Introduction

Sedum zokuriense Nakai (Sok-ri-gi-rin-cho) is a vascular plant that is endemic in various rich flora areas and regions within South Korea and even within the demilitarized areas of the Korean peninsula (Kim et al. 2016;Lee et al. 2003). This stonecrop glabrous herb possesses short woody tuberous rhizomes with slender stems, and alternate, obovate, and spatulate leaves (Eggli 2003). Although there is a continued taxonomic debate regarding this species, S. zokuriense has also been known as a member of the Phedimus genus (Mayuzumi and Ohba 2004;Nakai 1939;RBGK and MBG 2012). A bulk of the Crassulaceae species in the Korean peninsula were investigated by Joo et al. (2010) and S. zokuriense was found to be a medicinal plant with curative and detumescent properties.

However, endemic plants are prone to anthropogenic threats and natural changes and have been vulnerable to high extinction risks (Coelho et al. 2020). Because of this, efforts of various countries have been found to restore ecosystems and advances the capacity to propagate and cultivate highly threatened endemic species (Santelices et al 2011). Part of these efforts is investigating optimal conditions for cultivation which involves various environmental factors including lighting conditions, soil media, and fertilization. In domesticating and cultivating these plants, germplasm facilities of ornamental plants are generally indoors and plant species are grown under greenhouse conditions.

During the summer season, shading is considered a key element in controlling light intensities and has been found to be an important factor in growing succulent plants like that of S. zokuriense (Cabahug et al. 2017). In regulating light intensities, it is essential to consider how this factor affects plant morphology, productivity, and photosynthetic activities (Kim et al. 2011). Likewise, different types of succulent species would require various potting mixes as each species has a preference on underground conditions for root development (Cabahug et al. 2018).

Various soil mixtures are available and have been recommended, however, there is a need to properly investigate the combinations that would provide sufficient support to this endemic species (Kohlschreiber 1998). As a general rule of thumb, potting mixes are ideally able to provide proper drainage, aeration, support, and protection (Graham 1987). Readily available and commonly used soil components are decomposed granite, river sand, fertilizer-amended media, vermiculite, perlite, and burnt husk (Stephenson 1994).

Since S. zokuriense are vegetatively propagated, fertilization is deemed important for re-establishment of roots, and have been found that liquid feeding would be able to enhance root growth and improve soil condition (Bach 1998), however, fertilization rates should be investigated as the need of plants differ from one species to another. The use of a liquid fertilizer has been commonly used and investigated in various ornamental species such as that of Phalaenopsis spp. (Endo and Ikusima 1989), Orostachys iwarenge for. magnus (Jeong et al. 2012), Doritaenopsis hybrids (Shin et al. 2014), among others.

Thus, this study aims to investigate and provide optimization of shading levels, potting media, and fertilization rates on the vegetative growth of S. zokuriense in order to facilitate propagation and proper cultural management practices for endemic species in South Korea.

Materials and Methods

Plant materials

Sedum zokuriense Nakai (genetic resource No. IT317349) plants were taken from the National Agrobiodiversity Center. Experimental plants were brought to the experimental greenhouse facility of the Department of Environmental Horticulture, Sahmyook University. From the mother plants, stem cuttings were procured using sharp pruning shears. To standardize the cuttings, about 3.5 cm of length were removed from the terminal buds. A slanted cut at a 45-degree angle is then made, to maximize the available area for rooting, securing 1.5 cm tip stem cuttings with around 2 - 3 nodes. Tip stem cuttings were then planted in 11 cm x 10.5 cm pots to a depth of 1.5 cm with their respective soil media.

Experimental design and treatment

Plants were subjected to various factors affecting the growth of S. zokuriense plants were subjected to various relative shading levels of 50% (- 1,200 μmol m^-2 s^-1), 65% (- 670 μmol m^-2 s^-1), 80% (- 450 μmol m^-2 s^-1), 95% (- 60 μmol m^-2 s^-1), and 98% (- 15 μmol m^-2 s^-1). At 98% shading levels, plants were placed under-bed conditions. These measurements were taken during prior to the experiment wherein there were clear skies at 1 o’clock in the afternoon using the SpectraPen mini (Photon Systems Instruments, Czech Republic). Decomposed granite (DG), fertilizer-amended media (FM) (Hanareumsangto, Shinsung Mineral, South Korea), perlite (PL) (New Pearl Shine No. 1, GFC, South Korea), river sand (RS), burnt husk (BH) (Gangjin National Agricultural Cooperative Federation, South Korea), and vermiculite (VL) (Verminuri, GFC, South Korea) were used to create potting media treatments (ratio of components, v/v/v): DG:FM:PL (5:2:3), DG:RS:BH (4:5:1), RS:FM:VL (7:2:1), RS:VL:PL (6:2:2), FM:VL:PL (2:3:5), and FM:DG:BH (2:7:1). A liquid fertilizer (New Raysio 6-10-5, HYPONeX, Japan) was used in the study at different concentrations: no fertilizer application (control, 0 ppm), 500, 750, 1000, and 2000 ppm. The respective concentrations were applied at 50 ml per pot once a week.

