Introduction

The family Lauraceae comprises approximately 55 genera and 2,200–2,500 species, with the highest diversity concentrated in tropical and subtropical regions (Renner 2011). Most members are of tropical origin and have adapted to warm and humid environments, a legacy reflected in their seed biology (Farnsworth 2000). In the warm-temperate forests of East Asia, Camphora officinarum Nees and Neolitsea sericea (Blume) Koidz. are representative evergreen broad-leaved tree species distributed along the southern coast of Korea, including Jeju Island and Ulleungdo (Iwatsuki et al. 2001;Lee et al. 2010;NIBR 2017; Wu et al. 2008). These species play essential ecological roles in maintaining forest structure, regulating microclimates, storing carbon, and supporting biodiversity. However, habitat fragmentation, urbanization, and climate change have led to population declines and genetic erosion (Hou et al. 2025;Lee et al. 2013;Zhong et al. 2019). In Korea, a study analyzed the fine-scale spatial genetic structure of a Neolitsea sericea population, informing dispersal and genetic patterns (Chung et al. 2000). A detailed understanding of their reproductive biology and seed physiology is therefore a prerequisite for developing effective ex situ conservation strategies (Jaganathan et al. 2019).

Seeds of Lauraceae exhibit distinctive structural and physiological characteristics that determine their storage behavior, being typically recalcitrant with high moisture content and large cotyledons surrounding a fully developed embryo (Berjak and Pammenter 2008;Bewley et al. 2013;Hong and Ellis 1996;Chin et al. 1988). The fruits of C. officinarum and N. sericea are drupaceous, containing a single exalbuminous seed enclosed by a lignified endocarp (Chin et al. 1988;Lin and Chien 1999). This moisture-retaining structure, combined with metabolically active embryos, maintains high internal moisture and metabolic activity, reinforcing desiccation sensitivity rather than tolerance (Pammenter and Berjak, 2014;Walters et al., 2005). Consequently, C. camphora and Neolitsea spp. show rapid viability loss during drying and under low-temperature storage (Chen et al. 2007;Lin and Chien 1999;Walters et al. 2005).

The germination physiology of Lauraceae also reflects adaptation to moist environments. Most species lack morphological dormancy (MD) because their embryos are fully developed at dispersal (Fenner and Thompson 2005). Physiological dormancy (PD) is therefore the predominant type, arising from endogenous inhibitors or hormonal imbalance, and can be alleviated by cold stratification or gibberellic acid (GA₃) treatment (Baskin and Baskin 2014). However, poor germination in many Lauraceae species is often misinterpreted as loss of viability rather than dormancy (Jaganathan et al. 2019). Accurate dormancy diagnosis is thus essential for ex situ propagation. Such physiological patterns have been documented for Lindera communis (Lang et al. 2011), N. sericea (Lee et al. 2023), and Laurus nobilis (Güngör et al. 2024), while C. camphora exhibits delayed germination under prolonged dormancy and moisture limitation (Chen et al. 2004b).

Despite their ecological and economic importance, research on the seed-storage physiology of Korean native Lauraceae remains limited. Recent studies indicate that N. sericea seeds are highly desiccation-sensitive and rapidly lose germination capacity under drying or after-ripening conditions (Lee et al. 2023). However, comprehensive evaluation of desiccation sensitivity in C. officinarum and related taxa remains poorly characterized. The probabilistic model of Daws et al. (2006), which predicts desiccation sensitivity from seed-coat ratio (SCR) and seed dry mass, offers a practical quantitative framework for evaluating storage behavior without prolonged storage trials. Understanding the desiccation sensitivity and dormancy physiology of C. officinarum and N. sericea is therefore critical for conserving warm-temperate Lauraceae species and developing ex situ seed-bank and cryostorage strategies under changing climatic conditions.

Materials and Methods

Seed collection and preparation

Mature fruits of Camphora officinarum Nees and Neolitsea sericea (Blume) Koidz were collected from natural populations in Jeju-si and Seogwipo-si, Korea, during October to November 2022. Immediately after collection, the fleshy pericarp (exocarp + mesocarp) was removed. Only morphologically intact seed units, consisting of the seed enclosed by the lignified endocarp and containing a fully matured embryo were used for experiments. Seeds were surface-sterilized by immersion in 1% sodium hypochlorite (NaOCl) solution for 10 min, followed by three rinses with sterile distilled water for 30 minutes each. After surface-sterilization, the endocarp-intact seeds were stored at 4℃ until use, and this endocarp-intact form was used for all subsequent assays.

