Anatomical Insights into Megasporogenesis, Microsporogenesis, and Gametophyte Development in Michelia alba: Causes of Reproductive Failure

Anatomical Insights into Megasporogenesis, Microsporogenesis, and Gametophyte Development in Michelia alba: Causes of Reproductive Failure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Anatomical Insights into Megasporogenesis, Microsporogenesis, and Gametophyte Development in Michelia alba: Causes of Reproductive Failure Lijun Zhao, Haini Xu, Yang Sun, Guangli Wu, Liqiong Zhu This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7327064/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 Background Michelia alba DC. (Magnoliaceae), economically significant tree species in Southeast Asia, extremely low natural fruit production due to reproductive failure. This study aims to characterize the anatomical causes of this phenomenon by investigating megasporogenesis, microsporogenesis, and gametophyte development. Results Anatomical analysis revealed that microspore tetrads were arranged tetrahedrally, isobilaterally, or in T-shapes and that mature pollen was bicellular. Ovules were anatropous, forming linearly arranged megaspore tetrads; only the chalazal megaspore developed into a functional Polygonum-type embryo sac (7 cells, 8 nuclei). Critical abnormalities were identified: 1. Male gametophyte failure (>90% abortion): Premature tapetum degeneration and abnormal anther wall contraction caused microspore adhesion and pollen collapse. 2. Female gametophyte failure (>90% arrest): Most embryo sacs are arrested before maturity lacking essential cells (egg cells, synergids). Only 2 of the 200 examined sections contained mature embryo sacs. Conclusions Concurrent defects in male and female gametophyte development critically impair gametophyte functionality, explaining the low fruit set in M. alba. This study provides the first cytological elucidation of reproductive failure in this species and establishes a foundation for the conservation and breeding of endangered Magnoliaceae. Developmental anatomy Gametophyte abortion Megasporogenesis Microsporogenesis Michelia alba Magnoliaceae Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction The formation and development of male and female gametophytes constitute a critical stage in plant reproduction. Understanding these processes provides a crucial foundation for reproductive biology, facilitates the selection of superior tree varieties, and underpins yield-enhancing cultivation techniques [ 1 ]. Developmental abnormalities in gametophytes were prevalent within the Magnoliaceae family. For instance, pollen abortion in Liriodendron has been attributed to abnormal cytokinesis in microspore mother cells [ 2 ], while developmental arrest at the mid-uninucleate stage leads to maturation failure in 61.3% of pollen grains in Magnolia baohua [ 3 ]. Michelia alba DC. (Magnoliaceae), a tall evergreen tree native to Southeast Asia, is widely cultivated in southern China. Its white, fragrant flowers bloom for 2–3 seasons annually and were extensively utilized in perfumery [ 4 ], tea scenting [ 5 ], landscaping, and ecological forestry [ 6 ]. Current research on M. alba primarily focuses on volatile oil composition [ 7 ], biochemical characterization of extracts [ 8 ], aroma release mechanisms [ 9 ], cultivation practices [ 10 ], and genetic traits [ 11 ]. However, in-depth studies on its flowering biology, particularly gametophyte development, remain scarce. Natural reproduction of M. alba is challenging in most regions of China. Propagation relies predominantly on grafting or cuttings, which are methods that suffer from low survival rates and limited genetic diversity. Furthermore, efficient tissue culture protocols are currently underdeveloped. Consequently, the scarcity of viable seed sources significantly hinders broader cultivation and application of this species. This study characterizes anomalies in male and female gametophyte development in M. alba to elucidate the causes underlying its reproductive failure, thereby establishing a foundation for its reproductive biology. Moreover, significant progress has been made in cross-breeding within Michelia spp. [ 12 , 13 ]. A detailed understanding of the reproductive traits of M. alba would enable its effective utilization as parental material, expanding possibilities for varietal improvement and optimization, and enhancing its role in ornamental horticulture, ultimately supporting conservation efforts for this economically significant species. Materials and methods Study site description The study site was the campus of Guangxi University. Guangxi University is located in Xixiangtang District, Nanning City, Guangxi Zhuang Autonomous Region, at longitude 108°17′E, latitude 22°50′N, and an average elevation of 76.5m. Nanning City has a humid subtropical monsoon climate type, with abundant sunshine, a type mild climate, abundant rainfall, and humid air. The average annual temperature is 21.7°C, the average temperature in January is 12.8°C, and the average temperature in July and August reaches 28.2°C; the average annual rainfall is 1304.2 mm, and the relative air humidity is large throughout the year; the soil type is mainly reddish loam, with a pH of 4.5–5.5 [ 14 ]. Thirty adult M. alba trees (age: ~11 years; height: ~16 m) with intact crowns and uniform growth were selected from three environmentally homogenous sites on the Guangxi University campus. From July 2021 to January 2023, they were subjected to 19 consecutive months of phenological observations, and detailed phenological observation records were made. Flower buds at various stages in batches from the upper part of robust new shoots that were free of pests and diseases and full of flowers during bud differentiation and development were collected in batches at the later stage of development and classified into developmental stages on based size, with larger grades representing larger size and more mature development. The species is very common and widely cultivated in this region. The material was formally identified by Lei Wang based on morphological characteristics. Due to the commonality of the species and the fact that the sampled trees are located in a public, permanent landscape, we did not create a new voucher specimen for this study. However, the sampled trees are marked and can be reliably re-located for verification. Representative photographic vouchers of the sampled trees and floral organs are available from the corresponding author upon request. Experimental Methods The longitudinal and transverse diameters of the flower buds were recorded. All tepals and petals were removed, and the buds were then fixed in FAA fixative (formalinacetic acidalcohol; 70% ethanol: glacial acetic acid: formaldehyde = 18:1:1, v/v/v) for at least 24 hours. Paraffin sections Paraffin sections were prepared following the method of Zhu Liqiong et al. [ 15 ], with modifications. Briefly, samples were: gradually dehydrated through an ethanol series; samples were dehydrated through a graded ethanol series, followed by stepwise clearing and paraffin infiltration using a TO solvent-based clearing agent mixed with absolute ethanol (paraffin wax melting point: 52–62°C). After embedding, the sections were cut at thickness 6–8 µm. The sections were then dewaxed through a graded ethanol series with baking, rehydrated, stained with hematoxylin-eosin (H&E), and finally mounted in neutral gum. For each developmental stage, a minimum of 30 flower buds were processed, yielding approximately 300 randomly selected sections for microscopic examination. The above-prepared sections were observed and photographed under a light microscope, focusing on the development of the anther wall, microsporogenesis, and the development of male gametophytes. Data processing The polar and equatorial diameters of the pollen grains were measured using via ImageJ software (NIH, USA). Experimental data were statistically analyzed, graphs were generated, and figure plates were prepared using via SPSS (IBM, USA), Microsoft Excel (Microsoft, USA), and Adobe Photoshop (Adobe, USA). Results and analyses Development and formation of the anther wall in M. alba Cross-sectional observations showed that the young anther consisted of primary epidermal cells enclosing a mass of sporogenous tissue (Fig. 1 . A). As development proceeded, large size and archesporial cells with dense cytoplasm appeared at the four corners of the anther (Fig. 1 . B). The archesporial cell divided to form an outer primary parietal cell (future anther wall), and an inner primary sporogenous cell (future microsporangium) divided periclinally (Fig. 1 ). C). The primary parietal cells divided periclinally to form four layers of wall cells. These layers initially appeared similar in shape and size (Fig. 1 . D). After further development, differentiation occurred, and the shape, size, and arrangement of the cells varied: a layer of cells close to the epidermis was the largest, nearly square, and developed into the endothecium; the two layers of cells within were the smallest, flat, vesicular and obvious and were the middle layer; the innermost layer was initially small and rectangular in a neat and tightly packed, initially small and rectangular in form, but later in the development of the volume increased significantly and the direction of the vertical axis of the long axis, and this layer was the tapetum. At this point, the anther wall development is complete, comprising the epidermis, endothecium, middle layer, and tapetum, which enclose the anther locule. Anther cross-sections at this stage exhibited bulges at the four corners and locules of nearly equal diameter. As