In Vitro Plantlet Regeneration and Accumulation of Ginkgolic Acid in Leaf Biomass of Ginkgo biloba L.

Abstract

This study established an efficient in vitro regeneration system using stem nodes from root collar suckers as explants. Subsequently, regenerated shoots were used to establish an in vitro medicinal production protocol that achieved ginkgolic acid production. The self-developed Ginkgo biloba medium (GBM), first reported in this study, was pivotal to system establishment. The plantlet propagation system showed that the bases of stem nodes dipped in GBM with 2 mg·L⁻¹ 6-benzyladenine (BA) and 0.2 mg·L⁻¹ 1-naphthaleneacetic acid (NAA) achieved near-complete axillary bud induction (99.56%). Adventitious shoot induction reached 82.22% (3.5 shoots/explant) using GBM with 0.2 mg·L⁻¹ BA, 0.02 mg·L⁻¹ kinetin (Kin) and 0.2 g·L⁻¹ proline (Pro). Maximum adventitious shoot elongation (92.22%, average 3.35 cm) was observed on GBM containing 0.1 mg·L⁻¹ zeatin (ZT) and 0.01 mg·L⁻¹ BA. After 3-week preculture with 15 mg·L⁻¹ phloroglucinol (PG), treatment with 0.6 mg·L⁻¹ indole-3-butyric acid (IBA) and 0.2% activated carbon (AC) yielded 96.67% rooting (6.19 roots/explant) and 85% acclimatization survival. For medicinal resource production, bud cluster induction at 94.44% (20.89 buds/explant) on GBM with 1 mg·L⁻¹ BA, 0.03 mg·L⁻¹ Kin, and 0.2 g·L⁻¹ Pro. Leaf organs in GBM with 0.3 mg·L⁻¹ BA, 0.01 mg·L⁻¹ Kin, 0.01 mg·L⁻¹ IBA, 0.3 g·L⁻¹ Pro, and 0.01 mg·L⁻¹ glutamine (Gln) accumulated 20.64 g fresh weight and 41.910 mg·g⁻¹ DW ginkgolic acids, representing a 4.93-fold increase over mother plants. This system enables large-scale Ginkgo biloba L. propagation and provides an in vitro strategy for producing medicinal compounds in endangered plants.

1. Introduction

Ginkgo biloba L. is a deciduous tree of the genus Ginkgo in the family Ginkgoaceae [1]. As a “living fossil” in the plant world, it is one of the oldest relict plants surviving after the Quaternary glacial movement, and its unique phylogenetic position endows it with high value for conservation biology and research [2]. G. biloba is originally from China, and was later introduced to Japan, South Korea and other countries [3]. It has a tall and straight trunk and unique fan-shaped leaves, and is often planted in large areas as an excellent landscape tree [4]. G. biloba is adaptable and resistant to a variety of pollutants, so it can effectively purify the air, so it is widely used in urban streets, industrial areas and other heavily polluted areas [5]. Meanwhile, G. biloba forests provide stable habitats for insects and microorganisms, playing an important role in maintaining ecosystem balance and protecting biodiversity [6]. Owing to its rarity and ecological significance, G. biloba is listed as Endangered (EN) on the IUCN Red List and included in the List of Rare and Endangered Plants in China [7].

G. biloba is a dual-use plant of significant medicinal and nutritional value [8]. Its leaves are extensively utilized in both traditional medicine and modern pharmacology. Traditional Chinese Medicine considers that G. biloba leaves has the effect of activating blood circulation, unblocking meridians, antibacterial and anti-inflammatory, relieving cough and asthma, and stopping leukorrhea [9,10]. Modern pharmacological studies have demonstrated that the main active components in G. biloba leaf extract include flavonoids (quercetin, kaempferol, isorhamnetin), terpenoid lactones (ginkgolide A, B, C), and phenolic acids (ginkgolic acid) [11,12]. These compounds exhibit a wide range of biological activities, including antibacterial, antioxidant, antiviral, anti-allergic, anti-aging, and anti-tumor effects [13,14,15,16,17]. It is worth noting that ginkgo acid, as a unique active ingredient of G. biloba, has shown significant application potential in the field of ophthalmology. It can not only reduce the frequency of retinal detachment, prevent inflammation related to retinal diseases, but also effectively alleviate age-related macular degeneration (MD) and glaucoma [5]. At present, ophthalmic diseases have become common clinical diseases, so the development of ginkgo acid as a drug has broad market prospects.

However, G. biloba grows extremely slow, with a juvenile period of 20–30 years, which has become the main obstacle to its large-scale propagation [18]. Traditional in vivo propagation is not only a long cycle, but also difficult to overcome its inherent slow growth problem. It also faces significant challenges such as sex differentiation (especially because the market prefers male plants) and genetic variation. Male G. biloba trees are favored for their lack of unpleasant falling fruit in urban greening, landscape and road planting. The existing in vitro regeneration research [19,20] mainly focuses on embryo and stem segment culture, with extremely low regeneration efficiency, producing an average of only 1.56 regenerated buds per explant, and there are problems such as difficulty in rooting, low shoot survival rate, and long regeneration cycle, which seriously restrict industrial application. In order to meet the demand of male shoots, promote the industrialization of G. biloba and solve the problem of seed source shortage, it is imperative to establish an efficient, rapid and controllable propagation system. In vitro regeneration technology is the key to solving this dilemma and has already achieved significant results in the propagation of various relict plants such as the Bretschneidera sinensis Hemsl. [21] and Metasequoia glyptostroboides Hu & W. C. Cheng [22]. Therefore, overcoming the bottleneck of in vitro regeneration technology and establishing an efficient rapid propagation system for male G. biloba trees is the key to solving the problem of seed shortage and realising industrial development.

The acquisition of G. biloba leaves, especially as a stable supply of medicinal sources, also faces severe challenges. Although artificial large-scale planting can relieve the pressure on wild resources, the content of effective medicinal components in the leaves is easily affected by tree age, planting environment and seasonal changes, which makes it difficult to ensure the uniformity and stability of medicinal substances. In vitro culture technology has become an important direction to break through the traditional restrictions. In the existing research on G. biloba medicinal sources, the main methods are to induce callus tissue and suspension cell culture of leaves and embryos. The main researches focus on the production of flavonoids and terpenyl esters, while the production of ginkgolic compounds through organ regeneration is relatively less. Previous studies have generally shown that there are some limitations in the production of active ingredients using callus or suspension cell system, such as seasonal limitation of explant source, genetic instability and difficulty in maintaining secondary metabolite content [23,24,25]. Therefore, developing a controllable tissue culture leaf organ regeneration technology that is not subject to seasonal and regional constraints and can achieve efficient production throughout the year has become a breakthrough path to solve the sustainable and high-quality supply of G. biloba medicinal resources.

To address the critical shortage of G. biloba germplasm resources and the unstable supply of medicinal ingredients, this study developed an integrated in vitro regeneration system. Stem nodes from root collar suckers were used as innovative explants, and growth regulators were applied by dipping to induce axillary buds. The elongated axillary buds were then used to efficiently induce adventitious shoots. By using additives and auxins in combination, the rooting bottleneck was overcome, enabling large-scale propagation of shoots. Sterile shoot stem nodes, produced on a large scale, were used as medicinal sources for producing explants. This process involved inducing bud cluster formation and proliferation, expanding leaf organ biomass, and investigating the effects of plant growth regulators and additives on the enrichment of ginkgolic acids. Therefore, this study aims to achieve the in vitro regeneration of high-quality male shoots and the targeted enrichment of ginkgolic acids for medicinal use.

