Approaches to increasing the salt tolerance of Indian Mustard (Brassica juncea L.

Czern and Coss) Jogendra Singh and Vijayata Singh Central Soil Salinity Research Institute, Karnal (Haryana)-132001, India Around the globe 932.2 million hectare area is affected with salinity and sodicity stresses (Metternicht and Zinck, 2003), out of which, nearly 6.73 million hectare area is affected by these stresses in India. Further, the arid and semiarid areas in different states are associated with saline underground water, which have to be used for irrigation, due to unavailability or diversion of good quality water to other than agricultural purpose. Use of such water is further rendering the soils unfit for crop cultivation. Rapeseed mustard is the third most important source of vegetable oil in the world and is grown in more than 50 countries across the globe. China, Canada, India, Germany, France, UK, Australia, Poland and USA are the major cultivators of its different species of Brassica. During 20122013, the estimated area, production and yield of rapeseed mustard in the world was 30.74 mha, 60.43 mt and 1.95 tonnes/ha, respectively. Globally, India account for 21.7% area and 10.7% production (USDA 2013). Brassica rapa, B. napus and B. juncea are grown predominantly for oil and seed meal. India is the second largest country in rapeseed mustard production and more than 85% of its area under rapeseed mustard is occupied by Indian mustard B. juncea (L.) alone. The most common adverse effects of salinity on Brassica are the reduction in plant height, size and yield as well as deterioration of the product quality (Zamani et al., 2011). Improved genotypes of mustard with tolerance to high salt along with consumers acceptance and good oil quality are required for obtaining optimum yield and expansion of cultivated area under such stress situation. These concerns prompted an intensive breeding program to develop high yielding cultivars with salinity tolerance at Central Soil Salinity Research Institute (CSSRI). Only halophytes (plants adapted to saline habitats) will continue to grow at salinities over 250 mM NaCl. However, some are useful forage species for saline land. Saltbushes (Atriplex spp.) are very salt tolerant, and can lower water-tables that have reached the surface, and restore saline land for animal production (Barrett-Lennard, 2002). However, in the present scenario information on approaches to increasing the salt tolerance be quite useful for the breeders in the improvement programme to make this crop globally competitive. This article describes physiological mechanisms and selectable indicators of gene action, with the aim of promoting new screening methods to identify genetic variation for increasing the salt tolerance of mustard. Physiological mechanisms that underlie traits for salt tolerance could be used to identify new genetic sources of salt tolerance. Important mechanisms of tolerance involve Na+ exclusion from the transpiration stream, sequestration of Na+ and Cl- in the vacuoles of root and leaf cells, and other processes that promote fast growth despite the osmotic stress of the salt outside the roots. Screening methods for these traits are discussed in relation to their use in breeding. Precise phenotyping is the key to finding and introducing new genes for salt tolerance into crop plants. Salt stress mechanisms According to Stavarek and Rains (1984), plants exposed to saline environments encounter three basic problems: 1. A reduction in water potential of the surrounding environment results in water becoming less available. 2. Toxic ions can interfere with the physical and biochemical processes of the organism. 3. Required nutrient ions must be obtained despite the predominance of other ions.

Different strategies have been adopted by plants to overcome the difficulties of water stress. According to Jones (1987), plants avoid salt stress through osmotic adjustment. This may be achieved primarily by: o Restriction of inorganic ion uptake and the biosynthesis of organic solutes. o Absorption of inorganic solutes from the soil environment where features minimizing ion toxicities must be brought into play. This raises the question of whether selection should be made for ion exclusion or accumulation. In part this will be depend.ent upon the availability of genetic resources for either mechanism. The mechanism, of salt exclusion has been one of the most frequently reported to differentiate between saltsensitive and tolerant crop cultivars (Shannon, 1982). Strategies in osmoregulation research According to Moore (1984), there are three major research strategies in developing plants with increased tolerance to saline conditions, although they are not mutually exclusive or exhaustive: i) Plant breeding from existing gene pools. ii) Cell culture with subsequent plant breeding. iii) Genetic engineering. Plant breeding from existing gene pools This approach involves utilizing existing genotypes, including wild species, and subjecting them to a highly saline environment. Plants that survive and produce economic fields are considered tolerant and are used in further breeding work to develop varieties acceptable for cultivation on a commercial scale (Ramage, 1980). This approach has one negative factor: the potential increase in salt tolerance in a species is limited by the variability of the existing gene pool. According to Jones (1987), improvement in plants requires the presence of genetic variability and the expression of this variability through phenotype. The most common approach to identify sources of variability for breeding for salt tolerance has been looking among primitive cultivars, landraces, wild species, and world collections for those which exhibit characteristics for salt tolerance. The wild progenitors have been, and will continue to be, successfully exploited by plant breeders as sources of useful genes for crop improvement. The majority of plant breeders working on bio-saline problems have treated salinity stress as NaCl or mixed with CaC12. While convenient, such artificial formulations do not reflect the major ion compositions of naturally occurring saline soils (Epstein and Rains, 1986). Expectations that a line selected for tolerance to NaCl alone will exhibit equal performance under ionically more complex saline conditions are unreasonable. Jones (1987) believes that considerations must be given to breed for production in a specific saline environment, mimicking this environment. In any given environment, the concentration of soluble salts changes temporally and spatially. Sources of irrigation water are also likely to change in their quality during the course of the growing season. These represent important variables that must be monitored and assessed for the development of appropriate breeding strategies. Continuous efforts of CSSRI resulted in development and release of three high yielding salt tolerant varieties of mustard; CS 52, CS 54 and CS 56 for the country and several other advanced breeding lines/germplasms are in the pipe lines of testing and development.

