Advanced Breeding Strategies and Future Perspectives of Salinity Tolerance in Rice

: Rice, generally classiﬁed as a typical glycophyte, often faces abiotic stresses such as excessive drought, high salinity, prolonged submergence, cold, and temperature, which signiﬁcantly affects growth, development, and ultimately, grain yield. Among these negative impacts of abiotic factors in rice production, salinity stress is a major constraint, followed by drought. There is considerable research on the use of marker-assisted selection (MAS), genome editing techniques, and transgenic studies that have profoundly improved the present-day rice breeders’ toolboxes for developing salt-tolerant varieties. Salinity stresses signiﬁcantly affect rice plants during seedling and reproductive stages. Hence, greater understanding and manipulation of genetic architecture in developing salt-tolerant rice varieties will signiﬁcantly impact sustainable rice production. Rice plants’ susceptibility or tolerance to high salinity has been reported to be the result of coordinated actions of multiple stress-responsive quantitative trait loci (QTLs)/genes. This paper reviews recent literature, updating the effects of salinity stress on rice plants and germplasm collections and screening for salinity tolerance by different breeding techniques. Mapping and identiﬁcation of QTLs salt tolerance genes are illuminated. The present review updates recent breeding for improvement in rice tolerance to salinity stress and how state-of-the-art tools such as MAS or genetic engineering and genome editing techniques, including mutagenesis and conventional breeding techniques, can assist in transferring salt-tolerant


Introduction
Rice (Oryza sativa L.) is the most important staple food crop for more than 3.5 billion people worldwide, particularly in Asia and Africa. By the year 2050, the world's population is expected to reach 9.6 billion people [1]. Hence, there is an urgent need to enhance rice production to meet the rising global food demand. This target must be achieved in the face of rising biotic and abiotic stresses caused by climate change and further overwhelmed by growing competition for already scarce resources such as land and water. Conditions of drought, excessive salinity, cold, and heat are common causes of abiotic stresses that rice plants endure as growth proceeds in the ever-changing environment [2]. Numerous studies have cited that salinity stress is one of the most common abiotic stresses in rice, inhibiting crop growth, development, and ultimate yield production. It has been estimated that more than 1000 million hectares of land are saline or sodic, with records between 25% and 30% of irrigated areas (about 70 million hectares) being salt impacted and, in commercial terms, practically unproductive [3]. Previous studies have cited that soil salinity severely affected 50% of the world's population (around 4.03 billion people) in 13 countries. By the year 2050, seawater intrusion and inundation have been predicted to impact about 5.02 billion of the world population [4]. Rice is one of the most salt-susceptible cereal crops, characterized as having severely stunted plant growth and yield at a salinity level of 30 mmol L −1 NaCl (3 dSm −1 ) [5]. Recent studies have indicated that rice is more salttolerant at germination and vegetative stages than seedling and reproductive stages due to a weak association between the latter two salt-susceptibility stages. Recent studies have also noted that rice plants have adopted several coping strategies with salinity stress at the various development stages. About 12% yield reduction was recorded in modern high yielding rice varieties above a salinity level of 3 dSm −1 . At a salinity level of 6 dSm −1 , about 50% yield reduction has been recorded [6].
Rice has a small genome size and wide genetic diversity. With the availability of whole-genome sequences and promising genome editing techniques, rice has been acknowledged as a model plant for genomic and molecular research [7]. In recent years, development in salt-tolerant varieties and studies on mechanisms of salinity tolerance have made essential contributions. Nonetheless, because of complications in plants' responses to salts stresses, which include intrinsic components of osmotic, ionic, and oxidative stresses, the development of salt-resistant rice varieties remains challenging [8]. It has been documented that coordinated actions of multiple stress-responsive quantitative trait loci (QTL) genes and metabolic pathways may determine salinity tolerance in rice. Although several genes/QTLs associated with salinity stress have been comprehensively mapped in rice, none have been incorporated into commercial cultivars [9,10]. Studies have extensively shown that salt stress inhibits growth and photosynthesis, resulting in reduced biomass and partial grain sterility, leading to decreases in rice production [11].
Several researchers have reviewed response mechanisms in salinity-stressed rice. However, only a few have focused on advances made in developing salt-resistant varieties. Consequently, the development of salinity-resistant rice varieties is regarded as the most promising and effective way to ensure food security. In this review, the effects of salinity stress on rice plants, rice germplasm collections and screening for salt tolerance, and fine mapping and identification of QTLs/genes for improving salt tolerance are discussed. The review is also complemented with different and recent advancements in breeding methods for improving and developing salt-tolerant rice varieties. Biotechnological approaches for salt tolerance are also reviewed, including the state-of-the-art tools such as markerassisted selection (MAS) or genetic engineering and genome editing techniques such as mutagenesis and conventional breeding, which may enhance introgression of QTLs/genes associated with salinity into elite rice genotypes in an attempt to accelerate breeding for salt-resistant cultivars.

