【目的】探索低温层积不同阶段油用牡丹‘凤丹’种子种皮结构、胚形态以及营养物质、酶活性的动态变化,为其休眠解除以及促进萌发提供理论参考。【方法】以低温层积5个阶段的‘凤丹’种子为材料,用扫描电镜(SEM)和体视显微镜观察种皮结构、种胚形态的变化,测定各层积阶段种子大小、吸水率的变化,同时测定种子中粗脂肪、可溶性蛋白、可溶性糖、淀粉含量的变化以及过氧化物酶(POD)、酸性磷酸酶(APA)活性的变化,分析营养物质含量、酶活性与‘凤丹’种子休眠解除的关系。【结果】‘凤丹’种皮不存在吸水障碍,浸种96 h时,吸水率为54.2%,达到吸水平衡。层积30 d时,种子纵径由8.97 mm显著增加至10.44 mm,增加了16.4%;横径由6.42 mm显著增加至8.87 mm,增加了15.0%;之后种子大小增加缓慢。SEM观察结果表明:种皮主要由角质层、栅栏层和薄壁细胞层3层结构组成,成熟种子、种皮栅栏组织中长柱状细胞排列整齐紧密,但栅栏层、薄壁细胞层中很多细胞间有较小的空隙;层积30 d后,角质层内侧出现了明显的孔洞,栅栏层细胞排列较之前疏松,透性加大。成熟种子的胚较小,层积30 d后子叶略有膨大,下胚轴略有增粗;胚根突破种皮后子叶进一步增大,胚根、下胚轴明显伸长和增粗,但上胚轴仍需层积2个多月才能得以延伸。‘凤丹’种子含油率高,层积处理前种子中粗脂肪含量占种子鲜质量的32.3%,是胚乳中的主要贮藏物质。种子休眠解除过程中,其可溶性糖含量呈下降—上升—下降的变化趋势,而淀粉、可溶性蛋白和粗脂肪含量则一直呈下降趋势;层积过程中,POD和APA活性逐渐增强。【结论】‘凤丹’种皮不存在吸水障碍,透水性不是限制种子萌发的重要原因;成熟的种子胚极小,需在10 ℃条件下层积一段时间,使胚进一步分化发育完全,完成生理后熟作用;胚根突破种皮后仍存在上胚轴休眠现象,需在5 ℃下低温层积较长时间以解除其休眠;层积过程中随着酶活性的不断增强,贮藏物质逐渐被分解,为种子的各种代谢活动提供能量,且粗脂肪的分解与转化是此过程中的主要供能物质。‘凤丹’种子层积过程中其生理生化指标的变化与种胚形态变化呈现高度的一致性。

【Objective】 Paeonia ostii ‘Feng Dan’ is an edible woody oil crop. Propagation is primarily carried out via seeds. However, under natural conditions, the germination of matured seeds normally takes at least four months, and this strong dormancy is a major restriction on its reproduction. Many studies have been carried out on the dormancy of ‘Feng Dan’ seeds which found that the germination was limited by endogenous inhibitors. However, the effects of the seed coat on dormancy and embryo morphological changes during dormancy breaking have not been studied. In addition, researches on the changes in seed nutrients have rarely been reported. Therefore, we explored changes in the seed coat structure, the morphology of the embryo and the nutrients of ‘Feng Dan’ seeds at different stratification stages to provide a theoretical reference for releasing its dormancy and promoting its germination. 【Method】 The ‘Feng Dan’ seeds were stratified at five different stages and were used as materials for the study. The morphology at each stage was observed by stereomicroscopy. Matured seeds (stage Ⅰ) and seeds stratified for 30 days (stage Ⅱ) were used to observe the seed coat structure in three different regions of the longitudinal section of the seed hilum, the chalazal region, and middle part with the help of an S-3000 scanning electron microscope (SEM). Additionally, during the stratification process, the changes in seed size (transverse and longitudinal diameters) and water absorption were measured. Indexes such as crude fat, soluble protein, soluble sugar, starch content, peroxidase (POD) and acid phosphatase (APA) activity were also studied. 【Result】 The seed coat of ‘Feng Dan’ did not hinder its water uptake. When soaked for 96 h, the water absorption was 54.2%, which was very close to the maximum result of 56.9% (144 h). After stratification for 30 days, the longitudinal diameter of seeds increased significantly from 8.97 mm to 10.44 mm, and the transverse diameter increased significantly from 6.42 mm to 8.87 mm. During the subsequent stratification, the seed size remained almost unchanged. The SEM images showed that the seed coat was mainly composed of three layers: the cuticle, the palisade and the parenchymatous. The seed coat structure of matured seeds showed that the long columnar cells in the palisade layer were arranged neatly and