NUCLEAR MIGRATION IN GERMINATING SPORES OF ONOCLEA SENSIBILIS: THE PATH AND KINETICS OF MOVEMENT

Nuclear migration was observed in individual germinating spores of Onoclea sensibilis from the onset of movement to the completion of mitosis. About 16 hr after the initiation of germination, the nucleus migrated from its initial position in the center of the spore to the proximal side. It then appeared to migrate along the raphe to one end of the spore where an asymmetric division occurred. The average velocity of migration was measured at 0.256 ± 0.065 μm/min. During migration the nucleus underwent changes in shape. No migrations or movement other than that by the nucleus were observed.     

STORAGE PRODUCTS IN SPORES OF ONOCLEA SENSIBILIS L

Lipids and proteins in substantial quantities are present as storage products in spores of Onoclea sensibilis. They fill the sparce spore cytoplasm and ultrastructurally are indistinguishable from reserve materials observed in storage tissue of higher plants. Hydrolysis of both products is correlated with early stages in spore germination.     

AN ULTRASTRUCTURAL STUDY OF CELL DIVISION IN THE CAMBIUM

The fine structure of dividing cambial cells of Ulmus americana and Tilia americana has been studied in material fixed in glutaraldehyde followed by osmium tetroxide. The cambia examined consisted of 7–9 rows of unexpanded fusiform cells, all of which had similar ultrastructural components. The fine structure and sequence of events of mitosis and cytokinesis in the dividing cambial cells apparently are similar to those of dividing cells in root tips and leaves. Of special interest was the observation that during cytokinesis, a broad cytoplasmic plate or phragmosome precedes the developing phragmoplast and cell plate through the dividing cambial cell. Smooth and coated vesicles derived from dictyosomes are associated with cell plate formation in these cells, smooth vesicles primarily with earlier stages of plate formation, and coated vesicles in later stages.     

THE EFFECT OF AUXIN AND GUANINE ON CELL EXPANSION AND CELL DIVISION IN THE GAMETOPHYTE OF THE FERN,ONOCLEA SENSIBILIS

Miller , J. H. (Yale U., New Haven, Conn.) The effect of auxin and guanine on cell expansion and cell division in the gametophyte of the fern, Onoclea sensibilis. Amer. Jour. Bot. 48(9): 816–819. Illus. 1961.—Auxin and guanine promote cell expansion in 0. sensibilis gametophytes. The optimum concentration of auxin for total expansion is 10^−-5 M, but the optimum for elongation is 10^−-6 M. Above this concentration the cells expanded isodiametrically. Guanine is active at higher concentrations than auxin. Increasing concentrations of auxin progressively inhibit red light-induced cell division, while guanine has no effect on cell division. Neither kinetin nor adenine promotes cell expansion or cell division.     

DETERMINATION OF RHIZOID ORIENTATION BY LIGHT AND DARKNESS IN GERMINATING SPORES OF ONOCLEA SENSIBILIS

When spores of the fern, Onoclea sensibilis L., are allowed to germinate in darkness, the rhizoid and the protonema are positioned at close to a right angle. If the spores are exposed initially to light and allowed to germinate, the rhizoid and protonema are positioned nearly axially, at opposite ends of the spore. The greater the duration and intensity of the initial illumination, the greater the tendency towards axial arrangement. All colors of light are active to some degree, and the effects are intensity-dependent. The response occurs in a uniform light field and is not dependent on a directional stimulus; the phenomenon reflects the relative arrangement of one part of the gametophyte to another part but not the orientation of growth with respect to an external stimulus. Direct tests show that neither the relative rhizoid orientation nor initial polarity of germination are affected by unilateral white light or polarized red light; the subsequent growth of the protonema, however, is oriented perpendicular to the plane of light polarization. The effects of light in determining the positional relationship between rhizoid and protonema are interpreted in terms of a hypothesis proposing light-induced changes in the structure and mechanical properties of the spore wall.     

