Induction and Enlargement of Bulblets in Lilium brownii var. viridulum In Vitro

Abstract

Lilium brownii var. viridulum is a popular natural health product that combines medicine and food. However, the lack of high-quality and efficient propagation technology has been a significant impediment to the development of the L. brownii var. viridulum industry. In this study, bulb scales of L. brownii var. viridulum were used as materials to investigate the induction of bulblets from scales, as well as the proliferation and enlargement of the induced bulblets. An in vitro regeneration system for L. brownii var. viridulum was successfully established. The most effective methods for scale disinfection were found to be 75% alcohol disinfection for 30 s and 10% Sodium Hypochlorite (NaClO) disinfection for 15 min. The recommended medium for scale differentiation was Murashige and Skoog (MS) + 0.05 mg·L⁻¹ 1-Naphthaleneacetic acid (NAA) + 0.25 mg·L⁻¹ Thidiazuron (TDZ) + 0.05 mg·L⁻¹ kinetin (KIN) + 30 g·L⁻¹ sucrose + 6 g·L⁻¹ Activated Charcoal (AC). The recommended medium for bulblet proliferation was MS + 1.5 mg·L⁻¹ 6-benzylaminopurine (6-BA) + 0.5 mg·L⁻¹ NAA + 30 g·L⁻¹ sucrose. The recommended medium for bulblet enlargement was Modified MS Medium (potassium dihydrogen phosphate (KH2PO4, KDP) doubled) + 90 g·L⁻¹ sucrose. Bulblets with diameters of 1.5 cm were transplanted, and the germination rate was 100%. This study established a systematic in vitro regeneration system for L. brownii var. viridulum, which will provide the foundation for industrial production of L. brownii var. viridulum bulbs and improvement through genetic transformation.

1. Introduction

Lilium brownii F. E. Brown var. viridulum Baker, known locally as the Longya lily, belongs to the genus Lilium of the family Liliaceae. Its bulbs are rich in sugars, proteins, minerals, and active ingredients (such as steroidal saponins, phenolic acids, polysaccharides, alkaloids, etc.), making it a popular natural health product that combines medicine and food, with economic benefits that are three to ten times greater than those of other crops [1,2,3]. In production, the Longya lily is mainly propagated asexually through scale cutting, bulb division, and stem bulblets. However, these propagation methods not only have a long cycle and low propagation coefficient, but also cause issues, especially the accumulation of viruses and the deterioration of species, which ultimately results in a decrease in yield and quality [4]. High-quality and efficient bulb propagation is the only way to solve the development problems of the Longya lily industry.

Plant tissue culture technology is an effective technique to obtain a large number of identical plants in a short span of time and produce disease-free planting material [5,6]. In the early 1950s, Robb first achieved success in tissue culture using the bulb scales of Lilium speciosum [7]. Since then, the tissue culture of lilies has gradually gained popularity and is currently in lilies for successful applications, such as Lilium leucanthum [8], Lilium longiflorum [9], Lilium monodelphum var. Armenum [10], Lilium regale [11], Lilium fargesii [12]. Although many parts may be used in the in vitro propagation of Lilium, scales are most frequently used [13,14]. At present, the tissue culture technology system of lily is mainly completed through two processes: the differentiation and proliferation of adventitious buds from scales and the formation and enlargement of bulblets [15]. The successful culture of explants requires complex nutritional and hormonal requirements. In the in vitro culture of lily, commonly used media include Murashige and Skoog (MS), N6, B5, White, etc. Among them, the MS medium is the most frequently used in lily tissue culture and can also achieve excellent results [16]. Han’s research also indicated that MS was the optimal medium, as it could efficiently induce bulb formation and result in the largest number of bulblets produced [17]. Plant hormones or plant growth regulators (PGRs) and sucrose play important roles in the induction and development of bulblets. During the bulblet induction stage, the sucrose concentration is typically 30 g·L⁻¹ [16,18]. A relatively higher sucrose concentration is typically used for bulblet enlargement or growth. In the study on the growth of the Lilium oriental hybrid ’Casablanca’, Lian et al. found that larger bulblets formed on media containing higher concentrations (90 g·L⁻¹) of sucrose [19]. The contribution of PGRs on the induction, proliferation, and development of bulblets has been studied in various lily plants. Prior research findings have suggested that auxins 1-Naphthaleneacetic acid (NAA), and cytokinins 6-benzylaminopurine (6-BA) and Thidiazuron (TDZ) are predominantly used in the bulblet induction of lilies, such as L. longiflorum [20], Lilium orientalis [21], and Oriental Lilium Hybrid Cv. ‘Ravenna’ [18]. During the proliferation stage, plant hormones can increase the number of bulblets, enabling propagation. It has been reported that the combined application of 1.5 mg·L⁻¹ NAA and 2 mg·L⁻¹ BA significantly induces bulblet regeneration in Oriental hybrid lilies [22]. In Lilium rosthornii, the greatest proliferation ratio was from the combination of 0.1 mg·L⁻¹ NAA and 0.5 mg·L⁻¹ TDZ [23]. In the stage of bulblet enlargement, studies have reported that low concentrations of jasmonic acid (JA) can promote the growth and development of bulblets [12]. The application of a certain concentration of N-(2-Chloro-4-pyridyl)-N’-phenylurea (CPPU) in Asiatic hybrid lily ‘Matrix’ can promote bulblet initiation and development [24]. However, the impact of PGRs on the enlargement of L. brownii var. viridulum remains to be further studied.

The establishment of a tissue culture technology system is one of the key links for the efficient and high-quality propagation of lilies. At present, most of the tissue culture studies on L. brownii var. viridulum focus on the differentiation research using scales as explants, and there is a lack of systematic studies on its sterilization, browning, differentiation, proliferation, and enlargement [25]. Moreover, the research mainly focuses on 6-BA and NAA, with a lack of application of other growth regulators, which leads to problems such as a long differentiation cycle, a low differentiation coefficient, and the non-enlargement of bulbs [26].

