A study on pollen characteristics of bisexual and functionally male flowers in pomegranate

[Objective] Pollen is the genetic carrier of the male parent in plant sexual reproduction. Pollen germination on the stigma of female flower and fertilization is the prerequisite for successful reproduction for plants. Therefore, the determination of pollen morphological differences is the basis for plant hybridization and breeding. According to morphology, pomegranate flowers are divided into two types: bisexual flowers and functional male flowers. Bisexual flowers, also known as “tubular flowers”, have normal pistil and eventually develop into fruit. Functional male flowers, which also known as “bell-shaped flowers”, have aborted pistil and will fall off eventually. Pollen characteristics plays a critical role in pomegranate production, influencing the fruit sets. The objectives of this study were to explore the effects of anther quality and different culture conditions on pollen germination rate of bisexual flowers and functionally male flowers in different pomegranate varieties, which will provide reference for pollen selection and preservation for pomegranate breeding. [Methods] Pollen samples were collected from the National Horticultural Germplasm Resource Center. The number of anthers of bisexual flowers and functionally male flowers were collected in cultivars including Suanmeiren, Mollar, Tianshihong, Zhongshiliu 4, Huaguang and Turkmenistan. On June 8th, 2022, at the initial period of the flowering, 50 flower buds of uniform size that were about to open were selected and sampled. The anthers were peeled in a dry carton indoors and the filaments were removed. They were put in a place at room temperature with dark ventilation to allow cracking of the anthers. The pollen grains were completely released and packed into a dry small glass bottle and sealed for later analyses. Anther quality and pollen germination rate of bisexual flowers and functionally male flowers of different pomegranate varieties were analyzed. The differences in anther number, single anther yield, stigma, pollen tube and pollen germination rate between bisexual flowers and functionally male flowers were compared. The effects of sucrose concentration on germination rate and storage temperature on pollen viability were also analyzed. [Results] When the sucrose concentration was 300 g·L-1, the pollen germination rate reached 83.5%, exhibiting no significant difference when compared to the treatment with a sucrose concentration of 250 g·L-1. Sucrose at concentrations of 250 g·L-1 and 300 g·L-1 was beneficial for the germination of pomegranate pollen. There was no significant difference in the number of stamens between bisexual flowers and functionally male flowers in the same cultivar. The number of stamens of Turkmenistan in fertile flowers was significantly lower than that of Suanmeiren, Mollar, Tianshihong, Zhongshiliu 4 and Huaguang (p<0.05). The pollen number in each anther of the bisexual flowers (31-48) was significantly lower than that of the functionally male flowers (85-91). Otherwise, the pollen germination rate of the bisexual flowers (90%-93%) was significantly higher than that of the functionally male flowers (41%-43%). Pollen tube growth of the bisexual flowers was faster than that of the functionally male flowers, and the length of pollen tube was higher than that of the functionally male flowers. There was no correlation between the number of stamens, the number of pollens produced by anthers and the pollen germination rate in the bisexual flowers and the functionally male flowers. Pollen from the bisexual flowers began to germinate 12 hours after pollination, and the number of germinated pollens was small at 24 hours, and some of the pollen tubes had grown into the pistil. By 36 hours, pollen from the bisexual flowers had germinated in large quantities and pollen tubes had extended in the pistil. In contrast, germination of the pollen from the functionally male was not observed until 36 h, when only partial pollen germination was observed on the stigma surface, and only a very small number of pollen was able to protrude into the pistil. Although the functionally male flowers could accept pollen and the pollen on the stigma surface could germinate, only a few pollens could germinate with pollen tube successfully growing into the pistil. The massive germination of pollen from the bisexual flowers at 36 h post-pollination indicates that pollen needs at least 36 h of contact time to fully interact with the stigma for successful and reliable pollination. Storage temperatures had an effect on pomegranate pollen activity, and the pollen germination rate was significantly higher at -20 ℃ than at 4 ℃ and 25 ℃. The pollen was completely inactivated after 3 days of storage at 25 ℃, but it could maintain pollen viability for a longer time when stored at -20 ℃. The pollen germination rate of different varieties was different, with the highest germination rate observed in Tianshihong pollen (93.3%) and the lowest in Zhongshiliu 4 (82.7%). [Conclusion] There were differences in anther quality and pollen germination rate between the bisexual flowers and the functionally male flowers in different cultivars. But there was no significant difference in the number of stamens between the two flower types in the same cultivar. The functionally male flowers have a large amount of pollen but a low germination rate. The storage conditions of -20 ℃ could maintain high pollen viability.