Acta Physiologiae Plantarum

, Volume 35, Issue 12, pp 3437–3446 | Cite as

In vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Indian Kino tree (Pterocarpus marsupium Roxb.)

  • Radhika Tippani
  • Rajesh Yarra
  • Mallesham Bulle
  • Mahendar Porika
  • Sadanandam Abbagani
  • Christopher ThammidalaEmail author
Original Paper


The objective of the present study was to develop a protocol for in vitro plantlet regeneration and Agrobacterium tumefaciens-mediated genetic transformation using immature cotyledon explants of Indian Kino tree (Pterocarpus marsupium Roxb.). Immature cotyledon explants excised from 9-day-old axenic seedlings produced optimal callus on Murashige and Skoog (MS) medium supplemented with 1.07 μM α-naphthalene acetic acid (NAA), after 2 weeks of culture. When the above said callus was incubated on MS + 8.90 μM 6-benzylaminopurine (BAP) + 1.07 μM NAA, a regeneration frequency of 60.41 % with shoot number and length 12.2 ± 0.85 and 1.4 ± 0.13, respectively, was observed. For further shoot multiplication and elongation, these cultures were transferred onto MS + 4.40 μM BAP. Elongated shoots dipped in 19.60 μM indole-3-butyric acid (IBA) for 24 h and then cultured on ½MS + 2.85 μM IBA, 75 % shoots developed roots and 95 % of plantlets survived in field condition. Organogenic callus was co-cultivated with the A. tumefaciens strain LBA4404 harboring the binary plasmid pCAMBIA1301with ß-glucuronidase (uidA) and hygromycin phosphotransferase (hpt) genes and grown on MS + 8.90 μM BAP + 1.07 μM NAA (RM) + 200 μM acetosyringone for 2 days and then transferred to MS + 8.90 μM BAP + 1.07 μM NAA + 20 mg/l hygromycin + 250 mg/l cefotaxime (SIM) and 4.40 μM BAP + 15 mg/l hygromycin + 200 mg/l cefotaxime (SEM). The putatively transformed shoots were subsequently rooted on ½MS + 2.85 μM IBA + 20 mg/l hygromycin (SRM), after pulse treatment for 24 h with 19.60 μM IBA. Successful gene transfer into putatively transformed plantlets was confirmed by histochemical GUS assay, PCR and RT-PCR analysis. Southern blot analysis of regenerated plantlets confirmed the integration of hpt gene in transgenic plantlets. In the present study, a rate of 20.92 % transformation frequency was achieved and the genetic transformation protocol presented here may pave way for genetic manipulation of this multipurpose legume tree.


Agrobacterium tumefaciens Cotyledons Genetic transformation Pterocarpus marsupium Transgenic plantlets 



RT is thankful to Jawaharlal Nehru Memorial Fund, New Delhi, India for financial support. The authors gratefully acknowledge Prof. Peng Zhang, Institute of Plant Physiology and Ecology, Shanghai Institutes for Biological Sciences, China for providing pCAMBIA1301`construct.