tightly, but some obvious gaps were found in the palisade and parenchymatous layers. After 30 days of stratification, the cells in the palisade layer were loosely arranged, and some small scattered holes between the cuticle and palisade layers were observed. These two obvious changes in seed coat structure may further increase the permeability of ‘Feng Dan’. The embryo of the matured seed was very small. After 30 days of stratification, the cotyledons became slightly larger and the hypocotyl became slightly thicker. When the radicle broke through the seed coat, the cotyledons were further enlarged, the radicle was obviously elongated, and the hypocotyls were thickened, but the seed still needed to be stratified for more than two months for the epicotyl to extend. Therefore, the undeveloped hypocotyl and epicotyl of ‘Feng Dan’ seeds could hinder its germination. During the dormancy breaking procedure, the content of soluble sugar in the seed showed a significant decrease initially (from 63.4 mg/g to 53.8 mg/g), followed by an increase (stages Ⅱ to Ⅲ) and then another decrease. In stage Ⅴ, the content of soluble sugar was 44.6 mg/g, a decrease of 29.8% from stage Ⅰ. The contents of starch decreased by 45.2%, from 26.7 mg/g (stage Ⅰ) to 14.7 mg/g (stage Ⅴ). Soluble proteins decreased by 57.8% from 93.4 mg/g (stage Ⅰ) to 39.3 mg/g (stage Ⅴ), and decreased rapidly during stages Ⅱ and Ⅲ. Before the stratification (stage Ⅰ), the crude lipid content of ‘Feng Dan’ seeds accounted for 32.3% of the fresh weight, which was the main storage substance in the endosperm. In stage Ⅴ, the content decreased by 65.0% to 11.3%, and the most rapid decrease occurred in stages Ⅲ to Ⅳ. During the stratification stages, POD activity increased rapidly, peaking at 52.1 μmol/(min·g) in stage Ⅲ, then decreased slightly (in stages Ⅲ to Ⅳ), followed by a slow increase (stage Ⅳ to stage Ⅴ). APA activity showed an increasing tendency, and the greatest value was 25.1 U/(min·g) in stage Ⅴ. The most rapid increase was during stage Ⅲ and Ⅳ when the radicle enlarged quickly. Overall, these indexes showed different changes during the stratification. The contents of starch, soluble protein and crude lipid showed a gradually decreasing trend coupled with an increase in POD and APA activities. Owing to the higher activities of enzymes, the metabolism in the seed was accelerated, and the nutrition and energy required for germination were produced continuously. 【Conclusion】 The seed of ‘Feng Dan’ had good water permeability and was, therefore, able to take in water successfully during germination; the permeability of the seed coat was not the reason for its dormancy. The embryo size of mature seeds was very small, and the embryo gradually differentiated and developed when stratified at 10 ℃; therefore, the observations of the morphology of embryos indicated that the hypocotyl and epicotyl of ‘Feng Dan’ were the main reasons for its dormancy. During the stratification, the nutrition contents decreased with an increase in the activities of POD and APA. Crude lipids were the main source of energy during dormancy breaking. The dynamic changes in these physiological and biochemical indexes in ‘Feng Dan’ seeds were highly consistent with the morphological changes of the embryo.