A data‐driven image extraction and analysis pipeline for plant phenotyping in controlled environments

Abstract Advances in automation, imaging, and artificial intelligence have enabled large‐scale plant phenotyping, but image analysis remains a critical bottleneck for crop improvement and biological discovery. We developed an integrated multispectral phenotyping framework using imagery from the Texas A&M AgriLife Automated Precision Phenotyping Greenhouse and expanded plant growth and phenotyping (PGP v2) data across maize ( Zea mays L.), cotton ( Gossypium hirsutum ), rice ( Oryza sativa ), and sorghum ( Sorghum bicolor L.). The pipeline integrates pseudo‐red‐green‐blue composite image generation, plant detection and segmentation, image stitching, vegetation‐index analysis, texture analysis, morphological trait extraction, and temporal comparison of image‐derived features to quantify changes in plant structure, spectral reflectance, and texture over time. Among the evaluated segmentation approaches, segment anything model (SAM) v3 provided the highest and most consistent accuracy across diverse crop structures, although it required greater computational time than classical methods. Segment anything model for long sequences (SAM2Long)maintained plant‐instance associations across vertically stacked frames, while scale‐invariant feature transform–based stitching reconstructed plant mosaics when individual plants extended beyond a single field of view. For each plant and imaging date, the pipeline generated an 863‐dimensional feature vector spanning vegetation indices, spectral statistics, texture descriptors, and morphological traits. The framework was evaluated through two case studies: treatment‐level temporal analysis of mutagenized sorghum lines and cold‐stress phenotyping of maize using a separate imaging system. In both studies, the extracted features supported statistical and multivariate analyses of phenotypic variation and enabled separation of plants based on treatment‐ or stress‐related responses. The combined dataset and workflow provide structured, automated, and well‐documented phenotypic analysis across multiple crops, experimental settings, and imaging systems for controlled‐environment plant science and crop improvement.