Tissue culture of Juniperus chinensis: effects of sex and leaf morphology on in vitro regeneration

Abstract

Background and Aims

In tissue culture of Juniperus chinensis, issues such as inadequate sterilization, severe browning and poor rooting have significantly hindered the widespread application of techniques of its asexual propagation. Meanwhile, J. chinensis presents three sexes: female (FP), male (MP) and monoecious (MOP), with leaf morphological differentiation into scale leaves (SL) and spiny leaves (SPL). Variations in sexes and leaf morphogenesis may affect the establishment of tissue culture systems. However, there are currently no studies that have established tissue culture systems by integrating both sex and leaf morphogenesis, and the differences in responses of explants with different sexes and leaf morphogenesis during culture remain unclear.

Methods

Using SL and SPL from FP, MP and MOP as explants, we optimized explant disinfection protocols, screening of culture medium, selection of anti-browning agents, determination of antioxidant enzyme activities, proliferation and rooting culture, plant hardening and transplantation. We comprehensively explore the differences in the process of establishing tissue culture among J. chinensis with different sexes and leaf morphogenesis.

Key Results

Disinfection of explants with two types of leaf morphogenesis from three sexes can be achieved by treating them with sodium hypochlorite (NaClO) for 18 min. The basal medium suitable for the early growth of explants of the three sexes and two leaf morphogenesis types is Douglas-fir Cotyledon Revised medium (DCR). Polyvinylpyrrolidone (PVP) can inhibit browning by reducing the activity of oxidases. During the proliferation stage, explants derived from MOP demonstrate consistent hormone requirements. The efficiency of adventitious bud proliferation of SPL is notably higher than that of SL, and all proliferated adventitious buds are SPL. At the rooting stage, the rooting ability of adventitious buds from SPL is inferior to that of SL. Conversely, MOP still maintain consistent hormone demands during the rooting stage. After transplantation, sterile plants display vigorous growth, with stem segments undergoing progressive lignification, and the survival rate shows variability influenced by both sexes and leaf morphogenesis.

Conclusions

This study established a multi-dimensional tissue culture system for J. chinensis based on three sexes (FP, MP and MOP) and their corresponding SL and SPL. It analysed the differences and preferences among different sexes and leaf forms at various stages of tissue culture, providing technical support for the targeted breeding, germplasm resource innovation and industrial development of J. chinensis. Meanwhile, it offers novel perspectives for research on sex differentiation, mechanisms of leaf form transformation and other related areas in polygamous or heterophyllous tree species.

INTRODUCTION

Juniperus chinensis, an evergreen tree belonging to the genus Juniperus in the family Cupressaceae, is predominantly dioecious and rarely monoecious. Leaves are all spiny leaves when young, and then scale leaves gradually differentiate (Yao, 2022; Cui et al., 2025). Juniperus chinensis is highly stress-resistant, tolerating barrenness and drought. It has straight trunks, with yellow–brown heartwood, pale yellow to white sapwood, light soft texture and neat grain, making it ideal for construction, home decoration and carving. Juniperus chinensis is native primarily to East Asia, with a wide distribution across temperate to subtropical zones. It grows in China from southern Northeast China to mountainous areas in the southwest (below 2000 m elevation), and is also native to Korea and Japan. It has since been introduced to Europe, North America and other regions (Singh et al., 2018).

Juniperus chinensis has scarce wild resources and slow artificial growth. Its main propagations are seed propagation and softwood cutting (Jin et al., 2012). Seed propagation, prone to natural hybridization, fails to retain superior varieties and even causes their degradation. Softwood cutting, highly affected by material genotype and environment, shows low rooting rates and uncontrollable variations in cuttings from some male and female plants. Grafting, though preserving superiority, depends on rootstock compatibility; its efficiency cannot meet large-scale needs and causes significant damage to mother plants (Abshahi et al., 2022; Yao, 2022). Plant tissue culture enables the acquisition of regenerated plants via in vitro culture of explants, and achieves large-scale propagation in a short timeframe, thereby meeting the requirements of industrial production (Pence, 2011; Hazubska-Przybył, 2019). Additionally, it features convenient and sufficient explant sourcing and freedom from environmental and seasonal constraints and effectively reduces genetic variation risks, ensures seedling quality (Berlyn et al., 1986; Dhiman et al., 2010), and thus opens new avenues for the asexual propagation of J. chinensis.

Research on J. chinensis tissue culture is limited, unlike J. vulgaris and J. formosana (Shi, 2009; Liu et al., 2022a, b). Also, tissue culture systems have been established using axillary buds or leaves as explants for species such as J. excelsa, J. navicularis, J. virginiana and J. thulifera. However, these studies still suffer from issues such as incomplete disinfection, severe browning, poor rooting or low rooting rates, all of which impede the normal growth of plants (Castro et al., 2011; Kašparová et al., 2016; Hazubska-Przybył, 2019).

The right disinfectant and disinfection time are the first step to successful tissue culture. Ethanol at 75 % combined with mercury chloride (HgCl₂) is commonly used for disinfecting Cupressaceae plants, such as Platycladus orientalis and J. vulgaris (Zhou et al., 2020; Chang et al., 2023). However, NaClO and hydrogen peroxide (H₂O₂) are more often used for Juniperus species such as J. navicularis and J. polycarpos (Castro et al., 2011; Ahani et al., 2013; Hazubska-Przybył, 2019). In terms of anti-browning, adding activated carbon (AC), polyvinylpyrrolidone (PVP) and silver nitrate (AgNO₃) to the medium can effectively inhibit browning (Liu et al., 2024a, b). However, anti-browning formulas vary with plant species, requiring further research.

Rooting culture is an extremely challenging, slow and inefficient process in conifers, and its effectiveness directly determines the feasibility of large-scale propagation of coniferous tree species. The rooting rates of softwood cuttings of J. excelsa, J. phoenicea and J. navicularis range from 18.5 to 60 %. In DCR medium, adventitious buds from P. orientalis spiny leaves root well, while those from its scale leaves fail to root. For J. chinensis, most studies have only yielded rootless plants, with failure to root or extremely low rooting rates (Castro et al., 2011; Ezz et al., 2012; Zhou et al., 2020; Fathollahi et al., 2025). It is thus evident that different plant species require different rooting media, and even for the same species, the rooting media suitable for its rooting may also vary due to differences in sexes and leaf morphogenesis.

Sex differentiation and leaf morphogenesis are important research contents in plant developmental biology. Tissue culture serves as an important technical means for studying plant developmental mechanisms. The sexual attributes and organ types of its explants can directly affect the efficiency of culture system establishment and regeneration capacity (Ślesak et al., 2017; Hazubska-Przybył, 2019). Male Ginkgo biloba plants exhibited a 18.7 % higher rooting rate than their female counterparts (Qaderi et al., 2021). In terms of leaf morphology, Thuja acuta spiny leaves demonstrated a 32.5 % higher rate of adventitious bud induction compared to scale leaves. While scale leaves could root in the same medium, spiny leaves were unable to do so. These findings suggest a notable association between sexes, leaf morphologies and tissue culture regeneration (Zhou et al., 2020). Juniperus chinensis has three sexes and two leaf morphologies. Directional propagation of low-pollen female plants can mitigate spring pollen allergies, while utilizing the high regenerative capacity of scale leaves enables efficient propagation of rare germplasms. Establishing a tissue culture system for monoecious plants will facilitate understanding of sex differentiation plasticity. Currently, there is no tissue culture system available for investigating sexes and leaf morphology in J. chinensis. This limitation hinders germplasm innovation and the understanding of developmental mechanisms.

We used stem segments of scale leaves (SL) and spiny leaves (SPL) from female (FP), male (MP) and monoecious (MOP) plants of J. chinensis. Through a series of experiments, including optimizing disinfection protocols, screening primary media and anti-browning agents, determining antioxidant enzymes, selecting the most suitable media and hormones for proliferation and rooting, and finally conducting hardening and transplanting plants, we established a multi-dimensional tissue culture system encompassing different sexes and leaf morphologies. This study offers valuable support for investigating the molecular mechanisms governing sexual differentiation and leaf morphogenesis in J. chinensis. Furthermore, it establishes a solid theoretical and technical basis for germplasm innovation, targeted breeding efforts and the industrial advancement of J. chinensis.

