Among the plant genetic resources at the National Agrobiodiversity Center at Rural Development Administration in South Korea, we selected variegated baby sun rose (Mesembryanthemum cordifolium f. variegata) (genetic resource number: IT345915) as the experimental plant. Prior to the study, we cut the stems, limiting the number of leaves to 5 leaves, with a size of 3 cm, to ensure consistent plant size. For the study, we used plants with axillary buds developed by at least 2 cm after one month.

The study was conducted for a total of 8 weeks from May 2 to June 28, 2022, at the experimental greenhouse (37°38′40″ N 127°06′25″ E) in the Department of Environmental Horticulture, Sahmyook University. Plants were planted in round plastic pots with a diameter and height of 11 × 10.5 cm. We used the substrate for succulent plants by mixing decomposed granite (1–2 mm) with river sand (0.5–1 mm) and horticultural substrate (Hanareumsangto, Shinsung Mineral, South Korea) at a ratio of 6: 3: 1 (v/v/v) by volume, and planted each plant one by one at the center of the pot. We designed six levels of shading treatment: no shade (i.e., direct sunlight) (0%), greenhouse glass (35%), greenhouse glass and 1 layer of clear polyethylene (PE) shading film (45%), greenhouse glass and 1 layer of white PE shading film (60%), greenhouse glass and 2 layers of white PE shading film (75%), and greenhouse glass and 1 layer of black PE shading film (99%). For the photo-synthetic photon flux density (PPFD) of each shading level, we calculated the mean and standard deviation of the measurements taken at 1 p.m. once a week on sunny days using a portable spectral radiometer (SpectraPen Mini, Photon Systems Instruments, Czech Republic) (Table 1).

The plants of all treatments including the control (0% shading level) were arranged on beds that are 1 m high, and the average temperature in the greenhouse during the study was 21.2 ± 3.1°C, and the average temperature of the outdoor area where the control was placed was 20.1 ± 2.8°C (Fig. 1A). Here, the greenhouse facility where the study was conducted was ventilated by fully opening the top and sides for 24 hours. The relative humidity in the greenhouse was 63.2 ± 13.5%, and the relative humidity of the outdoor area was 64.6 ± 13.2% (Fig. 1B). The average cloud cover during the study was 5.2 ± 2.7 okta, and this was evaluated by visual observation, which is the same as the measurement method of Korea Meteorological Administration, at 1 p.m. every day (Fig. 1C). The plants were watered twice a week. In this study, watering continued until gravitational water drainage occurred.

To investigate the effects of shading levels on the growth and leaf color of M. cordifolium f. variegata, various parameters were examined, including shoot height, shoot width, root length, leaf length, leaf width, fresh weight, dry weight, plant moisture content, chlorophyll content (SPAD units), and leaf color. The CIELAB color space, developed by the International Commission on Illumination in 1976, was utilized for assessing leaf color. The color coordinates, represented by L^* (lightness), a^* (green-red opponent colors), and b^* (blue-yellow opponent colors) values, as well as ΔE^*_ab and RHS values, were analyzed. The shoot height was measured as the vertical distance from the ground to the highest point of the plant, and the shoot width by measuring the broadest area of the plant seen from above. For root length, we measured the longest of the plant roots. Secondary analysis was conducted on the SH/RL ratio, which represents the ratio of shoot height to root length, and the LL/LW ratio, which represents the ratio of leaf length to leaf width. Fresh weight was measured after washing the plant and naturally drying it for 24 hours in an enclosed space. Dry weight was measured after hot air drying of the plants for 24 hours at 85°C using a heat dryer (HK-DO135F, HANKUK S&I, South Korea). Here, we performed a secondary analysis by contrasting fresh weight with dry weight to analyze the plant moisture content, and equation (1) is as follows.

