Microbial Inoculation Is Crucial for Endocarp Opening of Panax ginseng Seeds in Warm Indoor Stratification

Abstract

Panax ginseng Meyer is one of the most popular traditional medicinal plants in Korea. Since ginseng seeds are morpho-physiologically dormant and have a very short lifespan, the harvested seeds need outdoor warm and cold stratification for 100 days each. The seeds were covered by a fruit coat (endocarp), which opened during warm stratification. Farmers must, therefore, dehisce (open the endocarp) seeds annually. The conditions for embryo growth, dehiscence percentage, and endocarp hardness were temperature, watering, stratification substances, solution scarification, and microbial inoculation of the seed endocarp. Watering, temperature (17.5 °C), and aeration are crucial for embryo growth as a germination condition. Moreover, microbial-mediated endocarp decomposition is necessary for dehiscence and embryonic development. This study suggests that a combination of embryo growth and microbial-mediated decomposition of the endocarp during warm stratification is a prerequisite for the dehiscence of ginseng seeds, implying physical and morpho-physiological dormancy. Any microbes (fungi, actinomycetes, and bacteria) tested with high or low cellulose-decomposing ability increased the dehiscence percentage by 66% compared to the untreated control. Seeds of three varieties of P. ginseng and one variety of P. quinquefolius were successfully dehisced by fungal inoculation of seeds. This approach opens the door for year-round indoor dehiscence of ginseng seeds without substrates, such as sand.

1. Introduction

Panax ginseng Meyer is one of the most popular traditional medicinal plants in Korea. Despite its seed-propagating behavior as a self-pollinating species, the Korean ginseng germplasm has been maintained in field collections due to its short lifespan and extended cultivation period (three to four years) for fruiting [1]. Considering ginseng seeds are covered by a fruit coat (endocarp), they should be opened for embryo growth and germination. This process is known as dehiscence (i.e., endocarp opening). The dehiscence percentage of un-dehisced ginseng seeds decreased to 38.7% after one year of cold storage, and the seeds did not germinate [2]. Therefore, farmers need freshly harvested seeds to prepare seedlings. The cryopreservation protocol and cryobanking of un-dehisced (with or without attached fruits), dehisced, and cold-acclimated (ready to germinate) ginseng seeds at the after-ripening stage have been established [3,4]. However, genebank staff encountered difficulties in germinating cryopreserved seeds because germination is season-dependent.

The ginseng seeds have immature embryos (0.35–0.4 mm long) at maturity in late summer, germinating in the second spring after dispersal, i.e., 18–21 months in natural conditions [5,6]. After removing the fruits, the seeds were traditionally mixed with river sand (1:3 ratio) and subjected to underground watering 1–3 times a day for approximately 100 days (late July to early November) to shorten the after-ripening period. During the warm stratification at moderate temperature, embryos grow from the embryo-to-endosperm length ratio (EER) of 0.09 to 0.4, and endocarps are opened (dehiscence).

Following the warm stratification, the seeds need cold stratification for another 100 days at 4–5 °C before germination due to their physiological dormancy [7,8]. In practice, seeds are sown in the field, allowing chilling in winter and germination in the following spring [8]. Pre-soaking in gibberellic acid (GA₃) may improve ginseng seeds’ embryo development and dehiscence but increase seed decay [6,9].

Ginseng seeds have morpho-physiological (MPD) based on seed dormancy classification [10,11]. However, ginseng seeds are considered to have no physical dormancy [12], and microbial action-mediated endocarp opening is considered less likely [13]. However, preliminary experiments have raised the possibility that ginseng seeds have mechanical (physical) dormancy due to the endocarp. Kwon et al. [8] argued that ginseng seeds exhibit triple dormancy, that is, morphological, physiological, and physical.

This study aimed to develop artificial dehiscence (by warm stratification) of ginseng seeds using automatic machinery instead of traditional stratification facilities. I examined the effects of temperature, watering, and diverse microbial inoculation on endocarp hardness, embryo growth, and dehiscence percentage. This study suggests that the combination of embryo growth and microbial-mediated decomposition of the endocarp during warm stratification is a prerequisite for ginseng seed dehiscence.

2. Materials and Methods

2.1. Plant Material

Korean ginseng (Panax ginseng C.A. Meyer) landrace ‘Jakyungjong’ was used in this study. The red-purple-colored berries with seeds were harvested randomly from a population of four-year-old plants from late July to early August (Figure 1A). The seeds contained immature embryos at harvest with an embryo-to-endosperm length ratio (EER) of 0.09 (Figure 1B). The harvested fruits were pulped, and the un-dehiscent seeds were soaked in running water for a day. The seeds of no less than 4 mm in diameter were stored at 10 °C before the experiments. For the year-round experiments, un-dehisced seeds were cryopreserved (vapor phase) at a moisture content (MC) of 6% [3]. After cryopreservation, the seeds were moistened with MC 45% before the warm stratification experiments.

