Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants

Abstract

S. 1992. Comparison of three DNA fluorochromes for flow cytometric estimation of nuclear DNA content in plants. -Physiol. Plant. 85:'625-631.Flow cytometric estimation of nuclear DNA content was performed in sis plant species employing three fluorochromes showing different DNA base preferences: propidium iodide (no base preference), 4',6-diamidino-2-phenylindole (DAPI; AT preference), and mithramycin (GC preference). Nuclei isolated from human leukocytes were used as a primary reference standard. While nuclear DNA contents estimated using propidium iodide were in agreement with published data obtained using other techniques, the values obtained using fluorochromes showing base preference were significantly different. It was found that the differences were caused by the differences in overall AT/GC ratios, and by the spedes-specific differences in binding of these fluorochromes to DNA. !t was coneluded that nuclear DNA content estimations performed with fluorochromes showing base preference should be interpreted with caution even when AT/GC ratios of the reference and the sample are equal. The use of intercalating dyes (e.g. propidium iodide) is recommended for this porpose. On the other hand, comparison of the staining behaviour of intercalating dyes with that of dyes showing base preference may give additional information on chromatin structural differences and arrangement of molecule pairs in DNA.

Genome size and base composition of Bromeliaceae species assessed by flow cytometry

“…In addition to the target species, Solanum lycopersicum L. (2C = 1.96 pg and AT = 64.5%; Doležel et al 1992;Meister and Barow 2007) was also cultivated and used as primary internal standard. Seeds were generously supplied by Jaroslav Doležel (Experimental Institute of Botany, Olomouc, Czech Republic).…”

Assessment of genetic stability of two micropropagated wild olive species using flow cytometry and microsatellite markers

“…Ploidy level was estimated according to Brito et al (2008) using Pisum sativum cv. Ctirad as the internal standard (2C = 9.09 pg; Doležel et al 1992). Briefly, nuclei were released by chopping with a razor blade half of a leaf of the olive plant and of P. sativum, in LB01 buffer (Doležel et al 1989).…”

Genome Size and its Uses: The Impact of Flow Cytometry

“…7.6.1.1 Choice of Fluorochromes Following the publication of Galbraith et al's paper (1983), a diverse range of fluorochromes was employed for genome size estimation, which is broadly divided into two groups: (i) the DNA intercalating dyes, including propidium iodide and ethidium bromide, and (ii) base-specific fluorochromes, including mithramycin, chromomycin, olivomycin, DAPI and Hoechst. However, in a series of studies Doležel and colleagues (Doležel et al 1992(Doležel et al , 1998 showed that only intercalating dyes were suitable for the accurate estimation of genome size. Further, in a largescale comparative study involving four laboratories, Doležel et al (1998) demonstrated that there was excellent agreement between results obtained using either Feulgen microdensitometry or FCM if propidium iodide was used as the fluorochrome.…”