Bicellular Pollen Grains Research Articles

DEVELOPMENT OF MALE REPRODUCTIVE STRUCTURES IN <i>CAMPANULA ALLIARIIFOLIA</i> (CAMPANULACEAE)

A cytoembryological study of the formation of microsporangium wall and the development of pollen grains in Campanula alliariifolia Willd. was carried out. A significant similarity in the structure of male embryonic structures in this species with the other representatives of Campanulaceae was revealed, namely: simultaneous formation of tetrads of microspores, bicellular pollen grains with relatively large vegetative cell, cellular secretory tapetum, bilayered endothecium. Characteristic features of this species are as follows: the absence of placentoids which indicates its evolutionary advancement, since the placentoids are characteristic of the most primitive representatives of the family; long-term, in comparison with previously studied species of the family, preservation of the tapetum, namely up to the opening of the anther, in the form of a cord of deformed tapetal cells located in a row and tightly adjacent to each other; relatively late appearance of fibrous bands.

Embryology of Ficus tuerckheimii (subg. Spherosuke, Moraceae) and its relevance in interactions with its pollinator wasps, Pegoscapus sp. (Agaonidae)

• Ficus tuerckheimii has two female floral morphs with dimorphic stigmas. • Both floral morphs form showed cohesive synstigmas. • Wet stigmas are described for the first time within the genus Ficus. • The development of the anther wall in male flowers is a basic type. • Several evidence suggest that F. tuerckheimii is actively pollinated. Species in the Ficus (Moraceae) genus carry an inflorescence called syconium, formed by a fleshy, urn-shaped receptacle. This study aims to i) describe the embryology of Ficus tuerckheimii flowers at different developmental stages and ii) determine whether this species is pollinated actively or passively. The species is monoecious, with protogynous syconia and diclinous flowers. The syconia contain two pistillate floral morphs: one sessile with a long style and a symmetrically bilobed stigma, the other pedicellate with a short style and asymmetrical stigmatic lobes. The ovule of each flower type can develop into a seed or a (parasitic or pollinator) wasp. The gynoecium is pseudomonomerous; the flowers form a cohesive synstigma due to the proximity of the stigmatic papillae, which facilitates pollen concentration and pollen tubes growth in neighboring flowers. The staminate flowers are pedicellate and unistaminate. The anther dehiscence is longitudinal, rotating horizontally to open and release bicellular pollen grains. The syconium includes three different trichome types whose functions remain unknown. The drupelet fruit has a pericarp conformed of a tanniferous exocarp, a parenchymatic mesocarp, and a schlerenchymatic endocarp. We detected the following embryological characteristics that were previously undescribed for the genus and are potentially informative for the systematics of the family: i) wet stigmas, ii) a basic anther wall with a non-persistent epidermis and a multiplicative middle layer, iii) pollen grains that can germinate within the anther, iv) a profuse nucellus that later forms a perisperm, and v) a persistent perisperm. Altogether, the synstigma, the low anther-to-ovule ratio (0.12 SD ± 0.031), the smooth pollen ornamentation, and the specialized pollen collection and transport structures (corbiculae) of its pollinator wasps ( Pegoscapus sp., Agaonidae) suggest that F. tuerckheimii is actively pollinated.

Microgametophyte Development in Cannabis sativa L. and First Androgenesis Induction Through Microspore Embryogenesis.

Development of double haploids is an elusive current breeding objective in Cannabis sativa L. We have studied the whole process of anther and pollen grain formation during meiosis, microsporogenesis, and microgametogenesis and correlated the different microgametophyte developmental stages with bud length in plants from varieties USO31 and Finola. We also studied microspore and pollen amyloplast content and studied the effect of a cold pretreatment to excised buds prior to microspore in vitro culture. Up to 476,903 microspores and pollen grains per male flower, with in vivo microspore viability rates from 53.71 to 70.88% were found. A high uniformity in the developmental stage of microspores and pollen grains contained in anthers was observed, and this allowed the identification of bud length intervals containing mostly vacuolate microspores and young bi-cellular pollen grains. The starch presence in C. sativa microspores and pollen grains follows a similar pattern to that observed in species recalcitrant to androgenesis. Although at a low frequency, cold-shock pretreatment applied on buds can deviate the naturally occurring gametophytic pathway toward an embryogenic development. This represents the first report concerning androgenesis induction in C. sativa, which lays the foundations for double haploid research in this species.

