Enhancement of cardenolide production in transgenic Digitalis purpurea L. by expressing a progesterone-5β-reductase from Arabidopsis thaliana L.

Abstract

Metabolic engineering of Digitalis purpurea L. has emerged as a powerful biotechnological tool to enhance in vitro cardenolide biosynthesis. Thereto, Agrobacterium tumefaciens-mediated transformation protocols have been developed using nptII as selectable marker gene and uidA as reporter gene. In this work, the Arabidopsis thaliana L. VEP1 gene, encoding progesterone-5β-reductase, was expressed in cardenolide-producing D. purpurea plants. Resistance to hygromycin B was efficiently used as a selectable marker in callus induction and multiplication media. Successful transformation was confirmed in nine transgenic lines by PCR analyses using primers for the selectable marker gene (hpt) and the VEP1 gene. The number of copies of the transgene construct was estimated between one and three in different lines by quantitative PCR and inverse PCR. VEP1 expression was confirmed in eight transgenic lines by reverse transcription-quantitative PCR. Digitoxin and digoxin content were increased up to 3.8-fold (757 μg/gDW) and 2.2-fold (199 μg/gDW) respectively in transgenic plants cultivated in vitro. However, for plants grown in the greenhouse, digitoxin content was not significantly different from non-transgenic plants and digoxin production was enhanced up to 1.8-fold (87 μg/gDW). Genetic transformation thus allowed to enhance cardenolide production in vitro with higher efficiency than with other biotechnological strategies reported so far.