L. insularis seeds were collected from habitats in Ulleung-gun, Gyeongsangbuk-do, South Korea in September 2021. At the time of collection, the temperature in the region ranged from 15.8 – 29.1°C, and only seeds that were sufficiently mature were harvested. These seeds were provided from the National Institute of Biological Resources and only those that were sufficiently mature at the time of collection were harvested. The seeds collected immediately after harvest were dried in the shade, subsequently selected, and sealed in a plastic bag with silica gel, then stored at stored in the dark at 4°C until used in the experiment.

The basic characteristics of the seeds, including 100-seed weight, length and width, E:S ratio, and viability, were assessed. The 100-seed weight was determined by weighing 100 seeds in four replicates using a microbalance (AS200; Radwag, Radom, Poland). The length and width of 10 seeds were measured using a stereomicroscope (SZ61; OLYMPUS, Tokyo, Japan) equipped with a complementary metal-oxide-semiconductor (CMOS) camera (eXcope F-630; Dixi Sci., Daejeon, Korea). To assess the presence of MD, 10 seeds were cross-sectioned using a double-edged razor, and the E:S ratio was measured using a stereomicroscope, with the average value recorded. Seed viability was assessed by soaking 50 seeds in distilled water at 20°C in the dark for 24 h, followed by immersion in 1% tetrazolium solution (Triphenyl tetrazolium chloride, St. Louis, MO, USA) in the dark at 30°C for 24 h. Subsequently, the seeds were cut in half, with those stained red classified as viable and those unstained classified as dead, and the results were expressed as a percentage.

To determine the presence of PY based on the initial moisture content and water absorption, a water absorption test was conducted. The weight of 100 seeds was measured in four replicates and subsequently soaked in distilled water at 4°C in the dark. The surface moisture of the seeds was removed using paper towels every 24 h, and the seeds were weighed. This process was repeated for seven days, with the seeds re-immersed in distilled water after each measurement. Following the final measurement, the seeds were placed in mesh bags and dried in an oven at 70°C for 48 h to determine the initial moisture content based on the dry weight.

To determine the temperatures required for embryo growth, seeds were sown at various temperatures and observed. The seeds were surface-sterilized by soaking them in a 1.4% sodium hypochlorite solution for 10 min and subsequently rinsing them with distilled water. The sterilized seeds were placed on two layers of filter paper in Petri-dishes (10010; SPL Life Science Co. Ltd, Korea), with 4 mL of distilled water, and 50 seeds were sown in six replicates. Embryo growth was observed at constant temperatures of 4, 15, 20, 25, and 30°C [30 µmol·m^-2·s^-1 photosynthetic photon flux density (PPFD), white-light emitting diode (LED)] under light conditions, and at alternating temperatures of 15/6 (14/10 h), 20/10 (14/10 h), and 25/15°C (16/8 h) (30 µmol·m^-2·s^-1 PPFD, white-LED). The E:S ratio was measured daily at all temperatures by cutting cross sections with a razor blade, and embryo growth was observed for 30 days.

To observe germination characteristics and determine the presence of PD, an initial germination test was conducted. The germination test involved sowing 25 seeds in four replicates at constant temperatures of 4, 15, 20, 25, and 30°C under light or dark conditions and at alternating temperatures of 15/6, 20/10, and 25/15°C. Germination was observed daily for 30 days under light or light/dark cycle conditions, and the final germination (FG, %) was calculated for seeds sown under dark conditions. Additionally, seeds that did not germinate in any of the experiments were tested for viability using 1% tetrazolium solution to distinguish between viable and non-viable seeds.

To break dormancy and expand the germination temperature range, GAs treatment was conducted. GA₃ (Cas No. 77-06-5; Sigma-Aldrich, St. Louis, MO, USA) and GA_{4 + 7} (2:1, Cat No. 468-44-0/510-75-8; Kisan Bio, Seoul, Korea) were prepared at concentrations of 0, 100, 200, and 500 mg·L^-1. The GAs were initially dissolved in a small amount of 100% ethanol and distilled water was subsequently added to achieve the desired concentrations. L. insularis seeds were immersed in each GAs solution and treated for 24 h at 4°C in the dark. After GAs treatment, the seeds were sown in four replicates of 25 seeds each at constant temperatures of 4, 15, 20, 25, and 30°C under light conditions and at alternating temperatures of 15/6, 20/10, and 25/15°C under light/dark cycle conditions. Germination was observed daily for 30 days to determine the effects of GAs.

