The Effect of Low-Pressure Dielectric Barrier Discharge (LPDBD) Plasma in Boosting Germination, Growth, and Nutritional Properties in Wheat

Plasma agriculture is an emerging technology, although the application of non-thermal plasma in wheat productivity is still in its early stage. This study deciphers the effect and mechanistic basis of non-thermal air-generated LPDBD (low-pressure dielectric barrier discharge) plasma in boosting germination, growth and nutritional properties in wheat. Seeds treated with LPDBD plasma exhibited cracked periphery and discernible expansion during seed germination. LPDBD plasma applied for 6 min showed a 22.11% increase in the germination percentage and a substantial increase in iron content in grains compared to non-treated controls. At the cellular level, the concentration of H2O2 in leaves significantly increased (3.56 µM g−1 FW) due to LPDBD treatment compared to controls. This increased level of H2O2 may act as a stimulating agent to trigger the physiological functions in wheat plants. In addition, plants sprouted from air-treated seeds exhibited a marked elevation in CAT and SOD activity accompanied by the increased expression of TaCAT and TaSOD genes in roots of wheat. Interestingly, the grain yield of wheat increased by 27.06% in response to plasma treatment compared to control. Further, grains harvested from plasma-treated plants showed a substantial elevation in iron and fat content as well as decreased moisture content that may contribute to the increased shelf life. The study will open up a new avenue for practical application of plasma in agriculture.


Introduction
For several years a huge number of people around the globe can not meet their daily food needs without charitable assistance, primarily driven by two factors: persistent instability in food price and adverse climatic events [1]. Food prices rushed in 2008, sinking millions into hunger and triggering riots in developing countries [2]. The United Nations Food and Agriculture Organization (FAO) indicated that in 2010, the entire global production of grain was accounted for about 2.216 billion tons against a consumption rate of 2.254 billion tons. This resulted in the starvation of nine million people, consequentially an elevation in demand of food is occurring on a par with the rapid population expansion globally [3]. An analysis of the Food and Agriculture Organization (FAO) predicted a remarkable extension in the world population by 2050. Based on this forecast, FAO specified that agronomic yield should be extended in the upcoming year by 60% globally [4,5]. As the cultivatable land is steadily decreasing, food safety can only be ensured by an exponential increment of crop per unit production. Fertilization [6] and Irrigation [7] is accessible technology to progress production, although sometimes these technologies are limited by the farmers' economy [8] and they do not ensure an uninterrupted ecological equilibrium [9,10]. Molecular breeding [11] and genetic engineering [12] are more modern approaches to increase gross production, however, they are time consuming. The application of cold plasma technology is new in agriculture as a viable, cost effective, eco-friendly, and non-hazardous measure by creating a new way for developing grain yield. It could act as a stimulant for the vigor of seed keeping the risk of genetic mutation at minimum, as it is based on non-ionizing lowlevel radiation. In many countries, extensive research has been carried out on the agricultural application of plasma technology. It was found the growth and yield of lettuce [13], cucumber [14], tomato [2], soybean [15], rice [16], spinach [17], peanut [18], eggplant [19], maize [20], rape [21,22] oat [23], radish [24] black gram [25,26] and Andrographis paniculata [27,28] were improved after treated with plasma. Although wheat (Triticum aestivum L.) is a staple crop in many countries, it's yield is being steadily reduced because of several environmental factors (changes in global temperature, humidity, and drought, etc.) and manmade adversities [15,29,30] leading to malnutrition which is a current crisis worldwide. Hydrogen peroxide (H 2 O 2 ) is an important REDOX (reduction-oxidation reaction) metabolite and causes oxidative damage to biomolecules at high concentrations, which culminates in cell death. However, H 2 O 2 acts as a signalling molecule at concentrations in the nanomolar range and resembles phytohormones in certain ways. A number of excited, reactive oxygen and nitrogen (ROS, RNS) molecules, free radicals, ions, photons are created by plasma treatment, and the high temperature of electrons helps in generating these reactive species. Plants activate their defense mechanisms such as enzymatic and non-enzymatic antioxidants when encounter stress such as ROS and RNS (O 3 , OH − , H 2 O 2 , NO, NO 2 , O 2 , O) [31,32]. The antioxidant enzyme's coordinated action scavenges the activity of ROS and RNS [33,34]. The interaction of plasma with cell metabolites could enhance the activities of seedling germinating enzymes [35] and accelerate the decomposition of the inner nutrients of the seed, which could lead to increased use of the seed reserve and growth of the seedling. As research on the effect of dielectric barrier discharge (DBD) or non-thermal air plasma is new in wheat, the fundamental purpose of the current study is to inquire the following questions a) studying the effect of air plasma on seed germination that reduces the total number of seeds required for cultivation; b) investigating the mechanistic basis of plasma-induced improvement of wheat, and c) studying the effect of plasma on physicochemical and nutritional composition in wheat.

Seed Collection
Mature dried wheat seeds (Bari 21) were collected from the local market. They were dried and stored at room temperature. Seeds were sensitively selected by bare eyes with the help of a simple magnification glass so that all the seeds are similar in size and have no scratches. The temperature of the laboratory is maintained at 26 °C and the initial seed temperature was also the same measured by a IRT25 Infrared Thermometer (Extech, Nashua, NH 03063, USA).

