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Effect of nitrogen and carbon sources on in vitro shoot multiplication, root induction and withanolides content in Withania somnifera (L.) Dunal

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Abstract

Effect of nitrogen and carbon sources on multiple shoot regeneration and withanolides contents of Withania somnifera were evaluated. Inclusion of l-glutamine (20 mg/l) in medium fortified with the optimal levels of 1.5 mg/l 6-benzyladenine (BA) and 0.3 mg/l indole-3-acetic acid (IAA) resulted in the production of 58 shoots/explant. Sucrose at 4 % and 20 mg/l l-glutamine with idéal concentrations of BA and IAA improved shoot multiplication (62 shoots/explant) while 6 % sucrose enhanced withanolides contents in regenerated multiple shoots. All the shoots were rooted (26 roots/shoot) when cultured in MS medium amended with 15 mg/l ammonium nitrate, 2 mg/l IBA, and 2 % sucrose. The present study resulted in the production of 4.42-fold higher multiple shoots and 2.6-fold higher roots as well as enhancing the contents of all major withanolides (withaferin A, withanone, withanolide A and B) in regenerated plants when compared to previous reports on W. somnifera regeneration.

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Abbreviations

2,4-D:

2,4-Dichlorophenoxy acetic acid

BA:

6-Benzyladenine

DW:

Dry weight

HPLC:

High performance liquid chromatography

IAA:

Indole-3-acetic acid

IBA:

Indole-3-butyric acid

KN:

Kinetin

MS:

Murashige and Skoog

NN:

Nitsch and Nitsch

PGRs:

Plant growth regulators

References

  • Cuenca B, Vieitez M (2000) Influence of carbon source on shoot multiplication and adventitious bud regeneration in in vitro beech cultures. Plant Growth Regul 32:1–12

    Article  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 Plant 54:357–360

    Article  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  CAS  PubMed  Google Scholar 

  • Gamborg OL (1970) The effects of amino acids and ammonium on the growth of plant cells in suspension culture. Plant Physiol 45:372–375

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Heble MR (1985) Multiple shoot cultures: a viable alternative in vitro system for the production of known and new biologically active plant constituents. In: Neumann KH, Barz W, Reinhard E (eds) Primary and secondary metabolism of plant cell cultures. Springer, Berlin, pp 281–289

    Chapter  Google Scholar 

  • Jain N, Babbar SB (2003) Regeneration of ‘juvenile’ plants of black plum, Syzygium cuminii Skeels, from nodal explants of mature trees. Plant Cell Tissue Organ Cult 73:257–263

    Article  CAS  Google Scholar 

  • Jang JC, Leon P, Zhou L, Sheen J (1997) Hexokinase as a sugar sensor in higher plants. Plant Cell 9:5–19

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • 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 

  • Masuda H, Takahashi T, Sugawara S (1988) Acid and alkaline invertases in suspension cultures of sugar beet cells. Plant Physiol 86:312–317

    Article  PubMed Central  CAS  PubMed  Google Scholar 

  • Mishra LC, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (ashwagandha): a review. Altern Med Rev 5:334–346

    CAS  PubMed  Google Scholar 

  • Murashige T (1974) Plant propagation through tissue culture. Annu Rev Plant Biol 25:135–166

    Article  CAS  Google Scholar 

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

    Article  CAS  Google Scholar 

  • Murthy HN, Praveen N (2012) Influence of macro elements and nitrogen source on adventitious root growth and withanolide A production in Withania somnifera (L.) Dunal. Nat Prod Res 26:466–473

    Article  CAS  PubMed  Google Scholar 

  • Nitsch JP, Nitsch C (1969) Haploid plants from pollen grains. Science 163:85–87

    Article  CAS  PubMed  Google Scholar 

  • Patil G, Patel R, Jaat R, Pattanayak A, Jain PK, Srinivasan R (2009) Glutamine improves shoot morphogenesis in chickpea (Cicer arietinum L.). Acta Physiol Plant 31:1077–1084

    Article  CAS  Google Scholar 

  • Praveen N, Murthy HN (2011) Effects of macroelements and nitrogen source on biomass accumulation and withanolide-A production from cell suspension cultures of Withania somnifera (L.) Dunal. Plant Cell Tissue Organ Cult 104:119–124

    Article  Google Scholar 

  • Praveen N, Murthy HN (2012) Synthesis of withanolide A depends on carbon source and medium pH in hairy root cultures of Withania somnifera. Ind Crop Prod 35:241–243

    Article  CAS  Google Scholar 

  • Ray S, Jha S (2001) Production of withaferin A in shoot cultures of Withania somnifera. Planta Med 67:432–436

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  Google Scholar 

  • 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 Pharm Bull 55:1371–1375

    Article  CAS  PubMed  Google Scholar 

  • Sechley K, Yamaya T, Oaks A (1992) Compartmentation of nitrogen assimilation in higher plants. Int Rev Cytol 134:85–163

    Article  CAS  Google Scholar 

  • Selvaraj N, Vasudevan A, Manickavasagam M, Kasthurirengan S, Ganapathi A (2007) High frequency shoot regeneration from cotyledon explants of cucumber via organogenesis. Sci Hortic 112:2–8

