Identifying Optimal Parts of Oriental Lily Bulbs for Large-Scale Propagation Using Tissue Culture Bulb Induction

Abstract

Lily (Lilium spp.) is a perennial ornamental plant valued for its striking ornamental value and the edible and medicinal properties of its bulbs. Compared to other lilies, Oriental lilies are characterized by their large flowers and strong fragrance, making them one of the most popular types of lilies on the market. It is important to identify optimal parts of lily bulbs for rapid propagation of tissue culture seedlings. In this study, bulb scales of the Oriental lily ‘Pacific Ocean’ were used to explore the optimal parts for the tissue culture bulb induction. After 30 days of inoculation of lily scales in the induction medium, our results showed that Middle Central regions of the Inner layer scales (MCI) had the highest induction rate of 90.27 % among all regions from all layers of scales. In scales of all layers, the basal parts had the best longitudinal induction effects, followed by the middle and apical parts. Among all layers, the inner layers also had the lowest rates of contaminations. When taking both induction rate and contamination rate into account, MCI also represents the best choice among all compared parts. In summary, our results formed a practical guide for large-scale propagation of Oriental lily using tissue culture seedlings.

1. Introduction

Lily (Lilium spp.) is a perennial herbaceous flowering plant in the family Liliaceae, comprising over 110 accepted species across 7 taxonomic sections [1,2]. Owing to their large, fragrant, and colorful flowers with extended vase life, lilies are among the most economically valuable ornamental plants globally, widely cultivated as cut and potted flowers [3,4]. They are also commonly used in home gardens for aesthetic purposes [5], ranking fourth among the top ten cut flowers in international floriculture trade [6]. In response to growing demand, breeding programs have intensified to develop dual-purpose Lilium cultivars with ornamental and edible value, particularly in China [7]. For example, Yunnan province produced over 8.5 billion cut stems in 2022 across 17,247 hectares, making lilies a dominant floricultural crop. However, 70.24% of bulb flowers used in China are still imported highlighting the need for high-quality domestic bulb production and germplasm resource development [8,9]. Beyond their ornamental value, lily bulbs are also prized for their edible and medicinal properties. Extracts from lily bulbs have demonstrated antioxidant and anti-inflammatory effects [10]. However, some wild species contain compounds like colchicine, which can oxidize into toxic dimers in the human body [11,12]. Consequently, most commercial lilies are products of long-term domestication and hybridization, resulting in nine major hybrid groups, among which Asiatic hybrids, Oriental hybrids, Lilium longiflorum, and trumpet lilies dominate the global market [13,14,15]. China, a major center of lily biodiversity, is home to around 55 species and 18 varieties, primarily in Sichuan and Yunnan provinces [16]. However, edible germplasm remains limited, with cultivars like Lanzhou lily (Lilium davidii var. willmottiae), Yixing lily (Lilium lancifolium Thunb), and Longya lily (Lilium brownie var. viridulum Baker) being the primary examples.

Lily can be propagated through several methods, including seed propagation, bulblet division, bulbils, scale cutting, and tissue culture [17,18]. Seed propagation, though mainly used in breeding, is limited in commercial use due to its long growth cycle and genetic instability [19]. Asexual methods dominate cultivation, with bulblet division offering low multiplication efficiency and potential genetic degradation over time [20]. Bulbil propagation, while convenient and high-yielding, is restricted to certain species such as Lilium lancifolium. Tissue culture, though technically demanding and relatively costly, is a superior propagation method due to its rapid multiplication rate and ability to produce virus-free, genetically uniform plantlets within approximately 70 days [21]. It has become essential for the large-scale production and breeding of elite cultivars. Micropropagation enables the efficient regeneration of high-quality plants, with a single large bulb capable of producing up to one million bulblets over two years [22,23]. Among various explants used in Lilium tissue culture, bulb scales are most commonly employed due to their strong regenerative capacity, typically yielding 3–4 bulblets per scale, depending on cultivar and size [24]. However, bulb scales fleshy, moisture-retentive nature makes them susceptible to microbial contamination, necessitating the use of optimized sterilization protocols tailored to each cultivar [25]. Additionally, studies have shown that regenerative efficiency varies across different scale regions. Receptacles and outer scales, in particular, have demonstrated high differentiation potential, making them ideal for in vitro regeneration [26]. There are significant morphological and physiological differences between the middle layer scales and the outer layer scales, which affect the formation of lily buds [27]. Under high temperature conditions, the outer and middle scales of Lilium longiflorum ‘White American’ have a more significant inducing effect [28]. Under 25 °C conditions, the outer, middle, and inner layers of Lilium longiflorum bulbs have different differentiation conditions. The middle layer is the ideal starting material for bulb propagation experiments, with the largest bulb weight, surface area, fresh weight, and dry weight [29].

