Paeonia ostii ‘Feng Dan’ plant regeneration through direct organogenesis and direct meristematic nodule culture

Paeonia ostii ‘Feng Dan’ plant regeneration through direct organogenesis and direct meristematic nodule culture | 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 Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Paeonia ostii ‘Feng Dan’ plant regeneration through direct organogenesis and direct meristematic nodule culture Chengcheng fan, kexin li, Li Xu, zhijun deng, shiming deng, jitao Li, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4062314/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract Tissue culture is preferred for solving the shortcoming of low efficiency in terms of conventional propagation ways in tree peony, an economically important woody plant in China with various purposes. However, callus differentiation is hard to obtain during in vitro regeneration. Meristematic nodule (MN) is a favorable way capable of overcoming this problem, but possesses a lengthy process. Direct organogenesis excluding the callus step is needed to simplify the procedure. This study firstly presented a protocol of direct organogenesis and direct MNs induction and differentiation using cotyledon explant for in vitro regeneration of P.ostii ‘Feng Dan’. The highest direct MNs induction rate (41.67%) and frequency of direct organogenesis (DO) (66.67%) was achieved under the following procedure. The explants were pretreated in dedifferentiation induction medium (DIM) [Murashige and Skoog (MS) medium with 2.27 µMthidiazuron (TDZ)+5.37 µM α-naphthylacetic acid (NAA)] for 10 days, and then the cotyledons without callus induced were transferred to differentiation medium (DM) [Woody plant medium (WPM) containing 2.02 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)+2.27 µM TDZ and 4.04 µM CPPU+4.54 µM TDZ] respectively, with 6 subcultures, 90 days in total. The regenerated shoots rooted and transplanted successfully. Histological study confirmed the process of DO and direct MNs induction, and revealed that shoots and MNs were originated from increased division of meristematic cell under cortical tissue, as well as from actively divided meristematic cells around vascular center. Moreover, shoots regenerated through MNs differentiation were originated from the epidermal and subepidermal cells. This study is an innovation and supplement in the field of in vitro regeneration in tree peony, and will be conductive to clonal micropropagation, fundamental studies of developmental biology and genetic transformation. Tree peony Cotyledon Direct organogenesis Meristematic nodule Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Tree peony ( Paeonia sect. Moutan ) is valued as an ornamental plant rich in colors and patterns, also used as medicinal and oil production plant (Yu et al. 2016 ). However, mass propagation of this species is limited by the characteristics of its conventional propagation methods, low efficiency and long period. Tissue culture is a useful way to solve this problem. The research of tree peony concerned with tissue culture date back to 1984 (Li et al. 1984 ), and over the past four decades, it has been widely recognized that difficulty in differentiation of callus is a key issue that hinders in vitro regeneration (Du et al. 2020b ; Wen et al. 2020 ). For example, several studies on somatic embryogenesis of tree peony have been reported, but no available regeneration system published because of rare differentiation from callus in indirect pathway (Zhu et al. 2018 ; Du et al. 2020b ), so did organogenesis from callus (Wen et al. 2020 ). Breakthrough, an available regeneration system of tree peony via meristematic nodules (MNs) culture, derived from callus, has been described previously (Xu et al. 2022a ). MN shown resemblance in appearance with somatic embryo, but could be distinguished from histological aspect (Xu et al. 2022b ; Haensch 2004 ). It possessed high potential in micropropagation, genetic transformation, and bioreactor for producing chemical components (McCown et al. 1988 ; Batista et al. 2008 ). Nevertheless, the system yielded the disadvantage of complex procedure and long period as a result from the cumbersome process of callus and MNs induction. Hence, the regeneration system via MNs culture not involving a strict callus phase is needed to streamline the lengthy process. Similar protocols have already been announced in some literature (Piéron et al. 1993 , 1998 ; Trindade and Pais 2003 ; Moyo et al. 2009 ). Direct organogenesis (DO) originated from explant without callus period have been declared in some plants, such as Anaphalis hancockii (Geng et al. 2023 ), Solanum melongena (García-Fortea et al. 2020 ), Hypericum perforatum (Ravindran et al. 2023 ). This system is relatively plainer and possesses a shorter cycle. Whereas there were scarce reports carried out about this protocol in tree peony. Overall, innovative breakthroughs concerning in vitro shoot organogenesis excluding the callus step are needed to simplify the procedure. The present work aimed to achieve shoot organogenesis with two novel pathways avoiding callus step, shoots emerged directly from explants or explants-derived MNs. Histological analyses were undertaken to reveal the characteristics of develop stages in morphogenetic process. This study could be a significant innovation and supplement in the field of in vitro regeneration in tree peony. Materials and methods Plant material In August 2022, seeds at 90 days after anthesis from five-year-old P. ostii ‘Feng Dan’ plants were obtained from Beijing Guose Peony Garden in Beijing, China (40°45′N, 115°97′E). The collected seeds were placed in the refrigerator (-4℃) for at least 2 months. Explant sources After washing under running tap water for 10 mins, the seeds were soaked in commercial liquid detergents (1% v/v; 5 min). Afterwards, seeds with clean surface were sterilized in clean bench by dipping in ethanol (70% v/v; 1 min) first, and then NaOCl (2% v/v; 15 min). Finally, the seeds were rinsed with sterile distilled water for at least three times before inoculation. According to the method adapted from Xu et al. ( 2022a ), zygotic embryos were inoculated on MS medium containing 2.57 µM 6-benzyladenine (BA) and 2.89 µM gibberellin (GA 3 ) for germination, the cotyledons after 15 days of culture were used as explants. Medium and culture conditions All the cultures were grown on Woody plant medium (WPM), Murashige and Skoog medium (MS) and 1/2 MS (half-strength macroelements). The basal media containing 3% sucrose and 0.7% agar (Biosharp, Beijing, China). The pH of the media was pre-adjusted to 5.8-6.0 prior to sterilisation (at 120°C and 115 kPa for 20 min). All cultures were kept in a growth room at temperature of 23 ± 2°C under a 16/8 h photoperiod using cool white light (25 µmol·m − 2 ·s − 1 ). Direct organogenesis (DO) and direct meristematic nodule (MN) induction To study the effects of culture time and plant growth regulators (PGRs) in dedifferentiation induction medium (DIM) on the DO and direct MN induction, the explants were cut into pieces (1×1 cm) and inoculated on DIM [MS + 2.57 µM BA + 5.37 µM α-naphthylacetic acid (NAA)] for different days (0, 5, 10 and 15 days) firstly. The excised cotyledon explants were placed with their abaxial face down in contact with the medium, and kept in dark conditions. Secondly, DIM containing different PGR combinations [2.57 µM BA + 5.37 µM NAA, 2.02 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU) + 5.37 µM NAA, 2.27 µM thidiazuron (TDZ) + 5.37 µM NAA] was used. The excised cotyledon explants were cultured for 10 days respectively. Afterwards, the cotyledons were transferred from DIM to differentiation medium (DM) [WPM + 2.27 µM TDZ], with subculture times of 15 days. The frequency of DO (%) and direct MN induction rate (%) were evaluated 6 subcultures after transfer. Each treatment consisted of 16 explants, and the experiment was repeated three times. The frequency of DO is expressed as the average percentage of explants/cotyledons that differentiated shoots directly over total number of explants/cotyledons. The direct MN induction rate is presented as the mean number of explants/cotyledons induced MNs directly over total number of explants/cotyledons. The cotyledons from best DIM after cultured for optimal culture time obtained above were subculture in DM. To screen optimal PGRs in DM for DO and direct MN induction, three cytokinins [2.57 µM BA, 2.02 µM CPPU and 2.27 µM TDZ] were employed respectively. After preliminary screening of suitable hormones, an orthogonal test involving two factors (CPPU and TDZ) and three levels of concentration (1.00, 2.02, 4.04 µM; 1.14, 2.27, 4.54 µM) was undertaken. Shoot elongation, rooting and acclimatization The nodules induced directly from explants were transferred to medium [WPM + 2.02 µM CPPU + 2.27 µM TDZ] (Xu et al. 2022a ) for leaf clusters differentiation with 3 subcultures, 30 days in total. The cotyledons with leaf clusters (DO), as well as nodules with leaf clusters (direct MN induction and differentiation) were placed in shoot elongation medium [WPM + 1.29 µM BA + 0.58 µM GA 3 ] respectively with 2 subcultures, 60 days in total. For in vitro root formation, micro-shoots about 1–3 cm in length were transferred to 1/2 MS basal medium supplemented with 4.92 µM indole-3-butyric acid (IBA) and 11.34 µM putrescine (Wang et al. 2016 ). Micro-shoots on root induction medium were cultured in the dark for the first 8 days at 4°C and then 30 days at 24 ± 1°C, prior to being cultured in 1/2 MS basal medium supplemented with 0.4% activated carbon for 20 days under a 16/8 h photoperiod using cool white light (25 µmol·m − 2 ·s − 1 ). Plantlets with well-developed shoots and roots were then removed from the agar medium and potted in plastic pots containing autoclaved substrate (vermiculite, peat, and perlite in a 1:1:1 volumetric ratio). Agar was removed from the roots thorough carefully washed with running water prior to transplanting. The pots were placed in a culture chamber at 20 ± 1°C under a 16/8 h photoperiod using cool white light (25 µmol·m − 2 ·s − 1 ). Histological analysis To study the development stage of direct organogenesis and direct MN induction and differentiation, fresh samples [cotyledons cultured in DIM for 15 days, 1×1 cm; cotyledons cultured in DM for different times of subcultures (1, 2, 3, 4, 5 and 6 times), cotyledons with nodules, cotyledons with leaf clusters, nodules with leaf clusters, 1×1×1 cm] were fixed for 48 h in the FAA solution (50% alcohol, glacial acetic acid, and formaldehyde at a ratio of 18:1:1). The permanent preparation was made based on the method adapted from Xu et al. ( 2022b ). Sections 8–10 µm in thickness were obtained using a rotary microtome, and stained with fast green (0.1%) and safranin (0.1%). The prepared slides were studied with Leica model DM500 microscope. Statistical analysis The data were subjected to analysis of variance (ANOVA) following Duncan’s multiple range test to detect significant differences (p ≤ 0.05) in the mean using SPSS 23.0 (SPSS Inc., Chicago, USA), after transforming the percentage values using arcsine transformation. Variability in the data was expressed as the mean ± standard deviation. Results M orphological and histological study on DO from cotyledons The detailed morpho-histological