Introduction

Mung bean (Vigna radiata), a member of the family Fabaceae, is becoming a popular food, and the consumption of sprouts is increasing because of their health benefits and high nutritional value. During germination, important changes in macro and micronutrient content and bioactive components occur, such as increases in protein, mineral content, dietary fiber, and antioxidant capacity, while fat content decreases1,2. The consumption of mung beans helps ameliorate metabolic disorders due to the unsaturated nature of the lipid composition, insoluble fiber and digestible carbohydrate contents, antioxidant activity, and vitamin C and phenolic contents3,4,5,6,7. In recent years, consumption of mung beans has increased due to a growing awareness of their health benefits. However, plant diseases and abiotic stresses (such as drought, heat, waterlogging, and salinity) can significantly affect their nutritional quality and economic yield8. Unlike traditional methods (e.g., natural sunlight exposure, seed treating by rubbing, use of chemicals), atmospheric or low-pressure plasma technology is an eco-friendly, low-energy, waste-free, and environmentally benign innovation to enhance seed germination and plant growth.

Some studies have explored the effects of atmospheric plasma-activated water (PAW) on mung bean seed germination, plant growth, sterilization, and enzymatic activity9,10,11,12,13,14,15,16,17. PAW has been extended to agricultural applications to regulate seed germination and plant growth. In one study, mung bean seeds treated with PAW showed improved vitality and hypocotyl length, with enhanced overall growth9. The germination rate, growth characteristics, and total phenolic and flavonoid levels were all maximized when mung bean seeds were treated with PAW for 15 s, followed by a decline in effectiveness during prolonged plasma activation (30–90 s)10. Another study showed significant growth improvement compared to untreated seeds with atmospheric plasma treatment at doses from 200 to 1600 mA18. Notably, effects of PAW on germination and growth have also been observed in wheat19,20,21,22, soybean23,24,25,26, radish27, tomato28,29,30, lentil31, and chickpea32.

In other studies, atmospheric plasma-treated seeds showed higher germination rates and increased sprout length compared to untreated seeds. Mung bean seeds treated with atmospheric plasma at 60 W RF (Radio Frequency) and 20 min of irradiation exhibited a 36.2% increase in germination rate after 24 h and a 30% higher sprout length after 48 h compared to untreated mung beans11. Atmospheric plasma treatment also enhanced hydrolytic enzymes (amylase, protease, and phytase) while reducing anti-nutritional properties like trypsin11. The effects of plasma duration and different reactive species ratios in cold atmospheric-pressure plasma treatment were investigated; enhanced physical and endogenous hormones were observed, impacting the germination and growth of mung bean seeds12,33. Plasma treatment induced up to three times higher regulation of the natural plant growth hormone GA3, which plays a key role in radicle growth in mung beans12,13.

Atmospheric-pressure microplasma arrays (using N2, He, air, and O2) applied to mung bean seeds significantly influenced germination and growth14. Air microplasma arrays outperformed other gases, enhancing both germination rates and seedling growth of mung bean by interactions with plasma-generated reactive oxygen and nitrogen species14. Both direct and indirect plasma treatments positively impacted plant development, including seed germination, growth, and disease prevention, and regulated the vegetative growth phase with long-term effects34,35,36,37,38. A linear relationship was found between vitamin C content and the germination time and sprouting of mung beans and chickpea39,40. In a study examining pressure variations, Raphanus sativus seeds treated with low-pressure O2 plasma (RF power of 60 W and pressures ranging from 20 to 80 Pa) exhibited varying plant lengths27,41. Oxygen plasma treatment also impacts the reduction-type thiol content. This alteration in antioxidative activity, induced by active oxygen species from the plasma, contributes to the production of plant growth factors. Researchers found an association between thiol content in plasma-treated seeds and plant growth34.

