中国水仙遗传转化体系研究及NTLEAFY基因在花芽分化过程中的表达
|
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~(-1)NaH2PO4+0.003g·L~(-1)6-BA+0.001g·L~(-1)NAA+0.2g·L~(-1)Ad+30g·L~(-1)sucrose。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。
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~(-1)sucrose with1g·L~(-1)NaH2PO4+0.003g·L~(-1)6-BA+0.001g·L~(-1)NAA+0.2g·L~(-1)Ad 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~(-1)AS 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.
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~(-1)NaH2PO4+0.003g·L~(-1)6-BA+0.001g·L~(-1)NAA+0.2g·L~(-1)Ad+30g·L~(-1)sucrose。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。
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~(-1)sucrose with1g·L~(-1)NaH2PO4+0.003g·L~(-1)6-BA+0.001g·L~(-1)NAA+0.2g·L~(-1)Ad 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~(-1)AS 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.
引文
曹尚银，张秋明，吴顺.果树花芽分化机理研究进展.果树学报，2003，20(5):345～350.
蔡润，黄伟华，潘俊松，等.甜瓜子叶离体培养直接再生不定芽的形态学和解剖学观察.武汉植物学研究，2002,20(5):338～342.
蔡文燕.根癌农杆菌介导人乳铁蛋白基因转化中国水仙的初步研究.福建师范大学硕士学位论文，2005.
陈怡平，丁兰，赵敏桂.用紫斑牡丹不同外植体诱导愈伤组织的研究.西北师范大学学报，2001,3(3):66～69.
陈大明，金勇丰，张上隆.柑橘LEAFY同源基因片段分离及特性研究.园艺学报，2001,28(4):295～300.
陈段芬，高健，彭镇华.水仙属植物研究进展.林业科学，2008,44(3):140～146.
陈段芬.中国水仙花型、花色发育基因（NTMADS1、NtMADS3、NTPDS1和NTPZDS1）的克隆与转化.中国林业科学研究院博士论文，2008,1～5.
陈林姣，缪颖，陈德海，等.中国水仙种质资源的遗传多样性分析.厦门大学学报(自然科学版)，2002,41(6):810-814.
柴红梅，张绍松，万萌，等. GNA和α2PAP双抗表达载体的构建及对烟草的遗传转化.西南农业学报，2002,15(4):35～37.
崔凯荣，陈克明，王晓哲，等.植物体细胞胚胎发生的某些现状.植物学通报，1993,10(3):14～20.
崔广荣，侯喜林，张子学，等.蝴蝶兰叶片离体培养胚状体的发生及组织学观察.园艺学报，2007,34(2):431～436.
崔广荣，刘士勋，何玉华，等.文心兰试管苗丛生芽高效增殖体系的建立.西北植物学报，2005,25(3):562～567.
邓馨，胡文玉.草莓叶片再生芽及遗传转化系统的建立.植物学通报，2000,17(2):174～178.
狄翠霞,张满效,谢忠奎，等.百合组织培养和遗传转化的研究进展.西北植物学报，2006,26(4):858～863.
傅永福，孟繁静.植物成花转变过程的基因调控.植物生理学通讯，1997,33(5):393-400.
顾亨森，高翠华，王淑芬.由水仙愈伤组织产生再生植株.植物学报，1987,28(3):336～339.
桂敏，郑凌，陈敏.四种球根花卉的生育特性，植物学性状及花期调控初探.中国花卉，2000,8～10.
高健.60Co-γ射线辐照中国水仙的诱变效应和机理研究.北京:中国林业科学研究院博士学位论文,2000,1～4.
郭蕊，赵祥云，王文和，陈伟之.百合花芽分化的形态学观察.沈阳农业大学学报,2006,37(1):31～34.
韩晓玲，王玉华，李红民，等.菊苣高效不定芽直接发生及其植株再生.核农学报，2006,20(6):482～485.
郝敬虹,齐红岩,阎妮,王昊翔.园艺作物花芽分化的研究进展.农业科技与装备,2008,1,7～9.
贺丹，王政，何松林.牡丹试管苗生根过程解剖结构观察及相关激素与酶变化的研究.园艺学报，2011,38(4):770～776.
洪维廉，沈明山，郭洁，等.中国水仙花梗组织培养及脱分化的电镜观察.厦门大学学报(自然科学版)，1993,32(增刊1):117～122.
姜长阳，宁淑香，于淼，等.蕨菜愈伤组织高效再生体系的建立.园艺学报，2003,30(3):343～345.
蒋玉宝，于元杰.农杆菌在单子叶植物上的应用.中国农学通报，2005,21(10):47～52.
李钱峰，蒋美艳，于恒秀.水稻胚乳RNA定量RT-PCR分析中参照基因选择.扬州大学学报(农业与生命科学版)，2008,29(2):61～66.
李洪清，李美茹.影响农杆菌介导植物基因转化的因素问题.植物生理学通讯，1999,35(2)：145～151.
李瑾，董美芳，尚富德.丹桂和籽银桂花芽分化的研究.植物学通报，2007,24(5):620～623.
李云，冯慧，田砚亭.红地球葡萄叶片、叶柄不定芽再生体系的建立.园艺学报，2002,29(1):60～62.
李一琨，范云，王金发.籼粳杂交水稻FI种子胚愈伤组织诱导及再生体系建立.植物学通报，1999,16(4):416～419.
李笑寒，李莉，宋风顺，等.根癌农杆菌介导的水稻转基因技术体系的优化.生物学杂志，2011,28(2):13～16.
林益明.福建漳州竹类资源分布与开发利用.亚热带植物通讯，1999,28(2):15～19.
栾爱业，徐海峰，曾黎辉.中国水仙生物技术研究进展.生物技术，2006,16(6):77-80.
栾爱业.中国水仙不同花器官离体培养和鳞茎盘EMS离体诱变的研究.福建农林大学硕士论文.2008.
龙岳林，唐前瑞，许春英.中国水仙花芽形成数量的初步观察.湖南农业大学学报（自然科学版），1996,22(2):214～216.
刘凡，莫东发，姚磊，等.遗传背景及活性炭对白菜小孢子胚胎发生能力的影响.农业生物技术学报，2001,9(3):297～300.
刘庆法，唐克轩，叶建明，等.农杆菌介导的小麦遗传转化条件的研究.复旦学报(自然科学版)，1998,5(4):569～572.
刘文凤.中国水仙LEAFY及NTA基因的克隆与表达分析.北京：北京林业大学2009.
权冬玲，常永义.葡萄不同器官直接再生不定芽研究甘肃农业大学学报，2005,4(2):173～177.
苗红梅，琚铭，魏利斌，等.芝麻愈伤组织诱导与植株再生体系的建立.植物学报，2012,47(2):162～170.
宁云芬，龙明华，陶劲，等.新铁炮百合花芽分化过程的形态学观察.园艺学报，2008,35(9):1368～1372.
秦凡，周吉源.不同生长调节剂对蝴蝶兰快速繁殖的影响.武汉植物学研究，2003,21(5):452～456.
