, Volume 249, Issue 1, pp 53–63 | Cite as

ER disruption and GFP degradation during non-regenerable transformation of flax with Agrobacterium tumefaciens

  • Juraj Bleho
  • Bohuš Obert
  • Tomáš Takáč
  • Beáta Petrovská
  • Claudia Heym
  • Diedrik Menzel
  • Jozef ŠamajEmail author
Original Paper


Flax is considered as plant species susceptible to Agrobacterium-mediated genetic transformation. In this study, stability of flax transformation by Agrobacterium rhizogenes versus Agrobacterium tumefaciens was tested by using combined selection for antibiotic resistance and visual selection of green fluorescent protein (GFP)-fusion reporter targeted to the endoplasmic reticulum (ER). Transformation with A. rhizogenes was stable for over 2 years, whereas transformation by A. tumefaciens resulted in non-regenerable stable transformation which was restricted solely to transgenic callus and lasted only 6–8 weeks. However, shoots regenerated from this callus appeared to be non-transgenic. Importantly, callus and root cells stably transformed with A. rhizogenes showed typical regular organization and dynamics of ER as visualized by GFP-ER marker. On the other hand, callus cells transformed with A. tumefaciens showed disintegrated ER structure and impaired dynamics which was accompanied with developmental degradation of GFP. Consequently, shoots which regenerated from such callus were all non-transgenic. Possible reasons for this non-regenerable flax transformation by A. tumefaciens are discussed.


Agrobacterium rhizogenes Agrobacterium tumefaciens Endoplasmic reticulum Flax GFP Non-regenerable transformation Stable transformation 



We thank Dr. Bekir Ülker (IZMB Bonn) for critical reading of the manuscript and useful suggestions. This work was supported by Grant No. ED0007/01/01 Centre of the Region Haná for Biotechnological and Agricultural Research.

Conflict of interest

The authors declare that they have no conflict of interest.