Therefore, this study explored the effects of different types and concentrations of sterilizing agents as well as hormone treatment combinations on scale differentiation and optimized the differentiation and proliferation system of L. brownii var. viridulum. It also investigated the impact of exogenously added substances on bulb enlargement and studied the bulb enlargement system. The aim is to establish an efficient propagation system for L. brownii var. viridulum and provide technical support for its industrial production.

2. Materials and Methods

2.1. Plant Materials

Bulbs of L. brownii var. viridulum were collected from the lily planting base of Jiangxi Woqi Ecological Agriculture Development Co., Ltd., Wanzai, Jiangxi Province, China, which is situated in a subtropical humid climate zone, with geographic coordinates of 28°9′ N latitude and 114°31′ E longitude. The climate is warm, with four distinct seasons, and the annual average temperature ranges from 16.9 °C to 18.29 °C. The region is characterized by black sandy loam soil (pH 5.7–6.3) and is situated at an elevation of 100 m above sea level. Fresh bulbs weighing approximately 200 g were collected in July 2023, when temperatures averaged around 35 °C and the leaves of the aerial plant parts had withered. Healthy scales without disease spots or damage were separated from the bulbs and used as explants.

2.2. Surface Disinfection of Explants

The middle scales were washed with sterile distilled water and transferred to sterile conditions for further sterilization. The scales were immersed in 75% alcohol for 30 s. They were then soaked in different concentrations of Sodium Hypochlorite (NaClO, Shanghai Acmec Biochemical Co., Ltd., Shanghai, China) (2%, 5%, 10%, and 20% v/v) for 15 min (A1–A5) and with sterile water treatment as the control. The final step involved four additional washes with sterile distilled water. Following the surface disinfection process, the outer layers of the scales were carefully removed, leaving only the inner fleshy layer. This inner layer of the scale was then cut into small squares, approximately, using a sterile scalpel. The squares were cultured in MS medium with the concave surface facing upward, cultured at 25 ± 1 °C in the dark, and the contamination was observed and counted.

$$\mathrm{Contamination} \mathrm{rate}\left(\%\right)={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{contaminated} \mathrm{scales}}{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{inoculated} \mathrm{scales}}}\times 100\%$$

2.3. Effects of PGRs on Scale Differentiation

To determine the optimal medium for scale differentiation, scales were inoculated into the media with different concentrations of PGRs, including 6-BA (Caisson Labs, Smithfield, UT, USA), NAA (Aladdin Biochemical Technology Co., Ltd. Shanghai, China), TDZ (Solarbio Science & Technology Co., Ltd., Beijing, China), and kinetin (KIN) (Solarbio Science & Technology Co., Ltd., Beijing, China). The test utilized 16 combinations of 0, 0.5, 1, and 1.5 mg·L⁻¹ of 6-BA; 0, 0.05, 0.1, and 0.2 mg·L⁻¹ of NAA; 0, 0.25, 0.5, and 1 mg·L⁻¹ of TDZ; and 0, 0.05, 0.1, and 0.2 mg·L⁻¹ of KIN; according to orthogonal experimental design (

Table 1. Different combinations of PGRs for scale differentiation of L. brownii var. viridulum.

Treatment	PGRs (mg·L−1)
	6-BA	NAA	TDZ	KIN
B1	-	-	-	-
B2	-	0.05	0.25	0.05
B3	-	0.10	0.50	0.10
B4	-	0.20	1.00	0.20
B5	0.50	-	0.25	0.10
B6	0.50	0.05	-	0.20
B7	0.50	0.10	1.00	-
B8	0.50	0.20	0.50	0.05
B9	1.00	-	0.50	0.20
B10	1.00	0.05	1.00	0.10
B11	1.00	0.10	-	0.05
B12	1.00	0.20	0.25	-
B13	1.50	-	1.00	0.05
B14	1.50	0.05	0.50	-
B15	1.50	0.10	0.25	0.20
B16	1.50	0.20	-	0.10

). MS (Caisson Labs, Smithfield, UT, USA) with 30 g·L⁻¹ of sucrose and 5.8 g·L⁻¹ of agar (Beijing Juhua Tech Co., Ltd., Beijing, China) was used as the basal medium for the B1-B16 treatments. In each medium, 30 explants were inoculated. All explants were cultured at 25 ± 1 °C in constant darkness. The experiment was repeated three times. The differentiation rate (number of differentiated scales/total number of scales × 100%) of each treatment was recorded after 30 days.

2.4. Effects of Activated Charcoal (AC) on Bulblet Induction

Due to the browning problem at the cut of L. brownii var. viridulum scales, we carried out an AC experiment. Different concentrations (0, 2, 4, 6, and 8 g·L⁻¹) of AC (325 mesh, Tianjin Fuchen Chemical Reagent Factory, Tianjin, China) were added to the optimal differentiation medium (C1–C5). In each medium, 30 explants were inoculated. All scales were cultured at 25 ± 1 °C in constant darkness. The experiment was repeated three times. The browning and differentiation of the scales were observed when cultured for 30 days. Bulblets inducted from scales were measured at 45 d (Figure 1).

