Laser light and magnetic field stimulation effect on biochemical, enzymes activities and chlorophyll contents in soybean seeds and seedlings during early growth stages

    Laser light and magnetic field stimulation effect on some biochemical, enzymes activity and chlorophyll contents in soybean seeds and seedlings during early growth stages Tehseen Asghar, Yasir Jamil, Munawar Iqbal, Zia-ul-Haq, Mazhar Abbas PII: DOI: Reference:

S1011-1344(16)30654-6 doi: 10.1016/j.jphotobiol.2016.10.022 JPB 10621

To appear in: Received date: Revised date: Accepted date:

9 August 2016 15 October 2016 18 October 2016

Please cite this article as: Tehseen Asghar, Yasir Jamil, Munawar Iqbal, Zia-ul-Haq, Mazhar Abbas, Laser light and magnetic field stimulation effect on some biochemical, enzymes activity and chlorophyll contents in soybean seeds and seedlings during early growth stages, (2016), doi: 10.1016/j.jphotobiol.2016.10.022

This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT Laser light and magnetic field stimulation effect on some biochemical, enzymes activity and chlorophyll contents in soybean seeds and seedlings during early growth stages

PT

Tehseen Asghara, Yasir Jamila, Munawar Iqbalb,*, Zia-ul-Haqc and Mazhar Abbasd a

NU

SC

RI

Bio-Electromagnetics and Laser Laboratory, Department of Physics, University of Agriculture, Faisalabad b Department of Chemistry, The University of Lahore, Lahore, Pakistan c Department of Physics, University of Agriculture, Faisalabad, Pakistan d Institute of Molecular Biology and Biotechnology, The University of Lahore, Lahore, Pakistan, *Corresponding author E-mail: [email protected]

MA

Abstract

D

Laser and magnetic field bio-stimulation attracted the keen interest of scientific community in

TE

view of their potential to enhance seed germination, seedling growth, physiological, biochemical

AC CE P

and yield attributes of plants, cereal crops and vegetables. Present study was conducted to appraise the laser and magnetic field pre-sowing seed treatment effect on soybean sugar, protein, nitrogen, hydrogen peroxide (H2O2) ascorbic acid (AsA), proline, phenolic and malondialdehyde (MDA) along with chlorophyll contents (Chl “a” “b” and total chlorophyll contents). Specific activities of enzymes such as protease (PRT), amylase (AMY), catalyst (CAT), superoxide dismutase (SOD) and peroxides (POD) were also assayed. The specific activity of enzymes (during germination and early growth), biochemical and chlorophyll contents were enhanced significantly under the effect of both laser and magnetic pre-sowing treatments. Magnetic field treatment effect was slightly higher than laser treatment except PRT, AMY and ascorbic acid contents. However, both treatments (laser and magnetic field) effects were significantly higher versus control (un-treated seeds). Results revealed that laser and magnetic field pre-sowing seed treatments have potential to enhance soybean biological moieties, chlorophyll contents and 1

ACCEPTED MANUSCRIPT metabolically important enzymes (degrade stored food and scavenge reactive oxygen species). Future study should be focused growth characteristics at later stages and yield attributes.

PT

Keywords: Soybean seeds; Laser; magnetic field; stimulation, biomolecules; enzymes activity;

RI

chlorophyll contents

SC

1. Introduction

NU

Pre-sowing laser and magnetic bio-stimulation of seeds beneficial effect on germination, seedling growth and yield have attracted the attention agriculturists [1]. The mobilization of seed

MA

storage proteins represents one of the most important post-germination events and seedling growth. Enzymes play a central role in the metabolic pathways during germination and the the

TE

D

accelerated enzymatic activities under the effect of laser and magnetic field pre-sowing seed treatment have been reported [2-7]. It is hypothesized that laser light can increase free radicals in

AC CE P

irradiated seeds, which can react with oxygen and this process leads to the formation of peroxides. The activity of hydrolytic enzymes increased under the influence of laser irradiation and resultantly, fast mobilization of reserve substances accelerates the seed germination, emergence and seedling growth [8]. Vashisth and Nagarajan [9] reported enhanced enzymatic activities as a results of exposure to magnetic field and Chen et al. [10] also documented that HeNe laser has also accelerating effect enzyme activities during germination and early growth stages. Similarly, [5, 11] also reported accelerated enzymatic activities along with fast germination and seedling growth. It is reported that pre-treatment of seed with laser have apparent inductions on the enzymatic activities, thermodynamic properties, altered physiological and biochemical metabolism pathways [10]. Similarly, the enhanced plant characteristics in response of magnetic field treatment have been correlated with change in ferromagnetic

2

ACCEPTED MANUSCRIPT properties of particle, change in energy level, changes in electron spins at atom and molecular level, which collectively affect physiological and biochemical metabolic pathways [2-4, 7, 12-

PT

14]. Under the current scenario of environmental pollution and soil chemistry alteration [15-51],

RI

different seed stimulating techniques are used to enhance germination and related plant characteristics. However, environmental pollution cannot be limited where primary use of

SC

chemical additives is frequent, which are harmful to biological systems. Clean, eco-friendly,

NU

more efficient and less expensive techniques are viable alternative and laser and magnetic field pre-sowing seed treatments is of great interest since these non-destructive, safer and cheap [2-7,

MA

11, 14, 52].

Soybean is one of valuable commodity in the global market as oil crop and is beneficial

TE

D

for health as is good source of unsaturated fatty acids and free from cholesterol [53]. The major components of soybean are proteins (40%) and carbohydrates and lipids (20%) along with

AC CE P

important secondary metabolites (phenolic components, saponins and isoflavones). Among all legumes and cereal crops, soybean is a good source of protein for human and animals [54, 55]. The fermented soybean foods are very popular in Asian countries as food [53]. Soybeans are planted worldwide and ~75 million acres are planted with soybeans every year in United States and in comparison, Pakistan, covers ~2300 to 6000 (variable and depends upon weather conditions each year) hectares/year since 1978 for soybean cultivation. Germination is considered the sensitive stage in the life cycles of soybean and under drought stress, the germination of soybean affected badly [56]. The drought stress affects soybean germination at planting. For normal germination, 50 percent water absorption is needed of seed weight. As the water contents drops below this limit, germination decreased significantly and also mean germination time is increased [57]. In Pakistan, KPK is the major produced and 59% of total

3

ACCEPTED MANUSCRIPT soybean is cultivated in Abbottabad and Mansehra districts (Hazara division) and small area is also cultivated in Malakand division and FATA. In Sindh, Hyderabad division (Badin and

PT

Hyderabad districts) is main cultivated area of soybean and Sanghar and Thatta districts also

RI

cover small area for soybean cultivation. In Punjab, very small area in Multan district in under the cultivation of soybean and this scattered distribution of soybean cultivation is due to weather

SC

conditions of specific area because soybean germination affected under dry conditions [58-60].

NU

In view of low germination of soybean in Pakistan, different strategies (sowing and management) have been investigated to enhance germination and yield of soybean [56, 58, 61,

MA

62]. However, effect of laser and magnetic field pre-sowing seed treatments on soybean have not

TE

the plant characteristics [52, 63-68].

