Abstract
Brachypodium distachyon has emerged as the model species for important temperate grass crops such as wheat and barley and the genome of the B. distachyon community inbred line Bd21 has been sequenced. Methods for tissue culture and Agrobacterium-mediated transformation have been developed for this model grass as a resource for reverse genetics and functional genomic analyses. In order to obtain a high quantity and quality of compact embryogenic callus (CEC) in B. distachyon, it is important to examine and optimize the optimal concentration of the auxin 2,4-D (dichlorophenoxyacetic acid) to use in both callus induction and callus proliferation media. Here, we investigated the effects of different concentrations of 2,4-D on callus induction and callus proliferation of B. distachyon Bd21. Our results showed that 2.5 mg l−1 2,4-D is an optimal concentration to use for both callus induction and proliferation, although 5.0 mg l−1 may also be used for callus proliferation. Additionally, the suitability of hygromycin or bialaphos as selectable markers was examined and results indicated that hygromycin is significantly more efficient than bialaphos when using the Agrobacterium-mediated transformation system.
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Alves, S.C., Worland, B., Thole, V., Snape, J.W., Bevan, M.W., Vain, P. 2009. A protocol for Agrobacterium-mediated transformation of Brachypodium distachyon community standard line Bd21. Nat. Protoc. 4:638–649.
An, T., Cai, Y, Zhao, S., Zhou, J., Song, B., Bux, H., Qi, X. 2016. Brachypodium distachyon T-DNA insertion lines: a model pathosystem to study nonhost resistance to wheat stripe rust. Sci. Rep. 6:25510.
Bragg, J.N., Anderton, A., Nieu, R., Vogel, J.P. 2015. Brachypodium distachyon. In: Wang, K. (ed), Agrobacterium Protocols. Springer. New York, USA. pp. 17–33.
Bragg, J.N., Wu, J., Gordon, S.P., Guttman, M.E., Thilmony, R., Lazo, G.R., Gu, Y.Q, Vogel, J.P., 2012. Generation and characterization of the western regional research center Brachypodium T-DNA insertional mutant collection. PLoS ONE 7:e41916.
Bablak, P., Draper, J., Davey, M.R., Lynch, P.T. 1995. Plant regeneration and micropropagation of Brachypodium distachyon. Plant Cell Tiss. Org. 42:97–107.
Cheng, M., Hu, T., Layton, J., Liu, C.N., Fry, J.E. 2003. Desiccation of plant tissues post-Agrobacterium infection enhances T-DNA delivery and increases stable transformation efficiency in wheat. In Vitro Cell. Dev.-Pl. 39:595–604.
Cheng, M., Lowe, B.A., Spencer, T.M., Ye, X., Armstrong, C.L. 2004. Factors influencing Agrobacterium-mediated transformation of monocotyledonous species. In Vitro Cell. Dev.- Pl. 40:31–45.
Christiansen, P., Andersen, C.H., Didion, T., Folling, M., Nielsen, K.K. 2005. A rapid and efficient transformation protocol for the grass Brachypodium distachyon. Plant Cell Rep. 23:751–758.
Collier, R., Bragg, J., Hernandez, B.T., Vogel, J.P., Thilmony, R. 2016. Use of Agrobacterium rhizogenes strain 18r12v and paromomycin selection for transformation of Brachypodium distachyon and Brachypodium sylvaticum. Front. Plant Sci. 7:716.
Draper, J., Mur, L.A., Jenkins, G., Ghosh-Biswas, G.C., Bablak, P., Hasterok, R., Routledge, A.P. 2001. Brachypodium distachyon. A new model system for functional genomics in grasses. Plant Physiol. 127:1539–1555.
Eamens, A.L., Blanchard, C.L., Dennis, E.S., Upadhyaya, N.M. 2004. A bidirectional gene trap construct suitable for T-DNA and Ds-mediated insertional mutagenesis in rice (Oryza sativa L.). Plant Biotechnol. J. 2:367–380.
