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

  • Claude Y. Hamany Djande
  • Paul A. Steenkamp
  • Lizelle A. Piater
  • Ntakadzeni E. Madala
  • Ian A. DuberyEmail author
Original Article


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.

Key message

Moringa oleifera callus developed the ability to grow without added stimulatory growth regulators, but retained responsiveness towards external growth regulators, resulting in distinct metabolomes.


Auxin Cytokinin Callus Moringa oleifera Organogenesis Secondary metabolites 



Base peak intensity


Chlorogenic acid




Electrospray ionisation


Folin–Ciocalteu reagent


Gallic acid equivalents


Hierarchical clustering analysis


Mass to charge ratio


Murashige and Skoog medium


Metabolomic Standards initiative


Multivariate data analyses


Orthogonal projection to latent structures-discriminant analysis


Principal component analysis


Plant growth regulators


Soft independent modeling of class analogy


Retention time


Ultra-high performance liquid chromatography–quadrupole time-of-flight high definition mass spectrometry



The South African National Research Foundation (NRF) is thanked for grant support to NEM (Grant No. 107067) and IAD (Grant No. 95818) and fellowship support to CHD.

Author Contributions

IAD and NEM conceived and supervised the project. YHD performed the experimental work and PAS the instrumental analysis. CHD and NEM analysed the data. All authors contributed to writing and editing the manuscript. All authors have read and approved the final version of the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare no conflict of interest.

Supplementary material

11240_2019_1565_MOESM1_ESM.pdf (1 mb)
Supplementary material 1 (PDF 1073 KB)


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© Springer Nature B.V. 2019

Authors and Affiliations

  1. 1.Centre for Plant Metabolomic Research, Department of BiochemistryUniversity of JohannesburgJohannesburgSouth Africa

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