| Seedless grapes are preferred by the consumers for table or raisin around the world. For the fertilized embryos often aborted during their development in the stenospermic grapes, the conventional strategies only could adopt seeded×seedless method, with a breeding cycle of more than 10 years. Wheareas, the development of embryo rescue makes stenospermic grapes serve as females directly, reducing the cross generations efficiently and raising the percentage of seedless hybridizations. It is well known that most of the European seedless cultivars (Vitis vinifera) are susceptible to fungul diseases. Thus, breeding for new resistant cultivars has been one of the most important objectives for the worldwide vine breeders. Some forgein breeders have tried to introgress the traits of disease resistance from Vitis rotundifolia to Vitis vinifera. But the different chromosomes (V. vinifera, 2n=38; V. rotundifolia, 2n=40) of the two subgenera, often lead to crossing incompatibility, which largely limited the breeding efficiency. China is one of the origin centers of Vitis species, with many wild disease resistant germplasms more compatible to V. vinifera compared to V. rotundifolia. To combine the fungul disease resistance with the good-quality traits of the European stenospermic females, it should be feasible for new germplasm innovation and genetic improvement in seedless grape breeding if using embryo rescue as a tool..In this research, the whole breeding process and influence factors was studied systemically, by using embryo rescue of 20 crosses (concluding 4 selfed and 2 open pollinated hybrid materials). An improvement of breeding efficiency and a novel medium for embryo rescue was developed. A successful disease resistant seedless grape breeding system comprised of crossing, ovule culture, embryo excise and plant formation etc was innonatied. The highest plant percentage over 65% was obtained in the hybridizations of stenospermic females×disease resistant males. The main results are in the following:1. The influence of different cross combinations on the embryo rescue was studied. It showed that the female parents play the most important role in the success of embryo rescue. Among the stenospermic cultivars, Delight, Emerald Seedless and Ruby Seedless are excellent female condidates. Whereas Monukka, Thompson Seedless and Youngle are inferior cultivars for their lower production in embryo rescue breeding. At the same time, the pollen donors play a crucial role as well. The cultivar Zixia showed superior to Xinyu significantly when Ruby Seedless served as the female parent. Although the plant development rate decreased in Flame Seedless, no significant difference was observed in other cultivars such as Delight, Emerald Seedless and Thompson Seedless when selfed, showing it should be strongly controlled by the female genotype.2. The influence of different medium types, medium phase, PH value, carbohydrate sources, concentrations of cysctine, casein hydrolysate and activated charcoal on the embryo rescue was investigated during in-ovule embryo culture. It showed that ER medium are significantly superior to NN and MS media. By the percentages of embryo development and plant establishment, no significant difference was detected among the PH value of 5.0, 6.0 and 7.0 if liquid medium was used. Among the carbohydrate sources of 6%, sucrose showed better than glucose and fructose. The optimum concentration of cysteine was 1.0mM. And casein hydrolysate of 500mg l-1 showed most suitable for embryo rescue. It seemed that the concentration of activated charcoal of 1.5g l-1 resulted in the largest proportion of plants. Based on the aboved results and some ajugements on mineral components, a novel more suitable medium(MM4) for disease resistant grape breeding was proposed as well (Applying for patent, no.200610043024.0).3. The influence of supplements of mineral elements, amino acids, plant hormones and anti-oxygents on the embryo rescue during in-ovule embryo culture was evaluated. It showed that the impact of these compounds relied on the different cross combinations. In the seedless grape breeding, the minor element zinc (10.0μmol l-1) showed superior to selenium (0.5μmol l-1). Among the amino acids (2.0 mmol l-1), glycine and glutamine show positive effects in this breeding system. Phyto-hormone of ABA shows significant positive impact on the embryo rescue. And mannitol (100 mmol l-1) added medium enhanced the embryo recovery and plant develiopment. In this study, the highest normal plantlet percentage of 65.3% was obtained from hybridizations of Delight×Beichun, when ovules cultured in the medium of MM4+ABA0.01μmol l-1.4. The process of in vitro embryo germination and plant formation was also studied. It was observed that not all germinated embryos could develop to normal plants in this study. In some cases, embryos formed plantlets abnormal in morphology, which was mainly influenced by genotypes and media. The females of Ruby Seedless and Emerald Seedless showed more abnormal plantlets than Delight and 00-3-1, with the highest percentage of 22.3% based on the germinated embryos. During the in-ovule culture period, zinc or selenium supplemented medium reduced the abnormal plantlet production while GA3 combined with IAA and putrescine supplemented media enhanced the production. Fruits pre-chilled 4℃for 20d before in vitro culture produced lower abnormal plantlet percentage. When the excised embryos were cultured on the WPM supplemented with 1.0μmol l-1 6-BA and 1/2MS supplemented with 0.5μmol l-1 IBA, higher abnormal plantlet proportion was obtained from the former, despite higher embryo germination rate as well. No significant difference was determined among the ovule culture time of 8, 10 and 12 weeks.5. The breeding efficiency was raised by chemical treatment in vivo combined with the innovation of other parameters of medium and sampling time. It showed that spraying of putrescine (10.0 mmol l-1) 6 weeks after pollination inhibited the process of embryo abortion. The best result was obtained from Emerald Seedless×Beichun, with 44.7% ovules generated plants when cultured on 10.0μmol l-1ZnSO4 supplemented MM4 medium. According to the results, a strategy composed of crossing, post-bloom treatment and embryo rescue was recommended. Moreover, The polyembryony was influenced by genotype and chemical treatment. Delight showed more easily to form polyembryos than 00-3-1, with the highest polyembryo rate reaching 22.0% when ovules from the cross of Delight×Beichun cultured in ER medium.6. The technique for rescued plants transplanting in green house was studied. The highest survival rate reached 90% when the autoclaved mixture (perlite: peat: soil=4: 1: 1; V/V) was used. The best age of the plantlets was 40 days of in vitro culture. The concentration of the nutrient liquid of 1/16MS showed better for the growth of the potted plants. It was also discovered that the survival rate was higher when the disease resistant parents was involved in the cross combinations. After 2-3 month's hardening, the survival plantlets could be planted in the field successfully. Thus, an efficient transplant method for large scale breeding system was proposed in correspondence.7. Some of the new hybrids were evaluated in the field and in the lab conditions. Disease resistance by natural identification showed that dorminant resistant genes existed in the Chinese wild Vitis species could be inherited to the progenies by crossing with seedless Vitis vinifera. Embryo rescue was confirmed to be an effient tool for new germplasms with traits of disease resistance and seedlessness. RAPD analysis was also carried out, using a grape probe of GSLP1 for seedlessness and a SCAR marker of SCO10-835 for downy mildew resistance identification. The results showed that 13.3~33.3% hybrids had the specific band of 569bp. In the cross of Sultanina Rose×1-9-6, 46.7% hybrids showed disease-resistant without the specific band of 835bp. By this means of earlier screening, the efficiency of disease resistant seedless grape breeding was improved significantly. |
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