# Non-Thermal Plasma: A Promising Technology for the Germination Enhancement of Radish (Raphanus sativus) and Carrot (Daucus carota sativus L.)

Authors: Rajesh Prakash Guragain; Hom Bahadur Baniya; Bikash Shrestha; Deepesh Prakash Guragain; Deepak Prasad Subedi
Journal: Journal of Food Quality
 (2023)

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## Abstract

Cold plasma is an innovative and promising technology that is developing in a variety of fields, and recently it has been getting a lot of attention in the agricultural industry. The influence of cold atmospheric pressure plasma (CAPP) exposure on germination parameters and vigor of radish (Raphanus sativus) and carrot (Daucus carota sativus L.) seeds was investigated in the present study. A custom-designed plasma driver utilizing 11.32 kV rms and 50 Hz was used for the generation of the discharge. Seeds were treated using a dielectric barrier discharge (DBD) in an argon atmosphere at exposure periods of 1–4 minutes. The estimate of plasma parameters was done using optical emission spectroscopy and electrical measurements. Germination-related measures such as the final germination percentage, germination index, germination value, coefficient of velocity of germination, vigor index, and chlorophyll content were all improved in the case of CAPP-treated seeds as compared to control seeds. Similarly, CAPP treatment changed the in vitro radical scavenging capabilities, total phenolic, and total flavonoid levels of the seedlings. Our results indicated that the seeds being treated by CAPP for 3 minutes seemed to have a favorable impact on seed germination and sprouting development.


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## Body

## 1. Introduction

Agriculture is currently one of the most researched and supported scientific disciplines. Food, being the basic need for human survival, is one of the major areas of investment in recent economies. With the rise of the global population and an increase in the demands associated with changing lifestyles, it is getting harder to fulfill the requests of supply chains. New and better methods in agronomy are sought after every day with the hope of finding an ideal solution for healthier and better-growing plants. Because cultivable land is diminishing at an alarming rate, the only way to assure food safety is to increase crop production per unit of land area by an exponential factor. The importance of food quality in the context of sustainable, environmentally friendly agriculture drives seed-plasma treatment research. Food prices spiked in 2008, putting millions of people in danger of starvation and triggering riots in developing countries [1]. According to the Food and Agriculture Organization, the world’s population is expected to grow to 10 billion by 2050 [2, 3]. Even if there is not enough food available, it’ is important to improve the quality of the food that is already being made. The cold plasma technique is being used in agronomy as a practical, cost-effective, and environmentally friendly approach to increase grain production [4–6]. CAPP has also been proven to boost seedling growth and survival, potentially reducing the need for chemical seed priming and increasing the production of a variety of essential crops [7–11].The complicated process of sprouting is initiated by water absorption, in which water enters the seed coat during fertilization, resulting in the weakening of dry interior tissue [12]. The seed absorbs moisture from the soil and swells due to the weakening of its seed coat. The seed coat then splits, enabling moisture to enter the seed more quickly. Water absorption rate on seed surfaces, which can be modified by CAPP, is regulated by surface energy/surface charges, implying that surface characteristics are important in material-environment interactions (e.g., seed and moisture) [13]. Furthermore, the CAPP treatment significantly reduces organics that are located on the surface of seeds [14–17]. Despite extensive research, some concerns remain unanswered about plasma seed disinfection. For example, plasma sources, seed species, and treatment settings may all impact disinfection results and efficiency. Thus, a standardized application dosage and its effects have yet to be defined. Also, the infection probability in germinated seedlings treated with plasma is seldom studied. More research and knowledge are needed in this area.Recent published research on the use of plasma technology indicates that appropriate CAPP treatment has a beneficial influence on seed germination and plant physiology [18–24]. Many nations have conducted substantial studies on plasma technology in agriculture. Plasma increased the growth and productivity of lettuce, melons, cucumbers, soybeans, and rice [25–29]. Low-pressure plasma (LPP) has been suggested by various researchers to minimize pathogen infection, promote sprouting, and hence boost productivity. For example, low-pressureradio-frequency argon discharge increased safflower sprouting by 50% [30]. Similarly, brown rice exhibited a 62% rise in germination rate after being exposed to low-pressure plasma [31]. Similarly, when treated with atmospheric plasma, the germination of chili and cucumber seeds improved significantly [27]. Also, wheat, maize, and lupine yields were all enhanced by 2.3%, 1.7%, and 26.8%, respectively, after LPP treatment [32]. A similar experiment was performed on carrot seeds and the significant enhancement in seed germination and seedling development was observed [18].Throughout this investigation, we analyzed the influence of CAPP on radish (Raphanus sativus) and carrot (Daucus carota sativus L.) seed germination and their subsequent effects on plant growth. Seed samples were placed directly in contact with discharge filaments, with the exposure period varying while various parameters (HV power and frequency, gas flow rate) remained constant.


