Plant hormones and Agrobacterium tumefaciens strain 82.139 induce efficient plant regeneration in the cardenolide-producing plant Digitalis minor

Shoot formation in explants of Digitalis minor was achieved, through axillary bud proliferation and adventitious bud differentiation, by varying the amount and source of plant hormones. Shoot regeneration was also obtained after infection of D. minor with the wild-type Agrobacterium tumefaciens strain 82.139, which induced shooty tumours. These shoots were not transgenic, as revealed by nopaline assays and the use of a C58pMP90/T139GUS-INT strain harbouring the intron inactivated gusA gene. Plants were easily rooted and transplanted into the greenhouse. Shoot cultures of D. minor accumulated up to 226 μg cardenolides per g dry mass when cultured on agar-solidified medium. Cardenolide content was significantly lower in submerged shoot cultures, the effect being related to a high incidence of hyperhydricity. Greenhouse-grown plants presented the highest amounts of these metabolites. This is the first report on micropropagation of the Balearic endemic medicinal plant species D. minor. The induction of caulogenic galls by A. tumefaciens wild strain 82.139 opens new possibilities for the biotechnological improvement of Digitalis spp.