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Article

Regeneration Capability Comparison of Leaves Between Nodal Cuttings from Young Stems and Suckers and Its Histological Analysis in Triadica sebifera

by
Yuan Chen
1,2,†,
Yumei Xie
1,2,3,†,
Keyuan Zheng
1,2,
Yanru Fan
4,
Huijing Zhou
1,2 and
Mulan Zhu
1,2,*
1
Shanghai Key Laboratory of Plant Functional Genomics and Resources, Shanghai Chenshan Botanical Garden, Shanghai 201602, China
2
National Key Laboratory of Plant Molecular Genetics (NKLPMG), CAS Center for Excellence in Molecular Plant Sciences, Chinese Academy of Sciences, Shanghai 200032, China
3
School of Life Sciences, Shanghai Normal University, Shanghai 200223, China
4
Research Institute of Subtropical Forestry, Chinese Academy of Forestry, Hangzhou 311400, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Forests 2025, 16(6), 992; https://doi.org/10.3390/f16060992
Submission received: 23 April 2025 / Revised: 22 May 2025 / Accepted: 30 May 2025 / Published: 12 June 2025
(This article belongs to the Section Forest Ecophysiology and Biology)
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Versions Notes

Abstract

:
Triadica sebifera, an economically and medicinally valuable tree species native to China, was investigated for its in vitro regeneration potential using leaf explants from nodal cuttings of young stems and sprouts. This study evaluated the effects of basal media, plant growth regulators (PGRs), explant sources, and incision methods on adventitious shoot induction, supplemented by histological analysis. The highest shoot regeneration frequency (98.89%) and maximum shoot number (72) were achieved via direct organogenesis using sucker-derived nodal cuttings cultured on MS medium with 2 mg/L 6- benzyladenine (6-BA), 0.3 mg/L kinetin (KT), and 0.2 mg/L α-naphthaleneacetic acid (NAA). Under identical conditions, branch-derived explants showed lower regeneration (84.44%, 64 shoots). Transverse midvein incision proved most effective, with sucker-derived leaves exhibiting superior regeneration. Shoots elongated completely (100%) on Murashige and Skoog (MS) medium containing 0.3 mg/L 6-BA, 0.03 mg/L NAA, and activated charcoal. Rooting was optimal on MS medium with 0.3 mg/L indole-3-butyric acid (IBA), yielding a 98% acclimatization survival rate. Histological analysis revealed de novo meristem formation from parenchyma cells, confirming direct organogenesis without callus intermediation, further validating the enhanced regenerative capacity of sprout-derived explants. This efficient in vitro regeneration system provides a foundation for large-scale propagation and germplasm conservation of T. sebifera, while offering insights for woody plant regeneration studies.

1. Introduction

Triadica sebifera, a deciduous tree belonging to the genus Triadica of the family Euphorbiaceae, is commonly known as Chinese tallow tree [1]. As a traditional and economically significant species native to China, T. sebifera serves as an important industrial oil crop and a source of raw materials, contributing to its considerable economic value [2,3]. The seeds of T. sebifera are rich in high-calorie oils, with the white waxy layer surrounding the seeds being refined for the production of candles, wax paper, and soaps [4,5,6,7]. The seed kernels yield oil also serves as a primary raw material for paints and inks, while the aril is utilized in the manufacture of candles and soaps [8,9,10]. Beyond its industrial applications, this species is a medicinal plant recorded in Chinese classical pharmacopeia works. The leaves secrete a white sap upon removal, which is traditionally used for its heat-clearing and detoxifying properties [11,12]. Furthermore, the root bark is employed in the treatment of venomous snake bites [13,14], underscoring the plant’s multifaceted value. In addition to its economic and medicinal importance, T. sebifera is prized for its ornamental appeal, displaying vibrant reddish foliage in spring and autumn [13,15,16]. It is widely used in plantation because of its vigorous vitality [17], strong adaptability to soil, and high planting survival rate [18,19,20]. Collectively, these attributes highlight T. sebifera as a species of significant ecological, economic, and cultural importance.
T. sebifera is a cross-pollinated plant, and genetic recombination and hybridization among different species groups [21,22] result in offspring with significant variability, leading to uneven economic benefits among individuals. Although the propagation of cuttings or grafting can preserve the desirable traits of the parent plant, the reproduction coefficient is low [23], which severely hinders the promotion of improved varieties [10,24,25]. The use of tissue culture to propagate T. sebifera has the characteristics of short propagation cycle, fast propagation speed, and the potential for seedling detoxification [26,27]. Moreover, the cultivation conditions allow it be artificially controlled and factory bred. Using tissue culture technology, a large number of high-quality clonal plantlets can be rapidly produced in vitro, effectively addressing the low propagation rates associated with cutting or grafting. At the same time, it also accelerates the dissemination of improved varieties, playing a critical role in the preservation of genetic resources and the study of genetic variation in T. sebifera. At present, several studies [2,28,29,30] have explored tissue culture and rapid propagation techniques of T. sebifera. However, research comparing the regeneration performance of leaves remains limited. Most tissue culture protocols for T. sebifera rely on stem segments as explants [31,32]. Furthermore, studies have demonstrated that immature embryos [33] and endosperm [2,34] can also serve as effective explants for tissue culture. Although, leaves have been successfully used as explants to induce callus formation and rooting [30]. However, the regeneration frequency and proliferation coefficient of isolated plants remain suboptimal. The absence of an efficient regeneration system for superior clonal lines, coupled with the lack of a reliable genetic transformation system, poses significant challenges to the advancement of T. sebifera biotechnology.
In this study, we compared the regeneration performance of T. sebifera leaves from adult macrophytes and sprouting shoots by examining the effects of the leaf incision method, explant source, and plant growth regulators (PGRs) on the induction of adventitious shoots. This work establishes an efficient regeneration system for T. sebifera using isolated leaves as explants. In addition, we conducted a histological analysis of the different developmental stages of adventitious shoots during regeneration, providing insights into the mechanisms underlying morphogenesis. To the best of our knowledge, the morphological formation process of regenerating buds in T. sebifera has not been previously reported. The regeneration system in this study enhances the technical foundation for the factory-scale production of high-quality T. sebifera plantlets. Furthermore, it also offers critical technical support for the genetic improvement of superior varieties and contributes to the preservation and propagation of elite genotypes.

2. Materials and Methods

2.1. Plant Material and Culture Conditions

Mother plants of T. sebifera were collected from Shanghai Chenshan Botanical Garden (121°18′ E, 31°08′ N). Young stem-derived nodal cutting of the current year and suckers-derived nodal cutting under the mother plant were used as the explants (Figure 1A–C).
Unless mentioned otherwise, all the media in our experiments consisted of 30 g/L sucrose, 4.5–5.0 g/L agar powder, with pH adjusted to 5.8–6.0. The cultures were sterilized for 20 min under the conditions of 121 °C, 103 kPa. The plants were exposed to a photoperiod of 16 h of light/8 h of darkness, a light intensity of 2000–2200 lx at 25 ± 2 °C, and 70% relative humidity.

