【作者】 高英；

【导师】 裴东；

【作者基本信息】 中国林业科学研究院 ， 林木遗传育种， 2014， 博士

【摘要】 核桃的产量由其树体花芽分化的数量和质量决定，而激素是调控花芽分化的重要因子。因此，前人开展了许多关于激素与花芽分化关系的研究，并在生产实践中施用生长调节剂对花芽分化进行调节。然而，目前即使最先进的激素含量测定方法，也需要一定量的组织材料，所得结果是成百上千个细胞的平均值，而不能对单个细胞或亚细胞中的激素进行精准定位，同时，也不能较准确的分析激素在细胞间的运输与代谢途径。所以本研究采用免疫胶体金定位新技术，以国内主栽早实核桃品种‘辽宁1号’和美国品种‘爱米格’为试材，研究了激素（生长素、脱落酸）在核桃花芽分化过程各组织中的原位分布及可能的运输途径。另外，以液质联用的方法测定茎尖生长素、脱落酸含量的变化，辅助分析了激素对核桃花芽分化的作用。取得的主要结果如下：（1）以核桃雄花芽为试材，利用形态观察和组织切片的方法研究了雄花芽与树体的变化，发现雄花芽外部形态及内部结构变化与核桃树的物候期之间存在密切的相关性。雄先型核桃的雄花芽分化和发育过程是：春季雄花开放始期，可见到幼嫩鳞片包裹的小芽贴生于叶腋，此时雄花芽已进入鳞片分化；在新梢伸长物候期，雄花序呈扁平状，花序内部沿花序轴自下而上逐渐形成苞片原基，并在苞片原基靠近花序轴的基部形成雄花原基；在生理落果物候期，雄花序伸长，并突破鳞片，苞片鲜绿色，此时雄花序组织内部包含了雄花原基，花被原基和雄蕊原基等若干分化时期；在果实迅速生长物候期，雄花序呈松果状，苞片呈黄绿色，尖端褐色，此时花序全部进入雄蕊分化期；在果实缓慢生长物候期，苞片呈褐色，各单体雄花间排列更加紧密，雄花序内部开始花药分化，此后进入分化停滞状态，一直持续到第二年春天；在第二年萌芽物候期，雄花序伸长增粗，花被和花药显现，花序内部开始花粉母细胞分化；新梢抽生物候期，雄花序伸长约10cm，花药变成黄色；此时花粉发育成熟，中层压缩、解体，绒毡层退化解体，待开花后进入新一年的花芽分化进程。雌先型核桃‘爱米格’的花芽分化经历的各时期与雄先型相同，所不同的是各阶段开始分化的时间和进程。雌先型的雄花与雄先型的雌花同时开放，雄花芽的各分化阶段落后于雄先型，至休眠期只进行到雄蕊分化期。（2）以核桃雄花芽为试材，采用免疫胶体金定位技术分析了雄花序中生长素的分布和变化，发现在雄花序分化初期顶端分生组织聚集大量的生长素，此时顶端分生组织可能是生长素的合成部位。当花器官分化时，苞片原基、雄花原基和雄蕊原基中也分布较多的生长素，研究结果证明生长素可能对形态分化有重要的影响。另外，在细胞特化过程中，花粉母细胞和绒毡层仍分布较多的生长素，说明该部位可能是生长素合成的重要部位。当花粉成熟时，花粉萌发孔分布较明显的生长素信号，说明生长素有利于花粉的萌发。（3）以核桃雌雄花芽为试材，利用免疫胶体金定位技术对花芽分化过程脱落酸进行定位分析，结果在花芽分化过程中花柄、幼叶和维管组织始终分布大量的脱落酸，表明这些部位可能是脱落酸重要的源，或为积累源，或为原位源。花芽分化过程中脱落酸的时空分布变化明显。雌雄花芽分化所需的脱落酸水平不一致。对核桃花芽分化过程中脱落酸的原位分析发现由叶芽状态向雌花芽转化时顶端分生组织中脱落酸的水平明显降低。脱落酸在原基细胞（苞片和花被）中信号明显，而当器官分化完成时明显降低。然而，在雄花序分化过程中脱落酸分布广泛，与生殖细胞的发生密切相关。这些证据表明雌花芽的诱导和形态分化需要低于叶芽水平的脱落酸，而雄花序的分化可能与脱落酸关系更密切，分化活跃的细胞中分布较高水平的脱落酸。（4）以早实核桃茎尖为试材，采用免疫胶体金定位技术和液质联用方法对核桃花芽分化过程的生长素和脱落酸进行定量比较分析，结果表明胶体金定位法可以直观的反应植物激素在组织中的时空分布，同时达到定量分析的目的，优于液质联用法。对生长素和脱落酸的平衡分析，可以看出雌花芽的诱导需要较高的脱落酸/生长素，达1.9，而雄花序的分化与生长素和脱落酸的平衡关系不密切。（5）以河南汝阳地区栽植的早实核桃品种‘爱米格’为试材，利用摘叶、去除顶芽和施用生长调节剂的方法，发现去除顶芽会完全抑制二次花的发生；摘叶时间不同则会不同程度的抑制二次花的发生，4月17日摘叶完全抑制二次花的形成，5月7日摘叶处理，二次开花率为21%；而生长素运输抑制剂在核桃二次花的诱导中起到促进作用，两次处理后二次成花率分别为65.6%和61.1%。更多还原

