A study on the application of seedless grapevine breeding for cold-hardness and disease-resistance using embryo rescue

Abstract: 【Objective】Our research involved obtaining the heterozygous plant of the hybridized combina⁃tion of European seedless grape × Chinese wild grape using embryo rescue technology, so as to study theeffect of the parental genotype on the embryo rescue, as well as to conduct greenhouse seedling transplant⁃ing. In addition, we conducted hybrid identification and assisted selection on the filial generation usingmolecule marker technology, and then selected the new seedless grape plants found among them, provid⁃ing new materials for the future breeding of the new seedless grape strain.【Methods】8 seedless grape varieties were treated as the female parent, while the Chinese wild grape with cold and disease resistance wastreated as the male parent. 9 hybridized combinations were designed centering on the seedless and resis⁃tance breeding objective; artificial emasculation and pollination were conducted in early May, the hybridfruit was chosen timely to pick off its ovule, which was placed onto the embryo growth medium under asep⁃tic conditions, and cultured in the dark for 8 weeks, after which it was picked off from its embryo and putonto the embryo germination medium so that it could develop into seedlings. Then the subculture propagation was conducted after the seedling had grown in the test tube. The vigorous seedlings on the subculturemedium were selected for greenhouse transplanting and seedling exercising , the DNA of the filial genera⁃tion leaves was extracted through embryo rescue technology, then hybrid identification and assisted selec⁃tion was conducted on the filial generation, and finally they were transplanted to the field.【Results】Theseedless grape varieties were treated as the female plant and the European seedless grapes were hybrid⁃ized with the Chinese wild grapes through the embryo rescue technology; all together 9 hybridized combi⁃nations were designed, and 3 617 hybrid ovules were obtained from the field for culture; finally, 32 embryo rescue hybrid strains and 394 seedlings were obtained. Greenhouse transplanting and seedling exer⁃cising were conducted on the 32 embryo rescue hybrid filial generation strains obtained through the exper⁃iment, and 394 single plants were transplanted and the seedlings exercised, among which 321 singleplants survived, with the survival rate of the greenhouse seedling exercising of 81.1%. Three types of genetic molecular markers GLSP1, SCF27 and SCC8 were selected for detecting the cross parent and the fil⁃ial generation, and the GLSP1 detection results of the parents showed that the 569 bp specific strips couldonly be amplified by the‘Beauty Seedless’and‘Flame Seedless’, and it was also applicable to the filialgeneration of the‘Delight’בTaishan-2’when detecting the filial generations. Detecting the 32 embryorescue filial generation strains through GLSP1, SCF27 and SCC8 could preliminarily determine 11, 18,and 21 seedless strains, respectively. There were 4 out of the 32 strains that could amplify specific stripsin the three types of markers and 18 that could amplify specific strips in two and three types of markers;while seedless phenotypic traits could be detected in 17 strains by applying SCF27 and SCC8 simultane⁃ously. Seedless markers could be detected in 28 strains when integrating the detection results of the threetypes of markers on the 32 hybrid filial generation strains.【Conclusion】In the experiments conducted inthis paper, the seedling rates of the combinations with the‘Flame Seedless’‘Aishenmeigui’and‘Centen⁃nial Seedless’being the female plants were relatively higher, which was consistent with the relativelyhigher germination rates and seedling rates obtained through ovule culture with the‘Flame Seedless’asthe female plant in the research performed by other scholars; however, the combination of‘Flame Seedless’בBeichun’was associated with a relatively low embryo development rate and seedling rate, thus itcould be speculated that the male parent showed certain influences on the embryo rescue effects. The combinations with the‘Delight’and‘Sultanina Rose’being the female parents had relatively high embryogermination rates, which were 41.67% and 72.72%, respectively. Among them,‘Delight’was the brandwith a large seed scar as well as high seedling rate, thus it was the excellent material as a female parent,which was consistent with the previous research results. The‘Delight’בTaishan-2’was the seedlessgrape × Chinese wild grape combination, the embryo germination rate of which was higher than that of the‘Delight’בJiangxi-2’(34.57%) and‘Delight’בJiangxi-3’(36.61%) studied by Tang Dongmei. Therefore, the selection of the female parent was critical when conducting cross breeding and the selection ofthe male parent also had certain influences on the embryo rescue effects.