In Vitro Culture Systems of Rosa damascena Mill. and Their Role in Secondary Metaolite Production

1. Introduction

Rosa damascena Mill. is widely regarded as one of the most valuable aromatic species within the genus Rosa, primarily due to its long-standing economic importance and its role as a major source of high-value secondary metabolites. Cultivation is largely devoted to the production of rose essential oil and rose water, which remain central to the perfumery and cosmetic industries. In parallel, a growing number of studies have pointed to the pharmaceutical and nutraceutical potential of Damask rose, extending its relevance beyond traditional fragrance-related applications [1,2]. Commercial cultivation is geographically restricted to a limited number of traditional production areas, including Bulgaria, Turkey, and Iran, where R. damascena represents a key crop within specialized agro-industrial systems. This restricted distribution has contributed to the development of relatively uniform cultivation practices, but it has also raised questions regarding genetic diversity and long-term breeding potential. At the biochemical level, petals constitute the primary site of accumulation of bioactive compounds. They are rich in volatile secondary metabolites responsible for the characteristic aroma of Damask rose. The essential oil is dominated by monoterpenoid alcohols such as citronellol, geraniol, nerol, and phenethyl alcohol, while a wide array of minor constituents contributes to overall oil quality and commercial value [2,3] (Table 1). Petal tissues also contain substantial amounts of phenolic compounds and flavonoids. These non-volatile metabolites are often overlooked in aroma-focused studies, despite their relevance for antioxidant capacity and stress-related functions in planta. A broad spectrum of biological activities has been associated with R. damascena, including antioxidant, antimicrobial, anti-inflammatory, and anticancer effects [4]. Rather than being attributable to single dominant compounds, these activities are generally considered the result of synergistic interactions among multiple phytochemicals (Figure 1). This aspect is particularly relevant when interpreting biological data derived from complex plant extracts, as reductionist approaches may fail to capture the functional properties of the whole metabolic profile. Despite the substantial body of literature available, the regulation of secondary metabolite biosynthesis in R. damascena remains incompletely understood. This variability reflects the complex regulation of secondary metabolism in aromatic plants, where environmental cues, developmental stage, and genetic background interact to shape metabolite profiles [5,6,7]. Metabolic output is strongly influenced by genetic background, environmental conditions, agronomic practices, and harvest timing, often resulting in pronounced variability in essential oil yield and composition [1]. Genetic studies indicate a relatively narrow genetic base in many cultivated populations, especially in long-established production regions, which may partly explain this variability and represents a limitation for targeted breeding strategies [8]. From a plant biotechnology perspective, Damask rose should therefore be regarded as an underexploited system rather than a fully characterized model species. The integration of genetic, molecular, and metabolic approaches appears essential to improve yield stability, enhance metabolite consistency, and support the development of standardized plant-derived products suitable for industrial applications.

