Review and Progress

Effects of Different Medium Formulations on the Proliferation Rate and Growth Performance of Anoectochilus roxburghii Tissue-Cultured Seedlings  

Zonghui Liu , Meifang Li
Tropical Medicinal Plant Research Center, Hainan Institute of Tropical Agricultural Resources, Sanya, 572025, Hainan, China
Author    Correspondence author
Plant Gene and Trait, 2025, Vol. 16, No. 2   
Received: 19 Mar., 2025    Accepted: 21 Apr., 2025    Published: 30 Apr., 2025
© 2025 BioPublisher Publishing Platform
This is an open access article published under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
Abstract

This study summarized how the carbon source, nitrogen source and plant hormones acted in the medium of Anoectochilus roxburghii, compared the effects of different formulations on the proliferation and growth of seedlings, sorted out the physical and chemical characteristics of the substrate, and saw if they were related to whether the seedlings grew well or not. This study also summarized the successful experiences and problems encountered in the optimization of culture media at home and abroad, and discussed how to use new technologies such as intelligent monitoring and precise regulation to assist. This study aims to provide theoretical support and operational reference for the efficient tissue culture of Anoectochilus roxburghii, and lay the foundation for its future industrialization.

Keywords
Anoectochilus roxburghii; Medium formulation; Tissue-cultured seedling; Proliferation rate; Growth quality

1 Introduction

A. roxburghii, known as the “King of Medicines”, is a well-known medicinal plant and is widely used in traditional Chinese medicine. It contains components such as flavonoids, polysaccharides and kinsenoside, and has the functions of antioxidation, anti-inflammation and liver protection. The wild resources of A. roxburghii are becoming increasingly scarce due to excessive digging and the destruction of its growth environment. It has become very necessary to find an efficient planting method to meet the market demand. Li et al. (2017) proposed that tissue culture technology can cultivate a large number of healthy seedlings, ensuring that resources are not damaged and can be continuously utilized.

 

Compared with traditional seed or cutting methods, tissue culture is faster, has a higher success rate, can avoid diseases and maintain genetic consistency. Zhang et al. (2015) found that it played a significant role in promoting the large-scale cultivation of Anoectochilus roxburghii, protecting the genetic resources of Anoectochilus roxburghii and meeting the needs in medicine and industry.

 

Wang et al. (2022) indicated that the formulation of the culture medium would directly affect the proliferation rate of the seedlings, the growth of the roots and the overall growth condition during this process. Components such as carbon sources, nitrogen sources, hormones and substrates can affect nutrient absorption and metabolism. Xu et al. ’s research in 2016 holds that the reasonable combination of these factors is the key to breeding healthy seedlings and is also related to the survival and field growth of the seedlings after transplantation.

 

This study tested different medium formulations to explore which combination was most suitable for the proliferation and growth of Anoectochilus roxburghii seedlings, and clarified how these components affected the development of the seedlings. Optimizing the medium formula is the core to improving the quantity and quality of Anoectochilus roxburghii seedlings. This research aims to promote the improvement of the cultivation technology of Anoectochilus roxburghii, meet the expanding market demand, and also provide references for the protection and utilization of other rare medicinal plants.

 

2 Biological Characteristics of Tissue-Cultured Seedlings of A. roxburghii

2.1 Growth habits and active components of A. roxburghii

Anoectochilus roxburghii is a very precious traditional Chinese medicine plant. It has medicinal effects such as anti-diabetes, anti-inflammation and liver protection, and is widely used in traditional medicine. The components it contains, such as polysaccharides, flavonoids, glycosides and kinsenoside, are the key to its medicinal effect (Ye et al., 2017). The research by Zhang et al. (2021) found that the contents of polysaccharides and flavonoids in tissue culture seedlings were similar to those in the wild, and some were even higher than those in the wild. Light is also crucial for its growth. Exposing it to specific LED lights can make it grow better and accumulate more flavonoids.

