Five varieties: IR29, Horkuch, I-14, I-71 and Pokkali were selected based on their functional response towards salinity. Horkuch from the coastal region (Lisa et al., 2011) and Pokkali have shown tolerance to salt condition (Xie et al., 2000). IR29 is a modern rice variety from IRRI which is highly susceptible to salinity. I-14 and I-71 are Recombinant Inbred Lines of the F₇ generation from the reciprocal crosses between IR29 and Horkuch (Haque et al., 2022). I-14 behaves more like Horkuch in terms of maintaining K⁺/Na⁺ homeostasis as Horkuch is the donor plant of the Potassium-QTL on chromosome 3 for I-14. On the contrary, in most cases I-71 behaves more like IR29 as IR29 is the donor plant of Potassium-QTL for I-71. BRRI dhan28 (http://dhcrop.bsmrau.net/rice-variety-brri-dhan-28/), a salt sensitive high-yielding modern variety of rice was used to overexpress the TPKa gene. BRRI dhan28 was used for transformation because it is a commercially popular variety liked by farmers. Moreover, it is genotypically very similar to IR29 (Rahman et al., 2019).

As the genome of Horkuch and IR29 is unannotated, the gene sequences are not available in rice databases. To extract the sequence from raw data of Horkuch and IR29 genome, (Unpublished work, Zeba I. Seraj) local BLAST was performed. In the BLAST program, the reference sequence of Oryza sativa Nipponbare was used as a reference to detect the chromosomal location of specific sequences of Horkuch and IR29. For the extraction of the sequences and the CDS (coding sequence), 5′ UTRs (5′-untranslated regions), 3′ UTRs (3′-untranslated regions) of candidate genes from salt-tolerant Horkuch and salt-sensitive IR29 transcriptome was extracted. Expasy’s translate tool (https://web.expasy.org/translate/) was used to translate and predict amino acid sequences of the genes. To find out the functional differences of candidate protein between Horkuch and IR29, Provean (http://provean.jcvi.org/seq_submit.php) and TMHMM (http://www.cbs.dtu.dk/services/TMHMM/) and for detection of putative motif in promoter regions of candidate gene PLACE (http://www.dna.affrc.go.jp/PLACE/) was used.

Total RNA was extracted from leaf and root of 18-day old seedlings after 0, 12, 24, 36, 48 and 72 h of 150 mM NaCl stress using TRIZOL reagent (Invitrogen, Carlsbad, CA, USA). Total RNA concentration and purity was determined using a Nanodrop ND1000 spectrophotometer (Thermo SCIENTIFIC). The cDNA was synthesized from 1.5μg of total extracted RNA using the Invitrogen Superscript III reverse transcription (RT)-PCR following the manufacturer’s protocol (Invitrogen, USA) in a final volume of 20μl. The final cDNA products of 1500-1700 ng/µl concentration were diluted 4-fold prior to use in real-time PCR.

Primers for qRT-PCR were designed using Primer3web program (http://bioinfo.ut.ee/primer3). The primers are listed in Supplementary Table 1 . In quantitative Real-Time PCR analysis, a total of 3 replicates for all the samples of both leaf and root were used. As a control for genomic DNA contamination, an equivalent amount of total RNA without reverse transcription was tested for each sample per gene. Quantitative PCR was done by a 6μl reaction using SYBR Green (Bio-Rad, USA) with gene-specific primers in CFX96 TM Real-Time PCR detection system (Bio-Rad, USA). Using the comparative cycle threshold method, the relative abundance of transcripts was calculated. Elongation Factor- α (EF- α) was used as the normalization control. The thermal profile of the reaction was 95°C for 3 min activation and denaturation, followed by 41 cycles of 95°C for 10 sec, and 62°C for 1 min. At, 62°C step the amplification was determined by SYBR Green. Finally, a dissociation curve was generated by increasing temperature starting from 65 to 95°C to create the melting temperature curve for amplicon validation. For statistical analysis mean variables of gene expression between different genotypes and at different time points were compared with the two-way analysis of variance (ANOVA) followed by Tukey’s HSD post hoc test. The p < 0.05 and p < 0.01 were considered as significant and highly significant change with respect to control.