The experiment was conducted using a completely randomized design. Each factor was analyzed separately. There were five replications per treatment with 7 plants in each replication.

As a control, the shading conditions were set at 50%, and DG:RS:FM:VL (4:3:1:2, v/v/v/v) was used as soil media for other said factors not involving their respective treatments. The watering of plants was done twice a week. The relative humidity and temperature in the greenhouse and under-bed conditions during the experiment (May to July) were recorded and data were graphed as shown in Fig. 1. The average temperature and humidity within the greenhouse facility was 25.8 ± 7.9°C, 69.8 ± 16.7% and under-bed conditions was 25.2 ± 8.5°C, 71.5 ± 17.3% throughout the duration of the study.

Plant growth parameters

The survival rate was determined at the end of the experiment by counting the number of cuttings that successfully produced roots and shoots over the total number of leaf cuttings multiplied by 100 in order to obtain the percentage. Growth and quality evaluation was done by collecting plant height, width, and fresh weight of shoots and roots. To determine leaf color, this was assessed using the CIELAB color value using a spectrophotometer (CM-2600d, Konica Minolta, Japan) which utilizes L*, a*, b* color space values indicating the lightness, hue, and saturation and RHS values were processed through a CIELAB–RHS converter (CIELAB-RHS Colour Converter, Oregon State University, USA).

SPAD chlorophyll content

Chlorophyll content was determined using a portable SPAD chlorophyll meter (SPAD-502, Konica Minolta, Japan). Calibration was done by carefully clamping the instrument onto the central part of the leaf to obtain chlorophyll readings. SPAD readings were done by using three leaves that were tagged as representatives per plant and served as replications.

Data Analysis

Data from plant growth parameters and chlorophyll content were organized using Microsoft Excel (Microsoft, USA) and subjected to standard analysis of variance (ANOVA) using SPSS Statistics 22.0 (IBM, USA). To compare mean differences, Duncan’s multiple range test was applied with a 5% level of significance.

Results and Discussion

Survival rate and plant growth

The plant growth parameters and survival rates of S. zokuriense as influenced by shading levels, potting media, and different fertilization rates is shown in Table 1.

Results showed that the survival rate decreased as the shading levels increased. According to Stephenson (1994), Sedum plants vary in their light requirements and their responses reflect their natural habitat indicating that this species may be cold-hardy and should be grown under indirect sunlight. Results suggest that S. zokuriense plants are able to tolerate shaded conditions up to 80%, however, at 95%-98%, there would be a significant decline in its survival rate. On plant growth parameters, the use of 65% shading had the tallest plants (13.11 cm) which were comparable to those grown under 80% shading level (Fig. 2A). Plant width was also higher than those grown under 65% shading (8.65 cm) and were followed by those at 50% shading (7.90 cm). However, plants grown at 65% and 50% shading levels had comparable results gaining the highest shoot and root fresh weight with 4.76 g, 0.69 g, and 4.28 g, 0.58 g, respectively, which did not significantly differ from each other. Similar results were also found from S. sarmentosum species which increased fresh shoot weight and other local accessions when placed under 50% shading level (Lee et al. 2007). At higher shading levels (95% and 98%), had the lowest plant and fresh weigh parameters. Although they are generally known to be shade-loving plants, well-established and mature stonecrop plants are transplanted and used as green roof systems as some species can withstand extremely low temperatures and limited watering (Butler and Orians 2011).

For potting media, the use of DG:FM:PL had the lowest survival rate (50.5%) while those grown under other treatments integrated with compost or organic materials had 70 - 97% survival rates. Many stonecrops have been reported to have grown to thrive with compost alone (Stephenson 1994), while other species have been known to do well under soil mixtures integrating composts or organic matter when they are being vegetatively propagated (Kim and Kim 2015). Likewise,the tallest plants were taken from those planted in RS:VL:PL with 13.16 cm which did not significantly differ from those of RS:FM:VL (12.57 cm) and DG:RS:BH (11.91 cm), however, wider plants were taken from those of RS:FM:VL (9.18 cm) which significantly differed from the rest of the treatments (Fig. 2B). Interestingly, the highest shoot and root fresh weight were taken from those plants grown in RS:VL:PL with 7.57 g and 1.81 g, respectively. Rowley (1953) emphasized the importance and remarkable differences of growth for succulents citing that unsuitable soil mixes would have a high tendency to delay growth, impede drainage and induce rooting. Common to the high vegetative growth of S. zokuriense is the use of river sand in combination with organic composts. The use of sandy soil coupled with compost has been reported to protect Sedum spp. from rot, facilitate proper drainage and aeration from previous studies of Graham (1987) and Monterusso et al. (2005).

