Keywords

1 Introduction

Abiotic stresses such as drought, salinity, submergence, extreme temperatures, mineral toxicities, and deficiencies impair crop growth and productivity and threaten global food security (Gao et al. 2007; Witcombe et al. 2008). Among these stresses, salinity is impacting more farm lands worldwide due to poor quality irrigation water, inadequate drainage, salt water flooding of coastal land, and salt accumulation in dry areas (Kijne 2006). Around 21% of world’s irrigated land is estimated to be affected by salinity (Ghassemi et al. 1995) and it continues to be a major problem in the arid and semi-arid regions. The negative impact of climate change on food crops is well recognized. Global temperatures are estimated to rise between 1.1°C and 6.4°C during the next century (IPCC 2007). The increased temperatures will disrupt weather patterns, leading to regular occurrence of floods, drought, and salinity. Melting ice caps and glaciers are expected to cause a rise in sea level (Wassmann et al. 2004; Melloul and Collin 2006), which may seriously affect crop productivity in coastal areas due to increased soil salinity.

World population is increasing at an alarming rate and it is expected to grow from 6 billion today to nearly 8.3 billion by 2030 (FAO 2010). With no prospect of expanding arable land due to urbanization, rapid industrialization, and water scarcity in many populous developing countries of the world (Rengasamy 2010), providing food security for the world population will require at least 57% increase in food grain production by 2050 (Wild 2003). Although most major crops have witnessed increased productivity in the past, productivity has been stagnant in recent years and replicating the success of the past to increase food production further may not be easy. Increasing salinity tolerance of the world’s major food crops is an important goal of plant scientists as the world’s population is increasing more quickly than the area of agricultural land to support it (FAO 2010). To alleviate the negative impact of salinity on food production, use of halophytic species, improved water management, and enhancing salinity tolerance in major field crops have been advocated. But improving adaptation of crop plants in saline environments remains a challenging task due to complex genetic basis of salt tolerance mechanisms. In this chapter, we discuss about different approaches for the development of salinity-tolerant crop plants to boost food grain production.

2 Effect of Salinity on Crop Plants

Salinity inhibits seed germination and alters the physiology and anatomy of the plants resulting in reduced crop yield. Salt accumulates in soil due to movement of salty water from adjacent areas during flooding or poor quality irrigation water. When water recedes after flooding, salt from lower soil profiles comes to the soil surface due to the capillary movement. Salinity is a soil condition characterized by a high concentration of soluble salts, mostly chloride and sulfates of sodium in the soil. Soils are classified as saline when the electrical conductivity (EC) is 4 dS/m or more, which is equivalent to approximately 40 mM NaCl and generates an osmotic pressure of approximately 0.2 MPa (USDA-ARS 2008). Both chloride and sodic salts cause damage to the root system of crops. The chloride-triggered injury is identifiable by the extensive leaf blade scorching symptoms whereas the accumulation of sodic salts results in leaf mottling and leaf necrosis. Soil salinity creates both osmotic and ionic stresses in plants. Presence of salts in the soil solution reduces the ability of the plant to take up water, and this leads to reduction in the growth rate, referred as the osmotic or water-deficit effect of salinity. If an excessive amount of salt enters the plant in the transpiration stream, it causes injury to cells in the transpiring leaves, resulting in further reduction in plant growth. This is called the salt-specific or ionic effect of salinity (Greenway and Munns 1980). Salinity stress is finally quantified as a decrease in water potential. Plants resist low water potential in different ways by accumulating osmolytes and modifying the properties of cell walls through production of protective proteins (Verslues et al. 2006). Plants differ greatly in their tolerance to salinity, as reflected in their different growth responses. Most of the commonly used food crops are sensitive to salinity (Flowers and Colmer 2008). Among cereals, rice (Oryza sativa) is the most sensitive and barley (Hordeum vulgare) is the most tolerant. Bread wheat (Triticum aestivum) is moderately tolerant than durum wheat (Triticum turgidum ssp. durum). The variation in salinity tolerance in dicotyledonous species is even greater than in monocotyledonous species. Rice, wheat, and barley are the most extensively studied cereal crop plants for understanding the physiological and molecular basis of salt tolerance. The comprehensive survey of salt tolerance for crops and pasture species published by the US Salinity Laboratory (Maas and Hoffman 1977; USDA-ARS 2005), presents a threshold salinity below which there is no reduction in yield, and then a linear reduction in yield with increasing salinity. Salinity reduces the rate of leaf expansion, and closes stomata and thereby reduces photosynthesis, through the soil water deficit caused by the osmotic stress (Rahnama et al. 2010). Upon exposure to soil salinity, plants accumulate toxic concentrations of Na+ in leaves, which impose an additional limitation to growth by reducing the longevity of photosynthetic tissues (Munns 2002). The control of Na+ transport and its effective exclusion from the mesophyll cells of leaves is therefore an important requirement for salinity tolerance. Na+ exclusion from leaves is associated with salt tolerance in cereal crops including rice (Asch et al. 2000; Haq et al. 2010), durum wheat (Munns and James 2003), bread wheat (Cuin et al. 2009, 2010), barley (Shavrukov et al. 2010), and its wild relatives (Garthwaite et al. 2005), tall wheatgrass (Colmer et al. 2006), and Triticum tauschii (Schachtman et al. 1991). The major components that govern salt tolerance are reduced salt uptake or salt exclusion, enhanced K+/Na+ ratio, tissue tolerance, closure of stomata, upregulation of antioxidant system for protection against reactive oxygen species (ROS), synthesis of osmolytes, water use efficiency (WUE), early flowering, and vigorous growth to dilute the salt concentration in plant tissue (Colmer et al. 2005; Ismail et al. 2007).

