Aust. J. Agric. Rex, 1988, 39, 759-72

An Examination of Selection Criteria for Salt Tolerance in Wheat, Barley and Triticale Genotypes

H. M. Rawson, R. A. Richards and Rana Munns

Division of Plant Industry, CSIRO, P.O. Box 1600, Canberra, A.C.T. 2601.

Abstract This study of 20 genotypes of barley, wheat, durum wheat and triticale had three aims: (1) To determine whether simple measurements on plants grown in salinity tanks in a glasshouse would reflect the documented reputations for salinity tolerance of the genotypes; (2) to test whether rapid development, commonly associated with barleys, is linked with salinity tolerance; (3) to assess several types of measurements as screening tools for salinity tolerance.

Measurements of whole-plant leaf area expansion rates were well correlated with biomass production and ranked the genotypes largely in accord with their documented reputations. There was no evidence, either from experimental manipulation of rate of development, or from regression analysis amongst genotypes, that rapid development was linked with salinity tolerance. The origins of tolerance were twofold, deriving from (1) a physiological tolerance - this was defined as a small relative reduction in growth due to salinity, and (2) an absolute tolerance - this was shown as an intrinsic high growth rate of the genotype, i.e. apparent both in and out of salinity. A good indicator of high absolute tolerance, and of potential for screening purposes, was large area of seedling leaves. Regression analysis indicated that absolute tolerance contributed more to productivity in saline conditions than physiological tolerance. Indeed, in one study the latter failed to correlate significantly with productivity. C1- concentration also was a poor general indicator of productivity in salinity, as was extension rate of single leaves during 10 days after NaCl was applied. It is proposed that screening for intrinsic high growth rate and physiological tolerance should go hand in hand, with more emphasis on the former. This is the reverse of the usual situation.

Introduction

Screening large numbers of genotypes on naturally saline soils for improved salinity tolerance is not feasible becauke of the extreme variability in soil salinity both spatially and temporally (Hajrasuliha et al. ,1980; Richards 1983). To avoid this variability in screening programmes, plants are often g:own in nutrient solution to which NaCl has been added. Selection then is frequently based on ability to germinate or survive, often under extreme NaCl concentrations (Epstein et al. 1980; McGuire and Dvorak 1981). Unfortunately, germination and survival in salinized nutrient solutions may bear no relationship to growth and yield of plants in saline soils (e.g. Ayers et al. 1952; Richards et al. 1987), though Schaller et al. (1981) showed that variation in emergence rate amongst barley cultivars was correlated with performance in saline fields. Germination involves processes not found in established seedlings, and very young roots before the Casparian strip is formed cannot exclude NaCl like mature roots. Survival involves processes which may be counter-productive to rapid growth, such as stornatal closure and osmotic adjustment. Thus, to compare salinity tolerance of a range of genotypes, it may be necessary to measure plant growth throughout development.

The purpose of the current study was to evaluate some simple procedures to screen temperate cereals for salinity tolerance and three questions were addressed:

(1) Can genotypes of cereals be ranked in accord with field observations for salt tolerance when they are grown in artificial culture? The study included barleys, wheats (both I: aestivum and 7: turgidum), and triticales with documented reputations for NaC1-tolerance or

0004-9409/88/050759$03.00

H. M. Rawson et al.

NaC1-sensitivity. (2) Can simple, cheap measurements be made at a relatively early growth stage which reflect

the plants' long-term NaCl tolerance? The simple, early measurements used here were length and breadth measurements of all leaves on plants on three occasions starting 10 days after NaCl was first added. Leaf areas and relative leaf expansion rates (RLER) were calculated for the two intervals. Ruler measurements of selected leaves were also made daily during the initial 10-day period. Long-term NaCl tolerance was assessed by total-plant dry weight measurements made when leaf 7 on the main shoot had emerged. A further simple measurement, that of leaf C1- concentration, was made by using an inexpensive ion-selective electrode. Na+ measurements were not made because of the relative high cost or complexity of the instruments required.

(3) What impact does rate of development have on NaCl tolerance? Barleys have a reputation for greater tolerance than wheats, and barleys also are commonly of shorter duration than wheats. It is conceivable that the reputation of the barleys may partly derive from their rapid early growth associated with the fast progression of phenological development. The effect of development on tolerance was tested by seed vernalizing all genotypes and then growing them under long days and high radiation and temperature. This had the effect of accelerating rates of development and reducing the range in the timing of ear emergence amongst genotypes to approximately 1 week. The results from this study were compared with those from a study in which development was not accelerated.

Materials and Methods

Plant Culture There were two glasshouse experiments, one sown in late April and harvested in early June when leaf

7 was fully emerged (normal floral development; Experiment I), and the other sown in early March and harvested in late April when leaf 7 and most ears had emerged (hastened floral development; Experiment 2). Experiment 1 used natural daylength (10.9 h decreasing to 9.8 h), whilst Experiment 2 used incandescent lamps to extend the natural photoperiod (12.3 h decreasing to 11.3 h) to 14 h. Experiment 2 was designed to accelerate floral development in the later genotypes and consequently make the progression of floral development more similar amongst genotypes. Delays in development were also minimized in the second study by vernalizing imbibed, planted, seeds at 2-5'C f& 26 days in a controlled temperature room before the plants were moved to the glasshouse. Radiation in the first study averaged 9.4 MJ day-' and in the second 17.9 MJ m-2 day-l and the 12 h thermoperiods approximated 23/15"C and 27/18"C respectively.

