Genetic and physiological characteristics of salt-tolerance in nine rice varieties at seedling stage

Saline-alkali land is widespread in China and poses a significant limitation to crop growth. Screening for salt-tolerant crop varieties is a key strategy to enhance the utilization of saline-alkali soils. In this study, 40 radiation-induced wheat-<italic>Thinopyrum intermedium</italic> lines, three conventional wheat varieties, and the wheat-<italic>Th. intermedium</italic> disomic addition line SN6306 were used as experimental materials. Chinese Spring (CS) was used as a salt-sensitive control, and Shanrong 3 (SR3) served as a salt-tolerant control. Phenotypic traits were comprehensively analyzed under hydroponic conditions, and the optimal salt treatment concentrations for both germination and seedling stages were determined. Using multivariate statistical analysis, salt tolerance of the 44 experimental materials was evaluated at the seedling and germination stages. Based on the results, the materials were classified into four salt tolerance levels: five highly salt-tolerant lines (e.g., A275, A051), thirty-one moderately salt-tolerant lines (e.g., A079, A262), seven salt-sensitive lines (e.g., A140, A284), with CS identified as highly sensitive. A stepwise regression model was established using the salt tolerance scores and the relative salt tolerance coefficients of physiological and biomass indices: <italic>D</italic>-value = 0.033-0.050×K⁺-S+0.002×Na⁺-R+0.237×K⁺-R+0.176×GP+0.139×GR-0.005×PC+0.197×SFW+0.088×WCS-0.110×RDW+0.242×SL+0.019×MRL (<italic>R</italic>²= 0.999). Significant indicators for identifying salt-tolerant wheat included potassium ion content in shoots and roots, sodium ion content in roots, germination potential and rate, proline content, seedling fresh weight, water content, root dry weight, seedling height, and maximum root length. This study established a reliable regression model for evaluating wheat salt tolerance and identified both salt-tolerant and salt-sensitive lines, providing valuable genetic resources for further research on the molecular mechanisms of salt tolerance and for breeding salt-tolerant wheat varieties.

Raising crops for dry and saline lands: Challenges and the way forward.

Salinity is one of the main environmental factors influencing rice production. Many genes affecting salt tolerance (ST) have been cloned in rice so far. In the present study, four genes negatively regulating ST, including HST1, LRRK1, STRK2, and PC1, were edited by CRISPR-Cas9 technology in six rice varieties (three in indica (xian) and three in japonica (geng) backgrounds), and three two-gene editing combinations, including hst1-lrrk1, hst1-strk2, and hst1-pc1, were created. All combinations of hst1-pc1, hst1-lrrk1, and hst1-strk2 significantly improved the ST of all the tested materials in both xian and geng backgrounds and had much better ST than single-gene editing lines. The combination of hst1-pc1 had the poorest ST in CH70 and 8TX23 backgrounds but showed almost the same level of ST as the combinations of hst1-strk2 and hst1-lrrk1 in the C199S background for 17 days after salinization, which clearly brought out the background effect on ST and its utilization in ST breeding. As a comparison of the recipient varieties, almost all gene-edited lines except hst1-pc1 in the CH70 background showed significantly reduced grain weight owing to reduced seed setting rate in normal conditions. The hst1-strk2 showed the highest level of ST at the seedling stage and a relatively higher grain yield among all the lines; thus, it is feasible to enhance the ST of high-yielding rice varieties by simultaneously gene-editing against the two loci or pyramiding these two alleles with the other major ST genes of rice. Our results provide valuable gene resources and germplasms for improving rice salt tolerance and high yield.

Physiological and biochemical parameters for evaluation and clustering of rice cultivars differing in salt tolerance at seedling stage

