Investigation On Reproductive Barriers In Distant Hybridization Of Chrysanthemum And Germplasm Enhancement

Chrysanthemum is one of the most important and popular ornamentals over the world. However, most of chrysanthemum cultivars have low resistance to diseases, pests and other environmental stress, which seriously influences their output and quality. Therefore, it is very necessary to improve chrysanthemum. Wide distant hybridization has become a promising strategy to improve tolerance of chrysanthemum through introducing tolerance trait from the wild species in Dendranthema into it. However, reproductive barriers often exist in chrysanthemum distant hybridization and seriously influence the utilization of the these excellent germplasm resources. In response to it, several wide crosses were carried out using D. grandiflorum ’Jinlinghuangyu’ and ’Yuhuaxingchen’ as female parents, and D. lavandulifolium, D. nankingense, D. indicum and D. zawadskii as male parents in this study. Factors influencing seed set of the wide crosses were particularly investigated from the aspect of plant reprouductive biology. In addition, the effects of mentor pollen, gibberellic acid treatment and delayed pollination on the cross between ’Yuhuaxingchen’ and D. nankingense were examined. Finally, interspecific hybrids between ’Yuhuaxingchen’ and D. indicum were identified and their drought tolerance was evaluated. The main results and conclusions are listed as follows:(1) In the cross between ’Jinlinghuangyu’(female parent) and D. lavandulifolium (male parent), pollen viability of male parent was11.2%just before pollination. No pollen grains germinated on the stigmas during4h after pollination and only7.5pollen grains germinated on each stigma at8h after pollination. At12h after pollination, the number reached the highest value,16.1. At24h and48h after pollination, the number was quickly decreased to3.2and2.4, respectively. In addition, most pollen grains germinated abnormally on the stigmas and many pollen tubes stayed on the stigmas’ surface and failed to enter ovaries. At8d after pollination, normal globular embryos were observed only in12.0%ovaries. Thereafter, only abnormal embryos were observed in some ovaries, and as a consequence, no seeds were obtained. However, seed set of female parent under open pollination conditions was as high as42.3%. These results suggest that both pre-and post-fertilization barriers, i.e. only a few germinated pollen grains on the stigmas and abnormal germination of most pollen grains before fertilization as well as degeneration of a large number of embryos after fertilization, may be the main factors resulting in infertility in the cross.(2) In the three crosses between ’Yuhuaxingchen’(female parent) and three wide species (D. nankingense, D. indicum and D. zawadskii)(male parents), pollen viability of three wild species ranged from20%to25%. In the cross between ’Yuhuaxingchen’ and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in12.2%ovaries at8d after pollination and thereafter all the embryos aborted. In other two crosses, many pollen grains germinated on stigmas and pollen tubes penetrated stigmas normally after pollination. Moreover, normal embryos were observed in over50%ovaries from8to15d after pollination in the cross between ’Yuhuaxingchen’ and D. indicum, and seed set was58.9%. In the cross between ’Yuhuaxingchen’ and D. zawadskii, normal embryos were observed in51.8%ovaries at8d after pollination. After that, however, most embryos degenerated and seed set was only9,2%. These data suggest that pollen viability has no significant effects on seed set of the three crosses. Very few germinated pollen grains on stigmas and abnormal growth of most pollen tubes before fertilization, and embryo abortion are the main factors causing failure of the cross between ’Yuhuaxingchen’ and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between ’Yuhuaxingchen’ and D. zawadskii and no barriers occur in the cross between ’Yuhuaxingchen’ and D. indicum.(3) In the cross between ’Yuhuaxingchen’(female parent) and D. nankingense (male parent), the average number of pollens germinating on stigmas at24h after pollination and the percentage of embryo were significantly higher in the treatments of mentor pollen and gibberellic acid than in the control. Although the average number of pollen grains germinating on stigmas24h after pollination was slightly lower in the treatment of delayed pollination than that in the control, the percentage of embryo in the treatment of delayed pollination was significantly higher than that in the control. These data suggest that the mentor pollen, delayed pollination and gibberellic acid treatment are three efficient pollination methods to overcome some reproductive barriers in the cross between ’Yuhuaxingchen’ and D. nankingense.(4) In the cross between ’Yuhuaxingchen’(female parent) and D. indicum (male parent),282seeds were obtained and only155seeds germinated. Finally,132seedlings survived. Six putative hybrid lines with most drought tolerance were first screend from the132hybrid lines by withholding water. Chromosome counting and morphological analysis showed that the six putative hybrids were real hybrids. The density of leaf epidermal hair, proline content, and plant survival rate after PEG6000treatment were the highest in D. indicum and the lowest in ’Yuhuaxingchen’ among the six hybrids and their parents. In contrast, EL value and MDA content after PEG6000treatment were the highest in ’Yuhuaxingchen’ and the lowest in D. indicum. These results suggest that some true hybrids with improved drought tolerance can be obtained through interspecific hybridization and these excellent germplasm resources are important intermediate materials in the future drought breeding of chrysanthemum. In addition, the association of EL and MDA contents, proline accumulation after PEG6000treatment and the density of leaf epidermal hair may be useful selection criteria for drought-tolerant evaluation in future chrysanthemum breeding programs with the aim of improvement of plant drought tolerance.