水稻雌蕊与胚囊中钙的超微细胞化学定位

用焦锑酸盐沉淀法对水稻（OryzasativaL．）授粉前后雌蕊和胚囊中的钙进行了超微细胞化学定位。结果表明,柱头乳突细胞表面和花柱薄壁细胞中均含钙沉淀;开花前1d,整个胚囊中含钙较少,两个助细胞中钙分布无差异;临近开花时,1个助细胞已退化,其钙含量明显增加;开花后6h,胚囊已受精,退化助细胞中钙含量进一步增加;受精前卵细胞中钙主要分布在液泡中,核和胞质中较少;受精后,其钙含量明显增加,主要分布于核中。重点讨论了钙与助细胞退化和卵细胞激活的关系。     

鹤顶兰胚囊发育过程中Ca~（2＋）分布的超微细胞化学定位（英文）

用焦锑酸盐沉淀法对鹤顶兰（Phaius tankervilliae）胚囊发育过程中的Ca2＋状态进行超微细胞化学定位。观察结果发现：功能大孢子时期,珠孔端的胚囊壁上开始出现小颗粒的Ca2＋沉淀,但功能大孢子细胞内未见明显的Ca2＋标记;四核胚囊时期胚囊壁上的Ca2＋沉淀明显增多,液泡膜上有Ca2＋沉淀出现,珠孔处的Ca2＋沉淀颗粒较大;成熟胚囊时期,胚囊壁上的Ca2＋沉淀进一步增多,且胚囊内Ca2＋分布明显增多,且极性明显,珠孔端助细胞、卵细胞比合点端反足细胞有更多的Ca2＋沉淀。鹤顶兰成熟胚囊内Ca2＋积累的来源有：（1）在胚囊成熟前主要由珠被细胞、珠细胞通过胞间连丝向胚囊运输;（2）以沉淀有大量Ca2＋的小泡形式跨过胚囊壁进入胚囊。     

甘蓝型油菜授粉前后珠孔和胚囊中钙分布的超微细胞化学定位

用焦锑酸盐沉淀法对甘蓝型油菜（ＢｒａｓｉｃａｎａｐｕｓＬ．）授粉前后珠孔和胚囊中的钙进行了超微细胞化学定位。Ｘ射线能谱微区定性分析（ＥＤＸＡ）表明前法所得沉淀确系焦锑酸钙。同时利用图像处理系统对胚囊成员细胞中钙沉淀颗粒的体密度和大小进行了定量分析。结果显示,授粉前后内、外珠孔处钙沉淀明显较珠被其它部位为多,钙主要分布在细胞壁和胞间基质等质外体系统中。授粉前２个助细胞中均含有较其它细胞为多的钙沉淀,约为卵细胞的２５倍,中央细胞的１９倍;但沉淀颗粒的直径仅为它们的２／３左右。助细胞中含钙最多的部位是丝状器和细胞核。授粉后１对姊妹助细胞均明显退化,钙含量显著增高,约为授粉前的２４倍;钙颗粒明显减小,不足授粉前的１／３。讨论了钙的分布与助细胞的退化及其功能的关系。     

Structure of Embryo Sac Before and After Fertilization and Distribution of Transfer Cells in Ovules of Green Gram

The structure of embryo sac before and after fertilization, embryo and endosperm development and transfer cell distribution in Phaseolus radiatus were investigated using light and transmission electron microscopy. The synergids with distinct filiform apparatus have a chalazal vacuole, numerous mitochondria and ribosomes. A cell wall exists only around the micropylar half of the synergids. The egg cell has a chalazally located nucleus, a large micropylar vacuole and several small vacuoles. Mitochondria and plasrids with starch grains are abundant. No cell wall is present at its chalazal end. There are no plasma membranes between the egg and central cell in several places. The zygote has a complete cell wall, abundant mitochondria and plastids containing starch grains. Both degenerated and persistent synergids migh.t serve as a nutrient supplement to proembryo. The wall ingrowths occur in the central cell, basal cell, inner integumentary cells, suspensor cells and endosperm cells. These transfer cells may contribute to embryo nutrition at different developmental stages of embryo.     