MATERIALS AND METHODS

Plant material

The J. chinensis material used in this study was provided with germplasm resources support by Shanxi Agricultural University located in Jinzhong City, China (112°58′E, 37°42′N). Its original introduction record was traced back to 1985, with the introduction site being the Taihang mountain forest region in Jinzhong City, Shanxi Province, China (112°15′E, 35°27′N). This region, characterized by a temperate continental monsoon climate with four distinct seasons, provides a unique natural environment for J. chinensis growth.

The experiments were conducted from May to October 2024. We selected 40-year-old J. chinensis plants on sunny mornings, collecting current-year, undamaged stem segments with SL (Fig. 1A) and SPL (Fig. 1B) from the sunlit side of FP, MP and MOP. We placed the collected explants in a foam box with ice cubes and took them back to the laboratory for tissue culture.

Fig. 1.

Sampling explants of FP, MP and MOP. (A) Stem segments with SL; (B) stem segments with SPL.

Culture conditions

All culture media in different experiments were supplemented with 30 g L⁻¹ sucrose and 7 g L⁻¹ agar (Biosharp, Beijing, China), adjusted to pH 5.8–6.0 and sterilized at 121 °C for 15 min using an autoclave (Boxun, Shanghai, China). We inoculated explants into 240-mL glass bottles, with three explants inoculated per bottle. For each different treatment, we inoculated 20 explants as one replicate, and set up a total of three replicates. The explants were placed in an illuminated incubator (Boxun) for culture, at 25 ± 2 °C, under cool white LED lighting (Boxun) with a 16 h d⁻¹ photoperiod and a light intensity of 7000 lx.

Explant disinfection

Initially, we first rinsed surface dust off the stem segments using clean water. Subsequently, after eliminating any adhering pollen and fruits, the segments were fragmented into 1- to 2-cm sections, immersed in a detergent solution for 15 min and thoroughly rinsed with running water for 30 min. The segments were then subjected to disinfection and sterilization within a laminar flow hood (Solarbio, Beijing, China). After soaking in 75 % ethanol for 5 min, the segments were rinsed three times with sterile water. They were then immersed in 0.1 % HgCl₂, 0.3 % NaClO and 0.3 % H₂O₂ for 16, 17 and 18 min respectively, followed by five or six rinses with sterile water. The portions of the disinfected stem segments that had contacted the disinfectants were appropriately excised, after which the segments were inoculated into hormone-free Murashige and Skoog medium (MS) medium. The contamination rate, browning rate and survival rate were recorded 15 d later.

$$\begin{array}{rl}\mathrm{Contamination}\mathrm{rate}(\text{\%})& ={\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{contaminated}\mathrm{explants}}{\mathrm{Total}\mathrm{inoculated}\mathrm{explants}}}\\ & \times 100\end{array}$$

$$\mathrm{Browning}\mathrm{rate}(\text{\%})={\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{browned}\mathrm{explants}}{\mathrm{Total}\mathrm{inoculated}\mathrm{explants}}\times 100}$$

$$\mathrm{Survival}\mathrm{rate}(\text{\%})={\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{surviving}\mathrm{explants}}{\mathrm{Total}\mathrm{inoculated}\mathrm{explants}}\times 100}$$

Screening of primary culture medium

After subsequent disinfection with 0.3 % NaClO for 18 min, stem segments were individually cultured on MS, half-strength Murashige and Skoog medium (½MS), Woody plant medium (WPM), White medium (White) and Douglas-fir Cotyledon Revised medium (DCR) (Solarbio) devoid of exogenous hormones. After a 30-d incubation period, the survival rate and browning rate in J. chinensis were assessed.

Screening of anti-browning agents

Following disinfection with 0.3 % NaClO for 18 min, stem segments were cultured on DCR medium supplemented with varying concentrations of AC (0, 0.5, 1.0, 2.0 and 3.0 g L⁻¹), PVP (0, 0.5, 1.0, 2.0 and 3.0 mg L⁻¹) andAgNO₃ (0, 0.5, 1.0, 2.0 and 3.0 mg L⁻¹). The DCR medium contained no cytokinins or auxins. Subsequently, the survival rate and browning rate of J. chinensis were assessed after a 30-d incubation period.

Antioxidant determination

We selected stem segments treated with different concentrations of PVP (0.5, 1.0, 2.0 and 3.0 mg L⁻¹) as test materials, using the group without PVP addition (0 mg L⁻¹) as the control. The dynamic changes in peroxidase (POD), polyphenol oxidase (PPO) and phenylalanine ammonia-lyase (PAL) activities during the anti-browning process were measured in the SL and SPL of the three sexes of J. chinensis.

PPO activity was determined using a modified catechol method (Syamsurizal et al., 2025). Randomly selected 2.0-g leaf samples were homogenized with 15.0 mL pre-chilled phosphate buffer (50 mmol L⁻¹, pH 6.4) in an ice bath. The homogenate was centrifuged at 13 000 rpm (4 °C, 15 min), and the supernatant was retained as the enzyme extract. Two millilitres of acetate buffer (pH 5.4) was pipetted into a cuvette, followed by sequential addition of 1.0 mL catechol (0.04 mol L⁻¹) and 0.5 mL enzyme extract. The reaction mixture was vortexed immediately and absorbance at 420 nm was recorded at 30-s intervals for 2 min.

$$\mathrm{PPO}(\mathrm{U}\cdot {\mathrm{g}}^{-1}\cdot {min}^{-1})={\displaystyle \frac{\mathrm{\Delta }{A}_{420}}{0.001\times m\times t_4}\times D}$$

where ΔA₄₂₀ is the change in absorbance value during reaction time; t₄ is the reaction time, min; D is the dilution multiple (the total enzyme solution extracted is the multiple of enzyme solution in reaction solution); and m is the fresh mass of the sample (g).

Determination of POD activity followed the guaiacol method (Syamsurizal et al., 2025): take 0.1 mL of enzyme solution, add 1.0 mL of 0.05 mmol L⁻¹ guaiacol, 2.9 mL of phosphate buffer solution and 1.0 mL of 2 % H₂O₂ in turn, then immediately determine A₄₇₀ record once every 30 s, for a total of 2 min.

$$\mathrm{POD}(\mathrm{U}\cdot {\mathrm{g}}^{-1}\cdot {min}^{-1})={\displaystyle \frac{\mathrm{\Delta }{A}_{470}\times V_{\mathrm{t}}}{0.01W\times V_{\mathrm{s}}\times t}}$$

where Δ₄₇₀ is the absorbance change during the reaction time, W is the sample weight (g), t is the reaction time (min), V_t is the total volume of the extract (mL), and V_s is the sample volume used in the determination (mL).

PAL activity was determined as follows: 2.0-g leaf samples were homogenized in 15.0 mL extraction buffer (containing 2 mmol L⁻¹ EDTA, 5 mmol L⁻¹ β-mercaptoethanol and 40 g L⁻¹ PVP) using an ice bath. The homogenate was centrifuged at 12 000 rpm (4 °C, 30 min), and the supernatant was stored on ice for analysis. Three millilitres of borate-borax buffer (50 mmol L⁻¹, pH 8.8) and 0.5 mL L⁻¹ phenylalanine (20 mmol L⁻¹) were mixed and pre-incubated at 37 °C for 10 min. After adding 0.5 mL enzyme extract, the initial absorbance at 290 nm (A₂₉₀) was measured immediately. The reaction mixture was incubated at 30 °C for 60 min, after which the final A₂₉₀ was recorded.

$$\mathrm{PAL}(\mathrm{U}\cdot {\mathrm{g}}^{-1}\cdot {min}^{-1})={\displaystyle \frac{(\mathrm{O}{\mathrm{D}}_1-\mathrm{O}{\mathrm{D}}_0)\times V}{0.01\times V_{\mathrm{s}}\times t\times m}}$$

where OD₁ is the absorbance value of the reaction solution in the sample tube or initial absorbance value of the reaction solution before incubation; OD₀ id absorbance value of the reaction solution in the control tube or final absorbance value of the reaction solution after incubation; V is the total volume of sample extraction solution (mL); V_s is the extraction volume of sample taken during determination (mL); t is enzymatic reaction time (h); and m is sample mass (g).

