【作者】 孙丹；

【导师】 朴钟云；

【作者基本信息】 沈阳农业大学 ， 药用植物学， 2014， 博士

【摘要】 北五味子是我国东北地区珍贵的道地药材,在国内外市场深受消费者的青睐。随着对北五味子开发利用的不断深入,其原料的需求量也逐年增加,但由于北五味子的人工栽培起步较晚,育成的优良品系较少,植株间的生物学特性和品质差异较大,丰产稳产性极差,严重制约了北五味子栽培产业的发展。为了更好地发挥北五味子的资源优势,本研究对不同基因型北五味子资源的果实表型和木脂素含量进行了鉴定分析,从而为北五味子种质资源评价提供参考；为了进行良种繁育,本研究以具有优良果实性状的北五味子的休眠芽为材料,建立了北五味子具有遗传稳定性的体细胞胚途径植株再生系统,并在此基础上针对内源激素的动态变化对其发生过程中各个阶段所产生的影响进行较全面的研究。主要研究成果如下：1.综合比较11份北五味子样本果实大小、果穗鲜果重、干果重、果实干鲜比、果穗长、果实数目以及紧实度,结果表明不同样本的这些表型存在广泛变异,并且这些表型在不同年份间存在差异。三种颜色的北五味子果实中,粉果的平均鲜果重、干果重、干鲜比和紧实度均最大,红果果粒直径最大。筛选出表型较好的种源编号为B、D、E、G、P1和P2。2.通过多次试验条件的摸索,本研究确立了北五味子总木脂素的最佳高效检测体系：Symmetry(?)C18色谱柱(4.6mm×250mm,5μm),流动相为乙腈-水梯度洗脱：0～15min,40:60→50:50；15～20min,50:50→75:25；20～25min,75:25→95:5；流速0.5mL·min-1,检测波长254nm,柱温：35℃,进样量20μL。本试验可在同一色谱条件下,可同时测定五味子甲素、五味子乙素、五味子丙素、五味子醇甲、五味子醇乙、五味子酯甲等六种木脂素类成分的含量,方法简便,且精密度高,重现性好。3.以11份北五味子果实样本为试材,对其六种主要木脂素含量进行了测定。七份红色北五味子样品在2011、2012年间除了五味子甲素含量相对稳定以外,五味子乙素、五味子丙素、五味子醇甲、五味子醇乙和五味子酯甲的含量均存在较大差异并呈现出不规律的变化,七份红色北五味子果实之间木脂素含量也存在一定差异。不同颜色北五味子果实木脂素含量存在较显著差异,红色北五味子果实六种木脂素总含量最高,白色果实次之,粉色果实最低。红色北五味子果实富含五味子醇甲、五味子乙素和五味子丙素,而白色北五味子果实中五味子醇乙和五味子甲素较高,粉色北五味子果实中五味子酯甲含量的平均值高于白果W和红果。通过果实表型以及果实品质两方面相结合,北五味子样品B,D,E的果实性状较好,适宜进行育种。4.利用筛选到的4对SRAP引物组合对七种北五味子样本A～G进行PCR扩增,共产生22个位点,其中有13个多态性位点,总的位点多态性比率为59.09%,说明这七种北五味子具有不同的基因型。5.本研究采用固液培养相结合的培养方式建立了高效稳定的北五味子植株再生系统,结果表明不同基因型的北五味子休眠芽愈伤组织诱导率不同,北五味子休眠芽在MS+3.0mg·L-12,4-D+0.2mg·L-1TDZ培养基上愈伤组织诱导率较高；将愈伤组织置于MS+1.0mg·L-1TDZ+0.2mg·L-1ZT+50mg·L-1AgNO3培养基中适宜胚性愈伤组织的产生；对胚性愈伤组织进行细胞悬浮培养,当50mLl/2MS培养基接种3g胚性愈伤组织时有利于球形胚的形成,将所获得的球形胚转入1/2MS固体培养基培养,能较好的完成后期分化；1/2MS+0.2mg·L-1IBA培养基最有利于北五味子再生植株根部的形成,将根系发达的北五味子小植株进行移栽,其移栽成活率为90%。本研究利用筛选到的4对SRAP引物组合对七株北五味子母本及再生植株进行PCR扩增,其产物均没有产生特异性条带,这说明本研究建立的北五味子离体再生体系遗传稳定,可以应用于北五味子再生植株的大量生产。6.本研究对内源激素的动态变化对北五味子体细胞胚的发生和发育所产生的影响进行了研究,结果表明在几种不同类型的愈伤组织中,胚性愈伤组织内源IAA、ABA、GA3含量均为最高,这与其胚性能力相关。北五味子体细胞胚发育过程中,内源IAA含量在球形胚时期达到最大值,内源ABA含量在子叶形胚阶段较高,内源GA3含量最高点出现在心形胚阶段,与球形胚阶段无显著性差异。在北五味子畸形胚中,内源IAA和GA3含量均显著高于正常体细胞胚,而ABA含量则显著低于正常体细胞胚。经过0.2mg·L-1IBA处理后的生根再生植株中,内源IAA含量显著上升,ABA和GA3含量均显著下降。更多还原