Ultracytochemical Localization of Calcium in the Stigma,Style and Micropyle of Sunflower

Calcium was localized ultracytochemically in the stigma, style and micropyle of sun-flower (Helianthus annuus L. ) by pyroantimonate precipitation technique. To identify the element constitution of the pyroantimonate deposits, wave-dispersive X-ray microanalysis (WDX) method was employed in addition to the energy-dispersive X-ray microanalysis (EDX) and the deposits observed were directly proved as calcium pyroantimonate. In the stigma, calcium was more abundant on the receptive surface, especially outside and inside the papillae, than on the noo-receptive surface. In the style, more calcium was seen in the transmitting tissue as compared with the adjacent parenchymatous tissue, and was concentrated at the intercellular matrix and the boundary between the cell wall and the intercellular matrix. In micropyle region, the transmitting tissue on the side proximal to the funicle contained large amount of calcium, where as more calcium was localized in the intercellular matrix. As for the pollen tubes growing along the gynoecium, calcium was mainly localized at the pectin layer of the tube wall after 1.5 hours of pollination.     

秋水仙碱对甘蓝型油菜离体小孢子胚胎发生的影响

研究了秋水仙碱不同浓度和处理时间对甘蓝型油菜23个基因型离体小孢子胚胎发生的影响.3个基因型的小孢子被10、50和100mg/L秋水仙碱处理24h或48h,胚产量是2.55～14.75胚/蕾,10～50mg/L处理72h则是0.94～2.43胚/蕾.这表明处理72h对小孢子胚发生有抑制作用.用200、400、500和800mg/L处理2个基因型小孢子16～48h,胚产量为0.6～1.33胚/蕾,未处理对照是6.25和9.36胚/蕾.可见200～800mg/L浓度对胚再生有不同程度的阻碍效应.结果还证明,小孢子对秋水仙碱的反应与其基因型有关.当用10、20、50和100mg/L处理48h时,22B5-6和903-3小孢子的胚产量为37.09～69.47胚/蕾,而F1-29、W592和SF10-12是0.28～1.45胚/蕾,相互之间差异很大.秋水仙碱处理小孢子的目的是使其再生植株的染色体高频率加倍,因此应根据胚产量和染色体加倍率来确定秋水仙碱浓度和处理时间.本试验中,采用10～50mg/L处理48h或者用100mg/L处理24h,约80%基因型的小孢子胚产量在5胚/蕾以上,约70%基因型的再生植株加倍率达60%以上,可有效地用于油菜遗传和育种研究等领域.     

多花地宝兰胚囊和胚发育的细胞学研究

为探讨多花地宝兰(Geodorum recurvum)胚胎发育的系统分类学意义,采用石蜡制片法对多花地宝兰胚囊和胚的发育进行解剖学观察。结果表明,在开花前,多花地宝兰胚珠原基发育缓慢,开花授粉后胚珠原基快速发育成"树状二杈分枝结构",随后在"分枝结构"末端形成孢原细胞,开始胚囊发育。多花地宝兰的胚囊发育属于单孢蓼型胚囊,胚珠具有双层珠被。孢原细胞形成后,经过细胞膨大延长发育形成胚囊母细胞,胚囊母细胞经过减数分裂形成线性四分体,在珠孔端形成1个功能大孢子,功能大孢子经过3次有丝分裂形成8核胚囊。多花地宝兰的胚发育具有藜型和紫苑型两种方式。双受精完成后,多花地宝兰合子进行一次橫裂后形成基细胞和顶细胞;基细胞经过多次分裂形成细胞团,细胞团中的细胞向不同方向膨大延长形成多个胚柄细胞;顶细胞有两种分裂方式,一种是横裂形成藜型胚,一种是纵裂形成紫苑型胚。因此,推测多花地宝兰在兰科植物系统分类学上属于较为原始种。     

Study on Degeneration of Unfertilized Aposporous Embryo Sac in Pennisetum squamulatium (Gramineae)

To understand the degeneration of unfertilized aposporous embryo sac in an aposporous species Pennisetum squamulatum, the central cell in the unfertilized embryo sac was characterized ultrastructurally . The most prominent sign of degeneration is that the central cell nucleus produced nuclear vacuoles . These nuclear vacuoles can be classified into singleanddouble- layered types . Single- layered nuclear vacuoles are found in the cytoplasm, while the double-layered are inside the nucleus . There are two ways to produce nuclear vacuoles . One is that the outer membrane of the nuclear envelope distends towards the cytoplasm to form nuclear vacuoles ; and the other is the double-layered nuclear envelope derives vacuoles by depressing inwards . Nuclear envelope types are in relation to the ways they are produced . All nuclear vacuoles absorb cytoplasm or nuclear matrix , and trap organelles such as mitochondria .     

Calcium distribution in fertilized and unfertilized ovules and embryo sacs of Nicotiana tabacum L.