Effect Salinity Stress on Rice Growth, Development and Grain Yield
Plant responses to salinity stresses are considered natural and random processes to increase rice grain yield under salt-stressed conditions. In the literature, salt tolerance is a complex quantitative trait governed by multiple gene interactions [12]. Rice crops are highly susceptible to salt stresses, particularly in the early growth stages [5] and in the reproductive stages [13], leading to significant grain yield reduction. Several studies have recorded that Indica rice is more salt-tolerant than japonica rice [14]. Morpho-physiological, biochemical, and molecular parameters have been reported to be affected by salinity stress. The effects of salinity stress on morpho-physiological parameters include inhibition in seed germination, stunting of growth of roots and shoots, reduction in tiller number per plant, number of grains per panicle, and pollen viability, with delays in seed setting and occurrence of sterile spikelets, decreased total dry matter accumulation, poor leaf area development, and crop area establishment ( Figure 1) [15]. Several researchers have also reported the effects of salt stress on rice seedling establishment and seedling growth and reductions in yield and yield-related traits such as grain number per hill and effective tiller number per hill [16]. Salinity stress in rice has also been reported to induce biochemical and physiological changes, causing growth inhibition and consequential yield losses [17]. Evaluation of salinity-tolerant lines based on morpho-physiological parameters such as plant height, dry matter accumulation, leaf injury, and ratio of Na + /K + content in leaves and roots has been investigated to understand the drastic effects of the accumulation of salts at the physiological level [18]. Three main effects on rice plants have been reported and include lowering of water potential, impaired uptake of essential nutrients, and direct toxicity of sodium (Na + ) and chlorine (Cl − ) [19]. Uptake of sodium (Na + ), kalium (K + ), and calcium (Ca 2+ ) ions; total cation and osmotic stress; relative growth rate; and transcription efficiency are some of the physiological characteristics that are altered by salinity stress [20]. Studies have also cited that proline, anthocyanins, peroxidase activity, calcium, sodium, potassium, chlorophyll, and hydrogen peroxide concentrations are all biochemical characteristics of rice affected by salt-stress [21]. The nutritional content of rice grains includes a range of minerals, including calcium, magnesium, and phosphorus, and trace elements such as iron, copper, zinc, and manganese. Vitamins including thiamine, riboflavin, and niacin are also found in rice and are also affected by salinity stress [22]. Several studies have demonstrated that salinity stress affects macro-and micronutrients elements absorption and uptake [23]. A study evaluated macronutrient and micronutrients contents in rice grains of Pokkali and KDML105 under saline circumstances and observed that salt stress caused substantial decreases in macronutrient components but increased Ca 2+ content of both rice varieties. In another study, quantities of manganese, copper, and zinc were higher in Pokkali than in KDML105 [24]. Salinity has also been reported to increase sodium, iron, and zinc concentrations and decrease phosphorous and manganese. Nitrogen, magnesium, copper, potassium, and calcium contents were observed to be unaffected in the genotypes studied [24]. These investigations revealed that the impact of salinity stress on rice grain quality is complex and varied depending on salt concentrations and rice genotypes.

Mining and Screening of Salt-Tolerance in Rice Germplasm
Rice has numerous natural genetic variations, within both species and genera. The International Rice Research Institute (IRRI) has preserved more than 130,000 rice accessions from different countries and regions worldwide. Some rice accessions are wild types or wild relatives or landraces, including cultivated species (https://www.irri.org/internationalrice-genebank, accessed on 30 December 2019). As has been previously reported in the literature, wild rice relatives can improve cultivated rice's salt tolerance, and integrating QTLs/genes resistant to salt-stress characteristics from wild species into cultivated rice is seen as a viable strategy to increasing rice's salinity tolerance [25]. Under various salinity treatments, thousands of rice genotypes have been screened at the seedling stage using different parameters [26]. Germination index, germination percentage [27], plant survival, salt injury score and dry matter indicator at the vegetative stage [28], water use efficiency and chlorophyll content [29], relative growth rate, and concentrations of Na + and K + in shoots and roots [30] were among the indicators observed.
A previous study revealed a wide range of genetic variabilities between rice accessions O. sativa and O. glaberrima in salinity tolerance [31]. Indian accessions O. rufipogon and O. nivara were reported to have moderate tolerance to salinity [32]. Salt screenings were reported to have been applied at various salt levels and short periods (10)(11)(12)(13)(14) at seedling stages, focusing on osmoprotectants in plants for salt stress [33], when ion toxicity was not present. A recent study was conducted on two landraces adapted to seawater, namely Changmaogu [34] and Sea Rice 86 [35], both discovered in the coastal region of China. At the germination stage, the salt-tolerant rice variety Pokkali showed lower tolerance to salinity than Changaogu, with 18.7% germination in 140 mmol L −1 (~0.8%) NaCl treatment [34]. A recent study reported that, at 0.8% NaCl salinity treatments, 98% germination was recorded by Sea Rice 86, with no germination recorded above this level [35]. Salinity tolerance existed among wild rice, but the tolerance level was reported to have been lost [25]. Recently, Oryza coarctata species appeared more promising towards salinity tolerance with KKLL gene [36]. Understanding the molecular and physiological mechanisms underlying its enhanced salt tolerance might pave the way for certain essential features to be included in breeding programs [37]. For example, in an allele mining program, L-myo-inositol 1-phosphate synthase (MIPS) protein was reported to increase salinity tolerance in rice, which was a unique example of molecular salinity tolerance [38]. In addition to natural diversity, various mutants for salt tolerance in rice have been found. After screening >270,000 ethyl methanesulfonate (EMS)-mutagenized M 2 rice seedlings of Zhonghua 11 at 140 mmolL −1 NaCl, the dst mutant was discovered. Drought and salt tolerance (DST) is a crucial gene that encodes a zinc finger transcription factor that regulates stomatal aperture by directly manipulating the modulation of genes related to H 2 O 2 homeostasis in guard cells. It was found to be a negative regulator of drought and salt tolerance in rice [39]. From 6000 EMS mutant lines of the local elite cultivar, Hitomebore, a salt-tolerant mutant with hitomebore salt-tolerant 1 gene (hst1), was chosen. The loss-of-function of OsRR22, a cytokinin signaling protein, was discovered to be the cause of the salinity-tolerance trait of hst1 [40].

