Visualization by fluorescence of chloroplast DNA in higher plants by means of the DNA-specific probe 4'6-diamidino-2-phenylindole.

Abstract

The DNA in isolated chloroplasts [Nicotiana tabacum, Spinacia oleracea, Beta vulgaris and N. excelsior] was visualized by the fluorescent probe 4''6-diamidino-2-phenylindole (DAPI). When excited with light of 360 nm, the DNA-DAPI complex fluoresces brilliantly at 450 nm. Nuclei also fluoresce but their nucleoli do not. RNase and Pronase treatment of chloroplasts did not affect the fluorescence, but both pre- and post-treatment of DAPI-stained chloroplasts with DNase specifically destroyed the fluorescence. DNA-DAPI complexes in the chloroplasts show up as bright dots. These are distributed uniformly within the chloroplast except for the outer margins. The fluorescent dots can be seen at different focal levels. The number of DNA dots is roughly proportional to chloroplast area which, in turn, is a function of leaf size. The number of fluorescent dots also gave the impression that large leaves with large chloroplasts contain more chloroplast DNA than nuclear DNA.