From previous studies, we have identified that all those researches have investigated only the germination rate, growth, and antioxidant level enhancement following PAW at atmospheric pressure plasma treatment of mung bean seeds. The present study diverges from conventional atmospheric PAW treatment. To clarify the effect of plasma on plants (seeds), we used low-pressure oxygen plasma, which is very simple and has been thoroughly investigated for this purpose. While in recent years PAW has been actively researched and featured in many papers, its basic characteristics remain unclear. Even the fundamental aspects, such as the type of active species exist in PAW and their energy levels, lack definitive consensus. Therefore, using PAW to study to clarify unknown plant characteristics is challenging. In low-pressure oxygen plasma, only oxygen (O2) gas contributes to the discharge, generating atomic oxygen and excited oxygen molecules. These particle’s generation and annihilation cross-sections are also known allowing us to understand immediately the particle species and energy levels in the oxygen plasma we produced. Therefore, in this paper, we are trying to clarify unknown plant characteristics by using this known plasma.

When PAW is used for seed treatment, the treatment volume is large, but the seeds become wet and germinate, necessitating treatment immediately before sowing. Therefore, seed manufacturers cannot adopt this method in their seed processing factories, and each farmer must own this device, which is a major problem in terms of cost-effectiveness. Low-pressure plasma can treat large quantities of seeds at manufacturing facilities because the plasma volume can be expanded to about 1,000 times that of atmospheric pressure by reducing the pressure. In addition, since it is a completely dry process, it allows for long-term seed storage, with the effects of plasma treatment lasting several weeks.

Currently there is no available information regarding the direct impact of oxygen plasma irradiation on mung bean seed germination, growth, and antioxidative potential, particularly with respect to variations in pressure and exposure time. This research is unique in using pure oxygen plasma directly on seeds at various pressure and exposure times to investigate germination rate, growth, vitamin C content, thiol concentration and antioxidant activity. According to this experiment, the oxygen plasma irradiation conditions for promoting the production of vitamin C and antioxidant active substances are similar to the irradiation conditions for promoting the growth of sprouts by irradiating mung bean seeds with oxygen plasma that has begun to be put to practical use. Since the same low-pressure chamber is used and the parameters such as the oxygen gas pressure range, required high-frequency power, and treatment time are also the same, it is possible to realize and put into practical use a method for improving vitamin C and antioxidant activity in seeds by slightly modifying the operating parameters of a practical mung bean plasma treatment device. Presently, tests are underway to put this method into practical use for enhancing vitamin C production in mung bean sprouts by irradiating seeds with the oxygen plasma.

This study aims to investigate the effects of direct oxygen plasma treatment under different pressure conditions and exposure times on the physicochemical properties of mung bean.

Experimental apparatus and methods

Plasma device

In this study, a low-pressure plasma device was used. Figure 1 shows a schematic diagram of the low-pressure capacitively coupled plasma apparatus42,43,44,45,46. The vacuum chamber is a stainless-steel cylindrical container with an inner diameter of 210 mm, a length of 480 mm, and a volume of 20 L. A high-frequency electrode is installed in the upper part of the chamber, and a sample holder on which the object to be processed is placed in the diffused region. The distance between the electrode and the inner wall of the chamber is 3 cm, and the distance between the electrode and the sample holder is 5–6 cm. The material gas for plasma production is pure oxygen, which is introduced into the chamber through the fine needle valve. The inside of the vessel is filled with oxygen gas at a specific oxygen gas pressure (10, 20, 40, 60, 80, and 100 Pa), and high-frequency power of 13.56 MHz, as shown in Fig. 1, is used to generate a volume of oxygen plasma. Plasma is produced in the gap between the high-frequency electrode and the inner wall of the grounded chamber. Oxygen gas is introduced from the gas cylinder to the vacuum pump through a gas regulator and a fine variable needle bulb. When the flow rate of oxygen gas is controlled using a variable needle bulb, and the evacuation velocity of the vacuum pump is kept constant, the oxygen gas pressure varies. Input high-frequency electrical power from a radio frequency power supply to the low-pressure plasma is kept constant at 60 W. Discharge occurs in a space of 1 to 2 cm between the electrode and the inner wall of the vacuum vessel, and the generated oxygen ions become neutral active species by charge exchange while diffusing toward the center of the vacuum vessel. Neutral reactive oxygen species (excited oxygen atoms O(1D), O(5P), singlet oxygen molecules 1∑g+, etc.) in the oxygen plasma were measured by optical light emission spectroscopy.

Fig. 1
figure 1

Schematic of the low-pressure plasma device.

