Application of Tissue Culture and Transformation Techniques in Model Species Brachypodium distachyon

  • Bahar Sogutmaz OzdemirEmail author
  • Hikmet BudakEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1667)


Brachypodium distachyon has recently emerged as a model plant species for the grass family (Poaceae) that includes major cereal crops and forage grasses. One of the important traits of a model species is its capacity to be transformed and ease of growing both in tissue culture and in greenhouse conditions. Hence, plant transformation technology is crucial for improvements in agricultural studies, both for the study of new genes and in the production of new transgenic plant species. In this chapter, we review an efficient tissue culture and two different transformation systems for Brachypodium using most commonly preferred gene transfer techniques in plant species, microprojectile bombardment method (biolistics) and Agrobacterium-mediated transformation.

In plant transformation studies, frequently used explant materials are immature embryos due to their higher transformation efficiencies and regeneration capacity. However, mature embryos are available throughout the year in contrast to immature embryos. We explain a tissue culture protocol for Brachypodium using mature embryos with the selected inbred lines from our collection. Embryogenic calluses obtained from mature embryos are used to transform Brachypodium with both plant transformation techniques that are revised according to previously studied protocols applied in the grasses, such as applying vacuum infiltration, different wounding effects, modification in inoculation and cocultivation steps or optimization of bombardment parameters.

Key words

Brachypodium distachyon Microprojectile bombardment (Biolistics) Agrobacterium-mediated transformation Plant tissue culture Plant transformation Mature embryo-derived callus culture 



This work was supported by TÜBA-GEBİP and Sabancı University.


  1. 1.
    Draper J, Mur LAJ, Jenkins G et al (2001) Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol 127:1539–1555CrossRefPubMedPubMedCentralGoogle Scholar
  2. 2.
    Ozdemir BS, Hernandez P, Filiz E, Budak H (2008) Brachypodium genomics. Int J Plant Genomics 2008:Article ID 536104. doi: 10.1155/2008/536104 CrossRefGoogle Scholar
  3. 3.
    International Brachypodium Initiative (2010) Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463:763–768CrossRefGoogle Scholar
  4. 4.
    Bablak P, Draper J, Davey MR, Lynch PT (1995) Plant regeneration and micropropagation of Brachypodium distachyon. Plant Cell Tissue Organ Cult 42:97–107CrossRefGoogle Scholar
  5. 5.
    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 Org Cult 84:199–211CrossRefGoogle Scholar
  6. 6.
    Christiansen P, Didion T, Andersen C, Folling M, Nielsen K (2005) A rapid and efficient transformation protocol for the grass Brachypodium distachyon. Plant Cell Rep 23:751–758CrossRefPubMedGoogle Scholar
  7. 7.
    Pacurar DI, Thordal-Christensen H, Nielsen KK, Lenk I (2007) A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L. Transgenic Res 17:965–975CrossRefPubMedGoogle Scholar
  8. 8.
    Vogel J, Hill T (2007) High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep 27:471–478CrossRefPubMedGoogle Scholar
  9. 9.
    Vain P, Worland B, Thole V, McKenzie N, Alves SC, Opanowicz M, Fish LJ, Bevan MW, Snape JW (2008) Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis. Plant Biotechnol J 6(3):236–245CrossRefPubMedGoogle Scholar
  10. 10.
    Alves SC, Worland B, Thole V, Snape JW, Bevan MW, Vain P (2009) A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat Protoc 4(5):638–649CrossRefPubMedGoogle Scholar
  11. 11.
    Lee MB, Jeon WB, Kim DY, Bold O, Hong MJ, Lee YJ, Park JH, Seo YW (2011) Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21 with two binary vectors containing hygromycin resistance and GUS reporter genes. J Crop Sci Biotechnol 14(4):233–238CrossRefGoogle Scholar
  12. 12.
    Fursova O, Pogorelko G, Zabotina OA (2012) An efficient method for transient gene expression in monocots applied to modify the Brachypodium distachyon cell wall. Ann Bot. doi: 10.1093/aob/mcs103
  13. 13.
    Trabucco GM, Matos DA, Lee SJ, Saathoff AJ, Priest HD, Mockler TC, Sarath G, Hazen SP (2013) Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon. BMC Biotechnol 13(1):61CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Filiz E, Ozdemir BS, Budak F, Vogel JP, Tuna M, Budak H (2009) Molecular, morphological and cytological analysis of diverse Brachypodium distachyon inbred lines. Genome 52(10):876–890CrossRefPubMedGoogle Scholar
  15. 15.
    Nadolska-Orczyk A, Orczyk W, Przetakiewiez A (2000) Agrobacterium-mediated transformation of cereals- from technique development to its application. Acta Physiol Plant 22:77–88CrossRefGoogle Scholar
  16. 16.
    Jones HD, Doherty A, Wu H (2005) Review of methodologies and a protocol for the Agrobacterium-mediated transformation of wheat. Plant Methods 1(5):1–9Google Scholar
  17. 17.
    Murashige T, Skoog F (1962) A revised medium for rapid growth bioassays with tobacco tissue cultures. Physiol Plant 15:473–497CrossRefGoogle Scholar
  18. 18.
    Jefferson RA (1987) Assaying chimeric genes in plants: The GUS gene fusion system. Plant Mol Biol Rep 5:387–405CrossRefGoogle Scholar
  19. 19.
    Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure from small quantities of fresh leaf tissues. Phytochem Bull 19:11–15Google Scholar
  20. 20.
    Li L, Li R, Fei S, Qu R (2005) Agrobacterium-mediated transformation of common bermudagrass (Cynodon dactylon). Plant Cell Tissue Org Cult 83:223–229CrossRefGoogle Scholar
  21. 21.
    Luo H, Hu Q, Nelson K, Longo C et al (2004) Agrobacterium tumefaciens-mediated creeping bentgrass (Agrostis stolonifera L.) transformation using phosphinothricin selection results in a high frequency of single-copy transgene integration. Plant Cell Rep 22:645–652CrossRefPubMedGoogle Scholar

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© Springer Science+Business Media LLC 2018

Authors and Affiliations

  1. 1.Faculty of Engineering, Department of Genetics and BioengineeringYeditepe UniversityIstanbulTurkey
  2. 2.Faculty of Engineering and Natural Sciences, Molecular Biology, Genetics and Bioengineering ProgramSabanci UniversityIstanbulTurkey
  3. 3.Department of Plant Sciences and Plant PathologyMontana State UniversityBozemanUSA

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