Dynamic of polysaccharide and lipid in the developing microsporangiums of Chinese fir (Cunninghamia lanceolata)

Microsporongium development in Chinese fir (Cunninghamia lanceolata) was investigated using cytochemical methods with a special attention to the fluctuations (in amount and distribution) of polysaccharide and lipid reserves along the development of the microsporangium. Semi-thin sections of microsporangia at different developmental stages were stained with periodic acid–Schiff (PAS) reagent and Sudan Black B to detect insoluble polysaccharides and neutral lipids, respectively. In young microsporangia, microspore mother cells began to accumulate starch grains and lipids, which disappeared during microspore development. Following microspore division, the starch grains present in bicellular pollen disappeared and abundant lipid deposits were accumulated. In mature pollen, only abundant lipids accumulated as storage material. The pollen wall of C. lanceolata is predominantly composed of polysaccharidic intine, and the sporopollenin-containing exine is weakly developed and only forms a thin layer covering the intine.     

Study of male sterility in Taiwania cryptomerioides Hayata (Taxodiaceae)

Summary. A study of male sterility over a period of three consecutive years on a conifer species endemic to Taiwan, Taiwania cryptomerioides Hayata (Taxodiaceae), was done for this article. With the aids of fluorescence and electron microscopic observations, the ontogenic processes in the fertile and sterile microsporangia are compared, using samples collected from Chitou Experimental Forest and Yeou-Shoei-Keng Clonal Orchard of the National Taiwan University, Nantou, Taiwan. The development of male strobili occurred from August to the end of March. Microsporogenesis starts with the formation of the archesporium and ends with the maturation of 2-celled pollen grains within the dehiscing microsporangium. Before meiosis, there was no significant difference in ultrastructure between the fertile and sterile microsporangia. Asynchronous pollen development with various tetrad forms may occur in the same microsporangium of either fertile or sterile strobili. However, a callose wall was observable in the fertile dyad and tetrad, but not in the sterile one. After dissolution of the callose wall, the fertile microspores were released into the locule, while some sterile microspores still retained as tetrads or dyads with intertwining of exine walls in the proximal faces. As a result, there was no well developed lamellated endexine and no granulate ectexine or intine in the sterile microspores. Eventually, the intracellular structures in sterile microspores were dramatically collapsed before anthesis. The present study shows that the abortion in pollen development is possibly attributed to the absence of the callose wall. The importance of this structure to the male sterility of T. cryptomerioides is discussed. Correspondence and reprints: Department of Life Science, National Taiwan University, 106 Taipei, Taiwan.     

STUDIES IN THE BIGNONIACEAE. I. ONTOGENY OF DIMORPHIC ANTHER TAPETUM IN PYROSTEGIA

Development of the anther wall was studied with special reference to the tapetum in Pyrostegia ignea. The archesporium in each microsporangium is horseshoe-shaped. The inner tapetum develops earlier from the vegetative cells of the connective region while the outer differentiates a little later from the parietal layers. Thus, the tapetum has a distinct dual origin. The two tapetal layers exhibit a pronounced structural dimorphism. Sometimes, sterile septae, partitioning the sporogenous tissue, develop in microsporangia. A prominent membrane with Ubisch granules (orbicules) is organised on the inner tangential surface of the tapetal protoplasts facing the uninucleate microspores.     

POLLEN AND TAPETUM DEVELOPMENT IN DESMODIUM GLUTINOSUM AND D. ILLINOENSE (PAPILIONOIDEAE; LEGUMINOSAE)

Each of the four microsporangia has three or four wall layers, a uninucleate tapetum of various cell shapes with nuclei that remain in prophase, and 12-24 pollen mother cells (PMCs). A sterile transverse septum sometimes bisects the microsporangium. PMCs secrete callose but not uniformly, and contact among them continues through meiosis. Simultaneous cytokinesis by furrowing isolates each microspore in callose, which later disperses. The separated microspores become vacuolate, undergo mitosis to become pollen, and later become filled with food reserves. Endothecial wall thickening and tapetal dissolution occur after pollen engorgement. Calcium oxalate crystals form in tapetal cells during the sporogenous stage, reach maximum size during early meiosis, and remain prominent until tapetal dissolution.     

