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Annals of Biological Research, 2011, 2 (4) :490-497 (http://scholarsresearchlibrary.com/archive.html)
ISSN 0976-1233 CODEN (USA): ABRNBW

Effect of salt (NaCl) stress on germination and early seedling growth of Spinach (Spinacia oleracea L.)
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Mohammad Hosein Bijeh keshavarzi*, 2Mehrnaz S. Ohadi Rafsanjani, 1S. Mohsen. Moussavinik and 3Amir Parviz Lak
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Department of Agronomy, Zabol University, Iran Dept. Biotechnology, F/O- Science, Jamia Hamdard, New Delhi, India, India 3 M.Sc. Post graduated in industrial management, Islamic Azad University, Tabriz Branch ______________________________________________________________________________ ABSTRACT Salinity is one of the major environmental factors that lead to a deterioration of agricultural land and reduction in crop productivity worldwide. This research was carried out in order to test the effects of different salinity levels on germination and early growth of Spinach seedlings. The experiment was carried out using completely randomized design in four replication in 2011 at Hamdard University laboratory in India. Experimental treatment includes 4 levels of NaCl concentration (o, 50, 100 and 150 mM). Result showed that the percentage and speed of germination, plumule length, radicle length and heaviest wet and dry seedling weights were higher in control treatment. At 150 mM and more concentration, germination decreased significantly. This reduction in germination indicates this plants extreme insensitivity to salinity, so it isnt advisable to cultivate it in saline soil. All the result data analyzed by SAS software and comparison of means had been done with Duncan test in 0.05% probable level. Key words: germination, NaCl, salinity stress, seedling, Spinacia oleracea L. ______________________________________________________________________________ INTRODUCTION More than 900 million hectares of land world-wide, approx. 20 % of the total agricultural land, are affected by salt, accounting for more than 6% of the worlds total land area. NaCl is the predominant salt causing salinization, and it is unsurprising that plants have evolved mechanisms to regulate its accumulation [1].

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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ Seed germination is an important and vulnerable stage in the life cycle of terrestrial angiosperms and determines seedling establishment and plant growth. Despite the importance of seed germination under salt stress [2], the mechanism (s) of salt tolerance in seeds is relatively poorly understood, especially when compared with the amount of information currently available about salt tolerance physiology and biochemistry in vegetative plants [3, 4, 5, 6]. In vegetative plants, salt stress causes reduced cell turgor and depressed rates of root and leaf elongation [7, 8], suggesting that environmental salinity acts primarily on water uptake. Furthermore, high intracellular concentrations of both Na+ and Cl- can inhibit the metabolism of dividing and expanding cells [9], retarding germination and even leading to seed death. The different results were dedicated from the effect of salinity stress on the quantitative and qualitative parameters. For instance, it was found that increasing of salinity stress decreased almost all of growth parameters in Nigella sativa some growth parameters and essential oil amount in chamomile [10]. Also Younis et al. [11] reported that enhancing salinity treatments lead to growth reduction. It also reduces germination amounts and seedling weight. Ashraf and Orooj [12] reported that salinity treatment lead to reduction of growth and plant developments. Overall, salinity through enhancement of osmotic pressure leads reduction of water absorbance and disturbance in metabolic and physiological processes will be under its effect. So it cause more delay in germination following by enhancing seed germination duration [13]. Spinach (Spinacia oleracea L.) is a vegetable and belongs to the family Amaranthaceae. It is native to central and southwestern Asia. Spinach has a high nutritional value and is extremely rich in antioxidants, especially when fresh, steamed, or quickly boiled. It is a rich source of vitamin A (and especially high in lutein), vitamin C, vitamin E, vitamin K, magnesium, manganese, folate, betaine, iron, vitamin B2, calcium, potassium, vitamin B6, folic acid, copper, protein, phosphorus, zinc, niacin, selenium and omega-3 fatty acids. Recently, opioid peptides called rubiscolins have also been found in spinach. Polyglutamyl folate (Vitamin B9 or folic acid) is a vital constituent of cells and spinach is a good source of folic acid, but boiling spinach can more than half the level of folate left in the spinach, though microwaving does not affect folate content [14]. MATERIALS AND METHODS The experiment was carried out using completely randomized design in four replication and 4 salinity levels (0, 50, 100 and 150 mM) in 2011 at Hamdard University laboratory in India. Each experimental unit includes 1 Petri dish with 100 150 mm dimension each contains 15 healthy and homogenous seeds which were put on the No1 Watman filter paper. First of all, to disinfect seeds, we put them in 10% Hypochlorite Sodium solvent then we washed them 3 times by distilled water. Next, we added 6 ml NaCl solvent to each Petri dish in this way filter water was weltered by NaCl completely. Eventually, their lids were closed by parafilm and had been located in growth room. The temperature adjusted in 25o C. This experiment took 7 days. The following characteristics were studied: Germination Percentage (GP): From second day, the germinated seeds were counted daily in specific time. At that time, those seeds were considered germinated which their radical length was more than 3 mm. Counting continued till we could count more germinated seeds and the resulted final counting considered as final germination percentage.
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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ GP: Ni / N 100 Ni: number of germinated seed till ith day N= total number of seeds. Germination Race (GR): In order that, from the second day to 7th once in 24 hours we counted germinated seeds and its race was determined by Maguire equation [15]:

