THE RESPONSE TO NaCl AND IONIC CONTENT OF SELECTED SALT-TOLERANT AND NORMAL LINES OF THREE LEGUME FORAGE SPECIES IN SAND CULTURE

New Phytol. (1986) 104, 463-471 463 THE RESPONSE TO NaCl AND IONIC CONTENT OF SELECTED SALT-TOLERANT AND NORMAL LINES OF THREE LEGUME FORAGE SPECIES IN SAND CULTURE BY M. A S H R A F, T. M C N E I L L Y* AND A. D. BRADSHAW Department of Botany, University of Liverpool, P.O. Box 147, Liverpool L69 3BX, UK {Accepted 15 July 1986) SUMMARY Selected NaCl-tolerant lines of Trifolium alexandrinum L., Medicago sativa L., and Trifolium pratense L. produced significantly greater dry matter than unselected control lines after 4 weeks of growth in sand culture with different NaCl concentrations. Tolerant lines of all three species generally contained less Na^ but more Cl^ in their shoots than normal lines, although these differences were not statistically significant. Selected lines also contained less K""" in their shoots than unselected lines. Ca"^ distribution was similar in T. "lexandrinum and M. sativa but not in T. pratense, in which the tolerant line had significantly higher Ca^+ in the shoot than the unselected line. Key words: Ion content, salinity, selection, NaCl tolerance, legume species. INTRODUCTION The need to produce crops with enhanced tolerance to high salt levels in the soil Has been emphasized by many workers (Dewey, 1962; Shannon, 1979; Epsteiti, 1985). The success of this approach depends upon the occurrence of appropriate genetic variability in crop species and the ability to exploit such variability using convenient selection techniques. It is important that selection at one stage of the life-cycle for increased tolerance should select those individuals also tolerant in the remaining stages. It has, however, been argued (Shannon, 1979) that the response of plants to salinity varies at different stages of the life cycle. In some species, tolerances exhibited at all growth stages are highly correlated, as for example in Medicago sativa L. (Noble, 1983). In others this may not be the case. The work described here assesses both the response to NaCl and the pattern of inorganic ion distribution in normal and salt-tolerant lines of Trifolium alexandrinum L., M. sativa L., and Trifolium pratense L. produced by selection at the seedling stage after 2 weeks of growth in saline nutrient solution. These three species are commonly used as forage crops in several countries where soil salinity is a major problem for agriculture. T. alexandrinum has been categorized as moderately tolerant, while M. sativa and T. pratetise are intermediate in salt tolerance compared with other legumes (Lauchli, 1984). Patterns of distribution of Na+, K+, Ca-+, Mg^^ and Cr within the plant after 19 days of growth in NaCl solution culture have previously been described by Winter & Lauchli (1982) for normal material of T. alexandrinum and T. * To whcun reprint requests should be addressed. 0028-646X/86/tlO463 + 09 $03.00/0 1986 The New Phytologist

