In vitro conservation of Bacopa monnieri (L.) using mineral oil

Abstract

Bacopa monnieri (L.) Wettst., a traditional Indian medicinal plant with high commercial potential, is used as a potent nervine tonic. A slow growth protocol was developed for medium-term conservation using mineral oil (MO) overlay. Nodal segments of B. monnieri (two genotypes; IC249250, IC468878) were conserved using MO for 24 months. Single node explants were implanted on MS medium supplemented with 0.2 mg l−1 BA and were covered with MO. Subculture duration could be significantly enhanced from 6 to 24 months, on the above medium. Normal plants regenerated from conserved cultures were successfully established in soil. On the basis of 20 random amplified polymorphic DNA and 5 inter-simple sequence repeat primers analyses and bacoside A content using HPLC, no significant reproducible variation was observed between the controls and in vitro-conserved plants. The results demonstrate the feasibility of using MO for medium-term conservation of B. monnieri germplasm without any adverse genetical and biochemical effects.

This is a preview of subscription content, access via your institution.

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6

Abbreviations

BA:

6-Benzyladenine

MO:

Mineral oil

MS:

Murashige and Skoog’s medium

NS:

Nodal segment

UPGMA:

Unweighted pair group method with arithmetic mean

RAPD:

Random amplified polymorphic DNA

ISSR:

Inter-simple sequence repeat

References

  1. Anonymous (1988) The wealth of India: raw materials, vol 2. Council of Scientific and Industrial Research, New Delhi

    Google Scholar 

  2. Augereau JM, Courtois D, Petiard V (1986) Long term storage of callus cultures at low temperatures or under mineral oil overlayer. Plant Cell Rep 5:372–376

    Article  Google Scholar 

  3. Caplin SM (1959) Mineral oil for conservation of plant tissue cultures. Am J Bot 46:324–329

    Article  Google Scholar 

  4. Ceasar AS, Maxwell SL, Prasad KB, Karthigan M, Ignacimuthu S (2010) Highly efficient shoot regeneration of Bacopa monnieri (L.) using a two stage culture procedure and assessment of genetic integrity of micropropagated plants by RAPD. Acta Physiol Plant 32:442–452

    Google Scholar 

  5. Chandel KPS, Shukla G, Sharma N (1996) Biodiversity in medicinal and aromatic plants in India. National Bureau of Plant Genetic Resources, New Delhi

    Google Scholar 

  6. Deepak M, Sangli GK, Arun PC, Amit A (2005) Quantitative determination of major saponin mixture Bacoside A in Bacopa monnieri. Phytochem Anal 16:24–29

    PubMed  Article  CAS  Google Scholar 

  7. Dekkers AJ, Rao AN, Goh CJ (1991) In vitro storage of multiple shoot cultures of gingers at ambient temperatures of 24–29 °C. Sci Hort 47:157–167

    Article  Google Scholar 

  8. Elangovan V, Govindasamy S, Ramamoorthy N, Balasubramanian K (1995) In vitro studies on the anticancer activity of Bacopa monnieri. Fitoterapia 66:211–215

    Google Scholar 

  9. Engelmann F (1997) In vitro conservation methods. In: Callow JA, Ford-Lloyd BV, Newbury HJ (eds) Biotechnology and plant genetic resources- conservation and use, biotechnology in agriculture series, no. 19, CAB International, pp 119–161

  10. Jayanthi M, Mandal PK (2001) Plant regeneration through somatic embryogenesis and RAPD analysis of regenerated plants in Tylophora indica (Brm.F.Merrii). In Vitro Cell Dev Biol Plant 37:576–580

    Article  CAS  Google Scholar 

  11. Karki M, Williams JT (1999) Priority species of medicinal plants in south asia. In: Medicinal and Aromatic Plants Program in Asia (MAPPA), International Development Research Center (IDRC), South Asian Regional Office (SARO)—A report of an expert consultation on medicinal plant species prioritization for South Asia, New Delhi, India, pp 1–63

  12. Lattoo SK, Bamotra S, Sapru DR, Khan S, Dhar AK (2006) Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—an endangered medicinal herb. Plant Cell Rep 25:499–506

    PubMed  Article  CAS  Google Scholar 

  13. Mandal BB, Tyagi RK, Pandey R, Sharma N, Agarwal A (2000) In vitro conservation of germplasm of agri-horticultural crops at NBPGR: an overview. In: Razdan MK, Cocking EC (eds) Conservation of plant genetic resources in vitro. Science Publishers, Inc, USA, pp 297–307

    Google Scholar 

  14. Mannonen L, Toivonen L, Kauppienen V (1990) Effects of long-term preservation on growth and productivity of Panax ginseng and Catharanthus roseus cell cultures. Plant Cell Rep 9:173–177

    Article  CAS  Google Scholar 

  15. Mathur S, Kumar S (1998) Phytohormone self sufficiency for regeneration in the leaf and stem explants of Bacopa monnieri. J Med Arom Plant Sci 20:1056–1059

