| Grape is one of the major fruit trees. Seedless grape breeding has become an importantaspect of grape research, with increasing proportion of seedless grapes in fresh grapes. Atpresent, cultivated seedless grapes are most from Vitis vinifera L., which own fine quality, butsusceptible to pathogens. Therefore, for seedless grapes, the selection of high quality, bigberry, and high resistance has become one of important goals of grape breeding. Moreover,the embryo rescue is a main technique to improve breeding efficient in seedless grapes.In present research,36crossings with European seedless grape cultivars （including,Thompson Seedless, Flame Seedless, Heshi Seedless, Ruby Seedless） as the female parentsand3types cultivars （wild Chinese Vitis species: Tangwei, Xuefeng, Shuangyou, Beichun.Seedless grapes: Thompson Seedless, Flame Seedless, Heshi Seedless, Ruby Seedless, andseeded grapes: Muscat Hamburg, Red Globe） as the male parents are conducted. To reveal thephysiological mechanism of embryo abortion of seedless grape, the content of mineral, activeoxygen, enzyme, endogenous polyamine and hormone was measured in pulps and in-vitroovules. To obtain a new batch of hybrid progenies of seedless grape, the improvement of therate of hybridization fruit and embryo rescue was conducted. Meanwhile, we gained somenew grape germplasm resources by studying the affecting factors of abnormally germinatedembryos in the process of embryo rescue and the conditions of transforming into normalseedlings. Moreover, to improve the rate of transplantation, the standardization operation ofembryo rescue seedlings was studied in the process of transplantation and acclimation. Theresults obtained were as follows:1. Among the same variety, the contents of endogenous polyamine and hormone weredifferent in different pulps and in different kinds of in vitro ovules, suggesting that theendogenous hormones and polyamines had a certain influence on embryo abortion of seedlessgrape cultivars. We also found that spraying Put50mmol·l-1+Spd50mmol·l-1and IAA30mg·l-1+ABA30mg·l-1was helpful to improve the development of the ovules in fruit berryduring the fruit setting. Adding Put2mmol·l-1+Spm2mmol·l-1+Spd2mmol·l-1andspraying IAA1.0mg·l-1+6-BA2.0mg·l-1+GA32.0mg·l-1could increase the rate of embryorescue seedlings in seedless grapes.2. The fruit setting rate was different in hybridized combinations of different parents, so hybridized combination was a key factor to control fruit setting rate. Our results showed thatfor the fruit setting rate, Ruby Seedless×Beichun was higher （15.8%） among16combinations of seedless grapes×wild Chinese Vitis species; Ruby Seedless×HeshiSeedless was higher （9.7%） among12combinations of seedless grapes×seedless grapes;Ruby Seedless×Muscat Hamburg reached higher （16.8%） among8combinations of seedlessgrapes×seeded grapes.3. The different appropriate culture mediums were conducted during the process ofembryo rescue. For ovule development, improved MM3as a solid medium and ER as a liquidmedium were helpful to improve embryo development under the dark culture at roomtemperature; for embryo germination, the medium with WPM+IAA1.0mg·l-1+6-BA2.0mg·l-1+GA32.0mg·l-1adding Put2mmol·l-1+Spm2mmol·l-1+Spd2mmol·l-1couldincrease embryo germination rate and plantlet development rate. In addition, our resultshowed that the rate of embryo rescue was different in hybridized combinations. Such as, theembryo germination rate was higher （28.1%） and plantlet development rate was higher（61.0%） in Ruby Seedless×Beichun among combinations of seedless grapes×wild ChineseVitis species; among combinations of seedless grapes×seedless grapes, the embryogermination rate of Thompson Seedless×Ruby seedless was higher （37.5%）, plantletdevelopment rate was33.3%, followed by embryo germination rate of Ruby Seedless×FlameSeedless （17.1%） and plantlet development rate （28.6%）; among combinations of seedlessgrapes×seeded grapes, embryo germination rate of Ruby Seedless×Red Globe was higher,34.5%and plantlet development rate was33.3%.4. The genotype of parents and culture condition of in-vitro affected the rate of abnormalseedlings induced. The medium WPM+6-BA2.0mg·l-1+IAA2.0mg·l-1was conducted totransform from abnormal seedlings to normal seedlings, and24.6%abnormal seedlings wastransformed into normal. For excessive growth-seedlings, the better transform rate was33.3%by inoculating every14days. We also investigated all induced albino seedlings and vitrifiedshoots, and they failed to transform into normal finally, indicating that the two kinds ofmalformed seedlings was difficult to change into normal ones.5. The factors influencing transplantation and acclimation, including the rooting oftest-tube plantlet, plant growth potential, matrix, and nutrient solution were investigated in2011and2012. We summarized a set of normalized procedures of the transplantation andacclimation of embryo-rescue seedlings. Such as, the medium1/2×MS+0.3mg·l-1IAA asthe most suitable culture medium for the rooting and seedling; transplanting10cm highseedling into pots （18cm×16cm） with M1（peat: vermiculite: coconut shell=1:4:1） andspraying nutrient solution （1/10×MS macroelement+1×MS other culture medium）, helpful for improving the seedling formation rate of transplantation and acclimation of embryo-rescueseedlings. After seedlings adaption,371lines were transplanted and survived in the field.In summary, the physiological changes during the berry development of seedless grapehad a decisive effect on young embryo development and abortion; meanwhile, genotypes ofparents played a primary role in deciding the success of embryo rescue. Secondly, thecomponents in medium were an important factor influencing embryo development by in vitroculture. Moreover, rooting of test-tube plantlet, plant growth potential, matrix and nutrientsolution became main factors improving seedling rate during transplantation and acclimation. |
