白头翁的受精及组织化学研究

成熟花粉为二细胞,生殖细胞的蛋白质染色较营养细胞的深,淀粉粒充满营养细胞。花粉管常见穿人正在退化中的助细胞,并释放出两个精子,大量稠浓的蛋白质和淀粉,个别的释放在助细胞与胚囊壁之间。在一些卵细胞核,次生核(或极核)中,受精前后有1—3个小核仁(约1微米);助细胞具丝状器;反足细胞宿存,具多核和多核仁。胚囊中各个细胞的原生质稠浓程度和蛋白质染色深浅不同,其中以反足细胞和助细胞的原生质最稠浓,蛋白质染色最深。淀粉十分贫乏,绝大多数卵细胞,中央细胞和几乎全部助细胞和反足细胞均不含淀粉。双受精属于有丝分裂前类型,两性核融合步骤为:精子核接近和贴附在卵核和次生核上(或极核上),两性核的核膜溶解,其染色质沉入融合核,并随之松解,同时出现雄性核仁,最终,雄性核仁和雌性核仁合并,形成具单核和单核仁的合子和初生胚乳细胞。另外,雄性核仁同卵核仁合并较雄性核仁和次生核的晚,所以卵细胞完成受精较次生核晚。     

野生与栽培白头翁药用部位解剖结构和皂苷组织化学定位

利用解剖学和组织化学的方法,研究了野生和栽培白头翁（Pulsatilla chinensis）主根的解剖结构和皂苷组织化学定位。结果表明：野生白头翁根部韧皮纤维散在,而栽培白头翁的则多个聚集成环状或其它形状;栽培白头翁根的次生木质部导管管腔内富含侵填体;野生白头翁根中分泌腔和分泌道丰富;分泌腔与分泌道的数量及大小是决定白头翁皂苷物质含量高低的主要因子。因此,分泌腔与分泌道大小和数量的多寡可以作为判断所选育的白头翁品种是否优良的结构性指标。     

Antitumor activity of Pulsatilla chinensis (Bunge) Regel saponins in human liver tumor 7402 cells in vitro and in vivo

Pulsatilla chinensis (Bunge) Regel is a Chinese medicinal herb for "blood-cooling" and detoxification. Now it is used for the treatment of malignant tumor, but the antitumor mechanisms and toxic side effects of P. chinensis are unclear. The present study was undertaken to investigate if P. chinensis saponins (PRS) possesses anticancer effects and toxic side effects in human liver tumor 7402 cells in vitro and vivo. 7402 cells were treated with different concentrations of PRS for 24h. Cell viability was measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Cell apoptosis was assessed by flow cytometry. The in vivo effect of PRS on 7402 tumor cells transplanted in athymic nude mice was investigated. 15 saponins were isolated and identified from PRS. PRS inhibited the proliferation of human liver tumor 7402 cells in vitro by apoptosis. 19 days after administration of PRS (100, 200mg/kg), the weight of tumor mass was markly decreased in nude mice. The anti-tumor effect of PRS in vivo was associated with a significant increase in the 7402 apoptosis rate. Although PRS inhibited the weight of mice, it showed almost no effect on leukocyte number, liver and spleen weight index. Light microscopic histopathological examination showed that PRS had no specific lesion in organ. These results suggested that P. chinensis saponins exert potential anticancer activity in treating tumors in nude mice and no toxic side effects.     

Development and Histochemical Observations of Tapetum and Peritapetal Membrane in Anther of Pulsatilla chinensis

