Temperature dependence of pollen germination in Douglas-fir (Pseudotsuga menziesii): A machine learning-based detection of pollen viability from microscopic images

Background and Aims: Pollen germination and tube growth are critical stages of plant reproduction that are highly sensitive to temperature but remain labor-intensive to quantify. This study aimed to develop a deep learning-based approach for pollen phenotyping and to characterize the temperature dependence of pollen germination and elongation in Douglas-fir (Pseudotsuga menziesii). Methods: A convolutional neural network (CNN) was trained to segment pollen grains and classify germination status from microscopic images. Germination percentage and pollen length were quantified across a range of incubation temperatures from 5 to 40{degrees}C. Gamma functions were fitted to temperature response curves to estimate the optimal temperatures for pollen germination and elongation among Douglas-fir populations collected across an elevational gradient. Key Results: The CNN achieved high segmentation accuracy (intersection over union = 0.846), accurately distinguishing pollen grains from the background but showing moderate accuracy in separating germinated from ungerminated pollen during early elongation. Both pollen germination and elongation exhibited bell-shaped temperature response curves with distinct thermal optima. Pollen elongation consistently reached its optimum at higher temperatures than pollen germination. Estimated optimal temperatures fell within a narrow range, approximately 19 to 23{degrees}C. No significant relationship was detected between elevation and thermal optima, although some higher-elevation populations exhibited lower optimal temperatures. Comparisons with previous analyses of three western North American conifers showed that each species occupied a distinct reproductive thermal niche corresponding to the spring temperatures of its native habitat. Conclusions: Deep learning provides an efficient approach for high-throughput quantification of pollen germination and elongation from microscopic images. The narrow thermal range for reproductive performance suggests that Douglas-fir pollen is sensitive to temperature variation and that warming climates may alter reproductive success. These findings improve our understanding of the thermal sensitivity of conifer reproduction and provide a scalable framework for assessing impacts of climate warming on forest regeneration.