Apart from the differences already described, cultural conditions for the two studies were similar. Seeds were sown into 150 mm high by 75 mm diameter pots containing a coarse gravel. Groups of 105 pots were arranged in plastic-lined, metal tanks, 1 tank per NaCl treatment. Seedlings were thinned to one per pot, leaving approximately 150 plants m-2; a second thinning reduced the density to 110 plants m-2. There were five plant-replicate blocks in each tank, two blocks on the edge and three in the centre, with the genotypes being randomly distributed within the blocks. There was some concern that competition for light at these relatively high densities would favour the genotypes with rapid early growth. If this were the case, the coefficient of variation (c.v.) for biomass production would become greatest in the slow- starting genotypes, as the edge plants of these genotypes grew well and the inner canopy plants were suppressed with development of the canopy. Our data showed no difference in C.V. at the final harvest between genotypes grouped for rapid or slow early growth.

The pots were subirrigated initially with tap water, with 0.25 strength Hoagland's solution (No. 2; Hoagland and Arnon 1938) on the first day after emergence, 0.5 strength on the second, and full strength on the third. NaCl was added from the fifth day in daily increments of 25 mol m-3 until the eight final concentrations were reached (0, 25, 50, 75, 100, 125, 175, and 250 mol m-3 NaCI) by which time two to three leaves were fully emerged. Solutions were pumped into and drained from the tanks twice every hour, ensuring that the roots were immersed in solution for a third of the time. They were changed at least once each week and were regularly replenished with water to maintain the electrical conductivity (FT 1-10 Mini Digital conductivity meter).

Selection Criteria for Salt Tolerance

Genotypes Genotypes used in the two experiments were chosen for their presumed tolerance or susceptibility to

salinity. Presumed tolerant barleys (Hordeum vulgare, L.) were: California Mariout ((6-row), cf. Ayers et al. (1952), Epstein et al. (1980), Richards et al. (1987)); Beecher ((6-row), W. J. Boyd quoted in Delane et al. (1982)); ELB 14, ELB 48, and ELB 58 ((2-row); tolerant selections from Syria, E. Weltzien personal communication). The presumed sensitive barleys were: Arivat ((6-row), Epstein et al. (1980), Richards et al. (1987)); Clipper (2-row), and the Danish variety Bomi (2-row) which has never been exposed to selection pressure under salinity. Tolerant wheats (Triticum aestivum L.) were: Kharchia (PI 322280) and PI 180988 (McCuire and Dvorak 1981; Kingsbury and Epstein 1984), WW15 (Anza) (Kingsbury and Epstein 1984; Rawson 1986; Richards et al. 1987), whilst the sensitive wheats were Baart, Sonora 64 (Richards et al. 1987) and 461 (Rawson 1986). Ramona was selected as a wheat with intermediate sensitivity (Ayers et al. 1952; Richards et al. 1987), and an oligoculm (AUS 20431) as a wheat with unknown tolerance. Two durum wheats (Triticum turgidum L.) were included: Kamilaroi, chosen for its capacity to osmotically adjust (Hare 1983) and the salt-sensitive Modoc (Richards et al. 1987). Also included were two hexaploid triticales (~Triticosecale Wittmack), Currency and the tolerant Siskiyou (Richards et al. 1987). Where the genotypes are identified individually on the figures they are numbered as follows: Barleys: (1) Arivat, (2) Beecher, (3) Bomi, (4) California Mariout, (5) Clipper, (6,7,8) ELB 14, 40, 58; bread wheats: (9) Baart, (10) Kharchia, (11) Oligoculm, (12), PI. (13) 461, (14) Ramona, (15) Sonora 64; durum wheats: (17) Kamilaroi, (18) Modoc; triticales: (19) Currency, (20) Siskiyou.

Measurements Leaf areas (A) were determined non-destructively on all plants on three occasions a week apart by

measuring the length (L) and greatest breadth (B) of each leaf blade (A = L * B * 0.73) and summing the data for each plant. They were also determined at a destructive harvest, when leaf 7 on the main stem had emerged, by means of a video planimeter (Delta-T instruments). At this harvest the plants were washed from the gravel, separated into roots and shoots, and the green leaf blades were removed for fresh weight and area measurement; dead leaves were considered separately. Leaf blades were dried at 70°C for 2 days, and weighed. Leaf 4 was then extracted with boiling 100 mol HN03 for C1- measurements with a C1- selective electrode (Orion 96-17). Further destructive harvests were made of individual genotypes in Experiment 1 when ears emerged. Studies were terminated prior to grain filling because genotypes differed widely in anthesis date in the main study. This would have led to very different post-anthesis radiation conditions and biased comparisons.

Relative leaf expansion rates (RLER: m2 m-2 day-') were calculated for each leaf, tiller and plant for the intervals between measurements as

where t = time in days. The first whole-plant leaf area measurements used in this paper occurred on the first day that the 250 mol m-3 NaCl treatment was reached, by which time plants in both experiments had four leaves.

