(A) Overview of greenhouse experiments for the salt tolerance evaluation of tomato. (B) Leaf injury score (1 = healthy, rich green; 2 = early chlorosis, paler green; 3 = increased chlorosis, fading green; 4 = extensive chlorosis, green and yellow; 5 = complete chlorosis, fully yellow; 6 = initial necrosis, yellow with brown spots; and 7 = widespread necrosis, brown and wilted). (C) Seedling growing under both normal and salt stress conditions. Yellowing of leaves was observed in plants under salt stress.
Distributions of the leaf injury score among 71 tomato genotypes.
Distributions of the leaf chlorophyll content [soil plant analysis development (SPAD)] among 71 tomato genotypes: (A) leaf chlorophyll content under nonsalt treatments (C_NS) and (B) leaf chlorophyll content under salt treatments (C_S) using 200 mM NaCl for 2 weeks.
Distributions of leaf chlorophyll content [soil plant analysis development (SPAD)] among 71 tomato genotypes: (A) absolute decrease in the leaf chlorophyll content (cm) (AD_C); (B) inhibition index for chlorophyll content (%) (II_C); and (C) relative salt tolerance for the chlorophyll content (%) (RST_C).
Distributions of seedling height (cm) among 71 tomato genotypes: (A) seedling height (cm) of plants irrigated with deionized water (SH_NS) and (B) those that were salt-stressed with 200 mM NaCl for 2 weeks (SH_S).
Distributions of parameter values for assessing seedling height reduction upon salt treatment among 71 tomato genotypes: (A) absolute decrease (cm) in seedling height (AD_SH); (B) inhibition index of the seedling height (%) (II_SH); and (C) relative salt tolerance of seedling height (RST_SH).
(A) Two-way dendrogram and (B) constellation plot of 71 tomato accessions according to the hierarchical cluster analysis using JMP Pro 17 based on five salt tolerance-related traits: leaf injury score (LIS), absolute decrease in the leaf chlorophyll content (AD_C), II_C, absolute decrease in seedling height (AD_SH), and inhibition index of the seedling height (II_SH). The top seven salt-tolerant accessions were grouped into one cluster I.
Phylogenetic tree created by MEGA 11 based on 2398 single nucleotide polymorphisms (SNPs) distributed on 12 chromosomes in 71 USDA Germplasm Resources Information Network (GRIN) tomato accessions. The seven tomato accessions with a score <3.0 are marked with red rectangle.
Principal component analysis (PCA) of 71 tomato accessions by JMP Genomics based on five salt tolerance-related traits, leaf injury score (LIS), absolute decrease in the leaf chlorophyll content (AD_C), II_C, absolute decrease in seedling height (AD_SH), and inhibition index of the seedling height (II_SH). (A) Bioplot. (B) Scree plot. (C) PCA with three clusters.
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Soil salinity is a significant abiotic factor that impedes sustainable crop production in key agricultural regions worldwide. Saline cultivation adversely affects soil quality, whereas the use of saline water for irrigation disrupts the physiological and biochemical processes of plants. Continuous irrigation with high salt concentrations leads to a gradual buildup of soil salinity, thus hindering optimal plant growth and development. Consequently, there is a growing emphasis on breeding salinity-tolerant cultivars of various crops. In this study, 71 tomato accessions sourced from 20 countries and provided by the US Department of Agriculture were evaluated under controlled greenhouse conditions and subjected to saline stress (200 mM NaCl). The experiment used a split-plot design, with the salt treatment serving as the main plot and the tomato accession as the subplot, which were arranged in a completely randomized design with three replications. Results identified nine accessions (PI 109837, PI 127820, PI 270256, PI 634828, PI 636205, PI 636255, PI 647143, PI 647528, and PI 647556) as salt-tolerant. Additionally, high broad-sense heritability was observed for the leaf injury score and leaf chlorophyll content. Furthermore, positive correlations were found among parameters related to the leaf injury score and leaf chlorophyll content (soil plant analysis development value). These findings offer valuable insights for tomato breeding programs, particularly those focused on enhancing salt tolerance of elite cultivars of this crucial crop.
Tomato (Solanum lycopersicum L.), which is a member of the Solanaceae family, originates from Central America and South America and has a pivotal role in global agriculture (Rezk et al. 2021). Despite its botanical classification as a fruit, it is predominantly used as a vegetable in culinary applications (Rao et al. 2020). Tomatoes rank as the second most important vegetable in the United States, only trailing behind potatoes, and they are integral to a diverse array of raw, cooked, and processed foods (Meena and Kumar 2020). The recent increase in tomato consumption can be attributed to their rich composition of bioactive compounds, including carotenoids, flavonoids, vitamins, and tocopherols, as well as their low fat and cholesterol contents, which make them advantageous to human health (Tommonaro et al. 2012). Tomatoes are cultivated extensively and encompass approximately 4.85 million hectares globally, with an annual yield of 182.3 million tons. The United States contributes approximately 20% of the world’s total production (Quinet et al. 2019). Despite their global agricultural significance, tomatoes are especially susceptible to saline conditions, thus significantly impacting their yield and quality (Roșca et al. 2023).