2.2. Regeneration of Plantlets

2.2.1. Acquisition of Sterile Leaves

Young stem-derived nodal cutting and suckers-derived nodal cutting of the current year were taken and cut into 1.5–2 cm long stem segments. The peeled stem segments were rinsed under running water with detergent for 2 h and then absorbed the surface water of the stems. After that, stems were sterilized under sterile conditions using 75% alcohol for 30 s, then treated to disinfect with 0.1% HgCl2 for 8 min, and washed six times with sterile water. Dry stem segments, which were blotted dry with filter paper, were inoculated into a Murashige and Skoog (MS) medium [35] supplemented with 1 mg/L 6-Benzyladenine (6-BA) and 0.1 mg/L α-naphthaleneacetic acid (NAA) (Figure 1D,G). For each treatment, three replications were performed, and each experiment consisted of 30 explants. The response of the definitive shoots were observed.
The first-generation sterile plantlets were inoculated for successional culture in the MS medium with the addition of 2 mg/L 6-BA until a large number of spreading leaves were obtained.

2.2.2. Selection of Basic Culture Media

Under aseptic conditions, sterile leaves of young stem-derived nodal cutting and suckers-derived nodal cutting were individually cut, with a scalpel 3–5 times perpendicular to the direction of leaf veins. After that, the leaves were laid flat, adaxial side up, in the wood plant medium (WPM), MS, and Douglas fir cotyledon revised medium (DCR) medium, fortified with 1 mg/L of 6-BA and 0.1 mg/L of NAA. A total of three replications of each treatment were performed, and each experiment included 30 explants. Adventitious shoot induction rate was counted after 45 d so that the most optimum medium for adventitious shoot induction could be determined.

2.2.3. Treatment of the Method of Incision

A study on the effect of leaf incision method on the regeneration frequency of T. sebifera was analyzed to establish an effective T. sebifera plant regeneration system. Aiming to evaluate the impact of leaf explant integrity on regeneration frequency and the number of shoots per explant, as well as to identify the optimal leaf explant type. Three treatments were applied to leaf explants: (1) whole leaves, (2) transversely cut midvein leaves (with two to three incisions), and (3) unmargined leaves (with four to five incisions). Leaf explants of nodal cuttings from both young stems and sprouts were inoculated onto the optimized induction medium. For each treatment, three replications were performed, and each experiment consisted of 30 explants. After 45 d of incubation, the frequency and number of adventitious shoots regenerated from each explant were determined.

2.2.4. Effect of Cytokinin KT Concentration

Sterile leaves from young stem-derived nodal cutting and suckers-derived nodal cutting were taken with 3–5 wounds created perpendicular to the direction of the veins to probe the effect of the concentration of kinetin (KT) on the induction of adventitious shoots from leaf explants. Adaxial surface with leaf explants were then placed in medium containing various concentrations of KT (0, 0.1, 0.2, 0.3, 0.4, and 0.5 mg/L), 6-BA (1 mg/L), and NAA (0.1 mg/L) for optimal adventitious shoot induction. For each treatment, three replications were performed and each experiment consisted of 30 explants. The percentage of induced adventitious shoots was counted after 45 d to obtain the optimum KT concentration for the induction of adventitious shoots.

2.2.5. Effects of Plant Growth Regulator Combinations

Screened leaves of sterile plantlets were used as recipient material for inducing adventitious shoots. Under aseptic conditions, the leaves from well-grown young stem-derived nodal cutting and suckers-derived nodal cutting were cut and the leaves were optimally incised. Afterwards, the leaves were spread adaxially on the optimal induction medium added with diverse levels of 6-BA (1, 2, and 3 mg/L), NAA (0.1, 0.2, and 0.3 mg/L), and 0.3 mg/L KT. Without the hormone-added medium was utilized as a control. The percentage of adventitious shoots was counted after 45 d to determine the optimal composition of the medium. For each treatment, three replications were performed, and each experiment consisted of 30 explants.

2.2.6. Adventitious Shoot Elongation

The adventitious shoots induced from isolated leaves were inoculated onto MS medium with the addition of cytokinin 6-BA (0.1, 0.2, 0.3, 0.4, and 0.5 mg/L) and the growth hormone NAA (0.01, 0.02, 0.03, 0.04, and 0.05 mg/L) and active carbon (AC) (0.2 g/L). At the same time, adventitious shoots elongation was carried out using the phytohormone-free medium as control cultivation. The growth of shoots was observed periodically. After 45 d, the percentage of elongation and the mean seedling height of the regenerated shoots were counted. For each treatment, three replications were performed, and each experiment consisted of 30 explants. The optimum medium for elongation of adventitious shoots was determined.

2.2.7. Adventitious Root Induction

Healthy elongated plantlets in good growth condition were inoculated into the MS and WPM containing Indole-3-butyric acid (IBA) (0, 0.1, 0.2, 0.3, 0.4, 0.5 mg/L) for in vitro rooting experiments. For each treatment, three replications were performed, and each experiment consisted of 30 explants. The rooting rate was counted after 30 d to select the best rooting medium.

2.2.8. Acclimatization

The bottle with healthy rooted plantlets was filled with 0.5–1 cm of water, uncapped and placed indoors for 2 d to refine the plantlets. Subsequently, the plantlets were washed off the medium attached to them and planted in a substrate containing vermiculite, perlite, and peat soil (1:1:1). A transparent plastic bag was used to cover the plantlets to ensure adequate humidity. The plantlets were grown under 16 h photoperiod (33.73 µmol∙m−2∙s−1 light intensity) with the conditions of 25 °C and 70% relative humidity. Transplant survival was tallied after 60 d.

2.3. Histological Analysis

Adventitious shoots of leaves from sucker-derived nodal cutting at different developmental stages were subjected to histological analysis. Samples were placed in dehydration cassettes and processed through a sequential gradient alcohol dehydration series in a dehydrator. Using the order of increasing from low to high concentration, from 70%, 85%, 95%, to pure alcohol (anhydrous ethanol), with each step lasting 1–2 h. After that, the samples were immersed in an alcohol–benzene mixture for 15–30 min, followed by a wax–benzene mixture at 62 °C for 2 h, and finally melted paraffin wax twice at 62 °C for 4 h. The wax-dipped tissues were then transferred to an embedding machine for embedding. A small amount of melted paraffin wax was added to the embedding frame. Before solidification, the sample were placed into the frame and labeled. After the wax completely solidified, the wax blocks were removed, trimmed, and mounted on a paraffin slicer for sectioning at a thickness of 4–8 μm. Wax strips were floated on a flotation machine in warm water at 40 °C to flatten the tissue; then, the tissue was lifted up on an anti-dehiscence slide and exposed to an oven at 60 °C. Prior to staining, the dried sections underwent dewaxing and rehydration. This involved sequential immersion in xylene (twice for 15 min each), anhydrous ethanol (twice for 5 min each), 85% alcohol (5 min), 75% alcohol (5 min), and finally, distilled water. Depending on the degree of lignification, the sample slides were stained by immersing them into Senka staining solution for 2–4 h, and then rinsed slightly with tap water to wash away the excess staining solution. Decolorization was performed using 50%, 70%, and 80% ethanol, each for 5–8 s. The sections were then counterstained with solid green staining solution for 6–20 s and dehydrated through three changes in anhydrous ethanol. Finally, the slides were cleaned in xylene for 5 min and mounted with neutral gum for sealing. Relevant data were collected and analyzed under a microscope.

2.4. Statistical Analysis

It is indicated that all data are the mean value of each treatment. In this study, SPSS 27.0 software (IBM SPSS Statistics 27.0) was used to analyze all data by one-way ANOVA with Duncan’s Multiple Extreme Differences. The means were considered statistically significant at p ≤ 0.05.

