Abstract
Abiotic stresses have considerable negative impact on Mediterranean plant ecosystems and better comprehension of the genetic control of response and adaptation of trees to global changes is urgently needed. The single cell gel electrophoresis (SCGE) assay could be considered a good estimator of DNA damage in an individual eukaryotic cell. This method has been mainly employed in animal tissues, because the plant cell wall represents an obstacle for the extraction of nuclei; moreover, in Mediterranean woody species, especially in the sclerophyll plants, this procedure can be quite difficult because of the presence of sclerenchyma and hardened cells. On the other hand, these plants represent an interesting material to be studied because of the ability of these plants to tolerate abiotic stress. For instance, holm oak (Quercus ilex L.) has been selected as the model plant to identify critical levels of O3 for Southern European forests. Consequently, a quantitative method for the evaluation of cell injury of leaf tissues of this species is required. Optimal conditions for high-yield nuclei isolation were obtained by using protoplast technology and a detailed description of the method is provided and discussed. White poplar (Populus alba L.) was used as an internal control for protoplast isolation. Such a method has not been previously reported in newly fully developed leaves of holm oak. This method combined with SCGE assay represents a new tool for testing the DNA integrity of leaf tissues in higher plants under stress conditions.
Highlights
There are a lot of hazards connected to global change at a global scale, and air pollution is considered one of the single largest environmental risks because it leads to a multitude of adverse effects on human health and ecosystems (European Environment Agency [EEA], 2015)
Due to constraints in obtaining suitable nucleoids (Collins, 2004; Ventura et al, 2013), plant tissues are considered difficult for the employment of single cell gel electrophoresis assay (SCGE), a routine procedure for the detection of genotoxicity in animal cells
The axenic in vitro growth facilitates the release of viable protoplasts starting from plant organs, seedlings and cell suspension cultures in contrast to in vivo and glasshousegrown plants, which are influenced by contaminations and seasonal variations; Jones et al (2012) demonstrated that phenylpropanoids confer resistance to enzymatic digestion of plant tissues as they lead to an increase of lignin and other polyphenols that harden the cell wall (2012) and inhibit cellulase activity (Ximenes et al, 2010)
Summary
There are a lot of hazards connected to global change at a global scale, and air pollution is considered one of the single largest environmental risks because it leads to a multitude of adverse effects on human health and ecosystems (European Environment Agency [EEA], 2015). Comet assay is a rapid and sensitive procedure applicable, in principle, to any organism or tissue from which analyzable single cell/nuclei suspensions can be derived and recently increased use of this method has been suggested in plant toxicology (Santos et al, 2015) and functional genomics studies (Brunner et al, 2014). In this context protoplasts could be considered a new source for obtaining nuclei from leaf tissues. This is true for mature leaves, which are very interesting for testing the effects of air pollution, as they are the primarily affected tissues
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