【作者】 李金凤；

【导师】 章镇； 陶建敏；

【作者基本信息】 南京农业大学 ， 果树学， 2007， 硕士

【摘要】 本研究以葡萄砧木‘5BB’(V.berlandierixV.riparia)为材料，建立了叶柄不定芽离体再生的体系，采用农杆菌介导法和超声波辅助农杆菌介导法两种方法将rolC基因导入葡萄砧木‘5BB’中，系统地探讨了提高‘5BB’再生率和影响遗传转化效率的因子，优化了农杆菌介导法的主要技术参数，建立了超声波辅助农杆菌介导法的遗传转化体系。研究结果如下：1．以葡萄砧木‘5BB’试管苗离体叶柄为外植体诱导不定芽再生。研究了不同基本培养基、外植体类型、植物生长调节剂的种类及其浓度组合等因素对‘5BB’再生的影响。结果表明，MS基本培养基为较适合的叶柄再生基本培养基，着生于顶端第2、3节位的叶柄为适宜的外植体，适于叶柄再生的植物生长调节剂种类及浓度组合为BA2.5mg／L，IBA0.05mg／L，叶柄再生频率最高可达到43.33％。2．以葡萄砧木‘5BB’的叶柄为农杆菌介导转化受体，通过对GUS基因瞬时表达率的分析，优化了农杆菌介导的遗传转化技术体系。实验表明，侵染4min、共培养2d、预培养3d，卡那霉素选择压2.5mg／L，头胞霉素浓度250mg／L时可获得40％的GUS瞬时表达率。3、建立了超声波辅助农杆菌介导法转化葡萄砧木‘5BB’的遗传转化体系：茎段为适合的外植体类型，超声波处理时间为8s，功率为100W，AS为75mg／L时GUS瞬时表达率最高可达40％。4、将rolC基因转入葡萄砧木‘5BB’中，经GUS组织化学染色和PCR扩增检测，初步断定rolC基因已整合到‘5BB’2个株系的基因组中。更多还原

【Abstract】 An effective petiole adventitious bud regeneration system of grape rootstock’5BB’(Vitis berlandieri xV. riparia) in vitro was developed in this study.Agrobacterium-mediated and Sonication-assisted Agnobacterium-mediated transformation(SAAT) were adopted to introduce the roIC gene into ’5BB’. In this research, factors thatcan increase the efficiency of grape plant regeneration and transformation were studiedsystematically, the major technique parameters of agrobacterium-mediated transformationwere optimized, and built the genetic transfrormation system of SAAT. The detailed resultswere listed as follows:1. Adventitious buds were induced from petiole of grape rootstock ’5BB’. The effectsof diferent basic media, explant types, plant growth regulators and their concentrationcombinations on regeneration frequency of grape rootstock ’5BB’ were studied in vitro. Theresults indicated that the preferable basic medium for inducing adventitious bud frompetiole was MS basic medium; The better explant type was petiole which nodal position isthe second and the third; The felicitous growth regulator combination was BA 2.5mg/L andIBA 0.05mg/L; The highest regeneration frequency of petiole was 43.33％.2. Transgenic plants were generated from petioles of grape rootstock ’5BB’ byagrobacterium-mediated transformation, and the percentages of GUS transient expression wereanalyzed to study the optimum parameters of this transformation system. The resultsshowed that 40 percentage of GUS transient expression could be realized under the followingcondition, namely explants being dipped into agrobacterium suspension for 4 minutes, twodays of co-cultivation and pre-culture on MS with Km 2.5mg/L, and Cef 250mg/L. Resistantbuds were obtained after maintainable culture on MS supplemented with BA2.5mg/L+IBA0.05mg/L+Cef250mg/L+Km2.5mg/L.3. The genetic transformation of SAAT was built: The tiptop percent (40％) GUStransient expression was obtained when the better explant type was stem, Sonication treatmenttime was 8s., Sonication treatment power was 100W and AS concentrateion was 75mg/L.4. The rolC gene was transformed into grape rootstock ’5BB’ for the first time. The new idioplasm of grape rootstock ’5BB’ with fastness of kanamycin was obtained. One linewith fastness of kanamycin had approved that roIC gene was conformitied into ’5BB’genome by GUS histochemistry coloration and PCR.更多还原