The Development of Microsporangium and Microspore in Azolla

This paper presents a detailed report on the developmental progresses of the microsporangium and its microspores in Azolla filiculoides Lam., and shows the morphologicaI structures of the respective developmental stares with the aid of scanning electron photographs. The entire developmental progress may be divided into six stages: ( 1 ) The microspore mother cell initiating stage The microsporangium initial on the placenta of the sporocarp gave rise a sporogenous cell, and then divided four times to form sixteen microspore mother cells; (2) The meiotic stage–The microspore mother cells initiated meiosis inside their calIose walls. The radial and inner tangential walls of the tapetum were dissolved at the same time and followed by the formation of a sporoplasmodium; (3) The microspore shrinking Ⅰ–After the callose walls of tetrads was dissolved, those microspores that just released from the callose walls shrunk intensely and became spherical later again. The sporoderm of microspores was principally synthesized in this stage, and the volume of microspores became evidently increased. The microspores then gradually moved to the periphery of the sporoplasmodium; (4) The microspore shrinking Ⅱ-Each microspore formed a large vacuole and gave rise the second contraction. The periphery of the sporoplasmodium was gradually dissolved; (5) The massulae forming stage–The sporoplasmodium was dissolved successivelly, and the undissolvable granules and organelle membrane residues. became aggregated into the compartmental layer, and the microsporangium was divided into several large vesicles, each vesicle will form a massulae; (6) The microspore germinating stage–The ,natured microspores inside the massulae each gave rise an androgonial initial which divided two times to form four antherozoid mother cells and then gave rise the antherozoids. The relationships between the various morphological structures and their functions in the microsporangium developmental progress have breify discussed. In addition, our viewpoints have compared with those of previous investigations.     

Characterization of callase (β-1,3-d-glucanase) activity during microsporogenesis in the sterile anthers of Allium sativum L. and the fertile anthers of A. atropurpureum

We examined callase activity in anthers of sterile Allium sativum (garlic) and fertile Allium atropurpureum. In A. sativum, a species that produces sterile pollen and propagates only vegetatively, callase was extracted from the thick walls of A. sativum microspore tetrads exhibited maximum activity at pH 4.8, and the corresponding in vivo values ranged from 4.5 to 5.0. Once microspores were released, in vitro callase activity peaked at three distinct pH values, reflecting the presence of three callase isoforms. One isoform, which was previously identified in the tetrad stage, displayed maximum activity at pH 4.8, and the remaining two isoforms, which were novel, were most active at pH 6.0 and 7.3. The corresponding in vivo values ranged from pH 4.75 to 6.0. In contrast, in A. atropurpureum, a sexually propagating species, three callase isoforms, active at pH 4.8-5.2, 6.1, and 7.3, were identified in samples of microsporangia that had released their microspores. The corresponding in vivo value for this plant was 5.9. The callose wall persists around A. sativum meiotic cells, whereas only one callase isoform, with an optimum activity of pH 4.8, is active in the acidic environment of the microsporangium. However, this isoform is degraded when the pH rises to 6.0 and two other callase isoforms, maximally active at pH 6.0 and 7.3, appear. Thus, factors that alter the pH of the microsporangium may indirectly affect the male gametophyte development by modulating the activity of callase and thereby regulating the degradation of the callose wall.     

