Isolation of Mesophyll Protoplasts from Mediterranean Woody Plants for the Study of DNA Integrity under Abiotic Stress

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.

Osmotin: A Cationic Protein Leads to Improve Biotic and Abiotic Stress Tolerance in Plants

“…Plants come across various biotic and abiotic stresses during their crop cycle due to constantly changing environmental conditions that lead to different morphological, physiological, and biochemical changes, resulting in severe effects on their growth, production, and even survival [ 1 ]. Therefore, a better comprehension of the response and adaptation of plants to global changes is urgently needed in a climate changing scenario [ 2 ]. Biotic factors that provoke stress conditions are fungi, viruses, and bacteria, whereas abiotic factors consist of high or low temperature, salinity, drought, hypoxia, pollution, and ultraviolet radiations.…”

Isolation and Purification of Mesophyll Protoplasts from <i>Ginkgo biloba</i> L.

“…The amount of mannitol and the pH of the enzyme solution may differ depending on the type of original plant material. In this work, the optimal mannitol concentration and pH for ginkgo were 0.7 M and 5.7, which were different from the conditions for holm oak mesophyll protoplasts of 0.6 M and 5.0, respectively (Kuzminsky et al 2016). Interestingly, leaves of Arbutus unedo collected in spring needed to be digested with 0.6 M mannitol, while those collected in November required 0.8 M mannitol to obtain partial protoplasts (Choury et al 2017).…”

Protoplast: A Valuable Toolbox to Investigate Plant Stress Perception and Response

“…In these procedures, protoplasts are used as a direct source of nuclei to perform gel electrophoresis with ethidium bromide staining in order to detect DNA laddering ( Poot-Poot et al, 2016 ). Identically, single cell gel electrophoresis assay (SCGE), also called Comet assay, allows the study of DNA damage on protoplasts at the single cell or nuclei level ( Kuzminsky et al, 2016 ; Badaró Costa et al, 2018 ; Choury et al, 2018 ). While this technique is amply used on animal cell cultures which are easily lysed, the presence of the CW makes it technically difficult to transpose on plant tissue or cell culture.…”