Abstract: The European seedless grape varieties and the progeny of cold-hardy Chinese wild Vitis amurensis crossed with ‘Zuoyouhong’ and ‘Beibinghong’,as well as other cold resistant materials were used as female and male parents to hybrid in the field.Immature berries were gathered before embryo abortion, and hybrid progenies were obtained in vitro embryo rescue.Additionally, the molecular markers were applied to screen the new seedless and cold resistance grape from hybrid seedling.To improve the efficiency of breeding seedless and cold resistance grapes by embryo rescue, the factors of hybrid ovules sampling time, parental genotype, different kinds and concentrations of amino acids in embryo germination medium were studied, in order to improve embryo rescue efficiency of cold-resistant grapevine.The results showed that 235 hybrid plants were obtained.The hybridization combination of ‘Ruby Seedless’ and ‘Kunxiang Seedless’ as the parent had better embryo rescue efficiency and were more suitable as the parent in seedless grape embryo rescue.It was better for embryo rescue efficiency to add glutamine on MM3 at the isolated developmental stage.There were 32 hybrids carried specific bands detected by seedless molecular marker SCF27-2000,38 hybrids carried specific bands detected by cold resistant molecular markers S241-717 and S238-800.