Potassium antimonate was used to localize Ca²⁺ in tobacco ovules from 0 to 7 d after anthesis in pollinated and emasculated flowers. Antimonate binds “loosely bound” Ca²⁺ into calcium antimonate; less-soluble forms are unavailable and free calcium usually escapes. Ovules are immature at anthesis. Abundant calcium precipitates in nucellar cells surrounding the micropylar canal. A difference between calcium in the two synergids emerges at 1 d, which is enhanced in pollinated flowers. The future receptive synergid accumulates more precipitates in the nucleus, cytoplasm and cell walls. After fertilization, micropyle precipitates diminish, and the ovule is unreceptive to further tube entry. In emasculated flowers 6 d after anthesis, ovular precipitates essentially disappear; however, flowers pollinated at 4–5 d and collected 2 d later largely restore their prior concentration of precipitates. Ovular precipitates occur initially in the nucellus, then the embryo sac, and finally the synergid and micropylar filiform apparatus. Possibility, calcium is released from the embryo sac, although no structural evidence of exudate formation was observed. Calcium precipitates in the ovule correlate with the ability of the ovule to be fertilized, suggesting that successful pollen tube entry and later development may require calcium of the class precipitated by antimonate. Received: 14 August 1996 / Accepted: 9 October 1996     

The Use of a Whole Stain-Clearing Technique for Observations on Embryo Sac,Embryo, Endosperm and Embryoid

A new stain-clearing procedure has been developed for embryological observations on whole mounted specimens. Ovules of Helianthus annus and Nicotiana tabacum as well as ovaries of Oryza sativa were stained with diluted Ehrlich's hematoxylin for a proper short time, followed by steps of washing and dehydration, and finally cleared and mounted in methyl salicylate. When observed by ordinary bright-field microscopy, the embryo sacs before fertilization and the embryos and endosperms after fertilization were clearly visible. The gynogenic embryoids induced in unpollinated rice ovaries in vitro were also finely detectable. The Ehrlich's hematoxylin-methyl salicylate technique has the merits of rapidity in specimen preparation, high contrast and three dimensional view, needlessness of phase- or interference-contrast equipment, and the feasibility for a wide range of materials. The special significance of this technique for in vitro embryological studies is emphasized.     

非洲狼尾草未受精无孢子生殖胚囊退化研究

为理解植物无孢子生殖胚囊未受精条件下的退化,对无孢子生殖植物非洲狼尾草未受精成熟胚囊中央细胞退化做了细胞形态学研究。没有受精的中央细胞退化时最显著的特点是细胞核产生核膜囊泡。核膜囊泡有两种类型:单层膜的囊泡和双层膜的囊泡,单层膜囊泡在细胞质中,双层膜囊泡在细胞核内。核膜囊泡有两种发生方式:1)核膜的外膜向细胞质一侧膨胀产生囊泡,囊泡进入细胞质;2)核膜向核内凹陷形成囊泡,囊泡进入细胞核。核膜囊泡类型与产生方式密切关联。核膜囊泡吞噬并消化包括线粒体在内的细胞质和核质。     

The Ultracytochemical Localization of ATPase Activity in the Ovules of Sunflower

The ultracytochemical localization of ATPase activity was determined employing the method of lead precipitation in the ovules of sunflower (Helianthus annuus L.). No ATPase activity is observed in the egg and synergids except some at the filiform apparatus. Much ATPase activity is localized on the plasma membrane and wall of the central cell. In the antipodal cells, ATPase activity is also found on the plasma membranes, but only a little in their walls. In the integumentary tapetum, besides the plasma membranes, most of the nuclei are rich in ATPase. Between the integumentary tapetum and uncontinuous cuticle surrounding the embryo sac, there is a gap where a lot of ATPase are found. These ATPases are continuously linked with those in the central cell wall throuth the intervals of the cuticle. At the sites of the wall ingrowths of the central celT, abundant vesicles and other structures with high ATPase activity aggregate noticeably in the gap region. According to the ATPase distribution in the ovules, we propose that the whole surface of embryo sac functions in absorbing nutrients directly from the apoplast outside the cuticle, especially via the wall-membrane apparatus of 'he central cell.     

