JXB Advance Access published online on February 5, 2007
Journal of Experimental Botany, doi:10.1093/jxb/erl293
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RESEARCH PAPER |
Salt tolerance in a Hordeum marinum–Triticum aestivum amphiploid, and its parents
1School of Plant Biology, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
2CRC for Plant-based Management of Dryland Salinity, The University of Western Australia, 35 Stirling Highway, Crawley, WA 6009, Australia
3School of Agriculture and Wine, The University of Adelaide, Waite Campus, Glen Osmond, SA 5064, Australia
* To whom correspondence should be addressed. E-mail: tdcolmer{at}cyllene.uwa.edu.au
Received 24 August 2006; Revised 28 November 2006 Accepted 4 December 2006
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Abstract |
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Growth, grain production, and physiological traits were evaluated for Hordeum marinum, Triticum aestivum (cv. Chinese Spring), and a H. marinum–T. aestivum amphiploid, when exposed to NaCl treatments in a nutrient solution. H. marinum was more salt tolerant than T. aestivum and the amphiploid was intermediate, both for vegetative growth and relative grain production. H. marinum was best able to ‘exclude’ Na+ and Cl–, particularly at high external NaCl. At 300 mM NaCl, concentrations of Na+ (153 µmol g–1 dry mass) and Cl– (75 µmol g–1 dry mass) in the youngest fully-expanded leaf blade of H. marinum were, respectively, only 7% and 4% of those in T. aestivum; and in the amphiplolid the Na+ and Cl– concentrations were 39% and 36% of those in T. aestivum. Glycinebetaine and proline concentrations in the youngest fully-expanded leaf blade of plants exposed to 200 mM NaCl were highest in H. marinum (128 and 60 µmol g–1 dry mass, respectively), lowest in T. aestivum (85 and 37 µmol g–1 dry mass), and intermediate in the amphiploid (108 and 54 µmol g–1 dry mass). Thus, salt tolerance of H. marinum was expressed in the H. marinum–T. aestivum amphiploid.
Key words: Glycinebetaine, halophyte, ion ‘exclusion’, leaf Cl–, leaf K+, leaf Na+, proline, salinity tolerance, sap osmotic potential, sea barleygrass, Triticeae, wheat, wide hybridization, wild relative
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Introduction |
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About 7% of the world's land area, which amounts to 930 million ha, was affected by salinity in 1999, and the area is increasing (Szabolcs, 1994). Salinity reduces the ability of plants to take up water by lowering soil water potential, which leads to internal water deficits and changes in hormone production (Munns, 2002). Growth is also reduced due to the salt-specific effect of high Na+ and/or Cl– concentrations in tissues resulting in ‘ion toxicity’ (Munns, 2002). Inhibition of the uptake of nutrients such as K+ and Ca2+, causing nutrient imbalances in the plant, might also impede growth (Greenway and Munns, 1980).
Salt tolerance depends on a number of traits, expressed at several levels of organization (Greenway and Munns, 1980; Munns, 2002). ‘Exclusion’ of toxic Na+ and Cl– from the shoots and maintenance of K+ uptake are regarded as important traits. In order to maintain low cytoplasmic concentrations of Na+ and Cl–, these ions can be sequestered in vacuoles. The accumulation of organic solutes, that do not inhibit biochemical reactions in the cytoplasm, maintains equal osmotic potential in the cytoplasmic and vacuolar compartments (Greenway and Munns, 1980). As one example of an organic solute, glycinebetaine is found in a wide range of plant families, including the Graminaceae, to which wheat belongs (McCue and Hanson, 1990; McNeil et al., 1999). Glycinebetaine contributes to cytoplasmic osmotic adjustment, and might also protect enzymes against ion toxicity (Greenway and Munns, 1980; Volkmaar et al., 1998).
Wheat (Triticum aestivum L.) is considered to be moderately tolerant of salinity (Maas, 1986). Salinity tolerance in cereals is associated with a capacity to restrict the rate of entry of Na+ into the shoots (Munns, 2002). In wheat, there appears to be little genetic variation for Cl– concentrations in leaves, whereas there is variation for leaf Na+ concentrations and for selectivity of K+ over Na+ (Gorham et al., 1990). For the Na+ that does enter shoots of cereals, its accumulation in old leaves, and continued transport of K+ to young leaves, also contributes to salt tolerance (Greenway et al., 1965; Yeo and Flowers, 1984; Wolf et al., 1991). Furthermore, glycinebetaine accumulates in the young leaves of salt-tolerant cereals (Colmer et al., 1995; Nakamura et al., 1996). In addition to these physiological traits, other characteristics also determine salt tolerance (i.e. grain production) of cereals in saline fields (summarized in Colmer et al., 2005a).
