【作者】 刘琴；

【导师】 李式军；

【作者基本信息】 南京农业大学 ， 蔬菜学， 2002， 硕士

【摘要】 山葵(Wasabi japonica Matsum)又名山嵛菜，为十字花科山嵛菜属多年生草本植物，具有强烈的香辛味。由其根状茎制成的山葵膏作为一种高级调味品已风靡欧美、日本和东南亚地区。最新的研究表明，山葵中的某些活性成分具有杀菌、防腐、利尿、清血、抗感染、抗癌等多种药用活性，其产品受到越来越多人们的青睐。 目前在山葵生产中存在的一个最大问题即健壮种苗的供给。在生产上山葵多采用种子繁殖和分株繁殖，前者山葵种子难以长期保存；后者则存在多代营养繁殖种性退化的问题，由此导致种苗繁殖系数低、繁殖速度慢，难以生产大量优质种苗的问题。为解决这一难题，人们试图通过山葵的茎尖培养建立组培快繁体系，虽已取得一定进展，但这一体系也存在着很多不足之处，其中最为突出的有两点： (1)初代培养时，由于污染严重而使成苗率很低，茎尖生长缓慢而使初代培养时间很长 (2)增殖培养时，增殖系数较低，山葵苗长势较弱而使得继代周期较长。 本文针对生产实践的要求，在前人工作的基础上，从山葵茎尖快繁体系优化和山葵愈伤组织培养初探两个方面着手，进行了一系列的研究。 1．山葵茎尖初代培养 1．1利用室外生长的山葵植株进行茎尖初代培养 考察了不同消毒处理程序、供体植株栽培方式和生长时期对成苗率及生长的影响，研究结果表明：选用在消毒基质上生长4个月的植株较为适宜；进行顶、侧芽的消毒处理前，宜先将其切剥成0.1-0.2cm大小；0.1％升汞消毒时间为5min。以上改进措施使初代培养的成苗率上升到52％，同时茎尖生长加快。 1．2利用组培条件下山葵种子无菌苗进行茎尖初代培养 考察了不同种子状态、升汞消毒时间对无菌苗率的影响，以及种子无菌苗的苗龄、培养基、BA、蔗糖浓度、pH、温度等对茎尖生长的影响。研究结果表明：应选用未露白的种子，0.1％升汞消毒10min；种子成苗后，取苗龄在35-50d的山葵苗的茎尖效果最好，茎尖生长的适宜条件为：1／2MK(0.5 6-BA)，30g／L蔗糖，pH5.8，温度18度。成苗率达到72％，且茎尖快速萌发，无菌苗生长旺盛。 2．山葵的增殖培养 2．1在山葵的增殖培养中，考察了培养基、TDZ和 2 i P对山葵生长和增殖的影响，同时首次将无机基质作为一种新的培养基介质引入了山葵的组培体系。以上研究的结果表明：MK培养基比较适宜山葵的生长；TDZ不能有效促进山葵的增殖和生长，Zip在1．0的浓度水平上能够很好的促进山葵的生长，但对增殖的作用不显著；以无机基质取代琼脂进行山葵的增殖培养，有利于山葵的生长，但对增殖无促进作用。 2．2 在山葵的增殖培养中，首次采用分阶段培养的方法使山葵的生长和增殖水平得到了显著提高。研究结果表明：将山葵单芽接种于 MK（6－BA 5＋NAAO．05）的液体培养基中，pH6．0，置于 100r．min“’的摇床上旋转培养 Zd，然后转接于 MK（l刀Zip＋0．05NAA）的琼脂固体培养基中培养，30d后能使繁殖系数达到7．33，且植株生长较快；若转入相同的无机基质培养基中进行第二阶段的培养，虽然繁殖系数有所下降（为6．33），但植株生长更加旺盛、健壮。3．山葵的生根、驯化和移栽 在生根培养中，研究了IAA、IBA、NAA以及无机基质培养对生根的影响，结果表明；0．Zing几的 NAA对生根的效果最佳；以无机基质取代琼脂进行生根培养显著促进了根系的发育，即使在无激素的无机基质培养基上，生根率也达到了 100％。 在驯化移栽的阶段，考察了不同生根培养时间和移栽基质对成活率和生长的影响，研究表明：生根培养时间不同的山葵苗在成活率上没有差别，但生长状况却明显不同，其中未经生根培养和仅经历7d生根培养的山葵小苗生长健壮，叶色正常；而随着生根培养时间的延长，山葵大苗反倒出现徒长现象，且叶色变浅。不同的移栽基质组成对成活率没有影响，但对生长情况而言，含有苇末的基质组合显著促进组培苗生长，其余三种处理间没有显著差异。4．山葵的愈伤组织培养 研究了不同类型外植体、激素浓度及配比、环境条件、外源添加物和培养方式等对山葵愈伤组织诱导、增殖和分化的影响，结果表明；山葵的幼叶叶柄比较适合进行愈伤组织诱导，诱导宜在 25 C。黑暗条件下进行，TDZ能有效促进山葵愈伤组织的产生；以 1／ZMS（0．ZTDZ＋0．4NAA）为基本培养基，外源添加 3mg／L的 AgNO。，进行固液交替培养最利于山葵愈伤组织的增殖和分化。更多还原