Two single sgRNA were designed against OsTPKa (LOC_Os03g54100) and OsHAK_like (LOC_Os03g55370) using CRISPR-P web base resource (http://crispr.hzau.edu.cn/CRISPR2/) (Xie et al., 2014). The PAM sequence was set to be 5´-NGG-3´ (SpCas9 from Streptococcus pyogenes). The guide sequence length was defined to be 20 nucleotides and the locus tag of the above-mentioned genes were also provided. In the preliminary step, the sgRNA sequences ( Supplementary Table 2 ) with an on-score of greater than 0.05, a GC content between (30-80%) and a lower off-target were chosen.

pRGEB32 plant binary expression vector was obtained from Addgene (Addgene plasmid # 63142) (Xie et al., 2015) which contains rice snoRNA U3 (Os U3p) promoter and UBI promoter (UBIp) for simultaneous sgRNA and Cas9 expression respectively. The 20 bp long multiple cloning site was removed by BsaI restriction enzyme and the designed sgRNA was inserted into the vector by restriction, digestion and ligation reaction according to provider’s instructions (Xie et al., 2014). The sgRNA_pRGEB32 constructs were transformed into E.coli using heat shock transformation.

Specific sgRNA inserted plasmids were isolated from the E.coli colonies using Promega plasmid isolation kit protocol. Then the plasmids were confirmed by PCR reaction using Primer sets 4, 5, 6, 7 ( Supplementary Table 1 ). PCR analyses were carried out in a 25 μl reaction mixture containing 100 ng of plant DNA, 100 μM of each dNTP, 2.4 ng each of primers, 1 unit of Taq DNA polymerase (Invitrogen, USA), 1.5 mM MgCl₂, 2.4% DMSO and 1 × PCR Buffer(-MgCl₂) (Invitrogen, USA). The optimized reaction was: Initial denaturation at 95°C for 5 min, 35 cycles of denaturation at 95°C for 1 min, annealing at 62.5°C for 1 min and extension at 72°C for 1 min following a final extension at 72°C for 10 min. The PCR-positive colonies were sequenced using primer sets-8 ( Supplementary Table 1 ) by First Base DNA sequencing services from Malaysia.

The pRGEB32_sgRNA vector was transformed into Agrobacterium tumefaciens strain LBA4404 by applying standard protocols (Sambrook et al., 1989). The insertions were also confirmed by gene specific PCR. Transformed Agrobacterium strain (LBA4404) containing specific sgRNA construct was cultured and prepared for in planta transformation by following the standard protocol discussed in Parvin et al. (2015) and adapted according to Ahmed et al. (2018). Following the protocols mentioned by Amin et al. (2012), mature seeds of Horkuch were sterilized and two-days-old, germinated seeds were used for Agrobacterium inoculation. During the transformation events, each time embryonic apical meristem of germinated seeds of Horkuch were infected with the transformed Agrobacterium strain. Infected seeds were then incubated in the dark at 28°C for 6–7 days. Later, the seedlings were treated with carbenicillin solution (250 mg/l) for 1 hour to kill and remove the remnants of Agrobacterium. After that, seedlings were kept in light for 16 hours and in dark for 8 hours at 28°C.

When the seedlings turned green, these were transferred to hydroponic solution. After 2-3 days, the hydroponic pots were transferred to a net-house. When the seedlings were 15-20 days old, they were transferred to soil and allowed to grow and set seeds.

For the hygromycin resistant assay, the plant leaves were cut into 3-6 pieces with a length of about 2 cm at the heading stage and then immediately soaked in Petri dish containing selected solution (20 mg/L hygromycin). The plates were incubated under both dark and light condition (16h day/8 h night) at 25± 2°C.

