Indian Journal of Plant Physiology

, Volume 21, Issue 1, pp 1–7 | Cite as

Establishment and standardization of in vitro regeneration protocol in Nothapodytes nimmoniana Graham and evaluation of camptothecine (CPT) in tissue culture plants

  • Ugraiah Amilineni
  • Vinod Gangal
  • Ravikanth Gudasalamani
  • Nataraja N. Karaba
  • Uma Shaanker Ramanan
Original Article

Abstract

Nothapodytes nimmoniana Graham is an important medicinal tree species occurring in the Western Ghats, a mega diversity hotspot in Southern India. The inner stem bark of the tree contains an important anti-cancer alkaloid, camptothecine (CTP), for which the natural population of the trees is heavily extracted. In this paper, we report the development of a rapid, high frequency regeneration protocol from leaf and nodal explants of N. nimmoniana. Multiple shoot induction was carried out using leaf and nodal explants on Murashige and Skoog (MS) medium supplemented with different concentration/combination of phyto-hormones. N6-benzyladenine (BA) was the most effective cytokinin for the induction of multiple shoots. The MS medium with 8.87 µM BA yielded the highest number of shoots from leaf and nodal explants, respectively. Further, proliferation and elongation of adventitious buds were observed in secondary medium containing MS supplemented with 4.44 µM BA and 0.87 µM gibberellic acid (GA3). Shoots were rooted on half strength MS medium containing 4.9 µM indole-3-butyric acid. The plantlets were acclimatized in a growth chamber at 25 °C, 60 % relative humidity, with 16/8 h light/dark photoperiod. Regenerated plants were free of any noticeable phenotypic variability and showed a survival rate of 90 %. The in vitro regenerated plants accumulated substantial amount of camptothecine (ranging from 0.08 to 0.2 %). These results suggest the possibility of using in vitro regenerated plants as a possible alternative source of CPT. This is the first report of direct regeneration in N. nimmoniana with significantly high plant regeneration frequency and with high CPT yield.

