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.     

Microphotometric Determination of DNA Contents of Early Developmental Pollen Grains in Tobacco Anther Culture

The Feulgen-DNA contents of microspores, vegetative and generative nuclei of tobacco pollen grains in vivo and in anther culture have been determined by microphotometry. 1. The values of DNA content of vegetative and generative nuclei of the pollen grains selected at definite developmental stages vary between 1C and 2C levels, which coincide with the role of the dynamics of DNA in haploid cell cycle. This method applied in the study of androgenesis in anther culture is proved successful and valid. 2. By the cytomorphological investigation on androgenesis, the pollen embryoid in this experiment results from repeated divisions of the vegetative cell within the pollen grains. 3. In mature pollen grains of the same variety of tobacco in vivo, DNA replication has not occured in vegetative nuclei, in which the level of DNA remains in 1C. 4. In the cultured anthers after 8 days innoculation, 30% of the total pollen grains measured indicate that the vegetative nuclei have completed DNA replication and show 2C level. The pollen grains which have the potential to differentiate into the embryogenie pollen grains, may be distinguished from non-embryogenie ones by this method before any cytomorphological sign appears. The significance of this method in the study of the mechanism of androgenesis is discussed.     

Patterns of DNA synthesis during pollen embryogenesis in henbane

Continued DNA synthesis in the generative cell nucleus, followed by mitosis and cytokinesis, results in the formation of pollen embryoids in cultured anthers of H. niger. In contrast, the nucleus of the vegetative cell undergoes no DNA synthesis after it is cut off, or synthesizes DNA only during a limited number of cell cycles. DNA synthetic patterns in the generative and vegetative cell nuclei confirm the ontogeny of embryoids described in this plant.     

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.     

Initial Patterns of Androgenesis in Wheat Anther Culture

1. There are mainly two modes of first divisions from pollen cells diverted into sporophytes in anther culture of wheat, i.e. equal and unequal divisions. According to Feulgen reaction of its daughter nuclei and whether they participate in the formation of multicellular pollen or not, we distinguished four basic types of the abnormal pollen, i.e. type A, B. C and D. C and D are the types in which their generative nuclei were involved in the formation of multicellular pollen. 2. Before inoculation, the excised anthers on Ne liquid medium supplemented with 10% sucrose were subjected to a pretreatment for 72 h at 3–5 ℃, then the anthers were suspended on N6 liquid medium containing 12 mg/L IAA, 2 mg/L kinetin, 300 mg/L casein hydrolysate and 10% sucrose. Under these conditions the mean amount of multicellular pollen grains per anther might be increased to 21.42. 3. We also found that the early development of pollens was related to the viability of anther wall tissue. Comparatively, higher exogenous hormones could keep viability and prolong the life of anther wall cells.     

Nuclear fusion in cultured microspores of barley

Fusion of the generative and vegetative nuclei physically separated by a wall has been observed in cultured microspores of barley. The generative cell appears to play an active role in fusion as it elongates toward the vegetative nucleus, becomes detached from the microspore wall, and finally completely encloses the vegetative nucleus. The generative cell wall disappears before nuclear fusion takes place. Since these events have been known to occur during pollen development in vivo, it is hypothesized that the occurrence of nuclear fusion in cultured microspores is the result of continued expression of the genes for gametophytic development.     

In vitro development of plants from microspores of rice

Summary Rice (Oryza sativa L., 2n=24) anthers containing microspores in the early-uninucleate to first-mitosis stages were induced successfully to develop into plants in vitro through an intermediary step of callus formation. Callus initiation occurred with highest frequency in anthers containing mid-uninucleate microspores. The callus derived from different stages of microspore development differed in the potential to differentiate into plants. The plants regenerated from pollen callus were predominantly haploid or diploid; polyploid and aneuploid plants were relatively infrequent. The first division of the uninucleate microspores was asymmetrical, resulting in the formation of large vegetative and small generative nuclei. The vegetative nucleus divided repeatedly and assumed the major role in the formation of callus, whereas the generative nucleus degenerated rapidly. Simultaneous division of the two nuclei was observed in a few pollen grains. Nuclear fusion during the very initial stages of pollen development was postulated to account for the occurrence of the diploid and polyploid plants. This work was supported by the National Science Council, Republic of China.     