QTLs/Genes Mapping to Improve Salt-Tolerant Rice
Soil salinity tolerance is a complex physiological trait and is genetically controlled by QTLs [41]. Many QTLs have been identified and linked to salinity resistance in rice (Table 1). Presently, only a few significant QTL mappings or isolations were successfully done by genomic methods. Two major QTLs, qSNTQ-7 and qSKC-1, from crossing between salt-susceptible Koshihikari and salt-tolerant Nona Bokhra, were identified for Na + and K + concentrations in shoots [42], as qSNTQ-7 and qSKC-1 QTLs regulate K + /Na + homeosta-sis under saline conditions. The qSNTQ-7 and qSKC-1 QTLs recorded 48.5 and 40.1% of total phenotypic variation, respectively [42]. The qSKC-1 gene includes a member of the HKT-type transport family isolated by map-based cloning [18]. The Saltol QTLs on chromosome 1 controls Na + /K + homeostasis in the Pokkali cultivar resistant to salinity, mapped from crossing Pokkali and IR29 [43]. The qSE3 QTLs represent OsHAK21, a potassium transporter gene that influences rice seedling establishment and seed germination under salt stress [44]. The qST1 and qST3 QTLs are identified on chromosome 1 and 3, which confer salinity tolerance at the seedling stage and 36.9% phenotypic variations recorded in the recombinant inbred line (RIL) population [45]. The qST1 QTLs plays a vital role in salinity tolerance and recorded 62.6% phenotypic variations in Sea Rice (SR86) [46]. Mapping, cloning, and identification of the gQTLs in rice are the steps forward for associating salinity resistance. In light of reported studies on salinity tolerance, it is conceivable that further research should focus on discovering the major salinity resistance genes and their salt responsive mechanisms in rice. Focus should be on the markers that assist the incorporation/introgression of these QTLs into elite rice varieties through marker-assisted backcrossing (MABC) to develop salt-resistant rice varieties.
Considerable progress has been made through genetic dissection and molecular analyses in responses to salt stress to identify and map the genes involved in salinity stress in rice (Table 2). It is noted that researchers are more interested in identifying the salttolerant genes associated with reactive oxygen species (ROS) metabolism, as maintaining a proper level of ROS is essential for plant survival under salt stress conditions. The level of ROS in rice plants depends on ROS biosynthesis and ROS scavenging traits. Salinity induces the expression of respiratory burst of oxidase homologus (Rbohs), which catalyzes the conversion of O 2 to O 2− [47]. Rbohs are candidate genes that improve salinity tolerance. Genes associated with ROS biosynthesis and ROS scavenging improve salinity tolerance in rice. For instance, ascorbate peroxidase (APX) converts H 2 O 2 and O 2 , where APX enzyme acts as a specific electron donor [48]. Overexpression of other antioxidative genes such as OsECS, OsVTE1, and OsMSRA4.1 in transgenic rice had shown high salt tolerance [49]. The overexpression of APX genes enhances salinity tolerance by reducing ROS in plants. The knockdown of OsVTC1-1 and OsVTC1-3 genes enhances salinity tolerance by synthesizing ascorbate enzyme and increasing ROS level accumulation in plants [50]. Genes associated with Na + /K + homeostasis are also candidate genes for increasing salinity tolerance in rice. The changing ratio of Na + /K + in rice plants leads to metabolic changes. Na + ion inhibits the growth and development of rice plants through Na + toxicity. K + ion promotes the growth and development of rice plants. Therefore, the prohibition of intercellular accumulation of Na + toxic ions is essential for rice plants' survival under salinity resistance. High-affinity K + transporter (HKT) family genes are Na + , Na + /K + transporters.
In rice, HKT genes also restrict the accumulation of toxic Na + ions in roots and ultimately change intercellular Na + accumulation content and increase salt tolerance [51]. This suggests that modification of the ion channel is an option to develop rice variety resistance to salt stress. Genes associated with Na + /K + homeostasis and ROS also significantly regulate salinity resistance [52], such as overexpression of OsEXPA7, a cell wall-loosening protein gene increasing salinity tolerance by coordinating sodium transport, cell-wall loosening, and ROS scavenging [53]. The OsRR22, a response regulatory protein, acts as a transcription factor (TF) that regulates genes associated with osmotic stress or ion transport in the roots [40]. The OsCYP71D8L negatively regulates salinity resistance and affects cytokinin and gibberellin homeostasis [54]. A ubiquitin-binding protein acts as a dominant suppressor of KAR2 (OsDSK2a), enhancing rice seedling growth and salinity responses [55]. Many salt tolerance genes have been successfully identified, mapped, and cloned. Overexpression of these genes has also been carried out. The focus is now on the introgression of those genes for developing salt-tolerant rice varieties using marker aided selection breeding methods.

