Plant Physiology and Biochemistry 51 (2012) 139e144

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Plant Physiology and Biochemistry

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Research article

Pulsed magnetic eld: A contemporary approach offers to enhance plant growth and yield of soybean
Ramalingam Radhakrishnan*, Bollipo Diana Ranjitha Kumari
Stress Physiology and Plant Biotechnology Unit, Department of Plant Science, School of Life Science, Bharathidasan University, Tiruchirappalli 620024, Tamil Nadu, India

a r t i c l e i n f o
Article history: Received 14 September 2011 Accepted 26 October 2011 Available online 7 November 2011 Keywords: Enzymes Protein prole Pulsed magnetic eld Seedlings Yield

a b s t r a c t
The possible involvement of pulsed magnetic eld (PMF) pretreatment in development and yield of soybean was investigated. Seeds were subjected to 20 days with 1500 nT at 10.0 Hz of PMF for 5 h per day. PMF pretreatment increased the plant height, fresh and dry weight, and protein content with the changes of protein prole in 8 days old seedlings. In addition, activity of enzymes such as b-amylase, acid phosphatase, polyphenol oxidase and catalase was enhanced while a-amylase, alkaline phosphatase, protease and nitrate reductase activities declined due to PMF exposure. However, a considerable increment of Fe, Cu, Mn, Zn, Mg, K and Na contents with reduced level of Ca was found in PMF treated seedlings. The number of leaves, pods, seeds and length of pods, and weight of seeds were also remarkably higher in PMF treatment in contrast to controls. The results suggest that pretreatment of PMF plays important roles in improvement of crop productivity of soybean through the enhancement of protein, mineral accumulation and enzyme activities which leads to increase the growth and yield. 2011 Elsevier Masson SAS. All rights reserved.

1. Introduction Agricultural productivity is minimized by the number of factors such as soil salinity, droughts, soil erosion and wide spread of disease. Soybean is one of the important commercial crops in many countries. It is rich in nutrient content such as 40% of protein, 18e22% of oil, 35% of carbohydrates, minerals and isoavonoids such as genistein and daidzein are useful to prevent heart disease, cancer, diabetes and hypertension. The yield of soybean is affected by both biotic and abiotic factors. In recent years, the farmers expect alternative fertilizers against chemical fertilizers, due to the increase of cost and harmful effects of chemical fertilizers. Usage of MF to improve the crop productivity is a recent method and it gives better plant growth and yield than chemical fertilizers. In addition, it is ecologically friendly and nonpolluting to the soil base, specifically the consumption of electric power levels is very low which can be no hazard to environment. This PMF technology when it becomes fully developed for commercial use, it should offer the attraction of being affordable to farmers [1]. Pre-sowing seed treatment of MF can be used in practical agriculture to improve the plant growth and yield [2,3]. Since, MF is one of the natural components in the earth. Plants and other living things are interacted with magnetic eld in day to
* Corresponding author. E-mail address: ramradhakrish@gmail.com (R. Radhakrishnan). 0981-9428/$ e see front matter 2011 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.plaphy.2011.10.017

day life. Generally, earth acts as magnet with their south and north poles and the natural effects of magnetic eld have been changing the plant growth and yield in the globe. Specically, the electromagnetic spectrum of sunlight stimulate the growth of plants by the process of photosynthesis. The possible mechanism would be a change in the electrostatic balance of the plant system at the cell membrane level, as it is the primary site for action of any inhibition or enhancement of plant growth. Certain mechanisms for action of extremely low frequency electromagnetic elds on biological systems have been reported in earlier [4]. Galland and Pazur [5] described the effects of magnetic eld by radical pair mechanism and ion cyclotron resonance mechanisms. In addition, geomagnetic eld strength could affect variety of enzymes in biological organisms. The Ca2/calmodulin dependent cyclic nucleotide phosphodiesterase [6] and cytochrome C oxidase [7] changed due to the effect of MF. Electric or magnetic treatments enhanced seed vigour by inuencing the biochemical processes, which stimulate the activity of proteins and enzymes [2,8] and then some studies reported that MF had a positive effect on the number of owers and yield [9], nutrient [10,11] and water uptake [12]. Some reports are available that higher intensity of magnetic eld is useful to increase the seed germination and plant growth [2,3]. Our present research work in magnetic eld induced soybean plant development differed from previous research work of Shine et al. [3] by the application of pulsed magnetic eld with low intensity (1500 nT) and frequency (10.0 H) for 5 h per day upto 20