D

been investigated previously since these methods have been successfully employed to enhance

Therefore, present investigation was conducted to appraise the laser and magnetic field pre-

AC CE P

sowing seed treatments on enzymatic activities and bio-biomolecule during early growth stages. The principle objective was to compare the effects of both laser and magnetic field pre-sowing seed treatments effect on total soluble sugar, reduced sugar, protein, nitrogen content, H2O2, AsA, proline, phenolic, MDA contents, CAT, SOD POD and chlorophyll contents.

2. Materials and Methods 2.1.

Chemicals and Reagents

All chemical and reagents used were of analytical grade i.e., trichloro-acetic acid, dichloroindophenol, casein, methionine, nitroblue tetrazolim, riboflavin, ninhydrin and toluene were purchased from Sigma Chemical Co. (St. Louis, Mo, USA), whereas thiobarbituric acid, Folin-ciocalteau’s reagent, anthron, potassium iodide, glacial acetic acid and Rochelle salt were 4

ACCEPTED MANUSCRIPT purchased from Merck (Darmstadt, Germany). Bovine serum albumin and Triton X and potassium diphosphate and hydrogen peroxide were purchased from Bio red (USA), MP

Laser and magnetic field setup

RI

2.2.

PT

Bioforma (France) and Fluka (USA), respectively.

SC

Portable He-Ne laser was used for seed irradiation (Model No. 1508P-1256, JDS Uniphase USA,

NU

wavelength 632.8 nm and beam diameter 1.5 mm), which emits continuous laser light of wavelength 632.8 nm. During irradiation of seeds, the laser output power was measured using

MA

power/energy meter (Quantel, France). The seeds were irradiated following the procedure reported by Chen et al. [10]. A general laser set up used for irradiation is shown in Fig. 1(A).

D

Whereas magnetic field set up is shown in Fig. 1(B). Briefly, the electromagnet used for seed

TE

treatments contained four cylindrical coils (each coil has 4000 turns of 0.41 mm enameled

AC CE P

copper wire) and resistance of each coil was 26 Ω. Each pair of coils was wound 10 cm apart on an iron bar, which were placed vertical to each other on both sides and metallic support was used to hold each bar at fixed position. The coils were connected in series and fed through a variable power supply (0 to 220 V) of 50 Hz full-wave rectified sinusoidal voltage. The magnetic field strength was adjusted by passing variable voltage through coils. Magnetic flux meter (ELWE, Germany) was used for magnetic field strength measurement. 2.3.

Seeds and treatment procedure

Healthy and uniform soybean seeds were selected (collected from ayyub Agriculture Research Institute) for experimentation. For laser, seeds were exposed to He-Ne laser of wavelength 632.8 nm, power density 1 mW/cm2 for 3 and 5 min. For Magnetic seed stimulator was used as sources of magnetic fields with magnetic field induction values of 50, 75 and 100 milli Tesla (mT) for an 5

ACCEPTED MANUSCRIPT exposure time of 3 and 5 min. The seeds were treated and shown in triplicate for each dose both for laser and magnetic. Un-treated seed were used as control. Biochemical parameters measurement

PT

2.4.

RI

Laser and magnetic bio-stimulation effects on biochemical parameters (hydrogen peroxide

SC

(H2O2), ascorbic acid (AsA), proline and phenol content and the content of malondialdehyde

Measurement of protein and nitrogen contents

MA

2.5.

NU

(MDA) were measured as precisely reported previously [5, 11].

Protein content was measured by Bredford method [69] and nitrogen contents was measured by

TE

2.5.1. Malondialdehyde

D

following modified micro-Kjeldhal’s method [70].

AC CE P

Malondialdehyde (MDA) was analyzed following Carmak & Horst [71] method. This method is based on the reaction with thiobarbituric acid. Briefly, fresh leaves (1.0 g) were ground properly in 20 mL of 0.1% trichloroacetic acid solution and centrifuged for 10 min at 12000 rpm. One mL of the supernatant was reacted with 4 mL of 20% TCA solution comprising 0.5% thiobarbituric acid and heated for 30 min at 100°C in a water bath and immediately cooled on ice bath. After centrifugation for 10 min at 12000 rpm, the absorbance of the supernatant was read at 532 and 600 nm. The contents of MDA were worked out using the extinction coefficient of 155/ (mM/cm) using the relation shown in Eq. 1, where A is the absorbance. MDA level (nmol) = Δ (A 532nm-A 600nm)/1.56×105

(1)

2.5.2. Total phenolics

6

ACCEPTED MANUSCRIPT Total phenolics were estimated following Julkenen-Titto [72] method. Fresh leaf samples (50 mg) were homogenized in 80% acetone. After centrifugation for 10 min at 12000 rpm, the

PT

supernatant was separated. To 100 μL of the supernatant 2.0 mL of distilled water and 1 mL of

RI

Folin–Ciocalteau’s phenol reagent was added. Then the final volume was raised to 10 mL by

SC

adding distilled H2O. After mixing thoroughly, the absorbance was read at 755 nm.

NU

2.5.3. Hydrogen Peroxide (H2O2)

Hydrogen peroxide (H2O2) was determined by Harinasut et al. [73] method. Leaf tissues (0.5 g)

MA

were homogenized in an ice bath with 5 mL 0.1% (w/v) trichloroacetic acid. The homogenate was centrifuged at 12000 rpm for 15 min and 0.5 mL of the supernatant was added to 0.5 mL of

D

10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI. The absorbance of the

AC CE P

2.5.4. Proline content

TE

supernatant was measured at 390 nm.

Proline content was measured following Unyayar et al. [74] method. A 0.1 g of plant material was homogenized in 5 mL of 3% 5-sulfosalicylic acid the extract was reacted with 2 mL of glacial acetic acid and 2 mL acid ninhydrin (1.25 g ninhydrin in 30 mL glacial acetic acid and 20 mL 6 M phosphoric acid until dissolved), for 1 h at 100 0C and the reaction was terminated in an ice bath. At the end, 1 mL of toluene was added, and optical density measured at 520 nm. 2.5.5. Reducing sugar To determine total reducing sugar, a modified method given by Sadasiven and Manickam [75] was adopted, for this 100 µL of extract was taken and digested with 900 µL of dist followed by water and 1.5 mL of DNS reagent addition. The mixture was mixed well and boiled for 5 min

7

ACCEPTED MANUSCRIPT until dark brown colour was appeared. Then 1 mL of rochelle salt solution was added in test tubes with slight heating. The tubes were cooled under running water and absorbance was taken

PT

at 510 nm. The amount of reducing sugar in the sample was calculated using a standard graph

RI

prepared from working standard glucose solution.

SC

2.5.6. Measurement of total soluble sugar

NU

Total soluble sugars was determined following method given by Sadasivam and Manickam [75], 100 µL of extract was taken and 900 µL distill H20 was added, mixed and 1mL anthrone reagent

MA

was added and heated for 8 min. Then the reaction mixture was cooled and absorbance was taken at 630 nm. The amount of soluble sugar in the sample was calculated using a standard graph

AC CE P

2.5.7. Ascorbic Acid

TE

D

prepared by plotting concentration of the standard.