彭镇华，汪政科，孙振元.辐射转基因水仙花育种取得突破.中国花卉园艺，2001,(7):30-31.
沈周高，项艳，蔡诚，等.3个杨树品种叶片再生体系的建立.林业科学，2006,22(11):90～96.
邵寒霜，李继红，郑学勤，等.拟南芥LFY cDNA的克隆及转化菊花的研究.植物学报，1999,41(3):268～271．
宋建军，龙岳林，李莹.薰烟和持续高温处理对中国水仙生长发育的影响.河南科技大学学报(农学版)，2003,23(3):21～23.
孙美莲，王云生，杨冬青，等.骆洋茶树实时荧光定量PCR分析中内参基因的选择.植物学报，2010,45(5):579～587.
汤绍虎，孙敏，周启贵，等.‘雪青’梨叶片高频再生体系的建立.园艺学报，2005,32(6):1084～1087.
魏开发，刘逸，林子英，等.农杆菌介导单子叶植物遗传转化问题与对策植物学通报2008,25(4):491-496.
王关林，方宏筠.单子叶植物基因工程的研究进展及新策略.辽宁师范大学学报(自然科学版)，1996,19(3):223-231.
王关林，方宏筠.植物基因工程.第2版.北京:科学出版社.2002.
王关林，李春斌，方宏筠.提高银杏悬浮培养细胞内酯合成的研究.园艺学报，2002,29(4):321～325.
王瑞，张亚楠，王雅英，田惠桥.中国水仙六倍体的诱导和染色体数目的变异.分子细胞生物学报，2007,40(3):263-270.
王静，娄玉霞，郝再彬.大量元素、有机添加物、激素对蝴蝶兰原球茎增殖的影响.上海农业科技，2004(3):21～23.
汪政科.水仙转化系统建立与基因的克隆及油菜素内酯应答基因鉴定与分析.中国林业科学研究院博士论文，2000.
汪志军，李康，处格鲁克·艾尼，等.胡杨的组织培养.新疆林业科学，1997(4):185～186.
魏琴，王丽，傅体华.周黎军油樟悬浮培养及次生代谢产物的诱导.植物学通报，2008,25(5):591～596.
王关林，方宏筠.植物基因工程原理与技术.北京:科学出版社，1998，598～625.
吴文凯，刘成前，周志刚.用于莱茵衣藻荧光定量PCR分析的内参基因选择.植物生理学通讯，2009(7):667～672.
吴昌银，叶志彪，李汉霞，等.雪花莲外源凝集素基因转化番茄.植物学报，2000,42(7):719～723.
武文琪，沈强，吴艳涛，等.荷兰水仙鳞茎花芽分化与开花调节.上海交通大学学报(农业科学版)，2007,25(5):445～454.
谢友菊，陈璐，戴景瑞.玉米体细胞胚状体诱导及其植株再生的研究.遗传学报，1986,13(2):113～119.
谢嘉华,袁建军.中国水仙(Narcissus tazetta var. Chinensis)的组织培养.生物学杂志，2002,19(3):30-36.
熊莉君，李小芳，王洋，等.崇明水仙不同外植体分化能力与鳞茎增大初探.华东师范大学学报（自然科学版），2008(6):103～109.
阎芳.朱顶红(Hippeastrum vittatum)花芽分化与发育的形态学观察研究.陕西杨凌:西北农林科技大学2009.
杨剑波，许智宏，卫志明和白永延.影响根癌农杆菌附着禾谷类作物培养细胞的因素.实验生物学报，1993(1):1～9.
杨秋生，高致明，萧蓉萍，等.郁金香花芽分化的解剖学研究.河南农业大学学报，1997,31(3):251～254.
姚丽娟，徐晓薇，林绍生，等.蝴蝶兰原球茎增殖分化影响因子探讨.亚热带植物科学，2004,33(3):42～44.
叶兴国，Shirley Sato，徐惠君，等，小麦农杆菌介导转基因植株的稳定获得和检测.中国农业科学，2001.34(5):465～468.
叶祖云，郑丽屏，贺静，等.中国水仙基因转化受体系统的建立.云南大学学报（自然科学版），2001,23(3):235～237.
袁鹰，李启云，郝文媛，等.农杆菌介导玉米遗传转化影响因子的研究.分子植物育种，2006,4(2):228～232.
余望.植物激素对水仙愈伤组织形成及分化的影响.福州师专学报（自然科学版），2001,21(5):55-57.
曾荣华，陈亮，沈明山，等.农杆菌介导的中国水仙遗传转化体系的建立.厦门大学学报（自然科学版），2001,40(6):1145～1149.
赵华燕，卢善发，晁瑞堂.杨树的组织培养及其基因工程研究.植物学通报，2001,18(2):171～176.
赵术珍,阮圆,王宝山.盐地碱蓬幼嫩花序的组织培养及植株再生.植物学通报，2006,23(1):52～55.
赵利铭，刘树君，宋松泉.甜高粱再生体系的建立.植物学通报，2008,25(4):465-468.
张智乾，何秀丽，义鸣放.唐菖蒲离体直接再生技术的研究.华中农业大学学报，2008,27(1):110～116.
张乔松，杨伟儿.中国水仙花芽分化及贮藏期外界因子对花序数的影响.园艺学报，1987,14(2):139～143.
张荣，王国英，张晓红，等.根癌家杆菌介导的玉米遗传转化体系的建立.农业生物技术学报，2001,9(1):45～48.
张清国，黄敏玲，叶秀仙.中国水仙花药培养及植株再生体系建立.分子植物育种，2010,8(2):399～404.
周俊彦.植物体细胞的胚状体发生.植物生理学报，1981,7(4):389～397.
周索，杜丽，褚学英，等.翠菊‘花束绯红’叶片的植株再生体系建立.园艺学报，2010,37(10):1667～1672.
周国宁，应求是，陈绍云，等.温度对郁金香花芽分化的影响.浙江农业学报，1995,7(2):146～148.
钟衡.中国水仙花芽分化研究.园艺学报，1984,11(3):195～201.
朱至清.体细胞无性系变异与植物改良.植物学通报，1991,8（增刊）:1～8.
庄晓英.中国水仙遗传转化及离体诱变的研究.浙江大学硕士论文，2005.
邹清成，庄晓英，卢钢，江鸿飞.反义PSY基因植物表达载体的构建及其对中国水仙的转化.浙江林业科技，2006,26(3):25-30.
Angenent GC, Franken J, Busscher M, et al. A novel class of MADS box genes is involved in ovuledevelopment in petunia. Plant Cell,1995(7):1569～1582.
Bowman J L, Smyth D R, Meyerowitz E M. Genes directing flower development in Arobidopsis. Plant Cel1,1989,1(1):37～52．
Blazquez M A, Soowal L N, Lee I, et al. LEAFY expression and flower initiation in Arabidopsis.Development,1997,124(19):3835～3844.
Bhattarcharjee S. Effect of growth regulating substance on in vitro seed germination of Phalaenopsis hybrid.Indian Agriculturist,1999,43(1/2):79～83.
