Recently, atmospheric plasma treatment of proper dose on plant have shown to promote growth (Kalachova et al. 2024; Motrescu et al. 2024; Tsuboyama et al. 2024). These effects include key phase transitions such as seed germination and fruit development (Kobayashi et al. 2024; Lee et al. 2021; Lim et al. 2023; Mujahid et al. 2020). The growth promoting effects are believed to be due to the generation of reactive oxygen species and nitrogen species (RONS) by the plasma treatment in a dose-dependent manner (Priatama et al. 2022). Thus, recent studies have focused on the dose-dependent effects of plasma treatment on various plant species. Additionally, the chemically active nature of RONS prompts researchers to study their potential to influence secondary metabolite production (Prasad et al. 2023).

Some medicinal plants, such as Panax ginseng, Medicago truncatula, and Angelica gigas, accumulate high levels of secondary metabolites possessing antimicrobial, anticancer, and antioxidant activities. These metabolites are often found in plant storage organs such as fruits and roots, with many medicinal plants specifically storing these compounds in their roots as a potential defense mechanism (Kim et al. 2023). Thus, studies have increasingly focused on root development to enhance the production of these beneficial metabolites.

Hairy roots are axillary roots that develop in response to wounding. Thus, hairy roots were considered as a primary sign of pathogen invasion in plants (Doran 2013). The bacterium Agrobacterium is a representative bacterial agent that induces hairy root formation through invasive gene transfer called hairy root (HR) syndrome (Chilton et al. 1982). After this discovery, researchers developed hairy root culture systems to study the production of medicinal metabolites by activating transcription factors involved in HR syndrome (Bulgakov et al. 2016; Chilton et al. 1982). These studies were enforced by findings that hairy roots are abundant in pharmaceutical metabolites such as ginsenosides (Chen et al. 2020).

To date, little study has focused on the effects of plasma treatment on root systems, although plasma treatment applied to entire plants has been shown to promote plant growth (Ji et al. 2020). To reveal the effects of plasma on root tissue, we treated the roots of Arabidopsis thaliana seedlings with atmospheric plasma jet. Our results showed that plasma treatment at the root tip inhibited primary root length growth while significantly increasing the number of axillary roots. Further analysis revealed the similar effects in other plant species such as Petunia axillaris and Nicotiana benthamiana. Microscopic analysis revealed that plasma treatment triggered ROS accumulation in both the root tip and elongation zone. These findings suggest that atmospheric plasma could provide a pathogen-safe method for inducing axillary root development in plants, with potential applications for enhancing secondary metabolite production in medicinal species.

Plant materials and plasma treatment

Arabidopsis thaliana cv. Col-0, Nicotiana benthamiana, and Petunia axillaris were grown in a 16 h light/ 8 h dark cycle at 22°C for general growth and seed harvesting. All the seeds were harvested and ripened in a dry chamber of 22°C for at least 2 months to ensure a fully ripened seed stage. The plasma device was utilized in the previous study (Seol et al. 2017). In brief, a needle-shaped plasma jet using He gas was utilized. Plasma treatment on plants was performed on a 1/2 MS agar plate (1/100 MS, 0.8% phytoagar, and 0.05% MES, pH 5.7). The treatment duration of plasma was 10 seconds. Cutting of root tip was performed with Dorco Procut DN52 gil and indirect plasma treatment was done by covering the plant with MARIENFELD Microscope Cover Glasses Thickness No. 1 (Lauda-Königshofen, Germany).

Root growth analysis

All the plants were grown on 1/2 MS agar plate for 3 to 4 days and subjected to plasma treatment. After treatment, plants were grown for another 3 to 4 days till growth was measured. ImageJ software was utilized to measure the length. Light conditions for growth analysis utilized fluorescent light bulb [FL40EX-D], 100 µmol m^-2 s^-1 of continuous light condition.

Confocal microscopic analysis

Seedlings were mounted with distilled water and observed. Zeiss LSM 880 model was utilized for microscopic analysis. Cell walls and nuclei were visualized by staining with propidium iodide (PI; 30 µM in distilled water) for 5 minutes and washed with distilled water. To visualize the ROS signal, seedlings were 10 µM 2′,7′–dichlorofluorescin diacetate (DCF-DA, SIGMA-Aldrich, D6883-50MG, Darmstadt, Germany) solution in 10 mM Tris-Cl buffer (pH 7.4) for 10 min. The PI signal was manually changed into magenta.

