The Dormancy Types and Germination Characteristics of the Seeds of Berberis koreana Palibin, an Endemic Species of Korea

Abstract

Berberis koreana Palibin is an endemic plant native to Korea. In this study, we aimed to study the seed germination of this species using a water imbibition experiment, gibberellic acid (GA₃) treatment (0, 10, 100, or 1000 mg·L⁻¹), cold stratification (0, 2, 4, 8, or 12 weeks at 4 °C), move-along experiment, and phenology studies. In the water imbibition experiment, the weight of the seeds increased by more than 120% in 24 h. An analysis of the internal and external morphological characteristics of the seed revealed that the embryo was already fully grown from the fruit and did not grow thereafter. The final germination percentages for the cold stratification at 0, 2, 4, 8, and 12 weeks at 4 °C were 12 ± 3.65, 32 ± 9.09, 59 ± 1.00, 59 ± 9.59, and 71 ± 1.91%, respectively. In the move-along experiment and phenology studies, a longer low-temperature treatment period resulted in a higher germination percentage. However, the GA₃ treatment had little effect on the seed germination. Our results indicate that B. koreana exhibits an intermediate physiological seed dormancy.

1. Introduction

Plant propagation methods include various methods such as sowing, stem cutting, tissue cultures, and layering. The sowing method has the following advantages: (1) a large number of plants can be secured at once, (2) it reduces labor, and (3) it does not require a relatively large number of auxiliary facilities, in comparison to those required by a tissue culture facility. (4) In addition to these, there are various other advantages. However, some species must have the conditions required for seed germination. “Seed dormancy”, an innate seed property, defines the environmental conditions that must be met before a seed can germinate [1]. Thus, to propagate plants using the sowing method, a suitable dormancy-breaking technique specific to their seeds must first be determined [2].

Various types of seed dormancy exist depending on the life cycle of the plants, the ambient environmental conditions, and their geographical distribution; seed dormancy has been studied and classified according to plant species and genera [3]. Lang [4] classified seed dormancy into three types: eco-dormancy, para-dormancy, and endo-dormancy. Baskin and Baskin [3] classified seed dormancy into five types by comprehensively considering physiological and morphological factors: physiological dormancy (PD), where inhibitory compounds inside and outside the seeds prevent germination; morphological dormancy (MD), where the seeds contain underdeveloped or immature embryos; morphophysiological dormancy (MPD), which is a combination of PD and MD; physical dormancy (PY), which involves the suppression of water absorption by the seeds; and combinational dormancy (PY + PD), which is a combination of PY and PD.

Berberis koreana Palibin, commonly known as Korean barberry, is a deciduous shrub of Berberidaceae, which are endemic to Korea and used in oriental medicine for their anti-inflammatory, analgesic, anti-cancer, anti-conjunctivitis, and antibacterial properties [5,6], and as a source of functional foods [7]. Berberis spp. is also a spinous shrub that is highly ornamentally valued for its evergreen or multicolored leaves, brilliant flowers, and often showy fruit, and is often used as garden trees or hedges [8].

Berberis seeds are known for their physiologically non-deep dormancy [9]. Baskin and Baskin [3] reported that the seeds of five Berberis species, namely, B. aristata, B. dictrophylla, B. dubia, B. kansuensis, and B. vernae, displayed PD. Thakur et al. [10] reported that the dormancy of B. aristata seeds was broken under light conditions during the growth phase at 20 °C. Wang et al. [11,12] reported that B. dictrophylla and B. kansuensis seeds were subjected to a low-temperature wet treatment for 80 days to break their dormancy. The dormancy of B. dubia and B. vernae seeds was broken after culturing at 20/15 °C in the growth phase after 168 days of a low-temperature wet treatment [3]. Deb at al. [13] proved that the effect of chilling and light can break the seed dormancy of B. manipurana seeds. The seeds of most plants in the Berberis genus have PD and reports have suggested that that their dormancy is broken under cold stratification treatments and temperature conditions of 20 °C [14,15,16,17].

Some studies have reported on the inter- and intraspecific variability in the fruit and/or seed traits in the Berberis genus [18,19,20,21,22]. These studies have suggested that the differences between the populations for all the analyzed fruit and seed traits depend on environmental factors and species-related differences. Therefore, in this study, we present the external morphological measurements of B. koreana seeds.