## 2. Experimental Set-Up

The laboratory arrangement used in the investigation is shown in Figure1. The reactor chamber is rectangular in shape and made from polycarbonate, measuring 357.0 mm × 200.0 mm × 150.0 mm in length, breadth, and height, respectively. A gap of 5 mm was maintained between two rectangular electrodes (made from copper with dimension: 75.4 mm × 49.8 mm × 10.0 mm) throughout this study. A dielectric (polycarbonate plate) measuring 150.0 mm × 120.0 mm × 2.0 mm in length, breadth, and width covers the lower electrode. Argon discharge was created by applying a sinusoidal voltage and frequency of 11.32 kV and 50 Hz, respectively. To measure the voltage and current across the electrodes, a PINTEK high-voltage probe (capable of sensing 28 kV voltage with a division ratio of 1000 : 1) and an oscilloscope probe over a 10 kΩ shunt resistor were used, respectively. Both the probes are connected to a Tektronix (TDS 2002) digital oscilloscope. The emission spectra were measured using a spectrometer (Ocean Optics, USB 2000+). Argon gas flow rate was maintained at four litres/minute. During the test, 200 radish and carrot seeds were carefully placed above the dielectric into the active discharge zone of CAPP for periods of 1 to 3 minutes and 1 to 4 minutes, respectively.Figure 1
Image of the experimental setup.


## 3. Material and Methods

### 3.1. Analysis of Germination Characteristics

Seeds were collected from “the Nepal Agriculture Research Council (NARC), Nepal.” Only good and undamaged seeds were taken for the investigation. A cocopeat was cleaned with deionized water and allowed to dry at room temperature. Three replicates of 200 seeds each of radish and carrot were sown in different trays filled with cocopeat to study the rate of germination and growth. A sprout is regarded to have germinated when the radicle emerges from the seed coat [5, 33]. Many germination characteristics like final germination percentage (FGP), relativized percentage (RPG), mean germination time (MGT), mean germination rate (MGR), coefficient of velocity of germination (CVG), germination index (GI), uncertainty of the germination process (U), synchronization index (Z), mean daily germination percentage (MDG), germination (G)-value, and vigor index (I) were explored using the methodology available from different investigators [5, 34–37].


### 3.2. Statistical Investigation

Tests were done in three replicates, and the results were represented as the mean ± standard deviation. GraphPad Prism 8.0.2 was used to assess the significant difference in the mean germination parameters using one-way Anova and Tukey’s multiple comparison test. The values indicated with different letters (A–E) in the graphs are statistically significant atp<0.05.


## 3.1. Analysis of Germination Characteristics

Seeds were collected from “the Nepal Agriculture Research Council (NARC), Nepal.” Only good and undamaged seeds were taken for the investigation. A cocopeat was cleaned with deionized water and allowed to dry at room temperature. Three replicates of 200 seeds each of radish and carrot were sown in different trays filled with cocopeat to study the rate of germination and growth. A sprout is regarded to have germinated when the radicle emerges from the seed coat [5, 33]. Many germination characteristics like final germination percentage (FGP), relativized percentage (RPG), mean germination time (MGT), mean germination rate (MGR), coefficient of velocity of germination (CVG), germination index (GI), uncertainty of the germination process (U), synchronization index (Z), mean daily germination percentage (MDG), germination (G)-value, and vigor index (I) were explored using the methodology available from different investigators [5, 34–37].


## 3.2. Statistical Investigation

Tests were done in three replicates, and the results were represented as the mean ± standard deviation. GraphPad Prism 8.0.2 was used to assess the significant difference in the mean germination parameters using one-way Anova and Tukey’s multiple comparison test. The values indicated with different letters (A–E) in the graphs are statistically significant atp<0.05.


## 4. Results and Discussions

### 4.1. Electrical Signal Analysis

Figure2 illustrates the voltage and current waveform of the DBD. The current signal consists of many microdischarges. CAPP-DBD is identified by the presence of microdischarge [38]. The power consumed can be estimated by the integration of current and voltage [39].(1)P=1T∫0TItVtdt.Figure 2
I(t)–V(t) signal of the DBD argon plasma.From (1), the power consumed was found to be 17.9 W. Similarly, the energy consumed per cycle was found to be 0.36 J.


### 4.2. Optical Characterization of the Discharge

Figure3 illustrates the optical emission spectra (OES) of the DBD using argon as the working gas. From the spectrum, five suitable lines of Ar I were chosen, and electron excitation temperature were estimated using the Boltzmann plot method [40, 41].(2)lnIλAjigj=−EjkTexc+C.Figure 3
OES of the discharge.The wavelengthλ and intensity I of the spectral lines are taken from observation in (2), while the values of transition probability Aji, statistical weight gj, and energy Ej, of the spectral lines, respectively, are acquired from “National Institute of Standards and Technology (NIST) atomic spectra database” [42].A straight line is obtained when a graph oflnIλ/Ajigj on the y-axis was plotted with respect to Ej along x-axis. The slope of the obtained straight line gives the excitation temperature Texc. In this study, the slope was found to be 1.13 (Figure 4). This corresponds to Texc of about 0.88 eV which approximately equals to 10208 K.Figure 4
Estimation of excitation temperature.


