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.
Schulz
Alexander
A
Plant Anatomy and Physiology Laboratory, The Royal Veterinary and Agricultural University, Copenhagen, Denmark.
Knoetzel
Jürgen
J
Scheller
Henrik V
HV
Mant
Alexandra
A
D017360:Arabidopsis / Q000378:metabolism* / Q000648:ultrastructure; D001205:Ascorbic Acid; D002021:Buffers; D002469:Cell Separation; D002736:Chloroplasts / Q000378:metabolism*; D005452:Fluoresceins / Q000378:metabolism; D005456:Fluorescent Dyes / Q000378:metabolism*; D008856:Microscopy, Fluorescence; D015388:Organelles / Q000378:metabolism*; D018532:Pisum sativum; D012987:Soil
D002021:Buffers; D005452:Fluoresceins; D005456:Fluorescent Dyes; D012987:Soil; C084577:5-carboxyfluorescein diacetate; D001205:Ascorbic Acid