Macro elements in inoculation and co-cultivation medium strongly affect the efficiency of Agrobacterium-mediated transformation in Lilium

  • Pejman Azadi
  • Dong Poh Chin
  • Kiyo Kuroda
  • Raham Sher Khan
  • Masahiro MiiEmail author
Original Paper


A highly efficient Agrobacterium-mediated transformation system for Lilium × formolongi was established by modifying the medium used for inoculation and co-cultivation. Meristematic nodular calli of Lilium were inoculated with an overnight culture of A. tumefaciens strain EHA101 containing the plasmid pIG121-Hm harboring an intron-containing β-glucuronidase (GUS), hygromycin phosphotransferase, and neomycin phosphotransferase II genes. The effects of ten different types of media and carbohydrates (sucrose, d-glucose, and l-arabinose) in both inoculation and co-cultivation media were evaluated. Interestingly, a dramatic increase in the frequency of transformation (25.4%) was observed when Murashige and Skoog (MS) medium containing sucrose and lacking KH2PO4, NH4NO3, KNO3, and CaCl2 was used. Hygromycin-resistant transgenic calli were obtained only in medium supplemented with sucrose. The effects of this modified medium were also investigated for Lilium cultivars ‘Acapulco’, ‘Casa Blanca’, and ‘Red Ruby’. The highest frequency of transformation (23.3%) was obtained for cv. Acapulco. Hygromycin-resistant calli were successfully regenerated into plantlets on plant growth regulator-free MS medium. Transgenic plants were confirmed by GUS histochemical assay, polymerase chain reaction (PCR), and Southern blot analyses.


Agrobacterium tumefaciens Transformation Co-cultivation Macro elements Carbohydrate Lilium 