Cell culture techniques Plant cell culture is one of the methods scientists have for studying how plants tolerate stress and for producing and selecting genetically superior plants. This technique is often praised a means to perform selection in several Petri dishes which would take hundreds of acres if performed at the whole plant level. Moreover, in this approach, cells can be subjected to mutagenic agents in order to expand the variability of the gene pool beyond that available in nature. Cell and tissue culture techniques have been used to obtain salt tolerant plants employing two in vitro culture approaches. The first approach is selection of mutant cell lines from cultured cells and plant regeneration from such cells (somaclones). In vitro screening of plant germplasm for salt tolerance is the second approach, and a successful employment of this method in durum wheat is presented here. Doubled haploid lines derived from pollen culture of F1 hybrids of salt-tolerant parents are promising tools to further improve salt tolerance of plant cultivars. Enhancement of resistance against both hyper-osmotic stress and ion toxicity may also be achieved via molecular breeding of salt-tolerant plants using either molecular markers or genetic engineering. While assessing cellular responses of two rapid-cycling Brassica species, B. napus and B. carinata, to seawater salinity, He and Cramer (1993) showed callus tolerance similar to that of whole plants. B. napus was more salt-tolerant than B. carinata, consistent with the response of whole plants of the same species to seawater salinity. The pattern of accumulation of Na+, Cl, K+, and Ca2+ in both B. napus and B. carinata at the cellular level was similar to that at whole plant. Sunita et al. (1996) selected some highly salt-tolerant lines of B. juncea using in vitro selection procedures. They compared the in vitro selected salt-tolerant lines with a non-selected somaclone with regard to vegetative growth and seed yield, and accumulation of ions and organic osmotica under salt stress. The in vitro salt-tolerant lines had higher biomass and seed yield and maintained lower levels of Na+ and Cl and higher of K+ as compared to nonselected somaclone under salt stress. In contrast, no consistent pattern of accumulation of organic solutes such as quaternary ammonium compounds, glycine betaine, and choline was found in the lines differing in salt tolerance. The above-mentioned reports indicate that the lines of different brassicas selected using different in vitro protocols maintained their salt tolerance when tested as adult under saline conditions. Maintenance of a degree of salt tolerance by cell lines as adult is of great practical value. In addition, since very few brassicas have been examined in order to uncover the mechanism of salt tolerance at cellular level, it is not yet possible to draw a parallel between salt tolerance and different physiological/ biochemical attributes. Thus, an extensive amount of work is necessary to be carried to elucidate the mechanism of salt tolerance in brassicas at cellular level. Advantages and disadvantages are given by Rains (1981). This includes increased control of environmental factors, a greatly expanded number of treatments and replications with reduced manpower, and a vastly increased potential for selection of salt tolerant variants. Disadvantages include the difficulty of selecting for characteristics that are manifested in subsequent growth stages such as yields. Thus the cells must be regenerated and grown out, thereby providing additional breeding material for standard breeding and selection work. Genetic engineering The ability to introduce DNA sequences in different cells and to monitor their expression opens new methods for plant breeding. Proper expression of introduced genes is not the only major barrier to the improvement of crops via recombinant DNA technology. Identification and isolation of genes that specify salt tolerance are requisites for directed genetic engineering of crop plants. Recent advances raise the

