In Vitro Bulb‑Scale Propagation of Lilium leichtlinii var. maximowiczii for Urban Landscaping

Rapid urbanization in Seoul and the surrounding metropolitan area has made establishing urban green networks a key strategy for mitigating environmental degradation and the impacts of climate change [1,2,3]. In recent years, roadside flowerbeds, median strips, neighborhood green spaces, and citizen-led flower road projects have been actively expanded in Seoul [4,5]. Among these, roadside flowerbeds, which citizens encounter most frequently in their daily lives, are a core element determining the quality of urban landscapes [3,6,7]. As a vital component of green infrastructure, roadside flowerbeds serve aesthetic purposes for urban beautification and perform various ecological functions, including reducing fine dust, alleviating the urban heat island effect, and enhancing pedestrian safety [3,6,8]. However, current roadside flowerbeds in Seoul rely heavily on annual bedding plants that are replaced every season. To compensate for this, bulbous plants such as tulips and daffodils have been introduced. Still, these bulbs are largely imported, resulting in high costs and poor persistence under local environmental conditions and, consequently, requiring substantial annual budgets [5,9].

Lilium leichtlinii var. maximowiczii is a promising native bulbous geophyte that is well-adapted to the climate and soils of the Seoul region and blooms in summer, thereby complementing seasonal landscapes [10,11,12]. As a perennial bulbous plant belonging to the Liliaceae family, it is distributed throughout the Korean Peninsula, Japan, northeastern China, and the Ussuri region of Russia [11,12,13]. It develops numerous roots from an ovoid bulb (3–4 cm in diameter), with a stem that grows upright up to 1 m and branches at the top. It bears 2–10 nodding, yellowish-red flowers with strongly recurved tepals densely spotted with dark purple from July to August, providing high ornamental value for use in urban flowerbeds and roadside plantings. In Korea, this species predominantly inhabits well-drained grassy or rocky slopes and forest margins, where it grows under cool, moist conditions with full sun to light shade during the growing season. However, despite its high ornamental potential and good adaptation to local conditions, the use of L. leichtlinii var. maximowiczii in urban flowerbeds has remained limited because its bulbs must be propagated individually, making large-scale distribution difficult. The morphological characteristics and geographical distribution of L. leichtlinii var. maximowiczii in Korea are shown in Figure 1. In addition to its ornamental value, this taxon has recently been recognized as a high-value edible and medicinal lily, and colchicine-induced mutants of L. leichtlinii var. maximowiczii have been developed to improve agronomic traits [14].

For large-scale use of L. leichtlinii var. maximowiczii in roadside flowerbeds and other urban green spaces, it is essential to ensure uniformity of planting material and establish an economically viable mass-production system [2,15,16,17]. Low multiplication rates and slow growth limit natural bulb propagation. Seed propagation is unsuitable for landscaping sites because of low germination rates and considerable genetic variability among individuals, which results in differences in plant height and flowering time in the propagated material [10,12,18,19]. In vitro scale culture technology is an economical way to rapidly increase bulblet production from multiple scales of a single bulb via various organogenic or embryogenic pathways [19,20,21,22,23,24]. This method has the advantage of providing pathogen-free, high-quality, genetically uniform plantlets through clonal propagation [13,25].

In the micropropagation of Korean native lilies, regeneration pathways vary significantly depending on the species. For instance, Lilium hansonii is often propagated through indirect organogenesis using callus-inducing media [26,27]. However, L. leichtlinii var. maximowiczii exhibits a strong tendency for direct bulblet formation from bulb scales rather than callus differentiation [10,15,28]. This developmental characteristic necessitates a specialized approach to optimize combinations of plant growth regulators (PGRs), as the efficiency of direct bulblet induction and subsequent growth is highly sensitive to the specific balance of cytokinins and auxins. Therefore, establishing a tailored protocol that deviates from conventional callus-based methods is essential for the stable mass production of high-quality L. leichtlinii var. maximowiczii plantlets [19.26].

