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Plant Cell, Tissue and Organ Culture

, Volume 54, Issue 3, pp 161–171 | Cite as

Agrobacterium-mediated stable transformation of cell suspension cultures of barley (Hordeum vulgare)

  • Huixia Wu
  • Alex C. McCormac
  • Malcolm C. Elliott
  • Dong-Fang Chen
Article

Abstract

Barley (Hordeum vulgare L. cvs. Igri and Dissa) cell suspension cultures, which had been initiated from immature embryo-derived (IED) and microspore-derived (MSD) callus, were co-cultivated with various Agrobacterium tumefaciens strains. The T-DNA vectors contained visually-detectable marker genes (C1/Lc orgusA-intron), as reporters of transient T-DNA transfer, and also drug resistance genes (hph or bar) to facilitate selection of stably transformed cell lines. A set of normal binary vectors in a super-virulent Agrobacterium strain [EHA101(pBECKS)] and also a super-binary vector [LBA4404(pTOK233)] were used in this study. Cells of the suspension cultures which received T-DNA were able to proliferate under selection regimes and a number of hygromycin- or phosphinothricin-resistant barley callus lines were isolated which expressed a co-transferred gusA gene. To ensure homogeneity of the cell lines, prolonged tissue culture regimes were used but these resulted in a loss of the capacity to regenerate plants from the transgenic callus lines. The frequency of recovery of transformed callus lines ranged from 0.3% to 2.9%. Southern blot analyses of the transformed callus lines confirmed the presence of the marker genes and demonstrated them to be associated with DNA which was distinct from that of the original Agrobacterium plasmid. Furthermore, independent transgenic lines showed diverse patterns of hybridising bands. These data suggest that the T-DNA fragment was stably maintained through integration into the genomes of the barley cell lines.

bar hph immature embryo microspore pBECKS pTOK233 

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References

  1. Aldemita RR & Hodges TK (1996) Agrobacterium tumefaciens-mediated transformation of japonica and indica rice varieties. Planta 199: 612–617CrossRefGoogle Scholar
  2. Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner T & Wan Y (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115: 971–980PubMedGoogle Scholar
  3. Chilton M-D, Currier TC, Farrand SK, Bendich AJ, Gordon MP & Nester EW (1974) Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumours. Proc. Nat. Acad. Sci. USA 71: 3672–3676PubMedCrossRefGoogle Scholar
  4. Chu CC & Hill RD (1988) An improved anther culture method for obtaining higher frequency of pollen embryoids in Triticum aestivum L. Plant Science 55: 175–181CrossRefGoogle Scholar
  5. Creissen G, Smith C, Francis R, Reynolds H & Mullineaux P (1990) Agrobacterium-and microprojectile-mediated viral DNA delivery into barley microspore-derived cultures. Plant Cell Reports 8: 680–683CrossRefGoogle Scholar
  6. Dong J, Teng W, Buchholz GB & Hall TC (1996) Agrobacterium-mediated transformation of Javanica rice. Molecular Breeding 2: 267–276CrossRefGoogle Scholar
  7. Hansch R, Koprek T, Mendel RR & Schulze J (1995) An improved protocol for eliminating endogenous β-glucuronidase background in barley. Plant Science 105: 63–69CrossRefGoogle Scholar
  8. Hiei Y, Ohta S, Komari T & Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6: 271–282PubMedCrossRefGoogle Scholar
  9. Hoekema A, Hirsch PR, Hooykaas PJJ & Schilperoort RA (1983) A binary vector strategy based on separation of vir-and T-region of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179–180CrossRefGoogle Scholar
  10. Hood EE, Helmer GL, Fraley RT & Chilton M-D (1986) The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. of Bacteriology 168: 1291–1301Google Scholar
  11. Ishida Y, Saito H, Ohta S, Hiei Y, Komari T & Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnology 14: 745–750PubMedCrossRefGoogle Scholar
  12. Jähne A, Becker D, Brettschneider R & Lörz H (1994) Regeneration of transgenic, microspore-derived fertile barley. Theor. Appl. Genet. 89: 525–533CrossRefGoogle Scholar
  13. Jefferson RA (1987) Assaying chimeric genes in plants: the GUS gene fusion system. Plant Mol. Biol. Rep. 5: 387–405Google Scholar
  14. McCormac AC, MC Elliott & Chen DF (1997) pBECKS: a flexible series of binary vectors for Agrobacterium-mediated plant transformation. Molecular Biotechnology 8: 199–213Google Scholar
  15. McCormac AC, Wu H, Bao M, Wang Y, Xu R, Elliott MC & Chen DF (1998) The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and Barley (Hordeum vulgare L.). Euphytica 99: 17–25CrossRefGoogle Scholar
  16. Mooney PA, Goodwin PB, Dennis ES & Llewellyn DJ (1991) Agrobacterium tumefaciens-gene transfer into wheat tissues. Plant Cell Tiss. Org. Cult. 2: 209–218Google Scholar
  17. Murashige T & Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiology Plantarum 15: 473–497CrossRefGoogle Scholar
  18. Odell JT, Nagy F & Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313: 810–812PubMedCrossRefGoogle Scholar
  19. Ohta S, Mita S, Hattori T & Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol. 31: 805–813Google Scholar
  20. Peng JY, Wen FJ, Lister RK & Hodges TK (1995) Inheritance of gusA and neo genes in transgenic rice. Plant Mol. Biol. 27: 91–104PubMedCrossRefGoogle Scholar
  21. Rashid H, Yokoi S, Toriyama K & Hinata K (1996) Transgenic plant production mediated by Agrobacterium in Indica rice. Plant Cell Reports 15: 727–730CrossRefGoogle Scholar
  22. Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual, 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory PressGoogle Scholar
  23. Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S & Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant Journal 11: 1369–1376CrossRefGoogle Scholar
  24. Vancanneyt G, Schmidt R, O'Conner-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. Gen. Genet. 220: 245–250PubMedCrossRefGoogle Scholar
  25. Wan Y & Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol. 104: 37–48PubMedGoogle Scholar
  26. Zhang J, Xu R, Elliott MC & Chen DF (1997) Agrobacterium-mediated transformation of élite indica and japonica rice cultivars. Molecular Biotechnology 8: 223–231PubMedGoogle Scholar

Copyright information

© Kluwer Academic Publishers 1998

Authors and Affiliations

  • Huixia Wu
    • 1
  • Alex C. McCormac
    • 1
  • Malcolm C. Elliott
    • 1
  • Dong-Fang Chen
    • 1
  1. 1.The Norman Borlaug Institute for Plant Science Research, De Montfort University, ScraptoftLeicesterUK

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