V. pyrethrina Nakai seeds used in the experiment are those gathered and carefully selected from the greenhouse (37°28′45.2″N, 127°35′51.4″E) in Useful Plant Resources Center located in Yongmun-myeon, Yangpyeong-gun, Gyeonggido in November 2018, which were dried and stored for 27 months at 4°C.

To identify growth of V. pyrethrina Nakai depending on cell size of plug trays, we used 72-cell, 128-cell, and 200-cell plug trays (Bumnong Co., Ltd, Korea) and the size of plug trays was the same regardless of cell size (W × L × H : 280 × 540 × 45 mm). The cell size used was 72-cell (34 mL/cell), 128-cell (21 mL/cell), and 200-cell (10 mL/cell). We filled each cell with horticultural substrates (Baroker, SeoulBio, Korea) and sowed 2–3 seeds in each cell. We applied bottom watering 1–2 times a week so that the medium does not go dry. We rogued the seeds a week after sowing so that the plants grow evenly in the trays. The experiment was conducted for 6 weeks in a phyto-garden where the temperature of 25±1°C, relative humidity of 70±5%, photosynthetic photon flux density of 100±10 μmol • m⁻² • s⁻¹, and day length of 9/15h (day/night) can be controlled. We used LED for light source, and the wavelength was 406–770 nm, and we used 458 nm white light for maximum wavelength.

For the experiment to identify the growth difference depending on substrate type, we used different types of substrates in the plug tray with 10mL cells. We mixed horticultural substrate, peat moss (Profi2, Durpeta, Lithuania), and perlite (Supergreenpamix, Misung, Yesan, Korea) in the volume ratio of 1:1, 3:1, and 4:1(v:v) for the substrates, and sowed 2–3 seeds in each cell. The experiment was conducted for 6 weeks in a phyto-garden where the temperature and humidity are maintained. Before the experiment, we measured pH, EC, and water holding capacity of each substrate. We used the effluent obtained from diluting air-dried substrate and distilled water in the ratio of 1:5(v:v) and mixing them for 1 hour, and measured pH and EC using the PH-meter (Seven EasypH meter, Mettler Toledo, Switzerland) and EC-meter (EC Testr 11+, Eutech Instruments, Singapore). For measurement of water holding capacity of the substrate, we filled the pot with 15–20g of air-dried substrates and applied bottom watering for 12 hours, after which we discharged water for 30 minutes and measured the weight. The formula is as follows.

We filled the plug tray with 10mL cells with horticultural substrates to determine the effect of fertilizer concentration on growth when raising seedlings, and sowed 2–3 seeds each. We used Peters (Peters professional, ICL Specialty Fetilizers, USA) for the fertilizer, composed of nitrogen 20%, phosphoric acid 20%, potassium 20%, magnesium 0.05%, boron 0.0125%, iron 0.05%, manganese 0.025%, copper 0.0125%, zinc 0.025%, and molybdenum 0.005%. The concentration of each treatment was 500, 1000, 2000 mg • L⁻¹, and EC of each was 0.484, 0.886, 1.604 dS • m⁻¹. The untreated plot was the control plot, and fertilization was done by bottom watering for an hour once a week so that the substrates can sufficiently absorb the diluted fertilizer. The experiment was conducted for 5 weeks from April 16 to May 21, 2021.

We measured plant length, plant width, leaf length, leaf width, number of leaves, root length, shoot and root fresh weight and dry weight, and maximum quantum yield of V. pyrethrina Nakai. We measured plant length from the soil surface to the highest part of the plant body, and plant width with the thickest part of the plant body. We measured leaf length and leaf width with the biggest leaf out of completely bloomed ones. We measured the number of all leaves except those not fully bloomed and seed leaves. The growth survey was conducted with a completely randomized design repeating 3 times with 20 entities per treatment. We measured shoot and root fresh weight using an electronic scale (Shimadzu Analytical Balance AUW2200, Shimadzu, Japan) right after measurement, and we measured dry weight after measuring fresh weight and drying in a 70°C constant temperature drying oven for 72 hours. For chlorophyll fluorescence analysis, we selected 6 leaves per treatment, applied dark adaptation for 30 minutes, and used a chlorophyll fluorometer (Maxi Imaging-PAM-series, Heinz Walz GmbH, Effeltrich, Germany) to measure maximum quantum yield (Fv/Fm).