the internal tissue grows and increases in size, the wall of the anther locule expands via mitosis to accommodate developing tissues. However, changes in the components of the anther wall were inconsistent, with the cells of the tapetum having an irregular shape, a marked increase in volume, a slightly thickened radial wall, a deepened cytoplasmic color, and the observation of 2 or more nuclei (Fig. 1 . N, O). The endothecium and middle layer were compressed by increased cell numbers and became flattened (Fig. 1 . H). The middle layer was short-lived and degenerated during the late stage of anther development. After the decomposition of the middle layer, the tapetal cells detached from the anther wall and became disorganized (Fig. 1 . I), and by the vacuolated uninucleate stage (Fig. 1 . K), and most tapetum cells had degenerated and been absorbed. At pollen maturity, the tapetum was nearly absent. The anther wall of Michelia alba did not undergo any displacement or intercellular fusion during development and thus belonged to the glandular tapetum. At this time, the endothecium also continued to develop on the one hand, the radial wall was extended, the volume increased, while the protoplasts degenerated and disappeared, and the radial cell wall was thickened with fibrous thickenings, and at this time, the endothecium could be called the fibrous layer. At this point, only the fibrous layer and the degenerated epidermis remain in the endothecium (Fig. 1 . L). The fibrous layer on the same side of the anther locule in the adjacent position has a small number of unthickened cells when drying here, it is easy to be mechanically pulled, prompting the dehiscence of the anther for and allowing the pollen grains to open the channel. In some cases, the anther wall contracted prematurely, compressing the anther locule into a U shape (Fig. 4 . D, E). Processes of the formation of microspores and male gametophytes in M. alba The archesporial cell is first divided into a primary parietal cell and a primary sporogenous cell. The latter further divided to form microspore mother cells (also known as pollen mother cells), which were characterized by large nuclei and distinct intercellular spaces. Over time, the microspore mother cells became polygonal, larger than the surrounding parietal cells, with a dense arrangement, a dark cytoplasm, and a prominent round nucleus. As the anther chamber enlarged, the spaces between the microspore mother cells also grew larger, signaling the imminent onset of meiosis. The prophase, metaphase, anaphase, and telophase stages of meiosis I were similar to those of mitosis in general: the nucleolus and nuclear membrane disappear (Fig. 4 . F), the chromatin spirals and thickens to form chromosomes, and the spindle is formed; the chromosomes align at the metaphase plate during metaphase, and in the late stage, the centromere splits in two, the chromosomes move to the poles by the pull of spindle filaments, and the cytoplasm underwent cytokinesis but without cell plate formation at the end of this division in the formation of a new cell plate and a new cell wall. However, at the end of th3 stage. Cytokinesis occurred lacking cell plate formation, and the cells directly entered meiosis II (Fig. 4 . G-1), the chromosomes were separated again and moved to the two poles (Fig. 4 . H), and the cytoplasm was divided again, forming a tetrad (Fig. 4 . I). The tetrads in M. alba were arranged in three types: isobilateral, tetrahedral, and T-shaped. Microspore development within the same locule was largely synchronous, with occasional differences of one to two developmental stages (Fig. 4 . J-3,4). A small number of tetrads appeared contracted and reniform but regained a spherical shape upon maturation, similar to other microspores that developed into spherical or oblong pollen grains. Upon separation of the tetrads, each becomes a separate microspore and is released into the anther locule. Initially, the microspores have a centered nucleus and a light-colored cytoplasm with only xine layer, at the early uninucleate microspore stage (Fig. 4 . K). With growth, the microspore cell size increases, and vacuolation becomes prominent. At this vacuolated uninucleate stage, the cytoplasm and nucleus were peripherally displaced, generating the inner pollen wall, which later forming the two-layered pollen wall (Fig. 4 . L). Subsequent microspore development involved nutrient absorption, cytoplasmic thickening, and mitotic division into a generative cell and a vegetative cell. This culminated in the formation of bicellular pollen grains. During this maturation, the cytoplasm thickened and deepened in color. Subsequently the microspore underwent mitosis, giving rise to a generative cell and a vegetative cell, each containing a nucleus: the larger generative cell nucleus and the smaller vegetative cell nucleus (Fig. 4 . O). At this stage, the microspore reached maturity, forming the male gametophyte (pollen grain), which was of the bicellular type. Processes of the formation of white orchid megaspores and female gametophytes At the same time as the formation of microsporocytes, the placenta in the pistil ovary also bulges upwards, gradually forming the ovule primordium (Fig. 3 . A). A layer of epidermal primordium wrapped around the archesporial cell, constituting the nucellus (Fig. 4 . B), the archesporial cell divided in the periphery to form parietal cells and inward divides inwardly to form sporogenous cells, the former further divided to form 2–4 layers of new cells to add to the nucellus, and the latter developed into a megaspore mother cell. Simultaneously, tissue at the outer flank of the primordium initiated the formation of the integument base on both sides. The integument rapidly divided, growing upwards and downwards to envelop the central nucellus tissue, leaving only the micropyle open (Fig. 3 . C, D). Due to the inconsistent development rate of the integument on both sides, the ovule gradually tilts, and bends, and finally forms an anatropous ovule. After meiosis, the mother cell of the megaspore in the nucellus forms a tetrad, which is arranged in a straight line in the center of the nucellus, and one daughter cell at the end of the chalazal-most daughter cell is a functional megaspore, which is able to continue to develop and form a uninucleate embryo sac, while the remaining three daughter cells gradually were degenerated and absorbed (Fig. 3 ). E, F). The uninucleate embryo sac enlarges rapidly, accompanied by further thickening of the surrounding nucellus tissue (Fig. 3 . G). Subsequently the uninucleate embryo sac undergoes three mitotic divisions: the two nuclei formed by the first division each move to the two ends of the embryo sac (Fig. 3 . H) and then each divided two more times to form an eight-nucleate embryo sac and entered the cellularization stage (Fig. 3 . J): the nuclei from each pole migrated to the center of the embryo sac to form polar nuclei, which then developed together with the surrounding cytoplasm to form a central cell; three nuclei at the chalazal end formed antipodal cells, which were antipodal cells. The three cells near the end of the micropyle were arranged in a zigzag pattern, comprising a larger, pear-shaped egg cell in the middle flanked by two synergids; together, these three cells constituted the egg apparatus. At this point, the 7-cell 8-nucleated embryo sac of Michelia alba matured. From the above, it can be seen that the embryo sacs of Michelia alba belong to the typical single-spore polygonum type embryo sacs. Only a few mature embryo sacs with seven cells and eight nuclei were observed during the study—just 2 out of 200 sections at the same developmental stage. Most embryo sacs arrested development before initiating the mitotic divisions following uninucleate embryo sac formation. A smaller proportion of embryo sacs arrested development at the two-nucleate, four-nucleate, or eight-nucleate stages, exhibiting abnormalities such as the absence of functional cells or missing synergids. In some abnormal sacs, synergids lacked a filiform apparatus and/or were positioned abnormally distant from the micropylar end. The majority of embryo sacs appeared largely devoid of cellular contents (Fig. 4 . I, J). At flowering, the stigma becomes receptive to pollen, and the perianth segments senesce, but the pistil does not; however, but the ovary fails to expand because of the failure of fertilisation, and the integuments and nucellus were shriveled up and separated from the embryo sac, and the pistil terminates its development and fails to set fruit. Correspondence between the external characteristics of flower buds and the internal developmental processes of female and male gametophytes In Michelia alba, male and female gametophytes develop asynchronously, with stamens mature earlier than before pistils. Internal gametophyte development closely correlates with flower bud morphology and size, as shown in Table 1 . At the onset of microsporocyte meiosis, pistils were only beginning to undergo archesporial cell differentiation. As stamen development progresses to the late MMC stages, ovule primordia begins to form. By the time microspores are released and reach the uninucleate stage, internal differentiation of the megasporangium is underway within the ovule. Subsequently the development of the embryo sac accelerates. At anthesis, both male and female gametophytes were fully mature, coinciding with pollen release (Fig. 5 . ). Table 1 Correlation between floral bud size and developmental stages of stamens and pistils in Michelia alba Average size/mm External morphological features Microspore and Male Gametophyte Stage Megaspore and Female