Data visualization and statistical analysis

Physiological data was taken from at least 10 replicates and visualized with the ggplot2 package of project R (Villanueva and Chen 2019). For statistical analysis, either the student’s T-test or Tukey HSD test was applied with project R. In the case of applying the student’s T-test, the asterisk was shown to reveal statistical significance while the alphabets were denoted to show the physiological values are the same in the case of the Tukey HSD test. All the statistical significance was annotated by P value 0.05.

Identification of the plasma-responsive site in the root system

To analyze how the root system responds to the plasma treatment, we utilized 3-day-grown A. thaliana Col-0 seedlings. After plasma treatment, the plants were grown for an additional 4 days. We treated plasma on two different root regions: the elongation zone and the root tip, for a duration of 10 seconds (Fig. 1A). As a result, plasma treatment on the elongation zone caused a slight alteration in the length of the primary root while had negligible effects on the number of axillary roots (Fig. 1). Meanwhile, plasma treatment on the root tip resulted in a threefold reduction in primary root growth while a significant increase in the number of axillary roots (Fig. 1). These results prompted us to focus on plasma treatment at the root tip to investigate its role in axillary root development.

Figure 1. Determination of the effect of plasma treatment on root tips. A. Schematic representation of plasma treatment on elongation zone and root tip of Arabidopsis thaliana roots. B. Schematic representation of experimental plan. Purple arrow indicates plasma treatment on plants grown for 3 d. C. Representative picture of plasma-treated or control plants. Yellow bar indicates scale bar of 5 mm. The left plant represents the Cont. plant, middle plant represents the Elon. plant, and the right plant represents the Tip plant. Yellow arrowhead indicates the original position of root tip before plasma treatment. D. Primary root length phenotype. Each dot indicates individual data and characters above the bar indicate statistical significance determined via Tukey’s honestly significant difference test. E. Number of axillary roots. Each dot indicates individual data and characters above the bar indicate statistical significance determined via Tukey’s honestly significant difference test. Cont., control; Elon., sample treated with plasma on elongation zone; Tip, sample treated with plasma on tip

To determine whether this physiological response was consistent across different plant species, we treated plasma on the root tips of P. axillaris and N. benthamiana (Fig. 2A). Similar to A. thaliana, both plant species showed comparable responses, with plasma treatment at the root tip inhibiting primary root growth and promoting axillary root development, though with varying sensitivities (Fig. 2B, C). Notably, in N. benthamiana, plasma treatment also induced the formation of hairy axillary roots (Fig. 2A). Considering N. benthamiana is widely used in studies of secondary metabolite due to its abundance of precursors (Yao et al. 2022), it is possible that plasma-mediated signals promote hairy root formation, especially in metabolite-rich plants. These results demonstrate that atmospheric plasma treatment specifically at the root tip effectively inhibits primary root growth while promoting axillary root development in diverse plant species.

Figure 2. Effect of plasma treatment on different plants. A. Representative photographs of plasma-treated or control plants. The yellow bar indicates a scale bar of 5 mm. For each species, two Cont. and two Pla. plants were visualized from the left. Yellow arrowhead indicates the original position of the root tip before plasma treatment. The red arrow indicates the hair-root phenotype observed. The inlet image in the bottom right panel shows an example of hairy-root phenotype after plasma treatment on the root tip of Nicotiana benthamiana from a different sample. B. Primary root length phenotype. Characters above the box indicate statistical significance determined via the Student’s t-test, ***, p < 0.0001. C. Number of axillary roots. Characters above the box indicate statistical significance determined via the Student’s t-test, ***, p < 0.0001. Con., control samples; Pla., plasma-treated samples

ROS generation and axillary root development

To investigate the cellular effects of plasma treatment on root tip tissues, we used confocal microscopy to observe the plasma-treated root tip at the cellular level (Fig. 3). Compared to the control samples, plasma-treated roots showed a strong propidium iodide (PI) signal throughout the cells (Fig. 3), which indicates necrosis of cells (Fulcher and Sablowski 2009). The observed morphological changes induced by the plasma treatment were similar to those found in both DNA damage and cell death (Fulcher and Sablowski 2009). Previous study indicated that bleomycin treatment induces Double Strand Break (DSB) in DNA, which is able to inhibit primary root growth with minimal impact on axillary root development (Zhang et al. 2015). Thus, it is likely that plasma-induced DNA damage in the root tip contributed to the inhibition of primary root growth observed in our experiment.