For statistical analysis, we conducted an analysis of variance using SPSS (SPSS 23.0, IBM Corporaton, USA), and conducted a post-hoc test at a 5% significance level using Duncan’s Multiple Range Test when the statistical difference is significant. The graph was developed using Sigma plot 10.0 (SPSS Inc., IL, USA).

As a result of the experiment on cell size of plug trays, plant length after 6 weeks was highest at 2.2 cm in 34 mL, but plant width was 5.8 cm, leaf length 2.1 cm, leaf width 1.8 cm, number of leaves 9, root length 10.0 cm, and shoot and root fresh weight and dry weight 234, 37, 24, and 5 mg, which were all higher in 10 mL. However, there was no statistically significant difference by cell size (Table 1), and there was also no significant difference in maximum quantum yield (Fig. 1A). Previous studies on number of cells and size of individual plug trays proved that there was greater growth in bigger cells (Shin et al., 2000; Kim et al., 2001; Nam et al., 2003), whereas smaller cells of trays lead to smaller volume, which reduces nourishment and thus not enough for long-term growth. However, the root zone of the plant body grows and there can be root pruning. V. pyrethrina Nakai showed the most root development in 10 mL, the smallest cells, but there was no significance in shoot growth depending on cell size. This indicates that 6 weeks are when the root zone develops, and at least 6 weeks are required in raising seedlings to show shoot growth. However, Kim et al. (2019) reported that smaller cell size leads to quicker root pruning after sowing, thereby moving up the time for planting, which as a result was effective in growth after planting. Hwang et al. (2018) reported that silver grass for landscaping did not show good growth with smaller cells when raising seedlings in plug trays, but seedlings raised in small cells showed excellent growth about 3 months after planting. The results of this study showed that V. pyrethrina Nakai did not show a difference in shoot growth when raising seedlings in plug trays for 6 weeks, but there was sufficient root pruning, and it seems effective to use 10 mL with small yet many cells to increase the yield after planting.

We measured pH and EC depending on the type of substrates used in the experiment. The horticultural substrate showed the lowest pH at 5.1 and highest EC at 0.96, and water holding capacity was high at 81.8% (Table 2). On the other hand, perlite showed the highest pH at 6.9 and lowest EC at 0.01, and water holding capacity was 64.7%. In general, perlite with thick particles shows about 20% of water holding capacity, but the type of perlite used in this experiment had small particles, which is why water holding capacity was higher. Peat moss showed higher EC than provided by the manufacturer, which is due to the different substrate storage conditions or measurement method. In the mixed substrate of peat moss and perlite (1:1, 3;1, 4:1), higher ratio of peat moss led to increased pH, EC, and water holding capacity. The results of the experiment showed that using the horticultural substrate led to excellent shoot growth, with plant length 1.7 cm, plant width 4.4 cm, leaf length 1.7 cm, leaf width 1.5 cm, and high fresh weight and dry weight (Table 3, Fig. 2). For shoot growth depending on the ratio of peat moss and perlite, the 1:1 mixed substrate showed lowest growth with plant length 1.4 cm, plant width 3.4 cm, leaf length 1.3 cm, leaf width 1.1 cm, and fresh weight and dry weight, while there was no significant difference in overall shoot growth in 3:1 and 4:1 mixed substrate. However, root length, fresh weight, and dry weight were lowest in the horticultural substrate. Lee et al. (1998) reported that, in the growth of Codonopsis lanceolata according to soil pH, higher pH led to increased root weight. Ku et al. (2019) claimed that there is a difference in growth of cultivated chives and wild chives depending on soil pH concentration. V. pyrethrina Nakai showed lower root growth when the pH concentration was low. Moreover, maximum quantum yield was lowest in the 1:1 mixed substrate, while there was no difference in the horticultural substrate and mixed substrate (3:1, 4:1) (Fig. 1B), which is because water holding capacity decreased when the ratio of perlite increased in the mixed substrate of peat moss and perlite, which relatively caused water stress. Peat moss has many micropores inside and thus has high water retentivity (Choi et al., 2000), but plant growth becomes poor due to difficult gas diffusion with the formation of perched water tables when raising seedlings in plug trays (Nelson, 1991). To make up for this deficiency, the porosity and size inside the substance are generally increased by mixing perlite. Lee et al. (2020b) claimed that mountain spike speedwell did not show favorable growth in the mixed substrate of peat moss and perlite, but Yoo and Roh (2012) argued that roots of chrysanthemum ‘Baekma’ were long in the mixed substrate of peat moss and perlite (2:2, 3:1). Lee et al. (2005) also reported that raising seedlings of Miscanthus sacchariflorus Benth in the mixed substrate of peat moss and perlite (1:1) resulted in favorable growth of the root zone. V. pyrethrina Nakai also showed higher length, fresh weight, and dry weight of the root zone in the mixed substrate of peat moss and perlite. However, all parts of the shoot measured in the horticultural substrate showed excellent growth, and there was a significant difference with the mixed substrate, which was consistent with the result by Lee et al. (2020b) that using horticultural substrates is most effective when raising seedlings of mountain spike speedwell in plug trays. Moreover, horticultural substrates include nitrate nitrogen (NO₃-N), available phosphate (P₂O₅), ammonium nitrogen (NH₄-N), and microelements (K, Ca, Mg, Fe, Cu, etc.), along with other types of substrates mixed in such a scoco peat and vermiculite aside from perlite and peat moss included in the horticultural substrates. Therefore, it is efficient to use horticultural substrates when raising seedlings of V. pyrethrina Nakai in a tray, and manufacturing components must be considered in soil experiments since soils or microelements included vary depending on the manufacturer of horticultural substrates.

