Status of sturgeon aquaculture and sturgeon trade in China: a review based on two recent nationwide surveys

The authors reviewed the aquacultural history of Acipenseriformes in China, related the legal status and examined the current status of the cultured species or hybrids, origins of seedlings, quantities of production, geographic distribution in farming, and the sustainability for both restocking programmes and human consumption. The census shows that since 2000, the production of cultured sturgeons in China appears to have become the largest in the world. As of 2000, the rapid growth of sturgeon farming in China mainly for commercial purposes has shifted harvests in the Amur River from caviar production to the artificial culture of sturgeon seedlings. This dramatic development has also caused a series of extant and potential problems, including insufficient market availability and the impact of exotic sturgeons on indigenous sturgeon species. Annual preservation of sufficient higher-age sturgeons should be a national priority in order to establish a sustainable sturgeon-culture industry and to preserve a gene pool of critically endangered sturgeon species to prevent their extinction.

Artificial interspecific hybridization between Macrobrachium species

Viable F-1 hybrids were obtained from crosses of female Macrobrachium nipponense and male Macrobrachium hainanense involving spermatophore transfer and artificial insemination. This represents the first successful known case of hybridization of two Macrobrachium species by means of artificial insemination. The hatching rate was over 90%. About 20-60% of newly hatched larvae metamorphosed to postlarvae. The morphological characteristics of the hybrids resembled a combination of features of both parents. Malate dehydrogenase (MDH) and esterase (EST) isozyme electrophoresis indicated parents and F-1 hybrids showed co-dominant expression of the paternal and maternal alleles controlling the isozymes and confirmed the hybridization. (C) 2004 Elsevier B.V. All rights reserved.

Margulis' theory on division of labour in cells revisited

Division of labour is a marked feature of multicellular organisms. Margulis proposed that the ancestors of metazoans had only one microtubule organizing center (MTOC), so they could not move and divide simultaneously. Selection for simultaneous movement and cell division had driven the division of labour between cells. However, no evidence or explanation for this assumption was provided. Why could the unicellular ancetors not have multiple MTOCs? The gain and loss of three possible strategies are discussed. It was found that the advantage of one or two MTOC per cell is environment-dependent. Unicellular organisms with only one MTOC per cell are favored only in resource-limited environments without strong predatory pressure. If division of labour occurring in a bicellular organism just makes simultaneous movement and cell division possible, the possibility of its fixation by natural selection is very low because a somatic cell performing the function of an MTOC is obviously wasting resources. Evolutionary biologists should search for other selective forces for division of labour in cells.

长俊木瓜体细胞胚胎诱导的初步研究

以长俊木瓜为材料,研究了其体细胞胚胎诱导过程中各个环节的影响因素。结果表明:愈伤组织诱导的适宜外植体是叶片,培养基为MS+6-BA1.0 mg.L-1+2,4-D0.2 mg.L-1,黑暗培养;非胚性愈伤组织向胚性愈伤组织转化的适宜培养基是MS+6-BA1.0 mg.L-1+NAA1.0 mg.L-1;胚性愈伤组织的保持与增殖应在黑暗条件下进行,培养基为MS+6-BA1.0 mg.L-1+2,4-D0.2 mg.L-1;长俊木瓜叶片体胚的发生以加入ABA2.0 mg.L-1的MS培养基最为有利。

溶胶-凝胶法制备导电聚苯胺杂化材料

针对有机一无机杂化材料制备过程中致命的体积收缩问题，本论文相继选用一系列具有亲水性官能团的聚倍半硅氧化物类前驱体为基体，采用溶胶一凝胶方法，以区域限制方式把水溶性导电聚苯胺固定在三维无机网络内，获得了耐水型自支撑杂化导电膜，该自支撑膜呈现出一定的机械强度与耐磨性。同时，为了进一步增加有机、无机组分之间的作用力，获取分子级别杂化材料，本文尝试了在导电聚苯胺与无机网络之间引入共价键、离子键等较强相互作用，得到了一些有意义的结果，具体如下：（1）亲水性的倍半硅氧烷前驱体、桥联倍半硅氧烷前驱体均能与水溶性导电聚苯胺形成平整的自支撑膜，该导电膜呈现出较好的耐”水性。（2）通过一步掺杂法制备的化学键接型水溶性导电聚苯胺／无机杂化膜除显示出良好的耐水性外，其热稳定性也得到了提高，从而为耐热型导电杂化膜的开发提供了思路。（3）有机一无机组分间通过离子键接引入静电相互作用亦是提高体系相容性，获取耐水型自支撑杂化膜的另一有效的方法。但由于导电膜中含有大量的离子键，与上述两体系相比，机械强度显得稍脆。（4）带有长链状亲水基团的掺杂剂酸性磷酸酷具有很强的自组装能力，不仅能在杂化体系中指导聚苯胺纳米管的构筑，而且能通过聚合单体法直接生成大批量聚苯胺纳米管材料。

猕猴体细胞核移植影响因素的研究：着床前胚胎表观遗传重编程和供体细胞周期同步化

体细胞核移植（somatic cell nuclear transfer）克隆技术的成功，特别 是运用终末分化的淋巴细胞和嗅觉神经元细胞成功克隆出小鼠，证实了分化的体 细胞核潜在的发育全能性。该技术已经在多个物种上成功地得到克隆后代，在转 基因动物、基因敲除动物和疾病模型动物生产中也得到成功应用，在结合干细胞 技术的治疗性克隆和再生医学方面也取得了初步成果，展现出了具有深远意义的 应用前景。但是，目前该领域仍然存在着很多急待解决的重要问题：克隆成功率 低，克隆胚和克隆动物经常呈现发育异常，妊娠和出生前后的高死亡率。对哺乳 动物早期胚胎发育过程中DNA 甲基化、组蛋白修饰等表观遗传重编程 （epigenetic reprogramming）机制的深入了解，有助于研究体细胞核在去核卵 母细胞中的表观遗传重编程事件，进而改善克隆胚重编程效率和发育能力。 猕猴是一种重要的实验动物，在人类疾病模型和生物医药研究中有重要的意 义。本研究主要围绕猕猴体细胞克隆胚胎早期发育过程中的表观遗传重编程事件 和核移植前体细胞同步化处理这两方面展开。1），首次详细地了描绘了猕猴着床 前胚胎发育过程中整体水平的DNA 甲基化表观遗传重编程事件，研究发现在受精 卵中父本基因组形成原核后迅速地发生了去甲基化，在2 细胞期后的卵裂过程 中，母本基因组才开始逐渐地去甲基化，到桑葚胚达到最低水平，然后开始重新 （de novo）甲基化,到囊胚期时形成不对称的甲基化模式，滋养外胚层（TE）呈 现高甲基化状态，而内细胞团（ICM）呈现低甲基化状态,这一不对称模式可能是 灵长类动物特有的，其他哺乳动物呈现正好相反的不对称模式。2），研究发现， 大多数猕猴克隆胚胎的DNA 甲基化重编程存在异常,效率低。很多2 细胞期克隆 胚（67%）和8 细胞期克隆胚（50%）的核DNA 甲基化水平显著高于对应的体 外受精胚，8 细胞克隆胚之间呈现多种不同的表观遗传特征。大多数克隆囊胚的 ICM 细胞核的甲基化水平显著高于IVF 囊胚，这些异常可能是导致克隆胚胎移 植到代孕母体后发育时间不长就失败的原因。3），在核移植前对猕猴成纤维细 胞同步化处理的研究中发现，血清饥饿，细胞周期阻断剂DMSO（二甲基亚砜）、 roscovitine、aphidicolin 和indirubin 的处理都有显著的同步化效果，提高了G0+G1 期细胞的比例。经过BrdU 标记法证实了这几种处理方法抑制细胞增殖的效果，并且证实了这种周期阻滞作用是可逆的。用原位末端标记法（TUNEL）分析证 实，血清饥饿1 到4 天后细胞凋亡比例显著上升，在贴壁的细胞中约有6%发生 凋亡，而正常对照只有1%左右，而周期阻断剂处理没有增加细胞凋亡率，这提 示这些周期阻断剂可能是一种相对安全且有效的猕猴成纤维细胞处理方法。核移 植前对猕猴成纤维细胞进行处理，有助于优化体细胞核移植技术，也是改善体细 胞核在克隆胚中重编程效率的重要途径。

