【作者】 张晓晴；

【导师】 彭镇华；

【作者基本信息】 中国林业科学研究院 ， 园林植物与观赏园艺， 2012， 博士

【摘要】 中国水仙（Narcissus tazetta var．chinensis）是我国十大传统名花之一，栽植历史悠久。但由于其为三倍体植株，不结实，传统的杂交育种无法进行，千年来只有‘金盏银台’，‘玉玲珑’等少数几个品种，限制了中国水仙产业的发展。随着生物技术的不断成熟，特别是基因工程技术的发展为中国水仙的品种创新提供了有效途径。建立有效的遗传转化体系、发掘中国水仙中潜在的基因资源是中国水仙基因工程育种的必要前提。通过对中国水仙‘金盏银台’品种的各花器官愈伤组织的诱导，筛选出花梗为最佳外植体。其优点有：灭菌方法简便；无愈伤组织诱导、继代难的问题；鳞茎芽再生时间短。筛选出花梗愈伤组织诱导及鳞茎芽分化的最佳培养基为MS+1g·L^-1NaH2PO4+0.003g·L^-16-BA+0.001g·L^-1NAA+0.2g·L^-1Ad+30g·L^-1sucrose。愈伤诱导率达43.33%，愈伤组织诱导到鳞茎芽分化的时间一共需要30～35d，比其它组织通过愈伤组织途径诱导芽再生时间缩短约50d。组织学观察表明小鳞茎是由愈伤组织再分化得来的。通过对几种不同农杆菌菌株侵染率的比较，选择菌株LBA4404；侵染时100mg·L^-1的乙酰丁香酮对农杆菌具有较好的活化作用；最佳预培养时间为15d；最佳共培养时间为3d；最佳抑菌抗生素为头孢霉素，浓度控制在400mg·L^-1。GUS染色结果显示农杆菌LBA4404转化花梗外植体得到了阳性的鳞茎芽。利用该方法进行NTLEAFY基因的转化，PCR检测及Southern杂交检测证明得到转NTLEAFY基因已经整合到中国水仙基因组中。LEAFY基因是成花相关基因，与中国水仙的花芽分化相关。切片观察表明从7月上旬开始，经叶芽时期、花序原基形成期、佛焰状总苞形成期、花原基形成期、花被片形成期、雄蕊形成期、雌蕊形成期7个时期，到9月中旬形成雌蕊结束。分别取中国水仙花芽分化各阶段的组织，用qRT-PCR方法进行NTLEAFY的表达模式分析，主花序中表达量在花芽分化中、后期比花芽分化初始期高，主花序中第一朵小花花冠形成期，基因相对表达量最高。鳞片中的表达量随着花芽分化过程逐渐降低。NTLEAFY基因的表达在花芽中高于鳞片中。证明该基因在中国水仙中调控花芽的启动并参与整个花发育过程。更多还原

【Abstract】 Narcissus tazetta var. chinensis is a famous traditional Chinese Flower with higheconomic and ornamental value. As a homologous triploid plant, it is difficult to createdesirable varieties by traditional hibridization. For thousand years, only two varieties such as‘jinzhanyintai’ and ‘yulinglong’ have cultured in China. As the development of biotechnology,transgenic technology provides an effective way for the Chinese narcissus varieties innovation.The estabilishment of genetic transformation system and the exploration the potential geneticresources in the narcissus is a necessary prerequisite for genetic engineering breeding ofnarcissus.Peduncle was the best explants compared with other organs in this study. The bestexplants was peduncles. It has three advantages in easy sterilization, easy for callus inductionand subculture and the time of duration short. The media for callus induction and shootsregeneration from peduncles were based of MS+30g·L^-1sucrose with1g·L^-1NaH2PO4+0.003g·L^-16-BA+0.001g·L^-1NAA+0.2g·L^-1Ad and the rate of callus induction was43.33%. The process of callus from peduncles to the bulbs bud differentiation required30～35d, which was shorter50d than that from other explants. Shoots were regenerated bypeduncles callus for histological examination in the process of callus from peduncles todifferentiation.Agrobacterium tumefaciens（LBA4404） was the best for infection of peduncles callusamong four kinds of Agrobacterium stains.100mg·L^-1AS was good for A. tumefaciensactivation.15days co-cultivation and3days preculture were good for transformation.Cefotaxime was suit for inhibitting A. tumefaciens and the concentration was400mg·L^-1.Positive shoots got through the above conditions by GUS coloration analysis from peduncles.The results of PCR detection and Southern blot showed that NTLEAFY were inserted intonarcissus genome through the above-mentioned methods. The observation started from early July and went through seven phases, which were leafbud formation, inflorescence primordium differentiation phase, spathe primordia informationphase, sepal differentiation phase, petal differentiation phase, stamen differentiation phase andpistil differentiation phase and is over at pistil fromed in mid-september. Tissues were selectedin each phases and real-time quantitative PCR was used to analyze NTLEAFY gene expression.The expression level of NTLEAFY gene in the period of petal differentiation phase was muchhigher than the initial period. In the scales the gene expression level fell gradually during theflower bud differentiation with higher expression in the inflorescences. The results suggestedthat NTLEAFY gene controled flowering time and related to the flower development.更多还原