Improving embryo rescue efficiency in seedless grape breeding with zeatin

Embryo rescue is a crucial technique for developing new seedless grape germplasm; however, its current efficiency remains suboptimal. Optimizing the culture medium is essential for the success of embryo rescue, as it directly influences the development and germination of in vitro embryos. Therefore, this study systematically investigated the effects of parental genotypes and the addition of zeatin (ZT) to the culture medium on the efficiency of embryo rescue, using four self-pollinated maternal parents and 23 seedless×seeded (or seedless) cross combinations. The results showed that among the four self-pollinated parental lines, hybrid ovules exhibited the highest development rate in the ER medium, while a higher proportion of normal plantlets was obtained in the MM3 medium. Although the addition of ZT at various concentrations did not significantly enhance the embryo development rate in the self-pollinated maternal parents, it significantly improved germination outcomes: compared to the control, the rate of normal plantlet formation increased by 8.84-19.03%, while the proportions of abnormal plantlets and ungerminated embryos were significantly reduced. Further validation in hybrid combinations revealed that the addition of an appropriate concentration of ZT significantly increased both embryo development and plantlet formation rates in both MM3 and ER media. Specifically, in the MM3 medium supplemented with 0.5 mg L^-¹ ZT, the hybrid combination 'Huozhoucuiyu × Yan73' achieved the highest embryo development rate (63.33%) and plantlet formation rate (26.67%). Across the 23 hybrid combinations, the average embryo development rate reached 39.57%, yielding a total of 1,554 hybrid progeny. Parental screening indicated that 'Huozhoucuiyu' and 'Ruby Seedless' performed excellently as maternal materials, while 'Yan73', 'Italy', and 'Ruidukemei' were suitable as paternal materials. Using the KASP_VviAGL11 molecular marker, 290 potential seedless lines were identified from 420 hybrid progeny across six combinations, accounting for 69.05% of the total. This study confirms that the appropriate addition of ZT to the culture medium significantly enhances the efficiency of embryo rescue breeding in seedless grapes, providing a practical foundation for establishing a highly efficient and stable embryo rescue system.