GridFree: a python']python package of imageanalysis for interactive grain counting and measuring

Grain characteristics, including kernel length, kernel width, and thousand kernel weight, are critical component traits for grain yield. Manual measurements and counting are expensive, forming the bottleneck for dissecting these traits' genetic architectures toward ultimate yield improvement. High-throughput phenotyping methods have been developed by analyzing images of kernels. However, segmenting kernels from the image background and noise artifacts or from other kernels positioned in close proximity remain as challenges. In this study, we developed a software package, named GridFree, to overcome these challenges. GridFree uses an unsupervised machine learning approach, K-Means, to segment kernels from the background by using principal component analysis on both raw image channels and their color indices. GridFree incorporates users' experiences as a dynamic criterion to set thresholds for a divide-and-combine strategy that effectively segments adjacent kernels. When adjacent multiple kernels are incorrectly segmented as a single object, they form an outlier on the distribution plot of kernel area, length, and width. GridFree uses the dynamic threshold settings for splitting and merging. In addition to counting, GridFree measures kernel length, width, and area with the option of scaling with a reference object. Evaluations against existing software programs demonstrated that GridFree had the smallest error on counting seeds for multiple crop species. GridFree was implemented in Python with a friendly graphical user interface to allow users to easily visualize the outcomes and make decisions, which ultimately eliminates time-consuming and repetitive manual labor. GridFree is freely available at the GridFree website (https://zzlab.net/GridFree).