Application of a site-specific DNA excision strategy for marker gene removal during gene transfer in Vitis spp.

Site-specific DNA excision strategy for marker gene removal was exploited for gene transfer into grape. Nicotiana benthamiana leaf sections and embryogenic calli of V. vinifera (‘Chardonnay’ and ‘Brachetto’) were co-cultured with Agrobacterium tumefaciens carrying the chemically-inducible pX6 vector with the Green Fluorescent Protein gene (GFP). This vector provides a reliable system for marker gene (nptII) excision, based on Cre/loxP and regulated by 17-β-estradiol. Transgenic cultures were selected on kanamycin, and plantlets were regenerated. Preliminary molecular assays showed the transfer of the GFP gene into the plant genome. After induction on different concentrations and exposition time on 17-β-estradiol, observations at the fluorescence stereomicroscope showed the expected GFP gene expression. To exploit the effectiveness of this strategy for achieving Pathogen Derived Resistance, the GFP gene was replaced with a sequence of the GVA virus coat protein gene in sense and antisense orientation for transcription of a hairpin RNA (pX6-pKcpGVA). Gene transfer experiments on tobacco and grapes produced plantlets containing the foreign genes