Comparison of the genetic maps of Brassica napus and Brassica oleracea

 The genus Brassica consists of several hundreds of diploid and amphidiploid species. Most of the diploid species have eight, nine or ten pairs of chromosomes, known respectively as the B, C, and A genomes. Genetic maps were constructed for both B. napus and B. oleracea using mostly RFLP and RAPD markers. For the B. napus linkage map, 274 RFLPs, 66 RAPDs, and two STS loci were arranged in 19 major linkage groups and ten smaller unassigned segments, covering a genetic distance of 2125 cM. A genetic map of B. oleracea was constructed using the same set of RFLP probes and RAPD primers. The B. oleracea map consisted of 270 RFLPs, 31 RAPDs, one STS, three SCARs, one phenotypic and four isozyme marker loci, arranged into nine major linkage groups and four smaller unassigned segments, covering a genetic distance of 1606 cM. Comparison of the B. napus and B. oleracea linkage maps showed that eight out of nine B. oleracea linkage groups were conserved in the B. napus map. There were also regions in the B. oleracea map showing homoeologies with more than one linkage group in the B. napus map. These results provided molecular evidence for B. oleracea, or a closely related 2n=18 Brassica species, as the C-genome progenitor, and also reflected on the homoeology between the A and C genomes in B. napus. Received: 14 June 1996 / Accepted: 11 October 1996     

Purification and characterization of a nitrilase from Brassica napus

In germinating seedlings of Brassica napus glucosinolate levels decrease and are potentially degraded to nitriles by a myrosinase. Little is known about the metabolism of glucosinolate aglycone products and the objective of this work was to investigate nitrilase activity and carry out a purification of the enzyme from seedlings of B. napus . A nitrilase capable of converting phenylpropionitrile to phenylpropionic acid was purified to apparent homogeneity from seedlings of B. napus . The protein has a molecular mass of approximately 420 kDa made up of 38 kDa subunits. The pI of the native protein was found to be 4.6. Under denaturing conditions on an isoelectric focusing (IEF) gel a major and minor protein was observed with pI in the range of 5.4-5.9, suggesting the presence of isoforms. Apart from the potential role of the nitrilase in indole-3-acetic acid (IAA) synthesis a developmental study with seedlings indicates that the increase in activity observed may be linked to the in vivo degradation of glucosinolates.     

甘蓝型油菜矮秆突变材料99CDAM的发现及遗传分析

随着杂种优势的利用,油菜株高增加了20cm以上,导致油菜生长后期倒伏的风险加大。通过利用特殊矮秆基因来控制株高将是解决倒伏问题的有效方法。在甘蓝型油菜自交多代品系中发现了一个株高85cm左右的矮秆突变株99CDAM,该突变株具有开花早、分枝多等优良特性,产量性状和品质性状较好,各性状都能稳定遗传,具有重要的育种价值。对99CDAM和高秆品系2091、7045和7350的正反交F1以及99CDAM和2091的F2、BC1及F2:3代的遗传分析结果表明： 99CDAM的矮秆性状受3对隐性基因控制,存在母体细胞质效应,与以往的甘蓝型矮秆油菜有明显的区别。     

An Ultrastructural Study on the Development of Egg- apparatus in Rice Embryo Sac

The development of the egg-apparatus (consisted of an egg cell and two synergids) of rice ( Oryza sativa L. ) was studied at the uhrastructural level. The walls of the egg cell and synergids, immediately after their formation, possessed numerous plasmodesmata. Plasmodesmata were also present on walls between the egg cell and synergids. During the enlargement phase of the egg cell and synergids, the walls at the tip region began to loosen and vesiculate. By the time the embryo sac became mature, the part of the wall of the egg cell and synergids, facing the chalaza, disappeared. Consequently, the tip regions of the egg cell and synergids were only protected by a plasma membrane. When the embryo sac reached full maturity, the upper and middle region of the wall of the synergids broke up into pieces. At that time one synergid began to degenerate. Plasmodesmata persisted at the hook region of the wall of both the egg cell and synergids. Most plastids in the egg cell contained starch grains that persisted throughout the period of the embryo sac development. Starch grains in the plastids of the synergids appeared only before the time when the two polar-nuclei moved into the region above the egg-apparatus. They then disappeared and did not appear again until the embryo sac had reached full maturity. The size and location of the vacuoles in the egg cell were different from those in the synergids. The time of formation was also different. Vacuoles in the egg cell formed late in comparison with the synergids. Vacuoles in the chalazal region of the egg cell (especially at the early stage of the embryo sac development) were much larger than those in the micropylar region. Vacuoles in the synergids tended to concentrate mainly in the chalazal region. There was a peak period of lipid formation in the two synergids. The peak appeared when the embryo sac neared maturity. At the early stage of development, the nuclei of the synergids were elliptical in shape and were situated at the central region near the micropyle. The shape of the nuclei at the late stage of development became less regular and tended to move more towards the micropylar region. Changes in the uhrastructure of the egg cell and synergids of rice appeared to be closely related to the metabolic processes controlling the embryo sac formation and development.     