Gametophyte Stage Longitudinal Transverse diameter 11.30 ± 0.75 3.79 ± 0.25 Outer 3 layers of tepals, thickly leathery, Light green, and leathery Archesporial cell - 13.08 ± 0.65 4.22 ± 0.44 Outer 3 layers of tepals, thickly leathery, Light green, and leathery Primary parietal cell and primary sporogenous cell - 16.78 ± 0.31 4.73 ± 0.58 Outer 3 layers of tepals, thinly leathery, Light green and leathery Microspore mother cell (MMC) Ovule primordium 19.21 ± 2.41 5.31 ± 0.37 First outermost whorl of tepals deciduous, greenish green Meiosis in MMCs Archesporial cell 23.57 ± 2.48 5.89 ± 0.70 Outer 3 layers of tepals-second layer of tepals dehiscent Tetrad stage Sporogenous cells - megaspore mother cell (MMC) 26.36 ± 1.67 7.03 ± 0.79 Outer 2 layers of tepals, thinly leathery, green Uninucleate microspore stage Megaspore mother cell (MMC) 27.11 ± 2.82 7.92 ± 1.03 Outer 1 outermost whorl of tepals, thinly leathery, green Vacuolated uninucleate microspore Uninucleate embryo sac 29.21 ± 1.54 8.12 ± 1.24 Outer 1 outermost whorl of tepals, thinly leathery, green Uninucleate microspore Binucleate embryo sac - quadrinucleate embryo sac 34.58 ± 2.52 8.86 ± 1.56 Tertiary annular stipule cracked or green silk white bud stage Bicellular pollen grain Eight-nucleate embryo sac 44.67 ± 2.04 9.05 ± 1.45 White buds with green buds with green outer tepals state Dinucleate mature pollen Mature embryo sac Note: Data are presented as mean ± standard deviation. Discussion In Michelia alba, the development and maturation of stamens and pistils generally followed patterns typical of angiosperms and closely resembled those of related species such as Michelia figo [ 16 ], Michelia maudiae [ 17 ], and Michelia glauca [ 18 ]. The pollen sac wall is composed of four layers: the epidermis, endothecium, middle layer, and tapetum. Microspore tetrads were arranged in three configurations: cross-shaped, isobilateral, and “T”-shaped, while the mature pollen grains were of the bicellular type. In the anatropous ovule, the megaspore tetrads were linearly arranged, and the chalazal megaspore—farthest from the micropyle—developed into a functional uninucleate embryo sac. This process followed a monosporic pattern and resulted in a Polygonum-type mature embryo sac. The initiation of stamen development precedes that of the pistil, but both structures reached maturity simultaneously, potentially enabling self-compatibility. Interestingly, unlike in many other plants, a transient contraction of microspores was observed during the early tetrad stage in M. alba. This phenomenon has also been reported in other Magnoliaceae species, such as Michelia guangxiensis [ 19 ], Tsoongiodendron odorum [ 20 ], and Manglietia insignis [ 21 ], suggesting that such microspore contraction may be a common feature during microsporogenesis in this family. Similar to other Magnoliaceae species, Michelia alba exhibits poor sexual reproductive success. The few seeds that were occasionally formed typically exhibit poor development and fail to germinate, a manifestation of reproductive failure or sterility. Reproductive failure in plants can stem from both extrinsic and intrinsic factors. External causes often include imbalances in nutrient availability and environmental stresses, such as abnormal temperature, moisture, or light conditions. In Magnolia championii [ 22 ] and Woonyoungia septentrionalis [ 23 ], for example, pollen abortion has been attributed to environmental fluctuations. Intrinsic factors encompass disruptions in genetic regulation, physiological processes, and metabolic pathways. In M. alba, multiple abnormalities were identified during the development of male and female gametophytes. In M alba. male reproductive organs, early shrinkage and inward folding of the anther wall during pollen mother cell formation reduces locule space. Alternatively, premature degradation of the tapetum may lead to nutrient insufficiency for microspores. During meiosis, chromosomal anomalies may arise, resulting in some microspores arresting at the uninucleate stage. Some microspores show clumping, abnormal shapes, thickened walls, or hollow pollen grains. Similar phenomena have been observed in Michelia guangxiensis [ 24 ], where delayed or incomplete degeneration of the tapetum leads to pollen abortion. Abnormal pollen grains and extremely low germination rates (sometimes below 0.01%) have also been reported in Manglietia aromatica [ 25 ], Liriodendron chinense [ 26 ], and Magnolia officinalis subsp. Biloba [ 27 ]. On the female side, the proportion of gametophytes that develop to maturity is extremely low. Developmental failure mainly occurrs during the three mitotic divisions after the uninucleate embryo sac forms. Arrests have been observed at the one-, two-, and four-nucleate stages. Even in embryo sacs that reach the eight-nucleate stage, key functional cells—such as antipodals, egg cells, or synergids, were frequently absent or only partially developed. In some cases, synergids were positioned too far from the egg cells to function properly, or the embryo sac developd as a hollow structure. Severe embryo sac abortion has also been reported in other Magnoliaceae species, including Michelia maudiae [ 17 ], Michelia xichouensis [ 28 ], and Manglietia insignis [ 29 ], with sterility rates of approximately 25%, 70%, and 87%, respectively. Compared to stamens, developmental abnormalities in the pistils of M. alba were particularly pronounced. This observation supports the resource allocation theory in angiosperms, which proposes that under resource constraints, monoecious species favor male flower production to optimize biomass and reproductive output [ 30 ], since male reproduction is simpler and more efficient in terms of resource usage. Consequently, female gametophyte abortion poses a more critical constraint on reproductive success in endangered Magnoliaceae species [ 31 ]. The anatomical features underlying these developmental failures were often governed by complex internal factors. For instances, Nie et al. [ 32 ] suggested that disruptions in endogenous hormone balance and differential gene expression under heat stress may play significant roles in sterility. Similarly, Li et al. [ 33 ] identified the MawuAP1 gene as a key regulatory element in floral organ development in Magnolia wufengensis. Therefore, future studies on plant reproduction should adopt integrative approaches, and hybrid breeding efforts must account for parental reproductive traits and genetic compatibility. Conclusion This study, through anatomical analysis, provides the first systematic elucidation of the cytological mechanisms underlying reproductive abortion in Michelia × alba. The research has revealed that in normally developing male gametophytes, microspore tetrads were arranged in crossed, symmetrical, or T-shaped configurations, and mature pollen was of the bicellular type. The female gametophyte developed as a monosporic Polygonum-type embryo sac, maturing into a 7-celled, 8-nucleate structure. The maturation timing of male and female gametophytes was synchronized, providing a basis for self-pollination. However, the primary causes of abortion were identified as abnormalities in both gametophytes. Abnormal male gametophyte development was characterized by premature contraction of the anther wall and precocious degradation of the tapetum, leading to microspore adhesion and the formation of collapsed pollen grains. Concurrently, abnormal female gametophyte development manifested as embryo sacs frequently being arrested at the uninucleate or binucleate stages, or exhibiting the absence of functional cells (such as the egg cell and synergids) during the 8-nucleate stage, with hollow embryo sacs commonly observed. This research clarifies the cytological basis of abortion in Magnoliaceae plants, fills a significant gap in the reproductive biology studies of Michelia × alba, provides key technical support for the conservation and propagation of endangered Magnoliaceae species, holds substantial importance for maintaining species genetic diversity, and offers both a theoretical foundations and practical guidance for breeding innovation and the population restoration of economically important Magnoliaceae trees. Declarations Supplementary Information No supplementary material was generated for this study. All supporting data are presented in the main manuscript figures and tables. Acknowledgment This work was supported by the National Natural Science Foundation of China (Grants 31260093 and 31560061), the Guangxi Forestry Science and Technology Promotion Demonstration Project (Guilin Scientific Research [2022] No. 1), and the Guangxi State-owned Qinlian Forestry Research Project. We thank the Guangxi Key Laboratory of Forest Ecology and Conservation for providing laboratory facilities. We are grateful to Dr. Zhu from Guangxi Vocational University of Agriculture for providing technical assistance in histological sectioning. Special thanks to the Guangxi University Campus Management Office for permitting plant sample collection. Author Contributions Conceptualization: L.-J.Z. and L.-Q.Z.; Methodology: H.-N.X. and Y.S.; Formal analysis: G.-L.W.; Investigation: L.-J.Z., H.-N.X. and Y.S.; Resources: L.-Q.Z.; Data curation: H.-N.X. and G.-L.W.; Writing—original draft preparation: L.-J.Z.; Writing—review and editing: L.-Q.Z. and G.-L.W.; Supervision: L.-Q.Z.; Funding acquisition: L.