Full size image

Mung bean seed treatment and sprout production

As the sample, mung bean (V. radiata) seeds were used in this experiment. The seeds were collected from Daisey Co. Ltd., Japan. Seeds were placed for irradiation in the afterglow region, 5 cm away from the linear discharge electrode fixed on the inner surface of the vacuum vessel of the plasma device (Fig. 2). The seeds were irradiated at 10, 20, 40, 60, 80 and 100 Pa of oxygen gas pressure under different exposure times of 10, 30 and 60 min. After irradiation, the seeds were placed in a seedling tray floated on distilled water in a container (Fig. 3). To evaluate the growth of seedlings from each treatment under similar conditions, the seeds were incubated in an artificial climate chamber at 24 °C under darkness. To keep the water level constant in the container, water was added as needed. A control (no plasma) experiment was carried out under identical conditions to compare the effect of plasma treatment.

Fig. 2
figure 2

Plasma irradiation of mung bean seed.

Full size image
Fig. 3
figure 3

Seeds sown on a seedling tray.

Full size image

Calculation of germination rate

The emergence of a radicle elongated to around 1 mm from the seed coat was taken as the criteria for germination. Six, 12, 18, 24, and 30 h after seed sowing, the germination rate was calculated as: Germination rate (%) = (N1/N0) × 100%, where N0 is the total number of seeds in each treatment and N1 is the number of germinated seeds.

Growth measurement

The stem and root length of each mung bean sprout was measured with a graded ruler 108 h (5th day) after sowing. The stem and root length were considered as the length of the hypocotyl and the length of a radicle, respectively. The final results were expressed as the average length of stem and roots in each group. The weight of sprouts and roots was also measured using an analytical balance.

Preparation of sample extract

For measurement of vitamin C and thiol contents, 1 g of sample (fresh weight (FW)) was ground with a mortar and pestle. The samples mixed with 10 ml distilled water were thoroughly shaken and then the supernatant was collected by centrifuging at 4000 rpm (26 × 100 g) for 15 min. For determination of antioxidants, ethanol was used as the solvent instead of distilled water and extractions were centrifuged at 1500 rpm (3 × 100 g) for 10 min.

Analysis of Vitamin C

Vitamin C content of the stem and root was measured by the Folin-Dennis colorimetric method47. The sample extract (1.5 ml) was reacted with the test reagent to produce a blue color product and its absorbance was measured spectrophotometrically at 760 nm. In the same procedure, the absorbance was measured using different known concentrations of ascorbic acid to generate a standard curve. By plotting the absorbance value of the sample in the linear equation (y = 48.754x + 0.1928, R2 = 0.9967) of the standard curve, the content of vitamin C was measured and expressed as mg/gm FW.

Analysis of Thiol Concentration

The thiol concentration in stem and root was measured using a thiol quantification kit, a colorimetric assay for measuring thiol48,49. The assay was performed in a 96-well microplate. In the microplate, 170 µL of thiol quantification coloring reagent was added to 30 µL of sample and incubated for 30 min at room temperature to complete the reaction. The yellow colored final product was detected spectrophotometrically at 450 nm using a microplate reader. A glutathione standard curve was prepared with known concentrations of serially diluted glutathione (100, 50, 25, 12.5, 6.25, 3.12, and 1.56 µM) using the same procedure. By plotting the absorbance value of the sample in the linear equation (y = 0.0011x + 0.0021, R2 = 0.9999) of the glutathione standard curve, the concentration of thiol (µM) was measured.

Determination of antioxidant activity and IC50 value

The antioxidant activity of the sample was measured by a DPPH scavenging assay protocol50,51. The sample was diluted for a range of concentrations. The different sample volumes (50, 100, 200, 400, 600, 800, and 1000 µL) were taken and adjusted to 1000 µL by adding the required amount of ethanol. Then, each 1000 µL diluted sample was added with an equal volume of 0.1 mM DPPH solution in a cuvette to initiate the reaction. A control experiment (solvent and DPPH) was also carried out. After gentle vortexing, the solution was incubated for 30 min in darkness and the absorbance was measured spectrophotometrically at 517 nm.