GR: Germination Race (number of germinated seed in each day) Si: number of germinated seeds in each numeration Di: number of days till nth numeration. n: number of numeration times. Seed vigor (SV): This index was determined by the following formula and with the help of Abdul-baki and Anderson [16] method: Strong seed index = {germination percentage means of seedling length (radicle + plumule) mm} / 100 At the end of experiment, we had selected 10 plants from each Petri dish, separated their radicle and plumule and measure each plats radicle and plumule length separately. Then we put each repetition on the filter separately. In order to make them dry and measure its dry weight, we put them in oven with 75oC temperature for 24 hours. Thereafter, we weighed them and data was analyzed using SAS and Excel software. RESULTS AND DISCUSSION
Table 1: result of variance analysis on 7 seed germination and growth of seedling characteristics under NaCl concentration Mean Square S.O.V df GP (%) GR RL (cm) PL (cm) Seed vigor WW (g) DW (g) Replication 3 30.73ns 0.64ns 0.055ns 0.058ns 0.18ns 0.0003ns 0.000091ns Treatment 3 2634.9** 48.92** 13.17** 4.15** 34.2** 0.086** 0.0045** Error 12 27.6 0.45 0.07 0.048 0.14 0.00026 0.000057 C.V. (%) 8.37 8.86 8.13 11.74 9.71% 6.25 14.85 Note: *and ** indicate significant difference at 5% and 1% probability level, respectively ns is not significant. GR: Germination rate, GP: Germination percentage, PL: Plumule length, RL: Radicle length, SV: Seed vigor, WW: Wet weight, DW: Dry weight. Table 2: Effect of different NaCl concentration on seed germination and growth of seedling characteristics NaCl concentration (mM) GP (%) GR RL (cm) PL (cm) SV WW (g) DW (g) 0 86.25a 10.32a 5.52a 3.15a 7.47a 0.41a 0.086a 50 72.5b 9.31a 3.72b 2b 4.14b 0.308b 0.057b 100 61.25c 6.04b 3.1c 1.35c 2.72c 0.183c 0.03c 150 26.25d 2.6c 1.12d 0.77d 0.5d 0.07d 0.0087d Note: Similar letters in each column hadnt any significant statistical difference.; GR: Germination rate, GP: Germination percentage, PL: Plumule length, RL: Radicle length, SV: Seed vigor, WW: Wet weight, DW: Dry weight.

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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ Results of statistical analysis of experimental data have been given in table 1 and results of comparison between considered characteristics means have been written in table 2. As table 1 show, salinity made significant differences on all considered characteristics. Germination percentage and race: According to results of variance analysis, effect of salinity stress level on germination percentage and race were significant (P < 0.01) (Table 1). Comparison between means of different level of salinitys effects on germination race and percentage has been showed in table 2. As you see in salinity stress, the most germination percentage was high (86.25%) and reduced at 150mM NaCl concentration. (26.25%) (Figure1).
Figure 1: Effect of different levels of NaCl on germination percentage of Spinacia oleracea L.