LPDBD Plasma Generation and Seed Treatment
Two copper electrodes (diameter 9 mm, thickness 0.5 mm) were placed axially at the two ends of the test tube (diameter 12 mm, length 50 mm) as shown in (Fig. 1). The powered electrode was covered with a pyrex glass disk used as dielectric layer. The gaps between the electrodes were kept at 40 mm. Wheat seeds were kept in the space between two electrodes. To ensure uniform seed treatment, a servo motor capable of rotation in both forward and reverse direction was employed to rotate the plasma chamber. The glass plasma chamber was positioned horizontally to which ensures flipping of the seeds with movement of the motor. A vacuum pump decreased the pressure inside the chamber and the pressure was maintained at ∼ 10torr . The humidity of the chamber was not controlled. However, the humidity of the laboratory which was air-conditioned (General AOGA24FMTBH Split Air Conditioner, 24,000 BTU, Thailand) was between 35 and 55%. The gas temperature inside the chamber and seeds surface temperature was range between 28.77-40.0 °C and 26.7-32.7 °C respectively (Supplementary file 1), which was measured using an IRT25 Infrared Thermometer (Extech, Nashua, NH 03063, USA). A high voltage (5-10 kV, 3-8 kHz) was provided to the electrodes for plasma generation and treating the seeds. During seed treatment, atmospheric air was supplied to the chamber, and flow was controlled by a gas flow controller Yamato, KIT (Yamato Scientific Co. Ltd., Tokyo, Japan) and was maintained at 1l∕m. Cytiva Whatman EPM 2000 air filter (Cytiva, Marlborough, MA, USA) was used (Air retention efficiency of 99.95% of 0.3 µm size particulates, thickness 450 µm). The wavelength of the discharge, voltage (HVP-08), and current (CP-07C) were recorded in a digital oscilloscope (GDS-1000B). The emitted spectra produced in the plasma were recorded with spectrometers (USB2000 + XR1, slit size 25 μm, grating 800 lines/mm, optical resolution (107 nm) in the wavelength range from 200 to 1100 nm for the identification of species. High resolution dual-channel spectrometer (AVASpec-2018, slit size 10 μm, grating 2400 lines/ mm, optical resolution 0.07 nm) was used in the range from 200 to 500 mm for the estimation of plasma parameters. Seeds were treated for 1 min, 3 min, 6 min, 9 min, and 12 min by air plasma.

Scanning Electron Microscopy (SEM)
Control and treated Seeds were dried in an oven at 30 °C overnight to remove moisture for carrying out the SEM. Subsequently, plasma treatment was performed and immediately carried out the scanning electron microscopy (SEM) by FEI S50 scanning electron microscope (FEI Technologies Inc., Oregon, United States) using ZEISS software at 10 µm scales, and changes found in the seed coat were compared to the non-treated controls.

In Vitro Seed Germination Assay
Forty (40) control (non-treated) and treated seeds were immersed for 5-h in deionized water and subsequently kept in petri dishes for germinating. Two layers of wet filter paper were placed into sterile petri dishes (90 mm), and 40 wheat seeds were placed in each petri dish. The petri dishes were kept in a seed germination chamber (Dimension 735 mmW × 915 mmD × 2085 mmH, Digital PID humidity controller, 4 nos of 15 W T5 LED light with > 1500 lumens output) at 25 °C, 12 h light/12 h dark photoperiod (flux intensity 120 µmol m −2 s −1 ), and 75-95% humidity. Upon treatment seeds were placed on a moist filter paper inside a 90 mm petri dish. The petri dish lid was closed for retaining moisture and placed inside the germination chamber. Although the humidity of the chamber is controlled by Digital PID humidity controller, additionally, 5 ml of deionized H 2 O was sprayed daily twice in each petri dish to sustain an adequate amount of moisture for germination. After 4 days, the germinated seeds were separated and the germination percentage (GR, %) was calculated by the formula mention bellow (the radicle projection at one mm was considered as the criterion for germination): where GR Germination percentage SG Number of germinated seeds ST Total number of seeds.

Field Preparation and Seeds Sowing
In this study, the randomized complete block design (RCBD) method was followed for field preparation. Plot length was 4 m 2 , line-line spacing (72 cm) and every condition had three replications and the total number of the plot was 18 with control. The seeds for control and each treatment were taken in separate container, mixed and picked blindly for sowing in the field to ensure randomization. The soil type of the field was clayey loam. Ground water was irrigated in the field as needed. The soil of the field was not allowed to dry at any point of time. Excess watering was also checked.

Growth Parameter
Germinated plantlets were collected from the field for growth parameter analysis, such as roots length, shoots length, number of tiller, fresh weight, and dry weight after 30 days of sowing. Plants were taken out of the soil carefully, washed and placed on the flat table top and root and stem length was measured using a tailoring tape. Three replications from each plot were taken and mean value was considered for each trait. Sufficient amounts of roots and leaves were stored at minus 20 °C for further analysis. After 40 days of sowing, plant height, stem diameter and after 90 days length of panicle, the diameter of panicle, and plant height with panicle were determined using tailoring tape scale and vernier caliper whichever applicable. Chlorophyll content was also measured at LEAF CHL STD Chlorophyll meter (FT GREEN, USA).

