{"paper_id":"01ebb001-b1cf-4647-86b1-1cf3bc673000","body_text":"Studies on floral organ Structure and female and male gametophyte development of Pseudosasa subsolida S. L. Chen & G. Y. 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L. Chen & G. Y. Sheng Bonan Jiang, Zhihua Cao, Dejia Yang, Yongmei Wang, Yingchun Ma, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4600448/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Pseudosasa subsolida belongs to Poaceae Pseudosasa, Because of its unique flowering cycle of bamboo plants and the physiological characteristics of asexual reproduction, The acquisition of the floral material from Pseudosasa subsolida poses significant challenges. A comprehensive anatomical study on the floral organs and the development of female and male gametes of P. subsolida was conducted in 2021 at Southwest Forestry University (Kunming,Yunnan,China) to investigate the influencing factors of its low seed setting rate, utilizing routine paraffin section methods. The results revealed that the spikelet of P. subsolida exhibited the characteristics of a pseudospikelet with a latent bud, while the inflorescence displayed traits of an infinite inflorescence. Each spikelet contained approximately 10–16 florets and was accompanied by two bracts at its base. The fundamental structure of the florets comprised one lemma, one palea, three lodicules, three stamens, and one pistil. Many anomalies were still observed at later stages of anther development. These abnormalities included the failure of pollen grain formation, shrinkage deformation in the drug sac and tapetum cells, and the absence of middle layer cells. Additionally, microspores appeared hollow with no discernible contents. The primary factor contributing to the low seed setting rate of P. subsolida was the aberrant development of male gametophytes. The significance of this study lay in its pioneering exploration of the reproductive structure of P. subsolida ,and provide a theoretical reference for the fundamental examination of flower structure. Pseudosasa subsolida abortion flower morphology anther ovary Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Background Bamboo, belonging to the Bambusoideae subfamily of the Gramineae family, represents one of the most significant forest resources, along with timber[ 1 ]. China possesses abundant bamboo resources, encompassing approximately 43 genera and 800 species. Bamboo forests cover approximately one-third of its overall land area[ 2 ]. The flowering cycle of bamboo plants is typically prolonged, shortcycle bamboo plants take 10–15 years to flower, such as Melocanna baccifera [ 3 ], the Shimen Moso Bamboo Forest in Fenghua, Zhejiang exhibits an exceptionally prolonged flowering period, with no documented instances of flowering occurring for a span exceeding 200 year[ 4 ]. The majority of bamboo species exhibit a low seed-setting rate or even the complete absence of fruiting[ 5 ]. Collecting flower and seed materials from fully developed bamboo plants therefore poses challenges that hinder research into their embryology. Currently, only a limited number of studies have provided detailed descriptions of the flower morphology and structure, as well as of megaspore occurrence and male/female gametophyte formation in bamboo species including Bambusa multiplex [ 6 ], Bambusa rigida [ 7 ], Bambusa eutuldoides [ 8 ], Dendrocalamus sinicus [ 9 ], Fargesia yuanjiangensis [ 10 ], Neomicrocalamus prainii [ 11 ], Shibataea chinensis [ 12 ], Tongpeia fungosa [ 13 ], etc. Pseudosasa subsolida belongs to the Poaceae Pseudosasa and is characterized by its shrublike or small treelike growth and its rapid development. Additionally, it possesses significant ornamental value. The occurrence of this species is restricted to the Daliyu Mountain in Yiyang County, Hunan, China (112.327023,28.590358). Being a natural wild species, it thrives exclusively in hilly terrains and yellow soil[ 14 , 15 ]. About the morphological characteristics of its adult bamboo, slight grooves can be observed at the base of branching internodes, where the rod wall is thick and nearly solid, with a medullary spongy structure. These characteristics contribute to exceptional firmness, resilience, and durability. P. subsolida is the preferred material for furniture and carving handicrafts[ 16 ]. Bamboo species infrequently undergo flowering, and their floral structure generally aligns with that of other plants in the Gramineae family. However, certain bamboo flowers exhibit a regression into scales, while other components regress into membranes, resulting in relatively diminutive sizes. The bamboo flowers were distinguished by their absence of vibrant hues and fragrant aromas, as well as their unique shapes. Angio-sperms utilize flowers as the fundamental structural unit for the formation of inflorescences, whereas bamboo inflorescences exhibit a higher level of complexity, being composed of spikelets. Thus, spikelets are the constituents of bamboo inflorescences[ 17 ]. Currently, the research on P. subsolida primarily focuses on species classification and morphological description, leaving significant gaps in other areas of study. There is a lack of records regarding flowering and other aspects of P. subsolida research. Examining its microstructure using the paraffin section method is even more limited. Because of the characteristics of bamboo plants, such as difficulty in flowering, and only flowering but no fruity, there are great obstacles in the breeding research of P. subsolida . For its special physiological characteristics, the reasons for the abortion of the floreus of its species should be first revealed. Then further solutions should be found, to provide effective help for breeding. It is known from the Flora of China[ 18 ] that P. subsolida has been evaluated as a vulnerable species, and the change in its ecological environment has led to the continuous decline of its species population. Therefore, studying the floret, the reproductive organ of P. subsolida , to find out the reason for its low seed-setting rate, will be hopeful to change the physiological characteristics of asexual reproduction in further research. To save endangered species. The morphological structure of the floral organs of P. subsolida is described in this study, aiming to complement the known classification characteristics of P. subsolida . Additionally, the development of male and female gametophytes, as well as the changes in the anther wall structure, is investigated in P. subsolida flowers to further elucidate the factors contributing to its low seed-setting rate. This study provides original embryological data for future research on P. subsolida and establishes a foundation for breeding efforts. Results 3.1. Flower morphology and anatomy of P. subsolida The spikelet base of P. subsolida exhibited latent buds (Fig. 1 a), also known as pseudo spikelets. The inflorescence of P. subsolida consists of pseudo-spikelet growth on various levels of vegetative branches, called indefinite inflorescences.Anatomical observations of the flowers of P. subsolida revealed that two bracts were present in close proximity to the floret at the base of the spikelet (Fig. 1 b). The average length of twenty randomly selected spikelets was 6.42 cm, with each spikelet containing 10–16 florets. The uppermost floret among these exhibited sterility, characterized by a young ovary devoid of stamens. Subsequently, a total of 50 florets were randomly selected, and their average length was determined to be 1.36 cm. Adjacent florets within the same spikelet were interconnected through the rachilla (Fig. 1 c), exhibiting alternate arrangement along the spikelet axis with cilia present. Furthermore, the mean length of 50 spikelet axes was measured to be 0.47 cm. The anatomical examination of a complete floret revealed that it comprised (from outside to inside) one lemma, one palea, three lodicules, three stamens, and one pistil (Fig. 1 d). The average length of the lemma was 1.35 cm, and it had 9–12 longitudinal veins, with the thickest and longest vein positioned at the center (Fig. 1 e). Additionally, cilia were observed on the surface, with long cilia present at the edge of the apex, which featured a sharp tip (Fig. 1 f,g). The palea was found to be paired with the internal growth of the lemma. the length of the lemma in the mature florets of P. subsolida was slightly greater than that of the palea (Fig. 1 h), whereas in immature florets, there was minimal difference in length between them (Fig. 1 i), except for a few instances where the palea extended beyond the lemma (Fig. 1 j). The palea exhibited two prominent ridges accompanied by four veins positioned between the ridges and three to four veins on each side. Notably, the surface of the palea was adorned with cilia, which were particularly elongated at the apex edge (Fig. 1 k). Three bracts (Fig. 1 l) surrounded the ovary; two were located near the lemma and one was covered by the palea. These bracts were thin and possessed white transparent membranous structures. During flowering, the bracts absorbed water and expanded, causing the lemma to spread open. The floret comprised three stamens (Fig. 1 m). The stamens were typically light yellow but showed browning in some instances (Fig. 1 n). The surface of the ovary exhibited a smooth and glabrous texture, appearing white in its juvenile stage (Fig. 1 o). Subsequently, as it reached maturity, the ovary underwent a gradual browning process (Fig. 1 p). The style was characterized by its short length, while the stigma displayed a trilobed structure with feathery appendages. 3.2 Microsporogenesis and male gamete development of P. subsolida 3.2.1 Microsporogenesis P. subsolida possesses three stamens, each consisting of four chambers. During the early stages of anther development, the outer epidermis comprises a layer of flattened cells, while inside the epidermis lies a cluster of cells with similar morphological structures. Notably, the cells located at the four corners divide more rapidly than other cells, leading to the gradual differentiation of sporogenous cells beneath each corner’s epidermis. Subsequently, these sporogenous cells initiated periclinal divisions, dividing outward to form the primary parietal layer and inward to generate primary sporogenous cells (Fig. 2 a). The primary sporogenous cells continue dividing and differentiate into secondary sporogenous cells (Fig. 2 b), which subsequently differentiate into larger microspore mother cells devoid of distinct vacuoles (Fig. 2 c). These microspore mother cells undergo subsequent developmental stages involving meiosis. At the conclusion of the first meiotic division, a dyad was formed by the microspore mother cell (Fig. 2 d), and a symmetrical tetrad was subsequently generated after meiosis II (Fig. 2 e). Consequently, the cytoplasmic division mode exhibited by the microspore mother cell adheres to a continuous type. 3.2.2 Development of male gametes The anthers progressed through the tetrad stage before transitioning into the microspore stage. Upon the dissolution of callose, the four daughter cells within the tetrad underwent separation and gave rise to autonomous mononuclear pollen grains, known as microspores. Immediately following release from the tetrad stage, the microspore exhibited a dense cytoplasm, with the nucleus occupying the central region of the cell, while vacuole formation had not yet occurred. This developmental phase was referred to as the systolic or early microspore phase (Fig. 2 f). As the volume of the microspores increased, vacuoles appeared in the cytoplasm, and the nucleus began to migrate towards one side of the cell. Simultaneously, gradual cell wall formation took place until complete nuclear relocation and extensive vacuolation in the middle region of the cell were achieved. The appearance of the germination hole (Fig. 2 f) was referred to as the mononuclear margination stage (Fig. 2 g). Subsequently, gradual cytoplasmic filling occurred throughout the entire cell. Concurrently, the nuclear division took place through mitosis, resulting in the formation of a binucleate pollen grain where one nucleus was divided into two nuclei (Fig. 2 h). The division resulted in two distinct cells: one, located adjacent to the pollen wall, functioned as a reproductive cell; the other, situated in the central part of the cell, served as a nutritive cell. In mature pollen grains observed from P. subsolida florets, predominantly binucleate pollen grains were found, with a small proportion consisting of trinucleate ones (Fig. 2 i). 