3. Results

3.1. Acquisition of Aseptic Leaves

The study investigated the primary culture of stem segments with buds from different explants. The result showed that sprouting stem segments responded faster to bud set than branching stem segments. The stem segments from T. sebifera young stem-derived nodal cuttings were inoculated into the primary medium and started to sprout in about 14 d, and the buds sprouted to 3 mm in about 21 d (Figure 1D); the leaves were tip-shaped by 35 d (Figure 1E); in contrast, the stem segments taken from the suckers-derived nodal cuttings began to sprout within 7 d. As a result, all three bud points sprouted about 14 d (Figure 1G). The germination of the central buds were 6 mm and the uppermost buds were 3 mm. Furthermore, the buds that located in the basal part of the bud point expanded into a sphere with a diameter of 2 mm. It was observed that the leaves of the middle buds began to stretch after 35 d (Figure 1H). At this stage, the buds were cut off and transferred to the following medium for bud proliferation culture. Tender, green leaves with clear veins were observed for 28 d (Figure 1F,I).
During the process of primary culture, leaves and shoot tips of the young stem-derived nodal cutting were transparent or translucent water-soaked with a tendency to vitrification. The activated charcoal was added to the culture medium in this study in order to further improve the survival rate of sterile plantlets during tissue culture. The sterile plantlets grew normally after adding 2 g/L of activated charcoal [36].

3.2. Effect of Basic Medium on the Induction of Adventitious Shoots

Different types of basal medium showed different regeneration efficiencies. It can be shown from Table 1 and Figure 2 that the MS basic medium had the best induced adventitious shoots (81.83% and 56.50%), with an average of 25 and 21.83 adventitious shoots, respectively (Figure 2B,E). This was followed by the WPM (Figure 2A,D), whereby the induction rate of adventitious shoots was 80.00% and 56.50%, respectively, and the average number of adventitious shoots was 25.00 and 13.33, respectively. Finally, the poorest induction effect was observed in the DCR (Figure 2C,F), where 74.83% of adventitious shoots were induced from sprouting shoot leaves, while only 51.93% of adventitious shoots and 11.17 adventitious shoots per explant were induced from leaves of young stem-derived nodal cuttings. At the same time, we could also see that the adventitious bud induction rate and average adventitious bud number with leaves from suckers-derived nodal cutting were higher than those from leaves of young stem-derived nodal cutting (p < 0.05). Therefore, the regeneration ability of leaves from suckers-derived nodal cutting is stronger than that of leaves from young stem-derived nodal cutting.

3.3. Effect of KT Concentration on the Induction of Adventitious Shoots

In the study, we investigated the effect of cytokinin KT on adventitious shoots induction (Table 2). The maximum induced rate of adventitious shoots (83.67% and 59.67%) was observed when the KT was 0.5 mg/L. The average number of adventitious shoots was the highest when the KT was 0.3 mg/L, with an average of 29.67 and 22.83 regenerated shoots per explant, respectively. Under the constant concentration of 6-BA and NAA, the adventitious shoot induction rate gradually increased with the increase in KT concentration. The number of adventitious shoots, on the other hand, indicated a tendency of increasing followed by decreasing with the growth of KT concentration. Among the two leaf explants cultured at the same conditions, leaves from suckers-derived nodal cutting were significantly stronger induced than leaves from young stem-derived nodal cutting.

3.4. Effect of the Method of Incision on the Induction of Adventitious Shoots

The leaves of the three treatments showed different regeneration efficiencies (Figure 3 and Table 3). Leaf explants with transverse cuts to the midrib were the most effective in inducing adventitious shoots. Transverse midvein leaf explants from sucker-derived nodal cutting proliferated and began to swell along the incision after 7 d of culture; a large number of shoot primordia formed at the margins of the leaf and at the incision after 21 d of culture (Figure 3B). By contrast, transverse cuts to midvein leaves from young stem-derived nodal cutting required 10 d to initially swell at the incision, and a small percentage of bud primordia formed after 21 d (Figure 3E). Whole-leaf explants of budding shoots swelled and formed bud primordia at the leaf margins after 21 d of culture (Figure 3A); whole-leaf explants from young stem-derived nodal cutting formed a small quantity of bud primordia close to the petiole site after 21 d of culture (Figure 3D). A significant amount of dense cellular tissue was formed after 21 d of incubation of leafless marginal leaves from sucker-derived nodal cutting (Figure 3C), whereas adventitious shoots were not induced after 21 d of incubation of leafless marginal leaves from young stem-derived nodal cutting. Remarkably, the leaf explants would die and failed to induce adventitious shoots if the inoculated leaf area was too small during the process of adventitious shoot induction (Figure 3F).
Regarding the induction of adventitious shoots, a significantly higher percentage of adventitious shoots was induced in the transverse cut midrib leaves than in the whole and marginless leaves, with a maximum of 98.89% and 84.44%, respectively, and producing an average of 71.75 and 64.00 adventitious shoots per leaf explant, respectively. Among the three explants, adventitious shoot induction was the lowest in whole leaf explants with 53.33% and 38.89, producing an average of 23.75 and 20.42 adventitious shoots per explant, respectively. In all treatments, the induction ability of leaves from sucker-derived nodal cutting was stronger than those from young stem-derived modal cutting, and transverse cut midvein leaves from sucker-derived nodal cutting were most effective in inducing adventitious shoots.

3.5. Effect of Plant Growth Regulators on Adventitious Shoots Induction from Leaves of Nodal Cuttings from Both Young Stems and Sprouts

It was found that the addition of different concentrations of plant growth regulators had a statistically significant effect on the ability to induce adventitious shoots from leaf explants derived from both young stems and sprouts (Table 4). At a fixed concentration of 6-BA, increasing the NAA concentration initially enhanced the shoot induction frequency and the average number of shoots per explant, followed by a decline. The maximum induction rate (98.89%) and the highest number of adventitious shoots (72) were observed in sprouting shoot explants cultured on medium containing 2 mg/L 6-BA, 0.3 mg/L KT, and 0.2 mg/L NAA. In contrast, branch-derived explants exhibited significantly lower induction rates (84.44%) and fewer adventitious shoots (64) under the same conditions. When leaves were cultivated in the medium that contained 6-BA and lower concentration of NAA, it differed significantly in the adventitious shoot induction rate of branching leaf explants compared to leaf explants from suckers-derived nodal cutting (p < 0.05). Furthermore, at a fixed NAA concentration, increasing the 6-BA concentration led to significant differences in both the induction rate and the number of adventitious shoots between explants derived from sprouting shoots and those from branches.
Sterile seedling leaves were maintained in the MS medium augmented with 2 mg/L 6-BA, 0.3 mg/L KT, and 0.2 mg/L NAA, both of which successfully induced adventitious shoots. The leaves from sucker-derived nodal cutting (Figure 4A) and leaves from young stem-derived modal cutting (Figure 4D) were cultured for about 35 d, and both of them formed a considerable amount of adventitious shoots. However, the adventitious shoots derived from sprouting shoot leaves exhibited more vigorous growth, as evident from the bottom view (Figure 4B,E). By 45 d, the adventitious shoots induced by leaves from sucker-derived nodal cutting (Figure 4C) were significantly more numerous than those induced by leaves from young stem-derived modal cutting (Figure 4F).
To sum up, plant growth regulators were of great importance in inducing adventitious shoots from leaf explants. Moreover, the induction rate and the numbers of adventitious shoots were significantly higher in leaf explants derived from sucker nodal cuttings compared to those from young stem nodal cuttings. The most suitable medium was the MS medium that was added with 2 mg/L 6-BA and 0.2 mg/L NAA. Consequently, subsequent experiments focusing on shoot elongation and rooting were carried out on adventitious shoots induced from transverse midvein cuttings of sucker-derived nodal explants.

