Plants were regenerated from leaf, cotyledon, and hypocotyl explants of tomato cv Moneymaker. Various phenotypic alterations were observed among regenerated plants (R1), but were not transmitted to the progenies, except for ploidy variation. Variation in ploidy level, mainly tetraploidy, occurred in R1 plants and their R2 progenies, and the frequency of polyploid plants depended on the explant source. More than 50% of the regenerants derived from hypocotyl explants were found to be polyploid. A correlation was observed between the percentage of polyploid cells present in the explant material in vivo and the frequency of polyploid plants. Several monogenic mutations were recovered in the R2, four of which were shown to be allelic to known, recessive, single-gene mutants. No significant effect of explant source or duration of tissue culture period on mutant frequency or spectrum was found. For several mutant types that could be scored unambiguously, somaclonal variation was compared to variation induced by treatment of seeds with ethyl methane sulphonate (EMS). The results showed that the mutant frequencies were higher after EMS treatment than those generated through tissue culture. With respect to the mutant spectrum, no clear differences were observed between the spectra obtained after EMS treatment and those after tissue culture. However, tissue culture gave rise to polyploid plants, whereas no ploidy variants occurred after EMS treatment.

The viability of Clavibacter michiganensis subsp. michiganensis (Cmm) was determined by measuring the intracellular pH (pH in ) as a viability parameter.This was based on the observation that growth of Cmm was inhibited at pH 5Á5 and below.Therefore, viable cells should maintain their pH in above this pH value.The pH in of Cmm was determined using the £uorescent probe 5(and 6 -)-carboxy£uorescein succinimidyl ester (cFSE).The pH in of Cmm cells exposed to acid treatments was determined using £uorescence spectro£uorometry, and for cells exposed to elevated temperatures, the pH in was determined using £uorescence spectro£uorometry and £ow cytometry (FCM). A good correlation was found between the presence of a pH gradient and the number of colony-forming units (cfu) observed in plate counts. However, with the spectro£uorometry technique, the analysis is based on the whole cell population and the detection sensitivity of this technique is rather low, i.e., cell numbers of at least10 7 cfu ml À1 are needed for the analysis. Using FCM, heat-treated and non-treated Cmm cells could be distinguished based on the absence and presence of a pH gradient, respectively. The major advantage of FCM is its high sensitivity, allowing analysis of microbial populations even at low numbers, i.e., 10 2 À103 cfu ml À1

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Background: Xanthomonas campestris pv. campestris (Xcc) is a seed-transmitted plant pathogenic bacterium that causes black rot of crucifers. Seed lots and plants are screened for contamination with this pathogen using plating or serological assays. These methods, however, are time consuming and not very sensitive, respectively. Therefore, flow cytometry (FCM) was evaluated as a tool for the rapid detection and quantification of Xcc cells labeled with a mixture of specific fluorescein isothicyanate (FITC)-monoclonal antibodies (mAb) in pure culture, in mixed cultures of Xcc with either the common saprophyte Pseudomonas fluorescens (Psf) or a nonpathogenic X. campestris isolate (Xc), and in crude seed extracts.

Determination of the viability of bacteria by the conventional plating technique is a time-consuming process. Methods based on enzyme activity or membrane integrity are much faster and may be good alternatives. Assessment of the viability of suspensions of the plant pathogenic bacterium Clavibacter michiganensis subsp. michiganensis (Cmm) using the fluorescent probes Calcein acetoxy methyl ester (Calcein AM), carboxyfluorescein diacetate (cFDA), and propidium iodide (PI) in combination with flow cytometry was evaluated. Heat-treated and viable (non-treated) Cmm cells labeled with Calcein AM, cFDA, PI, or combinations of Calcein AM and cFDA with PI, could be distinguished based on their fluorescence intensity in flow cytometry analysis. Non-treated cells showed relatively high green fluorescence levels due to staining with either Calcein AM or cFDA, whereas damaged cells (heat-treated) showed high red fluorescence levels due to staining with PI. Flow cytometry also allowed a rapid quantification of viable Cmm cells labeled with Calcein AM or cFDA and heat-treated cells labeled with PI. Therefore, the application of flow cytometry in combination with fluorescent probes appears to be a promising technique for assessing viability of Cmm cells when cells are labeled with Calcein AM or the combination of Calcein AM with PI.