In China, the classification of flower types within the Paeonia exhibits some differences compared to other countries. According to Qin and Li (1990), the flower types of Paeonia were divided into three levels according to the series, class, and type. According to the differences between the wild species of tree peony, cultivars were divided into peony series, spotted peony series, yellow peony series and purple peony series. According to the differences between wild species or populations of herbaceous peony, cultivars were divided into Chinese peony series and European peony series.

Based on the differences in flower structure, tree peonies and herbaceous peonies were further classified into two major categories within each group: single-flower type and double-flower type. Within the single-flower type, they were further divided into the Thousand-layer Subclass and the Lou-zi Subclass based on the origin of the petals, totaling nine flower types. These included Single form, Lotus form, Chrysanthemum form, Rose form, which belong to the Thousand-layer Subclass, and Golden-slamen form, Anemone form, Golden-circle form, Crown form, Globular form, which belong to the Lou-zi Subclass. The double-flower types were divided into the Thousand-layer Tai-ge Subclass and the Lou-zi Tai-ge Subclass, totaling four flower types, including Primary Proliferation form, Color-petalled Proliferation form, Stratified Proliferation form, and Globular Proliferation form. Primary Proliferation form belonged to the Thousand-layer Tai-ge Subclass, while Color-petalled Proliferation form, Stratified Proliferation form, and Globular Proliferation form belonged to the Lou-zi Tai-ge Subclass (Qin and Li, 1990).

Different flower types exhibit distinct morphological differences. Single-petal type: 2-3 rounds of petals, normal development of stamens and pistils ( Figure 2A ). Lotus type: 4-5 rounds of petals, normal development of stamens and pistils ( Figure 2B ). Chrysanthemum type: Over 6 rounds of petals, reduced number of stamens, some stamens petaloid ( Figure 2C ). Rose type: Extreme proliferation of petals, complete loss of stamens or several small stamens in the flower center ( Figure 2D ). Golden stamen type: Outer petals expanded, 2-3 rounds of petals, stamens dome-shaped ( Figure 2E ). Tulip type: Outer petals 2-3 rounds, stamens completely petaloid, pistils normal ( Figure 2F ). Golden ring type: Wide outer petals, stamens transformed into elongated inner petals, a circle of normal stamens remaining between inner and outer petals ( Figure 2G ). Crown type: Wide outer petals, stamens almost completely petaloid, rounded and prominent ( Figure 2H ). Hydrangea type: Stamens completely petaloid, forming petals, resembling a hydrangea ( Figure 2I ).

Tai-ge flowers consist of two flowers: an upper flower and a lower flower ( Figure 2J ). In the initial Tai-ge type, both upper and lower flowers develop normal stamens and pistils, with little difference in petal morphology between inner and outer petals. In the variegated petal Tai-ge type, a few stamens are petalized, some stamens and pistils develop normally in the upper flower, while the pistils of the lower flower are almost completely petalized, with some differences in petal shapes between inner and outer petals. In the layered Tai-ge type, stamens in the upper flower are almost completely petalized or few remain, while pistils develop normally; in the lower flower, both stamens and pistils are almost completely petalized, with noticeable differences in petal shapes between inner and outer petals. In the spherical Tai-ge type, stamens and pistils in both upper and lower flowers are almost completely petalized, with differences in petal shapes between inner and outer petals. The classification of flower types is based on their growth and development stages; once the flowers are fully open, it becomes more difficult to accurately identify the type of Tai-ge flower.

The American Peony Society classifies the flower forms of the Paeonia into five types based on floral structure, including Single form ( Figure 2K ), Semi-double form ( Figure 2L ), Japanese form ( Figure 2M ), Bomb form ( Figure 2N ), and Double form ( Figure 2O ).

