【作者】 丁霞；

【导师】 陈晓阳；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2006， 博士

【摘要】 本研究以胡杨当年生枝条的带芽茎段为外植体,首先建立了它的离体快繁及植株再生体系,并就植物生长调节剂等因素对胡杨叶片再生不定芽的影响进行了详细的研究;然后利用NPTⅡ筛选基因对影响根癌农杆菌介导胡杨遗传转化的条件进行了深入探索,通过优化转化条件,建立了胡杨遗传转化系统;最后在此基础上,进行rolB-pttGA20ox双价基因的转化,并由PCR、PCR-Southern杂交分子检测,验证rolB-pttGA20ox基因的插入情况。研究结果摘要如下:1.胡杨组培再生体系的建立消毒的最佳方法为:夏季采集的材料: 70%的酒精浸泡30sec后,以0.1%氯化汞分别灭菌7min、8min;初春水培枝条:试剂同上,茎尖灭菌0.5min,茎段灭菌1.0min。最适宜的启动培养基为MS+6-BA0.5mg/L+NAA0.1mg/L,MS+6-BA0.5mg/L+NAA0.2mg/L+糖20 g/L为增殖培养的最佳配方,生根培养的最适培养基为1/2MS+IBA1.0mg/L+糖25g/L+琼脂6g/L+AC 0.2 g/L,生根率达到81%。移栽炼苗以蛭石:珍珠岩(2:1)或者沙子:土壤(1:1)作为最佳基质。2.在组培中黄化现象产生的规律以及避免的方法在一个继代周期内,随着时间的推移,试管苗的黄化率越来越高,整体呈现线性上升的趋势;经生理生化指标研究发现,当继代时间超过20d后,叶绿素含量尤其以叶绿素a的降幅较大,建议可以接种20d为一个继代周期;ABA为IAA、ZR的拮抗物,它的消长过程大体上与生长过程是相反的;采用SDS-聚丙烯酰胺凝胶电泳法对蛋白质进行分析,发现黄化试管苗电泳谱带均出现缺失等异常现象。这说明黄化苗在基因表达调控上出现差异,其内部机制可能已发生变化。研究影响胡杨黄化率的培养条件因子发现:培养基的pH值在6.0～7.0之间、昼夜变温处理、不同波段的光质中以蓝光对降低胡杨黄化率有较好效果。3.胡杨遗传转化体系的建立胡杨叶片再生的最适培养基是MS+BA0.5mg/L+NAA0.1～0.2mg/L+腺嘌呤40mg/L+水解乳蛋白500mg/L +白砂糖25g/L+琼脂6.0g/L,再生频率达100%;叶片培养的最佳取材位置是自茎尖展叶始第1～3片叶;叶片正面向上或正面向下接种培养,对其更多还原

【Abstract】 The major objective of this study was to breed new Populus euphratica varieties with improved rooting ability and apical dominance through transformation of the rolB-pttGA20ox two genes by using Agrobacterium tumefaciens-mediate transformation technology. For this purpose, first, an efficient in vitro propagation system and plant regeneration system were developed for Populus euphratica. Factors that affecting shoots regeneration via plant growth regulators etc., were investigated in details. Subsequently, parameters that affecting the efficiency of Agrobacterium tumefaciens-mediate transformation of Populus euphratica were comprehensively studied and optimized by accessing NPTⅡexpression, and a transformation protocol was established. Finally, on the basis of the established transformation system, pttGA20ox、rolB-pttGA20ox genes were transferred to Populus euphratica by PCR and PCR-Southern blotting tests.The results of this study were introduced separately as following:1. Establishment of plant egeneration system of Populus euphraticaThe optimum sterilized measure for summer material is to be immerged in 70% ethanol for 30seconds and then in 0.1% HgCl2 for 7 and 8 minutes in succession. For stem apexes, it’s better to be immerged in 70% ethanol for 30seconds and then in 0.1% HgCl2 for 0.5 minutes, but for stem segments with axillary buds the optimum time in 0.1% HgCl2 is 1.0 minutes. The optimal induction medium was MS+6-BA0.5mg/L+NAA0.1mg/L, the proliferation medium was MS+6-BA1.0mg/L+NAA0.2mg/L+sugar20g/L, and the most optimal rooting medium was 1/2MS+IBA1.0mg/L+surar 25g/L+AC0.2g/L, rooting rate being up to 81%. And the appropariate substance for seeding transplantation is vermiculite and perlite in 2:1 or sand and soil in 1:1.2. Etiolation in the vitro of Populus euphratica.and measures:The etiolated rate was increased with the increasing of days obviously within a subculture circle. With some index of physio-biochemical the results showed that: when the subculture time was more than 20 days, chlorophyll content falled much especially更多还原