3.2.3. Development of the anther wall The young stamens consisted of an epidermal layer and sporogenous cells when observed from a transverse perspective, while the epidermal cells maintained a consistent monolayer throughout the entire developmental process. The formation of primary parietal cells and primary sporogenous cells occurred both inwardly and outwardly during peripheral division (Fig. 3 a). The inner layer of the primary wall differentiated into the middle layer and tapetum, while the outer layer gave rise to the endothecium. Through secondary spore production, all cell layers including the epidermis, endothecium, middle layer, and tapetum underwent differentiation during anther development (Fig. 3 b). In the later stages of pollen maturation, a small aperture formed between two adjacent locules to connect the two chambers (Fig. 3 c), resulting in the formation of four longitudinal chambers for the efficient release of pollen grains. The epidermis persisted throughout development and underwent periclinal division to accommodate the expansion of internal tissues within the locule walls while providing them with protection. During the spore production phase, The epidermis assumed a rectangular shape with a discernible nucleus (Fig. 3 d). However, during microspore development, its morphology became irregular, accompanied by the near disappearance of the nucleus and the formation of a cuticle layer (Fig. 3 e). The inner wall of the locules underwent radial elongation through striplike additions on its internal surface. As the anthers matured, their fibers progressively thickened as they underwent radial elongation, leading to longitudinal splitting in this region to facilitate the release of pollen grains (Fig. 3 d). During another developmental stage, the middle layer existed transiently, with its constituent cells assuming a rectangular morphology during the secondary sporogenesis phase (Fig. 3 b). During microspore meiosis, the middle layer underwent significant reduction and eventually disappeared completely upon anther maturation. The development of the tapetum was intricately linked to microspore formation and male gametophyte development. During the microspore stage, the tapetum played a crucial role in providing essential nutrients for micro-spore development. During the secondary sporulation stage, there was a noticeable increase in density within the tapetum cytoplasm (Fig. 3 f). Afterward, the microspore initiated disintegration during the later stage and ultimately underwent complete disintegration upon pollen maturation, leaving behind only residual thin layers in their original position. Consequently, the tapetum layer of P. subsolida anthers should classified as the glandular type. 3.3. Megasporogenesis and anatomical structure of the ovary The ovary of P. subsolida was superior (Fig. 1 o), and the ovule ovulation was inverted (Fig. 4 a,b) with two layers of integument (Fig. 4 c,d). When the sporogenous cells initiated differentiation, an outer integument was formed surrounding the inner integument, while the archesporial cells continued to grow and directly developed into megasporocytes (Fig. 4 e). Subsequently, each megasporocyte underwent two rounds of meiosis, resulting in the formation of a dyad and a tetrad, respectively. With each tetrad, one functional megaspore developed adjacent to the micropyle, while the other three degenerated. The functional megaspore underwent multiple mitotic divisions until it reached maturity as an embryo sac. During embryo sac maturation, there was a central cell composed of two parallel polar nuclei positioned between the egg apparatus and antipodal cells (Fig. 4 f). This central cell served as a precursor to the endosperm and represented the largest cell within the embryo sac. 3.4. Anther abortion types of P. subsolida florets Although P. subsolida underwent flowering, its fruiting rate was extremely low, resulting in a scarcity of harvestable seeds. Sliced observations of the anthers of P. subsolida revealed a phenomenon of sterile anthers. These sterile anthers were classified into several types based on our analysis: complete absence of pollen grains throughout the locule (Fig. 5 a); shrinkage and deformation of the locules (Fig. 5 b); failure to form a tapetum layer and middle layer cells (Fig. 5 c); the presence of empty microspores lacking cell nuclei or undergoing incomplete vacuolation or contraction phase (Fig. 5 d); normal pollen grains but some without a cell nucleus and cytoplasm to provide nutrients for normal pollen grains (Fig. 5 e); and sterile pollen grains that became hollow, lost their protoplasm, and ultimately underwent shrinkage deformation, resulting in a crescentshaped contracted state (Fig. 5 f). Discussion Comparison of the morphological structure of floral organs Bamboo species exhibit significant variation in terms of the size and shape of their bamboo florets, as well as the spikelets, cobs, stamens, pistils, and bracts of the inflorescence structure. There were disparities in the morphology and length of flower branches between bamboo species. The flower branches of P. subsolida are densely arranged, with clustered spikelets distributed flatly, resembling the morphological characteristics observed in Bambusa oldhami [ 19 ] and D. sinicus [ 9 ]. The morphological features of Chimonobambusa utilis [ 20 ] and Sasaela kongosanensis [ 21 ] were different. In examining the morphological anatomy of floret organs, it was observed that a majority of bamboo plants had bisexual florets with either three or six stamens. P. subsolida , similar to Phyllostachys praecox [ 22 ], Chimonobambusa utilis [ 20 ], and Shibataea chinensis [ 12 ], possesses three stamens in its floret organ. The number of stamens in Dendrocalamus hamiltonii [ 23 ], Bambusa intermedia [ 24 ], B. multiplex [ 6 ], and D. sinicus [ 9 ] was six. P. subsolida possesses a pistil with a smooth and glabrous ovary, similar to the conspecific P. viridula [ 25 ], which exhibits densely ciliated and nondesquamated apices, in contrast to D. hamiltonii [ 23 ]. The six-stamen bamboo species included Dendrocalamus and Bambusa. The styles of P. subsolida are short, as indicated by the length of its stigma[ 26 ]. In bamboo plants, florets can be divided into long and short styles; therefore, P. subsolida was indicated rather than the congeneric P. viridula type because of the short styles. The mature lemma of the floret was slightly longer than the palea, while the length of the immature lemma did not differ significantly. The lemma exhibits a sharp tip and ciliated edges, with the palea positioned internally. These characteristics were similar to those of the conspecific P. viridula [ 25 ]. In P. subsolida , Bambusa oldhami , Bambusa multiplex , Bambusa intermedia , Bambusa rigida , Shibataea chinensis , Pseudosasa viridula , and Chimonobambusa utilis , the shape of the pistil was mostly a three-branched stigma; in Dendrocalamus hamiltonii and Dendrocalamus sinicus , there was an unbranched pistil, while the stigma of Arundinaria simonii contains two branches. Bamboo plants are distinguished by their towering stature and predominantly wind mediated dispersal. In terms of pollination mechanisms, it is more difficult for wind dispersed plants to achieve widespread distribution compared to insect pollinated counterparts. Therefore, bamboo plants exhibit sporadic flowering patterns. Flowering and subsequent seed production in bamboo are rare, with limited or nonexistent seed collection even during sporadic bamboo flowering. As for the flower type, P. subsolida has continuous bloomsplants that do not decline after flowering but instead undergo asexual reproduction to renew the bamboo forest. The low seed-setting rate can be attributed to the varying types and periods of flowering observed in bamboo plants, along with potential insect infestation (in studies of P. viridula )[ 25 ]. In the field of bamboo plant reproductive biology, McClure[ 27 ] initially proposed the concept of “pseudo” spikelets and subsequently classified bamboo plant inflorescences into two categories based on the presence or absence of dormant buds located at the base of spikelets: determinate inflorescence and indeterminate inflorescence. In 1986, Geng Bojie provided a more precise definition of bamboo inflorescence as a finite true inflorescence occurring only once and an infinite false inflorescence with successive occurrences[ 28 ]. According to Zhang Zuxin's research literature, employing gene editing technology to knock out genes in the inflorescence can significantly reduce the abortion rate of maize florets, suggesting a potential association between inflorescence and this phenomenon[ 29 ]. No existing literature has been found regarding the correlation between the structure of false spikelets and inflorescences and abortion. According to the flowering dynamics of florets in bamboo plants, upon blooming, water absorption by the pulp leads to lemma expansion. This facilitates the emergence of both male and female stamens from the lemma sheet while ensuring their simultaneous maturation. Upon completion of the powdering process, the pulp undergoes desiccation and contraction, resulting in the closure of the lemma sheet and retraction or abscission of stamens. This floral morphology corresponds to an open type. When in bloom, the lemma remains closed, with simultaneous maturation of both the pistil and stamen. This phenomenon can be classified as a closed type[ 17 ]. The most apparent distinction between the two types lies in the presence of a pulp sheet in the open type, whereas the closed type lacks such a component. In this study, P. subsolida was found to have pulp flakes and to be an open floret. In this respect, its floral features were very similar to those of Bambusa. By contrast, Dendrocalamus sinicus and D. hamiltonii did not have pulp and are of the closed type. In this study, P. subsolida was found to exhibit infrequent blooming and a significantly low seed-setting rate within its native habitat, with no naturally occurring seedlings observed. The cytokinesis mode of the pollen mother cell was classified as the continuous type. This type was completely consistent with the spore development of M. sichuanensi [ 30 ] and was the same as that of D. sinicus [ 9 ]. However, most of the microspore tetraploids produced by D. sinicus are tetrahedral. The resulting dizygomorphic tetraploid was similar to that of B. multiplex [ 6 ], although the process of formation was not the same. The cytokinesis type of B. multiplex was simultaneous, resulting in the absence of diploid formation at the end of the first division of meiosis and direct tetraploid formation during the second division. During secondary sporulation, anther wall development involves four layers of cells: the epidermis, anther chamber wall, mesosphere, and tapetum. The anther walls of B. eutuldoides [ 31 ] and B. intermedia [ 24 ] were also fully differentiated during the secondary sporulation period, which was consistent with the development of the anther wall of P. subsolida . In contrast, the anther walls of D. sinicus [ 9 ], M. sichuanensi [ 30 ], and B. multiplex [ 6 ] were fully differentiated at the microspore mother cell stage. The anther wall of P. praecox differs from that of P. subsolida in that it lacks an inner layer, which typically consists of (from outer to inner) the epidermis, middle layer, and tapetum. The majority of mature pollen grains in P. subsolida exhibited binucleate characteristics, while a minority displayed trinucleate features. The pollen of M. sichuanensi [ 30 ] and S. chinensis [ 12 ] was similar to that of P. subsolida . Reasons for the low seed-setting rate The potential causes of spontaneous abortion in P. subsolida included the presence of an in-distinct or absent demarcation between tapetal cells and intermediate cells. The primary role of the tapetum was to provide nourishment and structural components for microspores [ 32 ]. During the later stages of anther development, the tape-tum undergoes deformation and releases lipids or phenols that were essential for proper anther development [ 33 ]. During the pollen grain development of P. subsolida , similar to that in most bamboo species (e.g., B. multiplex [ 6 ], B. sinospinosa [ 34 ], S hibataea chinensis [ 35 ], there was abnormal tapetum development. In contrast to D. sinicus [ 41 ], in which the tapetum is normally developed, in D. sinicus , the tapetum begins to disgroup at the stage of microspore mother cells, but it remains in its original position and does not disappear. However, in P. subsolida , although the tapetum and midlayer persist during the microspore mother cell stage, they completely disappear during meiosis and cannot provide the necessary nutrients for normal microspore development. Phyllostachys edulis [ 36 , 37 ] has also been reported to have flowering (flowering in all seasons, all developmental stages) and normal fruit-bearing. Still, the anatomical structure of the female and male gametes in Phyllostachys edulis had not been described. The second point pertains to the presence of hollow microspores, which do not possess characteristics typical of pollen grains. In this study, anther shrinkage was observed during the development of P. subsolida florets; a similar phenomenon was observed in Neomi-crocalmus praini [ 38 ]and B. tuldoides [ 39 ]. Additionally, B. intermedia [ 40 ]and N. prainii [ 38 ]exhibited the contraction of other compartments, as observed in P. subsolida . However, it is hypothesized that S. chinensis exhibits limited and potentially negligible seed setting due to factors such as concealed stigma hindering effective pollination, self-pollination, and challenges associated with dioecious maturation. No instances of abnormal structural development induced abortions were observed in female or male gametophytes of D. sinicus [ 41 ] through paraffin continuous section analysis. It was worth noting that potential factors contributing to the low seed-setting rates in P. subsolida , similar to those observed in the aforementioned bamboo species, cannot be completely disregarded. Thus, there was a need for further research. The application uses and characteristics for future research The morphological examination and dissection of the female and male gametes in P. subsolida can fill the research gap regarding the reproductive structure of this genus and its conspecifics, while also serving as a reference for anatomical investigations into other floret structures. Simultaneously, it elucidates the reasons behind the low or even absent seed setting rate in P. subsolida , thereby explaining the physiological characteristics of bamboo plants with diminutive flowers that solely reproduce asexually. In future endeavors, advancements in bamboo plant breeding and transitioning from cuttings and transplantation to sexual reproduction can further enhance our understanding of P. subsolida 's floret sterility causes. Conclusions Based on the anatomical study of the florets of P. subsolida , the results obtained were as follows: The inflorescence of the P. subsolida floret was classified as an indeterminate inflorescence with dormant buds located at the base of the spikelet, exhibiting a pseudo-spikelet morphology. The inflorescence consists of 10–16 florets accompanied by two bracts positioned at the base of the spikelet. Each floret comprises a lemma, a palea, three lodicules, three stamens, and one pistil. The stamens were within a four-compartment longitudinal fissure, with the anther wall comprising four layers of cells: the epidermis, inner anther wall, middle layer, and tapetum. Among these layers, the tapetum was glandular in nature. The cytokinesis of the pollen mother cell was classified as continuous type. The ovary was one locule and superior, featuring a feathery three forked stigma, anatropous ovule, parietal placenta, and two integuments, with partial browning observed in certain fertile florets. The maturity of florets gradually decreases from the base to the top of the spikelets, where the uppermost floret exhibits infertility due to a young ovary and the absence of stamens. The majority of pollen grains were binuclear or trinuclear; however, abnormal development of their anthers and the occurrence of brown stamens can be observed in fertile florets. Three distinct types of spontaneous abortion can be distinguished. For example, the observed abnormalities included the absence of tapetum formation, hollow microspores, the shrinkage and deformation of the capsule, and the presence of hollow capsules. Among, anther abortion was the primary contributor to the low seed-setting rate observed in P. subsolida . This study will significantly contribute to the morphological investigation of bamboo florets, establish a solid foundation for the taxonomic classification of bamboo plants, and provide a theoretical reference for the fundamental examination of flower structure. The significance of this study lay in its pioneering exploration of the reproductive structure of P. subsolida. Furthermore, it offered a theoretical framework for future investigations into flower structures within the same genus. However, one limitation was the scarcity of studies on flower structures within this particular genus, which hinders clear comparisons and impedes obtaining more precise common features. Material and methods In 2018, 60 spikelets of P. subsolida from six P. subsolida plants in flowering stage were collected at the Shahe Forest Seed Breeding Center of National Long term Research Base, Anhui Province (31.851156,117.181445)（Figure 6abc）. The development status of materials was preliminarily judged according to the size and color of the materials. The materials were placed in a 50% FAA stationary solution (50% ethanol: 40% formaldehyde: glacial acetic acid = 18: 1: 1) and pumped using a vacuum pump for 8 hours for later use. The FAA-fixed spikelets were transported to the laboratory under low-temperature conditions, and a total of 15 spikelets exhibiting good developmental status and an average length of 6.42cm were carefully selected for subsequent anatomical experiments. The P. subsolida spikelets were taken from the FAA fixation solution and dissected under an anatomical microscope (Olympus H011). Before dissection, the spikelet, floret, and spikelet axis were photographed and their lengths measured; then, the lemma, palea, pulp, pistil, and stamen of the spikelet were dissected, photographed, and measured, and the data were statistically processed using Excel software. The anatomized stamens and pistils were dissected according to Li Zhengli’s paraffin continuous section method[42]. After dissection, the florets, anthers, and ovaries were dehydrated, immersed in paraffin, and cut into 7μm thick sections using a Leica RM2165 microtome. After that, the slices were stained twice, with 1% saffron and 1% solid green, and dehydrated in 50% xylene and 100% xylene. The slice images were selected under a Nikon-ECLIPSE 50 microscope and measured with DS-3000 two-dimensional measurement software. To ensure accuracy, each microscopic morphological feature was observed and measured three times, and a total of 60 slides were prepared to minimize the potential errors in microscopic observation. Declarations Acknowledgments Thanks to Professor Cao Zhihua from Anhui Academy of Forestry for providing experimental materials and pictures. Author Contributions: B. J. contributed to the experimental design and writing of the paper; Z. C. provided the experimental materials; S. W. supervised experimental studies; D. J., S. Z., Y. W., H. Z., L. Y. and Y. M. and J. L. designed the experiments and revised the manuscript. All the authors have read and approved the manuscript for publication. And to have agreed both to be personally accountable for the author's own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, are appropriately investigated, resolved, and the resolution documented in the literature. Funding This work was jointly supported by the Key Laboratory of Forest Resources Conservation and Utilization in the Southwest Mountains of China Ministry of Education, Southwest Forestry University, Yunnan Provincial Key Laboratory for Conservation and Utilization of In-forest Resource, Key Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Southwest China (LXXK-2023M02); the Yunnan Province Xingdian Talents Support Plan of 2022 (XDYC-QNRC-2022-0229); the Yunnan Provincial Joint Special Project for Basic Research in Agriculture (202401BD070001-110). Data Availability Statement: [REAGENTS/TOOLS/MATERIALS] generated in this study are available from the corresponding author upon request. Data sharing is not applicable to this article as all new created data is already contained within this article. Conflicts of Interest: The authors declare no conflict of interest. Availability of data and materials materials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant confidentiality. Ethics approval and consent to participate Not applicable. The authors declared that experimental research works on the plants described in this paper comply with institutional, national and international guidelines. Use of plant material has been permitted. Consent for publication : Not applicable. Conflicts of Interest: The authors declare no conflict of interest. A statement of the location of the extant specimen and the information of the depositor Appropriate permission has been obtained for spikelet material from plants collected for this study. The plant material was collected by Cao Zhihua in Shahe Forest Seed Breeding Center of National Long term Research Base, Chuzhou, Anhui 230000, China, and the bamboo species belongs to the base. Has obtained the unit permission, can be used for scientific research; The present type specimen is deposited in the Bamboo and Rattan Research Center of Southwest Forestry University, where it was identified and preserved by Professor Wang Shuguang.This study covered plant species and did not address ethical issues. References Zhang QiSheng, Z.Q. Thinking on development of the bamboo industry in China at present. 2000 , 19 ,16-19. Long, L.; Minghui, Y.; Wenjing, Y.; Yulong, D.; Shuyan, L. Research advance in growth and development of bamboo organs. Industrial Crops and Products 2023 , 205 , 117428. Ramanayake, S.M.S.D.; Weerawardene, T.E. Flowering in a bamboo, Melocanna baccifera (Bambusoideae: Poaceae). Botanical Journal of the Linnean Society 2015 , 143 , 287-291. Zhen-Lin, C.; Yu-Chuan, Q.; Xi-Qi, H.; Zu-Jun, W.; Qi, W. Advance of Studies on Bamboo Flowering Causes. Journal of Zhejiang Forestry ence & Technology 2006 , 26 ,53-57. Yifa, X. Present Situation and Outlook of Bamboo Resources Utilization in China. Chinese Journal of Tropical Agriculture 2004 , 24 ,46-52. Shuyan, L.; Jie, L.I.; Rong, Z.; Xiaobo, D.; Yulong, D. The development of flowering bud differentiation and male gametophyte of Bambusa multiplex. Journal of Nanjing Forestry University(Natural Sciences Edition) 2015 , 39 ,51-56. Yang, N.; CUI, Y.; Wang, Q.; WANG, S. A study on the morphology and anatomical structure of Bambusa rutila spiklets. JOURNAL OF NANJING FORESTRY UNIVERSITY 2021 , 45 , 90. Tang, G.; Yang, J.; Ding, Y.; Zhan, H.; Zhao, J.; Wang, Y.; Wang, S. Studies on the flower morphology and structure in Bambusa eutuldoides McClure var. viridi-vittata (WT Lin) Chia. JOURNAL OF NANJING FORESTRY UNIVERSITY 2016 , 59 , 71. Shu-Guang, W. The structures of reproductive organs and development of the female and male gametophyte of Dendrocalamus sinicus. Bulletin of Botanical Research 2006 , 03 ,270-274. Huang, L.; Deng, L.; Chu, C.-h.; Zhan, H.; Wang, S.-g. Morphological and anatomical observations of floral organs and sterility analysis of Fargesia yuanjiangensis. 2020 , 56 ,64-73. Caihua, C.; Ling, H.; Shuguang, W. Floral Morphology and Development of Female and Male Gametophytes of Neomicrocalamus prainii. Acta Botanica Boreali-Occidentalia Sinica 2019 , 39 ,763-769. Lin, S. Studies on the Reproductive Biology of Shibataea chinensis and Arundinaria simonii f. albostriatus. Nanjing Forestry University Nanjing, China, 2009. Lin, D.; Ling, H.; Cai-Hua, C.; Qian, W.; Hui, Z.; Shuguang, W. Study on the Flower Morphology and Structure of Fargesia fungosa. Bulletin of Botanical Research 2019 , 39 , 801. Wu, Z.; Raven, P.H.; Hong, D. Flora of China. Volume 9: Pittosporaceae through Connaraceae ; Science Press: 2003. Chen, G.C.; Ma, N.X. Advances in Studies on Genetics and Breeding of Bamboos. Forest Research 2005 , 18 , 749-754. Shi, J.; Zhou, D.; Zhang, Y.; Ma, L.; Yang, L. Illustrated Flora of Bambusoideae in China ; Illustrated Flora of Bambusoideae in China: 2020. Shuyan, L.; Wenwen, S.; Binbin, M.; Yulong, D. Research Advances in Reproduction Biology of Bamboos. World Bamboo and Rattan 2010 , 8 , 1-6. Board, E. Flora of China Vol.2-25. 