Karyogamy after Electrofusion of Single Egg and Sperm Cell Protoplasts from Maize: Cytological Evidence and Time Course

In maize, in vitro fusion of isolated male gametes with isolated egg cell protoplasts can be induced by electric pulses. Until now, karyogamy has not been demonstrated. In this study, we cytologically examined fusion products fixed at different times after electrofusion with phase contrast microscopy and transmission electron microscopy. We obtained a precise timetable from 23 samples studied during the first 3 hr. The sperm nucleus was integrated within the egg cell protoplast, migrated toward the egg cell nucleus, and fused with it within 1 hr, as demonstrated by ultrastructural observations, three-dimensional reconstructions of nuclei, and subsequent nuclear volume estimates. Fusion of nuclei occurred before zygotic mitosis, as is the case in vivo. These findings demonstrate karyogamy during in vitro fertilization of maize.     

Observations on the time course of wall development at the surface of isolated protoplasts

The formation of cell wall fibres at the surface of isolated leaf protoplasts has been studied by scanning electron microscopy. Fibres are not formed on incubated protoplasts until a lag period has elapsed. This period is about 8 h for leaf protoplasts of Nicotiana tabacum and about 45 h for leaf protoplasts of Antirrhinum majus. In the case of Antirrhinum protoplasts the length of the lag period is dependent on the concentration of osmoticum present during the incubation period. If regenerating protoplasts are briefly treated with dilute cellulase, the newly formed wall is completely digested. Such protoplasts are capable of producing new fibres at the surface within minutes of their return to a nutrient medium. These results are discussed in terms of the likely source of the lag period and its significance in wall regeneration studies.Abbreviations MS culture medium used at full strength - 0.1 MS culture medium used at one tenth full strength     

THE FINE STRUCTURE OF OOGENESIS IN MARCHANTIA POLYMORPHA

Archegonium development, beginning with the archegonial initial and culminating in the mature egg, was studied with the electron microscope. The ultrastructural features of the beginning stages in development of the archegonium are relatively similar to one another. Plasmodesmata occur between all adjacent cells at this time. After the secondary central cell is formed these protoplasmic connections are lost, and both axial and parietal cell lineages begin to show signs of ultrastructural differentiation. The mature egg is characterized by cytoplasm rich in ribosomes and larger organelles. Mitochondria and simplified plastids commonly display a juxtaposed association. As far as could be ascertained the numerous plastids and mitochondria in the egg of Marchantia arise through division of preexisting organelles and are not formed anew from evaginations of the nucleus. Blebbing of the nucleus produces polymorphic organelles which appear to be pinched off into the cytoplasm. The mature egg also contains vacuoles and lipid bodies toward its periphery, while dictyosomes and extensive endoplasmic reticulum occur throughout. The space between the wall cells and the mature egg appears to contain an amorphous substance. No extra membrane was observed around the mature egg.     

Early cytological events after induction of cell division in egg cells and zygote development following in vitro fertilization with angiosperm gametes

Early events, such as formation of the cell wall, first nuclear division and first unequal division of the zygote, were examined following in vitro fusion of single egg and sperm protoplasts of maize ( Zea mays L.). The time course of these events was determined. The formation of cell wall components was observed 30 sec following egg—sperm fusion and proceeded continuously thereafter. Within 15 h after fusion most of the organelles became more densely grouped around the nucleus of the zygote. In the in vitro produced zygote the location of the cell organelles and of the dividing nucleus showed polarity. Two nucleoli were first observed 18 h after gamete fusion. The zygotic nucleus remained undivided for about 40 h. The first cell division was observed 40–60 h, generally 42–46 h, after egg—sperm fusion. The non-fused egg cell could be triggered to sporophytic development in vitro by pulses of high amounts of 2,4-D. Without such a treatment, cultured egg cells of different maize lines did not divide. Although nuclear fusion seemed to occur, fusion products of two egg cells also did not divide. Cell wall formation was incomplete and non-uniform, showing a polarity of cultured egg cells and fusion products of two egg protoplasts. Cell division was also induced after fusion of maize egg with sperms of genetically remote species, such as Coix, Sorghum, Hordeum or Triticum . These gametic heterologous fusion products developed to microcalli. Moreover, cell division occurred in fusion products of an egg and a diploid somatic cell-suspension protoplast from maize.     

