Integrating Chlorophyll Fluorescence Imaging and Kinetic Analysis for the Identification of Freezing-tolerant Citrus Rootstocks

As citrus production expands into northern Florida and neighboring states, where environmental conditions are considered marginal due to increased exposure to freezing events, identifying cold-tolerant genotypes has become a critical priority. This study addresses the urgent need for climate-resilient agriculture by using high-throughput plant phenotyping to evaluate the freezing tolerance of the ‘Marathon’ mandarin (cv. UF 1420) grafted onto 11 diverse citrus rootstocks. Using a controlled stress environment of −4 °C, we integrated chlorophyll fluorescence imaging and kinetics analysis in citrus plants to map the functional and structural stability of the photosynthetic apparatus under stress. Multivariate principal component analysis followed by linear discriminant analysis effectively discriminated tolerance levels, pinpointing four key parameters: electron transport flux per active reaction center (ET 0 /RC), initial slope of the relative variable fluorescence ( M 0 ), absorption flux per active reaction center (ABS/RC), and dissipation flux per active reaction center (DI 0 /RC), as the most powerful physiological indicators of freeze-induced damage. Our results identified UFR-2 as a standout freezing-tolerant rootstock, characterized by superior photochemical efficiency, minimal cellular damage (47% electrolyte leakage compared with >90% in sensitive varieties), and rapid poststress recovery. In contrast, rootstocks such as KH, Rich 16-6, UFR-4, and US 897 exhibited significant reaction center inactivation and severe photoinhibition, leading to high mortality rates 6 weeks after stress. This research provides an analytical approach for citrus breeding programs based on the characterization of photosynthetic responses, demonstrating that the stability of photosynthetic processes is a key determinant of cold resilience. By identifying freezing-resilient citrus rootstocks, this work provides actionable insights for selecting rootstocks adapted to North Florida conditions through the characterization of photosynthetic responses, thereby supporting the long-term viability of the regional citrus industry.