【作者】 孙宁；

【导师】 孙建设；

【作者基本信息】 河北农业大学 ， 果树学， 2004， 硕士

【摘要】 苹果是重要的果树树种之一，主要靠嫁接繁殖，其耐盐性主要取决于砧木。因此，选择利用具有较高耐盐能力的砧木是提高苹果耐盐力的关键。利用组织培养与诱变技术相结合选育抗性突变体，可极大提高耐盐砧木育种效率，获得优良的新种质。但是经诱变和耐盐性反复筛选而获得的突变体，不排除部分个体是由环境胁迫而产生的适应性改变，而并非稳定的遗传变异。因此，突变体的鉴定是诱变育种的关键环节之一。 本试验以苹果砧木M₇、78-48及其耐盐诱变株系为材料，对苹果砧木耐盐诱变体进行了筛选和RAPD分子标记分析，主要研究结果如下： 1．采用高浓度NaCl 0.8％和1.0％作为选择压，对2个砧木的耐盐诱变株系进行多代交替盐筛选，获得耐盐性稳定的突变株系。 2．在盐培养下对M₇及其5个耐盐突变株系Z₁-Z₅和78-48及其2个耐盐突变株系L₁、L₂的形态指标进行观察和测定，结果表明：各突变株系的存活率高于对照，受害率和受害指数低于对照，表现较高的耐盐性。 3．建立了苹果砧木稳定、高效的RAPD反应体系，即20μL体系中，模板DNA的用量约40ng，Taq DNA聚合酶用量为1.25U，引物浓度0.25μM，dNTPs浓度为0.2mM，Mg²⁺浓度为3.0mM，1×Buffer。45个扩增循环为宜。 4．本试验对M₇、78-48及其耐盐突变株系进行了RAPD分析。用153个引物检测到M₇的突变株系Z₂的2个多态性位点、Z₃的4个多态性位点和Z₅的1个多态性位点，而78-48的突变株系L₁、1₂多态性位点数分别为42和32个。对2个砧木及其各突变株系的多态性和遗传背景进行统计分析，结果表明，各突变株系与对照的遗传背景高度一致，但在DNA分子水平上确认发生了变异。从而证明了突变的真实性。但所检测出的多态性位点那些与耐盐性突变有关，还需作进一步研究。 5．分析了苹果砧木耐盐突变体耐盐性鉴定的形态和生理生化指标，认为：受害指数能够综合表现苹果砧木受盐害的程度，较准确地反映出盐胁迫对砧木的生长抑制作用，可作为描述苹果砧木耐盐性的形态学指标；细胞膜透性、MDA和Cl^-含量、SOD和POD酶活性等生理生化指标在苹果耐盐突变筛选初期，可作为耐盐性鉴定的参考指标。利用分子标记技术，从DNA分子水平上探查突变体，是鉴定环境适应性改变和遗传突变的最准确的手段。更多还原

【Abstract】 Apple rootstocks were decisive in apple salt tolerance. Screening and breeding salt tolerant mutants could improve apple salt tolerant ability. Recently tissue culture and mutagenesis technique were used in screening salt tolerant mutant successful in many crops. But the mutant’s reality was often doubtful, because it might not be genotype. So it is the most important to identify the genotype of the mutants.The research was carried out with two apple rootstocks: My and 78-48, as well as their salt tolerant regenerations. The mutants were screened were screende out by salt-stressed culture for many generations. The mutants were identified through RAPD technique. The main results were as follows:1.To obtain salt tolerant rootstocks, high level NaCl (0.8% & 1.0%) was added into the culture medium directly. Though salt stress culture for six successive generations, the salt tolerant mutants were obtained.2.The survival rate of two apple rootstocks and their mutants cultured on salt medium were higher than control, but the injury rate and injury index of mutants were lower than control. The mutants showed higher level of salt tolerance.3.A consistent amplication reaction system were established by adding 20uL mixture containing 1.25 units Taq DNA polymerase, 20ng template DNA, 0.25uM primer, 0.2mM dNTPs, 3.0mM Mg + and 1-fold Buffer. 45 amplified cycles were carried out with a set of PCR.4.Through RAPD analysis of rootstock M7 and its mutants, 5 out of 153 randomized primers could be detected to have DNA polymorphism of three mutated lines Z2, Z3 and Z5; 36 random primers could be detected to have DNA polymorphism of 78-48’s mutated lines (L1, L2) definitely. Results showed the mutants varied at the DNA molecular level, which provided an evidence of the mutation. Further study is required to obtain the molecular marker of RAPD closely linked to the salt tolerant mutation locus.5.There were many indicators for salt tolerance identification. Discriminatory analysis showed that injury index could reflect the injuried degree of apple rootstocks under salt stress. So the injury index could be used as a morphological index to accurately evaluate the levels of salt tolerance of rootstocks. In earlier screening, the cell membrane permeability, MDA and Cl content, SOD and POD activity, and some other biochemical indicators could be used as references to assess salt tolerance. However, application ofmolecular marker techniques is a precise method to detect the mutant.更多还原