Mesembrine alkaloid production in in vitro culture morphotypes of Sceletium tortuosum (L.) NE Br

Different routes of access to mesembrine alkaloids uniquely produced by the medicinal succulent Sceletium tortuosum (Aizoaceae) are thus highly sought after in the natural products sector. In order to develop a biotechnological method, the establishment of in vitro microshoot and callus suspension cultures was investigated using in vitro seedlings of S. tortuosum. The production of mesembrine and other alkaloid derivatives in vitro was monitored using high-throughput ultra-high performance liquid chromatography-mass spectrometry in regenerated microplant and callus cultures. Microshoots were transferred to a shoot multiplication medium after initiation on media with factorial combinations of 1-naphthylacetic acid and N-6-benzyladenine at 0-1 mg/L and 1-5 mg/L, respectively. Persistent carryover effects associated with the initiation procedures led to four dominant morphotypes. A drier Micropore (TM) seal environment reduced the number of propagules but the cultures exhibited greater proportions of healthy plantlets (88%) even though the incidence of a red coloured pigment was higher, recorded at 100% in some of the lines. Using a semi-in vitro technique, 96% of in vitro derived plants survived ex vitro. Microshoot cultures, pre-treated with a dehydration step, accumulated four key biomarkers with mesembrine (3270.9 mg/kg), mesembranol (4738.8 mg/kg), mesembrenol (333.1 1 mg/kg), and Delta(4) mesembrenone (29.9 mg/kg) (dry weight) at levels comparable to wild types. Delta(7)-Mesembrenone, while detected in the microshoots, was not present in callus cultures. The reported mass propagation strategies are opportune for offering an alternative source to the mesembrine alkaloids currently used in clinical herbal formulations.