Agrobacterium-mediated transformation of the medicinal plant Podophyllum hexandrum Royle (syn. P. emodi Wall. ex Hook.f. & Thomas)

  • Manoharan Rajesh
  • Murugaraj Jeyaraj
  • Ganeshan Sivanandhan
  • Kondeti Subramanyam
  • Thankaraj Salammal Mariashibu
  • Subramanian Mayavan
  • Gnanajothi Kapil Dev
  • Vasudevan Ramesh Anbazhagan
  • Markandan Manickavasagam
  • Andy GanapathiEmail author
Original Paper


An efficient Agrobacterium-mediated genetic transformation method has been developed for the medicinal plant Podophyllum hexandrum Royle, an important source of the anticancer agent podophyllotoxin. Highly proliferating embryogenic cells were infected with Agrobacterium tumefaciens harbouring pCAMBIA 2301, which contains npt II and gusA as selection marker and reporter genes, respectively. The transformed somatic embryos and plantlets were selected on Murashige and Skoog (MS) basal medium containing kanamycin and germination medium, respectively. GUS histochemical analysis, polymerase chain reaction and Southern blot hybridisation confirmed that gusA was successfully integrated and expressed in the P. hexandrum genome. Compared with cefotaxime, 200 mg l−1 timentin completely arrested Agrobacterium growth and favoured somatic embryo development from embryogenic cells. Among the different Agrobacterium strains, acetosyringone concentrations and co-cultivation durations tested, embryogenic callus infected with A. tumefaciens EHA 105 and co-cultivated for 3 days on MS basal medium containing 100 μM acetosyringone proved to be optimal and produced a transformation efficiency of 29.64 % with respect to germinated GUS-positive plantlets. The Agrobacterium-mediated genetic transformation method developed in the present study facilitates the transference of desirable genes into P. hexandrum to improve the podophyllotoxin content and to enhance other useful traits.


Acetosyringone Agrobacterium tumefaciens EHA 105 Podophyllum hexandrum Podophyllum peltatum Podophyllotoxin Timentin 



The authors are thankful to the Life Science Research Board, Defence Research and Development Organisation (DRDO), Govt. of India for financial support (DLS/81/48222/LSRB–171 BTB/2008) used to carry out the present work. The corresponding author is thankful to the University Grants Commission (UGC), Govt. of India, for providing an Emeritus fellowship under the BSR scheme. The authors are also thankful to Prof. S.K. Nandi, G.B. Pant Institute of Himalayan Environment and Development, Almora, Uttarakhand, India and Prof. M.C. Nautiyal, H.N.B. Garhwal University, Garhwal Srinagar, Uttarakhand, India, for help with the collection of Podophyllum hexandrum seeds. The authors are thankful to Dr. A.S. Rao for his valuable suggestions to improve the manuscript.


