ORIGIN AND DEVELOPMENT OF POLLEN EMBRYOIDS AND POLLEN CALLUSES IN CULTURED ANTHER SEGMENTS OF HYOSCYAMUS NIGER (HENBANE)

The dedifferentiation of pollen grains of Hyoscyamus niger (henbane) into embryoids and calluses was examined by culturing identical segments of the same anther in a mineral salt-sucrose basal medium and in the basal medium supplemented with 2.0 mg/l 2,4-dichlorophenoxyacetic acid, respectively. Addition of auxin enhanced anther efficiency but did not affect the number of embryogenic pollen grains of an anther segment transformed into calluses. In anther segments cultured in the basal medium, the organogenetic part of the pollen embryoid was formed by the division of the generative cell alone, or by the division of both generative and vegetative cells. More or less similar pathways were followed by pollen grains of anther segments cultured in a medium containing auxin to form calluses. Culture of anther segments in a medium containing a high concentration of auxin (50.0 mg/l) led to a significant reduction in the yield of calluses which were formed almost entirely by the division of both generative and vegetative cells. The bearing of these observations on the role of auxin in determining the pathway of differentiation of embryogenic pollen grains in cultured anther segments is considered. The appearance of embryogenic pollen grains in close proximity to the tapetum as seen in longitudinal sections of cultured anther segments has suggested a role for a gradient of tapetal factors in embryogenic induction.     

ULTRASTRUCTURE OF ANOMALOUS POLLEN DEVELOPMENT IN EMBRYOGENIC ANTHER CULTURES OF HYOSCYAMUS NIGER

The formation of anomalous, binucleate pollen grains and their subsequent embryogenic development, induced by anther culture in Hyoscyamus niger, were analyzed by transmission electron microscopy (TEM). In culture, uninucleate pollen grains occasionally divided symmetrically giving rise to two apparently identical nuclei sharing a common cytoplasm. These nuclei divided once or twice unaccompanied by cell wall formation. After the daughter nuclei organized into cells, their subsequent division products contributed to embryoid formation. In conjunction with previous studies of pollen embryogenesis in H. niger, it appears that in contrast to the principle mode of embryogenesis (i.e., first asymmetric division forms typical two-celled pollen grain and the generative cell acts as the embryogenic precursor), anomalous pollen show no carry-over of gametophytic influences following embryogenic induction. This suggests that specific pathways of embryogenesis are correlated with the rate at which gametophytic gene activity is repressed following induction.     

A CYTOLOGICAL ANALYSIS OF MICROSPORES OF TRITICUM AESTIVUM (POACEAE) DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Uninucleate microspores of Triticum aestivum cv. Pavon can be induced in vitro to alter their development to produce embryoids rather than pollen. Microspores expressed their embryogenic capacity through one of two division pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen grain. Here the generative cell detached from the intine, migrated to a central position in the pollen grain, and underwent a second haploid mitosis as the vegetative cell divided to give rise to the embryoid. In the second pathway, the first division was symmetric and both nuclei divided repeatedly to form the embryoid. This comparative analysis of normal pollen ontogeny and induced embryogenesis provided no evidence for the existence of predetermined embryogenic microspores in vitro or in vivo. Instead, microspores are induced at the time of culture, and embryogenesis involves continued metabolic activity associated with the gradual cessation of the gametophytic pathway and a redifferentiation into the sporophytic pathway. In conjunction with a previous study, it appears that embryogenic induction of wheat microspores involves switching off gametophytic genes and derepressing sporophytic genes.     

Nuclear DNA synthesis during the induction of embryogenesis in cultured microspores and pollen of Brassica napus L.

