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Article

Efficient In Vitro Propagation of Turpinia arguta and Quantitative Analysis of Its Ligustroflavone and Rhoifolin Content

by
Jiangmei Hu
1,2,†,
Junhuo Cai
1,†,
Xinrui Hu
1,2,
Lijun Wang
1,2,
Qiangqiang Cheng
2,* and
Xiuhua Tao
2,*
1
College of Forestry, Jiangxi Agricultural University, Nanchang 330022, China
2
Jiangxi Academy of Forestry, Nanchang 330022, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(6), 587; https://doi.org/10.3390/horticulturae10060587
Submission received: 9 May 2024 / Revised: 31 May 2024 / Accepted: 1 June 2024 / Published: 4 June 2024
(This article belongs to the Section Propagation and Seeds)
Browse Figures
Versions Notes

Abstract

:
Turpinia arguta is an excellent medicinal plant mainly used for the treatment of pharyngitis, tonsillitis, and tonsillar abscesses. However, an efficient regeneration protocol using tissue cultures for T. arguta does not exist. Its main medicinal constituents are flavonoids, particularly ligustroflavone and rhoifolin. Here, we aimed to establish a tissue culture system for T. arguta for the first time using annual stem segments with axillary buds harvested from the field of the Jiangxi Academy of Forestry as explants by dynamically determining the accumulation of effective functional components in the tissue culture plantlets. Orthogonal tests were conducted to compare the effects of different explant disinfection times, media, and exogenous hormone ratios on the induction of the axillary bud growth, successional proliferation, and rooting of T. arguta stem segments. The best explant disinfection effect was achieved by disinfecting the T. arguta explant with 75% ethanol for 50 s, followed by 0.1% mercuric chloride (HgCl2) for 6 min, and the optimal media for successional proliferation and rooting were Murashige and Skoog (MS) + 0.2 mg/L of 6-benzyladenine (6-BA), + 0.03 mg/L of naphthaleneacetic acid (NAA), and ½ MS + 2.5 mg/L of indole-3-butyric acid + 0.5 mg/L of NAA, respectively. The detection of ligustroflavone and rhoifolin in tissue culture plantlets 0, 3, and 5 months after transplanting showed a significant increasing trend and eventually exceeded the content requirements of the 2020 Edition ofChinese Pharmacopoeia for T. arguta. Our findings provide, for the first time, an effective tissue culture system for T. arguta, thereby providing important information to support the germplasm preservation, innovation, and application of T. arguta in the future.

