Introduction
Lilyturf (Liriope muscari ( Decne.) L.H. Bailey; also referred to as Liriope platyphylla F.T. Wang and T. Tang) is a perennial, evergreen herbaceous species in the family Asparagaceae (formerly classified within Liliaceae) and holds significant medicinal and horticultural value in East Asia (Zhang et al., 2020). The roots of L. muscari have long been used in traditional medicine for their expectorant, antitussive, and immune-boosting properties. More recently, this species has attracted increasing interest as a promising ingredient for functional foods and pharmaceutical products owing to its antioxidant and anti-inflammatory activities (Lei et al., 2021; Seong et al., 2018; Shang et al., 2017; Wu et al., 2017). In addition, L. muscari exhibits strong shade tolerance (Zhang et al., 2021) and drought resistance (Pichakum and Pichakum, 2021), and can be used as a groundcover plant. These horticultural traits are considered to have great potential for use in landscaping and urban horticulture, such as urban greening and green-roof systems (Fantz, 2008; Ju and Yoon, 2014; Park and Park, 2019).
Many farmers rely on vegetative propagation of L. muscari, primarily through clump division (Fantz, 2008). Although this method is simple and practical for field application, it has notable limitations, particularly the restriction of genetic diversity within cultivated populations (Lee et al., 2023, 2025c; Megersa, 2017). Therefore, the development of technologies that improve seed propagation efficiency is crucial for large-scale production and for the advancement of breeding programs. Efficient and uniform seed germination, along with the production of vigorous seedlings, represents an essential foundation for commercial horticultural success (Finch-Savage and Bassel, 2016; Zulfiqar, 2021). However, previous studies have reported that L. muscari seeds exhibit low germination rates and relatively weak early seedling growth (Fagan et al., 1981; Hruska et al., 1982a, 1982b), which have long been bottlenecks to seed-based seedling production. In this context, improving germination performance and ensuring stable early seedling development may serve as key strategies for reducing growers’ dependence on vegetative propagation.
Seed germination can be constrained by endogenous hormonal balance as well as by mechanical and chemical inhibition imposed by the pericarp, pulp, and testa, together with environmental cues (Finch-Savage and Leubner-Metzger, 2006). In particular, L. muscari seeds possess a relatively thick pericarp and pulp that restrict water uptake and gas exchange. These structures have been reported to delay germination, partly due to residual substances inhibitory to germination (Chimera and Drake, 2010; Fagan et al., 1981; Hruska et al., 1982b). Therefore, pretreatments that remove the pericarp and pulp may enhance germination performance. Conversely, sowing intact seeds—retaining both pericarp and pulp—may offer advantages for storage and adaptation to natural environmental conditions (Radchuk and Borisjuk, 2014; Raviv et al., 2017). Thus, comparing these two sowing approaches is essential for establishing effective and practical nursery techniques.
Wet-cold stratification has traditionally been used to break seed dormancy, and it has been employed in various studies (Lee et al., 2025a; Pergolotti et al., 2023; Šulitka et al., 2025; Yang et al., 2023). However, this approach requires prolonged treatment under high-humidity conditions, which can increase the risk of pathogen contamination and seed decay, thereby limiting its applicability in large-scale commercial seedling production. In contrast, dry-cold stratification (also referred to as dry-cold treatment in this study) minimizes seed moisture content, reducing the likelihood of disease incidence and improving stability during storage and transport. This method has also been reported to enhance germination rates and post-germination seedling vigor (Ervin and Wetzel, 2002; Kim et al., 2024a; Peng et al., 2021).
Dry-cold treatment can induce physiological changes necessary for germination by promoting the breakdown of endogenous abscisic acid (ABA) and the accumulation of gibberellic acid (GA) (Rodríguez-Gacio et al., 2009). Several studies have also reported that storing seeds under dry conditions can effectively break dormancy (Kim et al., 2024a; Nelson et al., 2023; Tuttle et al., 2015). However, while some species respond to short-term treatment, others require prolonged exposure to low temperatures, making it necessary to carefully determine the optimal treatment duration for each species.