The highest germination race was related to control also with (10.32), while lowest with 150 mM with (2.6) (Figure 2). Its cause could be more than usual presence of anion, cation which in addition to toxication, decreased water potential that is because of its solubability in water. Ions so plant cant absorb water and encount to lake of water [17]. We also can say that this reduction in germination race relies on salinity could be because of its bad effect on physiological processes which are effective on seed germination [18].
Figure 2: Effect of different levels of NaCl on germination rate of Spinacia oleracea L.

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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ Radicle and plumule length: The effects of salinity stress on radicle and plumule length have been showed in table 2 Results a significant difference in radicle and plumule length in 0.05% probable level (Table 1). Comparison of radicle and plumule length means in salinity different level (0, 50, 100 and 150 mM) showed that when salinity level increase, seedlings radicle and plumule length decrease. The most reduction in radicle length (Figure 3) and plumule (Figure 4) related to 150 mM. In this relation Munns and Termaat [19] suggested that salinity decrease radicle and plumule growth and if we increase salinity level, the amount of reduction will increase. Also Salinity, declines plumule and radicle growth, and by increasing salinity these reduction increase. Salinity which is result of osmotic pressure leads reduction in water absorbance so cell division and differentiation reduce and reduction of plumule and radicle length will be Explainable.
Figure 3: Effect of different levels of NaCl on radicle length of Spinacia oleracea L.

Salinity causes shorter plumule, which is more in case of NaCl more than other salinity factors gas deterrent impact on embryo tissues appearance [20].
Figure 4: Effect of different levels of NaCl on plumule length of Spinacia oleracea L.

In addition, Hajar et al. [21] by studding Nigella sativa L. different salinity treatment till 300 mM NaCl. They conclude that, in Nigella sativa L. root growth will decrease if salinity increase till 150 mM NaCl. Some studies showed that germinated seeds in salinity environments have short
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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ root and shoot and NaCl, has on extreme deterrence effect on embryo development rather than other salinity factors [22, 23]. Seed vigor: In strong seed vigor index, had been observed that there exists a significant difference (P 0.01) between different salinity levels (table 1). By increasing NaCl concentration, seed vigor index declines (Table 2, Figure 5). The most seed vigor index was related to control treatment (7.47) and the least at was related to 150 mM (0.5) (Figure 5). Generally, race and percentage of germination and seed vigor index is related to special impact of ions and reduction of environmental water potential in the presence of salinity. Result showed that if salinity increases (reduction of environmental osmotic potential), seed characteristics will decrease these results are in accordance with the founding of Kader and Jutzi [24].
Figure 5: Effect of different levels of NaCl on seed vigor of Spinacia oleracea L.

Figure 6: Effect of different levels of NaCl on dry and wet weight of Spinacia oleracea L.