Flower bud differentiation refers to the process in which the growth status of a plant changes from vegetative bud to reproductive bud. The apical meristem of the bud undergoes a transition from vegetative growth state to reproductive growth state under suitable conditions. When the plant receives a signal to initiate flowering, the apical meristem of the bud transforms into an inflorescence meristem, and then the floral organs begin to develop (Coen and Meyerowitz, 1991). During the nutritional growth process of Paeonia, the apex of the stem (or bud) is pointed in the vegetative growth stage. Upon entering the reproductive growth stage, the apex of the stem (or bud) becomes relatively blunt. The protrusion formed at the edge of the apical meristem is the bract primordium. After the formation of the bract primordium, protuberances gradually appear on its inner side, which are the sepals primordium. As the floral bud continues to develop, the primordia of petals gradually appear on the inner side of the sepals primordium, and the layers of petal primordia correspond to the number of petals. The stamen primordium begins to appear after the formation of petal primordia. Before the stamen primordium is fully formed, the carpel primordium also begins to develop. The process can be divided into six stages: floral bud differentiation initiation stage, bract primordium differentiation stage, sepal primordium differentiation stage, petal primordium differentiation stage, stamen primordium differentiation stage, and carpel primordium differentiation stage ( Table 1 ) (Zhang et al., 2019). Herbaceous peony and tree peony have the same floral bud differentiation process, but the tree peony floral bud differentiation period is earlier and the process takes less time compared to herbaceous peony (He et al., 2014). In terms of research methods, the main approaches used to observe floral bud differentiation include paraffin sectioning, cryosectioning, and stereomicroscopy (Zhang et al., 2019). Some studies suggest that paraffin sectioning provides clearer images than cryosectioning, while stereomicroscopy is the simplest and quickest among the three methods (Zhang et al., 2019). Research on floral bud differentiation not only allows for an understanding of the process in Paeonia from a morphological perspective but also summarizes the regularities in floral pattern formation. It provides reference for sampling in further molecular biology studies (Fan et al., 2021). Additionally, studying floral bud differentiation helps in understanding the floral types of Paeonia, providing guidance for selecting parents in hybrid breeding programs (He et al., 2014).

In Paeonia, the different flower colors are closely associated with the types of pigments present in petal cells, cellular pH values, epidermal cell shapes, and intracellular physiological environments. The main determinants of flower color are the types and concentrations of pigments. Paeonia flowers primarily contain anthocyanins, flavonoids, flavonols, and other flavonoid substances, among which anthocyanins play a key role in determining flower color (Wang et al., 2023). With the advancement of molecular biology technologies, applications of omics such as transcriptomics, proteomics, microRNA analysis, Simplified Genome-Specific Locus Fragment Sequencing (SLAF-seq), DNA methylation profiling, and others have provided effective research methods for elucidating the biosynthesis pathways and regulatory genes involved in flavonoid and anthocyanin glycoside production related to Paeonia flower colors. These approaches also facilitate the discovery of new genetic resources and the clarification of genetic evolution patterns.