Salt tolerance in wheat might be increased by using wild species within the Triticeae as sources of genes for wheat improvement (Forster et al., 1987; Omielan et al., 1991; King et al., 1997a; Colmer et al., 2006). Wheat–Lophopyrum and wheat–Thinopyrum amphiploids have displayed considerable improvements in salt tolerance above that in the wheat parent (Omielan et al., 1991; Mahmood and Quarrie, 1993). Na+ ‘exclusion’, particularly from young leaves, contributed to salt tolerance in a wheat–Lophopyrum amphiploid (Colmer et al., 1995). Recently, a Hordeum marinum–wheat amphiploid has been produced by AKMR Islam (Colmer et al., 2005b). Hordeum marinum (sea barleygrass) inhabits salt marshes (von Bothmer et al., 1991) and is tolerant to salinity (Garthwaite et al., 2005), being regarded as even more salt tolerant than L. elongatum (Colmer et al., 2005a). The present report describes, for the first time, salt tolerance in this recently produced H. marinum–T. aestivum amphiploid, by contrast with its parents. Growth and grain production, as well as ion concentrations in different-aged leaves and the accumulation of compatible solutes, were evaluated in greenhouse experiments.
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Materials and methods |
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Production of the amphiploid
Hordeum marinum (accession H21) was used as the female parent in crosses to produce F1 hybrids with Triticum aestivum (cv. Chinese Spring). The small anther size made H. marinum unsuitable to use as the pollen parent. The F1 hybrids produced had 28 somatic chromosomes, confirmed from chromosome counts in root tip cells. These hybrid plants were then treated with colchicine to double their chromosome number. Seeds produced in the doubled sectors were germinated, 56-chromosome presumptive amphiploid plants were selected from root tip chromosome counts, and these were planted and their amphiploid status confirmed from observing pairs of chromosomes (28'') at meiosis. Seed produced by these confirmed amphiploids was used in all experiments.
Experiment 1: Responses of growth and leaf blade ion concentrations to increasing NaCl
Plant culture
The experiment was conducted to evaluate the NaCl dose–responses of growth and leaf blade ion concentrations, for the amphiploid and its parents. The experiment used a completely randomized design, with 4 NaCl treatmentsx3 genotypesx4 replicates, and was conducted in a 20/15 °C day/night phytotron.
Hordeum marinum–Triticum aestivum amphiploid (Genome AABBDDXX), Hordeum marinum (Accession H21) (XX), and Triticum aestivum (cv. Chinese Spring) (AABBDD) were grown. Seeds were surface-sterilized with 0.04% bleach and rinsed thoroughly with deionized water. Seeds were then imbibed in aerated 0.5 mM CaSO4 for 3 h before being placed on plastic mesh floating on aerated 0.1 concentration nutrient solution in the dark for germination (full-strength solution composition is given below). To ensure plants were at similar developmental stages at the time treatments were imposed, seeds of H. marinum were germinated 3 d before the other two genotypes. Four days (H. marinum) or 3 d (other two genotypes) after germination, the seedlings were exposed to light and the aerated nutrient solution was refreshed with 0.25 concentration solution. Two days later, seedlings were transplanted into 4.2 l pots of aerated, full-concentration nutrient solution (four seedlings of the same genotype per pot). Seedlings were held in the lids of pots using polystyrene foam. The outside of each pot was covered with aluminium foil to prevent light penetration into the solution.
The nutrient solution at full-strength contained the macronutrients (mM): K+ 5.80, Ca2+ 5.0, Mg2+ 0.4, 
0.625, 
4.375, 
5.4, 
0.2, and the micronutrients (µM): Cl 50, B 25, Mn 2, Zn 2, Ni 1, Cu 0.5, Mo 0.5, and Fe-EDTA 50. The solution also contained 0.1 mM Na2SiO3 (as recommended by Epstein, 1994) and 1.0 mM 2-(N-morpholino) ethanesulphonic acid (MES) and the pH was adjusted to 6.5 with KOH (to give the final K+ concentration as listed above). Fe2SO4 was added at 5 µM a few days before treatments were imposed to prevent symptoms of slight Fe-deficiency that can occur if Fe2SO4 was not routinely added.
NaCl treatments
Treatments of control (0.2 mM NaCl), 100, 200, and 300 mM NaCl were imposed in 50 mM steps per d. Pots (three genotypesxfour treatmentsxfour replicates=48 pots) were arranged randomly on benches. Each pot contained four plants; one plant from each pot was sampled at the time treatments were imposed (for an initial harvest) and one after the final NaCl treatments were reached (i.e. on the sixth day when the highest concentration of 300 mM was reached) leaving two plants in each pot for the final harvest at the end of treatments. The plants had 2–2.5 leaves (14-d-old for H. marinum, 11 d for wheat and the amphiploid) at the time treatments were imposed. The treatment period was 28 d (after the final concentration was reached for the 300 mM NaCl treatment). All nutrient solutions were renewed every 7 d throughout the experiment.
