Indirect organogenesis and plant regeneration in Helicteres isora L., an important medicinal plant

  • Varsha Shriram
  • Vinay Kumar
  • Mahadeo G. ShitoleEmail author


Helicteres isora is a medicinal plant effective against asthma, diabetes, hypolipidemia, HIV, polio besides a good source of diosgenin. Seed dormancy and low natural fruit production rate make this plant a perfect candidate for developing an in vitro regeneration method. However, to date, no such work has been procured in this plant. An efficient method for plant regeneration via shoot organogenesis from callus cultures has been developed using nodal explants in H. isora. Murashige and Skoog (MS) media counting 2,4-Dichlorophenoxyacetic acid (2,4-D, 2.26 to 13.57 μM), Indole-3-acetic acid (IAA, 2.85 to 17.13 μM), Indole-3-butyric acid (IBA, 2.46 to 14.70 μM), 6-Benzylaminopurine (BA, 2.22 to 13.32 μM) and Kinetin (Kin, 2.32 to 13.92 μM) either singly or in the following combinations (IAA + BA; IAA + Kin, and BA + Kin) produced granular callus except BA + Kin which resulted in compact, hard, greenish-white (CHGW) callus. The optimum CHGW callus (2.62 g fresh weight/ explant) was produced on MS media with 13.32 μM BA + 2.32 μM Kin with over 93% callus induction frequency. Optimum shoot organogenesis (67% frequency) was achieved in CHGW callus with lower level of BA (2.22 μM) and Kin (2.32 μM) and produced 3.2 shoots/0.5 g callus within 35 d of culture. Microshoots were rooted successfully (62% frequency) after 35 d of culture on 1/2MS containing 4.90 μM IBA and hardened off. Antioxidant enzymes such as catalase, peroxidase, polyphenol oxidase, and biochemical parameters viz. hydrogen peroxide, reducing and nonreducing sugars, starch, proteins, phenols, and proline contents were studied in regenerating and nonregenerating CHGW calluses to establish a correlation between these parameters and shoot morphogenesis. All the enzyme activities and biochemical parameters were found more in regenerating callus than in nonregenerating except phenols.