2.2. Warm Stratification of Un-Dehisced Seeds

2.2.1. Warm Stratification

The un-dehisced seeds surrounded by endocarp (hereafter “seeds”) were loaded into an onion net (10 × 20 cm²) and placed in a pot (15 × 25 cm²). The seeds were maintained in a ventilated room at 18–20 °C and manually watered daily using tap water for 3–4 months until dehiscence (opening of the endocarp) was observed (Figure 1C), where the EER was around 0.4 (Figure 1D).

During warm stratification, diverse options were tested, including stratification temperatures, watering intervals, stratification substrates, sodium hydroxide solution scarifications, microbial inoculations, and application to seeds of Korean and American ginseng varieties.

2.2.2. Warm Stratification Treatments

• Warm Stratification Temperature

Un-dehisced seeds located within the onion net were stratified in incubators controlled at 10 °C, 15 °C, 17.5 °C, 20 °C, 22.5 °C, 25 °C or alternating temperature of 15–25 °C (one cycle consists of 20 °C, 8 h → 25 °C, 4 h → 20 °C, 8 h → 15 °C, 4 h) for 24 weeks.

• Stratification Substrates and Fungi Inoculation

Un-dehisced seeds were mixed with rice husks, vermiculite, or river sand (1:3 ratio). Intact seeds that were not mixed with the substrates were used as controls. These four substrate options were inoculated with a fungi mix (four strains of Talaromyces flavus), which was isolated from the dehisced seed endocarp of the KT&G farm (the government agency for ginseng and tobacco) (

Table 1. List of microbial strains inoculated with un-dehisced ginseng seeds.

Group	No.	Microbial Strains *	Source	Code
Fungi	1	Talaromyces flavus (F1)	Isolated	fungi #1
	2	Talaromyces flavus (F2)	Isolated	fungi #2
	3	Talaromyces flavus (F3)	Isolated	fungi #3
	4	Talaromyces flavus (F4)	Isolated	fungi #4
	5	A mix of the four strains of T. flavus	Isolated	fungi mix
	6	Trichoderma harzianum (40509)	Non-decomposition	fungi 40509
Actinomycetes	1	Kitasatospora gansuensis (CMM1-1)	Decomposing	actino CMM1-1
	2	Micromonospora matsumotoense (CMM7-5)	Decomposing	actino CMM7-5
	3	Strptomyces sp. (CMM1-28)	Decomposing	actino CMM1-28
	4	Mix of three strains of Actinomycetes	Decomposing	actino-mix
Bacteria	1	Bacillus sp. (B5001)	Decomposing	bacter B5001
	2	Bacillus sp. (B5002)	Decomposing	bacter B5002
	3	Bacillus sp. (B2390)	Decomposing	bacter B2390
	4	A mix of three strains of bacteria	Decomposing	bacter-mix
	5	Bacillus amyloliquefaciens (amylo 12067)	Non-decomposition	bacter amylo

* Microbial strains were cultured in rice bran in vitro. The 1% (w/v) microbial inoculum was inoculated with ginseng seeds by co-culture at 25 °C for a day and then kept at 18–20 °C room for 3–5 days without watering.

). The strains were cultured in rice bran obtained by milling rice for one week at 27 °C. The mixture of seeds and fungal inoculum (1% w/v) was co-cultured for a day at 25 °C and loaded in a pot maintained at 18–20 °C room without water for 3–5 days. The seeds were watered once daily (1/d) during warm stratification. Watering was compared three times a day (3/d) or once every three days (1/3d).

• NaOH soaking and Fugi Inoculation

From the preliminary experiments, soaking the seeds in 5% sodium hydroxide (NaOH) for 25 min, followed by washing with running tap water overnight, decreased endocarp hardness. NaOH-soaked seeds were subjected to warm stratification with a combination of control (intact), sand (sand), or fungi mix (fungi).

• Sterilization of Seeds and Fungi Inoculation

The combined effects of intact/sterile and no-fungi/fungi-inoculation were tested. Seeds were sterilized with 1.5% NaOCl for 12 min and washed three times with tap water. The seeds were inoculated with a fungi mix of 1% w/v, co-cultured a day at 25 °C, and loaded in a pot, maintained at 18–20 °C room without water for 3–5 days. The seeds were watered once daily during warm stratification.