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days. However, very limited studies have been conducted in biology to describe the role of low frequency and intensity of magnetic eld. Therefore, there is a need to elucidate the effect of PMF on changes of seed germination, seedling growth and yield in crop plant. To date, very few reports have explored the interaction of MF with protein, enzyme activity and uptake of minerals in plants. The aim of the present investigation is an attempt to evaluate the ability of PMF on soybean seed germination, plant growth and yield, and the positive effects of PMF are conrmed by the analysis of protein, activities of enzymes and uptake of minerals.

2. Materials and methods 2.1. Seed pretreatment with PMF Seeds of soybean (Glycine max (L.)) cv. CO-3 were obtained from Tamil Nadu Agricultural University, Coimbatore, India. The healthy uniform dry seeds (8.6% of moisture content) were selected and each 100 g seeds kept at the geometric centre of coil assemblies were subjected to 10.0 Hz at 1500 nT as per the method of Leelapriya et al. [1]. Control seeds were kept under similar condition (local geomagnetic eld only) but in the absence of pulsed magnetic eld. Exposure was 20 days for 5 h per day and control seeds were kept under the similar condition in the absence of the PMF. The magnetic treatment of seeds was applied using an electromagnet highly improved version of the classical Helmholtz coil. This consisted of four-coil system that are made up of two larger (inner) coil frames and two smaller (outer) coil frames, where instead of the classical two identical coils there are now two pairs of coils, each pair having a different diameter (Fig. 1). The four coils have the same number of turns and connected in series-aiding conguration. The four coils are mounted co-planar and co-axial. The ratios between the radii of inner and outer coil frames and

the spacing between them are pre-determined according to the parametrical equations of Fanselau [13] and Brauenbeck [14]. The coil assemblies designed and fabricated at Madras Institute of Magnetobiology (Chennai, Tamil Nadu, India) have been carefully calibrated using high precision magnetometers (the size of the magnetometer is approximately 3.0 cm) and current measuring devices in the Magnetic Standardization Lab of the Institute. The high precision measurements involved calibration of the coils for arriving at the coil constant expressed in nano teslas (nT) per milli ampere (mA) and the generated magnetic eld (a few hundred nano teslas) determined correct to the order of 1e2 nT. The magnetometer used for these measurements is a magnetic observatory eld standard called the Zero-Balance Magnetometer manufactured by the Danish Meteorological Institute, Copenhagen, Denmark. The magnetometer has provision for determining the correction for temperature variation so that the nal eld measurements are free of any contribution arising from any temperature changes. Pulsed electric current from a signal generator (Fig. 2) energizes the coil system such that the strength, frequency and waveform of the output current can be controlled to any set of desired values, thus offering along the axis of the coil system a magnetic eld of any desired frequency, intensity and waveform. 2.2. Seed germination and seedling development PMF pretreated and control seeds were surface sterilized with 0.1% mercuric chloride solution for 5 min, washed thoroughly 5 times with distilled water and then propagated in clay pots containing air dried clay and sand mixture (3:1). The pots were maintained under natural photoperiod with 35% (w/w) soil moisture content. Seed germination and viability was observed at 4 month intervals upto 2 years, and germinated seedlings were uprooted and measured the length, fresh and dry weight of 8 days old both control and treated seedlings. 2.3. Protein contents and SDS PAGE Seedlings were homogenized with 50 mM TriseHCl buffer pH 7.2, 5% sucrose and 14 mM mercaptoethanol, and then centrifuged at 10,000 rpm for 15 min at 4  C. The protein concentration of the supernatant was determined by a standard method of Bradford [15]. The samples were denatured for 1 min in a boiling water bath with sodium dodecylsulfate (SDS), sample buffer and equal

Fig. 1. Magnetic eld enclosure.

Fig. 2. Function generator.

