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Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids

Part of the Methods in Molecular Biology book series (MIMB,volume 2287)

Abstract

In plant research and breeding, haploid technology is employed upon crossing, induced mutagenesis or genetic engineering to generate populations of meiotic recombinants that are themselves genetically fixed. Thanks to the speed and efficiency in producing true-breeding lines, haploid technology has become a major driver of modern crop improvement. In the present study, we used embryogenic pollen cultures of winter barley ( Hordeum vulgare ) for Cas9 endonuclease-mediated targeted mutagenesis in haploid cells, which facilitates the generation of homozygous primary mutant plants. To this end, microspores were extracted from immature anthers, induced to undergo cell proliferation and embryogenic development in vitro, and were then inoculated with Agrobacterium for the delivery of T-DNAs comprising expression units for Cas9 endonuclease and target gene-specific guide RNAs (gRNAs). Amongst the regenerated plantlets, mutants were identified by PCR amplification of the target regions followed by sequencing of the amplicons. This approach also enabled us to discriminate between homozygous and heterozygous or chimeric mutants. The heritability of induced mutations and their homozygous state were experimentally confirmed by progeny analyses. The major advantage of the method lies in the preferential production of genetically fixed primary mutants, which facilitates immediate phenotypic analyses and, relying on that, a particularly efficient preselection of valuable lines for detailed investigations using their progenies.

Key words

  • cas9
  • Cereals
  • CRISPR
  • Genome editing
  • Targeted mutagenesis

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Acknowledgements

We wish to thank Carola Bollmann and Andrea Müller for her expert technical assistance. We thank the German Federal Ministry for Science and Education for funding our research in frame of the IdeMoDeResBar project (FKZ 031B0199C and 031B0887C). We are also grateful to the Leibniz Institute of Plant Genetics and Crop Plant Research (IPK) Gatersleben for providing our research group with excellent working conditions.

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Correspondence to Jochen Kumlehn .

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Hoffie, R.E., Otto, I., Hisano, H., Kumlehn, J. (2021). Site-Directed Mutagenesis in Barley Using RNA-Guided Cas Endonucleases During Microspore-Derived Generation of Doubled Haploids. In: Segui-Simarro, J.M. (eds) Doubled Haploid Technology. Methods in Molecular Biology, vol 2287. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1315-3_9

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  • DOI: https://doi.org/10.1007/978-1-0716-1315-3_9

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