• Inoculation with Fungi, Actinomycetes, Bacteria

Seeds were inoculated with 1% (w/v) of fungi mix, actinomycetes-mix, and bacteria-mix (Table 1), and co-cultured for a day at 25 °C, loaded in a pot, maintained at 18–20 °C room without water for 3–5 days. The seeds were watered once daily during warm stratification.

• Inoculation with Fungi, Actinomycetes, and Bacteria of Cellulose Dissociation Activity

Seeds were inoculated with microbial strains with high and low cellulose dissociation activity (Table 1). The seeds were inoculated individually or in the mixture (total concentration of 1%, w/v) of fungus, actinomyces, or bacteria and co-cultured for a day at 25 °C, loaded in a pot, and maintained at 18–20 °C room without water for 3–5 days. Subsequently, the seeds were watered once daily during warm stratification.

2.2.3. Application of Fungi Inoculation to Seeds of Ginseng Varieties

Three varieties of Korean ginseng (Panax ginseng) landrace ‘Jakyeong (JK),’ the newly released variety ‘Yeonpoong (YP),’ and ‘Cheonpoong (CP)’ seeds and one American ginseng variety (Panax quinquefolius) seeds were stratified as non-treated (intact) or fungi #4 inoculated.

2.2.4. Measurement of Endocarp Hardness, Embryo Growth and Dehiscence

The seeds for the measurement of endocarp hardness, embryo-to-endosperm length ratio (EER), and dehiscence percentage at 0 weeks were selected from the original batches of fresh seeds before the stratification. Measurement was performed periodically at 0 (fresh), 4, 8, 12, and 16 weeks. For the experiment on the effect of stratification temperature, EER measurement was extended to 20 and 24 weeks because embryo growth was slower than expected in the controlled chambers where aeration was not supplied.

• Endocarp Hardness

The seed endocarp’s hardness (kg cm⁻¹) was measured periodically using fifteen seeds by a Rockwell Hardness Tester (Navimro, Seoul, Korea). Seeds (with an endocarp) were mounted vertically on a tester plate. The hardness was measured when the endosperm was split. The hardness of the open (dehisced) seeds was recorded as zero.

• Embryo-to-Endosperm Length Ratio (EER)

Embryo and endosperm lengths were measured at each period using 15 seeds per condition. Seeds were cut longitudinally in half using a surgical knife, and the length of the embryo and endosperm were measured using analytical software loaded into a Leica microscope (Wetzlar, Germany). The EER was expressed as the ratio of the embryo length to the endosperm length.

• Dehiscence Percentage

Seeds were considered dehiscent when the endocarp was open (no less than 3–4 mm). Dehiscence was expressed as the percentage of seeds with an open endocarp among the total number of seeds.

2.3. Statistical Analysis

To measure endocarp hardness and EER in all treatments, 15 seeds were randomly selected for each period of 0 (fresh), 4, 8, 12, and 16 weeks during warm stratification. A total of 100 seeds with three to five replicates (n = 300–500) were monitored for dehiscence percentages. During each period, dehisced seeds were removed from the onion net.

Data from all experiments in the final period (16 weeks) were analyzed by analysis of variance (ANOVA) and Duncan’s multiple range test (p < 0.05) using SAS on Demand for Academics software (SAS Institute Inc., Cary, NC, USA).

3. Results

3.1. Effect of Warm Stratification Treatments

3.1.1. Effect of Warm Stratification Temperature

• Embryo-to-Endosperm Length Ratio (EER)

After pulping, un-dehisced ginseng seeds were incubated in the growth chamber at 10–25 °C for 24 weeks and watered once a day. The embryo size is less than 0.4 mm, and the embryo-to-endosperm length ratio (EER) was 0.09 at the harvest stage.

Warm stratification temperatures critically affect embryo development. The optimum temperature for embryo growth was 15–20 °C, with the highest growth at 17.5 °C; embryo growth was highly retarded below (10 °C) or above (22.5 °C, 25 °C) this temperature (Figure 2). At 15 °C and 17.5 °C, the EER increased rapidly after 16–24 weeks, aligning with the dehiscence increase.

The temperature alternating cycle of 15–25 °C (20 °C, 8 h → 25 °C, 4 h → 20 °C, 8 h → 15 °C, 4 h) was beneficial for embryo growth at the early stage (4–16 weeks); thus, EER reached 0.38 after 16 weeks. However, afterward, the EER of alternating temperatures of 15–25 °C was retarded to 0.44 at 24 weeks. This retardation was partly on account of a lower dehiscence percentage compared to the 15–20 °C. It is likely that fluctuating temperatures were not beneficial for microbial growth and eventually lowered dehiscence, resulting in slower embryo growth. In contrast, rapid embryo growth was observed at 17.5 °C, and the EER reached 0.54 at 24 weeks due to a higher dehiscence percentage benefit.

