Approaches To Increasing The Salt Tolerance of Wheat and Other Cereals

Journal of Experimental Botany, Vol. 57, No. 5, pp.
1025–1043, 2006
Plants and Salinity Special Issue
doi:10.1093/jxb/erj100 Advance Access publication 1 March, 2006
Approaches to increasing the salt tolerance of wheat and

other cereals
Rana Munns1,*, Richard A. James1 and André Läuchli2

1
CSIRO Plant Industry, GPO Box 1600, Canberra, ACT, Australia
2
Department of Land, Air and Water Resources, University of California, One Shields Avenue, Davis,
CA 95616-8627, USA
Received 1 August 2005; Accepted 16 December 2005
Downloaded from http://jxb.oxfordjournals.org/ at Fordham University Library on December 14, 2012

Abstract plants to extract more water. Salt tolerance may have its
greatest impact on crops growing on soils with natural
This review describes physiological mechanisms and
salinity as, when all the other agronomic constraints have
selectable indicators of gene action, with the aim of
promoting new screening methods to identify genetic
been overcome (e.g. disease resistance and nutrient de-
variation for increasing the salt tolerance of cereal ficiency), subsoil salinity remains a major limitation to
crops. Physiological mechanisms that underlie traits agriculture in all semi-arid regions. Even where clearing
for salt tolerance could be used to identify new genetic of land in higher rainfall zones has caused water-tables to
sources of salt tolerance. Important mechanisms of rise and salt to move, improved salt tolerance of crops will
tolerance involve Na1 exclusion from the transpiration have a place. The introduction of deep-rooted perennial
stream, sequestration of Na1 and Cl2 in the vacuoles species is necessary to lower the water-table, but salt toler-
of root and leaf cells, and other processes that pro- ance will be required not only for the ‘de-watering’ species,
mote fast growth despite the osmotic stress of the but also for the annual crops that follow, as salt will be
salt outside the roots. Screening methods for these left in the soil when the water-table is lowered.
traits are discussed in relation to their use in breed- Wheat (Triticum aestivum) is a moderately salt-tolerant
ing, particularly with respect to wheat. Precise phe- crop (Maas and Hoffman, 1977). In the field, where the
notyping is the key to finding and introducing new salinity rises to 100 mM NaCl (about 10 dS m1), rice
genes for salt tolerance into crop plants. (Oryza sativa) will die before maturity, while wheat will
produce a reduced yield. Even barley (Hordeum vulgare),
Key words: Barley, durum wheat, plant breeding, rice, salinity, the most-tolerant cereal, dies after extended periods at salt
sodium. concentrations higher than 250 mM NaCl (equivalent to
50% seawater). Durum wheat (Triticum turgidum ssp.
durum) is less salt tolerant than bread wheat, as are maize
Introduction (Zea mays) and sorghum (Sorghum bicolor) (Maas and
Increased salt tolerance of crops is needed to sustain food Hoffman, 1977; Salt Tolerance Database reproduced on
production in many regions in the world. In irrigated USDA-ARS, 2005).
agriculture, improved salt tolerance of crops can lessen Only halophytes (plants adapted to saline habitats) will
the leaching requirement, and so lessen the costs of an continue to grow at salinities over 250 mM NaCl. Domes-
irrigation scheme, both in the need to import fresh water tication of halophytes as new crops has been reviewed by
and to dispose of saline water (reviewed by Pitman and Colmer et al. (2005), who point out that few species have
Läuchli, 2002). Salt-tolerant crops have a much lower reached the status of crop plant and none has a wide usage.
leaching requirement than salt-sensitive crops. In dry-land However, some are useful forage species for saline land.
agriculture, improved salt tolerance can increase yield on Saltbushes (Atriplex spp.) are very salt tolerant, and can
saline soils. In areas where the rainfall is low and the salt lower water-tables that have reached the surface, and restore
remains in the subsoil, increased salt tolerance will allow saline land for animal production (Barrett-Lennard, 2002).
* To whom correspondence should be addressed. E-mail: rana.munns@csiro.au
ª The Author [2006]. Published by Oxford University Press [on behalf of the Society for Experimental Biology]. All rights reserved.
For Permissions, please e-mail: journals.permissions@oxfordjournals.org
1026 Munns et al.
The halophytic relative of wheat, tall wheatgrass (Thinopy- starting earlier in the former studies, and reducing the yield
rum ponticum, syn. Agropyron elongatum), is grown for potential through the reduction in tiller formation, or it
forage on saline soils. Distant halophytic relatives of barley, may have been due to the number of irrigations being
such as sea barleygrass (Hordeum marinum), are even more less or the ambient conditions hotter and drier so that
salt tolerant (Garthwaite et al., 2005), but are not useful for the salt concentration around the roots was greater (for
forage. The salt tolerance in these halophytic relatives of a given EC) than in the experiments reported by Maas and
wheat and barley has not yet been used to improve the Hoffman (1977). This raises the problem that screening in
tolerance of the related crop species, as the mechanisms one environment may not select the right genotypes for
conferring tolerance to the wild relatives are unknown, and a different environment. Field conditions vary from site to
the genomes do not recombine at meiosis, so a forward site, not only in soil salinity, but also in soil physical and
genetic approach cannot be used. The potential of wild chemical properties such as sodicity, high pH, and possibly
relatives to improve the salt tolerance of wheat is reviewed toxic trace elements such as boron (Rengasamy, 2002).
by Colmer et al. (2006). Further, there are differences across seasons in temperature
and drought, which, particularly in dry-land agriculture,
Relationship between salinity and yield in grain crops will directly affect the build-up of salts around the roots.

Estimates of grain yield bring another complexity to the Differences in flowering or maturity times between geno-
salinity response, not just because the crops must be grown types can cause large differences in yield if the ambient
in uncontrolled environments for long periods of time, but conditions are variable during the flowering or grain-filling
because the conversion of shoot biomass to grain biomass periods.
is complex. The harvest index (the proportion of total For many soils, waterlogging and salinity are inextrica-
shoot mass that is found in grain) can vary from 0.2 to bly linked. In countries like Pakistan, the use of irrigation
0.5, depending on the timing and severity of the salt treat- water of high ‘sodium hazard’ (high sodium absorption
ment (Francois et al., 1994; Husain et al., 2003). A low ratio) causes a decline in the structure of the fine-textured
level of salinity may not reduce grain yield even though the soils, and poor infiltration of water results in salinization
leaf area and shoot biomass is reduced, which is re- (through evaporation of irrigation water at the soil surface)
flected in a harvest index that increases with salinity, and and waterlogging (Qureshi and Barrett-Lennard, 1998). In
the fact that grain yield may not decrease until a given Australia, secondary salinity arises where water-tables rise
(‘threshold’) salinity is reached. The comprehensive survey to within 2 m of the soil surface, i.e. close to the root zone.
of salt tolerance of crops and pasture species published Furthermore, as the soil has air-filled porosities of about
by the US Salinity Laboratory (Maas and Hoffman, 1977; 10%, it takes only 100 mm of rain for water-tables to rise
USDA-ARS, 2005) presents for each species a threshold to the surface, and plants to experience simultaneous salt
salinity below which there is no reduction in yield, and then and waterlogging stresses (Barrett-Lennard, 2002). Rarely
a linear reduction in yield with increasing salinity (a ‘bent are genotypes ranked for salt tolerance in contrasting
stick’ relationship). In that survey, the yield of rice starts to environments. The extent of genotype3environment in-
decline at 3 dS m1 (30 mM NaCl) compared with 6–8 dS teractions is not fully understood, but is likely to be quite
m1 for wheat (60–80 mM NaCl), and the subsequent large.
linear yield decline of rice with increasing salinity is double
that of wheat. The survey highlights the genetic variation Past approaches to improving salt tolerance in wheat
between species. However, in most cases, the data are for Various approaches have been taken to improve the salt
a single cultivar of the species or a limited number of tolerance of wheat by introducing genes for salt tolerance
cultivars at a single site, so they are not necessarily repre- into adapted cultivars, including screens of large inter-
sentative of the species, which can show a large genetic national collections, detailed field trials of selected culti-
diversity. For example, large genetic variation was found vars, conventional breeding methods, and unconventional
with barley and durum wheat grown in the field with drip crosses with wheat relatives. The aim has been to exploit
irrigation of different salinity levels up to 26 dS m1 (Royo variation in salt tolerance within wheat and its progenitors
et al., 2000; Royo and Abió, 2003). These and other studies or close relatives to produce new wheats with more toler-
present a sigmoidal rather than a ‘bent stick’ relationship ance than modern wheat cultivars.
between yield and salinity, leading to the suggestion that Large international collections have been screened in
an EC50 (EC that results in a 50% yield decline) is hydroponic or sand culture by Kingsbury and Epstein
a more useful comparison between genotypes than a linear (1984), Srivastava and Jana (1984), Sayed (1985), and
rate of decline (Royo et al., 2000; Steppuhn et al., 2005). Martin et al. (1994). For example, Kingsbury and Epstein
The studies of Royo et al. (2000) with barley, and (1984) screened 5000 accessions of bread wheat and found
of Royo and Abió (2003) with durum wheat, report a 29 that produced seed in 50% seawater. The results of
30–40% lower salt tolerance than those of Maas and these extensive screens have been summarized by Colmer
Hoffman (1977). This may have been due to treatments et al. (2005), who comment that they were not followed by
Increasing salt tolerance in monocotyledonous plants 1027
+ +
yield trials and that new cultivars have not been developed high selectivity for K over Na (Gorham et al., 1987,
from the outstanding genotypes identified. Jafari-Shabestari 1990). Bread wheats (hexaploid, ABD genomes) have a
et al. (1995) screened 400 Iranian wheats in an irrigated low rate of Na+ accumulation and enhanced K+/Na+ dis-
field site in California and identified several accessions that crimination, a character controlled by a locus (Kna1) on
were consistently high for grain yield in both low and high chromosome 4D (Dubcovsky et al., 1996). The gene or
salinity treatments, but no new salt-tolerant wheat cultivar genes associated with this locus have not been identified.
was developed through breeding as a consequence of the Durum wheats (tetraploid, AB genomes) have higher rates
screening work (CO Qualset, personal commmunication). of Na+ accumulation and poor K+/Na+ discrimination
Little work has been done on breeding for salt tolerance (Gorham et al., 1987; Munns et al., 2000b) and are less
in wheat. Many plant breeders in Australia are aware of the salt tolerant than bread wheat. A locus (Nax1) on chromo-
need for salt tolerance but this is only one of a number of some 2A controlling Na+ accumulation has been found
constraints, the major one being drought, so salt tolerance in an unusual durum genotype (Lindsay et al., 2004), and
is not specifically targeted. Targeted breeding has been a tightly linked molecular marker is being used to intro-
largely confined to India and Pakistan. The most success- duce the trait for low Na+ accumulation into durum culti-
ful releases have been the Indian KRL1-4 and KRL 19, vars in a breeding programme, as described in a following