Soil salinity profoundly affects tomato physiology by inducing osmotic stress and ionic toxicity, which reduce water availability, inhibit growth, and disrupt cellular balance because of excessive sodium and chloride ions (Yang et al. 2014). Salinity also interferes with the uptake and distribution of essential nutrients, thus compounding plant stress (Zhang et al. 2017). A major consequence of this stress is the reduction of photosynthetic pigments such as chlorophyll, thus limiting the plant’s ability to capture light and perform photosynthesis efficiently (Alam et al. 2021). This aligns with the findings by Mousa et al. (2013), who observed that high salinity levels reduced growth parameters in various tomato genotypes, thereby signaling a decline in overall physiological functions, including photosynthesis. The detrimental effects of salinity extend further to the chloroplast structure and function, thus impacting energy production (Hameed et al. 2021). Seedling traits such as leaf area, root length, shoot length, and biomass also suffer, thus impairing early growth and establishment (Alam et al. 2021). In response to these stresses, hormonal changes, particularly those involving abscisic acid, trigger key defense mechanisms. (Yang et al. 2014). Genetic analyses have identified key morphophysiological and molecular traits associated with salt tolerance in tomato introgression lines, thus providing valuable insights for breeding programs (Ali et al. 2021). Addressing these challenges through breeding programs is critical to sustaining tomato production in regions that are increasingly affected by soil salinity (Gong et al. 2020).
Mitigating the effects of salinity on tomato production requires a comprehensive approach that integrates breeding programs, genomic tools, and agronomic practices. Marker-assisted selection and conventional breeding have leveraged genes from wild relatives such as Solanum pimpinellifolium, S. habrochaites, and S. peruvianum, which possess traits that confer resistance to both biotic and abiotic stresses, including salinity (Zhang et al. 2022). Wild tomato species from the Galapagos Islands, including Solanum cheesmaniae and Solanum galapagense, exhibit superior salinity tolerance compared with that of commercial tomato cultivars by maintaining growth under salt stress, thus making them valuable genetic resources for improving salinity tolerance in cultivated tomatoes (Pailles et al. 2020). Advanced genomic tools such as genotyping by sequencing (GBS) and single nucleotide polymorphism (SNP) discovery have enabled the precise identification of genes involved in ion regulation and osmotic balance, thus accelerating the development of salt-tolerant cultivars (Ali et al. 2021). Technologies such as CRISPR-Cas9 and genome-wide association studies have further enhanced breeding programs, thus facilitating the creation of cultivars that are more resilient in saline environments (Tran et al. 2021). Key mechanisms, including sodium exclusion, tissue tolerance, and osmotic regulation, observed in the adaptive responses of Galapagos tomato accessions provide essential pathways for improving the salinity resilience of commercial tomato cultivar (Munns and Tester 2008).
In addition to these genetic advancements, agronomic practices such as optimized irrigation, soil amendments (e.g., gypsum and organic matter), and biostimulants support plant health and mitigate the adverse effects of salinity on crop yields. By combining these practical measures with advanced breeding technologies, a sustainable and comprehensive strategy can be implemented to maintain tomato production in regions that are increasingly affected by soil salinity (Ghanem et al. 2011; Ma et al. 2022; Sattar et al. 2021).
This study focused on the often-overlooked seedling stage by evaluating the US Department of Agriculture (USDA) tomato germplasm to understand salt tolerance mechanisms. Leaf injury scores and chlorophyll content [soil plant analysis development (SPAD) values] were measured to assess tolerance at this critical stage, which serves as a key indicator of the plant’s future performance under salt stress and its overall robustness throughout the growth cycle. A total of 71 USDA tomato accessions were evaluated to identify potential salt-tolerant cultivars for breeding programs that seek to enhance genetic resistance to salt stress. Additionally, the study also integrated a genetic diversity analysis based on GBS to help identify genetic variability and understand the genetic architecture of this evaluation population.
A total of 71 tomato accessions were obtained from the USDA Agricultural Research Service Germplasm Resources Information Network. The valuation of salt tolerance considered both the genetic diversity of the accessions and the significance of the American accessions. Notably, 35 accessions (49.3%) were from the United States, thus underscoring the considerable genetic diversity within the domestic collection. The remaining accessions, which represent a broad spectrum of genetic variation, were sourced from 19 other countries (Supplemental Table S1).
The evaluation of tomato accessions was performed in a greenhouse at the Arkansas Agricultural Research and Extension Center in Fayetteville, AR, USA (lat. 36°4′55″N, long. 94°10′18″W). The controlled environment maintained a daytime temperature of 21 °C, nighttime temperature of 18 °C, and consistent humidity level of 73% throughout the 2023 experiment. The experimental design and methodology closely followed the procedure outlined previously (Chiwina et al. 2024) for tomato drought evaluation, with minor modifications. Six seeds from each accession were planted in individual pots and then placed in larger trays. Each tray (length × width × height: 52 cm × 26 cm × 6 cm) housed 12 square pots (height, 8.5 cm; top length, 8.5 cm; base length, 5.8 cm), and filled to a depth of 8 cm with commercial compost (BM 6; Berger, Saint-Modeste, Quebec City, Canada). Initially, 300 mL of water was added to each pot and 2 L was added to each tray. According to the initial irrigation, a regular watering schedule was established, with 180 mL of water added to each pot every 3 d for 35 d. Additionally, starting 10 d after seeding, the pots received a fortnightly application of Miracle-Gro Water-Soluble All-Purpose Plant Food 24–8–16 containing nutrients such as ammoniacal nitrogen, urea nitrogen, available phosphate, soluble potash, and trace elements (boron, copper, iron, manganese, molybdenum, and zinc).
The experimental design used a completely randomized design with three replications organized in a split-plot manner, with salt treatment as the main plot and tomato genotypes as the subplot (Dong et al. 2019; Ravelombola et al. 2021). The evaluation involved two groups: the salt-treated and the control (nonsalt-treated) groups. Salt treatment began 5 weeks after seeding and consisted of a daily 200-mM NaCl solution application for 6 h, followed by rinsing, over a 14-d period. In contrast, control plants were irrigated normally without salt. Plant thinning occurred 15 d after planting to standardize plant vigor and height. Each pot maintained three plants per tomato accession. The treatment continued until the susceptible genotypes wilted (typically by day 14), thus revealing varying salt tolerance levels across the tomato genotypes (Fig. 1A).