1. Introduction

In many countries, medicinal plants are the most widely used form of medicine and play an important role in traditional medicine, because they are safer and more effective than other treatments [1]. Natural products from medicinal plants, including flavonoids, alkaloids, and terpenoids, play pivotal roles as sources of pharmaceutical compounds. Flavonoids, a large group of polyphenolic secondary metabolites widely distributed in the plant kingdom, have also attracted the attention of most phytopharmacologists, owing to their significant anti-hypertensive [2], neuroprotective [3], cardioprotective [4], wound-healing [5], anti-cardiovascular [6], cancer-preventive [7], antioxidant [8], anti-tumour [9], anti-bacterial, and anti-viral properties [10] that are beneficial to human health. The commercial demand for flavonoids is increasing, and strategies for the sustainable production and efficient manufacturing of flavonoids have attracted the interest of many researchers.
Turpinia arguta, a woody medicinal plant belonging to the Staphyleaceae turpinia family, is mainly distributed in southern China. It has a height of up to 100–150 cm and is well-branched. It produces small white flowers, and the leaves are dark green when ripe. The dry leaves are called Turpiniae folium and can be used medicinally [11]. Turpiniae folium was used in a Chinese patented drug by Jiangxi Pharmaceutical Enterprises in the 1970s, included in the Jiangxi Provincial Chinese Materia Medica Standard in the 1990s, and included in the Pharmacopoeia of the People’s Republic of China in 2010, owing to its effectiveness in the treatment of tonsillitis and pharyngitis [12]. Various chemical constituents have been identified in Turpiniae folium, including flavonoids, triterpenoids, megaterpenoids, and phenolic acids [13], with flavonoids being the most widely studied and pharmacologically active constituents [14,15,16]. Previous studies have also shown that the secondary metabolites of flavonoids, represented by ligustroflavone and rhoifolin, are the main effective medicinal components of T. arguta [15,17], with anti-bacterial, anti-inflammatory, analgesic, and immune-boosting pharmacological properties [18], as well as medicinal effects such as clearing heat, removing toxins, relieving swelling, activating blood circulation, and relieving pain [11].
Ligustroflavone is one of the main compounds contained in the fruits of ligustroflavone [19], which prevents osteoporosis, improves hepatic fibrosis [20], and possesses anticancer [21] and anti-ischaemic effects [22]. It is an ideal anti-cancer drug with a high degree of selectivity, allowing it to distinguish between cancerous and non-cancerous cells and kill only the cancerous cells and overcoming the side effects of chemotherapy [23]. There are no reports that both ligustroflavone and rhoifolin can be produced by biosynthetic techniques; therefore, species such as T. arguta, Citrus reticulata Blanco, and Ligustrum lucidum are currently the only sources of ligustroflavone and rhoifolin. T. arguta is one species that produces high levels of ligustroflavone and rhoifolin, and Li et al. [24] reported that ligustroflavone and rhoifolin levels in samples from the Jiangxi Province could reach 0.6% and 0.4%, respectively. Yu et al. [25] found that the average rhoifolin content of Citrus sinensis from nine different origins was 0.15%. Guo et al. [26] reported that over different growth periods, the levels of ligustroflavone in Ligustrum lucidum fruits fluctuated within the range of 0.08–0.23%. Thus, in the future, the commercial production of ligustroflavone and rhoifolin will largely depend on the cultivation of T. arguta and an optimal process for drying its leaves and extracting active ingredients.
The chemical constituents of the Turpiniae folium extract exhibit good antioxidant and anti-inflammatory activities [27]; they can be used as a natural ingredient for the development of functional foods and pharmaceuticals. Functional foods are foods with documented health benefits, such as a reduced risk of chronic diseases or benefits that otherwise optimize health [28]. For example, blackberries are recognized worldwide as a functional food due to their high content of polyphenolic compounds as well as antioxidant activities [29]. Also, modern Chinese patented drugs, such as Shanxiang Yuan tablets [30], Shanxiang Yuan granules, and Shanxiang Yuan tablets, have been introduced. T. arguta is also a natural plant feed material, with abundant natural resources and only minor side effects [31]. Its nutritional and pharmaceutical properties can enhance immunity, improve resistance to viruses, improve feed intake, promote growth, and prevent diseases in livestock and poultry. It is safe for use in livestock and poultry and for human beings; furthermore, it does not pollute the environment and is considered an environmentally friendly green feed additive. Song Wenjing et al. [31] showed that the appropriate amount of Turpiniae folium extract inhibited the reproduction of harmful microorganisms and increased the abundance of beneficial microorganisms in the intestinal tract of Wenchang chickens, thereby improving the intestinal micro-ecological environment and the growth performance of Wenchang chickens.
Recently, with the increasing use of T. arguta as a raw ingredient in several proprietary Chinese medicine products, wild T. arguta resources are on the verge of extinction, and, therefore, production is not meeting the demand of the T. arguta medicinal materials market. Jiangxi pharmaceutical enterprise (Qingfeng Pharmaceutical, MinKang Pharmaceutical, and BaiJi Pharmaceutical) is currently experiencing a shortage of 1000 t/year; these shortages are also reported in other provinces and other industries outside of the beverage pharmaceutical industry [32]. Whilst the traditional artificial seedling method is employed, it is restricted by many factors, such as temperature, soil, and region, and does not maintain the excellent traits of a single plant. Therefore, there is an urgent need to develop a fast-breeding technology suitable for the T. arguta industry to address the scarcity of T. arguta medicinal resources, laying a firm foundation for the subsequent commercial production of active ingredients.
Plant tissue culturing is a technique that allows for the aseptic propagation and cultivation of plants in a specific environment, allowing for the large-scale production of plants with the same excellent traits of normally grown plants [33]. The establishment of plant tissue cultures typically involves the following stages: explant disinfection induction, successional proliferation, rooting induction, and transplant domestication [34]. During these phases, information on the type and combination of plant growth regulators is an important part of the overall tissue culture system [35]. In vitro tissue culture techniques have been shown to be effective in propagating plant species with medicinal value, laying the foundation for the commercial extraction and production of plant-contained secondary metabolites, e.g., Codonopsis Pilosula (flavonoids) [36] and Hypericum perforatum (phenols) [37]. In this context, the establishment of a comprehensive large-scale propagation method using tissue culture techniques is essential to increase the population size and promote the conservation of T. arguta.
However, no study has reported an efficient regeneration protocol for T. arguta. Therefore, this study aimed to develop an efficient in vitro plant regeneration protocol using stem segment induction for the mass propagation of T. arguta.

