【作者】 刘华英；

【导师】 肖浪涛；

【作者基本信息】 湖南农业大学 ， 植物学， 2003， 博士

【摘要】 柑橘是世界上有重要经济价值的果树之一，利用柑橘胚性愈伤组织可有效地保存其种质资源。但植物愈伤组织在保存过程中会逐渐丧失胚性能力。体胚发生是植物细胞全能性的一种表达方式，是快速繁殖的新途径。体胚发生的机理及其调控以及胚性能力的长期保持是体胚发生中的重要研究课题。以具有不同体胚发生能力的长期继代培养的柑橘愈伤组织为材料，分别从组织细胞学、超微细胞化学、可溶性蛋白含量及组分分析、相关酶活性及同工酶分析、内源激素水平和内源多胺水平等方面对柑橘体胚发生的机理进行了研究。研究结果如下： 1．长期继代培养的柑橘胚性愈伤组织由胚性细胞和非胚性细胞组成。胚性细胞核大，质浓，富含各种细胞器。而非胚性愈伤组织由无规则状态的细胞组成，高度液泡化，胞质稀少。柑橘体胚发生以单细胞外起源为主。分化培养10d时形成多细胞原胚，20d时形成大量球形胚，30d时由具有一定分化的球形胚和少量心形胚组成。胚性细胞和球形胚都积累大量淀粉粒，代谢活跃。球形胚转移到新培养基上，能进一步发育并再生植株。 2．超微细胞化学研究表明，ATP酶和Ca²⁺的时空分布随细胞的生理状态而异。非胚性愈伤组织细胞的ATP酶定位于液泡，与液泡的水解功能有关。胚性愈伤组织细胞的ATP酶活性高，定位于细胞壁、胞间隙、质膜及细胞核，为物质的吸收和积累提供能量。胚性细胞和球形胚细胞具有较高的质膜ATP酶活性，子叶胚时降低。而Ca²⁺的分布及水平在非胚性愈伤组织分化培养前后无明显变化，而胚性愈伤组织分化培养后胞质Ca²⁺浓度增加，推测其参与了诱导胚性细胞启动胚胎发育。球形胚中，Ca²⁺集中分布于质膜和胞质一定区域。子叶胚中，Ca²⁺主要分布于细胞壁和胞间隙。 3．可溶性蛋白含量和抗氧化酶活性与体胚发生能力密切相关。体胚发生能力强的愈伤组织具有较高的可溶性蛋白含量和抗氧化酶活性。高SOD活性参与调节胚性细胞的分化和发育过程，高POD活性和蛋白水平为大量球形胚形成提供物质和能量代谢基础。POD和淀粉酶同工酶在体胚发生的不同时期有特征酶带，可作为跟踪体胚发生的分子标志。POD同工酶的P₃带是胚性愈伤组织特有的酶带，表明胚性存在。SDS-PAGE结果显示，胚性愈伤组织比非胚性愈伤组织增加88.1KD的蛋白，而体胚发生不同时期，蛋白质组分无明显变化，仅在表达量上有差异。 4.内源激素水平和激素平衡对柑橘体胚发生起重要的调节作用。愈伤组织在长期继代培养过程中胚性能力的保持需要较高水平的CTK、IAA和ABA，GA3具有负作用。具有不同胚性能力的材料之间，随着胚性能力的下降，其内源CTK、IAA和ABA水平也下降，GA3则升高。ABA与CTK、IAA之间的平衡使胚性愈伤组织在继代培养时保持在胚性状态，不向体胚发生方向进行。分化培养后，GA3、IAA明显降低，ABA明显升高，形成AB刀CTK和ABAjIAA峰值，有利于胚性能力的表达，形成多细胞原胚。20一30d，较低水平的ABAjIAA比值和IAA/CTK水平的回升，导致球形胚难以进一步发育和次生胚形成。体胚发生能力越弱的材料其内源激素变化规律越接近非胚性愈伤组织。 本研究还表明，IAA氧化酶活性与内源IAA代谢密切相关;向培养基中释放是内源激素代谢的另一种途径。此外，采用梯度洗脱在柑橘愈伤组织内源激素的HPLC分析中的分离效果较好。 5.优化了植物多胺的HPLC测定条件，确定在37℃苯甲酞化反应30min，检测波长230lun，流速0.7ml·min一，效果最佳。在此基础上，对柑橘愈伤组织内源多胺进行了分析，发现柑橘体胚发生能力与内源多胺水平正相关，无体胚发生能力的非胚性愈伤组织内源多胺水平低。分化培养早期，较高的Put及多胺含量的增加有利于柑橘体胚发生，外源Put处理的结果为此提供了更多证据。多胺含量的增加与ADC活性升高同步。随着体胚发育，Put降低，Spd、SPm先后达到最大值，推测Spd主要参与球形胚形成，Spm与球形胚的进一步发育有关。后期维持较高的Put水平和P叫Spd比值，引起不正常生长，是次生胚形成的又一重要原因。更多还原

【Abstract】 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更多还原