【作者】 董宁光；

【导师】 裴东；

【作者基本信息】 中国林业科学研究院 ， 森林培育， 2008， 硕士

【摘要】 不定根发生既是植物器官分化的重要理论问题,又是无性繁殖和完整植株再生等林业重要实践的关键。成功地诱导不定根,对实现无性繁殖获得最大遗传增益以及转基因材料的完整植株再生都具有重要的实践意义。本研究以741杨试管苗为试材,系统地研究了影响不定根发生的因素,探讨最优化的不定根发生的研究体系,并且着重从激素调控、解剖学、IAA免疫组织化学定位等方面对不定根发生调控机制进行了研究。主要结果如下:1.建立了高效、同步和稳定性好的741杨叶不定根发生体系。对741杨不同发育状态、不同外植体以及生长调节剂IBA水平进行了系统的研究,确定了以741杨生根试管苗上的叶为材料,在1/2MS附加0.5mg/L IBA培养基中进行生根诱导。利用这一体系,生根时间集中诱导后6-8天,第8天生根率达到100%,平均生根条数为3.7条/叶,重复试验的结果表明该体系具有较好的稳定性。2.施用生长素极性运输抑制剂TIBA,明显地地抑制了741杨叶不定根发生,表明IAA极性运输在不定根发生中发挥重要作用。采取培养基中添加和叶片涂抹方法进行TIBA处理,TIBA为5mg/L时,第8天生根率为52.2%,比对照降低了47.8%;TIBA浓度为10mg/L时,第8天生根率为5.6%,比对照降低了94.4%;TIBA浓度为20mg/L时,第8天生根率为0,且叶柄基部长出了愈伤;TIBA浓度为100mg/L时,叶受到毒害,叶片变黄。因此,TIBA抑制不定根发生的较适宜浓度为10mg/L.3.解剖学试验结果表明:741杨叶不定根属于诱生根原基类型,根原基起源于形成层,其发生过程为:形成层细胞纵向和横向分裂加快形成分生组织细胞群,分生组织进一步分裂分化形成根原基,根原基生长发育形成根,根穿过皮层突破表皮,形成不定根。4.以741杨嫩茎和核桃幼胚为材料,优化和改进了木本植物IAA免疫胶体金定位体系。1)针对木本植物组织结构复杂、抗原往往深藏在蛋白质的网络中的特点,用尿素-胰蛋白酶联合消化技术对抗原进行了修复。使修复后的抗原充分暴露出来,抗体很容易接近; 2)提高了缓冲液中Triton X-100的用量。从而增强了细胞膜的通透性,使抗体更容易渗透到细胞内部并与IAA反应;3)在缓冲液中加入了2.5%牛血清白蛋白,消除了背景染色。牛血清白蛋白不仅能降低非特异性吸附,抑制非特异性染色,还可封闭抗血清中混杂的白蛋白抗体; 4)抗原抗体反应由4℃反应过夜改为37℃反应2h,缩短了反应时间,提高了效率。通过染色技术的改进,获得了很好的结果:敏感性高,特异性强,背景清晰,耗时少。5. 741杨叶不定根发生过程中IAA免疫胶体金定位结果显示,不定根诱导阶段叶柄形成层有明显的IAA积累,即IAA在叶柄形成层中的积累与不定根的分化密切相关,施用10mg/L TIBA,明显地延缓了IAA在叶柄形成层中积累,延缓了生根时间,降低了生根率。具体的结果是:诱导前(0d),叶柄中IAA分布较少,表皮和皮层的金颗粒密度为4.8个/100um2,维管组织的金颗粒密度为5.1个/100um2;不定根诱导初期(1~3d),IAA主要分布在加厚的形成层及其周缘维管束,其金颗粒密度为216.8个/100um2;不定根原基形成阶段(3~5d),IAA大量积累在根原基细胞群,其金颗粒密度为345.7个/100um2;不定根伸长阶段(6~7d),IAA集中分布根尖生长点和根中柱鞘,其金颗粒密度分别为210.5和32.3个/100um2;施用生长素极性运输抑制剂TIBA,显著地推迟了生根时间,降低了生根率,TIBA施用后第3天,叶柄中的IAA含量仍然很少,表皮和皮层的金颗粒密度为6.9个/100um2,维管组织的密度为8.7个/100um2;第6天,IAA集中分布在维管组织,其金颗粒密度为150.2个/100um2;第8天,IAA集中分布在根原基,其金颗粒密度为300.5个/100um2;第10天,IAA集中分布在根尖和根维管,其金颗粒密度分别为220.4个/100 um2和30.1个/100um2。6.利用免疫胶体金电镜技术对叶不定根发生过程中的IAA亚细胞分布进行了分析。在不定根诱导初期和根原基形成阶段,IAA主要分布在细胞壁、细胞质、液泡、质体、高尔基体及其囊泡中,没有发现在特定细胞器中积累的特点,间接反映出调控不定根的IAA是在其它组织器官运输而来,IAA质膜上的受体可能在调控不定根发生上发挥重要作用。更多还原