For the estimation of ascorbic acid (AsA), 900 µL distilled H2O, 100 µL sample extract, 1mL dichloroindophenol (DCIP), 100 µL 0.1% metaphosphoric acid (H3PO4) were mixed in a test tube and absorbance was taken at 520 nm [76]. 2.6.

Chlorophyll contents

The chlorophyll a and b were measured following already reported the method [3, 5, 6, 77]. Fresh leaves of soybean were cut out and extracted with 80% acetone at 0-4°C. Extract were centrifuged at 12000 rpm for 5 min. Absorbance of the supernatant was read at 645, 663 and 480 nm by spectrometer (Hitachi-U2001, kyoto, Japan). 2.7.

Enzymes activities

2.7.1. Protease specific activity 8

ACCEPTED MANUSCRIPT The PRT specific activity was determined by the casein digestion assay [78]. The extracted sample was centrifuged at 14000 rpm and 4 °C for 5 min. The supernatant (0.5 mL) was mixed

PT

with 2 mL of 1% casein solution preincubated for 30 min. The mixture was incubated for 30 min

RI

and 2 mL of 10% TCA (trichloroacetic acid) solution was added to the mixture immediately to stop the reaction. The reaction mixture was centrifuged at 10000 rpm and 4 °C for 5 min. The

SC

PRT specific activity was units per mg of protein. Protein concentration was measured by

NU

Bradford (1976) method and bovine serum albumin (BSA) served as standard.

MA

2.7.2. Catalyse (CAT) and Peroxidase (POD) Activities Catalase (CAT) and peroxidase (POD) was determined using the methods of Chance and Maehly

D

[79]. A 1 mL reaction mixture contained 50 mM phosphate buffer, pH 7, 20 mM 2-methoxy

TE

phenol (guaiacol), 40 mM hydrogen peroxide with 100 µL of enzyme extract were mixed and

AC CE P

after 3 min. One unit of peroxidase activity represents in units/µg protein and the CAT reaction solution contained 50 mM phosphate buffer (PH 7); 5.9 mM H2O2 and 100 µL enzyme extract. 2.7.3. Superoxide dismutase (SOD) Superoxide dismutase activity (SOD) was assayed by monitoring the inhibition of the photochemical reduction of nitroblue tetrazolium, according to the method of Giannopolitis and Ries [80]. For total the superoxide dismutase assay, the 1 mL reaction mixture contained, 222 mg methionine, 0.0375 mL Triton X in 17.5 mL of distilled water, 15 mg nitroblue tetrazolium, 13.2 gm riboflavin and an appropriate aliquot of enzyme extract. The reaction mixtures were illuminated for 15 min at a light intensity of 350 µmol m-2s-1. One unit of superoxide dismutase activity (SOD) was defined as the amount of enzyme required to cause 50% inhibition of nitroblue tetrazolium reduction, which was monitored at 560 nm. 9

ACCEPTED MANUSCRIPT 2.7.4. α- amylase The activity of α- AMY was determined by following Varavinit et al. [81] method with slight

PT

modification. 100 µL extract was taken in test tubes then 1.5 mL soluble potato starch solution

RI

containing 500 ppm of calcium ion (cofactor) and then added 1mL of 100 mM tri (hydroxyl

SC

methyl amino methane/Hcl buffer). The mixture was then incubated in a water bath with

min and absorbance was measured at 540 nm. Statistical Analysis

MA

2.8.

NU

constant shaking at 40 0C for 30 min. add 1 mL of 3, 5 dinitrosalicylic acid and boiling for 10

The statistical analysis of the data was computed using SPSS 13.0 software at 95% confidence

TE

D

interval and P < 0.05 was considered significant.

AC CE P

3. Results and Discussion

Laser and magnetic treatments enhanced the biochemical, enzymatic activity and chlorophyll contents significantly. The laser and magnetic field pre-sowing seed treatment effects on soybean various biochemical parameters are shown in Figs. 1 and 2. It was observed that biochemical moieties were considerably higher in laser and magnetic field treatments versus control. The effects on biochemical parameters were variable at different laser energy levels and magnetic field doses (MF strength and exposure duration). The total soluble protein (µg/mL) values are shown in Fig. 2(A). The magnetic field of 100 mT strength for 3 min exposure showed highest total sugar contents followed by 50 mT for 3 min exposure and 75 mT for 3 min exposure, whereas 50, 75 and 100 mT for 5 min magnetic field treatments effect on sugar contents was slightly low in comparison to 50, 75 and 100 mT for 3 min exposure. In case of laser treatment, 3 min exposure showed higher sugar contents than 5 min exposure. Overall, magnetic field 10

ACCEPTED MANUSCRIPT treatment effect on sugar contents was higher versus laser treatment. The responses of proline (µmole/g) in response of laser and magnetic field treatments are depicted in Fig. 2(B). The

PT

magnetic field doses (75 mT for 3 min, 100 mT for 3 min, 75 mT for 5 min and 50 mT for 3

RI

min) was significantly higher than control, whereas laser effect on proline contents was insignificant and slightly greater than control. In case of total phenolic contents (µg/mL), the

SC

laser effect was higher for 5 min and magnetic field showed slightly lower phenolic contents,

NU

however, both laser and magnetic field treatments effects on phenolic contents were significantly higher than control (Fig. 2(C)). Total soluble sugar contents were recorded significantly higher in

MA

magnetic field treated seedlings versus laser irradiation. Overall, the soluble sugar contents (%) were recorded in following order; 50 mT for 3 min exposure > 100 mT for 5 min exposure > 100

TE

D

mT for 3 min exposure > 75 mT for 3 min exposure > 75 mT for 5 min exposure > 50 mT for 3 min exposure, 5 min laser irradiation > 3 min laser irradiation (Fig. d(D)). Reducing sugar

AC CE P

contents (%) were also recorded higher in seedlings received magnetic field treatment. Among magnetic field treatments, 75 mT for 3 min exposure showed higher reducing sugar content followed by 100 mT for 5 min exposure, 50 mT for 3 min exposure, 100 mT for 3 min exposure, and 50 mT for 5 min exposure, whereas laser effect on reducing sugar content was insignificant (Fig. 2(E)). The nitrogen and protein contents (%) were recorded higher in magnetic field exposed seedlings versus laser and overall, both treatments enhanced the nitrogen and protein contents versus control (Fig. 2(F)). As it can be seen in Fig. 3(A), the hydrogen peroxide (U/mg) did not increase significantly, only 100 and 50 mT for 3 min exposure and 3 min laser irradiation showed the hydrogen peroxide contents comparable with control, while all other magnetic field and laser treatments did not affect the hydrogen peroxide contents. The malodialdehyde contents (nM) of laser and magnetic field treated seedlings are shown in Fig. 3(B). The magnetic field

11

ACCEPTED MANUSCRIPT treatments of 50 mT for 3 min exposure showed higher malodialdehyde contents, whereas 75 and 100 mT for 3 min exposure, 50 and 75 mT for 5 min exposure showed malodialdehyde

PT

contents comparable with control and laser irradiation did not affect the malodialdehyde