哺乳动物体细胞核移植影响因素的研究

题。 在已有的基础上，建立了基于单个植入前胚胎的逆转录-聚合酶链反应（RT-PCR）技术，成功实现了针对单个猕猴植入前胚胎进行可重复的，多个特定基因的表达检测。在此基础上，对一些重要的早期胚胎发育相关基因在猕猴体细胞核移植胚胎和体外受精胚胎中的表达进行了比较研究。这其中包括核仁相关蛋白基因nucleolin，nucleophosmin，fibrillarin，PAF53，UBF和mRNA早期装配相关蛋白snrpn，这些基因被认为与植入前胚胎的再程序化有着非常密切的关系。我们的研究结果发现，相对于体外受精胚胎，被检测基因在核移植胚胎中的转录出现了不同程度的异常，表现为表达丰度相对较低，这意味着体细胞核移植植入前胚胎的再程序化，或者说供体核的再程序化可能出现了异常。通过进一步对fibrillarin（胚胎合子基因组启动的标志基因）的表达研究，比较了合子基因组启动这一再程序化分子事件在核移植胚胎和体外受精胚胎中的发生时序。研究结果证实，体细胞核移植胚胎的合子基因组启动出现了明显的滞后。我们还发现这与核移植胚胎的异常卵裂速度有着一定的关联，同时说明某些源自核移植操作技术和方法的未知因素可能是造成核移植胚胎再程序化异常的主要原因。为了验证合子基因组滞后对核移植胚胎后续发育的影响，本论文研究分析了着床相关基因Mamu-AG在猕猴核移植胚胎中的表达。研究首次发现Mamu-AG mRNA只在囊胚期开始出现，这与在人类中的情况有所不同（始于8细胞时期）。在正常体外受精囊胚中，不论发育时间如何，第6和第7天的囊胚均正常表达Mamu-AG mRNA。而在核移植囊胚中，仅有第7天的囊胚检测到该mRNA，第6天的囊胚中则没有，这进一步说明了胚胎合子基因组启动的延迟是影响核移植胚胎发育的重要环节。 本研究从分子水平上对猕猴植入前胚胎基因转录和表达进行了分析研究，首次发现了在猕猴体细胞核移植胚胎中发育相关基因表达异常，以及合子基因组启动滞后的分子证据，这为我们改进现有的猕猴核移植操作程序提供了新的思路，同时也为进一步研究猕猴植入前胚胎发育的再程序化奠定了基础。

体细胞核移植影响因素的研究

尽管体细胞核移植(somatic cell nuclear transfer, SCNT)技术仍然处于 起步发展阶段，但是随着体细胞核移植技术在近些年来的飞速发展，人们已经得 到了多种哺乳动物体细胞核移植的存活后代。体细胞核移植技术在生物医药、农 业及其它领域的应用显示了这项技术的巨大发展前景。另一方面，人们发现体细 胞核移植效率低下并且体细胞克隆动物存在许多缺陷，这些主要是由于核移植技 术，供体细胞选择，体外培养系统以及卵母细胞的状态等的差异导致的。本研究 主要围绕着这些因素在体细胞核移植过程中对于克隆胚胎植入前后发育的影响 而开展，目的在于提高体细胞核移植的效率。 实验一，研究了几个因素对克隆胚胎发育和克隆效率的影响，为提高体细胞 核移植效率提供一些依据。这部分研究主要包括四倍体半克隆小鼠研究和蛋白酶 体抑制剂在克隆胚胎和孤雌激活胚胎发育中的影响。四倍体体细胞半克隆 （tetraploid semi-cloned , TSC）胚胎的体外发育结果表明，虽然TSC 胚胎可 能避免二倍体半克隆胚胎发育过程中的非整倍体现象，但是仍然不能形成胎儿。 另外，利用蛋白酶体抑制剂MG132 处理克隆胚胎的结果表明，虽然MG132 通过抑 制成熟促进因子（maturation promoting factors，MPF）活性的降低可以提高 克隆胚胎的体外发育率，但是没有改变克隆胚胎的质量。 实验二，研究了克隆技术在克隆胚胎构建和发育上的影响。首先，我们研究 了完整颗粒细胞注射到卵母细胞中所引起的变化。我们发现完整的颗粒细胞注入 到卵母细胞中之后很快引起细胞膜裂解和细胞核碎裂，从而导致激活后重构胚胎 的碎裂。这一实验表明完整供体细胞核注入的方法不适用于小鼠体细胞克隆。接 下来我们研究了不同的核移植技术――利用piezo 的直接注射法（piezoelectric microinjection，PEM）和电融合法（Electrofusion，EF）――对植入前后的克隆胚 胎以及出生后的克隆动物的影响。研究的结果发现，在PEM 法中，提高piezo 脉 冲的强度会导致供体细胞核DNA 断裂增加，使植入前克隆胚胎的细胞数目减少， 凋亡增加，从而降低克隆胚胎的质量。相反，在用EF 法产生的胚胎中细胞核DNA 的断裂很少，克隆胚胎质量相对较好。实验的结果表明，由于两种克隆技术利用 的原理不同，会对克隆胚胎的发育产生一定的影响，从而改变克隆的效率。 实验三，研究了猕猴体细胞克隆胚胎早期发育过程中细胞核内有丝分裂器蛋 白[Nuclear Mitotic Apparatus Protein，NuMA]的表达和分布。尽管在有些猕猴体 细胞核移植胚胎中没有检测到NuMA 的正确表达和分布并且有些胚胎出现了微 管组装异常，但是大多数猕猴体细胞核移植胚胎具有正常的NuMA 蛋白表达和 正常的核型。通过改进操作技术我们得到了可以发育到正常囊胚的猕猴体细胞核 移植胚胎。这些结果提示猕猴SCNT 胚胎发育失败的原因不是NuMA 等重要蛋 白的缺失。

猕猴胚胎干细胞自我更新机理及WNT 信号通路在猕猴胚胎早期发育中作用的研究

尽管大部分动物实验是在啮齿类动物上开展的，我们仍然相信涉及人类的许多问题，如胚胎干细胞的体内功能、调亡和肿瘤形成等只有在非人灵长类模型上才能得到最好的回答。猕猴(标准的非人灵长类动物模型)在解剖、生理和代谢方面都和人类非常相似。人类很多神经疾病，如阿尔茨海默氏病、帕金森病，只能在非人灵长类模型上才能精确建模。所以研究猕猴胚胎干细胞自我更新的原理及猕猴胚胎早期发育，对研究免疫排斥，检测基于胚胎干细胞的治疗的可行性，安全性和有效性具有重要意义。本文一方面对胚胎干细胞维持自我更新和多潜能性的机理研究进行了综述，另一方面对以下两个方面的内容进行了研究： 1）运用寡核苷酸芯片和定量PCR 验证的方法来分析五株猕猴饲养层细胞的表达模式，期望发现在支持性和非支持性的饲养层细胞中差异性表达的基因。我们着重定位于饲养层胞外空间和细胞膜上的细胞因子，因为这些因子可以通过直接接触或通过膜结合受体激活下游信号通路，并最终促进猕猴胚胎干细胞的自我更新。我们发现在支持性的饲养层中有八个基因是高表达的，他们是GREM2， bFGF，KITLG，DKK3，GREM1，AREG，SERPINF1 和LTBP1； 经定量PCR 验证的SCF，bFGF 和GREM2 的表达情况都和芯片数据吻合。 2）为了描述在IVF (in vitro fertilized, 体外受精)，ICSI (intracytoplasmic sperm injection, 单精注射)，SCNT (somatic cell nuclear transfer, 体细胞核移植)和孤雌生殖猕猴囊胚中WNT 信号通路的表达情况，我们运用了信号通路特异性PCR Array 系统及免疫细胞化学来检测mRNA 和蛋白表达水平。其中，ICSI 作为IVF 胚胎的参照组，以排除显微操作对胚胎质量的影响。结果，我们发现非经典WNT/JNK 信号，而不是经典WNT 信号通路，在IVF 正常胚胎发育中起作用。而体细胞核移植和孤雌生殖的胚胎的WNT 信号通路基因表达明显高于正常胚胎。WNT 信号通路基因的表达模式可以作为胚胎质量的一个指示标准，有助于回答为什么猕猴 SCNT 和孤雌生殖胚胎发育异常。

