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Spike-Dip Transformation Method of Setaria viridis

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Genetics and Genomics of Setaria

Part of the book series: Plant Genetics and Genomics: Crops and Models ((PGG,volume 19))

Abstract

Conventional Agrobacterium-mediated transformation of monocots involves labor-intensive and time-consuming in vitro tissue culture methodology to regenerate T0 plants. To overcome these difficulties, a simple in planta Agrobacterium-mediated genetic transformation method for the emerging monocot model Setaria viridis was developed. Initial standardization of transient and stable transformations was performed using A. tumefaciens strain AGL1 harboring the β-glucuronidase (GUS) reporter gene driven by the cauliflower mosaic virus 35S (CaMV35S) promoter to transform preanthesis developing spikes. The method was further optimized by using A. tumefaciens strain EHA105 carrying β-glucuronidase plus (GUSplus), green fluorescent protein (GFP), and Discosoma sp. red fluorescent protein (DsRed) reporter genes driven by either CaMV35S or an intron-interrupted maize ubiquitin (Ubi) promoters to develop stable transgenic lines from S. viridis. Dipping of 5-day-old S3 spikes into Agrobacterium cultures containing S. viridis spike-dip medium supplemented with 0.025 % Silwet L-77 and 200 μM acetosyringone for 20 min produced stable transformants at the rate of 0.8 ± 0.1 %. Transgenic lines showed stable integration of transgenes into the genome, and inherited transgenes followed the Mendelian segregation pattern and were expressed in subsequent generations. This spike-dip method will facilitate high-throughput translational research in a monocot model.

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Acknowledgements

Authors are thankful to Dr. Ellen Tumimbang, Dr. Hiromi Tajima, Elham Abed, and Kevin Abernathy for technical support. This work was funded by the United States Agency for International Development (USAID) to support the Feed the Future Innovation Lab for Climate-Resilient Millet under the Grant No. APS M/OAA/GRO/EGAS-11-002011.

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Authors and Affiliations

  1. Department of Plant Sciences, University of California, One Shields Avenue, Davis, CA, 95616, USA

    Prasenjit Saha & Eduardo Blumwald

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  1. Prasenjit Saha
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  2. Eduardo Blumwald
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Correspondence to Eduardo Blumwald .

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Editors and Affiliations

  1. Department of Plant Biology, Ecology, and Evolution, Oklahoma State University, Stillwater, Oklahoma, USA

    Andrew Doust

  2. Institute of Crop Sciences, Chinese Academy of Agricultural Sciences, Beijing, China

    Xianmin Diao

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Saha, P., Blumwald, E. (2017). Spike-Dip Transformation Method of Setaria viridis . In: Doust, A., Diao, X. (eds) Genetics and Genomics of Setaria. Plant Genetics and Genomics: Crops and Models, vol 19. Springer, Cham. https://doi.org/10.1007/978-3-319-45105-3_21

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