Treatment of Pea Seeds with Plasma Activated Water to Enhance Germination, Plant Growth, and Plant Composition

Abstract

The present study has been carried to investigate the interaction and effect of plasma activated water (PAW) on pea seeds. PAW is produced with the interaction of air plasma with water that forms reactive oxygen–nitrogen species in it. Our results with surface morphological study shows that PAW treatment removes the wax from the surface of peas and modifies their hydrophobic surface to hydrophilic. Wettability study shows decrease in water contact angle with seed surface after PAW treatment. Also, PAW treatment improves germination rate, viability index and mean germination time compared to control. Further, the study reveals that the grown plants have higher roots and shoots length, fresh and dry weight, increased chlorophyll ‘a’ and higher sugar and protein concentration compared to control. Although electrolytic and phenolic leakage from pea leaves did not show any significant difference in PAW and control-treated seeds, results obtained from antioxidant analysis clearly show an increased antioxidant enzymatic (SOD (EC no. 1.15.1.1), CAT (EC no. 1.11.1.6), APX (EC no. 1.11.1.11), and POD (EC no. 1.11.1)) activity mainly in the roots in seedlings grown from seeds treated with PAW. However, no significant difference in H2O2 concentration in the pea plant was observed. Hence, our study indicates potential role of seed pre-treatment with PAW to improve germination and plant growth.

Full text

Vol.:(0123456789)
Plasma Chemistry and Plasma Processing
https://doi.org/10.1007/s11090-021-10211-5
1 3
ORIGINAL PAPER
Treatment ofPea Seeds withPlasma Activated Water
toEnhance Germination, Plant Growth, andPlant
Composition
VikasRathore1,3· BudhiSagarTiwari2· SudhirKumarNema1,3
Received: 6 August 2021 / Accepted: 12 October 2021
© The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature 2021
Abstract
The present study has been carried to investigate the interaction and effect of plasma acti-
vated water (PAW) on pea seeds. PAW is produced with the interaction of air plasma with
water that forms reactive oxygen–nitrogen species in it. Our results with surface morpho-
logical study shows that PAW treatment removes the wax from the surface of peas and
modifies their hydrophobic surface to hydrophilic. Wettability study shows decrease in
water contact angle with seed surface after PAW treatment. Also, PAW treatment improves
germination rate, viability index and mean germination time compared to control. Further,
the study reveals that the grown plants have higher roots and shoots length, fresh and dry
weight, increased chlorophyll ‘a’ and higher sugar and protein concentration compared
to control. Although electrolytic and phenolic leakage from pea leaves did not show any
significant difference in PAW and control-treated seeds, results obtained from antioxidant
analysis clearly show an increased antioxidant enzymatic (SOD (EC no. 1.15.1.1), CAT
(EC no. 1.11.1.6), APX (EC no. 1.11.1.11), and POD (EC no. 1.11.1)) activity mainly in
the roots in seedlings grown from seeds treated with PAW. However, no significant dif-
ference in H2O2 concentration in the pea plant was observed. Hence, our study indicates
potential role of seed pre-treatment with PAW to improve germination and plant growth.
Keywords Plasma activated water· Reactive oxygen–nitrogen species· Seeds germination
and plant growth· Surface morphology and wettability study· Agronomy traits and
antioxidant enzymes
* Vikas Rathore
vikas.rathore@ipr.res.in
* Budhi Sagar Tiwari
bstiwari@iar.ac.in
1 Institute forPlasma Research (IPR), Gandhinagar, Gujarat382428, India
2 Institute ofAdvanced Research (IAR), Gandhinagar, Gujarat382426, India
3 Homi Bhabha National Institute, Mumbai, Maharashtra400094, India

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Introduction
Continuously increasing population and reduction in crop-land due to rapid urbaniza-
tion all over the world has created a threat to meet global food demand. In addition, the
industrialization is sharply changing our climate and making the situation much worse.
Hence, to meet the global food demand, various techniques are being used to increase
the crop yields, for example using various fertilizers (potassium, nitrogen, phosphorus,
etc.), acid treatment (e.g., H2SO4), pesticides, hot water treatment, heat treatment, and
bacterial and fungal disinfection, etc. [1–5]. However, these methods possess several
problems such as negative impact on crops health, causing post-treatment pollution,
adding toxicity to crops that lead to various health issues in animals and humans and
economically unviable, etc. [5–7]
Hence, in search of an eco-friendly and economically viable solution to enhance
seedling and plant growth, our research led us to explore plasma activated water
(PAW). PAW can be defined as plasma modified water which carries various reactive
oxygen–nitrogen species (RONS) in it due to plasma-water interaction [8–10]. When
cold plasma that contains various high energy radicals, species, free electron, and radia-
tions (e.g. UV) comes in contact with water (direct or indirect) results in the production
of various short-lived species (O, ∙OH, ONOOH, and NO, etc.) and long-lived species
(NO3‾, NO2‾, H2O2, and dissolved O3, etc.) in water. The presence of these reactive
radicals and species in PAW makes it a promising product that can be used in numerous
applications such as disinfection agent for killing various pathogens (bacteria, fungi,
virus, and pest, etc.), selective killing of cancer cells, food preservation, seeds germina-
tion and plant growth, etc. [8, 11–16]
Plasma activated water (PAW) or plasma activated tap water (PATW) technology is
still new in the agriculture field [8, 9, 17–25]. Past reported work of various researchers
has shown the effect of PAW on seeds germination and plant growth [8, 9, 21, 22, 25].
Sajib etal. [8] describes significant improvement in agronomic traits of a black gram when
treated with PAW produced using O2 plasma. They have reported that PAW treatment of
seeds enhances the catalase (CAT) activity of the roots of the grown plant. It also helps in
the upregulation of CAT activity that result in increased germination, growth and devel-
opment of black gram. The role of plasma and PAW (air) on seeds germination rate and
plant growth on three different seeds (radish, tomato, and sweet pepper) were explored by
Sivachandiran etal. [9]. They reported the positive effect of plasma and PAW on seed ger-
mination and seedling growth. However, they also emphasized on that longer exposure of
seeds and water with plasma, resulted in a negative effect on seeds germination and plant
growth. This is due to excessive exposure of seeds to plasma and highly reactive (higher
ORP i.e. higher oxidizing tendency) PAW that may damage the seeds and plants. Porto
etal. [21] showed a combination of ultrasound and PAW treatment with soybean seeds
enhances its rate of germination and plant growth. The role of plasma-water exposure time
on mung bean sprouts was discussed by Fan etal. [22]. They showed PAW prepared by
15s of plasma exposure showed higher germination rate and plant growth compared to
30 s, 60 s, and 90 s respectively. Hence, they also recommended optimization of PAW
properties to prevent damage to seeds and plants.
The role of different gases (O2 and Ar), used for PAW production in rapeseed seeds
germination and plant growth has been explored by Islam etal. [17]. They have shown
that PAW (O2) treatment induces faster germination. In addition, PAW (O2) and PAW
(Ar) showed notable improvement in roots and shoots characteristics.