    Article  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 

  • 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 51:161–164

    Article  CAS  Google Scholar 

  • Sharada M, Ahuja A, Vij SP (2008) Applications of biotechnology in Indian ginseng (ashwagandha): progress and prospects. In: Kumar A, Sopory SK (eds) Recent advances in plant biotechnology and its applications. I. K. International Pvt Ltd, New Delhi, pp 645–667

    Google Scholar 

  • Sivanandhan G, Mariashibu TS, Arun M, Rajesh M, Kasthurirengan S, Selvaraj N, Ganapathi A (2011) The effect of polyamines on the efficiency of multiplication and rooting of Withania somnifera (L.) Dunal and content of some withanolides in obtained plants. Acta Physiol Plant 33:2279–2288

    Article  CAS  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Mariashibu TS, Manickavasagam M, Selvaraj N, Ganapathi A (2012a) Chitosan enhances withanolides production in adventitious root cultures of Withania somnifera (L.) Dunal. Ind Crop Prod 37:124–129

    Article  CAS  Google Scholar 

  • Sivanandhan G, Arun M, Mayavan S, Rajesh M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012b) Optimization of elicitation conditions with methyl jasmonate and salicylic acid to improve the productivity of withanolides in the adventitious root culture of Withania somnifera (L.) Dunal. Appl Biochem Biotechnol 168:681–696

    Article  CAS  PubMed  Google Scholar 

  • Sivanandhan G, Rajesh M, Arun M, Jeyaraj M, Kapil Dev G, Manickavasagam M, Selvaraj N, Ganapathi A (2012c) Optimization of carbon source for hairy root growth and withaferin A and withanone production in Withania somnifera. Nat Prod Commun 7:1271–1272

    CAS  PubMed  Google Scholar 

  • Sivanandhan G, Rajesh M, Arun M, Jeyaraj M, Kapil Dev G, Arjunan A, Manickavasagam M, Muthuselvam M, Selvaraj N, Ganapathi A (2013a) Effect of culture conditions, cytokinins, methyl jasmonate and salicylic acid on the biomass accumulation and production of withanolides in multiple shoot culture of Withania somnifera (L.) Dunal using liquid culture. Acta Physiol Plant 35:715–728

    Article  CAS  Google Scholar 

  • Sivanandhan G, Kapil Dev G, Jeyaraj M, Rajesh M, Muthuselvam M, Selvaraj N, Manickavasagam M, Ganapathi A (2013b) A promising approach on biomass accumulation and withanolides production in cell suspension culture of Withania somnifera (L.) Dunal. Protoplasma 250:885–898

    Article  CAS  PubMed  Google Scholar 

  • Stepan-Sarkissian G, Fowler MW (1986) The metabolism and utilization of carbohydrates by suspension cultures of plant cells. In: Morgan MJ (ed) Carbohydrate metabolism in cultured cells. Plenum Publishing Corporation, New York, pp 151–182

    Chapter  Google Scholar 

  • Su WW (1995) Bioprocessing technology for plant cell suspension cultures. Appl Biochem Biotechnol 50:189–230

    Article  CAS  Google Scholar 

  • Vidal EA, Gutierrez RA (2008) A systems view of nitrogen nutrient and metabolite responses in arabidopsis. Curr Opin Plant Biol 11:521–529

    Article  CAS  PubMed  Google Scholar 

  • Yu PLC, El Olemy MM, Stohs ST (1974) A phytochemical investigation of Withania somnifera tissue cultures. J Nat Prod 37:593–597

    CAS  Google Scholar 

  • Zhao J, Lawrence CD, Verpoorte R (2005) Elicitor signal transduction leading to production of plant secondary metabolites. Biotechnol Adv 23:283–333

    Article  CAS  PubMed  Google Scholar 

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Acknowledgments

Prof. A. Ganapathi is thankful to University Grants Commissions (UGC), Government of India, for the award of UGC-BSR (Basic Scientific Research Fellowship). The first author gratefully acknowledges the Council of Scientific and Industrial Research (CSIR), Government of India, for the award of CSIR-SRF. The authors are thankful to Dr. R. Boopathy, Head of the Department and Dr. S. Girija, Associate Professor, Department of Biotechnology, Bharathiar University, Coimbatore for providing HPLC facility.

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Authors and Affiliations

  1. Department of Biotechnology and Genetic Engineering, School of Biotechnology, Bharathidasan University, Tiruchirappalli, 620 024, Tamil Nadu, India

    Ganeshan Sivanandhan, Andy Ganapathi & Markandan Manickavasagam

  2. Department of Botany, Periyar E. V. R College (Autonomous), Tiruchirappalli, 620 023, Tamil Nadu, India

    Natesan Selvaraj

Authors
  1. Ganeshan Sivanandhan
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  2. Natesan Selvaraj
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  3. Andy Ganapathi
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  4. Markandan Manickavasagam
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Correspondence to Ganeshan Sivanandhan or Markandan Manickavasagam.

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Communicated by K.Y. Paek.

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Sivanandhan, G., Selvaraj, N., Ganapathi, A. et al. Effect of nitrogen and carbon sources on in vitro shoot multiplication, root induction and withanolides content in Withania somnifera (L.) Dunal. Acta Physiol Plant 37, 12 (2015). https://doi.org/10.1007/s11738-014-1758-7

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  • DOI: https://doi.org/10.1007/s11738-014-1758-7

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