released by the Central Soil Salinity Research Institute section.
(CSSRI) at Karnal, the Pakistani LU26S and SARC-1, Correlations between grain yield and Na+ exclusion from
released by the Saline Agriculture Research Cell (SARC) leaves, along with the associated enhanced K+/Na+ dis-
at Faisalabad, and the Egyptian Sakha 8, released by the crimination, have been shown in wheat (Chhipa and Lal,
Agricultural Research Centre at Giza. 1995; Ashraf and O’Leary, 1996; Ashraf and Khanum,
In India, almost all salt-tolerant wheat germplasm is 1997), although the relationship does not hold across all
derived from Kharchia 65, a line developed from selections genotypes (Ashraf and McNeilly, 1988; El-Hendawy
from farmers’ fields in the sodic-saline soils of the Kharchi- et al., 2005), showing that Na+ exclusion is not the only
Pali area of Rajasthan (Rana, 1986). KRL1-4, a cross of mechanism of salt tolerance.
Kharchia 65 with WL711, has done well on the saline soils
of northern India, but not in Pakistan, possibly because Improving salt tolerance in other cereals
of the heavier soils and greater problems of waterlog- Barley is one of the most salt-tolerant crops (Maas and
ging (Hollington, 2000). Another derivative of Kharchia 65 Hoffman, 1977). Its greater salt tolerance in the field may
was developed in the UK by SA Quarrie and A Mahmood: derive partly from its rapid growth and fast phenological
a doubled haploid line, KTDH 19, from a cross of development, leading to an early maturity date. When
Kharchia 65 with a line identified with exceptional sodium developmental differences are eliminated, the difference
exclusion, TW161. This derivative performed well in Spain in salt tolerance between barley and wheat becomes less
(Hollington et al., 1994) but in India and Pakistan, al- clear. In a glasshouse comparison of the response to salin-
though highly tolerant in terms of total dry matter, its grain ity in various cereals at the vegetative stage, Rawson et al.
yield was very low due to it maturing around 2 weeks (1988b) found only small differences in biomass pro-
later than local genotypes (Hollington, 2000). Mutation duction in salinity between several barley, wheat, and
breeding has been used to reduce its time to maturity by triticale cultivars. All the same, the barley cultivars were,
3 weeks without adverse effects on yield at 150 mM on the whole, more salt tolerant than the bread-wheat culti-
NaCl (Mahar et al., 2003). This material is now being vars in conditions of both normal and accelerated develop-
further tested in southern Pakistan (PA Hollington, ment, and some barley cultivars were more salt-tolerant
personal communication). than others (Rawson et al., 1988b). Varietal differences for
The Pakistan selection LU26S showed improved yields yield in saline conditions have been shown in several
on saline soils in Pakistan (Qureshi et al., 1980), but it is studies in both glasshouse (Greenway, 1962) and field
susceptible to rust and not adapted to dense saline-sodic (Richards et al., 1987; Slavich et al., 1990).
soils where there is the possibility of waterlogging (PA The Kna1 locus appears to be absent in barley, as judged
Hollington, personal communication). LU26S was crossed by the high Na+ and low K+ concentrations compared with
with Kharchia, and two salt-tolerant genotypes, S24 and wheat (Gorham et al., 1990). There are varietal differences
S36, were selected from F3 seed at salinity levels of 24 and in the extent of accumulation of Na+ and Cl in leaves
36 dS m1, respectively (Ashraf and O’Leary, 1996). S24 (Greenway, 1962; Forster et al., 1994), but the relationship
had high salt tolerance, as high as Kharchia and SARC-1, between Na+ or Cl accumulation and salt tolerance has
possibly due to its low leaf Na+ accumulation (Ashraf, 2002). not been established in barley to the same extent as in
Other approaches to improving salt tolerance in wheat wheat and rice (Colmer et al., 2005).
are based on mechanisms for salt tolerance, using physio- Progress in breeding for salt tolerance in rice has been
logical traits to select germplasm. In wheat, salt tolerance is reviewed by Gregorio et al. (2002). Salt-tolerant rice
associated with low rates of transport of Na+ to shoots, with varieties such as Pokkali and Nona Bokra originated in
1028 Munns et al.
coastal areas of India, and are the source of salt tolerance seen on the more sensitive genotype, and the rate of leaf death
in most rice breeding programmes. Recent approaches to of old leaves was clearly greater on the sensitive than the
improve the salt tolerance of modern rice cultivars include tolerant genotype. Once the number of dead leaves increased
somaclonal variants, anther culture-derived lines, and mol- above about 20% of the total, plant growth slowed down
ecular marker-assisted selection (Gregorio et al., 2002). and many individuals started to die (Munns et al., 1995).
A region on the long arm of chromosome 1 has been shown The first phase of the growth response results from the
to contain a quantitative trailt locus (QTL) for Na+ exclu- effect of salt outside the plant. The salt in the soil solution
sion and K+/Na+ discrimination (Koyoma et al., 2001; (the ‘osmotic stress’) reduces leaf growth and, to a lesser
Bonilla et al., 2002; Lin et al., 2004), independently desi- extent, root growth (Munns, 1993). The cellular and meta-
gnated Saltol and SKC1. Just recently, OsHKT8 has been bolic processes involved are common to drought. The rate
identified as a candidate gene for SKC1 by Ren et al. (2005). at which new leaves are produced depends largely on the
Major increases in salt tolerance of modern cereal water potential of the soil solution, in the same way as for
cultivars will come from introducing new genes by either a drought-stressed plant. Salts themselves do not build up
crossing with new donor germplasm or by transformation in the growing tissues at concentrations that inhibit growth,
with single genes. In either case, the progeny must be as the rapidly elongating cells can accommodate the salt

