In Vitro Plant Regeneration and Bioactive Metabolite Production of Endangered Medicinal Plant Atractylodes lancea (Thunb.) DC

Abstract

The rhizome of Atractylodes lancea (Thunb.) DC. is a traditional Chinese medicine used extensively owing to its antimicrobial properties. It is utilized to treat nyctalopia and problems related to the gastrointestinal tract. However, its yield is limited because of its endangered status, long growth period, and restricted reproductive ability. Ancillary approaches have not been established to ensure sustainable resource utilization by applying efficient plant regeneration technologies and producing bioactive metabolites via genome editing. This study reports the effects of explants, hormones, and culture conditions on embryogenic callus induction, plant regeneration, adventitious and hairy root cultivation, and essential oil production. Embryogenic calli were successfully induced in MS and 2.0 mg/L 2,4-D and 1.0 mg/L NAA and 1/2MS medium supplemented with 4.0 mg/L 6-BA and 0.4 mg/L NAA, which were optimal for callus differentiation. Maximum proliferation (12-fold) of cluster buds was observed with a select combination of hormones [NAA (0.2 mg/L) and 6-BA (2.0 mg/L)]. “Efficient plant regeneration and bioactive metabolite production” can provide technical support for the protection and sustainable utilization of A. lancea germplasm resources in terms of resource preservation and new variety breeding, natural product production, and industrial breeding of medicinal plants.

1. Introduction

Atractylodes lancea (Thunb.) DC is a perennial herbaceous plant belonging to the genus Atractylodes in the Asteraceae family, which is endemic to East Asia and widely distributed in China. A. lancea plants cultivated in the Maoshan region of Jiangsu are Dao-di herbs with the best quality [1]. A. lancea exerts the effects of drying dampness and strengthening the spleen, expelling wind and removing cold, and improving vision [2]. Modern pharmacological experiments have proved that Atractylodes rhizomes exhibit hepatoprotective, hypoglycemic, diuretic, and antihypoxic effects. The primary substance basis for its pharmacological effects is the presence of volatile oils, including atractylodin, atractylon, β-eudesmol, and hinesol [3]. In recent years, the reserves of wild A. lancea resources have declined significantly owing to habitat alterations and increases in market demand. Wild resources of Mao-A. lancea are endangered because of excessive excavation, a low seed-set rate, poor resistance to adversity, etc. According to the list of key protected wild plants in Jiangsu Province (first batch), as one of the four endangered medicinal plants under key protection in Jiangsu Province, there has been no collection or purchase of commercial medicinal materials in the past 40 years [4]. A. lancea can be propagated via tuber transplantation or direct seeding. Artificial cultivation is an effective way to protect and utilize the resources of A. lancea. However, a key issue is the severe shortage of seedling sources [5]. The application of tissue culture technology for rapidly propagating rare and endangered medicinal plants can overcome several problems in seed germination and the disadvantage of variety degeneration in conventional asexual reproduction [6,7].

The tissue culture of medicinal plants offers prominent advantages such as accelerating breeding, enhancing quality, not being limited by regional seasons, and industrial breeding. This technology is indispensable in solving the shortage of traditional Chinese medicine (TCM) resources, preserving excellent germplasm, and promoting the sustainable development of TCM resources in China. Moreover, it is the most widely used biotechnology, aiding in synthesizing secondary metabolites from medicinal plants and breeding new varieties. This technology is acclaimed as the fourth green revolution in the history of agricultural development [8]. Since its inception, the tissue culture system has matured to the point where different concentration ratios of auxin and cytokinin are critical to regenerating adventitious roots and shoots [9]. Recently, a series of breakthroughs have been made in studying medicinal plant regeneration. Huang et al. systematically investigated the induction of embryogenic calli, differentiation of shoot buds and roots, suspension cell culture, and production of active substances in Angelica sinensis. An effective and complete system of suspension cell culture and plant regeneration via somatic embryos of A. sinensis was established for the first time [10]. Furthermore, Zhang et al. designed an in vitro regeneration and cell suspension culture system for Fritillaria cirrhosa bulbs using tissue culture techniques [11]. Costa-Pérez et al. examined the combined effects of cytokinin and UV-C radiation on the phenolic pattern in Ceratonia siliqua shoot cultures. The findings indicate that the accumulation of secondary metabolites in adventitious shoot balls formed in vitro was significantly higher than that in soil cultivation [12].

