Medicinal Plant Research 2025, Vol.15, No.3, 119-128 http://hortherbpublisher.com/index.php/mpr 122 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).
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