Catalase (CAT), Superoxide Dismutase (SOD), Ascorbate Peroxidase (APX), and Glutathione Reductase (GR) Activities in Roots and Leaves
Enzymes activities were evaluated as per earlier report with minor changes [36]. In brief, tissues were crushed in phosphate buffer (100 mM, ph = 7.0) using mortar and pestle and centrifuged for 8 min (10,000 rpm) before separating the aliquot in a fresh tube. The activity of antioxidant enzymes (CAT, SOD, GR, and APX) was evaluated using spectrophotometric method, as illustrated earlier [36].

Relative Gene Expression Investigation
The relative expression of TaSOD, and TaCAT transcripts was carried out in roots by qPCR following the previous reports [37,38]. 50 mg roots were crushed with mortar and pestle into liquid nitrogen. Complete RNA was isolated using the SV Total RNA Isolation kit (Cat. no. Z3100, Promega Corporation, USA) using the manufacturer's protocol. The Integrity of RNA samples was examined on formamide gel electrophoresis. RNA was quantified and purity of RNA was checked using NanoDrop2000 (Thermo Scientific, USA). 1 μg of total RNA was reverse transcribed into first-strand cDNA using GoScriptTM Reverse Transcription kit (Cat no. A5001, Promega Corporation, USA). To eradicate the risk of any RNA carryover cDNA was treated with RNase H. Finally, real-time PCR analysis was performed in an EcoTM real-time PCR (Illumina, USA) device operated by Eco Software v4.0.7.0. Sequences of gene specific primers used in real time PCR are given in Supplementary file 2. As an internal control, expression analysis was standardized with β-Actin.

3
The PCR software in real time was used as follows: 10 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 55 °C and 15 s at 72 °C.

Analysis H 2 O 2 in Plant Tissues
After through washing, the fresh roots and leaves, tissues were crushed in 0.1% trichloroacetic acid [39] with the aid of mortar and pestle and centrifuged for 15 min at 10,000 rpm. The supernatant was then mixed with potassium iodide (1 M) and phosphate buffer (10 mM, ph 7.0) and kept for 1 h in the dark. Subsequently, the optical density (OD) was measured at 390 nm by Genesys 10S UV-VIS spectrophotometer (Thermo Scientific, USA).

Root and Shoot NO Concentration Analysis
The concentration of nitric oxide (NO) in wheat roots and leaves was measured on the basis of alterations in hemoglobin absorption and subsequent transformation from oxyhemoglobin (HbO 2 ) to methemoglobin (metHb) in the presence of NO [40,41]. Plant tissue samples were homogenized in 1 ml of cooled NO buffer containing 0.1 M sodium acetate, 1 M NaCl, and 1% (w/v) ascorbic acid (pH 6.0). The admixture was then centrifuged for 5 min (10,000 rpm) at 4 °C, and the supernatants are transmitted to a fresh tube. Subsequently, the HbO 2 solution stock (5 mM) was added to the samples and incubated for 5 min at room temperature. The transmission rate of HbO 2 to metHb was assessed at 401 nm.

Estimation of Total Soluble Protein and Sugar in Plant Tissues
The concentration of total soluble protein in root and leaf was estimated by measuring the optical density at 595 nm in a GENESYS 10S UV-Vis spectrophotometer (Thermo Scientific, USA). Using different concentrations of bovine serum albumin (BSA) a calibration curve was prepared from which the concentration of the unknown sample was calculated [42]. Besides, the total soluble sugar was estimated in root and shoot as described earlier [43].

Elemental Analysis in Tissue and Grain
Wheat seeds were ground by blander and 2 g powder were weighed. In a glass beaker, 2 g of samples were then taken and 5 ml of HNO 3 and 2 ml of H 2 O 2 were added and heated in a microwave oven for 1 min. The Flame Atomic Absorption Spectroscopy (AAS) connected to an ASC-6100 auto-sampler air-acetylene atomization gas mixture device (Model No. AA-6800, Shimadzu) was used to estimate the concentrations of Mn, Fe and Zn. A standard solution was also made from their subsequent stock solutions for Fe, Mn and Zn [43]. The remaining flour was stored for further analysis. The concentrations of Fe, Mn and Zn in leaves were calculated as earlier described [37].

Determination of Moisture Contents
Moisture content was determined using AOAC (2000) method. Briefly, a piece of aluminum foil was dehydrated in an oven at 105 °C for 1 h and then transferred to the dryer to cool down and the blank foil was weighed. Subsequently, 2 g of flour were weighed through an electronic balance and the sample was spread to uniformity. The samples were dried in an oven at 105 °C for 3 h and shifted to the desiccant to cool down the foil, and its desiccated sample was reweighted. Moisture content was determined by the following formula, where W 1 A sample's weight (g) prior to desiccating W 2 Weight (g) of the sample after desiccating.

