Skip to main content
SpringerLink
Log in

Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal

  • Research Article
  • Published:
Physiology and Molecular Biology of Plants Aims and scope Submit manuscript

Abstract

Withania somnifera (L.) Dunal, is an important medicinal plant being the source of extremely important compounds like withanolides and withaferin. Influence of different plant growth regulators (PGRs) were evaluated for induction of callus, callus mediated regeneration and production of secondary metabolites in them. Explants for callusing were collected from plants grown in vitro and maximum callusing (98 %) was obtained on MS medium supplemented with a combination of 2,4-dichlorophenoxy acetic acid (2,4-D) (0.5 mg l-1) and kinetin (KN) (0.2 mg l−1). Among different types of calli, best shoot regeneration was observed on green, compact calli produced on MS medium with a combination of 6-benzylamino purine (BAP) and indole butyric acid (IBA). MS medium supplemented with BAP (2 mg l−1) showed highest frequency (98 %) of shoot bud regeneration. The micro-shoots were efficiently rooted on MS media supplemented with 0.5 mg l−1 IBA. Rooted plants were transferred to soil-vermi-compost (1:3; w/w) medium in greenhouse for acclimatization. Presence of withanolide A and withaferin A in calli was validated through high performance thin layer chromatography (HPTLC). It was interesting to observe that the PGRs showed significant influence on the secondary metabolites production in callus and 2,4-D having the least effect. Histological studies revealed the origin of shoot tip in the callus during regeneration.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

Abbreviations

PGR:

Plant Growth Regulator

MS:

Murashige and Skoog (1962) medium

GA3 :

Gibberelic Acid A3

2,4-D:

2,4-dichlorophenoxy Acetic Acid

KN:

kinetin

BAP:

6-benzylamino Purine

IBA:

indole-3-butyric Acid

HPTLC:

High Performance Thin Layer Chromatography

References

  • Abbasi BH, Ahmad N, Fazal H, Mahmood T (2010) Conventional and modern propagation techniques in Piper nigrum. J Med Plants Res 4(1):007–012

    CAS  Google Scholar 

  • Agarwal B, Singh U, Banerjee M (1992) In vitro clonal propagation of tea (Camellia sinensis (L.) Kuntze). Plant Cell Tissue Organ Cult 30:1–5

    Article  CAS  Google Scholar 

  • Banerjee S, Naqvi AA, Mandal S, Ahuja PS (1994) Transformation of Withania somnifera (L.) Dunal by Agrobacterium rhizogenes: infectivity and phytochemical studies. Phytother Res 8:452–455

    Article  CAS  Google Scholar 

  • Banerjee M, Gantait S, Pramanik BR (2011) A two step method for accelerated mass propagation of Dendrocalamus asper and their evaluation in field. Physiol Mol Biol Plants 17(4):387–393

    Article  CAS  Google Scholar 

  • Blakesley D, Constantine D (1992) Uptake and Metabolism of 6- Benzyladenine in shoot cultures of a range of species. Plant Cell Tissue Organ Cult 28:1183–1186

    Google Scholar 

  • Ciddi V (2006) Withaferin A from cell cultures of Withania somnifera. Indian J Pharm Sci 68(4):490–492

    Google Scholar 

  • Das A, Datta AK, Ghosh S, Bhattacharyya A (2009) Growth and cultivation of two varieties of Withania somnifera. J Trop Med Pl 10:225–230

    Google Scholar 

  • Das A, Ghosh S, Bhattacharyya A, Datta AK (2010) In vitro biogeneration of alkaloids and withanolides in Withania somnifera (L.) Dunal (Solanaceae) var. ‘Poshita’ and ‘Jawahar 22’. Int J Plant Dev Biol 4(1):42–46

    Google Scholar 

  • Devi PU, Sharada AC, Solomon FE, Kamath MS (1992) In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180. Indian J Exp Biol 30:169–172