OPTICAL-PHONON MODES AND FROHLICH POTENTIAL IN ONE-DIMENSIONAL QUANTUM-WELL WIRES

By extending our microscopic model on optical-phonon modes in quantum wells to one-dimensional (1D) quantum-well wires (QWW), the optical displacements and associated electrostatic potentials of optical-phonon modes in 1D QWW are calculated. The modes can be clearly divided into confined LO bulklike, TO bulklike modes, and extended interfacelike modes provided the bulk phonon dispersion is ignored. The character of each type of mode is illustrated with special attention to the interfacelike modes, which are hybrids of longitudinal- and transverse-optical waves from the corresponding bulk materials. Based on the numerical results, approximate analytical formulas for bulklike modes are presented. As in 2D wells, both the optical displacements and Frohlich potentials for the bulklike modes vanish at the interfaces. The finite dispersion of bulk phonons has a more pronounced effect on the 1D phonon modes because interfacelike modes show mixed characteristics of 2D interface and bulklike modes.

小麦—偏凸山羊草6Mv/6B代换系的研究

小麦条锈病（Puccinia striiformis f. sp. tritici）是世界性小麦病害，可导致受害小麦减产30%以上，甚至绝收。小麦条锈病在我国西南、华北麦区危害严重，四川麦区是小麦条锈病发病最重的地区之一，每年因条锈病流行造成小麦产量损失巨大。利用抗条锈病品种是控制该病害最安全、经济的有效途径，因此挖掘利用抗病新基因，开展抗病遗传基础研究是当前育种工作中面临的重要任务。 偏凸山羊草（Aegilops ventricosa，DDMvMv，2n=28）是一年生草本植物，起源于地中海西部沿岸地区，具有对小麦白粉病、锈病等高抗或免疫、耐盐、抗寒、蛋白质含量高等优良性状，是小麦遗传育种很好的种质资源。本研究以高抗条锈病的小麦—偏凸山羊草6Mv/6B代换系（Moisson 6Mv/6B）为材料，对其含有的带条锈病抗性基因的偏凸山羊草6Mv染色体在四川小麦背景中的传递情况、与小麦—簇毛麦双端体附加系所具有的白粉病抗性的聚合以及对Moisson 6Mv/6B进行电离辐射诱变筛选抗条锈病的小麦—偏凸山羊草易位系三个方面进行了研究。取得的主要研究结果如下： 1. Moisson 6Mv/6B与高感条锈病的四川地区普通小麦品种绵阳26、绵阳93-124和SW3243的杂种F1与其普通小麦亲本分别作为父、母本回交，通过对其BC1和F2的结实率、根尖细胞有丝分裂中期染色体的观察以及对条锈病抗性的鉴定，发现含6Mv染色体的F1植株作母本时的回交结实率（83.10%）普遍高于含6Mv染色体的F1植株作父本（48.61%），结实率与普通小麦基因型密切相关（χ2=34.15>>χ20.05=5.99（df=2））；6Mv染色体在三种四川小麦中通过雌、雄配子传递的传递方式与其传递率间没有显著相关性，其传递率与普通小麦基因型呈显著相关性（χ2=6.42>χ20.05=5.99（df=2））。 2. Moisson 6Mv/6B与高抗白粉病的小麦—簇毛麦双端体附加系Pana（2n=42+2t）正反杂交，希望在聚合两者抗性的同时观察不同受体背景下的抗性反应。对Moisson 6Mv/6B和Pana正反杂交的结实率、杂交后代的农艺性状进行观察，并对杂交后代进行基因组荧光原位杂交（GISH）分析及条锈病和白粉病的抗性鉴定。结果表明Moisson 6Mv/6B作母本时杂交结实率（80.56%）高于Pana作母本时（58.33%），结实率与杂交方式间紧密相关（χ2=4.96>χ20.05=3.84（df=1））；Moisson 6Mv/6B和Pana杂交后代株高比最高亲本高约10cm，成熟期也较两亲本提前两个星期左右；正反杂交后代中具有偏凸山羊草6Mv染色体的植株具有条锈病抗性，具有簇毛麦端体的植株具有白粉病抗性，同时筛选到4株含有偏凸山羊草和簇毛麦遗传物质并对条锈病和白粉病兼抗的材料，证明来自偏凸山羊草6Mv染色体的条锈病抗性与来自簇毛麦端体的白粉病抗性已经聚合在一起，且没有产生相互抑制的作用，暗示通过这两个抗性基因的聚合是完全能获得兼抗条锈病和白粉病的小麦新种质。 3. 对Moisson 6Mv/6B在减数分裂时期的成株进行总剂量为6Gy、辐射频率为120rad/min的60Co-γ射线辐射，对辐射植株自交后代进行农艺性状及根尖细胞有丝分裂中期染色体形态观察和条锈病抗性鉴定。结果为辐射植株自交结实率为2.22%，根尖细胞有丝分裂中期的染色体存在明显碎片，辐射自交后代植株对条锈病具有成株期抗性。 小麦—偏凸山羊草6Mv/6B代换系对条锈病抗性稳定，是培育条锈病抗性品种的良好供体。本研究证明在四川小麦背景中要利用该品种抗性，在结实数满足需要时，可将其作父本，亦可作母本，但关键是要选择好一个优良的受体基因型；同时其条锈病抗性与来自簇毛麦的白粉病抗性没有相互抑制作用，可将两者抗性有效聚合用于小麦育种中。 Wheat stripe rust (Puccinia striiformis f. sp. Tritici) is a worldwide disease of wheat, and could lead to victims of 30 percent or even total destruction of wheat production. Wheat stripe rust harms badly in China's southwest and North China. Sichuan province is one of the regions damaged by wheat stripe rust heavily. The use of resistant varieties is the most secure and economical way to control the wheat stripe rust. Therefore, it is essential to identify new disease-resistant genes and genetically research of disease resistance. Aegilops ventricosa (DDMvMv, 2n = 28) is an annual herbaceous plant, originating in the coastal areas of the western Mediterranean, with good characters such as resistance of wheat powdery, rust, salt, cold and high protein content. It is a good germplasm resource. In this study, the wheat- Aegilops ventricosa 6Mv/6B substitution line Moisson 6Mv/6B (highly resistant to the wheat stripe rust) was used to study on the transmission of chromosome 6Mv of Aegilops ventricosa in different genetic background of Sichuan wheat varieties, hybridization with wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew) and screening of wheat- Aegilops ventricosa translocation line by exposuring Moisson 6Mv/6B under ionizing radiation. The main results are as following: 1. Moisson 6Mv/6B was crossed with Sichuan wheat varieties mianyang26, mianyang93-124 and SW3243 (highly susceptible to stripe rust), respectively. Their F1 hybrids were further backcrossed as male and female to corresponding wheat varieties. The seed-setting rate, chromosomes confirmation in the mitotic metaphase of root tip cells, and resistance to stripe rust of the subsequent BC1 and F2 plants were investigated. The average seed-setting rate of backcross via 6Mv as female donor (83.10%) was higher than that of backcross via 6Mv as male donor (48.61%), suggesting that the seed-setting rate was associated with the wheat genotypes（χ2=34.15>>χ20.05=5.99（df=2））. In all analyzed populations, transmission frequencies of chromosome 6Mv were not correlated with the ways of 6Mv through male or through female. However, transmission frequencies of chromosome 6Mv were significantly correlated with Sichuan wheat genotypes（χ2=6.42>χ20.05=5.99（df=2））. 2. To aggregating the resistances to stripe rust and powdery mildew, as well as research on the resistance reactions in different genetic background, Moisson 6Mv/6B was reciprocally hybrided with the wheat- Haynaldia villosa ditelosomic addition line Pana (highly resistant to the powdery mildew). The seed-setting rate, agronomic characters, genomic in situ hybridization (GISH) of hybrid progenies，and resistances to stripe rust and powdery mildew were investigated. The results showed that the seed-setting rate of hybridization via Moisson 6Mv/6B as female donor (80.56%) was significant higher than that via Pana as female donor (58.33%). The seed-setting rate was associated with the hybrid methods (χ2 = 4.96> χ20.05 = 3.84 (df = 1)). The plant height of hybrid progenies was about 10 cm higher than Pana, the parent with maximum height. And the maturity of hybrid progenies was about two weeks earlier than that of the parents. In the hybrid progenies, the plants with the 6Mv chromosome have the resistance to stripe rust and the plants with the telosome from Haynaldia villosa have the resistance to powdery mildew. It was found that four plants with both the 6Mv chromosome and the telosome from Haynaldia villosa were resistant to stripe rust and powdery mildew. It indicated that the resistance to stripe rust and powdery mildew aggregated, and no mutual inhibition was found. It implied that the aggregation of the two resistance genes was able to provide the new wheat germplasm with the resistances to stripe rust and powdery mildew. 3. Moisson 6Mv/6B was irradiated with 60Co-γ rays of 6Gy (120rad/min) during meiosis. The agronomic characters and chromosomes confirmation in the mitotic metaphase of root tip cells，as well as resistance to stripe rust were investigated. The seed-setting rate of irradiated plants was only 2.22%. The chromosomes in mitotic metaphase had clear fragments. The resistance to stripe rust of progeny of irradiated plants was the adult-plant resistance. The wheat- Aegilops ventricosa 6Mv/6B substitution line is a good stripe rust resistance donor for its stabile resistance. Our study demonstrated that the key for use the resistance is to choose a good receptor. There is no difference between Moisson 6Mv/6B be the female and be the male if the seed number meets the requirement. At the same time, the stripe rust resistance of Moisson 6Mv/6B did not have the mutual inhibition with the powdery mildew resistance from Haynaldia villosa. It is able to aggregate the two resistances for wheat breeding.

