【作者】 蒋桂华；

【导师】 马逾英；

【作者基本信息】 成都中医药大学 ， 中药学， 2012， 博士

【摘要】 川芎为伞形科植物川芎Ligusticum chuanxiong Hort的根茎,为我国的传统中药,也是著名的川产道地药材,具有近两千年的栽培和使用历史,长期以来生产上均以无性方式进行繁殖,其繁殖材料为膨大的地上茎节(苓种)。经过上千年的栽种,加上缺乏选种育种,存在一定程度的种质退化。四川主产区药农对苓种繁育仍然停留在自繁自用,缺乏统一的生产技术规程和科学的组织管理,且没有苓种分级标准和检验规程,导致川芎苓种质量良莠不齐。另外,目前生产上仍然采用传统的自然存放方法贮存苓种,不能久贮；种质保存只能采用传统的田间保存方法,存在耗费人力物力,容易损失等诸多缺点,而相关研究尚属空白。中药材质量稳定需要中药材生产的规范化,中药材生产规范化需要中药材种子种苗的标准化。优良品种和优质的种子种苗是实现中药材规范化生产的基础和源头。本文针对川芎规范化生产中的薄弱环节,以苓种标准化问题为切入点,开展了川芎苓种的标准化研究和种质保存技术研究,以推动川芎苓种的标准化进程,为川芎规范化生产提供源头保障。以生产出优质、高产、稳定、可控的川芎药材,促进川芎产业的可持续发展。研究结果如下：1.川芎苓种的调查、收集在前期全国川芎种质资源调查的基础上,本文重点对四川省内川芎主要产区及苓种繁育地所涉及的5个市县20余个乡镇进行了实地调研,基本弄清了四川川芎苓种的分布和主产区的演迁、当地传统繁育技术、苓种保存方法、质量评判及产销情况等；调查表明,近年来,尤其是5.12汶川大地震后,发生了一些变迁,目前主要集中在都江堰市中兴镇、都江堰市青城后山红岩、都江堰市虹口乡,彭州市小渔洞镇、龙门山镇、通济镇,汶川县水磨镇、三江口镇,崇州市三郎镇,什邡市冰川镇等,海拔在900m-1500m。其中彭州小鱼洞镇的川芎苓种繁育面积较大。主产区苓种的来源主要是坝区药农定点委托山区药农代为自繁自用,收获的苓种多数供坝区自我栽种,少数流入集市销售供其他栽种所需。川芎苓种市场上质量良莠不齐,无统一标准规范。另外,近年在一些川芎主产区和苓种集市上出现了少量“坝苓种”、“冻苓种”和“山区自栽苓种”,已在四川省内或调往省外作为川芎生产用种。调查同时,收集了不同产地、不同集市的川芎苓种共34分,部分保留在前期建立的川芎种质资源圃中,部分提供给国家药用植物种质资源库,部分作为课题研究用。2.川芎苓种贮藏及种质保存技术研究针对川芎生产用苓种,首次进行了常温保存及低温贮藏技术研究,弄清了苓种在常温条件下存放6～10天,脱去的水分适中,不会影响发芽率,为川芎苓种采收后的短期贮藏提供了科学依据；建立了川芎苓种冷藏保存方法,可使川芎苓种短期保存时间延长至3个月,为该方法的进一步的推广应用,以满足川芎不同栽种地区对川芎苓种不同贮藏时间的需求及改变川芎苓种自繁自用,促使其商品化奠定了基础。针对川芎种质中长期保存,首次采用限制生长保存法,以川芎茎段为组培材料,以MS为固体培养基,建立了川芎试管苗培养方法,对比了改变培养环境、调整培养基养分水平、添加渗透性化合物和生长抑制剂等实验,得到了川芎种质限制生长保存最佳方案。恢复生长实验表明保存6月后的种质能生长成活,保存苗和对照苗的形态特征与同工酶检测分析,没有发现变异现象,保持了良好的遗传稳定性,为实现川芎种质材料的中长期保存奠定了基础。同时采用玻璃化法对川芎种质超低温保存的可行性进行探索,找到了适合的川芎玻璃化法的外植体是川芎茎尖,建立了川芎组培再生苗的培养方法,为进一步深入研究打下了基础。3.川芎新品种选育研究在前期川芎种质资源鉴定的基础上,构建成了由1300个株系,约8000个单株组成的育种用群体。利用该群体,采用系统选育的方法进行了川芎优良新品种选育,从优良单株选择、优良株系的筛选、品系比较试验、品种(系)多点试验,最终选育出川芎优良新品种“绿芎1号”,并通过四川省农作物品种审定委员会的药用植物新品种审定。该品种具有成株叶色浓绿,持绿期长,生长旺盛,群体整齐性、一致性较好,抗病性强的特点,并表现出良好的丰产性、稳定性和适应性,其药材质量符合《中国药典》的相关规定。为逐步实现川芎品种的标准化奠定了基础,为川芎药材的规范化生产提供了优良种源。4.川芎苓种繁育技术研究在前期研究的基础上,深入进行了川芎苓种的繁育技术规程的研究,包括适宜栽种区域、产地环境条件、选地与整地、抚芎的选择与处理、栽种、田间管理、病虫害防治、采收与采后处理等,确定了川芎苓种繁育各环节最适宜的方法和措施。在此基础上制订了川芎苓种繁育技术操作规程(SOP),为山区苓种繁育地优质苓种的生产提供了技术保障。5.川芎苓种质量标准研究首次进行了川芎苓种质量检验规程的研究,针对川芎繁殖材料的特殊性,结合产区药农传统经验,创新性的提出了以外观形态、百粒重、苓子系数、混杂率、病虫害等作为川芎苓种质量评价的综合指标,并制定了川芎苓种质量检验规程,内容包括真实性鉴定、形态指标、重量测定方法、苓子系数、混杂率、病虫害。为川芎苓种的质量评判提供了技术支撑。首次进行了川芎苓种分级标准的研究,通过对比不同等级川芎苓种对其植株生长发育、药材产量和质量的影响,结果表明,不同等级苓种对川芎的生长有显著性的影响,并且在川芎的不同生长时期其影响因素有差异,一、二等苓种的川芎在植株生长发育及药材产量上均明显优于三等苓种的川芎；药材质量方面,不同等级苓种的川芎在药材性状、薄层色谱、主成分含量上无明显差异。在总结药农长期实践经验和前期研究的基础上,根据不同等级苓种对植株生长发育和药材产量、质量影响的实验研究结果,并结合生产实际,拟订了川芎苓种等级划分标准。在上述研究基础上,起草了《川芎苓种质量标准(草案)》。为川芎苓种生产、流通、检验和使用提供了标准依据。更多还原

【Abstract】 Chuanxiong Rhizoma, the rhizome of Ligusticum Chuanxiong Hort., is a famous trueborn Chinese medicinal materials in Sichuan, China which has been cultivating and using for two thousands years. Ligusticum Chuanxiong Hort. is an asexual reproductive plant. Its overground swollen node called Lingzhong is used as reproductive material in production. Because of lacking stain selection and breeding, its genetic characterization degenerated to some extent over thousands years of cultivation. In Sichuan, Chuanxiong Rhizoma’s main production area, Lingzhong breeding is still absent of unified technology protocol and scientific organization and management. And the absence of classification criterion and testing rules for Lingzhong result in its uneven quality. Meanwhile, traditional natural conservation method is still widely used in production that can not preserve the medicinal herb for long. Traditional field conservation method consumes much manpower and material resources and may lead to the loss of germplasm, while relevant germplasm conservation technology is still lack of research.Seed and seedling of good variety and good quality are warranties to realize good agricultural practice for Chinese medicinal materials which can ensure herb’s quality in return. In view of the bottleneck in standardized production of Chuanxiong Rhizoma, the article took Lingzhong’standardization as the breakthrough point, studied the standardization of seedling of Ligusticum Chuanxiong Hort.