Effects of Different Levels of Salinity on Germination, Proline Contents ...

International Journal of Agriculture and Crop Sciences. Available online at www.ijagcs.com IJACS/2012/4-15/1055-1059 ISSN 2227-670X ©2012 IJACS Journal

Effects of Different Levels of Salinity on Germination, Proline Contents and A-, B- Chlorophylls in Rapessed (Brassica napus L.) Mehdi Hooshi Alavi1, Gholam Ali Ranjbar2 1. MSC of Plant Breeding, Sari Agricultural Sciences and Natural Resources University, Sari, Iran 2. Associate Professor, Sari Agricultural Sciences and Natural Resources University, Sari, Iran

Corresponding author email: [email protected],[email protected] ABSTACT: Soil salinity is a major limitation for crop production in many areas of the world. Germination and seedling responses of nine rapeseed cultivars (Hayola 401, Zarfam, Sarigol, Rgs003, L3006, Lph9, Lph12, Lph22 and Lph23) to different levels of salinity stress (0, 100, 200 and 300 mM) evaluated in a RCBD base on factorial experiment with three replicates in glasshouse. Results showed that the germination of rapeseed cultivars were significantly affected by salinity. Cultivars Sarigol and Lph23 exhibited the highest and the lowest germination under salinity stress conditions. Amount of total chlorophyll, chlorophyll a and b were significantly reduced by increasing salinity. The results showed that increasing salinity levels significantly reduced chlorophyll a and b. Sarigol showed the most decrease in the amount of total chlorophyll. Leaf and root proline content of cultivars have been significantly increased by increasing the NaCl level, so that, the most increasing was happened using 300 mM NaCl treatments. Keywords: Rapeseed, salinity stress, germination, chlorophyll and praline INTRODUCTION Salinity is one of the major abiotic stresses in arid and semi-arid regions that substantially reduce the average yield of major crops by more than 50% (Bray, 2000). Salinity affects 7% of the world's land area for around 930 million ha (Munns, 2006). Every year more and more lands become non-productive due to salt accumulation. Therefore, understanding the mechanisms of plant tolerance to salinity stress is important (Bartels et al., 2005). Although salt stress affects all growth stages of a plant, seed germination and seedling growth stages are known to be more sensitive in most plant species (Cuartero, 2006). Furthermore, germination and seedling stage is predictive of plant growth responses to salinity (Blum, 1985). Therefore, seeds with more rapid germination under salt stress and/or normal conditions may be expected to achieve a rapid seedling establishment and more salt tolerance, resulting in good stand establishment and hence higher yields (Munns, 2002). Successful seedling establishment depends on the frequency and the amount of precipitation as well as on the ability of the seed species to germinate and grow while soil moisture and osmotic potentials decrease (Roundy, 1985). Moreover, salt stress has also been found responsible for an increased respiration rate, ion toxicity (Sudhir et al., 2004), decreased biosynthesis of chlorophyll (Khan et al., 2009) and inefficiency of photosynthesis (Munns, 2002), all of which ultimately leading to lowered economic productivity. One of the effects of salinity stress is reducing photosynthetic activity which caused to decreasing b and a chlorophylls and reducing Co2 uptake and photosynthetic capacity (Francois et al., 1993). Proline is used as an enzymic protector that contributes in macromolecules structure and is main source of energy and nitrogen to confront salinity. According to studies, Iqbal et al (2006) reported that chlorophyll is reduced under saline conditions. Nazarbeygi et al (2011) reported that with increasing salinity, reduction in the rate of chlorophyll and increase in proline in various rapeseed cultivars were significantly different. Rapeseed 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. Present study was conducted to consider effects of salinity on seed germination, seedling growth, Proline and a-, b- Chlorophylls of nine rapeseed genotypes cultured under salinity conditions.

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MATERIALS AND METHODS Germination experiment Nine rapeseed cultivars (Brassica napus L. c.v's Hayola 401, Zarfam, Sarigol, Rgs003, L3006, Lph9, Lph12, Lph22 and Lph23) were collected from Agricultural Research Center of Sari, Iran. The same size seeds from each cultivar were surface-sterilized for 5 min in sodium hypochloride solution (10%) and then rinsed with distilled water for 3 to 5 times. After sterilization, 25 seeds were transferred into 9 cm sterile petri dishes on filter paper and then were wetted with 7 ml distilled water (control) or saline water solution at 0, 100, 200 and 300 mM NaCl levels. To prevent infection and evaporation of solution, all plates were sealed using parafilm and culture operations were performed under a laminar air flow cabinet in sterile conditions. The Petri dishes were labeled and incubated in a germinator at 25 °C and 12/12 h day/night photoperiod. Measurement of germinated seed was done daily until end of seventh day. At the end of germination, root length were tested and shoot and root and shoot fresh weight were measured. The germination percentage (GP) and final germination percentage (FGP) were calculated for all cultivars as follow:

Where, n: Number of germinated seed at seventh day; N: number of seeds

Where, n: Number of germinated seed at lightly day; t: days after beginning Glasshouse experiment Ten seeds of each cultivar were sown directly in plastic pots containing 4 kg of field soil. The bottom of each pot was delved for drainage of extra water. Pots were transferred to glasshouse under conditions of 25/18 °C day/night temperature and natural light. After full germination, the number of plants was reduced to three seedlings per pot. Salinity stress induction was done when fourth leaf was completely expanded. Different concentrations of NaCl solution was added to each pot for 15 days. In order to assess leaf chlorophyll, leaf samples were harvested and dried for 48 hours at a temperature of 75 °C. Hiscox method (1979) was used to evaluate the amount of chlorophyll and Lichtenthaler equation (1987) was exploited to calculate them numerically. For measuring Proline content, Bates metod (1973).was used. The experiment was carried out using a factorial experiment based on randomized block design with three replications. Analysis of variance (ANOVA) was carried out using general linear model (GLM) in SAS (6.12 version) statistical software. Also, for comparison of means Duncan's Multiple Range Test (DMRT) was used. RESULTS AND DISCUSSION The results of analysis of variance related to the percentage, germination speed, root length and shoot lenght showed that these traits were influenced by genotype (Table 1). The interaction of salinity cultivar on germination rate and germination speed significant and root length and shoot lenght non-significant. These figures indicate that the effect of different concentrations of salinity in terms of response rate and percentage germination were different. Mean comparisons of different canola genotypes showed that Varieties Sarigol and Lph23 highest and lowest germination under salt stress conditions, respectively. Fast decreasing of germination of genotypes Lph22and Lph12 by increasing salinity showed sensitivity of these genotypes to salinity. (Table 2). These results are in agreement with research of Demir (2003). Increasing salinity levels, reduced germination rate and reduced final germination percentage was followed (fig 1 and 2). Results showed the negative effects of salinity on root and shoot length (fig 3 and 4). In addition, according to the results of Werner and Finkelstein (1995) salinity decreases water absorption and growth of root and shoot. The results of analysis of variance related to prolin, clorophyll a and b showed that Interaction of cultivar × salinity on proline, chlorophyll a and b in different amounts of rapeseed cultivars and salinity levels of chlorophyll a, chlorophyll b, chlorophyll a to b ratio and total chlorophyll was significant (Table 3). Results showed that amount of total chlorophyll, chlorophyll a and b significantly reduced by increasing salinity levels (Table 4). Cultivar Sarigol showed the highest decrease in total chlorophyll than the other genotypes. The decrease in chlorophyll contents

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under saline conditions was reported by Ashraf et al (2005). Also, results indicated that proline content of leaf and root has significantly increased due to increasing in NaCl rate. So that, treatment 300 mM NaCl demonstrated more proline content than the other treatments (fig 5). Valia et al (1993) suggested that increasing of proline content under saline conditions caused less existing glutamate in biosynthesizing chlorophyll, thus, it helped to producing proline for plant bearing more salinity conditions. Table 1. Analysis of variance on germination traits under salinity stress S. O. V

DF

MS Germination speed

Repeat Salinity Cultivar Cultivar Salinity Eror

2 3 8 24 69

2.79 ** 26.31 ** 6.10 ** 0.72 0.31

Germination percentage

**

**

Shoot length (cm) **

2836.93 ** 19962.70 ** 3860.13 * 555.68 377.17

Root length (cm) **

0.7 ** 14.15 ** 0.43

ns

9.57 ** 140.50 ** 2.71

1.38 0.06

ns

1.46 1.02

*, ** significant at the 0.05 and 0.01 probability levels, respectively. ns, not significan. Table 2. Comparison of main effect of cultivars on germination traits Cultivar

Germination speed

Hayola401 Lph9 Lph12 Lph22 LpH23 RGS003 L3066 Zarfam Sarigol

ab

Mean Germination percentage

Shoot length (cm)

a

2.66 b 2.29 c 1.64 c 1.68 d 0.63 ab 2.53 ab 2.68 b 2.24 a 2.89

Root length (cm)

a

76.00 ab 66.00 bc 52.67 c 49.33 d 21.08 a 72.67 a 73.00 abc 63.67 a 76.42

abc

1.22 ab 1.00 b 0.98 ab 1.00 c 0.53 b 0.98 ab 1.04 ab 1.04 ab 1.10

2.34 c 1.72 a 3.02 abc 2.26 bc 1.75 a 2.98 abc 2.64 abc 2.65 ab 2.67

For a given means within each colum followed by the same letter are not significantly differences (P < 0.01). Table3. Analysis of variance on prolin, chlorophyll a and b under salinity stress S. O. V

DF

Repeat

2

Salinity Cultivar Cultivar Salinity Eror

8 3 24 69

MS Prolin 128.13

Chla **

2413.32 ** 403.88 ** 52.75 2.32

2.27 **

Chlb

ns

12.65 **

698.54 ** 107.25 ** 12.28 2.57

Chla+Chlb *

4.22 **

797.35 ** 125.58 ** 23.41 4.09

ns

2960.44 ** 406.38 ** 46.84 5.38

Chla/Chlb 1.13

**

** **

6.70 ** 2.38 ** 0.88 0.2

*, ** significant at the 0.05 and 0.01 probability levels,respectively. ns, not significan.

Table 4 . Mean comparisons of chlorophyll content of rapeseed treated with NaCl NaCl treatment (mM) 0 100 200 300

Mean Chla

Chlb a

22.31 b 19.57 c 14.45 d 10.86

Chla+Chlb a

17.28 b 11.91 c 8.10 d 4.47

a

39.59 b 31.48 c 22.55 d 15.33

Chla/Chlb c

1.47 b 1.81 b 2.02 a 2.67

For a given means within each colum followed by the same letter are not significantly differences (P < 0.01).

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Figure 1 . Effects of different salinity levels on germination germination rate of rapeseed cultivars.

Figure 2 . Effects of different salinity levels on germination percentage of rapeseed cultivars.

Figure 3 . Effects of different salinity levels on radicle length of rapeseed cultivars. cultivars

Figure 4 . Effect of different salinity levels on plumule length of rapeseed cultivars. cultivars

Intl J Agri Crop Sci. Sci Vol., 4 (15), ( 1055-1059 1059, 2012

Figure 5 . Proline content changes in rapeseed cultivars under different NaCl concentrations. concentrations

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