2. Materials and Methods

2.1. Plant Materials and Reagents

Plants were obtained from the field of the Jiangxi Academy of Forestry on 1 No-vember 2023 and identified as Turpinia arguta by Senior Engineer Liang Yuelong of the Jiangxi JiuLian Mountain National Nature Reserve Administration. The active ingredient content (ligustroflavone and rhoifolin) was tested in the leaves of group-cultivated plantlets (0, 3, and 5 months). 6-benzyladenine (6-BA, purity 99%, Aladdin Company, Shanghai, China); indole-3-butyric acid (IBA, purity 98%, Aladdin Company, Shanghai, China); and naphthaleneacetic acid (NAA, purity 96%, Aladdin Company, Shanghai, China). And standard ligustroflavone (purity ≥ 98%, Shanghai Yuanye Company, Shanghai, China) and rhoifolin (purity ≥ 98%, Shanghai Yuanye Company, Shanghai, China) products. Chromatographically pure MeOH (purity ≥ 99.9%, Merck Company, Rahway, NJ, USA), All other reagents were of analytical-grade purity.
Unless otherwise stated, all media in this experiment contained 0.6% agar (purity ≥ 98%, Aladdin Company, Shanghai, China) and 3% sucrose (purity ≥ 98%, Aladdin Company, Shanghai, China) at a pH of 5.8–6.2 and were then placed under 12 h of light per day with a light intensity of 2100–2500 lx and incubation temperatures of 23–25 °C. In our study, tissue culture test tube flasks (30 mm × 120 mm) made of a high borosilicate glass (Bikeman Bio Company, Changsha, China) were used as in vitro containers for initiation, propagation, and rooting.

2.2. Methods

2.2.1. Explant Disinfection and Induction

Semi-woody pest- and disease-free stem segments with robust growth were harvested from pot-grown plants and selected as explants, and all leaves were cut off with scissors and cut into 1–2 cm stem segments with 2 buds. They were soaked and washed with a detergent solution for 30 min, rinsed with running water for 2–3 h, placed onto an ultra-clean bench, soaked with 75% alcohol for 50 s, and then washed with sterile water three times. They were then soaked in mercuric chloride for 4, 6, 8, 10, and 12 min and washed with sterile water six times for 20 s. All experiments were performed in triplicate, and each time, 17 stem segments were inoculated in a Murashige and Skoog (MS) primary medium without hormone addition. The contamination, survival, and mortality rates of the explants were counted after 30 days of culture.

2.2.2. Successional Proliferation

Different basic media: After the best treatments were established in the primary culture, a large number of sterile tissue culture plantlets were obtained by large-scale in-flasking. The lower healing tissue and the upper shoot portion were cut off, plantlets were cut into single-shooting stem segments of 1.0–1.5 cm, which were inoculated into (Douglas fir cotyledon revised) DCR [38], WPM [39], MS [40], and ½ MS (half-strength of MS) media, with a total of 4 treatments, 3 biological replicates, and 20 inoculations in each replicate. After 30 days, the growth status of the tissue culture plantlets for the different treatments was observed, and the proliferation coefficients were counted to determine the best proliferation medium.
Different plant growth regulators: Stem segments with single shoots were vertically inoculated into an MS medium supplemented with various concentrations of 6-BA (0, 0.1, 0.2, and 0.5 mg/L); NAA (0.01, 0.03, and 0.5 mg/L); and gibberellic acid (GA3) (1 mg/L). There were 9 formulations in total (Table 1), which were repeated three times, with each replicate consisting of 21 explants. After 30 days, the proliferation coefficients were counted, and the best phytohormone formulations were selected.

2.2.3. In Vitro Rooting and Acclimatization

In this experiment, a ½ MS medium was used as the basic medium, and robustly grown tissue culture plantlets were inoculated on a ½ MS medium supplemented with different concentrations of IBA (0, 0.5, 1.0, 1.5, 2.0, and 2.5 mg/L); 0.5 mg/L of NAA; and 0.4 mg/L of activated charcoal. A rooting culture was performed in 3 replications with 20 inoculations per replication. After 30 days, the growth status of the tissue culture plantlets for each treatment was observed, and the rooting rate and average number of roots for each treatment were counted to select the best rooting medium.
Rooted T. arguta plantlets were selected and removed from the culture flasks. The agar and other components attached to the root system were rinsed with water and transplanted in a greenhouse at 25 °C with 3000 lx. The transplanting substrate was mixed with peat soil and perlite (1:1, v/v), and the substrate was disinfected with a 20% carbendazim solution 1 week before transplanting, after which the plants were watered thoroughly, covered with a plastic film for heat and humidity preservation, and removed after 1 week for normal management.

