【作者】 乔琦；

【导师】 肖娅萍；

【作者基本信息】 陕西师范大学 ， 植物学， 2005， 硕士

【摘要】 体细胞胚发生途径是细胞全能性表达的最完全方式,深入研究体细胞胚的发生发育过程对于揭示细胞分化、发育、形态发生与合子胚发育等重大理论问题的机制以及真核细胞中基因表达与调节控制等具有十分重要的意义。 防风(Saposhnikovia divaricata(Turcz.)Schischk.)系伞形科多年生草本植物,为国家重点保护的野生药材之一。药用部位是植物的干燥根,具有解热、镇痛、镇静、抗炎等作用。目前关于防风体细胞胚发生发育的研究主要集中于超微结构以及核型分析等方面,关于组织培养中畸形胚的发生与控制、体细胞胚发生发育的生化基础以及基因表达和调控等方面尚未见相关报道。 本论文在组织培养的基础上首次探讨了防风畸形胚的发生和控制;观察到罕见的多年生植株组培苗的开花现象和畸形胚形成果实的现象,并初步探讨了其发生机制;研究了防风体细胞胚发生发育过程中抗氧化代谢系统、多糖等物质的含量动态变化;淀粉粒、蛋白体和脂体等后含物的动态分布等,为防风种质资源的保护以及体细胞胚的研究提供基础资料。 本实验以降低畸形胚的发生率为目的优化了防风的组织培养体系。研究发现防风体细胞胚的培养过程需要较复杂的培养基顺序变化。生长素是诱导胚性细胞发生的关键因素,细胞分裂素起协同作用,ABA能有效控制防风畸形胚的发生。分化培养基中的蔗糖浓度适当提高至4%～5%可增加渗透势,能减少玻璃化胚的发生和抑制早熟萌发的趋势。减少接种密度并将愈伤组织按其颗粒的自然状态分割成较小的块,或采用将愈伤组织悬浮培养24h后再转入固体培养基中,均能降低畸形胚的发生率。 长期继代培养使防风染色体数目发生变异,但是通过体细胞胚发生途径形成的再生植株仍大多是二倍体,说明染色体组的相对完整性是体细胞胚发生潜力实现的重要基础。 利用连续石蜡切片技术研究了防风体细胞胚发生发育的显微结构。发现体细胞胚与合子胚不同的是,胚柄分化不明显,说明该结构在体细胞胚发育中并非不可代替,推测离体培养条件与细胞的相互作用代替了胚柄与胚之间的相互作用,也可能是造成畸形体细胞胚的发生率远高于合子胚的原因之一。在发育过程中,体细胞胚始终与周围细胞存在明显界限,体细胞的壁显著加厚,而周围细胞处于半解体和解体状态。 利用半薄切片技术能较清楚的观察到细胞内淀粉粒、蛋白体和脂体等后含物的变化。在组织培养中淀粉粒的出现是胚性细胞开始分化的一个标记,淀粉的代更多还原

【Abstract】 The way of somatic embryogenesis is the best expression of cell totipotency. Study on the somatic embryogenesis and development of Saposhnikovia divaricata is significant for the best understanding of mechanism of the differentiation, development, occurrence of morphology, zygote embryogenesis, the theory of gene modulation and expression as well.Sapcshnikovia divaricata (Turcz.) Schischk. belongs to Umbelliferae, perennial herbaceous, it is one of most critical natural medicines in China. Its root can be used to relieve pain and cold, and has the ability of antibacterial, anti-allergy as well as tranquilizer. Current research on the somatic embryogenesis of Saposhnikovia divaricata has been focused on the ultrastructure and the stability of chromosome. Both the problem of occurrence and control of abnormal embryo in tissue culture, mechanism of the biochemical and molecular haven’t been studied and reported.The aim of this paper is to study the occurrence and growth of abnormal somatic embryos, and to observe and discuss the rare phenomena of blooming and bearing fruit in the abnormal somatic embryo of perennial herbaceous. The activities of superoxide dismutase (SOD), peroxidase (POD), polyoses have been studied in this thesis, and the distribution of ergastic substance such as starch, protein and oil have also been explored here. These studies just aim at keeping the germplasm resources of Saposhnikovia divaricata and providing the academic data for the field of somatic embryogenesis.The regeneration of tissue culture is optimized in order to control the occurrence of abnormal somatic embryos at first. The study shows that it is necessary to adjust the complex procedure of tissue culture and to seek the best combination of mediums. Auxin is key factor for initiating embryogenic cell, and cytokinin is cooperating. ABA can control abnormal somatic embryos efficiently. 4%-5% sucrose can be used to heighten osmotic pressure and decrease the rate of vitrification embryo and precociousness. In addition, lower the density of inoculation and divide the callus into granules according natural form, or utilize suspension culture for 24h all can decrease the rate of abnormal embryos.Though long-term subculture will cause chromosome variation, diploid plant is the更多还原