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Plant Cell, Tissue and Organ Culture (PCTOC)

, Volume 105, Issue 1, pp 135–140 | Cite as

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

  • Rohit Jain
  • Arunima Sinha
  • Devendra Jain
  • Sumita Kachhwaha
  • S. L. KothariEmail author
Research Note

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.

Keywords

Micropropagation HPLC TLC RAPD Withania coagulans Withanolides 

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

Notes

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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Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Rohit Jain
    • 1
  • Arunima Sinha
    • 1
  • Devendra Jain
    • 1
  • Sumita Kachhwaha
    • 1
    • 2
  • S. L. Kothari
    • 1
    • 2
    Email author
  1. 1.Department of BotanyUniversity of RajasthanJaipurIndia
  2. 2.Centre for Converging Technologies (CCT)University of RajasthanJaipurIndia

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