Spatially resolved quantification of wheat kernel vitreousness using hyperspectral imaging and spectral unmixing.

Introduction: Wheat kernel hardness, vitreousness, and creaseness are key determinants of milling performance, yet they reflect different physical scales of grain structure and are not necessarily coupled. Methods: We developed a digital phenotyping framework based on hyperspectral imaging and spectral unmixing to quantify these traits at both kernel and cultivar levels in a diverse panel of common wheat. Pixel-level spectral unmixing resolved glassy, intermediate, and mealy endosperm components within individual kernels, enabling vitreousness to be expressed as a continuous spatial index. Results: The hyperspectral-derived vitreousness index showed moderate associations with kernel protein content and the protein-to-starch ratio, consistent with variation in endosperm packing density, but weak relationships with kernel hardness and crease geometry. Kernel hardness, primarily determined by puroindoline genotype, showed limited association with bulk protein and starch composition. Crease geometry, quantified using composite indices from RGB images, captured macroscopic grain features largely independent of both hardness and vitreousness. Discussion: These results demonstrate that hardness, vitreousness, and creaseness represent complementary but largely independent dimensions of grain quality, corresponding to molecular-scale adhesion, mesoscale packing, and macroscopic geometry, respectively. The proposed framework provides a scalable, non-destructive approach for resolving intra-kernel heterogeneity, enabling improved digital phenotyping for wheat breeding and quality assessment.