Bowman J L, Smyth D R, Meyerowitz E M. Genes directing flower development in Arabidopsis. The PlantCell,1989,1(1):37～52.
Bowman J L, Alvarez J, Weigel D, et al. Control of flower development in Arabidopsis thaliana byAPETALA1and interacting gene. Development,1993,119:721～743．
Chain J L, Joachims M L, Hooker S W, et al. Real-time PCR method for the quantitative analysis of humanT-cell receptor γ and β gene rearrangements. Journal of Immunological Methods,2005,300(1):12～23.
Catlin D, Ochoa O, Mc Cormick S, et al. Celery transformation by Agrobacterium tumefaciens Cytologicaland genetic analysis of transgenic plants. Plant cell Rep.1988,7(2):100～103.
Critz L, Davies J. Plasmid-encoded hygromycin B resistance the sequence of hygromycin Bphosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene,1983,25(2):179～188.
Chow Y N, Selby C, Fraser T W, et al. Basal plate tissue in Narcissus bulbs and in shoot clump cultures:Itsstructure and role in organogenic potential of single leaf cultures. Annals of Botany.1993,71(5):437～443.
Colasanti J, Sundaresan V. Control of the transition to flowering. Curr Opin Biotech,1996(7)：145～149．
Coen E S, Carpenter R. The metamorphosis of flower. Plant Cell,1993,5(10):1175～1181.
Carmona MJ, Cubas P, Martinez-Zapater J M. The Grapevine FLORICAULA/LEAFY ortholog is expressedin meristematic regions independently of their fate (Vitis vinifera). Plant Physiology,2002,130(1):68～77.
Cheng Z M, He X Y, Chen C Z, et al. Barley yellow dwarf virus coat protein gene and transgenic wheatplants obtained by pollen tube pathway. Plant physiol,1993,102:1077～1084.
Chavarriage-Aguirre P, Schopke C, Sangare A, et al. Tranformation of cassava (Manihot esculenta Crantz)embryogenic tissues using Agrobacterium tumefaciens. CIAT Working Document,1993,123:222～228.
Cardarelli M, Spano L, Mariotti D, et al. The role of auxin in hairy root induction. Mol Gen Genet,1987,208(3):457～463.
Duijs J G, Voorrips R.E., Visser D L., et al. Microspore culture is successful in most crop types of Brassicaoleracea L. Euphytica,1992,60(1):45～55.
Delzer B W, Somer D A, Orf J H. Agrobacterium tumefaciens susceptibility and plant regeneration of10soybean gencotypes in maturity groups. Crop Science,1990,30(2):320～322.
De Bruyn MH, Ferreira DI, Slabbert MM, Pretorius J. In vitro propagation of Amaryllis belladonna. Plantcell, tissue and organ cult,1992,31(3):179～184.
Etschmann B, Wilcken B, Stoevesand K, et al. Selection ofreference genes for quantitative real-time PCRanalysis in canine mammary tumors using the GeNorm algorithm. Veterinary Pathology,2006,43(6):934～942.
F.Murray,R. Brettell,P. Matthews, et al. Comparison of Agrobacterium-mediated transformation of fourbarley cultivars using the GFP and GUS reporter genes. Plant Cell Reports,2004,22(6):397～402.
Gang Lu, Qingcheng Zou, Deping Guo, et al. Agrobacterium tumefaciens-mediated transformation ofNarcissus tazzeta var. chinensis. Plant Cell Reports,2007,26(9):1585～1593.
Gocal G F W, King R W. Changes in gene expression following long-day floral induction in Loliumtermulentum. Plant Physiol,1996,111(2):150～681.
Hiei Y, Ohta S, Komsri T, et al. Efficient transformation of rice (Oryza satics) mediated by Agrobacteriumand sequence analysis the boundaries of the T-DNA. Plant Journal,1994,6(2):271～282.
Hussey G. Totipotency in tissue explants and callus of some members of the Liliaceae, Iridaceae andAmaryllidaceae. Journal of Experimental Botany,1975,26(2):253～262.
Hosoki T, Asahira T. In vitro propagation of narcissus. Hort Science,1980,15(5):602～603.
Hussey G. Totipotency in tissue explants and callus of some members of the Liliaceae, Iridaceae andAmaryllidaceae. Journal of Experimental Botany,1975,26(2):253～262.
He Z, Zhu Q, Dabi T, et al. Transformat ion of rice with theArabidopsis floral regulator LEAFY causes earlyheading. Transgenic Research,2000,9(3):223～227.
Hempel F D, Weigel D, Mandel M A. Floral determination and expression of floral regulatory genes inArabidopsis. Development,1997,124:3845～3853.
Hofer J, Turner L, Hellens R, et al. UNIFOLIATA regulates leaf and flower orphogenesis in pea. CurrentBiology,1997,7:581～587.
Hookaas-Van, Slogteren G M S. Nature,1984,311:763～764.
Hussey G. Totipotency in tissue explants and callus of some members of the Liliaceae, Iridaceae andAmaryllidaceae. Journal of Experimental Botany,1975,26(2):253～262.
James D J, Passey A J, Babara J. Agrobacterium tumefaciens mediated transformation of the Cultivatedstrawberry (Fragaria×Anannassa Duch.) using disarmed binary vectors. Plant Science,1990,69(1):79～94.
Jack T. Molecular and genetic mechanisms of floral control. Plant Cell,2004,16(1): S1～S17．
Katen A, Jacobson H J, Agrobacterium tumefaciens mediated transformation of Pium sativum L. usingbinary and cointergrate vectors. Plant cell Reports.1990,9:276-279.
Koike Y, Ohbiki A, Mori G, Imanishi H. Effects of bulb storage temperatures and duration on the floweringof Narcissus tazetta var. Chinensis. Jourmal of Japanese Society for Horticultural Science,1994,63(3):639～644.
Kelly AJ, Bonnlander MB, Meeks-Wagner DR. NFL the tobacco homolog of FLORICAULA and LEAFY, istranscriptionally expressed in both vegetative and floral meristems. Plant Cell,1995,7(2):225～234.
Kyozuka J, Konishi S, Nemoto K, et al. Down-regulation of RFL, the FLO/LFY homolog of rice,accompanied with panicle branch initiation. Proceedings of the National Academy of Sciences of USA,1998,95(5):1979～1982.
Lam Chan L T, Lee Sm. Inflorescence culture of some tropical orchid varidties singapore. Journal ofPrimary Industries,1996,24:35.
Li XQ, Liu CN, Ritche SW et al. Factor influencing Agrobacterium tumefaciens mediated transientexpression of gusA in rice. Plant Mol Biol,1992,20(6):1037～1048.
LinJiao Chen, XueYi Zhu, Li Gu, Jian Wu. Efficient callus induction and plant regeneration from anther ofChinese narcissus (Narcissus tazetta var. chinensis). Plant Cell Reports,2005,24(7):401～407.
Gang Lu, Qingcheng Zou, Deping Guo et al. Agrobacterium tumefaciens-mediated transformation ofNarcissus tazzeta var. chinensis. Plant Cell Reports,2007,26(9):1585–1593.