In studies of flower color in Paeonia, transcriptomics was the most widely utilized approach, while proteomics, microRNA, DNA methylation, and SLAF-seq were less commonly applied. Research indicated that multiple genes were involved in tree peony flower color formation, including flavanone 3-hydroxylase gene (F ₃ H), dihydroflavonol 4-reductase (DFR), anthocyanidin synthase (ANS), anthocyanidin 3-O-glucosyltransferase (3GT), THC 2′-glucosyltransferase (THC2’GT), SQUAMOSA promoter-binding protein-like (SPL2), flavone synthase, (FNS II), chalcone synthase (CHS), chalcone isomerase (CHI), F₂H, flavanone-3-hydroxylase (F₃′H) and PsDFR, among others. These genes regulated the synthesis of flavonoids and anthocyanins, thereby influencing flower color presentation (Shi et al., 2015; Luo et al., 2022; Zou et al., 2023; Zhang et al., 2018a). In addition, some transcription factors MYB, bHLH and WD40 were also involved in the regulation of floral color (Gu et al., 2019. PoMYB2 and PoSPL1 were reported to negatively regulate the accumulation of anthocyanins by modulating the activity of the MYB-bHLH-WDR complex (Gao et al., 2016). Among different-colored peonies, numerous genes showed differential expression, potentially directly or indirectly participated in the formation and regulation of floral color. UDP-glucosyltransferases (UGTs) such as PhUGT78A22 was reported to play important roles in floral color formation, participating in glycosylation reactions of flavonoids and anthocyanins (Li et al., 2022a). Methylation modifications played crucial regulatory roles in the formation of spots on tree peony petals, potentially influencing the expression of structural genes (Zhu et al., 2023) ( Table 4 ). Key genes and regulatory mechanisms influencing the formation of flower color in herbaceous peonies primarily focus on the biosynthetic pathways of flavonoids and anthocyanins, as well as the regulatory roles of transcription factors. Genes such as phenylalanine ammonialyase gene (PlPAL), flavonol synthase gene (PlFLS), PlDFR, PlANS, Pl3GT, and anthocyanidin 5-O-glucosyltransferase gene (Pl5GT) were involved in the biosynthetic pathways of flavonoids and anthocyanins, playing crucial roles in the formation of yellow flower color (Zhao et al., 2014). Transcription factors PsMYB111, PsMYB4 and PsSPL9 regulated the expression of structural genes, influencing the synthesis and accumulation of pigments in herbaceous peony flowers. Research had found that by modulating the expression levels of specific genes, it was possible to induce or inhibit the accumulation of particular pigments, thereby affecting the presentation of herbaceous peony flower colors (Luo et al., 2021a). Transcriptome sequencing and association analysis have revealed SNPs closely associated with herbaceous peony flower color, providing crucial information for genetic improvement of herbaceous peony coloration (Liu et al., 2022). miR156e-3p might participate in the regulation of yellow flower color formation by modulating specific genes such as SPL1 (Zhao et al., 2017).

Research indicated that glutathione S-transferases transporter of anthocyanin, PsGSTF3, interacted with PsDFR, jointly promoting petal coloring in P. suffruticosa ‘Zhao Fen’ (Han et al., 2022). PsDFR and PsANS played crucial roles in the synthesis of flower pigments in P. suffruticosa, leading to the transformation of flower color from white to red ( Figure 4A ) (Zhao et al., 2015). PsMYB58 interacted with PsbHLH1 and PsbHLH3 in P. suffruticosa ‘Er qiao’. When expressed heterologously in tobacco, PsMYB58 promoted the expression of anthocyanin biosynthetic genes and upregulates the expression of the anthocyanin regulatory bHLH, NtAN1b, indicating that PsMYB58 functioned as a regulator of anthocyanin biosynthesis in tree peony. PsMYB58 played a significant role in the formation of tree peony flower color (Zhang et al., 2021a). PsMYB44 negatively regulated anthocyanin biosynthesis by directly binding to the PsDFR promoter and inhibiting petal spot formation in P. suffruticosa ‘High noon’, thus elucidating the molecular regulatory network mediated by anthocyanins in plant spot formation (Luan et al., 2023). In the petals of P. suffruticosa, PsMYBPA2 and PsbHLH1-3 activated the expression of PsF3H, providing precursor substrates for anthocyanin biosynthesis and blotch formation. Meanwhile, PsMYB21 activated the expression of both PsF3H and PsFLS, promoting flavonol biosynthesis. Its significantly high expression in the non-blotch region inhibited blotch formation by competing for substrates involved in anthocyanin biosynthesis. Conversely, its lower expression in the blotch region facilitated blotch development. Additionally, PsMYB308 interferes with the expression of PsDFR and PsMYBPA2 by competitively binding to PsbHLH1-3 with PsMYBPA2, thereby preventing anthocyanin biosynthesis and blotch formation in P. suffruticosa petals ( Figure 4B ) (Luan et al., 2024). Exogenous glucose improved the coloration of cut flowers of P. suffruticosa ‘Tai Yang’ by affecting the accumulation of flavonoids and anthocyanins through signal transduction pathways. Studies indicated that ectopic expression of PsMYB2 in tobacco led to strong pigmentation in petals and stamens, while in Arabidopsis, ectopic expression enhances the ability to accumulate anthocyanins induced by glucose in seedlings (Zhang et al., 2022b). anthocyanin O-methyltransferase (PsAOMT) and PtAOMT, two homologous genes, exhibited similar substrate preferences for anthocyanins in vitro. Transgenic tobacco expressing PsAOMT showed significant differences in flower color compared to wild-type tobacco. PsAOMT played a crucial role in flower color formation in P. suffruticosa ‘Gunpohden’ (purple-flowered) (Du et al., 2015).