3 Approaches for Developing Salt-Tolerant Crop Plants

3.1 Conventional Approach

3.1.1 Germplasm Screening and Classical Breeding

The success of the crop-breeding program largely depends on the availability of natural genetic variation among the germplasm resources. Large number of cultivated and wild germplasm in major crops, preserved in the Consultative Group on International Agricultural Research (CGIAR) institutions and national centers, provide unique resources for systematic screening for discovery of novel variability to improve adaptation of crop plants in saline environments. Particularly, the wild relatives, land races, and traditional cultivars are the potential reservoirs of novel alleles to improve abiotic stress tolerance. Accurate phenotyping procedures are critical for identifying useful germplasm for crop improvement program as well as for deciphering the genetic basis of the mechanisms associated with salinity tolerance. Several parameters for salinity tolerance are studied by growing the germplasm in a variety of culture techniques such as hydroponics, pot culture, and field screening. Plant materials are evaluated from germinating seeds through seedlings up to mature plants. Salinity causes not only ion toxicity and imbalance, but also reduces photosynthesis in plants. Classical screening methods are based on assessment of yield responses to salt stress. Although screening based on yield represents the combined genetic and environmental effects on plant growth and includes integration of the physiological mechanisms conferring salinity tolerance at the whole plant level, it is more convenient and practical if indirect indicators of salt tolerance can be employed at the whole plant, tissue, or cellular levels (Ashraf and Harris 2004). Faster screening methods can be employed for identification of potential parents in a breeding program through selection for high leaf K+/Na+ ratios in the presence of salinity, and high K+/Na+ discrimination that has been described as a physiological index for salinity tolerance in bread wheat (Dvorak et al. 1994), durum wheat (Munns et al. 2000), and rice (Asch et al. 2000).

Gregorio et al. (1997) used Standard Evaluation System (SES) for rice seedling salinity screening based on the percent of leaf damage, which in turn is used to assign the SES scores. These SES scores measure overall survival and/or vigor of the plant and are therefore good indicators of performance of the plant under stress. SES scores as well as low Na+ uptake, K+ uptake and low Na+/K+ ratio have been reported to be tightly linked to seedling salinity tolerance (Lee et al. 2003a, Lisa et al. 2004). Analysis of Na+ uptake, K+ uptake and Na+/K+ ratio of rice seedlings under salt stress, however, are difficult to measure in the large populations associated with breeding programs. Therefore, screening for salinity tolerance at seedling level using SES method is ideal.

Artificial salinized soil in pots or irrigation with saline water under field condition has been used in rice (Aslam et al. 1993). Absolute shoot fresh and dry weights along with percent mortality at seedling stage in rice under salinity stress revealed an efficient, reproducible, reliable, and simple method for assessing relative salinity tolerance in breeding program (Aslam et al. 1993). Physiological characters such as leaf area index (LAI), measurement of Na+, K+, and Ca+ along with amount of photosynthetically active radiation (PAR) absorbed by plants under salinity stress have been used to evaluate salt tolerance in rice (Zeng et al. 2003b). Leaf area index (LAI) was found to be significantly contributing to the yield of grain than other physiological parameters under salt stress. Salt tolerance as defined by the grain yield and amount of PAR absorbed by a plant was found to be strongly related to LAI (Zeng et al. 2003a). Nutrient solution popularly known as Yoshida medium (Yoshida et al. 1976), supplemented with different concentrations of NaCl, is commonly used for salt tolerance screening in rice. A number of cultivars, landraces, and advanced breeding lines such as Pokkali, Nonabokra, SR26B, Damodar, Cheriviruppu, CSR11, Getu, FL378, FL 478, IR 51500-AC17, IR 51500-AC11-1, IR 4595-4-1-13, IR 51491-AC10 have been identified as useful sources for salt tolerance in rice (Dwivedi et al. 2010). Using these lines as donors, few salt-tolerant lines have been released (Gregorio et al. 2002; Ismail et al. 2007). Large number of introgression lines with enhanced abiotic stress tolerance have been developed in a massive backcross breeding program using three recurrent parents and 203 donor lines with tolerance to several abiotic stresses in rice (Ali et al. 2006).

Genetic variation in two key physiological traits, leaf Na+ and K+/Na+ discrimination ratio, among the genotypes of barley and wheat, indicates the possibility of genetic improvement of salt tolerance. Bread wheat, which maintains a lower shoot Na+ concentration than durum wheat, restricts Na+ transport to leaf tissues through Na+ exclusion and maintains high selectivity of K+ over Na+, while barley is not so efficient with respect to these traits. However, the adverse effects of Na+ within leaves of barley are minimized by its compartmentalization into vacuoles (with Cl-) in leaves by a mechanism known as tissue tolerance (Munns and James 2003; Colmer et al. 2005) and the production of organic solutes to osmotically balance the cytosol (Garthwaite et al. 2005). Khan et al. (2006) compared performance of 16 wheat genotypes under saline condition using gravel culture technique in lysimeters with four salinity levels, i.e., control (1.5 dS m−1), low saline (6.0 dS m−1), medium saline (9.0 dS m−1), and highly saline (12.0 dS m−1). On the basis of less than 50% reduction in yield and yield components, they found five genotypes viz. LU-26 s, HT-45, ESW-9525, V-8319, Sarsabz were tolerant, whereas Bhittai, Marvi, Chakwal-86, DS-17, Sussi (SD-66), Zardana were medium tolerant, SD1200/51, Khirman, V-7012 medium sensitive, and RWM-9313, SH-43 sensitive. Tolerant wheat genotypes were successful in maintaining low Na+ and high K+ uptake and high K+/Na+ ratio. A durum wheat line 149 has low Na+ concentrations and high K+/Na+ ratios in the leaf blade (Munns et al. 2000) due to presence of two genes, Nax1 and Nax2, which are responsible for exclusion of salts from leaves and roots, respectively (Munns and James 2003). However, the concentration of Na+ in shoots of line 149 as a whole was not as low as bread wheat (Husain et al. 2004), suggesting retention of Na+ in the leaf sheath. The physiological traits associated with Na+ accumulation include the rate of transfer of Na+ from the root to the shoot (net root xylem loading) and this was much lower in Line 149 than the durum cv Tamaroi (Davenport et al. 2005). The genotypes did not differ in unidirectional root uptake of Na+. The major differences in Na+ transport between the genotypes were in the rate of transfer to the shoot and the preferential accumulation of Na+ in the leaf sheath versus the leaf blade (Davenport et al. 2005). The K+/Na+ ratio at early seedling stage has been shown to be a convenient and reliable indicator of salt tolerance in wheat and barley (Tajbakhsh et al. 2006; Thalji and Shalaldeh 2007). Stomatal conductance and chlorophyll content in leaves can be measured by a non-destructive, rapid, and simple technique using a porometer and SPAD meter, respectively. El-Hendawy et al. (2007) used net photosynthetic rate, stomatal conductance, and SPAD values to screen wheat genotypes for salinity tolerance and reported genetic variation for these traits. Based on this study, SPAD reading could be used as a potential tool for large-scale screening for salt tolerance.