-Q.Z. Funding This research was funded by the National Natural Science Foundation of China (Grants 31260093 and 31560061), the Guangxi Forestry Science and Technology Promotion Demonstration Project (Guilin Scientific Research [2022] No. 1), and the Guangxi State-owned Qinlian Forestry Research Project. Data Availability Statement All data supporting the findings of this study are available within the paper and within its Supplementary Materials published online. Ethics approval and consent to participate Not applicable. Conflicts of interest. The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Consent for publication Not applicable. Publisher's Note Springer Nature remains neutral with regard to junisdictional caimsinpublished mapsand intitutionalaffliations. References Li HR, Zhou C, Wei W et al. 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Morphological observations on flower bud differentiation in Cornus hongkongensis. Plant Sci J (2018), *36*(4): 501–7. Hu SY. Reproductive biology of angiosperms [M]. Higher Education Press; 2005. Zhao XF, Sun WB, Yang HB et al. Megasporogenesis, microsporogenesis and gametophyte development in critically endangered Michelia coriacea. Acta Bot Yunnanica (2008), *30*(5): 549–56. Wang J, Ren XL, Huang YP, et al. Morphological and cytological characterization of cytoplasmic male sterile line 09-05A in spring Brassica juncea. Journal of Zhejiang University (Agriculture & Life Sciences; 2018. pp. 588–93. 5. Paterno GB, Silveira CL, Kollmann J et al. The maleness of larger angiosperm flowers. Proceedings of the National Academy of Sciences (2020), *117*(20): 10921–10926. https://doi.org/10.1073/pnas.1910631117 Tucker SC. Phyllotaxis and vascular organization of the carpels in Michelia fuscata. Am J Bot (1961), *48*(1): 60–71. Nie T, Jiang Z, Sun L, Trees et al. (2022), *36*(5): 1515–28. https://doi.org/10.1007/s00468-022-02299-9 Wu W, Chen F, Jing D et al. Isolation and characterization of an AGAMOUS-like gene from Magnolia wufengensis (Magnoliaceae). Plant Molecular Biology Reporter (2012), *30*(3): 690–698. https://doi.org/10.1007/s11105-011-0373-7 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. 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-7327064","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":516532223,"identity":"49276e23-cba8-4cb7-a38d-f168c8bac21a","order_by":0,"name":"Lijun Zhao","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Lijun","middleName":"","lastName":"Zhao","suffix":""},{"id":516532224,"identity":"ff74a00d-54fa-4b25-abb6-1e00872a485f","order_by":1,"name":"Haini Xu","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Haini","middleName":"","lastName":"Xu","suffix":""},{"id":516532225,"identity":"c783d2ff-26a6-4d81-ab60-7cba1a2ee390","order_by":2,"name":"Yang Sun","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Yang","middleName":"","lastName":"Sun","suffix":""},{"id":516532226,"identity":"34c4c781-1e6a-458f-ad7d-4ec7bb508263","order_by":3,"name":"Guangli Wu","email":"","orcid":"","institution":"Guangxi Academy of Agricultural Sciences","correspondingAuthor":false,"prefix":"","firstName":"Guangli","middleName":"","lastName":"Wu","suffix":""},{"id":516532227,"identity":"721e74c1-0a51-438e-847d-ccf5de65494a","order_by":4,"name":"Liqiong Zhu","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAr0lEQVRIie3PvwrCMBDH8ZRApytZG/AhAg4ilPoqBcGps3QMFLK5p+BzOF/oWuIq9CXSN/DP5CJcN4f7zveB3wnBcf9YKQSmrgKlLJ1kwU+njfZIJ3Is3FgZ2xCFmS8YBncHIzBLS0sg+hobTHGGnbRSDzcCUWVrgj/PsLeYy4JC8hcZIY9gsCES9SEOVxDt38OmI2gfetov5tFuU+rqg1J9SAuFfJXZdfccx3Hc757qXjoN4nFqnAAAAABJRU5ErkJggg==","orcid":"","institution":"3Urban-Rural Construction College, Guangxi Vocational University of Agriculture","correspondingAuthor":true,"prefix":"","firstName":"Liqiong","middleName":"","lastName":"Zhu","suffix":""}],"badges":[],"createdAt":"2025-08-08 12:08:16","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-7327064/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-7327064/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":91639344,"identity":"bbdb412c-e354-4ef5-8365-98726e2390ff","added_by":"auto","created_at":"2025-09-18 14:33:13","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":433673,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe development of anther walls of M. alba. \u003c/strong\u003eA, B: The spore mother cells initiate development, and the corners of the anther protrude; C: The primary parietal cells and primary sporogenous cells were produced; D: The secondary parietal layer and sporogenous tissue were formed; E: The microsporocyte gradually takes shape; F, G: The anther wall begins to form, consisting of 4 layers of cells; H, I: The mature anther wall; J, K: The middle layer and tapetum begin to disintegrate; L: The tapetum and middle layer disappear, while the inner walls were formed. Middle layer disappears, while the inner walls of the locules thicken longitudinally;. M: The anther wall ruptures, resulting in the release of pollen;. N: Tapetal cells with two nuclei. The tapetal cells with two nuclei; O: The tapetal cells with three nuclei; ps: Primary sporogenous cell; ac: Archesporial cell; pp: Primary parietal cell; ps: Primary sporogenous cell; sp: Secondary parietal layer; ss: Secondary sporogenous cell; MMC: Microspore mother cell; ep: Epidermis; en: Endothecium; ml: Middle layer; ta: Tapetum; te: Tetrad; mp: Mature pollen; bt: Binucleate tapetal cell; tt: Trinucleate tapetal cell.\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7327064/v1/dc629f02371746d8d96572c0.jpeg"},{"id":91639347,"identity":"656332f0-ac19-48a0-8949-02910dbc5a54","added_by":"auto","created_at":"2025-09-18 14:33:13","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":360496,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMicrosporogenesis and male gametophyte development of M. alba.\u003c/strong\u003e A: E: Microspore mother cells start meiosis; C~D: Microspore mother cell; F: Meiotic metaphase Ⅰ; G: Meiosis telophase Ⅰ; H: Anaphase of meiosis Ⅱ; I. Meiosis telophase Ⅱ; J: Tetrad stage; K: Microspores released from tetrad; L: Vacuolated uninucleate stage; N: Early uninucleate microspore; O: Mature two-cell pollen. gc: Generative cell; vc: Vegetative cell.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7327064/v1/340a57c7314da8a3bab64f7d.jpeg"},{"id":91639349,"identity":"834ad601-fd6d-4f48-bb70-909c6cd08d2b","added_by":"auto","created_at":"2025-09-18 14:33:13","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":490473,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eMegasporogenesis and the development of female gametophytes of M. alba. \u003c/strong\u003eA, B: Ovule primordium differentiation; G: Uninucleate embryo sac; H: Two-nucleate embryo sac; I, J: Eight-nucleate embryo sac; K: Center cell with two polar nuclei; L: Anatropous ovule. cc: Central cell; sy: Synergids; ant: Antipodal cells.\u003c/p\u003e","description":"","filename":"floatimage3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7327064/v1/cd8f7ea1a2344ac507a6fa9d.jpeg"},{"id":91640299,"identity":"838af620-d113-461c-8b94-991c6af26da6","added_by":"auto","created_at":"2025-09-18 14:41:13","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":458897,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe abnormal phenomenon in the process of male and female gametophyte development. \u003c/strong\u003eA, B: Abnormal development of the premeiotic tapetum in microsporocyte; C: Adhesion during tetrad release; D~G: Aberrant anther wall and empty pollen grain; H: Overly contracted microspore; I, J: Incomplete embryo sac at flowering; K: An incomplete embryo sac after flowering; L: Degenerated integument of the ovule.\u003c/p\u003e","description":"","filename":"floatimage4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7327064/v1/8628c6e4c22c087649fb92b9.jpeg"},{"id":91639350,"identity":"f9b30534-1e6d-4db6-a636-882f971b4880","added_by":"auto","created_at":"2025-09-18 14:33:13","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":144674,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDevelopmental synchrony of stamens and pistils in M. alba\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA: When the ovule primordium of the ovary differentiates, the microspore mother cells begin developing. B: At the early stage of embryo sac mother cell formation, the uninucleate pollen begin to develop.\u003c/p\u003e","description":"","filename":"floatimage5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7327064/v1/141ed4d0878705d67d870d73.jpeg"},{"id":92243466,"identity":"7031c643-ff70-486c-9c88-557105528195","added_by":"auto","created_at":"2025-09-26 09:09:22","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2829902,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7327064/v1/8b21793b-7c3c-42e0-b7d3-5112152da8a3.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Anatomical Insights into Megasporogenesis, Microsporogenesis, and Gametophyte Development in Michelia alba: Causes of Reproductive Failure","fulltext":[{"header":"Introduction","content":"\u003cp\u003eThe formation and development of male and female gametophytes constitute a critical stage in plant reproduction. Understanding these processes provides a crucial foundation for reproductive biology, facilitates the selection of superior tree varieties, and underpins yield-enhancing cultivation techniques [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Developmental abnormalities in gametophytes were prevalent within the Magnoliaceae family. For instance, pollen abortion in Liriodendron has been attributed to abnormal cytokinesis in microspore mother cells [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], while developmental arrest at the mid-uninucleate stage leads to maturation failure in 61.3% of pollen grains in Magnolia baohua [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eMichelia alba DC. (Magnoliaceae), a tall evergreen tree native to Southeast Asia, is widely cultivated in southern China. Its white, fragrant flowers bloom for 2\u0026ndash;3 seasons annually and were extensively utilized in perfumery [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], tea scenting [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], landscaping, and ecological forestry [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Current research on M. alba primarily focuses on volatile oil composition [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], biochemical characterization of extracts [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e], aroma release mechanisms [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e], cultivation practices [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e], and genetic traits [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. However, in-depth studies on its flowering biology, particularly gametophyte development, remain scarce.