A Data-Driven Image Extraction and Analysis Pipeline for Plant Phenotyping in Controlled Environments
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 Precision Automated Phenotyping Greenhouse and expanded Plant Growth and Phenotyping (PGP v2) data across maize, cotton, rice, and sorghum. The pipeline integrates pseudo-RGB 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, SAM v3 provided the highest and most consistent accuracy across diverse crop structures, although it required greater computational time than classical methods. SAM2Long maintained plant-instance associations across vertically stacked frames, while Scale-Invariant Feature Transform (SIFT)-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 treatmentor 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 controlledenvironment plant science and crop improvement. Plain Language SummaryTemporal imaging of plants in controlled environments helps scientists better understand growth and biological processes. However, analyzing large volumes of images has been limited by a lack of automated tools. Multispectral imagery captures additional information about plant pigments, structure, and stress beyond standard color images. We developed an automated analysis pipeline that identifies individual plants, tracks their growth over time, and measures traits such as height, area, shape, texture, and vegetation indices. Using artificial intelligence, the system efficiently processes thousands of images to provide consistent and repeatable measurements. By integrating engineering and plant biology, this work supports data-driven decisions for crop improvement and agricultural research.