Creation Of New Seedless Grape Germplasm With Cold Resistance And Cloning Of Related Cold Resistance Genes

Seedless grapes have fine quality and are convenient. Therefore, breeding fine qualityseedless grape varieties has become an important research topic in the grape breeding.However, most grape cultivars belong to Vitis vinifera, which have good characters but withlow stress-resistance, especially with the low chilling resistance, which limit the developmentof grape industry in the cold regions. Chilling injury caused huge economic losses, which isthe ubiquitous prominent problems in grape production. Therefore, introgressing the traits ofcold resistance from Vitis amureusis to Vitis vinifera by hybridization or genetically modifiedmethods to breeding new seedless grape varieties is the basic way to solve the chilling injuryproblems in production. In this study we used the Chinese wild Vitis amurensis withresistance to cold-stress and it’s F₁（Vitis vinifera×Vitis amurensis） as male parents; seedlesscultivars of Vitis vinifera as female parents, for the cold-resistance embryos rescue breeding.In addition we analysed the cold related EST sequences form the Vitis Amurensis coldresistance genes cDNA library by semiquantitative PCR. Obtained the full length of VaERDgene and VaGLU gene by cloning and analysed the expression preliminary. Specific resultsare as follows:1. Embryo rescue of seedless grape with cold resistance（1）Defination of the best timing of the three female parents for embryo rescueTo improve the embryo development and germination in embryo rescue, the threestenospemocarpic seedless grape cultivars, Sultanina Rose, Perlette and Autumn Royal wereused as experiment materials, the best timing of them for embryo rescue was studied undernatural pollination conditions. The results show that the best sampling time for Sultanina Rose,Perlette and Autumn Royal were51-53d,55d and66d after full bloom, respectively.（2） The impact of the medium to embryo rescueThe ovules from Sultanina Rose, Perlette and Autumn Royal were cultivated on sevendifferent media respectively, the optimum media selected is the MM4+0.5mg/L GA3+1.5mg/L IAA+500mg/L hydrolyzed casein+60.0g/L sucrose+1.5g/L Activated Carbon+10.0μmol/L ZnSO4+1.0mmol/L glycine+1.0mmol/L cysteine+100.0mmol/L mannitol.The development rate and germination rate of ovules from Sultanina Rose, Perlette andAutumn Royal was15.00%and6.66%,24.00%and10.00%,3.75%and2.50%respectively, and an average of15.26%and6.84%. Total98hybrid lines were obtained in this study.（3） The effect of male and female parents on seedless grape embryo rescueHybridization was carried using two stenospemocarpic seedless grape cultivars SultaninaRose and Perlette as female parents, two clones of Chinese wild Vitis amurensis HeilongjiangSeedling and Shuangyou, as well as two interspecific hybrids Beichun （Muscat Hamburg×V.amurensis） and00-1-10（Muscat Hamburg×V. amurensis clone Heilongjiang Seedling） asmale parents. After fifty-one days of pollination, ovules were cultivated on ER and MM4medium by embryo rescue technique, and sixty days later germinated on WPM+BA0.2mg/Lmedium. The results showed that two female cultivars suit to different medium. The propermedium for Perlette was ER, for Sultanina Rose was MM4. The effect of development rateand germination rate of two female parents is insignificant, and Perlette is a little better thanSultanina Rose. However, four male parents have a greater impact on embryo rescue, twointerspecific hybrids00-1-10and Beichun were suitable for male parents than Chinese wildVitis amurensis Heilongjiang Seedling and Shuangyou. Totally fifty lines which belonged tonew grape germplasm were obtained in this study.2. Cloning and sequence analysis of the full length cDNAs and genes （VaERD andVaGLU） related to cold-resistance from V. amurensis Heilongjiang seedlingThe specific primers of the VaERD and VaGLU were designed basiced on their ESTssequences. The full length cDNAs and the genes （ERD and GLU） related to cold-resistancefrom V. amurensis Heilongjiang seedling were cloned. The sequence of ERD and GLU was685bp and1019bp respectively, and named VaERD, and VaGLU （GenBank accession number:JQ687321and JQ687320）. Based on the complete sequence of the open reading frame regions,part DNA sequences of VaERD and VaGLU were cloned.The introns size of VaERD was88bp,VaGLU had no introns sequences.3. The expression changes in mRNA level of the cold-related genes （VaERD、VaGLU、VaECH and VaRD22） from Heilongjiang SeedlingWith the Semi-quantitative PCR technique, the expression change in mRNA levels ofcold-related genes from Heilongjiang seedling was analyzed. The results show that theexpression of four genes were raised in leaf after cold teatments, VaERD reached the highestlevel at12h, VaGLU started to increase at2h, then after the decline the expression levels risedagain in24-48h, VaECH and VaRD22reached the highest level at24h; the expression in stemsof VaERD and RD22reached highest level at48h and2h, respectively; VaGLU and VaRD22did not express in the roots, the expression level of VaERD and VaECH in the roots reachedthe highest at24h and6h respectively. This study shows that the four genes can involved incold response.