Tapetum of Pulsatilla chinensis is of secretory type. Its development proceeds rapidly in following sequence: (1) The stage of initiation-differentiation. At this stage cytological and histochemical features have been described in detail in this paper. (2) The stage of growth- synthesis: This stage appears to be the most important anabolic phase during the development of the tapetum. The salient features are that the tapetal cells become relatively enlarged and form two polyploid nuclei or aberrent polyploid nuclei resulting in synthetizing maximum proteins, fluorescing substances and maximum fluorescent Pro-Ubisch bodies in the tapetal cytoplasm. (3) The stage of secretion-disorganization: After the disintegration of the tapetal wall the enlarged naked cells appear at once. This is an important secretion period in which Pro-Ubisch bodies as well as all other fluorescing substances, carbohydrate or some enzymes are released into anther loculus. The naked cell layer becomes disorgnized until the beginning divition of the pollen grains into two ceils. As to peritapetal membrane of P. chinensis, mainly based on the membrane being on the outer side of the tapetum enclosing both the pollen, tapetal cytoplasm and Ubisch bodies, and the cellular configurations facing the pollen, Authors postulate that peritapetal membrane might be survival of the cytoplasmic membrane of tapetal cells. However, the peritapetal membrane of P. chinensis is similar to that of plasmodial, tapetum reported in certain Compositae and that of secretory tapetum reported in Pinus banksiana. Heslop-Harrison and Gupta et al. had conceded that the tapetal and peritapetal membrane belong to the general class of sporopollenin. On the contrary in P. chinensis the sporopollenin property of peritapetal membrane is only confined to its inner surface. But the thin mem- brane itself with the reticulate sporopollenin attched on its inner side appears negative staining reactions for sporopollenin though it has an ability to resist the acetolysis as well. In P. chinensis the Ubisch body is short necked flask shaped and their size is very similar. Ubisch body is either single or 2–5 in a group, resulting in compound bodies. When the Pro-Ubisch body is still within the tapetal cell it shows positive fluorescent reaction, while it eomletely unstains with Teluidine blue O. So Authors infer that the sporopollenin precur- sors may have permeated through Pro-Ubisch bodies. Finally, How sporopollenin precursor is synthesized in the tapetal cells, transported to pollen locula and polymerized into the sporopollenin on pollen, Ubisch body and peritapetal membrane? Future works along these problems may yield fruitful results.     

Investigation on Fertilization of Cephalotaxus

The structure of spermatogenous cell of Cephalotaxus is unique among the gymnosperms. While towards the mature stage, its nucleus is close to one side of the spermatogenous cell, and on the other side there is abundant and prominent . cytoplasm, which contain a group of the aggregate cytoplasms of radial arrangement similar to blepharoplast of spermatogenous cell of Ginkgo. But, there are two opposite blepharoplasts at either side of the nucleus in Ginkgo, and while there is only one blephareplast at one side in Cephalotaxus. This is one feature of the sexual process in Cephalotaxus. When the pollen tubes approach the top of the archegonia, the division of the spermatogenous cell takes place and there are two almost similar sperm cells both in size and morphology. It is interesting to note that the cytoplasm of the sperm cell contains certain granules of nucleolus-like structure, which appears to be a rare phenomenon among the gymnosperms. This is another feature of the sexual process in Cephalotaxus. These two features are the important characters of Cephalotaxaceae. The egg morphology of Cephalotaxus is also unique among the conifers, its outline looks like a carrot. The upper part of the egg is rather wide and is about 85 to 108 μm in width. On the other hand, the opposite end is gradually becoming narrow and about 910 to 1100 μm in total length. So the ratio of the length and width in the egg of Cephalotaxus is about 10:1. The structure of the egg in Celhalotaxus fortunei and C. oliveri have the following common feature: 1. When their eggs mature the cytoplasm of the egg at lower part of the nucleus possesses deep- staining and fine granules of 2 to 3 groups of aggregate cytoplasm. 2. During maturation of the egg, some of the granules of nucleolus-like structure are scattered in the cytoplasm. As fer- tilization takes place the number of these granules reaches the peak. This condition has been encountered in the egg of Amentotaxus argotaenia. Therefore we could conclude that they are closely related between Cephloraxaceae and Taxaceae. The fertilization of Cephalotaxus fortunei occured on May 10 to 24 (1983), and that of C. oliveri took place on May 28 to June 13 (1983). The fertilization of the genus belong to the type of undergoing mitosis prior to complete fuse of both male and female nuclei. This type of fertilization has been found only in Pinaceae and Cephalotaxaceae. After fertilization the structure of fertilized egg becomes prominent in polar organization. In other words, the cytop- lasm at upper part of the fertilized egg becomes highly vacuolated and that at lower portion, conversely, is rich in abundant proteinous vacuoles and certain granules of nucleolus-like structure dispersed in the cytoplasm. Because the division and differentiation of the proembryo are proceeding at the base of the archegonium, the large inclusions and the nucleolus-like granules may be involved in the nourishing and development of the proembryo.     

