EVALUATING THE SALINITY TOLERANCE OF COTTON GENOTYPES USING PHYSIOLOGICAL MARKERS

SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 11 EVALUATING THE SALINITY TOLERANCE OF COTTON GENOTYPES USING PHYSIOLOGICAL MARKERS J. Shavkiev1, A. Azimov2, Z. Mamarasulov3, X. Asatova4, F. Abdurasulov5, S. Nabiev6 Institute of Genetics and Plant Experimental Biology, Academy of Sciences of the Republic of Uzbekistan1,2,3,4,5,6 https://doi.org/10.5281/zenodo.17606577 Abstract. Soil salinity remains a primary constraint to cotton production in arid irrigated zones. To enable rapid, physiology-based screening, we evaluated 17 Gossypium hirsutum genotypes under moderate (0.3–0.6%) and high (0.6–0.9%) field salinity using a randomized block design. Leaf relative water content (RWC), excised-leaf water loss (ELWL, 0–3 h) and chlorophyll index (SPAD) were measured, and two-way ANOVA (Genotype, Salinity, Genotype×Salinity) was applied. Salinity decreased RWC, increased ELWL, and lowered SPAD with strong main effects and interactions for all traits (p < 0.001), evidencing genotype-specific physiological responses. Based on integrated performance, Yulduz-2 and T-1002 were consistently superior; T-1003, T1090, and L-217/L-218 were also promising. Sensitive checks included Orzu, T-1050, and T-2000. A practical selection signature emerged: high RWC + low ELWL, with ELWL ≤ ~10% at 3 h prioritizing tolerant lines. For pigment stability, SPAD ≥ ~52 units under high salinity (or ≤10% decline vs. moderate salinity) efficiently flagged robust genotypes. We conclude that combining RWC, ELWL, and SPAD provides a fast, reliable toolbox for screening salt tolerance and identifying donor parents for breeding targeted at stable yield on saline soils. Keywords: cotton; salinity tolerance; RWC; ELWL; SPAD; physiological markers; breeding; Uzbekistan. Introduction Cotton cultivation is one of the leading sectors in the agriculture of the Republic of Uzbekistan, playing a key role in economic stability, supplying raw material for the textile industry, and increasing export potential. However, agro-ecological factors—particularly soil salinity—adversely affect yield and plant development in cotton production. Soil salinization is widespread in many regions of Uzbekistan, especially on irrigated lands, and it significantly impacts photosynthesis, growth, flowering cycles, nutrient uptake, and ultimately yield. Therefore, assessing the physiological and morphological responses of cotton genotypes under salinity is a pressing scientific and practical task. Tolerance to salt stress in plants is ensured by complex physiological and biochemical mechanisms. Among the most important are indicators of plant water status: relative water content (RWC) and excised-leaf water loss (ELWL), which are widely used to evaluate plant responses to salinity. These parameters reflect not only the capacity of leaves to retain water and regulate transpiration but also, at the cellular level, osmotic adjustment, ion homeostasis, and the functional integrity of the photosynthetic apparatus [4,9,18,20,21,23,24,3]. Accordingly, assessing the ability of different genotypes to maintain water balance under salinity enables the identification of tolerant sources for breeding. This approach provides physiological markers necessary for developing varieties with stable productivity in saline conditions [4,40]. The literature emphasizes that, in the initial (osmotic) phase of salt stress, leaf water potential declines, leading to reduced turgor and restricted transpiration; in the subsequent SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 12 (ionic) phase, toxic accumulation of Na⁺ and Cl⁻ further disrupts photosynthesis and growth [28,38]. RWC is considered a primary physiological indicator of salt tolerance: tolerant genotypes maintain higher RWC and thus sustain photosynthetic activity, whereas sensitive genotypes lose water rapidly and exhibit increased membrane damage [1,33,38,49]. ELWL is an effective marker for discriminating tolerance, where lower ELWL values indicate prolonged