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The use of tap water instead of distilled water, ultrapure milli-Q water, or demineralized
water were also reported previously. However, commenting on the chemistry of plasma
interaction of tap water is quite difficult due to different concentrations of salts and organic
impurities present in different origins of tap water. Kostoláni etal. [20], Kučerová etal.
[25], Judée etal. [18] and Gao etal. [19] used plasma activated tap water (PATW) to study
its effect on pea seeds, barley seeds, wheat seeds, and lentils seeds. Kostoláni etal. [20]
reported PATW treatment with pea seedlings to show stimulation of amylase activity with-
out hindering germination. The effect of PAW on wheat seeds growth characteristics was
shown by Kučerová etal. [25]. They reported PAW improves the rate of germination, ear-
lier seedling, soluble protein, and photosynthetic pigments. In addition, suppressed the
antioxidant activity of enzymes. Judée etal. [18] was observed a significant increase in
seedling length of lentil seeds after irrigating with PATW. Also, PATW irrigation improves
the growth parameters (germination rate, seedling height, dry weight, and chlorophyll
content) in pea seeds by more than 50% [19]. The studies made so for and summarized
above were restricted to evaluate effect of PAW or PATW on visible outcomes of seed
germination process, however, change in other seed properties like wettability, morphology
changes, electrolytic and phenolic leakage, oxidant and antioxidant in collective manner
were remained unexplored.
The present work uses atmospheric pressure pencil plasma jet (PPJ) to produce PAW.
The generated PAW was exposed to white dried pea (Pisum sativum L.) seeds to study
the various phenotypic, growth parameters, nutritional (sugar and protein), and enzymatic
changes, etc. that occur in it. White dried pea seeds were chosen for this study due to their
nutritional aspect. Peas are a rich source of various nutrients such as protein, carbohy-
drates, minerals, and vitamins for people mainly in Asian countries[26].
The following novel aspects are presented in the present study that was not reported
before to the best of our knowledge:
• Role of PAW in changing surface morphology and wettability properties of pea seeds
• Change in sugar and protein concentration in pea plant leaves and roots after seeds
treatment with PAW
• Change in chlorophyll ‘a’, Chlorophyll ‘b’, and carotenoids ‘cc+x’ concentration of pea
leaves after PAW seeds treatment
• Change in H2O2 concentration in pea plant leaves and roots, and electrolytic and phe-
nolic leakage from pea leaves after PAW seeds treatment
• Change in antioxidant enzyme (SOD, CAT, APX, and POD) activity present in plant
leaves and roots after PAW seeds treatment
Materials andMethods
Experimental Setup ofPencil Plasma Jet andMeasurement ofOptical Emission
andElectrical Properties
The schematic of pencil plasma jet (PPJ) and picture of plasma activated water (PAW) pro-
duction is shown in Fig.1. A high voltage high-frequency power supply (Vmax = 10 kV,
fmax = 40 kHz) powered this PPJ, more details about PPJ setup and PAW production are
found in Rathore etal.[10]. The voltage across PPJ was measured using a 1000 × voltage
probe (Tektronix P6015A) and oscilloscope (Tektronix TDS 2014C). A 31-Ω (R) resistor

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or 1 nF capacitor (C) was connected in series with the ground (10 × voltage probe, Tek-
tronix TPP0201) electrode to measure the discharge current or transported charge during
plasma production. Lissajous curve between applied voltage and transported charge was
used to calculate the discharge power during PAW production.
An optical fiber and spectrometer (Model EPP2000-UV from StellarNet Inc.) was used
to measure the air emission spectrum. The optical fiber was placed just below the PPJ
(Fig.1).
PAW Production andPea Seeds PAW Exposure
To study the PAW effect on pea (Pisum sativum L.) seeds germination. A 50ml of ultrapure
milli-Q water (DM water or control) was taken in 600ml of a glass beaker. This water was
exposed to plasma with two different plasma-water exposure times namely 5min (PAW–5)
and 10min (PAW–10) (Fig. 2) (movie of production of PAW shown in Fig. S1 of sup-
plementary information). In the present study, higher plasma-water interaction time was
avoided as it had shown negative impact on seeds germination and plant growth (results
were not shown in present work)[9].
White dried pea seeds were purchased from the local market of Gandhinagar, Gujarat
(India). A total number of 600 seeds (3 groups’ × 4 replicate × 50 seeds) were divided into
three groups named control, PAW–5, and PAW–10. Each group was further subdivided
into 4 sets of 50 seeds. Each set (50 seeds) of the group soaked in 50ml of sample (con-
trol, PAW–5, and PAW–10) for 24h in the dark (24°C, 40% Relative humidity). After
that, seeds were transferred to a petri dish carrying a wet paper towel with corresponding
sample (control, PAW–5, and PAW–10) for another 24h in the dark (24°C, 40% Relative
humidity). Then seeds were transferred to pots carrying soil (germination day 0) purchased
from a local nursery (pH ~ 7) and irrigated with ultrapure milli-Q water (Demineralized
water or DM water) to avoid any role of nutrient in germination and plant growth (24°C,
40% Relative humidity, and 16–8h light–dark cycle (irradiance 44W m−2, Philips Fluores-
cent Tube Light Bulb)). Schematic shown in Fig.2.
The germination of seeds was monitored every day till day 5. To compare the
germination data among the groups, following parameters were used: Cumulative
Fig. 1 Schematic of the experimental setup for the electrical and optical emission characterization of pencil
plasma jet and picture of plasma activated water production

