Medicinal Plant Research 2025, Vol.15, No.3, 119-128 http://hortherbpublisher.com/index.php/mpr 120 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. Figure 1 Morphological characteristics of diploid and tetraploid A. roxburghii: whole plants (A–C), leaves (D) and stems (E). Diploids on the left and tetraploids on the right in each picture. 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
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