\u003c/p\u003e\u003cp\u003eNatural reproduction of M. alba is challenging in most regions of China. Propagation relies predominantly on grafting or cuttings, which are methods that suffer from low survival rates and limited genetic diversity. Furthermore, efficient tissue culture protocols are currently underdeveloped. Consequently, the scarcity of viable seed sources significantly hinders broader cultivation and application of this species.\u003c/p\u003e\u003cp\u003eThis study characterizes anomalies in male and female gametophyte development in M. alba to elucidate the causes underlying its reproductive failure, thereby establishing a foundation for its reproductive biology. Moreover, significant progress has been made in cross-breeding within Michelia spp. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. A detailed understanding of the reproductive traits of M. alba would enable its effective utilization as parental material, expanding possibilities for varietal improvement and optimization, and enhancing its role in ornamental horticulture, ultimately supporting conservation efforts for this economically significant species.\u003c/p\u003e"},{"header":"Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eStudy site description\u003c/h2\u003e\u003cp\u003eThe study site was the campus of Guangxi University. Guangxi University is located in Xixiangtang District, Nanning City, Guangxi Zhuang Autonomous Region, at longitude 108\u0026deg;17\u0026prime;E, latitude 22\u0026deg;50\u0026prime;N, and an average elevation of 76.5m. Nanning City has a humid subtropical monsoon climate type, with abundant sunshine, a type mild climate, abundant rainfall, and humid air. The average annual temperature is 21.7\u0026deg;C, the average temperature in January is 12.8\u0026deg;C, and the average temperature in July and August reaches 28.2\u0026deg;C; the average annual rainfall is 1304.2 mm, and the relative air humidity is large throughout the year; the soil type is mainly reddish loam, with a pH of 4.5\u0026ndash;5.5 [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e\u003cp\u003eThirty adult M. alba trees (age: ~11 years; height: ~16 m) with intact crowns and uniform growth were selected from three environmentally homogenous sites on the Guangxi University campus. From July 2021 to January 2023, they were subjected to 19 consecutive months of phenological observations, and detailed phenological observation records were made. Flower buds at various stages in batches from the upper part of robust new shoots that were free of pests and diseases and full of flowers during bud differentiation and development were collected in batches at the later stage of development and classified into developmental stages on based size, with larger grades representing larger size and more mature development.\u003c/p\u003e\u003cp\u003eThe species is very common and widely cultivated in this region. The material was formally identified by Lei Wang based on morphological characteristics. Due to the commonality of the species and the fact that the sampled trees are located in a public, permanent landscape, we did not create a new voucher specimen for this study. However, the sampled trees are marked and can be reliably re-located for verification. Representative photographic vouchers of the sampled trees and floral organs are available from the corresponding author upon request.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eExperimental Methods\u003c/h3\u003e\n\u003cp\u003eThe longitudinal and transverse diameters of the flower buds were recorded. All tepals and petals were removed, and the buds were then fixed in FAA fixative (formalinacetic acidalcohol; 70% ethanol: glacial acetic acid: formaldehyde\u0026thinsp;=\u0026thinsp;18:1:1, v/v/v) for at least 24 hours.\u003c/p\u003e\n\u003ch3\u003eParaffin sections\u003c/h3\u003e\n\u003cp\u003eParaffin sections were prepared following the method of Zhu Liqiong et al. [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e], with modifications. Briefly, samples were: gradually dehydrated through an ethanol series; samples were dehydrated through a graded ethanol series, followed by stepwise clearing and paraffin infiltration using a TO solvent-based clearing agent mixed with absolute ethanol (paraffin wax melting point: 52\u0026ndash;62\u0026deg;C). After embedding, the sections were cut at thickness 6\u0026ndash;8 \u0026micro;m. The sections were then dewaxed through a graded ethanol series with baking, rehydrated, stained with hematoxylin-eosin (H\u0026amp;E), and finally mounted in neutral gum. For each developmental stage, a minimum of 30 flower buds were processed, yielding approximately 300 randomly selected sections for microscopic examination. The above-prepared sections were observed and photographed under a light microscope, focusing on the development of the anther wall, microsporogenesis, and the development of male gametophytes.\u003c/p\u003e\n\u003ch3\u003eData processing\u003c/h3\u003e\n\u003cp\u003eThe polar and equatorial diameters of the pollen grains were measured using via ImageJ software (NIH, USA). Experimental data were statistically analyzed, graphs were generated, and figure plates were prepared using via SPSS (IBM, USA), Microsoft Excel (Microsoft, USA), and Adobe Photoshop (Adobe, USA).\u003c/p\u003e"},{"header":"Results and analyses","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eDevelopment and formation of the anther wall in M. alba\u003c/h2\u003e\u003cp\u003eCross-sectional observations showed that the young anther consisted of primary epidermal cells enclosing a mass of sporogenous tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. A). As development proceeded, large size and archesporial cells with dense cytoplasm appeared at the four corners of the anther (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. B). The archesporial cell divided to form an outer primary parietal cell (future anther wall), and an inner primary sporogenous cell (future microsporangium) divided periclinally (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). C).\u003c/p\u003e\u003cp\u003eThe primary parietal cells divided periclinally to form four layers of wall cells. These layers initially appeared similar in shape and size (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. D). After further development, differentiation occurred, and the shape, size, and arrangement of the cells varied: a layer of cells close to the epidermis was the largest, nearly square, and developed into the endothecium; the two layers of cells within were the smallest, flat, vesicular and obvious and were the middle layer; the innermost layer was initially small and rectangular in a neat and tightly packed, initially small and rectangular in form, but later in the development of the volume increased significantly and the direction of the vertical axis of the long axis, and this layer was the tapetum. At this point, the anther wall development is complete, comprising the epidermis, endothecium, middle layer, and tapetum, which enclose the anther locule. Anther cross-sections at this stage exhibited bulges at the four corners and locules of nearly equal diameter. As the internal tissue grows and increases in size, the wall of the anther locule expands via mitosis to accommodate developing tissues. However, changes in the components of the anther wall were inconsistent, with the cells of the tapetum having an irregular shape, a marked increase in volume, a slightly thickened radial wall, a deepened cytoplasmic color, and the observation of 2 or more nuclei (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. N, O). The endothecium and middle layer were compressed by increased cell numbers and became flattened (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. H). The middle layer was short-lived and degenerated during the late stage of anther development. After the decomposition of the middle layer, the tapetal cells detached from the anther wall and became disorganized (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. I), and by the vacuolated uninucleate stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. K), and most tapetum cells had degenerated and been absorbed. At pollen maturity, the tapetum was nearly absent. The anther wall of Michelia alba did not undergo any displacement or intercellular fusion during development and thus belonged to the glandular tapetum. At this time, the endothecium also continued to develop on the one hand, the radial wall was extended, the volume increased, while the protoplasts degenerated and disappeared, and the radial cell wall was thickened with fibrous thickenings, and at this time, the endothecium could be called the fibrous layer. At this point, only the fibrous layer and the degenerated epidermis remain in the endothecium (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. L). The fibrous layer on the same side of the anther locule in the adjacent position has a small number of unthickened cells when drying here, it is easy to be mechanically pulled, prompting the dehiscence of the anther for and allowing the pollen grains to open the channel. In some cases, the anther wall contracted prematurely, compressing the anther locule into a U shape (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. D, E).