Effect of H e Ne Laser Irradiation and Fe 3 O 4 N P s Foliar Spray on Growth and Yield of Sweet Basil Plant

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Effect of H e Ne Laser Irradiation and Fe 3 O 4 N P s Foliar Spray on Growth and Yield of Sweet Basil Plant
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  14102 , Issue.46, Vol.4263- N:2051ISS, Indigenous Medicinal PlantsJournal of International   © RECENT SCIENCE PUBLICATIONS ARCHIVES| November 2013|$25.00 | 27702817 |*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions* Effect of He Ne Laser Irradiation and Fe 3 O 4 NPs Foliar Spray on Growth and Yield of Sweet Basil Plant Mohammed. A. M. El-Kereti   Department of Laser Applications in Metrology, Photochemistry and Agriculture. National Institute of Laser Enhanced Science (NILES), Cairo University, Egypt. 12613   Souad. A. El-feky   Department of Laser Applications in Metrology, Photochemistry and Agriculture. National Institute of Laser Enhanced Science (NILES), Cairo University, Egypt. 12613   Mohammed S. Khater    Department of Laser Applications in Metrology, Photochemistry and Agriculture. National Institute of Laser Enhanced Science (NILES), Cairo University, Egypt. 12613. Department of Chemistry, University of Bath, Claverton Down, BA2 7AY, United Kingdom. Yasser A. H. Osman   Medicinal and Aromatic Plants Department, Desert Research Center (DRC), Egypt.   El-Sayed. El-Sherbini   Department of Laser Applications in Metrology, Photochemistry and Agriculture. National Institute of Laser Enhanced Science (NILES), Cairo University, Egypt. 12613 Corresponding Author E-mail: sae21@bath.ac.uk    ABSTRACTT his Study Was Conducted To Evaluate The Effectiveness Of An Iron Nanoparticles Strategy On Sweet Basil Yield, Through Alone Application Or Combined With Pre-Sowing Laser Irradiation. Furthermore, Evaluate The Growth Of Plant And The Level Of Active Essential Oil Constituents. Furthermore, Evaluate The Growth Of Plant And The Level Of Active Essential Oil Constituents. Iron Oxide (Fe3O4) Nanoparticles (NPs) Were Synthesized, And Transmission Electron Micrography Revealed Particle Size Of Approximately 12.6 nm. Fe3O4 NPs Were Applied To Sweet Basil Plants By Foliar Spray At Varying Concentrations (10, 20 And 30 Mg/L) ; He-Ne Laser Of Power 3mw Was Used For Red Light Irradiation Of Ocimum Basilicum  Seeds For 2 Min. Exposure Time. Total Chlorophyll, Total Carbohydrate, Essential Oil Levels, Iron Content, Plant Height, Branches/Plant, Fresh Weight And Dry Weight Were Measured. In general, the combined foliar spray application of Fe 3 O 4   NPs with pre-sowing He Ne laser irradiation showed more effectiveness than Fe 3 O 4  NPs treatment alone and 30 mg/L concentration gave the highest results of all measured traits. Statistical analysis (t-test) showed significant differences among the effects of the various concentrations of Fe 3 O 4  NPs on these attributes. Our results showed an inverse relationship between total carbohydrate content and the  percentage of essential oil in the leaves. Together these findings support the usefulness and effectiveness of iron oxide nanoparticles and laser irradiation treatment of  plants to enhance growth and yield of sweet basil plants. Keywords-  Basil, yield, carbohydrate, chlorophyll, Fe 3 O 4 nanoparticles, essential oil, He Ne laser 1. INTRODUCTION S weet basil ( Ocimum basilicum L.  )  is a popular annual herb of the Lamiaceae (or Labiatae) family. Basil is a tropical species native to India and East Africa  [1]  Essential oils are extracted from basil by steam distillation from the leaves and flowering tops, and used for food flavoring, dental and oral products, fragrances and in traditional rituals and medicines  [2] . The essential oils have also been shown to contain biologically active constituents with insecticidal  [3] , antimicrobial  [4]  and antifungal properties  [5] . These properties can be attributed to Predominant essential oil constituents, such as methyl chavicol, eugenol, linalool, camphor, and methyl cinnamate  [6] . Two minor components,  juvocimene I and II have been reported as potent juvenile hormone analogs [7] . Several studies have reported these essential oil components can be enhanced with some herbs through iron foliar fertilization  [8], [9] and [10].   Nano-agriculture involves the employment of nanoparticles in agriculture with the particles imparting specific beneficial effects to the crops  [11] . The success of nanoparticles may be due to, a greater density in reactive areas, or increased reactivity of these areas on the  particles surfaces. Nanotechnology is a potential solution for increasing the value of agricultural products and environmental problems. For example, with the use of nanoparticles and nanopowders, researchers can produce controlled or delayed release fertilizers  [12] . In addition, several studies have shown that nanoparticles can have a  beneficial effect on seedlings growth and development    14112 , Issue.46, Vol.4263- N:2051ISS, Indigenous Medicinal PlantsJournal of International   © RECENT SCIENCE PUBLICATIONS ARCHIVES| November 2013|$25.00 | 27702817 |*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions* [13] . Iron, a cofactor for approximately 140  enzymes  [14] ,  plays an important role in the photosynthetic reactions and is one of the essential elements for plant growth and development, including chlorophyll synthesis, thylakoid synthesis and chloroplast development  [15] . Iron contributes to RNA synthesis and improves the  performance of photosystems  [16] . Application of iron in low-iron soils can increase grain yield in soybean  [17] . Sala, one of the first researchers to demonstrate the effects of magnetic nanoparticles in  plants, showed an increase of chlorophyll levels and  photosynthesis rate in seven-day-old bean seedlings following the addition of 0.1% magnetite-based magnetic fluid in the culture medium [18] . Laser irradiation is considered as a new branch of agriculture; especially, in medicinal plants. The effect of different types of laser on seed germination and the rate of the growth of plants were carried out. The results showed improvements in sowing qualities of the seeds, shortened the phases of plant development, produced more vigorous  plants, increased the yields of both stems and seeds to a considerable extent and increasing the germination by 10-15% [19]  and [20] . The stimulating effect of radiation was also observed on plant height, stalk thickness, height of the first ear formation, number of leaves and leaf area size [21]  and [22] . It was reported that the mutation frequency of plants derived from wet seeds being higher than that of  plants derived from irradiated dry seeds  [23]  and [24] .  In this study, we applied the iron oxide nanoparticle strategy alone and with the combined application of pre-sowing He Ne laser irradiation to examine the possible improvement of sweet basil growth and yield. We also examined the effects of laser irradiation and Fe 3 O 4  NPs on the levels of the active constituents, with the aim of developing a technical approach for the agricultural application of economical nano-materials. 2. MATERIALS AND METHODS 2.1. Synthesis of Fe 3 O 4  magnetic nanoparticles (MNPs) The Fe 3 O 4  magnetic nanoparticles were prepared by co- precipitation of Fe 3+  and Fe 2+  at a molar ratio of 3:2 with aqueous ammonia (0.3mol/L) as precipitating agent  [25] .   