Haploid Embryogenesis in Isolated Microspore Culture of Carrots (Daucus carota L.).

The process of embryogenesis in isolated microspore culture was studied in eight carrot accessions of different origin. The ½NLN-13 medium supplemented with 0.2 mg/L 2,4D and 0.2mg/L kinetin was used to induce embryogenesis. The temperature treatment was performed at 5–6 °C for three days, followed by cultivation at 25 °C in darkness. As was shown, the first embryogenesis was only observed in microspores at the late vacuolated stage when the nucleus moved from the center to one pole following the long cell axis. Depending on the nucleus position, the microspore can divide into two equal or two different sized cells. Following divisions occurred either in one of these cells or in two. However, microspores that divided into two unequal cells were morphologically different form bi-cellular pollen grain. Embryogenic divisions in bi-cellular pollen grains were not observed. First divisions began by the third day of cultivation, and continued until the globular embryoid stage that was well-seen after the fourth week of cultivation. The already-formed embryoids can develop the secondary embryoids on their surface. Depending on the genotype, up to 1000 secondary embryoids can be produced from one embryoid in the liquid MSm medium supplemented with 0.1 mg/L of kinetin for regeneration. All carrot accessions studied were split into three groups: responsive genotypes, weakly responsive genotypes, and reluctant genotypes. The highest yield was 53 initial embryoids per a 6 cm diameter petri dish. Thus, the Nantskaya 4 cultivar totally produced 256 initial embryoids, out of which 94 developed into green plantlets and 162 into albino plantlets, whereas 97 initial embryoids with 45 albino plantlets formed from them were obtained from Chantenay cultivar.

Calcium distribution during anther development in Impatiens balsamina

The subcellular localization of loosely bound calcium in developing anthers of Impatiens balsamina (rose balzam) was investigated using potassium antimonate to precipitate the ions. At the sporogenous stage, few calcium precipitates were found in the young anthers. During meiosis of the microspore mother cell, the amount of calcium precipitates increased both inside these cells and in their cell walls. After meiosis, the resulting microspores accumulated abundant calcium precipitates in their cytoplasm and vacuoles. The microspores underwent mitosis to produce a bicellular pollen grain, in which the large vacuoles disintegrated, and the calcium precipitates of the vegetative cell were again localized in the cytoplasm. The amount of precipitates decreased during pollen maturity and finally disappeared from the inside of the pollen grains and accumulated on the pollen walls.

Dynamic distribution of ARGONAUTE1 (AGO1) and ARGONAUTE4 (AGO4) in Hyacinthus orientalis L. pollen grains and pollen tubes growing in vitro

The transcriptional and posttranscriptional AGO-mediated control of gene expression may play important roles during male monocot gametophyte development. In this report, we demonstrated dynamic changes in the spatiotemporal distribution of AGO1 and AGO4, which are key proteins of the RNA-induced silencing complex (RISC) in Hyacinthus orientalis male gametophyte development. During maturation of the bicellular pollen grains and in vitro pollen tube growth, the pattern of AGO1 localization was correlated with previously observed transcriptional activity of the cells. During the period of high transcriptional activity, AGO1 is associated with chromatin while the clustered distribution of AGO1 in the interchromatin areas is accompanied by condensation of chromatin and the gradual transcriptional silencing of both cells in mature, dehydrated pollen. During pollen tube growth and the restarting of RNA synthesis in the vegetative nucleus, AGO1 is dispersed in the chromatin. Additionally, the gradual increase in the cytoplasmic pool of AGO1 in the elongating pollen tube indicates the activation of the posttranscriptional gene silencing (PTGS) pathway. During pollen tube growth in the generative cell and in the sperm cells, AGO1 is present mainly in the areas between highly condensed chromatin clusters. Changes in the distribution of AGO4 that indicated the possibility of spatiotemporal organization in the RNA-directed DNA methylation (RdDM) process (cytoplasmic and nuclear steps) were also observed during hyacinth male gametophyte development. Based on our findings, we propose that in the germinating pollen tube, the cytoplasmic assembly of AGO4/siRNA takes place and that the mature complexes could be transported to the nucleus to carry out their function during the next steps of pollen tube growth.