characterization of explants during in vitro direct organogenesis was conducted for the first time in tree peony to determine the timing and tissue origin of the regenerants. Compared to the original state, active-divided subepidermal cells and meristematic cells around vascular centers were observed in cotyledon after pretreated in DIM (Fig. 1A). Incubation in DM for 1-2 times resulted in obviously swelled and elongated of cotyledon (Fig. 1B). In particular, the occurrence of DO was observed at two positions. Strip protuberances gradually apparent on the surface of cotyledons unevenly (Fig. 1C), and visible swell formed at the cotyledon petiolar cut edge (Fig. 1D). Sections revealed subepidermal cells vigorously proliferated with large and clear nuclei leading to the formation of strip protuberances (Fig. 1E). Meanwhile, transverse sections of swell showed rapid division of meristematic cell under cortical tissue and around vascular bundles filling up the expansive intercellular spaces (Fig. 1F). The volume of protuberances in both positions was increased irregularly after 3-4 times of incubation, and primordia were shown up. Direct connections between vascular tissue inside primordia and explants were detected in strip protuberances (Fig. 2A, B, C). However, histological observation of swell demonstrated that primordia was initiated inside and developed towards the surface of cotyledons (Fig. 2D, E). Leaf clusters occurred at two positions after 5-6 subcultures, protuberances on the surface of cotyledon (Fig. 3A, B) and swell on the edge (Fig. 3C). Subsequently, apical meristems could be found out (Fig. 3D). Shoots developed from leaf clusters successively after transferring to shoot elongation medium (Fig. 3E). M orphological and histological study on direct MNs induction and differentiation Histological examination confirmed direct MNs induction. The process of direct MNs induction and DO was occurred simultaneously. Rapid cell division were initiated in the adaxial portion of subepidermal cells, leading to small globular protuberances arose from the surface of cotyledons within 1-2 times of incubation in DM (Fig. 4A, B). The protuberances significantly expanded during 3-4 subculture (Fig. 4C, D), accompanied with massively formation of vascular tissue (Fig. 4E). Under histological observation, the large protuberances gradually developed into MNs, composed of cortical, epidermal layer cells, and various organization centers, like nested and linear (Fig. 4F, G). During 5-6 times of incubation, nodules proliferation occurred with a special way like budding. Therefore, nodular clusters were appeared since different sizes of nodules were gathered (Fig. 5A). Owing to vigorous division of the epidermal and subepidermal cells, primordia emerged from nodules (Fig. 5B, C, D). When nodular clusters were transferred to leaf cluster differentiation medium, leaf clusters gradually observed on the surface (Fig. 5E). Similarly, shoots arose from leaf clusters in succession after incubated in shoot elongation medium (Fig. 5F). The eligible shoots induced both from DO pathway and direct MN culture developed roots after placement on the rooting medium (Fig. 5G), and the rooted plantlets were acclimatized successfully (Fig. 5H). Effect of culture time in DIM on DO and direct MNs induction DO or direct MN induction were failed to be observed from the cotyledons not cultured in DIM for dedifferentiation (Fig. 6). Pre-culture in DIM positively affected the formation of DO and direct MNs induction. the frequency of DO and the direct MN induction rate were gradually increased with dedifferentiation time, and the highest frequence of DO (43.75%) was achieved when cotyledons explants were pretreated in DIM for 10 days, but with no significant difference between 10-15 days of treatment. The direct MN induction rate of all treatments was generally low, but treatments of 10-15 days were higher than that of 0-5 days. Effect of PGRs in DIM on DO and direct MNs induction As shown in figure 7, the frequency of DO from cotyledons pre-cultured in DIM supplemented with 2.02 µM CPPU+5.37 µM NAA (50.00%) and 2.27 µM TDZ+5.37 µM NAA (52.08%) was significantly higher that of 2.57 µM BA+5.37 µM NAA (39.58%). Combination of TDZ and NAA was the treatment that showed the highest frequence of DO. However, there was no significant difference on the direct MNs induction rate among treatments (＜10%). Effect of PGRs in DM on DO and direct MNs induction In the preliminary experiment, DO and direct MNs induction only occurred in DM containing TDZ or CPPU, but not in presence of BA (Fig. 8). The results indicated that the frequency of DO in treatment with CPPU (41.67%) and TDZ (45.83%) was conspicuously higher than BA (0%), but present no significant difference between two of them. Meanwhile, the direct MN induction rate of treatment with CPPU (10.42%) superior to TDZ (4.17%). In subsequent steps, the best performance of direct MN induction rate (41.67%) was obtained at 2.02 µM CPPU and 2.27 µM TDZ, while the optimal concentration of CPPU and TDZ was 4.04 µM and 4.54 µM respectively in terms of the frequency of DO (66.67%) (Table 1). Variance analysis demonstrated that there was a significant response for CPPU and TDZ concentration on the frequency of DO ( p ＜0.01), as well as their interaction (Table 2). In terms of the direct MN induction rate, CPPU and its combination with TDZ remained significant effect, but TDZ alone not influenced ( p ＞0.05). The number of regenerated shoots originated from both DO and direct MNs culture pathway varying from three to seven per explant. The rooting rate remains around 50%, and the survival rate was kept between 30-40%. Discussion This is the first report of DO from cotyledon explants without callus period in tree peony. In this experiment, leaf cluster initiation was observed at cut site of the proximal end of cotyledon, as well as the paraxial surface, which in agreement with description in some literature (Huang et al. 2014 ; Debnath et al. 2018 ). Simultaneously, the occurrence of direct organogenesis was confirmed by the histologic analysis that shoots originated from increased division of meristematic cell under cortical tissue, as well as meristematic cells around vascular centers inside of swell on the edge of the cotyledon. Similar phenomenon was revealed previously, but shoot initiation was varied. For instance, from the upper epidermal cells and their inside parenchyma cells in Capsicum annuum (Gao et al. 2021 ), from the epidermal tissue in Neolamarckia cadamba (Huang et al. 2014 ), from the epidermal and subepidermal cells in Cucumis melo (Cai et al. 2002 ), which was called for exogenous initiation. Besides, buds could regenerate from endogenous meristematic cells around vascular centers (Sarkar and Jha 2017 ). Thus, the origin of shoots through the DO pathway is endogenous and exogenous coexist in tree peony. On the other hand, this is also the first report of direct MNs induction and differentiation without callus phase in tree peony. Histological examination confirmed this morphogenic response. This is contrary to previous conclusion that callus formation was necessary for MNs induction in tree peony (Xu et al. 2022a ). In fact, there were two morphogenesis pathways has been reported on MNs induction, direct (Moyo et al. 2009 ; Ferreira et al. 2009 ; Piéron et al. 1998 ) and indirect with callus formation (Fortes and Pais 2000 ; Batista et al. 2000 ). Therefore, two pathways were existed side by side in tree peony. In this model of direct MNs culture on tree peony, cells competent for nodule induction was located mainly in meristematic cell under cortical tissue with histological observation, and new formed vascular system developed inside nodules subsequently, which was basically consistent with results in some reports (Moyo et al. 2009 ; Ferreira et al. 2009 ). However, there were different conclusion in Cichorium intybus that the cambium of nodule was originated from the procambium of leaf, and the parenchyma and periderm cork cell layers were originated from the fascicular parenchyma and bundle sheath tissues of leaf in direct (Piéron et al. 1998 ). These distinctions might be related to species differences. Additionally, it was documented that shoots originated from epidermis or cortex tissue of nodules in Humulus lupulus (Batista et al. 2000 ; Fortes and Pais 2000 ), or from parenchymal cells around the vascular center in Cichorium intybus (Piéron et al. 1993 , 1998 ). In contrast to previous text that shoots were regenerated from endogenous parenchyma cells around nodule vascular (Xu et al. 2022b ), our histological examination revealed that increased division of the epidermal and subepidermal cells of nodules led to shoot regeneration. Similar observation has been recognized by Qin et al. ( 2012 ) in P. lemoinei ‘Golden Era’. This difference might attribute to insufficient number of sections. In our studies, DO and direct MNs induction took place synchronously, and highly parallel at the early stage. Both induced protuberances initiated from subepidermal cells, but shared different shapes. When it comes to DO pathway, strip protuberances became apparent on the upper part surface of cotyledons, and the appearance of swell growth with irregular shape was observed on the edge. On the contrary, the nodules exhibited a global-shaped structure at the beginning of their development, similar to globular somatic embryos. The histological analysis could provide detail evidence for distinguishing (Xu et al. 2022b ; Haensch 2004 ). Subsequently, the volume of swell at the cut site of cotyledon gradually increased, so did the nodules. Nevertheless, they were remarkable distinct in terms of appearance (Fig. 2D; Fig. 4D) and location. The swell located just at the cut end of cotyledon explants, same as Garcinia mangostana (Qosim et al. 2015 ), but nodules were not restricted. In addition, there were more plenty of autogenetic vascular tissues inside the nodules compared to swell (Fig. 2E; Fig. 4E). Pretreatment in DIM (dedifferentiation) before callus formation played a dominant role in this protocol. With the enhancement of dedifferentiation time (0–15 days), the differentiation rate in DM improved. It was speculated that cells with high activity after losing their original characteristic structure and function within dedifferentiation time, could trigger a particular developmental fate when recognized a single inductive signal. Specifically, dedifferentiated subepidermal cells and meristematic cells around vascular centers could developed into primordia or nodules after transferring cotyledons from DIM to DM supplement with highly active cytokinin. The results emphasized the importance of dedifferentiation step of explants in DIM enriched with auxin. It is well known that auxin is essential for apical meristem formation. In detail, auxins and downstream transcriptional regulation interfere with the structural elements of the cell wall to induce specific morphogenetic events, the absence of auxins in the pretreatment inhibited the morphogenic process (Traas 2019 ). Furthermore, cross-talk with other signaling pathways, cytokinin in particular, is crucial in organ regeneration (Vernoux et al. 2011 ; Huang et al. 2014 ). Similarity, the procedure was developed in Eucalyptus nitens (Ayala et al. 2019 ) and in Prunus cerasifera (Carmona-Martin and Petri 2020 ), that pre-treatment with auxins under dark condition and subsequent transfer to medium rich in cytokinin in light conditions, and considered to be appropriate in recalcitrant specie for direct regeneration. Effect of PGRs in DM on direct regeneration was dependent on PGRs type and concentration (Bao et al. 2017 ; Huang et al. 2014 ), genotype (García-Forte et al. 2020) and explant type (Geng et al. 2023 ). According to Xu et al ( 2022a ), CPPU and TDZ, two kinds of cytokinin with high activity, played remarkably positive roles in MNs induction and shoots regeneration from callus. Meanwhile, DO and direct MNs induction only occurred in DM containing TDZ or CPPU in this research, but not in presence of BA, which further supported previous speculation that high level of cytokinin was indispensable for differentiation (Xu et al. 2022a ; Meng et al. 2017 ). The result that TDZ + NAA, CPPU + NAA treatment in DIM had a better effect on the induction of MNs and DO than BA + NAA treatment also consistent with this conclusion. In contrast, the cytokinin suitable for direct regeneration was varied among other plant species, like BA for Garcinia mangostana (Qosim et al. 2015 ), ZR for Solanum melongena (García-Fortea et al. 2020 ), TDZ for Rhododendron (Hebert et al. 2010 ), and combination of KT and BA for Anaphalis hancockii (Geng et al. 2023 ). Besides, mixed use of CPPU and TDZ was more effective than single one in direct regeneration, which was in coincidence with other result (Gao et al. 2021 ). The developmental direction of differentiation was varied with cytokinin type and concentration. For example, the epidermal cells of leaves developed into nodular callus if stimulated by combination TDZ and BA, while shoot originated with BA stimulation alone. What’s more, the optimum treatment for direct shoots induction was achieved at a concentration of 22.2 µM BA, but the use of BA (> 44.4 µM) inhibited the formation and elongation of shoots (Qosim et al. 2013 , 2015 ). In the present study, both of CPPU and TDZ concentration had significant effect on the frequency of DO ( p 0.05). Similar result has been concluded in tree peony that CPPU was considerably effective in promoting MNs induction from callus (Xu et al. 2022a ), and in herbaceous peony that TDZ was appropriate for direct organogenesis from cotyledon explants (Zhao et al. 2017 ). Concurrently, the best performance of DO and direct MNs induction was presented on CPPU and TDZ combination at different level of concentration in our study, and higher level suitable for DO. Thus, researchers can make different choices in concentration formulas based on varied purposes. The protocol achieved in this essay can greatly shorten the period needed for regeneration by skipping the callus stage. More specifically, it takes approximately 6 months to obtain differentiated shoots in DO pathway, and this regeneration system through direct MNs induction from explants and differentiation can save approximately 2 months compared to previous cycle (Xu et al. 2022a ). Moreover, MNs of tree peony in vitro work as storage organs like corms or tubers, and scale-up multiplication of MNs can be reached in liquid medium (Zhong et al. 2011). Therefore, optimization of in vitro regeneration can be achieved with procedure that MNs induced from explant directly multiplicated in liquid environment, and then differentiated in solid medium. This hypothesis has been realized in Ananas comosus var. comosus (Scherer et al. 2013 ), Vriesea reitzii (Dal Vesco and Guerra 2010), and Charybdis numidica (Kongbangkerd and Wawrosch 2003 ). We have obtained preliminary evidence that nodules proliferated in liquid medium could finish differentiate in solid medium, but further optimization of culture conditions still underway. Conclusion This study firstly presented a protocol of in vitro regeneration through DO and direct MNs culture in P. ostii ‘Feng Dan’. This protocol includes two pathways at the same time. The explants need pretreated in DIM in both ways, and then occurred simultaneously in DM. Shoots regenerated directly from explants (DO pathway) and from explants-derived MNs could rooted, and the rooted plantlets were acclimatized successfully. In addition, histological study revealed the developmental sequence and tissue origin of the regenerants. This protocol simplified the differentiation process and will be beneficial to the clonal micropropagation, fundamental studies of developmental biology and genetic improvement of tree peony. Abbreviations BA, 6-benzyladenine; CPPU, N-(2-chloro-4-pyridyl)-N-phenylurea; DM, Differentiation medium; DIM, dedifferentiation induction medium; DO, Direct organogenesis; GA 3 , Gibberellin; IBA, Indole-3-butyric acid; MN, Meristematic nodule; MS, Murashige and Skoog; WPM, Woody plant medium; NAA, α-naphthylacetic acid; PGR, Plant growth regulator; TDZ, Thidiazuron; Declarations Compliance with ethical standards Funding The study was supported by the National Natural Science Foundation of China (32302598), and Natural Science Foundation of Hubei Province of China (2023AFB509). Conflict of interest The authors declare that they have no conflict of interest. Ethical approval This article does not contain any studies with human participants or animals performed by any of the authors. Data availability The data supporting the findings of this study are available with Li Xu and can be made available upon request. 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J Horticult Sci Biotechnol 78(5):650-655. http://dx.doi.org/10.1080/14620316.2003.11511679 Li YL, Wu DY, Pan SL, Xu SL, Wei ZM (1984) Chinese Science Bulletin (8):500-502. (In Chinese) McCown BH, Zeldin EL, Pinkalla HA, Dedolph R (1988) Nodule culture: a developmental pathway with high potential for regeneration, automated micropropagation, and plant metabolite production from woody plants. In: Hanover JW, Keathley DE (Editors) Genetic manipulation of woody plants. Plenum, New York, pp.149-166. Meng WJ, Cheng ZJ, Sang YL, Zhang MM, Rong XF, Wang ZW, Tang YY, Zhang XS (2017) Type-B Arabidopsis response regulators specify the shoot stem cell niche by dual regulation of WUSCHEL . Plant Cell 29:1357-1372. https://doi.org/10.1105/tpc.16.00640 Moyo M, Finnie J F, Van Staden J (2009) In vitro morphogenesis of organogenic nodules derived from Sclerocarya birrea subsp. caffra leaf explants. 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Moutan ): Influence of genotype, explant developmental stage and position, and plant growth regulators. Propag Ornam Plants 12(2):117-126． Ravindran BM, Rizzo P, Franke K, Fuchs J, D'Auria J (2023) Simple and robust multiple shoot regeneration and root induction cycle from different explants of Hypericum perforatum L. genotypes Plant Cell Tissue Organ Cult 152(1):1-15. https://doi.org/10.1007/s11240-022-02370-w Sarkar S, Jha S (2017) Morpho-histological characterization and direct shoot organogenesis in two types of explants from Bacopa monnieri on unsupplemented basal medium. Plant Cell Tissue Organ Cult 130(2):435-441. https://doi.org/10.1007/s11240-017-1231-6 Scherer RF, Garcia, Garcia AC, Fraga HP, Vesco LLD, Steinmacher DA, Guerra MP (2013) Nodule cluster cultures and temporary immersion bioreactors as a high performance micropropagation strategy in pineapple ( Ananas comosus var. comosus ). Sci Hortic 151:38-45. http://dx.doi.org/ 10.1016/j.scienta.2012.11.027 Traas J (2019) Organogenesis at the shoot apical meristem. Plants 8(1):6. https://doi.org/10.3390/plants8010006 Trindade H, Pais MS (2003) Meristematic nodule culture: A new pathway for in vitro propagation of Eucalyptus globulus . Trees 17(4):308-315. http://dx.doi.org/10.1007/s00468-002-0240-0 Vernoux T, Brunoud G, Farcot E, Morin V, Van den Daele H, Legrand J, Oliva M, Das P, Larrieu A, Wells D, Guéon Y, Armitage L, Picard F, Guyomarc’h S, Cellier C, Parry G, Koumproglou R, Doonan JH, Estelle M, Godin C, Kepinski S, Bennett M, De Veylder L, Traas J (2011) The auxin signalling network translates dynamic input into robust patterning at the shoot apex. Mol Syst Biol 7(1):508.https://doi.org/10.1038/msb.2011.39 Huang X, Chen J, Bao Y, Liu L, Jiang H, An X, Dai L, Wang B, Peng D (2014) Transcript profiling reveals auxin and cytokinin signaling pathways and transcription regulation during in vitro organogenesis of ramie ( Boehmeria nivea L. Gaud). Plos One 9(11):e113768https://doi.org/10.1371/journal.pone.0113768 Wang X, Cheng FY, Zhong Y, Wen SS, Li LZM, Huang LZ (2016) Establishment of in vitro rapid propagation system for tree peony ( Paeonia ostii ). Scientia Silvae Sinicae 52:102-110.https://doi.org/10. 11707/j.1001-7488.20160512 Wen SS, Chen L, Tian RN (2020). Micropropagation of tree peony ( Paeonia sect. Moutan ): A review. Plant Cell Tissue Organ Cult 141(1):15. https://doi.org/10.1007/s11240-019-01747-8 Xu L, Cheng FY, Zhong Y (2022a) Efficient plant regeneration via meristematic nodule culture in Paeonia ostii ‘Feng Dan’. Plant Cell Tissue Organ Cult.149(3):599-608. https://doi.org/10.1007/s11240-021-02216-x Xu L, Cheng FY, Zhong Y (2022b) Histological and cytological study on meristematic nodule induction and shoot organogenesis in Paeonia ostii ‘Feng Dan’. Plant Cell Tissue Organ Cult. 149(3):1-12. https://doi.org/10.1007/s11240-021-02208-x 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 Zhao D, Xue Y, Shi M, Tao J (2017) Rescue and in vitro culture of herbaceous peony immature embryos by organogenesis. Sci Hortic 217:123-129. http://dx.doi.org/10.1016/j.scienta.2017.01.040 Zhong Y. (2011) Induction and Culture of Meristematic Nodules in Paeonia rockii . Beijing Forestry University, Beijing. (In Chinese) 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 Tables Table 1 Screening of CPPU and TDZ concentration for the frequency of DO and the direct MN induction rate CPPU (µM) TDZ (µM) The frequency of DO (%) The direct MN induction rate (%) 1.00 1.14 8.33±3.61 e 4.17±3.61 g 1.00 2.27 12.50±6.25 de 12.50±0.00 f 1.00 4.54 16.67±6.25 cd 27.08±3.61 bcd 2.02 2.27 20.83±3.61 c 41.67±3.61 a 2.02 4.54 22.92±3.61 c 20.83±3.61 de 2.02 1.14 22.92±3.61 c 22.92±3.61 cde 4.04 4.54 66.67±3.61 a 29.17±3.61 bc 4.04 1.14 20.83±3.61 c 31.25±6.25 b 4.04 2.27 43.75±6.25 b 16.67±3.61 ef Different letters within a column show significant differences by Duncan’s multiple range tests (p ≤ 0.05). Each data represent mean ± standard error. Table 2 Variance analysis of CPPU and TDZ concentration on the frequency of DO and the direct MN induction rate Source of variance The frequency of DO (%) The direct MN induction rate (%) df F test p value df F test p value CPPU 2 122.385 0.00 2 33.60 0.00 TDZ 2 21.77 0.00 2 2.10 0.15 CPPU*TDZ 4 32.50 0.00 4 33.00 0.00 Error 18 18 Cite Share Download PDF Status: Under Review Version 1 posted Reviewers agreed at journal 08 Apr, 2024 Reviewers invited by journal 07 Apr, 2024 Editor assigned by journal 02 Apr, 2024 First submitted to journal 01 Apr, 2024 Editorial decision: Major revisions 21 Mar, 2024 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. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board 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-4062314","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":288425986,"identity":"97914d28-7091-4c3a-a164-e83393e29c12","order_by":0,"name":"Chengcheng fan","email":"","orcid":"","institution":"Hubei minzu university","correspondingAuthor":false,"prefix":"","firstName":"Chengcheng","middleName":"","lastName":"fan","suffix":""},{"id":288425987,"identity":"e2412968-3b6f-4144-a3d7-cbb55aac5aa9","order_by":1,"name":"kexin li","email":"","orcid":"","institution":"[email protected]","correspondingAuthor":false,"prefix":"","firstName":"kexin","middleName":"","lastName":"li","suffix":""},{"id":288425988,"identity":"f65353bb-064c-4d66-a72c-6f0d9ed176fa","order_by":2,"name":"Li Xu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA7klEQVRIie3QsWoCQRDG8TkW1mbAdkVMXmFkwTSSvMoeC1pdb2kQziIktYIPscFC0q1skebshVhYXWVxYKGVUbET2TNdiv2XH/NrBiAU+odVGZuvVe/wQOIyRP0yUhummorMyvsJZVmrNk5tbO4msFRUR77qzupveovQbhjL8rVPRCOlJGKefE0WRiJ0pLH8iXyECWU1CpaYZWI0gouNRS58hIu475BYl07EIfyWE0QXvY6UUyfyOUCw5URUUgaF7TTNajGNJqTl2PGWl7y46m4fH9qP9PM+LTa958bH9yD3kqvOr2J/uA+FQqHQ7Y6SYU/5KqIRNAAAAABJRU5ErkJggg==","orcid":"https://orcid.org/0000-0003-4587-4011","institution":"Hubei minzu university","correspondingAuthor":true,"prefix":"","firstName":"Li","middleName":"","lastName":"Xu","suffix":""},{"id":288425989,"identity":"947741b9-d609-4bd3-ab15-70f972b1a0cd","order_by":3,"name":"zhijun deng","email":"","orcid":"","institution":"Hubei minzu university","correspondingAuthor":false,"prefix":"","firstName":"zhijun","middleName":"","lastName":"deng","suffix":""},{"id":288425990,"identity":"59e70d9c-5582-4d8a-8e01-53fe41687787","order_by":4,"name":"shiming deng","email":"","orcid":"","institution":"Hubei minzu university","correspondingAuthor":false,"prefix":"","firstName":"shiming","middleName":"","lastName":"deng","suffix":""},{"id":288425991,"identity":"b8456931-238b-486c-9d24-89dfb432785d","order_by":5,"name":"jitao Li","email":"","orcid":"","institution":"Hubei minzu university","correspondingAuthor":false,"prefix":"","firstName":"jitao","middleName":"","lastName":"Li","suffix":""},{"id":288425992,"identity":"4c71a7d7-b431-498f-82c7-ce7a14e385ae","order_by":6,"name":"jiaolin mou","email":"","orcid":"","institution":"Hubei minzu university","correspondingAuthor":false,"prefix":"","firstName":"jiaolin","middleName":"","lastName":"mou","suffix":""}],"badges":[],"createdAt":"2024-03-10 06:28:52","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4062314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4062314/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":54411970,"identity":"e9aa3ba8-3d5f-4099-badb-000aa2769ed4","added_by":"auto","created_at":"2024-04-10 05:48:49","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49808344,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and histological observation of direct organogenesis (Early stage)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Active-divided subepidermal cells (black arrow) and meristematic cells around vascular centers (hollow black arrows) were observed in cotyledon after pretreated in DIM; B. Cotyledons obviously swelled and elongated after cultured in DM for 1-2 times; C. Strip protuberances were observed on the surface of cotyledons unevenly (black arrow); D. Visible swell formed at the cotyledon petiolar cut edge (black arrow); E. Subepidermal cells proliferated extensively to form protuberances (black arrow); F. Rapid division of meristematic cell under cortical tissue (black arrow) and around vascular bundles (hollow black arrows) filling up the intercellular spaces of swell. Abbreviations: V. Vascular bundle; EP. Epidermis.\u003c/p\u003e","description":"","filename":"Fig1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/195b95e32d9312941e829c94.jpg"},{"id":54412528,"identity":"3c2b87ec-d0c1-4c7d-9785-ea5f0160047b","added_by":"auto","created_at":"2024-04-10 05:56:49","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":45506355,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and histological observation of direct organogenesis (Middle stage)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. After 3-4 times of culture in DM, the volume of protuberances was increased, and leaf primordia were appeared on the surface of cotyledon (black arrow), and direct connections between vascular tissue inside primordia and explants were detected (hollow black arrows); C. Enlarged details of vascular tissue in Figure A (hollow black arrows); D-E. After 3-4 times of culture in DM, the volume of swell was increased irregularly, and bud primordia were observed inside, and developed towards the surface of cotyledon. Abbreviations: V. Vascular bundle; LP. leaf primordia; P. bud primordia.\u003c/p\u003e","description":"","filename":"Fig2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/e0d0f8a68ca506182875784e.jpg"},{"id":54411973,"identity":"fc8ad1ab-9e0b-4a90-930e-0218dfebde19","added_by":"auto","created_at":"2024-04-10 05:48:50","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":32946107,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and histological observation of direct organogenesis (Later stage)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA-B. Early stage of leaf clusters observed from the surface of cotyledon after 5-6 subcultures in DM; C. Leaf clusters occurred from the swell at the edge of cotyledon; D. Apical meristems (black arrow) can be detected; E. Shoots developed from leaf clusters successively after transferring to shoot elongation medium.\u003c/p\u003e","description":"","filename":"Fig3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/f2827040a6a47398b1449fc1.jpg"},{"id":54411976,"identity":"fdcc019d-b7c2-4632-a216-074a58efa5d7","added_by":"auto","created_at":"2024-04-10 05:48:50","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":60607005,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and histological observation of direct MNs induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Small globular protuberance (black arrow) arose from the surface of cotyledons within 1-2 times of incubation in DM; B. Rapid cell division were initiated in the abaxial portion of subepidermal cells leading to globular protuberance (black arrow); C-D. The protuberances (black arrow) significantly expanded after 3-4 subculture; E. MN composing of cortical, epidermal layer cells, and various organization centers; F. Nested types of organization center; G. Linear types of organization center. Abbreviations: V. Vascular bundle; EP. epidermal layer cells; CP. cortical cells; OC. organization centers.\u003c/p\u003e","description":"","filename":"Fig4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/2747f8d9dc6b6d49a1937edc.jpg"},{"id":54411975,"identity":"9ed70eb9-c13b-49a2-ad2f-baa330157e30","added_by":"auto","created_at":"2024-04-10 05:48:50","extension":"jpg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":59774439,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMorphological and histological observation of nodular cluster differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Explants with nodular cluster induced directly after 5-6 subculture in DM; B. Primordia (black arrow) occurred on the surface of nodule; C. Nodular cluster with primordia initiated (black arrow); D. Bud primordia (black arrow); E. Leaf clusters were developed from nodule; F. Shoots developed from leaf clusters successively after transferring to shoot elongation medium.; G. Shoots developed root; H. The rooted plantlets were acclimatized successfully. Abbreviations: EP. epidermal layer cells; CP. cortical cells; OC. organization centers.\u003c/p\u003e","description":"","filename":"Fig5.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/8db250e1fa20b931c12d8d05.jpg"},{"id":54411974,"identity":"24bd5c5c-8980-4205-93d2-0f68792236d1","added_by":"auto","created_at":"2024-04-10 05:48:50","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":386706,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/1e7ff921f228c5a3f22189c1.jpg"},{"id":54411972,"identity":"9dac29e1-04b6-40e2-957a-eca5a05ec6f0","added_by":"auto","created_at":"2024-04-10 05:48:50","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":425875,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/2593d4e46ffca2a6d4662dfd.jpg"},{"id":54411971,"identity":"20903779-6720-4ff8-a0b3-385bd6473e37","added_by":"auto","created_at":"2024-04-10 05:48:49","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":419764,"visible":true,"origin":"","legend":"\u003cp\u003eLegend not included with this version\u003c/p\u003e","description":"","filename":"Fig8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/9ba9b4489ad9f31b0bfffe1b.jpg"},{"id":54412529,"identity":"c6011a87-0b2e-45b5-b23a-e1a3c9fe806d","added_by":"auto","created_at":"2024-04-10 05:56:58","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2586264,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4062314/v1/c29f506c-1f6a-438a-81e0-c45a0625e230.pdf"}],"financialInterests":"","formattedTitle":"Paeonia ostii ‘Feng Dan’ plant regeneration through direct organogenesis and direct meristematic nodule culture","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTree peony (\u003cem\u003ePaeonia\u003c/em\u003e sect. \u003cem\u003eMoutan\u003c/em\u003e) is valued as an ornamental plant rich in colors and patterns, also used as medicinal and oil production plant (Yu et al. \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). However, mass propagation of this species is limited by the characteristics of its conventional propagation methods, low efficiency and long period. Tissue culture is a useful way to solve this problem.\u003c/p\u003e \u003cp\u003eThe research of tree peony concerned with tissue culture date back to 1984 (Li et al. \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e1984\u003c/span\u003e), and over the past four decades, it has been widely recognized that difficulty in differentiation of callus is a key issue that hinders in vitro regeneration (Du et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e; Wen et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). For example, several studies on somatic embryogenesis of tree peony have been reported, but no available regeneration system published because of rare differentiation from callus in indirect pathway (Zhu et al. \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Du et al. \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e2020b\u003c/span\u003e), so did organogenesis from callus (Wen et al. \u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Breakthrough, an available regeneration system of tree peony via meristematic nodules (MNs) culture, derived from callus, has been described previously (Xu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). MN shown resemblance in appearance with somatic embryo, but could be distinguished from histological aspect (Xu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Haensch \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). It possessed high potential in micropropagation, genetic transformation, and bioreactor for producing chemical components (McCown et al. \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e1988\u003c/span\u003e; Batista et al. \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). Nevertheless, the system yielded the disadvantage of complex procedure and long period as a result from the cumbersome process of callus and MNs induction. Hence, the regeneration system via MNs culture not involving a strict callus phase is needed to streamline the lengthy process. Similar protocols have already been announced in some literature (Pi\u0026eacute;ron et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e; Trindade and Pais \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2003\u003c/span\u003e; Moyo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDirect organogenesis (DO) originated from explant without callus period have been declared in some plants, such as \u003cem\u003eAnaphalis hancockii\u003c/em\u003e (Geng et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e), \u003cem\u003eSolanum melongena\u003c/em\u003e (Garc\u0026iacute;a-Fortea et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), \u003cem\u003eHypericum perforatum\u003c/em\u003e (Ravindran et al. \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This system is relatively plainer and possesses a shorter cycle. Whereas there were scarce reports carried out about this protocol in tree peony. Overall, innovative breakthroughs concerning in vitro shoot organogenesis excluding the callus step are needed to simplify the procedure.