1 Introduction

Anoectochilus roxburghii, also known as "Jewel Orchid", is a highly regarded medicinal and edible plant in traditional Chinese medicine, renowned for its efficacy in treating hypertension, diabetes, liver diseases, and as a tonic ingredient (Gam et al., 2020; Ye et al., 2020). Its pharmacological benefits are mainly attributed to its rich bioactive components, including flavonoids, kinsenoside and polysaccharides (Ye et al., 2017; 2020). However, excessive digging and slow natural reproduction rate have led to its endangered status, and conservation and sustainable cultivation efforts are urgently needed (Gam et al., 2020).

Tissue culture has become an important technical means for the large-scale propagation and resource conservation of A. roxburghii. It can not only obtain high-quality and disease-free seedlings, but effectively preserve valuable germplasm resources (Gam et al., 2020; Zhang et al., 2025a). By optimizing specific growth regulators and medium formulations, researchers enhanced bud cluster proliferation, root induction efficiency, and transplanting survival rate, thereby simultaneously supporting commercial production and germplasm conservation (Zhang et al., 2025a).

Root development is the foundation of plant health, and directly affects the absorption of nutrients and water, biomass accumulation and overall growth performance (Ye et al., 2020; Zhang et al., 2025a; Zhong et al., 2025). A well-developed root system, manifested as more lateral roots and root hairs, has been proven to contribute to enhancing plant vitality and the accumulation of secondary metabolites (Ye et al., 2020).

Antioxidant capacity plays a role in the process of plants adapting to adverse conditions, mainly relying on the activities of antioxidant enzymes, like superoxide dismutase, catalase and peroxidase. These enzymes can alleviate oxidative stress, and enhance the stress resistance of plants in adverse environments, such as drought, salt stress and extreme temperature (Sun et al., 2023; Zhong et al., 2025). In A. roxburghii, the enhancement of antioxidant enzyme activity is closely related to its stress tolerance, and the maintenance of physiological functions (Ye et al., 2017; Cui et al., 2023; Zhong et al., 2025).

This study explored the effects of different growth regulators, such as auxin, cytokinin, and novel compounds, on the root development of tissue culture seedlings of A. roxburghii, with the aim of optimizing the culture protocol for inducing robust root systems, and establishing complete plant growth. It will also clarify how different regulatory treatments regulate the activity of antioxidant enzymes and the overall antioxidant capacity, thereby enhancing the stress resistance and quality of tissue culture seedlings. This study will provide beneficial theoretical and practical references for improving the cultivation efficiency, protection and utilization, and medicinal value of A. roxburghii.

2 Biological Characteristics of A. roxburghii and Tissue Culture Propagation

2.1 Botanical features of A. roxburghii

A. roxburghii belongs to the genus Anoectochilus of the Orchidaceae family. It is a perennial herb with both medicinal and ornamental values (Huang et al., 2022). Its morphological characteristics are mainly manifested as soft ovate leaves, with veins in a distinct reticular pattern, slender and creeping stems, and a rhizome type of root system. This plant can produce small and beautiful flowers, and it shows significant differences in the size of its root system and leaves, especially between diploid and tetraploid plants, the latter often having stronger root systems and larger stomata (Figure 1).

The natural habitat of A. roxburghii is mostly distributed in the shady, and moist forest understory environment. The soil is loose, well-drained and rich in organic matter (Shao et al., 2014; Huang et al., 2022). This plant is adapted to low-light environments and has a certain ecological adaptability. But, it is extremely sensitive to environmental disturbances and excessive digging, resulting in its endangered status in the wild (Shao et al., 2014). Studies show that A. roxburghii grows best in an environment with about 30% shading. This condition helps it maintain healthy photosynthesis and promote root development.

2.2 Advances in tissue culture techniques

Recent years, some progress has been made in the study of tissue culture of A. roxanensis. Stem segments, terminal buds and pseudo-embryos (PLBs) have been confirmed to be excellent explant materials for rapid propagation (Wang et al., 2022; Yu et al., 2025). During the culture initiation stage, strict aseptic techniques are of crucial importance, including disinfection with 70% alcohol and sodium hypochlorite, to ensure successful culture and prevent contamination (Yu et al., 2025). Besides, the selection of midsection stem tissues without terminal buds and without basal roots, has been proven to have a higher survival rate and proliferation rate.

