Tetraploidization is a critical pathway for fertility restoration in inter-subsectional hybrid tree peonies (Paeonia × lemoinei). However, mechanisms underlying the male sterility or reduced fertility observed in some allotetraploid tree peonies remain unclear. In this study, three allotetraploid tree peonies (07-LB-30, 07-LB-22, and 07-LB-56) with different fertility levels were investigated by observing meiosis behavior and anther development, characterizing pollen morphology and viability, and analyzing chromosomal composition via genomic in situ hybridization (GISH). We found that the fertility differences among the three materials were not significantly correlated with their overall frequency of meiotic irregularities, suggesting that meiotic disturbance could not necessarily lead to fertility defects in these allotetraploids. Although 07-LB-22 formed 17.57% normal tetrads, its defective tapetum development in anthers, which led to pollen abortion, gave rise to its male sterility. 07-LB-56 generated a higher proportion (38.90%) of normal tetrads, yet the disorders in its chromosomal pairing likely resulted in a high frequency of aneuploid gametes, contributing to its low fertility. GISH revealed that 07-LB-56 possesses an AAAB type chromosomal composition, distinct from the near-AABB configuration of the other two materials. This asymmetric subgenomic composition could cause genomic imbalance, thereby give rise to pairing failure and low fertility. The AAAB-type allotetraploid tree peony might originate from backcrossing between inter-subsectional hybrid tree peonies (P. × lemoinei, AABB or AB) and common tree peonies (P. suffruticosa, AA), suggesting a novel pathway for the tetraploidization in tree peonies. This study elucidated the mechanisms underlying fertility differences in three allotetraploid tree peonies, and uncovered a backcrossing pathway for tetraploidization, providing valuable theoretical bases for polyploid breeding in tree peony.

Tree peony (Paeonia sect. Moutan) is an important ornamental species with notable economic and ecological value. However, its large-scale cultivation is constrained by difficulties in propagation. Girdling has recently emerged as an effective method to promote adventitious root (AR) formation in tree peony, yet the underlying regulatory mechanisms remain insufficiently understood. In this study, we examined the effects of girdling time on rooting and elucidated the molecular basis of girdling-induced AR formation using multi-omics approach. We determined that 50 days after flowering (DAF) was the optimal stage for girdling. Transcriptome analysis identified 6 375 differentially expressed genes (DEGs), primarily enriched in plant hormone signal transduction and starch and sucrose metabolism pathways. A total of 1 511 metabolites were detected and classified into 13 categories. Weighted gene co-expression network analysis (WGCNA) revealed 48 hormone-related genes and 20 transcription factors associated with hormone signaling. Integrated transcriptomic and metabolomic analyses further indicated that seven transcription factors (including PrSCL8, PrSHR2, and PrERF109) play key in AR formation. Functional validation demonstrated that overexpression of PrSAUR32 and PrSCL8 in Arabidopsis significantly enhanced AR development, increasing both root number and length. Overall, our study elucidates the regulatory network underlying girdling-induced AR formation in tree peony and provides a theoretical foundation to support improved cultivation practices and industrial scale propagation through softwood girdling.

Tree peony (Paeonia rockii) is a promising woody oilseed crop valued for its seed oil, which is rich in abundant unsaturated fatty acids beneficial to human health. However, the transcription factors (TFs) responsible for high-quality oil accumulation in tree peony remain largely unexplored. Here, integrated histological and transcriptomic analyses of P. rockii seeds revealed a vital role for ERF TFs in seed development and oil accumulation. Characterization and expression profiling of 12 ERF genes identified PrRAP2.1, which is predominantly expressed in developing seeds and functions as a key positive regulator of seed oil accumulation. Our results show that PrRAP2.1 displays a specific, endosperm-predominant, development-dependent expression pattern and encodes a protein localized in the nucleus. Overexpression of PrRAP2.1 in Arabidopsis significantly promoted seed development, and regulated oil accumulation by increasing the content of the triacylglycerol (TAG) precursor diacylglycerol (DAG), thereby enhancing the total seed oil content by up to 5.57 %; Furthermore, it improved the fatty acid composition by elevating the substrate PC (18:2/18:2) pool, resulting in an increased C18:2-PC/C18:3-PC ratio and a significant accumulation of C18:2. Collectively, these findings provide novel insights into the role of PrRAP2.1 in promoting seed oil accumulation and lay the groundwork for enhancing oil yield in tree peony and other oilseed crops.