3.6. Elongation of Adventitious Shoots

For leaf explants from sucker-derived nodal cutting (Figure 5A), the maximum (100%) shoot elongation and the longest average shoot length up to 6.05 cm were observed in MS medium fortified with 0.3 mg/LBA, 0.03 mg/L NAA, and AC (Figure 5C). A percentage of 95.57% of shoot elongation and 5.80 cm of average stem length were observed for sprouting shoot leaf explants treated in the same medium without AC (Figure 5B). Compared with the culture of MS without AC (control), the differences in stem elongation rate and mean stem length were significant (p < 0.05) (Table 5). Thus, we found that a significant increase in adventitious shoot elongation rate and mean shoot length was observed with AC (Figure 5D). Also from Table 3, lower concentrations of pgr promoted stem elongation, but also resulted in poorer elongation when pgr was reduced to a certain level.

3.7. Root Formation

Elongated adventitious shoots from multiple shoot clusters were isolated by transferring them to MS and WPM media supplemented with 0–0.5 mg/L IBA for root induction studies. As shown from Table 6, both media were able to induce root growth of the regeneration plantlets. Yet there were large differences in rooting rate and rooting quality (p < 0.05). The white conical root tips started to emerge at the base after 5 d of incubation of the histocultures in MS medium, whereas the bases of the sprouts expanded after 10 d of incubation of the histocultures in WPM, and white root dots could be observed. In our study, the highest induced percentage of adventitious roots was 98.89% and the highest mean number of roots was 14.00 under the MS medium by adding 0.3 mg/L IBA. The regenerated plantlets under this condition also showed better growth in terms of robust root system, color of dark green, and larger leaves (Figure 6A,B). In contrast, the highest adventitious root induction rate of 84.44% and the highest average root number of 11.67 roots were observed in the WPM under the same conditions. As seen in Table 6, with the rise in IBA concentration, root number decreased significantly, and the rate of adventitious root induction also decreased. Moreover, the rooting rate was at 65.56% when IBA was 0.4 mg/L, and with relatively weak roots.

3.8. Acclimatization

After 60 d of acclimatization, a survival rate of 98% was achieved for isolated regenerated T. sebifera plantlets. The acclimatized T. sebifera plantlets showed good growth and exhibited normal growth characteristics with typical plant morphology (Figure 6C).

3.9. Histological Analysis

During in vitro regeneration, various proliferative structures of adventitious shoots were histologically analyzed to elucidate a underlying comprehension of the regeneration pattern. The micromorphology of the initial adventitious shoot structure showed that it consisted of equidistant, small-sized, densely packed parenchyma cells with dense cytoplasm and early signs of vascular tissue development (Figure 7A). Notably, the early vascular tissue exhibited irregular, non-directional, and non-axial development. Under the influence of exogenous hormones, these dense cells formed a narrow band of meristematic tissue, characterized by multiple zones of meristematic activity dividing in various directions, gradually establishing an organized structure. It represented an initial establishment of meristematic organization, which consisted of an aggregation of large-nucleated small cells, with densely packed small meristematic tissue cells surrounded by large cells (Figure 7B). Approximately 10 d later, morphologically distinct globular clumps of meristematic tissue, termed meristematic nodules were produced (Figure 7C). These nodules underwent further division and differentiation, giving rise to the early stages of organ primordia (Figure 7D). With the elongation of the bud, it was observed that the cells of the callus tissue gradually divided and continuously differentiated into new thin-walled cells towards the inner part of the tissue, some of which began to differentiate into vascular tissues (Figure 7E). Frequent cell divisions led to the organization of meristematic tissue clusters, culminating in the formation of a well-defined apical meristem surrounded by leaf primordia (Figure 7F), representing the apical meristem of the bud primordium. Furthermore, cell divisions on both sides of the shoot meristematic tissues facilitated the formation of leaf primordia, ultimately resulting in the development of adventitious shoots (Figure 7G).

