【作者】 李文龙；

【导师】 田义新；

【作者基本信息】 吉林农业大学 ， 中药学， 2007， 硕士

【摘要】 平贝（Bulbs Fritillaria Ussuricnsis）是四种药用贝母药材（浙贝、川贝、平贝、伊贝）的一种，原植物为百合科贝母属平贝母（Fritillariac ussuricnsis Maxim），以干燥鳞茎入药，有清热润肺，化痰止咳，开郁散结的功效。平贝是常用大宗中药，在中医临床中用于治疗气管炎、肺结核、肺炎、百日咳等上呼吸道疾病，多种中成药的复方中也都含有平贝母。近年来，随着药用量的不断增加，野生资源已不能满足用药需求。人工种植则与我国很多药材生产一样，主要以众多农户分散种植为主体，存在的问题是：分散零星，科技含量低，生产规范化和集约化程度差，导致平贝母质量不稳定，难以供应等。本研究就是针对这些问题，利用近代新发展起来的先进的组织培养技术进行平贝母的有效成分——生物碱的诱导生产研究，并通过前体物的饲喂研究开发出高有效成分生产培养基。以此摆脱传统的平贝母生产方法中的田间各种环境条件、气候条件、生产技术措施等因素的制约。本次试验根据植物生物技术的原理，首先进行了4种培养基（MS、B₅、N₆、H）对平贝母愈伤组织诱导能力的研究。在此基础上，用正交试验设计研究了激素种类和浓度对平贝母愈伤组织诱导和继代培养的影响，并进一步开展了碳源、氮源、磷酸盐、光照条件有无及前体物质对平贝母愈伤组织中总生物碱含量的影响研究。培养过程中，以平贝母愈伤组织的生物量和总生物碱含量为主要指标，确定了平贝母愈伤组织生长和进行其有效成分生产的最佳条件。其中前体物质饲喂对平贝母愈伤组织中总生物碱含量的影响研究属创新性工作。通过调查与愈伤组织中总生物碱生产有关的诸多指标，包括干（鲜）重增长率、生物碱含量等，综合评价结果表明：（1）平贝母最佳诱导培养基为MS+NAA0.5mg／L+2，4-D1.0mK／L+BA1.5mg／L，诱导率为68.7％；（2）平贝母生物量生产最佳培养基为MS+2.4-D0.5mg／L+BA0.05mg／L，其中蔗糖浓度以4％、NH₄⁺∶NO₃^-=1∶1、总氮浓度以60 mmol／L、KH₂PO₄浓度以1.5 mmol／L为宜；（3）首次发现了三种氨基酸饲喂物均对平贝母愈伤组织中总生物碱的含量有诱导提高作用，其最佳饲喂浓度和提高能力按从高到低排列如下：①L-酪氨酸10mg／L，诱导平贝母愈伤组织中总生物碱的含量提高到0.4235％，比对照高41.2％；②L-苯丙氨酸10mg／L，诱导平贝母愈伤组织中总生物碱的含量提高到0.4122％，比对照高37.4％；③L-色氨酸20mg／L，诱导平贝母愈伤组织中总生物碱含量提高到0.4025％，比对照高34.2％。更多还原

【Abstract】 Pingbeimu, Bulbus Fritiilariae ussuriensis, is one of the four Fritillaria medicinal materials （Bulbus Fritillariae thunbergii、Bulbus Fritillariae cirrhosae、Bulbus Fritiilariae ussuriensis、Bulbus Fritillariae pallidiflorae）. Its original plant is the Fritillaria ussuricnsis Maxim belong to Fritillaria genus, Liliaceae family, and the medicinal part is its bulb whose functions as a traditional Chinese medicine （TCM） are cleaning up fever, nourishing lung, expectorant, releasing stasis. Pingbeimu is one of common and great consumed TCM in China, it is usually used to cure upper respiratory disease, such as tracheitis, tuberculosis, pneumonia and chincough, and there are plenty of compounding prescriptions contained Pingbeimu. In recent years, the wild resource provide too little amount of the Pingbeimu to meet the demand. Similar to most of our country’s cultivated TCM, it is mainly cultivated with individual, that result in the following problems: spreading around, low techniques, short of standard and intensive farming, the Pingbeimu quality, thus, has been not stable and difficult to supply, etc.The research aim to those problems, to induce the Pingbeimu’s effective component, the alkaloid, with the advanced technique of tissue culture, and develop the producing media of high effective component. The methods may get rid of the restriction of the field conditions, climate conditions, techniques in traditional cultivation.The experiment is based on biotechnological theory, the optimum medium is screened first from the four media of MS, B₅, N₆ and H according to the induced ratio of the bulb’s callus in the media. After the optimum medium is screened, the Orthogonal experimental design was applied to study the effects of the proportion and concentration of the plant hormones to the callus inducing and the subculture, and it is fatherly researched that what kinds of effects of different carbohydrates, nitrogen substances, phosphate, light or dark culture and adding precursor substances they are to the alkaloid content in the callus. During the culture, the main targets were biomass and the total alkaloid content in the callus, the optimum cultural conditions of the Pingbeimu callus growth and the total alkaloid production has been identified. It is the first reported that adding precursor substances induced the alkaloid production in the callus.After investigating the targets, such as the fresh or dry weight, the total alkaloid content in the callus, the whole results indicate: 1. the optimum Fritillaria callus induced medium is MS+ NAA0.5mg/L+2, 4-D1.0mg/L+BA1.5mg/L, the induced rate is 68.7%; 2. the optimum Fritillaria callus production medium is MS+2.4-D0.5mg/L+ BA0.05mg/L, and the optimum sugar concentration is 4%, NH4+:N03-=1:1, the total nitrogen concentration is 60 mmol/L、KH2P04 concentration is 1.5 mmol/L; 3. It is first discovered that the three kinds of amino acids as precursor substances could induce the alkaloid content rise in the callus, the increasing abilities as the following order:①L-tyrosine 10mg/L could induce the alkaloid content in the callus to 0.4235%, 41.2% more than control;②L-phenylalanine 10mg/L could induce the alkaloid content in the callus to 0.4122%, 37.4% more than control;③L- tryptophan 20mg/L could induce the alkaloid content in the callus to 0.4025%, 34.2% more than control.更多还原