\u003c/p\u003e \u003cp\u003eThe present work aimed to achieve shoot organogenesis with two novel pathways avoiding callus step, shoots emerged directly from explants or explants-derived MNs. Histological analyses were undertaken to reveal the characteristics of develop stages in morphogenetic process. This study could be a significant innovation and supplement in the field of in vitro regeneration in tree peony.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003ePlant material\u003c/h2\u003e \u003cp\u003eIn August 2022, seeds at 90 days after anthesis from five-year-old \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Feng Dan\u0026rsquo; plants were obtained from Beijing Guose Peony Garden in Beijing, China (40\u0026deg;45\u0026prime;N, 115\u0026deg;97\u0026prime;E). The collected seeds were placed in the refrigerator (-4℃) for at least 2 months.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003eExplant sources\u003c/h2\u003e \u003cp\u003eAfter washing under running tap water for 10 mins, the seeds were soaked in commercial liquid detergents (1% v/v; 5 min). Afterwards, seeds with clean surface were sterilized in clean bench by dipping in ethanol (70% v/v; 1 min) first, and then NaOCl (2% v/v; 15 min). Finally, the seeds were rinsed with sterile distilled water for at least three times before inoculation. According to the method adapted from Xu et al. (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), zygotic embryos were inoculated on MS medium containing 2.57 \u0026micro;M 6-benzyladenine (BA) and 2.89 \u0026micro;M gibberellin (GA\u003csub\u003e3\u003c/sub\u003e) for germination, the cotyledons after 15 days of culture were used as explants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003eMedium and culture conditions\u003c/h2\u003e \u003cp\u003eAll the cultures were grown on Woody plant medium (WPM), Murashige and Skoog medium (MS) and 1/2 MS (half-strength macroelements). The basal media containing 3% sucrose and 0.7% agar (Biosharp, Beijing, China). The pH of the media was pre-adjusted to 5.8-6.0 prior to sterilisation (at 120\u0026deg;C and 115 kPa for 20 min). All cultures were kept in a growth room at temperature of 23\u0026thinsp;\u0026plusmn;\u0026thinsp;2\u0026deg;C under a 16/8 h photoperiod using cool white light (25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003eDirect organogenesis (DO) and direct meristematic nodule (MN) induction\u003c/h2\u003e \u003cp\u003eTo study the effects of culture time and plant growth regulators (PGRs) in dedifferentiation induction medium (DIM) on the DO and direct MN induction, the explants were cut into pieces (1\u0026times;1 cm) and inoculated on DIM [MS\u0026thinsp;+\u0026thinsp;2.57 \u0026micro;M BA\u0026thinsp;+\u0026thinsp;5.37 \u0026micro;M α-naphthylacetic acid (NAA)] for different days (0, 5, 10 and 15 days) firstly. The excised cotyledon explants were placed with their abaxial face down in contact with the medium, and kept in dark conditions. Secondly, DIM containing different PGR combinations [2.57 \u0026micro;M BA\u0026thinsp;+\u0026thinsp;5.37 \u0026micro;M NAA, 2.02 \u0026micro;M N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)\u0026thinsp;+\u0026thinsp;5.37 \u0026micro;M NAA, 2.27 \u0026micro;M thidiazuron (TDZ)\u0026thinsp;+\u0026thinsp;5.37 \u0026micro;M NAA] was used. The excised cotyledon explants were cultured for 10 days respectively. Afterwards, the cotyledons were transferred from DIM to differentiation medium (DM) [WPM\u0026thinsp;+\u0026thinsp;2.27 \u0026micro;M TDZ], with subculture times of 15 days. The frequency of DO (%) and direct MN induction rate (%) were evaluated 6 subcultures after transfer. Each treatment consisted of 16 explants, and the experiment was repeated three times. The frequency of DO is expressed as the average percentage of explants/cotyledons that differentiated shoots directly over total number of explants/cotyledons. The direct MN induction rate is presented as the mean number of explants/cotyledons induced MNs directly over total number of explants/cotyledons.\u003c/p\u003e \u003cp\u003eThe cotyledons from best DIM after cultured for optimal culture time obtained above were subculture in DM. To screen optimal PGRs in DM for DO and direct MN induction, three cytokinins [2.57 \u0026micro;M BA, 2.02 \u0026micro;M CPPU and 2.27 \u0026micro;M TDZ] were employed respectively. After preliminary screening of suitable hormones, an orthogonal test involving two factors (CPPU and TDZ) and three levels of concentration (1.00, 2.02, 4.04 \u0026micro;M; 1.14, 2.27, 4.54 \u0026micro;M) was undertaken.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003eShoot elongation, rooting and acclimatization\u003c/h2\u003e \u003cp\u003eThe nodules induced directly from explants were transferred to medium [WPM\u0026thinsp;+\u0026thinsp;2.02 \u0026micro;M CPPU\u0026thinsp;+\u0026thinsp;2.27 \u0026micro;M TDZ] (Xu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e) for leaf clusters differentiation with 3 subcultures, 30 days in total. The cotyledons with leaf clusters (DO), as well as nodules with leaf clusters (direct MN induction and differentiation) were placed in shoot elongation medium [WPM\u0026thinsp;+\u0026thinsp;1.29 \u0026micro;M BA\u0026thinsp;+\u0026thinsp;0.58 \u0026micro;M GA\u003csub\u003e3\u003c/sub\u003e] respectively with 2 subcultures, 60 days in total.\u003c/p\u003e \u003cp\u003eFor in vitro root formation, micro-shoots about 1\u0026ndash;3 cm in length were transferred to 1/2 MS basal medium supplemented with 4.92 \u0026micro;M indole-3-butyric acid (IBA) and 11.34 \u0026micro;M putrescine (Wang et al. \u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Micro-shoots on root induction medium were cultured in the dark for the first 8 days at 4\u0026deg;C and then 30 days at 24\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, prior to being cultured in 1/2 MS basal medium supplemented with 0.4% activated carbon for 20 days under a 16/8 h photoperiod using cool white light (25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e). Plantlets with well-developed shoots and roots were then removed from the agar medium and potted in plastic pots containing autoclaved substrate (vermiculite, peat, and perlite in a 1:1:1 volumetric ratio). Agar was removed from the roots thorough carefully washed with running water prior to transplanting. The pots were placed in a culture chamber at 20\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C under a 16/8 h photoperiod using cool white light (25 \u0026micro;mol\u0026middot;m\u003csup\u003e\u0026minus;\u0026thinsp;2\u003c/sup\u003e\u0026middot;s\u003csup\u003e\u0026minus;\u0026thinsp;1\u003c/sup\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003eHistological analysis\u003c/h2\u003e \u003cp\u003eTo study the development stage of direct organogenesis and direct MN induction and differentiation, fresh samples [cotyledons cultured in DIM for 15 days, 1\u0026times;1 cm; cotyledons cultured in DM for different times of subcultures (1, 2, 3, 4, 5 and 6 times), cotyledons with nodules, cotyledons with leaf clusters, nodules with leaf clusters, 1\u0026times;1\u0026times;1 cm] were fixed for 48 h in the FAA solution (50% alcohol, glacial acetic acid, and formaldehyde at a ratio of 18:1:1). The permanent preparation was made based on the method adapted from Xu et al. (\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e). Sections 8\u0026ndash;10 \u0026micro;m in thickness were obtained using a rotary microtome, and stained with fast green (0.1%) and safranin (0.1%). The prepared slides were studied with Leica model DM500 microscope.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003eStatistical analysis\u003c/h2\u003e \u003cp\u003eThe data were subjected to analysis of variance (ANOVA) following Duncan\u0026rsquo;s multiple range test to detect significant differences (p\u0026thinsp;\u0026le;\u0026thinsp;0.05) in the mean using SPSS 23.0 (SPSS Inc., Chicago, USA), after transforming the percentage values using arcsine transformation. Variability in the data was expressed as the mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eorphological and histological study on DO from cotyledons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe detailed morpho-histological characterization of explants during in vitro direct organogenesis was conducted for the first time in tree peony to determine the timing and tissue origin of the regenerants. Compared to the original state, active-divided\u0026nbsp;subepidermal cells\u0026nbsp;and\u0026nbsp;meristematic cells around\u0026nbsp;vascular centers were observed in cotyledon after pretreated in DIM (Fig. 1A). Incubation in DM for 1-2 times resulted in obviously swelled and elongated of cotyledon (Fig. 1B). In particular, the occurrence of DO was observed at two positions. Strip protuberances gradually apparent on the surface of cotyledons unevenly (Fig. 1C), and visible swell formed at the cotyledon petiolar cut edge (Fig. 1D). Sections\u0026nbsp;revealed\u0026nbsp;subepidermal cells\u0026nbsp;vigorously proliferated with large and clear nuclei leading to the formation of strip protuberances (Fig. 1E). Meanwhile, transverse sections of swell\u0026nbsp;showed\u0026nbsp;rapid division of meristematic cell under cortical tissue and around vascular bundles filling up the expansive intercellular spaces (Fig. 1F). The volume of protuberances in both positions was increased\u0026nbsp;irregularly\u0026nbsp;after 3-4 times of incubation, and\u0026nbsp;primordia were shown up. Direct\u0026nbsp;connections between vascular tissue inside\u0026nbsp;primordia\u0026nbsp;and explants were detected in strip protuberances (Fig. 2A, B, C). However,\u0026nbsp;histological observation of swell demonstrated that primordia was initiated inside\u0026nbsp;and developed towards the surface of\u0026nbsp;cotyledons\u0026nbsp;(Fig. 2D, E). Leaf clusters occurred at two positions after 5-6 subcultures, protuberances on the surface of cotyledon (Fig. 3A, B) and swell on the edge (Fig. 3C). Subsequently, apical meristems could be found out\u0026nbsp;(Fig. 3D). Shoots developed from leaf clusters successively after\u0026nbsp;transferring to shoot elongation medium\u0026nbsp;(Fig. 3E).