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


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.


Micropropagation HPLC TLC RAPD Withania coagulans Withanolides 





Choline chloride


Diode array detector


Indole–3–acetic acid


Indole–3–butyric acid




Murashige and Skoog


α–naphthaleneacetic acid


Phenylacetic acid




Random amplification of polymorphic DNA


Thin layer chromatography



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.


  1. Atta-ur-Rahman A, Choudhary MI, Qureshi S, Gul W, Yousaf M (1998) Two new ergostane–type steroidal lactones from Withania coagulans. J Nat Prod 61:812–814PubMedCrossRefGoogle Scholar
  2. Baskaran P, Jayabalan N (2005) An efficient micropropagation system for Eclipta alba available medicinal herb. In Vitro Cell Dev Biol Plant 41:532–539CrossRefGoogle Scholar
  3. Bhandari MM (1995) Flora of the Indian desert. MPS Repros, JodhpurGoogle Scholar
  4. Corredoira E, Ballester A, Vieitez AM (2008) Thidiazuron-induced high-frequency plant regeneration from leaf explants of Paulownia tomentosa mature trees. Plant Cell Tissue Org Cult 95:197–208CrossRefGoogle Scholar
  5. Dayal S, Lavanya M, Devi P, Sharma KK (2003) An efficient protocol for shoot regeneration and genetic transformation of pigeonpea [Cajanus cajan (L.) Millsp.] using leaf explant. Plant Cell Rep 21:1072–1079PubMedCrossRefGoogle Scholar
  6. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  7. Faivre-Rampant O, Kevers C, Gaspar T (2004) IAA–oxidase activity and auxin protectors in nonrooting, rac, mutant shoots of tobacco in vitro. Plant Sci 153:73–80CrossRefGoogle Scholar
  8. Feeney M, Bhagwat B, Mitchel JS, Lane WD (2007) Shoot regeneration from organogenic callus of sweet cherry (Prunus avium L.). Plant Cell Tissue Org Cult 90:201–214CrossRefGoogle Scholar
  9. Gentile A, Monticelli S, Damiano C (2002) Adventitious shoot regeneration in peach (Prunus persica). Plant Cell Rep 20:1011–1016CrossRefGoogle Scholar
  10. Gomez KA, Gomez AA (1984) Statistical procedures for agricultural research. Wiley, New York, p 680Google Scholar
  11. Jain P, Kachhwaha S, Kothari SL (2009a) Improved micropropagation protocol and enhancement in biomass and chlorophyll content in Stevia rebaudiana (Bert.) Bertoni by using high copper levels in the culture medium. Sci Hort 119:315–319CrossRefGoogle Scholar
  12. Jain R, Sinha A, Kachhwaha S, Kothari SL (2009b) Micropropagation of Withania coagulans (Stocks) Dunal: a critically endangered medicinal herb. J Plant Biochem Biotechnol 18:249–252Google Scholar
  13. Kachhwaha S, Kothari SL (1996) Plant regeneration from immature embryo explants of Hordeum spontaneum and Hordeum vulgare. Cer Res Comm 24:27–32Google Scholar
  14. Koroch A, Juliani HR, Kapteyn J, Simon JE (2002) In vitro regeneration of Echinacea purpurea from leaf explants. Plant Cell Tissue Org Cult 69:79–83CrossRefGoogle Scholar
  15. Kothari SL, Joshi A, Kachhwaha S, Ochoa-Alejo N (2010) Chilli peepers—a review on tissue culture and transgenesis. Biotechnol Adv 28:35–48PubMedCrossRefGoogle Scholar
  16. Kulkarni AA, Thengane SR, Krishnamurthy KV (2000) Direct shoot regeneration from node, internode, hypocotyls and embryo explants of Withania somnifera. Plant Cell Tissue Org Cult 62:203–209CrossRefGoogle Scholar
  17. Liu X, Pijut PM (2008) Plant regeneration from in vitro leaves of mature black cherry (Prunus serotina). Plant Cell Tissue Org Cult 94:113–123CrossRefGoogle Scholar