4. Discussion

In this research, the study was conducted to set up an efficient and rapid regeneration program using the leaves of adult large branches and budding shoots of T. sebifera. A significant effect of the type of medium, type, and concentration of plant growth regulator, source of explant, and method of incision on the regeneration response is observed.
The selection of initial explants is the first step and a critical part in establishing an efficient in vitro regeneration system [37,38,39]. Ideal explants should be both easily accessible and possess strong regenerative properties. The success rate of in vitro induction varies greatly depending on the explant type [2,40]. Previous studies on T. sebifera regeneration have predominantly utilized stem segments as explants [8,9,15,26], of which some studies have reported that the stem segments of the middle non-woody portion of T. sebifera sprouting sapodermis are optimal explant materials [24], with a low reproduction coefficient. In the present work, adventitious shoot output was as high as 72 buds/explant using leaves from suckers-derived nodal cuttings as explants. Although leaves are highly differentiated tissues that are generally difficult to regenerate in vitro, we successfully established an efficient regeneration system for T. sebifera by optimizing the basal medium, plant growth regulators (PGRs), and incision methods for adventitious shoot induction using nodal cuttings from both young stems and sprouting leaves as explants. During the collection of T. sebifera leaf explants, we observed that young stem-derived nodal cutting sprouting occurred later than suckers-derived nodal cutting, and the adventitious buds from tiller sprouting were more robust and vigorous compared to those from young stem-derived nodal cutting (Figure 1D,G). In our study, we observed that sucker-derived explants exhibited a superior regeneration capacity compared to branch-derived explants. We propose several possible reasons for this observed superiority. Firstly, suckers grow at the base of mature trees where the nutrient and water supply is richer, leading to more vigorous growth and enhanced regeneration. Secondly, suckers are located close to large vascular bundles, possibly resulting in higher endogenous hormone levels than distal branches. This result is also consistent with the results of earlier findings [24]. This study is the first to report that suckers-derived nodal cutting exhibit stronger regenerative capacity than young stem-derived nodal cutting in T. sebifera. This result also provides valuable theoretical support for molecular breeding applications, including in vitro seedling production, transgenesis, and gene editing, not only for T. sebifera but also for other tree species.
Different tissues and organs from the same plant are differently adapted to the basic medium, making the selection of a suitable basic medium crucial for the successful regeneration of T. sebifera. In this study, we compared the efficacy of three basal media—WPM, MS, and DCR—in inducing adventitious shoots from T. sebifera leaves. It was observed that MS medium exhibited the highest percentage of induced adventitious shoots and number of average adventitious shoots among the three types of mediums. WPM is widely used for woody plant tissue culture [9,15,16,26]. However, its nutritional composition differs from MS medium in trace elements. Specifically, WPM has lower and less balanced trace element content, leading to reduced shoot regeneration rates and fewer shoots per explant. The DCR failed to provide adequate levels of essential nutrients and trace elements, thus inhibiting shoot induction in broad-leaved plants like T. sebifera. The component ratios in the DCR also failed to fully meet the requirements of T. sebifera compared to the MS, which performed more ideally. In the DCR, studies on specific plant species such as creosote bush have shown significant results [41]. At the same time, it may be less effective in inducing adventitious shoots due to its unsuitable composition for the biological characteristics of T. sebifera. In contrast, the MS medium, with its relatively rich sources of nitrogen, phosphorus, and trace elements, effectively meets the nutritional demands of plant cells during adventitious shoot induction [42]. Previous studies have utilized various media for T. sebifera regeneration, including MS, WPM, and DCR. Chen et al. [2] reported moderate success using MS medium but noted that regeneration rates were significantly lower than in our findings. Similarly, studies [9,16] indicated that while WPM was effective, it did not achieve the same levels of shoot multiplication as seen with MS medium in our work. And, according to Hou et al. [30], the MS medium is also more suitable for the regeneration of T. sebifera leaves, a conclusion supported by the results of this study (Table 1). In summary, the varying effectiveness of different basal media in inducing adventitious shoots in T. sebifera leaves underscores the importance of medium composition diversity in plant regeneration [43].
During the present investigation, the contribution provided by the cytokinin KT in the induction of adventitious shoots was explored. KT, as a kind of cytokinin, can effectively promote the division and expansion of plant cells. Adventitious shoot induction increased gradually with growing KT concentration at 1 mg/L for 6-BA and 0.1 mg/L for NAA. It suggests that the elevation of KT may enhance the activity of cell division under the condition that the concentrations of 6-BA and NAA remain stable, which in turn increases the induced rate of adventitious shoots [44]. Higher induction rates usually imply enhanced regeneration of meristematic tissues, demonstrating that KT plays a facilitating role in this process [45]. Synergistic effects of cytokinins and growth factors, especially under the joint influence with 6-BA and NAA, created a favorable environment for the formation of adventitious shoots [46,47]. However, with continued increase in KT concentration, the average number of adventitious shoots significantly decreased although the rate of adventitious shoot induction still increased. It could be a potential inhibitory effect of cytokinin. When KT concentration reaches a certain threshold, it may trigger a negative feedback response of plant cells to hormone excess, resulting in inhibition of cell division and regeneration processes [48].
In this paper, we conducted three treatments on the leaves, among which the cross-cut midvein leaves showed the best induced adventitious shoots (Figure 2B), with a high adventitious shoot induction rate of 98.89% (Table 3). Our findings indicated that the carving treatment promoted the regeneration response and improved the regeneration efficiency in leaf explants to a certain extent. This is also the same as the results in the ex vivo regeneration of some plant leaf explants [49,50,51]. After a short injury to the leaves, it may cause a significant augmentation in the levels of cytokinin and auxin in plants. The increase in these growth hormones promotes the emergence of adventitious shoots, indicating that cutting or injury of leaves can effectively activate the plant growth responses [41,52,53,54].
Plant growth regulators (PGRs) are a critical factor to induce adventitious shoot formation in plant tissue culture [55]. Studies have shown that combinations of different types and concentrations of PGRs significantly influences the induction of adventitious shoots in leaf explants [56]. In this work, we found that the combination of cytokinin and auxins markedly enhanced adventitious shoot induction (p < 0.05) compared to cytokinin on its own. Similar observations have been reported for other species within the Euphorbiaceae family, such as Jatropha [57,58]. It is shown that the synergistic action of growth hormones and cytokinins is essential for adventitious shoot induction. Although the addition of cytokinins and growth hormones to culture medium promotes adventitious shoot formation [48,59,60], the rate of adventitious shoot induction depends on the specific interactions between these growth regulators [61]. Previous studies have shown that 6-BA can promote the differentiation of non-differentiated tissues [62]. In this study, the frequency of adventitious shoot regeneration increased when KT concentration was 0.3 mg/L and higher concentration of 6-BA was added at constant NAA concentration. This indicates that increasing the 6-BA concentration significantly increased the adventitious shoot induction rate at insufficient KT concentration, which reached 96.67% at 2 mg/L. In contrast, high concentrations of cytokinin negatively affected leaf regeneration [48]. Consistent with this, we observed that the induction rate and the number of adventitious shoots were higher in the medium with lower concentrations of 6-BA (below 2 mg/L) compared to those with higher concentrations of 6-BA (>2 mg/L), aligning with findings by Chen and Dang [26,63]. In this study, the rate of induction and the average number of adventitious shoots gradually increased by increasing the concentration of 6-BA at a certain concentration of KT. This suggests that 6-BA as an indispensable hormone performs a key position in the formation of adventitious shoots, whereas KT assists in the process [45]. These findings highlight the importance of optimizing PGR combinations and concentrations to achieve efficient adventitious shoot induction in T. sebifera.
Adventitious shoot elongation is a critical phase in the leaf regeneration process, which directly affects plant growth and development. In general, higher concentrations of auxin-like regulators promote the elongation of adventitious shoots, as opposed to higher concentrations of cytokinin-like regulators that may inhibit this process [30,59]. From our studies, the elongation rate of adventitious shoots was found to be as high as 100%, with the highest mean shoot length (6.05 cm) observed in the MS medium supplemented with 0.3 mg/L 6-BA, 0.03 mg/L NAA and activated charcoal (AC). This may be attributed to the role of cytokinins in promoting cell expansion and division, thereby accelerating shoot growth [61]. In this study, activated charcoa played a crucial role in enhancing the efficiency of our tissue culture process. Specifically, activated charcoa was used to rapidly reduce cytokinin levels in adventitious buds, which is essential for shortening the elongation culture period. Moreover, AC effectively adsorbed harmful phenolic compounds and other toxins that are commonly released during tissue culture [16,36,64]. Additionally, activated charcoal was used mainly to reduce endogenous cytokinin levels in the regenerated shoots, thereby creating a more favorable physiological condition for subsequent adventitious root induction. Rooting of adventitious shoots represents the final and crucial step in leaf regeneration, which is directly related to the survival rate and growth of plants. For woody plants, achieving effective rooting remains one of the most challenging aspects of the cultivation process. Though all rooting responses in this study were good, the best rooting effect was achieved when 0.3 mg/L IBA was added to MS. Consistent with our findings, several studies have reported the effectiveness of IBA for rooting of many isolated regenerated plant species, such as Artemisia vulgaris, Larix olgensis [41,65]. These results underscore the importance of optimizing growth regulator concentrations and medium composition to enhance adventitious shoot elongation and rooting in T. sebifera.
In this study, we describe the in vitro formation of adventitious buds from leaf explants of Triadica sebifera (Chinese tallow). Adventitious bud formation through organogenesis involves a process of dedifferentiation, wherein mature plant cells undergo a reversible transition from a differentiated state to a meristematic stage [66,67]. Histological studies provide valuable insights into plant development by revealing cellular and tissue-level changes during in vitro regeneration [63,68,69]. Through histological analysis of leaf explants, we observed direct organogenesis, as adventitious shoots directly from the leaf explants without visible or histological evidence of callus formation. In contrast, other explants such as mature woody stem segments or leaves mostly regenerate via indirect organogenesis involving callus formation, which may delay regeneration and reduce efficiency [63,66]. Furthermore, the vascular tissues of the adventitious shoots were found to be connected to those of the leaf explants, with no intermediate callus observed. Histological examination of the various developmental stages of adventitious shoots confirmed direct organogenesis, further highlighting the superior regeneration capacity of sprouting buds.