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eM\u003c/strong\u003e\u003cstrong\u003eorphological and histological study on direct\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eMNs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;induction and differentiation\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHistological examination confirmed direct MNs induction. The process of direct MNs induction and DO was occurred simultaneously. Rapid cell division were initiated in the adaxial portion of\u0026nbsp;subepidermal cells,\u0026nbsp;leading to small\u0026nbsp;globular protuberances\u0026nbsp;arose from the surface of cotyledons within 1-2 times of\u0026nbsp;incubation\u0026nbsp;in DM\u0026nbsp;(Fig. 4A, B). The\u0026nbsp;protuberances\u0026nbsp;significantly expanded during 3-4 subculture (Fig. 4C, D), accompanied with massively formation of\u0026nbsp;vascular tissue (Fig. 4E). Under histological observation, the\u0026nbsp;large\u0026nbsp;protuberances gradually developed into MNs,\u0026nbsp;composed of cortical, epidermal layer cells, and various organization centers,\u0026nbsp;like nested and linear\u0026nbsp;(Fig. 4F, G). During 5-6\u0026nbsp;times of\u0026nbsp;incubation,\u0026nbsp;nodules proliferation occurred with a special way like budding. Therefore,\u0026nbsp;nodular clusters\u0026nbsp;were appeared since\u0026nbsp;different sizes of nodules were gathered\u0026nbsp;(Fig. 5A). Owing to vigorous division of\u0026nbsp;the epidermal and subepidermal cells,\u0026nbsp;primordia emerged from nodules (Fig. 5B, C, D). When\u0026nbsp;nodular clusters were\u0026nbsp;transferred to\u0026nbsp;leaf cluster differentiation medium, leaf clusters gradually observed on the surface\u0026nbsp;(Fig. 5E).\u0026nbsp;Similarly, shoots arose from leaf clusters in succession after incubated in\u0026nbsp;shoot elongation medium\u0026nbsp;(Fig. 5F).\u003c/p\u003e\n\u003cp\u003eThe eligible shoots induced both from DO pathway and direct MN culture developed roots after placement on the rooting medium\u0026nbsp;(Fig. 5G), and the\u0026nbsp;rooted plantlets were acclimatized successfully\u0026nbsp;(Fig. 5H).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;culture time in DIM\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDO and direct MNs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDO or direct MN induction were failed to be observed from the cotyledons not cultured in DIM for dedifferentiation (Fig. 6). Pre-culture in DIM positively affected the formation of DO and direct MNs induction.\u0026nbsp;the frequency of DO and the direct MN induction rate\u0026nbsp;were gradually increased with dedifferentiation time, and the highest\u0026nbsp;frequence of DO (43.75%) was achieved when cotyledons explants were pretreated in DIM for\u0026nbsp;10 days,\u0026nbsp;but with no significant difference between 10-15 days of treatment. The direct MN induction rate of all treatments was generally low, but treatments of 10-15 days were higher than that of 0-5 days.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;PGRs in DIM\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDO and direct MNs\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;induction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAs shown in figure 7, the frequency of DO from\u0026nbsp;cotyledons pre-cultured in DIM supplemented with\u0026nbsp;2.02 µM\u0026nbsp;CPPU+5.37 µM\u0026nbsp;NAA (50.00%) and 2.27 µM\u0026nbsp;TDZ+5.37 µM\u0026nbsp;NAA (52.08%)\u0026nbsp;was significantly higher that of 2.57 µM\u0026nbsp;BA+5.37 µM\u0026nbsp;NAA (39.58%). Combination of TDZ and NAA was the treatment that showed the highest frequence of DO. However, there was no significant difference on the direct MNs induction rate among treatments (＜10%).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEffect of\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;PGRs in DM\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eDO and direct MNs\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003einduction\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn the preliminary experiment, DO and direct MNs induction only occurred in DM containing TDZ or CPPU, but not in presence of BA (Fig. 8). The results indicated that the frequency of DO in treatment with CPPU (41.67%) and TDZ (45.83%) was conspicuously higher than BA (0%), but present no significant difference between two of them. Meanwhile, the direct MN induction rate of treatment with CPPU (10.42%) superior to TDZ (4.17%).\u003c/p\u003e\n\u003cp\u003eIn subsequent steps, the best performance of direct MN induction rate (41.67%) was obtained at 2.02 µM CPPU and 2.27 µM TDZ, while the optimal concentration of CPPU and TDZ was 4.04 µM and 4.54 µM\u0026nbsp;respectively\u0026nbsp;in terms of the frequency of DO (66.67%) (Table 1).\u0026nbsp;Variance analysis demonstrated that there was a significant response for CPPU and TDZ concentration on the frequency of DO (\u003cem\u003ep\u003c/em\u003e＜0.01), as well as their interaction (Table 2). In terms of the direct MN induction rate, CPPU and its combination with TDZ remained significant effect, but TDZ alone not influenced (\u003cem\u003ep\u003c/em\u003e＞0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003eThe number of regenerated shoots originated from both DO and direct MNs culture pathway varying from three to seven per explant. The rooting rate remains around 50%, and the survival rate was kept between 30-40%.\u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis is the first report of DO from cotyledon explants without callus period in tree peony. In this experiment, leaf cluster initiation was observed at cut site of the proximal end of cotyledon, as well as the paraxial surface, which in agreement with description in some literature (Huang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Debnath et al. \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Simultaneously, the occurrence of direct organogenesis was confirmed by the histologic analysis that shoots originated from increased division of meristematic cell under cortical tissue, as well as meristematic cells around vascular centers inside of swell on the edge of the cotyledon. Similar phenomenon was revealed previously, but shoot initiation was varied. For instance, from the upper epidermal cells and their inside parenchyma cells in \u003cem\u003eCapsicum annuum\u003c/em\u003e (Gao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e), from the epidermal tissue in \u003cem\u003eNeolamarckia cadamba\u003c/em\u003e (Huang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), from the epidermal and subepidermal cells in \u003cem\u003eCucumis melo\u003c/em\u003e (Cai et al. \u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e2002\u003c/span\u003e), which was called for exogenous initiation. Besides, buds could regenerate from endogenous meristematic cells around vascular centers (Sarkar and Jha \u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Thus, the origin of shoots through the DO pathway is endogenous and exogenous coexist in tree peony.\u003c/p\u003e \u003cp\u003eOn the other hand, this is also the first report of direct MNs induction and differentiation without callus phase in tree peony. Histological examination confirmed this morphogenic response. This is contrary to previous conclusion that callus formation was necessary for MNs induction in tree peony (Xu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). In fact, there were two morphogenesis pathways has been reported on MNs induction, direct (Moyo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Pi\u0026eacute;ron et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e) and indirect with callus formation (Fortes and Pais \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Batista et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e). Therefore, two pathways were existed side by side in tree peony.\u003c/p\u003e \u003cp\u003eIn this model of direct MNs culture on tree peony, cells competent for nodule induction was located mainly in meristematic cell under cortical tissue with histological observation, and new formed vascular system developed inside nodules subsequently, which was basically consistent with results in some reports (Moyo et al. \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2009\u003c/span\u003e; Ferreira et al. \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2009\u003c/span\u003e). However, there were different conclusion in \u003cem\u003eCichorium intybus\u003c/em\u003e that the cambium of nodule was originated from the procambium of leaf, and the parenchyma and periderm cork cell layers were originated from the fascicular parenchyma and bundle sheath tissues of leaf in direct (Pi\u0026eacute;ron et al. \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). These distinctions might be related to species differences. Additionally, it was documented that shoots originated from epidermis or cortex tissue of nodules in \u003cem\u003eHumulus lupulus\u003c/em\u003e (Batista et al. \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Fortes and Pais \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2000\u003c/span\u003e), or from parenchymal cells around the vascular center in \u003cem\u003eCichorium intybus\u003c/em\u003e (Pi\u0026eacute;ron et al. \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e1993\u003c/span\u003e, \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e1998\u003c/span\u003e). In contrast to previous text that shoots were regenerated from endogenous parenchyma cells around nodule vascular (Xu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e), our histological examination revealed that increased division of the epidermal and subepidermal cells of nodules led to shoot regeneration. Similar observation has been recognized by Qin et al. (\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e2012\u003c/span\u003e) in \u003cem\u003eP. lemoinei\u003c/em\u003e \u0026lsquo;Golden Era\u0026rsquo;. This difference might attribute to insufficient number of sections.\u003c/p\u003e \u003cp\u003eIn our studies, DO and direct MNs induction took place synchronously, and highly parallel at the early stage. Both induced protuberances initiated from subepidermal cells, but shared different shapes. When it comes to DO pathway, strip protuberances became apparent on the upper part surface of cotyledons, and the appearance of swell growth with irregular shape was observed on the edge. On the contrary, the nodules exhibited a global-shaped structure at the beginning of their development, similar to globular somatic embryos. The histological analysis could provide detail evidence for distinguishing (Xu et al. \u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e2022b\u003c/span\u003e; Haensch \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). Subsequently, the volume of swell at the cut site of cotyledon gradually increased, so did the nodules. Nevertheless, they were remarkable distinct in terms of appearance (Fig.\u0026nbsp;2D; Fig.\u0026nbsp;4D) and location. The swell located just at the cut end of cotyledon explants, same as \u003cem\u003eGarcinia mangostana\u003c/em\u003e (Qosim et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), but nodules were not restricted. In addition, there were more plenty of autogenetic vascular tissues inside the nodules compared to swell (Fig.\u0026nbsp;2E; Fig.\u0026nbsp;4E).