Innovation Of Cold-resistant Germplasm By Hybridized Of Grape With Different Ploidy And Embryo Rescue

Seedless grape is an excellent raw material for fresh food,drying and canning.It is the development direction and trend of international grape production and consumption.Seedless grapes are mostly Eurasian grapes.Although seedlessness is a good characteristic of Seedless grapes,early embryo abortion and low resistance seriously restrict the breeding and cultivation of Seedless grapes.Therefore,breeding triploid seedless grape varieties with strong resistance has become one of the important objectives of grape breeding.In this study,two tetraploid grapes and five diploid grapes were used as female parents,and 12 grape varieties with strong resistance were used as male parents,and 38 combinations were set up to cross.The pollen viability of hybrid paternal parents was determined by FDA fluorescence staining,TTC staining and in vitro culture.The best method for determining pollen viability of grape was screened and the cross compatibility of different cross combinations was analyzed.The effects of endogenous hormones on embryo abortion in seedless grapes were studied with Wuhe Cuibao,Lihongbao and seeded Cabernet Sauvignon grapes as materials.Using embryo rescue technology and molecular marker-assisted technology,cold-resistant triploid hybrid plants were bred,and the deformed seedlings in the experiment were classified and transformed,so as to provide new materials with reference value for triploid cold-resistant grape breeding.The main results are as follows:（1）FDA staining is the most suitable method for determining grape pollen viability.The cross combinations with Hejin as male parent belonged to strong affinity combination,while the four cross combinations with Wuhe Cuibao as female parent belonged to weak affinity combination,among which Hutai No.8×Hejin hybrid had the highest affinity.Six cross combinations,Beibinghong×Hutai No.8,Zaoheibao×Huozhou Ziyu,Zaoheibao×SP115,Wuhe Cuibao×Shanhe No.1,Wuhe Cuibao×Xinyu and Wuhe Cuibao×Huozhou Hongyu, had a seed rate of 0,which was a cross incompatible combination.The pollen viability of parents is closely related to the fruit setting rate of hybridization.For grape varieties with high or medium pollen viability,the fruit setting rate of hybridization as male parent is higher.On the contrary,the pollen viability of grape varieties is lower,and the fruit setting rate of hybridization as male parent is lower.（2）The high content of IAA,ABA,GA₃,GA₄,i PA and the low ratio of（IAA+GA₃）/ABA and IAA/（ZR+i PA）in the ovules of seedless grapes are not conducive to the development of Wuhe Cuibao and Lihongbao ovules and embryos.Moreover,they are highly correlated with embryo abortion in seedless grapes.（3）The effect of parental genotype on embryo rescue was different.The embryo formation rate of Tetraploid Grape Zaoheibao and Hutai No.8 was the highest,20.1% and 13.9% respectively.The embryo germination and seedling formation rate of Hutai No.8×Beibinghong was the highest,44.7%.The embryo formation rate of the combination of diploid grape Ruby Seedless was the highest,and the embryo formation rate of the combination of Ruby Seedless and Beibinghong was the highest（13.7%）and the germination and seedling formation rate was the highest（59.8%）.（4）Twelve hybrid progenies of two cross combinations were identified by cold-resistant molecular marker S241-670,and four cold-resistant lines were preliminarily identified.Sixteen hybrid progenies of three cross combinations were identified by using nuclear-free molecular marker SCC8-1018 combined with root tip chromosome observation and flow cytometry.Four seedless lines were preliminarily identified,including one triploid line.（5）Adding 500 mg/L banana mud to embryo formation medium and embryo development medium could significantly improve embryo formation rate and Germination Seedling rate.（6）According to the morphology of deformed seedlings,it can be divided into six categories:（1）epicotyledon has only one cotyledon;（2）neither cotyledon nor root;（3）cotyledon folds and curls;（4）hypocotyls form young roots and no cotyledons;（5）cotyledons but no roots;（6）early seedling development stagnates.The transformation rate of deformed seedlings with cotyledon curl type was the highest,and the transformation rate of deformed seedlings without cotyledon and root type was the lowest.