Abstract: 【Objective】In this experiment, seedless grapes were used as the female material for the creation of new seedless grape materials. In the experiment, Flame Seedless, Zicui Seedless, Crimson Seedless, Ruby Seedless were used as the female materials to explore the suitable female materials for grape embryo rescue breeding; When using Flame Seedless, Zicui Seedless, Crimson Seedless, and Ruby Seedless grape cultivars as female parents, different sampling times were set for young fruits to explore the optimal sampling time for embryo rescue breeding using Flame Seedless Crimson Seedless and Ruby Seedless grape cultivars as female parents; Identification of seedless traits in hybrid offspring using the seedless molecular marker p3-VvAGL11, GLSP1-569, SCF27-2000, SCC8-1018 to improve the efficiency of seedless grape breeding. 【Methods 】Flame Seedless, Zicui Seedless, Crimson Seedless, Ruby Seedless were selected as female parents for hybridization, and the sampling time gradients for young fruits were set for the female parent cultivars. In the hybrid combination, the sampling time for young fruits with Flame Seedless as the female parent was 36 d, 38 d, and 40 d after the first pollination, the sampling time for young fruits with Crimson Seedless as the female parent was 44 d, 46 d, 48 d, and 50 d after the first pollination, and the sampling time for young fruits with Ruby Seedless as the female parent was 53 d, 55 d, and 57 d after the first pollination, the young fruits were taken back to the laboratory for sterilization. After sterilization, they were placed in a super clean environment. The seeds were taken out from the young fruit in the clean bench and cultured in MM3 medium for 8-12 weeks in the dark.; After the dark cultivation was completed, the seeds were placed in an ultraclean workbench. Under a dissecting microscope, the embryos were removed with a surgical knife and placed in WPM medium. They were cultured at room temperature and under light for 40-60 days. The embryo development rate and embryo seedling rate of different cultivars as female parents were recorded, as well as the embryo development rate and embryo seedling rate at different sampling times of young fruits when Flame Seedless, Crimson Seedless, and Ruby Seedless were used as female parents; Using the nuclear free molecular marker p3-VvAGL11, GLSP1-569, SCF27-2000, SCC8-1018, the hybrid offsprings were identified for their nuclear free traits. Among them, all descendants of the Flame Seedless × Sunshine Muscat, Flame Seedless × Jumeigui, Crimson Seedless × Sunshine Muscat, and Zicui Seedless × Zuijinxiang were identified. 50, 58, and 120 hybrid offsprings of Ruby Seedless × Hutai No. 8, Ruby Seedless × Zuijinxiang, and Ruby Seedless × Jingxiangyu were selected for nuclear free identification. 【Results】The embryo development rates of the three hybrid combinations using Ruby Seedless as the female parent were significantly higher than those of other combinations, with rates of 25.40%, 22.99%, and 30.23%, respectively; Moreover, the embryo seedling rates of these three combinations were significantly higher than those of other combinations, at 12.48%, 13.59%, and 15.46%, respectively. A sampling time gradient was set for hybrid combinations using Flame Seedless, Crimson Seedless, and Ruby Seedless as female parents. It was found that the two combinations of Flame Seedless × Jumeigui and Flame Seedless × Sunshine Muscat had embryo development rates of 10.74% and 8.25%, embryo seedling rates of 4.03% and 3.10%, respectively, 38 d after the first pollination. The embryo development rates and seedling rates of both combinations were higher than those of the first pollination at 36 d and 40 d. The hybrid combination of Crimson Seedless × Sunshine Muscat had embryo development rates of 7.69% and seedling rates of 4.07%, respectively, 48 days after the first pollination. The development rate and embryo seedling rate were higher than those of the first pollination at 44 d, 46 d, and 50 d. Three combinations of Ruby Seedless × Hutai No. 8, Ruby Seedless × Zuijinxiang, and Ruby Seedless × Jingxiangyu had embryo development rates of 28.25%, 24.78%, and 33.88%, respectively, at 55 d after the first pollination. The embryo seedling rates were 17.14%, 18.42%, and 20.09%, respectively. The embryo development rate and embryo seedling rate of the three combinations were higher than those at 53 d and 57 d after the first pollination. Using the nuclear free molecular marker p3-VvAGL11, GLSP1-569, SCF27-2000, SCC8-1018, a random sample of 58 hybrid offsprings of Ruby Seedless × Zuijinxiang was used for early identification of nuclear free traits. Among them, 37 strains amplified nuclear specific bands, with a nuclear free rate of 63.79%. 120 hybrid offsprings of Ruby Seedless × Jingxiangyu were randomly selected for early identification of nuclear free traits, with 71 strains amplified nuclear free specific bands, with a nuclear free rate of 59.17%. Randomly sampling 50 hybrid offsprings of Ruby Seedless × Hutai No. 8 for early identification of seedless traits, 31 of them amplified nuclear specific bands, with a seedless rate of 62.00%. All hybrid offsprings of Crimson Seedless × Sunshine Muscat, Flame Seedless × Jumeigui, Flame Seedless × Sunshine Muscat, Zicui Seedless × Zuijinxiang were identified, including 16, 7, 4, and 2 plants that amplified nuclear specific bands, with seedless rates of 72.73%, 58.33%, 100.00%, and 100% respectively. 【Conclusion】Ruby Seedless was suitable as a maternal material for embryo rescue breeding; The optimal sampling time for hybrid young fruits was 38 days after the first pollination when using Flame Seedless as the female parent, 48 days after the first pollination when using Crimson Seedless as the female parent, and 55 days after the first pollination when using Ruby Seedless as the female parent; By using the nuclear free molecular marker p3-VvAGL11, GLSP1-569, SCF27-2000, SCC8-1018 for nuclear free identification, early prediction of the nuclear free traits of hybrid offspring can be made, avoiding the impact of excessive grape seedling stage on the selection of nuclear free grapes and accelerating the speed of grape breeding.
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