2.2.4. Quantitative Detection of the Active Ingredient of T. arguta

Preparation of sample solutions and standard solutions: In the transplanting process, fresh leaves from different periods of growth (0, 3, and 5 months) in the greenhouse for acclimatization (Figure 1) were collected, washed, and oven-dried (60 °C) to a constant weight. Liquid nitrogen was applied, and the leaves were ground into powder and extracted according to the Chinese Pharmacopoeia [11]. The specific process was as follows. First, 0.3 g of ground powder of the above samples was passed through the No. 3 sieve, weighed, and placed in a stoppered flask. Fifty percent methanol (50 mL) was added, the sample was weighed, treated with ultrasound (power 250 W and frequency 25 kHz) for 1 h, allowed to cool, and weighed. Then, 50% methanol was added to compensate for the loss of weight, and the sample was filtered to obtain the renewed filtrate. Ultrasonic treatment (power 250 W and frequency 25 kHz) was applied again for 1 h, the sample was allowed to cool, and weighed again, and 50% methanol was added to compensate for the loss of weight. The sample was shaken and filtered to obtain the filtrate. The sample solution was filtered through a 0.22 μm filter membrane and pinhole syringe, and the filtered extract was used for a quantitative assay. The ligustroflavone control and rhoifolin control samples were dried using phosphorus pentoxide under a reduced pressure for 24 h for each 10 mg, precision weighed, and placed in a 100 mL flask. Then, 50% methanol was added to dissolve and dilute the samples to scale (that is, each l ml contained 100 μg of ligustroflavone and 100 μg of rhoifolin) and shaken to create the standard master batch. These batches were diluted to 50, 25, 12.5, and 6.25 μg/mL and other series of standard solutions in preparation for HPLC detection.
Quantitative detection of active ingredients: The HPLC analyser (Shimadzu Corporation, Kyoto, Japan) used was equipped with a UV-visible detector, SunFireTMC18 (250 mm × 4.6 mm, 5 μm; detection wavelength, 335 nm; Shimadzu Corporation). The mobile phase was a methanol–0.5% phosphoric acid aqueous solution (43:57), and elution was performed at a flow rate of 1 mL/min, with an injection volume of 1 mL, and the column temperature box was set to 35°, which was kept constant throughout the process. The samples and standards were injected three times, and the ligustroflavone and rhoifolin contents of the samples were determined by comparing the retention times and peak areas with those of the standards.

2.3. Statistical Analysis

In the different experiments, various parameters were calculated to evaluate the effects of treatments, as described below.
Contamination rate = contaminated explants/explants inoculated;
Mortality rate = dead explants/explants inoculated;
Survival rate = viable explants/explants inoculated;
Proliferation coefficient = number of induced regeneration shoots/number of inoculated shoots;
Average number of roots = number of roots of rooted plantlets/number of rooted plantlets;
Rooting rate = number of rooted tissue culture plantlets/number of inoculated tissue culture plantlets.
All experiments were conducted using a randomised method. Microsoft Excel 2010 (Microsoft Corp., Redmond, WA, USA) was used to tabulate the data, and IBM SPSS Statistics 25 (IBM Corp., Armonk, NY, USA) was used to perform a one-way, two-way analysis of variance (ANOVA) and multiple range tests (LSDs).

3. Results

3.1. Explant Disinfection and Induction

Disinfection times with mercuric chloride (HgCl2) had a significant effect on the contamination, survival, and mortality rates of stem segments with axillary buds (Table 2). Under a constant disinfection time with 75% ethanol, with an increase in the mercury disinfection time, the contamination rate gradually decreased, the mortality rate increased, and the survival rate first increased and then decreased. At 4 min of mercury disinfection, the contamination rate was the highest at 68.01%, the mortality rate was the lowest at 7.97%, and the survival rate was 24.02%. At 12 min of mercury disinfection, the contamination rate was the lowest at 50.00%, the mortality rate was the highest at 30.51%, and the survival rate was only 20.47%. An increased number of explant deaths occurred when exposed to prolonged HgCl2-disinfection treatments (12 min). On the other hand, a short duration of 4 min in HgCl2 leads to incomplete disinfection. After 6 min of mercury disinfection, the HgCl2 contamination level was 58.09%, and the lowest mortality rate (6.00%) and highest survival rate (35.91%) were observed. The surviving stem segment explants are shown in Figure 2a.

3.2. Successional Proliferation

3.2.1. Effect of the Culture Media on the Proliferation of Tissue Culture Plantlets

Culture media have a significant effect on the proliferation coefficient of T. arguta cultures (Table 3). The proliferation coefficients of the four basic media reached a level of significant difference, indicating that the different basic media significantly affected the proliferation coefficients. Among them, the MS medium had the highest proliferation coefficient (3.12), followed by WPM (2.27), and the lowest proliferation coefficient was that of ½ MS (1.42). In addition, the tissue culture plantlets grew vigorously in the MS medium with a thick and sturdy seedling height, which was suitable for multiple successive proliferation. In contrast, tissue culture plantlets in the DCR, WPM, and ½ MS media were shorter, and plantlets in the WPM medium showed a stunted growth, with noticeable browning of the callus, and they were not suitable for the successional proliferation of T. arguta.

3.2.2. Effect of Different Hormone Combinations on the Subproliferation of Tissue Culture Plantlets

The addition of different combinations of hormones at different concentrations significantly affected the successional proliferation of T. arguta when MS was used as the basic medium. Table 4 shows that in the proliferation culture, when the hormone ratio was 0.1 mg/L of 6-BA and 0.1 mg/L of NAA, the proliferation coefficient was the lowest at 1.33, and the plantlets were relatively shorter with thin stems. When the hormone ratio was 0.2 mg/L of 6-BA + 0.03 mg/L of NAA, the proliferation coefficient was the highest at 3.13, which was significantly higher than other treatment combinations, indicating that the growth condition was good, and the seedling growth was vigorous with good seedling heights, strong stems, and relatively more growth, as per Figure 2b. The ANOVA results for the proliferation coefficient are shown in Table 4. Different concentrations of 6-BA and NAA had a significant effect on the proliferation coefficients of T. arguta group-cultured plantlets (p < 0.05), and there are significant interactions between them. Therefore, the optimal medium for the proliferation of T. arguta was MS + 0.2 mg/L of 6-BA + 0.03 mg/L of NAA (Table 5).