Isolation and separation of S-competent and S-incompetent nuclei from Tradescantia pollen grains

A method is described, using discontinuous sucrose density gradients, for the separation of generative and vegetative nuclei from young Tradescantia pollen grains during the period of DNA synthesis. The nuclei are obtained in 20–30% yield from the pollen grains; they are 85–95% pure (generative) and 70–80% pure (vegetative).     

Cytophotometric study of nuclear differentiation during pollen development in Tradescantia Paludosa

Summary During pollen development the dry weight, total protein, histone, DNA, arginine, and lysine content were analysed by cytophotometric methods in partially isolated nuclei. The amount of analysed substances increased from the end of the meiosis to the mitosis of the microspores to the double of the initial values. After mitosis the ratio histone/DNA remained almost unchanged in both vegetative and generative nuclei. On the other hand a large difference in the ratio non-histone protein/DNA could be observed, the vegetative nucleus containing more non-histone protein than the generative nucleus. The rate of RNA synthesis being higher in the vegetative nuclei, these non-histone proteins may have some function in nuclear activation. The DNA of the generative nucleus is duplicated before anthesis, whilst in the vegetative nucleus the DNA content remains constant.     

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.     

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.     

Ultrastructural studies on pollen embryogenesis in maize (Zea mays L.)

Maize anthers have been induced on modified N6 medium to produce embryoids. Different stages from the cultures were sampled and prepared for microscopical examination. The microspores at the onset of culture were in an early developmental stage, with the nucleus and numerous organelles centred in the middle, surrounded by many small vacuoles with a lipid content. The binuclear pollen grains contained small vesicles and much starch. The partially condensed vegetative nucleus indicated participation of the vegetative component in the formation of multicellular pollen grains (MPGs). Several MPGs have been observed which differed in morphology. We suggest, on the basis of these ultrastructural observations, that in maize mainly the vegetative cell contributes to the MPG which further develops directly into embryoids.     

Maturation of maize pollen in vitro

Summary Maturation of maize pollen was obtained in male reproductive structures cultured in vitro. Immature tassels containing microspores at the mid-uninucleate to late-binucleate stage of development were excised and spikelets, anthers, and/or isolated microspores were cultured on a medium capable of supporting pollen maturation. Microspore mitosis, culminating in the production of starch-filled, trinucleate pollen capable of germination, was observed after 7–15 days, depending on the genotype and stage at which the cultures were initiated. Up to 100%, 70%, and 20% of the cultured spikelets, anthers, and isolated microspores, respectively, produced mature pollen, which germinated, however, at different frequencies (i.e., spikelets, 50–70%; anthers, 5–10%; microspores, <1%). Mature kernels were produced following fertilization with pollen from cultured spikelets and anthers. These procedures provide methods for the in vitro manipulation of a significant phase of the maize life cycle.     

Regulation of developmental pathways in cultured microspores of tobacco and snapdragon by medium pH

The regulation of developmental pathways in cultured microspores of tobacco (Nicotiana tabacum L) and snapdragon (Antirrhinum majus L) by medium pH is described for the first time. Unicellular tobacco and snapdragon microspores developed into normal, fertile pollen when cultured in media T1 and AT3 at pH 7.0 and 25°C for 6 and 8 days, respectively. First, pollen mitosis was asymmetric and mature pollen grains were filled with starch granules and germinated upon transfer to a germination medium. However, when tobacco and snapdragon microspores were cultured in media T1 and AT3, respectively, at pH 8.0–8.5 for 4–6 days at 25 °C, the frequency of symmetric division increased significantly with the formation two nuclei of equal size, and the gametophytic pathway was blocked, as seen by the lack of starch accumulation and the inhibition of pollen germination. The transfer of these microspores to embryogenesis medium AT3 at pH 6.5 resulted in the formation of multicellular structures in both species and, in tobacco, in the formation of embryos and plants. In order to understand the possible mechanisms of the action of high pH, sucrose metabolism was analysed in isolated microspores of tobacco cultured at various pH values. Invertase (EC 3.2.1.26) activity in microspores was maximal at pH 5.0 and strongly decreased at higher pH, leading to a slow-down of sucrose cleavage. At the same time the incorporation of ¹⁴C-labelled sucrose from the medium into microspores was drastically reduced at high pH. These data suggest that isolated microspores are not able to metabolise carbohydrates at high pH and thus undergo starvation stress, which was shown earlier to block the gametophytic pathway and trigger sporophytic development.     

