【作者】 牛遇达；

【导师】 刘桂丰； 李成浩；

【作者基本信息】 东北林业大学 ， 林木遗传育种， 2007， 硕士

【摘要】 北五味子是传统中药材，近年来在制药、食品和饮料等工业中的需求不断增加。但是，盲目的开采使野生北五味子急剧减少；选育出的北五味子无性系新品种，也因为没有成熟的无性繁殖体系而得不到推广。通过体细胞胚发生快速繁殖再生植株是有效的解决扩大北五味子资源量的措施之一，具有重要的应用价值。本文对北五味子的体细胞胚发生进行了研究，为实现北五味子的快速繁殖、种质保存打下基础。主要结果如下：以北五味子种子发芽后的子叶和下胚轴为实验材料，进行体细胞胚发生研究，结果表明：培养温度为30℃，以下胚轴为外植体，2，4-D浓度为4.0 mg·L^-1时，胚性愈伤组织诱导率最高，为18.3％；添加0.5 mg·L^-1 BA或2％蔗糖的1／3 MS培养基中形成的体细胞胚数量较多，3 mg胚性愈伤组织最多可产生349个体细胞胚；添加2％蔗糖的1／3MS培养基适合于体细胞胚的萌发和形成再生植株，发芽数和植株再生数分别为63个和19个；再生植株移栽成活率为95％。以北五味子的优良品种“红珍珠”冬芽为外植体，在添加4.0 mg·L^-1 2，4-D的MS培养基上可诱导出体细胞胚；25℃，GA₃浓度为1.0mg·L^-1，处理10 d可促进体细胞胚的形成、萌发和植株再生；4℃低温处理对于形成的体细胞胚数量影响显著，但是对体细胞胚萌发和植株再生却没有显著影响。对子叶阶段的畸形体细胞胚进行组织解剖学观察，认为筒形胚的顶端分生组织缺失，可能是导致其不能正常发育的主要原因。对北五味子“红珍珠”的体细胞胚形成的再生植株进行RAPD分析，结果表明，体细胞胚再生植株遗传上较稳定。30℃有利于添加2，4-D的培养基中体细胞胚直接诱导次生胚发生。对北五味子胚性细胞进行悬浮培养，添加1.0 mg·L^-1 2，4-D的MS液体培养基中，初始浓度为30 mL液体培养基中培养30 mg（鲜重）细胞可大量增殖，并具有很好的分散性。1／3 MS液体培养基有利于体细胞胚的发育和成熟，每30 mL悬浮培养的胚性细胞可产生大约3 400个体细胞胚。将子叶形胚转至不添加植物生长调节物质的1／2 MS半固体培养基中，可发育成植株，植株再生率为11.2％。更多还原

【Abstract】 The fruits of S. chinensis have long been used as traditional Chinese medicine. Recently, its demands increased dramatically for employing in pharmaceutical, food and drink industries. As a result, over exploitation has led to a rapid decline of S. chinensis in nature. The new breed of S. chinensis also didn’t have proper organized cultivation. Somatic embryogenesis is considered by many as a cost-efficient method for producing uniform plants. In this paper, we study for somatic embryogenesis in S. chinensis.Somatic embryogenesis were induced from cotyledonary leaf and hypocotyl explant-derived of S. chinensis. When culture temperature was 30℃, the induction rate of friable embryogenic callus was best from hypocotyls explants on MS semi-solid medium supplymented with 4.0 mg·L^-1 of 2,4-dichlorophenoxyacetic acid. The more number of somatic embryo was obtained when embryogenic callus transferred to MS medium containing 0.5 mg·L^-1 BA or 1/3 MS medium with 2% sucrose, the most number of somatic embryo was 349 per 3 mg. Both the number of germinated somatic embryos and regenerated plantlets were best when somatic embryos developed in 1/3 MS medium with 2 % sucrose, which were 63 and 19. Approximately 95% of the plantlets were successfully transplanted to soil and grew into fertile plants.Resting bud of S. chinensis Baill cultured on medium supplemented with 4 mg·L^-1 2,4-D produced somatic embryos directly from the surface of explants without intervening callus formation. When culture temperature was 30℃, the most number of somatic embryos was obtained in 1 mg·L^-1 concentration of gibberellic acid which cultured 10 days. It also had the most germination and regeneration in this treatment. There was significant difference in the number of somatic embryo with low temperature treatment which was 4℃, but there were no effect in germination and regeneration. Cotyledonary somatic embryos were observed by histological method. The results indicate most abnormal cotyledonary somatic embryos laking apical meristem differentiation, it maybe the reason why abnormal somatic embryos couldn’t develop into plantlets. Random amplified polymorphic DNA analyses found no evidence of genetic variation in the embryogenic lines. The embryogenic system used in this study appeared to be suitable for true-to-type clonal propagation of mature tissue of Schisandra chinensis Baill. It was avail for second somatic embryogenesis in 2,4-D when cultured at 30℃.An efficient somatic embryogenesis and plant regeneration protocol was developed for S. chinensis Baill using embryogenic cell suspensions and optimized media conditions. Fast growing and well dispersed embryogenic cell suspensions were developed within two months when embryogenic calli were transferred to MS Iiquid medium containing 1.0 mg·L^-1 2,4-D. 1/3 MS medium was the best for both overall growth and development of somatic embryos in liquid culture. Over 3 400 viable somatic embryos were produced from one 150 mL flask with an initial cell density of 30 mg in 30 mL medium. Germinated somatic embryos developed in liquid medium converted into plantlets at a low frequency（11.2%） after transferred to halfstrength MS semi-solid medium.更多还原