5. Conclusions

In this study, an efficient and comprehensive organ regeneration system (Figure 8) for T. sebifera was established, using leaves as explants. The regenerative capacity of leaves from nodal cuttings derived from both young stems and sprouts of mature T. sebifera trees was systematically compared. The effects of plant type, cytokinin concentrations, and incision methods on the regeneration efficiency of T. sebifera were investigated. We also evaluated histologically the developmental stages of adventitious shoots induced by leaves from sucker-derived nodal cutting. The cellular and tissue-level changes observed during adventitious shoot formation provide valuable insights that can be leveraged to enhance the rate of new shoot regeneration. Moreover, this optimized regeneration system not only facilitates the production of high-quality plantlets for large-scale breeding but also supports advanced plant improvement technologies, including genetic transformation and gene editing.

Author Contributions

Investigation, Methodology, Validation, Formal analysis, Writing—original draft, Writing—review and editing, Y.C.; Methodology, Software, Validation, Formal analysis, Investigation, Y.X.; Conceptualization, Methodology, Investigation, K.Z.; Formal analysis, Investigation, Y.F.; Software, Investigation, H.Z.; Conceptualization, Funding acquisition, Investigation, Supervision, M.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by National Key Research and Development Program of China (2023YFD2200604) and Special Fund for Scientific Research of Building National Botanical Garden (XM04-10).

Data Availability Statement

All data generated or analyzed during this study are included in this published article.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

MS medium, Murashige and Skoog medium; DCR, Douglas fir cotyledon revised medium; WPM, wood plant medium; 6-BA, 6-Benzyladenine; IBA, Indole-3-butyric acid; KT, Kinetin; NAA, a-naphthaleneacetic acid; PGRs, Plant growth regulators; Active charcoal, AC.