越南篦齿苏铁小孢子发生及其系统学意义

运用常规石蜡切片方法,结合显微荧光技术对越南篦齿苏铁Cycas elongata 小孢子发生和花粉个体发育进行了研究。结果表明：其小孢子叶球5月中下旬开始萌动,小孢子囊着生在小孢子叶远轴面,且3-5小孢子囊以辐射状排列方式聚生成聚合囊。小孢子囊壁由6-7层细胞组成,包括表皮、中层及绒毡层。绒毡层来源于成熟造孢组织的外围细胞,其退化形式为分泌型。6月中旬,小孢子母细胞进入减数分裂I,至6月下旬形成四分体。母细胞减数分裂后胞质分裂的方式与其他苏铁类植物不同,具有连续型与同时型两种类型。7月中旬,小孢子经过2次有丝分裂后,形成3细胞的成熟花粉粒。7月下旬进入散粉状态。在花粉发育过程中,母细胞内淀粉粒的积累及其壁上胼胝质的沉积均呈现规律性变化。     

Expression of ogu cytoplasmic male sterility in cybrids of Brassica napus

Summary A light and electron microscopic investigation revealed that ogu cytoplasmic male sterility (CMS) in cybrids of Brassica napus is primarily a deficiency of the tapetum and clearly time and site specific. Three patterns of ogu CMS were found, and specific conclusions drawn. First, the partially male fertile cybrid 23 was highly variable. It sometimes produced heterogeneous stamens with an endothecium formed exclusively around the fertile locules, thus delineating each microsporangium as a functional unit. The second type, including cybrids 27, 58 and 85, on the contrary, was stable and completely male sterile. In the four locules of normal length, microspores were observed to die at the vacuolate polarized stage while the tapetum disappeared prematurely through excessive vacuolization by the end of meiosis followed by a rapid autolysis during the tetrad or early free microspore stage. The subepidermal layer of the locule wall failed to form characteristic thickenings. The male-sterile stamens were completely indehiscent. At the time of anthesis they contained only collapsed empty exines adhering to each other. These cybrids, 27, 58 and 85, were closest to the ogu CMS trait of radish and seemed to be the best suited for further use in plant breeding. The third pattern was found in cybrids 77 and 118, which besides showing abortion of the microsporangia also showed a feminization of the stamens. We suggest that this feminization might be due to an alloplasmic situation associating Brassica napus nuclear genes with the mitochondrial DNA of radish.     

Evolution of microsporangium numbers in Microseris (Asteraceae: Lactuceae)

The genus Microseris contains species with disporangiate stamens and species with tetrasporangiate stamens. We determined the number of microsporangia per stamen in serial sections of heads from all 13 species of Microseris, its close relative Uropappus lindleyi, and the two allopolyploid species of Slebbinsoseris. Four Microseris species, three diploid and one tetraploid, have two microsporangia per stamen; all other species investigated have four. The most parsimonious assumption is that the disporangiate condition is derived and arose once in the evolution of Microseris. The inheritance of the number of microsporangia per stamen in crosses between M. bigelovii (disporangiate) and M. douglasii (tetrasporangiate) was determined. Segregation of microsporangium number per stamen in F2s derived from these crosses is quantitative rather than Mendelian. The average number of microsporangia per stamen in the F2 plants ranges from 2.0 to 4.0. There is a predominance of tetrasporangiate stamens in the F1 and in most F2 plants. The observed pattern of inheritance suggests a major gene with dominance and quantitative modifiers.     

矮牡丹小孢子发生和雄配子体发育及其与该种濒危的关系

研究了矮牡丹Paeonia jishanensis Hong et W．Z．Zhao的小孢子发生及雄配子体的形成。矮 牡丹花药具4个小孢子囊,药壁结构属双子叶型,腺质绒毡层,小孢子母细胞减数分裂后胞质分裂为 同时型,四分体多为四面体形,少左右对称形,成熟花粉为2-细胞。对芍药属木本类型的雄性发育进行 了全面研究,还对小孢子母细胞减数分裂和单核小孢子发育时期的异常现象进行了观察,对能育花粉 与不育花粉的百分比进行了测定,结果表明,能育花粉为45.03％～84.18％,它们在不同花中,不同花 药中,甚至同一花药的不同花粉囊中表现都不完全一致。联系矮牡丹的致濒原因进行了讨论,认为雄配子体形成过程中的异常现象,并不是导致矮牡丹濒危的主要因素。     

