Impact of pulsed electric field treatments on the growth parameters of wheat seeds and nutritional properties of their wheat plantlets juice

Abstract This study was designed to explore the impacts of the pulsed electric field (PEF; 2 to 6 kV/cm; a number of pulses 25 and 50) on wheat (Tritium aestivum L.) seeds before imbibition to improve the germination, growth, and their nutritional profile in juice form. It was observed that the PEF treatment at 6 kV/cm at 50 pulses increased water uptake, germination of seeds, and growth parameters of seedlings. A significant increase in total phenolic contents, DPPH, chlorophylls, carotenoids, soluble proteins, minerals, and amino acids in PEF‐treated seeds plantlets juice as compared to the untreated seeds plantlets juice was observed. The results indicate that the PEF may effectively stimulate the growth of the wheat kernels and positively affect their metabolism, optimize the nutrients, and enhance the strength of the wheat kernels plantlets.

also known as a good source of bioactive compounds (ferulic, benzo(a)pyrene, caffeic, p-coumaric acid, gallic, and syringic), minerals (iron, magnesium, and calcium), and vitamins (A, B, C, and E; Akbas et al., 2017). Wheat plantlets are nutrient-dense widely consumed as raw juice immediately after production, as well as tablets and capsules at the commercial level. Wheat plantlets have various health beneficial properties such as antioxidant, neuroprotective, antidiabetic, and anticancerous (Mujoriya & Bodla, 2011).
Due to public awareness, the demands for functional foods and especially nutrient-dense foods have been increased. Quality optimization techniques are necessary for the quality enhancement of functional foods . Nowadays, the plantlets of different crops are considered an excellent source of bioactive compounds, which makes them the best functional food (Marton, Mandoki, Csapo-Kiss, & Csapo, 2010). However; the market demand for plantlets is decreased due to the quality of plantlet metabolites. To overcome these problems, extrinsic compounds are used but they are evenly distributed to the kernels and could cause undesirable changes (Pérez-Balibrea, Moreno, & García-Viguera, 2011).
The pulsed electric field (PEF) is a nonthermal processing technology commercially used, due to power proficient, less energy consumption, and trifling processing technique Siddeeg, Faisal Manzoor, Haseeb Ahmad, et al., 2019).
This can alter the structural features of the agriculture products, through the assistance of bioactive metabolites discharge and/ or improve the shelf life of foods, without the use of chemicals (Manzoor, Ahmad, Aadil, et al., 2019;Toepfl, Mathys, Heinz, & Knorr, 2006). PEF has been considered to be a sublethal promising technique that enhances cell membrane permeability, the content of food material to the high voltage pulses of short repetition can cause temporary or everlasting pore formation in the cell membrane (Angersbach, Heinz, & Knorr, 2000;. Seeds treatment by PEF has been revealed the growth response in different plants such as lettuce, barley, Arabidopsis thaliana, and many other plants. The inhibitory and stimulatory growth effects caused by PEF are based on the physiological state of seeds, electric field (EF) strength and intensity. Since the methods of seed germination and imbibition include a series of significant biochemical and physiological differences, the PEF technique could be applied to manipulate these processes (Leong, Burritt, & Oey, 2016). The plant cell has responses against stress condition, the common one is reactive oxygen species (ROS). The permeabilization of the cell membrane due to PEF usage is evidence of the production of ROS, the ROS lead to oxidative stress, which leads to an elevated level of antioxidants and many other metabolic components (Sabri, Pelissier, & Teissié, 1996). The aim of this study was to assess the impact of PEF on the seed. We monitored the plantlet's germination, growth, vigor index, water uptake, juice yield, soluble proteins, chlorophyll, amino acids, minerals, and bioprotective properties.

| Seeds selection
The nongenetically modified (NON-GMO) wheat (Tritium aestivum L.) kernels were purchased from the Cereal Crop Research Institute (CCRI) Nowshehra, Pakistan from a single batch for the regular size, shape, and color. Debris and damaged seeds were removed from the experimental sample.

