The effects of NaCl priming on salt tolerance in canola (Brassica napus L.) seedlings grown under saline conditions

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1 THE ROYAL SOCIETY OF CRO SCIENCE Indian J. Crop Science, 1(1-2): (2006) The effects of NaCl priming on salt tolerance in canola (Brassica napus L.) seedlings grown under saline conditions RSCS 2006 Rozbeh Farhoudi and Farzad Sharifzadeh 1 Effects of NaCl priming in canola Agronomy Department, Faculty of Agriculture, Islamic Azad University, Shoushtar Branch, Iran 1 Agronomy Department, Faculty of Agricultre,Tehran University, Karaj, Rozbeh Iran Farhoudi and Farzad Sharifzadeh Abstract Seeds of canola (Brassica napus) cultivars Hayola401 and Zarfam were primed with 14 ds m -1 NaCl solution for 24 hours at 20 C. After priming, non-primed (N) and primed () seeds were sown in germination boxes containing perlite. The germination boxes were placed in greenhouse and treated with five different NaCl solutions (0.4 (control), 4, 8, 12 and 16 ds m -1 ), for a period of 3 weeks. Total emergence and dry weight were higher in canola seedlings derived from seeds and they emerged earlier than N seeds. Moreover, seeds from N groups could tolerate up to 8 ds m -1 NaCl salinity, while the total emergence values of groups in Hayola401 and Zarfam did not decrease below 50% at 12 and 16 ds m -1, respectively. NaCl priming enhanced proline accumulation and prevented toxic and nutrient deficiency effects of salinity because less Na + but more K + and especially Ca 2+ was accumulated in canola seedlings. As a matter of fact, Na: Ca 2+ balances of seedlings derived from seeds were significantly lower than those of N seeds under similar salinity levels. Key words: Introduction Brassica napus, NaCl priming, Salt tolerance, Ion balance. The need to develop crops with higher salt tolerance has increased strongly, within the last decade, due to increased salinity problems. In general, plants do not develop salt tolerance unless they are grown in saline conditions. This means that they must be hardened to salt stress (Levitt, 1980). Salt tolerance of plants could be increased by treatment of seeds with NaCl solutions prior to sowing. Although priming (osmoconditioning) is one of the physiological methods, which improves seed performance and provides faster and synchronized germination (Sivritepe and Dourado, 1995), it has been shown that NaCl priming could be used as an adaptation method to improve salt tolerance of seeds. Wiebe and Muhyaddin (1987), Cano et al. (1991) and Cayuela et al. (1996) working with tomatoes, ill et al. (1991) working with asparagus and tomatoes, assam and Kakouriotis (1994) working with cucumber have concluded that seed priming improves seed germination, seedling emergence and growth under saline conditions. Nevertheless, beneficial effects of NaCl priming for later growth and development stages of plants remain unclear. assam and Kakouriotis (1994) reported that benefits of NaCl priming did not persist beyond the seedling stage in cucumber, while Cano et al. (1991) pointed out that NaCl priming had positive effects on mature plants and on yield of tomato due to cultivars and salinity level. Although NaCl priming is a useful technique to increase salt tolerance of plants, it is very important to understand the physiological effect of this technique on plants, which mediate their responses to salinity. However, physiological changes induced by NaCl priming have barely been studied in plants. Cayuela et al. (1996) have concluded that the higher salt tolerance of plants from primed () seeds seems to be the result of a higher capacity for osmotic adjustment since plants from seeds have more Na + and Cl - in roots and more sugars and organic acids in leaves than plants from non-primed (N) seeds. Canola is an important oil crop often cultivated in arid and semiarid regions of the world such as Iran where salinity threatens to become, or already is, a problem. The present study was conducted to examine the effect of NaCl priming on salt tolerance of canola at the seedling stage and to evaluate the physiological effects of priming. Materials