Wild relatives of wheat and barley exhibit genetic variation for Na+ and Cl- exclusion (Colmer et al. 2005) which provides opportunity to improve salt tolerance through interspecific hybridization. In case of wheat, the diploid progenitors, Aegilops tauschii (DD) and Triticum urartu (AA), and synthetic hexaploid wheat involving Triticum urartu, were superior in K+/Na+ discrimination. Similarly, wild Hordeum species including Hordeum vulgare subsp. spontaneum showed high leaf K+ concentration and enhanced ability to exclude Na+ and Cl-. Wild relatives of wheat, Tall wheatgrass (Agropyron elongatum) and Lophopyrum elongatum provide a source of novel genes for improvement of salt tolerance of bread wheat. A comprehensive list of salt-tolerant germplasm in important cereal crops is reported by Dwivedi et al. (2010).

3.1.2 Mutation Breeding

Creation of novel and useful genetic variation in important agronomic traits is the most important prerequisite for a crop-breeding program. Mutagenic agents, such as X-rays, gamma rays, fast neutrons, and chemical mutagens such as ethyl methane sulfonate have been used to induce mutations in seeds to generate genetic variation for crop improvement (Fig. 4.1). It offers the ­possibility of inducing desired characters that either cannot be found in nature or have been lost during evolution. The mutagen treatment breaks the nuclear DNA and during the process of DNA repair, new mutations are induced randomly. The mutants with abiotic stress tolerance can be selected by plant breeders to develop salinity-tolerant crop plants. The purpose of induced mutations is to enhance the mutation frequency rate in order to select appropriate variants for salinity tolerance. The FAO/IAEA Mutant Variety Database (MVD) collects information on plant mutant varieties (cultivars) released officially or commercially worldwide. However, with international collaborative effort coordinated by IAEA and FAO, more than 2,700 mutant varieties with one or more useful traits from induced mutations (mainly from X-rays and γ-rays) have been released in 170 different plant species all over the world. Diamant variety in barley, created by irradiation of dormant seeds with X-rays, has very high yield, short stem, very good grain, and malting quality as well as lodging resistance. Calrose 76 was the first semi-dwarf table rice variety released in the US produced by irradiation with γ-rays. Some outstanding examples of mutant rice cultivars are VND 95-20 and VND 99-3 which have long grains with excellent grain quality and wide adaptation to acid sulphate soil and salinity. These mutants not only increased biodiversity, but also provided valuable breeding material for crop improvement. The database of mutant varieties can be found at the web http://mvgs.iaea.org/AboutMutantVarieties.aspx, maintained by Plant Breeding and Genetics section of joint IAEA/FAO program.

Fig. 4.1
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A generalized approach for developing salinity-tolerant crop plants using mutation breeding

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3.2 Tissue Culture Approach

Plant tissue culture techniques provide a promising and feasible approach to develop salt-tolerant crop plants. Haploid culture, double haploidy, somaclonal variation, and in vitro-induced mutagenesis has been used to create variability to improve salinity tolerance in crop plants. Cell and tissue culture techniques have been used to obtain salt-tolerant plants through in vitro culture approaches: selection of mutant cell lines from cultured cells and subsequent plant regeneration (Zair et al. 2003; Gandonou et al. 2006; Lu et al. 2007; Queirs et al. 2007) and in vitro screening of plant germplasm for salt tolerance (Arzani and Mirodjagh 1999; Dziadczyk et al. 2003; Lee et al. 2003b; Wheatley et al. 2003; Dasgupta et al. 2008). Cereal tissue culture is of great economic importance for selection of improved cell lines under in vitro condition (Fig. 4.2). Tissue culture has been used as a breeding tool for rapid screening of genetic materials for salt tolerance in wheat (Arzani and Mirodjagh 1999). Houshmand et al. (2005) evaluated salt-tolerant genotypes of durum wheat derived from the in vitro culture in field experiments under both saline and non-saline field conditions as well as under greenhouse condition using salinized solution culture. In spite of the smaller range of genotypes used from the in vitro method, tolerant genotypes performed comparably with those of the field-derived tolerant genotypes for grain yield under saline field conditions. Overall, in vitro selected tolerant genotypes showed significantly better performance for biomass production under high salinity condition than field-derived tolerant genotypes.

Fig. 4.2
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An in vitro procedure for regeneration of salinity tolerant crop plants

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3.2.1 Somaclonal Variants

Somaclonal variation refers to the variation seen in plants that have been originated through plant tissue culture. It is particularly common in plants regenerated from callus. The amount of variation that can be expected under in vitro condition may vary with the clone, age of the clone, and use of selection pressure applied to single cells for salinity stress. Somaclonal variations are stable and occur at high frequencies. Novel gene mutations may result during the tissue culture process. It can be performed in vegetatively or sexually or asexually propagated plants. This approach reduces the time required for the release of new variety compared to mutation breeding and has been useful in breeding programs (Zhu et al. 2000).