Garvin, D., Gu, Y., Hasterok, R., Hazen, S., Jenkins, G., Mockler, T., Mur, L., Vogel, J. 2008. Development of genetic and genomic research resources for Brachypodium distachyon, a new model system for grass crop research. Crop Sci. 48:S69–S84.
Hanahan, D. 1983. Studies on the transformation of Escherichia coli with plasmids. J. Mol. Biol. 166:557–580.
Hunt, D., Chambers, J.P., Behpouri, A., Kelly, S.P., Whelan, L., Piettrzykowska, M., Downey, F., Mccabe, P.F., Ng, C.K.Y. 2014. Brachypodium distachyon cell suspension cultures: establishment and utilization. Cereal Res. Commun. 42:58–69.
Lee, M.B., Jeon, W.B., Kim, D.Y., Bold, O., Hong, M.J., Lee, Y.J., Park, J.H., Seo, Y.W. 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. 14:233–238.
Mohammadhassan, R., Kashefi, B., Shabanzadeh, Delcheh, K. 2014. Agrobacterium-based vectors: a review. Intl. J. Farm. Allied Sci. 3:1002–1008.
Păcurar, D.I., Thordal-Christensen, H., Nielsen, K.K., Lenk, I. 2008. A high-throughput Agrobacterium-mediated transformation system for the grass model species Brachypodium distachyon L. Transgenic Res. 17:965–975.
Perochon, A., Jianguang, J., Kahla, A., Arunachalam, C., Scofield, S.R., Bowden, S., Wallington, E., Doohan, F.M. 2015. TaFROG encodes a pooideae orphan protein that interacts with SnRK1 and enhances resistance to the mycotoxigenic fungus Fusarium graminearum. Plant Physiol. 169:2895–906.
Steinwand, M.A., Young, H.A., Bragg, J.N., Tobias, C.M., Vogel, J.P. 2013. Brachypodium sylvaticum, a model for perennial grasses: transformation and inbred line development. PLoS ONE 8:e75180.
The International Brachypodium Initiative. 2010. Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature 463:763–768.
Thole, V., Peraldi, A., Worland, B., Nicholson, P., Doonan, J.H., Vain, P. 2011. T-DNA mutagenesis in BrachyIpodium distachyon. J. Exp. Bot. 63:567–576.
Thole, V., Vain, P. 2012. Agrobacterium-mediated transformation of Brachypodium distachyon. Transg. Plants 847:137–149.
Vain, P., Thole, V. 2009. Gene Insertion Patterns and Sites. In: Jones, H.D., Shewry, P.R. (eds), Transgenic Wheat, Barley and Oats: Production and Characterization Protocols Humana Press. Totowa, NJ, USA. pp. 203–226.
Vain, P., Worland, B., Thole, V., McKenzie, N., Alves, S.C., Opanowicz, M., Fish, L.J., Bevan, M.W., Snape, J.W. 2008. Agrobacterium-mediated transformation of the temperate grass Brachypodium distachyon (genotype Bd21) for T-DNA insertional mutagenesis. Plant Biotechnol. J. 6:236–245.
Vogel, J.P., Gu, Y.Q., Twigg, P., Lazo, G.R., Laudencia-Chingcuanco, D., Hayden, D.M., Donze, T.J., Vivian, L.A., Stamova, B., Coleman-Derr, D. 2006a. EST sequencing and phylogenetic analysis of the model grass Brachypodium distachyon. Theor. Appl. Genet. 113:186–195.
Vogel, J.P., Hill, T. 2008. High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep. 27:471–478.
Vogel, J.P., Garvin, D.F., Leong, O.M., Hayden, D.M. 2006b. Agrobacterium-mediated transformation and inbred line development in the model grass Brachypodium distachyon. Plant Cell Tiss. Org. 84:199–211.
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Behpouri, A., Perochon, A., Doohan, F.M. et al. Optimizing callus induction and proliferation for Agrobacterium-mediated transformation of Brachypodium distachyon. CEREAL RESEARCH COMMUNICATIONS 46, 221–231 (2018). https://doi.org/10.1556/0806.46.2018.04
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DOI: https://doi.org/10.1556/0806.46.2018.04