In P. lactiflora ‘Hong Yan Zheng Hui’ (purplish-red), ‘Huang Jin Lun’ (yellow), and ‘Yu Lou Hong Xing’ (white), research divided the flower development process into flower-bud stage, initiating bloom, bloom stage, and withering stage. Phenylalanine ammonia-lyase (PlPAL) showed high expression levels in the flower-bud stage and withering stage. PlCHS and PlCHI maintain high expression levels throughout all developmental stages. PlF₃H, PlF₃’H, PlDFR, PlANS, and UDP-glucose: flavonoid 5-o-glucosyltransferase (PlUF₅GT) gradually decreased as the flowers develop. PlUF₅GT exhibited a gradual increase during flower development. Among them, PlCHI promoted the accumulation of chalcones, playing a crucial role in the formation of yellow flowers. The significant expression of upstream genes and low expression of DFR led to the synthesis of abundant anthoxanthins and a small amount of colorless anthocyanins. Due to high expressions of PlDFR, PlANS, and PlUF₃GT, numerous colored anthocyanins were converted from anthoxanthins (Zhao et al., 2012a). The reduced expression of PlF₃′H, PlDFR, and PlANS following daminozide treatment might have decreased anthocyanin accumulation, ultimately resulting in reduced red color intensity in P. lactiflora ‘Fen Zhu Pan’ (Tang et al., 2018b). Research showed that ATP-citrate lyase (ACL) PlACLB2 promoted anthocyanin accumulation by increasing the abundance of its precursor substrate acetyl-CoA, thereby regulating the formation of red petals in P. lactiflora (Luan et al., 2022). Melatonin played a crucial role in the formation of herbaceous peony flower color. The tryptophan decarboxylase gene (TDC) gene was the rate-limiting gene in melatonin biosynthesis. Herbaceous peony petals were rich in melatonin, with the highest content found in white cultivars, followed by black, red, and pink cultivars. Melatonin levels peak during flowering, and the expression pattern of the TDC correlated positively with melatonin production (Zhao et al., 2018a). These studies can provided important theoretical foundations for understanding the molecular mechanisms regulating Paeonia flower coloration, thereby offering valuable insights for flower color improvement and breeding. Future research could further explore the differences in flower color formation among different cultivars, uncover additional key genes and transcription factors, and elucidate their regulatory networks.