For instance, in the wetland species Juncus effusus, both germination percentage and seedling vigor varied depending on the length of dry-cold storage (Ervin and Wetzel, 2002). In Japanese white birch (Betula platyphylla var. japonica), seed germination and survival rates improved after 30 days of dry-cold treatment, whereas a 15-day exposure primarily enhanced morphological and physiological seedling traits (Kim et al., 2024a). Meanwhile, seeds of Primula secundiflora and P. sikkimensis stored under dry-cold conditions exhibited higher germination compared with fresh seeds ( Peng et al., 2021). Studies on a range of forb species have also revealed substantial interspecific variation in response, highlighting the importance of determining the optimal storage duration for each species (Kildisheva et al., 2019). These previous studies indicate that dry-cold treatment affects not only germination rates but also the morphological and physiological traits of seedlings, underscoring the necessity of species-specific investigations to optimize treatment protocols.
Furthermore, seed germination characteristics, beyond germination percentage and speed, are critical factors influencing the physiological health and vigor of seedlings after germination (Kim et al., 2024a). The integrity of morphological indicators and biomass parameters during early seedling growth directly relates to cultivation stability and productivity. In addition, chlorophyll fluorescence parameters and remote sensing vegetation indices have become established as robust, non-destructive (non-invasive) indicators for evaluating photosynthetic efficiency and physiological status in seedlings (Garbulsky et al., 2011; Jang et al., 2023; Kim et al., 2024b; Park et al., 2024). Kim et al. (2024a) noted that many studies on seed germination have focused exclusively on germination-related metrics such as germination percentage and energy. Even in some studies that extended their scope to include seedling growth traits, the limited diversity of parameters often resulted in insufficient insights. Therefore, an analytical approach that integrates various morphological and physiological indicators represents a scientifically sound way to improve assessments of post-germination seedling health.
This study investigated the effects of two factors— post-harvest pericarp removal and the duration of dry-cold treatment—on the seed germination percentage of L. muscari, as well as the subsequent morphological and physiological characteristics of the seedlings. These findings are expected to provide fundamental information for establishing efficient sowing practices and stable seedling production techniques for L. muscari, thereby enhancing its industrial applicability and supporting the advancement of seed-based propagation technologies.
Research Methods
Experimental Materials and Pre-Experimental Preparation
This experiment was conducted using mature lilyturf (Liriope muscari (Decne.) L.H. Bailey) fruits collected in November 2023 from cultivated plants on the campus of Sahmyook University (37°38′39″N 127°06;23″E). Fruits exhibiting damage or irregular morphology were discarded, and only healthy, well-developed fruits were selected for the analyses. Representative images of the fruits and seeds used in the experiment are presented in Fig. 1. Pure seeds were obtained by completely removing both the pericarp and pulp from the fruits. The seeds were spherical, with an average diameter of 0.40 × 0.40 cm, and a hundred-seed weight of approximately 6.12 g (Table 1). After selection, the fruits were thoroughly rinsed with purified water, surface-dried using filter paper, and air-dried under cool, dark, and dry conditions for approximately one week. Following this pretreatment, 50 fruits were sealed in zipper storage bags ( approximately 5 .0 × 5 .5 cm) for storage.
Experimental Design and Cultivation Conditions
Fruits stored in zipper storage bags were subjected to dry-cold treatment at a constant temperature of 3°C. The treatment durations were 0, 15, 30, 45, and 60 days, resulting in a total of five treatment periods. After each period, fruits were divided into two groups: a control group, in which no mechanical or structural modification was applied, and a pericarp-removal treatment group, in which both the pericarp and pulp were removed immediately following the dry-cold treatment. The experiment was conducted in March 2024 using seedling trays containing 105 cells. Ten fruits or seeds from each treatment group were sown per replicate, with five replicates in total. One fruit or seed was placed in each cell and covered with approximately 2 mm of growth media. A fertilized horticultural substrate (Hanareumsangto; Shinsung Mineral, South Korea) was used as the growing medium.
Fruit and seed management after sowing, as well as seedling cultivation, were carried out in the experimental greenhouse of the Department of Environmental Horticulture, Sahmyook University (37°38;39″N 127°06;23″E). Irrigation was applied three times per week until gravitational water drained from the seedling trays. The greenhouse was maintained at approximately 25% shading. Seedlings were cultivated for about 13 weeks from the date of sowing. During the experimental period, the average air temperature and relative humidity in the greenhouse were 22.3 ± 3.8°C and 62.8 ± 13.2%, respectively.