甘蓝型油菜若干农艺性状与单株产量的关系分析

以38个甘蓝型油菜品种为材料,对单株产量、有效分枝部位高度、主轴花序长度等11个农艺性状进行了方差分析、通径分析和聚类分析．结果显示:所有调查性状在品种间存在显著的差异;主花序长度、主序有效角果数与单株产量都有显著的遗传相关性,两者都通过有效分枝部位高度对单株产量产生较大的正向间接作用;有效分枝部位高度对单株产量的影响也较显著,高产品种具有相对较高的有效分枝部位高度;总角果数对单株产量的直接遗传通径系数虽小,但它具有最大的直接表型通径系数,它通过主序有效角果数和有效分枝数对单株产量产生了两个较大的间接作用;要获得3130kg/hm~2的产量,重点应具备果多、枝多的特征,总角果数不少于657个,有效分枝数不少于18个．     

Cytological Observation on Megasporogenesis and Embryo Sac Development of Regenerated Ovule in Hyacinth

The morphogenesis of regenerated ovule and cytological changes of its megasporogenesis and embryo sac development were studied. Results showed as follows: 1. the differentiation of the regenerated ovule had followed a normal process in the order of inner integument , outer integument and then funiculus. But the form of the regenerated ovules in vitro was quite different from that of ovule in vivo. Most of the regenerated ovules were orthotropous and hemianatropous , only a few were anatropous which are the same with that in vivo. 2. the megasporogenesis and the embryo sac development also had normal cytological process ,and the Polygonum type-embryo sac consisted of one egg, two synergids , one central cell and three antipodals could be seen in mature regenerated ovule. These ex-perimental results make clear that the regenerated ovule differentiated directly from explant could accomplish the complex processes of megasporogenesis and embryo sac development. By this fact ,authors infer that once the differentiation of ovule primordium, the complex biochemical programs for the megasorogenesis and embryo sac development can be controlled by the ovule itself and need no more information from flower bud and /or plant.     

甘蓝型油菜ZF-HD基因家族的鉴定与系统进化分析

ZF-HD是一类植物特有的转录因子, 在植物生长发育及胁迫响应过程中发挥重要作用。利用生物信息学方法, 在甘蓝型油菜(Brassica napus)基因组中鉴定到62个ZF-HD基因, 其中83.9%的基因缺乏内含子, 93.5%的BnZF-HD等电点大于7, 预测定位于细胞核的蛋白大多由100个以上氨基酸组成。根据进化关系可将其分为6个亚群, 在每个亚群中, 甘蓝(B. oleracea)和白菜(B. rapa)的ZF-HD基因数量相等或近似相等, 而甘蓝型油菜的ZF-HD基因数量接近或等同于甘蓝和白菜的ZF-HD基因数量之和。同一亚群的motif数量和类型高度相似。共线性分析结果显示, 全基因组三倍体化使ZF-HD基因在二倍体祖先得到扩张, 而异源多倍体化又进一步使甘蓝型油菜ZF-HD基因家族扩张。K_a/K_s值说明大多数ZF-HD基因在进化过程中受到了纯化选择。所有BnZF-HD基因都具有光响应元件, 2/3的基因具有MeJA、ABA和厌氧诱导顺式作用元件, 推测这些基因可能参与相关生物学过程。研究结果为进一步挖掘该家族基因的生物学功能奠定基础, 同时为揭示多基因家族在异源多倍体中的进化式样提供借鉴。     

Microspore Culture of Hybrids Between Brassica napus and B. campestris

Embryos and regenerated plants were produced by isolated microspore culture of inter-specific hybrids between Brassica napus and B. campestris. The NLN media with different sucrose concentrations and pH values were tested and a protocol for optimal microspore culture of B. carnpestris was identified. The reciprocal hybrids between UM921 (B. campestris) and 911186 (B. napus) had significant higher embryo yield than other cultured hybrids. Obvious improvement of embryo yield and quality was achieved when hybrid plants of reciprocal UM921 × 911186 were grown under 10 ℃/5 ℃ (day/night) condition. There was significant correlation between embryo yield and seeds per pod on hybrid plants but no correlation between pollen fertility and embryo yield was detected among cultured.hybrids. The majority of microspore-derived plants from the reciprocal B. napus × B. campestris hybrids are aneuploids and 22.8% of the plants observed originated from the microspores with parent′s chromosome numbers, almost all n = 19. The factors affecting the embryogenesis in microspore culture of interspecific hybrids and the possible applications of the technique are discussed.