华中科技大学 硕士学位论文 甘草离体再生及毛状根诱导研究 姓名：雷呈 申请学位级别：硕士 专业：生物化工 指导教师：余龙江;付春华 20070207 I 摘摘 要要 甘草是常用的中草药, 具有补脾益气、清热解毒、润肺止咳、缓急止痛、调和 诸药之功效由于人为破坏性地对野生甘草采挖，对甘草的保护性利用迫在眉睫 离体再生体系和发根培养技术的建立，对甘草资源的保护、基因工程研究和活性成 分利用具有重要意义 本研究以乌拉尔甘草、胀果甘草、光果甘草、黄甘草和野生光果甘草为试验材 料，对甘草离体再生及毛状根诱导进行了研究，主要研究结果如下： 1. 建立了甘草下胚轴离体再生体系， 为基因工程进行甘草品种改良及优良品种 快速繁殖奠定了基础 以甘草下胚轴为外植体，对影响下胚轴离体再生不定芽的因素进行了研究，结 果表明, KT 具有显著作用，与 6-BA、IBA 结合，可高效诱导下胚轴直接产生不定 芽，优化的分化培养基为 MS + 6-BA 2.0 mg/L + KT 0.5 mg/L + IBA 0.5 mg/L，芽苗 分化率平均为 41.5％同时，胀果甘草下胚轴离体再生频率最高，达 44.7％；预培 养 4 天和 AgNO3 6 mg/L 对甘草下胚轴再生不定芽具有促进作用。
2. 建立了甘草快速繁殖技术体系， 为优良甘草品种的进一步开发和利用打下了 基础 以甘草子带子叶茎段和带叶茎段作外植体，结果表明：乌拉尔甘草诱导的丛生 苗数较多，是三个供试材料中最好的；子叶茎段是甘草快速繁殖最适宜的外植体； 甘草快速繁殖体系的适宜培养基是： 诱导培养基 MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA0.1 mg/L 复壮培养基 MS + 6-BA 2.0 mg/L + KT 0.5 mg/L + IBA0.5 mg/L 生根培养基 MS + IBA0.5 mg/L 3. 建立了甘草胚性愈伤组织诱导和体细胞胚胎发生体系， 为甘草遗传转化和人 工种子制种提供了材料 以乌拉尔甘草、胀果甘草、光果甘草、黄甘草为材料，经愈伤组织诱导、胚性 愈伤组织的形成和胚状体的产生，从基因型、外植体、激素、附加物等因素研究和 II 分析了胚状体发生和发育的过程，结果表明，甘草胚状体发生方式有三种：一是从 下胚轴形态学上端直接发生，不经过愈伤组织阶段，但诱导的胚状体少；二是经过 愈伤组织阶段再分化为体胚，诱导的胚状体多；三是从游离的悬浮细胞发生，诱导 的胚状体多。
甘草胚状体三种发生方式分化成再生植株都比较困难用石蜡切片和 半薄切片对胚性愈伤组织和非胚性愈伤组织形态和结构作了进一步的分析，发现胚 性愈伤组织、胚状体和非胚性愈伤组织、非胚状体形态和结构有很大差异 几种适宜的培养基是： 愈伤组织诱导 MS + 6-BA 2.0 mg/L + 2,4-D 0.5 mg/L 愈伤组织继代 MS + 6-BA 0.5 mg/L + NAA 0.2 mg/L + 2,4-D 0.2 mg/L 胚性愈伤诱导 MS + 6-BA 0.5mg/L + KT 0.5 mg/L + IBA 0.1 mg/L 胚性愈伤组织继代和胚状体分化培养基 MS + 6-BA 0.5mg/L + KT 0.5 mg/L + IBA 0.1 mg/L + ME 500 mg/L 胚状体增殖 MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA 0.1 mg/L + ME 500 mg/L 4.建立了甘草毛状根诱导的技术平台，获得了甘草毛状根，为甘草毛状根次生 代谢产物的生产工业化奠定了基础 对影响毛状根诱导的因素（不同菌株，不同外植体,不同菌液浓度，不同侵染时 间）进行了系统研究，结果表明：菌株 LBA9402 易诱导毛状根，是乌拉尔甘草的敏 感菌株。
下胚轴、子叶和真叶均诱导出毛状根，下胚轴转化率最高用 LBA9402 发根农杆菌菌悬液稀释两倍后侵染乌拉尔下胚轴，在 5~10 min 左右达到最好诱导 获得的毛状根嫩、白、细、生长速度快、多分枝、多根毛、呈丛生状，并失去向地 性 关键词：关键词：甘草 离体再生 体细胞胚胎发生 下胚轴 毛状根 快速繁殖 III Abstract The Glycyrrhiza is one of the most common Chinese herbal medicine which has many drug action, such as cleaning up heat detoxification, moistening lung to arrest cough, relieving pain, coordinating the drug actions of a prescription. Due to the ruinously spading of the grow wide Glycyrrhiza, now the quantity of the Glycyrrhiza is very cut down, so it’s very urgent to protect the Glycyrrhiza. The establishments of vitro plant regeneration system and hairy root cultivation have a very important significance in protection of the Glycyrrhiza resource, the researching of genetic engineering and the utilization of the active constitutents. Glycyrrhiza uralensis Fisch, Glycyrrhiza inflata Bat. Glycyrrhiza glabra