同源四倍体水稻三系亲本的遗传差异、细胞遗传学比较研究及低直链淀粉含量突变体Wx基因序列分析

同源四倍体水稻（2N=4X=48,AAAA）是由二倍体水稻（2N=2X=24,AA）通过秋水仙素诱导染色体加倍后得到的新品系，具有优良的抗病性以及较高的蛋白质含量。因此，在四倍体水平上挖掘水稻的增产潜力成为水稻育种的新手段。同源四倍体水稻具有很强的遗传可塑性和很弱的遗传保守性，利用其作为水稻远缘杂交的桥梁，从野生物种中不断地引进有益的基因，这将有助于杂交水稻的多代利用和固定水稻的杂种优势。但是迄今为止，还没有关于同源四倍体水稻遗传多样性，遗传背景的报道。目前世界关于同源四倍体水稻的研究主要集中在中国，主要研究方向为培育、筛选结实正常的亲本材料，配置和筛选结实率正常或接近正常的组合。经过几十年研究，虽然在材料构建，细胞学研究等方面取得了较大进展，但同样由于结实率低的瓶颈问题未解决，而使多倍体水稻育种未能取得实质性进展。而近年来一些关于同源四倍体水稻低结实率机理的细胞学研究也由于缺乏统计学数据而缺乏说明性。本文用SSR标记，对选取的36个结实率正常同源四倍体水稻三系亲本和14个来源二倍体亲本，分析他们的遗传差异和群体遗传结构。本文还利用我们培育的高、低结实率的同源四倍体水稻恢复系、优良保持系和杂种F1及二倍体对照为材料，进行系统深入的细胞遗传学研究，进一步探讨同源四倍体水稻有性传递后代的发育过程，探索分裂期染色体行为特征与遗传性状稳定性的关系，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%，为研究其直链淀粉含量下降的原因，本文还根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析，并根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 SSR标记具有基因组分布广泛、数量丰富、多态性高、容易检测、共显性、结果稳定可靠、实验重现性好、操作简单、经济、易于高通量分析等许多优点，被认为是用于遗传多样性、品种鉴定、物种的系统发育、亲缘关系及起源等研究的非常有效的分子标记。本研究选取了中国科学院成都生物所培育的同源四倍体和二倍体水稻亲本，并用36个微卫星标记进行了遗传差异和种群遗传结构分析。在50个品系中，我们观察到较高水平的多态性，每基因等位基因数（Ae）分布于2至6之间（平均值3.028），多态性信息含量（PIC）分布于0.04至0.76之间（平均值0.366）；期望杂合度（He）分布于0.04至0.76之间（平均值0.370），Shannon指数（I）分布于0.098至1.613之间（平均值0.649）。同源四倍体品系的等位基因数，期望杂合性和Shannon指数都比二倍体品系高。在供试50个品系中，较多材料均发现Rare基因，根据SSR多态性指数我们构建了同源四倍体和二倍体水稻的核心指纹库。F-统计值表明遗传差异主要存在于同源四倍体品系中（Fst=0.066）。聚类分析结果表明50个品系可以分为4个组。I组包括所有的同源四倍体和二倍体籼稻保持系，以及一个同源四倍体籼稻雄性不育系及其来源二倍体。II组仅包括IR来源的品系。III组比II组和IV组更复杂，包括同源四倍体和二倍体籼稻恢复系品系。IV组包括同源四倍体和二倍体粳稻品系。此外,由于等位基因及配子的遗传差异,同源四倍体与二倍体品系中存在单位点和双位点的遗传差异。分析结果表明,二倍体和四倍体水稻基因库的不同,其中遗传变异可以区分四倍体与二倍体水稻。同源四倍体水稻具有长期而独立的遗传性，我们能够选育并得到与二倍体亲本相比有特殊优良农艺性状的品系。 本研究以高结实率的同源四倍体水稻恢复系DTP-4、D明恢63及优良保持系D46B为材料进行农艺性状及细胞遗传学比较研究。DTP-4、D明恢63及保持系D46B的的染色体组成均为2N=4X=48，花粉母细胞具有较为理想的减数分裂行为，配对染色体的比率在99%以上，这与理论染色体组构成相符。DTP-4和D明恢63PMC减数分裂各个时期单价体和三价体的比例都非常低，而在MI， PMC观察到较多的二价体和四价体且四价体多以环状形式出现，其最大频率的染色体构型分别为12II 6IV和10II 7IV。恢复系DTP-4和D明恢63在MI四价体频率分别为2.00/PMC和2.26/PMC，而保持系D46B在MI四价体频率为6.00/PMC，极显著地高于恢复系品系，表明保持系D46B具有更好的染色体配对性质；AI保持系D46B的染色体滞后频率为10.62%，远低于恢复系材料DTP－4和D明恢63的19.44％和23.14％，接近二倍体对照明恢63的7.30%水平；TI保持系D46B具有比恢复系更低频率的微核数。而在TII，D46B的正常四分小孢子比率不但高于恢复系品系甚至高于二倍体对照。对高低结实率的同源四倍体水稻恢复系和杂种F1代的花粉育性，结实率和细胞遗传学行为进行了比较研究。DTP-4, D明恢63, D46A´DTP-4和D46A´D明恢63的花粉育性和结实率比D什香和D46A´D什香显著提高。减数分裂分析的结果表明，DTP-4，D明恢63，D什香，D46A´DTP-4，D46A´D明恢63和D46A´D什香其减数分裂染色体构型分别为：0.05I +19.96 II (9.89棒状+10.07环状) +0.01III + 2.20 IV, 0.11I +19.17 II (8.90 棒状+10.37 环状) +0.09III + 2.26 IV + 0.01 VI, 1.33I +9.46 II (4.50 棒状+4.96 环状) +0.44III + 6.02 IV + 0.09VI + 0.09 VIII, 0.02I +14.36 II (6.44 棒状+7.91 环状) +0.01III + 4.80IV + 0.01VIII, 0.06 I +17.67 II (11.01 棒状+6.67 环状) +0.06 III + 3.10 IV + 0.01 VI and 1.11 I +11.31 II (5.80 棒状+5.51 环状) +0.41 III + 5.63 IV+0.03VI+0.03VIII。在同源四倍体水稻恢复系和杂种F1代材料中，最常见的染色体构型为16II +4IV和12II +6IV。在减数分裂过程中，结实率较高的材料染色体异常现象较少而结实率较低的材料染色体异常现象较严重。在杂种F1代中，二价体的比例要低于其相应的恢复系亲本，同样的，单价体，三价体和多价体的比例相比其恢复系亲本也偏低。然而，在减数分裂MI，杂种F1代中四价体的比例要显著高于其恢复系亲本。在中期I，每细胞单价体的比例和花粉育性呈现出极高的负相关(-0.996)，当单价体数目升高时，花粉育性下降。其次是每细胞三价体的比例(-0.987)，之后则是每细胞多价体的比例与花粉育性的负相关(-0.948)。但是统计分析表明，二价体和四价体的比例对花粉育性和结实率没有显著影响。这一结果表明出了花粉育性和细胞减数分裂行为的相关性，同源四倍体的减数分裂行为为筛选高结实率的同源四倍体种系提供了理论依据。 突变体是遗传学研究的基本材料。利用突变体克隆水稻基因,并进而研究基因的生物学功能是水稻功能基因组学的重要研究内容。本课题组在多年的四倍体水稻育种研究中已获得多个低直链淀粉含量突变体，其中一些突变体在直链淀粉含量下降的同时，胚乳外观也发生了显著改变，呈半透明或不透明。同源四倍体水稻突变株D4063-1直链淀粉含量比来源二倍体明恢63下降一半,即其直链淀粉含量为5.23%。为研究其直链淀粉含量下降的原因，我们根据普通水稻Wx基因设计引物，扩增测序获得了D4063-1Wx基因的全序列，与已报道Wx基因进行比对分析；同源四倍体水稻D4063-1Wx基因最显著变化为在外显子序列中发生了碱基缺失，导致移码突变，在第9外显子终止密码子提前出现。D4063-1Wx基因碱基位点的变化还导致了其序列上的酶切位点的变化,对常用限制性内切酶位点分析分析结果表明同源四倍体水稻相对于籼稻和粳稻多了2个sph1酶切位点，相对于粳稻减少了6个Acc1，增加了4个Xba1，1个Xho1，1个Pst1和1个Sal1酶切位点。聚类分析表明D4063-1Wx基因序列与籼稻亲源关系较近，由此推测D4063-1Wx基因来源于籼稻的Wxa基因型。另外，根据D4063-1Wx基因的碱基差异，我们推测D4063-1Wx基因外显子碱基变化导致的RNA加工障碍是其直链淀粉降低的主要原因，并可能与其米饭较软等品质相关。本文还根据D4063-1和籼稻、粳稻的序列差异并根据D4063-1在该片段上的特征序列位点设计了用于识别D4063-1的寡核苷酸片段,并作为PCR反应的引物命名为AUT4063-1,将该引物与我们设计的扩增普通籼稻、粳稻的Wx基因引物F5配合使用建立了识别D4063-1的显性和共显性两种检测方式的分子标记,为快速、准确的鉴别低直链淀粉的D4063-1创造了条件。 研究同源四倍体水稻基因组的遗传差异，探索同源四倍体水稻的遗传规律，研究分裂期染色体行为特征与遗传性状稳定性的关系,旨在揭示四倍体水稻中同源染色体配对能力的遗传差异，为进一步选育多倍体水稻品种并将其应用于生产提供理论依据。 Autotetraploid rice (2N=4X=48, AAAA) is a new germplasm developed from diploid rice (2N=2X=24, AA) through chromosomes doubling with colchicines and is an excellent resource for desirable resistance genes to the pathogens and high protein content. Therefore, heterosis utilization on polyploidy is becoming a new strategy in rice breeding. At present, the main research on autotetraploid rice centralizes in China. Breeding effort has been made to improve autotetraploid rice genetically, however, the progresses are limited due to higher degree of divergence between hybrid sterility and polygenic nature. But to date, almost nothing is reported about the genetic diversity, original and genetic background of autotetraploid rice. Despite several reports on cytological analysis of the mechanisms of low seed set in autotetraploid rice still the results are inconclusive due to lack the statistical evaluation. Therefore, the study on the mechanisms of low seed set in autotetraploid is a priority for rice breeding. Microsatellites or simple sequence repeats (SSRs) are the widely used marker for estimating genetic diversity in many species, including wild, weedy, and cultivated rice. In our research, genetic diversity and population genetic structure of autotetraploid and diploid populations collected from Chengdu Institute of Biology, Chinese Academy of Sciences were studied based on 36 microsatellite loci. For the total of 50 varieties, a moderate to high level of genetic diversity was observed at population levels with the number of alleles per locus (Ae) ranging from 2 to 6 (mean 3.028) and PIC ranging from 0.04 to 0.76 (mean 0.366). The expected heterozygosity (He) varied from 0.04 to 0.76 with the mean of 0.370 and Shannon’s index (I) ranging from 0.098 to 1.613 (mean 0.649). The autotetraploid populations showed a slightly higher level of effective alleles, the expected heterozygosity and Shannon’s index than that of diploid populations. Rare alleles were observed at most of the SSR loci in one or more of the 50 accessions and core fingerprint database of the autotetraploid and diploid rice was constructed. The F-statistics