The efficiency of bulblet regeneration and proliferation in scale culture is highly dependent on the type and concentration ratio of PGRs added to the medium [15,26,29,30,31]. In tissue culture of Lilium species, the interaction between cytokinins, which promote cell division and adventitious bud formation, and auxins, which induce root formation and bulblet enlargement, plays a crucial role [1,26]. Previous studies have reported that benzyladenine (BAP) increases the number of bulblet-forming explants and that its combinations with auxins, such as naphthaleneacetic acid (NAA) or indole-3-acetic acid (IAA), can enhance the bulblet formation efficiency [16,23,29,30]. With increasing urbanization, establishing urban green networks is crucial for mitigating environmental degradation and climate change. Urban greening provides essential ecosystem services, including the mitigation of the heat island effect and the improvement of air quality [1,6,8]. However, plant species currently used for roadside greening are often limited to exotic species, necessitating the introduction of native plants adapted to local ecosystems [9,32].

Bulb‑scales of L. leichtlinii var. maximowiczii used in this study were obtained in 2023 from plants cultivated at Seoul Botanic Park (37.78625° N, 127.7424472° E). The plant material originated from wild individuals collected in September 2014 from Gangwon-do, Korea (37°35′34.1″ N, 128°28′22.8″ E). Subsequently, it propagated at the Shingu University Botanical Garden. We acquired these plants in November 2020 and have since maintained it ex situ for conservation and propagation (manager: Hye-min Park, accession No. 10001913). This study was conducted from June 2023 to December 2024 using these ex situ conserved plants as the source of bulb materials. For experiments, healthy, vigorously growing bulbs free of visible pests and diseases were selected and used for explant preparation.

Dry outer scales of the bulbs were removed, and the whole bulbs were rinsed under running tap water for 10 min. Individual scales were then separated and cut into segments of approximately 1.0–1.5 cm in both length and width. To determine the optimal surface sterilization method, three sterilization protocols were tested: (I) Immersion in 70% (v/v) EtOH for 30 seconds (s); followed by 2% (v/v) sodium hypochlorite (NaOCl) for 20 min; (II) Immersion in a 0.1% (w/v) benomyl solution for 1 h; (III) Immersion in 0.1% (w/v) benomyl solution for 1 h, followed by 70% (v/v) EtOH for 30 s, then sequential surface sterilization in 1% (v/v) NaOCl for 20 min and 2% (v/v) NaOCl for 10 min (Figure 2).

After sterilization, the segments were cleaned three times with sterile distilled water in a laminar airflow cabinet and blotted dry on sterile filter paper. To evaluate disinfection efficiency, the segments were cultured on Murashige and Skoog (MS) basal medium [23] supplemented with 87.6 mM sucrose (pH 5.7). Ten explants were placed in each 90 mm Petri dish, and three dishes (30 explants) were used per treatment, with each dish considered as one independent replicate (n = 3). Contamination rate and survival rate were assessed 1 week after inoculation.

Bulb-scale segments sterilized according to the optimal method identified in Section 2.2 were used for bulblet induction. Explants were placed on 90-mm Petri dishes at a density of two explants per dish, with nine dishes (18 explants) per treatment. Two groups of PGRs combinations were tested for bulblet induction, which were designed based on previous reports in Lilium species [15,26,29]: (I) BAP + IAA: 2.2 or 4.4 µM BAP combined with 1.7 or 5.7 µM IAA; (II) BAP + NAA: 4.4 or 8.9 µM BAP combined with 1.6 or 2.7 µM NAA. The MS basal medium without plant growth regulators was used as the control treatment.

All media were supplemented with 87.6 mM sucrose, 0.8% (w/v) agar, and the pH was adjusted to 5.7 before autoclaving. Unless otherwise specified, all cultures described in Sections 2.2–2.4 were maintained in a culture room at 25 ± 1 °C, 60 ± 5% relative humidity, and a photosynthetic photon flux density of 40–60 μmol·m⁻²·s⁻¹ under a 16 h light/8 h dark photoperiod provided by daylight-white T5 LED lamps (15 W, 900 mm, Dooyoung Lighting Co., Ltd., Siheung-si, Gyeonggi-do, Korea) [16,33]. After 3 weeks of culture, the number of bulblets per explant was recorded.

For further bulblet enlargement and proliferation, induced bulblets were subcultured into culture tubes (30 × 150 mm) medium containing double-strength MS mineral salts and 175.2 mM sucrose (pH 5.7) and maintained for an additional 7 weeks [19,22,30].

Plantlets with well-developed shoots and roots were removed from the culture medium, and residual agar was gently washed off with distilled water. The plantlets were then transplanted into cutting boxes filled with a 1:1:1 (v/v/v) mixture of peat moss, perlite, and commercial potting soil [19,30].