Investigation of Germination and Seedling Development Characteristics
To assess the effects of dry-cold treatment on the improvement of germination in L. muscari fruits and seeds, several germination parameters were measured, including germination percentage (GP), survival rate (SR), germination energy (GE), mean germination time (MGT), time to reach 25% germination (T25), and vigor index (VI) (Table 2). GP and SR were calculated at the end of the experiment, corresponding to 13 weeks after sowing. GE was determined as the ratio of GP at 10 weeks to that at 13 weeks. Germination was recorded when the emerging sprout exceeded 0.2 cm in length.
To analyze seedling development following germination, basic growth parameters and biomass-related indices were evaluated, including shoot height, shoot width, stem diameter, root length, leaf number, leaf length, leaf width, leaf area, and the fresh and dry weights of shoots and roots. In addition, relative water content (RWC), the shoot-to-root dry weight ratio (S/R ratio), compactness, and Dickson quality index (DQI) were assessed. Changes in shoot height and width between 9 and 13 weeks after sowing—when seedling growth was vigorous—were presented as line graphs. Measurement methods for seedling growth parameters followed the criteria described by Park et al. (2023). Dry weight was determined after oven-drying samples at 85°C for 24 hours using a drying oven (HK-DO135F, HANKUK S&I, South Korea). RWC was estimated by comparing fresh and dry weights of individual seedlings.
Basic qualitative parameters, including visual score and chlorophyll content (SPAD units), were also assessed. The visual scoring reference was based on the methodology described by Kim et al. (2024a, 2024c), in which visual sensory evaluation was performed on germinated L. muscari seedlings using a 0–10 scale. Chlorophyll content was measured using a portable chlorophyll meter (SPAD-502Plus, Konica Minolta, Japan).
Investigation of Physiological Indices
A suite of physiological indices was examined to evaluate the physiological responses and overall health status of germinated seedlings. Among these, remote sensing vegetation indices were measured with a portable spectroradiometer (PolyPen RP410, Photon Systems Instruments, Czech Republic), including the normalized difference vegetation index (NDVI), photochemical reflectance index (PRI), and modified chlorophyll absorption ratio index (MCARI). For plants with leaves smaller than the device’s measurement aperture, reflectance was recorded by horizontally aligning two leaves together, following the procedure described by Silva-Perez et al. (2018).
Chlorophyll fluorescence parameters were quantified using a portable fluorometer (FluorPen FP 110/D, Photon Systems Instruments, Czech Republic). Measurements included fundamental fluorescence parameters (Fv, Vj, and Vi), the maximum quantum yield ( Fv/Fm), the slope of the fluorescence transient (Mo), quantum yield parameters (ΦPo, Ψo, ΦEo, and ΦDo), specific energy fluxes per reaction center (RC; ABS/RC, TRo/RC, ETo/RC, and DIo/RC), and the performance index on an absorption basis (PIABS).
For fluorometric measurements, a detachable dark-adaptation clip was attached to leaves at the sampling site in accordance with the manufacturer’s instructions, and leaves were dark-adapted for 15 min prior to measurement (PSI, 2025). Following the method of Shin et al. (2024), the fluorometer was set to an excitation wavelength of 455 nm, and leaves were exposed to an actinic light intensity of 1,500 μmol·m−2·s−1—equivalent to 50% of the super pulse— to induce Fm, the maximum fluorescence level required for the JIP-test.
Statistical Analysis
All statistical analyses were performed using SAS software ver. 9.4 (SAS Institute, USA). One-way and two-way analyses of variance (ANOVA) were conducted, and both main effects and interaction effects were evaluated. Post hoc comparisons among means were carried out using Duncan’s multiple range test, with a significance level set at p < .05. A total of 15 key indices (shoot length, shoot width, stem diameter, root length, SPAD units, dry weights of shoots and roots, relative water content, S/R ratio, compactness, DQI, GP, Fv/Fm, PIABS, and NDVI) were selected for analysis. Pearson correlation coefficients were computed to generate a correlation matrix to assess pairwise relationships among the indices. In addition, hierarchical cluster analysis was performed by calculating Euclidean distances from the correlation matrix and applying average linkage to evaluate similarities in the correlation profiles of the indices. The correlation matrix was visualized as a heatmap in which positive correlation coefficients were represented by black (darker) shades and negative coefficients by white (lighter) shades, with color intensity indicating the magnitude of the correlation.