showed that genetic variability mainly existed among autotetraploid populations rather than among diploid populations (Fst=0.066). Cluster analysis of the 50 accessions showed four major groups. Group I contained all of the autotetraploid and diploid indica maintainer lines and a autotetraploid and its original diploid indica male sterile lines. Groups II contained only original of IR accessions. Group III was more diverse than either group II or IV and comprised of both autotetraploid and diploid indica restoring lines. Group IV included japonica cluster of the autotetraploid and diploid rices. Furthermore, genetic differences at the single-locus and two-locus levels, as well as components due to allelic and gametic differentiation, were revealed between autotetraploid and diploid varieties. This analysis indicated that the gene pools of diploid and autotetraploid rice are somewhat dissimilar, which made a variation that distinguishes autotetraploid from diploid rices. Using this variation, we can breed new autotetraploid varieties with some new important agricultural characters but the diploid rice has not. Cytogenetic characteristics in restorer lines DTP-4, DMinghui63 and maintainer line D46B of autotetraploid rices were studied. DTP-4, DMinghui63 and D46B showed the advantage of high seed set and biological yield. The meiotic chromosome behavior was slightly irregular in DTP-4, DMinghui63 and D46B. We observed less univalent, trivalent and multivalent at MI, but more bivalent and quadrivalent were observed. The most frequent chromosome configurations were 12II 6IVand 10II 7IV in restorer and maintainer lines, respectively. The quadrivalent frequency of DTP-4 and Dminghui63 at metaphase(MI) was respectively 2.00/PMC and 2.26/PMC. However that frequency of D46B was 6.00/PMC, which was greatly significantly higher than DTP-4 and Dminghui63. That indicates the maintainer D46B has better chromosome pairing capability in metaphase (MI). The frequency of lagging chromosomes of the maintainer D46B at anaphaseI (AI) was 10.62%, which was significantly lower than that of DTP-4(19.44%) and Dminghui63(23.14%) and nearly reaching the level of diploid CK(7.30%). In telophaseI (TI) maintainer D46B showed lower frequency of microkernel at TI and lower frequency of abnormal spores at telophaseII(TII). We also studied pollen fertility, seed set and cytogenetic characteristics of restorer lines and F1 hybrids of autotetraploid rice. DTP-4, DMinghui63, D46A´DTP-4 and D46A´DMinghui63 showed significantly higher pollen fertility and seed set than DShixiang and D46A´DShixiang. Pairing configurations in PMC of DTP-4, DMinghui63, DShixiang, D46A´DTP-4, D46A´DMinghui63 and D46A´DShixiang were 0.05 I+19.96 II (9.89 rod+10.07 ring)+0.01 III+2.20 IV, 0.11 I+19.17 II (8.90 rod+10.37 ring)+0.09 III+2.26 IV+0.01 VI, 1.33 I+9.46 II (4.50 rod+4.96 ring)+0.44 III+6.02 IV+0.09 VI+0.09 VIII, 0.02 I+14.36 II (6.44 rod+7.91 ring)+0.01 III+4.80 IV+0.01V III, 0.06 I+17.67 II (11.01 rod+6.67 ring)+0.06 III+3.10 IV+0.01 VI and 1.11 I+11.31 II (5.80 rod+5.51 ring)+0.41 III+5.63 IV+0.03 VI+0.03 VIII, respectively. Configuration 16 II+4 IV and 12 II+6 IV occurred in the highest frequency among the autotetraploid restorers and hybrids. Meiotic chromosome behaviors were less abnormal in the tetraploids with high seed set than those with low seed set. The hybrids had fewer frequencies of bivalents, univalents, trivalents and multivalents than the restorers, but higher frequency of quatrivalents than the restorers at MI. The frequency of univalents at M1 had the most impact on pollen fertility and seed set, i.e., pollen fertility decreased with the increase of univalents. The secondary impact factors were trivalents and multivalents, and bivalents and quatrivalents had no effect on pollen fertility and seed set. The correlative relationship between pollen fertility and cytogenetic behaviors could be utilized to improve seed set in autotetraploidy breeding. The amylose content of autotetraploid indica mutant Rice D4063-1 dropped by half than diploid Minghui 63, that is, its amylose content of 5.23%.The whole sequence of Waxy gene of D4063-1 is amplified and sequenced. And the discrepancy of bases is found comparing to the reported Waxy gene. The Waxy gene of autotetraploid Rice D4063-1 had a base deletion in exon sequence, which resulted frameshift mutation in exon 9 and termination codon occur early. The mutation of Wx also led to the change of some common restriction endonuclease sites. Results showed compared to indica and japonica, D4063-1 had two adding sph1 sites. Compared to japonica, D4063-1 had six decreasing Acc1, a adding Xho1, Pst1 and Sal1 restriction sites. Phylogeny analysis shows that the DNA sequence of Waxy gene of D4063-1 is closer to Indica, and we suppose that the Waxy gene of D4063-1 is origin from genotype Wxa. In addition, according to the base differences of Wx in D4063-1, we deduce that RNA processing obstacle led by base change of intron is the main cause to low the amylose content, and related to phenotype of its soft rice. Based on analysis of fragments of D4063-1, indica and japonica and according to the special point of the three species, primers as markers-AUT4063-I were designed for distinguishing the D4063-1 from other rice. Combining with primer pair F5, dominant and codominant ways were established for discriminating them., rapid and correct identification of D4063-1 from other rice could be done. The genetic analysis is important to ensure the original of autotetraploid rice, for maintaining the “distinctiveness” of autotetraploid varieties, and to differentiate between the various genetic background of autotetraploid rice. The autotetraploid breeding will benefit from detailed analysis of genetic diversity in the germplasm collections. Further investigation on mechanisms of meiotic stability should benefit polyploid breeding. These findings demonstrated opportunity to improve meiotic abnormalities as well as grain fertilities in autotetraploid rice.