Dynamic changes in insoluble polysaccharides and neutral lipids in the developing anthers of an endangered plant species, Pancratium maritimum

Dynamic changes in the distribution of lipid and insoluble polysaccharide reserves of Pancratium maritimum L. (Amaryllidaceae) anthers were investigated throughout the successive stages of pollen development, using cytochemical methods, to determine whether the synthesis, transformation, and mobilization of reserve materials in developing anthers follow the regular pathway in angiosperms and support the physiological activities in developing pollen. Polysaccharides and lipid reserves exhibited a variable pattern of distribution from the sporogenous cell stage to the anthesis. Starch granules and lipid bodies were scarce in the cytoplasm of sporogenous cells, but their number increased significantly at the premeiotic stage. Conversely, starch and lipid reserves of meiocytes reduced at the early prophase of the first meiotic division, and then their amount showed fluctuations during the microsporogenesis. The cytoplasm of free and vacuolated microspores was poor regarding the polysaccharide and lipid reserves. However, at the late vacuolated microspore stage, small insoluble polysaccharides began to appear in the microspore cytoplasm, and their number increased remarkably in the cytoplasm of the bicellular pollen grain. During the maturation of pollen grains, polysaccharide reserves were replaced with lipids. The starch and lipid reserves of the staminal envelope also showed variations at different stages of the anther development. The dynamic changes in the polysaccharide and lipid reserves of P. maritimum anthers were consistent with the physiological activities such as differentiation, cell division and material synthesis that occur in the anther tissue at different stages of the male gametophyte development, and supported the normal pollen development.

Occurrence of polytene chromosomes in the bicellular and mature pollen grains of endangered plant species Pancratium maritimum L. (Amaryllidaceae)

Endoreduplication is a modified form of mitotic cycle in which repeated rounds of DNA replication occur without chromosome segregation and cell division, leading to formation of larger and thicker chromosomes called polytenics. In the present study, spontaneous formation of polytene chromosomes in the generative nuclei of pollen grains of Amaryllidaceae, and presumably in angiosperms, was described for the first time. For this purpose, microspores and mature pollens of the endangered species Pancratium maritimum L. were examined by light and transmission electron microscopy with a special reference to the observation of polytenic nuclei. Polytene chromosomes were detected in most of the generative cells of young bicellular pollen grains and mature pollen grains found in some anther locules, but they were rare or absent in the other anther locules. Polytenization of chromosomes did not follow the same pattern in all of the pollen grains. Polytene structures found in the developing pollen grains of P. maritimum exhibited variation from a diffuse to a condensed state and from reticulate to cable-like structures with different degrees of bonding. In addition to cryptic polyteny, cable-like polytene chromosomes with bands resembling classic polytene chromosomes were also detected. However, some nuclei did not exhibit polytenic structure clearly. Morphological changes in polytene chromosomes that are caused by the formation of functional structures such as DNA amplification, puffing and looping were also recognized. Other reasons for morphological differences of polytene chromosomes are discussed in detail.

Pollen development in Rhododendron in relation to winter dormancy and bloom time.