【Abstract】 Schisandra chinensis （Turcz.） Baill. is the precious Chinese drugs in northeast China which favored by consumers in domestic and overseas. With the deepening of the development and utilization of S. chinensis, the demand for raw materials also increased in recent years. However, because of late start for artificial cultivation, less fine improved varieties, biological characteristics and quality diversity among the plants and poor yield stability seriously restrict the cultivation industry development of S. chinensis. In order to better utilize the resource advantage of S. chinensis, this research also studies the fruit phenotype and main lignan content in fruit of different genotypes, which provides foundation for the germplasm resources evaluation of S. chinensis. To breed improved variety, this study established a stable and efficient plant regeneration system for S. chinensis with good fruit characters via somatic embryogenesis, and the dynamic changes of endogenous hormones at various stages in this process are researched comprehensively. The main research results are as follows:1. Comprehensive comparison for11samples of S. chinensis, the fruit phenotype were extensive variation including size, average fresh and dry weight of fruit, dry-fresh ratio, cluster length, number of fruits and fruits compactness, moreover, these fruit phenotype were instable between two years. The average fres hweight, dry weight, the dry-fresh ratio and the compactness were greater in pink fruits than that in red fruits and white fruits. The size of red fruits was larger. The fruit phenotypes of B, D, E, G, P1and P2were better.2. A Symmetry（?） C18column （4.6×250mm,5μm） was maintained at35℃. Detection wavelength was set at254nm, The mobile phase consisted of acetonitrile （A） and water （B） at a flow-rate of0.5mL·min-1. The gradient programme was as follows:0～15min,40:60→50:50;15～20min,50:50→75:25;20～25min,75:25→95:5; injection volume:20μ L. schisandrin, schisantherin A, deoxyschizandrin, γ-schissandrin, schisandrol B and schisandrin C could be detected at the same time. This method was simple, repeatability and precision.3. We determined the contents of six lignans in11samples of S. chinensis, the results showed that most of the lignan contents varied between two years except the contents of deoxyschizandrin were relatively stable. The total contents of lignans in fruits with different colors presented large differences, which was found highest in red fruits and lowest in pink fruits. High contents of schizandrol, γ-schizandrin and schisandrin C in red fruits were observed while white fruit was rich in schisandrol B and deoxyschizandrin. The contents of schisantherin A in pink fruits were higher than that in W and the average contents in red fruits. Considering the factors of phenotype and fruit quality, fruit character of samples B, D and E were good and suitable for breeding.4. This study used4pairs of SRAP primer for PCR amplification of seven samples of S. chinensis, which produced22amplification bands and13bands were polymorphism. The results showed that the genotypes of these seven samples of S. chinensis were difference.5. This research adopted the combination of solid and liquid cultivation for the establishment of stable and efficient plant regeneration system for S. chinensis. Different genotype influenced the callus induction rate of S. chinensis. The callus induction rate was highest when the sterilized dormant bud of S. chinensis cultured on MS+3.0mg·L-12,4-D+0.2mg·L-1TDZ. MS+1.0mg·L-1TDZ+0.2mg·L-1ZT+5.0mgL-1AgN₃was beneficial to embryonic callus induction.3g embryonic callus cultured in50mLl/2MS liquid medium was suitable for the formation of globular embryo.1/2MS+0.2mg·L-1IBA was most favorable for the roots formation of the regeneration plant of S. chinensi and the survival rate was90%after2months transplanting. This study used4pairs of SRAP primer for PCR amplification of seven S. chinensis female parent and the regeneration plant via somatic embryogenesis system, and there were no specific bands produced by four pairs of primers, suggesting that the plant regeneration system we established in this study was stable and reliable, which could be applied to mass production for plant regeneration of S. chinensis.6. The effect of dynamic changes of endogenous hormones on the formation and development of somatic embryos was studied.The contents of endogenous IAA, ABA and GA3related to the embryonic ability,which were highest in embryonic callus among several different types of callus of S. chinensis. During the development of somatic embryos of S. chinensis, the content of endogenous IAA peaked in globular embryo period and the content of endogenous ABA was higher in cotyledon embryo phase. In the whole process, the content of endogenous GA3peaked at heart shaped embryo stage, but no significant difference with the content in globular embryo stage. The contents of endogenous IAA and GA3were significantly higher in malformed embryo of S. chinensis than that in normal somatic embryo, but the content of ABA in malformed embryo were significantly lower than that in normal somatic embryo. The content of IAA was increased significantly when the contents of ABA and GA3declined in rooted plant treated with0.2mg·L-1IBA.更多还原