Non-thermal plasma modified growth and differentiation process of Capsicum annuum PP805 Godiva in in vitro conditions

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1 2017 Hefei Institutes of Physical Science, Chinese Academy of Sciences and IOP Publishing Printed in China and the UK Plasma Science and Technology Plasma Sci. Technol. 19 (2017) (6pp) Non-thermal plasma modified growth and differentiation process of Capsicum annuum PP805 Godiva in in vitro conditions Nasrin SAFARI 1, Alireza IRANBAKHSH 1,3 and Zahra ORAGHI ARDEBILI 2 1 Department of Biology, Science and Research Branch, Islamic Azad University, Tehran, Iran 2 Department of Biology, Garmsar Branch, Islamic Azad University, Garmsar, Iran iranbakhsh@iau.ac.ir Received 16 October 2016, revised 28 December 2016 Accepted for publication 30 December 2016 Published 31 March 2017 Abstract With the aim of evaluating the possible impacts of cold plasma on the structure and growth pattern of Capsicum annuum, the current study was carried out. The seeds were exposed to an argon-derived plasma (0.84 W cm 2 surface power densities) for 0, 1 or 2 minutes. Plasmatreated seeds were grown in the Murashige and Skoog (MS) medium or MS medium supplemented with BA and IAA. The presence of purple stems was recorded in plasma-treated plants grown in the medium supplemented with hormones. The recorded morphological differences were dependent on the exposure time of plasma treatments and/or the presence of hormones in the culture media. Plasma treatment of 1 minute had an improving effect on the shoot and root lengths as well as total leaf area, whereas plasma treatment of 2 minutes had an adverse effect. In contrast to the 1 minute treatment, plasma treatment of 2 minutes significantly impaired growth and hence reduced the total biomass. Alterations in stem diameter and differences in tissue patterns (especially in the vascular system) occurred, and were mainly dependent on the plasma exposure time and/or the presence of hormones. This is a first report on the effects of cold plasma on plant growth in in vitro conditions. Keywords: applied physics, cold plasma, differentiation, nitric oxide, growth (Some figures may appear in colour only in the online journal) 1. Introduction There have been many efforts to make various generating instruments of cold plasmas facilitate the treatment of living tissues to achieve different aims in various fields, including medicine, agriculture, biology, and industry. One of these tools is known as dielectric barrier discharge plasma (DBD), which has been greatly developed for different ranges of medicinal purposes [1], and in just the last few years for seed treatments. Some biologically active substances and signaling molecules (such as superoxide, singlet oxygen, ozone, nitric oxide (NO), and hydrogen peroxide), in combination with UV radiation, are produced in DBD [2]. Therefore, it may be supposed that cold plasma may play the role of an effective elicitor or inhibitor to alter plant growth, differentiation, 3 Author to whom any correspondence should be addressed. development, and metabolism. Various kinds of non-thermal plasma treatments have been introduced as fast, novel, economic and pollution-free alternatives to promote seed germination, seed wall modification, and plant early growth [3 7]. There is limited research focusing on the possible impacts of plasma on the various plant parameters. The most widely described effects of plasma on the different studied plants are limited to decreases in seed microbial contamination [8], modifications in seed coat structures [9], stimulation of seed germination [9 11], modifications in the physiological characteristics [4, 9, 12], increases in seed permeability [9], and effectis on early plant growth [13, 14]. Findings about and understanding of plant plasma interactions are rare and more studies are required to elucidate the plasma triggered responses. In vitro cultures provide a suitable method for a convincing study of different proposed hypothesizes in plant /17/ $