possibility of the development of new plant germplasm through the introduction of any gene from any organism into plants. Several leading laboratories have achieved the transfer and expression of bacterial and foreign plant genes in plant cells. Several predictions can be made regarding the near-term developments of recombinant DNA technology for plant genetic engineering. With the availability of halophytes, it is easy to speculate that the potential shift in the response curve to salinity tolerance for some economical crops. Physiologic or metabolic adaptations to salt stress at the cellular level are the main responses amenable to molecular analysis and have led to the identification of a large number of genes induced by salt (Ingram and Bartels, 1996; Bray, 1997; Shinozaki and Yamaguchi-Shinozaki, 1997). These genes can be classified in groups related to their physiologic or metabolic function predicted from sequence homology with known proteins. Functional groups of genes/proteins activated in salt stress with potential for providing tolerance are; Carbon metabolism and energy production/photosynthesis, Cell wall/membrane structural components, Osmoprotectants and molecular, chaperons, Water channel proteins, Ion transport, Oxidative stress defences, Detoxifying enzymes, Proteinases, Proteins involved in signalling and Transcription factors. Recent advances in genetic and molecular analysis of Arabidopsis thaliana mutants, ion transporters and stress signalling proteins have improved our understanding of the mechanisms of cellular ion homeostasis and its regulation in plants. Since Na toxicity is the principal stress component in saline soils, much research has focused on the identification of ion transporters and regulatory mechanisms that mediate Na+ homeostasis and maintenance of a high cytoplasmic K /Na ratio. The SALT OVERLY SENSITIVE (SOS) signalling pathway, composed of the SOS1, 2 and 3 proteins, has emerged as a key factor in the detection of and tolerance to salt stress. Recent evidence suggests that the SOS pathway may regulate several ion transport mechanisms critical for salt tolerance.
+ +

Fig. Mechanism of salt tolerance: Regulation of ion homeostasis by SOS and related pathways in relation to salt stress adaptation. Salt stress is perceived by an unknown receptor present at the plasma membrane (PM) of the cell. This induces a

cytosolic calcium perturbation, which is sensed by SOS3 and accordingly changes its conformation in a Ca 2+-dependent manner and interacts with SOS2. This interaction relieves SOS2 of its auto-inhibition and results in activation of the enzyme. Activated SOS2, in complex with SOS3 phosphorylates SOS1, a Na +/H+ antiporter resulting in efflux of excess Na+ ions. SOS3SOS2 complex interacts with other salt mediated pathways resulting in ionic homeostasis. This complex inhibits HKT1 activity (a low affinity Na+ transporter) thus restricting Na+ entry into the cytosol. SOS2 also interacts and activates NHX (vacuolar Na+/H+ exchanger) resulting in sequestration of excess Na + ions, further contributing to Na+ ion homeostasis. CAX1 (H+/Ca+ antiporter) has been identified as an additional target for SOS2 activity reinstating cytosolic Ca 2+ homeostasis [Mahajan and Tuteja (2005)].

Marker Assisted Selection (MAS) Strategies to supplement the conventional breeding programs MAS has been seen as a means of improving the speed and efficiency of plant breeding programs because it is growth stage independent, unaffected by environment; no dominance effect and efficient to use in early generations. Most widespread use of MAS to date has done in the marker assisted backcrossing (MAB) of major genes to into already established varieties, mega-varieties (which occupies a large area within the country on across the countries) or elite cultivars. These markers could reduce the linkage may around the target gene, and also recover the recurrent parent background within less number of generation in comparison to conventional breeding. MAS in early generation is most useful for relatively less number of genes but which are affecting the important traits and difficult to phenotype. Two important factor need to be satisfied for effective MAS strategy, first: the markers are tightly linked (1-2 cM) to loci with large effects on trait which are difficult or costly or appear lately (maturity) for accurate phenotyping. Second, specific marker alleles are associated with desired alleles at target loci consistently across the different breeding populations. But unfortunately both of these two situations are not applicable for most traits and most populations (Luby and Shaw, 2001). Most wide use of markers in conventional breeding have been in back crossing purpose where previously evolved varieties or elite material through conventional breeding is augmented with selected alleles with major effects for which they are lacking. Young and Tanksley (1989), demonstrated that large amount of DNA from the donor can remain around the target gene even after many generation of backcrossing. This surrounding material contributes toward linkage drag especially if the donor parent is a wild relative. So markers are used to select the same progeny in which recombination near the target gene have as little chromosome segment as possible. This is called fore-ground selection. Most of the traits of economic importance like yield and stresses are controlled by polygenes and considerably influenced by environment and g x e interaction for their expression. These traits are most difficult to breed conventionally and using nonconventional techniques like MAS as well. DNA markers could be of great importance to plant breeding if they are used to aid selection for quantitative traits. Now -a- days microsatellites or simple sequence repeats (SSR) markers are the first choice of molecules biologist while single nucleotide polymorphism (SNPs) are going to be the most preferred markers of the future. Major difficulty in the employing MAS for polygenic traits is the limited phenotyping accuracy of the quantitative trait loci (QTL) because QTLs do not have direct phenotypic variation hence their chromosomal location is typically inferred by calculating the LOD (likelihood of odds) value. The chromosomal location with maximum LOD value has the likelihood for the QTLs of the trait but there is always a good possibility that the QTL is not located precisely at the maximum likelihood position. This precision could be increased by increasing size of mapping population (Stuber 1998, Zamir, 2001). The detection of QTLs for stress tolerance also represent an important means toward cloning of stress tolerance genes, an achievement that would be very helpful for use in the analysis of the underlying physiological and biochemical mechanisms. A gene, AtNHX1, coding for Na+/H+ antiport from Arabidopsis, was inserted in the genome of cv. Westar of canola (B. napus L.) by Zhang et al. (2001). The transgenic plants of canola so produced showed vigorous growth as compared to the wild-type plants