Estimation of Fat Amount
Fat was extracted as earlier described [42]. Fat determination required uninterrupted extraction by n-hexan in a Soxhlet apparatus. Briefly, the container and cover were placed in an oven at 105 °C for 12 h to confirm the weight of the container is unchanging. 10 g of sample were weighed using electric balance and placed into extraction thimble, transfer into soxhlet and poured 250 ml n-hexan into the pot and placed it on the boiler veil. Soxhlet apparatus was connected and turned on the water to cool and then turned on the boiler veil. The sample was heated for 6 h (heat rate of 150 drops/min) and evaporated the solvent with the help of a vacuum condenser and incubated the bottle at 80-90 °C for 1 h. After that, the bottle was transferred to the desiccators and reweighted the bottle and its dried content. Fat content was calculated using the subsequent equation, The fat free sample was stored for the estimation of crude fiber.

Determination of Crude Fiber
Two (2) gram fat-free sample was weighted through electric balance and the sample was put into a beaker attached to a condenser. 200 ml hot H 2 SO 4 (0.125 M) was added to the beaker and boiled for 30 min. Then the sample was filtered with boiling distilled water and transferred the residue into the beaker. Subsequently, 200 ml hot NaOH (0.313 M) was added to the beaker and boiled for 30 min followed by filtration using boiling deionized water, 1% HCL and boiling dH 2 O, respectively. Finally, the sample was again filtered twice through 100% ethanol. The sample was dried in an oven at 100 °C overnight and cooled in a desiccator. The sample was placed into the moisture-free crucible, weighted and ignited for 3 h at 500 °C in a muffle furnace, cooled and reweighted. Crude fiber content was determined through the following formula, Fat content(%) = (Weight of fat∕weight of sample) × 100 where W 1 Weight of silica crucible with contents before ashing W 2 Weight of silica crucible with contents after ashing W 0 Weight of sample.

Estimation of Ash Content
Ash content was determined using AOAC (2000) method. Briefly, crucibles with the lid were placed into a furnace at 600 °C overnight and cooled down in a desiccator (30 min). The weight of the crucible with lid was measured and 5 g samples were put into it. Afterward, crucibles were burnt in a muffle furnace at 600 °C overnight, cooled, and reweighed. Ash content was determined using the following equation,

Yields
Three panicles were collected from each plot, separating the grain from it, counted the number of grain per panicle and calculated their mean for yield analysis per plots and the total yield of every plot was converted as g/m 2 . 1000 grain weight of control and treatments were also taken randomly for comparison.

Estimation of Protein in Wheat Grain
Protein concentration was quantified as previously described [43] with slight modification. Standard curve was made using 0%, 0.05%, 0.1%, 0.2%, 0.4% and 0.8% BSA solution. 0.03 g flour was diluted in 10 ml deionized water and 2 ml Bradford reagent was added. The sample was centrifuged 2 times at 4000 rpm for 5 min, and the supernatant was taken. The optical density (OD) was taken at 595 nm by a spectrophotometer (Genesys 10S UV-VIS Spectrometer, Thermo Scientific, USA).

Statistical Analysis
All the examinations were conducted in three autonomous repetitions. The significance of all groups of data was investigated statistically at P ≤ 0.05 by one-way ANOVA which was carried out by Duncan's Multiple Range Test (DMRT) by SPSS Statistics 23 software. The graphs existing in this report were prepared via GraphPad Prism 6.

