Improvement of Seed Dehiscence using Plant Growth Regulators and Its Effect on Subsequent Germination and Growth of Panax ginseng C. A. Meyer

INTRODUCTION

Korean ginseng (Panax ginseng C. A. Meyer), a perennial herbaceous plant, is a representative medicinal plant in Korea that has been proven effective in treating various diseases due to its excellent pharmacological properties. Although new propagation methods based on plant tissue culture have been developed (Lee et al., 2023a, b, c), the primary propagation method for ginseng remains seed propagation.

Ginseng requires at least 3 years to produce seeds from the mother plant due to its juvenility (Kim et al., 2016). Ginseng seeds have a morphologically dormant characteristic, in which the seed coat is very hard and closed (Min et al., 2022). Additionally, the zygotic embryo of the seed is too small to germinate. Therefore, after-ripening is essential for embryo development (Kim et al., 2014a). The development of the undifferentiated embryo in ginseng is highly influenced by the flesh and endocarp (Lim et al., 2008).

Ginseng seed ripening takes approximately 18 months under natural conditions. To shorten this period, seeds are artificially subjected to stratification through a process known as dehiscence (Lee et al., 2018). Even after dehiscence, the zygotic embryo remains immature and requires additional cold treatment for more than 3 months to break its physiological dormancy. This cold treatment is known as cold stratification (Suh et al., 2022). Various factors affect the dehiscence rate of ginseng seeds, including variety, seed condition, and seed harvest time (Kim et al., 2014a).

Seeds are an important stage in the plant life cycle (Soppe and Bentsink, 2020). Seed dormancy and germination are complex physiological processes controlled by both endogenous and environmental factors (Yang et al., 2020).

Plant growth regulators, such as abscisic acid (ABA) and gibberellin (GA), have a direct effect on the regulation of seed dormancy and germination, and their balance is an essential factor (Tuan et al., 2021). Exogenous plant growth regulators can improve the dehiscence and germination of ginseng seeds.

For example, GA and cytokinins directly affect the dormancy and germination of ginseng seeds (Kwon and Lee, 1997). In particular, GA has a significant impact on the development of early embryos (Hu et al., 2018), and treatment with GA₃ is known to improve the seed dehiscence rate and promote the breaking of dormancy in ginseng (Kim et al., 2014a; Lee et al., 2016).

In addition, the treatment of ginseng seeds with exogenous GA₃ promotes embryo development (Lee et al. 2018). Impro- vements in germination rates due to GA₃ treatment have been reported in other medicinal plants (Han et al., 2022).

Cytokinins are essential plant growth regulators involved in cell differentiation, the movement of inorganic and organic nutrients, and senescence (Skoog and Armstrong, 1970). Also, cytokinins can affect germination and break dormancy (Prasad et al., 1983; Kabar, 1998).

In previous studies, kinetin, a cytokinin, was found to have a positive effect on dormancy breaking and sprouting in one-year-old ginseng (Park et al., 1979), and improved the dehiscence rate of ginseng (Lee et al., 2018). However, there has been no research thus far on the effects of treatment with plant growth regulators before dehiscence on subsequent seed germination and plant growth, and further research on this is needed.

In this study, the effects of GA₃ and kinetin treatment on the dehiscence of ginseng seeds were confirmed. Additionally, the germination of seeds treated with GA₃ for dehiscence and their subsequent growth characteristics were investigated to evaluate the stability of treatment with growth regulators before dehiscence.

MATERIAL AND METHOD

3. Seed dehiscence

The treated indehiscent seeds were stratified according to a previously described method (Lee et al., 2018). Irrigation was performed twice a day for 45 days after the start of stratification, once a day from days 46 to 80, and once every two days thereafter. After 90 days of stratification, the seeds were considered dehiscent once the seed coats had split. In addition, the contamination rate of the seeds by fungi and bacteria was assessed. Dehiscent seeds were classified into three groups according to the length of the seed coat (Fig. 1): seeds with a width less than 0.9 ㎝ were classified as mild type, between 0.9 ㎝ - 1.5 ㎝ as moderate type, and greater than 1.5 ㎝ as a hyper-type.

Fig. 1.