Keywords

Leaf and nodal explants Nothapodytes nimmoniana Propagation Camptothecine 

References

  1. Ciddi, V., & Shuler, M. L. (2000). Camptothecine from callus culture of Nothapodytes foetida. Biotechnology Letters, 22, 129–132.CrossRefGoogle Scholar
  2. Dandin, V. S., & Murthy, H. N. (2012). Enhanced in vitro multiplication of Nothapodytes nimmoniana Graham using semisolid and liquid cultures and estimation of camptothecine in the regenerated plants. Acta Physiologiae Plantarum, 34, 1381–1386. doi:10.1007/s11738-012-0934.CrossRefGoogle Scholar
  3. Emmanuel, S., Ignacimuthu, S., & Kathiravan, K. (2000). Micropropagation of Wedelia calendulacea Less., a medicinal plant. Phytomorphology, 50(2), 195–200.Google Scholar
  4. Gomez, K. A., & Gomez, A. A. (1976). Statistical procedures for agricultural research with emphasis on rice. Los Banos: International Rice Research Institute.Google Scholar
  5. Govindachari, T. R., & Viswanathan, N. (1972). Alkaloids of Mappia foetida. Phytochemistry, 11, 3529–3531.CrossRefGoogle Scholar
  6. Hombe Gowda, H. C., Vasudeva, R., Georgi, P. M., Uma, S. R., & Ganeshaiah, K. N. (2002). Breeding types in Nothapodytes nimmoniana Graham. Current Science, 83, 1077–1078.Google Scholar
  7. Hsiang, Y. H., Hertzberg, R., Hecht, S., & Liu, L. F. (1985). Camptothecin induces protein-linked DNA breaks via mammalian DNA topoisomerase-I. Journal of Biological Chemistry, 260(27), 14873–14878.PubMedGoogle Scholar
  8. Kai, G., Dai, L., Mei, X., Zheng, J., Wang, W., Qian, Z., & Zhou, G. (2008). In vitro plant regeneration from leaf explants of Ophiorrhiza japonica. Biologia Plantarum, 52(3), 557–560.CrossRefGoogle Scholar
  9. Kathiravan, K., & Ignacimuthu, S. (1999). Micropropagation of Canavalia virosa (ROXB) Wight & Arn. A medicinal plant. Phytomorphology, 49, 61–66.Google Scholar
  10. Kawiak, A., Krolicka, A., & Lojkowska, E. (2003). Direct regeneration of Drosera from leaf explants and shoot tips. Plant Cell, Tissue and Organ Culture, 75, 175–178.CrossRefGoogle Scholar
  11. Lattoo, S. K., Bamotra, S., SapruDhar, R., & Khan, S. (2006). Rapid plant regeneration and analysis of genetic fidelity of in vitro derived plants of Chlorophytum arundinaceum Baker—An endangered medicinal herb. Plant Cell Reports, 25, 499–506. doi:10.1007/s00299-005-0103-4.CrossRefPubMedGoogle Scholar
  12. Lorence, A., & Craig L. N. (2004) Camptothecin: over four decades of surprising findings. Phytochemistry, 65(20), 2731–2841.CrossRefGoogle Scholar
  13. Lorence, A., Medina, B. F., & Nessler, C. L. (2004). Camptothecin and 10-hydroxycamptothecin from Camptotheca acuminata hairy roots. Plant Cell Reports, 22, 437–441.CrossRefPubMedGoogle Scholar
  14. Martin, K. P., Sunandakumari, C., Chithra, M., & Madhusoodanan, P. V. (2005). Influence of auxins in direct in vitro morphogenesis of Euphorbia nivulia, a lectinacious medicinal plant. In Vitro Cellular & Developmental Biology-Plant, 41(3), 314–319.CrossRefGoogle Scholar
  15. Murashige, T., & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum, 15, 473–497.CrossRefGoogle Scholar
  16. Namdeo, A. G., Priya, T., & Bhosale, B. B. (2012). Micropropagation and production of camptothecine from in vitro plants of Ophiorrhiza mungos. Asian Pacific Journal of Tropical Biomedicine, 2(2 supplement), S662–S666.CrossRefGoogle Scholar
  17. Neeti, D., & Kothari, S. L. (2005). Micropropagation of Eclipta alba (L.) Hassk—An important medicinal plant. In Vitro Cellular & Developmental Biology-Plant, 41, 658–6661.CrossRefGoogle Scholar
  18. Padmanabha, B. V., Chandrashekar, M., Ramesha, B. T., Hombe, G. H. C., Gunaga, R. P., Suhas, S., et al. (2006). Pattern of accumulation of Camptothecin, an anticancer alkaloid in Nothapodytes nimmoniana Graham. In the Western Ghats, India: Implications for identifying high yielding sources of alkaloid. Current Science, 90(1), 95–99.Google Scholar
  19. Priel, E., Showalter, S. D., & Blair, D. G. (1991). Inhibition of human immunodeficiency virus (HIV-I) replication in vitro by noncytotoxic doses of camptothecine, a topoisomerase I inhibitor. AIDS Research and Human Retroviruses, 7, 65–72.PubMedGoogle Scholar
  20. Purohit, D., & Dave, A. (1996). Micropropagation of Sterculia aurens Roxb.—An endangered tree species. Plant Cell Reports, 15, 704–706.CrossRefPubMedGoogle Scholar
  21. Rai, R. V. (2002). Rapid clonal propagation of Nothapodytes foetida (Wight) Sleumer—A threatened medicinal tree. In Vitro Cellular & Developmental Biology-Plant, 38, 347–351.CrossRefGoogle Scholar
  22. Ramadevi, T., Ugraiah, A., & Pullaiah, T. (2012). In vitro shoot multiplication from nodal explants of Boucerosia diffusa Wight—An endemic medicinal plant. Indian Journal of Biotechnology, 11, 344–347.Google Scholar
  23. Ramesha, B. T., Amna, T., Ravikanth, G., Rajesh, P. G., Vasudeva, R., Ganeshaiah, K. N., et al. (2008). Prospecting for camptothecine from Nothapodytes nimmoniana in the Western Ghats, South India: Identification of yielding sources of camptothecine and new families of camptothecines. Journal of Chromatographic Science, 46, 362–368.CrossRefPubMedGoogle Scholar
  24. Ramesha, B. T., Suma, H. K., Senthilkumar, U., Priti, V., Ravikanth, G., Vasudeva, R., et al. (2013). New plant sources of the anti-cancer alkaloid, camptothecine from the Icacinaceae taxa, India. Phytomedicine, 20, 521–527.CrossRefPubMedGoogle Scholar
