Image-Based Phenotyping for Early Assessment of Radiosensitivity of Cowpea (Vigna unguiculata L. Walp.) Seedlings Irradiated with Gamma Rays

Calibrating the mutagenic dose is the first practical step of any radiation mutation-breeding programme, and it is usually summarised by the median lethal dose (LD50) or the median growth-reduction dose (GR50). We asked whether an accessible, image-based phenotyping pipeline can quantify the early radiation response of cowpea (Vigna unguiculata L. Walp.) seedlings finely enough to estimate GR50 and to rank organ- and pigment-level sensitivities. Seeds of the traditional Paraguayan landrace kumandá pyta’i were exposed to Cobalt-60 gamma rays at 0, 100, 200, 300, 400, 500, 600, and 700 Gy, grown in a greenhouse, and photographed at the early seedling stage. A single calibrated photograph (5.1 px mm−1) of 83 seedlings was segmented in Fiji/ImageJ 1.54p and analysed with Python to extract morphometric traits (total, root, and shoot length, root:shoot ratio, tortuosity, and a two-dimensional biomass proxy) and colorimetric traits (CIE L*a*b*, a normalised greenness index, and colour-class pixel fractions). Because the data departed from normality, dose effects were tested with Kruskal–Wallis, Spearman rank correlation, and Dunn post hoc tests, and GR50 was estimated by regression of each trait expressed as a percentage of the control. Total length, shoot length, and the biomass proxy declined significantly with dose (Spearman ρ = −0.40, −0.51, and −0.47; all p < 0.001), preceded by a low-dose stimulation at 100 Gy. Estimated GR50 values were ≈390 Gy for shoot length, ≈510 Gy for total length, and ≈550 Gy for the biomass proxy, within the range reported for other cowpea genotypes. Shoot elongation was more radiosensitive than root elongation, so the root:shoot ratio did not decline; tortuosity showed no dose response. Among pigment traits, the loss of greenness was the most robust signal (a* increased, ρ = +0.62, p = 5 × 10−10; green pixel fraction fell from 0.32 to near zero by 500 Gy). These results show that single-photograph phenotyping resolves a coherent, statistically supported dose response and yields a GR50 estimate usable for dose calibration. For kumandá pyta’i, doses of roughly 300–400 Gy (below GR50) are the most defensible starting window for mutation induction. The framework is reproducible and low-cost, but it is based on one greenhouse experiment and a single genotype, and should be validated across independent trials and cultivars.