Results and Discussion

Development Phenological (floral) development was different in the two experiments. In Experiment 1,

with natural photoperiod, the genotypes differed considerably in final leaf number and consequently time to ear emergence. The earliest wheat had its flag leaf at position 7, whereas the latest barley still had not reached the flag position at leaf 13. By contrast, in Experiment 2, with its vernalization and extended photoperiod designed to accelerate phenological development, all genotypes produced only 7 or 8 leaves before ear emergence. Salinity also affected floral development. In Experiment 1, phenological development was accelerated with increasing salinity, as shown by anthesis occurring up to 3 weeks earlier in the 250 mol m-3 NaCl treatment than in control plants. For the wheats and triticales, the effect of NaCl (X, mol m-3) on days from sowing to anthesis (Y) was well described by the regression Y = 78.4 - 6.92X; r2 = 0.95. This acceleration was less evident in the barleys than the other species (cf. Greenway (1962) for earlier maturity in NaC1-treated barleys). In Experiment 2, phenological development was considerably advanced by the vernalization and long-day treatments, and effects of salinity on development were not evident.

H. M. Rawson et al.

Responses of Plant Weight and Leaf Area to Salinity: Main Effects Plant dry weight (root plus shoot) declined linearly with increasing salinity in both experiments

(3 = 0.97), with a reduction of approximately 0.24% (5-6 mg at the leaf 7 stage) for each mol m-3 NaCl increase. The linear reduction contrasts with results from field experiments (e.g. Richards et al. 1987) where a threshold response sometimes precedes the linear reduction (Richards 1954; Mass and Hoffman 1977). Greenway (1962) argued that the discrepancy between field and glasshouse responses must be due in part to the prolific growth of control plants in the glasshouse, where water and nutrients are not limiting.

Unlike plant weight, plant leaf area was reduced non-linearly with increasing salinity with the effect being proportionately less at high NaCl (Fig. I). The different patterns of reduction

Fig. 1. Plant leaf area when leaf 7 on the main shoot had emerged in Experiments 1 (normal development; 0 ) and 2 (hastened development; A). Mean data for all genotypes (+ s.e.).

0 5 0 1 0 0 1 5 0 2 0 0 2 5 0

Salmty (mol I I - ~ NaCI)

in leaf area and dry weight with salinity were partly due to the effects of salinity on specific leaf weight discussed later. Leaf areas in Experiment 1 (normal development) were much greater than in Experiment 2 (hastened development) at the same stage of leaf number development. This is shown on Fig. 1 for data collected when leaf 7 on the main shoot had emerged in both experiments (respectively 5.5 and 5 weeks after plant emergence). A semi-logarithmic plot of log leaf area v. time (not presented), showed a linear reduction in both studies (? = 0.99). This analysis indicated that the effects of salinity on leaf area were greater in Experiment 2 than in Experiment 1 (P < 0.01).

A plant's leaf area at any time is a reflection of the integrated effects of the environment over the plant's life, whereas relative leaf expansion rate (RLER, m2 m-2 day-') largely reflects the plant's current responses to its environment. RLER was therefore measured to examine

Fig. 2. Salinity effects on relative leaf expansion rate per plant (m2 m-2 day-'), averaged over genotypes, 4 (0) and 5 (0) weeks after emergence in Experiment 1 and 5 weeks after emergence in Experiment 2 (A). The bars are s.e. of the mean.

Salinity (mol mJ NaCI)

Selection Criteria for Salt Tolerance

the changes in response to salinity with plant ontogeny. This sensitivity of RLER is demonstrated in Fig. 2, where, by comparing the two curves for Experiment 1, the net effect of reduced leaf production and death in the 250 mol m-3 NaCl treatment can be seen (i.e. 5 weeks after plant emergence). Data for Experiment 2 are included in the figure to show that RLERs were higher in that study at a n equivalent time after emergence, partially as a consequence of a lower leaf area and more light penetration into the canopy, but also because temperatures were higher. Furthermore, the reduction in RLER with salinity was greater in Experiment 2 than in Experiment 1, confirming the conclusion from the logarithmic analysis of total leaf area described above, that plants were more sensitive to NaCl in the hastened-development study.

Responses of Plant Weight and Leaf Area to Salinity: Species Effects As described above, plant dry weight declined almost linearly with increase in salinity (Fig. 3

Salinity (mol m" NaCI)

4

- 0) - *

shows data for Experiment 1 only), but surprisingly, the slopes of this relationship did not differ significantly between the grouped barleys, the grouped wheats and the two durums within

-

Plant leaf area in control regime (cm2)

Fig. 3. Plant root plus shoot dry weight change with salinity in Experiment 1, at a harvest taken when leaf 7 had emerged, averaged for the barleys, the wheats, and

- am '-9 the durum wheats. The bars \.-. 1 - are s t . of the mean.

Fig. 4. Plant leaf area in the absence of salt expressed against area in the 100 and 250 mol m-3 NaCl regimes (Experiment 1). Measurements were made when leaf 7 on the main shoot had emerged. The numbers identify each genotype (see key in Materials and Methods).