Regeneration pathways in seed plants can be categorized into tissue repair, somatic embryogenesis, and de novo organogenesis [13]. Currently, the cultivation of A. lancea tissues and cells focuses on in vitro organ culture and industrial breeding. A few studies have been conducted on callus induction, protoplast isolation, and polyploid induction. At present, there are many limitations in the selection and pollution control of explants, optimization of culture media, severe browning phenomenon, and poor adaptability of tissue culture seedlings to a transplantation environment in A. lancea tissue culture. These limiting factors result in a production cost 2–3 times higher than traditional seedling cultivation, which restricts the large-scale application of A. lancea tissue culture seedlings. The latest breakthrough direction is focused on establishing a three-dimensional cell embryogenesis system and developing anti-browning culture media. An efficacious and comprehensive approach for constructing regeneration systems via the induction of A. lancea somatic embryos and organs in vitro has not been established. Therefore, an effective tissue and organ culture system must be urgently developed to produce bioactive metabolites, regenerate plants, and develop new germplasms of A. lancea. This study examined the effects of different explants, exogenous hormones, and culture conditions on the induction of embryogenic calli, the culture of organs, differentiation of shoot buds and roots, and acclimatization of plantlets. The contents of bioactive metabolites in different cultures of A. lancea (calli, adventitious roots, and hairy roots) were determined. This study provides a key technical system and platform for applying modern biotechnology to create new germplasms of A. lancea, verify important functional genes, and perform fermentation studies to produce active substances.

2. Materials and Methods

2.1. Plant Materials and Establishment of Aseptic Seedlings

Mature seeds, apical buds, and axillary buds of A. lancea were obtained from Huang Mei Town, Jurong City, Zhenjiang City, Jiangsu Province. The species was identified by Professor Lanping Guo of the National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.

Aseptic seedlings were established in accordance with the literature [14], albeit with some modifications. Briefly, the explants were first washed with running water (30 min) and then successively rinsed with ethanol (75%, v/v; 30 s), HgCI₂ (0.1%, v/v; 8 min), sterile water (5 times); and then disinfected with the abovementioned rinsing steps. The disinfected explants (i.e., seeds, apical buds, and axillary buds) were inoculated on Murashige and Skoog (MS) medium (pH 5.86) [15] containing 30 g/L sucrose and 7.6 g/L agar in a 125-mL flask and incubated at 23 °C under 16/8-h light/dark cycle of 3000 lx white light, and 75% relative humidity to allow germination. After 30 days, aseptic seedlings containing one radicle and stem were obtained.

2.2. Effect of Different Exogenous Hormones on the Induction and Proliferation of Calli

To investigate the effects of different explants and three different plant growth regulators on callus induction, an L₉ (3⁴) orthogonal experiment was designed, as shown in

Table 1. Factor levels (mg/L) of an orthogonal experiment for inducing callus tissues in Atractylodes lancea.

Level	Factor
	2,4-D (A)	NAA (B)	KT (C)	Explant (D)
1	2.0	0	0.3	Leaf
2	2.0	1.0	0	Petiole
3	1.5	0	0.5	Stem

.

The sterile seedlings of A. lancea were cut into 0.4 × 0.4-cm leaf pieces (blade back), 0.3–0.5-cm leaf stalk segments, and 0.3–0.5-cm stem segments. Each Petri dish was inoculated with 10 leaf explants and 20 leaf stalk and stem explants. Each experiment was repeated thrice. During inoculation, the leaves were placed downward, and the leaf stalks and stem segments were spread flat on the surface of the medium. A slight force was applied to ensure full contact with the medium. The culture temperature was 25 ± 2 °C, with a light intensity of 2000–3000 lx and a light duration of 16 h each day. The callus induction rate was recorded every 10 days, and the statistics were completed after 30 days. The callus induction rate was calculated as the number of explants forming callus divided by the total number of inoculated explants, multiplied by 100%.

2.3. Effect of Different Exogenous Hormones on the Differentiation of Calli

The commonly used auxin NAA and cytokinin 6-BA were selected as plant growth regulators in the culture medium. Considering the uncertainty about whether these two factors independently affected the process, the interaction between them was assessed. Based on the abovementioned analysis, an L₉ (3⁴) orthogonal experiment was designed to investigate the effects of the two plant growth regulators, NAA and 6-BA, and the components of the basic medium (MS) on the differentiation and sprouting of callus. The experimental design is displayed in

Table 2. Factor levels of an orthogonal experiment for the differentiation of Atractylodes lancea callus (mg/L).

Level	Factor
	6-BA (A)	NAA (B)	Medium (C)
1	2.0	0.1	MS
2	4.0	0.4	1/2MS
3	6.0	0.6	1/4MS

.

During inoculation, vigorous callus tissues of size 5 × 5 mm² were selected and uniformly distributed on a differentiation medium (7.6 g/L agar, 30 g/L sucrose, pH 5.86). Five pieces were inoculated in each bottle, and six bottles were set up for each treatment. The culture conditions were as follows: 25 ± 2 °C temperature, 2000–3000 lx light intensity, and 16 h/day light duration. After 30 days, the bud formation rate of the callus tissues was determined.

2.4. Effect of Different Conditions on Bulblet and Adventitious Bud Induction

The uniform design method was adopted to screen the proliferation medium for the adventitious buds of A. lancea. First, the L₇ (7²) table was selected for the experiment design, and 6-BA and NAA were selected as the candidate factors (marked as E and B, respectively). The experimental design is shown in

Table 3. L₇ (7²) The factors and levels for uniform design.

