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

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.