Extracts from virus infected hypersensitive tobacco leaves are detrimental to protoplast survival

Abstract

Tobacco mesophyll protoplasts cultured in vitro were killed when extracts, prepared from the leaves of Nicotiana tabacum L. cv. Xanthi nc (hypersensitive host) infected with tobacco mosaic virus (TMV), were included in the medium. Protoplast survival was not significantly diminished by similar extracts from plants of N. tabacum L. cv. Xanthi (systemic host) infected with TMV or from tobacco plants sham inoculated with water only. Protoplasts from the hypersensitive host were more susceptible to being killed by toxic leaf extracts than protoplasts from systemic hosts. Protoplasts were not killed immediately upon resuspension in toxic leaf extracts when the criterion for viability was their ability to reduce the vital stain methylene blue. Instead, there was a lag phase of 8 to 10 h; the killing reaction was virtually complete by 1 day. Toxicity of leaf extracts was detected 24 h before lesions appeared, and was related to both lesion number and lesion development. Toxicity of leaf extracts was apparently due to a non-dialysable, thermolabile substance or group of substances. The toxicity was reduced by Ca ++ ions, and increased by pre-treating extracts with ethylenediaminetetra-acetic acid (EDTA) or incubating protoplasts in the dark, in culture medium containing toxic leaf extract. The significance of these results is discussed in relation to necrogenesis in virus infected plants responding hypersensitively.