Level	E: 6-BA (mg/L)	B: NAA (mg/L)
1	1.00	0.10
2	1.50	0.20
3	2.00	0.30
4	2.50	0.50
5	3.00	1.00
6	3.50	1.50
7	4.00	2.00

. The aseptic seedlings obtained after seed germination were inoculated into the proliferation medium. Each treatment material was inoculated in 10 bottles, with five young buds in each bottle. After 30 days of culture, the number of differentiated buds in each treatment was determined.

2.5. Effect of Different Conditions on Root Induction

The rooting medium was designed using the L₃ (3² × 2) uniform design table, with NAA, activated carbon, and basic medium serving as the selected factors (marked as A, B, and C, respectively). The levels of the test factors are shown in

Table 4. L₃ (3²) The factors and levels for uniform design.

Level	A: NAA (mg/L)	B: Activated Carbon (g/L)	C: Basic Medium
1	0.1	0.2	1/2MS
2	0.5	0.5	1/2MS
3	1.0	1.0	1/2MS

. A rooting culture was performed when the sprouts reached ≥ 4 cm. The rooted seedlings were first cultivated in the medium containing auxin for 5–7 days and then transferred to the basic medium. After 2 weeks, the total number of roots and the growth status of the plants were determined.

2.6. Establishment of Field Seedlings

In mid-May, when the A. lancea seedlings in test tubes reached a height of 5 cm and had 3–4 long new roots, they could be hardened off and transplanted. The bottle cap was loosened and placed in a greenhouse for 3 days and allowed to harden off under natural light for 4–5 days. The test tube seedlings were then removed, the adhering culture medium was washed off, and the seedlings were transplanted into a seedling substrate (vermiculite: nutrient soil = 2:1) and placed in a plastic greenhouse covered with 60% shading nets for routine management. After 30 days, the transplant survival rate and growth status of the seedlings were investigated.

2.7. Establishment of Hairy Root and Adventitious Root Cultures of A. lancea

Hairy root cultures of A. lancea were established in accordance with the method of Zhang et al. [16], albeit with some modifications. The C58C1 (pRiA4) Agrobacterium rhizogenes was cultured in Luria–Bertani medium supplemented with 50 mg·L⁻¹ rifampicin (w/v) with shaking (180 rpm) at 26 °C in the dark. Single bacterial colonies were inoculated in 50 mL of liquid YEB medium, and the culture was incubated with rotation (180 rpm) at 26 °C for 18 h until the OD₆₀₀ was approximately 0.6–0.8 through the NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, MA, USA). The bacterial suspension was centrifuged at 5000 rpm for 10 min. The cell pellet was resuspended in 50 mL of MS liquid medium and used for subsequent explant co-cultivation experiments. Leaf, petioles, and stem explants isolated from A. lancea aseptic seedlings were used. After 3 days of co-cultivation, the explants were cleaned 3–5 times with the MS liquid medium containing 200 mg·L⁻¹ timentin to kill any residual Agrobacterium. The timentin concentration was halved every 7 days. After approximately 2 months, several hairy roots appeared on the A. lancea explants. The hairy roots, which arose mainly from the cut surfaces, were separated from the explants when they reached a length of 4–5 cm and then placed on MS medium for further growth.

Aseptic seedlings of A. lancea with consistent growth were selected to investigate the induction of adventitious roots under different influencing factors. Each experimental group contained 10 aseptic seedlings of A. lancea, including callus tissue, leaves, stem segments, and petioles. The culture medium used in this study was based on 1/2MS, with sucrose serving as the carbon source. Different concentrations of indoleacetic acid (IAA, 0 and 0.5 mg/L), naphthylacetic acid (NAA, 0.3, 0.4, and 0.5 mg/L), and indolebutyric acid (IBA, 0.5, 1.0, and 1.5 mg/L) were added, and the induction of adventitious roots in A. lancea was calculated after 21 days of cultivation.

2.8. Determination of Essential Oils

2.8.1. Extraction of Essential Oils

The extracts were prepared as described previously [11]. Briefly, the dried calli were finely ground and 0.5 g aliquots were extracted in ethanol (95% v/v, 20 mL) on a shaker (20 °C, 120 rpm for 72 h). The homogenate was centrifuged (4 °C, 5000 rpm, and 10 min), and the supernatant was increased to 20 mL with ethanol (95% v/v) and kept at 4 °C. Differently, the extraction method of volatile oil from hairy and adventitious roots was performed as per the method of Zhang et al. [16], with some modifications. Approximately 500 mg of each hairy root and adventitious root sample was placed into a 50-mL centrifuge tube to which 20 mL of hexane was added. The mixture was subjected to ultrasonic extraction (40 kHz, 30 min) and centrifugation (3000 rpm, 10 min) at room temperature, and the resulting clear supernatant was decanted. The solid residue was re-extracted by repeating the process with an additional 20 mL sample of hexane. After a total of two centrifugations, the supernatants were combined, the volume was adjusted to 50 mL with hexane, and 1 μL of the filtered extract was processed by GC-MS. Prior to chemical analyses, freeze-dried root samples were ground into a fine powder and filtered through a sieve with a screen mesh size of 60, defined as a size number 3 sieve by the Chinese Pharmacopoeia (2020).

