Optimization of In Vitro Germination, Viability Tests and Storage of Daylily (Hemerocallis spp.) Pollen

Daylily (<i>Hemerocallis</i> spp.) are perennial herbaceous flowers with high ornamental and medicinal value. Currently, the breeding of new daylily cultivars was mainly achieved through hybrid breeding, but issues such as self-incompatibility, hybridization barriers, and asynchronous reproductive phenology severely hinder the breeding process. Understanding pollen viability was essential for daylily breeding and cultivar improvement. In this study, we systematically investigated the effects of pollen viability determination methods, collection time, medium combinations, culture temperature and storage conditions on the pollen germination characteristics of daylily, using five daylily cultivars introduced in the Zhanjiang region of China as materials. Comparing the Iodine-potassium iodide (I₂-KI) staining and Acetocarmine staining, the results of 2,3,5-Triphenyltetrazolium Chloride (TTC) staining showed a significant positive correlation (<i>p</i> < 0.05) with the in vitro germination rate, which is suitable for the rapid detection of daylily pollen vigor. The daylily variation of pollen vigor was significant in different cultivars, and most cultivars had the highest vigor at 9:00-12:00 a.m., which was suitable for artificial pollination. The in vitro germination experiment showed that sucrose concentration was the key factor for daylily pollen germination and pollen tube growth, and the optimal medium for pollen in vitro germination was 50 g/L^-1 sucrose + 0.1 g/L^-1 H₃BO₃ + 0.06 g/L^-1 KNO₃ + 0.2 g/L^-1 Ca(NO₃)₂. The temperature experiment showed that the optimum temperature for pollen germination was 24.1-26.7 °C, and the optimum range for pollen tube growth was 24.1-25.7 °C, and the high temperature significantly inhibited the elongation rate of pollen tube. Storage experiments showed that low temperature (-40 °C) combined with drying treatment could significantly prolong pollen life, and the "Water Dragon" variety still maintained 41.29% vigor after 60 days of dry storage. This study provides theoretical basis and technical support for the introduction and domestication of daylily in South China, hybridization and garden application.