Novel sporophyte-like plants are regenerated from protoplasts fused between sporophytic and gametophytic protoplasts of<Emphasis Type="Italic"> Bryopsis plumosa</Emphasis>

Protoplasts of the marine coenocytic macrophyte Bryopsis plumosa (Hudson) C. Agardh. [Caulerpales] can easily be obtained by cutting gametophytes or sporophytes with sharp scissors. When a protoplast isolated from a gametophyte was fused with a protoplast isolated from a sporophyte of this alga, it germinated and developed into either one of two completely different forms. One plant form, named Type G, appeared quite similar to a gametophyte, and the other, named Type S, looked similar to a sporophyte. While the Type G plant contained many small nuclei of gametophyte origin together with a single giant nucleus of sporophyte origin, the Type S plant contained many large nuclei of uniform size. These large nuclei in the Type S plant had metamorphosed from the gametophytic nuclei, and were not formed through division of the giant nucleus of sporophyte origin. Fragments of the Type S plant, each having such a large nucleus, developed into creeping filaments that look very similar to sporophytes. While cell walls of gametophytes and Type G plants were stained by Congo-red, those of the thalli of regenerated Type S plants and sporophytes were not stained by the dye. This indicated that the large nuclei of the Type S plant did not express genes for xylan synthesis, which are characteristic of gametophytes. Two-dimensional gel electrophoretic analysis revealed that most of the proteins synthesized in the Type S plant were identical to those of sporophytes. These results strongly suggest that in the Type S plant, the gametophytic nuclei are transformed into sporophyte-like nuclei by an unknown factor(s) produced by the giant nucleus of sporophyte origin and that the transformed nuclei express the set of genes characteristic of sporophytes. Despite morphological similarity, however, the regenerated Type S plant could not produce zoospores, because its large nuclei did not divide normally. The transformed large nuclei of gametophyte origin still seemed to be in the haploid state.Abbreviations DAPI 4,6-Diamidino-2-phenylindole - DIC Differential interference contrast - IEF Isoelectric focusing - PES Provasolis enriched seawater     

Visualization of cytoskeletal changes through the life cycle inAcetabularia

Summary The cytoskeleton in the siphonous, marine green algaAcetabularia is visualized by immunocytochemistry using antibodies against plant alfa tubulin and animal smooth muscle actin. In the vegetative phase of the life cycle, when the cell grows a cylindrical stalk and until the reproductive cap is completed, actin forms continuous, parallel bundles that extend through the entire length of the stalk and cap rays respectively. Microtubules (MTs) cannot be detected until the primary nucleus, located in the rhizoid of the giant cell, divides to form thousands of secondary nuclei. MTs can then be seen radiating from each secondary nucleus that is encountered in the stalk on its migration upwards into the cap rays. They are oriented mostly parallel to the long axis of the cell. At arrival in the cap rays up to the white spot stage, when nuclei assume equidistant positions in the cap ray cytoplasm, a radiating system of MTs forms around each nucleus and dramatically increases until impressive radial arrays have developed. This phase coincides with a disappearance of actin bundles in the cap rays, but they are retained in the stalk cytoplasm. Shortly after that additional MTs appear around the disk like partitions of cap ray cytoplasm. Concomitantly, bundles of actin reappear colinearly with the circumferrential MTs eventually forming complete rings around each disk of cap ray cytoplasm. During this process the compartments of the future cysts are gradually bulging outwards and simultaneously the rings of actin sink inwards until domes are formed with the nuclei fixed in the top centers of the domes. At this stage the peripheral areas of the radiating MT systems around the nuclei start to break down, whereas the circumferrential MT systems remain intact. Subsequently, the rings of both actin and MTs decrease in diameter, and finally contract to a spot opposite the nucleus, while the cysts continue to develop their oval shape. After the cysts have become separated, they round up and enter several rounds of nuclear divisions. MTs form short radial arrays around each nucleus with minor changes due to a reduction of MTs during division followed by a reappearance after completion of each division. Actin is rearranged in the cysts to a cortical network of randomly oriented, short bundles, that is maintained until gamete formation sets in.These findings accentuate the involvement of Cytoskeletal elements in the key steps of morphogenesis inAcetabularia to an extent that is unknown in higher plants.     

