Morpho-Histological and Cytological Study on Meristematic Nodule Induction and Shoot Organogenesis in Paeonia Ostii ‘Feng Dan’

Morpho-Histological and Cytological Study on Meristematic Nodule Induction and Shoot Organogenesis in Paeonia Ostii ‘Feng Dan’ | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Morpho-Histological and Cytological Study on Meristematic Nodule Induction and Shoot Organogenesis in Paeonia Ostii ‘Feng Dan’ Li Xu, Cheng Fang Yun*, Yuan Zhong This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-685307/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 10 Jan, 2022 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted 4 You are reading this latest preprint version Abstract This is the first report concerning the sequence of morpho-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 (TEM) observation. Histological studies revealed a complex developmental process of morphogenesis that including five stages: (1) callus originated from cell division in both cambial and cortical regions, and type I - yellow compact callus with densely arranged clumps were 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 complete vascular system and axillary bud primordia were developed. SEM observations showed three types of extracellular matrix (ECM), a smooth membranous layer, fibrillar structures and granular mucilage-like secretions on embryogenic callus, and recorded 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. Plant Molecular Biology and Genetics Paeonia ostii ‘Feng Dan’ Meristematic nodule Organogenesis Histology Ultrastructure Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tree peony ( Paeonia sect . Moutan ) is well-known in China because of its medical, ornamental and oil value (Yu et al. 2016). P. ostii is main option for oil tree peony because of the high yield and adaptability. However, the low efficiency and long cycle of conventional propagation methods, such as grafting and division, severely constrain its breeding and are insufficient to address the increasing commercial demands. Thus, an efficient and stable in vitro regeneration system is urgently needed. In general, no regeneration system published to date can meet the needs for the propagation and genetic manipulation of tree peonies due to various obstacles, such as vitrification, low multiplication, poor rooting and difficult acclimatization in micropropagation (Beruto et al. 2004; Wen et al. 2020), rare differentiation in callus culture (Zhu et al. 2018), and high deformity rates and low germination in somatic embryogenesis (Du et al. 2020); hence, innovative breakthroughs are needed to overcome this problem. Meristematic nodules (MNs) are a special structure comprising organization centers (OCs), a cortical-like area of parenchymatous cells and an epidermal-like area under histological observation, which have high regeneration potential, genetic stability and long-term cellular dynamics, making them an attractive alternative for plant regeneration via organogenesis, in vitro phytochemical production and plant transformation (McCown et al. 1988; Batista et al. 2008). Successful in vitro regeneration through MN culture has been reported in several woody and herbaceous plants, such as Eucalyptus globulus (Dobrowolska et al. 2017), Liquidambar orientalis (Bayraktar et al. 2015), Populus euphratica (Ferreira et al. 2009), Humulus lupulus (Fortes and Pais 2000), Sclerocarya birrea (Moyo et al. 2009) and Billbergia zebrina (Dal Vesco et al. 2011). There are scarce reports of MNs in Paeonia . Zhong (2011) established a nodule induction and multiplication system of P. rockii through calli induced from petiole sections. Subsequently, Qin et al (2012) further optimized the system in P. lemoinei ‘Golden Era’ and P. itoh ‘Barzella’ but failed to regenerate complete shoots. Histological and cytological analysis about the MN culture system have been performed in several plants (Aitken-Christie et al. 1988; Batista et al. 2000; Ferreira et al. 2009; Dal Vesco et al. 2010), while the origin of MN and their developmental pattern are highly variable according to the species and explant type. Meanwhile, histological research is necessary to provides detail evidence for distinguishing MN from somatic embryo since they are superficially resembled (Haensch 2004). Organogenesis from nodules is the major limiting step of the morphogenesis process in tree peony, thus little information on the correlation of developmental stages with histological and cytological events available. Recently, we have developed an efficient and reproducible regeneration system via MN culture (unpublished), which can be useful as a model for studying different aspects of nodule morphogenesis. Therefore, this study aims to reveal the morpho-histological and cytological pattern addressing developmental sequence for induction and differentiation process of MN in P. ostii ‘Feng Dan’, providing critical references for optimization of this system and application of gene transfer. Materials And Methods Plant materials and sterilization Mature seeds of P. ostii ‘Feng Dan’ at 90 days after anthesis (DAA) were collected in August 2018 from living adult plants grown in Beijing Guose Peony Garden in Beijing, China (40°45′N, 115°97′E) (Fig. 1A, B), and stored in a freezer (-4℃) for 2 months before use to break dormancy. The seeds were washed under running tap water for 15 mins before soaking in commercial liquid detergents (1% v/v; 5 min). Then, seeds were sterilized by a dipping in ethanol (70% v/v; 30 s), followed by dipping in a solution of NaOCl (0.2% v/v; 5 min) and three rinses with sterile distilled water. Medium and culture conditions The basal mediums were modified Murashige and Skoog medium (mMS, half-strength macroelements and full-strength Ca 2+ ) (Murashige and Skoog 1962),1/2 MS (all macroelements at half-strength) and modified woody plant medium (mWPM, double strength of Ca 2+ ) (Lloyd and McCown 1980). All media were supplemented with 3% sucrose and 0.7% agar, and the pH was adjusted to 5.8-6.0 before autoclaving (at 118 kPa and 121°C for 20 min). All reagents were supplied by Biodee (Beijing, China). The cultures, without additional description, were maintained at 24 ± 1°C under a 16 h photoperiod of 50 µmol·m -2 ·s -1 illumination intensity provided by an LED light (70% red light + 30% blue light) (TLD 36 W Philips, Beijing, China). Callus induction Zygotic embryos were aseptically isolated from seeds and inoculated on germination medium [mMS+2.57 µM 6-benzyladenine (BA)+2.89 µM gibberellin (GA 3 )] for 15 days (Fig. 1C). Their expanded cotyledons were cut into 1×1 cm pieces and inoculated on callus induction medium (CIM) [mMS+2.57 µM BA+5.37 µM α-naphthylacetic acid (NAA)] under dark condition with the abaxial side touching the medium. Cultured explants were collected on 0, 5, 10, 15, 20 and 25th day respectively from CIM for morphological examine and histological analysis. Embryogenic callus (EC) and non-embryonic callus (NEC) cultured in CIM for 30 days were distinguished with morpho-histologic and ultrastructural analyses. MN induction and shoots differentiation ECs induced in CIM after 30 days of culture derived from cotyledons were inoculated into differentiation induction medium (DIM) [mWPM containing 2.02 µM N -(2-chloro-4-pyridyl)- N -phenylurea (CPPU)] under light condition for MN induction and shoot differentiation with 10 days of subculture. Nodules with leaf clusters developed from DIM after 12 times of subculture were transferred onto mWPM containing 1.29 µM BA and 0.58 µM GA 3 for shoot elongation with subculture time of 30 days. The materials were taken every generations for morpho-histologic and ultrastructural analyses. Rooting and acclimatization Shoots elongated to 1-3 cm were successively excised and cultured for rooting following the protocol of Wang et al. (2016). Shoots were cultured on root induction medium [1/2 MS+4.92 µM indole-3-butyric acid (IBA)+11.34 µM putrescine (Put)] for 38 days in the dark and then transferred to root expression medium [Plant growth regulator-free 1/2 MS+0.4% activated carbon (AC)] for 20 days in the light. During the root induction phase, the shoots were subjected to cold treatment (4°C, 8 days) in the dark and then cultured at 24 ± 1°C for another 30 days. The rooted plantlets were excised carefully from the medium, washed thoroughly with tap water, and finally transferred to pots containing a mix of an autoclaved vermiculite, peat, and perlite (1:1:1, v/v/v) substrate. The plantlets were grown in a culture chamber at 20 ± 1℃ under a 16 h photoperiod of 50 µmol·m -2 ·s -1 photosynthetic photon flux density provided by fluorescent lamps. Histological analysis Samples were fixed in a mixture of formaldehyde, glacial acetic acid, and 50% alcohol (1:1:18) for 48 h. The fixed samples were dehydrated in a series of graded alcohol (50, 70, 85, and 95 to 100%), at 1 h per level, submerged in absolute ethanol and xylene (1:1) for 2 h followed by dipping in pure xylene for 2 h. The samples were embedded in paraffin wax overnight and sectioned at 8 to 10 µm on a rotary microtome. The sections were dried overnight and exposed to a xylene-ethanol series to remove the paraffin and stained with 0.1% safranin and 0.1% fast green. A Leica model DM500 microscope was used for histological analysis. Scanning electron microscopy (SEM) observation Samples were prefixed in 2.5% buffered glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 2 h at room temperature. After dehydration through a graded ethanol series (30, 50, 70, 80, 90, 95, and 100%) and gradual replacement through a graded tert-butanol series (50%, 70% and 100%), 20 minutes per level. The samples were dried with Freeze dryer (JEOL JFC-320), sputter-coated with gold (JEOL JFC-160) and observed with a SEM (S-3400N). Transmission electron microscopy (TEM) observation Samples of 2×2×2 mm size were prefixed in 4% buffered glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 2 h at room temperature. The samples were rinsed in the same buffer with four changes (15 min each) and post fixed in buffered 1% OsO4 at 4℃ overnight. After rinsing in distilled water, the samples were dehydrated in a graded acetone series (50, 70, 80, 90, and 100%) with 20 minutes per level and embedded in Epoxy resin. Ultrathin sections were cut to 70 nm on a LKB-5 ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a TEM (JGE-1200 EX). Results Callus induction Five days after cultured on CIM, there was no obvious difference in morpho-histological observation of the explants. The cotyledons turned white and became swollen (Fig. 2A) due to vigorously division of cortex parenchyma cells (Fig. 2D) after 10 days of culture. Meanwhile, meristematic cell mass with rapid cell division was observed locally around the area of the vascular bundle (Fig. 2E). After 15 days of culture, visible calli formed on the edge of the cotyledons (Fig. 2B). Sectional observation revealed that callus formation was unevenly along the explant tissue so that the explant not kept a regular shape (Fig. 2F). The original tissue structure and morphology of some parts of the explant disappeared, and a small proportion of calli expanded into clumps after 20 days (Fig. 2C). The proportion of clumpy calli increased, and gradually covering the entire explant after 25 days. There were two morphologically distinct types of calli after 30 days of induction: type I-yellow compact calli with densely arranged clumps (Fig. 3A) and type II-transparent watery loose calli (Fig. 3E). Identification of ECs and NECs It was observed through SEM that surface cells of type I callus showed globular shape and tightly packed structure (Fig. 3B), while type II callus exhibited disorganized, elongated and tubular cells (Fig. 3F). Cells of type I callus were small, isodiametric and densely arranged with large and clear nuclei under histological analysis (Fig. 3C). While type II callus were shown to be large, irregular, and highly vacuolated cells with abundance of intercellular space (Fig. 3G). Regarding cytological analysis，type I callus cells had large nucleus containing prominent nucleoli and chromatin that showed even distribution and little or no condensation, a few small scattered vacuoles, abundant mitochondria , and chloroplasts were degraded to plastids with starch grains accumulation in which the endometrial system was disassembled (Fig. 3D). In contrast, cells of type II callus showed a large vacuole occupying nearly the whole cytoplasmic space, the nucleus and other cytoplasmic organelles located in a narrow strip of cytoplasm between the cell wall and the large vacuole (Fig. 3H). Besides, no starch grain was observed. Type I callus cultured in CIM obtained regenerated shoots ultimately, while Type II callus gradually browning without differentiation. In summary, type I callus were identified as ECs. MN induction and organogenesis Morpho-histological study revealed a developmental sequence leading to the formation of MNs and shoot differentiation. ①Pre-nodular structures: The yellow callus turned green after subculture in DIM for 1-2 generations (Fig. 4A). Correspondingly, cells on the surface of the callus were organized into small units under SEM observation (Fig. 4B). The external layer of the callus were composed of small, isodiametric, densely stained meristematic cells which were organized into abundant meristematic cell masses in peripheral regions (Fig. 4C). During 3-4 generations, neo-formed tracheary elements developed inside of meristematic cell mass and organization centers (OCs) consisting of a central area of vascularization surrounded by meristematic cell layers with vigorous division were observed in histological analysis. Later, the OCs became autonomous and developed an epidermis-like layer (Fig. 4F). These visible small protuberances were termed as pre-nodular structures (Fig. 4D, E). ② MNs: After subculture for 5-6 generations, the pre-nodular structures greatly increased in diameter due to vigorous divisions taking place in meristematic cells surrounding vascularized centers, and rapidly formed conspicuous large protuberances (Fig. 4G, H) differentiated a more defined internal structure. Histological sections revealed that the large protuberance comprising of OCs, a cortical-like area of parenchymatous cells and an epidermal-like area (Fig. 4I). These typical features account for their classification as MNs. Smaller nucleus and larger vacuoles were observed under TEM, and less starch grains exhibited in the plastids, where containing moderately developed lamellar structures (Fig. 6A, B). ③Nodular clusters: After 7-8 generations, enlargement of nodules in size were accompanied by the formation of indentations created by differential expansion of multiple OCs，that appeared initially as small groove on the surface of the nodules, and then progressively deepened, yet nodule break-up was never observed. In the same way, smaller ‘daughter nodules’ were produced without detachment. Thus, several MNs displaying different levels of development were loosely attached to each other and developed into nodular clusters in appearance (Fig. 4A, B). During 7-10 generations, meristematic cells of OCs inside the nodules intensely divided and showed relative movement towards the nodules periphery (Fig. 4C), which resulted in the formation of primordia established vascular connection with the nodule (Fig. 4D, E, F). Smaller nucleus and larger vacuoles were also observed under TEM, nevertheless, plastids contained moderately developed lamellar structures without starch grain (Fig. 6C, D). There were lots of mitochondria and extensive rough endoplasmic reticulum (RER) adjacent to dictyosome that were active in producing vesicles (Fig. 6E). ④ Shoots differentiation: After 11-12 generations, early stage of leaf clusters formed with development and elongation of primordia (Fig. 4G, H), and apical meristems establishing vascular connection with the nodule (Fig. 4I). Nodules with leaf clusters were not conducive to promoting shoot elongation when cultured in the same DIM (Fig. 7C), but new elongated shoots were able to successively developed after transferring to medium containing BA and GA 3 (Fig. 7D), which had complete vascular system and axillary bud primordia (Fig. 7A, B). At this stage, chloroplasts developed a well-organized internal membrane system (Fig. 6F). These regenerated shoots were developed into plantlets through rooting culture (Fig. 7E) and transplanted to culture chamber successfully (Fig. 7F), which supporting the usefulness of this in vitro regeneration protocol in tree peony. The extracellular matrix (ECM) SEM revealed the presence of a discontinuous amorphous secretions outside the callus surface，and varied in structure, which termed as ECM. Compared to ECs covered with numerous compact membranous layers, fibrillar structure and abundant granular mucilage-like secretions (Fig. 8A, B), cells of NECs provided a ‘‘peeling’’ appearance (Fig. 8C). Enlargement and multiplication of the surface cells of nodules resulted in the rupture at various sectors of membranous layer, consequently, transition from membranous layers to fibrillar structures were exhibited on the nodules surface (Fig. 8D, E). The structure of superficial cells become slightly elongated and regularly arranged leading to the formation of epidermis-like surface under SEM at stage of nodular clusters (Fig. 8F), and the ECM structures on the surface gradually decreased and presented fragments appearance (Fig. 8G). However, dense granular mucilage-like secretions were exposed on the top surface of the primordia regions exclusively (Fig. 8H). No ECM performed on the smooth surface of newly formed leaves (Fig. 8I). Discussion Histological analysis To our knowledge, this is the first report about histologic and ultrastructural analyses of in vitro regeneration via MNs culture in tree peony. The selection of ECs is prerequisite for in vitro regeneration. In order to distinguish ECs and NECs, morpho-histological and ultrastructural studies were conducted, respectively. There were distinct differences between the two tissues and we identified type I callus as ECs, making it easier to select for subsequent experiment in view of its morphological characteristics, which was basically consistent with previous observations (Moura et al. 2008; Shang et al. 2009; Maadon et al. 2016). MNs could initiated directly from explants in Cichorium intybus (Piéron et al. 1993,1998), Eucalyptus globulus (Trindade and Pais 2003), Populus euphratica (Ferreira et al. 2009), Sclerocarya birrea (Moyo et al. 2009) or indirectly from inducted calli, like Pinus radiata (Aitken-Christie et al. 1988), Humulus lupulus (Batista et al. 2000; Fortes and Pais 2000) and Acacia mangium (Xie and Hong 2001). Induction of MNs in tree peony