 

2.2 Application status of tissue culture technology in A. roxburghii cultivation

Tissue culture technology can rapidly and in large quantities propagate Anoectochilus roxburghii seedlings, providing a stable source for both medicinal and commercial use. It can also help cultivate some new materials, such as tetraploid Anoectochilus roxburghii, which grows faster and has more medicinal components than ordinary diploid ones (Figure 1). Huang et al. (2022) also improved the methods of micropropagation, and made considerable progress in the induction and propagation techniques of protocorm-like bodies (PLBs).

 


Figure 1 Morphological characteristics of Anoectochilus roxburghii under tissue culture: whole plant (A-C), leaves (D), and stem (E) (Adopted from Huang et al., 2022)

 

2.3 Nutritional requirements of tissue-cultured seedlings

In 2020, Gam et al. found that MS medium containing plant hormones (such as 6-benzylaminopurine (6-BA) and naphthaleneacetic acid (NAA)) could significantly promote the induction and expansion of protocorm-like bodies. Coexisting with beneficial fungi (such as Epulorhiza sp.) can further enhance its growth effect. This symbiosis is helpful for the plant to absorb more nutrients and make the plant stronger. The early research by Li et al. (2012) also found that the use of specific light (such as blue and red leds) can also optimize the growth environment, promote the growth of plants, and enable them to produce more medicinal components.

 

3 Key Factors in Medium Formulations

3.1 Effects of carbon and nitrogen sources and their ratios on proliferation rates

Zeng et al. (2017) found that using sucrose as a carbon source helps seedlings grow faster and also promotes the accumulation of effective components such as polysaccharides and kinsenoside in rhizomes. The best effect is achieved when the sucrose concentration is 35 g/ L. Combined with a 45 mM nitrogen source, the content of the active ingredient can reach the maximum value. Datta et al. (2022) attempted to use industrial wastewater rich in carbon and nitrogen resources as microbial fertilizer and also found that it could increase the yield of Anoectochilus roxburghii (Figure 2) (Huang et al., 2020).

 


Figure 2 Growth performance of Anoectochilus roxburghii under different medium formulations (Adopted from Huang et al., 2020)

Image caption: A: Control group treated with water; B: A. roxburghii treated with wastewater fermentation liquid once; C: A. roxburghii treated with wastewater fermentation liquid twice; D: A. roxburghii treated with wastewater fermentation liquid three times (Adopted from Huang et al., 2020)

 

3.2 Role of hormones in medium formulations

The influence of plant hormones on the proliferation and differentiation processes in the tissue culture of Anoectochilus roxburghii is very obvious. Adhikari and Pant (2019) found that when 3 mg/L of 6-BA and 0.5 mg/L of NAA were added to the culture medium, it could effectively induce and expand protocorm-like bodies (PLBs) and improve the proliferation efficiency. Huang et al. (2020) demonstrated that the combination of 6-BA with kinetin (KT) and NAA has also been proven to be beneficial for bud proliferation and root growth.

 

3.3 Regulation of seedling growth by substrate type and physical-chemical properties

Jin et al. ’s research in 2018 found that adding an appropriate concentration of hormones to a half-concentration MS medium could better promote bud formation and root growth. Adding a little banana pulp and activated carbon to the culture medium can also increase the rooting rate and the success rate of seedling domestication. The physical and chemical properties of the substrate can also affect the growth of seedlings. Only by providing a suitable environment can seedlings develop healthily and grow better.

 

4 Effects of Different Medium Formulations on the Proliferation Rate of Tissue-Cultured Seedlings

4.1 Impact of carbon sources on proliferation efficiency

Sucrose is one of the most commonly used carbon sources in tissue culture, and multiple studies have confirmed that it can enhance the proliferation effect. In the early experiments of Goldman and Dennett (2000), using a medium containing 30 g/L sucrose, the proliferation rate of Adventist buds reached 23 times within 90 days. The research of Adetunji et al. (2018) indicates that the formula of 28 g/L sucrose combined with other components can also achieve a proliferation coefficient of 2.7254. Sucrose is a very effective carbon source that can promote the rapid proliferation of Anoectochilus roxburghii tissue culture seedlings.

