Brassica Breeding for Salt stress Tolerance: Mendelian to Molecular Approaches

Vijayata Singh and Jogendra Singh

ICAR-Central Soil salinity Research Institute, Karnal-132001, Haryana (India)
Production and productivity of Brassica in the world is affected by number of abiotic stresses
[drought (17%), high temperature (20%) and salinity (50-90%)] which causes significant
reduction in production. Salt stress affects normal growth and development of crops by
disturbing the cellular osmotic status and ion homeostasis. Salinity tolerance is a very complex
trait regulated by several independent and/or inter-dependent pathways and acquired by specific
modulation of gene expression leading to multitude of changes in physiology and biochemistry
at the cellular level. Direct selection for salt tolerance is difficult as the soil shows patches and
many times selection may be misleading.
Cellular ion homeostasis and photosynthesis are two key components of salt stress
tolerance which often gets perturbed under stress by activating a complex network of genes.
DNA markers permit mapping of polygenic traits or quantitative trait loci (QTL) which is not
possible through conventional plant breeding techniques. However, limited sequence information
and genomic resources available, exploiting genetic potential for genome-assisted breeding and
trait improvement in B. juncea is greatly hampered. Recently highthroughput genotyping
techniques are developed which allow marker aided screening of many genotypes. In this
context, whole transcriptome analysis and Genome-by-Sequencing is a pre-requisite to
investigate the global changes in gene expression in response to salinity stress and understand the
functional genetic basis of salinity tolerance in B. juncea.
1. Mendelian or classical approaches
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/germplasm 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 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.
Molecular approaches
Genotyping By Sequencing (GBS)
GBS is a method that uses Illumina HiSeq2000/ 2500 nextgeneration sequencing platform to
discover and score tens to hundreds of thousands of SNP markers in hundreds of individuals in
model or non model organisms. It is a complexity reduction technique designed to reliably
interrogate a segment of genome, the method involves cutting down a genome anywhere from
0.1 to 15% with at least one restriction enzyme and sequencing the ends of the resulting
fragments for either genetic marker discovery or genotyping. It allows you to interrogate a
scalable number of loci even in a highly heterozygous species that leads to study of QTL
mapping and population genomics. GBS based on the Illumina HiSeq 2000 platform has proven
to be a fast and costeffective means of SNP discovery.
Whole Transcriptome Sequencing
RNASeq refers to the use of high throughput next generation sequencing technologies to
sequence complementary DNA (cDNA) sequences. Successful whole transcriptome analysis
depends on RNA quality and efficient conversion into cDNA libraries. The entire pool of RNA
present in the sample is converted into cDNA copies, sequenced and mapped on to a reference
genome. The reference based approach is preferred for model organisms whose genomes are
available along with proper annotations.
Marker Assisted Selection (MAS)
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 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. Once traits have been
mapped and closely linked markers have been found, it is possible to screen large numbers of
samples for rapid identification of progeny that carry desirable characteristics in early stages
which allows a breeder to reject undesirable genotypes from the program more quickly for
salinity tolerance and also oil seed production. This will help breeders to curtail in the time of
varietal development and also in shifting traditional breeding to marker aided selection.
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 (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, Osmoprotectant and molecular, chaperons, Water
channel proteins, Ion transport, Oxidative stress defenses, Detoxifying enzymes, Proteinases,
Proteins involved in signaling and Transcription factors.

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 saltaffected soils. In another study, it was observed that transgenic Arabidopsis over-expressing the
Cod A 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.
Recent advances in genetic and molecular analysis of Arabidopsis thaliana mutants, ion
transporters and stress signaling 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) signaling pathway, composed of the
SOS1, SOS2 and SOS3 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.
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.
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