Selection of proliferation medium

Using adventitious buds from the SL and SPL of FP, MP and MOP J. chinensis plants obtained after anti-browning treatment as explants, single buds (1–2 cm) were excised and inoculated into DCR medium supplemented with different concentrations of 6-benzylaminopurine (6-BA) (1.0, 2.0 and 3.0 mg L⁻¹), trans-zeatin (Trans-ZT) (0.1, 0.5 and 1.0 mg L⁻¹) and 1-naphthaleneacetic acid (NAA) (0.05, 0.1 and 0.5 mg L⁻¹) (Table 1). Among these, 1.0 mg L⁻¹ PVP is supplemented in the different treatments for adventitious bud proliferation of SL, while 2.0 mg L⁻¹ PVP is supplemented for SPL for anti-browning. After 30 d, we counted the proliferation rate, multiplication coefficient and plant height of adventitious buds:

Table 1.

Experimental design different hormone concentrations used in proliferation medium for adventitious buds of SL and SPL from FP, MP and MOP. Specifically, 1.0 mg L⁻¹ PVP was supplemented in the treatment for SL, while 2.0 mg L⁻¹ PVP was added in that for SPL.

No.	6-BA (mg L−1)	Trans-ZT (mg L−1)	NAA (mg L−1)
1	1.0	0.1	0.05
2	1.0	0.5	0.1
3	1.0	1.0	0.5
4	2.0	0.1	0.5
5	2.0	0.5	0.05
6	2.0	1.0	0.1
7	3.0	0.1	0.1
8	3.0	0.5	0.5
9	3.0	1.0	0.05

$$\mathrm{Bud}\mathrm{proliferation}\mathrm{rate}(\text{\%}){\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{proliferated}\mathrm{buds}}{\mathrm{Total}\mathrm{inoculated}\mathrm{explants}}\times 100}$$

$$\mathrm{Proliferation}\mathrm{coefficient}{\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{proliferated}\mathrm{buds}}{\mathrm{Number}\mathrm{of}\mathrm{inoculated}\mathrm{buds}}}$$

Plant height (cm) was measured vertically from the basal part of the sterile plant stem (where it contacts the medium) to the highest growth point using a ruler with a precision of 1 mm.

Selection of rooting medium

After SPL were proliferated on medium No. 7 (DCR + 3.0 mg L⁻¹ 6-BA + 0.5 mg L⁻¹ Trans-ZT + 0.5 mg L⁻¹ NAA + 1.0 mg L⁻¹ PVP) and SL were proliferated on medium No. 8 (DCR + 3.0 mg L⁻¹ 6-BA + 0.1 mg L⁻¹ Trans-ZT + 0.1 mg L⁻¹ NAA + 2.0 mg L⁻¹PVP), we cut them into individual buds of about 1–2 cm. These buds were inoculated respectively into MS, ½MS, WPM, White and DCR medium supplemented with 0.1 mg L⁻¹ NAA, where the medium for SL and SPL were additionally supplemented with 1.0 and 2.0 mg L⁻¹ PVP respectively. After 30 d, the rooting rate, root length, number of roots and plant height were recorded:

$$\mathrm{Rooting}\mathrm{rate}(\text{\%})={\displaystyle \frac{\mathrm{Number}\mathrm{of}\mathrm{rooted}\mathrm{explants}}{\mathrm{Total}\mathrm{inoculated}\mathrm{explants}}\times 100}$$

Selection of rooting hormone species and concentration

After screening out the optimal rooting medium, the adventitious buds were cut into single buds, which were then inoculated respectively into ½MS and DCR basal medium supplemented with different concentrations of NAA, indole-3-butyric acid (IBA) and ABT rooting powder (ABT) (0, 0.5, 1.0 and 2.0 mg L⁻¹ each), as well as 1.0 or 2.0 mg L⁻¹ PVP. After 60 d, the rooting rate, root length, number of roots, plant height and growth status were recorded.

Transplantation of sterile plants

When the 2–3 cm tall sterile plants were taken out of the incubator, their bottle caps were loosened and they were placed at room temperature for 2 d, after which the caps were removed for plants hardening for 3 d. The sterile plants were then taken out, and the culture medium attached to their roots was rinsed off with sterile water. They were transplanted into a substrate (peat: vermiculite = 1:1) (Solarbio) and watered immediately afterwards. The rooted plants were placed in an incubator for cultivation, with their growth status observed regularly, and the survival rate after plant hardening was calculated.

Data analysis

The present study observed and measured the characteristics and data of explants of different sexes and leaf shapes of J. chinensis at each stage of tissue culture. After calculating and summarizing core indicators (including contamination rate, browning rate, survival rate, proliferation rate, proliferation coefficient and rooting rate) according to the above equations, ANOVA was conducted using SPSS 23.0. All data were expressed as (mean ± s.e.), and post-hoc multiple comparisons were performed via Duncan’s new multiple range test. Different lowercase letters were used to indicate significant differences among different treatment groups (P < 0.05) (Ezz et al., 2012; Zhou et al., 2020; Fathollahi et al., 2025). Finally, bar charts were plotted using Origin 2021, where the x-axis was set as treatment groups and the y-axis as experimental indicators. Error bars (representing mean ± s.e.) and significance labels were added, with the significance arranged in descending order. All data represented the average values of three replicate experiments.

RESULTS

Explant disinfection

After culturing explants on MS medium for 15 d, we observed significant differences in sterilization effects between different disinfectants and disinfection durations. Overall, the contamination rate of explants decreased as disinfection duration extended, while the browning rate showed an increasing trend (Fig. 2). SL and SPL disinfected with H₂O₂ had a survival rate of less than 10 % and their contamination rate was higher than that of the other two disinfectants (Fig. 2A, D, G). Disinfection with NaClO for 18 min effectively disinfected SL and SPL of all three sexes, achieving peak survival rates that were significantly higher than those with other treatment durations (P < 0.05). It also reached the lowest contamination rates, ranging from 7.65 to 11.67 % for SL and 7.33 to 15.00 % for SPL (Fig. 2B, E, H). For SL of FP and MP with 18 min of disinfection with HgCl₂ achieved good efficacy, with a survival rate around 20 % – significantly higher than that with other treatments (P < 0.05). For SPL, the survival rate was relatively higher at 16 and 17 min of disinfection (Fig. 2C, F). For SL and SPL in MOP, 16  min of disinfection with HgCl₂ was more appropriate (Fig. 2I). Notably, when SL and SPL were disinfected with HgCl₂ for 18 min, they showed almost no contamination, but this was accompanied by a high browning rate. For SPL in particular, the browning rate was consistently over 90 %. Thus, the disinfection efficacy of the three disinfectants was of the order of 0.1 % HgCl₂, 0.3 % NaClO and 0.3 % H₂O₂.

Fig. 2.

Effects of different disinfectant combinations on stem segment disinfection of SL and SPL of FP, MP and MOP. (A) FP disinfected with H₂O₂; (B) FP disinfected with NaClO; (C) FP disinfected with HgCl₂; (D) MP disinfected with H₂O₂; (E) MP disinfected with NaClO; (F) MP disinfected with HgCl₂; (G) MOP disinfected with H₂O₂; (H) MOP disinfected with NaClO; (I) MOP disinfected with HgCl₂. Bar graphs represent the mean ± s.e. Different lowercase letters indicate significant differences in survival rate, contamination rate and browning rate among different disinfectants and disinfection times (P < 0.05), which are arranged from high to low according to the significance of differences.

However, HgCl₂ is highly toxic, environmentally polluting and cumbersome to handle. In contrast, NaClO not only ensures disinfection efficacy but is more environmentally friendly and allows one-step operation. In conclusion, 0.3 % sodium hypochlorite (18 min) is the optimal choice for explant disinfection. Nevertheless, the survival rate of explants remained low, which required further investigation.

Screening of primary culture medium

Different culture media showed significant differences in the effects on explants (Fig. 3). The results showed that: the survival rate of stem segments with SL and SPL of the three sexes in DCR medium was significantly higher than that in other media (P < 0.05), but the difference between groups was small, especially the survival rate of SL of MP in DCR medium. MS, ½MS and WPM medium had little difference in survival rate and browning rate of explants. Browning rate of stem segments with SL and SPL reached the peak and survival rate was the lowest in White medium.

Fig. 3.

Effects of different medium types on stem segments of SL and SPL of FP, MP and MOP. (A) FP, (B) MP and (C) MOP. Bar graphs represent the mean ± s.e. Different lowercase letters indicate significant differences in survival rate and browning rate among different media (P < 0.05), which are arranged from high to low according to the significance of differences.