Harvest
All plants in replicate 1 were harvested first, then those in replicates 2, 3, and 4. The roots and stem base of the plants were rinsed three times, for 
10 s each, with deionized water to remove surface ions. The leaf blades on the main stem were excised and separated according to age, the oldest leaf blade being number 1, the second oldest leaf blade being number 2, and so on. The remaining green leaf blades (on tillers) were then excised and bulked together. Sheaths, which were the base of main stem and tillers with sheaths and young leaves not yet emerged, were another sample. Dead leaf tissues were collected as a separate category. The roots were also collected. Dry masses of all samples were recorded after being oven-dried at 65 °C.
Growth analyses
Growth of roots and shoots was determined by measuring the dry mass of the two components at the start (i.e. after final NaCl treatment concentrations had been reached) and end of the 28 d treatment and calculating the relative growth rates (RGR), using the formula
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The rate of increase in length of the youngest expanding leaf on the main stem of the three genotypes was measured for 10 d, starting 13 d after the final NaCl treatment concentrations were reached.
Analyses of Na+, K+ and Cl–
Oven-dried samples were ground (ball mill grinder, manufactured by the combined workshop UWA) and then subsamples (50 mg or 20 mg, if sample was limited) were extracted in 5 ml of 0.5 M HNO3 by shaking for 2 d in a dark room at 25 °C. Concentrations of Na+ and K+ were determined in dilutions of extracts using a flame photometer (Corning, Model 410, Halstead, Essex, UK) and Cl– by amperometric titration with silver ions using a Buchler–Cotlove Chloridometer (Buchler Instruments, Model 4–2000, New Jersey, USA). Blanks and reference tissue standards were taken through the same procedures.
Experiment 2: Effects of 200 mM NaCl on leaf blade ion and organic solute concentrations
Experimental design and NaCl treatment
A total of 24 pots (three genotypesxtwo NaCl treatmentsxfour replicates) were used. Plants were grown using the procedures as described above for Experiment 1. The 200 mM NaCl treatment was imposed in 50 mM steps per day, and plants were exposed to the treatment for 28 d (after the final concentration of 200 mM NaCl was reached).
Harvest
Harvests were taken as described above for Experiment 1; however, tissues were processed in various ways depending on the measurements. The youngest fully-expanded leaf blade on the main stem was excised and wrapped in Al-foil, and frozen in liquid N2, and then stored at –70 °C. These samples were later freeze-dried (FD 4.0, Heto-Holten A/S, Denmark) before processing for the measurement of organic solutes. Youngest fully-expanded leaves from tillers 1 and 2 were sealed immediately after excision in an air-tight cryovial and frozen in liquid N2 and placed in dry ice for later measurement of leaf sap osmotic potential. The remaining green leaves, dead leaves, and sheaths, were then excised and processed as separate categories. Fresh masses these of tissues were recorded, and so were dry masses after being freeze-dried, or oven-dried at 65 °C.
Leaf blade water content
Water content of the youngest fully-expanded leaf blade was determined as
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Leaf blade sap osmotic potential
Osmotic potential (
) of the sap expressed from frozen and thawed tissues was measured using a calibrated freezing-point depression osmometer (ONE-TEN, Fiske Associates, Massachusetts, USA). Leaf tissues were thawed in the cryovial, crushed in a stainless steel press, and the sap was then immediately analysed.
Organic solutes in leaf blades
The concentrations of glycinebetaine, proline, and asparagine in the youngest fully-expanded leaf blade from the main stem were determined using high performance liquid chromatography (HPLC) (Naidu, 1998). The freeze-dried tissues were ground (ball and mill grinder) and extracted for metabolites using ice-cold 5% (w/v) perchloric acid and then neutralized to pH 3.5 by slowly adding potassium carbonate while in a vial on ice (Fan et al., 1993). The extract was then filtered through a disposable 0.45 µm PTFE filter and stored on ice, or stored frozen at –70 °C, before being injected into the HPLC system. The HPLC system (Waters Corporation, Milford, MA, USA) consisted of a 600E pump, 717 auto-sampler, 996 photodiode array detector, and Millennium Chromatography Manager software. A Sugar-Pak column (300 mm length, 6.5 mm diameter) (Waters Corporation) was housed in a column heater at 90 °C. Recoveries of glycinebetaine, proline, and asparagine from spiked samples of leaf tissues were 96, 94, and 82%, respectively.