OsTPS1
Enhances salinity, drought, and cold tolerance in transgenic rice Overexpression [18,78] OsSPL10 SBP-box gene, negatively controls salt tolerance but positively controls trichome formation in rice Overexpression [79] OsEXPA7 Improves salt stress tolerance in rice by regulating ion homeostasis, ROS scavenging, and cell wall-loosening Overexpression [53] OsMYB6 MYB family gene, increases drought and salinity stress tolerance in transgenic rice Overexpression [80] OsMADS25 Transcriptional regulator that regulates the root growth and confers salinity tolerance in rice via the ABA-mediated regulatory pathway and ROS scavenging Overexpression [81] OsSOS1 Regulate ion transport regulation under salt stress Overexpression [82] OsDOF15 Contributes to ethylene-inhibited primary root elongation under salt stress Overexpression [83] OsGA2ox5 A gibberellin metabolism enzyme, GAs homeostasis, development, gravity responses, and stress tolerance in rice Ectopic expression [84] OsCDPK7 Positive regulator involved in both cold and salt/drought tolerance in rice Overexpression [85] OsCPK21 A calcium-dependent protein kinase, confers salinity tolerance in rice Overexpression [86] OsCPK12 Calcium-dependent protein kinase, negatively regulate salt-stress tolerance Overexpression [87] OsCam1-1 ABA biosynthesis and salt tolerance in rice Overexpression [88] OsMSR2 Enhances drought and salt tolerance and increases ABA sensitivity Overexpression [89] OsRPK1 Receptor-like protein kinase genes, negatively regulate salt tolerance Overexpression [90]

Genome-Wide Association Study (GWAS) Technique
The genome-wide association study (GWAS) is a powerful approach in understanding the genetic basis of complex traits and identified allelic variations of candidate genes in rice [91]. GWAS analyzed the statistical association between traits and genetic variants on the whole genome sequence and has been applied to identify causal genetic variability for targets traits [92]. GWAS enables high-resolution mapping, utilizing single nucleotide polymorphisms (SNPs) as genetic markers instead of the QTL linkage mapping approach [93]. In the indica subspecies of rice, GWAS has been conducted for agronomic characteristics such as tiller number, grain width, grain length, and spikelet number using single nucleotide polymorphisms (SNPs) discovered by whole-genome sequencing [94]. GWAS has been applied for mapping traits associated with loci responsible for salinity tolerance. A previous study reported that a total of 23 marker-trait associations (MTAs) were mapped for salinity tolerance at the early vegetative stage using GWAS. These MTAs were found on rice chromosomes 1, 2, 5, 6, 7, 9, and 12 and observed 13.98 to 29.88 percent of the trait phenotypic variations [95]. There are numerous GWAS applications under saline conditions in rice, with distinct growth phases and characteristics. Another study reported using 6000,000 SNPs to conduct a GWAS on the germination stage of salt-treated rice and recorded that 11 loci, including 22 significant SNPs, were responsible for stresssusceptibility indicators such as vigor index and germination time [27]. In another recent GWAS study, 27 QTLs for moderate salinity-related characteristics were mapped on 12 rice chromosomes. GWAS also studied 20 QTLs for 11 salt-resistant traits in rice at the germination and seedling stages [96]. GWAS identified two genes related to auxin biosynthesis as potential genes for salinity resistance from a large sample of 478 rice accessions [97]. Under salt stress conditions, 15 potential genes, transcription factors (TFs), and cation transporters were recently discovered to be connected with grain production and associated characteristics [98]. Several potential genes, including two SUMO proteases (OsSUMO1 and OsSUMO2) and a K + transporter gene (OsHAK6) have been associated with salinity tolerance in rice [99]. These identified material transfer agreements (MTAs), QTLs, and genes pave the path for molecular breeding to combine salinity tolerance with high yield characteristics in rice. In GWAS, the candidate genes Os09g0278000, Os09g0279100, Os09g0279400, Os09g0279500, and Os09g0280300 at locus 167 control early seedling vigor (ESV), conferring salinity tolerance in rice [26].

Conventional/Traditional Breeding
Genome and whole-genome sequences have contributed to the genomic study, proteomics, transcriptomics, metabolomics, and QTLs/gene mapping to improve salinity tolerance in rice. The collection of germplasms, including wild species, has provided important genetic diversity for salinity tolerance, and the success of a breeding method depends largely on the choice of germplasms. Screening rice germplasms to identify salt tolerance genotypes has led to the development of salt-tolerant rice by transferring genes. Genetic diversity analyses of 107 rice accessions were conducted using 376 single nucleotide polymorphism (SNP) markers to characterize the allelic variations on a major QTL, Saltol. The results indicated that rice landraces, e.g., Akundi, Ashfal, Capsule, Chikirampatnai, Jatai Balam, Kalarata, and Kutipatnai, tolerated salinity through the accumulation of more K + ion with less Na + ion, and lower levels of Na + /K + ratio in leaves [100]. These landraces provide valuable resources for developing salinity-tolerant rice and other abiotic stresses.
Conventional breeding methods such as hybridization, recurrent selection, pedigree selection, backcrossing, and induced mutations have been applied to develop salt-tolerant rice. Several researchers have developed salt-tolerant rice varieties by conventional breeding methods [101]. Since the 1970s, the International Rice Research Institute (IRRI) has developed more than 30 salinity-resistant varieties using sexual hybridization approaches. Varietal development through traditional/conventional breeding requires six to seven years or more. The rice varieties developed through traditional breeding methods contain unwanted deoxyribonucleic acid (DNA) from donor parents. Such a variety fails to achieve a well-known exclusive cultivar. Conventional breeding methods are slower, more timeconsuming, inefficient, unreliable, and laborious [99]. Success in conventional breeding depends on the proper mapping/identification of genes for new varietal development process. Wild species and landraces are the prime considerations for the success of conventional breeding due to their greater genetic variations in response to salinity [102]. Salinity resistance varietal development relies on identifying QTLs/genes linked to target genes and tightly linked molecular markers applied in marker-assisted selection (MAS) [103].

