• Open Access

The chloroplast transformation toolbox: selectable markers and marker removal

Authors

  • Anil Day,

    1. Faculty of Life Sciences, The University of Manchester, Manchester, UK
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  • Michel Goldschmidt-Clermont

    Corresponding author
    1. Departments of Plant Biology and of Molecular Biology, University of Geneva, Genève, Switzerland
      (Tel +41 22 379 6188; fax +41 22 379 6868; email michel.goldschmidt-clermont@unige.ch)
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(Tel +41 22 379 6188; fax +41 22 379 6868; email michel.goldschmidt-clermont@unige.ch)

Summary

Plastid transformation is widely used in basic research and for biotechnological applications. Initially developed in Chlamydomonas and tobacco, it is now feasible in a broad range of species. Selection of transgenic lines where all copies of the polyploid plastid genome are transformed requires efficient markers. A number of traits have been used for selection such as photoautotrophy, resistance to antibiotics and tolerance to herbicides or to other metabolic inhibitors. Restoration of photosynthesis is an effective primary selection method in Chlamydomonas but can only serve as a screening tool in flowering plants. The most successful and widely used markers are derived from bacterial genes that inactivate antibiotics, such as aadA that confers resistance to spectinomycin and streptomycin. For many applications, the presence of a selectable marker that confers antibiotic resistance is not desirable. Efficient marker removal methods are a major attraction of the plastid engineering tool kit. They exploit the homologous recombination and segregation pathways acting on chloroplast genomes and are based on direct repeats, transient co-integration or co-transformation and segregation of trait and marker genes. Foreign site-specific recombinases and their target sites provide an alternative and effective method for removing marker genes from plastids.

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