### 4.3. Influence of CAPP on Germination Parameters

On the 30th day from the day of sowing, the observation of the seedlings was studied to find the final germination percentages. As indicated in Figure 5, the final germination percentage of CAPP-exposed seeds was found to be higher than that of the control in both cases. It was found that the germination percentage of 1-minute, 2-minute, and 3-minute treated radish seeds rose by 3% when compared to untreated seeds. However, a statistically significant difference was not identified for radish seeds exposed to plasma at different times.Figure 5
Influence of CAPP exposure on the germination of (a) radish and (b) carrot.
(a)(b)Similarly, the FGP for 1, 2, 3, and 4-minute carrot sprouts rose by 13.66%, 22.24%, 25.8%, and 8.7%, respectively, as compared to untreated. Compared to 1, 2, and 3 minutes, carrot seeds exposed to CAPP for 4 minutes showed a lower FGP. This may have occurred as a result of the deposition of excessively energetic reactive species generated in DBD after 3 minutes of treatment. This species has an effect on plant structure due to the fact that prolonged exposure promotes surface degradation and nutrient loss in seeds. These alterations demonstrate the advantages of exposing seeds to CAPP for a suitable amount of time to considerably improve germination and growth-related parameters [43].RPG (relativized percentage of germination) is a measurement of “equivalent treatment comparisons where the amount of dormancy disruption varies” [18]. The measurement of RPG also revealed no statistically significant difference among radish seeds treated to CAPP for 1 minute, 2 minutes, and 3 minutes (Figure 6). Compared to untreated seeds, the RPG of CAPP-exposed radish seeds rose by 3.1%. However, the assessment of RPG revealed statistically significant differences among carrot seeds exposed to CAPP for 1 minute, 2 minutes, 3 minutes, and 4 minutes, with RPG increasing by 13.6%, 23.4%, 26.4%, and 9.6%, respectively, compared to untreated seeds.Figure 6
Influence of CAPP exposure on the relativized germination of (a) radish and (b) carrot.
(a)(b)MGT (mean germination time) is a measure of “the average time it takes for a seed to germinate or emerge,” and MGR (mean germination rate) is defined as “the reciprocal of the MGT” [37]. CAPP-exposed radish and carrot seeds germinated substantially faster than untreated seeds, with the exception of carrot seeds treated for 1 minute.Compared to untreated seeds, the MGT of radish seeds treated for 1 minute, 2 minutes, and 3 minutes was lowered by 18.6%, 29.1%, and 30.5%, respectively. Similarly, compared to the control, the MGT values of treated carrot seeds at 2, 3, and 4 minutes decreased by 0.8%, 1.2%, and 5.9%, respectively. Statistically significant difference was not identified among 2 minutes and 3 minutes treated radish and carrot seeds (Figure7).Figure 7
Influence of CAPP exposure on the mean germination time of (a) radish and (b) carrot.
(a)(b)The MGR of radish seeds treated for 1 minute, 2 minutes, and 3 minutes increased by 22.2%, 40.7%, and 40.7%, whereas that of carrot seeds treated for 3 minutes and 4 minutes increased by 2.5% and 6.5%, respectively (Figure8). There was no statistically significant difference in MGR values between untreated carrot seeds and those exposed to CAPP for 1 or 2 minutes. The data clearly demonstrate that radish and carrot seeds exposed to CAPP germinated more quickly than untreated seeds.Figure 8
Influence of CAPP exposure on the mean germination rate of (a) radish and (b) carrot.
(a)(b)In the case of carrot seeds, the germination index value rose by 15.01%, 24.98%, 31.11%, and 15.69%, respectively, compared to the control. However, no significant differences were seen in the cases of 1-minute and 4-minute treated seeds. Also, a noticeable variation in GI value was noted among the control and LTP-treated radish seeds (Figure9). A greater GI value suggests a higher proportion of sprouting [44].Figure 9
Effect of CAPP treatment on the GI of (a) radish and (b) carrot.
(a)(b)The coefficient of germination velocity (CVG) measures the rate at which seeds germinate. Its usefulness increases as the quantity of sprouting seeds grows and the time it takes for them to grow lowers [25, 45]. Figure 10 illustrates that when radish seeds were treated by CAPP for 1–3 minutes, CVG improved by 23.90%, 41.36%, and 43.70%, respectively, when compared with untreated seeds. A similar increase in CVG appears when carrot seeds are exposed to CAPP for 1–4 minutes, respectively.Figure 10
Influence of CAPP treatment on the CVG of (a) radish and (b) carrot.
(a)(b)U (uncertainty of the germination process) is defined as “the degree of uncertainty associated with the relative frequency of germination dispersion,” and Z (synchrony of the germination process) is defined as “the amount to which members of a certain demographic overlap” [34]. Radish seeds treated for 2 and 3 minutes had 6.3% and 13.9% lower germination uncertainty than untreated seeds, while seeds treated for 1 minute had 2.1% higher uncertainty values (Figure 11). For carrot seeds treated for 4 minutes, uncertainty in germination was 11.1% lower than for untreated seeds, but it rose by 8.0%, 16.4%, and 15.5% for seeds treated for 1, 2, and 3 minutes, respectively. However, synchronization of germination was dramatically reduced in both radish and carrot seeds subjected to CAPP (except for 4 minutes treated carrot seeds) (Figure 12). Carrot seeds treated for 4 minutes exhibited a 25.0% improvement in synchrony of germination.Figure 11
Effect of CAPP on uncertainty of germination process of (a) radish and (b) carrot.
(a)(b)Figure 12
Effect of CAPP on synchronization index of (a) radish and (b) carrot.
(a)(b)Time to 50% germination (T50) is the number of days required for 50% of the total number of sown seeds to sprout. It was discovered that the radish seed exposed to CAPP for 2 and 3 minutes and carrot seeds exposed to CAPP for 4 minutes took less time for 50% of the total sown seeds to sprout as compared to the untreated one (Figure 13).Figure 13
Time to 50% germination of (a) radish and (b) carrot seeds.
(a)(b)MDG (mean daily germination) is a measure of “the daily average of how many seeds germinate” [37]. Radish seeds exposed to CAPP for 1, 2, and 3 minutes did not change substantially. The MDG for treated radish seeds increased by 2.9% compared to untreated seeds. Similarly, the MDG values of carrot seeds treated for 1, 2, 3, and 4 minutes rose by 13.7%, 23.0%, 25.9%, and 9.3%, respectively, compared to the control group (Figure 14). The results indicate that the daily average germination of radish and carrot seeds exposed to CAPP is considerably greater than that of untreated seeds.Figure 14
Mean daily germination of (a) radish and (b) carrot seeds.
(a)(b)For 1, 2, and 3 minutes, CAPP treated radish seed germination value rose by 22.71%, 40.23% and 45.23% compared to the untreated seeds. Moreover, CAPP-treated radish seedlings showed significant differences. Similarly, the germination values improved by 26.17%, 35.93%, 45.01%, and 26.9% for 1, 2, 3, and 4 minutes of CAPP-treated carrot seeds, respectively, as indicated in Figure15.Figure 15
Effect of plasma on germination value of (a) radish and (b) carrot.
(a)(b)The several RONS, including NO, nitrates, and hydroxyl radicals (OH), created in the production of CAPP, as depicted in Figure3, get deposited in exposed seeds and function as a signaling molecule. This species interacts with plant hormones and is essential for the alleviation of seed dormancy in plant species. Furthermore, the CAPP disinfects seeds by inhibiting microorganisms that adhere to their surface and contaminate them. This results in a significant improvement in the germination of exposed seeds to CAPP [46, 47].


