| Phellodendron amurense Rupr. is of genus Phellodendronand family Rutaceae. Its bark is the source of traditional Chinese drug "Guan Huang Bai", i.e., Cortex phellodendron amurensis. This medicine is traditionally used to clear away heat and dry dampness, mainly attacks dampness-heat in lower energizer and is the main drug of some certain prescriptions. Currently this medicine comes mainly from the wild populations. The natural resources of P. amurense has largely decreased and this plant has been considered an endangered species, being listed as first-class national key protective wild medicinal species, second-class national key protective wild plants, and China’s rare plants. Because P. amurense is precious timber species, important Chinese medicinal materials and ancient relic plants, the protection and research on P. amurense is of scientifically significant.Basic research is important to protect the wild populations and conserve the genetic diversity of P. amurense, for example, further exploring the biological and ecological characteristics, analyzing the endangered factors, finding the effective conservation methods, building a quantitative index system and interpreting the inherent rule of growth and renewal with modern scientific ways. To explore the characteristics and detect the reasons of P. amurense seed dormancy, analyzing the dynamic changes of endogenous hormones in the process of seed germination is very necessary.In this study, we discussed the methodology of high performance liquid chromatographic (HPLC) with four kinds of endogenous hormones in P. amurense seeds. We then optimized the extraction and purification methods of endogenous hormones, and set up HPLC detection method of four kinds of endogenous hormones. By this method, the average recovery rates were79.37%,86.70%,77.31%, and90.07%for GA3, IAA, ABA, and ZT, respectively.We discussed the relationship between the endogenous hormones content changes and the seed dormancy breaking and seed germination rate by measuring the endogenous hormone content changes in the process of dormancy breaking of different dormancy degree seeds. We found that for non-deep dormant seeds in the process of dormancy breaking, the content of GA3, IAA and ABA are highly linearly related to the seed germination, with the correlation coefficient being RGA3=0.812,RIAA=0.834, and RABA=-0.906. The total hormone contents that promote seed germination are highly linearly related to the seed germination, with the correlation coefficient being RZ+G+I=0.894, and RZ+G=0.894; the ratio of hormones of promoting germination and inhibiting germination are highly linearly correlated to the seed germination, with the correlation coefficient being RZT/ABA=0.875, RGA3/ABA=0.949, RIAA/ABA=0.935, RZ+G+I/A=0.938, and Rz+G/A=0.938. Thus, the content changes of GA3, IAA, ABA and the ratios between ABA and hormones of promoting germination are of great significant to dormancy breaking of non-deep dormancy seed. The content of GA3, ABA are highly linearly related to the seed germination for moderate-deep dormant seeds in the process of dormancy breaking, with the correlation coefficient being RGA3=0.775, and RABA=-0.527; the ratio of hormones of promoting germination and inhibiting germination is highly linearly correlated to the seed germination, the correlation coefficient:RZT/ABA=0.516, RGA3/ABA=0.901, Rz+G/A=0.808, and RZ+G+I/A=0.808, respectively. Thus, the content changes of GA3, ABA and the ratio between ABA and hormones of promoting germination are important to dormancy breaking of moderate-deep dormancy seed. For the dormancy breaking of deep dormancy seeds, such as the seeds came from Qiqihar, northern China, the content of IAA and ABA significantly influences the seed germination, with the correlation coefficient being RIAA=0.689, and RABA=-0.650; the ratio of hormones of promoting germination and inhibiting germination and the seed germination is highly correlated, the correlation coefficient:RZT/ABA=0.700, RIAA/ABA=0.691, and Rz+G+I/A=0.590, respectively. Thus, the content changes of IAA, ABA and the ratio between ABA and hormones of promoting germination is of great significance to dormancy breaking of seed from Qiqihar. For the seed from Beijing (Institute of medicinal plant development, IMPLAD), the content of IAA, and GA3is highly linearly correlated with the seed germination, the correlation coefficient: RIAA=0.928,RGA3=0.667; IAA/ABA, G+I, G+I/A and seed germination has highly linear correlation, the correlation coefficient:RIAA/ABA=0.812, RG+I=0.812, RG+I/A=0.812. Thus, the content changes of IAA, GA3and the ratio between ABA and hormones of promoting germination is of great significance to dormancy breaking of seed from IMPLAD, Beijing. We concluded that the relationship between dormancy breaking of non-deep dormancy seed and GA3, IAA, ABA and the ratio of hormone were more close; the relationship between dormancy breaking of moderate-deep dormancy seed and GA3, ABA and the ratio of hormone were more close; the relationship between dormancy breaking of deep dormancy seed and IAA, ABA and the ratio of hormone were more close; The conclusion was similar to the former results that the effect was not the same when we used the same exogenous hormone to break dormancy of different dormant degree seeds.Further, we observed the embryo of seed with4℃low temperature stratification, and found that the transparent degree of seed embryo and seed dormancy breaking has a certain relevance. Paraffin section microscopic observation showed that, with the increase of stratification time, the activities of hypocotyl gradually became obvious and hypocotyl gradually differentiated into column; oil body of storage cells in embryo became bigger, and evenly distributed, and then concentrated in the center of the cell. We believe that the morphology changes of embryo are mainly embodied in the internal cell differentiation in the process of dormancy breaking. |
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