3.2.2 Double Haploids

A major limitation of the conventional breeding is the long-time frame in developing varieties. Double haploid breeding enables breeders to produce genetically uniform lines within one generation. This effectively bypasses the lengthy process of self-pollination and selection normally required to produce true breeding genotypes. Double Haploids (DH) are plants that have undergone chromosome duplication from haploid plants. The production of haploids and DHs through gametic embryogenesis is the most effective way for the development of complete homozygous lines from heterozygous parents in comparison with the conventional breeding methods that employ several generations of selfing for getting homozygous plants. DH technique is well established in a range of economically important crop species, including major cereals (Wedzony et al. 2009). Various methods such as chromosome elimination subsequent to wide hybridization, pollination with irradiated pollen, selection of twin seedlings, in vivo or in vitro pollination with pollen from a triploid plant, gynogenesis and pollen embryogenesis through in vitro anther or isolated microspore culture were used to obtain DHs (Forster and Thomas 2005). Anther culture and DH production is influenced by various internal and external factors. A generalized method for haploid and DH production is shown in Fig. 4.3. A list of haploid-derived varieties of asparagus, barley, brassica, eggplant, melon, pepper, rapeseed, rice, tobacco, triticale, and wheat are available at http://www.scri.sari.ac.uk/assoc/COST851/Default.htm. The development of ­protocols to produce haploid and DH plants has significant impact on agricultural systems. Mostly in vitro-derived anther or isolated microspore culture method are preferably used to obtain haploids and DHs from diploid plants (Germanà 2010). Recently, a simple method for synthesizing DHs (SynDH) especially for allopolyploid species has been reported by utilizing meiotic restitution genes (Zhang et al. 2011a ). This method involved three steps: hybridization to induce recombination, interspecific hybridization to extract haploids, and spontaneous chromosome doubling by selfing the interspecific F1s. Zhang et al. (2011a ) used Triticum turgidum L. and Aegilops tauschii Coss, the two ancestral species of common wheat (Triticum aestivum L.) to demonstrate the SynDH method using molecular markers. DHs produced in this way contain recombinant chromosomes in the genome(s) of interest in a homogeneous background. This method does not require special equipment or treatments involved in the DH production, and it can be easily applied in any breeding program. Lee et al. (2003b) produced salt-tolerant DHs rice using anther culture techniques with different genotypes in six F1 hybrids obtained by backcross or three-way cross between indica and japonica differing in salt tolerance. It was found that the efficiency of callus induction and plant regeneration was decreased by NaCl concentration and salt tolerance of donor variety, whereas induction in japonicas was higher than those in indicas. The percentages of callus induction in Gyehwa 5 (japonica, tolerant) and IR61633-B-2-2-1 (japonica, sensitive) were 21.1% and 13.5% on agar medium containing 0.3% NaCl, respectively. In four F1 hybrids, the frequencies of high salt-tolerant DHs were 21.4% and 8.9% in 0.3% NaCl medium and the control, respectively. Therefore, the high frequency of salt-tolerant DHs could be selected in the callus induction medium (0.3% NaCl) and in the combinations crossed with salt-tolerant japonica as the third parent. F1 anther culture has become an effective tool to attain homozygosity of recombinants within the shortest possible time. The technique also offers the opportunity to screen haploid materials at the early stage of tissue culture. This allows recessive mutants to be identified under a variety of selection pressures. This approach was used to develop salt-tolerant homozygous recombinants from diverse cross combinations, which led to the identification of the promising rice varieties IR51500-AC-17, IR51485-AC-1, IR51500-AC11-1, and AC6534-4 for salinity, AC6533-3 for sodicity, and AC6534-1 for dual tolerance (Singh et al. 1992; Singh and Mishra 1995; Senadhira et al. 2002). Rahman et al. (2010) developed DH lines from the crosses involving salt-tolerant IRRI-derived lines using anther culture and in a field study one line AC-1 was promising for cultivation in saline areas of Bangladesh.

Fig. 4.3
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A common procedure for haploid and dihaploid production in crop plants

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3.3 Genomics Approach

Genome-mapping techniques are accelerating identification of exact position and function of individual genes controlling agronomic traits including tolerance to salinity. Striking similarities among the genomes of different crop species has been helpful in expanding the genetic variability of important traits for crop improvement. The scope and precision of current breeding programs are enhanced due to use of linked markers for selection of desirable alleles for the target traits.

3.3.1 QTLs for Components of Salinity Tolerance

Plant adaptation to unfavorable environments is governed by morphological, physiological, and unique genetic architecture. By integrating physiological and genetic strategies, we can obtain a better understanding of the molecular basis of crop adaptation thus paving the way toward a more targeted breeding approach for enhancing abiotic stress tolerance in crop plants. QTL mapping is revealing genetic components of salt tolerance for genetic improvement of existing varieties. QTLs controlling salinity tolerance related traits have been mapped in several mapping populations in major field crops: rice (Table 4.1), wheat (Table 4.2), and barley (Table 4.3). To date, there are over 10,000 mapped QTLs reported for rice and maize in the Gramene database (http://www.gramene.org).

Table 4.1 Molecular markers associated with quantitative trait loci for salt tolerance in rice
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Table 4.2 Molecular markers associated with quantitative trait loci for salt tolerance in wheat
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Table 4.3 Molecular markers associated with quantitative trait loci for salt tolerance in barley
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Bonilla et al. (2002) identified a major QTL, designated Saltol, on chromosome 1, using an RIL population between the highly tolerant landrace Pokkali and sensitive IR29. The QTL accounts for about 45% of the variation for seedling and shoot Na+/K+ ratio. Ismail et al. (2007) mapped the Saltol QTL within 1.2 Mb, which is currently being introgressed into several popular salt-sensitive rice cultivars. But multiple alleles were identified at the Saltol locus when several Pokkali accessions and near-isogenic lines were analyzed (Thomson et al. 2010). Using an RIL population developed from the cross Co39 X Moroberekan, Haq et al. (2010) identified a major effect on QTL for leaf Na+ concentration and K+:Na+ ratio on chromosome 1 which may harbor multiple genes for salt tolerance whose impact varies with stress duration, leaf age, and leaf type. Lang et al. (2001) reported a micro-satellite marker, RM223 on rice chromosome 8, associated with salt tolerance at both vegetative and reproductive stages. Recently, Alam et al. (2011) identified salinity-related QTLs at seedling stage using SES scores and identified Saltol as well as non-Saltol related QTLs at seedling-stage in Pokkali, which may assist in QTL pyramiding and marker-assisted breeding programs. A study by Lin et al. (2004) employing the tolerant indica landrace Nonabokra with the susceptible japonica Koshihikari, identified several large-effect QTLs, including the SKC1 QTL and a QTL for shoot Na+ concentration. While the salt-tolerant landraces Pokkali and Nonabokra were routinely used in the past for breeding, the level of tolerance attained by new lines is always lower than traditional donors (Gregorio et al. 2002), and the existing tolerant varieties seem to be superior in only few traits associated with tolerance. The QTL SKC1, originally detected by its effect on K+ concentration, was cloned by map-based cloning and was identified as the sodium transporter OsHKT8 (Ren et al. 2005). Combining superior alleles underlying the component traits could potentially result in higher levels of tolerance. Based on QTL-linked marker profile, Manneh et al. (2007) identified superior salt-tolerant genotypes of rice to improve selection efficiency while selecting for yield in stress environments. Wang et al. (2011) mapped 16 QTLs for salt tolerance of rice at the seed germination stage and four QTLs with major effect could be useful to improve stand establishment under saline areas. QTLs associated with tolerance at various developmental stages will be needed for more stable performance in salt affected areas.