Formation of microspores and development of male gametes in Paeonia jishanensis,with an analysis of factors of endangerment of this entity

Investigated in the present work were development of microspores and formation Of male gametes in Paeonia jishanensis T．Hong et W．Z．Zhao)．Its anthers are 4-sporran giate;structure of anther wall is of the Dicotyledonous type,with glandular tapetum;cy tokinesis at meiosis of microspore mother cells is simultaneous;tetrads are mostly tetrahe dral,less frequently isobilateral,and mature pollen grains are 2-celled． The overall observa tion on formation of microspores and development of male gametes made in this work is the first for woody peonies．In addition,we observed in the present work abnormal phenomena at meiosis of microspore mother cells and uninucleate microspores,and also made measure ments of fertility of pollen grains．The results show that their fertility ranges from 45.0％ to 84.2％ and varies among flowers,among anthers in a flower and among microsporangia in an anther． The abnormal processes found in development of male gametes are not considered as an important factor responsible for the endangerment of the species．     

Ornamentation ofIsoetes (Isoetaceae, Lycophyta) microspores

More than any other taxonomic character, megaspores have been used in the genusIsoetes (known by the English common name of “quillwort”), despite the fallacy of a single-character taxonomy. Microspores, on the other hand, have been largely neglected in taxonomic schemes. Like megaspores, terms for microspore ornamentation (also known as “sculpturing”) have not been standardized. I examined microspore ornamentation, including both macroornamentation and microornamentation, of 52 taxa from Africa, Asia, Australasia, Europe, North America, and South America with the scanning electron microscope. Macroornamentation is discernible with light microscopy; microornamentation requires scanning electron microscopy. Ornately sculptured spores were much more frequent than were laevigate or psilate patterns: 21 taxa had an echinate pattern; 19 had an aculeate pattern; 6 were cristate; 5 were psilate; and 1 was laevigate. The proximal and distal ridges and surfaces may vary in both the type and density of ornamentation. Distinct macroornamentation patterns characterize certain species groups. Microornamentation types include granulate, bacillate, fimbriate, and filamentose: of the microspores I examined, virtually all were partially granulate; 11 were bacillate; 4 were fimbriate; and 1 was filamentose. Based on this limited sampling, species with a higher ploidy level often have larger microspores, but no clear relationship between microspore ornamentation and ploidy level was established, nor were any geographical or ecological trends clear. Like megaspores, microspore ornamentation is strongly convergent. Although microspores are often attached to megaspores, the role of spore ornamentation in coordinated dispersal remains unclear.     

罗汉松雄球花及其雄配子体发育的形态结构

该研究采用光学显微镜和扫描电镜,观察罗汉松雄球花、小孢子及其配子体发育过程的形态结构特征,以揭示罗汉松小孢子的发生和雄配子体的发育规律,为罗汉松的生殖和杂交组合提供胚胎学证据。结果发现：(1)罗汉松花芽于每年的7月开始分化,至次年5月花粉成熟散粉,雄球花由单生的卵圆形转为2～3个葇荑花序并生,小孢子叶螺旋状着生于圆柱状的花序轴上,每一小孢子叶远轴面基部并列着生2个小孢子囊。(2)小孢子囊壁发育过程中由外及里出现各由1层薄壁细胞组成的表皮、药室内壁、中层和绒毡层,至散粉前,后两者基本被分解吸收。(3)同一小孢子囊内的造孢细胞发育在时间上存在差异,小孢子母细胞减数分裂后形成的四分体有四面体型和十字交叉型两种排列方式,成熟的雄配子体包括生殖细胞和粉管细胞,发育过程中出现的第一和第二原叶细胞大部分被分解消失。(4)电镜下罗汉松花粉粒为典型的松花型花粉,两侧各具1个气囊,远极面具一萌发沟,花粉粒表面具纹理或皱褶。     