Microsporogenesis and microgametogenesis of Rhododendron ledebourii (semi-deciduous), Rhododendron luteum (deciduous), and Rhododendron catawbiense (evergreen) were studied by light and electron microscopies in order to determine the stages of pollen development in relation to period of winter dormancy and bloom time throughout an annual growth cycle. Development of generative organs starts in June in R. ledebourii and in July in R. luteum and R. catawbiense and reaches completion about 11 months later. R. luteum and R. catawbiense microspores undergo meiosis at the end of the August and spend winter at the vacuolization stage. Mitosis with the formation of bicellular pollen grain occurs shortly before flowering at the beginning of June. R. ledebourii develops two types of flowers which differ in the timing of microgametogenesis. The first type is characterized by early microspore meiosis and mitosis leading to development of bicellular pollen grains by the end of August, and is prone to fall blooming during warm autumn temperatures. Microspores of the second flower type have a more prolonged vacuolization stage with mitosis and subsequent bicellular pollen grains occurring in November. By winter, flower buds in R. ledebourii are more advanced developmentally than in R. catawbiense and R. luteum, and bloom about 1 month earlier. The different strategies of pollen development identified both within and between these three Rhododendron species were recognized which are not associated with leaf drop during winter but appear to be related to the time of spring flowering and the frequency of autumn flowering.

Cytochemical study of developing anthers of Amomum villosum Lour.

We analyzed anther development in Amomum villosum Lour. (Zingiberaceae) using the periodic acid-Schiff's technique and Sudan black staining to test for the presence of starch and lipids, respectively. Our analyses showed that microspore mother cells of A. villosum lack typical callose walls, and numerous lipid granules appear in the cells early in development. Some starch granules are present in anther wall cells, but not in tapetal cells. After meiosis, numerous lipid granules remain unchanged in the microspores. During microspore development, some small starch granules first appear in the central cell region, and then the starch granules increase in size. After microspore division, the bicellular pollen grains become filled with starch and lipids, and remain in this state until the pollen grains reach maturity. At anthesis, the anther wall of A. villosum consists of several layers of endothecium cells with an evidently thickened radial wall, and some layers of parenchyma cells containing numerous starch granules.

Pollen

Pollen

Diplospory and obligate apomixis in Miconia albicans (Miconieae, Melastomataceae) and an embryological comparison with its sexual congener M. chamissois

Apomixis, or asexual reproduction through seeds, has been reported for species of the tribe Miconieae, Melastomataceae, but details of the process have yet to be described. We analyzed and compared sporogenesis and gametogenesis in the apomictic Miconia albicans and the sexual M. chamissois. The results point to some differences between species, which were related to the apomictic process. In M. albicans microsporogenesis, problems during meiosis and degeneration of its products led to total pollen sterility, while M. chamissois presented normal bicellular pollen grains in the mature anther. The absence or abnormality of meiosis in M. albicans megasporogenesis led to the formation of an unreduced embryo sac and also to egg cell parthenogenesis, which gave rise to the apomictic embryo. Embryo and endosperm development were autonomous, resulting in seeds and fruits independent of pollination and fertilization. Thus, in this species, apomixis can be classified as diplosporic and obligate. In contrast, meiosis was as expected in the sexual M. chamissois, and led to the development of a reduced embryo sac. Despite the divergent pathways, many embryological characteristics were similar between the studied species and other Melastomataceae and they seem to be conservative character states for the family.

Cytochemical Observation of Fertile and Sterile Anthers of Thermo-Sensitive Genic Male-Sterile Wheat

Cytochemical observation of fertile and sterile anthers of thermo-sensitive genic male-sterile wheat (Triticum astivum L.) indicated that lipids in both fertile and sterile anthers were very few during their development. Starches in both anthers were few too before microspore division. In fertile anthers, after microspore division and forming a bi-cellular pollen grain, the big vacuole in vegetative cell of pollen was disintegrated and disappeared, and the cytoplasmic inclusion and starch grains increased persistently. The mature pollen accumulated many starch grains. In sterile anthers, the structure of pollen was similar to that of fertile pollen before microspore division, without much difference between them. After forming a bi-cellular pollen, however, the big vacuole in vegetative cell was not disintegrated and disappeared, the cytoplasmic inclusion decreased, and the accumulated starches in pollen also disappeared. Finally, the pollen grains aborted with its cytoplasmic inclusion disintegration in bi-cellular state. Pollen abortion in this male sterile wheat is related to starch metabolism.