Improving salt tolerance and productivity wheat is a major challenge in breeding program. In studies involving hexaploid, tetraploid, and diploid types, it was suggested that the D genome of wheat carries gene Kna1 that controls the relative concentration of K+ and Na+ in the shoots of plants grown in saline hydroponic culture (Shah et al. 1987; Gorham et al. 1987, 1997; Gorham 1994). This gene was mapped on to the chromosome 4DL (Dvorak et al. 1994) and then fine mapped as a single gene (Dubcovsky et al. 1996). Ma et al. (2007) used a wheat RIL population derived from the cross Opata85  ×  W7984 (international mapping population) and identified 47 QTLs based on salt tolerance index, salt injury index, biomass, shoot length, root length, chlorophyll content, and proline content on all wheat chromosomes except 1B, 1D, 4B, 5D, and 7D. Ten of these QTLs were effective during germination stage and 37 QTLs were important at the seedling stage. In another study, genetic analysis of wheat Line 149 identified two major genes Nax1 and Nax2 for Na+ exclusion (Munns et al. 2003). Nax1 was located on chromosome 2A by QTL analysis (Lindsay et al. 2004) and has been fine mapped as an Na+ transporter of the HKT gene family, HKT7 (Huang et al. 2006). Nax2 located on chromosome 5A was identified as HKT8 (Byrt et al. 2007). Nax1 removes Na+ from the xylem in roots and the lower parts of leaves, the leaf sheaths, while Nax2 removes Na+ from the xylem only in the roots (James et al. 2006). Nax2 has the same phenotype as Kna1, the QTL for Na+ exclusion and enhanced K+/Na+ selectivity in bread wheat, T. aestivum (Dvorak et al. 1994). Nax2 was shown to be homologous to Kna1 in T. aestivum, namely TaHKT8 (Byrt et al. 2007). The HKT gene family encodes transporters in the plasma membrane that mediate the uptake of Na+ or K+ from the apoplast (Hauser and Horie 2010). They are important for cellular Na+ and K+ homeostasis, and their expression in the stele, particularly in the xylem parenchyma cells lining the xylem vessels, helps in retrieving Na+ from the transpiration stream and thus contributing to Na+ exclusion from leaves (Munns and Tester 2008; Hauser and Horie 2010). James et al. (2011) analyzed a population derived from crossing of a tetraploid durum wheat (Triticum turgidum ssp. durum) and hexaploid bread wheat (Triticum aestivum) by marker-assisted selection (MAS) for hexaploid plants containing either Nax1, Nax2 or both Nax1 and Nax2. Nax1 line decreased the leaf blade Na+ concentration by 50%, whereas Nax2 line decreased it by 30%, and both genes together decreased it by 60%. High Na+ sheath:blade ratio in Nax1 lines conferred extra advantage under waterlogged and saline conditions. The effect of Nax2 on lowering the Na+ concentration in bread wheat was surprising as this gene is already present in bread wheat, putatively at the Kna1 locus. The results indicate that both Nax genes have the potential to improve the salt tolerance of bread wheat. A list of salinity-related QTLs for wheat and barley is given in Tables 4.2 and 4.3, respectively.

3.3.2 Marker-Assisted Selection

The application of QTL mapping provided the means to genetically dissect tolerance traits into discrete QTLs that can then be pyramided into high-yielding varieties using marker-assisted selection (MAS). Marker-assisted selection is the use of molecular markers linked to useful traits to select individuals with desirable genetic makeup during the variety development process. It provides a dramatic improvement in the efficiency with which breeders can select plants with desirable combination of genes. DNA markers should enhance the recovery rate of the isogenic recurrent genome after hybridization and facilitate the introgression of quantitative trait loci necessary to increase stress tolerance (Fig. 4.4). Molecular marker techniques were used successfully to transfer alleles of interest from wild relatives into commercial cultivars (Tanksley and McCouch 1997). Use of permanent mapping populations, such as recombinant inbred lines (RILs) or chromosomal segment substitution lines (CSSLs) enables testing stress-tolerant traits in replicated experiments across different environments, which can help in differentiating the QTLs based on their effectiveness at different stress levels. Once important QTL targets are identified, it can be used as single introgressions in a set of near-isogenic lines (NILs), which will help in identifying the complexity of different traits by limiting the variation between lines to focus only on the locus of interest. The recent advances in genomics have paved the way for clear and reliable methods for MAS in plants starting from QTL identification, NIL development, and fine-mapping to transferring the QTL into popular varieties using a precise marker-assisted backcrossing (MABC) strategy (Collard et al. 2005, 2008 Collard and Mackill 2008; ). MABC involves the manipulation of genomic regions involved in the expression of particular traits of interest through DNA markers, and combines the power of a conventional backcrossing program with the ability to differentiate parental chromosomal segments (Fig. 4.4).