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eProcesses of the formation of microspores and male gametophytes in M. alba\u003c/h3\u003e\n\u003cp\u003eThe archesporial cell is first divided into a primary parietal cell and a primary sporogenous cell. The latter further divided to form microspore mother cells (also known as pollen mother cells), which were characterized by large nuclei and distinct intercellular spaces. Over time, the microspore mother cells became polygonal, larger than the surrounding parietal cells, with a dense arrangement, a dark cytoplasm, and a prominent round nucleus. As the anther chamber enlarged, the spaces between the microspore mother cells also grew larger, signaling the imminent onset of meiosis. The prophase, metaphase, anaphase, and telophase stages of meiosis I were similar to those of mitosis in general: the nucleolus and nuclear membrane disappear (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. F), the chromatin spirals and thickens to form chromosomes, and the spindle is formed; the chromosomes align at the metaphase plate during metaphase, and in the late stage, the centromere splits in two, the chromosomes move to the poles by the pull of spindle filaments, and the cytoplasm underwent cytokinesis but without cell plate formation at the end of this division in the formation of a new cell plate and a new cell wall. However, at the end of th3 stage. Cytokinesis occurred lacking cell plate formation, and the cells directly entered meiosis II (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. G-1), the chromosomes were separated again and moved to the two poles (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. H), and the cytoplasm was divided again, forming a tetrad (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. I). The tetrads in M. alba were arranged in three types: isobilateral, tetrahedral, and T-shaped. Microspore development within the same locule was largely synchronous, with occasional differences of one to two developmental stages (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. J-3,4). A small number of tetrads appeared contracted and reniform but regained a spherical shape upon maturation, similar to other microspores that developed into spherical or oblong pollen grains.\u003c/p\u003e\u003cp\u003eUpon separation of the tetrads, each becomes a separate microspore and is released into the anther locule. Initially, the microspores have a centered nucleus and a light-colored cytoplasm with only xine layer, at the early uninucleate microspore stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. K). With growth, the microspore cell size increases, and vacuolation becomes prominent. At this vacuolated uninucleate stage, the cytoplasm and nucleus were peripherally displaced, generating the inner pollen wall, which later forming the two-layered pollen wall (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. L). Subsequent microspore development involved nutrient absorption, cytoplasmic thickening, and mitotic division into a generative cell and a vegetative cell. This culminated in the formation of bicellular pollen grains. During this maturation, the cytoplasm thickened and deepened in color. Subsequently the microspore underwent mitosis, giving rise to a generative cell and a vegetative cell, each containing a nucleus: the larger generative cell nucleus and the smaller vegetative cell nucleus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. O). At this stage, the microspore reached maturity, forming the male gametophyte (pollen grain), which was of the bicellular type.\u003c/p\u003e\n\u003ch3\u003eProcesses of the formation of white orchid megaspores and female gametophytes\u003c/h3\u003e\n\u003cp\u003eAt the same time as the formation of microsporocytes, the placenta in the pistil ovary also bulges upwards, gradually forming the ovule primordium (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. A). A layer of epidermal primordium wrapped around the archesporial cell, constituting the nucellus (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. B), the archesporial cell divided in the periphery to form parietal cells and inward divides inwardly to form sporogenous cells, the former further divided to form 2\u0026ndash;4 layers of new cells to add to the nucellus, and the latter developed into a megaspore mother cell. Simultaneously, tissue at the outer flank of the primordium initiated the formation of the integument base on both sides. The integument rapidly divided, growing upwards and downwards to envelop the central nucellus tissue, leaving only the micropyle open (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. C, D). Due to the inconsistent development rate of the integument on both sides, the ovule gradually tilts, and bends, and finally forms an anatropous ovule.\u003c/p\u003e\u003cp\u003eAfter meiosis, the mother cell of the megaspore in the nucellus forms a tetrad, which is arranged in a straight line in the center of the nucellus, and one daughter cell at the end of the chalazal-most daughter cell is a functional megaspore, which is able to continue to develop and form a uninucleate embryo sac, while the remaining three daughter cells gradually were degenerated and absorbed (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). E, F). The uninucleate embryo sac enlarges rapidly, accompanied by further thickening of the surrounding nucellus tissue (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. G). Subsequently the uninucleate embryo sac undergoes three mitotic divisions: the two nuclei formed by the first division each move to the two ends of the embryo sac (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. H) and then each divided two more times to form an eight-nucleate embryo sac and entered the cellularization stage (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. J): the nuclei from each pole migrated to the center of the embryo sac to form polar nuclei, which then developed together with the surrounding cytoplasm to form a central cell; three nuclei at the chalazal end formed antipodal cells, which were antipodal cells. The three cells near the end of the micropyle were arranged in a zigzag pattern, comprising a larger, pear-shaped egg cell in the middle flanked by two synergids; together, these three cells constituted the egg apparatus. At this point, the 7-cell 8-nucleated embryo sac of Michelia alba matured. From the above, it can be seen that the embryo sacs of Michelia alba belong to the typical single-spore polygonum type embryo sacs. Only a few mature embryo sacs with seven cells and eight nuclei were observed during the study\u0026mdash;just 2 out of 200 sections at the same developmental stage. Most embryo sacs arrested development before initiating the mitotic divisions following uninucleate embryo sac formation. A smaller proportion of embryo sacs arrested development at the two-nucleate, four-nucleate, or eight-nucleate stages, exhibiting abnormalities such as the absence of functional cells or missing synergids. In some abnormal sacs, synergids lacked a filiform apparatus and/or were positioned abnormally distant from the micropylar end. The majority of embryo sacs appeared largely devoid of cellular contents (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e. I, J). At flowering, the stigma becomes receptive to pollen, and the perianth segments senesce, but the pistil does not; however, but the ovary fails to expand because of the failure of fertilisation, and the integuments and nucellus were shriveled up and separated from the embryo sac, and the pistil terminates its development and fails to set fruit.\u003c/p\u003e\u003cp\u003e\u003cb\u003eCorrespondence between the external characteristics of flower buds and the internal developmental processes of female and male gametophytes\u003c/b\u003e\u003c/p\u003e\u003cp\u003eIn Michelia alba, male and female gametophytes develop asynchronously, with stamens mature earlier than before pistils. Internal gametophyte development closely correlates with flower bud morphology and size, as shown in Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. At the onset of microsporocyte meiosis, pistils were only beginning to undergo archesporial cell differentiation. As stamen development progresses to the late MMC stages, ovule primordia begins to form. By the time microspores are released and reach the uninucleate stage, internal differentiation of the megasporangium is underway within the ovule. Subsequently the development of the embryo sac accelerates. At anthesis, both male and female gametophytes were fully mature, coinciding with pollen release (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e. ).