Electron and immunofluorescence microscopy on the fertilization ofFucus distichus (Fucales,Phaeophyceae)

Summary Processes of fertilization and zygote development inFucus distichus were studied by indirect immunofluorescence microscopy using anti- tubulin antibody and electron microscopy. Just after plasmogamy, sperm aster formation occurs during migration of a sperm nucleus toward an egg nucleus at the center of cytoplasm. Only sparse microtubules (MTs) exist around the egg nucleus. The sperm aster can be observed till karyogamy, but afterwards vanishes. Accompanying sperm aster formation, cortical MTs which are reticulately arranged develop further in the zygotes. In 4 h-old zygotes, characteristic structures which are composed of fine granular masses and consist of intermixed dense and lighter staining areas appear around the nucleus. These structures cannot be detected with anti- tubulin immunofluorescence microscopy. The two centrioles derived from the sperm separate and migrate to both poles. In 4 h-and 8 h-old zygotes, there are no defined MT foci around the zygote nucleus and MTs radiate from the circumference of it. In 12 h-old zygotes, each centriole has migrated to the poles and derivative centrioles are generated. The fine granular masses also migrate to both poles and finally disappear accompanying the appearance of numerous MTs radiating from the poles. Therefore, two distinct MT foci appear from 12 h onwards. Progressive stages of nuclear division were also examined with electron and immunofluorescence microscopy in 16 h-old zygotes. The sperm chloroplast with an eyespot and the sperm mitochondria with an intercristal tubular structure, which are distinctive from those of egg, can be detected after plasmogamy and karyogamy. The sperm chloroplast is still present in 16 h-old zygotes.     

Microtubule formation on the nuclear surface during meiosis inAcetabularia acetabulum

Summary Microtubules (MT) are a feature of all eukaryotic cells. However, they have not been observed in the cytoplasm of the vegetative phase ofAcetabularia acetabulum. Previous investigators have reported that, in the propagative phase, MTs function as anchors in the transport of secondary nuclei to the cap. They also form elaborate arrays around nuclei during cyst formation. The life history ofA. acetabulum is marked by changes in chromatin, the nucleolus, and the perinuclear cytoplasm. In this study light microscopical features of the nucleolus and changes in chromatin, labelled with anti-histon antibodies, were used to define the developmental stages. Anti-tubulin antibodies have been used to trace the origin and development of MTs, MTs are formed on the surface of the primary nucleus. They are organized first into short thick sticks and then later elongate into thinner strands which enclose the nucleus in a dense network. Following these events on the surface of the nucleus, the spindle develops inside the nuclear membrane which remains intact throughout the mitotic division.     

The fine structure of isolated Acetabularia nuclei

Summary Acetabularia nuclei were isolated in solutions of sucrose and fixed, dehydrated and embedded for electron microscopy using a number of techniques. The dimensions of the nucleic changed during all the procedures used, but the addition of calcium to the fixative prevented swelling. The structure observed in isolated nuclei was in good agreement with that seen in nuclei in intact cells and amputated rhizoids. A layer of cytoplasm about 0.1 thick was found on the outside of even the cleanest nuclei — this layer probably gives the nucleus protection when it is isolated.     

Reproduction by Binary and Multiple Fission in Gigantomonas*

SYNOPSIS. Gigantomonas usually exists in the multinucleate stage. Hence, multiple fission is more common than binary fission. In its flagellate as well as its aflagellate state it is always an amoeboid cell which possesses from one to several large, clear pseudopodia. Fairly often in the multinucleate, as well as the uninucleate, stage no extranuclear organelles are present except large plain centrioles, two of which are always closely associated with each nucleus. During nuclear reproduction four centrioles, two old ones and two new ones, are always present with each nucleus. During all nuclear reproductions, regardless of the number of nuclei present, extranuclear organelles, such as flagella, axostyle, undulating membrane, and costa when present are discarded. If they are renewed, it is by the new centrioles at the same time that the old ones produce new central spindles, two always cooperating in the process. Thus, Gigantomonas, like other genera of the Devescovinidae, the family to which it belongs, never has for each nucleus present more than one set of extranuclear organelles, a characteristic which Devescovinidae and Lophomonadidae have in common. It is the only genus of Devescovinidae without a parabasal body. Owing to the liquidness of the cytoplasm, the central spindle, which becomes very long indeed, often extends beyond the cytoplasm and thus pushes the nucleus fastened to this end of it completely out of the cell. Mainly because of this situation, multinucleate forms with an odd number of nuclei occur often; otherwise the nuclear numbers would be 2, 4, 8, 16, 32, etc., because, no matter how many nuclei are present, they all reproduce simultaneously or nearly so. An unusual situation occurs in which Gigantomonas ingests and digests a small species of Holomastigotoides, while several hundred individuals of the latter become attached to and destroy Gigantomonas.     