Despite progress in Lilium micropropagation, comparative studies on the regeneration potential of different bulb parts, such as outer and inner scales, basal plates, and root zones, remain limited. Traditional propagation methods like seed sowing, bulb division, and scale cutting are constrained by genetic variation, disease susceptibility, and low commercial viability. In contrast, tissue culture enables rapid, uniform, and disease-free plantlet production, supporting large-scale cultivation and cultivar development. Oriental lilies occupy a unique premium position in the floral market due to their large, vibrant blooms and rich, fragrant aroma. Oriental lilies occupy a unique premium position in the floral market due to their large, vibrant blooms and rich, fragrant aroma [5]. Consequently, there is significant market demand for Oriental lily bulbs. Investigating the key factors in lily bulb propagation is crucial for enhancing bulb propagation efficiency. This study uses the Oriental lily cultivar ‘Pacific Ocean’ to evaluate the in vitro induction efficiency of different scale regions, aiming to identify the most responsive part for regeneration and provide a theoretical basis for efficient commercial propagation.

2. Materials and Methods

2.1. Test Material

The test material selected was the Oriental lily ‘Pacific Ocean’. Lily bulbs were obtained from Wode Plant Horticulture Co., Ltd., Kunming, China, on 20 December 2024. Lily scales from bulbs that were plump, undamaged, free of pests and diseases, and 10–12 cm in diameter were selected as the test material.

2.2. Selection and Disinfection of Scales for External Grafting

Taked out the lily bulbs obtained by the research team, removed the roots, and selected healthy scales without yellow spots. First, divided these scales into outer, middle, and inner layers, placed them in three separate bottles, labeled them O, M, and I (Figure 1a,b). Respectively, washed them 2–3 times with dishwashing liquid, rinsed them 2–3 times with sterile water, and then placed them in a super-clean workbench for sterilization. Added 75% ethanol and soaked for 1 min. Then transfered the material to 0.1% mercury chloride [30] and soaked for 12 min. During the soaking process, continuously agitated the sterilization container. After sterilization was completed, rinsed three times with sterile water and set aside for later use.

2.3. Induction of Different Regions of Scales

Used forceps, removed the sterilized scales and placed them on sterile kraft paper. The scales were cut, normally with sanitized scalpels, at the one-third points along both the longitudinal and transverse axes, dividing them into six sections. The left and right edges along the longitudinal axis were considered parts of the same sections. The vertical axis divides the scale into two parts: the Side (S) and the Center (C), while the horizontal axis divides it into three parts: the Apical (A), Middle (M), and Basal (B) regions. The diagram of different regions of the outer scales of the lily is shown in Figure 1. Then, the cut pieces of scales were inoculated into the induction medium. The formula was as following: 4.43 g/L Murashige and Skoog medium [31] (MS, Basebio, Hangzhou, China) + 30 g/L sucrose (Xilong Science, Shanghai, China) + 5.5 g/L agar (Aladdin Reagent, Shanghai, China) + 1 mg/L 6-BA (Shanghai Yuanye Bio-Technology, Shanghai, China) + 0.1 mg/L NAA (Shanghai Yuanye Bio-Technology, Shanghai, China), pH = 5.8. Each bottle contained 6 explants, with 6 bottles inoculated per region. Two bottles constituted one replicate, with a total of 3 biological replicates. Then, cultures were maintained at 24 ± 1 °C under a 16-h photoperiod with cool white fluorescent lighting at 40 μmol/m²/s, in a growth room with 50–65% relative humidity.

2.4. Observation of Differences Between Different Induction Methods

Thirty days after inoculation, the status and morphology of tissue culture balls induced in different locations were observed, and the number of contaminated samples was counted to calculate the Induction Rate (IR) and Contamination Rate (CR).

$$IR={\displaystyle \frac{S\times 100}{U}},$$

(1)

where S is the number of sprouts, and U is the total number of uncontaminated explants.

$$CR={\displaystyle \frac{C\times 100}{I}},$$

(2)

where C is the number of contaminated explants, and I is the total number of inoculated explants.

