【作者】 皮伟；

【导师】 李名扬；

【作者基本信息】 西南农业大学 ， 园林植物与观赏园艺， 2004， 硕士

【摘要】 据史料记载，早在三千年前，古埃及和叙利亚就已开始栽培蔷薇和铃兰，我国关于花卉栽培的文献记载最早的是吴王夫差曾在会稽营建梧桐园，广植花木。现在，花卉产业已成为世界上最具活力的产业之一。由于经济的发展和市场的刺激，花卉育种越来越受到植物育种工作者的重视，对新品种、高品质花卉的要求促进了花卉育种工作的不断发展。人们通过常规杂交育种、辐射育种、组织培养育种、多倍体育种、航天育种等手段培育了大量的花卉新品种。尤其是上个世纪90年代以来，随着分子生物学和基因工程技术的发展，利用转基因技术培育新品种成为育种工作者研究的热点。将目的基因定向导入植物细胞或组织中，培育成植株，则可获得人们预期的新材料(新品种)，从而为花卉的定向育种提供了一条更有效的新途径。目前已有数十种花卉植物获得了转基因植株。 菊花是我国原产的十大名花之一。关于菊花的文字记载最早见于《礼记》中，距今已有两千余年的历史。菊花不仅为我国人民所喜爱，而且早已传至国外，现为世界各国广泛栽培。虽然全世界菊花品种已有7000多个，我国也多达3000多个，但绝大多数品种自然花期集中在11月前后，仅少数花型较小、花色单调的品种能在夏季或早秋开花。因此，丰富的菊花品种资源并未得到充分的利用。菊花的花期问题在一定程度上制约了菊花生产的发展。在菊花的育种工作中，国内外育种者多集中在花色、花形及抗逆性等方面的研究，而改变花期则主要通过人为光照处理的方式来解决。人为光照处理不仅成本高，技术上要求严格，而且并不是所有品种都适合这种处理。这也是至今切花市场上菊花品种单一的原因之一。我国南京农业大学的李鸿渐为了攻克菊花花期局限的难题，在1986年开始利用常规杂交育种法进行切花菊新品种的选育工作，培育了上百个新品种。虽然花形、花色各异，但并为从根本上解决花期的问题。传统的杂交育种方法存在随意性大、杂交难度大、周期太长、难以打破基因连锁等不足。 据研究，植物的开花期是由基因控制的，环境因子以及细胞自身的生长状况对这些基因的表达起着调控作用。环境因子对植物开花时间决定基因的调控是通过光周期和春化作用来实现的。有的植物对日照长度非常敏感，必须经历一定时间的长日照或短日照才能开花，因而相应地分为长日照植物和短日照植物；有的植物必须经过一定时间的低温诱导才能开花，低温对开花的促进作用被称为春化作用；还有的植物营养生长到一定阶段后，即使没有适宜的环境因子的诱导也会开花。菊花是典型的短日照植物，植株在营养生长到一定阶段后，必需经历一段时间的短日照诱导，才能进行西南农业人学硕士学位论文摘要花芽分化和开花。 从拟南芥和金鱼草中，人们利用突变体己经克隆到了一系列与开花时间有关的基因，如:尸尸Fl、CO、卢t洲、LD、API、LFY等开花促进基因和五人勇Fl、乙HY、TFLI等开花抑制基因，并对这些基因的结构、功能以及表达调控进行了深入研究，有的己经进入了应用研究阶段。如:华志明通过控制月尸1基因的超表达导致转基因烟草的花期明显提前;杨树正常开花需要8一10年，couPland将拟南芥LFY基因转入杨树中，6一7个月就开花;安利忻将A尸l基因转入矮牵牛，转基因植株表现出持续不断开花的特性。 FPFI基因是1990年Melze:等人从拟南芥cDNA文库中分离得到的序列长约33obP的促进开花基因，他们检测到该基因在光周期诱导后很快在拟南芥顶端分生组织中表达。后来，Kaniat，3l等将尸尸Fl基因连上CaMV35S启动子再导入拟南芥中，使转基因植株的花期明显提前。黄琼华(2002)将该基因转入油菜中，也获得了具有明显早花、早熟性状的转化植株。 本研究旨在通过对菊花再生体系及农杆菌介导的遗传转化体系的研究，将FPFI基因导入到菊花中，以期获得花期提前的菊花新材料，并为利用转基因技术改良菊花品种的广泛运用打F基础。 主要工作和结果如下: 1.高频再生体系的研究 通过对5个菊花品种幼叶再生的研究，获得了“黄秀芳”以幼叶为外植体的高频再生体系，在以Ms十1.om眺BA+0.!m叭NAA的分化培养基中再生率可高达94%。在l趁MS培养基中添加0.05m妙NAA可使再生芽出根快、整齐、根粗壮。 2.建立了以“黄秀芳”幼叶为受体的转化体系 通过对外植体的Cef脱菌浓度、KM筛选浓度以及影响转化频率因素的研究，获得了根癌农杆菌介导的“黄秀芳”幼叶转化体系:叶盘在00、=0.5的根癌农杆菌菌液中浸染3Omin、45min共培养2一4d、延迟筛选3d;以100m留LCef为脱菌浓度，!sm留LKM为几卜盘分化抗性筛选浓度，!om醉KM为生根抗性筛选浓度。 3.根癌农杆菌介导F尸尸1基因转化菊花及转基因植株的田间鉴定 获得了一批尸尸Fl基因的转基因植株，平均转化率为2.75%。通过转基因植株园艺性状的田间观察，与对照相比，部分植株开花期明显提前，且其它园艺性状也发生了很大改变。主要表现在:植株变矮、叶片变小、叶色加深、分枝节位降低等，初步证明外源FPFI基因己在转基因植株中进行了表达。 4.转基因植株的PCR检测 对转基因植株?更多还原