H. M. Rawson et al.

an experiment. However, the intercepts of the y-axis, i.e. the dry weight without NaCI, did differ. Thus, biomass at any NaCl concentration was lower in the wheats and durums than in the barleys (89% and 59% of the barleys respectively in the control regime of Experiment 1, and 84% and 66% in Experiment 2). These linear relationships implied that salinity tolerance was largely a reflection of the ability of the species to grow in the absence of NaCl. The same conclusion could be drawn from the leaf area data: plants which produced most leaf area in the control regime also invariably produced more area in NaCl regardless of species (Fig. 4). The coefficient of determination for this relationship ranged between 9 = 0.66 and 0-71 (P < 0.05). Ability to grow 'well' either in or out of salinity was in part related to the size of the seedling leaves. For example, plant leaf area at the leaf 7 stage was significantly correlated with the area of leaf 3 on the main shoot (Fig. 5; r2 = 0.74 and 0.69 in Experiments 1 and

Area of leaf 3 (cm2)

Contrc J

Area of leaf 3 (cm2)

Fig. 5. Relationship between the area of the plant and the area of leaf 3 in control and 250 mol-3 NaCl regimes. (a) Experiment 1. (b) Experiment 2. Measurements in both experiments were made when leaf 7 on the main shoot had emerged. The numbers identify each genotype (see key in Materials and Methods).

Selection Criteria for Salt Tolerance

2 respectively at 250 mol m-3 NaCI. Note that leaf 3 was much smaller in Experiment 2 than in Experiment 1 and was reduced in area far more by salinity: 23% 1 cm2 for control leaves v. 20k1 cm2 at 250 mol m-3 NaCl in Experiment 1, and 12.8'0.6 cm2 v. 8.7'04 cm2 in Experiment 2.). Barleys tended to have the largest seedling leaves under the more normal conditions of Experiment 1 (cf. numbers within Fig. 5a). Plant weight at harvest in the 250 mol m-3 regime was also significantly correlated with the area of leaf 3 in both experiments. Thus, the differences in early leaf area were associated to a large degree with differences in final biomass production both between experiments and between genotypes.

Proportional allocation of dry weight to roots and shoots did not differ amongst the species groupings. However, partitioning changed with salinity with an increasing relative distribution to roots between 0 and 125 mol m-3 NaCl (Fig. 6) . As already described, there were clear

0.30

0.28 Experiment 2

a , Experoment 1 Fig. 6. Effects of salinity on root/shoot

0.24 dry weight ratio in Experiments 1 (a)

0.22 and 2 (A); all genotypes averaged.

0.20

Salinity (mol m" NaCI)

differences amongst species in shoot production; the barleys produced more leaf weight than the wheats in all treatments but, in addition, less leaf died at the higher NaCl concentrations (Fig. 7). Furthermore, specific leaf weight (SLW, weight per unit leaf area) differed significantly

600

wheats

200 - 20

0 50 100 150 200 250

Salinity (mol m-3 NaCI)

Fig. 7. Effects of salinity on live (open symbols) and dead leaf dry weight (closed symbols) in the wheats (triangles) and barleys (squares) in Experiment 1. Note the change of scale for live and dead leaf.

H. M. Rawson et al.

amongst species in the order barleys <wheats <durums (Fig. 8). So, of all the species, the barleys had the 'thinnest', largest leaves, which also had the greatest likelihood of survival

- N Durums

w E

.- Fig. 8. Effects of salinity on specific leaf weight in the grouped barleys (M), wheats (A) and durum

5 - 3-/* wheats (0) in Experiment 1. The bars are * s.e. of the mean.

0 50 1 0 0 150 200 250 Salinity (rnol m-3 NaCI)

in NaC1. In all species SLW increased significantly with salinity at approximately 60 mg m-2 leaf for every mol m-3 NaC1. Approximately half of this increase would be accounted for by accumulation of Na+ and C1- ions as shown in the following example for the barleys. At 175 mol m-3 NaCl, C1- concentration in most genotypes was about 2 mmol g-l dry weight, and, on assuming that Na+ concentration was similar (Greenway 1962, and our own measurements), 12% of leaf dry weight would be due to NaCl. An increase of 1 mg ~ m - ~ in SLW between 0 and 175 moI m-3 NaCl (Fig. 8) would therefore have included 0.45 mg NaCI.

Responses of Plant Weight and Leaf Area to Salinity: Genotype Effects Rankings on plant weight The most realistic estimate of 'absolute salinity tolerance' during vegetative growth in these

studies is plant weight at harvest in Experiment 1 (Table 1). In this experiment results were remarkably similar to previously documented data on these genotypes. Amongst the barleys, California Mariout, ELB 14 and Beecher produced more biomass than Arivat and Clipper, as expected from the literature (see Materials and Methods). Also, as expected, WW15 (Anza) and Kharchia ranked highest amongst the wheats, whilst Siskiyou was the higher-ranking triticale. The only genotype that did not accord with expectations was the supposed-tolerant PI 180988 (see Kingsbury and Epstein 1984), though McGuire and Dvorak (1981) had indicated earlier that it was somewhat sensitive.

