The stable 3St(3D) substitution line offers promising genetic potential for improving drought tolerance in wheat during critical reproductive stages. The flowering stage is highly susceptible to drought, which significantly reduces wheat grain yield globally. Low genetic diversity in wheat further limits the discovery of optimal gene variants for breeding climate-resilient varieties. The substitution of chromosome 3D by a group 3 chromosome pair from Thinopyrum intermedium × Th. ponticum artificial hybrid was identified using in situ hybridization and genotyping-by-sequencing. This homoeologous substitution showed good functional compensation for grain yield and fertility, similar to the wheat parents ('Mv9kr1' and 'Mv Karizma') in field and greenhouse trials. The substitution line exhibits a semidwarf phenotype due to the Rht8 and Rht2 dwarfing alleles. Automated shoot phenotyping after a 10-day water withdrawal at flowering revealed efficient water preservation allowing to maintain photosynthetic functions, sustained photosynthetic activity, and less chlorophyll degradation, indicated by Normalized Difference Vegetation Index (NDVI) and modified Normalized Difference Index (mND705) values and moderate level of protective functions shown by the expression of stress-related genes. Compared to the wheat parents, the substitution line developed thicker roots with increased volume under drought, resulting in a lower surface-to-volume ratio. This may enhance water storage efficiency and help reduce yield loss under drought conditions.

Centre for Agricultural Research Hungarian Research Network Agricultural Institute 2462 Martonvásár Hungary

Institute of Experimental Botany of the Czech Academy of Sciences Centre of Plant Structural and Functional Genomics Olomouc 779 00 Czech Republic

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