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In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers

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Abstract

An efficient large-scale clonal propagation protocol has been described for Withania somnifera (L.) Dunal, a valuable medicinal plant, using cotyledonary nodes derived from axenic seedlings. Murashige and Skoog’s (Physiol Plant 15:473–497, 1962) (MS) medium supplemented with 1.0 mg l−1 N 6-benzyladenine (BA) was found to be optimum for production of multiple shoots (100 % shoot proliferation frequency and 16.93 shoots per explant). Successive shoot cultures were established by repeatedly sub-culturing the original cotyledonary node on a fresh medium after each harvest of newly formed shoots. Multiple shoot proliferation was also achieved from nodal segments derived from in vitro raised shoots on MS medium augmented with 1.0 mg l−1 BA. Regenerated shoots were best rooted (95.2 %, 38.7 roots per shoot) in half-strength MS medium supplemented with 1.0 mg l−1 indole-3-butyric acid. The plantlets were successfully acclimated and established in soil. Random amplified polymorphic DNA and inter-simple sequence repeats analysis revealed a homogeneous amplification profile for all micropropagated plants analyzed validating the genetic fidelity of the in vitro regenerated plants.

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Abbreviations

BA:

N 6-Benzyladenine

IBA:

Indole-3-butyric acid

ISSR:

Inter-simple sequence repeats

Kin:

Kinetin

MS:

Murashige and Skoog’s (1962) medium

PCR:

Polymerase chain reaction

RAPD:

Random amplified polymorphic DNA

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Acknowledgments

Fund support under FIST program by Department of Science and Technology (DST), Government of India to Department of Botany, Ravenshaw University, Cuttack-753 003, India is gratefully acknowledged (SR/FST/LSI-017-2010).

Conflict of interest

The authors state that they have no conflict of interest.

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Authors and Affiliations

  1. Department of Botany, Ravenshaw University, Cuttack, 753 003, Odisha, India

    S. A. Nayak, S. Kumar, A. Moharana, B. Behera, D. P. Barik, P. K. Mohapatra, P. K. Jena & S. K. Naik

  2. Center of Biotechnology, Siksha ‘O’ Anusandhan University, Bhubaneswar, 751 030, Odisha, India

    K. Satapathy & L. Acharya

Authors
  1. S. A. Nayak
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  2. S. Kumar
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  3. K. Satapathy
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  4. A. Moharana
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  5. B. Behera
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  6. D. P. Barik
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  7. L. Acharya
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  8. P. K. Mohapatra
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  9. P. K. Jena
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  10. S. K. Naik
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Corresponding authors

Correspondence to L. Acharya or S. K. Naik.

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Communicated by B. Borkowska.

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Nayak, S.A., Kumar, S., Satapathy, K. et al. In vitro plant regeneration from cotyledonary nodes of Withania somnifera (L.) Dunal and assessment of clonal fidelity using RAPD and ISSR markers. Acta Physiol Plant 35, 195–203 (2013). https://doi.org/10.1007/s11738-012-1063-2

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  • DOI: https://doi.org/10.1007/s11738-012-1063-2

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