The family Araliaceae comprises approximately 55 genera and 1,500 species, most of which are utilized as medicinal crops (Wen et al., 2001). Among them, Panax ginseng C. A. Meyer is a representative medicinal crop in Korea, and its root has been used as traditional medicine in East Asia for thousands of years owing to its beneficial effects on human health (Potenza et al., 2022). P. ginseng grows naturally only in eastern Asia, particularly between 33° and 48° N latitude (Woo et al., 2004). This perennial herb loses its aerial parts in winter, while the root with rhizome survives and produces new shoots the following spring. Flowering is more strongly influenced by the vegetative growth period than by photoperiod or temperature, requiring more than three years after sowing for the roots to reach a size suitable for flowering (Kwon et al., 1998). Furthermore, the seed production cycle is long and the yield is low (Lee et al., 2023a), limiting the distribution of new cultivars to farmers.

Plant tissue culture techniques offer a promising solution for crops with low propagation efficiency (Koufan et al., 2022). Somatic embryogenesis is an effective method that enables the rapid propagation of genetically uniform plants (Guan et al., 2016), and direct somatic embryogenesis carries a lower risk of genetic variation than organogenesis (Gaj, 2001). In P. ginseng, direct somatic embryogenesis can produce about 40 plantlets from a single seed in one year (Lee et al., 2023a), and acclimatization in artificial soil under greenhouse conditions can achieve survival rates close to 90% (Lee et al., 2021).

However, the emergence, flowering, growth, and ginsenoside content of acclimatized In vitro-grown roots (IGRs) after transplantation under field conditions have rarely been reported. To address this gap, the present study evaluated the adaptability of IGRs produced via direct somatic embryogenesis under field conditions. Specifically, we compared the emergence rates and flowering of IGRs of different weights with seed-derived controls, examined phenotypic traits of aerial and underground parts, and assessed the equivalence of ginsenoside content between acclimatized IGRs and controls.

For the practical application of micropropagation of new ginseng cultivars through plant tissue culture, high adaptability of tissue-cultured plantlets is a prerequisite. In the study of Choi et al. (1998), the survival rate of regenerated ginseng plantlets acclimatized to soil was only 36%. Kim et al. (2013a) reported that the survival rate in the field condition was limited to 46.7 – 53.3% when regenerated plantlets had a root length greater than 4 ㎝ or a weight above 0.4 g. Kim et al. (2016) proposed IGRs as a novel source for soil transplantation, and their high survival rate was later demonstrated by Lee et al. (2021). However, to date, no studies have documented the emergence and growth of acclimatized IGRs after transplantation under field conditions.

In the present study, acclimatized IGRs were classified according to root fresh weight and subsequently transplanted into the field, where the emergence rate was found to increase in proportion to initial root weight (Fig. 4, Table 1). In particular, the acclimated IGRs with a root weight greater than 1.4 g exhibited a very high emergence rate of 88.0%. This finding is in agreement with the report of Lee et al. (2023c), who demonstrated that the emergence rate increased with IGR weight prior to acclimatization in the greenhouse. Our findings provide the first empirical evidence that transplantation of acclimatized IGRs with greater root weight (> 1.4 g) is particularly effective in enhancing survival rates under field conditions.

In ginseng plants germinated from seeds, it is generally typical for young plants of 1 – 2 years of age to produce only a single stem (Lee et al., 2021). In seed-derived ginseng plants, it is generally observed that individuals two years of age or younger produce only a single stem (Lee et al., 2021). Consistent with this, in the present study the control group derived from one-year-old seedlings was predominantly exhibited the single-stem type (Table 1). In contrast, acclimatized IGRs showed a markedly higher proportion of multi-stemmed individuals, and this proportion increased progressively with root weight. Similarly, several studies have reported that ginseng plantlets derived from IGRs exhibit a high frequency of multi-stem type emergence (Kim et al., 2016; Lee et al., 2021). Given that the cultivar ‘Cheonryang’ used in this study is typically characterized by a single stem (Kim et al., 2013b), the multi-stem type observed in the acclimatized IGRs may be attributed to alterations in endogenous plant hormone composition under In vitro conditions, which likely promoted the concurrent development of multiple dormant buds. Kwon et al. (2023) suggested that cytokinin serves as a key regulatory factor in bud initiation and the increase of stem number in P. ginseng. Further research is needed on quantitative profiling of endogenous cytokinins and other plant hormones in IGRs, along with the expression of associated genes.

Consistent with previous reports indicating that flowering in P. ginseng generally occurs only in plants older than three years (Kim et al., 2016), no flowering was observed in the two-year-old control plants in the present study (Fig. 5). Interestingly, some acclimatized IGRs with an initial root weight exceeding 0.8 g exhibited flowering. Flowering has previously been reported in regenerated ginseng plants older than three years (Kim et al., 2013b); however, our study is the first to demonstrate flowering in acclimatized IGRs during the same year of transplantation. Lee et al. (1990) reported the induction of flowering In vitro through ginseng tissue culture, attributing the response primarily to the action of 6-benzyladenine (BA) and GA₃. In the present study, although flowers that bloomed from acclimatized IGRs in the year of transplantation did not produce seeds, they successfully yielded normal seeds in the subsequent year (data not shown). For successful seed production following flowering, sufficient nutrient reserves stored in the root are essential. Thus, it is plausible that acclimatized IGRs lacked sufficient storage capacity to support seed development in the transplantation year. If IGRs with a root weight of more than 2 g can be produced in the future, it is expected that seeds could be obtained immediately after field transplantation, and subsequent studies should be conducted to verify this possibility.