Dormancy is the primary obstacle to flowering in Paeonia, and studying the dormancy mechanisms of tree peonies and herbaceous peonies is of great significance for understanding their flowering mechanisms. In tree peonies, Huang et al. (2008a) screened for genes that were highly expressed in dormancy-released buds, ultimately identifying 31 unique genes, of which 25 sequences were identified as potentially involved in the dormancy release process. DNA methylation was identified as an important epigenetic regulatory factor influencing gene expression. Chilling accumulation promoted bud dormancy release and sprouting through DNA methylation modification of specific genes (Zhang et al., 2021b). Studies indicated that the SUPPRESSOR OF OVEREXPRESSION OF CO1 (PsSOC1) exhibits high expression in early flowering leaves of tree peonies and showed a high hybridization signal in tender leaves of dormant buds. PsSOC1 accelerates bud dormancy release and flowering, and its overexpression in Arabidopsis resulted in early flowering (Zhang et al., 2015b). Long-term cold exposure (0-4°C) activated the Pentose Phosphate Pathway (PPP), promoting dormancy release in P. suffruticosa ‘Lu he hong’ (Zhang et al., 2018b). The mitochondrial phosphate transporter gene of P. suffruticosa ‘Lu He Hong’, mitochondrial phosphate transporters (PsMPT), when ectopically expressed in Arabidopsis, accelerated growth and advanced flowering time. PsMPT played a crucial role in the dormancy release process of peony buds (Huang et al., 2008b). The transcription factor MYB-like protein, PsMYB1, was expressed in all tissues of P. suffruticosa ‘Lu He Hong’ and responds to 4°C cold temperatures. PsMYB1 was implicated not only in dormancy release and eco-dormancy but also in tissue development (Zhang et al., 2015c). In P. suffruticosa ‘Lu He Hong’, PsmiR172b negatively regulated bud dormancy release, whereas TARGET OF EAT (PsTOE3) promoted bud dormancy release. Genes associated with bud dormancy release, including EARLY BUD-BREAK 1 (PsEBB1), EARLY BUD-BREAK 3 (PsEBB3), D-type cyclins (PsCYCD) and β-1,3-glucanase (PsBG6), were upregulated. Ectopic expression of PsmiR172b-PsTOE3 in Arabidopsis conservatively regulated flowering (Zhang et al., 2023).

In herbaceous peony, the molecular mechanisms underlying dormancy release in herbaceous peony buds were studied later compared to tree peony. Zhang et al. (2015d) pioneered the construction of transcriptome libraries using RNA-seq technology for different stages of underground bud dormancy in the low-chill P. lactiflora ‘Hang Bai Shao’, providing a comprehensive insight into the transcriptome profiles during dormancy release. A set of target genes related to bud dormancy release were identified, involving genes associated with environmental response, energy and substance metabolism, and cell growth and development. Subsequently, analysis focused on 66 candidate genes related to light and temperature during herbaceous peony bud dormancy release, with expression pattern analysis of 12 genes across different dormancy stages, suggesting the involvement of SOC1 and WRKY33 in the low-temperature dormancy release process of P. lactiflora ‘Hang Bai Shao’ buds (Zhang et al., 2017). Subsequently, researchers utilized P. lactiflora ‘Da Fu Gui’ to conduct transcriptome sequencing of underground buds at three different stages: physiological dormancy, ecological dormancy, and sprouting. They identified a set of differentially expressed genes related to dormancy and focused particularly on annotating and analyzing genes associated with hormones (Guo et al., 2017).

Treatment of P. suffruticosa ‘Qiu Fa No. 1’ with gibberellin (GA) plus nutrient can advance its autumn reflowering time by 38 days. During this process, GA induced peony reflowering, while the fertilizer provided sustained nutrients for the flowering process. At the transcriptional level, SUPPRESSOR OF CONSTANS OF OVEREXPRESSION1 (PsSOC1) and LEAFY (PsLFY) played critical integrating roles in gibberellin-induced bud differentiation and nutrient supply from fertilization, respectively. GA20 OXIDASE (PsGA20ox) was primarily involved in the GA signaling pathway, while GA INSENSITIVE (PsGAI) potentially modulated flower formation post-fertilization. Sugar signaling pathways also participated in this process, emphasizing the requirement for adequate nutrient supply during early gibberellin-induced autumn reflowering of tree peonies (Xue et al., 2022). SHORT VEGETATIVE PHASE (PsSVP) negatively regulated flowering and positively regulated leaf growth in P. suffruticosa ‘Luoyang Hong’. Gibberellic ACID 3 (GA₃) improved floral bud development by suppressing PsSVP expression. High expression of the PsSVP gene may contributed to floral bud abortion in tree peonies (Wang et al., 2014).