Marker-Assisted Selection
The use of molecular markers linked to agronomic traits of interest controlled by individual genes or QTL is called MAS [104]. This is the most promising and successful method for developing new salt-tolerant rice lines [105]. The use of MAS can shorten the breeding cycle by allowing for the selection of plants with the target genes at the early growth stages saving labour, time and agricultural inputs such as fertilizer, pesticides and irrigation and is not influenced by environmental factors. Marker-assisted backcross (MABC) breeding is faster and more precise to introgress/transfer linked genes for salinity tolerance. MABC allows selecting plants in each breeding season to confirm the introgressed target genes and recovery of parental genome (RPG) and reduced linkage drag [106]. The recovery of parental genome of developed lines can be estimated. The distinctness, uniformity, and stability (DUS) test of developed rice lines confirms the percentage of RPG of parental traits with SNP marker genotype [107]. A variety of molecular markers are available to identify various QTLs and introgression of QTLs into elite rice genotype through MAS. SNPs simple sequence length polymorphisms (SSLPs), restriction fragment length polymorphisms (RFLP), amplified fragment length polymorphisms (RFLP), sequence-tagged sites (STS), and simple sequence repeats (SSRs), or microsatellites, are all DNA markers used for genotyping in molecular mapping studies and MAS [108]. Utilizing the SSR marker, the QTLs of pollen fertility; Na + concentration; and calcium (Ca 2+ ), sodium (Na + ), and potassium (k + ) accumulation in the F 2 rice populations have been reported [109]. QTLs for yield and yield contributing characteristics and physical traits were recorded using F 2 rice populations as plant materials and SSRs as a DNA marker [109]. QTLs for potassium and sodium absorption for improving salt tolerance in rice have been determined using AFLP, RFLP, and SSR [110]. SNP has been used to identify the QTLs of salt stress-sensitive genes in rice [111]. SSRs markers linked to Saltol QTL include RM3412, AP3206, RM10796, RM8094, RM493, RM10793, some functional markers, and SNP [112]. RAPD markers have been developed, identified, and applied into marker-assisted selection breeding programs [113]. The availability of molecular marker development for MAS is still in progress. MAS utilizes novel sources of genetic materials provided by allelic variations in the gene of interest and can create, design, and develop a cheap molecular marker for salt tolerance [114]. By applying MABC, Saltol QTLs was introgressed into a popular elite variety in different countries: Saltol QTLs was introgressed into two Bangladeshi varieties, BR11 and BRRI dhan28 [115], AS996 and BT7 in Vietnam [116], Rassi in West Africa [117], and Pusa Basmati 1121 and PB6 in India [103,118]. In India, a multi-institutional program on the introgression of Saltol QTLs into elite mega-rice varieties is in progress [106,119]. Marker-assisted gene/QTLs pyramiding has been proven to be a powerful tool to introgress multiple genes/QTLs for broad-spectrum resistance to abiotic and biotic stresses [120]. Studies pyramiding multiple QTLs/genes in rice for tolerance to drought have been carried out [121], as have studies investigating salinity and submergence tolerance through MAS [122] by pyramiding genes for disease resistance, submergence tolerance, and aromatic fragrance [123]. Additionally, genes have been pyramided for bacterial leaf blight under deep water rice through MAS. Presently, marker-assisted gene pyramiding for developing salinity tolerance rice variety using MAS has been proven to be a promising technique.

Transgenic Approach
Genetic engineering has become a powerful technique in modern plant breeding. It allows the introduction of the gene of interest into elite cultivars without compensating for desirable characteristics. The introduction of an effective agrobacterium-mediated transformation technique has contributed significantly to genetic improvement in rice [124]. Generally, salinity tolerance through genetic modification focused on genes that encode late embryogenesis abundant and heat-shock proteins, transcription and signal transduction factor, programmed cell death, detoxification of ROS (antioxidants), compatible organic solutes, and ion transport [124]. In addition, all identified techniques used by rice plants in coping with salinity have been utilized in genetic modification to develop rice salinity tolerance. The expression of salt-responsive genes such as phosphatase 1a (OsPP1a) contributes to salinity tolerance in rice. The nRK1A, OsNAC5, and OsNAC6 genes are also upregulating in transgenic lines, viz, OsPP1a-2, OsPP1a-3, and OsPP1a-6 [125]. Transgenic rice demonstrates salinity tolerance through the accumulation of Na + /H + in both shoots and roots under salinity treatments. Sodium proton antiporter is one of the potential gene families in rice showing multiple stress responses under abiotic stress conditions. OsNHX 1 genes were derived from landraces Pokkali, a vascular Na + /H + antiporter gene overexpression that enhances the level of salinity tolerance in transgenic rice [126]. The accumulations of Na + /H + in shoots and roots changed biomass production and germination, which also improved by overexpression of OsNHX1 genes [127]. The overexpression of treholose-6-phosphate synthase, associated with OsTPS1 genes, was reported to enhance salinity, drought, and cold tolerance in transgenic rice. It also increases the accumulation of compatible solutes by reducing wilting and maintaining photosynthetic activities in transgenic rice [78]. Ectopic expression of the PtCYP 714 A 3 gene in transgenic rice promotes active tillering, with reduced seed size and semi-dwarfed phenotypes. These findings provide the essential roles of molecular foundation in transgenic research in rice responses to salinity and the roles of PtCYP 714 A 3 in shoot responses to salinity [128]. Li et al. reported that the overexpression of SIDP361 genes enhances salinity tolerance in rice at the seedling and reproductive stages. The genes have high potential as a tool for genetic engineering for salt-tolerant rice [129]. Previous studies on OsSUV3 overexpression in transgenic rice increased the concentrations of gibberellic acid (GA 3 ), zeatin (Z), and indole-3-acetic acid (IAA) in leaves, stems, and roots as compared to wild-types, with reduced adverse effects of salinity on the growth and development of rice plants [130]. Pea DNA helicase 45 (PDH 45 ) genes in transgenic rice accumulate lower concentrations of Na + , ROS, and calcium homeostasis in roots under salinity stress by maintaining cell viability [131]. High-salt induction of OsPP1a in transgenic plants upregulate the expression of SOD significantly [125]. Salinity tolerance transgenic rice was developed through over-expression of the OsIF genes from wild rice (Porteresia coarctata) native to Bangladesh, India, and Myanmar.