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quantities of protein (50 mg) were analyzed on 12% polyacrylamide gel with 5% stacking gel, using the discontinuous buffer system, as reported by Laemmli [16]. Quantication of the protein bands was performed using a densitometer (Gs 300 Transmittance/reectance scanning densitometer, Hoefer Scientic Instruments, USA). 2.4. Activities of enzymes Activity of a-amylase (EC 3.2.1.1) and b-amylase (EC 3.2.1.2) was determined by the procedure adopted by Dure [17]. Seedlings were ground with distilled water and centrifuged to collect the supernatant which was used to study the above enzymes activity. It was expressed as mg of maltose1 mg protein1 h1. In addition, fresh seedlings were homogenate with 0.1 M acetate buffer and after centrifugation the supernatant was used for analysis of the activity of acid phosphatase (EC 3.1.3.2) and alkaline phosphatase (EC 3.1.3.1) according to the modied method of Ikawa et al. [18]. The enzyme activity was expressed as mg of nitrophenol released1 mg protein1 h1. Activity of protease (EC 3.4.24.4) enzymes was studied according to the method followed by Mahadevan and Sridhar [19], and activity was denoted as mg1 mg protein1 h1. Seedlings homogenized with 0.1 M potassium phosphate buffer containing 1% PVP were centrifuged and supernatant was used to measure the activity of polyphenol oxidase (EC 1.14.18.1) as per the method of Sadasivam and Manickam [20]. It was expressed as mg of quinone released1 mg protein1 h1. However, catalase (EC 1.11.1.6) activity was determined according to the method of Sinha [21] by seedlings ground with phosphate buffer and it was reported as mg of H2O2 released1 mg protein1 min1. Nitrate reductase (EC 1.6.6.2) activity was analysed by the method of Hageman and Huckles [22], and expressed as mg of nitrate released1 mg protein1 h1. 2.5. Minerals Minerals (Fe, Cu, Mn, Zn, Ca, Mg, K and Na) were determined by Association of Ofcial Analytical Chemists [23] method using atomic absorption spectrometry. Seedlings were dried at 60  C for 36 h and digested with triple acid. The digested samples were used to determine the Fe, Cu, Mn, Zn, Ca, Mg, K and Na by using atomic absorption spectrometry. 2.6. Analysis of yield and yield contributing characters After seed maturation, number of leaves/plant, number of pods/ plant, length of pod, number of seeds/plant and weight of 100 seeds were measured. 2.7. Statistical analysis The data obtained from the experiments were analysed and calculated the mean SE of 6 replicates three times. As the experimental design is completely randomized design and data for each experiment were analyzed by one-way ANOVA with factorial arrangement to determine the effects of magnetic treatment. Means were compared using Duncans multiple-range test at a 5% level of signicance by SPSS 11 software package (SPSS Inc., Chicago, Illinois, USA). 3. Results and discussion The effect of PMF treatments along with different intervals of exposure time and frequencies on rate of seed germination was previously checked in soybean and the signicant positive result

was observed in seeds pretreated into 20 days with 1500 nT at 10.0 Hz of PMF for 5 h per day (data not shown). The present ndings conrm that all growth and biochemical variables of soybean drastically altered due to the stimulatory effects of PMF. Previous studies suggested that magnetic eld might be involved in the improvement of soybean plant growth [3]. However, very little is known about how MF enhances the plant growth. In this study, in an attempt to understand the mechanism by which PMF enhances the growth and yield of crop plants. PMF pretreated soybean seeds accelerated the rate of germination and enhanced the seed viability (Fig. 3), and PMF untreated seeds lost their viability after 20 months. The positive effect of MF on seed germination was reported by many researchers [2,3,24], and suggested that enhanced rate of seed germination and seed vigour under the treatment of magnetic eld would inuence the biochemical processes that involve free radicals formation and stimulating the activity of proteins and enzymes. Efcacy of PMF was evaluated on 8 days old seedling in height and biomass (Fig. 4). Seedling height increased 10% in PMF treatment over than control, and fresh and dry biomass also increased to 28% and 44% respectively. Cotton plants exposed to pulsed magnetic elds might be attributed to change the transport of assimilates, enzymic activity, growth regulators, ions and water uptake, which leads to regulate the overall pattern of plant growth and yield [1]. Florez et al. [25], observed that total length of maize plant was greater than control plants of maize exposed to magnetic eld. Sunower seedlings exposed to experimental MF showed signicant increases in shoot and root fresh weights [2,26]. The present results were correlated with Shine et al. [3] results, who found enhanced level of shoot length, fresh and dry weight of shoot in one month old soybean plants when seed pretreated with magnetic eld. In recently, Bilalis et al. [27] observed a positive effect on root length, fresh and dry weight in PMF exposed oregano plants and suggested that PMF may be of useful in organic agriculture and which should be able to replace the usage of chemical hormones. The polypeptide distribution and translational pattern were altered in bacterial and eukaryotic cells in response to different levels of electromagnetic and thermal stress [28,29]. In our study, PMF exposure signicantly accelerated the synthesis of total protein in soybean seedlings (Fig. 5), and led to modulate the pattern of protein proles. Novitsky et al. [30] had shown the two fold increase of protein contents in onion leaves subjected with magnetic eld. In control and treated groups of seedlings invariably exhibited 12 polypeptides in Coomasive Brilliant Blue stained SDS PAGE gel. The number and staining intensity of polypeptides in the