    PubMed  CAS  Google Scholar 

  • Dewir YH, Chakrabarty D, Lee SH, Hahn EJ, Paek KY (2010) Indirect regeneration of Withania somnifera and comparative analysis of withanolides in In vitro and greenhouse grown plants. Biol Planta 54(2):357–360

    Article  Google Scholar 

  • Easu K (1977) Anatomy of seed plants. John Wiley and Sons, New York, pp 271–285

    Google Scholar 

  • Fatima N, Anis M (2012) Role of growth regulators on in vitro regeneration and histological analysis in Indian ginseng (Withania somnifera L.) Dunal. Physiol Mol Biol Plants 18(1):59–67

    Article  CAS  Google Scholar 

  • Furmanowa M, Gajdzis-Kuls D, Ruszkowska J, Czarnocki Z, Obidoska G, Sadowska A, Rani R, Upadhyay SN (2001) In vitro propagation of Withania somnifera and isolation of withanolides with immunosuppressive activity. Planta Med 67:146–149

    Article  PubMed  CAS  Google Scholar 

  • Gamble JS (1918) Flora of presidency of Madras 2. Botanical survey of India, Calcutta

    Google Scholar 

  • Ghimire BK, Seong ES, Kim EH, Lamsal K, Yu CY, Chung M (2010) Direct shoot organogenesis from petiole and leaf discs of Withania somnifera (L.) Dunal. African. J Biotechnol 9(44):7453–7461

    CAS  Google Scholar 

  • Jaffer HJ, Jawad ALM, Saber HS, Al-Naib A (1988) Evaluation of antimicrobial activity of Withania somnifera extracts. Fitoterapia 59:497–500

    Google Scholar 

  • Johansen DA (1940) Plant Microtechnique. McGraw-Hill, NewYork, pp 126–154

    Google Scholar 

  • Kiritikar KR, Basu BD (1975) Indian Medicinal Plants 3, 1 st Edn. Dehra Dun

  • Kulkarni AA, Thengane SR, Krishnamurthy KV (2000) Direct shoot regeneration from node, internode, hypocotyl and embryo explants of Withania somnifera. Plant Cell Tissue Organ Cult 62:203–209

    Article  Google Scholar 

  • Manickam VS, Mathavan RE, Antonisamy R (2000) Regeneration of Indian ginseng plantlets from stem callus. Plant Cell Tissue Organ Cult 62:181–185

    Article  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiol 15:473–497

    Article  CAS  Google Scholar 

  • Murthy HN, Dijkstra C, Anthony P, White DA, Davey MR, Power JB, Hahn EJ, Paek KY (2008) Establishment of Withania somnifera hairy root cultures for the production of withanolide A. J Int Plant Biol 50:975–981

    Article  CAS  Google Scholar 

  • Nagella P, Murthy HN (2010) Establishment of cell suspension cultures of Withania somnifera for the production of withanolide A. Bioresource Technol 101:6735–6739

    Article  CAS  Google Scholar 

  • Pati PK, Sharma M, Salar RK, Sharma A, Gupta AP, Singh B (2008) Studies on leaf spot disease of Withania somnifera and its impact on secondary metabolites. Indian J Microbiol 48:432–437

    Article  PubMed  CAS  Google Scholar 

  • Rani G, Grover IS (1999) In vitro callus induction and regeneration studies in Withania somnifera. Plant Cell Tissue Organ Cult 57:23–27

    Article  Google Scholar 

  • Rani G, Virk GS, Nagpal A (2003) Callus induction and plantlet regeneration in Withania somnifera (L.) Dunal. In Vitro Cell Dev Biol Plant 39:468–474

    Google Scholar 

  • Ray S, Jha S (1999) Withanolide synthesis in cultures of Withania somnifera transformed with Agrobacterium tumefaciens. Plant Sci 146:1–7

    Article  CAS  Google Scholar 

  • Ray S, Jha S (2001) Production of withaferin A in shoot cultures of Withania somnifera Dunal. Planta Med 67:432–437