AN ULTRASTRUCTURAL STUDY OF VEGETATIVE CELL DIVISION IN OEDOGONIUM BORISIANUM12

Vegetative cell division in Oedogonium borisianum is initiated by the formation of a 3-layered ring adjacent to the wall in the upper portion of the cell. This structure enlarges by the coalescence of vesicles. When the ring is fully developed, the parent wall splits adjacent to the ring, and the ring expands into a cylinder, which becomes the cuticle of the upper daughter cell. The lateral wall then forms between this cuticle and the plasmalemma of the cell. Concurrent with ring development and expansion, the nucleus migrates to a position in the center of the cell and karyokinesis occurs. Commencing with late telophase, evidence of transverse wall formation becomes apparent. The zone between the daughter nuclei contains a layer of microtubules in a plane parallel to the plane in which the transverse wall will develop. Subsequently a random coalescence of vesicles occurs along this plane. During the latter stages of this process, the ring expands and the plane of the transverse wall moves upward to the base of the ring cylinder. The completed transverse wall then fuses at is periphery with the newly formed lateral wall.     

CO-REQUIREMENT FOR CALCIUM AND POTASSIUM IN THE GERMINATION OF SPORES OF THE FERN ONOCLEA SENSIBILIS

Spores of Onoclea sensibilis L. do not germinate on distilled H₂O if they are pretreated for sufficient time with dilute NaClO solution. However, spores will germinate, after NaClO pretreatment, on a simple mineral medium containing the major and trace elements. Complete germination after pretreatment also is obtained on a solution containing only Ca²⁺ and K⁺ as the cations, but neither ion by itself is sufficient. Rb⁺, but not Li⁺ or Na⁺, can replace K⁺. Hypochlorite-treated spores do not require the continuous presence of Ca²⁺ and K⁺ to germinate; exposure during the first 4 hr of culture, with the remainder of the time on distilled H₂O, is sufficient. Extraction of spores with ethylene glycol bis(aminoethyl ether) tetraacetic acid [EGTA] makes their germination dependent on Ca²⁺, as reported by other workers, but it does not produce a co-requirement for K⁺. Colorimetric analysis with arsenazo III confirms that Ca²⁺ is extracted from Onoclea spores by NaClO. Extractable Ca²⁺ amounts to about 78 nmol/mg spore dry wt. Of this amount, 31% is contained in the perispore. The perispore comprises 13% of the total spore dry wt.     

ANTHERIDIAL FORMATION IN ONOCLEA SENSIBILIS L.: GENESIS OF THE JACKET CELL WALLS

Antheridial initiation in Onoclea sensibilis L., an advanced leptosporangiate fern, begins with the production of a small, wedge-shaped cell within the anterior region of the vegetative cell. This is in contrast to previous reports claiming that the initials are formed by a localized protuberance in the cell wall of the vegetative cell (Campbell, 1886; Davie, 1951; Leung and Naf, 1979; Nayar and Kaur, 1971). The mature antheridium of Onoclea is composed of three uniquely shaped jacket cells surrounding spermatogenous cells. The two funnel-shaped jacket cell walls are shown to form in a lateral circular manner. Except for the production of the antheridial initial cell, jacket cell formation in Onoclea proceeds in accordance with the classical concept of antheridial development in advanced ferns accredited in part to Atkinson (1894), Campbell (1886), Kny (1869), and Strasburger (1869). The classical concept has been contested in more recent years by Davie (1951), Leung and Näf (1979), and Verma and Khullar (1966).     