Chromosome behavior in the primary nucleus ofAcetabularia calyculus as revealed by epifluorescent microscopy

Summary Chromosome behavior preceding secondary nuclei formation within a giant primary nucleus (50–100 m in diameter) inAcetabularia calyculus was observed by the fluorescence emitted from 4-6-diamidino-2-phenylindole (DAPI)-stained DNA.Throughout the period when the large nucleolus was present in the primary nucleus, thin chromonemata were observed twining around the nucleolus. Nuclear division was initiated by degeneration of the sausage-shaped nucleolus into a number of spherical subunits soon after the initiation of cap formation. On the fourth day of cap development, the chromonemata became thicker and chromomeres appeared. They accumulated adjacent to the single spherical nucleolus. The lump of chromosomes became loosened and thick chromosomes were scattered in the nucleus. The peculiar shapes of chromosomes which suggest the existence of chiasmata were frequently observed until the chromosome segregation started. This sequence of chromosome behavior seems to be the prophase of meiotic division. Chromosome segregation, the first meiotic division, occurred on the seventh day of cap development, probably being accompanied by the second meiotic division. Immediately after nuclear division of the primary nucleus, secondary nuclei were formed and cyst formation started 24 hours after repeated mitoses of the secondary nuclei.     

Apertural chambers inGeranium: Development and ultrastructure

Pollen grains ofGeranium robertianum andG. pratense are tricolpate. At the time of the vacuolated microspore stage intine protrusions are formed at each aperture. Each aperture becomes separated from the vegetative cytoplasm by a thick ectintine layer. Starch grains are enclosed in the protrusions and do not participate in pollen tube growth.     

Mitosis in the fungusBasidiobolus ranarum as revealed by electron microscopy

Summary Mitosis of nuclei in vegetative hyphae of the fungusBasidiobolus ranarum has been studied by electron microscopy. Cells fixed with glutaraldehyde and OsO⁴ were embedded in Vestopal. Sections were obtained of single cells whose mitotic status was known. Attention was paid to the behaviour of the microtubules, the nuclear envelope and the nucleolus. Nuclear division begins with the dilution and rearrangement of nucleolar material and the gradual breakdown of the nuclear envelope. At this stage the nucleus is surrounded by a sheet of closely packed microtubules. Some of these penetrate into the nucleus through gaps in the envelope. Dissolution of the envelope is followed or accompanied by the development of an extensive labyrinth of membranous cisternae which persists at the periphery of the division site through mitosis and probably contributes material to the envelopes of the daughter nuclei. The drum-shaped spindle of metaphase is composed of large numbers of microtubules aligned parallel to each other. Many of them are associated with chromosomes. Metaphase is soon followed by the movement of dense masses of nucleolar material and chromosomes to the poles of the division figure to form the socalled end plates. Microtubules extend into the end plates but not beyond. Neither centrioles nor centriolar plaques have been seen.     

MITOSIS IN THE AQUATIC FUNGUS RHIZOPHYDIUM SPHEROTHECA (CHYTRIDIALES)

The structure of centric, intranuclear mitosis and of organelles associated with nuclei are described in developing zoosporangia of the chytrid Rhizophydium spherotheca. Frequently dictyosomes partially encompass the sides of diplosomes (paired centrioles). A single, incomplete layer of endoplasmic reticulum with tubular connections to the nuclear envelope is found around dividing nuclei. The nuclear envelope remains intact during mitosis except for polar fenestrae which appear during spindle incursion. During prophase, when diplosomes first define the nuclear poles, secondary centrioles occur adjacent and at right angles to the sides of primary centrioles. By late metaphase the centrioles in a diplosome are positioned at a 40° angle to each other and are joined by an electron-dense band; by telophase the centrioles lie almost parallel to each other. Astral microtubules radiate into the cytoplasm from centrioles during interphase, but by metaphase few cytoplasmic microtubules are found. Cytoplasmic microtubules increase during late anaphase and telophase as spindle microtubules gradually disappear. The mitotic spindle, which contains chromosomal and interzonal microtubules, converges at the base of the primary centriole. Throughout mitosis the semipersistent nucleolus is adjacent to the nuclear envelope and remains in the interzonal region of the nucleus as chromosomes separate and the nucleus elongates. During telophase the nuclear envelope constricts around the chromosomal mass, and the daughter nuclei separate from each end of the interzonal region of the nucleus. The envelope of the interzonal region is relatively intact and encircles the nucleolus, but later the membranes of the interzonal region scatter and the nucleolus disperses. The structure of the mitotic apparatus is similar to that of the chytrid Phlyctochytrium irregulare.     