Results and Discussion
Evaluation of Germination Indices
Seed germination is a complex process regulated by the interplay between endogenous hormonal balance and external environmental conditions, with the physical barrier imposed by the pericarp and the physiological effects of cold stratification serving as major determinants (Gao et al., 2022; Kim et al., 2024a; Walker et al., 2021). In this study, pericarp removal and dry-cold treatment exerted diverse effects on the germination of L. muscari seeds ( Table 3). The germination percentage (GP) reached its maximum value of 88.0% in the pericarp-removal treatment combined with 45 days of dry-cold treatment, whereas the control group exhibited markedly low GP values of 10.0–12.0% under 0–15 days of treatment. The survival rate (SR) followed a trend similar to that observed for GP. Germination energy (GE), defined as the proportion of GP at 10 weeks after sowing relative to the final GP, was comparatively high (52.2–59.3%) in pericarp-removed seeds subjected to 30–45 days of dry-cold treatment, indicating that pericarp removal effectively enhanced GE.
The mean germination time (MGT) was prolonged in the control group across the 0–45-day dry-cold treatment, ranging from 11.9 to 12.6 weeks. Similarly, the time to reach 25% germination (T25) in the control group was also extended, ranging from 11.2 to 12.0 weeks. These results suggest that sowing intact fruits, with both the pericarp and pulp retained, substantially delays germination in L. muscari. Meanwhile, the vigor index (VI) was highest (4913) in the pericarp-removal treatment combined with 45 days of dry-cold exposure.
In summary, pericarp removal combined with an appropriate duration of dry-cold treatment significantly improved the germination performance and overall seed vigor of L. muscari. These results align with the findings of Kim et al. (2024a), who reported that short-term dry-cold treatment enhanced germination in Japanese white birch (Betula platyphylla var. japonica).
Although this study did not directly analyze the chemical composition of the pericarp or pulp, the beneficial effects associated with pericarp removal support previous reports suggesting that a thick pericarp or pulp can impede water uptake and gas exchange, and can retain germination- inhibitory compounds such as abscisic acid (ABA) (Fagan et al., 1981; Hruska et al., 1982b). Earlier studies have also shown that phenolic acids and tannin-like polyphenols present in the pulp of L. muscari exert strong inhibitory effects on germination (Hruska et al., 1982a). Therefore, removal of the pericarp and pulp can be regarded as a key pre-treatment strategy for improving the germination percentage of L. muscari seeds.
Evaluation of Seedling Development After Germination
Representative images of seedlings 13 weeks post-sowing for both the control and pericarp-removed treatments are presented in Fig. 2. Analysis of the results revealed that both shoot height and width differed significantly (p < .001) depending on whether the pericarp was removed. These traits also showed significant differences (p < .05) across different durations of the dry-cold treatment. Notably, in the control group, a 60-day dry-cold treatment tended to increase shoot height and width to 3.17 and 3.75 cm, respectively, at 13 weeks post-sowing compared with other treatment durations. In the pericarp-removed group, the 0-day dry-cold treatment produced the greatest shoot height (3.80 cm), whereas shoot width reached its maximum (7.83 cm) under the 60-day treatment, exhibiting a pattern similar to that observed in the control group.
Overall, in the control group, certain dry-cold treatment durations (30 and 45 days) resulted in a gradual decline in shoot height over the growth period, while shoot width continued to increase. In contrast, seedlings in the pericarp-removed treatment generally exhibited increasing shoot height up to week 12, followed by a slight decrease at week 13. Shoot width in this group, as in the control, steadily increased throughout the observation period.
As seedlings advance through the post-germination growth stages, the decline in shoot height accompanied by the continuous expansion of shoot width likely reflects a morphological strategy that prioritizes lateral development of aboveground structures (e.g., improved light interception) to enhance plant stability and assimilate accumulation (García-Fernández et al., 2015; Poorter et al., 2012). This trend aligns with previous reports indicating that the duration of dry-cold treatment can influence shoot elongation and stem thickening during subsequent growth stages (García-Fernández et al., 2015; Kim et al., 2024a; Poorter et al., 2012).