2. In Vitro Culture Systems for Secondary Metabolite Production

Plant in vitro culture systems, including callus cultures, cell suspension cultures, and differentiated organ cultures, represent powerful experimental platforms for investigating and modulating secondary metabolism in medicinal plants. By uncoupling metabolite biosynthesis from environmental variability, these systems enable controlled analysis of biosynthetic pathways, regulatory mechanisms, and accumulation dynamics of bioactive compounds [9,10]. Similar limitations have been widely reported in other medicinal species, where undifferentiated cultures often exhibit reduced metabolic specialization and altered pathway fluxes compared to intact plants [11,12,13]. In medicinal species, in vitro cultures have been widely applied to produce pharmacologically relevant secondary metabolites such as phenolic compounds, flavonoids, alkaloids, and terpenoids. However, metabolite yield and profile are strongly influenced by the degree of tissue differentiation. Undifferentiated systems, such as callus and suspension cultures, frequently exhibit reduced accumulation of compounds whose biosynthesis depends on specialized structures or cellular compartmentalization, whereas differentiated organ cultures often retain higher biosynthetic competence [14,15]. For R. damascena, secondary metabolites are primarily associated with medicinal properties including antioxidant, anti-inflammatory, and antimicrobial activities [16]. Although field-grown plants remain the main source of these compounds, in vitro systems offer a complementary approach for studying metabolic regulation and ensuring reproducible production. Previous studies emphasize that phenolic compounds, rather than volatile constituents, are particularly amenable to accumulation under in vitro conditions, making tissue culture a promising tool for medicinal applications of R. damascena [2,8]. A summary of the main published studies on in vitro cultures of R. damascena is reported in Table 2. This trend is consistent with observations across plant cell cultures, where phenolic pathways remain active under in vitro conditions, while terpenoid biosynthesis is frequently downregulated or spatially constrained [17,18]. Recent advances in plant biotechnology highlight the role of culture type optimization, nutrient composition, and metabolic regulation strategies in enhancing secondary metabolite production. Manipulation of biosynthetic pathways through precursor feeding, growth regulator adjustment, and metabolic signalling has proven effective across a wide range of medicinal plants [19,20]. These approaches are especially relevant for species such as R. damascena, where genetic diversity and metabolic plasticity influence both qualitative and quantitative aspects of metabolite accumulation [8,21]. Moreover, scalable in vitro systems, including bioreactor-based cultures, are gaining attention as sustainable alternatives for producing high-value secondary metabolites. Although their application to Rosa species remains limited, experiences from other medicinal plants demonstrate the feasibility of transferring optimized in vitro cultures to larger-scale production systems, bridging the gap between experimental research and industrial application [9,22]. Overall, in vitro culture technologies constitute a versatile and increasingly refined toolkit for studying and enhancing secondary metabolite production in medicinal plants. For R. damascena, these systems not only support fundamental investigations into metabolic regulation but also offer insights for the development of standardized sources of bioactive compounds, independent of agronomic and environmental constraints.

3. Biological Activities of R. damascena Secondary Metabolites

3.1. Antioxidant Activity

Antioxidant activity represents one of the most consistently reported biological properties of secondary metabolites derived from Rosa damascena Mill. This activity is primarily attributed to phenolic acids, flavonoids, and related polyphenolic compounds, which contribute to free radical scavenging and cellular protection against oxidative stress [2,16]. Several in vitro studies have demonstrated that extracts obtained from petals, as well as from callus cultures, retain significant antioxidant capacity. Callus-derived extracts of Rosa damascena were shown to accumulate phenolic compounds associated with measurable radical scavenging and antibacterial activity, indicating that undifferentiated tissues can still serve as sources of bioactive metabolites [25]. These findings are particularly relevant for biotechnological applications, as phenolic compounds are among the secondary metabolites most amenable to accumulation under in vitro culture conditions.

3.2. Anti-Inflammatory Effects

Anti-inflammatory activity is another key biological feature associated with secondary metabolites of Rosa species. Although direct studies on R. damascena in vitro systems remain limited, bioactive compounds isolated from Rosa leaves and petals have demonstrated inhibitory effects on inflammatory mediators in cellular models. Yan et al. [27] identified several secondary metabolites with pronounced anti-inflammatory activity, highlighting the relevance of non-volatile constituents in modulating inflammation-related pathways. For R. damascena, pharmacological evidence suggests that phenolic-rich extracts exert anti-inflammatory effects through the modulation of oxidative stress and inflammatory signaling cascades [16]. While most data derive from extract-based assays, these results provide a functional framework for evaluating metabolites produced under in vitro conditions, particularly in callus and cell culture systems enriched in phenolic compounds.

3.3. Antimicrobial Activity

Secondary metabolites from R. damascena have also been reported to exhibit antimicrobial activity against a range of bacterial and fungal strains. Extracts from petals and other tissues show inhibitory effects that are largely attributed to phenolics and flavonoids rather than volatile oil components [16]. Importantly, antimicrobial activity has been demonstrated for metabolites produced in vitro. Callus cultures of R. damascena were reported to synthesize compounds with detectable antibacterial activity, supporting the concept that in vitro systems can generate biologically functional metabolites even in the absence of full tissue differentiation [25]. These observations strengthen the case for tissue culture as a complementary approach to field cultivation for medicinal applications.

3.4. Antiproliferative and Cytoprotective Effects

Beyond antioxidant and antimicrobial properties, secondary metabolites from R. damascena have been associated with antiproliferative and cytoprotective activities in vitro. Recent studies using cell-based assays reported that extracts derived from R. damascena callus cultures exert inhibitory effects on cancer cell proliferation and migration, suggesting the presence of bioactive compounds capable of modulating cellular growth pathways (e.g., Caco-2 cell models). In addition, phenolic-rich extracts obtained from petals and processing by-products have demonstrated protective effects against oxidative damage in cellular systems, further supporting their therapeutic potential [28]. Although these studies are not always directly linked to tissue culture optimization, they provide important biological validation of metabolites that could be targeted in in vitro production systems.