Ultrastructural characterization of apospory in Panicum maximum

The nucellar ultrastructure of apomictic Panicum maximum was analyzed during the meiocytic stage and during aposporous embryo sac formation. At pachytene the megameiocyte shows a random cell organelle distribution and sometimes only an incomplete micropylar callose wall. The chalazal nucellar cells are meristematic until the tetrad stage. They can turn into initial cells of aposporous embryo sacs. The aposporous initials can be recognized by their increased cell size, large nucleus, and the presence of many vesicles. The cell wall is thin with few plasmodesmata. If only a sexual embryo sac is formed, the nucellar cells retain their meristematic character. The aposporous initial cell is somewhat comparable to a vacuolated functional megaspore. It shows large vacuoles around the central nucleus and is surrounded by a thick cell wall without plasmodesmata. In the mature aposporous embryo sac the structure of the cells of the egg apparatus is similar to each other. In the chalazal part of the egg apparatus the cell walls are thin and do not hamper the transfer of sperm cells. Structural and functional aspects of nucellar cell differentiation and aposporous and sexual embryo sac development are discussed.     

Immunofluorescence and electron microscopic studies of microtubule organization during the cell cycle ofDictyota dichotoma (Phaeophyta,Dictyotales)

Summary Interphase cells ofDictyota dichotoma (Hudson) Lamour. lack cortical microtubules (Mts) but display an impressive network of cytoplasmic microtubules (c-Mts). These are focussed on two opposed perinuclear centriolar sites where centrin or a centrin-homologue is localized. Some of the Mts surround the nucleus, but the majority traverse the cytoplasm as bundles variously directed towards the plasmalemma. In apical cells, and to a lesser extent in the square or slightly elongated meristematic cells, Mts are more or less evenly arranged. In elongated cells they form thick bundles longitudinally traversing the cytoplasm; a pattern maintained in differentiated cells. In early prophase the non-perinuclear Mts disappear but by late prophase a bi-astral arrangement of short Mts is observed. They enter polar nuclear depressions and attach to differentiated regions of the nuclear envelope where polar gaps open. By metaphase the spindle Mts converge on the centrioles at the polar gaps. At anaphase, interzonal Mts are evident and the asters start to reassemble. After telophase disruption of the interzonal Mts, the daughter nuclei approach each other, but move apart again before cytokinesis. The latter movement keeps pace with the development of two interdigitating Mt systems, ensheathing both daughter nuclei. The partition membrane bisects this Mt cage. Between telophase and cytokinesis the centrosomes separate, finally occupying opposed perinuclear sites. New Mts arise at the new centrosomes, some terminating on the consolidating partition membrane. Our data show thatD. dichotoma vegetative cells display a prominent cytoplasmic Mt cytoskeleton, which undergoes continual, but definite, change in organization during the cell cycle.     

Development of the resting sporangia ofSynchytrium endobioticum,the causal agent of potato wart disease