In summary, dry-cold treatment appears to direct resource allocation toward the early development of horizontally oriented light-intercepting structures during the initial post-germination growth stage. The combination of pericarp removal and an optimal dry-cold treatment duration not only enhanced GP in L. muscari but also improved morphological balance, thereby contributing to higher overall seedling quality.
Based on the seedling growth results, stem diameter did not differ significantly among treatments (Fig. 3). In contrast, root length was shortest under the 15-day dry-cold treatment in the control group (2.66 cm), whereas it was longest under the 0- and 45-day treatments in the pericarp-removed group, measuring 9.42 and 9.36 cm, respectively. The number of leaves was relatively higher in the pericarp-removed group under the 0-, 30-, and 45-day dry-cold treatments, with 4.1, 3.9, and 4.2 leaves, respectively. In the control group, however, the 15-day dry-cold treatment resulted in the fewest leaves (1.6 leaves). Leaf length was greatest in the pericarp-removed group under the 0- and 60-day treatments, measuring 8.99 and 9.00 cm, respectively. Leaf width was widest under the 30-day dry-cold treatment in the pericarp-removed group (0.28 cm). Leaf area exhibited a trend similar to that observed for leaf length.
The fresh and dry weights of both shoots and roots were highest under the 0-day dry-cold treatment in the pericarp- removed group. Water content plays a critical functional and physiological role in plants (Tran et al., 2024) and can vary depending on environmental conditions and plant developmental status. The highest relative water content (81.8%) was observed under the 60-day dry-cold treatment in the control group. The visual score was highest under the 45-day dry-cold treatment in the pericarp-removed group, with a rating of 8.1.
Based on these findings, the combination of the control group and the 15-day short-term dry-cold treatment appeared to reduce the initial vigor of L. muscari seeds, potentially exerting long-term negative effects on post-germination morphological traits and biomass accumulation. In contrast, the pericarp-removed treatment seemed to promote stable seedling growth following germination, likely due to the removal of germination-inhibitory substances and consequent changes in endogenous hormone levels (Kim et al., 2024a). Similar results have been reported in several studies demonstrating that varying durations of cold treatment applied to seeds can directly affect seedling developmental characteristics after germination (Kim et al., 2024a; Poff et al., 2016; Wang et al., 2025). In this context, optimizing the duration of dry-cold treatment applied to fruits and seeds is considered essential for producing vigorous L. muscari seedlings. Considering that the optimal treatment period differed among morphological parameters and bio-mass even within the pericarp-removed group, a strategic approach to determining treatment duration is required.
Evaluation of Plant Quality Indices and Physiological Changes
According to the results for chlorophyll content (SPAD units), SPAD values in the pericarp-removed group remained generally high, with means of 51.04–52.60 SPAD units under the 0-, 30-, 45-, and 60-day treatments. The 15-day dry-cold treatment showed a numerically lower mean value (44.04 SPAD units), although this difference was not statistically significant. Overall, no significant differences in SPAD units were detected among the dry-cold periods within the pericarp-removed group (Table 4). In contrast, the 15-day dry-cold treatment in the control group showed a markedly lower chlorophyll content, recording 15.10 SPAD units. The shoot-to-root dry weight ratio (S/R ratio) was highest (2.42) under the 0-day dry-cold treatment in the pericarp-removed group, whereas the lowest value (1.33) was recorded under the 30-day treatment in the control group. Overall, these results indicate that applying an appropriate dry-cold treatment in conjunction with pericarp removal increases chlorophyll content per unit leaf area. In addition, pericarp removal alone—without dry-cold treatment—appears to promote preferential allocation of resources to the shoot rather than the root. Compactness and DQI showed no significant differences among the ten treatment combinations according to DMRT (Table 4). However, for DQI, the main effect of pericarp removal was statistically significant in the two-way ANOVA (p < .05).