Thirty days after inoculating the scales onto the culture medium, for statistical convenience, we defined any scale exhibiting bacterial or fungal contamination as a contaminated scale, regardless of the extent of the contaminated area. A scale on which a bulbous growth appeared on its surface was considered a successfully induced scale.

2.5. Data Statistics

The experimental data were organized using Microsoft Excel 2021 software (Microsoft, Redmond, NY, USA), and IBM SPSS Statistics 26 (IBM, Armonk, NY, USA) and Duncan’s multiple range test and t-test was applied to determine significant differences at $p<0.05$. GraphPad Prism 10 (Dotmatics, Boston, MA, USA) were used for statistical analysis and graphing.

2.6. The Induction-Contamination Line for Identifying the Optimal Region

Firstly, the Induction rate and contamintation rate values of different regions from different layers were plotted in a scatter plot. In the scatter plot, x-axis represented the induction rate of a sample, while y-axis indicated the pollution rate of the sample. We then performed a linear fit for all data points. We named this line as Induction-Contamination line (IC line). Theoretically, the intersecting point between x-axis and the IC line represented the best experimental setting, since the highest Induction rate and the lowest Contamination rate (0) were achieved at this point. Therefore, the IC line offered a simple model for identifying the optimal experimental setting by choosing the real sample that was closest to the x-intersecting point of the IC line.

3. Results

3.1. The Effects of Different Layers on the Induction of Lily Scales

In tissue culture of lily, different layers of scales for tissue culture balls exhibit different induction capacity and distinct differentiation phenomenon. To identify the optimal layers for tissue culture, we chose the Oriental lily “Pacific Ocean” bulb. As shown in Figure 1a,b, the outer layers are those from the largest distances to the centers of bulbs. Scales of outer layers are normally larger than those from middle and inner layers (see Figure 1c).

During the induction of tissue culture balls, our results showed that the induction rate of Inner layer (I) scales was significantly higher than that of Outer layer (O) and Middle layer (M) scales, with an average induction rate as high as 54.5%. In contrast, the induction rate of outer layer scales was only 21.2%, and that of middle layer scales was 27.2% (Figure 2a).

The contamination rate of lily scales is extremely high, mainly due to fungal or bacterial infection. As shown in Figure 2b, the contamination rate of Inner layer (I) scales is 28.67%, which is significantly lower than that of Outer (O) and Middle layer (M) scales. Thirty days after inoculation, adventitious buds grew on the lily scales. It could be observed that the inner layer (Figure 2e) of the lily scales grew better than the outer layer (Figure 2c) and middle layer (Figure 2d).

3.2. The Effects of Horizontal Differences on the Induction of Lily Scales

As shown in Figure 3a, the scales were divided vertically into Side (S) and Central (C) parts. The induction rates of the Outer layer (O) scales at the Side (S) and Central (C) portions were approximately 21.22% (Figure 3b), with an average contamination rate of 74.63% (Figure 3c). For middle layers, the induction rates of the Side (S) and Central (C) sections were around 27.19% (Figure 3d), with an average contamination rate of 64.18% (Figure 3e). For inner layers, the induction rates of the Side (S) and Central (C) parts are around 54.51% (Figure 3f), with an average contamination rate of 28.65% (Figure 3g). In summary, our results indicated that within the same layer of scales, there was no significant difference in the induction rate and contamination rate between the central and side portions of the transverse scales.

3.3. The Effects of Different Longitudinal Locations on the Induction of Lily Scales

The induction effects of different longitudinal regions of the same lily scales also exhibit relative differences. As shown in Figure 4a, the scales were longitudinally divided into three parts: Apical (A), Middle (M), and Basal (B). The results of our study showed that the average induction rate of the Basal region (B) of the Outer layer (O) scales was 29.93%, significantly higher than that of the Middle layer (M) and Apical layer (A) scales (Figure 4b). However, the contamination rates of the scales from the three regions (Figure 4c) showed no significant differences, all around 75%. The average induction rate of the Basal part (B) of the Middle layer (M) scales was 38.72%, which was significantly higher than that of the Middle (M) and Apical (A) scales (Figure 4d). Additionally, the contamination rate of the basal scales was 43.83%, which was significantly lower than that of the Middle (M) and Apical (A) scales (Figure 4e). There were no significant differences in the induction rates of the Apical (A), Middle (M), and Basal (B) regions of the Inner layer (I) scales, with average induction rates around 50% (Figure 4f). However, the contamination rate of the Basal (B) scales was low, at only 17.33%, significantly lower than that of the Apical (A) scales (Figure 4g).