leaf water retention and efficient stomatal regulation [13,25,26,45]. Overall, integrated analysis of RWC and ELWL under salt stress enables accurate evaluation of genotype tolerance levels. Previous studies confirm that these indicators can serve as practical physiological markers in breeding programs [5,6,11,47]. In essence, salt stress first induces an osmotic phase that lowers leaf water potential and turgor, followed by an ionic phase in which Na⁺/Cl⁻ accumulation disrupts ion homeostasis, compromises membrane stability, and damages the photosynthetic apparatus. Consequently, water-status markers—RWC and ELWL— are among the primary tools for rapid and reliable assessment of salinity tolerance [7,12,28]. RWC directly reports tissue water status (FW, TW, DW) and thus the effectiveness of turgor maintenance, osmotic adjustment, and sustaining a favorable K⁺/Na⁺ ratio. Tolerant genotypes typically maintain higher RWC, whereas sensitive ones show sharp declines; RWC often correlates positively with growth and yield, making it suitable for rapid field screening [1,10,33,39,48,51]. ELWL quantifies the rate of water loss from excised leaves over a set interval; low ELWL reflects a strong cuticular barrier, fast and reversible stomatal responses, and accumulation of compatible solutes (proline, soluble sugars), and is frequently inversely related to RWC [13,45,47]. Chlorophyll content (SPAD) is likewise a key phenotypic indicator for discriminating salt tolerance. SPAD offers a simple, rapid proxy for leaf chlorophyll in cotton, allowing assessment of photosynthetic performance and overall physiological status. Accordingly, this study examined growth traits (e.g., plant height) and leaf chlorophyll (SPAD) of diverse cotton genotypes grown under moderately saline conditions. The results provide actionable information for screening tolerant genotypes and supporting breeding decisions. Globally, soil salinity poses a major threat to agriculture: according to FAO (2021), more than 20% of terrestrial land is affected by salinity to varying degrees; in Uzbekistan’s irrigated zones, the figure may reach 30–50% [8,17,19]. Genotypic differences in cotton salinity tolerance are substantial [1,31,35]. Salinity commonly reduces growth rates, root length, stem height, and leaf greenness (chlorophyll) [32,41]. SPAD has been widely used to detect declines in photosynthetic activity under stress [27], and in some studies correlates positively with yield [2,30], broadening its utility in selection. Local institutions—including the Academy of Sciences of Uzbekistan and agricultural research institutes—are conducting extensive work to develop salttolerant cultivars [52], proposing phenotypic, morphological, and physiological criteria for screening. Building on this foundation, the present study aims to provide a scientific basis for selection by evaluating chlorophyll content (SPAD) and water-balance dynamics (RWC, ELWL) as indicators of cotton genotype adaptability to salinity. Materials and Methods Study site and conditions The experiment was conducted at the Institute of Genetics and Plant Experimental Biology. Field work was performed during the 2025 growing season at a field experimental plot located in a designated area of the Tashkent Region, using soils with moderate sulfate-type salinity originating from the Syrdarya. The study was carried out on saline soils under Uzbek conditions. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 13 Two salinity backgrounds were used in the field: moderately saline (0.3–0.6% total salts) and highly saline (0.6–0.9% total salts). Experimental design The trial was laid out as a randomized block design with two replications. Each replication consisted of four-row plots with 60 cm inter-row spacing and 10–15 cm within-row spacing. For observations, 15–20 plants per genotype were selected. Irrigation, fertilization and other agronomic practices followed local agro-climatic recommendations. Salinity was natural; no additional salt was applied—field-inherent salinity was monitored. Note: In a