Screening of anti-browning agents

To improve survival and alleviate severe browning, explants were inoculated into DCR medium supplemented with varying concentrations of AC, PVP and AgNO₃ (Fig. 4A, D). In medium without anti-browning agents, browning initiated at the base of explants on day 15, with subsequent gradual diffusion of brown pigments across the bottom of the medium (Fig. 4B, E). This led to a browning rate exceeding 60 % for SL and approaching 70 % for SPL (Fig. 5).

Fig. 4.

Browning resistance stages in FP, MP and MOP of J. chinensis: (A) inoculation of SL stem segments; (B) browning of SL stem segments; (C) anti-browning of SL stem segments; (D) inoculation of SPL stem segments; (E) browning of SPL stem segments; (F) anti-browning of SPL stem segments.

Fig. 5.

Effects of different anti-browning agents on stem segments of SP and SPL of FP, MP and MOP. (A) Effect of PVP on browning of FP; (B) effect of AC on browning of FP; (C) effect of AgNO₃ on browning of FP; (D) effect of PVP on browning of MP; (E) effect of AC on browning of MP; (F) effect of AgNO₃ on browning of MP; (G) effect of PVP on browning of MOP; (H) effect of AC on browning of MOP; (I) effect of AgNO₃ on browning of MOP. Bar graphs represent the mean ± s.e. Different lowercase letters indicate significant differences in survival rate and browning rate among different anti-browning agents and concentrations (P < 0.05), with significance arranged from high to low.

In contrast, media supplemented with anti-browning agents exhibited different outcomes. The survival rates of stem segments with SL and SPL of the three sexes of J. chinensis initially decreased and then increased with rising concentrations of PVP and AC. Conversely, the browning rate first increased and subsequently declined with increasing AgNO₃ concentration. The optimal anti-browning agents for SL and SPL of J. chinensis in order were PVP, AC and AgNO₃. Compared to the control, adding different concentrations of PVP to the culture medium effectively inhibited explant browning. Among the three sexes, stem segments of SL exhibited the lowest browning rate in the medium supplemented with 1.0 mg L⁻¹ PVP, whereas SPL required a higher PVP concentration (2.0 mg L⁻¹).

Antioxidant determination

To investigate the dynamic changes of oxidative enzymes during explant browning, stem segments with SL and SPL of FP, MP and MOP of J. chinensis were used as explants. The activities of POD, PPO and PAL of SL and SPL were measured under different PVP concentrations. The results demonstrated that in PVP-free medium, the activities of POD, PPO and PAL in both SL and SPL of J. chinensis across the three sexes peaked, with significantly higher enzyme activities observed in SPL compared to SL (Fig. 6). Following PVP treatment, the activities of POD, PPO and PAL in both leaf morphotypes were lower than those in the control group. With increasing PVP concentrations, all enzyme activities in SL and SPL exhibited a trend of initial decrease followed by an increase. Specifically, at 1.0 mg L⁻¹ PVP, the activities of POD, PPO and PAL in SL reached their minimum levels, whereas a PVP concentration of 2.0 mg L⁻¹ was necessary to achieve significant inhibition of enzyme activities in SPL.

Fig. 6.

Determination of antioxidant activity of adventitious buds of FP, MP and MOP. (A) antioxidant activity of SL of FP; (B) antioxidant activity of SL of MP; (C) antioxidant activity of SL of MOP; (D) antioxidant activity of SPL of FP; (E) antioxidant activity of SPL of MP; (F) antioxidant activity of SPL of MOP. Bar graphs represent the mean ± s.e. Different lowercase letters indicate significant differences in POD, PPO and PAL among different PVP concentration treatments (P < 0.05), with significance arranged in descending order.

Selection of proliferation medium

The proliferation rate was fastest in MOP (5–7 d), followed by MP (8–10 d) and slowest in FP (10–15 d). Significant differences in adventitious bud proliferation among the three sexes of J. chinensis SL and SPL were observed after 15 d (Fig. 7B, E). By 30 d of culture, a large number of adventitious buds had proliferated (Fig. 7C, F). It was of note that whether SL or SPL were inoculated, the adventitious buds proliferated from them were all SPL.

Fig. 7.

Adventitious bud proliferation stage of FP, MP and MOP J. chinensis. (A) Inoculation of SL adventitious buds; (B) adventitious buds at 15 d post-inoculation of SL; (C) adventitious buds at 30 d post-inoculation of SL; (D) inoculation of SPL adventitious buds; (E) adventitious buds at 15 d post-inoculation of SPL; (F) adventitious buds at 30 d post-inoculation of SPL.

The results showed that under treatment with low-concentration 6-BA (1.0 mg L⁻¹, No. 1, 2 and 3), the proliferation coefficient of SL adventitious buds of the three sexes was only about 1-fold compared with the initially inoculated adventitious buds, while that of SPL was about 2-fold and plant height was less than 3.0 cm. This indicated that their proliferation efficiency was relatively low (Table 2). When the 6-BA concentration was increased to 2.0 mg L⁻¹ (No. 4, 5 and 6), the proliferation effect improved compared to 1.0 mg L⁻¹. At a 6-BA concentration of 3.0 mg L⁻¹ (No. 7, 8 and 9), the proliferation rate of adventitious buds in both SL and SPL reached 80 %, with proliferation coefficients exceeding 4-fold compared with the initially inoculated adventitious buds, demonstrating the most effective proliferation outcome. Under the same concentration conditions, the MOP of J. chinensis exhibited the highest proliferation efficiency, followed by MP and finally FP showing the lowest. Comparing the two leaf morphologies, SPL demonstrated significantly superior proliferation effects compared to SL. Additionally, the proliferation rates of SL adventitious buds from FP and MP in medium No. 8 (DCR + 3.0 mg L⁻¹ 6-BA + 0.5 mg L⁻¹ Trans-ZT + 0.5 mg L⁻¹ NAA + 1.0 mg L⁻¹ PVP) were 88.93 and 90.17 %, respectively, with plant heights of 4.79 and 4.27 cm. The proliferation coefficients exceeded 4-fold. In contrast, medium No. 7 (DCR + 3.0 mg L⁻¹ 6-BA + 0.1 mg L⁻¹ Trans-ZT + 0.1 mg L⁻¹ NAA + 2.0 mg L⁻¹ PVP) was more suitable for the proliferation of SPL adventitious buds, achieving proliferation rates of 93.42 and 94.14 %, respectively and plant heights of 5.37 and 5.67 cm. For MOP, medium No. 7 was optimal for both SL and SPL proliferation, with proliferation rates of 96.13 and 98.24 %, plant heights of 4.62 and 5.94 cm, and proliferation coefficients of 5.0–6.0.

Table 2.

Effects of different hormone concentrations on adventitious bud proliferation rate, proliferation coefficient and plant height in SL and SPL of FP, MP and MOP. Data are presented as mean ± s.e. Different lowercase letters indicate significant differences among different treatments (P < 0.05). A ‘+’ indicates the proliferation coefficient of adventitious buds, with each ‘+’ representing a doubling of adventitious bud proliferation. Specifically, 1.0 mg L⁻¹ PVP was supplemented in the treatment for SL, while 2.0 mg L⁻¹ PVP was added in that for SPL.