back-crossed into adapted cultivars before the donor genes that arrives in the xylem within their expanding vacuoles.
are of use to farmers. An understanding of physiological So, the salt taken up by the plant does not directly inhibit
and genetic mechanisms is necessary for a breeding pro- the growth of new leaves.
gramme, in order to select the desired trait in the different The second phase of the growth response results from the
genetic backgrounds. With either method of introducing toxic effect of salt inside the plant. The salt taken up by the
new genes for salt tolerance, the expressed trait must be plant concentrates in the old leaves; continued transport
selected in back-crossed lines that undergo seed multipli- of salt into transpiring leaves over a long period of time
cation before field trials can take place. eventually results in very high Na+ and Cl concentrations,
Characteristics of a salt-tolerant variety include Na+ ‘exclu- and the leaves die. The cause of the injury is probably due
sion’, K+/Na+ discrimination, retention of ions in the leaf to the salt load exceeding the ability of the cells to com-
sheath, tissue tolerance, ion partitioning into different-aged partmentalize salts in the vacuole. Salts then would rapidly
leaves, osmotic adjustment, transpiration efficiency, early build up in the cytoplasm and inhibit enzyme activity.
vigour and early flowering leading to a shorter growing season, Alternatively, they might build up in the cell walls and
the latter increasing water use efficiency (summarized by dehydrate the cell (Flowers et al., 1991), but Mühling and
Colmer et al., 2005). This paper discusses the physiolog- Läuchli (2002) found no evidence for this in maize culti-
ical mechanisms; the individual genes that regulate these vars that differed in salt tolerance.
processes have been reviewed recently (Munns, 2005). The rate that leaves die is crucial for the survival of
a plant. If new leaves are continually produced at a rate
greater than that at which old leaves die, then there are
Processes involved in the osmotic versus enough photosynthesizing leaves for the plant to produce
salt-specific effects on growth flowers and seeds, although in reduced numbers. However,
if old leaves die faster than new ones develop, then the plant
The two phases of the growth response may not survive to produce seed. For an annual plant there
Salt in the soil water inhibits plant growth for two reasons. is a race against time to initiate flowers and form seeds
First, the presence of salt in the soil solution reduces the while the leaf area is still adequate to supply the necessary
ability of the plant to take up water, and this leads to photosynthate. For perennial species, there is an opportu-
slower growth. This is the osmotic or water-deficit effect of nity to become dormant, and thus survive a period of stress
salinity. Second, excessive amounts of salt entering the that will be relieved later by rainfall. These results illustrate
transpiration stream will eventually injure cells in the the principle that the initial growth reduction is due to the
transpiring leaves and this may further reduce growth. osmotic effect of the salt outside the roots, and that what
This is the salt-specific or ion-excess effect of salinity. distinguishes a salt-sensitive plant from a more tolerant
In wheat, the two responses occur sequentially, giving rise one is its inability to prevent salt from reaching toxic levels
to a two-phase growth response to salinity (Munns, 1993). in the transpiring leaves.
For example, comparisons between genotypes with con-
trasting rates of Na+ uptake, and long-term differences in Cause of the Phase 1 response: water stress,
salt tolerance (Schachtman et al., 1991), showed that both not salt toxicity
genotypes had the same growth reduction for 4 weeks in The mechanisms controlling this phase of the growth
150 mM NaCl, and it was not until afterwards that a growth response are not specific to salinity. Reductions in the
difference between the genotypes was clearly seen (Munns rate of leaf and root growth are probably due to factors
et al., 1995). However, within 2 weeks, dead leaves were associated with water stress rather than a salt-specific effect
(Munns, 2002). This is supported by the evidence that those extracted from beans (Phaseolus vulgaris) or peas
Na+ and Cl are below toxic concentrations in the grow- (Pisum sativum) (Flowers, 1972; Greenway and Osmond,
ing cells themselves. For example, in wheat growing in 1972; Flowers et al., 1977). Even enzymes from the pink
120 mM NaCl, Na+ in the growing tissues of leaves was salt-lake alga Dunaliella parva, which can grow at salinities
at most only 20 mM, and only 10 mM in the rapidly 10-fold higher than those of seawater, are as sensitive to
expanding zones, and Cl only about 50 mM (Hu et al., NaCl as those of the most-sensitive glycophytes (reviewed
2005). Similarly, in maize growing in 80 mM NaCl, Neves- by Munns et al., 1983). Generally, Na+ severely inhibits
Piestun and Bernstein (2005) found that Na+ and Cl were, most enzymes at a concentration above 100 mM. More
at most, only 40 mM in the most rapidly growing tissues than 50 enzymes require K+ as a cofactor, and these are
(20 mm from the leaf base), and that the extent of inhibition particularly susceptible to high Na+ and high Na+/K+ ratios.
by salinity of either the elongation rate or the total volume The concentration at which Cl becomes toxic is even
expansion rate did not correlate with the Na+ or Cl in the less well defined, but is probably in the same range as that
tissues. Further, Fricke (2004) found only 38 and 49 mM for Na+. Even K+ salts inhibit enzymes when at high
Na+ in mesophyll and epidermal cells, respectively, in the concentrations (Greenway and Osmond, 1972). Some
growing cells of barley after 24 h of exposure to 100 mM halophytes may have slightly modified forms of some

NaCl. That this Na+ was not inhibitory to growth, but was enzymes (Flowers and Dalmond, 1992), but even so, most
probably beneficial as it might be taken up into the ex- of the Na+ must be compartmentalized in vacuoles.
panding vacuole for osmotic adjustment, was indicated Mechanisms for the salt-specific features of salt tolerance
by the fact that the growth rate increased with time over are therefore of two main types: those minimizing the entry
24 h (after a temporary decline when the salt was applied) of salt into the plant, known as ‘salt exclusion’, and those
while the cellular Na+ increased (Fricke, 2004). In roots minimizing the concentration of salt in the cytoplasm,
also, there is evidence that Na+ concentrations in dividing known as ‘tissue tolerance’. Halophytes have both types
or rapidly elongating cells are low and well below toxic of mechanisms; they ‘exclude’ salt well, but the cells can
levels (Jeschke, 1984; Jeschke et al., 1986). For example, compartmentalize the salt in vacuoles. These mechanisms,
in root tips of saltbush (Atriplex amnicola), Na+ was only together with salt glands or bladders that excrete salt, allow
40 mM at external NaCl concentrations of 400 mM them to grow for long periods of time in saline soil. The
(Jeschke et al., 1986). The rapid expansion of the grow- physiological processes involved in ‘salt exclusion’ and
ing cells would help to keep the salt from building up ‘tissue tolerance’ are reviewed, followed by discussion of
to high concentrations. the processes involved in the response to the osmotic stress
Results of experimental manipulation of shoot water- of the salt in the soil solution. Screening methods to identify
relations suggest that hormonal signals, probably induced genetic variation in these three processes will be suggested.
by the osmotic effect of the salt outside the roots, are con-
trolling the rate of cell elongation growth (Munns et al.,
2000a). These results were obtained using specialized root The mechanism of ‘salt exclusion’
pressure chambers, and are described in a later section. There is a strong correlation between salt exclusion and salt
tolerance in many species (reviewed by Läuchli, 1984;
Flowers and Yeo, 1986; Munns and James, 2003), and
Cause of the Phase 2 response: salt toxicity recently reported for rice (Lee et al., 2003; Zhu et al., 2004)
Species which cannot effectively exclude salt from the and wheat (Poustini and Siosemardeh, 2004). In those
transpiration stream must have ways to handle the salt species that retain Na+ in woody roots or stems, there is
arriving in leaves as the water evaporates and salt gradually a strong correlation between Cl exclusion and salt
builds up with time. The salt concentrations in older leaves tolerance; for example, citrus (Storey and Walker, 1999).
are much higher than in younger leaves at a given time. In Figure 1 shows the relationship between leaf Na+ con-
the older leaves, the salt concentration eventually becomes centration and salt tolerance of a range of durum wheat
high enough to kill the cells, unless they can compartmen- genotypes. Salt tolerance was assessed as shoot dry matter
talize the salt in vacuoles, thereby protecting the cytoplasm after nearly 4 weeks of salt treatment (Munns and James,
from ion toxicity. 2003). In general, the genotypes with the lowest Na+
The concept that salt must either be excluded from tissues concentrations produced the greatest dry matter. These low-
or compartmentalized in cell vacuoles, derives from the Na+ genotypes had fewer injured leaves, and a greater
early discovery by biochemists that enzymes of halophytes proportion of living to dead leaves. The effect on growth
are no more tolerant of high concentrations of NaCl than are was probably due to a better carbon balance in the
those of non-halophytes (also called glycophytes, or plants genotypes with less Na+. A similar relationship between
requiring sweet water). For example, in vitro activities of shoot dry matter and leaf Na+ was found in a population
enzymes extracted from the halophytes Atriplex spongiosa from a cross between high- and low-Na+ genotypes.
or Suaeda maritima were just as sensitive to NaCl as were There was a strong correlation between shoot dry matter
1030 Munns et al.
50 retained in the shoot for every gram of water transpired, i.e.
Salt tolerance (biomass in salt, % control)
the shoot retains only 2% of the water transpired. In order

40 to prevent the salt concentration in the shoot increasing
above that in the soil, then only 2% of the salt should be
allowed into the shoot, i.e. 98% should be excluded.
30 A soil salinity of 100 mM NaCl or ;10 dS m1 is about
as high as most crops will tolerate without a significant
20 reduction in growth or yield, and a concentration of 100
mM NaCl on a whole shoot basis is about as high as is
desirable because it will include some old leaves with
10
much higher salt concentrations, as well as younger leaves
or other tissues with lower concentrations. So for plants
0 to grow for extended periods of time in soils with salinity
0.0 0.5 1.0 1.5 2.0 2.5 3.0 of this order of magnitude, no more than 2% should get to
Leaf 3 Na+ concentration (mmol gDW-1) the shoots.