Summary An ultrastructural study of the development of the resting sporangium ofSynchytrium endobioticum (Schilb.) Perc. infecting potato cells is presented. The resting sporangium is found to have a single large, centrally placed nucleus with a prominent nucleolus through its entirein situ development. The cytoplasmic organization of the resting sporangium is further characterized by numerous membrane-bound lipid bodies and osmiophilic bodies. The latter have a characteristic sieve-like appearance, probably because certain storage components have been extracted during preparation for electron microscopy. Because of the similar location and appearance of these osmiophilic bodies it is suggested that they are identical to what has earlier (based on light microscopy) been described as chromatin granules; and the ultrastructural studies presented here show that nucleolar discharge which was described from light microscopic observations as leading to chromatin granules in the cytoplasm, and finally forming the nuclei of the zoospores (bally 1912,curtis 1921,percival 1910) simply does not occur.The appearance of dense fibrillar-like structures on the sporangial surface at an early stage of resting sporangium development ultrastructurally distinguishes the resting sporangium from the zoosporangium. The development of the layered portion of the thick sporangial wall is shown to be due to the fusion of vacuoles containing pre-made wall fibrils with the cell membrane. It is suggested that the inner compact wall layer which is essentially substructureless is formed by the membrane itself.The characteristic wings of the matureS. endobioticum resting sporangium originate from the potato host cell wall. Remnants of host cell organelles in the outermost layer of the resting sporangium wall show that degradation of the host cell cytoplasm contributes to wall formation of the parasite.     

中国鲎精子发生的研究:Ⅱ.精子形成

应用电技术研究中国鲎精子形成过程和特点,早期精子细胞核圆,染色质呈网状分布,胞质中有许多线粒体和高尔基液泡,精子形成期间,出现合胞体现象,核分裂而胞质不分裂,细胞通过胞质桥相联系,随后出现隔板,逐渐将细胞核分开。细胞随访核向后移动而呈现极化现象。核的顶部出现一个高尔基－线粒体区,顶体由高在液泡演变而。精子形成期间,核中央位置形成贯穿整个的中央通道,并在核中形成植入窝,中心粒位于其中,成熟精子胞质极     

Disruption of microtubules and retardation of development ofDictyostelium with ethylN-phenylcarbamate and thiabendazole

Summary The effects of ethylN-phenylcarbamate (EPC) and thiabendazole (TB) onDictyostelium discoideum andD. mucoroides cells were examined as a step toward purifying tubulin and clarifying the function of microtubules in cellular slime molds. EPC (1.5 × 10^–3M) or TB (5 × 10^–5M) inhibited the development ofDictyostelium, inducing the formation of aberrant fruiting bodies with stalks irregular in shape and sori containing spores of various sizes and shapes.EPC and TB inhibited cell division but not cell growth, resulting in the production of giant cells up to ten times larger than untreated cells. The giant cells either had a single huge nucleus of irregular shape or contained multiple nuclei. The effects of the inhibitors were reversible. After the removal of the inhibitors, the giant cells underwent successive cell divisions producing many daughter cells. Interestingly, most of the giant cells induced by EPC treatment contained gigantic secondary lysosomes probably produced by extensive lysosomophagy.Light microscopy using Nomarski optics revealed that these inhibitors caused the round-up of the cells resulting in the inhibition of cell locomotion, whereas non-Brownian movement of the cytoplasmic granules was not affected. Indirect immunofluorescence using anti--tubulin revealed that networks of microtubules were apparently destroyed by the EPC or TB treatment.These results show both EPC and TB are potent inhibitors of microtubules inDictyostelium and are effective tools for studying the function of microtubules either in cellular or multicellular organization throughout its life cycle.     

Selective disappearance of maternal centrioles after fertilization in the anisogamous brown alga Cutleria cylindrica (Cutleriales, Phaeophyceae): Paternal inheritance of centrioles is universal in the brown algae

The behavior of centrioles in zygotes and female gametes developing parthenogenetically in the anisogamous brown alga Cutieria cyiindrica Okamura was studied using electron and immunofluorescence microscopy. Two pairs of centrioles, detected using anti-centrin antibody, were observed in the vicinity of the male and female nuclei, respectively, just after plasmogamy. The fluorescence intensity of one of the two centrin foci became weak 6 h after plasmogamy and finally disappeared. It was impossible to determine whether the male- or female-derived centrioles disappeared in zygotes, because there was nothing to detect morphological differences between the two centrioles. However, a prominent anti-centrin staining focus was located at the condensed male nucleus in zygotes in which karyogamy had not occurred yet. As a result, it was considered that the maternally inherited centrioles had selectively disappeared during development in C. cylindrica. The paternal inheritance of centrioles in zygotes was also confirmed by electron microscopy. Considering previous observations from oogamous and isogamous species of brown algae, we concluded that the paternal inheriance of centrioles could be universal in the brown algae.     