A change in rankings from the control to the NaCl treatments indicates the relative resistance or sensitivity to salinity of each genotype, and is one measure of 'physiological salt tolerance'. This contrasts with the absolute tolerance defined above. In Experiment 1, four of five barleys ranked higher with increased salinity, substantiating previous studies (Clipper moved down). WW15 and Kharchia wheats also demonstrated relative tolerance using this basis for comparison, whilst Sonora 64 and Baart showed sensitivity. Kharchia was the only wheat genotype with 100% survival in 250 mol m-3 NaCI, confirming the observation of McGuire and Dvorak (1981) that Kharchia has high survival, whilst Clipper was the only barley with plant deaths in this treatment (confirming earlier observations in this laboratory). A second measure of physiological NaCl tolerance is the percentage reduction in biomass production with increase in salinity (data in Table 1). Using this basis for comparison, California Mariout and ELB 14 ranked highest amongst the barleys and WW15, Kharchia and PI 180988 ranked highest in the wheats. This method did not differentiate between the remaining wheats (sensitive) and the supposed-

Selection Criteria for Salt Tolerance

sensitive Arivat and Clipper barleys. With the exception of the triticales, this measure of tolerance matched expectations from the literature (see the 070 column in Table 1).

Table 1. Rankings of genotypes for plant weight at a harvest taken when leaf 7 had emerged in Experiment 1 (normal development) and in Experiment 2 (hastened development)

Rankings are given for the control treatment and the mean of the 175 and 250 mol m-3 NaCl treatments. The barleys are underlined, the durums double underlined, the triticales are in italics and the wheats in normal script. TheTjEEEntages column refers to the mean of the 175 and 250 regimes expressed in relation to control data, and the letter in that column

is the expectation of sensitivity (S) or tolerance (T) according to the literature

Experiment 1 Experiment 2 Control 175/250 To Control 175/250 To

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 Mean (%)

Range (g)

CalMariout ELB 14 Baart Sonora Siskiyou Beecher Ramona Clipper

Kharchia WW 15 PI Currency Kamilaroi Modoc -

3.71-1.50

CalMariout - ELB 14 Beecher -- WW 15B ArivatA -- Kharchia BaartB SiskiyouB SonoraAB RamonaB ClipperB PIB Currency* KamilaroiB -- -- ModocB

1.97-0.47

ELB 58 -- E m U v a t Oligoculm Sonora Baart Sisk~you Kharchia WW 15 ELB 14 --

Q 61 PI Currency Ramona Kamilaroi Modoc CalMariout Clipper Beecher -- --

3.22-1.28

ELB 40 --

E D WW 15B A- *B

-hiaB SonoraAB U e r B ELB 14B OligoculmB Q 61* RamonaB PIB SiskiyouB CurrencyB ClipperB KamilaroiB CalMariout BaartB ModocB -- --

0.80-0.20

*In both studies, Arivat and Sonora 64 changed their rankings considerably between the 175 and 250 mol m-3 NaCl treatments, the barley showing increased resistance and the wheat decreased resistance to salinity. BIndicates that some plants were dead at the final harvest.

The effects of seed vernalization followed by long days and higher radiation and temperatures (Experiment 2) were quite dramatic on some genotypes. California Mariout barley grew poorly, both with and without NaCl, after performing so well under the conditions of Experiment 1. Otherwise, there were some similarities to Experiment 1; the genotypes which moved up in ranking with increased NaCl were WW 15, Kharchia and Beecher, and the genotypes which moved down were Sonora and Baart (Sonora 64 wheat moved down considerably in ranking between 175 and 250 mol m-3 NaCl). The only genotypes for which all plants survived in 250 mol m-3 NaCl in Experiment 2 were the barleys, California Mariout, ELB 58 and ELB 40. Some plants died even in the 175 mol m-3 NaCl treatment in Modoc and Kamilaroi durums, in PI wheat and in Siskiyou triticale. The persistance of California Mariout at 250 mol m-3 NaCl, in spite of its very poor growth, demonstrates that survival does not imply healthy growth and vice versa.

Rankings on leaf growth Biomass production depends on the cumulative responses of the plant to its environment,

whereas relative growth rate estimates current responses. RLER was measured in both studies in the period between the emergence of leaves 4-7. The rankings of RLER in Experiment

H. M. Rawson et a/.

1 for the highest salinity levels (175-250 rnol m-3 NaCl, Table 2) almost matched the rankings for biomass production at harvest (Table 1). This implies that genotypes differed little in their

Table 2. Rankings of genotypes for plant relative leaf expansion rate (RLER; m2 m-2 day-' in Experiment 1 (normal development) and Experiment 2 (hastened development)

Rankings are given for the control treatment and the mean of the 175 and 250 rnol m-3 NaCl treatments. The b w are underlined, the &rums double underlined, the triticales

are in italics and the wheats in normal script

Experiment 1 Experiment 2 Control 175/250 Control 175/250

1 -- ELB 14 CalMariout ELB 40 ELB 40 2 CalMariout E L U -4 Kharchia 3 Kharchia &her -- CalMariout E w 4 Szskryou WW 15 Baart ELB 14 5 Sonora 64 Q 61 Currency 6 Baart KharchiaA Kharchia Beecher 7 Be& BaartB ELB 58 Q 61 8 WW 15 SiskryouB WW 15 WW 15 9 PI Ramona Currency Sonora 64

10 &iv& CurrencyA Oligoculm Oligoculm 11 Ramona Sonora 64B Ramona SlskiyouB 12 m e r PI Sonora 64 Baart 13 Currency m r -- -- Kamilaroi CalMariout 14 -- Kamilaroi -- Kamilaroi M A C Clipper -- 15 M& _Mo& PI A-A 16 Szskiyou Ramona 17 Clipper PI 18 -- Beecher Kamilaroi 19 A r i a -- Modoc Range 0.12:0.09 0.08: - 0.07 0.22:O. 15 0.08: - 0.03

