Cytokinesis block micronucleus assay in field plants for monitoring radiation-induced genotoxicity of the environment

• We developed and tested a cytokinesis block MN assay for plants.
• High precision and efficiency was confirmed in root meristems of wild cedar seeds.
• High accuracy was demonstrated in clear dose responses to ionizing irradiation.

Effective biomonitoring for detection of radiation-induced genotoxicity of contaminants in natural environments involves testing of field plants for cytogenetic changes. To increase the efficiency and precision of cytogenetic analyses of field plants that have naturally high individual variability, an improved micronucleus assay is proposed that employs a cytokinesis block technique similar to the lymphocyte test system used in mammals. In seed embryonic meristems of the Japanese cedar, application of a methylxanthine derivative, 3-isobutyl-1-methylxanthine (IBMX), was found to be effective in inhibiting cytokinesis to make once-divided cells easily recognizable by their binucleate appearance. In the meristem of IBMX-treated seminal roots from X-ray-irradiated seeds, variation in micronucleus frequency in the binucleate cell population was reduced compared to that in the total cell population. The highest efficiency of measurement of micronucleus frequencies was obtained in the root meristems where 0.2- to 1.5-mm-long seminal roots were incubated with IBMX for 24 h. This result indicated that this root elongation stage corresponded to the first divisions of the root meristematic cells, and was therefore suitable for obtaining reliable estimations of accumulated genetic damage in the seeds. This cytokinesis block assay applied specifically at the root elongation stage was then used to examine dose–response relationships in Japanese cedar seeds irradiated either acutely with X-rays or chronically with γ-rays. The resulting dose–response curve for the acute X-ray irradiation was fitted onto a linear–quadratic regression curve, whereas the dose–response curve for the chronic γ-irradiation matched a linear regression line better. Both dose–response curves were consistent with the target theory of classical radiation biology. The good agreement of the micronucleus data to a simple dose–response model indicates the proposed accuracy of the cytokinesis block micronucleus assay for plant monitoring.