Biologia Plantarum

, 53:637 | Cite as

Production of transgenic Podophyllum peltatum via Agrobacterium tumefaciens-mediated transformation

  • V. R. Anbazhagan
  • Y. -S. Kim
  • Y. -E. Choi
Original Papers


Transgenic Podophyllum peltatum plants were successfully produced by Agrobacterium tumefaciens-mediated transformation. Embryogenic callus was co-cultivated with Agrobacterium tumefaciens harboring a binary vector pBI 121 carrying β-glucuronidase (GUS) and neomycinphosphotransferase (NPT II) gene. GUS-histochemical analysis revealed that, 50 µM acetosyringone treatments during Agrobacterium infection and 3 d co-cultivation with Agrobacterium showed enhanced transformation efficiency. Percentage of GUS positive callus increased rapidly as the subculture time proceeded on selection medium containing 100 mg dm−3 kanamycin. Kanamycin resistant somatic embryos were formed from embryogenic callus after cultivation with 11.35 µM abscisic acid (ABA) for 3 weeks and then on hormone-free selection medium. Somatic embryos were germinated and converted into plantlets on medium containing 2.89 µM gibberellic acid (GA3). The integration of GUS and NPT II gene into transgenic plants was confirmed by polymerase chain reaction and Southern analysis.

Additional key words

abscisic acid embryogenic callus gibberellic acid GUS gene kanamycin resistance NPT II gene 