Floral fragrance is a crucial biological and ornamental trait in plants. Flare tree peony (FTP), originating from Paeonia rockii (S.G.Haw & Lauener) T.Hong & J.J.Li ex D.Y.Hong, exhibits diverse flower scents, which enhance its ornamental and ecological value. However, the regulatory mechanisms governing floral volatile compounds biosynthesis in FTP remain elusive. In this study, we identified geraniol as the most abundant volatile compound and found its accumulation to be strongly correlated with the expression of an R2R3-MYB transcription factor, PrMYB5b. Transient silencing of PrMYB5b in FTP petals resulted in a significant reduction in geraniol accumulation and downregulated the expression of PrDXS and PrGES genes. In contrast, stable overexpression of PrMYB5b in tobacco (Nicotiana tabacum L.) remarkably upregulated the transcript levels of endogenous NtDXS and heterologous PrGES genes, thereby leading to a substantial increase in geraniol production. Electrophoretic mobility shift assay (EMSA), yeast one-hybrid (Y1H), and dual-luciferase (Dual-LUC) assays revealed that PrMYB5b directly binds to and activates the promoters of PrDXS and PrGES. These findings confirm PrMYB5b as a core regulator of geraniol biosynthesis in FTP, providing both mechanistic insights and direct genetic targets for breeding fragrant varieties and engineering geraniol production.

Introduction:Polyploidization is not only an important driving force for plant speciation and evolution, but also an effective approach for plant domestication and improvement. Polyploid taxa are quite common in Paeonia section Paeonia (herbaceous peonies), but very rare in section Moutan (tree peonies), which are known as the 'king of flowers'. Methods:In this paper, we studied the origination of a tetraploid tree peony, P. × lemoinei 'Golden Era' ('GE', AABB), by comparing its karyotype with its seed parent, P. × lemoinei 'Golden Isles' ('GI', AB), based on genomic in situ hybridization (GISH) and rDNA fluorescence in situ hybridization (FISH). The meiosis behaviors of 'GI' were observed to make clear the cytogenetic process of unreduced (2n) gamete generation. Results:Four chromosomes with inter-genome translocations were identified in 'GE', two of which might be reciprocal translocations. The 2n female gametes via first division restitution (FDR) from 'GI' might play an important role in tetraploidization of 'GE'. Disucssion:The distant hybridization between intersterile species from different subsections of section Moutan probably promotes the tetraploidization of tree peonies by facilitating 2n gamete production. The mechanism of tetraploidization in section Moutan is highly consistent with that in section Paeonia, but is inseparable with the assist from mankind. The divergence of life history between tree peonies and herbaceous peonies is speculated to contribute to the different level of polyploidization, and distinct tempo of speciation and evolution, between section Moutan and section Paeonia in genus Paeonia. These findings bring new insights to polyploid breeding, speciation and evolution in genus Paeonia.