Fig. 1. Relationship between salinity tolerance (% growth of controls)
Most plants, in fact, do exclude about 98% of the salt in
and leaf Na+ concentration in durum wheat (Triticum turgidum ssp. the soil solution, allowing only 2% to be transported in the
durum) selections. Na+ concentrations were measured on leaf 3 after xylem to the shoots. Differences between cereal genotypes
10 d in 150 mM NaCl and shoot biomass after 24 d. Values are expressed with contrasting rates of Na+ uptake, when grown in 50 mM
as a percentage of shoot biomass in control conditions (r2=0.74). All
values are means (n=5). Reproduced from Munns and James (2003), NaCl, range from 99% for Janz to 98% for other bread
with kind permission of Springer Science and Business Media. wheats (Munns, 2005). Durum wheat, rice, and barley are
not such good excluders, yet they still exclude at least
production and Na+ concentration in leaves between 94% of the soil Na+ from the transpiration stream (Munns,
families from a cross between the genotypes with the 2005). Roots themselves do not accumulate excessively
highest and lowest Na+ shown in Fig. 1 (S Husain, R high concentrations of salt. The Na+ and Cl concentration
Munns, unpublished results). in roots is rarely much higher than in the external solution,
and often is lower (Munns, 2005).
Do all plants ‘exclude’?
Roots must exclude most of the Na+ and Cl in the soil Control of salt exclusion at the whole-plant and
solution or the salt will gradually build up with time in the cellular level
shoot and become so high that it kills the leaves. To prevent
salt building up with time in the shoot, roots should exclude Physiological mechanisms conferring exclusion that oper-
98% of the salt in the soil solution, allowing only 2% to be ate at the cellular and whole-plant level have been described
in previous reviews (Greenway and Munns, 1980; Läuchli,
transported in the xylem to the shoots. The concentration at
1984; Pitman, 1984; Storey and Walker, 1999), and with
which NaCl accumulates in the shoot depends on the salt
particular reference to selectivity for K+ over Na+ (Jeschke,
concentration in the soil solution, the percentage of salt
1984; Jeschke and Hartung, 2000; Tester and Davenport,
taken up by roots, and the percentage of water retained in
2003). Salt tolerance of monocotyledonous species without
the leaves, as shown in Equation 1:
salt glands depends on the control of Na+ transport at four
½NaClshoot = ½NaClsoil major points: (i) selectivity of uptake by root cells in the
ð1Þ cortex and stele; (ii) loading of the xylem by xylem
3 ð% NaCl taken up=% water retainedÞ
parenchyma cells in roots; (iii) removal of salt from the
Plants retain only about 2% of the water they transpire, i.e. xylem in the upper part of the roots, the stem, or leaf sheaths
they take up about 50 times more water from the soil than by xylem parenchyma cells; (iv) loading of the phloem.
they retain in their shoot tissues. The percentage of These four control points for Na+ exclusion were examined
transpired water that is retained in the shoot can be in a recent study with two genotypes of durum wheat
calculated from the product of the water use efficiency (Triticum turgidum L. ssp. durum) known to differ in rates
(WUE; mass of shoot produced per mass of H2O tran- of Na+ accumulation, Line 149 and the cultivar Tamaroi
spired) and the shoot water content (WC; shoot H2O per (Munns et al., 2000b; see also below). Genetic studies had
shoot mass), i.e. WUE3WC3100. Water use efficiencies indicated two major gene loci controlling leaf-blade Na+
of plants growing at moderate evaporative demand are accumulation (Munns et al., 2003). The physiological
usually in the range of 3–6 mg g1, the variation being due traits determined by these genetic differences were invest-
to extremes of evaporative demand rather than a peculiarity igated using measurements of unidirectional 22Na+ trans-
of the species. For a water use efficiency of 4 mg g1 and port and net Na+ accumulation. The genotypes did not
a shoot H2O:dry weight ratio of 5:1, about 20 mg of water is differ significantly at the first control point, i.e. in the
+
unidirectional root uptake of Na (Davenport et al., 2005). that xylem loading and leaf sheath sequestration are sep-
The major differences in Na+ transport between the arate genetic traits that interact to control leaf-blade Na+.
genotypes were at the second control point, the rate of
transfer from the root to the shoot (xylem loading), which
was much lower in the salt-tolerant genotype (Davenport Root uptake and transport
et al., 2005), and the third control point, the capacity of Roots have a remarkable ability to control their Na+ and
the leaf sheath to extract and sequester Na+ as it entered the Cl concentrations, which do not increase in proportion to
leaf (Fig. 2). There was no substantial recirculation of Na+ the external concentration; rather, they seem to plateau at
from shoots to roots (Davenport et al., 2005). It is likely about 50 mM NaCl, as illustrated in Table 1. Roots also
have the ability to regulate their turgor over a wide range
of salinity levels, at about 0.6 MPa (Pritchard et al.,
1991). In wheat, once the external NaCl exceeds 50 mM
300 (A) leaf sheath
(0.25 MPa), organic solutes must make a significant con-
250
tribution to turgor maintenance, because the internal Na+
and Cl concentrations are not increasing in proportion to

200 the external solution and there is not enough salt inside to
Na+ (mM)
balance the osmotic pressure of the salt outside (Table 1).

150 K+ is usually only 100 mM or less in roots, and declines
with salinity. Table 1 indicates that there must be increasing
100 concentrations of organic solutes. Remarkably little work
has been done on the production of organic solutes in roots.
50 Proline and glycinebetaine concentrations on a root fresh-
weight basis were only one-quarter of that in shoots of
0 barley grown at 100–200 mM NaCl, even though the Na+
(B) leaf blade
and Cl concentrations were much lower in roots than
300
shoots (Wyn Jones and Storey, 1978). This suggests that
unknown organic solutes are involved, possibly sucrose
Tamaroi
and other sugar-related compounds; however, this would
Na+ (mM)
200 impose a real carbon cost on the plant especially as the

production of photosynthates would have been reduced at
150 mM NaCl by both stomatal closure and smaller leaf
area (James et al., 2002).
100
Line 149 Cell-specific localization of Na+ in the root can provide
insight into the major control points that limit Na+ loading
of the xylem. Cryo-SEM X-ray microanalysis was used to
0 characterize cell-specific ion profiles in roots of the two
(C) roots
80 durum wheat genotypes Line 149 and Tamaroi that differ in
Na+ exclusion (Läuchli et al., 2005). The results from this
X-ray microanalysis study indicate that the root cortex is
60 the barrier to Na+ transport into the stele, rather than the
endodermis, and that the major control of Na+ entry was
Na+ (mM)
from the outer two layers of cells in the cortex, the

40
epidermis and hypodermis. The localization pattern for
the Na+-excluding durum Line 149 is shown in Table 2.
20
The highest concentration of Na+ was in the cells of the
pericycle (the outermost cell layer of the stele, immediately
within the endodermis), indicating that it may provide
0 a major control point in limiting xylem loading of Na+,
0 2 4 6 8 10 especially as this accumulation was less marked in cv.
Time in 50 mM NaCl (d) Tamaroi, which is less able to exclude Na+ from the xylem.
Fig. 2. Increase in Na+ concentrations in (A) leaf sheath, (B) leaf blade, The K+-accumulation distribution pattern was the reverse
and (C) roots of durum wheat Line 149 (closed symbols) and Tamaroi of the Na+ distribution (Läuchli et al., 2005).
(open symbols) during 10 d of exposure to 50 mM NaCl. Data represent Very high Na+ in the pericycle was also found by Storey
mean 6standard error of the mean. The leaf shown is the first leaf.
Modified from Davenport et al. (2005). ª American Society of Plant et al. (2003) in grapevine roots grown in 25 mM NaCl,
Biologists. much higher than in the endodermis, again suggesting that
1032 Munns et al.
Table 1. Root ion concentrations in wheat grown for 2 weeks in 1–150 mM NaCl
Data shown are for a durum landrace, Line 141, which was typical of other cereal genotypes measured (Husain et al., 2004). The balancing anion
(assumed to be monovalent) is calculated from the negative charge to balance the Na+ and K+ (i.e. the concentration of Na+ plus K+ minus Cl). ‘D ions’
is the difference between the internal concentration of those ions (i.e. Na+, K+, Cl, and a balancing monovalent anion), and the external concentration of
ions, in osmol l1. The ‘unknown solutes’ are those extra solutes to generate 0.6 MPa of turgor, i.e. the organic solutes needed along with the ions to
make up an internal concentration of 240 mOsmol l1 above that of the external solution.
NaCl (mM) Na+ (mM) K+ (mM) Cl (mM) Balancing anion (mM) D ions (mOsmol l1) Unknown solutes (mOsmol l1)
1 11 103 4 110 +226 14

10 26 76 10 92 +184 56
25 36 65 15 86 +152 88
50 45 77 23 99 +144 96
75 47 63 21 89 +70 170
100 53 61 28 86 +28 212
150 65 54 28 91 –62 302