Among remote sensing vegetation indices, the normalized difference vegetation index (NDVI) typically ranges from approximately 0.6 to 0.8 in healthy vegetation (Gascon et al., 2016). In this study, NDVI values ranged from 0.674 to 0.716 across all treatments, indicating that the seedlings possessed a dark green coloration characteristic of well-pigmented, healthy plants. Notably, the pericarp-removed group under the 0-day dry-cold treatment exhibited the highest NDVI value (0.716). The photochemical reflectance index (PRI), which reflects variations in xanthophyll cycle activity and photosynthetic light-use efficiency (Wong et al., 2022), responded sensitively to differences in the duration of dry-cold treatment. The lowest PRI value (−0.003) was observed in the control group subjected to 15 days of dry-cold treatment, suggesting a reduction in photochemical efficiency. The modified chlorophyll absorption ratio index (MCARI), an indicator associated with chlorophyll content, exhibited trends consistent with measured chlorophyll levels. The control group under the 15-day dry-cold treatment showed the highest MCARI value (0.372), demonstrating an inverse relationship between chlorophyll content and MCARI. This result is also in agreement with previous studies reporting that MCARI tends to increase under unfavorable treatment conditions or suboptimal growth environments where plant vigor declines (Kim et al., 2024b; Lee et al., 2025c; Park et al., 2024).
Taken together, the results for plant quality indices and physiological parameters suggest that indices such as compactness and Dickson quality index (DQI), although not exhibiting statistically significant differences in this study, effectively served as indirect indicators of the structural stability and morphological balance of seedlings. Vegetation indices, on the other hand, demonstrated sensitivity in capturing physiological changes. Accordingly, the integrated interpretation of these two categories of indicators proved valuable for a comprehensive understanding of both morphological and physiological responses to dry-cold treatments. This integrative approach supports the concept that germination and early seedling development are regulated by the complex interplay of morphological and physiological factors rather than by a single determinant (Bewley et al., 2013; Kim et al., 2024a).
Chlorophyll fluorescence analysis provides a rapid assessment of photosystem II (PSII) efficiency and is characterized by its high sensitivity, non-destructive nature, and robust data reliability (Gorbe and Calatayud, 2012; Lee et al., 2021, 2022; Stirbet and Govindjee, 2011). The results of the chlorophyll fluorescence analysis obtained in this study, expressed as the four-phase OJIP induction curve, are presented in Fig. 4. The results show that the duration of dry-cold treatment elicited differential effects on OJIP induction in the control group. Notably, a 15-day dry-cold treatment tended to increase both the initial fluorescence (Fo) and the maximum fluorescence (Fm). This pattern suggests that dormancy induction and metabolic downregulation triggered by short-term dry-cold exposure (15 days) imposed indirect physiological stress that had a prolonged effect on seedlings after germination. Meanwhile, in the pericarp-removed group, the dry-cold treatment appeared to have only a relatively limited effect on changes in the chlorophyll fluorescence induction curves (OJIP transients) themselves.
The chlorophyll fluorescence parameters derived from the OJIP transients indicated that the initial fluorescence (Fo) reached its highest value under the 15-day dry-cold treatment in the control group, with a maximum of 6630, which is commonly interpreted as an early indicator of physiological stress in plants (Table 5). In contrast to Fo, no statistically significant differences were detected among treatments in the maximum fluorescence (Fm) or variable fluorescence (Fv). The relative variable fluorescence at the J-step (Vj) and I-step (Vi) exhibited patterns similar to those of Fo, with the highest values (0.430 and 0.793, respectively) also recorded in the control group subjected to the 15-day dry-cold treatment.
Previous reports have shown that the maximum quantum efficiency of PSII (Fv/Fm) in non-stressed higher plants typically ranges from 0.78 to 0.84 (Ahn et al., 2024; Asadi-Sanam et al., 2015; Lee et al., 2024, 2025b; Muniz et al., 2014). In this study, Fv/Fm ranged from 0.753 to 0.785 across all treatments, indicating values slightly below the generally accepted optimal range. Meanwhile, the parameter Mo, which reflects the initial slope of the OJIP fluorescence transient, exhibited a trend similar to that of Fo, Vj, and Vi, with the highest value (0.651) recorded in the control group subjected to the 15-day dry-cold treatment. Given that both germination and growth indices were relatively low under this treatment, the elevated Mo value further supports previous reports indicating that this parameter is closely linked to stress responses elicited by environmental factors or treatment conditions (Kim et al., 2024b; Liu et al., 2024).
