Uptake of a Fluorescent Dye as a Swift and Simple Indicator of Organelle Intactness: Import-competent Chloroplasts from Soil-grown Arabidopsis

We developed a rapid and reliable technique for specifically staining intact chloroplasts using the fluorescent dye carboxyfluorescein diacetate. Intact, import-competent chloroplasts were isolated simply and rapidly from soil-grown Arabidopsis thaliana plants, with yields of 20 +/- 5 micro g chlorophyll per g FW, greater than previously reported yields from soil-grown Arabidopsis. Traditional chloroplast isolation buffers sometimes contain low concentrations (<10 mM) sodium ascorbate as a general-purpose anti-oxidant, but we found that only Arabidopsis chloroplasts isolated in the presence of high concentrations (50-100 mM) of sodium ascorbate in the initial grinding buffer were import-competent.