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 culti-vars are the potential reservoirs of novel alleles to improve abiotic stress tolerance. Accurate pheno-typing 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, Naxl 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+

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

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 fDD) 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. spon-taneum 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 g-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 g-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.

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, soma-clonal 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 salttolerant 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.

( In vitro generation of calU

I Proliferation and maintenance of calli |

Selection of callus on salt containing media

Isolation of salt tolerant calli]

Regeneration of salt tolerant crop plants |

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

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 salttolerant 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.

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

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

Pepper Dihaploid 2012

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 chromo some 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 hap-loids and DHs from diploid plants (Germana 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 back-cross 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.

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).

Bonilla et al. (2002) identified a major QTL, designated Saltol, on chromosome 1, using an RIL population between the highly tolerant lan-drace 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

Table 4.1 Molecular markers associated with quantitative trait loci for salt tolerance in rice

Molecular markers

QTLs

Cross

References

Chr 1

RM8094, RM10793,

Standard Evaluation System

Pokkali X IR29

Alam et al. (2011)

SKC1, RM493

(SES) score

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