$$\begin{array}{l}\mathrm{Browning}\mathrm{rate}\left(\%\right)={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{browned} \mathrm{scales}}{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{inoculated} \mathrm{scales}}}\times 100\%\\ \mathrm{Differentiation}\mathrm{rate}\left(\%\right)={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{differentiated} \mathrm{scales}}{\mathrm{number} \mathrm{of} \mathrm{scales}}}\times 100\%\\ \mathrm{Differentiation} \mathrm{coefficient}={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{differentiated} \mathrm{bulblets}}{\mathrm{number} \mathrm{of} \mathrm{differentiated} \mathrm{scales}}}\times 100\%\\ \mathrm{Bulblet} \mathrm{length}\left(\mathrm{cm}\right)={\displaystyle \frac{\mathrm{Sum} \mathrm{of} \mathrm{the} \mathrm{lengths} \mathrm{of} \mathrm{all} \mathrm{bulblets} }{\mathrm{number} \mathrm{of} \mathrm{bulblets}}}\\ \mathrm{Bulblet} \mathrm{width}\left(\mathrm{cm}\right)={\displaystyle \frac{\mathrm{Sum} \mathrm{of} \mathrm{the} \mathrm{widths} \mathrm{of} \mathrm{all} \mathrm{bulblets}}{ \mathrm{number} \mathrm{of} \mathrm{bulblets}}}\\ \mathrm{Bulblet} \mathrm{weight}\left(\mathrm{g}\right)={\displaystyle \frac{\mathrm{Sum} \mathrm{of} \mathrm{the} \mathrm{weights} \mathrm{of} \mathrm{all} \mathrm{bulblets}}{\mathrm{number} \mathrm{of} \mathrm{bulblets}}}\end{array}$$

2.5. Effects of PGRs and Sucrose on Bulblet Proliferation

To standardize bulblet proliferation, a multifactor experimental design was employed with 16 combinations of 0, 0.5, 1, and 1.5 mg·L⁻¹ of 6-BA; 0, 0.25, 0.5, and 1.0 mg·L⁻¹ of NAA; and 0, 30, 60, and 90 mg·L⁻¹ of sucrose were conducted (

Table 2. Different combinations of PGRs and sucrose for bulblet proliferation of L. brownii var. viridulum.

Treatment	PGRs (mg·L−1)		Sucrose (g·L−1)
	6-BA	NAA
D1	-	-	-
D2	0.50	0.25	30
D3	1.00	0.50	60
D4	1.50	1.00	90
D5	-	0.25	30
D6	0.50	-	30
D7	1.00	1.00	30
D8	1.50	0.50	30
D9	-	0.50	60
D10	0.50	1.00	60
D11	1.00	-	60
D12	1.50	0.25	60
D13	-	1.00	90
D14	0.50	0.50	90
D15	1.00	0.25	90
D16	1.50	-	90

). MS with 5.8 g·L⁻¹ agar was used as the basal medium for the D1-D16 treatments. Bulblets (of 0.5 cm in width) differentiated from scales were inoculated onto the above-mentioned medium. In each medium, 30 bulblets were inoculated and cultured at 25 ± 1 °C in constant darkness. The experiment was repeated three times. After two months, the proliferation of bulblets was observed, the proliferation coefficient was counted, and the size of the bulblets was measured.

$$\mathrm{Proliferation} \mathrm{coefficient}={\displaystyle \frac{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{bulblets} \mathrm{obtained} \mathrm{after} \mathrm{culturing}}{\mathrm{initial} \mathrm{number} \mathrm{of} \mathrm{inoculated} \mathrm{bulblets}}}$$

2.6. Effects of Macronutrients and PGRs on Bulblet Enlargment

To promote the rapid enlargement of bulblets, bulblets with an average diameter of 0.7 × 0.63 cm (average weight of 0.23 g) were selected for enlargement cultivation. A total of 14 treatments were set up for enlargement (

Table 3. Different media used for bulblet enlargement of L. brownii var. viridulum.

Treatment	Media Used for Bulblet Enlargement Culture
E1	MS
E2	2MS
E3	Modified MS (KDP doubled)
E4	MS + 0.25 mg·L−1 Me-JA
E5	MS + 0.50 mg·L−1 Me-JA
E6	MS + 1.00 mg·L−1 Me-JA
E7	MS + 0.05 mg·L−1 BR
E8	MS + 0.10 mg·L−1 BR
E9	MS + 0.20 mg·L−1 BR
E10	MS + 0.50 mg·L−1 CPPU
E11	MS + 1.00 mg·L−1 CPPU
E12	MS + 2.00 mg·L−1 CPPU

Note: The sucrose concentration in all treatments was 90 g·L⁻¹.

), including MS + 90 g·L⁻¹ of sucrose, 2MS + 90 g·L⁻¹ of sucrose, Modified MS Medium (potassium dihydrogen phosphate (KH2PO4, KDP) doubled) + 90 g·L⁻¹ of sucrose, MS + methyl jasmonate (Me-JA, Solarbio Science & Technology Co., Ltd., Beijing, China) (0.25, 0.5, 1 mg·L⁻¹) + 90 g·L⁻¹ of sucrose, MS + CPPU (Solarbio Science & Technology Co., Ltd., Beijing, China) (0.5, 1, 2 mg·L⁻¹) + 90 g·L⁻¹ of sucrose, and MS + Brassinolide (BR, Tianjin Alta Scientific Co., Ltd., Tianjin, China) (0.05, 0.1, 0.2 mg·L⁻¹) + 90 g·L⁻¹ of sucrose. After 5 months, the enlargement of bulblets were observed, and the size and weight of bulblets were measured.

$$\begin{array}{l}\mathrm{Rooting} \mathrm{rate}\left(\%\right)={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{bulblets} \mathrm{with} \mathrm{induced} \mathrm{roots}}{\mathrm{initial} \mathrm{number} \mathrm{of} \mathrm{bulblets}}}\times 100\%\\ \mathrm{Rooting} \mathrm{coefficient}={\displaystyle \frac{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{roots}}{\mathrm{number} \mathrm{of} \mathrm{bulblets} \mathrm{with} \mathrm{induced} \mathrm{roots}}}\\ \mathrm{Bulblet} \mathrm{length} \mathrm{enlargement} \mathrm{multiple}={\displaystyle \frac{\left(\mathrm{length} \mathrm{of} \mathrm{bulblet} \mathrm{after} \mathrm{enlargement}-\mathrm{length} \mathrm{of} \mathrm{bulblet} \mathrm{before} \mathrm{enlargement}\right)}{\mathrm{length} \mathrm{of} \mathrm{bulblet} \mathrm{before} \mathrm{enlargement}}}\\ \mathrm{Bulblet} \mathrm{width} \mathrm{enlargement} \mathrm{multiple}={\displaystyle \frac{\left(\mathrm{Width} \mathrm{of} \mathrm{bulblet} \mathrm{after} \mathrm{enlargement} - \mathrm{width} \mathrm{of} \mathrm{bulblet} \mathrm{before} \mathrm{enlargement}\right)}{\mathrm{width} \mathrm{of} \mathrm{bulblet} \mathrm{before} \mathrm{enlargement}}}\\ \mathrm{Bulblet} \mathrm{weight} \mathrm{enlargement} \mathrm{multiple}={\displaystyle \frac{\left(\mathrm{Weight} \mathrm{of} \mathrm{bulblet} \mathrm{after} \mathrm{enlargement} - \mathrm{weight} \mathrm{of} \mathrm{bulblet} \mathrm{before} \mathrm{enlargement}\right)}{\mathrm{weight} \mathrm{of} \mathrm{bulblet} \mathrm{before} \mathrm{enlargement}}}\end{array}$$

