1 Introduction

Anoectochilus roxburghii, also known as “Medicine King”, is a perennial herb belonging to the Orchidaceae family. It is mainly distributed in subtropical and tropical regions, especially in Southeast Asia and southern China. Anoectochilus roxburghii contains many active components such as polysaccharides, flavonoids, glycosides and polyphenols, which have various pharmacological effects such as antioxidation, anti-inflammation and immune regulation. Anoectochilus roxburghii is often used in traditional Chinese medicine to treat diabetes, liver diseases and problems of the immune system (Chen et al., 2020a). Modern pharmacology and the field of health supplements are paying more and more attention to it, which has also made people more interested in the cultivation and research of Anoectochilus roxburghii.

The medicinal demand for Anoectochilus roxburghii is increasing, but its wild population has been greatly reduced due to long-term excessive digging and habitat destruction. Wang et al. (2021) found that tissue culture technology provides an effective method to solve this problem. This technology can cultivate a large number of genetically identical seedlings under laboratory conditions, reducing the reliance on Anoectochilus roxburghii. In their 2022 study, Zhang et al. proposed that advancements in somatic embryogenesis, microreproduction, and the induction of protocorm-like body (PLB) have also significantly enhanced the tissue culture efficiency of Anoectochilus roxburghii. Some studies have also optimized conditions such as the medium formula, light intensity and temperature, which have increased the growth rate of the seedlings and the accumulation of active ingredients. However, Li et al. (2019) believe that there are still many difficulties in improving the survival rate of transplanted seedlings and the stability of the quality of medicinal products during in vitro acclimation.

This study explored the regulatory role of plant hormones in the tissue culture process of Anoectochilus roxburghii, analyzed the effects of different types and concentrations of hormones on the growth, metabolic activities and accumulation of secondary metabolites of seedlings, and hoped to further optimize tissue culture techniques through these studies to solve the existing problems such as low root growth rate and poor survival rate after transplantation. This study aims to provide new references for the application of plant hormones in tissue culture and offer lessons for the tissue culture and protection of other rare medicinal plants.

2 Growth and Metabolic Characteristics of Anoectochilus roxburghii Tissue Culture Seedlings

2.1 Current status and technical applications of tissue culture in Anoectochilus roxburghii seedlings

Tissue culture has become a key method for the propagation and protection of Anoectochilus roxburghii. The research by Xu et al. (2016) found that lighting with LED lights can promote the growth of Anoectochilus roxburghii seedlings and also increase the accumulation of flavonoids. The light source combining blue and red light (BR LED) is more effective than other lighting conditions in increasing plant height, stem thickness, leaf number and flavonoid content, and is regarded as a promising in vitro propagation technique. The research conducted by Zhang et al. in 2020 demonstrated that optimizing the formula of the culture medium could also enhance the induction, proliferation and regeneration efficiency of the protocorm-like body (PLBs), thereby increasing the success rate of micropropagation of Anoectochilus roxburghii.

2.2 Dynamic changes in major growth indicators of Anoectochilus roxburghii

The research by Wang et al. (2018) found that the seedlings of the Anoectochilus roxburghii grew better under the combined lighting of blue and red light (BR LED), with taller plants, thicker stems and larger leaf areas (Figure 1). Li et al. (2017) demonstrated that some endophytic fungi, such as Chaetomium globosum and Colletotrichum gloeosporioides, could promote the growth of plants. They can not only increase biomass, but also regulate the expression of genes related to plant growth. These research results indicate that environmental conditions and beneficial microorganisms play a very important role in the cultivation of Anoectochilus roxburghii.

Figure 1  Cultivation of Anoectochilus roxburghii under supplemental lighting with various light qualities (Adopted from Wang et al., 2018) Image caption: A: Red light; B: Blue light; C: Yellow light; D: Green light; E: White light; F: Control (40 days) (Adopted from Wang et al., 2018)

2.3 Characteristics and functions of secondary metabolites in Anoectochilus roxburghii

Gam et al. (2020) hold that flavonoids in the secondary metabolites of Anoectochilus roxburghii are of great significance. They accumulate the most during the soil cultivation stage, indicating that this stage is crucial for the formation of beneficial components. Ye et al. ’s research in 2020 found that after symbiosis with some mycorrhizal fungi (such as Ceratobasidium sp. AR), it could activate certain genes for synthesizing flavonoids, allowing for a greater accumulation of flavonoids and enhancing the medicinal quality of the plant. The protocorm-like body (PLBs) of Anoectochilus roxburghii can also be used to produce kinsenoside, and some flavonoids also show differential accumulation characteristics in different tissues or at different growth time points.