Tree peony is well-known for its ornamental value, medicine function, oil, and edible use. However, the difficulty in propagating tree peony impedes its cultivation and industrial advancement. Softwood cutting is an effective method to promote the propagation of tree peony. This research investigated the effects of several factors (cultivar, cutting phenological stage, auxin type, polyamine, and substrate) on the rooting of softwood cutting in tree peony. The results showed that rooting ability varied with cultivars and cutting phenological stages, with the highest rooting rates being for ‘High noon’ and ‘Jinghua Qingxue’ during the vigorous growth stage, reaching 50% and 53.33%, respectively. IBA 2000 mg·L−1 was optimal for rooting in ‘High noon’ cuttings, with the maximum root number (5.67) and root length (6.3 cm). Putrescine of 1.0 mM could significantly improve the rooting rate and rooting quality of ‘Jinghua Qingxue’ cuttings, which had the highest rooting rate of 54.17% in the cocopeat/perlite substrate (v:v 1:1). Anatomical observation showed that most adventitious roots were generated from callus meristem nodules differentiated from cortical parenchyma cells while a few came from stem bark, as well as integrated root induction. This study is an innovation in and supplement to tree peony propagation research, and a propagation protocol was primarily established for softwood cuttings in tree peony.

Flower color is an essential biological and ornamental trait in plants. Paeonia rockii (flare tree peony, FTP) exhibits diverse flower colors, characterized by a distinctive basal flare in petals, which enhances its ornamental and ecological value. However, while previous research has mainly focused on flare formation, the regulatory mechanisms controlling the background color of petals remain unclear. This study identifies a novel regulatory module governing petal background coloration in FTP. Within this module, PrMYB75a acts as the central regulator to promote anthocyanin accumulation, as evidenced by stable transformation in Arabidopsis thaliana and tobacco (Nicotiana tabacum), as well as virus-induced gene silencing in FTP. Furthermore, yeast one-hybrid, dual-luciferase reporter, and electrophoretic mobility shift assays collectively demonstrated that PrMYB75a directly activates two key anthocyanin structural genes, PrCHS1 and PrANS, by interacting with MYB-binding sites nearest to the ATG start codon in their promoters. Additionally, we identified an upstream regulator, PrFRS2, which activates both PrMYB75a and PrANS by binding to the FAR1/FHY3-binding sites in their promoters. Modulation of PrFRS2 expression levels through gene silencing and overexpression resulted in alterations in flower pigmentation in both FTP and tobacco. In summary, within the PrFRS2-PrMYB75a module, PrFRS2 indirectly activates PrCHS1 and PrANS by regulating PrMYB75a, or directly activates PrANS, leading to anthocyanin accumulation in FTP purple petals. This module represents a novel regulatory mechanism of petal background coloration in FTP, providing new perspectives on color variation in flowering plants and offering genetic resources for the improvement of the flower color trait in tree peonies.

[Objective]Most intersectional hybrids in Paeonia are triploids and generally sterile,producing a small number of hybrid offspring.Observing the meiotic chromosome behaviors of'Bartzella',an inter-sectional hybrid with high ornamental value and several hybrid offspring,can provide insight into the for-mation mechanism of its low fertility.[Methods]The study utilized the anthers of'Bartzella'as materials to observe the meiotic chromosome behaviors of pollen mother cells(PMCs)through the squash method.[Results](1)In metaphase Ⅰ,a significant number of univalents were formed in'Bartzella'PMCs.In anaphase Ⅰ,various types of chromosomal aneuploid segregation were observed,accounting for up to 89.13％.(2)During the first meiotic division,chromosome bridges and fragments were found in 27.20％of the PMCs;during the second meiotic division,this percentage was increased to 47.68％.(3)In meta-phase Ⅱ,spindle positioning abnormalities were relatively common,with fused spindles accounting for 27.06％and tripolar spindles accounting for 12.84％.The meiotic products included up to 72.41％of dy-ads and 13.43％of triads,which could generate unreduced gametes accounting for about 67％.[Conclusion]Chromosome aneuploidy segregation and loss of chromosome segments during meiosis may be the primary causes of the high sterility of'Bartzella'.Fused spindles,tripolar spindles,and abnormal cytokinesis at the end of second meiotic division may result in the production of unreduced gametes.The significant num-ber of unreduced gametes might give'Bartzella'the potential polyploid breeding.However,interploidy hybridization barriers could restrict its hybrid affinity.