Analysis of chlorophyll fluorescence parameters related to quantum yields and specific energy fluxes revealed distinct responses depending on the factors examined (Fig. 5 and Table 6). The parameter representing maximum quantum efficiency of PSII photochemistry (ΦPo) was significantly affected by the duration of dry-cold treatment (p < .05). In the control group, ΦPo reached its highest value under the 30-day dry-cold treatment (0.785), whereas in the pericarp-removed group, the highest value was observed under the 60-day treatment (0.784). The probability that trapped excitons transfer electrons beyond QA to the electron transport chain (Ψo), as well as the quantum yield of electron transport (ΦEo), differed significantly depending on whether the pericarp was removed (p < .001). In contrast, the duration of dry-cold treatment had no significant effect on either parameter. The quantum yield representing the probability that absorbed photons are dissipated as heat, fluorescence, or via non-photochemical pathways (ΦDo) was highest under the 60-day dry-cold treatment in the control group (0.246) and under the 15-day treatment in the pericarp-removed group (0.229). Overall, an appropriate duration of dry-cold treatment for fruits and seeds appears to enhance the physiological status of seedlings during post-germination development, whereas short-term dry-cold treatment (15 days) may induce prolonged physiological stress after germination.
The parameters representing absorbed energy per reaction center (ABS/RC), trapped energy per reaction center (TRo/RC), and electron transport flux from QA to QB per reaction center (ETo/RC) were significantly affected by the duration of dry-cold treatment (p < .01). The ABS/RC values were highest under the 15-day dry-cold treatment in both the control (1.95) and pericarp-removed (1.76) groups, suggesting a higher proportion of inactive or damaged reaction centers under this condition. The pattern observed for TRo/RC was similar to that of ABS/RC. In contrast, ETo/RC reached its highest value under the 60-day dry-cold treatment in the control group (0.878), whereas the highest value in the pericarp-removed group (0.914) occurred under the 15-day treatment, indicating that photochemical electron transport capacity was partially maintained. Meanwhile, the parameter indicating the relative increase in energy dissipation (DIo/RC) showed the highest value under the 60-day dry-cold treatment in the control group (0.478), whereas the highest value (0.406) in the pericarp-removed group was observed under the 15-day treatment.
Taken together, these results suggest that the duration of dry-cold treatment applied to fruits or seeds exerts a long-term effect on PSII energy fluxes in seedlings after germination. Under certain conditions, the proportion of trapped energy dissipated as fluorescence or heat, rather than being efficiently utilized for photochemical processes, appears to increase.
Among chlorophyll fluorescence parameters, PIABS serves as a comprehensive index that integrates photochemical efficiency, energy fluxes, and electron transport processes. It is considered a more sensitive tool than Fv/Fm for detecting stress responses and assessing changes in plant vitality (Dai et al., 2019; Lee et al., 2025b; Srivastava et al., 1999; Živčák et al., 2008). The results showed no significant differences in PIABS across the different durations of dry-cold treatment within the control group (Fig. 6). In contrast, the pericarp-removed group exhibited PIABS values ranging from 4.04 to 4.98, with a significant decline to 4.04 under the 15-day dry-cold treatment compared with the other treatment durations. This suggests that short-term dry-cold treatment (15 days) exerts long-term negative effects on the photochemical performance of post-germination seedlings originating from seeds that underwent pericarp removal. These results are consistent with previous studies reporting statistically significant reductions in PIABS in treatment groups exhibiting lower growth and biomass indices due to adverse treatment conditions or unfavorable growth environments (Kim et al., 2024a, 2024b; Lee et al., 2025c; Shin et al., 2023).
Correlation and Cluster Analysis
Pearson correlation coefficient analysis revealed strong positive correlations among several major indices (Fig. 7). In total, 15 primary parameters and indices were evaluated. The analysis showed that compactness and DQI exhibited a notably high positive correlation (r = 0.94), indicating that these two indices capture similar characteristics in L. muscari. Shoot dry weight and root dry weight also showed a strong positive correlation (r = 0.86), suggesting coordinated accumulation of shoot and root biomass. Among the morphological indices, shoot height and shoot width also demonstrated a positive association (r = 0.74), implying that size-related traits are closely integrated within the same cluster. Meanwhile, root dry weight and germination percentage were positively correlated (r = 0.70), indicating a direct relationship between germination percentage and subsequent root development in L. muscari.