belongs to latter. In addition, callus originate from cell division in both cambial and cortical regions of explant, same as clarification in Qin et al (2012). Our morpho-histological observation of MNs development in tree peony was similar to descriptions in Cichorium intybus (Piéron et al. 1993,1998), Humulus lupulus (Batista et al. 2000; Fortes and Pais 2000), Populus euphratica (Ferreira et al. 2009) and Vriesea reitzii (Dal Vesco and Guerra 2010). There were three developmental stages leading to the formation of nodules before organogenesis, including pre-nodular structures, MNs and ‘polycenter’ nodules in sequence. Concurrently, MNs have been characterized by distinct internal structures involving a central area of vascularization surrounded by actively dividing meristematic cells (OCs), a cortical-like area of parenchymatous cells and an epidermal-like area. In opposition to descriptions in Populus euphratica (Ferreira et al. 2009), Cichorium intybus (Piéron et al. 1993,1998) that expansion of ‘polycenter’ nodules in diameter was accompanied by the formation of cracks in the parenchymatous tissue where break up observed along the surfaces of voids, thus multiplying the number of independent nodules, we never observed nodules division. In this report, the nodules increased number with groove on the surface and developed into nodular cluster structure in appearance without nodules detachment. This was consistent with demonstration in Humulus lupulus (Batista et al. 2000; Fortes and Pais 2000). It was reported that shoots originate from epidermis or cortex tissue of nodules in Humulus lupulus (Batista et al. 2000; Fortes and Pais 2000), while histological examination revealed that it developed from parenchymal cells around the vascular center in Cichorium intybus (Piéron et al. 1993,1998). In our research, shoots were proved to regenerate from endogenous parenchyma cells in the vicinity of nodule vascular centers. These distinctions may be related to species differences. Abundant vascular tissues, a predominance feature of nodules, might be closely related to regeneration since the neovascularization following shoots origination was frequent in numerous cases of organogenesis (Piéron et al. 1998; Fortes and Pais 2000; Ferreira et al. 2009). Presence of vascular centers may improve individualization of nodules and can be sign of regeneration. The histological analysis provided detail evidence for hypothesis that the nodule developmental pathway was distinct to the embryogenic pathway, but highly parallel (McCown et al. 1988). The MNs morphogenetic resulted in the development of monopolar axes, in contrast to the bipolar pattern of somatic embryos differentiation in tree peony (Du et al. 2020). Ultrastructural changes The most striking ultrastructural feature was the changes of chloroplast morphology and starch content during development under TEM observation. Chloroplasts were degraded to plastids with large amount of starch grains accumulation in ECs cells. Plastids gradually developed into mature chloroplasts as well as starch grain decreased and disappeared step by step. Similar phenomenon had been demonstrated previously in Bauhinia forficate (Appezzato-da-Glória and Machado 2004), Papaver somniferum (Oveűka et al. 2000) and Solanum melongena (Fournier et al. 1995). Starch, one kind of carbohydrate, considered to be the primary source of energy for cellular proliferation and growth, thus being consumed during in vitro morphogenesis (Moura et al. 2008). In addition, the storage of starch, abundant in ECs and shortage in NECs, probable indicated the acquisition of embryogenic potential (Pinto et al. 2010; Ribas et al. 2011), and they might function as signal molecules in signal transduction and gene regulation (Luis and Scherwinski-Pereira 2014). There was also a remarkable observation during differentiation that secretory dictyosome located around abundant extensive RER, surrounded with plentiful complete or dividing mitochondria. This phenomenon had been intensively reported (Aitken-Christie et al. 1988; Appezzato-da-Glória and Machado 2004; Diego et al. 2012). The active operation of inner membrane systems may reveal a high synthesis capacity associated with in vitro morphogenesis. SEM observations SEM observation in this report revealed the presence of ECM, which consist of membranous, fibrillar and granular structures. Similar structure had also been reported in Centella asiatica (Lai et al. 2011), Actinidia deliciosa (Popielarska-Konieczna et al. 2006, 2010) and Triticum aestivum (Konieczny et al. 2005). The chemical compositions and functions of ECM are still uncertain. It might be pectin polymers (Verdeil et al. 2001; Konieczny et al. 2007), arabinogalactan proteins (Konieczny et al. 2007) and lipid (Popielarska-Konieczna et al. 2008) that composing of ECM in previous reports. In addition, the formation of ECM might be a stress response to in vitro conditions or resulted from unsuitable pre-treatment (Konieczny et al. 2005). On the other hand, ECs could be identified with the ECM since it was suggested to be an indicator of cells potential for regeneration (Namasivayam et al. 2006; Popielarska-Konieczna et al. 2006; Lai et al. 2011; Yusoff et al. 2012). However, ECM was observed both on the ECs and NECs in Oryza sativa (Bevitori et al. 2014) and Helianthus tuberosus (Pilarska et al. 2014) regardless of its morphogenetic competence. This discrepancy may result from genotype differences. In our study, three kinds of ECM structures were abundant on ECs, but absent from NECs. Furthermore, dense granular secretions were exposed on the surface of the primordia exclusively, which was aligned with result of some thesis that the appearance of ECM covering the surface was linked to the induction of morphogenesis (Konieczny et al. 2005) and can served as a structural marker of somatic embryogenesis (Namasivayam 2007) or organogenesis (Popielarska-Konieczna et al. 2006). The ECM was also considered to play a vital role in regulation of signal, movement of nutrients, protection of surface structure during plant development and morphogenesis (Popielarska-Konieczna et al. 2010). The earliest symptom of differentiation was the distinctive presence of nodular clusters with elongated and regularly arranged superficial cells under SEM observation, leading to the formation of epidermis-like surface, where primordia initialed soon after. This corresponded with findings in Oryza sativa (Bevitori et al. 2014) and Actinidia deliciosa (Popielarska-Konieczna et al. 2011). The noticeable structure might be closely related to the origin of primordia and could be serviced as an indicator of differentiation (Brisibe et al. 1992). Conclusion This is the first report covering morpho-histological and cytological analyses involved in MNs morphogenesis system in P. ostii ‘Feng Dan’. The histological study revealed a developmental sequence leading to the formation of MNs and shoots regeneration, including callus induction, pre-nodular structures, MNs, nodular clusters, shoots differentiation. In addition, the knowledge obtained by SEM and TEM about the developmental states of MNs morphogenesis can be useful for optimization of the in vitro regeneration protocol and form the basis for further molecular analysis or genetic transformation. Abbreviations AC, activated carbon; BA, 6-benzyladenine; CIM, Callus induction medium; CPPU, N-(2-chloro-4-pyridyl)-N-phenylurea; DAA, Days after anthesis; DIM, Differentiation induction medium; EC, Embryogenic callus; GA 3 , Gibberellin; IBA, Indole-3-butyric acid; MN, Meristematic nodule; mMS, Modified Murashige and Skoog; mWPM, Modified woody plant medium; NAA, α-naphthylacetic acid; OC, Organization center; Put, putrescine; RER, Rough endoplasmic reticulum; SEM, Scanning electron microscopy; TEM, Transmission electron microscopy Declarations Compliance with ethical standards Funding The study was supported by National key R&D Program of China (2020YFD1000503). 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Plant Cell Tissue Organ Cult 66:167-173 http://dx.doi.org/10.1023/A:1010632619342 Yu SY, Du SB, Yuan JH, Hu YH (2016) Fatty acid profile in the seeds and seed tissues of Paeonia L. species as new oil plant resources. Sci Rep 6:26944-26944. https://doi.org/10.1038/srep26944 Yusoff NFM, Alwee SSRS, Abdullah MO, Chai-Ling H, Namasivayam P (2012) A time course anatomical analysis of callogenesis from young leaf explants of oil palm ( Elaeis guineensis Jacq.). J Oil Palm Res 24:1330-1341 Zhong Y (2011) Induction and Culture of Meristematic Nodules in Paeonia rockii . Beijing Forestry University, Beijing Zhu X, Li XQ, Ding WJ, Jin SH, Wang Y (2018) Callus induction and plant regeneration from leaves of peony. Hortic Environ Biotechnol 59:575-582 https://doi.org/10.1007/s13580-018-0065-4 Cite Share Download PDF Status: Published Journal Publication published 10 Jan, 2022 Read the published version in Plant Cell, Tissue and Organ Culture (PCTOC) → Version 1 posted Reviews received at journal 12 Jul, 2021 Reviewers invited by journal 06 Jul, 2021 Editor assigned by journal 04 Jul, 2021 First submitted to journal 03 Jul, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-685307","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":38031992,"identity":"16ba3343-5ce0-430c-8e0c-b7f9931a8dec","order_by":0,"name":"Li