Table 2. Cell-specific Na+ profiles across the seminal root of phloem sap is difficult to obtain, analyses of apical or
durum wheat genotype Line 149, an efficient Na+ excluder reproductive tissues are a good indication of whether or
Plants were grown at 50 mM NaCl for 9 d. The Na+ profiles were not salt is excluded from the phloem.
determined by quantitative cryo-SEM X-ray microanalysis (n=16).
Detection limit for Na+ was 5–10 mM (Läuchli et al., 2005).
Other factors—growth rates and shoot:root ratios
Cell type Na+ concentration (mM) Other factors that contribute to low rates of salt accumu-
Epidermis 4869 lation in leaves are high shoot:root ratios and high relative
Sub-epidermis (hypodermis) 5165 growth rates (Pitman, 1984; Munns, 2005).
Cortex (outer layer) 3569
Cortex (middle layer) 2365 The shoot ion concentration is the result of the rate at
Cortex (inner layer) 2265 which ions arrive in the shoot, the rate at which they are re-
Endodermis 39610 exported back to the root, and the rate at which the shoot
Pericycle 85614
Xylem parenchyma 3465 expands (the relative growth rate). For ions such as Na+ and
Metaxylem vessels Not detectable Cl, for which export from the shoot is a small proportion
of import in the xylem, the concentration in the shoot
results essentially from the uptake rate divided by the
the pericycle is an important control point in the radial relative growth rate, as shown in Equation 2:
transport of Na+.
Shoot ion concentration ðmol g1 Þ
Phloem export = ½shoot ion uptake rate ðmol g1 d1 Þ= ð2Þ
Rates of retranslocation of salt from leaves are low in shoot relative growth rate ðg g
1 1
d Þ
relation to rates of import in the transpiration stream, as
shown by the continued presence of salt in leaves long after This equation shows that the relative growth rate (g g1
the salt around the roots is removed. Estimates of fluxes d1), not the absolute growth rate (g d1), determines shoot
of Na+ from phloem sap collected through aphid stylets ion concentration.
indicated that, in barley, phloem export from a leaf was The shoot ion uptake rate is not determined by the
only about 10% of the import in the xylem (Munns et al., transpiration rate. The flux of ions to the shoot is largely
1986; Wolf et al., 1990). independent of the flux of water; the transport pathways
Measurements of ions in phloem sap indicate that the for water and ions are separate. Water moves across root
more-salt-tolerant species exclude Na+ and Cl from the membranes through aquaporins (Tyerman et al., 2002) and
phloem to a greater extent than less-tolerant ones (reviewed ions move across root membranes through ion channels
in Wolf et al., 1991). Exclusion of salt from the phloem or transporters (Amtmann and Sanders, 1999), so when
ensures that salt is not redirected to growing tissues of the transpiration rates fall, ion concentrations in the xylem sap
shoot. As shown by [14C]urea feeding studies, lower leaves increase (Fig. 3). It is well established that K+ uptake is
supply the root, upper leaves feed the shoot apex, and mid- not influenced by transpiration rates in non-saline soil
leaves feed both shoot apex and root (Layzell et al., 1981), (Smith, 1991), and Na+ and Cl fluxes are also independent
and the same has been shown for K+ and Na+ (Wolf et al., of transpiration rates in saline soil (Munns, 1985; Ball,
1991, and references therein). Na+ and Cl concentrations 1988). The independence of Cl flux over a wide range of
in shoot apices and reproductive tissue of wheat and barley transpiration rates is shown in Fig. 3, and a similar relation-
were very low compared with the leaves, and K+/Na+ ratios ship was found for Na+ and K+ (Munns, 1985). However,
were particularly high (Munns and Rawson, 1999). As there is evidence for a ‘bypass’ pathway in rice roots, in
which water can bypass all membranes and enter the xylem 1990; Husain et al., 2004). In order to introduce the trait of
by an apoplastic route, which can account for a large part Na+ exclusion into current durum wheat varieties, genetic
of the Na+ delivered to the shoot (Garcia et al., 1997). variation in salt tolerance was investigated across a wide
Improvements in ion exclusion could be made by range of ancient durum-related accessions and landraces
selecting genotypes with lower rates of transport from representing five Triticum turgidum subspecies. Selections
root to shoot, higher relative growth rates or, in the case of were screened non-destructively for low Na+ concentra-
rice, minimization of the bypass pathway through which tion in leaves, and the associated enhanced K+/Na+ dis-
ions leak into the xylem. crimination (Munns et al., 2000b). Wide genetic variation
in Na+ accumulation (and K+/Na+ discrimination) was
found, and a particular landrace named Line 149 was sel-
Genetic improvement in salt tolerance of durum ected for breeding.
wheat using the trait for sodium exclusion Proof of the concept that Line 149 would provide
Cultivated durum (pasta) wheat (Triticum turgidum ssp. a source of salt tolerance for modern durum wheat was
durum) is more salt sensitive than bread wheat (Triticum obtained by comparing it with another durum landrace,
aestivum), a feature that restricts the production of durum Line 141 with a high rate of sodium transport to leaves, to

wheat on farms with sodic or saline soils. To increase the assess the effects of the sodium exclusion trait on prevent-
salt tolerance of durum wheat, attempts have been made to ing leaf injury and death in saline soil (Husain et al., 2003).
improve its sodium exclusion, building on the earlier work Leaves of Line 149 lived longer than leaves of Line 141, the
of John Gorham and Jan Dvořák (Gorham et al., 1990; start of leaf senescence being delayed by 1 week or more.
Dvořák et al., 1994). In the bread and durum wheat, salt Figure 4 illustrates that the high Na+ lines lost chlorophyll
tolerance is associated with low rates of transport of Na+ to more rapidly and died earlier than the low Na+ lines. Other
shoots with high selectivity for K+ over Na+; there is little leaves showed similar results (Husain et al., 2003), so by
genotypic variation in rates of Cl transport (Gorham et al., the time the grain was developing, all the leaves of Line
141 were dead but some were still alive in Line 149.
25 The low sodium trait improved yield by over 20% in
A saline soil in glasshouse trials at moderate salinity (75 mM
20
NaCl); the low Na+ Line 149 suffered a yield reduction
of 12% but the high Na+ Line 149 suffered a reduction of
Cl- concentration (mM)
30% compared with their respective controls (Husain et al.,

15 2003). However, at high salinity (150 mM NaCl), there was
no advantage to having the Na+-excluding trait; the yield
10 was severely reduced in both genotypes (Husain et al.,
2003). The differences in yield were similar to the differ-
ences between bread and durum wheat reported by Maas
5 and Grieve (1990). At a salinity level equivalent to about
0
B
Chlorophyll content (SPAD units)
50
100
40
Cl- flux (mol m-2 s-1)
80
30
60
20
40
10
20
0
0
0 1 2 3 30 35 40 45 50 55
Water flow (mmol m-2 s-1) Time after salt added (d)
Fig. 3. The relationship between ion concentrations in the xylem, ion Fig. 4. Effect of 150 mM NaCl on chlorophyll content in leaf 6 of low
fluxes to the shoot, and transpiration rates (Munns, 1985). The data sodium (circles) and high sodium (squares) genotypes. Leaf 6 emerged
shown are for Cl, but Na+ and K+ concentrations and fluxes showed 21 d after the salt treatment started. Bars show the standard error of the
a similar relationship to transpiration rate (Munns, 1985). mean. Modified from Husain et al. (2003).
1034 Munns et al.
100 mM NaCl (0.45 MPa) they showed that the yield of the builds up with time. In the older leaves, the salt concen-
(low Na+) bread wheat was reduced by only 7% compared tration will soon become high enough to kill the cells,
with a 38% reduction for the (high Na+) durum wheat. At unless they can compartmentalize the salt in vacuoles,
a higher salinity, equivalent to about 150 mM NaCl (0.65 thereby protecting the cytoplasm from ion toxicity.
MPa), there was less difference between genotypes, the This compartmentation is exemplified by halophytes,
bread wheat being reduced by 43% and the durum wheat which hold concentrations of over 500 mM on a leaf tissue
by 54% (Maas and Grieve, 1990) basis, but which show no sign of injury. Barley leaves can
Does Na+ exclusion pose a problem for turgor mainte- tolerate concentrations close to this without showing injury
nance? The low Na+ trait did not restrict turgor maintenance (Greenway, 1962; Rawson et al., 1988a), as can numerous
as K+ uptake was enhanced (Rivelli et al., 2002). Four other species. In these species, salt must be sequestered in
wheat genotypes with contrasting degrees of Na+ exclusion vacuoles. This is difficult to measure directly, but it must
were selected to see if low Na+ uptake adversely affected happen when the leaf contains at least 100 mM on a tissue
water relationships or growth rates during exposure to saline water basis (i.e. 0.5 mmol g1 DW), as this concentration
conditions. Plants were grown in supported hydroponics, cannot be tolerated by most enzymes, as described above.
with and without 150 mM NaCl, and sampled for measure- To understand the physiology of tolerance to high