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germination rate, Final germination percentage, Mean germination time, Germination
index, Coefficient of velocity of germination, Germination rate index, First day of ger-
mination, Last day of germination, Time spread of germination. The expression used
to calculate the above parameters are taken from published literature[27]. To charac-
terize germination in terms of viability (Vi), mean germination time (Me), dispersion
(Qu), and skewness (Sk), Richards function was fitted in the germination data. These
population parameters of Richards function were taken from Hara[28].
Water Holding Capacity
The water holding capacity of pea seeds after control and PAW treatment can be calcu-
lated from the following expression:
Wf = Weight of seeds after sample (control and PAW) soaked for 24h. Wi = Initial weight
of seeds.
Water holding capacity
(%)=
W
f
−W
i
Wi
×
100
Fig. 2 Schematic of generation of plasma activated water (PAW–5 and PAW–10) and pea seeds treatment
using PAW and control

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Measurement ofPhysicochemical Properties ofPAW andRONS Present inPAW
Physicochemical properties of PAW such as pH, oxidation–reduction potential (ORP),
electrical conductivity (EC), and total dissolved solids (TDS) were measured using
pH meter (Hanna Instruments), ORP meter (Contech COR-01)), EC meter (Contech
CC-01), and TDS meter (HM digital) respectively.
A semi-quantitative O3 test strip (QUANTOFIX, MN filters) was used to quantify the
gases O3 during air discharge. Similarly, semi-quantitative test strips were used to deter-
mine Nitrite (NO2‾) ions (QUANTOFIX, MN filters) and hydrogen peroxide (H2O2)
(QUANTOFIX, MN filters) present in PAW. Nitrate (NO3‾) ions colorimetry test kit
(VISOCOLOR alpha, MN filters) was used to determine the approximate concentration
of NO3‾ ions in PAW. Dissolved O3 test kit were used to determine an approximate dis-
solved O3 in PAW.
The UV–visible spectroscopy (UV–visible spectrophotometer, SHIMADZU
UV-2600) was used to determine the concentration of RONS present in PAW. Stand-
ard curves of NO3‾ ions (NaNO3 salt, HPLC lab reagents), NO2‾ ions (NaNO2 salt,
HPLC lab reagents) and, H2O2 (HPLC lab reagents) were prepared to determine the
unknown concentration of these compounds in PAW. The molar extinction coefficient
of NO3‾ ions, NO2‾ ions, and H2O2 were given as 0.0602 (mg l−1)−1 cm−1, 0.0009 (µg
l−1)−1 cm−1, and 0.4857 (mM)−1 cm−1 respectively. Dissolved O3 in PAW was measured
using the indigo calorimetry method [10].
Surface Morphological andWettability Study ofPea Seeds After PAW Treatment
Pea seeds were soaked in control, PAW–5, and PAW–10 for 8h in the dark (24°C and 40%
RH). Then seeds were taken out and excess water (control and PAW) was soaked using a
paper towel. Before conducting surface morphological and wettability studies, seeds were
completely dried in an oven at 70°C for 72h. For morphological analysis, seeds were
placed on the sample holder using double-sided carbon tape. To make the seeds conduct-
ing for morphological analysis, seeds were gold coated using gold sputtering for 120s
(Quorum Q150R ES). A high-resolution scanning electron microscope (HR-SEM, Zeiss
MERLIN Series) was used to examine the morphology of seeds. Images were taken in
secondary electron mode with electron high tension voltage 5.0kV. In addition, Energy-
dispersive X-ray spectroscopy (EDS) was performed to analyze the change in surface ele-
mental weight percentage after PAW and control treatment of seeds.
For wettability analysis, above mentioned dried seeds from the oven were cooled to
room temperature (25°C). The seeds wettability analysis (sessile drop method) was per-
formed using a digital goniometer (OCA 15EC, Dataphysics) by measuring the contact
angle (CA) between water and seeds surface.
Growth Analysis ofPea Plants
The analysis of the length of roots and shoots of the pea plants, change in chlorophyll
‘a’, ‘b’ and carotenoids, dry and fresh weight of pea plants were determine the growth
of pea plants.
Two-week-old plants were harvested from the soil, and roots and shoots were washed
with running water. The excess washing water was absorbed using tissue paper. The

