Grapevine is a crop of vegetative propagation with great economical importance which is distributed worldwide in temperate zones. It is mainly used for wine and table grape production. Despite the fact thousands of varieties exist only few are commonly used, producing a loss of variability. Since the arrival of the phylloxera aphid the grapevine culture is performed using resistant rootstocks. The Directive Commission 2005/43/EC amending the Directive Council annexes 68/193/EEC, indicates that grapevine material should be free of these viruses: Grapevine fanleaf virus (GFLV), Grapevine leafroll associated virus 1 (GLRaV-1), Grapevine leafroll associated virus 3 (GLRaV-3), Grapevine fleck virus (GFkV) and Arabis mosaic virus (ArMV). In this work different methodologies related with in vitro culture were evaluated and developed with the aim of sanitate, mutiplicate and preserve grapevine germplam. In vitro preservation of grapevine varieties is a complementary stratregy of field preservation. In a preservation program it is important the selection of the starting material, determine its sanitary status, sanitate if it is necessary and, establish the appropriate in vitro conditions for its preservation. In the first chapter of this thesis (Chapter 1) the assays carried out for inducing somatic embryogenesis in 16 grapevine varieties which include six that were infected by the viruses GFLV, GLRaV-3 and GFkV are described. In grapevine, somatic embryogenesis has been described as a usefull methodology for virus sanitation and is the common pathway to adventitious regeneration. Efficient regeneration prototocols are necessary to address breeding by genetic transformation. In this work, cut seed are used as starting material and virus-free plants and high precentages of regeneration were obtained in all the varieties evaluated. The use of microsatellite markers has been useful to select those plants with the same genotype than the mother plant. From the other side, in this work, a micropropagation protocol starting from a healthy plant of the cultivar 'Monastrell' was developed and high multiplication rates with good adaptation to field conditions were obtained (Chapter 2). The culture medium MW was evaluated in 16 grapevine varieties as well as the following modifications: reduction in a half of the sugar content and elimination of the Indolebutyric Acid. The aim of these assays was to determine for each variety which conditions are the most appropiate to maintain in active in vitro culture this germplasm, lengthen the time between subcultures for reducing costs (Chapter 3). In Chapter 4 it is included a divulgation report that summarized the steps followed in the MINECO proyect CGL2015-70843-1.5.1.5. Crioconservación/Crioterapia 1.5.2 Micropropagación X 1.5.3 Conservación in vitro 19 1.5.4 Mejora genética y transformación genética 20 2. Objetivos 39 3. Capítulos 41 Capítulo 1: Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants 43 Capítulo 2: In vi...