Fig. 3. Inuence of PMF on rate of seed germination of soybean. Bars represent means plus standard error (n 15). Means followed by the same letter are not signicantly different (P < 0.05) as determined by Duncans multiple-range test.

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Fig. 4. Effect of PMF on soybean seedling height and biomass. Bars represent means plus standard error (n 15). Means followed by the same letter are not signicantly different (P < 0.05) as determined by Duncans multiple-range test.

seedling of MF pretreated seeds was found to be altered when compared to control (Fig. 5). The staining intensity of polypeptides with molecular mass of 53 kDa and 14 kDa was found to be higher in PMF treated seedlings. Shine et al. [3] also observed the similar kind of results in static magnetic eld pretreated soybean plants and suggested that the concentration of photosynthetic enzyme Rubiso was enhanced due to higher efciency of carbon xation. These results strongly support the hypothesis that magnetic eld is involved in the photosynthetic enzyme Rubisco subunits. Rubisco is the most important enzyme to x carbon dioxide from the atmosphere, and this enzyme activity is inefcient in unfavourable climatic condition and leads to limits agricultural productivity. Ellis et al. [31] made attempts to improve the properties of this key enzyme of plants have failed because it proved impossible to reconstitute Rubisco in active form in vitro from its unfolded subunits in high yield. The remarkable increase of larger (53 kDa) and smaller (14 kDa) subunits of Rubisco enzyme by PMF treatment which might be the reason to enhance growth and yield of soybean. The number of enzyme activities was changed at various extends in soybean seedlings when pretreated with PMF (Table 1). Plant metabolism such as photosynthesis, activity of plant growth regulators and enzyme balances were affected by magnetic eld. The combined effect of amplitude, gradient and high frequency of

the non-uniform MF applied to the dry seeds for a brief exposure could induce or alter the biological material so-called ponderomotive effects. This effect may lead to change one or more biological parameters that affect the enzymatic activity, transport of assimilates and growth regulators [32]. Carbohydrate metabolism involving enzymes a-amylase and b-amylase activity were extremely decreased (50%) and slightly increased (2%) by the effect of PMF treatment, respectively. a-Amylase plays an important role in hydrolyzing the starch into simple sugars, which provide the energy for the growth of roots and shoots [33]. The activity of aamylase decreased during growth of seedlings, while b-amylase activity increased [34]. PMF application enhanced the seedling growth by the stimulation of starch degradation; this might be the reason of decreased level of a-amylase and elevated level of bamylase activity. However, altered level of acid phosphatase (9% of increase) and alkaline phosphatase (57% of decrease) enzyme activities was found in seedlings subjected with PMF. Acid phosphatase implicates in hydrolysis and absorption of organic phosphate compounds from soil and also plays a role in tissue differentiation [35]. Increase of acid phosphatase activity could be related to either de nova synthesis of protein or activation of protein [36]. Maheshwari and Dubey [37] found that alkaline phosphate activity declined gradually after 24 h of germination. The high concentration of Zn, Cd, Cu, Hg and Mo might inhibit the activity of alkaline phosphatase [38]. In the comparison with control, PMF exposed seedlings showed a decline in protease activity (10%). This result suggests that PMF application could either induce the synthesis of proteins or prevent the degradation of proteins. Recently, Vashisth and Nagarajan [2] reported the enzyme activities of a-amylase, dehydrogenase and protease were signicantly higher in treated seeds of sunower in contrast to controls, when exposed to static magnetic elds of strength 50 and 200 mT for 2 h. However, Table 1 shows the drastically enhanced activity of enzyme polyphenol oxidase (41%) in seedling subjected to PMF treatment. Increase of polyphenol oxidase activity represents the tolerance of plants [39]. Catalase activity also enhanced (95%) in PMF pretreated seedling over than the control. This result is consistent with Xiao-ju and Guo [40], found an increase in the activity of catalase and peroxidase in tomato plants when the seeds pretreated with MF. In addition, nitrogen metabolism related enzyme of nitrate reductase activity signicantly declined (30%) in

