Horticulture, Environment, and Biotechnology

, Volume 54, Issue 6, pp 531–537 | Cite as

Adventitious shoot regeneration from immature zygotic embryos of Indian Kino tree (Pterocarpus marsupium Roxb.) and genetic integrity analysis of in vitro derived plants using ISSR markers

  • Radhika Tippani
  • Anil Kumar Vemunoori
  • Rajesh Yarra
  • Rama Swamy Nanna
  • Sadanandam Abbagani
  • Christopher ThammidalaEmail author
Research Report Tissue Culture/Biotechnology


A simple and highly efficient protocol has been developed for in vitro regeneration of Pterocarpus marsupium using immature zygotic embryo (IZE) as explant. The highest frequency of shoot regeneration (93.8%) was obtained on Murashige and Skoog (MS) medium supplemented with 3.0 mg·L−1 6-benzylaminopurine (BA) and 0.5 mg·L−1 indole-3-acetic acid (IAA), wherein maximum of 17.3 ± 0.9 shoots per explant was induced. When these cultures were sub-cultured on MS medium supplemented with 1.0 mg·L−1 BA, more shoots (27.2 ± 1.1) with an average shoot length of 4.5 cm were observed. The highest rooting (70.8%) and maximum number of roots (3.2 ± 0.3) per shoot were obtained when shoots were dipped in 3.0 mg·L−1 indol-3-butyric acid (IBA) solution for 24 hours and further cultured on half-strength MS medium. Plantlets obtained in vitro were transferred to the field after being hardened with a 74% survival rate. Analysis of regenerated plantlets using inter-simple sequence repeat (ISSR) markers confirmed that there was no genetic variability. All ISSR banding profiles from regenerated plantlets were monomorphic and similar to those of the mother plant. This protocol might be helpful for the mass multiplication and in vitro conservation of P. marsupium.

Additional key words

genetic variability legume tree malabar kino multiple shoots plant growth regulators 


Unable to display preview. Download preview PDF.

Unable to display preview. Download preview PDF.