The combination of culture medium and growth regulator, is a key factor affecting bud cluster proliferation and rooting efficiency. Murashige and Skoog (MS) media are usually used in combination with cytokinins (6-BA) and auxin (like NAA, IBA) (Wang et al., 2022; Zhang et al., 2025a). On this basis, additives such as banana puree and activated carbon can further promote root growth and the vigorous growth of seedlings. Light quality also has a significant impact on plant growth and secondary metabolism, especially the combination light of red and blue leds, which helps promote biomass accumulation and metabolite synthesis (Wang et al., 2018; Gam et al., 2020; Wu et al., 2024). Leds with a 1:1 ratio of red and blue light have been proven to simultaneously optimize plant growth and polyamine metabolism (Wu et al., 2022; 2024).

2.3 Bottlenecks and improvement needs

Despite the continuous advancement of tissue culture propagation techniques, domestication and transplantation remain the difficult links in the expansion and propagation of A. roxanensis. The survival rate of tissue culture seedlings when transplanted into soil is often limited by poor root development and environmental stress. Even under the optimized scheme, the survival rate can reach 80% to 95%, but it often decreases significantly under uncontrolled conditions (Yu et al., 2025; Zhang et al., 2025a).

The current main problems are concentrated on insufficient root development and weak stress resistance, which limit the successful establishment and long-term growth of propagating plants (Yu et al., 2025; Zhang et al., 2025a; b). Compared with wild plants, the root system of tissue culture seedlings is weaker, and their tolerance to drought, salt and extreme temperature environments is poorer (Zhang et al., 2025b; Zhong et al., 2025). So, in the future, it is urgently necessary to further optimize the application strategies of growth regulators, light quality regulation and domestication techniques, etc., in order to improve the root structure and stress resistance (Wang et al., 2018; Wu et al., 2022).

3 Types and Mechanisms of Growth Regulators

3.1 Auxin-based regulators

Auxin, especially indole-3-acetic acid (IAA) and indole-3-butyric acid (IBA), play a fundamental role in root induction in the tissue culture of A. roxanensis. In which, the effect of IBA is particularly obvious. Studies have shown that when 1.0 mg/L IBA and 1.0 mg/L NAA were added to the MS medium, the rooting rate could reach 92%, and an average of 2.62 roots were produced per plant, which was superior to other auxin treatments (Zhang et al., 2025a). IAA can also promote root formation, but since IBA is more stable and has a better induction effect on adventite roots under in vitro conditions, it is usually more favored (Hong et al., 2020). These auxin mainly promote the elongation and division of cells at the root origin, eventually forming a strong root system.

The concentration of auxin directly affects the length and quantity of roots. The optimal concentration, like 1.0 mg/L IBA, can maximize the rooting efficiency, while concentrations that are too high or too low may inhibit root growth or cause abnormal root morphology (Zhang et al., 2025a). Excessive auxin can cause callus formation or root dwarfing, while insufficient concentration leads to low rooting efficiency. Therefore, precisely regulating the auxin concentration is the key to achieving a uniform and robust root system in the rapid tissue propagation of A. roxanensis.

3.2 Cytokinin-based regulators

6-benzylaminopine (6-BA) is an artificially synthesized cytokinin that also plays a key role in the proliferation and differentiation of bud clusters in A. roxanensis. Studies have shown that when the concentration of 6-BA is 1.0-2.0 mg/L and combined with a low concentration of NAA, the best bud cluster proliferation effect can be achieved, with a high induction rate and an increase in the number of buds produced by a single explant (Zhang et al., 2025a). Although cytokinin mainly promotes bud formation, it also affects root structure in rooting culture media. Therefore, it is often necessary to maintain a reasonable balance with auxin. For instance, 1.0 mg/L 6-BA + 0.05 mg/L NAA is most suitable for bud cluster proliferation, while high concentrations of cytokinin may inhibit rooting.