Stratification was conducted to determine whether it can expand the germination temperature range of L. insularis seeds, whose embryos were grown through stratification. The experimental seeds were placed on two layers of filter paper in Petri-dishes, containing 4 mL of distilled water. Twenty-five seeds were sown in four replicates and subsequently stratifed at 15°C under light conditions for 0, 1, or 2 weeks. Distilled water was added regularly to prevent drying during the stratification period. After stratification, the seeds were transferred to constant temperatures of 4, 15, 20, 25, and 30°C under light conditions, and at alternating temperatures of 15/6, 20/10, and 25/15°C under light/dark cycle conditions. Germination was observed daily for 30 days, and the FG (%), mean germination time (MGT, days), and time for 50% germination (T₅₀, days) were calculated. The germination indices were calculated using the following formula:

Where n i is the number of seeds germinated at time t i .

Where t b 50 is the time just before 50% germination, N is the total number of germinated seeds, G a 50 is the cumulative number of seeds germinated just after 50%, G b 50 is the cumulative number of seeds germinated just before 50%, and t a 50 is the time just after 50% germination.

DR priming was conducted to apply priming to dormant seeds and confirm the commercial potential of seeds with expanded germination temperature range. DR priming involved stratifying L. insularis seeds at 15°C under light conditions for 1 and 2 weeks, followed by drying at room temperature for 48 h to restore their initial moisture content. Subsequently, untreated seeds (control), seeds stratified at 15°C under light conditions for 1 week (1W) or 2 weeks (2W), and seeds that completed DR priming for 1 week (1DR) or 2 weeks (2DR) were sown under light/dark cycle conditions at 20/10 and 25/15°C. Germination was observed daily for 30 days, and the FG, MGT, and T₅₀ were calculated.

To confirm seedling emergence in the soil, 25 seeds each from the control, 1DR, and 2DR priming seeds groups were sown in four replicates in transparent plastic boxes filled with horticultural soil (Hs, Hanareum No.2; Shinsung Mineral Co., Ltd., Seongnam, Korea). The seeds were sown under light/dark cycle conditions at 20/10 (14/10 h) and 25/15°C (16/8 h). Seedling emergence was calculated at 4 and 8 weeks.

The weight of 100 seeds, size of seeds, E:S ratio, and germination experiments (FG, MGT, and T₅₀), and seedling emergence are expressed as the mean and standard error (SE). Significance was assessed using analysis of variance to test the null hypothesis, followed by Duncan’s Multiple Range Test to compare the means, with a significance level of p< 0.05. Analyses were performed using the Statistical Analysis Software (SAS, Version 9.4; SAS Institute Inc., Cary, NC, USA).

The weight of 100 L. insularis seeds measured 0.44 ± 0.01 g, with a seed length of 3.87 ± 0.07 mm and a seed width of 2.64 ± 0.05 mm ( Table 1 , Figure 1 ). Seeds exhibited an E:S ratio of 0.24 ± 0.01, indicating an underdeveloped embryo, whereas the E:S ratio of seeds immediately before germination was 0.81 ± 0.05. A significant difference was observed between the seeds and seeds immediately before germination. The seed viability test using a 1% tetrazolium solution indicated 84.0% viability. The initial moisture content of L. insularis seeds was 9.83 ± 0.07%, reaching a maximum moisture content of 43.9% on the first day of water absorption ( Figure 2 ).