水稻同源四倍体三系法杂种优势利用研究

水稻是重要的粮食作物，其产量的增加和品质的改良都是关系国计民生的大事。就我国现阶段的国情而言，水稻产量在现有水平上稳步提升仍是未来十几年甚至几十年农业生产最重要的目标之一。尽管根据“超级杂交水稻育种”的战略设想和水稻育种实践，通过不断地改进育种技术可望在更高的产量水平上进行水稻杂种优势利用，在稻属植物内还具有很大的产量潜力可以挖掘。然而，仅仅从现有的种质基础出发，要更大幅度提高水稻单产，实现“超级杂交稻”的目标也存在一些困难：现有的推广品种是二倍体，尽管种类众多，但是其基因组的来源相对单一；同时，水稻基因组DNA含量也是作物中最少的，基因组内寻求开发潜力有一定困难；水稻作为C3植物，光合利用效率不高也是制约水稻产量提高的因素之一。因此，寻求常规手段以外的技术突破或者方法创新，是实现“超级杂交稻”的目标的迫切需求。本研究利用秋水仙素能抑制细胞分裂中纺锤丝的收缩、使细胞染色体加倍的作用，对水稻幼穗诱导的愈伤组织细胞进行加倍，并分化出再生植株；创制出水稻同源四倍体新的种质材料，在此基础上选育水稻同源四倍体雄性不育三系材料，并实现水稻同源四倍体的三系配套，开展水稻同源四倍体杂种优势利用和四倍体杂交水稻选育研究，建立水稻同源四倍体杂种优势利用的新技术体系。这不仅有助于倍性水平杂种优势的开拓和利用，同时也将为我国新世纪“超级稻”育种研究开辟一条新的技术途径。 水稻幼穗诱导愈伤组织并分化成苗是一项成熟、简单的组织培养技术。本研究以普通二倍体水稻亲本为材料，用秋水仙素进行水稻的多倍体化诱导，创制同源四倍体水稻三系亲本材料并对其进行鉴定。多倍体化以秋水仙素诱导的愈伤组织培养为基础，研究不同秋水仙素浓度梯度和愈伤组织诱导培养基组合对诱导四倍体植株的影响。结果表明在MS+2,4 D 1.0mg/L+ KT0.2mg/L+ IAA0.2mg/L 和500mg/L的秋水仙素处理下，水稻愈伤组织染色体加倍（有最高的效率）效果较好，平均加倍频率可达25.26%，其中，材料CDR22和IR26诱导较易成功，加倍频率分别达到75%和26.5%；相对材料94109 1.3%加倍频率和冈46B 10.8%加倍频率，诱导率差异极显著。 对水稻四倍体材料进行了形态学鉴定结果表明，与二倍体水稻对照相比其株高、穗长、花粉育性等主要农艺性状，确定四倍体材料在穗长和千粒重两方面极显著提高，种子的长度和宽度也显著增长。对花粉育性鉴定，确认水稻四倍体不育系材料仍为不育，保持系材料自交和杂交可育，恢复系材料自交和杂交可育。对四倍体材料进行细胞形态、染色体数目等方面进行细胞学鉴定，经核型分析表明水稻四倍体材料具有48条染色体，是二倍体水稻的两倍。水稻四倍体材料根尖分生组织细胞与二倍体的根尖分生组织细胞相比，细胞体积、细胞核和核仁显著增大。四倍体三系材料在细胞有丝分裂中期均可规则排列在赤道板，并能均等地移向两极；后期观察中没有发现染色体分离滞后现象，分裂末期细胞能够形成大小相对均一的子细胞。水稻同源四倍体三系材料细胞分裂未见异常，植株生长发育正常。 从1996年至2006年，针对结实率、有效分蘖、着粒数和穗长等主要农艺性状，通过系谱选育的方法，对培育的同源四倍体水稻亲本材料进行了连续选择和改良，取得较好成效。表现为结实率的改良效果极佳，所有改良材料的平均结实率均呈上升趋势，如D237（29.70％→72.70％）、DTB（19.55％→53.21％）等。有效分蘖总体呈现上升趋势，但在不同的年份，如1998和2002存在较大的负向波动。部分材料改良效果明显，如D19B（5.87→13.50）、D什香 (7.00→12.00)等；同时一些材料如DTB和D明恢63虽然总体略有提高，但在不同的年份波动很大，因此存在较大改良阻力，原因还有待进一步研究。着粒数的改良上升趋势比较显著，除保持系的DTB之外，其余材料的平均着粒数有显著提高。穗长的改良阻力较大，虽然不同材料总体上有所提高，但效果并不显著，并且不同年份有较大负向波动（2001）。此外还对株高、剑叶长等性状也进行了选择，但效果不显著，原因有待进一步提高。同源四倍体材料产量相关性状遗传改良幅度不一致，保持系和恢复系间的遗传改良效果也存在差异。这为同源四倍体水稻的进一步利用打下了良好的基础。 籼稻和粳稻亚种间杂交及杂种优势利用的主要障碍就是其低的结实率。而同源四倍体杂交水稻的研究为提高杂交水稻的杂种优势利用创造了新的途径。本研究通过随机区组设计方案，挑选性状优良的二倍体水稻材料，包括雄性不育系，保持系和恢复系进行秋水仙素诱导加倍，从而获得同源四倍体水稻对应的三系材料。利用选育的优良水稻同源四倍体三系材料，配制7个杂交组合，杂交F1代与其恢复系亲本进行比较，用于计算超亲优势（HB）；而杂交F1代与生产上大面积推广的二倍体杂交品种汕优63进行比较，用于计算杂种优势。结果显示，同源四倍体杂交水稻的超亲优势表现为：每株有效穗变化幅度为1.4%至105.9%，总粒数为0.5%至74.3%，每穗实粒数为17.6%至255.7%，结实率为9.6%至130.4%。这些农艺性状的改良使得这7个杂种F1的理论产量的超亲优势高达64.8%至672.7%。小区试验中四倍体杂交水稻组合T461A/T4002和T461A/T4193分别比二倍体对照汕优63提高46.3%和38.3%以上，除一个品种以外所有品种产量均接近或高于汕优63的产量。同源四倍体水稻强大的杂种优势表明，亚种间杂交育性低的问题可通过四倍体化及强化选择来解决。此外，同源四倍体杂交水稻器官的巨大性也是其产量提高的有利因素，水稻同源四倍体三系杂种优势利用研究具有一定的理论价值和商业生产潜力。 Rice is one of the major food crops, the improvement of the production and quality of it is an important thing related to the people's livelihood. On China's current national conditions, steadily increase of the rice yield based on the current level is still one of the most important goals in the next decade or even decades of agricultural production. According to the "super hybrid rice breeding" the strategic and rice breeding practice, improvement of the use of hybrid rice heterosis through continuous improvements in breeding technology is expected to get a higher level of rice yield, there are also a great yield potential can be exploited. However, there are also some difficulties to increase rice yield obviously and implement the goal of "super hybrid rice" based on the existing germplasm: Rice varieties in promotion are diploid, although there are many varieties, but their genome are from a comparatively single source; Meanwhile, the rice genome DNA are the least among the crops, it is difficult to exploit the development potential within the genome; Rice as C3 plants, photosynthetic efficiency is not high, it is one of the factors constraint rice yield. Therefore, seeking technological breakthroughs or innovative methods different from conventional means is the urgent needs to reach the target of "super hybrid rice". Using colchicine inhibit spindle contraction during cell division, double the cell chromosome, we induced callus cells from rice panicle to be doubled, and differentiated regeneration; we created a new autotetraploid rice germplasm material, and on that basis we bred male sterility three line autotetraploid rice materials, and the achieved the three line rice autotetraploid matchmaking, researched in autotetraploid rice heterosis usage and tetraploid hybrid rice breeding, constituted a new technology system of autotetraploid hybrid rice heterosis utilization. This not only helps the tetraploid rice heterosis exploration and use, but also inaugurates a new technical means for China in the new century "super rice" breeding research. We chose ordinary diploid rice as materials, using colchicine to induce the polyploidization, created the autotetraploid rice three-line materials and identified them. The polyploidization was based on the colchicine-induced callus tissue culture, and