2.2. Characterization of Fe 3 O 4  magnetic nanoparticles (MNPs) The size and shape of Fe 3 O 4  magnetic nanoparticles were observed directly by transmission electron microscope (TEM) using an electron acceleration voltage of 60 kV. The TEM samples were prepared by placing a few drops of the solution on a carbon-coated copper grid (Okenshoji Co., Ltd., microgrid B). 2.3. He-Ne laser irradiation He-Ne laser of power 3mW was used for red light irradiation of Ocimum basilicum  seeds. The spot diameter of He-Ne laser was 2 mm, thus it is suitable for direct irradiation to basil seeds without any divergence. The  power density was calculated as (95 mW/cm 2 ). The 2mm spot diameter was enough for one seed exposure in each time fig (1). 2.4. Seed treatment Ocimum basilicum seeds were treated before cultivation with fungicides (Topsin M 70% w.p./vitavax 300% w.p.) at 2.5 g/kg. This treatment is generally used to prevent any fungal damage during cultivation  [26] . All treatment experiments were repeated three times. 2.5. Field cultivation A total of 1500 Ocimum basilicum  seeds was used for the study. The seeds were divided into three main groups for field cultivation; the first group was considered the control, the second group for Fe 3 O 4  NPs foliar spray alone and the last group for Fe 3 O 4  NPs foliar spray combined with the pre-sowing He Ne laser irradiation. Ocimum basilicum  seeds were first sown on foam trays (84 cells) on February 15 th . The media for seed germination contained peat moss: vermiculite (1:3 by volume). On April 1 st  uniform seedlings of approximately 15 cm in height were transplanted to an open field. The uniform seedlings were transplanted into plots 3x3.5m on rows with 30 cm between the seedlings .  Morphological characteristics such as plant height (cm), number of branches, number of leaves per plant, and both fresh and dry matter of the herb were recorded. 2.6. Leaf anatomy study Leaves and stem samples of Ocimum basilicum L. were collected from the mature leaves (fourth node on the shoot from the terminal end) during September 2012. The specimens were taken from the leaf between the mid vein and the leaf margin and then fixed in formalin-acetic acid-alcohol (FAA)) using 70% ethanol. The specimens were gradually dehydrated in a tert-butyl alcohol (TBA) series  [27], and embedded in paraffin wax (m.p. 56 °C). Sections were cut on a rotary microtome at a thickness of 8-10 µm (Model RM2245, Leica Microsystems). Paraffin was removed with xylol and slides were stained with safranin FCF methanol and fast green, and then mounted in Canada balsam  [27] . The selected sections were Figure (1) . Set up of He-Ne laser irradiation of sweet basil seeds .    14122 , Issue.46, Vol.4263- N:2051ISS, Indigenous Medicinal PlantsJournal of International   © RECENT SCIENCE PUBLICATIONS ARCHIVES| November 2013|$25.00 | 27702817 |*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*examined and photographed using a light microscope (Model BX51; Olympus Optical). 2.7. Essential oil extraction Oil was extracted from the plants by hydro distillation. A laboratory distillation of essential oil from plant herb is often necessary in order to evaluate the raw material to be used in large-scale distillation. A sufficient quantity of the  plant (25 g) and water was added in a short neck round  bottom 250 ml flask. The proper essential oil trap and condenser were attached to the flask, and water was added to fill the trap. The flask was placed in an oil bath, and electrically heated to approximately; 130 о C. The temperature of the bath was adjusted so that a condensate of about one drop per second was obtained. The distillation was continued until collection of oil ceased (approximately three hours). When the distillation was complete, the oil was left undisturbed so that good separation was obtained. The volume obtained was determined and the yield was expressed as a volume/weight percentage, i.e., volume of oil per 100 g of  plant herb. The crude oil was dried over pure anhydrous sodium sulfate (120  –  150 g/L of oil). The mixture was stored at 4 o C and kept in closed bottles in the dark to  prevent light and oxygen exposure  [28] . 