2010 . Shuyan, L.; Huajun, F.U.; Yawen, W.; Shixing, Z.; Ruji, Z.; Fusheng, W.; Yulong, D. Anther development and floral morphology characteristics of Bambusa oldhami 'Xia Zao' ZSX. Journal of Nanjing Forestry University(Natural Sciences Edition) 2019 , 43 ,7-13. Yang, M.; Zhang, Y.; Ding, Y.-l.; Yao, W.-j.; Lin, S.-y. Flowering characteristics and floral organ development characteristics of Chimonobambusa utilis. 2022 , 50 ,7-13. Yao, W.-J.; Jiang, M.-Y.; Wang, X.; Shi, W.-S.; Ding, Y.-L.; Lin, S.-Y. Biological analysis of flowering and pollen germination in Sasaella kongosanensis,'Aureostriatus'. 2020 , 03 ,003. Jianqin, H.; Zhongju, L.; Changhong, P.; Shiqiang, L. The Formation of Microspore and the Development of Male Gametophyte of Phyllostachys praecox. JOURNAL OF BAMBOO RESEARCH 1999 , 03 ,55-58. Yang, D.; Li, J.; Wang, S.; Yu, L.; Zhan, H.; Ma, Y. Abnormalities in Stamen and Ovary Development Responsible for Low Seed Set of Dendrocalamus hamiltonii Nees et Arn. ex Munro. Forests 2023 , 14 , 2282. Yu-Jun, W.; Jian, L.; Nan-Nan, C.; Shu-Yan, L.; Yu-Long, D.; Shu-Guang, W. Floral Morphology and Development of Female and Male Gametophyte of Bambusa intermedia Hsueh et Yi. Bulletin of Botanical Research 2017 , 37 ,492-498. Wan-qi, Z.; Zheng-chun, W.; Jiao, X.; Chun-ce, G.; Guang-yao, Y.; Fen, Y. Flowering biological characteristics of Pseudosasa viridula. Forest Research 2020 , 33 , 31-38. Liu, W.; Hui, C.; Wang, F.; Wang, M.; Liu, G. Review of the Resources and Utilization of Bamboo in China. Bamboo-current and future prospects 2018 , 133-142. Meclure, F.A. Reproductive Phase of Bamboos. Journal of Bamboo Research 1983 , 02 ,119-136. Paichieh, K. A PRELIMINARY STUDY OF THE INFLORESCENCE TYPE ARISING FROM BAMBOOS AND ITS VARIATION. Journal of Wuhan Botanical Research 1986 , 04 ,323-336. Ning, Q.; Jian, Y.; Du, Y.; Li, Y.; Zhang, Z. An ethylene biosynthesis enzyme controls quantitative variation in maize ear length and kernel yield. Nature Communications 2021 , 12 . Shu-Yan, L.; Juan-Juan, H.; Hua, X.; Yu-Long, D. The megasporogenesis,microsporogenesis and the development of their femaleand male gametophyte in Menstruocalamus sichuanensis. Journal of Nanjing Forestry University(Natural Sciences Edition) 2009 , 33 ,9-12. Guojian, T.; Jinmei, Y.; Yulong, D.; Hui, Z.; Jingwei, Z.; Yujun, W.; Shuguang, W. Studies on the flower morphology and structure in Bambusa eutuldoides McClure var. viridi-vittata(W.T.Lin) Chia. Journal of Nanjing Forestry University(Natural Sciences Edition) 2016, 40 ,71-75. Zhang Ying-tao, Y.H.-d.a.C.Z. Advances on the Study of Tapetum. Chinese Bulletin of Botany 1996 , 13(04): 6-13 . Zhou, J.; Zhang, S.; Hui, D.; Vancov, T.; Fang, Y.; Tang, C.; Jiang, Z.; Ge, T.; Cai, Y.; Yu, B. Pyrogenic organic matter decreases while fresh organic matter increases soil heterotrophic respiration through modifying microbial activity in a subtropical forest. Biology and Fertility of Soils 2024 , 60 , 509-524. Wang, S.G.; Pu, X.L.; Lin, S.Y.; Ding, Y.L. Reproductive characteristics of three bamboo species. 2015 ,47,2301-2308. Shu-Yan, L.; Yu-Long, D. Development of the Male and Female Gametophytes in Shibataea chinensis(Bambusoideae). Acta Botanica Boreali-Occidentalia Sinica 2012 , 32 ,907-914. Feiyan, Q.J., G; Yuan wen, C; Zhongyuan, S. A study on Biological Characteristics in Blooming Period of Moso Bamboo. In Proceedings of the The 7th China Bamboo Industry Academic Conference, 2011. Guo, Q.; Zhou, J.; Sun, L.; Lian, C.; Feng, Y.; Ran, H.; Zhang, Y. Development of Phyllostachys edulis inflorescences. Plant Science Journal 2015 , 33 , 19-24. Chu CaiHua, C.C.; Huang Ling, H.L.; Wang ShuGuang, W.S. Floral morphology and development of female and male gametophytes of Neomicrocalamus prainii. 2019 ,763-769. Long, H.; Chu, C.-h.; Jin, D.-k.; Lv, Z.; Wang, S.-g. Anatomical observation and analysis on floral of Bambusa tuldoides. 2022 , 174-182. Yu-Jun, W.; Jian, L.; Nan-Nan, C.; Shu-Yan, L.; Yu-Long, D.; Shu-Guang, W. Floral morphology and development of female and male gametophyte of Bambusa intermedia Hsueh et Yi. Bulletin of Botanical Research 2017 , 37 , 492. Shu-Guang, W.; Xiao-Lan, P.; Yu-Long, D. The structures of reproductive organs and development of the female and male gametophyte of Dendrocalamus sinicus. Bulletin of Botanical Research 2006 , 26 , 270. Zheng LI, L. Plant tissue production. Peking University Press 1996 , 15-50. Additional Declarations No competing interests reported. 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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-4600448\",\"acceptedTermsAndConditions\":true,\"allowDirectSubmit\":true,\"archivedVersions\":[],\"articleType\":\"Research Article\",\"associatedPublications\":[],\"authors\":[{\"id\":326885533,\"identity\":\"6d26b4aa-11ad-474b-aeef-da2dfc079530\",\"order_by\":0,\"name\":\"Bonan Jiang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Bonan\",\"middleName\":\"\",\"lastName\":\"Jiang\",\"suffix\":\"\"},{\"id\":326885538,\"identity\":\"481ba34c-41e2-4656-a5f9-d4af2370177b\",\"order_by\":1,\"name\":\"Zhihua Cao\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Shahe Forest Seed Breeding Center of National Long term Research Base\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Zhihua\",\"middleName\":\"\",\"lastName\":\"Cao\",\"suffix\":\"\"},{\"id\":326885539,\"identity\":\"5519abe9-9560-4217-ba36-1adacfd91145\",\"order_by\":2,\"name\":\"Dejia Yang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Dejia\",\"middleName\":\"\",\"lastName\":\"Yang\",\"suffix\":\"\"},{\"id\":326885542,\"identity\":\"ea0c2c0d-0c3c-40ee-adc4-d9a43bd10c08\",\"order_by\":3,\"name\":\"Yongmei Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yongmei\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":326885544,\"identity\":\"565916c5-3a6b-4685-8510-215f2be5a8a4\",\"order_by\":4,\"name\":\"Yingchun Ma\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Yingchun\",\"middleName\":\"\",\"lastName\":\"Ma\",\"suffix\":\"\"},{\"id\":326885545,\"identity\":\"433a8a0b-be02-4c50-8857-74cfaaab20dc\",\"order_by\":5,\"name\":\"Shiqi Zhang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shiqi\",\"middleName\":\"\",\"lastName\":\"Zhang\",\"suffix\":\"\"},{\"id\":326885546,\"identity\":\"12b0ee6b-dede-4aaf-bc74-b8e42c457ecd\",\"order_by\":6,\"name\":\"Hui Zhan\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Hui\",\"middleName\":\"\",\"lastName\":\"Zhan\",\"suffix\":\"\"},{\"id\":326885547,\"identity\":\"b9afceb6-de00-487a-921e-cd76643044fa\",\"order_by\":7,\"name\":\"Lixia Yu\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Lixia\",\"middleName\":\"\",\"lastName\":\"Yu\",\"suffix\":\"\"},{\"id\":326885548,\"identity\":\"e9308367-fe58-46d8-afa5-0a5d726e7161\",\"order_by\":8,\"name\":\"Shuguang Wang\",\"email\":\"\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":false,\"prefix\":\"\",\"firstName\":\"Shuguang\",\"middleName\":\"\",\"lastName\":\"Wang\",\"suffix\":\"\"},{\"id\":326885549,\"identity\":\"2573486b-c331-445e-ae80-21f121141b78\",\"order_by\":9,\"name\":\"Juan Li\",\"email\":\"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAyElEQVRIiWNgGAWjYBACPmaGBCBlw8DAfABIsxGhhQ2iJQ3ISiBWC4Q6TIoWdoZnEj93nLc3OMZjwPCh7DAD/+wGgg5Lk+w9cztxA1AL44xzhxkk7hwgrEWCt+12gtn9HgNm3rbDDAYSCUTY8rftnL0Z0Bbmv8RqkeZtO8C4DaSFkUgtydaybcmJ+4+xFRzsOZfOI3GDgBZ+/jOJN9+22dlLtjFvfPCjzFqOfwYBLQwMPAgVB0BcQuqBgP0AEYpGwSgYBaNgRAMAnnw60ZhfeKIAAAAASUVORK5CYII=\",\"orcid\":\"\",\"institution\":\"Southwest Forestry University\",\"correspondingAuthor\":true,\"prefix\":\"\",\"firstName\":\"Juan\",\"middleName\":\"\",\"lastName\":\"Li\",\"suffix\":\"\"}],\"badges\":[],\"createdAt\":\"2024-06-18 13:47:27\",\"currentVersionCode\":1,\"declarations\":\"\",\"doi\":\"10.21203/rs.3.rs-4600448/v1\",\"doiUrl\":\"https://doi.org/10.21203/rs.3.rs-4600448/v1\",\"draftVersion\":[],\"editorialEvents\":[],\"editorialNote\":\"\",\"failedWorkflow\":false,\"files\":[{\"id\":60443139,\"identity\":\"3578eada-da91-4b99-bfc6-80e36d471d60\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:16:11\",\"extension\":\"jpg\",\"order_by\":1,\"title\":\"Figure 1\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":119543,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe morphological anatomy of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. (a) LB: Latent bud. (b) BR: Bracts. (c) RA: Rachilla. (d) Whole floret. L: Lemma. P: Palea. NS: Normal stamens. BS: Brown stamens. YO: Young ovary. (e) Lemma, CR: coarsest ridge. (f) AS: apex sharp. (g) SC: surface ciliated. (h) Lemma was longer than the palea. (i) Lemma was equal in length to the palea. (j) Palea was slightly longer than the lemma. PA: palea. LE: lemma. (k) Palea had two ridges, ciliated; CR: coarsest ridge. (l) lodicules. (m) Normal stamens. (n) Brown stamens. (o) Young ovary, FA: feathery appendages. (p) MO: Mature ovary.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"1.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/4a1621c14d0689f837d752d8.jpg\"},{\"id\":60442639,\"identity\":\"c4b3b18e-c68a-464e-874b-054522f91ad6\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:08:11\",\"extension\":\"jpg\",\"order_by\":2,\"title\":\"Figure 2\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":272587,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eThe anatomical structure of the anther of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. (a) PSC: Primary sporogenous cells. (b) SSC: Secondary sporogenous cells; (c) MMC: Microspore mother cell; (d) Dichotomous period, DY: dyad; (e) TE: Tetrad period; (f) MPG: Mature pollen grains, GH: germination holes; (g) Large vacuoles in the center of cells; VA: vacuoles; (h) BPG: Binucleate pollen grains; (i) TPG: Tri-nuclear pollen grains. Note: The scale in the figure is 50μm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"2.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/1724460f319f1bb1a2722f4f.jpg\"},{\"id\":60442643,\"identity\":\"49575148-6a1d-41a5-8c83-268aa86fa083\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:08:11\",\"extension\":\"jpg\",\"order_by\":3,\"title\":\"Figure 3\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":182688,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnther wall development and abortive anthers of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. (a) Primary wall and primary sporulation cells. (b) The septum of adjacent drug chambers disappeared, and the drug chambers communicated. (c) The secondary sporulation stage had four layers of walls; the epidermis cells were shown in the sporulation stage. (d) Microspore stage. (e) Tapetum degradation and the disappearance of the middle layer. (f) Secondary sporulation period. PW: Primary wall. PSC: primary sporulation cells; DC: drug chambers; EN: endothecium; EP: epidermis; MI: middle layer; SSC: secondary sporogenous cell; TL: Thin layer, FL: Fiber layer.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"3.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/bb0ba1b57e87064efc0d1f44.jpg\"},{\"id\":60442641,\"identity\":\"ce6f934c-8d84-4f0f-8fa5-65c45f5186c1\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:08:11\",\"extension\":\"jpg\",\"order_by\":4,\"title\":\"Figure 4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":171905,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eMegaspore and female gametophyte of \\u003cem\\u003eP. subsolida. \\u003c/em\\u003e(a,b) Anatropous ovule; (c,d) double-layer integument; (e) Megasporocyte; (f) Central cell. PN: Polar nucleus.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/d011cc4a62cc673988092e1d.jpg\"},{\"id\":60443140,\"identity\":\"1194a0ff-c750-45fc-8e6d-0cbd963a9a46\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:16:11\",\"extension\":\"jpg\",\"order_by\":5,\"title\":\"Figure 5\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":145826,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAnther abortion type of \\u003cem\\u003eP. subsolida. \\u003c/em\\u003e(a) The capsule was hollow and no pollen grains formed; (b) Shrinkage deformation of pollen grains; (c) The tapetum and middle layer cells degenerated and no microspore formed; (d) Some microspores had no nucleus; (e) Pollen grains had no nucleus or cytoplasm; (f) Contracted and abortive pollen grains.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"5.