  1. Adinarayana D, Syamasundar KV (1982) A new sesquiterpene alcohol from Pterocarpus marsupium. Phytochem 21:1083–1085CrossRefGoogle Scholar
  2. Al Abdallat A, Sawwan J, Al Zoubi B (2011) Agrobacterium tumefaciens-mediated transformation of callus cells of Crataegus aronia. Plant Cell Tiss Org Cult 104:31–39CrossRefGoogle Scholar
  3. Anis M, Kasif HM, Anwar S (2005) In vitro plantlet regeneration of Pterocarpus marsupium Roxb., an endangered leguminous tree. Curr Sci 88:861–863Google Scholar
  4. Chakraborty A, Gupta N, Ghosh K, Roy P (2010) In vitro evaluation of the cytotoxic, anti-proliferative and anti-oxidant properties of pterostilbene isolated from Pterocarpus marsupium. Toxicol In Vitro 24:1215–1228PubMedCrossRefGoogle Scholar
  5. Chand S, Singh AK (2004) In vitro shoot regeneration from cotyledonary node explants of a multipurpose leguminous tree, Pterocarpus marsupium Roxb. In Vitro Cell Dev Biol Plant 40:464–466CrossRefGoogle Scholar
  6. Chaudhuri AB, Sarkar DD (2002) Biodiversity endangered: India’s threatened wildlife and medicinal plants. Sci Publ, Jodhpur, pp 169–172Google Scholar
  7. Devgun M, Nanda A, Ansari SH (2009) Pterocarpus marsupium Roxb.-a comprehensive review. Pharmacog Rev 3:359–363Google Scholar
  8. Doyle JJ, Doyle JL (1990) Isolation of plant DNA from fresh tissue. Focus 12:13–15Google Scholar
  9. Giri CC, Shyamkumar B, Anjaneyulu C (2004) Progress in tissue culture, genetic transformation and applications of biotechnology to trees: an overview. Trees 18:115–135CrossRefGoogle Scholar
  10. Gorpenchenko TY, Kiselev KV, Bulgakov VP, Tchernoded GK, Bragina EA, Khodakovskaya MV, Koren OG, Batygina TB, Zhuravlev YuN (2006) The Agrobacterium rhizogenes rolC-gene-induced somatic embryogenesis and shoot organogenesis in Panax ginseng transformed calluses. Planta 223:457–467PubMedCrossRefGoogle Scholar
  11. Husain MK, Anis M, Shahzad A (2007) In vitro propagation of Indian Kino (Pterocarpus marsupium Roxb.) using Thidiazuron. In Vitro Cell Dev Biol Plant 43:59–64Google Scholar
  12. Husain MK, Anis M, Shahzad A (2008) In vitro propagation of a multipurpose leguminous tree (Pterocarpus marsupium Roxb.) using nodal explants. Acta Physiol Plant 30:353–359CrossRefGoogle Scholar
  13. Husain MK, Anis M, Shahzad A (2010) Somatic embryogenesis and plant regeneration in Pterocarpus marsupium Roxb. Trees 24:781–787CrossRefGoogle Scholar
  14. Igasaki T, Mohri T, Ichikawa H, Shinohara K (2000) Agrobacterium tumefaciens-mediated transformation of Robinia pseudoacacia. Plant Cell Rep 19:448–453CrossRefGoogle Scholar
  15. James DJ, Uratsu S, Cheng J, Negri P, Viss P, Dandekar AM (1993) Acetosyringone and osmoprotectants like betaine or proline synergistically enhance Agrobacterium-mediated transformation of apple. Plant Cell Rep 12:559–563PubMedCrossRefGoogle Scholar
  16. Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions: ß-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  17. Jube S, Borthakur D (2009) Development of an Agrobacterium-mediated transformation protocol for the tree-legume Leucaena leucocephala using immature zygotic embryos. Plant Cell Tiss Org Cult 96:325–333CrossRefGoogle Scholar
  18. Kanwar K, Bhardwaj A, Agarwal S, Sharma DR (2003) Genetic transformation of Robinia pseudoacacia by Agrobacterium tumefaciens. Indian J Exp Biol 41:149–153PubMedGoogle Scholar
  19. Kirana H, Girish HN, Srinivasan BP (2010) The study of aqueous extract of Pterocarpus marsupium Roxb. on cytokine TNF-α in type 2 diabetic rats. Ind J Pharmacol 42:392–396CrossRefGoogle Scholar
  20. Li ZN, Fang F, Liu GF, Bao MZ (2007) Stable Agrobacterium-mediated genetic transformation of London plane tree (Platanus acerifolia Willd.). Plant Cell Rep 26:641–650PubMedCrossRefGoogle Scholar
