Genetic transformation of Fragaria with candidate genes involved in drought stress

Agrobacterium-mediated transformation of Fragaria was performed in F. vesca, F. chiloensis and F. ananassa 'Ventana' and 'EL02.2011' to allow a functional analysis of CAT (catalase), P5CS (Delta¹-pyrroline-5-carboxylate synthetase) and FaAIV (sucrose acid invertase) genes under drought stress. These genes were targeted independently for the silencing by RNAi and over-expression in Fragaria. In vitro grown plantlets of F. vesca, F. chiloensis and F. ananassa 'EL02.2011' and in vivo grown plantlets of F. x ananassa 'Ventana' were transformed with 3 different RNAi constructs coding for a hairpin RNA containing inverted repeat of F. vesca sequences for these 3 genes.They were also transformed with 1 construct coding for over-expression of FaAIV whole coding sequences from F. x ananassa. Transformation for all constructs was confirmed by visible GFP expression from the same T-DNA. There was no callusing and regeneration of F. chiloensis. Callus formation was obtained in F. x ananassa 'Ventana' but shoot proliferation was weak. F. vesca presented reasonable callusing, shoot regeneration and transformation using kanamycin (25 mg/L) selection. F. x ananassa 'EL02.2011' showed high regeneration and transformation efficiency with stable GFP expression in shoots. The occurence of transgenic plants was confirmed by PCR analysis of genomic DNA in F. vesca and F. x ananassa 'EL02.2011'. Consistent with previous observations, here direct comparison of wild species and cultivars shows that the transformation efficiency in Fragaria can be affected by the type genotype, gene, construct and explant as well as the concentration of A. tumefaciens suspension culture.