Development of an automated scoring system for plant comet assay

Abstract

In plants, an increasing interest for the Comet Assay was shown in the last decade and this versatile technique appears to be promising to detect the genotoxic effect of pollutants and to monitor the environment. However, its use in plant studies was rather limited compared to animal studies because of (i) the difficulty to isolate intact nuclei compared to animal systems and the lack of a standardized protocol, (ii) the low throughput of current nucleus extraction, and (iii) the lack of a high throughput comet assay scoring method. In order to deal with these issues, we recently identified the key steps of the comet assay on plant models and proposed an optimized protocol to increase its reliability (Pourrut et al.; 2015). At the same time, in the frame of the French-Norwegian project ComPack (2014-2017), we have worked on: - the development a new nucleus extraction technique compatible with the high-throughput comet assay scoring methods; - the automation of the scoring method based on the automated scoring system Pathfinder™, developed by IMSTAR. Details and results from the preliminary experiments will be presented and discussed. Major issues have been: - scoring system: specific adaptation of the automated scoring system Pathfinder™ is crucial as it was initially set up for human/animal cells; - background: optimization of the protocol to reduce the presence of debris and increase background quality; - nucleus density: increase the density of nuclei is of importance to increase scoring reliability (Sharma et al., 2012). In conclusion, increasing plant nucleus extraction yield and automated scoring of nuclei do represent big challenges. However, our promising preliminary results open up the perspective of an automated high-throughput scoring of plant nuclei