2. Materials and Methods

2.1. Preparation of Culture Media

Prepare the media according to the components and concentrations listed in

Table 1. Components and concentrations of the media.

Medium Component	MS	GBM	WPM
		Concentrations (mg·L−1)
KNO3	1900	1425	—
K2SO4	—	—	990
NH4NO3	1650	1237.5	400
KH2PO4	170	170	170
MgSO4·7H2O	370	370	370
CaCl2	332.5	249	—
Ca(NO3)2·4H2O	—	—	556
KI	0.83	0.83	—
H3BO3	6.2	6.2	6.2
MnSO4·4H2O	22.3	22.3	22.3
ZnSO4·7H2O	8.6	8.6	8.6
Na2MoO4·2H2O	0.25	0.25	0.25
CuSO4·5H2O	0.025	0.25	0.25
CoCl2·6H2O	0.025	0.025	0.025
Inositol	100	100	100
Glycine	2	2	1
Fat-soluble vitamin	0.5	0.5	0.5
Vitamin B6	0.5	0.5	0.5
Vitamin B1	0.1	0.1	0.1
Na2·EDTA	37.3	37.3	37.3
FeSO4·7H2O	27.8	27.8	27.8

, add sucrose (Xilong Scientific Co., Ltd., Shantou, Guangdong, China) (30 g·L⁻¹) and agar (Shanghai Shize Biotechnology Co., Ltd., Shanghai, China) (5 g·L⁻¹), adjust the pH to 6.0 using 1 mol·L⁻¹ HCl or 1 mol·L⁻¹ NaOH (Sartourius, Göttingen, Germany) solution, and autoclave (SHENAN, Shanghai, China) at 121 °C and 103 kPa for 20 min. The GBM prepared and used in this study is a basic medium specially designed and optimized for G. biloba tissue culture, and its name is directly derived from the species name.

2.2. Initial Explant Preparation

Fifty healthy male G. biloba trees over 30 years old were randomly selected from the Shanghai Chenshan Botanical Garden (121°18′ E, 31°08′ N) (Figure 1A), and their annual root collar suckers were collected (Figure 1B). The leaves were removed from the root collar suckers, and stem nodes were cut as explants (Figure 1C). The stem nodes were immersed in water with detergent to clean the surface dust, then rinsed continuously with running tap water for 60 min, and finally cleaned with an ultrasonic cleaner (Xiaomei Ultrasonic Instruments Co., Ltd., Kunshan/Jiangsu, China) for 30 min. Transfer to a laminar flow hood for surface disinfection: first soak in 75% [v/v] ethanol solution (Shanghai Titan Technology Co., Ltd.) for 40 s, then soak in 20% sodium hypochlorite (NaClO) solution (Shanghai Titan Technology Co., Ltd.) for 10 min, and rinse with sterile water five times. Dry the surface moisture of the stem nodes with sterile filter paper, then inoculate them into the culture medium. Cultivate under conditions of 25 ± 2 °C, 16 h of light (2000–2200 lx)/8 h of darkness. After two weeks of cultivation, select sterile stem segment explants that are free from contamination and show good growth for subsequent experiments.

2.3. Plant Regeneration for Plantlet Resource

2.3.1. Selection of Basic Medium

To determine the optimal basic culture medium for the proliferation of axillary bud on G. biloba stem nodes, disinfected stem nodes were inoculated into hormone-free MS, GBM, and WPM culture media. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (100 mL triangular glass bottle with 40 mL of medium). Axillary bud proliferation time was recorded during the cultivation period. After 5 weeks, a comprehensive statistical analysis of parameters, including the average number of leaves per bud and growth status was performed on all 90 explants.

2.3.2. Induction and Elongation of Axillary Buds

In order to further establish an effective G. biloba plant regeneration system, the effects of different plant growth regulators (PGRs) on axillary bud induction and subsequent elongation of stem nodes from root collar suckers were evaluated. Two treatments were used for axillary bud induction, one was directly inoculated in GBM supplemented with graded concentrations of BA (0.1, 0.5, 1.0 mg·L⁻¹) and NAA (0.01, 0.03, 0.05 mg·L⁻¹), and medium without PGRs was set up as a control, and the other was to first immerse the bases of stem nodes in BA (1.0, 2.0, 3.0 mg·L⁻¹) and NAA (0.1, 0.2, 0.3 mg·L⁻¹) mixture for 30 s and then transferred to GBM without PGRs. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (100 mL triangular glass bottle with 40 mL of medium). After 4 weeks of incubation, a comprehensive measurement and statistical analysis of the axillary bud induction rate (the number of axillary bud induced explants/the number of initial explants × 100%) was performed on these 90 explants.

To study the effect of PGRs on axillary bud elongation. Well-grown axillary buds were selected for elongation, cut off with old stumps and inoculated into GMB medium containing BA (0.01, 0.02, 0.1, 0.2 mg·L⁻¹) and zeatin (ZT) (0.1, 0.2, 1.0 mg·L⁻¹). A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each of the 3 explants was inoculated into a container (100 mL triangular glass bottle with 40 mL of medium). After 2 weeks of incubation, the plants were subcultured into the same medium, with 1 explant per container. After 4 weeks of incubation, a comprehensive measurement and statistical analysis of the axillary bud elongation rate (the number of elongated buds/the number of buds on the elongation medium × 100%) and average shoot height was performed on these 90 explants.

2.3.3. Induction of Adventitious Shoots from Elongated Axillary Buds

The effects of PGRs on the induction of adventitious shoots from elongated axillary buds were studied. Cut stem nodes approximately 2 cm in length from elongated axillary buds and inoculate them onto GBM-based shoot regeneration medium. The induction medium contains Kin (0.01–0.03 mg·L⁻¹), BA (0.1–0.3 mg·L⁻¹) and 0.2 g·L⁻¹ proline (Pro). A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each of the 3 explants was inoculated into a container (100 mL triangular glass bottle with 40 mL of medium). After 5 weeks of culture, a comprehensive measurement and statistical analysis of the frequency of adventitious shoot induction (the number of explants with adventitious shoots/the number of initial explants × 100%) and the average number of shoots was performed on these 90 explants.

2.3.4. Elongation of Adventitious Shoots

To investigate the effect of PGRs on the elongation of adventitious shoots. Transfer the adventitious buds obtained after 5 weeks of cultivation to GBM containing 10 mg·L⁻¹ phloroglucinol (PG) for a 2-week preculture. Subsequently, the explants were transferred to GBM containing combinations of BA (0, 0.01, 0.02 mg·L⁻¹) and ZT (0.05, 0.1, 0.2 mg·L⁻¹) to induce shoot elongation. After 3 weeks of culture, the elongated shoots were cut and transferred to the same culture medium for further culture. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (100 mL triangular glass bottle with 40 mL of medium). After 5 weeks of culture, a comprehensive measurement and statistical analysis of the shoot elongation rate (the number of elongated shoots/the number of shoot on the elongation medium × 100%) and average shoot height was performed on these 90 explants.