The dynamics of nuclear DNA synthesis were analysed in isolated microspores and pollen of Brassica napus that were induced to form embryos. DNA synthesis was visualized by the immunocytochemical labelling of incorporated Bromodeoxyuridine (BrdU), applied continuously or as a pulse during the first 24 h of culture under embryogenic (32 °C) and non-embryogenic (18 °C) conditions. Total DNA content of the nuclei was determined by microspectrophotometry. At the moment of isolation, microspore nuclei and nuclei of generative cells were at the G1, S or G2 phase. Vegetative nuclei of pollen were always in G1 at the onset of culture. When microspores were cultured at 18 °C, they followed the normal gametophytic development; when cultured at 32 °C, they divided symmetrically and became embryogenic or continued gametophytic development. Because the two nuclei of the symmetrically divided microspores were either both labelled with BrdU or not labelled at all, we concluded that microspores are inducible to form embryos from the G1 until the G2 phase. When bicellular pollen were cultured at 18 °C, they exhibited labelling exclusively in generative nuclei. This is comparable to the gametophytic development that occurs in vivo. Early bicellular pollen cultured at 32 °C, however, also exhibited replication in vegetative nuclei. The majority of vegetative nuclei re-entered the cell cycle after 12 h of culture. Replication in the vegetative cells preceded division of the vegetative cell, a prerequisite for pollen-derived embryogenesis.     

Origin of Multicellular Pollen and Pollen Embryos in Cultured Anthers of Pepper (Capsicum Annuum)

Anthers of Capsicum annuum L. were cultured on Murashige and Skoog (MS) medium containing 0.1 mg l⁻¹ NAA and 0.1 mg l⁻¹ kinetin. Inoculated anthers were subjected to 31 °C and development of microspores in anthers of varying stages was observed cytologically using 4′-6-diamidino-2-phenylindol-2HCl (DAPI). Pepper was characterized by a strong asynchrony of pollen development within a single anther. Percentage of pollen at different stages changed with the culture period, and the proportion of dead pollen increased drastically from day 2 after culture. Microspores that were cultured at the late-uninucleate stage followed one of two developmental pathways. In the more common route, the first sporophytic division was asymmetric and produced what appeared to be a typical bicellular pollen. Embryogenic pollen was formed by repeated divisions of the vegetative nucleus. In the second pathway, which occurred in fewer microspores, the first division was symmetric and both nuclei divided repeatedly to form embryogenic pollen. In early-bicellular pollen, sporophytic pollen was produced through division of the vegetative nucleus. In mid-bicellular pollen, the generative nucleus may undergo division to produce two or more sperm-like nuclei. However, division of the generative nucleus alone to form the embryo was never observed. The anther stage optimal for embryo production contained a large proportion (>75%) of early-binucleate pollen. Associations were found among the percentage of early-binucleate pollen, the frequency of embryogenic multinucleate pollen, and the yield of pollen embryos.     

Structural changes during early embryogenesis in wheat pollen

Summary We have made a detailed cytological examination of the development of wheat embryoids, monitoring their initial divisions from two to ten cells by both light and electron microscopy. According to our observations the first embryogenic division is symmetrical. After the androgenesis induction treatment, there is a decrease in ribosome population with cells that have inactive nucleoli made up almost exclusively of a dense fibrillar component. This population is restored after initial embryogenic divisions. During the initial divisions the embryogenic pollen grains do not appear to change in size and the pollen wall remains intact. The exine undergoes no modification but the intine thickens, and we have observed that the thickness of the intine can be used as a cytological marker of androgenesis. The walls separating the cells obtained after embryogenic division contained numerous plasmodesmata. The beginnings of embryo polarization and cell differentiation could be made out in the very early pollen embryoids.     