Overall, embryo growth in the non-ventilated chambers was slower than in traditional outdoor warm stratification facilities. Additional side experiments determined that the chamber’s aeration (oxygen supply) was crucial for embryo development. For instance, dipping seeds in shallow water retards embryo growth, but forced aeration in a controlled room facilitates embryo growth.

3.1.2. Effect of Dehiscence Substrate, Fungi Inoculation, and Watering Period

• Embryo-to-Endosperm Length Ratio (EER)

Effective dehiscence materials (stratification substrates) were selected to increase dehiscence (endocarp opening) because dehiscence is a prerequisite for germinating ginseng seeds. Un-dehisced ginseng seeds mixed with dehiscence substrates were inoculated with fungi mix and incubated in a growth room at 18–20 °C for 16 weeks with watering once a day.

During warm stratification using dehiscence substrates, embryos free of dehiscence substrates (intact) grew comparably well onto rice husk and vermiculite, and the EER reached 0.60 at 16 weeks (Figure 3). River sand effectively stimulated embryo growth among the dehiscent materials tested, while vermiculite was inadequate for embryo growth, implying that aeration is critical for embryo growth. Faster embryo growth with sand-treated seeds may be related to the fact that sand provides sufficient aeration to the seeds, and the surface of the sand holds more microbial organisms, which play a pivotal role in endocarp opening. Moreover, faster embryo growth in sand-treated seeds was linked to a higher percentage of dehiscence than in other conditions. In Figure 4, the dehiscence of sand-treated seeds started at eight weeks (23.3%) and reached 69.5% at 12 weeks, as dehiscent seeds may facilitate water supply and aeration and permit more expansive space for embryo growth.

The fungi-treated endocarp surface was darker than the non-inoculated seeds. Inoculation of fungi on the surface of the seed endocarp slightly inhibited embryo growth (EER) in dehiscence substrate treatments compared to the non-inoculated conditions at 16 weeks (0.54–0.64 → 0.49–0.56). Particularly in the sand treatment, inoculation with fungi inhibited embryo growth at 16 weeks (0.68 → 0.49). Despite a variable EER range of 0.29–0.45 at 12 weeks, the embryo growth reached a stable EER range of 0.49–0.56 at 16 weeks.

Watering three times a day for seeds without a dehiscent substrate and inoculated fungi {intact-fungi mix(3/d)} significantly stimulated embryo growth at 8–12 weeks compared to watering once a day (intact-fungi mix). Still, there was no difference at 16 weeks. Significant inhibition of embryo growth was observed in watering once in three days for sand-treated seeds and fungi-inoculated {sand-fungi mix(1/3d)}, compared to once a day (sand-mix) (0.49 → 0.37).

• Dehiscence Percentage

The dehiscence substrates (rice husk, vermiculite, and sand) were effective in increasing the dehiscence percentage of ginseng seeds compared to that of non-treated (intact) seeds (at 16 weeks, 67.5–85.0% vs. 43.3%; Figure 4). Among the dehiscent substrates, sand was the most effective at 8–12 weeks and, finally, the most effective at 16 weeks (85%), followed by rice husks (71.5%) and vermiculite (67.5%). Sand can facilitate drainage and provide aeration.

The inoculation of fungi with non-substrate-treated (intact-fungi mix) seeds drastically increased the dehiscence percentage from 43.3% to 80.5% after 16 weeks (Figure 4), which was comparable to the dehiscence percentage of dehiscent substrate-treated and fungi-inoculated seeds (67.5–85.0%).

The inoculation of fungi with seeds of dehiscence substrates was not so effective on dehiscence; only a slight increase of dehiscence was observed in rice husk (rice husk-fungi mix, 71.5% → 81.0%) and vermiculite treated (vermiculite-fungi mix, 67.5% → 71.0%) seeds, but even significantly decrease in sand-treated (sand-fungi mix) seeds (85.0% → 71.5%). The effect of dehiscent substrates during warm stratification is primarily related to providing a beneficial environment for fungal growth (watering and aeration) and the opening of the endocarp rather than embryo growth.

The inoculated fungi may break down fibrin near the endocarp hole (hilum) and promote endocarp dehiscence. There is also an interaction between embryo growth and dehiscence percentage since embryo growth and expansion of the endosperm stimulate endocarp opening and vice versa by providing aeration and moisture in the opened endocarp.

