Genotypic difference in salinity tolerance of green gram cultivars

Plant Science 166 (2004) 1135–1142

Genotypic difference in salinity tolerance of green gram cultivars Neelam Misra∗ , U.N. Dwivedi Department of Biochemistry, Lucknow University, Lucknow, U.P., India Received 8 September 2003; received in revised form 17 November 2003; accepted 17 November 2003

Abstract Responses of two green gram (Phaseolus aureus) cultivars differing in salt tolerance ability were compared for seed germination efficiency, seedling vigor (root and shoot length), plant growth (dry weight (DW) and fresh weight (FW)), water uptake and intracellular Na+ /K+ contents during germination under the conditions of absence as well as presence of various levels of salinity. In cultivar SML-32 increasing levels of salinity remarkably decreased seed germination and caused pronounced decrease in seedling vigor, plant growth and water uptake in shoot and root compared to cultivar T-44. Intracellular sodium content of the root and shoot tissues of the cultivar SML-32 was found to be increased several folds in presence of salinity while that of the cultivar T-44 exhibited only moderate increase even at high salinity level (200 mM NaCl). However intracellular K+ content of the root and shoot tissues of both the cultivars did not much increased in presence of salinity. Results suggest possible different behaviors of cultivars differing in salt tolerance with respect to germination, seedling vigor, plant growth, water content (WC) and Na+ /K+ contents. Based on these criteria, cultivar T-44 was considered as salt tolerant while that of SML-32 salt sensitive. © 2003 Elsevier Ireland Ltd. All rights reserved. Keywords: Ion content; Salt stress; Salinity; Phaseolus aureus

1. Introduction Germination of seeds, one of the most critical phases of plant life, is greatly influenced by salinity. Both growths as well as metabolism are reported to be altered under saline stress [1,2]. Salinity is reported to decrease as well as delay germination of most of the crops. Lower levels of salinity delayed germination whereas higher levels in addition, reduced the final percentage of seed germination [3]. Based on a number of physiological and biochemical parameters, various workers have tried to ascribe genotype differences between salt tolerant and sensitive plants in an effort to develop rapid screening methods for salt tolerance [4]. It is well established that salt tolerance ability depends on genetic and biochemical characteristics of the species and sufficient genetic variability in relation to salinity exist in many agricultural crops including mung bean [5]. Mechanism of the responses to salt (NaCl) treatment has been Abbreviations: FW, fresh weight; DW, dry weight; WC, water content Corresponding author. Present Address: Department of Biochemistry, Bundelkhand University, Jhansi, U.P., India. Fax: +91-517-232-1761. E-mail address: neelam [email protected] (N. Misra). ∗

examined in many species. The understanding of salt stress, however, still remains incomplete because of the complexity of the process presenting an ionic component on the one hand and an osmotic component on the other, which involves morphological, physiological and metabolic change [6]. These changes permit the plant to restore conditions, permitting continued growth under salinity stress [7]. Some of the morphological changes are reduction of shoot [8] and root length [9] and restricted rooting [10]. Reduced growth rates as a result of salt stress were initially related to loss of turgor and its accompanying relaxation of cell wall tension [11]. However, the relationship appears to be more complex. Reductions of growth rate occur even without loss of turgor [12] and it appears likely that growth is actively controlled, independently of the sensing of turgor pressure, via changes in cell wall extensibility [13]. Such changes appear to be related to changes in protein composition of cell walls [13]. Thus, adverse effects of salinity on growth and metabolism may be due to osmotic inhibition of water availability (dehydration), toxic effects of high salt ions and disturbance of the uptake and translocation of nutritional ion [14]. Excess of Na+ and Cl− , the predominant ions in saline soils, creates high ionic imbalances that may impair the selectivity of root membrane [15].

