烟草花发育过程中花药和花粉IAA分布规律的研究

以烟草（Nicotiana tabacumL.）花药为材料,通过4’,6-二脒基-2-苯基吲哚（DAPI）染色详细观察花粉发育过程,获得了花药发育时期与花蕾大小的对应关系;通过吲哚乙酸（IAA）单克隆抗体、免疫组织化学技术以及DR5∶∶GUS转基因植株的GUS活性对花药和花粉发育过程中生长素的分布规律进行了研究。免疫酶标记结果表明,在不同的花药发育时期IAA水平呈现出明显的差别。小孢子母细胞时期,IAA在整个花药中均有分布,并且在小孢子母细胞发育晚期,IAA信号集中在小孢子母细胞的细胞核中;随着小孢子母细胞减数分裂后形成四分体,IAA信号逐渐减弱,四分体中几乎没有信号;单核花粉期的花药中IAA信号进一步减弱,仅存在于花药壁中;待小孢子继续发育为成熟二核期时,花粉和整个花药组织中均出现较强的IAA信号。GUS活性检测结果表明,烟草DR5∶∶GUS转基因植株中花药和花粉粒的GUS信号与IAA免疫酶定位结果基本一致。总的来说,IAA在烟草花药和花粉中的积累呈现出由强到弱、再由弱到强的分布规律,暗示IAA在被子植物花药和花粉发育过程中可能起着较为重要的作用。     

小麦胞质不育系花药中脂氧合酶及抗坏血酸过氧化物酶活性的变化(简报)

在花粉单核期和二核期,K型不育系花药的脂氧合酶（LOX）活性低于而T型不育系则高于保持系,两者的抗坏血酸过氧化物酶（AsA-POD）活性均高于保持系。花粉三核期两种类型不育系的LOX活性均明显高于而ASA－POD活性则明显低于保持系。不育系花药的丙二醛（MDA）含量在各发育时期均高于保持系,三核期尤为明显。     

小麦胞质不育系花药中脂氧合酶及抗坏血酸过氧化物酶活性的…

在花粉单核期和二核期,Ｋ型不育系花药的脂氧合酶（ＬＯＸ）活性低于而Ｔ型不育系则高于保持系,两者的抗坏血酸过氧化物酶（ＡｓＡ－ＰＯＤ）活性均高于保持系。花粉三核期两种类型不育系的ＬＯＸ活性均明显于高于而ＡｓＡ－ＰＯＤ活性则明显低于保持系。不育系花药的丙二醛（ＭＤＡ）含量在各发育时期均高于保持系,三核期尤为明显。     

小麦不同发育时期花药对离体培养的反应

花药的发育时期是决定花药培养时诱导频率高低的重要因素。据文献报道,绝大多数植物只在花药中小孢子处于单核期至二核期时才对离体培养有反应(即产生愈伤组织),只有少数植物能从四分体时期的花药获得花粉胚,如烟草、毛地黄、油菜、大麦、野生二粒小麦、玉米     

小麦不同发育时期花药对离体培养的反应

花药的发育时期是决定花药培养时诱导频 率高低的重要因素。据文献报道,绝大多数植 物只在花药中小抱子处于单核期至二核期时才 对离体培养有反应（即产生愈伤组织）,只有少 数植物能从四分体时期的花药获得花粉胚,如 烟草、毛地黄、油菜、大麦、野生二粒小麦、玉米 等。也有一些作者培养过减数分裂时期的花药, 但只有Gresshoff和Doy在番茄、拟南芥菜及 葡萄[11l有过成功的报道。较晚的花药发育时期 方面,Nitzsche[1a1曾报道从多花黑麦草及苇状 羊茅杂种“将近成熟”的花药获得少量愈伤组 织,Kameya等[123曾从Brassica属的成熟花粉 获得愈伤组织,但没有分化成苗。到目前为止, 还没有一种植物能从上述各个时期的花药产生 花粉胚。     

