Introduction
Plant tissue culture techniques are reliable methods for the rapid multiplication of rare and endangered plants (An, 2019). Moreover, in vitro culture techniques can be used not only to grow plants in every season, but also to produce plant seedlings year-round. Significantly, plant tissue culture has been applied to the propagation of various plant species due to its advantage of enabling high plant production efficiency. For the propagation of endangered plant species using in vitro culture systems in South Korea, the mass propagation and restoration of Dendrobium moniliforme (L.) Sw. was reported by Kim et al. (2016), and a micropropagation method via callus for Ranunculus kazusensis Makino was established by Park et al. (2017). In addition, regeneration from embryogenic callus of old wild ginseng (Panax ginseng) via somatic embryogenesis has been reported (Bae and Choi, 2014). Lee et al. (2015) also showed the efficiency of micropropagation of a rare and endangered plant, Abeliophyllum distichum Nakai, and Bae et al. (2014) reported the in vitro propagation of Dendrobium moniliforme through protocorm-like body induction from the culture of leaf, stem and root explants. Recent studies on the successful in vitro propagation of endangered plants native to Korea, namely, Daphne pseudomezereum var. koreana, Iris koreana Nakai, and Lilium dauricum were reported by Bae et al. (2021), Chu et al. (2020), and Kim et al. (2019), respectively.
The genus Kirengeshoma (Hydrangeaceae) is distributed mainly in parts of eastern Asia. In South Korea, there are two plant species endemic to the Korean Peninsula that grow in temperate forests, Kirengeshoma koreana Nakai and K. palmata Yatabe. K. koreana is an important species of forest ecosystems, but native populations have declined due to climate change and other factors, such as insects and human activities. Furthermore, K. koreana has been classified as a class II endangered species according to the South Korean Ministry of Environment (Kang et al., 2007), and also has been designated as an endangered species B2ab (ii, iii, v) on the International Union for Conservation of Nature (IUCN) Red List (IUCN, 2012). Despite the importance of this species, only a few studies related to its environmental characteristics (Cho et al., 2013), vegetation structure (Kang et al., 2007), and genetic diversity (Chung et al., 2013) have been reported. To date, there are no studies on the propagation of K. koreana.
The aim of this study was to develop an in vitro system of stem cutting protocols for clonal propagation of K. koreana. We conducted experiments to determine the effects of nine different basal culture media on the growth of K. koreana, and established the effects of five different carbohydrate sources on its growth.
Research Methods
Plant materials, explant sterilization, and culture conditions
K. koreana seeds collected from Mt. Baegun (35°04′N, 127°39′E) in South Korea were provided by Seoul National University Forests (Fig. 1). Seeds of K. koreana were immersed in 70% ethanol for 1 min and rinsed three times with sterile distilled water. Next, the seeds were disinfected for 5 min in 1% sodium hypochlorite, and rinsed five times with sterile distilled water. Seed coats were removed with a surgical knife and tweezers, and the seeds were cultured in vitro on half-strength Murashige and Skoog (MS) medium (Murashige and Skoog, 1962) containing 20 g·L−1 sucrose and 3 g·L−1 Phytagel (Sigma) to induce germination. Germinants were moved, four per vessel, to square vessels (60 × 60 × 100 mm, SPL Life Sciences, Korea) containing 50 ml of half-strength MS medium supplemented with 30 g·L−1 sucrose and 3 g·L−1 Phytagel. The pH of the medium was adjusted to 5.7 with 0.1 N HCl or 0.1 N NaOH before autoclaving at 121°C for 20 min. All in vitro tissue cultures were maintained at 25 ± 1°C with a 16-h photoperiod of 24 μmol·m−2·s−1 (cool white fluorescent tubes).