Types of dehiscent seed according to the size of the seed coat width of P. ginseng C. A. Meyer. (A) mild type, seed coat width: < 0.9 ㎝, (B) moderate type, seed coat width: 0.9 ㎝ - 1.5 ㎝, (C) hyper type, seed coat width: > 1.5 ㎝. Scale bar; 1 ㎝

RESULTS

1. Seed dehiscence

The dehiscence rate of the seeds was investigated after 90 days of stratification. The highest dehiscence rate of 97.2% was observed in the group treated with 10 ㎎/ℓ of GA₃ (Fig. 2A).

Fig. 2.

Effects of plant growth regulators on dehiscence after 90 days of warm stratification in P. ginseng C. A. Meyer. (A) dehiscence and contamination rates according to plant growth regulators treatment, (B) characteristics of dehiscence by GA3 300 ㎎/ℓ treatment before warm stratification. Red arrow; seeds with detached seed coat due to excessive dehiscence, yellow arrow; early germinated seeds, green arrow; contaminated seeds. (C) ratio of dehiscence types by concentration of plant growth regulators.

In the 50 ㎎/ℓ GA₃ treatment group, the dehiscence rate was 89.4%, but there was no significant difference compared to the control group (88.9%). The 100 ㎎/ℓ GA₃ treatment group exhibited a dehiscence rate of 81.8%, which was significantly lower than that of the control group. In the 300 ㎎/ℓ GA₃ treatment group, the dehiscence rate was 42.8%.

No seed contamination was observed in the control group. However, in the GA₃ treatment group, the contamination rate increased with the concentration. In particular, in the GA₃ 300 ㎎/ℓ treatment group, 42.7% of the seeds were contaminated (Fig. 2A), and as the seeds grew excessively, the seed coat fell off naturally; in severe cases, early germination was also observed (Fig. 2B).

There was no significant difference compared to the control group in kinetin concentration from 10 to 100 ㎎/ℓ. However, the 300 ㎎/ℓ kinetin treatment group had a significantly lower dehiscence rate than the control group. Seed contamination was not observed in the kinetin-treated groups.

The effect of the concentration of treated plant growth regulators on the type of dehiscent seeds was investigated. In the control group, the moderate type was most frequently identified, followed by the mild type; the hyper-type was not observed (Fig. 2C).

As the GA₃ concentration increased, the proportion of hyper- types increased. In the 100 ㎎/ℓ GA₃ treatment group, the proportion of hypertype was exceeded 50%, and in the 300 ㎎/ℓ GA₃ treatment group, most seeds were observed to be hypertype. The moderate type was maximized at 83.5% in the 10 ㎎/ℓ GA₃ treatment group, followed by the 50 ㎎/ℓ GA₃ treatment group and the control group. In contrast, in the kinetin-treated groups, the hyper-type was not observed, and the proportion of the moderate type was lower in the kinetin- treated groups than in the control group.

2. Embryo growth

The lengths of the endosperm and embryo were measured for each treatment on seeds for which the dehiscence investigation was completed. GA₃ treatment before dehiscence had a significant effect on both endosperm and embryo length. The length of the endosperm was 4.7 ㎝ in the control group and increased with GA₃ concentration, reaching 5.8 ㎝ in the 300 ㎎/ℓ GA₃ treatment group (Fig. 3A). On the other hand, kinetin treatment slightly increased the size of the endosperm at 50 ㎎/ℓ and 100 ㎎/ℓ compared to the control, but no clear difference was observed.

Fig. 3.

Effects of plant growth regulators on endosperm and embryo length after 90 days of warm stratification in P. ginseng C. A. Meyer. (A) endosperm length, (B) embryo length, (C) ratio of embryo to endosperm length.

Embryo length increased significantly with an increasing GA₃ concentration (Fig. 3B). The embryo length was 2.2 ㎝ in the control group, but it reached 4.3 ㎝ in the 300 ㎎/ℓ GA₃ treatment group. However, no significant differences were observed between the kinetin-treated and control groups.

In the control group, the embryo-to-endosperm length ratio was 0.46 (Fig. 3B). In the GA₃ treatment groups, the ratio was significantly higher than in the control and increased with increasing GA₃ concentration. The ratio was maximized at 0.74 in the 300 ㎎/ℓ GA₃ treatment group. However, no significant differences were observed between kinetin-treated and control groups.

