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Approaches to increasing the salt tolerance of Indian Mustard (Brassica junceaL. 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 stressesin 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 ofBrassica. 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 andB. 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 otherprocesses 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 lessavailable.

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.

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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 salt-

sensitive 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.

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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 ofthe gene pool beyond that available in nature. Cell and tissue culture techniques have beenused 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 andB. 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 bothB. 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 ofB. 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 non-

selected 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 theimprovement 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

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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

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cytosolic calcium perturbation, which is sensed by SOS3 and accordingly changes its conformation in a Ca2+

-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 non-

conventional 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 QTLsdo 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

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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 werecompared 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 identifythe transcriptional factors that regulate functionally related genes since these factors may also be involved

in regulation of quantitative traits such as salinity tolerance.

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