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Article

Influence of Chlorella sorokiniana and Plant Growth Regulators During the Micropropagation of Callicarpa peichieniana

by
Yiteng Zhang
1,2,
Manna Guo
1,2,
Jinfeng Xu
1,2,
Yuping Xiong
1,
Junyu Liu
1,2,
Guohua Ma
1,
Songjun Zeng
1,
Kunlin Wu
1,* and
Lin Fang
1,*
1
Guangdong Provincial Key Laboratory of Applied Botany, South China Botanical Garden, Chinese Academy of Sciences, Guangzhou 510650, China
2
University of Chinese Academy of Sciences, Beijing 100049, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(9), 1016; https://doi.org/10.3390/horticulturae11091016
Submission received: 17 July 2025 / Revised: 18 August 2025 / Accepted: 25 August 2025 / Published: 27 August 2025
(This article belongs to the Section Propagation and Seeds)
Browse Figures
Versions Notes

Abstract

Callicarpa peichieniana is an important traditional Chinese medicinal plant with pharmacological benefits for digestive system diseases and wounds, as well as high ornamental value. The goal of this study is to establish an effective in vitro regeneration system in order to satisfy the expanding market demand. Extracts from algae can enhance the proliferation and rooting effect of adventitious buds and can improve the survival rate of transplantation. This study developed an in vitro regeneration system using apical bud explants of C. peichieniana associated with Chlorella sorokiniana (an alga species). Inter simple sequence repeat (ISSR) molecular markers confirmed the genetic fidelity of the regenerated plantlets. The highest number of adventitious buds (5.00 buds) was induced from the apical buds with 0.5 mg/L 6-BA in a Murashige and Skoog (MS) medium, and the highest proliferation coefficient (5.83) was achieved with 2.0 mg/L 6-BA. A rooting rate of 100% was achieved by using 0.1 mg/L NAA, MS with 50% macroelements, and 20 g/L sucrose, averaging 6.36 roots per explant and a root length of 1.32 cm. In all micropropagation stages, C. sorokiniana coexisted and proliferated alongside C. peichieniana materials. ISSR showed that the genetic fidelity of C. peichieniana regenerated plants was 95.45%. Coconut coir/perlite = 1∶1 (v/v) was identified as the optimal transplantation substrate, achieving a 100% survival rate. The “C. peichieniana–C. sorokiniana association” in vitro regeneration system established in this study not only enables the mass production of high-quality regenerated plantlets but provides new ideas and demonstrations for culturing multiple species in the same in vitro system.

