In addition to studying the effects of HC toxin on growth and regeneration of susceptible and resistant callus, we looked at the effects of HC toxin on protoplasts. The objective was to study the effects of HC toxin on protoplasts derived from susceptible and resistant near-isogenic genotypes in an effort to provide information which might prove useful for understanding the mechanism of action of the toxin

Mesophyll protoplasts were isolated from leaves of 10 to 12 day-old susceptible (Pr x K61) and resistant (Pr1 x K61) corn plants by incubating leaf pieces in an enzyme solution containing 2% cellulysin, 0.5M sorbitol, and 10 mM CaCl2.2H20 for 3 to 4 hours. Isolated protoplasts were cultured in a minimal medium containing 0.5 M sorbitol + 10 mM CaCl2 + 5 mM MES buffer at a density of 1.0 x 105 protoplasts/ml unless otherwise stated. The toxin used in these studies was the same preparation as that used for the callus work (see previous article).

The most striking differential effect of the toxin was in distribution of chloroplasts. Populations of susceptible protoplasts treated with low toxin concentrations had more protoplasts with chloroplasts well distributed throughout the cytoplasm than control (untreated susceptible and resistant) or resistant protoplasts treated with low toxin concentrations (Figure 1). That effect occurred after a minimum of 36 hours of toxin treatment. Control and resistant (low toxin levels) protoplast populations had chloroplasts localized and clustered within the cytoplasm. Like previously reported assays, resistant genotypes showed a similar toxin effect, but at higher toxin concentrations. A greater percentage of resistant protoplasts treated with high toxin concentrations had well-distributed chloroplasts. Protoplasts with well-distributed chloroplasts looked similar to protoplasts cultured in a complete nutrient medium. For that reason we compared the effects of HC toxin on susceptible and resistant protoplasts when cultured in a complete medium (D.S. Brar, S. Rambold, O.L. Gamborg, and F. Constabel, Z. Planzenphysiol. 96:269). In those experiments, a differential response for chloroplast distribution was not as pronounced as that which occurred in a minimal medium and was noted only after a much longer toxin exposure.

In addition to studying differences in chloroplast distribution, we looked at differences in viability by staining with fluorescein diacetate (FDA) or Evans Blue. Low toxin concentrations increased long-term viability of susceptible protoplasts whereas high toxin concentrations increased long-term viability of resistant protoplasts. Like differences in chloroplast distribution, viability differences were not immediate (Table 1). Similar differences in long-term viability were not noted when susceptible and resistant protoplasts were cultured in a complete nutrient medium. Additional studies are underway to determine components of the complete medium which influence the toxin effect.

Table 1. Viability rating (based on fluorescence) of populations of susceptible and resistant protoplasts cultured with various concentrations of toxin in minimal medium for 8, 11, 14 and 18 days.

Figure 1. Percent protoplasts with clustered chloroplasts after exposure to various toxin concentrations. Protoplasts were cultured in the dark in minimal medium for 60 hours. Greatest standard deviation is 10.

S.J. Wolf and E.D. Earle

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