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/e4fa427dea865467e6c27ced.jpg\"},{\"id\":60442638,\"identity\":\"45f0cb1e-7c56-4726-9414-34ef4d9b5197\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:08:11\",\"extension\":\"jpg\",\"order_by\":6,\"title\":\"Figure 6\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":73022,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eFloater morphology of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e in the wild. (a, b, c) Spikelet morphology.\\u003c/p\\u003e\\n\\u003cp\\u003eNote: The scale bars are uniformly 1 cm.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"6.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/2cd64e53a73fccf57790212e.jpg\"},{\"id\":66158867,\"identity\":\"4f2345db-8c05-4be1-b17c-8388f32bd884\",\"added_by\":\"auto\",\"created_at\":\"2024-10-08 09:02:30\",\"extension\":\"pdf\",\"order_by\":0,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"manuscript-pdf\",\"size\":1603804,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"manuscript.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/94e868da-4816-4364-9ce8-d40a520d5100.pdf\"},{\"id\":60442644,\"identity\":\"9ab30856-bd77-4a0d-8eca-e8f664a75713\",\"added_by\":\"auto\",\"created_at\":\"2024-07-16 20:08:12\",\"extension\":\"rar\",\"order_by\":8,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":79697476,\"visible\":true,\"origin\":\"\",\"legend\":\"\",\"description\":\"\",\"filename\":\"picture.rar\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-4600448/v1/0afc0c29f834224ca8959bd1.rar\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"\\u003cp\\u003e\\u003cstrong\\u003eStudies on floral organ Structure and female and male gametophyte development of Pseudosasa subsolida S. L. Chen \\u0026amp; G. Y. Sheng\\u003c/strong\\u003e\\u003c/p\\u003e\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eBamboo, belonging to the Bambusoideae subfamily of the Gramineae family, represents one of the most significant forest resources, along with timber[\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. China possesses abundant bamboo resources, encompassing approximately 43 genera and 800 species. Bamboo forests cover approximately one-third of its overall land area[\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. The flowering cycle of bamboo plants is typically prolonged, shortcycle bamboo plants take 10\\u0026ndash;15 years to flower, such as \\u003cem\\u003eMelocanna baccifera\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e], the Shimen Moso Bamboo Forest in Fenghua, Zhejiang exhibits an exceptionally prolonged flowering period, with no documented instances of flowering occurring for a span exceeding 200 year[\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e]. The majority of bamboo species exhibit a low seed-setting rate or even the complete absence of fruiting[\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. Collecting flower and seed materials from fully developed bamboo plants therefore poses challenges that hinder research into their embryology. Currently, only a limited number of studies have provided detailed descriptions of the flower morphology and structure, as well as of megaspore occurrence and male/female gametophyte formation in bamboo species including \\u003cem\\u003eBambusa multiplex\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], \\u003cem\\u003eBambusa rigida\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e], \\u003cem\\u003eBambusa eutuldoides\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e], \\u003cem\\u003eDendrocalamus sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], \\u003cem\\u003eFargesia yuanjiangensis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e], \\u003cem\\u003eNeomicrocalamus prainii\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e], \\u003cem\\u003eShibataea chinensis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], \\u003cem\\u003eTongpeia fungosa\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR13\\\" class=\\\"CitationRef\\\"\\u003e13\\u003c/span\\u003e], etc.\\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\u003cp\\u003e \\u003cem\\u003ePseudosasa subsolida\\u003c/em\\u003e belongs to the Poaceae \\u003cem\\u003ePseudosasa\\u003c/em\\u003e and is characterized by its shrublike or small treelike growth and its rapid development. Additionally, it possesses significant ornamental value. The occurrence of this species is restricted to the Daliyu Mountain in Yiyang County, Hunan, China (112.327023,28.590358). Being a natural wild species, it thrives exclusively in hilly terrains and yellow soil[\\u003cspan citationid=\\\"CR14\\\" class=\\\"CitationRef\\\"\\u003e14\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. About the morphological characteristics of its adult bamboo, slight grooves can be observed at the base of branching internodes, where the rod wall is thick and nearly solid, with a medullary spongy structure. These characteristics contribute to exceptional firmness, resilience, and durability. \\u003cem\\u003eP. subsolida\\u003c/em\\u003e is the preferred material for furniture and carving handicrafts[\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eBamboo species infrequently undergo flowering, and their floral structure generally aligns with that of other plants in the Gramineae family. However, certain bamboo flowers exhibit a regression into scales, while other components regress into membranes, resulting in relatively diminutive sizes. The bamboo flowers were distinguished by their absence of vibrant hues and fragrant aromas, as well as their unique shapes. Angio-sperms utilize flowers as the fundamental structural unit for the formation of inflorescences, whereas bamboo inflorescences exhibit a higher level of complexity, being composed of spikelets. Thus, spikelets are the constituents of bamboo inflorescences[\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eCurrently, the research on \\u003cem\\u003eP. subsolida\\u003c/em\\u003e primarily focuses on species classification and morphological description, leaving significant gaps in other areas of study. There is a lack of records regarding flowering and other aspects of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e research. Examining its microstructure using the paraffin section method is even more limited. Because of the characteristics of bamboo plants, such as difficulty in flowering, and only flowering but no fruity, there are great obstacles in the breeding research of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. For its special physiological characteristics, the reasons for the abortion of the floreus of its species should be first revealed. Then further solutions should be found, to provide effective help for breeding. It is known from the Flora of China[\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e] that \\u003cem\\u003eP. subsolida\\u003c/em\\u003e has been evaluated as a vulnerable species, and the change in its ecological environment has led to the continuous decline of its species population. Therefore, studying the floret, the reproductive organ of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, to find out the reason for its low seed-setting rate, will be hopeful to change the physiological characteristics of asexual reproduction in further research. To save endangered species. The morphological structure of the floral organs of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e is described in this study, aiming to complement the known classification characteristics of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. Additionally, the development of male and female gametophytes, as well as the changes in the anther wall structure, is investigated in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e flowers to further elucidate the factors contributing to its low seed-setting rate. This study provides original embryological data for future research on \\u003cem\\u003eP. subsolida\\u003c/em\\u003e and establishes a foundation for breeding efforts.\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.1. Flower morphology and anatomy of P. subsolida\\u003c/h2\\u003e\\n \\u003cp\\u003eThe spikelet base of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e exhibited latent buds (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ea), also known as pseudo spikelets. The inflorescence of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e consists of pseudo-spikelet growth on various levels of vegetative branches, called indefinite inflorescences.Anatomical observations of the flowers of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e revealed that two bracts were present in close proximity to the floret at the base of the spikelet (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eb). The average length of twenty randomly selected spikelets was 6.42 cm, with each spikelet containing 10\\u0026ndash;16 florets. The uppermost floret among these exhibited sterility, characterized by a young ovary devoid of stamens. Subsequently, a total of 50 florets were randomly selected, and their average length was determined to be 1.36 cm. Adjacent florets within the same spikelet were interconnected through the rachilla (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ec), exhibiting alternate arrangement along the spikelet axis with cilia present. Furthermore, the mean length of 50 spikelet axes was measured to be 0.47 cm.\\u003c/p\\u003e\\n \\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\n \\u003cp\\u003eThe anatomical examination of a complete floret revealed that it comprised (from outside to inside) one lemma, one palea, three lodicules, three stamens, and one pistil (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ed). The average length of the lemma was 1.35 cm, and it had 9\\u0026ndash;12 longitudinal veins, with the thickest and longest vein positioned at the center (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ee). Additionally, cilia were observed on the surface, with long cilia present at the edge of the apex, which featured a sharp tip (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ef,g). The palea was found to be paired with the internal growth of the lemma. the length of the lemma in the mature florets of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e was slightly greater than that of the palea (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eh), whereas in immature florets, there was minimal difference in length between them (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ei), except for a few instances where the palea extended beyond the lemma (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ej). The palea exhibited two prominent ridges accompanied by four veins positioned between the ridges and three to four veins on each side. Notably, the surface of the palea was adorned with cilia, which were particularly elongated at the apex edge (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ek). Three bracts (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003el) surrounded the ovary; two were located near the lemma and one was covered by the palea. These bracts were thin and possessed white transparent membranous structures. During flowering, the bracts absorbed water and expanded, causing the lemma to spread open. The floret comprised three stamens (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003em). The stamens were typically light yellow but showed browning in some instances (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003en). The surface of the ovary exhibited a smooth and glabrous texture, appearing white in its juvenile stage (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eo). Subsequently, as it reached maturity, the ovary underwent a gradual browning process (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003ep). The style was characterized by its short length, while the stigma displayed a trilobed structure with feathery appendages.