Pollen Protoplast Culture Leading to Embryogenic Divisions in Hemerocollis fufva

Pollen protoplasts were isolated from the cold-pretreated flower buds containing uninucleate microspores and were inoculated in K3 basic medium supplemented with various additives. A part of the pollen protoplasts were triggered to embryogenic divisions and further to formation of proembryos and clusters. Microscopical observations showed that the first cell division might be equal or unequal and the subsequent growth might be organized or unorganized. This is the first example of pollen protoplast culture in which a sporophytic, or embryogenic, pathway is triggered     

AN ULTRASTRUCTURAL AND STEREOLOGICAL ANALYSIS OF POLLEN GRAINS OF HYOSCYAMUS NIGER DURING NORMAL ONTOGENY AND INDUCED EMBRYOGENIC DEVELOPMENT

Selected nuclear and cytoplasmic changes of pollen grains of Hyoscyamus niger during normal gametophytic development and embryogenic development, induced by anther culture, were analyzed and compared ultrastructurally using stereological methods. Potentially embryogenic, uninucleate pollen could be identified within 6 hr of culture by an increased ratio of the volume density of the nucleolar granular zone to the volume density of the fibrillar zone and an increased ratio of dispersed to condensed chromatin in the nucleoplasm. Nonembryogenic pollen in vitro and in vivo possessed prominent nucleolar fibrillar zones and low ratios of dispersed to condensed chromatin. These differences may reflect changes in nuclear activity in potentially embryogenic pollen grains during early stages of culture. Following the first haploid mitosis, in potentially embryogenic pollen the generative cell maintained its large granular nucleolus and high ratio of dispersed to condensed chromatin through its first division to form a proembryoid. The volume fraction of the cytoplasm occupied by mitochondria and plastids and the area fraction occupied by RER and Golgi cisternae differed in the generative cells of potentially embryogenic and nonembryogenic pollen. Those changes only detected in generative cells of potentially embryogenic pollen include: increased area and complexity of cytoplasmic membranes, increased mitochondrial volume, and the presence of plastids at all stages of development. These results support the idea that embryogenic induction of H. niger takes place at the uninucleate stage of development and that subsequent nuclear and cytoplasmic changes are essential for continued sporophytic development.     

Changes in synthesis and localization of members of the 70-kDa class of heat-shock proteins accompany the induction of embryogenesis inBrassica napus L. microspores

Elevation of the culture temperature to 32°C for approximately 8 h can irreversibly change the developmental fate of isolatedBrassica napus microspores from pollen development to embryogenesis. This stress treatment was accompanied by de-novo synthesis of a number of heat-shock proteins (HSPs) of the 70-kDa class: HSP68 and HSP70. A detailed biochemical and cytological analysis was performed of the HSP68 and HSP70 isoforms. Eight HSP68 isoforms, one of which was induced three fold by the stress treatment, were detected on two-dimensional immunoblots. Immunocytochemistry revealed a co-distribution of HSP68 with DNA-containing organelles, presumably mitochondria. Six HSP70 isoforms were detected, one of which was induced six fold under embryogenic culture conditions. During normal pollen development, HSP70 was localized in the nucleoplasm during the S phase of the cell cycle, and predominantly in the cytoplasm during the remainder. Induction of embryogenic development in late unicellular microspores was accompanied by an intense anti-HSP70 labeling of the nucleoplasm during an elongated S phase. In early bicellular pollen the nucleus of the vegetative cell, which normally does not divide and never expresses HSP70, showed intense labeling of the nucleoplasm with anti-HSP70 after 8 h of culture under embryogenic conditions. These results demonstrate a strong correlation between the phase of the cell cycle, the nuclear localization of HSP70 and the induction of embryogenesis. As temperature stress alone is responsible for the induction of embryogenic development, and causes an altered pattern of cell division, there might be a direct involvement of HSP70 in this process.Abbreviations HSP heat-shock protein - 2-D two-dimensional - DAPI 4,6-diamidino-2-phenylindole. 1-D = one-dimensional - pI isoelectric point     

Effects of temperature on the mode of pollen development in anther culture of Brassica campestris

