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In our earlier study, pre-fertilization barrier was one of the main factors causing the failure of the wide hybridization between Chrysanthemum grandiflorum ‘Yuhuaxingchen’ and C. nankingense, and seriously restricted the utilization of the excellent chrysanthemum germplasm, C. nankingense. In order to overcome the pre-fertilization barrier and obtain some hybrids, we carried out wide hybridization between them again in this study, and tentatively adopted three pollination techniques including mentor pollen, delayed pollination, and gibberellic acid treatment. The average number of pollen grains germinating on stigma at 24 h after pollination and the percentage of embryo were investigated with a temporary mount method and the technique of fluorescence microscopy. It was found that, compared with the control, delayed pollination, gibberellic acid treatment, and mentor pollen significantly increased the average number of pollen grains germinating on each stigma at 24 h after pollination by approximately 152, 235, and 182%, respectively. In addition, all the three pollination techniques significantly increased the percentage of normal embryos at different periods after artificial pollination in comparison to the control. Furthermore, seed sets of the crosses using the three special pollination techniques were 1.1, 1.4, and 2.0%, respectively, whereas we did not obtained any seeds in the control. Taken together, these results suggest that delayed pollination, gibberellic acid treatment, and mentor pollen are three useful pollination techniques for overcoming pre-fertilization barriers and increasing seed set in the wide hybridization between C. grandiflorum ‘Yuhuaxingchen’ and C. nankingense, and they may be widely applied for increasing breeding efficiency in other chrysanthemum wide hybridization facing similar pre-fertilization barriers.

Chrysanthemum grandiflorum ‘Yuhuaxingchen’ is an important commercial chrysanthemum cultivar with excellent ornamental quality but low drought tolerance, whereas C. indicum has exceptional drought tolerance. In our earlier study, many hybrid seeds between them were obtained through interspecific hybridization. In the present study, we selected six putative hybrid lines with most drought tolerance from all the hybrid lines by withholding water, indentified their facticity by chromosome counting, and then evaluated their drought tolerance through determining foliar electrolyte leakage (EL), contents of malondialdehyde (MDA) and proline, and plant survival rate after 20% polyethylene glycol 6000 treatment. It was found that 155 out of 282 seeds germinated and only 132 seedlings survived. In addition, chromosome and morphological analysis showed that the six putative hybrids were real hybrids and their morphological features were intermediate between their parents. Furthermore, the density of leaf epidermal hair, proline content, and plant survival rate were the highest in C. indicum and the lowest in C. grandiflorum among the six hybrids and their parents. In contrast, EL value and MDA content were the highest in C. grandiflorum and the lowest in C. indicum. These results suggest that some true hybrids with improved drought tolerance can be obtained through interspecific hybridization in chrysanthemum breeding. Therefore, interspecific hybridization between chrysanthemum cultivars and their wild species may become a promising way to improve their biotic and abiotic resistance in the future breeding.

Reproductive barriers often exist in the crosses between Dendranthema grandiflorum (Ramat.) Kitamura and its wild species and seriously result in low seed set, consequently reducing breeding efficiency. For the purpose of revealing the factors leading to low seed set, we investigated pollen viability, germination behavior of pollen grains on stigmas and embryo development in the crosses between D. grandiflorum and three wild species, D. nankingense (Nakai) Tzvel., D. indicum (L.) Des Moul. and D. zawadskii (Herb.) Tzvel. using technique of paraffin section, and light, fluorescence and scanning electron microscopy. The results indicated pollen viability of three wild species ranged from 20 to 25%. In the cross between D. grandiflorum and D. nankingense, very few pollen grains germinated on stigmas after pollination and most of them germinated abnormally. In addition, normal embryos were observed in 12% ovaries at 8 days 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 over 50% ovaries from 8 to 15 days after pollination in the cross between D. grandiflorum and D. indicum, and seed set was 59%. In the cross between D. grandiflorum and D. zawadskii, normal embryos were observed in 52% ovaries at 8 days after pollination. After that, however, most embryos degenerated and seed set was only 9%. 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 D. grandiflorum and D. nankingense, whereas only embryo abortion is a main factor resulting in low seed set in the cross between D. grandiflorum and D. zawadskii and no barriers occur in the cross between D. grandiflorum and D. indicum.

Embryo abortion is the main cause of failure in chrysanthemum cross breeding, and the genes and proteins associated with embryo abortion are poorly understood. Here, we applied RNA sequencing and isobaric tags for relative and absolute quantitation (iTRAQ) to analyse transcriptomic and proteomic profiles of normal and abortive embryos. More than 68,000 annotated unigenes and 700 proteins were obtained from normal and abortive embryos. Functional analysis showed that 140 differentially expressed genes (DEGs) and 41 differentially expressed proteins (DEPs) were involved in embryo abortion. Most DEGs and DEPs associated with cell death, protein degradation, reactive oxygen species scavenging, and stress-response transcriptional factors were significantly up-regulated in abortive embryos relative to normal embryos. In contrast, most genes and proteins related to cell division and expansion, the cytoskeleton, protein synthesis and energy metabolism were significantly down-regulated in abortive embryos. Furthermore, abortive embryos had the highest activity of three executioner caspase-like enzymes. These results indicate that embryo abortion may be related to programmed cell death and the senescence- or death-associated genes or proteins contribute to embryo abortion. This adds to our understanding of embryo abortion and will aid in the cross breeding of chrysanthemum and other crops in the future.

Background Breeding programs for the water lotus (Nelumbo nucifera) are hampered by an inability to account for variation in seed set associated with crosses between different cultivars. We studied seed set in two reciprocal crosses between lotus cultivars (‘Guili’ × ‘Aijiangnan’ and ‘Molingqiuse’ × ‘Qinhuaiyanzhi') to obtain insights into factors that govern fecundity in these experimental hybrids. Pollen viability, stigma receptivity and embryo development were compared for each hybrid and reciprocal cross. Results Pollen viability of the individual cultivars ranged from 4.1% to 20.2%, with the highest level (>11.9%) for all cultivars observed from the earliest collected grains (05:00–06:00 a.m.). Stigmatic pollen germination peaked at 4 h after pollination and varied from 4.8 to 60.6 grains per stigma among the crosses. Production of normal embryos ranged from 7.6% to 58.8% at 1 d after pollination and from 0 to 25% by 11 d after pollination. Seed set in crosses (0.2–23.3%) was generally lower than in open-pollinated plants (8.4–26.5%). Similar to the germination results, seed set was substantially reduced in both reciprocal crosses. Conclusions These results suggested that poor pollen fertility, low stigma receptivity, and embryo abortion were responsible for the failure of the crosses ‘Molingqiuse’ × ‘Qinhuaiyanzhi’, ‘Qinhuaiyanzhi’ × ‘Molingqiuse’, and ‘Aijiangnan’ × ‘Guili’.