A study on embryo after-ripening development of Euscaphis konishii seeds during natural sand stratification

<bold>Objective</bold> <italic>Euscaphis konishii</italic> Hayata is a plant species unique in China，valued for both medicinal and ornamental purposes.Its seeds exhibit complex dormancy characteristics，typically requiring natural sand stratification until the spring of the third year to meet production requirements（achieving a germination rate of around 75%）.Exploring the developmental process and physiological mechanisms of its embryo after-ripening can provide a theoretical basis for the research on dormancy breaking and seedling propagation techniques. <bold>Method</bold> Seeds from naturally matured <italic>E.konishii</italic> fruits were subjected to natural sand stratification for 0，30，60，90，120 and 150 days.Morphological changes in the embryo，germination characteristics，storage substances，reactive oxygen species，and endogenous hormone levels were measured at each stage.Furthermore，principal component analysis（PCA）was employed to systematically analyze the physiological dynamics throughout the stratification process. <bold>Result</bold> （1）The germination rate and germination index of <italic>E. konishii</italic> seeds increased significantly with prolonged stratification time.The germination rate was 0% at 0 d，which increased to （24±2.16）% after dehulling.After 150 d of stratification，the germination rate reached （35±1.29）%，and notably increased to （89.5±1.26）% upon dehulling.The embryo rate also showed an increasing trend，with a significant increase observed at 60 d compared to 0 d and 30 d.（2）The embryo after-ripening development could be divided into three stages：Stage I（0-30 d），characterized by a relatively compact embryonic tissue structure；Stage II（60-90 d），featuring elongation of radicle and hypocotyl cells and expansion of the plumule；Stage III（120-150 d），marked by rapid growth of embryonic organs and degradation of the endosperm.（3）Soluble sugar and soluble protein contents increased significantly during stratification，with α-amylase activity peaking at 60 d.The contents of abscisic acid（ABA），jasmonic acid（JA），zeatin riboside（ZR），and superoxide anion（O₂·⁻）decreased significantly，whereas the contents of indole-3-acetic acid（IAA）and hydrogen peroxide（H₂O₂），as well as the ratios of IAA/ABA，（GA₁₊₃）/ABA，（GA₄₊₇）/ABA，and （GA₁₊₃+IAA+ZR）/ABA，increased continuously.（4）PCA revealed that ABA and JA exhibited high negative loadings on PC1，while IAA/ABA，（GA₁₊₃+IAA+ZR）/ABA，germination rate，GA/ABA，embryo rate，soluble sugars，and（α+β）-amylase activity exhibited high positive loadings on PC1.The time points of 60 d and 90 d were identified as the turning points where sample points shifted from the negative to the positive axis of PC1. <bold>Conclusion</bold> <italic>E. konishii</italic> seeds complete after-ripening after 150 d of natural sand stratification（autumn-winter-spring），with the period of 60-90 days identified as the critical phase.The degradation and conversion of storage substances provide essential energy support for embryo development.The increases in IAA，H₂O₂，IAA/ABA，and GA/ABA ratios，coupled with the decrease in ABA，JA，and O₂·⁻ contents，collectively create a favorable internal environment for the after-ripening development of <italic>E. konishii</italic> seeds.