ments of water relationships, biomass, stomatal conduc- internal salt concentrations, two genotypes that differ in
tance, and ion accumulation. After 4 weeks of exposure to the degree of salt-induced leaf injury, the durum cultivar
salt, there was little difference between genotypes in the Wollaroi and the durum-related landrace Line 455, were
effect of salinity on water relationships, as indicated by their grown in 150 mM NaCl for 4 weeks (James et al., 2002).
relative water content (RWC) and estimated turgor. The shoot biomass of both genotypes was substantially
Osmotic adjustment occurred in all genotypes. In the low reduced by salinity, but genotypic differences appeared
Na+ genotypes, osmotic adjustment depended on higher after 3 weeks, when the durum cultivar Wollaroi showed
K+ and high organic solute accumulation. These data in- greater leaf injury and a greater reduction in biomass than
dicate that selecting lines with low Na+ accumulation for the landrace Line 455. Ion accumulation, water relation-
the purpose of improving salt tolerance is unlikely to ships, chlorophyll fluorescence, and gas exchange were
introduce adverse effects on plant–water relationships or followed throughout the life of a leaf. Salinity caused
growth (Rivelli et al., 2002). Thus the Na+ exclusion trait a large decrease in stomatal conductance in both genotypes.
in Line 149 reduces the rate of leaf death, and improves This was not due to poor water relationships, as leaf turgor
plant growth and grain yield under saline conditions. of both genotypes was not affected by the salt treat-
Genetic analysis of the segregating populations devel- ment (James et al., 2002), so was presumably due to ‘root
oped from crosses between genotypes with low and high signals’ as discussed in the following section. Photosyn-
rates of Na+ uptake indicated two dominant and interacting thesis per unit leaf area was not initially reduced by salinity,
genes of major effect (Munns et al., 2003). It is likely that particularly in the more-tolerant Line 455, as the chloro-
one gene controls the loading of Na+ in the xylem in the phyll per unit area was higher in saline than non-saline
roots, while the other controls the retrieval of Na+ from the conditions (the leaves were narrower, the cells were
xylem in the lower part of the leaves (Davenport et al., smaller, and so the chloroplast density was greater), but
2005). These processes would work together to produce photosynthesis per plant was reduced as the leaves were
very low Na+ concentrations in the leaf blades, and so fit smaller in area. With time, photosynthesis per unit area
the Mendelian analysis of two interacting genes. decreased in both genotypes due to reductions in stomatal
A locus for one gene, designated Nax1 (Na+ exclusion), conductance, and later there were non-stomatal limitations
was mapped to the long arm of chromosome 2A using associated with a build-up of Na+ and Cl in the whole
a QTL approach with AFLP, RFLP, and microsatellite tissue above 250 mM. Chlorophyll fluorescence measure-
markers (Lindsay et al., 2004). This locus had a LOD score ments showed that the efficiency of PSII photochemistry in
of 7.5 and accounted for 38% of the phenotypic variation Line 455 was unaffected throughout. However, in Wollaroi,
in the mapping population. One particular microsatellite the potential and actual quantum yield of PSII photochem-
marker, Xgwm312, was closely linked to the low Na+ trait istry began to decline as the leaf aged and the thermal
in other populations with different genetic backgrounds, energy dissipation of excess light energy (NPQ) increased.
and is being used to select low Na+ progeny in a durum This coincided with high Na+ and Cl concentrations in the
breeding programme (Lindsay et al., 2004). leaf (250 mm) and with chlorophyll degradation, indicating
that these later reductions in CO2 assimilation in Wollaroi
were a likely consequence of a direct toxic ion effect. The
The mechanism known as ‘tissue tolerance’
fluorescence parameters, other than NPQ, were no more
Species which cannot exclude 98% of the salt from the sensitive than chlorophyll itself. The more easily measured
transpiration stream must have ways to handle the salt fluorescence parameter Fv/Fm decreased only when chloro-
arriving in leaves as the water evaporates, and salt gradually phyll content decreased. The physiological mechanism of
tolerance in Line 455 was in delaying the onset of non- identify genotypes with the least leaf injury associated
stomatal effects on photosynthesis, probably by delaying with highest leaf Na+ concentration (Munns and James,
the time at which Na+ or Cl reached a critical toxic level. 2003). Barley was included as a benchmark, because of its
In a recent study, the relationship between photosyn- established reputation for salinity tolerance coupled with
thetic capacity and the cellular and subcellular distribution high rates of salt accumulation, and previous observations
of Na+, K+, and Cl was studied in a salt-sensitive durum that it was slow to develop leaf injury. Significant variation
wheat, as well as a salt-tolerant barley, to see if barley’s in percentage dead leaf (weight of dead leaf as a proportion
superior salt tolerance was associated with compartmen- of total leaf dry weight) was found between individual
talization of salt in the vacuole (R James and R Munns, tetraploid lines, the percentage dead leaf ranging from 2 to
unpublished results). Vacuolar concentrations were meas- 29 (Munns and James, 2003). The barley cultivar had a
ured in mesophyll and epidermal cells using cryo-SEM X- low degree of leaf injury as expected, only 3%.
ray microanalysis. Cytoplasmic Na+ and K+ concentrations The total leaf Na+ content of individual genotypes did
were calculated from these data, and from whole tissue not correlate with the percentage dead leaf, suggesting there
analyses and volume fractions of vacuoles in different might be genotypic variation in the ability to tolerate the
cell types. Efficient cellular and subcellular partitioning of Na+ at the tissue or cellular level. The ratio of Na+ content

both Na+ and K+ in barley led to the preservation of a to percentage dead leaf (whole shoot basis) was calculated
favourable K+:Na+ ratio in the cytoplasm at high leaf as an index of tolerance to Na+ in the leaves. A higher Na+
Na+ concentrations (200–300 mM) by contrast to durum content per percentage dead leaf might indicate a higher
wheat (Fig. 5). The photosynthetic capacity of durum degree of tissue tolerance to Na+. This ratio ranged from 15
wheat declined at lower leaf Na+ concentrations than barley to 108 lmol Na+ per percentage dead leaf, with the barley
(R James and R Munns, unpublished results), indicating cultivar Skiff at the high end of that range with a value of
that the maintenance of photosynthetic capacity (and the 107 (Fig. 6). The bread-wheat cultivars, while excluding 2–
resulting greater salt tolerance) was due to the maintenance 3 times the amount of Na+ from the leaves, displayed
of high K+, low Na+, and the resulting high K+:Na+ in the similar levels of leaf injury as a number of tetraploid
cytoplasm of mesophyll cells. selections, indicating greater sensitivity to tissue Na+ levels.
This experiment revealed five tetraploid genotypes with an
Screens for tissue tolerance of Na+
exceptional combination of high Na+ accumulation and low
leaf injury, indicating they may have an exceptional ability
In an attempt to find genotypes of durum wheat as tolerant to tolerate high Na+ levels in tissues (Munns and James,
as barley to high salt concentrations in leaves, over 50 2003). Other screening methods have been based on loss of
genotypes of durum wheat and its closely related tetraploid chlorophyll with increasing salt concentration (Yeo and
relatives were harvested, after 21 d of salt treatment, to Flowers, 1983), but could assess the health of a leaf with
ageing, such as its rate of photosynthesis or turgidity at
2.5 a given age. However, most methods have drawbacks.
barley
12
Leaf cytoplasm K+:Na+ ratio
2.0
10 LSD (0.05)
1.5
No of selections
8
1.0
6 Wollaroi
durum wheat
0.5
4 Skiff
(barley)
0.0 2
100 150 200 250 300 350 400 450
Leaf Na+ concentration (mM)
0
0 20 40 60 80 100
Fig. 5. Relationship between leaf Na+ concentration and the estimated
K+:Na+ ratio in the cytoplasm of leaves from barley (cv. Franklin) and Na+ per %DL (µmol)
durum wheat (cv. Wollaroi) grown in a range of high salinities leading
to different leaf Na+ concentrations. Cytoplasmic Na+ and K+ concen- Fig. 6. Frequency distribution of Na+ content per percentage dead leaf
trations were estimated from vacuolar concentrations measured using of 47 tetraploid wheat selections, grown in 150 mM NaCl for 21 d. The
cryo-SEM X-ray microanalysis, whole tissue analyses, and volume bars represents LSDs at P=0.05 for selection comparisons. Reproduced
fractions of cell compartments in different cell types (R James, R Munns, from Munns and James (2003), with kind permission of Springer Science
unpublished results). and Business Media.
1036 Munns et al.
Photosynthesis Osmotic tolerance: mechanisms and
screening methods
Screening methods based on gas exchange are not feasible
as the measurements are too slow to handle large numbers. The osmotic or water stress effect of salt in the soil quickly
The chlorophyll fluorescence measurement of Fv/Fm can reduces the growth rate in proportion to the salinity level
be made quickly and can handle larger numbers but, as (the Phase 1 effect; see Fig. 7). In species that produce
mentioned earlier, it may not be significantly better than multiple stems such as wheat, the growth reduction occurs
chlorophyll level as measured with a SPAD meter. The mainly in the reduction of tiller number (Maas and Grieve,
more easily measured fluorescence parameter Fv/Fm de- 1990; Francois et al., 1994; Husain et al., 2003) which, for
creased only when chlorophyll content decreased, indicat- a farmer, means fewer spikes and therefore less poten-
ing that a simple meter for measuring chlorophyll density in tial yield per plant. Developing salt injury will cause the
leaves (such as the SPAD meter) is a more cost-effective sensitive genotypes to grow even slower than the more
measure of photosynthetic capacity than chlorophyll tolerant (Phase 2 effect; see Fig. 7). In the example shown
fluorescence. in Fig. 7, the osmotic effect of 150 mM NaCl reduces the
biomass after 40 d by roughly 75% and the salt-specific

Water relationships effect by another 20%.
Despite the fact that the osmotic effect on growth of the
Plant water status is difficult to assess as it can change so
more-tolerant species such as wheat and barley is much
much from minute to minute, as much as stomatal
greater than the salt-specific effect, the mechanisms that
conductance on which it is entirely dependent in the short
regulate the growth rate are not understood. Whether water
term, and psychometric or pressure chamber measurements
status, hormonal regulation, or supply of photosynthate
are tricky to do accurately. Relative water content (RWC) is
exerts the dominant control over growth rate of plants in
easier to measure, but not valid when osmotic adjustment
dry or saline soil is still unresolved. Over the timescale of
occurs (Lafitte, 2002). RWC, although a convenient and
days, hormonal signals rather than water relationships are
widely used method of assessing plant water status, is not
controlling growth in saline soils. The evidence for this is
useful for salt-treated plants, at least not with the conven-
that leaf expansion in saline soil at the timescale of days
tional method of detaching leaves and rehydrating on
does not respond to an increase in leaf water status. Water
distilled water. This is because in most plants, osmotic
status was manipulated by growing plants in sand in pots
adaptation has occurred; i.e. the solute content of cells is
that could be placed in pressure chambers, watering with
higher in saline than non-saline conditions, due largely to
saline solution (100 mM NaCl), and then pressurizing the
the accumulation of Na+, Cl, and also to organic solutes.
root systems in chambers with a pressure equal to that of
The increased solute content of the cells in the salt-treated
the salt concentration, to compensate for the suction of the
plants causes more water to be taken up than in the con-
soil solution (Termaat et al., 1985). No lasting effect of
trol leaves, resulting in an apparent low RWC in the salt
treatment. This was shown in water relationship measure-
ments in durum wheat when the turgor pressure (calculated 10
Phase 1 Phase 2
from the difference between total water potential and
osmotic potential) was unchanged by salinity, but the
8
RWC was significantly decreased (James et al., 2002;
Total dry weight (g)
Rivelli et al., 2002).