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
The leaf injury score (LIS) has been used as an indicator during the evaluation of salt tolerance in tomato seedlings (Ahsan et al. 2022). The study by Ravelombola et al. (2021) further corroborated the validity of the LIS, which uses a graded scale ranging from 1 to 7 (1 = healthy plants and 7 = entirely deceased plants; the score sequentially increased through signs of leaf chlorosis, extensive chlorosis, complete chlorosis, initial necrosis, and widespread necrosis) (Fig. 1B). This scale was applied at the point when susceptible specimens exhibited complete mortality. This approach not only streamlined the assessment process but also provided a cost-effective and accessible method of evaluating salt tolerance in tomato seedlings, which is particularly relevant in large-scale breeding programs.
The chlorophyll content (SPAD value) in trifoliate leaves was quantified across three distinct regions for every plant in each genotype under salt treatment and nonsalt treatment. Measurements were performed 14 d after the salt treatment began, and fully developed trifoliate leaves were selected from the upper part of the plant. This was executed using the SPAD-502 Plus Chlorophyll Meter (Spectrum Technologies, Inc., Plainfield, IL, USA). Separate recordings were performed for each region on the leaves. The data gathered were methodically documented and analyzed using the following five indexes, as outlined in previous studies (Phiri et al. 2024; Ravelombola et al. 2021) (Supplemental Table 1):
C_S: leaf chlorophyll content under salt treatment measured by the SPAD-502 Plus Chlorophyll Meter (Spectrum Technologies, Inc.).
C_NS: leaf chlorophyll content under nonsalt treatments.
AD_C: absolute decrease in the leaf chlorophyll content (cm) = leaf SPAD chlorophyll under the nonsalt treatment – leaf SPAD chlorophyll under salt treatment.
II_C: inhibition index for chlorophyll content (%) = 100 * [(leaf SPAD chlorophyll under the nonsalt treatment − leaf SPAD chlorophyll under salt treatment)/leaf SPAD chlorophyll under nonsalt treatment].
RST_C: relative salt tolerance for chlorophyll content (%) = (100 * leaf SPAD chlorophyll under salt treatment/leaf SPAD chlorophyll under nonsalt treatment).
The seedling height was measured for each accession under both salt-treated and nonsalt-treated conditions 14 d after initiating the salt treatment. Following the methodology of Dong et al. (2019), measurements of the base of the shoot to the growing point were obtained, coinciding with the complete mortality of the susceptible control. Height data for each plant were recorded, and an average was calculated for each pot (Fig. 1C). This streamlined approach allowed for a clear comparison between plants under salt and nonsalt treatments, thus providing insights into their respective tolerance levels. The following indexes were collected and computed (Ravelombola et al. 2018, 2021; Saad et al. 2013).
SH_S: seedling height under salt treatment (cm).
SH_NS: seedling height under nonsalt treatment (cm).
AD_SH: absolute decrease in seedling height (cm) = seedling height under nonsalt treatment − seedling height under salt treatment.
II_SH: inhibition index for seedling height (%) = 100 * [(seedling height under nonsalt treatment − seedling height under salt treatment)/seedling height under nonsalt treatment]
RST_SH: relative salt tolerance for seedling height (%) = 100 * (seedling height under salt treatment/seedling height under nonsalt treatment).
The data analysis of the tolerance ranking was conducted using JMP PRO 17 software. This involved an analysis of variance via the general linear model procedure and visualization of data distribution using the software’s ‘Distribution’ feature. Descriptive statistics were obtained using the ‘Tabulate’ function, whereas Pearson’s correlation coefficients and their P values were calculated using the ‘Multivariate Methods’ option. A cluster analysis was also performed using JMP PRO 17. Additionally, broad-sense heritability (H2) was estimated according to the work of Holland et al. (2003) using the following formula:
where σ2G represents the total genetic variance, σ2e is the residual variance, and r is the number of replications. The estimates for σ2G were obtained as [EMS(G) − Var (residual)]/r, where EMS(G) and Var (residual) were extracted from the analysis of variance table.
A principal component analysis and dendrogram phylogenetic tree were constructed based on the five salt tolerance-related traits (LIS, AD_C, II_C, AD_SH, and II_SH) among the 71 tomato accessions using the hierarchical cluster method in JMP Pro 17.
The 71 tomato accessions were ranked from 1 to 71 for each of the seven traits (LIS, AD_C, II_C, RST_C, AD_SH, II_SH, and RST_SH), where 1 indicated the highest level of salt tolerance and 71 represented the most vulnerable accession. Because the value of II (injury index) is calculated as 100 minus the relative salt tolerance (RST) value (II = 100 – RST), the ranking order of RST was the same as the ranking order of II. Consequently, RST_SH and RST_C were not explicitly listed.
The CTAB/SDS method was used to extract DNA from fresh tomato leaves. Subsequently, the extracted genome was sequenced using the GBS approach, following the protocol outlined by Elshire et al. (2011), and using pair-end sequencing. The sequencing libraries were processed using an Illumina NovaSeq platform at the University of Wisconsin-Madison Biotechnology Center (https://biotech.wisc.edu/; accessed 6 Mar 2024). The obtained short-read sequences were aligned with the tomato genome reference Solanum lycopersicum ITAG_4.0 (https://phytozome-next.jgi.doe.gov/info/Slycopersicum_ITAG4_0; accessed 6 Mar 2024). A pipeline integrating TASSEL-GBS (Glaubitz et al. 2014) and Stacks 2 (https://catchenlab.life.illinois.edu/stacks/; accessed 6 Mar 2024) was used for SNP identification (Rochette et al. 2019). This process resulted in the discovery of 392,496 SNP markers across 287 tomato genotypes distributed over 12 chromosomes; genotypic data were provided by the University of Wisconsin-Madison Biotechnology Center. The phylogenetic tree was constructed using the maximum likelihood method in MEGA 11 (Tamura et al. 2021) based on 2398 SNP markers distributed across 12 chromosomes. The SNP markers were selected based on criteria including a minor allele frequency more than 2.0%, less than 5% missing alleles, and a heterozygosity rate of 8% or lower. This dual approach that integrated both phenotypic and genotypic data enhanced the understanding of the genetic diversity present in the tomato accessions.