(Lingzhong) and its germplasm conservation technology and aimed to promote Lingzhong’s standardization, to provide Lingzhong of high quality for production of Chuanxiong Rhizoma, and finally to produce Chuanxiong Rhizoma with high, stable and controllable quality as well as high yield thus to promote the continuable development of Chuanxiong Rhizoma production industry. The research results are as follows:1Lingzhong search and collectingOn the basis of early germplasm investigation around China, the article did field survey at Chuanxiong Rhizoma’s main production and breeding area including more than20villages and towns belonging to5cities and counties of Sichuan. Its distribution, evolution and migration of main production area, local cultivation technology, germplasm breeding and conservation technology, quality evaluation method and marketing situation of Lingzhong in Sichuan are basically made clear. The search showed that its production area had alternated at resent years especially after Wenchuan earthquake. Lingzhong in Sichuan are mainly gathered at Zhongxing town, Hongyan at back peak of Qingcheng mountain, Hongkou village of Dujiangyan city, Xiaoyudong town, Longmenshan town, Tongji town of Pengzhou city, Shuimo town, Sanjiangkou town of Wenchan county, Sanlang town of Pengzhou city, Bingchuan town of shifang city, whose average elevation are between900m and1500m. Among them, Lingzhong’s breeding area at Xiaoyudong town of Pengzhou city is bigger.In Chuanxiong Rhizoma’s major production area, Lingzhong is mainly bred by famers in mountainous area who are spot authorized by farmers in plain area. Most collected Lingzhong are planted at plain while few flew into the market for else need. Good and bad Lingzhong at the market are intermingled without unified criterion. Recent years, it was discovered that a few Ba Lingzhong, Dong Lingzhong, Moutain Lingzhong for own need in Chuanxiong Rhizoma’s main production area as well as the market had flown into many planting area of Chuanxiong Rhizoma in and out of Sichuan province.Lingzhong germplasm coming from34production areas and markets are colleted in the investigation which are partly preserved in early-established germplasm bank, partly provided to National Germplam Bank and partly stayed behind for project research.2Lingzhong storage and germplasm conservation technologyStudied normal temperature storage technology and low temperature storage technology to conserve Lingzhong used for produciton. Results showed that Lingzhong could be conserved for6to10days under normal temperature which will not influence its germination percentage, which provided scientific references for its short term conservation after recovered. Established refrigeration conservation method that could prolong Lingzhong’s short term conservation time to3months. This method lay a solid foundation for its further spreading and commercialization which can satisfy the need of different area’s farmers for Lingzhong’s different storage time.Growth restriction conservation method was first applied to Chuanxiong tube seedling for its middle term conservation. Taking its stem as explant and MS as solid medium, Chuanxiong tube seedling’s culture method was established. The article studied the influences on Chuangxiong tube seedlings by altering culture circumstance, adjusting medium nutrient composition, adding permeable compound and growth inhibitor. And the best growth restriction conservation method had been established. The recovering growth experiment showed that Chuanxiong germplasm could survive after six months’reservation. Variation was not found between