Nitsch J P, Nitseh C. Haploid plants from pollen grains. Science,1969,163(3862):85～87.
Mukhopadhyay A, Arumugan N, Nandakumor PBA, et al. Agrobacterium mediated transformation ofoilseed Brassica campestris transformation frequence is strongly influenced by the mode of shootregeneration. Plant cell Reports.1992,11(10):506～513.
M Sellés, S Bergonón, F Viladomat, et al. Effect of sucrose on growth and galanthamine production inshoot-clump cultures of Narcissus confusus in liquid-shake medium.Plant Cell Tissue and OrganCulture,1997,49(2):129～136.
Malik M. Comparison of different liquid/solid culture systems in the production of somatic embryos fromNarcissus ovary explants. Plant Cell Tissue and Organ Culture,2008,94(3):337～345.
Michaels S D，Amasino R M. Memories of winter：Vernalization and the competence to flower．Plant CellEnviron,2000,23(11):1145～1148.
Mandel MA, Gustafson-Brown C, Savidge B, et al．Molecular characterization of the Arabidopsis floralhomeotic gene APETALA1. Nature,1992,360:273～277.
Mizukami Y, Ma H.Determination of Arabidopsis floral meristem identity by AGAMOUS. Plant Cell,1997,9(3):393～408.
Mellerowicz E J, Horgan K, Walden A et al. PRFLL-a Pinus radiate homologue of FLORICAULA andLEAFY is expressed in buds containing vegetative shoot and undifferentiated male cone primordial.Plant,1998,206:619～629.
Ohl F, Jung M, Radonic A, et al. Identification and validation of suitable endogenous reference genes forgene expression studies of human bladder cancer. Urology,2006,175(5):1915～1920.
Papini-Terzi F S, Rocha F R, Vêncio R Z, et al. Transcription profiling of signal transduction-related genesin sugarcane tissues. DNA Res,2005,12(1):27～38.
Popescu A N，Isac V. High Frequency shoot regeneration from leaf-derived callus in rasphberry（Rubusidaeus L.）. Acta Horticulturae，2000,538(2):667～670.
Pena L, Mart in Trillo M, Juarez J, et al. Constitutive expression of Arabidopsis LEAFY or APETALA1genes in ci trus reduces their generation time. Nature Biotechnology,2001,19:263～267.
Parcy F, Nilsson O, Busch MA, et al. A genetic framework for noral patterning．Nature．1998,395:561～566.
Pelaz S, Ditta GS, Baumann E, et al. B and C noral organ identity functions requires EPALL A MADS-boxgenes．Nature．2000,405:200～203.
Pike LM, Yoo KS. A tissue culture technique for the clonal propagation of onion using immature flower buds.Scentiai Horticulturae,1990,45(1-2):31～36.
Pneuli L, Carmel-Goren, Hareven D et al. The SELFPRUNING gene of tomato regulates vegetative toreproductive switching of sympodial meristems and is the ortholog of CEN and TFL1. Development,1998,125:1979～1989.
Pouteau S, Nicholls D, Tooke F, et al. The induction and maintenance of flowering in impatiens.Development,1997,124:3343～3351.
Raemakers CJJM, Jacobsen E, Visser RGF. Histology of somatic embryogenesis and evaluation ofsomaclonal variation in cassava. CIAT Working Document,1994,150:336～346.
Ray A，Lang J D．Golden short integument(SINI)，a gene required for ovule development in A robidopsis，also controls flowering time. Development,1996,122:263l～2638．
Rottmann W H, Meilan R, Sheppard L A, et al. Diverse effects of overexpression of LFY and PTLF, a poplar(Populus)homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis. Plant Journal,2000,22(3):235～245.
Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques,2000,29:332～337.
Suzuki K，Hattori Y, Uruji M，et al. Compplete nucleotide sequence of a plant tumor-indueing Ti plasmid.Gene,2000,24(2):331.
Stone O M. The elimination of viruses from Narcissus tazetta cv. Grand Soleil d’Or and rapid multiplicationof virus-free clones. Annals of Applied Bio1ogy.1973,73(1):45～52.
Santos J, Santos I, Salema R. In vitro production of bulbs of Narcissus bulbocodium flowering in the firstseason of growth. Scientia Horticulturae.1998,76(3-4):205～217.
Sage D, Hammatt N. Somatic Embryogenesis and Transformation in Narcissus pseudonarcissus Cultivars.Acta Horticulturae,2002,570:247～249.
Sellés M, Bergonon S, Viladomat F et al. Effect of sucrose on growth and galanthamine production inshoot-clump cultures of Narcissus confusus in liquid-shake medium. Plant Cell Tissue and OrganCulture,1997,49(2):129～136.
Sellés M, Viladomat F, Bastida J et al. Callus induction somatic embryogenesis and organogenesis inNarcissus confuses:correlation between the state of differentiation and the content of galanthamine andrelated alkaloids. Plant Cell Reports,1999,18(7-8):646～651.
Sage DO, Lynn J,Hammatt N. Somatic embryogenesis in Narcissus pseudonarcissus‘Golden Harvest andSt.Keverne’. Plant Science,2000,150(2):209～216.
Shannon S. Meeks-Wagne rDry. Genetic in teraction that regulates inflorescence development in Arabidopsis.Plant Cell,1993,5(6):639～655.
Souer E, van der Krol, Kloos D, Spelt C, Bliek M. Genetic control of branching pattern andfloral identityduring Petunia in florescence development. Development,1998,125:733～742.
Southerton S G, Strauss S H, Olive M R et al. Eucalyptus has a functional equivalent of the Arabidopsisfloral meristem identity gene LEAFY. Plant Molecular Biology,1998,37(6):897～910.
Shimada T, Yoshio Yamada and Haruhiko Takeshita. On the formation of bulb and flower in clusterflowering jonqui plant Narcissus Jonquilla L. Agric. Bull. Saga. Univ,1959,6:71～81.
ThellinO, ZorziW, Lakaye B,et al. Housekeeping genes as internal standards: use and limits. Biotechnology,1999,75(2-3):29～295.
Vicentini R, Sassaki F T, Gimenes M A, et al. In silico evaluation of the Eucalyptus transcriptome. Geneticsand Molecular Biology,2005,28(3):487～495.
Van Roekel JSC, Damm B, Melchers LS et al. Factor influencing transformation of tomato (Lycopersiconesculentum). Plant cell Reports,1993,12(11):644～647.
Wang Zhengke, Gao Jian, Li Lubin and Peng Zhenhua. Isolation and characterization of the Agamoushomologus gene NTAG in chinese narcissus (Narcissus tazetta var. Chinensis). Forestry Studies inChina,2006,8(1):21～26.
Weigel D, Nilsson O. A developmental switch sufficient for flower initiation in diverse plants. Nature,1995,377:495～500.
Weigel D, Meyerowitz E M. Activation of floral homeotic genes in Arabidopsis. Science,1993,261(5129):1723～1726．
Willems E, Mateizel I, Kemp C, et al. Selection of reference genes in mouse embryos and in differentiatinghuman and mouse ES cells. Int. J. Dev. Biol.,2006,50:627～635.