Embryo growth was monitored over 30 days at various temperatures (15, 20, 20/10, and 25/15°C), whereas no growth was observed at other temperatures ( Figure 3A ). After 30 days, the E:S ratio ranged from 0.39 – 0.62 at 20, 20/10, and 25/15°C. At 15°C, the E:S ratio reached 0.81 after 18 days, indicating complete embryo growth. Initial germination test results indicated germination ranging from 0.0 – 85.0% under light and light/dark cycle conditions, whereas no germination was observed under dark conditions ( Figure 3B ). At 20, 20/10, and 25/15°C, germination was 52.0, 38.0, and 2.0%, respectively, which did not reach the viability level (84.0%). The FG at 15°C was 85.0%, which is significantly higher than that at all other temperatures. Therefore, 15°C under light conditions was identified as optimal for embryo growth and germination and was used in subsequent stratification experiments.

Treatment of L. insularis seeds with 0, 100, 200, and 500 mg·L^-1 of GA₃ and GA_{4 + 7} for 24 h did not significantly affect dormancy breaking and germination temperature range ( Figure 4 ). Germination was observed only at 15, 20, and 20/10°C, with FG ranging from 46.0 – 55.0% at 20°C and 35.0 – 42.0% at 20/10°C; however, no germination was observed at other temperatures. In contrast, FG was the highest at 15°C, ranging from 78.0 – 86.0%, but no significant differences were observed among the concentrations at any temperature.

L. insularis seeds began embryo growth at 7 days at 15°C, completing the growth by 18 days with an E:S ratio of 0.81; however, germination started at 15 days ( Figure 3 ). Therefore, stratification was conducted for 14 days, from the onset of embryo growth (7 days) until immediately before germination. Stratification of L. insularis seeds at 15°C for 0, 1, and 2 weeks resulted in different patterns across various temperatures, based on the stratification period ( Figures 5 , 6 ). A longer stratification period at 15°C significantly increased FG and reduced MGT and T₅₀. Seeds with 0 week of stratification at 15°C exhibited germination ranging from 2.0 – 85.0% at certain temperatures, including 15, 20, 20/10, and 25/15°C ( Figures 5A , 6A ). In contrast, seeds stratified for 1 week at 15°C exhibited germination ranging from 12.0 – 86.0% at all temperatures, except at 4°C ( Figures 5B , 6A ). Stratification for 2 weeks at 15°C significantly expanded the germination temperature range, with germination ranging from 24.0 – 82.0% at all temperatures, except at 4°C and significantly increased the FG at 20, 25, 30, 15/6, 20/10, and 25/15°C ( Figures 5C , 6A ). Additionally, seeds stratified for 2 weeks at 15°C significantly facilitated the germination rates regarding MGT and T₅₀ ( Figures 6B, C ).

Significant differences in FG, MGT, and T₅₀ were observed among the control, 1WS, 2WS, 1DR, and 2DR treatments at 20/10 and 25/15°C ( Figures 7 , 8 ). FG ranged from 2.0 – 44.0 and 38.0 – 69.0% at 25/15°C, and 20/10°C. At both 20/10 and 25/15°C, all treatments exhibited significantly higher FG than that of the control. Additionally, longer stratification periods enhanced FG, MGT, and T₅₀ in all the treatments ( Figure 8 ). FG for 1WS and 1DR at 20/10°C was 54.0 and 51.0%, respectively, with no significant difference in FG. In contrast, the MGT and T₅₀ values for 1WS were 20.6 and 19.2, respectively, whereas those for 1DR were 22.3 and 22.4, respectively, indicating delayed germination rates for 1DR. At 25/15°C, the FG values for 1WS and 1DR were 32.0 and 27.0, respectively, indicating no significant difference. However, the MGT for 1WS and 1DR was 23.15 and 27.4, respectively, and the T₅₀ was 22.8 and 27.3, respectively, indicating a significantly delayed germination rate for 1DR. Similarly, no significant difference was observed in FG between 2WS and 2DR at 20/10 and 25/15°C, but 2DR exhibited delayed germination rates regarding MGT and T₅₀.

At 20/10°C, seedling emergence was observed in the control, 1DR, and 2DR treatments. At 4 weeks, seedling emergence in the control, 1DR, and 2DR was 0, 44.0, and 54.7%, respectively, with significantly higher emergence observed in 2DR ( Figure 9A ). By 8 weeks, the emergence in the control, 1DR, and 2DR was 52.0, 57.3, and 76.0%, respectively, with significantly higher emergence in 2DR ( Figure 9B ).

