A New Vertical Mesh Transfer Technique for Metal-Tolerance Studies in Arabidopsis (Ecotypic Variation and Copper-Sensitive Mutants)

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Abstract
A new vertical mesh transfer (VMT) technique has been developed to facilitate the rapid isolation of plant metal-tolerance mutants. The technique is quantitative, allowing comparisons of the growth responses of different strains or ecotypes. Using the VMT technique, we have characterized the dose responses of 10 ecotypes of Arabidopsis thaliana to Cu2+, Zn2+, Ni2+, Cr3+, Cd2+, and Al3+. Ecotypic variations in the highest concentration causing no inhibition and the lowest concentration causing complete inhibition for the six metals were observed. Two ecotypes, Ws and Enkheim, exhibited an inducible tolerance mechanism in response to copper. Pretreatment of Ws with the highest concentration causing no inhibition for copper resulted in a shifting of the lowest concentration causing complete inhibition to a higher value. Partial cross-induction and cross-tolerance between Cu2+ and Zn2+ were demonstrated. In addition, ethyl methanesulfonate-mutagenized Columbia seeds were screened for copper-sensitive (cus) mutants using the VMT procedure. Thus far, 59 putative cus mutants have survived retesting to the M4 or M5 generation. When grown on gellan gum supplemented with 30 [mu]M CuCl2, cus mutants develop marked toxicity symptoms. A copper dose-response curve of the cus1 mutant showed that the metal-sensitive phenotype is specific for the lower concentration range.