不同倍性柿花芽分化进程观察

Morphological Observation on floral bud differentiation of Diospyros with different ploidies

摘要

【目的】通过对柿属植物二倍体君迁子、四倍体德阳柿和六倍体小果甜柿花芽分化过程的观察及分析，阐明不同倍性柿属植物间花器官原基形成时间与形态差异，揭示德阳柿早花特性的形态学基础。【方法】以多年生君迁子、德阳柿和小果甜柿雌株为材料，于花芽分化期定期采样，使用体视显微镜、扫描电子显微镜和石蜡切片技术，观察其混合芽及雌花的外部形态与内部结构发育过程。【结果】三份试材花芽分化均始于5月下旬，经历苞叶、萼片、花瓣、雄蕊和心皮原基的分化跨年度进行，均需经历休眠期。柿不同倍性花芽分化关键差异时期为花瓣原基分化期：德阳柿与小果甜柿于休眠前（当年7月下旬）完成，而君迁子于休眠解除（次年3月）后完成。德阳柿及小果甜柿的花瓣原基分化时间早于君迁子7个月，由此导致二者后续各花器官分化进程与初花期均较后者明显提前。此外，花蕾的大小与倍性呈显著正相关，且三者在外部形态（如芽鳞颜色、茸毛、蜡质）上存在明显差异。【结论】德阳柿的早花特性与其花芽分化启动早、休眠前分化进程快密切相关。花瓣原基分化期开始早晚是决定不同倍性柿花期进程的关键阶段。

Abstract

Abstract: 【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 multi-dimensional 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 primordi a 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), whereas D. lotus L. did not complete this stage until the following March after dormancy release. Consequently, the subsequent differentiation of all floral organs and the initial flowering time of D. deyangensis and ‘Xiaoguo Tianshi’ were significantly earlier than those of D. lotus L. 【Conclusion】The early flowering trait of D. deyangensis is closely related to its early initiation of floral bud differentiation and rapid differentiation progress before dormancy. The timing of petal primordium differentiation is the key stage determining the flowering process of Diospyros with different ploidies.

关键词

柿 / 德阳柿 / 倍性 / 花芽分化 / 形态观察 / 扫描电镜

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基金资助

国家自然科学基金青年基金(42406181)

国家重点研发计划(2022YFD2200403)

渭南市科技局渭南试验站建设专项(2024WNXNZX-3)