### 4.4. Estimation of Seedling Length

The cocopeat from the roots of the seedlings was carefully removed from the germination tray to assess their length. Figure16 depicts photographs of radish and carrot seedlings that sprouted from untreated and plasma-treated seeds. As seen from Figure 16, seedlings sprouted from plasma-treated seeds had a longer length than those from untreated seeds.Figure 16
Photograph of the seedlings of (a) radish and (b) carrot.
(a)(b)Researchers have shown that subjecting seeds to CAPP treatment improves water imbibition rate, lowers contact angle, and changes the seed surface by generating novel chemical interactions [5, 19, 48]. The physiology of germinating seeds is thought to be altered by plasma reactive species interactions with seeds. It may also enhance the seeds’ surface with an oxygen-containing functional group [49]. These groups may promote surface wettability, which benefits nutritional absorption, and seed physiology and sprouting [49, 50].


### 4.5. Calculation of Vigor

“Vigor is used to describe a seed’s potential degree of activity and performance during germination and seedling emergence” [5].The plasma treatment boosted the vigor index (Figure17). Compared to control, the Vigor index (I), of treated seeds of radish and carrot improved by 1.2–1.5 times. However, the carrot seed treated for 4 minutes had a 1.12 drop in vigor index (I) than untreated one. These gains were linked to sprouts length and greater GI, in line with other studies [51, 52]. The interaction of CAPP with vegetal cells may activate natural signals, hormones, and enzymes, explaining the observed variations in germination and fast sprout germination [53].Figure 17
Influence of CAPP exposure on the vigor index (I) of (a) radish and (b) carrot.
(a)(b)


### 4.6. Estimation of Mass Loss and Water Uptake Capacity of Seed due to Plasma Treatment

100 seeds were exposed to CAPP for a specified time to estimate mass loss and water uptake capacity. In this study, a weighing machine (MG124Ai, Bel Instrument) was used to measure the mass. The mass loss (%) and water uptake capacity of seeds due to plasma treatment were calculated using standard protocols as described by various researchers [5, 37].There was a substantial difference in mass loss (%) between seeds treated with CAPP and untreated seeds. Radish seeds exposed to CAPP for 1 minute showed a maximum mass loss of around 33.6% compared to untreated seeds. Compared to those exposed for 1 minute, the mass loss (%) of radish seeds exposed to CAPP for 2 and 3 minutes decreased gradually. In contrast, mass loss (%) in the case of carrot seeds increased steadily from 21.9% to 41.1% as exposure duration increased. No significant difference in mass loss (%) between carrot seeds exposed to CAPP for 2 and 3 minutes was noticed (Figure18).Figure 18
Estimation of mass loss in (a) radish and (b) carrot seeds due to CAPP exposure.
(a)(b)Radish seeds have a softer seed covering than carrot seeds. Due to the weaker seed coat, maximal etching may occur within 1 minute of contact with CAPP, resulting in a greater mass loss (%) for radish. The subsequent accumulation of reactive species seen in CAPP may result in a progressive reduction in mass loss (%) [37]. Due to the harder seed coat of carrots, however, etching by RONS occurs at a slower rate, resulting in a steady rise in mass loss as exposure duration increases.Similarly, the water uptake rate of control and CAPP-exposed seeds was determined by dipping them in a petri dish containing 50 ml of distilled water for a predetermined amount of time (Figure19). From Figure 19, it can be shown that CAPP-exposed radish and carrot seeds absorbed more water than untreated seeds.Figure 19
Evolution of Imbibition rate in (a) radish and (b) carrot seeds due to CAPP exposure.
(a)(b)CAPP-exposed radish seeds absorbed about 29.3% more water than untreated seeds when soaked for 6 hours in distilled water. Similarly, the water absorption capacity of carrot seeds exposed to CAPP for 3 minutes is 30.1% higher than the control when soaked for 3 hours in distilled water.Typically, seeds are hydrophobic by nature. When seeds are exposed to CAPP for a certain period of time, reactive species (RONS) present in the CAPP may be implicated in the surface etching of the seed coat, which enhances the roughness of the seeds and causes them to become more granular, as reported by several researchers using SEM micrographs [37, 54, 55]. When the reactive species interact with the seed coat, they may result in the etching of a waxy layer from the surface of the seed coat (testa). This increases the hydrophilicity of the seeds. According to our study, seeds exposed to plasma for a specific period absorbed more water, resulting in an increase in water uptake compared to untreated seeds. Increased water uptake stimulates the development of the hypocotyl and radicle, thereby accelerating the germination of seeds compared to the control when immersed in distilled water for a specific period of time.


