**Background:** Water deficit stress (WDS) is a major constraint to maize productivity, particularly in rainfed systems. In India, about 80% of Kharif maize is grown under rainfed conditions, and drought affected 60% of cropped areas in 2002, impacting 85 million people. While most breeding efforts have focused on above-ground traits (anthesis-silking interval, yield components), root traits—critical for water uptake—have been neglected due to the difficulty of field phenotyping. This study aimed to decipher variability in root architecture of diverse maize germplasm under controlled hydroponic conditions, simulate osmolyte-induced water deficiency effects, and identify candidate drought-tolerant donor lines using drought tolerance index (DTI).
**Methods:** Seventy-one diverse maize inbred lines were grown in a hydroponic system under controlled polyhouse conditions (16 h light/8 h dark, 25°C/18°C day/night, 65% relative humidity). Seeds were germinated in coco-coir, transferred to plastic containers with clay balls, and supplied with Hoagland's nutrient solution. At 30 days, WDS was induced using 10% PEG 6000. Thirteen seedling traits were recorded: fresh root weight (FRW), fresh shoot weight (FSW), dry root weight (DRW), dry shoot weight (DSW), root projection area (RPA), total root length (TRL), maximum root length (MRL), number of root tips (RT), number of forks, average root diameter (ARD), root volume (TRV), vigour (0–4 scale), and chlorophyll content (µmol/m²). Roots were scanned using Biovis PSM-R2000 for image-based phenotyping. Principal component analysis (PCA) reduced the dataset to 10 uncorrelated traits. DTI was calculated as (YP × YS)/Ŷp². A core set of 20 lines with differential DTI was validated under 15% (T1) and 20% (T2) PEG 6000 in pot experiments. For anatomical studies, the most tolerant line (LM22) and most susceptible line (CML494) were selected. Primary root samples (20 days old) were fixed in 2.5% glutaraldehyde, post-fixed in 2% osmium tetraoxide, dehydrated through graded ethanol series, critical point dried, gold-sputtered, and examined under SEM (JSM-7610FPlus: JOEL) at 180×–200× magnification.
**Key Results:** ANOVA revealed wide variability among lines under both control and WDS conditions. Mean values for all traits decreased under WDS. The highest chlorophyll content was recorded for PML 98 (19.55 µmol/m²), LM22 (18.5), PML296 (14.2), LM6 (13.7), and LM23 (13.15); lines PML 48, CML494, CML387, and CML444 recorded below 5 µmol/m². LM22 showed the highest vigour (4). PCA yielded five components with eigenvalues >1, explaining 77% cumulative variability (PC1: 25.5%, PC2: 18.7%, PC3: 14.3%, PC4: 10.2%, PC5: 8.2%). Ten traits were identified for effective selection: FRW, FSW, DRW, DSW, chlorophyll, vigour, TRL, RT, forks, and ARD. Heatmap clustering grouped lines into four clusters; Cluster IV had highest means for FRW, FSW, DRW, DSW, vigour, and chlorophyll. DTI ranged from 0.01 (CML533) to 14.6 (LM22) for FRW; 0.02 (PML55) to 18.6 (LM22) for FSW; 0.01 (PML73) to 11 (PML95) for DRW; 0.01 (CML533) to 8.5 (LM22) for DSW; 0.07 (PML53) to 7.62 (PML73) for TRL; 0.07 (PML276) to 3.7 (LM22) for RT; and 0.2 (PML296) to 18 (PM207) for ARD. LM22 ranked first for DTI in FRW, FSW, DSW, RT, and ARD. In validation experiments (Experiment II), MRL increased under T1 and T2 for tolerant lines, while shoot length decreased (range: 5.5–23 cm in T1; 3.5–18.8 cm in T2). TRL successively decreased with increasing osmolyte concentration, while root tips increased in T1. SEM analysis showed that under control conditions, LM22 exhibited higher numbers of xylem and metaxylem with larger diameters compared to CML494. Under WDS (20% PEG), LM22 maintained vessel number with slight diameter reduction (average 85.9 µm), while CML494 showed marked reduction in both number and diameter of metaxylem and xylem (average 76.7 µm). Vessel walls were not suberized after osmolyte treatment, confirming that diameter reduction was due to stress response rather than osmolyte artifact.
**Clinical Implications:** This study provides a rapid, low-cost hydroponic screening protocol for identifying WDS-tolerant maize genotypes at the seedling stage, which can accelerate breeding programs. The identification of LM22 as a superior donor line for multiple drought-tolerance traits (biomass, vigour, root plasticity, vascular integrity) offers a valuable genetic resource for hybrid development. The finding that root anatomical plasticity—specifically maintenance of xylem and metaxylem number with moderate diameter reduction under stress—is a key adaptive trait provides a refined phenotyping target. The recommendation to incorporate xylem and metaxylem characteristics under controlled conditions as part of precise phenotyping could improve selection efficiency for WDS tolerance in maize, with potential applicability to other cereal crops facing increasing climatic volatility.