Optimizing <i>Brassica oleracea</i> L. Breeding Through Somatic Hybridization Using Cytoplasmic Male Sterility (CMS) Lines: From Protoplast Isolation to Plantlet Regeneration

The <i>Brassica oleracea</i> L. species embrace important horticultural crops, such as broccoli, cauliflower, and cabbage, which are highly valued for their beneficial nutritional effects. However, the complexity of flower emasculation in these species has forced breeders to adopt biotechnological approaches such as somatic hybridization to ease hybrid seed production. Protoplasts entail a versatile tool in plant biotechnology, supporting breeding strategies that involve genome editing and hybridization. This review discusses the use of somatic hybridization in <i>B. oleracea</i> L. as a biotechnological method for developing fusion products with desirable agronomic traits, particularly cytoplasmic male sterile (CMS) condition. These CMS lines are critical for implementing a cost-effective, efficient, and reliable system for producing F1 hybrids. We present recent studies on CMS systems in <i>B. oleracea</i> L. crops, providing an overview of established models that explain the mechanisms of CMS and fertility restoration. Additionally, we emphasize key insights gained from protoplast fusion applied to <i>B. oleracea</i> L. breeding. Key steps including pre-treatments of donor plants, the main tissues used as sources of parental protoplasts, methods for obtaining somatic hybrids and cybrids, and the importance of establishing a reliable plant regeneration method are discussed. Finally, the review explores the incorporation of genome editing technologies, such as CRISPR-Cas9, to introduce multiple agronomic traits in <i>Brassica</i> species. This combination of advanced biotechnological tools holds significant promise for enhancing <i>B. oleracea</i> breeding programs in the actual climate change context.