection was decreased sixfold when CaC12 was omitted. Inoculation at temperatures below l o °C also decreased infection substantially. With SBMV a maximum of 3o to 35 % of the protoplasts were infected when o'4 M-sorbitol, 2 to 2"5/~g virus/ml, 2 #g PLO/ml, ro mm-tris-HC1 buffer, pH 8.o, I mM-MgSO4 and 0. 5 mM-CaC12 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.

Infection of Soybean Protoplasts from Cell Suspension Culture with Bean Pod Mottle Virus

“…By 18 h, over half of the developing fluorescence had appeared. At 24 h, the rate of increase in fluorescence began to level off markedly until little increase was seen between 36 to 60 h. The fluorescence was diffuse and was similar to the type seen for CPMV in this system (Jarvis & Murakishi, 1980) rather than the discrete crystalline pinpoint fluorescence as reported for TMV in tobacco protoplasts (Takebe & Otsuki, 1969). Non-inoculated control protoplasts showed no development of fluorescence during the entire incubation period.…”

“…'Harosoy 63' were initiated from soybean hypocotyl and maintained as cell suspension culture as described previously (Jarvis & Murakishi, 1980). Protoplast isolation.…”

“…Inoculation and incubation procedures were as previously described (Jarvis & Murakishi, 1980) with the following variations. Preincubation of the inoculation medium was for 15 min at 22 °C.…”

“…Protoplast isolation. Protoplasts were isolated as previously described (Jarvis & Murakishi, 1980) with the following modification. Just before inoculation, protoplasts were pelleted, resuspended in 0.6 i-sorbitol (to cause some shrinkage) then passed through a 25 am mesh stainless steel screen to remove cell wall debris and non-digested cells.…”

“…Recently, the first workable system for studying plant virus infection with soybean protoplasts derived from liquid suspension culture was developed (Jarvis & Murakishi, 1980). Optimum infection with cowpea mosaic virus (CPMV), a comovirus, required poly-L-ornithine (PLO) and CaC12 in the inoculum.…”