3.4. The Effect of Different Parts of Each Layer on the Induction of Lily Scales

The induction rate and contamination rate vary significantly across different regions of the same layer of lily scales. This experiment divided the lily scales into six regions (Figure 1d). The induction rate and contamination rate for the these regions of each scale layer are shown in Figure 5. In the Outer layer (O) scales, the induction rates of the Basal Side (BS) scales were the highest at 34.37%, significantly higher than that of the Apical Side (AS), Middle Side (MR), Acpical Central (AC), and Middle Central (MC) scales (Figure 5a). There were no significant differences in contamination rates among the six regions of the outer layer scales, all around 74.63% (Figure 5b). In the middle layer scales, the Basal Central (BC) scales had the highest induction rate at 41.1%, significantly higher than the Apical Side (AS), Middle Side (MR), Apical Central (AC), and Middle Central (MC) scales (Figure 5c). The Basal Central (BC) has the lowest contamination rate at only 41.1% (Figure 5d). In the inner scales, the Middle Central (MC) scales had the highest induction rate at 90.27%, significantly higher than other regions (Figure 5e), and the contamination rates of the Basal Side (BR) and Middle Central (MC) scales were low, at only 14% (Figure 5f).

In Figure 5g, we prepared a scatter point using all available regions in the Outer, Middle and Inner layers. As shown in Figure 5g, samples from Inner layer located on the lower-right corner, the samples from Outer layer located on the upper-left corner, and samples from Middle layer located between those from Inner and Outer layers. As stated in Materials and Methods, we then performed a linear fit for all data points to obtain the Induction-Contamination line. MCI was the closest point to the x-intersecting point of the IC line. Therefore, as introduced in Section 2.6, MCI, with the highest induction rate and the second lowest contamination rate, was chosen as the optimal region for inducing bulbous growth.

4. Discussion

Lily scales are important organs for reproduction and vegetative growth in lilies, providing nutrients for their growth processes. Differences exist in their formation, and the induction capacity varies depending on the location where the scales grow [32,33,34,35]. Lilies are typical polyploid plants in nature, possessing complex genetic backgrounds. Different lily cultivars exhibit distinct growth characteristics, and variations in the development of lily scales result in slightly different outcomes when being propagated using scales [2]. Oriental lilies and Lilium longiflorum belong to different cultivars. In this study, scales from the Oriental lily ‘Pacific Ocean’ were used to induce tissue culture bulbs, with the overall induction rate following the pattern: inner layer > middle layer > outer layer. This result differs from those of previous studies that middle-layer scales from Lilium longiflorum ‘White American’ exhibit the best induction efficacy [28,29]. The discrepancy may stem from differences in the cultivated varieties and hormone concentrations used in the induction medium, leading to variations in scale induction and differentiation [36,37]. Therefore, it is more reliable to examine the optimal bulb regions in large-scale commercial production using similar strategies of this study for other Oriental lily cultivars.

In this study, the induction rates at the bases of the outer and middle scales were significantly higher than those in the middle and upper regions, while there was no significant difference in induction rates among the inner scales. This result may be due to differing hormone levels in different regions, which in turn affect their efficacy on scale growth. Basal regions may contain higher cytokinin activity, which promotes bud initiation, while the upper and apical regions, being more mature, likely have reduced hormonal responsiveness and diminished regenerative capacity. This spatial difference in endogenous hormone balance could therefore explain the variation in induction efficiency observed across scale regions. In previous studies, the balance of endogenous hormone ratios has been shown to influence the germination of tulip bulbs. Since scales at different layers of lilies are at distinct developmental stages, their endogenous hormone levels may vary, leading to differing induction effects among scales at various layers [38,39,40]. Alternatively, it could be resulted from the fact that the inner scales of the lily, particularly their bases, are closer to the bulb base and contain more meristematic cells, with densely distributed vascular bundles, facilitating the transport of nutrients and hormones from the culture medium to the explants. Vascular bundles serve as the circulatory system of plants, primarily composed of two types of conducting tissues: xylem and phloem, which play crucial roles in transporting nutrients and plant hormones [41,42]. In a previous work [43], the development of vascular bundles influences the transport of plant nutrients, thereby affecting the yield of various crops. We speculate that the growth of lily bulbs may also follow this pattern. Among the six regions of the same scale, the central middle region exhibited the highest induction efficiency, significantly higher than other regions. This may be because the middle region scales are thicker, containing the most nutrients, making it relatively easier to form buds [24,27,44,45,46]. In contrast, the upper region, due to its high maturity and cellular differentiation, presents greater induction challenges, is prone to contamination, and has a low induction rate of only 10%. This indicates that higher tissue maturity and advanced cell differentiation reduce cellular competence for regeneration, as highly differentiated cells have limited ability to re-enter the cell cycle or respond effectively to growth regulators. Therefore, when conducting industrial-scale propagation of lilies, it is advisable to remove the upper part of the scales before proceeding with tissue culture propagation.