 

4.2 Optimization of nitrogen source forms

Nowadays, the commonly used inorganic nitrogen sources mostly come from the formulations in Murashige and Skoog (MS) media. Cui et al. (2021) found that many studies have adopted such nitrogen sources in combination with hormones and other additives to promote the induction and propagation of buds. Chakrawal et al. (2022) indicated that there are currently few studies on the comparative effects of organic nitrogen and inorganic nitrogen. In the future, a more in-depth exploration can be conducted on the specific differences between these two nitrogen source forms in the tissue culture of Anoectochilus roxburghii.

 

4.3 Influence of hormone ratios on multiplication rates

6-BA and NAA are common combinations that can promote the formation and expansion of Adventist buds. The study by Ml et al. in 2018 pointed out that the formula containing 2.0 mg/L 6-BA and 0.5 mg/L NAA had a very good effect on inducing adventative buds. In another experiment, 3 mg/L 6-BA combined with 0.8 mg/L NAA achieved a proliferation coefficient of more than 4.5 within two months. Reasonable combination of hormone ratios is an important step to improve the proliferation efficiency of tissue culture seedlings of Anoectochilus roxburghii.

 

5 Effects of Different Medium Formulations on the Growth Performance of Tissue-Cultured Seedlings

5.1 Regulation of root growth by substrate aeration and water retention

The growth condition of tissue culture seedlings of Anoectochilus roxburghii is closely related to the aeration and water retention capacity of the culture medium. Adding activated carbon to the culture medium can improve air permeability and water retention, and promote the rooting effect. The study by Neocleous and Savvas (2019) successfully achieved a 100% rooting rate using a combination of 1/2 MS medium, NAA and activated carbon. Liu et al. (2021) hold that adding some banana juice is also beneficial for root development, perhaps because banana juice improves the structure of the culture medium and provides more nutrients.

 

5.2 Impact of nutrient supply on leaf growth and photosynthetic efficiency

The nutritional components of the culture medium are crucial for leaf development and photosynthesis. Adding plant hormones 6-BA and NAA to the MS medium can promote the growth of leaves, enhance the vitality of plants and improve the proliferation efficiency. In 2019, Pissolato et al. found that adding some rare earth elements (such as La(NO3)3 and Ce(NO3)3) to the culture medium can also increase chlorophyll content and enzyme activity, and enhance photosynthetic efficiency.

 

5.3 Effects of medium formulations on biomass and active compound accumulation

Xiong et al. (2022) demonstrated that adjusting the concentrations of BA, kinetin (KT), and NAA, such as using a 0.75× MS basal medium combined with these hormones, can effectively increase the contents of polysaccharides and kinsenoside. Ye et al. discovered in 2021 that if yeast extract is added as an additive, the accumulation of these useful components can be further increased, indicating that the formula of the culture medium is crucial in enhancing medicinal value.

 

6 Successful Cases and Challenges in Medium Research Domestically and Internationally

6.1 Progress in optimizing A. roxburghii medium formulations in China

Many studies are striving to identify the optimal concentrations of plant hormones and other culture medium components in order to make Anoectochilus roxburghii seedlings grow faster and have a higher proliferation rate. Some experiments have found through orthogonal tests that the concentrations of plant hormones and sucrose are the key factors affecting bud proliferation. The most effective formula is to add 6-BA and NAA to the MS medium. Another study optimized the induction of the protocorm-like bodies (PLBs) and the proliferation of Adventist buds, achieving a 23-fold proliferation rate within 90 days. Quiroz et al. (2024) also found that adding rare earth elements such as La(NO3)3 and Ce(NO3)3 to the culture medium could promote bud formation and make the seedlings grow better, indicating that rare earth elements have potential in optimizing the culture conditions.

 

6.2 Experiences from tissue culture studies of similar medicinal plants abroad

Many tissue culture studies on similar medicinal plants abroad can draw on the research of Anoectochilus roxburghii. Adhikary et al. (2021) developed large-scale in vitro propagation techniques suitable for medicinal and ornamental plants, with a focus on optimizing the conditions for bud formation, proliferation and rooting. Marthe (2018) indicated that these studies usually combine the use of cytokinin and auxin to enhance proliferation efficiency and facilitate the smooth domestication of plants. Some techniques have also been introduced into the breeding of Anoectochilus roxburghii, demonstrating their application value in achieving large-scale reproduction and protecting endangered resources.