Sex	No.	6-BA (mg L−1)	Trans-ZT (mg L−1)	NAA (mg L−1)	SL			SPL
					Proliferation rate (%)	Proliferation coefficient	Plant height (cm)	Proliferation rate (%)	Proliferation coefficient	Plant height (cm)
FP	1	1.0	0.1	0.05	23.48 ± 4.32c	+	1.41 ± 0.12c	42.37 ± 6.89c	++	2.34 ± 0.19c
	2	1.0	0.5	0.1	29.83 ± 3.41c	+	2.05 ± 0.33c	46.91 ± 8.23c	++	2.96 ± 0.33c
	3	1.0	1.0	0.5	17.71 ± 3.30c	+	2.74 ± 0.15bc	44.71 ± 7.39c	++	2.14 ± 0.52c
	4	2.0	0.1	0.5	49.17 ± 5.41bc	++	2.83 ± 0.52b	66.51 ± 9.32b	+++	4.34 ± 0.48b
	5	2.0	0.5	0.05	52.80 ± 5.19bc	+++	2.96 ± 0.44b	70.25 ± 9.88b	+++	4.56 ± 0.69b
	6	2.0	1.0	0.1	73.26 ± 9.77b	+++	2.76 ± 0.17bc	84.22 ± 9.03ab	++++	4.11 ± 0.40b
	7	3.0	0.1	0.1	81.42 ± 6.55a	++++	3.07 ± 0.59b	93.42 ± 8.71a	+++++	5.37 ± 0.69a
	8	3.0	0.5	0.5	88.93 ± 8.71a	++++	4.79 ± 0.41a	90.22 ± 7.32a	+++++	5.18 ± 0.82a
	9	3.0	1.0	0.05	80.13 ± 5.49a	++++	4.09 ± 0.61a	87.56 ± 8.06a	+++++	5.09 ± 0.92a
MP	1	1.0	0.1	0.05	36.76 ± 3.69c	+	1.93 ± 0.21c	44.69 ± 6.34c	++	2.52 ± 0.23c
	2	1.0	0.5	0.1	42.13 ± 3.41bc	+	2.55 ± 0.45bc	50.14 ± 4.52c	++	3.09 ± 0.93c
	3	1.0	1.0	0.5	29.19 ± 2.22c	+	2.68 ± 0.19b	47.61 ± 3.94c	++	2.52 ± 0.27c
	4	2.0	0.1	0.5	57.84 ± 4.09bc	++	2.77 ± 0.14b	69.27 ± 5.41b	+++	4.69 ± 0.38b
	5	2.0	0.5	0.05	61.08 ± 5.15bc	+++	2.84 ± 0.52b	76.13 ± 4.20b	+++	4.73 ± 1.21b
	6	2.0	1.0	0.1	74.29 ± 5.71b	+++	3.02 ± 0.73b	85.91 ± 5.66ab	++++	4.25 ± 1.03b
	7	3.0	0.1	0.1	84.58 ± 5.98a	++++	3.86 ± 0.19a	94.14 ± 5.92a	+++++	5.67 ± 1.09a
	8	3.0	0.5	0.5	90.17 ± 6.53a	++++	4.27 ± 0.98a	91.28 ± 6.11a	+++++	5.39 ± 1.04a
	9	3.0	1.0	0.05	83.22 ± 6.65a	++++	3.22 ± 0.28ab	90.43 ± 7.81a	+++++	5.28 ± 0.96a
MOP	1	1.0	0.1	0.05	42.17 ± 4.10c	+	2.19 ± 0.42c	43.19 ± 4.90c	++	3.52 ± 1.09bc
	2	1.0	0.5	0.1	49.75 ± 5.13c	+	2.38 ± 0.77c	52.04 ± 3.85c	++	3.74 ± 1.20bc
	3	1.0	1.0	0.5	33.19 ± 4.46c	+	2.87 ± 0.92c	40.93 ± 4.82c	++	2.89 ± 0.78c
	4	2.0	0.1	0.5	60.19 ± 5.02b	+++	3.11 ± 0.45bc	66.19 ± 4.59b	+++	4.29 ± 1.09b
	5	2.0	0.5	0.05	73.11 ± 3.19b	++++	3.49 ± 0.63b	77.01 ± 6.28b	+++++	4.69 ± 1.23b
	6	2.0	1.0	0.1	77.18 ± 6.77ab	++++	3.88 ± 0.28b	79.84 ± 4.11b	+++++	4.30 ± 0.95b
	7	3.0	0.1	0.1	96.13 ± 7.10a	+++++	4.62 ± 1.03a	98.24 ± 6.29a	++++++	5.94 ± 1.07a
	8	3.0	0.5	0.5	92.16 ± 7.84a	+++++	4.53 ± 0.84a	96.77 ± 6.15a	++++++	5.80 ± 1.23a
	9	3.0	1.0	0.05	89.04 ± 7.53a	+++++	4.09 ± 1.09ab	90.28 ± 4.97a	++++++	5.62 ± 1.20a

Selection of rooting medium

The results demonstrated that different media significantly influenced the rooting of adventitious buds from SL and SPL of FP, MP and MOP (Table 3). Adventitious buds from SL and SPL of J. chinensis across all three sexes failed to root, yet the plantlets grew normally; among them, those cultured on White medium showed the poorest growth. The optimal medium for rooting of adventitious buds from SL of FP and MP was ½MS medium, whereas the rooting rate of adventitious buds from SPL was higher in DCR medium. For MOP, the medium requirement was consistent for adventitious buds of SL and SPL, with DCR medium being optimal. After screening for suitable rooting medium, we found that the rooting rate of adventitious buds remained poor; therefore, further investigation was required by supplementing appropriate rooting hormones.

Table 3.

Effects of different medium types on rooting rate, root length, root number and plant height of adventitious buds from SL and SPL of FP, MP and MOP. Data are presented as mean ± s.e. Different lowercase letters indicate significant differences among different treatments (P < 0.05). Specifically, 1.0 mg L⁻¹ PVP was supplemented in the treatment for SL, while 2.0 mg L⁻¹ PVP was added in that for SPL.

Sex	Type	SL				SPL
		Rooting rate (%)	Root length (cm)	Root number	Plant height (cm)	Rooting rate (%)	Root length (cm)	Root number	Plant height (cm)
FP	MS	0.00c	0.00b	0.00b	1.69 ± 0.15b	0.00c	0.00c	0.00c	2.37 ± 0.34bc
	½MS	7.31 ± 1.04a	1.53 ± 0.22a	1.31 ± 0.11a	2.29 ± 0.31a	4.26 ± 0.33b	1.85 ± 0.22b	1.27 ± 0.14bc	2.65 ± 0.11ab
	WPM	0.00c	0.00b	0.00b	1.93 ± 0.12ab	0.00c	0.00c	0.00c	1.28 ± 0.10c
	White	0.00c	0.00b	0.00b	1.27 ± 0.14b	0.00c	0.00c	0.00c	1.39 ± 0.21c
	DCR	2.68 ± 0.41b	1.21 ± 0.20ab	1.29 ± 0.14ab	2.17 ± 0.16ab	6.17 ± 0.42a	2.09 ± 0.19a	1.65 ± 0.18a	2.77 ± 0.29a
MP	MS	0.00c	0.00c	0.00b	1.87 ± 0.26b	0.00c	0.00c	0.00b	2.47 ± 0.15b
	½MS	8.39 ± 1.06a	1.29 ± 0.23a	1.03 ± 0.06a	2.35 ± 0.24a	2.65 ± 0.53b	1.14 ± 0.22b	1.26 ± 0.23ab	2.07 ± 0.33bc
	WPM	0.00c	0.00c	0.00b	2.05 ± 0.12ab	0.00c	0.00c	0.00b	1.74 ± 0.28bc
	White	0.00c	0.00c	0.00b	1.43 ± 0.31c	0.00c	0.00c	0.00b	1.33 ± 0.11c
	DCR	2.45 ± 0.25b	0.65 ± 0.04b	1.07 ± 0.05a	1.57 ± 0.22bc	9.65 ± 0.14a	1.41 ± 0.24a	1.32 ± 0.12a	2.85 ± 0.27a
MOP	MS	0.00c	0.00b	0.00c	1.32 ± 0.14c	0.00c	0.00c	0.00b	1.29 ± 0.11bc
	½MS	4.51 ± 0.32b	1.97 ± 0.41ab	1.23 ± 0.12bc	1.71 ± 0.21b	3.49 ± 0.13b	1.39 ± 0.18b	1.49 ± 0.23ab	1.85 ± 0.32b
	WPM	0.00c	0.00b	0.00c	1.28 ± 0.17c	0.00c	0.00c	0.00b	1.31 ± 0.08bc
	White	0.00c	0.00b	0.00c	1.66 ± 0.22b	0.00c	0.00c	0.00b	0.96 ± 0.05c
	DCR	8.54 ± 1.89a	2.03 ± 0.13a	2.12 ± 0.14a	2.19 ± 0.18a	5.78 ± 0.41a	2.01 ± 0.13a	1.96 ± 0.11a	2.61 ± 0.26a

Selection of rooting hormone species and concentration

After the adventitious buds were inoculated onto culture media containing different hormone concentrations, white adventitious roots began to appear at the base of the plants around 35 d later (Fig. 8A, D), and the root systems began to gradually elongate after 60 d (Fig. 8B, C, E, F).