To evaluate the stress tolerance potential of a plant, leaf disc senescence (LDS) assay was done after the maturation of plants. Flag leaf of around 1.0-cm dimension were cut from fully developed T₀ transgenic lines and wild type (WT) plants and then allowed to float in a 20 mL of 150 mM NaCl solution (Sanan-Mishra et al., 2005) for 3-7 days at 25°C temperature. There was no added K⁺ or K⁺ adjustment.

To compare the germination level under salinity stress, both the non-transformed and OsTPKa_sgRNA and OsHAK_like_sgRNA transformed Horkuch seeds were subjected to 100 mM salt-stress at germination. After 8 days, shoot and root length were measured and compared.

The salt-stress screening of non-transformed and sgRNA transformed Horkuch was done according to the protocol described by Amin et al. (2012). 50mM salt-stress was applied to the 14 days old seedling with a gradual increment of 50 mM up to 150 mM stress. After 9 days of salt application the samples were collected and different parameters like electrolyte leakage, H₂O₂ concentration, chlorophyll content, root, shoot length, and weight were measured. These protocols were followed for salt stress screening of the overexpressed TPKa transgenic lines as well with gradual increment of 30 mM up to 120 mM salt stress.

Total RNA was isolated from 16 days-old salt (NaCl) stressed (100 mM) O. sativa cv. Horkuch using Trizol and first strand cDNA was synthesized using oligodT following manufacturer’s protocol (Invitrogen, USA). PCR was performed to amplify the target sequences of OsTPKa. Primer set 9 were designed for the whole fragments (1.0 kb) of OsTPKa ( Supplementary Table 1 ). The forward primers were designed adding CACC overhang at the 5′ end to ensure correct orientation while cloning into the entry vector (pENTR-D-TOPO). PCR reactions for the target fragment was performed at 95°C for 5 min, 35 cycles of 1 min at 95°C, 30 s at 61°C, 2 min at 72°C followed by a final extension of 10 min at 72°C. The resulting fragments were gel purified using Qiagen Gel Purification system and later cloned to pENTR-D-TOPO vector following the manufacturer’s protocol (Invitrogen, USA). Positive clones were initially selected based on the lysate PCR confirmation from kanamycin resistant clones of transformed E. coli cells. Plasmid isolation was done from confirmed colonies using Promega plasmid isolation kit (Promega, USA). Isolated plasmids were digested with PstI to check the insertion of the desired fragments into the entry vector. Then, digestion-positive colonies were sequenced with M13 forward and reverse primers Primer set 12 by First Base DNA sequencing services from Malaysia. Clones with the confirmed sequences were then recombined with the destination vector (pH7WG2.0) by LR reaction (Invitrogen, USA) (Karimi et al., 2002). Positive clones were selected by gene specific PCR and restriction digestion. The constructs were then transformed into Agrobacterium and used to infect the embryonic apical meristem of germinated seeds of salt-sensitive variety BRRI dhan28 using in planta transformation method as described above.

No sequence variation in CDS (Coding Sequence) and amino acid sequence was found but a significant variation was observed for both genes in the promoter region between the tolerant and sensitive genotypes which may result in variation in the expression of these genes.

For functional characterization of the differences in the promoter region, promoter sequences isolated from salt tolerant Horkuch and sensitive IR29 were scanned using the PLACE (Higo et al., 1999) programs. For OsTPKa, differences in the pattern of motifs between these two varieties were observed, among which auxin, jasmonate, abscisic acid, water stress, and dehydration responsiveness motifs were found in higher amounts in tolerant Horkuch whereas a negative regulatory element for the inducible expression of WRKY18 was present in sensitive IR29. Similarly, for OsHAK_like promoter, auxin and dehydration responsive motifs were more abundant in Horkuch. Additionally, tissue-specific expression related motifs and gibberellin-responsive motif was found to be present in Horkuch but absent in IR29. As shown below, salt stress induces increased expression of OsTPKa and OsHAK_like in the root of the tolerant genotype.

