Optimization of the embryo rescue system for seedless grapes from different hybrid combinations

【Objective】Most seedless grape cultivars are stenospermocarpic: fertilization proceeds normally, but the embryos abort during early development, which severely limits the recovery rate of hybrid progeny when seedless grape cultivars are used as female parents. Embryo rescue is therefore essential for seedless grape breeding. However, its efficiency is strongly affected by multiple factors, including parental genotype, sampling time after pollination, medium composition and exogenous nutrient supply. This study aimed to establish an efficient and stable embryo rescue system for the mid-tolate ripening seedless grape cultivar Zhongpu 18, and clarify the key factors affecting embryo rescue efficiency across different hybrid combinations, so as to provide technical support for seedless grape germplasm innovation.【Methods】Six hybrid combinations were constructed by using Zhongpu 18 and Ruby Seedless as female parents, which were crossed with seeded and seedless male parents: the seeded male parents included Muscat Hamburg and Xiangfei, while the seedless male parents included Zhong-pu 18 and Ruby Seedless. Ovules were sampled at different days after pollination (DAP) and cultured in vitro on NN-based media. We systematically evaluated the effects of sampling time (46-54 DAP), medium culture type (solid medium versus solid-liquid two-phase medium), and exogenous amino acid supplementation (proline or serine at 1-5 mmol·L-1 ) on embryo development. We recorded and statistically analyzed embryo development rate, embryo germination rate and seedling establishment rate to identify the optimal technical parameters.【Results】Embryo rescue efficiency varied significantly across different hybrid combinations, and was jointly regulated by maternal genotype, paternal genotype, sampling time after pollination, culture medium type, and exogenous amino acid supplementation. Across all six hybrid combinations, 8582 ovules were cultured, and 1518 developing embryos were obtained, with an average embryo development rate of 12.42% . In total, 534 seedlings were obtained, with an average seedling establishment rate of 50.09%, and the optimal sampling time differed significantly among maternal lines. For crosses with Zhongpu 18 as the female parent, embryo development peaked at 50 days after pollination (DAP), with the rate ranging from 18.18% to 21.43%. Sampling that was either too early or too late led to a marked reduction in the embryo development rate. In contrast, for hybrid combinations with Ruby Seedless as the female parent, the embryo development rate generally reached its maximum at 52 days after pollination, with the peak rate hitting 18.40%. Parental genotypes have a significant effect on the efficiency of embryo rescue. With the same maternal genetic background, hybrid combinations derived from seeded male parents consistently showed higher embryo development rates and seedling survival rates than those derived from seedless male parents. The phase state of the culture medium has a significant influence on embryo development. Compared with traditional solid media, solidliquid dual-phase media improved embryo development rates in most hybrid combinations. Exogenous amino acid supplementation further improved embryo rescue efficiency, although responses varied among different hybrid combinations. The addition of 5 mmol·L-1 proline or 3 mmol·L-1 serine significantly promoted embryo development in multiple hybrids, with the Zhongpu 18 × Xiangfei hybrid combination reaching an embryo development rate of 25.00% . In contrast, simultaneous supplementation with proline and serine failed to produce additive or synergistic effects. In some cases, it even led to lower embryo development and germination rates compared with treatments using a single amino acid. These findings indicate that optimizing embryo rescue efficiency depends on selecting appropriate amino acid types and concentrations, rather than relying on mixed amino acid supplementation.【Conclusion】Sampling time after pollination, parental genotype, culture medium composition, and appropriate amino acid supplementation are all key factors determining the embryo rescue efficiency of seedless grapes. The optimized embryo rescue system established in this study is highly suitable for mid- to lateripening seedless grape cultivars such as Zhongpu 18 and Ruby Seedless, which would provide a reliable technical basis for improving hybrid seedling recovery efficiency and accelerating the process of seedless grape breeding.