Studies on the growth and development of protoplasts of the moss,Physcomitrella patens,and its control by light

Protoplasts prepared from protonemal cultures of the moss Physcomitrella patens begin to regenerate a new cell wall within 1 h of removal from cellulase. The wall is seen as a gradually thickening mat of fibres when examined by scanning electron microscopy. Development of filaments from protoplasts takes place in the majority of cases only after one or more cell divisions have occurred. The direction of emergence of filaments is random in uniform light, but strongly negatively phototropic in bright unidirectional horizotal light. Filament growth is also strongly negatively phototropic. The influence of unidirectional light can be destroyed by incubating protoplasts in the presence of colchicine. Filaments growing in unidirectional light have cytoplasmic microtubules running along their long axes and in close association with large organelles. These results are discussed in terms of the potential for this system for the study of polarity in plants.     

High pressure freezing and freeze substitution reveal new aspects of fine structure and maintain protein antigenicity in barley aleurone cells

High pressure freezing and freeze substitution (HPF-FS) were used to prepare barley ( Hordeum vulgare L. cv Himalaya) aleurone protoplasts for transmission electron microscopy (TEM). We show that HPF-FS is superior to conventional chemical fixation and dehydration techniques for the preservation of cellular fine structure and antigenicity of proteins in barley aleurone protoplasts. HPF-FS extracted fewer proteins from the cytosol and organelles of aleurone protoplasts and maintained the details of cellular structure. The cortical cytoskeleton, made up of microtubules, was observed for the first time by TEM in barley aleurone protoplasts prepared by HPF-FS. Organelles such as protein storage vacuoles retained their proteinaceous contents, and other cellular organelles (including the Golgi apparatus, the nucleus and mitochondria) were also well preserved in protoplasts fixed by HPF-FS. Antibodies to the vacuolar enzyme nuclease I, the tonoplast aquaporin α-TIP and the glyoxysomal enzyme malate synthase showed that the antigenicity of organellar enzymes and membrane proteins was preserved in cells prepared by HPF-FS. We conclude that HPF-FS is superior to chemical fixation for the preparation of plant protoplasts for TEM and is the method of choice for the preservation of aleurone protoplasts for structural and immunochemical studies.     

Ultrastructure and microtubule organization of isolated generative cells ofAllemanda neriifolia at interphase and prophase

Summary The ultrastructure of isolated generative cells ofAllemanda neriifolia at interphase and prophase was studied. The microtubule organization of the isolated cells was also investigated by immunofluorescence microscopy with a monoclonal anti--tubulin. After the generative cells had been isolated from the growing pollen tubes by osmotic shock, most of the cells were at prophase and only a few were at interphase. The interphase cell is spindle shaped and contains an ellipsoidal nucleus. In addition to the usual organelles, the cytoplasm of the interphase cell contains numerous vesicles (each measuring 40–50 nm in diameter) and two sets of longitudinally oriented microtubule bundles — one in the cortical region and the other near the nucleus. Most of the prophase cells are spherical in shape. Based on the ultrastructure and the pattern of microtubule cytoskeleton organization three types of prophase cells can be recognized. (1) Early prophase cell, which contains the usual organelles, numerous vesicles, and a spherical nucleus with condensed chromosomes. Longitudinally oriented microtubule bundles can no longer be seen present in the early prophase cell. A new type of structure resembling a microtubule aggregate appears in the cytoplasm. (2) Mid prophase cell, which has a spherical nucleus containing chromosomes that appear more condensed than those seen in the early prophase cell. In addition to containing the usual organelles, the cytoplasm of this cell contains numerous apparently randomly oriented microtubules. Few vesicles are seen and microtubule aggregates are no longer present. (3) Late prophase cell, typified by the lack of a nuclear envelope. Consequently, the chromosomes become randomly scattered in the cytoplasm. Microtubules are still present and some become closely associated with the chromosomes. The changes in the ultrastructure and in the pattern of microtubule organization in the interphase and prophase cells are discussed in relation to the method of isolation of the generative cells.     