2.3.5. Effects of IBA and PG on Root Induction

The effects of indole-3-butyric acid (IBA) and PG on root induction were systematically evaluated through two consecutive experiments. For IBA treatment, elongated shoots were cultured in WPM medium containing an IBA gradient (0.2–1.2 mg·L⁻¹) and 0.2% activated carbon (AC), with hormone-free WPM medium used as the control. After 8 weeks of cultivation, the average number of roots and rooting rate (number of rooted seedlings/number of initial buds × 100%) were recorded. For PG treatment, elongated shoots were first pre-cultured in WPM medium containing a PG gradient (5–30 mg·L⁻¹) for 3 weeks, then transferred to the optimal IBA-containing medium (determined in previous experiments) for 5 weeks. The average number of roots and rooting rate were recorded. Both groups were cultured for 8 weeks. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (each consisting of one explant in a 350 mL wide-mouth jar containing 100 mL of medium).

2.3.6. Acclimatization

The rooted regenerated plantlets were filled with sterile water placed under natural light for 3 d and then the medium was thoroughly removed by rinsing under running water. Subsequently, plantlets were transplanted into plastic pots with a substrate of peat: vermiculite: perlite = 3:1:1 volume ratio, covered with transparent polyethylene bags, and placed in a growth chamber with temperature of 25 ± 2 °C, light intensity of 2000–2200 lx, light period of 16/8 h, and relative humidity of 70%. A three-phase acclimatization programme was implemented: total enclosure for 3 weeks, followed by gradual ventilation for 1 week, and finally bag removal was completed after week 4. Regenerated plantlets survival was assessed 2 months after transplantation.

2.4. Plant Regeneration for Medicinal Resource

Scale-cultivated regenerated shoots were used as initial materials to study their bioactive metabolites.

2.4.1. Induction and Proliferation of Bud Clusters

The effect of PGRs on the induction and proliferation of bud clusters was investigated. Stem nodes of G.biloba sterile shoots were cut as explants and pre-cultured in GBM basal medium containing BA (0.5, 1, 2, 3 mg·L⁻¹), Kin (0.01, 0.03, 0.05 mg·L⁻¹), Pro (0.2 g·L⁻¹), and PG (10 mg·L⁻¹). After 2 weeks, the material was transferred to GBM without PG but maintaining the original concentration gradient of BA, Kin and 0.2 g·L⁻¹ Pro for 8 weeks. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (100 mL triangular glass bottle with 40 mL of medium). A comprehensive measurement and statistical analysis of the induced rate of adventitious shoots (the number of bud clusters induced explants/the number of initial explants × 100%) and the average number of shoots was performed on these 90 explants at the end of this stage (bud clusters induction stage). Subsequently, stem nodes with bud clusters produced by induction were selected as explants in the proliferation stage, and the effect of BA concentration gradient (0.1, 0.3, 0.5, 0.7, 1.0 mg·L⁻¹) on the proliferation of adventitious buds was investigated using GBM as the basic medium. The fresh weight of each explant was recorded before inoculation. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (350 mL wide-mouth jar with 100 mL of medium). After 8 weeks of cultivation, a comprehensive measurement and statistical analysis of the fresh weight was performed on these 90 explants.

2.4.2. Effect of PGRs on Biomass Expansion of Leaf Organs

The proliferating leaf organs were cut and inoculated in GBM containing a gradient concentration combination (BA: 0.1–0.5 mg·L⁻¹; NAA: 0.01–0.03 mg·L⁻¹). Before inoculation, the fresh weight of each explant was weighed and recorded. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (100 mL triangular glass bottle with 40 mL of medium). After 5 weeks of cultivation, a comprehensive measurement and statistical analysis of the fresh weight was performed on these 90 explants.

2.4.3. Effects of PGRs and Additives on Ginkgolic Acid Content and Biomass Accumulation

The effects of PGRs and additives on ginkgolic acid content and biomass accumulation were investigated simultaneously. Leaf organs from Section 2.4.2 were cut and inoculated into a medium containing different concentrations of BA (0.1–0.5 mg·L⁻¹), Kin (0–0.02 mg·L⁻¹) and IBA (0.01–0.05 mg·L⁻¹) with GBM as the basic medium. Leaf organs from Section 2.4.2 were inoculated into GBM with different concentrations of Pro (0.1–0.5 g·L⁻¹). The initial fresh weight of each explant was weighed and recorded before inoculation. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (150 mL triangular glass bottle with 50 mL of medium). After 6 weeks of cultivation, a comprehensive measurement and statistical analysis of the fresh weight was performed on these 90 explants. The ginkgolic acid content was determined by HPLC for the optimal treatment of PGRs and additives.

2.4.4. Effect of Combination Formulas on Ginkgolic Acid Content and Biomass Accumulation

Cut the leaf organs in Section 2.4.2 and inoculate them with the three optimal PGR combinations and Pro in Section 2.4.3, using GBM as the basic medium and adding 0.01 mg·L⁻¹ glutamine (Gln) to each group of medium. Prior to inoculation, the initial fresh weight of each explant was weighed and recorded. A total of 90 explants were used per treatment, constituting 3 independent biological replicates with 30 explants each. Each explant was inoculated individually into a container (150 mL triangular glass bottle with 50 mL of medium). After 6 weeks of cultivation, a comprehensive measurement and statistical analysis of the fresh weight was performed on these 90 explants. The ginkgolic acid content of the optimal treatment was determined by HPLC.

2.4.5. Detection of Ginkgolic Acid Content

Sample pretreatment: Fresh tissue samples were freeze-dried to obtain dry weight samples, ground and crushed, weighed 300 mg, added 3.0 mL of methanol (Shanghai Titan Technology Co., Ltd., Shanghai, China), and extracted by ultrasonic extraction for 30 min. 1 mL of the extract was centrifuged at room temperature for 10 min at 15,000 rpm, and 300 μL of the supernatant was taken. The sample was diluted 10-fold with methanol solution, 100-fold with methanol solution, and finally 2-fold with an equal volume of water to obtain the test solution, which was used for UPLC-MS analysis (instrument: XEVO-TQS WAA529 mass spectrometer; Waters Corporation, Milford, MA, USA; controlled by MassLynx V4.1 software, Waters Corporation, Milford, MA, USA).

Ultra-high performance liquid chromatography-mass spectrometry (UPLC-MS) analysis: Samples were sent to Shanghai Institute of Materia Medica, Chinese Academy of Sciences for qualitative and quantitative detection of ginkgolic acid.

2.5. Statistical Analysis

Data were subjected to analysis of variance (ANOVA), and significantly different means were separated using Duncan’s multiple range test in SPSS (version 27.0).

3. Results

3.1. Plant Regeneration for Plantlet Resource

3.1.1. Effects of Basic Medium on Axillary Bud Proliferation and Growth Status

GBM had the shortest axillary bud proliferation time (7 d) and the best growth condition, with an average of 7 leaves per bud, robust stems, and tender green leaves with good expansion (Figure 2A); MS medium performed second best, with an axillary bud proliferation time of 8 d, an average of 7 leaves, and slender stems, mildly yellowed leaves, and relatively poor growth condition (Figure 2B); WPM medium performed the worst, with axillary bud proliferation delayed to 10 d, an average of only 5 leaves per bud, weak stems, and most leaves failing to unfold (Figure 2C).

Based on axillary buds proliferation time, average number of leaves per bud, and growth condition (

Table 2. Effects of basic medium on axillary bud proliferation and growth status.