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length of the roots and shoots were measured using ImageJ software. Simultaneously,
the weight of the fresh roots and shoots were measured (weight balance of least count
0.1mg). The dry weight of roots and shoots was measured after roots and shoots was
dried in an oven at 70°C for 72h.
To measure the chlorophyll and carotenoids concentration of fresh leaves, liquid nitro-
gen (LN2) was added to 100mg of leaves to make a dry powder using a mortar pestle.
This leaves power grounded in 2ml of freshly prepared chilled 80% acetone (HPLC lab
reagents) solution with the help of an ice-cooled mortar pestle. The homogenized mixture
was transferred to a 2ml centrifuge tube and the mixture was centrifuged at 12,000rpm
for 10min. The supernatant was collected to measure the chlorophyll and carotenoids con-
centration using a UV visible spectrophotometer (SHIMADZU UV-2600). The expression
used for measurement of chlorophyll and total carotenoids concentration [29] is given as:
where Ca, Cb, and Cx+c represent the Chlorophyll ‘a’, Chlorophyll ‘b’, and total carotenoids
respectively.
Estimation ofSugar andProtein
For total soluble sugar estimation in plant leaves and roots, 100 mg of fresh leaves or
100mg of the fresh roots were added in 2ml of warm aqueous 80% (v/v) C2H5OH (Sigma-
Aldrich). This solution with leaves or roots were heated in a boiling water bath for 10min
so the extraction of sugar from leaves or roots could take place. The mixture was centrifuge
at 12,000rpm for 5min, and the supernatant was collected. After that, a 0.2% freshly pre-
pared ice-cold anthrone reagent (HPLC lab reagents) was added in collected supernatant.
Then the solution was incubated in a boiling water bath for 10min and the solution was
placed on ice after incubation. The absorbance of the ice-cooled solution was measured at
590nm[30]. The quantification of extracted sugar was performed using a standard sugar
curve with a molar extinction coefficient 0.0064 (mg l−1)−1 cm−1. The different concentra-
tions of dextrose (HPLC lab reagents) were used to make the standard curve of sugar.
Lowry etal.[31] method was used to determine the protein concentration in plants leaves
and roots. 100mg of leaves or roots were converted to dry power using LN2 and mortar pes-
tle. Protein extraction buffer (lysis buffer) was prepared using 10% trichloroacetic acid (TCA)
(HPLC lab reagents) and 0.07% β-Mercaptoethanol (β-ME) (HPLC lab reagents) in acetone.
Leaves or roots powder were grounded in a 2ml of extraction buffer using an ice-cooled mor-
tar pestle. The mixture was centrifuged (REMI centrifuge) at 12,000rpm for 10min and the
supernatant was collected. A 0.1ml of 2 N sodium hydroxide (NaOH) (HPLC lab reagents)
was added in 0.1ml of supernatant. The solution was hydrolysed in a boiling water bath for
10min. Then the sample was cooled to room temperature (25°C), and a 1ml of freshly pre-
pared complex reagent (2% Na2CO3 (HPLC lab reagents), 1% CuSO4.5H2O (HPLC lab rea-
gents), 2% KNaC4H4O6.4H2O (HPLC lab reagents) in the following composition; 100:1:1)
C
a
(
𝜇
g
ml )
=12.25A663 −2.79A
647
C
b
(𝜇g
ml )
=21.5A647 −5.1A
663
C
x+c
𝜇g
ml 
=

1000A470 −1.82Ca−85.02Cb

∕
198

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was added. The solution was incubated for 10min at room temperature, and a 0.1ml of folin
reagent (HPLC lab reagents) was added, and left for 30 to 60min. Finally, the absorbance
of the solution was measured at 750nm. The different concentrations of bovine serum albu-
min (HPLC lab reagents) were used to make a standard curve of protein solution. The molar
extinction coefficient of the standard curve was 0.7 (µg l−1)−1 cm−1.
Estimation of H2O2, Electrolytic, andPhenolic Leakage
For estimation of H2O2, dry powder of leaves or roots was prepared as discussed previ-
ously. A 2ml of 0.1% TCA was added in this dry powder for extraction of H2O2. The
extract was centrifuged at 12,000rpm for 10min and the supernatant was collected. A
0.25 ml of this supernatant was taken, and a 0.25 ml of 100 mM potassium phosphate
buffer (HPLC lab reagents) and 1ml of 1 M potassium iodide reagent (HPLC lab rea-
gents) were added. Then the solution was placed in the dark for 1h, and absorbance was
measured at 390nm[32]. The different concentrations of 30% H2O2 solution (HPLC lab
reagents) was used to prepare the standard curve of H2O2. The molar extinction coefficient
of the standard curve was 0.9µM−1 cm−1.
100mg of plant leaves were cut in half to measure the electrolytic and phenolic leak-
age[33]. The cut leaves were washed with DM water to remove any impurities on them.
The cut leaves were placed in 50ml of DM water and incubated in a rotary shaker for 24h.
The EC meter (Contech CC-01) and UV visible spectrophotometer was used to measure
the electrolytic leakage and phenolic leakage (absorbance at 260nm).
Enzyme Activity
As disccused above, dry powder of leaves and roots were prepared to study the enzyme
activity of superoxide dismutase (SOD, EC no. 1.15.1.1), catalase (CAT, EC no. 1.11.1.6),
peroxidase (POD, EC no. 1.11.1), and Ascorbate peroxidase (APX, EC no. 1.11.1.11) in
plant leaves and roots.
A 2ml of an ice-cooled extraction buffer (100 ml buffer contains 12.5 ml of 0.8 M
phosphate buffer (HPLC lab reagents), 250µl of 0.2 N EDTA solution (HPLC lab rea-
gents), 0.0176 gms of ascorbic acid (HPLC lab reagents), and 87.5ml of DM water) was
added in powder, and grounded with ice-cooled mortar pestle. The homogenate was centri-
fuged at 12,000rpm for 20min, and collected the supernatant (enzyme extract) for enzyme
activity[34]. The SOD test kit (19,160 SOD Determination Kit, Sigma-Aldrich) and CAT
test kit (Catalase Assay Kit, Item No. 707002, Cayman Chemical) were used to determine
the SOD and CAT activity by following the procedure given in the user manual.
To determine the POD activity, 3ml of buffer solution, 0.05 ml of guaiacol solution
(HPLC lab reagents), and 0.03ml of H2O2 (HPLC lab reagents) were added in 0.1ml of
enzyme extract. The reaction start as soon as the enzyme was added. The absorbance was
measured at a difference of 30s up to 3min at 436nm. The time required to increase the
absorbance by 0.1 (Δt)[35] was noted. The following expression was used to determine the
POD activity:
To determine the APX activity, 1ml of the reaction mixture was added in 0.1 ml of
enzyme extract. The reaction start as soon as enzyme extract came in contact with the
SOD activity
(Ul−1)=
500
Δt