An Ultrastructural Study on Triggering of Cell Division in Young Pollen Protoplast Culture of Hemerocallis fulva L.

The ultrastructural changes of young pollen protoplasts under culture condition in Hemerocallis fulva were studied. In comparison with the original pollen grains, the pollen protoplasts had been completely deprived of pollen wall, but kept the internal structure intact, including a large vacuole, a thin layer of cytoplasm and a peripherally located nucleus. After 8 days of culture a few pollen protoplasts were triggered to cell division: some of them were just undergoing mitosis with clearly visible chromosomes and spindle fibers; the others already divided into 2-celled units. The two daughter cells were equal or unequal in size but with similar distribution of organelles inside. Besides cell division, there were also free nuclear division, amitosis and formation of micronuclei indicating a diversity of division modes in pollen protoplast culture, A series of changes occurred during the process of induction of cell division, such as locomotion of the nucleus toward the central position, disappearence of the large vacuole, increase of electron density of cytoplasm, increase and activation of organelles, diminishing of starch granules in plastids, etc. However, the regeneration of surface wall was not sufficient it contained mostly vesicles with only a few microfibrits. The wall separating the two daughter cells were either complete or incomplete. The weak capability of wall formation is supposed to be one of the major obstacles which has so far restricted sustained cell divisions of young pollen protoplasts under current culture condition.     

ORGANIZATION OF ISOLATED EMBRYO SACS AND EGGS OF PLUMBAGO ZEYLANICA (PLUMBAGINACEAE) BEFORE AND AFTER FERTILIZATION

The organization of isolated embryo sacs and eggs of Plumbago zeylanica was described before and after fertilization using microscopic cytochemistry and scanning electron microscopy. Major developmental events of fertilization, including preferential fertilization and early embryogenesis, are described in isolated embryo sacs. The two sperms, one unassociated with vegetative nucleus (S_ua) and the other physically associated with the vegetative nucleus (S_vn), fuse with nuclei of egg and central cell, respectively. The zygote divides asymmetrically to form a two-celled embryo, consisting of a massive suspensor occupying most of the micropylar portion of the embryo during early embryogenesis. Plastids are distributed in the perinuclear and micropylar regions of the egg cell and in cytoplasmic strands of the central cell before fertilization. Calcofluor white-positive fibrillar material in the filiform apparatus (presumed β-1,4 linked glucans) was investigated using scanning electron microscopy. The egg of P. zeylanica can easily be divided into three cytologically distinct regions: 1) perinuclear cytoplasm, 2) lateral cytoplasm, and 3) micropylar cytoplasm. Cytological differences are evident in the organization of the cell walls, general degree of vacuolization, and the distribution of heritable organelles, storage bodies, and microtubules. The present study supports the concept that the egg of P. zeylanica plays combined synergid and gamete functions.     

Fertilization and rejection of spermatozoids by egg cells in artificially pollinated ovules ofEncephalartos (Zamiaceae)

The structure and behaviour of free female, male and proembryonal nuclei ofEncephalartos villosus Lem. were studied during a light-microscopical investigation of serially sectioned archegonia in successfully pollinated ovules. Before spermatozoids were released from the pollen tubes into the archegonial chamber, the ventral canal nucleus had disintegrated in the neck region of the egg cell among minute, amoeboid bodies with PAS-positive granules. In archegonia containing multiple spermatozoids, the egg nucleus was unobtrusive and syngamy followed by proembryo formation regularly resulted. The egg cell usually reacted violently in archegonia penetrated by a single spermatozoid. These reactions were regarded as rejection phenomena and considered as indicators that the egg cell can differentiate between compatible and incompatible male gametes.     

