【作者】 马和平；

【导师】 李毅；

【作者基本信息】 甘肃农业大学 ， 森林经理， 2005， 硕士

【摘要】 枸杞(Lycium barbarum L.)属茄科,是多年生落叶灌木,也是宁夏地区主要经济树种之一。近年来随着枸杞药用保健价值的进一步开发,栽培面积不断扩大,常规育种方法已不能满足现代农业对枸杞品种改良的需求。70 年代发展起来的植物组织培养技术,为作物抗逆品种的选育提供了新的手段。植物组织合理化因素的处理,可大大提高变异的范围和频率。而且,组织培养便于操作和控制,成本低,周期短,所以利用此项技术进行植物培养近年来受到国内外学者的广泛重视。据此,本试验以萌发的枸杞枝条为外植体,进行了组培快繁的研究,对外植体的采取、初代培养、继代培养、壮苗培养、生根培养和移栽以及简化培养基成分的研究,从有利于工厂化育苗的目的出发,试图为枸杞生物技术育种提供技术和方法。本研究以宁夏农科院选育的新品种宁杞2 号为试材,采用其幼年型和成年型枝条的带芽茎段,以多元设计进行试验,用多元分析法进行数据分析,着重研究并筛选枸杞组织培养最佳外植体、培养程序及培养基配方。结果表明: 1. 选取外植体,以幼苗为接种材料时,其枝条中部腋芽及下部腋芽作外植体,对减少污染有较好效果;而以成年树冠外围抽生的枝为接种材料时,以顶芽为外植体效果较好;外植体采集最佳时间为4 月底或5 月初。2. 在初代培养中,以幼树枝条下部腋芽接种于培养基: MS+6-BA1.0mg/L+IBA0.2mg/L+AC0.5g/L 上对枸杞的启动最好。3. 在继代培养中,培养基MS+6-BA1.0mg/L+IBA1.0mg/L对增殖效果最好,30d时增殖系数可达6.03。4. 在壮苗培养中,采用培养基1/2MS+6-BA0.5mg/L+KT0.5mg/L。5. 在生根培养中,其中以1/2MS+IBA0.1mg/L+NAA0.1mg/L最好,生根率为87%。6. 在炼苗培养中,基质以腐质土:蛭石:河沙=1:1:1 对炼苗最好。7. 简化培养基成分,去除有机物,可降低试管苗成本;采用自然光代替日光灯,可以减少设备投入,有利于工厂化育苗生产。8. 以MS 为基本培养基,附加6-BA1.0mg/L和IBA0.5mg/L 对体细胞胚胎诱导最有效;在MS+1.0~1.5mg/L2,4-D 的培养基上胚性愈伤组织诱导率为51.0%和56.7%;黑暗低温处理(10℃)5d 不但可以促进细胞胚胎的发生,而且可以提高其发生的频率(60.0%)。更多还原

【Abstract】 Lycium barbarum L. is a perennial deciduous bush in the solanaceous family, and is an important tree for commerical fruit growing in Ningxia areas. With the further development for medical use and health care recently, the cultivated areas have increased continuously. The convential breeding cannot meet the requirements for Lycium barbarum L improvement of varieties. The techniques, developed in 1970, for tissue culture provide the selection of resistant varieties with an advanced method. The range and frequency of somaclonal variation will be extended widely by combing these techniques with treatment of rational factors. The application of tissue culture become a highlight increasingly because of its easy operation an control, lower costs and short period of breeding. According this, the micropropagation procedure of Lyciun barbarum L.had been researched, including the establishment of selecting explant, indiniation breeding, subculture breeding, stronger nurslings breeding, rooting breeding, transplation and simplifying composition of culture medium which are benefical to the production of factory seedlings. In this study, the stems with buds of “No.2”of Lycium barbarum L.plantlet and mature tree were used for to establish the technich system of tissue culture. Multydesign and multyanalysis was used to find out the best explant proceeding of cultuning and the best composition of medium. The results were as follows: 1. Selecting explant: bottom axillarry bud as explant is effect to childhood. Butterminal buds are effect to mature age. It is the best time which April ending or May beinning that explants are collected. 2. Iniastion culture: MS medium with 6-BA1.0mg/L, IBA0.2mg/L,AC0.5g/L,is the best iniation medium to sprout to childhood. 3.Subculture culture: the MS medium with the 6-AB1.0mg/L and IBA1.0mg/Lwas applied as the subculturing medium. After 30 days culture,the multiplication rate of shoot clumps could reach 6.03. 4.Using 1/2MS with 6-BA0.5mg/L and KT0.5mg/L, stronger nurslings could be got. 5. Rooting culture: 1/2MS medium with NAA 0.1mg/L, IBA0.1mg/L is the best one in rooting culture. The rooting rate is 87%. 6.Transplantion: the transplanting medium in rotten quality soil, vermiculite and river sand by 1 to 1 to 1, is useful. 7.By simplifying composition of culture medium, removing the organic compound, the cost of test-tube seedling can be reduced;Using the natural daylight instead of the fluorescent lamp, conjoning darknees, the culturing can decrease equipment which are benefical to the production of factory seedlings. 8.The induction of somatic embryos were best when the embryonic callus were cultured on MS medium supplemented with 1.0mg/L 6-BA and 0.5mg/L IBA; the embryogenic callus (EC) frequency on the 2,4-D1.0~1.5mg/L medium was 51.0% and 56.7%. Dark and low temperature treatment (10℃) for 5 days can not only promote the taking place of the somatic embryogenesis, but also can improve its frequency (60.0%). 9.The experimental results showed that leaf situation could greatly affect “No.2”of Lycium barbarum regeneration frequency. The best explants were those 35 days apical leaves from the cultural plantlets. The high frequency of shoot regeneration was observed when leaf explants were cultured on 1/2MS medium supplemented with 1.0mg/L 6-BA and 1.0mg/L IBA. Firstly, the leaves were cultured in dark. After 8 days, the explants were transferred under light and began to directly regenerate adventitious buds in about 28 days. The maximum number of the adventitious buds was observed within 35 to 45 days. The shoot differentiation frequency was more than 90.0%. When the shoots grow to 1~2cm high, they were cut from leaves, then transferred on 1/2MS+NAA0.6mg/L+IBA0.2mg/L, and developed into whole plants. This regeneration system could apply to Lycium barbarum transgenic manipulation.更多还原