| Pulsed electric field (PEF) treatment
Wheat kernels were not immersed in water before treatment, they were divided into groups according to the PEF-treated and untreated kernels. For the treatment, a laboratory-scale PEF (EX-1900, SCUT, PEF-Team) was used ( Figure 1). The treatment chamber has 10 cm of length and 11 cm of width with two corresponding electrodes.
The chamber was filled with 40 g wheat kernels and 160 g distilled water, submerged the kernels and facilitated the equal EF strength in the course of PEF treatment. In this experiment, we used different EF strength of 2, 4, and 6 kV/cm for (25 and 50 pulses) with a pulse width of 100 μs at a stable frequency of 1 Hz. The temperature was maintained for all the treatments (<30°C).
For the calculation of voltage and specific energy used, following formulae 1-2 were used and values are presented in Table 1. where d is the resistance between lower and upper electrodes of the sample solution container in cm; U is oscilloscope (kV) to estimate the voltage (10 load resistances of the electrodes at both ends of the sample solution. (1) E = 10U m ∕d F I G U R E 1 The Schematic diagram of PEF extraction s system where n is the number of pulses, W is a pulse as per pulse energy, and m is the mass of the sample.

| Water uptake index
Wheat kernels water uptake was estimated by weighing seeds before treatment. The seeds (3.20 g) were measured by almost 100 seeds/treatment. PEF treatment with different intensities (2, 4 and 6 kV/cm) and different numbers of pulses (25 and 50) was applied.
The water uptake determined gravimetrically at the scheduled time, the surface water removed with tissue paper. The electric balance (error of ±0.1 g) weighed the kernels and then returned to the dish and reweighed at the scheduled time. Kernels imbibed for 3, 6, 20, and 24 hr at 20 ± 2°C in the dark condition. The increase in the weight of the kernel stated the amount of water uptake.

| Cultivation
The cultivation was performed in the petri dish at filter paper, taking 25 kernels/sample. The dish was filled with 3 ml of deionized water and left for 3 days. On the third day, 5 ml of deionized water was added and left for three days more, for 6 days cultivation in dark at 22°C.

| Germination rate
The germination rate was estimated as a ratio between a number of germinated kernels and the total number of kernels per dish. The kernels were determined as germinated when the length of a 1 mm radical sprout from the kernel appeared. For the calculation of the germination rate, following equation was used;

| Vigor index
The plantlet's length was measured by selecting 10 shoots randomly with mm scale. The germinated kernels shoots were measured freshly, and for drying purpose packed into aluminum foil separately at 60°C for three days with the known weight. The weight of one seedling fresh and dry plantlet was measured. The data were used vigor index calculation:

| Leaf area, sencent, and green leaf
For the determination of sencent, and green, selected 14 leaves and divided as green (healthy) or senescent (physiological aging) as described by Chanda and Singh (2002). The leaf area measurement was done with the help of a simple following formula: where Y, L, and W are leaf area, length (cm), and width (cm), respectively.

| Germination for Juice preparation
Wheat organic seeds of variety (Inqilab-91) were procured from CCRI Nowshehra, Pakistan. The reason for choosing this variety was highly nutritive value among the other 16 varieties available in Khyber Pakhtunkhwa, Pakistan. The wheatgrass was grown inside the laboratory where sufficient light and airflow were present.
Warm water was used to wash the wheat seeds for the removal of microbial load and undesirable adhered material. Wheat seeds were then soaked for 24 hr in distilled water, then drained. After that, seeds were wrapped in a muslin cloth and left for sprouting at least for 12 hr. Grains were spread on soil trays, and the water was sprinkled every day. Wheat plantlets were cut with scissors after 14 days, 2 cm above the soil. Wheat plantlets juice was extracted using specifically designed manual juice extractor suitable being fibrous in nature. It pressed the plantlets employing centrifugal force; manual plantlets juicers also prevent oxidation and heat. Then, wheat plantlets juice was extracted by the masticating process after washed and air-dried.