3.3. In Vitro Rooting and Acclimatization

Different concentrations of IBA had significant effects on the rooting rate and average number of rooting strips of T. arguta (Table 6). The rooting rate and average number of rooting strips of the plantlets decreased with increasing IBA concentrations, and the plantlets grew vigorously with spreading leaves and stout stems, as shown in Figure 2c. The rooting rate and average number of rooting strips of the plantlets were significantly affected by the different IBA concentrations (Table 6). The highest rooting rate of 66.60% was observed at 2.5 mg/L, along with the second highest number of roots (3.50). Rooted bottle plantlets were transplanted into a pre-prepared substrate, watered thoroughly, and managed by covering the membrane. After 1 month, the survival rate was above 90%, and the plants grew robustly, with spreading and enlarged green leaves and a well-developed root system, as shown in Figure 2d.

3.4. Determination of Ligustroflavone and Rhoifolin Contents

Based on the method described in Section 2.2.4 above for determining ligustroflavone and rhoifolin standards, as well as period samples, the standard of ligustroflavone and rhoifolin curves were plotted, and the regression equations were y (ligustroflavone) = 12,966x – 19,421, R2 = 0.9998, indicating a good linear relationship with the ligustroflavone concentration gradient, and y (rhoifolin) = 21,489x – 13,507, R2 = 0.9999, indicating a good linear relationship with the rhoifolin concentration gradient. The ligustroflavone and rhoifolin contents during the growth process were calculated according to the standard ligustroflavone and rhoifolin curves, and the specific results are shown in Figure 3 and Table 7. The ligustroflavone and rhoifolin contents increased gradually with the continuous growth of the plantlets, and the contents of the two active ingredients differed significantly during seedling growth. When the plantlets were grown for 5 months in a transplanting substrate placed in a greenhouse, the highest levels of ligustroflavone and rhoifolin were 0.469% and 0.188%.