    Article  PubMed  CAS  Google Scholar 

  • Ray S, Ghosh B, Sen S, Jha S (1996) Withanolide production by root cultures of Withania somnifera transformed with Agrobacterium rhizogens. Planta Med 62:571–573

    Article  PubMed  CAS  Google Scholar 

  • Roja G, Heble MR (1991) Tissue cultures of Withania somnifera: morphogenesis and withanolide synthesis. Phytother Res 5:185–187

    Article  CAS  Google Scholar 

  • Sabir F, Sangwan NS, Chaurasiya ND, Misra LN, Tuli R, Sangwan RS (2007) Rapid micropropagation of Withania somnifera L. accessions from axillary meristems. J Herbs Spices Med Plants 13(4):123–133

    Google Scholar 

  • Salvi ND, Singh H, Tivarekar S, Eapen S (2001) Plant regeneration from different explants of neem. Plant Cell Tissue Organ Cult 65:159–162

    Article  CAS  Google Scholar 

  • Sangwan RS, Chaurasiya ND, Misra LN, Lal P, Uniyal GC, Sharma R, Sangwan NS, Suri KA, Qazi GN, Tuli R (2005) Process for isolation of withaferin A from plant materials and products therefrom. US Patent (AppFT) 20050226950

  • Sangwan RS, Chaurasiya NS, 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 Pharm Bull 55:1371–1375

    Article  PubMed  CAS  Google Scholar 

  • Sen J, Sharma AK (1991) Micropropagation of Withania somnifera from germinating seeds and shoot tips. Plant Cell Tissue Organ Cult 26:71–73

    Article  CAS  Google Scholar 

  • Singh NK, Sehgal CB (1999) Micropropagation of Holy Basil (Ocimum sanctum Linn.) from young inflorescences of mature plants. Plant Growth Regul 29:161–166

    Article  CAS  Google Scholar 

  • Sivanesan I (2007) Direct regeneration from apical bud explants of Withania somnifera Dunal. Indian J Biotechnol 16:125–127

    Google Scholar 

  • Sivanesan I, Murugesan K (2005) In vitro adventitious shoot formation from leaf explants of Withania somnifera Dunal. Plant Cell Biotechnol Mol Biol 6:163–166

    Google Scholar 

  • Sivanesan I, Murugesan K (2008) An efficient regeneration from Nodal Explants of Withania somnifera Dunal. Asian J Plant Sci 7(6):551–556

    Article  CAS  Google Scholar 

  • Vitali G, Conte L, Nicoletti M (1996) Withanolide composition and in vitro culture of Italian Withania somnifera. Planta Med 62:287–288

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

The authors are indebted to Dr. Chhanda Mondal, Ramakrishna Mission Quality Testing Laboretory, Narendrapur, Kolkata for chemical analysis of the samples.

Author information

Authors and Affiliations

  1. West Bengal State Council of Science and Technology, Bikash Bhavan, North block, 4th Floor, Salt Lake, Kolkata, 700091, India

    Nilanjan Chakraborty, Debarupa Banerjee, Moumita Ghosh & Maitreyi Banerjee

  2. Molecular and Applied Mycology and Plant Pathology Laboratory, Department of Botany, University of Calcutta, Kolkata, 700019, India

    Nilanjan Chakraborty, Debarupa Banerjee, Moumita Ghosh, Prakash Pradhan, Namrata Shanu Gupta & Krishnendu Acharya

Authors
  1. Nilanjan Chakraborty
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Debarupa Banerjee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Moumita Ghosh
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Prakash Pradhan
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Namrata Shanu Gupta
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Krishnendu Acharya
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Maitreyi Banerjee
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maitreyi Banerjee.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chakraborty, N., Banerjee, D., Ghosh, M. et al. Influence of plant growth regulators on callus mediated regeneration and secondary metabolites synthesis in Withania somnifera (L.) Dunal. Physiol Mol Biol Plants 19, 117–125 (2013). https://doi.org/10.1007/s12298-012-0146-2

Download citation

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s12298-012-0146-2

Keywords

Search

Navigation