Fig. 4.4
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A schematic representation of marker-assisted backcross breeding procedure for development of introgression lines with enhanced salt tolerance. RP recurrent parent, DP donor parent, MAS Marker assisted selection

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3.4 Transgenic Approach

Progress in genomics is instrumental in discovery and characterization of large number of salt stress-related candidate genes offering unique opportunity for exploiting transgenic technology to enhance salinity tolerance in crop plants. Development of transgenic plants with improved tolerance to abiotic stresses has brought some hope for sustainable agriculture under harsh environmental conditions. Genetic engineering is an attractive option when genes of interest are present in cross barrier species, distant relatives, or non-plant sources (Bhatnagar-Mathur et al. 2008). It is also faster to introduce beneficial genes than the conventional or molecular breeding. Due to the complexity associated with salt tolerance mechanism, effort should be made to introduce and fine tune multiple genes with synergistic effect under suitable stress inducible promoters for controlling their expression at a specific time, in a specific organ, or under specific conditions of stress.

3.4.1 Discovery of Candidate Genes

When a plant is subjected to abiotic stresses, expressions of a number of genes are changed, resulting in altered levels of several proteins and metabolites inside the cell. Altered expression of these genes may be responsible for conferring protection or susceptibility to abiotic stresses. Candidate genes for salinity stress tolerance have been identified from prokaryotic extremophiles, lower eukaryotes, tolerant wild relatives, landraces, cultivars, and T-DNA mutant plants using high throughput techniques such as differential display polymerase chain reaction (DD-PCR), cDNA-amplified fragment length polymorphism (cDNA-AFLP), suppression subtractive hybridization (SSH), serial analysis of gene expression (SAGE), DNA microarray, and two-dimensional gel electrophoresis.

Differentially expressed genes under osmotic stress have been identified by DDRT-PCR in barley, sunflower, and pak-choi (Wei et al. 2001; Liu and Baird 2003; Qiu et al. 2009). Wei et al. (2001) identified a gene encoding the barley vacuolar ATPase subunit B (BSVAP) from salt-sensitive barley cultivar Maythorpe. In barley roots, this enzyme may be involved in the sequestration of Na+ and Ca2+ ions into the vacuole, since the proton gradient produced by the ATPase is used by Na+/H+ and Ca2+/H+ antiporters to drive the uptake of Na+ and of Ca2+ (Taiz 1992). Liu and Baird (2003) isolated 17 cDNA clones that are differentially expressed in sunflower under drought or salinity and 13 of these cDNAs were confirmed by quantitative RT-PCR to be expressed differentially in response to osmotic stress. Qiu et al. (2009) generated 101 cDNA fragments differentially expressed under salt stress in Pak-choi (Brassica campestris L. ssp. chinensis (L.) Makino var. communis Tsen et Lee), and found seven cDNA sequences highly homologous to some known expression genes or the genes related to the signaling pathways in plants under different abiotic stress.

Kumari et al. (2009) applied SSH technique in a salt-sensitive rice cultivar IR64 and a salt-tolerant landrace Pokkali and isolated 1,194 salinity-regulated cDNAs. Gene expression analysis of selected genes using macroarrays and Northern blots indicated that salinity tolerance of Pokkali may be due to constitutive overexpression of many salt responsive genes which are stress inducible in IR64. Few clones mapped near Saltol locus on chromosome 1. Using global gene expression analyses, Walia et al. (2005, 2007) observed gene expression changes in large number of genes under salinity stress in salt-sensitive genotypes compared with the tolerant lines. A total of 164 ESTs were upregulated in the tolerant line (FL478) under salinity stress during vegetative stage (Walia et al. 2005), whereas during panicle initiation stage, 292 genes were upregulated and 346 genes were downregulated in salt-sensitive japonica M103. Interestingly, in the salt-tolerant japonica only 54 were upregulated and 54 downregulated (Walia et al. 2007). Cotsaftis et al. (2011) carried out root-specific transcriptome studies in contrasting genotypes of rice (FL478, IR29, Pokkali and IR63731) under salinity stress and identified several gene families with known links to salinity tolerance.

Chen et al. (2003) used cDNA-AFLP to analyze differentially expressed genes in wheat under salinity stress and found a large number of gene fragments related to salt stress. One of the cDNA encoding glycogen synthase kinase-shaggy kinase (TaGSK1) was induced by NaCl stress as a part of the signal transduction component. This technique has also been used to isolate differentially expressed ESTs under salinity stress in soybean (Akoi et al. 2005) and Spartina alterniflora (Baisakh et al. 2008). An expressed sequence tag (EST) analysis in a grass halophyte Spartina alterniflora at early stages of salt stress (Baisakh et al. 2008) produced 1,227 quality ESTs of which 27% of ESTs represented genes for stress response. Transcript abundance analysis of eight known genes of various metabolic pathways and nine transcription factor genes showed temporal and tissue-dependent variation in expression under salinity stress.

A combination of genetic mapping and bulked transcriptome profiling was used by Pandit et al. (2010) resulting in identification of two genes, an integral transmembrane protein DUF6 and a chloride cotransporter, which colocalized within the QTL interval. Walia et al. (2006) performed expression analysis of genes in barley (Hordeum vulgare L.) during salinity stress at the seedling stage of barley using microarray. Genes, differentially regulated by salinity, were also associated with various abiotic stresses such as low temperature, heat stress, and drought stress. Expression level of genes related to jasmonic acid (JA) biosynthesis and jasmonic acid-responsive genes (JRGs) was found to be differentially regulated. Jasmonic acid may function as a “master switch” for stress-induced signaling pathway leading to changes in gene expression (Wasternack et al. 1998). It has been reported that JA and ABA affect gene expression in a synergistic manner through more or less independent signaling pathways (Ortel et al. 1999). This indicates that ABA signaling pathway is also activated along with JA pathway in response to salinity stress in barley. Ozturk et al. (2002) analyzed drought and salinity-induced responses in barley (Hordeum vulgare L. cv. Tokak) transcriptome using cDNA microarray and reported alteration in 5% of the genes under salinity stress compared to 15% under drought stress. Upregulation under both drought and salt stress was restricted to ESTs for metallothionein-like and LEA proteins, while increases in ubiquitin-related transcripts characterized salt stress.

