Anther development, microsporogenesis and microgametogenesis in Heliconia (Heliconiaceae, Zingiberales)

Anther development, microsporogenesis and microgametogenesis in several species of Heliconia were investigated as part of a complementary embryological study of the Heliconiaceae. All studied Heliconia species present bithecate and tetrasporangiate anthers with fertile pollen grains; only H. rivularis, a natural hybrid, presented sterile pollen grains of variable size and no content. The anther wall has an uniseriate epidermis and endothecium, the latter with helicoidal thickenings, although some cells of the middle layers also showed thickenings; the biseriate tapetum is of amoeboid non-syncytial type, since the tapetum cells did not fuse together forming a true plasmodium. The microsporogenesis is successive leading to isobilateral tetrads. The inaperturate pollen grains had a very reduced exine consisting of a thin, more or less continuous layer with small spines upon; the pollen grain shape is variable among the species, all of them presenting heteropolar pollen, except H. angusta with isopolar ones. Most of these characteristics were shared with other studied Zingiberales, although more studies need to be done.     

New acritarch species from Holocene sediments in central West Greenland

Anther development, microsporogenesis, and microgametogenesis were studied using both light and TEM microscopy in the six accessible subdioecious/cryptically dioecious species of Consolea (Cactaceae). Anther wall development, microsporogenesis, and microgametogenesis are uniform in staminate flowers of all six species, and are typical for Cactaceae. Breakdown of microsporogenesis in male‐sterile anthers occurs early, at the onset of meiosis, and results in anthers bearing no pollen grains. The abortive process follows a common pattern in all investigated species. The tapetum is the first layer to deviate from normal male‐fertile anther development. Tapetal cells in male‐sterile anthers elongate at an early stage and have abundant rER with atypical configurations. Ultimately, the tapetum becomes hypertrophied and non‐functional. Male‐sterility in pistillate flowers appears to be directly related to these anomalies. In addition, other anther layers and tissues are affected, and normal patterns of programmed cell death (PCD) are disrupted. The relationship between these patterns and the pattern of PCD in normal male‐fertile anthers is discussed. We hypothesize a single origin for the cryptically dioecious/subdioecious breeding system of Consolea based on the uniformity of the anther's abortive processes in pistillate flowers.     

濒危植物水松小孢子发生和雄配子体发育的研究

水松(Glyptostrobus pensilis)是我国特有的单种属孑遗植物,是极度濒危物种,被列为国家一级重点保护植物。为了从生殖生物学方面探讨水松的濒危机制,采用石蜡切片法对水松小孢子发生及雄配子体发育过程进行了系统地观察研究。结果表明:水松雄球花于10月中旬开始分化,11月初小孢子囊壁形成,12月初小孢子母细胞形成,12月中旬减数分裂。翌年1月中旬形成四面体形和左右对称形四分体,1月下旬四分体解体,释放出游离小孢子。2月中旬花粉粒发育成熟,并以二细胞形态进行传粉,散粉期约为2周左右。3月萌发花粉管,3月下旬出现精原细胞、管核和不育核,5月下旬花粉管到达颈卵器顶部,精原细胞分裂成两个精细胞。水松小孢子和雄配子体发育过程中败育和变形现象很少,据此我们认为,水松小孢子发生与雄配子体发育正常,不是致其濒危的主要原因。     

A Fluorescence Staining-Methyl Salicylate Clearing Technique for Demonstrating Pollen Nuclei

Recently several DNA-binding fluotochromes have been used for demonstrating pollennuclei. However, the autofluorescence of pollen wall often obscured the fluorescence of nuclei, thus limited the use of this method. Methyl salicylate (MS) as a clearing agent has shownexcellent effect for observing embryo sac in whole-mounted ovules. This aroused me to trya combination of fluorescent staining with MS clearing in orded to make a better demonstration of the pollen nuclei. Mature 2-celled or 3-celled pollen of several angiosperm species stained with Hoechst 33258(H33258) and cleared (via ethanol dehydration) with MS showed clearcut fluorescence oftheir generative or sperm nuclei and vegetative nucleus. MS greatly decreased the wall fluorescence and increased the transparency of the pollen contents, meanwhile maintained the H33258stained fluorescence, consequently made the nuclei brighter under a darkened background. For example, in sunflower pollen a pair of elongated and winding sperm nuclei whichcould not be identified after simple H33258 staining were quite visible after MS clearing, inartificially germinated pollen tubes, the locomotion of nuclei from pollen grain into the tube,the sequence of generative and vegetative nucle travelling along the tube and the division of generative nucleus into two sperm nuclei could be well followed by this method. The present technique may be adoptable for observations on the processes of microsporogenesis and male gametophyte development, and rogenesis in cultured anthers, and also possiblyfor tracing the nuclear events during pollination-fertilization.     

