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Comet assay in plants

The popularity of the comet assay in plants has been steadily increasing and, naturally, the production of a standardised protocol would be of benefit to research and industry. Researchers in France have been investigating this situation and a case study of their findings is summarised here.

The researchers aim was to identify key factors affecting comet assay performance in plants and to improve its reliability and reproducibility.

Plants are sedentary organisms. They are continuously exposed to environmental and chemical stresses. Plants have been widely used for the evaluation of chemical compound toxicity and for the biomonitoring of both aquatic and terrestrial ecosystems. The scientists believe that the versatile comet assay appears to be a promising technique to detect the genotoxic effect of pollutants and to monitor the environment. 

The comet assay, in its neutral version, was used for the first time on plant tissues approximately two decades ago. However, the absence of free cells in plants and the presence of a cell wall (which has proved to be a resistant barrier to cell lysis) cause technical issues for performing the comet assay on plant tissues. Over the past few years, several research groups have investigated and improved the methodology for the comet assay on plant cells (for full references, please refer to the original publication). The influence of both internal (different nucleus isolation methods, presence or absence of filtration and lysis steps) and external (room temperature, light intensity) parameters were evaluated during this project.

The research group aimed to identify key factors affecting comet assay performance and to improve its reliability and reproducibility. The scientists examined the effect of varying several experimental parameters on four different plant species: broad bean (Vicia faba), white clover (Trifolium repens), English ryegrass (Lolium perenne) and miscanthus (Miscanthus x giganteus).

The researchers hoped to:

(i) identify the various factors affecting comet assay application in higher plants,

(ii) improve the reliability and reproducibility of the comet assay and

(iii) increase nucleus extraction yield.

The extensive and comprehensive details of the methods used in the investigation can be found in the original publication.  The publication includes details for the standard comet assay protocol, the modified comet assay  protocol and the influence of external parameters on the comet assay.

Typically, after electrophoresis, the slides were rinsed three times with Tris and stained with SYBR Gold for 5 min.  For each slide, 50 randomly selected nuclei were analysed using an Olympus fluorescence microscope.  A computerised image analysis system (Comet Assay IV; Perceptive Instruments) was employed to score the comets. The “DNA percent in tail” was used as the primary measure of DNA damage.  The researchers evaluated three slides per plant and six plants were analysed in each treatment. From the repeated experiments, the averaged mean DNA percent in tail value was calculated for each treatment group from the DNA percent value of each slide.

For a comprehensive description of the results and conclusions drawn from this investigation, please refer to the original publication.  The authors state that the results clearly indicated that “short chopping” is more efficient to isolate nuclei than the “standard slicing method”. They also conclude that filtration and lysis steps were unnecessary and can be skipped. Data also demonstrated that high room temperatures and light could induce DNA damage in isolated nuclei. The paper also showed that calibration tests with H2O2 or ethyl methanesulfonate revealed that special attention should be paid to plant growing stage, leaf position and exposure duration.

In summary, the scientists identified the key parameters (internal and external) that have an impact on comet assay results and throughput in plants. They recommend new standards for obtaining reproducible and reliable comet assay data.  However, they concluded the nucleus isolation step is clearly operator-dependent and comet assay parameters could be plant/organ-dependent, they suggested that laboratories should carry out their own tests to obtain optimal conditions.

In conclusion, the scientists involved in this investigation suggest that other researchers should always specify their comet assay parameters, as well as nucleus extraction method, calibration procedures, plant stage (if relevant) and external factors (room temperature and light intensity) as part of the experimental conditions of a comet assay.

This case study is based upon:
Recommendations for increasing alkaline comet assay reliability in plants
Bertrand Pourrut, Eric Pinelli, Vanessa Celiz Mendiola, Jérôme Silvestre and Francis Douay
Mutagenesis (2015) 30 (1): 37-43.