Studies on Microspore Development in Liliaceous Plants III. Pollen Tube Development in Lily Pollens Cultured from the Uninucleate Microspore Stage

Uninucleate microspores of Lilium longiflorum isolated at theGi phase of the cell cycle were cultured on a solid nutrientmedium for 12 days. The typical pollens produced in the culturegerminated on a germination medium and developed a structureresembling a pollen tube. The elongating pollen tubes occasionallycontained dividing generative nuclei or two sperm nuclei. (Received September 29, 1980; Accepted November 25, 1980)     

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

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

AN AUTORADIOGRAPHIC STUDY OF RNA SYNTHESIS DURING POLLEN EMBRYOGENESIS IN HYOSCYAMUS NIGER (HENBANE)

RNA synthesis during pollen embryogenesis in cultured anther segments of Hyoscyamus niger (henbane) has been followed by autoradiography of ³H-uridine incorporation. Embryogenic divisions were initiated in binucleate pollen grains in which the generative nucleus or both generative and vegetative nuclei synthesized RNA. When the first haploid mitosis in culture resulted in pollen grains with two nearly identical nuclei, those in which both nuclei synthesized RNA became embryogenic. Binucleate pollen grains in which ³H-uridine incorporation was confined exclusively to the vegetative nucleus gradually became starch-filled and nonembryogenic. Based on the degree of involvement of the vegetative nucleus in embryoid formation, some differences were noted between the counts of autoradiographic silver grains over cells cut off by the generative and vegetative nuclei during progressive embryogenesis. The possible significance of RNA synthesis in the nuclei of binucleate pollen grains in determining the pathway of embryogenic divisions is discussed.     

Anther culture of papaya (Carica papaya L.)

Papaya (Carica papaya L.) anther containing microspores in tetrad to early-binucleate stages were successfully cultured on 1/2 strength MS salts and vitamins with full strength Na-Fe-EDTA supplemented with 2 mg/l NAA, 1 mg/l BA and 6% sucrose for callus initiation and formation. Highest frequencies of callus induction were obtained when anthers at the uninucleate stage were cultured in the dark. Haploid plantlets and pollen-derived embryoids were obtained from anthers cultured at the uninucleate stage on solidified MS medium containing 3% sucrose without any growth regulators under a low light intensity (1,500 lux). Large quantities of embryoids were obtained when the original embryoids were transferred to MS medium with 3% sucrose and no growth regulators. Cytology of root tips of embryoid-derived plants confirmed the haploid chromosome number of 9 indicating that the embryoids originated from pollen.Abbreviations MS Murashige and Skoog (1962) - MAA naphthaleneacetic acid - BA 6-benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid     

AN ATTEMPT TO DETECT UTILIZATION OF DNA BREAKDOWN PRODUCTS FROM THE TAPETUM FOR DNA SYNTHESIS IN THE MICROSPORES OF LILIUM LONGIFLORUM

Takats , Stephen T. (Brookhaven Natl. Lab., Upton, N. Y.) An attempt to detect utilization of DNA breakdown products from the tapetum for DNA synthesis in the microspores of Lilium longiflorum. Amer. Jour. Bot. 49(7): 748–758. Illus. 1962.—The tapetum in anthers of Lilium longiflorum encloses the developing microspores and when it degenerates is a possible source of precursor material for DNA⁴ synthesis in the microspores. To check this, time-course experiments were carried out tracing the fate of label introduced into the tapetal DNA. H³-thymidine was given in vivo to anthers during late pachytene of meiosis, when the tapetum can be selectively labelled. Growth was then followed by sampling anthers until the tapetum degenerated and the microspores synthesized DNA and divided. Autoradiographs indicated that the label in tapetal nuclei was lost shortly before DNA synthesis in the microspores. The microspore walls were transiently labelled, but the microspore nuclei did not incorporate a detectable amount of label. The results are discussed in the light of related biochemical findings, and are explained on the basis of: (1) complete catabolism of tapetal DNA (or tapetal thymine); and (2) the existence of a non-tapetal pool of precursors.