subsequent description, the trial is also stated to have been arranged with 3–4 replications in a randomized block design, and measurements were taken on fully expanded mainstem leaves between 09:00 and 11:00. Plant material The experiment used 17 cotton (Gossypium hirsutum L.) genotypes representing diverse genetic sources developed within Uzbek cotton breeding programs. The panel included, among others: Yulduz-2, T-1002, T-1003, T-1004, T-1005, T-1050, T-1080, T-1090, T-2000, Orzu, Ishonch, Mehnat, L-217, L-218, L-421. Measured physiological traits 1) Relative water content (RWC, %) RWC was calculated from fresh weight (FW), fully turgid weight (TW), and dry weight (DW) (Slavík, 1974): RWC (%) = (FW – DW) / (TW – DW) × 100 Procedure: FW—disks were weighed immediately to 0.1 mg precision; TW—disks were floated on distilled water at 25 ± 1 °C under dim light for 4–6 h, blotted gently and weighed; DW— disks were oven-dried at 70 °C for 48 h to constant mass and weighed. RWC was computed for each replicate and reported as mean ± SE. 2) Excised-leaf water loss (ELWL, %; 0–3 h) ELWL was determined from the initial fresh mass of leaf disks and their mass after 3 h at room conditions, and expressed as a percentage: ELWL₀–₃h (%) = (FW₀ – FW₃) / (FW₀ – DW) × 100 Conditions: FW₀—initial mass at 0 h; disks held 3 h on dry filter paper at 25 ± 1 °C, RH 45–55%; FW₃—mass at 3 h; DW—70 °C for 48 h to constant mass. 3) Chlorophyll (SPAD) Leaf chlorophyll was measured with a SPAD-502 Plus chlorophyll meter (Konica Minolta, Japan). For each genotype, readings were taken at 2–3 positions on the upper, healthy leaf of 10 plants, and the mean SPAD value was calculated. Measurements were made between 10:00 and 11:00 on sunny days. Data analysis The obtained data were processed using Microsoft Excel and STATISTICA 13.3, and a two-way analysis of variance (ANOVA) was performed. F-statistic values were calculated for genotype, salinity levels, and their interaction. Differences were considered statistically significant at p < 0.05 and p < 0.001. Results and Discussion The experiment showed that as salinity increased, the relative water content (RWC) decreased in all genotypes; however, the magnitude of decline differed significantly among genotypes, indicating that their ability to retain water in leaf tissues varies. Salt-sensitive genotypes SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 14 included L-218 (74.2 → 64.9%), L-421 (65.5 → 64.7%), and Orzu (72.9 → 64.8%). These lines exhibited a sharp reduction in RWC, suggesting weaker water-retention mechanisms. Under salt stress, this likely leads to rapid loss of cell turgor and restrictions on photosynthetic activity. Genotypes with moderate tolerance were Ishonch (74.0 → 64.1%), Mehnat (78.7 → 66.1%), T-1004 (75.1 → 68.2%), and T-2000 (74.8 → 73.4%). Although they partly maintained water balance under stress, their stability was not sufficient; nonetheless, their physiological responses make them suitable intermediate sources in breeding. The highest RWC values were recorded for L-217 (82.4 → 74.2%), T-1002 (79.4 → 74.0%), T-1003 (80.7 → 69.1%), T-1090 (77.6 → 75.9%), and especially Yulduz-2 (93.0 → 70.7%). Notably, Yulduz-2 maintained high water content at both salinity levels. This points to effective osmotic adjustment, superior leaf water retention, and mechanisms that mitigate ion toxicity. As reported in the literature, genotypes with higher RWC under salt stress tend to sustain growth and photosynthesis more stably, creating favorable conditions for yield component formation [11,28]. Overall, the RWC analysis indicates that the capacity to retain water in leaf tissues is a key physiological indicator of salinity tolerance. Based on our results, Yulduz-2, T-1002, and T-1090 were among the most tolerant, whereas L-218 and L-421 were the most sensitive. These differences expand opportunities to use tolerant lines as donors in subsequent breeding and genetic studies. Salt stress can sharply reduce leaf water retention. In our trial, RWC declined for all genotypes under both