3. Germination

Dehiscent seeds were sown in the soil 90 days after cold treatment, and germination was observed for 30 days. The first germination was observed 3 days after sowing in the 100 ㎎/ℓ and 300 ㎎/ℓ GA₃ treatment groups (Fig. 4A).

Fig. 4.

Characteristics of germination by GA3 treatment before warm stratification in P. ginseng C. A. Meyer. (A) cumulative germination, (B) germination rate, (C) mean germination time, (D) T50.

In the 10 ㎎/ℓ and 50 ㎎/ℓ GA₃ treatment groups, the first germination was observed 7 days after sowing, and the control group germinated 8 days after sowing. The germination rate in the GA₃ treatment group was significantly higher than that in the control group (65.3%). The highest rate was 92.0% in the 100 ㎎/ℓ GA₃ treatment group, and no significant difference was observed depending on the GA₃ concentration (Fig. 4B).

The mean germination time was shortest at 14.3 days and 14.9 days in the 100 ㎎/ℓ and 300 ㎎/ℓ GA₃ treatment groups, respectively. As the GA₃ concentration decreased, the mean germination time gradually increased, and the longest germination time of 19.1 days was observed in the control group (Fig. 4C).

The T₅₀ was also lowest in the 100 ㎎/ℓ and 300 ㎎/ℓ GA₃ treatment groups at 13.2 days and 13.3 days, respectively. As the GA₃ treatment concentration decreased, the T₅₀ increased, reaching the longest time in the control group at 18.3 days (Fig. 4D).

4. Growth of aerial and underground parts

Germinated seeds were examined for the growth of aerial parts 60 days after transplantation into the greenhouse. Stem length was significantly increased in the 50 ㎎/ℓ and 100 ㎎/ℓ GA₃ treatment groups compared to the control group, and the other GA₃ treatment groups were similar to the control group (Table 1).

Table 1.

Characteristics of aerial parts in P. ginseng C. A. Meyer by GA3 treatment before warm stratification.

No significant difference was observed in the stem diameter. In contrast, leaf length was significantly increased in the 50 ㎎/ℓ and 100 ㎎/ℓ GA₃ treatment groups compared with that in the control group. The leaf length of the 10 ㎎/ℓ GA₃ treatment group was similar to that of the control group, whereas in the 300 ㎎/ℓ GA₃ treatment group, the leaf length was significantly reduced compared to the control group. Leaf widths were similar in all treatments except in the 300 ㎎/ℓ GA₃ treatment group.

The characteristics of the underground parts were also investigated. The longest root length was observed in the 50 ㎎/ℓ GA₃ treatment group, although no significant difference was observed compared to the other treatment groups, including the control (Table 2).

Table 2.

Characteristics of underground parts in P. ginseng C. A. Meyer by GA3 treatment before warm stratification.

The root diameter was largest in the 10 ㎎/ℓ GA₃ treatment group, but there was no significant difference compared to the other treatments. Similarly, the root weight was highest in the 50 ㎎/ℓ GA₃ treatment group, although no significant difference was observed between the treatments.

DISCUSSION

Dehiscence, the starting point of seed production, is an important process in ginseng cultivation. Ginseng seeds are hard seeds that can take up to 21 months to germinate without artificial treatment (Lee et al., 2018). Therefore, the after-ripening process of seeds is essential, and various attempts have been made to improve their dehiscence rate. The dehiscence rate has been improved by endophytic microbes (Kim et al., 2017), as well as by treatment with plant growth regulators (Kim et al., 2014a; Lee et al., 2018). Some farmers treat seeds with GA₃ for stable dehiscence. However, research on the appropriate concentrations of plant growth regulators for seed dehiscence and their effects on plant growth remains scarce. In addition, in South Korea, the use of plant growth regulators for the dehiscence of ginseng seeds requires an official safety evaluation and registration of the substances. However, there is a lack of information regarding this process.

