| In this experiment, mature embryos were used as explants to established in vitro regeneration system in Citrus microcarpa Bunge, and in vitro conservation of plantlets without subculture was also conducted. At the same time, ultrastructural observation and changes of calcium distribution through transmission electron microscopy were performed using ultra-thin sections of leaves preserved at all stages during conservation for trying to reveal the in vitro conservation mechanism of Citrus microcarpa plantlets that could be conserved for a long time without subculture from a microcosmic angle. The main results were as follows:1 Establishment of in vitro propagation system of Citrus plantletsCitrus microcarpa plantlets were used as materials to induce adventitious buds, and in vitro propagation system was then established. Moreover, the effects of different material parts of plantlets, MS salt concentrations, different sucrose concentrations, different hormones and their proportions on induction and regeneration culture of adventitious buds were compared. The results showed that after cultured in the dark for two weeks, basal morphological part of hypocotyls was inserted into the medium MS + 6-BA 1 mg ? L-1 + NAA 0.1 mg ? L-1 medium, which hold the greatest budding rate, up to 85.6%. Explants cultured in the dark for some time was facilitate to form callus rapidly, which increased the budding rate of epicotyls. However, high concentration of 6-BA and NAA inhibited the formation of epicotyls. The best medium for shoot proliferation was 1/2MS + 1.0 mg ? L-1 BA +0.2 mg ? L-1 KT, and the proliferation rate reached 7.29. But the optimum shoot rooting medium was 1/2 MS + 0.2 mg ? L-1 IAA +0.5 mg ? L-1 IBA with 77.8% rooting.2. Long-term in vitro conservation of plantlets without subculture in Citrus microcarpaThe mature embryos were used as explants to preserve Citrus microcarpa germplasm. In this study, citrus plantlets were cultured on MS medium without hormones. When the medium nearly completed water loss, and then 3g L-1 semi-liquid agar MS medium was added into the bottle. The results showed that citrus could be conservated for a long time without adding any growth regulator, the plantlets was preserved up to 3 year. The survival rate of plantlets conserved for 1 year was 88.9%; but the survival rate of plantlets conserved for 2 years was 73.3%;while the survival rate of plantlets sconserved for 3 years was 44.4%.3 Ultrastructural observation during in vitro conservation of Citrus microcarpa plantletsThe plantlets of Citrus microcarpa obtained from nucellar embryo culture were used as materials, which were divided into 4 stages: one month, one year, two years and three years, according to the length of conservation time. And the leaves were selected to perform ultrastructure observation and comparison among these four stages. The results showed that the cell senescence pace gotten slower during conservation. There were no significantly senescence characteristics of chloroplast in the first tow years. It was observed that some chloroplast's adventitia expanded and ruptured when preserved for three years. The slower senescence pace of cell provided evidence for citrus plantlets conserved for a long time without subculture from a microcosmic angle.4 Studies of calcium distribution changes during in vitro conservation of Citrus microcarpa plantletsThe concentration and distribution of Ca2+ in cells had some correlation with plant senescence. The changes of Ca2+ could reflect the senescence condition of cell. Citrus microcarpa plantlets were divided into four stages: one month, one year, two years, and three years, according to the length of conservation time, which was studied by ultrastructural observation of the leaves. The results showed that with the increase of conserved time, Ca2+ was transferred to the cytosol, nucleoplasm and cell gap from cell sap. Cell could regulate the concentration of Ca2+ itself to make it maintain at a low level to delay the aging of the cell.
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