大花红景天组培快繁及指纹图谱构建
文献类型：学位论文
| 作者 | 赵燕 |
| 学位类别 | 博士 |
| 答辩日期 | 2011-04-02 |
| 授予单位 | 中国科学院研究生院 |
| 导师 | 刘春朝 |
| 关键词 | 大花红景天 组培再生 冷冻保存 间歇浸没式反应器 中药指纹图谱 |
| 其他题名 | Establishment of Fingerprints of Rhodiola crenulata and Tissue Culture |
| 学位专业 | 生物化工 |
| 中文摘要 | 红景天为传统的藏药，具有抵抗高原反应、抗缺氧、抗疲劳和抗氧化的功效，但是由于生长环境特殊，人工采挖严重，造成红景天资源日益减少；红景天种子自然萌发率极低，采用根茎进行营养繁殖时，腐烂现象十分严重，至今没有大面积人工栽培成功的报道。本论文以解决大花红景天 (Rhodiola crenulata) 资源短缺为目的，建立红景天的指纹图谱，对大花红景天进行组培快繁、种质资源冷冻保存及生物反应器大规模培养的研究，获得结果如下：建立了大花红景天指纹图谱的高效液相色谱方法，并用此方法分析了其它种的红景天的指纹图谱。以相对峰面积和相对保留时间分析了不同种的红景天色谱图，结果表明，各种红景天共有峰的相对保留时间差异不大，但共有峰的相对峰面积差别较大。作为药典指定的红景天属的唯一种大花红景天，其红景天甙及酪醇含量均高于其他种，因此选择大花红景天作为研究的对象。以野生大花红景天的叶片为外植体进行芽再生，将叶片外植体在黑暗处理5天后可显著提高不定芽的出芽数，暗处理5天的植物材料的内源玉米素 (Zeatin，ZT) 和赤霉素的含量及ZT与IAA的比值显著提高，同时黑暗预处理5天还降低了内源的多酚氧化酶的活力，从而抑制了褐化现象。建立了大花红景天玻璃化法超低温冷冻保存的工艺。在超低温保存前将大花红景天愈伤组织置于含褪黑素的培养基上预处理，浓度为0.1 µM的褪黑素预处理5天，大花红景天愈伤组织的超低温保存成活率由46.6%提高到72.2%，再生率由30.5%提高到53.0%。经过褪黑素预处理的大花红景天愈伤组织的过氧化物酶 (Peroxidase, POD) 和过氧化氢酶 (Catalase, CAT) 的活力比未经处理的愈伤组织的酶活高，褪黑素预处理后的红景天愈伤组织的丙二醛 (Malondialdehyde, MDA) 含量比对照减少。采用间歇浸没式生物反应器对大花红景天丛生芽进行增殖培养，间隔300 min浸没3 min为最佳间歇浸没周期，丛生芽的增殖率达到最大为46.8，玻璃化率为35.4%；在间歇浸没式生物反应器内增加强制通风，在间歇浸没周期为3 min/180 min的周期下，强制通风速率为40 L/h时，丛生芽的增殖率最大达到了55.7，玻璃化率降低至26.1%。 |
| 英文摘要 | Rhodiola is genus belongs to the family Crassulaceae and mainly grows in Tibet. It has been used in Tibet as an important traditional herbal for a long time. Extracts from the roots of Rhodiola are found to have a number of physiological functions including adaptogenic, antioxidant and anti-cancer effects. A growing commercial demand and over harvest of Rhodiola have caused a serious reduction in native population. The natural resource of Rhodiola is on the edge of exhaustion. The major constraint in conventional propagation of Rhodiola through seeds is the high mortality of seedlings in early stages; meanwhile, the destructive harvesting of the rhizome for vegetative propagation seems to be unfeasible because it is of high possibility to lose the mother plant. A simple and reliable high performance liquid chromatographic (HPLC) method was developed and validated for the study of fingerprint chromatograms of extracts from the roots of R. crenulata. Then we developed a rapid and reproducible method for regeneration of the plant via shoot organogenesis of R. crenulata. Furthermore, a simple and convenient cryopreservation procedure of callus of R. crenulata was established. At last, shoot culture of R. crenulata in large scale in temporary immersion bioreactor was realized. A simple and reliable HPLC method was developed and validated for the study of fingerprint chromatograms of extracts from the roots of Rhodiola crenulata (RC). The properties of HPLC fingerprints were validated by analyzing related herbals including R. yunnanesis (RY), R. rosea (RR), R. sachdlinensis (RSA) and R. sacra (RS). The relative retention time and relative peak area of characteristic peaks were calculated. The relative retention time of five species resembled each other, but the relative peak area varied dramatically for the five species fingerprints. The simulative mean chromatograms of the five species were generated by the professional software named Similarity Evaluation System for Chromatographic Fingerprint of Traditional Chinese Medicine (version 2004A). All of the similarities of the simulative mean chromatogram of RY, RR, RSA and RS to that of RC were below 0.630. Our results showed that the chromatographic fingerprint combining similarity evaluation could efficient distinguish RC from other species. Shoot organogenesis occurred from R. crenulata leaf explants inoculated on a medium supplemented with a combination of plants growth regulators. The optimal medium for the shoot induction is the medium containing 15 µM 6-benzylaminopurine (6-BA) and 2.5 µM gibberellic acid (GA3). Regenerated shoots formed complete plantlets on a medium containing 5 μM indole-3-acetic acid (IAA). The leaf explants pretreated in darkness for 5 days had an average of 9.1 shoots per leaf explant which was 2.5-fold of those without dark preincubation. During the culture period, the contents of zeatin (ZT), GA3 and ratio of ZT/ IAA were highest when the explants were incubated under the pretreatment of 5 days darkness. After 5 days dark pretreatment, polyphenol oxidase (PPO) activity did not change when compared to that under light condition. |
| 语种 | 中文 |
| 公开日期 | 2013-09-24 |
| 页码 | 115 |
| 源URL | [http://ir.ipe.ac.cn/handle/122111/1747]  |
| 专题 | 过程工程研究所_研究所（批量导入） |
| 推荐引用方式 GB/T 7714 | 赵燕. 大花红景天组培快繁及指纹图谱构建[D]. 中国科学院研究生院. 2011. |
入库方式： OAI收割
来源：过程工程研究所
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