In vitro propagation of Alkanna tinctoria Tausch.: a medicinal plant of the Boraginaceae family with high pharmaceutical value

Abstract

Alkanna tinctoria Tausch. (Boraginaceae), commonly known as alkanet or dyers’ bugloss/alkanet, is a perennial plant rich in naphthoquinone enantiomers, such as Alkannin and Shikonin (A/S), which possess a wide range of pharmaceutical properties and are used as cosmetics, food additives, and natural dyes. This plant is mostly exploited from the wild, increasing the risk of its extinction as reported for other A/S producing plants extracted from their natural environment. Its cultivation under controlled conditions remains difficult and the need for alternative production systems both for preserving this endangered species and for increasing the production of A/S at a marketable level, has become a necessity. In the present study, a protocol for the in vitro production of A. tinctoria plants using shoot-tip explants was developed. Several culture media, concentrations of hormones, sugar, and gelling agents were tested to improve proliferation, rooting, and acclimatization of micropropagated shoot-tip explants from plants collected in the wild. Surface disinfection was optimal after immersion of shoot-tips and/or nodal explants in Signum® fungicide (30 min), ethanol 70% (1 min), and sodium hypochlorite 3% (10 min). Shoot proliferation was the highest on Murashige-Skoog basal medium enriched with 1.1 μM 6-benzyladenine, 0.15 μM α-naphthalene acetic acid, and 0.3 μM gibberellic acid with a proliferation rate of 3.9 every two weeks. For rooting, the Root Culture 1 (RC1) modified medium free of ammonium nitrate and enriched with 2.85 μΜ indole-3-acetic acid was the more adequate with 80% of roots formation after 30 days. Finally, acclimatization was optimal (100% survival rate) following transfer of the rooted explants in pots containing a peat moss:perlite (1:1, v/v) mixture, kept under a 90% relative humidity fog system for 10 days, followed by a decrease in relative humidity of 5% every day until 40% and a gradual increase in light intensity. The protocol developed allowed the production under in vitro culture conditions of a sufficient number of A. tinctoria plants with high levels of ex vitro survival, opening the door to industrial exploitation of its secondary metabolites and to the conservation of this important medicinal plants.