2.8. Determination of carbohydrates The percentage of carbohydrate content was determined  by 3, 5-dinitrosalicylic acid (DNS) [29] . A mixture of 1 g dinitrosalicylic acid, 200 mg crystalline phenol and 50 mg sodium sulfate in 100 ml 1% NaOH was dissolved by stirring, and stored at 4°C. Leaf samples (100 mg) were extracted with hot 80% ethanol two times (5 ml each time). The supernatant was collected and evaporated by incubating the sample in the 80°C water bath. Water (10 ml) was added to dissolve the sugars, and three samples (each 3 ml) were elected in three separate tubes, together with an additional 3 ml of water in each tube. DNS reagent (3 ml) was added, and the samples were heated in a boiling water bath for 5 min. with the contents of the tubes still warm, 1 ml of 40% Rochelle salt solution was added. After cooling, the intensity of the dark red color absorption was read at a wavelength of 510 nm on a spectrophotometer. 2.9. Measurement of chlorophyll content   Measurement of chlorophyll content was performed using the SPAD-502 meter (Konica-Minolta, Japan). The instrument measures the transmission of red light at 650 nm, at which chlorophyll absorbs light, and transmission of infrared light at 940 nm, at which no absorption occurs  [30] .   2.10. Determination of plant macronutrient content A half gram of each plant sample was digested using the H 2 SO 4  and H 2 O 2  [31] . The extracted samples were used to determine the nitrogen content in the digested solution by the modified microkjeldahl method  [32] . Crude  phosphorus content was determined colorimetrically  [33] . Potassium and sodium content were determined against a standard using flame photometer   [34] . 2.11. Determination of plant micronutrient content Each ashed sample was dissolved in 0.1N HCl and the volumes were completed to the mark (100ml). This solution was used for the quantitative determination of Fe, Zn and Cu by atomic absorption spectrophotometer (Thermo Jarrell Ash Model AA SCAN1). All the determinations were carried out with an air-acetylene gas mixture at a rate of 5L/min according to the method described by Reuter (1980). 2.12. T-test statistical analysis Average values and standard deviations were calculated; t-test was applied to assess the statistical significance of the differences between the control and magnetic nanoparticle treated plants using srcinPro. 3. RESULTS 3. 1. Characterization of magnetite nanoparticles Physicochemical properties of Fe 3 O 4  nanoparticles were characterized via TEM imaging Fig (2). The images of the synthesized magnetite nanoparticles reveal a spherical shape and an average particle size of 12.6.0±2.0 nm. 3. 2. The effect of foliar applied iron oxides NPs on the growth and plant elements content.   Fig (3) shows that the application of Fe 3 O 4  NPs (30mg) increased tissue iron content to level two times higher than controls. Fe 3 O 4  NPs foliar spray alone of 10, 20 and 30 mg/l significantly increased shoot iron concentrations  by about 1.68-, 1.71- and 1.88-fold, respectively; however, pre-sowing He Ne laser irradiation combined with Fe 3 O 4  NPs foliar spray of the same concentration of Fe 3 O 4  NPs increased levels by approximately 1.85-, 1.91- and 1.97-fold, respectively, compared to controls. Figure 2:  TEM imaging of the prepared magnetite nanoparticles revealed a spherical shape of the  particles, with an average size of 12.6±2 . 0 nm (inset shows electron diffraction pattern).  