Microsporogenesis, Pollination, Pollen Germination and Male Gametophyte Development in Taxus brevifolia

Taxus brevifolia(Nutt.), commonly known as Pacific or westernyew, is a conifer native to the Pacific northwest of North America.Contrary to other Taxus species, T. brevifolia staminate strobiliare usually located on 2-year-old foliage although they mayoccur on foliage from 1 to 5-years-old. This delayed staminatestrobilus development may be an adaptation to the low lightenvironment where T. brevifolia grows. Microsporogenesis occurredin the autumn preceding pollination. Successive divisions producedisobilateral tetrads visible as early as mid-October. Over-winteringstaminate strobili usually contained separate microspores. In1996 to 1999, pollination occurred in March and April in twonatural forest sites on southern Vancouver Island, British Columbia,Canada. The low amounts of airborne pollen and prolonged pollinationperiod indicated low pollination success within T. brevifolia.Female receptivity was measured by the presence of a pollinationdrop. Protandry up to 18 d was observed. In vitro pollen germinationwas moderate to good, ranging from 65 to 88% depending on thetree and year. DAPI fluorescence staining showed successfulmale gametophyte development in vitro. The microspore dividedforming a tube nucleus and generative cell within 3 d of culture.The generative cell then divided forming a sterile nucleus andspermatogenous nucleus after 17 d. The spermatogenous nucleusacquired a cell wall then divided forming two equal sperm after24 d. Copyright 2000 Annals of Botany Company Taxus brevifolia, Pacific yew, microsporogenesis, pollination, pollen germination, male gametophyte development     

Ultrastructural study of pollen and anther development in Luehea divaricata (Malvaceae,Grewioideae) and its systematic implications: Role of tapetal transfer cells,orbicules and male germ unit

Developmental processes of microsporogenesis and microgametogenesis in Luehea divaricata (Malvaceae, Grewioideae) were analyzed with transmission electron microscopy. The species studied has perfect flowers. The young anthers are bithecal and tetrasporangiate; microspore mother cells undergo simultaneous meiosis, forming tetrads with a tetrahedral arrangement. The development of the anther wall conforms to the basic type and the tapetum is secretory. The results highlight the presence of multinucleate tapetal cells, which acquire ultrastructural features characteristic of transfer cells at the young pollen grain stage, and which are associated with the presence of orbicules. Tapetal transfer cells have not been previously investigated in detail for other species of angiosperms; their function during pollen development is discussed. The present work is the first contribution to the knowledge of ultrastructural pollen development in the genus Luehea, as well as in the subfamily Grewioideae.     

Why are plastids maternally inherited in epilobium? Ultrastructural observations during microgametogenesis

We have investigated the sequential stages of microgametogenesis by electron microscopy, to determine the basis of maternal inheritance of plastids in Epilobium. The development of both the vegetative and generative cells has been followed using a semi-artificial growth system for pollen tubes. The generative cells inside the pollen grain contains numerous mitochondria, 5–8 proplastids, and, in contrast to the vegetative cytoplasm, only a few vacuoles. When the generative cell has divided into the two sperm cells inside the pollen tube, small vesicles deriving from dicytosome cisternae become abundant. These vesicles appear to form vacuoles by fusion which then contain remnants of fibrillar, globular or membranaceous material. It is suggested that this material derives from proplastids as the proplastids disappear either before or shortly after the generative cell has divided, concurrently with the appearance of the ‘remnants’ in the vacuoles. The mitochondria of the sperm cells remain intact.     