\u003c/p\u003e \u003cp\u003ePretreatment in DIM (dedifferentiation) before callus formation played a dominant role in this protocol. With the enhancement of dedifferentiation time (0\u0026ndash;15 days), the differentiation rate in DM improved. It was speculated that cells with high activity after losing their original characteristic structure and function within dedifferentiation time, could trigger a particular developmental fate when recognized a single inductive signal. Specifically, dedifferentiated subepidermal cells and meristematic cells around vascular centers could developed into primordia or nodules after transferring cotyledons from DIM to DM supplement with highly active cytokinin. The results emphasized the importance of dedifferentiation step of explants in DIM enriched with auxin. It is well known that auxin is essential for apical meristem formation. In detail, auxins and downstream transcriptional regulation interfere with the structural elements of the cell wall to induce specific morphogenetic events, the absence of auxins in the pretreatment inhibited the morphogenic process (Traas \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Furthermore, cross-talk with other signaling pathways, cytokinin in particular, is crucial in organ regeneration (Vernoux et al. \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2011\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). Similarity, the procedure was developed in \u003cem\u003eEucalyptus nitens\u003c/em\u003e (Ayala et al. \u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e2019\u003c/span\u003e) and in \u003cem\u003ePrunus cerasifera\u003c/em\u003e (Carmona-Martin and Petri \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), that pre-treatment with auxins under dark condition and subsequent transfer to medium rich in cytokinin in light conditions, and considered to be appropriate in recalcitrant specie for direct regeneration.\u003c/p\u003e \u003cp\u003eEffect of PGRs in DM on direct regeneration was dependent on PGRs type and concentration (Bao et al. \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2017\u003c/span\u003e; Huang et al. \u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e2014\u003c/span\u003e), genotype (Garc\u0026iacute;a-Forte et al. 2020) and explant type (Geng et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). According to Xu et al (\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), CPPU and TDZ, two kinds of cytokinin with high activity, played remarkably positive roles in MNs induction and shoots regeneration from callus. Meanwhile, DO and direct MNs induction only occurred in DM containing TDZ or CPPU in this research, but not in presence of BA, which further supported previous speculation that high level of cytokinin was indispensable for differentiation (Xu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e; Meng et al. \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The result that TDZ\u0026thinsp;+\u0026thinsp;NAA, CPPU\u0026thinsp;+\u0026thinsp;NAA treatment in DIM had a better effect on the induction of MNs and DO than BA\u0026thinsp;+\u0026thinsp;NAA treatment also consistent with this conclusion. In contrast, the cytokinin suitable for direct regeneration was varied among other plant species, like BA for \u003cem\u003eGarcinia mangostana\u003c/em\u003e (Qosim et al. \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e), ZR for \u003cem\u003eSolanum melongena\u003c/em\u003e (Garc\u0026iacute;a-Fortea et al. \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2020\u003c/span\u003e), TDZ for \u003cem\u003eRhododendron\u003c/em\u003e (Hebert et al. \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2010\u003c/span\u003e), and combination of KT and BA for \u003cem\u003eAnaphalis hancockii\u003c/em\u003e (Geng et al. \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Besides, mixed use of CPPU and TDZ was more effective than single one in direct regeneration, which was in coincidence with other result (Gao et al. \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2021\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe developmental direction of differentiation was varied with cytokinin type and concentration. For example, the epidermal cells of leaves developed into nodular callus if stimulated by combination TDZ and BA, while shoot originated with BA stimulation alone. What\u0026rsquo;s more, the optimum treatment for direct shoots induction was achieved at a concentration of 22.2 \u0026micro;M BA, but the use of BA (\u0026gt;\u0026thinsp;44.4 \u0026micro;M) inhibited the formation and elongation of shoots (Qosim et al. \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e2013\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). In the present study, both of CPPU and TDZ concentration had significant effect on the frequency of DO (\u003cem\u003ep\u003c/em\u003e\u0026lt;0.01), but TDZ showed no significant effect on the direct MN induction rate (\u003cem\u003ep\u003c/em\u003e\u0026gt;0.05). Similar result has been concluded in tree peony that CPPU was considerably effective in promoting MNs induction from callus (Xu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e), and in herbaceous peony that TDZ was appropriate for direct organogenesis from cotyledon explants (Zhao et al. \u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Concurrently, the best performance of DO and direct MNs induction was presented on CPPU and TDZ combination at different level of concentration in our study, and higher level suitable for DO. Thus, researchers can make different choices in concentration formulas based on varied purposes.\u003c/p\u003e \u003cp\u003eThe protocol achieved in this essay can greatly shorten the period needed for regeneration by skipping the callus stage. More specifically, it takes approximately 6 months to obtain differentiated shoots in DO pathway, and this regeneration system through direct MNs induction from explants and differentiation can save approximately 2 months compared to previous cycle (Xu et al. \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e2022a\u003c/span\u003e). Moreover, MNs of tree peony in vitro work as storage organs like corms or tubers, and scale-up multiplication of MNs can be reached in liquid medium (Zhong et al. 2011). Therefore, optimization of in vitro regeneration can be achieved with procedure that MNs induced from explant directly multiplicated in liquid environment, and then differentiated in solid medium. This hypothesis has been realized in \u003cem\u003eAnanas comosus\u003c/em\u003e var. \u003cem\u003ecomosus\u003c/em\u003e (Scherer et al. \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eVriesea reitzii\u003c/em\u003e (Dal Vesco and Guerra 2010), and \u003cem\u003eCharybdis numidica\u003c/em\u003e (Kongbangkerd and Wawrosch \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). We have obtained preliminary evidence that nodules proliferated in liquid medium could finish differentiate in solid medium, but further optimization of culture conditions still underway.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study firstly presented a protocol of in vitro regeneration through DO and direct MNs culture in \u003cem\u003eP. ostii\u003c/em\u003e \u0026lsquo;Feng Dan\u0026rsquo;. This protocol includes two pathways at the same time. The explants need pretreated in DIM in both ways, and then occurred simultaneously in DM. Shoots regenerated directly from explants (DO pathway) and from explants-derived MNs could rooted, and the rooted plantlets were acclimatized successfully. In addition, histological study revealed the developmental sequence and tissue origin of the regenerants. This protocol simplified the differentiation process and will be beneficial to the clonal micropropagation, fundamental studies of developmental biology and genetic improvement of tree peony.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eBA, 6-benzyladenine; CPPU, N-(2-chloro-4-pyridyl)-N-phenylurea; DM, Differentiation medium; DIM, dedifferentiation induction medium; DO, Direct\u0026nbsp;organogenesis;\u0026nbsp;GA\u003csub\u003e3\u003c/sub\u003e, Gibberellin; IBA, Indole-3-butyric acid; MN, Meristematic nodule; MS, Murashige and Skoog; WPM, Woody plant medium; NAA, \u0026alpha;-naphthylacetic acid; PGR, Plant growth regulator; TDZ, Thidiazuron;\u0026nbsp;\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 the National Natural Science Foundation of China (32302598), and Natural Science Foundation of Hubei Province of China (2023AFB509).\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\u003eData availability\u003c/strong\u003e The data supporting the findings of this study are available with Li Xu and can be made available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contribution\u003c/strong\u003e CCF and KXL conducted the experiments and written the manuscript. 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Trees 17(4):308-315. http://dx.doi.org/10.1007/s00468-002-0240-0 \u003c/li\u003e\n\u003cli\u003eVernoux T, Brunoud G, Farcot E, Morin V, Van den Daele H, Legrand J, Oliva M, Das P, Larrieu A, Wells D, Gu\u0026eacute;on Y, Armitage L, Picard F, Guyomarc\u0026rsquo;h S, Cellier C, Parry G, Koumproglou R, Doonan JH, Estelle M, Godin C, Kepinski S, Bennett M, De Veylder L, Traas J (2011) The auxin signalling network translates dynamic input into robust patterning at the shoot apex. Mol Syst Biol 7(1):508.https://doi.org/10.1038/msb.2011.39\u003c/li\u003e\n\u003cli\u003eHuang X, Chen J, Bao Y, Liu L, Jiang H, An X, Dai L, Wang B, Peng D (2014) Transcript profiling reveals auxin and cytokinin signaling pathways and transcription regulation during in vitro organogenesis of ramie (\u003cem\u003eBoehmeria nivea\u003c/em\u003e L. Gaud). Plos One 9(11):e113768https://doi.org/10.1371/journal.pone.0113768\u003c/li\u003e\n\u003cli\u003eWang X, Cheng FY, Zhong Y, Wen SS, Li LZM, Huang LZ (2016) Establishment of in vitro rapid propagation system for tree peony (\u003cem\u003ePaeonia ostii\u003c/em\u003e). Scientia Silvae Sinicae 52:102-110.https://doi.org/10. 