Skip to main content
Log in

A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.

  • Original Paper
  • Published:
Transgenic Research Aims and scope Submit manuscript

An Erratum to this article was published on 01 July 2008

Abstract

In the ongoing process of developing Brachypodium distachyon as a model plant for temperate cereals and forage grasses, we have developed a high-throughput Agrobacterium-mediated transformation system for a diploid accession. Embryogenic callus, derived from immature embryos of the accession BDR018, were transformed with Agrobacterium tumefaciens strain AGL1 carrying two T-DNA plasmids, pDM805 and pWBV-Ds-Ubi-bar-Ds. Transient and stable transformation efficiencies were optimised by varying the pre-cultivation period, which had a strong effect on stable transformation efficiency. On average 55% of 17-day-old calli co-inoculated with Agrobacterium regenerated stable transgenic plants. Stable transformation frequencies of up to 80%, which to our knowledge is the highest transformation efficiency reported in graminaceous species, were observed. In a study of 177 transgenic lines transformed with pDM805, all of the regenerated transgenic lines were resistant to BASTA®, while the gusA gene was expressed in 88% of the transgenic lines. Southern blot analysis revealed that 35% of the tested plants had a single T-DNA integration. Segregation analysis performed on progenies of ten selected T0 plants indicated simple Mendelian inheritance of the two transgenes. Furthermore, the presence of two selection marker genes, bar and hpt, on the T-DNA of pWBV-Ds-Ubi-bar-Ds allowed us to characterize the developed transformation protocol with respect to full-length integration rate. Even when not selected for, full-length integration occurred in 97% of the transformants when using bialaphos as selection agent.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3

Similar content being viewed by others

Abbreviations

2,4-D:

2,4-Dichlorophenoxyacetic acid

GUS:

Glucuronidase

MG/L liquid medium:

Garfinkel and Nester (1980) medium

References

  • Bablak P, Draper J, Davey MR, Lynch PT (1995) Plant regeneration and micropropagation of Brachypodium distachyon. Plant Cell Tissue Organ Cult 42:97–107

    Article  Google Scholar 

  • Bajaj S, Ran Y, Phillips J, Kularajathevan G, Pal S, Cohen D, Elborough K, Puthigae S (2006) A high throughput Agrobacterium tumefaciens-mediated transformation method for functional genomics of perennial ryegrass (Lolium perenne L.). Plant Cell Rep 25/7:651–659

    Article  CAS  Google Scholar 

  • Bettany AJE (2003) Agrobacterium tumefaciens-mediated transformation of forage grasses. ISB News Report

  • Binns AN, Thomashow MF (1988) Cell biology of Agrobacterium infection and transformation of plants. Annu Rev Microbiol 42:575–606

    Article  CAS  Google Scholar 

  • Cao X, Liu Q, Rowland LJ, Hammerschlag FA (1998) GUS expression in blueberry (Vaccinium spp.): factors influencing Agrobacterium-mediated gene transfer efficiency. Plant Cell Rep 18:266–270

    Article  CAS  Google Scholar 

  • Chateau S, Sangwan RS, Sangwan-Norreel BS (2000) Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormone. J Exp Bot 51/353:1961–1968

    Article  Google Scholar 

  • Cheng M, Fry JE, Pang S, Zhou H, Hironaka CM, Duncan DR, Conner TW, Wan Y, Cheng M, Pang SZ, Zhou HP, Wan YC (1997) Genetic transformation of wheat mediated by Agrobacterium tumefaciens. Plant Physiol 115:971–980

    PubMed  CAS  Google Scholar 

  • Christiansen P, Andersen CH, Didion T, Folling M, Nielsen KK (2005) A rapid and efficient transformation protocol for the grass Brachypodium distachyon. Plant Cell Rep 23:751–758

    Article  PubMed  CAS  Google Scholar 

  • D’Halluin K, De Block M, Denecke J, Janssens J, Leemans J, Reynaerts A, Botterman J (1992) The bar gene as selectable and screenable marker in plant engineering. Methods Enzymol 216:415–426

    Article  PubMed  CAS  Google Scholar 

  • Devos KM, Beales J, Nagamura Y, Sasaki T (1999) Arabidopsis-rice: will colinearity allow gene prediction across the eudicot-monocot divide? Genome Res 9:825–829

    Article  PubMed  CAS  Google Scholar 

  • Draper J, Mur LAJ, Jenkins G, Ghosh-Biswas GC, Bablak P, Hasterok R, Routledge APM (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol 127:1539–1555