and methods Seeds of canola cultivars Hyola401 and Zarfam were used. Canola seeds were primed with 14 ds m -1 NaCl solution for 24 hours at 20 C as described by Sivritepe et al. (1999). After priming seeds washed with tap water for 3 min and then rinsed with distilled water. Following this, seeds were dried between two filter papers and set to germinate. Non primed (N) and primed () seeds were sown in germination boxes filled with perlite. The germination boxes were placed in greenhouse where temperature ranged between 18 and 25 C for a period of 3 weeks. The boxes irrigated daily with five different Hoagland solutions by the use of NaCl. Electrical conductivities (EC) at 25 C of the five salinity levels were 0.4(control), 4, 8, 12 and 16 ds m -1, respectively. Surplus water drained naturally from the bottom of boxes to avoid build-up of salt in the growth media. The layout of the experiment was a Factorial complete block Design. There were four replicates in each

2 Effects of NaCl priming in canola [ 75 ] treatment group and 150 seeds in each replicate. In order to collect satisfactory amount of plant materials for chemical analyses in N groups of 16 ds m -1 salinity level, extra seedlings were grown along with the main experiment. Germination boxes were inspected daily and seedling emergence recorded as the appearance of the cotyledons. Total number of emerged seedlings in each replicate was determined and evaluated as percentage, in calculation of total emergence. Mean emergence time (MET) was calculated according to the equation of Ellis and Roberts (1981). At the end of 3 weeks, seedlings were harvested and evaluated for their response to salinity. In order to determine dry weight and ion concentrations of seedlings, plant materials dried for 48 h at 70 C. Na +, K + and Ca 2+ concentrations of seedlings were determined by the use of a flame-photometer following nitric perchloric acid digestion. roline contents of seedlings was determined according to Bates et al. (1973) and Dimler et al. (1952), respectively. lant material was extracted with 3% aqueous 5-sulfosalicylic acid for proline determination. The electrolyte leakage determined according to Sreenivasulu et al. (2000). The data were subjected to two-factor ANOVA and the means were compared by Duncan s tests at 0.05 confidence level by the use of MSTAT-C computer programs, respectively. Results and discussion In general, increased NaCl salinity decreased total emergence of seedlings derived from and N seeds in both cultivars (Table 1). However, total emergence percentages in seedlings of the groups were higher compared with those of the N ones. Regarding groups of canola cultivars, similar responses were observed in seedlings of 0.3, 4 and 8 ds m -1 salinity treatments. Significant decreases in total emergence of groups were obtained at 12 ds m -1, but at 8 ds m -1 salinity with the N groups. The most inhibiting salinity level for total emergence was 16 ds m-1. Nevertheless, in both cultivars, total emergence values were significantly higher in than in N groups. In both cultivars, MET of seedlings derived from N seeds significantly increased due to an increase in NaCl salinity (Table 1). However, NaCl priming had a positive effect on MET of seedlings of both cultivars. Seedlings from seeds emerged earlier than N seeds under saline conditions. Regarding groups of cv. Hyola401, MET values obtained from 0.4, 4 and Table 1. The effects of NaCl salinity on total emergence, MET and dry weight of canola seedlings derived from N and seeds. Cultivar 0.4 N NaCl riming Emergency(%) MET(day) Dry Weight(g) 4 N Hayola401 8 N 12 N 16 N 0.4 N 4 N Zarfam 8 N 12 N 16 N 94 a 92 a 76 b 95 a 42 c 72 b 7 d 51 c 96 a 9 96 a 77 b 95 a 48 c 71 b 9 d 52 c 5.1 a b 5.9 a 6.9 b 5.1 a 12.1 d 7.2 c 15.1 e 9.1 c 5.2 a 5.2 a 6.9 b 5.5 a 7.1 b 4.9 a 11.8 e 7.0 c 15.4 f 8.7 d a b c c d c d d d d a b c c d c d d d d