Weigel D, Alvarez D, Smyth DR, et al. LEAFY controls floral meristem identity in Arabidopsis. Cell,1992,69(5):843～859.
Xia G M, Li Z Y, He C X, et al. Trangenic plantregeneration from wheat (Tritium asetivam L) mediated byAgrobacterium tumefaciens. Acta phytophysiologica Sinica,1999,25(1):22～28.
蔡润，黄伟华，潘俊松，等.甜瓜子叶离体培养直接再生不定芽的形态学和解剖学观察.武汉植物学研究，2002,20(5):338～342.
蔡文燕.根癌农杆菌介导人乳铁蛋白基因转化中国水仙的初步研究.福建师范大学硕士学位论文，2005.
陈怡平，丁兰，赵敏桂.用紫斑牡丹不同外植体诱导愈伤组织的研究.西北师范大学学报，2001,3(3):66～69.
陈大明，金勇丰，张上隆.柑橘LEAFY同源基因片段分离及特性研究.园艺学报，2001,28(4):295～300.
陈段芬，高健，彭镇华.水仙属植物研究进展.林业科学，2008,44(3):140～146.
陈段芬.中国水仙花型、花色发育基因（NTMADS1、NtMADS3、NTPDS1和NTPZDS1）的克隆与转化.中国林业科学研究院博士论文，2008,1～5.
陈林姣，缪颖，陈德海，等.中国水仙种质资源的遗传多样性分析.厦门大学学报(自然科学版)，2002,41(6):810-814.
柴红梅，张绍松，万萌，等. GNA和α2PAP双抗表达载体的构建及对烟草的遗传转化.西南农业学报，2002,15(4):35～37.
崔凯荣，陈克明，王晓哲，等.植物体细胞胚胎发生的某些现状.植物学通报，1993,10(3):14～20.
崔广荣，侯喜林，张子学，等.蝴蝶兰叶片离体培养胚状体的发生及组织学观察.园艺学报，2007,34(2):431～436.
崔广荣，刘士勋，何玉华，等.文心兰试管苗丛生芽高效增殖体系的建立.西北植物学报，2005,25(3):562～567.
邓馨，胡文玉.草莓叶片再生芽及遗传转化系统的建立.植物学通报，2000,17(2):174～178.
狄翠霞,张满效,谢忠奎，等.百合组织培养和遗传转化的研究进展.西北植物学报，2006,26(4):858～863.
傅永福，孟繁静.植物成花转变过程的基因调控.植物生理学通讯，1997,33(5):393-400.
顾亨森，高翠华，王淑芬.由水仙愈伤组织产生再生植株.植物学报，1987,28(3):336～339.
桂敏，郑凌，陈敏.四种球根花卉的生育特性，植物学性状及花期调控初探.中国花卉，2000,8～10.
高健.60Co-γ射线辐照中国水仙的诱变效应和机理研究.北京:中国林业科学研究院博士学位论文,2000,1～4.
郭蕊，赵祥云，王文和，陈伟之.百合花芽分化的形态学观察.沈阳农业大学学报,2006,37(1):31～34.
韩晓玲，王玉华，李红民，等.菊苣高效不定芽直接发生及其植株再生.核农学报，2006,20(6):482～485.
郝敬虹,齐红岩,阎妮,王昊翔.园艺作物花芽分化的研究进展.农业科技与装备,2008,1,7～9.
贺丹，王政，何松林.牡丹试管苗生根过程解剖结构观察及相关激素与酶变化的研究.园艺学报，2011,38(4):770～776.
洪维廉，沈明山，郭洁，等.中国水仙花梗组织培养及脱分化的电镜观察.厦门大学学报(自然科学版)，1993,32(增刊1):117～122.
姜长阳，宁淑香，于淼，等.蕨菜愈伤组织高效再生体系的建立.园艺学报，2003,30(3):343～345.
蒋玉宝，于元杰.农杆菌在单子叶植物上的应用.中国农学通报，2005,21(10):47～52.
李钱峰，蒋美艳，于恒秀.水稻胚乳RNA定量RT-PCR分析中参照基因选择.扬州大学学报(农业与生命科学版)，2008,29(2):61～66.
李洪清，李美茹.影响农杆菌介导植物基因转化的因素问题.植物生理学通讯，1999,35(2)：145～151.
李瑾，董美芳，尚富德.丹桂和籽银桂花芽分化的研究.植物学通报，2007,24(5):620～623.
李云，冯慧，田砚亭.红地球葡萄叶片、叶柄不定芽再生体系的建立.园艺学报，2002,29(1):60～62.
李一琨，范云，王金发.籼粳杂交水稻FI种子胚愈伤组织诱导及再生体系建立.植物学通报，1999,16(4):416～419.
李笑寒，李莉，宋风顺，等.根癌农杆菌介导的水稻转基因技术体系的优化.生物学杂志，2011,28(2):13～16.
林益明.福建漳州竹类资源分布与开发利用.亚热带植物通讯，1999,28(2):15～19.
栾爱业，徐海峰，曾黎辉.中国水仙生物技术研究进展.生物技术，2006,16(6):77-80.
栾爱业.中国水仙不同花器官离体培养和鳞茎盘EMS离体诱变的研究.福建农林大学硕士论文.2008.
龙岳林，唐前瑞，许春英.中国水仙花芽形成数量的初步观察.湖南农业大学学报（自然科学版），1996,22(2):214～216.
刘凡，莫东发，姚磊，等.遗传背景及活性炭对白菜小孢子胚胎发生能力的影响.农业生物技术学报，2001,9(3):297～300.
刘庆法，唐克轩，叶建明，等.农杆菌介导的小麦遗传转化条件的研究.复旦学报(自然科学版)，1998,5(4):569～572.
刘文凤.中国水仙LEAFY及NTA基因的克隆与表达分析.北京：北京林业大学2009.
权冬玲，常永义.葡萄不同器官直接再生不定芽研究甘肃农业大学学报，2005,4(2):173～177.
苗红梅，琚铭，魏利斌，等.芝麻愈伤组织诱导与植株再生体系的建立.植物学报，2012,47(2):162～170.
宁云芬，龙明华，陶劲，等.新铁炮百合花芽分化过程的形态学观察.园艺学报，2008,35(9):1368～1372.
秦凡，周吉源.不同生长调节剂对蝴蝶兰快速繁殖的影响.武汉植物学研究，2003,21(5):452～456.
彭镇华，汪政科，孙振元.辐射转基因水仙花育种取得突破.中国花卉园艺，2001,(7):30-31.
沈周高，项艳，蔡诚，等.3个杨树品种叶片再生体系的建立.林业科学，2006,22(11):90～96.
邵寒霜，李继红，郑学勤，等.拟南芥LFY cDNA的克隆及转化菊花的研究.植物学报，1999,41(3):268～271．
宋建军，龙岳林，李莹.薰烟和持续高温处理对中国水仙生长发育的影响.河南科技大学学报(农学版)，2003,23(3):21～23.
孙美莲，王云生，杨冬青，等.骆洋茶树实时荧光定量PCR分析中内参基因的选择.植物学报，2010,45(5):579～587.