During the initial acclimatization period, the trays were covered with transparent lids and intermittently misted to maintain high humidity. After 2–3 days, the lids were gradually opened to lower humidity and increase light penetration. Plantlets were acclimatized in a greenhouse at 20–25 °C for 1 week and then slowly exposed to ambient conditions [18,30].

Contamination rate (%) was calculated as (number of contaminated explants/total explants) × 100. Survival rate (%) was calculated as (number of survived explants/total explants) × 100.

All experiments were conducted using a completely randomized design (CRD) with three replicates (n = 3). For bulblet number and bulblet induction rate, statistical analysis was performed on the mean values, and data are presented as mean ± standard error (SE). Data were analyzed using SAS 9.1 (SAS Institute Inc., Cary, NC, USA), and differences among treatment means were determined using Duncan’s Multiple Range Test (DMRT) at a 5% significance level (p < 0.05). Means within a column followed by the same letter are not significantly different according to DMRT.

The effectiveness of surface sterilization treatments for in vitro establishment of L. leichtlinii var. maximowiczii scale explants differed markedly among the methods tested. The treatment combining 70% (v/v) EtOH and 1% (v/v) NaOCl, which is commonly used in lily tissue culture, resulted in a survival rate of only 33.3%, while explants treated with 0.1% (w/v) benomyl solution alone for 1 h showed the lowest survival rate of 6.7%. In the benomyl-only treatment, the contamination rate reached 93.3%. In comparison, the combined EtOH and NaOCl treatment also showed a high contamination rate of 60.0%, indicating that both conventional treatments were unsuitable for scale sterilization in this species.

In contrast, the combined protocol consisting of 0.1% (w/v) benomyl solution pretreatment followed by stepwise sterilization with 1% and 2% (v/v) NaOCl achieved the highest survival rate of 63.3% with the lowest contamination, and statistical analysis confirmed that this value was significantly higher than those of the other treatments (DMRT, p < 0.05), suggesting that this protocol is the optimal surface sterilization method for effectively reducing contamination while maintaining high explant survival in L. leichtlinii var. maximowiczii (Table 1).

Scale explants of L. leichtlinii var. maximowiczii were cultured under various combinations of PGRs to evaluate bulblet induction and proliferation. Significant differences were observed among treatments (Figure 3). Specifically, the 2.2 µM BAP + 5.7 µM IAA treatment produced the highest number of bulblets (2.5 ± 0.12 per survived explant, total 38), followed by 4.4 µM BAP + 2.7 µM NAA (2.0 ± 0.00 per survived explant, total 24). The mean number of bulblets per explant was calculated as the total number of bulblets divided by the number of surviving explants in each treatment (Table 2).

After 7 weeks of subculturing the differentiated bulblets on MS medium, the plantlets developed into healthy individuals with shoot elongation, dark green foliage, and vigorous white roots (Figure 4). While the 2.2 µM BAP + 5.7 µM IAA combination was most effective in terms of the number of induced bulblets, plantlets obtained from BAP + NAA media were used for subsequent proliferation and acclimatization (Figure 4b; Table 2, treatments 6–8).

The conventional sterilization treatments reduced contamination to some extent but resulted in low survival of the scale explants, most likely because high NaOCl concentrations or benomyl applied alone imposed excessive chemical stress on the fleshy scale tissues, leading to tissue damage. As a result, the protocol combining benomyl solution pretreatment with stepwise NaOCl sterilization can be interpreted as a balanced sterilization strategy that suppresses contamination while maintaining tissue viability [23,34,35].

Similar multi-step sterilization approaches have been reported as effective for other lily species and bulbous plants, where excessive chemical stress can easily damage fleshy scale tissues [18,28]. In this context, our findings that a benomyl solution pretreatment combined with stepwise NaOCl sterilization improves explant survival while controlling contamination are in good agreement with those previous reports. Therefore, the present protocol can be considered a practical and reproducible method for in vitro introduction of L. leichtlinii var. maximowiczii, and it may also be adaptable to related Korean native lilies requiring careful control of both microbial load and explant injury.