2.7. Transplantation of Tissue-Cultured Bulbs

Bulblets of L. brownii var. viridulum with diameters of 1.5 cm were selected for transplanting in November 2024. The tissue-cultured bulblets were taken out of the medium and rinsed off the medium on the roots with clean water. Afterwards, bulblets were soaked in carbendazim diluted 1000 times for 20 min. After draining the water, bulblets were transplanted into the substrate, with the top of the bulblets covered by 3 cm of substrate. The seedling emergence was observed, and the emergence rate was counted after 5 months.

$$\mathrm{Seedling} \mathrm{emergence} \mathrm{rate}\left(\%\right)={\displaystyle \frac{\mathrm{number} \mathrm{of} \mathrm{emerged} \mathrm{bulblets} \mathrm{after} \mathrm{transplantation}}{\mathrm{total} \mathrm{number} \mathrm{of} \mathrm{transplanted} \mathrm{bulblets}}}\times 100\%$$

2.8. Data Analysis

Statistical significances were tested using one-way analysis of variance (ANOVA) at p < 0.05, and multiple comparisons were conducted using the least significant difference (LSD) method and Duncan’s (D) test. All statistical analyses were carried out using SPSS version 27.0 software (SPSS, Chicago, IL, USA). The results were expressed as the mean ± standard deviation (SD) of triplicates.

3. Results

3.1. Surface Disinfection of Lilium brownii var. viridulum Scales

Scales of L. brownii var. viridulum treated with different concentrations of NaClO were inoculated onto MS medium. It was observed that the contamination rates of NaClO treatments were significantly lower than those of the control group. The rate of contamination of the scales showed significant differences among the four different disinfection treatments, with a continuous decrease as the concentration of NaClO increased (

Table 4. Effects of different disinfection treatments on contamination of L. brownii var. viridulum.

Treatment	NaClO Concentrations (%)	Number of Contaminated Scales	Contamination Rate (%)
A1	0	55	91.67 ± 2.89 a
A2	2	42	70.00 ± 5.00 b
A3	5	28	46.67 ± 7.64 c
A4	10	14	23.33 ± 2.89 d
A5	20	12	20.00 ± 5.00 d

Note: Number of scales in different treatments was 60. Different lowercase letters indicate the significant differences among the different treatments (p < 0.05, n = 3).

). Among them, the contamination rate of disinfection with 20% NaClO was the lowest, at 20%, followed by 10% NaClO at 23.33%, and there was no significant difference between the two. However, it was found that the browning of the scales became more and more serious as the concentration of NaClO increased. Considering the above factors comprehensively, the recommended disinfection method for L. brownii var. viridulum scales was finally determined as follows: 75% alcohol disinfection for 30 s and 10%NaClO disinfection for 15 min.

3.2. Effects of Different Combinations of PGRs on Scale Differentiation

There is no difference in the time when the scales start to differentiate. Bulges began to form on the adaxial side near the scale wound of L. brownii var. viridulum at 15 days, after which small bulblets gradually formed in the bulged parts after 30 days (Figure S1). The B2, B3, and B4 treatments were combinations without 6-BA. With the continuous increase in the concentrations of NAA, TDZ, and KIN, the differentiation rate of the scales kept decreasing. B2 was a combination of NAA (0.05 mg·L⁻¹), TDZ (0.25 mg·L⁻¹), and KIN (0.05 mg·L⁻¹), with the highest differentiation rate (83.33%) compared with other treatments and the best growth state of bulblets (

Table 5. Scale differentiation of L. brownii var. viridulum under different combinations of PGRs for 30 days.

Treatment	Differentiation Rate (%)	Differentiation Coefficient	Status of Scale Differentiation
B1	31.67 ± 5.77 g	1.27 ± 0.25 c	Small bulblets, small quantity
B2	83.33 ± 2.89 a	3.31 ± 0.21 abc	Large bulblets, numerous, best growth
B3	53.33 ± 5.77 bc	4.54 ± 0.32 a	Small bulblets, numerous
B4	51.67 ± 2.89 bcd	3.56 ± 0.68 abc	Loose bulblets
B5	31.67 ± 7.64 fg	2.98 ± 0.92 abc	Bulblets with small quantity
B6	40.00 ± 5.00 efg	3.86 ± 0.46 ab	Small bulblets, slow development
B7	45.00 ± 5.00 cdef	2.78 ± 0.32 abc	Small bulblets, slow development
B8	41.67 ± 2.89 defg	4.46 ± 2.92 a	Vitrification, numerous
B9	50.00 ± 8.66 bcde	4.46 ± 0.77 a	Small bulblets, numerous, slow development
B10	46.67 ± 7.64 cdef	3.32 ± 1.21 abc	Small bulblets, non-spherical shape
B11	60.00 ± 10.00 b	2.19 ± 1.20 abc	Small quantity, slow development, browning
B12	38.33 ± 2.89 fg	2.63 ± 1.73 abc	Slow development, non-spherical shape
B13	20.00 ± 5.00 h	2.06 ± 1.00 abc	Non-spherical shape, browning
B14	43.33 ± 2.89 cdef	1.83 ± 1.67 bc	Non-spherical shape
B15	45.00 ± 5.00 cdef	3.07 ± 2.10 abc	Small bulblets, slow development
B16	50.00 ± 8.66 bcde	3.15 ± 1.17 abc	Small bulblets, slow development

Note: The initiation time of scale differentiation was 15 days across all different treatments. Different lowercase letters indicate significant differences among treatments (p < 0.05, n = 3). The analysis of the experimental design was limited in this work to a one-way ANOVA.