Cross-incompatibility is the major challenge in inter-sectional distant hybridization between tree peonies (Paeonia sect. Moutan) and herbaceous peonies (Paeonia sect. Paeonia). Most of the intersectional cross combinations rarely produce hybrid progenies or even seeds. However, a combination with Paeonia × lemoinei Rehd. (P. delavayi × P. suffruticosa, tree type) as the pollen parent and P. lactiflora (herbaceous) as the seed parent resulted in many progenies. This research focuses on cytogenetics of two highly compatible intersectional combinations, P. lactiflora ‘Martha W.’ × P. × lemoinei ‘Golden Era’ (‘MW’ × ‘GE’) and P. lactiflora ‘Fen Yun Fei He’ × P. × lemoinei ‘L’Esperance’ (‘FYFH’ × ‘LE’). Karyotype analysis combined with genomic in situ hybridization (GISH) showed that the seed parents, ‘MW’ and ‘FYFH’, are both diploids (2n = 2x = 10, CC), while the pollen parents, ‘GE’ and ‘LE’, are tetraploid (2n = 4x = 20, AABB) and diploid (2n = 2x = 10, AB) respectively. Four progenies from ‘MW’ × ‘GE’, and another four progenies from ‘FYFH’ × ‘LE’, are all triploids (2n = 3x = 15, ABC). P. × lemoinei contributes two sets of chromosomes (AB) showing distinct signals in all tested triploids. The essential of a successful intersectional hybridization is to obtain 2x gametes from tree peony. P. × lemoinei has better compatibility in intersectional hybridization than other tree peony species, probably because of its higher frequency of 2x gamete (unreduced in diploid, or reduced in tetraploid) generation. Tetraploid tree peonies, such as ‘GE’, might produce abundant 2x gametes through normal meiosis, which is the possible reason for their better fertility and superior intersectional compatibility than diploid tree peonies. The mechanism of compatibility in intersectional hybridization is supposed to be a rebalance between the distant divergent parents, tree peonies and herbaceous peonies, triggered by ploidy increase of gametes from tree peonies. The discovery of tetraploid tree peony also provides valuable materials to study the mechanism of polyploidization in sect. Moutan, and to promote the distant hybridization breeding in Paeonia.

Polyploid breeding is one of the most important approaches to improve the traits of cultivated plants, but there has been no feasible method to get polyploid tree peony. In this study, we revealed a sexual triploidization approach from a diploid intersubsectional hybrid tree peony, Paeonia x lemoinei 'High Noon', to an allotriploid one, P. x lemoinei 'Oukan', by accurate karyotyping based on genomic in situ hybridization (GISH) and fluorescence in situ hybridization (FISH). The ancestor genomes from P. suffruticosa and P. delavayi, which were defined as A-genome and B-genome respectively, were clearly distinguished by GISH in 'High Noon' and 'Oukan'. Nonhomologous chromosomes were successfully recognized by means of 5S and 45S rDNA FISH. Karyotype comparation based on GISH and FISH indicated that the B-genome and one of the two A-genomes in 'Oukan' (2n=3x=15, AAB) were highly consistent to the corresponding genomes in 'High Noon' (2n=2x=10, AB), implying the triploid origination via 2n gametes produced by first division restitution (FDR). In addition, it was demonstrated for the first time that the dual in series sites of 5S rDNA located on the short arm of chromosome 3 in both A and B-genomes. Besides the familiar strong 45S rDNA signals observed at terminal region of short arms, single weak 45S rDNA signal was newly detected at terminal region in the short arm of certain chromosome 1 in A-genome, and at interstitial-distal region in the long arm of chromosome 5 in both genomes. These findings provide a proof and theoretical support for tree peony polyploid breeding, and an accurate karyotyping method for genetic research of plants in Paeonia.