SPAD units were measured using a portable chlorophyll meter (SPAD-502, Konica Minolta, Japan). CIELAB values were measured by setting the spectrophotometer (CM-2600d, Konica Minolta, Japan) to CIELAB D65/10° with reference to the leaf color measurement method by Lee et al. (2022a), after which we collected data including specular components. Here, to determine SPAD units and CIELAB, we randomly selected 3 leaves from each plant and measured the central part of the leaves. To compare the leaf color differences of M. cordifolium f. variegata at each shading level, we calculated the color differences by setting CIE76 ΔE^*_ab with reference to each shading level, and the equation (2) about the CIE76 color differences used is as follows (CIE, 2004).

For RHS values, we compared the results of each of L^*, a^*, and b^* with the Royal Horticultural Society (RHS) colour charts edition V and evaluated the leaf colors by selecting 2 approximate RHS values for each treatment. For leaf colors, we converted the means of CIELAB L^*, a^*, and b^* to converted colors using Converting Colors developed by Zettl (2023).

Analysis of variance (ANOVA) was conducted using SAS 9.4 (SAS Institute, USA) to analyze the results of the study. To compare the means, statistical analysis was conducted using Duncan’s multiple range test at p < .05. The study was in a completely randomized design, with 5 plants assigned to each treatment in 3 replications.

The plant sizes of Mesembryanthemum cordifolium f. variegata affected by the shading levels showed various differences (Fig. 2). The results showed that shoot height was tallest at 8.28cm under the 60% shading level (Table 2). This is similar to the results of previous shading study on Sedum zokuriense (Lee et al., 2021b) and Hylotelephium sieboldii cv. Mediovariegatum (Nam et al., 2022) discovered that shoot height was highest under the 65, 60% shading levels, respectively. Phenotypic plasticity is the ability of a plant to change its morphological characteristics so that it can adapt to the changing growth environment (Bradshaw, 1965; DeWitt et al., 1998; Sultan, 1987). The change in plasticity shown in shades is referred to as shade-induced phenotype (Weijschede et al., 2006). Here, in a shade-induced phenotype, we can expect to see an increase in the plant’s stems, shoot width, cover area, and leaf area (Lee and Nam, 2022). For shoot width of M. cordifolium f. variegata, the result of the 60, 75% shading levels in Duncan’s multiple range test showed the same significance level with 26.22 and 25.70 cm, respectively. Here, M. cordifolium f. variegata showed relatively low shoot width under 0, 35, and 99% shading levels compared to other treatments. Under the 0% shading level, factors such as light stress (Demmig-Adams and Adams, 1992), temperature rise, and water loss from soil may have had an effect. Additional research may be needed about light stress and water loss from soil by direct sunlight. On the contrary, under the 99% shading level, the growth rate may have decreased significantly because carbon dioxide assimilation is difficult, and the plant showed a shade avoidance response, implying that the amount of light received was extremely insufficient to overcome the low-level light environment. A previous study showed that Echeveria agavoides had relatively higher shoot height and shoot width in partial shading and shading compared to no shade, showing similar results as this study (Cabahug et al., 2017). Root length was longest under the 99% shading level at 21.26 cm. Some studies reported that the biomass allocated to the roots of Betula pendula was reduced under shading conditions (Van Hees and Clerkx, 2003), and Quercus robur and Fagus sylvatica also showed root growth inhibition under shading conditions (Van Hees, 1997). However, this study proved that shading treatment did not have a negative effect on the root length of M. cordifolium f. variegata, showing contrary results to the aforementioned studies. The SH/RL ratio that represents the ration of shoot height and root length was highest at 0.44 under the 60% shading level. Therefore, when cultivating M. cordifolium f. variegata under adequate shading level, shoot growth tends to be promoted relatively more than root growth.

Leaf length was longest at 5.93 cm under the 75% shading level, and leaf width was also widest at 3.24 cm under the 75% shading level, indicating that we can expect the most increase in leaf area through shade avoidance response under the 75% shading level when cultivating M. cordifolium f. variegata. This is similar to the results of previous study where the leaf length and leaf width of H. telephium cv. Lajos was highest under the 75% shading level (Nam et al., 2022). Meanwhile, there was no significant difference in the LL/LW ratio that represents the ratio of leaf length to leaf width.