464 M. ASHRAF, T. MCNEILLY AND A. D. BRADSHAW pratense. This paper compares the distribution of these ions in selected NaCltolerant lines and unselected control lines of these two species, and of M. sativa in response to NaCI after 28 days of growth in sand culture. MATERIALS AND METHODS Plant material Salt-tolerant lines were selected from 10000 seeds of commercial varieties of T. alexandrinum L. cv. Multifoliate, M. sativa L., cv. Euver and T. pratense L. cv. Altaswede on the basis of differences in shoot length after 2 weeks of growth in saline solution cultures (Ashraf, McNeilly & Bradshaw, unpublished data). Twenty plants were raised at the same time from unselected seeds of the same three cultivars. The plants of both the selected and unselected lines were grown to maturity in a glasshouse at 18 C and a 16 h photoperiod, and they were manually crossed at random in separate polycrosses. The resulting progeny of each line were harvested separately. Sand culture Silica sand was soaked in 2% (v/v) HCl for 5 days and then thoroughly washed with tapwater and rinsed three times with distilled deionized water. Eighteen centimetre diameter plastic pots were filled with 3-54 kg dry sand. The main object of these experiments was the comparison of lines within species and not species comparisons, so each species was considered independently. The concentrations of NaCI used were 0, 100 and 175 mol m"^. The experiment had three blocks for each species, each block having two lines and three concentrations of NaCI. One hundred seeds of each line progeny of each species were germinated on moist filter paper in Petri dishes, and five randomly selected seedlings of each line were transplanted equidistant from each other into each pot. The seedlings of both lines were of comparable size when transplanted into the pots. The experiment was conducted in a heated glasshouse at 18 C and a 16 h day length. The seedlings were grown for 2 weeks, irrigated with half-strength nutrient (Rorison) solution (Hewitt, 1966), after which time NaCI treatments were begun by adding aliquots of a 50 mol m"-' solution of NaCI in 1 1 of the half-strength nutrient solution as above, on alternate days, until the appropriate NaCI concentrations were reached. Treatments continued with addition of 1 1 of the appropriate solution on alternate days to each pot. This volume was sufficient to wash through solution already present in the sand as determined from the electrical conductivity of the effluent solution from the pots. The plants were harvested 28 days after the start of the salt treatment. Plant roots were removed carefully from the sand, shoots and roots were separated and washed with distilled deionized water. Plant material was dried at 65 C for 3 days, divided into shoots and roots and weighed. Chemical analysis Cations. Ten to twenty milligram samples of leaf, stem and root dry matter were digested in 1 ml concentrated HNO3. After digestion was complete, the sample volume was made up to 5 ml with distilled deionized water, and all the cations were determined by atomic absorption spectrometry using a Varian 1275.

Ion uptake and salt tolerance 465 s Z c o in * Z i I S O c.2 " O 2 6 ta t; _ Z S 7 1 ro ca b do i'^ 00 ^ CO S 3 c U c.-i to <u o o V ft, ^ t3 QJ U O C O - O > -S o o o a, Iv oi. Po» 6 ^.^ Z*

466 M. ASHRAF, T. MCNEILLY AND A. D. BRADSHAW Chloride. Ten milligram subsamples were extracted by boiling in distilled deionized water for 3 h, and Cl^ content of the extracts were determined with a CMT 10 chloride titrator (Radiometer). Analyses of variance were carried out separately for the ion content of each plant part within each species. 0 100 175 O 100 175 0 100 175 NaCl concentrations in sand culture (mol m"^) Fig. 1. Mean dry weights of plants from selected and unselected lines of three legume species [Trifolium alexandrinum (a), Medicago sativa (b), Trifolium pratense (e)] after 4 weeks of growth in sand culture at different NaCl concentrations. %, selected line; O, unselected line. RESULTS Mean dry weight per plant The results for mean dry weight per plant (Fig. 1) and their analyses of variance (Table 1) show that dry matter yield of both lines of all three species declined linearly with increasing NaCl concentration {P < 0-001). However, the selected line of each species produced significantly greater dry matter than the unselected line (T. alexandrinum, P<0-05; M. sativa, P < 0-001; T. pratense, P < 0-01). The selected lines of T. alexandrinum, M. sativa and T. pratense yielded about 25 %, 30 % and 45 % respectively more dry matter than the unselected lines at 175 mol m^'. The lines x concentration interaction was only significant in M. sativa (P < 0-05). This may have been due to the large differences in dry weight between the lines at 0 and 100 mol m ' NaCl. Shoot/root ratio Dry weight was also expressed as shoot/root ratios (Table 2). There were no significant differences in shoot/root ratio in T. alexandrinum and T. pratense, but a significant difference between the lines (P < 0-01) was found in M. sativa. The unselected line of M. sativa had a higher shoot/root ratio than the selected line, suggesting a more adverse effect in root dry weight in the former. The shoot/root ratio of the T. pratense unselected line was also greater than that of the selected line at 175 mol m^'', although the difference was not statistically significatit. Ion content Sodium. The Na+ content (Fig. 2) of the roots of the selected tolerant line of T. alexandrinum was significantly greater than in the unselected line (P < 0-01), and the leaves of the selected line contained less Na"*" than those of the unselected