### 4.7. Estimation of the Change in Flavonoid, Phenolics, Radical Scavenging, Chlorophyll, and Carotenoid Contents

The estimation of flavonoids, phenolics, scavenging, and chlorophyll contents were done using standard protocols as done by various researchers [56–58].Plants’ defense systems mainly depend on phenolic and flavonoid chemicals, which have several health benefits. Antioxidants provide electrons to injured cell, preventing, and stabilizing free radical damage. Radical scavenging is crucial in diseases prevention [57]. Plant extracts are often tested for their antioxidant activities using the DPPH technique. Our findings suggest that in case of radish, 2 minutes of plasma treatment increases the amounts of flavonoids and phenolic (Figures 20(a) and 21(a)). In the first 2 minutes of plasma treatment, plant extracts demonstrated better radical scavenging activity against both DPPH and ABTS free radicals (Figure 22). Similarly in the case of carrot, CAPP exposure period enhanced the phenolics and flavonoids for up to 3 minutes (Figures 20(b) and 21(b)). This may aid in the improvement of defensive reactions in difficult situations [59]. Similarly, 3 minutes of plasma treatment increased DPPH scavenging while 4 minutes of exposure lowered it (Figure 23). This may be because after 3 minutes of treatment, excessively energetic reactive species formed in DBD might have been deposited on the surface of the seeds. This active species affects plant structure because prolonged exposure causes surface degradation and causes seeds to lose all of their nutritional content. These changes reveal the benefits of exposing seeds to CAPP at a suitable treatment time for significantly enhancing germination and growth related measures [60, 61].Figure 20
Estimation of flavonoid contents in untreated and plasma treated seeds of (a) radish and (b) carrot.
(a)(b)Figure 21
Estimation of phenolics content in untreated and plasma treated seeds of (a) radish and (b) carrot.
(a)(b)Figure 22
Estimation of ABTS/DPPH scavenging in radish seedlings.Figure 23
Estimation of DPPH/ABTS in carrot seedlings.Plant pigmentation is essential for photosynthetic activity, converting solar energy to chemical energy. Chlorophyll a, b, and carotenoids are essential pigments in plant physiological activity [62]. Also, chlorophyll pigments in plants are thought to be stress and habitat indicators. Generally, chlorophyll gives green pigmentation while carotenoids offer red, yellow, and orange pigmentation [4]. Plant pigments may be used to examine plant physiology and health. In the case of radish, the leaf extract germinated from 2 minute-treated seeds had the maximum chlorophyll-a concentration. Changes in chlorophyll-a concentration in untreated and treated seedling extracts show that the plasma treatment helps seedlings increase their overall biochemical and physiological activity [5, 63]. The CAPP treatment, on the other hand, had no noticeable effects on chlorophyll-b and carotenoid levels (Figure 24(a)). Similarly, in the case of carrot, our findings demonstrated a progressive rise in chlorophyll concentration with CAPP exposure duration up to 3 minutes, then a drop at 4 minutes. Similarly, total carotenoid levels fell as plasma treatment duration increase (Figure 24(b)).Figure 24
Estimation of chlorophyll and carotenoids contents in the leaves of seedlings germinated from control and plasma treated seeds of (a) radish and (b) carrot.
(a)(b)


## 4.1. Electrical Signal Analysis

Figure2 illustrates the voltage and current waveform of the DBD. The current signal consists of many microdischarges. CAPP-DBD is identified by the presence of microdischarge [38]. The power consumed can be estimated by the integration of current and voltage [39].(1)P=1T∫0TItVtdt.Figure 2
I(t)–V(t) signal of the DBD argon plasma.From (1), the power consumed was found to be 17.9 W. Similarly, the energy consumed per cycle was found to be 0.36 J.


## 4.2. Optical Characterization of the Discharge

Figure3 illustrates the optical emission spectra (OES) of the DBD using argon as the working gas. From the spectrum, five suitable lines of Ar I were chosen, and electron excitation temperature were estimated using the Boltzmann plot method [40, 41].(2)lnIλAjigj=−EjkTexc+C.Figure 3
OES of the discharge.The wavelengthλ and intensity I of the spectral lines are taken from observation in (2), while the values of transition probability Aji, statistical weight gj, and energy Ej, of the spectral lines, respectively, are acquired from “National Institute of Standards and Technology (NIST) atomic spectra database” [42].A straight line is obtained when a graph oflnIλ/Ajigj on the y-axis was plotted with respect to Ej along x-axis. The slope of the obtained straight line gives the excitation temperature Texc. In this study, the slope was found to be 1.13 (Figure 4). This corresponds to Texc of about 0.88 eV which approximately equals to 10208 K.Figure 4
Estimation of excitation temperature.