  1. Al Abdallat AM, Sawwan JS, Al Zoubi B (2011) Agrobacterium tumefaciens-mediated transformation of callus cells of Crataegus aronia. Plant Cell Tissue Org Cult 104:31–39CrossRefGoogle Scholar
  2. Anbazhagan VR, Ahn CH, Harada E, Kim YS, Choi YE (2008) Podophyllotoxin production via cell and adventitious root cultures of Podophyllum peltatum. In Vitro Cell Dev Biol Plant 44:494–501CrossRefGoogle Scholar
  3. Anbazhagan VR, Kim YS, Choi YE (2009) Production of transgenic Podophyllum peltatum via Agrobacterium tumefaciens-mediated transformation. Biol Plantarum 53:637–642CrossRefGoogle Scholar
  4. Badhwar RL, Sharma BK (1963) A note on the germination of Podophyllum seeds. Indian For 89:445–447Google Scholar
  5. Bastos JK, Burandt CL, Dhammika NP, Bryant L, McChesney JD (1996) Quantization of aryltetralin lignans in plant parts and among different populations of Podophyllum peltatum by reversed-phase high-performance liquid chromatography. J Nat Prod 59:406–408CrossRefGoogle Scholar
  6. Canel C, Moraes RM, Dayan FE, Ferreira D (2000) Molecules of interest podophyllotoxin. Phytochemistry 54:115–120PubMedCrossRefGoogle Scholar
  7. Canel C, Dayan FE, Ganzera M, Khan IA, Rimando A, Burandt CL, Moraes RM (2001) High yield of podophyllotoxin from leaves of Podophyllum peltatum by in situ conversion of podophyllotoxin 4-O-b-D glucopyranoside. Planta Med 67:97–99PubMedCrossRefGoogle Scholar
  8. Chattopadhyay S, Mehra RS, Srivastava AK, Bhojwani SS, Bisaria VS (2003) Effect of major nutrients on podophyllotoxin production in Podophyllum hexandrum suspension cultures. Appl Microbiol Biotechnol 60:541–546PubMedGoogle Scholar
  9. Cheng ZM, Schnurr AA, Kapaun JA (1998) Timentin as an alternative antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. Plant Cell Rep 17:646–649CrossRefGoogle Scholar
  10. Dai SH, Zheng P, Marmey P, Zhang SP, Tian WZ, Chen SY, Beachy RN, Fauquet C (2001) Comparative analysis of transgenic rice plants obtained by Agrobacterium-mediated transformation and particle bombardment. Mol Breed 7:25–33CrossRefGoogle Scholar
  11. Damayanthi Y, Lown JW (1998) Podophyllotoxins: current status and recent developments. Curr Med Chem 5:205–252PubMedGoogle Scholar
  12. Dangi B, Kachhwaha S, Kothari SL (2012) Regeneration and Agrobacterium-mediated genetic transformation of Terminalia bellerica Roxb.: a multipurpose tree species. In Vitro Cell Dev Biol Plant 48:304–312Google Scholar
  13. Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA mini preparation: version II. Plant Mol Biol Rep 1:19–21CrossRefGoogle Scholar
  14. Gelvin SB (2003) Agrobacterium-mediated plant transformation: the biology behind the ‘gene-jockeying’ tool. Microbiol Mol Biol Rev 67:16–37PubMedCrossRefGoogle Scholar
  15. Gelvin SB (2009) Agrobacterium in the genomics age. Plant Physiol 150:1665–1676PubMedCrossRefGoogle Scholar
  16. Giri A, Lakshmi Narasu M (2000) Production of podophyllotoxin from Podophyllum hexandrum: a potential natural product for clinically useful anticancer drugs. Cytotechnology 34:17–26PubMedCrossRefGoogle Scholar
  17. Giri A, Giri CC, Dhingra V, Narasu ML (2001) Enhanced podophyllotoxin production from Agrobacterium rhizogenes transformed cultures of Podophyllum hexandrum. Nat Prod Lett 15:229–235PubMedCrossRefGoogle Scholar
  18. Goldberg JB, Ohman DE (1984) Cloning and expression in Pseudomonas aeruginosa of a gene involved with the production of alginate. J Bacteriol 158:1115–1121PubMedGoogle Scholar
  19. Han JL, Wang H, Ye HC, Liu Y, Li ZQ, Zhang Y, Zhang YS, Yan F, Li GF (2005) High efficiency of genetic transformation and regeneration of Artemisia annua (L.) via Agrobacterium. Plant Sci 168:73–80CrossRefGoogle Scholar
  20. He C, Zhang J, Chen J, Ye X, Du L, Dong Y, Zhao H (2007) Genetic transformation of Aloe barbadensis Miller by Agrobacterium tumefaciens. J Genet Genomics 34:1053–1060PubMedCrossRefGoogle Scholar
  21. Hoekema A, Hirsch PR, Hooykaas PJJ, Schilperoort RA (1983) A binary plant vector strategy based on separation of vir and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303:179–180CrossRefGoogle Scholar
  22. Hood EE, Helmer GC, Fraley RT, Chilton MD (1986) The hypervirlence of Agrobacterium tumefaciens A281 is encoded in the region of pTiBo542 outside the T-DNA. J Bacteriol 168:1291–1301PubMedGoogle Scholar