RI

contents. The ascorbic acid contents (µg/mL) are depicted in Fig. 3(C). Here, laser effect on ascorbic acid contents was promising and 3 min laser irradiation showed highest concentration

SC

followed by 5 min exposure. The magnetic field treatment also enhanced the ascorbic acid

NU

contents in comparison to control and ascorbic acid contents in case of magnetic field treatments were found in following order; 75 mT for 5 min exposure > 75 mT for 3 min > 50 mT for 5 min

MA

> 50 mT for 3 min, 100 mT for 5 min and 100 mT for 3 min exposure. Enzyme has central role in seed germination and seedling growth and their activities can

TE

D

be accelerated by employing regulator externally or changing the internal environment [5, 11]. Therefore, the response of enzymes involved in germination and seedling growth were also

AC CE P

evaluated in soybean under the effect of laser and magnetic field and results, thus observed are shown in Fig. 4. The PRT (IU/mL) accelerated significantly under the effect of both laser and magnetic field treatments; however, laser irradiation for 5 min enhanced the PRT activity exponentially (Fig. 4(A)). All other laser and magnetic field treatments also showed higher PRT activities versus control. Among magnetic field treatments, 75 mT for 3 min exposure furnished higher PRT activity followed by 75 mT for 5 min exposure, 100 mT for 3 min and 50 mT for 3 min. The AMY activities (IU/mL) of laser and magnetic field seedlings are depicted in Fig. 4(B). The AMY activity was also recorded higher for laser 5 min exposure; however, magnetic field treatment also enhanced the AMY activity in exposed seedlings and found in following order; 75 mT for 3 min exposure > 100 mT for 3 min exposure > 50 mT for 3 min exposure > 100 mT for 5 min exposure > 75 mT for 5 min > 50 mT for 5 min exposure. Superoxide dismutase activity

12

ACCEPTED MANUSCRIPT (IU/mL) was recorded higher in seedling exposed to magnetic field in comparison to laser treatment (Fig. 4(C)). Among magnetic field treatments, 75 mT for 3 and 5 min exposure showed

PT

higher superoxide dismutase contents and laser 5 min irradiation showed greater superoxide

RI

dismutase activity versus 3 min irradiation. Both laser and magnetic field showed superoxide dismutase activity considerably higher than control. Catalase (IU/mL) activity observed higher in

SC

magnetic field treated seedlings and 50 mT for 3 min treatments effect was promising followed

NU

by 75 mT for 3 min exposure, 100 mT for 3 min exposure. In case of laser treatment, 3 min laser irradiation showed higher catalase activity versus 5 min exposure (Fig. 4(D)). In case of peroxide

MA

activity, the laser effect was higher for 5 min exposure. In case of magnetic field treatments, 100, 75 and 50 mT for 3 min exposure showed higher peroxidase activity, while other treatments

TE

D

furnished peroxidase activity comparable with control (Fig. 4(E)). Chlorophyll plays a key role in plant growth and adaptation to different environmental

AC CE P

conditions, which depends upon photosynthetic efficiency of leaves. The effect of laser and magnetic field pre-sowing seed treatment on soybean chlorophyll contents was also studied since this pigment is highly sensitive to external factors affecting the biosynthesis of chlorophyll and the leaf chlorophyll vary significantly under different environmental conditions. The soybean chlorophyll contents under the effect of laser and magnetic field are shown in Fig. 5(ABC) for chlorophyll a, chlorophyll b and total chlorophyll, respectively. Both laser and magnetic field treatments enhanced the chlorophyll “a” contents significantly and magnetic field effect was slightly higher than laser and similar trends were recorded for chlorophyll “b” and total chlorophyll contents (Fig. 4(BC)). Overall, magnetic field effect on soybean chlorophyll contents was higher than laser; however, both laser and magnetic field furnished significantly higher chlorophyll contents versus control.

13

ACCEPTED MANUSCRIPT Laser irradiation of seeds at different energy levels and magnetic field specific doses (exposure time and mT) have been proved to be beneficial for stimulating the plant biological

PT

characteristics [10]. The enhanced enzyme activities are responsible for viable germination and

RI

seedling growth at lateral stages of development. Moreover, the antioxidant enzymes also protect the seedlings and plant from unfavorable conditions [82]. In response of enhanced enzymatic

SC

activities and biological moieties, the process behind germination of seed and seedling growth

NU

alters, which results in higher plant productivity and ultimately, better yield. The AMY and PRT take part in seed germination process i.e., AMY enzyme hydrolysis stored starch into sugars,

MA

whereas PRT converts protein into amino acids and germinated seeds are nourished since enzymes breakdown the reserved food and simple sugars and amino acids are transported to the

TE

D

growing seedlings [9, 83]. Here, both AMY and PRT activities were recorded significantly higher under the effect of laser and magnetic field pre-sowing seed treatment and therefore,

AC CE P

metabolic reactions are expected to be fast and viable. Previous reports also revealed that AMY activity in wheat was enhanced under the effect magnetic field treatment [11]. Similarly, the enhanced PRT activity has also been reported higher in sunflower germinating [5]. The SOD, POD and CAT enzymes are regarded as antioxidant enzymes. These enzymes protect plant tissue from harmful external factor as well as oxidative species produced inside the cell organelles [2, 3]. The reactive oxygen species (ROS) are produced in plant tissues under stress conditions and the antioxidant enzymes reactive species with these species and preclude them from oxidative reaction with biomolecule. So far, antioxidant enzymes cope against stresses and resultantly, protect the plant tissues from negative effects of ROS. The SOD, POD and CAT activities were found significantly higher in seedlings raised from magnetic field and laser treated seeds. Therefore, seedling growth can be enhanced under accelerated antioxidant enzymes activities [4,

14

ACCEPTED MANUSCRIPT 5, 11]. These findings are in line with reported studies that magnetic field i.e., pre-sowing seed treatment protects the cucumber seedlings against UV-B radiation [84], laser light treatment

PT

effect on various biochemical and physiological was also found to be promising for in white

RI

lupine and Vicia faba [85], Isatis indogotica [10] and brinjal [86]. It is well known that the enzymes, which are necessary for seed germination at particular stages of germination were

SC

found higher in treated seeds as compared to during germination [9]. The enhanced growth,

NU

development at lateral stages and enhanced physiological attributes have been correlated well with the enhanced activities of antioxidant enzymes [11, 87]. The leaf area also found to be

MA

enhanced, which intercept greater light and was responsible for higher chlorophyll contents and photosynthetic rates. Hoff [88] found an increase in photosynthetic rate and influx of water as a

TE

D

result of magnetic field treatment and in another study is reported that synthesis of chlorophyll and carotenes is useful for seedling nutrition [89]. The results of present investigation and

AC CE P

literature survey strongly prove the potential of laser and magnetic field pre-sowing soybean seed treatment to enhance the biochemical, enzymes and synthetic process, which are prerequisite for biomass productivity and higher yield. However, magnetic field treatment effect was slightly higher than laser treatment except AMY, PRT activities and ascorbic acid concentration. The slight higher effect of magnetic field on biochemical and oxidative enzymatic system can be explained on the basis of strengths or dose used for seed treatment (laser and magnetic stimulation). The exposure time was same (3-5 min) for both treatments. Based on present findings, the light treatment may be effective for shorter duration and that of magnetic field longer duration. In case of magnetic field treatment, the particles havening ferromagnetic properties, susceptible to energy level amendment and changes in electron spins in atom and molecules are responsible for changing the physiological pathways and metabolism of

15

ACCEPTED MANUSCRIPT biochemical. Whereas, laser stimulation mechanism is based on the synergism between the polarized monochromatic laser beam and the photoreceptors, activate biological reactions and

PT

control development in plants. So, laser activates the bio-energetic potential, fitochrome,

RI

fitohormone and fermentative systems [52] and magnetic field can effect chemical reactions by altering electron spin location and resultantly, cause biological effects [12, 13]. It has been

SC

reported that electromagnetic induction cause more water assimilation and intensifies synthesis

NU

process [90]. Moreover, an increased photosynthetic rate and influx of water as a result of magnetic treatments has also been observed in response of magnetic field treatment [88].

