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Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences

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Chloroplast Biotechnology

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2317))

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Abstract

Excision of marker genes using DNA direct repeats makes use of the efficient native homologous recombination pathway present in the plastids of algae and plants. The method is simple, efficient, and widely applicable to plants and green algae. Marker excision frequency is dependent on the length and number of directly repeated sequences. When two repeats are used a repeat size of greater than 600 bp promotes efficient excision of the marker gene. A wide variety of sequences can be used to make the direct repeats. Only a single round of transformation is required and there is no requirement to introduce site-specific recombinases by retransformation or sexual crosses. Selection is used to maintain the marker and ensure homoplasmy of transgenic plastid genomes (plastomes). Release of selection allows the accumulation of marker-free plastomes generated by marker excision, which is a spontaneous and unidirectional process. Cytoplasmic sorting allows the segregation of cells with marker-free transgenic plastids. The marker-free shoots resulting from direct repeat mediated excision of marker genes have been isolated by vegetative propagation of shoots in the T0 generation. Alternatively, accumulation of marker-free plastomes during growth, development and flowering of T0 plants allows for the collection of seeds that give rise to a high proportion of marker-free T1 seedlings. The procedure enables precise plastome engineering involving insertion of transgenes, point mutations and deletion of genes without the inclusion of any extraneous DNA. The simplicity and convenience of direct repeat excision facilitates its widespread use to isolate marker-free crops.

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Acknowledgments

This work was supported by research grants BB/E020445 and BB/I011552 from the Biotechnology and Biological Sciences Research Council (UK).

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Authors and Affiliations

  1. School of Biological Sciences, The University of Manchester, Manchester, UK

    Elisabeth A. Mudd, Elena Martin Avila & Anil Day

  2. Institute of Applied Biosciences, CERTH, Thessaloniki, Greece

    Panagiotis Madesis

Authors
  1. Elisabeth A. Mudd
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  2. Panagiotis Madesis
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  3. Elena Martin Avila
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  4. Anil Day
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Correspondence to Anil Day .

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Editors and Affiliations

  1. Waksman Institute of Microbiology, Rutgers University, Piscataway, NJ, USA

    Pal Maliga

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Mudd, E.A., Madesis, P., Avila, E.M., Day, A. (2021). Marker-Free Transplastomic Plants by Excision of Plastid Marker Genes Using Directly Repeated DNA Sequences. In: Maliga, P. (eds) Chloroplast Biotechnology. Methods in Molecular Biology, vol 2317. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1472-3_4

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  • DOI: https://doi.org/10.1007/978-1-0716-1472-3_4

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1471-6

  • Online ISBN: 978-1-0716-1472-3

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