4. Implications for In Vitro Culture–based Production

Collectively, available evidence indicates that the medicinal relevance of Rosa damascena is largely associated with non-volatile secondary metabolites exhibiting antioxidant, anti-inflammatory, antimicrobial, and cytoprotective activities. While the number of studies specifically addressing metabolite production in R. damascena in vitro cultures remains limited, existing data from callus cultures and extract-based assays support the feasibility of producing biologically active compounds under controlled conditions. These findings justify further development of in vitro culture systems aimed at enhancing phenolic accumulation and biological efficacy, bridging the gap between metabolic studies and practical pharmaceutical applications.

5. Rosa damascena as a Challenging and Underexplored In Vitro Model

Despite the economic and medicinal interest in the species, its exploitation as an in vitro model for secondary metabolite production remains comparatively underdeveloped relative to other medicinal plant systems. Early work in plant biotechnology demonstrated that tissue culture extracts from R. damascena contain the key enzymes for monoterpene biosynthesis, suggesting intrinsic metabolic potential even in undifferentiated cultures. In vitro extracts derived from calli were able to convert isopentenyl pyrophosphate (IPP) into monoterpenoid precursors such as geraniol and nerol at rates far exceeding those from extracts of field-grown plants, indicating enzymatic capacity albeit uncoupled from full biosynthetic output in culture [30]. Work on R. damascena suspension cultures further illustrates the difficulty of achieving high yields of target metabolites in vitro. A comprehensive analysis of volatile and polar compounds from cell suspension cultures found that hydrocarbons, free acids, and esters dominated, with only trace levels of terpenoids detected under standard cultivation conditions, even when various culture regimes were applied (e.g., flask, bioreactor, two-phase systems with adsorbents). These findings underscore the challenge of replicating the full spectrum of secondary metabolism outside the intact plant context. At the molecular level, recent RNA-sequencing studies on long-term rose suspension cultures have begun to reveal the transcriptional landscape of cells propagated in vitro [31]. Transcriptome profiling highlighted that, although many coding RNAs related to translation and peptide synthesis are highly expressed in suspension cells, genes associated with organelle-specific functions (chloroplasts, mitochondria) show markedly reduced expression. Moreover, stress and detoxification pathways are dynamically regulated as cultures progress, suggesting a cellular reprogramming that may influence metabolic fluxes relevant to secondary metabolite production. Such transcriptional reprogramming is commonly observed in long-term plant cell cultures and has been associated with shifts toward primary metabolism and stress-response pathways at the expense of specialized metabolite biosynthesis [5,32]. While these data do not yet provide direct metabolite quantification, they establish a foundation for understanding the regulatory complexity of rose cell cultures at the gene expression level. Callus cultures of R. damascena have provided some of the most direct evidence of in vitro production of bioactive secondary metabolites, including phenolic compounds with antibacterial activity [25] and those exhibiting antiproliferative and anti-migratory effects against human colorectal cancer cell lines. In the latter, the secondary metabolite content was enhanced by supplementing the medium with ascorbic acid, which improved both callus growth parameters and the biological efficacy of the extracts, illustrating that culture conditions can modulate biomass and metabolite output in vitro.

Despite these accomplishments, several challenges remain intrinsic to R. damascena as an in vitro model:

• Limited terpenoid accumulation in vitro: Classic tissue culture systems struggle to produce monoterpenes characteristic of field-grown plants, pointing to the absence of specialized structures or differentiation states required for full pathway expression. In many aromatic species, monoterpene biosynthesis is associated with glandular trichomes or other differentiated tissues, whose absence in vitro represents a major limitation for volatile metabolite production [6,33].
• Metabolic complexity vs culture simplicity: Suspension cultures tend toward simplified metabolite profiles dominated by primary metabolic products, complicating efforts to target specific medicinal secondary metabolites.
• Regulatory and developmental bottlenecks: Transcriptomic data reveal substantial reprogramming in vitro that likely impacts pathways of interest, but functional links between gene expression and metabolite biosynthesis remain poorly defined.

