染色体加倍对忍冬生物学性状及其生态适应性的影响

忍冬（Lonicera japonica Thunb.）属忍冬科忍冬属，是一种重要的药用植物，其花蕾称为金银花（Flos Lonicerae），在我国已有1000多年的药用历史，具有清热解毒、凉散风热等功效。我们利用秋水仙素诱导二倍体“大毛花”品种茎尖选育出同源四倍体“九丰一号”品种，并在生产中发现花蕾产量显著提高，而药效成份含量是否发生变化了呢？四倍体忍冬表现出典型的器官巨大性，这些变化是否与其较强生态适应性之间存在联系呢？染色体加倍增强了忍冬的生态适应性，其生态修复功能如何呢？在本研究中，主要从以下几个方面进行了探讨：（1）染色体加倍对其叶片，茎、花蕾和花蕾产量及其药效成份等生物学性状的影响;（2）染色体加倍后植物对水分胁迫的响应;（3）染色体加倍后植物对热胁迫的响应;（4）除上述两个品种外，增加一个变异品种“红银花”，探讨3个忍冬品种对退化生态系统的修复功能。主要结果如下： 1. 通过测定根尖细胞染色体数目和使用流式细胞仪分析茎尖细胞DNA含量，表明四倍体忍冬（2n = 4x = 36）确实来自二倍体忍冬（2n = 2x = 18）的染色体加倍。四倍体忍冬气孔细胞大小显著大于二倍体，而气孔密度显著低于二倍体。四倍体忍冬没有光合“午休”，而二倍体存在明显的光合“午休”，这可能与其存在抗高温的叶片解剖结构特性有关。四倍体忍冬叶片较二倍体变大、变厚、变浓绿（较高叶绿素含量）。与二倍体相比，四倍体忍冬单位叶面积重量显著增高，表明其具有较强的生态适应性。四倍体忍冬单个花蕾的鲜重和干重均显著大于二倍体。连续3年的花蕾产量调查表明，四倍体显著高于二倍体。染色体加倍使其茎干粗壮、节间变短、新梢上着生花蕾数目增多及单个花蕾变大，这是其花蕾高产的生物学基础。对金银花的药效成份而言，染色体加倍不影响绿原酸的含量，但增加了木犀草苷的含量。结果表明，染色体加倍能增加金银花的产量和药效成份的含量，建议四倍体忍冬在药材生产中推广应用。 2. 水分胁迫显著降低二倍体和四倍体忍冬叶片的净光合速率、气孔导度和蒸腾速率。水分胁迫也降低电子传递速率、光系统II实际量子产量和光化学猝灭，而增加非光化学猝灭、总可溶性糖、脯氨酸和丙二醛的含量。四倍体忍冬对水分胁迫的响应表现为其叶片水势、气体交换、叶绿素荧光和有关代谢物含量的变化程度低于二倍体，并且复水后其恢复能力快于二倍体，表明染色体加倍增强了忍冬的抗旱能力。分析其主要原因，可能是由于四倍体植株总叶面积减少、单位叶面积重量增加、叶片表皮细胞和栅栏组织增大以及叶片表皮毛较浓密等形态解剖结构特性有关。结果表明，染色体加倍能增加忍冬植物的抗旱能力，而使其具有较强的生态适应性。 3. 二倍体和四倍体忍冬受48 ºC热胁迫处理6 h和恢复10 h，以及离体叶片45 ºC，50 ºC，55 ºC 水浴热胁迫3 min，应用叶绿素荧光成像系统研究了它们对热胁迫的响应。热胁迫显著降低了两个品种叶片的最大光化学效率、电子传递速率、光系统II实际量子产量和光化学猝灭，降低了四倍体的非光化学猝灭，而增加了二倍体的非光化学猝灭。热胁迫增加了两个品种叶片的总可溶性糖、脯氨酸和丙二醛的含量。四倍体受热胁迫的叶绿素荧光参数和有关代谢物的响应程度低于二倍体，以及其恢复程度快于二倍体，表明染色体加倍提高了抗热性。此外，叶绿素荧光成像的异质性也表明，四倍体的抗热能力大于二倍体。进一步的叶片解剖结构分析表明，四倍体叶片的表皮细胞变大、栅栏组织增厚、表皮毛较浓密等特点，是其抗热性强的主要原因。 4.根据以上的研究结果，通过四倍体忍冬生态修复功能的野外试验证明染色体加倍后其生态适应性变化。在本研究中，针对北京市门头沟区大面积不同类型的废弃地急需恢复植被和景观的问题，在恢复生态学理念的指导下，综合运用集成技术，将3个忍冬品种植物用于这些退化生态系统的恢复。在王平镇的公路下边坡（以碎石和矿渣为主）、煤矿、石灰窑和采石场4种类型废弃地建立生态修复的试验示范区。结果表明，在4个废弃地类型上引进的3个金银花品种，具有使示范区快速复绿、当年成景和群落快速形成的潜力，并具有对不同退化迹地的适应能力和恢复效果，其中四倍体忍冬效果更好些，这主要与其形态解剖结构和较强的生态适应性有关。