Experiment 3: Effect of 150 mM NaCl on growth to maturity and grain yield
To evaluate salt tolerance in terms of biomass and grain yield, the H. marinum–T. aestivum amphiploid and its parents were grown in 20.0 l tubs (pots) in temperature-controlled tanks at 20 °C in a greenhouse during the winter–spring growing season in south-western Australia (seeds imbibed 10 May 2004). Seed germination and plant establishment were carried out following the procedure described above.
Experimental design and NaCl treatments
A total of 18 pots (three genotypesxtwo NaCl treatmentsxthree replicates) were used. Three temperature-controlled tanks were used so that the experiment was set up as a completely randomized block design (6 pots per tank). Pots were arranged randomly within each tank. Each pot contained 10 plants; two plants were sampled at the time treatments were imposed (for an initial harvest) and two after the final NaCl concentration was reached (second ‘initial’ harvest) leaving 6 plants in each pot. Treatments were: control (0.2 mM NaCl) and 150 mM NaCl (imposed in 50 mM steps every d). In this experiment, 150 mM NaCl was used since Forster et al. (1987, 1988) showed the ability to set seed was arrested for Chinese Spring at 200 mM NaCl, whereas at 150 mM NaCl, seven out of eight plants were able to set at least some seed (average of 29.5 grains per plant). Two plants from each pot were harvested after 28 d and 56 d of treatments (final concentrations) leaving two plants in each pot for the final harvest at maturity. At each harvest (28 d and 56 d) the shoot was divided into expanding leaf blades, youngest fully-expanded leaf blade from the main stem, other green leaves (bulked together), and the remaining dead leaves (bulked), sheaths or stem bases, and roots. Fresh and dry masses were measured. At maturity, the spike number, grain number per spike, grain mass, and root and shoot dry masses, were measured. Spikes were threshed by hand and grain numbers were counted for each replicate by taking a random subsample of 200 grains, measuring the mass, and scaling up based on total grain mass.
Statistical analyses
Genotype and treatment comparisons of the parameters measured were examined by analyses of variance using the statistical software Genstat, 6th edition. Genotype and tissue means were compared using Fischer's least significance difference (LSD), 
=0.05.
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Results |
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Experiment 1: Responses of growth and leaf blade ion concentrations to increasing NaCl
Plant growth over a range of NaCl concentrations
The three genotypes did not differ in shoot and root RGRs when grown in non-saline solution for 28 d (shoot, P=0.84 and root, P=0.35) (Fig. 1). Increased salinity caused reductions in shoot and root RGRs of all three genotypes (P <0.001), but the genotypes differed in their responses (genotypextreatment interaction, P <0.001) (Fig. 1). At 100 mM NaCl, the shoot RGR was 90% of the control in T. aestivum, while it was unaffected in H. marinum and was 95% in the amphiploid. At 200 mM NaCl, the shoot RGR was only 68% of the control in T. aestivum, 79% in amphiploid, whereas growth was still unaffected in H. marinum. At 300 mM NaCl, the shoot RGR was only 30% of the control in T. aestivum, 70% of the control in H. marinum and 57% in the amphiploid. The percentage reductions in root RGR (Fig. 1B) under salinity reflected the trends in shoot RGR (Fig. 1A).
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There was no dead leaf material in any of the three genotypes when grown in non-saline solutions (Fig. 2). At 100 mM NaCl, the amount of dead leaf material of all the genotypes was 1–1.5% (as a percentage of shoot dry mass). At 200 mM NaCl, the percentage of dead leaf material in T. aestivum was higher (7%) than in both H. marinum (0.5%) and the amphiploid (3.7%). At 300 mM NaCl, the percentage of dead leaf material in T. aestivum was
15%, being almost 7-fold higher than in H. marinum, and 3-fold higher compared with the amphiploid.
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Main stem leaf number was similar in all three genotypes when grown in non-saline solution (Fig. 3A). Plants exposed to the NaCl treatments had reductions in the development of the main stem, as compared with controls (P <0.001) (Fig. 3A). The reduction in main stem leaf number became larger as NaCl was increased (Fig. 3A) and there were differences amongst the genotypes (P <0.01). At 100 mM NaCl, the number of main stem leaves in T. aestivum was 93% of the control, while it was unaffected in both H. marinum and the amphiploid. At 200 mM NaCl, it was 87% of the control in T. aestivum and 92% of the control in the amphiploid, but it was still unaffected in H. marinum. At 300 mM NaCl, main stem leaf number was only about half of the control in T. aestivum, 88% of the control in H. marinum, and 73% of the control in the amphiploid.