Previous studies report that Lauraceae seeds commonly possess PD and that cold stratification increases final germination or shortens mean germination time (MGT) (Chen et al. 2006;Chen et al. 2004b;Lee et al. 2023;Smith et al. 2002). Baskin and Baskin (2014) further noted that most Lauraceae exhibit PD. Accordingly, all seeds in this study were cold-stratified for 12 weeks at 4℃ and 40% relative humidity (RH) in the short-term storage of the Korea National Arboretum (KNA) Seed Bank prior to sowing.

Seed morphology and water absorption

To examine the internal structure of seeds, a 3D digital microscope (KH-8700, Hirox, Japan) was used. For imaging, the stone and seed coat were carefully opened to expose the embryo. Both species are exalbuminous; no endosperm tissue is present. The seed coat and endocarp were carefully dissected to expose the embryo.

Moisture content determination

Seed moisture content (MC) was determined following the guidelines of the International Seed Testing Association (ISTA 2023) using the oven-drying method. For each species, four replicates of ten seeds were randomly selected. Fruits were depulped, and seeds were surface-dried with filter paper and held in a short-term storage vault (4℃) for approximately 24 hours to stabilize moisture before the initial fresh-mass measurement. Seeds were then dried at 103 ± 2℃ for 17 hours in a forced-air oven, cooled in a desiccator for 30 minutes, and weighed immediately after removal. MC was expressed as the percentage of fresh weight lost during drying following ISTA (2023) guidelines:

where W_f is fresh weight and W_d is dry weight.

Prediction of desiccation sensitivity

Desiccation sensitivity was predicted using the logistic model of Daws et al. (2006), adapted from the KEW Technical Information Sheet 10 (TIS-10). The model estimates the probability of desiccation sensitivity (P) from seed-coat ratio (SCR) and seed dry mass (g). The lignified endocarp was cracked to isolate endocarp-free seeds for analysis. For this prediction analysis only, 10 seeds per species were dissected into two components: the outer complex (lignified endocarp firmly adherent to the testa) and the inner tissue (embryonic axis and cotyledons). These components were dried to constant mass and weighed. SCR was defined as:

The following logistic equation was used to estimate P:

where a is SCR and b is seed dry mass (g). All input masses were within the model’s validated range (0.01– 10 g).

Imbibition test

To assess seed-coat permeability, 20 seeds per species (four replicates) were immersed in sterile distilled water. Fresh mass was recorded at 6, 12, 24, 48, and 72 hours; imbibition was expressed as:

where W₀ is initial weight and W_t is weight at time t.

To visualize pericarp and endocarp permeability, seeds with pericarps intact were immersed in 0.1% (w/v) methylene blue solution for 1, 2, 3, 4, 24, 72, and 168 hours, following the method of Mahadevan and Jayasuriya (2013). After each imbibition period, seeds were sectioned longitudinally and examined under a 3D digital microscope (KH-8700, Hirox, Japan) for dye penetration through the pericarp and endocarp layers.

Germination test

A germination experiment was conducted to evaluate the effects of temperature and gibberellic acid (GA₃) on seed germination. Two alternating temperature regimes, 25/15℃ and 30/20℃ (14 hours light / 10 hours dark photoperiod), were applied to simulate natural thermal conditions. Four concentrations of GA₃ (Sigma-Aldrich, St. Louis, MO, USA; 0, 100, 500, and 1000 ppm) were used as hormonal treatments. All seeds were soaked in the respective GA₃ solutions for 24 hours; the 0 ppm control was soaked in distilled water. For each combination of temperature and GA₃ treatment, three replicates of twenty seeds were used. Seeds were placed on 0.8% agar-only medium (MB Cell, Korea) prepared with distilled water in sterile Petri dishes and incubated under controlled environmental conditions. Germination was recorded daily for 60 days, and a seed was considered germinated when the radicle protruded at least 2 mm beyond the seed coat. This experimental design enabled a systematic assessment of the effects of temperature and GA₃ on dormancy release and germination performance. Germination performance was evaluated based on three parameters: final germination percentage (GP), mean germination time (MGT), and time to 50% germination (T₅₀).