Xu","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Li","middleName":"","lastName":"Xu","suffix":""},{"id":38031993,"identity":"f393420d-aec4-4524-9be8-7bdd6c558cb7","order_by":1,"name":"Cheng Fang Yun*","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA2klEQVRIiWNgGAWjYBAC/hkg0oZBjp+9+fADorRI3ACRaQzGkj3H0gxAAgS1GERAtCRuuJGjIEGcFunmYw+/JBw2Nj6Qw2DAuMOmjrAWmWPpxjIJh+XMDpw98IDxTBoRtkjkmElL/jhsbHawL8GAse0wMVryv0lLJBxO3NzMYyDB2PafGO/nsEl+AGrZwAbWcoCwFokbaWbSDAnpxhJn2NIMEtuSJRsIaeGfkfxM8keCtRz//MeHH3xss+MnaAsIMPMwNENYCUSpBwLGHwyEY2MUjIJRMApGMAAAAPs+CfwVAUMAAAAASUVORK5CYII=","orcid":"https://orcid.org/0000-0002-3928-5731","institution":"Beijing Forestry University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Cheng","middleName":"Fang","lastName":"Yun*","suffix":""},{"id":38031994,"identity":"8cd2e8b9-5427-47c9-a273-7a2250eb131b","order_by":2,"name":"Yuan Zhong","email":"","orcid":"","institution":"Beijing Forestry University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Yuan","middleName":"","lastName":"Zhong","suffix":""}],"badges":[],"createdAt":"2021-07-04 04:23:02","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-685307/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-685307/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1007/s11240-021-02208-x","type":"published","date":"2022-01-11T01:50:05+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":11306281,"identity":"bd138d6e-676a-4d23-8462-e1e418fe9329","added_by":"auto","created_at":"2021-07-09 16:25:17","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":7244352,"visible":true,"origin":"","legend":"Explants used for study\na Flowers of P. ostii ‘Feng Dan’; b Mature seeds and zygotic embryos of P. ostii ‘Feng Dan’; c Cotyledons of P. ostii ‘Feng Dan’ seedlings cultured in germination medium for 15 days used as explants","description":"","filename":"OnlineFig1.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/f92e2066a08d8d7cc0c96fa5.png"},{"id":11306531,"identity":"afd560f5-05b7-4083-bc6d-692c956c2493","added_by":"auto","created_at":"2021-07-09 16:28:17","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":14735196,"visible":true,"origin":"","legend":"Morpho-histological observation of callus induction\na Cotyledons turned white and swollen after cultured in CIM for 10 days; b Visible callus was formed on the edge of the cotyledon after 15 days of culture; c Callus expanded into clumps after 20 days; d Vigorously division of cortex parenchyma cells and meristematic cells around vascular bundle; e Meristematic cell mass (white star) around area of the vascular bundle; f Callus formed unevenly on explant and initiated from cortex parenchyma cells and meristematic cells around vascular bundle; Abbreviations: P Parenchyma cells, VB Vascular bundle, CA Callus","description":"","filename":"OnlineFig2.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/7bccb0afdbbad27ec2201c2f.png"},{"id":11306960,"identity":"48f34b0f-6f35-4cf1-b447-7a2c80c3145f","added_by":"auto","created_at":"2021-07-09 16:31:17","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":12815194,"visible":true,"origin":"","legend":"Identification of embryogenic callus and non-embryogenic callus \na Type I-yellow compact callus with densely arranged clumps; b-d Characteristics of type I callus cells; b Surface cells showed globular shape and tightly packed structure; c Small, isodiametric and densely arranged cells with large and clear nuclei; d Cellular ultrastructure of type I callus; e Type II-transparent watery loose callus; f-h: Characteristics of type II callus cells; f Surface cells exhibited disorganized, elongated and tubular cells; g Large, irregular, and highly vacuolated cells with abundance of intercellular space; h Cellular ultrastructure of type II callus; Abbreviations: N Nuclear, NU Nucleoli, V Vacuole, P Plastid, M Mitochondria, S Starch grain, CW Cell wall","description":"","filename":"OnlineFig3.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/9169bcb73bee1dcfc2542fb4.png"},{"id":11306285,"identity":"6914a664-eb85-4f9d-8948-84769c4cb22d","added_by":"auto","created_at":"2021-07-09 16:25:17","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":15143444,"visible":true,"origin":"","legend":"Morpho-histological observation of meristematic nodules induction and organogenesis \na, b Callus with small units (black arrows); d, e Pre-nodular structures; g, h Meristematic nodules; c meristematic cell masses; f OCs consisting of a central area of vascularization surrounded by meristematic cell layers and developing an epidermis-like layer; i MNs comprised of OC, a cortical-like area of parenchymatous cells and an epidermal-like area; Abbreviations: PN Pre-nodular structures, MN Meristematic nodule, OC organization centers, EP Epidermal-like area, CP Cortical-like area of parenchymatous cells","description":"","filename":"OnlineFig4.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/ad3aa11299322f826c84f05b.png"},{"id":11306530,"identity":"16f13e64-00dc-4556-9f45-efd15b79c140","added_by":"auto","created_at":"2021-07-09 16:28:17","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":12883750,"visible":true,"origin":"","legend":"Morpho-histological observation of meristematic nodules organogenesis\na, b Nodular clusters; d, e Nodular clusters with dome-shaped primordia (black asterisks) and leave primordia (black arrows); g, h Leaf clusters; c meristematic cells of OCs inside the nodules intensely divided and generated a relative movement towards the nodules periphery; f Primordia established vascular connection with the nodule; i Early stage of leaf cluster was formed with development and elongation of primordia，and apical meristem establishing vascular connection with the nodule; Abbreviations: NC Nodular cluster, LC Leave cluster, OC organization centers, P primordia, LP Leaf primordia, AM Apical meristem, V vascular system","description":"","filename":"OnlineFig5.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/48a405ec572a5c92ce0cbf83.png"},{"id":11306288,"identity":"a3148ed0-2524-4fa8-acd5-0978a65172f9","added_by":"auto","created_at":"2021-07-09 16:25:21","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":116475134,"visible":true,"origin":"","legend":"TEM observation of cells during meristematic nodules induction and organogenesis\na Cellular ultrastructure of MNs cells; b Less starch grains exhibited in the plastids, where contained moderately developed lamellar structures; c Cellular ultrastructure of nodular cluster cells; d Plastids contained moderately developed lamellar structures without starch grain; e Numerous mitochondria and extensive rough endoplasmic reticulum (RER) adjacent to secretory dictyosome; f Chloroplasts developed a well-organized internal membrane system; Abbreviations: N Nuclear, NU Nucleoli, V Vacuole, P Plastid, C Chloroplast, R Rough endoplasmic reticulum, M Mitochondria, D Dictyosome, S Starch grain, CW Cell wall, G Grana, L Lamella structure, IS Intercellular spaces","description":"","filename":"OnlineFig6.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/d3548d9b29de5793087d5921.png"},{"id":11306282,"identity":"23a61d9a-62a1-4685-90cb-1ec0bc9e2702","added_by":"auto","created_at":"2021-07-09 16:25:17","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":12595478,"visible":true,"origin":"","legend":"Morpho-histological observation of shoots regeneration, rooting and acclimatization\na Leaf clusters with axillary bud primordia (black arrow); b Elongated shoots with axillary bud primordia (black arrow); c Leaf clusters; d Shoots elongation; e Rooted plantlets; f Plantlets transplanted to culture chamber successfully\n","description":"","filename":"OnlineFig7.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/40d97a4e4331d813177dae1d.png"},{"id":11306287,"identity":"197f4ac4-2d29-46da-8760-82dc67dc0cd2","added_by":"auto","created_at":"2021-07-09 16:25:20","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":82779118,"visible":true,"origin":"","legend":"SEM observations of ECM structures\na Membranous layer (white asterisks) and fibrillar structures (black arrows); b Granular mucilage-like secretions (black arrows) on embryogenic callus surface; c Cells on surface of non-embryogenic callus provided a ‘‘peeling’’ appearance; d, e ECM structures on MNs show transition from membranous layers to fibrillar structures (black arrows); f Epidermis-like surface of nodular clusters; g ECM structures gradually decreased and presented fragments appearance on nodular cluster; h Dense granular structures on the primordium region; i Smooth leaf surface cells showing the absence of ECM structures\n","description":"","filename":"OnlineFig8.png","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/145c7632dd181e15e73c0490.png"},{"id":17185704,"identity":"4743be12-324b-4671-b343-07e4335f2e67","added_by":"auto","created_at":"2022-01-11 01:50:10","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":9196559,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-685307/v1/0ff9229b-2112-49a6-a304-edf14747761f.pdf"}],"financialInterests":"","formattedTitle":"\u003cp\u003eMorpho-Histological and Cytological Study on Meristematic Nodule Induction and Shoot Organogenesis in Paeonia Ostii ‘Feng Dan’\u003c/p\u003e","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTree peony (\u003cem\u003ePaeonia\u0026nbsp;\u003c/em\u003esect\u003cem\u003e. Moutan\u003c/em\u003e) is well-known in China because of its medical, ornamental and oil value (Yu et\u0026nbsp;al. 2016).