The expression pattern of OsTPKa and OsHAK_like was analyzed in leaf and root tissue of IR29, Horkuch, I-14, I-71 and Pokkali at 24 hours under 150 mM salt stress condition. Both genes showed higher expression in the root compared to the leaf region in all the genotypes. In addition, the expression in root was found to be much higher in salt-tolerant Horkuch, Pokkali and I-14 compared to IR29 under salt-stress ( Figures 1A, B )

For a more detailed understanding of the differences in the expression pattern of OsTPKa and OsHAK_like gene between tolerant and sensitive genotypes, differential gene expression analysis was carried out at different times in both leaf and root of IR29 and Horkuch in 150 mM salt stress.

In leaf samples, for both OsTPKa and OsHAK_like genes no significant difference was observed between tolerant Horkuch and sensitive IR29. However, in the roots of IR29, OsTPKa showed a significant reduction in expression level at various time points under salt stress compared to non-stress, whereas a significant escalation in the expression was observed in Horkuch ( Figure 1C ). Similarly, OsHAK_like showed a reduction in expression level at all the observed time points under salt stress conditions compared to control in IR29. But for Horkuch, there was a gradual increase in expression compared to control at all time points under salt stress ( Figure 1D ).

The expression of OsTPKa significantly increased up to 24 hours under salt compared to OsHAK_like, followed by a sharp reduction in the next 48 hours. In contrast, there was a gradual increase in expression of OsHAK_like and it was significantly higher than that of OsTPKa at 48 and 72 hours of salt stress, indicating that OsTPKa and OsHAK_like may work in a coordinated pattern in Horkuch to provide salt tolerance ( Figure 2A ). In IR29, no such relationship was observed ( Figure 2B ). A proposed mechanism of how the coordinated function might help Horkuch to fight saline stress has been discussed ( Figure 3 ).

The expression of OsTPKa and OsHAK_like is upregulated in tolerant Horkuch under salt-stress. Therefore, to ascertain the role of these two genes in conferring tolerance to Horkuch, a loss of function CRISPR/Cas9 mediated mutagenesis of OsTPKa and OsHAK_like was done in Horkuch. Successful transformation was achieved and is shown in Supplementary Figures 1, 2 .

Transformation was confirmed by hygromycin resistant assay which was used as the selection marker. Due to absence of hygromycin resistant gene in wild type plant, leaf pieces from the positive transformed lines remained green, whereas the wild type ones turned yellow ( Figure 4A ). The transformation rate was 64.63% and 69.83% for OsTPKa and OsHAK_like respectively. For double confirmation, we performed PCR amplification of the hygromycin positive lines using the primer sets designed to specifically amplify Cas9 and hptII sequences. 77.35% and 75% of the hygromycin positive lines contained the targeted sequences for OsTPKa and OsHAK_like respectively ( Figure 4B ).

Phenotypical assessment was performed at the T₀ stage using leaf disc senescence assay. Flag leaf pieces of non-inoculated Horkuch plants (wild type) remain mostly green after 5 days in salt solution. However, about 68% of OsTPKa_sgRNA and 63% of OsHAK_like_sgRNA transformed lines became almost yellow indicating sensitivity in salt ( Figures 4C ).

The lines which performed poorly in LDS assay were selected for the salt-stress screening at the seedling stage. The 21-day-old seedlings of transformed Horkuch and wild type were exposed to 150 mM salt-stress for OsTPKa_sgRNA ( Figure 5A ), where more than 70% of the transformed plants performed poorly, showing yellowing and wilting. For the OsHAK_like_sgRNA ( Figure 5B ) about 55% resulted in visually wilted, rolled, and yellow leaves after 9 days. The score for seedling injury (SES score) was recorded after 9 days. The transformed plants had higher SES score compared to wild type plants ( Figures 5C, D ). The lines showed other salt-sensitive phenotypes with respect to several physiological parameters.

After 8 days of 100 mM salt-stress, the transgenic T₁ seeds showed higher sensitivity, poor germination rate, and significantly decreased shoot length for both OsTPKa ( Figures 6A, C ) and OsHAK_like ( Figures 6B, D ) compared to wild type indicating the loss in tolerance of transgenic Horkuch.