Final germination percentage (GP) was calculated as the ratio of germinated seeds to the total number of seeds × 100.

Mean germination time (MGT) was determined following the formula described by Ellis and Roberts (1981):

where nᵢ is the number of seeds germinated on day tᵢ. This parameter represents the average time required for seed germination.

Time to 50% germination (T₅₀) was calculated as the time required to reach 50% of the final cumulative germination, determined by linear interpolation between the days immediately before and after the 50% point, following Coolbear, Francis, and Grierson (1984).

Statistical analysis

The germination parameters (GP, MGT, and T₅₀) were expressed as mean ± standard deviation. Germination percentages were arcsine square-root transformed prior to analysis of variance (ANOVA) to satisfy assumptions of normality and homogeneity of variance. Two-way ANOVA was performed to evaluate the main and interactive effects of temperature and GA₃ concentration, and significant differences among means were determined using Duncan’s multiple range test (p < 0.05) in R version 4.5.1 (R Core Team 2025). However, non-transformed mean values (%) are presented in figures and tables for clarity.

Results and discussion

Seed morphology

The fruits of Camphora officinarum Nees and Neolitsea sericea (Blume) Koidz are drupaceous, consisting of a thin exocarp, a fleshy mesocarp, and a lignified endocarp that encloses a single seed. Although the endocarp is part of the pericarp rather than the true seed coat, it plays an important role in regulating water movement and the timing of germination under natural conditions (Jaganathan et al. 2019). Microscopic observations revealed that both species possessed fully developed embryos at the time of dispersal, indicating the absence of MD. The embryos consisted of a short embryonic axis and two large, fleshy cotyledons that occupied most of the seed volume and dry mass (Fig. 1). The mean thousand-seed weight (TSW) was 182.4 ± 2.4 g in C. officinarum and 369.5 ± 19.7 g in N. sericea, showing that the latter species produces nearly twice heavier seeds. This difference corresponds to the general trend observed in other Lauraceae species with desiccation-sensitive seeds, such as Cinnamomum camphora (L.) J. Presl (the basionym for C. officinarum), Litsea glutinosa, and Persea americana (Berjak and Pammenter 2008;Pritchard et al. 2004;Tweddle et al. 2003), and is consistent with the typical morphological pattern reported for the family.

Both species share the common structural feature of having seeds that lack endosperm and are dominated by large cotyledons, which serve as the primary storage tissue (Baskin & Baskin, 2014). A high cotyledon proportion combined with a short embryonic axis is regarded as an adaptive trait to warm and humid environments, promoting high water dependence and metabolic activity during germination (Berjak and Pammenter 2008;Farnsworth 2000). However, these traits also make the seeds highly susceptible to desiccation, leading to a rapid decline in viability upon moisture loss—an attribute characteristic of recalcitrant seeds. The lignified endocarp surrounding the seed (Fig. 1) can delay water exchange and create a localized microenvironment that slightly postpones germination, although it does not induce physical dormancy (PY) (Jaganathan et al. 2019). Such structural modifications, including the function of the lignified endocarp, work in concert with other seed tissues to influence water relations and dormancy behavior in Lauraceae. Taken together, these structural and anatomical features of C. officinarum and N. sericea indicate the absence of MD but provide a physiological and anatomical basis for their high water dependence, complex dormancy patterns, and pronounced desiccation sensitivity. These characteristics are consistent with the recalcitrant storage behavior commonly reported for Lauraceae species (Baskin and Baskin 2014).

Desiccation sensitivity

The moisture content (MC) of mature seeds was 8.3% for C. officinarum and 11.0% for N. sericea (Table 1). Although these values might appear relatively low for typical tropical recalcitrant species (Berjak and Pammenter 2008), both were classified as desiccation-sensitive according to the probabilistic model proposed by Daws et al. (2006). This logistic model estimates the probability of desiccation sensitivity (P) based on the seed-coat ratio (SCR = seed-coat dry mass / total seed dry mass) and oven-dried seed mass (g). The SCR was 0.357 in C. officinarum and 0.377 in N. sericea, yielding predicted probabilities (P) of 0.706 and 0.774, respectively. Because P > 0.5 indicates recalcitrant behavior, both species are predicted to be highly sensitive to drying and therefore unsuitable for long-term storage (-18℃) under conventional seed-bank conditions (Walters et al. 2005).