  25. Ravi kumar, K., & Ved, D. K. (2000). 100 Red listed medicinal plants of conservation concern in Southern India (pp. 261–263). Bangalore: FRLHT.Google Scholar
  26. Roja, M., & Heble, M. R. (1994). The quinoline alkaloids camptothecine and 9-methoxycamptothecine from tissue culture and mature trees of Nothapodytes foetida. Phytochemistry, 36, 65–66.CrossRefGoogle Scholar
  27. Rout, G. R. (2005). Direct plant regeneration of Curry leaf tree (Murraya koenigii Koenig.), an aromatic plant. In Vitro Cellular & Developmental Biology-Plant, 41(2), 133–136.CrossRefGoogle Scholar
  28. Sajeevan, R. S., Singh, S. J., Nataraja, K. N., & Shivanna, M. B. (2011). An efficient in vitro protocol for multiple shoot induction in mulberry, Morus alba L variety V1. International Research Journal of Plant Sciences, 2(8), 254–261.Google Scholar
  29. Shahanaz, B. A., Martin, K. P., Zhang, Chun-Lai, Nishitha, I. K., Ligimol, Slater A., & Madhusoodanan, P. V. (2007). Organogenesis from leaf and internode explants of Ophiorrhiza prostrata, an anticancer drug (Camptothecine) producing plant. Electronic Journal of Biotechnology, 10(1), 1–10.Google Scholar
  30. Tavares, A. C., Salgueiro, L. R., & Canhoto, J. M. (2010). In vitro propagation of the wild carrot Daucus carota L. subsp. halophilus (Brot.) A. Pujadas for conservation purpose. In Vitro Cellular & Developmental Biology-Plant, 46, 47–56.CrossRefGoogle Scholar
  31. Thejavathi, D. H., Raveesha, H. R., & Shobha, K. (2011). Evaluation of different treatments to improve the seed germination among the populations of Nothapodytes foetida (WT.) Sleumer. Indian Journal of Fundamental and Applied Life Sciences, 4, 187–192.Google Scholar
  32. Thengane, S. R., Kulkarni, D. K., Shrikhande, V. A., Joshi, S. P., Sonawane, K. B., & Krishnamurthy, K. V. (2003). Influence of medium composition on callus induction and camptothecine accumulation in Nothapodytes foedita. Plant Cell, Tissue and Organ Culture, 72, 247–251.CrossRefGoogle Scholar
  33. Thengane, S. R., Kulkarni, D. K., Shrikhande, V. A., & Krishnamurthy, K. V. (2001). Effect of thidiazuron on adventitious shoot regeneration from seedling explants of Nothapodytes foetida. In Vitro Cellular & Developmental Biology-Plant, 37, 206–210.CrossRefGoogle Scholar
  34. Thomas, T. D., & Shankar, S. (2009). Multiple shoot induction and callus regeneration in Sarcostemma brevistigma Wight & Arnott, a rare medicinal plant. Plant Biotechnology Reports, 3, 67–74.CrossRefGoogle Scholar
  35. Ugraiah, A., Raja Sreelatha, V., Krishna Reddy, P. V., Rajasekhar, K., Sandhya Rani, S., Karuppusamy, S., & Pullaiah, T. (2011). In vitro shoot multiplication and conservation of Caralluma bhupenderiana Sarkaria—An endangered medicinal plant from South India. African Journal of Biotechnology, 10(46), 9328–9336.Google Scholar
  36. Ulukan, H., & Swaan, P. W. (2002). Camptothecine: A review of their chemotherapeutic potential. Drugs, 62(14), 2039–2057.CrossRefPubMedGoogle Scholar
  37. Uma, S. R., Ramesha, B. T., Ravikanth, G., Gunaga, R., Vasudeva, R., & Ganeshaiah, K. N. (2008). Chemical profiling of Nothapodytes nimmoniana for camptothecine, an important anticancer alkaloid: Toward the development of a sustainable production system. In K. G. Ramawat & J. M. Merillon (Eds.), Bioactive molecules and medicinal plants (pp. 197–213). UK: Springer Publishing.Google Scholar
  38. Verma, S., Magotra, R., & Koul, A. K. (2002). In vitro multiplication of Eremurus persicus Boiss. (Liliaceae)—An endangered species. Phytomorphology, 52, 315–321.Google Scholar
  39. Wall, M. E., Wani, M. C., Cook, C. E., Palmer, K. H., McPhail, A. T., & Sim, G. A. (1966). Plant antitumor agents. I. The isolation and structure of camptothecine, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. Journal of the American Chemical Society, 88, 3888–3890.CrossRefGoogle Scholar
  40. Wang, H. M., Zu, Y. G., Wang, W. J., Wu, S. X., & Dong, F. L. (2006). Establishment of Camptotheca acuminate regeneration from leaf explants. Biologia Plantarum, 50, 725–728.CrossRefGoogle Scholar
  41. Yamazaki, Y., Sudo, H., Yamazaki, M., Aimi, N., & Saito, K. (2003). Camptothecine biosynthetic genes in hairy roots of Ophiorrhiza pumila: Cloning, characterization and differential expression in tissues and by stress compounds. Plant and Cell Physiology, 44, 395–403.CrossRefPubMedGoogle Scholar
  42. Zhang, L., Kai, G., Xu, T., Pi, Y., Zhang, H., Sun, X., & Tang, K. (2004). Efficient regeneration of tetraploid Isatis indigotica plants via adventitious organogenesis from hypocotyls explants. Biologia Plantarum, 48, 121–124.CrossRefGoogle Scholar

Copyright information

© Indian Society for Plant Physiology 2015

Authors and Affiliations

  • Ugraiah Amilineni
    • 1
    • 2
  • Vinod Gangal
    • 1
    • 2
  • Ravikanth Gudasalamani
    • 1
    • 2
    • 3
  • Nataraja N. Karaba
    • 2
  • Uma Shaanker Ramanan
    • 1
    • 2
    • 3
  1. 1.School of Ecology and ConservationUniversity of Agricultural SciencesBangaloreIndia
  2. 2.Department of Crop PhysiologyUniversity of Agricultural SciencesBangaloreIndia
  3. 3.Ashoka Trust for Research in Ecology and the EnvironmentBangaloreIndia

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