珍稀濒危植物大花万代兰的花粉团发育及其分类学意义

万代兰属的属间界限划定及其亲缘关系重建是兰科分类系统中的难解之谜。该研究采用常规石蜡切片技术观察了珍稀濒危植物大花万代兰的一对深裂花粉团的形成机制、花药壁发育模式、小孢子发生及雄配子体发育等的胚胎学特征。结果表明：(1)大花万代兰早期的花药原基分化出一对侧生药室,每个药室的小孢子囊中央分化出一条在花药成熟时会降解的不育隔膜组织,形成两个不等深裂的花粉团。(2)发育完整的花药壁有5~9层,包括2~6层药室内壁,符合多层型花药壁发育类型;绒毡层细胞为单核,腺质型,在花药成熟时,表皮、中层和绒毡层皆降解,仅留下2~6层纤维性加厚的药室内壁。(3)小孢子母细胞经过连续型胞质分裂形成正四面体和左右对称的小孢子四分体,小孢子四分体继续保持在同一个胼胝质内,完成有丝分裂形成了2 细胞型的四合花粉;四合花粉两两紧密排列,且由于隔膜组织的降解,最终发育为一对深裂的花粉团。根据现有兰花花药发育资料,分析了大花万代兰花粉团发育的胚胎学特征的分类学意义,为万代兰属错综复杂的系统分类提供了新资料。     

Genetic manipulation of microspores and microspore-derived embryos

Summary Recent advances in plant cell and molecular biology have furthered the genetic manipulation of many plant species and advanced the options for crop improvement. Among the many targets for genetic manipulation, microspores offer several unique advantages: they are haploid, single-celled, and highly synchronized. In many plant species microspores develop into haploid embryos, and eventually haploid and doubled haploid plants, after in vitro anther or microspore culture. This induced in vitro developmental pathway of microspores, termed microspore embryogenesis, can be used to recover individual homozygous plants from microspores and microspore-derived embryos after genetic manipulation such as mutagenesis and gene transfer. The highly efficient microspore embryogenesis system inBrassica napus has been used successfully to obtain various mutants after microspore mutagenesis, and to achieve gene transfer mediated byAgrobacterium tumefaciens. Presented in the Session-in-Depth In Vitro Gametophyte Biology at the 1991 World Congress on Cell and Tissue Culture held in Anaheim, California, June 16–20, 1991.     

Ultrastructure and development during meiosis and the tetrad period of sporogenesis in the leptosporangiate fern Alsophila setosa (Cyatheaceae) compared with corresponding stages in Psilotum nudum (Psilotaceae)

The pre-meiotic, meiotic and tetrad stages of development in microsporangia of Alsophila setosa were studied with particular emphasis on the early establishment of patterning in the microspore wall and the subsequent development of the sporoderm. The data obtained were compared with corresponding ontogenetic stages of Psilotum nudum. Tapetal behaviour was also examined. During the tetrad period, only one layer, a thin undulating sheet, appeared alongside the plasma membrane of the tetraspores, and this was evidently formed on a pre-patterned structure – a fibrillar layer, corresponding to a kind of primexine matrix. The early free microspores had a wavy plasma membrane with a parallel, sinusoidal, thin initial sporoderm layer. The proximal apertural fold was observed to be an extended outgrowth of this initial spore envelope. Sporoderm ontogeny during the tetrad period in Alsophila and Psilotum show some common points, but also fundamental differences, mainly in the relative timing of events: in Alsophila the end of the tetrad period is the starting point for exospore development, whereas in Psilotum the exospore is already complete at this stage. Considerable differences were also observed in the tapetum of the two species.     

Pro-embryos induction from <Emphasis Type="Italic">Olea europaea</Emphasis> L. isolated microspore culture

Studies were undertaken with one olive (Olea europaea L.) cultivar to identify buds with microspores competent to embryogenesis in vitro. Isolated microspore cultures were performed for the induction of gametic embryogenesis. Different pollen development stages and stress conditions (heat or cold shock) were evaluated. The correlation of inflorescence, anther morphology and the suitable stage of microspore development were analysed. The morphology of responsive buds was identified which corresponded with microspores from the late uni-nucleate to early bi-nucleate pollen stages. Symmetrical divisions of microspores as well as resulting multinucleate structures and pro-embryos were observed. In this paper, a new method of isolated microspore culture that leads to cell division and pro-embryos in olive, is reported.     

