K.R. Wood

The isolation and culture of cucumber mesophyll protoplasts

Abstract Large numbers of viable protoplasts were obtained from the first leaves of two cultivars of cucumber, Cucumis sativus L. cv. Ashley and China. Cell wall regeneration was observed in liquid culture within 3 days of isolation, and division within 5 days.

Improved isolation and culture methods for cucumber mesophyll protoplasts

Abstract Improved methods for the isolation of large numbers of viable protoplasts from cotyledons and first leaves of two cultivars of cucumber, Cucumis sativus L. cv. Ashley and China are described. Short-term isolation methods (STM), and long-term isolation methods (LTM), using a variety of commercial enzymes are outlined. Liquid culture and subsequent solid culture of isolated first leaf protoplasts resulted in sustained cell division and eventual compact callus formation, which has been induced to form roots. No shoot or whole plant regeneration has, as yet, been demonstrated.

Virus multiplication and peroxidase activity in leaves of cucumber (Cucumis sativurs L.) cultivars systemically infected with the W strain of cucumber mosaic virus

The rate of multiplication of cucumber mosaic virus (W strain) was assessed in the systemically infected leaves of the three cucumber cultivars, Ashley, National Pickling and China. Cyclic multiplication of the virus was observed, with maxima at 10–11 and 14–17 days after infection at 20°C. The highest virus titres were recorded from Ashley, particularly second true leaves, establishing Ashley as the most susceptible and China the most resistant cultivar. The low titres from National Pickling and China could not be accounted for by a greater concentration of inhibitors, which might have affected infectivity assays. Increased peroxidase activities of extracts from infected leaves were correlated with symptom severity and virus multiplication.

Changes in the soluble protein constitution of cucumber cotyledons following infection with two strains of cucumber mosaic virus

Abstract Changes in the soluble protein constitution of cucumber cotyledons following inoculation with two strains of cucumber mosaic virus (W and N), which differe in the severity of symptoms they induce, were investigated by polyacrylamid gel electrophoresis and isoelectric focusing. Inoculation with either strain resulted in similar quantitative changes in soluble protein consitution as revealed by electrophoresis on 5, 7, 10 and 15% polyacrylamide gels. These changes were related both to the appearance of symptoms and of infective virus. Isoelectric focusing revealed an increase in a protein of pI approximately 4·0. Both strains elicited similar quantitative changes in peroxidase and polyphenoloxidase isoenzymes.

Peroxidase isoenzymes in leaves of cucumber (Cucumis sativus L.) cultivars systemically infected with the W strain of cucumber mosaic virus

Abstract The isoperoxidases in leaf extracts of healthy and cucumber mosaic virus infected cucumber cultivars of differing susceptibility to infection and of healthy tobacco plants have been examined by disk electrophoresis. The relative intensities of the principal isoperoxidases in zymograms of extracts from systemitically infected cucumber leaves, compared with those of healthy control leaves, were correlated with symptom severity and peroxidase activity. Thus, extracts from infected leaves of the susceptible cultivars Ashley and Marketer, with pronounced symptoms, showed greater intensities of the principal isoperoxidase bands in comparison with extracts of the resistant, almost symptomless cultivar China, and had greater peroxidase activities. The changes in isoperoxidase patternsappeared to be nonspecific responses to infection, although zymograms of extracts from senescing detached leaves of the four cucumber cultivars were not identical to those from virus infected plants. Cucumber mosaic virus infection of both cucumber and tobacco cultivars resulted in increased activity or stimulated synthesis of host isoperoxidases rather than induction of virus specific ones, in support of conclusions reached by other workers, based on experiments with several viruses including tobacco mosaic, tobacco streak, tobacco necrosis and tobacco ringspot viruses.