The leaves of P. ostii ‘Fengdan’ were sprayed with brassinolide at concentrations of 25 μg/L, 50 μg/L, 100 μg/L, and 200 μg/L. Significant flowering delay was observed with brassinolide at 50 μg/L. Transcriptome and miRNA sequencing of P. ostii ‘Fengdan’ leaves treated with different brassinolide concentrations identified six miRNAs and their target genes potentially involved in regulating the flowering of P. ostii. These include four novel miRNAs targeting SPA1, miR156b targeting SPL, and miR172 targeting AP2 (Zhang et al., 2022c). Additionally, research indicated that in P. ostii ‘Fengdan’, transgenic plants with pCAMBIA2300-PoVIN3 exhibit significantly earlier flowering compared to the wild type, suggesting a role for the PoVIN3 gene in promoting flowering (Li et al., 2022b). Overexpression of the PoFLC gene in Arabidopsis resulted in significantly delayed bolting and flowering compared to the wild type, indicating an important role for the PoFLC gene in delaying flowering processes (Fan et al., 2023b). Overexpression of the gene CONSTANS (PlCO) from P. × lemoinei ‘High Noon’ in Arabidopsis resulted in earlier flowering, indicating its promotive role in tree peony flowering. PlCO protein localized to the nucleus and exhibited transcriptional activity, suggesting that PlCO may function as a transcription factor (Chang et al., 2022). Transcriptome sequencing was conducted on P. lemoinei ‘High Noon’ and non-reblooming P. suffruticosa ‘Luo Yang Hong’, identifying 59,275 and 63,962 unigenes, respectively. Integrated analysis and RT-PCR validation identified several genes potentially playing crucial roles in tree peony reblooming, including FLOWERING LOCUS T (PsFT), VERNALIZATION INSENSITIVE 3 (PsVIN3), PsCO and GIBBERELLIN 20-OXIDASE (PsGA20OX) (Zhou et al., 2013).

Dormancy breaking treatment of P. lactiflora ‘Taebaek’ in November (0°C for 6 weeks) advanced flowering to early spring. However, similar treatments in August, September, or October resulted in flower bud abortion or deformities. Research indicated that a 2-week pre-chilling at 10°C followed by dormancy breaking treatment was the optimal method for promoting autumn or winter flowering of herbaceous peonies without bud abortion (Park et al., 2015).

Research indicated that the duration of cut flower bloom period was closely related not only to harvest time and storage methods but also to the addition of exogenous substances post-harvest, which could enhance cut flower quality and prolong the bloom period. During the cut flower opening process of P. suffruticosa ‘Luoyang Hong’, studies have found that ethylene inhibits the expression of Ethylene resistant (Ps-ETR1-1) and Ethylene insensitive (Ps-EIN3-1) genes. Additionally, Ps-ETR1-1 expression was significantly inhibited by 1-MCP, with a slight induction observed in Ps-EIN3-1 gene expression. This suggests that the proteins encoded by Ps-ETR1-1 and Ps-EIN3-1 played important roles in ethylene sensitivity and response processes in tree peony cut flowers. These findings were crucial for extending the cut flower bloom period (Zhou et al., 2010). Furthermore, research indicated that PsEIL2, PsEIL3, and PsERF1 might also have played roles in regulating ethylene response in P. suffruticosa ‘Luoyang Hong’ (Wu et al., 2017).

Treatment of fresh-cut flowers of P. lactiflora ‘Hongyan Zhenghui’ with nano-silver increased the soluble protein content inside the flowers. This enhancement promoted the activities of antioxidant enzymes such as superoxide dismutase (SOD), catalase (CAT), and ascorbic acid peroxidase (APX), thereby reducing the accumulation of reactive oxygen species such as malondialdehyde (MDA), superoxide radicals (O₂ ^·−), and hydrogen peroxide (H₂O₂). Furthermore, nano-silver treatment exerted inhibitory effects on the growth and reproduction of microorganisms at the stem base of cut flowers, thereby delaying stem blockage. Additionally, AQUAPORIN genes (AQPs) induced by nano-silver treatment played a crucial role in maintaining water balance in cut flowers (Zhao et al., 2018b). Moreover, research has shown that both sucrose transporter genes and invertase genes were involved in maintaining vase quality in cut flowers, specifically in P. lactiflora ‘Yang Fei Chu Yu’ (Xue et al., 2018).