0168-9452/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.plantsci.2003.11.028

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Measurement of the Na+ and K+ content of salt tolerant and sensitive plants has revealed differential accumulation of Na+ and K+ characteristic to the cultivar variety. Salt tolerant plants are reported to contain low Na+ and high K+ content as opposed to salt sensitive, which possess high levels of Na+ and low K+ under salinity conditions [16,17]. Salinity exposure (140 mM NaCl) to Lotus plants caused a reduction in total dry weight (DW) of and increased absorption and accumulation of ions in the leaf tissue leading to toxic effects and tissue dehydration [18]. A number of mechanisms contribute to salt tolerance [19]. Commonly proposed mechanism includes compartmentation of ions in vacuole [20], accumulation of compatible solutes in the cytoplasm [21,22], as well as genetic salt resistance [20]. Such type of osmotic adjustment lowering the toxic concentration of ions in the cytoplasm by restriction of Na+ influx or its sequestration into vacuole/and/or its extrusion [23,24]. The aim of the present study was to characterize the effect of salinity on the two cultivar of Phaeolus aureus, namely, T-44 and SML-32 were evaluated for their salt tolerance/sensitivity on the basis of magnitude of seed germination, seedling vigor (length of root and shoot), plant growth (DW and FW) and Na+ and K+ content during seed germination and seedling growth stage.

2. Materials and methods 2.1. Plant material and stress treatments Seeds of green gram (Phaseolus aureus, family Leguminosae) cultivar T-44 (salt tolerant; hybrid between T-1 and T-49) and SML-32 (salt sensitive; hybrid between T-1 and G-39) were well screened in lab. Seeds of green gram cultivars T-44 and SML-32 were surface sterilized with 1% sodium hypochlorite and germinated as described by Misra and Dwivedi [25]. Four concentrations of NaCl namely, 50, 100, 150 and 200 mM, were used for tolerant cultivar T-44 while three concentrations of NaCl namely, 1, 10 and 50 mM were used for sensitive cultivar SML-32. Starting with 4 h socked seeds (0 h of seed germination) the germinated seeds were taken out at 24 h intervals up to 5 days, root and shoot (along with cotyledons) were separated from the seeds. The experiments were performed with three replicates of each. Seedling vigor (length of root and shoot) and plant growth (DW and FW) were evaluated using ten seeds from each cultivar in triplicate. At specified periods of germination, seeds were taken out and root and shoot (along with cotyledons) were separated. Length of root, shoot and fresh weight (FW) of these seed parts was measured. For the determination of dry weight these seed parts (root and shoot) were dried at 70 ◦ C for 3 days in an oven (or till there is no decrease in weight). Water content (WC) as percentage of fresh weight

was calculated using following formula: 
FW − DW × 100 RWC (%) = FW 2.2. Ion content and chemical analysis The oven dried root and shoot tissues (≈20 g) were ground to fine powder and 100 mg of this transferred to a digestion flask (50 ml) containing a acid mixture of HNO3 and H2 SO4 , in the ratio 10:1 (v/v) [26]. The flask was heated gently over a sand bath. The cooled digest was then diluted by adding double distilled water and the volume was made up as required. The estimation of Na+ and K+ contents in the acid extracts were carried out on a flame photometer (Systronics, India make). 2.3. Statistical analysis Each treatment was analyzed with at least three replicates and standard deviation (S.D.) was calculated.

3. Results 3.1. Effect of salinity levels on seed germination It was observed that in absence of salinity almost 100% germination was observed from day 1 onwards in both the cultivars T-44 and SML-32 (Fig. 1). However in the presence of salinity the seed germination decreased in both the cultivars at all salinity levels. The decrease was more prominent at the beginning, which progressively became less prominent during subsequent days of germination at all salinity levels in both the cultivars. Furthermore, with increasing salt concentration the germination of seeds decreased progressively in both the cultivars through this inhibitory effect of salinity on germination was more pronounced on the cultivar SML-32 as compared to that of T-44 (data not shown). Thus, the cultivar T-44 germinated even in the presence of 200 mM NaCl exhibiting a fair degree of salt tolerance. On the other hand cultivar SML-32 was unable to germinate above 50 mM NaCl salinity exhibiting quite sensitivity towards salt stress. The germination of SML-32 seeds was found to decrease significantly even at 50 mM NaCl salinity (Fig. 1). 3.2. Effect of salinity on seedling vigor The seedling vigor (length of root and shoot) increased gradually in both the cultivars with 1–5 days of seed germination under the conditions of absence (control) and presence of various levels of salinity (data not shown). However, salinity treatment resulted in decreasing the root and shoot in both the cultivars as compared to their respective control