Tree peony ( Paeonia sect. Moutan ) is an economically important multipurpose woody plant in terms of its medical, ornamental and oil values, but its breeding and industrial development are severely limited due to inefficient traditional propagation methods and existing in vitro regeneration systems. Meristematic nodules (MNs) are an attractive alternative to solve this problem. This study first presented a protocol for in vitro regeneration of P. ostii ‘Feng Dan’ via MN culture with four consecutive steps, including embryogenic callus (EC) formation, MN induction and leaf cluster differentiation, shoot elongation, rooting and acclimatization. The highest EC induction rate (81.25%) was achieved when cotyledons were cultured on modified Murashige and Skoog (mMS) medium with 4.04 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 5.37 µM α-naphthylacetic acid (NAA) for 30 days. The optimal MN induction rate (100%) and leaf cluster differentiation rate (45.83%) were obtained when ECs were cultured on modified woody plant medium (mWPM) supplemented with 2.02 µM CPPU + 2.27 µM thidiazuron (TDZ) for a subculture time of 10 days. The combination of 1.29 µM 6-benzyladenine (BA) + 0.58 µM gibberellin (GA 3 ) yielded the best shoot elongation (13.40 shoots per nodule), rooting rate (43.33%) and consequently survival rate (45.83%). The study will be beneficial to the mass propagation, breeding and genetic improvement of tree peony.

This is the first report concerning the sequence of histological and cytological events occurring during organogenesis from cotyledon-derived meristematic nodules (MNs) in Paeonia ostii ‘Feng Dan’. Sections were made and studies were carried out with dissecting microscope, light microscope, scanning electron microscopy (SEM) and transmission electron microscopy observation. Histological studies revealed a complex developmental process of morphogenesis including five stages: (1) callus originated from cell division in both cambial and cortical regions; type I—yellow compact callus with densely arranged clumps was identified as embryogenic callus. (2) pre-nodular structure consisted of organization center (a central area of vascularization surrounded by meristematic cell layers) and an epidermis-like layer; (3) independent MNs comprised of organization center, a cortical-like area of parenchymatous cells and an epidermal-like area; (4) nodular clusters displayed vigorously internal meristematic cell division and generated a relative movement towards the nodules periphery, establishing vascular connection with primordia; (5) successive new elongated shoots with axillary bud primordia were developed. SEM observations showed three types of extracellular matrix, a smooth membranous layer, fibrillar structures and granular mucilage-like secretions on embryogenic callus, demonstrating its dynamic morphological changes. Ultrastructural analysis revealed striking changes of chloroplast morphology and starch content during MNs morphogenesis. This study allows a better understanding of in vitro regeneration via MN culture and provides references for protocol optimization and genetic transformation. This report firstly revealed a developmental sequence, dynamic changes of extracellular matrix and ultrastructural characteristics during meristematic nodules morphogenesis in Paeonia ostii ‘Feng Dan’ with morpho-histological and cytological analyses.

Tree peony (Paeonia sect. Mouton) is a world-fa-mous ornamental and economically important spe-cies, but its conventional propagation methods se-verely limit industrial development. When tissue culture is an attractive option, the acclimatization becomes a bottleneck due to the poor root system architecture (RSA) with limited lateral roots and ar-rested growth of seedlings during ex vitro establish-ment. In this paper, we studied the seedling growth of P rockii 'Ding Hong' during acclimatization under various treatments with Arbuscular mycorrhizal fungi (AMF) inoculation or/and putrescine (Put) ap-plication. We found that AMF or Put alone optimized seedling growth and RSA in a species-dependent and concentration-dependent manner, respectively, and the combined uses of Claroideoglomus etunicatum (C.E.) with exogenous Put promoted additionally survival, growth and RSA traits of the seedlings ver-sus individual treatment, and the effects were de-pendent on concentration, with 0.05 mM Put in the C.E.-inoculated treatments being the most effective. Our studies also suggested that AMF inoculation and exogenous Put application improved morphological parameters via alterations of endogenous polyam-ines (PAs). The most obvious change was the dra-matic increase in spermidine (Spd), thus leading to a significant increase in both total PAs and the ratio of [spermine (Spm)+Spd]/Put and a decrease in Put/ PAs. Correlation analysis indicated that the overall balance of endogenous PM might be more import-ant than a specific one in root development, with the number of correlated parameters ranked as Put/ PAs > Spm+Spd/Put> Spd, total PM, Put> Spm. These results provide additional information for an im-proved ex vitro establishment of peony seedlings and enrich PA regulation theory.