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Tiller number was similar in all three genotypes when grown in non-saline solution (Fig. 3B). The number of tillers (Fig. 3B) of all genotypes decreased with increasing salinity (P <0.001), but it was least affected in H. marinum and did not differ significantly between T. aestivum and the amphiploid. At 100 mM NaCl, the tiller numbers of T. aestivum were reduced to 84% of the control, were unaffected in H. marinum, and were 88% of the control in the amphiploid. At 200 mM NaCl, the tiller numbers were reduced to almost half in T. aestivum and 60% in the amphiploid, whereas it was 93% of the control value in H. marinum. At 300 mM NaCl, tiller numbers were only 14% of the control in T. aestivum, 27% in the amphiploid and 50% in H. marinum.
The rate of leaf elongation (Fig. 4) during the linear phase for control T. aestivum was 5.1 cm d–1, and this was reduced to 78% of the control at 100 mM NaCl, 56% at 200 mM NaCl, and 19% at 300 mM NaCl. The leaf elongation rate of H. marinum was not different between the control and 100 mM NaCl treatment, at 3.5 cm d–1, whereas at 200 mM and 300 mM NaCl the rates were reduced to 77% and 45% of the control, respectively. The amphiploid showed a similar rate of leaf elongation to H. marinum at both control and 100 mM NaCl (3.5 cm d–1), and at 200 mM and 300 mM NaCl, it was reduced to 65% and 37% of the control, respectively. Increasing salinity resulted in shorter leaves for all three genotypes (Fig. 4).
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Na+ concentrations in leaf blades
Leaf blade Na+ concentrations of plants grown in non-saline solution were not significantly different for the three genotypes (Fig. 5A). Na+ concentrations increased with increasing salinity (P <0.001).
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T. aestivum had higher Na+ concentrations in the youngest fully-expanded leaf blade at all NaCl treatments, compared with the other two genotypes (Fig. 5A). At 100 mM and 200 mM NaCl, leaf blade Na+ concentrations in T. aestivum were 2-fold higher compared with the amphiploid, and 4.7-fold and 3.8-fold higher, respectively, at these two NaCl concentrations, compared with H. marinum (Fig. 5A). For plants in 300 mM NaCl, the Na+ concentrations in the youngest fully-expanded leaf blade of T. aestivum (leaf 5), H. marinum (leaf 6) and the amphiploid (leaf 6) were 2203, 153, and 866 µmol g–1 dry mass, respectively.
Cl– concentrations in leaf blades
Cl– concentrations in the youngest fully-expanded leaf blade of all three genotypes were lowest in plants grown in non-saline solution (Fig. 5B). Leaf blade Cl– concentrations were, however, 10–30-fold higher than those of Na+ for the plants grown in non-saline solution. Cl– concentrations were lower in T. aestivum than in the two other genotypes when grown in non-saline solution. When exposed to salinity, leaf blade Cl– concentrations increased in all three genotypes (P <0.001). The youngest fully-expanded leaf of T. aestivum had 1.8-fold higher Cl– compared with H. marinum and 1.7-fold higher compared with the amphiploid, in both 100 mM and 200 mM NaCl treatments (Fig. 5B). For plants in 300 mM NaCl, the Cl– concentrations in the youngest fully-expanded leaf blade of T. aestivum (leaf 5), H. marinum (leaf 6), and the amphiploid (leaf 6), were 2065, 75, and 738 µmol g–1 dry mass, respectively.
K+ concentrations in leaf blades
For all three genotypes, leaf blade K+ concentration was highest when grown in non-saline solution (Fig. 5C) and it decreased with increasing salinity (P <0.001). K+ concentrations were higher in the youngest fully-expanded leaf blade of H. marinum compared with those in the other two genotypes, except at 300 mM NaCl (Fig. 5C).
Leaf blade K+:Na+
K+:Na+ in the youngest fully-expanded leaf blade became lower with increasing NaCl in the growth medium (data calculated from Fig. 5). At 100 mM NaCl, the K+:Na+ in T. aestivum was 2.5 and it decreased to 0.2 at 300 mM NaCl. H. marinum was able to maintain higher K+:Na+ (e.g. 3.2 at 300 mM NaCl). The amphiploid showed an intermediate response to its parents at 300 mM NaCl, with a K+:Na+ of 0.5.
Experiment 2: Effects of 200 mM NaCl on leaf blade ion and organic solute concentrations
Growth of plants at low and high NaCl concentrations
Growth of the three genotypes in non-saline and saline (200 mM NaCl) conditions was similar to the results described above for Experiment 1. To avoid repetition, the growth data of Experiment 2 are not presented.