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values (maximum concentration of salinity, 200 mM NaCl for T-44 and 50 mM NaCl for SML-32, Fig. 2). Furthermore, root was found to be more sensitive towards salinity than that of shoot as decrease in root length was more pronounced as compared to that of shoot in both the cultivar at 200 mM NaCl (T-44) and 50 mM NaCl (SML-32) of salinity. 3.3. Growth kinetics and physiological parameters

Fig. 1. Effect of salinity on germination percentage of green gram cultivar T-44 (a) control 200 mM NaCl and SML-32 (b) control 50 mM NaCl at day of germination. Each value represents mean of three independent observations and S.D. determined.

The plant growth trends in terms of biomass (fresh weight, dry weight, water content and Na+ :K+ ratio) in both the cultivars in absence and presence of salinity was studied (Table 1). With the progress of plant growth, in the root, there was a gradual increase in the FW, DW and WC under both the conditions of absence as well as presence of NaCl salinity in both the cultivars. However, salinity treatment resulted in decreasing the FW, DW and WC of the root tissue as compared to their respective non-saline control values in both the cultivars at different levels of salinity (Table 1 and Fig. 3(a, b) T-44; Fig. 3(c, d) SML-32). In case of shoot a similar pattern as that for root was obtained with only difference that in shoot the salinity treatment resulted in increasing the DW as compared to their respective non-saline controls (Table 1 and Fig. 3(b, d). Decrease in the FW and WC at various levels of salinity in both root and shoot was more in cultivar SML-32 as compared to that of T-44 (Fig. 3(a, b), compare values at 50 mM in both cultivars). Note that even at 200 mM salinity level, cultivar T-44

Fig. 2. Effect of salinity on seedling vigor as measured by lengths of root (R) and (S) of green gram cultivars T-44 (a, b) control; 200 mM NaCl and SML-32 (c, d) control; 50 mM NaCl at 1–5 days of seed germination. Each value represents mean of three independent observations based on average lengths of 10 roots/shoots and S.D. determined.

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Table 1 Effect of salinity on fresh weight, dry weight, water content and Na+ : K+ ratio of root and shoot of green gram cultivars T-44 and SML-32 at day 5 of seed germination Cultivar

T-44 SML-32

NaCl salinity (mM)

Root

Shoot

Fresh weight (mg)

Dry weight (mg)

Water content (%)

0 (control) 200

245 ± 0.5 51 ± 0.3

32 ± 0.7 15 ± 0.3

0 (control) 50

235 ± 3.5 65 ± 1.7

21 ± 1.2 15 ± 0.6

Na:K ratio

Fresh weight (mg)

Dry weight (mg)

Water content (%)

Na:K ratio

87 ± 1.2 71 ± 1.0

0.62 0.60

1790 ± 9.0 921 ± 5.0

178 ± 2.0 273 ± 1.8

90 ± 1.0 70 ± 2.1

0.34 0.43

91 ± 2.0 77 ± 3.8

0.84 12.27

1693 ± 6.0 605 ± 4.0

218 ± 1.7 246 ± 2.4

87 ± 3.6 59 ± 1.6

0.87 9.76

Each value represents mean of three replicates. Values are rounded up to nearest whole figure and S.D. determined.

was able to maintain the water content at a higher salinity level as compare to that of SML-32 at 50 mM salinity in both root as well as in shoot (Table 1, Fig. 3(a, b) T-44; Fig. 3(c, d) SML-32).

Plant exposure to salinity induced the uptake and accumulation of considerable amount of Na+ and K+ , which was cultivar dependent and organ specific. Cultivar T-44 (tolerant) accumulated the highest amount of K+ and least amount

Fig. 3. Changes in fresh weight (䊉), dry weight ( ), water content (䉱) and lengths of (䉬) root (a) and shoot (b) in cultivar T-44, and (c) root and (d) shoot in cultivar SML-32 on day 2 of seed germination at different levels of salinity (50, 100, 150, 200 mM NaCl in T-44 and 1, 10, 50 mM NaCl in SML-32). Data are expressed as percent relative change over non-saline control (control as 100%) and S.D. determined.