  1. Abbadi A, Domergue F, Bauer J, Napier JA, Welti R, Zahringer U, Cirpus P, Heinz E (2004) Biosynthesis of very-long-chain polyunsaturated fatty acids in transgenic oilseeds: constraints on their accumulation. Plant Cell 16:2734–2748PubMedCrossRefGoogle Scholar
  2. Basiran N, Armitage P, Scott RJ, Draper J (1987) Genetic transformation of flax (Linum usitatissimum) by Agrobacterium tumefaciens regeneration of transformed shoots via a callus phase. Plant Cell Rep 6:396–399CrossRefGoogle Scholar
  3. Beranová M, Rakouský S, Vavrová Z, Skalický T (2008) Sonication assisted Agrobacterium-mediated transformation enhances the transformation efficiency in flax (Linum usitatissimum L.). Plant Cell Tissue Organ Cult 94:253–259CrossRefGoogle Scholar
  4. Boisson-Dernier A, Chabaud M, Garcia F, Becard G, Rosenberg C, Barker DG (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatulata for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant-Microbe Interact 14:695–700PubMedCrossRefGoogle Scholar
  5. Bretagne-Sagnard B, Chupeau Y (1996) Selection of transgenic flax plants is facilitated by spectinomycin. Trans Res 5:131–137CrossRefGoogle Scholar
  6. Chalfie M, Tu Y, Euskirchen G, Ward WW, Prasher DC (1994) Green fluorescent protein as a marker for gene-expression. Science 263:802–805PubMedCrossRefGoogle Scholar
  7. Chiu W, Niva Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter in plants. Curr Biol 6:325–330PubMedCrossRefGoogle Scholar
  8. Ditt RF, Kerr KF, de Figueiredo P, Delrow J, Comai L, Nester EW (2006) The Arabidopsis thaliana transcriptome in response to Agrobacterium tumefaciens. Mol Plant Microbe Interact 19:665–681PubMedCrossRefGoogle Scholar
  9. Dong JZ, McHughen A (1993a) An improved procedure for production of transgenic flax plants using Agrobacterium tumefaciens. Plant Sci 88:61–71CrossRefGoogle Scholar
  10. Dong JZ, McHughen A (1993b) Transgenic flax plants from Agrobacterium tumefaciens transformation—incidence of chimeric regenerants and inheritance of transgenic plants. Plant Sci 91:139–148CrossRefGoogle Scholar
  11. Duan YX, Liu X, Fan J, Li DL, Wu RCh, Guo WW (2007) Multiple shoot induction from seedling epicotyls and transgenic citrus plant regeneration containing the green fluorescent protein gene. Bot Stud 48:165–171Google Scholar
  12. Fahraeus G (1957) The infection of clover root hairs by nodule bacteria studied by a simple glass slide technique. J Gen Microbiol 16:374–381PubMedGoogle Scholar
  13. Franklin G, Conceicao LF, Kombrink E, Dias AC (2008) Hypericum perforatum plant cells reduce Agrobacterium viability during co-cultivation. Planta 227:1401–1408PubMedCrossRefGoogle Scholar
  14. Franklin G, Conceicao LF, Kombrink E, Dias AC (2009) Xanthone biosynthesis in Hypericum perforatum cells provides antioxidant and antimicrobial protection upon biotic stress. Phytochemistry 70:65–73CrossRefGoogle Scholar
  15. Gamborg OL, Shyluk JP (1976) Tissue culture, protoplast, and morphogenesis in flax. Bot Gaz 137:301–306CrossRefGoogle Scholar
  16. Genre A, Ortu G, Bertoldo C, Martino E, Bonfante P (2009) Biotic and abiotic stimulation of root epidermal cells reveals common and specific responses to arbuscular mycorrhizal fungi. Plant Physiol 149:1424–1434PubMedCrossRefGoogle Scholar
  17. Hano C, Martin I, Fliniaux O, Legrand B, Gutierrez L, Arroo RRJ, Mesnard F, Lamblin F, Laine E (2006) Pinoresinol-lariciresinol reductase gene expression and secisolariciresinol diglucoside accumulation in developing flax (Linum usitatissimum) seeds. Planta 224:1291–1301PubMedCrossRefGoogle Scholar
  18. Haseloff J, Siemering KR, Prasher DC, Hodge S (1997) Removal of a cryptic intron and subcellular localization of green fluorescent protein are required to mark transgenic Arabidopsis plants brightly. Proc Natl Acad Sci USA 94:2122–2127PubMedCrossRefGoogle Scholar
  19. Heim R, Cubitt AE, Tsien RY (1995) Improved green fluorescence. Nature 373:663–664PubMedCrossRefGoogle Scholar
  20. Hepburn AG, Clarke LE, Blundy KS, White J (1983) Nopaline Ti-plasmid, pTiT37, T-DNA insertions into flax genome. J Mol Appl Genet 2:211–224PubMedGoogle Scholar
  21. Hraška M, Heřmanová V, Rakouský S, Čurn V (2008) Sample topography and position within plant body influence the detection of the intensity of green fluorescent protein fluorescence in the leaves of transgenic tobacco plants. Plant Cell Rep 27:67–77PubMedGoogle Scholar
  22. Hraška M, Rakouský S, Čurn V (2009) Green fluorescent protein as a vital marker for non-destructive detection of transformation events in transgenic plants. Plant Cell Tissue Organ Cult 86:303–318Google Scholar
  23. Hurkman WJ, Tanaka CK (1986) Solubilization of plant membrane proteins for analysis by two-dimensional gel-electrophoresis. Plant Physiol 81:802–806PubMedCrossRefGoogle Scholar
  24. Jordan MC, McHughen A (1988a) Glyphosate tolerant flax plants from Agrobacterium mediated gene transfer. Plant Cell Rep 7:281–284CrossRefGoogle Scholar
  25. Jordan MC, McHughen A (1988b) Transformed callus does not necessarily regenerate transformed shoots. Plant Cell Rep 7:285–287CrossRefGoogle Scholar
