Chromosome CPD(PI/DAPI)- and CMA/DAPI-Banding Patterns in Allium cepa L.

Chromosome banding patterns of Allium cepa L. were obtained by using fluorescent dye combinations chromomycin A₃ (CMA) + 4",6-diamidino-2-phenylindole (DAPI), DAPI + actinomycin D (AMD) and propidium iodide (PI) + DAPI. In A. cepa,telomeric heterochromatin displayed dull fluorescence after staining with DAPI and DAPI/AMD. After joint staining with the GC-specific CMA and AT-specific DAPI, the CMA-positive fluorescence of the NOR region and the telomeric bands of C-heterochromatin was observed. In combination with DAPI, PI, a dye with low AT/GC specificity, produced almost uniform fluorescence of chromosomal arms and heterochromatin, whereas the NOR-adjoining regions displayed bright fluorescence. Denaturation of chromosomal DNA (2 × SSC, 95°C for 1–3 min) followed by renaturation (2 × SSC, 37°C, 12 h) altered the chromosome fluorescence patterns: specific PI-positive bands appeared and the contrast of CMA-banding increased. Bright fluorescence of NOR and adjoining regions was also observed in the case. Three-minute denaturation led also to a bright PI-positive fluorescence of telomeric heterochromatin. The denaturation of chromosomal DNA before staining results in changes of the DAPI fluorescence pattern and in the appearance of bright DAPI fluorescence in GC-rich NOP regions. The mechanisms underlying the effects of denaturation/renaturation procedures on chromosome banding patterns obtained with different fluorochromes are discussed.