GRAPEGENFUN · Grapevine genetic Transformation to study gene function
FP6 — Marie Curie Actions (Human Resources and Mobility)
- Duration
- 2006-04-01 → 2008-03-31
- EU contribution
- €80,000
- Participants
- 2
- Scheme
- IRG
Lines connect the coordinator with its partners.
Results in brief
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.
Data: CORDIS, © European Union
Project objective
Grapevine genetic transformation is a potential tool for both functional genomics and molecular breeding projects. Genomic resources of grapevine have been very little exploited and little is known on grapevine biology at the molecular level. Here we focus on the development of the methodology for efficient transformation of European cultivars to study gene function. There are three key points to approach grapevine transformation: The ability to produce plant tissue competent for transformation and regeneration, the choice of the quick method for transformation and regeneration and the design of DNA constructs for transgene integration. Anthers and ovaries will be used to initiate embryogenic cell suspensions, which will be assayed with novel cryopreservation techniques for keeping long-term regeneration capacity. We will select the biolistic device as strategy for transformation because of the advantage of the genegun for grape transformation avoiding the use of Agrobacterium, which causes hypersensitive reaction in grapes.Whole circular plasmids will be compared to the novel minimal linear cassette technology for over-expression and silencing strategies of target grape genes. In this proposal, we will focus on grape genes involved in flower transition. We wi ll over-express VFL under control of the 35S-CaMV promoter as a possible strategy to reduce flowering time as well as the over-expression of the mutated VvGAI gene to obtain early flowering plants producing only inflorescences. We will approach the novel RNA interference (RNAi) technology as a tool for functional genomics studies by using the VAP1 gene, which silencing could affect tendril development. We expect to see early in vitro the effect of the expression strategy on the phenotype of recovered transgenic plants. The availability of an efficient grapevine transformation technology will allow approach in the future the study of genes involved in quality traits as well as approach molecular breeding programs.
Original text from CORDIS.
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Data: CORDIS, © European Union