水稻不育花药中H_2O_2的积累与膜脂过氧化的加剧

水稻7017、二九矮细胞质雄性不育系及其保持系花药的POD,CAT和SOD活性研究的结果表明,单核早期时不育及可育花药的酶活性差异不明显,单核晚期、二核及三核期的不育花药显著低于可育花药。在不育花药中缺少两条Cu-Zn SOD同工酶带,而且O_2~ 产生效率为可育的4.1～5.5倍,并有H_2O_2和MDA的积累。不育花药中H_2O_2的积累和膜脂过氧化的加剧可能与花粉败育有关。     

甘蓝型油菜隐性上位互作核不育材料1665花药败育的细胞学研究

采用石蜡切片方法,对甘蓝型油菜隐性上位互作核不育材料1665的可育株与不育株花药进行细胞学观察.结果显示:(1)不育株花药在花粉母细胞减数分裂时期出现异常,部分花粉母细胞细胞分裂相不均等分裂或分裂异常.导致部分四分体形状异常.(2)不育株绒毡层细胞在四分体时期开始生长膨大,单核花粉时期出现液泡化和巨型化,侵占药室,使得小孢子不能正常释放或无法继续发育;部分释放出的小孢子未及时形成花粉壁,阻碍花粉继续发育.不能发育形成二核期和三核期花粉,导致花药败育.     

水稻不育系与其保持系花药中的内源多胺和抗氧化代谢

7017、东野CMS两不育系花药与其相应的保持系比较,在花粉单核、二核和三核发育时期均含有较低的腐胺、精胺、工精胺、ASA和GSH含量;单核早期的ASA—POD、GR活性有所提高,但在单核晚期、二核及三核期AsA—POD、GR和6—GPDH活性明显低于保持系,并少1～3条6—GPDH同工酶带。表明不育花药有较低的内源多胺含量和较弱的抗氧化代谢效能。     

细胞质雄性不育水稻幼穗花药的呼吸酶活性研究

研究珍汕９７Ａ和珍汕９７Ｂ的雌雄蕊原基形成期、花粉母细胞形成期和花粉母细胞减数分裂期的幼穗及单核期、二核期和三核期的花药中呼吸代谢三羧酸循环（ＴＣＡ）的苹果酸脱氢酶（ＭＤＨ）和异柠檬酸脱氢酶（ＩＤＨ）及戊糖途径（ＰＰＰ）的磷酸葡萄糖脱氢酶（Ｇ６ＰＤＨ）、磷酸葡萄糖酸脱氢酶（６ＰＧＤＨ）和５－磷酸核糖异构酶（Ｒ５ＰＩ）的活性。结果表明：可育花药的５种酶活性皆高于同期不育花药;而幼穗中,ＴＣＡ途径中的     

高粱雄性不育与可育花药同工酶及游离组蛋白的比较研究

本文研究了高粱细胞质雄性不育花药、可育花药不同发育时期的COD、PPO、MDH及游离组蛋白变化特征,结果表明,在花粉母细胞减数分裂期,COD、PPO未呈现差异,但到了小孢子单核期,不育花药与可育花药间COD、PPO出现明显差异,并且这种差异一直保持至花粉粒双核—三核期。COD、PPO的变化时期与花粉败育的关键时期(小孢子单核期)相一致。不育花药与可育花药的MDH两者相同,但游离组蛋白在花药发育的不同时期均呈现明显差异。本文作者将不育花药、可育花药的COD、PPO及游离组蛋白中出现的差异归因于不育花药中的细胞质不育基因对核基因表达的调控作用。     

Microspore development inBrassica napus and the effect of high temperature on division in vivo and in vitro