Characterization of a Novel Pollen-Specific Promoter from Wheat (Triticum Aestivum L.)

PSG076 is a pollen-specific gene isolated from wheat. The 1.4-kb promoter upstream of the ATG start codon was isolated by inverse-PCR (IPCR). To determine its activity, the PSG076 promoter was fused with the β-glucuronidase (GUS) reporter gene and introduced into tobacco. Histochemical analysis in transgenic tobacco showed that GUS activity was detected in late bicellular pollen grains and increased rapidly in mature pollen. GUS activity was also detected in pollen tubes of transgenic tobacco. No GUS activity was found in other floral and vegetable tissues. These results indicate that the PSG076 promoter directs pollen-specific activity at late stages of pollen development and pollen tube growth. Deletion analysis showed that a 0.4 kb fragment of the promoter was enough to confer pollen-specific expression.

Understanding male sterility in Miconia species (Melastomataceae): a morphological approach

Pollen abortion occurs in virtually all species and often does not prejudice reproductive success. However, large numbers of abnormal pollen grains are characteristic of some groups. Among them is Miconia, in which partial and complete male sterility is often related to apomixis. In this study, we compared the morphology of pollen grains over several developmental stages in Miconia species with different rates of male sterility. Our aim was to improve the knowledge of mechanisms that lead to male sterility in this ecologically important tropical group. Routine techniques for microscopy were used to examine anthers in several developmental stages collected from the apomictic species Miconia albicans and M. stenostachya. Both species are completely male sterile since even the pollen grains with apparently normal cytoplasm were not able to develop a pollen tube. Meiosis is a rare event in M. albicans anthers and happens in an irregular way in M. stenostachya, leading to the pollen abortion. M. albicans has more severe abnormalities than M. stenostachya since even the microspores and pollen grain walls were affected. Moreover, in M. stenostachya, most mitosis occurring during microgametogenesis was also abnormal, leading to the formation of bicellular pollen grains with two similar cells, in addition to the formation of pollen grains of different sizes. Notably, abnormalities in both species did not reach the production of Übisch bodies, suggesting little or no tapetum involvement in male sterility in these two species.

Ultrastructural study on dynamics of plastids and mitochondria during microgametogenesis in watermelon

Dynamics of plastids and mitochondria during microgametogenesis in watermelon were examined by means of transmission electron microscopy. Plastids are present as proplastids in the microspore and as amyloplasts in the vegetative cell of the bicellular pollen grain, whereas the generative cell is completely devoid of plastids, suggesting that microspore plastids are excluded from the generative cell during the microspore mitotic division. Therefore, watermelon is classified as Lycopersicon type, where plastids exclusion from the generative cell leads to purely maternal plastid inheritance. Mitochondria in the generative cell show noticeable alterations in size and cristae during microgametogenesis. The diameter of mitochondria is about 0.5 μm in the newly born generative cell, while only about 0.16 μm in the spindle-shaped generative cell. Numerous cristae are present in mitochondria in the spherical generative cell, but, in contrast, mere two or three cristae retain in the spindle-shaped generative cell in the mature pollen grain. In conclusion, the size and cristae number of mitochondria in the generative cell are reduced significantly during microgametogensis in watermelon.