After 9 days of salt stress, it was observed that the percent increase in both electrolyte leakage and H₂O₂ content was significantly higher for 6 OsTPKa and 5 OsHAK_like mutated lines compared to wild type indicating stress injury ( Figures 7A –D ).

The percent increase in root length and weight was significantly lower whereas the percent reduction in shoot length and weight was higher in the mutated lines compared to wild type Horkuch indicating severe salt susceptibility ( Figures 8A –D ).

The chlorophyll content was greatly reduced in the transgenic lines compared to wild type under 150 mM salt-stress indicating mutation of these genes makes a tolerant plant susceptible to chlorophyll degradation under salt stress ( Figures 9A, B ).

The percentage increase in Na⁺/K⁺ ratio was higher in transgenic lines in comparison with wild type under salt stress which indicates that the lowering of OsTPKa and OsHAK_like gene expression disrupts the cytosolic balance of Na⁺/K⁺ ( Figures 9C –F )

OsTPKa gene of Horkuch was selected for cloning into the salt sensitive modern high-yielding farmer popular genotype BRRI dhan28 to test for gain of function. Molecular confirmation of the transformed plants is provided in the supplementary section ( Supplementary Figure 3 ). The transformed plants showed enhanced growth and improved phenology at the T₀ generation, compared to the wild type plants, both under 80 mM salt stress as well as without stress ( Supplementary Figure 4A ). Significant plant growth was observed in the putative transgenics in 9 days old plants but the difference between wild type and transgenics’ height reduced over subsequent days ( Supplementary Figure 4B ). In terms of effective tiller, panicle length, flag leaf length, filled grain number and filled grain weight, the transgenic plants performed better compared to the wild type plant without stress. Transgenic status was confirmed with PCR using hpt gene primers from the vector construct. Leaf Disc Senescence (LDS) assay from T₀ plant flag leaves under 150 mM salt stress and PCR positive results were used to select T₁ seeds from specific panicles for generation advancement. Leaf sections that showed greener texture under salt stress for 3 days compared to the wild type, were considered positive.

Phenotypic screening of T₁ plants at seedling stage under 120 mM salt stress showed lower SES (salt injury), higher survival rate, lesser reduction in shoot and root length as well as shoot and root biomass ( Figures 10A , B , 11A –D ). Leaf tissue measurements of peroxide, reduction in chlorophyll content and electrolyte leakage, were found to be less but not significantly so in the transgenic plants compared to the wild type. However, in the putative transgenic plants’ shoots, both wild type and transgenic plants showed increase in K⁺/Na⁺ ratio under salt stress but transgenic lines showed significant increase compared to no stress condition ( Figure 11 E ). In root tissue, a significant reduction in K⁺/Na⁺ ratio was observed in the wild type plants under salt stress, which was opposite in case of the transgenic lines, specifically a higher increase was observed in line 39 ( Figure 11F ).

Relative expression analysis of the TPKa gene in the overexpresser T₁ plants RNA at seedling stage also showed higher expression of the potassium channel genes in transgenic lines 3, 21 and 39 under 150 mM salt stress for 24 hr ( Supplementary Figure 5 ) compared to the wild type plants. LDS assay in 3 replicates of the T₁ plant flag leaves also confirmed better performance of the transgenic plants ( Supplementary Figure 6 ). The selected transgenic plants are being advanced to T₂ generation and will be followed up in subsequent generations for their performance.

In the study of In silico promoter analysis, for both OsTPKa and OsHAK_like, there were significant differences in stress-related cis regulatory element in promoter region such as- presence of higher number of Auxin, Jasmonate, Gibberellin, Abscisic acid responsive and dehydration responsive motifs in Horkuch. Phytohormones like auxin, jasmonate, gibberellin, abscisic acid regulates plant growth adaptation under salt stress. Additionally, these hormones help plant build a defense system to fight against stress (Yu et al., 2020). Therefore, the presence of these motifs in higher number might account for the greater responsiveness of those genes to salt-stress stimuli in the tolerant rice landrace.