An underdeveloped embryo is characterized by a relatively small embryo size and an E:S ratio of< 0.5, and is classified as MD (Baskin and Baskin, 2014). Seeds with MD have embryos that are either undifferentiated or underdeveloped at harvest, necessitating time for further development before germination (Rhie et al., 2016). The seeds of L. insularis exhibited an E:S ratio of 0.24, indicating an underdeveloped embryo, thereby confirming the presence of MD. These underdeveloped embryos of L. insularis seeds were able to achieve an E:S ratio of 0.81 through incubation at 15°C under light conditions. Seed water absorption is the result of long-term adaptation to natural habitats and represents the initial stage of seed germination (Yang et al., 2023). Seeds with PY, such as Vicia villosa (Renzi et al., 2016), Senna multijuga (Rodrigues-Junior et al., 2018), Delonix regia (Jaganathan et al., 2017), and Melilotus albus (Zhang et al., 2020), often encounter hindrance in water uptake owing to their impermeable seed coat that negatively affects seed germination. PY is caused by the impermeability of palisade cells in the seed or fruit coats (Tang et al., 2022). Seeds are considered permeable if their weight increases by over 20% upon water absorption (Baskin and Baskin, 2004). Because L. insularis seeds reached a maximum moisture content of 49.9% on the first day of water absorption, the characteristics of PY owing to impermeability were not observed.

Seeds with PD do not germinate within 4 weeks, even after water absorption (Baskin and Baskin, 2022). Approximately 70% of the seed plants produce dormant seeds, and PD is the most common class of dormancy (Soltani et al., 2017). However, L. insularis seeds exhibited 85.0% germination within 30 days at 15°C, indicating the absence of PD characteristics. In general, seeds with only MD complete embryo growth in a relatively short period and germinate within 4 weeks (Baskin et al., 2008). Additionally, seeds with MD require appropriate temperatures for embryo growth and germination (Soltani et al., 2019). According to previous studies, L. insularis seeds have underdeveloped embryos and were classified as MD, with 94.4% germination at 15°C in 4 weeks (Park et al., 2019). In this study, the L. insularis seeds also demonstrated embryo growth at 15°C, with germination observed from day 15, indicating the absence of PD. Therefore, the seeds of L. insularis were classified as exhibiting only MD, with underdeveloped embryos and germination within 4 weeks at 15°C.

In this study, L. insularis seeds germinated only under light conditions. Although the seeds of numerous species successfully germinate in both light and darkness, certain species may germinate only under light or dark conditions (Baskin and Baskin, 2014). Light serves as a crucial environmental signal that controls diverse biological processes, such as seed dormancy and germination, photomorphogenesis, phototropism, shade avoidance, and flowering (Yang et al., 2020). Additionally, light is an important environmental factor that helps seeds determine whether the surrounding environment is suitable for germination and subsequent seedling emergence (Yan and Chen, 2020). Therefore, the role of light in regulating the germination of L. insularis seeds highlights the species’ potential reliance on light as a cue for optimal germination conditions.