Taken together, these studies illustrate that Rosa damascena possesses inherent metabolic potential that is not fully exploited in current culture systems. The disconnect between enzymatic capability (e.g., monoterpene precursor synthesis) and the actual accumulation of target metabolites highlights the need for deeper molecular and metabolic engineering strategies. Integrative approaches combining transcriptomics, metabolic flux analysis, and optimized culture conditions may be necessary to realize the full biotechnological promise of R. damascena as an in vitro model for medicinal secondary metabolite production.

6. Current Knowledge Gaps and Future Research Directions

Despite long-standing interest in R. damascena Mill. as a source of high-value secondary metabolites, significant knowledge gaps persist regarding its behavior under in vitro conditions. One of the most evident limitations is the scarcity of systematic studies addressing the relationship between tissue differentiation and metabolite accumulation. Although early work demonstrated monoterpene biosynthesis in cultured tissues, subsequent research has rarely revisited this aspect using modern analytical or molecular tools, leaving unresolved questions about the regulation of volatile biosynthesis in undifferentiated systems. Another major gap concerns the limited exploration of culture-optimization strategies. Compared to other medicinal plants, R. damascena in vitro systems have been only marginally investigated in terms of elicitation, precursor feeding, or controlled stress application, despite evidence that these approaches can significantly enhance secondary metabolite production. Indeed, elicitation strategies based on jasmonates, salicylic acid, or abiotic stress have proven effective in activating silent biosynthetic pathways in vitro [18,20]. The few available studies on callus and suspension cultures suggest that phenolic compounds with biological activity can be accumulated, yet comparative analyses across culture types, growth stages, and metabolic classes remain largely absent. Furthermore, the lack of integrated omics-based approaches represents a critical bottleneck. Transcriptomic, proteomic, and metabolomic datasets for R. damascena in vitro cultures are extremely limited, preventing a comprehensive understanding of the regulatory networks controlling secondary metabolism. Integrative omics approaches have already demonstrated their value in linking gene expression to metabolite accumulation in other plant systems, highlighting regulatory bottlenecks and pathway constraints [34,35]. This limitation hampers both the rational design of culture strategies and the reproducibility of experimental outcomes across laboratories. Addressing these open questions would not only clarify whether R. damascena can be effectively exploited as an in vitro production platform but would also provide valuable insights into the developmental and regulatory constraints governing secondary metabolism in aromatic plants. In this context, the species offers a unique opportunity to investigate why certain high-value metabolites remain tightly linked to whole-plant organization, challenging current paradigms in plant cell biotechnology.

7. Conclusions and Future Perspectives

Despite its long-standing relevance as a medicinal and aromatic species, R. damascena Mill. remains a surprisingly underexplored model in the context of in vitro secondary metabolite production. The limited number of available studies spanning more than four decades consistently indicates that rose tissues cultured in vitro retain intrinsic biosynthetic potential but fail to fully reproduce the metabolic complexity observed in vivo [5,11,36]. This discrepancy is particularly evident for monoterpenes, whose accumulation appears tightly linked to tissue differentiation and to specialized cellular structures that are absent in most vitro systems [6,7,36].

At the same time, evidence from callus and suspension cultures demonstrates that biologically active phenolic compounds can be produced in vitro and that their accumulation responds to culture conditions [12,13,18]. These findings suggest that R. damascena should not be regarded as a recalcitrant species, but rather as a system whose metabolic regulation under artificial growth conditions remains insufficiently understood. In this sense, the challenges associated with rose tissue culture reflect broader issues in plant secondary metabolism, including the frequent uncoupling between gene expression, enzymatic capacity, and final metabolite accumulation [5,17,37].

From a plant cell biotechnology perspective, R. damascena represents a promising yet largely untapped model for integrative studies combining tissue culture, molecular regulation, and metabolite profiling. Future research should focus on linking developmental status with metabolic output, applying omics-based approaches to identify regulatory bottlenecks, and designing culture systems that better mimic key aspects of in vivo organization [32,34,35]. Addressing these aspects may not only improve the in vitro production of bioactive metabolites but also contribute to a deeper understanding of secondary metabolism regulation in medicinal plants [5,13].