The bulk of transgenic rice is developed in the United States (11.5 percent), Europe (8.9%), Oceania (1.1 percent), and Africa (1%), while the majority of field trials have taken place in Asian countries (77.5%), primarily in China (47.8%) and Japan (1%). Developing countries such as Bangladesh, Myanmar, Thailand, Nepal, and Bhutan are still developing transgenic rice due to lack of modern laboratory facilities and technical knowledge. Salinity tolerance in rice is physiologically and genetically complex due to some critical aspects, e.g., ion homeostasis and compartmentation, programmed cell death, signal transduction, and transcription factors that are regulated by respective sets of genes. No single cultivar has been released to farmers for commercial cultivation despite the number of salinity tolerance rice cultivars developed through the transgenics approach. The extension of this rice variety is still the prime obstacle due to complicated procedures for transgenic rice. Genetically modified rice must adhere to the worldwide Cartagena Protocol, which is applied in each country by its own biosafety rules [132]. The primary hindrances to the extension of transgenic rice are asynchronous approval and release processes, propagation of illegal seeds, and contamination in non-labelled transgenic rice producers in countries with zero tolerance [133]. Therefore, the primary obstacles to implementing a transgenic method include limited public acceptability in different countries, prior knowledge of gene sequences, necessary laboratory infrastructures, and strict rules and regulations.

Mutation Breeding
In recent years, huge progress in functional genomics research among cereal crops has been recorded [134]. Mutation breeding can be applied as a vital tool for creating genetic variability in rice crops and functional gene study [135]. Mutagenesis is an important technique for increasing the frequency of mutants, which can help in functional genomic research and lead to the development of new genotypes [136]. Mutation occurs through the application of biological, chemical, or physical mutagens [137]. In rice, the level of dose and exposure time regulate the degree of mutations [138]. Mutagenesis in rice is advantageous because of its small genome, which requires a smaller population to saturate the entire genome and provide a wider allelic series sequence [139]. Several rice mutants have been developed in different countries using random mutations such as Amaroo rice mutant variety in Australia [140]; Binasail, Iratom-24, and Binadhan-6 rice mutants in Bangladesh; 26 Zhaizao and Zhefu 802 rice mutants in China [141]; PNR-381 rice mutants in India [141]; Basmati 370 rice mutant in Pakistan [141]; Calrose 76 rice in the United States [142]; and VND 95-20, VND-99-1, and VN121 rice mutants in Vietnam. While efforts to increase salt stress resistance by genetic changes have achieved significant results, the mutation technique is a primary strategy for obtaining salt-tolerant varieties and other desirable characteristics [143]. Mutation breeding has an important role in improving high yield and rice resilience to salt stress [144]. There are several successful applications of mutation breeding to increase salt tolerance. For instance, carbon (C) and neon (Ne) irradiated rice seeds have developed a mutant type with excellent salt tolerance [145]. The IAEA (2003) has reported the identification of eight salt-tolerant mutants with higher salinity tolerance than the parents. Six mutants have been produced from parent Bicol and two from salt sensitive parent IR29 [146]. Lee at al. [14] has developed salt-tolerant and sensitive mutants by applying gamma irradiation in the callus of a Korean rice variety, Dongjinbyeo. Salt-tolerant mutants have also been developed through in vitro anther cultures and double haploids by Sathish [147]. Lin et al. [148] developed a salt hypersensitive 1 (shs1) mutant using sodium azide, which plays a vital role in Na + homeostasis and antioxidant metabolism. Azolla varieties resistant to high salinity, hazardous aluminium levels, and pesticides were developed via mutation breeding [149]. Two mutant lines, ST-87 and ST-301, were developed using gamma-rays for salinity tolerance in rice by Song et al. [150]. Several salt-tolerant rice varieties, such as A-20, Shua92, Basmati370, Changwei19, Atomita2, Nipponbare, Fuxuan No. 1, Mohan-CSR4, Emai No. 9, and Changwei 19, have been developed by applying gamma rays and released in many countries all over the world [151]. By application of target mutations in rice, several genes have been modified for salinity tolerance via CRISPR/Cas9 and other genome editing approaches [152]. However, the application of mutagenesis breeding is restricted due to the randomness of mutation and problems with the regeneration of entire plants [153]. In addition, the high population size needed to screen phenotypes, occurrence of accidents, availability of mutagens, and specific infrastructures are all obstacles to the application of mutations.