In anther culture of Brassica campestris L., the yield of pollen-derived embryoids is greatly stimulated by a high-temperature (35°C) tratment for the first 1 to 3 days of the culture. We have inestigated the effects of the high-temperature treatment on the mode of pollen division in cultured anthers.
Anthers containing late uninucleate pollen were cultured on modified B5 medium. High-temperature treatment for the first 24 h inhibited the normal development of the pollen and induced abnormal symmetrical division. Which is the first step in androgenesis. This symmetrical division was rarely observed in pollen developed in vivo. In anthers cultured without high-temperature treatment, the mode of pollen development was similar to that in vivo. This suggests that the normal differentiation of the pollen is blocked by high temperatures, and sporophtic growth is induced. Sucrose (0.29 M) was essential for the induction of this symmetrical division, though neither plant growth regulator nor any other nurient was necessary. Pollen division could not be induced effectively if sucrose was replaced by either mannitol or sorbitol plus a lower concentration of sucrose. Therefore, it seems that sucrose actively influences the embryogenic division of pollen, and does not have only an osmotic effect.     

Pollen Ultrastructure in Anther Cultures of Nicotiana tabacum: III . THE FIRST SP.OROPHYTIC DIVISION

Ultrastructure of embryogenic tobacco pollen was examined atthe first division of the vegetative cell. Structurally emptyzones formed before division by the degradation of gametophyticcytoplasm were rapidly re-populated with ribosomes, mitochondria,dictybsomes, and organelles interpreted as lipid centres. Thelatter appeared to be active in synthesis, and were associatedwith a vesicular component. Starch accumulation also began atthe first division in plastids carried over from the mothercell. Each daughter cell possessed a typical primary wall confluentwith the inner layers of the intine. Plasmodesmata traversedthe wall between the two cells. The cells were either equalor unequal; equal divisions occurred in both the longitudinaland transverse planes. In many proembryoids, the generativecell showed signs of degeneration. The cell was either attachedto the intine or detached from it. Detached cells sometimesdivided. The data, are discussed in relation to other embryogenicand morphogenic systems induced from normally quiescent or highlydifferentiated cells.     

A co-culture system leads to the formation of microcalli derived from microspore protoplasts ofBrassica napus L. cv. Topas

Summary This paper describes a procedure in which protoplasts are obtained from microspores and pollen of rapeseed to induce callus formation aided by a feeder cell system with embryogenic microspores. Microspores at late unicellular stage and pollen at early bicellular stage were isolated and precultured for 24 h at 32 °C before enzymatic treatment. Eleven enzymes were tested in various combinations and concentrations. The optimal enzyme combination was 1.0% cellulase, 0.8% pectinase, 0.3% macerozyme, and 0.02% pectolyase, in which 26.3% of the microspores released protoplasts. A successful co-culture system was set up by employing embryogenic microspores as feeder cells. To this end, microspores were cultured in a medium with high osmotic pressure at 32 °C. Up to 37% of the microspores exhibited cell division and embryos developed to the heart-shape stage without changing medium. Microspore protoplasts were cultured in Millicells surrounded by the embryogenic microspores as feeder. In growth-regulator-free medium 14.5% of the protoplasts divided but only formed budding-like multicellular structures. Only after pretreatment with 4 mg of 2,4-dichlorophenoxyacetic acid and 1 mg of naphthaleneacetic acid per liter protoplasts divided and formed microcalli. Pollen tubes or tubelike structures were not observed. The experiments reveal that selection of the specific developmental stage of microspores, which is a prerequisite for microspore embryogenesis, is also important in microspore protoplast culture. Compared to other methods used before, microculture fed with embryogenic microspores has obvious superiority.Abbreviations CPW basic protoplast washing medium according to Power and Chapman - CPW972 CPW basic medium supplemented with 9% mannitol and 7.2% sorbitol - DAPI 4,6-diamidino-2-phenylindole - NLN nutrient medium according to Lichter modified by Pechan and Keller - NLN13 NLN medium supplemented with 13% sucrose - NLNP NLN13 supplemented with 7.2% sorbitol     

Studies on Conditions for Cell Division and Embryogenesis in Isolated Pollen Culture of Nicotiana rustica

