The development of crop varieties with higher nitrogen use efficiency is crucial for sustainable crop production. Combining high-throughput genotyping and phenotyping will expedite the discovery of novel alleles for breeding crop varieties with higher nitrogen use efficiency. Digital and hyperspectral imaging techniques can efficiently evaluate the growth, biophysical, and biochemical performance of plant populations by quantifying canopy reflectance response. Here, these techniques were used to derive automated phenotyping of indicator biomarkers, biomass and chlorophyll levels, corresponding to different nitrogen levels. A detailed description of digital and hyperspectral imaging and the associated challenges and required considerations are provided, with application to delineate the nitrogen response in wheat. Computational approaches for spectrum calibration and rectification, plant area detection, and derivation of vegetation index analysis are presented. We developed a novel vegetation index with higher precision to estimate chlorophyll levels, underpinned by an image-processing algorithm that effectively removed background spectra. Digital shoot biomass and growth parameters were derived, enabling the efficient phenotyping of wheat plants at the vegetative stage, obviating the need for phenotyping until maturity. Overall, our results suggest value in the integration of high-throughput digital and spectral phenomics for rapid screening of large wheat populations for nitrogen response.

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Efficient, precise and timely measurement of plant traits is important in the assessment of a breeding population. Estimating crop biomass in breeding trials using high-throughput technologies is difficult, as reproductive and senescence stages do not relate to reflectance spectra, and multiple growth stages occur concurrently in diverse genotypes. Additionally, vegetation indices (VIs) saturate at high canopy coverage, and vertical growth profiles are difficult to capture using VIs. A novel approach was implemented involving a fusion of complementary spectral and structural information, to calculate intermediate metrics such as crop height model (CHM), crop coverage (CC) and crop volume (CV), which were finally used to calculate dry (DW) and fresh (FW) weight of above-ground biomass in wheat. The intermediate metrics, CHM (R2 = 0.81, SEE = 4.19 cm) and CC (OA = 99.2%, Κ = 0.98) were found to be accurate against equivalent ground truth measurements. The metrics CV and CV×VIs were used to develop an effective and accurate linear regression model relationship with DW (R2 = 0.96 and SEE = 69.2 g/m2) and FW (R2 = 0.89 and SEE = 333.54 g/m2). The implemented approach outperformed commonly used VIs for estimation of biomass at all growth stages in wheat. The achieved results strongly support the applicability of the proposed approach for high-throughput phenotyping of germplasm in wheat and other crop species.

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