Somaclonal variation of cardenolide content in Heywood's foxglove, a source for the antiviral cardenolide glucoevatromonoside, regenerated from permanent shoot culture and callus

Abstract

Cardiac glycosides are plant natural compounds used to treat cardiac insufficiency in humans. Recent findings suggest a further medical use in oncology and virology. Since species of the genus Digitalis are the major source of these compounds, in vitro culture techniques can be useful tools for the propagation and selection of elite genotypes. Digitalis mariana ssp. heywoodii, an endemic and endangered species in Portugal, is known to have a very high content of cardiac glycosides, especially glucoevatromonoside. Here, D. mariana ssp. heywoodii plants were cloned from a seed-derived shoot explant through direct or indirect in vitro propagation. Multiple shoot formation was induced from a seed grown on agar-solidified Murashige and Skoog (MS)-based medium supplemented with 9.8 μM indole-3-butyric acid (IBA). Shoots were used to regenerate plants directly, and some were then used as a source of root explants for callus induction and growth by transferring to MS medium supplemented with 9.3 μM kinetin (KIN), 5.7 μM indoleacetic acid (IAA), and 1.1 μM 2,4-dichlorophenoxyacetic acid (2,4–D). Callus that was 3, 6, or 24 mo old efficiently developed adventitious shoots when placed on MS medium containing 13.3 μM 6-benzylaminopurine (BAP) and 1.7 μM IAA. Plants regenerated from either callus (R plants) or shoot cultures (S plants) were transferred to the greenhouse and propagated for 3, 4, or 10 mo. Regenerated plants were analyzed for cardenolide content and accumulation pattern. The highest cardenolide content, about 2.0 mg/100 g DW in terms of digitoxin equivalents (Dteq), was found in S10 plants (10 mo in the greenhouse), whereas plants regenerated from 24-mo-old callus (R24/10; 10 mo in the greenhouse) had the lowest cardenolide content of about 1.2 mg Dteq/100 g DW.