Studies on Fertilization and Embryogenesis of Red Vinespinach (Basella rubra L.)

This paper presents detailed report on the process of fertilization and the develop- ment of embryo and endosperm of Basella rubra L. The results obtained are summarized as follows: About 4–6 hours after anthesis a great deal pollen grains germinated on the stig- ma; 6–11 hours, the pollen tube passed through the style; 11–16 hours, the pollen tube reached the ovary cavity. About 16–18 hours, one sperm nucleus entered into the egg and the other one entered into the secondary nucleus. In most cases, after 16–24 hours the double fertilization had completed. After 2–8 days, two-celled proembryo was first shown. Finally, the proembryo gave rise to multicellular globular embryo proper. The development of the embryo of Basella rubra L. conforms to the Asterad type. Whether the Asterad type had a high frequency needs further to be studied. Although the fertilization of the sperm nucleus with the secondary nucleus began later, the fusion of two sexual nuclei and the development of the endosperm proceeded often quickly. After 20 hours, the free nuclei period began, and after 2–4 days the free nuclei of endosperm were rapidly formed. The endosperm of Basella rubra L. is a nuclear type. And at the time When the seed had ripened the endosperm tissues were all absorbed by the developing embryo. The author observed that there are different distributions of the vegetative nucleus and two sperms in the pollen tube, and that two sperms entered a egg or secondary nucleus. The changes of the starch accumulated and distributed et al. in the floral organs are also studied and discussed.     

银杏精细胞结构及受精作用的细胞学研究

采用常规石蜡制片技术和环氧树脂半薄切片技术,对银杏的精细胞结构及受精过程进行了研究。结果表明,临近受精前,精原细胞分裂形成两个半球形的精细胞,每个精细胞内含液泡状结构、生毛体和纤维性颗粒体各一个。两精细胞的液泡状结构其位置可同时或分别位于近极面和远极面,这种位置的变动可能是鞭毛摆动导致精细胞的旋转所引起,这说明银杏的精细胞在花粉管内也许即可以旋转运动。在银杏受精前后,珠孔端颈卵器室附近的珠心组织表现出向上隆起、出现受精滴、皱褶等规律性变化,这些现象是判断银杏受精时期的良好形态特征。在银杏受精时带有鞭毛的完整精细胞进入颈卵器,随后鞭毛带及精细胞质遗留于颈卵器口下方、卵细胞上面,仅精核进入卵细胞。进入卵细胞的精核直径约30μm,小于成熟精细胞的精核直径（约40μm）。这些对探讨银杏的系统地位及裸子植物的生殖演化具有一定意义。     

Fertilization of the eggs ofCynops pyrrhogaster (japanese newt) after immersion in water

Summary Cynops pyrrhogaster oviducal eggs with and without jelly envelopes (jelly egg and dejellied egg respectively) were immersed in water, and then inseminated artificially. After 1 h of immersion in water, more than half the dejellied eggs were fertilized and developed, but no jelly eggs developed. The rapid decrease in the ability of jelly eggs to be fertilized after immersion in water is not due to a deficiency in the egg. Our results make it clear that hydrated jelly envelopes prevent the eggs from fertilizing. The ability of the egg to be fertilized decreases for a long time in water and this decrease proceeds more slowly in De Boer's solution or Holtfreter's balanced salt solution than in water.     

The Development of Gynoecium After Anthesis and Fertilization in Eleutherococcus brachypus (Araliaceae)

Eleutherococcus brachypus Harms. is a protandrous plant because the female gametophyte delays its maturation until the fifth day after anthesis and pollen shelling. On the fifth day after anthesis, about 57.69% of the embryo sacs matured and the rest degenerated or failed to develop. Fertilization began in the embryo sac on the fifth day. On the tenth day fertilization took place in 53.37 % of the total of embryo sacs. The stigma became receptible after 3 to 4 days of anthesis. It took 2 to 3 days from the germination of pollen grains on stigma to the fusion of male and female nuclei. The process of fertilization in E. brachypus is not different from most other angiosperms. It belonged to the type of premitotic syngamy. The observations and statistical analysis were made on the number feature of male and female nucleoli in the zygote. The result indicated that it took three days or so from the appearance of male nucleolus in the zygote to its fusion with the female nucleotus. Refering to the number of free nuclei of the endosperm, the fusion of male and female nucleoli in most of the zygotes occurred in the stage of 32 to 128 nuclei of the endosperm. Most zygotes con-tained a big nucleolus resulting from the fusion of male and female nucleolus and proceeding to mitosis. A few without fusion could also proceed to the mitotic stage. Features of multiple sperms entering the embryo sac or entering the egg cell and the degeneration of mature embryo sacs were observed as well. The sign of the termination of fertilization in angiosperms was discussed.     