The leaf injury score is a reliable indicator that can be used to assess salt tolerance in seedlings. Among 71 tomato genotypes, scores ranged from 2.10 to 7.00 [average, 5.97, standard deviation (SD), 1.32], indicating significant variability and a left-skewed distribution (Fig. 2, Supplemental Table 2). A statistical analysis showed notable genotypic differences (F = 8.32; P < 0.0001) (Supplemental Table 3); lower scores indicated higher tolerance to stress. The five most tolerant genotypes were PI 647556 (2.1), PI 634828 (2.4), PI 109837 (2.6), PI 647528 (2.6), and PI 636255 (2.7) (Supplemental Table 1). Conversely, genotypes with the highest susceptibility, PI 97538, PI 128586, PI 270239, PI 270228, and PI 270236, each had a score of 7 (Supplemental Table 1). The estimated broad-sense heritability for this trait was 88.0% (Supplemental Table 3).
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
Chlorophyll content measurements under nonsalt conditions were performed for individual leaves 14 d after salt treatment, and each leaf was tested three time. The SPAD values ranged from 31.80 to 39.01 (average, 34.71; SD, 1.54), and the distribution appeared normal (Fig. 3A). Significant differences were noted among the 71 tomato genotypes (F = 3.49; P < 0.0001) (Supplemental Table 3), with the highest values in genotypes PI 647143 (39.01), PI 647556 (38.54), and PI 647528 (38.35) (Supplemental Table 1). The lowest values were in PI 636277 (33.20) and PI 647486 (32.93). The heritability for chlorophyll content (SPAD value) under nonsalt conditions was estimated at 71.4%.
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
Under salt treatment, the chlorophyll content (SPAD value) varied widely from 12.71 to 22.50 (mean, 15.06; SD, 2.41). This distribution was right-skewed (Fig. 3B), with significant genotypic differences (F = 10.85; P < 0.0001) (Supplemental Tables 1 and 2). The highest values were observed in PI 647556 (22.50) and PI 647143 (22.21), whereas the lowest were in PI 109834 (13.66) and PI 645390 (13.64). The heritability under salt conditions was 90.8%.
The AD_C in the leaf chlorophyll content (SPAD value), calculated by subtracting the nonsalt value from the salt treatment value, ranged from 12.39 to 23.43 cm (average, 19.65; SD, 2.10) (Supplemental Table 2). The distribution was nearly normal (Fig. 4A), with significant genotypic differences (F = 3.92; P < 0.0001). The highest decreases were in genotypes PI 109113 (23.43) and PI 286426 (23.18), whereas the smallest were in PI 636205 (12.39) and PI 634828 (14.23). Heritability for AD_C was estimated at 74.5% (Supplemental Table 3).
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
The inhibition index (II_C), indicating the percentage reduction in the leaf chlorophyll content (SPAD value) after salt treatment compared with that after nonsalt conditions, ranged from 36.38 to 64.22 (average, 56.63; SD, 5.92) (Supplemental Table 2). The distribution was right-skewed (Fig. 4B). Significant differences among genotypes were noted (F = 6.74; P < 0.0001) (Supplemental Table 3). Genotypes like PI 109113 and PI 129033 (64.22) showed high susceptibility, whereas PI 636205 and PI 634828 indicated greater tolerance. The estimated heritability for II_C was 85.2%.
The RST_C quantifies the change in the chlorophyll content (SPAD value) between salt-treated and nontreated plants. The RST_C values ranged from 35.78 to 63.62 (mean, 43.37; SD, 5.92) and showed significant variability and a right-skewed distribution (Fig. 4C, Supplemental Table 2). Notable differences were observed among genotypes 14 d after initial salt exposure (F = 6.74; P < 0.0001) (Supplemental Table 3). Genotypes with the highest RST_C, such as PI 636205 (63.62) and PI 109936 (63.00), demonstrated robustness, whereas those such as PI 645390 (38.52) and PI 128586 (38.51) were less tolerant. The heritability for RST_C was also 85.2%.
Seedling height measurements were recorded for tomato plants under both nonsalt and salt treatments 14 d after salt exposure. Under nonsalt conditions, seedling heights varied between 32.17 and 54.80 cm (mean, 41.7 cm; SD, 4.51 cm), showing an approximately normal distribution (Fig. 5A). Significant genotypic differences were observed (F = 2.93; P < 0.0001) (Supplemental Table 3), with heights ranging from 35.67 cm for PI 109835 to 24.07 cm for PI 647518. Heritability under nonstress conditions was 65.8%.
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
For salt-treated seedlings, heights ranged from 21.53 to 35.67 cm (average, 28.89 cm; SD, 3.48 cm, displaying a near-normal distribution (Fig. 5B). Significant variability was also found (F = 3.25, P < 0.0001) (Supplemental Table 3), with the tallest genotypes such as PI 270236 reaching 54.80 cm, and the shortest genotypes such as PI 647447 reaching 36.57 cm. Heritability under salt treatment was 69.2%.
The AD_SH between nonsalt-treated and salt-treated plants ranged from 8.23 to 28.10 cm (mean, 12.81 cm) and had a right-skewed distribution (Fig. 6A). Significant genotypic differences were noted (F = 5.87; P < 0.0001) (Supplemental Table 3), with high susceptibility indicated by genotypes such as PI 270236 (28.1 cm) and high tolerance indicated by genotypes such as PI 647513.