| Soluble protein content
The plantlet juice was accurately measured and mixed according to the ratio of weight (g): volume (ml) = 1:9. The solution was centrifuged for 10 min at 10000 g, then the supernatant was removed.
Take 1 ml of protein (Coomassie bright blue method No. A045-2) reagent, 0.5 ml of the standard mixed well and left for 10 min then The energy input (kJ/kg) of the pulsed electric field in the different treatment conditions Electric fields strength (kV/cm) measured the absorbance at 595 nm as a standard value, the same procedure was used for absorbance measurement of samples.

| Pigments content estimation
The plantlets were used to estimate the pigments. The sample of average 0.5 g weight was homogenized with sand, MgCO 3 , and 80% acetone. The solution was filtered and diluted to the absorbance range. The chlorophyll a, chlorophyll b, and carotenoids were measured by spectrophotometer at 664, 648, and 470 nm accordingly (Lichtenthaler, 1987).

| Determination of total phenolic
The total phenolic contents of wheat plantlet juice were determined by a method proposed by  with little modification. Briefly, 1 ml of Folin-Ciocalteu reagent was added to a 250 µl of wheat plantlet juice. After the addition of 2 ml sodium carbonate (20%), the mixture was mixed well and then left for 12 min at ambient temperature in a dark. Then, spectrophotometric (UV-1800, SHIMADZU) absorbance was measured at 760 nm. A suitable calibration curve was prepared before analyzing the sample using a standard solution (1 mg/ml) of gallic acid, and the results were expressed as µg of gallic acid equivalents (GAE) per ml of sample.

| 2,2-diphenyl-1-picrylhydrazyl (DPPH) activity
The DPPH activity of the wheat plantlets juice was measured according to the method of . Take 250 µl of each sample and then mixed with 50 µl of 1 mM DPPH solution prepared in methanol. After that incubated for 30 min in dark at room temperature. Spectrophotometric absorbance was measured at 517 nm against the blank. DPPH (%) was calculated by the following equation; The DPPH percentage values were plotted against the Trolox concentration and the antioxidant capacity measured from the linear equation.

| Free amino acids
The free amino acids of treated seeds plantlets were distinguished by an A300 amino acid analyzer (membraPure GmbH). The software iPeak and iControl (membraPure) were used. In brief, 4 ml of each sample supplemented with 3, 5-dinitrosalicylic acid (1 ml) was stored for 1 hr at 4°C to invert the protein and centrifuged twice at 10000 g for 15 min. Diluents comprising acetic acid, formic acid, trifluoroacetic acid lithium acetate, and ethanol to the final amino nitrogen of 0.008%-0.01% were used to dilute the supernatant. Liquid chromatography with a column (TS263, membraPure) was used to separate the amino acids and detected by ninhydrin reaction, and then the absorbance was recorded at 570 and 440 nm for proteins. Each amino acid concentration was measured using external standards (Sigma-Aldrich). All values were expressed in mg/100 ml.

| Mineral elements
Mineral contents of wheat plantlets juice were estimated using atomic absorption spectroscopy (Hitachi Z, 2000, Polarized, Zeman) by the method of Aadil et al. (2015). One milliliter of wheat plantlets juice sample was poured into a Teflon vessel. After that digested on a microwave work station with 1 ml of 30% H 2 O 2 and 7 ml of 65% HNO 3 and acid digested sample was diluted. A hollow-cathode lamp was used as the radiation source. For each mineral compound, the standards were prepared within the rage of mineral elements concentration contained in the sample. The results were described in mg/L of samples.

| Statistical analysis
All experiments in the present study were carried out in triplicate.
The experimental data were statistically analyzed using SPSS software version 24 (IBM SPSS Statistics). In this research, p-value < .05 was considered statistically significant.