). High concentrations of 6-BA (≥1.0 mg·L⁻¹) generally led to delayed development, browning (such as B11, B13), or deformity (B12, B14) of the bulblets. The bulblets induced by the treatments of B5, B9, and B13 without NAA showed low quantities, slow development, and browning on the bulb surface. B6, B11, and B16 treatments were combinations without TDZ, and the bulblets were spherical but small. However, when TDZ was ≥0.5 mg·L⁻¹, it was easy to cause structural abnormalities, such as the scales being loosely clasped in B4 or failing to form a spherical bulb in B10 (Table 5). B7, B12, and B14 treatments were combinations without KIN, and all of them showed that the scales failed to form spherical bulblets. B8, B9, B10, and B15 treatments were combinations of 6-BA, TDZ, NAA, and KIN. They had a high differentiation coefficient, but they showed several issues, such as the slow growth of bulblets, a failure to form spherical shapes, and a low differentiation rate. When there was no NAA or KIN in the medium, most of the bulblets had low quantities, slow development, and a non-spherical shape. Therefore, the recommended medium for scale differentiation of L. brownii var. viridulum was MS + 0.05 mg·L⁻¹ of NAA + 0.25 mg·L⁻¹ of TDZ + 0.05 mg·L⁻¹ of KIN + 30 g·L⁻¹ of sucrose.

3.3. Effects of AC on Bulblet Induction and Growth

Scales were inoculated into media with different concentrations of AC, and significant differences in the browning, bulblet induction, and growth were observed (

Table 6. Effects of AC on scale browning, bulblet induction, and growth of L. brownii var. viridulum.

Treatment	Cultivate for 30 Days			Cultivate for 45 Days
	Browning Rate (%)	Differentiation Rate (%)	Differentiation Coefficient	Bulblet Length (cm)	Bulblet Width (cm)	Bulblet Weight (g)
C1	95.56 ± 3.85 a	83.33 ± 5.77 a	3.92 ± 1.16 a	0.14 ± 0.02 d	0.10 ± 0.03 c	0.04 ± 0.01 c
C2	42.22 ± 3.85 b	80.00 ± 5.00 a	2.61 ± 0.89 a	0.35 ± 0.06 c	0.24 ± 0.04 b	0.06 ± 0.01 c
C3	33.33 ± 6.67 b	76.67 ± 7.64 a	2.58 ± 0.47 a	0.43 ± 0.08 bc	0.29 ± 0.05 b	0.16 ± 0.03 b
C4	15.56 ± 3.85 c	71.67 ± 10.41 a	3.67 ± 1.37 a	0.50 ± 0.03 ab	0.41 ± 0.04 a	0.22 ± 0.05 b
C5	13.33 ± 6.67 c	50.00 ± 5.00 b	2.06 ± 0.51 a	0.56 ± 0.08 a	0.44 ± 0.07 a	0.32 ± 0.04 a

Note: Different lowercase letters indicate the significant differences among the different treatments (p < 0.05, n = 3).

, Figure 2). The medium with AC effectively reduced the browning of scales, and the degree of browning gradually decreased as the concentration of AC increased. When the concentration of AC was 6 g·L⁻¹ and 8 g·L⁻¹, the browning rates were 15.56% and 13.33%, respectively, with no significant difference (Table 6). However, AC can affect the differentiation of scales. With the increase in AC concentration, the differentiation rate of scales decreased. The concentrations of AC at 2, 4, 6 g·L⁻¹ showed no significant difference from the control, while AC at 8 g·L⁻¹ was significantly lower than the others (Table 6). In addition, the addition of AC promoted the growth of bulblets compared with the control group. The diameter and weight of bulblets increased with the increase in AC concentration (Figure 2). Taking all factors into account, adding 6 g·L⁻¹ AC to the culture medium is the recommended treatment for reducing the browning rate of Lilium brownii var. viridulum scales.

3.4. Effects of Different Combinations of PGRs on Bulblet Proliferation

The small bulblets with a diameter of approximately 1 cm induced from scales were transferred to different media, and the bulblet proliferation was observed. The proliferation coefficients of different treatments (D2–D16) were higher than the control group (D1) (

Table 7. Bulblet proliferation of L. brownii var. viridulum under different combinations of PGRs and sucrose.

Treatment	Proliferation Coefficient	Status of Bulblets Proliferation
D1	0.07 ± 0.12 d	No proliferation
D2	1.27 ± 0.81 b	Abnormal roots
D3	0.27 ± 0.23 cd	Slender roots, low proliferation coefficient
D4	0.93 ± 0.23 bc	Abnormal roots, low proliferation coefficient
D5	0.33 ± 0.12 cd	Slender roots, low proliferation coefficient
D6	0.33 ± 0.12 cd	Low proliferation coefficient
D7	0.60 ± 0.20 bcd	Abnormal roots, low proliferation coefficient
D8	3.60 ± 1.04 a	Higher proliferation coefficient, normal roots, best growth
D9	0.33 ± 0.31 cd	Abnormal roots, low proliferation coefficient
D10	0.73 ± 0.42 bcd	Abnormal roots, low proliferation coefficient, slow development
D11	0.47 ± 0.31 bcd	Slender roots, low proliferation coefficient
D12	0.33 ± 0.31 cd	Few short roots, low proliferation coefficient
D13	0.73 ± 0.31 bcd	Abnormal roots, low proliferation coefficient
D14	0.67 ± 0.46 bcd	Abnormal roots, low proliferation coefficient
D15	0.87 ± 0.31 bcd	Short and thick roots, low proliferation coefficient
D16	0.40 ± 0.20 cd	Slender roots, low proliferation coefficient

Note: Different lowercase letters indicate the significant differences among the different treatments (p < 0.05, n = 3).