\u003c/p\u003e\u003cp\u003e\u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e\u003ccaption language=\"En\"\u003e\u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\u003cdiv class=\"CaptionContent\"\u003e\u003cp\u003eCorrelation between floral bud size and developmental stages of stamens and pistils in Michelia alba\u003c/p\u003e\u003c/div\u003e\u003c/caption\u003e\u003ccolgroup cols=\"5\"\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e\u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e\u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e\u003cthead\u003e\u003ctr\u003e\u003cth align=\"left\" colspan=\"2\" nameend=\"c2\" namest=\"c1\"\u003e\u003cp\u003eAverage size/mm\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c3\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eExternal morphological features\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c4\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMicrospore and Male Gametophyte Stage\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c5\" morerows=\"1\" rowspan=\"2\"\u003e\u003cp\u003eMegaspore and Female Gametophyte Stage\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003ctr\u003e\u003cth align=\"left\" colname=\"c1\"\u003e\u003cp\u003eLongitudinal\u003c/p\u003e\u003c/th\u003e\u003cth align=\"left\" colname=\"c2\"\u003e\u003cp\u003eTransverse diameter\u003c/p\u003e\u003c/th\u003e\u003c/tr\u003e\u003c/thead\u003e\u003ctbody\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e11.30\u0026thinsp;\u0026plusmn;\u0026thinsp;0.75\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e3.79\u0026thinsp;\u0026plusmn;\u0026thinsp;0.25\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 3 layers of tepals, thickly leathery, Light green, and leathery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eArchesporial cell\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e13.08\u0026thinsp;\u0026plusmn;\u0026thinsp;0.65\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 3 layers of tepals, thickly leathery, Light green, and leathery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003ePrimary parietal cell and primary sporogenous cell\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003e-\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e16.78\u0026thinsp;\u0026plusmn;\u0026thinsp;0.31\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e4.73\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 3 layers of tepals, thinly leathery, Light green and leathery\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMicrospore mother cell (MMC)\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eOvule primordium\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e19.21\u0026thinsp;\u0026plusmn;\u0026thinsp;2.41\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.31\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eFirst outermost whorl of tepals deciduous, greenish green\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eMeiosis in MMCs\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eArchesporial cell\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e23.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.48\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e5.89\u0026thinsp;\u0026plusmn;\u0026thinsp;0.70\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 3 layers of tepals-second layer of tepals dehiscent\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eTetrad stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eSporogenous cells - megaspore mother cell (MMC)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e26.36\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.03\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 2 layers of tepals, thinly leathery, green\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUninucleate microspore stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMegaspore mother cell (MMC)\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e27.11\u0026thinsp;\u0026plusmn;\u0026thinsp;2.82\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e7.92\u0026thinsp;\u0026plusmn;\u0026thinsp;1.03\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 1 outermost whorl of tepals, thinly leathery, green\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eVacuolated uninucleate microspore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eUninucleate embryo sac\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e29.21\u0026thinsp;\u0026plusmn;\u0026thinsp;1.54\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.12\u0026thinsp;\u0026plusmn;\u0026thinsp;1.24\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eOuter 1 outermost whorl of tepals, thinly leathery, green\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eUninucleate microspore\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eBinucleate embryo sac - quadrinucleate embryo sac\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e34.58\u0026thinsp;\u0026plusmn;\u0026thinsp;2.52\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e8.86\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eTertiary annular stipule cracked or green silk white bud stage\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eBicellular pollen grain\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eEight-nucleate embryo sac\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003ctr\u003e\u003ctd align=\"left\" colname=\"c1\"\u003e\u003cp\u003e44.67\u0026thinsp;\u0026plusmn;\u0026thinsp;2.04\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e\u003cp\u003e9.05\u0026thinsp;\u0026plusmn;\u0026thinsp;1.45\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c3\"\u003e\u003cp\u003eWhite buds with green buds with green outer tepals state\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c4\"\u003e\u003cp\u003eDinucleate mature pollen\u003c/p\u003e\u003c/td\u003e\u003ctd align=\"left\" colname=\"c5\"\u003e\u003cp\u003eMature embryo sac\u003c/p\u003e\u003c/td\u003e\u003c/tr\u003e\u003c/tbody\u003e\u003c/colgroup\u003e\u003ctfoot\u003e\u003ctr\u003e\u003ctd colspan=\"5\"\u003eNote: Data are presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation.\u003c/td\u003e\u003c/tr\u003e\u003c/tfoot\u003e\u003c/table\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eIn Michelia alba, the development and maturation of stamens and pistils generally followed patterns typical of angiosperms and closely resembled those of related species such as Michelia figo [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], Michelia maudiae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], and Michelia glauca [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The pollen sac wall is composed of four layers: the epidermis, endothecium, middle layer, and tapetum. Microspore tetrads were arranged in three configurations: cross-shaped, isobilateral, and \u0026ldquo;T\u0026rdquo;-shaped, while the mature pollen grains were of the\u003c/p\u003e\u003cp\u003ebicellular type. In the anatropous ovule, the megaspore tetrads were linearly arranged, and the chalazal megaspore\u0026mdash;farthest from the micropyle\u0026mdash;developed into a functional uninucleate embryo sac. This process followed a monosporic pattern and resulted in a Polygonum-type mature embryo sac. The initiation of stamen development precedes that of the pistil, but both structures reached maturity simultaneously, potentially enabling self-compatibility. Interestingly, unlike in many other plants, a transient contraction of microspores was observed during the early tetrad stage in M. alba. This phenomenon has also been reported in other Magnoliaceae species, such as Michelia guangxiensis [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], Tsoongiodendron odorum [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], and Manglietia insignis [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], suggesting that such microspore contraction may be a common feature during microsporogenesis in this family.\u003c/p\u003e\u003cp\u003eSimilar to other Magnoliaceae species, Michelia alba exhibits poor sexual reproductive success. The few seeds that were occasionally formed typically exhibit poor development and fail to germinate, a manifestation of reproductive failure or sterility. Reproductive failure in plants can stem from both extrinsic and intrinsic factors. External causes often include imbalances in nutrient availability and environmental stresses, such as abnormal temperature, moisture, or light conditions. In Magnolia championii [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] and Woonyoungia septentrionalis [\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], for example, pollen abortion has been attributed to environmental fluctuations. Intrinsic factors encompass disruptions in genetic regulation, physiological processes, and metabolic pathways. In M. alba, multiple abnormalities were identified during the development of male and female gametophytes. In M alba. male reproductive organs, early shrinkage and inward folding of the anther wall during pollen mother cell formation reduces locule space. Alternatively, premature degradation of the tapetum may lead to nutrient insufficiency for microspores. During meiosis, chromosomal anomalies may arise, resulting in some microspores arresting at the uninucleate stage. Some microspores show clumping, abnormal shapes, thickened walls, or hollow pollen grains. Similar phenomena have been observed in Michelia guangxiensis [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], where delayed or incomplete degeneration of the tapetum leads to pollen abortion. Abnormal pollen grains and extremely low germination rates (sometimes below 0.01%) have also been reported in Manglietia aromatica [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], Liriodendron chinense [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], and Magnolia officinalis subsp. Biloba [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. On the female side, the proportion of gametophytes that develop to maturity is extremely low. Developmental failure mainly occurrs during the three mitotic divisions after the