AGenotypes which ranked several positions higher in the 250 than the 175 mol m-3 NaCl treatment (i.e. resistant genotypes). BGenotypes which ranked several positions lower in the 250 than the 175 rnol m-3 NaCl treatment (i.e. sensitive genotypes).

response to salinity. There were, however, more shifts in the rankings between the 175 and 250 rnol m-3 NaCl treatments in the RLER measurements than in the biomass data (data not shown). Arivat, Kharchia and Currency were relatively more tolerant of the highest salinity treatment, in that they moved up in ranking, whilst Baart and Sonora showed sensitivity.

In Experiment 2, rankings for RLER did not match those for biomass in the 175 and 250 mol m-3 NaCl or in the control treatment. Though the general pattern of the biomass rankings held, the wheat Baart, and the barley Arivat, ranked much lower, whilst Currency triticale ranked much higher.

Absolute leaf expansion rates (LER cm2 day-') for an interval are dependent on the size of the plant at that time (i.e. the cumulative responses of the plant to its environment) and also on the current responses. Thus, LER falls between RLER and a single dry matter harvest for indicating the plant's response to salinity. The rankings for genotypes are given in Table 3a. For both experiments the overall pattern was that the barleys ranked highest followed by the wheats, the triticales and the durums last. However, California Mariout ranked poorly in Experiment 2, a consequence of the treatment effects on its early growth.

A simpler measure to rank genotypes for salinity tolerance was also tested in Experiment 1. This was the daily elongation rate of the expanding leaf on the main shoot, over the 10 days that the NaCl solutions were increased at 25 rnol m-3 day-' from 0 to 250 rnol m-3 NaCl. Over those 10 days elongation rates of the NaC1-treated plants dropped linearly to about 50% of control values. The method, however, failed to distinguish between genotypes. For example,

Selection Criteria for Salt Tolerance

a comparison of the performance of the two tolerant barleys (California Mariout and ELB 14) against the two sensitive durums, over the 10-day period, showed no differences either

Table 3. Rankings of genotypes for (a) plant absolute leaf expansion rate (LER; cm2 day-l) in Experiment 1 (normal development) and Experiment 2 (hastened development) and (b) leaf chloride concentration (mmol g dry wt-1) in

Experiment 1 Rankings are given for the mean of the 175 and 250 mol m-3 NaCl treatments. The barleys are underlined, the durums double underlined, the triticales are

in italics and the wheats in normal script

(a) LER mean 175/250

Expt 1 Expt 2

(b) Chloride 175 Expt 2

1 CalMariout -- Bomi Mo& --

2 -- ELB 14 ELB 40 Clipper 3 Beecher ELB 58 CalMariout 4 & i a A Kharchia E u 5 WW 15 Beecher 6 Ramona WW 15B Beecher 7 BaartB &B 1 4 B Kamilaroi 8 KharchiaA Clipper" ELB40-- 9 PIC Q 61 ~r i va t

10 Sisk~youB Sonora 64 Sonora 64 11 Clipper Ramona Q 61 - -- 12 Currency Currency Oligoculm 13 Sonora 64C PI Baart 14 Karnilaroic Oligoculm* WW 15 15 ModocC -- CalMarioutA Ramona 16 ELB 14 17 SiskiyouBC Siskiyou 18 Baart Currency 19 Kamilaroic PI 20 ModocC Kharchia Range 20.1: - 3.56 4.25: -0.33 1.78:0.73

*Genotypes which ranked several positions higher in the 250 than the 175 mol m-3 NaCl treatment (i.e. resistant genotypes). BGenotypes which ranked several positions lower in the 250 than the 175 rnol m-3 NaCl treatment (i.e. sensitive genotypes). CGenotypes which had negative rates of growth in the 250 mol rn-3 NaCl treatment.

in slope, intercept, or per cent reduction in elongation rate. Indeed, no genotype comparisons were significantly different. Possibly, salt uptake by the genotypes did not differ by this stage, but even if it did, there is no evidence that Naf and C1- concentrations in expanding leaves directly affect growth rates (Munns et a/. 1988). Salt concentrations in rapidly expanding tissues are unlikely to ever reach toxic concentrations, and any genotypic differences in leaf osmotic adjustment leading to turgor maintenance would not affect growth. Termaat et al. (1985) and Munns and Termaat (1986) showed that artificially increased leaf turgor did not increase leaf expansion rate in saline media.

Rankings on CI- concentrations As it has been established for several species that NaC1-tolerance is associated with salt

exclusion from leaves (e.g. Greenway 1962 for barleys), C1- concentrations were measured at the leaf 7 stage. In Experiment 1 there was a significant correlation between C1- concentration and RLER and biomass production, but this did not apply in Experiment 2 (rankings for leaf chloride concentrations in Experiment 2 are given in Table 36). All barleys but one ranked

H. M. Rawson et al.

above the wheats whilst the triticales tended towards low concentrations. The durum wheat, Modoc, ranked above the barleys.