Yan et al. (2005) investigated the salt stress-responsive proteins in the root of rice (Oryza sativa L. cv. Nipponbare) in proteomics approach using 3-week-old seedlings treated with 150 mM NaCl for 24, 48 and 72 h. Two-dimensional gel electrophoresis showed 34 upregulated and 20 downregulated proteins. Mass spectrometry analysis could identify 12 spots representing 10 different proteins involved in regulation of carbohydrate, nitrogen, and energy metabolism, reactive oxygen species scavenging, mRNA and protein processing, and cytoskeleton stability. Comparative proteomic analysis in two contrasting hybrid rice variety by Ruan et al. (2011) led to identification of new components involved in salt-stress signaling. One protein that was upregulated during salt stress was homologous to cyclophilin 2 (OsCYP2). Chitteti and Peng (2007) reported the differential expression of phosphoproteome under salinity stress in the root of rice. They identified 17 differentially upregulated and 11 differentially downregulated putative phosphoproteins. Witzel et al. (2009) conducted two-dimensional gel electrophoresis using a series of hydroponics-based salinity stress experiments in contrasting genetic mapping parents of barley cvs Steptoe and Morex. The proteome analysis of roots from both genotypes revealed cultivar-specific and salt stress-responsive protein expression. Twenty-six proteins could be identified by mass spectrometry. Among those, two proteins involved in the glutathione-based detoxification of reactive oxygen species (ROS) were more abundant in the tolerant genotype.

The identification of various abiotic stress-specific changes in gene expression has been achieved by comparing gene expression in non-induced and salinity stress-induced tissues or by comparing contrasting cultivars (Sahi et al. 2003; Baisakh et al. 2008; Karan et al. 2009; Kumari et al. 2009). In one such study, it was noted that less number of genes were induced under salt stress in Thellungiella halophila (a salt-tolerant relative of Arabidopsis) in comparison to Arabidopsis (Inan et al. 2004). This indicated that the stress tolerance of Thellungiella halophila may be due to constitutive overexpression of a few salt tolerance related genes which are stress inducible in Arabidopsis (Taji et al. 2004). Sengupta and Majumder (2009) analyzed changes in leaf protein expression under salt stress in the wild halophytic rice Porteresia coarctata and salt-sensitive Oryza sativa and identified 16 proteins involved in osmolyte synthesis, photosystem functioning, RubisCO activation, cell wall synthesis, and chaperone functions. These differentially regulated genes in different plant species may serve as candidates to improve salinity tolerance in crop plants using transgenic approach.

3.4.2 Transformation in Crop Plants

Most common genes used for genetic engineering of stress-tolerant plants include transcription factors, signal transduction genes, water channel proteins, ion transporters, detoxifying genes, molecular chaperones, dehydrins, and osmoprotectants (Table 4.4).

Table 4.4 Examples of transgenic intervention in crop plants to improve salt tolerance
Full size table

Calcium acts as a secondary messenger in various signal transduction pathways in plants.

Xu et al. (2011) isolated a calcium-binding multi-stress-responsive gene OsMSR2 from rice which is strongly upregulated by a wide spectrum of stresses, including cold, drought, and heat in different tissues at different developmental stages of rice. Expression of OsMSR2 gene into Arabidopsis conferred enhanced tolerance to high salt and drought through ABA mediated pathway. Calcium-dependent protein kinases (CDPKs) regulate downstream components in calcium signaling pathway. Rice has 29 genes for CDPKs and constitutes a large multigene family. Asano et al. (2011) used mini-scale full-length CDNA overexpressor (FOX) gene hunting system and generated 250 independent transgenic rice plants overexpressing individual rice CDPKs. Transgenic rice plants over expressing OsCDPK21-FOX had more survival rate under salinity stress than wild type plants and also found to be involved in the positive regulation of ABA and salt stress signaling pathways.

The members of NAM, ATAF, and CUC (NAC) transcription factor family has been used to improve abiotic stress tolerance through genetic engineering. Transgenic rice plants constitutively overexpressing OsNAC6 showed tolerance to dehydration, salinity, and blast disease (Nakashima et al. 2007). However, the plants exhibited growth retardation and low reproductive yields. By utilizing stress-inducible promoters, such as the native OsNAC6 promoter, it may be possible to reduce negative impact on plant growth and reproduction. SNAC1 (Stress responsive NAC1), which is expressed in guard cells under drought stress, improved both salt and drought tolerance in transgenic rice plants with upregulation of many stress-responsive genes (Hu et al. 2006). OsNAC5 is induced by abiotic stresses such as salinity, drought, cold, abscisic acid, and methyl jasmonic acid and interacts with OsNAC5, OsNAC6 and SNAC1. Transgenic plants overexpressing OsNAC5 had improved tolerance to high salinity compared to control plants (Takasaki et al. 2010).

Li et al. (2010a) isolated and characterized a basic helix-loop-helix (bHLH) protein gene OrbHLH001 from wild rice (Oryza rufipogon), which encodes an ICE1-like protein containing multiple homopeptide repeats and is induced by salt stress in the shoots. Transgenic Arabidopsis expressing OrbHLH001 enhanced salt and freezing tolerance independent of a CBF/DREB1 cold-response pathway. Oh et al. (2005) reported that DREB1A and ABF3 rice transgenic lines did not show any stunted growth despite constitutive expression. However, transgenic plants exhibited drought and salinity stress tolerance but low level of freezing stress tolerance. DREB homologs have been isolated from rice: OsDREB1A and OsDREB1B are cold inducible, OsDREB2A is induced by dehydration and salinity stress (Dubouzet et al. 2003). Over-expression of the OsDREB1 gene in rice and Arabidopsis correlated with improved salt, drought, and low temperature tolerance (Ito et al. 2006). OsDREB1F is one of the most upregulated genes isolated from upland rice. The transgenic plants harboring OsDREB1F gene led to enhanced tolerance to salt, drought, and low temperature in both rice and Arabidopsis (Wang et al. 2008). The HARDY (HRD) gene, an AP2/ERF-like transcription factor from Arabidopsis enhanced drought resistance and salt tolerance, accompanied by an enhancement in the expression of abiotic stress associated genes (Karaba et al. 2007).

