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Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato

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Abstract

Main conclusion

An efficient method of DNA-free gene-editing in potato protoplasts was developed using linearized DNA fragments, UBIQUITIN10 promoters of several plant species, kanamycin selection, and transient overexpression of the BABYBOOM transcription factor.

Abstract

Plant protoplasts represent a reliable experimental system for the genetic manipulation of desired traits using gene editing. Nevertheless, the selection and regeneration of mutated protoplasts are challenging and subsequent recovery of successfully edited plants is a significant bottleneck in advanced plant breeding technologies. In an effort to alleviate the obstacles related to protoplasts’ transgene expression and protoplasts’ regeneration, a new method was developed. In so doing, it was shown that linearized DNA could efficiently transfect potato protoplasts and that UBIQUITIN10 promoters from various plants could direct transgene expression in an effective manner. Also, the inhibitory concentration of kanamycin was standardized for transfected protoplasts, and the NEOMYCIN PHOSPHOTRANSFERASE2 (NPT2) gene could be used as a potent selection marker for the enrichment of transfected protoplasts. Furthermore, transient expression of the BABYBOOM (BBM) transcription factor promoted the regeneration of protoplast-derived calli. Together, these methods significantly increased the selection for protoplasts that displayed high transgene expression, and thereby significantly increased the rate of gene editing events in protoplast-derived calli to 95%. The method developed in this study facilitated gene-editing in tetraploid potato plants and opened the way to sophisticated genetic manipulation in polyploid organisms.

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Data availability

The data underlying this article are available in the article and in its online supplemental material.

Abbreviations

BBM:

BABYBOOM

GB:

GoldenBraid

GMO:

Genetically modified organisms

NPT2:

NEOMYCIN PHOSPHOTRANSFERASE 2

TALEN:

Transcription activator-like effector nucleases

YFP:

Yellow fluorescent protein

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Acknowledgements

We are very thankful to Dr. Tzahi Arazi for his advice and critical reading of this manuscript. We are grateful to Per Hofvander and Mariette Andersson for their assistance in achieving regeneration from protoplast. We also thank Amit Gal-on and Diana Leibman for providing the pSAT vector used to knockout StVINV.

Funding

This study was financially supported by the Chief Scientist—Ministry of Agriculture and Rural Development NO. 20-01-0209 as part of the National Center for Genome Editing in Agriculture.

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Authors and Affiliations

  1. Department of Ornamental Plants and Agricultural Biotechnology, The Institute of Plant Sciences, The Volcani Center, ARO, Rishon LeTsiyon, Israel

    Gulzar A. Rather, Dana Ayzenshtat, Manoj Kumar, Zohar Forotan & Samuel Bocobza

  2. Department of Postharvest Science, The Institute of Postharvest and Food Sciences, The Volcani Center, ARO, Rishon LeTsiyon, Israel

    Paula Teper-Bamnolker & Dani Eshel

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  1. Gulzar A. Rather
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  2. Dana Ayzenshtat
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  4. Manoj Kumar
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  6. Dani Eshel
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  7. Samuel Bocobza
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Correspondence to Samuel Bocobza.

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Rather, G.A., Ayzenshtat, D., Teper-Bamnolker, P. et al. Advances in protoplast transfection promote efficient CRISPR/Cas9-mediated genome editing in tetraploid potato. Planta 256, 14 (2022). https://doi.org/10.1007/s00425-022-03933-z

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