Paeonia ostii 'Feng Dan' is an economically important, multipurpose woody plant in terms of its medical, ornamental, and oil values; however, there is a noticeable contradiction between the increasing demands and the lack of excellent germplasm resources because of traditional breeding and propagation approaches. In vitro embryo culture is an attractive option for this issue. This study presents a protocol for in vitro immature embryo culture in P. ostii `Feng Dan', which involves two steps: 1) immature seeds at 30 days after anthesis (DAA) (cellularization stage of endosperm, proembryo stage) or after being cultured in vitro for cotyledon embryo formation (upward micropyle with placenta was the best inoculation method with the highest ratio of seed with cotyledon embryo of 66.67%); and 2) seedling establishment was realized within 7 months via embryo (at 40 DAA or after) germination, shoot induction, rooting, and acclimatization. The multiplication potential was increased with embryo maturity. This protocol provides an available reference for embryo rescue and propagation of tree peony and will be beneficial to shortening the breeding cycle.

Allelic variation in floral quantitative traits, including the elements of flowers and fruits, is caused by extremely complex regulatory processes. In the genetic improvement of flare tree peony (Paeonia rockii), a unique ornamental and edible oil woody species in the genus Paeonia, a better understanding of the genetic composition of these complex traits related to flowers and fruits is needed. Therefore, we investigated the genetic diversity and population structure of 160 P. rockii accessions and conducted single-marker association analysis for 19 quantitative flower and fruit traits using 81 EST-SSR markers. The results showed that the population had a high phenotypic diversity (coefficients of variation, 11.87–110.64%) and a high level of genetic diversity (mean number of alleles, NA = 6.09). These accessions were divided into three subgroups by STRUCTURE analysis and a neighbor-joining tree. Furthermore, we also found a low level of linkage disequilibrium between these EST-SSRs and, by single-marker association analysis, identified 134 significant associations, including four flower traits with 11 EST-SSRs and 10 fruit traits with 32 EST-SSRs. Finally, based on the sequence alignment of the associated markers, P280, PS2, PS12, PS27, PS118, PS131, and PS145 may be considered potential loci to increase the yield of flare tree peony. These results laid the foundation for further analysis of the genetic structure of some key traits in P. rockii and had an obvious potential application value in marker-assisted selection breeding.

[目的]染色体核型信息是植物遗传育种研究的重要基础.亚组间远缘杂交育种是现代牡丹育种的重要方向.牡丹组植物染色体形态相似度高,远缘杂种的核型分析需要大量高质量的染色体制片.预处理是影响染色体制片质量的关键环节,通过改良预处理方法,提高染色体制片质量和效率,对获得牡丹远缘杂种准确的核型数据、研究牡丹远缘杂交育种的遗传规律具有重要意义.[方法]本研究以'正午'牡丹幼嫩花蕾中的雌蕊为材料,量化分析了4℃条件下不同预处理液和预处理时间对染色体收缩程度和分散程度的影响,统计了不同预处理效果下核型数据的差异.[结果]用对二氯苯饱和水溶液或对二氯苯-α-溴萘混合液进行预处理,均能取得良好的效果.在4℃下用对二氯苯饱和水溶液预处理36～72 h,或对二氯苯-α-溴萘混合液预处理24～48 h,可稳定获得大量收缩适度、分散良好的染色体制片.利用大量高质量的染色体制片获得了'正午'牡丹更多的核型信息:其核型公式为2n=2x=10=8m(2SAT)+2st(2SAT),随体位于第4、8、9、10号染色体短臂上,其中9、10号染色体上的随体比4、8号染色体上的随体更容易观察到.[结论]通过改进预处理效果能有效提高'正午'牡丹染色体制片质量和效率,获得更加丰富、准确的核型信息.本研究采用的量化方法能从染色体收缩程度和分散程度两个方面有效评价染色体制片质量,筛选出的预处理方法稳定、高效,为远缘杂种牡丹染色体核型分析和进一步遗传学研究奠定了良好的基础.