Wet and dry weight: Impact of salinity stress treatments, on dry and wet weight of Spinacia oleracea L. seedling was significant (P 0.01) (Table 1). Impact of salinity stress on dry and wet weight had been showed in Table 2. As you see by enhancing salinity levels, seedlings wet weight amounts decrease extremely. In this case in 150 mM we have 0.07 gr also in other treatments (100, 50 and 0 mM),
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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ dry weights were 0.183, 0.308 and 0.41 in orderly. (Table 2, Figure 6). In addition, dry weight of seedling have similar results which when we increase salinity level till 150 mM dry weight decreases, means that it decrease from 0.086 gr to 0.0087 gr. (Table 2, Figure 6). Etesami and Galeshi [25] reported that salinity is the cause of reduction in germination percentage, race and homogeneity of germination and dry weight of barley (Hordeum vulgare) seedling. Massai et al. [26] say that salinity is delaying plant growth under reduction of photosynthesis effects, it is cause of closing stomata and reduction of water entrance into the plant and so that it cause duplicate reduction in plant weight. Redman et al. [27] showed that this reduction in dry weight of plumule and radicle which is results of enhancing the salinity concentration is a normal phenomenon and probably it is the result of low water absorbance by germinating seeds. ACKNOWLEDGMENT Authors are thankful to M.Z. Abdin, Head, CTPD, Dept. of Biotechnology, Jamia Hamdard University, New Delhi, India for providing laboratory facilities REFERENCES [1] R. Munns, M. Tester. Annual Review of Plant Biology., 2008, 59, 651-681. [2] IA. Ungar. In Seed germination and seed-bank ecology of halophytes Marcel,, Dekker, New York, 1995; pp. 599-627. [3] MW. Hester, IA. Mendelssohn, KL. McKee. Environmental and Experimental Botany., 2001, 46, 277-297. [4] V. Hu, H. Lu, QL. Liu, XM. Chen, XN. Jiang. Tree Physiology., 2005, 25, 1273-1281. [5] AJ. Garthwaite, R. von Bothmer, TD. Colmer. Journal of Experimental Botany., 2005, 56, 2365-2378. [6] M. Kanai, K. Higuchi, T. Hagihara, T. Konishi, T. Ishii, N. Fujita, Y. Nakamura, Y. Maeda, M. Yoshiba, T. Tadano. New Phytologist., 2007, 176, 572-580. [7] IE. Werner, RR. Finkelstein. Physiologia Plantarum., 1995, 93, 659-666. [8] W. Fricke, G. Akhiyarova, WX. Wei, E. Alexandersson, A. Miller, PO. Kjellbom, A. Richardson, T. Wojciechowski, L. Schreiber, D. Veselov, G. Kudoyarova, V. Volkov. Journal of Experimental Botany., 2006, 57, 1079-1095. [9] P. Neumann. Plant Cell and Environment., 1997, 20, 1193-1198. [10] K. Razmjoo, P. Heydarizadeh, MR. Sabzalian. Int. J. Agri. Biol., 2008, 10, 451-454. [11] ME. Younis, MNA. Hasaneen, AR. Ahmed, DMA. El-Bialy. Australian Journal of Crop Science., 2008, 2(2), 83-95.. [12] M. Ashraf, A. Orooj. Journal of Arid Environments., 2006, 64, 209-220. [13] HM. Kang, ME. Saltveit. Physiol. Plantarum., 2002, 115, 571-576. [14] GFM. Ball. In Vitamins in foods: analysis, bioavailability, and stability., CRC Press, 2006; pp. 236. [15] ID. Maguire. Crop Sci., 1962, 2, 176-177. [16] AA. Abdul-baki, JD. Anderson. Crop Science., 1970, 10, 31-34. [17] KN. Singah, DK. Sharma, RK. Chillar. J. Agric. Sci. Camb., 1988, I11, 459-463. [18] MA. Khan, B. Gul, DJ. Weber. Biological Plant., 2002, 45, 133-135. [19] R. Munns, A. Termaat. Aus. J. Plant Physiol., 1986, 13, 143-160. [20] MA. Khan, IA. Ungar. Seed Science and Technology., 1997, 25, 83-91..
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M. H. Bijeh keshavarzi et al Annals of Biological Research, 2011, 2 (4):490-497 _____________________________________________________________________________ [21] AS. Hajar, MA. Zidan, HS. Al-Zahrani. Persian Gulf. J. Sci. Res., 1996, 14, 445-454. [22] N. Katergi, JW. Van Hoorn, A. Hamdy, F. Karam, M. Mastrortilli. Agricultural Water Management., 1994, 26, 81-91. [23] MA. Khan, IA. Ungar. Physiology Plantarum., 1985, 63, 109-113. [24] MA. Kader, SC. Jutzi. J. Agron. Crop Sci., 2004, 190, 35-38. [25] M. Etesami, S. Galeshi. Journal of agriculture science and natural resource., 2008, 15, 5. [26] R. Massai, D. Remorin, H. Tattini. Plant and Soil., 2004, 259, 153-162. [27] RE. Redmann, M. Belyk. Can. J. Plant Sci., 1994, 74(4), 797-799.

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