Summary Brassica napus cv. Topas microspores isolated and cultured near the first pollen mitosis and subjected to a heat treatment develop into haploid embryos at a frequency of about 20%. In order to obtain a greater understanding of the induction process and embryogenesis, transmission electron microscopy was used to study the development of pollen from the mid-uninucleate to the bicellular microspore stage. The effect of 24 h of high temperature (32.5 °C) on microspore development was examined by heat treating microspore cultures or entire plants. Mid-uninucleate microspores contained small vacuoles. Late-uninucleate vacuolate microspores contained a large vacuole. The large vacuole of the vacuolate stage was fragmented into numerous small vacuoles in the late-uninucleate stage. The late-uninucleate stage contained an increased number of ribosomes, a pollen coat covering the exine and a laterally positioned nucleus. Prior to the first pollen mitosis the nucleus of the lateuninucleate microspore appeared to be appressed to the plasma membrane; numerous perinuclear microtubules were observed. Microspores developing into pollen divided asymmetrically to form a large vegetative cell with amyloplasts and a small generative cell without plastids. The cells were separated by a lens-shaped cell wall which later diminished. At the late-bicellular stage the generative cell was observed within the vegetative cell. Starch and lipid reserves were present in the vegetative cell and the rough endoplasmic reticulum and Golgi were abundant. The microspore isolation procedure removed the pollen coat, but did not redistribute or alter the morphology of the organelles. Microspores cultured at 25 °C for 24 h resembled late-bicellular microspores except more starch and a thicker intine were present. A more equal division of microspores occurred during the 24 h heat treatment (32.5 °C) of the entire plant or of cultures. A planar wall separated the cells of the bicellular microspores. Both daughter cells contained plastids and the nuclei were of similar size. Cultured embryogenie microspores contained electron-dense deposits at the plasma membrane/cell wall interface, vesicle-like structures in the cell walls and organelle-free regions in the cytoplasm. The results are related to embryogenesis and a possible mechanism of induction is discussed.Abbreviations B binucleate - LU late uninucleate - LUV late uninucleate vacuolate - M mitotic - MU mid-uninucleate - RER rough endoplasmic reticulum - TEM transmission electron micrograph     

青葙花药发育的结构和组织化学观察

对苋科植物青葙Celosia argentea花药发育的结构和组织化学（多糖和脂滴）特征进行观察。青葙小孢子发生为同时型,四分体为四面体型。药壁为典型四层,绒毡层属于同型绒毡层。成熟花粉为二胞型。早期花药中的淀粉粒和脂滴均较少,绒毡层细胞至小孢子晚期退化为体积较大的脂块。二胞花粉时期的中层细胞退化为脂滴。早期二胞花粉中先出现多糖颗粒,晚期的成熟花粉中积累大量淀粉粒和较少的脂滴为营养储存物。     

洋葱花药发育中钙离子分布特征

采用焦锑酸钾沉淀钙离子技术,对洋葱（Alliumcepa）花药发育中Ca^2＋分布进行了研究。在小孢子母细胞时期,小孢子母细胞中的钙沉淀颗粒很少,但绒毡层细胞的内切向壁已出现明显的钙沉淀颗粒。在四分体时期,四分体小孢子的胼胝质壁中出现较多的钙沉淀颗粒;绒毡层细胞内切向壁的钙沉淀颗粒消失,而在外切向壁和径向壁部位的钙沉淀颗粒增加。在小孢子早期,小孢子中也出现了钙沉淀颗粒,而绒毡层细胞内切向壁表面出现了很多絮状物,其上附有细小钙沉淀颗粒。到小孢子晚期,小孢子中出现一些小液泡,细胞质中的钙沉淀颗粒有所下降。此时绒毡层细胞已明显退化,但在绒毡层膜上仍有一些乌氏体和钙沉淀颗粒。在二胞花粉早期,营养细胞中的液泡收缩、消失,细胞质中又出现了较多的钙沉淀颗粒,在质体和其内部的淀粉粒表面上附有较多的钙沉淀颗粒。到二胞花粉晚期,花粉中的钙沉淀颗粒已明显下降,仅在花粉外壁中还有一地钙沉淀颗粒．     

