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The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants

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Abstract

An efficient mass multiplication protocol was developed for Withania somnifera (L.) Dunal from nodal explants of field-grown plants on Murashige and Skoog medium (MS) supplemented with 6-benzyladenine (BA) [1.5 mg L−l], indole-3-acetic acid (IAA) [0.3 mg L−l] and with the addition of polyamine, spermidine (20 mg L−l) (shoot multiplication medium). A total of 46.4 shoots were obtained from nodal explants and they were elongated in the same medium in a culture duration of 6 weeks. The elongated shoots produced roots in MS medium fortified with putrescine (20 mg L−l) after 4 weeks, and all the rooted plants were successfully hardened and acclimatized with a survival rate of 100%. An average of 276 shoots (46 × 6) was produced when at least six nodal explants obtained from each of the 46 in vitro grown shoots were cultured by microcutting method in the same shoot multiplication medium. On an average, 12,696 plants could be produced from all the shoots (276 × 46) by microcuttings in a period of 7 months. HPLC revealed a significant increase in the quantities of withanolide A, withanolide B, withaferin A and withanone in the leaves, stems, and roots of in vitro regenerated plants compared to the field-grown parent plants. Ploidy analysis using flow cytometry revealed genetic stability of in vitro regenerated plants. This protocol will be useful for scale-up production of withanolides on commercial scale.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

BA:

6-Benzyladenine

DNM:

De novo meristems

HPLC:

High-performance liquid chromatography

IBA:

Indole-3-butyric acid

IAA:

Indole-3-acetic acid

MS:

Murashige and Skoog medium

NN:

Nitsch and Nitsch medium

PAs:

Polyamines

PEM:

Pre-existing meristems

TDZ:

Thidiazuron

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Acknowledgments

We gratefully acknowledge the Council of Scientific and Industrial Research (CSIR) [38(1180/08/EMR-II)], Government of India, for providing financial support for a part of this work.

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Authors and Affiliations

  1. Department of Biotechnology, School of Life Sciences, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Ganeshan Sivanandhan, Thankaraj Salammal Mariashibu, Muthukrishnan Arun, Manoharan Rajesh & Andy Ganapathi

  2. Temasek Life Sciences Laboratory Limited, 1 Research Link National University of Singapore, Singapore, 117604, Singapore

    Sampath Kasthurirengan

  3. Periyar E.V.R College, Tiruchirappalli, 620 023, India

    Natesan Selvaraj

Authors
  1. Ganeshan Sivanandhan
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  2. Thankaraj Salammal Mariashibu
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  3. Muthukrishnan Arun
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  4. Manoharan Rajesh
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  5. Sampath Kasthurirengan
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  6. Natesan Selvaraj
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  7. Andy Ganapathi
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Correspondence to Andy Ganapathi.

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Communicated by B. Borkowska.

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Sivanandhan, G., Mariashibu, T.S., Arun, M. et al. The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants. Acta Physiol Plant 33, 2279–2288 (2011). https://doi.org/10.1007/s11738-011-0768-y

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