The ratio of cytokinin to auxin is the key to determining the direction of organ differentiation: a higher cytokinin/auxin ratio is conducive to bud formation, while a higher auxin/cytokinin ratio is conducive to root formation (Zhang et al., 2025b). It was reported that, 1.0 mg/L 6-BA + 0.05 mg/L NAA is most suitable for bud cluster proliferation, while 1.0 mg/L IBA + 1.0 mg/L NAA is most conducive to root induction. This interaction is the foundation for the success of tissue culture, and can achieve the expected effect of plant regeneration through precise regulation.

3.3 Other types of regulators

Gibberellin (GA) mainly participates in promoting cell elongation and can enhance the growth of roots and buds. In A. roxanensis, gibberellin-related genes, such as GA20-oxidases, are closely related to growth regulation. Their down-regulation often leads to decreased plant height and abnormal root development (Liu et al., 2015; Zhang et al., 2025b). Although the exogenous addition of GA in tissue culture is not common, its endogenous regulation still plays an important role in the overall vitality of plants and root elongation, especially under adverse conditions or during the seed germination stage.

Salicylic acid (SA) and jasmonic acid (JA) are important regulatory factors, for plant defense and stress response. In A. roxanensis, the SA response pathway is activated under specific light conditions, which can promote the increase of antioxidant enzyme activity, and the accumulation of secondary metabolites (Li et al., 2024). These hormones improve the quality of tissue culture seedlings by regulating the antioxidant metabolism of plants, and enhancing their tolerance to abiotic stress.

4 Evaluation of Root Development: Phenotypic and Physiological Indicators

4.1 Root morphological measurements

In the tissue culture study of A. roxburghii, root development is usually evaluated by measuring root length, the number of roots per plant and root surface area. For instance, in vitro studies on tetraploid A. roxburghii have shown that, under the optimal rooting medium conditions (MS + 1.0 mg/L IBA + 1.0 mg/L NAA), each plant can form an average of 2.62 roots, with a rooting rate as high as 92%. And the improvement in seedling height and root robustness was more significant, compared with other treatments (Zhang et al., 2025a). Meanwhile, the root systems of tetraploid plants are more robust and show stronger vitality compared to diploid plants, indicating that the ploidy level can affect the morphological structure of roots, and overall growth performance (Huang et al., 2022).

Microscopic observation of the root tip meristem can reveal cellular activities, and the growth potential of the root system. Plants with larger root meristems and higher cell division rates tend to exhibit stronger root elongation and nutrient absorption capabilities, especially in those with improved genetic backgrounds or optimized hormone conditions (Huang et al., 2022).

4.2 Physiological functions of roots

The physiological functions of roots can be indirectly evaluated by measuring the accumulation levels of amino acids, mineral elements and soluble proteins in plant tissues. For instance, tetraploid A. roxburghii exhibited higher amino acid and mineral contents than diploid A. roxburghii, indicating its advantages in nutrient absorption and transport efficiency (Huang et al., 2022). Under phosphorus deficiency conditions, the application of strigolactone can increase the content of soluble protein and promote root elongation, further supporting the close connection between root development and nutrient absorption (Zhong et al., 2025).

Root vitality, as an important indicator of metabolic level, is often determined by the TTC (2,3, 5-triphenyltetrazolium chloride) reduction method. This method reflects the vitality of roots by detecting the ability of root tissue to reduce TTC to Formazan. Studies have shown that, different light qualities and hormone treatments have significant effects on root viability. Among them, supplementary light, like yellow light, can enhance the root viability of A. roxburghii (Wang et al., 2018).

4.3 Relationship between root development and seedling survival

Robust root systems are closely related to a higher transplanting survival rate. In the tissue culture of A. roxburghii, the survival rate of plants with good root systems after domestication can reach 80%, highlighting the importance of root vitality in the process of soil formation (Zhang et al., 2025a). In addition, the enhanced root development demonstrated by the quadruple system further improved the transplanting survival rate, and subsequent growth performance (Huang et al., 2022).