2.8.2. Quantitative Analysis of Embryogenic Calli, Hairy Root, and Adventitious Root Essential Oils

The essential oil contents of embryogenic calli, adventitious roots, and hairy roots were determined by GC-MS, and the metabolites were analyzed with ultra-performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS). The test samples subjected to these analyses were cultured for 30 days and the fresh weight of the test samples was 40 mg. GC-MS analyses were performed on the Thermo Fisher Scientific gas chromatography system (Waltham, MA, USA) coupled to a triple quadrupole mass spectrometer (TP-8030; Agilent Technologies, Santa Clara, CA, USA). The GC was fitted with a DB-5MS capillary column (0.25 mm × 30 m, 0.25-μm particle size) with helium as a carrier gas at a flow rate of 1 mL·min⁻¹. The injection mode was split-flow (split ratio, 50:1). The column temperature was held at 120 °C for the first 2 min and then increased at 5 °C·min⁻¹ to 240 °C, at which temperature it was held for 5 min. The MS was operated in electron ionization mode at 70 eV, with an iron source temperature of 230 °C, and an MS temperature of 150 °C, and the MS scanned from m/z 40 to 500 in the MSD data acquisition mode.

2.9. Statistical Analysis

Statistical Analysis: Data preprocessing was performed using R language (v4.3.1). The mean (Mean) and standard deviation (SD) of the proliferation indices for each treatment group were calculated using the dplyr package. Significance tests for differences: one-way analysis of variance (ANOVA) were conducted to assess the inter-treatment differences. For significant effects (p < 0.05), a Tukey’s HSD multiple comparison test (implemented via agricolae: HSD.test) was subsequently applied. Statistically distinct groups were labeled with lowercase letters (α = 0.05) and uppercase letters (α = 0.01) to denote significance levels. Response surface modeling: A quadratic polynomial regression model was established: [Equation placeholder]. Significant terms (p < 0.05 retention threshold) were selected through stepwise regression using the MASS: step function (AIC criterion). Model fitness was evaluated via adjusted R² and AIC values, with significance verification through F-test (α = 0.05).

3. Results and Discussion

3.1. Effects of Different Exogenous Hormones on the Induction Rate and Differentiation of Calli

3.1.1. Analysis of Variance

Most plants must go through the callus stage to produce regenerated plants under in vitro culture conditions. Hence, callus culture is one of the most common forms of plant tissue culture [17]. The core mechanism of A. lancea callus induction involves the activation of cellular dedifferentiation potential via physical/chemical signals, coupled with optimizing hormone ratios, explant selection, and environmental parameters to achieve high-efficiency regeneration. Studies have reported that using a combination of plant growth regulators considerably influences A. lancea tissue culture outcomes [18]. 2,4-Dichlorophenoxy acetic acid (2,4-D), a synthetic auxin analog, is widely utilized owing to its robust efficacy in promoting callus induction. At optimized concentrations, 2,4-D effectively activates cellular dedifferentiation and enhances callus proliferation. It exerts these effects by suppressing light-dependent differentiation pathways and stabilizing auxin signaling. Although this hormone excels in initiating undifferentiated cell clusters, excessive concentrations may inhibit subsequent organogenesis (e.g., root/shoot formation). This dual effect emphasizes the need for precise hormonal modulation tailored to specific developmental stages in tissue culture protocols [19,20].

In plant tissue culture, 2,4-D is often combined with other auxins or cytokinins to optimize callus induction efficiency. This strategy leverages the complementary effects of various plant growth regulators to enhance the dedifferentiation and proliferation of plant cells. In a study, Mamdouh et al. observed that combining 2,4-D and NAA optimally induced callus formation in Lycium schweinfurthii [21]. In addition, suitable explants aid in callus tissue induction. After 7 days of culturing, semitranslucent callus masses emerged at the incision sites of explants. Over time, these callus tissues displayed distinct morphological and color transitions. The callus induction rates after 30 days of cultivation across different treatments are summarized in

Table 5. Orthogonal experiment results of different plant growth regulators on the induction of calli in Atractylodes lancea.

Test Number	Factor				Induction Rate of Calli/%
	2,4-D (mg/L)	NAA (mg/L)	KT (mg/L)	Explant
1	2.0	0	0.3	1	73.67 ± 3.51 bB
2	2.0	1.0	0	2	91.00 ± 2.00 aA
3	2.0	0	0.5	3	64.33 ± 1.53 dC
4	2.0	0	0	3	34.67 ± 2.52 gF
5	2.0	1.0	0.5	1	53.00 ± 2.00 eD
6	2.0	0	0.3	2	72.33 ± 0.58 bcB
7	1.5	0	0.5	2	67.00 ± 1.00 cdBC
8	1.5	1.0	0.3	3	45.33 ± 1.53 fE
9	1.5	0	0	1	18.00 ± 1.00 hG
k1	43.44	55	47.89	48.22
k2	64.83	63.11	63.78	76.78
k3	-	-	61.44	48.11
R	21.39	8.11	15.89	28.67

Note: Different small letters indicate significant differences at the 5% level, and different capital letters indicate significant differences at the 1% level.