Therefore, we hypothesize as follows: (a) it is expected that B. koreana seeds will also have PD, (b) B. koreana seeds will absorb water, (c) a low-temperature wet treatment will break the dormancy of B. koreana seeds, and (d) a hormone treatment (GA₃) will break the dormancy of B. koreana seeds. The results of this research can be used as reference data for further research on the morphological characteristics of barberry plants in Korea.

2. Materials and Methods

2.1. Experimental Materials

The seeds of B. koreana used in this study were obtained from wild plants growing near Samneung in Paju (37°44′35″ N, 126°49′27″ E), Republic of Korea, on 17 October 2019. The harvested fruit was removed using the repair method and the selected seeds were shade-dried for 7 days in a well-ventilated space. The seeds were then stored under refrigeration (4–5 °C) until further use.

2.2. Investigation of Internal and External Characteristics of Seeds

To investigate the external morphological characteristics of the seeds, images were acquired using a scanning electron microscope (SEM; CX-200, COXEM, Daejeon, Republic of Korea). The weights of the dried seeds were measured per 1000 dried seeds in triplicate.

To investigate the internal morphological characteristics, the seeds were cut in half using a double-edged razor (stainless steel blade, Dorco, Seoul, Republic of Korea) and photographed using a digital microscope (DVM6, Leica, Land Hessen, Germany). Changes in the embryos and endosperms were observed before and after the seed germination.

2.3. Seed Disinfection and Setting

Before the start of the experiment, the seeds were surface-sterilized via soaking in 1000 mg·L⁻¹ of benomyl (FarmHannong, Seoul, Republic of Korea) for 24 h; the seeds were then washed three times with distilled water. The surface-sterilized seeds were placed over two sheets of filter paper (Whatman No. 1; GE Healthcare, Buckinghamshire, UK) in a Petri dish containing 5 mL of distilled water.

After the GA₃ and cold stratification treatments (described below), the germination percentage was investigated in a growth chamber (TGC-130H, Espec Mic Corp., Aichi, Japan), where the seeds were cultured at a constant temperature of 20 °C. The experiment was performed with four replicates and 25 seeds per treatment group. If microorganisms were present during the culture period, they were disinfected by being soaked for 24 h in 1000 mg·L⁻¹ of benomyl (FarmHannong), followed by being washed in distilled water; the distilled water was replenished in the Petri dishes to prevent the drying of the filter paper.

The seeds were considered germinated when the radicle emergence through the seed coat was >1 mm; the germination percentages were recorded at 1-week intervals [23]. Seeds that died due to decay during the experiment were removed immediately and excluded while calculating the germination percentage.

2.4. Water Imbibition Test

To determine whether the B. koreana seeds were physically dormant, the moisture absorption percentages of the seeds were measured. A Petri dish containing two sheets of filter paper (Whatman No. 1) soaked in distilled water was plated with 100 seeds; three replicates were maintained. The initial weight before water absorption and the weights at 3, 6, 9, 12, 24, 36, and 48 h after settling were measured. The water absorption percentage was calculated using the following formula [24]:

$$\%\mathrm{Ws} =\left[\left(\mathrm{Wh}-\mathrm{Wi}\right)/\mathrm{Wi}\right]\times 100$$

(1)

where Ws is the relative increase in the weight of the seeds due to moisture absorption, Wh is the weight of the seeds per hour after the water supply, and Wi is the initial weight of the seeds in the dried state. The water imbibition test was conducted at the Seed Germination Laboratory of Baekdudaegan National Arboretum from 2–4 December 2019.

2.5. Effect of Temperature on Germination: A Move-Along Experiment

According to Baskin and Baskin [25], the “move-along experiment” provides the dormancy-breaking temperature required for germination in most species. Based on the move-along experiment, the temperature for each season was set and the changes in the germination rates were measured. For the temperature treatments, the four seasons of natural environmental conditions were set as spring (15 °C), summer (25 °C), autumn (20 °C), and winter (5 °C). The treatment groups were subjected to temperature changes from winter to spring and summer (T1: 5→15→20→25 °C) and temperature changes from summer to autumn and winter (T2: 25→20→15→5 °C). In the T1 and T2 treatment groups, the dwell times at each temperature were set to 12, 4, 4, and 12 weeks (

Table 1. Outline of the modified temperature treatments [25].

