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玛咖组织培养过程调控和质量评价
Alternative TitleTissue Culture Process Regulation and Quality Evaluation of Lepidium meyenii
王亚丽
Subtype博士
Thesis Advisor王玉春
2007-06-04
Degree Grantor中国科学院过程工程研究所
Place of Conferral过程工程研究所
Degree Discipline生物化工
Keyword玛咖 快繁 悬浮培养 抗氧化活性 指纹图谱相似度评价
Abstract玛咖(Lepidium meyenii. Walp)是当今世人瞩目的药食两用植物,有许多独特药理作用。但由于生长环境独特,限制了玛咖的大量种植。为了缓解供需矛盾,本文借助植物细胞组织培养技术,系统研究了玛咖快繁和细胞培养过程的影响因素,为达到驯化、引种或人工生产玛咖有用次生代谢产物的目的做出了有益的探索。同时对试种地的玛咖质量进行了评价。 首次建立了玛咖愈伤组织高效诱导和繁殖体系,考察了玛咖细胞固体培养基上的生长周期。在优化后的愈伤组织诱导条件下,叶外植体愈伤组织诱导率为76%;根外植体愈伤组织诱导率为100%。在优化后的愈伤组织继代条件下,经过30天的培养,愈伤组织平均干重分别达0.472g /flask (根愈伤)和0.507g /flask(叶愈伤),分别是接种时的11.8倍和12.68倍。 首次建立了玛咖丛生芽诱导和继代体系。在优化后的丛生芽诱导和继代条件下,丛生芽诱导率达80%以上,继代后丛生芽存活率为33%。 首次建立了玛咖丛生芽两步生根体系。优化后的生根条件下玛咖丛生芽生根率为63%。 首次研究了稀土元素对玛咖丛生芽诱导和生长的影响。在丛生芽诱导培养基中添加稀土元素(La3+, Ce3+或Nd3+)可有效降低丛生芽玻璃化发生率,显著提高丛生芽的存活率。当诱导培养基中的稀土元素浓度为0.1mM时,丛生芽存活率达70%左右。并考察了玛咖丛生芽诱导和继代过程中玻璃化现象的发生机制,以及稀土元素缓解玻璃化的作用机制。 首次研究了光质对玛咖愈伤组织生长和分化的影响,探索了不同光质与细胞中糖代谢关键酶活性的关系。研究表明,白光、红光和黄光下,玛咖愈伤组织生长较快、分化率高,此时糖代谢中的关键酶活性偏高;绿光和蓝光下,玛咖愈伤组织生长较慢、分化率低,此时糖代谢中的关键酶活性偏低。 首次考察了人工培养玛咖细胞的抗氧化活性,并建立了玛咖细胞摇瓶悬浮培养体系和周期浸没气升式悬浮培养体系。在优化后的摇瓶培养体系中,玛咖细胞培养周期为15d,细胞的增殖率为2.05,细胞甲醇提取物抗氧化能力达89.05%;在优化后的周期浸没气升式悬浮培养体系中,玛咖细胞的培养周期为25d,细胞的增殖率为1.94,细胞甲醇提取物抗氧化能力最高达58.21%。 首次考察了水杨酸甲酯对玛咖细胞生长和抗氧化活性的影响,并探索了作用机制。在固体培养基中添加水杨酸甲酯虽然可影响玛咖细胞的生长,但同时能显著提高玛咖抗氧化活性。水杨酸甲酯的适宜添加浓度为0.05M,在这种浓度下,玛咖细胞的抗氧化活性是对照的1.81倍。 通过HPLC-MS/MS分离鉴定出了玛咖中的特征成分,即玛咖烯和玛咖酰胺。对试种地所产玛咖和秘鲁产玛咖成分进行了比较。首次利用指纹图谱相似度评价对试种地玛咖的质量作了分析、评价。
Other AbstractMaca (Lepidium meyenii. Walp) is a plant which can be used as food and medicine. It has lots of special pharmacological functions. Due to its growth in special growth environment, the output of maca has been restricted. In this thesis with the help of plant cell and organ culture technique, the influence factors in the process of maca rapid regeneration and cell culture were investigated in order to domesticate, artificially transplant maca and produce valuable secondary metabolites. The quality of maca transplanteded in Yunnan province was evaluated also. The efficient maca callus induction and reproduction systems were firstly established. And maca cell growth period on the solid culture medium was researched. Under the optimal conditions of maca callus induction, the induction rate of the calli induced from the leaves and the roots, were 76% and 100% separat ely. Under the optimized conditions of maca callus subculture, the callus dry weight was 0.472g/flask (the callus induced from the roots) and 0.507g/flask (the callus induced from the leaves) after 30 days, which is 11.8 and 12.68 times of the inoculation size. Maca adventitious shoot induction and subculture system were established firstly. Under the optimized conditions of adventitious shoot induction and subculture, the shoot induction rate was above 80%, the shoot survival rate was 33%. Bi-staged rootage system was established firstly. Under optimized conditions of rootage, the rootage rate of maca shoot was 63%. The effects of rare earth elements on maca shoot induction and growth were studied. Rare earth elements (La3+, Ce3+ and Nd3+) added to shoot induction culture medium reduced hyperhydric rate and improved shoot survival rate efficiently. After addition 0.1mM rare earth elements, the shoot survival rate was about 70%. The mechanism of hyperhydricity occurred in the process of maca shoot induction and subculture, as well as the lightening the hyperhydricity mechanism by rare earth elements were researched. The mechanism of light quality influencing maca callus growth and differentiation were investigated. The results showed that under white light, red light and yellow light, maca callus grew more quickly and well differentiated, the activities of key enzymes in glycolysis were higher; But under green light and blue light, the results were opposite. In the optimized suspension culture system in conical flasks, maca cell growth period was 15d, the cell multiplication rate was 2.05, the antioxidative activity of methanol extract of maca cell was 89.05%. In the optimized suspension culture system in periodically submerged airlift bioreactor, maca cell growth period was 25d, the cell multiplication rate was 1.94, the antioxidative activity of methanol extract of maca cell was 58.21%. The effects of methyl salicylate on maca cell growth and antiioxidative activity and its mechanism were investigated. Although methyl salicylate addition to the solid culture media inhibited maca cell growth, it improved maca cell antioxidative activity remarkably. The suitable concentration of methyl salicylate added to the medium was 0.05M. Under this condition, maca cell antioxidative activity was 1.81 times of that in the control. The characteristic compounds of maca, i.e. macaene and macamide, were separated and analyzed by HPLC-MS/MS. The chemical compounds of maca harvested in the Andes and transplanted in Yunnan province were compared. HPLC fingerprints chromatogram was used to evaluate the similarity of above maca from different area.
Pages166
Language中文
Document Type学位论文
Identifierhttp://ir.ipe.ac.cn/handle/122111/1040
Collection研究所(批量导入)
Recommended Citation
GB/T 7714
王亚丽. 玛咖组织培养过程调控和质量评价[D]. 过程工程研究所. 中国科学院过程工程研究所,2007.
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