A method for the induction of a high rate of cell division and embryogenesis of Nicotiana rustica pollen was developed. Binucleate pollen grains were fractionated by Percoll density gradient (35/45%) centrifugation and cultured in 0.4 molar mannitol at 30°C (the first culture). After 3 days in culture pollen was recollected by a second Percoll fractionation (0/30%) and transferred to and cultured in a medium containing the Murashige-Skoog macro-elements, 0.4 molar mannitol, 40 millimolar galactose, 3 millimolar glutamine, and 5 micromolar ABA for 10 days (the second culture). The cell population consisting of about 80% dividing pollen was transferred to a Murashige-Skoog medium containing 0.4 molar mannitol, 3 millimolar glutamine, and no phytohormone (the third culture), where about 40% of dividing pollen developed into embryos or embryogenic calli.     

Pollen ultrastructure in anther cultures of Datura innoxia. I. Division of the presumptive vegetative cell.

Ultrastructural features of embryogenic pollen in Datura innoxia are described, just prior to, during, and after completion of the first division of the presumptive vegetative cell. In anther cultures initiated towards the end of the microspore phase and incubated at 28 degrees C in darkness, the spores divide within 24 h and show features consistent with those of dividing spores in vivo. Cytokinesis is also normal in most of the spores and the gametophytic cell-plate curves round the presumptive generative nucleus in the usual highly ordered way. Further differentiation of the 2 gametophytic cells does not take place and the pollen either switches to embryogenesis or degenerates. After 48-72 h, the remaining viable pollen shows the vegetative cell in division. The cell, which has a large vacuole and thin layer of parietal cytoplasm carried over from the microspore, divides consistently in a plane parallel to the microspore division. The dividing wall follows a less-ordered course than the gametophytic wall and usually traverses the vacuole, small portions of which are incorporated into the daughter cell adjacent to the generative cell. The only structural changes in the vegetative cell associated with the change in programme appear to be an increase in electron density of both plastids and mitochondria and deposition of an electron-dense material (possibly lipid) on the tonoplast. The generative cell is attached to the intine when the vegetative cell divides. Ribosomal density increases in the generative cell and exceeds that in the vegetative cell. A thin electron-dense layer also appears in the generative-cell wall. It is concluded that embryogenesis commences as soon as the 2 gametophytic cells are laid down. Gene activity associated with postmitotic synthesis of RNA and protein in the vegetative cell is switched off. The data are discussed in relation to the first division of the embryogenic vegetative cells in Nicotiana tabacum.     

An autoradiographic study of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger

Summary The pattern of RNA synthesis during maturation and germination of pollen grains ofHyoscyamus niger was studied using³H-uridine autoradiography. Incorporation of label during pollen maturation was periodic with peak RNA synthesis occurring in the uninucleate, nonvacuolate pollen grains and in the vegetative cell of the bicellular pollen grains. During the early stages of germination, isotope incorporation occurred predominantly in the nucleus of the vegetative cell with little or no incorporation in the generative cell. With the appearance of the pollen tube, incorporation of³H-uridine in the vegetative cell nucleus decreased and completely disappeared at later stages of germination. No incorporation of isotope was observed in the sperms formed in the pollen tube by the division of the generative cell. From a comparison of the results of this study with those of previous works on RNA synthesis during pollen embryogenesis in cultured anthers ofH. niger, it is concluded that in contrast to embryogenic development, there is no requirement for sustained RNA synthesis by the generative cell nucleus for normal gametophytic development.     

Control of the developmental pathway of tobacco pollen in vitro

We developed a new method for culture of isolated pollen. Using highly homogeneous populations of immature pollen grains of Nicotiana tabacum L. prepared by means of Percoll density gradient centrifugation, we could direct their developmental pathway by regulating certain culture conditions. When the pollen population was cultured in basal medium with glutamine, most pollen grains underwent normal maturation. On the other hand, when first cultured in basal medium without glutamine, most pollen grains did not mature but after transfer to medium with glutamine and sucrose began to divide. This method for inducing pollen cell division was possible only with midbinucleate pollen grains which are characterized by having no central vacuole and no or only a few starch grains. Evidently, some essential changes necessary for the embryogenic response can be induced by glutamine starvation only in pollen grains at a specific stage.     

