Establishment of an efficient explants surface disinfection protocol is essential for in vitro cell and tissue culture as well as germplasm conservation, such as the case of Grapevine (Vitis spp.) culture. In this research, different procedures for disinfection and regeneration of field-grown grapevine cv. ‘Flame seedless’ axillary buds were evaluated. The buds were disinfected using either NaOCl or allyl, benzyl, phenyl and 2-phenylethyl isothiocyanates. Two different media for shooting and four media for rooting were tested. Shoot and root development per buds were registered. The best disinfection procedure with 90 % of tissue survival involved shaking for 60 min in a solution containing 20 % Clorox with 50 drops/L Triton® X-100. These tissues showed the potential to regenerate a complete plant. Plant regeneration was conducted using full strength Murashigue and Skoog (MS) medium supplemented with 8 µM benzyl aminopurine for shoot induction and multiplication, whereas rooting was obtained on half strength MS supplemented with 2 mg L−1 of indole-3-butyric acid and 200 mg L−1 of activated charcoal. In this work, it was designed the protocols for obtaining sterile field-grown grapevine buds and in vitro plant development. This methodology showed potential to produce vigorous and healthy plants in 5 weeks for clonal grapevine propagation. Regenerated plants were successfully established in soil.

Cryopreservation is used for the long-term conservation of plant genetic resources. This technique very often induces lethal injury or tissue damage. In this study, we measured indicators of viability and cell damage following cryopreservation and vitrification-cryopreservation in Vitis vinifera L. axillary buds cv. “Flame seedless” stored in liquid nitrogen (LN) for: three seconds, one hour, one day, one week and one month; after LN thawed at 38 °C for three minutes. The enzymatic activity of catalase (CAT) and superoxide dismutase (SOD), as well as the amount of malondialdehyde (MDA), total protein and viability were assayed.

Grapevine (Vitis spp.) is an economically important fruit crop worldwide. In Mexico, Sonora State leads the table grape production and exportation to international markets. In this regard, it is important to preserve the grape varieties during long time without phenotypical or genotypical changes. Cryopreservation is a good alternative, although it very often can induce changes in genome and phenotype. In this study, grapevine cv. "Flame Seedless" axillary buds were cryopreserved by vitrification using the plant vitrification solution 2 (PVS2) and stored in liquid nitrogen (LN) for one hour, one week and one month, respectively. Genetic stability of buds cryopreserved under all treatments was evaluated using inter-simple sequence repeats (ISSR) markers. Ten ISSR primers were evaluated, but only two primers were possible to amplify distinct and reproducible bands with sizes between 300 bp and 2,000 bp. Different ISSR fragment patterns were recorded in cryopreserved buds as compared with control. These results suggest that cryopreservation by LN and vitrification-cryopreservation affect genetic stability in grapevine.

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La crioconservación ha revolucionado el campo de la biotecnología. Congelar en nitrógeno líquido (NL) preserva células por largo tiempo. En ese sentido, en este trabajo se evaluaron tres condiciones de crioconservación basados en la vitrificación de yemas de vid. Las yemas fueron sometidas a PVS2, PVS3 y glicerol por 0-420 min, y colocadas en NL por una hora. De modo posterior a cada tiempo de incubación se cuantificó la pérdida de iones como medida de viabilidad y se evaluó el daño mediante observación en estereoscopio. Basados en el porcentaje de viabilidad el mejor método fue empleando PVS3 (30% viabilidad), seguido de glicerol (25%) y PVS2 (<10%). Las imágenes de las yemas expuestas a PVS3 no muestran daño en el tejido, a diferencia de PVS2 y glicerol, los cuales resultaron insuficientes para preservar el tejido.