2 science. One of the critical issues in plant tissue culture is seed-borne contamination. Despite good documentation of the antimicrobial effects of plasma, data on the effects of seed plasma treatments on the behavior, morphogenesis, organogenesis, differentiation, and growth pattern of seedlings in in vitro conditions are rare. Capsicum annuum L. is regarded as the most economically vital species of the genus Capsicum, which belongs to Solanaceae. The current study was carried out to evaluate possible impacts of plasma treatments on the structure and growth pattern of Capsicum annuum in in vitro conditions. 2. Material and methods 2.1. Preparation, experimental design, and treatments Seeds of Capsicum annuum PP805 Godiva were purchased from a reliable industrial center (Pop Vriend Seeds, Netherlands). The experimental apparatus was DBD plasma. DBD plasma at atmospheric pressure is generated between two glass plates as dielectric barriers covering the two powered circular plate copper electrodes of 5.5 cm in diameter with a gap of 3 mm between dielectrics. Argon was applied as a functional gas between dielectrics. The dielectric acts as a stabilizing material when the potential across the gap reaches the breakdown voltage leading to the formation of a large number of micro-discharges. To generate DBD plasma, a modified AC high voltage power supply was used. Applied voltage was measured by a high voltage probe (Pintek HVP40) connected to an oscilloscope (Tektronix TDS1012B). During all experiments throughout this study, the frequency and the applied voltage of the device were fixed at 23 khz and 11 kv, respectively. The instrument power was 80 W, so for cm 2 plasma treatment areas, the surface power density was equal to 0.84 W cm 2. Dry seeds were exposed to plasma with three different exposure times, 0, 1, and 2 minutes. Then, the seeds were thoroughly washed three times with sterile distilled water and grouped into six different treatments with three replications (each containing three seeds). Murashige and Skoog (MS) medium [15] was applied to the experiments. The treated seeds were cultured in two different kinds of prepared culture media, including the hormone-free MS medium or MS medium supplemented with hormones (MSH), including 2mgl 1 BA and 0.5 mg l 1 IAA. All cultures were incubated in a germinator at 25 C under illumination of 1500 Lux (16 h of light and 8 h of dark). The treatments were grouped as follows. MS-C: control samples cultured in hormone-free MS. MS-P1: samples treated with plasma for 1 min cultured in hormone-free MS. MS-P2: samples treated with plasma for 2 min cultured in hormone-free MS. MSH-C: control samples cultured in MS medium containing hormones (2mgl 1 BA and 0.5 mg l 1 IAA). MSH-P1: samples treated with plasma for 1 min cultured in MS supplemented with hormones. MSH-P2: samples treated with plasma for 2 min cultured in MS supplemented with hormones Growth analysis 42 days after plasma treatment, the seedlings were harvested for further analyses. Shoot and root lengths were determined. Total leaf area was measured using a digital leaf area meter (Leaf Area Meter- AM-200; ADC BioScientific Ltd, UK). Total fresh weights were assessed Histological procedure Ethanol:glycerol (80:20) was applied as fixing solution. Cross sections of the basal stems were prepared. Cross sections (stained with carmine and methylene blue) of the basal stem were observed with a light microscope and photographed. Determination of the stem diameter was performed on the microscopic images using the ImageJ software (1.42l, Wayne Rasband, National Institutes of Health, USA, a public domain Java-based image processing program) Statistical analysis The experimental design was a completely randomized. The obtained data were analyzed as a factorial experiment in a completely randomized design with three replications by analysis of variance (ANOVA) using SPSS software, and mean separation was performed with Duncan s multiple range test, at the level of P < Results and discussion 3.1. Morphological differences, growth rates and biomass accumulation The presence of purple stems, as a probable sign of anthocyanin accumulation, was observed in cold plasma-treated seedlings grown in MS medium supplemented with hormones. These reactions may be triggered by the emission of UV during plasma treatment. This evidence may indicate plasma as a new alternative possible elicitor for inducing the production of various desirable secondary metabolites in the plant cell and tissue culture. Anthocyanins are pigments responsible for the pink, red, violet and blue colors in plant organs, and have antioxidant activity [16]. Anthocyanin (a phenolic compound) is a secondary metabolite and may act as a crucial component in the plant defense system against various biotic or abiotic stress conditions, in particular UV. It has been recorded that UV radiation remarkably promoted the accumulation of secondary metabolites, especially phenolics, in the calli of Vitis vinifera L. Öküzgözü [17]. The plasma treatment of 2 min led to the production of deformed leaves (figure 1). The application of hormones (2mgl 1 BA and 0.5 mg l 1 IAA) resulted in the accelerated formation of 2