| Water uptake
The results of water uptake after the treatment of seeds at different EF intensities (2, 4, and 6 kV/cm) and the number of pulses (25 and 50) are shown in Figure 2. The rapid water uptake leads to stimulate faster germination and strengthens growth. The maximum impact of PEF treatment was inspected in seeds treated at 6 kV/cm with 50 pulses; the water uptake was 56.56% at 24 hr. The water uptake in untreated seeds was 50.93% at 24 hr. It was observed that by increasing the EF intensities and the number of pulses, the water uptake capacity was increased. The highest water uptake was observed during the first 3 hr after the PEF treatments ( Figure 2). The accretion in the water uptake capacity of PEF-treated seeds at 6 kV/cm with 50 pulses guides to enhance the ability to absorb nutrients that assist plantlet growth and strength (Bhardwaj, Anand, & Nagarajan, 2012).
For germination, the water uptake is the initial step that activates the enzymes which affect the catabolism of seed reserves. Rapid hydration of seeds leads to enhance amylase and protease activity during seed germination. These enzyme's activity was reported in cucumber seeds with magnetopriming (Bhardwaj et al., 2012).

| Germination and juice yield
The germination percentage of seeds after different PEF treatments is presented in Figure 3 after 24 hr. Our results showed that the PEF treatment at 6 kV/cm increased the rate of germination as compared to the other treatments and untreated seeds. The seed's germination inclination reveals the relativity with the water uptake data that more hydration led to faster germination. The stimulating effect of PEF 6 kV/ cm on germination was probably due to hydration and the bran stress associated with EF strength. The positive outcome of PEF on seeds germination was already described for various plant species (Costanzo, 2008 Juice yield was estimated on the bases of the same wheat plantlet's weight for all treatments (Figure 3). The obtained yield was varied with different PEF intensities; a significant increase was observed as untreated (59.57%) to the highest PEF intensity-treated seeds (kV/cm) (78.72%). The PEF treatments increased the juice yield from 2.13% to 19.15%. This is possibly associated with more water uptake, fast germination associated large leaf area, resulting in strength of the plantlet, and finally increased the juice yield. Earlier, Eshtiaghi and Knorr (2000) stated that a 4.5% increment in juice yield treated with PEF.

| Growth parameters
The important growth factors of wheat plantlets after 6 days of seed growth, such as the weight, length, and vigor indices A and B, are presented in Table 2. Our results indicate that the most significant effect of PEF treatment was observed at 6 kV/cm with 50 pulses and the length of plantlets increased from 3% to 9% for untreated to the highest intensity-treated seeds. The fresh weight of plantlets increased from 8% to 42% for untreated to the highest intensity treatment. The PEF influence was considered as the change in the fresh and dry weight of the plantlets. The dry weight represents the biomass and indicates fewer errors as compared to fresh weight. The PEF induces the pore formation in seed coat and affects the metabolic process, it is an efficient technique for the transfer of external materials inside the cell, as well as for maintaining good simulative effects on plant intracellular organelles (Kandušer & Miklavčič, 2009). Therefore, the highest intensity of PEF treatment causes significant differences in the results.

| Growth parameters (leaf area, green, and senescent leaves)
The results of leaf area, number of green and senescent leaves in untreated seed plantlets, and PEF-treated plantlets are shown F I G U R E 2 Impact of PEF treatment (2, 4, and 6 kV/cm, with 25 and 50 pulses) on the water uptake capacity of wheat seeds during the 3, 6, 20, and 24 hr. Values are shown as mean ± SD (p < .05). PEF 2-25, treated at 2 kV/cm with 25 pulses; PEF 2-50, treated at 2 kV/cm with 50 pulses; PEF 4-25, treated at 4 kV/cm with 25 pulses; PEF 4-50, treated at 4 kV/cm with 50 pulses; PEF 6-25, treated at 6 kV/cm with 25 pulses; PEF 6-50, treated at 6 kV/cm with 50 pulses F I G U R E 3 Impact of PEF treatment (2, 4, and 6 kV/cm, with 25 and 50 pulses) on the germination (%) and juice yield (%) of wheat seed plantlets. Values are shown as mean ± SD (p < .05). PEF 2-25, treated at 2 kV/cm with 25 pulses; PEF 2-50, treated at 2 kV/cm with 50 pulses; PEF 4-25, treated at 4 kV/cm with 25 pulses; PEF 4-50, treated at 4 kV/cm with 50 pulses; PEF 6-25, treated at 6 kV/ cm with 25 pulses; PEF 6-50, treated at 6 kV/cm with 50 pulses in Figure 4. The PEF-treated seeds at 6 kV/cm with 50 pulses had significant leaf area (46.47%) as compared to untreated and other low-intensity-treated samples. In the case of greener and less senescent leaves, PEF treatment at 6 kV/cm with 50 pulses had a significant effect. The early aging of plantlets caused by the deficiency of many elements needed for rapid growth (Kučerová, Henselová, Slováková, & Hensel, 2019). The results show a significant impact on the growth; however, it can optimize the density of nutrients in the plantlets, and it can be further enriched with increment in the cultivation time for more than 15 days. The results indicated that PEF treatment enhances nutrient intake, such as nitrogen for the integration of biomolecules yield and active photosynthesis. Earlier Maniruzzaman, Sinclair, Cahill, Wang, and Dai (2017) described that the H 2 O 2 and nitrate produced in water by electric discharge caused a beneficial impact on plant growth, increase biomass, and leaves length in wheat plantlets. The PEF experimental procedure can be declined the plant tissue, which gives more compounds beneficial for growth parameters (Ersus, Oztop, McCarthy, & Barrett, 2010).