Basic Medium	The Time of Axillary Bud Proliferation (d)	The Average Number of Leaves per Bud	Growth Status
MS	8	7	Average growth, slender stems, leaves spread out, and leaves are slightly yellowed.
GBM	7	7	Good growth, sturdy stems, leaves spread out, and leaves are light green.
WPM	10	5	Poor growth, weak stems, most leaves unspread, and leaves are yellowish.

), GBM was determined to be the most suitable basal medium and was used in subsequent experiments.

3.1.2. Effect of Different PGRs and Treatment Methods on Axillary Buds Induction and Elongation

The effect of two different treatment methods on axillary buds induction was investigated. As shown in

Table 3. Effect of different concentrations of PGRs and treatments on axillary bud induction.

Treatments	Plant Growth Regulators (mg·L−1)		Axillary Buds Induction Rate (%)
	BA	NAA
Directly inoculated	0	0	86.67 ± 1.34 a
	0.1	0.01	77.78 ± 0.77 b
	0.1	0.03	69.45 ± 1.35 c
	0.1	0.05	62.22 ± 1.54 d
	0.5	0.01	54.90 ± 2.13 e
	0.5	0.03	47.51 ± 2.10 f
	0.5	0.05	40.89 ± 2.04 g
	1.0	0.01	31.11 ± 1.54 h
	1.0	0.03	24.77 ± 1.85 i
	1.0	0.05	16.89 ± 0.77 j
Dipping	1.0	0.1	68.44 ± 0.77 e
	1.0	0.2	76.89 ± 2.04 d
	1.0	0.3	61.34 ± 2.31 f
	2.0	0.1	92.89 ± 1.54 b
	2.0	0.2	99.56 ± 0.77 a
	2.0	0.3	85.33 ± 1.34 c
	3.0	0.1	45.33 ± 2.67 h
	3.0	0.2	54.66 ± 2.31 g
	3.0	0.3	38.39 ± 1.77 i

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

, the axillary bud induction rate decreased in medium containing hormones (BA and NAA). The highest rate of axillary buds induction (86.67%) was observed in GBM without the addition of BA and NAA, yet with less leaf unfolding and weak stems (Figure 3A). Under BA (0.1 mg·L⁻¹) and NAA (0.03 mg·L⁻¹), the axillary buds induction rate was 69.45%, and some buds showed basal bursting and necrosis (Figure 3B). The addition of high concentration of BA (1.0 mg·L⁻¹) decreased the rate of axillary bud induction, the rate of the combination of 1.0 mg·L⁻¹ BA + 0.01 mg·L⁻¹ NAA was only 31.11%, and the stem nodes were severely burst (Figure 3C). When the base of stem nodes was dipped in PGRs and then transferred to GMB medium, the induction rates of different combinations of BA and NAA were significantly different (p < 0.05, Table 3). When the concentration of one hormone remains constant, as the concentration of another hormone increases, the induction rate gradually rises, reaches a peak, and then gradually decreases. The induction rate was 61.34–76.89% at 1.0 mg·L⁻¹ BA, and was lower than 54.66% at 3.0 mg·L⁻¹ BA, accompanied by the formation of callus wounds. Healthy axillary buds cultured under dip 2.0 mg·L⁻¹ BA + 0.2 mg·L⁻¹ NAA for 10 days (Figure 4A) and vigorous growth of buds and tender green unfolding of leaf blades at 4 weeks (Figure 4B) were the optimal axillary bud induction protocols, with an induction rate of 99.56%.

At axillary bud elongation, the elongation ranged from 22.22% to 87.78%, and the mean bud height ranged from 0.73 to 6.17 cm. At a fixed value of ZT, the elongation increased and then decreased with the increase in BA concentration (

Table 4. Effects of different concentrations of PGRs on axillary bud elongation.

Plant Growth Regulators (mg·L−1)		Axillary Buds Elongation Rate (%)	Average Shoot Height (cm)
BA	ZT
0.01	0.1	66.67 ± 3.34 d	3.47 ± 0.42 d
0.01	0.2	72.22 ± 1.92 c	4.60 ± 0.26 c
0.01	1.0	54.45 ± 3.85 ef	2.50 ± 0.17 e
0.02	0.1	80.00 ± 3.33 b	5.53 ± 0.21 b
0.02	0.2	87.78 ± 1.92 a	6.17 ± 0.15 a
0.02	1.0	58.89 ± 5.09 e	2.80 ± 0.26 e
0.1	0.1	45.56 ± 1.93 g	1.73 ± 0.06 f
0.1	0.2	50.00 ± 0.00 fg	2.03 ± 0.06 f
0.1	1.0	27.78 ± 1.92 j	1.00 ± 0.17 gh
0.2	0.1	33.33 ± 3.34 i	1.20 ± 0.10 g
0.2	0.2	38.89 ± 1.92 h	1.37 ± 0.15 g
0.2	1.0	22.22 ± 5.09 k	0.73 ± 0.21 i

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). The highest elongation (87.78%) was observed at 0.2 mg·L⁻¹ ZT + 0.02 mg·L⁻¹ BA and the lowest (22.22%) at 1.0 mg·L⁻¹ ZT + 0.2 mg·L⁻¹ BA. Shoot height was significantly inhibited at 0.1 mg·L⁻¹ and 0.2 mg·L⁻¹ BA concentrations, with mean shoot heights ranging from 0.73–2.03 cm, which differed significantly (p < 0.05) from that of the lower concentration group (0.01–0.02 mg·L⁻¹ BA). The combination 0.2 mg·L⁻¹ ZT + 0.02 mg·L⁻¹ BA was the most effective, achieving 87.78% elongation and the best mean shoot height (6.17 cm), with robust shoot morphology and physiological integrity, most suitable for subsequent experiments (Figure 4C, D).

3.1.3. Effect of PGRs on the Induction of Adventitious Shoots from Elongated Axillary Buds

In adventitious shoot induction culture, 0.2 g·L⁻¹ Pro was added to the basal medium to promote the induction of adventitious shoots. The combination of two hormones, BA and Kin, differed significantly on inducing adventitious shoots (p < 0.05,

Table 5. Effect of PGRs on the induction of adventitious shoots from elongated axillary buds.

Plant Growth Regulators (mg·L−1)		Shoot Regeneration Frequency (%)	Shoots per Explant
BA	Kin
0.1	0.01	36.67 ± 3.34 f	0.58 ± 0.07 f
0.1	0.02	44.45 ± 3.85 e	0.77 ± 0.04 e
0.1	0.03	24.44 ± 1.93 g	0
0.2	0.01	71.11 ± 5.09 b	2.56 ± 0.05 b
0.2	0.02	82.22 ± 3.85 a	3.50 ± 0.07 a
0.2	0.03	60.00 ± 3.33 c	1.78 ± 0.09 c
0.3	0.01	45.56 ± 1.93 e	0.83 ± 0.07 e
0.3	0.02	52.22 ± 3.85 d	1.44 ± 0.05 d
0.3	0.03	12.22 ± 5.09 h	0

