Cherry blossoms, belonging to the genus Cerasus, are renowned for their elegant morphology, exceptional ornamental value, and cultural significance. They play a vital role in enhancing urban greening, improving environmental quality, and boosting tourism development. China has a long history of cherry cultivation and boasts the largest number of cultivated cherry trees in the world. However, research on cherry breeding has long lagged behind, resulting in relatively limited diversity among cultivated varieties, most of which are primarily introduced from Japan (Liu et al., 2018). Moreover, the exploitation and utilization of native characteristic cherry germplasm resources remain inadequate, making it difficult to meet the diversified market demands Cerasus campanulata Maxim., as an indigenous cherry blossom tree species in China, has an early flowering period, rich flower colors, strong adaptability, anti-pollution ability, and high ornamental value. It is more tolerant to high temperatures and has stronger stress resistance compared to Japanese cherry blossoms. It is suitable for planting in tropical or subtropical regions of southern China (Wang et al., 2024a, 2024b; Zou et al., 2013). Given its broad market application prospects, there is an urgent demand for large-scale seedling propagation.

However, conventional propagation methods for C. campanulata face notable limitations. Seed propagation often leads to trait segregation in offspring, while vegetative techniques such as cutting and grafting—although capable of preserving genetic integrity—are constrained by seasonal dependence, limited scion availability, and an under−optimized technical framework. These factors collectively hinder the standardization and large−scale production of uniform plantlets (Hu et al., 2019). Currently, the qualification rate for domestically produced C. campanulata seedlings remains as low as 58.2%, falling short of the ≥85% threshold required for industrial-scale production (State Forestry and Grassland Administration of China, 2021). Tissue culture has a high propagation coefficient and a short seedling formation cycle. It can maintain the stability of varieties and preserve their excellent characteristics, making it an effective approach for the large-scale seedling cultivation of cherry blossoms (Zhang and Jiang, 2016). As early as 1974, Boxus and Quoirin (1974) began to attempt to propagate cherry blossoms using tissue culture technology. Since then, researchers have continuously carried out research related to the tissue culture of cherry blossoms. However, existing studies have primarily focused on establishing basic regeneration systems, with objectives largely confined to small-scale laboratory cultivation. The medium formulations and culture conditions lack systematic optimization for industrial-scale application. Consequently, a standardized, reproducible technical system suitable for industrial propagation remains underdeveloped, particularly with respect to proliferation efficiency, rooting consistency, verification of genetic stability across multiple subcultures, and the integrated optimization of the complete workflow from inoculation to acclimatization.

The core requirements for industrial-scale micropropagation are high stability, high proliferation efficiency, standardized operation, and scalable integration with field production (Honda and Kobayashi, 2004; Abdalla et al., 2022). However, existing protocols are generally plagued by issues such as unstable proliferation coefficients, high vitrification rates, and low transplant survival rates, which fail to meet the industrial demand for reliable batch-output of elite plantlets. For instance, in the culture system established by Sanson et al. (2024), the rooting rate was only 63%, with an acclimatization period as long as 120 days. Chen et al. (2023) merely screened the culture media for the tissue culture stage of C. campanulata but did not involve acclimatization and transplantation, making direct industrial application difficult. Luo et al. (2021) developed a regeneration system for Cerasus serrulata var. lannesiana cv. ‘Grandiflora’, yet they did not verify its genetic stability; after multiple subcultures, leaf yellowing and increased mortality were observed. The market still lacks high-quality, genetically uniform standardized tissue-cultured plantlets, which severely restricts the large-scale application of this species in landscaping and related industries. Therefore, establishing an optimized, reproducible, and industrially applicable micropropagation system for C. campanulata, along with the promotion of high-quality tissue-cultured plantlets, is now crucial for overcoming this key industrial development bottleneck.

Therefore, the present study took C. campanulata as the experimental material, aiming to establish a highly efficient, stable, and reproducible micropropagation system toward industrial-scale production. It systematically optimized key culture factors for proliferation and rooting using an orthogonal experimental design. Beyond establishing an efficient regeneration protocol, it also verified the genetic stability of the regenerated plantlets over subculture generations and accomplished the complete cycle from in vitro culture to pot cultivation. This integrated approach provides a scalable and verified protocol, directly addressing the gap between laboratory research and the commercial need for standardized, high-quality plantlets.

This study systematically evaluated the effects of basal medium and 6-BA on the in vitro proliferation of C. campanulata. The results showed that among the three basal media tested—½MS, MS, and WPM—½MS yielded the best proliferation outcome, outperforming both MS and WPM. This ranking inversely correlated with their ammonium nitrate content (MS > ½MS > WPM), indicating that the superior performance of ½MS is closely associated with its moderate ammonium nitrate concentration. This finding not only aligns with the view of Chen et al. (2011) that reducing ammonium nitrogen can mitigate vitrification but further suggests the existence of an “optimal range” for nitrogen supply, rather than a simple “lower is better” approach. This differs from the results of Chen et al. (2023), who reported that WPM was more suitable for the micropropagation of Cerasus campanulata var. wuyiensis, possibly reflecting interspecific or genotypic differences in nutritional requirements.Regarding hormonal regulation, 6-BA significantly promoted proliferation, consistent with findings from most tissue culture studies on cherry blossoms (Li et al., 2014, 2020; Wang, 2021). However, this study further revealed that excessively high concentrations of 6-BA (e.g., 1.0 mg/L) led to thin shoots and leaf curling—symptoms often attributed in previous studies to high salt-induced phenolic oxidation in the basal medium (Zhang and Jiang, 2015). By comparing different treatments, we propose that in the proliferation culture of C. campanulata, morphological abnormalities are primarily associated with cytokinin overload, while the influence of salt stress may be partially masked after medium selection. Therefore, this study identifies 0.5 mg/L as the suitable concentration of 6-BA, promoting proliferation while maintaining robust shoot growth. Additionally, supplementation with low concentrations of NAA (0.1 mg/L) and BP (2.5–5 g/L) exhibited a synergistic promotive effect on proliferation. This suggests that, beyond optimizing the basal medium and cytokinin, appropriate organic additives and auxin can further enhance culture efficiency, potentially through mechanisms involving carbon source supplementation and the modulation of endogenous hormone networks (Zhang and Jiang, 2016).

