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DNA Diffusion Assay Applied to Plant Cells

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Plant Programmed Cell Death

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1743))

Abstract

DNA diffusion assay is a simple, sensitive and reliable technique which allows the assessment of programmed cell death (PCD) or necrosis events based on nuclear morphology. It consists in isolating nuclei from plant material, which are then embedded in agarose and subjected to lysis in alkaline buffers. Under these conditions, and due to the presence of abundant alkali-labile sites in the DNA, small pieces of DNA diffuse in the agarose gel giving a specific halo appearance when stained with fluorescent dyes like DAPI (4′,6-diamidino-2-phenylindole). Here, we describe an optimized protocol for DNA diffusion assay applied to different types of plant cells/tissues, indicating all the critical steps required for a successful experimental procedure.

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Acknowledgments 

This work was carried out within the integrated project “Advanced Priming Technologies for the Lombardy Agro-Seed Industry-PRIMTECH” supported by grants from Regione Lombardia (D.G. Attività Produttive Ricerca e Innovazione - Struttura Asse 1 POR FSE 2007-2013, Project ID 4344853) and CARIPLO Foundation (Action 3, Code 2013-1727).

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Authors and Affiliations

  1. Department of Biology and Biotechnology ‘Lazzaro Spallanzani’, University of Pavia, Pavia, Italy

    Anca Macovei, Daniela Carbonera & Alma Balestrazzi

  2. Gregor Mendel Institute (GMI), Vienna Biocenter (VBC), Austrian Academy of Science, Vienna, Austria

    Mattia Donà

Authors
  1. Anca Macovei
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  2. Mattia Donà
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  3. Daniela Carbonera
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  4. Alma Balestrazzi
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Corresponding author

Correspondence to Alma Balestrazzi .

Editor information

Editors and Affiliations

  1. Food Sciences and Human Nutrition Unit, Università Campus Bio-Medico di Roma, Roma, Italy

    Laura De Gara

  2. Food Sciences and Human Nutrition Unit, Università Campus Bio-Medico di Roma, Roma, Italy

    Vittoria Locato

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Macovei, A., Donà, M., Carbonera, D., Balestrazzi, A. (2018). DNA Diffusion Assay Applied to Plant Cells. In: De Gara, L., Locato, V. (eds) Plant Programmed Cell Death. Methods in Molecular Biology, vol 1743. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7668-3_10

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  • DOI: https://doi.org/10.1007/978-1-4939-7668-3_10

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7667-6

  • Online ISBN: 978-1-4939-7668-3

  • eBook Packages: Springer Protocols

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