| Photosynthetic pigments
The chlorophylls and carotenoids were increased with the PEF treatments; the stress may promote respiration or initiate metabolic reactions, which might lead to polyphenolic accumulation in plantlets.
Many researchers reported that stress increases metabolites (Cantos, García-Viguera, de Pascual-Teresa, & Tomás-Barberán, 2000). The contents of chlorophyll and carotenoids of PEF-treated seed plantlets are displayed in Figure 5. Results indicate that the increase in PEF treatment led to increase chlorophyll and carotenoids content.
In the case of total chlorophyll contents, a significant increment was TA B L E 2 Growth parameters of PEF-treated and untreated wheat seed plantlets

| Total phenolic component (TPC) and antioxidant activity (DPPH)
PEF-treated seeds wheat plantlets were observed to be rich in total phenolic contents in 14-day-old wheat plantlets ( possibly has a scale of practical application in sprouts production with modified nutritional characteristics. We found that the contents of some metabolites were changed after PEF treatment, and these differences support the hypothesis that PEF penetrates into the caryopses and affects metabolic processes, because of the direct PEF treatment of the seed coat and influence on cells inside the seed. As we know the shoots of wheatgrass are good sources of phenolic and chlorophyll, all with strong antioxidant features, we observed whether the juice of wheat plantlets from PEF-treated seeds, with improved antioxidant metabolism, had a better antioxidant ability than the juice of wheat plantlets from the untreated seeds. The increase in phenolic and antioxidant activity of wheat plantlets from PEF-treated seeds might be due to the interaction between PEF and cells, which causes the cell wall fracture, DNA damage, change enzymatic activity, protein structure, and excite natural signal (e.g., opening the calcium channel and stimulation of growth components).
Earlier, Zhang and Björn (2009) and Petit et al. (2009) reported these kinds of effects after the UV treatment to different plant seeds.

| Soluble protein
Wheat is widely grown as a significant crop in the world; the one reason is its high amount of protein. The soluble protein plays a vital role in the growth of the plants and a substantial part of many plant enzymes that indicted the metabolism. The uncombined proteins in the cell and or organelle membrane represent the soluble protein (Sinha, 2004). The PEF-treated wheat seeds plantlets and their protein concentrations are presented in Table 2. The significant increase (16.76%) in soluble protein was observed in PEF-treated wheat seed plantlets with PEF6-50 as compared to untreated wheat seed plantlets. Earlier,  stated that the increase in soluble protein was due to the voltage stress or voltage-sensitive channel, the channel activated at a deficient level of voltage, in comparison to a critical transmembrane potential which might open due to electrical damage, the easy availability of nutrients caused the increment. Protein synthesis was found to be stimulated in electroporated plant protoplasts of Dactus carotta L., Nicotiana tabacum L., and Beta vulgaris L. after a single rectangular pulse of 400 μs at 1,700, 1,200, and 1,000 V/cm, respectively (Joersbo & Brunstedt, 1990).

