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Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred

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Maize

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1676))

Abstract

Genetic transformation of maize inbred genotypes remains non-routine for many laboratories due to variations in cell competency to induce embryogenic callus, as well as the cell’s ability to receive and incorporate transgenes into the genome. This chapter describes two transformation protocols using Agrobacterium- and biolistic-mediated methods for gene delivery. Immature zygotic embryos of maize inbred B104, excised from ears harvested 10–14 days post pollination, are used as starting explant material. Disarmed Agrobacterium strains harboring standard binary vectors and the biolistic gun system Bio-Rad PDS-1000/He are used as gene delivery systems. The herbicide resistant bar gene and selection agent bialaphos are used for identifying putative transgenic type I callus events. Using the step-by-step protocols described here, average transformation frequencies (number of bialaphos resistant T0 callus events per 100 explants infected or bombarded) of 4% and 8% can be achieved using the Agrobacterium- and biolistic-mediated methods, respectively. An estimated duration of 16–21 weeks is needed using either protocol from the start of transformation experiments to obtaining putative transgenic plantlets with established roots. In addition to laboratory in vitro procedures, detailed greenhouse protocols for producing immature ears as transformation starting material and caring for transgenic plants for seed production are also described.

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Acknowledgments

The authors wish to thank Marcy Main, Haleigh Summers, Sarah Salmon, Stephanie Widener, Aaron Brand, and Katey Warnberg for their contributions to this work. This project was partially supported by the USDA National Institute of Food and Agriculture, Hatch project number # IOW05162, and by State of Iowa funds.

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Authors and Affiliations

  1. Department of Agronomy, Iowa State University, Ames, IA, 50011-1010, USA

    Jennifer A. Raji, Bronwyn Frame, Daniel Little, Tri Joko Santoso & Kan Wang

  2. Center for Plant Transformation, Plant Sciences Institute, Iowa State University, Ames, IA, 50011-1010, USA

    Jennifer A. Raji, Bronwyn Frame, Daniel Little, Tri Joko Santoso & Kan Wang

  3. Indonesian Center for Agricultural Biotechnology and Genetic Resources Research and Development (ICABIOGRAD-IAARD), Bogor, Indonesia

    Tri Joko Santoso

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  1. Jennifer A. Raji
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  2. Bronwyn Frame
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  4. Tri Joko Santoso
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  5. Kan Wang
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Correspondence to Kan Wang .

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Editors and Affiliations

  1. Department of Agronomy and Horticulture, University of Nebraska – Lincoln, Lincoln, Nebraska, USA

    L. Mark Lagrimini

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Raji, J.A., Frame, B., Little, D., Santoso, T.J., Wang, K. (2018). Agrobacterium- and Biolistic-Mediated Transformation of Maize B104 Inbred. In: Lagrimini, L. (eds) Maize. Methods in Molecular Biology, vol 1676. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7315-6_2

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  • DOI: https://doi.org/10.1007/978-1-4939-7315-6_2

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7314-9

  • Online ISBN: 978-1-4939-7315-6

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