Effects of culture media on the growth of K. koreana
The apical segments (at least 1 cm long) collected randomly from a six-week-old K. koreana plantlet grown in vitro were used as explants to determine the effect of basal culture media on the growth of K. koreana. The explants were transferred into square vessels containing 50 ml of nine different basal culture media: full-strength MS, half-strength MS, one-third-strength MS, full-strength woody plant medium (WPM) (Lloyd and McCown, 1980), half-strength WPM, one-third-strength WPM, full-strength Schenk and Hildebrandt (SH) (Schenk and Hildebrant, 1972), half-strength SH, and one-third-strength SH, supplemented with 30 g·L−1 sucrose and 3 g·L−1 Phytagel. Each experiment contained 4 cultures, and the experiments were repeated four times to enhance reproducibility. The data on stem length, shoot fresh weight, leaf width, leaf length, root count, and root length were collected at the end of 8 weeks of culture.
Effects of carbohydrate sources on the growth of K. koreana
To determine the effect of carbohydrate sources on the growth of K. koreana, we used apical segments (at least 1 cm long) collected randomly from a six-week-old K. koreana plantlet grown in vitro. The explants were transferred into square vessels containing 50 mL half-strength SH medium supplemented with 30 g·L−1 five different carbohydrate sources: fructose, galactose, glucose, maltose, and sucrose. Each medium was solidified with 3 g·L−1 Phytagel. Each experiment contained 4 cultures, and the experiments were repeated four times to enhance reproducibility. The data on stem length, shoot fresh weight, leaf width, leaf length, root count, and root length were collected at the end of 8 weeks of culture.
Acclimatization of regenerated plants
Completely regenerated plants with well-developed shoot and root systems were removed from the solid half-strength SH medium, and traces of media on the roots of regenerated K. koreana plantlets were gently removed by thoroughly rinsing them under running tap water. Then, the plantlets were planted into a dome-covered tray containing a sterile mixture of coco peat, peat, and perlite (5 : 1 : 3), and the trays were placed in a culture room with a 16-h photoperiod and a light intensity of 24 μmol·m−2·s−1 provided by cool white fluorescent lamps at 25 ± 1°C. After 10 days, the plantlets were gradually exposed as they acclimatized by gradually opening the vents in the dome covering the tray. The plantlets were watered twice weekly using tap water and then three times weekly after complete removal of the dome. After two weeks, the plantlets (approximately 10 cm in length) were transferred to the greenhouse and potted, into pots containing a mixture of coco peat, decomposed granite, peat, and perlite (5 : 1 : 1 : 3). Finally, the survival rate of the plants was evaluated after acclimatization for 4 weeks.
Statistical analysis
All collected data were analyzed by analysis of variance (ANOVA) using the statistical programming environment R (version 4.2.1), and the normality of the residuals was checked with the Shapiro–Wilk test. Mean values were calculated with standard errors. Significant differences between means were identified using Tukey’s HSD (honestly significant difference) test at a significance level of 5%.
Results and Discussion
Effects of basal culture media on the growth of K. koreana
A basal culture medium is an important element for in vitro culture systems. MS and SH basal media have been widely used and are well-established basal culture media. WPM is the most commonly used basal culture medium for woody plants (Liu et al., 2022). Among the Hydrangeaceae family, micropropagation studies of two Hydrangea macrophylla cultivars have been reported, and the explants were cultured on B5 basal culture medium (Khaing et al., 2018). In addition, in vitro propagation of H. quercifolia and H. macrophylla was cultured on MS basal culture medium (Ledbetter and Preece, 2004; Malyarovskaya and Samarina, 2017). Various media have been used for the Hydrangeaceae family.
To determine the effect of different basal culture media on the growth of K. koreana, explants were cultured for 8 weeks and observed (Fig. 2). The results showed a significant variation in growth (Table 1). The ANOVA results showed that the basal culture medium had a significant effect on the growth of K. koreana (p < .001). The half-strength SH medium was the best condition for stem length, shoot fresh weight, leaf width and root length (3.76 ± 0.12, 0.60 ± 0.06, 1.19 ± 0.05, and 2.83 ± 0.13, respectively). However, the highest percentage increase in root count was observed in half-strength WPM (13.00 ± 0.90). Our study confirmed that the basal culture medium had a significant effect on the growth of K. koreana. In particular, half-strength SH medium was more efficient than any other salt composition for the growth of K. koreana.