Literature Cited

  1. Agrawal, V. and P.R. Sardar. 2007. In vitro regeneration through somatic embryogenesis and organogenesis using cotyledons of Cassia angustifolia Vahl. In Vitro Cell Dev. Biol. Plant 43:585–592.CrossRefGoogle Scholar
  2. Anis, M., M.K. Husain, and S. Anwar. 2005. In vitro plantlet regeneration of Pterocarpus marsupium Roxb., an endangered leguminous tree. Curr. Sci. 88:861–863.Google Scholar
  3. Anuradha, M. and T. Pullaiah. 1999. In vitro seed culture and induction of enhanced axillary branching in Pterocarpus santalinus and Pterocarpus marsupium: A method for rapid multiplication. Phytomorphology 49:157–163.Google Scholar
  4. Balaraju, K., P. Agastian, S. Ignacimuthu, and K. Park. 2011. A rapid in vitro propagation of red sanders (Pterocarpus santalinus L.) using shoot tip explants. Acta Physiol. Plant. 33:2501–2510.CrossRefGoogle Scholar
  5. Bhatia, R., K.P. Singh, T.R. Sharma, and T. Jhan. 2011. Evaluation of the genetic fidelity of in vitro-propagated gerbera (Gerbera jamesonii Bolus) using DNA-based markers. Plant Cell Tissue Organ Cult. 104:131–135.CrossRefGoogle Scholar
  6. Bonga, J.M., K.K. Klimaszewska, and A.P. Von. 2010. Recalcitrance in clonal propagation, in particular of conifers. Plant Cell Tissue Organ Cult. 100:241–254.CrossRefGoogle Scholar
  7. Chakraborty, A., N. Gupta, K. Ghosh, and P. Roy. 2010. In vitro evaluation of the cytotoxic, anti-proliferative and anti-oxidant properties of pterostilbene isolated from Pterocarpus marsupium. Toxicol. In Vitro 24:1215–1228.PubMedCrossRefGoogle Scholar
  8. Chand, S. and A.K. Singh. 2004. In vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, Pterocarpus marsupium Roxb. In Vitro Cell Dev. Biol. Plant 40: 167–170.CrossRefGoogle Scholar
  9. Chandrika, M. and V.R. Rai. 2009. Genetic fidelity in micropropagated plantlets of Ochreinauclea missionis an endemic, threatened and medicinal tree using ISSR markers. Afri. J. Biotech. 8:2933–2938.Google Scholar
  10. Chaturvedi, R., M.K. Razdan, and S.S. Bhojwani. 2004. In vitro morphogenesis in zygotic embryo cultures of neem (Azadirachta indica A. Juss.). Plant Cell Rep. 22:801–809.PubMedCrossRefGoogle Scholar
  11. Devgun, M., A. Nanda, and S.H. Ansari. 2009. Pterocarpus marsupium Roxb.-A Comprehensive Review. Phcog. Rev. 3:359–363.Google Scholar
  12. Dimitrov, B., K. Tashera, N. Zagorska, and L. Evstatiera. 2003. In vitro cultivation of Rhodiola rosea L. Genet. Breed. 32:3–6.Google Scholar
  13. Distabanjong, K. and R. Geneve. 1997. Multiple shoot formation from cotyledonary node segments of Eastern redbud. Plant Cell Tissue Organ Cult. 47:247–254.CrossRefGoogle Scholar
  14. Doyle, J.J. and J.L. Doyle. 1990. Isolation of plant DNA from fresh tissue. Focus 12:13–15.Google Scholar
  15. Husain, M.K., M. Anis, and A. Shahzad. 2007. In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using Thidiazuron. In Vitro Cell Dev. Biol. Plant 43:59–64.Google Scholar
  16. Husain, M.K., M. Anis, and A. Shahzad. 2008. In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants. Acta Physiol. Plant. 30:353–359.CrossRefGoogle Scholar
  17. Igasaki, T., T. Sato, N. Akashi, T. Mohri, E. Maruyama, I. Kinoshita, C. Walter, and K. Shinohara. 2003. Somatic embryogenesis and plant regeneration from immature zygotic embryos of Cryptomeria japonica D. Don. Plant Cell Rep. 22:239–243.CrossRefGoogle Scholar
  18. Iram, S. and M. Anis. 2007. In vitro shoot multiplication and plantlet regeneration from nodal explants of Cassia angustifolia (Vahl.), a medicinal plant. Acta Physiol. Plant. 29:233–238.CrossRefGoogle Scholar
  19. Jahromi, M.A. and A.B. Ray. 1993. Antihyperlipidemic effects of flavonoids from Pterocarpus marsupium. J. Nat. Prod. 56:989–994.PubMedCrossRefGoogle Scholar