The % DPPH scavenging activity or inhibition rate was calculated for each sample using the formula: % Scavenging activity = [(Abscontrol – Abssample) / Abscontrol] x 100, where Abscontrol is the absorbance of control (DPPH + solvent) and Abssample is the absorbance of the sample incubated with DPPH. The % scavenging activity vs. sample amount was plotted for each treatment. Then, the IC50 value (sample amount that causes 50% inhibition) was determined.

Statistical analysis

All experiments were repeated at least three times. The significance of differences was statistically analyzed with one-way analysis of variance (ANOVA) with a confidence level at P < 0.05. All statistical analysis was performed using Microsoft Excel 2019.

Results and discussion

Production of active oxygen species

Figure 4 shows a typical light emission spectrum in the diffused region of low-pressure oxygen plasma at pressures of 10, 20, 40, 60, 80, and 100 Pa. The exposure time was 500 ms, with spectra accumulated 10 times and a slit width of 25 μm. The light fiber, connected to the spectrometer, is positioned axially 110 mm from the RF antenna set along the inner wall of the vacuum chamber. Obvious peaks were observed at 309, 762, and 777 nm at each pressure, even though the intensity at 100 Pa is lower than that at 10 Pa. These peaks correspond to active oxygen species of the OH radical, singlet oxygen molecule O2(1∑g+), and atomic oxygen O(5P)42,44,52,53, respectively. Moreover, the observed peaks at 527, 559, and 636 nm were attributed to oxygen molecule ions O2+, which require relatively higher electron energy for generation. These peaks cannot be observed at a higher pressure of 80–100 Pa, where the electron temperature decreases. Figure 5 shows the pressure dependence of light emission intensity from atomic oxygen O(5P) and singlet oxygen molecule O2(1∑g+). The light emission intensity of O(5P) decreases with increasing oxygen gas pressure, which is due to the decrease of mean electron energy from the discharge by the increase of electron—neutral collisions. The excitation energy of O(5P) from the ground state is around 10.7 eV. The number of energetic electrons decreases with the pressure, and then the amount of atomic oxygen monotonically decreases. The pressure dependencies of OH radical, O(3P), O(1S), and O(1D) are almost similar to O(5P). On the other hand, the light emission intensity of singlet oxygen molecule O2(1∑g+ →3∑g‒) increases with pressure up to 60 Pa, then decreases above 60 Pa. The excitation energy of O2(1∑g+) is relatively lower, 1.63 eV. When the pressure is in the range of 10 to 60 Pa, the production of atomic oxygen decreases and the production of O2(1∑g+) increases. Above 60 Pa, electrons lose energy due to an increase in collisions, and the proportion of electrons that do not exceed the oxygen excitation energy increases, followed by a decrease in O2(1∑g+) production.

Fig. 4
figure 4

Typical UV-Vis light emission spectra of low-pressure oxygen plasma at different pressures.

Full size image
Fig. 5
figure 5

Light emission intensities from active oxygen species in low-pressure oxygen plasma.

Full size image

Effects of plasma on germination rate

To study the effect of oxygen plasma on mung bean seed germination at different gas pressures (10, 20, 40, 60, 80, and 100 Pa) under different plasma exposure times (10, 30, and 60 min), the germination rate was determined 6, 12, 18, 24 and 30 h after seed sowing. The obtained data suggested that oxygen plasma accelerated and enhanced seed germination (Fig. 6). Compared to the control, oxygen plasma was able to shorten the germination time and improve the germination rate at every gas pressure and exposure time. Plasma-treated seed reached more than 90% germination rate by 18 h after seeding, whereas the control (no plasma) reached this level by around 30 h. After 12 h, the maximum germination rate was observed when the oxygen gas pressure was 60 Pa, which was 48, 44, and 36% higher compared to the control at an exposure times of 10, 30 and 60 min, respectively (P < 0.05); after 18 h, it was 40 to 44% higher compared to the control. This could be attributed to the relatively high density of reactive oxygen species generated at 60 Pa of oxygen gas (Fig. 6). It was revealed that the germination rate accelerated between 6 and 18 h under various gas pressures during both 10- and 30-min exposure times. However, when exposed for 60 min, the germination rate peaked between 12 and 18 h. This suggests that short-duration plasma treatment enhances seed germination more rapidly, while prolonged treatment initially exhibits inhibitory effects. Notably, the highest germination rates occurred at 12 and 18 h with a 10-min exposure time compared to the 30-min treatment, and the rate was even lower with 60-min plasma treatment.