The results of the present study highlight the importance of the balance between cytokinins and auxins for efficient bulblet induction in L. leichtlinii var. maximowiczii. Among the treatments tested, 2.2 µM BAP + 5.7 µM IAA produced the highest bulblet induction (2.5 ± 0.12 bulblets per survived explant; Table 2), suggesting that moderate cytokinin levels combined with an appropriate auxin concentration favor direct organogenesis in this species. This pattern agrees with previous reports that BAP in combination with auxins promotes bulblet initiation in Lilium species. The enhanced shoot initiation observed under cytokinin-rich conditions in the present study is in agreement with findings reported for L. longiflorum and oriental hybrids [7,30,36].

The combination of 4.4 µM BAP + 2.7 µM NAA was applied during the development of induced bulblets into plantlets (total 24; Table 2). This response is consistent with previous reports indicating that an appropriate cytokinin–auxin balance plays a crucial role in morphogenesis and bulb development in Lilium species [15,26,29].

Although BAP + IAA combinations, particularly 2.2 µM BAP + 5.7 µM IAA, produced the highest number of bulblets, plantlet development and subsequent acclimatization were observed under the 4.4 µM BAP + 2.7 µM NAA condition. Similar use of different cytokinin–auxin combinations during micropropagation has been reported in other Lilium species [10,11,25,28,37]. These findings suggested that the hormonal requirements for bulblet induction and later growth stages may differ in L. leichtlinii var. maximowiczii.

Regenerated plantlets were transferred to soil and maintained under nursery conditions, demonstrating their potential for further cultivation. The use of different PGR combinations during micropropagation has been widely reported in Lilium and other bulbous ornamental species [16,18,24,30].

Future research should prioritize optimizing cultivation management to promote bulblet enlargement, an essential step for successful flowering in L. leichtlinii var. maximowiczii [10,17,18]. Furthermore, it is critical to conduct field performance trials in practical settings, such as roadsides and urban parks, to evaluate the floral characteristics, viability, and long-term persistence of micropropagated plantlets [5,9,15]. Such studies will provide a more comprehensive assessment of their ecological resilience and functional value within sustainable landscape architecture [1,6,17,32]. In addition, detailed quantitative evaluations of plant growth, bulb enlargement, and flowering performance will be carried out in future field and greenhouse studies [14,25].

This in vitro bulb‑scale propagation system can also support future breeding programs and cultivar development by providing large numbers of genetically uniform bulbs as standardized material for selection and evaluation of native Lilium adapted to urban landscapes.

The present study established an in vitro propagation system for the Korean native bulbous species L. leichtlinii var. maximowiczii using bulb‑scale explants. An optimized surface-sterilization protocol with 0.1% (w/v) benomyl solution pretreatment followed by stepwise NaOCl treatment reduced contamination.

Different PGR requirements were observed for bulblet induction and subsequent growth stages. For bulblet induction, 2.2 µM BAP + 5.7 µM IAA produced the highest number of bulblets (2.5 ± 0.12 per surviving explant). Induced bulblets were subsequently developed into plantlets under the 4.4 µM BAP + 2.7 µM NAA condition.

The protocol developed in this study provides a practical framework for propagating uniform planting materials and may support the potential use of L. leichtlinii var. maximowiczii in urban greening and roadside planting.

During the preparation of this work, the authors used Grammarly and Perplexity (AI-assisted tools) to improve readability and language. After using these tools, the authors reviewed and edited the content as needed and took full responsibility for the publication's content.

The authors thank Wan‑Hee Lee, Head of the Plant Management & Research Division, and Su‑Mi Park, Director of Seoul Botanic Park, for administrative support and facilitation of this project. The authors also thank the staff of Seoul Botanic Park for their assistance with field sampling, cultivation and in vitro propagation of Lilium accessions.

I.‑J.C.: Conceptualization, data curation, formal analysis, supervision, writing—original draft, writing—review & editing. H.‑M.P.: data curation, monitoring, ex situ conservation of Lilium, and management of the Lilium culture green house at Seoul Botanic Park. M.‑J.P.: investigation, tissue culture experiments, investigation and ongoing bulb‑scale culture and maintenance of Lilium accessions.

Not applicable. This study did not involve humans or animals. The plant materials used in this study are not listed in the IUCN Red List, and no endangered or protected species were harmed during sample collection. All plant sampling and subsequent experiments were conducted in accordance with the guidelines and regulations of Seoul Botanic Park.

Not applicable.

All data supporting the findings of this study are included in this article.

This research was funded by the Seoul Metropolitan Government.

The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.