    Google Scholar 

  16. Mathur J, Mukunthakumar S, Gupta SN, Mathur SN (1991) Growth and morphogenesis of plant tissue cultures under mineral-oil. Plant Sci 74:249–254

    Article  Google Scholar 

  17. Mishra J, Singh M, Palni LMS, Nandi SK (2011) Assessment of genetic fidelity of encapsulated microshoots of Picrorhiza kurrooa. Plant Cell Tiss Org Cult 104:181–186

    Article  Google Scholar 

  18. Mohanty S, Parida R, Singh S, Joshi RK, Subudhi E, Nayak S (2011) Biochemical and molecular profiling of micropropagated and conventionally grown Kaempferia galanga. Plant Cell Tiss Org Cult 106:39–46

    Article  CAS  Google Scholar 

  19. Moriguchi T, Kozaki I, Matsuta N, Yamaki S (1988) Plant regeneration from grape callus stored under a combination of low temperature and silicone treatment. Plant Cell Tiss Org Cult 15:67–71

    Article  Google Scholar 

  20. 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 

  21. Rastogi S, Pal R, Kulshreshtha DK (1994) Bacoside A3 – A triterpenoid saponin from Bacopa monniera. Phytochemistry 36:133–137

    PubMed  Article  CAS  Google Scholar 

  22. Rout GR, Das G (2002) An assessment of genetic integrity of micropropagated plants of Plumbago zeylanica by RAPD markers. Biol Plant 45:27–32

    Article  CAS  Google Scholar 

  23. Saghai-Maroof MA, Soliman KM, Jorgensen RA, Allard RW (1984) Ribosomal DNA spacer-length polymorphisms in barley: mendelian inheritance, chromosomal location, and population dynamics. Proc Natl Acad Sci USA 81:8014–8018

    PubMed  Article  CAS  Google Scholar 

  24. Sharma N, Sharma B (2003) Cryopreservation of shoot tips of Picrorrhiza kurroa Royle ex Benth., an indigenous endangered medicinal plant through vitrification. CryoLetters 24:181–190

    PubMed  Google Scholar 

  25. Sharma N, Satsangi R, Pandey R, Vimala Devi S (2007a) In vitro clonal propagation and medium term conservation of Brahmi [Bacopa monnieri (L.) Wettst.]. J Plant Biochem Biotechnol 16:139–144

    CAS  Google Scholar 

  26. Sharma N, Vimala Devi S, Satsangi R (2007b) Biotechnological approaches in multiplication and conservation of plants of medicinal value. In: Shukla PK, Chaubey OP (eds) Threatened wild medicinal plants: assessment, conservation and management. Anmol Publications Pvt Ltd., New Delhi, pp 248–257

    Google Scholar 

  27. Singh HK, Dhawan BN (1997) Neurophycopharmacological effects of the ayurvedic nootropic Bacopa monniera Linn (Brahmi). Indian J Pharmacol 29:8359–8365

    Google Scholar 

  28. Singh S, Kuanar A, Mohanty S, Subudhi S, Nayak S (2011) Evaluation of phytomedicinal yield potential and molecular profiling of micropropagated and conventionally grown turmeric (Curcuma longa L.). Plant Cell Tiss Org Cult 104:263–269

    Article  CAS  Google Scholar 

  29. Starisky G, Dekkers AJ, Louwaars NP, Zandvoort EA (1986) In vitro conservation of aroid germplasm at reduced temperatures and osmotic stress. In: Withers LA, Alderson PG (eds) Plant tissue culture and its agricultural applications. Butterworths, London, pp 277–283

    Google Scholar 

  30. Tejavathi DH, Rao NN (1998) Comparative studies between normal and tissue cultured plants of Bacopa monnieri (L.) Pennell. In: Khan IA, Khanum A (eds) Role of biotechnology in medicinal and aromatic plants, vol I. Ukaaz Publications, Hyderabad, pp 239–247

    Google Scholar 

  31. Tiwari V, Tiwari KN, Singh BD (2001) Comparative studies of cytokinins on in vitro propagation of Bacopa monniera. Plant Cell Tiss Org Cult 66:9–16

    Article  CAS  Google Scholar 

Download references

Acknowledgments

We thank Director, National Bureau of Plant Genetic Resources, New Delhi for facilities and encouragement. Thanks are due to Sh. Ramesh Chandra for technical assistance. Financial support provided by the Department of Biotechnology, Ministry of Science and Technology, is gratefully acknowledged.

Author information

Affiliations

Authors

Corresponding author

Correspondence to Neelam Sharma.

Rights and permissions

Reprints and Permissions

About this article

Cite this article

Sharma, N., Satsangi, R., Pandey, R. et al. In vitro conservation of Bacopa monnieri (L.) using mineral oil. Plant Cell Tiss Organ Cult 111, 291–301 (2012). https://doi.org/10.1007/s11240-012-0194-x

Download citation

Keywords

  • Bacopa
  • Bacoside A
  • ‘Brahmi’
  • Genetic stability
  • ISSR
  • Medicinal plant
  • Medium-term conservation
  • Mineral oil overlay
  • RAPD
  • Slow growth