The objectives of this study were to select Thai rice that are tolerant at the seedling stage and investigate their growth, physiological and yield responses at the reproductive stage in comparison with the standard salt-tolerant Pokkali (PK). Twenty-two local, commercial and improved Thai rice genotypes along with PK and salt-susceptible IR29 were evaluated at the seedling stage for salt tolerance using a 15 dS m−1 saline solution with five replications. Ten selected genotypes were grown in pots with four replications and exposed to a 15 dS m−1 saline level from early booting to the flowering stage. During the grain-filling stage, salt treatments decreased from 15 to 12 dS m−1 and were sustained at this level until harvest. The experimental design for both experiments was a randomized complete block design (RCBD). Based on the physiology of flag leaves, almost all genotypes exceled in the protection of chlorophyll, relative water content (RWC), membrane integrity and lipid peroxidation. In contrast, the photosynthesis, growth and grain yield of all were dramatically reduced. The rice genotypes exhibited varying degrees of osmotic adjustment (OA), ranging from 1.598 to 2.541 MPa. The cultivar RD73 and line TSKC1–144, which were genetically improved from KDML105 by the introgression of a salt-tolerant QTL/gene from PK, showed the least reduction in grain yield (60 and 53% reduction, respectively) along with PK (60%). Among the five Thai local rice varieties, Go Main Surin (GMS) showed the least reduction in grain weight (58%), total plant dry weight (28%) and green leaf dry weight (1%), while Khao Gaew (KG) and Leuang Puang Tawng (LPT) were the most reduced. PK and RD73 showed a high level of tolerance at both the seedling and reproductive stages. In contrast, KG and LPT, which exhibited high tolerance at the seedling stage, showed high susceptibility in growth, yield and most physiological traits. On the other hand, TSKC1–144 was sensitive at the seedling stage but showed increased tolerance at the reproductive stage. This result implies that suitable cultural practices should be performed to obtain the best field conditions for growing rice genotypes having different levels of salt tolerance at the seedling and reproductive stages. Future research should focus on molecular characterization of tolerance mechanisms of the promising local genotypes and the potential to use them as tolerance gene donors.

Changes in the antioxidant enzymes, lipid peroxidation, sodium and potassium, chlorophyll, H2O2 and proline content were monitored in the leaves of 42 rice varieties which were not yet well-documented for the salinity tolerance under different salinity levels. The tolerant varieties (FL478, Hassani, Shahpasand, Gharib and Nemat) showed signs of tolerance (lower Na+/K+ ratio, high proline accumulation, less membrane damage, lower H2O2 production, and higher superoxide dismutase and catalase activity) very well. The positive relationship between the level of salt tolerance and the amount of proline accumulation in the rice varieties support the important role of proline under the salt stress. The varieties were genotyped for 12 microsatellite markers that were closely linked to SalTol QTL. The results of association analysis indicated that RM1287, RM8094, RM3412 and AP3206 markers had the high value of R2 for the regression models of the studied traits. It shows the important role of SalTol in controlling physio-biochemical traits. The results can be used in the future marker assisted selection (MAS) directly, if the results are confirmed.

Salt and heavy metal tolerance and expression levels of candidate tolerance genes among four extremophile Cochlearia species with contrasting habitat preferences.

Turfgrasses show a wide range of salinity tolerance. In this study, twenty wild turfgrasses were collected from coastal regions in Japan, and their species; evolutionary lineage; salt tolerance levels; shoot and root K+, Na+, and proline contents; and amounts of ions secreted from their salt glands were determined. Among them, eighteen turfgrass species were determined based on the internal transcribed spacer 1 sequences. All collected wild turfgrasses were identified as halophytes and were divided into two salt-tolerant levels. They maintained the shoot relative water contents and suppressed excess Na+ accumulation in their shoots and roots and K+ content homeostasis compared with rice, resulting in the maintenance of a higher K+/Na+ ratio under salt stress. These characteristics must be part of the salt tolerance mechanisms. Among the four turfgrasses with salt glands, three selectively secreted Na+ from their salt glands; however, interestingly, one secreted K+ over Na+, although it still maintained a K+/Na+ ratio comparable to that of the other turfgrasses. A significant amount of proline synthesis was observed in most of the turfgrasses in response to salt stress, and the proline content was highly correlated with the salt tolerance, suggesting its key role in the salt tolerance mechanisms. These wild turfgrasses with such diverse ion control mechanisms and proline synthesis profiles are useful materials for investigating the salt tolerant mechanisms and breeding salt tolerant turfgrasses.

Response of broomcorn millet (Panicum miliaceum L.) genotypes from semiarid regions of China to salt stress