小鼠四倍体半克隆胚胎发育研究

半克隆U-9/G;7Q":9V2胚胎是通过注射体细胞核到未去核的卵母细胞中产生的# 在半克隆胚胎中!体细胞被用来作为精子的替代物# 然而!由于异常的染色体分离!构建的半克隆胚胎在激活后形成了非整倍体而导致胚胎发育受到严重影响!不能发育到期# 本研究通过抑制小 鼠半克隆胚胎在激活过程中染色体数目减半!避免非整倍体胚胎形成!研究四倍体半克隆$W9XKMY#"GV -9/G;Z#":9V!W-7%胚胎的发育和体细 胞核的掺入对胚胎发育的影响& 结果显示!W-7 胚胎的体外发育率显著高于二倍体半克隆胚胎!与正常受精卵及孤雌激活对照无显著性差异! 但W-7 胚胎的细胞数在桑椹胚和囊胚期比正常二倍体受精胚胎和孤雌激活胚胎少& 通过AZX;> 染色发现!W-7 胚胎囊胚期内细胞团$5::9K 79## [M\\]57[%细胞很少或者没有& 移植1& 个四倍体半克隆胚胎到& 只假孕母鼠体内!得到%* 个胎盘!但没有得到胎儿& 组蛋白乙酰化和 4’@ 甲基化检测显示!部分W-7 胚胎在囊胚期没有形成正常受精胚胎在57[ 和滋养外胚层$WK"Y89ZX"V9K/!WE%之间的差异分布& W-7 胚 胎的基因表达不依赖于细胞分裂次数而依赖于发育时间& 虽然W-7 胚胎避免了二倍体半克隆胚胎形成非整倍体现象!但由于W-7 胚胎没有 57[ 细胞或57[ 细胞很少!所以只能形成胎盘而不能形成胎儿& 本实验第一次较为全面地研究了W-7 胚胎的发育!同时也为研究体细胞核再 程序化’基因打靶技术提供了一种新的途径#

Phylogeny of east Asian bufonids inferred from mitochondrial DNA sequences (Anura : Amphibia)

We investigated the relationships of Asian bufonids using partial sequences of mitochondrial DNA genes. Twenty-six samples representing 14 species of Bufo from China and Vietnam and 2 species of Torrentophryne from China were examined. Three samples of Bufo viridis from Armenia and Georgia were also sequenced to make a comparison to its sibling tetraploid species B. danatensis. Bufo americanus, from Canada, was used as the outgroup. Sequences from the 12S ribosomal RNA, 16S ribosomal RNA, cytochrome b, and the control region were analyzed using parsimony. East Asian bufonids were grouped into two major clades. One clade included B. andrewsi, B. bankorensis, B. gargarizans, B. tibetanus, B. tuberculatus, its sister clade B. cryptotympanicus, and the 2 species of Torrentophryne. The second clade consisted of B. galeatus, B. himalayanus, B. melanostictus, and a new species from Vietnam. The placement of three taxa (B. raddei B. viridis, and its sister species, B. danatensis) was problematic. The genus Torrentophryne should be synonymized with Bufo to remove paraphyly. Because B. raddei does not belong to the clade that includes B. viridis and B. danatensis, it was removed from the viridis species group. The species status of B bankorensis from Taiwan is evaluated. (C) 2000 Academic Press.