Recent work with isopiestic psychrometry on wheat and 6
osmotic effect
barley in a range of saline solutions confirmed that turgor
was unchanged, but RWC decreased (JS Boyer, RA James, 4
R Munns, unpublished results). This was due to abnormal
water uptake by leaves with a high solute content when
2
floated on distilled water, which caused a leakage of specific ion effect
cytoplasmic solution into the apoplast. Thus, osmotic ad-
justment shifts the relationship between turgor and RWC, 0
0 10 20 30 40 50
and so RWC is not an indicator of turgor in plants under- Time after NaCl added (d)
going osmotic adjustment. Screening methods for leaf
water status should use rehydrating conditions for the Fig. 7. Two accessions of the D-genome diploid wheat progenitor
Aegilops tauschii grown in control solution (closed symbols) and in
whole plant, such as a dark humidified atmosphere while 150 mM NaCl (open symbols). Circles denote the tolerant accession, tri-
roots are still in saline soil, rather than detached leaves angles the sensitive one (modified from Munns et al., Australian Journal
floating in distilled water when abnormal water uptake of Plant Physiology 24, 1995, http://www.publish.csiro.au/journals.fpb).
The osmotic effect of the NaCl in the soil reduces the biomass after 40 d
occurs (JS Boyer, RA James, R Munns, unpublished by about 75% and the salt-specific effect by another 20%, as indicated by
results). the arrows.
pressurization on growth was found over periods up to For instance, when plants were grown at 95% relative
8 d, with species as diverse as barley, wheat, white lupin, humidity to maximize the leaf water status of both
Egyptian clover, and saltbush (summarized in Munns, genotypes, there was no significant effect of salt on shoot
1993). More recently, pressurization was done at ‘balancing dry weight of either genotype. However, older leaves of
pressures’, where the chambers were pressurized suffi- sitiens died due to desiccation, whereas no visible injury
ciently to keep the xylem at atmospheric pressure (i.e. appeared in the wild type (Mäkelä et al., 2003). These
a small cut on a leaf was kept on the point of bleeding). This results confirm that ABA promotes rather than inhibits
meant that the leaf water potential was maintained close to plant growth under stress, and has a major effect on
its maximum during both day and night, while the soil was preservation of older leaves.
watered with 100 mM NaCl. This treatment also did not That the hormonal control of cell division and differen-
make plants grow faster (Munns et al., 2000a), indicating tiation is affected by salinity is clear from the appearance
that hormonal signals, and not leaf water deficit or ion of leaves: leaves are smaller in area but greener, i.e. the
toxicity, were controlling growth. The results in every way density of chloroplasts has increased, indicating that cell
were reminiscent of experiments done with plants in dry size and shape has changed. Leaves have a higher specific
soil; these had shown no response of leaf growth to an leaf weight (higher dry weight:area ratio) which means that

increase in shoot water status brought about by pressuri- their transpiration efficiency is higher (more carbon fixed
zation at balancing pressures. Further, ‘split-root’ experi- per water lost), a feature that is common in plants adapted
ments with plants whose root systems were divided to both dry or saline soil.
between wet and dry soils showed that leaf expansion de-
creased while leaf water status was unaffected (reviewed Screens for tolerance of the osmotic effect of salt
by Davies and Zhang, 1991; Munns, 2002).
The regulation of growth rates in leaves and roots under
These experiments indicated that there are chemical
stress is so complex, and the mechanisms so little un-
signals coming from roots in dry or saline soil that reduce
derstood, that the idea of improving salt tolerance by the
leaf growth. Abscisic acid (ABA) has been considered the
manipulation of individual genes or even pathways con-
obvious candidate for this signal as it is found in xylem sap
cerned with growth rates is not feasible at this stage.
and increases after drought and salinity stress (reviewed by
Screening methods that have been used or tested to select
Munns and Cramer, 1996). However, there is still no
for genetic improvements in the growth response to osmo-
conclusive proof that ABA is the only signal from the roots
tic stress are related to growth or survival and have the
(reviewed by Dodd, 2005). Moreover, the origin of the
following disadvantages.
ABA in the xylem sap is not known, for it moves readily in
the phloem and recirculates from leaves to roots (reviewed (i) Growth rate is difficult to replicate because of the
by Munns and Cramer, 1996), and may be synthesized large environmental effects and the length of time
in situ in leaves. ABA could regulate leaf cell expan- over which plants must be grown. The maintenance
sion through a signal transduction pathway that controls of optimal conditions for growth of plants in non-
the activity of ion channels that take up essential solutes saline conditions is particularly difficult, especially
for growth, such as K+ and amino acids, or it could work when genotypes with different heights or develop-
through other hormones. ABA may affect the synthesis mental patterns are being compared, as the tallest or
of gibberellins that control the rate of cell expansion, or it more vigorous ones compete successfully for light
may affect the synthesis of other hormones such as cyto- with those with dwarfing genes, thus giving a false
kinins and auxins that are known to control cell division. idea of salt tolerance. It is not possible to accom-
ABA’s reputation as a ‘growth inhibitor’ under stress modate large numbers of genotypes in greenhouses,
may be undeserved. There is strong evidence that the especially if the effect on grain yield needs to be
increased production of ABA under drought suppresses measured. On the other hand, the field is a complex
accumulation of ethylene that would otherwise inhibit and unpredictable environment.
growth (LeNoble et al., 2004). Further indication of (ii) Germination rate is by far the easiest thing to
a positive role of ABA under salt stress is indicated by measure, but the least likely to predict the ability
studies with the ABA-deficient tomato mutant sitiens. The of plants to grow in saline soil. No correlation has
mutant grows slower under optimal conditions, probably been observed between salt tolerance at germination
because of excessive ethylene production (Sharp et al., and the seedling stage (reviewed by Shannon, 1997;
2000). To see whether ABA accumulation inhibits or Munns and James, 2003), nor between germination
promotes shoot growth under salinity stress, sitiens and and grain yield (Ashraf and McNeilly, 1988).
its wild type were grown at 75 mM NaCl for 2 weeks under Furthermore, in the field, germination rarely takes
conditions of moderate or high relative humidity (Mäkelä place in high salt concentrations. In irrigated
et al., 2003). The major difference between genotypes was agriculture, salt would normally be leached from
in the degree of desiccation injury suffered by older leaves. the surface at sowing, and in dry-land agriculture,
1038 Munns et al.
the crop is normally planted after rain. In those sumably due to variation in soil moisture. Thus, field
salt-affected situations where the crop is sown trials give information of performance only at
without rain or leaching irrigation, the soil in the a particular site, and in a particular season. Field
top 10 cm is likely to be sodic as well as saline, and experiments may be more appropriate at the final
the main constraint to emergence might be the stages of a breeding programme, rather than at initial
hardness of the soil as much as the salts in the soil stages when screening and selection for novel
solution. Seeds that germinate on filter paper wetted germplasm or for specific traits is best done under
with a highly saline solution may be too weak to controlled environments. This is particularly impor-
break a soil crust and establish as viable plants tant when the putative donors of specific traits are
(Shannon, 1997). Emergence rate might be a more foreign genotypes not adapted to local conditions.
practical screening criterion than germination rate,
The most extensive screen for salt tolerance in the field
and seedling vigour may also be a useful screening
has been done by Jafari-Shabestari et al. (1995), who evalu-
factor for soils that form hard crusts.
ated 400 Iranian wheats on one site in California over two
(iii) Desiccation tolerance is also easy to score: potted
seasons, irrigated with water at three salinity levels (1, 5,
plants can be left to dry out and then rewatered so