No. of Weeks at Treatment Temperatures		4	4	4	4	4	4
Move along	T1	5 °C winter	5 °C winter	5 °C winter	15 °C early spring	20 °C late spring	25 °C summer
	T2	25 °C summer	25 °C summer	25 °C summer	20 °C early autumn	15 °C early autumn	5 °C winter

). For all the treatment groups, 25 seeds were used in four replicates and the measurements were obtained at 1-week intervals. In addition, to observe the changes in the embryo and endosperm based on the temperature changes, the surfaces of the seeds were cut at 1-month intervals and photographed using a digital microscope. A controlled temperature shift experiment was conducted at the Seed Germination Laboratory of Baekdudaegan National Arboretum from 19 December 2019 to 9 July 2020.

2.6. Effect of Cold Stratification Experiment on Germination

For the cold stratification treatment, the surface-sterilized seeds placed into a Petri dish were subjected to 4 °C in a growth chamber for 0, 2, 4, 8, and 12 weeks. At the end of each low-temperature treatment duration, the Petri dishes were shifted to a 20 °C growth chamber; the germination percentage was recorded while culturing the treatment groups at 20 °C and the experiment interval was set to 1 week. For all the treatment groups, 25 seeds were used in four replicates and the measurements were obtained at 1-week intervals. The experiments were conducted at the Seed Germination Laboratory of Baekdudaegan National Arboretum from 19 December 2019 to 9 July 2020.

2.7. Experiment to Determine the Effect of GA₃ on Germination

The seeds were soaked in solutions with 0 (distilled water, control), 10, 100, 500, or 1000 mg·L⁻¹ of GA₃ for 24 h at room temperature and then incubated in a growth chamber at 20 °C. The germination rates were determined at 1-week intervals. For all the treatment groups, 25 seeds were used in four replicates and the measurements were obtained at 1-week intervals. The GA₃ treatment experiments were conducted at the Seed Germination Laboratory of Baekdudaegan National Arboretum from 1 April 2021 to 3 June 2021.

2.8. Effect of Light Conditions on Seed Germination

For the cold stratification, GA₃ treatment, and move-along experiment, similar light (12 h light/dark photoperiod) and dark conditions (24 h dark) were utilized. For all the treatment groups, 25 seeds were used in four replicates and the measurements were obtained at 1-week intervals.

The light conditions inside the growth chamber were maintained using fluorescent lamps with 40 ± 10 μmol·m⁻²·s⁻¹ PPFD. The dark conditions were maintained by wrapping the Petri dishes with aluminum foil.

2.9. Phenology of Embryo Growth, Germination, and Seedling Emergence under Natural Environmental Conditions

To observe the seasonal changes in the seeds under natural environmental conditions, the ground (almost loamy sand) was dug to a depth of 5 cm and a tray was planted in the nursery field of the Baekdudaegan National Arboretum. The tray was filled with potting soil with a mixing ratio of 64.3% cocopeat, 15% peatmoss, 2.5% nitrogen, 10% pearlite, 8% zeolite, 0.19% fertilizer, and 0.01% of a wetting agent. The phenology of the embryo growth, germination, and seedling emergence was investigated from 1 December 2019 to 1 August 2020.

2.9.1. Embryo Growth

Approximately 400 seeds were placed in fine-mesh polyester bags filled with river sand and buried in a tray filled with potted soil. The trays were placed at ground level in the nursery field. Every 2 or 4 weeks, a bag was exhumed, and 10 seeds were randomly selected for the embryo growth measurement. The seeds were cut into thin sections using a razor blade; the lengths of the seeds and embryos were measured using a digital microscope. The ratio of the embryo length to the seed length (E:S ratio) was calculated to correct for the positive correlation between the seed and embryo lengths.

2.9.2. Germination

Four replicates of 25 seeds were sown in 8 cm plastic pots filled with potting soil and placed in trays filled with the same potting soil. The trays were placed at the ground level in the experimental garden. The seeds with emerged radicles were counted and removed every week. The seeds were considered to have “germinated” when the radicle protrusion length was at least 1 mm. The intact seeds that did not germinate were buried in the field.