Among fertilization rates, control plants and those applied treated with 500 ppm had a 91.6% survival rate followed by those of the 750 ppm and 2,000 ppm (95.8%), while application of 1,000 ppm gave the highest survival rate (100%). Since the plants were propagated using stem cuttings, the use of 1,000 ppm has provided an optimum rate in which cuttings were able to successfully establish roots contributing to the high survival percentage. However, fertilization rates did not statistically affect plant height, width, and fresh weight of shoot parameters. In the study of Lee et al. (2020) on potted Veronica rotunda var. subintegra. using the same fertilizer amendment, its application, likewise, had no significant effect on plant growth. However, it may be noted that the use of 2,000 ppm had the tallest (11.28 cm) and widest (8.66 cm) plants which are evident in Fig. 2C. This may be attributed that at higher concentrations, the plants would likely respond positively including those of root growth as seen in the results. The high amounts of N, P, K, Mg, and Mn found in Hyponex have affected the growth which was also true for other native species to South Korea such as Thalictrum spp. (Lee et al. 2015), Cypripedium macranthum (Kim et al. 1997), Veronica rotunda var. subintegra (Lee et al. 2019), etc. It would also be noted that studies with the use of the liquid fertilizer had significant improvement with the use of 1,000 ppm on the vegetative growth of the endemic plants. Similarly, studies of Jeong et al. (2013) on potted Orostachys malacophyllus plants affected above ground plant parameters (i.e., plant height and width, and fresh weight), although the differences in fresh weigh of shoots were evident, comparable results were found from those of the control and the application of fertilizer at 1,000 ppm.

Chlorophyll content and CIELAB values

The chloropyll content and color reading values, their equivalent RHS value and color group are presented in Table 2. Results revealed that shading levels and potting media had significantly affected color reading values of S. zokuriense while only shading levels significantly affected chloropyll content. However, fertilization rates did not significantly affected both parameters wherein the non-significance in the chloropyll content also manifested in the same RHS color value of 146A indicating that leaves at all fertilization rates were classified as yellow-green.

S. zokuriense was significantly affected by shading treatments on both chlorophyll content and CIELAB color reading. The highest chlorophyll content was observed from those at 80% shading level (34.87) which did not significantly differ from those at 65% (34.21) and 50% (33.57) shading levels. However, at 98%, the lowest chlorophyll content was observed with 27.73. Plants grown under 50% shading had the lightest pigment with an L* value of 42.83 which was significantly different from the rest of the treatments. On the a* value, 50 – 80% shading values were found to be comparable at -8.25 – -8.52 indicating lighter hues than those at 95 – 98% shading levels. However, b* values at 98% shading levels significantly provided higher saturation of 23.34. Plotting these values in the color quadrant, RHS values suggest that 50%, 65%, and 98% shading levels had a 146A RHS value indicating the yellow-green color of leaves, while those at 80 – 95% had an N137D RHS value indicating green leaf color. At 98% shading levels, plants were found to be stunted in growth with etiolation, compared to those at lower shading levels (50% and 65%) in which plants appeared to have healthy and vibrant colors.

Results were found similar in the study of S. sarmentosum wherein chlorophyll values were significantly higher from 50 – 90% shading compared to the control (Lee et al. 2007). While it is true that shaded leaves produce higher chlorophyll content, at a certain point, dark conditions would produce etiolation lowering chlorophyll a and b altogether. This is further explains that at the limited light source the state of etiolation will have non-green plastids in plant tissues which would have normally had chloroplasts when under well-lit conditions (Armarego-Marriott et al. 2019). Likewise, as a consequence of shading levels, those subjected at 80 - 95%, leaves may have enhanced chlorophyll b and a ratio wherein darker colors are observed when chlorophyll b has a much higher concentration (Khumaida 2002).

In summary, optimal conditions for S. zokuriense vegetative growth involve subjecting plants to shading conditions that facilitate plant growth without compromising chlorophyll and visual quality of plants, potting media that provides proper anchorage, and development of roots and shoots, and fertilization rates that would enhance and support nutrient plant requirement. Although the use of 1,000 ppm fertilization rate had the highest survival rate, it did not significantly affect other plant growth parameters. On the other hand, the use of 65% shading and RS:VL:PL potting media have substantially produced a high survival rate in propagation while providing sufficient vegetative growth, green and healthy plants with high chlorophyll content.