. The findings demonstrate that the highest callus induction rate (91%) in A. lancea was achieved when petioles were used as explants and cultured on MS medium supplemented with 2.0 mg/L 2,4-D and 1.0 mg/L NAA. The leaf explants of A. lancea exhibit significantly lower callus induction efficiency compared to petiole explants, primarily due to high cellular differentiation, accumulation of physiological barrier compounds, and structural/functional constraints.

Two main pathways are available for regenerating complete A. lancea plants from detached organs: organogenesis and somatic embryogenesis. This study exclusively investigated the somatic embryogenesis pathway in A. lancea as it exhibits high efficacy and serves as a stable technical platform for constructing genetic transformation systems [22]. This pathway is aligned with CRISPR/Cas9-mediated trait stacking strategies and facilitates the advancement of A. lancea molecular breeding for pharmaceutical applications.

Although the stages of somatic embryogenesis vary across plant species, the process relies universally on cellular pluripotency and requires sterile conditions, optimized culture media, and hormonal regulation for successful regeneration. After the dedifferentiation of in vitro tissues or cells into a callus, embryo-like structures (e.g., globular and heart-shaped embryos) are induced via hormonal regulation, ultimately developing into intact plantlets. 6-Benzylaminopurine (6-BA) is a crucial plant growth regulator in callus differentiation. When used alone, it can induce callus differentiation, and its efficacy is substantially augmented when combined with other phytohormones. Studies have documented that 6-BA, a cytokinin, maintains callus vitality by promoting cell division and inhibiting chlorophyll degradation. 6-BA acts synergistically with other hormones (e.g., IBA and IAA), regulating the differentiation direction (shoot/root) and enhancing callus regeneration capacity [23]. In a postharvest yellowing inhibition study of pak choi, 6-BA + gibberellin (GA) co-treatment delayed leaf senescence and preserved green quality by modulating sugar metabolism and chloroplast autophagy, indirectly sustaining callus activity [24]. Niu et al. showed that callus induction was optimally achieved in potato stem explants in MS medium + 6-BA 1.5 mg/L + NAA 0.5 mg/L. GA3 2.0 mg/L + ZT 1.0 mg/L was added to subsequent differentiation media, significantly promoting shoot regeneration [25]. Once appreciable amounts of embryogenic callus tissue (Figure 1) were obtained from A. lancea, the germination rate of adventitious buds was counted after 7 days of cultivation. The rates for each treatment after 30 days are listed in

Table 6. Orthogonal experiment on induction and differentiation of adventitious buds from callus of Atractylodes lancea explants.

Level	6-BA (mg/L)	NAA (mg/L)	Medium	Budding Rate/%
1	2.0	0.1	1.0	8.5
2	2.0	0.4	1.0	7.2
2	2.0	0.6	1.0	3.1
4	2.0	0.1	0.5	16.3
5	2.0	0.4	0.5	14.7
6	2.0	0.6	0.5	13.8
7	2.0	0.1	0.25	15.2
8	2.0	0.4	0.25	12.7
9	2.0	0.6	0.25	10.8
10	4.0	0.1	1.0	11.3
11	4.0	0.4	1.0	9.2
12	4.0	0.6	1.0	8.3
13	4.0	0.1	0.5	22.7
14	4.0	0.4	0.5	19.1
15	4.0	0.6	0.5	18.3
16	4.0	0.1	0.25	19.8
17	4.0	0.4	0.25	16.3
18	4.0	0.6	0.25	15.2
19	6.0	0.1	1.0	3.6
20	6.0	0.4	1.0	4.2
21	6.0	0.6	1.0	5.1
22	6.0	0.1	0.5	7.6
23	6.0	0.4	0.5	6.2
24	6.0	0.6	0.5	5.7
25	6.0	0.1	0.25	6.1
26	6.0	0.4	0.25	2.3
27	6.0	0.6	0.25	0
k1	11.37	12.34	10.93
k2	15.58	10.21	13.82
k3	4.53	8.92	6.72
R	11.05	3.42	7.1

. The findings demonstrate that the optimal medium composition for the differentiation of adventitious buds from A. lancea callus tissue was 1/2MS + 4.0 mg/L 6-BA + 0.4 mg/L NAA. This study laid the foundation for the tissue culture, rapid propagation, and cell culture techniques of A. lancea.