Genetic diversity is important for developing salt-tolerant rice varieties. This research used the existing whole-exome sequences of eight Thai rice varieties, including the standard checks for tolerance and susceptibility, and the previously reported salt-tolerant genes to understand the mechanisms of salt tolerance in Thai rice, as well as the bulk-segregant analysis (BSA) to identify salt-tolerant QTLs in ‘Jao Khao’, the salt tolerant Thai rice. Salt responsive characteristics were used to classify tolerant and susceptible genotypes at the seedling stage. Phylogenetic analysis of SNPs from exome sequences showed that the Thai rice varieties were monophyletic and distantly related to 'Pokkali' and IR29. 'Lai Mahk' and 'Luang Pratahn', which were characterized as Thai salt tolerant varieties, were closely related based on either the exome SNPs or SNPs from 164 salt tolerant genes. Genomic regions conferring salt tolerance traits were identified in an F2 population of the Thai landrace salt tolerant variety, 'Jao Khao', and the salt susceptible line, IR29, via BSA. In total, 600 F2 individuals were phenotyped in the hydroponic system. The 14-day old seedlings were treated with salt stress in a stepwise manner to reach 12 dS.m-1 for 12 days. The F2 members showed a high level of distribution in the standard salt injury evaluation system (SES), cell membrane stability (CMS), relative water content (RWC), whole plant fresh weight (WFW), plant height (PH), and root length (RL). SES showed a strong negative correlation with CMS, RWC, and WFW, but it showed less of a negative correlation with PH and RL. The BSA predicted 4 QTLs harboring the putative salt-tolerant regions on chromosome 3 (CMS trait) and chromosomes 2, 6, and 12 (RWC trait). Three of the identified QTLs overlapped with some reported salt-tolerant QTLs, while the QTLs on chromosome 6 overlapped with drought-tolerant QTLs. The validation of the QTLs was carried out by designing four molecular markers. Their possible involvement in salt tolerance was assessed in an F3 population. The findings revealed that F3 individuals with the ‘Jao Khao’ allele were more tolerant to salt stress than the ones with the allele from IR29 or containing the heterozygous allele, indicating that these QTLs are important in salt tolerance. In the future, the 4 salt stress-tolerant Thai rice varieties can be employed in rice breeding. Moreover, 4 salt tolerant QTLs identified in the study will be used to breed salt tolerant rice, and the developed genetic markers can be used in the marker assisted-breeding program.

Rice is a staple food for more than half of the world’s population. However, the pervasive problem of salinity is severely undermining rice production, especially in coastal and low-lying areas where soil salinization is widespread. This stress, exacerbated by climate change, necessitates the development of salt-tolerant rice varieties to ensure food security. In this study, an F2:3 population (n = 454) from a cross of KDML105 and its chromosome segment substitution line (CSSL) was used to identify genomic regions associated with salt tolerance at the seedling stage. Using the QTL-seq approach, a QTL significantly associated with salt tolerance was identified on chromosome 1. Annotation of candidate genes in this region revealed the potential regulators of salt tolerance, including MIKC-type MADS domain proteins, calmodulin-binding transcription factors, and NB-ARC domain-containing proteins. These and other identified genes provide insights into the genetic basis of salt tolerance. This study underscores the importance of using advanced genomics tools and CSSL populations in the study of complex traits such as salt tolerance in rice. Several candidate genes identified in this study could be used in further studies on molecular or physiological mechanisms related to the salt response and tolerance mechanism in rice. Additionally, these genes could also be utilized in plant breeding programs for salt tolerance.

Genetic diversity is important for developing salt-tolerant rice (Oryza sativa L.) cultivars. Certain Thai rice accessions display salt tolerance at the adult or reproductive stage, but their response to salinity at the seedling stage is unknown. In this study, a total of 10 rice cultivars/line, including eight Thai cultivars and standard salt-tolerant cultivar and susceptible line, were screened using a hydroponic system to identify salt-tolerant genotypes at the seedling stage. Different morphophysiological indicators were used to classify tolerant and susceptible genotypes. Phylogenetic analyses were performed to determine the evolutionary relationships between the cultivars. Results showed that 'Lai Mahk', 'Jao Khao', 'Luang Pratahn', and 'Ma Gawk' exhibited salt stress tolerance comparable with the standard salt-tolerance check 'Pokkali'. Whole-exome single-nucleotide polymorphism (SNP)-based phylogenetic analysis showed that the Thai rice cultivars were monophyletic and distantly related to Pokkali and IR29. Lai Mahk and Luang Pratahn were found closely related when using the whole-exome SNPs for the analysis. This is also the case for the analysis of SNPs from 164 salt-tolerance genes and transcription regulatory genes. The tolerant cultivars shared the same haplotype for 16 genes. Overall, the findings of this study identified four salt-stress-tolerant Thai rice cultivars, which could be used in rice breeding programs for salinity tolerance.