Effects of a severe (P6) and a mild (W) strain of cucumber mosaic virus on tobacco leaf chlorophyll, starch and cell ultrastructure

The nature of the symptoms induced in tobacco (Nicotiana tabacum cv. Xanthi nc.) by a severe (P6) or a mild (W) strain of cucumber mosaic virus (CMW) were compared. Under conditions optimal for symptom expression, leaf chlorophyll was reduced to 38 or 87% in systemically infected leaves after infection with CMV-P6 or -W respectively. Dark treatment did not affect virus replication but was only partially effective for suppressing the severe chlorosis. Iodine staining of leaves revealed no correlation between the severe CMV-P6-induced chlorosis and increased starch levels. However in CMV-W-infected plants only, starch ringspots, and sometimes necrotic ringspots, were seen in inculated leaves and narrow bands of tissue with a low starch accumulating activity were found in systemically infected leaves. Electron microscopy revealed that the severe chlorosis induced after systemic infection with CMV-P6 was principally associated with a reduction in the size of chloroplasts which contained fewer grana, rather thair disruption and/or excess accumulation of starch. In some cases myelin-like chloroplast-related structures were also seen.

Virus multiplication, peroxidase and polyphenoloxidase activity in leaves of two cucumber (Cucumis sativus L.) cultivars inoculated with cucumber mosaic virus

Abstract The multiplication of the W strain of cucumber mosaic virus in inoculated cotyledons of the two cucumber cultivars Ashley and China followed a cyclical pattern, with maxima at 4·5±0·5, 8±1 and 14±2 days after infection, but the virus attained much smaller concentrations in China than in Ashley. Peroxidase activities were correlated with virus multiplication and symptom development rather than resistance. Polyphenoloxidase activities were estimated both in inoculated and in systemically infected leaves, using either chlorogenic acid or l -3, 4-dihydroxyphenylalanine as substrate. The only significant deviation from control levels was a rise in infected cotyledons of the cultivar Ashley, beginning at about the time of the second peak of virus infectivity, when symptoms appeared. This rise was unconnected with the subsequent fall in virus infectivity.

Preliminary studies on the RNA components of three strains of cucumber mosaic virus

Abstract In investigations of the causal relationships between disease symptoms and virus infection of plants, three strains of cucumber mosaic virus (viz. N, Prices No. 6 and W) which caused different symptoms in several plant species were studied. The ribonucleic acid components of the three strains were resolved by electrophoresis in 2·4% polyacrylamide/0·5% agarose gels and their sizes relative to those of the two ribosomal RNAs of Escherichia coli estimated. All three strains contained the four major RNA species.

The influence of actinomycin D on cucumber mosaic virus (strain W) multiplication in cucumber cultivars

Abstract Actinomycin D was demonstrated, by its effect on the incorporation of [ 3 H]uridine into acid-precipitable material, to curtail RNA synthesis in cucumber cotyledons. Treatment of China cucumber plants, normally resistant to infection by cucumber mosaic virus (CMV), with actinomycin D allowed an enhanced accumulation of virus infectivity, measured 4 days after infection, compared to that in control plants, the maximum observed increase occurring following injection of an actinomycin solution containing 10 μg/ml 1 h after infection. No significant influence on infectivity in susceptible Ashley plants was observed. While not ruling out alternative possibilities, the results are consistent with the hypothesis that the China cultivar responds to CMV infection with the synthesis of one or more antiviral compounds, a process restricted by actinomycin treatment. Although a different virus and cultivar were employed, these observations agree in principle with those reported by Nachman et al.

The infection of cucumber mesophyll protoplasts with tobacco mosaic virus

SummaryProtoplasts from the first leaf mesophyll of cucumber plants were isolated by an 18 hour combined petinase/cellulase treatment. Conditions favouring the infection of these protoplasts with tobacco mosaic virus (TMV), and the accumulation of infective virus up to 96 hours after inoculation have been studied. Infection of approximately 5–10 per cent of the protoplasts, revealed by indirect fluorescent antibody staining, was achieved by pre-treatment of the cells in 0.01m citratebuffered mannitol (CBM), pH 5.2 with 2 µg/ml poly-L-ornithine followed by centrifugation and direct resuspension of the cells in the same mixture together with 2 to 4 µg/ml TMV. Higher concentrations of the polycation and buffer were toxic to the protoplasts. Under the best conditions, virus yields of approximately 10–20 µg TMV/106 protoplasts were attained, while after 72 hours incubation, significant amounts of virus could often be recovered from the incubation medium. Addition of actinomycin D to cultures of protoplasts 2 hours post-inoculation partially inhibited development of infectivity.