Helicteres isora Anti-HIV Antidiabetic Antioxidant enzymes Organogenesis 


  1. Anonymous (1959) The wealth of India: raw material. CSIR, New Delhi, Vol. V, pp 27–29.Google Scholar
  2. Atluri, J. B.; Rao, S. P. and Reddi, C. S. Pollination ecology of Helicteres isora Linn (Sterculiaceae). Curr Sci. 78(6): 713–718; 2000.Google Scholar
  3. Badave, G. N. and Jadhav, S. C. Germination studies in local plants from Koyana Valley, I: Murudsheng- Helicteres isora L. Ayurveda Update. 1(2): 10; 1998.Google Scholar
  4. Barik, B.; Dey, A. K. and Das, P. C. Helicteres isora Linn, a new source of diosgenin. Indian J Chem. 20(B): 938; 1998.Google Scholar
  5. Bates, L. S.; Waldren, R. P. and Teara, I. D. Rapid determination of free proline for water stress studies. Plant Soil. 39: 205–207 1973.CrossRefGoogle Scholar
  6. Bean, M. F.; Antoun, M.; Abramson, D.; Chang, C. J.; McLaughlin, J. L. and Cassady, J. M. Cucurbitacin B and isocucurbitacin B: cytotoxic components of Helicteres isora. J Nat Prod. 48(3): 500; 1985.PubMedCrossRefGoogle Scholar
  7. Benson, E. E. Do free radicals have a role in plant tissue culture recalciterance? In vitro Cell Dev Biol – Plant. 36: 163–170; 2000.CrossRefGoogle Scholar
  8. Chakrabarti, R.; Vikramadithyan, R. K.; Mullangi, R.; Sharma, V. M.; Jagadheshan, H.; Rao, Y. N.; Sairam, P. and Rajagopalan, R. Antidiabetic and hypolipidemic activity of Helicteres isora in animal models. J Ethnopharmacol. 81(3): 343–349; 2002.PubMedCrossRefGoogle Scholar
  9. Chen, J. and Ziv, M. The effect of ancymidol on hyperhydricity, regeneration, starch and antioxidant enzymatic activities in liquid cultured Narcissus. Plant Cell Rep. 22: 22–27; 2001.CrossRefGoogle Scholar
  10. Esterbauer, H.; Schwarz, E. and Hayn, M. A rapid assay for catechol oxidase and laccase using 2-nitro-5-thio benzoic acid. Anal Biochem. 77: 486–494; 1977.PubMedCrossRefGoogle Scholar
  11. Fracaro, F. and Echeverrigaray, S. Micropropagation of Cunila galioides, a popular medicinal plant of south Brazil. Plant Cell Tiss Org Cult. 64: 1–4; 2001.CrossRefGoogle Scholar
  12. Gasper, T. The concept of cancer in in-vitro plant cultures and the implication of habituation to hormones and hyper hydricity. Plant Tiss Cult Biotechnol. 1: 126–136; 1995.Google Scholar
  13. Gupta, S. D. and Datta, S. Antioxidant enzyme activities during in in vitro morphogenesis of gladiolus and the effect of application of antioxidants on plant regeneration. Biol Plantarum. 47(2): 179–183; 2004.CrossRefGoogle Scholar
  14. Hedge, J. E. and Hofreiter, B. T. Methods of estimating starch and carbohydrates. In: Whistler, R. L. and Miller, J. N. (eds.) Carbohydrate Chemistry for Food Scientists. Eagan Press, St. Paul Minn; pp 163–201. 1962.Google Scholar
  15. Huang, W. L. and Liu, L. F. Carbohydrate metabolism in rice during callus induction and shoot regeneration induced by osmotic stress. Bot Bull Acad Sinica. 43(2): 107–113; 2002.Google Scholar
  16. Joshy, M. K.; Mathew, L. and Joseph, R. Studies on short isora fibre-reinforced polyester composites. Composite Interfaces. 13(4): 377–390; 2006.CrossRefGoogle Scholar
  17. Komalavalli, N. and Rao, M. V. In vitro micropropagation of Gymnema sylvestre – A multipurpose medicinal plant. Plant Cell Tiss Org Cult. 61: 97–105; 2000.CrossRefGoogle Scholar
  18. Kumar, G.; Murugesan, A. G. and Pandian, M. R. Effect of Helicteres isora barks extract on blood glucose and hepatic enzymes in experimental diabetes. Pharmazie. 61(4): 353–355; 2006.PubMedGoogle Scholar
  19. Kusumoto, I. T.; Shimada, I.; Kakiuchi, N.; Hattori, N.; Namba, T. and Supriyatna S Inhibitory effects of Indonesian plant extracts of reverse transcriptase of an RNA tumor virus- I. Phytother Res. 6 (5): 241-244; 1992.CrossRefGoogle Scholar
  20. Larson, R. A. The antioxidants of higher plants. Phytochemistry 27: 969–978; 1988.CrossRefGoogle Scholar