The Na+/H+ antiporters play an important role in the maintenance of cellular ion homoeostasis, cytoplasmic pH regulation, and cell turgor leading to salt tolerance in plants (Horie and Schroeder 2004). Ohta et al. (2002) used a NHX1 gene from a halophyte Atriplex gmelini to generate transgenic rice lines which showed eightfold higher activity of this gene compared to the wild rice. These transgenic lines survived in 300 mM NaCl for 3 days but the wild types failed to survive. Over-expression of vacuolar Na+/H+ antiporter gene OsNHX1 enhanced salt tolerance in rice (Fukuda et al. 2004), whereas over-expression of the same gene from wheat together with H+-pyrophosphatase (TVP1) improved both salt and drought tolerance in Arabidopsis (Brini et al. 2007). A field trial of wheat transgenic lines expressing AtNHX1 gene exhibited higher grain yield, heavier and larger grains in saline conditions and there was less Na+ and more K+ accumulation in leaves in transgenic lines compared with the non-transgenic lines (Xue et al. 2004).

Garg et al. (2002) overexpressed E. coli trehalose bisynthetic genes (otsA and otsB) under the control of either tissue-specific or stress responsive promoter in transgenic rice plants which accumulated 3–10 times more trehalose and showed better growth, less photo-oxidative damage and favorable mineral balance under salt, drought, and low temperature stress conditions. Transgenic rice overexpressing OsCYP2 encoding a cyclophilin 2 protein showed increased salinity tolerance with lower levels of lipid peroxidation products and higher activities of antioxidant enzymes than wild type seedlings (Ruan et al. 2011).

Halophytes represent an ideal target for understanding the genetic and molecular basis of their adaptation in saline environments (Subudhi and Baisakh 2011). Several types of genes belonging to different metabolic functions have been identified and used for overexpression into glycophytic plants to enhance salinity stress tolerance. Ohta et al. (2002) reported enhanced salinity tolerance in transgenic rice using the vacuolar Na+/H+ antiporter (AgNHX1) from Atriplex gmelini. Similarly, transgene expression of Na+/H+ antiporter (SsNHX1), SsNHX2 (an alternative splicing variant of SsNHX1) from Suaeda salsa in Arabidopsis resulted into higher salt tolerance as well as vigorous growth of transgenics along with higher fresh, dry weights, and Na+ and K+ accumulation compared with the wild type plants (Li et al. 2009). Tau class glutathione transferases genes are specific to plant and important for protecting plants against oxidative damage. Expression of SbGSTU from a halophyte Salicornia brachiata in tobacco led to suppressed growth of transgenic seedlings at higher salt, but these transgenic seedlings had continuous growth significantly better than WT seedlings below 300 mM NaCl (Jha et al. 2010). Jithesh et al. (2006) isolated a cDNA (Sod1) encoding a cytosolic Cu/ZnSOD that accumulated during oxidative stress in the mangrove species, Avicennia marina. Transgenic Pusa Basmati-1 rice plants with Sod1 were more tolerant to ­oxidative, salinity (150 mM NaCl) and drought stresses indicating mangrove as a potential source for other abiotic stress tolerance candidate genes (Prashanth et al. 2008). Majee et al. (2004) isolated and characterized a novel salt-tolerant gene L-myo-inositol 1-phosphate synthase from halophytic rice, Porteresia coarctata. Incorporating this gene into rice and other plant species increased synthesis of inositol under salinity stress and enhanced tolerance to salinity stress (Das-Chatterjee et al. 2006). Jacobs et al. (2011) identified a sodium pumping ATPase gene PpENA1 from the moss Physcomitrella patens, which is not found in higher plants. Expression of PpENA1 gene increased the salinity tolerance of transgenic rice.

4 Conclusions and Future Perspective

Salinity stress causes extensive crop losses in many parts of the world due to lack of salt tolerance in major field crops. Enhancing tolerance to salinity in crops will be an important goal of plant breeders in future to ensure food supply for the growing world population (Flowers 2004). Wide range of variation in level of salt tolerance found in halophytes, wild species, and crop germplasm clearly demonstrates the genetic basis of salt tolerance. Although it is widely recognized that the genetic and physiological basis of salt tolerance in plants is inherently complex due to involvement of multigene controlled traits or mechanisms, lack of thorough understanding of these mechanisms and their contribution toward salt tolerance is a major limitation to develop salt-tolerant plants. Additionally, lack of proper screening methods, low heritability, and low selection efficiency of component traits, and limited knowledge of the interactions among the genotype, plant growth stage, and the timing of stress also contributed to slow progress in breeding salinity-tolerant crops (Flowers and Flowers 2005; Ismail et al. 2007).

Conventional breeding technologies coupled with molecular genetic analysis particularly the QTL mapping studies are revealing important genetic components to enhance salt tolerance in field crops. Salt-tolerant germplasm have been identified and developed by breeding, mutation, and tissue culture techniques. QTL mapping and marker-assisted breeding will be increasingly employed with the effort for systematic dissection and utilization of natural variation in the available germplasm for improving crop performance in saline environments (Collins et al. 2008). Particularly, the wild relatives of crops and landraces hold enormous promise to mine the superior alleles for enhancing crop adaptation under salinity (Tanksley and McCouch 1997; Feuillet et al. 2008).

A large number of candidate genes involved in different salt tolerance mechanisms from diverse sources have been identified. It is expected that modern genomics tools will continue to be employed in model organisms such as Arabidopsis and rice, salt-tolerant crop accessions and wild relatives, and halophytes to provide genetic clues for plant adaptation to salinity through identification and verification of candidate genes (Collins et al. 2008; Subudhi and Baisakh 2011). Genomic technology is expected to contribute significantly toward discovery of useful candidate genes for various component traits which can be targeted to improve elite cultivars using transgenic pyramiding (Takeda and Matsuoka 2008). The utility of transgenic technology can be further enhanced through discovery and exploitation of stress inducible promoters which could enhance salt tolerance with minimum undesirable pleiotropic effect on crop growth and yield. It is conceivable that designing crop genotypes with improved salinity tolerance may be difficult but not impossible. However, collaboration of geneticists, molecular breeders, physiologists, and genomicists will be needed to implement an integrated approach to discover, test, and introgress the superior alleles to enhance salt tolerance in major food crops.