Screening gamma-induced Leucaena tarramba mutants for drought tolerance under PEG-simulated osmotic stress in hydroponics

Abstract Drought tolerance remains a major challenge in improving Leucaena tarramba, a legume widely cultivated in arid and semi-arid tropics. Effective screening for drought tolerance is critical for identifying Leucaena tarramba putative mutants with improved adaptation to water-limited environments. A hydroponic system combined with polyethylene glycol (PEG)-induced osmotic stress provides a controlled and reproducible platform for evaluating drought-related responses. This study aimed to screen M₂ mutants and identify promising drought-tolerant putative mutants based on estimated estimates of genetic variability and key morpho-physiological responses under drought-simulating osmotic stress induced by polyethylene glycol (PEG) 6000 in a hydroponic deep-water culture system. Seeds from the Livestock Breeding and Forage Crops Center, West Nusa Tenggara, Indonesia were irradiated with Co-60 gamma rays at Center for Isotope and Radiation Application under the National Nuclear Energy Agency of Indonesia (PAIR-BATAN Indonesia) at doses ranging from 50 to 1000 Gy, with an estimated LD₅₀ of 820 Gy based on survival at 21 days after planting. Following phenotypic evaluation of the M₁ generation, materials derived from 0, 250, 500, and 750 Gy were advanced to the M₂ generation. A split-plot design was arranged with PEG levels consisted of 0, 10, 20% as main plot and irradiation doses included 0, 250, 500, and 750 Gy as subplots. The putative mutants of M₂ were screened under hydroponic PEG stress until 42 days after planting (DAP), followed by post-hydroponic greenhouse evaluations at 60 and 90 DAP. Interaction between PEG and irradiation dose significantly affected several morpho-physiological traits. Under 10% PEG stress, the 250- and 500-Gy-derived M₂ putative mutants showed relatively better maintenance of several growth-related traits together with adaptive physiological responses, whereas 20% PEG caused more pronounced reductions in growth and physiological performance. Drought-tolerance screening based on individual dry-weight values revealed considerable variation among M₂ putative mutants derived from 250, 500, and 750 Gy. Stress selection indices varied among the M₂ irradiation-dose groups under PEG-induced osmotic stress. Stress intensity increased from 0.68 under 10% PEG to 0.85 under 20% PEG. Under 10% PEG, STI increased from 0.23 in the wild type to 0.41 in the 750 Gy group, while SSI decreased from 1.10 to 0.90. The same trend was observed under 20% PEG, although STI values were lower overall. The 500 and 750 Gy groups had SSI values below 1 under both stress conditions, indicating lower relative susceptibility than the wild type and 250 Gy groups. PCA based on individual morpho-physiological traits explained 89.5% of the total variation and separated entries with maintained and reduced performance. Several M₂ putative mutants under 10% PEG, including 250Gy_2P1, 250Gy_3P1, 250Gy_4P1, and 500Gy_1P1–500Gy_4P1, showed maintained performance and were identified as promising candidates. Preliminary broad-sense heritability estimates ranged from 0.24 to 0.60 during the hydroponic phase and from 0.11 to 0.80 during the post-hydroponic evaluations at 60 and 90 DAP. Hydroponic PEG screening provides a useful preliminary framework for identifying promising gamma-induced M₂ putative mutants with maintained morpho-physiological performance under stress, which can be prioritized for further progeny testing and molecular validation.