【作者】 于海武；

【导师】 张志毅；

【作者基本信息】 北京林业大学 ， 林木遗传育种， 2004， 硕士

【摘要】 本研究以八个毛白杨优良无性系植株为试验材料。在盐胁迫条件下，调查了八个毛白杨优良无性系的存活状况，受害症状及苗高等生长性状，同时测定了植株叶片内叶绿素，脯氨酸，MDA的含量等理化指标。进而，利用农杆菌介导的基因工程方法将mtlD基因导入毛白杨中，获得了转基因植株。经研究获得如下主要结论： 1．经盐胁迫处理后，毛白杨各个无性系的耐盐能力存在差异，主要表现在苗木存活状况，受害特征出现的时间和受害程度，以及高生长量等方面。 2．经盐胁迫处理后，随着盐浓度的升高，毛白杨无性系植株的叶片内叶绿素含量逐渐降低，MDA含量逐渐升高，脯氨酸含量先上升后下降。 3．根据盐胁迫后毛白杨优良无性系植株的生长表现，再辅以生理生化指标，评定八个毛白杨优良无性系的耐盐性，由强到弱可分为三个类型：L5和L16耐盐能力较强；L11、L14和L27耐盐能力中等；L12、L13和L24对盐胁迫敏感。 4．建立了毛白杨优良无性系的最佳再生体系：最佳增殖培养基是MS+6—BA0.3mg／L+NAA0.1mg／L附加糖3.0％和琼脂0.5％；最佳叶片分化培养基是MS+6—BA3.0mg／L+NAA0.1mg／L附加糖3.0％和琼脂0.5％；最佳生根培养基是1／2MS基本培养基附加NAA0.3mg／L，另加入糖2.0％和琼脂0.5％。 5．同龄同代的无根组培苗叶片和生根组培苗叶片作外植体，不定芽生成率有差异；不同类型培养基（WPM、MS）对毛白杨叶片再生率的影响无显著差异；在培养基中加入不同浓度梯度的AgNO₃溶液，结果发现4mg／L的AgNO₃能够提高叶片产生不定芽的数量。 6．按照以下优化的转化体系：菌液OD₆₀₀=0.3-0.5；侵染时间为10—20min；叶片在黑暗下共培养2-3天；卡那霉素的临界浓度是50mg／L，对筛选出的抗性植株进行PCR及PCR-Southern检测。结果表明，成功获得了转入mtlD基因的毛白杨植株。更多还原

【Abstract】 In this study, the eight clones of Populus tomentosa were used for evaluating their salt-resistance abilities through the experiments. Under the condition of salt stress, the survival performance, stress appearance and height growth rates of clones were investigated; at the same time, chlorophyll content, proline content and MDA content were measured. Furthermore, the mtlD gene was transformed into the plantlet of Populus tomentosa successfully. The results were as follows:1. Under the condition of salt stress, it was all difference among 8 clones indicated by the survival performance, stress appearance and height growth rates.2. With the increase of salt concentration, the chlorophyll content decreased; The proline content fluctuated and the MDA content climbed gradually.3. According to growth performance of eight clones affiliated with their physiological and biochemical parameters, the salt resistance abilities of clones were judged three types: L5 and L16 had a good tolerance to salt stress; the abilities of LI 1, L14 and L27 to salt resistance were moderate; LI2, L13 and L24 were the most sensitive to salt stress.4. The optimum regeneration system of Populus tomentosa was founded as follows: proliferation medium was MS+ 6-BA 0.5mg/L+NAA0.1mg/L+sugar 30g/L+agar 4.5g/L; regeneration medium of leaf-explant was MS+6-BA 3.0mg/L+NAA0.1mg/L+ sugar30g/L+agar 4.5g/L; rooting medium was MS+NAA0.3mg/L+sugar 20g/L+agar 4.5g/L.5. The test demonstrated the best leaves for adventitious bud regeneration stem from rooted plant. And, there were significant effects on adventitious bud regeneration by the way of adding different concentrations of AgNO3. At the same time, the effects ofIdifferent types of media on the regeneration of adventitious shoots were studied, indicating that there was no difference in adventitious shoot induction. 6. The optimized condition for transformation of Populus tomentosa was co-cultured with an engineered A.tumefaciens strain carrying mtlD gene from E. coli. for 2-3days and infected with A.tumefaciens concentration of OD6oo=0.3-0.5 about 10-20min. The buds were screened in the differentiation culture media containing Km 50mg/L. At last, the integration of the mtlD gene into plant genomes of transgenic P. tomentosa was confirmed by PCR-Southern hybridization and PCR analysis.更多还原