【作者】 陈菁；

【导师】 孙威江； 赖钟雄；

【作者基本信息】 福建农林大学 ， 茶学， 2008， 硕士

【摘要】 本研究以武夷名丛中的早生种金凤凰、中生种金锁匙以及晚生种肉桂的成年茎段为材料，对武夷名丛的离体培养进行了研究，建立了武夷名丛的无菌系，并对其无菌芽苗进行增殖、生根研究，比较3个品种间的差异，同时还探讨了提取测定茶叶内源激素的一些基本条件参数，并对外植体以及离体培养过程中的培养物进行内源激素的HPLC分析，最后对离体培养前后的材料进行RAPD分析检测其遗传变异性。主要研究结果如下：1、武夷名丛成年材料离体培养研究。试验比较了不同消毒方法、不同切割方式以及光照强度、不同的生长素种类、GA₃浓度、不同的激素水平以及盐和糖的浓度等因素对武夷名丛腋芽诱导的影响，此外，还比较了不同激素水平对增殖的影响以及不同盐浓度和生长素种类对生根的影响。结果表明：以取春季以及一芽二叶时期二叶位的茎段材料为接种外植体最为合适；用0．1％升汞消毒剂浸泡9-11min为最佳消毒方法，但应视不同品种以及老嫩度而定，将肉桂接种于MS+2．0 mg·L^-1BA+0．5 mg·L^-1IAA+0．5 mg·L^-1 GA₃中适合其启动培养；用培养基1／2MS+1．0-1．5 mg·L^-1BA+0．2-0．8 mg·L^-1IAA+0．2 mg·L^-1 GA₃继代培养武夷名丛可以达到一定的增殖效果，金凤凰在培养基中的适应能力比金锁匙和肉桂好；培养基1／4MS+4mg·L^-1NAA中含低矿质盐浓度和较高的生长素，有利于生根。本试验通过增殖培养，平均增殖系数为3，可以预计在这个培养体系情况下，一年内从一个腋芽可以增殖培养729个腋芽，这在茶叶生产中具有广阔的应用前景。2、武夷名丛茶树内源激素变化的HPLC研究。（1）茶叶内源激素的检测，结果表明：GA₃、IAA选择波长分别为207 nm和280 nm，ABA、ZT选择波长分别为280 nm和254nm；用于GA₃分析的流动相为甲醇-乙腈-KH₂PO₃缓冲液系统（15：15：70，v／v，pH 3．5）;用于检测IAA和ABA的流动相为甲醇-乙腈-KH₂PO₃缓冲液系统（20：20：60，v／v，pH 3．5）;用于检测Zeatin的流动相为甲醇-乙腈-磷酸缓冲液系统（15：15：70，v／v，pH 7．0）;（2）武夷名丛茶树外植体与试管苗的内源激素的测定，结果表明：品种（系）间的内源激素含量及含量变化幅度差异可能与其遗传特性有关，GA₃是茶树生长发育不可缺少的内源激素之一；肉桂品种不同生长时期二叶位叶片以及同一生长时期不同部位的叶片含量，以二叶位的新梢生长旺盛，内源激素变化幅度比较大，春季是促进芽苗生长的最合适的季节。添加了外源激素BA、IAA、GA₃情况下茶叶芽苗增殖的效果比无添加任何激素的茶叶芽苗效果好，内源IAA和ZT的含量上升了，但是GA₃的含量反而逐渐下降，在整个离体培养过程中ABA的含量变化不大。3、武夷名丛试管苗遗传稳定性的RAPD检测。结果表明：肉桂品种离体培养前后存在变异，但变异率仅为4．13％，从生产角度考虑，在田间存在4．13％的变异植株并不影响茶叶产品的品质。因此离体培养材料选用茎切段分生组织比较合理。同时发现，组培苗的纯度更高，且外植体和试管苗的DNA浓度相当，含量稳定，也可作为方便取材，提取茶叶DNA的理想材料。更多还原