14132 , Issue.46, Vol.4263- N:2051ISS, Indigenous Medicinal PlantsJournal of International   © RECENT SCIENCE PUBLICATIONS ARCHIVES| November 2013|$25.00 | 27702817 |*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions* Table (1) shows that the foliar application of Fe 3 O 4  NPs results in significantly increasing of plant N, P and K, Fe , Zn and Cu through the combined application of pre-sowing laser irradiation with Fe 3 O 4  NPs foliar spray than Fe 3 O 4  NPs foliar spray treatment alone and their levels increased whenever Fe 3 O 4  NPs concentration increased. 3.3 Study the effect of Fe 3 O 4  NPs treatment on the anatomical structure of sweet basil leaf As shown in (Fig. 4a) the epidermis cells of the control were similar in their shape and size, while the epidermal cells of the NPs-treated leaves became larger in size and reached a maximum size at 30 mg/L Fe 3 O 4  NPs (Fig. 4b). In addition, the thickness of mesophyll tissue, which is specialized photosynthetic tissue that contains chloroplasts in the palisade and spongy parenchyma tissue, was greater in pre-sowing laser irradiation combined with Fe 3 O 4  NPs treatment at 30 mg/L compared to Fe 3 O 4  NPs treatment alone and control leaves (Fig. 4c). The air spaces were largest in laser irradiation combined with Fe 3 O 4  NPs compared to Fe 3 O 4  NPs treatment alone and control leaves. These findings were clearly based on the chlorophyll concentration, which was higher in leaves receiving pre-sowing laser irradiation combined with Fe 3 O 4  NPs than Fe 3 O 4  NPs treatment alone. (a) (b) (c) 3.4. Study the effect of Fe 3 O 4  NPs treatment on chlorophyll content As shown in fig. (5) The foliar treatment of basil with varying concentrations of Fe 3 O 4  NPs (10, 20 and 30 mg/L) significantly increased the chlorophyll content in the experimental plants than the control ones (untreated). While the application of Fe 3 O 4  NPs at 30 mg/L for the  plants, which cultivated from He Ne laser irradiated seeds had a marked enhancement of the total chlorophyll content compared to the plants treated by Fe 3 O 4  NPs alone. The results were (48 SPAD units for plants cultivated from the combined application of pre-sowing laser 050010001500control10mg/L20mg/L30mg/L    F   e    (   p   p   m    ) NPs materials concentrations Fe3O4 NPs + He NeFe3O4 NPs0204060control10mg/L20mg/L30mg/L    T   o   t   a    l   c    h    l   o   r   o   p    h   y    l    l    (   S   P   A   D    ) NPs materials concentrations Fe3O4 NPs + He NeFe3O4 NPs Treatment N (%) P (%) K (%) Cu (ppm) Zn (ppm) Fe3O4 NPs Control. 1.22 0.108 2.21 14 132 10mg/L 3.31 0.114 4.42 22 281 20mg/L 3.47 0.222 4.84 29 344 30mg/L 3.83 0.244 5.44 37 318 Fe3O4 NPs + He Ne laser Control. 1.22 0.108 2.21 14 132 10mg/L 3.73 0.402 5.98 28 338 20mg/L 3.88 0.506 7.31 35 404 30mg/L 4.25 0.624 8.79 46 355 Table (1):  The Effect of Different Concentrations of Fe 3 O 4  NPs Foliar Spray on N, P, K, Fe, Zn and Cu % of Ocimum basilicum  plants Cultivated from Seeds Treated with and without Pre-sowin He Ne Laser Irradiation.     Figure (5)  Effect of different concentrations of Fe 3 O 4  NPs foliar spray on leaves total chlorophyll (SPAD units) of Ocimum basilicum  plants cultivated from seeds treated with and without pre-sowing He Ne laser irradiation. Figure (3):   The effect of different concentrations of Fe 3 O 4   NPs foliar spray on Fe content (ppm) of Ocimum basilicum    plants with and without pre-sowing He Ne laser irradiation. Figure (4) (4a ): Leaf cross section in Ocimum basilicum control plants. ( 4b ): 30 mg/L Fe 3 O 4  by foliar spray application. ( 4c ): cultivated from pre-sowing He Ne laser irradiation and Fe 3 O 4 foliar spray (30 mg /L). ( EP :Epidermis, P : Palisade, Sp : Spongy, as : Air spaces ).    