3 Figure 1. The effects of high intensity of plasma treatments on leaf performance (deformed leaves in P2 treated plants). (A) MS-control; (B) MS-P1 (plasma of 1 min); (C) and (D) MS-P2 (plasma of 2 min). Figure 2. The effects of different plasma treatments on the growth of pepper seedlings grown in two hormone-free MS or MS containing 2mgl 1 BA and 0.5 mg l 1 IAA (35 days after plasma treatment). (A) MS-control; (B) MS-P1; (C) MS-P2; (D) MSH-control; (E) MSH-P1; (F) MSH-P2. 3

4 Figure 3. The effects of different cold plasma on the morphology of pepper plants (42 days after seed plasma treatment) grown in a hormonefree MS medium or MS medium containing 2 mg l 1 BA and 0.5 mg l 1 IAA (MSH). (A) MS-control; (B) MS-P1; (C) MS-P2; (D) MSHcontrol; (E) MSH-P1; (F) MSH-P2. Table 1. The effects of different argon-derived plasma treatments on the various characteristics related to the growth of pepper (Capsicum annuum PP805 Godiva) grown in a hormone-free MS medium or supplemented with 2 mg l 1 BA and 0.5 mg l 1 IAA (MSH) (42 days after seed plasma treatment). Shoot length (mm) Root length (mm) Total leaf area (mm 2 ) Total fresh mass (g) MS-C ± 0.66 b ± 2.6 b 1279 ± c 0.62 ± 0.03 b MSH-C ± 0.65 b ± 0.95 b ± a 0.85 ±.04 a MS-P ± 1.67 a ± 7.31 a ± b 0.81 ±.02 a MSH-P ± 0.80 a ± 0.68 b ± a 0.78 ± 0.02 a MS-P ± 0.71 c ± 2.40 c ± d 0.28 ± 0.03 c MSH-P ± 0.85 d ± 2.85 c ± 9.53 d 0.30 ± 0.01 c * Mean values followed by different letters are significantly different at P < 0.05 according to Duncan s multiple range test. ** MS-C-control grown in hormone-free MS medium; MSH-C-control grown in MS medium supplemented with hormones (2 mgl 1 BA and 0.5 mg l 1 IAA); MS-P1-plasma of 1 min grown in hormone-free MS medium; MSH-P1-plasma of 1 min grown in MS containing hormones; MS-P2-plasma of 2 min grown in hormone-free MS; MSH-P2-plasma of 2 min grown in MS supplemented with hormones. adventitious roots (figure 2). The recorded morphological differences were dependent on the intensity of plasma treatment and/or the presence of hormones in the culture media (figure 3). The data presented in table 1 clearly indicated that plasma treatment of 1 min had a significant improving effect on the shoot and root lengths (table 1), whereas these parameters were significantly reduced by the highest applied plasma intensity (2 min). NO has been known to be either harmful or helpful to plant growth and development [18]. The destructive impacts of plasma of 2 min could be attributed to the disturbances occurring in the meristem zone, caused by the high levels of NO, ozone, other bioactive agents, and/or UV produced during plasma treatments. The presence of a high concentration of NO results in disturbance of the root apical meristem, mainly by affecting the expression patterns of some auxin-related genes [19]. The deformed leaves, inhibited growth, and thickening of the stem found in P 2 -treated seedlings could be triggered by the impaired hormonal balances, especially ethylene with its gaseous nature and the closed in vitro condition. It is well known that ethylene is implicated in controlling cell division, growth direction and plant reactions to various stress conditions. In contrast to Figure 4. The effects of different intensities of argon-derived plasma treatments on the stem diameter of pepper (Capsicum annuum PP805 Godiva) grown in a hormone-free MS medium (MS) or supplemented with 2 mg l 1 BA and 0.5 mg l 1 IAA (MSH) (42 days after seed plasma treatments). MS-C: control grown in a hormone-free MS medium; MSH-C: control grown in MS medium supplemented with hormones (2mgl 1 BA and 0.5 mg l 1 IAA); MS-P1: plasma of 1 min grown in a hormone-free MS medium; MSH-P1: plasma of 1 min grown in MS containing hormones; MS- P2: plasma of 2 min grown in a hormone-free MS; MSH-P2: plasma of 2 min grown in MS supplemented with hormones. 4

5 Figure 5. The effects of argon-derived plasma treatment on tissue patterns in basal stems of the treated pepper (Capsicum annuum PP805 Godiva) grown in a hormone-free MS medium (MS) or supplemented with 2 mg l 1 BA and 0.5 mg l 1 IAA (MSH) (42 days after seed plasma treatments). (A) MS-control; (B) MS-P1; (C) MS-P2; (D) MSH-control; (E) MSH-P1; (F) MSH-P2. plasma of 1 min, plasma treatment of 2 min significantly impaired growth, and hence reduced the total biomass in plants grown in a hormone-free MS medium (MS) or culture medium supplemented with hormones (MSH) (table 1). The first applied levels of plasma treatments (1 min) had enhancing effects on the total leaf area, whereas plasma treatment of 2 min adversely affected the mentioned characteristics (table 1). The observed differences in the morphological traits and notable plasma induced changes in growth related characteristics, mainly dependent on the plasma exposure time, clearly indicated that plasma treatment of 1 min was an effective treatment to improve the growth rates in the this cultivar of pepper, in contrast with 2 min exposure. However, plasma treatment of 2 min had no 5