The coordinated development of roots, stems and leaves, is the key to ensuring the excellent traits of seedlings. Treatment measures (specific light schemes, hormone combinations, etc.), that can simultaneously promote the growth of roots and stems and leaves can cultivate more robust plants, which perform better in biomass accumulation and physiological functions, and have enhanced adaptability and stress resistance (Wang et al., 2018; Huang et al., 2022; Zhang et al., 2025a).

5 Measurement and Analysis of Antioxidant Capacity in A. roxburghii

5.1 Antioxidant enzyme activity assays

Generally, the antioxidant capacity of A. roxburghii, is evaluated by detecting the activities of superoxide dismutase (SOD), peroxidase (POD), and catalase (CAT). Studies have shown that, in both in vitro and in vivo experiments, the extracts and polysaccharides of A. roxburghii can increase the activities of SOD and CAT, and reduce the level of malondialdehyde (MDA), indicating a strong protective effect against oxidative stress (Zeng et al., 2016; 2017; Wang et al., 2020). For instance, pretreatment with ARPP80 polysaccharide can restore the levels of SOD and CAT in the liver and serum of mice, and its effect is comparable to that of standard antioxidant drugs (Zeng et al., 2016). Light quality and growth regulator treatment can also regulate the activities of these enzymes. Among them, blue light can increase the activities of SOD, POD and CAT in plant tissues (Ye et al., 2017; Wu et al., 2024).

Comparative analysis reveals that, different extraction methods, dosages, and environmental conditions (such as light quality) can lead to differences in the activity of antioxidant enzymes. Medium and low doses of phenolic extracts can enhance the activities of SOD and glutathione peroxidase (GSH-Px), and reduce the MDA level in oxidative stress models, while high doses may have poor effects or even negative effects (Xu et al., 2017; Wang et al., 2020).

5.2 Non-enzymatic antioxidant compounds

A. Roxburghii is rich in non-enzymatic antioxidant substances, mainly including phenols and flavonoids. Quantitative determinations (Folin-Ciocalteu method for total phenol determination, the NaNO₂-Al (NO₃)₃-NaOH method for flavonoid determination, etc.) indicated that, the contents of total phenol and total flavonoids were the highest under the residue extract and specific light quality or hormone treatment (Xu et al., 2017; Xie et al., 2017; Ye et al., 2017). Quercetin, kaempferol and rutin are the main contributing compounds of antioxidant activity, and their contents increase under stress conditions or after exogenous application (Xie et al., 2017; Bin et al., 2022; Cui et al., 2023).

The accumulation of secondary metabolites such as phenols, flavonoids and polysaccharides is correlated with the overall antioxidant capacity. These compounds can not only directly eliminate free radicals, but synergistically enhance the function of the enzymatic antioxidant system, improving the ability of plants to resist oxidative damage (Yang et al., 2017; Liu et al., 2020; Wu et al., 2021; Qiu et al., 2023).

5.3 Antioxidant capacity and stress tolerance

Growth regulators that can enhance the activity of antioxidant enzymes and the accumulation of secondary metabolites, as well as environmental factors (such as light quality), can also improve the stress resistance of A. Roxburghii. For instance, exogenous quercetin treatment can enhance antioxidant capacity, and also increase the heat tolerance of plants. Meanwhile, polysaccharide and flavonoid extracts can effectively reduce oxidative damage in animal and cell models (Wang et al., 2020; Cui et al., 2023).

Enhanced root antioxidant capacity, such as higher SOD, CAT activities and non-enzymatic antioxidant levels, helps to increase the survival rate, growth rate and environmental stress resistance of seedlings. This further emphasizes the importance of optimizing the application scheme of growth regulators, to maximize root development and antioxidant defense simultaneously (Ye et al., 2017; Wu et al., 2024).