moderate and high salinity, but the extent of decline reflected their physiological adaptability. Again, L-218 (74.2 → 64.9%), L-421 (65.5 → 64.7%), and Orzu (72.9 → 64.8%) emerged as sensitive genotypes; their steep RWC losses likely reflect weak maintenance of osmotic balance and greater susceptibility to ion toxicity, leading to reduced turgor, photosynthesis, and growth. Among the moderately tolerant genotypes were Ishonch (74.0 → 64.1%), Mehnat (78.7 → 66.1%), T-1004 (75.1 → 68.2%), and T-2000 (74.8 → 73.4%). These genotypes partially maintained water balance under salt stress, yet their stability under severe conditions was insufficient. Their physiological responses indicate intermediate adaptability, making them suitable as secondary sources in breeding. The salt-tolerant group comprised L-217 (82.4 → 74.2%), T-1002 (79.4 → 74.0%), T-1003 (80.7 → 69.1%), T-1090 (77.6 → 75.9%), and especially Yulduz-2 (93.0 → 70.7%). Sustaining high RWC even under strong salinity points to effective osmotic adjustment mechanisms—including accumulation of compatible solutes (proline, sugars), vacuolar ion sequestration, and maintenance of cell turgor. In particular, Yulduz-2 reaching RWC ≈ 93% and remaining >70% under severe salinity supports its value as a genetically salt-tolerant donor. Overall, RWC clearly captured genotype-dependent differences in water retention under salinity. Our results indicate Yulduz-2, T-1002, T-1003, and T-1090 as promising tolerant genotypes, whereas L-218 and L-421 can be regarded as sensitive. Hence, using RWC as a key physiological criterion for assessing salinity tolerance is warranted. Salt stress manifests as disruption of plant water balance and reduced leaf water retention. In this study, RWC decreased across all genotypes under severe salinity, but the magnitude varied by genotype, reflecting differences in osmotic adjustment and tolerance to ion toxicity. The sensitive genotypes were L-218 (74.2 → 64.9%), L-421 (65.5 → 64.7%), and Orzu (72.9 → 64.8%). Their sharp RWC declines indicate weaker leaf water-retention mechanisms. As reported in the literature, sensitive genotypes rapidly lose turgor under salt stress, photosynthetic activity drops, and membrane damage increases [1,33]. The moderately tolerant set—Ishonch, SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 15 Mehnat, T-1004, T-2000—showed slight RWC decreases under severe salinity yet remained moderately stable. Such genotypes typically partially accumulate compatible solutes but cannot fully sustain stability under high ion toxicity [43,47]. The most tolerant genotypes—L-217, T-1002, T-1003, T-1090, and especially Yulduz-2— maintained RWC ≥ 70% even under strong salinity; notably, Yulduz-2 reached 93% at moderate salinity and 70.7% under severe salinity, indicating effective control of osmotic balance, vacuolar compartmentalization of ions, and stable turgor ([28];[34]). As widely reported in the literature, genotypes that maintain high RWC under salinity can sustain photosynthesis more effectively and form yield components more stably ([11];[12];[42]). Our observations corroborate this conclusion: genotypes such as Yulduz-2, T-1002, and T-1090 are suitable for use as salt-tolerant donors in breeding. Overall, RWC proved to be a key physiological indicator for assessing salinity tolerance. Whereas sensitive genotypes exhibited weak water-retention mechanisms, tolerant genotypes demonstrated the capacity to maintain leaf turgor and water balance under stress. Therefore, incorporating RWC analysis as a physiological marker in breeding pipelines is warranted. Fig. 1. Relative water content (RWC) of leaves in cotton genotypes grown under different salinity levels. Two-way ANOVA showed that Genotype (p < 0.001), Salinity level (p < 0.001), and their interaction (p < 0.001) had significant effects on RWC. As salinity increased, RWC declined in all genotypes; however, several lines—Yulduz-2, T-1090, T-1003, and T-1080—maintained comparatively higher leaf water status even under severe salinity, indicating robust physiological