3 Relationship Between Hormone Types and Growth of Anoectochilus roxburghii Tissue Culture Seedlings

3.1 Regulation of root development by auxins in Anoectochilus roxburghii

Auxin, especially naphthylacetic acid (NAA), plays an important role in the root development of Anoectochilus roxburghii. The research of Saini et al. (2013) found that the most suitable concentration of NAA is 0.5 mg/ L. When NAA is used together with other hormones such as indole-3-butyric acid (IBA), it can also promote root growth more significantly. Adding NAA to the culture medium is the key to inducing root growth, and the combination of NAA and IBA is considered to achieve the best rooting effect.

3.2 Effects of cytokinins on tissue division and differentiation in Anoectochilus roxburghii

Cytokinins, such as 6-Benzylaminopurine (6-BA), are crucial in the division and differentiation of Anoectochilus roxburghii tissues. In 2018, Qin and Huang discovered that the combined use of 6-BA and naphthylacetic acid (NAA) could significantly enhance the proliferation of adventing buds and the differentiation efficiency of stem segments. When the concentration of 6-BA is 2.0 mg/L, it has the best effect on the induction and differentiation of buds and can also significantly increase the reproduction rate of tissue culture.

3.3 Role of gibberellins in plant elongation of Anoectochilus roxburghii

Gibberellin has a significant effect in promoting the elongation of plant stems. It is generally believed that gibberellin can help stems grow longer and may, along with other hormones, affect the growth of tissue culture seedlings. Olatunji et al. (2017) indicated that adding gibberellin to the culture medium might have a certain promoting effect on the elongation of plants, but more experiments are needed to confirm its actual effect on Anoectochilus roxburghii.

3.4 Regulation of stress adaptation by abscisic acid in Anoectochilus roxburghii

Abscisic acid (ABA) plays an important role in plants’ response to adverse conditions, and the the same is true for Anoectochilus roxburghii. It is generally believed that it can help plants resist drought and salt stress by regulating the opening and closing of stomata and balancing water. Cao et al. (2024) hold that if ABA is added to tissue culture, it may enhance the adaptability of Anoectochilus roxburghii seedlings to adverse environments.

3.5 Comprehensive effects of jasmonic acid and other hormones on the growth of Anoectochilus roxburghii

Jasmonic acid usually works together with other hormones in the growth of Anoectochilus. Previous studies have shown that jasmonic acid can interact with hormones such as auxin, cytokinin, and gibberellin to regulate plant growth, resistance responses, and the formation of secondary metabolites. Li et al. (2012) indicated in an early study that the participation of jasmonic acid in tissue culture might be helpful in improving the overall growth performance and developmental effect of Anoectochilus roxburghii.

4 Hormonal Regulation Mechanisms of Secondary Metabolism in Anoectochilus roxburghii

4.1 Effects of hormones on the synthesis and accumulation of polysaccharides in Anoectochilus roxburghii

Different light exposure can affect the activity of hormones, thereby altering the content of polysaccharides. The content of soluble sugar and polysaccharides in the body of Anoectochilus roxburghii is significantly higher under yellow light than under normal light conditions, indicating that hormone regulation can promote the synthesis of polysaccharides under specific light conditions. The presence of endophytic fungi can also affect the hormone balance in plants, thereby promoting the generation and accumulation of polysaccharides. Yang et al. (2022) demonstrated that these results indicated a complex relationship between hormone signals and secondary metabolites.

4.2 Role of hormones in flavonoid and polyphenol metabolic pathways

The expression of genes such as chalcone synthase (CHS) and flavonol synthase (FLS), which are related to flavonoid synthesis, is affected by hormone signals, and hormone signals can be regulated by environmental factors such as light intensity. The research by Chen et al. (2020b) found that blue light can promote the accumulation of total flavonoids and polyphenols in Anoectochilus roxburghii. This might be because blue light enhances the expression of these key genes through hormonal pathways (Figure 2) (Zhang et al., 2020). The participation of mycorrhizal fungi can also change the hormone levels in plants, thereby increasing the content of flavonoids, indicating that hormones play a bridging role between fungal action and flavonoid synthesis.