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
The II_SH, representing the percentage reduction after salt treatment, varied from 22.33% to 51.29% (average, 30.40%) and had a slightly right-skewed distribution (Fig. 6B). Significant differences among genotypes were detected (F = 6.00; P < 0.0001) (Supplemental Table 3). The highest indices, which indicated increased susceptibility, included PI 270236 (51.29%), whereas the lowest, which suggested better tolerance, included PI 129033 (22.33%). The estimated heritability for II_SH was at 83.3%.
The RST_SH for tomato seedlings was assessed by comparing seedling height under salt treatment to that under nonsalt treatment. RST_SH values ranged from 48.71% to 77.67% (mean, 69.59%; SD, 6.68%), indicating significant genotypic variability (F = 6.00; P < 0.0001) (Supplemental Table 3). The distribution was left-skewed (Fig. 6C).
The genotypes that demonstrated the highest RST_SH and, thus, greater salt tolerance, included PI 129033 (77.67%), PI 647532 (77.55%), and PI 109837 (76.44%) (Supplemental Table 1). In contrast, genotypes such as PI 270236 (48.71%) and PI 99782 (53.07%) showed the lowest RST_SH, indicating increased susceptibility to salt stress. The broad-sense heritability for RST_SH was estimated at 83.3% (Supplemental Table 3), underscoring a strong genetic influence on this trait.
A strong positive correlation between the II_C (SPAD value) and the AD_C (SPAD value) was observed (r = 0.92; highly significant P = 5.76E-30), indicating a consistent physiological response to salt stress (Table 2). Similarly, the II_SH and the AD_SH also demonstrated a strong positive correlation (r = 0.92; P = 6.11E-30), highlighting the predictive value of early seedling responses under salt stress (Table 2). Additionally, significant correlations were found between the LIS and both AD_C (r = 0.82; P = 1.09E-18) and II_C (r = 0.94; P = 2.664E-33), suggesting shared physiological mechanisms.
Conversely, weaker correlations were noted between the seedling height and chlorophyll content (SPAD value), with marginal correlations observed between LIS and both AD_SH and II_SHI (r = 0.09 and 0.07, respectively), between AD_C and both AD_SH and II_SH (r = 0.05 and 0.01, respectively), and between II_C and chlorophyll-related parameters (r = 0.04 and 0.02, respectively) (Table 2). These lower correlations, combined with less significant P values, implied that height is a less reliable predictor of salt tolerance, thus reflecting potential influences from external factors (Supplemental Fig. 1).
The correlation among all 11 indexes, including LIS, C_SC_NS, AD_C, II_C, RST_C, SH_S, SH_NS, AD_SH, II_SH, and RST_SH, is shown on Supplemental Table 4.
During the study, several genotypes consistently ranked among the top for salt tolerance across various traits, with genotype PI 109837 standing out because of its exceptional performance. It consistently appeared within the top 10 across traits, securing number 3 in terms of the LIS, AD_C (SPAD value), and II_SH, number 4 in terms of the II_C (SPAD value), and number 9 in terms of the AD_SH (Table 1). The consistent high rankings of PI 109837 across all measured categories underscore its robustness and establish it as a prime candidate for breeding programs aimed at enhancing salt tolerance characteristics in plants.
Furthermore, eight other genotypes—PI 647528, PI 647556, PI 636205, PI 636255, PI 270256, PI 647143, and PI 127820—also showed excellent performance, consistently ranking in the top nine salt-tolerant accessions based on the following three traits: LIS, AD_C, and II_C (Table 1). The repeated high rankings of these genotypes suggest a possible correlation among the traits, indicating a shared genetic mechanism that influences both the chlorophyll content (SPAD value) and leaf injury score under salt stress. These 10 accessions were collectively grouped as cluster I (Fig. 7), reflecting their notable salt tolerance.
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
The 71 tomato accessions were classified into three clusters (I, II, and III) based on five salt tolerance-related traits (LIS, AD_C, II_C, AD_SH, and II_SH) (Fig. 7). Notably, nine accessions—PI 634828, PI 109837, PI 647556, PI 636205, PI 636255, PI 270256, PI 647528, PI 647143, and PI 127820—demonstrated the highest salt tolerance (Supplemental Table 5) and were all placed in cluster I, reflecting similar salt tolerance characteristics. In contrast, clusters II and III grouped together less tolerant accessions, highlighting their greater susceptibility to salt stress.
For the trait analysis, AD_SH and II_SH were grouped together, reflecting a correlation in how seedling height is affected by salt stress. Conversely, AD_C, II_C, and LIS formed another cluster, with LIS and II_C further categorized together, suggesting a closer relationship between the LIS and II_C (SPAD value) under salt conditions, distinct from the impact on seedling height.
The nine salt-tolerant accessions were positioned in different sections of the phylogenetic tree (Fig. 8), indicating a diverse genetic base among these accessions. Furthermore, the unique placement of PI 647556 highlighted its distinctiveness (Fig. 8).
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
The biplot (Fig. 9A) illustrated consistent correlations among AD_C, II_C, and LIS, which clustered together, whereas AD_SH and II_SH were closely associated with each other but distinct from the other three traits. The scree plot (Fig. 9B) and principal component analysis plot (Fig. 9C) further supported the delineation of three clusters among the 71 tomato accessions based on the five salt tolerance-related traits.