【Abstract】 Adventitious root is an important topic both on the theoretical aspect of plant organogenesis and on the practical side of plant propagation and excised tissues or organs in vitro regeneration. Factors affecting rhizogenesis were investigated, technical-platform of adventitious root was optimized and established, and anatomical and IAA immunolocalized characteristics in the process of rhizogenesis were studied with Poplar 741 in vitro in this research. The main results were as follow:1. A stable, efficient, and synchromic technical-platform of rhizogenesis was established. Leaves, stems, leaf discs and petioles from rooted plantlets and subcultured cultures of Poplar 741 were taken to compare their rooting difference, and the effect of IBA concentration on the rhizogenesis was also studied. The achieved results were that leaves from rooted plantlets were obviously better than other explants in aspects like time of rooting, rooting rate and synchronism in 1/2MS medium with 0.5mg/L IBA. The adventitious root started at the 6th day, and the rooting rate was up to 100% at the 8th day, and the average number of root was 3.7 in this system.2. TIBA application inhibited rhizogenesis of Poplar 741 leaves in vitro significantly, suggesting IAA polar transport plays an important role in the rhizogenesis. The rooting rate was 52.2% at the 8th day in the treatment of 5mg/L TIBA, decreased 47.8% compared with the control.The rooting rate was 5.6% at the 8th day in the treatment of 5mg/L TIBA, decreased 94.4% compared with the control. When TIBA increased to 20mg/L, the basal of petiole had no root and grew out callus. When the concentration of TIBA increased to 100mg/L, the leaves was hurt and the laminae get yellow. So the suitable concentration of TIBA to inhibit the adventitious root was 10mg/L.3. Anatomic result showed: root primordium was induced root primordium in the Poplar 741 leaves.The root primordium originated from the cambium cells.The process of adventitious root was: the cambium cells divided to form cell groups, the cell groups developed into root primordium and the primordium cells grew and developed to adventitioud root.4. The technique of immuno-gold localization of endogenous auxin was improved with embryo of walnut and poplar tender shoots. (1) considering the woody plants characteristics of perennial gowth, compound complexity, antigen hiding in the networks, urea together with trypsin was used to unmask the antigens of tissue sections. (2) the concentration of Triton X-100 in the buffer was improved to increase the permeability of cell membrane. (3) 2.5% BSA was added to the buffer to eliminate the background staining. (4) the reaction condition between antigen and antibody was at 37℃for 2h instead of 4℃overnight. The improved methods have characters of high sensitivity, strong speciality and clear background.5. The results of immuno-gold localization of IAA in the rhizogenesis showed that IAA was accumulated at the cambium during the induction stage. Application of 10mg/L TIBA delayed the accumulation of IAA. The details of the results were: little IAA was found in the basal of petiole before induction, the density of gold particles in cortex and vascular tissues were 4.8/100um2 and 5.1/100um2. At the beginning stage of induction, gold particles mainly distributed in vascular tissues and the density was 216.8/100um2. At the stage of root primordium formation, gold particles mainly distributed in the primordium and the density was 345.7/100um2. At the development stage of root, IAA mainly distributed in the root tip and stele, and the densitys were 210.5/100um2 and 30.1/100um2. At the 3th day after application TIBA, little IAA was found still in the basal of petiole, the density of gold particles in the cortex and vascular tissues were 6.9/100um2 and 8.7 /100um2. At the 6th day, gold particles maily distributed in the vascular tissues and the density was 150.2/100um2. At the 8th day, gold particles mainly distributed in the root primordium and the density was 300.5/100um2. At the 10th day, mainly distributed in the root tip and stele, and the density were 220.4/100um2 and 30.1/100um26 Subcellular localization of IAA in the rhizogenesis of Poplar 741 leaves was studied with immuno-gold microscope technique. Cytoplasm, cell wall, vacuole, plastid and golgi body were labeled with gold particles at the differentiation and primordium formation stages. It was not found that gole particles accumulate in the specific cell organ, indicating IAA regulated rhizogenesis was transported to the destination tissue from other tissues organs.更多还原