MA

Accelerated ions movement across plasma membranes and amino acid have also been reported [91], which collectively enhance the germination and seedling growth. Whereas, laser

TE

D

stimulation of seed, specific energy is used, this may not be effective to enhance the specific parameter during early growth stages since laser cause photo-stimulations that may not be so

AC CE P

effective in enhancing the biological moieties at early growth stages. In previous studies, it was observed that the enhancement in biochemical, physiological and yield parameters was not in correlation with laser energy i.e., 100 and 300 mJ laser energy levels showed variable effect on various parameters [5] and similar observation have been reported by other researchers i.e., Rybinski and Garczynski [50] observed He-Ne laser at the wavelength of 632 nm was effective in enhancing photosynthetic rate, transpiration rate and gas exchange efficiency. Govil et al. [92] reported that 337.1 nm (Laser radiation) for 30 min irradiation enhanced the growth parameters, for 20 min irradiation was effective to enhance protein and chlorophyll did not change in seedling treated 337.1 nm irradiation. Similarly, Hernández et al. [93] found 660 nm for 30 s and 60 s with intensities of 3.2 and 20 mW/cm2 gave better response. An intensity of 20 mW/cm2 for 1 min exposure was excellent to enhance the germination and growth parameters. Osman et al.

16

ACCEPTED MANUSCRIPT [94] revealed that laser treatments for 5, 10 and 20 min with power density of 95 mW/cm2 for 20 min significantly enhanced the growth and biochemical parameters, whereas highest fruit yield

PT

was recorded from 5 min of exposure. Saghafi et al. [95] found that the laser He-Ne laser (632

RI

nm) for 10 min irradiation (40 mJ/cm2) gave positive response in wheat. Rybinski and Garczynski [96] found that the wavelength of 632 nm for 180 min irradiation was more effective

SC

than 60 min irradiation. In case of magnetic field, it have been observed and reported that

NU

specific doses (magnetic strength and exposure duration) was effective for most of the measured parameters [2-4, 6]. So far, it is concluded that laser at same energy level and wavelength was

MA

not effective for the enhancement of different parameters, whereas in case of magnetic field treatment, no such observation have been reported and also in present study consistent effect of

TE

D

magnetic field treatment was observed in comparison to laser treatment. In view of positive effect of laser and magnetic field treatment on plant characteristics [52, 63-68], the application of

AC CE P

laser and magnetic field is an eco-friendly, economical and can be easily employed at farm level for those crops and plant. In spite of recommendations, these methods have not been applied practically, especially in Pakistan and reason is the lack of awareness as well as lack of interest of agricultural organization and lack of facilities provision in the country. Therefore, there is need to conduct experiment at farm level with the participation of farmers and agricultural organization, which will be highly effective for practical application of magnetic and laser in agriculture sector.

4. Conclusions

Pre-sowing soybean seed laser and magnetic field treatments effect was studied on total soluble sugar, reduced sugar, protein, nitrogen contents, H2O2, AsA, proline, phenolic and MDA along

17

ACCEPTED MANUSCRIPT with chlorophyll contents. Specific activity of PTR, AMY, CAT, SOD and POD were also assessed. Both laser and magnetic field seed bio-stimulation enhanced the nutrient contents,

PT

enzymatic activities and chlorophyll contents. Magnetic field individual treatments (different

RI

strength and time of seed exposure) showed variable effect on measured parameters. Higher magnetic field strength was more effect and exposure time effect was found to be insignificant.

SC

Laser treatments duration effect was insignificant, which was also slightly lower than magnetic

NU

field treatment except PRT, AMYL and ascorbic acid contents. However, both treatment effects were highly significant versus control in enhancing biomolecule, enzymatic activities and

MA

chlorophyll contents. So far, both treatments could be used to enhance soybean biological

AC CE P

References

TE

D

characteristics. Future studies should be focused on growth at lateral stages and yield attribute.

[1] S. Pietruszewski, S. Muszynski, A. Dziwulska, Electromagnetic fields and electromagnetic radiation as noninvasive external stimulants for seeds (selected methods and responses), Int. Agrophys. 21 (2007) 95-100. [2] M. Iqbal, Z.u. Haq, Y. Jamil, J. Nisar, Pre-sowing seed magnetic field treatment influence on germination, seedling growth and enzymatic activities of melon (Cucumis melo L.), Biocatal. Agric. Biotechnol. 6 (2016) 176183. [3] M. Iqbal, Z. ul Haq, A. Malik, C.M. Ayoub, Y. Jamil, J. Nisar, Pre-sowing seed magnetic field stimulation: A good option to enhance bitter gourd germination, seedling growth and yield characteristics, Biocatal. Agric. Biotechnol. 5 (2016) 30-37. [4] Y. JAMIL, M.R. AHMAD, Effect of pre-sowing magnetic field treatment to garden pea (Pisum sativum L.) seed on germination and seedling growth, Pak. J. Bot. 44 (2012) 1851-1856. [5] R. Perveen, Y. Jamil, M. Ashraf, Q. Ali, M. Iqbal, M.R. Ahmad, He-Ne Laser-Induced Improvement in biochemical, physiological, growth and Yield characteristics in sunflower (Helianthus annuus L.), Photochemistry and photobiology, 87 (2011) 1453-1463. [6] Z. ul Haq, M. Iqbal, Y. Jamil, H. Anwar, A. Younis, M. Arif, M.Z. Fareed, F. Hussain, Magnetically treated water irrigation effect on turnip seed germination, seedling growth and enzymatic activities, Informat. Process. Agric. 3 (2016) 99-106. [7] Z. ul Haq, Y. Jamil, S. Irum, M.A. Randhawa, M. Iqbal, N. Amin, Enhancement in the germination, seedling growth and yield of radish (Raphanus sativus) using seed pre-sowing magnetic field treatment, Pol. J. Environ. Stud. 21 (2012) 369-374. [8] J. Podlesny, The effect of drought on the development and yielding of two different varieties of the fodder broad bean (Vicia faba minor), J. Appl. Genet. 42 (2001) 283-287. [9] A. Vashisth, S. Nagarajan, Effect on germination and early growth characteristics in sunflower (Helianthus annuus) seeds exposed to static magnetic field, J. Plant Physiol. 167 (2010) 149-156.