3 [ 76 ] Rozbeh Farhoudi and Farzad Sharifzadeh 8 ds m -1 treatments were not significantly different. However, the MET in groups of cv. Zarfam reduced while the salinity level increased up to 12 ds m-1. Dry weight of seedlings decreased due to an increase in NaCl salinity (EC) in both cultivars (Table 1). Under saline conditions, the seedlings of the group had a higher dry weight than the N group. At 0.4dS m -1, dry weight of the seedlings of the group in each cultivar was less than in the N group. roline content significantly increased with EC in and N groups of each cultivar (Table 2). However, the increase in proline contents was higher in the groups than in the N groups. Moreover, sharp increases in proline contents were observed above 8 ds m -1 in the groups and above 12 ds m -1 in the N groups. Electrolyte leakage (EL) significantly increase with EC in and N groups of each cultivars (Table 2) but the increase in EL was higher in the N groups than in the groups in both cultivars. Na + concentration significantly increased with an increase in salinity level in and N groups of each cultivar (Table 3). In general, the increase in Na + concentration was lower in groups relative to N groups. Increased NaCl salinity caused significant decreases in K + concentration of seedlings derived from and N seeds in both cultivars. However, seedlings of groups had significantly higher K + concentration than N groups at each salinity level. Similar to the general results obtained in K + concentration, increased NaCl salinity decreased Ca 2+ concentration of seedlings both in and N groups. However, seedlings of groups had significantly higher Ca 2+ concentration than N groups at each salinity level. Due to the effects of NaCl priming on ion accumulation of canola seedlings, the groups in both cultivars had lower Na + : Ca 2+ ratio than the N ones. However, NaCl priming did t cause significant changes in terms of K + : Na + ratio. In seedlings of each canola cultivar, the ratio of K + : Na + decreased due to an increase in NaCl salinity, both in and N groups, but the groups in both cultivars had higher K + : Na + ratio than the N ones (Table 3). It is evident from the results that NaCl salinity caused growth inhibition in canola seedlings due to an increase in MET and decreases in total emergence and dry weight. These effects of salinity on canola cultivars supported the previous findings of Botia et al. (1998) and ill et al. (1991) working with different plants such as melon and tomato. However, in the present study, it was concluded that NaCl priming diminished inhibiting effect of salinity on seed germination and seedling growth in canola, as has been shown in other priming treatments in Table 2. The effects zof NaCl salinity on rolin content and electrolyte leakage in canola seedlings derived from N and seeds. cultivar NaCl riming 0.4 N 4 N Hayola401 8 N 12 N 16 N 0.4 N 4 N Zarfam 8 N 12 N 16 N roline content (mg/ e 0.54 e 0.41 e 0.59 e 0.94 d 1.32 d 1.15 d 2.10 b 1.42 c 3.18 a 0.42 e 0.50 e 0.40 e 0.58 e 0.89 d 1.41 c 1.21 d 2.25 b 1.62 c 3.21 a EL (%) 2 a 4 a 5 a 8 b 12 c 15 c 23 d 18 c 34 e 4 b 5 b 9 b 14 c 14 c 25 d 17 c 35 e

4 Effects of NaCl priming in canola [ 77 ] cucumber and tomato (Weibe and Muhyaddin, 1987; Cano, 1991; ill 1991; assam and Kakouriob's 1994 and Cayuela et al. 1996). The total emergence and dry weight were higher in canola seedlings derived from seeds and they emerged earlier than N seeds. Moreover, seeds from N groups could tolerate 8 ds m -1 NaCl salinity, while the total emergence values of groups in cvs. Hayola401 and Zarfam did not decrease below 50% at 12 and 16 ds m -1, respectively (Table 1). These results suggest that in both canola cultivars, seedlings derived from seeds have higher adaptation capacity to salinity. Levitt (1980) states that all salt resistant plants must possess adaptation, originating from osmoregulation, by dehydration avoidance, which is the basis of their tolerance of the salt induced osmotic stress. Osmoregulation can occur in plants by active uptake of inorganic ions (such as Na +, K + and Cl - ) or synthesis of organic solutes (such as sugars, organic acids, free amino acids and proline) depend on species. The results of this study clearly showed that NaCl priming enhanced proline accumulation in canola seedlings. increase electrolyte leakage in canola seedling but this increase was lower in seeds