Watering three times a day {intact-fungi mix(3/d)} improved embryo growth and dehiscence at 8–12 weeks, compared to once a day {intact-fungal mix(1/d)}: at 12 weeks EER 0.31 → 0.40, dehiscence 43.5% → 73.5%. However, at 16 weeks, there were no significant effects on the EER (Figure 3) or dehiscence percentage (Figure 4).

Watering once every three days {sand-fungi mix(1/3d)} decreased embryo growth (Figure 3) and dehiscence percentage (Figure 4) compared with daily watering {sand-fungi mix(1/d)}. In this treatment, the endocarp’s surface turned darker than in the other treatments, indicating that the higher fungal population induced by sparse watering inhibited embryo growth and decreased dehiscence. This implies that the dehiscence of ginseng seeds is modulated by the interaction between embryo growth and microbial (fungal) decomposition.

3.1.3. Effect of Sand, Fungi Inoculation, and NaOH

• Endocarp Hardness

The endocarp hardness of intact seeds decreased from 5.25 to 2.41 after 16 weeks of warm stratification. Inoculating the fungi mix before warm stratification was the most effective in reducing the endocarp hardness (0.27 at 16 weeks), followed by the application of sand as a dehiscent substrate (0.43 at 16 weeks) (Figure 5).

To facilitate endocarp opening by decreasing endocarp hardness, non-dehisced seeds were soaked in sodium hydroxide (NaOH) because sulfonic acid (H₂SO₄) is ineffective in reducing endocarp hardness. Based on the preliminary screening, un-dehisced seeds were soaked in 5% NaOH for 25 min, which decreased the endocarp hardness from 5.25 (intact) to 3.16 (NaOH-intact). NaOH-soaking reduces the endocarp hardness, reaching 0.09–0.43 at 16 weeks. Among the treatments tested, combining NaOH soaking with an inoculating fungi mix (NaOH-fungi) was the most effective in decreasing endocarp hardness (0.99 and 0.09 at 8 and 16 weeks, respectively).

• Embryo-to-Endosperm Length Ratio (EER)

Soaking with NaOH facilitated embryo growth at eight weeks during warm stratification. However, the EER at 16 weeks was not significantly different among the conditions tested (Figure 6).

• Dehiscence Percentage

Inoculating fungi before warm stratification resulted in the fastest and highest dehiscence percentage (90.3% at 16 weeks) compared with intact seeds (28.8%) (Figure 7). Mixing sand as a dehiscence substrate also facilitated dehiscence.

Soaking un-dehisced seeds with NaOH (NaOH-intact) increased the dehiscence percentage by 18% compared to intact seeds. However, soaking the seeds with NaOH and then applying sand as a dehiscent substrate (NaOH-sand) or inoculating fungi (NaOH-fungi) negatively affected the dehiscence percentage; that is, there was an approximately 15% decrease at 16 weeks. Although soaking the seeds in NaOH effectively reduced endocarp hardness, it sterilized the seed surface and negatively affected the dehiscence percentage, implying a crucial role for fungi in endocarp opening. Therefore, inoculating un-dehisced seeds with fungi or mixing them with sand before warm stratification is a prerequisite for the dehiscence of ginseng seeds.

3.1.4. Combinational Effect of Fungi Inoculation and Sterilization of Un-Dehisced Seeds

• Embryo-to-Endosperm Length Ratio (EER)

A mixture of fungi was inoculated onto non-sterilized (intact) or sterilized ginseng seeds to confirm the effect of the fungi on dehiscence. Embryo growth was not significantly different (Figure 8).

• Dehiscence Percentage

Inoculation with fungi benefited intact ginseng seeds without dehiscence substrates (20.6% vs. 83.0%, Figure 9). The dehiscence percentage of surface sterilized seeds without fungal inoculation was 31.5%, and that of fungal inoculation was 66.7% after 16 weeks (Figure 9). Sterilization of the seed surface before warm stratification negatively affected fungal inoculation (growth) on the seed surface and endocarp opening.

The surface area of the non-sterilized and fungi-inoculated (intact-fungal mix) seeds turned dark brown during warm stratification. Sterilization of the seed surface and subsequent fungal inoculation (sterile-fungal mix) also turned the endocarp surface dark brown (Figure 10A-below-right). In general, the endocarp color of the dehisced (opened) seeds was often light black, whereas that of the un-dehisced seeds was light brown.