1. Introduction

Callicarpa peichieniana is a Chinese endemic plant belonging to the Callicarpa of Verbenaceae (Engler system) [1,2]. Its leaves are rhombic–elliptical, and the fruits turn purplish–red upon maturation. The plant is distributed in temperate to subtropical regions and grows on hills, mountains, and in valleys at an altitude of 200–700 m. Guangdong, Guangxi, and Hunan are the main distribution areas in China [1,2]. The species has medicinal, ornamental, economic, and scientific research value. C. peichieniana is used in the treatment of digestive system diseases [3], and its leaves are applied topically to treat wounds [4]. Phosphodiesterase 4 (PDE4) is a key enzyme in the degradation of cyclic adenosine monophosphate (cAMP), and PDE4 inhibitors are considered to be important targets for a variety of inflammatory diseases by inhibiting PDE4 enzyme activity and increasing cAMP levels [5]. C. peichieniana contains components that inhibit the activity of PDE4. When its crude drug concentration reached 6.46 g/L, it showed a 95.70% inhibition rate against the D2 subtype target of PDE4 (PDE4D2), indicating its potential as a novel PDE4 inhibitor and its involvement in the treatment of inflammatory diseases, such as asthma, intestinal inflammation, and arthritis [5]. Additionally, it is a popular landscaping plant and one of the dominant shrub species in the evergreen broadleaf forest community of the Heishiding Nature Reserve, Zhaoqing, China [6]. Therefore, C. peichieniana holds significant potential for medicinal market applications and resource utilization.
However, wild C. peichieniana resources exhibit low reproductive capacity in the natural environment, and the overharvesting of the plant has led to a sharp decline in its yield. At present, C. peichieniana is mainly propagated by seeds and cutting propagation, but the tissue culture technology has not been applied on a large scale. The seed propagation coefficient of C. peichieniana is low, the growth cycle is long, and it is highly susceptible to environmental factors [7]. Asexual propagation in most Callicarpa species is primarily achieved through cuttings, but the inconsistent growth of cuttings hinders large-scale production, making it difficult to satisfy the increasing market demand. Plant tissue culture techniques can significantly improve the propagation coefficients and efficiently produce healthy, high-quality plantlets in a short period, alleviating the pressure on wild populations due to medicinal use [8]. Therefore, establishing an in vitro regeneration system through plant tissue culture is particularly suitable for the conservation of the C. peichieniana germplasm and satisfying the demands of high economic value.
Inducing plant regeneration through adventitious buds serves as a key approach for important germplasm conservation and propagation. Despite the existence of established in vitro regeneration protocols for multiple Callicarpa species, such as C. pedunculata [9], C. nudiflora [10], C. macrophylla [11], C. bodinieri [12], C. japonica [13], C. dichotoma [14], and C. mollis [15], no regeneration system has been reported for C. peichieniana to date. The regeneration efficiency of plants is influenced by explant, basic medium, plant growth regulators (PGRs), carbon source, and transplantation substrate [16]. Due to the totipotency of plant cells, adventitious buds can be induced from any part of the plant. Stem segments are the primary explants for adventitious bud induction in Callicarpa [9,10,12,17], followed by new buds [13,18]. The basic medium is integral to the entire tissue culture process, with a Murashige and Skoog (MS) medium [19] widely used for adventitious bud induction and proliferation, and half MS medium commonly used for rooting [20]. Most micropropagation studies of Callicarpa also adhere to this principle [12,14,21,22]. Sucrose serves as the carbon source in the medium, with typical concentrations ranging from 2% to 5% in plant tissue culture [23]. However, the existing Callicarpa micropropagation studies have not investigated the impact of explant type on adventitious buds induction, and the influence of specific medium components (such as macroelements and sucrose concentration) on adventitious root induction has not been considered.
The establishment of an ideal in vitro regeneration system primarily depends on the appropriate combination of PGRs. Different PGRs, such as auxins, cytokinins (CKs), gibberellins, ethylene, and abscisic acid, play a crucial role in determining outcomes, like morphogenesis, callus induction, and the regeneration of buds and roots, depending on their type and concentration [24]. The most commonly used PGRs in Callicarpa micropropagation are auxins and CKs. Most studies tend to use a combination of both. For example, C. nudiflora had optimal adventitious bud induction with 2.0 mg/L 6-Benzylaminopurine (6-BA) + 0.1–0.3 mg/L a-Naphthaleneacetic acid (NAA) [25], while 1.5 mg/L 6-BA and 0.1 mg/L NAA were effective for adventitious bud proliferation [22], and best rooting was achieved with 0.05 mg/L NAA + 0.5 mg/L Indole-3-butyric acid (IBA) [25]. C. macrophylla showed excellent adventitious bud induction and proliferation with 0.8 mg/L 6-BA + 0.02 mg/L IBA [21]. However, there are still differences in the PGR concentration effects among the different species within Callicarpa, and further specific investigations are required.
Transplantation is a key stage in in vitro propagation [26]. The transplantation substrate for Callicarpa mainly consists of a mixture of more than three substrates, including river sand, perlite, and vermiculite, but survival rates rarely reach 100% [12,27,28,29]. It should be noted that tissue culture could lead to genetic variation, and the genetic fidelity of in vitro regenerated plantlets must be determined. Currently, DNA molecular markers for genetic diversity analysis mainly include random amplified polymorphic DNA (RAPD) and inter simple sequence repeat (ISSR) [30,31]. ISSR offers more advantages, such as lower DNA requirements, simpler procedures, and greater polymorphism [32,33], making it more efficient than RAPD. To date, reports on the genetic fidelity evaluation of Callicarpa regenerated plantlets remain scarce.
Traditionally, plant tissue culture is carried out under sterile conditions with the goal of cultivating a single target. Furthermore, during the establishment of an in vitro regeneration system, it is essential to surface-disinfect explants to eliminate all harmful microorganisms. In this study, the target species, C. peichieniana, had Chlorella sorokiniana (an alga species) closely attached to its explant surface. Previous studies have shown that algae secrete various bioactive compounds, including PGRs, which significantly enhance adventitious bud proliferation, root formation, transplantation survival rate, as well as microtuber proliferation and fresh weight [34,35,36]. However, the existing studies have primarily focused on the application of algal extracts or filtrates, with no reported investigations on the effects of a live alga-associated culture in plant micropropagation. Thus, the in vitro regeneration system of C. peichieniana associated with C. sorokiniana was established for the first time based on the experience of Callicarpa tissue culture.
This study explored several factors affecting the in vitro regeneration of C. peichieniana associated with C. sorokiniana, including different explants, concentrations, and ratios of 6-BA, Thidiazuron (TDZ), NAA, and IBA, as well as the concentrations of macroelements and sucrose in the basic medium and transplantation substrate. The aim is to establish an efficient regeneration system and provide sufficient seedlings for the economic market and for scientific research. Additionally, an ISSR marker analysis was employed to assess the genetic fidelity of regenerated plantlets. Given the potential applications of C. peichieniana in various fields, the successful establishment of its in vitro regeneration system will lay the foundation for its germplasm research, resource development, in vitro conservation, and genetic breeding.

2. Materials and Methods

2.1. Explant Source, Decontamination, and Bulking

A 5-year-old C. peichieniana, from Heishiding Nature Reserve, Zhaoqing, China, is the original mother plant from which we obtained the explants for research. The mother plant was maintained in a greenhouse at the South China Botanical Garden, Chinese Academy of Sciences, Guangzhou, China. The new shoots of the same year were collected and used immediately. Their surfaces were gently wiped with cotton dipped in 75% (v/v) ethanol, followed by surface disinfection in a laminar flow cabinet. The shoots were immersed in 75% (v/v) ethanol for 30 s, washed once with sterile water, then soaked in 0.1% (w/v) HgCl2 containing 1 drop of Tween 20 (Merck KGaA, Darmstadt, Germany) for 5 min, and finally rinsed three times with sterile water. The shoots were dried with sterile filter paper, and the browned portions were removed. The shoots were inoculated on MS + 0.1 mg/L 6-BA. This disinfection method can obtain a large number of explants to meet the requirements of subsequent experiments. Microscopic examination revealed that C. sorokiniana is closely attached to the stems and leaves of the mother plant of C. peichieniana, and it is found to be difficult to remove when disinfecting explants. When explants were cultured on the MS medium supplemented with 0.1 mg/L 6-BA, both organisms showed active proliferation, yielding abundant C. peichieniana experimental materials associated with C. sorokiniana. Therefore, the explants used were associated with C. sorokiniana, and no C. sorokiniana extract or filtrate was added to the medium.
Unless otherwise specified, the medium used in the experiments was MS (Coolaber Technology Co. Ltd., Beijing, China) + 30 g/L sucrose (Guangshi Reagent Technology Co. Ltd., Guangzhou, China) + 4.8 g/L agar (Jige Biotechnology Co. Ltd., Guangzhou, China) + 5 mL/L coconut water, with pH adjusted to 5.80, sterilized under high pressure at 121 °C for 20 min. The culture containers were 250 mL glass bottles, and the cultivation temperature was maintained at 25 ± 2 °C under a 12 h/d light cycle using cool white, fluorescent lights with light intensity set to 40 μmol m−2 s−1.