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。     

ANALYSIS OF STARCH ACCUMULATION AND GERMINATION IN ONOCLEA SPORES

Previous studies have shown that low fluences of light accelerate starch accumulation and enhance germination of Onoclea spores. Fluence response curves for induction of starch accumulation were compared with fluence response curves for enhancement of germination in order to determine if the two photoresponses in Onoclea spores have a common photoreceptor. Fluence response curves indicate that both responses were proportional to the log of the fluence and that the relative fluence efficiencies of the four wavelength regions tested were similar for induction of both germination and starch accumulation. Red (600–720 nm) irradiation was the most efficient, while green (500–600 nm), blue (400–520 nm), and far-red (720–900 nm) irradiations showed a decreasing order of efficiency for induction of the responses. A correlation coefficient between the amount of starch accumulated as a result of red irradiation and the final percent germination was calculated to be 0.964. These results support the hypothesis that a common photoreceptor mediates both photoinduced germination and starch accumulation. 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU) inhibits photosynthesis by blocking the flow of electrons from Photosystem II to Photosystem I. At 0.1 mM DCMU failed to inhibit both photoinduced starch acumulation and germination. This result and the greater efficiency of red than blue light, the low fluence functional for induction, and the fluence dependency argue against the participation of photosynthesis in photoinduced starch accumulation. A similar conclusion has been previously drawn for photoenhancement of Onoclea spore germination. Additionally, the effects 0.01–1.0 mm cycloheximide and 100 μl/l ethylene on photoinduced starch accumulation were investigated. Neither agent inhibited starch accumulation, whereas both substances inhibited germination 70–90% when applied at a time coincidental with the period of rapid starch accumulation. These results indicate that the photoinduction of starch accumulation does not have an ethylene sensitive stage nor does it require protein synthesis as does photoenhancement of germination of Onoclea spores.     

BIOCHEMISTRY OF FERN SPORE PROTEINS: GLOBULIN STORAGE PROTEINS IN ONOCLEA SENSIBILIS AND OSMUNDA CINNAMOMEA

Onoclea sensibilis was found to contain globulin storage proteins of 2.0S and 11.3S. These globulins, comparable in size and subunit composition to the spore storage proteins characterized in Matteuccia struthiopteris (Templeman, DeMaggio, and Stetler, 1987), declined during imbibition and the initial stages of spore germination. Osmunda cinnamomea, a fern that is only distantly related to Matteuccia, also contained globulin proteins, but these had S values of 5.5 and 11.3. SDS-PAGE analysis revealed extensive differences in banding patterns of the 11.3S protein between Onoclea and Osmunda. This work indicates that while globulin proteins are important storage materials in a variety of ferns they exhibit considerable molecular heterogeneity. The observed heterogeneity in the globulin proteins may be a useful tool to explore evolutionary relationships in the ferns.     

ON THE CYTOLOGY OF ANTHERIDIUM FORMATION IN THE FERN SPECIES ONOCLEA SENSIBILIS. I. CYTOLOGY OF CELL PLATE AND CELL WALL FORMATION

In the four-celled antheridium of the fern species Onoclea sensibilis a central spermatogenous cell is enveloped by a jacket of three cells. Starting from the base, the jacket comprises the cup-shaped basal cell, the ring cell—both of which encircle the spermatogenous cell—and the cap cell. The lower wall of the spermatogenous cell has the configuration of a funnel; its upper wall is dome shaped. The choice of whole antheridia for study instead of sectioned ones has, for the first time, made it possible to study the formation of the uniquely shaped antheridial cell plates step by step. The cell plate antecedent of the funnel wall has the configuration of a funnel. This conclusion conflicts with Davie's contention that this cell wall is oriented transversely at first and acquires funnel-shape secondarily. The present studies further show that the funnel cell plate forms from base to rim. This finding contrasts with a report that in another fern species this cell plate begins to form on one side of the initial and then proceeds circularly around it. The base of the funnel cell plate attaches to the basal wall of the antheridium initial in a separate event. The genesis of the dome-shaped upper wall of the spermatogenous cell is described for the first time.     

Biochemistry of Fern Spore Germination: GLYOXYLATE AND GLYCOLATE CYCLE ACTIVITY IN ONOCLEA SENSIBILIS L

In chlorophyll-containing spores of Onoclea sensibilis, depletion of lipid reserves during germination is correlated with increases in the activity of the glyoxylate cycle enzymes isocitrate lyase and malate synthase. In Onoclea, the heterotrophic activity associated with lipid catabolism occurs at the same time that autotrophic activity is taking place. Increases in chlorophyll content and in the activity of glycolate oxidase were recorded during the earliest stages of spore germination. In this species, there is no temporal separation of heterotrophic and autotrophic reactions. Concurrent increases in glyoxylate and glycolate cycle activities appear to occur naturally.     