mechanisms of salt tolerance and making them promising donors for breeding. Consistent with the ANOVA summary (Table 1), both Genotype (F = 20.87; p < 0.001) and Salinity (F = 25.63; p < 0.001) exerted strong main effects on RWC, confirming marked differences among genotypes in their ability to retain water in leaves. The Genotype × Salinity interaction was also significant (F = 9.74; p < 0.001), indicating genotype-specific response patterns to salinity. Tolerant genotypes such as Yulduz-2, T-1002, and L-217 sustained higher RWC values under strong salinity, whereas sensitive genotypes—Orzu, T-1050, and T-2000—showed pronounced water loss from leaf tissues. In summary, RWC is a reliable physiological marker for assessing salinity tolerance and for prioritizing genotypes in screening and selection pipelines. SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 16 Table 1 Two-way ANOVA for relative water content (RWC): effects of genotype, salinity level and their interaction Factor SS (sum of squares) df F p-value Genotype 1452.381 16 20.87 <0.001 Salinity level 112.476 1 25.63 <0.001 Genotype × Salinity 689.205 16 9.74 <0.001 Residual 447.892 102 – – Note: Two-way ANOVA model (factors: Genotype, Salinity, and their interaction). All main effects and the interaction were significant for RWC (p < 0.001). Excised Leaf Water Loss (ELWL) is a key physiological indicator that reflects the capacity of plants to maintain water balance under saline conditions. In this study, ELWL was evaluated for each genotype under moderate and high salinity. The results revealed significant differences among genotypes. Salt-sensitive genotypes. Orzu (18.0% → 15.0%), T-1005 (15.0% → 10.5%), and T-1050 (18.0% → 12.0%) showed the highest ELWL, indicating poor ability to retain water in leaves and, thus, greater sensitivity to salt stress. In these genotypes, transpiration control is likely weak and osmotic adjustment is limited. Intermediate genotypes. Ishonch (13.0% → 9.0%), L-217 (10.0% → 9.0%), Mehnat (11.0% → 11.0%), T-1003 (9.0% → 9.0%), T-1024 (13.0% → 10.0%), and T-2000 (12.5% → 10.0%) exhibited moderate ELWL, with slight reductions as salinity increased. These values indicate a capacity to retain water for some time, although stability under severe stress remains limited. Salt-tolerant genotypes. L-218 (12.0% → 9.0%), L-421 (9.0% → 9.0%), T-1001 (12.0% → 9.0%), T-1002 (14.0% → 8.0%), T-1080 (9.0% → 9.0%), T-1090 (12.0% → 9.0%), and especially Yulduz-2 (14.0% → 9.0%) maintained low ELWL even under high salinity. Low ELWL reflects effective regulation of transpiration, prolonged water retention in leaf tissues, and better osmotic balance. T-1002 and Yulduz-2 are therefore promising donor genotypes for salttolerance breeding. Overall. ELWL clearly differentiated genotypes by their ability to retain water under salinity. High-loss genotypes (Orzu, T-1050) formed the sensitive group; those with intermediate values (Mehnat, T-1024, T-2000) showed moderate tolerance; and those with the lowest ELWL (T-1002, T-1090, Yulduz-2) were tolerant. Thus, ELWL is an effective physiological marker for assessing salinity tolerance. Methodological note. In this experiment, leaf water-retention capacity was assessed as ELWL (% at 3 h)—the fraction of water lost by an excised leaf over a fixed period. Lower ELWL indicates efficient control of stomatal and cuticular conductance and better tissue water conservation. The literature shows that high ELWL is typically associated with poorer water retention, greater membrane damage, and lower yield stability under stress ([12],[13],[45]). Intermediate group. Ishonch (≈13 → 9%), L-217 (≈10 → 9%), Mehnat (≈11 → 11%), T1024 (≈13 → 10%), and T-2000 (≈12.5 → 10%) showed moderate water loss. In these genotypes, adaptation to the osmotic component of salinity is only partial: stomatal closure is rapid, but cuticular and mesophyll water conductance are not fully constrained. Such a profile is valuable as an intermediate source of tolerance for breeding ([11]; [44]). Tolerant group (low ELWL). L-218 (≈12 → 9%), L-421 (≈9 → 9%), T-1001 (≈12 → 9%), T-1003 (≈9 → 9%), T-1080 (≈9 → 9%), SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 17 T-1090 (≈12 → 9%), and especially T-1002 (≈14 → 8%) and Yulduz-2 (≈14 → 9%) maintained low leaf water loss under stress. Low ELWL typically reflects fast and reversible stomatal response, low cuticular diffusive conductance, accumulation of osmoprotectants (proline, soluble sugars), and vacuolar ion compartmentalization ([21];[28];[47]). Accordingly, these genotypes are physiologically salt-tolerant and promising donors for selection. Fig. 2. Excised Leaf Water Loss (ELWL) of cotton genotypes grown under different salinity levels. Integrated interpretation. When low ELWL is evaluated together with previously noted high RWC/TWC, the salt-tolerant phenotype can be discriminated more reliably. For the next stage, we recommend assessing ELWL–RWC correlations (r), regressions of ELWL on yield components, and integrated analyses with stomatal conductance, cuticle thickness, proline/sucrose content, and K⁺/Na⁺ ratio. Practically, genotypes with ELWL ≤ ~10% (at 3 h)—T-1002, Yulduz2, T-1090, T-1003, L-421, T-1080—should be prioritized for testing on saline breeding sites. Twoway ANOVA showed that both Genotype and Salinity level had significant effects on ELWL (see Table 2). The Genotype effect was large (F = 12.85; p < 0.001; η² = 0.39), indicating clear differences among genotypes in their ability to retain water in leaves. Salinity also played an important role (F = 15.20; p < 0.001; η² = 0.07): under high salinity, some genotypes increased water loss (e.g., Yulduz-2, Mehnat), whereas others decreased it (e.g., T-1050, T-1005, L-217), reflecting genotype-specific mechanisms of turgor regulation under stress. The Genotype × Salinity interaction was also significant (F = 5.88; p < 0.001; η² = 0.23), indicating differential adaptive responses. Tolerant genotypes (T-1002, T-1003, L-218, Ishonch) maintained low ELWL even under strong salinity, while sensitive genotypes (T-1050, L-421, T-1090) exhibited high water loss. Some lines (Yulduz-2, Mehnat) showed elevated ELWL under stress. In summary, ELWL is a useful physiological marker for assessing salinity tolerance; genotypes in the tolerant group are promising donors for breeding. Table 2 Two-way ANOVA for Excised Leaf Water Loss (ELWL): effects of genotype, salinity, and their interaction Factor SS (sum of squares) df F p-value η² Genotype 1536.7 16 12.85 <0.001 0.39 Salinity level 86.9 1 15.20 <0.001 0.07 SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 18 Genotype × Salinity 703.5 16 5.88 <0.001 0.23 Residual 583.2 102 – – – Note: All main effects and their interaction were significant for ELWL (p < 0.001); η² denotes effect size. The chlorophyll index (SPAD) is a key indicator of the functional status of the photosynthetic apparatus in leaves. Salt stress typically induces pigment degradation and a decline in chlorophyll content. In this study, SPAD values were compared across genotypes under moderate and high salinity. Salt-sensitive genotypes. In Orzu (44.5 → 41.6), T-1050 (49.6 → 46.2), T-1090 (48.0 → 45.9), and T-2000 (46.0 → 44.9) the chlorophyll index decreased markedly under strong salinity. This indicates accelerated pigment breakdown and a higher sensitivity of the photosynthetic machinery to ion toxicity, leading to reduced photosynthetic rates and constraints on yield component formation. Moderately tolerant genotypes. In Ishonch (49.1 → 51.0), L-217 (53.1 → 50.2), Mehnat (51.5 → 49.2), T-1001 (49.7 → 47.0), and T-1024 (52.0 → 47.7), SPAD values declined only slightly under high salinity and remained at moderate levels overall. This pattern suggests relatively slower chlorophyll degradation and the operation of partial protective mechanisms against ionic stress. Salt-tolerant genotypes. L-218 (57.1 → 53.0), L-421 (53.0 → 51.2), T-1003 (59.8 → 55.2), T-1004 (58.7 → 54.2), T-1080 (52.0 → 50.0), and especially Yulduz-2 (51.5 → 50.0) maintained high and relatively stable SPAD. Notably, T-1003 and T-1004 preserved chlorophyll at ~54–55 units