| Seedless grape breeding and production is an important goal of table grapes and drying in the world today.Most of the seedless varieties cultivated in the world belong to the European grapes（Vitis vinifera L）,which has good quality but not cold-resistant.Cultivation of seedless grapes in cold regions is prone to frost damage,which not only causes high production and burial costs,but also leads to a decline in the yield and quality of grapes.Therefore,the selection of seedless and cold-resistant grape variety is an effective measure to expand the scope of grape cultivation and prevent frost damage.In this study,high-quality European grape seedless varieties were used as the female parent,and cold-resistant wild Chinese grapes and their progeny were used as the male parent,a total of 16 hybrid combinations and 1 selfing combination were configured.Embryo rescue technology was used to culture hybrid ovules in vitro,the medium composition of embryo development was optimized,and the traits of hybrid progeny were identified with non-nuclear molecular markers and cold-resistant molecular markers.The results are as follows:1.Obtained new seedless and cold-resistant germplasm:16 hybrid combinations and 1selfing combination were configured,obtained 6319 ovules,1455 developing embryos,and embryo rescued 416 hybrid seedlings;‘Ruby Seedless’בBeibinghong’had the best embryo rescue effect,the embryo development rate reached 56.29%,and the seedling rate was the highest up to 21.92%.2.Determining the sampling period of embryo-saving young fruit:‘Crimson Seedless’and‘Flame Seedless’as the hybrid female parent,and‘Zuoyouhong’as the male parent.Determine the best sampling time for the hybrid,set 5 sampling time points,and take hybrid young fruits at 37 d,39 d,41 d,43 d,and 45 d after pollination.The seedling rate of‘Crimson Seedless’בZuoyouhong’was 2.6%.when sampling and culturing 43 days after pollination period.The embryo development rate and seedling rate reached the highest when inoculated and cultured 41 days after pollination period of‘Flame Seedless’בZuoyouhong’,which were 26.7%and 6.7%.3.Optimization of the addition of amino acids to MM3 embryo development medium:MM3 is used as the basic medium,add 1 mmol·L-1,3 mmol·L-1 and 6 mmol·L-1glutamine and phenylalanine.The effect of‘Ruby Seedless’בBeibinghong’is better when cultured in MM3+3 mmol·L-1glutamine medium,the embryo development rate is（69%）,and the seedling rate is（32%）,which is higher than the embryo development of the control group.Rate（49%）and seedling rate（29%）.The effect of‘Perlette’בBeibinghong’was better when cultured in MM3+3 mmol·L-1glutamine medium,the embryo development rate reached（20%）,and the seedling rate reached（5%）.The embryo development rate（8%）and the seedling rate（1%）were higher than those of the control group.The effect of‘Jupiter’בJupiter’is better when cultured in the medium of MM3+6 mmol·L-1 glutamine,the embryo development rate reaches（62%）,and the seedling rate reaches（28%）;it is higher than the embryo development rate of the control group（42%）and seedling rate（8%）.When cultured in embryo development medium supplemented with different concentrations of phenylalanine,the effect of embryo development and seedling formation is not ideal.4.Early-assisted selection of molecular markers for salvage hybrid offspring of seedless cold-resistant embryos:The seedless molecular marker SCF27-2000 was used to detect 111lines of 6 hybrid combinations,of which 64 lines carried seedless specific bands;The cold-resistant molecular markers S241-717 and S238-800 detected 70 strains with cold-resistant specific bands.obtained 46 seedless cold-resistant strains.5.Identification of cold resistance genes in the hybrid progeny plants rescued by seedless cold resistance embryos:cold resistance genes Va CBF1,Vv COR27 and Va ERD15were used to identify cold resistance genes.Set 24℃,16℃,8℃,4℃and 0℃,respectively,5 temperature gradients,followed by low temperature treatment,the treatment time of each gradient is 24 h.The expression levels of the cold hardiness genes Va CBF1 and Vv COR27 in the cold hardy plants of the hybrid progeny of‘Ruby Seedless’בBeibinghong’and‘Ruby Seedless’בXuelanhong’are higher than those in non-cold hardy plants expression level;the expression level of the cold-resistant gene Va ERD15 in cold-resistant plants is lower than that in non-cold-resistant plants.In summary,16 cross and 1 selfing combination,obtain 416 embryo-saving hybrid plants.Using molecular marker-assisted selection,64 out of the 111 hybrid progeny lines were found to carry the seedless trait,and 70 lines carried the cold-resistant trait.Seedless varieties are compared as female parents,and‘ruby seedless’is the most suitable hybrid female parent.The best young fruit sampling periods for‘Crimson Seedless’בZuoyouhong’and‘Flame Seedless’בZuoyouhong’were 43 days and 41 days after pollination period,respectively.Embryo development medium MM3+3mmol·L-1 or MM3+6mmol·L-1glutamine is effective as embryo development medium.Analysis of the differential expression of three related cold hardiness-related genes Va CBF1,Vv COR27 and Va ERD15showed that Va CBF1 and Vv COR27 are effective in the cold hardiness strains of‘Ruby Seedless’בBingbinghong’and‘Ruby Seedless’בXuelanhong’,the expression levels were higher than those of non-cold-resistant strains. |
