title: 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.
authors: Laboratory of Forest Biotechnology, Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia Viterbo, Italy.
authors: Elena
authors: E
authors: Kuzminsky
authors: Laboratory of Molecular Cytogenetic and Mutagenesis, Department of Ecological and Biological Science, University of Tuscia Viterbo, Italy.
authors: Roberta
authors: R
authors: Meschini
authors: Laboratory of Forest Biotechnology, Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia Viterbo, Italy.
authors: Serena
authors: S
authors: Terzoli
authors: Laboratory of Forest Biotechnology, Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia Viterbo, Italy.
authors: Liliana
authors: L
authors: Pavani
authors: Laboratory of Tissue Culture and Biotechnology of Woody Plants, Department of Agricultural and Forestry Sciences, University of Tuscia Viterbo, Italy.
authors: Cristian
authors: C
authors: Silvestri
authors: Laboratory of Forest Biotechnology, Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia Viterbo, Italy.
authors: Zineb
authors: Z
authors: Choury
authors: Laboratory of Forest Biotechnology, Department for Innovation in Biological, Agro-food and Forest systems, University of Tuscia Viterbo, Italy.
authors: Giuseppe
authors: G
authors: Scarascia-Mugnozza
keywords: DNA integrity; SCGE assay; abiotic stress; holm oak; protoplast isolation