Observation of Androgenesis in Cultured Wheat Anthers at Meiosis,Tetrad, Early Uninucleate and Trinucleate Stage

The obtaining of calluses and plantlets from cultured wheat anthersat the stages from pollen mother cell to trinucleate microspore has been reported previously. Haploids as well as diploids existed among the regenerated plantlets derivedfrom anthers at these stages. Present paper reports the study on androgenesis patter-ns of cultured anthers at meiosis, tetrad, early mid- and late uninucleate and trinucleate stage. Cytological evidence of pollen-origin of calluses produced by anthers atthese stages was given. Observation showed that meiosis of wheat anthers was able tocomplete under culture conditions, resulting in releasing microspores, from which multinucleate and multicellular pollen grains formed. In meiosis anthers, abnormal cells,including syncytium and two kinds of binueleate calls were sometimes observed. Theymight be products of abnormal meiosis and abnormal development of tapetum cells. Itwas noted that failure and/or uncomplction of forming callus wall and/or pollen wallin in vitro anthers at meiosis, tetrad and early uninucleate stage occured often. Itmight lead to the low frequency of callus induction. Mature wheat anthers (trinucleate stage) contained both normal and abnormal pollen grains (pollen dimorphism); onlythe abnormal pollen grains developed into embryoids while all the normai trinucleatepollen grains degenerated rapidly. However, the date of the frequency of equal divisionof microspores suggested that abnormal pollen (N pollen, small pollen) could not be theonly source of androgenic pollens in cultured anthers at late uninucleate and other earlier stages.     

A short severe heat shock is required to induce embryogenesis in late bicellular pollen of Brassica napus L.

 Until now it has been considered that in rape seed (Brassica napus) only late uninucleate microspores and early bicellular pollen are competent for induction of in vitro embryogenesis. Here we describe that pollen isolated at the late bicellular stage can also be induced to undergo embryogenesis. By the application of an additional short and more severe heat stress treatment, DNA synthesis was initiated in both generative and vegetative nuclei, but only vegetative cells were able to complete the cell cycle and to divide further. The ability of late bicellular pollen to respond to embryogenic induction treatment was accompanied by rearrangements of the microtubulular cytoskeleton and by the nuclear localization of 70 kDa heat shock proteins (HSP70). These findings confirm earlier observations that there is a strong correlation between the induction of embryogenesis and the synthesis and nuclear localization of HSP70. Received: 9 January 1997 / Revision accepted: 23 May 1997     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.     

Distribution of insoluble polysaccharides, neutral lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. (Bignoniaceae)

In this study, distribution of polysaccharides, lipids, and proteins in the developing anthers of Campsis radicans (L.) Seem. was examined from sporogenous cell stage to mature pollen, using cytochemical methods. To detect the distribution and dynamic changes of insoluble polysaccharides, lipid bodies, and proteins in the anthers through progressive developmental stages, semi-thin sections of anthers at different developmental stages were stained with periodic-acid-Schiff (PAS) reagent, Sudan black B, and Coomassie brilliant blue, respectively, and examined under light microscope. Ultrastructural observations with TEM were also carried out to determine the storage form of starch in the connective tissue, and storage form of lipids in the tapetal cells. In sporogenous cell stage, anther wall contains numerous insoluble polysaccharides. However, from the sporogenous cell stage to the vacuolated microspore stage, the amount of insoluble polysaccharides in the anther wall decreases gradually. At bicellular pollen stage, tapetum degenerates completely and polysaccharides are not seen in the anther wall. Lipid bodies are observed in the cytoplasm of both middle layer and tapetal cells at tetrad stage, whereas they disappear in the vacuolated microspore stage. Compared with polysaccharides, proteins are limited in the anther wall at early stages of development. During pollen development, polysaccharides, proteins, and lipid bodies are scarce in the cytoplasm of sporogenous cells, but their amount increases at premeiotic stage. From tetrad stage to bicellular pollen stage, microspore cytoplasm contains variable amount of insoluble polysaccharide grains, lipid and protein bodies. At bicellular pollen stage, plentiful amount of starch granules are stored in the cytoplasm of the pollen grains. Proteins and lipid bodies are also present in the cytoplasm.     