3.1.5. Inoculation of Fungi, Actinomycetes, and Bacteria (Bacillus) Mix

• Endocarp Hardness, EER, and Dehiscence Percentage

A mixture of fungi, actinomycetes, and bacteria was inoculated into the seeds to compare the microorganisms’ efficiency in the ginseng seed dehiscence. Inoculation with these three microbial mixtures accelerated the decrease in endocarp hardness (Figure 11) and had no significant effect on EER (Figure 12). All inoculation treatments significantly increased the dehiscence percentage (Figure 13) compared with the control seeds (75.2–79.9% vs. 47.2%). However, no significant differences were observed among the three microbial mixtures.

3.1.6. Inoculation of Fungi, Actinomycetes, and Bacteria with High or Low Cellulose Dissociation Activity

• Endocarp Hardness

Seeds were inoculated with a broad range of microorganisms with high or low cellulose dissociation activity to validate the effects of microbial inoculation. All the microbial inoculation treatments tested, including fungi, actinomycetes, and bacteria, significantly decreased endocarp hardness, and there were no significant differences among the three groups tested (Figure 14). Interestingly, no significant difference was observed in the endocarp hardness between the strains with high cellulose dissociation activity in the field (fungi #4) or in vitro (actino CMM1-1, bacter 2390) and the two strains with low cellulose dissociation activity in the in vitro assay (fungi 40509, bacter amylo). The mixture of four fungi strains (fungi mix) more effectively decreased the endocarp hardness after eight weeks (1.50 vs. control 3.54).

• Embryo-to-Endosperm Length Ratio (EER)

The EER of intact (control) seeds increased linearly, and there was no difference in the EER of control and microbially-inoculated seeds during 0–8 weeks (Figure 15). However, the EER in microbial inoculated seeds rose rapidly, compared to non-inoculated seeds, during 12–16 weeks. This rapid embryo growth at 12–16 weeks coincides with the rapid increase in the percentage of dehiscence in microbially-inoculated seeds during this period (Figure 16). There was no difference in EER among the microbially-inoculated seeds, implying that the cellulose dissociation activity of microorganisms may not be specific for decomposing the seed endocarp.

• Dehiscence Percentage

Endocarp opening (dehiscence) started after eight weeks, particularly during inoculation with a mixture of four fungal strains (fungi mix 32% vs. other treatments 2–17%) (Figure 16). This result coincides with Figure 14, which shows a sharp decrease in endocarp hardness after eight weeks. Dehiscence rapidly increased in all the microbial-inoculated seeds during 12 weeks (67–83%), except for bacteria-inoculated seeds (bacter 2390), which presented relatively lower dehiscence (56%) (Figure 16). After 16 weeks, all microbially-inoculated seeds presented 60% higher dehiscence than the control (75–83 vs. 17%).

3.2. Application of Fungi Inoculation to Seeds of Three Korean Ginseng Varieties and One American Ginseng

• Endocarp Hardness

Three varieties of P. ginseng seeds (one landrace ‘JK’, two varieties of ‘YP’ and ‘CP’) and P. quinquefolius seeds with (-fungi #4) or without (-control) fungi inoculation were subject to warm stratification in pots.

Inoculation of fungi facilitated a decline in endocarp hardness in ‘YP’and ‘JP’ varieties of P. ginseng seeds (2.3 and 2.0, respectively) (Figure 17). The drop of endocarp hardness in ‘CP’ and P. quinquefolius (American) seeds (1.1 and 1.2, respectively) was slower than the former, which is related to the lower endocarp opening (dehiscence percentage).

• Embryo-to-Endosperm Length Ratio (EER)

Inoculation of fungi on the seeds of P. ginseng significantly increased the EER at 16 weeks; ‘YP’ 0.51 → 0.65, ‘CP’ 0.38 → 0.70, ‘JK’ 0.40 → 0.75 (Figure 18). Significant embryo growth at 8–16 weeks was associated with the opening of seeds (dehiscence), facilitating water and aeration. However, fungi inoculation in P. quinquefolius seeds (American-fungi #4) slightly increased the EER (0.25→0.32), reflecting the lower dehiscence (Figure 19).

• Dehiscence Percentage

Intact seeds of P. ginseng (two varieties of ‘YP’ and ‘CP’, one landrace ‘JK’) and P. quinquefolius without fungi inoculation presented lower dehiscence at 16 weeks (15.2–20.5%) (Figure 19). Inoculation of fungi (#4) significantly facilitated the dehiscence of P. ginseng (‘YP’ 82.7%, ‘CP’ 53.3%, ‘JK’ 58.8%) and P. quinguefolius seeds (41.5%). Facilitation of endocarp opening in fungi-inoculated seeds was associated with a decline in endocarp hardness due to microbial inoculation during the 0–12 weeks, and the opening of the endocarp accelerated embryo growth.

