Successful application of isolated microspore culture to create doubled haploid lines in ‘XiangQingCai’ pak choi

• Isolated microspore culture is a rapid and efficient breeding method for obtaining doubled haploid lines. • This study successfully applied isolated microspore culture to create fragrant pak choi doubled haploids. • The DH lines obtained in this study can be directly used for hybrid breeding of fragrant pak choi and improve breeding efficiency. Aroma is an important sensory quality for evaluating the flavor of pak choi, and ‘XiangQingCai’ (XQC) is the only precious germplasm resource of pak choi with a unique fragrance. In production, XQC varieties with high uniformity, high purity, and strong heterosis are typically required. However, the traditional self-pollination method is time-consuming, which limits the speed of breeding fragrant pak choi varieties. The isolated microspore culture (IMC) method can rapidly create doubled haploid (DH) pure lines. Currently, there are no reports on the application of IMC to create XQC pure lines for hybrid breeding. Therefore, this study conducted IMC on three XQC varieties: ‘yellow-leaf XiangQingCai’ (XQC-Y), ‘black-leaf XiangQingCai’ (XQC-B), and ‘Xiuhuajin’ (XHJ). Embryoids were obtained from XQC-Y and XQC-B, but not from XHJ. The embryoid induction rate varied significantly among genotypes, with XQC-B showing a 3.5-fold higher rate than XQC-Y. However, no significant differences were observed in direct seedling formation rate, callus rate, or mortality between the two materials. A total of 65 regenerated plants and 36 DH lines were obtained from XQC-Y and XQC-B, with natural doubling rates exceeding 50 % for both varieties. Additionally, this study created hybrid combinations using superior XQC DH lines and self-incompatible lines of komatsuna, and conducted horticultural trait evaluation and aroma sensory assessment on the obtained XQC-B DH lines and hybrid combinations. These results provide new insights into the practical application of IMC technology in the breeding of fragrant pak choi.