Detection of hybridization between two loach species (Paramisgurnus dabryanus and Misgurnus anguillicaudatus) in wild populations

Artificial interspecific hybrids between large scale loach P. dabryanus and tetraploid pond loach M. anguillicaudatus (Cobitidae, Cypriniformes) are viable. To detect the occurrence of possible natural hybridization, genetic analyses by using microsatellite markers were performed for natural populations of large scale loach and pond loach, the reciprocal laboratory hybrids, and "supposed hybrids" with ambiguous morphology. The fertility of the artificial hybrids was also tested. At one diagnostic microsatellite (Mac50), one out of 20 "supposed hybrids" was identified to be F-1 hybrid between the two loach species because it had the same genotype as that of the laboratory hybrids. The triploid hybrids between the two species were confirmed to be female-sterile. The results show that rare hybridization has occurred between diploid large scale loach and tetraploid pond loach in nature although it may have little effect in genetic introgression. This study is helpful for fish conservation and encourages further investigation on natural hybridization and introgression of loaches.

Genetic Stability of Progeny from an Artificial Allotetraploid Carp Using Sperm from Five Fish Species

Microsatellite markers and D-loop sequences of mtDNA from a female allotetraploid parent carp and her progenies of generations 1 and 2 induced by sperm of five distant fish species were analyzed. Eleven microsatellite markers were used to identify 48 alleles from the allotetraploid female. The same number of alleles (48) appeared in the first and second generations of the gynogenetic offspring, regardless of the source of the sperm used as an activator. The mtDNA D-loop analysis was performed on the female tetraploid parent, 25 gynogenetic offspring, and 5 sperm-donor species. Fourteen variable sites from the 1,018 bp sequences were observed in the offspring as compared to the female tetraploid parent. Results from D-loop sequence and microsatellite marker analysis showed exclusive maternal transmission, and no genetic information was derived from the father. Our study suggests that progenies of artificial tetraploid carp are genetically stable, which is important for genetic breeding of this tetraploid fish.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

灌浆期干旱对不同倍性小麦光合和产量的影响

为揭示灌浆期水分亏缺对不同倍性小麦光合特性和产量的影响,选用二倍体野生一粒、栽培一粒小麦,四倍体野生二粒、栽培二粒小麦,六倍体小麦"长武134"和"陕253"等6个小麦品种作为供试材料,通过盆栽控水方式,对不同倍性小麦旗叶净光合速率、瞬时水分利用效率和产量进行了研究。结果表明,在正常供水、轻度干旱和严重干旱3种水分处理下,不同倍性小麦旗叶净光合速率、水分利用效率和产量差异极显著。在灌浆过程中,水分亏缺对不同倍性小麦净光合速率变化趋势的影响不明显。而最大净光合速率和水分利用效率随水分胁迫的加重而减小。六倍体小麦平均最大净光合速率为22.03μmol CO2.m-2.s-1),高于二倍体和四倍体小麦。六倍体小麦平均最大水分利用效率约为7.12μmol CO2/mmol H2O,分别是四倍体和二倍体的1.63倍和2.05倍,并且在灌浆开始时就达到最大。因此,小麦长期进化过程中,六倍体小麦花后较强的光合能力和较高的水分利用效率是提高小麦产量的重要生理基础。

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。

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.

不同倍性小麦材料对水分和密度条件的响应

以栽培一粒(2n)、栽培二粒(4n)和长武134(6n)为材料在大型活动防雨棚条件下研究不同倍性小麦材料在不同密度和水分条件下的产量适应性变化。结果发现,在两种水分条件下随着染色体倍性从2n→6n的增加,产量、千粒重、水分利用效率(WUE)和收获指数均呈增加趋势,在水分胁迫下各材料穗粒数和穗数则呈降低趋势,而在正常供水下穗粒数则呈增加趋势。在水分胁迫下栽培一粒、栽培二粒和长武134最高产量分别出现在中、低、高密度群体,而同一材料不同密度群体间变异系数分别为6.73%,1.98%,9.07%;不同倍性材料千粒重均随着密度增加而减小,而穗数则逐渐增加,二倍体的穗粒数以中密度最高,四倍体的穗粒数随着群体密度的增加而减小,六倍体则相反;三种材料WUE和收获指数分别以低、高、低密度最高。正常供水下随着染色体倍性从2n→6n的增加,三个倍性材料最高产量分别出现在高、低、低密度群体,而同一材料不同密度群体间变异系数分别为6.01%,17.12%,2.46%;千粒重表现为中密度>低密度>高密度,而穗粒数均以低密度群体最高,二倍体和四倍体以高密度群体最低,六倍体则以中密度群体最低,穗数则随着密度群体增加而增加;二倍体WUE以高...