4. Discussion

The successful disinfection of explant material, and thus the establishment of a sterile environment during tissue culturing, is a prerequisite for in vitro plant propagation techniques [41]. For explant disinfection, the choice of reagents used for disinfection and the time of disinfection are of great importance. Incomplete disinfection can lead to the growth of bacterial and fungal organisms in the explants, which in turn can lead to the death of the inoculated explants. Prolonged disinfection may also lead to the death of explants.
Existing studies have shown that the use of a single type of disinfectant reagent in the disinfection process can reduce the degree of damage to the explant, but the results are relatively unsatisfactory compared with the use of a mixture of two or more disinfectant reagents; therefore, common methods of explant disinfection involve ethanol and mercuric chloride disinfection or a combination of ethanol and sodium hypochlorite (NaClO) disinfection [42,43,44,45]. In this experiment, the optimal mercuric chloride disinfection time was determined to be 4 min. By screening the optimal disinfection time of HgCl2 on the explants, disinfection for 12 min resulted in the lowest contamination rate, but it negatively affected the survival rate. This is because the HgCl2 disinfection time was extremely long, and the exosome material showed a certain degree of browning. Our results, showing an increase in the mortality rate of the explants and a decrease in the survival rate with the extension of disinfection time, are consistent with the results of Gu et al. [46] and Romadanova et al. [47], who investigated the effect of disinfection time on the survival rate of the stem segments and axillary buds of Cnidoscolus aconitifolius and Vitis spp., respectively. In our study, optimizing the HgCl2 disinfection time improved the disinfection efficiency and survival rate of Turpinia arguta explants, and better results were obtained when explants were exposed to 6 min of HgCl2.
A basic medium provides sufficient nutrients for the growth of plants in tissue cultures; however, the nutrient composition of different media varies greatly, which can directly affect the growth and development of explants. Plants have different nutrient requirements because of the different biological characteristics and genetic traits [48]. MS, WPM, and DCR are all widely used for the tissue culturing of woody plants [49,50,51]. In this experiment, the four basic media had a significant effect on the successional proliferation of T. arguta, and the MS basic medium had the greatest effect on the successional proliferation of the tissue cultured plantlets, and the plantlets grew vigorously. Compared with the MS medium, ½ MS, DCR, and WPM are all low inorganic salt media [52,53], containing significantly lower total nitrogen contents. Studies have shown that nitrogen plays an important role in the growth, development, and physiological metabolism of histocultures [54,55], and nitrogen deficiency in plants can lead to dwarf plants, leaf yellowing, and poor growth [56,57], which was observed in the present study. This suggests that T. arguta successional proliferative cultures should typically have a high N content. Generally, an MS medium with a high nitrate content has the right amount and proportion of nutrients to meet the nutritional and physiological needs of the target cells; therefore, it is suitable for the tissue culturing of most organisms [45,58,59].
Phytohormones can effectively regulate the cell cycle [60], tissue and organ differentiation [61,62], morphogenesis [63,64,65], and plant growth and development [66,67]. The most commonly used phytohormones in plant proliferation cultures are 2-(isopentinyl)-adenine (2-IP), 6-BA, zeatin (ZT), and NAA [68]. In this study, we selected two hormones, 6-BA and NAA, and compared the degree of effect of the two hormones on plant proliferation, and the results show that the effect of 6-BA was greater than that of NAA. 6-BA showed a more significant promotional effect on T. arguta, consistent with the results of hormone screening in previous studies [69]. When the 6-BA concentration was 0.1–0.5 mg/L, the treatment supplemented with 0.03 mg/L of NAA had better proliferation than the treatment supplemented with 0.01 mg/L of NAA or 0.05 mg/L of NAA, indicating that T. arguta was not sensitive to too high or too low NAA levels. Many plants, such as Jatropha curcas [70], Hevea brasiliensis [71], and tomatoes [72], exhibit similar effects. At these suitable concentrations, T. arguta group plantlets produce a large number of adventitious shoots, which grow vigorously and can be used for rooting at a later stage with high utilisation.
As rooting is more difficult in woody plants than in other plants, the application of exogenous growth hormones has become the main method for optimising in vitro rooting [73]. Rooting induction is an extremely important part of plant tissue culturing [73], and the addition of growth auxins, such as IBA and NAA, to the rooting medium can effectively promote root development in many plant species [74,75,76]. According to previous studies, IBA was found to be an important factor in Castanea dentata [77], Vaccinium corymbosum [78], and Juniperus foetidissima Willd [79], and the addition of IBA could promote the rooting of tissue culture plantlets. In the present study, IBA increased and then decreased the rooting percentage and number of rooted strips with increasing the IBA concentration within a certain range, which is consistent with the findings of Upadhayay [80] and Khan [81], among others. Here, the highest rooting rate (66.60%) and the highest number of rooted strips (3.50) were recorded at an IBA concentration of 2.5 mg/L. The rooting rate and average number of rooted strips gradually decreased at an IBA concentrations greater than 2.5 mg/L. This may be attributed to the use of high concentrations of auxin, which inhibits the continuing differentiation of root primordia [82], leading to a decrease in the rooting rate and the number of rooted strips. In this study, the rooting rate of T. arguta was still low, which was related to the plant species in one way and the type of medium, hormone type, and concentration used in another way.
Acclimatisation is a key stage in the micropropagation process, during which plants are transitioned from a controlled laboratory environment to natural conditions [83], i.e., from in vitro-grown plantlets to an in vitro environment. Appropriate acclimatisation techniques, such as gradually exposing plantlets to the external environment and providing appropriate humidity and light, facilitate plant growth in the new environment [84]. This stage helps the plantlets to adapt to changes in their surroundings as well as to develop the structures needed to grow and survive in their natural environment [85]. In our study, T. arguta tissue culture plantlets successfully adapted and thrived in the in vitro environment, which is important to ensure the long-term survival of micropropagated plants [86]. The micropropagation protocol we have developed is essential for the efficient propagation of T. arguta species [87] and is an alternative way to conserve the genetic resources of the species [88].
Xiaolian et al. [12] showed that Turpiniae folium has high efficiency and low toxicity as a traditional Chinese medicine derived from T. arguta. Leaves are the main plant organs involved in photosynthesis, and they play an important role in plant life. Therefore, in the present study, we collected leaves and identified ligustroflavone and rhoifolin during the growth of transplanted plantlets using HPLC [89]. The ligustroflavone and rhoifolin contents observed in 5-month-old tissue culture T. arguta plantlets was greater than the requirements of the 2020 edition of the Chinese Pharmacopoeia. There was a significant difference in the active ingredient content in the leaves at different stages of growth. This is consistent with the results of Deng et al. [90], who showed that the flavonoid content in the leaves of Canada montana differed significantly during the nutritive growth, blooming, and post-flowering maturation periods and that the highest flavonoid content was noted during the blooming period. Guo et al. [91] showed that the developmental stage is a key factor in the differences in secondary metabolites of Ginkgo biloba flavonoids, based on the LC–MS metabolomics approach of Guo et al. [91]. Additionally, the leaf age [92], harvest season [93], and objective environmental factors [94,95] can affect active ingredient contents in the leaves of medicinal plants, as well as different parts of the medicinal plants (bark, leaves, seeds, roots, flowers, and stems); the extraction process (methods and solvents); and the setting of the mobile phase. Gori et al. [93] found that the leaves of the Mediterranean plant Phillyrea latifolia had the highest flavonoid content in the summer (July), which was higher than that in the spring (May) and fall (October), and the summer was the lowest. Lezoul et al. [96] showed that within the different organs of Passiflora caerulea, the flavonoid content was ranked in the following order: leaf > root > stem > flower. In Physalis peruviana, under a maceration condition, ethanol and acetone were almost the highest extraction solvents for extracting flavonoids. For Solanum muricatum, among the different extraction modes, the flavonoid content obtained from maceration was greater than that obtained via decoction. The results of this study, along with some exploratory findings on the accumulation trend of the active ingredient content of T. arguta in the growing stage, can provide a reference basis for the quality evaluation of T. arguta tissue culture plantlets and their rational development.