18

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

[10] Y.-P. Chen, M. Yue, X.-L. Wang, Influence of He–Ne laser irradiation on seeds thermodynamic parameters and seedlings growth of Isatis indogotica, Plant Sci. 168 (2005) 601-606. [11] Y. Jamil, R. Perveen, M. Ashraf, Q. Ali, M. Iqbal, M.R. Ahmad, He–Ne laser-induced changes in germination, thermodynamic parameters, internal energy, enzyme activities and physiological attributes of wheat during germination and early growth, Laser Phys. Lett. 10 (2013) 045606. [12] A. De Souza, D. García, L. Sueiro, F. Gilart, Improvement of the seed germination, growth and yield of onion plants by extremely low frequency non-uniform magnetic fields, Sci. Hortic. 176 (2014) 63-69. [13] M. Iqbal, Z. Haq, Y. Jamil, M. Ahmad, Effect of presowing magnetic treatment on properties of pea, Int. Agrophys. 26 (2012) 25-31. [14] Z.u. Haq, M. Iqbal, Y. Jamil, H. Anwar, A. Younis, M. Arif, M.Z. Fareed, F. Hussain, Magnetically treated water irrigation effect on turnip seed germination, seedling growth and enzymatic activities, Informat. Process. Agric. 3 (2016) 99-106. [15] S. Amadi, C. Ukpaka, Role of molecular diffusion in the recovery of water flood residual oil, Chem. Int. 2 (2015) 103-114. [16] A. Babarinde, Ogundipe, Kemi, K.T. Sangosanya, B.D. Akintola, A.-O. Elizabeth Hassan, Comparative study on the biosorption of Pb(II), Cd(II) and Zn(II) using Lemon grass (Cymbopogon citratus): kinetics, isotherms and thermodynamics, Chem. Int. 2 (2016) 89-102. [17] A. Babarinde, G.O. Onyiaocha, Equilibrium sorption of divalent metal ions onto groundnut (Arachis hypogaea) shell: kinetics, isotherm and thermodynamics, Chem. Int. 2 (2016) 37-46. [18] R. Gangadhara, N. Prasad, Studies on optimization of transesterification of certain oils to produce biodiesel, Chem. Int. 2 (2016) 59-69. [19] M. Iqbal, R.A. Khera, Adsorption of copper and lead in single and binary metal system onto Fumaria indica biomass, Chem. Int. 1 (2015) 157b-163b. [20] S. Jafarinejad, Control and treatment of sulfur compounds specially sulfur oxides (SO x) emissions from the petroleum industry: a review, Chem. Int. 2 (2016) 242-253. [21] M.A. Jamal, M. Muneer, M. Iqbal, Photo-degradation of monoazo dye blue 13 using advanced oxidation process, Chem. Int. 1 (2015) 12-16. [22] A.O. Majolagbe, A.A. Adeyi, O. Osibanjo, Vulnerability assessment of groundwater pollution in the vicinity of an active dumpsite (Olusosun), Lagos, Nigeria, Chem. Int. 2 (2016) 232-241. [23] R. Mouhamad, A. Atiyah, M. Iqbal, Behavior of Potassium in Soil: A mini review, Chem. Int. 2 (2016) 47-58. [24] M. Asif Tahir, H.N. Bhatti, M. Iqbal, Solar Red and Brittle Blue direct dyes adsorption onto Eucalyptus angophoroides bark: Equilibrium, kinetics and thermodynamic studies, J. Environ. Chem. Eng. 4 (2016) 2431-2439. [25] H.N. Bhatti, Q. Zaman, A. Kausar, S. Noreen, M. Iqbal, Efficient remediation of Zr(IV) using citrus peel waste biomass: Kinetic, equilibrium and thermodynamic studies, Ecol. Eng. 95 (2016) 216-228. [26] I.A. Bhatti, M.A. Hayat, M. Iqbal, Assessment of thorium in the environment (a review), J. Chem. Soc. Pak. 34 (2012) 1012-1022. [27] M. Iqbal, Vicia faba bioassay for environmental toxicity monitoring: a review, Chemosphere. 144 (2016) 785802. [28] M. Iqbal, M. Abbas, M. Arshad, T. Hussain, A.U. Khan, N. Masood, M.A. Tahir, S.M. Hussain, T.H. Bokhari, R.A. Khera, Gamma radiation treatment for reducing cytotoxicity and mutagenicity in industrial wastewater, Pol. J. Environ. Stud. 24 (2015) 2745-2750. [29] M. Iqbal, I.A. Bhatti, Gamma radiation/H2O2 treatment of a nonylphenol ethoxylates: degradation, cytotoxicity, and mutagenicity evaluation, J. Hazard. Mater. 299 (2015) 351-360. [30] M. Iqbal, J. Nisar, Cytotoxicity and mutagenicity evaluation of gamma radiation and hydrogen peroxide treated textile effluents using bioassays, J. Environ. Chem. Eng. 3 (2015) 1912-1917. [31] M. Mushtaq, H.N. Bhatti, M. Iqbal, S. Noreen, Eriobotrya japonica seed biocomposite efficiency for copper adsorption: Isotherms, kinetics, thermodynamic and desorption studies, J. Environ. Manage. 176 (2016) 21-33. [32] R. Nadeem, Q. Manzoor, M. Iqbal, J. Nisar, Biosorption of Pb(II) onto immobilized and native Mangifera indica waste biomass, J. Ind. Eng. Chem. 35 (2016) 185-194. [33] J. Nisar, R. Razaq, M. Farooq, M. Iqbal, R.A. Khan, M. Sayed, A. Shah, I.u. Rahman, Enhanced biodiesel production from Jatropha oil using calcined waste animal bones as catalyst, Renew. Energy. 101 (2017) 111-119. [34] J. Nisar, M. Sayed, F.U. Khan, H.M. Khan, M. Iqbal, R.A. Khan, M. Anas, Gamma–irradiation induced degradation of diclofenac in aqueous solution: Kinetics, role of reactive species and influence of natural water parameters, J. Environ. Chem. Eng. 4 (2016) 2573-2584. [35] A. Rashid, H.N. Bhatti, M. Iqbal, S. Noreen, Fungal biomass composite with bentonite efficiency for nickel and zinc adsorption: a mechanistic study, Ecol. Eng. 91 (2016) 459-471.