Tree peonies native to China are a precious crop with ornamental, medicinal and edible oil properties, of which flare tree peony (Paeonia rockii) is one of the most significant germplasms in Paeonia. The development and application of expressed sequence tag-simple sequence repeat (EST-SSR) markers are very valuable for genetic and breeding applications, but EST-SSR resources for the genus Paeonia are still limited. In this study, we first reported the development of SSRs within transcription factors (TFs) in P. rockii based on next-generation sequencing (NGS) and single-molecule long-read sequencing (SMLRS). A total of 166 EST-SSRs containing six nucleotide repeat types were identified from 959 candidate TFs associated with yield, with an average of one SSR per 5.83 unigenes. In total, 102 (61.45%) pairs of primers produced amplification products in the two RNA-seq cultivars. Among them, 58 (56.86%) pairs of primers from 18 gene families (AP2, bHLH, HSF, etc.) were identified to be polymorphic both in the parents of a linkage mapping population and in eight randomly selected accessions of P. rockii. Further, the 58 EST-SSRs indicated a high level of informativeness with PIC values ranging from 0.32 to 0.91 (mean 0.70) after assessment in 37 tree peony accessions. Transferability studies indicated that the amplification ratio of the 58 pairs of primers ranged from 89.66 to 100% across seven species of Paeonia. In addition, a genetic relationship study was performed in 62 accessions. Cluster analysis using the neighbour-joining (NJ) tree demonstrated that major clusters corresponded to the known pedigree trees. Taken together, these newly developed EST-SSRs have a potential use in the conservation of tree peony germplasm and marker-assisted selection (MAS) breeding.

[目的]牡丹微繁殖技术已在20多个品种中建立,但污染率高、增殖系数低、生根难和移栽成活率低等问题仍然存在,并制约着该技术的推广应用,因此开展微繁殖技术体系的优化研究具有重要意义.[方法]以'正午'牡丹为材料,通过对不同外植体启动培养效果比较、增殖培养阶段LED(发光二级管)光源应用以及生根方式改进等试验,对微繁殖技术体系进行优化改进.[结果]启动培养阶段,黄化嫩茎外植体优于鳞芽外植体,其污染率较低(19.59％),存活率较高(80.41％),且基本不褐化.增殖培养阶段,与荧光灯光源相比,LED光源(光质配比70％红光+30％蓝光,光照强度50μmol·m-2 s-1)可以显著促进试管苗的增殖和生长,增殖系数3.79,且试管苗生长健壮.生根培养阶段,基于维生素B2(VB2)在黑暗条件下可以和IBA共存、但在光照条件下分解IBA的特性,将试管苗先在附加IBA 1.0 mg·L-1和VB21.0～5.0 mg·L-1的培养基中暗培养30天诱导根原基,再转至光照培养20天,利用VB2加速IBA分解、消除IBA对根原基伸长生长的抑制,可以实现试管苗一步生根,其生根率(71.07％～74.23％)和根数(3.17～3.34条)与两步生根无显著差异.与两步生根法获得的生根苗相比,一步生根法获得的生根苗基部愈伤组织较少,生根苗质量更佳,其一级苗占比为75.00％,移栽成活率为85.00％.[结论]在'正午'牡丹微繁殖中,黄化嫩茎是一种优于鳞芽的外植体来源,其污染率较低、诱导成活率较高;光质配比70％红光+30％蓝光、光照强度50μmol·m-2 s-1的LED光照培养,可以显著促进试管苗增殖和生长;在生根培养基中添加维生素B2,结合光照条件控制(暗培养30天后转至光照培养20天),可实现试管苗的一步生根,其操作流程简单、生根成本低、试管苗质量佳、移栽成活率高.本研究所建立和优化的'正午'牡丹微繁殖技术体系,不仅可以促进该品种快繁技术的产业化应用,还可为其他牡丹品种微繁殖技术研究提供参考.

