| Citrus is one of the most important economic fruit trees, and its germplasm resources can be preservated effectively with embryogenic calli. However, embryogenic capability may be gradually lost during the preservation of calli. Somatic embryogenesis is a way of plant cell totipotency, and a new pathway of rapid propagation. Mechanism of somatic embryogenesis as well as its regulation and long-term preservation of embryogenic capability are very important. To explore the mechanism of somatic embryogenesis in Citrus, cytology, ultracytochemistry, soluble protein contents and its composition analysis, activities of some relative enzymes and isoenzyme analysis, endogenous hormonal levels and endogenous polyamines levels were studied by using the long-term (about 8 years) subcultured calli with different embryogenic capability as materials. The results are as follows:1. In Citrus, the embryogenic calli consisted of embryogenic and non-embryogenic cells. The embryogenic cells were characterized by large nucleus, dense cytoplasm, abundant organelles. While the non-embryogenic calli consisted of non-regular shaped cells with big central vacuole and thin cytoplasm. Somatic embryos were found to be mainly originated from single embryogenic cells on the surface of embryogenic calli. 10 days after being transferred to the differentiation medium, multi-cellular proembryos were formed, then great amount of globular embryos were observed at day 20, specific differentiated globular embryos and a few heart-shaped embryos were formed at day 30. Both embryogenic cells and globular embryos had strong metabolism, and accumulated plenty of starch grains. Globular embryos developed cotyledonary embryos and regenerated plantlets when transferred to other mediums.2. It was showed by ultracytochemical method that the spatiotemporal distribution and level of ATPase and calcium varied with the cellular physiologic state. In the cells of non-embryogenic calli, ATPase located in vacuoles and involved in the function of the hydrolysis of vacuole. The cells of embryogenic calli had higher ATPase activities, which located on cell wall, intercellular space, plasma membrane and nucleus, providing energyembryo. However, no obvious changes were observed regarding distribution and levels of calcium in cells of non-embryogenic callus before and after being transferred to differentiation medium, but there was an increase of calcium in cytoplasm of embryogenic cells, it was suggested that the increase of calcium involved in inducing the embryogenic cells to initiate embryogenesis. At globular embryo stage, calcium concentrated on plasma membrane and certain areas of cytoplasm; at cotyledonary embryo stage, calcium presented on cell wall and intercellular space.3. There were close relationship between embryogenic capability and soluble protein contents as well as anti-oxidase activity. It was showed that the higher somatic embryogenic capability was, the higher soluble protein contents and anti-oxidase activities would be. During 0-10d, SOD activity was found to be increased, it might involve in regulating the differentiating and developing process of embryogenic cells. At day 20, POD activity and soluble protein contents reached their maximum, which provided the metabolic substances and energy for the formation of globular embryos. Isoenzyme analysis indicated that there were specific bands at different stages of somatic embryogenesis, which could be molecular markers. Band P3 of POD isoenzyme was the specific band of embryogenic callus, and it might show the embryogenic capability. SDS-PAGE results showed that embryogenic callus had a new 88.1KD protein comparing with non-embryogenic callus, but only the difference of the express of protein compositions was observed at different stages of somatic embryogenesis.4. Endogenous hormonal levels and their balance played a key role in regulating somatic embryogenesis in Citrus. To keep the embryogenic capability of callus during long-term subcultured period, relat...
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