  23. Hood EE, Gelvin SB, Melchers LS, Hoekema A (1993) New Agrobacterium helper plasmids for gene transfer to plants. Transgenic Res 2:208–218CrossRefGoogle Scholar
  24. Ieamkhang S, Chatchawankanphanich O (2005) Augmentin as an alternative antibiotic for growth suppression of Agrobacterium for tomato (Lycopersicon esculentum) transformation. Plant Cell Tissue Org Cult 82:213–220CrossRefGoogle Scholar
  25. Jackson DE, Dewick PM (1984) Aryltetralin lignans from Podophyllum hexandrum and Podophyllum peltatum. Phytochemistry 23:1147–1152CrossRefGoogle Scholar
  26. Jefferson RA, Kavanagh TA, Bevan NW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  27. Kondo T, Hasegawa H, Suzuki M (2000) Transformation and regeneration of garlic (Allium sativum L.) by Agrobacterium-mediated gene transfer. Plant Cell Rep 19:989–993CrossRefGoogle Scholar
  28. Le VQ, Belles-Isles J, Dusabenyagasani M, Tremblay FM (2001) An improved procedure for production of white spruce (Picea glauca) transgenic plants using Agrobacterium tumefaciens. J Exp Biol 52:2089–2095Google Scholar
  29. Lee SH, Lee DG, Woo HS, Lee KW, Kim DH, Kwak SS, Kim JS, Kim H, Ahsan N, Choi MS, Yang JK, Lee BH (2006) Production of transgenic orchard grass via Agrobacterium-mediated transformation of seed-derived callus tissues. Plant Sci 171:408–414PubMedCrossRefGoogle Scholar
  30. Li DD, Shi W, Deng XX (2002) Factors influencing Agrobacterium-mediated embryogenic callus transformation of Valencia sweet orange (Citrus sinensis) containing the pTA29-barnase gene. Tree Physiol 23:1209–1215CrossRefGoogle Scholar
  31. Lièvre K, Hehn A, Tran TLM, Gravot A, Thomasset B, Bourgaud F, Gontiet E (2005) Genetic transformation of the medicinal plant Ruta graveolens L. by an Agrobacterium tumefaciens-mediated method. Plant Sci 168:883–888CrossRefGoogle Scholar
  32. Manickavasagam M, Ganapathi A, Anbazhagan VR, Sudhakar B, Selvaraj N, Vasudevan A, Kasthurirengan S (2004) Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharum species hybrids) using axillary buds. Plant Cell Rep 23:134–143PubMedCrossRefGoogle Scholar
  33. Mishra S, Sangwan RS, Bansal S, Sangwan NS (2012) Efficient genetic transformation of Withania coagulans (Stocks) dunal mediated by Agrobacterium tumefaciens from leaf explants of in vitro multiple shoot culture. Protoplasma. doi: 10.1007/s00709-012-0428-0 Google Scholar
  34. Mondal TK, Bhattacharya A, Ahuja PS, Chand PK (2001) Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium-mediated transformation of somatic embryos. Plant Cell Rep 20:712–720CrossRefGoogle Scholar
  35. Moraes RM, Burandt C, Ganzera M, Li XL, Khan I, Canel C (2002) The American may apple revisited: Podophyllum peltatum: still a potential cash crop? Econ Bot 54:471–476CrossRefGoogle Scholar
  36. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  37. Naing AH, Kim CK, Yun BJ, Jin JY, Lim KB (2012) Primary and secondary somatic embryogenesis in Chrysanthemum cv. Euro. Plant Cell Tissue Organ Cult. doi: 10.1007/s11240-012-0243-5 Google Scholar
  38. Palomo-Ríos E, Barceló-Muňoz A, Mercado JA, Pliego-Alfaro F (2012) Evaluation of key factors influencing Agrobacterium-mediated transformation of somatic embryos of avocado (Persea americana Mill.). Plant Cell Tissue Organ Cult 109:201–211CrossRefGoogle Scholar
  39. Sagar PS, Zafar R (2005) In vitro enhanced production of podophyllotoxinin in phytohormonal induced and regenerated roots of Podophyllum hexandrum. Pharm Biol 43:404–410CrossRefGoogle Scholar
  40. Sambrook J, Fritch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring, HarborGoogle Scholar
  41. Selvaraj N, Kasthurirengan S, Vasudevan A, Manickavasagam M, Choi CW, Ganapathi A (2010) Evaluation of green fluorescent protein as a reporter gene and phosphinothricin as the selective agent for achieving a higher recovery of transformants in cucumber (Cucumis sativus L. cv. Poinsett76) via Agrobacterium tumefaciens. In Vitro Cell Dev Biol Plant 46:329–337CrossRefGoogle Scholar
  42. Srivastava T, Das S, Sopory SK, Srivastava PS (2009) A reliable protocol for transformation of Catharanthus roseus through Agrobacterium tumefaciens. Physiol Mol Biol 15:93–98CrossRefGoogle Scholar
  43. Subramaniam S, Rathinam X (2010) Emerging factors that influence efficiency of T-DNA gene transfer into Phalaenopsis violacea orchid via Agrobacterium tumefaciens-mediated transformation system. Int J Biol 2:64–73Google Scholar