Silencing PlZFP in tobacco petals prolonged flower opening time, while overexpressing PlZFP shortened flower lifespan. This process involved downregulation of genes related to plant hormone biosynthesis, including PlACO1, PlACO3, PlACS1, PlNCED2 and PlAAO, along with increased transcript levels of PlGA3ox1. Conversely, overexpression of PlZFP in tobacco flowers exhibited opposite effects on these genes. Additionally, PlZFP directly bound to the PlNCED2 promoter. Silencing PlNCED2 delayed senescence in petal discs. Reduction of PlZFP decreased ethylene and ABA levels but increased GA levels in herbaceous peony petal discs. Furthermore, exogenous ABA, ethephon, and paclobutrazol (PAC), a GA inhibitor, accelerated senescence in PlZFP-silenced discs. Collectively, the PlZFP-PlNCED2 regulatory checkpoint likely enhanced ABA production and modulates the interplay of ABA with GA and ethylene during flower senescence in cut P. lactiflora ‘Hangshao’ (Ji et al., 2023).

Paeonia are known for their rich floral fragrances, with various aromatic profiles identified in tree peony flowers, including grassy scent, fruity scent, woody scent, rose scent, lily of the valley scent, phenolic scent, and unidentified scent (Li et al., 2012, 2021). In herbaceous peony fragrance research, six types of fragrances have been identified, namely woody scent, fruity scent, lily scent, rose scent, orange blossom scent, and mixed scent (Song et al., 2018; Zhao et al., 2023).

The composition of floral fragrance was complex and diverse, primarily including alcohols, aldehydes, terpenes, esters, and volatile phenols, which often exhibit significant diversity in plants (Li et al., 2012; Luo et al., 2020). Currently, common methods for aroma collection and component analysis in Paeonia included Headspace Solid-Phase Microextraction (HS-SPME) and Dynamic Headspace Sampling (DHS). Widely used analytical methods included artificial olfactory systems, GC-MS, and Automated Thermal Desorber-Gas Chromatography-Mass Spectrometry (ATD-GC/MS) ( Table 5 ) (Zhao et al., 2012b; Li et al., 2023). The headspace technique was favored in fragrance preparation due to its automation, ability to minimize sample matrix interference, and simplicity in sampling, thus it was widely applied in volatile compound preparation. GC-MS offered high sensitivity and simultaneous qualitative and quantitative analysis capabilities, contributing to its widespread use in fragrance component analysis.

The fragrance composition of Paeonia was complex and diverse ( Table 5 ). In tree peonies, the main components responsible for the grassy scent in petals were ocimene, for fruity scent it was 2-ethyl hexanol, for musky scent it was cis-ocimene/longifolene, for rose scent it was D-citronellol, for lily scent it was linalool, for phenolic scent it was 1,3,5-trimethoxybenzene, and for unknown scent it was Pentadecane (Li et al., 2012, 2021). In herbaceous peonies, the primary components responsible for woody scent were β-caryophyllene, for fruity scent it was phenylethyl alcohol, for lily scent it was linalool, for rose scent it was (R)-citronellol, and for orange blossom scent it was nerol (Song et al., 2018; Zhao et al., 2023). Additionally, terpenes were the most abundant compounds in peony petal volatiles (Li et al., 2012), and major fragrance components in peonies also include geraniol and 2-phenylethyl alcohol (Zhao et al., 2023). In conclusion, the fragrance components of Paeonia are complex and diverse, and further research is needed to understand their biosynthetic pathways and gene regulation mechanisms.