## 4.3. Influence of CAPP on Germination Parameters

On the 30th day from the day of sowing, the observation of the seedlings was studied to find the final germination percentages. As indicated in Figure 5, the final germination percentage of CAPP-exposed seeds was found to be higher than that of the control in both cases. It was found that the germination percentage of 1-minute, 2-minute, and 3-minute treated radish seeds rose by 3% when compared to untreated seeds. However, a statistically significant difference was not identified for radish seeds exposed to plasma at different times.Figure 5
Influence of CAPP exposure on the germination of (a) radish and (b) carrot.
(a)(b)Similarly, the FGP for 1, 2, 3, and 4-minute carrot sprouts rose by 13.66%, 22.24%, 25.8%, and 8.7%, respectively, as compared to untreated. Compared to 1, 2, and 3 minutes, carrot seeds exposed to CAPP for 4 minutes showed a lower FGP. This may have occurred as a result of the deposition of excessively energetic reactive species generated in DBD after 3 minutes of treatment. This species has an effect on plant structure due to the fact that prolonged exposure promotes surface degradation and nutrient loss in seeds. These alterations demonstrate the advantages of exposing seeds to CAPP for a suitable amount of time to considerably improve germination and growth-related parameters [43].RPG (relativized percentage of germination) is a measurement of “equivalent treatment comparisons where the amount of dormancy disruption varies” [18]. The measurement of RPG also revealed no statistically significant difference among radish seeds treated to CAPP for 1 minute, 2 minutes, and 3 minutes (Figure 6). Compared to untreated seeds, the RPG of CAPP-exposed radish seeds rose by 3.1%. However, the assessment of RPG revealed statistically significant differences among carrot seeds exposed to CAPP for 1 minute, 2 minutes, 3 minutes, and 4 minutes, with RPG increasing by 13.6%, 23.4%, 26.4%, and 9.6%, respectively, compared to untreated seeds.Figure 6
Influence of CAPP exposure on the relativized germination of (a) radish and (b) carrot.
(a)(b)MGT (mean germination time) is a measure of “the average time it takes for a seed to germinate or emerge,” and MGR (mean germination rate) is defined as “the reciprocal of the MGT” [37]. CAPP-exposed radish and carrot seeds germinated substantially faster than untreated seeds, with the exception of carrot seeds treated for 1 minute.Compared to untreated seeds, the MGT of radish seeds treated for 1 minute, 2 minutes, and 3 minutes was lowered by 18.6%, 29.1%, and 30.5%, respectively. Similarly, compared to the control, the MGT values of treated carrot seeds at 2, 3, and 4 minutes decreased by 0.8%, 1.2%, and 5.9%, respectively. Statistically significant difference was not identified among 2 minutes and 3 minutes treated radish and carrot seeds (Figure7).Figure 7
Influence of CAPP exposure on the mean germination time of (a) radish and (b) carrot.
(a)(b)The MGR of radish seeds treated for 1 minute, 2 minutes, and 3 minutes increased by 22.2%, 40.7%, and 40.7%, whereas that of carrot seeds treated for 3 minutes and 4 minutes increased by 2.5% and 6.5%, respectively (Figure8). There was no statistically significant difference in MGR values between untreated carrot seeds and those exposed to CAPP for 1 or 2 minutes. The data clearly demonstrate that radish and carrot seeds exposed to CAPP germinated more quickly than untreated seeds.Figure 8
Influence of CAPP exposure on the mean germination rate of (a) radish and (b) carrot.
(a)(b)In the case of carrot seeds, the germination index value rose by 15.01%, 24.98%, 31.11%, and 15.69%, respectively, compared to the control. However, no significant differences were seen in the cases of 1-minute and 4-minute treated seeds. Also, a noticeable variation in GI value was noted among the control and LTP-treated radish seeds (Figure9). A greater GI value suggests a higher proportion of sprouting [44].Figure 9
Effect of CAPP treatment on the GI of (a) radish and (b) carrot.
(a)(b)The coefficient of germination velocity (CVG) measures the rate at which seeds germinate. Its usefulness increases as the quantity of sprouting seeds grows and the time it takes for them to grow lowers [25, 45]. Figure 10 illustrates that when radish seeds were treated by CAPP for 1–3 minutes, CVG improved by 23.90%, 41.36%, and 43.70%, respectively, when compared with untreated seeds. A similar increase in CVG appears when carrot seeds are exposed to CAPP for 1–4 minutes, respectively.Figure 10
Influence of CAPP treatment on the CVG of (a) radish and (b) carrot.
(a)(b)U (uncertainty of the germination process) is defined as “the degree of uncertainty associated with the relative frequency of germination dispersion,” and Z (synchrony of the germination process) is defined as “the amount to which members of a certain demographic overlap” [34]. Radish seeds treated for 2 and 3 minutes had 6.3% and 13.9% lower germination uncertainty than untreated seeds, while seeds treated for 1 minute had 2.1% higher uncertainty values (Figure 11). For carrot seeds treated for 4 minutes, uncertainty in germination was 11.1% lower than for untreated seeds, but it rose by 8.0%, 16.4%, and 15.5% for seeds treated for 1, 2, and 3 minutes, respectively. However, synchronization of germination was dramatically reduced in both radish and carrot seeds subjected to CAPP (except for 4 minutes treated carrot seeds) (Figure 12). Carrot seeds treated for 4 minutes exhibited a 25.0% improvement in synchrony of germination.Figure 11
Effect of CAPP on uncertainty of germination process of (a) radish and (b) carrot.
(a)(b)Figure 12
Effect of CAPP on synchronization index of (a) radish and (b) carrot.
(a)(b)Time to 50% germination (T50) is the number of days required for 50% of the total number of sown seeds to sprout. It was discovered that the radish seed exposed to CAPP for 2 and 3 minutes and carrot seeds exposed to CAPP for 4 minutes took less time for 50% of the total sown seeds to sprout as compared to the untreated one (Figure 13).Figure 13
Time to 50% germination of (a) radish and (b) carrot seeds.
(a)(b)MDG (mean daily germination) is a measure of “the daily average of how many seeds germinate” [37]. Radish seeds exposed to CAPP for 1, 2, and 3 minutes did not change substantially. The MDG for treated radish seeds increased by 2.9% compared to untreated seeds. Similarly, the MDG values of carrot seeds treated for 1, 2, 3, and 4 minutes rose by 13.7%, 23.0%, 25.9%, and 9.3%, respectively, compared to the control group (Figure 14). The results indicate that the daily average germination of radish and carrot seeds exposed to CAPP is considerably greater than that of untreated seeds.Figure 14
Mean daily germination of (a) radish and (b) carrot seeds.
(a)(b)For 1, 2, and 3 minutes, CAPP treated radish seed germination value rose by 22.71%, 40.23% and 45.23% compared to the untreated seeds. Moreover, CAPP-treated radish seedlings showed significant differences. Similarly, the germination values improved by 26.17%, 35.93%, 45.01%, and 26.9% for 1, 2, 3, and 4 minutes of CAPP-treated carrot seeds, respectively, as indicated in Figure15.Figure 15
Effect of plasma on germination value of (a) radish and (b) carrot.
(a)(b)The several RONS, including NO, nitrates, and hydroxyl radicals (OH), created in the production of CAPP, as depicted in Figure3, get deposited in exposed seeds and function as a signaling molecule. This species interacts with plant hormones and is essential for the alleviation of seed dormancy in plant species. Furthermore, the CAPP disinfects seeds by inhibiting microorganisms that adhere to their surface and contaminate them. This results in a significant improvement in the germination of exposed seeds to CAPP [46, 47].


