Vol.:(0123456789) 1 3 Plant Cell, Tissue and Organ Culture (PCTOC) https://doi.org/10.1007/s11240-019-01565-y ORIGINAL ARTICLE Habituated Moringa oleifera callus retains metabolic responsiveness to external plant growth regulators Claude Y. Hamany Djande 1  · Paul A. Steenkamp 1  · Lizelle A. Piater 1  · Ntakadzeni E. Madala 1  · Ian A. Dubery 1 Received: 26 July 2018 / Accepted: 28 January 2019 © Springer Nature B.V. 2019 Abstract Undiferentiated 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 rediferentiation/organogenesis and hence changes in metabolic profles. The aim of the study was to investigate the efects of combining auxin (2,4-dichlorophenoxyacetic acid) and cytokinin (kinetin) at concen- trations of 2, 4, 6 and 9 µM on undiferentiated 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 diferent 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 profles. While the anti-oxidant capacity of extracts from the various treatments exhibited little variation, the TPC difered signifcantly. Despite the observed habitua- tion 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 refected by a number of phytochemicals that were annotated as biomarkers from PGR-treated calli. These fndings demonstrate the diferential infuence of 2,4-D and kinetin on M. oleifera callus for the production of secondary metabolites. Key message Moringa oleifera callus developed the ability to grow without added stimulatory growth regulators, but retained responsive- ness towards external growth regulators, resulting in distinct metabolomes. Keywords Auxin · Cytokinin · Callus · Moringa oleifera · Organogenesis · Secondary metabolites Abbreviations BPI Base peak intensity CGA Chlorogenic acid DPPH 2,2′-Diphenyl-1-picrylhydrazyl ESI Electrospray ionisation F–C Folin–Ciocalteu reagent GAE Gallic acid equivalents HCA Hierarchical clustering analysis m/z Mass to charge ratio MS Murashige and Skoog medium MSI Metabolomic Standards initiative MVDA Multivariate data analyses OPLS-DA Orthogonal projection to latent structures-discriminant analysis PCA Principal component analysis PGRs Plant growth regulators SIMCA Soft independent modeling of class analogy Communicated by Nokwanda Pearl Makunga. Electronic supplementary material The online version of this article (https://doi.org/10.1007/s11240-019-01565-y) contains supplementary material, which is available to authorized users. * Ian A. Dubery idubery@uj.ac.za 1 Centre for Plant Metabolomic Research, Department of Biochemistry, University of Johannesburg, Auckland Park, Johannesburg 2006, South Africa