Fig. 6
figure 6

Germination rate at different oxygen gas pressures and exposure times.

Full size image

Seed germination is a physiological process. During the early period of germination, the seed coat acts as physical protection against the environment and as a constraint to root protrusion. Therefore, any structural modification of this coating could affect seed germination. The plasma directly induced changes in the seed coating. The interaction of the electrons and ions of the plasma with the seed coat may create cracks and reduce the water contact angle of the seed to modulate seed surface hydrophilicity, increasing water uptake and initiating subsequent biochemical processes to enhance the germination rate and promote sprout radicle growth22. Another possible mechanism for plasma-induced enhanced seed germination is the biochemical and molecular processes inside the seed that are activated by plasma-generated reactive oxygen species. This facilitates oxidation of the aleurone layer and mobilization of food reserves during seed germination54.

Effects of plasma on mung bean seedling growth

Stem and root length

To observe the growth characteristics of mung bean, the length and weight of both the stem and root were recorded on 5th day after seeding. We observed that both the average length and weight of the stem and root increased at all exposure times (10, 30, and 60 min) and different gas pressures compared to the untreated seed. Compared to the control, the stem length increased significantly ranges from 35.62 to 52.69%, 30.43 to 47.31%, and 25.42 to 41.93% (P < 0.05) at plasma exposure times of 10, 30 and 60 min, respectively, depending on the oxygen gas pressure (Fig. 7a). On the other hand, in the case of root length, compared to control, values increased by the ranges from 8.80 to 37.16%, 10.51 to 33.01%, and 8.19 to 22.98% at 10, 30, and 60 min, respectively (P < 0.05), depending on the pressure (Fig. 7b). Maximum stem length was observed at 60 Pa for each exposure time as 52.69, 47.31, and 41.93% greater than the control at 10, 30, and 60 min exposure time, respectively (Fig. 7a). For the root, compared to control, the highest root length was 37.16, 33.01, and 22.98% greater at 10, 30, and 60 min exposure time, respectively (Fig. 7b). However, at 30-min exposure time, the maximum root length was observed when pressure was 80 Pa. Le et al. found maximum stem length when mung bean seeds were treated for 10 min with atmospheric pressure plasma12. Conversely, Sadhu et al. observed the highest sprout length with a 20 min atmospheric plasma treatment11.

Fig. 7
figure 7

Changes in (a) stem and (b) root length at different oxygen gas pressures and exposure times.

Full size image

Stem and root weight

Stem and root weight were also recorded to observe the effect of oxygen plasma under different gas pressures and exposure times. Compared to control, stem weight increased significantly by a range from 39.37 to 52.57%, 30.60 to 43.33%, and 25.07 to 41.34% at exposure times of 10, 30, and 60 min (P < 0.05), respectively (Fig. 8a). In the case of root weight, compared to control, values increased at a range from 22.64 to 38.84%, 14.21 to 33.88%, and 10.58 to 34.21% at 10, 30, and 60 min (P < 0.05), respectively, depending on the pressure (Fig. 8b). Both stem and root weight showed a tendency to be higher at 10-min exposure time and a tendency to be lower at 60-min exposure time (Fig. 8). At each plasma exposure time, the highest stem and root weight were observed at 60 Pa gas pressure, except for the 30-min exposure time, where the maximum root weight was found at 80 Pa pressure.

Fig. 8
figure 8

Changes in (a) stem and (b) root weight at different oxygen gas pressures and exposure times.

Full size image

A possible reason for the maximum growth of mung bean at 60 Pa gas pressure might be due to increased stimulation of growth hormones. Plasma treatment of seeds can affect growth hormone levels, thereby affecting vegetative growth28. Phytohormones like gibberellic acid (GA3) and abscisic acid (ABA) play key roles in seed germination and seedling growth55. ABA induces seed dormancy and inhibits germination, while GA3 promotes dormancy release, cell growth, and stem elongation. These hormones counteract each other, and their activity depends on the concentrations of reactive species56; however, their mechanisms are not fully understood.