Wheat Seed Germination and Plant Growth Characteristics
The germination of wheat seeds was prompted by LPDBD plasma treatment. The mean germination, root length, shoot length, fresh weight, and dry weight were shown in Ash (%) = (Weight of ash∕Weight of sample) × 100 ( Table 1). The mean germination percentage was significantly improved to 89.17%, 96.67%, 90.83%, and 88.33%% for the 3 min, 6 min, 9 min, and 12 min of plasma treatments respectively compared to the control. However, the highest germination percentage achieved by 6 min treated seeds showed significant difference with 1 min, 3 min, 9 min, and 12 min treated seeds. Thus, cold plasma treatment increased the germination percentage by 4.21%, 12.63%, 22.11%, 14.74%, and 11.58% in 1 min, 3 min, 6 min, 9 min and 12 min treatment, respectively, compared to the control. All the treatments improved plant growth characteristics. The mean root length increased to 19.01 cm, 19.06 cm, and 19.48 cm for the 1 min, 3 min, and 12 min treatments, respectively, and these treatments presented no significant differences compared to the control (the root length 15.98 cm). The mean root length was 19.94 cm, 20.16 cm for the 6 min, and 9 min treatment, which exhibited a significant difference compared to the control. The shoot length was 65.39 cm, 70.67 cm, 76.06 cm, 76.66 cm, 80.07 cm, and 74.51 cm in control, 1 min, 3 min, 6 min, 9 min, and 12 min treatments, respectively. Among them 3 min, 6 min, and 9 min treatments presented significant difference compared to the control. The highest shoot length and root length reached 80.07 cm, 20.16 cm in 9 min treatment, which was significantly higher compared to the controls by 22.45% and 26.15% respectively. The dry weight of plants from the 1 min, 3 min, 6 min, 9 min, and 12 min treated seeds were increased by 3.92%, 28.23%, 98.04%, 76.86%, and 18.43% respectively, compared to control in which 6 min and 9 min treatment were statistically significant compared to the control. However, a slight increase was found in fresh weight, but that was not statistically significant. The average chlorophyll contents, numbers of tiller, stem diameter, plant height, panicle length, panicle diameter were shown in (Table 3). The uppermost mean chlorophyll content was acquired in the 6 min treatment, which represented a significant difference compared to the control, and increased by 27.10% compared to the control. The uppermost mean number of tillers was 7.78 for 6 min treatments, which significantly differed from the control, 1 min, 3 min, and 12 min treatments. While the average number of the tiller for 9 min treatments was 6.56, that is significantly different from the control, but exhibited no significant difference with 1 min, 3 min, 6 min, and 12 min treatments. The mean stem diameter was improved to 4.93 mm and 4.22 mm for 6 min and 9 min treatments, that was exhibited significant difference compared to the control (the mean stem diameter 3.46 mm), in which the uppermost stem diameter obtained in 6 min treatments exhibited significant difference from all others treatment. The uppermost mean plant height was 94.88 cm for 6 min treatment that exhibited significant difference compared to the control (73.31 cm), but showed no significant difference compared to the 6 min treatment and between them. However, the uppermost mean panicle length was 20.72 cm for 6 min treatment, which was significant compared to control, 1 min, and 3 min treatments. The mean panicle length was increased by 11.79%, 21.26%, 31.7%, 22.18%, and 18.43% for 1 min, 3 min, 6 min, 9 min, and 12 min treatment correspondingly compared to the control. The average panicle diameter was 14.98 mm, and 13.62 mm for 6 min and 9 min treatment, which was significantly different compared to the control and 1 min (9.91 mm), and significantly higher than the control (8.06 mm) by 85.93% and 69.10%, but exhibited no significant difference compared to the 3 min and 12 min treatments and between them. The mean panicle diameter for 3 min (11.89 mm) and 12 min (12.24 mm) exhibited significant differences compared to the control. The average panicle diameter for 1 min treatment exhibited no significant difference than control.
The uppermost mean number of grains per panicle was 287, which is significantly higher compared to the control (210). Thus, 6 min plasma treatment caused 36.67% increase in grain number per panicle. Seeds treated for 1 min, 3 min 9 min, and 12 min also showed marked improvement in terms of grains per panicle which were 247.67, 270.33, 234.33 and 221.67 respectively compared to the ( Table 3). The average number of grain per panicle was 270.33 for 3 min treatments, which presented a significant difference compared to the control but exhibited no significant difference compared to the 1 min, 6 min, and 9 min treatments ( Table 3). The average number of grains per panicle was improved by 17.94%, 28.73%, 11.59%, and 0.79% for 1 min, 3 min, 9 min, and 12 min treatments, respectively, compared to the to the control. The mean thousand grains weight was 39 g for 6 min treatment, which presented a significant difference compared to the control (32 g) and increased by 22.92% compared to control ( Table 3). The average thousand grains weight improved to 35 g, 36 g, 37.67 g, and 36.67 g, for 1 min, 3 min, 9 min, and 12 min treatments respectively, presented no significant difference compared to the control and increased by 9.38%, 12.5%, 17.71%, and 14.58% respectively compared to control ( Table 3). The uppermost mean yield m −2 was 272.3 g for 6 min treatment, which was significantly higher than the control (214.33 g) by 27.06% (Table 3). The average yield per meter square was 225.33 g, 228.33 g, 245.67 g, and 238.67 g for 1 min, 3 min, 9 min, and 12 min treatments, respectively, which were higher than the control by 5.13%, 6.53%, 14.62%, and 11.35% respectively, but they were not statistically significant (Table 3).

Scanning Electron Microscope (SEM) Analysis of Wheat Seeds Surface
The wheat seed surface exhibited a rectangular shape of sub-domain with a distinct edge before the air plasma treatment (Fig. 2a). In contrast, the rectangular shape sub-domain was completely disappeared after the air plasma treatment (Fig. 2b). Besides, cracks were noticed on the seed coat after the plasma treatment (Fig. 2b arrowed). The crack in seed coat was due to plasma treatment, because, control seeds were similarly dried before SEM but did not show any crack. So, the crack is caused by plasma treatment only. We have similar observation published in in Scientific Reports journal, Nature (https:// doi. org/ 10.