【Abstract】 The rosebush and the lily of the valley were planted in ancient Egypt and Syria before three thousands years according to historical materials. The earliest recordation about flower planting in China is that the Wu Guo’s king-fu chai had ever constructed the phoenix tree garden to planted a great deal of flowers and trees at hui ji, China.Flower industry has become the one of the livingest industry of the world. The flower breeding has been gotten more and more recognition by plant breeders because of the development of economy and the stimulation of market. It promotes the continued development of flower breeding that the people’s request for new varieties and high character. Breeders have bred a great deal of new flower varieties by taking routine cross breeding, radiation breeding, tissue culture breeding, polyploidy breeding, and spaceflight breeding etc. . Especially since 1990s, with the development of molecular biology and genetic engineering technique, it has become a study hotspot using transgenic technique breed new varieties. We can get anticipatory new materials (new varieties) of plants cultivated after the target gene is inducted directly into plant cell or tissue. They provide a more effective new method for direct breeding of flower. There are several tens kinds of flowers have gotten transgenic plants at present.Chrysanthemum is one of the ten famous flower originated in China with two thousands years history. And the earliest words recordation about chrysanthemum was in Li Ji. Chrysanthemum is not only population in China, but also far and wide in many countries of world. There are 7000 varieties of chrysanthemum in the world and more than 3000varieties in China, but abundance resources of chrysanthemum haven’t been made full use. One cause is that most of them bloom regularly about November; a few of them bloom at summer or early autumn. Another factor is that most of varieties blooming early have small flowers and a little kinds and colors. The flowering time is the key for the development of chrysanthemum on some aspects. Most of breeders in the world are engaged in researches of flower color, flower shape, adverse-resistance in breed of chrysanthemum. The treatment of illumination as a normally method for changing flowering time with high cost and ark for strict technique but does not fit all kinds of chrysanthemum. This is one of the reasons for cut chrysanthemum is lack ofabundant kinds in the market of at present. In order to overcome the difficulties of flowering time, Li Hongjian in Nan Jing agricultural university began breed new varieties of cut chrysanthemum at 1986 with routine hybridization method, which varieties cover many flower shape and color with breeding more than hundred varieties of chrysanthemum, but the problem of chrysanthemum’s flowering time is not been solved completely. There are several difficulties in the traditional breeding methods, such as the great uncertainty, the highly difficult hybridization, the longer breeding periods and the difficulty of breaking up gene linkage. The excellent target genes can be transferred directly if you adopt transgenic method and take the foreign gene into chrysanthemum and make the flowering time of it produce anticipate changes. Furthermore, the advantages of more stability and shorter breeding even enriched the hereditary resources by this method.The flowering time of plants is controlled by genes according to other researches. The expression of the genes is modulated by environmental factors and the growth status of cell itself. Environmental factor modulate the grnes of flowering time depend on photoperiod and jarovization. Some plants are sensitive to the length of illumination, and they are divided into the long-day plant and the short-day plant depend on flowering must undergo a certain long-day or a certain short-day. Some plants’ flowering must under go a certain time induction of lower temperature, and the promotive effect of lower temperature is named jarovization. The other plants can flower without更多还原