uninucleate embryo sac forms. Arrests have been observed at the one-, two-, and four-nucleate stages. Even in embryo sacs that reach the eight-nucleate stage, key functional cells\u0026mdash;such as antipodals, egg cells, or synergids, were frequently absent or only partially developed. In some cases, synergids were positioned too far from the egg cells to function properly, or the embryo sac developd as a hollow structure. Severe embryo sac abortion has also been reported in other Magnoliaceae species, including Michelia maudiae [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e], Michelia xichouensis [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e], and Manglietia insignis [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e], with sterility rates of approximately 25%, 70%, and 87%, respectively. Compared to stamens, developmental abnormalities in the pistils of M. alba were particularly pronounced. This observation supports the resource allocation theory in angiosperms, which proposes that under resource constraints, monoecious species favor male flower production to optimize biomass and reproductive output [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], since male reproduction is simpler and more efficient in terms of resource usage. Consequently, female gametophyte abortion poses a more critical constraint on reproductive success in endangered Magnoliaceae species [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. The anatomical features underlying these developmental failures were often governed by complex internal factors. For instances, Nie et al. [\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e] suggested that disruptions in endogenous hormone balance and differential gene expression under heat stress may play significant roles in sterility. Similarly, Li et al. [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] identified the MawuAP1 gene as a key regulatory element in floral organ development in Magnolia wufengensis. Therefore, future studies on plant reproduction should adopt integrative approaches, and hybrid breeding efforts must account for parental reproductive traits and genetic compatibility.\u003c/p\u003e"},{"header":"Conclusion","content":"\u003cp\u003eThis study, through anatomical analysis, provides the first systematic elucidation of the cytological mechanisms underlying reproductive abortion in Michelia \u0026times; alba. The research has revealed that in normally developing male gametophytes, microspore tetrads were arranged in crossed, symmetrical, or T-shaped configurations, and mature pollen was of the bicellular type. The female gametophyte developed as a monosporic Polygonum-type embryo sac, maturing into a 7-celled, 8-nucleate structure. The maturation timing of male and female gametophytes was synchronized, providing a basis for self-pollination. However, the primary causes of abortion were identified as abnormalities in both gametophytes. Abnormal male gametophyte development was characterized by premature contraction of the anther wall and precocious degradation of the tapetum, leading to microspore adhesion and the formation of collapsed pollen grains. Concurrently, abnormal female gametophyte development manifested as embryo sacs frequently being arrested at the uninucleate or binucleate stages, or exhibiting the absence of functional cells (such as the egg cell and synergids) during the 8-nucleate stage, with hollow embryo sacs commonly observed. This research clarifies the cytological basis of abortion in Magnoliaceae plants, fills a significant gap in the reproductive biology studies of Michelia \u0026times; alba, provides key technical support for the conservation and propagation of endangered Magnoliaceae species, holds substantial importance for maintaining species genetic diversity, and offers both a theoretical foundations and practical guidance for breeding innovation and the population restoration of economically important Magnoliaceae trees.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Information\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNo supplementary material was generated for this study. All supporting data are presented in the main manuscript figures and tables.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgment\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the National Natural Science Foundation of China (Grants 31260093 and 31560061), the Guangxi Forestry Science and Technology Promotion Demonstration Project (Guilin Scientific Research [2022] No. 1), and the Guangxi State-owned Qinlian Forestry Research Project. We thank the Guangxi Key Laboratory of Forest Ecology and Conservation for providing laboratory facilities. We are grateful to Dr. Zhu from Guangxi Vocational University of Agriculture for providing technical assistance in histological sectioning. Special thanks to the Guangxi University Campus Management Office for permitting plant sample collection.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConceptualization: L.-J.Z. and L.-Q.Z.; Methodology: H.-N.X. and Y.S.; Formal analysis: G.-L.W.; Investigation: L.-J.Z., H.-N.X. and Y.S.; Resources: L.-Q.Z.; Data curation: H.-N.X. and G.-L.W.; Writing\u0026mdash;original draft preparation: L.-J.Z.; Writing\u0026mdash;review and editing: L.-Q.Z. and G.-L.W.; Supervision: L.-Q.Z.; Funding acquisition: L.-Q.Z.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was funded by the National Natural Science Foundation of China (Grants 31260093 and 31560061), the Guangxi Forestry Science and Technology Promotion Demonstration Project (Guilin Scientific Research [2022] No. 1), and the Guangxi State-owned Qinlian Forestry Research Project.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Availability Statement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAll data supporting the findings of this study are available within the paper and within its Supplementary Materials published online.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of interest.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003ePublisher\u0026apos;s Note\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSpringer Nature remains neutral with regard to junisdictional caimsinpublished mapsand intitutionalaffliations.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLi HR, Zhou C, Wei W et al. 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Plant Molecular Biology Reporter (2012), *30*(3): 690\u0026ndash;698. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1007/s11105-011-0373-7\u003c/span\u003e\u003cspan address=\"10.1007/s11105-011-0373-7\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\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":"[email protected]","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":"Developmental anatomy, Gametophyte abortion, Megasporogenesis, Microsporogenesis, Michelia alba, Magnoliaceae","lastPublishedDoi":"10.21203/rs.3.rs-7327064/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7327064/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003e\u003cstrong\u003eBackground\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMichelia alba DC. (Magnoliaceae), economically significant tree species in Southeast Asia, extremely low natural fruit production due to reproductive failure. This study aims to characterize the anatomical causes of this phenomenon by investigating megasporogenesis, microsporogenesis, and gametophyte development.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResults\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAnatomical analysis revealed that microspore tetrads were arranged tetrahedrally, isobilaterally, or in T-shapes and that mature pollen was bicellular. Ovules were anatropous, forming linearly arranged megaspore tetrads; only the chalazal megaspore developed into a functional Polygonum-type embryo sac (7 cells, 8 nuclei). Critical abnormalities were identified:\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e1. Male gametophyte failure (\u0026gt;90% abortion): \u003c/strong\u003ePremature tapetum degeneration and abnormal anther wall contraction caused microspore adhesion and pollen collapse.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2. Female gametophyte failure (\u0026gt;90% arrest): \u003c/strong\u003eMost embryo sacs are arrested before maturity lacking essential cells (egg cells, synergids). Only 2 of the 200 examined sections contained mature embryo sacs.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConclusions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eConcurrent defects in male and female gametophyte development critically impair gametophyte functionality, explaining the low fruit set in M. alba. This study provides the first cytological elucidation of reproductive failure in this species and establishes a foundation for the conservation and breeding of endangered Magnoliaceae.\u003c/p\u003e","manuscriptTitle":"Anatomical Insights into Megasporogenesis, Microsporogenesis, and Gametophyte Development in Michelia alba: Causes of Reproductive Failure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-09-18 14:33:08","doi":"10.21203/rs.3.rs-7327064/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","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":"806e28e3-57db-41b1-b0e4-76d95f69747b","owner":[],"postedDate":"September 18th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-09-26T09:08:41+00:00","versionOfRecord":[],"versionCreatedAt":"2025-09-18 14:33:08","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7327064","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7327064","identity":"rs-7327064","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}