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Figure 1. Acquisition of sterile leaves. (A) T. sebifera adult tree, bar = 120 cm. (B) Young branch, bar = 1.50 cm. (C) Suckers-derived nodal cutting, bar = 6.00 cm. (D–F) Stem segment from young stem-derived nodal cutting cultured in MS medium: (D)—for 21 d, bar = 1.00 cm; (E)—for 35 d, bar = 1.00 cm; (F)—for 28 d, bar = 1.00 cm. (G–I) Stem segments from suckers-derived nodal cutting cultured in MS medium: (G)—for 14 d, bar = 1.00 cm, (H)—for 35 d, bar = 1.00 cm, (I)—for 28 d, bar = 1.00 cm.
Figure 1. Acquisition of sterile leaves. (A) T. sebifera adult tree, bar = 120 cm. (B) Young branch, bar = 1.50 cm. (C) Suckers-derived nodal cutting, bar = 6.00 cm. (D–F) Stem segment from young stem-derived nodal cutting cultured in MS medium: (D)—for 21 d, bar = 1.00 cm; (E)—for 35 d, bar = 1.00 cm; (F)—for 28 d, bar = 1.00 cm. (G–I) Stem segments from suckers-derived nodal cutting cultured in MS medium: (G)—for 14 d, bar = 1.00 cm, (H)—for 35 d, bar = 1.00 cm, (I)—for 28 d, bar = 1.00 cm.
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Forests 16 00992 g002
Figure 2. Effect of basal medium on regeneration of T. sebifera. (A–C) Stem segments from suckers-derived nodal cutting cultured in different basic medium: (A)—in WPM basic medium, bar = 0.20 cm; (B)—in MS basic medium, bar = 0.20 cm; (C)—in DCR basic medium, bar = 0.20 cm. (D–F) Leaves from suckers-derived nodal cutting in different basic medium: (D)—in WPM basic medium, bar = 0.20 cm; (E)—in MS basic medium, bar = 0.20 cm; (F)—in DCR basic medium, bar = 0.20 cm.
Figure 2. Effect of basal medium on regeneration of T. sebifera. (A–C) Stem segments from suckers-derived nodal cutting cultured in different basic medium: (A)—in WPM basic medium, bar = 0.20 cm; (B)—in MS basic medium, bar = 0.20 cm; (C)—in DCR basic medium, bar = 0.20 cm. (D–F) Leaves from suckers-derived nodal cutting in different basic medium: (D)—in WPM basic medium, bar = 0.20 cm; (E)—in MS basic medium, bar = 0.20 cm; (F)—in DCR basic medium, bar = 0.20 cm.
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Figure 3. Effect of incision method on the induction of adventitious shoots in T. sebifera leaves. (A–C) Leaf explants from suckers-derived nodal cutting with different incision method were cultured in MS medium with 2 mg/L 6-BA and 0.2 mg/L NAA for 21 d: (A)—transverse leaf explants, bar = 1.00 cm; (B)—whole leaf explants, bar = 1.00 cm; (C)—leafless explants, bar = 0.50 cm. (D–F) Leaf explants from young stem-derived nodal cutting were cultured in MS medium with 2 mg/L 6-BA and 0.2 mg/L NAA for 21 d: (D)—transverse leaf explants, bar = 1.00 cm; (E)—whole leaf explants, bar = 1.00 cm; (F)—leafless explants, bar = 0.30 cm.
Figure 3. Effect of incision method on the induction of adventitious shoots in T. sebifera leaves. (A–C) Leaf explants from suckers-derived nodal cutting with different incision method were cultured in MS medium with 2 mg/L 6-BA and 0.2 mg/L NAA for 21 d: (A)—transverse leaf explants, bar = 1.00 cm; (B)—whole leaf explants, bar = 1.00 cm; (C)—leafless explants, bar = 0.50 cm. (D–F) Leaf explants from young stem-derived nodal cutting were cultured in MS medium with 2 mg/L 6-BA and 0.2 mg/L NAA for 21 d: (D)—transverse leaf explants, bar = 1.00 cm; (E)—whole leaf explants, bar = 1.00 cm; (F)—leafless explants, bar = 0.30 cm.
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Figure 4. Effect of leaf source on adventitious shoot induction. (A–C) Transverse midvein-cut leaves from sucker-derived nodal cutting cultured on MS medium supplemented with 2 mg/L 6-BA and 0.2 mg/L NAA: (A)—for 35 d, bar = 0.50 cm; (B)—bottom view of (A), bar = 0.50 cm; (C)—for 45 d, bar = 0.50 cm. (D–F) Transverse midvein-cut leaves from young stem-derived nodal cutting cultured on MS medium with 2 mg/L 6-BA and 0.2 mg/L NAA: (D)—for 35 d, bar = 0.50 cm; (E)—bottom view of (D), bar = 0.50 cm; (F)—for 45 d, bar = 0.50 cm.
Figure 4. Effect of leaf source on adventitious shoot induction. (A–C) Transverse midvein-cut leaves from sucker-derived nodal cutting cultured on MS medium supplemented with 2 mg/L 6-BA and 0.2 mg/L NAA: (A)—for 35 d, bar = 0.50 cm; (B)—bottom view of (A), bar = 0.50 cm; (C)—for 45 d, bar = 0.50 cm. (D–F) Transverse midvein-cut leaves from young stem-derived nodal cutting cultured on MS medium with 2 mg/L 6-BA and 0.2 mg/L NAA: (D)—for 35 d, bar = 0.50 cm; (E)—bottom view of (D), bar = 0.50 cm; (F)—for 45 d, bar = 0.50 cm.
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Figure 5. Adventitious shoot elongation of sprouting shoot leaves from sucker-derived nodal cutting. (A) The adventitious shoots were directly induced by adding 2 mg/L 6-BA and 0.2 mg/L NAA to the MS medium of the sprouting shoot leaves, bar = 0.50 cm. (B) The shoot clusters induced in A were taken and elongated in MS medium supplemented with 0.3 mg/L IBA, bar = 0.80 cm. (C) Shoot clusters were elongated in MS medium supplemented with 0.3 mg/L IBA and AC, bar = 1.00 cm. (D) Further elongation of shoots, bar = 1.00 cm.
Figure 5. Adventitious shoot elongation of sprouting shoot leaves from sucker-derived nodal cutting. (A) The adventitious shoots were directly induced by adding 2 mg/L 6-BA and 0.2 mg/L NAA to the MS medium of the sprouting shoot leaves, bar = 0.50 cm. (B) The shoot clusters induced in A were taken and elongated in MS medium supplemented with 0.3 mg/L IBA, bar = 0.80 cm. (C) Shoot clusters were elongated in MS medium supplemented with 0.3 mg/L IBA and AC, bar = 1.00 cm. (D) Further elongation of shoots, bar = 1.00 cm.
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Figure 6. Rooting and acclimatization. (A) Rooting of in vitro regenerated shoots from sucker-derived nodal cutting in MS medium supplemented with 0.3 mg/L IBA, bar = 1.00 cm. (B) Roots of fully developed plant, bar = 1.00 cm. (C) Acclimatized potted plant, bar = 2.00 cm.
Figure 6. Rooting and acclimatization. (A) Rooting of in vitro regenerated shoots from sucker-derived nodal cutting in MS medium supplemented with 0.3 mg/L IBA, bar = 1.00 cm. (B) Roots of fully developed plant, bar = 1.00 cm. (C) Acclimatized potted plant, bar = 2.00 cm.
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Figure 7. Histological assessment and analysis of in vitro regeneration of T. sebifera leaves from sucker-derived nodal cutting. (A) Early stage of development of adventitious shoots (swelling). (B) Meristem initiation, with meristem clumps containing a small area of dense small cells. (C) Globular meristem clumps; (D) Pre- and meristem nodules. (E) Formation of vascular tissues. (F) Formation of near-surface bud progenitors. (G) Fully developed shoots, with bud progenitor and leaf progenitor/healing tissue surface forming developmentally Well-developed adventitious shoots. (Red arrows show small protrusions on the surface of the meristematic tissue mass. Lp, leaf primordia; Am, apical meristem; Mn, meristematic nodule; Ac, annular catheter; Mc, meristematic cell; Ep, epidermis).
Figure 7. Histological assessment and analysis of in vitro regeneration of T. sebifera leaves from sucker-derived nodal cutting. (A) Early stage of development of adventitious shoots (swelling). (B) Meristem initiation, with meristem clumps containing a small area of dense small cells. (C) Globular meristem clumps; (D) Pre- and meristem nodules. (E) Formation of vascular tissues. (F) Formation of near-surface bud progenitors. (G) Fully developed shoots, with bud progenitor and leaf progenitor/healing tissue surface forming developmentally Well-developed adventitious shoots. (Red arrows show small protrusions on the surface of the meristematic tissue mass. Lp, leaf primordia; Am, apical meristem; Mn, meristematic nodule; Ac, annular catheter; Mc, meristematic cell; Ep, epidermis).
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Forests 16 00992 g008
Figure 8. An efficient leaf regeneration system from sucker-derived nodal cutting of T. sebifera.