19

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

[36] S. Shoukat, H.N. Bhatti, M. Iqbal, S. Noreen, Mango stone biocomposite preparation and application for crystal violet adsorption: A mechanistic study, Micropor. Mesopor. Mater. DOI http://dx.doi.org/10.1016/j.micromeso.2016.10.004(2016). [37] N. Tahir, H.N. Bhatti, M. Iqbal, S. Noreen, Biopolymers composite with peanut hull waste and application for Crystal Violet adsorption, Int. J. Biol. Macromole. 94 (2017) 210–220. [38] R. Mouhamad, A. Atiyah, R. Mohammad, M. Iqbal, Decomposition of organic matter under different soil textures, Curr. Sci. Perspect. 1 (2015) 22-25. [39] S. Padmavathy, N.A. Devi, M. Alagar, R.D. Babu, Characterization and antibiogram pattern of marine bacteria against multi drug resistant pathogens, Curr. Sci. Perspect. 1 (2015) 62-68. [40] C. Ukpaka, E. Wami, S. Amadi, Effect of pollution on metal corrosion: a case study of carbon steel metal in acidic media, Curr. Sci. Perspect. 1 (2015) 107-111. [41] S. Ehsan, S. Sarfraz, B. Khan, S.M. Hassan, M. Iqbal, A rapid solid supported synthesis of 4-amino-5-(pyridin4-YL)-4H-1, 2, 4-triazol-3-thiol and its derivatives, Chem. Int. 2 (2016) 262-266. [42] M. Pervaiz, K.M. Butt, M.A. Raza, A. Rasheed, S. Ahmad, A. Adnan, M. Iqbal, Extraction and applications of aluminum hydroxide from bauxite for commercial consumption, Chem. Int. 1 (2015) 99-102. [43] U.C. Peter, U. Chinedu, Model prediction for constant area, variable pressure drop in orifice plate characteristics in flow system, Chem. Int. 2 (2016) 80-88. [44] K. Qureshi, M. Ahmad, I. Bhatti, M. Iqbal, A. Khan, Cytotoxicity reduction of wastewater treated by advanced oxidation process, Chem. Int. 1 (2015) 53-59. [45] M. Sayed, Efficient removal of phenol from aqueous solution by the pulsed high-voltage discharge process in the presence of H2O2, Chem. Int. 1 (2015) 81-86. [46] H. Shindy, Basics in colors, dyes and pigments chemistry: A review, Chem. Int. 2 (2016) 29-36. [47] C. Ukpaka, Development of model for bioremediation of crude oil using moringa extract, Chem. Int. 2 (2016) 19-28. [48] C. Ukpaka, Predictive model on the effect of restrictor on transfer function parameters on pneumatic control system, Chem. Int. 2 (2016) 128-135. [49] C. Ukpaka, Empirical model approach for the evaluation of pH and conductivity on pollutant diffusion in soil environment, Chem. Int. 2 (2016) 267-278. [50] C. Ukpaka, BTX Degradation: The concept of microbial integration, Chem. Int. 3 (2016) 8-18. [51] Y. Zhao, D. Wang, X. Li, Y. Liu, Determination of rutin, chlorogenic acid and quercetin in solidaginis by large volume sample stacking with polarity switching and acid barrage stacking, Chem. Int. 2 (2016) 121-127. [52] A. Hernandez, P. Dominguez, O. Cruz, R. Ivanov, C. Carballo, B. Zepeda, Laser in agriculture, Int. Agrophys. 24 (2010) 407-422. [53] M. Yang, J. Fu, L. Li, Rheological characteristics and microstructure of probiotic soy yogurt prepared from germinated soybeans, Food Technol. Biotechnol. 50 (2012) 73-80. [54] B.F. Gibbs, A. Zougman, R. Masse, C. Mulligan, Production and characterization of bioactive peptides from soy hydrolysate and soy-fermented food, Food Res. Int. 37 (2004) 123-131. [55] G. Sakthivelu, M. Akitha Devi, P. Giridhar, T. Rajasekaran, G. Ravishankar, M. Nikolova, G. Angelov, R. Todorova, G. Kosturkova, Isoflavone composition, phenol content, and antioxidant activity of soybean seeds from India and Bulgaria, J. Agric. Food Chem. 56 (2008) 2090-2095. [56] B. Rasaei, M.-E. Ghobadi, M. Khas-Amiri, M. Ghobadi, Effect of osmotic potential on germination and seedling characteristics of soybean seeds, Int. J. Agric. Crop Sci. 5 (2013) 1265. [57] C.J. Willenborg, R.H. Gulden, E.N. Johnson, S.J. Shirtliffe, Germination characteristics of polymer-coated Canola (L.) seeds subjected to moisture stress at different temperatures, Agron. J. 96 (2004) 786-791. [58] M.B. Khan, A. Khaliq, Production of soybean (Glycine max L.) as cotton based intercrop, J. Res. Sci. 15 (2004) 79-84. [59] Z. Fatima, M. Zia, M.F. Chaudhary, Effect of Rhizobium strains and phosphorus on growth of soybean Glycine max and survival of Rhizobium and P solubilizing bacteria, Pak. J. Bot. 38 (2006) 459-464. [60] P. PAR, http://edu.par.com.pk/wiki/oil-seeds/soybean/. [61] A. Wahid, E. Milne, S.R.A. Shamsi, M.R. Ashmore, F.M. Marshall, Effects of oxidants on soybean growth and yield in the Pakistan Punjab, Environ. Poll. 113 (2001) 271-280. [62] C. Liu, Y. Zhao, J. Liu, W. Gen, Y. Cheng, The effects of ethylene on the HCl-extractability of trace elements during soybean seed germination, Elect. J. Biotechnol. 18 (2015) 333-337. [63] M.E. Maffei, Magnetic field effects on plant growth, development, and evolution, Front. Plant Sci. 5 (2014) 445. DOI 10.3389/fpls.2014.00445.

