Alkalinity and salinity tolerance during seed germination and early seedling stages of three alfalfa (Medicago sativa L.

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1 Legume Research, ()217 : Print ISSN:2-371 / Online ISSN: AGRICULTURAL RESEARCH COMMUNICATION CENTRE Alkalinity and salinity tolerance during seed germination and early seedling stages of three alfalfa (Medicago sativa L.) cultivars Huihui Zhang, Xin Li, Xu Nan 1*, Guangyu Sun 2, Minglong Sun 3, Dunjiang Cai 4 and Siyu Gu* College of Resources and Environment, Northeast Agricultural University, Harbin, Heilongjiang Province-3, P R China. Received: Accepted: DOI:1.18/lr.vi.81 ABSTRACT The germination of halophytes in saline-alkali substrates is a useful criterion for selection tolerant alfalfa (Medicago sativa L.) cultivars. The experimental conditions consisted of five levels of salinity (24, 48, 72, 96 and 12 mm) and four ph levels (7, 8, 9 and 1). The results showed that under saline conditions, the germination percentage (G p ), plumule length (P l ) and radical length (R l ) also decreased with increasing concentration. However, under low or moderate saline conditions, P l and fresh weight decreased sharply but R l was the longest at 48 and 72 mm and ph 7.1, which indicated that low salinity may promote radical growth appreciably. Salinity and alkalinity stresses had synergetic effects on G p but not on P l and fresh weight. The results confirmed G p and G e are important indices for assessing germinated energy of alfalfa in salinity-alkalinity soils. Moreover, three alfalfa cultivars tested were relatively tolerant of salinity and alkalinity and Stockpile was the most tolerant cultivar in the present experiment. Key words: Alfalfa, Alkalinity, Salinity, Seed germination. INTRODUCTION Alkalization and salinization of soils are two of the most important factors limiting global agricultural productivity (Liu et al., 21). The area of grasslands influenced by salt and alkali are estimated to be hm 2 in Songnen Plains in Northeast China. Salinity stress refers to neutral salts (NaCl and Na 2 ), whereas alkalinity stress is related to alkaline salts (NaH and Na 2 ). Saline soils negatively impact plant growth due to osmotic inhibition of water absorption (Munns, 22). The pernicious effects of alkalinity on plant growth are related to high ph, which disrupts ionic balance (Shi and Zhao, 1997) and poor micronutrient availability in soils (Alam et al., 1999). Furthermore, soil salinization-alkalization may be more severe problem than soil salinization alone (Yang et al., 28). It is known that alfalfa (Medicago sativa L.) not only is high quality forage grass for animals but also could improve soil fertility (Jin et al., 21). It is resistant to various stresses, such as saline (Abusuwar et al., 2), heavy metals (Zhou et al., 28) and drought (Naya et al., 27). The germination response of plants to environmental parameters could limit species distribution in saline and alkaline conditions (Tobe et al., 2). Therefore, tolerance to salinity and alkalinity during germination is central to the establishment of plants growing in saline-alkaline soils (Sosa et al., 2). Since alfalfa resistance to abiotic factors is mainly controlled by genotype, such resistance is expressed during the early growth stage. In our experiment, in order to investigate the physiological adaptive mechanisms of germinating seeds under salinity-alkalinity stress, we compared the resistance of three different alfalfa cultivars based on seed germination and seedling growth under 21 combinations of salinities and ph values. Such an investigation may help in early evaluation of criteria for selection of adopted cultivars in the laboratory, which may be related to subsequent performance in salinity-alkalinity soils. MATERIALS AND METHODS Seed materials: Seeds of three alfalfa (Medicago sativa L.) cultivars, WL33 HQ, PGI427 and Stockpile, weight of 1 seeds was 2.1 g, 2.4 g and 2.17g, respectively. Alfalfa seeds were supplied by the Institute of Crops, Heilongjiang Academy of Land Reclamation and Agricultural Sciences, Jiamusi, Heilongjiang Province, China. Design of simulated salt and alkaline conditions: Two neutral salts (NaCl and Na 2 ) and two alkaline salts (NaH and Na 2 ) were selected based on the *Corresponding author s xunan41@126.com; gusiyu777@163.com 1 Natural Resources and Ecology Institute, Heilongjiang Academy of Sciences, Harbin,, P R China. 2 College of Life Science, Northeast Forest University, Harbin, Heilongjiang Province,, P R China. 3 Institute of Crop Breeding, Heilongjiang Academy of Agriculture Sciences, Harbin, 86, P R China. 4 Institute of Crops, Heilongjiang Academy of Land Reclamation and Agriculture Sciences, Jiamusi, Heilongjiang Province, 7, P R China.