11707/j.1001-7488.20160512\u003c/li\u003e\n\u003cli\u003eWen SS, Chen L, Tian RN (2020). Micropropagation of tree peony (\u003cem\u003ePaeonia\u003c/em\u003e sect. \u003cem\u003eMoutan\u003c/em\u003e): A review. Plant Cell Tissue Organ Cult 141(1):15. https://doi.org/10.1007/s11240-019-01747-8\u003c/li\u003e\n\u003cli\u003eXu L, Cheng FY, Zhong Y (2022a) Efficient plant regeneration via meristematic nodule culture in \u003cem\u003ePaeonia ostii\u003c/em\u003e \u0026lsquo;Feng Dan\u0026rsquo;. Plant Cell Tissue Organ Cult.149(3):599-608. https://doi.org/10.1007/s11240-021-02216-x\u003c/li\u003e\n\u003cli\u003eXu L, Cheng FY, Zhong Y (2022b) Histological and cytological study on meristematic nodule induction and shoot organogenesis in \u003cem\u003ePaeonia ostii\u003c/em\u003e \u0026lsquo;Feng Dan\u0026rsquo;. Plant Cell Tissue Organ Cult. 149(3):1-12. https://doi.org/10.1007/s11240-021-02208-x\u003c/li\u003e\n\u003cli\u003eYu SY, Du SB, Yuan JH, Hu YH (2016) Fatty acid profile in the seeds and seed tissues of \u003cem\u003ePaeonia \u003c/em\u003eL. species as new oil plant resources. Sci Rep 6:26944-26944. https://doi.org/10.1038/srep26944\u003c/li\u003e\n\u003cli\u003eZhao D, Xue Y, Shi M, Tao J (2017) Rescue and in vitro culture of herbaceous peony immature embryos by organogenesis. Sci Hortic 217:123-129. http://dx.doi.org/10.1016/j.scienta.2017.01.040\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. (In Chinese)\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. https://doi.org/10.1007/s13580-018-0065-4\u003c/li\u003e\n\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"567\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"96.11992945326278%\" colspan=\"4\" style=\"width: 66.4234%;\"\u003e\n \u003cp\u003eTable 1 Screening of CPPU and TDZ concentration for the frequency of DO and the direct MN induction rate\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003eCPPU (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003eTDZ (\u0026micro;M)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" valign=\"top\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003eThe frequency of DO (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" valign=\"top\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003eThe direct MN induction rate (%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" valign=\"top\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e8.33\u0026plusmn;3.61\u003csup\u003ee\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" valign=\"top\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e4.17\u0026plusmn;3.61\u003csup\u003eg\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e2.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" valign=\"top\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e12.50\u0026plusmn;6.25\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" valign=\"top\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e12.50\u0026plusmn;0.00\u003csup\u003ef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e1.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e4.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" valign=\"top\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e16.67\u0026plusmn;6.25\u003csup\u003ecd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" valign=\"top\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e27.08\u0026plusmn;3.61\u003csup\u003ebcd\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e2.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" valign=\"top\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e20.83\u0026plusmn;3.61\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" valign=\"top\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e41.67\u0026plusmn;3.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e4.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e22.92\u0026plusmn;3.61\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e20.83\u0026plusmn;3.61\u003csup\u003ede\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e22.92\u0026plusmn;3.61\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e22.92\u0026plusmn;3.61\u003csup\u003ecde\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e4.54\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e66.67\u0026plusmn;3.61\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e29.17\u0026plusmn;3.61\u003csup\u003ebc\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e1.14\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e20.83\u0026plusmn;3.61\u003csup\u003ec\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e31.25\u0026plusmn;6.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"15.845070422535212%\" style=\"width: 10.9679%;\"\u003e\n \u003cp\u003e4.04\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.43661971830986%\" style=\"width: 9.982%;\"\u003e\n \u003cp\u003e2.27\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"33.62676056338028%\" style=\"width: 23.1681%;\"\u003e\n \u003cp\u003e43.75\u0026plusmn;6.25\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.91549295774648%\" style=\"width: 24.7701%;\"\u003e\n \u003cp\u003e16.67\u0026plusmn;3.61\u003csup\u003eef\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"4\" style=\"width: 69.7507%;\"\u003e\n \u003cp\u003eDifferent letters within a column show significant differences by Duncan\u0026rsquo;s multiple range tests (p \u0026le; 0.05). Each data represent mean \u0026plusmn; standard error.\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable border=\"0\" cellspacing=\"0\" cellpadding=\"0\" width=\"375\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"7\" valign=\"top\"\u003e\n \u003cp\u003eTable 2 Variance analysis of\u0026nbsp;CPPU and TDZ concentration\u0026nbsp;on the frequency of DO and the direct MN induction\u0026nbsp;rate\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.066666666666666%\" rowspan=\"2\" valign=\"top\"\u003e\n \u003cp\u003eSource of variance\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"42.666666666666664%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eThe frequency of DO\u0026nbsp;\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"40.266666666666666%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003eThe direct MN induction rate\u003c/p\u003e\n \u003cp\u003e(%)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"18.971061093247588%\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.112540192926046%\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"17.363344051446944%\"\u003e\n \u003cp\u003e\u003cem\u003ep\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"13.183279742765274%\"\u003e\n \u003cp\u003edf\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.434083601286174%\"\u003e\n \u003cp\u003e\u003cem\u003eF\u003c/em\u003e test\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"19.935691318327976%\"\u003e\n \u003cp\u003e\u003cem\u003ep\u0026nbsp;\u003c/em\u003evalue\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.066666666666666%\"\u003e\n \u003cp\u003eCPPU\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.733333333333333%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.533333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e122.385\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.4%\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.933333333333334%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.8%\"\u003e\n \u003cp\u003e33.60\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.533333333333335%\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.066666666666666%\"\u003e\n \u003cp\u003eTDZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.733333333333333%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.533333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e21.77\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.4%\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.933333333333334%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.8%\"\u003e\n \u003cp\u003e2.10\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.533333333333335%\"\u003e\n \u003cp\u003e0.15\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.066666666666666%\"\u003e\n \u003cp\u003eCPPU*TDZ\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.733333333333333%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.533333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e32.50\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.4%\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.933333333333334%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.8%\"\u003e\n \u003cp\u003e33.00\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.533333333333335%\"\u003e\n \u003cp\u003e0.00\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"17.066666666666666%\"\u003e\n \u003cp\u003eError\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"15.733333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.533333333333333%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"14.4%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"10.933333333333334%\" valign=\"top\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"12.8%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"16.533333333333335%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\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":false,"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":"Tree peony, Cotyledon, Direct organogenesis, Meristematic nodule","lastPublishedDoi":"10.21203/rs.3.rs-4062314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4062314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eTissue culture is preferred for solving the shortcoming of low efficiency in terms of conventional propagation ways in tree peony, an economically important woody plant in China with various purposes. However, callus differentiation is hard to obtain during in vitro regeneration. Meristematic nodule (MN) is a favorable way capable of overcoming this problem, but possesses a lengthy process. Direct organogenesis excluding the callus step is needed to simplify the procedure. This study firstly presented a protocol of direct organogenesis and direct MNs induction and differentiation using cotyledon explant for in vitro regeneration of \u003cem\u003eP.ostii\u003c/em\u003e ‘Feng Dan’. The highest direct MNs induction rate (41.67%) and frequency of direct organogenesis (DO) (66.67%) was achieved under the following procedure. The explants were pretreated in dedifferentiation induction medium (DIM) [Murashige and Skoog (MS) medium with 2.27 µMthidiazuron (TDZ)+5.37 µM α-naphthylacetic acid (NAA)] for 10 days, and then the cotyledons without callus induced were transferred to differentiation medium (DM) [Woody plant medium (WPM) containing 2.02 µM N-(2-chloro-4-pyridyl)-N-phenylurea (CPPU)+2.27 µM TDZ and 4.04 µM CPPU+4.54 µM TDZ] respectively, with 6 subcultures, 90 days in total. The regenerated shoots rooted and transplanted successfully. Histological study confirmed the process of DO and direct MNs induction, and revealed that shoots and MNs were originated from increased division of meristematic cell under cortical tissue, as well as from actively divided meristematic cells around vascular center. Moreover, shoots regenerated through MNs differentiation were originated from the epidermal and subepidermal cells. This study is an innovation and supplement in the field of in vitro regeneration in tree peony, and will be conductive to clonal micropropagation, fundamental studies of developmental biology and genetic transformation.\u003c/p\u003e","manuscriptTitle":"Paeonia ostii ‘Feng Dan’ plant regeneration through direct organogenesis and direct meristematic nodule culture","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-04-10 05:48:44","doi":"10.21203/rs.3.rs-4062314/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-04-08T07:31:03+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-04-07T11:21:30+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-03T03:45:34+00:00","index":"","fulltext":""},{"type":"submitted","content":"Plant Cell, Tissue and Organ Culture (PCTOC)","date":"2024-04-01T22:59:16+00:00","index":"","fulltext":""},{"type":"decision","content":"Major revisions","date":"2024-03-21T06:19:43+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":"b4f9b7b6-af25-4cdb-af16-7d30866627c8","owner":[],"postedDate":"April 10th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2024-05-22T14:16:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-04-10 05:48:44","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4062314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4062314","identity":"rs-4062314","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}