Distribution of Starch and Neutral Lipids in the Developing Anthers ofLpomoea cairica

The Periodic Acid-Schiff (PAS) reaction was used to test for the presence of starch, and Sudan black was applied to detect neutral lipids (lipid drops) during anther development in Ipomoea cairica. Starch was observed in sporogenous and anther wall cells. Prior to meiosis in microspore mother cells (MMCs), starch was not evident, although small lipid drops appeared. A substantial amount of starch accumulated in the epidermal and endothecium cells, but starch and lipid drops were not identified in the tapetum. At the tetrad stage, starch appeared in microspores, and the amount of starch remained constant in anther cells. Early in the microspore stage, the tapetum degenerated, starch remained in the microspores, and lipid drops were completely lost. A large and conspicuous vacuole was formed in the microspores and lipid drops reformed in the cells at the late microspore stage. Following microspore division, the vegetative cell of bicellular pollen grains was filled with starch and lipid drops, and the inner middle layer degenerated. Finally, at anthesis, lipid drops and starch constituted the stored nutritive material in the mature pollen.

Precocious pollen germination in Arabidopsis plants with altered callose deposition during microsporogenesis

Pollination is essential for seed reproduction and for exchanges of genetic information between individual plants. In angiosperms, mature pollen grains released from dehisced anthers are transferred to the stigma where they become hydrated and begin to germinate. Pollen grains of wild-type Arabidopsis thaliana do not germinate inside the anther under normal growth conditions. We report two Arabidopsis lines that produced pollen grains able to in situ precociously germinate inside the anther. One of them was a callose synthase 9 (cs9) knockout mutant with a T-DNA insertion in the Callose Synthase 9 gene (CalS9). Male gametophytes carrying a cs9 mutant allele were defective and no homozygous progeny could be produced. Heterozygous mutant plants (cs9/+) produced approximately 50% defective pollen grains with an altered male germ unit (MGU) and aberrant callose deposition in bicellular pollen. Bicellular pollen grains germinated precociously inside the anther. Another line, a transgenic plant expressing callose synthase 5 (CalS5) under the CaMV 35S promoter, also contained abnormal callose deposition during microsporogenesis and displaced MGUs in pollen grains. We also observed that precocious pollen germination could be induced in wild-type plants by incubation with medium containing sucrose and calcium ion and by wounding in the anther. These results demonstrate that precocious pollen germination in Arabidopsis could be triggered by a genetic alteration and a physiological condition.

Calcium Distribution in Developing Anthers of Lettuce (Lactuca sativa)

Potassium antimonite was used to locate calcium in the anthers of lettuce (Lactuca sativa). There are no calcium precipitates in young anthers. After meiosis of microspore mother cells, calcium precipitates first appear in the tapetal cells, from which some small secretive vesicles containing many calcium precipitates are secreted into the locule. At a late stage of the microspore, tapetal cells completely degenerate and their protoplasts move into the locule with many calcium precipitates. The calcium precipitates increase in the early microspores, and in the exine. When the microspores form some small vacuoles containing calcium precipitates, and those vacuoles then fuse to form a large one, the calcium precipitates evidently decrease. The large vacuole of bicellular pollen grain discomposes and calcium precipitates again appear in the cytoplasm and then decrease. When the pollen matures, most calcium precipitates are located in its exine with only a few in the cytoplasm.

Standardization of the fluorochromatic reaction test to assess pollen viability

The fluorochromatic reaction that viable pollen grains exhibit when exposed to fluorescein diacetate (FDA) is an easy, quick and accurate tool for assessing pollen viability in many plants. Despite its widespread use, the method as initially proposed by Heslop-Harrison and Heslop-Harrison lacks specificity in some respects that are essential for comparing simultaneously the viability of different pollen sources. We have determined the time needed for the fluorochromatic reaction to take place and fade, and the lifetime and most effective concentration of the FDA working solution. Our results show that consistent records of pollen viability in samples of olive (bicellular pollen grains) and cherimoya (tricellular pollen grains) can be obtained within minutes, but that measurement must be completed in the first hour after pollen exposure to FDA. The lifespan of the FDA working solution is 1 h, whereas concentrations between 1.5×10−5 M and 1.8×10−3 M are equally valid. Pollen grains of both species responded similarly, although some differences were observed in the persistence of fluorochromasia.