2 84 LEGUME RESEARCH An International Journal composition of the saline-alkaline soil over the northeast of China (Ge and Li, 199). Four treatment groups (labeled as A, B, C and D) were selected with gradually increasing alkalinity, and the composition is shown in Table 1. All treatment groups had a 1:1 molar ratio of monovalent salts (NaCl+NaH ) to divalent salts (Na 2 +Na 2 ); therefore, if the individual molar concentrations were the same then the total ion concentrations were the same throughout the treatments. The groups A, B, C and D featured five levels of saline concentration 24, 48, 72, 96 and 12 mm, respectively. Using a full combination design and one control with distilled water, a total of 21 combinations of salinity and ph were established. Table 1: The salt composition and its molar ratio of various treatments. Treatment Salt composition and molar proportions group NaCl Na 2 NaH Na 2 A 1 1 B C D Seed germination tests: Average-sized seeds were placed in 9-cm Petri dishes with two layers of filter paper, moistened with 1 ml of distilled water or with simulated mixed saltalkali saturated solution. The seed germination experiments were repeated four times with seeds per treatment. The dishes were maintained at 2 C±1 C Total weight was maintained constant every day by adding distilled water to compensate for the water lost. The germinated seeds were counted every day for 7 days. Lengths of plumule and radicals of 1 seeds were chosen to measure. The seed samples were oven-dried at 1 C for 1 min and then dried at C to constant weight and the dry weights (DW) were recorded. The germination percentage, index and energy were estimated according to the following formula (Hu et al., 2). Statistical data analysis: All data were obtained from the average of four replicates. Statistical analyses on two-way variance analysis (ANOVA) were carried out using the Statistical Package for Social Sciences (SPSS 13.), and the significance level was set at P. RESULTS AND DISCUSSION Germination percentages: Germination percentages of alfalfa seeds in mixed salinity-alkalinity stress treatments were lower than that of the control, with the exception that 24 mm salt concentration was rather similar to that of the control in Groups X1, Y1 and Z1 (ph=7.1) (Fig. 1.). The germination percentages of all treatment groups decreased with increased salinity and ph, respectively. The germination percentages were sharply decreased when both the ph was higher than 7.1 and the salinity was more than 24 mm. At Fig-1: Simulated logistical curves showing the dynamic germination percentages under mixed salinity-alkalinity stress. X, Y, and Z respectively represent the cultivars WL33 HQ, PGI427 and Stockpile. the same salinity concentration, the germination percentages markedly decreased with increases in ph. Furthermore, alfalfa seeds could not germinate in high salinity combined with high alkalinity (ph 9, salinitye 96 mm). The germination percentages of all three cultivars showed a similar dynamic tendency. However, the three cultivars of alfalfa were significantly different in terms of the germination percentage, which was in the descending order Stockpile>PGI427>WL33 HQ. Germination energy: Germination energy (G e ) in the same alkalinity treatment groups was decreased with rising salinity (Fig.2). Similarly in the same salinity treatment group tended to be lower with increasing alkalinity. The decrease in Ge was steep when salinity and alkalinity were both high (salinity>24 mm, ph>7.1). The G e of the cultivar WL33 HQ significantly decreased with increasing salinity when ph was 7.1, whereas that of the cultivar Stockpile had no significant difference when compared with the control. WL33 HQ was the only cultivar showing a small G e when the ph was 1 and the salinity was 48 mm, showing that WL33 HQ had more alkalinity tolerance than the other two cultivars. Germination index: Like G e was not significantly altered by low salinity ( 24 mm), though slightly lower than the control (Fig. 3.). However significantly decreased as salinity and ph increased, a higher salinity at a higher ph level resulting in a more obvious decrease in G i. The analysis of variance indicated that salinity and ph significantly affected seed G i individually, salinity and ph yielding a pairwise effect (Table 2). G i levels were: PGI427> Stockpile>WL33 HQ in saline and alkaline stress. Like G e, G i decreased most steeply when both salinity and alkalinity were both high (salinity>24 mm, ph>7.1).

