Background: Grains make up a large proportion of both human and animal diets. With threats to food production, such as climate change, growing sustainable and successful crops is essential to food security in the future. Germination is one of the most important stages in a plant’s lifecycle and is key to the success of the resulting plant as the grain undergoes morphological changes and the development of specific organs. Micro-computed tomography is a non-destructive imaging technique based on the differing x-ray attenuations of materials which we have applied for the accurate analysis of grain morphology during the germination phase. Results: Micro Computed Tomography conditions and parameters were tested to establish an optimal protocol for the 3-dimensional analysis of barley grains. When comparing optimal scanning conditions, it was established that no filter, 0.4 degrees rotation step, 5 average frames, and 2016 × 1344 camera binning is optimal for imaging germinating grains. It was determined that the optimal protocol for scanning during the germination timeline was to scan individual grains at 0 h after imbibition (HAI) and then the same grain again at set time points (1, 3, 6, 24 HAI) to avoid any negative effects from X-ray radiation or disruption to growing conditions. Conclusion: Here we sought to develop a method for the accurate analysis of grain morphology without the negative effects of possible radiation exposure. Several factors have been considered, such as the scanning conditions, reconstruction, and possible effects of X-ray radiation on the growth rate of the grains. The parameters chosen in this study give effective and reliable results for the 3-dimensional analysis of macro structures within barley grains while causing minimal disruption to grain development.

Summary: Cereal grain is a global food staple. Filial tissues in grain, such as the embryo and endosperm, grow embedded in the maternal ovule and surrounding ovary wall. Maternal tissues provide nutrition and protection but are progressively reduced and degraded as filial tissues expand, driving grain expansion between floret hulls. While we know some enzymatic players, regulation of this process remains less understood. However, our previous work in barley, Hordeum vulgare spp. vulgare suggested a role for the phytohormone jasmonate (JA). We edited the barley ALLENE OXIDASE CYCLASE ( HvAOC ) gene encoding a JA biosynthetic enzyme. We examined hvaoc mutants for developmental phenotypes, genetic interactions and effects on the transcriptome. Defective HvAOC alleles reduced JA levels, and caused enhanced vegetative growth, faster flowering and lengthened grain, all phenotypes rescued by MeJA treatment. HvAOC control of grain length appears parentally‐derived, consistent with enriched HvAOC expression in maternal compared to filial tissues. Grain dimension changes were uncoupled from hull size and correlated with larger endosperm cavities, more persistent maternal tissues and higher expression of genes linked to sucrose transport. This work provides the first genetic dissection of JA in barley and highlights roles in coordinating maternal tissues within grain.

Quantitative imaging of plant tissues by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is hindered by the lack of matrix-matched calibration standards. Established approaches, such as gelatine or homogenised tissue blocks, do not replicate plant matrices accurately. Here, we introduce a nano-dispenser-based calibration strategy that deposits nanolitre volumes of elemental standards directly onto paraffin-embedded grain sections, exploiting the low endogenous metal content of the endosperm to generate in situ calibration curves. Calibration performance for Mg, Mn, Cu, Zn, and Mo was assessed using LA-ICP-MS imaging and Iolite 4 data processing. The method demonstrated excellent linearity ( R ² > 0.98), reproducibility across multiple grains, and sub-ppm limits of detection. Comparative analysis with in-house homogenised blocks and NIST wheat reference material confirmed superior accuracy and reproducibility of the nano-dispenser approach. As a proof-of-concept, we have applied the method for the quantitative imaging of metals in a whole barley grain section, and the results show excellent agreement with published data obtained by conventional liquid-mode ICP MS. The technique reported here provides a robust, scalable, solution for quantitative metallomics studies of plant tissues, enabling improved assessment of nutrient distribution and supporting the development of standardised protocols for LA-ICP-MS imaging.