Fig. 8.

Adventitious root formation stages of SL and SPL from FP, MP and MOP. (A) Adventitious root formation of SL at 35 d; (B, C) adventitious root formation of SL at 60 d; (D) adventitious root formation of SPL at 35 d; (E, F) adventitious root formation of SPL at 60 d.

Different concentrations of hormones exerted significant effects on adventitious buds from SL and SPL. Adventitious buds of the three sexes produced no adventitious roots in medium without NAA, IBA or ABT (Tables 4 and 5). Overall, ABT was suitable for the rooting of SL adventitious buds of FP and MP, while IBA was more suitable for the rooting of SPL; for MOP, IBA was suitable for the rooting of both SL and SPL adventitious buds. Among the three sexes, the adventitious buds of MP showed the best rooting effect, followed by MOP and FP last. In terms of leaf morphogenesis, all rooting indicators of SPL during the rooting process were slightly lower than those of SL. Medium No. 8 (½MS + 1.0 mg L⁻¹ ABT + 1.0 mg L⁻¹ PVP) was suitable for the rooting of SL adventitious buds of FP and MP, with rooting rates of 73.82 and 82.13 % respectively. Meanwhile, medium No. 6 (½MS + 2.0 mg L⁻¹ IBA + 2.0 mg L⁻¹ PVP) was suitable for the rooting of SPL, with rooting rates of 70.29 and 77.93 % respectively. Rooting rates in these two media were significantly higher than those in other treatments (P < 0.05), and indices such as root length, number of roots and plant height also reached maximum values. For MOP, medium No. 6 was suitable for the rooting of both SL and SPL adventitious buds simultaneously, and all rooting indices reached a maximum.

Table 4.

Effects of different hormone concentrations on rooting rate, root length, root number and plant height of adventitious buds from SL of FP, MP and MOP. Data are presented as mean ± s.e. Different lowercase letters indicate significant differences among different treatments (P < 0.05). A ‘–’ indicates no addition of the hormone under the corresponding treatment. Different treatments were all supplemented with 1.0 mg L⁻¹ PVP.

Sex	No.	NAA (mg L−1)	IBA (mg L−1)	ABT (mg L−1)	Rooting rate (%)	Root length (cm)	Root number	Plant height (cm)
FP	0	–	–	–	0.00c	0.00c	0.00c	1.49 ± 0.23c
	1	0.5	–	–	5.19 ± 1.03c	0.79 ± 0.07c	1.03 ± 0.13c	1.51 ± 0.14c
	2	1	–	–	7.62 ± 1.21c	1.13 ± 0.13c	1.24 ± 0.10c	1.69 ± 0.21bc
	3	2	–	–	4.81 ± 1.05c	1.76 ± 0.21bc	1.65 ± 0.31bc	1.57 ± 0.34c
	4	–	0.5	–	11.24 ± 1.71c	1.83 ± 0.19b	1.92 ± 0.24b	1.95 ± 0.15bc
	5	–	1	–	23.81 ± 2.09bc	1.92 ± 0.31b	2.07 ± 0.51b	2.09 ± 0.32b
	6	–	2	–	19.93 ± 3.62c	2.09 ± 0.46b	2.15 ± 0.15b	2.32 ± 0.59b
	7	–	–	0.5	56.41 ± 3.79b	2.85 ± 0.29a	2.78 ± 0.43a	2.87 ± 0.51a
	8	–	–	1	73.82 ± 3.92a	2.97 ± 0.41a	2.99 ± 0.46a	2.92 ± 0.44a
	9	–	–	2	59.81 ± 4.53b	2.48 ± 0.24ab	2.76 ± 0.65a	2.61 ± 0.28a
MP	0	–	–	–	0.00c	0.00c	0.00c	1.31 ± 0.23c
	1	0.5	–	–	5.62 ± 0.15c	0.92 ± 0.08c	1.09 ± 0.02c	1.63 ± 0.19c
	2	1	–	–	9.68 ± 0.16c	1.08 ± 0.21c	1.65 ± 0.34c	1.72 ± 0.14c
	3	2	–	–	8.57 ± 0.61c	1.23 ± 0.11c	1.49 ± 0.20c	2.54 ± 0.29bc
	4	–	0.5	–	23.47 ± 1.09bc	1.84 ± 0.17bc	2.01 ± 0.25bc	2.36 ± 0.43bc
	5	–	1	–	32.19 ± 2.85bc	2.41 ± 0.38b	2.66 ± 0.35b	3.01 ± 0.51b
	6	–	2	–	19.28 ± 3.07bc	2.51 ± 0.74b	1.56 ± 0.23c	3.32 ± 0.18b
	7	–	–	0.5	64.93 ± 4.51b	3.14 ± 0.81b	2.94 ± 0.71b	3.49 ± 0.64b
	8	–	–	1	82.13 ± 5.98a	4.61 ± 0.93a	4.18 ± 0.87a	5.73 ± 0.58a
	9	–	–	2	74.61 ± 5.30b	3.84 ± 0.72ab	3.96 ± 0.98a	5.29 ± 0.42a
MOP	0	–	–	–	0.00c	0.00c	0.00c	1.51 ± 0.43c
	1	0.5	–	–	5.29 ± 0.21c	0.85 ± 0.03c	1.31 ± 0.23c	1.69 ± 0.14c
	2	1	–	–	7.83 ± 0.19c	1.04 ± 0.10c	1.39 ± 0.17c	1.98 ± 0.25bc
	3	2	–	–	6.15 ± 0.33c	1.17 ± 0.17bc	1.61 ± 0.25bc	1.74 ± 0.29bc
	4	–	0.5	–	65.41 ± 5.09a	3.50 ± 0.87a	2.96 ± 0.32a	4.21 ± 0.24a
	5	–	1	–	70.18 ± 5.76a	3.82 ± 0.68a	3.02 ± 0.65a	4.53 ± 0.19a
	6	–	2	–	76.91 ± 4.86a	3.91 ± 0.77a	3.81 ± 0.51a	4.91 ± 0.53a
	7	–	–	0.5	19.87 ± 1.27b	1.65 ± 0.21b	1.97 ± 0.14bc	1.82 ± 0.16bc
	8	–	–	1	28.33 ± 1.55b	1.82 ± 0.33b	2.32 ± 0.44b	2.15 ± 0.51b
	9	–	–	2	20.56 ± 2.03b	1.41 ± 0.26bc	2.09 ± 0.29b	2.69 ± 0.22b

Table 5.

Effects of different hormone concentrations on rooting rate, root length, root number and plant height of adventitious buds from SPL of FP, MP and MOP. Data are presented as mean ± s.e. Different lowercase letters indicate significant differences among different treatments (P < 0.05). A ‘–’ indicates no addition of the hormone under the corresponding treatment. Different treatments were all supplemented with 2.0 mg L⁻¹ PVP.