4. Discussion

Ginseng seeds have a short life span, and it takes 3–4 years to harvest them. After three years of storage at −2 °C, the ginseng seed’s dehiscence percentage dropped to 45% from the 84% of fresh unstored seeds [14]. The hydration window for cryopreservation of ginseng seeds (excluding the endocarp) varies during the after-ripening stage: un-dehisced seeds (EER 0.1) 4–8%, dehisced (EER 0.45) 7–10%, and fully developed embryos (EER 0.9) 9–11% moisture content [4]. Seeds damaged by desiccation and cryo-injury failed to dehiscence. However, dehiscent seeds germinate after cold stratification, implying that dehiscence is a significant hurdle for ginseng seed germination [4].

The moisture content of fresh seeds (embryo and endosperm) increased from 45.9% for un-dehisced seeds after harvest to 58.5% after dehiscence and 66.3% for fully developed embryos after cold stratification. This implies that the endocarp hinders moisture imbibition heavily in the un-dehisced stage and slightly in the dehisced stage; the physiological state of seeds varies during the after-ripening stages.

The dormancy of ginseng seeds is classified as MPD [10,11]. Therefore, seeds require warm and cold stratification for further embryonic development and germination [7,8]. Because physical dormancy is defined as water-impermeable layers of palisade cells in the seed or fruit coat [12], ginseng seeds are considered to have no physical dormancy. However, this study suggests that ginseng seeds undergo mechanical (physical) dormancy due to the fruit coat (endocarp). The hard endocarp of ginseng seeds has a hole (hilum) that partially allows the imbibition of water and gas exchange. Therefore, the endocarp can be opened from the hilum from summer to autumn via microbial decomposition under traditional outdoor dehiscence facility conditions. However, the endocarp may not open indoors, especially during the winter, thereby preventing further embryo growth and germination.

Although embryo growth may facilitate endocarp opening, embryo growth does not directly determine this. In summer, the sand used as a dehiscent material (stratification substrate) contains sufficient microorganisms for the traditional farming practice of endocarp opening. During the warm stratification period, microorganisms are inoculated into the endocarp of the ginseng seed and eat away the fibers of the endocarp, thereby allowing the opening to occur in association with the seed embryo and endosperm expansion.

The beneficial effects of sand as a stratification substrate may include (1) providing aeration (oxygen supply) via facilitated drainage, (2) providing fungal inoculation to the endocarp surface, and (3) balancing the inhabitation of microorganisms and after-ripening of the seeds. Oxygen is a pivotal factor in embryonic development and seed germination. High-pressure oxygen favors dormancy breakage and germination of Crataegus mollis seeds [15].

These findings suggest that endocarp dehiscence requires embryonic development and microbial-assisted decomposition of endocarp holes (hilum). Therefore, a dehiscence model of ginseng seeds during warm stratification at a traditional dehiscence facility is proposed (Figure 20). Three conditions (water, oxygen, and temperature) are essential for seed stratification and germination. This study emphasized the importance of microbial inoculation on the surface area of the endocarp for microbial-assisted decomposition (from the hilum to raphe) and dehiscence (opening of the endocarp). Dehiscence is a combination of the microbial-assisted decomposition of the endocarp and extension of the seed embryo and endosperm.

In traditional outdoor warm stratification, sand as a stratification substrate may provide a source of fungal inoculum and appropriate conditions for moisturization and aeration (supplying oxygen). Seed embryos can grow under such conditions, and fungi on the endocarp surface can inhabit them. It has been hypothesized that moisture and aeration at an appropriate temperature are prerequisites for embryo growth, similar to seed germination conditions. Under these conditions, fungi can also multiply and may decompose the cellulose of the endocarp and allow it to open through the expansion of the embryo and endosperm.

During warm stratification, seed embryos grow in water and oxygen and prefer moderate temperatures. The temperature effect is critical for developing immature embryos, and 17.5 °C was the optimum temperature. Contrary to the results of previous studies [8], the constant optimum temperature was superior to the alternating temperature for embryo development and dehiscence, indicating that earlier endocarp opening was beneficial for further embryo development. Moreover, the opening of the endocarp facilitates embryo growth at a later stage of warm stratification, implying that a hard endocarp is a limiting factor for embryo development.

The optimum after-ripening temperature for ginseng seeds is variable during development stages—warming stratification (dehiscence treatment, 100 days) 17 °C, middle stage (30 days after dehiscence) 10 °C, physiological dormancy at terminal stage (60–90 days) 5 °C, germination 15 °C [16,17].