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reaction mixture. Hence, the decrease in absorbance was measured from 10 to 30s at
290nm (molar extinction coefficient 2.8 mM−1 cm−1)[36]. A 50mM of potassium phos-
phate (pH 7.0) (HPLC lab reagents), 0.5 mM ascorbate (HPLC lab reagents), 0.1 mM
H2O2, and 0.1mM EDTA respectively was used to prepare the reaction mixture.
Data Analysis
All the experiments were performed at least three times (n ≥ 3) and the results were
expressed as mean ± standard deviation (µ ± σ). The one-way ANOVA followed by Fish-
er’s Least significant difference (LSD) test using the MS Excel 2016 software package was
used to determine the significant difference (p < 0.05) among the groups.
Results
Optical Emission andElectrical Properties ofPPJ
To characterize the generated plasma in PPJ during air discharge, the current–voltage
waveform of PPJ was studied as shown in Fig.3a. The current waveform showed the air
plasma was the combination of several filamentary micro-discharges. The charge–volt-
age Lissajous figure was used to calculate the power consumption during PAW production
(Fig.3b). The calculated discharge power was 39W.
The identification the various reactive species and radicals present in air plasma were
performed by recording emission spectra of air plasma as shown in Fig.3c. It showed
a high intensity second positive N2 vibrational bands (C 3Πu → B 3Πg) along with weak
intensity N2+ (B 2Σu+ → X 2Σg+) first negative vibrational band[37]. It was due to elec-
tronic transition of the second positive and first negative system of from high energy states
to low energy states (ν′ → ν″).
RONS andPhysicochemical Properties ofPAW
These high-energy electronic transition in vibrational bands in air plasma when interact-
ing with water surface resulted in the formation of various RONS in it. Detail mechanism
of the formation of RONS in PAW shown in our previous reported work[10, 11]. These
excited N2 molecules reacted with O2 in the air to form NOx. Generated NOx dissolved in
water to form stable reactive nitrogen species (RNS) such as NO2¯ and NO3¯ ions (nitrous
and nitric acid) as shown in Table1, due to which pH of PAW decreased as shown in
Table2. Observed pH of PAW–5 and PAW–10 was significantly (p < 0.05) lower than con-
trol. The increase in PAW electrical conductivity (EC) and total dissolved solids (TDS)
compared to control confirmed the presence of these inorganic ions (NO2¯ and NO3¯ ions).
Table2 showed significant (p < 0.05) increases in the EC and TDS of PAW–5 and PAW–10
compared to control that supported the results of Table1. In addition, increasing plasma-
water interaction time significantly increased (p < 0.05) the NO2¯ and NO3¯ ions concen-
tration in PAW (RNSPAW-10 > RNSPAW- 5) which also supported by the results of EC, TDS,
and pH (Table2) (semi-quantitative determination of RONS in PAW shown supplementary
information Fig. S2).
Beside RNS, plasma-water interaction also formed reactive oxygen species (ROS) in
PAW such as H2O2, and dissolved O3, etc. The measured concentration of gaseous O3 in

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air plasma was greater than 210µg m−3 (Fig. S2). This gaseous O3 dissolved in water and
increased the oxidizing tendency of PAW. We observed a significant (p < 0.05) increase in
the dissolved O3 concentration of PAW compared to the control (Table1). Plasma interac-
tion with air moisture or water dissociated the water molecule to give hydroxyl radicals.
These hydroxyl radicals reacted with each other to form H2O2 in PAW. Table1 showed the
H2O2 concentration in PAW. The next combination of all stable oxidising species (NO2‾,
Fig. 3 a Voltage-current characteristics, b Charge–voltage (Lissajous curve) characteristics, and c Optical
emission spectrum of air plasma of pencil plasma jet
Table 1 Reactive oxygen–nitrogen species present in PAW
Different lowercase letters showed a statistically significant (p < 0.05, n = 3) difference among column
Sample NO2‾ (mg l−1)NO3‾ (mg l−1) H2O2 (mg l−1)Dissolved O3 (mg l−1)
Control 0.0c ± 0.0 0.0c ± 0.0 0.0c ± 0.0 0.0c ± 0.0
PAW–5 5.1b ± 0.4 34b ± 2.6 0.5b ± 0.1 2.8a ± 0.1
PAW–10 10.5a ± 1.1 53.9a ± 3.6 1.5a ± 0.2 2.1b ± 0.3

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NO3‾, dissolved O3, and H2O2) in PAW measured as oxidation–reduction potential (ORP)
(Table2). Increasing plasma-water interaction times increased the ORP value that showed
more formation of RONS in PAW (Table1).
Surface Morphology andWettability ofPea Seeds After PAW Treatment
PAW treatment with seeds changed the surface morphology of seeds as shown in Fig.4a–c.
PAW treatment remove the hydrophobic wax structure on the surface of seeds. Figure4a
showed the naturally occurring wax on the surface of the seeds. The presence of this wax
on the seeds surface made them hydrophobic due to which time taken by seeds to soaked
water gets delayed. However, PAW treatment remove this wax structure and made the
PAW-treated seeds more hydrophilic (Fig.4b,c). The presence of RONS such as H2O2,
dissolved O3, NO3¯ ions, and NO2¯ ions oxidized this wax structure and hence helped in
the removal of wax from the seeds surface. Sajib etal. [8] also reported that PAW treat-
ment helped in removal of wax from seeds surface. In addition, EDX analysis showed that
the carbon weight % (wax content) of seeds surface decreased (Table3). However, this
decrease in carbon weight % was not significant (p > 0.05) (plot of EDX analysis shown in
Fig. S3 of supplementary information).
Table 2 Physicochemical
properties of PAW
Different lowercase letters showed a statistically significant (p < 0.05,
n = 3) difference among column
Sample ORP (mV) pH EC (µS cm−1) TDS (ppm)
Control 251.7c ± 12.6 6.8a ± 0.1 1.7c ± 0.6 0.0c ± 0.0
PAW–5 423.3b ± 11.7 6.5b ± 0.1 99.0b ± 13.5 36.3b ± 2.1
PAW–10 483.3a ± 12.6 6.0c ± 0.2 177.3a ± 11.0 68.3a ± 5.5
Fig. 4 Morphological (HR-SEM images) changes in pea seeds surface after PAW and control treatment. a
Control, b PAW–5, c PAW–10, and d Change in wettability properties of pea seeds after PAW and control
treatment. DM water (control), PAW–5, and PAW–10