| Effect on minerals composition
The results of elemental analysis of the untreated and PEF-treated seeds plantlets are shown in Table 4. The elemental analysis indicated variations in mineral concentration in plantlets, according to treatment conditions. According to the obtained results, Ca, Zn, and Mn were significantly higher in PEF6-50 than the untreated and other PEF-treated seed plantlets, while Fe and K were lower as compared to the other PEF-treated seed plantlets. Zn and Ca increased by 20.45% and 15.55%, with increasing PEF intensities PEF6-50 as compared to untreated seed plantlets. Earlier, the Ca and Mg contents of wheat plantlets were more in those plantlets cultivated just on water (Kulkarni et al., 2006) and K, Mn, Zn, and Fe level increased in those plantlets cultivated in soil (Kulkarni, Acharya, Rajurkar, & Reddy, 2007). The fundamental stress differences of soil and hydroponic growth to release elements influenced by soil medium, as the exchange cation complex and the outward conditions of soil, could change the availability of the elements. The interaction of seed and water and PEF polarized the seeds, could influence to extract the elements from water and soil (Ade-Omowaye, Angersbach, Eshtiaghi, & Knorr, 2000). Mn is a micronutrient that serves as the enzyme activator; it is the constitution of some enzymes. Mn is related to photosynthesis, oxygen-producing reactions, water splitting, and structural improvement of the chloroplast lamellae (Shenker, Plessner, & Tel-Or, 2004). The PEF polarized seeds have rapid development, so the initial manganese uptake was quick, via xylem pathways; that is why the high PEF-treated seeds plantlets had higher manganese as compared to untreated seeds plantlets. The Mn had a positive interaction with Ca; in the current study, PEF-treated seeds increased the Ca and Mn in plantlets (Millaleo, Reyes-Díaz, Ivanov, Mora, & Alberdi, 2010). The results of K show that the increase of Mn reduces the level of K, and the PEF treatment enhances Mn, and ultimately photosynthesis due to large leaf area. High PEF-treated seeds showed similarity for the K level with untreated seed plantlets. The Ca, Mn, Zn, and Fe are divalent, and the uptake of these minerals is less rapid than others, such as K is more readily available. The PEF creates stress that improves permeabilization water uptake, germination, vigor index, and overall plant growth due to facilitated the seeds and enhanced the nutrients uptake in quite systematic way (Vorobiev & Lebovka, 2009). The world population has a diet deficient in Fe (60%), Zn (30%), Ca, Mn, and K, so treatment of raw material such as plantlets is a very effective way to fulfill these deficiencies (Martínez-Ballesta et al., 2010). Wheat plantlets juice consumption may contribute toward meeting daily mineral requirements.

| CON CLUS ION
The present study demonstrates the use of PEF technology to manipulate a living plant system. In this study, the aim of the PEF application was to enhance the functional characteristics of wheat plantlets. The variations in seed water content influence the ability of a PEF treatment to induce significant and sustained changes in the metabolism of the resultant wheatgrass seedlings. PEF increased the activities of antioxidant enzymes in the resultant seedlings by stimulation and increasing the bioprotective capacity of harvested shoots.
"Electro priming" of seeds using PEF, if appropriately optimized, could be used as a relatively simple method to produce high-quality and nutritious sprouts without using chemical treatments. While the present study concentrated on the potential use of PEF treatment on imbibed seeds for the production of wheatgrass shoots with greater bioprotective capacity for human consumption, the resultant electro-stimulated shoots could also be valuable food for human as well as a supplement for animal feed.

CO N FLI C T O F I NTE R E S T
The authors do not have any conflicts of interest.

Zahoor Ahmed carried out the experimental work and Muhammad
Faisal Manzoor did the preparation of the manuscript. Abdul Rahaman, Ume Roobab, Rabia Siddique, Abdul Qayyum, Zia ud Din, and Azhari Siddeeg helped in the results discussion and statistical analysis. Xin-An Zeng and Nazir Ahmad revised the final paper.

E TH I C A L A PPROVA L
This article does not contain any studies with human participants or animals performed by any of the authors.

I N FO R M E D CO N S E NT
For this type of study, formal consent is not required.