General Discussion

This study set out to answer three questions. The first was: would genotypes grown in artificial conditions rank realistically for NaCl tolerance in terms of biomass production? The results showed that rankings of plants grown in solution culture in a glasshouse correlated well with published field data, as long as a pre-emergence vernalization treatment was not given and radiation and temperature conditions were not too high (Experiment I). Surprisingly, the rankings were generally similar in the presence and absence of NaCl. Thus, the amount of biomass produced by a genotype in saline conditions, which we term its 'absolute salt tolerance', was largely dependent on its intrinsic growth rate. Differences between genotypes in 'physiological salt tolerance', which was estimated as the change in ranking for biomass production with increasing salinity, was not the sole factor in determining the final amount of biomass. Multiple regression was used to gauge the relative importance of intrinsic growth rate and our second measure of physiological salt tolerance (percentage reduction in biomass production from control to the 175/250 mol m-3 NaCl treatment) in determining biomass production in salinity. In Experiment 1 the two independent variables accounted for 84% of the variation, and the individual coefficients of determination were 0.63 (intrinsic growth rate) and 0-60 (physiological tolerance). In Experiment 2, 87% of the variation was accounted for by these variables, though only intrinsic growth rate was significantly correlated with biomass production in salinity (46% of the variation accounted for). Physiological tolerance accounted for only 24% of the variation.

Table 4. Regressions fitted between biomass production per plant (mg) in salt and several potential indicators of salt tolerance (also in salt) using genotype data, meaned for the 175 and 250 mol m-3 NaCl treatments, to provide

variation Experiments 1 and 2 are shown separately. The relationships between biomass production in NaCl and leaf 3 area (cm2), both in NaCl and in the absence of NaC1, are shown. Physiological tolerance is mean plant weight for the 175 and 250 mol m-3 NaCl treatments expressed as a percentage of control plant

weight

Screening indicator Expt. r2 F-value

Leaf 3 area (in NaC1) 1 0.55 15.8*'* 1A 0.70 28.1***

Leaf 3 area (control) 1 0.51 13.2*** 1A 0.65 21.9***

RLER (m2 m2 day-l) 1 0.60 19.3*** LER (cm2 day-]) 1 0.82 95.2*** Dead leaf number 1 0.73 35.0*** C 1 (mmol g dry wt-1) 1 0.40 8.7'" Physiol. tolerance 1 0.60 19.6*** Phasic development 1 0.06 NS

Leaf 3 area (in NaC1) 2 0.39 10.7** 2A 0.48 14.4***

Leaf 3 area (control) 2 0.47 15.9*** 2" 0.59 24.4***

RLER (m2 m2 day-]) 2 0.48 15.9*** LER (cm2 day-l) 2 0.66 33.6*** Dead leaf number 2 (-1 0.31 7.5* C1- (mmol g dry wt-I) 2 0.00 NS Physiol. tolerance 2 0.24 NS

AWW 15 was excluded from these regressions as it has a very small leaf yet tillers freely.

Selection Criteria for Salt Tolerance

The second question addressed was: can simple measurements be made during the vegetative stage of growth that correlate with final biomass production in saline conditions? We found that the area of leaf 3 in salinity was a good predictor of absolute NaCl tolerance particularly under the conditions of Experiment 1 (see the 3 and F values in Table 4). Furthermore, the correlations were significant between the area of leaf 3 on control plants and biomass production in the presence of NaCI. Other methods, which would estimate relative physiological NaCl tolerance rather than absolute NaCl tolerance, were not as successful, though the RLER measurements came very close (Table 4). We measured C1- concentrations in fully expanded leaves as there is a correlation between exclusion of C1- from leaves and NaCl tolerance for several species. There is no established correlation between unrelated species in C1- exclusion and salt tolerance, and we did not observe one here (Table 4). But perhaps unexpectedly, there was no correlation between C1- concentration and biomass production for the wheats or triticales in either experiment. We also measured injury, in this case dead main-shoot leaf number, considering only leaves 1 to 3 at harvest. This was highly significantly negatively correlated with biomass production in Experiment 1 but not in Experiment 2 (Table 4).

The third question addressed was: is the rate of floral development important in determining the NaCl tolerance of certain species or cultivars, it. are genotypes which begin floral development earlier than others more NaC1-tolerant? We believe not, primarily because there was no correlation between the rate of phasic development and biomass production in the high salinity treatments of Experiment 1 (Table 4). Certainly, the conditions of Experiment 2, which were designed to eliminate differences in phenology amongst genotypes, resulted in a genotypic ranking for NaCl tolerance very different from that in Experiment 1. However, the longer duration genotypes as a group showed no improvement in ranking from Experiment 1 to 2. One key to the changed rankings between experiments may lie in the fact that vernalization resulted in very small seedling leaves regardless of the salinity treatment, and that genotypes were not all affected equally. For example, California Mariout had the largest leaf 3 of all genotypes in Experiment 1, but was ranked 12th in Experiment 2; its biomass production declined almost in proportion. This suggests that even in Experiment 2 seedling growth was of primary importance to later productivity.