REPRODUCTIVE FEATURES OF DENTARIA LACINIATA AND D. DIPHYLLA (CRUCIFERAE), AND THE IMPLICATIONS IN THE TAXONOMY OF THE EASTERN NORTH AMERICAN DENTARIA COMPLEX

Reproductive features including ovule development, megasporogenesis, megagametogenesis, microsporogenesis, microgametogenesis, pollen tube growth, embryogeny, and natural seed germination were studied in a single population each of Dentaria laciniata Muhl. ex. Willd. and D. diphylla Michx. to test for possible agamospermy. The population of D. laciniata studied is sexual. The archesporial cell functions directly as the megasporocyte. It undergoes two meiotic divisions, but the micropylar cell of the dyad fails to undergo meiosis II, and a linear triplet of three cells is formed. The chalazal megaspore divides to form an eight-nucleate, seven-celled megagametophyte of the Polygonum type. Simultaneous cytokinesis follows the second meiotic division of the microsporocyte yielding a tetrahedral tetrad of microspores. A three-celled pollen grain is formed prior to anther dehiscence. Following apparent fertilization, the Capsella-variation of the Onagrad type of embryogeny results in a conduplicate embryo. Endosperm is initially nuclear, but eventually becomes cellular. Seeds readily germinate in nature. Similar events are documented in one population of D. diphylla up to the organization of the embryo-sac, which disintegrates before cellularization. These reproductive events and other data indicate that the eastern North American species of Dentaria may form a sexual polyploid complex with some sexual populations and some sterile ones.     

Reproductive biology of the “Brazilian pine” (Araucaria angustifolia – Araucariaceae): Development of microspores and microgametophytes

Araucaria angustifolia Bert. (O. Ktze), also known as the “Brazilian pine”, is native to the South of Brazil. This species has a long reproductive cycle, taking about 29–34 months. Its cones begin to develop in early January and remain dormant from March to July. In August, they become active again and microsporogenesis occurs, which proceeds until September. From September to October, microgametogenesis is established and pollination occurs from October to November. Meiosis is asynchronous, with simultaneous cytokinesis, and the tetrads are of the tetrahedral and isobilateral type. During gametogenesis, microgametophytes gradually develop an axial row of cells that are isolated by internal callose and undergo four mitotic cycles until pollen dispersal. In mature pollen grains, the vegetative cells do not possess a cell wall, but maintain strong internal polarization. The pollen of A. angustifolia is suboblate, without apertures or air sacs. Histochemical analysis of the sporoderm was also performed and when compared to other conifer families, showed the most simplified intine structure among the group. Embryological characteristics analyzed during the phenological phases of this species showed certain peculiarities, knowledge about which may be helpful contributing to the management and conservation of A. angustifolia.     

Microsporogenesis,microgametogenesis, and pollen morphology of <Emphasis Type="Italic">Ambrosia artemisiifolia</Emphasis> L. in China

Ambrosia artemisiifolia L. from Ambrosia of the Heliantheae of the Asteraceae family is a recognized harmful weed worldwide and one of the major invasive foreign plants in China. In this study, we investigated its reproductive features, focusing on its microsporogenesis, microgametogenesis, and pollen morphology. The results show that (1) Ambrosia artemisiifolia L. is a dicotyledonous plant and has spherical, tricolpate pollen grains with spiny outer wall; (2) its anther wall comprises four layers, namely epidermis, endothecium, middle layers, and amoeboid tapetum; (3) cytokinesis of microspore mother cells is successive; (4) most of tetrads are tetrahedral; and (5) mature pollen grains are three-celled. In conclusion, although Ambrosia artemisiifolia L. is a dicotyledonous plant with tricolpate pollen, its microsporogenesis is successive, which is different from typical dicots.     

Developmental and cytogenetic analyses of pollen sterility in Valeriana scandens L.