Osmotic potential of sap expressed from the youngest fully-expanded leaf blade
In non-saline solution there was no difference among the three genotypes in sap osmotic potential of the youngest fully-expanded leaf blade (Table 1). Salinity decreased sap osmotic potential in all three genotypes (P <0.001), but there were no significant differences among the genotypes (Table 1). The decline in sap osmotic potential was –0.61 to –0.80 MPa, compared with an external medium change of –0.98 MPa. Exposure to salinity also resulted in a decline (P <0.001) in tissue water content by 30–36% in all three genotypes (Table 1). The reduced tissue water content contributed more to decline in sap osmotic potential in response to NaCl, than did accumulation of additional solutes.
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Glycinebetaine, proline and asparagine concentrations in the youngest fully-expanded leaf blade
In non-saline solution, glycinebetaine concentration in the youngest fully-expanded leaf blade of H. marinum was 2.6-fold higher than in T. aestivum and 2.2-fold higher than in the amphiploid (Table 2). Exposure to 200 mM NaCl increased the glycinebetaine concentration in all three genotypes, compared with the control plants (P <0.001), but the concentration differed amongst the genotypes (P <0.001) (Table 2). In T. aestivum, the concentration of glycinebetaine was lower than in the two other genotypes.
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In non-saline solution, proline and asparagine concentrations in the youngest fully-expanded leaf blade of H. marinum were, respectively, 3.6-fold and 1.5-fold higher than in T. aestivum and 2.4-fold and 2.0-fold higher than in the amphiploid (Table 2). Salinity increased the proline and asparagine concentrations in all three genotypes (P <0.001), but for both solutes the levels remained lower than those of glycinebetaine (Table 2). Proline concentrations differed significantly among the three genotypes (P <0.001); the concentrations were lower in T. aestivum compared with those in the two other genotypes.
Experiment 3: Effect of 150 mM NaCl on growth to maturity and grain yield
Growth and grain production at low and high NaCl concentrations
In this experiment, samples were taken at the commencement of treatments and after 28 d, 56 d, and at maturity. At all three times after the 150 mM NaCl treatment was imposed, H. marinum was least affected, and T. aestivum was the most affected, and the amphiploid showed an intermediate response. Only the data for growth and yield at maturity have been presented here (Table 3). Na+, K+, and Cl– data have been presented only from the 56 d harvest, as the 28 d data showed similar patterns.
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In non-saline solution, at maturity, H. marinum had a shoot dry mass almost 1.5-fold higher than in T. aestivum and 1.3-fold higher than in the amphiploid (Table 3). H. marinum produced 1114 spikes per plant, T. aestivum produced 108, and the amphiploid 159. The number of grains was highest in H. marinum, and the amphiploid produced the least due to its smaller production of seeds per spike (almost 60% lower than in T. aestivum) (Table 3). The 100-grain mass of T. aestivum was 12 times higher than for H. marinum, and 1.3 times higher than for the amphiploid. Thus, in non-saline conditions all plants showed vigorous growth; for example, the 108 tillers with spikes produced by Chinese Spring was higher than usually reported for wheat. It is hypothesized that the abundant nutrients and unlimited water supplied (full nutrient solution refreshed weekly), combined with ideal growth temperatures, would have promoted the luxurious growth in the present experiment [for comparison, Chinese Spring grown in a nutrient solution produced
25 tillers in Experiment 1 by Gorham et al. (1986), even though the authors state that at times the nutrient solution became depleted of ]. At 150 mM NaCl, shoot and root dry masses were reduced most in T. aestivum, the values were respectively only 34% and 36% of those in the control; whereas in H. marinum the shoot and root dry masses were, respectively, 68% and 66% of the control, and in the amphiploid 52% and 53% of the control (Table 3). At 150 mM NaCl, the number of spikes per plant decreased significantly in T. aestivum (74% of control), but not in H. marinum or the amphiploid (95% of controls) (Table 3). Grain numbers per plant decreased significantly in all three genotypes when grown in 150 mM NaCl (P <0.01), but was reduced least in H. marinum (92% of the control), then the amphiploid (71% of the control), and then T. aestivum (54% of the control). Salinity decreased both 100-grain mass and total grain mass in all three genotypes (P <0.01), but the genotypes differed in their response (P <0.001). The 100-grain mass was reduced to 78% of the control in T. aestivum, 91% in H. marinum, and 95% in the amphiploid. The mass of total grains was reduced to 54% of the control in T. aestivum, to 84% in H. marinum, and 67% in the amphiploid.