6 destructive effects on another studied cultivar of pepper (data not shown). Therefore, there are variable responses dependent on the plant species. This is the first report on the effects of seed treatment with plasma in in vitro conditions. The modifications in phytohormones [9], some other physiological characteristics, especially defense-related responses [4], and alterations in the rate of plant early growth [13] caused by plasma treatments have been reported Anatomical changes and tissue pattern Alterations in the stem diameter occurred, mainly dependent on the plasma intensities and the presence of the applied hormones (figure 4). The highest stem diameters were recorded in the P 2 - treated plants, while the smallest were found in the MSH-P 1 group (figure 4). As shown in figure 5, plasma treatments affected differentiation processes in the treated seedlings. The patterns of vascular systems were considerably altered by the plasma treatments (figure 5). The plasma treatment of 1 min led to modifications in the vascular system, where the pattern of tracheids and vessels was different from the control samples (figure 5). Plasma-induced alterations in tissue patterns recorded in the basal stem of the treated-seedlings are additional complementary evidence, confirming the possible potential of plasma to influence growth and differentiation processes. These results could be attributed to the triggering or toxic concentrations of the plasma-derived active compounds (especially NO) and/or the UV emissions generated during plasma treatment. NO is considered as a key controller of development and a critical signaling agent, implicated in the various aspects of plant growth and physiology [20 22]. Therefore, the recorded enhancing effects of plasma on plant growth [4] could be attributed to the possible improving effects of plasma on the differentiation of the vascular system via modifying the balances of phytohormones. 4. Conclusions This is the first study on the effects of plasma treatment on seedlings grown in in vitro conditions. Based on the findings of the current research, it seems that plasma treatments, mainly dependent on the plasma intensity, exposure time to plasma, and/or plant species, as well as the applied device producing the plasma, might have significant desirable promoting effects or destructive impacts on the plant differentiation, metabolism, and growth pattern, probably by altering the signaling pathways and/or the hormonal balances. The following hypothesis and mechanisms are presented. 1. Plasma treatment may have the potential to affect the accumulations of secondary metabolites, which may be considered as a new alternative possible elicitor in the plant cell and tissue culture. 2. Plasma treatments, mainly dependent on the applied intensity and exposure time, may induce some differences in growth rates, differentiation process and the tissue patterns, especially the vascular system. 3. The high intensities of plasma treatments may result in disturbances in the meristem zone. 4. Plasma treatments may impair the hormonal balances, especially ethylene. 5. High intensities of plasma treatment may trigger some changes in the pattern of gene expression, thereby inhibiting growth rates and inducing morphological and physiological differences. 6. Plant reactions and behavior to plasma treatments are dependent on the plant species and even variety. It is obvious that more precise studies are required to clarify the exact mechanisms involved in these reactions. Non-thermal atmospheric plasma technology may be introduced as a new alternative approach for decontaminating seeds and modifying differentiation processes in the plant cell and tissue culture. Understanding of the plasma-triggered reactions in plants is low, so the findings presented in this research could be path-finding in this rapidly developing area. Acknowledgments The authors would like to thank Professor M Ghoranneviss, Dr Narges Oraghi, and MSc. Hamed Nikmaram for their benevolent and professional collaborations in the research procedure. The corresponding author especially would like to acknowledge the Plasma Physics Research Center, Science and Research Branch, Islamic Azad University, Tehran, Iran. References [1] Kalghatgi S et al 2008 Applications of non thermal atmospheric pressure plasma in medicine Plasma Assisted Decontamination of Biological and Chemical Agents (Netherlands: Springer) pp [2] Bußler S et al 2015 Food Res. Int [3] Alekseev O et al 2014 Transl. Vis. Sci. Technol. 3 2 [4] Li L et al 2014 Sci. Rep [5] Šerá B et al 2009 Plasma Sci. Technol [6] Šerá B et al 2013 Plasma Sci. Technol [7] Yin M Q et al 2005 Plasma Sci. 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