## 4.4. Estimation of Seedling Length

The cocopeat from the roots of the seedlings was carefully removed from the germination tray to assess their length. Figure16 depicts photographs of radish and carrot seedlings that sprouted from untreated and plasma-treated seeds. As seen from Figure 16, seedlings sprouted from plasma-treated seeds had a longer length than those from untreated seeds.Figure 16
Photograph of the seedlings of (a) radish and (b) carrot.
(a)(b)Researchers have shown that subjecting seeds to CAPP treatment improves water imbibition rate, lowers contact angle, and changes the seed surface by generating novel chemical interactions [5, 19, 48]. The physiology of germinating seeds is thought to be altered by plasma reactive species interactions with seeds. It may also enhance the seeds’ surface with an oxygen-containing functional group [49]. These groups may promote surface wettability, which benefits nutritional absorption, and seed physiology and sprouting [49, 50].


## 4.5. Calculation of Vigor

“Vigor is used to describe a seed’s potential degree of activity and performance during germination and seedling emergence” [5].The plasma treatment boosted the vigor index (Figure17). Compared to control, the Vigor index (I), of treated seeds of radish and carrot improved by 1.2–1.5 times. However, the carrot seed treated for 4 minutes had a 1.12 drop in vigor index (I) than untreated one. These gains were linked to sprouts length and greater GI, in line with other studies [51, 52]. The interaction of CAPP with vegetal cells may activate natural signals, hormones, and enzymes, explaining the observed variations in germination and fast sprout germination [53].Figure 17
Influence of CAPP exposure on the vigor index (I) of (a) radish and (b) carrot.
(a)(b)


## 4.6. Estimation of Mass Loss and Water Uptake Capacity of Seed due to Plasma Treatment

100 seeds were exposed to CAPP for a specified time to estimate mass loss and water uptake capacity. In this study, a weighing machine (MG124Ai, Bel Instrument) was used to measure the mass. The mass loss (%) and water uptake capacity of seeds due to plasma treatment were calculated using standard protocols as described by various researchers [5, 37].There was a substantial difference in mass loss (%) between seeds treated with CAPP and untreated seeds. Radish seeds exposed to CAPP for 1 minute showed a maximum mass loss of around 33.6% compared to untreated seeds. Compared to those exposed for 1 minute, the mass loss (%) of radish seeds exposed to CAPP for 2 and 3 minutes decreased gradually. In contrast, mass loss (%) in the case of carrot seeds increased steadily from 21.9% to 41.1% as exposure duration increased. No significant difference in mass loss (%) between carrot seeds exposed to CAPP for 2 and 3 minutes was noticed (Figure18).Figure 18
Estimation of mass loss in (a) radish and (b) carrot seeds due to CAPP exposure.
(a)(b)Radish seeds have a softer seed covering than carrot seeds. Due to the weaker seed coat, maximal etching may occur within 1 minute of contact with CAPP, resulting in a greater mass loss (%) for radish. The subsequent accumulation of reactive species seen in CAPP may result in a progressive reduction in mass loss (%) [37]. Due to the harder seed coat of carrots, however, etching by RONS occurs at a slower rate, resulting in a steady rise in mass loss as exposure duration increases.Similarly, the water uptake rate of control and CAPP-exposed seeds was determined by dipping them in a petri dish containing 50 ml of distilled water for a predetermined amount of time (Figure19). From Figure 19, it can be shown that CAPP-exposed radish and carrot seeds absorbed more water than untreated seeds.Figure 19
Evolution of Imbibition rate in (a) radish and (b) carrot seeds due to CAPP exposure.
(a)(b)CAPP-exposed radish seeds absorbed about 29.3% more water than untreated seeds when soaked for 6 hours in distilled water. Similarly, the water absorption capacity of carrot seeds exposed to CAPP for 3 minutes is 30.1% higher than the control when soaked for 3 hours in distilled water.Typically, seeds are hydrophobic by nature. When seeds are exposed to CAPP for a certain period of time, reactive species (RONS) present in the CAPP may be implicated in the surface etching of the seed coat, which enhances the roughness of the seeds and causes them to become more granular, as reported by several researchers using SEM micrographs [37, 54, 55]. When the reactive species interact with the seed coat, they may result in the etching of a waxy layer from the surface of the seed coat (testa). This increases the hydrophilicity of the seeds. According to our study, seeds exposed to plasma for a specific period absorbed more water, resulting in an increase in water uptake compared to untreated seeds. Increased water uptake stimulates the development of the hypocotyl and radicle, thereby accelerating the germination of seeds compared to the control when immersed in distilled water for a specific period of time.


