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Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal

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Abstract

A micropropagation system through leaf explant culture has been developed for Withania coagulans. Shoot bud proliferation occurred through both adventitious and de novo routes depending on the hormonal regime of the culture medium. Green compact nodular organogenic callus developed on Murashige and Skoog (MS) medium supplemented with 2.3 μM kinetin (Kn) and lower levels of 6–benzyladenine (BA) (13.3 μM) while multiple adventitious shoot bud differentiation occurred on medium fortified with 2.3 μM kinetin (Kn) and higher levels of BA (22.2 μM). Shoot buds were transferred to proliferation medium containing 2.2 μM BA, 2.3 μM Kn, and 3.9 μM phloroglucinol (PG) for further growth and development of shoot system. Elongated shoots were rooted using a two-step procedure involving pulse treatment of 7 days in a medium containing 71.6 μM choline chloride (CC) and 3.9 μM PG and then transferred to rooting medium containing ½ MS, 1.2 μM IBA, 3.6 μM PAA, and 14.3 μM CC for 3 weeks. Well-rooted plants were transferred to a greenhouse for hardening and further growth. Random amplification of polymorphic DNA (RAPD) showed monomorphic bands in all the plants thereby confirming clonality of the regenerants. Thin layer chromatography (TLC) showed the presence of withanolides in the regenerated plants. Quantification through reverse-phase HPLC revealed increased concentration of withanolides in the regenerated plants compared to the field-grown mother plant. Accumulation of withaferin A and withanolide A increased up to twofold and that of withanone up to tenfold. Direct regeneration via leaf explants will be useful for Agrobacterium-mediated genetic transformation, and will facilitate pathway manipulation using metabolic engineering for bioactive withanolides.

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Abbreviations

BA:

6–benzyladenine

CC:

Choline chloride

DAD:

Diode array detector

IAA:

Indole–3–acetic acid

IBA:

Indole–3–butyric acid

Kn:

Kinetin

MS:

Murashige and Skoog

NAA:

α–naphthaleneacetic acid

PAA:

Phenylacetic acid

PG:

Phloroglucinol

RAPD:

Random amplification of polymorphic DNA

TLC:

Thin layer chromatography

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Acknowledgments

Financial support from Council of Scientific and Industrial Research (CSIR) in the form of R&D project: CSIR–38(1178) EMR–II/2007 is gratefully acknowledged. Rohit Jain, Arunima Sinha and Devendra Jain thank CSIR for the award of Senior Research Fellowships.

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Authors and Affiliations

  1. Department of Botany, University of Rajasthan, Jaipur, 302004, India

    Rohit Jain, Arunima Sinha, Devendra Jain, Sumita Kachhwaha & S. L. Kothari

  2. Centre for Converging Technologies (CCT), University of Rajasthan, Jaipur, 302004, India

    Sumita Kachhwaha & S. L. Kothari

Authors
  1. Rohit Jain
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  2. Arunima Sinha
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  3. Devendra Jain
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  4. Sumita Kachhwaha
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  5. S. L. Kothari
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Correspondence to S. L. Kothari.

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Jain, R., Sinha, A., Jain, D. et al. Adventitious shoot regeneration and in vitro biosynthesis of steroidal lactones in Withania coagulans (Stocks) Dunal. Plant Cell Tiss Organ Cult 105, 135–140 (2011). https://doi.org/10.1007/s11240-010-9840-3

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