Fig. 5. Effect of PMF on protein content, electrophoretic distribution of total protein (SDS PAGE 12%) and densitometric pattern of protein prole in PMF treated seedlings (L1 e marker, L2 e control, L3 e MF treated). Bars represent means plus standard error (n 6). Means followed by the same letter are not signicantly different (P < 0.05) as determined by Duncans multiple-range test.

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Table 1 Effect of PMF on the activities of enzymes in soybean seedlings SE. a-Amylase (mg of maltose released1 mg protein1 h1), b-amylase (mg of maltose released1 mg protein1 h1), acid phosphotase (mg of nitrophenol released1 mg protein1 h1), alkaline phosphotase (mg of nitrophenol released1 mg protein1 h1), protease (mg1 mg protein1 h1), polyphenol oxidase (mg of quinone released1 mg protein1 h1), catalase (mg of H2O2 released1 mg protein1 min1), nitrate reductase (mg of nitrate released1 mg protein1 h1). Values in the table refer to mean SE (n 6). Means followed by the same letter are not signicantly different (P < 0.05) as determined by Duncans multiple-range test. Treatment Activity of enzymes

a-Amylase
Con PMF 175.29 1.86a 87.52 0.03b

b-Amylase
37.15 0.01ab 38.04 0.01a

Acid phosphotase 1.16 0.03ab 1.23 0.20a

Alkaline phosphotase 0.44 0.02a 0.19 0.01b

Protease 0.63 0.02a 0.57 0.01b

Polyphenol oxidase 0.0016b 0.0021a

Catalase 37.07 0.22b 72.30 0.15a

Nitrate reductase 5.24 0.10a 1.58 0.11b

seedlings subjected to PMF treatment. The nitrate reductase activity may have been limited by low concentration of Ca in seedling tissues [41]. Therefore, current study elucidates that PMF improved the seedling growth by the positive alteration of enzyme activities. The measurement of Cu, Mn and Zn contents increased more than 25% in MF treated seedlings over than control (Table 2). Similarly, PMF pretreated seedling showed that over accumulation of Fe and Na contents near 15% with slight increment (1 and 2%) of Mg and K when compared to control. The application of MF to irrigation water enhanced the plant nutrient elements of tomato plants [10]. However, the concentrations of N, K, Ca, Mg, Fe, Mn and Zn were signicantly increased, but Cu and Na were not affected by MF treatment in leaves of strawberry [11]. Deak et al. [42] observed overproducing ferritin in transgenic plants exhibited a tolerance against viral and fungal infections. In addition, the level of Ca in PMF exposed seedlings was highly reduced (30%) than that of the control experiment. Thus, this result is agreement with Hajnorouzi et al. [43], who noticed that reduction of Ca ions in MF treated maize seedlings, but the limited accumulation of Fe ions in maize is controversy with our present nding. MF caused temporary changes in the internal Ca2 ions and that this phenomenon can be explained according to ion cyclotron resonance [44]. However, the electromagnetic eld (EMF) affects the membranes and Ca2 signalling in plant cell, and magnetic effects in living organisms were probably related to the alterations in membrane-associated calcium ux [5]. Specically, the diamagnetic anisotropic properties of membrane phospholipids in magnetic eld treatment reorient the phospholipids causing deformation of membrane channels, whereas Na channels were also affected to a lesser degree than Ca2 channels [45]. Hajnorouzia et al. [43] suggested that the decreased level of Ca content according to the effects of MF increased the membrane integrity or by direct effect on the calcium channels and pumps, and thus the excess Ca2 removal from cytosol to extracellular medium by Ca2eATPases and/or Ca2/H antiporters might be a reason to lower level of calcium in MF treated plants. Magnetic elds could be inactivating calmodulin so that the transport of calcium is affected in the cell [46]. This result strongly suggests that PMF application can able to increase the uptake of minerals which leads to improve the plant growth. PMF exposure signicantly altered the number of leaves, pods per plant, length of pods, number and weight of seeds in soybean (Fig. 6). The signicant increase (16%) of number of leaves showed