【Abstract】 Walnut production is decided by the quality and quantity of flower-bud differentiation.While plant hormones are the important regulatory factors. Many previous studies have beendone about the relationship between plant hormones and flower-bud differentiation. Growthregulators were used to control flower-bud differentiation in production practice. However,even the most advanced method for the determination of hormones also need amount ofmaterial tissue and the result was the average of hundreds or thousands cells, but not accuratepositioning in a single cell or subcellular cell. At the same time, this method could not analysisthe transport and metabolic pathway of hormones accurately. Then we used new technology ofimmuno-colloidal gold localization to monitor the temporal and spatial pattern of IAA andABA and their transport during flower bud differentiation in the walnut (Juglans regia L.)cultivar Liaoning1of Chnia and Amigo from American. The HPLC-MS used to analysis thequantification of IAA and ABA for supporting role. The main results obtained are as follows:(1) The methods of morphology observation and tissue sections were used in staminateflower study. There is a close correlation among phenology, external morphology and internalanatomical structure. For the protandrous walnuts, as the staminate flowers beginning open, thetender and little bud wrapped with squama pasted in axils. Now in the microscope we can seethe staminate flower bud has entered squama differentiation. During shoot elongationphenology, the staminate flower was flat and covered with squama as the only externallyvisible portion of the catkin. The bract primordia formed from bottom to top and the floretprimordia formed at the base of bract. When fruit drop stage beginning, the length of catkinswas elongated, with catkins protruding from the squama and the floret, perianth, and stamenprimordia formed basipetally. The staminate catkins like pine cones and bracts’ color variedfrom yellow-green to brown. The whole catkins turned into stamens differentiation during theperiod of fruit grown quickly. When the fruit began to grow slowly, the length of catkin did not change evidently. The bracts were brown and each floret arranged tightly. The anthers began todifferentiation and lasted to the spring of next year. When turned into germination stage, thecatkin elongared and thicked, the perianth and anther became visible externally, pollen mothercells begin to differentiate in catkin. As shoots growing coming, the catkin elongated to10cmand the color of anther turned into yellow. As the anthers became matured, middle layercompressing and disintegrating, the tapetum dissoluted and degradated. At this point the entirebud differentiation completed, until flowering a new flower bud differentiation process willbegin.Each differentiation stage of protogynous walnut is the same as the protandrous walnut.However, there are also some differences, such as the time and process of the variousdifferentiation stages. The pistillate flower of protogynous walnut and the staminate flower ofprotandrous walnut opened at the same time. While the staminate flower differentiation is fallbehind the protandrous just staied at stamen differentiation period.(2) The distribution and variation of IAA was analysised using immuno-colloidal goldlocalization in staminate flower of walnut. In staminate flower the auxin signal was strongest inthe shoot apical meristem (SAM) during early differentiation of catkin; thus, the SAM may bea site of auxin production. When the floral organs began centralized differentiation, auxin wasdistributed mainly in the differentiating tissues. Our findings indicate that a high level of auxinmay strongly affect morphogenesis. Additionally, the tapetal and reproductive cells that ariseduring cellular specialization may be important for auxin production. The distribution of auxinwas centralized in germ pores at the pollen grain surface, indicating that a high level of auxininduces pollen germination.(3) The distribution and variation of ABA was monitored using immuno-colloidal goldlocalization in walnut pistillate and staminate flowers. During flower differentiation, highlevels of ABA were consistently indentified in pedicle, young leaf and vascular bundle,indicating there might be the important ABA source caused either by accumulation ororigination. Obvious changes in temporal and spatial pattern were observed during flower-bud differentiation. The differentiation of pistillate and staminate flower needed different level ofABA. The levels of ABA in SAM decreased obviously as the transition from leaf bud topistillate flower-bud. ABA preferentially localized at primordial cells. As the tissues (bract andperianth) became highly differentiated, ABA was significantly and uniformly decreased.However, during stamen development, ABA accumulation was instead restricted to cells thatare closely linked to the formation of reproductive cells. This direct evidence indicates that lowlevel ABA may be needed during pistillate flower induction and morphogenesis, instead, ABAmay strongly affect staminate flower differentiation.(4) We used immunogold localization and HPLC-MS to analysis the quantification of IAAand ABA during flower-bud differentiation in precocious walnut. The result indicated thatimmunogold localization is better than HPLC-MS, because it can respond the spatial andtemporal distribution of plant hormones and achieve the purpose of quantitative analysissimultaneously. To analysis the balance of auxin and abscisic acid, we found that the pistillateflower-bud differentiation requires a higher ratio of ABA/IAA, about1.9. However, thestaminate inflorescence differentiation may have little correlation with the balance of auxin andabscisic acid.(5) The variety ‘Amigo’ of precocious walnut planted in Henan Ruyang area was tested.The methods of pick leaves, remove the terminal bud and application of exogenous growthregulators were used. It is found that the secondary flower inhibited completely by removingterminal bud and the time of pick leaves will affect the occurrence of secondary flowers indifferent degrees. Pick leaves in April17th inhibited secondary flowers completely，while inMay7secondary flowering rate is21%. The auxin transport inhibitor (NPA) plays animportant role in promoting secondary flowers. The secondary flowering rate is65.6%and61.1%respectively.更多还原