we experimented different colchicine concentrations and culture mediums to induce tetraploid plants, confirmed that the optimal concentration for inducement was 500 mg/L, the average induce rate was 25.26 %. Among all the materials, CDR22 and IR26 had higher induced rate; in contrary, 94109 and GANG46B had lower induced rate, the difference was significant. Autotetraploid materials was identified of both morphological and cytological, compared plant height, length of pollen sterility, and other major agronomic traits with a diploid rice as the control plant, identified that the autotetraploid materials had very significant advantages in ear length and thousand-grain weight, as well as the size of the seeds. Cytology identification included observation of the cell morphology, the number of chromosomes, and karyotype analysis on the autotetraploid materials confirmed that their chromosome number was 48, twice of the diploid rice. Mitoses in the three lines were common: chromosomes arrayed normally in metaphase and separated balanced into the two poles, chromosome moved without lagging in anaphase and daughter cells normally formed in telophase except one. It has been proved that tetraploid rice has normal meiosis as their diploid relatives, which usually including series of sub-phases as interphase, prophase I (five sub-phases), prophase II, metaphase I, II, anaphase I, II and telophase I, II. However, abnormal phenomena, such as formation of tetravalent, trivalent and univalent, chromosome lagging and so on, which would finally block meiosis. Configurations of chromosome in metaphaseⅠwere versatile in structure and form accept the bivalent. That condition varied in different strain, suggesting more complex paring configurations and more versatile genetic characters in tetraploid rice. All these abnormalities in meiosis contributed to low fertility of gamete and might consequently resulted in low seed setting. Successive selection and improvement on seed set, productive tiller per plant, total grains per panicle, panicle length and so on had been carried out from 1996 to 2006. The raise of seed sets was significant in both restorers and maintainers. Seed sets of some strains were improved more significantly than others, for example D237（29.70％→72.70％）、DTB（19.55％→53.21％）and et al.. Productive tiller per plant was improved to some extant. The tendency of improvement was rising on the whole but changed in some years such as 1998 and 2002. Part of the stains increased greatly, such as D19B（5.87→13.50）、Dshixiang (7.00→12.00) and so on, but some strains including DTB and Dminghui63 only increased little and decreased in some years by unknown reason. Total grains per panicle increased significantly and all strains except DTB increased. Improvement of panicle length termed to be hard. Different strains showed different capacities for improvement and floating existed in different years for example 2001. It has been proved that other agronomical traits including plant length, flag leaf length and so on could be improved but not significantly by selection also. In a word, agronomical traits could be raised by successive selection that is prerequisite for further utility of autotetraploid rice. Poor fertility is the main barrier for utilizing heterosis between the two rice (Oryza stiva L.) sub-species, indica and japonica. Recently, the development of autotetraploid hybrids (2n=4x=48) has been suggested as a new method for increasing heterosis in hybrid rice. Using standard experimental protocols, the elite diploid rice male sterile, maintainer, and restorer lines were colchine-doubled and autotetraploid counterparts were obtained. Seven resulting hybrids were analyzed for heterobeltiosis (HB), where the F1 was compared to the male parent, and the degree of heterosis, where the F1 was compared to the diploid commercial hybrid, Shanyou 63. The HB among the autotetraploid hybrids ranged from 1.4 to 105.9% for the productive panicles per plant, 0.5 to 74.3% for total kernels per panicle, 17.6 to 255.7% for filled kernels per panicle, and 9.6 to 130.4% for seed set. Improvements in these yield components resulted in the HB for kernel yield ranging from 64.8 to 672.7% among the seven hybrids. Hybrids T461A/T4002 and T461A/T4193 yielded 46.3 and 38.3% more, respectively than Shanyou 63, and all other hybrids but one yielded the same or more than Shanyou 63. The high heterosis for yield suggests that hybrid sterility between two rice sub-species may be overcome by using tetraploid lines followed by intensive selection. Also, the gigantic features of the autotetraploid hybrids may establish a plant structure able to support the higher yield.