in PMF treated plants than control group. Similarly, the more number of pods (15%) were also observed in plants subjected to PMF, whereas pod length, number of seeds per pod and weight of seeds were also slightly enhanced in MF pretreated plants. This result supports the previous report of Matsuda et al. [9] that MF had a positive effect on yield in strawberry. In addition, similar effects were also reported on ax, wheat, tomato, pepper, soybean, cotton and strawberry [1,11,47e50]. The enhancement in growth and yield of tomato plants derived from magnetically treated seeds may be attributed to an energetic excitement of one or more parameters of the cellular substratum (proteins and carbohydrates) or water

Fig. 6. Inuence of PMF on yield and its contributing characters of soybean plant. Bars represent means plus standard error (n 6). Means followed by the same letter are not signicantly different (P < 0.05) as determined by Duncans multiple-range test.

Table 2 Effect of PMF on mineral accumulation in soybean seedlings. Values in the table refer to mean SE (n 6). Means followed by the same letter are not signicantly different (P < 0.05) as determined by Duncans multiple-range test. Treatment Fe Con PMF 3.34 0.05ab 3.83 0.04a Cu 0.38 0.00b 0.53 0.01a Mn 1.15 0.00b 1.45 0.01a Zn 2.07 0.02b 2.72 0.03a Minerals (mg/kg Dwt) Ca 87.32 0.39a 63.46 1.57b Mg 4.41 0.01ab 4.49 0.04a K 16.64 0.16ab 16.75 0.15a Na 43.88 0.12b 49.42 0.38a