Effects of plasma on vitamin C content

Vitamin C content in the stem and root of mung bean was observed on the 5th day after sowing plasma-treated seeds. The content varied with the changing pressure of oxygen gas under different plasma exposure times. It was observed that vitamin C content increased greatly compared to the control (no plasma) under every pressure and exposure time. In the stem, vitamin C content increased significantly with increasing pressure up to 60 Pa, then decreased with increasing pressure at both 30- and 60-min exposure times (P < 0.05). However, in the case of 10-min exposure time, vitamin C content increased up to 80 Pa and decreased thereafter. However, in the case of the root, at every exposure time, vitamin C content increased up to 60 Pa and then decreased with increasing pressure (P < 0.05). On the other hand, vitamin C content also varied with plasma exposure time. Vitamin C content was reduced with longer plasma exposure time.

In the stem, the maximum vitamin C content was 1.97-fold greater than the control at 80 Pa and 10-min exposure time. At 30- and 60-min exposure times, maximum vitamin C content was observed at 60 Pa, which was 1.87 and 1.81-fold higher compared to the control, respectively (Fig. 9a). However, in the root, vitamin C content was higher at 60 Pa, which was 2.04, 1.81, and 1.68-fold higher compared to the control at exposure times of 10, 30, and 60 min, respectively (Fig. 9b). The increasing rate of vitamin C in the stem was less prominent with changes in pressure between 20 and 80 Pa.

Fig. 9
figure 9

Changes in vitamin C content of (a) stem and (b) root at different oxygen gas pressures and exposure times.

Full size image

The oxygen gas pressure dependence of vitamin C content shows the same tendency as the oxygen gas pressure dependence of the amount of reactive oxygen species generated in plasma (Fig. 5). Up to an oxygen gas pressure of about 80 Pa, the collision frequency of electrons and neutral particles in plasma is small, electrons are less likely to lose energy, and singlet oxygen molecules are effectively generated. When the oxygen gas pressure increases, electrons lose energy through collisions and singlet oxygen molecules cannot be generated. On the other hand, at high pressure, it is thought that the generation of reactive species such as superoxide (O22−) increases, which are thought to not contribute to vitamin C production. As a result, the production of vitamin C and antioxidants decreases.

In particular, around 80 Pa, the reactive oxygen species related to vitamin C production and the reactive oxygen species that do not contribute to vitamin C production increase at the same time, and it is presumed that long-term irradiation for 30–60 min increases the oxidative damage to seeds caused by reactive oxygen species that do not contribute to vitamin C production. Moreover, vitamin C showed a greater reduction at the highest pressure and higher exposure times, which could be attributed to structural changes in the seed during germination, making vitamin C more accessible to degradation7.

Effects of plasma on thiol concentration

Thiol compounds, like thioredoxin and glutathione (GSH), are key antioxidative substances in cells and maintain the redox state of the cell57,58,59. In our study, we examined changes in thiol compounds, particularly GSH, within the stem and root of mung bean after oxygen plasma irradiation of seeds at varying pressures and exposure times. Notably, the GSH levels in both the stem and root increased following irradiation compared to the control. Increased antioxidative substances may boost reduction-type thiol compounds in cells. Optimal plant growth depends on balanced active oxygen species levels, as both an excess and a deficiency can hinder cell proliferation and development. Therefore, thioredoxin levels were measured following plasma irradiation of seeds. To investigate the relationship between thiol content and plant growth under varying pressure and plasma exposure time, thiol content was measured in the stem and root of mung bean on the 5th day after sowing of plasma-treated mung bean seeds.

