Analysis of stomatal characteristics of maize hybrids and their parental inbred lines during critical reproductive periods

The stomatal phenotype is a crucial microscopic characteristic of the leaf surface, and modulating the stomata of maize leaves can enhance photosynthetic carbon assimilation and water use efficiency, thereby playing a vital role in maize yield formation. The evolving imaging and image processing technologies offer effective tools for precise analysis of stomatal phenotypes. This study employed Jingnongke 728 and its parental inbred to capture stomatal images from various leaf positions and abaxial surfaces during key reproductive stages using rapid scanning electron microscopy. We uesd a target detection and image segmentation approach based on YOLOv5s and Unet to efficiently obtain 11 phenotypic traits encompassing stomatal count, shape, and distribution. Manual validation revealed high detection accuracies for stomatal density, width, and length, with R2 values of 0.92, 0.97, and 0.95, respectively. Phenotypic analyses indicated a significant positive correlation between stomatal density and the percentage of guard cells and pore area (r=0.36), and a negative correlation with stomatal area and subsidiary cell area (r=-0.34 and -0.46). Additionally, stomatal traits exhibited notable variations with reproductive stages and leaf layers. Specifically, at the monocot scale, stomatal density increased from 74.35 to 87.19 Counts/mm2 from lower to upper leaf layers. Concurrently, the stomatal shape shifted from sub-circular (stomatal roundness = 0.64) to narrow and elongated (stomatal roundness = 0.63). Throughout the growth cycle, stomatal density remained stable during vegetative growth, decreased during reproductive growth with smaller size and narrower shape, and continued to decline while increasing in size and tending towards a rounded shape during senescence. Remarkably, hybrid 728 differed notably from its parents in stomatal phenotype, particularly during senescence. Moreover, the stomatal density of the hybrids showed negative super parental heterosis (heterosis rate = -0.09), whereas stomatal dimensions exhibited positive super parental heterosis, generally resembling the parent MC01. This investigation unveils the dynamic variations in maize stomatal phenotypes, bolstering genetic analyses and targeted improvements in maize, and presenting a novel technological instrument for plant phenotype studies.