In this study, the effects of treatment with GA₃ and kinetin on dehiscence in P. ginseng C. A. Meyer were evaluated. The dehiscence rate in the 10 ㎎/ℓ GA₃ treatment group improved by 9.3% compared to the control group (Fig. 2A). On the other hand, the 50 ㎎/ℓ GA₃ treatment group showed no significant difference compared to the control. When treated with more than 100 ㎎/ℓ GA₃, the dehiscence rate significantly decreased compared to the control. In particular, when 300 ㎎/ℓ of GA₃ was applied, the dehiscence rate sharply decreased as the contamination rate increased. Our results contradict a previous study, which found that the dehiscence rate increased as the GA₃ concentration increased up to 300 ㎎/ℓ. These varying results may be due to differences in cultivar and harvested seed conditions, as well as differences in the environmental conditions under which the seed-mother plants grew during the test year, resulting in differences in the maturity state of the seeds. Additionally, in this study, contaminated seeds were considered indehiscent, which is thought to have influenced the difference in dehiscence rates between the present study and those reported in previous studies. A similar study was reported in Eleutherococcus senticosus, a close relative of ginseng, where the dehiscence rate increased with the GA₃ concentration up to 500 ㎎/ℓ, but at 700 ㎎/ℓ, the dehiscence rate decreased as the contamination rate increased (Lim et al., 2008). The increase in contamination is believed to be due to excessively high GA₃ concentrations, which overly stimulated the cell development of the zygotic embryo, hindering its normal differentiation and maturation. Further research is required to elucidate this issue.

In contrast, kinetin treatment did not cause a significant difference in seed dehiscence or contamination rates compared to the control group (Fig. 2A). Contrast to the results of the present study, Lee et al. (2018) found that treatment with 50 ㎎/ℓ of GA₃ or 100 ㎎/ℓ of kinetin significantly increased the dehiscence rate compared to the control. Kim et al. (2014b) reported that the treatment of indehiscent seeds with more than 100 ㎎/ℓ of GA₃ significantly increased the dehiscence rate, while treatment with 10 ㎎/ℓ of GA₃ showed no difference from the control.

An evaluation of the effect of plant growth regulators on the dehiscence type revealed clear differences between the GA₃ treatment groups (Fig. 2C). In the control group, most of the dehiscent seeds were of the moderate or mild type, whereas very few were hyper-types. In contrast, in the GA₃ treatment groups, as the concentration increased, the proportion of hyper-types increased, and in the 100 ㎎/ℓ GA₃ treatment group, more than half of the seeds were hyper-types.

This study is the first to report on the effect of GA₃ treatment on the type of dehiscent seeds, demonstrating that GA₃ treatment has a significant effect not only on the dehiscence rate but also on the type of dehiscent seeds. In contrast, no significant difference was observed between the kinetin treatment groups and the control group before dehiscence.

The size of the endosperm in ginseng seeds is known to grow rapidly until 30 days after fertilization, and does not change significantly thereafter. Before seed dehiscence, the length of the embryo is about 500 ㎛, increasing rapidly after 1 month (Kim et al., 2014a). However, there have been no previous studies on changes in embryo and endosperm size caused by treatment with plant growth regulators before dehiscence.

In this study, the effects of GA₃ and kinetin treatments on endosperm and embryo growth before dehiscence were evaluated. GA₃ treatment before dehiscence increased the endosperm size, and as the concentration of GA₃ increased, the endosperm size significantly increased (Fig. 3A).

For example, the endosperm size in the 300 ㎎/ℓ GA₃ treatment group increased by over 20% compared to the control group. Embryo size also increased after GA₃ treatment before dehiscence, increasing with the concentration (Fig. 3B). The embryo size of the 300 ㎎/ℓ GA₃ treatment group increased by approximately two-fold compared to the control group. The ratio of embryo length to endosperm length also increased as the GA₃ concentration increased, reaching 0.74 in the group treated with 300 ㎎/ℓ GA₃ (Fig. 3C).

After dehiscence, in seeds exposed to low temperatures, the abscisic acid (ABA) concentration decreases as the GA₃ concentration rapidly increases (Kim et al., 2014b). External GA₃ treatment increased GA and decreased ABA in the seeds, thereby promoting embryo development in the GA₃ treatment group. Similarly, Lee et al. (2018) found that GA₃ treatment had a positive effect on seed maturation. However, kinetin treatment showed no differences, except for some parts of the endosperm length, compared to the control group (Fig. 3A, B, C).