Studies have shown that the rooting culture of cherry blossoms typically uses ½MS as the basal medium (Li et al., 2020; Wang, 2021; Liu et al., 2020; Duţă, 2012), while some studies employ modified MS or WPM media (Huang et al., 2002; Sanson et al., 2024). Notably, distinct strategies exist across studies regarding auxin application: for example, Lv et al. (2006) supplemented ½MS with 1.0 mg/L NAA and 0.2 mg/L IBA during the rooting stage of C. campanulata, significantly enhancing rooting efficiency; whereas Zhang and Jiang (2015) suggested that a short-term high-concentration auxin treatment followed by subculture onto hormone-free medium can avoid sustained inhibition of rooting. These differences indicate that the effect of auxins may depend not only on concentration but also on the timing of treatment and the overall balance of the culture system. In this study, ½MS was identified as the optimal basal medium, and the addition of 0.5 g/L AC along with 0.1 mg/L NAA or IBA helped further improve the rooting rate and promote shoot elongation. However, in the treatment where only ½MS was used without any exogenous auxin, rooting initiated most rapidly, and both the final rooting performance and transplant survival rate were excellent, indicating that ½MS alone can meet the requirements for efficient rooting in this system. This finding aligns with results reported by Luo et al. (2021) for C. serrulata ‘Grandiflora’, whose plantlets achieved a 100% rooting rate on ½MS basal medium, while the addition of NAA or IBA inhibited rhizogenesis. We speculate that as the culture period extends, plant hormones may accumulate dynamically, leading to changes in the plant’s hormonal response thresholds and reducing the demand for exogenous regulators. Alternatively, the plants may have developed hormone-autotrophic adaptation (i.e., activation of endogenous hormone synthesis pathways), thereby acquiring regenerative capacity independent of exogenous PGRs (George et al., 2008). In summary, whether exogenous auxin is added is not the sole determinant of successful rooting in cherry species. Its effects may vary depending on genotype, basal medium composition, culture stage, and endogenous hormone levels.

While the consistent ISSR amplification profiles across three subculture generations indicate genetic stability, the limitations of the chosen methodology must be acknowledged. One key constraint is that, as dominant markers, ISSRs cannot distinguish between homozygous and heterozygous states. Furthermore, they sample only specific repetitive regions of the genome, offering a partial view rather than a comprehensive genomic scan (Debnath, 2012). Despite these well-recognized methodological limitations, ISSR analysis remains a widely validated and efficient tool for the initial screening of somaclonal variation in micropropagated plants. This includes its successful application in the rapid propagation of woody Rosaceae species, such as cherry (Prunus pseudocerasus), to verify genetic integrity (Zhang et al., 2023), as well as in the conservation of other plant species (Al-Qurainy et al., 2018). The complete absence of polymorphic bands across all ten loci in this study provides robust evidence for genetic fidelity within the regions surveyed under our culture conditions. An additional consideration is that the assessment was confined to three subculture cycles. This scope aligns with the validation phase of micropropagation protocols, where stability through early generations (typically 3rd to 5th) is considered critical, as these often serve as the “core mother stock” for production scaling (Hou et al., 2022). It should be noted, however, that this does not preclude the possibility of variation emerging in later, extended subcultures beyond the tested range. To address these limitations and further strengthen the conclusions for large-scale application, future work could: (1) employ a broader set of molecular markers (e.g., SSRs, SNPs) for more genome-wide coverage; (2) extend stability monitoring to later subculture generations (e.g., 5-10); and (3) integrate phenotypic, physiological, and cytological assessments with molecular data. Such a multidimensional approach would provide a more comprehensive validation of the long-term genetic stability of C. campanulata in commercial micropropagation systems.

In summary, an efficient micropropagation system for Cerasus campanulata was established in this study. The optimal proliferation medium was ½MS supplemented with 0.1 mg/L NAA, 0.5 mg/L 6-BA, and 2.5 g/L banana powder, yielding a survival rate of 100.00%, an induction rate of 96.67%, a proliferation coefficient of 4.03, and well-developed shoots averaging 4.57 cm in height. For rooting, ½MS basal medium alone achieved a rooting rate and transplant survival rate both exceeding 88%, with an average of 5 roots per plantlet. Regenerated plants acclimatized successfully and exhibited vigorous growth in subsequent pot cultivation. ISSR marker analysis across three subculture cycles showed no obvious genetic variation in the regenerated plantlets. This protocol has been successfully implemented for pilot-scale seedling production, demonstrating its practical applicability. Overall, this system provides a reproducible and effective platform for the propagation of C. campanulata, supporting both further physiological studies and potential horticultural production.