Standardized and rapid protocol for drought tolerance screening at seedling stage in small grain cereals and their wild relatives

Drought stress severely threatens the stability of production of cereals worldwide. Improving genetic tolerance to drought stress requires detailed characterization of global germplasm collections and their wild relatives under drought conditions. However, the lack of rapid screening methods remains a major obstacle in achieving this goal. Here, we present a rapid, reliable, and highly reproducible soil-based screening protocol for evaluating large germplasm collections under drought stress at the seedling stage. This protocol efficiently discriminates drought tolerant and sensitive genotypes with minimal environmental variability and is broadly applicable across various small grain cereals, enabling the evaluation of 1200 genotypes of wheat, oat, barley, and their wild relatives such as Aegilops tauschii and Ae. umbellulata. Additionally, evaluation of two contrasting wheat genotypes identified using this protocol under drought stress imposed at anthesis and grain filling stages provided evidence that their contrasting drought responses were also consistently maintained at the later developmental stages. Given its efficiency, reproducibility, and cross-species applicability, this protocol offers a practical approach for preliminary screening of large germplasm collections as well as for identifying contrasting genotypes to be included in subsequent breeding, physiological, and genetic studies.