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R. Radhakrishnan, B.D. Ranjitha Kumari / Plant Physiology and Biochemistry 51 (2012) 139e144 [22] R.H.G. Hageman, D.P. Hucklesby, Nitrate reductase in higher plants, Methods Enzymol. 17 (1971) 491e503. [23] AOAC, Ofcial Methods of Analysis, 16th ed. Association of Ofcial Analytical Chemists, Washingdon, DC, USA, 1995. [24] Y. Chen, R. Li, J.M. He, Magnetic eld can alleviate toxicological effect induced by cadmium in mungbean seedlings, Ecotoxicology 20 (2011) 760e769. [25] M. Florez, M.V. Carbonell, E. Martinez, Exposure of maize seeds to stationary magnetic elds: effects on germination and early growth, Environ. Exp. Bot. 59 (2007) 68e75. [26] G. Fischer, M. Tausz, M. Kock, D. Grill, Effects of weak 16 2/3 Hz magnetic elds on growth parameters of young sunower and wheat seedlings, Bioelectromagnetics 25 (2004) 638e641. [27] D. Bilalis, N. Katsenios, A. Efthimiadou, P. Efthimiadis, A. Karkanis, Pulsed electromagnetic elds effect in oregano rooting and vegetative propagation: a potential new organic method, Acta Agric. Scand., Sect. B-Soil. Plant Sci. (2011). doi:10.1080/09064710.2011.570374. [28] M. Blank, O. Khorkova, R. Goodman, Changes in polypeptide distribution stimulated by different levels of electromagnetic and thermal stress, Bioelectrochem. Bioenerg. 33 (1994) 109e114. [29] E.M. Goodman, B. Greenebaum, M.T. Marron, Magnetic elds alter translation in Escherichia coli, Bioelectromagnetics 15 (1994) 77e83. [30] Y.I. Novitsky, G.V. Novitskaya, T.K. Kocheshkoiva, G.A. Nechiporenko, M.V. Dobrovolskii, Growth of green onions in a weak permanent magnetic eld, Russ. J. Plant. Physiol. 48 (2001) 709e715. [31] R.J. Ellis, Biochemistry: tackling unintelligent design, Nature 463 (2010) 164e165. [32] A.D. Souza-Torres, D. Garcia, L. Sueiro, F. Gilart, E. Porras, L. Licea, Pre-sowing magnetic treatments of tomato seeds increase the growth and yield of plants, Bioelectromagnetics 7 (2006) 247e257. [33] M. Kaneko, H. Itoh, M. Ueguchi-Tanaka, M. Ashikari, M. Matsuoka, The aamylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium, Plant Physiol. 168 (2002) 1264e1270. [34] A. Kohno, T. Nanmor, Changes in a and b amylase activities during seed germination of clover (Trifolium repens), Bot. Mag. Tokyo 105 (1992) 167e170. [35] N.C. Juma, M.A. Tabatabai, Phosphatase activity in corn and soybean roots: conditions for assay and effects of metals, Plant Soil 107 (1988) 39e47. [36] N.A.T. Garcia, M. Olivera, C. Iribarne, L. Luch, Partial purication and characterization of a non-specic acid phosphatase in leaves and root nodules of Phaseolus vulgaris, Plant Physiol. Biochem. 42 (2004) 585e591. [37] R. Maheshwari, R.S. Dubey, Effect of nickel toxicity on the alteration of phosphate pool and the suppressing activity of phosphorolytic enzymes in germinating seeds and growing seedlings of rice, Int. J. Plant Physiol. Biochem. 3 (2011) 50e59. [38] K. Shah, R.S. Dubey, Cadmium suppresses phosphate level and inhibits the activity of phosphatases in growing rice seedlings, J. Agron. Crop. Sci. 180 (1998) 223e231. [39] S. Niranjan-Raj, B.R. Sarosh, H.S. Shetty, Induction and accumulation of polyphenol oxidase activities as implicated in development of resistance against pearl millet downy mildew disease, Funct. Plant Biol. 33 (6) (2006) 563e571. [40] M.M. Xiao-ju, Y.G. Guo, Study on the effect of tomato seeds physiology and biochemistry with magnetic eld treatment, Bull. Bot. Res. 99 (1999) 1e8. [41] J.E. Harper, G.M. Paulsen, Nitrogen assimilation and protein synthesis in wheat seedlings as affected by mineral nutrition. I. Macronutrients, Plant Physiol. 44 (1969) 69e74. [42] M. Deak, G.V. Horvath, S. Davletova, K. Torok, L. Sass, I. Vass, B. Barna, Z. Kiraly, D. Dudits, Plants ectopically expressing the iron-binding protein, ferritin, are tolerant to oxidative damage and pathogens, Nat. Biotechnol. 17 (1999) 192e196. [43] A. Hajnorouzia, M. Vaezzadeha, F. Ghanatib, H. Jamnezhada, B. Nahidianb, Growth promotion and a decrease of oxidative stress in maize seedlings by a combination of geomagnetic and weak electromagnetic elds, J. Plant Physiol. 168 (2011) 1123e1128. [44] A. Pazur, V. Rassadina, Transient effect of weak electromagnetic elds on calcium ion concentration in Arabidopsis thaliana, BMC Plant Biol. 9 (2009) 47e57. [45] A.D. Rosen, Mechanism of action of moderate intensity static magnetic elds on biological systems, Cell Biochem. Biophys. 39 (2003) 163e174. [46] W.X. Balcavage, T. Alvager, J. Swez, C.W. Goff, M.T. Fox, S. Abdullyava, M.W. King, A mechanism for action of extremely low frequency electromagnetic elds on biological systems, Biochem. Biophys. Res. Commun. 222 (1996) 374e378. [47] G.H. Gubbels, Seedling growth and yield response of ax, buckwheat, sunower and eld pea after preseeding magnetic treatment, Can. J. Plant Sci. 62 (1982) 61e64. [48] S. Pieturszewski, Effect of magnetic biostimulation of wheat, Seeds Sci. Technol. 21 (1993) 621e626. [49] K. Ogolnej, R. Uprawy, A. Rolnieza, The effect of magnetical biostimulation of sowing material od spring wheat on its development and crops, Folia Univ. Agric. Stetin. Agricultura 226 (2002) 77e82. [50] G. Vasilevski, Perspectives of the application of biophysical methods in sustainable agriculture, Bulgarian J. Plant Physiol. 2 (2003) 179e186.