Fertilization affects soil organic content, characteristics of microbes, and enzyme activity, thereby changing plant growth (Thirukkumaran and Parkinson, 2002; Compton et al., 2004; Frey et al., 2004), and the difference in plant species and nutrients in cultivating containerized seedlings results in deteriorated quality of seedlings (Juntunen et al., 2003; Hernández et al., 2009). The water-soluble fertilizer used in this study is 20-20-20 + TE with a balanced content of macroelements and microelements, and it promotes delivery of nutrients and absorption into the plant body (ICL, 2021). As a result of the experiment, V. pyrethrina Nakai seedlings showed improved growth overall in fertilizer application than the untreated control plot (Table 4). Plant length showed a 0.6 cm difference with 2.1 cm in 500, 2000 mg • L⁻¹ and 1.5 cm in the untreated plot, and plant width showed more than a 1.0cm difference in all fertilizer application plots than the untreated plot with 5.2 cm. Moreover, there was a significant difference from the untreated plot in leaf length and leaf width as well. There was almost no significance in maximum quantum yield (Fig. 1C). Lee et a l. (2015) reported that there was a difference in seedling growth from the control plot depending on fertilization level of Thalictrum uchiyamae Nakai and Thalictrum rochebrunianum var. grandisepalum (H. Lev.) Nakai, but there was not much difference in growth by concentration. The results of this study also revealed that growth was better in fertilizer application than the control, but there was not much difference in growth depending on concentration. Shim and Kwon (2010) conducted a nutrient solution experiment on foliage plants like Spathiphyllum and Hedera helix and reported that nutrient requirements varied depending on plat type and that clear fertilization standards for foliage plants are insufficient. V. pyrethrina Nakai did not show high nutrient requirements, and thus it does not seem to require more than 500 mg • L⁻¹ concentration in fertilizer application, and to increase the yield and consider the economic aspect, concentration below 500 mg • L⁻¹ may seem effective. Moreover, it is important to determine the optimal level of fertilizer concentration for plant bodies for cultivation, propagation, and supply of native plants in the future.

In sum, when raising seedlings of V. pyrethrina Nakai, it is effective to use 10 mL cell plug trays and horticultural substrate by itself and apply 500 mg • L⁻¹ fertilizers to increase seedling growth and yield as well as an economic effect.