than N seeds (Table 2). These results are supported by Cayuela et al. (1996) working with tomatoes. Therefore, the higher adaptation capacity of seedlings in groups to salinity could be due to osmoregulation induced by organic solutes and cell membranes stability. On the other hand, recent studies suggest that proline may play a role as an enzyme-stabilizing agent cell membranes stability under NaCl salinity (Demir and Kocacaliskan, 2001) and reduces peroxidative damage to the lipid membranes due to salt dependent oxidative stress (Jain et al., 2001). Salt induced injuries can occur not only due to osmotic and oxidative effects, but also toxic and nutrient deficiency effects of salinity. The results of the present study showed that NaCl treatments caused an increase in Na + concentration and a decrease in K + and Ca 2+ concentrations of canola seedlings derived from N seeds (Table 3). revious studies showed similar effects of salinity in melon (Botia et al, 1998 and Carvajal et al., 1998) as well as in celery (ardossi et al., 1999), eggplant (Chartzoulakis and Loupassaki, 1997), pepper (Chartzoulakis and Klapaki, 2000) and tomato (Alian et al, 2000 and Romero et al., 2001). In addition, accumulation of Na + ion changes ion balances such as Na + : Ca 2+ and K + : Na + in plant cells under saline conditions. While the change in Na + : Ca 2+ balance results in increased cell permeability, the change in K + : Na + balance causes to decreased use of Table 3. The effects of NaCl salinity on changes in Na +, K + and Ca 2+ concentrations and Na + :Ca 2+ and K + : Na + ratios of canola seedlings derived from N and seeds cultivar NaCl riming Na + K + Ca 2+ Na + :Ca 2+ K + : Na N 14 a 12 a 130 a 128 a a f f 9.28 a a 4 N 33 b 31 b 97 c 132 a 191 c 221 a 0.17d 0.14d 2.93 c 4.25 b Hayola401 8 N 48 d 39 c 91 c 112 b 169 d 193 c 0.28c 0.20c 1.85 d 2.87 c 12 N 59 e 54 e 75 d 89 c 141 f 165 d 0.41 b 0.32 c 1.27 f 1.64 e 16 N 69 f 53 e 45 e 74 d 139 f 112 e 0.45 b 0.47 b 0.65 h 1.39 e 0.4 N 1 14 a 128 a 132 a 231 a f 0.06 f 9.84 a 9.42 a 4 N 31 b 32 b 123 b 129 b 212 b 225 a 0.14 d 0.15 d 3.96 b 4.03 b Zarfam 8 N 45 d 37 c 119 b 81 c 171 d 214 b 0.26 c 0.17 d 2.64 c 2.18 d 12 N 71 f 56 e 71 c 79 c 143 f 114 e 0.49 e 0.49 b 1.02 g 1.46 e 16 N 77 f 54 e 40 e 62 d 98 g 113 e 0.78 a 0.47 b 0.52 h 1.14 f

5 [ 78 ] Rozbeh Farhoudi and Farzad Sharifzadeh metabolic energy (Levitt, 1980). Similarly, in canola seedlings from N seeds, Na + : Ca 2+ ratio increased while K + : Na + ratio decreased depending on increases in salinity levels (Table 3). The results regarding the K + : Na + ratio were in compatible with Cano et al. (1991), working with tomato. However, the results obtained from canola seedlings of groups showed that NaCl priming could be the cause of decreasing detrimental effects of salinity on ion metabolism. Therefore, NaCl priming induced avoidance of canola seedlings from toxic and nutrient deficiency effects of salinity on growth because of less Na + but more K + and especially Ca 2+ accumulation. Recently, numerous studies indicated that an increase in the concentration of Ca 2+ in plants challenged with salinity stress could ameliorate the inhibitory effects on growth (Navarro et al and Kaya et al. 2002). Furthermore, higher Ca 2+ accumulation capacity under saline conditions could provide the sustainability of Na + : Ca 2+ balance, which is responsible for the semi-permeability of cell membranes (Greenway and Munns, 1980). As a matter of fact, Na + : Ca 2+ balances of seedlings derived from the seeds were significantly lower than those of the N seeds under similar salinity levels. These results suggested that NaCl priming of canola seeds increased salt tolerance by promoting K + and Ca 2+ accumulation (Table 3), besides inducing osmoregulation by the accumulation of proline (Table 2). In conclusion, this study showed that NaCl priming of canola seeds could be used to increase salt tolerance of seedlings. Our studies are in progress to investigate whether the beneficial effects of NaCl priming persist beyond the later growth and development stages of canola. 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