3 Volume Issue (October 217) 8 Fig-2: Effects of salinity and ph on the germination energy. Length of plumules and radicals: The P l of the alfalfa were negatively correlated with high salinity and ph, which was not found when the salinity was 24 mm (Fig. 4) (Table 2). At same salinity level except for 24 mm, an increase in ph decreased P l. However, when salinity was 24 mm, the increase of ph from 7.1 to 8.3 did not decrease P l. P l were longer than the control, particularly for the cultivars PGI427 and Stockpile, whose Pl and Rl were significantly longer among all ph values. Within treatments at ph=7.1, P l slowly decreased along the salinity gradient, while they sharply decreased when ph>7.1. When the salinity and alkalinity exceeded 24 mm and ph 7.1, respectively, either increasing salinity or increasing ph sharply lowered the P l. The analysis of variance indicated that both the saline concentration and ph significantly affected the P l, respectively, but they were not significantly affected by interaction between salinity and alkalinity. Fresh weight: The fresh weight was not significantly decreased by slight stresses (salinity=24 mm, ph=7.1), but it was negatively correlated with the salinity and ph (Fig. ). Like in the control treatment, comparing the fresh weight of the three cultivars shows that PGI427>Stockpile>WL33 HQ when the ph was 7.1 or 9. Interestingly, the fresh weight of PGI427 and Stockpile were significantly higher than that of WL33 HQ when the ph was 8.3 or 1.7, but no significant difference was observed in PGI427 and Stockpile. The results of two-way ANOVA (Table 2) shows that the significant interaction effects of salinity and alkalinity on fresh weight was not significantly affected by interaction between salinity and alkalinity. Fig-3: Effects of salinity and ph on the germination index Gi. Responses of various measures of seeds to salinityalkalinity stresses: G p, P l and fresh weight (1 seedlings) decreased with increasing of salinity and ph (Fig. 6). When salinity 48 mm and ph 8.3, G p and G e were more than % (Fig. 6A, B). The seeds did not germinate under the condition of salinity 96 and ph 9. When salinity was 24 mm and ph was 7.1 or 8.3, P l was the longest (Fig. 6D), and P l and fresh weight were decreased with the increasing of salinity and ph (Fig. 6D, F). However, when salinity was 48 and 72 mm and the ph was 7.1 was the longest (Fig. 6E). Correlation analysis of parameters of seed germination: Correlations of parameters of alfalfa seed germination among salinity and alkalinity stress are listed in Table 3. The positive correlation is noted among G p (R=.93, P<.1). The analysis reveals that P l is positively correlated with R l (R=.97) and fresh weight (R=.91). However, the correlation coefficients between G p and P l was slightly low (.8<R<.9). Therefore, G p were identified as alfalfa seeds germination indices, and P l and biomass were seedling growth indices. Four salts (NaCl, Na 2, NaH and Na 2 ) were mixed in various proportions to simulate complex saline-alkaline conditions. Seed germination is one of the most critical periods in the life cycle of plants, which is often associated with high mortality rates in plants. Based on present experimental study, seed germination of alfalfa was relatively sensitive to salt and alkali stresses. G p decreased rapidly with increasing salinity and alkalinity

4 86 LEGUME RESEARCH An International Journal Table 2: Two-way ANOVA table presenting the effects of the salt concentration and ph on germination percentage, germination energy, germination index, radicallength, plumule length and biomass. *p<; **p<.1; NSp>. Gp Ge Gi Radical length Plumule length Fresh weight df F P F P F P F P F P F P Salinity <.1** <.1** 7.12 <.1** 1.19 <.1** <.1** <.1** ph <.1** 41. <.1** <.1** 64 <.1** 7 <.1** 12.9 <.1** Salinity ph <.1** 4.2 <.1** 6.93 <.1** 9 1 NS NS NS Fig-4: Effects of salinity and ph on the lengths of the plumules and radicals. Histogram above the abscissa represents the plumule length, and below is the radical length. Fig- : Effects of salinity and ph on the fresh weight. The lower case letters indicate differences at p< within the groups, while the capital letters indicate differences at p<.1 within the groups A 6 2 B 1 C D 1 E. F ph Fig- 6: Responses of various measures to salinity-alkalinity stresses. A to F represent the germination percentage, germination energy, germination index, plumule length, radical length and freshweight (1 seedlings). respectively.

