Abstract
Key message
We have developed and validated an efficient protocol for producing gene-edited carrot plants that do not result in the stable incorporation of foreign DNA in the edited plant’s genome.
Abstract
We report here a method for producing transgene-free, gene-edited carrot (Daucus carota subs. sativus) plants. With this approach, PEG-mediated transformation is used to transiently express a cytosine base editor and a guide RNA in protoplasts to induce targeted mutations in the carrot genome. These protoplasts are then cultured under conditions that lead to the production of somatic embryos which subsequently develop into carrot plants. For this study, we used the Centromere-Specific Histone H3 (CENH3) gene as a target for evaluating the efficiency with which regenerated, edited plants could be produced. After validating sgRNA performance and protoplast transformation efficiency using transient assays, we performed two independent editing experiments using sgRNAs targeting different locations within CENH3. In the first experiment, we analyzed 184 regenerated plants and found that 22 of them (11.9%) carried targeted mutations within CENH3, while in the second experiment, 28 out of 190 (14.7%) plants had mutations in CENH3. Of the 50 edited carrot lines that we analyzed, 43 were homozygous or bi-allelic for mutations in CENH3. No evidence of the base editor expression plasmid was found in the edited lines tested, indicating that this approach is able to produce transgene-free, gene-edited lines. The protocol that we describe provides an efficient method for easily generating large numbers of transgene-free, gene-edited carrot plants.
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Acknowledgements
We thank Aneta Malec for instruction in the method of carrot protoplast regeneration. We thank Phillip Simon and Richard Amasino for their assistance with vernalization of the mutant carrot plants.
Funding
This work was supported by the AFRI Exploratory Research Program [GRANT12854173 /project accession no. 1021280] from the USDA National Institute of Food and Agriculture, United States of America.
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All authors participated in planning the experiments. All authors provided critical feedback and helped shape the research, analysis and manuscript. CMM carried out the majority of the experiments with PJK also contributing. CMM collected and analyzed the data. CMM produced the figures for the manuscript with input from PJK. CMM took the lead in writing the manuscript with input from PJK. All authors participated in review and revision of the manuscript.
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Communicated by Zhanyuan Jon Zhang.
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Meyer, C.M., Goldman, I.L., Grzebelus, E. et al. Efficient production of transgene-free, gene-edited carrot plants via protoplast transformation. Plant Cell Rep 41, 947–960 (2022). https://doi.org/10.1007/s00299-022-02830-9
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DOI: https://doi.org/10.1007/s00299-022-02830-9