In the tissue specific expression analysis study ( Figure 1 ) and salt-responsive genotype specific expression analysis study ( Figure 2 ), OsTPKa and OsHAK_like, a hypothetical protein of HAK (High affinity K⁺ transporter) family were found to be upregulated in roots of the salt-tolerant Horkuch, Pokkali and I-14 compared to sensitive IR29, indicating that the increased expression of these 2 genes is perhaps important for the performance of tolerant varieties under saline conditions. During the onset of salt stress, the rapid increase in Ca²⁺ may lead to compensatory K⁺ efflux into cytoplasm from the vacuole through activation of TPK channels to replenish stress-induced cytosolic K⁺ depletion (Latz et al., 2007). So, the ideal scenario is to increase K⁺ selective TPK activity (Wu et al., 2018). Again, HAK is the only K⁺ uptake transporter that is known to operate in K⁺ uptake from soil at external concentrations well below 10 µM and this characteristic activity for high-affinity K⁺ transport renders plants expressing the HAK transporter genes very tolerant to low-K⁺ conditions (Assaha et al., 2017). Our hypothesis is strengthened by the fact that the separate downregulation of each of the genes, OsTPKa as well as OsHAK_like in the salt tolerant rice landrace led to poorer seed germination both without and with salt. The reduction in shoot length was statistically significant ( Figures 6A-D ). Therefore, upregulation of OsHAK_like in the tolerant varieties likely mediates the higher uptake of K⁺ from soil during salt-stress.

From the coordinated expression analysis study of OsTPKa and OsHAK_like ( Figures 2 , 3 ) in root of Horkuch under salt-stress it was found that there was a slow but gradual increase in expression of OsHAK_like whereas for OsTPKa, the expression was higher at the initial stage and decreased at the latter stages of salinity stress. It is possible that at the initial stage of salinity stress, the vacuolar TPKa is activated via the rapid increase in Ca²⁺ (Latz et al., 2007) and the rapid de-polarization of the membrane and K⁺ efflux is countered. Meanwhile, the released Ca²⁺ reduces the Na⁺ load by activating the SOS pathway, while the simultaneous H⁺ pump activity further re-polarizes the membrane which in turn may subsequently activate the HAK_like K⁺ transporter (Shabala and Cuin, 2008). Therefore, tolerant plants may rely on a rapid replenishment of K⁺ supply from vacuolar storage via TPK mediated K⁺ efflux into the cytoplasm to maintain osmotic balance and to normalize membrane potential. The activity of the TPK may allow the cells the time to complete the signaling processes and increase the number of transcripts for high affinity transporters (HAK) to further increase K⁺ uptake and restore the cellular K⁺ pool volume (Wu et al., 2018). Now, when HAK transporters take part in maintaining K⁺ in cytoplasm, there is no need for vacuolar storage support and finally, other vacuolar inward transporters help replenish the lost K⁺ in the vacuole. Therefore, from this study, it can be hypothesized that both genes may work in a coordinated manner to increase the chance of survival under salinity stress in Horkuch. However, this hypothesis needs to be proven with by evaluation of the role of both potassium and Ca²⁺ in this coordination which are being planned.

The expression analysis data of OsTPKa and OsHAK_like in the roots showed the overexpression of both genes in tolerant varieties like Horkuch and Pokkali compared to the sensitive one (IR29) under salt stress. However, the mechanism of how the upregulation of these two genes helps maintain a favorable ion gradient in tolerant varieties is not well characterized yet. So, to gain insight into the biophysical properties and physiological function of these genes, they were subjected to CRISPR/Cas9-based loss of function mutation in Horkuch.