Ion uptake and salt tolerance 467 line, but this difference was not statistically significant. The Na+ content in the stems of the two T. alexandrinum lines did not differ. By contrast, the different plant parts of selected and unselected lines of M. sativa had similar Na"*" contents. In T. pratense, the Na+ content was lower in both the leaves and the stems of the selected line than in the unselected line, although this difference was again not statistically significant. The Na"*" contents in the three species were in the order T. pratense > M. sativa > T. alexandrinum.. Chloride. The Cl" contents (Fig. 3) of the leaves and stems were generally higher in the selected than in the unselected lines of all three species, although the 3200 - o 2400 - S. 1600-800 - 100 175 0 100 175 100 175 NaCI concentrations in sand culture [mol m~^) Fig. 2. Na^ contents in different parts of plants from selected and unselected lines of three legume species [Trifolium alexandrinum (a), Medieago sativa (b), Trifotium pratense (c)] after 4 weeks of growth in sand culture at different NaCI concentrations. Selected (#) and unselected (O) leaves; selected ( ) and unselected (D) stems; selected (A) and unselected (A) roots. 3750-1800 - 175 0 100 175 O 100 175 NGCI concentrations in sand culture (mol m -) Fig. 3. Cl~ contents in different parts of plants from selected and unselected lines of three legume species [Trifolium alexandrinum (a), Medieago sativa (b), Trifolium pratense (c)]; details as in Figure 2.

468 M. AsHRAF, T. MCNEILLY AND A. D. BRADSHAW differences were not statistically significant. For the roots, the selected line of T. alexandrinum absorbed more CI" at 100 mol m~-* than the unselected line and the lines X concentration interaction was significant (P < 0-05). There were diflferences between species in patterns of CI" content within the plant. T. alexandrinum leaves contained less Cr than the stem, while in other species the reverse situation was found. Overall, Cl~ contents in the leaves of T. 2750 175 0 100 175 0 100 175 NoCI concent rolions m sond culture (mol Fig. 4. K+ contents in different parts of plants from selected and unselected lines of three legume species [Trifolium alexandrinum (a), Medicago sativa (b), Trifolium pratense (c)]; details as in Figure 2. m"^) alexandrinum were lower than those in M. sativa, which in turn were lower than those in T. pratense. Roots of all three species had lower Cl~ contents than the stems or leaves. T. alexandrinum and M. sativa retained almost the same amount of CI, i.e. lower than that found in T. pratense. Potassium. The leaves and stems of the selected lines of T. alexandrinum and M- sativa contained less K+ overall (Fig. 4) than those of unselected lines {P < 0-01). In T. pratense, all plant parts in the selected line again retained less K"*", but this difference was not statistically significant. The three species showed different patterns of K+ accumulation. In M. sativa, roots retained more K^ than the other parts, while in T. alexandrinufjj and T. pratense, shoots were the K"''-accumulating organs. The order of accumulation of K"'' was T. pratense > M. sativa > T. alexandrinum. Little difference in the accumulation of K+ between plant parts was found in T. alexandrinum and M. sativa, but the differences in accumulation by different plant parts were highly significant in T. pratense. Calcium. The T. alexandrinum selected line had less (not statistically significant) Ca^+ in its leaves at 175 mol m"'' than the unselected line but did not differ at 0 and 100 mol m"' NaCI (Fig. 5). In M. sativa, the selected line contained significantly less Ca''^^ than the unselected line in both leaves and stem {P < 0-01). In T. pratense, the leaves and stems of the selected line contained significantly greater amounts of Ca^* (leaves, P < 0-05; stem, P < 0-001) than the unselected