even under strong salinity. These responses point to effective mechanisms that slow chlorophyll breakdown—such as antioxidant defenses and vacuolar ion compartmentalization. Overall, the decline in SPAD under salt stress varied by genotype: rapid pigment loss characterized the sensitive group, whereas tolerant lines retained higher pigment levels. Consequently, T-1003, T-1004, L-218, and Yulduz-2 can be highlighted as donor genotypes for photosynthetic robustness under salinity. Mechanistically, salt stress usually reduces chlorophyll via pigment degradation, disruption of Mg-chelation, and ROS accumulation in photosynthetic membranes; SPAD provides a rapid, minimally invasive proxy for these processes. Our results corroborate this: most genotypes showed lower SPAD under severe salinity, but the magnitude of decline differed significantly among genotypes. Sensitive group: Orzu (≈44.5→41.6), T-1050 (≈49.6→46.2), T-1090 (≈48.0→45.9), and T-2000 (≈46.0→44.9) lost SPAD substantially, consistent with ion (Na⁺/Cl⁻) toxicity, oxidative damage to chloroplast membranes, and attenuation of the chlorophyllase/porphyrin pathway—effects typically associated with reduced photochemical efficiency and yield [1,12,33]. Moderate response. In Ishonch (≈49.1→51.0), L-217 (≈53.1→50.2), Mehnat (≈51.5→49.2), T-1001 (≈49.7→47.0), and T-1024 (≈52.0→47.7), SPAD declined slightly (with small positive shifts in a few cases). This “moderate” profile indicates that antioxidant protection and ion homeostasis operate partially effectively, yet are insufficient to fully stabilize pigments under strong salinity [22,28]. Tolerant group. L-218 (≈57.1→53.0), L-421 (≈53.0→51.2), T-1003 (≈59.8→55.2), T-1004 (≈58.7→54.2), T-1080 (≈52.0→50.0), and Yulduz-2 (≈51.5→50.0) maintained high and relatively stable SPAD. Notably, T-1003 and T-1004 retained pigments around 54–55 units even under severe salinity. This likely reflects better regulation of vacuolar ion compartmentalization (NHX/HKT/SOS), antioxidant systems, and nitrogen–magnesium SCIENCE AND INNOVATION INTERNATIONAL SCIENTIFIC JOURNAL VOLUME 4 ISSUE 11 NOVEMBER 2025 ISSN: 2181-3337 | SCIENTISTS.UZ 19 supply and chlorophyll biosynthesis ([28];[47]). Sustained SPAD typically aligns with stable Fv/Fm and ΦPSII, and with higher yield [12,16]. Integrated interpretation and practical criterion. Tolerant groups defined by SPAD coincide with the previously identified high RWC / low ELWL profiles, indicating that preservation of the photosynthetic apparatus under salinity is coupled with a stable water balance. For applied selection, we propose SPAD ≥ ~52 units under strong salinity (or ≤10% decline relative to moderate salinity) as a practical pigment-stability threshold; T-1003, T-1004, L-218, L-421, and Yulduz-2 meet this criterion. For future work, we recommend joint analyses of SPAD–Fv/Fm–RWC–Tr correlations and expression of genes involved in chlorophyll biosynthesis/antioxidant pathways. Fig. 3. Chlorophyll index (SPAD) in leaves of cotton genotypes grown under different salinity levels. Two-way ANOVA showed that both Genotype and Salinity level significantly affected SPAD (see Table 1). The Genotype effect was large (F = 21.63; p < 0.001), indicating clear differences among genotypes in their capacity to retain pigments. Salinity also had a strong impact (F = 24.12; p < 0.001): under high salinity, SPAD values declined markedly relative to moderate salinity, reflecting direct damage of salt stress to pigments of the photosynthetic apparatus. Table 3 Two-way ANOVA for chlorophyll index (SPAD): effects of genotype, salinity level, and their interaction Factor SS (sum of squares) df F p-value Genotype 1284.537 16 21.63 <0.001 Salinity level 92.846 1 24.12 <0.001 Genotype × Salinity 611.204 16 10.29 <0.001 Residual 392.517 102 – – Note: All main effects and the Genotype × Salinity interaction were significant for SPAD (p < 0.001). In addition, the Genotype × Salinity interaction was significant (F = 10.29; p < 0.001), meaning the effect of salinity on pigment retention was not uniform across genotypes. Some