abscisic acid


cauliflower mosaic virus


2,4-dichlorophenoxyacetic acid


gibberellic acid




Murashige and Skoog


nopaline synthase


neomycinphosphotransferase II


polymerase chain reaction




  1. Ayres, N.M., Park, W.D.: Genetic transformation of rice. — Crit. Rev. Plant Sci. 13: 219–239, 1994.CrossRefGoogle Scholar
  2. Cervera, M., Pina, J.A., Juarez, J., Navarvo, L., Pena, L.: Agrobacterium-mediated transformation of citrange: factors affecting transformation and regeneration. — Plant Cell Rep. 18: 271–278, 1998.CrossRefGoogle Scholar
  3. Chilton, M.D., Currier, T.C., Farrand, S.K., Bendich, A.J., Gordon, M.P., Nester, E.W.: Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors. — Proc. nat. Acad. Sci. USA 71: 3672–3676, 1974.CrossRefPubMedGoogle Scholar
  4. Damayanthi, Y., Lown, J.W.: Podophyllotoxins: current status and recent developments. — Curr. Med. Chem. 5: 205–52, 1998.PubMedGoogle Scholar
  5. Franklin, G., Lakshmi Sita, G.: Agrobacterium tumefaciensmediated transformation of egg plant (Solanum melongena L.) using root explants. — Plant Cell Rep. 21: 549–554, 2003.PubMedGoogle Scholar
  6. Giri, A., Giri, C.C., Dhingra, V., Narasu, M.L.: Enhanced podophyllotoxin production from Agrobacterium rhizogenes transformed cultures of P. hexandrum. — Nat. Prod. Lett. 15: 229–235, 2001.PubMedGoogle Scholar
  7. Godwin, I., Todd, G., Ford-Lloyd, B., Newbury, H.J.: The effects of acetosyringone and pH on Agrobacteriummediated transformation vary according to plant species. — Plant Cell Rep. 9: 671–675, 1991.CrossRefGoogle Scholar
  8. Han, K.H., Meilan, R., Ma, C., Strauss, S.H.: An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). — Plant Cell Rep. 19: 315–320, 2000.CrossRefGoogle Scholar
  9. Hiei, Y., Komari, T., Kubo, T.: Transformation of rice mediated by Agrobacterium tumefaciens. — Plant mol. Biol. 35: 205–218, 1997.CrossRefPubMedGoogle Scholar
  10. Howe, A., Sato, S., Dweikat, I., Fromm, M., Clemente, T.: Rapid and reproducible Agrobacterium-mediated transformation of Sorghum. — Plant Cell Rep. 25: 784–791, 2006.CrossRefPubMedGoogle Scholar
  11. Kim, Y.S., Lim, S., Choi, Y.E., Anbazhagan, V.R.: High frequency plant regeneration via somatic embryogenesis in Podophyllum peltatum L., an important medicinal plant for source of anticancer drug. — Curr. Sci. 92: 662–666, 2007.Google Scholar
  12. Li, X.Q., Liu, C.N., Ritchie, S.W., Peng, J., Gelvin, S.B., Hodges, T.K.: Factors influencing Agrobacterium-mediated transient expression of gusA in rice. — Plant mol. Biol. 20: 1037–1048, 1992.CrossRefPubMedGoogle Scholar
  13. Lichtenstein, C., Draper, J.: Genetic engineering of plants. — In: Glover, D.M. (ed.): DNA Cloning. Pp. 67–119. IRL Press, Oxford 1985.Google Scholar
  14. Manickavasagam, M., Ganapathi, A., Anbazhagan, V.R., Sudhakar, B., Selvaraj, N., Vasudevan, A., Kasthurirengan, S.: Agrobacterium-mediated genetic transformation and development of herbicide-resistant sugarcane (Saccharam species hybrids) using axillary buds. — Plant Cell Rep. 23: 134–143, 2004.CrossRefPubMedGoogle Scholar
  15. May, G.D., Afza, R., Mason, H.A., Wiecko, A., Novak, F.J., Arntzen, C.J.: Generations of transgenic banana (Musa acuminata) plants via Agrobacterium-mediated transformation. — Biotechnology 13: 486–492, 1995.CrossRefGoogle Scholar
  16. Murashige, T., Skoog, F.: A revised medium for rapid growth and bioassays with tobacco tissue cultures. — Physiol. Plant. 15: 473–497, 1962.CrossRefGoogle Scholar
  17. Nadeem, M., Palni, L.M.S., Purohit, A.N., Pandey, H., Nandi, S.K.: Propagation and conservation of Podophyllum hexandrum Royle: an important medicinal herb. — Biol. Conserv. 92: 121–129, 2000.CrossRefGoogle Scholar
  18. Saini, R., Jaiwal, P.K.: Efficient transformation of a recalcitrant grain legumes Vigna mungo L. Hepper via Agrobacteriummediated gene transfer into shoot apical meristem cultures. — Plant Cell Rep. 24: 164–171, 2005.CrossRefPubMedGoogle Scholar
  19. Saini, R., Jaiwal, P.K.: Agrobacterium tumefaciens-mediated transformation of blackgram: an assessment of factors influencing the efficiency of uidA gene transfer. — Biol. Plant. 51: 69–74, 2007.CrossRefGoogle Scholar
  20. Schroeder, H.E., Schotz, A.H., Wardley-Richardson, T., Spencer, D., Higgins, T.J.V. Transformation and regeneration of two pea (Pisum sativum) cultivars. — Plant Physiol. 101: 751–757, 1993.CrossRefPubMedGoogle Scholar
  21. Stachel, S.E., Messens, E., Montagu, M.V., Zambryski, P.: Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. — Nature 318: 624–629, 1985.CrossRefGoogle Scholar
  22. Tiwari, R.K., Trivedi, M., Guang, Z.-C., Guo, G.-O., Zheng, G.- C.: Agrobacterium rhizogenes mediated transformation of Scutellaria baicalensis and production of flavonoids in hairy roots. — Biol. Plant. 52: 26–35, 2008.CrossRefGoogle Scholar
  23. Van Wordragen, M.F., Dons, H.J.M.: Agrobacterium tumefaciens mediated transformation of recalcitrant crops. — Plant mol. Biol. Rep. 10: 12–36, 1992.CrossRefGoogle Scholar
  24. Xiao, K., Zhang, C., Harrison, M., Wang, Z.Y.: Isolation and characterization of a novel plant promoter that directs strong constitutive expression of transgenes in plants. — Mol. Breed. 15: 221–231, 2005.CrossRefGoogle Scholar
  25. Yu, T.A., Yeh, S.D., Yang, J.S.: Comparison of the effects of kanamycin and geneticin on regeneration of papaya from root tissue. — Plant Cell Tissue Organ Cult. 74: 169–178, 2003.CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2009

Authors and Affiliations

  1. 1.Division of Forest Resources, College of Forest SciencesKangwon National UniversityKangwon-doSouth Korea
  2. 2.Research and Education Center for Genetic InformationNara Institute of Science and TechnologyNaraJapan

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