\u0026nbsp;\u003cem\u003eP. ostii\u0026nbsp;\u003c/em\u003eis main option for oil tree peony because of the high yield and adaptability. However, the low efficiency and long cycle of conventional propagation methods, such as grafting and division, severely constrain its breeding and\u0026nbsp;are insufficient to address the increasing commercial demands.\u0026nbsp;Thus, an efficient and stable\u003cem\u003e\u0026nbsp;in vitro\u003c/em\u003e regeneration system is urgently needed. In general, no regeneration system\u0026nbsp;published to date\u0026nbsp;can meet the needs for the propagation and genetic manipulation of tree peonies due to various obstacles,\u0026nbsp;such as vitrification, low multiplication, poor rooting and difficult acclimatization in micropropagation (Beruto et al. 2004; Wen et al. 2020), rare differentiation in callus culture (Zhu et al. 2018), and high\u0026nbsp;deformity rates and\u0026nbsp;low germination in\u0026nbsp;somatic embryogenesis (Du et al. 2020);\u0026nbsp;hence, innovative breakthroughs\u0026nbsp;are\u0026nbsp;needed to\u0026nbsp;overcome this problem.\u003c/p\u003e\n\u003cp\u003eMeristematic nodules (MNs)\u0026nbsp;are a special structure\u0026nbsp;comprising\u0026nbsp;organization centers (OCs), a cortical-like area of parenchymatous cells and an epidermal-like area under\u0026nbsp;histological observation, which have high regeneration potential, genetic stability and long-term cellular dynamics,\u0026nbsp;making them an attractive alternative for\u0026nbsp;plant regeneration via organogenesis,\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e phytochemical production and plant transformation\u0026nbsp;(McCown et al. 1988; Batista et al. 2008). Successful \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003eregeneration through MN culture has been reported in\u0026nbsp;several\u0026nbsp;woody and herbaceous plants, such as\u0026nbsp;\u003cem\u003eEucalyptus globulus\u003c/em\u003e (Dobrowolska et al.\u0026nbsp;2017), \u003cem\u003eLiquidambar orientalis\u003c/em\u003e (Bayraktar et al. 2015),\u0026nbsp;\u003cem\u003ePopulus euphratica\u0026nbsp;\u003c/em\u003e(Ferreira et al. 2009), \u003cem\u003eHumulus lupulus\u003c/em\u003e (Fortes and Pais 2000),\u003cem\u003e\u0026nbsp;Sclerocarya birrea\u0026nbsp;\u003c/em\u003e(Moyo et al. 2009) and\u003cem\u003e\u0026nbsp;Billbergia zebrina\u0026nbsp;\u003c/em\u003e(Dal Vesco et al. 2011).\u0026nbsp;There are scarce reports of MNs in \u003cem\u003ePaeonia\u003c/em\u003e. Zhong (2011) established a nodule induction and multiplication system of \u003cem\u003eP. \u0026nbsp;rockii\u0026nbsp;\u003c/em\u003ethrough calli induced from petiole sections. Subsequently, Qin et al (2012) further optimized the system in \u003cem\u003eP. lemoinei\u0026nbsp;\u003c/em\u003e\u0026lsquo;Golden Era\u0026rsquo; and \u003cem\u003eP. itoh\u0026nbsp;\u003c/em\u003e\u0026lsquo;Barzella\u0026rsquo; but\u0026nbsp;failed to regenerate complete shoots.\u003c/p\u003e\n\u003cp\u003eHistological and cytological analysis about the MN culture system have been performed in several plants (Aitken-Christie et al. 1988; Batista et al. 2000;\u0026nbsp;Ferreira et al. 2009; Dal Vesco et al. 2010), while the origin of MN and their developmental pattern are highly variable according to the species and explant type. Meanwhile,\u0026nbsp;histological research is necessary to provides detail evidence for distinguishing MN from somatic embryo since they are superficially resembled (Haensch 2004). Organogenesis\u0026nbsp;from nodules is the major limiting step of the\u0026nbsp;morphogenesis\u0026nbsp;process in tree peony, thus little information on the correlation of developmental stages with\u0026nbsp;histological and cytological\u0026nbsp;events available.\u0026nbsp;Recently, we have developed an efficient and reproducible regeneration system via MN culture (unpublished), which can be useful as a model for studying different aspects of\u0026nbsp;nodule morphogenesis.\u0026nbsp;Therefore, this study aims to reveal the morpho-histological and cytological\u0026nbsp;pattern addressing\u0026nbsp;developmental sequence for induction and differentiation process of MN in\u0026nbsp;\u003cem\u003eP. ostii\u0026nbsp;\u003c/em\u003e\u0026lsquo;Feng Dan\u0026rsquo;, providing critical references for optimization of this system and application of gene transfer.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cp\u003e\u003cstrong\u003ePlant materials and\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003esterilization\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMature seeds of \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Feng Dan\u0026rsquo;\u0026nbsp;at 90 days after anthesis (DAA) were collected in August 2018 from living adult plants grown in Beijing Guose Peony Garden in Beijing, China (40\u0026deg;45\u0026prime;N, 115\u0026deg;97\u0026prime;E) (Fig. 1A, B), and stored in a freezer (-4℃) for 2 months before use to break dormancy. The seeds were washed under running tap water for 15 mins before soaking in commercial liquid detergents (1% v/v; 5 min). Then, seeds were sterilized by a dipping in ethanol (70% v/v; 30 s), followed by dipping in a solution of NaOCl (0.2% v/v; 5 min) and three rinses with sterile distilled water.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMedium and culture conditions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe basal mediums were modified Murashige and Skoog medium (mMS, half-strength macroelements and full-strength Ca\u003csup\u003e2+\u003c/sup\u003e) (Murashige and Skoog 1962),1/2 MS (all macroelements at half-strength) and modified woody plant medium (mWPM, double strength of Ca\u003csup\u003e2+\u003c/sup\u003e) (Lloyd and McCown 1980). All media were supplemented with 3% sucrose and 0.7% agar, and the pH was adjusted to 5.8-6.0\u0026nbsp;before autoclaving (at 118 kPa and 121\u0026deg;C for 20 min). All reagents were supplied by Biodee (Beijing, China). The cultures, without additional description, were maintained at 24 \u0026plusmn; 1\u0026deg;C under a 16 h photoperiod of 50 \u0026micro;mol\u0026middot;m\u003csup\u003e-2\u003c/sup\u003e\u0026middot;s\u003csup\u003e-1\u003c/sup\u003e illumination intensity provided by an LED light (70% red light + 30% blue light) (TLD 36 W Philips, Beijing, China).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCallus induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eZygotic embryos were aseptically isolated from seeds and inoculated on germination medium [mMS+2.57\u0026nbsp;\u0026micro;M\u0026nbsp;6-benzyladenine (BA)+2.89 \u0026micro;M gibberellin (GA\u003csub\u003e3\u003c/sub\u003e)] for 15 days (Fig. 1C). Their expanded cotyledons were cut into 1\u0026times;1 cm pieces and inoculated on callus induction medium (CIM) [mMS+2.57 \u0026micro;M BA+5.37 \u0026micro;M\u0026nbsp;\u0026alpha;-naphthylacetic acid\u0026nbsp;(NAA)]\u0026nbsp;under dark condition\u0026nbsp;with the abaxial side touching the medium.\u0026nbsp;Cultured explants were collected on 0, 5, 10, 15, 20 and 25th day respectively from CIM for morphological examine and histological analysis. Embryogenic callus (EC) and non-embryonic callus (NEC) cultured in CIM for 30 days were distinguished with morpho-histologic and ultrastructural analyses.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMN induction and shoots differentiation\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eECs induced in CIM after 30 days of culture derived from cotyledons were inoculated into differentiation induction medium (DIM) [mWPM containing 2.02 \u0026micro;M \u003cem\u003eN\u003c/em\u003e-(2-chloro-4-pyridyl)-\u003cem\u003eN\u003c/em\u003e-phenylurea (CPPU)]\u0026nbsp;under light condition for MN induction and shoot differentiation with 10 days of subculture.\u0026nbsp;Nodules with leaf clusters developed from DIM after 12 times of subculture were transferred onto mWPM containing 1.29 \u0026micro;M BA and 0.58 \u0026micro;M GA\u003csub\u003e3\u003c/sub\u003e for shoot elongation with subculture time of 30 days. The materials were taken every generations for\u0026nbsp;morpho-histologic and ultrastructural analyses.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eRooting and acclimatization \u0026nbsp;\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eShoots elongated to 1-3 cm were successively excised and cultured for rooting\u0026nbsp;following the protocol of Wang et al. (2016).\u0026nbsp;Shoots were cultured on root induction medium [1/2 MS+4.92\u0026nbsp;\u0026micro;M indole-3-butyric acid (IBA)+11.34\u0026nbsp;\u0026micro;M\u0026nbsp;putrescine\u0026nbsp;(Put)]\u0026nbsp;for 38 days in the dark and then transferred to root expression medium [Plant growth regulator-free 1/2 MS+0.4% activated carbon (AC)] for 20 days in the light. During the root induction phase, the shoots were subjected to cold treatment (4\u0026deg;C, 8 days) in the dark and then cultured at 24 \u0026plusmn; 1\u0026deg;C for another 30 days. The rooted plantlets were excised carefully from the medium, washed thoroughly with tap water, and finally transferred to pots containing a mix of an autoclaved vermiculite, peat, and perlite (1:1:1, v/v/v) substrate. The plantlets were grown in a culture chamber at 20 \u0026plusmn; 1℃\u0026nbsp;under a\u0026nbsp;16 h\u0026nbsp;photoperiod of 50\u0026nbsp;\u0026micro;mol\u0026middot;m\u003csup\u003e-2\u003c/sup\u003e\u0026middot;s\u003csup\u003e-1\u0026nbsp;\u003c/sup\u003ephotosynthetic photon flux density\u0026nbsp;provided by fluorescent lamps.