5 Volume Issue (October 217) 87 Table 3: Correlation among salt toxicity coefficient of indices under complex salt and alkali conditions. **Correlation significant at.1 level of probability. G p G e G i Plumular length Radical length Fresh weight G p 1 G e.93** 1 G i.93**.96** 1 Plumular length.**.87**.87** 1 Radical length.**.8**.87**.97** 1 Fresh weight.86**.88**.89**.92**.91** 1 (Figs. 1, 2 and 3). It is generally considered that salt stress inhibits plant growth by water deficiency and ion toxicity among other factors (de Lacerda et al., 23). However, alfalfa maintained a relatively high G p at a low alkaline ph at any salt concentration, which indicates its salt tolerance and moderate alkalinity tolerance. At the same salinity concentration, G p markedly decreased with increases in the ph. High ph slows down water uptake by seeds and inhibits their germination and seedling growth (Campbell and Nishio, 2). Bell et al. (1993) previously indicated that alkalinity tolerance under increasing levels of alkalinity stress appear to be associated with the ability of the plant to maintain root membrane function, nutrient uptake balance and root tissue structure. Quick-germinating species should have advantage over the slow-germinating ones on saline soils in semi-arid regions (Sinha et al., 1982). Our results show that alfalfa has good potential to become established under conditions of salinity and low ph because of its superior G e. Therefore, alfalfa is relatively tolerant of salinity-alkalinity, which is in agreement with previous studies (Li et al., 21). Excessively high salinity with high alkalinity (ph 9, salinity 96 mm) led to ungerminated seeds (Fig. 1). Ungerminated seeds caused by salt stress or combined salinity-alkalinity stress may be in a state of dormancy to escape from the harsh environment (Gulzar et al., 21). Plant roots are the key structures in contact with environmental soils. The abiotic stresses in environments may primarily injure the roots. The results concerning plant P l and plant fresh weight indicated that at ph=7.1, these indices slowly decreased along the increase in salinity, while they sharply decreased when ph>7.1 (Fig. 4 and ). When the salinity and alkalinity exceeded 24 mm and ph 7.1, respectively, either the salinity or the increasing ph sharply lowered P l (Fig. 4). This implies that synergetic effects of salinity and alkalinity exhibited cooperative effects at higher salt and alkali levels. However, when salinity was 24 mm, the increase of ph from 7.1 to 8.3 did not decrease P l, and the P l were in fact longer than the control. This indicates that salt stress inhibits plant growth by water deficiency and ion toxicity among other factors (Ghoulam et al., 22), but in salinity-alkalinity tolerant species, plant growth is only moderately inhibited, or even stimulated, by low salinity-alkalinity stress (Cramer et al., 1986). The salinity and alkalinity stresses had synergetic effects on G p, G e but not P l and fresh weight. The reason may be the phases of seed germination and root elongation were caused by different limiting factors. The results confirm that G p and G e are important indices for assessing germinated energy of alfalfa in salinity-alkalinity soils. The alfalfa cultivars were relatively salt-alkali tolerant; what is more, we also found Stockpile was the most tolerant cultivar in our experiment. ACKNOWLEDGEMENT This research was supported by The Twelfth Five- Year National Science and Technology Support Program of China (211BAD8B2-3), The Major Project for the Heilongjiang Province Science and Technology Program (GZ13B4), The Project for the Heilongjiang Province People s Government (WB13B14) and The National Natural Science Fund (3296). RFFERENCES Abusuwar, A.O. and Daur, I. (2). Effect of seed pelleting with organic manures and rhizobia on the performance of the two alfalfa cultivars grown in saline environment. Legume Res. 38:1-6 Alam, S.M., Naqvi, S.S.M. and Ansari, R. (1999). Impact of soil ph on nutrient uptake by crop plants. Handbook of Plant and Crop Stress Bell, D.T., Wilkins, C.F., Moezel P.G.V.D. and Ward, S.C. (1993). Alkalinity tolerance of woody species used in bauxite waste rehabilitation, western Australia. Restor Ecol. 1:1-8 Campbell, S.A. and Nishio, J.N. (2). Iron deficiency studies of sugar beet using an improved sodium bicarbonate-buffered hydroponic growth system. J Plant Nutr. 23: Cramer, G.R., Läuchli, A. and Epstein, E. (1986). Effects of NaCl and CaCl 2 on ion activities in complex nutrient solutions and root growth of cotton. Plant Physiol. 81:

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