Creation of new seedless grape materials via embryo rescue

Abstract: 【Objective】The experiment aimed to explore the optimum sampling time of the young berries for embryo rescue and to verify the feasibility of identification of seedless traits in hybrid offsprings using molecular markers in grape.【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. The sampling times for young berries of Flame Seedless were 36 d, 38 d, and 40 d after the first pollination, the sampling times for young berries of Crimson Seedless were 44 d, 46 d, 48 d, and 50 d after the first pollination, and the sampling times for young berries of Ruby Seedless were 53 d, 55 d, and 57 d after the first pollination. The young fruits were taken to the laboratory for sterilization. After sterilization, the fruits were placed in a super clean environment. The seeds were taken out from the young fruits and cultured in MM3 medium for 8-12 weeks in the dark. After the dark cultivation, 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 with light for 40-60 days. The embryo development rate and embryo seedling rate of different cultivars were recorded. The seedless trait of the hybrid offsprings wasidentified using the nuclear free molecular marker p3- VvAGL11, GLSP1- 569, SCF27- 2000, SCC8- 1018.【Results】The embryo development rates of the three crossing combinations of Ruby Seedless as the female parent were significantly higher than those of the other combinations. Moreover, the embryo seedling rates of these three combinations were significantly higher than those of the other combinations. A sampling time gradient was set for crossing combinations of Flame Seedless, Crimson Seedless, and Ruby Seedless as female parents. It was found that the two combinations of Flame Seedless × Jumeigui and Flame Seedless × Shine Muscat had embryo development rates of 10.74% and 8.25%, embryo seedling rates of 4.03% and 3.10%, respectively, when the berries were collected on 38th d after the first pollination, higher than those collected on 36th d and 40th d. The crossing combination of Crimson Seedless × Shine Muscat had embryo development rates of 7.69% and seedling rates of 4.07%, respectively, when the berries were collected on 48th days after the first pollination, higher than those collected on 44th d, 46th d, and 50th d. The 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, when the berries were collected on 55th d after the first pollination and the embryo seedling rates were 17.14%, 18.42%, and 20.09%, respectively, higher than those collected on 53th d and 57th d. The molecular marker p3-VvAGL11, GLSP1-569, SCF27-2000, SCC8- 1018, were successfully used for identification of seedless trait. The seedless rates of hybrid offsprings of Ruby Seedless × Zuijinxiang, Ruby Seedless × Jingxiangyu and Ruby Seedless × Hutai No. 8 were 63.79%, 59.17% and 62.00%, respectively. The seedless rates of the hybrid offsprings of Crimson Seedless × Shine Muscat, Flame Seedless × Jumeigui, Flame Seedless × Shine Muscat, Zicui Seedless × Zuijinxiang were 72.73%, 58.33%, 100.00%, and 100.00%, respectively.【Conclusion】Ruby Seedless was suitable as a maternal material for seedless breeding via embryo rescue in grape for higher seedless rate. The optimal sampling times for the young fruits of Flame Seedless, Crimson Seedless and Ruby Seedless were 38 days, 48 days and 55 days after the first pollination. Preselection of seedless trait of the grape hybrid offspring could be achieved using the molecular marker p3- VvAGL11, GLSP1- 569, SCF27-2000, SCC8-1018.