Application of Euclidean distance and average linkage hierarchical clustering resulted in the segregation of indices into several distinct clusters. Compactness and DQI clustered together owing to their strong correlation, indicating that both indices convey comparable information as plant quality indices integrating morphological and biomass attributes. Shoot dry weight and root dry weight formed an independent subgroup, demonstrating the close association between dry weight indices. shoot height and shoot width clustered together, consistently characterizing traits associated with shoot size. In addition, Fv/Fm clustered with PIABS, while SPAD units grouped with NDVI. Consistent with the findings of Gallegos-Cedillo et al. (2021), this study confirms that morphological and physiological indices independently contribute to the assessment of seedling quality. Their study treated indicator groups separately, which aligns with the distinct separations observed among indicator categories in this study. Furthermore, Dumschott et al. (2022) identified a distinct separation between photosynthesis-related physiological indices and morphological indicators and emphasized the complementary roles of these two indicator groups. Similarly, this study also revealed that physiological indices, such as SPAD units, Fv/Fm, PIABS, and NDVI, did not cluster with morphological traits, but were positioned independently, illustrating the differentiation of photosynthetic efficiency and physiological vitality from structural size and biomass indices. This separation among indicator groups is consistent with the multivariate analyses conducted by Mohi-Ud-Din et al. (2021) and with the principal component and cluster analyses reported by Abrar et al. (2024), all of which demonstrate the independent classification of different indicator categories.
These findings indicate that morphological and biomass indices, as well as physiological indicators, exhibit complementary yet independent characteristics. SPAD units, Fv/Fm, PIABS, and NDVI formed clusters distinct from morphological and biomass indices, suggesting that these physiological indices provide complementary assessments of physiological vigor and photosynthetic performance that cannot be captured by morphological or biomass indices. Therefore, a comprehensive evaluation of L. muscari seedling quality in practical horticultural settings should incorporate concurrent assessments of morphological, biomass, and physiological indices.
In summary, the combined application of pericarp removal and an appropriate duration of dry-cold treatment (45–60 days) comprehensively improved L. muscari seed germination, early seedling growth, photosynthetic efficiency, and overall physiological vigor. These pre-sowing treatments are essential for ensuring stable seedling establishment and post-germination productivity, thereby offering substantial practical value for seed-propagated horticultural production.
Conclusion
This study examined the effects of pericarp removal and the duration of dry-cold stratification on seed germination, seedling growth, and physiological traits of Liriope muscari (Decne.) L.H. Bailey. Overall, pericarp removal combined with approximately 45 days of dry-cold stratification significantly improved germination performance. In particular, 30–45 days minimized the time to germination, whereas 45–60 days further enhanced seedling vigor and morphophysiological stability. However, biomass components and remote sensing vegetation indices such as NDVI and PRI tended to reach their highest values in pericarp-removed seeds without dry-cold stratification (0–day treatment). In contrast, in the control group subjected to short-term dry-cold stratification for 15 days, relative declines in germination and early seedling growth indices were observed. Similar negative trends were detected in physiological indicators, including chlorophyll content (SPAD units) and vegetation indices such as NDVI, PRI, and MCARI. Hierarchical cluster analysis revealed that plant quality indices, vegetation indices, and chlorophyll fluorescence parameters represent distinct dimensions of seedling performance. Morphological and biomass indices reflected structural stability and balance in L. muscari seedlings, whereas vegetation indices and chlorophyll fluorescence parameters were more sensitive indicators of physiological status. From a practical perspective, pericarp removal followed by dry-cold stratification at 3°C for approximately 45–60 days appears to promote stable germination and early seedling vigor. The PIABS results further corroborate the association between this pretreatment regime and enhanced physiological performance. Thus, this combined pretreatment can be considered a promising standard protocol for the commercial propagation and large-scale cultivation of L. muscari. In summary, the concurrent application of pericarp removal and an appropriate period of dry-cold stratification can be viewed as a strategy that not only improves germination performance but also reinforces the morphological and physiological integrity of early seedlings. Furthermore, the complementary integration of vegetation indices and chlorophyll fluorescence parameters is expected to enhance the reliability of pretreatment evaluations and strengthen their applicability under field conditions. Future research should refine the seed pretreatment framework for L. muscari and related species by empirically evaluating combinations of temperature and stratification duration, comparing moisture conditions (dry vs. wet), assessing ABA-GA dynamics and associated gene expression, and examining varietal differences in response.
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