汤绍虎，孙敏，周启贵，等.‘雪青’梨叶片高频再生体系的建立.园艺学报，2005,32(6):1084～1087.
魏开发，刘逸，林子英，等.农杆菌介导单子叶植物遗传转化问题与对策植物学通报2008,25(4):491-496.
王关林，方宏筠.单子叶植物基因工程的研究进展及新策略.辽宁师范大学学报(自然科学版)，1996,19(3):223-231.
王关林，方宏筠.植物基因工程.第2版.北京:科学出版社.2002.
王关林，李春斌，方宏筠.提高银杏悬浮培养细胞内酯合成的研究.园艺学报，2002,29(4):321～325.
王瑞，张亚楠，王雅英，田惠桥.中国水仙六倍体的诱导和染色体数目的变异.分子细胞生物学报，2007,40(3):263-270.
王静，娄玉霞，郝再彬.大量元素、有机添加物、激素对蝴蝶兰原球茎增殖的影响.上海农业科技，2004(3):21～23.
汪政科.水仙转化系统建立与基因的克隆及油菜素内酯应答基因鉴定与分析.中国林业科学研究院博士论文，2000.
汪志军，李康，处格鲁克·艾尼，等.胡杨的组织培养.新疆林业科学，1997(4):185～186.
魏琴，王丽，傅体华.周黎军油樟悬浮培养及次生代谢产物的诱导.植物学通报，2008,25(5):591～596.
王关林，方宏筠.植物基因工程原理与技术.北京:科学出版社，1998，598～625.
吴文凯，刘成前，周志刚.用于莱茵衣藻荧光定量PCR分析的内参基因选择.植物生理学通讯，2009(7):667～672.
吴昌银，叶志彪，李汉霞，等.雪花莲外源凝集素基因转化番茄.植物学报，2000,42(7):719～723.
武文琪，沈强，吴艳涛，等.荷兰水仙鳞茎花芽分化与开花调节.上海交通大学学报(农业科学版)，2007,25(5):445～454.
谢友菊，陈璐，戴景瑞.玉米体细胞胚状体诱导及其植株再生的研究.遗传学报，1986,13(2):113～119.
谢嘉华,袁建军.中国水仙(Narcissus tazetta var. Chinensis)的组织培养.生物学杂志，2002,19(3):30-36.
熊莉君，李小芳，王洋，等.崇明水仙不同外植体分化能力与鳞茎增大初探.华东师范大学学报（自然科学版），2008(6):103～109.
阎芳.朱顶红(Hippeastrum vittatum)花芽分化与发育的形态学观察研究.陕西杨凌:西北农林科技大学2009.
杨剑波，许智宏，卫志明和白永延.影响根癌农杆菌附着禾谷类作物培养细胞的因素.实验生物学报，1993(1):1～9.
杨秋生，高致明，萧蓉萍，等.郁金香花芽分化的解剖学研究.河南农业大学学报，1997,31(3):251～254.
姚丽娟，徐晓薇，林绍生，等.蝴蝶兰原球茎增殖分化影响因子探讨.亚热带植物科学，2004,33(3):42～44.
叶兴国，Shirley Sato，徐惠君，等，小麦农杆菌介导转基因植株的稳定获得和检测.中国农业科学，2001.34(5):465～468.
叶祖云，郑丽屏，贺静，等.中国水仙基因转化受体系统的建立.云南大学学报（自然科学版），2001,23(3):235～237.
袁鹰，李启云，郝文媛，等.农杆菌介导玉米遗传转化影响因子的研究.分子植物育种，2006,4(2):228～232.
余望.植物激素对水仙愈伤组织形成及分化的影响.福州师专学报（自然科学版），2001,21(5):55-57.
曾荣华，陈亮，沈明山，等.农杆菌介导的中国水仙遗传转化体系的建立.厦门大学学报（自然科学版），2001,40(6):1145～1149.
赵华燕，卢善发，晁瑞堂.杨树的组织培养及其基因工程研究.植物学通报，2001,18(2):171～176.
赵术珍,阮圆,王宝山.盐地碱蓬幼嫩花序的组织培养及植株再生.植物学通报，2006,23(1):52～55.
赵利铭，刘树君，宋松泉.甜高粱再生体系的建立.植物学通报，2008,25(4):465-468.
张智乾，何秀丽，义鸣放.唐菖蒲离体直接再生技术的研究.华中农业大学学报，2008,27(1):110～116.
张乔松，杨伟儿.中国水仙花芽分化及贮藏期外界因子对花序数的影响.园艺学报，1987,14(2):139～143.
张荣，王国英，张晓红，等.根癌家杆菌介导的玉米遗传转化体系的建立.农业生物技术学报，2001,9(1):45～48.
张清国，黄敏玲，叶秀仙.中国水仙花药培养及植株再生体系建立.分子植物育种，2010,8(2):399～404.
周俊彦.植物体细胞的胚状体发生.植物生理学报，1981,7(4):389～397.
周索，杜丽，褚学英，等.翠菊‘花束绯红’叶片的植株再生体系建立.园艺学报，2010,37(10):1667～1672.
周国宁，应求是，陈绍云，等.温度对郁金香花芽分化的影响.浙江农业学报，1995,7(2):146～148.
钟衡.中国水仙花芽分化研究.园艺学报，1984,11(3):195～201.
朱至清.体细胞无性系变异与植物改良.植物学通报，1991,8（增刊）:1～8.
庄晓英.中国水仙遗传转化及离体诱变的研究.浙江大学硕士论文，2005.
邹清成，庄晓英，卢钢，江鸿飞.反义PSY基因植物表达载体的构建及其对中国水仙的转化.浙江林业科技，2006,26(3):25-30.
Angenent GC, Franken J, Busscher M, et al. A novel class of MADS box genes is involved in ovuledevelopment in petunia. Plant Cell,1995(7):1569～1582.
Bowman J L, Smyth D R, Meyerowitz E M. Genes directing flower development in Arobidopsis. Plant Cel1,1989,1(1):37～52．
Blazquez M A, Soowal L N, Lee I, et al. LEAFY expression and flower initiation in Arabidopsis.Development,1997,124(19):3835～3844.
Bhattarcharjee S. Effect of growth regulating substance on in vitro seed germination of Phalaenopsis hybrid.Indian Agriculturist,1999,43(1/2):79～83.
Bowman J L, Smyth D R, Meyerowitz E M. Genes directing flower development in Arabidopsis. The PlantCell,1989,1(1):37～52.
Bowman J L, Alvarez J, Weigel D, et al. Control of flower development in Arabidopsis thaliana byAPETALA1and interacting gene. Development,1993,119:721～743．
Chain J L, Joachims M L, Hooker S W, et al. Real-time PCR method for the quantitative analysis of humanT-cell receptor γ and β gene rearrangements. Journal of Immunological Methods,2005,300(1):12～23.
Catlin D, Ochoa O, Mc Cormick S, et al. Celery transformation by Agrobacterium tumefaciens Cytologicaland genetic analysis of transgenic plants. Plant cell Rep.1988,7(2):100～103.