Citation: HortScience 60, 1; 10.21273/HORTSCI18149-24
Salt tolerance in crops varies widely because of diverse influencing factors, thus making its comprehensive study complex. This variability extends across environmental conditions, crop cultivars, and developmental stages. As a result, researchers use several indicators to accurately assess salt tolerance that are crucial for identifying resilient cultivars suitable for cultivation and breeding programs. Although there has been progress, a significant knowledge gap remains in the development of salt-tolerant tomato cultivars. The mechanisms through which tomatoes respond to salinity stress and the genetic architecture that could be used for breeding salt-resistant cultivars are not well-understood, highlighting the need for thorough germplasm evaluation. This study addresses this gap by systematically evaluating tomato germplasm and offering critical insights into the differential responses of tomato accessions to salt stress
The evaluation of the LIS in this study clearly demonstrated its effectiveness as a reliable indicator of salt tolerance among tomato genotypes, echoing similar applications in other crops (Ledesma et al. 2016). The considerable variability observed in the LIS reflects the diverse physiological responses to salt stress across different genotypes. Genotypes such as PI 647556 and PI 634828, which exhibited lower LIS, were more resilient to salt stress, suggesting that these genotypes possess robust ion regulation mechanisms to cope with salinity (Ali et al. 2021; Wang et al. 2020). Recent studies support this finding, thus highlighting the role of ion transporters and their influence on salt tolerance in tomatoes and other crops (Wang et al. 2020). These findings underline the potential for selecting genotypes with a lower LIS for breeding programs aimed at improving salt tolerance.
In contrast, genotypes like PI 97538 and PI 128586, with higher LIS, were more vulnerable to salt-induced damage. The high broad-sense heritability (89.2%) of LIS, supported by Khaliluev et al. (2022), reinforces the utility of this trait in breeding programs targeting enhanced salt tolerance. This is further supported by research demonstrating that early-stage physiological traits, including injury scores, are critical indicators for assessing and selecting salt-tolerant cultivars (Choudhury et al. 2023). The left-skewed distribution of scores and the predominance of genotypes with higher tolerance highlight the valuable genetic diversity available for developing resilient tomato cultivars, which was a key finding of Ahsan et al. (2022), who demonstrated the importance of genetic variability in salinity tolerance.
The investigation of the leaf chlorophyll content (SPAD value) in tomato genotypes under salt stress highlights key physiological and genetic dynamics that mirror findings from recent research of plant responses to salinity. Salt stress disrupts ion homeostasis and increases intracellular osmotic pressure, leading to a decline in chlorophyll content (SPAD value). This reduction is largely attributed to the plant’s regulatory adjustments in ion channels and transporters, which help to maintain ionic balance, coupled with the accumulation of osmolytes to mitigate osmotic stress (Zhao et al. 2021). Loudari et al. (2020) similarly found that salt stress induces nutrient imbalances that impair chlorophyll synthesis, further diminishing photosynthetic efficiency. Studies by Munns and Tester (2008) and Negrão et al. (2017) emphasized that salt-induced stress directly impacts photosynthetic pigments, which are critical to maintaining plant growth under saline conditions. The high heritability of the chlorophyll content (SPAD value) under salinity stress underscores the genetic potential for breeding programs focused on salt-tolerant cultivars. Genotypes capable of sustaining higher levels of chlorophyll under stress conditions are particularly valuable for enhancing photosynthetic efficiency and overall growth performance in saline environments. Understanding the molecular and physiological mechanisms governing these traits is essential for the development of robust breeding programs aimed at improving salt tolerance in tomatoes. By identifying and selecting genotypes that sustain higher chlorophyll content (SPAD value) under salt stress, breeders can cultivate cultivars that not only exhibit enhanced photosynthetic efficiency but also show improved growth performance in saline conditions. The high heritability of the chlorophyll content (SPAD value) under salt stress conditions provides a robust foundation for the genetic improvement of tomatoes toward enhanced salt tolerance. This study’s findings underscore the importance of integrating physiological and genetic insights into the development strategies of resilient agricultural varieties.
This study’s exploration of salt tolerance in tomato genotypes at the seedling stage, particularly through variations in seedling height, significantly enriches our understanding of plant responses to salinity stress. The findings corroborate recent research, highlighting the intricate interplay between physiological and genetic factors that shape salt tolerance. Observed variations in seedling height under salt stress reveal the diverse physiological strategies used by different tomato genotypes to cope with salinity. Da Silva Oliveira et al. (2022) have noted that salt stress impacts various growth parameters of tomatoes, including seedling height and yield components. Certain genotypes that have the ability to maintain higher seedling heights under salt stress may be linked to superior ion regulation, particularly in managing sodium and potassium ions, and efficient osmotic adjustment through the accumulation of compatible solutes, as discussed by Alam et al. (2021).
The substantial broad-sense heritability values for seedling height under salt treatments underscore a strong genetic basis for salt tolerance, thus aligning with findings from previous studies. Ali et al. (2021) identified specific introgression lines in tomatoes showing enhanced salt tolerance supported by the upregulation of salt-responsive genes. Additionally, Wang et al. (2020) highlighted the genetic complexity of salt tolerance by identifying a Na+/K+ transporter gene, SlHAK20, as a key player in this trait.
The significant variation in salt tolerance among tomato genotypes observed in this study echoes findings by Ahsan et al. (2022), who reported a wide range of diversity in tomato genotypes under salinity stress. This variability is essential for breeding programs and provides a diverse pool of genotypes with potential salt tolerance traits for breeding salt-tolerant tomato cultivars. Identifying genotypes with high RST based on seedling height offers a practical approach for selecting parent lines in breeding programs. Furthermore, the high heritability of seedling height traits under salt stress conditions, as observed in this study, suggests their effectiveness in breeding programs, as corroborated by Fatima et al. (2022), who evaluated tomato germplasm for salinity tolerance. This study’s insights are instrumental to advancing our strategic breeding approaches to enhance salt tolerance in tomatoes.