The content of ginseng seed and endocarp’ abscisic acid (ABA) gradually decreased, whereas the GA₃ content increased during warm stratification [18]. However, the effect of exogenous treatment of GA₃ before stratification remains controversial. Soaking of Eleutherococus senticosus (Araliaceae family) seeds in around 300 mg L⁻¹ GA₃ for a day increased the dehiscence percentage (from 58.8% to 80.7%), but the seed decay rate also increased [19,20].

Ginseng seed endocarp is partially water-permeable through the hilum (Figure 1A). This may also partially allow respiratory gas exchange. However, the endocarp impedes embryo development during initial warm stratification and prevents further cellular extension during embryo development, preventing germination. When the endocarp was manually removed using a nail cutter, most seeds decayed during warm stratification. This suggests that ginseng seed endocarp functions as a seed coat, inhibits embryonic development, and protects the seeds.

Ginseng endocarp is composed of cellulose and hemicellulose. Likewise, the hard endocarp (fruit coat) of Lannea microcarpa seeds contains lignin and hemicellulose [21]. The split (dehiscence) and germination of American ginseng seeds depended more on plumpness (seed kernel area/seed coat area) than on seed size [22].

Kim et al. [23] observed that the water supply for embryo growth was accomplished by vascular bundles entering the endocarp through the opened hilum. The thickness of the raphe region of the endocarp is thinner (200 µm) than the other regions (300–600 µm), and sclereids in that region are regularly arranged. Hence, the seed coat of the ginseng seed opens, and it always occurs along the raphe, centering on the hilum [23].

Eight endophytic bacteria and ten fungi have been isolated from the endocarp of dehiscent ginseng seeds [24,25]. Kim et al. [26] identified five fungal strains of Talaromyces flavus from dehiscent seeds in traditional ginseng seed stratification facilities. They found that T. flavus-treated seeds showed more than 40% greater dehiscence than untreated seeds. T. flavus (Penicillium dangeardii or P. vermiculatum) is a notable biocontrol agent for soil-borne plant pathogens [27]. This species is also known for its ability to decompose various organic materials, including lignocellulosic substrates of plant materials [27]. Specific studies directly investigating its activity in the endocarp (the hard inner layer of the fruit) decomposition were not extensively documented. Fungi such as Talaromyces are generally crucial in nutrient cycling within ecosystems and can participate in the decomposition of rigid plant materials. Ginseng seed-associated fungi rarely colonize ginseng roots in the soil [28].

Watering of microbially-inoculated seeds should start a few days after inoculation to allow the inhabitation of the surface areas of the endocarp. Excessively early or frequent watering during the initial stage resulted in failed inoculation. However, watering that is too scarce increases microbial density, inhibits embryo growth, and even causes seed decay.

Some studies have reported dormancy breaking in seeds with water-impermeable fruit coats due to soil microbial action. For example, microbial inoculation promotes the germination of Rosa seeds [29]. However, Baskin and Baskin [13] found “only weak support for breakage physical dormancy by soil microbial action”. Yang et al. [19] used a traditional warm stratification facility in the summer–autumn season, and microbial inoculation of bacteria or fungi on the ginseng seed surface lowered the dehiscence percentage compared to intact seeds. In this case, additional inoculation of microorganisms may damage the embryo and hinder dehiscence, as the authors already used sand as a substrate.

When the seeds were treated with the fungicide Captan, the hardness of the seed endocarp merely decreased from 10.9 kg cm⁻¹ to 8.0 kg cm⁻¹, compared to the non-treated seeds, which dropped to 4.9 kg cm⁻¹ during the 80 days of warm stratification [25]. The EER and dehiscence of fungicide-treated seeds were 0.34 and 5%, respectively, in contrast to the EER of untreated seeds (0.43) and dehiscence (79%) [25]. Lee et al. [25] argued that embryo growth was associated with decreased seed hardness. Moreover, the frequency of fungal isolation from the endocarp was negatively correlated (r= −0.984) with hardness of the seed endocarp [25]. The endocarp surface of fungicide-treated seeds remained yellowish-brown compared to non-treated seeds, which turned dark brown [25].

5. Conclusions

This study provided comprehensive insights into the crucial role of microbial inoculation on the opening of hard-structured ginseng seed endocarp, which is a prerequisite for further embryo development and germination. Inoculation of seeds with any microbes (fungi, actinomycetes, and bacteria) with high or low cellulose-decomposing ability increased the dehiscence percentage by 66% compared to the untreated control. Using the fungal inoculation, seeds of three varieties of P. ginseng and one variety of P. quinquefolius were successfully dehiscent in indoor warm stratification. Ginseng seeds have triple dormancy, i.e., morpho-physiological and physical. This indoor warm stratification approach may contribute to the year-round germination of ginseng seeds and pave the road for producing processed ginseng seedlings.