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.

芸苔属包括西兰花、卷心菜、花椰菜、羽衣甘蓝和大头菜等蔬菜，这些蔬菜在经济上很重要，因为它们含有有益的化合物，如硫代葡萄糖苷。成簇规则间隔短回文重复序列-Cas9 (CRISPR-Cas9) 是一种新的育种技术，有望取代传统育种技术，因为它能产生具有改良性状的非转基因品系。因此，需要开发原生质体介导的再生方法。迄今为止，已经在芸苔属植物中发现了几种分离和再生原生质体的方法蔬菜。然而，这些方法的效率因物种和基因型而异。此外，原生质体再生效率不足以满足CRISPR-Cas9技术的常规应用。在这项研究中，我们发现西兰花、卷心菜、花椰菜、羽衣甘蓝和大头菜的第三和第四片真叶是分离完整原生质体的良好来源。此外，针对这些蔬菜优化了改良的原生质体分离和微愈伤组织培养方案。此外，还开发并优化了卷心菜和花椰菜中真正的叶源原生质体的再生方案。我们相信该协议可以应用于其他芸苔属蔬菜，例如西兰花、羽衣甘蓝和大头菜。