Genome Editing Techniques
Genome modification through targeted genome editing has revolutionized plant genomics for improving plants' characteristics against biotic and abiotic stressors [154]. Mega nucleases, transcription activator-like effector nucleases (TALENs), zinc finger nucleases (ZFNs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas) systems have all been used to alter genomes [134]. CRISPR-Cas9 has been widely utilized to transform plants by inducing mutations via either homology-dependent repair (HDR) or non-homologous end-joining of double-stranded breaks (NHEJ) [155]. Previous studies revealed that CRISPR-Cas9 best targets specificity because the Watson and Crick model recognizes target sites, and sequence analyses identify off-target regions [156]. CRISPR/Cas9 splits foreign DNA via two components, Cas9 and a single guide RNA. The guide RNA consists of the CRISPR-derived RNA (crRNA) and trans-activating RNA (tracrRNA) systems. Cas9 is a DNA endonuclease obtained from several bacteria, including Brevibacillus laterosporus, Staphylococcus aureus, Streptococcus pyogenes, and S. thermophilus, with S. pyogenes [157] being the most commonly utilized for Cas9 isolations. Two domains are included in Cas9, the HNH domain and the domain RucV-like. The HNH breaks off the CRISPR RNA complementary strand (crNA), while RucV cuts off the opposite strand to the double-stranded DNA. The sgRNA is a synthetic RNA, approximately 100 nt in length. The 5 -end sequence has a 20-nt sequence acting as the guide to identify the target sequence, which is frequently the NGG consensus, along with the PAM sequence (N, any nucleotide; G, guanine). At the end 3 of the sgRNA, the loop structure can attach the target sequence by means of the sequence guide and creates a Cas9 complex, which splits the double-stranded DNA and causes a double-strand break (DSB). When a DSB is created, DNA repair processes commences, either nonhomologous end-joining (NHEJ) or homology-directed repair (HDR). In most cases, the DSB is generally repaired by NHEJ and it is an easy way to produce malfunctions and deletions (indeles), resulting in knock-out. HDR causes certain replacement of gene or foreign DNA knock-ins when oligo template is available [158].
Since 2016, the sophisticated genome editing method utilized for plant gene editing has been the clustered regular interspaced short palindrome repeats of Prevotella and Francisella 1 (CRISPR/Cpf1) [159]. This makes the CRISPR/Cpf1 system the most advanced and efficient genome editing technology, with several major benefits over the CRISPR/Cas9 system. Cpf1-crRNA complex can itself efficiently cleave the target DNA without needing tracrRNA I the CRISPR/Cpf1 system. For example, a 40-45 nt crRNA with repetition and spacer is sufficient to assist gene editing compared to the 100 nt sgRNA employed in the CRISPR/Cas9 system [160]. Presently, three CRISPR/Cpf1 systems, FnCpf1, LbCpf1, and AsCpf1, are available for genome editing applications in rice [161].
Although CRISPR/Cas9 and CRISPR/Cpf1 based gene editing can be a convenient method for gene replacement, template DNA transport and targeted insert or gene replacement are relatively low in frequency and efficiency. A CRISPR/Cas9 based base editor technique is the latest and most sophisticated method that can be applied as an alternative method, which permits a direct and irreversible conversion of one target base into another without the need for a DSB or donor template [162]. The base editor merges the dormant Cas9 domain with a cytosine deaminase domain, which changes the base G-C pairings to A-T base pairs [163]. Base editing methods in grain crops such as rice, wheat, and maize have been optimized and have been shown to be effective [164]. A base editing construct known as nCas9-PBE, consisting of rat Cytidine deaminase APOBEC1 and a Cas9 variant Cas9-D10A nickase (nCas9), has been applied for editing the OsCDC48 gene in rice for cell death and senescence [165]. The results showed that rice editing of site-specific C to T base might be utilized efficiently with nCas9-PBE. Subsequently, the use of nCas9 coupled with a cytidine deaminase enzyme revealed a targeted point mutation in rice [166].
Due to its simplicity, flexibility, and great accuracy, CRISPR/Cas9 technology has been utilized effectively in key crops and model plants [167]. In rice, genome editing may be combined with innovation to create new varieties with greater yield and quality and a high resistance to abiotic and biotic stressors. CRISPR/Caa s9 has been widely utilized in several crops, including rice, where it has shown great promise in producing desired alterations in response to biotic and abiotic stressors [168]. There are several successful applications of CRISPR-Cas9 gene editing techniques in rice, viz, editing of transcriptomic factor MYB family genes, OsMYB1-OsMYB5, editing of the OsSPP gene for early seedling leaf chlorosis, the pleotropic phenotype controlling gene, OsMSH1, and the photoperiod sensitive male sterility responsive gene, OsPMS3, and also the drought resistance controlling gene, OsDERF1 [169]. Recently, [168] has developed rice lines for abiotic stresses by applying the CRISPR-Cas9 gene editing techniques, such as editing a transcription factor gene (TIFY1b) using rice variety Nipponbare. Improved blast resistance rice using Kuiku131 has also been developed by editing the OsERF922 gene, and herbicide resistance rice by editing the acetolactate synthase 1 gene (ALS1) [170]. The TMS5 [171], OsHAK1 [172], and Badh2 [173] genes have also been edited through CRISPR/Cas9 for thermo sensitive male sterility, low-Cs+ rice plants, and fragrance characteristics in rice and yielded the expected phenotypes. It was reported that salinity-tolerant rice was improved by engineering a Cas9-OsRR22-gRNA expressing vector to edit the targeted gene OsRR22 [174]. The application of the CRISPR/Cas9 system for simultaneous editing of three genes, OsPIN5b, GS 3, and OsMYB30 was undertaken and reported that the generated rice mutant's lines show outstanding cold tolerance with high grain yield [175]. The application of CRISPR/Cas9 to create new OsBADH2 alleles in order to induce fragrance in any non-aromatic rice was also reported [174].
