Automated estimation of stomatal number and aperture in haskap (Lonicera caerulea L.)

This study developed the reliable Mask R-CNN model to detect stomata in Lonicera caerulea. The obtained data could be utilized for evaluating some characters such as stomatal number and aperture area. The native distribution of haskap (Lonicera caerulea L.), a small-shrub species, extends through Northern Eurasia, Japan, and North America. Stomatal observation is important for plant research to evaluate the physiological status and to investigate the effect of ploidy levels on phenotypes. However, manual annotation of stomata using microscope software or ImageJ is time consuming. Therefore, an efficient method to phenotype stomata is needed. In this study, we used the Mask Regional Convolutional Neural Network (Mask R-CNN), a deep learning model, to analyze the stomata of haskap efficiently and accurately. We analyzed haskap plants (dwarf and giant phenotypes) with the same ploidy but different phenotypes, including leaf area, stomatal aperture area, stomatal density, and total number of stomata. The R-square value of the estimated stomatal aperture area was 0.92 and 0.93 for the dwarf and giant plants, respectively. The R-square value of the estimated stomatal number was 0.99 and 0.98 for the two phenotypes. The results showed that the measurements obtained using the models were as accurate as the manual measurements. Statistical analysis revealed that the stomatal density of the dwarf plants was higher than that of the giant plants, but the maximum stomatal aperture area, average stomatal aperture area, total number of stomata, and average leaf area were lower than those of the giant plants. A high-precision, rapid, and large-scale detection method was developed by training the Mask R-CNN model. This model can help save time and increase the volume of data.