2.9.3. Seedling Emergence

The timing of the seedling emergence was monitored by sowing four replicates of 25 seeds at a depth of 3 cm in plastic pots filled with potting soil, which were placed in the trays described above. The emerged seedlings were counted and removed every week during the field experiments. The pots were covered with nets to prevent disturbance from wild animals.

2.10. Statistical Analyses

The statistical analyses were performed using SPSS version 21 (SPSS Inc., Chicago, IL, USA). The results of the germination experiment were subjected to an analysis of variance and Duncan’s multiple range test (p ≤ 0.05).

3. Results

3.1. Investigation of Internal and External Characteristics of Seeds

To investigate the morphological characteristics of the B. koreana seeds, they were photographed using scanning electron and digital microscopes (Figure 1). The color of the seed coat was reddish-brown (Figure 1c). When part of the seed coat was magnified and photographed using an electron microscope, it revealed a curvature (Figure 1a,b). The examination of the dissected seeds using a digital microscope confirmed the development of the embryo in the seed of the ripe fruit (Figure 1d).

The mean length of the embryo was 4.00 ± 0.04 mm (mean ± standard error); the mean seed length was 6.48 ± 0.03 mm; and the E/S ratio (embryo: seed ratio) was 0.62 ± 0.05. In addition, the mean weight of 1000 seeds was 11.514 ± 0.392 g.

3.2. Water Imbibition Test

The water imbibition test, performed to evaluate the permeability of the B. koreana seeds, showed that the seed weight increased by 34.10 ± 0.54% in 24 h and 57.13 ± 0.58% in 48 h (Figure 2).

3.3. Effect of GA₃ Treatment on Seed Germination

The germination percentages of the B. koreana seeds subjected to the GA₃ treatment for 30 weeks at 20 °C under light/dark conditions were as follows: at GA₃ concentrations of 0, 10, 100, 500, and 1000 mg/L, the final germination percentages under the light/dark cycle conditions were 7.00 ± 3.00, 5.00 ± 1.91, 4.00 ± 1.63, 7.00 ± 1.91, and 13.00 ± 2.52%, respectively, and the final germination percentages under the dark conditions were 4.00 ± 1.63, 1.00 ± 1.00, 0, 11.00 ± 3.42, and 6.00 ± 3.46%, respectively. Both conditions showed a significant difference when the GA₃ concentration was 500 mg/L or higher (Figure 3).

3.4. Effect of Cold Stratification Experiment on Seed Germination

The seeds treated with cold stratification for 0, 2, 4, 8, and 12 weeks were cultured in a growth chamber (light/dark conditions) at 20 °C for 30 weeks. The final germination percentage of the seeds treated with cold stratification for 12 weeks was the highest at 71.00 ± 1.91%, regardless of the light conditions (Figure 4a).

Under the light conditions, the final germination percentage of the seeds was the highest at 71 ± 1.91% when treated with cold stratification for 12 weeks. The germination rates at 4, 8, and 12 weeks of cold stratification were statistically identical. Under the dark conditions, the final germination percentage of the seeds was the highest at 70.00 ± 3.83% when treated with cold stratification for 12 weeks.

In addition, the cold stratification for 12 weeks under the light conditions and the cold stratifications for 8 and 12 weeks under the dark conditions were observed to initiate germination during the cold stratification treatment period.

3.5. Seed Germination Based on Temperature Conditions: A Move-along Experiment

A move-along experiment indicated final germination percentages of 76.00 ± 4.32% at T1 (5→15→20→25 °C) and 12.00 ± 4.62% at T2 (25→20→15→5 °C), indicating that the temperature change from winter to spring and summer showed better results (Figure 5). The Berberis koreana seeds germinated under the T1 temperature conditions as follows: (1) the seeds started to germinate in 6 weeks under the winter temperature conditions (5 °C); the germination percentage was 66.0 ± 4.16% until 12 weeks; (2) the germination percentage increased to 73.00 ± 4.43% under the early spring temperature conditions (15 °C, 13–16 weeks); (3) after changing the temperature conditions in late spring (20 °C), the final germination percentage was 76.00 ± 4.32% at 17 weeks; and (4) further germination of the seeds was not observed.