These successful applications of CRISPR-Cas9 techniques and improvement of rice varieties resistant to abiotic and biotic stresses clearly indicate that genome editing approach such as CRISPR-Cas9 has a huge potential as an accurate, promising, and effective technique for improving more traits [176]. However, there is a need to focus on improving saltresistant rice by editing salinity tolerance genes. SNPs found in GWAS studies that are functionally relevant can be exploited for genome editing [177]. Suppose GWAS is utilized to find the so-called non-phenotypic variations such as expression QTL, methylation QTL, and metabolite QTL, as well as novel and useful alleles. In that case, genome editing can be utilized to assist the combination of new alleles [178]. Such an approach has the potential to revolutionize rice breeding, particularly through genomics-driven crop design. In this review, we emphasize different breeding strategies which can lead to the development of salt-tolerant rice. Figure 2 gives the outline of the integration of different breeding tools for developing salt-tolerant rice varieties.

Development of Salt-Resistant Rice Varieties
The most commercially feasible and ecologically acceptable approach in effectively using saline soil is to develop exceptional salt-tolerant rice cultivars. Yield is an essential agronomic trait for evaluating the benefits of rice types as a food crop. Rice salinity tolerance is described as the capacity to grow with a 0.3 percent saline-alkali concentration and to produce above 4500 kg rice per hectare. Breeders have spent years scouring the globe for salt-tolerant rice resources, which they have gathered, assessed, and improved. Sri Lanka is the first country to research salinity to screen and identify salt-tolerant rice cultivars. Firstly, Sri Lanka introduced the salt-tolerant rice variety Pokkali in 1939. The IRRI have preserved more than 130,000 rice accessions from different countries around the world. The Philippines has developed salinity-resistant rice varieties. The USA has developed a salt-tolerant variety; American Rice. Bangladesh also breeds salt-tolerant rice cultivars, namely BRRI dhan47, BRRI dhan54, BRRI dhan55, BRRI dhan61, Binadhan-8, and Binadhan-10. Thailand breeds the salt-tolerant variety FL530. Japan breeds salinity-resistant rice cultivars for saline soil management. South Korea has also researched salinity resistance and bred a series of salinity-resistant rice varieties. India largely contributed to managing saline soil by introducing many salt-tolerant rice varieties through the application of conventional and molecular breeding methods. China has also screened and bred salt-tolerant rice varieties, including wild rice, namely Sea Rice 86 and Changmaogu (Table 3). Improved white ponni (IWP) Saltol MABC India [191] Improved IWP Saltol or qSKC1, qDTY1.1, qDTY2.1) MABC India [121] 7PcINO1, 4PcINO1 and 3PcINO1-lines PcINO1, PcIMT1 Transgenic approach India [192] OsPP1a-2, OsPP1a-3, OsPP1a-6 transgenic lines OsPP1a Transgenic approach India [125] Abp57-transgenic rice Abp57 Transgenic approach Malaysia [193] * MABC denotes Marker-assisted backcrossing.

Conclusions and Future Perspectives
Salinity stress is the most critical factor among the abiotic stresses influencing rice growth, development, and grain yield production. Developing salt-tolerant rice varieties is one of the most effective and promising solutions to tackle the salinity problem. Researchers have identified some salt-tolerant rice genotypes from the collection and screening of germplasm. This information has built a foundation for rice breeders in understanding salt responsive mechanisms, mapping or identifying QTLs/genes associated with salt stresses for improving salinity resistance rice. The present review addresses recent breeding progress in improving salt tolerance in rice and discusses state-of-the-art tools such as MAS or genetic engineering and genome editing techniques, including mutagenesis and conventional breeding that can help transfer salt-tolerance QTLs/genes into elite rice genotype to accelerate breeding of salinity-stress resistant cultivars. Generally, phenotypic characteristics are employed to measure salt tolerance; however, this method requires thoroughness and precision. As a result, salinity tolerance evaluation differs from actual field performance. Indeed, a consistent technique is critical for determining rice salt tolerance. Numerous QTLs/genes associated with salinity tolerance have been discovered from various mapping populations, but only a few key salt tolerance genes have been identified. QTLs have been limited in breeding programs due to their variability and inconsistency in different genetic origins and environments. Researchers have developed several salt-tolerant rice cultivars using classic breeding methods, but the salt tolerance mechanism remains unknown. These concerns and how plants detect salinity stress, how these signals are converted into adaptive responses, and how numerous salt tolerance genes coordinately regulate salt tolerance in rice require further research. Traditional breeding procedures are inefficient and time-consuming. Molecular breeding methods have been substantially improved by SNP marker-assisted selection and genetic engineering technologies. As a result, more systematic and theoretical analysis on salinity stress is needed to utilize the essential QTLs/genes linked to salt tolerance by transferring into elite rice cultivars through marker aided selection.
Obtaining salt-resistant rice varieties for commercial production by incorporating a single or many genes is difficult. Multiple salinity stress-resistant genes must be transferred into elite rice varieties through marker-assisted gene pyramiding with a view to improving important salt-tolerant rice genotypes. Another problem to overcome is how to transfer these genes quickly and effectively into elite genotypes. Many novel breeding methods, such as genome editing techniques, gene pyramiding, mutmap, and knock-out/in could improve salt-tolerant rice varieties for commercial cultivation. More research should be done on cloning salinity-resistant genes and understanding the regulatory mechanisms of salt responsive genes and looking into how several genes can be introgressed simultaneously with stable inheritance. One of the challenges for all researchers is the limited parental resources for the conventional approach and the complexity of salinity stress. Wild rice species can contribute to breeding salt-tolerant rice varieties using genome editing techniques. Modern breeding tools and strategies such as marker aided selection, genome-wide association, genome editing techniques, and genetic engineering are all rapid advancements in genomic research that can be utilized to improve salt-tolerant rice varieties to minimize the effect of salinity.