A robust positive correlation between the II_SH and the AD_SH in tomato plants under salt stress, supplemented by correlations among various chlorophyll content (SPAD value) measures, was elucidated. These findings echo the observations of Loudari et al. (2020), who noted significant decreases in the chlorophyll content (SPAD value) along with reductions in the shoot and root dry weights under saline conditions, showcasing a consistent physiological response to salinity stress. The strong correlation coefficients suggest that seedling height and chlorophyll content (SPAD value) could potentially serve as reliable predictors of plant responses to salt stress in later growth stages, reflecting a broader understanding of plant adaptations to salinity. However, the study also found weaker correlations between seedling heights and chlorophyll contents (SPAD value), which may be influenced by additional factors impacting plant growth and chlorophyll synthesis under stress. This complexity echoes the findings of Amjad et al. (2014), who explored the role of phytohormones in modulating plant physiology under saline conditions, further underscoring the multifaceted nature of plant responses to environmental stress.
Notably, significant relationships between the salt tolerance LIS and chlorophyll contents (SPAD value) were uncovered, resonating with the findings of Gong et al. (2020), who found a beneficial impact of a halotolerant actinomycete on growth and physiological traits such as the chlorophyll content (SPAD value) in tomato seedlings under salt stress. This relationship suggests that the chlorophyll content (SPAD value) might serve as an effective indicator of salt tolerance in tomato plants; therefore, further investigation is merited. These findings have substantial implications for breeding programs aimed at enhancing salt tolerance in tomato plants. Correlations between early growth indicators, the chlorophyll content (SPAD value), and salt tolerance scores provide valuable insights for selecting genetically resilient varieties. Supporting this perspective, Hernández-Hernández et al. (2018) demonstrated that applying chitosan–PVA and copper nanoparticles enhanced growth and activated stress response genes in tomato plants under salt stress, suggesting a viable strategy for developing salt-tolerant tomato cultivar. Despite these promising insights, this study had some limitations, such as weaker correlations among certain physiological traits, suggesting that other unaccounted factors may also influence plant responses to salt stress. Future research could delve deeper into these aspects and examining the genetic basis of salt tolerance or the influence of additional environmental stressors on plant physiology.
This study’s analysis of the genotype landscape provides insightful revelations about the genetic underpinnings of salt tolerance in tomato plants. The consistently high performance of genotype PI 109837 across multiple traits, such as AD_SH, II_SH, AD_C, II_C, and LIS, underscores a robust genetic constitution that confers broad-spectrum salt tolerance. This observation is in line with contemporary advancements in plant genetics and the study of salt stress responses. García-Abellan et al. (2014) demonstrated that specific gene overexpression in tomatoes leads to reduced shoot Na+ accumulation and enhanced salt tolerance, thus shedding light on potential genetic mechanisms that might underlie the superior performance of PI 109837.
Furthermore, the recurrent appearance of genotypes such as PI 636255, PI 127820, and PI 647528 among the top ranks for the II_C (SPAD value) and LIS indicates a possible shared genetic mechanism. This notion is supported by Yang et al. (2014), who emphasized the role of abscisic acid in tomato salt tolerance, suggesting a potential pathway for further exploration. Additionally, genotypes such as PI 636277 and PI 129026, which have good AD_SH, II_SH, and LIS, suggest a genetic correlation between the seedling height response and overall salt tolerance. Wang et al. (2022) identified genetic variations in an Na+/K+ transporter gene associated with tomato salt tolerance, further supporting the genetic basis of salt tolerance observed in these genotypes.
The diversity in performance among different tomato genotypes, with some excelling in specific traits, suggests varied genetic adaptation mechanisms to salt stress. Research by Kou et al. (2019) explored the salt tolerance mechanism of specific genes in tomatoes, providing a theoretical basis for genetic improvement efforts. This underscores the importance of understanding the genetic basis of salt tolerance to enhance breeding strategies. Moreover, the identification of top-performing cultivars in terms of the RST_C (SPAD value) and LIS, which were not among the best for seedling height traits, suggests that different genetic mechanisms influence these traits. This observation aligns with the findings of Kashyap et al. (2020), who emphasized the importance of comprehensive gene expression studies for understanding tomato salinity stress tolerance mechanisms, highlighting the need for further exploration of the genetic underpinnings of salt tolerance in tomatoes.
The delineation of tomato accessions into genetic clusters, particularly the salt-tolerant cultivars within cluster I, underscores the genetic basis of salt tolerance. This observation resonates with the research by Wang et al. (2022), which highlighted the significance of ion transport genes such as SlHAK20 in the response to salt stress.
The genetic diversity exhibited by the susceptibility of other clusters to salt stress has crucial implications for breeding programs. As emphasized by Kashyap et al. (2020), the intricate hormonal and signaling pathways involved in the salt stress response underscore the complexity of salt tolerance mechanisms.
The differentiation of traits such as the LIS and leaf chlorophyll content (SPAD value) from seedling height offers a nuanced understanding of trait selection for breeding. Ali et al. (2021) demonstrated that specific traits in tomato introgression lines could enhance salt tolerance, suggesting the potential of targeted genetic traits in breeding programs.
The unique positioning of PI 647556 in the phylogenetic analysis unveils genetic diversity among salt-tolerant cultivars, offering valuable insights for breeding strategies. This diversity is pivotal for developing resilient tomato cultivars in response to increasing soil salinization and climate change.
This study assessed the salt tolerance of 71 tomato accessions from the USDA germplasm collection with a focus on enhancing cultivation in salt-affected soils. Nine accessions—PI 109837, PI 127820, PI 270256, PI 634828, PI 636205, PI 636255, PI 647143, PI 647528, and PI 647556—were identified as particularly salt-tolerant, demonstrating significant genetic potential for breeding programs aimed at developing cultivars suited for high-salinity environments. The substantial variation in salt tolerance observed across these accessions highlights the importance of genetic diversity in future breeding efforts. Further research should focus on uncovering the molecular and physiological mechanisms that contribute to salt tolerance in these accessions, which will enable the development of improved tomato cultivars and promote sustainable farming in regions affected by salinity.
