Factors affecting pollen viability and and its associated physiological responses in Shine Muscat grape

【Objective】Shine Muscat (Vitis labrusca × V. vinifera) is a globally dominant table grape cultivar, valued for its rose aroma, crisp texture, and excellent storage performance. Pollen viability, a critical factor in reproductive biology, directly affects pollination efficiency, fruit set, and hybrid breeding success. However, systematic studies on the influencing factors and regulatory mechanisms of its pollen viability remain limited, hindering improvements in breeding efficiency and fruit quality. This study aimed to investigate factors affecting pollen viability of Shine Muscat grape and related physiological mechanisms, optimize pollen collection storage, and pollination techniques, and provide theoretical and technical support for hybrid breeding and production.【Methods】Healthy 4-year-old Shine Muscat grape plants in the grape germplasm nursery of Henan Institute of Science and Technology were selected. Anthers were collected from plump inflorescences at the early messenger flower stage, and dried at 25 ℃ to release pollen. Pollen quantity was determined using a hemocytometer: 30 anthers were dried at 50 ℃, suspended in 20% sodium hexametaphosphate solution, and counted under a microscope. Pollen viability was evaluated via orthogonal test design and fluorescent staining. Based on the L4 (23 ) orthogonal experimental design, four media (T1-T4) were formulated with specific combinations of sucrose, boric acid, and calcium chloride. The detailed matrix compositions are as follows: T1 (10% sucrose + 50 mg·L-1 boric acid + 20 mg·L-1 calcium chloride), T2 (10% sucrose + 100 mg·L-1 boric acid+40 mg ·L-1 calcium chloride), T3 (15% sucrose + 50 mg ·L-1 boric acid + 40 mg ·L-1 calcium chloride), and T4 (15% sucrose + 100 mg·L-1 boric acid + 20 mg·L-1 calcium chloride). All media were prepared by first dissolving 7 g·L-1 agar powder, then sequentially adding the above components. After adjusting the volume, the pH was adjusted to 6.0, and the media were dispensed into Petri dishes while hot to form germination beds for in vitro pollen culture. The orthogonal test included the four media with varying concentrations of sucrose (10% or 15%), boric acid (50 mg ·L-1 or 100 mg ·L-1 ), and calcium chloride (20 mg · L- 1 or 40 mg · L- 1 ); in vitro germination rate was calculated after culturing at 25 ℃ with 60%-70% relative humidity. Fluorescein diacetate (FDA) staining was used for rapid viability assessment: pollen was stained with 0.02 mg·L-1 FDA working solution, observed under a fluorescence microscope, and the proportion of viable pollen (with strong green fluorescence) was counted. For storage experiments, pollen was stored at 4 ℃ or -80 ℃ for 5 d or 7 d (with fresh pollen as CK) before viability check, with 3 biological replicates per treatment. Physiological indices measured included soluble sugars (SS) which was measured via anthrone colorimetry, soluble proteins (SP) via Coomassie Brilliant Blue G- 250 method, proline (Pro) via ninhydrin colorimetry, and activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT) via NBT photochemical reduction, guaiacol, and potassium permanganate titration methods, respectively.【Results】Significant differences in pollen quantity were observed among the three plants (P＜0.05). Plant 1 produced the highest pollen quantity (24 × 104 grains/anther), which was 28.57% higher than Plant 2 (18.67 × 104 grains/anther) and 38.46% higher than Plant 3 (17.33 × 104 grains/anther). Intra-plant variation was minimal, with Plant 1 showing a standard deviation of 0 across replicates, indicating stable pollen production within individual plants. The orthogonal test revealed significant differences in germination rates among media (P＜0.05). T4 medium yielded the highest germination rate for fresh pollen (95.79%), significantly exceeding T1 (89.37%), T2 (91.24%), and T3 (92.86%). This indicated a synergistic effect of high sucrose (15%) and boric acid (100 mg · L- 1 ) in promoting germination, with calcium chloride (20 mg · L- 1 ) playing a secondary role. Storage temperature and duration significantly affected pollen viability (P＜0.05). After 5 days of storage, germination rate in treatment at - 80 ℃ (73.73%- 88.26% ) was 15%- 20% higher than at 4 ℃ (63.67%-73.54% ). After 7 days of storage, treatment at -80 ℃ maintained higher viability (79.23%- 87.98%) compared to 4 ℃ (55.75%-68.75%), with the T4 treatment showing a 57.8% higher germination rate at -80 ℃ than at 4 ℃. FDA staining confirmed that viable pollen (strong green fluorescence) was more abundant and stable under -80 ℃, consistent with germination rate result. Fresh pollen exhibited the highest levels of SS, SP, Pro, and antioxidant enzyme (SOD, POD and CAT) activities. All indices decreased with storage time, but the decline was significantly slower at -80 ℃ than at 4 ℃. For example, SS content in fresh pollen (1.2 mg·g-1 ) dropped to 0.8 mg·g-1 after 7 days at -80 ℃ but to 0.5 mg·g-1 at 4 ℃. Similarly, SOD activity (150 U · g- 1 in fresh pollen) decreased to 100 U · g- 1 at -80 ℃ (7 days) versus 80 U·g-1 at 4 ℃. Pearson correlation analysis showed extremely significant positive correlations (P＜0.001) between pollen viability and all measured indices. Correlation coefficients ranged from 0.856 (SP) to 0.915 (SOD), indicating that nutrient contents and antioxidant enzyme activities were strongly associated with the maintenance of pollen viability.【Conclusion】This study demonstrates significant intra-varietal variation in pollen quantity among Shine Muscat plants, highlighting the importance of selecting high-pollen-yield plants as paternal parents in breeding. T4 medium (15% sucrose + 100 mg ·L-1 boric acid + 20 mg ·L-1 CaCl2) is optimal for in vitro pollen germination. -80 ℃ ultra-low temperature storage effectively preserves pollen viability by slowing the degradation of nutrients (SS, SP and Pro) and maintaining antioxidant enzyme activities. The established“pollen viability-physiologi-cal indices”correlation model provides a quantitative tool for assessing pollen quality. These findings optimize pollen management techniques, which may improve hybrid breeding efficiency.