【Abstract】 In this experiment the adult stem nodes were used as materials to carry out in vitro culture of Wuyimingcong cultivar group such as the early-sprouting line Jin Fenghuang, the middle-sprouting line Jin Suoshi and the late-sprouting cultivar Rou Gui. The aseptic lines were first established, and then the studies on the bud proliferation and rooting were conducted, and finally the difference among the three cultivars was compared. In the meanwhile, the basic condition parameters of extraction and determination on endogenous hormones in tea trees were investigated, and the changes of the endogenous hormones in both explants and in vitro materials were analysed by HPLC. Morover, the genetic stability of the materials of pre and post in vitro culture were performed by RAPD analysis. The main results were as follows:1. In vitro culture of adult stems of Wuyimingcong cultivar group. Experiment with different sterilization methods, as well as cutting mode and different light intensity, the different types of growth hormone, GA₃ concentration of the different hormone levels, as well as salt and sugar concentration on Wuyimingcong axillary buds were induced, and also compared the different hormone levels and the proliferation of different types of salt and and the impact on the root. The results showed that the most suitable explants were two-leaf-located stem materials in spring and one-bud-two-leaf period, the most appropriate disinfection method was using 0.1 % mercuric chloride disinfectant for 9～11 minutes which depended on different cultivars as well as their tender extent, and medium MS+2.0 mg·L^-1BA+0.5 mg·L^-1IAA+0.5 mg·L^-1 GA₃ was suitable for initiation culture of variety Rou Gui, medium 1/2MS+1.0～1.5 mg·L^-1BA+0.2 0.8 mg·L^-1IAA+0.2 mg·L^-1 GA₃ reached fair propagation effects on subculture of Wuyimingcong, and the ordinal adaptability to medium was Jin Fenghuang, Jin Suoshi and Rou Gui, medium 1/4 MS+4 mg·L^-1 NAA containing lower minery and higher auxin concentrations was beneficial to rooting. The average propagation coefficient was 3.00 by propagation culture, and under this culture system it might be predicated that 729 axillary buds be regenerated and propagated from one bud within a whole year, which opened up broad prospect of applications to tea production.2. Changes of endogenous hormones in Wuyimingcong by HPLC.（1）On investigating the endogenous hormones in tea trees, the selected wavelength of GA₃ was 207 nm, and that of IAA, ABA and ZT was respectively 280 nm, 280 nm and 254 nm, the mobile phase for the analysis of GA₃ was methanol- acetonitrile-KH₂PO₃ buffer system （15:15:70, v/v, pH 3.5）, the mobile phase for the analysis of both IAA and ABA was methanol-acetonitrile-KH₂PO₃ buffer system （20:20:60, v/v, pH 3.5） , the mobile phase for the analysis of zeatin methanol - acetonitrile -phosphate buffer （15:15:70, v/v, pH 7.0）. （2） On investigating endogenous hormones in both explants and plantlets, the between-changers contents and content variation ranges might be relevant to genetic characters of the cultivars, and GA₃ was one of the indispensible endogenous hormones during the growth and development of tea trees. The two-leaf-located leaves in different growth phases, different leaf-located leaves in the same growth phases and two-leaf-located leaves grew vigorously, whose variation ranges of endogenous hormones were relevatively larger, moreover, spring was the suitable season for the growth of bud seedlings. The propagation effect of medium adding exogenous hormones BA, IAA and GA₃ was better than free hormones, and under that circumstances the content of IAA and ZT increased while the content of GA₃ declined gradually, where the content of ABA varied little.3. Genetic stability test on in vitro plantlets of Wuyimingcong by RAPD analysis. The main results showed that it varied between pre and post in vitro culture of Rou Gui by 4.13 %, From the production point of view, the existence of 4.13 percent in the fields of plant variation did not affect the quality of tea products, which was stable so that meristems were reasonably used for in vitro culture. On the other hand, the purity of in vitro plantlets was higher while the DNA concentrations of plantlets and explants were almost equivalent and of stable contents, both convenient for material access and DNA extraction.更多还原