  20. Kalimuthu, K. and K.K. Lakshmanan. 1995. Effect of different treatments on pod germination of Pterocarpus species. Ind. J. Forestry 18:104–106.Google Scholar
  21. Kanita, A. and S.L. Kothari. 2002. High efficiency adventitious shoot bud formation and plant regeneration from leaf explants of Dianthus chinensis L. Sci. Hortic. 90:205–212.Google Scholar
  22. Manickam, M., M. Ramanathan, M.A. Jahromi, J.P. Chansouria, and A.B. Ray. 1997. Antihyperglycemic activity of phenolics from Pterocarpus marsupium. J. Nat. Prod. 60:609–610.PubMedCrossRefGoogle Scholar
  23. Mankani, K.L., V. Krishna, B.K. Manjunatha, S.M. Vidya, S.D.J. Singh, Y.N. Manohara, A.U. Raheem, and K.R. Avinash. 2005. Evaluation of hepatoprotective activity of stem bark of Pterocarpus marsupium Roxb. Ind. J. Pharmacol. 37:165–168.CrossRefGoogle Scholar
  24. Murashige, T. and F.A. Skoog. 1962. Revised medium for growth and bioassay with tobacco tissue culture. Physiol. Plant. 15:473–479.CrossRefGoogle Scholar
  25. Nanda, R.M. and G.R. Rout. 2003. In vitro somatic embryogenesis and plant regeneration in Acacia Arabica. Plant Cell Tissue Organ Cult. 73:131–135.CrossRefGoogle Scholar
  26. Pal, A., V.S. Negi, and D. Borthakur. 2012. Efficient in vitro regeneration of Leucaena leucocephala using immature zygotic embryos as explants. Agroforest Syst. 84:131–140.CrossRefGoogle Scholar
  27. Radhika, T., P. Mahendar, A. Venkatesham, A.R.N. Reddy, Y.N. Reddy, D.R. Krishna, T. Christopher, and A. Sadanandam. 2010a. Antioxidant and Analgesic Activities of Pterocarpus marsupium Roxb. J. Herbs Spices Med. Plants 16:63–68.CrossRefGoogle Scholar
  28. Radhika, T., P. Mahendar, A. Venkatesham, A.R.N. Reddy, Y.N. Reddy, A. Sadanandam, and T. Christopher. 2010b. Hypoglycemic activity of red Kino tree in normal and streptozotocin induced diabetic rats. Int. J. Pharmacol. 6:301–305.CrossRefGoogle Scholar
  29. Rajeswari, V. and P. Kailash. 2006. In vitro propagation of Albizia odoratissima L.F. (Benth.) from cotyledonary node and leaf nodal explants. In Vitro Cell Dev. Biol. Plant 42:399–404.CrossRefGoogle Scholar
  30. Rout, G.R. 2005. In vitro somatic embryogenesis in callus cultures of Azadirachta indica A. Juss-A multipurpose tree. J. Forest Res. 10:263–267.CrossRefGoogle Scholar
  31. Satu, L., L. Amparo, and A.M. Scott. 2006. In vitro regeneration of Salix nigra from adventitious shoots. Tree Physiol. 26:969–975.CrossRefGoogle Scholar
  32. Sinha, R.K., K. Majumdar, and S. Sinha. 2000. In vitro differentiation and plant regeneration of Albizia chinensis (Osb) Merr. In Vitro Cell Dev. Biol. Plant 36:370–373.CrossRefGoogle Scholar
  33. Tang, W. and Z. Guo. 2001. In vitro propagation of loblolly pine via direct somatic organogenesis from mature cotyledons and hypocotyls. Plant Growth Reg. 33:25–31.CrossRefGoogle Scholar
  34. Thomas, T.D. 2003. Thidiazuron induced multiple shoot induction and plant regeneration from cotyledonary explants of mulberry. Biol. Plantarum 46:529–533.CrossRefGoogle Scholar
  35. Tiwari, S., S. Pankaj, and S. Kanchan. 2004. In vitro propagation of Pterocarpus marsupium Roxb. An endangered medicinal tree. Indian J. Biotechnol. 3:422–425.Google Scholar
  36. Wilkins, C.P. 1991. Conservation of tree crops, p. 151–237. In: J.H. Dodd (ed.). In vitro methods for conservation of plant genetic resources. Chapman and Hall, London.CrossRefGoogle Scholar

Copyright information

© Korean Society for Horticultural Science 2013

Authors and Affiliations

  • Radhika Tippani
    • 1
  • Anil Kumar Vemunoori
    • 2
  • Rajesh Yarra
    • 3
  • Rama Swamy Nanna
    • 1
  • Sadanandam Abbagani
    • 1
  • Christopher Thammidala
    • 1
    • 3
    Email author
  1. 1.Department of BiotechnologyKakatiya UniversityWarangalIndia
  2. 2.Department of BiochemistryKakatiya UniversityWarangalIndia
  3. 3.Department of BotanyKakatiya UniversityWarangalIndia

Personalised recommendations