The fresh weight of M. cordifolium f. variegata was relatively higher under the 45, 60, 75% shading levels at the same significance level compared to other treatments as a result of statistical analysis, each showing 38.65, 38.61, and 41.33 g (Fig. 3A). Meanwhile, dry weight was highest under the 45, 60% shading levels at the same significance level, each showing 2.16, 2.20 g (Fig. 3B). Plant moisture content was highest under the 75% shading level at 95.1% (Fig. 3C). Considering these results, the 75% shading level seems to have increased the cell size of M. cordifolium f. variegata compared to other shading levels, and thus the plants could contain high moisture content. In summary of growth parameters, shading films prevented the abiotic stress of plants and had a positive effect on plant growth. In particular, better growth was shown in shading compared to growing M. cordifolium f. variegata under direct sunlight, indicating that it is better to use shades for the mass production of plants. Therefore, it is recommended to cultivate M. cordifolium f. variegata in about 60–75% shading levels in order to significantly increase the plant sizes and biomass.

Chlorophyll content (SPAD units) of M. cordifolium f. variegata were highest at 22.87 under the 60% shading level, indicating that chlorophyll content was the highest in the same leaf area compared to other treatments (Fig. 4A). This result is similar to the results of shoot width and consistent with the reports of previous studies that S. zokuriense (Lee et al., 2021b), Hoya carnosa and Spathiphyllum wallisii (Lee et al., 2021a), and H. telephium cv. Lajos (Nam et al., 2022) showed an increase in chlorophyll content was observed concomitant with high shoot width under conditions of optimal shading level and light intensity. Therefore, creating a suitable growth environment for the plant species is an important factor to increase the shoot width and chlorophyll content of plants at the same time. Leaf color is a factor that enables intuitive evaluation of the external quality of plants and is important as it affects marketability (Lee and Nam, 2023). In previous studies, the increase in CIELAB L^* that is the color space coordinate representing lightness showed a negative correlation with the growth parameters of a few succulent species (Lee et al., 2022b; 2022c), and L^* was highest at 55.39 under the 99% shading level where the growth was lowest, thereby showing consistent results with previous studies (Fig. 4B). CIELAB a^* that represents green-red opponent colors was highest at −6.24 under the 0% shading level, indicating that leaves were being colored significantly under direct sunlight (Fig. 4C). Moreover, CIELAB b^* that represents blue-yellow opponent colors was highest at 21.89 under the 35% shading level (Fig. 4D). Meanwhile, some studies showed that L^* and b^* had a positive correlation (Kim et al., 2022; Lee et al., 2022b, 2022c), and similar trends were found under the 35, 99% shading levels.

Leaf color difference analyzed with CIE76 (ΔE^*_ab) ranged from 1.35 to 9.64 by shading levels (Table 3). As for Royal Horticulture Society (RHS) values, the leaf colors of M. cordifolium f. variegata were N137D, 147B under the 60, 75% shading levels, indicating that they were closest to green, whereas the leaf colors in other treatments were 147B, 147C, 148B, indicating that they were relatively closer to yellow. Here, the leaf color difference under the 75% shading level showing the vigorous growth state and the 99% shading level showing the lowest growth state was highest at ΔE^*_ab = 9.64, indicating that there was ‘big color difference’.

As a result of comprehensively evaluating the growth parameters, changes in leaf colors, and color differences, it was found that M. cordifolium f. variegata preferred the 60–75% shading levels compared to direct sunlight. Meanwhile, M. cordifolium f. variegata seems to have good shade tolerance so that it can show some growth under even the 99% shading level, but the SPAD units was the lowest and the lightness of leaves was the highest, thereby showing the low ornamental value of leaves. Accordingly, it is recommended to cultivate M. cordifolium f. variegata under 60–75% shading levels to significantly increase the plant sizes, biomass and improve the external quality of leaf colors.