2.2. Adventitious Bud Induction

The explants were divided into apical buds and stem segments (containing one node and two leaves) and inoculated onto the MS medium supplemented with different concentrations of 6-BA (0.1, 0.3, 0.5, and 1.0 mg/L). The MS medium without PGRs was used as the control treatment.
Average adventitious bud number = Total number of induced adventitious buds/Number of explants inducing adventitious buds.

2.3. Adventitious Bud Proliferation

Adventitious buds of similar growth were inoculated onto the MS medium containing various concentrations of TDZ (0.02, 0.04, 0.08 mg/L), 6-BA (1.5, 2.0, 3.0 mg/L), and combinations of 6-BA with NAA (0.1, 0.2, 0.4 mg/L). The MS medium without PGRs was used as the control treatment.
Adventitious bud proliferation coefficient = Total number of adventitious buds after proliferation/Number of inoculated explants.
Average adventitious bud height (cm) = Total height of proliferated adventitious buds/Total number of adventitious buds after proliferation.

2.4. Adventitious Root Induction

Different concentrations of IBA (0.1, 0.5, 1.0 mg/L) and NAA (0.05, 0.1, 0.2 mg/L) were added to the MS medium to investigate their effects on adventitious root induction, and the MS medium without PGRs was used as the control treatment. Macroelement concentrations were set at three levels (12.5%, 25%, and 50% MS), and 100% MS medium was used as the control treatment. Sucrose concentrations were tested at three levels (10, 20, and 30 g/L), and the MS medium without sucrose was used as the control treatment. The macroelements and sucrose experiment both included 0.05 mg/L NAA. Adventitious buds with a height of 3–4 cm were inoculated onto the aforementioned media.
Rooting rate (%) = Number of adventitious buds with induced adventitious roots/Number of inoculated adventitious buds × 100.
Average adventitious root number = Total number of induced adventitious roots/Number of adventitious buds with induced adventitious roots.
Average adventitious root length (cm) = Total length of induced adventitious roots/Number of adventitious buds with induced adventitious roots.

2.5. Acclimatization and Transplantation

The rooting regenerated plantlets with a height of 4–5 cm were placed in a greenhouse under strong light for one week and then removed from the culture bottle, and the culture medium of the roots was washed with tap water. Before transplantation, the base of the plant was soaked in 1.0 g/L carbendazim solution (Macklin, Shanghai, China) for 30 s, and then slightly dried and transplanted into the substrate. The transplantation substrate was set as peat, coconut coir, peat/perlite = 1∶1 (v/v), coconut coir/perlite = 1∶1 (v/v), peat/coconut coir/perlite = 1∶1∶1 (v/v/v).
Survival rate (%) = Number of viable plantlets/Number of transplanted plantlets × 100.

2.6. Genetic Fidelity Assessment Through ISSR Molecular Markers

The leaf samples of the mother plant and the regenerated plantlets were ground into powder in liquid nitrogen and then extracted by a high-efficiency plant genomic DNA extraction kit (Tsingke Biotechnology Co. Ltd., Beijing, China). The specific method followed the product instruction. A DNA quality detector microvolume spectrophotometer (K5600C, Kaiao Technology Development Co. Ltd., Beijing, China) was used to determine the concentration of DNA samples. The DNA sample was diluted to 20 ng/μL and stored at −20 °C for use. ISSR primers, which were designed by the University of British Columbia (UBC), were screened with reference to the studies of de Sena Filho et al. [37] and Julião et al. [38]. A total of 9 primers (UBC809, UBC818, UBC820, UBC830, UBC835, UBC842, UBC845, UBC848, UBC850) were finally selected for analysis and genetic fidelity was calculated. PCR amplification was performed using a 22 μL reaction mixture consisting of 1 μL genomic DNA (20 ng), 1 μL primer (10 μM), and 20 μL 1.1× T3 Super PCR Mix (Tsingke Biotechnology Co. Ltd., Beijing, China). The PCR procedure was as follows: initial denaturation at 98 °C for 2 min, followed by 34 cycles including 98 °C for 10 s, 50–53 °C (depending on the primers used) for 10 s, 72 °C for 10 s, and final extension at 72 °C for 5 min. The amplified PCR products were separated and detected on 1% agarose gel electrophoresis, and the gel was photographed and recorded by the gel recording system. The ISSR-PCR reaction was performed at least 3 times under the same PCR conditions to confirm the consistency of the DNA band pattern.
Genetic fidelity (%) = Total number of monomorphic bands/Total number of amplified fragments × 100.

2.7. Experimental Design and Data Analysis

Each treatment contained three replications with 12 explants each. The growth of cultures was monitored weekly and the data were collected after 4 weeks. Statistical analyses were performed using Excel 2019 and SPSS 19.0 software, including analysis of variance (ANOVA) followed by Tukey’s test (p < 0.05). Data are presented as mean ± standard error.

3. Results

3.1. Effect of Explant Types and 6-BA Concentrations on Adventitious Bud Induction

It can be seen from Table 1 that explant type and 6-BA had significant effects on adventitious bud induction. The growth of adventitious buds induced by apical buds was better than that induced by stem segments, and the average number of adventitious buds from apical buds was significantly higher than that from stem segments (Figure 1A). Under different 6-BA concentrations, there are significant differences in the average number of adventitious buds induced by apical buds, and the number was the highest at 0.5 mg/L 6-BA, which was 5.00, and the adventitious buds were thick with robust growth. However, there was no significant difference in the average number of adventitious buds induced by stem segments. Thus, the most suitable explant type and PGR treatment for adventitious bud induction were apical bud and 0.5 mg/L 6-BA.

