Optimization of Cultivation System and Transcriptome Analysis: From Unpollinated Chinese Chive Ovules to Plant Formation

Unpollinated ovary culture is an effective approach for generating haploid and doubled haploid lines, but its application in Chinese chive (Allium tuberosum) breeding has been constrained by strong genotype dependence and low regeneration efficiency. This study evaluated an efficient gynogenesis induction system and analyzed transcriptomic changes associated with embryogenesis. Among 20 evaluated genotypes, the commercial cultivar ‘21-CJ46’ showed the strongest response. The optimized induction conditions utilized ovaries collected 1 day before anthesis, cultured on Murashige and Skoog medium supplemented with 90 g/L sucrose, 1.0 mg/L 6-BA, and 0.2 mg/L 2,4-D at 25 °C. Under this system, ‘21-CJ46’ achieved a maximum embryo induction rate of 86.67%. Flow cytometry and chromosome counting indicated frequent spontaneous chromosome doubling, with regenerants mainly distributed as diploids and tetraploids. RNA-seq analysis comparing pre-induction (0 d) and 14 d ovaries showed extensive transcriptional reprogramming, including significant enrichment of phenylpropanoid biosynthesis, plant–pathogen interaction, and plant hormone signal transduction pathways. Differential expression analysis demonstrated that key embryogenesis regulators, such as BBM2, WUSCHEL9, LEC, PLT2, and ABI3, were regulated at 14 d. These results provide an induction protocol and molecular indications relevant to accelerating Chinese chive breeding.