Grapevine gene transfer for gene function studies: an ethylene responsive factor study

The completion of the grapevine genome has predicted the presence of about 30,000 genes in its sequence. Among the tools to study gene function, genetic transformation is of fundamental importance but this approach in Vitis has been impaired by the difficulties in transformation and the long time for regeneration and growth. Using a grapevine ethylene responsive factor (ERF) gene which is transcriptionally modulated during fruit ripening, we have tested different methods of grapevine transformation. The biolistic approach was employed to transfer the ERF overexpression and silencing constructs in ‘Chardonnay’ and ‘Pixie’ varieties. Pixie is a dwarf Vitis vinifera L. which looks a promising tool for functional genomics studies. Agrobacterium tumefaciens mediated transformation was instead used to transfer the same constructs in 'Brachetto' embryogenic callus. 25 embryos have been obtained from the ‘Brachetto’ callus transformed for ERF1 over-expression. 14 of them were regenerated into plantlets and were validated for the trans-gene presence by end-point PCR, qPCR and Southern blot analysis. Morphological evaluations and gene expression analysis are in progress. In the biolistic experiments, the regeneration process have produced 11 ‘Chardonnay’ and 12 ‘Pixie’ putatively ERF1 over-expressing plants, and 3 ‘Chardonnay’ and 9 ‘Pixie’ ERF1 silencing plants which are currently under evaluation