3.1.2. Stepwise Regression Analysis

Stepwise regression analysis was performed to validate the reliability of experimental results. While the original experimental design assumed independence among factors (A, B, and C), the analysis explicitly incorporated interaction effects and focused on the A × B interaction (Table 2) [26,27]. A quadratic stepwise regression model with the following initial expression was developed: Budding rate = β₀ + β₁A + β₂B + β₃C + β₄A² + β₅B² + β₆C² + β₇AB + β₈AC + β₉BC. Nonsignificant terms (B² and AB) were eliminated via iterative optimization, yielding the following final model: Budding rate = −4.3508 + 11.9222A − 10.8816B + 23.6439C − 1.9069A² − 34.3407C² + 2.7857AC + 6.8847BC. When A = 4 mg/L (6-BA concentration), B = 0.1 mg/L (NAA concentration), and C = 1/2MS medium, the optimal model calculated a germination rate of 20.55%. This rate was close to the callus differentiation rate of 22.7% under this level (Table 6), confirming the reproducibility of the optimal conditions selected by the uniform design.

3.2. Effects of Different Exogenous Hormones on the Bud Differentiation Rate

A. lancea can regenerate via direct organogenesis, a pathway by which explants such as apical or axillary buds bypass the callus formation phase to directly induce shoot and root organogenesis, ultimately developing into complete plantlets [28,29]. Multiple shoot induction is a core technology in plant in vitro regeneration systems and is extensively applied in rapid propagation, genetic transformation, and germplasm conservation [30]. Phytohormonal regulation is the core driver of adventitious shoot differentiation. The precise coordination of cytokinins (e.g., 6-BA) and auxins (e.g., NAA) regulates adventitious shoot differentiation via their concentration ratios and signaling crosstalk [31]. When inducing adventitious shoots in this study, A. lancea aseptic seedlings were categorized into hypocotyls, radicles, cotyledons, and true leaves. These were subsequently transferred to distinct differentiation media. After culturing for 30 days, hypocotyls exhibited the highest shoot induction capability, consistent with prior findings on the superior regenerative potential of embryonic-axis-derived explants in plant tissue culture systems [32]. The adventitious shoots induced from the hypocotyl explants of A. lancea (or those differentiated from the callus) were separated into single shoots. These were inoculated onto seven distinct media formulations (

Table 7. Effect of different hormone combination on cluster bud differentiation.

Test Number	6-BA (mg/L)	NAA (mg/L)	Proliferation Coefficient
1	1.0	1.0	5.47 ± 0.25 cCD
2	1.5	0.2	4.10 ± 0.10 dD
3	2.0	2.0	12.00 ± 0.75 aA
4	2.5	0.3	7.57 ± 0.21 bB
5	3.0	1.5	6.17 ± 0.15 cBC
6	3.5	0.1	5.33 ± 0.58 cCD
7	4.0	0.5	2.30 ± 0.26 eE

Note: Different small letters indicate significant differences at the 5% level, and different capital letters indicate significant differences at the 1% level.

). After 30 days of cultivation, shoot proliferation rates and growth vigor were estimated. The findings revealed significant intertreatment variability in both shoot multiplication efficiency and individual shoot morphology (Figure 2). Of the various tested media, Treatment 3 (MS medium supplemented with 0.2 mg/L NAA and 2.0 mg/L 6-BA) resulted in the highest proliferation of adventitious shoots. The proliferation coefficient was 12, significantly surpassing other treatments (Figure 1 and Table 7). The shoots demonstrated optimal growth vigor, with robust stem elongation and completely expanded true leaves. The following optimal predictive formula was derived from uniform design analysis: mean proliferation coefficient = −11.8538 + 13.4859A + 9.8821B − 2.3958A² − 3.3983AB, where A and B denote the concentrations of 6-BA and NAA (mg/L), respectively. Substituting A = 2 and B = 2 (matching Treatment 3), the predicted value (11.71) was closely aligned with the experimental result (12.0). This observation confirms the model’s reliability for A. lancea shoot optimization. This outcome corroborates prior studies in which cytokinin-dominant media (6-BA > 1.5 mg/L) maximized shoot multiplication in medicinal plants. This study further attempted to increase the proliferation coefficient via callus differentiation. The findings signify that adding different concentrations of 2,4-D resulted in varying degrees of vitrification in the differentiated buds of the callus tissue. Furthermore, the time needed to obtain complete A. lancea plants from the callus tissue was increased. In addition, the cultivation program was complex and prone to variation, which was not conducive to maintaining the characteristics of the parents [33,34]. Therefore, direct induction of shoot formation from explants should be used in the factory cultivation of A. lancea seedlings.

3.3. Effects of Different Hormone Ratios on the Rooting of Seedlings Cultured In Vitro

In vitro propagated shoot clusters, protocorms, and juvenile tissues require root induction after propagating sufficiently to obtain intact plantlets. This process is particularly crucial for medicinal herbs in which roots are used as pharmacological components. The underlying reason is that only rooted in vitro plantlets possess medicinal value [35,36]. Root induction is a core component of plant in vitro regeneration systems, and its efficacy directly determines the acclimatization and large-scale production of tissue-cultured seedlings. The totipotency of plant cells serves as the biological foundation for adventitious root formation in tissue-cultured seedlings. Once explants such as apical meristems and juvenile leaves dedifferentiate to form a callus, root primordia develop via hormone-mediated redifferentiation processes [37]. The number of roots in various culture media was statistically analyzed, and the results are presented in

Table 8. Effect of different hormone combination on the rooting of test tube seedlings.