, Figure 3). The control group shows almost no proliferation. The diameter of proliferated bulblets in different treatment groups ranged from 0.2 to 0.3 cm, with no significant differences. D8 was a combination of 6-BA (1.50 mg·L⁻¹), NAA (0.50 mg·L⁻¹) and sucrose (30 mg·L⁻¹), with the highest proliferation coefficient (3.60) compared with other treatments and the best growth state (Table 7). The D5, D9, and D13 treatments, which were combinations without 6-BA. There is no difference in the proliferation coefficient in D5, D9, and D13, and both were relatively low. The D6, D11, and D16 treatments, which were combinations without NAA. There is no difference in the proliferation coefficient with the continuous increase in the concentrations of 6-BA and sucrose, and the proliferation coefficient was relatively low. The D3, D12, and D15 treatments were high concentrations of sucrose, which showed short and thick roots and a low proliferation coefficient. In addition, the D2, D4, D7, D9, D10, D13, and D14 treatments showed abnormal root development (Figure 3). Therefore, the recommended medium for bulblets proliferation of L. brownii var. viridulum was MS + 1.5 mg·L⁻¹ of 6-BA + 0.5 mg·L⁻¹ of NAA+ 30 g·L⁻¹ of sucrose.

3.5. Effects of Macronutrients and PGRs on Bulblet Enlargement

The small bulblets with a size of 0.70 × 0.63 cm (average weight of 0.23 g) were transferred to different media for bulblet enlargement culturing. The results showed that 2MS (E2) could not promote the enlargement of bulblets (

Table 8. Bulblet enlargement of L. brownii var. viridulum under different media with macronutrients and PGRs.

Treatment	Length of Bulblet After Enlargement (cm)	Bulblet Length Enlargement Multiple	Width of Bulblet After Enlargement (cm)	Bulblet Width Enlargement Multiple	Weight of Bulblet After Enlargement (g)	Bulblet Weight Enlargement Multiple
E1	0.95 ± 0.07 d	0.36	0.73 ± 0.01 cd	0.16	0.29 ± 0.00 c	0.26
E2	0.95 ± 0.17 d	0.36	0.80 ± 0.20 bcd	0.27	0.44 ± 0.21 bc	0.91
E3	1.50 ± 0.15 a	1.14	1.22 ± 0.02 a	0.94	0.99 ± 0.31 a	3.30
E4	1.35 ± 0.11 abc	0.93	1.00 ± 0.09 b	0.59	0.68 ± 0.20 abc	1.96
E5	1.10 ± 0.10 cd	0.57	0.84 ± 0.12 bcd	0.33	0.52 ± 0.34 bc	1.26
E6	1.03 ± 0.17 d	0.47	0.68 ± 0.07 d	0.08	0.29 ± 0.09 c	0.26
E7	1.20 ± 0.14 bcd	0.71	0.99 ± 0.08 b	0.57	0.56 ± 0.13 bc	1.43
E8	1.12 ± 0.17 cd	0.60	0.88 ± 0.14 bcd	0.40	0.57 ± 0.21 bc	1.48
E9	1.06 ± 0.17 cd	0.51	0.82 ± 0.20 bcd	0.30	0.39 ± 0.16 bc	0.70
E10	0.94 ± 0.15 d	0.34	0.70 ± 0.04 d	0.11	0.27 ± 0.03 c	0.17
E11	1.23 ± 0.29 abcd	0.76	0.82 ± 0.14 bcd	0.30	0.52 ± 0.26 bc	1.26
E12	1.42 ± 0.07 ab	1.02	0.97 ± 0.22 bc	0.54	0.75 ± 0.31 ab	2.26

Note: Different lowercase letters indicate the significant differences among the different treatments (p < 0.05, n = 3).

). The growth of bulblets in modified MS Medium (KDP doubled) (E3) was significantly better than that of other treatments (Table 8). The length of the bulblet increased by 1.14 times (from 0.70 cm to 1.50 cm), its width increased by 0.94 times (from 0.63 cm to 1.22 cm). The fresh weight of the bulblet increased from 0.23 g to 0.99 g, increasing by 3.30 times. The rooting coefficient was 8.53 (

Table 9. Rooting status of L. brownii var. viridulum under different media with macronutrients and PGRs.

Treatment	Rooting Rate (%)	Rooting Coefficient	Root Length (cm)
E1	84.44 ± 5.10 b	4.13 ± 1.15 b	1.75 ± 0.44 d
E2	84.44 ± 3.85 b	3.58 ± 1.02 b	1.27 ± 0.44 d
E3	95.55 ± 3.85 a	8.53 ± 5.95 a	4.08 ± 1.74 bc
E4	84.44 ± 7.70 b	3.53 ± 0.64 b	2.70 ± 0.76 cd
E5	94.44 ± 9.62 a	3.22 ± 0.22 b	2.20 ± 0.70 cd
E6	71.11 ± 3.85 c	1.71 ± 0.42 b	2.08 ± 0.72 cd
E7	100.00 ± 0.00 a	4.86 ± 0.64 b	2.56 ± 0.48 cd
E8	100.00 ± 0.00 a	5.02 ± 0.46 b	1.28 ± 0.29 d
E9	91.11 ± 7.70 ab	3.82 ± 0.71 b	2.94 ± 0.38 cd
E10	97.78 ± 3.85 a	4.63 ± 0.78 b	2.03 ± 0.40 cd
E11	100.00 ± 0.00 a	4.74 ± 1.08 b	4.89 ± 0.82 b
E12	100.00 ± 0.00 a	4.37 ± 0.15 b	7.01 ± 1.25 a

Note: Different lowercase letters indicate the significant differences among the different treatments (p < 0.05, n = 3).