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHistological analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples were fixed in a mixture of formaldehyde, glacial acetic acid, and 50% alcohol (1:1:18) for 48 h. The fixed samples were dehydrated in a series of graded alcohol (50, 70, 85, and 95 to 100%), at 1 h per level, submerged in absolute ethanol and xylene (1:1) for 2 h followed by dipping in pure xylene for 2 h.\u0026nbsp;The samples were embedded in paraffin wax overnight and sectioned at 8 to 10 \u0026micro;m on a rotary microtome. The sections were dried overnight and exposed to a xylene-ethanol series to remove the paraffin and stained with 0.1% safranin and 0.1% fast green. A Leica model DM500 microscope was used for histological analysis.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;observation\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples\u0026nbsp;were prefixed in 2.5% buffered glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 2 h at room temperature. After dehydration through a graded ethanol series (30, 50, 70, 80, 90, 95, and 100%) and gradual replacement through a graded tert-butanol series (50%, 70% and 100%), 20 minutes per level. The samples were dried with Freeze dryer (JEOL JFC-320), sputter-coated with gold (JEOL JFC-160) and observed with a SEM (S-3400N).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTransmission electron microscopy (TEM)\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;observation\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSamples of 2\u0026times;2\u0026times;2 mm size were prefixed in 4% buffered glutaraldehyde (0.1 M phosphate buffer, pH 7.2) for 2 h at room temperature. The samples were rinsed in the same buffer with four changes (15 min each) and post fixed in buffered 1% OsO4 at 4℃ overnight. After rinsing in distilled water, the samples were dehydrated in a graded acetone series (50, 70, 80, 90, and 100%) with 20 minutes per level and embedded in Epoxy resin. Ultrathin sections were cut to 70 nm on a LKB-5 ultramicrotome, stained with uranyl acetate and lead citrate, and examined with a TEM (JGE-1200 EX).\u003c/p\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eCallus induction\u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFive days after cultured on CIM, there was no obvious difference in morpho-histological observation of the explants. The cotyledons turned white\u0026nbsp;and became swollen\u0026nbsp;(Fig. 2A)\u0026nbsp;due to vigorously division of cortex parenchyma cells\u0026nbsp;(Fig. 2D) after 10 days of culture. Meanwhile, meristematic cell mass with rapid cell division was observed locally around the area of the vascular bundle (Fig. 2E). After 15 days of culture, visible calli formed on the edge of the cotyledons (Fig. 2B). Sectional observation revealed that callus formation was unevenly along the explant tissue so that the explant not kept a regular shape (Fig. 2F). The original tissue structure and morphology of some parts of the explant disappeared, and\u0026nbsp;a small proportion of\u0026nbsp;calli expanded into clumps\u0026nbsp;after 20 days (Fig. 2C).\u0026nbsp;The proportion of clumpy calli increased, and gradually covering the entire explant after 25 days. There were two morphologically distinct types of calli after 30 days of induction: type I-yellow compact calli with densely arranged clumps (Fig. 3A) and type II-transparent watery loose calli (Fig. 3E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eIdentification of ECs and NECs\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIt was observed through SEM that surface cells of type I callus showed\u0026nbsp;globular shape and tightly packed structure\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e(Fig. 3B), while type II callus exhibited disorganized, elongated and tubular cells (Fig. 3F).\u0026nbsp;Cells of type I callus were small, isodiametric and densely arranged with large and clear nuclei\u0026nbsp;under histological analysis (Fig. 3C). While type II callus\u0026nbsp;were shown to be\u0026nbsp;large, irregular, and\u0026nbsp;highly\u0026nbsp;vacuolated cells with\u0026nbsp;abundance of intercellular space (Fig. 3G).\u0026nbsp;Regarding cytological analysis，type I callus cells\u0026nbsp;had large nucleus containing prominent nucleoli and chromatin that showed even distribution and little or no condensation, a few small scattered vacuoles, abundant mitochondria , and chloroplasts were degraded to plastids with starch grains accumulation in which the endometrial system was disassembled (Fig. 3D). In contrast, cells of type II callus showed a large vacuole occupying nearly the whole cytoplasmic space, the nucleus and other cytoplasmic organelles located in a narrow strip of cytoplasm between the cell wall and the large vacuole (Fig. 3H).\u0026nbsp;Besides, no starch grain was observed. Type I callus cultured in CIM obtained regenerated shoots ultimately, while Type II callus gradually browning without differentiation. In summary, type I callus were identified as ECs.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMN induction and organogenesis\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMorpho-histological study revealed a developmental sequence leading to the formation of MNs and shoot differentiation.\u003c/p\u003e\n\u003cp\u003e①Pre-nodular structures: The yellow callus turned green after subculture in DIM for 1-2 generations (Fig. 4A). Correspondingly, cells on the surface of the callus were organized into small units under SEM observation (Fig. 4B). The external layer of the callus were composed of small, isodiametric, densely stained meristematic cells which were organized into abundant meristematic cell masses\u0026nbsp;in peripheral regions (Fig. 4C). During 3-4 generations, neo-formed\u0026nbsp;tracheary elements\u0026nbsp;developed inside of meristematic cell mass and organization centers\u0026nbsp;(OCs)\u0026nbsp;consisting of a central area of vascularization surrounded by meristematic cell layers\u0026nbsp;with vigorous division were observed in histological analysis. Later, the OCs became autonomous and developed an epidermis-like layer (Fig. 4F). These visible small protuberances\u0026nbsp;were termed as pre-nodular structures\u0026nbsp;(Fig. 4D, E).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e②\u003c/strong\u003eMNs: After subculture for 5-6 generations, the pre-nodular structures greatly increased in diameter due to vigorous divisions taking place in meristematic cells surrounding vascularized centers, and rapidly formed conspicuous large protuberances (Fig. 4G, H) differentiated a more defined internal structure. Histological sections revealed that\u0026nbsp;the large protuberance\u0026nbsp;comprising of OCs, a cortical-like area of parenchymatous cells and an epidermal-like area\u0026nbsp;(Fig. 4I).\u0026nbsp;These typical features\u0026nbsp;account for their classification as MNs. Smaller nucleus and larger vacuoles were observed under TEM, and less starch grains exhibited in the plastids, where containing moderately developed lamellar structures (Fig. 6A, B).\u003c/p\u003e\n\u003cp\u003e③Nodular clusters: After 7-8 generations, enlargement of nodules in size were accompanied by the formation of indentations created by differential expansion of\u0026nbsp;multiple\u0026nbsp;OCs，that appeared initially as small groove on the surface of the nodules, and then progressively deepened, yet nodule break-up was never observed. In the same way, smaller \u0026lsquo;daughter nodules\u0026rsquo; were produced without detachment. Thus, several MNs\u0026nbsp;displaying different levels of development were\u0026nbsp;loosely attached to each other and developed into nodular clusters\u0026nbsp;in appearance (Fig. 4A, B).\u0026nbsp;During 7-10 generations, meristematic cells of OCs inside the nodules intensely divided and showed relative movement towards the nodules periphery\u0026nbsp;(Fig. 4C), which resulted in the formation of primordia\u0026nbsp;established vascular connection with the nodule\u0026nbsp;(Fig. 4D, E, F). Smaller nucleus and larger vacuoles were also observed under TEM, nevertheless, plastids contained moderately developed lamellar structures without starch grain (Fig. 6C, D). There were lots of mitochondria and extensive rough endoplasmic reticulum (RER) adjacent to dictyosome that were active in producing vesicles (Fig. 6E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e④\u003c/strong\u003eShoots differentiation: After 11-12 generations, early stage of leaf clusters formed with development and elongation of primordia (Fig. 4G, H), and apical meristems establishing vascular connection with the nodule\u0026nbsp;(Fig. 4I).\u0026nbsp;Nodules with leaf clusters were not conducive to promoting shoot elongation when cultured in the same DIM (Fig. 7C), but\u0026nbsp;new\u0026nbsp;elongated\u0026nbsp;shoots were able to successively developed after\u0026nbsp;transferring to medium containing BA and GA\u003csub\u003e3\u0026nbsp;\u003c/sub\u003e(Fig. 7D),\u0026nbsp;which had complete vascular system and axillary bud primordia (Fig. 7A, B). At this stage, chloroplasts developed a well-organized internal membrane system (Fig. 6F). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThese regenerated shoots were developed into plantlets through rooting culture (Fig. 7E) and transplanted to\u0026nbsp;culture chamber\u0026nbsp;successfully (Fig. 7F), which supporting the usefulness of this \u003cem\u003ein vitro\u003c/em\u003e regeneration protocol in tree peony.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eThe extracellular matrix (ECM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSEM revealed the presence of a discontinuous amorphous secretions outside the callus surface，and varied in structure, which termed as ECM. Compared to ECs covered with numerous compact membranous layers, fibrillar structure and abundant granular mucilage-like secretions (Fig. 8A, B), cells of NECs provided a \u0026lsquo;\u0026lsquo;peeling\u0026rsquo;\u0026rsquo; appearance (Fig. 8C). Enlargement and multiplication of the surface cells of nodules resulted in the rupture at various sectors of membranous layer, consequently, transition from membranous layers to fibrillar structures were exhibited on the nodules surface (Fig. 8D, E). The structure of superficial cells become slightly elongated and regularly arranged leading to the formation of epidermis-like surface under SEM at stage of nodular clusters (Fig. 8F), and the ECM structures on the surface gradually decreased and presented fragments appearance (Fig. 8G). However, dense granular mucilage-like secretions were exposed on the top surface of the primordia regions exclusively (Fig. 8H). No ECM performed on the smooth surface of newly formed leaves (Fig. 8I).\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003e\u003cstrong\u003eHistological analysis\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo our knowledge,\u0026nbsp;this is the first report about histologic and ultrastructural analyses of \u003cem\u003ein vitro\u003c/em\u003e regeneration via MNs culture in tree peony.\u0026nbsp;The selection of ECs is prerequisite for\u0026nbsp;\u003cem\u003ein vitro\u003c/em\u003e regeneration.\u0026nbsp;In order to distinguish ECs and NECs, morpho-histological and ultrastructural studies were conducted, respectively. There were distinct differences between the two tissues and we\u0026nbsp;identified type I callus as\u0026nbsp;ECs, making it easier to select for subsequent experiment in view of its morphological characteristics, which\u0026nbsp;was basically consistent with previous observations (Moura et al. 2008; Shang et al. 2009;\u0026nbsp;Maadon et al. 2016).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eMNs could initiated directly from explants in \u003cem\u003eCichorium intybus\u0026nbsp;\u003c/em\u003e(Pi\u0026eacute;ron\u0026nbsp;et al. 1993,1998), \u003cem\u003eEucalyptus globulus\u0026nbsp;\u003c/em\u003e(Trindade and Pais 2003), \u003cem\u003ePopulus euphratica\u003c/em\u003e (Ferreira et al. 2009),\u0026nbsp;\u003cem\u003eSclerocarya birrea\u003c/em\u003e (Moyo et al. 2009) or indirectly from inducted calli, like \u003cem\u003ePinus radiata\u0026nbsp;\u003c/em\u003e(Aitken-Christie et al. 1988),\u003cem\u003e\u0026nbsp;Humulus lupulus\u0026nbsp;\u003c/em\u003e(Batista et al. 2000; Fortes and Pais 2000) and\u0026nbsp;\u003cem\u003eAcacia mangium\u0026nbsp;\u003c/em\u003e(Xie and Hong 2001).\u0026nbsp;Induction of MNs in tree peony belongs to latter. In addition,\u0026nbsp;callus originate from cell division in both cambial and cortical regions of explant, same as clarification in Qin et al (2012).\u003c/p\u003e\n\u003cp\u003eOur morpho-histological observation of MNs development in tree peony was similar to descriptions in \u003cem\u003eCichorium intybus\u0026nbsp;\u003c/em\u003e(Pi\u0026eacute;ron\u0026nbsp;et al. 1993,1998), \u003cem\u003eHumulus lupulus\u0026nbsp;\u003c/em\u003e(Batista et al. 2000; Fortes and Pais 2000),\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003cem\u003ePopulus euphratica\u0026nbsp;\u003c/em\u003e(Ferreira et al. 2009)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand\u0026nbsp;\u003cem\u003eVriesea reitzii\u0026nbsp;\u003c/em\u003e(Dal Vesco and Guerra 2010). There were\u0026nbsp;three developmental stages leading to the formation of nodules before organogenesis, including pre-nodular structures, MNs and \u0026lsquo;polycenter\u0026rsquo; nodules in sequence.\u0026nbsp;Concurrently,\u0026nbsp;MNs have been\u0026nbsp;characterized by distinct internal structures\u0026nbsp;involving\u0026nbsp;a central area of vascularization surrounded by actively dividing meristematic cells (OCs), a cortical-like area of parenchymatous cells and an epidermal-like area.\u0026nbsp;In opposition to descriptions in \u003cem\u003ePopulus euphratica\u003c/em\u003e (Ferreira et al. 2009),\u003cem\u003e\u0026nbsp;Cichorium intybus\u0026nbsp;\u003c/em\u003e(Pi\u0026eacute;ron\u0026nbsp;et al. 1993,1998) that\u0026nbsp;expansion of \u0026lsquo;polycenter\u0026rsquo; nodules in diameter was accompanied by the formation of cracks in the parenchymatous tissue\u0026nbsp;where\u0026nbsp;break up observed along the surfaces of voids, thus multiplying the number of independent nodules,\u0026nbsp;we\u0026nbsp;never observed\u0026nbsp;nodules\u0026nbsp;division. In this report, the nodules increased number with groove on the surface and developed into nodular cluster structure in appearance without nodules detachment.\u0026nbsp;This\u0026nbsp;was\u0026nbsp;consistent with\u0026nbsp;demonstration in\u0026nbsp;\u003cem\u003eHumulus lupulus\u0026nbsp;\u003c/em\u003e(Batista et al. 2000; Fortes and Pais 2000).\u003c/p\u003e\n\u003cp\u003eIt was reported that shoots originate from\u0026nbsp;epidermis or cortex\u0026nbsp;tissue of nodules\u0026nbsp;in\u003cem\u003e\u0026nbsp;Humulus lupulus\u0026nbsp;\u003c/em\u003e(Batista et al. 2000; Fortes and Pais 2000), while histological examination revealed that it developed from parenchymal cells around the vascular center in\u0026nbsp;\u003cem\u003eCichorium intybus\u0026nbsp;\u003c/em\u003e(Pi\u0026eacute;ron\u0026nbsp;et al. 1993,1998). In our research, shoots were proved to regenerate from endogenous parenchyma cells in the vicinity of nodule vascular centers. These\u0026nbsp;distinctions may be related to species differences.\u0026nbsp;Abundant vascular tissues, a predominance feature of nodules,\u0026nbsp;might be closely related to regeneration since the neovascularization following shoots origination was frequent in numerous cases of organogenesis (Pi\u0026eacute;ron\u0026nbsp;et al. 1998; Fortes and Pais 2000; Ferreira et al. 2009). Presence of vascular centers may improve individualization\u0026nbsp;of nodules and can be sign of regeneration.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe histological analysis provided detail evidence for hypothesis that the nodule developmental pathway was distinct to the embryogenic pathway, but highly parallel (McCown et al. 1988). The MNs morphogenetic resulted in the development of monopolar axes, in contrast to the bipolar pattern of somatic embryos differentiation in tree peony (Du et al. 2020).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eUltrastructural changes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe most striking ultrastructural feature was the changes of chloroplast morphology and starch content during development under TEM observation. Chloroplasts were degraded to plastids with large amount of starch grains accumulation in ECs cells. Plastids gradually developed into mature chloroplasts as well as starch grain decreased and disappeared step by step. Similar phenomenon had been\u0026nbsp;demonstrated previously\u0026nbsp;in\u0026nbsp;\u003cem\u003eBauhinia forficate\u003c/em\u003e (Appezzato-da-Gl\u0026oacute;ria and Machado 2004),\u0026nbsp;\u003cem\u003ePapaver somniferum\u0026nbsp;\u003c/em\u003e(Oveűka\u0026nbsp;et al. 2000) and \u003cem\u003eSolanum melongena\u003c/em\u003e (Fournier et al. 1995).\u0026nbsp;Starch, one kind of carbohydrate, considered to be the primary source of energy for cellular proliferation and growth, thus being consumed during \u003cem\u003ein vitro\u003c/em\u003e morphogenesis (Moura et al. 2008). In addition, the storage of starch, abundant in ECs and shortage in NECs,\u0026nbsp;probable\u0026nbsp;indicated the acquisition of embryogenic potential (Pinto et al. 2010;\u0026nbsp;Ribas et al. 2011), and they might function as signal molecules in signal transduction and gene regulation (Luis and Scherwinski-Pereira 2014).\u003c/p\u003e\n\u003cp\u003eThere was also a remarkable observation during differentiation that secretory dictyosome located around\u0026nbsp;abundant extensive RER, surrounded with plentiful complete or dividing mitochondria. This\u0026nbsp;phenomenon had been intensively reported (Aitken-Christie et al. 1988; Appezzato-da-Gl\u0026oacute;ria and Machado 2004; Diego et al. 2012).\u0026nbsp;The active operation of inner membrane systems\u0026nbsp;may reveal a high synthesis capacity associated with \u003cem\u003ein vitro\u003c/em\u003e morphogenesis.