5. Conclusions

Our study established a rapid and effective protocol for the in vitro propagation of Turpinia arguta. And the contents of ligustroflavone and rhoifolin were significantly higher than the requirements of the Chinese Pharmacopoeia medicinal herbs in tissue culture plantlets of T. arguta. This protocol is of great significance for the large-scale production of T. arguta herbs with high contents of medicinal active ingredients.

Author Contributions

Conceptualization, methodology, software, validation, formal analysis, data curation, and writing—original draft preparation, J.H. and J.C.; investigation and resources, X.H.; L.W.; writing—review and editing, visualization, supervision, project administration, and funding acquisition, Q.C. and X.T. All authors have read and agreed to the published version of the manuscript.

Funding

This research was funded by the Basic Research and Talent Research Special Project of the Jiangxi Academy of Forestry (2023522001, 2022511603, and 2023512802) and the Jiangxi Provincial Forestry Bureau of Science and Technology Innovation Project “Selection and breeding of Chinese herbal medicine Turpinia arguta and research on planting mode” (Innovation Special [2023] No. 1).

Data Availability Statement

All data are presented in the article. Further inquiries can be directed to the corresponding author.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Different periods of growth of Turpinia arguta tissue culture plantlets after transplanting: (a) 0 month; (b) 3 months; (c) 5 months.
Figure 1. Different periods of growth of Turpinia arguta tissue culture plantlets after transplanting: (a) 0 month; (b) 3 months; (c) 5 months.
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Figure 2. The different stages of the micropropagation protocol of Turpinia arguta: (a) Explant disinfection and induction; (b) successional proliferation; (c) in vitro rooting; (d) in vitro acclimatization. Graph paper grid size = 5 mm × 5 mm. Bars = 1 cm.
Figure 2. The different stages of the micropropagation protocol of Turpinia arguta: (a) Explant disinfection and induction; (b) successional proliferation; (c) in vitro rooting; (d) in vitro acclimatization. Graph paper grid size = 5 mm × 5 mm. Bars = 1 cm.
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Figure 3. Chromatogram generated using high-performance liquid chromatography (HPLC). I. ligustroflavone; II. rhoifolin. Separate solutions of the two standards (a). Sample solutions for three different growth periods of Turpinia arguta: (b) 0 month; (c) 3 months; (d) 5 months.
Figure 3. Chromatogram generated using high-performance liquid chromatography (HPLC). I. ligustroflavone; II. rhoifolin. Separate solutions of the two standards (a). Sample solutions for three different growth periods of Turpinia arguta: (b) 0 month; (c) 3 months; (d) 5 months.
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Table 1. Successional proliferation media with different concentrations of PGRs.
Table 1. Successional proliferation media with different concentrations of PGRs.
Treatments
(Combination of 6-BA and NAA—mg/L)
0.1 + 0.01
0.1 + 0.03
0.1 + 0.05
0.2 + 0.01
0.2 + 0.03
0.2 + 0.05
0.5 + 0.01
0.5 + 0.03
0.5 + 0.05
Table 2. Effect of disinfection times with mercuric chloride (HgCl2) from explants of Turpinia arguta observed after 30 days.
Table 2. Effect of disinfection times with mercuric chloride (HgCl2) from explants of Turpinia arguta observed after 30 days.
Disinfection HgCl2 Time (min)Contamination Rate (%)Mortality Rate (%)Survival Rate (%)
468.01 ± 3.01 a7.97 ± 3.30 c24.02 ± 0.85 bc
658.09 ± 4.82 b6.00 ± 0.21 c35.91 ± 4.99 a
856.00 ± 2.95 bc16.05 ± 3.76 b27.94 ± 2.55 b
1051.96 ± 1.70 bc25.98 ± 3.06 a22.06 ± 3.89 bc
1250.00 ± 2.94 c30.51 ± 5.19 a20.47 ± 3.97 c
Note: Different lowercase letters in the same column indicate significant differences (p < 0.05).
Table 3. Effect of basic media on the proliferation of Turpinia arguta tissue culture plantlets after 30 days.
Table 3. Effect of basic media on the proliferation of Turpinia arguta tissue culture plantlets after 30 days.
Medium Type Proliferation CoefficientGrowth Situation
DCR1.65 ± 0.18 cPoor growth and wilted sprout leaves