Critz L, Davies J. Plasmid-encoded hygromycin B resistance the sequence of hygromycin Bphosphotransferase gene and its expression in Escherichia coli and Saccharomyces cerevisiae. Gene,1983,25(2):179～188.
Chow Y N, Selby C, Fraser T W, et al. Basal plate tissue in Narcissus bulbs and in shoot clump cultures:Itsstructure and role in organogenic potential of single leaf cultures. Annals of Botany.1993,71(5):437～443.
Colasanti J, Sundaresan V. Control of the transition to flowering. Curr Opin Biotech,1996(7)：145～149．
Coen E S, Carpenter R. The metamorphosis of flower. Plant Cell,1993,5(10):1175～1181.
Carmona MJ, Cubas P, Martinez-Zapater J M. The Grapevine FLORICAULA/LEAFY ortholog is expressedin meristematic regions independently of their fate (Vitis vinifera). Plant Physiology,2002,130(1):68～77.
Cheng Z M, He X Y, Chen C Z, et al. Barley yellow dwarf virus coat protein gene and transgenic wheatplants obtained by pollen tube pathway. Plant physiol,1993,102:1077～1084.
Chavarriage-Aguirre P, Schopke C, Sangare A, et al. Tranformation of cassava (Manihot esculenta Crantz)embryogenic tissues using Agrobacterium tumefaciens. CIAT Working Document,1993,123:222～228.
Cardarelli M, Spano L, Mariotti D, et al. The role of auxin in hairy root induction. Mol Gen Genet,1987,208(3):457～463.
Duijs J G, Voorrips R.E., Visser D L., et al. Microspore culture is successful in most crop types of Brassicaoleracea L. Euphytica,1992,60(1):45～55.
Delzer B W, Somer D A, Orf J H. Agrobacterium tumefaciens susceptibility and plant regeneration of10soybean gencotypes in maturity groups. Crop Science,1990,30(2):320～322.
De Bruyn MH, Ferreira DI, Slabbert MM, Pretorius J. In vitro propagation of Amaryllis belladonna. Plantcell, tissue and organ cult,1992,31(3):179～184.
Etschmann B, Wilcken B, Stoevesand K, et al. Selection ofreference genes for quantitative real-time PCRanalysis in canine mammary tumors using the GeNorm algorithm. Veterinary Pathology,2006,43(6):934～942.
F.Murray,R. Brettell,P. Matthews, et al. Comparison of Agrobacterium-mediated transformation of fourbarley cultivars using the GFP and GUS reporter genes. Plant Cell Reports,2004,22(6):397～402.
Gang Lu, Qingcheng Zou, Deping Guo, et al. Agrobacterium tumefaciens-mediated transformation ofNarcissus tazzeta var. chinensis. Plant Cell Reports,2007,26(9):1585～1593.
Gocal G F W, King R W. Changes in gene expression following long-day floral induction in Loliumtermulentum. Plant Physiol,1996,111(2):150～681.
Hiei Y, Ohta S, Komsri T, et al. Efficient transformation of rice (Oryza satics) mediated by Agrobacteriumand sequence analysis the boundaries of the T-DNA. Plant Journal,1994,6(2):271～282.
Hussey G. Totipotency in tissue explants and callus of some members of the Liliaceae, Iridaceae andAmaryllidaceae. Journal of Experimental Botany,1975,26(2):253～262.
Hosoki T, Asahira T. In vitro propagation of narcissus. Hort Science,1980,15(5):602～603.
Hussey G. Totipotency in tissue explants and callus of some members of the Liliaceae, Iridaceae andAmaryllidaceae. Journal of Experimental Botany,1975,26(2):253～262.
He Z, Zhu Q, Dabi T, et al. Transformat ion of rice with theArabidopsis floral regulator LEAFY causes earlyheading. Transgenic Research,2000,9(3):223～227.
Hempel F D, Weigel D, Mandel M A. Floral determination and expression of floral regulatory genes inArabidopsis. Development,1997,124:3845～3853.
Hofer J, Turner L, Hellens R, et al. UNIFOLIATA regulates leaf and flower orphogenesis in pea. CurrentBiology,1997,7:581～587.
Hookaas-Van, Slogteren G M S. Nature,1984,311:763～764.
Hussey G. Totipotency in tissue explants and callus of some members of the Liliaceae, Iridaceae andAmaryllidaceae. Journal of Experimental Botany,1975,26(2):253～262.
James D J, Passey A J, Babara J. Agrobacterium tumefaciens mediated transformation of the Cultivatedstrawberry (Fragaria×Anannassa Duch.) using disarmed binary vectors. Plant Science,1990,69(1):79～94.
Jack T. Molecular and genetic mechanisms of floral control. Plant Cell,2004,16(1): S1～S17．
Katen A, Jacobson H J, Agrobacterium tumefaciens mediated transformation of Pium sativum L. usingbinary and cointergrate vectors. Plant cell Reports.1990,9:276-279.
Koike Y, Ohbiki A, Mori G, Imanishi H. Effects of bulb storage temperatures and duration on the floweringof Narcissus tazetta var. Chinensis. Jourmal of Japanese Society for Horticultural Science,1994,63(3):639～644.
Kelly AJ, Bonnlander MB, Meeks-Wagner DR. NFL the tobacco homolog of FLORICAULA and LEAFY, istranscriptionally expressed in both vegetative and floral meristems. Plant Cell,1995,7(2):225～234.
Kyozuka J, Konishi S, Nemoto K, et al. Down-regulation of RFL, the FLO/LFY homolog of rice,accompanied with panicle branch initiation. Proceedings of the National Academy of Sciences of USA,1998,95(5):1979～1982.
Lam Chan L T, Lee Sm. Inflorescence culture of some tropical orchid varidties singapore. Journal ofPrimary Industries,1996,24:35.
Li XQ, Liu CN, Ritche SW et al. Factor influencing Agrobacterium tumefaciens mediated transientexpression of gusA in rice. Plant Mol Biol,1992,20(6):1037～1048.
LinJiao Chen, XueYi Zhu, Li Gu, Jian Wu. Efficient callus induction and plant regeneration from anther ofChinese narcissus (Narcissus tazetta var. chinensis). Plant Cell Reports,2005,24(7):401～407.
Gang Lu, Qingcheng Zou, Deping Guo et al. Agrobacterium tumefaciens-mediated transformation ofNarcissus tazzeta var. chinensis. Plant Cell Reports,2007,26(9):1585–1593.
Nitsch J P, Nitseh C. Haploid plants from pollen grains. Science,1969,163(3862):85～87.
Mukhopadhyay A, Arumugan N, Nandakumor PBA, et al. Agrobacterium mediated transformation ofoilseed Brassica campestris transformation frequence is strongly influenced by the mode of shootregeneration. Plant cell Reports.1992,11(10):506～513.
M Sellés, S Bergonón, F Viladomat, et al. Effect of sucrose on growth and galanthamine production inshoot-clump cultures of Narcissus confusus in liquid-shake medium.Plant Cell Tissue and OrganCulture,1997,49(2):129～136.