Cloning of cDNA encoding NtEPc, a marker protein for the embryogenic dedifferentiation of immature tobacco pollen grains cultured in vitro

We partially purified three Nicotiana tabacum L. embryogenic pollen-abundant phosphoproteins (NtEPa to c) which appeared in the cells undergoing a dedifferentiation process from immature pollen grains to embryogenic cells, caused by glutamine-deficiency in vitro. All the NtEPs had a highly conserved N-terminal amino acid sequence. Using degenerate oligonucleotide probes designed from the amino acid sequences, the cDNA for NtEPc was isolated from a cDNA library of pollen cultured in glutamine-free medium The cDNA sequence showed moderate homology with several type-1 copper-binding glycoproteins and with a kind of early nodulin though its function could not be predicted. Expression analysis revealed that the level of mRNA for NtEPc was high during the dedifferentiation and also in the very early period of pollen embryogenesis but it was low in the developmental process of microspores/pollen in anthers, in the in vitro maturation process and both in the stational and logarithmic growth phases of tobacco BY-2 cells. Furthermore, an acidic medium pH, which promoted the induction of dedifferentiation increased the level of mRNA for NtEPc, whereas the presence of 6-benzylaminopurine, which inhibited it, decreased the level. These results suggest that the expression of NtEPc gene is correlated with the dedifferentiation but not with pollen development or cell division.     

Differentiation of embryogenic pollen in cold-treated buds ofNicotiana tabacum var. Badischer burley and nutritional requirements of the isolated pollen to form embryos

Summary Embryogenic pollen were selectively isolated from buds after cold treatment at 10 °C for 10 days; it was immaterial whether the buds were taken from short day and low temperature (SD and LT; 8 hours light, 18 °C) or long day and high temperature (LD and HT; 16 hours light, 24 °C) plants. However, in buds from SD and LT plants the differentiation of embryogenic pollen could be detected as early as 7 days after the cold treatment, and pollen from these plants formed embryos at higher frequency (up to 4% of cultured pollen) than those from LD and HT plants (up to 1% only).The embryogenic pollen, in isolated buds, differentiated by way of pollen dimorphism. During cold treatment a fraction of pollen remained small, retained clear cytoplasm and was capable of embryogenesis in comparison to gametophytic pollen which enlarged and acquired granular cytoplasm. In our experiments cold treatment was a key factor in the induction of pollen dimorphism. This aspect of cold treatment in pollen embryogenesis is reported for the first time and was possible on the basis of selection of embryogenic pollen by density gradient centrifugation. The ratio of embryogenic pollen was about one fifth of the total population.The nutritional requirements of isolated pollen for embryogenesis were rather simple. These pollen formed embryos which readily developed into plantlets on a mineral medium supplemented with sucrose provided the pH was 6.8.     

Nutrient uptake and growth of an embryogenic and a non-embryogenic cell line of birch (Betula pendula Roth.) in suspension culture

The nutrient uptake of an embryogenic and of a non-embryogenic cell line of birch (Betula pendula Roth.) during cell growth and embryo production was studied in suspension culture. The embryogenic and non-embryogenic cell suspensions grew differently in the same medium. The non-embryogenic cell line started to grow without any lag period after the inoculation. It rapidly hydrolyzed sucrose in the medium to glucose and fructose and consumed the glucose as carbon source. The concentration of fructose in the medium decreased only after the depletion of glucose. The embryogenic cell line also rapidly hydrolyzed the sucrose to glucose and fructose, but the monosaccharides were consumed only after the embryos started to germinate after three weeks of culture. Both monosaccharides were then taken up at the same rate.