导　　师： 崔德才

学科专业： 071010

授予学位： 硕士

作　　者： ;

机构地区： 山东农业大学

摘　　要： 悬铃木/(Platanus acerifolia Willd./)是著名的行道树和庭荫树，在城市绿化中起着重要的作用。本研究课题以悬铃木为实验材料，以其叶片为外植体进行再生培养，系统地研究了激素浓度、叶片因素、光照条件等诸多因素对叶片再生的影响，建立了悬铃木叶片外植体的不定芽高频再生体系。在此基础上，用携带反义磷脂酶D/_r基因/(PLD/_r/)的根癌农杆菌介导转化悬铃木叶片外植体，通过对转化方法和条件的探索，建立了悬铃木的遗传转化体系。悬铃木遗传转化体系的建立对利用基因工程对悬铃木进行品种改良起着重要的作用。 研究结果表明悬铃木的叶片因素对悬铃木再生频率有重要影响，以40天苗龄、处于无菌苗顶部充分伸展的叶片为外植体再生效果好。在MS基本培养基中附加1.5mg／L 6-BA，0.5mg╱L IBA，0.5mg╱L KT的分化培养基可以高频诱导不定芽的产生。在叶片远轴面上划三刀，造成伤口，以该面接触培养基，接种在上述培养基上诱导芽分化，外植体经7天暗培养后转光下培养，15天后开始不经过或很少经过愈伤组织直接分化出不定芽，不定芽出现的高峰期在接种后的20-30天。通常不定芽成簇密集生长，不定芽散布在叶片的各个部位，以切口部位较多，芽分化率高达98％以上。待小芽长至1-2cm，将其从叶片上切下，转到芽伸长培养基/(MS+0.3mg／L6-BA+0.05mg／L NAA+30mg／L Ad/)上使小芽长大成苗，最后移到生根培养基/(1／2MS+0.1mg／L NAA/)上，培养基中琼脂的含量对生根的速度有影响，在低琼脂浓度/(5g／L/)的生根培养基上生根快，14天左右即产生根，最终得到完整的植株。 在建立了悬铃木叶片外植体的高频再生体系的基础上，通过抗生素筛选优化农杆菌介导的悬铃木遗传转化体系。具体过程是：将培养至对数生长期的农杆菌/(OD/_/(6 London plane-tree /(Platanus acerifolia Willd./) is a famous shade tree, which plays a very important role in city afforestation. The leaves of London plane-tree were used as the material and several factors such as hormone concentration, explant condition, illumination condition, and so on were investigated to optimize the regeneration system in vitro. A high frequency regeneration system in culture has been established. This culture system was adapted to Agrobaceriiffn-mediated transformation. The leaf explants of London plane-tree were inoculated with Agrobacerium tumefaciens strains harboring binary vectors, which contained the neomycin phosphotransferase II gene and the antisense of phospholipase D , /(PLD r /) gene. By systematically studying of transformation protocol, we have established plane tree genetic transformation system which is very helpful on modification of London plane-tree's cultivar. The experimental results show that leafs situation can greatly effect on London plane-tree regeneration frequency. The best explants are those apical leaves which from the 40 days cultured plantlets. The high frequency of shoot regeneration was observed when explants were cultured on MS medium supplemented with 1.5mg//L 6-BA, 0.5mg//L IBA and 0.5mg//L KT. Leaves were placed on the medium which is mentioned above for shoot induction. After 7 days cultured in dark, the explants were transferred under light and began to directly regenerate adventitious buds in about 15 days. The maximum of the adventitious buds were observed within 20 to 30 days. The shoot differentiation frequency was more than 98/%. When the shoots grow to 1-2 cm high, cut them from leaves, then transferred on to shoot elongation medium /(MS+0. 3 mg//L 6-BA+O.05mg//L NAA+30 mg//L Ad/) until plantlets coming into being. Finally, the plantlets were rooted on the root differentiation medium /(1//2MS+0. 1 mg//L NAA/) and developed into whole plants within 14 days. Based on the regeneration system, transformation system was estab

关 键 词： 悬铃木 叶片 再生 反义磷脂酶 基因 根癌农杆菌 遗传转化

领　　域： [生物学]