  21. Manickam M, Ramanathan M, Jahromi MAF, Chansouria JPN, Ray AB (1997) Anti hyperglycemic activity of phenolics from Pterocarpus marsupium. J Nat Prod 60:609–610PubMedCrossRefGoogle Scholar
  22. Maruthupandian A, Mohan VR (2011) Antidiabetic, antihyperlipidaemic and antioxidant activity of Pterocarpus marsupium Roxb. In alloxan induced diabetic rats. Int J Pharm Tech Res 3:1681–1687Google Scholar
  23. Maurya R, Singh R, Mundkinajeddu D, Handa SS, Yadav PP, Mishra PK (2004) Constituents of Pterocarpus marsupium: an ayurvedic crude drug. Phytochem 65:915–920CrossRefGoogle Scholar
  24. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  25. Niu X, Li X, Veronese P, Bressan RA, Weller SC, Hasegawa PM (2000) Factors affecting Agrobacterium tumefaciens-mediated transformation of peppermint. Plant Cell Rep 19:304–310CrossRefGoogle Scholar
  26. Radhika T (2012) Studies on medicinal properties and its in vitro multiplication of Pterocarpus marsupium (Roxb,). Kakatiya University, IndiaGoogle Scholar
  27. Radhika T, Mahendar P, Venkatesham A, Anreddy RNR, Narsimha RY, Krishna DR, Christopher T, Sadanandam A (2010a) Antioxidant and analgesic activities of Pterocarpus marsupium Roxb. J Herbs Spices Med Plants 16:63–68CrossRefGoogle Scholar
  28. Radhika T, Mahendar P, Venkatesham A, Reddy ARN, Reddy YN, Sadanandam A, Christopher T (2010b) Hypoglycemic activity of red kino tree in normal and streptozotocin induced diabetic rats. Int J Pharmacol 6:301–305CrossRefGoogle Scholar
  29. Sarria R, Calderon A, Thro AM, Torres E, Mayer JE, Roca WM (1994) Agrobacterium-mediated transformation of Stylosanthes guianensis and production of transgenic plants. Plant Sci 96:119–127CrossRefGoogle Scholar
  30. Seshadri TR (1972) Polyphenols of Pterocarpus and Dalbergia woods. Phytochem 11:881–898CrossRefGoogle Scholar
  31. Shimoda N, Toyoda YA, Nagamine J, Usami S, Katayama M, Sakagami Y, Michida Y (1990) Control of expression of Agrobacterium vir genes by synergistic action of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci USA 87:6684–6688PubMedCrossRefGoogle Scholar
  32. Thangjam R, Sahoo L (2012) In vitro regeneration and Agrobacterium tumefaciens-mediated genetic transformation of Parkia timoriana (DC.) Merr.: a multipurpose tree legume. Acta Physiol Planta 34:1207–1215CrossRefGoogle Scholar
  33. Tiwari S, Shah P, Singh K (2004) In vitro propagation of Pterocarpus marsupium Roxb: an endangered medicinal tree. Ind J Biotechnol 3:422–425Google Scholar
  34. Tournier V, Grat S, Marque C, Elkayal W, Penchel R, De Andrade G, Boudet AM, Teulieres C (2003) An efficient procedure to stably introduce genes into an economically important pulp tree (Eucalyptus grandis X Eucalyptus urophylla). Transgen Res 12:403–411CrossRefGoogle Scholar
  35. Vander Fits L, Deakin EA, Hoge JH, Memelink J (2000) The ternary transformation system: constitutive virG on a compatible plasmid dramatically increases Agrobacterium-mediated plant transformation. Plant Mol Biol 43:495–502CrossRefGoogle Scholar
  36. Vats V, Yadav SP, Biswas NR, Grover JK (2004) Anti-cataract activity of Pterocarpus marsupium bark and Trigonella foenum-graecum seeds extract in alloxan diabetic rats. J Ethnopharmacol 93:289–294PubMedCrossRefGoogle Scholar
  37. Yang M, Xie X, Zheng C, Zhang F, He X, Li Z (2008) Agrobacterium tumefaciens-mediated genetic transformation of Acacia crassicarpa via organogenesis. Plant Cell Tiss Org Cult 95:141–147CrossRefGoogle Scholar

Copyright information

© Franciszek Górski Institute of Plant Physiology, Polish Academy of Sciences, Kraków 2013

Authors and Affiliations

  • Radhika Tippani
    • 1
  • Rajesh Yarra
    • 1
  • Mallesham Bulle
    • 1
  • Mahendar Porika
    • 1
  • Sadanandam Abbagani
    • 1
  • Christopher Thammidala
    • 1
    Email author
  1. 1.Department of BiotechnologyKakatiya UniversityWarangalIndia

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