Estimation of Soluble Protein, Soluble Sugar, H 2 O 2 Activity, and NO Activity
The highest mean hydrogen peroxide (H 2 O 2 ) activity in leaves and roots were 3.56 µmol g −1 FW and 4.55 µmol g −1 FW respectively for 6 min treatment in which leaves H 2 O 2 activity were significantly higher compared to the control (1.25 µmol g −1 FW) and 1 min (1.81 µmol g −1 FW) treatment; while root H 2 O 2 activity exhibited no significant difference compared to the control (3.12 µmol g −1 FW) and other treatments (Fig. 3a, b). The average H 2 O 2 activity in leaves were 2.45 µmol g −1 FW, 2.08 µmol g −1 FW, and 2.66 µmol g −1 FW for 3 min, 9 min, and 12 min treatments respectively, which presented significant difference compared to the control and 1 min treatments (Fig. 3a). The upper most mean nitric oxide (NO) activity in leaves and roots was 6.68 µmol g −1 FW and 5.83 µmol g −1 FW for 6 min treatment respectively, in which leaves NO activity presented significant difference compared to the control (2.95 µmol g −1 FW) and 1 min (3.67 µmol g −1 FW), 3 min (3.75 µmol g −1 FW), and 12 min (4.71 µmol g −1 FW) treatments while roots NO activity presented no significant difference compared to the control (3.77 µmol g −1 FW) and other treatments (Fig. 3c, d). The uppermost mean soluble protein in leaves was 36.15 mg g −1 Fig. 2 Scanning electron microscopy (SEM) images of wheat seed a untreated control seed; b seeds after 6 min plasma treatment at10.0 kV. Crack on the seed coat layer is shown with arrow. Scale bar is 10 µm Fig. 3 Changes in H 2 O 2 activity in a leaves, b roots; NO activity in c, leaves d roots; contents of soluble protein in e leaves, f roots; contents of soluble sugar in g leaves, h roots of wheat plants grown from the seed treated with 0-12 min of air plasma treatment. The alphabets at the top of the bar indicate significant difference among mean ± SD (n = 3) at P < 0.05 level concerning treatments FW, which presented a significant difference compared to the control (23.92 mg g −1 FW), and 1 min (24.74 mg g −1 FW) treatments (Fig. 3e). The average soluble protein in leaves for 3 min, 9 min, and 12 min treatments presented no significant difference among them and other treatment (Fig. 3e). The uppermost mean soluble protein in roots was 47.59 mg g −1 FW for 6 min treatment, which showed a significant difference compared to the control (21.32 mg g −1 FW), 1 min (24.03 mg g −1 FW), 3 min (33.82 mg g −1 FW), and 12 min (34.28 mg g −1 FW) treatments (Fig. 3f). Average roots soluble protein activity in 9 min treatment presented substantial difference compared to the control and 1 min treatments, while roots soluble protein activity in 12 min treatment presented a significant difference only compared to the control (Fig. 3f). The uppermost mean soluble sugar in leaves and roots were 3.04 mg g −1 .
FW and 3.6 mg g −1 FW correspondingly, which did not present any significant difference compared to the control (Fig. 3g, h).

Antioxidant Enzymes
The Catalase (CAT) activity in the leaves and roots is improved gradually up to 6 min and thereafter it is decreased (Fig. 4a, b). The maximum mean catalase (CAT) activity in leaves and roots was 1.03 nmol min −1 (mg protein −1 ) and 0.32 nmol min −1 (mg protein −1 ) for 6 min treatment respectively, in which leaves CAT activity presented significant difference compared to the control and increased by 222.02% compared to the control (Fig. 4a). In contrast, maximum mean CAT activity in roots exhibited no significant difference compared to the control (Fig. 4b) and increased by 77.77% compared to the control (Fig. 4b). However, no other treatments CAT activity in leaves and roots presented significant difference compared to the control (Fig. 4a, b). The expanding tendencies of SOD activity were witnessed together in leaves and roots as displayed in (Fig. 4c, d) correspondingly, where the maximum SOD concentrations were 7.71 nmol min −1 (mg protein −1 ) and 3.76 nmol min −1 (mg protein −1 ) respectively, achieved by the plantlets produced from the seeds treated for 6 min, in which leaves SOD activity presented significant difference compared to the control and increased by 162.44% compared to control (Fig. 4c). In comparison, roots SOD activity showed no significant difference compared to the control and other treatments and increased by 27.81% compared to control (Fig. 4d). In other treatments, SOD activity in leaves and roots presented no significant difference compared to control and among them (Fig. 4c, d). The expanding tendencies of APX and GR activity were witnessed together in leaves and roots as displayed in (Fig. 4e, f) and (Fig. 4g, h) correspondingly, where the maximum APX concentrations were 0.80 nmol min −1 (mg protein −1 ), and 0.86 nmol min −1 (mg protein −1 ) and GR concentrations were 0.26 nmol min −1 (mg protein −1 ) and 0.54 nmol min −1 (mg protein −1 ) respectively, achieved by the plantlets produced from the seeds treated for 6 min. However, none of these treatments statistically significant compared to control.

Gene Expression Correlated with Antioxidant Activities
Our study exhibited noteworthy up regulation of TaCAT and TaSOD expression in roots of wheat seedlings grown from 6 min air plasma treated seeds related to controls (Fig. 5).

Evaluation of Food Values
The lowest mean moisture content was 9% for 6 min treatment, which presented significantly decreased compared to the control and other treatments. In contrast, other treatments demonstrated no significant decrease compared to the control and among them ( Table 4). The uppermost mean fat content was 1.73% for 6 min treatment, which presented a significant difference compared to the control, and increased by 70.62% compared to control, while other treatments also presented significant difference compared to the control and exhibited no significant difference among them ( Table 4). The mean fat content was increased by 43.79%, 54.07%, 53.78%, and 40.72% for 1 min, 3 min, 9 min, and 12 min treatments respectively, compared to control. There were no significant changes found in mean crude fiber, ash, and protein content in any treatments compared to control and among them. The highest mean crude fiber, ash, and protein content were 0.93%, 2.65%, and 11.85 mg g -1 for 6 min, 1 min, and 9 min treatments respectively (Table 4).