  21. Laukkanen, H.; Haggman, H.; Soppela, S. K. and Hohtola, A. Tissue browning of in vitro cultures of Scots pine: role of peroxidase and polyphenol oxidase. Physiol Plant. 106: 337-343; 1999.CrossRefGoogle Scholar
  22. Libik, M.; Konieczny, R.; Pater, B.; Slesak, I. and Miszalski, Z. Differences in the activities of some antioxidant enzymes and in H2O2 content during rhizogenesis and somatic embryogenesis in callus cultures of the ice plant. Plant Cell Rep. 23: 834–841; 2005.PubMedCrossRefGoogle Scholar
  23. Lowry, O. H.; Rosebrough, N. J.; Farr, A. L. and Randall, R. J. Protein measurement with the folin phenol reagent. J Biol Chem. 193: 265–75; 1951.PubMedGoogle Scholar
  24. Luck, H. In: Bergmeyer HU, Gawehn K (eds) Methods of Enzymatic Analysis 2. Academic Press, New York; pp 885–894. 1974.Google Scholar
  25. Malick, C. P. and Singh, M. B. Plant enzymology and histoenzymology. Kalyani Publishers, New Delhi. 1980.Google Scholar
  26. Marks, T. R. and Simpson, S. E. Factors affecting shoot development in apically dominant Acer cultivars in in vitro. J Hort Sci. 69: 543-551; 1994.Google Scholar
  27. Martin, K. P. Rapid in vitro multiplication and ex vitro rooting of Rotula aquatica Lour, a rare rhoeophytic woody medicinal plant. Plant Cell Rep. 21: 415-420; 2003.PubMedGoogle Scholar
  28. Miller, G. L. Use of dinitro salicylic acid reagent for determination of reducing sugar. Anal Chem. 31: 426; 1972.CrossRefGoogle Scholar
  29. Murashige, T. and Skoog, F. A revised medium for rapid growth and bioassays with tobacco tissue culture. Physiol Plant. 15: 473–479; 1962.CrossRefGoogle Scholar
  30. Otake, T.; Mori, H.; Morimoto, M.; Ueba, N.; Sutardjo, S.; Kusomoto, I.; Hattori, M. and Namba, T. Screening of Indonesian plant extracts for anti human immuno deficiency virus type1 (HIV-1) activity. Phytother Res. 9(1): 6–10; 1995.CrossRefGoogle Scholar
  31. Pohocha, N. and Grampurohit, N. D. Antispasmodic activity of the fruits of Helicteres isora Linn. Phytother Res. 15(1): 49–52; 2001.PubMedCrossRefGoogle Scholar
  32. Preece, J. E.; Hutterman, C. A.; Ashby, W. C. and Roth, P. L. Micro and cutting propagation of silver maple. I. Results with adult and juvenile propagules. J Am Soc Hortic Sci. 116: 142–148; 1991.Google Scholar
  33. Purohit, S. D. and Dave, A. Micropropagation of Sterculia urens Roxb.: An endangered tree species. Plant Cell Rep. 15(9): 704–706; 1996.CrossRefGoogle Scholar
  34. Purohit, S. D.; Singhavi, A. and Tak, K. Biochemical characteristics of differentiating callus cultures of Feronia limonia L. Acta Physiol Plant. 18(1): 47–52; 1996.Google Scholar
  35. Putter, J. In: Bergmeyer HU, Gawehn K (eds) Methods of Enzymatic Analysis 2. Academic Press, New York; pp 685. 1974.Google Scholar
  36. Qin, Y.; Zhang, S.; Zhang, L.; Zhu, D. and Syed, A. Response of in vitro strawberry to silver nitrate (AgNO3). Hortscience. 40(3): 747–751; 2005.Google Scholar
  37. Saini, R. and Jaiswal, P. K. In In vitro multiplication of Peganum harmala – an important medicinal plant. Indian J Exp Biol. 38: 499–503; 2000.PubMedGoogle Scholar
  38. Thaker, J. and Bhargava, S. Seasonal variation in antioxidant enzymes and the sprouting response of Gmelina arborea Roxb nodal sectors cultures in in vitro. Plant Cell Tiss Org Cult. 59: 181–187; 1999.CrossRefGoogle Scholar
  39. Venkatesh, S.; Reddy, G. D.; Reddy, Y. S.; Sathyavathy, D. and Reddy, B. M. Effect of Helicteres isora root extracts on glucose tolerance in glucose-induced hyperglycemic rats. Fitoterapia. 75(3–4): 364–367; 2004.PubMedCrossRefGoogle Scholar

Copyright information

© The Society for In Vitro Biology 2008

Authors and Affiliations

  • Varsha Shriram
    • 1
    • 2
  • Vinay Kumar
    • 1
  • Mahadeo G. Shitole
    • 1
    Email author
  1. 1.Department of BotanyUniversity of PunePuneIndia
  2. 2.Department of BotanyAnnasaheb Magar CollegePuneIndia

Personalised recommendations