Determining leaf stomatal properties in citrus trees utilizing machine vision and artificial intelligence

Abstract

Identifying and quantifying the number and size of stomata on leaf surfaces is useful for a wide range of plant ecophysiological studies, specifically those related to water-use efficiency of different plant species or agricultural crops. The time-consuming nature of manually counting and measuring stomata have limited the utility of manual methods for large-scale precision agriculture applications. A deep learning segmentation network was developed to automate the analysis of stomatal density and size and to distinguish between open and closed stomata using citrus trees grafted on different rootstocks as a model system. A novel method was developed utilizing the Mask-RCNN algorithm, which allows identification, quantification, and characterization of stomata from leaf epidermal peel microscopic images with an accuracy of up to 99%. Moreover, this method permits the differentiation of open and closed stomata with 98% precision and measurement of individual stomata size. In the citrus model system, significant differences in the size and density of stomata and diurnal regulation patterns were detected that were associated with the rootstock cultivar on which the trees were grafted. Nearly 9000 individual stomata were analyzed, which would have been impractical using manual methods. The novel automated method presented here is not only accurate, but also rapid and low-cost, and can be applied to a variety of crop and non-crop plant species.