Valeriana scandens presents perfect and pistillate flowers, the latter with sterile anthers. The species is composed of two varieties with different ploidy; V. scandens var. scandens (2n = 28) and V. scandens var. candolleana (2n = 56), both of which occur in RS, Brazil. Crosses between these varieties may give rise to hybrids with pollen sterility. In this study, we analyzed the microsporogenesis and microgametogenesis of sterile and fertile anthers, and also investigate whether pollen sterility is caused by an irregular meiotic process. Developmental analysis using light microscopy and scanning electron microscopy showed that sterile anthers develop similarly to fertile anthers until the end of meiosis. After this stage, sterile tetrads do not separate as a consequence of exine fusion between adjacent microspores, which is similar to sterile pollen of Brassica ms-cdl1 mutants. In addition, vacuolated immature pollen grains degenerate after separation. The cytogenetic analysis of the microspore mother cell (MMC) showed that the diploid population of V. scandens var. scandens (2n = 28) has pollen sterility that is not caused by a cytogenetic disturbance. The MMCs analyzed from prophase I to tetrad stage showed a regular meiotic process, indicating the phenotype of V. scandens sterile pollen is a postmeiotic process formed by fusion of exine between opposite microspores.     

Empirical assessment of the reproductive fitness components of the hybrid pine Pinus densata on the Tibetan Plateau

Pinus densata is distributed on the Tibetan Plateau, where it forms extensive forests at high elevations. Genetic studies have provided evidence that P. densata originated through hybridization between P. yunnanensis and P. tabuliformis. To clarify the relationships among these pines, and assess their reproductive fitness in their respective habitats, we conducted a comparative analysis of eight cone and seed morphometric traits and six reproductive traits in them. Among the eight morphometric traits examined, six appeared to be intermediate in P. densata between those of P. yunnanensis and P. tabuliformis. There were significant differences among the three pines in all of the morphometric traits, and P. densata showed greater variability in these traits than the other two pines. In contrast to the morphometric traits, the reproductive traits (including the proportions of filled and empty seeds, ovule abortion rate, seed efficiency, meiotic abnormalities during microsporogenesis and pollen viability) differed little among the three pines, indicating that they have similar overall rates of effective pollination and fertilization in their respective natural environments. Despite their location on the high plateau, natural populations of P. densata appeared to have normal levels of reproductive success, comparable to those of the two parental species in their natural habitats. This study provides empirical data characterizing the reproductive success and adaptation of a stabilized homoploid hybrid in a novel habitat that is ecologically and spatially inaccessible to its parental species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Changing patterns of nucleic acids, basic and acidic proteins in generative nuclei during pollen germination and pollen tube growth in Hippeastrum belladona

Generative and vegetative nuclei of mature and germinated pollen grains from Hippeastrum belladonna were separated in a continuous Ficoll gradient. Less than 3% contamination was observed between the generative and vegetative nuclear fractions. The vegetative nuclei were composed of two populations; the larger population consisted of nuclei with 1C levels of DNA and the smaller with 2C levels. The generative nuclei consisted of a homogeneous population composed of nuclei possessing 2C levels of DNA. Histone synthesis did not occur in vegetative nuclei. Changes appeared in the gel-electrophoretic banding patterns of the F1 histones of vegetative nuclei during germination. Changes were not observed in the generative nuclei. A reduction of general proteins and RNA was observed in vegetative nuclei by 20 h of germination. The phenol-soluble nuclear proteins of vegetative nuclei revealed transitions in electrophoretic banding patterns during pollen germination that were greater than those shown by the histones. These changes in the PSNP primarily involved reduced concentrations of certain proteins rather than synthesis of new ones. However, a new band was observed in the electrophoretic pattern of the PSNP of vegetative nuclei after 12 h of pollen tube growth. No transition was seen in the PSNP of generative nuclei during pollen germination and tube growth. The regulatory role of the PSNP in cell differentiation is discussed in the light of these findings.     

