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Grapevine genetic Transformation to study gene function

Final Activity Report Summary - GRAPEGENFUN (Grapevine genetic Transformation to study gene function)

In this two-year project, several parameters involved in the biolistic transformation of grapevine were optimised. Parameters including induction medium, floral explant and development stage were assayed to determine the best combination to induce somatic embryogenic cultures among eight grapevine genotypes. Ovaries at the development stage IV on Nitsch and Nitsch-derived medium gave the best percentage of embryogenesis for most of the genotypes assayed. Embryogenic calli were suitable for efficient initiation of cell suspensions and for embryo development from them.

The average conversion from emerged embryos to regenerated plants was 62.5 % thus shown a high capacity of plant regeneration. Also, embryogenic cell suspensions were successfully cryopreserved by the encapsulation-dehydration method. The 100 % of the cryopreserved cell beads gave a 50 % of viability and a vigorous re-growing.

This cryopreservation method is an advance to store ready-to-use competent embryogenic tissue for grapevine genetic transformation projects. Preliminary biolistic experiments using cell suspensions as a target tissue and the gus gene as a reporter maker gave up to 6 000 integration events based on the GUS assay.

Once, the competence of the cell suspension for transformation and regeneration was confirmed, we carried out the transformation of grapevine Albariño and Tempranillo with genes involved in flower transition such as VFL and FT (Arabidopsis Floricaula/LEAFY (FLO/LFY) and Flowering locus T (FT) orthologs, respectively). The overexpression of those genes is a strategy to produce short cycle lines with early flowering to be used by self-crossing to generate homozygote lines. Pure lines are important tools to approach genetic studies of grapevine, which are impossible to perform by traditional breeding due to the high heterozygote of the grapevine and to the juvenility of grapevine plantlets.