    Article  PubMed  CAS  Google Scholar 

  • Engvild KC (2005) Mutagenesis of the model grass Brachypodium distachyon with sodium azide Risoe-R-1510 (EN). Report, Risoe National Laboratory, Roskilde, Denmark

  • Garfinkel DJ, Nester EW (1980) Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism. J Bacteriol 144:732–743

    PubMed  CAS  Google Scholar 

  • Ge Y, Norton T, Wang ZY (2006) Transgenic zoysiagrass (Zoysia japonica) plants obtained by Agrobacterium-mediated transformation. Plant Cell Rep 25(8):792–798

    Article  PubMed  CAS  Google Scholar 

  • Gordon-Kamm W, Dilkes BP, Lowe K, Hoerster G, Sun X, Ross M, Church L, Bunde C, Farrell J, Maddock S, Snyder J, Sykes L, Li Z, Woo YM, Bidney D, Larkins BA (2002) Stimulation of the cell cycle and maize transformation by disruption of the plant retinoblastoma pathway. Proc Natl Acad Sci USA 99:11975–11980

    Article  PubMed  CAS  Google Scholar 

  • Han KH, Meilan R, Ma C, Strauss SH (2000) An Agrobacterium tumefaciens transformation protocol effective on a variety of cottonwood hybrids (genus Populus). Plant Cell Rep 19:315–320

    Article  CAS  Google Scholar 

  • Hasterok R, Draper J, Jenkins G (2004) Laying the cytotaxonomic foundations of a new model grass, Brachypodium distachyon (L.) Beauv. Chromosome Res 12:397–403

    Article  PubMed  CAS  Google Scholar 

  • Hasterok R, Marasek A, Donnison IS, Armstead I, Thomas A, King IP, Wolny E, Idziak D, Draper J, Jenkins G (2006) Alignment of the genomes of Brachypodium distachyon and temperate cereals and grasses using bacterial artificial chromosome landing with fluorescence in situ hybridization. Genetics 173:349–362

    Article  PubMed  CAS  Google Scholar 

  • 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 J 6:271–282

    Article  PubMed  CAS  Google Scholar 

  • Hu T, Metz S, Chay C, Zhou HP, Biest N, Chen G, Cheng M, Feng X, Radionenko M, Lu F, Fry J (2003) Agrobacterium-mediated large-scale transformation of wheat (Triticum aestivum L.) using glyphosate selection. Plant Cell Rep 21:1010–1019

    Article  PubMed  CAS  Google Scholar 

  • 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. Nat Biotechnol 14:745–750

    Article  PubMed  CAS  Google Scholar 

  • Jefferson RA, Kavanagh TA, Bevan MW (1987) Gus fusions—beta-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6(13):3901–3907

    PubMed  CAS  Google Scholar 

  • Jenkins G, Mur LAJ, Bablak P, Hasterok R, Draper J (2005) Prospects for functional genomics in a new model grass. In: Leister D (ed) Plant functional genomics. Haworth Press, Binghampton, NY, pp 305–325

    Google Scholar 

  • Kartzke S, Saedler H, Meyer P (1990) Molecular analysis of transgenic plants derived from transformations of protoplasts at various stages of the cell-cycle. Plant Sci 67:63–72

    Article  CAS  Google Scholar 

  • Keller B, Feuillet C (2000) Colinearity and gene density in grass genomes. Trends Plant Sci 5:246–251

    Article  PubMed  CAS  Google Scholar 

  • Lucca P, Ye X, Potrykus I (2001) Effective selection and regeneration of transgenic rice plants with mannose as selective agent. Mol Breed 7:43–49

    Article  CAS  Google Scholar 

  • Montoro P, Rattana W, Pujade-Renaud V, Michaux-Ferriere N, Monkolsook Y, Kanthapura R, Adunsadthapong S (2003) Production of Hevea brasiliensis transgenic embryogenic callus lines by Agrobacterium tumefaciens: roles of calcium. Plant Cell Rep 21:1095–1102

    Article  PubMed  CAS  Google Scholar 

  • Popelka JC, Altpeter F (2003) Agrobacterium tumefaciens-mediated genetic transformation of rye (Secale cereale L.). Mol Breed 11:203–211

    Article  CAS  Google Scholar 

  • Routledge APM, Shelley G, Smith JV, Talbot NJ, Draper J, Mur LAJ (2004) Magnaporthe grisea interactions with the model grass Brachypodium distachyon closely resemble those with rice (Oryza sativa). Mol Plant Pathol 5:253–265

