Freezing tolerance is a critical factor affecting the productivity and sustainability of citrus cultivation in subtropical regions. Photoperiod and cold acclimation work together to enhance a plant’s freezing tolerance by triggering specific biochemical and molecular pathways that help it withstand low temperatures and avoid cellular damage during freezing events. This study aims to investigate the interactive effects of photoperiod and cold acclimation on the freezing tolerance of Valencia orange (Citrus sinensis) plants grafted onto US942 rootstock, focusing on how these environmental factors modulate physiological and molecular responses to freezing stress. We hypothesize that varying photoperiods, when combined with cold acclimation, will synergistically enhance the freezing tolerance of Valencia orange plants by modulating biochemical and physiological traits associated with cold hardiness. Two-year-old Valencia orange plants will be grown under different photoperiods (8, 10, 12, 14, and 16 hours) for four weeks. After the photoperiod treatment, plants from each photoperiod group were divided into two treatments: one group undergo cold acclimation by being exposed to 4°C for 16 hours, while the other group was placed at 25°C for 16 hours (non-cold-acclimated). Following this, both cold-acclimated and non-cold-acclimated plants were exposed to freezing stress at -6°C in walk-in freezing chambers for 1 hour. The freezing temperature reached by gradually lowering the temperature by 1°C per hour, starting from 0°C to -6°C. We measured the photosynthesis, chlorophyll content and ELL. These results showed that the freezing stress showed the photosynthesis limitation, there are problem in enzymatic machinery in carbon dioxide assimilation. According to ELL and spad index GBT3R2 shows lowest cellular damage and the results show preserved the chlorophyll content. This research identifies how photoperiods and cold acclimation interact to enhance freezing tolerance in Valencia oranges, providing key markers for breeding more freeze resilient citrus cultivars.