Research Highlights: This study, based on the first collection of cultivated Paeonia rockii (flare tree peony, FTP) germplasm across the main distribution area by our breeding desires, comprehensively evaluates these accessions by using phenotypic traits, expressed sequence tag (EST)-simple sequence repeat (SSR) markers and chloroplast DNA sequences (cpDNA). The results show that these accessions collected selectively by us can represent the genetic background information of FTP as a germplasm of tree crops. Background and Objectives: FTP has high cultural, ornamental and medicinal value traditionally, as well as recently presenting a significance as an emerging edible oil with high α-linolenic acid contents in the seeds. The objectives of this study are to reveal the characteristics of the genetic diversity of FTP, as well as to provide scientific suggestions for the utilization of tree peony breeding and the conservation of germplasm resource. Materials and Methods: Based on the phenotypic traits, EST-SSR markers and chloroplast DNA sequence variation, we studied the diversity of a newly established population of 282 FTP accessions that were collected and propagated by ourselves in our breeding project in recent years. Results: (1) There was an abundant variation in phenotype of the accessions, and the phenotypic variation was evenly distributed within the population, without significant hierarchical structure, (2) the EST-SSR data showed that these 282 accessions had relatively high genetic diversity, in which a total of 185 alleles were detected in 34 pairs of primers. The 282 accessions were divided into three distinct groups, and (3) the chloroplast DNA sequences (cpDNA) data indicated that these accessions had a higher genetic diversity than the population level and a lower genetic diversity than the species level of wild P. rockii, and the existing spatial genetic structure of these accessions can be divided into two branches. Conclusions: From the results of the three analyses, we found that these accessions can fully reflect the genetic background information of FTP germplasm resources, so their protection and utilization will be of great significance for genetic improvement of woody peonies.

Tree peony (Paeonia sect. Moutan) is a world famous ornamental and economically important species, but it is recalcitrant to in vitro regeneration. Here, we present a protocol for induction of direct somatic embryogenesis (SE) and direct shoot organogenesis (SO) from zygotic embryos (ZEs) of Paeonia rockii and P. ostii. The results showed that explant genotypes, ZEs stages, sucrose and plant growth regulators (PGRs) have greatly influences on in vitro morphogenesis. The highest frequency of SE (48%) and mean number of somatic embryos (5) was obtained from mature ZE of P. rockii ‘Jing Hong’ when cultured on modified Murashige and Skoog (mMS) medium supplemented with 2.22 µM BA and 0.23 M sucrose.The proliferation can be achieved by recurrent production of embryogenic callus and somatic embryos on mMS medium supplemented with 0.89 µM BA. The germinated rate of somatic embryos was 45% on WPM medium containing 2.22 µM BA and 1.44 µM GA3 after dormancy release at 4 °C under dark for 20 days. In addition, direct SO was obtained firstly in tree peony by using a variety of cytokinins. Morpho-histological analysis confirmed the initiation, development and reserve accumulation of somatic embryos and shoots. The study will be benificial to the propagation and breeding of tree peony. Tree peony (Paeonia sect. Moutan) is a world famous ornamental and economically important species which is recalcitrant to in vitro regeneration. Direct somatic embryogenesis protocol of P. rockii ‘Jing Hong’ was developed and morpho-histological analysis confirmed the initiation, development and reserve accumulation during in vitro morphogenesis. The study will provide valuable reference for propagation and breeding of tree peony.