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High-efficiency generation of fertile transplastomic Arabidopsis plants

Abstract

The development of technologies for the stable genetic transformation of plastid (chloroplast) genomes has been a boon to both basic and applied research. However, extension of the transplastomic technology to major crops and model plants has proven extremely challenging, and the species range of plastid transformation is still very much limited in that most species currently remain recalcitrant to plastid genome engineering. Here, we report an efficient plastid transformation technology for the model plant Arabidopsis thaliana that relies on root-derived microcalli as a source tissue for biolistic transformation. The method produces fertile transplastomic plants at high frequency when combined with a clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9)-generated knockout allele of a nuclear locus that enhances sensitivity to the selection agent used for isolation of transplastomic events. Our work makes the model organism of plant biology amenable to routine engineering of the plastid genome, facilitates the combination of plastid engineering with the power of Arabidopsis nuclear genetics, and informs the future development of plastid transformation protocols for other recalcitrant species.

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Fig. 1: Biolistic nuclear and plastid transformation of A. thaliana.
Fig. 2: Construction of plastid transformation vectors and selection of transplastomic Arabidopsis plants.
Fig. 3: Homoplasmy of transplastomic lines obtained with vectors pCH8, pJF1153 and pJF1151, and demonstration of maternal transgene inheritance.
Fig. 4: Expression of the YFP reporter in transplastomic At-Δa-JF1151 plants.
Fig. 5: Immunoblot analysis of YFP accumulation in transplastomic Arabidopsis plants.

Data availability

The data supporting the findings of this study are available within the paper and its Supplementary Information files. Annotated sequences of plastid transformation vectors pCH8, pJF1151 and pJF1153 have been deposited in GenBank (accession numbers MH590891, MH590893 and MH590894). Nature Plants thanks Francis Quétier, Spencer M. Whitney and other anonymous reviewers for their contributions to the peer review of this work.

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Acknowledgements

We thank M. M. Bednarska, C. Runge and M. Rößner for excellent technical assistance, K. Kiemel and R. Narawitz for media preparation and help with tissue culture, D. Kleinschmidt for microscopy, and J. Zhang and D. Karcher for help with vector design. This research was financed by the Max Planck Society and a grant from the European Research Council under the European Union’s Horizon 2020 research and innovation programme (ERC-ADG-2014; grant agreement 669982) to R.B.

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S.R., J.F. and R.B. designed the research. C.H., X.K., S.S., A.S., L.S. and J.F. performed the experiments. All authors participated in data evaluation. R.B. wrote the manuscript, with input from S.R. and J.F.

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Correspondence to Ralph Bock.

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Journal peer review information: Nature Plants thanks Francis Quétier, Spencer M. Whitney and other anonymous reviewers for their contributions to the peer review of this work.

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Supplementary Figures 1–8 and Supplementary Table 1.

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Ruf, S., Forner, J., Hasse, C. et al. High-efficiency generation of fertile transplastomic Arabidopsis plants. Nat. Plants 5, 282–289 (2019). https://doi.org/10.1038/s41477-019-0359-2

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