A FAST COMET ASSAY VARIANT FOR SOLID TISSUE-CELLS - THE ASSESSMENT OFDNA-DAMAGE IN HIGHER-PLANTS

The single-cell gel electrophoresis or comet assay, under high alkalin e conditions, detects low levels of DNA damage. In it, broken DNA migr ates from the nucleus to the anode providing images similar to comets. To adapt this assay to solid tissue cells, nuclei were directly obtai ned from Allium cepa L. roots. The surface of each single fresh sharpl y cut meristem was exposed to a small drop of 50 mM Sorensen buffer at pH 6.8. placed on a regular agarose-coated slide. By immediately addi ng low melting point agarose at 30 degrees C, nuclei resulted embedded in agarose. A final layer of this agarose ended the preparative steps . Conventionary prepared leukocytes were used as a control. The treatm ent with detergent (lysis step of the conventional assay) proved to be unnecessary for the nude nuclei. A 20 min-long electrophoresis (at 0. 65 V . cm(-1), 230 mA and 10 degrees C) was more sensitive than a 10 m in-long one for detecting the differential response of plant nuclei to 2 and 4 Gy of gamma-irradiation. A short fixation in methanol transfo rmed the preparations into semi-permanent ones, without altering their later DNA staining by ethidium bromide. The use of instantaneously is olated nuclei simplifies and expands the use of this technique to any eukaryotic cell from solid tissues.