• B. JANARTHANAM Poonga Biotech Research Centre
  • E. SUMATHI University of Madras



Objective:  The objective of the present study was to evaluate the optimization of callus biomass culture yield and high-performance liquid chromatography (HPLC) analysis of
L-DOPA compound in the callus culture from cotyledonary leaves of Mucuna pruriens

Methods : M. pruriens seed explants were inoculated onto Murashige and Skoog medium supplemented with different concentrations of 2-isopentenyl adenine (2iP) and Gibberellic acid (GA3)  for germination of plants. The in vitro cotyledonary leaf and hypocotyl explants were cultured on MS basal media containing various concentrations of 2,4-dichlorophenoxyacetic acid (2,4-D), 1- Naphthaleneacetic acid (NAA), 6-Benzylaminopurine (BA) and 2iP for callus induction. A standard approach of Latin square method was followed for screening of media to establish optimum culturing of callus by manipulating the concentration of auxins (2, 4-D, Indole-3-acetic acid (IAA) and NAA) and cytokinins (BA and 2iP) alone and in combinations. The harvested callus biomass was screened for a major metabolite namely L- Dopa compound using HPLC.

Results: Cotyledonary leaf explants showed better callus initiation than hypocotyl explants. Maximum callus induction was observed in Murashige and Skoog (MS) medium containing 4.92µM 2iP. Further screening of callus culture was carried out on MS medium supplemented with different concentrations and combinations of 2, 4-D, NAA,  IAA, (BA)  and 2iP individually and in combinations. Optimum callus biomass of 21.63 g/L dry weight (246.31 g/L fresh weight) was developed on MS media containing 2.26µM 2, 4-D, 2.22µM BA and 4.92µM 2iP. The harvested callus biomass was subjected to extraction and purification of L- Dopa compound.

Conclusion: The present study concludes that  HPLC analysis of cell biomass extracts in comparison  with extracts from seeds of mother plants of Mucuna prurienss showed main component of L- Dopa was present in sufficiently large amounts in the undifferentiated cultured cells.

Keywords: Mucuna pruriens, Callus biomass, L-Dopa, HPLC analysis

Author Biographies

B. JANARTHANAM, Poonga Biotech Research Centre

Plant Biotechnology Division,

E. SUMATHI, University of Madras

National Centre for Nanosciences and Nanotechnology


1. Singh U, Wadhwani AM, Johri BM. Dictionary of economic plants in India. Indian Council of Agricultural Research, New Delhi.1996; 45-46.

2. Vadivel V, Janardhanan, K. Nutritional and antinutritional composition of velvet bean: an under-utilized food legume in South India, International Journal of Food Science and Nutrition. 2000; 51: 279-287.

3. Anon. The Wealth of India: A Dictionary of Indian Raw Materials and Industrial Products, New Delhi, India: CSIR, 1962;439-444.

4. Ali S, Haq I, Qadeer MA, Rajoka MI. Double mutant of Aspergillus oryzae for improved production of L-dopa (3,4- dihydroxyphenyl-L-alanine) from L-tyrosine. Biotechnol. Appl. Biochem 2005;42:143-149.

5. Bapat VA, Suprasanna P, Ganapathi TR, Rao PS. In vitro production of L-DOPA in tissue cultures of banana. Pharm Biol 2000; 38: 271–273.

6. Caius JF. The Medicinal and Poisonous Legumes of India. Jodhpur, India: Scientific Publishers. 1989; 70-71.

7. Farooqi AA, Khan MM, Asundhara M. Production Technology of Medicinal and Aromatic Crops, Bangalore, India: Natural Remedies Pvt. Ltd 1999; 26–28.

8. Verma DM, Balakrishnan NP, Dixit RD. Flora of Madhya Pradesh, Lucknow, India: Botanical Survey of India 1993; 190–191.

9. Krishnamurthy RP, Suvagia P, Pathak JM, Parikh KM, Bhikne NS. Selection of Mucuna lines for higher L-Dopa and improved yield through conventional breeding. In Proceedings of the Conference on Breeding Research on Medicinal Plants. Quedlinburg, Germany. Bundesanstalt-fur Zuchtungsforschung an Kulturpflanzen 1996; 2, 231–234.