  26. Lacoux J, Klein D, Domon JM, Burel C, Lamblin F, Alexandre F, Sihachakr D, Roger D, Lamblin F, Aime A, Hano Ch, Roussy I, Domon J-M, Droogenbroeck BV, Laine E (2007) The use of phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum L.). Plant Cell Rep 26:765–772CrossRefGoogle Scholar
  27. Lamblin F, Aime A, Hano CH, Roussy I, Domon JM, Droogenbroeck BV, Laine E (2007) The use of phosphomannose isomerase gene as alternative selectable marker for Agrobacterium-mediated transformation of flax (Linum usitatissimum L.). Plant Cell Rep 26:765–772Google Scholar
  28. McCubbin AG, Chung YY, Kao Th (1997) A Mutant S3 RNase of Petunia inflata lacking RNase activity has an allele-specific dominant negative effect on self-incompatibility interactions. Plant Cell 9:85–95PubMedCrossRefGoogle Scholar
  29. McHughen A, Jordan M, Feist G (1989) A preculture period prior to Agrobacterium tumefaciens inoculation increases production of transgenic plants. J Plant Physiol 135:245–248Google Scholar
  30. Mlynárová L, Bauer M, Nap JP, Preťová A (1994) High efficiency Agrobacterium-mediated gene transfer to flax. Plant Cell Rep 13:282–285CrossRefGoogle Scholar
  31. Molinier J, Himber C, Hahne G (2000) Use of green fluorescent protein for detection of transformed shoots and homozygous offspring. Plant Cell Rep 19:219–223CrossRefGoogle Scholar
  32. Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassay with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  33. Musialak M, Wrobel-Kwiatkowska M, Kulma A, Starzycka E, Szopa J (2007) Improving retting of fibre through genetic modification of flax to express pectinases. Trans Res 17:133–147CrossRefGoogle Scholar
  34. Ooms G, Hooykaas PJ, Van Veen RJ, Van Beelen P, Regensburg-Tuink TJ, Schilperoort RA (1982) Octopine Ti-plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7:15–29PubMedCrossRefGoogle Scholar
  35. Prasher DC, Eckenrode VK, Ward WW, Prendergast FG, Cormier MJ (1992) Primary structure of the Aequorea victoria green-fluorescent protein. Gene 111:229–233PubMedCrossRefGoogle Scholar
  36. Pruss GJ, Nester EW, Vance V (2008) Infiltration with Agrobacterium tumefaciens induces host defense and development-dependent responses in the infiltrated zone. Mol Plant Microbe Interact 21:1528–1538PubMedCrossRefGoogle Scholar
  37. Quandt N, Stindl A, Keller U (1993) Sodium dodecyl-sulfate polyacrylamide-gel electrophoresis for M(r) estimation of high-molecular weight polypeptides. Anal Biochem 214:490–494PubMedCrossRefGoogle Scholar
  38. Rakouský S, Tejklová E, Wiesner I, Wiesnerová D, Kocábek T, Ondřej M (1999) Hygromycin B—an alternative in flax transformant selection. Biol Plant 42:361–369CrossRefGoogle Scholar
  39. Saika H, Toki S (2009) Visual selection allows immediate identification of transgenic rice calli efficiently accumulating transgene products. Plant Cell Rep 28:619–626PubMedCrossRefGoogle Scholar
  40. Siemering KR, Golbik R, Sever R, Haseloff J (1996) Mutations that suppress the thermosensitivity of green fluorescent protein. Curr Biol 6:1653–1663PubMedCrossRefGoogle Scholar
  41. Stewart CN Jr (2001) The utility of green fluorescent protein in transgenic plants. Plant Cell Rep 20:376–382PubMedCrossRefGoogle Scholar
  42. Stewart CN Jr (2005) Monitoring the presence and expression of transgenes in living plants. Trends Plant Sci 8:390–396CrossRefGoogle Scholar
  43. Wang YS, Yoo ChM, Blancaflor EB (2008) Improved imaging of actin filaments in transgenic Arabidopsis plants expressing a green fluorescent protein fusion to the C- and N-termini of the fimbrin actin-binding domain 2. New Phytol 177:525–535PubMedGoogle Scholar
  44. Wrobel M, Zebrowski J, Szopa J (2004) Polyhydroxybutyrate synthesis in transgenic flax. J Biotechnol 107:41–54PubMedCrossRefGoogle Scholar
  45. Xu SX, Cai XD, Tan B, Guo WW (2010) Comparison of expression of three different sub-cellular targeted GFPs in transgenic Valencia sweet orange by confocal laser scanning microscopy. Plant Cell Tissue Organ Cult. doi: 10.1007/s11240-010-9819-0 Google Scholar
  46. Zhan XC, Jones DA, Kerr A (1988) Regeneration of flax plants transformed by Agrobacterium rhizogenes. Plant Mol Biol 11:551–559CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 2011

Authors and Affiliations

  • Juraj Bleho
    • 3
  • Bohuš Obert
    • 3
  • Tomáš Takáč
    • 1
  • Beáta Petrovská
    • 4
  • Claudia Heym
    • 2
  • Diedrik Menzel
    • 2
  • Jozef Šamaj
    • 1
    Email author
  1. 1.Centre of the Region Haná for Biotechnological and Agricultural Research, Department of Cell Biology, Faculty of SciencePalacký UniversityOlomoucCzech Republic
  2. 2.Institute of Cellular and Molecular BotanyUniversity of BonnBonnGermany
  3. 3.Institute of Plant Genetics and BiotechnologySlovak Academy of SciencesNitraSlovak Republic
  4. 4.Institute of Experimental BotanyAcademy of Sciences of the Czech RepublicSokolovská 6OlomoucCzech Republic

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