Tissue Na+ concentrations
For plants in non-saline solution, tissue Na+ concentrations were very low, and there were no differences among the three genotypes (Fig. 6A). When exposed to 150 mM NaCl, tissue Na+ concentrations increased in all three genotypes (P <0.001) (Fig. 6B). There were, however, differences amongst the genotypes in response to the salt treatment (P <0.001). After 56 d of treatments, Na+ concentrations in all the tissues of T. aestivum were higher than those in the corresponding tissues of the other two genotypes; the values in T. aestivum were almost 1.8–2.6-fold higher than in the corresponding tissues of H. marinum and 1.1–2.2-fold higher than in those of the amphiploid.
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Tissue Cl concentrations
In the control plants of all three genotypes, tissue Cl– concentrations were 10–40-fold higher than tissue Na+ concentrations in these same plants (Fig. 6C). Cl– concentrations in control T. aestivum were 19% lower than those in the amphiploid and in H. marinum. When grown at 150 mM NaCl, Cl– concentrations increased in all the leaf tissues of the three genotypes (P <0.001), but the genotypes differed in their response (genotypextreatment interaction, P <0.01) (Fig. 6D). Cl– concentrations were highest in T. aestivum and least in H. marinum. As examples, Cl– concentrations in other bulked green leaves and the youngest leaf tissues were 1.6–2.1-fold higher in T. aestivum than those in H. marinum, and 1.4-fold higher in T. aestivum than those in the amphiploid.
Tissue K+ concentrations
K+ concentrations were high in the control plants of all three genotypes (Fig. 6E), but T. aestivum had lower K+ concentrations in all the leaf tissues (700–950 µmol g–1 dry mass) compared with those in the other two genotypes. K+ decreased significantly in all the tissues of plants exposed to 150 mM NaCl (Fig. 6F) (P <0.001) and the genotypes differed in response (P <0.001). At 150 mM NaCl, leaf K+ concentrations decreased to be 56–65% of the values in controls for T. aestivum, to 80–90% in H. marinum, and to 75–90% in the amphiploid.
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Discussion |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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Salt tolerance and physiological traits were evaluated in a H. marinum–T. aestivum amphiploid, and its parents. In saline conditions, growth and grain production of the amphiploid were maintained better than in T. aestivum (Table 3). The amphiploid demonstrated superior ‘exclusion’ of Na+ and Cl– from young leaf blades, compared with T. aestivum, but not to the same capacity as in H. marinum (Fig. 5). These results show that salt tolerance from H. marinum was expressed in the H. marinum–T. aestivum amphiploid. Similarly, amphiploids of T. aestivum with other salt tolerant members of the Triticeae, specifically tall wheatgrass species (i.e. T. aestivum–Th. bessarabicum and T. aestivum–L. elongatum amphiploids), also possess salt tolerance superior to that of the bread wheat parent (Gorham et al., 1986; Omielan et al., 1991).
Hordeum marinum (sea barleygrass) inhabits salt marshes (von Bothmer et al., 1991) and is tolerant to salinity (Garthwaite et al., 2005). Salt tolerance in H. marinum, as compared with wheat, was evident in the present study by better maintenance of growth and grain production when exposed to NaCl treatments. For example, when grown to maturity at 150 mM NaCl, the number of grains per plant was reduced much less (92% of the control) in H. marinum than in T. aestivum (50% of the control). Significantly, salt tolerance in the amphiploid was intermediate (grains per plant 70% of the control) to its parents (Table 3). Thus, both H. marinum and the amphiploid were better able to tolerate NaCl, compared with T. aestivum, during prolonged exposure of both the vegetative and reproductive stages. Nevertheless, the present H. marinum–T. aestivum amphiploid did not out-perform T. aestivum (same cultivar, Chinese Spring) to the same degree as a T. aestivum–Th. bessarabicum amphiploid (at 150 mM NaCl the number of grains per plant was 92% of control, whereas T. aestivum produced 36.5% of control; Gorham et al., 1986). The various amphiploids, now available, should be evaluated side-by-side to provide a more definitive ranking of their salt tolerances.
The H. marinum–T. aestivum amphiploid has lower fertility than T. aestivum, and therefore had a lower grain production (total grain mass per plant; due also in part to a moderately lower 100-grain mass in the amphiploid) than T. aestivum, in both control and the 150 mM NaCl treatment (Table 3). The reduced fertility in the amphiploid is most likely due to cytoplasmic male sterility induced by the Hordeum cytoplasm, as due to the very small anther size of H. marinum, it had to be used as the female parent and T. aestivum as the male parent in the crossing. By comparison, fertility in the wheat–tall wheatgrass amphiploids, in both of which the tall wheatgrass was used as the male parent, can also be very poor (18% in a T. aestivum–Th. bessarabicum amphiploid; Gorham et al., 1986) or at best reasonable (83% in a T. aestivum–L. elongatum amphiploid; Dvo
ák and Sosulski, 1974). In the case of the H. marinum–T. aestivum amphiploid, it is expected that fertility can be improved by transferring back to a wheat cytoplasm. High fertility will be essential if this, or other, amphiploids are to be used as a new salt-tolerant cereal (cf. King et al., 1997b; Colmer et al., 2006).