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This hydrophilic changes in seeds due to PAW treatment further confirmed by the wet-
tability study. Figure4d shows the decrease in the contact angle between seed surface and
water droplet after PAW treatment (movie of PAW wettability on seeds surface shown in
Fig. S4 of supplementary information). The observed contact angle in control treatment
was > 90° (hydrophobic) and in PAW treatment < 90° (hydrophilic). In addition, increase in
plasma-water interaction from 5 to 10min, significantly reducing the contact angle signi-
fies seeds surface more hydrophilic.
Seed Germination andWater Holding Capacity ofPea Seeds
The water holding capacity of seeds when exposed to PAW–5, PAW–10, and control
shown in Fig.5a. Obtained results showed no significant difference (p > 0.05) (5% and 3%
increase in water holding capacity of PAW–5 and PAW–10 compared to control) among
the groups. This was due to the saturation of water holding capacities of seeds.
Although, PAW treatment of seeds improved the germination rate compared to control
(Fig.5b). Figure5b showed a significant (p < 0.05) increase in % cumulative germination
at days 2 and 3 in PAW compared to control (germination test results shown in TableS1
of supplementary information). At day 2, the increase in cumulative germination was 37%
Table 3 Change in elemental
weight (EDX analysis) of pea
seeds surface after PAW and
control treatment
Different lowercase letters showed a statistically significant (p < 0.05,
n = 3) difference among column
Element Normalized Weight (%)
Control PAW–5 PAW–10
Carbon (C) 53.676a ± 4.97 48.005a ± 2.53 49.597a ± 2.3
Nitrogen (N) 0.834a ± 0.31 0.522a ± 0.12 0.348b ± 0.09
Oxygen (O) 43.184a ± 4.15 47.210a ± 2.52 44.032a ± 2.08
Potassium (K) 1.061b ± 0.05 2.229a ± 0.06 2.17a ± 0.06
Calcium (Ca) 1.244c ± 0.06 2.034b ± 0.06 3.854a ± 0.08
Fig. 5 a Water holding capacity and b cumulative germination of pea seeds after PAW and control treat-
ment. Different lowercase letters showed statistically significant (p < 0.05, n = 3) difference among groups

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and 26% in PAW–5 and PAW–10 compared to control respectively. Sajib et al.[8] also
reported a positive effect of PAW on black gram seed germinations.
The observed viability of seeds after PAW treatment were comparatively high com-
pared to control as shown in Table4. Also, the mean germination time of PAW treated
seeds found lower than control (Richard fitted plot is shown in Fig. S5 of supplementary
information).
Length andWeight ofPea Plant
The observed change in average length and weight of the pea plants grown after seeds
treatment with PAW and control shown in Fig. 6a,b. Figure 6a showed a significant
increase in the average length of roots and shoots compared to control. We observed 26%
and 38% increase in roots length, and 57% and 95% increase in shoots length in PAW–5
and PAW–10 grown pea plants compared to control. In addition, the average length of the
shoots in PAW–10 was greater than PAW–5.
Also, the observed fresh and dry roots and shoots weight in PAW was significantly
higher (p < 0.05) compared to control as shown in Fig. 6b. The observed percentage
increase in fresh and dry roots and shoots weight in PAW–5 and PAW–10 grown pea plants
compared to control was given as 62% and 91% (fresh roots), 75% and 125% (fresh shoots),
141% and 224% (dry roots), and 128% and 216% (dry shoots) respectively. These increased
length, fresh and dry weight of the plants signified higher growth of the plants in PAW
treated seeds compared to control. Previously reported work of Islam etal.[17] and Sajib
etal. [8] showed the positive effect of PAW on grown plant length and weight. Islam etal.
[17] showed PAW generated using argon (Ar) plasma had the higher length of roots and
shoots, and dry weight of grown mustard plant compared to control. In addition, Sajib
etal. [8] used oxygen (O2) plasma to prepare PAW and reported a higher yield of the roots,
shoots, and dry weight in the black gram plants compared to control.
Chlorophyll, Protein, andSugar Present inthePea Plants
We observed a significantly higher concentration of chlorophyll ‘a’ (31% in PAW–5 and
28% in PAW–10) in the leaves grown after PAW treated seeds compared to control as
shown in Fig.6c. Beside, no change in the ratio of chlorophyll ‘a’ to ‘b’ (increase in Ca/
Cb ratio was 5% in PAW–5 and 3% in PAW–10 compared to control), also greenness (ratio
of (Ca + Cb)/Cc+x) of leaves did not show any change in PAW and control grown pea plants
leaves. Islam etal. [17] and Sajib etal.[8] also reported an increase in chlorophyll content
in leaves after PAW treatment compared to control.
The observed variation in protein concentration in pea plant leaves and roots after seeds
were treated with PAW and control is shown in Fig.6d. No significant (p > 0.05) difference
Table 4 Richards population
parameters for germination of
pea seeds after PAW and control
treatment
Seeds Treatment Population parameters
Vi (%) Me (Day) Qu (Day) Sk (%) Err. (%)
Control 84.65 2.16 0.58 46.61 3.10
PAW–5 92.79 1.92 0.35 46.61 0.43
PAW–10 88.44 1.95 0.45 46.61 2.88

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was observed in the protein concentration of plants leaves. It was only 5% and 11% higher
in leaves grown using PAW–5 and PAW–10 compared to control. However, protein con-
centration in the roots was significantly higher (100% in PAW–5 and 152% in PAW–10)
for seeds treated with PAW compared to control. Inverse behaviour of protein observed in
Sajib etal.[8] reported work on the black gram. They showed an increased in protein con-
centration in leaves with increased plasma-water interaction time up to 6min, then leaves
protein concentration decreased, becoming comparable to control. However, they reported
Fig. 6 Change in agronomic traits of pea plant after pea seeds treatment with PAW and control, a Average
length, b Average weight, c Change in pea plant leaves chlorophyll and carotenoids concentration, d Protein
and e Sugar concentration. Different lowercase letters showed statistically significant (p < 0.05, n = 3) differ-
ence among groups

