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Development of a general protoplast-mediated regeneration protocol for Brassica: cabbage and cauliflower as examples

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Abstract

The genus Brassica includes vegetables such as broccoli, cabbage, cauliflower, kale, and kohlrabi, which are economically important because they contain beneficial compounds, such as glucosinolates. Clustered regularly interspaced short palindromic repeats-Cas9 (CRISPR-Cas9) is a new breeding technology that is expected to replace traditional breeding technologies because it generates transgene-free lines with improved traits. Therefore, protoplast-mediated regeneration methods need to be developed. To date, several methods for isolating and regenerating protoplasts have been identified in Brassica vegetables. However, the efficiency of these methods varies between species and genotypes. Furthermore, protoplast regeneration efficiency was found to not be sufficient for the routine application of CRISPR-Cas9 technology. In this study, we identified that the third and fourth true leaves of broccoli, cabbage, cauliflower, kale, and kohlrabi were good sources for the isolation of intact protoplasts. In addition, modified protoplast isolation and microcallus culture protocols were optimized for these vegetables. Furthermore, a regeneration protocol for true leaf-originating protoplasts in cabbage and cauliflower was developed and optimized. We believe that this protocol could be applied to other Brassica vegetables such as broccoli, kale, and kohlrabi.

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Acknowledgements

We would like to thank Editage (www.editage.co.kr) for English language editing. The authors acknowledge Asia Seed Co. for supplying the seeds for this study. This study was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01652901) of the Rural Development Administration of the Republic of Korea.

Funding

This research was supported by the Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01652901) of the Rural Development Administration of the, Republic of Korea.

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Authors and Affiliations

  1. Plant Genomics Laboratory, Department of Bio-resource Engineering, College of Life Sciences, Sejong University, 209 Neungdong-ro, Gwanjing-gu, Seoul, 05006, Republic of Korea

    Muhammad Hussain, Haisu Li, Mahdi Badri Anarjan & Sanghyeob Lee

  2. Plant Engineering Research Institute, Sejong University, Seoul, 05006, Republic of Korea

    Sanghyeob Lee

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  1. Muhammad Hussain
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  2. Haisu Li
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  3. Mahdi Badri Anarjan
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  4. Sanghyeob Lee
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SL planned the experiments, analyzed the data, and wrote the manuscript. LH and BA analyzed the data. MH conducted the experiments, analyzed the data, and wrote the manuscript.

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Correspondence to Sanghyeob Lee.

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Hussain, M., Li, H., Badri Anarjan, M. et al. Development of a general protoplast-mediated regeneration protocol for Brassica: cabbage and cauliflower as examples. Hortic. Environ. Biotechnol. 65, 313–321 (2024). https://doi.org/10.1007/s13580-023-00557-4

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