3.2. Effect of 6-BA, TDZ and Combination of 6-BA and NAA Concentrations on Adventitious Bud Proliferation

After 4 weeks of culture, different numbers of adventitious buds were obtained on the proliferation medium with various types and concentrations of CKs and auxins. Both 6-BA (0.5–3.0 mg/L) and TDZ (0.02–0.08 mg/L) significantly enhanced adventitious bud proliferation when applied individually. However, 6-BA demonstrated markedly greater efficacy in stimulating adventitious bud proliferation compared to TDZ (Table 2). Obviously, the combination of NAA and 6-BA inhibited the proliferation (Figure 1B,C). Table 2 shows that the highest adventitious bud proliferation coefficient of 5.83 and an average height of adventitious buds of 0.73 cm were observed at 2.0 mg/L 6-BA across all treatments, the adventitious buds were thick and long with robust growth. In the combination of low NAA concentration with varying 6-BA levels, 0.1 mg/L NAA and 1.5 mg/L 6-BA resulted in a higher adventitious bud proliferation coefficient (3.84) and an average height of the adventitious buds (0.51 cm), which was similar to the effect observed with 0.02 mg/L TDZ, yielding an adventitious bud proliferation coefficient of 3.80 and an average height of adventitious buds of 0.52 cm. In conclusion, the most suitable PGR treatment for adventitious bud proliferation was 2.0 mg/L 6-BA.

3.3. Effect of IBA, NAA Concentrations on Adventitious Root Induction

The effects of different types and concentrations of auxins on adventitious root induction are shown in Table 3. After 4 weeks of culture, all treatments resulted in rooting of the adventitious buds, with significant differences in the average number and length of the adventitious roots. The rooting rate reached 100% under 0.1 mg/L and 0.2 mg/L NAA. In the IBA treatment, the average number and length of the adventitious roots decreased with the increasing IBA concentration. By contrast, the average number and length of the adventitious roots peaked at 0.1 mg/L and then began to decline in the NAA treatment (Table 3). Among all treatments, the best rooting rate, average number, and length of adventitious roots were observed under 0.1 mg/L NAA, reaching 100%, 6.36 roots, and 0.66 cm, respectively. The adventitious roots were thick with robust growth (Figure 1D).

3.4. Effect of Macroelements Concentrations on Adventitious Root Induction

Table 4 shows that the concentrations of macroelements in the MS basic medium significantly affected rooting. The rooting rate and the average number and length of adventitious roots reached the highest values, with 100%, 5.42 roots, and 1.32 cm, respectively, and then began to decline (Table 4). Thick adventitious roots and robust growth were observed under the MS medium with 50% macroelements (Figure 1E).

3.5. Effect of Sucrose Concentrations on Adventitious Root Induction

The results in Table 5 indicate that the concentration of sucrose in the MS basic medium had a significant effect on rooting. As the sucrose concentration increased to 20 g/L, the rooting rate and the average number and length of the adventitious roots showed a positive correlation with the sucrose concentration. Subsequently, the average number of adventitious roots decreased as the sucrose concentration increased, while the rooting rate and the average length of the adventitious roots slightly increased (Table 5). At a sucrose concentration of 20 g/L, the average number of adventitious roots (2.91) was the highest, and the rooting rate (88.89%) and average length of the adventitious roots (0.46 cm) showed no significant differences compared to the 30 g/L sucrose treatment (Table 5, Figure 1F).

3.6. Transplantation

For rooting regenerated plantlets, the growth status was greatly different in different transplantation substrates, and the survival rate was significantly different. Among all substrates, the survival rate of peat and coconut coir/perlite = 1∶1 (v/v) was 100%. After 4 months of transplantation, it was found that the survival rate in all substrates decreased due to plant infections and leaf chlorophyll loss, except for the coconut coir/perlite = 1∶1 (v/v), where the plantlets exhibited the best growth and robust root systems (Table 6, Figure 1G,H). Therefore, coconut coir/perlite = 1∶1 (v/v) can be selected as the preferred substrate for transplantation.

3.7. Genetic Fidelity Analysis by ISSR

In this study, the fidelity between the mother plant and the regenerated plantlets was evaluated by ISSR molecular markers. For all 9 ISSR primers, 1–4 bands were generated for each primer. A total of 22 bands were detected in 10 samples (1 mother plant and 9 regenerated plantlets), 1 band was polymorphic while the remaining 21 bands were monomorphic (Table 7), so the genetic fidelity was 95.45%. Up to four monomorphic bands can be amplified by primers UBC845 and UBC850 (Figure 2), the similar amplification profiles of the mother plant and the regenerated plantlets proved that there was no large variation between them, and genetically similar. Thus, the regenerated plantlets had high genetic fidelity.