Effects of NaCl on the growth, ion content, root cap structure and Casparian band development were examined in four rice (Oryza sativa L.) cultivars with different salt resistance (salt-sensitive indica-type IR 24 and japonica-type Nipponbare and salt-resistant indica-type Nona Bokra and Pokkali). Experiments were conducted to find the differences in salinity resistance during early seedling and developed seedling stages among the cultivars. For salinity treatment, sodium chloride (NaCl) was added to nutrient solution at concentrations of 0, 25 and 50 mM for 7 days from germination to the 7th day (early seedling stage) or from the 7th day to 14th day (developed seedling stage). Growth inhibition by salinity was more prominent in the early seedling stage than in the developed seedling stage. Based on the growth, the order of the sensitivity was IR24 > Nipponbare > Nona Bokra > Pokkali. The growth of NaCl-treated rice cultivars relative to control was significantly and negatively correlated with the Na+ content and Na+/K+ ratio in roots and shoots in both stages. Scanning electron microscopic observation revealed that the root cap tissues proliferated and extended to the basal part of the root tip by salinity. The length of root cap was, however, reduced by 50 mM NaCl in sensitive cultivars due to peeling off. An endodermal Casparian band was formed in the basal region of the root tip. Development of the Casparian band was more prominent in sensitive cultivars than in tolerant cultivars. Root cap proliferation might be related to NaCl resistance in rice seedlings, but the Casparian band may not function efficiently in Na+ exclusion. Essentially the present results suggest that exclusion of Na+ from roots plays a critical role in expression of Na+ resistance in rice seedlings and the root cap is important for Na+ exclusion.

Salt tolerance in cotton is highly imperative for improvement in the response to decreasing farmland and soil salinization. However, little is known about the genetic basis underlying salt tolerance in cotton, especially the seedling stage. In this study, we evaluated two salt-tolerance-related traits of a natural population comprising 713 upland cotton (Gossypium hirsutum L.) accessions worldwide at the seedling stage and performed a genome-wide association study (GWAS) to identify marker-trait associations under salt stress using the Illumina Infinium CottonSNP63K array. A total of 23 single nucleotide polymorphisms (SNPs) that represented seven genomic regions on chromosomes A01, A10, D02, D08, D09, D10, and D11 were significantly associated with the two salt-tolerance-related traits, relative survival rate (RSR) and salt tolerance level (STL). Of these, the two SNPs i46598Gh and i47388Gh on D09 were simultaneously associated with the two traits. Based on all loci, we screened 280 possible candidate genes showing different expression levels under salt stress. Most of these genes were involved in transcription factors, transporters and enzymes and were previously reported as being involved in plant salt tolerance, such as NAC, MYB, NXH, WD40, CDPK, LEA, and CIPK. We further validated six putative candidate genes by qRT-PCR and found a differential expression level between salt-tolerant and salt-sensitive varieties. Our findings provide valuable information for enhancing the understanding of complicated mechanisms of salt tolerance in G. hirsutum seedlings and cotton salt tolerance breeding by molecular marker-assisted selection.

BackgroundSaline land in coastal areas has great potential for crop cultivation. Improving salt tolerance in rice is a key to expanding the available area for its growth and thus improving global food security. Seed priming with salt (halopriming) can enhance plant growth and decrease saline intolerance under salt stress conditions during the subsequent seedling stage. However, there is little known about rice defense mechanisms against salinity at seedling stages after seed halopriming treatment. This study focused on the effect of seed halopriming treatment on salinity tolerance in a susceptible cultivar, IR 64, a resistant cultivar, Pokkali, and two pigmented rice cultivars, Merah Kalimantan Selatan (Merah Kalsel) and Cempo Ireng Pendek (CI Pendek). We grew these cultivars in hydroponic culture, with and without halopriming at the seed stage, under either non-salt or salt stress conditions during the seedling stage.ResultsThe SES scoring assessment showed that the level of salinity tolerance in susceptible cultivar, IR 64, and moderate cultivar, Merah Kalsel, improved after seed halopriming treatment. Furthermore, seed halopriming improved the growth performance of IR 64 and Merah Kalsel rice seedlings. Quantitative PCR revealed that seed halopriming induced expression of the OsNHX1 and OsHKT1 genes in susceptible rice cultivar, IR 64 and Merah Kalsel thereby increasing the level of resistance to salinity. The expression levels of OsSOS1 and OsHKT1 genes in resistant cultivar, Pokkali, also increased but there was no affect on the level of salinity tolerance. On the contrary, seed halopriming decreased the expression level of OsSOS1 genes in pigmented rice cultivar, CI Pendek, but did not affect the level of salinity tolerance. The transporter gene expression induction significantly improved salinity tolerance in salinity-susceptible rice, IR 64, and moderately tolerant rice cultivar, Merah Kalsel. Induction of expression of the OsNHX1 and OsHKT1 genes in susceptible rice, IR 64, after halopriming seed treatment balances the osmotic pressure and prevents the accumulation of toxic concentrations of Na+, resulting in tolerance to salinity stress.ConclusionThese results suggest that seed halopriming can improve salinity tolerance of salinity-susceptible and moderately tolerant rice cultivars.