  44. Subramanyam K, Subramanyam K, Sailaja KV, Srinivasulu M, Lakshmidevi K (2011) Highly efficient Agrobacterium-mediated transformation of banana cv. Rasthali (AAB) via sonication and vacuum infiltration. Plant Cell Rep 30:425–436PubMedCrossRefGoogle Scholar
  45. Sultan P, Shawl AS, Ramteke PW, Jan A (2006) In vitro propagation for mass multiplication of Podophyllum hexandrum: a high value medicinal herb. Asian J Plant Sci 5:179–184CrossRefGoogle Scholar
  46. Suma B, Keshavachandran R, Nybe EV (2008) Agrobacetrium tumefaciens-mediated transformation and regeneration of ginger (Zinger officinale Rosc.). J Trop Agr 46:38–44Google Scholar
  47. Tang H, Ren Z, Krczal Z (2000) An evaluation of antibiotics for the elimination of Agrobacterium tumefaciens from walnut somatic embryos and for the effects on the proliferation of somatic embryos and regeneration of transgenic plants. Plant Cell Rep 19:881–887CrossRefGoogle Scholar
  48. Thiruvengadam M, Chung IM (2011) Establishment of an efficient Agrobacterium tumefaciens-mediated leaf disc transformation of spine gourd (Momordica dioica Roxb. ex Willd). Afr J Biotechnol 10:19337–19345Google Scholar
  49. Thiruvengadam M, Praveen N, Chung IM (2012) An efficient Agrobacterium tumefaciens-mediated genetic transformation of bitter melon (Momordica charantia L.). Aust J Crop Sci 6:1094–1100Google Scholar
  50. Travella S, Ross SM, Harden J, Everett C, Snape JW, Harwood WA (2005) A comparison of transgenic barley lines produced by particle bombardment and Agrobacterium-mediated techniques. Plant Cell Rep 23:780–789PubMedCrossRefGoogle Scholar
  51. Van Uden W, Pras N, Visser JF, Malingre TM (1989) Detection and identification of podophyllotoxin produced by cell cultures derived from Podophyllum hexandrum royle. Plant Cell Rep 8:165–168CrossRefGoogle Scholar
  52. Vancanneyt G, Schmidt R, O’Connor-Sanchez A, Willmitzer L, Rocha-Sosa M (1990) Construction of an intron-containing marker gene: splicing of the intron in transgenic plants and its use in monitoring early events in Agrobacterium-mediated plant transformation. Mol Genet Genomics 220:245–250Google Scholar
  53. Vengadesan G, Ganapathi A, Anbazhagan VR (2002) Somatic embryogenesis in cell suspension cultures of Acacia sinuata (Lour.) Merr. In Vitro Cell Dev Biol Plant 38:52–57CrossRefGoogle Scholar
  54. Wang HM, To KY (2004) Agrobacterium-mediated transformation in the high-value medicinal plant Echinacea purpurea. Plant Sci 166:1087–1096CrossRefGoogle Scholar
  55. Wei X, Gou X, Yuan T, Russell SD (2006) A highly efficient in vitro plant regeneration system and Agrobacterium-mediated transformation in Plumbago zeylanica. Plant Cell Rep 25:513–521PubMedCrossRefGoogle Scholar
  56. Yan YP, Wang ZZ (2007) Genetic transformation of the medicinal plant Salvia miltiorrhiza by Agrobacterium tumefaciens-mediated method. Plant Cell Tissue Org Cult 88:175–184CrossRefGoogle Scholar
  57. Zhi-neng L, Guo-feng L, Fang F, Man-zhu B (2007) Adventitious shoot regeneration of Platanus acerifolia Willd. facilitated by timentin, an antibiotic for suppression of Agrobacterium tumefaciens in genetic transformation. For Stud China 9:14–18CrossRefGoogle Scholar
  58. Zhu Q, Wu F, Ding F, Ye D, Chen Y, Li Y, Zhifan Y (2009) Agrobacterium-mediated transformation of Dioscorea zingiberensis Wright, an important pharmaceutical crop. Plant Cell Tissue Org Cult 96:317–324CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media Dordrecht 2013

Authors and Affiliations

  • Manoharan Rajesh
    • 1
  • Murugaraj Jeyaraj
    • 1
  • Ganeshan Sivanandhan
    • 1
  • Kondeti Subramanyam
    • 1
  • Thankaraj Salammal Mariashibu
    • 2
  • Subramanian Mayavan
    • 3
  • Gnanajothi Kapil Dev
    • 1
  • Vasudevan Ramesh Anbazhagan
    • 1
    • 2
  • Markandan Manickavasagam
    • 1
  • Andy Ganapathi
    • 1
    Email author
  1. 1.Department of Biotechnology and Genetic Engineering, School of BiotechnologyBharathidasan UniversityTiruchirappalliIndia
  2. 2.Temasek Life Sciences Laboratory Limited, 1 Research LinkNational University of SingaporeSingaporeSingapore
  3. 3.Synthetic Biology and Biofuel GroupInternational Center for Genetic Engineering and Biotechnology (ICGEB)New DelhiIndia

Personalised recommendations