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of Na+ as compared to cultivar SML-32 (sensitive) (Figs. 4 and 5). The calculated Na+ :K+ ratio was significantly increased in SML-32 in both root and shoot as compared to T-44 (Table 1). There was a steady increase in the concentration of Na+ content in NaCl-tolerant cultivar T-44 with the corresponding increase in NaCl level with progress of plant growth till day 4 and thereafter declined both in the absence and presence of salinity in both the cultivars (Fig. 4(a, b) T-44; Fig. 4(c, d) SML-32). The K+ concentration in both root and shoot of T-44 cultivar was increased with increasing concentration of NaCl till day 5 of plant growth in absence and in presence of NaCl salinity (Fig. 5(a, b)) whereas, in cultivar SML-32 (Fig. 5(c, d)) a slight increase (not significant) in both root and shoot in absence and in presence of 50 mM NaCl salinity.

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In the cultivar T-44, the Na+ content of both root as well as shoot did not increased much over the control, even at 200 mM external NaCl salinity. Thus in cultivar T-44 the maximum increase of Na+ in the presence of 200 mM NaCl salinity in both the tissues were less than three-fold over the control value (in absence of salinity). In contrast to this even in presence of 50 mM NaCl the cultivar SML-32, Na+ content had 15-fold over the non-saline control in both tissues. Thus, cultivar T-44 was able to maintain a relatively low level of intracellular Na+ content and high level of K+ content (1.6-fold), even at high external NaCl salinity, as compared to that of cultivar SML-32 (sensitive) (not significant). The fluctuations in the content of K+ were relatively less in SML-32. The calculated Na+ :K+ ratio was significantly increased in SML-32 but much less in T-44 (Table 1).

Fig. 4. Effect of salinity on intracellular sodium content of root (a) and shoot (b) during seed germination in cultivar T-44 and root (c) and (d) shoot in cultivar SML-32. (䊉) in absence of salinity (control); ( ) in presence of 50 mM NaCl; (䉱) 200 mM NaCl salinity. Data are expressed as percent relative change over non-saline control value on day 1 (control as 100%) and S.D. determined.

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Fig. 5. Effect of salinity on intracellular potassium content of root (a) and shoot (b) during seed germination in cultivar T-44 and root (c) and (d) shoot in cultivar SML-32. (䊉) in absence of salinity (control); ( ), in presence of 50 mM NaCl; (䉱) 200 mM NaCl salinity. Data are expressed as percent relative change over non-saline control value on day 1 (control as 100%) and S.D. determined.

4. Discussion Genetic variability within a species offers a valuable tool for studying mechanism of salt tolerance. One of these mechanisms depends on the capacity for osmotic adjustment that allows growth to continue under saline conditions. With increasing salinity there was a decrease in germination of seeds, seedling vigor, plant growth and water uptake in both the cultivars. In the cultivars SML-32 the magnitudes of such decrease were more as compared to that of cultivars T-44. Inhibition of germination due to salinity has been reported earlier [3,27]. It is suggested that decrease in seed germination and depression in seedling vigor under saline stress is attributed to decrease water uptake followed by limited hydrolysis of food reserves from storage tissues as well as due to impaired translocation of food reserves from storage tissue to developing embryo axis [28–30]. It has been observed, under ex vitro conditions and after 60 days, a root volume reduction of 73% in 50 mM NaCl salt stressed tomato [10]. Our results indicate a pronounced decrease in water uptake in presence of salinity in root and shoot in sensitive cultivar SML-32 compared to that of tolerant cultivar T-44, which is well correlated with decreased seedling vigor in this cultivar. Higher water content in root and shoot of sensitive cultivar

