Integrated biochemical, ionic and ultrastructural analysis of salinity tolerance in novel citrus rootstocks

Salinity stress is a major constraint to citrus productivity, which is adversely affecting physiological, biochemical and cellular processes. The use of salt-tolerant rootstocks offers a sustainable strategy to mitigate these effects; however, rootstock breeding requires a comprehensive understanding of genotype-specific tolerance mechanisms operating across multiple biological levels.

Six citrus rootstocks (X9, JK, S19, C9, C13 and S12) were evaluated under control and 50 mM NaCl conditions. A comprehensive assessment included gas exchange traits (A, g_s, C_i and E), photosynthetic pigments, oxidative stress markers (H₂O₂ and MDA), antioxidant enzyme activities (SOD, APOX, CAT, GR and POX), osmolytes (LPC, TSS and TSP), TPh and mineral ion composition (Na⁺, Cl⁻, K⁺, Ca²⁺, and Mg²⁺) in leaves and roots. Chloroplast ultrastructure was analyzed using transmission electron microscopy. Multivariate analyses, including principal component analysis, hierarchical clustering and membership function value analysis, were used to establish a salinity tolerance ranking.

Salinity significantly affected all traits, with pronounced genotype-dependent variation. Under the present experimental conditions, the tolerance ranking was observed as: X9 > C13> S12 > S19 > C9 > JK. This hierarchy was supported by photosynthetic rate as tolerant genotypes exhibited comparatively lower reductions in rate (44-51%) than sensitive genotypes (62-86%), with X9 showing the least decline (44.3%). Chlorophyll loss ranged from 4.3% to 19.7%, in parallel with photosynthetic performance. Oxidative damage was substantially lower in tolerant genotypes, as indicated by reduced increases in H₂O₂ (8.1-83.3%) and MDA (31.9-86.6%). Enhanced antioxidant responses (SOD, APOX and GR) and greater accumulation of osmolytes and phenolics contributed to stress mitigation. X9 maintained the highest K⁺/Na⁺ ratio (4.5) through effective exclusion of Na⁺ and Cl⁻, while sensitive genotypes accumulated toxic levels. Ultrastructural analysis confirmed preservation of chloroplast integrity in tolerant genotypes, in contrast to severe structural damage in sensitive ones. Principal component analysis clearly distinguished tolerant and sensitive groups.

Salinity tolerance in citrus rootstocks is controlled by the coordinated integration of ion regulation, antioxidant defense, osmotic adjustment, photosynthetic maintenance and chloroplast integrity. X9 emerged as the most tolerant genotype, followed by C13 and S12. The multi-trait framework and quantitative analysis identified here provide practical criteria for breeding and selection of citrus rootstocks for salt-affected environments.