(A) Overview of greenhouse experiments for the salt tolerance evaluation of tomato. (B) Leaf injury score (1 = healthy, rich green; 2 = early chlorosis, paler green; 3 = increased chlorosis, fading green; 4 = extensive chlorosis, green and yellow; 5 = complete chlorosis, fully yellow; 6 = initial necrosis, yellow with brown spots; and 7 = widespread necrosis, brown and wilted). (C) Seedling growing under both normal and salt stress conditions. Yellowing of leaves was observed in plants under salt stress.
Distributions of the leaf injury score among 71 tomato genotypes.
Distributions of the leaf chlorophyll content [soil plant analysis development (SPAD)] among 71 tomato genotypes: (A) leaf chlorophyll content under nonsalt treatments (C_NS) and (B) leaf chlorophyll content under salt treatments (C_S) using 200 mM NaCl for 2 weeks.
Distributions of leaf chlorophyll content [soil plant analysis development (SPAD)] among 71 tomato genotypes: (A) absolute decrease in the leaf chlorophyll content (cm) (AD_C); (B) inhibition index for chlorophyll content (%) (II_C); and (C) relative salt tolerance for the chlorophyll content (%) (RST_C).
Distributions of seedling height (cm) among 71 tomato genotypes: (A) seedling height (cm) of plants irrigated with deionized water (SH_NS) and (B) those that were salt-stressed with 200 mM NaCl for 2 weeks (SH_S).
Distributions of parameter values for assessing seedling height reduction upon salt treatment among 71 tomato genotypes: (A) absolute decrease (cm) in seedling height (AD_SH); (B) inhibition index of the seedling height (%) (II_SH); and (C) relative salt tolerance of seedling height (RST_SH).
(A) Two-way dendrogram and (B) constellation plot of 71 tomato accessions according to the hierarchical cluster analysis using JMP Pro 17 based on five salt tolerance-related traits: leaf injury score (LIS), absolute decrease in the leaf chlorophyll content (AD_C), II_C, absolute decrease in seedling height (AD_SH), and inhibition index of the seedling height (II_SH). The top seven salt-tolerant accessions were grouped into one cluster I.
Phylogenetic tree created by MEGA 11 based on 2398 single nucleotide polymorphisms (SNPs) distributed on 12 chromosomes in 71 USDA Germplasm Resources Information Network (GRIN) tomato accessions. The seven tomato accessions with a score <3.0 are marked with red rectangle.
Principal component analysis (PCA) of 71 tomato accessions by JMP Genomics based on five salt tolerance-related traits, leaf injury score (LIS), absolute decrease in the leaf chlorophyll content (AD_C), II_C, absolute decrease in seedling height (AD_SH), and inhibition index of the seedling height (II_SH). (A) Bioplot. (B) Scree plot. (C) PCA with three clusters.
Contributor Notes
I.A. and H.X. contributed equally to this work.
(A) Overview of greenhouse experiments for the salt tolerance evaluation of tomato. (B) Leaf injury score (1 = healthy, rich green; 2 = early chlorosis, paler green; 3 = increased chlorosis, fading green; 4 = extensive chlorosis, green and yellow; 5 = complete chlorosis, fully yellow; 6 = initial necrosis, yellow with brown spots; and 7 = widespread necrosis, brown and wilted). (C) Seedling growing under both normal and salt stress conditions. Yellowing of leaves was observed in plants under salt stress.
Distributions of the leaf injury score among 71 tomato genotypes.
Distributions of the leaf chlorophyll content [soil plant analysis development (SPAD)] among 71 tomato genotypes: (A) leaf chlorophyll content under nonsalt treatments (C_NS) and (B) leaf chlorophyll content under salt treatments (C_S) using 200 mM NaCl for 2 weeks.
Distributions of leaf chlorophyll content [soil plant analysis development (SPAD)] among 71 tomato genotypes: (A) absolute decrease in the leaf chlorophyll content (cm) (AD_C); (B) inhibition index for chlorophyll content (%) (II_C); and (C) relative salt tolerance for the chlorophyll content (%) (RST_C).
Distributions of seedling height (cm) among 71 tomato genotypes: (A) seedling height (cm) of plants irrigated with deionized water (SH_NS) and (B) those that were salt-stressed with 200 mM NaCl for 2 weeks (SH_S).
Distributions of parameter values for assessing seedling height reduction upon salt treatment among 71 tomato genotypes: (A) absolute decrease (cm) in seedling height (AD_SH); (B) inhibition index of the seedling height (%) (II_SH); and (C) relative salt tolerance of seedling height (RST_SH).
(A) Two-way dendrogram and (B) constellation plot of 71 tomato accessions according to the hierarchical cluster analysis using JMP Pro 17 based on five salt tolerance-related traits: leaf injury score (LIS), absolute decrease in the leaf chlorophyll content (AD_C), II_C, absolute decrease in seedling height (AD_SH), and inhibition index of the seedling height (II_SH). The top seven salt-tolerant accessions were grouped into one cluster I.
Phylogenetic tree created by MEGA 11 based on 2398 single nucleotide polymorphisms (SNPs) distributed on 12 chromosomes in 71 USDA Germplasm Resources Information Network (GRIN) tomato accessions. The seven tomato accessions with a score <3.0 are marked with red rectangle.
Principal component analysis (PCA) of 71 tomato accessions by JMP Genomics based on five salt tolerance-related traits, leaf injury score (LIS), absolute decrease in the leaf chlorophyll content (AD_C), II_C, absolute decrease in seedling height (AD_SH), and inhibition index of the seedling height (II_SH). (A) Bioplot. (B) Scree plot. (C) PCA with three clusters.