Transfer of defined numbers of chloroplasts into albino protoplasts using an improved subprotoplast/protoplast microfusion procedure: Transfer of only two chloroplasts leads to variegated progeny

Summary A procedure is described by which it is possible to perform controlled microfusion of microscopically selected protoplast fusion partners with high efficiencies. The procedure is applied to fusion of Nicotiana tabacum (line 92V37, N. undulata cytoplasm) plastid albino protoplasts as a recipient and spontaneously formed subprotoplasts of green N. tabacum (line SRI) as donor. Products of individual electrofusion events are cloned via single cell nurse culture and the derived cell lines are analysed for the occurrence of variegated or green regenerating shoots, which are indicative of the establishment of the transferred organelles in the cell progeny. The plastid population in green regenerants recovered after the transfer of only two chloroplasts was demonstrated to have originated from the donor subprotoplast organelles by restriction analysis of total DNA using a plastome-specific probe.Some of the results described in this paper have been presented as posters at scientific meetings (Eigel and Koop 1989b; Eigel and Koop 1990)     

Nontranslation of foreign genetic information for fraction 1 protein under circumstances favorable for direct transfer ofNicotiana gossei isolated chloroplasts intoN. Tabacum protoplasts

Summary The nuclei and cytoplasm ofN. gossei andN. tabacum are compatible to the extent that reciprocal, interspecific F₁ hybrids can be produced by conventional breeding techniques. Conditions were established in which manyN. gossei isolated chloroplasts could be seen by phase and fluorescence microscopy to adhere to 40% of the population of protoplasts obtained from white tissue of variegatedN. tabacum plants and to remain attached after washing the protoplasts. Chloroplasts also could be seen to enter the interior of the protoplasts. After treating albino protoplasts withN. gossei chloroplasts, the protoplasts were subjected to further conditions whereby 65 calluses containing shoots developed. TwentyN. tabacum protoplasts not treated with foreign chloroplasts also produced calluses with shoots to serve as a control. All calluses developed chlorophyll irrespective of whether or not the albino protoplasts had been treated with isolatedN. gossei chloroplasts. The Fraction 1 protein ofN. tabacum has a different electrophoretic mobility from the protein ofN. gossei or anN. gossei xN. tabacum F₁ hybrid. The Fraction 1 protein large subunit is coded by chloroplast DNA, whereas the small subunit is coded by nuclear DNA. Fraction 1 protein was isolated from the variegated shoots of the 65 calluses obtained after treating albino protoplasts with foreign chloroplasts. Immunoelectrophoresis demonstrated the protein from each callus to have a mobility identical toN. tabacum protein. Therefore, under circumstances highly favorable for the direct transfer ofN. gossei isolated chloroplasts (and possibly nuclei also) intoN. tabacum protoplasts, no evidence was obtained to suggest that genetic information contained in the isolated foreign organelles was being translated into the polypeptides of either the large or small subunits of Fraction 1 protein contained in newly differentiated leaves derived from the protoplasts. Supported by Research Grant PCM-75-07368 from the National Science Foundation.     

Cytological evidence of biparental inheritance of plastids and mitochondria inPelargonium

Summary Based on the organelle differences between egg and sperm cells inPelargonium hortorum, the zygote, proembryo, and endosperm were examined under the transmission electron microscope. Plastids and mitochondria in the egg cell are significantly different from those of the sperm cell. Egg plastids are starch-containing and less electron dense. They appear circular, elliptical irregular elongate in sections. Sperm cell plastids are relatively electrondense, mostly cup-shaped or dumbbell and devoid of starch granules. Mitochondria of the egg cell are giant and mostly cup-shaped while sperm mitochondria are smaller and usually circular in section. Double fertilization is completed by 24 h after pollination and the pollen tube can be seen in the degenerated synergid. In the zygote, plastids and mitochondria from male and female gametes can be distinguished by their characteristic differences. Moreover, paternal and maternal organelles appear to be distributed at random in the zygote. Aside from the pollen tube and its released starch granules, there is no enucleated cytoplasmic body in the degenerated synergid. Two days after pollination, the zygote undergoes one transverse division to form a 2-celled proembryo which consists of one larger vacuolated basal cell and one smaller densely cytoplasmic apical cell. Paternal and maternal organelles can be detected in both cells of the proembryo and also in the endosperm at this stage. From these results, it can be concluded that plastids and mitochondria from both male and female gametes have been transmitted into the apical cell of the proembryo and most probably to the following generation.Abbreviations TEM transmission electron microscope - DAPI 4,6-diamidino-2-phenylindole - RFLP restriction fragment length polymorphism     