Note: Each group of culture medium was supplemented with 0.2 g·L⁻¹ Pro. The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). When the concentrations of BA and Kin were 0.2 mg·L⁻¹ and 0.02 mg·L⁻¹, respectively, and mixed at a ratio of 10:1, the number of adventitious buds regenerated from the explants was 82.22%, with an average of 3.5 shoots/explant, which induced the most shoots, with bright green leaves and better growth (Figure 5B). The medium containing the same concentration of BA and Kin concentration reduced to 0.01 mg·L⁻¹ was followed by 71.11% of shoot regeneration with 2.56 shoots/explant. At constant BA, increasing Kin to 0.03 mg·L⁻¹ reduced the bud regeneration rate to 60% and the average shoots per explant to 1.78, and at this time, the long-growth buds were average, with a little yellowing of the leaf blade and basal expansion (Figure 5A). Combined BA (0.3 mg·L⁻¹) and Kin (0.02 mg·L⁻¹) yielded 52.22% shoot regeneration and an average of 1.44 shoots per explant, yet the shoots were short and brittle, and the base appeared necrotic, which made it difficult to grow in the later stage of the season (Figure 5C). At 0.03 mg·L⁻¹ Kin, 0.1 or 0.3 mg·L⁻¹ BA, the induced rates of adventitious shoots were very low, 24.44% and 12.22%, respectively, and no shoots were differentiated. Therefore, 0.2 mg·L⁻¹ BA + 0.02 mg·L⁻¹ Kin + 0.2 g·L⁻¹ Pro was the optimal hormone combination for inducing adventitious buds, which resulted in the highest rate of adventitious shoot induction (82.22%) and 3.5 adventitious shoots per explant on average.

3.1.4. Effect of PGRs on the Elongation of Adventitious Shoots

Both ZT alone and ZT combined with BA promoted adventitious shoot elongation, with elongation rates ranging from 11.11% to 92.22% and average shoot heights ranging from 0.75 cm to 3.55 cm. In the treatment using only ZT, adventitious shoot elongation was initially stimulated and then inhibited as the ZT concentration increased, with significant statistical differences between treatments (p < 0.05,

Table 6. Effect of different concentrations of PGRs on the elongation of adventitious shoots.

Plant Growth Regulators (mg·L−1)		Adventitious Shoot Elongation Rate (%)	Average Shoot Height (cm)
BA	ZT
0	0.05	26.67 ± 0.00 h	0.98 ± 0.04 gh
0	0.1	33.33 ± 3.34 g	1.17 ± 0.13 e
0	0.2	11.11 ± 1.92 i	0.75 ± 0.02 hi
0.01	0.05	84.45 ± 3.85 b	2.94 ± 0.10 b
0.01	0.1	92.22 ± 1.92 a	3.55 ± 0.14 a
0.01	0.2	72.22 ± 3.85 c	1.75 ± 0.08 c
0.02	0.05	55.56 ± 5.09 e	1.08 ± 0.17 fg
0.02	0.1	65.56 ± 1.93 d	1.43 ± 0.01 d
0.02	0.2	42.22 ± 3.85 f	0.90 ± 0.02 h

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). Supplementing with BA enhances elongation capacity while maintaining a similar trend, and all BA and ZT combinations significantly outperform the pure ZT control. The optimal growth parameters were obtained at a plant growth regulator with 0.01 mg·L⁻¹ BA and 0.1 mg·L⁻¹ ZT. Therefore, the optimal adventitious shoot elongation formula was obtained by transferring the plants to a medium containing 0.01 mg·L⁻¹ BA and 0.1 mg·L⁻¹ ZT after pre-culturing them in GBM containing 10 mg·L⁻¹ PG for 2 weeks, resulting in 92.22% elongation and a mean height of 3.55 cm (Figure 6).

3.1.5. Effect of IBA and PG Concentrations on Root Induction

The rooting induction capacity was systematically evaluated using WPM medium supplemented with different concentrations of IBA and PG. The WPM medium without IBA failed to initiate root formation, while the addition of a small amount of IBA triggered adventitious root development (

Table 7. Effect of IBA and PG concentrations on root induction.

Plant Growth Regulators	Concentration (mg·L−1)	Rooting Rate (%)	Average Number of Roots
IBA	0	0	0
	0.2	68.89 ± 3.23 d	1.55 ± 0.14 f
	0.4	78.89 ± 1.42 c	1.93 ± 0.17 e
	0.6	94.44 ± 2.02 a	5.07 ± 0.19 a
	0.8	89.00 ± 2.39 b	4.74 ± 0.11 b
	1.0	86.89 ± 1.65 b	4.35 ± 0.15 c
	1.2	81.11 ± 4.13 c	2.93 ± 0.12 d
PG	5	72.22 ± 3.85 c	2.48 ± 0.14 e
	10	85.56 ± 1.93 b	4.20 ± 0.13 c
	15	96.67 ± 3.34 a	6.19 ± 0.22 a
	20	90.00 ± 3.33 b	5.43 ± 0.18 b
	30	75.56 ± 5.09 c	3.00 ± 0.17 d

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). At IBA levels of 0.2–0.4 mg·L⁻¹, statistically significant differences were observed (p < 0.05), with rooting rates and average root numbers per explant being 78.89% and 1.93 at 0.4 mg·L⁻¹ IBA, respectively. When the IBA concentration was 0.6 mg·L⁻¹, rooting efficiency reached its maximum, with a rooting success rate of 94.44% and an average of 5.07 roots after 56 days. Compared to the optimal treatment, super-optimal concentrations (0.8–1.2 mg·L⁻¹) exhibited an inhibitory effect, reducing rooting frequency and root number. Ultimately, 0.6 mg·L⁻¹ IBA was determined to be the critical concentration for maximising in vitro root induction efficiency in this experiment.

Elongated buds were pre-cultured in WPM medium supplemented with different concentrations of PG for 3 weeks, then cultured in WPM containing 0.6 mg·L⁻¹ IBA and 0.2% AC for 5 weeks, with evaluation conducted after 8 weeks of cultivation. Experimental data indicated that PG concentration significantly influenced root induction. Compared to the optimal treatment, both excessively high or low PG concentrations inhibited root growth (Table 7). Pre-culturing with 15 mg·L⁻¹ PG was the optimal treatment, yielding a rooting rate of 96.67% and 6.19 roots per explant (Figure 6D). Comparative analysis showed that, compared to the control group without PG addition, this protocol improved rooting efficiency by 2.23% and increased the average number of roots by 1.12 roots per explant within the same cultivation period. Therefore, initial induction for 3 weeks in WPM containing 15 mg·L⁻¹ PG, followed by induction in medium containing 0.6 mg·L⁻¹ IBA and 0.2% AC, is the optimal rooting induction strategy.

3.1.6. Acclimatization

After 3 months of acclimatization, the survival rate of G. biloba regenerated plantlets was 93%, and the acclimatised regenerated plantlets had healthy terminal buds that showed the typical morphological and growth characteristics of this plant species (Figure 6E). The leaves were still bright green at 12 months of transplantation (Figure 6F).

3.2. Plant Regeneration for Medicinal Source

3.2.1. Effect of PGRs on the Induction and Proliferation of Bud Clusters

In the study of the medicinal source system, 10 mg·L⁻¹ PG was used for pre-cultivation for 2 weeks to promote the lignification process of stem segment explants and lay a foundation for subsequent bud clusters induction and proliferation. Experimental data showed that the synergistic effect of BA and Kin significantly regulated bud regeneration (

Table 8. Effect of PGRs on the induction of bud clusters.