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec4\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.2 Microsporogenesis and male gamete development of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e\\u003c/h2\\u003e\\u003cspan\\u003e\\n \\u003cp\\u003e3.2.1 Microsporogenesis\\u003c/p\\u003e\\n \\u003c/span\\u003e\\n \\u003cdiv class=\\\"BlockQuote\\\"\\u003e\\n \\u003cp\\u003e\\u003cem\\u003eP. subsolida\\u003c/em\\u003e possesses three stamens, each consisting of four chambers. During the early stages of anther development, the outer epidermis comprises a layer of flattened cells, while inside the epidermis lies a cluster of cells with similar morphological structures. Notably, the cells located at the four corners divide more rapidly than other cells, leading to the gradual differentiation of sporogenous cells beneath each corner\\u0026rsquo;s epidermis. Subsequently, these sporogenous cells initiated periclinal divisions, dividing outward to form the primary parietal layer and inward to generate primary sporogenous cells (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ea). The primary sporogenous cells continue dividing and differentiate into secondary sporogenous cells (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eb), which subsequently differentiate into larger microspore mother cells devoid of distinct vacuoles (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ec). These microspore mother cells undergo subsequent developmental stages involving meiosis. At the conclusion of the first meiotic division, a dyad was formed by the microspore mother cell (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ed), and a symmetrical tetrad was subsequently generated after meiosis II (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ee). Consequently, the cytoplasmic division mode exhibited by the microspore mother cell adheres to a continuous type.\\u003c/p\\u003e\\n \\u003c/div\\u003e\\n \\u003cp\\u003e3.2.2 Development of male gametes\\u003c/p\\u003e\\n \\u003cp\\u003eThe anthers progressed through the tetrad stage before transitioning into the microspore stage. Upon the dissolution of callose, the four daughter cells within the tetrad underwent separation and gave rise to autonomous mononuclear pollen grains, known as microspores. Immediately following release from the tetrad stage, the microspore exhibited a dense cytoplasm, with the nucleus occupying the central region of the cell, while vacuole formation had not yet occurred. This developmental phase was referred to as the systolic or early microspore phase (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef). As the volume of the microspores increased, vacuoles appeared in the cytoplasm, and the nucleus began to migrate towards one side of the cell. Simultaneously, gradual cell wall formation took place until complete nuclear relocation and extensive vacuolation in the middle region of the cell were achieved. The appearance of the germination hole (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ef) was referred to as the mononuclear margination stage (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eg). Subsequently, gradual cytoplasmic filling occurred throughout the entire cell. Concurrently, the nuclear division took place through mitosis, resulting in the formation of a binucleate pollen grain where one nucleus was divided into two nuclei (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003eh). The division resulted in two distinct cells: one, located adjacent to the pollen wall, functioned as a reproductive cell; the other, situated in the central part of the cell, served as a nutritive cell. In mature pollen grains observed from \\u003cem\\u003eP. subsolida\\u003c/em\\u003e florets, predominantly binucleate pollen grains were found, with a small proportion consisting of trinucleate ones (Fig.\\u0026nbsp;\\u003cspan class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003ei).\\u003c/p\\u003e\\n \\u003cp\\u003e3.2.3. Development of the anther wall\\u003c/p\\u003e\\n \\u003cp\\u003eThe young stamens consisted of an epidermal layer and sporogenous cells when observed from a transverse perspective, while the epidermal cells maintained a consistent monolayer throughout the entire developmental process. The formation of primary parietal cells and primary sporogenous cells occurred both inwardly and outwardly during peripheral division (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ea). The inner layer of the primary wall differentiated into the middle layer and tapetum, while the outer layer gave rise to the endothecium. Through secondary spore production, all cell layers including the epidermis, endothecium, middle layer, and tapetum underwent differentiation during anther development (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb). In the later stages of pollen maturation, a small aperture formed between two adjacent locules to connect the two chambers (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ec), resulting in the formation of four longitudinal chambers for the efficient release of pollen grains. The epidermis persisted throughout development and underwent periclinal division to accommodate the expansion of internal tissues within the locule walls while providing them with protection. During the spore production phase, The epidermis assumed a rectangular shape with a discernible nucleus (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed). However, during microspore development, its morphology became irregular, accompanied by the near disappearance of the nucleus and the formation of a cuticle layer (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ee). The inner wall of the locules underwent radial elongation through striplike additions on its internal surface. As the anthers matured, their fibers progressively thickened as they underwent radial elongation, leading to longitudinal splitting in this region to facilitate the release of pollen grains (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ed).\\u003c/p\\u003e\\n \\u003cp\\u003eDuring another developmental stage, the middle layer existed transiently, with its constituent cells assuming a rectangular morphology during the secondary sporogenesis phase (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eb). During microspore meiosis, the middle layer underwent significant reduction and eventually disappeared completely upon anther maturation. The development of the tapetum was intricately linked to microspore formation and male gametophyte development. During the microspore stage, the tapetum played a crucial role in providing essential nutrients for micro-spore development. During the secondary sporulation stage, there was a noticeable increase in density within the tapetum cytoplasm (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003ef). Afterward, the microspore initiated disintegration during the later stage and ultimately underwent complete disintegration upon pollen maturation, leaving behind only residual thin layers in their original position. Consequently, the tapetum layer of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e anthers should classified as the glandular type.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec5\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.3. Megasporogenesis and anatomical structure of the ovary\\u003c/h2\\u003e\\n \\u003cp\\u003eThe ovary of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e was superior (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003eo), and the ovule ovulation was inverted (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ea,b) with two layers of integument (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ec,d). When the sporogenous cells initiated differentiation, an outer integument was formed surrounding the inner integument, while the archesporial cells continued to grow and directly developed into megasporocytes (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ee). Subsequently, each megasporocyte underwent two rounds of meiosis, resulting in the formation of a dyad and a tetrad, respectively. With each tetrad, one functional megaspore developed adjacent to the micropyle, while the other three degenerated. The functional megaspore underwent multiple mitotic divisions until it reached maturity as an embryo sac. During embryo sac maturation, there was a central cell composed of two parallel polar nuclei positioned between the egg apparatus and antipodal cells (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003ef). This central cell served as a precursor to the endosperm and represented the largest cell within the embryo sac.\\u003c/p\\u003e\\n\\u003c/div\\u003e\\n\\u003cdiv id=\\\"Sec6\\\" class=\\\"Section2\\\"\\u003e\\n \\u003ch2\\u003e3.4. Anther abortion types of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e florets\\u003c/h2\\u003e\\n \\u003cp\\u003eAlthough \\u003cem\\u003eP. subsolida\\u003c/em\\u003e underwent flowering, its fruiting rate was extremely low, resulting in a scarcity of harvestable seeds. Sliced observations of the anthers of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e revealed a phenomenon of sterile anthers. These sterile anthers were classified into several types based on our analysis: complete absence of pollen grains throughout the locule (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ea); shrinkage and deformation of the locules (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003eb); failure to form a tapetum layer and middle layer cells (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ec); the presence of empty microspores lacking cell nuclei or undergoing incomplete vacuolation or contraction phase (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ed); normal pollen grains but some without a cell nucleus and cytoplasm to provide nutrients for normal pollen grains (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ee); and sterile pollen grains that became hollow, lost their protoplasm, and ultimately underwent shrinkage deformation, resulting in a crescentshaped contracted state (Fig. \\u003cspan class=\\\"InternalRef\\\"\\u003e5\\u003c/span\\u003ef).\\u003c/p\\u003e\\n\\u003c/div\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cp\\u003eComparison of the morphological structure of floral organs\\u003c/p\\u003e \\u003cp\\u003eBamboo species exhibit significant variation in terms of the size and shape of their bamboo florets, as well as the spikelets, cobs, stamens, pistils, and bracts of the inflorescence structure. There were disparities in the morphology and length of flower branches between bamboo species. The flower branches of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e are densely arranged, with clustered spikelets distributed flatly, resembling the morphological characteristics observed in \\u003cem\\u003eBambusa oldhami\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e] and \\u003cem\\u003eD. sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. The morphological features of \\u003cem\\u003eChimonobambusa utilis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e] and \\u003cem\\u003eSasaela kongosanensis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e] were different. In examining the morphological anatomy of floret organs, it was observed that a majority of bamboo plants had bisexual florets with either three or six stamens. \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, similar to \\u003cem\\u003ePhyllostachys praecox\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e], \\u003cem\\u003eChimonobambusa utilis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], and \\u003cem\\u003eShibataea chinensis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e], possesses three stamens in its floret organ. The number of stamens in \\u003cem\\u003eDendrocalamus hamiltonii\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e], \\u003cem\\u003eBambusa intermedia\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e], \\u003cem\\u003eB. multiplex\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], and \\u003cem\\u003eD. sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e] was six. \\u003cem\\u003eP. subsolida\\u003c/em\\u003e possesses a pistil with a smooth and glabrous ovary, similar to the conspecific \\u003cem\\u003eP. viridula\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e], which exhibits densely ciliated and nondesquamated apices, in contrast to \\u003cem\\u003eD. hamiltonii\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e]. The six-stamen bamboo species included \\u003cem\\u003eDendrocalamus\\u003c/em\\u003e and \\u003cem\\u003eBambusa.