Machine learning-optimized spectral indices for high-throughput phenotyping of chlorophyll and yield of wheat breeding lines under salinity stress conditions

High-throughput phenotyping is key in modern breeding for rapidly and cost-effectively evaluating salt-adaptive traits. However, few studies have combined spectral reflectance indices (SRIs) with deep learning to assess field-grown wheat under salt stress. In this study, we developed optimized 2D and 3D SRIs integrated with artificial neural network (ANN) to assess chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophyll (TChl), and grain yield (GY) in 32 recombinant inbred lines (RILs) and four cultivars under 150 mM NaCl field conditions. ANOVA revealed that genotype contributed the largest proportion of the treatment sum of squares across all traits (60–80%), followed by the genotype × year interaction (8–15%), whereas year contributed the smallest proportion (1–5%). Heatmap clustering of these traits clearly distinguished salt-tolerant from salt-sensitive genotypes. The study findings highlight using four key traits as screening criteria for salt tolerance in wheat. Our optimized 2D/3D spectral indices showed moderate to strong predictive power (R 2 = 0.25–0.75), outperforming earlier indices. Multi-season data improved accuracy by 15–25%, with best predictions for Chl a and TChl (R 2 = 0.34–0.75) versus Chl b and GY (R 2 = 0.25–0.64). Top models included ANN-3D-SRIs-8 for Chl a (R 2 = 0.735/0.644), ANN-3D-SRIs-3 for Chl b (R 2 = 0.611/0.549), ANN-2D-3D-SRIs-3 for TChl (R 2 = 0.713/0.619), and ANN-2D-SRIs-2 for GY (R 2 = 0.648/0.553). This framework combines optimized indices and machine learning for scalable, high-throughput phenotyping to advance precision breeding of salt-tolerant wheat.