Paeonia flower fragrance is a quantitatively controlled trait influenced by multiple genes, with a complex genetic regulatory mechanism. Studying the biosynthetic pathways of floral fragrance compounds is crucial for understanding the molecular mechanisms underlying fragrance formation in peonies. In tree peonies, linalool was identified as one of the dominant volatile compounds in the fragrance of members of the subsection Delavayanae and subsect. Delavayanae hybrids. Five terpene synthases (TPSs) (PdTPS1, PdTPS2, PdTPS3, PdTPS4, and PdTPS5) were identified through global transcriptome sequencing analysis of 11 wild tree peony genotypes and 60 cultivars, potentially involved in the biosynthetic pathway of linalool. Among these, PdTPS1, PdTPS2, and PdTPS4 showed significant positive correlations with linalool emissions across tree peony cultivars. Biochemical data analyses confirmed that PdTPS1 and PdTPS4 were key genes determining linalool synthesis (Li et al., 2023). Furthermore, expression levels of enzymes crucial for terpene skeleton biosynthesis, including acetoacetyl-CoA thiolase (AACT), 3-Hydroxy-3-methylglutaryl-coenzyme A reductase (HMGR), phosphomevalonate kinase (PMK), 1-Deoxyxylulose-5-phosphate synthase (DXS), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR), 4-Hydroxy-3-methylbut-2-enyl diphosphate synthase (HDS), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (HDR), and geranylgeranyl diphosphate synthase (GGPS), were upregulated in the tree peony cultivar ‘Huangguan’ (strong fragrance) compared to the cultivar ‘Fengdan’ (faint fragrance). Additionally, transcription abundance of one linalool synthase gene (LIS) and one myrcene synthase gene (MYS) in ‘Huangguan’ petals was higher compared to ‘Fengdan’. Therefore, high expression of genes involved in terpene skeleton biosynthesis and monoterpene metabolism pathways was associated enhanced fragrance in tree peonies (Li et al., 2021).

In herbaceous peonies, analysis of aroma components and aroma thresholds across 17 cultivars identified linalool, geraniol, citronellol, and phenylethyl alcohol (2-PE) as primary aromatic substances. Subsequent qRT-PCR investigations into genes potentially regulating aroma synthesis in peony petals revealed significant candidates, including 1-DEOXY-D-XYLULOSE 5-PHOSPHATE SYNTHASE (PlDXS2), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (PlDXR1), 2-C-METHYL-D-ERYTHRITOL-2,4-CYCLODIPHOSPHATE (PlMDS1), 1-HYDROXY-2-METHYL-2-(E)-BUTENYL-4-DIPHOSPHATE REDUCTASE (PlHDR1), GERANYL PYROPHOSPHATE SYNTHASE (PlGPPS3 and PlGPPS4), as well as LINALOOL SYNTHASE (LIS) and GERANIOL SYNTHASE (GES) genes. These findings underscored the critical role of differential gene expression in the monoterpene and 2-PE synthesis pathways in shaping the aroma profile of herbaceous peonies (Zhao et al., 2023).

Furthermore, improvement of floral fragrance traits in Paeonia requires support from genomic marker resources. Molecular marker studies on floral fragrance traits in Paeonia have been shown to promote the breeding of fragrant cultivars. Research based on tree peony genomic data has developed microsatellite markers associated with major floral fragrance components. A total of 8443 EST-SSRs were identified, with genetic diversity and population structure analyses conducted on 31 polymorphic EST-SSR markers, yielding 176 alleles. Polymorphic information content (PIC) ranged from 0.31 to 0.87. Single marker-trait association analysis using a mixed linear model (MLM) identified 29 significantly associated loci, involving 18 SSR loci associated with 5 floral fragrance components, with phenotypic variance explained ranging from 4.57% to 23.46% (Luo et al., 2021b).