In the present study, the aerial parts of plants derived from acclimatized IGRs were consistently smaller than those of the control plants (Table 2). Consistent with these findings, a previous study reported that IGR-derived plants showed reduced stem length, stem diameter, leaf length, and leaf width compared with two-year-old seedlings (Lee et al., 2021). This difference is attributable to the higher proportion of multi-stemmed plants among tissue-cultured individuals. Because multi stems arise from a single root, they grow competitively and consequently exhibit smaller size than single-stemmed plants (Lee, 1996).

The present study revealed that, among the acclimatized IGR treatments, higher root weight was consistently associated with enhanced growth traits (Table 2). Lee et al. (2023c) likewise reported that plant growth tended to improve in positive correlation with the weight of IGRs. Although size differences were observed, no distinct differences in leaf morphology were found between the control plants and those derived from acclimatized IGRs (Fig. 6). Ginseng plantlets produced through somatic embryogenesis have been reported to be genetically stable in previous studies (Lee et al., 2021; Lee et al., 2023b,c).

After the aerial parts had senesced and the underground parts were harvested, the roots of acclimatized IGRs weighing less than 1.4 g were found to have significantly lower weight and length than those of the control. In contrast, acclimatized IGRs weighing more than 1.4 g displayed root weight and length comparable to the control, while exhibiting a significantly greater root diameter (Table 3). Consistent with our findings, Lee et al. (2021) reported that the roots of acclimatized IGRs cultivated under greenhouse conditions were significantly thicker than those of conventionally grown ginseng. Moreover, the number of rhizomes was higher in acclimatized IGR treatments than in the control, and plants with greater root weight tended to develop more rhizomes. An increased rhizome number in P. ginseng plants derived from tissue culture has also been reported in previous studies, consistent with our observations (Kim et al., 2016). This distinctive root morphology of acclimatized IGRs likely reflects the initial morphological differences between the IGRs used as transplanting material and the seed-derived control plants (Fig. 2). While the controls developed a long taproot with a single rhizome, the IGRs generated through somatic embryogenesis were characterized by a short, thick taproot with multiple rhizomes. A similar trend was observed even after one year of cultivation in the experimental field (Fig. 7), and this morphological tendency remained evident when the tissue-cultured plantlets were grown for up to three years, indicating that the trait is stably maintained over multiple cultivation years (Kim et al., 2013a). It should also be emphasized that these morphological differences were evident even before transplantation, suggesting that they are intrinsic features of somatic embryogenesis-derived plantlets. Previous studies have consistently reported enhanced lateral root development and shortened primary root structure in IGRs compared with seed-derived seedlings (Lee et al., 2021; Lee et al., 2023a). Such traits may result not only from hormonal alterations during In vitro culture but also from differences in culture environments, including high sucrose concentrations, limited gas exchange, light conditions, and relative humidity, all of which are known to affect root development and acclimatization capacity (Kim et al., 2023; Kim et al., 2025).

Ginsenosides are the primary bioactive constituents of P. ginseng. Approximately 300 ginsenosides have been identified in Panax species (Yang et al., 2014), and these compounds are generally classified into two major groups: PPD and PPT. The predominant ginsenosides, such as Rb1, Rb2, and Rg1, exhibit mechanisms of action comparable to those of steroid hormones (Lee et al., 2015). Various biological activities of ginsenosides have been continuously reported (Chung et al., 2016; Shi et al., 2019). To achieve large-scale micropropagation of new ginseng cultivars through plant tissue culture, it is therefore critical to confirm the stability of ginsenoside content in plants derived from tissue-cultured plantlets. In this study, acclimatized IGRs and seed-derived control plants that had been transplanted and cultivated for one year in the experimental field were compared for the contents of 10 ginsenosides, including Rb1 and Rb2 (Table 4). One-way ANOVA across IGR weight groups initially revealed significant differences in several ginsenosides, suggesting that compositional variation might be associated with root size at transplantation. However, ANCOVA with root weight as a covariate demonstrated that these differences disappeared after statistical adjustment. No significant differences remained in the levels of individual ginsenosides, the total ginsenoside content, or the PPD/PPT ratio between IGRs and seed-derived controls. This indicates that the apparent variation among IGR subgroups was largely attributable to weight effects rather than inherent metabolic instability of tissue culture-derived plantlets. These findings are consistent with previous reports indicating that the ginsenoside content of acclimatized IGRs does not differ significantly from that of conventionally grown ginseng roots (Lee et al., 2023b). Together, this provides further evidence that large-scale micropropagation of ginseng via tissue culture does not compromise the stability of its major bioactive constituents.

Through this study, it was demonstrated that acclimatized ginseng plantlets derived from plant tissue culture can stably emerge and flower under field conditions, while showing no compositional differences compared with seed-derived plants. Large-scale micropropagation of new ginseng cultivars via plant tissue culture is therefore expected to facilitate the rapid distribution of elite and stress-tolerant cultivars to farmers, thereby contributing to improved cultivation stability in P. ginseng production.