Plasma Chemistry and Plasma Processing
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no significant change in black gram roots protein concentration with increasing plasma-
water interaction time compared to control.
PAW treatment with seeds significantly (p < 0.05) increased the sugar concentration of
plants leaves and roots compared to control (Fig.6e). The sugar concentration of leaves
and roots were 56% and 94% (leaves), and 61% and 120% (roots) higher in PAW–5 and
PAW–10 grown plants compared to control. In addition, PAW–10 treated seeds grown
plants (leaves and roots) have significantly higher sugar concentration (24% and 36%) com-
pared to PAW–5. This increases in sugar concentration of pea plant leaves and roots after
PAW treatment of seeds supported by Islam etal. [17]. Islam etal.[17] showed an increase
in sugar concentration of roots and shoots of mustard grown after PAW (Ar) treatment with
mustard seeds compared to control.
Electrolytic andPhenolic Leakage, H2O2 inPlant
The amount of H2O2 present in plant leaves did not show a significant difference (p > 0.05)
in PAW and control-treated seeds. However, plant roots that had grown using PAW–10
treated seeds had significantly lower H2O2 than PAW–5 (53%) and control-treated seeds
(48%) (Fig.7a). Islam etal. [17] also showed no change in H2O2 concentration grown mus-
tard after PAW (O2 or Ar) treatment of seeds.
Also, the leaves grown using PAW and control-treated seeds did not show a significant
difference in electrolytic and phenolic leakage as shown in Fig.7b. The observed electro-
lytic and phenolic leakage in leaves were less than 11%.
Enzyme Activity
PAW treated seed-grown leaves and roots had higher enzyme activity (SOD, POD, APX,
and CAT) compared to control (Fig.7c–f). In addition, enzymatic SOD activity in roots
significantly (p < 0.05) increased with the increase in plasma-water interaction time with
seeds (Fig.7c). The POD, APX, and CAT enzymatic activity of root in PAW–5 signifi-
cantly higher compared to PAW–10 (Fig.7d-f). Increases plasma-water interaction time
with seeds significantly (p < 0.05) increased the POD activity of leaves. However, APX and
CAT activity in leaves of pea plant did not show any significant difference (p > 0.05) with
increasing plasma-water interaction time.
The present reported enzymatic activity of different antioxidant enzymes differed over
previously reported work of PAW effect of difference seeds treatment Sajib etal.[8] and
Islam etal. [17]. Sajib etal. [8] reported no significant change in enzymatic activity of
SOD, APX, CAT in leaves and roots of black gram plant with one exception (higher roots
CAT activity in 6 and 9min plasma-water treatment time compared to control, 12min,
and 15min). Similarly, Islam etal.[17] reported no significant change in antioxidant enzy-
matic activity SOD, APX, CAT in leaves of mustard. However, they reported a significant
increase in APX and CAT activity in the roots of mustard after PAW (Ar) treatment.
Discussion
In the present work we have studied the effect of PAW treatment on white dry pea seeds.
The wax structure that naturally forms on seeds surfaces made the seeds surface hydropho-
bic. Hence, it either reduces the water absorption by the seeds or delays water absorption

Plasma Chemistry and Plasma Processing
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by the seeds. Due to which the germination rate of seeds becomes considerably slow. The
present study shows the removal/degradation of this waxy structure after the treatment of
seeds using PAW evident from the observations made through scanning electron micros-
copy and EDX analysis (Fig.4a–c and Table3) that clearly demonstrate morphological
changes and wax degradation in the seed surface.
Wettability study of pea seeds after the treatment with PAW and control seeds has fur-
ther confirmed the results of morphological study. This may be due to change in nature of
seed surface i.e. becoming hydrophilic after PAW treatment compared to control pea seeds
that remained to be hydrophobic. However, no change in the water holding capacity of
seeds observed in PAW treated seeds and control seeds.
PAW treatment with pea seeds improved the germination and agronomic traits such
as roots and shoots length, fresh and dry weight compared to control. These observa-
tions indicate a possibility of involvement of reactive oxygen species (ROS) such as
H2O2, dissolved O3, etc. that damage the hydrophobic wax on seeds surface, and allowe
rapid absorption of water. In addition, PAW is a rich source of nitrogen, present in the
form of reactive nitrogen species (RNS) contained NO2‾, and NO3‾ ions, etc. (PAW
Fig. 7 a Change in H2O2 concentration of pea plant after pea seeds treatment with PAW and control, and b
Electrolytic and phenolic leakage from pea leaves after pea seeds treatment with PAW and control. Change
in enzyme activity of pea plant after pea seeds treatment with PAW and control. c SOD enzyme, d POD
enzyme, e APX enzyme, and f CAT enzyme. Different lowercase letters showed statistically significant
(p < 0.05, n = 3) difference among groups

