Stomatal observation and automatic stomatal detection are useful analyses of stomata for taxonomic, biological, physiological, and eco-physiological studies. We present a new clearing method for improved microscopic imaging of stomata in soybean followed by automated stomatal detection by deep learning. We tested eight clearing agent formulations based upon different ethanol and sodium hypochlorite (NaOCl) concentrations in order to improve the transparency in leaves. An optimal formulation—a 1:1 (v/v) mixture of 95% ethanol and NaOCl (6–14%)—produced better quality images of soybean stomata. Additionally, we evaluated fixatives and dehydrating agents and selected absolute ethanol for both fixation and dehydration. This is a good substitute for formaldehyde, which is more toxic to handle. Using imaging data from this clearing method, we developed an automatic stomatal detector using deep learning and improved a deep-learning algorithm that automatically analyzes stomata through an object detection model using YOLO. The YOLO deep-learning model successfully recognized stomata with high mAP (~0.99). A web-based interface is provided to apply the model of stomatal detection for any soybean data that makes use of the new clearing protocol.
The number of four-seeded pods is a plant trait that is of great interest in terms of increasing soybean production. The objective of this study was to understand the agronomic characteristics of four-seeded pods of FS1159, which contain a significantly higher ratio of four-seeded pods than do other genotypes. FS1159 showed a significantly lower ratio of one-and two-seeded pods and a significantly higher ratio of three-(39.6%) and four-(11.3%) seeded pods than did the four check soybeans. The average values of the traits of FS1159 in this study were: plant height, 58.1 cm; the number of nodes, 15.7; the number of branches, 6.5; and 100-seed weight, 20.3 g. These results indicate that FS1159 can be used as a new genetic resource to explore the traits of four-seeded-pod and improve the soybean yield.
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