4. Discussion

In the in vitro regeneration system of C. peichieniana, the type of explant, the composition of the basal medium, and the type, concentration, and ratio of PGRs are all key factors influencing regeneration. In this study, apical buds were found to be more suitable for adventitious bud induction than stem segments, which is consistent with the findings of Coelho et al. [39] on Aloysia gratisima. Both C. japonica [13] and C. nudiflora [18] successfully established their in vitro regeneration systems through the direct induction of adventitious buds from apical buds. This may be because apical buds have more meristematic tissue and stronger vitality. The meristem is the primary site for endogenous auxin synthesis, and its role is more prominent at the tip of the apical bud in cell division and elongation. The use of an appropriate concentration of CKs facilitates adventitious bud differentiation.
The culture medium provides the material foundation for plant tissue culture, with plants having different requirements at various developmental stages, especially the demand for macroelements and sucrose concentration during rooting. In rooting studies of the Verbena species, adventitious buds of Verbena officinalis rooted best in half MS + 2.0 mg/L IBA [40], while V. bonariensis showed a 100% rooting rate in MS + 0.05 mg/L IBA + 0.05 mg/L NAA [41]. In the Lippia species, excellent rooting characteristics were observed in both half MS and the MS media [42,43]. Sucrose provides energy for adventitious root formation. Marino et al. [44] found that Glandularia perakii had an 85.7% rooting rate at 20 g/L sucrose (without PGRs). C. mollis showed the highest rooting rate (100%) in a medium with 20 g/L sucrose [15]. The optimal rooting treatment obtained in this study was the same as the above experimental results, and it was in accordance with the low salt and low sugar principle during rooting. Combining the results of this study, macroelements mainly affect adventitious root induction efficiency by influencing the average number and length of the adventitious roots, while sucrose primarily affects the rooting rate and the average number of adventitious roots.
PGRs play a crucial role in plant tissue culture. In general, the ratio of auxin to CK controls the direction of plant morphogenesis. Auxins and CKs are versatile. A medium with high CKs and low auxins leads to shoot regeneration, while a medium with high auxins and low CKs leads to root regeneration [45]. In studies of the Callicarpa species, adventitious buds of C. dichotoma were induced by WPM + 1.0 mg/L 6-BA and then inoculated into MS + 1.0 mg/L 6-BA to obtain 4.3 adventitious buds [14]. C. macrophylla was induced and proliferated in the medium with 0.8 mg/L 6-BA + 0.02 mg/L IBA, achieving a high proliferation coefficient of 8–9 [21]. However, adding low concentrations of NAA to 6-BA inhibited the proliferation coefficient and height of the C. peichieniana adventitious buds, while the best induction and proliferation of adventitious buds was obtained when 6-BA was used alone. This may be because the explants contain some endogenous auxins, and additional auxins disrupt the PGRs balance. This study found that 0.5–3.0 mg/L 6-BA had a stronger effect on adventitious bud proliferation than 0.02–0.08 mg/L TDZ, consistent with the results of Huang et al. [10] on C. nudiflora. The effect of using 2.0 mg/L 6-BA to proliferate adventitious buds was better than that of zeatin (ZT), kinetin (KT), and TDZ at the same concentration [10,27]. The adventitious buds cultured with KT and TDZ grew poorly, and the leaves showed yellowing. The adventitious buds cultured with ZT were not neatly differentiated, and KT also led to vitrification of adventitious buds. This may be because the Callicarpa species are sensitive to ZT, KT, and TDZ.
This study showed that the addition of low concentrations of NAA and IBA to the medium can promote C. peichieniana rooting. In congeneric species, rooting was optimal at 0.05 mg/L NAA + 0.5 mg/L IBA for C. nudiflora, with a 100% rooting rate and robust root growth [25]. C. macrophylla induced four–six adventitious roots, with root lengths of 3–4 cm and a rooting rate above 90% in a medium with 0.05 mg/L IBA [21]. In contrast to herbaceous plants, the plant tissue culture of woody plants is more challenging due to their generally poor rooting ability. Callicarpa, as a shrub, typically exhibits excellent rooting under low auxin concentrations. As demonstrated in cutting propagation studies, the Callicarpa species predominantly exhibit cortex-rooting characteristics [46,47], displaying both rapid root initiation and consistently high survival rates [48]. Furthermore, it is speculated that the Callicarpa species have more endogenous auxin accumulation, and the addition of exogenous auxins can stimulate the continuous initiation of adventitious roots primordia. Because the content of endogenous IAA increased when the adventitious roots primordium started, the content of IAA gradually decreased to a lower level after the adventitious roots protruded from the epidermis [49].
The composition and ratio of the transplantation substrate directly determined key physicochemical properties, such as nutrient content, permeability, and water retention, which significantly affected the growth and development of the regenerated plantlets after transplantation. This study found that the survival rate of C. peichieniana in coconut coir/perlite = 1∶1 (v/v) substrate was 100%, a result consistent with the optimal ex vitro rooting effect obtained in C. mollis on peat or peat/perlite 1:1 (with 5 × 10−7 mol/L IAA or IBA in an MS liquid medium) [15]. This could be attributed to C. peichieniana preferring a humid and acidic environment, and coconut coir can effectively maintain soil humidity and aeration, and perlite can maintain its acidity.
Contamination is one of the major challenges in the plant tissue culture of woody plants. Many woody plants inevitably harbor various pathogenic microorganisms, including fungi, viruses, and bacteria, both on their surface and inside, which leads to a high contamination rate during their plant tissue culture process [50]. Traditional views suggest that once materials are contaminated by microorganisms, the culture process is bound to fail. It is noteworthy that the plant tissue culture of C. peichieniana does not follow the conventional proposal used in most studies, where the process is carried out in completely microbe-free conditions or in the presence of plant growth-promoting microbes (PGPMs). The plant regeneration process of C. peichieniana occurs in a microbial environment associated with C. sorokiniana. As shown in Figure 3, C. sorokiniana does not cause significant harm to the micropropagation of C. peichieniana, and an ISSR analysis showed that the “C. peichieniana–C. sorokiniana association” in vitro regeneration system was successfully established. Algae positively influence tissue culture outcomes. Supplementing the MS medium with 20% Oscillatoria tenuis extract significantly increased the adventitious bud proliferation and root formation of Phoenix dactylifera L., and the treatment resulted in the highest plant survival rate, plant height, and leaf number under irrigation conditions [34]. An MS medium supplemented with an algae filtrate improved microtuber formation in potato compared to the medium without an algae filtrate. Specifically, the MS medium containing 60% Aphanocapsa albida filtrate led to 5.7 microtubers, while the MS media containing 40% Scenedesmus obliquus filtrate and 80% A. albida filtrate resulted in the highest fresh weight of microtubers (305 mg and 300 mg, respectively) [35]. Algal supernatants have also been shown to enhance the bud proliferation rate in pea and tobacco cultures when supplemented with other plant hormones [36]. Some algae can produce a variety of substances, including PGRs [51], to enhance or replace the effects of synthetic PGRs on plant tissue culture [35]. During their growth, algae release vitamins, and an MS medium containing an algae filtrate can increase the content of macroelements, such as N, P, K, Ca, and Mg, as well as microelements, like Zn, Mn, and Na [52]. Therefore, even when the concentration of macroelements was reduced to below 25%, C. peichieniana can still maintain a root formation rate of over 83.33% and an average number of adventitious roots of more than 2.40 roots. In addition, excellent plant tissue culture outcomes were generally observed when C. sorokiniana was abundant and growing luxuriantly.
In the future, the application of algae will not only reduce the cost of expensive chemicals in micropropagation but will provide promising bioreactor applications for the production of medicinal components from medicinal plants like C. peichieniana. Many Callicarpa species, including C. peichieniana, contain active compounds, such as glycosides, flavonoids, and terpenoids, with notable pharmacological activities [53]. The use of bioreactors can ensure a stable and fast-growing permanent explant source to improve biomass and secondary metabolite production efficiency [54]. Previous studies have shown that C. sorokiniana FZU60 achieved ultra-high lutein yield and productivity in a bioreactor [55]. The “C. peichieniana–C. sorokiniana association” in vitro regeneration system established in this study has vast potential for bioreactor cultivation aimed at producing medicinal components.