Division of the generative nucleus in cultured pollen tubes of the Bromeliaceae

Pollen germination, division of the generative nucleus and position of the generative nucleus in the pollen tube during in vitro germination were examined for six bromeliad cultivars. The influence of mixed amino acids (casein hydrolysate) and individual amino acids (Arg, Asn, Asp, Glu, Gly, Met, Phe, Orn, Tyr) were tested. Aechmea fasciata and A. chantinii pollen tubes showed more generative nuclear division in cultured pollen tubes than the other four cultivars tested. Casein hydrolysate did not stimulate generative nuclear division. In general arginine (1 mM) improved division of the Aechmea generative nucleus and to a lesser extent this of Vriesea `Christiane', Guzmania lingulata and Tillandsia cyanea. A concentration of 2 mM arginine reduced pollen tube growth of Aechmea. The vegetative nucleus was ahead of the generative nucleus in approximately 50% of the pollen tubes of all cultivars studied. In about 25% of the pollen tubes, the generative nucleus was ahead and in ±25% pollen tubes the vegetative and generative nuclei were joined together. The distance between the two generative nuclei and the distance from the generative nuclei to the pollen tube tip differed significantly for Aechmea fasciata and A. chantinii. The influence of different amino acids for Aechmea fasciata and A. chantinii varied with respect to pollen germination and generative nuclear division. Arg and Met improved nuclear division of both Aechmea cultivars. Pollen germination and sperm cell production were not linked. This information is important to ameliorate in vitro pollination methods used to overcome fertilization barriers in Bromeliaceae and other higher plants.     

Autoradiographs of Pollen Tube Nuclei with Calcium-45

Autoradiography with Ca⁴⁵ has been used to obtain information about the relation between calcium and chromosomes. Labelled pollen from the Easter lily, Lilium longiflorum, was allowed to develop into pollen tubes between 5 and 6 cm. long in the styles of non-radioactive flowers. All of the nuclei, namely the tube nucleus and the two sperm nuclei, retain Ca⁴⁵ after this period of growth and development. Since the two sperm nuclei have formed during this interval by the mitotic division of the generative nucleus and growth of the tube has occurred under the influence of the tube nucleus, it is inferred that the calcium was bound in a stable nuclear component, the chromosomes.     

A COMPARATIVE LIGHT- AND ELECTRON-MICROSCOPIC STUDY OF MICROSPOROGENESIS IN MALE STERILE (MS,) AND MALE FERTILE SOYBEANS (GLYCINE MAX (L.) MERR.)

A comparative study of microsporogenesis in fertile and in male sterile (ms₁) soybean plants (Glycine max (L.) Merr.) was conducted by using various microscopic techniques. Once the developmental pattern for fertile microsporogenesis was established, it was compared with the developmental pattern in sterile plants to determine the time of microsporogenesis breakdown. Sterility of the ms₁ mutant is caused by failure of cytokinesis after telophase II. The four nuclei resulting from meiosis become enclosed in a single-celled structure, termed a coenocytic microspore. These microspores develop a pollen-like wall and become engorged with lipid and starch reserves. Coenocytic microspores usually degenerate after engorgement. This study of fertile and sterile (ms₁) microsporogenesis has shown that nuclear and cytoplasmic events must occur at precise times for the successful development of 1n pollen grains from 2n sporogenous cells. Any disruption during this process leads to sterility.     

Temperature stress interferes with male reproductive system development in clementine (Citrus clementina Hort. ex. Tan.)

Male gametophyte development is a critical phase of the plant life cycle due to its high sensitivity to environmental stresses. The rise in the average global temperature, often accompanied by extreme fluctuations, has an important impact on biological processes. Among those, male gametophytes are particularly sensitive to temperature stress during flower bud development and anthesis. Male gametophyte development was extensively studied in several plant species, but little information is available about the effects of temperature stress on male gametophyte development in the genus Citrus. We evaluated the effects of cold and hot temperatures during microsporogenesis and microgametogenesis on one of the most economically valuable citrus species, the “Comune” clementine (Citrus clementina Hort. ex. Tan.). The effect of constant temperature on the androecium was evaluated by a time course histological analysis performed on the anthers and by monitoring in vitro pollen germination. The results revealed how suboptimal hot and cold temperatures induce drastic alterations on the morphology of the tapetal cells, microspores and mature pollen grains. Shifting from the optimal temperature affected the timing of starch depletion in the anther walls, such as epidermis, endothecium and middle layer, influencing the pollen germination rate and pollen tube growth. To the best of our knowledge this is the first study attempting to assess how temperature stress affects male reproductive development in citrus. A better understanding of the mechanisms underlining male sterility will provide novel insights to elucidate the physiology of this agronomical important quality trait.     