It was observed that even at the T₀ generation, the mutant lines of Horkuch showed a remarkable sensitivity to 150 mM NaCl in leaf disc senescence assay indicating loss of tolerance in the transformed lines. Among selected lines that showed a sensitive response, 6 lines each for OsTPKa and 5 for OsHAK_like performed poorly with an extremely slower rate of seed germination, a significant reduction in shoot length and weight, and a very low percent increase in root length compared to wild type. Salinity-induced osmotic and ionic stresses are known to inhibit and delay seed germination along with interruption of nutrient uptake and growth retardation, especially and more prominently in sensitive varieties (Hakim et al., 2010). Therefore, our study indicates that due to mutation of the above-mentioned genes, tolerant Horkuch is behaving more like a sensitive variety which points to the importance of the two transporter genes in providing salinity tolerance.

To further verify the reduction in stress-tolerant characteristics of T₁ Horkuch OsTPKa and OsHAK_like, mutant lines, various tests including electrolyte leakage, H₂O₂ concentration and chlorophyll content were measured. Electrolyte leakage and H₂O₂ in shoots showed an increase with stress in the Horkuch mutant lines compared to the wild type indicating disturbed membrane integrity (Mishra et al., 2021). Percent increase in electrolyte leakage and peroxide (H₂O₂) content due to salt stress was lesser in the overexpressed lines under salinity compared to the wild type plants. For the mutant line, the content of the photosynthetic pigment in rice plant grown under salt condition declined significantly, being especially susceptible to salt. The chlorophyll pigment content in leaves of tolerant rice genotypes under stress is known to be maintained better than in sensitive varieties. In our study, the reduction of chlorophyll content was significantly higher in the mutant Horkuch lines which can lead to a decrease in photosynthetic efficiency and sugar content and a subsequently negative effect on plant survival under stress (Amirjani, 2011). There was striking increase in Na⁺/K⁺ ratio in OsTPKa and OsHAK_like downregulated lines compared to corresponding wild type which indicates the importance of above-mentioned genes in maintaining the appropriate K⁺ concentration under salt stress.

The overexpressed lines where the OsTPKa gene from Horkuch was cloned and transformed in a salt sensitive genotype BRRI dhan28, showed a significant increase in growth during germination as well as during initial growth at seedling stage. They looked healthy and taller than their wildtype counterpart even without any stress application ( Supplementary Figure 4A ). However, this significant difference with the wild type plants were observed only during the earlier growth of the plants. At 12 days old, wild type plants and putative transgenic plants were of similar height. Differences became evident between WT and transgenic plants, where the latter showed significantly lower reduction in plant height after salinity stress of 18 days ( Supplementary Figure 4B ). The putative transgenic plants’ survival rate was much higher under salt stress at the seedling stage. This interesting observation indicates that the overexpressed lines may have some kind of growth advantage due to supply of extra potassium during the early days compared to the wild type even without salt stress. The plants without stress were subjected to generation advancement and a panicle wise selection using LDS assay could select the lines for phenotypic screening at seedling stage in the T₁ generation.

Under salt stress, in the over-expressed lines of OsTPKa, the root length and weight were significantly higher and there was significant increase of K⁺/Na⁺ under salt stress in the transgenic lines compared to the wild type plants. They showed less percent reduction of chlorophyll content under salt stress compared to the wildtype plant. However, this change was not that significant indicating that the overexpressed lines get growth advantage for their survival and maintenance but may not have extra benefit for enhanced photosynthesis or chlorophyll production in the leaf tissues.

The overexpressed T₁ transgenic lines showed significantly higher K⁺/Na⁺ ratio in their shoot and root tissues compared to no stress condition. In the wild type plants’ root tissue, a significant reduction was observed under salt stress as opposed to the transgenic plants. The root tissues of transgenic line 39 showed a highly significant rise in K⁺/Na⁺ content compared to their control conditions, while the wild type plant showed a significant reduction. This trend was also supported by the higher gene expression level of OsTPKa in root tissue of line 39 compared to the other lines ( Supplementary Figure 5 ). Potassium is one of the major nutrients required for growth and development of plants. Not only does early vigor give the plant better survivability under salt stress, but the higher potassium also protects against the toxic effects of the sodium. Therefore, the study supports the possible role of OsTPKa and OsHAK_like in providing salt tolerance.