Habituated Moringa oleifera callus retains metabolic responsiveness to external plant growth regulators

Undifferentiated plant cells in culture represent a renewable system conducive to understanding biological processes and a valuable alternative for secondary metabolite production. Additionally, manipulation of these systems by plant growth regulators (PGRs) may result in redifferentiation/organogenesis and hence changes in metabolic profiles. The aim of the study was to investigate the effects of combining auxin (2,4-dichlorophenoxyacetic acid) and cytokinin (kinetin) at concentrations of 2, 4, 6 and 9 µM on undifferentiated Moringa oleifera callus cells, at a metabolome level. Results indicated that the callus became habituated, i.e. developed the ability to grow without added stimulatory PGRs, and no organogenesis was observed on any of the different PGR combinations under investigation. Methanolic extracts were screened for total phenolic content (TPC) and anti-oxidant activity, and further analysed using liquid chromatography coupled to mass spectrometry combined with multivariate data analysis to facilitate analysis of the metabolite profiles. While the anti-oxidant capacity of extracts from the various treatments exhibited little variation, the TPC differed significantly. Despite the observed habituation phenomenon, the calli retained responsiveness towards external PGRs and each of the 25 conditions generated a unique metabolome as found by principal component analysis. This was also reflected by a number of phytochemicals that were annotated as biomarkers from PGR-treated calli. These findings demonstrate the differential influence of 2,4-D and kinetin on M. oleifera callus for the production of secondary metabolites.