inside the dry seeds by direct effect of MF [49]. These results strongly prove the potentiality of PMF to enhance the soybean yield. In conclusion, PMF exposure to soybean seeds caused positive changes in protein, intensity of protein polypeptides, activities of various enzymes and uptake of minerals in seedlings of soybean compared to PMF untreated controls. However, the number of leaves, pods and seeds, and then length of pods with weight of seeds were also increased in soybean with the effect of PMF pretreatment. Our results suggest that PMF seed pretreatment can be used in agriculture to better growth and increase the yield. Acknowledgements The authors wish to thank Madras Institute of Magneto Biology, Chennai, India for providing the facility for magnetic eld treatments to soybean seeds and also thank Bharathidasan University, Tiruchirappalli, India for nancial support to successful completion of this work. References
[1] T. Leelapriya, K.S. Dilip, P.V. Sanker-Narayan, Effect of weak sinusoidal magnetic eld on germination and yield of cotton (Gossypium sp.), Electromagn. Biol. Med. 22 (2003) 117e125. [2] A. Vashisth, S. Nagaraja, Effect on germination and early growth characteristics in sunower (Helianthus annuus) seeds exposed to static magnetic eld, J. Plant Physiol. 167 (2010) 149e156. [3] M.B. Shine, K.N. Guruprasad, A. Anan, Enhancement of germination, growth, and photosynthesis in soybean by pre-treatment of seeds with magnetic eld, Bioelectromagnetics 32 (2011) 474e484. [4] W.R. Adey, Cell membranes: the electromagnetic environment and cancer promotion, Neurochem. Res. 13 (1988) 671e677. [5] P. Galland, A. Pazur, Magnetoreception in plants, J. Plant Res. 118 (2005) 371e389. [6] A.R. Liboff, S. Cherng, K.A. Jenrow, A. Bull, Calmodulin dependent cyclic nucleotide phosphodiesterase activity is altered by 20 mT magnetostatic elds, Bioelectromagnetics 24 (2003) 2e38. [7] B. Nossol, G. Buse, J. Silny, Inuence of weak static and 50 Hz magnetic elds on the redox activity of cytochrome-C oxidase, Bioelectromagnetics 14 (1993) 361e372. [8] E.G. Cho, S.J. Kweon, D.Y. Suh, H.S. Suh, S.K. Lee, J.K. Sohn, J.F. Oh, Studies of utilization of magnetic force in agricultural genetic engineering. Research reports of the rural development administration, Biotechnology 34 (1992) 10e14. [9] T. Matsuda, H. Asou, M. Kobayashi, M. Yonekura, Inuences of magnetic elds on growth and fruit production of strawberry, Acta Hortic. 348 (1993) 378e380. [10] C.E. Duarte-Diaz, J.A. Riquenes, B. Sotolongo, M.A. Portuondo, E.Q. Quintana, R. Perez, Effects of magnetic treatment of irrigation water on the tomato crop, Hortic. Abst. 69 (1997) 494. [11] A. Esitken, M. Turan, Alternating magnetic eld effects on yield and plant nutrient element composition of strawberry (Fragaria x ananassa cv. Camarosa), Acta. Agric. Scand., Sect. B-Soil Plant Sci. 54 (2004) 135e139. [12] F.G. Reina, L.A. Pascual, I.A. Fundora, Inuence of a stationary magnetic eld on water relations in lettuce seeds. Part II: experimental results, Bioelectromagnetics 22 (2001) 596e602. [13] G. Fanselau, The generation of large and homogenous magnetic eld through circuits, Zeitschr. Phys. 54 (1929) 260e269. [14] W. Brauenbeck, The egg-tool-LMG broad homogeneous magnetic eld by circuits, Zeitschr. Phys. 88 (1934) 399e402. [15] M.M. Bradford, A rapid and sensitive method for the quantication of microgram quantities of protein-dye binding, Anal Biochem. 72 (1976) 218e251. [16] U.K. Laemmli, Cleavage of structural proteins during the assembly of the head of bacteriophage T4, Nature 227 (1970) 680e685. [17] L.S. Dure, Site of origin and extent of activity of amylases in maize germination, Plant Physiol. 35 (1960) 924e936. [18] T. Ikawa, K. Nisizawa, T. Miwa, Specicities of several acid phosphatases from plant sources, Nature 203 (1964) 939e940. [19] A. Mahadevan, R. Sridhar, Methods of Physiological Plant Pathology. Sivakasi Publication, India, 1982, pp. 131e132. [20] S. Sadasivam, A. Manickam, Biochemical Methods. New Age International Private Publication, Coimbatore, India, 1996, pp. 108e110. [21] K.A. Sinha, Colorimetric assay of catalase, J. Biochem. 47 (1972) 389.