5. Conclusions

This study established an efficient “C. peichieniana–C. sorokiniana association” in vitro regeneration system, which not only enhances the yield of high-quality C. peichieniana regenerated plantlets but demonstrates the potential application of C. sorokiniana in micropropagation. The apical bud, as the most suitable explant, exhibited the best induction of adventitious buds when cultured on MS + 0.5 mg/L 6-BA. The optimal proliferation medium was MS + 2.0 mg/L 6-BA. The rooting rate and growth of adventitious roots were effectively promoted by 0.1 mg/L NAA, MS medium with 50% macroelements, and 20 g/L sucrose. The best transplantation substrate was coconut coir/perlite = 1∶1 (v/v), achieving a 100% survival rate. The ISSR marker analysis demonstrated a genetic fidelity of 95.45% between the mother plant and the regenerated plantlets. In summary, this study provides promising theoretical and technical insights for the future development and utilization of C. peichieniana, while also offering a reference for culturing multiple species within the same in vitro system.

Author Contributions

Conceptualization, Y.Z., L.F. and K.W.; methodology, Y.Z. and K.W.; formal analysis, Y.Z. and M.G.; investigation, Y.X. and J.L.; resources, K.W.; data curation, M.G.; writing—original draft preparation, Y.Z.; writing—review and editing, L.F., K.W. and S.Z.; visualization, J.X.; supervision, S.Z. and G.M.; project administration, K.W.; funding acquisition, L.F., K.W. and S.Z. All authors have read and agreed to the published version of the manuscript.

Funding

This work was funded by the Project of the Guangdong Province Key Area R&D Plan: Research on the Protection and Utilization of Important Strategic Wild Plant Resources in Guangdong Province (2022B1111040003); the Forestry Grassland Ecological Protection and Restoration Program (E536080011).

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

The authors express their gratitude to Qianqian Li for the contribution to the identification of associated algae.

Conflicts of Interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Abbreviations

6-BA: 6-Benzyladenine; CKs: Cytokinins; IBA: Indole-3-butyric acid; ISSR: Inter simple sequence repeat; KT: Kinetin; MS: Murashige and Skoog medium; NAA: α-Naphthaleneacetic acid; PGR: Plant growth regulator; RAPD: Random amplified polymorphic DNA; TDZ: Thidiazuron; UBC: Primers designed by University of British Columbia; ZT: Zeatin.

