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

Abstract

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.

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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

Rt:

Retention time

UHPLC–qTOF-HDMS:

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

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Acknowledgements

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.

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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.

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Correspondence to Ian A. Dubery.

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Hamany Djande, C.Y., Steenkamp, P.A., Piater, L.A. et al. Habituated Moringa oleifera callus retains metabolic responsiveness to external plant growth regulators. Plant Cell Tiss Organ Cult 137, 249–264 (2019). https://doi.org/10.1007/s11240-019-01565-y

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Keywords

  • Auxin
  • Cytokinin
  • Callus
  • Moringa oleifera
  • Organogenesis
  • Secondary metabolites