Figure 2  The flavonoid biosynthetic pathway in A. roxburghii (Adopted from Zhang et al., 2020)

4.3 Regulation of medicinal component content by hormone types and concentrations

Han et al. (2020) found in their research that the medicinal components such as flavonoids and polysaccharides in Anoectochilus roxburghii can be affected by the types and concentrations of hormones. The changes in hormone signals can lead to alterations in the expression of genes related to secondary metabolism under different environmental conditions. Specific light exposure can significantly increase the content of flavonoids and polysaccharides by adjusting hormone balance. The interaction between Anoectochilus roxburghii and endophytic fungi can also promote the accumulation of these active components through hormonal pathways. Wang et al. (2018) hold that to enhance the medicinal value of Anoectochilus roxburghii, precise regulation of hormones is required.

5 Molecular Mechanisms of Hormonal Regulation in Anoectochilus roxburghii Metabolism

5.1 Roles of hormonal signaling pathways in Anoectochilus roxburghii

Hormone signaling pathways regulate many physiological activities, including the synthesis of secondary metabolites such as anthocyanins (Zeng et al., 2017). Yuan et al. (2024) ’s recent research found that light intensity can affect the hormone levels in plants, thereby influencing the synthesis of anthocyanin glycosides. Hormone regulation helps Anoectochilus roxburghii adapt to changes in the external environment, indicating that hormone signals are an indispensable part of its metabolic regulation.

5.2 Hormonal regulation of key enzyme gene expression in Anoectochilus roxburghii Metabolism

In the study conducted by Aerts et al. in 2020, it was demonstrated that light intensity can affect the expression of genes such as chalkeone synthase (CHS), flavonol synthase (FLS), and flavonoid 3’-monooxygenase (F3’h), which play significant roles in anthocyanin synthesis. Xu et al. (2024) found that the transcription factors related to these enzyme genes contain many hormone-responsive cis-elements, indicating that hormones play a key role in regulating the expression of these metabolic pathway genes.

5.3 Molecular mechanisms of hormonal balance in growth and metabolism of Anoectochilus roxburghii

Light can affect the hormone levels in plants, causing changes in anthocyanin content, and anthocyanin is one of the important secondary metabolites of Anoectochilus roxburghii. Further studies have also found that hormone balance is achieved through the interaction between endogenous hormones and some specific transcription factors, which regulate the expression of genes related to metabolism (Liu et al., 2015). This hormone regulatory mechanism is beneficial for Anoectochilus roxburghii to maintain a good growth state and metabolic efficiency when facing environmental changes.

6 Optimized Application of Hormone Types and Concentrations in Anoectochilus roxburghii Tissue Culture

6.1 Practical application cases of different hormone combinations in Anoectochilus roxburghii transplantation

The combination of different hormones has been proven to effectively increase the survival rate of transplantation in the tissue culture of Anoectochilus roxburghii. Adding 6-BA, NAA and KT to the MS medium can well induce the formation of clustered buds, among which using stem segments as explants has the best effect. The study by Qie et al. (2021) also found that the incidence of buds was as high as 95.92% in the medium with 6-BA, NAA and ZT added, indicating that these hormones are useful for promoting the survival of transplanting. The selection of the right hormone combination is crucial for improving the transplantation success rate of Anoectochilus roxburghii (Jim, 2024).

6.2 Promotion of growth and metabolism by optimizing hormone concentrations

Different concentrations of hormones (such as 6-BA, NAA and 2,4-D) can affect the induction and reproductive effects of the protocorm-like body and adventitious bud. Delfosse et al. (2020) found that in the medium containing 6-BA (2.0 mg/L), NAA (0.5 mg/L) and TDZ (0.2 mg/L), the proliferation rate of Anoectochilus roxburghii reached 23 times within 90 days. Delfosse et al. ’s research in 2015 demonstrated that the use of low concentrations of hormones can also induce thicker buds, promote the differentiation of stems and leaves, thereby helping to enhance the overall growth and metabolic efficiency of plants.

6.3 Research on synergistic effects of hormones, substrates, and environmental conditions

The coordination among hormones, culture media and environmental conditions has a significant impact on the tissue culture effect of Anoectochilus roxburghii. The combination of 6-BA and 2,4-D performed best in callus induction, and the induction rates of rhizomes and young stems were higher than those of leaves and in vivo embryos. The light conditions can affect the degree of callus browning rates. The browning rate under long-term dark treatment is lower than that under alternating light and dark conditions (Holmstrup et al., 2010). When improving the efficiency of tissue culture, environmental factors such as hormones, substrates and light should all be taken into account (Zhang and Guo, 2009).