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Figure 1. Induction of adventitious bud and plant regeneration in Callicarpa peichieniana. (A) Induction of adventitious buds with the condition of 0.5 mg/L 6-BA. (B,C) Adventitious bud proliferation with the conditions of 2.0 mg/L 6-BA and the combination of 1.5 mg/L 6-BA and 0.1 mg/L NAA. (D–F) Induction of adventitious roots with the conditions of 0.1 mg/L NAA, MS with 50% macroelements, and 20 g/L sucrose. (G,H) Growth status of regenerated plantlets after 4 months in peat and coconut coir/perlite = 1∶1 (v/v), “a” and “b” represented the individual plant growth and overall growth. Bars = 1 cm.
Figure 1. Induction of adventitious bud and plant regeneration in Callicarpa peichieniana. (A) Induction of adventitious buds with the condition of 0.5 mg/L 6-BA. (B,C) Adventitious bud proliferation with the conditions of 2.0 mg/L 6-BA and the combination of 1.5 mg/L 6-BA and 0.1 mg/L NAA. (D–F) Induction of adventitious roots with the conditions of 0.1 mg/L NAA, MS with 50% macroelements, and 20 g/L sucrose. (G,H) Growth status of regenerated plantlets after 4 months in peat and coconut coir/perlite = 1∶1 (v/v), “a” and “b” represented the individual plant growth and overall growth. Bars = 1 cm.
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Figure 2. ISSR amplification profiles between the mother plant and the regenerated plantlets using primers UBC845 and UBC850. Lane M: Molecular ruler (2 kb). Lane MP: Mother plant. Lanes 1–9: Randomly selected regenerated plantlets.
Figure 2. ISSR amplification profiles between the mother plant and the regenerated plantlets using primers UBC845 and UBC850. Lane M: Molecular ruler (2 kb). Lane MP: Mother plant. Lanes 1–9: Randomly selected regenerated plantlets.
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Figure 3. “C. peichieniana–C. sorokiniana association” in vitro regeneration system work flow chart and growth condition of associated alga. (A) Mother plant shoots disinfected with 0.1% HgCl2. (B) Adventitious bud induction (4 weeks). (C) Adventitious bud proliferation (4 weeks). (D) Adventitious root induction (4 weeks). (E) The regenerated plantlets grew in the substrate of coconut coir/perlite = 1∶1 (v/v) (4 months). (F) Microscopic morphology of C. sorokiniana. Bars = 1 cm.
Figure 3. “C. peichieniana–C. sorokiniana association” in vitro regeneration system work flow chart and growth condition of associated alga. (A) Mother plant shoots disinfected with 0.1% HgCl2. (B) Adventitious bud induction (4 weeks). (C) Adventitious bud proliferation (4 weeks). (D) Adventitious root induction (4 weeks). (E) The regenerated plantlets grew in the substrate of coconut coir/perlite = 1∶1 (v/v) (4 months). (F) Microscopic morphology of C. sorokiniana. Bars = 1 cm.
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Table 1. Effect of explant types and 6-BA concentrations on the number of adventitious buds of Callicarpa peichieniana. The growth of the adventitious buds and Chlorella sorokiniana was evaluated after 4 weeks.
Table 1. Effect of explant types and 6-BA concentrations on the number of adventitious buds of Callicarpa peichieniana. The growth of the adventitious buds and Chlorella sorokiniana was evaluated after 4 weeks.
Explant Types6-BA (mg/L)Average Number of Adventitious BudsGrowth
Adventitious BudsChlorella sorokiniana
Apical bud01.00 ± 0.00 d+Δ
0.14.22 ± 0.40 a++
0.33.33 ± 0.19 b+++
0.55.00 ± 0.00 a++++
1.04.67 ± 0.19 a++
Stem segment02.00 ± 0.00 c+++Δ
0.12.00 ± 0.00 c+
0.32.55 ± 0.40 c+
0.52.44 ± 0.44 c++
1.02.44 ± 0.22 c++
Values were presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level: “+” indicates thin adventitious buds, worse growth; “++” indicates thin adventitious buds, favorable growth; “+++” indicates thick adventitious buds, favorable growth; “++++” indicates thick adventitious buds, robust growth; “Δ” indicates moderate growth and low abundance.
Table 2. Effect of 6-BA, TDZ, and a combination of 6-BA and NAA concentrations on the adventitious bud proliferation coefficient and the height of Callicarpa peichieniana. The growth of the adventitious buds and Chlorella sorokiniana was evaluated after 4 weeks.
Table 2. Effect of 6-BA, TDZ, and a combination of 6-BA and NAA concentrations on the adventitious bud proliferation coefficient and the height of Callicarpa peichieniana. The growth of the adventitious buds and Chlorella sorokiniana was evaluated after 4 weeks.
PGR Concentrations (mg/L)Proliferation CoefficientAverage Height of
Adventitious Buds (cm)		Growth
6-BANAATDZAdventitious BudsChlorella sorokiniana
0002.00 ± 0.16 hi0.91 ± 0.06 b+++Δ
0.5003.97 ± 0.09 cd1.20 ± 0.08 a+++Δ
1.0004.11 ± 0.15 c0.86 ± 0.05 b+++Δ
1.5004.94 ± 0.16 b0.54 ± 0.03 de+++++Δ
2.0005.83 ± 0.20 a0.73 ± 0.05 c+++++++ΔΔ
3.0005.58 ± 0.21 ab0.58 ± 0.03 d++++++ΔΔ
1.50.103.84 ± 0.15 cd0.51 ± 0.03 def+++++Δ
2.00.102.26 ± 0.18 ghi0.33 ± 0.01 f++Δ
3.00.103.07 ± 0.13 def0.41 ± 0.02 def++++Δ
1.50.203.27 ± 0.18 cde0.49 ± 0.03 def++++Δ
2.00.202.19 ± 0.15 ghi0.35 ± 0.01 ef++Δ
3.00.202.71 ± 0.19 fgh0.42 ± 0.02 def++Δ
1.50.401.91 ± 0.16 hi0.38 ± 0.01 ef++Δ
2.00.402.13 ± 0.17 ghi0.36 ± 0.01 ef++Δ
3.00.401.73 ± 0.08 i0.36 ± 0.01 ef+Δ
000.023.80 ± 0.09 cd0.52 ± 0.03 def++++Δ
000.042.72 ± 0.20 fgh0.37 ± 0.02 ef++++Δ
000.082.88 ± 0.19 efg0.44 ± 0.02 def++++Δ
Values were presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level: “+” indicates thin and short adventitious buds, worse growth; “++” indicates thin and short adventitious buds, moderate growth; “+++” indicates thin and long adventitious buds, moderate growth; “++++” indicates thick and short adventitious buds, moderate growth; “+++++” indicates thick and short adventitious buds, favorable growth; “++++++” indicates thick and short adventitious buds, robust growth; “+++++++” indicates thick and long adventitious buds, robust growth; “Δ” indicates moderate growth and low abundance; “ΔΔ” indicates strong growth and high abundance.
Table 3. Effect of IBA, NAA concentrations on rooting rate, number and length of adventitious roots of Callicarpa peichieniana. The growth of the adventitious roots and Chlorella sorokiniana was evaluated after 4 weeks.