Sex	No.	NAA (mg L−1)	IBA (mg L−1)	ABT (mg L−1)	Rooting rate (%)	Root length (cm)	Root number	Plant height (cm)
FP	0	–	–	–	0.00c	0.00c	0.00c	1.25 ± 0.11c
	1	0.5	–	–	4.32 ± 1.03c	0.57 ± 0.03c	1.14 ± 0.21c	1.42 ± 0.23c
	2	1	–	–	5.71 ± 0.25c	1.29 ± 0.10c	1.28 ± 0.13c	1.65 ± 0.31c
	3	2	–	–	11.43 ± 2.98c	1.86 ± 0.24c	1.32 ± 0.27c	2.07 ± 0.24bc
	4	–	0.5	–	39.86 ± 5.22bc	2.52 ± 0.20b	1.47 ± 0.21c	2.33 ± 0.28b
	5	–	1	–	57.29 ± 4.19b	3.03 ± 0.19a	2.05 ± 0.55b	2.86 ± 0.10ab
	6	–	2	–	70.29 ± 3.25a	3.42 ± 0.14a	2.69 ± 0.14a	3.47 ± 0.44a
	7	–	–	0.5	33.19 ± 2.38bc	2.86 ± 0.42b	2.29 ± 0.19ab	2.92 ± 0.52a
	8	–	–	1	32.28 ± 3.32bc	2.70 ± 0.17b	2.13 ± 0.22b	2.81 ± 0.65ab
	9	–	–	2	29.88 ± 3.86c	2.42 ± 0.63b	1.72 ± 0.14bc	2.73 ± 0.24b
MP	0	–	–	–	0.00c	0.00c	0.00c	1.62 ± 0.27c
	1	0.5	–	–	5.14 ± 1.09c	1.09 ± 0.10c	1.34 ± 0.15c	1.73 ± 0.41c
	2	1	–	–	5.73 ± 1.82c	1.23 ± 0.19c	1.52 ± 0.19c	1.95 ± 0.15c
	3	2	–	–	9.88 ± 1.91c	1.33 ± 0.13c	1.67 ± 0.20c	2.30 ± 0.18bc
	4	–	0.5	–	41.72 ± 1.04bc	1.14 ± 0.13c	2.41 ± 0.27b	1.77 ± 0.41c
	5	–	1	–	50.84 ± 5.49b	2.87 ± 0.28ab	2.49 ± 0.25b	3.07 ± 0.56b
	6	–	2	–	77.93 ± 6.82a	3.82 ± 0.44a	3.44 ± 0.13a	4.87 ± 0.37a
	7	–	–	0.5	34.58 ± 4.38bc	2.19 ± 0.32b	2.19 ± 0.18b	2.96 ± 0.19b
	8	–	–	1	40.91 ± 4.19bc	2.83 ± 0.17ab	2.52 ± 0.21b	3.40 ± 0.22b
	9	–	–	2	31.09 ± 3.27bc	2.04 ± 0.29b	2.03 ± 0.39bc	2.53 ± 0.45bc
MOP	0	–	–	–	0.00c	0.00c	0.00c	1.36 ± 0.27c
	1	0.5	–	–	4.95 ± 0.66c	1.27 ± 0.16c	1.19 ± 0.34c	1.52 ± 0.32c
	2	1	–	–	5.14 ± 0.67c	1.85 ± 0.38c	1.28 ± 0.10c	1.64 ± 0.15c
	3	2	–	–	7.75 ± 0.14c	1.55 ± 0.11c	1.36 ± 0.12c	1.90 ± 0.21bc
	4	–	0.5	–	32.35 ± 2.40b	2.68 ± 0.29b	3.40 ± 0.33ab	2.03 ± 0.17b
	5	–	1	–	61.43 ± 3.11ab	3.42 ± 0.42a	3.52 ± 0.25a	3.48 ± 0.64ab
	6	–	2	–	71.34 ± 3.18a	3.77 ± 0.40a	3.69 ± 0.19a	4.17 ± 0.37a
	7	–	–	0.5	24.44 ± 2.77b	2.51 ± 0.25b	2.11 ± 0.23b	2.39 ± 0.19b
	8	–	–	1	28.91 ± 2.54b	2.60 ± 0.48b	2.54 ± 0.41b	2.87 ± 0.20b
	9	–	–	2	37.62 ± 3.98b	2.72 ± 0.62b	2.19 ± 0.13b	3.41 ± 0.33ab

Transplantation of sterile plants

We transplanted 50 sterile plants each of SL and SPL of FP, MP and MOP each into a mixed substrate of peat and vermiculite (1:1, v/v). Around 15 d after transplantation, the plant leaves gradually grew larger and turned bright green (Fig. 9A, C). After 30 d, the sterile plants grew vigorously, with the total number of buds increasing, while the newly formed buds remained SPL, with their stem segments gradually lignifying (Fig. 9B, D). Among the FP, 27 SL sterile plants and 33 SPL sterile plants survived, with survival rates of 54 and 66 % respectively. For the MP, 38 SL sterile plants and 30 SPL sterile plants survived, with survival rates of 76 and 60 % respectively. For the MOP, 16 SL sterile plants and 19 SPL sterile plants survived, with survival rates of 32 and 66 % respectively.

Fig. 9.

Transplantation of sterile plants derived from SL and SPL of FP, MP and MOP. (A) Transplantation of SL sterile plants for 15 d; (B) transplantation of SL sterile plants for 30 d; (C) transplantation of SPL sterile plants for 15 d; (D) transplantation of SPL sterile plants for 30 d.

DISCUSSION

Response of explants with different sexes and leaf morphology to disinfectants

Juniperus chinensis is predominantly dioecious, rarely monoecious, with SP and SPL leaf forms. Owing to its distinct physiological properties, different sexes and leaf morphology types of J. chinensis differ in their requirements for disinfectants and treatment durations (Yao, 2022; Kang et al., 2024). From a physiological perspective of sexual differentiation, FP and MP exhibit markedly distinct responses to disinfectants. MP demonstrate a higher damage threshold to disinfectant stress, whereas FP due to continuous energy expenditure on reproductive development show significantly weaker damage resistance. Consequently, under identical disinfection treatments, FP explants are more susceptible to browning. As a transitional form in sexual differentiation, MOP individuals exhibit more consistent disinfection time patterns between SP and SPL (Ślesak et al., 2017). From a leaf morphology perspective, SL require extended treatment time, due to their thick waxy layer that significantly retards penetration of mercury ions (Hg²⁺). In contrast, SPL with thinner cuticles and less wax content permit faster Hg²⁺ permeation, thus needing shorter disinfection times (Jin et al., 2012; Kang et al., 2024). Compared to HgCl₂, which is highly toxic and penetrates rapidly, NaClO offers notable advantages: it gradually released hypochlorous acid (HClO), enabling mild yet sustained oxidative sterilization. The oxidation intensity and release rate of NaClO are well-adapted to the structural differences between the two leaf morphologies (Yildiz et al., 2012). For SL, the sustained release property compensates for the penetration delay caused by the thick cuticle, enabling HClO to accumulate to an effective bactericidal concentration within 18 min of disinfection. For SPL, the mild oxidation avoids explosive damage similar to that caused by HgCl₂, and the disinfection effect can still be achieved with 18 min of treatment. Thus, the optimal treatment time is the same for both leaf forms.

In terms of application value, the low toxicity, ease of removal and economic feasibility of NaClO overcome the limitations of high risks (operational safety hazards) and high costs (waste water liquid treatment costs) associated with HgCl₂-dependent disinfection in traditional tissue culture. Through precise disinfection time matching, this study provides an environmentally friendly yet effective standardized sterilization protocol for tissue culture of Juniperus and related Cupressaceae species. The established protocol offers a reference for developing in vitro systems in other gymnosperms exhibiting leaf dimorphism or sexual dimorphism (Ślesak et al., 2017; Yemiş and Harmancı, 2020).

Responses of explants of different sexes and leaf morphology to browning

Explant browning is a widespread phenomenon in plant tissue culture, attributed primarily to enzymatic browning. When explants are mechanically wounded during excision, their cellular integrity is disrupted, allowing oxidative enzymes (PAL, PPO, POD) to interact with phenolic compounds. Under aerobic conditions, these reactions trigger oxidative browning through redox processes (Amente and Chimdessa, 2021; Liu et al., 2024a, b). The high activity of PAL promotes accumulation of phenolic compounds, PPO accelerates the conversion of phenolics to quinones, and POD enhances oxidation efficiency. These three components form a coordinated cascade from substrate accumulation through catalytic conversion to oxidative intensification, ultimately resulting in severe explant browning (Chen et al., 2022; Liu et al., 2024a, b). To address explant browning, we screened three anti-browning agents, PVP, AC and AgNO₃, and their inhibitory effects on explant browning showed significant differences. As an adsorbent, AC not only absorbs harmful substances but takes up nutrients beneficial to explant growth, such as inorganic salts and hormones, consequently leading to a lower explant survival rate (Babaei et al., 2013; Xu et al., 2023). AgNO₃ suppresses enzyme activity through Ag⁺, but its strong toxicity resulted in a significantly lower explant survival rate compared to the PVP and AC treatment groups. PVP, with minimal interference on nutrient uptake, effectively inhibits browning while maintaining explant viability, demonstrating the best overall anti-browning performance.