We observed that thiol content varied with changing pressure and plasma exposure time both in the stem and root. Maximum thiol content was observed at a plasma exposure time of 10 min, while the lowest content was observed at 60-min exposure. Across all exposure times, both stem and root samples showed a significant increase in thiol content with rising oxygen pressure up to 60 Pa, followed by a decline at higher pressures (P < 0.05). Specifically, in the stem, the highest thiol content was observed at both 40 and 60 Pa, which was 2.76-fold greater compared to the control (Fig. 10a). However, in the case of 30 and 60 min of plasma treatment, the maximum thiol content was observed at 60 Pa (P < 0.05), which was 2.58 and 1.85-fold greater compared to the control, although the impact of pressure was not as obvious at an irradiation time of 60 min. On the other hand, in the root, the highest thiol content was found at 60 Pa at each plasma exposure time, which was 2.49, 1.89, and 1.61-fold higher than the control at exposure times of 10, 30, and 60 min, respectively (Fig. 10b).

Fig. 10
figure 10

Changes in thiol content in (a) stem and (b) root at different oxygen gas pressures and exposure times.

Full size image

The increase in stem and root length followed the same trend as the rise in thiol content with varying pressure and exposure time (Figs. 7 and 10). However, longer plasma irradiation reduced sprout length and thiol content to near control levels (Figs. 7 and 10). Excess active oxygen species from prolonged irradiation may convert thiol compounds into disulfide compounds34. Sprout growth varied with oxygen gas pressure, as observed for both stem and root lengths (Fig. 7) under the different irradiation times (10, 30, or 60 min) and 5 days of cultivation.

A thiol quantification reagent was used to measure reduced thiol compounds, including thioredoxin and GSH, in plant cells. Figures 7 and 10 show that stem or root length correlates with the thiol content, which increased up to 60 Pa of oxygen pressure before declining. Maximum growth and thiol levels were achieved with 10-min plasma irradiation. Consequently, thiol compounds are a key factor in enhancing plant growth among various antioxidative substances. The redox reaction of thiol compounds and optimization of the redox state in plant cells represent potential mechanisms for growth enhancement, with reactive plasma controlling this redox state34. Thus, the thiol content in seeds exposed to plasma irradiation may be associated with plant growth.

Effects of plasma on antioxidant capacity

The mung bean seeds were treated with oxygen plasma at different pressures and exposure times, and effects on the antioxidant capacity of the stem and root were observed. To study the effects of oxygen irradiation on mung bean, the IC50 value (sample amount required for 50% inhibition of DPPH) was measured from stem and root samples. A lower IC50 value indicates higher antioxidant activity.

Table 1 Effects of plasma treatment at different pressures and exposure times on the antioxidant capacity of mung bean.
Full size table

It was observed that plasma irradiation at different pressures and exposure times positively influenced the antioxidant capacity of mung bean stem and root. The antioxidant capacity of the stem and root generally increased with pressure up to 60 Pa (lower IC50 value), then decreased with increasing pressure (P < 0.05). This suggests that the antioxidative substances in plant seeds might be increased with exposure to the active oxygen species generated in the plasma. Higher antioxidant potency was most pronounced at 30-min plasma exposure, increasing from 10 to 30 min, but decreased with prolonged exposure time of 60 min (Table 1). While pressure changes had a minor effect, a significant increase in antioxidant potency was observed compared to the control.

Conclusion

Oxygen plasma treatment significantly affects the physicochemical properties of mung bean. The present study revealed that active oxygen species generated by plasma treatment at various pressures and exposure times influenced germination, plant growth, vitamin C and thiol contents, and antioxidant activity of mung bean. Our results show that: (a) Plasma treatment shortened the germination time up to 12 h to reach 100% germination and increased the germination rate up to 48% greater compared to the control; (b) Stem and root length generally increased with pressure up to 60 Pa, then declined. A similar trend was also found for stem and root weight; (c) Vitamin C content in the stem and root increased with pressure up to 60 Pa; (d) Thiol content in both the stem and root obviously increased with pressure up to 60 Pa at all exposure times; (e) Antioxidative potential varied with changing pressure and was maximum at 30-min exposure; (f) All physicochemical properties assessed were associated with pressure and exposure time. Notably, growth and antioxidative potential were reduced with longer irradiation exposure time. Overall, the findings of this study indicate significant effects of varying pressure and plasma exposure time on mung bean germination, growth, vitamin C content, thiol concentration, and antioxidant level in both stems and roots. However, the influence of plasma treatment at different pressures on seed genetics and other biological factors, which play roles in physicochemical enhancement, remains unclear. Further research is needed to explore these effects comprehensively.