Ultrastructure of germination and mucilage production inHalosphaeria appendiculata (Halosphaeriaceae)

The germination of ascospores of the marine fungusHalosphaeria appendiculata was investigated with transmission electron microscopy. Prior to germination, settled ascospores became surrounded by a fibro-granular layer. Small, membrane-bounded vesicles and larger electron-dense membrane-bounded vesicles aggregated at the site of germ tube formation where the plasmalemma adjacent to the aggregation was convoluted. The vesicles appeared to fuse with the plasmalemma, releasing their contents. Enzymatic digestion of the spore wall probably occurred at the time of germ tube emergence. After the nucleus had migrated into the newly formed germ tube, a septum was formed to delimit the germ tube from the ascospore. The growing germ tube can be divided into 3 morphological regions, namely the apical, sub-apical and vacuolated regions, and is typical of other fungi. A mucilaginous sheath was associated with the older mycelium. The germ tube displaced the polar appendage, and the ascospore, germ tube and appendage were enclosed in a mucilaginous sheath. In ascospores which subtended old germ tubes, the nucleus and lipid body became irregular in shape and the cytoplasm was more vacuolated. Microbody-like structures remained associated with the lipid throughout development, and were present in old ascospores.     

Structure and Development of the Endophyte in the Parasitic Angiosperm <Emphasis Type="Italic">Cuscuta japonica</Emphasis>

The endophyte, that is, the haustorial part within the tissues of the host plant Impatiens balsamina, of the parasitic angiosperm Cuscuta japonica was studied with light and electron microscopy. The endophyte consisted mainly of vacuolated parenchymatous axial cells and elongate, superficial (epidermal) cells. Then the elongate, epidermal cells separated from each other and transformed into filamentous cells, called searching hyphae. The hyphae grew independently either intercellularly or intracellularly in the host parenchyma. The apical end of the hyphal cells was characterized by conspicuous, large nuclei with enlarged nucleoli and very dense cytoplasm with abundant organelles, suggesting that the hyphal cells penetrating host tissue were metabolically very active. Numerous osmiophilic particles and chloroplasts were noted in the hyphae. The osmiophilic particles were assumed to be associated with elongation of the growing hyphe. Plasmodemata connections between the searching hyphal cells of the parasite and the host parenchyma cells were not detected. Hyphal cells that reached the host xylem differentiated into water-conducting xylic hyphae by thickening of the secondary walls. A xylem bridge connecting the parasite and the host was confirmed from serial sections. Some hyphal cells that reached the host phloem differentiated into nutrient-conducting phloic hyphae. Phloic hyphae had a thin layer of peripheral cytoplasm with typical features of sieve-tube members in autotrophic angiosperms, i.e., parallel arrays of smooth endoplasmic reticulum, mitochondria, and plastids with starch granules. Interspecific open connections via the sieve pores of the host sieve elements and plasmodesmata of the parasite phloic hyphae were very rarely observed, indicating that the symplastic translocation of assimilate to the parasite from the host occurred.     

Nuclear and cell migration during pollen development in rice (Oryza sativa L.)

Nuclear and cell migration during pollen development in rice were studied using semi-thin section light microscopy, differential interference contrast microscopy and epifluorescence microscopy. Four migrations of nuclei and cells were observed and described in detail here. The first nuclear migration occurs at the uninucleate microspore stage, when the nucleus of the microspore migrates from the center to the periphery of the cell, and then to the wall opposite the pollen aperture where pollen mitosis I takes place. The second migration occurs at the early bicellular pollen stage, with the vegetative nucleus migrating three-quarters of the circumference of the pollen wall, finally locating at the periphery of the wall where the microspore cell nucleus is positioned. The third migration occurs at the late bicellular pollen stage, with the vegetative nucleus migrating from the periphery of the cell to the central part of the pollen and the generative cell migrating from the opposite side of the aperture to a position between the aperture and the vegetative nucleus where pollen mitosis II takes place. The fourth migration appears at the mature pollen stage when the two sperm cells and the vegetative nucleus migrate to the opposite side of the aperture, finally becoming positioned in the cytoplasm of the vegetative cell distal to the aperture where the male germ unit forms. Cytological observations of pollen abortion resulting from allelic interaction at the S-a, S-b and S-c loci show that abnormalities in the first or second nuclear migration result in the formation of empty abortive pollen, whereas abnormalities in the third or fourth migrations cause production of stainable abortive pollen.