Optimizing pollen germination and subcellular dynamics in pollen tube of Torreya grandis

Torreya grandis, a dioecious Taxaceae species of significant economic value in southeast China, presents challenges for natural pollination due to asynchronous maturation of its sex organs and low pollen vitality. In order to enhance fertilization success through artificial pollination of T. grandis, this study investigated the optimal conditions for in vitro pollen germination and pollen tube growth of T. grandis. The optimal in vitro growth medium was found to contain 29 mM sucrose, 0.8 mM H₃BO₃, 0.72 mM CaCl₂, and 0.32 mM MgSO₄, supplemented with 4 μM NAA, 2 μM GA₃, and 5 μM 2,4-D at pH=5.6. Under these conditions, we achieved a maximum pollen germination ratio of 69.99 ± 5.17 % and a pollen tube length of 34.38 ± 6.04 µm after 6 days germination at 28°C. FM4-64 dye and Mitotracker Red staining revealed highly dynamics of vesicles and mitochondria during germination, which were accumulated at the tip of pollen tube and exhibited biphasic movement patterns. The total number, motion rate, and movement velocity of vesicles as well as mitochondria showed an initially increase followed by a gradual decrease pattern. The presence of sucrose in the medium significantly increased the dynamics and metabolic activity of both vesicles and mitochondria, which may relate with higher pollen germination ratio and faster pollen tube growth compared to sucrose-depleted conditions. Thus, these findings shed light on the physiological characteristics of Torreya pollen germination and provide scientific information for improving Torreya fruit yield through artificial pollination.