  18. Maurya R, Akanksha J (2010) Chemistry and pharmacology of Withania coagulans: an ayurvedic remedy. J Pharma Pharmacol 62:153–160CrossRefGoogle Scholar
  19. Mishra Y, Patel PK, Yadev S, Shirin F, Ansari SA (2008) A micropropagation system for cloning of Bambusa tulda roxb. Sci Hort 115:315–318CrossRefGoogle Scholar
  20. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  21. Murch SJ, Wierenga EJ, El-Demerdash MA, Saxena PK (2004) In vitro propagation of the Egyptian medicinal plant, Echinops spinosissimus turra. Plant Cell Tissue Org Cult 74:81–86CrossRefGoogle Scholar
  22. Rani V, Raina SN (2000) Genetic fidelity of organized meristem—derived micropropagated plants: a critical reappraisal. In Vitro Cell Dev Biol Plant 36:319–330CrossRefGoogle Scholar
  23. Ray S, Jha S (2001) Production of withaferin A in in vitro shoot cultures of Withania somnifera. Planta Med 67:432–436PubMedCrossRefGoogle Scholar
  24. Reddy PS, Rodrigues R, Rajasekharan R (2004) Shoot organogenesis and mass propagation of Coleus forskohlii from leaf derived callus. Plant Cell Tissue Org Cult 66:183–188CrossRefGoogle Scholar
  25. Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2004) Phytochemical variability in commericial herbal products and preparations of Withania somnifera (ashwagandha). Curr Sci 86:446–461Google Scholar
  26. Sangwan RS, Chaurasiya ND, Lal P, Misra L, Uniyal GC, Tuli R, Sangwan NS (2007) Withanolide A biogeneration in in vitro shoot cultures of ashwagandha (Withania somnifera Dunal), a main medicinal plant in ayurveda. Chem Pharma Bull 55:1371–1375CrossRefGoogle Scholar
  27. Sarkar D, Naik PS (2000) Phloroglucinol enhances growth and rate of axillary shoot proliferation in potato shoot tip cultures in vitro. Plant Cell Tissue Org Cult 60:139–149CrossRefGoogle Scholar
  28. Sharada M, Ahuja A, Suri KA, Vij SP, Khajuria RK, Verma V, Kumar A (2007) Withanolide production by in vitro cultures of Withania somnifera and its association with differentiation. Biol Plant 51:161–164CrossRefGoogle Scholar
  29. Sharma PK, Tyagi P, Sharma KC, Kothari SL (2003) Clonal micropropagation of Crataeva adansonii (DC.) Prodr: a multipurpose tree. In Vitro Cell Dev Biol–Plant 39:156–160CrossRefGoogle Scholar
  30. Sinha A, Jain R, Kachhwaha S, Kothari SL (2010) Optimization of the level of micronutrient copper in the culture medium improves shoot bud regeneration in Indian ginseng [Withania somnifera (L.) Dunal]. Natl Acad Sci Lett 33:11–16Google Scholar
  31. Sood A, Ahuja PS, Sharma M, Sharma OP, Godbole S (2002) In vitro protocols and field performance of elites of an important bamboo Dendrocalamus hamiltonii nees et arn. ex munro. Plant Cell Tissue Org Cult 71:55–63CrossRefGoogle Scholar
  32. Sreedhar RV, Venkatachalam L, Thimmaraju R, Bhagyalaxmi N, Narayanan MS, Ravishankar GA (2008) Direct organogenesis from leaf explants of Stevia rebaudiana and cultivation in bioreactor. Biol Plant 52:355–360CrossRefGoogle Scholar
  33. Tilkat E, Onay A, Yıldırım H, Ayaz E (2009) Direct plant regeneration from mature leaf explants of pistachio, Pistacia vera L. Sci Hort 121:361–365CrossRefGoogle Scholar
  34. Zhou H, Li M, Zhao M, Fan X, Guo A (2010) Plant regeneration from in vitro leaves of the peach rootstock ‘Nemaguard’ (Prunus persica × P. davidiana). Plant Cell Tissue Org Cult 101:79–87CrossRefGoogle Scholar

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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