3.6. Phenology of Embryo Growth, Germination, and Seedling Emergence

The cutting planes of the seeds observed at 2–4-week intervals showed that there was no increase in the E:S ratio (Figure 6). The E:S ratio was measured for the ungerminated seeds. The germination rate measurements based on seasonal changes showed that germination began in the 13th week (19 March 2020) and continued until the 18th week (23 April 2020); the final germination percentage was 80.00 ± 1.65%. The seedling emergence rate measurements were observed from the 17th week (16 April 2020) and were completed at 47.00 ± 3.42% in the 25th week (11 June 2020; Figure 7).

4. Discussion

The measurements of the cutting planes of the B. koreana seeds with time showed that there was no increase in the E/S ratio, and no embryo growth was observed (Figure 6). According to Baskin and Baskin [25], if a seed has an immature embryo, it will grow and germinate within 30 days of incubation under appropriate conditions, referred to as MD. The results of our study indicate that B. koreana seeds do not show MD.

The B. koreana seeds showed water permeability as the seed weight increased by 134% within 24 h of water imbibition (Figure 2). According to Baskin and Baskin [25], if the water absorption rate of the seed is ≤20% of its dry weight within 24 h, it is considered to be impermeable; this phenomenon is termed PY. Our results indicate that B. koreana seeds do not have PY.

According to Baskin and Baskin [26], the seeds of most Berberis plants have PD; seeds with PD can be divided into three types: deep PD, intermediate PD, and non-deep PD. In the case of non-deep PD, it has been reported that dormancy can be broken using a GA₃ treatment. The GA₃ treatment of the B. koreana seeds showed that there was no correlation between the GA₃ concentration and germination percentage, regardless of the light/dark cycle or dark conditions (Figure 3). In addition, the maximum germination percentage was 13.0%, which showed that GA₃ had a limited influence on the seed germination. Therefore, it was concluded that GA₃ did not affect the dormancy breakage; B. koreana seeds do not have non-deep PD.

The germination percentage of the B. koreana seeds increased with an increase in the period of the cold stratification treatment (Figure 4), indicating that it ended their dormancy. According to Zheng et al. [27], among the dormant types, dormancy can be broken with 2–3 months of a cold stratification treatment in the case of intermediate PD and 3–4 months in the case of deep PD. Our results indicate that B. koreana seeds are of the “intermediate PD” type. Similar results have been obtained by previous studies conducted using seeds of the same genus [15,17].

However, in all the experiments, there was no significant difference in the germination percentages between the seeds subjected to the light/dark cycles and dark conditions. Therefore, it appears that B. koreana seeds germinate regardless of the light or dark conditions. In addition, some seeds started germinating during the cold stratification treatment. From these results, it can be hypothesized that some B. koreana seeds have a life cycle that begins within the ground under natural conditions during winter and then germinate as the temperature rises.

Seeds with PD break their dormancy with a cold stratification treatment, but this dormancy may also be broken using a warm stratification treatment [26]. A move-along experiment indicated an increase in the germination percentage under the T1 treatment, whereas the seeds under the T2 treatment showed a poor germination percentage (Figure 5). These results indicate that the dormancy release of B. koreana seeds is affected by cold and not warm stratification treatment.

The phenology experiment showed that most seeds germinated during the period of 6–12 weeks at 5 °C, which is characteristic of winter (Figure 7). These results suggest that natural B. koreana seeds germinate in the ground during winter and seedling emergence occurs when the temperature rises in spring. These observations were similar to those observed in the cold stratification experiment and consistent with those of previous reports [9,12,28,29,30].

5. Conclusions

We hypothesized that (b) B. koreana seeds will absorb water, (c) a low-temperature wet treatment will break the dormancy of these B. koreana seeds, and (d) a hormone treatment (GA₃) will break the dormancy of these B. koreana seeds. Our results show that our (b) and (c) hypotheses were consistent and that a GA₃ treatment had little effect on the seed dormancy (hypothesis d). The most effective method for breaking the dormancy was a cold stratification treatment at 5 °C for 12 weeks. Collectively, the physiological characteristics indicated that, in the natural environment, the seeds of B. koreana begin to germinate at an average temperature of 20 °C after experiencing low-temperature conditions for over 8 weeks in the soil. Thus, the seeds of B. koreana Palibin exhibit intermediate PD among the dormant seed types (hypothesis a).