Malik M. Comparison of different liquid/solid culture systems in the production of somatic embryos fromNarcissus ovary explants. Plant Cell Tissue and Organ Culture,2008,94(3):337～345.
Michaels S D，Amasino R M. Memories of winter：Vernalization and the competence to flower．Plant CellEnviron,2000,23(11):1145～1148.
Mandel MA, Gustafson-Brown C, Savidge B, et al．Molecular characterization of the Arabidopsis floralhomeotic gene APETALA1. Nature,1992,360:273～277.
Mizukami Y, Ma H.Determination of Arabidopsis floral meristem identity by AGAMOUS. Plant Cell,1997,9(3):393～408.
Mellerowicz E J, Horgan K, Walden A et al. PRFLL-a Pinus radiate homologue of FLORICAULA andLEAFY is expressed in buds containing vegetative shoot and undifferentiated male cone primordial.Plant,1998,206:619～629.
Ohl F, Jung M, Radonic A, et al. Identification and validation of suitable endogenous reference genes forgene expression studies of human bladder cancer. Urology,2006,175(5):1915～1920.
Papini-Terzi F S, Rocha F R, Vêncio R Z, et al. Transcription profiling of signal transduction-related genesin sugarcane tissues. DNA Res,2005,12(1):27～38.
Popescu A N，Isac V. High Frequency shoot regeneration from leaf-derived callus in rasphberry（Rubusidaeus L.）. Acta Horticulturae，2000,538(2):667～670.
Pena L, Mart in Trillo M, Juarez J, et al. Constitutive expression of Arabidopsis LEAFY or APETALA1genes in ci trus reduces their generation time. Nature Biotechnology,2001,19:263～267.
Parcy F, Nilsson O, Busch MA, et al. A genetic framework for noral patterning．Nature．1998,395:561～566.
Pelaz S, Ditta GS, Baumann E, et al. B and C noral organ identity functions requires EPALL A MADS-boxgenes．Nature．2000,405:200～203.
Pike LM, Yoo KS. A tissue culture technique for the clonal propagation of onion using immature flower buds.Scentiai Horticulturae,1990,45(1-2):31～36.
Pneuli L, Carmel-Goren, Hareven D et al. The SELFPRUNING gene of tomato regulates vegetative toreproductive switching of sympodial meristems and is the ortholog of CEN and TFL1. Development,1998,125:1979～1989.
Pouteau S, Nicholls D, Tooke F, et al. The induction and maintenance of flowering in impatiens.Development,1997,124:3343～3351.
Raemakers CJJM, Jacobsen E, Visser RGF. Histology of somatic embryogenesis and evaluation ofsomaclonal variation in cassava. CIAT Working Document,1994,150:336～346.
Ray A，Lang J D．Golden short integument(SINI)，a gene required for ovule development in A robidopsis，also controls flowering time. Development,1996,122:263l～2638．
Rottmann W H, Meilan R, Sheppard L A, et al. Diverse effects of overexpression of LFY and PTLF, a poplar(Populus)homolog of LEAFY/FLORICAULA, in transgenic poplar and Arabidopsis. Plant Journal,2000,22(3):235～245.
Suzuki T, Higgins PJ, Crawford DR. Control selection for RNA quantitation. Biotechniques,2000,29:332～337.
Suzuki K，Hattori Y, Uruji M，et al. Compplete nucleotide sequence of a plant tumor-indueing Ti plasmid.Gene,2000,24(2):331.
Stone O M. The elimination of viruses from Narcissus tazetta cv. Grand Soleil d’Or and rapid multiplicationof virus-free clones. Annals of Applied Bio1ogy.1973,73(1):45～52.
Santos J, Santos I, Salema R. In vitro production of bulbs of Narcissus bulbocodium flowering in the firstseason of growth. Scientia Horticulturae.1998,76(3-4):205～217.
Sage D, Hammatt N. Somatic Embryogenesis and Transformation in Narcissus pseudonarcissus Cultivars.Acta Horticulturae,2002,570:247～249.
Sellés M, Bergonon S, Viladomat F et al. Effect of sucrose on growth and galanthamine production inshoot-clump cultures of Narcissus confusus in liquid-shake medium. Plant Cell Tissue and OrganCulture,1997,49(2):129～136.
Sellés M, Viladomat F, Bastida J et al. Callus induction somatic embryogenesis and organogenesis inNarcissus confuses:correlation between the state of differentiation and the content of galanthamine andrelated alkaloids. Plant Cell Reports,1999,18(7-8):646～651.
Sage DO, Lynn J,Hammatt N. Somatic embryogenesis in Narcissus pseudonarcissus‘Golden Harvest andSt.Keverne’. Plant Science,2000,150(2):209～216.
Shannon S. Meeks-Wagne rDry. Genetic in teraction that regulates inflorescence development in Arabidopsis.Plant Cell,1993,5(6):639～655.
Souer E, van der Krol, Kloos D, Spelt C, Bliek M. Genetic control of branching pattern andfloral identityduring Petunia in florescence development. Development,1998,125:733～742.
Southerton S G, Strauss S H, Olive M R et al. Eucalyptus has a functional equivalent of the Arabidopsisfloral meristem identity gene LEAFY. Plant Molecular Biology,1998,37(6):897～910.
Shimada T, Yoshio Yamada and Haruhiko Takeshita. On the formation of bulb and flower in clusterflowering jonqui plant Narcissus Jonquilla L. Agric. Bull. Saga. Univ,1959,6:71～81.
ThellinO, ZorziW, Lakaye B,et al. Housekeeping genes as internal standards: use and limits. Biotechnology,1999,75(2-3):29～295.
Vicentini R, Sassaki F T, Gimenes M A, et al. In silico evaluation of the Eucalyptus transcriptome. Geneticsand Molecular Biology,2005,28(3):487～495.
Van Roekel JSC, Damm B, Melchers LS et al. Factor influencing transformation of tomato (Lycopersiconesculentum). Plant cell Reports,1993,12(11):644～647.
Wang Zhengke, Gao Jian, Li Lubin and Peng Zhenhua. Isolation and characterization of the Agamoushomologus gene NTAG in chinese narcissus (Narcissus tazetta var. Chinensis). Forestry Studies inChina,2006,8(1):21～26.
Weigel D, Nilsson O. A developmental switch sufficient for flower initiation in diverse plants. Nature,1995,377:495～500.
Weigel D, Meyerowitz E M. Activation of floral homeotic genes in Arabidopsis. Science,1993,261(5129):1723～1726．
Willems E, Mateizel I, Kemp C, et al. Selection of reference genes in mouse embryos and in differentiatinghuman and mouse ES cells. Int. J. Dev. Biol.,2006,50:627～635.
Weigel D, Alvarez D, Smyth DR, et al. LEAFY controls floral meristem identity in Arabidopsis. Cell,1992,69(5):843～859.
Xia G M, Li Z Y, He C X, et al. Trangenic plantregeneration from wheat (Tritium asetivam L) mediated byAgrobacterium tumefaciens. Acta phytophysiologica Sinica,1999,25(1):22～28.