7 Case Studies: Application of Hormonal Optimization in Anoectochilus roxburghii Cultivation

7.1 Analysis of successful cases of hormonal treatments

Appropriate hormone combination treatment can significantly improve the induction and proliferation effect of the callus tissue of Anoectochilus roxburghii. Izuddin et al. (2018) found that the combined use of hormones such as 6-benzylaminopine (6-BA), naphthylacetic acid (NAA), and zeatin (ZT) could effectively promote the formation and expansion of callus tissue. Phillips et al. (2020) believed that the most effective medium formula was to add 0.2 mg/L of 2,4-D, 0.9 mg/L of NAA, 1.0 mg/L of 6-BA and 0.25 mg/L of ZT to the MS medium. The induction rate of this combination on rhizomes and young stems was much higher than that on leaves and in vivo embryos. The combined treatment with 6-BA, NAA, ZT and 2,4-D can successfully induce protocorm-like body (PLBs), increase the proliferation and regeneration rates, and achieve very good results (Jiang, 2024).

7.2 Practical application of hormone combinations and concentration optimization strategies

Researchers have made many attempts in the use of hormones to enhance the micropropagation efficiency of Anoectochilus roxburghii and achieved remarkable results. In large-scale propagation, the use of semi-intensity MS medium with 1.5 mg/L 6-BA added can achieve a high bud incidence rate of 91.67%, which is one of the best conditions for bud induction effect. The research conducted by Zhang et al. in 2015 found that adding 3.0 mg/L BA, 1.0 mg/L kinetin (Kn), and 0.5 mg/L NAA to the semi-intensity MS medium could achieve the highest bud proliferation rate.

7.3 Insights and potential for extending successful practices to other orchid species

The successful experience of hormone optimization in the cultivation of Anoectochilus roxburghii provides a good reference for the propagation of other orchid plants. The combination and dosage of these hormones have been proven to effectively enhance the growth, proliferation and regeneration efficiency of plants, which is beneficial for the protection and industrialized cultivation of endangered orchid plants. If these methods are drawn upon, the reproductive success rate and survival rate of other orchid plants can also be enhanced, promoting their protection and sustainable utilization. Chen et al. (2021) demonstrated in their study that the IPR-PLB technology developed for Anoectochilus roxburghii can serve as a reference model for the research of other orchid plants.

8 Concluding Remarks

Plant hormones are very important in the growth and metabolism of Anoectochilus roxburghii tissue culture seedlings. The changes of endogenous hormones can affect the synthesis of secondary metabolites like anthocyanins, and these substances play an important role in plants' adaptation to environmental conditions such as light. Hormones such as 6-benzylaminopine (6-BA), naphthylacetic acid (NAA), and kinetin (KT) can improve the growth environment of tissue culture and affect the number of leaves, the height of stems, and the formation of lateral branches. These hormones also play a crucial role in the stage of inducing and proliferating the protocorm-like body and adventitious buds, which is a very important step in the tissue culture process.

Reasonable adjustment of hormone concentration in tissue culture can promote the growth and development of Anoectochilus roxburghii and enhance its medicinal value. By precisely controlling hormone levels, scientists can significantly enhance reproductive efficiency, enabling plants to produce more active ingredients with medicinal effects. These components are precisely the key to the medicinal effects of the Anoectochilus roxburghii. A further understanding of how hormones regulate these metabolic processes is also helpful for increasing the production of important secondary metabolites, thereby enhancing the therapeutic effect of Anoectochilus roxburghii in traditional Chinese medicine.

Future research should pay more attention to how hormones regulate the synthesis of secondary metabolites of Anoectochilus roxburghii. Techniques such as metabolomics and transcriptomics can be used to identify the key genes and transcription factors involved in these processes. Whether there is a synergistic effect among different hormone combinations is also worth further exploration, which may be useful for optimizing the tissue culture program. In terms of practical application, it is recommended to develop more standardized cultivation methods to steadily enhance the medicinal effects of Anoectochilus roxburghii and ensure that its quality and efficacy remain consistent in herbal use.

Acknowledgments

We would like to express our heartfelt thanks to all colleagues who provided guidance and assistance in this study.

Conflict of Interest Disclosure

The authors affirm that this research was conducted without any commercial or financial relationships that could be construed as a potential conflict of interest.

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