L, Glycyrrhiza korshinski G. Grig, grow wild Glycyrrhiza glabra L are used as the test materials, this research contains Glycyrrhiza vitro plant regeneration and hairy root induction, the main results are as follows: 1.Establish the Glycyrrhiza hypocotyl vitro plant regeneration, provide the foundation of the Glycyrrhiza breed improvement and good breed intermediate propagation. Use the Glycyrrhiza hypocotyl as the explant, research the factor which effects hypocotyl vitro regeneration adventive bud, the results show that KT has a predominance effect when combine with 6-BA, IBA, which can induce adventive bud from the hypocotyl, the optimize medium is consist of MS, 6-BA 2.0 mg/L , KT 0.5 mg/L , IBA 0.5 mg/L. the average optimize rate is 41.5%, the G. inflate has the highest optimize rate, which is as high as 44.7%, preincubated for four days, using 6mg/L AgNO3 can promote the growth Glycyrrhiza hypocotyl regeneration adventive bud. 2. establish the Glycyrrhiza intermediate propagation technology syetem, prepare for the deeper development and utilization of Glycyrrhiza resource. IV using the stem apex of cotyledon and the stem apex of lateral bud as the explants, the results show: the induced G. uralensis regrowth is best among the three breeds, the stem apex of cotyledon is suitable for explant, the medium which suits the Glycyrrhiza intermediate propagation system are as follows: induction medium: MS + 6-BA 0.5 mg/L + KT 0.5 mg/L + IBA0.1 mg/L rejuvenescence medium: MS + 6-BA 2.0 mg/L + KT 0.5 mg/L + IBA0.5 mg/L root medium: MS + IBA 0.5 mg/L 3. Induction of Glycyrrhiza callus and somatic embryogenesis provide materials for transgenic and artificial seeds. when callus was induced, then the embryogenic callus and the embryoid were produced, we analysis the factors which have effect on the process of formation and development from genotype, explants, hormones, accessory, etc. Then make a further analysis to the morphous and structure of embryonic callus and non-embryonic callus with paraffin section and Semi-thin Sectioning. The results show that there are three different ways of Glycyrrhiza somatic embryos formation: the first is the formation from the hypocotyls superior extremity in morphology without callus phase, but embryoid were few induced; Second, the somatic embryoid was formatted through callus stage, and the embryoid were more induced; Third, the embryoid were formated from suspension cells, and they were more induced. It is difficult to formatt。