). E4–E12 refers to the addition of exogenous PGRs to the culture medium. Among them, E4, E5, and E6 are different concentrations of Me-JA added. A low concentration of Me-JA (E4) promoted bulblet growth, and the size of the bulblets reached 1.35 × 1.00 cm, the fresh weight of the bulblet was 0.68 g. With the increase in Me-JA concentration, the enlargement of the bulblets and the growth of roots were inhibited (Table 9, Figure S2). E7, E8, and E9 were treated with different concentrations of BR. Although BR could increase the rooting rate of bulblets (all rooting rates were above 90%), its inhibitory effect on the growth of bulblets significantly increased with the increase in concentration (Table 8). The length of bulblets decreased from 1.20 cm in E7 to 1.06 cm in E9, the width of the bulblets decreased from 0.98 cm in E7 to 0.82 cm in E9, and the fresh weight decreased from 0.56 g in E7 to 0.39 g in E9. E10, E11, and E12 were treated with different concentrations of CPPU. With the increase in CPPU concentration, the length, width, and fresh weight of bulblets continued to rise. The length of the bulblets increased from 0.94 cm to 1.42 cm, and the width increased from 0.70 cm to 0.97 cm. The fresh weight increased from 0.27 g to 0.75 g, second only to the E2 treatment group. Under CPPU treatment, the rooting rate was significantly higher than that of other treatment groups (all above 95%) (Table 9). However, the scales of bulblets were loosely adhered under CPPU treatment. Therefore, the recommended medium for bulblet enlargement of L. brownii var. viridulum was modified MS Medium (KDP doubled) +90 g·L⁻¹ of sucrose.

3.6. Transplanting of Tissue-Cultured Bulbs of L. brownii var. viridulum

Bulblets of L. brownii var. viridulum with diameters of 1.5 cm were selected for transplanting in November 2024. All transplanted tissue culture seedlings germinated after 150 days, with a germination rate of 100% (Figure 4). Seedlings grew well with 2.49 ± 0.10 cm of leaf width and 15.93 ± 0.09 cm of leaf length.

4. Discussion

The surface disinfection process of explants is the first step in the tissue culture, and its purpose is to eliminate the microorganisms carried by the explants [27,28]. The selection and optimization of the disinfection process are conducive to reducing the contamination rate of explants and improving the survival rate. HgCl₂ and NaClO are the most widely used in lilies [10,29,30]. However, HgCl₂ is toxic to both explants and the human body. As a disinfectant, NaClO is effective against many bacteria and viruses. In tissue culture, it is commonly used for the surface sterilization of explants in lilies [10,30], peonies [31], sugarcanes [32], etc. Different explants require different concentrations of NaClO and time, which is approximately 0.1% to 2% for 10–30 min, but higher concentrations are also used. In the in vitro culture of Lilium monodelphum var. Armenum, a 25% NaClO solution was used for 10 min for surface sterilization [10]. In our study, 10% was the recommended sterilization concentration. We found that 20% NaClO was also highly effective, but led to aggravated browning of scales, which also increased the mortality rate of scales. This is consistent with the research results of water lilies [33].

In plant tissue culture, the addition of PGRs plays a crucial role in plant morphology cultivation and development [34,35]. Auxins are mainly used to induce cell division and promote root differentiation. Cytokinins can promote cell division and shoot regeneration from calli or organs [36]. Auxins and cytokinins are often used in combination to exert their effects on organ differentiation [37]. Selecting PGR concentrations and the proper ratios of different PGRs are critical for tissue culture research [38]. BA is considered the main factor causing excessive water accumulation, and this issue can be avoided by reducing the concentration of BA or replacing it with other cytokinins such as KIN and TDZ [31]. TDZ is a proven effective and potent synthetic PGR, which can be used in organogenesis, regeneration, and development pathways, including axillary bud and adventitious bud proliferation, somatic embryogenesis, and in vitro flowering [10,39,40]. However, high concentrations of TDZ inhibit the development of explants. TDZ (>2.0 μM) induced undesirable changes in plant morphology, such as abnormal leaf morphology, fasciated shoots, and swollen shoot bases [40]. Abnormal bulblets with small bulb scales and swollen basal plates from bulb scales of the Lilium oriental hybrid ‘Casablanca’ formed in media containing 4.5 µM TDZ [17], which is consistent with the results of this study. In addition, our results showed that low concentrations of TDZ combined with certain concentrations of NAA and KIN can improve the differentiation rate of L. brownii var. viridulum scales, which has not been reported in previous studies on lily.

Browning was observed at the cut surface during the differentiation of L. brownii var. viridulum, which seriously affected the differentiation of scales. AC is considered to be able to adsorb inhibitory substances derived from the explants themselves that may exist in the culture medium, as well as inhibitory substances such as phenolics and other organic compounds contained in the culture medium [41,42]. AC can enhance the establishment of protoplast cultures, preventing the development of abnormal seedlings and bud formation [42]. It was found that when 200 mg·L⁻¹ AC was added to the culture medium, the bud formation of lettuce could be effectively induced, with an induction rate of 90.8 ± 7.9% [43]. Steinitz and Yahel and Peck and Cumming found that adding AC during the bulb culture of Narcissus and Pleurotus eryngii could effectively reduce browning and promote bulb formation [44,45]. This study has obtained similar results that adding AC can not only effectively improve browning but also promote the formation of bulblets in L. brownii var. viridulum.