The expression of alternative oxidase and alternative respiratory capacity in cytoplasmic male sterile common bean

Summary The alternative respiratory pathway is present in all plant species investigated to date. Yet, the role of the alternative pathway is not clear. Some evidence suggests an important role in pollen development. We undertook this study to investigate the expression of alternative oxidase, in comparison with expression of a component of cytochrome oxidase, during pollen formation in common bean (Phaseolus vulgaris L.). Expression was evaluated immunohistochemically. In addition, we compared both the alternative oxidase capacity in young seedling tissues and alternative oxidase expression in developing flower buds of isonuclear cytoplasmic male sterile and male fertile bean lines. We observed no evidence of an association between the abnormal pollen development of CMS bean and changes in alternative oxidase expression or capacity. We did observe a tissueand stage-specific pattern of expression of alternative oxidase, differing from the expression pattern of cytochrome oxidase subunit II, during anther development in normal bean lines. Although no association was evident between the cytoplasmic male sterility phenotype and differential expression of alternative oxidase, the regulated pattern of alternative oxidase expression in developing anthers does suggest that the alternative pathway may play a role in microgametogenesis and microsporogenesis.     

Cytological mechanism of pollen abortion resulting from allelic interaction of F1 pollen sterility locus in rice (Oryza sativa L.)

Pollen abortion is one of the major reasons causing the inter-subspecific F₁ hybrid sterility in rice and is due to allelic interaction of F₁ pollen sterility genes. The microsporogenesis and microgametogenesis of Taichung 65 and its three F₁ hybrids were comparatively studied by using techniques of differential interference contrast microscopy, semi-thin section light microscopy, epifluorescence microscopy and TEM. The results showed that there were differences among the cytological mechanisms of pollen abortion due to allelic interaction at the three F₁ pollen sterility loci. The allelic interaction at S-a locus resulted in microspores unable to extend the protoplasm membrane with the enlargement of the microspore at the middle microspore stage and finally producing empty abortive pollen. The allelic interaction at S-b locus caused asynchronous development of microspores at the middle microspore stage producing stainable abortive pollen. The allelic interaction at S-c locus mainly led to the non-dissolution of the generative cell wall and finally caused the hybrid F₁ mainly producing stainable abortive pollen. Genotypic identification indicated that the abortive pollen were those with S ^j allele.     

The developmental fate of angiosperm pollen is associated with a preferential decrease in the level of histone H1 in the vegetative nucleus

In angiosperm pollen, the vegetative cell is assumed to function as a gametophytic cell in pollen germination and growth of the pollen tube. The chromatin in the nucleus of the vegetative cell gradually disperses after microspore mitosis, whereas the chromatin in the nucleus of the other generative cell remains highly condensed during the formation of two sperm nuclei. In order to explain the difference in chromatin condensation between the vegetative and generative nuclei, we analyzed the histone composition of each nucleus in Lilium longiflorum Thunb. and Tulipa gesneriana immunocytochemically, using specific antisera raised against histones H1 and H2B of Lilium. We found that the level of histone H1 decreased gradually only in the vegetative nucleus during the development of pollen within anthers and that the vegetative nucleus in mature pollen after anther dehiscence contained little histone H1. By contrast, the vegetative nucleus contained the same amount or more of histone H2B than the generative nucleus. The preferential decrease in the level of histone H1 occurred in anomalous pollen with one nucleus (uninucleate pollen) or with two similar nuclei (equally divided pollen), which had been induced by treatment with colchicine. The nuclei in the anomalous pollen resembled vegetative nuclei in terms of structure and staining properties. The anomalous pollen was able to germinate and extend a pollen tube. From these results, it is suggested that the preferential decrease in level of histone H1 in pollen nuclei is essential for development of the male gametophytic cell through large-scale expression of genes that include pollen-specific genes, which results in pollen germination and growth of the pollen tube. Received: 9 May 1998 / Accepted: 4 June 1998