In conclusion, the present data suggest that absolute NaCl tolerance generally is not so much due to the greater ability to grow in the presence of NaCI, but to grow well per se. In many cases productivity in NaCl can be estimated from the size of seedling leaves on control plants. We suggest that the simple ruler measurement of length x width of either leaf 2 or leaf 3 would be a first indicator of productivity. (Leaves 1 and 4 are not suitable; the size of leaf 1 can be influenced by environmental conditions during seed growth prior to germination, and expansion of tiller leaves interacts with the final size of leaf 4; Rawson et al. 1983). Also, fresh or dry leaf weights are not as good as area, as there are marked genotypic differences in specific leaf weight. Apart from the finding that intrinsic growth rates, not physiological NaCl tolerance, was the main determinant of biomass production at high salinity, the ability to grow in the absence of NaCl is the logical factor to select for, especially in non-irrigated conditions, as Richards (1983) has shown that the most yield will usually come from the least saline parts of a typically salt-affected field.

Acknowledgments

We are grateful to Anne Gardner for tending the plants and associating in the harvests, to Delia Luke for ion analyses, and to Bernie Mickelsen, Bob Dunstone, Geoff Howe, Jan Hulse, Janus Grabowski, John Begg, Mark Rowland, Murray Long and Thomas Cobbleigh for occasional assistance with leaf area measurements. Drs. R. A. Fischer, R. W. Downes and J. L. Davidson made suggestions to improve the manuscript.

References

Ayers, A. D., Brown, J. W., and Wadleigh, C. H. (1952). Salt tolerance of barley and wheat in soil plots receiving several salinization regimes. Agron. J. 44, 307-10.

H. M. Rawson et al.

Delane, R., Greenway, H., Munns, R., and Gibbs, J. (1982). Ion concentration and carbohydrate status of the elongating leaf tissue of Hordeum vulgaregrowing at high external NaC1. I. Relationship between solute concentration and growth. J. Exp. Bot. 33, 557-73.

Dvorak, J., and Ross, K. (1986). Expression of tolerance of Na+, K+ , Mg2+, C1-, and Sod2- ions and sea water in the amphiploid of Triticum aestivum x Elytrigia elongata. Crop Sci. 26, 658-60.

Epstein, E., Norlyn, J. D., Rush, D. W., Kingsbury, R. W., Kelley, D. B., Cunningham, G. A,, and Wrona, A. E (1980). Saline culture of crops: a genetic approach. Science 210, 399-404.

Greenway, H. (1962). Plant response to saline substrates. I. Growth and ion uptake of several varieties of Hordeum during and after sodium chloride treatment. Aust. J. Biol. Sci. 15, 16-38.

Hajrasuliha, S. N., Baniabbassi, J., Metthey, J., and Nielson, D. R. (1980). Spacial variability in soil sampling for salinity studies in southwest Iran. Irrig. Sci. 1, 197-208.

Hare, R. A. (1983). Kamilaroi. Register of cereal cultivars in Australia. J. Aust. Inst. Agric. Sci 42. Hoagland, D. R., and Arnon D. I. (1938). The water-culture method for growing plants without soil.

Circ. 347, Univ. California, College of Agric., Berkley. Kingsbury, R. W., and Epstein, E. (1984). Selection for salt-resistant spring wheat. Crop. Sci. 24, 310-15. Mass, E. V., and Hoffman, G. J. (1977). Crop salt tolerance - current assessment. J. Irrig. Drainage

Division, Proc. Am. Soc. Civil Eng. 103, 115-34. McGuire, P. E. and Dvorak, J. (1981). High salt tolerance in wheat grasses. Crop Sci. 21, 702-5. Munns, R., Gardner, P. A., Tonnet, M. L., and Rawson H. M. (1988). Growth and development in NaCI-

treated plants. 11. Do Na+ or C1- concentrations in dividing or expending tissues determine growth in barley? Aust. J. Plant Physiol. 15, 00-00.

Munns, R., and Termaat, A. (1986). Whole-plant responses to salinity. Aust. J. Plant Physiol. 13, 143-60. Rawson, H. M., Hindmarsh, J. H., Fischer, R. A., and Stockman, Y. M. (1983). Changes in leaf photosynthesis

with plant ontogeny and relationships with yield per ear in wheat cultivars and 120 progeny. Aust. J. Plant Physiol. 10, 503-14.

Rawson, H. M. (1986). Gas exchange and growth in wheat and barley grown in salt. Aust. J. Plant Physiol. 13, 475-89.

Richards, L. A. (1954). Diagnosis and improvement of saline and alkali soils. Handb. U.S. Dep. Agric. No. 60. Richards, R. A. (1983). Should selection for yield in saline conditions be made on saline or non-saline

soils. Euphytica 32, 431-8. Richards, R. A., Dennett, C. W, Qualset, C. O., Epstein, E., Norlyn, J. D., and Winslow, M. D. (1987).

Variation in yield of grain and biomass in wheat, barley and triticale in a salt-affected field. Field Crops Res. 15, 227-87.

Schaller, C. W., Berdegue, J. A., Dennett, C. W., Richards, R. A., and Winslow, M. D. (1981). Screening the world barley collection for salt tolerance. Barley Genetics IV. Proc. Fourth Int. Barley Genet. Symposium, Edinburgh, 22-29 July 1981, pp. 389-93.

Termaat, A., Passioura, J. B., and Munns, R. (1985). Shoot turgor does not limit shoot growth of NaCI- affected wheat and barley. Plant Physiol. 77, 869-72.

Manuscript received 19 February 1988, accepted 10 May 1988