毛壳菌产抗真菌活性物质菌株筛选及种间原生质体融合研究

毛壳菌属很多种类具有重要生防价值，其生防机理包括对植物病原真菌的重寄生作用、诱导植物产生抗病性、产生抗真菌活性的次生代谢产物等。迄今，学界对毛壳菌的研究主要集中在毛壳菌的生防机理，毛壳菌活性次生代谢产物的分离等方面。本研究致力于产抗生素的毛壳菌的种间原生质体融合，从产抗生素毛壳菌菌株的筛选开始，进而对产抗生素的角毛壳菌进行诱变选育，最终用产不同抗生素的角毛壳菌与球毛壳菌进行种间原生质体融合。主要有以下五方面研究结果。 1、毛壳菌抗真菌活性物质产生菌株的筛选：不同毛壳菌菌株发酵液采用琼脂扩散法对植物病原真菌进行抑菌活性试验，结果显示，菌株CH08和CH23的发酵液对芒果炭疽、苹果炭疽和马铃薯晚疫菌具有抑制作用。菌株CH16和CH17的发酵液对芒果炭疽菌、苹果炭疽菌有抑制作用。菌株CH21发酵液对辣椒炭疽菌和西瓜枯萎菌有抑制作用。经形态学研究，菌株CH08、CH16、CH17和CH23鉴定为球毛壳菌，菌株CH21鉴定为角毛壳菌。对角毛壳菌与球毛壳菌菌株发酵液抑菌谱比较，发现角毛壳菌与球毛壳菌发酵液具有明显不同的抑菌谱，表明角毛壳菌与球毛壳菌产生不同的抗真菌活性物质。 2、角毛壳菌（CH21）和球毛壳菌（CH08）原生质体制备和再生条件研究：考察了菌龄、酶浓度、稳渗剂及其浓度、酶解温度、酶解时间及再生培养基对原生质体制备和再生的影响。用菌龄为生长54 h的角毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1.5 h，原生质体释放量2.02×107个/g；以PDA为再生培养基，0.7 mol/L的蔗糖再生稳渗剂，再生率可达51.45%。用菌龄为生长48 h的球毛壳菌菌丝，以0.06 M磷酸缓冲液（pH6.0）配制成含蜗牛酶15 mg/ml、溶壁酶10 mg/ml、蔗糖0.6 mol/L的酶解液，30℃酶解1 h，原生质体释放量达1.57×108个/g；以PDA为再生培养基，0.7 mol/L的蔗糖为再生稳渗剂，再生率可达41.48%。 3、角毛壳菌（CH21）原生质体紫外诱变选育：以CH21为出发菌株，制备原生质体进行紫外诱变，诱变条件为：15 w紫外灯，距离30 cm，照射90 s，致死率80%~85%。建立了诱变菌株初筛的双层平板筛选模型。经平板初筛和摇瓶复筛，获得一株突变菌株CH21-I-402，其发酵液抑菌活性较出发菌株提高18.3%。 4、抗性标记菌株的获得：菌株CH21-I-402和CH08抗生素药敏试验表明， CH21-I-402菌株对潮霉素有抗性、对G418（Geneticin）敏感，菌株CH08对潮霉素和G418都敏感。根癌农杆菌EHA105介导的新霉素磷酸转移酶基因转化球毛壳菌，经PCR检测，新霉素磷酸转移酶基因成功转化进菌株CH08-GR70，CH08-GR120。转化子对G418抗性提高3～4倍，对潮霉素仍然比较敏感。 5、以G418和潮霉素抗性为筛选标记的原生质体融合与融合菌株AFLP分析：制备角毛壳菌CH21-I-402和球毛壳菌CH08-GR70原生质体，以35％的PEG6000为助融剂进行原生质体融合，以65 μg/ml的潮霉素和60 μg/ml G418为抗性筛选标记，获得46个再生菌株。再生菌株连续传代5代后，再生菌株表现出多种形态类型。利用AFLP技术对再生菌株及亲本菌株基因组DNA分析表明，再生菌株PF1、PF26为融合菌株。抑菌活性测试表明，融合菌株PF26发酵液对芒果炭疽菌和苹果轮纹菌有强的抑制作用，且抑菌活性比亲本球毛壳菌明显提高。 Chaetomium spp. have great potentials as biocontrol agents against a range of plant pathogens on the basis of its mycoparasitism, induced plant disease resistance, production of antifungal metabolites, and so on. Previous researches on C. spp. mostly focused on the mechanisms of its biocontrol and the isolation of secondary metabolites. In this study, screening antifungal C. spp., mutation breeding of C. cupreum and interspecies protoplast fusion between C. cupreum and C. globosum were carried out, respectively. The corresponding results are as follows: Firstly, among more than 40 C. spp., the strains produced anti-fungal antibiotics were screened by agar diffusion experiments. Results showed that both CH08 and CH23 had inhibition against Colletotrichum gloeosporioides, Cladosporium fulvum, and Phytophthora infestans. Both CH16 and CH17 had inhibition against Colletotrichum gloeosporioides and Cladosporium fulvum. In addition, CH21 exhibited anti-fungal activity against Fusarium oxysporum f. sp niveum and Colletotrichum capsici. Furthermore, CH08, CH16, CH17 and CH23 were identified as C. globosum, CH21 was proved to be C. cupreum based on morphology. The comparison of the anti-fungal spectrum between C. cupreum and C. globosum, showed they could produce different antibiotics. Secondly, specified protocols for preparing and regenerating protoplasts from mycelia of C. cupreum CH21 and C. globosum CH08 were studied. The effects of the age mycelia, the concentration of enzyme, digestion temperature and time, kinds of osmotic stabilizer and regeneration medium on protoplasts preparation and regeneration were all optimized, respectively. In one protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1.5 h at 30 ºC, 2.02×107 protoplasts from each gram mycelia were obtained from cultures of C. cupreum CH21 grown in potato dextrose broth (PDB) medium for 54 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA (potato dextrose agar with osmotic stabilize), the regeneration efficiency of protoplasts was 51.45%. In another protocol, with 15 mg/mL snailase, 10 mg/mL lywallzyme, 0.6 M sucrose, in 0.06 M phosphate buffer (pH6.0), and digested for 1 h at 30 ºC, 1.57×108 protoplasts from each gram mycelia were obtained from cultures of C. globosum CH08 grown in PDB for 48 h. And when 0.7 M sucrose was used as osmotic stabilizer in the regeneration medium OPDA, the regeneration efficiency of protoplasts was 41.48%. Thirdly, the mutagenesis conditions and secondary screening model of C. cupreum CH21 were explored. An 80% to 85% death rate could be achieved when the protoplasts of C. cupreum CH21 were irradiated by 15 w UV lamp from 30 cm distance for 90 s. In addition, the doublelayer plate’s method for the primary screening of high-producing antibiotics strains was established. A high yielding antibiotic mutant CH21-I-402 was obtained through the primary screening on plate and the secondary selection in Erlenmeyer flask, compared to the original CH21 strain, the antifungal activity of the mutant CH21-I-402 was increased by 18.3%. Fourth, the sensitivity to antibiotics of both C. cupreum CH21-I-402 and C. globusm CH08 was detected. Results showed C. cupreum CH21-I-402 was sensitive to G418 (Geneticin) (Gs) and resistant to Hygromycin B(Hr), and C. globusm CH08 was sensitive to both G418 (Geneticin) (Gs) and Hygromycin B(Hs). At the same time, neomycin phosphotransferase II (npt II) gene was transformed into C. globusm CH08(Gs, Hs) mediated by Agrobacterium tumefaciens EHA105, and the npt II gene was verified by polymerase chain reaction in resistance to G418 strains CH08-GR70 and CH08-GR120. The transformants still showed sensitive to Hygromycin B(Hs). Finally, a selection system for hybrids was set up by interspecies protoplast fusion between C. cupreum and C. globusm using dominant selective drug resistance markers. At first, protoplasts of C. cupreum CH21-I-402 (Hr, Gs) and C. globusm CH08-GR70 (Hs, Gr) were prepared, then the protoplasts were fused in the presence of 35% polyethylene glycol 6000 and regenerated on OPDA medium with 65 μg/ml Hygromycin B and 60μg/ml G418, at last 46 colonies with Hr and Gr were obtained. Even after 5 generations’ subculture, most of the colonies displayed significant difference in taxonomic characteristics with their parental strains. Regenerated strains PF1 and PF26 were confirmed as fusants by amplified fragment length polymorphisms analysis with the genomic DNA as the model. PF26 showed higher inhibitory activity against Colletotrichum gloeosporioides and Macrophoma kuwatsukai than that of the parental strain C. globusm.

Europium complexes immobilization on titania via chemical modification of titanium alkoxide

We report a facile strategy to tether lanthanide complexes to organic-inorganic hybrid titania materials via sol-gel processing by employing chemically modified titanium alkoxide as the precursor where the organic ligand sensitizing the luminescence of lanthanide ions is bonded to titanium.