Engineering Strawberry Anthocyanin Levels by Transformation with Late Flavonoid Pathway Genes

Flavonoids are widespread plant secondary metabolites involved in many functions such as pigmentation, protection from biotic and abiotic stress, and plant-environment interactions. The anthocyanin subclass comprises the most important plant pigments, responsible for orange, red and blue colours in plants and having antioxidant activity. The last steps of anthocyanidin biosynthesis involve dihydroflavonol 4-reductase (DFR), catalysing the reduction of dihydroflavonols to leucoanthocyanidins, and anthocyanidin synthase (ANS), catalysing the formation of anthocyanidins. In this study, 35S-driven DFR and ANS constructs were introduced in two strawberry cultivars, 'Sveva' (June-bearing) and 'Calypso' (everbearing), by Agrobacterium tumefaciens-mediated transformation. A gene stacking experiment (DFR+ANS) has also been performed by transforming one 'Sveva' DFR line with the ANS gene, under double kanamycin and hygromycin selection. Transgenic lines were obtained using leaf disk regeneration and kanamycin selection, then in vitro proliferated and acclimatized. Antibiotic-resistant clones were confirmed as transgenic plants by PCR detection.