An evaluation of factors affecting the efficiency of Agrobacterium-mediated transformation of Citrus paradisi (Macf.) and production of transgenic plants containing carotenoid biosynthetic genes

An improved protocol for Agrobacterium-mediated transformation of Duncan grapefruit (Citrus paradisi Macf.) epicotyl explants was developed by examining the effects of six different factors on the efficiency of transformation and combining the best treatment for each factor. The preculturing of explants and the composition of the cocultivation medium were the factors that most influenced transformation efficiency. The optimized protocol was successfully employed in the production of transgenic grapefruit plants containing the carotenoid biosynthetic genes phytoene synthase, phytoene desaturase, or lycopene-β-cyclase under constitutive expression. With an eventual goal of metabolically engineering grapefruit with multiple genes, hygromycin as a selectable marker and BIBAC as a transformation vector for large pieces of DNA were also tested.