Transfer of organelles of the alga Chlamydomonas reinhardii into carrot cells by protoplast fusion

A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.Abbreviations PEG polyethylene glycol - TEM transmission electron microscopy - SEM scanning electron microscopy This study was undertaken during sabbatical leave in The Research School of Biological Sciences. Australian National University     

A light and electron microscopic study of the quadriflagellate green algaSpermatozopsis exsultans

The green flagellateSpermatozopsis exsultans Korshikov has been studied in culture by light and electron microscopy. The organism is naked, bears four flagella and is conspicuously spirally twisted. The ultrastructure and location of cell organelles (except the flagellar apparatus) has been investigated in detail using an absolute configuration analysis. With the exception of a doubling of the flagella and of the secondary cytoskeletal microtubule system,S. exsultans has the exact same complement of organelles occupying the same relative positions as has been described forS. similis. The two species are therefore correctly placed in the same genus. The usefulness of absolute orientations of cell organelles for green algal taxonomy and phylogeny is stressed.Dedicated to Prof.M. Mix on the occasion of her 60th birthday.     

Paternal cytoplasmic transmission in Chinese pine (Pinus tabulaeformis)

Summary. In this paper, the stages of normal sexual reproduction between pollen tube penetration of the archegonium and early embryo formation in Pinus tabulaeformis are described, emphasizing the transmission of parental cytoplasm, especially the DNA-containing organelles – plastids and mitochondria. The pollen tube growing in the nucellus contained an irregular tube nucleus followed by a pair of sperm cells. The tube cytoplasm contained abundant organelles, including starch-containing plastids and mitochondria. The two sperm cells differed in their volume of cytoplasm. The leading sperm, with more cytoplasm, contained abundant plastids and mitochondria, while the trailing one, with a thin layer of cytoplasm, had very few organelles. The mature egg cell contained a great number of mitochondria, whereas it lacked normal plastids. At fertilization, the pollen tube penetrated into the egg cell at the micropylar end and released all of its contents, including the two sperms. One of the sperm nuclei fused with the egg nucleus, whereas the other one was retained by the receptive vacuole. Very few plastids and mitochondria of male origin were observed around the fusing sperm and egg nuclei, while the retained sperm nucleus was surrounded by a large amount of male cytoplasm. The discharged tube cytoplasm occupied a large micropylar area in the egg cell. In the free nuclear proembryo, organelles of maternal and paternal origins intermingled in the neocytoplasm around the free nuclei. Most of the mitochondria had the same features as those of the egg cell, but some appeared to be from sperm cells and tube cytoplasm. Plastids were obviously of male origin, with an appearance similar to those of the sperm or tube cells. After cellularization of the proembryo, maternal mitochondria became more abundant than the paternal ones and the plastids enlarged and began to accumulate starch. The results reveal the cytological mechanism for paternal inheritance of plastids and biparental inheritance of mitochondria in Chinese pine. Correspondence and reprints: State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Science, China Agricultural University, Beijing 100094, People’s Republic of China.     