WPM2.27 ± 0.13 bPoor growth, severe browning of callus, and dwarf plants
½ MS1.42 ± 0.16 cThe leaves were curved, and the plant was short
MS3.12 ± 0.10 aPlant growth was vigorous, the leaves were stretched, and the plantlets were tall and stout
Note: Different lowercase letters in the same column indicate significant differences (p < 0.05). Abbreviations: MS, Murashige and Skoog; DCR, Douglas fir cotyledon revised; WPM, Woody Plant Medium.
Table 4. Effect of plant growth regulations on the proliferation of Turpinia arguta tissue culture plantlets after 30 days.
Table 4. Effect of plant growth regulations on the proliferation of Turpinia arguta tissue culture plantlets after 30 days.
Treatments
(Combination of 6-BA and NAA—mg/L)		Proliferation Coefficient
0.1 + 0.011.33 ± 0.13 d
0.1 + 0.032.08 ± 0.14 c
0.1 + 0.051.93 ± 0.23 c
0.2 + 0.011.90 ± 0.19 c
0.2 + 0.033.13 ± 0.10 a
0.2 + 0.052.57 ± 0.08 b
0.5 + 0.012.77 ± 0.29 b
0.5 + 0.033.12 ± 0.06 a
0.5 + 0.052.60 ± 0.17 b
Note: Different lowercase letters in the same column indicate significant differences (p < 0.05). Abbreviations: 6-BA, 6-benzyladenine; NAA, naphthaleneacetic acid.
Table 5. Analysis of variance of the effects of different growth regulators on the proliferation of Turpinia arguta tissue culture plantlets.
Table 5. Analysis of variance of the effects of different growth regulators on the proliferation of Turpinia arguta tissue culture plantlets.
Source of
Variation
(Factor)		Sum of Squares
(SS)		Degrees of Freedom
(df)		Mean Square
(MS)		F
6-BA5.2222.6190.446 *
NAA2.72521.36347.217 *
6-BA × NAA0.92440.2318.002 *
error0.519180.029
total 162.51827
Abbreviations: 6-BA, 6-benzyladenine; NAA, naphthaleneacetic acid. Note: * indicates a significant difference (p < 0.05).
Table 6. Effect of the concentrations of IBA on the rooting rate (%) and number of roots of Turpinia arguta tissue culture plantlets after 30 days.
Table 6. Effect of the concentrations of IBA on the rooting rate (%) and number of roots of Turpinia arguta tissue culture plantlets after 30 days.
IBA (mg/L)Rooting Rate (%)Number of Roots
0.522.41 ± 2.51 e1.75 ± 0.10 d
1.035.61 ± 1.06 d2.44 ± 0.16 c
1.540.60 ± 3.28 d3.26 ± 0.14 b
2.056.67 ± 2.89 b3.67 ± 0.20 a
2.566.60 ± 1.86 a3.50 ± 0.16 ab
3.050.79 ± 3.25 c3.34 ± 0.10 b
Note: Different lowercase letters in the same column indicate significant differences (p < 0.05). Abbreviations: IBA, indole-3-butyric acid.
Table 7. Effect of growth period on ligustroflavone and rhoifolin contents from Turpinia arguta tissue culture plantlets after being transplanted in a greenhouse.
Table 7. Effect of growth period on ligustroflavone and rhoifolin contents from Turpinia arguta tissue culture plantlets after being transplanted in a greenhouse.
Growth Period (Months)Ligustroflavone (%)Rhoifolin (%)
00.172 ± 0.026 a0.030 ± 0.039 a
30.237 ± 0.019 b0.118 ± 0.035 b
50.469 ± 0.018 c0.188 ± 0.013 c
Note: Different lowercase letters in the same column indicate significant differences (p < 0.05).
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MDPI and ACS Style

Hu, J.; Cai, J.; Hu, X.; Wang, L.; Cheng, Q.; Tao, X. Efficient In Vitro Propagation of Turpinia arguta and Quantitative Analysis of Its Ligustroflavone and Rhoifolin Content. Horticulturae 2024, 10, 587. https://doi.org/10.3390/horticulturae10060587

AMA Style

Hu J, Cai J, Hu X, Wang L, Cheng Q, Tao X. Efficient In Vitro Propagation of Turpinia arguta and Quantitative Analysis of Its Ligustroflavone and Rhoifolin Content. Horticulturae. 2024; 10(6):587. https://doi.org/10.3390/horticulturae10060587

Chicago/Turabian Style

Hu, Jiangmei, Junhuo Cai, Xinrui Hu, Lijun Wang, Qiangqiang Cheng, and Xiuhua Tao. 2024. "Efficient In Vitro Propagation of Turpinia arguta and Quantitative Analysis of Its Ligustroflavone and Rhoifolin Content" Horticulturae 10, no. 6: 587. https://doi.org/10.3390/horticulturae10060587

APA Style

Hu, J., Cai, J., Hu, X., Wang, L., Cheng, Q., & Tao, X. (2024). Efficient In Vitro Propagation of Turpinia arguta and Quantitative Analysis of Its Ligustroflavone and Rhoifolin Content. Horticulturae, 10(6), 587. https://doi.org/10.3390/horticulturae10060587

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