Plant Growth Regulators (mg·L−1)		Bud Cluster Induction Rate (%)	Average Number of Shoots
BA	Kin
0.5	0.01	84.44 ± 1.93 c	10.63 ± 0.93 d
0.5	0.03	85.56 ± 1.93 c	13.96 ± 0.94 c
0.5	0.05	83.33 ± 3.34 cd	9.04 ± 0.34 e
1.0	0.01	90.00 ± 0.00 b	16.22 ± 1.31 b
1.0	0.03	94.44 ± 1.93 a	20.89 ± 2.34 a
1.0	0.05	78.89 ± 3.85 de	7.78 ± 0.51 ef
2.0	0.01	81.11 ± 1.92 cde	6.70 ± 0.36 fg
2.0	0.03	76.67 ± 3.85 e	5.44 ± 0.20 gh
2.0	0.05	62.22 ± 1.92 g	3.52 ± 0.32 ij
3.0	0.01	70.00 ± 0.00 f	4.71 ± 0.85 hi
3.0	0.03	56.67 ± 3.34 h	2.52 ± 0.33 j
3.0	0.05	46.67 ± 3.34 i	0.60 ± 0.28 k

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). The optimal induction effect was achieved under the synergistic treatment of 1.0 mg·L⁻¹ BA and 0.03 mg·L⁻¹ Kin, with an explant bud regeneration rate of 94.44% and an average of 20.89 buds per explant, significantly higher than other treatment groups (p < 0.05). Dynamic observation showed that visible bud clusters formed after 4 weeks of cultivation (Figure 7A), and bud clusters grew on a large scale after 8 weeks (Figure 7B). The next best combination was 1.0 mg·L⁻¹ BA and 0.01 mg·L⁻¹ Kin, with a bud regeneration rate of 90.00% and an average number of buds of 16.22. Notably, when the BA concentration was reduced to 0.5 mg·L⁻¹, combined with a gradient treatment of 0.01–0.05 mg·L⁻¹ Kin, the bud regeneration rate remained stable within the range of 83.33–85.56%, with no significant differences between groups (p > 0.05). High concentrations of BA (3.0 mg·L⁻¹) significantly inhibited organogenesis, especially when combined with 0.05 mg·L⁻¹ Kin, resulting in a sharp decrease in bud regeneration rate to 46.67% and an average bud number of only 0.60. This phenomenon suggests that excessive cytokinin may cause hormonal imbalance, thereby inhibiting bud differentiation. The addition of different concentrations of BA can increase the average fresh weight of buds during the bud clusters proliferation stage, but the average fresh weight increase varies (

Table 9. Effect of BA on the proliferation of bud clusters.

BA (mg·L−1)	Average Fresh Weight Gain (g)
0.1	14.20 ± 0.63 c
0.3	16.81 ± 0.52 b
0.5	18.36 ± 0.56 a
0.7	15.96 ± 0.72 b
1.0	12.61 ± 0.65 d

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). At BA concentrations below 0.5 mg·L⁻¹, the average fresh weight increase was positively correlated with concentration. When the concentrations of BA between 0.7 and 1.0 mg·L⁻¹, the average fresh weight exhibited a significant dose-dependent negative effect. At 0.5 mg·L⁻¹ BA, the average fresh weight increase was highest, reaching 18.36 g. Therefore, the optimal bud clusters induction protocol was to use GBM containing 10 mg·L⁻¹ PG (1.0 mg·L⁻¹ BA + 0.03 mg·L⁻¹ Kin + 0.2 g·L⁻¹ Pro) for 2 weeks of pre-cultivation, followed by transfer to GBM without PG (same component concentrations as above) for cultivation. The optimal medium for bud clusters proliferation was GBM with 0.5 mg·L⁻¹ BA added (Figure 7C).

3.2.2. Effect of PGRs on Biomass Expansion of Leaf Organs

In order to promote the accumulation of in vitro medicinal sources, the effects of BA and NAA ratio on leaf organ biomass amplification were studied. The results showed (

Table 10. Effect of PGRs on biomass expansion of leaf organs.

Plant Growth Regulators (mg·L−1)		Average Fresh Weight Gain (g)
BA	NAA
0.1	0.01	1.64 ± 0.25 ef
0.3	0.01	2.07 ± 0.43 de
0.5	0.01	0.23 ± 0.21 h
0.1	0.02	3.11 ± 0.14 c
0.3	0.02	4.92 ± 0.34 a
0.5	0.02	2.60 ± 0.27 c
0.1	0.03	2.41 ± 0.25 cd
0.3	0.03	3.97 ± 0.34 b
0.5	0.03	1.41 ± 0.27 g

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

) that the growth of leaf organs was inhibited when BA was a constant value and the concentration of NAA was too high or too low. At BA:NAA ratio of 50:1, with concentrations of 0.5 mg·L⁻¹ and 0.01 mg·L⁻¹, respectively, the lowest average fresh weight increase observed was 0.23 g. The second lowest mean fresh weight gain of 1.41 g was observed when the concentrations of BA and NAA were 0.5 mg·L⁻¹ and 0.03 mg·L⁻¹, respectively. However, when the ratio of BA/NAA = 15:1 (0.3 mg·L⁻¹ + 0.02 mg·L⁻¹), the fresh weight accumulation reached a peak of 4.92 g, which was significantly higher (p < 0.01) than the other groups (Figure 8).

3.2.3. Effects of PGRs and Additives on Ginkgolic Acid Content and Biomass Accumulation

BA and Kin exhibit significant synergistic effects at low concentrations (0.1–0.3 mg·L⁻¹ BA). When the culture medium contained 0.1 mg·L⁻¹ BA and 0.01 mg·L⁻¹ IBA, the fresh weight of leaf organs in the treatment with 0.01 mg·L⁻¹ Kin was 13.19 g, which was 7.35 g higher than that in the control group without Kin (5.84 g) (

Table 11. Effects of PGRs on biomass accumulation.

Plant Growth Regulators (mg·L−1)			Average Fresh Weight Gain (g)
BA	Kin	IBA
0.1	0	0.01	5.84 ± 0.33 f
0.1	0.01	0.01	13.19 ± 0.57 b
0.1	0.02	0.02	10.86 ± 0.65 d
0.3	0	0.03	9.64 ± 0.46 e
0.3	0.01	0.01	14.15 ± 0.54 a
0.3	0.02	0.02	11.55 ± 0.51 cd
0.5	0.01	0.02	12.06 ± 0.65 c
0.5	0.02	0.03	4.84 ± 0.76 g
0.5	0.03	0.05	3.15 ± 0.30 h

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). The optimal hormone combination was 0.3 mg·L⁻¹ BA, 0.01 mg·L⁻¹ Kin and 0.01 mg·L⁻¹ IBA, with an average fresh weight increase of 14.15 g. The hormone combination of 0.1 mg·L⁻¹ BA, 0.01 mg·L⁻¹ Kin and 0.01 mg·L⁻¹ IBA had the second-highest average fresh weight increase, at 13.19 g. Notably, the combination of high-concentration IBA (0.05 mg·L⁻¹) with 0.5 mg·L⁻¹ BA and 0.03 mg·L⁻¹ Kin produced a significant inhibitory effect (increase of only 3.15 g). The effects of Pro concentration on ginkgolic acid content and biomass accumulation were investigated, and significant differences were observed between media containing 0.2, 0.3, and 0.4 g·L⁻¹ (p < 0.05) (

Table 12. Effect of additives on biomass amplification.

