Evaluating Citrus Rootstock Tolerance to Osmotic Stress Induced by PEG in Vitro

Abstract Citrus is one of the most widely planted fruit crops in the world, yet its growth and productivity are extremely susceptible to abiotic stresses, especially salinity and drought. With increasing climate change, water scarcity is becoming a major challenge, making the development of drought-tolerant rootstocks an important target in citrus biotechnology. The tolerance responses of four citrus rootstocks Citrus macrophylla, Volkameriana lemon, Cleopatra mandarin, and trifoliate orange under osmotic stress caused by polyethylene glycol (PEG) were assessed in this study. To replicate drought conditions, PEG-6000 shock treatments at doses of 0%, 4%, and 8% were applied to in vitro cells cultivated in 2024. Antioxidant enzyme activity, molecular characteristics, biochemical reactions (chlorophyll a, chlorophyll b, carotenoids, and relative water content), and morphological parameters (shoot number, shoot length, and leaf number) were all noted. PEG stress significantly reduced all growth and photosynthetic traits, while proline accumulation and antioxidant enzyme activity increased compared with the control, indicating activation of stress defense mechanisms. Genetic analysis using protein electrophoresis and peroxidase isozymes revealed banding pattern variations among rootstocks and treatments, demonstrating the usefulness of these biochemical markers in detecting genetic diversity under stress. Based on the combined results, Cleopatra mandarin showed the highest drought tolerance, followed by Citrus macrophylla, Volkameriana lemon, and Trifoliate orange. These findings suggest that the evaluated characteristics are effective for screening drought tolerance in citrus. The study highlights the potential of drought-tolerant rootstocks, particularly Cleopatra mandarin, for improving citrus adaptation and productivity under future water-limited environmental conditions.