Abstract
Markedly increased withanolide content was found in transformed roots (TR) of Withania somnifera germplasm grown in low mineral minimal media and withanolides showed high antioxidant potential when analysed using acidic potassium permanganate chemiluminescence. Transformation frequency of explants infected with Agrobacterium rhizogenes strain A4 varied between the three germplasms tested with the highest observed as 75 ± 0.9. Transformed root production was explant specific with leaves being the most productive among the different explants used. Withanolides, namely withaferin A, withanolide A, withanolide B and 12-deoxywithastramonolide were detected in TR cultures and differences in their content were found between germplasms. The highest concentrations of secondary metabolites were found in 4-week-old cultures and concentrations declined by the 8th and 12th week of culture. In 4-week-old cultures, the biomass of TR cultures was 4.5 fold higher than their respective non-transformed roots (NTR). Withaferin A was found in TR at levels that were 28–34 times higher than that found in NTR. A rapid method for the determination of the antioxidant potential of W. somnifera TR extracts was developed using post-column acidic potassium permanganate chemiluminescence (APPC) detection. The APPC chromatographic peaks for extract constituents showed strong alignment with those found for ultraviolet absorbance detection. The methods developed in this study for TR culture establishment and the use of a fast and sensitive way for the qualitative and quantitative determination of the antioxidant activity of their metabolites provides a new platform that will have use for similar studies in other species.
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Abbreviations
- ABTS˙ + :
-
2,2-azinobis-(3-ethylbenzothiazoline-6-sulphonic acid) radical cation
- APPC:
-
Acidic potassium permanganate chemiluminescence
- CL:
-
Chemiluminescence
- DPPH˙ :
-
2,2-diphenyl-1-picrylhydrazyl
- DW:
-
Dry weight
- HPLC:
-
High performance liquid chromatography
- M:
-
Minimal medium
- MS:
-
Murashige and Skoog medium
- MW:
-
Modified White’s medium
- NTR:
-
Non-transformed roots
- PCR:
-
Polymerase chain reaction
- PDA:
-
Photodiode array
- Ri:
-
Root inducing
- T-DNA:
-
Transfer DNA
- TR:
-
Transformed roots
- UV:
-
Ultraviolet
- WS:
-
Withania somnifera
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The authors acknowledge Deakin University, Australia, and The Energy and Resources Institute for financial assistance and infrastructure support. Leena Johny was the recipient of a Deakin University postgraduate scholarship.
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LJ conducted all experiments and wrote the first draft of the manuscript. XAC supervised the experiments, contributed to understanding analytical tools, to data interpretation and paper drafting. AA and DMC developed the scientific question, supervised the experiments and contributed to drafting the manuscript. All authors read, reviewed and approved the manuscript.
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Communicated by Ali R. Alan.
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Johny, L., Conlan, X.A., Adholeya, A. et al. Growth kinetics and withanolide production in novel transformed roots of Withania somnifera and measurement of their antioxidant potential using chemiluminescence. Plant Cell Tiss Organ Cult 132, 479–495 (2018). https://doi.org/10.1007/s11240-017-1344-y
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DOI: https://doi.org/10.1007/s11240-017-1344-y