Dormant seeds germinate within a narrower temperature range than seeds with alleviated dormancy (Baskin and Baskin, 2014). Additionally, dormant seeds germinate only within an appropriate temperature range (Geneve, 2003). Therefore, further research is required to expand the germination temperature range of dormant seeds for their utilization. The germination temperature range of Ribes multiflorum seeds was expanded through GA₃ treatment (Mattana et al., 2012). However, GA treatment did not significantly affect the germination of L. insularis seeds. This may be because of the presence of high levels of abscisic acid (ABA), which maintains dormancy (Finch-Savage and Leubner-Metzger, 2006). Cold or warm stratification breaks dormancy and induces germination through the interaction between various hormones, including ABA and GA (Leon et al., 2007). L. insularis seeds germinated only at specific temperatures of 15, 20, 20/10, and 25/10°C. The germination temperature range was enhanced for L. insularis seeds that had undergone embryo growth through stratification at 15°C for 0, 1, and 2 weeks because germination was observed at all temperatures, except at 4°C. Additionally, once embryo growth was completed through 2 weeks of stratification at 15°C, germination was effectively enhanced at previously non-germinating temperatures. This indicated that stratification at 15°C alleviated dormancy through the interaction of various hormones, thereby expanding the germination temperature range. In Pinus koraiensis seeds, cold stratification effectively expanded the germination temperature range (Song et al., 2023). Moreover, determining the optimal temperature and duration for stratification may help identify methods that can alleviate dormancy in historically challenging species, which can be crucial for restoration efforts (Just et al., 2023). Germination temperature range significantly affects the ecological environment of a species, and species that can germinate over a broader temperature range may have wider geographical distributions (Donohue et al., 2010). Research aimed at expanding the germination temperature range offers an alternative approach to nursery cultivation, thereby addressing climate crises for species conservation and propagation (Song et al., 2023). Therefore, in this study, L. insularis seeds effectively expanded their germination temperature range through 2 weeks of stratification at 15°C, suggesting this as a dormancy-breaking method for propagation, conservation, and utilization of the species.

Priming promotes seed germination metabolism, enhances germination, resistance to environmental stress, and dormancy breaking, and enhances seedling production capacity (Malek et al., 2022). Additionally, priming is an effective technique that breaks seed dormancy and enables immediate sowing (Bourioug et al., 2020). Moreover, primed seeds exhibit synchronous and fapid emergence, reduced dormancy, a broader wider range of germination temperatures, and enhanced competitiveness against weeds and pathogens (Paparella et al., 2015). However, despite various studies on enhancing priming, novel ideas are still lacking. Seeds with MD have underdeveloped embryos and require time for the embryos to grow to a critical size before germination (Soltani et al., 2018). Therefore, to apply priming to seeds with MD, the embryo should be grown and subsequently re-dried to complete the priming process. We confirmed that embryo growth occurred at 15°C, and 2 weeks stratification effectively expanded the germination temperature range, which was subsequently used for DR priming. The 2WS and 2DR treatments on L. insularis did not demonstrate significant differences in FG at 20/10 and 25/15°C. This indicates that the DR-priming technique, which expands the germination temperature range, may be commercially useful for L. insularis seeds.

In this study. the 2DR treatment applied to L. insularis seeds with MD demonstrated a slight delay in the germination rate compared to 2WS. For successful seed priming, water absorption should be halted before radicle protrusion; however, re-dried seeds can lose vigor (Pagano et al., 2023). Damage from priming has also been observed in Digitalis purpurea seeds, where redrying caused cellular damage, seed aging, and reduced vigor (Butler et al., 2009; Waterworth et al., 2024). Priming activates primary cellular processes before germination, such as the de novo synthesis of nucleic acids and proteins, ATP production, and DNA repair (Paparella et al., 2015). Seed repair response plays a crucial role in maintaining vigor, and DNA repair functions should be maintained at appropriate levels in the embryo to sustain seed vitality (Rajjou and Debeaujon 2008; Oge et al., 2008; Paparella et al., 2015). When DNA damage is effectively repaired, embryo cells resume the cell cycle progression and replicate DNA, but defective repair mechanisms, result in oxidative injury that causes cell death (Waterworth et al., 2010; Kranner et al., 2010; Balestrazzi et al., 2011; Ventura et al., 2012). Therefore, the delayed germination rate of DR-primed L. insularis seeds may be because of damage from redrying, and further research is required to address this issue, including identifying the specific causes of delayed germination resulting frome the priming process. Seed invigoration techniques, such as priming, shortens the seedling emergence time and achieves uniform seedling emergence, offering beneficial effects for numerous horticultural and agricultural crops (Jisha et al., 2013). In this study, 2DR-treated L. insularis seeds exhibited significantly higher seedling emergence rates at 4 and 8 weeks at 20/10°C. This indicates that DR-primed seeds with an expanded germination temperature range can be commercially useful. Therefore, research on seed propagation to secure seeds with dormancy for ornamental and medicinal purposes should continue, and the novel approach of DR priming technology can be applied to resource plant seeds with dormancy similar to L. insularis seeds.