Table 3. Effect of IBA, NAA concentrations on rooting rate, number and length of adventitious roots of Callicarpa peichieniana. The growth of the adventitious roots and Chlorella sorokiniana was evaluated after 4 weeks.
Auxin Concentrations (mg/L)Rooting Rate (%)Average Number of Adventitious RootsAverage Length of Adventitious Roots (cm)Growth
IBANAAAdventitious RootsChlorella sorokiniana
0083.33 ± 1.02 b3.80 ± 0.55 b0.72 ± 0.07 a+Δ
0.1094.44 ± 0.68 ab3.47 ± 0.22 b0.68 ± 0.04 a+Δ
0.5094.44 ± 0.70 ab3.41 ± 0.30 b0.47 ± 0.30 b+Δ
1.0094.44 ± 0.0.73 ab2.68 ± 0.23 c0.45 ± 0.02 b++Δ
00.0594.44 ± 0.68 ab2.09 ± 0.26 c0.43 ± 0.02 b+Δ
00.1100 ± 0.62 a6.36 ± 0.43 a0.66 ± 0.03 a+++ΔΔ
00.2100 ± 0.77 a3.47 ± 0.49 b0.48 ± 0.02 b+Δ
Values were presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level: “+” indicates thin adventitious roots, favorable growth; “++” indicates thick adventitious roots, moderate growth; “+++” indicates thick adventitious roots, robust growth; “Δ” indicates moderate growth and low abundance; “ΔΔ” indicates strong growth, and high abundance.
Table 4. Effect of macroelements concentrations on rooting rate, number and length of adventitious roots of Callicarpa peichieniana. The growth of the adventitious roots and Chlorella sorokiniana was evaluated after 4 weeks.
Table 4. Effect of macroelements concentrations on rooting rate, number and length of adventitious roots of Callicarpa peichieniana. The growth of the adventitious roots and Chlorella sorokiniana was evaluated after 4 weeks.
Macroelements Concentrations (%)Rooting Rate (%)Average Number of Adventitious Roots Average Length of Adventitious Roots (cm)Growth
Adventitious RootsChlorella sorokiniana
100%MS94.44 ± 1.68 a2.18 ± 0.37 b0.43 ± 0.02 c++Δ
50%MS100 ± 1.19 a5.42 ± 0.22 a1.32 ± 0.05 a+++
25%MS86.11 ± 2.43 b2.90 ± 0.51 b0.41 ± 0.02 c+
12.5%MS83.33 ± 2.43 b2.40 ± 0.50 b0.55 ± 0.04 b+
Values were presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level: “+” indicates thin adventitious roots, moderate growth; “++” indicates thin adventitious roots, favorable growth; “+++” indicates thick adventitious roots, robust growth; “Δ” indicates moderate growth and low abundance.
Table 5. Effect of sucrose concentrations on rooting rate, number and length of adventitious roots of Callicarpa peichieniana. The growth of the adventitious roots and Chlorella sorokiniana was evaluated after 4 weeks.
Table 5. Effect of sucrose concentrations on rooting rate, number and length of adventitious roots of Callicarpa peichieniana. The growth of the adventitious roots and Chlorella sorokiniana was evaluated after 4 weeks.
Sucrose
Concentrations (g/L)		Rooting Rate (%)Average Number of
Adventitious Roots		Average Length of
Adventitious Roots (cm)		Growth
Adventitious RootsChlorella sorokiniana
02.78 ± 1.35 c1.00 ± 0.00 c0.10 ± 0.00 c-×
1025.00 ± 1.65 b1.78 ± 0.48 bc0.40 ± 0.05 b+Δ
2088.89 ± 1.43 a2.91 ± 0.26 a0.46 ± 0.04 ab++Δ
3091.67 ± 1.43 a2.03 ± 0.31 b0.52 ± 0.06 a++Δ
Values were presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level: ”-” indicates almost no rooting, worse growth; “+” indicates thin adventitious roots, moderate growth; “++” indicates thin adventitious roots, favorable growth; “×” indicates bad growth and fewer abundance; “Δ” indicates moderate growth and low abundance.
Table 6. Effect of substrate types on survival rate of plantlets of Callicarpa peichieniana. The growth of the plantlets was evaluated after 4 weeks and 4 months.
Table 6. Effect of substrate types on survival rate of plantlets of Callicarpa peichieniana. The growth of the plantlets was evaluated after 4 weeks and 4 months.
Substrate TypesSurvival Rate (%)Plantlets Growth
4 Weeks4 Months
Peat100 ± 0.00 a91.67 ± 3.94 b++
Coconut coir97.22 ± 2.78 ab83.33 ± 5.73 c+
Peat/perlite = 1∶1 (v/v)94.44 ± 3.87 ab83.33 ± 5.79 c+
Coconut coir/perlite = 1∶1 (v/v)100 ± 0.00 a100 ± 0.00 a+++
Peat/coconut coir/perlite = 1∶1∶1 (v/v/v)88.89 ± 5.31 b77.78 ± 6.12 cd+
Values were presented as mean ± standard error. Different lowercase letters in the same column indicate significant differences at the p < 0.05 level: “+” indicates green plantlets, favorable root system and moderate growth; “++” indicates green plantlets, favorable root system and favorable growth; “+++” indicates bright green plantlets, strong root system and robust growth.
Table 7. ISSR analysis results for the genetic fidelity assessment.
Table 7. ISSR analysis results for the genetic fidelity assessment.
Primer
Name		Primer Sequence (5′→3′)Tm
(℃)		Number of Amplified FragmentsNumber of Monomorphic BandsNumber of Polymorphic BandsAmplification Length Range (bp)
UBC809AGAGAGAGAGAGAGAGG50110100–200
UBC818CACACACACACACACAG51330250–750
UBC820GTGTGTGTGTGTGTGTC51110250–500
UBC830TGTGTGTGTGTGTGTGG53211250–500
UBC835AGAGAGAGAGAGAGAGYC50220500–1000
UBC842GAGAGAGAGAGAGAGAYG50330250–750
UBC845CTCTCTCTCTCTCTCTRG50440100–1000
UBC848CACACACACACACACARG53220100–500
UBC850GTGTGTGTGTGTGTGTYC53440250–1000
Total 22211
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Zhang, Y.; Guo, M.; Xu, J.; Xiong, Y.; Liu, J.; Ma, G.; Zeng, S.; Wu, K.; Fang, L. Influence of Chlorella sorokiniana and Plant Growth Regulators During the Micropropagation of Callicarpa peichieniana. Horticulturae 2025, 11, 1016. https://doi.org/10.3390/horticulturae11091016

AMA Style

Zhang Y, Guo M, Xu J, Xiong Y, Liu J, Ma G, Zeng S, Wu K, Fang L. Influence of Chlorella sorokiniana and Plant Growth Regulators During the Micropropagation of Callicarpa peichieniana. Horticulturae. 2025; 11(9):1016. https://doi.org/10.3390/horticulturae11091016

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Zhang, Yiteng, Manna Guo, Jinfeng Xu, Yuping Xiong, Junyu Liu, Guohua Ma, Songjun Zeng, Kunlin Wu, and Lin Fang. 2025. "Influence of Chlorella sorokiniana and Plant Growth Regulators During the Micropropagation of Callicarpa peichieniana" Horticulturae 11, no. 9: 1016. https://doi.org/10.3390/horticulturae11091016

APA Style

Zhang, Y., Guo, M., Xu, J., Xiong, Y., Liu, J., Ma, G., Zeng, S., Wu, K., & Fang, L. (2025). Influence of Chlorella sorokiniana and Plant Growth Regulators During the Micropropagation of Callicarpa peichieniana. Horticulturae, 11(9), 1016. https://doi.org/10.3390/horticulturae11091016

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