Grain and Leaves Trace Elements
Grain Fe is improved gradually up to 6 min (Fig. 6a).The uppermost mean grain Fe was 30.75 mg/kg for 6 min treatments, which showed a significant difference compared to the control and increased by 36.89% compared to control, while other treatments presented no significant difference compared to the control and among them (Fig. 6a). The uppermost mean leaves Fe was 206.19 mg/kg for 6 min treatments, which showed a significant difference compared to the control and increased by 21.99% compared to control, while other treatments showed no significant difference compared to the control and among them (Fig. 6b). A slight enhancement is noticed in Zn and Mn concentration in grains and leaves as compared to control, while none of these treatments presented a significant difference compared to the control (Fig. 6c-f).

Discussion
Many nations of the universe are in a threat of food safety with growing need due to enhanced inhabitants and decrease in implanted area and adverse climatic changes. The production of wheat along with other crops is declining due to the alteration of weather [29,30] and severe reductions in implanted space globally. The current study revealed the effect of non-thermal plasma (generated from air) on the improvement of quantity and quality of wheat production. The 6 min Air plasma treatment increased the germination percentage up to 22.11% compared to the control, which will reduce the number of seeds required for cultivation (Table 1). Few other studies also have conveyed that non-thermal plasma significantly improved seed germination [15,[44][45][46]. Our result was coherent with those conveyed previously [28], a suitable air DBD plasma treatment could improve wheat seed germination in laboratory conditions. In the current experimental setup, LPDBD plasma also improved seedling growth such as the shoot length, root length; compared to the control for 9 min seed treatment. The tiller number, stem diameter, plant height, fresh weight, and dry weight improved significantly in 6 min treatment compared to the control (Tables 1 and 2). Similar studies in some other plants have revealed that non-thermal plasma treatments stimulate plantlet development [2,45,47]. Our experiment showed that chlorophyll content, panicle length, panicle diameter were also increased by 0.42%-21.1%, 11.79%-31.71%, 23.03%-85.93% respectively, compared to control due to the different duration of plasma treatments (Table 3). Higher chlorophyll content provides the plant added advantage for better photosynthesis which ultimately contributes in increasing grains size, panicle length, and diameter. Air plasma treatment increased grains per panicle, thousand grains weight, and yield m −2 by 0.79%-36.67%, 9.38%-22.92%, and 5.13%-27.06% respectively compared to control due to different duration of plasma treatments (Table 3) and the highest achievement was shown by 6 min of air plasma treatment (Table 3). Atmospheric pressure air plasma treatment causes increased gas temperature (~ 40 °C) inside the discharge vessel which release energy as heat by the plasma species such as ROS and RNS. This causes the seed coat starch and protein to interact with the generated oxygen and nitrogen related species, viz. reactive O, OH, NO 2 , N 2 O, NO, CO 2 , HNO 2 , HNO 3 [48], results in more permeable seed surface compared to the control (Fig. 2). These free radicals may induce series of reactions which is responsible for increased growth and grains production, and biochemical and molecular changes. Several scholars have suggested that air plasma prompted reactions on the seed surface might consequence in a greater invasion of ROS, RNS, and UV radiation, which in turn assists in various physiological responses [46,49]. These chemical reactions stimulate biological stimulation, which promotes seed germination [50]. It is found that air plasma treatment changes seed coat structure and roughness (Fig. 2b), which enhances water uptake ability results in greater germination and seedling growth [15]. Soluble protein and soluble sugar activity have important tasks in growth and adaptive responses [51]. Jiang et al. [52] reported that non-thermal plasma treatment significantly improved the absorption of nitrogen (12.7%), which in turn enhancing protein content. Soluble sugar is thought [53] to deliver an adaptive response to drought, low temperature, pathogen challenge, anoxic injury, and excess excitation energy. The activity of soluble sugar was improved in seedling (Fig. 3g, h) which might aid for the endurance of plantlets from anoxic injury. In the current studies, after LPDBD plasma treatment, the soluble protein content activity of wheat seedlings was enhanced compared to those of the controls (Fig. 3e, f). In corn seedlings, similar findings were reported by Wu et al. [54]. Table 2 Effects of plasma treatment on Chlorophyll content, number of tiller, stem diameter, panicle length, and panicle diameter Different alphabet indicates a significant difference among mean ± SD (n = 3) at P < 0.05 level. n-3 means 3 replicates for each experiment (total 18 × 3 plants The germination percentage of seed is found to increase with treatment duration and reaches the highest level after that it is reduced (Table 1). This phenomenon can be described as nitrogen (N) complex is a reserve compound in many seeds, which play a significant role in faster and increased germination [55] of wheat seeds. We also found the highest nitrogen activity after that it is reduced (Fig. 3c, d). This phenomenon is consistent with the seed germination percentage. Increased nitrogen content not only plays a significant role in enhanced germination [38] but also improve plantlet growth with the maximum extension of leaves [55]. The outcomes of this study concerning the improved root and shoot lengths because of enhanced contents of nitrogen in the seed as reported earlier [42]. Thus, the LPDBD air plasma treatments can enhance the N content in the seeds that function as reserved nitrogen. Further, the reserved N content of the seed is distributed [43] among proteins and amino acids. Subsequently, it is thought that the N enriched seeds can generate enough amino acids