【Abstract】 Wasab[(Wasabi japonica Matsum),which originally grows in Japan and China, is a rare worldwide vegetable used both for culinary and medicinal purpose, in recent years as the development of peoples’ living condition, the need for wasabi products greatly increases each year. Now one of the main problems in planting wasabi is the production of wasabi seedlings. In order to resolve this problem, a shoot-tip culture system has been established. However ,in this system , the contamination ratio of explants in initial culture is very high and the proliferation rate of plantlets is very low and the culture cycle is very long, all this limits its practical use.This study focuses on optimizing shoot-tip micropropogation system, some work on wasabi callus induction, multiplication and diffrentiation is also involved in it.1.optimizing shoot-tip micropropogation system1.1 In initial cultureEffects of sterilizing process, plant growth time and cultivation mode on contamination ratio and plantlet growth were studied, germ-free plantlet ratio was up to 52%.Shoot-tips and accessory buds for initial culture were often derived from field-grown wasabi plants, wasabi seeds were firstly used to culture for shoot-tip micropropogation. By this new method, the germ-free plantlet ratio was increased to 72% and plantlets grow better.1.2 In multiplication cultureIn order to improve plantlet propogation and growth, the effects of different media and new hormone (TDZ and 2ip) were investigated, inorganic substrate and two-phase culture method were firstly applied in wasabi multiplication culture. The results demonstrated that MK medium containing 2ip 1.0mg/L,NAA 0.05mg/L and the substitute of inorganic substrate(vermiculite/perlite = 2/l) for agar in culture medium were better for plantlet growth, and the two-phase culture method was excellent both for plantlet propogation and growth. The combination of MK medium(2ipl.O+NAA0.05) and two-phase culture method could advance proliferation rate from 3-4 to 7-8 andcould help plantiet grow best.1.3 In rooting and acclimatizationIn rooting culture, 0.2 NAA and inorganic substrate(vermiculite/perlite = 2/l) used in culture medium were suitable for root development and growth. In acclimatization, effects of rooting culture time and cultivate substrate on plantlet’s survival ratio and growth were studied, the results showed that plantiet without rooting culture was better for acclimatization, and the organic substrate (reed residue) was outstanding for plantiet growth.2. primary study on wasabi callus culturethe conditions of wasabi callus induction, multiplication and diffrentiation were investigated, the results demonstrated that young petiole was suitable for callus induction, which should be done under 25 癈 and in darkness, TDZ could efficiently induce callus; the combination of 1/2MS(0.2TDZ+0.4NAA) containing 3mg/L AgNo3 and solid-liquid alternation culture is best for callus multiplication and diffrentiation.更多还原