Deep learning for automated segmentation and counting of hypocotyl and cotyledon regions in mature Pinus radiata D. Don. somatic embryo images

In commercial forestry and large-scale plant propagation, the utilization of artificial intelligence techniques for automated somatic embryo analysis has emerged as a highly valuable tool. Notably, image segmentation plays a key role in the automated assessment of mature somatic embryos. However, to date, the application of Convolutional Neural Networks (CNNs) for segmentation of mature somatic embryos remains unexplored. In this study, we present a novel application of CNNs for delineating mature somatic conifer embryos from background and residual proliferating embryogenic tissue and differentiating various morphological regions within the embryos. A semantic segmentation CNN was trained to assign pixels to cotyledon, hypocotyl, and background regions, while an instance segmentation network was trained to detect individual cotyledons for automated counting. The main dataset comprised 275 high-resolution microscopic images of mature Pinus radiata somatic embryos, with 42 images reserved for testing and validation sets. The evaluation of different segmentation methods revealed that semantic segmentation achieved the highest performance averaged across classes, achieving F1 scores of 0.929 and 0.932, with IoU scores of 0.867 and 0.872 for the cotyledon and hypocotyl regions respectively. The instance segmentation approach demonstrated proficiency in accurate detection and counting of the number of cotyledons, as indicated by a mean squared error (MSE) of 0.79 and mean absolute error (MAE) of 0.60. The findings highlight the efficacy of neural network-based methods in accurately segmenting somatic embryos and delineating individual morphological parts, providing additional information compared to previous segmentation techniques. This opens avenues for further analysis, including quantification of morphological characteristics in each region, enabling the identification of features of desirable embryos in large-scale production systems. These advancements contribute to the improvement of automated somatic embryogenesis systems, facilitating efficient and reliable plant propagation for commercial forestry applications.