Inhibition of ethylene biosynthesis enhances embryogenesis of cultured microspores of Brassica napus

Procedures that induce microspore embryogenesis have been described for a range of Brassica species, but embryo yield remains low for a number of genotypes. We have carried out experiments with the microspores from a weakly responsive line of B. napus to determine the culture conditions that optimize their in vitro embryogenesis by treating them with effectors of ethylene synthesis and action. The results revealed that isolated microspores subjected to an initial heat stress in a medium supplemented with inhibitors of ethylene synthesis such as AVG and CoCl₂ exhibited significantly increased embryo yields. This suggested that regulatory effects are exerted by the ethylene produced by the isolated microspores on the early processes of gametogenesis. As a consequence, treatment of microspores with SAM, an ethylene synthesis precursor, or with the ethylene-releasing agent ethephon, led to decreases in embryo yield. A special response to ethylene during the early stages of microspore development was finally shown to occur through experiments where isolated microspores were treated for increasing periods of time with CoCl₂. Collectively, our data demonstrated that the induction of embryogenesis induced by heat stress can be enhanced by inhibitors of ethylene biosynthesis.     

MALE STERILITY IN SOYBEAN (GLYCINE MAX). I. PHENOTYPIC EXPRESSION OF THE ms2 MUTANT

The effects of a nuclear male-sterile mutant (ms2) of soybean, Glycine max (L.) Merr., on anther development were analyzed by means of light- and electron-microscopy. The structure of microspore mother cells (MMCs) in male-sterile plants was identical to that of male-fertile plants. Meiosis was completed, and tetrads of microspores formed. Microspores degenerated after the deposition of primexine and probacullae. The sheath of callose surrounding microspores did not dissolve. No structural abnormalities of the microspores were detected before the onset of degeneration. The tapetal and anther wall layers were characterized by aberrant development. Tapetal abnormalities included premature vacuolation, a persistent inner tangential cell wall, failure to differentiate normal concentrations of endoplasmic reticulum and dictyosomes, disruption of plastids, and premature degeneration. Malfunction of the tapetal layer preceded, and may have induced, microspore degeneration. Gross anther morphology was not influenced until advanced stages of development.     

A high throughput <Emphasis Type="Italic">Brassica napus</Emphasis> microspore culture system: influence of percoll gradient separation and bud selection on embryogenesis

Microspore culture for the purpose of developing doubled haploid plants is routine for numerous plant species; however, the embryo yield is still very low compared with the total available microspore population. The ability to select and isolate highly embryogenic microspores would be desirable for high embryo yield in microspore culture. To maximize the efficiency of canola microspore culture, a combination of bud size selection and microspore fractionation using a Percoll gradient was followed. This approach has consistently given high embryo yields and uniform embryo development. Microspores isolated from buds 1.5 to 4.4 mm in length of Brassica napus genotypes Topas 4079, DH12075, Westar and 0025 formed embryos at different frequencies. The most embryogenic bud size range varied with each cultivar: Topas 4079 3.5–3.9 mm, DH12075 2.0–2.4 mm, and Westar and 0025 2.5–2.9 mm. When the microspores from 2.0 to 2.4 mm buds of DH12075 were carefully layered on top of a discontinuous Percoll gradient of 10, 20 and 40%, and subsequently spun through the Percoll layers by centrifugation, bands were formed containing populations of microspores of uniform developmental stage. The middle layer of the gradient contained the late uninucleate and early binucleate microspores that were the most embryogenic. In addition, the relationship between the bud size, developmental stage of isolated microspores, Percoll gradient concentration and the embryogenic frequency of each cultivar were studied. Optimization of these factors is required for each genotype evaluated.