Plant growth regulators are natural or synthetic compounds that influence plant physiology and developmental processes. At the cellular level, plant growth regulators also play a crucial role in cell differentiation. During somatic embryogenesis, the ratio of auxin to cytokinin influences the induction of callus formation and organ development [47]. In previous studies, various plant growth regulators have been employed to enhance tissue culture efficiency in lilies, induce callus formation, develop somatic embryos, or directly form bulbils [48,49,50]. In this study, we employed 1 mg/L 6-BA and 0.1 mg/L NAA auxin formulation to directly induce bulb formation, observing notable variations in the induction outcomes. These results were based on a single medium formulation primarily aimed at identifying the optimal scale region and did not represent the ultimate induction capacity of each region. Higher induction efficiency may be achieved by using alternative auxin formulations.

We understand that temporary immersion system (TIS) represents a novel method in plant biotechnology. This method involves periodically immersing all or part of plant tissue in a nutrient medium, then draining the nutrient solution to expose the tissue to air for growth [51]. A recent study indicates that TIS demonstrates superior efficiency and cost-effectiveness in the production of Pennisetum × advena ‘Rubrum’ compared to agar culture [52]. Given this outstanding industrial benefit, it is advisable to employ TIS to identify optimal lily bulb parts to enhance commercial-scale production of lily bulb scales in future studies.

The disinfection and sterilization of lily scales is a critical issue in lily tissue culture. Under the same disinfection conditions, different parts of the lily scales often exhibit significant differences in disinfection efficacy [25]. In this study, the scales were first immersed in 75% ethanol for 1 minute, then transferred to a 0.1% mercury chloride solution for 12 minutes, rinsed with sterile water 2–3 times, and inoculated into induction medium. After 30 days of cultivation, it was clearly observed that the outer layer of scales had the poorest disinfection effect, with severe contamination, followed by the middle layer, while the inner scales showed the best disinfection efficacy, with the lowest contamination rate of 30%. The contamination rates of the outer scales reached as high as 93%, possibly because this layer of scales were directly in contact with the soil and were constantly exposed to a moist, pathogen-rich environment, leading to incomplete disinfection [53,54]. Additionally, within the same layer of lily scales, the contamination rate in the apical part was higher than in the basal and middle parts, which was potentially due to the upper tissue being more tender and having weaker stress resistance [20,22].

Currently, China’s lily bulb resources primarily rely on imports, with domestic propagation techniques still needing improvement. Utilizing tissue culture methods can enhance the propagation efficiency of lily bulbs and yield healthy, disease-free bulbs [55]. During the induction of lily tissue culture balls, it is important to select suitable regions of scales to enhance induction rates and to achieve rapid and efficient production of lily tissue culture balls. In this study, we investigated the induction efficiency of different parts of lily scales for in vitro culture. Overall, the Middle Central region of Inner layer (MCI) scales showed the best induction effect (Figure 6, Step 3, left). The induction rate of the entire inner layer scales was significantly higher than those of the corresponding regions of other layers. If the middle and outer layer scales were used as explants, the basal region scales could be selected (Figure 6, Step 3, right).

To examine the relationship between induction rate and pollution rate, we applied a scatter plot of induction rate and pollution rate for scales from different regions in different layers. Overall, MCI regions exhibited the highest induction rate and lower pollution rate among all regions from all different layers (as shown in Figure 5g), reflecting that MCI is the optimal choice for commercial expansion. In the future, it is still needed to validate the results in this study in commercial production of lily bulbs since the experimental environments are different from those in commercial production.

5. Conclusions

This study used different layers of scales from the Oriental lily variety ‘Pacific Ocean’ to identify the most suitable lily scale regions for in vitro culture ball induction. The results showed that the most suitable scales for induction were the inner layer scales; longitudinally, the most suitable scale regions were the basal region, followed by the middle region. Among the six regions of the inner layer scales, the middle central region exhibited the best induction efficacy and lower contamination rates. If middle or outer layer scales were used as explants, basal region scales could be selected to efficiently and rapidly obtain lily tissue culture balls.