Ion uptake and salt tolerance 469 900 750 - _ 600-175 0 100 175 NaCi concentrations in sand culture (mol Fig. 5. Ca" contents in different parts of plants from selected and unselected lines of three legume species [Trifolium alexandrinum (a), Medicago sativa (b), Trifolium pratense (c)]; details as in Figure 2. 175 (a) 350 280 210 A \ 140 70 m. '~ '~ ~O S ^ _j ^ 0 1-1 1 175 O 100 175 ^ 0 NaCI concentrations in sand culture Imol m Fig 6 Mg" contents in different parts of plants from selected and unselected lines of three legume species [Trifolium alexandrinum (a), Medicago sativa (b), Trifolium pratense (c)]; details as in Figure 2. ') line. A reverse situation was observed in the roots, the unselected line containing more Ca^^ than the selected line (P < 0-01). Comparing the species, the Ca2+ contents were in the order T. pratense > M. sativa > T. alexandrinum. Magnesium. The Mg contents (Fig. 6) of the leaves and roots of the two T. alexandrinum lines did not differ significantly, but the stems of the unselected line contained more Mg'^+ than the selected line (P < 0-001). Similarly, the selected line of M. sativa contained less Mg2+ in the leaves and stem than the unselected

47 M. ASHRAF, T. MCNEILLY AND A. D. BRADSHAW lines (leaves, P < 0-001; stem, P < 0-01), but accumulation in the roots of the two lines did not differ significantly. In T. pratense, the selected line contained less Mg^"*" in its leaves than the unselected line, and an increase of NaCI in the external medium had a very pronounced effect in reducing the internal Mg^"'' content by approximately 50% and 60% in the selected and unselected lines respectively. DISCUSSION The selected lines had been produced from the progeny of individuals showing superior lengths of shoot at the seedling stage after two weeks of growth in nutrient solution containing NaCI. These produced significantly greater yields of dry matter than the unselected lines in all three species. It has been argued that, in some species, tolerance at the seedling stage may not reflect tolerance at the adult stage (Akbar & Yabuno, 1974; Shannon, 1979). On the other hand, the seedling stage has been considered more sensitive than the adult stage (Pasternak, Twersky & De Malach, 1979; Noble, 1983), and it has been argued that to improve the salt tolerance of a species, tolerance at the most sensitive stage must be increased (Noble, 1983). Whether selection is carried out at the adult or at early stages, the important factor is the intensity of selection which can be applied for isolation of tolerant individuals. The NaCl-tolerant lines produced for use in this study were the product of selection of a very few individuals out of a total of at least 10000 seedlings of normal cultivars of the three species. The individuals selected were 0-32%, 0-76% and 0-80% of the total population in T. alexandritium, M. sativa and T. pratense, respectively. Differences in shoot length were used as the selection criterion in these dicotyledonous species following the proposal of Rozema & Visser (1981). Lines of M. sativa with enhanced NaCI tolerance produced by selection have recently been described by Noble (1983). These were the product of seleetion of adult plants from samples of only 500 individuals, but nevertheless they gave correlated NaCI tolerance at all other growth stages. The procedure outlined in the present work is based upon the screening of seedlings. Clearly, this method can also produce individuals whose progeny show enhanced tolerance in T. alexandrinum, T. pratense, as well as in M. sativa. Mesophytes respond to saline environments either by excluding salt and synthesizing organic solutes, or by accumulating high concentrations of electrolytes to maintain their turgor (Elzam & Epstein, 1969; Greenway & Munns, 1980). Although the selected line of T. alexandrinum had higher Na"*" and Cl" contents in the roots compared with those of the unselected line, it excluded Na^, but had higher Cl~ in the leaves. Similar results have been observed for the salt-tolerant soybean variety 'Lee' (Lauchli & Wieneke, 1979), although the leaves of tolerant T. alexandrinum line had a higher Cl~ content. The selected line also had lower K^, Ca^^\ and Mg^+ eontents in its tissues, in spite of the important role of the former two ions in maintaining the turgor of plants under saline stress. In M. sativa, the selected and unselected lines did not differ in Na"*" and K"*^ content, although the selected line had higher Cl~ in the leaves at high NaCI concentration. This line also had less Ca"'^^ in its shoot compared with the unselected line. The results for Cl" content agree with those of Croughan, Stavarek & Rains (1978) who found that, as in a typical halophyte, a selected NaCl-tolerant line of M. sativa from tissue culture accumulated Cl~. The tolerant line of T. pratense had less Na"*" in both leaves and stems, but took