Effect of carbohydrate source
Carbohydrates are of prime importance for in vitro organogenesis for plant growth and development. They are supplemented as a carbon source to maintain the osmotic potential of cells and supply carbon because the in vitro conditions are not suitable for photosynthesis (Sumaryono et al., 2012). Many types of carbohydrates, including monosaccharides (fructose, galactose, and glucose) and disaccharides (maltose and sucrose), have been used in plant tissue culture media. Among the disaccharides, sucrose is widely used due to its efficient growth induction and low cost.
In the present study, the results showed (Table 2) that the effect of different carbohydrate sources on the growth of K. koreana was significantly different ( p < .001), with the exception of stem length and leaf width (p = .26 and p = .09, respectively). Maltose was the best condition for shoot fresh weight (0.90 ± 0.09). Although there was no significant difference, sucrose was found to be best for leaf width, leaf length and root length (1.34 ± 0.07, 2.34 ± 0.10, and 3.86 ± 0.19, respectively). Fructose led to the highest root count (18.2 ± 0.70), and a significant difference was observed among carbohydrate sources (p < .001). However, fructose showed the shortest root length (1.14 ± 0.06). In a finding consistent with that of previous studies, sucrose not only was very favorable to the growth of plantlets but also favored better root growth in Centella asiatica L. (Anwar et al., 2005), Pogostemon cablin Berth (Swamy et al., 2010), and Solanum nigrum Linn (Sridhar and Naidu, 2011). In contrast, fructose gave better results than sucrose, maltose and glucose when used for the micropropagation of Stevia rebaudiana (Preethi et al., 2011). In terms of pollen germination, Hirsche et al. (2017) reported that most monosaccharides in the medium did not support pollen germination, whereas disaccharides (maltose and sucrose) differed considerably in their ability to support pollen germination (30.0 and 48.9%, respectively). In our study, the results showed that disaccharides strongly supported stem length of K. koreana, but most monosaccharides had an effect on the induction of short roots of K. koreana. These results can be rationalized assuming that monosaccharides and disaccharides trigger a change of the endogenous hormone metabolism in K. koreana. Further studies are clearly needed to understand how monosaccharides and disaccharides can control endogenous hormone metabolism in K. koreana.
Acclimatization of regenerated plants
Well-rooted plantlets (2–3 cm length with 6–10 leaves) produced in vitro (Fig. 3a) were washed under tap water to remove the solid medium from the roots and then transplanted to a dome-covered tray containing a sterile mixture of coco peat, peat, and perlite (5 : 1 : 3). To maintain high humidity in the culture environment, the plantlets on trays were placed in a culture room with a 16-h photoperiod and light intensity of 24 μmol m−2 s−1 provided by cool white fluorescent lamps at 25 ± 1°C. After 10 days, the plastic covers were removed (Fig. 3b), and plantlets were transferred to plastic pots (Fig. 3c). After 2 weeks, the plantlets were transferred to the greenhouse, where they showed approximately 90% survival after 4 weeks of growth in ex vitro conditions (data not shown).
Conclusion
The aim of this study was to develop protocols for using stem cutting for clonal propagation of K. koreana. We conducted experiments to determine the effects of nine different basal culture media and establish the effects of five different carbohydrate sources on the growth of K. koreana. In the present study, half-strength SH medium was more efficient than any other salt composition for the growth of K. koreana. In terms of carbohydrate sources, there was no efficient carbon source, but sucrose was suitable for the growth of K. koreana. This is the first report on an in vitro propagation and regeneration system for a member of the genus Kirengeshoma. This propagation and regeneration system for K. koreana shows promise in terms of scalability and could help greatly with germplasm conservation and restoration efforts for the species. In addition to the in-flight seedling propagation method, more efficient studies with experiments on multiple-shoot induction and somatic embryogenesis should be conducted.
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