Elicitation effects on Sceletium tortuosum growth, physiology, and mesembrine-type alkaloid production in vitro.

Abstract

Sceletium tortuosum is an immensely valuable South African succulent. The plant is wellknown for its versatility in traditional medicine. Its’ mesembrine-type alkaloids (mesembranol, mesembrenol, mesembrenone, and mesembrine) have shown remarkable potential as therapeutic agents for numerous disorders, including depression, anxiety, diabetes, epilepsy, and Alzheimer’s disease. Despite the tremendous value of these phytochemicals, investigations into improving alkaloid production in this species are virtually non-existent, and commercial alkaloid sources are not readily available. Hence, this research aimed to determine the effects of elicitation techniques on growth and mesembrine-type alkaloid production of S. tortuosum in vitro. Elicitors were applied to nodal explant cultures for 28 days. Growth was assessed by determining the mean number of new leaf pairs, mean number of roots, and the mean length of the longest root. Mesembrine-type alkaloids were extracted from plant material and quantified with UPLC-MS. Light (0, 8, 16, and 24 H), salt (0, 50, 100, and 200 µM NaCl), and osmotic stress (0, 25, 50, and 100 g/L polyethylene glycol (PEG)) were applied. Growth was optimized in 24 H light as plantlets produced the highest mean number of new leaf pairs (2.79 ± 1.89) and roots (2.60 ± 1.60). Total alkaloid contents were significantly increased by 0 H light (9.16 ± 0.12 µg/mg DW) and 100 g/L PEG (6.74 ± 0.30 µg/mg DW). Growth effects and conservation of secondary metabolite pools in the presence of salt confirmed a halophytic nature for S. tortuosum. Colorimetric methods were used to quantify primary metabolites (total carbohydrates, starch, proteins, and proline) in stress-treated plantlets. Treatment with 50 g/L PEG resulted in peak quantities of starch (130.10 ± 0.16 µg/mg FW) and proline (24.73 ± 0.06 µg/mg FW), while 100 g/L PEG produced the highest protein concentration (71.60 ± 0.30 µg/mg FW). Protein levels were sensitive to osmotic stress, suggesting enhanced synthesis of stress-response proteins. Salt treatments (100 and 200 µM NaCl) yielded the highest carbohydrate concentrations (383.3 ± 1.40 and 323.1 ± 0.72 µg/mg FW, respectively), but only slight increases in proline concentrations (17.07 and 16.10 µg/mg FW, respectively) relative to the control (15.40 µg/mg FW). This provided further evidence of a halophytic profile for the species. Plant growth regulators (indole-3-butyric acid (IBA), naphthaleneacetic acid (NAA), kinetin (KIN) and benzylaminopurine (BAP) were applied to tissue cultures at concentrations of 0, 2.5, 5, 10, 15, and 20 µM. Plantlets treated with IBA were the healthiest, with 20 µM IBA resulting in the highest mean number of roots (14.18 ± 4.66) and mean root length (6.28 ± 2.59 mm). Treatments with NAA and cytokinins caused unhealthy shoot and root proliferation, respectively. However, KIN treatments resulted in the highest total alkaloid contents (5.51–7.77 µg/mg DW), with the maximum amount achieved by 5 µM KIN. Various concentrations of biostimulants (smoke water (SW), Kelpak® (KEL), vermicompost leachate (VCL), sodium humate (SH)) were tested. Smoke water and SH showed the most potential for growth improvements. Treatment with 5 mg/L SH resulted in the highest mean number of new leaf pairs (1.67 ± 1.20), while 1:2000 SW resulted in the highest mean number of roots and mean root length (2.57 ± 2.95 and 4.14 ± 6.69 mm, respectively). Significant increases in total alkaloid contents were recorded for 1:1000 and 1:2000 SW treatments (4.95 ± 0.23 and 4.72 ± 0.06 µg/mg DW, respectively). Positive responses were inversely proportional to SW concentration and directly proportional to SH concentration. Callus cultures were successfully initiated with 2.5 µM 2,4-D. Cell suspension cultures were supplemented with PGRs (KIN, BAP, NAA, 2,4-dichlorophenoxyacetic acid (2,4-D) (NAA, 2,4- D, KIN, BAP) and amino acids (methionine, tyrosine, phenylalanine) at concentrations of 0, 2.5, 5, and 10 µM. After a 37-day incubation, culture growth and alkaloid contents were assessed. Treatments with 5 and 10 µM 2,4-D µM resulted in the highest mean total cell number and fresh weight (1.59 x 1011 ± 1.19 x 1011 cells/mL and 2.82 ± 0.95 g, respectively). Alkaloids were present in cells and culture media. Total alkaloid contents were optimized by BAP treatments (3.36–4.51 µg/mg DW) and 10 µM 2,4-D (3.70 ± 0.40 µg/mg DW). Among the amino acids tested, phenylalanine (2.5 µM) produced the highest mean fresh weight (2.36 ± 0.28 g), and total alkaloid content (3.32 ± 0.24 µg/mg DW). However, the efficacy of 2,4-D over phenylalanine was proven by the alkaloid contents obtained in a later experiment (2.73 ± 0.94 and 1.29 ± 0.35 µg/mg DW, respectively). Several treatments successfully improved growth and alkaloid production of S. tortuosum. These methods could prove valuable to small- and large-scale Sceletium growers, along with biotechnologists and pharmaceutical companies. The growing need for commercial availability of these alkaloids could be satiated with appropriate use of the methodologies described.