6 Case Studies

6.1 Combined effects of IBA and 6-BA on root development

Anoectochilus roxburghii, as a precious traditional Chinese medicine and ornamental plant, is endangered in its wild due to excessive collection and habitat destruction. Therefore, it is of great significance to establish an efficient artificial breeding system. Zhang et al. (2025a) found that, under different hormone combinations, culture media and photopolymer conditions, the proliferation effects of tetraploid plants of A. roxburghii varied significantly. Among them, the ratio of 1 mg/L 6-BA to 0.05 mg/L NAA could significantly promote bud cluster differentiation. The average number of proliferating buds per explant reached 3.06, and the induction rate exceeded 82% (Figure 2). In terms of the culture medium, the B5 formula performed better than MS and its diluted medium, with both the number of bud clusters and the induction rate reaching the optimal level. The light quality test showed that red light was most conducive to bud proliferation, with an induction rate as high as 92%, and the plants grew vigorously.

During the rooting stage, studies have shown that, MS medium combined with 1.0 mg/L IBA and 1.0 mg/L NAA can effectively promote rooting, with an average of 2.62 roots per plant and a survival rate exceeding 90%. Further domestication experiments have shown that transplanting with a 3:1 mixed substrate of peat soil and vermiculite can achieve a survival rate of up to 80%, providing feasibility for large-scale production. It established a complete tissue culture technology system for tetraploid A. roxburghii from proliferation to transplantation, providing a new approach for the protection of endangered resources, and laying a foundation for its medicinal and industrial utilization.

6.2 The effect of exogenous putretic amine on the antioxidant activity of A. roxburghii

A. roxburghii is rich in active substances such as polysaccharides, flavonoids and terpenoids, but its adaptability is relatively weak and it is extremely sensitive to environmental stress, especially drought. Drought conditions can lead to a large accumulation of reactive oxygen species (ROS) in the body, thereby triggering membrane lipid peroxidation, damaging cell structure, and weakening plant growth and medicinal material quality (Cui et al., 2023; Sun et al., 2023). The antioxidant defense system is an important mechanism for plants to alleviate water stress, and how to effectively enhance the function of this system has always been a research focus.

Sun et al. (2023) took A. roxburghii as the research object, to explore the role of exogenous spermidine (Spd) in alleviating water stress. The experiment used PEG6000 to simulate different degrees of drought. The results showed that under moderate water stress, the growth of A. roxburghii plants was restricted, manifested as a decrease in plant height, fresh weight, leaf area and stem diameter. At the same time, the activities of antioxidant enzymes (SOD, POD, CAT) and key enzymes of polyamine metabolism (ADC, SAMDC, PAO) decreased. However, the MDA, H2O2 and electrical conductivity of membrane lipid peroxidation products increased significantly.

However, after foliar spraying of an appropriate amount of Spd (0.5mM), the plants exhibited a significant stress relief effect. The contents of soluble protein and proline increased, MDA, H₂O₂ and electrical conductivity decreased, and the activity of antioxidant enzymes was enhanced, thereby effectively reducing oxidative damage and maintaining the integrity of cell membranes (Figure 3). Meanwhile, Spd treatment promoted the accumulation of endogenous polyamines (e.g., Put, Spd and Spm), improved metabolic balance and enhanced the overall drought resistance of plants. It is worth noting that, the effect of low-concentration Spd is superior to that of high-concentration SPD, showing a dose-dependent approach.

7 Discussion

7.1 Overall effects of growth regulators on root development

Studies have shown that the combined use of growth regulators (IBA and NAA or IBA and 6-BA), is generally more effective than single hormone treatment in promoting the number of roots, root length and fresh weight of A. roxburghii tissue culture. When 1.0 mg/L IBA and 1.0 mg/L NAA were added to the MS medium, the rooting rate could reach 92%, and the stem and leaf growth was better than that of the single hormone treatment group (Zhang et al., 2025a). Furthermore, the application of strigolactone under phosphorus deficiency conditions, can promote root elongation and reduce oxidative damage, further supporting the advantages of the application of compound or synergistic regulators (Zhong et al., 2025).