    Article  CAS  Google Scholar 

  • Sallaud C, Meynard D, van Boxtel J, Gay C, Bes M, Brizard JP, Larmande P, Ortega D, Raynal M, Portefaix M, Ouwerkerk PBF, Delseny M, Guiderdoni E (2003) Highly efficient production and characterization of T-DNA plants for rice (Oryza sativa L.) functional genomics. Theor Appl Genet 106:1396–1408

    PubMed  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press (ed), New York

  • Sangwan RS, Bourgeois Y, Brown S, Vasseur G, Sangwan-Norreel B (1992) Characterization of competent cells and early events of Agrobacterium-mediated genetic transformation in Arabidopsis thaliana. Planta 188:439–456

    Article  CAS  Google Scholar 

  • Spencer PA, Towers GHN (1991) Restricted occurrence of acetophenone signal compounds. Phytochemistry 30:2933–2937

    Article  CAS  Google Scholar 

  • Stachel S, Messens E, Van Montagu M, Zambryski P (1985) Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318:624–629

    Article  Google Scholar 

  • Suzuki S, Nakano M (2002) Agrobacterium-mediated production of transgenic plants of Muscari armeniacum Leichtl. ex Bak. Plant Cell Rep 20:835–841

    Article  CAS  Google Scholar 

  • Tikhonov AP, San Miguel PJ, Nakajima Y, Gorenstein NM, Bennetzen JL, Avramova Z (1999) Colinearity and its exceptions in orthologous adh regions of maize and sorghum. Proc Natl Acad Sci USA 96:7409–7414

    Article  PubMed  CAS  Google Scholar 

  • Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376

    Article  CAS  Google Scholar 

  • Trifonova A, Madsen S, Olesen A (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:871–880

    Article  CAS  Google Scholar 

  • Vain P, Afolabi AS, Worland B, Snape JW (2003) Transgene behaviour in populations of rice plants transformed using a new dual binary vector system: pGreen/pSoup. Theor Appl Genet 107:210–217

    Article  PubMed  CAS  Google Scholar 

  • Valvekens D, Van Montagu M, Van Lijsebettens M (1988) Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana roots explants by using kanamycin selection. Proc Natl Acad Sci USA 85:5536–5540

    Article  PubMed  CAS  Google Scholar 

  • Villemont E, Dubois F, Sangwan RS, Vasseur G, Bourgeois Y, Sangwan-Norreel BS (1997) Role of the host cell cycle in the Agrobacterium-mediated genetic transformation of Petunia: evidence of an S-phase control mechanism for T-DNA transfer. Planta 201:160–172

    Article  CAS  Google Scholar 

  • Vogel JP, Garvin DF, Leong OM, Hayden DM (2006) Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tissue Organ Cult 84:199–211

    Article  Google Scholar 

  • Wu C, Li X, Yuan W, Chen G, Kilian A, Li J, Xu C, Li X, Zhou DX, Wang S, Zhang Q (2003) Development of enhancer trap lines for functional analysis of the rice genome. Plant J 35:418–427

    Article  PubMed  CAS  Google Scholar 

  • Zhao T, Palotta M, Langridge P, Prasad M, Graner A, Schulze-Lefert P, Koprek T (2006) Mapped Ds/T-DNA launch pads for functional genomics in barley. Plant J 47:811–826

    Article  PubMed  Google Scholar 

Download references

Acknowledgements

We thank Dr. Thomas Koprek for providing the vector pWBV-Ds-Ubi-bar-Ds, and Drs. Marianne Folling, Caixia Gao and Dale Godfrey for critical reading of the manuscript. We are grateful to Dr. Niels Roulund for advice on statistical analysis. This research was financially supported by a Marie Curie Training Site (HPMT-CT-2000-00194) with HTC under the EU-FP5 in collaboration with DLF-TRIFOLIUM A/S.

Author information

Authors and Affiliations

Authors

Corresponding author

Correspondence to Ingo Lenk.

Additional information

An erratum to this article can be found at http://dx.doi.org/10.1007/s11248-008-9195-2

Electronic supplementary material

Rights and permissions

Reprints and permissions

About this article

Cite this article

Păcurar, D.I., Thordal-Christensen, H., Nielsen, K.K. et al. A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L.. Transgenic Res 17, 965–975 (2008). https://doi.org/10.1007/s11248-007-9159-y

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11248-007-9159-y

Keywords

Navigation