Improvement of polyploidy induction in Eriocheir sinensis

An incubating temperature of 15 degreesC is used to induce triploidy in Etiocheir sinensis through inhibition of the release of polar body H, and that of 18 degreesC to induce tetraploidy through inhibition of the first cleavage. Flow cytometry is used to identify the ploidy in different developmental stages. For induction of triploidy in fertilized eggs in vitro, the highest induction rate observed in blastula by cytochalasin B, 6-DMAP and KCI is 49.1%, 51.7% and 77.5%, respectively. In the KCI treatment of pregnant crabs with the fertilized eggs, the highest triploid induction rate observed in the zoea is 85.3%. For induction of tetraploidy, the highest induction rate observed in the blastula by cytochaslasin 13, 6-DMAP and KCI is 50.3%, 54.9% and 79.8% respectively. In the KCI treatment of pregnant crabs with the fertilized eggs, the highest induction rate in zoea is 27.3%. Through this study such difficulty as in vitro culture is overcome. Triploid zoea Etiocheir sinensis has been developed for the first time. The induction rate of tetraploid zoea has also been greatly improved.

Chromosomal rearrangement in Pectinidae revealed by rRNA loci and implications for bivalve evolution

Karyotype and chromosomal localization of major (18-5.8-28S) and minor (5S) ribosomal RNA genes were studied in two species of Pectinidae, zhikong (Chlamys farreri) and bay (Argopecten irradians irradians) scallops. using fluorescence in situ hybridization (FISH). C. farreri had a haploid number of 19 with a karyotype of 3m + 4sm + 7sm-st + 4st + 1st-t, and A. i. irradians had a haploid number of 16 with a karyotype of 5st + 11t. In C. farreri, the major and minor rRNA genes had one locus each and were mapped to the same chromosome-Chromosome 5. In A. i. irradians, the major rRNA genes had two loci, located on Chromosomes 4 and 8, and the 5S rRNA gene was found at a third chromosome-Chromosome 10. Results of this and other studies indicate that karyotype of A. i. irradians (n = 16, 21 arms) is secondary and derived from an ancestral karyotype similar to that of C. farreri (n = 19, 38 arms) through considerable chromosomal loss and rearrangements. The ability to tolerate significant chromosomal loss suggests that the modal karyotype of Pectinidae and possibly other bivalves with a haploid number of 19 is likely tetraploid; i.e., at least one genome duplication has occurred during the evolution of Bivalvia.

Reacción en plántula a oidio Blumeria graminis f. sp. tritici de cultivares de trigo tetraploide, cultivares diferenciales y especies emparentadas con trigo

p.111-117

Induction of tetraploidy through loss of p53 and upregulation of Plk1 by Human Papillomavirus type-16 E6

Cancer cells are insensitive to many signals that inhibit growth of untransformed cells. Here, we show that primary human epithelial cells expressing human papillomavirus (HPV) type-16 E6/E7 bypass arrest caused by the DNA-damaging drug adriamycin and become tetraploid. To determine the contribution of E6 in the context of E7 to the resistance of arrest and induction of tetraploidy, we used an E6 mutant unable to degrade p53 or RNAi targeting p53 for knockdown. The E6 mutant fails to generate tetraploidy; however, the presence of E7 is sufficient to bypass arrest while the p53 RNAi permits both arrest insensitivity and tetraploidy. We published previously that polo-like kinase 1 (Plk1) is upregulated in E6/E7-expressing cells. We observe here that abnormal expression of Plk1 protein correlates with tetraploidy. Using the p53 binding-defective mutant of E6 and p53 RNAi, we show that p53 represses Plk1, suggesting that loss of p53 results in tetraploidy through upregulation of Plk1. Consistent with this hypothesis, overexpression of Plk1 in cells generates tetraploidy but does not confer resistance to arrest. These results support a model for transformation caused by HPV-16 where bypass of arrest and tetraploidy are separable consequences of p53 loss with Plk1 required only for the latter effect.