Pollen Cryopreservation and Pollen Protoplast Isolation in Brassica carnpestris var.purpurea

The authors have investigated the factors affecting pollen cryopreservation in Brassica campestris var. purpurea, such as pollen development stages cryoprotectant and the process of freezing. A suitable procedure was established as follows :Pollen grains suspended in B5 medium containing 10X DMSO and 1SM sucrose were frozen by a three- –1℃/min – 1 ℃/min step method(0℃———→–10 ℃ ,standing for 15 min———→–40 ℃ , standing for 1 hr→liquid nitrogen)and later thawed in 40℃ water bath. During a period of 60, 90 days′preservation, the relative survival percentage of mature (at the day of anthesis)and nearly mature(2 days before anthesis, trinucleate stage)pollens maintained at ca. 91% that of young pollens(7-8 days before anthesis, late uninucleate stage to early binucleate stage)slightly declined from the original 91.6% to 84. 3%. Culture. experiment showed that the cryopreserved young pollen could be induced to cell division just as well as the fresh pollen. The method of isolating protoplasts from fresh mature pollen developed previously was improved and simplified. As a result, protoplasts were isolated more conveniently from mature pollen and young pollen for the first time. The protoplasts from cryopreserved mature and young pollen could be obtained as well with an isolation rate of 77.4% and 35.9% respectively. However, for isolation of protoplasts from preserved young pollen, an incubation in NLN medium at 35℃ after thawing was necessary.     

芡实绒毡层细胞发育的超微结构变化

芡实（ Ｅｕｒｙａｌｅｆｅｒｏｘ Ｓａｌｉｓｂ） 绒毡层细胞在小孢子母细胞时期, 质体出现明显的变形期,细胞中二核常相互贴近或呈嵌合状态, 细胞壁间层中胞间连丝发达。减数分裂期, 绒毡层细胞壁融解消失, 胞间连丝断离, 细胞间发育出现不同步现象。质体开始积累淀粉, 部分质体呈空泡状, 并出现质体膜内陷, 这与液泡具相似的功能。四分体时期, 绒毡层细胞内部结构开始解体。单核小孢子时期, 绒毡层细胞解体消失, 使小孢子后期发育的营养来源受到影响,作者认为这是生产上成熟花粉囊中花粉粒少而且发育不正常的主要原因之一。     

Anther culture and cold treatment of floral buds increased symmetrical and extra nuclei frequencies in soybean pollen grains

Androgenic response is characterized by a multinucleate or multicellular stage of pollen development. Histological sections stained with toluidine blue and squashes in propionic-carmine and in 4-6-diamidino-2-phenylindole (DAPI) were used for serial observations (0, 14 and 28 days) in soybean pollen grains from cultured anthers and floral buds submitted to cold treatment at 4 °C. In a total of 62,536 pollen grains, it were observed general averages of 2.06% of pollen grains with two symmetrical nuclei and of 1.41% pollen grains with typical extra nuclei (i.e. additional nuclei with typical morphology). Symmetrical and extra nuclei frequencies increased in both treatments but only the number of pollen grains with typical extra nuclei increased significantly with time of exposure to treatments. In addition, 8.59% of multinucleate pollen grains were recorded with atypical nuclei, smaller than vegetative or generative-types and with a fragmented shape. The frequency of these grains increased significantly with time of exposure to treatments. Thus, soybean multinucleate grains occurrence was not an exclusive response to culture. These preliminary results point to the need of further studies to clarify the relationship between typical and fragmented extra nuclei with both androgenesis and programmed cell death.