The H. marinum–T. aestivum amphiploid maintained lower Na+ and Cl– concentrations in its youngest fully-expanded leaf blade, compared with T. aestivum (Fig. 5A, B). At 300 mM NaCl, Na+ and Cl– concentrations in the youngest fully-expanded leaf blade of the amphiploid were 36–39% of those in T. aestivum. However, compared with H. marinum at 300 mM NaCl, the concentrations of Na+ and Cl– in the amphiploid were, respectively, about 5.6-fold and 9.8-fold higher. Nevertheless, the improved Na+ ‘exclusion’ from leaf blades of the H. marinum–T. aestivum amphiploid is reminiscent of the lower leaf Na+ concentrations reported for a T. aestivum–L. elongatum amphiploid (64% and 85% lower at 200 mM NaCl; Schachtman et al., 1989; Colmer et al., 1995, respectively) and in a T. aestivum–Th. bessarabicum amphiploid (Na+ concentration was about half of that in T. aestivum at 150 mM NaCl; Gorham et al., 1986). Together, these results support that salt-tolerant species in the Triticeae restrict the rate of entry of Na+, and in some cases Cl–, into their shoots (Colmer et al., 2006).
Organic solutes contribute to osmotic adjustment in the cytoplasm of cells of plants exposed to salinity (Greenway and Munns, 1980; Hanson and Wyse, 1982; Volkmaar et al., 1998). When grown at 200 mM NaCl, glycinebetaine accumulated to higher concentrations in the youngest fully-expanded leaf blade of H. marinum (40% higher) and the amphiploid (22% higher), compared with T. aestivum (Table 2). Increased glycinebetaine concentrations also occurred in young leaves of a T. aestivum–L. elongatum amphiploid (Colmer et al., 1995), but not in a T. aestivum–Th. bessarabicum amphiploid (Gorham et al., 1986). In the present study, when plants were exposed to 200 mM NaCl, proline and asparagine concentrations also increased in the three genotypes, but these two solutes did not reach the concentrations of glycinebetaine. On a tissue water basis, glycinebetaine concentrations were estimated at 43 mM in H. marinum, 25 mM in the amphiploid, and 21 mM in T. aestivum. If located in the cytoplasm (e.g. 10% of the tissue volume), then such concentrations (i.e. up to 430 mM in H. marinum) would be osmotically significant for that compartment. By contrast, there was no significant difference between the three genotypes in leaf sap osmotic potential (Table 1), supporting an earlier conclusion that salt-tolerant and salt-sensitive wheat genotypes might not differ in this regard (Munns et al., 1995). Previous studies by Kingsbury et al. (1984), Termaat et al. (1985), and Colmer et al. (1995) also found no significant differences in osmotic adjustment between salt-tolerant and salt-sensitive genotypes of wheat, whereas Saneoka et al. (1999) reported genotypic differences. Although osmotic potential might not differ, the identities of the solutes accumulated can differ substantially, with possible implications for cellular functioning; salt-sensitive wheat contained high concentrations of Na+, whereas a salt-tolerant T. aestivum–L. elongatum amphiploid contained low Na+ but relatively high glycinebetaine and K+ (Colmer et al., 1995).
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TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionsReferences |
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The present study on a H. marinum–T. aestivum amphiploid adds to earlier work on T. aestivum–tall wheatgrass amphiploids (Gorham et al., 1986; Schachtman et al., 1989; Omielan et al., 1991; Colmer et al., 1995; King et al., 1997a) showing wide-hybridization of halophytic wild relatives with wheat can enhance salt tolerance in wheat. The H. marinum–T. aestivum amphiploid maintained higher relative growth and yield under saline conditions, compared with T. aestivum. The higher salt tolerance in H. marinum (present study; Colmer et al., 2005b; Garthwaite et al., 2005), in addition to its waterlogging tolerance (McDonald et al., 2001; Garthwaite et al., 2003), make this a species of interest as a source of genes for improving salt and waterlogging tolerance in wheat.
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Acknowledgements |
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SI thanks AusAID for a MSc student scholarship. This research was supported by the Grains Research and Development Corporation and the CRC for Plant-based Management of Dryland Salinity. We thank Roland von Bothmer (Swedish University of Agricultural Sciences) for providing the H. marinum accession.
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