Cryopreservation of seeds of <i>Citrus indica</i> and <i>Citrus latipes</i>: enhancing post‐thaw recovery with sodium nitroprusside and tocopherol

BACKGROUND: Cryopreservation is a crucial technique for the long-term conservation of endangered plant germplasm. However, desiccation-sensitive seeds, such as those of Citrus indica Yu. Tanaka and Citrus latipes (Swingle) Yu. Tanaka, require optimized dehydration protocols to prevent cryo-injury. Enhancing post-thaw recovery through antioxidant supplementation is a promising approach to mitigate oxidative stress-induced damage. OBJECTIVE: This study aimed to establish an optimized dehydration-based cryopreservation protocol of seeds for C . indica and C . latipes while evaluating the protective effects of sodium nitroprusside (SNP) and tocopherol on post-cryopreservation recovery. MATERIALS AND METHODS: Mature seeds of C . indica and C . latipes were subjected to controlled dehydration to moisture contents of 22.7 and 30.1 using a laminar air flow chamber before liquid nitrogen storage. Post-thaw viability was assessed through germination studies, physiological assays (chlorophyll content and malondialdehyde estimation), and biochemical analyses (antioxidant enzyme activities). SNP (0.4 ??M) and tocopherol (25 ??M) were used as post cryopreservation supplements to enhance seedling recovery. RESULTS: Optimal seed moisture content prior to freezing was 24.9% for C . indica and 32.0% for C . latipes , maximizing post-thaw germination. SNP and tocopherol supplementation significantly reduced lipid peroxidation, improved chlorophyll retention, and enhanced superoxide dismutase, catalase, and peroxidase activities. CONCLUSION: SNP and tocopherol effectively mitigate cryo-induced oxidative stress, improving post-thaw recovery of C . indica and C . latipes seeds. These findings provide critical insights into cryopreservation strategies for conserving endangered citrus germplasm.