and proteins through the metabolization of the N complex and provide requisite nutrients that can improve plantlet development and chlorophyll concentration in the leaves. It was previously reported that cold plasma treatment enhanced N absorption in tomato plants which in turn plays a vital role in nutrients absorption and lateral growth [52]. Moreover, enough N content increases the chlorophyll content in the leaves [42] and enhances other nutrients uptake [52] that could help in increased production. In another study, it was reported that NO was a vital element for lateral root development in tomato plants, and the distinct presence of NO in lateral root tips significantly  [56]. NO has also been shown to mitigate the detrimental effects of reactive oxygen species (ROS) [57], and has a substantial influence on plant morphology, participating in aerenchyma formation, rhizogenesis, seed germination, and hypocotyl elongation [58]. In our study, NO activity slightly increased in plant tissue that also play role in the lateral development of wheat plants.
Hydrogen peroxide (H 2 O 2 ) concentration is increased in the seedlings due to plasma treatment (Fig. 3a, b). Although H 2 O 2 is an initiate of a stress factor in plants but the controlled [53] amount of H 2 O 2 function as the signal transduction for soluble sugar synthesis, therefore slightly increased up the soluble sugar content (Fig. 3g, h) in the seedlings. CAT and APX are mainly the scavengers of H 2 O 2 in which CAT exhibited significant changes in leaves subjected to air plasma (Fig. 4a). The leaves exposed to more oxidative stress rather than the roots due to air plasmas treatment or environmental stress. When cells are stressed for energy and are rapidly producing H 2 O 2 via catabolic processes, H 2 O 2 is not threatening or toxic to plants and H 2 O 2 is degraded by CAT enzymes in an energy-efficient way resulting from the expression of the gene TaCAT (Fig. 5). While APX activity is slightly increased in roots and shoots (Fig. 4e, f), but it is statistically not significant. These two antioxidant enzymes work redoxly to remove H 2 O 2 , while little APX activity would be led to the slight accumulation of H 2 O 2 in seedlings, which enable the plants to tolerate stress [25]. SOD is one of the major antioxidant enzymes elevated against oxidative stress produced by reactive oxygen. SOD is the only enzyme which acts on superoxide radical dismutase to hydrogen peroxide and oxygen. The SOD activity is found statistically significant in leaves compared to the control (Fig. 4c) which was further supported by the TaSOD expression (Fig. 5). Improved activities of SOD suggest enhanced production of superoxide anion in seedling which indicates that the plant's defense mechanism becomes enriched. This result is reliable with the findings of [59]. Glutathione reductase (GR) is an enzyme that catalyzes the reduction of glutathione disulfide (GSSG) to glutathione (GSH). GR activity is slightly improved in roots while in leaves it is almost similar compared to the control (Fig. 4g,  h).
Moisture contents were determined to measure the level of water in wheat grains, which is an important factor in terms of productivity [60]. Moisture content range between 9.0%-11.45% for different duration of treatment, among them moisture content of grain obtained from the plant from 6 min treated seed showed significant difference compare to others ( Table 4). The moisture content of different wheat varieties is estimated to range from 9.90 to 12.48% [61]. Thus, plasma treatments slightly reduce moisture content; protect the grain from the microorganism. It was previously reported that [62], plasma treatment increased the activity of oxidative marker, thus improved oxidation of lipid, which lead to the reduction of fat. On the contrary, in our experiments higher activity of antioxidants enzymes (CAT, SOD and APX) were observed. Therefore, we hypothesize that, higher antioxidative activity in the seeds down regulate the oxidative marker, which facilitated the accumulation fat in grains. Plasma treatment significantly increased fat content compared to the control. The mean fat content range between 1.02-1.73% (Table 4). This result is supported by the result of [63]. The highest fat content increased by 70.62% compared to control. The mean crude fiber, and ash content range from 0.81%-0.93%, and 2.29%-2.57% respectively (Table 4). This result is supported by the result of [64,65], respectively. The mean protein content ranges from 11.56 to 11.85 mg/g (Table 4). There are no significant changes found in mean crude fiber, ash, and protein content of wheat grain. Thus, plasma treatment only improves fat content and reduces moisture content in wheat grain, though the reason is indistinct. The higher-yield wheat genotypes have been reported to be associated with lower Fe, Mn, Zn, and Cu concentrations [66]. Fe and Zn the metals which are most often considered deficient in plants, and consequently in the human diet. In both leaves and grains of wheat plants subjected to air plasma treatment in seeds, we observed a substantial increase in Fe. Iron uptake depends on the ability of the plant to reduce Fe 3+ to Fe 2+ through the electrons at the surface of the cell [67]. While Zn and Mn concentration slightly increased in leaves and grains but it is statistically not significant compared control (Fig. 6c-f).

Conclusion
In the current study, the plasma generated from air by low pressure dielectric barrier caused oxidation of the wheat seed surface resulted in rough and cracked seed coat that piloted to the increased water acceptance and the permeability of the seeds, consequently promoting its germination. The active species perceived into the wheat seed caryopses and triggered their biological reactivity, resulting in improved soluble protein activity and supply nutrients to plantlets for growth enhancement. Increase activities of CAT and SOD not only improve plant defense system but also develop adaptive response of wheat plants. In conclusion, plasma treatment shows tremendous promise in practical application in increasing seed germination, different agronomic traits including yield and food value of wheat.