Production of tropane alkaloids by hairy root cultures of Duboisia leichhardtii transformed by Agrobacterium rhizogenes

Abstract

Hairy roots were induced by inoculating leaf disks and stem segments of Duboisia leichhardtii F. Muell with Agrobacterium rhizogenes strains 15834 and A4. Forty-five hairy root clones that had been derived from individual root meristems were established by culturing hairy roots for at least 5 passages in phytohormone-free HF (modified Heller's) medium. Characterization of the established hairy root clones showed that there was considerable variation in growth rate, alkaloid content and productivity from clone to clone. Clone DL-34, which produced the most scopolamine, was established from the selection of a number of hairy root clones. Properties such as rapid growth accompanied by extensive lateral branching, opine production and high scopolamine production were stable in DL-34 for 12 successive passages in HF medium; therefore, culture conditions for growth and alkaloid production were examined for this clone. After 4 weeks of culture at 25°C in the dark, its weight had increased 64-fold, and its scopolamine content was 1.8% on a dry weight basis in each optimum medium. This content was 2-fold that of the leaves of D. leichhardtii plants. Clone DL-34 produced 78 mg/1 of scopolamine after 4 weeks of culture. We show that selection and medium-composition are essential factors in obtaining good growth and scopolamine production.