Morphological Observation on floral bud differentiation of Diospyros with different ploidies

【Objective】Flower buds of Diospyros are mixed buds, and their differentiation involves complex morphogenetic and physio- biochemical changes that directly influence the success of subsequent flowering. Diospyros species feature diverse ploidy evolutionary trajectories, with marked developmental differences among germplasm of different ploidy levels. This study aimed to clarify the temporal formation dynamics and morphological differences of floral organ primordia among D. lotus L., D. deyangensis, and‘Xiaoguo Tianshi’through systematic observation and analysis of their floral differentiation processes. The primary objective was to reveal the morphological basis underlying early flowering in D. deyangensis, thereby providing an anatomical framework for understanding ploidy-dependent floral development in Diospyros.【Methods】Perennial female plants of D. lotus L., D. deyangensis, and‘Xiaoguo Tianshi’cultivated in the National Germplasm Repository for Diospyros of Northwest A & F University (Yangling, Shaanxi) were used as experimental materials. Sampling encompassed two critical phases of floral differentiation: the early differentiation stage of current-season flower buds (May to August 2025) and the late differentiation stage of previous-season flower buds (February to May 2025). At the early stage, five vigorously growing new shoots from each germplasm were selected in the morning and sampled weekly to ensure consistent material quality and consistent growing environment. In the late stage, three current-year branches were collected every 4 days in the morning before flower buds emerged; after flower buds emerged, five branches were sampled every 2 days to track floral maturation. Three complementary observation techniques were employed to capture multidimensional developmental data. First, continuous external morphological observation was conducted covering three key developmental stages: early differentiation, dormancy, and flowering. Overall branch morphology was photographed using a digital camera; external phenotypic traits of mixed buds were imaged via a stereofluorescence microscope; buds were dissected under the microscope with fine forceps and dissecting needles to obtain high-resolution images of internal anatomic structures. Secondly, for scanning electron microscopy (SEM) analysis, samples collected from February to August 2025 were fixed in 2.5% FAA fixative, rinsed with phosphate buffer, and dehydrated sequentially in 30% , 40%, 50%, 70%, 90%, 100%, and 100% ethanol (15 min per gradient). They were then subjected to solvent replacement with isoamyl acetate to preserve their structure, dried using a K850 critical point dryer, mounted on metal stubs with conductive adhesive, sputter-coated with gold for 90 s, and observed under a field emission SEM for fine-scale characterization of floral primordia. Thirdly, for paraffin sectioning, buds were incubated in a specialized fixative (85∶5∶5∶5, V/V of 70% tert-butanol, 35%-40% formaldehyde, propionic acid and glycerol), subjected to vacuum infiltration until all gas bubbles were eliminated and the materials were fully submerged, then stored at 4 ℃ for ≥48 h to ensure thorough fixation. After fixation, the samples were dehydrated through an ethanol gradient, infiltrated with molten paraffin wax, embedded in paraffin blocks, and sectioned into 5-8 μm slices using a microtome. The sections were then stained with hematoxylin-eosin (HE), mounted with neutral balsam, and observed under a light microscope to analyze internal bud development. These approaches collectively enabled systematic characterization of the dynamic development of external morphology and internal structure of mixed buds and female flowers.【Results】Comprehensive observations revealed that the three Diospyros germplasms shared a conserved centripetal sequence of floral organ differentiation and a consistent cross- annual developmental cycle: floral differentiation initiated in late May of the current year, followed by winter dormancy for low-temperature tolerance, and development resumed actively in the following spring until flowering. Inter-germplasm differences in external bud traits were observed: the bud scales of D. deyangensis and‘Xiaoguo Tianshi’were uniformly brown and densely pubescent, whereas those of D. lotus L. were dark brown to nearly black, with a thick, glossy waxy cuticle and rigid texture, suggesting enhanced abiotic resistance A distinct germplasm- specific marker was observed during the differentiation stage of stamen primordium: In D. deyangensis, the bases of stamen primordia and petal primordia displayed a stable pink hue, while those of D. lotus L. and‘Xiaoguo Tianshi’remained green. This color dimorphism persisted after flowering. The petals of D. lotus L. were creamy yellow, those of D. deyangensis were pink, and those of‘Xiaoguo Tianshi’were pale yellow. All three accessions produced solitary female flowers, but they exhibited significant differences in floral morphology. Correlation analysis showed a significant positive correlation between ploidy level and flower bud longitudinal diameter:‘Xiaoguo Tianshi’had the largest buds, followed by D. deyangensis, and D. lotus L. had the smallest. Floral differentiation initiated synchronously in late May across all three germplasms, with bract differentiation proceeding in parallel. D. deyangensis was the first to enter sepal differentiation, consistently advancing 3-5 days ahead of the other two accessions. Both bract differentiation and sepal differentiation were completed by June across all germplasms, with minimal temporal variation. However, a critical developmental divergence emerged at the petal differentiation stage: D. deyangensis and‘Xiaoguo Tianshi’completed petal primordium differentiation by late July (prior to dormancy initiation), whereas D. lotus L. remained at the sepal primordium stage until the onset of dormancy, and did not initiate petal differentiation until March of the following year (after dormancy release).Petal differentiation in D. deyangensis and‘Xiaoguo Tianshi’occurred 7 months earlier than in D. lotus L., which facilitated the accelerated differentiation of the subsequent stamen and carpel primordia. This temporal advantage ultimately led to an earlier initial flowering period for D. deyangensis and ‘Xiaoguo Tianshi’compared with D. lotus L.【Conclusion】The early-flowering trait of D. deyangensis is linked to three pivotal developmental features: early initiation of floral differentiation, rapid differentiation progression during the pre-dormancy period, and significantly advanced petal differentiation. Among these factors, petal differentiation acts as the critical regulatory checkpoint underlying temporal differences in floral differentiation among Diospyros germplasm of varying ploidies. The 7-month advance in this stage allows D. deyangensis to complete the development of key floral organ primordia before dormancy, shortening its developmental cycle and contributing to its early-flowering phenotype.