Proliferation culture is a key step in realizing the industrialization of plant tissue culture. An appropriate combination of growth regulators, usually auxins and cytokinins, can effectively improve the proliferation coefficient. In the proliferation culture of Acacia arabica, no proliferation occurred in media without growth regulators, and the highest average numbers of somatic embryos was 72.6 after 8 weeks of culture on medium containing 6.66 μM of BA and 6.78 μM of 2,4-D [46]. In the shoot regeneration of Sinningia Hybrida ‘Isa’s Murmur’, the combination of TDZ and NAA was more favorable for shoot proliferation. The highest shoot proliferation coefficient (24.5) was reached in 0.1 mg·L⁻¹ of NAA and 2.0 mg·L⁻¹ of TDZ [47]. Fu et al. found that MS medium with combinations of 0.5 mg·L⁻¹ of 6-BA and 1.0 mg·L⁻¹ of NAA was predominant for the callus growth of L. brownii var. viridulum, and the frequency of callus proliferation was 56.30% [4]. In our study, the highest proliferation rate was the combination of 6-BA (1.50 mg·L⁻¹) and NAA (0.50 mg·L⁻¹). However, our study found that 6-BA promotes proliferation within a certain concentration range, but when its concentration exceeds a specific threshold, the proliferation of bulblets is inhibited, which was consistent with the results obtained in Aeschynanthus pulcher [48], Cnidoscolus aconitifolius [49]. In addition, this study found that an excessively high sucrose concentration was not conducive to the proliferation of bulblets and would affect the normal development of bulblet roots. The same result was observed in the study on Moringa oleifera [50]. When the sucrose concentration was 30 g·L⁻¹, the shoot proliferation coefficient was 4.13. As the sucrose concentration increased, the proliferation coefficient decreased, which was significantly lower than that of the treatment with 30 g·L⁻¹ of sucrose, being 2.50 and 2.41, respectively.

At present, the tissue culture technology system of the lily is mainly completed through two processes: the differentiation and proliferation of adventitious buds from scales, and the formation and enlargement of bulblets [15]. Among them, the formation and enlargement of bulblets are the key to accelerating the production and localization of lily bulbs. However, slow development and the failure to enlarge bulblets are common in lily tissue culture. Studies have shown that macronutrients, such as N, P, and K, promote the growth of lily plants. Potassium is an essential nutrient for plant living cells and is crucial for plant osmotic regulation and phloem transport [51,52]. A single application of nitrogen and phosphorus fertilizer can promote the growth of lily plants, but its increase is lower than that of potassium fertilizer. Sha et al. conducted a study on the effects of potassium application on Lilium davidii var. unicolor growth, and the results showed that adding an appropriate K concentration could increase the bulb weight and bulb circumference [53], which is consistent with the results of this study. But the bulb weight and circumference decreased significantly with excessive K fertilizer application [54]. This is possibly because the application of an excessive amount of K hinders the absorption of other nutrients such as N and P, thereby negating the positive effects of K fertilization [55]. In addition, the application of PGRs, such as JA, salicylic acids, forchlorfenuron, and humic acids, can also promote the formation and expansion of bulbs [56,57]. Studies have shown that exogenous application of 5 µM or 7.5 µM of Me-JA can promote the enlargement of garlic bulbs [58]. However, it has been found that medium with high concentrations (300–1000 µL·L⁻¹) of Me-JA significantly inhibit the regeneration rate and fresh weight of bulbs in Lilium speciosum [30]. This is consistent with the result of the present study that excessively high concentrations of Me-JA have an inhibitory effect on the growth and development of bulblets. CPPU is known to be effective for enhancing fruit enlargement by stimulating cell division and/or cell expansion in many kinds of fruits and bulbous plants [56,59]. A total of 10 mg·L⁻¹ of CPPU promoted the increase in fruit size and weight of red-fleshed kiwifruit [60]. There is a significant positive correlation between bulb growth and starch accumulation. Spray treatment with an appropriate concentration of CPPU (1.0 mg·L⁻¹) could promote the activity of starch synthase and accelerate the accumulation of starch in bulblets, which was beneficial for the enlargement of Lycoris aurea bulblets [56], which was consistent with our results of CPPU promoting the size and fresh weight of Lilium brownii var. viridulum bulblets. BRs are a group of steroid hormones that play an essential role in promoting plant growth and rooting, enhancing stress resistance, increasing plant yield, and improving plant quality [61]. In the study of Bupleurum chinense, BR treatments increased fresh root weight, dry root weight, taproot length, and taproot diameter of B. chinense compared with the CK group [62]. In the study of bulbil formation and development in Pinellia ternata, the tuber and bulbil yield, starch, and soluble sugar content were significantly enhanced by BR treatment [63,64]. In our study, BRs at different concentrations could promote the enlargement of bulblets, with the concentration of 0.5 mg·L⁻¹ being recommended. Beyond this concentration, an inhibitory effect was observed as the BR concentration increased. This is consistent with the role of BR in increasing the flower diameter of Freesia hybrida ‘Hongshi’ that, within a certain concentration range, the promoting effect strengthens as the BR concentration increases, while inhibition occurs when the concentration exceeds a certain threshold [65].

5. Conclusions

This study successfully established an in vitro regeneration system for L. brownii var. viridulum. The recommended methods for scale disinfection were 75% alcohol disinfection for 30 s and 10% NaClO disinfection for 15 min. The recommended medium for scale differentiation was MS + 0.05 mg·L⁻¹ of NAA + 0.25 mg·L⁻¹ of TDZ + 0.05 mg·L⁻¹ of KIN + 30 g·L⁻¹ of sucrose + 6 g·L⁻¹ of AC. The recommended medium for bulblets proliferation was MS + 1.5 mg·L⁻¹ of 6-BA + 0.5 mg·L⁻¹ of NAA + 30 g·L⁻¹ of sucrose. The recommended medium for bulblets enlargement was modified MS Medium (KDP doubled) +90 g·L⁻¹ of sucrose. Bulblets with diameters of 1.5 cm were transplanted and the germination rate was 100%. In general, this study lays the foundation for the subsequent genetic improvement of Lilium brownii var. viridulum and provides a reference for the nursery factory.