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Isolation and Use of Protoplasts from Grapevine Tissues

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Methodologies and Results in Grapevine Research

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Abstract

Highly pure, intact and functional protoplasts can be obtained from plant tissues, which are readily amenable for challenging with exogenous sugars, acids, analogues, transport inhibitors and drugs. Thus, they may be used as models for both basic research and biotechnological approaches. Some of these studies require the regeneration of plants from protoplasts; however most agronomically important plant species, including grapevine, are recalcitrant to plant regeneration. Oxidative stress has been considered as a crucial factor accounting for the recalcitrance of grapevine protoplasts, as supported by the profiles of generated reactive oxygen species (ROS) and ROS-scavenging enzymes, the modified cell redox state, as well as the altered endogenous titers of polyamine levels. In the present work, methods for the purification of intact and functional protoplasts from grape berry mesocarp tissue and for the isolation and culture of mesophyll protoplasts are described. Methods for the detection of ROS in grapevine protoplasts, together with assays for antioxidant enzyme and antioxidant biomolecules are also detailed.

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Abbreviations

APO:

Ascorbate peroxidase

BAP:

6-Benzylaminopurine

CAT:

Catalase

cpm:

Counts per minute

DHAR:

Dehydroascorbate reductase

DTT:

Dithiothreitol

EDTA:

Ethylenediaminetetraacetic acid

FDA:

Fluorescein diacetate

GR:

Glutathione reductase

GSH:

Reduced glutathione

GSSG:

Oxidized glutathione

H2O2 :

Hydrogen peroxide

MDHAR:

Monodehydroascorbate reductase

MES:

2-(N-morpholino)ethanesulfonic acid

NAA:

1-Naphthaleneacetic acid

O2 .− :

Superoxide radical

PAs:

Polyamines

PCA:

Perchloric acid

PMSF:

Phenylmethanesulfonyl fluoride

ROS:

Reactive oxygen species

SOD:

Superoxide dismutase

TEMED:

Tetramethylethylenediamine

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Authors and Affiliations

  1. Departamento de Biologia, Centro de Investigação e de Tecnologias Agro-Ambientais e Biológicas (CITAB), Universidade do Minho, Braga, 4710-057, Portugal

    Natacha Fontes, Hernâni Gerós & Anastasia K. Papadakis

  2. UMR Ecophysiologie et Génomique Fonction nelle de la Vigne, Institut des Sciences de la Vigne et du Vin, Université de Bordeaux, Chemin de Leysotte CS 50008, 33882, Villenave d’Ornon, France

    Serge Delrot

  3. Department of Biology, University of Crete, Crete, Greece

    Kalliopi A. Roubelakis-Angelakis

Authors
  1. Natacha Fontes
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  2. Hernâni Gerós
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  3. Anastasia K. Papadakis
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  4. Serge Delrot
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  5. Kalliopi A. Roubelakis-Angelakis
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Corresponding author

Correspondence to Hernâni Gerós .

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Editors and Affiliations

  1. , UMR Ecophysiologie et Génomique Fonction, Université de Bordeaux, Chemin de Leysotte CS 50008, Villenave d'Ornon, 33882, France

    Serge Delrot

  2. , IMEDEA-Dept. de Biologia, Universitat de les Illes Balears, Carretera Valldemossa Km. 7,5, Palma de Mallorca, 01722, Spain

    Hipólito Medrano

  3. Agriculturall Research Organisation, The Volcani Center, Institute of Horticulture, Bet Dagan, 50250, Israel

    Etti Or

  4. , Istituto di Frutti-Vitico9ltura, Universita Cattolica del Sacro Cuore, Via E. Parmense 84, Piacenza, 29100, Italy

    Luigi Bavaresco

  5. , Fondazione Edmund Mach, IASMA Research and Innovation Centre, Via E. mach 1, San Michele alll'Adige (TN), 38010, Italy

    Stella Grando

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Fontes, N., Gerós, H., Papadakis, A.K., Delrot, S., Roubelakis-Angelakis, K.A. (2010). Isolation and Use of Protoplasts from Grapevine Tissues. In: Delrot, S., Medrano, H., Or, E., Bavaresco, L., Grando, S. (eds) Methodologies and Results in Grapevine Research. Springer, Dordrecht. https://doi.org/10.1007/978-90-481-9283-0_18

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