in 200 mol m3 NaCl. The transgenic plants produced flowers and seeds at this high salt concentration. In addition, the transgenic canola plants were found to be efficient Na+ accumulators. This suggests that despite their value as potential oilseed crop, they could be effectively used to reclaim salt-affected soils. In another study, it was observed that transgenic Arabidopsis over-expressing the CodA gene germinated at even up to 300 mol m3 NaCl (Hayashi et al., 1997), but transgenic Brassica could germinate only up to 150 mol m3 NaCl (Prasad et al., 2000). When transgenic Arabidopsis, B. napus, and tobacco were compared for the stress tolerance, drought tolerance was enhanced only in B. napus, while freezing tolerance was enhanced only in Arabidopsis (Huang et al., 2000). From these reports it is evident that significant improvement in salinity tolerance in brassica may be achieved by engineering of a single gene. Although the genetic engineering and molecular biology approaches have undoubtedly shown the possibilities of gene transfer across organisms and engineering abiotic stress tolerance by manipulation of genes, there are very few reports in the literature on the use of such techniques for the improvement of salt tolerance in potential oilseed brassicas. Conclusion The phenomenon of salt tolerance in mustard is very complex because of their genomic relationships. High salt tolerance of amphidiploids with respect to their diploid relatives, suggests that salt tolerance has been obtained from A and C genomes. This mode of inheritance of salt tolerance in mustard is different to what has been earlier observed in other crops. Selection and breeding, including the use of wide crosses, from one point of view, represent the best short term approach to the development of salt tolerant plants. Cell culture technology, from another point of view, offers several advantages. These include a well-defined media, a huge number of cells can be screened and evaluated and mutagenic agents can be applied to increase variability. Recent advances in cell culture and molecular genetics offer a great potential to expand the options available for plant improvement well beyond those of traditional plant breeding. Some cellular techniques such as embryo and pollen cultures and the efficient use of somaclonal variability should provide practical results in the short term. Protoplast fusion and organelle hybridization will probably be a longer term process, but greatly expand horizons concerning what is potentially feasible. Genetic engineering involves determining the mechanisms within the plant cell which control the plant's response to saline environment, then locating the genetic codes that control these mechanisms, and finally transferring the DNA molecules that control this process into the genome of the plant of interest. The major problems, however, centre on the lack of knowledge about exactly how the control mechanisms within the plant cell operate. Although there are some agreements, with respect to bacteria and yeast, gaining and understanding of complex plants will require further investigation. Efforts are also underway to identify the transcriptional factors that regulate functionally related genes since these factors may also be involved in regulation of quantitative traits such as salinity tolerance. References Barrett-Lennard E.G. 2002. Restoration of saline land through revegetation. Agricultural Water Management. 53: 213226. Bray E. A. 1997. Plant responses to water deficit. Trends in Plant Science. 2: 48-54. Epstein E., Rains D.W. 1986. Advances in salt tolerance. Second International Symposium on Genetic Aspects of Plant Mineral Nutrition. Madison, Wisconsin.

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