Pro (g·L−1)	Average Fresh Weight Gain (g)
0.1	1.14 ± 0.46 d
0.2	2.66 ± 0.22 b
0.3	3.52 ± 0.54 a
0.4	2.06 ± 0.35 c
0.5	0.56 ± 0.26 d

Note: The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

). Among these, the addition of 0.3 g·L⁻¹ Pro yielded the best results, with an average fresh weight increase of 3.52 g; the addition of 0.2 g·L⁻¹ Pro yielded the next best results, with an average fresh weight increase of 2.66 g.

3.2.4. Effect of Combination Formulation on Ginkgolic Acid Content and Biomass Accumulation

Leaf organs of Section 2.4.2 were cut and inoculated in a GBM-based culture system, and the experiment contained three optimised PGR combinations (0.3 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA, 0.1 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA, and 0.5 mg·L⁻¹ BA +0.01 mg·L⁻¹ Kin + 0.02 mg·L⁻¹ IBA) in combination with three proline concentration gradients (0.2, 0.3, and 0.4 g·L⁻¹) in cross treatment. The results showed (

Table 13. Effect of combination formulation on biomass accumulation.

Plant growth Regulators (mg·L−1)			Additives (g·L−1)	Average Fresh Weight Gain (g)
BA	Kin	IBA	Pro
0.1	0.01	0.01	0.2	15.75 ± 0.66 cd
0.1	0.01	0.01	0.3	16.23 ± 0.54 c
0.1	0.01	0.01	0.4	14.85 ± 0.88 d
0.3	0.01	0.01	0.2	18.14 ± 0.53 b
0.3	0.01	0.01	0.3	20.64 ± 0.65 a
0.3	0.01	0.01	0.4	16.43 ± 0.63 c
0.5	0.01	0.02	0.2	11.87 ± 0.95 f
0.5	0.01	0.02	0.3	13.53 ± 0.89 e
0.5	0.01	0.02	0.4	9.76 ± 0.77 g

Note: Each group of culture medium was supplemented with 0.01 mg·L⁻¹ Gln. The means with the same letters in the same column were not statistically significant after Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± standard deviation of three independent experiments with 30 explants per treatment.

) that when the hormone ration was 0.1 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA, the mean fresh weight increment of leaf organs did not show significant differences. In contrast, when two hormone combinations of 0.3 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA and 0.5 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.02 mg·L⁻¹ IBA were used, the fresh weight increment reached the level of significant difference (p < 0.05) among the treatment groups. Notably, the synergistic treatment of 0.3 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA + 0.3 g·L⁻¹ Pro showed the optimum biomass accumulation effect, with a mean fresh weight gain of 20.64 g, which obviously better than other treatment combinations (Figure 9).

3.2.5. Detection of Ginkgolic Acid Content

The results showed (

Table 14. Determination of ginkgolic acid content.

Sample Name	Enrichment Incubation Time	Medium Formulation	Ginkgolic Acid Contenta (mg·g−1 DW)
Natural material	—	—	8.498
Optimal plant growth regulator combinations	6 W	GBM + 0.3 mg·L−1 BA + 0.01 mg·L−1 Kin + 0.01 mg·L−1 IBA	13.127
Optimal proline concentration	6 W	GBM + 0.3 g·L−1 Pro	8.890
Optimal combination of formulas	6 W	GBM + 0.3 mg·L−1 BA + 0.01 mg·L−1 Kin + 0.01 mg·L−1 IBA + 0.3 g·L−1 Pro + 0.01 mg·L−1 Gln	41.910

) that 13.127 mg·g⁻¹ DW of ginkgolic acid was detected under the GBM with the plant growth regulator combination of 0.3 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA (Figure 10B), and 8.890 mg·g⁻¹ DW of ginkgolic acid was detected under the optimal proline concentration of 0.3 g·L⁻¹ (Figure 10C).

In the case of the combination of the formulation of GBM + 0.3 mg·L⁻¹ BA + 0.01 mg·L⁻¹ Kin + 0.01 mg·L⁻¹ IBA + 0.3 g·L⁻¹ Pro + 0.01 mg·L⁻¹ Gln, the ginkgolic acid content was detected as 41.910 mg·g⁻¹ DW in leaf organs cultured for 6 W, which is 4.93 times higher than that of natural materials (Figure 10D).

4. Discussion

This system can rapidly produce high-quality male shoots of G. biloba, avoiding the unpleasant odor of the white fruit, thus meeting market demand and promoting industrial planting (Figure 11). Additionally, it enables year-round, efficient production under sterile and controlled laboratory conditions, reducing the risk of heavy metal and pesticide contamination from soil, thereby obtaining cleaner medicinal materials (Figure 12).

Selecting the optimal basic medium is crucial for establishing an efficient in vitro plant regeneration system. The MS [26] medium provides ample nutrients for plant growth through high levels of nitrogen and potassium (KNO₃ 1900 mg·L⁻¹; NH₄NO₃ 1650 mg·L⁻¹), which aligns with its widespread use in the tissue culture of monocot and eudicot plants [27,28,29]. However, excessive nitrogen may interfere with the absorption of divalent ions such as calcium and magnesium, leading to nutritional unbalanced jaundice [30,31]. WPM is widely used in woody plant culture because of its good adaptability to woody plants [32,33,34,35]. The WPM medium was designed without KNO₃, and Ca(NO₃)₂·4H₂O (556 mg·L⁻¹) and K₂SO₄ (990 mg·L⁻¹) were used to provide nitrogen, potassium and calcium elements in combination [36]. However, Carrier et al. [37] observed in G. biloba suspension culture that the form of potassium ion provided by potassium sulfate in WPM may reduce the absorption efficiency of G. biloba cells to potassium. Given the aforementioned issues, this study developed a GBM that balances the concentrations of KNO₃ (1425 mg·L⁻¹), NH₄NO₃ (1237.5 mg·L⁻¹), and CaCl₂ (249 mg·L⁻¹) between MS and WPM (Table 1). This design addresses the high nitrogen stress in MS and the low potassium utilization efficiency in WPM. The moderate reduction in nitrogen concentration minimizes damage to cell membrane permeability, while the optimized ratio of calcium and potassium ions enhances cell wall stability and signal transduction efficiency [38]. For the trace elements in the culture medium, the GBM continues the high demand for copper ions (CuSO₄·5H₂O 0.25 mg·L⁻¹) from the WPM medium, with a content ten times higher than that of 0.025 mg·L⁻¹ in the MS medium. This is because copper ions act as cofactors for polyphenol oxidase (PPO), which plays a crucial role in lignin synthesis [39]. It is evident that the composition of elements in the culture medium is particularly important and is a key factor in plant regeneration. It is evident that the composition of elements in the culture medium is particularly important and is a key factor in plant regeneration. This critical role manifests specifically at different stages of cultivation. For instance, during the root induction phase, this study selected WPM medium rather than GBM precisely based on considerations of nutritional requirements at different stages. Compared to GBM, the lower salt concentration in WPM medium facilitates the transition of woody plants from the rapid proliferation phase to the root differentiation stage. This effectively avoids the inhibition of root primordia by high salt concentrations and promotes root elongation. In our preliminary experiments, we compared the rooting efficacy of three media: MS, GBM, and WPM. Results indicated that while root primordia could be induced in GBM, roots generally exhibited shorter, thicker growth accompanied by partial callus formation. Conversely, WPM medium yielded higher rooting rates, producing more robust, slender roots with significantly improved post-transplant survival rates. This finding that WPM medium enhances woody plant rooting aligns with multiple studies on woody plant regeneration [34,40,41].