Germination is an important process for plant development and is affected by endogenous GA and ABA (Chen et al., 2021). As the amount of GA in the seed increases, ABA activity decreases, thereby inducing germination (Chen et al., 2008). In ginseng seeds, the GA content increases rapidly starting 30 days after dehiscence is completed, after which the ABA content tends to decrease rapidly (Kim et al., 2014b).

In this study, the germination characteristics of the control and GA₃ treatment groups were evaluated, excluding the kinetin treatment group, which showed little improvement in dehiscence. In the GA₃ treatment group, as the treatment concentration increased, the initial germination period became shorter than that of the control, and GA₃ treatment before dehiscence was considered to have a positive effect on breaking dormancy (Fig. 4A). The germination rate was low in the control group (65.3%) (Fig. 4B), and cold treatment for 90 days was thought to be insufficient to break dormancy (Lee et al., 2018).

A previous study reported that the low-temperature period needed for breaking dormancy in ginseng seeds was more than 90 days, and that treatment at alternate temperatures was effective (Lee et al., 2016). The germination rate of the GA₃ treatment group was significantly higher than that of the control group (Fig. 4B).

Similarly, Lee et al. (2016, 2018) reported that treatment with 50–100 ㎎/ℓ GA₃ promoted breaking dormancy in ginseng seeds. Additionally, the mean germination time and T₅₀ of the GA₃ treatment group at 50 ㎎/ℓ or higher were significantly lower than those of the control group (Fig. 4C and Fig. 4D).

Lee et al. (2018) reported that GA₃ treatment in dehiscent seeds improved the germination rate and decreased the mean germination time and T₅₀ values. For seed germination, a certain concentration of gibberellins must be present in the seeds (Groot and Karssen, 1987; Karssen et al., 1989), and as the low-temperature treatment period passes, the concentration of synthesized in the seed increases, enabling seed germination (Ross and Bradbeer, 1971). Shortening the post-ripening period and promoting seed germination by GA₃ treatment are also known to occur in P. notoginseng, which is closely related to ginseng (Ge et al., 2023).

GA is a plant growth regulator that affects seed dormancy and germination, as well as growth and development. Although studies have been conducted to improve the dehiscence rate of ginseng seeds by GA₃ treatment before dehiscence (Kim et al., 2014a; Lee et al., 2018), research on its effects on subsequent growth is scarce.

The purpose of this study was to evaluate the effects of GA₃ treatment before dehiscence on the growth of germinated plants. In the treatment groups above 50 ㎎/ℓ GA₃, the lengths of stems and leaves were significantly increased compared to those in the control group, but no significant differences were observed in terms of stem diameter or leaf width (Table 1).

The effect of improving the growth of the aerial parts by GA₃ treatment on plants has been previously reported (Hong et al., 2021). When zygote embryos of P. quinquefolius were placed on a medium containing GA₃, stem length increased compared to that of the control (Hovius et al., 2007).

On the other hand, both the leaf length and width of the 300 ㎎/ℓ GA₃ treatment group decreased compared to the control, and excessively high GA₃ treatment before dehiscence was considered to have a negative effect on the growth of the aerial plant parts of ginseng.

GA₃ was previously found to promote the secondary growth of roots in 1-year-old ginseng (Hong et al., 2021). However, in the present study, the promotion of growth in the underground parts by treatment with gibberellin before dehiscence was not observed (Table 2). Based on these results, GA₃ treatment before dehiscence was considered to decompose as germination progressed, having no effect on the subsequent growth of the underground parts in P. ginseng C. A. Meyer.

In this study, the effects of plant growth regulators on the development and germination of ginseng seeds before dehiscence were evaluated. In addition, subsequent growth characteristics were evaluated, confirming that the plants were able to develop into normal plants. In conclusion, our research findings suggest that the use of plant growth regulators at appropriate concentrations during the dehiscence of ginseng seeds can be applied in agricultural practices. Subsequent processes are expected to be carried out to officially allow the use of plant growth regulators for ginseng seed stratification.

Acknowledgments

This work was carried out with the support of "Cooperative Research Program for Agriculture Science and Technology Development (Project No. PJ01476002)" Rural Development Administration, Republic of Korea.

This study was supported by 2024 the RDA Fellowship Program of National Institute of Horticultural & Herbal Science, Rural Development Administration, Republic of Korea.

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