Comparable desiccation-sensitive patterns have been widely reported for the Lauraceae family, including members of the Cinnamomum and Neolitsea genera (Jaganathan et al. 2019;Lin and Chien 1999). For instance, the classification of C. officinarum as ‘orthodox?’ was initially uncertain (Ellis and Roberts 1981;Hong and Ellis 1996). However, subsequent studies indicated that viability declined steeply when seed moisture content was reduced to below 20%, confirming an intermediate or recalcitrant storage behavior (Lin and Chien 1999). Likewise, the observed rapid post-harvest viability loss in N. sericea is consistent with the behavior of other Neolitsea species classified as recalcitrant (Lee et al. 2013).

Structurally, Lauraceae seeds have a thick, lignified endocarp and relatively high seed-coat ratios (SCR), which delay water loss but do not fully prevent the catastrophic cellular damage associated with desiccation (Farnsworth 2000). The desiccation sensitivity is fundamentally physiological: the fleshy cotyledons and metabolically active embryos of the seeds maintain high respiratory activity even under partial dehydration, making tissues highly prone to oxidative injury during drying (Berjak and Pammenter 2008;Pammenter and Berjak 2014). This combination—slowly permeable yet physiologically desiccation-sensitive—is characteristic of non-orthodox seeds (Baskin & Baskin, 2014). Consistent with this, the predicted probabilities of desiccation sensitivity (P ≈ 0.70–0.77) indicate that C. officinarum and N. sericea are non-orthodox (recalcitrant) and are unlikely to maintain viability after drying and storage at -18℃. Accordingly, alternative ex situ strategies, such as cryopreservation of embryonic axes or short-term moist storage, are strongly recommended to ensure germplasm conservation (Pammenter and Berjak 2014).

Germination behavior and dormancy characteristics

To further assess post-desiccation physiological responses, imbibition and germination experiments were conducted. Both C. officinarum and N. sericea exhibited a steady increase in seed mass within 72 hours of immersion, confirming that their seed coats are permeable to water and that PY is absent (Baskin and Baskin 2014). Although the lignified endocarp slightly delayed water uptake, it did not block it, consistent with findings in Cassytha filiformis (Lauraceae) (Jaganathan et al. 2019). Thus, delayed germination in both species is attributed primarily to physiological rather than structural constraints.

A two-way ANOVA revealed that neither temperature nor GA₃ concentration alone had a significant main effect on final germination (p > 0.05). However, their interaction was significant for both species (C. officinarum, p = 0.037; N. sericea, p = 0.025) (Table 2), indicating that seed responsiveness to GA₃ is temperature-dependent (Fig. 2). For C. officinarum, GA₃ treatment generally increased GP and reduced mean germination time (MGT) at 25/15℃. In contrast, at the higher temperature regime (30/20℃), GP declined as GA₃ concentration increased, with the 100 ppm treatment showing results similar to the control while higher concentrations proved detrimental. The crossing lines in the interaction plot illustrate this pattern and agree with previous reports of reduced germination above 30℃, underscoring the need to distinguish dormancy release from thermal inhibition at higher temperatures. This aligns with previous reports that germination is inhibited above 3 0℃, highlighting the need to distinguish between dormancy release and thermal inhibition under specific high-temperature regimes.

N. sericea displayed a distinct pattern. At 25/15℃, the control group showed 0% germination, whereas GA₃ treatment induced germination in a concentrationdependent manner. Conversely, at 30/20℃, the control group showed relatively high GP (mean 57.3%). However, adding GA₃ at this temperature shortened MGT but did not increase GP and in some cases reduced it, indicating that GA₃ at higher temperature did not improve completion of germination.

Overall, MGT was significantly shortened by GA₃ application in both species. After cold stratification at 4℃ for 12 weeks, the observed MGT was shorter than values reported for related Lauraceae species (Chen et al. 2006;Lee et al. 2023). This rapid germination following stratification and GA₃ application, together with fully developed embryos at dispersal, is consistent with non-deep PD (Baskin and Baskin, 2014). While C. officinarum shows PD that can be alleviated by GA₃ under suitable temperatures, N. sericea appears to require additional thermal cues (e.g., stratification or specific temperature fluctuation) for full release. Given the known complexity of dormancy in Lauraceae (Jaganathan 2019), longer-term storage and post-storage germination tests would further clarify desiccation tolerance and dormancy-breaking requirements in these species.