Cetyltrimethylammonium bromide






  1. Ahn JH, Lee JS (2003) Sugar acts as a regulatory signal on the wound-inducible expression of SbHRGP3::GUS in transgenic plants. Plant Cell Rep 22:286–293CrossRefPubMedGoogle Scholar
  2. Ahn BJ, Joung YH, Kamo KK (2004) Transgenic plants of Easter lily (Lilium longiflorum) with phosphinothricin resistance. J Plant Biotechnol 6:9–13Google Scholar
  3. Ankenbauer RG, Nester EW (1990) Sugar-mediated induction of Agrobacterium tumefaciens virulence genes: structural specificity and activities of monosaccharides. J Bacteriol 172:6442–6446PubMedGoogle Scholar
  4. Brencic A, Winans SC (2005) Detection of and response to signals involved in host–microbe interactions by plant-associated bacteria. Microbiol Mol Biol Rev 69:155–194CrossRefPubMedGoogle Scholar
  5. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980PubMedGoogle Scholar
  6. Citovsky V, Kozlovsky SV, Lacroix B, Zaltsman A, Dafny-Yelin M, Vyas S, Tovkach A, Tzfira T (2007) Biological systems of the host cell involved in Agrobacterium infection. Cell Microbiol 9:9–20CrossRefPubMedGoogle Scholar
  7. Cohen A, Lipsky A, Arazi T, Ion A, Stav R, Sandler-Ziv D, Pintea C, Barg R, Salts Y, Shabtai S, Gaba V, Gera A (2004) Efficient genetic transformation of Lilium longiflorum and Ornithogalum dubium by particle acceleration followed by prolonged selection in liquid medium. Acta Hort 651:131–138Google Scholar
  8. Danhorn T, Hentzer M, Givskov M, Parsek MR, Fuqua C (2004) Phosphorus limitation enhances biofilm formation of the plant pathogen Agrobacterium tumefaciens through the PhoR–PhoB regulatory system. J Bacteriol 186:4492–4501CrossRefPubMedGoogle Scholar
  9. de Oliveira R Machado L, de Andrade GM, Barreto Cid LP, Penchel RM, Brasileiro ACM (1997) Agrobacterium strain specificity and shooty tumour formation in eucalypt (Eucalyptus grandis × E. urophylla). Plant Cell Rep 16:299–303Google Scholar
  10. Dupré P, Lacoux J, Neutelings G, Mattar-Laurain D, Fliniaux MA, David A, Jacquin-Dubreuil A (2000) Genetic transformation of Ginkgo biloba by Agrobacterium tumefaciens. Physiol Plant 108:413–419Google Scholar
  11. Flego D, Pirhonen M, Saarilahti H, Palva TK, Palva ET (1997) Control of virulence gene expression by plant calcium in the phytopathogen Erwinia carotovora. Mol Microbiol 25:831–838CrossRefPubMedGoogle Scholar
  12. Hamill JD, Rounsley S, Spencer A, Todd G, Rhodes MJC (1991) The use of the polymerase chain reaction in plant transformation studies. Plant Cell Rep 10:221–224CrossRefGoogle Scholar
  13. Hoshi Y, Kondo M, Mori S, Adachi Y, Nakano M, Kobayashi H (2004) Production of transgenic lily plants by Agrobacterium-mediated transformation. Plant Cell Rep 22:359–364CrossRefPubMedGoogle Scholar
  14. Irifune K, Morimoto Y, Uchihama M (2003) Production of herbicide resistant transgenic lily plants by particle bombardment. J Jpn Soc Hortic Sci 72:511–516CrossRefGoogle Scholar
  15. Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907PubMedGoogle Scholar
  16. Kamo K, Han BH (2008) Biolistic-mediated transformation of Lilium longiflorum cv. Nellie White. Hortic Sci 43:1864–1869Google Scholar
  17. McCullen CA, Binns AN (2006) Agrobacterium tumefaciens and plant cell interactions and activities required for interkingdom macromolecular transfer. Annu Rev Cell Dev Biol 22:101–127CrossRefPubMedGoogle Scholar
  18. Mercuri A, Benedetti LD, Bruna S, Bregliano R, Bianchini C, Foglia G, Schiva T (2003) Agrobacterium-mediated transformation with rol genes of Lilium longiflorum Thunb. Acta Hort 612:129–136Google Scholar
  19. Mii M, Yuzawa Y, Suetomi H, Motegi T, Godo T (1994) Fertile plant regeneration from protoplasts of a seed-propagated cultivar of Lilium × formolongi by utilizing meristematic nodular cell clumps. Plant Sci 100:221–226CrossRefGoogle Scholar
  20. Montoro P, Teinseree N, Rattana W, Kongsawadworakul P, Michaux-Ferriere N (2000) Effect of exogenous calcium on Agrobacterium tumefaciens-mediated gene transfer in Hevea brasiliensis (rubber tree) friable calli. Plant Cell Rep 19:851–855CrossRefGoogle Scholar
  21. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  22. Murray MG, Thompson WF (1980) Rapid isolation of high molecular weight plant DNA. Nucleic Acids Res 8:4321–4325CrossRefPubMedGoogle Scholar
  23. Ogaki M, Furuichi Y, Kuroda K, Chin DP, Ogawa Y, Mii M (2008) Importance of co-cultivation medium pH for successful Agrobacterium-mediated transformation of Lilium × formolongi. Plant Cell Rep 27:699–705CrossRefPubMedGoogle Scholar
  24. Ogawa Y, Mii M (2007) Meropenem and moxalactam: novel β-lactam antibiotics for efficient Agrobacterium-mediated transformation. Plant Sci 172:564–572CrossRefGoogle Scholar
  25. Palmer AG, Gao R, Maresh J, Erbil WK, Lynn DG (2004) Chemical biology of multi-host/pathogen interactions: chemical perception and metabolic complementation. Annu Rev Phytopathol 42:439–464CrossRefPubMedGoogle Scholar
  26. Phelep M, Petit A, Martin L, Duhoux E, Tempé J (1991) Transformation and regeneration of a nitrogen-fixing tree, Allocasuarina verticillata Lam. Bio/Technology 9:461–466CrossRefGoogle Scholar
  27. Romantschuk M (1992) Attachment of plant pathogenic bacteria to plant surfaces. Annu Rev Phytopathol 30:225–243CrossRefPubMedGoogle Scholar
  28. Shimoda N, Toyoda-Yamamoto A, Nagamine J, Usami S, Katayama M, Sakagami Y, Machida Y (1990) Control of expression of Agrobacterium vir genes by synergistic actions of phenolic signal molecules and monosaccharides. Proc Natl Acad Sci USA 87:6684–6688CrossRefPubMedGoogle Scholar
  29. Stachel SE, Nester EW (1986) The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens. EMBO J 5:1445–1454PubMedGoogle Scholar
  30. Stomp AM (1992) Histochemical localization of β-glucuronidase. In: Gallagher SR (ed) GUS protocols: using the GUS gene as a reporter of gene expression. Academic Press, San Diego, pp 103–113Google Scholar
  31. Tzfira T, Yarnitzky O, Vainstein A, Altman A (1996) Agrobacterium rhizogenes-mediated DNA transfer in Pinus halepensis Mill. Plant Cell Rep 16:26–31CrossRefGoogle Scholar
  32. Watad AA, Yun DJ, Matsumoto T, Niu X, Wu Y, Kononowicz AK, Bressan RA, Hasegawa PM (1998) Microprojectile bombardment-mediated transformation of Lilium longiflorum. Plant Cell Rep 17:262–267CrossRefGoogle Scholar
  33. Winans SC (1990) Transcriptional induction of an Agrobacterium regulatory gene at tandem promoters by plant-released phenolic compounds, phosphate starvation, and acidic growth media. J Bacteriol 172:2433–2438PubMedGoogle Scholar
  34. Wise AA, Voinov L, Binns AN (2005) Intersubunit complementation of sugar signal transduction in VirA heterodimers and posttranslational regulation of VirA activity in Agrobacterium tumefaciens. J Bacteriol 187:213–223CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2010

Authors and Affiliations

  • Pejman Azadi
    • 1
  • Dong Poh Chin
    • 1
  • Kiyo Kuroda
    • 1
  • Raham Sher Khan
    • 1
  • Masahiro Mii
    • 1
    Email author
  1. 1.Laboratory of Plant Cell Technology, Faculty of HorticultureChiba UniversityMatsudo City, ChibaJapan

Personalised recommendations