\\u003c/em\\u003e The styles of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e are short, as indicated by the length of its stigma[\\u003cspan citationid=\\\"CR26\\\" class=\\\"CitationRef\\\"\\u003e26\\u003c/span\\u003e]. In bamboo plants, florets can be divided into long and short styles; therefore, \\u003cem\\u003eP. subsolida\\u003c/em\\u003e was indicated rather than the congeneric \\u003cem\\u003eP. viridula\\u003c/em\\u003e type because of the short styles. The mature lemma of the floret was slightly longer than the palea, while the length of the immature lemma did not differ significantly. The lemma exhibits a sharp tip and ciliated edges, with the palea positioned internally. These characteristics were similar to those of the conspecific \\u003cem\\u003eP. viridula\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e]. In \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, \\u003cem\\u003eBambusa oldhami\\u003c/em\\u003e, \\u003cem\\u003eBambusa multiplex\\u003c/em\\u003e, \\u003cem\\u003eBambusa intermedia\\u003c/em\\u003e, \\u003cem\\u003eBambusa rigida\\u003c/em\\u003e, \\u003cem\\u003eShibataea chinensis\\u003c/em\\u003e, \\u003cem\\u003ePseudosasa viridula\\u003c/em\\u003e, and \\u003cem\\u003eChimonobambusa utilis\\u003c/em\\u003e, the shape of the pistil was mostly a three-branched stigma; in \\u003cem\\u003eDendrocalamus hamiltonii\\u003c/em\\u003e and \\u003cem\\u003eDendrocalamus sinicus\\u003c/em\\u003e, there was an unbranched pistil, while the stigma of \\u003cem\\u003eArundinaria simonii\\u003c/em\\u003e contains two branches.\\u003c/p\\u003e \\u003cp\\u003eBamboo plants are distinguished by their towering stature and predominantly wind mediated dispersal. In terms of pollination mechanisms, it is more difficult for wind dispersed plants to achieve widespread distribution compared to insect pollinated counterparts. Therefore, bamboo plants exhibit sporadic flowering patterns. Flowering and subsequent seed production in bamboo are rare, with limited or nonexistent seed collection even during sporadic bamboo flowering. As for the flower type, \\u003cem\\u003eP. subsolida\\u003c/em\\u003e has continuous bloomsplants that do not decline after flowering but instead undergo asexual reproduction to renew the bamboo forest. The low seed-setting rate can be attributed to the varying types and periods of flowering observed in bamboo plants, along with potential insect infestation (in studies of \\u003cem\\u003eP. viridula\\u003c/em\\u003e)[\\u003cspan citationid=\\\"CR25\\\" class=\\\"CitationRef\\\"\\u003e25\\u003c/span\\u003e].\\u003c/p\\u003e \\u003cp\\u003eIn the field of bamboo plant reproductive biology, McClure[\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e] initially proposed the concept of \\u0026ldquo;pseudo\\u0026rdquo; spikelets and subsequently classified bamboo plant inflorescences into two categories based on the presence or absence of dormant buds located at the base of spikelets: determinate inflorescence and indeterminate inflorescence. In 1986, Geng Bojie provided a more precise definition of bamboo inflorescence as a finite true inflorescence occurring only once and an infinite false inflorescence with successive occurrences[\\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e]. According to Zhang Zuxin's research literature, employing gene editing technology to knock out genes in the inflorescence can significantly reduce the abortion rate of maize florets, suggesting a potential association between inflorescence and this phenomenon[\\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. No existing literature has been found regarding the correlation between the structure of false spikelets and inflorescences and abortion.\\u003c/p\\u003e \\u003cp\\u003e According to the flowering dynamics of florets in bamboo plants, upon blooming, water absorption by the pulp leads to lemma expansion. This facilitates the emergence of both male and female stamens from the lemma sheet while ensuring their simultaneous maturation. Upon completion of the powdering process, the pulp undergoes desiccation and contraction, resulting in the closure of the lemma sheet and retraction or abscission of stamens. This floral morphology corresponds to an open type. When in bloom, the lemma remains closed, with simultaneous maturation of both the pistil and stamen. This phenomenon can be classified as a closed type[\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e]. The most apparent distinction between the two types lies in the presence of a pulp sheet in the open type, whereas the closed type lacks such a component. In this study, \\u003cem\\u003eP. subsolida\\u003c/em\\u003e was found to have pulp flakes and to be an open floret. In this respect, its floral features were very similar to those of Bambusa. By contrast, \\u003cem\\u003eDendrocalamus sinicus\\u003c/em\\u003e and \\u003cem\\u003eD. hamiltonii\\u003c/em\\u003e did not have pulp and are of the closed type.\\u003c/p\\u003e \\u003cp\\u003eIn this study, \\u003cem\\u003eP. subsolida\\u003c/em\\u003e was found to exhibit infrequent blooming and a significantly low seed-setting rate within its native habitat, with no naturally occurring seedlings observed. The cytokinesis mode of the pollen mother cell was classified as the continuous type. This type was completely consistent with the spore development of \\u003cem\\u003eM. sichuanensi\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e] and was the same as that of \\u003cem\\u003eD. sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e]. However, most of the microspore tetraploids produced by \\u003cem\\u003eD. sinicus\\u003c/em\\u003e are tetrahedral. The resulting dizygomorphic tetraploid was similar to that of \\u003cem\\u003eB. multiplex\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], although the process of formation was not the same. The cytokinesis type of \\u003cem\\u003eB. multiplex\\u003c/em\\u003e was simultaneous, resulting in the absence of diploid formation at the end of the first division of meiosis and direct tetraploid formation during the second division. During secondary sporulation, anther wall development involves four layers of cells: the epidermis, anther chamber wall, mesosphere, and tapetum. The anther walls of \\u003cem\\u003eB. eutuldoides\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e] and \\u003cem\\u003eB. intermedia\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR24\\\" class=\\\"CitationRef\\\"\\u003e24\\u003c/span\\u003e] were also fully differentiated during the secondary sporulation period, which was consistent with the development of the anther wall of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. In contrast, the anther walls of \\u003cem\\u003eD. sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e], \\u003cem\\u003eM. sichuanensi\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e], and \\u003cem\\u003eB. multiplex\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e] were fully differentiated at the microspore mother cell stage. The anther wall of \\u003cem\\u003eP. praecox\\u003c/em\\u003e differs from that of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e in that it lacks an inner layer, which typically consists of (from outer to inner) the epidermis, middle layer, and tapetum. The majority of mature pollen grains in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e exhibited binucleate characteristics, while a minority displayed trinucleate features. The pollen of \\u003cem\\u003eM. sichuanensi\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e] and \\u003cem\\u003eS. chinensis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR12\\\" class=\\\"CitationRef\\\"\\u003e12\\u003c/span\\u003e] was similar to that of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e.\\u003c/p\\u003e \\u003cp\\u003eReasons for the low seed-setting rate\\u003c/p\\u003e \\u003cp\\u003eThe potential causes of spontaneous abortion in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e included the presence of an in-distinct or absent demarcation between tapetal cells and intermediate cells. The primary role of the tapetum was to provide nourishment and structural components for microspores [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e]. During the later stages of anther development, the tape-tum undergoes deformation and releases lipids or phenols that were essential for proper anther development [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e]. During the pollen grain development of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, similar to that in most bamboo species (e.g., \\u003cem\\u003eB. multiplex\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e], \\u003cem\\u003eB. sinospinosa\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], S\\u003cem\\u003ehibataea chinensis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e], there was abnormal tapetum development. In contrast to \\u003cem\\u003eD. sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e], in which the tapetum is normally developed, in \\u003cem\\u003eD. sinicus\\u003c/em\\u003e, the tapetum begins to disgroup at the stage of microspore mother cells, but it remains in its original position and does not disappear. However, in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, although the tapetum and midlayer persist during the microspore mother cell stage, they completely disappear during meiosis and cannot provide the necessary nutrients for normal microspore development. \\u003cem\\u003ePhyllostachys edulis\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e] has also been reported to have flowering (flowering in all seasons, all developmental stages) and normal fruit-bearing. Still, the anatomical structure of the female and male gametes in \\u003cem\\u003ePhyllostachys edulis\\u003c/em\\u003e had not been described. The second point pertains to the presence of hollow microspores, which do not possess characteristics typical of pollen grains. In this study, anther shrinkage was observed during the development of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e florets; a similar phenomenon was observed in \\u003cem\\u003eNeomi-crocalmus praini\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]and \\u003cem\\u003eB. tuldoides\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e]. Additionally, \\u003cem\\u003eB. intermedia\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]and \\u003cem\\u003eN. prainii\\u003c/em\\u003e [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]exhibited the contraction of other compartments, as observed in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. However, it is hypothesized that \\u003cem\\u003eS. chinensis\\u003c/em\\u003e exhibits limited and potentially negligible seed setting due to factors such as concealed stigma hindering effective pollination, self-pollination, and challenges associated with dioecious maturation. No instances of abnormal structural development induced abortions were observed in female or male gametophytes of \\u003cem\\u003eD. sinicus\\u003c/em\\u003e[\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e] through paraffin continuous section analysis. It was worth noting that potential factors contributing to the low seed-setting rates in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, similar to those observed in the aforementioned bamboo species, cannot be completely disregarded. Thus, there was a need for further research.\\u003c/p\\u003e \\u003cp\\u003eThe application uses and characteristics for future research\\u003c/p\\u003e \\u003cp\\u003eThe morphological examination and dissection of the female and male gametes in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e can fill the research gap regarding the reproductive structure of this genus and its conspecifics, while also serving as a reference for anatomical investigations into other floret structures. Simultaneously, it elucidates the reasons behind the low or even absent seed setting rate in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, thereby explaining the physiological characteristics of bamboo plants with diminutive flowers that solely reproduce asexually. In future endeavors, advancements in bamboo plant breeding and transitioning from cuttings and transplantation to sexual reproduction can further enhance our understanding of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e's floret sterility causes.