乌蕨孢子萌发研究

用随机区组试验和方差分析方法, 研究了培养温度、贮藏温度、GA 处理和光照强度对乌蕨( Sphenomeris chinensis) 孢子萌发的影响。与室温(20～25℃ ) 相比, 培养温度在28℃时, 孢子最大萌发率相近而萌发速率明显较高。贮藏温度(A) 极显著( P < 0 . 01 ) 影响孢子萌发率, - 20℃ 贮藏降低萌发率; GA (B) 对孢子萌发率无显著影响。光照强度(C) 极显著( P < 0.01) 影响孢子萌发率, 充足光照和弱光照无显著差异, 黑暗处理降低萌发率。A×B, A×C, B×C 及A×B×C 交互效应不显著。     

Tapetum Dimorphism and Its Histochemical Study in Sweet Pepper (Capsicum annuum L. Var. grossum)

In sweet pepper, the portion of tapetum toward the interior of the anther comprising large cells is derived from cells of connective of anther whereas the remaining tapetum on the outside of the anther comprising comparatively small cells is derived from the parietal layer. Those ceils of the former prosessing large vacuoles and large nuclei are stained weaker than the cells of the latter by methyl green-pyronin and mercuric-bromophenol blue staining. Large spherical grains which contain acid phosphatase appear in the vacuoles in both kinds of tapetum at sporogenesis stage. During meiosis of pollen mother cells, DNA, RNA and protein sysntheses increase in tapetum. The tapetum derived from connective accumulates more DNA than that derived from parietal layer. The activity of acid phosphatase becomes higher in tapetum when it degenerates. The degeneration of two kinds of tapetum is similar. There are no starch grains in tapetum through its whole course of development.     

乌蕨孢子萌发研究

用随机区组试验和方差分析方法,研究了培养温度、贮藏温度、GA处理和光照强度对乌蕨(Sphe-nomeris chinensis)孢子萌发的影响。与室温(20～25℃)相比,培养温度在28℃时,孢子最大萌发率相近而萌发速率明显较高。贮藏温度(A)极显著(P<0.01)影响孢子萌发率,-20℃贮藏降低萌发率;GA(B)对孢子萌发率无显著影响。光照强度(C)极显著(P<0.01)影响孢子萌发率,充足光照和弱光照无显著差异,黑暗处理降低萌发率。A×B,A×C,B×C及A×B×C交互效应不显著。     

Studies on north indian volvocales-III. Fertilization in Eudorina californica (shaw) goldstein

The present communication deals with the details of sexual reproduction including the process of fusion of gametes in Eudorina californica (Shaw) Goldstein (earlier described as E. indica lyengar). The present observations are compared with those on other species in which the process of fusion was recorded.     

Histochemical and ultrastructural effects of Enovid E on the endometrium of the baboon

Summary Endometrium from a group of baboons treated with the oral contraceptive, Enovid E, was studied histologically, histochemically and ultrastructurally, and compared to endometrium from normally cycling animals. All endometria were obtained by transcervical uterine biopsy between 10 and 14 days of the treatment cycle or the normal menstrual cycle. Histologically, no discernible differences between the control and experimental endometria were apparent. While observable differences were not evident between the endometrial alkaline phosphatase and succinate dehydrogenase activities of control and experimental animals, there was an increased acid phosphatase activity in the Enovid E-treated baboon endometrium. Ultra-structurally, the glandular cells of treated animals appeared to be more physiologically advanced than did those from the control endometria. These advances were evident from the prominent Golgi complex, increased development of the endoplasmic reticulum and increases in the size, number and complexity of mitochondria. The functional correlates of these morphological and histochemical observations are discussed and compared to human endometrial studies.The authors wish to express their appreciation to Mrs. Y. Insua and Mrs. M. St. Onge for technical, Mr. G. Kerimian for photographic, and Ms. S. Weaver for secretarial assistanceThis research was supported by NIH contract #NO1-HD-4-2862     