## 4.7. Estimation of the Change in Flavonoid, Phenolics, Radical Scavenging, Chlorophyll, and Carotenoid Contents

The estimation of flavonoids, phenolics, scavenging, and chlorophyll contents were done using standard protocols as done by various researchers [56–58].Plants’ defense systems mainly depend on phenolic and flavonoid chemicals, which have several health benefits. Antioxidants provide electrons to injured cell, preventing, and stabilizing free radical damage. Radical scavenging is crucial in diseases prevention [57]. Plant extracts are often tested for their antioxidant activities using the DPPH technique. Our findings suggest that in case of radish, 2 minutes of plasma treatment increases the amounts of flavonoids and phenolic (Figures 20(a) and 21(a)). In the first 2 minutes of plasma treatment, plant extracts demonstrated better radical scavenging activity against both DPPH and ABTS free radicals (Figure 22). Similarly in the case of carrot, CAPP exposure period enhanced the phenolics and flavonoids for up to 3 minutes (Figures 20(b) and 21(b)). This may aid in the improvement of defensive reactions in difficult situations [59]. Similarly, 3 minutes of plasma treatment increased DPPH scavenging while 4 minutes of exposure lowered it (Figure 23). This may be because after 3 minutes of treatment, excessively energetic reactive species formed in DBD might have been deposited on the surface of the seeds. This active species affects plant structure because prolonged exposure causes surface degradation and causes seeds to lose all of their nutritional content. These changes reveal the benefits of exposing seeds to CAPP at a suitable treatment time for significantly enhancing germination and growth related measures [60, 61].Figure 20
Estimation of flavonoid contents in untreated and plasma treated seeds of (a) radish and (b) carrot.
(a)(b)Figure 21
Estimation of phenolics content in untreated and plasma treated seeds of (a) radish and (b) carrot.
(a)(b)Figure 22
Estimation of ABTS/DPPH scavenging in radish seedlings.Figure 23
Estimation of DPPH/ABTS in carrot seedlings.Plant pigmentation is essential for photosynthetic activity, converting solar energy to chemical energy. Chlorophyll a, b, and carotenoids are essential pigments in plant physiological activity [62]. Also, chlorophyll pigments in plants are thought to be stress and habitat indicators. Generally, chlorophyll gives green pigmentation while carotenoids offer red, yellow, and orange pigmentation [4]. Plant pigments may be used to examine plant physiology and health. In the case of radish, the leaf extract germinated from 2 minute-treated seeds had the maximum chlorophyll-a concentration. Changes in chlorophyll-a concentration in untreated and treated seedling extracts show that the plasma treatment helps seedlings increase their overall biochemical and physiological activity [5, 63]. The CAPP treatment, on the other hand, had no noticeable effects on chlorophyll-b and carotenoid levels (Figure 24(a)). Similarly, in the case of carrot, our findings demonstrated a progressive rise in chlorophyll concentration with CAPP exposure duration up to 3 minutes, then a drop at 4 minutes. Similarly, total carotenoid levels fell as plasma treatment duration increase (Figure 24(b)).Figure 24
Estimation of chlorophyll and carotenoids contents in the leaves of seedlings germinated from control and plasma treated seeds of (a) radish and (b) carrot.
(a)(b)


## 5. Conclusion

In this study, the evaluation of several germination and growth-related parameters revealed that CAPP treatment promotes germination and growth without significantly harming the seeds. As per our investigation, seed exposed to CAPP for suitable time greatly improved germination traits and vigor whereas prolonged treatment resulted in the decrease in germination percentages. Reactive species may weaken the seed surface after plasma treatment, making it more sensitive to water absorption and promoting rapid sprouting. Plasma treatment dramatically increased chlorophyll levels, according to spectrophotometric measurements. However, there was no noticeable variation in carotenoid content. According to our results, seeds treated with plasma for 3 minutes have more flavonoids and phenolics. Extraction from plants demonstrated a superior ability to neutralize radicals for up to 2 minutes in radish and 3 minutes in carrot, but this capacity declined drastically after 4 minutes in the case of carrot. Using this plasma technology for seed treatment before sowing may minimize pesticide consumption throughout the crop cycle, boost agronomic yield, and reduce damage to the environment.



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*Source: 4131657-2023-03-04.xml*