and 8 dS m1). They measured final biomass and yield, and
that selections can be made from those that recover
calculated a ‘stress susceptibility index’ that relates grain
best. Although plant biologists have given an
yield in saline versus non-saline soils. They found little
enormous amount of attention to plant desiccation
correlation between grain yield at high salinity with
tolerance, these processes are largely irrelevant to
biomass, harvest index, or stress susceptibility, and noted
crop yield. If plant cells desiccate, crop yields will
that some genotypes with low stress susceptibility (i.e.
be negligible and even if yield is doubled by plant
apparent tolerance to stress) had low yield potential. They
manipulation, then it is still negligible (Serraj and
concluded that the susceptibility index is highly subject
Sinclair, 2002). One exception to this situation is the
to experimental errors, especially with small plots, and
combination of responses that allow a perennial
questioned its use. A lack of correlation between relative
crop plant to stay alive under desiccating conditions.
yield and absolute yield, in a comparison of 38 genotypes
This capacity to ‘live to fight another day’ can be
of wheat and other cereals, was also noted by Richards
highly advantageous for forage yield in succeeding
et al. (1987) who concluded that the most efficient way
growth seasons. The capacity to survive is often
to increase yields at high salinity was to select for the
irrelevant in annual grain crops as many are grown
best performers at low salinity.
in such short seasons that a stress-induced delay in
These results suggest that the field is not appropriate for
development can result in a complete loss of yield.
screening large numbers of genotypes, especially exotic or
(iv) Survival as a selection criterion, while rapid and
foreign genotypes, as their yield will be influenced strongly
simple, needs additional assessment. Survival at
by flowering and maturity time, as well as by other factors
high NaCl does not necessarily imply healthy
such as height and disease resistance, and so may not
growth at these high salinity levels, and it is
compare well with adapted cultivars for this reason alone.
important to evaluate yield and yield components
Further, extensive experiments involving replicated sites
of promising lines.
and seasons is not cheap. A more cost-effective way may
(v) Stomatal conductance measured by viscous flow
be to screen for specific traits in controlled environ-
porometry, or assessed by leaf temperature (thermal
ments, back-cross the traits (if from exotic germplasm) into
imaging) can measure several hundred genotypes
adapted cultivars, and test these breeding lines in the field.
per day, as long as ambient conditions are constant.
(vi) Yield needs to be measured in the field, as controlled
environment chambers or glasshouses cannot pro-
Taking salt tolerance from the laboratory to
vide the space required to maximize yield; in-
the field
adequate lighting and pot size will always limit
long-term growth and yield. Screening large num- Field performance is the ultimate test for salt tolerance, and
bers of genotypes for salt tolerance in the field is must be done at replicated sites and seasons. The differ-
difficult, as discussed in the Introduction, due to ences between seasons in temperature and drought which,
variation and unpredictability of weather, but also of particularly in dry-land agriculture, will directly affect the
soil moisture, soil type, spatial heterogeneity of soil build-up of salts around the roots, mean that tests must be
chemical and physical properties, and waterlogging. done over at least 3 years. Field tests may show up un-
For example, Slavich et al. (1990), after carefully expected responses, as described earlier for derivatives of
mapping the heterogeneity in soil conductivity with the salt-tolerant Indian landrace, Kharchia. KRL1-4 per-
an EM meter, found that barley rankings differed on formed well in India but not in Pakistan, possibly because
different soil types and in different seasons, pre- of the greater problems of waterlogging, and KTDH 19
+
performed well in Spain but not in India and Pakistan to high internal Na concentrations have been identified in
because it matured about 2 weeks later than local genotypes rice (Yeo and Flowers, 1983; Yeo et al., 1990) and durum
(Hollington, 2000). wheat (Munns and James, 2003), but it has not been easy
As mentioned in the Introduction, field conditions vary to transfer this into cultivars because of the lack of pre-
from site to site, not only in soil salinity, but also in soil cise phenotyping techniques.
physical and chemical properties such as sodicity, high pH, It is intriguing that so little is known about the
and boron, and interactions between these stresses can physiology of the Indian landrace Kharchia 65, universally
occur. High pH can cause reduced K+ uptake even though regarded as highly salt tolerant (Joshi, 1976; Kingsbury and
it might not affect Na+ uptake (Ahmad, 2002), and boron Epstein, 1984; Sharma et al., 1984; Ashraf, 2002), apart
can affect the subcellular distribution of salt in leaves and from an observation by Sharma et al. (1984) that it com-
hence the salt tolerance of the plant (Wimmer et al., 2003). bined low Na+ uptake rates with successful osmotic ad-
Waterlogging worsens the effects of salinity on wheat justment, and the finding of Richards and Lukacs (2002)
(Barrett-Lennard, 2003) and may be a major reason why that it has unusually high specific leaf area and early vigour.
wheat bred for salt tolerance has had little success in Yet it is still the mainstay of the Indian wheat breeding
farmers’ fields in some regions (Hollington et al., 2002). programme. Perhaps a combined physiological and mo-

When O2 deficiency occurs in roots in waterlogged soils in lecular analysis of this and the other highly salt-tolerant
species with little aerenchyma or adaptations such as genotypes such as SARC-1 and Sakha 8 could reveal
shallow roots, respiration is impaired, and may be the important mechanisms that could be used to develop salt-
cause of the higher salt uptake described by Barrett- tolerant cultivars for other countries.
Lennard (2003), especially now it is known that there are Also little is known about the mechanism of tolerance of
normally high rates of Na+ efflux from roots to the external barley in comparison to wheat, other than that barley
solution (Davenport et al., 2005) which is an energy- tolerates its high internal concentrations of salt almost as
demanding process. The roots of many wetland species well as halophytes, and must be able to compartmentalize
contain a barrier to radial O2 loss in addition to having the salt in vacuoles. This raises the questions of whether
extensive aerenchyma, and introduction of these traits barley could be even more tolerant of saline soil if it could
into wheat may increase its waterlogging tolerance and its exclude Na+ better, and what would happen if genes
ability to provide enough energy to exclude Na+ in responsible for the low Na+ transport rates in wheat were
waterlogged soils (Colmer et al., 2005). introduced.
All breeding lines developed with greater tolerance to Other traits are needed to improve tolerance of the
salt or any soil constraint should have the ability to yield osmotic effect of the salt outside the roots, which can have
well under optimal conditions. Most farmers’ fields are a greater effect on growth and yield than the salt-specific
heterogeneous for soil physical and chemical properties, component. Such traits include water use efficiency,
and most of the yield in the field comes from the least saline osmotic adjustment, and morphological or developmental
and most fertile areas, so yield potential is probably the patterns that conserve water; such traits are more impor-
most important trait of all. Richards (1993) argued that the tant for dry-land than irrigation agriculture.
best breeding strategy is to select for high yield on non- The largest gains from diversity within a crop species
saline soils, and it certainly follows that in a breeding or could be made by selecting for specific traits, and
backcrossing programme, the most productive genotype at recombining these from a series of donor parents, rather
the lowest salinity should always be used as the recurrent than selecting for salt tolerance per se, as discussed by Yeo
parent (Richards, 1993; Royo and Aragüés, 1999). That and Flowers (1986). This pyramiding approach might
argument was made in the context of yield penalties enable improvements in tolerance beyond that presently
associated with introducing new genes for salt tolerance available within a specific crop. This requires that the
from progenitors or wild relatives with little yield potential, underlying traits be identified; that there is a reliable and
and provides a caution to physiologists or cytogeneticists precise screening method, and that the traits are heritable.
to ensure that no yield penalty or ‘linkage drag’ is asso- When insufficient natural variation exists with in a spe-
ciated with the trait under study. cies or its close relatives, use of gene transformation
technology can provide novel and useful genetic material.
However, the novel gene has to be backcrossed into
Summary
commercial cultivars that are adapted to the environment
To increase the salt tolerance of cereals, attention in most of interest, and the value of the trait has to be measured in
laboratories has focused on identifying new genetic sources different genetic backgrounds. This process will be accel-
of low rates of Na+ uptake to leaves. Less attention has been erated by the knowledge of the physiological mechanisms
given to genetic sources of tolerance of high Na+ concen- and how to quantify their effect on salt tolerance. Selections
trations in leaves, probably because this trait is much more of breeding lines can be done most cost-effectively in the
difficult to quantify. Potential sources of tissue tolerance glasshouse, and field trials reserved for lines that are
1040 Munns et al.
known to express the gene or trait of interest. The field, with Davies WJ, Zhang J. 1991. Root signals and the regulation of
its inherent variability, can be used to assess the perform- growth and development of plants in drying soil. Annual Review
of Plant Physiology and Plant Molecular Biology 42, 55–76.
ance of the selected trait in adapted cultivars, and evaluate
Dodd IC. 2005. Root-to-shoot signalling: assessing the roles of ‘up’
breeding lines for commercial release. in the up and down world of long-distance signalling in planta.
Further improvements in salt tolerance will undoubtedly Plant and Soil 274, 251–270.
result from close interactions between molecular geneticists Dubcovsky J, Santa Maria G, Epstein E, Luo MC, Dvořák J.
and physiologists, and benefit from timely feedback from 1996. Mapping of the K+/Na+ discrimination locus Kna1 in wheat.
plant breeders and agronomists. Theoretical and Applied Genetics 2, 448–454.
Dvořák J, Noaman MM, Goyal S, Gorham J. 1994. Enhancement
of the salt tolerance of Triticum turgidum L. by the Kna1 locus
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87, 872–877.
AL received a McMaster Fellowship from CSIRO. The work on
El-Hendawy SE, Hu Y, Schmidhalter U. 2005. Growth, ion
durum wheat reported in this paper has been supported by the Grains
content, gas exchange, and water relations of wheat genotypes
Research and Development Corporation (GRDC) of Australia.
differing in salt tolerances. Australian Journal of Agricultural

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Journal of Experimental Botany, Vol. 57, No. 5, pp.

Approaches to increasing the salt tolerance of wheat and

Rana Munns1,*, Richard A. James1 and André Läuchli2

Received 1 August 2005; Accepted 16 December 2005

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* To whom correspondence should be addressed. E-mail: rana.munns@csiro.au

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the shoot retains only 2% of the water transpired. In order

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balance the osmotic pressure of the salt outside (Table 1).

200 impose a real carbon cost on the plant especially as the

from the outer two layers of cells in the cortex, the

1 11 103 4 110 +226 14

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30% compared with their respective controls (Husain et al.,

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Rivelli et al., 2002).

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