Histochemical and ultrastructural properties of myoid cells in the thymus of the frog

Summary Histochemical and ultrastructural properties of myoid cells in the thymus of the frog were investigated and compared with properties of skeletal muscle fibres. The histochemical reactions of phospholipids, phosphorylase, succinic dehydrogenase and adenosine triphosphatase activities in myoid cells were characterized by considerable variability. Individual myoid cells apparently possess different enzyme activities which correspond to different stages of development, maturity and degeneration of these cells. The mature mononucleated myoid cells have similar enzymatic properties to the fast muscle fibres of the frog. This finding has been extended by ultrastructural observations. Features, typical of fast muscle fibres of the frog, e.g. the presence of the M-line, straight and narrow Z-line and well developed triads were found in the majority of mature myoid cells.     

利用GIS模拟变量施肥尺度对施肥量的影响研究

以玉米氮肥变量施肥为例。采用GIS技术分别模拟不同肥力水平、不同空间变异和不同相关距条件下变量施肥尺度对施肥量的影响．首先用“Turning Band Method”方法随机生成符合指定均值、方差和空间分布(半方差模型)的一组表示最小施肥单元的土壤养分数据,每组10~4个数据,对每组数据进行了100个等级尺度单元的相关施肥量计算和分析．模拟结果表明：(1)随着变量施肥单元面积减小,施肥量不是减少,而是增加或保持不变;(2)相同土壤肥力,空间变异越大时,随着施肥尺度增大,平均施肥量增大越明显,而且施肥临界养分过渡带的变量施肥合理性受尺度影响最大．     