Figure 8. An efficient leaf regeneration system from sucker-derived nodal cutting of T. sebifera.
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Table 1. Effect of basic medium on the induction of adventitious shoots.
Table 1. Effect of basic medium on the induction of adventitious shoots.
OrderBasal MediumLeaf Explants from Suckers-Derived Nodal CuttingLeaf Explants from Young Stem-Derived Nodal Cutting
Shoot Regeneration Frequency (%)Shoots Per
Explant		Shoot Regeneration Frequency (%)Shoots Per
Explant
1WPM80.00 ± 1.00 b25.00 ± 1.00 b55.17 ± 0.76 a13.33 ± 0.58 b
2MS81.83 ± 0.29 a27.50 ± 0.50 a56.50 ± 0.87 a21.83 ± 0.77 a
3DCR74.83 ± 0.76 c15.50 ± 0.87 c51.93 ± 0.90 b11.17 ± 0.29 c
Means with the same letter in the same column were tested for statistically insignificant differences by Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± SD of three independent experiments; each independent experiment contains 30 explants.
Table 2. Effect of different KT concentrations on the induction of adventitious shoots.
Table 2. Effect of different KT concentrations on the induction of adventitious shoots.
OrderThe Concentration of KTLeaf Explants from Suckers-Derived Nodal CuttingLeaf Explants from Young Stem-Derived Nodal Cutting
Shoot Regeneration Frequency (%)Shoots Per
Explant		Shoot Regeneration Frequency (%)Shoots Per
Explant
1KT 0:BA 1.0:NAA 0.176.00 ± 1.00 e15.67 ± 0.58 d54.17 ± 0.76 e12.17 ± 1.04 e
2KT 0.1:BA 1.0:NAA 0.179.50 ± 0.50 d24.00 ± 1.00 c55.83 ± 0.76 d15.50 ± 0.50 d
3KT 0.2:BA 1.0:NAA 0.180.83 ± 0.29 c27.50 ± 0.50 b57.50 ± 0.50 c19.17 ± 0.76 b
4KT 0.3:BA 1.0:NAA 0.183.67 ± 0.58 b29.67 ± 0.58 a59.67 ± 0.58 b22.83 ± 0.76 a
5KT 0.4:BA 1.0:NAA 0.186.50 ± 0.50 a28.50 ± 0.50 ab61.17 ± 0.29 a20.00 ± 1.00 b
6KT 0.5:BA 1.0:NAA 0.187.00 ± 1.00 a24.83 ± 0.76 c61.33 ± 0.58 a17.67 ± 0.58 c
Means with the same letter in the same column were tested for statistically insignificant differences by Duncan’s multiple comparisons test (p < 0.05). Each value represent the mean ± SD of three independent experiments, each independent experiment contains 30 explants.
Table 3. Effect of different leaf types on the regeneration of T. sebifera leaves.
Table 3. Effect of different leaf types on the regeneration of T. sebifera leaves.
Explant TypeLeaf Explants from Suckers-Derived Nodal CuttingLeaf Explants from Young Stem-Derived Nodal Cutting
Shoot Regeneration Frequency (%)Shoots Per
Explant		Shoot Regeneration Frequency (%)Shoots Per
Explant
Whole leaf53.33 ± 1.92 c23.75 ± 1.25 c38.89 ± 1.11 c20.42 ± 1.76 c
Transected midvein blades98.89 ± 1.11 a71.75 ± 1.64 a84.44 ± 1.11 a64.00 ± 1.50 a
Leaf without margin85.56 ± 1.11 b51.92 ± 1.38 b70.00 ± 1.92 b45.08 ± 1.63 b
Means with the same letter in the same column were tested for statistically insignificant differences by Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± SD of three independent experiments; each independent experiment contains 30 explants.
Table 4. Effects of plant growth regulators on regeneration of isolated leaves from nodal cuttings from both young stems and sprouts.
Table 4. Effects of plant growth regulators on regeneration of isolated leaves from nodal cuttings from both young stems and sprouts.
OrderPlant Growth Regulators (mg/L)Leaf Explants from Suckers-Derived Nodal CuttingLeaf Explants from Young Stem-Derived Nodal Cutting
Shoot Regeneration Frequency (%)Shoots Per
Explant		Shoot Regeneration Frequency (%)Shoots Per Explant
16-BA 0:NAA 0:KT0.30.00 ± 0.00 g0.00 ± 0.00 m0.00 ± 0.00 j0.00 ± 0.00 l
26-BA 1.0:NAA 0:KT0.387.78 ± 1.92 b35.08 ± 0.14 g66.67 ± 3.33 ef28.25 ± 0.25 f
36-BA 1.0:NAA 0.1:KT0.390.00 ± 3.33 b40.33 ± 0.14 e71.11 ± 1.92 cd33.5 ± 0.25 d
46-BA 1.0:NAA 0.2:KT 0.391.11 ± 1.92 b45.00 ± 0.25 d83.33 ± 3.33 a33.42 ± 0.14 d
56-BA 1.0:NAA 0.3:KT0.380.00 ± 3.33 c30.83 ± 0.14 h63.33 ± 3.33 f23.75 ± 0.25 h
66-BA 2.0:NAA 0:KT0.396.67 ± 3.33 a49.83 ± 0.14 c72.22 ± 1.92 c40.00 ± 0.25 c
76-BA 2.0:NAA 0.1:KT0.397.78 ± 1.92 a60.08 ± 0.14 b77.78 ± 1.92 b51.92 ± 0.14 b
86-BA 2.0:NAA 0.2:KT0.398.89 ± 1.92 a72.00 ± 0.00 a84.44 ± 1.92 a64.00 ± 0.00 a
96-BA 2.0:NAA 0.3:KT0.391.11 ± 1.92 b37.67 ± 0.14 f67.78 ± 1.92 de29.50 ± 0.25 e
106-BA 3.0:NAA 0:KT0.353.33 ± 3.33 e23.75 ± 0.25 k38.89 ± 1.92 i20.25 ± 0.25 j
116-BA 3.0:NAA 0.1:KT0.365.56 ± 1.92 d26.75 ± 0.00 j44.44 ± 1.92 h22.58 ± 0.14 i
126-BA 3.0:NAA 0.2:KT0.367.78 ± 1.92 d30.25 ± 0.25 i53.33 ± 3.33 g25.00 ± 0 g
136-BA 3.0:NAA 0.3:KT0.348.89 ± 1.92 f20.75 ± 0.25 l35.56 ± 1.92 i17.00 ± 0.25 k
Means with the same letter in the same column were tested for statistically insignificant differences by Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± SD of three independent experiments; each independent experiment contains 30 explants.
Table 5. Effects of different concentrations of 6-BA and NAA on the elongation of adventitious shoots of T. sebifera leaves.
Table 5. Effects of different concentrations of 6-BA and NAA on the elongation of adventitious shoots of T. sebifera leaves.
Plant Growth Regulators with/Without AC (mg∙L−1)Average Shoot Length (cm)Rate of Elongation (%)
6-BANAA
0.10.014.68 ± 0.05 f66.67 ± 0.58 f
+AC4.87 ± 0.05 e76.67 ± 0.58 e
0.20.025.16 ± 0.05 d85.57 ± 0.33 d
+AC5.50 ± 0.04 c91.10 ± 0.33 c
0.30.035.80 ± 0.04 b95.57 ± 0.33 b
+AC6.05 ± 0.06 a100.00 ± 0.00 a
0.40.044.65 ± 0.04 f65.57 ± 0.33 f
+AC4.88 ± 0.05 e74.43 ± 0.33 e
0.50.053.67 ± 0.06 h55.57 ± 0.33 h
+AC3.94 ± 0.05 g61.10 ± 0.33 g
Means with the same letter in the same column were tested for statistically insignificant differences by Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± SD of three independent experiments; each independent experiment contains 30 explants.
Table 6. Effect of MS and WPM supplemented with IBA on root induction.
Table 6. Effect of MS and WPM supplemented with IBA on root induction.
Plant Growth
Regulators (mg/L)		Adventitious
Root Induction
Percentage (%)		Rooting Start
Time (d)		No. of Roots Per Shoot
WPMMSWPMMSWPMMS
016.67 ± 3.34 e20.00 ± 3.34 e16.33 ± 0.58 a13.33 ± 0.58 a3.33 ± 0.58 e4.00 ± 1.00 e
0.167.78 ± 1.92 c81.11 ± 1.92 c11.00 ± 1.00 cd7.33 ± 0.58 c8.67 ± 0.58 c9.33 ± 0.58 c
0.276.67 ± 3.33 b88.89 ± 1.92 b10.00 ± 1.00 de6.00 ± 1.00 d10.00 ± 1.00 b11.33 ± 0.58 b
0.384.44 ± 1.92 a98.89 ± 1.92 a9.00 ± 1.00 e4.67 ± 0.58 e11.67 ± 0.58 a14.00 ± 1.00 a
0.465.56 ± 1.92 c78.89 ± 1.92 c12.00 ± 1.00 c8.33 ± 0.58 c7.67 ± 0.58 c8.67 ± 0.58 c
0.561.11 ± 1.92 d73.33 ± 3.34 d13.67 ± 0.58 b10.67 ± 0.58 b6.33 ± 0.58 d7.00 ± 1.00 d
Means with the same letter in the same column were tested for statistically insignificant differences by Duncan’s multiple comparisons test (p < 0.05). Each value represents the mean ± SD of three independent experiments; each independent experiment contains 30 explants.
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Chen, Y.; Xie, Y.; Zheng, K.; Fan, Y.; Zhou, H.; Zhu, M. Regeneration Capability Comparison of Leaves Between Nodal Cuttings from Young Stems and Suckers and Its Histological Analysis in Triadica sebifera. Forests 2025, 16, 992. https://doi.org/10.3390/f16060992

AMA Style

Chen Y, Xie Y, Zheng K, Fan Y, Zhou H, Zhu M. Regeneration Capability Comparison of Leaves Between Nodal Cuttings from Young Stems and Suckers and Its Histological Analysis in Triadica sebifera. Forests. 2025; 16(6):992. https://doi.org/10.3390/f16060992

Chicago/Turabian Style

Chen, Yuan, Yumei Xie, Keyuan Zheng, Yanru Fan, Huijing Zhou, and Mulan Zhu. 2025. "Regeneration Capability Comparison of Leaves Between Nodal Cuttings from Young Stems and Suckers and Its Histological Analysis in Triadica sebifera" Forests 16, no. 6: 992. https://doi.org/10.3390/f16060992

APA Style

Chen, Y., Xie, Y., Zheng, K., Fan, Y., Zhou, H., & Zhu, M. (2025). Regeneration Capability Comparison of Leaves Between Nodal Cuttings from Young Stems and Suckers and Its Histological Analysis in Triadica sebifera. Forests, 16(6), 992. https://doi.org/10.3390/f16060992

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