Advancing radiation-induced mutant screening through high-throughput technology: a preliminary evaluation of mutant screening in Arabidopsis thaliana

Identifying mutant traits is essential for improving crop yield, quality, and stress resistance in plant breeding. Historically, the efficiency of breeding has been constrained by throughput and accuracy. Recent significant advancements have been made through the development of automated, high-accuracy, and high-throughput equipment. However, challenges remain in the post-processing of large-scale image data and its practical application and evaluation in breeding. This study presents a comparative analysis of human and machine recognition, with validation of a randomly selected mutant at the physiological level performed on wild-type Arabidopsis thaliana and a candidate mutant of the M₃ generation, which was generated through mutagenesis with heavy ion beams (HIBs) and ⁶⁰Co-γ radiation. The mutant populations were subjected to image acquisition and automated screening using the High-throughput Plant Imaging System (HTPIS), generating approximately 10 GB of data (4,635 image datasets). We performed Principal Components Analysis (PCA), scatter matrix clustering, and Logistic Growth Curve (LGC) analyses, and compared these results with those obtained from traditional manual screening based on human visual assessment, and randomly selected #197 candidate mutants for validation in terms of growth and development, chlorophyll fluorescence, and subcellular structure. Our findings demonstrate that as the confidence interval level increases from 75 to 99.9%, the accuracy of machine-based mutant identification decreases from 1 to 0.446, while the false positive rate decreases from 0.817 to 0.118, and the false negative rate increases from 0 to 0.554. Nevertheless, machine-based screening remains more accurate and efficient than human assessment. This study evaluated and validated the efficiency (greater than 80%) of high-throughput techniques for screening mutants in complex populations of radiation-induced progeny, and presented a graphical data processing procedure for high-throughput screening of mutants, providing a basis for breeding techniques utilizing HIBs and γ-ray radiation, and offering innovative approaches and methodologies for radiation-induced breeding in the context of high-throughput big data.