Ion uptake and salt toleranee up less K+ and Mg''^+ and had higher Ca^+ contents in its shoot. This species is more sensitive to salt than T. alexandrinum (data presented here, and that of Winter & Lauchli, 1982) and M. sativa. The lower Na+ content in the shoot of the tolerant line is again similar to the pattern in salt-tolerant soybean (Lauchli & Wieneke, 1979). If a forage species accumulates high quantities of salt in its tissue, it may not be suitable for fodder (Downton, 1984; Epstein, 1985). However, the tolerant lines of all the three species examined here showed a considerable variability in ion distribution compared with non-tolerant lines. Further investigation of two basic points of great importance is clearly needed. First, if further cycles of selection are carried out in the same way, lines of these species that have further increases in NaCI tolerance may show a greater tendency to exclude Na"*" from their tissues. Second, it is crucial to investigate plant-to-plant variability in Cl~ or Na"^ and to select CI" or Na+ excluders from tolerant lines for use in further breeding programmes. ACKNOWLEDGEMENTS The authors gratefully acknowledge financial support to M. Ashraf through an Overseas Research Studentship Award and a University of Liverpool Research Studentship. REFERENCES AKBAR, M. & YABUNO, T. (1974). Breeding for saline-resistant varieties of rice. U. Comparative performanee of some riee varieties to salinity during early development stages. Japan Journal of Breeding, 24, 176-181. CROUGHAN.T. P., STAVAREK, S. J. & RAINS, D. W. (1978). Selectionof a NaCI tolerant line of cultured alfalfa eells. Crop Scienee, 18, 959-963. DEWISY, D. R. (1962). Breeding crested vvheatgrass for salt tolerance. Crop Science, 2, 403-407. DOWNTON, W. J. S. (1984). Salt tolerance of food crops: perspectives for improvements. CRC Critical Review in Plant Sciences, 1, 183-201. ELZAM, O. E. & EPSTEIN, E. (1969). Salt relations of two grass species differing in salt tolerance. I. Growth and salt content at different salt concentrations. Agroctiimica, 13, 187-195. EPSTEIN, E. (1985). Salt-tolerant crops: origins, development, and prospects of the concept. Plant and Soil, 89, 187-198. GREENAWAY, H. & MuNNS, R. (1980). Mechanisms of salt tolerance in nonhalophytes. Annual Reviezv of Plant Physiology, 31, 149-190. HEWITT, E. J. (1966). Sand and Water Culture Methods used in ttie Study of Plant Nutrition, 2nd edn. Commonwealth Agricultural Bureau. Technical Communication No. 22. LAUCHLI, A. (1984). Salt exclusion: an adaptation of legumes for pastures and saline eonditions. In: Salinity Tolerance in Plants: Strategies for Crop Improvement. (Ed. by R. C. Staples & G. H. Toeniessen), pp. 171-187. John Wiley & Sons, New York. LAUCHM, A. & WIENEKE, ]. (1979). Studies on growth and distribution of Na+, K+ and CI" in soybean varieties differing in salt tolerance. Zeitschrift fiir Pflanzenernahrung und Bodenkunile, 142, 3-13. NOBLE, C. L. {\983). Genetical and ptiysiological aspects of.uilt-tolerance in lucerne {Medicafro sativa L.). Ph.D. thesis. University of Melbourne, Australia. PASTERNAK, D., TWERSKY, M. & DE MAI.ACH, Y. (1979). Salt resistance in agricultural crops. In: Stress Phvsiology in Crop Plants (Ed. by H. Mussel & R. C. Staples), pp. 127-142. John Wiley & Sons, New YoVk. RozEMA, J. & Vissia!, M. (1981). The applicability of the rooting technique measuring salt resistance in populations of Festuca rubra and Juncus species. Plant and Soil, 62, 479-485. SHANNON, M. C. (1979). In quest of rapid screening techniques for plant salt tolerance. HortScience, 14, 587-589. WINTER, E. & LAUCHLI, A. (1982). Salt tolerance of Trifolium alexandrinum L. 1. Comparison of salt response of T. alexandrinum and T. pratense. Australian Journal of Plant Physiology, 9, 221-226.