preserved seedling and controlled seedling by morphological characteristics comparison and isoenzyme analysis. Conserved seedling had kept good heredity stability. This had been lay a good foundation for Lingzhong’s middle term conservation.Explored the practicability of preserving Lingzhong under ultralow temperature adopting embedding vitrification method. The best explant for embedding vitrification method is the stem tip. This has been established a culture method for Chuanxiong tissue culture seedling and lay a foundation for its further study.3New variety breedingChuanxiong breed colony from1300fine individuals and8000single-plants is constructed on the basis of early Chuanxiong resouce identification. Using this colony, systematic selection method is adopted to breed new Chuanxiong variety of high quality. Over6six years, after good single-plant selection, good stains selection, strain comparison test and variety(stain) muti-location trials, Ivxiong1was finally selected out and certified by the Crops Variety Appraisal Committee of Sichuan Province. Lvxiong I’s mature plant grows dark green leaves that could be kept for a long time. It showed good colony regularity, strong disease-resistance, fine yielding ability, stability and adaptability. Quality of Chuanxiong Rhizoma from the strain fits to pharmacopeia’s need. It had lain a foundation for Chuanxiong variety’s standardization and provided good provenance for Chuanxiong Rizoma’s standardized production.4Lingzhong breeding technologyOn the basis of early research, Lingzhong breeding technical specification including suitable plant area, environmental condition of producing area, field selection and soil preparation, Fuxiong’s selection and treatment, planting, field management, extermination of disease and insect pest, processing in production place and so on were deeply researched. The best methods and measures in every process of Lingzhong breeding were determined. Standard operation practicee for Lingzhong breeding is formulated on basis of it, and this provide technical support for the production of fine Lingzhong at mountain breeding area.5Quality standard study of LingzhongStudied the quality testing protocol of Lingzhong. Taking account into Chuanxiong Rhizoma breeding material’s particularity and using medicinal herb grower’ traditional experience for references, comprehensive indexes were put forward to evaluate Lingzhong’s quality which include morphological appearance,100grain weight, seedling coefficient, mixture rate, insect pest and disease. And quality testing protocol of Lingzhong was formulated which contains authenticity identification, shape index, weight determination method, seedling coefficient, impurity rate and insect pest and disease. It provide technical support for the evaluation of quality of Lingzhong.First studied classification criterion of Lingzhong by compare influences of Lingzhong of different classification on its growth, yield and quality. The result showed that classification significantly influenced its growth and the influence factor varies at its different growth period. The first and second classification apparently excel third classification at Chuanxiong’s growth and yield. Different classification showed little differences at the herbal properties, Thin-Layer Chromatography, content of main component. Through summarizing herb farmer’s long practical experience and early research achievement, and referring to the research result above as well as actual production, Lingzhong classification criterion was drawn up.On the basis of above research results, Chuanxiong Lingzhong Quality Criterion (draft) was drawn up. It provide standard criterion for Chuanxiong Lingzhong’s production, circulation, examination and application.更多还原