Level	NAA (mg/L)	Activated Carbon (g/L)	Average Number of Roots
1	0.1	0.2	13.00 ± 1.00 efEF
2	0.1	0.5	9.33 ± 0.58 ghGH
3	0.1	1	8.00 ± 1.00 hH
4	0.5	0.2	17.00 ± 1.00 cdCD
5	0.5	0.5	23.00 ± 1.00 aA
6	0.5	1	21.00 ± 1.00 abAB
7	1	0.2	15.00 ± 1.00 deDEF
8	1	0.5	12.00 ± 1.00 fgFG
9	1	1	7.00 ± 1.00 hH
10	0	0.2	19.00 ± 1.00 bcBC
11	0	0.5	21.00 ± 1.00 abAB
12	0	1	16.00 ± 1.00 dCDE
k1	18.67	16
k2	10.11	16.33
k3	20.33	13
k4	11.33	NA
R	10.22	3.33

Note: Different small letters indicate significant differences at the 5% level, and different capital letters indicate significant differences at 1% level. NA: Not Available/Not Applicable.

. Significant variations in root number and growth morphology were observed under different hormone ratio treatments. Treatment 6 resulted in thicker roots, the highest average root count (e.g., 21.00 ± 1.00 roots/plant), and optimal seedling growth. The optimal medium for A. lancea tissue-cultured seedlings was 1/2MS + NAA 0.5 mg/L + activated carbon 1 g/L, with influence factors ranked as NAA > activated carbon (p < 0.05). Our research results suggest that adding activated carbon can help induce rooting in tissue culture seedlings, as activated carbon regulates the hormone balance and creates a dark environment. Research has indicated that 1/2MS medium alleviates osmotic stress on root elongation by reducing the concentrations of ammonium (NH₄⁺, 10.3 mM) and nitrate (NO₃⁻, 19.7 mM). This reduction facilitates root induction in woody plants (e.g., Eucalyptus and Pinus) by minimizing ionic toxicity to root meristem cells. Auxins such as IBA and NAA, at concentrations of 0.5–2.0 mg/L, initiate root primordium formation by activating ARF7/19-mediated auxin signaling, which in turn regulates auxin-responsive genes (e.g., LBD16 and WOX11). Nutrient stress, dark environments, and hormones regulate root initiation in in vitro cultures, whereas light, pH, and temperature further modulate the rooting efficiency [38]. The rooting efficiency of in vitro plantlets is regulated by several factors, including genotype specificity, hormone ratios (e.g., auxin/cytokinin balance), and environmental conditions (e.g., light cycles and temperature stability). Industrial application potential can be significantly enhanced by accurately designing the culture medium and combining gene editing with Nano delivery technology. In the future, limitations associated with species specificity should be overcome, and an intelligent and low-cost production system should be built.

Finally, A. lancea test tube seedlings were transplanted onto various substrates with different growth conditions. The substrate ratio of vermiculite: nutrient soil = 2:1 resulted in the highest growth and survival rates of the seedlings. However, the seedlings planted in garden soil exhibited weak growth and a few were rotten, considerably reducing the survival rate (Figure 2). This is mainly due to the poor permeability and air permeability of the garden soil, which can lead to soil compaction.

3.4. Effects of Different Conditions on Adventitious Root Induction in A. lancea

Adventitious root induction in medicinal plants (e.g., Panax ginseng and Codonopsis pilosula) has been substantially enhanced via systematic improvements in explant pretreatment, hormone synergy (NAA/IBA), and dark–light cyclic cultivation [39]. Plant roots are primary sites for synthesizing bioactive secondary metabolites (e.g., anticancer compounds in Dendrobium officinale). However, wild root collection faces challenges because of ecological constraints and compositional heterogeneity. Adventitious root induction overcomes these limitations by establishing homogeneous in vitro models with standardized growth conditions (e.g., optimized NH₄⁺/NO₃⁻ ratios in 1/2MS medium) [40]. When combined with KCRISPR-Cas9 gene editing or metabolomics technology, it can accurately analyze the secondary metabolism regulatory network, accelerate new drug development, and is the core technology for the sustainable development of TCM resources. Applying biotechnology for the mass production of bioactive compounds in medicinal plants is challenging owing to fluctuating secondary metabolite levels in conventional cell cultures. This difficulty is primarily attributed to the weak expression of biosynthetic pathways during cell proliferation. Culturing adventitious roots in A. lancea can solve this problem, and this approach exemplifies how organ-specific cultivation bridges the gap between metabolic stability and industrial feasibility in modernizing traditional medicine [41,42].