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seems to act as a nutrient under given circumstances like germination and initial devel-
opmental stages). Since, nitrogen is the building block of biomolecules such as amino
acids, etc.[38, 39]. The role of nitrogen species has been observed in the form of rapid
germination, increased length, and weight of pea plants in the present work.
It has been observed that the pea seeds treated with PAW shows increase in the solu-
ble sugar, and soluble protein (roots) in pea plant compared to control. A higher concen-
tration of sugar signifies that more energy is available that can support plant growth. In
addition to sugar, soluble protein also plays an important role in supporting better plant
growth and development. Soluble proteins contain several enzymes involved in cellu-
lar metabolic processes that eventaully enhance metabolism of plants in totality. Hence,
higher roots protein in pea plants in PAW treated pea seeds indicates better control over
the cellular metabolism of the plant [8]. The leaves grown after PAW treatment of seeds
have shown higher chlorophyll ‘a’ concentration compared to control. Chlorophyll ‘a’
acts as a molecule that sits in reaction center (PS I and PS II) of photosynthetic electron
transport machinary. Therefore higher amount of chlorophyll ‘a’ directly relates with
more light harvesting in PS I and PS II reaction center and higher oxygenic photosyn-
thesis [8, 40, 41].
Lower to moderate amount of hydrogen peroxide (H2O2) in plants acts as a signaling
molecule leading to adaptation against causing component. However, high H2O2 in plants
signifies high oxidative stress primarly menifested by increased superoxide (O2‾) level.
Leaves and roots grown after PAW and control treatment of seeds did not show increase in
the H2O2 concentration. This indicate that there is no significant change in ROS level in the
plant [8]. Also, PAW and control treatment of seeds did not show any significant difference
in electrolytic and phenolic leakage from pea leaves. It signifies membrane integrity in pea
leaves remains intact, also there was no lipid degradation [33]. However amplitude of ROS
accumulated within seed after PAW treatment may relay signal for better generation and
induced action of hormones like auxin and cytokinin that results to better growth of plant
after PAW treatment [42].
The oxidative stress created by intracellular reactive oxygen species (ROS) in the plant
causes the threat to living plant cells. In response to that, plants have an enzymatic defense
mechanism. These enzymes are antioxidants in nature, that regulate the level of these
ROS in plants [43, 44] since ROS also act as signaling molecules that help regulate plant
growth[45].
SOD enzyme acts as the first line of defense against oxidative stress. It converts O2‾
radical to O2 and H2O2 [45]. Increased level of SOD in pea plant roots in PAW treated
seeds in comparison to control signifies high oxidative stress in it. In addition, the SOD
activity of leaves also increases, however, it is relatively low. The observed SOD enzyme
activity in pea plant roots grown after PAW–10 treatment of seeds is higher compared to
PAW–5. It may be due to the high concentration of H2O2 in PAW–10 compared to PAW–5
(Table1). The presence of a higher concentration of H2O2 in PAW creates external oxi-
dative stress in seeds that resulted in elevated SOD enzyme activity in plants’ roots. In
addition, we observed elevated SOD enzyme activity of plant roots in PAW (PAW–5 and
PAW–10) compared to control. Hence, PAW treatment has shown a higher ability of plants
to tolerate oxidative stress which is created by O2‾ radical. In addition, low SOD enzyme
activity in plants may lead to weaker plant defense against O2‾ radicals [8, 46].
CAT enzyme catalyzing the H2O2 in the plant to O2 and H2O. Hence, it helps in regulat-
ing the H2O2 level in the plant [45]. PAW treatment of pea seeds increased the CAT activ-
ity in pea plant roots significantly. PAW–5 pea leaves had no significant difference com-
pared to control, but PAW-10 pea leaves had substantial difference. This increase in CAT

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activity in plant roots (black gram and mustard) after PAW treatment of seeds previously
reported by Sajib etal. [8] and Islam etal. [17].
APX plays an important role in controlling internal ROS in plants. It reduces the H2O2
to H2O. It is normally found in the H2O2-scavenging organelles [45]. APX activity in
pea plant roots was found higher after PAW treated seeds in comparison to control. APX
activity of leaves had no significant difference when grown using PAW and control. This
increase in APX activity of roots shown similarity with the work of Sajib etal. [8] and
Islam etal. [17].
POD is a heme (electron donor) protein enzyme. It catalyses the oxidation of organic
and inorganic compounds [47]. Also, POD plays an important role in plant defense mecha-
nism towards plant pathogens. PAW treatment of pea seeds has enhanced the POD activity
in roots and leaves in comparison to control. However, POD activity in roots was compara-
tively higher than in leaves. This shows high oxidative stresses in the roots of PAW grown
roots that were regulated by POD along with other antioxidant enzymes.
As discused above, POD, APX, and CAT activity of roots grown after PAW–5 treat-
ment of seeds is significantly (p < 0.05) higher compared to PAW–10 and control. In addi-
tion, POD and CAT activity of leaves grown after PAW–10 treatment of seeds is relatively
higher than seeds treated with PAW–5 and control. These elevated activities of enzymes
in PAW grown plants leaves and roots in comparison to control is due to triggering plant
defense mechanism against H2O2 present in plants cells by ROS (H2O2 and dissolved O3)
present in PAW. Increasing activities of these H2O2 scavenging enzymes implies elevated
induced H2O2 in plant cells after PAW seeds treatment. This induced H2O2 triggering faster
seeds germination and plant growth since H2O2 is a signaling molecule. The low activity of
H2O2 scavenging enzymes showed accumulation of H2O2 in plants cells [8, 46].
Conclusion
In conclusion we can say that, seeds treated with PAW have shown improvement in the
germination and plant growth parameters such as roots and shoots length, chlorophyll ‘a’,
fresh and dry weight of root and shoots. This improvement in plant growth parameters has
taken place due to the removal of waxy structure from the surface of the seeds after PAW
treatment leading to the improvement in the wettability properties of seeds compared to
control seeds.
Further the study reveals that, plants grown from PAW-treated seeds have higher protein
and sugar concentration compared to control. In addition, H2O2, electrolytic and phenolic
leakage after PAW treatment did not show significant difference in comparison to control.
Plants grown from the seeds treated with PAW have shown higher enzymatic activ-
ity (SOD, POD, APX, and CAT) in comparison to control. This shows higher the activ-
ity of antioxidant enzymes signifies higher pacification of damage that may cause due to
increased level of superoxide. In conclusion, we can say that pre-treatment of seeds with
PAW has shown potential benefits to be used in the agriculture sector to improve germina-
tion and plant growth and it may contribute in meeting future food demand.
Supplementary Information The online version contains supplementary material available at https:// doi.
org/ 10. 1007/ s11090- 021- 10211-5.
Acknowledgements This work is supported by the Department of Atomic Energy (Government of India)
graduate fellowship scheme (DGFS). The authors sincerely thank Mr. Chirayu Patil, Mr. Vivek Pachchigar,

Plasma Chemistry and Plasma Processing
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Mr. Adam Sanghariyat, and Dr. Vishal N. Jain for providing constant support and useful suggestions during
this work.
Author Contributions All authors contributed to the study conception and design. Material preparation,
data collection, and analysis were performed by VR. The first draft of the manuscript was written by VR
and all authors commented on previous versions of the manuscript. All authors read and approved the final
manuscript.
Data Availability The datasets generated during and/or analyzed during the current study are available from
the corresponding author on reasonable request.
Declarations
Confict of interest The authors declare that they have no confict of interest..
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