\\u003c/p\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eBased on the anatomical study of the florets of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e, the results obtained were as follows: The inflorescence of the \\u003cem\\u003eP. subsolida\\u003c/em\\u003e floret was classified as an indeterminate inflorescence with dormant buds located at the base of the spikelet, exhibiting a pseudo-spikelet morphology. The inflorescence consists of 10\\u0026ndash;16 florets accompanied by two bracts positioned at the base of the spikelet. Each floret comprises a lemma, a palea, three lodicules, three stamens, and one pistil. The stamens were within a four-compartment longitudinal fissure, with the anther wall comprising four layers of cells: the epidermis, inner anther wall, middle layer, and tapetum. Among these layers, the tapetum was glandular in nature. The cytokinesis of the pollen mother cell was classified as continuous type. The ovary was one locule and superior, featuring a feathery three forked stigma, anatropous ovule, parietal placenta, and two integuments, with partial browning observed in certain fertile florets. The maturity of florets gradually decreases from the base to the top of the spikelets, where the uppermost floret exhibits infertility due to a young ovary and the absence of stamens. The majority of pollen grains were binuclear or trinuclear; however, abnormal development of their anthers and the occurrence of brown stamens can be observed in fertile florets. Three distinct types of spontaneous abortion can be distinguished. For example, the observed abnormalities included the absence of tapetum formation, hollow microspores, the shrinkage and deformation of the capsule, and the presence of hollow capsules. Among, anther abortion was the primary contributor to the low seed-setting rate observed in \\u003cem\\u003eP. subsolida\\u003c/em\\u003e. This study will significantly contribute to the morphological investigation of bamboo florets, establish a solid foundation for the taxonomic classification of bamboo plants, and provide a theoretical reference for the fundamental examination of flower structure. The significance of this study lay in its pioneering exploration of the reproductive structure of \\u003cem\\u003eP. subsolida.\\u003c/em\\u003e Furthermore, it offered a theoretical framework for future investigations into flower structures within the same genus. However, one limitation was the scarcity of studies on flower structures within this particular genus, which hinders clear comparisons and impedes obtaining more precise common features.\\u003c/p\\u003e\"},{\"header\":\"Material and methods\",\"content\":\"\\u003cp\\u003eIn 2018,\\u0026nbsp;60\\u0026nbsp;spikelets of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e from six \\u003cem\\u003eP. subsolida\\u003c/em\\u003e plants in flowering stage were collected at the Shahe Forest Seed Breeding Center of National Long term Research Base, Anhui Province (31.851156,117.181445)（Figure 6abc）. The development status of materials was preliminarily judged according to the size and color of the materials. The materials were placed in a 50% FAA stationary solution (50% ethanol: 40% formaldehyde: glacial acetic acid = 18: 1: 1) and pumped using a vacuum pump for 8 hours for later use.\\u0026nbsp;The FAA-fixed spikelets were transported to the laboratory under low-temperature conditions, and a total of 15 spikelets exhibiting good developmental status and an average length of 6.42cm were carefully selected for subsequent anatomical experiments.\\u003c/p\\u003e\\n\\u003cp\\u003eThe \\u003cem\\u003eP. subsolida\\u003c/em\\u003e spikelets were taken from the FAA fixation solution and dissected under an anatomical microscope (Olympus H011). Before dissection, the spikelet, floret, and spikelet axis were photographed and their lengths measured; then, the lemma, palea, pulp, pistil, and stamen of the spikelet were dissected, photographed, and measured, and the data were statistically processed using Excel software. The anatomized stamens and pistils were dissected according to Li Zhengli\\u0026rsquo;s paraffin continuous section method[42]. After dissection, the florets, anthers, and ovaries were dehydrated, immersed in paraffin, and cut into 7\\u0026mu;m thick sections using a Leica RM2165 microtome. After that, the slices were stained twice, with 1% saffron and 1% solid green, and dehydrated in 50% xylene and 100% xylene. The slice images were selected under a Nikon-ECLIPSE 50 microscope and measured with DS-3000 two-dimensional measurement software. To ensure accuracy, each microscopic morphological feature was observed and measured three times, and a total of 60 slides were prepared to minimize the potential errors in microscopic observation.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgments\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThanks to Professor Cao Zhihua from Anhui Academy of Forestry for providing experimental materials and pictures.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor Contributions:\\u003c/strong\\u003e B. J. contributed to the experimental design and writing of the paper; Z. C. provided the experimental materials; S. W. supervised experimental studies; D. J., S. Z., Y. W., H. Z., L. Y. and Y. M. and J. L. designed the experiments and revised the manuscript. All the authors have read and approved the manuscript for publication. And to have agreed both to be personally accountable for the author\\u0026apos;s own contributions and to ensure that questions related to the accuracy or integrity of any part of the work, are appropriately investigated, resolved, and the resolution documented in the literature.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis work was jointly supported by the Key Laboratory of Forest Resources Conservation and Utilization in the Southwest Mountains of China Ministry of Education, Southwest Forestry University, Yunnan Provincial Key Laboratory for Conservation and Utilization of In-forest Resource, Key Laboratory of National Forestry and Grassland Administration on Biodiversity Conservation in Southwest China (LXXK-2023M02);\\u0026nbsp;the Yunnan Province Xingdian Talents Support Plan of 2022 (XDYC-QNRC-2022-0229); the Yunnan Provincial Joint Special Project for Basic Research in Agriculture (202401BD070001-110).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eData Availability Statement:\\u003c/strong\\u003e [REAGENTS/TOOLS/MATERIALS] generated in this study are available from the corresponding author upon request. Data sharing is not applicable to this article as all new created data is already contained within this article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of Interest:\\u003c/strong\\u003e The authors declare no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003ematerials described in the manuscript, including all relevant raw data, will be freely available to any scientist wishing to use them for non-commercial purposes, without breaching participant confidentiality.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable. The authors declared that experimental research works on the plants described in this paper comply with institutional, national and international guidelines. Use of plant material has been permitted.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e:\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConflicts of Interest:\\u003c/strong\\u003e\\u0026nbsp;\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare no conflict of interest.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eA statement of the location of the extant specimen and the information of the depositor\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eAppropriate permission has been obtained for spikelet material from plants collected for this study. The plant material was collected by Cao Zhihua in Shahe Forest Seed Breeding Center of National Long term Research Base, Chuzhou, Anhui 230000, China, and the bamboo species belongs to the base. Has obtained the unit permission, can be used for scientific research; The present type specimen is deposited in the Bamboo and Rattan Research Center of Southwest Forestry University, where it was identified and preserved by Professor Wang Shuguang.This study covered plant species and did not address ethical issues.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eZhang QiSheng, Z.Q. Thinking on development of the bamboo industry in China at present. \\u003cstrong\\u003e2000\\u003c/strong\\u003e,\\u003cem\\u003e19\\u003c/em\\u003e,16-19.\\u003c/li\\u003e\\n\\u003cli\\u003eLong, L.; Minghui, Y.; Wenjing, Y.; Yulong, D.; Shuyan, L. Research advance in growth and development of bamboo organs. \\u003cem\\u003eIndustrial Crops and Products \\u003c/em\\u003e\\u003cstrong\\u003e2023\\u003c/strong\\u003e, \\u003cem\\u003e205\\u003c/em\\u003e, 117428.\\u003c/li\\u003e\\n\\u003cli\\u003eRamanayake, S.M.S.D.; Weerawardene, T.E. 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Plant tissue production. \\u003cem\\u003ePeking University Press \\u003c/em\\u003e\\u003cstrong\\u003e1996\\u003c/strong\\u003e, \\u003cem\\u003e15-50.\\u003c/em\\u003e\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":true,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":false,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true},\"keywords\":\"Pseudosasa subsolida, abortion, flower morphology, anther, ovary\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-4600448/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-4600448/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cem\\u003ePseudosasa subsolida\\u003c/em\\u003e belongs to Poaceae Pseudosasa, Because of its unique flowering cycle of bamboo plants and the physiological characteristics of asexual reproduction, The acquisition of the floral material from \\u003cem\\u003ePseudosasa subsolida\\u003c/em\\u003e poses significant challenges. A comprehensive anatomical study on the floral organs and the development of female and male gametes of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e was conducted in 2021 at Southwest Forestry University (Kunming,Yunnan,China) to investigate the influencing factors of its low seed setting rate, utilizing routine paraffin section methods. The results revealed that the spikelet of \\u003cem\\u003eP. subsolida\\u003c/em\\u003eexhibited the characteristics of a pseudospikelet with a latent bud, while the inflorescence displayed traits of an infinite inflorescence. Each spikelet contained approximately 10–16 florets and was accompanied by two bracts at its base. The fundamental structure of the florets comprised one lemma, one palea, three lodicules, three stamens, and one pistil. Many anomalies were still observed at later stages of anther development. These abnormalities included the failure of pollen grain formation, shrinkage deformation in the drug sac and tapetum cells, and the absence of middle layer cells. Additionally, microspores appeared hollow with no discernible contents. The primary factor contributing to the low seed setting rate of \\u003cem\\u003eP. subsolida\\u003c/em\\u003ewas the aberrant development of male gametophytes. The significance of this study lay in its pioneering exploration of the reproductive structure of \\u003cem\\u003eP. subsolida\\u003c/em\\u003e,and provide a theoretical reference for the fundamental examination of flower structure.\\u003c/p\\u003e\",\"manuscriptTitle\":\"Studies on floral organ Structure and female and male gametophyte development of Pseudosasa subsolida S. L. Chen \\u0026amp; G. Y. Sheng\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2024-07-16 20:08:06\",\"doi\":\"10.21203/rs.3.rs-4600448/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"researchsquare\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":true,\"externalIdentity\":\"\",\"sideBox\":\"\",\"snPcode\":\"\",\"submissionUrl\":\"/submission\",\"title\":\"Research Square\",\"twitterHandle\":\"researchsquare\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"\",\"reportingPortfolio\":\"\",\"inReviewEnabled\":false,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"c12bf054-4101-4e9d-aa5c-1c7abca76378\",\"owner\":[],\"postedDate\":\"July 16th, 2024\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"posted\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2024-10-08T08:54:12+00:00\",\"versionOfRecord\":[],\"versionCreatedAt\":\"2024-07-16 20:08:06\",\"video\":\"\",\"vorDoi\":\"\",\"vorDoiUrl\":\"\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-4600448\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-4600448\",\"identity\":\"rs-4600448\",\"version\":[\"v1\"]},\"buildId\":\"_2-kVJe1T_tPrBINL-cwx\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}