This combined effect mainly stems from the complementary roles of different hormones in cell division, elongation and differentiation. Auxin (like IBA and NAA) mainly stimulate root induction, while cytokinins, such as 6-BA, promote cell division and bud cluster formation. Strigolactone and other novel regulators may further optimize root structure, and reduce oxidative stress by regulating hormone signaling and stress response (Zhang et al., 2025a; Zhong et al., 2025).

7.2 Relationship between root development and antioxidant capacity

Existing studies have shown that, robust root development is positively correlated with the increase of antioxidant enzyme activities (SOD, CAT, POD) and the accumulation of secondary metabolites (flavonoids, polysaccharides, etc.) (Ye et al., 2017; Wang et al., 2018). Treatments that can enhance root vitality, such as optimizing light quality or a reasonable combination of hormones, usually can also improve antioxidant capacity, indicating that there is a coordinated mechanism between root development and antioxidant response, jointly promoting plant growth and stress resistance (Ye et al., 2017; Wang et al., 2018; Sun et al., 2023).

High-level antioxidant capacity, can help reduce oxidative stress in the transplanting process of A. roxburghii seedlings, thereby increasing the survival rate. The application of exogenous antioxidants or growth regulators has been proven to enhance the stress tolerance of plants, and increase the survival rate after transplantation (Zeng et al., 2016; Sun et al., 2023).

7.3 Implications for A. roxburghii tissue culture production

Optimizing the types and concentrations of growth regulators, combined with environmental factors, like light quality, is the key to cultivating high-quality and healthy seedlings. Studies have found that the combined use of auxin and cytokinin, as well as the supplementation of blue or red light treatment, can maximize the promotion of root development and the accumulation of antioxidant substances (Wang et al., 2018; Zhang et al., 2025a).

The adoption of environmentally friendly regulators, such as strigolactone and natural polyamines, offers new prospects for the sustainable and large-scale production of A. roxburghii. These methods can enhance the growth and stress resistance of plants, but also comply with environmental protection and health safety standards, which is conducive to the resource protection and industrial utilization of this precious medicinal plant (Sun et al., 2023; Zhong et al., 2025).

8 Concluding Remarks

Growth regulators play a unique role in the root development of A. roxburghii and sometimes produce a synergistic effect. Common auxin (like IBA, NAA), cytokinins (6-BA), and novel hormones (strigolactone) perform outstandingly in promoting the number, length and rooting rate of root systems. Combined treatment (1.0 mg/L IBA + 1.0 mg/L NAA), is often more effective than a single hormone, with a rooting rate of up to 92% and vigorous growth of stems and leaves. Strigolactone can also alleviate the inhibition of stress on root growth and promote root elongation in a phosphorus-deficient environment. The effect is more obvious when hormone treatment is combined with environmental factors (light quality). Blue light and red light can not only improve the overall growth of plants, but also enhance the activity of antioxidant enzymes (SOD, CAT, POD), and promote the accumulation of secondary metabolites, like flavonoids and polysaccharides, thereby enhancing stress resistance and medicinal value.

But, it should be noted that most studies are still confined to in vitro or greenhouse conditions and may not fully reflect the actual situation in the complex field environment. Under natural conditions, root development and antioxidant responses may yield different results, thereby affecting the universality of research conclusions. Although there have been many achievements at the physiological and biochemical levels, there is still a lack of research at the molecular level that, links hormone treatment with gene expression and metabolic pathways. The latest advancements in transcriptomics suggest that, further exploration of its underlying mechanisms is needed.

Future research should integrate genomics, transcriptomics and metabolomics, to reveal the molecular networks induced by regulators, so as to precisely regulate plant growth and stress response. The optimized application of hormones and environmentally friendly treatments (e.g., strigolactone, polyamines, and customized photolithoplasm), are worthy of promotion in commercial seedling cultivation and the production of medicinal components, which will provide support for the sustainable cultivation and resource utilization of A. roxburghii.

Acknowledgments

The authors sincerely thank Dr. Li for reviewing the manuscript and providing valuable suggestions, which contributed to its improvement. Additionally, heartfelt gratitude is extended to the two anonymous peer reviewers for their comprehensive evaluation of the manuscript.

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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