 

6.3 Technical bottlenecks and improvement directions in current research

The low regeneration rate of traditional propagation methods limits the number of seedlings, which is not conducive to the large-scale production of important components such as kinsenoside (Huang, 2024). During the process of formula optimization, due to the different effects of various components on growth, a large number of repeated experiments are required. Espinosa-Leal et al. (2018) proposed that in the future, the research focus could be placed on refined formulas and attempts could be made to use new additives to improve the growth effect of plants. Niazian (2019) demonstrated that a thorough understanding of the physiological responses of Anoectochilus roxburghii under different culture conditions is also beneficial for finding more efficient propagation methods.

 

7 Future Research Directions and Application Prospects

7.1 Prospects of precise optimization and standardization of medium formulations

Optimizing and standardizing the formula of the tissue culture medium for Anoectochilus roxburghii is crucial in enhancing the consistency of propagation efficiency and planting quality. Trinh et al. ’s research in 2024 demonstrated that choosing the right combination of plant growth regulators and nutrients can significantly enhance the proliferation rate and growth effect of seedlings. Shan (2024) discovered in the same year that adding an appropriate amount of 6-BA, NAA and some additives to the MS medium could promote the induction of buds and the formation of roots. Future research can refine these formulas to make the effects more stable and reliable, laying a solid foundation for large-scale commercial cultivation in the future.

 

7.2 Enhancing culture efficiency through intelligent monitoring technology

Regulating environmental conditions such as temperature, humidity and light through an automated system can enhance growth efficiency and also provide real-time data feedback for precise adjustments. The research by Gong et al. (2022) indicates that the use of specific LED lighting can accelerate the growth of Anoectochilus roxburghii and promote the accumulation of active components such as flavonoids, demonstrating the significant value of intelligent lighting systems in tissue culture. The application of these technologies can enhance production efficiency and make organizational culture more suitable for large-scale promotion.

 

7.3 Contribution of efficient tissue culture techniques to the industrial development of A. roxburghii

By developing rapid propagation methods, such as induction and propagation using protocorm-like bodies (PLBs), a stable and reliable source of seedlings can be provided for commercial production. This can not only reduce the reliance on wild resources, which is helpful for protecting the wild population of A. roxburghii, but also meet the demand for high-quality plants in the pharmaceutical and horticultural markets. Luo et al. (2018) believe that with the in-depth research on these technologies, they will provide solid support for the development of A. roxburghii industry, enhancing its economic value and market potential.

 

8 Concluding Remarks

The formula of the culture medium has a significant impact on the proliferation efficiency and growth condition of the tissue culture seedlings of A. roxburghii. The concentrations of plant hormones, especially 6-BA and NAA, as well as the usage amount of sucrose, are the key factors affecting bud proliferation and seedling development. Other studies have found that adding rare earth elements such as La(NO3)3 and Ce(NO3)3 can increase chlorophyll content and enzyme activity, and enhance the regenerative capacity of plants. Some auxiliary components have also been proven to be helpful for the process of root growth and proliferation.

 

Selecting the right culture medium is the key to obtaining high proliferation rates and healthy seedlings. The most commonly used basic formula is MS medium, and specific concentrations of hormones and sugar are added as needed. This can effectively increase the proliferation multiple and the success rate of root growth. Seedlings at different developmental stages have different demands for nutrition and hormones, and the required formulas need to be adjusted accordingly. This is very important for improving the success rate of the entire seedling raising process.

 

These research results are of great significance for the large-scale breeding and resource conservation of A. roxburghii. The optimized technology can support its commercial cultivation in the fields of medicinal and ornamental purposes, and also help protect this endangered plant and ensure a more sustainable supply of its resources. Future research can further improve these techniques, enhance efficiency, and also attempt to introduce new hormones or additives to observe the effects. The genetic stability of tissue culture seedlings should be further studied to ensure the quality and consistency of each batch of propagation materials.

 

Acknowledgments

Thanks to Dr. Cheng for his modification suggestions.

 

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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