14142 , Issue.46, Vol.4263- N:2051ISS, Indigenous Medicinal PlantsJournal of International   © RECENT SCIENCE PUBLICATIONS ARCHIVES| November 2013|$25.00 | 27702817 |*This article is authorized for use only by Recent Science Journal Authors, Subscribers and Partnering Institutions*irradiation with Fe 3 O 4  NPs vs. 43 SPAD units for the  plants cultivated from Fe 3 O 4  NPs alone and 25 SPAD units for control plants.   3.5. The effect of MNPs (Fe 3 O 4  NPs) on total carbohydrates and essential oil (Figures 6 and 7) showed that plants treated with the highest iron oxide NPs concentration at (30 mg/L) had the lowest total carbohydrate concentrations (14.5% and 19.4%, of pre-sowing laser irradiation combined with Fe 3 O 4  NPs and Fe 3 O 4  NPs treatment alone, respectively. While the same plants had the highest essential oil content (1.4% and 0.77 %,) respectively, due to consumption of carbohydrate in the oil production process, which confirmed our proposed relationship between carbohydrate content and oil production. The total carbohydrate content of laser irradiation combined with Fe 3 O 4  NPs foliar spray (14.5%) was less than Fe 3 O 4  NPs treatment alone (19.4%) (Fig.6). This could be explained from the view of the higher essential oil in plants cultivated from the combined application of laser irradiation with Fe 3 O 4  NPs than the plants treated with Fe 3 O 4  NPs alone (1.4% and 0.77%) respectively(Fig.7).. 3.6. Morphological characterization and yield The data presented in Table (2) showed that pre-sowing laser irradiation combined with Fe 3 O 4  NPs and Fe 3 O 4  NPs treatment alone significantly increased plant height, number of leaves/plant and number of branches/plant, especially at 30 mg/L Fresh and dry weight of herb followed the same trend. In addition, the results presented in Table (2) show that pre-sowing laser irradiation and Fe 3 O 4  NPs foliar spray had an observable positive effect higher than Fe 3 O 4  NPs treatment alone on all measurements. Pre-sowing He Ne laser irradiation combined with Fe 3 O 4  NPs treatment at 30mg/L increases the plant height from 93 (control) to 138 cm, fresh weight from 268 (control) to 803gm, dry matter from 79.3 (control) to 84.9%, number of branches/plant from 45 (control) to 100 and the number of leaves/plant from 744 (control) to 1521. In case of Fe 3 O 4  NPs foliar treatment alone, 30mg/L , the plant height increases from 93 (control) to 132 cm, fresh weight of 268 (control) to 725gm, dry matter from 79.3 (control) to 82.5%, number of branches/plant from 45 (control) to 85 and the number of leaves/plant from 744 (control) to 1300.   0102030control10mg/L20mg/L30mg/L    T   o   t   a    l   c   a   r    b   o    h   y    d   r   a   t   e   % NPs materials concentrations Fe3O4 NPs + He NeFe3O4 NPs00.511.5control10mg/L20mg/L30mg/L    E   s   s   e   n   t   i   a    l   o   i    l   % NPs materials concentrations Fe3O4 NPs + He NeFe3O4 NPs Treatment Plant Height (cm) Fresh Weight (g) Dry Matter % Branches /plant Leaves/plant Fe3O4 NPs Control. 93 268 79.3 45 744 10mg/L 123 582 81.3 74 1264 20mg/L 127 669 82.1 79 1277 30mg/L 132 725 82.5 85 1300 Fe3O4 NPs + He Ne laser Control. 93 268 79.3 45 744 10mg/L 133 661 82.4 85 1365 20mg/L 134 722 83.6 96 1443 30mg/L 138 803 84.9 100 1521 Figure (6) Effect of different concentrations of Fe 3 O 4  NPs foliar spray on leaves total carbohydrate % of Ocimum basilicum  plants cultivated from seeds treated with and without pre-sowing He Ne laser irradiation.  Figure (7) Effect of different concentrations of Fe 3 O 4  NPs foliar spray on leaves essential oil % of Ocimum basilicum  plants cultivated from seeds treated with and without pre-sowin He Ne laser irradiation.   Table (2): The Effect of Different Concentrations of Fe 3 O 4  NPs Foliar Spray on Morphological Parameters of Ocimum basilicum Plants Cultivated from Seeds Treated with and without Pre-sowing He Ne Laser Irradiation  
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