20

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

[64] F. Dhawi, Why Magnetic Fields are Used to Enhance a Plant's Growth and Productivity?, Ann. Res. Rev. Biol. 4 (2014) 886-86. [65] J.A.T. da Silva, J. Dobránszki, Impact of magnetic water on plant growth, Environ. Exp. Biol. 12 (2014) 137– 142. [66] P. Galland, A. Pazur, Magnetoreception in plants, J. Plant Res. 118 (2005) 371-389. [67] R.W. Hunt, A. Zavalin, A. Bhatnagar, S. Chinnasamy, K.C. Das, Electromagnetic biostimulation of living cultures for biotechnology, biofuel and bioenergy applications, Int. Jo. Mole. Sci. 10 (2009) 4515-4558. [68] C. Hernández-Aguilar, A. Domínguez-Pacheco, A. Cruz-Orea, A. Podleśna, R. Ivanov, A. Carballo Carballo, M.C. Pérez Reyes, G. Sánchez Hernández, R. Zepeda Bautista, J.L. López-Bonilla, Bioestimulación láser en semillas y plantas, Gayana. Botánica, 73 (2016) 132-149. [69] M.M. Bradford, A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding, Anal. Biochem. 72 (1976) 248-254. [70] T. Ma, G. Zuazaga, Micro-Kjeldahl determination of nitrogen. A new indicator and an improved rapid method, Ind. Eng. Chem. Anal. Ed. 14 (1942) 280-282. [71] I. Cakmak, W.J. Horst, Effect of aluminium on lipid peroxidation, superoxide dismutase, catalase, and peroxidase activities in root tips of soybean (Glycine max), Phys. Plant. 83 (1991) 463-468. [72] R. Julkunen-Tiitto, Phenolic constituents in the leaves of northern willows: methods for the analysis of certain phenolics, J. Agric. Food Chem. 33 (1985) 213-217. [73] P. Harinasut, D. Poonsopa, K. Roengmongkol, R. Charoensataporn, Salinity effects on antioxidant enzymes in mulberry cultivar, Sci. Asia. 29 (2003). [74] S. Ünyayar, Y. Keleþ, E. Ünal, Proline and ABA levels in two sunflower genotypes subjected to water stress, Bulg. J. Plant Physiol. 30 (2004) 34-47. [75] S. Sadasivam, Biochemical methods, New Age International, New Age International, (1996) Pp. 256. [76] J. Tabart, C. Kevers, D. Evers, J. Dommes, Ascorbic acid, phenolic acid, flavonoid, and carotenoid profiles of selected extracts from Ribes nigrum, J. Agric. Food Chem. 59 (2011) 4763-4770. [77] M. Maqsood, M.A. Shehzad, S.N.A. Ali, M. Iqbal, Rice cultures and nitrogen rate effects on yield and quality of rice (Oryza sativa L.), Turk. J. Agric. For. 37 (2013) 665-673. [78] G.R. Drapeau, [38] Protease from Staphyloccus aureus, Method Enzymol. 45 (1976) 469-475. [79] B. Chance, A. Maehly, [136] Assay of catalases and peroxidases, Method Enzymol. 2 (1955) 764-775. [80] C.N. Giannopolitis, S.K. Ries, Superoxide dismutases I. Occurrence in higher plants, Plant Physiol. 59 (1977) 309-314. [81] S. Varavinit, N. Chaokasem, S. Shobsngob, Immobilization of a thermostable alpha-amylase, Sci. Asia. 28 (2002) 247-251. [82] Z. Qi, M. Yue, X.-L. Wang, Laser pretreatment protects cells of broad bean from UV-B radiation damage, J. Photochem. Photobiol. B: Biol. 59 (2000) 33-37. [83] M.M. Rahman, L.A. Banu, M.M. Rahman, U.F. Shahjadee, Changes of the enzymes activity during germination of different mungbean varieties, Bangladesh J. Sci. Ind. Res. 42 (2007) 213-216. [84] Y. Yao, Y. Li, Y. Yang, C. Li, Effect of seed pretreatment by magnetic field on the sensitivity of cucumber (Cucumis sativus) seedlings to ultraviolet-B radiation, Environ. Exp. Bot. 54 (2005) 286-294. [85] J. Podleśny, A. Stochmal, A. Podleśna, L.E. Misiak, Effect of laser light treatment on some biochemical and physiological processes in seeds and seedlings of white lupine and faba bean, Plant Growth Regulat. 67 (2012) 227233. [86] A. Muthusamy, P.P. Kudwa, V. Prabhu, K.K. Mahato, V.S. Babu, M.R. Rao, P.M. Gopinath, K. Satyamoorthy, Influence of Helium‐Neon laser irradiation on seed germination in vitro and physico‐biochemical characters in seedlings of Brinjal (Solanum melongena L.) var. Mattu Gulla, Photochem. Photobiol. 88 (2012) 1227-1235. [87] R. Perveen, Y. Jamil, M. Ashraf, Q. Ali, M. Iqbal, M.R. Ahmad, He‐Ne Laser‐Induced Improvement in Biochemical, Physiological, Growth and Yield Characteristics in Sunflower (Helianthus annuus L.), Photochem. Photobiol. 87 (2011) 1453-1463. [88] A. Hoff, Magnetic field effects on photosynthetic reactions, Quart. Rev. Biophys. 14 (1981) 599-665. [89] F. Dhawi, J.M. Al-Khayri, The effect of magnetic resonance imaging on date palm (Phoenix dactylifera L.) elemental composition, Comm. Biomet. Crop Sci. 4 (2009) 14-20. [90] J. Podleoeny, S. Pietruszewski, A. Podleoena, Efficiency of the magnetic treatment of broad bean seeds cultivated under experimental plot conditions, Int. Agrophys. 18 (2004) 65-71. [91] B. Stange, R. Rowland, B. Rapley, J. Podd, ELF magnetic fields increase amino acid uptake into Vicia faba L. roots and alter ion movement across the plasma membrane, Bioelectromagnetic. 23 (2002) 347-354.

21

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

RI

PT

[92] S.R. Govil, D. Agrawal, K. Rai, S. Thakur, Growth responses of Vigna radiata seeds to laser irradiation in the UV‐A region, Physiol. Plant. 63 (1985) 133-134. [93] A. Hernandez, C. Carballo, A. Artola, A. Michtchenko, Laser irradiation effects on maize seed field performance, Seed Sci. Technol. 34 (2006) 193-197. [94] Y.A. Osman, K.M. El-Tobgy, E. El-Sherbini, Effect of laser radiation treatments on growth, yield and chemical constituents of fennel and coriander plants, J. Appl. Sci. Res. 5 (2009) 244-252. [95] S. Saghafi, A. Heydai, M. Nami, B. Shadid, Influence of laser irradiation on wheat growth. Advanced Optoelectronics and Lasers, Proc. 2nd Int. Conf. CAOL (2005) pp. 12-17. [96] W. Rybiñski, S. Garczyñski, Influence of laser light on leaf area and parameters of photosynthetic activity in DH lines of spring barley (Hordeum vulgare L.), Int. Agrophys. 18 (2004) 261-268.

22

AC CE P

TE

D

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 1: Laser and magnetic field set up used for seed treatment (A) Laser and (B) Magnetic field

23

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

D

Fig. 2: Effect of laser and magnetic field treatment on soybean various biochemical parameters (a) total soluble protein (µg/mL), (b) proline (µmole/g), (c) phenolic contents (µg/mL), (d) total

AC CE P

TE

soluble sugar (%), (e) reducing sugar (%) and (f) nitrogen and protein (%)

24

RI

PT

ACCEPTED MANUSCRIPT

AC CE P

TE

D

MA

NU

SC

Fig. 3: Effect of laser and magnetic field treatment on soybean biochemical; (a) hydrogen peroxide (U/mg), (b) Malondialdehyde (nanomole) and (c) ascorbic acid (µg/mL).

25

MA

NU

SC

RI

PT

ACCEPTED MANUSCRIPT

Fig. 3: Effect laser and magnetic field pre-sowing seed exposure on soybean enzymatic activities

D

(a) protease (U/mL), (b) amylase (U/mL), (c) superoxidedismutase (IU/mg of protein), (d)

AC CE P

TE

catalase (U/mg protein) and (e) peroxidase (U/mL)

26

RI

PT

ACCEPTED MANUSCRIPT

SC

Fig. 4: Effect laser and magnetic field pre-sowing seed exposure on soybean chlorophyll contents

AC CE P

TE

D

MA

NU

(a) (a) chlorophyll a (mg/g), (b) chlorophyll b (mg/g), (c) total chlorophyll content (mg/g)

27

ACCEPTED MANUSCRIPT Highlights

TE

D

MA

NU

SC

RI

PT

Soybean seeds were undergone to laser and magnetic field pre-sowing treatments The biomolecule, enzymes and chlorophyll contents enhanced significantly Magnetically treated seeds showed slightly higher response versus laser irradiation Both treatments could be used to enhance the soybean biological characteristics

AC CE P

   

28