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSEM observations\u003c/strong\u003e\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eSEM observation in this report revealed the presence of ECM, which consist of membranous, fibrillar and granular structures. Similar structure had also been reported in \u003cem\u003eCentella asiatica\u003c/em\u003e (Lai et al. 2011), \u003cem\u003eActinidia deliciosa\u0026nbsp;\u003c/em\u003e(Popielarska-Konieczna\u0026nbsp;et al. 2006, 2010) and \u003cem\u003eTriticum aestivum\u003c/em\u003e (Konieczny et al. 2005). The chemical compositions and functions of ECM are still uncertain. It might be pectin polymers (Verdeil et al. 2001;\u0026nbsp;Konieczny et al. 2007), arabinogalactan proteins (Konieczny et al. 2007) and lipid (Popielarska-Konieczna\u0026nbsp;et al. 2008) that composing of ECM in previous reports. In addition, the formation of ECM might be a stress response to \u003cem\u003ein vitro\u0026nbsp;\u003c/em\u003econditions or resulted from unsuitable pre-treatment (Konieczny et al. 2005). On the other hand, ECs\u0026nbsp;could be identified with\u0026nbsp;the ECM since it was suggested to be an indicator of cells potential for regeneration (Namasivayam et al. 2006;\u0026nbsp;Popielarska-Konieczna\u0026nbsp;et al. 2006; Lai et al. 2011; Yusoff et al. 2012). However, ECM was observed both on the ECs and NECs in \u003cem\u003eOryza sativa\u0026nbsp;\u003c/em\u003e(Bevitori\u0026nbsp;et al. 2014) and \u003cem\u003eHelianthus tuberosus\u0026nbsp;\u003c/em\u003e(Pilarska et al. 2014) regardless of its morphogenetic competence. This discrepancy may result from genotype differences. In our study, three kinds of ECM structures were abundant on ECs, but absent from NECs. Furthermore, dense granular secretions were exposed on the surface of the primordia exclusively, which was aligned with result of some\u0026nbsp;thesis\u0026nbsp;that the appearance of ECM covering the surface was linked to the induction of morphogenesis (Konieczny et al. 2005)\u0026nbsp;and can served as a structural marker of somatic embryogenesis (Namasivayam 2007) or organogenesis (Popielarska-Konieczna et al. 2006). The ECM was also considered to play a vital role in regulation of signal, movement of nutrients, protection of surface structure during plant development and morphogenesis (Popielarska-Konieczna\u0026nbsp;et al. 2010).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe earliest symptom of differentiation was the distinctive presence of nodular clusters with elongated and regularly arranged superficial cells under SEM observation, leading to the formation of epidermis-like surface, where primordia initialed soon after.\u0026nbsp;This corresponded with findings in \u003cem\u003eOryza sativa\u003c/em\u003e (Bevitori\u0026nbsp;et al. 2014)\u003cem\u003e\u0026nbsp;\u003c/em\u003eand \u003cem\u003eActinidia deliciosa\u003c/em\u003e (Popielarska-Konieczna et al. 2011). The noticeable structure might be closely related to the origin of primordia and could be serviced as an indicator of differentiation (Brisibe et al. 1992).\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis is the first report covering morpho-histological and cytological analyses involved in MNs morphogenesis system in \u003cem\u003eP. ostii\u0026nbsp;\u003c/em\u003e\u0026lsquo;Feng Dan\u0026rsquo;.\u0026nbsp;The histological study revealed a developmental sequence leading to the formation of MNs and shoots regeneration, including callus induction,\u0026nbsp;pre-nodular structures, MNs, nodular clusters, shoots\u0026nbsp;differentiation. In addition,\u0026nbsp;the knowledge obtained by SEM and TEM about the developmental states of MNs\u0026nbsp;morphogenesis\u0026nbsp;can be useful for optimization of the \u003cem\u003ein vitro\u003c/em\u003e regeneration protocol and form the basis for further molecular analysis or genetic transformation.\u0026nbsp;\u003c/p\u003e"},{"header":"Abbreviations ","content":"\u003cp\u003eAC, activated carbon; BA, 6-benzyladenine; CIM, Callus induction medium; CPPU, N-(2-chloro-4-pyridyl)-N-phenylurea; DAA, Days after anthesis; DIM, Differentiation induction medium;\u0026nbsp;EC, Embryogenic callus;\u0026nbsp;GA\u003csub\u003e3\u003c/sub\u003e, Gibberellin; IBA, Indole-3-butyric acid; MN, Meristematic nodule; mMS, Modified Murashige and Skoog; mWPM, Modified woody plant medium; NAA, \u0026alpha;-naphthylacetic acid; OC, Organization center; Put, putrescine; RER, Rough endoplasmic reticulum; SEM, Scanning electron microscopy; TEM, Transmission electron microscopy\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompliance with ethical standards\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e The study was supported by National key R\u0026amp;D Program of China (2020YFD1000503).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e The authors declare that they have no conflict of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u003c/strong\u003e This article does not contain any studies with human participants or animals performed by any of the authors.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e LX conducted the experiments and written the manuscript. FYC and YZ revised the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n \u003cli\u003eAitken-Christie J, Singh AP, Davies H (1988) Multiplication of meristematic tissue: A new tissue culture system for \u003cem\u003eRadiata pine\u003c/em\u003e. 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Sci Rep 6:26944-26944.\u0026nbsp;\u003c/li\u003e\n \u003cli\u003e\u003ca href=\"https://doi.org/10.1038/srep26944%0d\"\u003ehttps://doi.org/10.1038/srep26944\u003c/a\u003e\u003c/li\u003e\n \u003cli\u003eYusoff NFM, Alwee SSRS, Abdullah MO, Chai-Ling H, Namasivayam P\u0026nbsp;(2012) A time course anatomical analysis of callogenesis from young leaf explants of oil palm (\u003cem\u003eElaeis guineensis\u003c/em\u003e Jacq.). J Oil Palm Res 24:1330-1341 \u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhong Y (2011) Induction and Culture of Meristematic Nodules in \u003cem\u003ePaeonia rockii\u003c/em\u003e. Beijing Forestry University, Beijing\u0026nbsp;\u003c/li\u003e\n \u003cli\u003eZhu X, Li XQ, Ding WJ, Jin SH, Wang Y (2018) Callus induction and plant regeneration from leaves of peony. Hortic Environ Biotechnol 59:575-582 \u003ca href=\"https://doi.org/10.1007/s13580-018-0065-4%0d\"\u003ehttps://doi.org/10.1007/s13580-018-0065-4\u003c/a\u003e\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Paeonia ostii ‘Feng Dan’, Meristematic nodule, Organogenesis, Histology, Ultrastructure","lastPublishedDoi":"10.21203/rs.3.rs-685307/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-685307/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis is the first report concerning the sequence of morpho-histological and cytological events occurring during organogenesis from cotyledon-derived meristematic nodules (MNs) in \u003cem\u003ePaeonia ostii\u003c/em\u003e\u003cstrong\u003e\u003cem\u003e \u003c/em\u003e\u003c/strong\u003e‘Feng Dan’. Sections were made and studies were carried out with dissecting microscope, light microscope, scanning electron microscopy (SEM) and transmission electron microscopy (TEM) observation. Histological studies revealed a complex developmental process of morphogenesis that including five stages: (1) callus originated from cell division in both cambial and cortical regions, and type I - yellow compact callus with densely arranged clumps were 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 complete vascular system and axillary bud primordia were developed. SEM observations showed three types of extracellular matrix (ECM), a smooth membranous layer, fibrillar structures and granular mucilage-like secretions on embryogenic callus, and recorded 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 \u003cem\u003ein vitro\u003c/em\u003e regeneration via MN culture and provides references for protocol optimization and genetic transformation.\u003c/p\u003e","manuscriptTitle":"Morpho-Histological and Cytological Study on Meristematic Nodule Induction and Shoot Organogenesis in Paeonia Ostii ‘Feng Dan’","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-07-09 16:25:15","doi":"10.21203/rs.3.rs-685307/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2021-07-12T09:04:32+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-07-07T00:00:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-07-05T00:00:00+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2021-07-04T00:22:42+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"plant-cell-tissue-and-organ-culture-pctoc","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pcto","sideBox":"Learn more about [Plant Cell, Tissue and Organ Culture (PCTOC)](https://www.springer.com/journal/11240)","snPcode":"11240","submissionUrl":"https://submission.nature.com/new-submission/11240/3","title":"Plant Cell, Tissue and Organ Culture (PCTOC)","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"c331dbf1-11b1-4b07-86ad-32914ac93b71","owner":[],"postedDate":"July 9th, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":5603072,"name":"Plant Molecular Biology and Genetics"}],"tags":[],"updatedAt":"2022-01-11T01:50:05+00:00","versionOfRecord":{"articleIdentity":"rs-685307","link":"https://doi.org/10.1007/s11240-021-02208-x","journal":{"identity":"plant-cell-tissue-and-organ-culture-pctoc","isVorOnly":false,"title":"Plant Cell, Tissue and Organ Culture (PCTOC)"},"publishedOn":"2022-01-11 01:50:05","publishedOnDateReadable":"January 11th, 2022"},"versionCreatedAt":"2021-07-09 16:25:15","video":"","vorDoi":"10.1007/s11240-021-02208-x","vorDoiUrl":"https://doi.org/10.1007/s11240-021-02208-x","workflowStages":[]},"version":"v1","identity":"rs-685307","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-685307","identity":"rs-685307","version":["v1"]},"buildId":"GqpaHPwrfC8PjnIFayRh5","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}