Figure 3. Microscopic observation of ROS in plasma-treated root tissues. Cell walls were visualized via PI staining and ROS signals were visualized via DCF-DA staining. PI is shown in magenta and the DCA-DA signal is shown in green. “Elongation” indicates root cells located above 5 to 15 mm from root tip. “Plasma” indicates samples immediately after plasma treatment, and “Plasma after” indicates samples at 48 h after treatment with plasma. All photographs present a merged channel of PI and DCF-DA. The scale bar is indicated in the upper-left panel as a white bar. ROS, reactive oxygen species; PI, propidium iodide; DCF-DA, 2′-7′-dichlorodihydrofluorescein diacetate

To reveal the underlying mechanism of axillary root development following plasma treatment, we examined ROS levels using DCF-DA (2′,7′–dichlorofluorescin diacetate) dye (Fig. 3). Although the sample preparation and observation took less than 30 minutes, we observed ROS signal not only in the root tip but also in the elongation zone (Fig. 3). Notably, the ROS signal in the elongation zone appeared to be highly localized and concentrated at the cellular level (Fig. 3). Two days after the plasma treatment, ROS signals were no longer detectable from the plants, but we observed the formation of secondary meristem-like tissue in the elongation zone of plasma-treated roots (Fig. 3). These observations suggest that plasma-mediated ROS may be transported to the elongation zone to form local maxima, which triggered the formation of secondary meristem, ultimately leading to axillary root development.

Surgical cutting could induce similar physiological effects with pathogenic risk

To determine whether cell death is correlated with the phenotypic changes induced by plasma treatment, we performed an indirect plasma treatment that blocked the generation of ROS, electric fields, and most ultraviolet light (Fig. 4A). The indirect plasma treatment resulted in only a slight change in primary root length with a negligible impact on the number of axillary roots (Fig. 4B, C), suggesting that plasma-induced ROS, electric fields and ultraviolet wavelength light are the key factors influencing root phenotypes. Previous reports have shown that both electric fields and ultraviolet wavelength light induce DNA damage (Ceron-Carrasco and Jacquemin 2013; Kciuk et al. 2020), which supports the idea that these elements contribute to cell death in root tip tissues and affect root development.

Figure 4. Effect of cell death analyzed using a simple surgical cut of root tip. A. Schematic representation of indirect treatment, surgical cutting, and plasma treatment on root tips of Arabidopsis thaliana. Indirect treatment was performed by placing a cover glass on root tips. A surgical cut was performed at 1 mm above the root cap. B. Primary root length phenotype. Each dot indicates individual data and characters above the bar indicate statistical significance determined via Tukey’s honest significant difference test. C. Number of axillary roots. Each dot indicates individual data and characters above the bar indicate statistical significance determined via Tukey’s honest significant difference test

We then mimicked the effects of cell death by surgically cutting the root tip. Interestingly, this simple surgical cutting of the root tip mimicked the physiological effects of plasma treatment in terms of axillary root formation, but it did not affect primary root length (Fig. 4B). Specifically, the number of axillary roots produced after root cutting was almost identical to that observed following plasma treatment (Fig. 4C). This indicates that cell death in the root tip plays a significant role in regulating the number of axillary roots (Fig 4C). However, plasma treatment offers an advantage over surgical cutting in agricultural applications, since plasma treatment has been shown to possess antimicrobial activities (Barjasteh et al. 2024; Cyganowski et al. 2024; Prasad et al. 2023). Thus, our findings suggest that plasma treatment on the root tip of young plants provides a safe and effective method to enhance axillary root development, including the formation of hairy roots.

In summary, we could identify where does cold plasma treatment could only affect root development by treatment on root tip tissue (Fig 1). This was applicable at least for tomato and tobacco, of which tobacco even developed hairy root phenotype (Fig 2). Fluorescent analysis and other analysis accessing on the components of plasma treatment revealed plasma alters the root architecture through ROS and physical damage which thus ensures the safe measure to enhance axillary root development in plant system (Fig 3, Fig 4).