马铃薯再生体系的建立及遗传转化的研究-作物遗传育种专业毕业论文.docxVIP

摘要本研究对马铃薯再生培养基、基因型、外植体类型进行了系统筛选，并对影响再生 摘要 本研究对马铃薯再生培养基、基因型、外植体类型进行了系统筛选，并对影响再生 率和农杆菌介导遗传转化主要因素进行了优化，建立了一个适于农杆菌介导遗传转化的 高效受体系统，同时对几丁质酶(chi)基因进行了遗传转化。 实验采用茎段和薯块两种外植体类型，茎段最佳愈伤培养基为MS+0．Img／12， 4-D+1mg／IBA，最佳分化培养基为MS+lmg／IZT+1ing／1GA3，较适宜侵染时间10min：薯 块最佳愈伤及分化培养基均为MS+2mg／IZT+Img／lIAA，较适宜侵染时间5min。 杀菌剂选用进口头孢噻肟钠，在浓度为200mIg仃时，既可完全抑制菌的生长，又对 外植体再生无影响。 选择剂卡那霉素(Kan)在愈伤形成和生根阶段最适浓度分别为50mg／l和75mg／I。 实验共接种转化1395个茎段和363个薯块外植体，获得4株转幽f基因东农303抗 性再生株，经PCR检测及PCR·Southern杂交检测，有3株呈阳性，转化率为3．2％。 关键词马铃薯；簿拿≥遗传转化；chi基因； 鞍二f V 东北农业大学农学硕士学位论文．Abstract 东北农业大学农学硕士学位论文 ．Abstract The systematic screenings of mediums for regenerating plants，genotypes and explant forms were conducted and main factors affecting regenerating rates and genetic transformation were optimized aS well．A high efficient receptor system adapted for genetic transformation by agro bacterium had been established．Genetie transformation of chitinase (chO gene was carried out in the study． Two types of explants，tuber pieces and stem cuRings，were used in the study．For tuber pieces．optimum medium for callus culture was MS+0．1 mg／l 2,4-D+I ml／IBA；the best medium for differentiation Was MS+I mg／l ZT+I mg／l GA3；optimum infection duration was 10 min．For stem cuttings．the best medium for both callus culture and differentiation was MS+2 mg／l ZT+I mg／l IAA，and optimum infection duration Was 5 min． Cefotaxime sodium was used as bactericide，which can restrain bacterium jnfeetion without any negative influence on regeneration of explants when the concentration was 200 ml／1． The best concentration of kanamycine used as selective agent WaS 50 mg／l for callus formation and 75 m∥l for radication respectively． In the experiment，1395 stem cuttings and 363 tuber pieces were infected by chi gene， and 4 transformed plants from variety NEA303 were obtained．By tests of PCR and PCR-Southern hybridization， 3 transformed plants were positive reaction， and the transformation rate was 3．2％． Key Words：potato；regeneration；genetic transformation；chi gene VI 1前言1．1研究的目的