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

11. Collin H. A. and Edwards S. The plant Cell Tissue Culture. In: D. Rickwood and C. Howe (ed.). Bios scientific publishers Limited, Oxford, UK), 1998; 32.

12. Janarthanam B, Gopalkrishnan M, Sekar T. Secondary metabolite production in callus cultures of Stevia redaudiana. Bangaldesh journal of science industrial research 2010; 45(3): 243-248.

13. Janarthanam B, Seshadri S. In vitro manipulations of Rosa bourboniana L. Acta Hort. 2008; 357-370.

14. Myhrman R. Detection and removal of L-DOPA in the legume Mucuna. In M. Flores, M. Eilitta¨ , R. Myhrman, L. Carew, & R. Carsky (Eds.), Mucuna as a food and feed: Current uses and the way forward. Proceedings in an international workshop, Tegucigalpa, Honduras: CIDICCO 2002; 142–163.

15. Selvaraj S, Chittibabu CV, Janarthanam B. Studies on phytochemical screening, antioxidant activity and extraction of active compound (swertiamarin) from leaf extract of Enicostemma littorale. Asian J Pharm Clin Res 2014; 7 (4): 240-244.

16. Khafagi IK. Variation of callus induction and active metabolite accumulation in callus cultures of two varieties of (Ricinus communis L). Biotechnology 2007; 6: 193-201.

17. Bhagya N Chandrashekar KR. Effect of growth regulators on callus induction from Cyclea peltata (Lam.) Hook. F. Thoms. Asian J Pharm Clin Res 2013;6(4) 85-88

18. Janarthanam B, Gopalkrishnan M, Sai GL, Sekar T. Plant regeneration from leaf derived callus of Stevia rebaudiana Bertoni. Plant Tiss. Cult. Biotech 2009; 19(2): 133-141.

19. Thomas TD, Maseena EA. Callus and plant regeneration in Cardiospermum halicacabum Linn. An important medicinal plant, Sci. Hortic 2006; 108: 332 -336

20. Reddy PS, Gopal GR, Sita GL. In vitro multiplication of Gymnema sylvestre R.Br. -An important medicinal plant. Curr. Sci 1998; 75: 843-845.

21. Gopi C, Vatsala TM. In vitro studies in effects of plant growth regulators on callus and suspension culture biomass yield from Gymnema sylvestre R. Br. Afr J Biotechnol (2006);5(12):1215–1219.

22. Siregar LAM, Keng LC, Lim PB. Selection of Cell Source and the effect of pH and MS Macronutrients on Biomass Production in cell cultures of Tongkat Ali (Euryocoma Longifoli Jack). J. Plant Biotechnology 2003; 5:131-135.

23. Brain KR. Accumulation of L‐DOPA in cultures from Mucuna pruriens. Plant Sci. Lett. 1976; 7: 157‐161.

24. Huizing HJ, Wijnsma R, Batterman S, Malingre Th M, Wichers HJ. Production of L-DOPA by cell suspension cultures of Mucuna pruriens. I. Initiation and maintenance of cell suspension cultures of Mucuna pruriens and identification of L-DOPA. Plant Cell Tissue Organ Cult. 1985;4: 61–73.

25. Wichers HJ, Visser JF, Huizing HJ, Pras N. Occurrence of L‐DOPA and dopamine in plants and cell cultures of Mucuna pruriens and effects of 2,4‐D and NaCl on these compounds. Plant Cell Tiss. Org. Cult. 1993; 33: 259‐264.
348 Views | 759 Downloads
How to Cite
JANARTHANAM, B., and E. SUMATHI. “OPTIMIZATION OF BIOMASS CULTURE YIELD AND L-DOPA COMPOUND IN THE CALLUS CULTURE FROM COTYLEDONARY LEAVES OF MUCUNA PRURIENS”. Asian Journal of Pharmaceutical and Clinical Research, Vol. 8, no. 4, July 2015, pp. 282-6, https://innovareacademics.in/journals/index.php/ajpcr/article/view/6680.
Original Article(s)