In this study, aseptic seedlings of A. lancea were categorized into different explant types (leaf blades, petioles, stem segments, and callus) and inoculated onto solid media containing varying hormone combinations. The findings proved that stem segments and callus exhibited superior capacity for adventitious root induction when cultured on 1/2MS solid medium supplemented with 0.5 mg/L IBA + 0.5 mg/L NAA, attaining a root induction rate of 72.30% (Figure 3). The adventitious root culture system developed in this study offers a novel and efficient platform for synthesizing bioactive sesquiterpenes (e.g., atractylodin, β-eudesmol, and hinesol) during in vitro propagation. The challenges associated with conventional cell suspension systems can thus be addressed.

3.5. Comparative Analysis of Essential Oils Content in Embryogenic Calli, Hairy Root, and Adventitious Root of A. lancea

The technology of plant calli, adventitious roots, and hairy roots enables the efficient and stable production of active ingredients via in vitro culture. This technology is valuable in resource conservation, drug development, and industrial production [43]. Our previous studies have suggested that the four A. lancea essential oil components studied here, namely, atractylodin, β-eudesmol, hinesol, and atractylon, could be utilized as chemical markers to authenticate and trace the geographic origins of plant samples [2,41].

The contents of atractylodin, atractylon, hinesol, and β-eudesmol, considered active components, were investigated in two root cultures (adventitious and hairy roots) and calli of A. lancea. As depicted in Figure 4, the contents of atractylodin and atractylon in hairy roots were significantly higher (p < 0.05) than those in adventitious roots and calli. Of these, the content of atractylon in hairy roots was the highest (0.02%). Calli had the lowest atractylon content of 0.0017%. The hinesol content in adventitious roots was significantly higher (p < 0.05) than that in hairy roots and calli. In contrast, the content in hairy roots was slightly higher than that in calli, but the difference was not significant. Similarly, the β-eudesmol content in adventitious roots was significantly higher than that in hairy roots and calli. These results reveal a clear difference in the essential oil synthesis ability of various root systems and tissue parts of A. lancea. The hairy and adventitious roots of A. lancea were significantly superior to calli in synthesizing active substances owing to their genetic stability, environmental responsiveness, and metabolic capacity. In the future, their industrial potential must be explored further via gene editing and process optimization [44].

3.6. A Proposed Approach to Produce Bioactive Metabolites and Regenerate Seedlings

Plant tissue culture, the most widely utilized biotechnology in plant science, has been applied on an industrial scale in horticultural and staple crops (e.g., ornamental flowers, rice, and wheat). Recent advancements have extended this technology to medicinal plant research and production, termed medicinal plant tissue culture, aligning with the societal demand for sustainable utilization and conservation of TCM resources [45,46]. Plant stem cells exhibit inherent advantages as a biosynthetic chassis for plant-derived natural metabolites, leveraging their intrinsic metabolic networks and pluripotency to synthesize complex secondary metabolites with high fidelity. Concurrently, the large-scale cultivation of medicinal plant adventitious roots and engineered microbial/plant cell factories under synthetic biology frameworks represents a pivotal pathway for advancing TCM modernization [47,48]. Therefore, by exploiting the “efficient plant regeneration adventitious root culture cell factory” technology system, technical support can be provided for the protection and sustainable utilization of medicinal plant germplasm resources. Various aspects include germplasm resource preservation and new variety breeding, natural product production, and the industrial production of medicinal plants.

Building upon previous studies, this research systematically deciphered the key regulatory pathways of in vitro organ regeneration in A. lancea and established a high-efficiency technical framework. This framework integrated organ regeneration, hairy root induction, and adventitious root amplification by optimizing hormone combinations and refining culture parameters (Figure 5). The findings indicate that the tissue culture technology of A. lancea can achieve efficient plant regeneration and active ingredient production by precisely regulating the explants, culture media, and environmental conditions. In the future, genotype limitations and metabolic bottlenecks should be overcome, artificial intelligence and synthetic biology should be integrated, and a new generation of intelligent production systems should be built.

4. Conclusions

Based on the above investigations, a strategy to produce bioactive metabolites and regenerate seedlings was established within 95 days under optimized culture conditions (e.g., hormones, light, and temperature). Essential oils (e.g., atractylodin, β-eudesmol, hinesol, and atractylon) were obtained via callus proliferation and root culture. Optimized culture media for A. lancea in vitro regeneration were determined. Callus induction: MS basal medium supplemented with 2.0 mg/L 2,4-D (auxin analog) and 1.0 mg/L NAA; callus differentiation: 1/2MS medium containing 4.0 mg/L 6-BA (cytokinin) and 0.4 mg/L NAA; bud proliferation: MS medium with 2.0 mg/L 6-BA and 0.2 mg/L NAA; root induction: 1/2MS medium fortified with 0.5 mg/L NAA and 2.0 g/L activated carbon (for inhibitor adsorption). Based on these results, an effective and complete in vitro approach was proposed to regenerate plants and produce bioactive metabolites in A. lancea. Furthermore, the establishment of “efficient plant-regeneration and bioactive metabolite production” can provide technical support for the protection and sustainable utilization of A. lancea germplasm resources in terms of germplasm resource preservation and new variety breeding, natural product production, and the industrial breeding of medicinal plants.