Novel Germplasm Innovation Of Seedless Grapes By Embryo Rescue And Technique Improvement

Development of seedless grapes is one of principle objectives of grape breeders worldwide. Since the major commercial seedless grape cultivars are derived from Vitis vinifera, a species susceptible to diseases, it is necessary to develop new resistant, big berries and high quality seedless cultivars for meeting the demand of consumers and grapevine growers. In conventional seedless grape breeding program, seeded grape varieties were used as female parents, while seedless cultivars were used only as male parents to transmit seedless trait due to the zygotic embryo abortion. But the proportions of seedless offsprings were quite low and the breeding programms were inefficient. The development of embryo rescue techniques had ensured more seedless progenies using seedless cultivars as female and male parents, and utilized to obtain triploid hybrid seedlings from crosses between diploid and tetraploid cultivars. To introgress disease resistance of China wild Vitis species as pollen parent into seedless Vitis vinifera as female parent through controlled crosses, and to produce more efficiently triploid hybrids from crosses between diploid and tetraploid grapes, it should be reliable to breed new seedless and disease resistance, large-sized berries cultivars through using embryo rescue technique.Thirteen seedless cultivars/line (including'Thompson Seedless','Flame Seedless','Deligh't,'Dawn Seedless','Ruby Seedless','Cenennial Seedless','Black Monukka','Autumn Royal','Orlando Seedless','Superior Seedless','Fantasy Seedless','Otilia Seedless','00-3-1') and twelve seeded cultivars (including'Tangwei','Xuefen'g,'Shuangyou','Beichun','Kyoho','Takatsuma','Zizhenxiang','Fujiminori','Xinyu','Red Globe','Zixia','Rizamat','Xinpu-1') were used in this study.'Tangwei'and'Xuefeng'are V. davidii cultivars.'Shuangyou'is V. amurensis cultivar.'Beichun'is European-Amur hybrid with V. vinifera and V. amurensis. Line'00-3-1'is European-Wild hybrid with V. vinifera and V. thunbergii,'Kyoho','Takatsuma','Zizhenxiang','Fujiminori'are tetraploid cultivars and European-American hybrids with V. vinifera and North American species (V. spp.), while the other cultivars are diploid and V. vinifera cultivars. Thirty-two crosses, including seedless×China wild Vitis species (and hybrid'Beichun'), seedless×seedless, seedless×seeded, interploid crosses between diploid and tetraploid cultivars, were carried out in order to obtain the novel seedless germplasm through embyo rescue. Meanwhile the cultivars and hybrid ovules above were used to improve the technique of embryo rescue on the basis of previous study, involved in demonstrating the effect of influence factors during different embryo rescue stages and the effect of exogenous application of plant growth regulators on development of ovule and subsequent embryo rescue of stenospermic grape. The main results were in the following:1. We had obtained 1337 hybrid seedlings from 32 crosses through embryo rescue technique, including 153 hybrid seedlings from 6 crosses of seedless×China wild Vitis species (or Beichun), 401 hybrid seedlings from 6 crosses of seedless×seedless, 353 hybrid seedlings from 9 crosses of seedless×seeded, 430 hybrid seedlings from 8 crosses of diploid×tetraploid and 4 crosses of tetraploid×diploid. The hybrid seedlings had acclimatized and transplanted into seedling nursery in batch es, 509 survival lines were obtained.2. The detection of the seedlessness in 191 hybrid lines was performed using seedless specific probe GSLP1, 77 lines (40.3%) amplified the 569 bp specific band. Assistant selection of RAPD marker S183-1354 of resistance to anthracnose was carried out for the cross of'Flame Seedless'×'Tangwei'. 41 lines (48.8%) amplified the 1354 bp specific band from 84 lines. After comprehensive detection using the two molecular markers, 24 lines of seedlessness and resistance to anthracnose as well were selected.3. Eight triploid hybrid progenies were obtained from 409 lines of the 12 crosses between diploid and tetraploid grapes by flow cytometry identification. The results showed that regeneration rate of hybrid seedings obtained by embryo rescue were obviously higher than that of obtained by sowing mature seeds. It could be confirmed that larger amount of hybrid seedlings were obtained from the cross form of 2x×4x than the cross form 4x×2x, while more triploid hybrid progenies were obtained from the cross form of 4x×2x than the cross form of 2x×4x.4. The investigation was conducted to evaluate the feasibilities and optimal sampling periods of 10 seedless grape cultivars. By measuring in terms of most number of developed embryos or germinated embryos per berries, the result showed that the order from ease to difficulty of embryo rescue was'Otilia Seedless','Fantasy Seedless','Flame Seedless','Superior Seedless','Black Monukka','Autumn Royal','Delight','Dawn Seedless','Thompson Seedless', and'Centennial Seedless'. The optimal sampling dates of above cultivars for embryo rescue were 40 DAB, 60 DAB, 70 DAB, 40 DAB, 50 DAB, 70 DAB, 70 DAB, 60 DAB, 55 DAB, 35 DAB, 40 DAB, respectively.5. The effects of various correlative factors on embryo rescue in vitro were investigated at every culture stage on the basis of previous experiment. At the stage of ovule culture, the results indicated that MM3 and Nitsch +GA3 0.5 mg l-1+IAA 1.5 mg l-1 medium were most suitable for embryo development, and the next was ER medium.?Solid-liquid double phase medium and room temperature had positive effect on embryos development in vitro. At the stage of embryo germination, WPM + BA 0.2 mg l-1 medium, and supplemented with 2~5 mM Put had positive effect on embryos germination. It showed that embryo germination correlated with size of embryo. At the stage of plantlet regeneration, the results indicated that rates of normal plantlets was relative to zogyic embryo development degree of grape cultivars, and the emergence of albino seedlings connected with maternal genotypes. The optimal medium of converting?abnormal plantlets was MS + BA 2.0 mg l-1 + IBA 0.5 mg l-1, the plantlets with normal shape stems and leaves produced roots on MS medium supplemented with 0.2 mg l-1 IBA, therefore normal plantlets were regenerated from abnormal plantlets. At the stage of plantlet acclimatization and transplantation, the suitable seedling age was 30 days. It was beneficial to survival of embryo rescue seedlings that mixture substrate was autoclave sterilized and the solution of 1/4 B5 macroelement irrigated seedlings every week.4. The effects of exogenous application of plant growth regulators on embryo rescue of high abortion cultivars'Centennial Seedless','Thompson Seedless'and'Flame Seedless'were discussed. The optimum concentration and period of application of plant growth regulators was determined: 100~500 mg l-1 chlormequat application in'Centennial Seedless'14 days before bloom, 500 mg l-1 chlormequat application in'Thompson Seedless'10 days before bloom, 30 mg l-1 benzyladenine application in'Flame Seedless'20 days before bloom.