萱草幼嫩花粉原生质体培养启动细胞分裂的超微结构研究

萱草(Hemerocallis fulva L.)幼嫩花粉,即后期小孢子原生质体在培养8天时进入有丝分裂或已形成二个细胞。此外,还观察到游离核分裂、无丝分裂、微核形成等现象。这显示了花粉原生质体分裂方式的多样性。在启动分裂时发生一系列变化:如细胞核移位、大液泡消失、细胞质电子密度增加、细胞器增多、质体不含淀粉等。再生的细胞壁含许多小泡,很少纤丝,表现出现有培养条件下壁的形成能力薄弱。这是今后改进培养技术需要特别注意的问题。     

Plant regeneration from the protoplasts of Solanum tuberosum, S. nigrum and S. bulbocastanum

The mesophyll protoplasts were isolated from the Solanum tuberosum (S. tbr) clones of different ploidy level (4x Bzura cv., 2x H-8105, and 2x ZEL-1136) as well as from the wild species: S. bulbocastanum (S. blb, 2x) and two accessions of S. nigrum (S. ngr, 6x). Additionally, the protoplasts were isolated from the cell suspensions of Bzura cv. and H-8105 clone. The conditions of protoplast isolation as well as the media for their culturing and regeneration, were selected and optimized for the studied genotypes. For mesophyll protoplasts, the shooting calli were produced by all the cultured protoclones except that of S. bulbocastanum. The shoots excised from the protoplast-derived calli developed into whole plants in all the studied potato clones but only in one accession of S. nigrum, i.e. S. ngr var. gigantea. As for suspension-cell-derived protoplasts, only H-8105 clone produced the regenerative type of calli, though normal shoots could not be obtained. The regenerative capacity of the protoplasts isolated from leaves and cell suspensions is compared and discussed. We regret to report the death of M. Sc. Maria Borkowska after the completion of this work.     

Ultrastructure of the cell wall regeneration of isolated protoplasts of Skimmia japonica thunb

Isolated protoplasts obtained from leaves and from stem callus cultures of Skimmia japonica were cultivated for 72 h to regenerate a new cell wall. During this process the structural changes in the protoplasts and at the surface of the plasmalemma were studied in ultrathin sections and after freeze-fracturing and deep-etching.The cultured protoplasts show an apparent increase in cell organelles compared to the freshly isolated protoplasts. In particular, mitochondria, endoplasmic reticulum, and ribosomes, many of them appear as polysomes, become numerous. Moreover, special connections between the ER and the plasmalemma are visible. Most important are the fracture faces of the plasmalemma with two different arrangements of membrane-bound particles: (1) particles in hexagonal arrays and (2) rows of ca. 14 particles. Their orientation usually conforms with that of the regenerated microfibrils of the cell wall. According to these results the following model for microfibril synthesis and orientation in higher plants is proposed: While the cytoplasmic activity is involved in the production of cellulose precursors and enzymes, the hexagonal arrays may respresent specialized regions for the outward passage of these cellulose precursors. The rows of membrane-associated particles may function as a linear enzyme complex (matrix) for microfibril biosynthesis and orientation.Abbreviations ER endoplasmic reticulum - IAA -indolylacetic acid - 2,4-D 2,4-dichlorophenoxy acetic acid     

Cytological Events during Zygote Formation of the Fern Ceratopteris thalictroides

The cytological events, including nuclear fusion, digestion of male organelles and rebuilding of the plasmalemma and cell wall, during zygote formation of the fern Ceratopteris thalictroides (L.) Brongn. are described based on the observations of transmission electron microscopy. When the spermatozoid enters the egg and contacts the cytoplasm, the male chromatin relaxes continually. The microtubular ribbon (MTr) is separated from the male nucleus and then an envelope reappears around the male nucleus. During nuclear fusion, the egg nucleus becomes highly irregular and extends some nuclear protrusions. It is proposed that the protrusions fuse with the male nucleus actively. After nuclear fusion the irregular zygotic nucleus contracts gradually. It becomes spherical before the zygote divides. The male chromatin is identifiable as fibrous structure in the zygotic nucleus in the beginning, but it gradually becomes diffused completely. The male organelles, including the MTr, multilayered structure, flagella and the male mitochondria are finally digested in the zygotic cytoplasm. Finally a new plasmalemma and cell wall are formed outside the protoplast. The organelles in the zygote are rearranged, which produces a horizontal polarity zygote. The zygote divides with an oblique-vertical cell plate facing the apical notch of the gametophyte.