A Contribution to Pollination and Fertilization of Amentotaxus argotaenta

The present investigation was conducted during 1980–1982, and mater- ials collected from Jin-Fo shan (Golden Buddha Mountain), at a height of 1400-1600 m, Sichuan province, China. Pollination of Amentotaxus argotaenia began to proceed last week of May, and came into bloom the first week of June. The male strobiles were almost entirely wilting at June 12–15. Thus, florescence of Amentotaxus spread over a period of 3 weeks. While the pollen grains approaching to maturity, most of the microspores divide to form a larger tube cell and a smaller antheridial initial. In this case the mature pollen grains of Amentotaxus consist of two cells. Then pollen grains are attracted down into the pollen chamber in the apex of the nucellus after pollination. The pollen chamber of Amentotaxus in longitudinal section looks like a flask in shape and is very much similar to that of Ginkgo biloba. As pollen grains at pollen chamber begin to germinate, the antheridial initials divide again to give rise to a spermatogenous cell and a sterile cell. At first, the spermatogenous cell is of a size only 11–13 μ in diameter. When the pollen tube reaches the middle part of the nucellus, the spermatogeneous cell is of a size about 30 μ. In the middle of July, pollen tube approaches the top of the female gametophyte. In this time, the spermatogenous cell has already been mature enough and is of 58–85 μ in diameter. The nuclei of spermatogenous ceils, 30–36 μ in size, are usually lying in the lateral side of the cytoplasm at its micropylar end. From the middle to the end of July, spermatogenous cells divide to form two unequal sperms, one of which is larger than the other and is the functional one. The large sperm is almost round in shape and about 56 μ in diameter. The small sperm is elliptic in shape, non-functional, and about 33 μ in diameter. The nuclei of the large and small sperms are about 40 μ and 26 μ, respectively. In some cases there are lateral pollen tube and sperms in the ovules of Amentotaxus, or the pollen tube even grows toward the lower part of female gametophyte in the chalazal end and there are well developed sperms in such a case. In the middle of July, nucleus of the central cell divides to form a ventral canal nucleus and an egg nucleus. The former then breaks down quickly and the latter continues to develope and moves toward the central part of the egg cell gradually. It is interesting to note that there are a number of nucleolus-like grains in the cytoplasm of the egg cell in Amentotaxus. The large nueleolus-like grains contain a larger central vacuole with several smaller vacuoles surrounding it. These grains show a positive reaction and blue colour by PAS and aniline blue black or coomassie brilliant blue, respectively. The above facts show that the nucleolus-like grains contain not only po- lysaccharides, but also protein. Similar grains may also found in the developing pollen tube. This is a unique feature in Amentotaxus and even in Gymnosperms. Otherwise, there are often two groups of the dense cytoplasm under the egg nucleus in Amentotaxus. Fertilization of Amentotaxus took place around July 20–29 (1980–1982). Interval between pollination and fertilization was about two months. After male nucleus fuses entirely with the female nucleus, the zygote begins to divide by mitosis. During fertilization, in addition that the large sperm enters the egg cell and fuses with the egg nucleus, the small sperm, tube nucleus, and sterile cell are often delivered into the egg cell. But they are disintegrated gradual]y and eventually. It is worthy to note that the nucleolus-like grains and the starches in pollen tube are also released into the egg cell. Then enlargement, fusion, and budding in the nucleolus-like grains may be found within the cytoplasm of the egg cell after fertilization. The history of investigating Amentotaxus found in 1883 has been lasting a long period of 100 years. But researches in sex production has never been studied before. The present work has shown that fertilization in Araentotaxus is very much similar to that in Taxus, Pseudotaxus, and Torreya. In other words, they all belong to the same type, that is, mitosis of zygote taking place after fusion of the two sexual nuclei. This condition constitutes one of the features of Taxaceae. But fertilization in Cephalotaxaceae is different from that of Taxaceae in having mitosis taking place before fusion of the two sexual nuclei. Pollination of Amentotaxus is similar to that of Cephalotaxus with dual-cell pollen grains at shedding stage. On the other hand, interval between pollination and fertilization in Austrotaxus lasts for 13.5 months, and this is the longest one in Taxaceae, and it is similar to that of Cephalotaxus proceeding for 14 months. To sum up, from the point of view of pollination, fertilization, and embryogenesis, Amentotaxus could be considered a primitive type in Taxaceae. Perhaps an order of systematic position of the genera belonging to Taxaceae can be arranged thus: Amentotaxus, Austrotaxus, Taxus, Pseudotaxus, and Torreya. And Cephalotaxaceae may be related to Taxaceae by way of Amentotaxus.     

Fertilization and rejection of spermatozoids by egg cells in artificially pollinated ovules ofEncephalartos (Zamiaceae)

The structure and behaviour of free female, male and proembryonal nuclei ofEncephalartos villosus Lem. were studied during a light-microscopical investigation of serially sectioned archegonia in successfully pollinated ovules. Before spermatozoids were released from the pollen tubes into the archegonial chamber, the ventral canal nucleus had disintegrated in the neck region of the egg cell among minute, amoeboid bodies with PAS-positive granules. In archegonia containing multiple spermatozoids, the egg nucleus was unobtrusive and syngamy followed by proembryo formation regularly resulted. The egg cell usually reacted violently in archegonia penetrated by a single spermatozoid. These reactions were regarded as rejection phenomena and considered as indicators that the egg cell can differentiate between compatible and incompatible male gametes.     

光处理对白头翁愈伤组织生长及代谢产物合成的影响

以白头翁试管苗叶片为外植体,于MS＋TDZ0.2 mg/L＋2,4-D 0.2 mg/L上进行愈伤组织诱导和增殖,进行不同光质照射,观察光处理下白头翁愈伤组织的生长和代谢产物合成情况.结果表明：黄光能提高愈伤组织的诱导率,产生的愈伤组织质地最好;光质对愈伤组织的鲜重增殖倍数依次为：黄（16.69）〉 红（11.41）〉 绿（8.97）〉 白（6.74）〉蓝（5.97）,延长光照时间会促进细胞生长;与白光相比,黄光和红光能显著促进干物质的积累,而蓝光明显抑制,绿光与白光两处理之间差异不明显.自然光照下,白头翁叶片中代谢产物极少,而黄、蓝和绿光均诱导合成了包括皂苷在内的数种代谢产物,而红光处理同白光一样只诱导出了一种代谢产物.由此可见,光处理可以调控白头翁愈伤组织的生长状态及皂苷等次生代谢物的积累.