The method of dipping PGRs ensures the growth of axillary buds, which is a crucial step in establishing plant regeneration. Currently, some studies on axillary bud induction primarily involve adding different concentrations of PGRs to the culture medium [42,43,44,45,46]. This study investigates axillary bud induction using both the addition and dipping methods of PGRs. When plant regulators are added, the young G. biloba root collar suckers stem nodes will continuously absorb and accumulate plant growth regulators, resulting in the main stem bursting from the base and severe necrosis. By dipping the sprout segments, the young stems can absorb the regulators while avoiding continuous hormone accumulation. By using the dipping method, it not only allows the young shoots of G. biloba to absorb plant growth regulators but also avoids the continuous accumulation of hormones. This experimental design not only evaluates the impact of treatment methods on bud induction but also suggests that G. biloba has high endogenous hormone concentrations, providing a theoretical reference for further exploring its growth and development mechanisms. This finding is consistent with previous research results [47].

The additive PG plays a key role in the regeneration of G. biloba. PG is a phenolic compound, which is the degradation product of phloridzin, it can promote plant growth and is used as a plant growth regulator [48,49,50]. Research by Ross et al. [51,52] found that PG accelerates the division and elongation of plant cells, thereby promoting lignification in plants. Cheng et al. [53] conducted an orthogonal experiment during tissue culture and concluded that adding 10 g·L⁻¹ PG enhances the rooting of papaya. Feng et al. [54] studied Taxus chinensis and discovered that adding PG increases the biomass of Taxus chinensis sprouts. This study fully drew on the previous experience and applied PG in the study of G. biloba in vitro regeneration system. During the growth phase of adventitious shoots, the shoots are pre-cultured for two weeks in a GBM containing 10 mg·L⁻¹ PG, followed by transplantation to a medium with 0.01 mg·L⁻¹ BA and 0.1 mg·L⁻¹ ZT. The shoot elongation rate is 92.22%, with an average height of 3.55 cm. In the critical rooting induction stage, the effects of different concentrations of PG, 0.6 mg·L⁻¹ IBA, and 0.2% AC on rooting induction were investigated. The results indicate that 15 g·L⁻¹ PG is the optimal concentration, achieving a rooting rate of 96.67%, with each explant producing 6.19 roots, and robust root development. This study successfully demonstrates the significant efficacy of PG in promoting plant in vitro regeneration.

Cytokinase plays a central role in the G. biloba in vitro regeneration system, and its type and concentration ratio directly determine the efficiency of bud induction and proliferation [55]. Our study demonstrates that the combined use of BA and Kin produces significant synergistic effects [56]. As the primary cytokin, BA effectively stimulates the formation of meristematic tissue and induces cellular dedifferentiation to initiate protoblast development [55]. Kin further enhances bud differentiation and subsequent growth. This dual-action combination not only increases induction rates quantitatively but also improves bud robustness qualitatively. During the proliferation phase, appropriate concentrations of BA are crucial for maintaining self-renewal and proliferative capacity in stem cell populations within leaf organs [57]. This provides sufficient cellular resources for continuous adventitious bud formation, significantly increasing the number of regenerative leaf organs and establishing a material foundation for large-scale biomass expansion.

Establishing an efficient in vitro medicinal plant production system is crucial for G. biloba variety improvement and active compound development [58]. This study reveals that the ginkgo acid content and biomass accumulation in leaf organs are synergistically regulated by PGRs and additives. The optimal ratio of cytokinin (BA, Kin) to auxin (IBA) maximizes cell division and elongation processes, thereby enhancing biomass yield. Notably, proline supplementation creates a stable internal environment for leaf organ growth. It improves osmotic regulation [59] and antioxidant capacity [60] to enhance hormone interactions, while boosting BA and Kin signal transduction to accelerate biomass increase. Additionally, exogenous Gln enhances efficacy through three mechanisms: first, supporting amino acid and nucleotide synthesis as a nitrogen metabolism hub [61]; second, regulating redox homeostasis to maintain cellular vitality [62]; and third, synergizing secondary metabolic pathways to accelerate active compound accumulation. In conclusion, the BA-Kin-IBA-Pro-Gln formulation systematically improves both biomass and active compound accumulation in G. biloba leaf organs, providing a strategic approach for industrial-scale application of in vitro medicinal plant systems.

The synthesis and accumulation of secondary metabolites (e.g., ginkgolic acid) represent a prototypical time-dependent dynamic process, typically requiring an extended cultivation period to stabilise or reach peak levels. A six-week cultivation cycle was determined as the critical time point for ginkgolic acid content measurement within the culture system established for this study. This six-week cultivation period provided a sufficient time window for ginkgolic acid synthesis within our experimental system, enabling its accumulation to levels amenable to accurate detection and possessing biological significance. Our preliminary observations indicate that under the established culture conditions, a stable phase following rapid growth is reached at 6 weeks. Whilst longer cultivation periods (such as 8–10 weeks) may theoretically yield further accumulation, these are concurrently associated with nutrient depletion, metabolic waste accumulation, and potential risks of enzymatic degradation or biotransformation. We hypothesise that ginkgolic acid accumulation kinetics may follow an S-shaped or parabolic curve model. Beyond six weeks, its accumulation rate is anticipated to gradually decelerate towards a plateau phase. Should cultivation persist beyond this point, ginkgolic acid content may either enter a decline due to aforementioned degradation mechanisms or stabilise at a lower level owing to complete nutrient depletion and a sharp decline in cellular viability.

Constructing an efficient and stable in vitro regeneration system is crucial for overcoming the bottleneck of sustainable production of G. biloba medicinal resources. As a significant medicinal plant, G. biloba leaves and seeds are known for their significant pharmacological activity and have a huge market demand. However, traditional propagation methods using shoots are time-consuming, and access to high-quality germplasm resources is limited, often affected by seasonal factors, making it challenging to meet the demands of large-scale and stable medicinal resource production. This study successfully developed an in vitro regeneration system for G. biloba, which efficiently induces adventitious buds from elongated axillary buds, followed by the elongation and rooting of these shoots. The establishment of this system provides a large number of stable sterile shoots, laying a solid foundation for large-scale and controlled production of medicinal materials. Therefore, the development of this efficient in vitro regeneration system serves as a vital bridge and essential guarantee between high-quality G. biloba germplasm resources and sustainable large-scale medicinal resource production.

5. Conclusions

In this study, for the first time, root collar suckers of G. biloba were used as explants to establish an efficient in vitro regeneration system and achieve in vitro directed production of ginkgolic acid. Based on the GBM developed for G. biloba in vitro culture, the key effects of plant growth regulator (PGR) concentrations on axillary bud induction, axillary bud elongation, adventitious shoot induction, adventitious shoot elongation, and rooting were systematically studied. The effects of PGRs and additive combinations on leaf organ biomass expansion and ginkgolic acid accumulation were also studied. The ginkgo acid content in regenerated leaf organs was significantly higher than that in the parent plant, confirming its potential for pharmaceutical production. This technological breakthrough not only provides a solution for the conservation of endangered G. biloba species and the large-scale supply of high-quality shoots but also serves as a reference for the in vitro resource development of other medicinal plants.