under control (absence of salinity) suggest varied behaviors for water uptake during germination of seeds differing in salt tolerance. Salt tolerance ability appears to be associated with decreased rate of water uptake during germination. Salt tolerant sorghum cultivar IS-1347 had relatively high osmolarities concurrent with high relative water uptake than that of salt sensitive cultivar IS-4807 [17]. In shoots of both the cultivars DW increased with increasing salinity. This may be attributed due to decreased mobilization of reserve food materials from the cotyledons at higher salinity levels (as shoot contains cotyledons). Similar suggestions have been made by others [28,31]. Under salt stress, mechanisms of salt tolerance depend on the capacity for osmotic adjustment, which allows growth of the plant to continue under salt stress. When both the cultivars were compared, very significant differences in Na+ and K+ ions were observed. A relatively very high level of intracellular Na+ and low level of K+ ion observed, in salt sensitive cultivar SML-32, as opposite trend was observed in cultivar T-44, Na+ ions may be toxic to cells making them unable to grow in presence of salt [30]. Under saline condition uptake and accumulation of Na+ ions accomplish this process. It was reported that the tolerant sorghum cultivar IS-1347 restricted the uptake of sodium and it was

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translocated to the shoot in limited amount while in the sensitive cultivar IS-4807, the reverse was the case [17]. The salt tolerant cultivar of reed plants always maintained low Na+ contents in the shoot [16]. The highly tolerant species (Casuarina equisetifolia and C. glauca) accumulated little Na+ in their shoots than that of lesser tolerant (sensitive) species [32]. Salinity induced a greater increase in cytoplasm Na+ content in the salt sensitive variety than those of tolerant varieties [30,33]. Consequently, the Na+ :K+ ratio was also significantly different (Table 1). The Na+ :K+ ratio was very small in the control plants, increased substantially in the two cultivars after the plants were exposed to high levels of NaCl, particularly in the sensitive cultivars SML-32 (Table 1). It was reported that the Na+ :K+ ratio might serve as an indicator of crop tolerance to stress as the increase of Na+ in salt tolerant species is generally associated with a decrease in K+ [4]. However, salt tolerance is actually often associated with particular capacities to maintain K+ content high. The low water content of SML-32 root and shoot as compared to those of T-44 may be due to the high Na+ content of these tissues in SML-32. The restricted growth of cultivar SML-32 under saline condition was mainly due to high Na+ content of the cells rather than loss of turgor (reduce water uptake). Under saline conditions the salt sensitive cultivar SML-32 not only accumulated more toxic Na+ ions, in both root and shoot than the salt tolerant T-44 (Fig. 4) showed larger reduction in both the tissues elongation (Figs. 2 and 3). In this experiment, the accumulation of toxic ion (Na+ ) coincided with a period of very intense growth (Table 1), suggesting that salt tolerance could be associated with the synchronization between the rate of ion transport to the shoot and the plant capacity to compartmentalize them in different tissues or cells [33]. Besides, a control mechanism of absorption and transport of toxic ion to leaves may be involved, at least in sorghum [34]. Furthermore the shoot and root that suffered the elongation inhibition as a result of stress (Fig. 2) were the ones that showed the greatest reduction in K+ content, especially in the salt sensitive cultivar (Fig. 5). Based on the growth parameter (FW, DW, seedling vigor and water content) cultivar T-44 germinated at the highest levels of NaCl (200 mM), FW and DW were highest, low levels of Na+ and high levels of K+ accumulation and high water content while just reverse was found in salt sensitive cultivar SML-32. It is generally accepted that the roots suffer first from exposure to environmental stress, followed by their injury. Thus, even slight root damage permits a large flux of ions to the shoot. As a result, comparative studies of genotypes or cultivars are more important if ion accumulation is determined in shoot. Ion accumulation, water content and dry matter production were highly correlated as far as salinity is concerned, suggesting that cultivar T-44 may serve as potential source for salt tolerance of cultivated green gram, and especially for those characterized by high vegetative growth and severe stress effects.

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From the present study it seems that salt tolerant cultivar T-44 has some unique Na+ /K+ transporter which maintains low intracellular Na+ . The future study may be directed towards the isolation and the characterization of this transporter.

Acknowledgements We wish to acknowledge the financial assistance for this project from Council of Science and Technology, U.P., India. The work was partially supported from the Department of Biotechnology and University Grant Commission, New Delhi.

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