Furthermore, the oxidative properties of the explants themselves further regulate browning. In the absence of PVP, the activities of PPO, POD and PAL in explants of the three sexes of J. chinensis reach peak levels, consistent with the browning mechanism involving the synergistic chain reaction from substrate catalysis to accelerated oxidation. SPL exhibited significantly higher activities of PPO, POD and PAL compared to SL, indicating stronger oxidative efficiency in SPL. If the PVP concentration is insufficient (e.g. 1.0 mg L⁻¹), although browning can be inhibited initially, only part of the phenolic substances are consumed, and the remaining phenolics will still be oxidized, eventually leading to browning. In contrast, PVP at 2.0 mg L⁻¹ achieves the optimal anti-browning effect (Babaei et al., 2013; Liu et al., 2024a, b).

Proliferation selectivity of explants varies by sexes and leaf morphology

The efficacy of proliferation culture is pivotal for establishing large-scale rapid propagation systems. A cytokinin–auxin combination is commonly used for adventitious bud proliferation, achieving both high multiplication coefficients and vigorous growth potential (Hazubska-Przybył, 2019). In general, a higher concentration of cytokinin favours shoot proliferation. At a low concentration of 6-BA (1.0 mg L⁻¹), the proliferation coefficient of adventitious buds in J. chinensis is only about 1-fold, with plant height less than 3.0 cm. When the concentration is increased to 3.0 mg L⁻¹ the proliferation rate exceeds 80 % and the proliferation coefficient reaches over 4-fold, demonstrating the critical role of 6-BA in the proliferation stage of adventitious shoots in J. chinensis. Trans-ZT, as a highly active natural cytokinin, is widely used in tissue culture of Cupressaceae plants. An appropriate concentration of Trans-ZT can significantly enhance the proliferation of adventitious shoots, which is consistent with research findings on adventitious bud proliferation in J. thurifera, J. excelsa, J. communis and P. orientalis (Shi, 2009; Hazubska-Przybył, 2019; Zhou et al., 2020; Fathollahi et al., 2025).

Moreover, there are significant differences in adventitious bud proliferation efficiency among J. chinensis of different sexes. Notably, MOP exhibit markedly superior proliferation compared to FP or MP, and this difference is not coincidental. From the perspective of proliferation initiation and sustainability, adventitious buds of MOP exhibited noticeable proliferation within 5–7 d after inoculation, forming dense shoot clusters by around 15 d. In contrast, MP showed a slightly delayed proliferation onset (8–10 d), while FP demonstrated an even more prolonged lag phase (12–15 d). This phenomenon may be attributed to the fact that FP must allocate substantial resources during the reproductive phase to support the differentiation, development and seed formation of female cones. This process probably suppresses physiological activities related to cell division (Ślesak et al., 2017). Although MP do not bear the resource burden of seed development, the concentrated growth of male cones still leads to a unidirectional resource allocation, thereby weakening the material basis for adventitious bud proliferation (Irish and Nelson, 1989; Liu et al., 2022a, b). In contrast, MOP possess both MP and FP reproductive structures with synchronized developmental rhythms. This balanced growth pattern effectively prevents excessive resource depletion, allowing adventitious shoots to continuously receive sufficient nutrients and hormones (Ślesak et al., 2017; Hazubska-Przybył, 2019; Chen et al., 2022). Consequently, these shoots exhibited faster responses to exogenous cytokinins and demonstrate superior proliferation efficiency (Liu et al., 2022a, b; Chen et al., 2025). SL and SPL of MOP require the same proliferation medium, and both can achieve efficient proliferation in the same formula. In contrast, SL and SPL of FP and MP require different proliferation medium. The reason may lie in the biased endogenous physiological status of individual plants. To achieve favourable proliferation effects, it is necessary to adjust components such as hormone ratios in the medium according to the characteristics of SL and SPL, as well as the allocation of nutrients to their own reproductive resources (Hesami et al., 2024).

Meanwhile, differentiated adventitious buds all develop into SPL, which is a typical age-dependent heterophylly, possibly related to environmental factors for leaf shape transition. Similar leaf morphology transformation phenomena are also found in plants such as J. communis, Thuja sutchuenensis and Populus euphratica. Subsequent studies can further explore the transformation mechanisms between different sexes and leaf morphology, providing a theoretical reference basis for the directional cultivation and regulation of heteromorphic leaves (Zhou et al., 2020; Song et al., 2021).

Selection of rooting culture from explants of different sexes and leaf morphology

Rooting culture is a bottleneck of gymnosperm tissue culture. Although many plants can be made sterile, it is often difficult to induce rooting or the rooting rate is very low, which restricts construction of the system (Hazubska-Przybył, 2019). Commonly used basal medium for gymnosperm rooting induction include MS, ½MS, WPM, White and DCR media (Jin et al., 2012). In the present study, suitable medium types for rooting of adventitious buds of SL and SPL from FP and MP are ½MS and DCR medium, respectively, while DCR medium is uniformly suitable for adventitious buds of SL and SPL from MOP. This indicates that different sexes of the same plant have different medium requirements, and the medium types required for different leaf morphology of the same sex also vary. This phenomenon has also been observed in studies of Thuja occidentalis and P. orientalis (Ślesak et al., 2017; Zhou et al., 2020). Furthermore, auxins such as NAA, IBA and ABT have demonstrated significant root-inducing effects on adventitious buds, though their optimal concentrations vary significantly among plant species (Castro et al., 2011).

The adventitious buds of SL from FP and MP exhibit the best rooting ability in medium containing 1.0 mg L⁻¹ ABT, but their rooting ability gradually weakens as the concentration increases. The adventitious buds of SPL have a stronger affinity for 2.0 mg L⁻¹ IBA, while high concentrations can inhibit their growth. This indicates that the rooting of adventitious buds of J. chinensis has a certain tolerance range to the concentration of auxins. Interestingly, the adventitious buds of both SL and SPL from MOP exhibit the best rooting performance in medium containing 2.0 mg L⁻¹ IBA, suggesting their more balanced requirement for exogenous hormones. In contrast, FP show poorer rooting performance, probably because they prioritize hormonal allocation for megastrobilus differentiation, development and seed formation during the growth period. It is noteworthy that among the three sexes of J. chinensis, the rooting efficiency of SL is significantly higher than that of SPL, which is related to their developmental stages: in J. chinensis, all leaves are SPL in the juvenile stage, and then differentiate into SL, the latter having more sufficient nutrient reserves, which can provide stable material substrates for the differentiation of root primordia. Since SPL are in the juvenile state, they accumulate fewer nutrients, and more resources are allocated to leaf morphology transformation, bud development and other aspects (Yao, 2022; Kang et al., 2024). These conclusions are consistent with the performance in the above-mentioned adventitious bud proliferation stage. Future studies could further investigate the resource allocation mechanisms between sexes and leaf morphology, providing a theoretical foundation for developmental regulation in adventitious rooting of heteromorphic leaves in gymnosperm tissue culture.

CONCLUSIONS

We have constructed a tissue culture system for J. chinensis based on sexes and leaf morphogenesis differences. By disinfecting with NaClO for 18 min and adding PVP to DCR medium, we analysed the browning mechanism and effectively resolved the issues of incomplete disinfection and severe browning during tissue culture. Furthermore, J. chinensis tissue culture exhibits significant differences in adaptability to sexes and leaf morphogenesis across various stages, necessitating targeted optimization of technical parameters. During the proliferation stage, explants derived from MOP show consistent hormone requirements. The adventitious bud proliferation efficiency of SPL is notably higher than that of SL, and all proliferated adventitious buds are SPL, indicating a strategy of prioritizing resource allocation for bud development. At the rooting stage, the rooting ability of adventitious buds from SPL is inferior to that of SL, further validating the preference for allocating resources primarily to bud development. Conversely, MOP still maintain consistent hormone demands during the rooting stage, reflecting their physiological uniformity under conditions of incomplete sexual differentiation. This study provides technical support for the targeted breeding, germplasm resource innovation and industrial development of J. chinensis. Future studies could further explore the differences among various stages of tissue culture for different genotypes of the same sex. Meanwhile, we provide a new perspective for research into sexual differentiation, mechanisms of leaf shape transition and other related fields of tree species with multiple sexes or of heterophyllous tree species.

Funding

This study was supported by the Postgraduate Practical Innovation Fund of Shanxi Agricultural University of China (2024SJ149) and the Science and Technology Innovation Fund of Shanxi Agricultural University of China (2020BQ37).

Author contributions

JLW and XPT designed the research plan; JLW and KJY performed the experiment and analysed the data; JLW wrote the draft; KQ, JJQ, HXL and HWK revised the manuscript. All authors approved the final version.

Data Availability

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