A simple non-toxic ethylene carbonate fluorescence in situ hybridization (EC-FISH) for simultaneous detection of repetitive DNA sequences and fluorescent bands in plants

Protoplasma. 2019 May;256(3):873-880. doi: 10.1007/s00709-019-01345-7. Epub 2019 Jan 17.


The major drawbacks of standard plant fluorescence in situ hybridization (FISH) designed for double-stranded DNA probes include requirement for experimentally determined heat denaturation of chromosomes at high temperatures and at least overnight hybridization. Consequently, processing with chromosomal preparations may easily result in heat-induced deterioration of chromosomal structural details, is time-consuming, and involves the use of toxic formamide and formaldehyde. Here, I have described a simple and appealing non-toxic procedure with ethylene carbonate (EC)-a formamide-substituting solvent and double-stranded repetitive DNA probes. Applying EC as a component of the hybridization solution at 46 °C not only allowed successful overnight hybridization but also gave a possibility to reduce the hybridization time to 3 h, hence converting the technique into a 1-day procedure. Importantly, the EC-FISH tended to preserve well chromosome structural details, e.g., DAPI-positive bands, thus facilitating simultaneous FISH mapping and chromosome banding on the same slide. The procedure requires no formaldehyde and RNA-se treatment of chromosomes, and no heat denaturation of chromosomal DNA. The key condition is to obtain high-quality cytoplasm-free preparations. The method was reproducible in all the plants studied (Allium, Nigella, Tradescantia, Vicia), giving a species-specific signal pattern together with clear DAPI bands on chromosomes. The procedure described here is expected to give a positive stimulus for improving gene-mapping approaches in plants.

Keywords: Chromosome DAPI banding; Ethylene carbonate; Fluorescence in situ hybridization; Heterochromatin; Plants; rDNA.

MeSH terms

  • Base Sequence
  • DNA Probes / metabolism
  • DNA, Ribosomal / metabolism
  • Dioxolanes / chemistry*
  • Fluorescence
  • In Situ Hybridization, Fluorescence / methods*
  • Indoles / chemistry
  • Plants / genetics*
  • Repetitive Sequences, Nucleic Acid / genetics*


  • DNA Probes
  • DNA, Ribosomal
  • Dioxolanes
  • Indoles
  • DAPI
  • ethylene carbonate