UNUSUAL DARK-GROWTH AND ANTHERIDIAL DIFFERENTIATION IN SOME GAMETOPHYTES OF THE FERN ONOCLEA SENSIBILIS

A fertile frond of O. sensibitis was found which yielded spores with unusual growth characteristics. About 25% of the gametophytes derived from the spores were able to undergo 2-dimensional development in darkness, in contrast to normal plants which are filamentous in darkness. When the aberrant spores were cultured in darkness under conditions of reduced ethylene concentration, the proportion of 2-dimensional plants rose to 75%, and, moreover, up to 50% of the gametophytes produced antheridia within 2 weeks. Under comparable conditions normal gametophytes produced no antheridia. The medium from antheridial cultures of the aberrant spores failed to induce the formation of antheridia in other plants.     

THE EFFECT OF METHANOL ON SPORE GERMINATION AND RHIZOID DIFFERENTIATION IN ONOCLEA SENSIBILIS

In germinating spores of Onoclea sensibilis, the nucleus migrates to one end prior to an asymmetric cell division that partitions each spore into two daughter cells of unequal size. The larger cell develops into a protonema, whereas the smaller cell immediately differentiates into a rhizoid. When spores were germinated in the presence of methanol, nuclear migration was inhibited and most nuclei moved only to the raphe on the proximal side of the spores. Subsequent cell division partitioned each spore into daughter cells of equal size of which both developed into a protonema and neither into a rhizoid. Spores became sensitive to methanol at a time just prior to or coincident with nuclear migration and the effects of the alcohol were rapidly reversible as long as the spores were removed from methanol prior to the completion of cell division. Exposure to methanol prior to, but not during, nuclear migration or after mitosis had no effect upon rhizoid differentiation. The alcohol disrupted the formation of crosswalls after mitosis and they were often convoluted and irregularly branched. These results are consistent with the interpretation that methanol may disrupt a membrane site that plays an essential role in nuclear movement and rhizoid differentiation.     

ROLE OF ASYMMETRIC CELL DIVISION IN PTERIDOPHYTE DIFFERENTIATION. II. EFFECT OF CA2+ ON ASYMMETRIC CELL DIVISION,RHIZOID ELONGATION,AND ANTHERIDIUM DIFFERENTIATION IN VITTARIA GEMMAE

Gametophytes of Vittaria graminifolia reproduce vegetatively by means of gemmae. Each gemma consists of a linear array of six cells: four body cells and a knob-shaped terminal cell at each end. When gemmae are shed from the gametophyte onto Knop's mineral medium, the two terminal cells do not divide, but elongate to form primary rhizoids. The body cells undergo asymmetric cell division, and the smaller daughter cells differentiate into either secondary rhizoids or prothalli. When gibberellic acid is included in the medium, antheridia are formed as a result of asymmetric cell division instead of vegetative structures. We studied the effect of Ca²⁺ on asymmetric cell division, rhizoid elongation, and antheridium formation in gemmae cultured on Knop's mineral medium and variations of Knop's medium. Ca²⁺ inhibited the onset of cell division and rhizoid elongation, but was required for differentiation of antheridia. Treatments which lowered the Ca²⁺ content of gemmae (EGTA and dilute HCl extraction, culture on verapamil-containing and Ca²⁺-deficient medium) caused an early onset of cell division and rhizoid elongation. The stimulation of growth was most pronounced when gemmae were deprived of Ca²⁺ during the first 24 hr of culture. The proportion of cell divisions which differentiated into antheridia in response to GA was greatly reduced when the Ca²⁺ status of gemmae was lowered with verapamil and Ca²⁺-EGTA buffers.     

枸杞胚性细胞分化的超微结构和ATP酶的细胞化学定位研究

枸杞的胚性细胞多由愈伤组织表层的薄壁细胞分化而来,与愈伤组织中未分化的细胞相比,胚性细胞呈卵圆形,细胞核大,核仁明显,细胞质浓厚并含有丰富的细胞器,细胞壁较薄,细胞间有胞间连丝相通;胚性细胞发育到晚期细胞壁加厚,胞间连丝逐渐消失,细胞核向一端偏移,有大液泡形成;胚性细胞的第一次分裂多为均等分裂,形成二细胞原胚,继续分裂形成多细胞原胚;组成多细胞原胚胚体的细胞核大,核形状不规则,细胞质浓厚,细胞器丰富,在质体中出现淀粉的积累。在胚性细胞发育的早期,ATP酶活性主要位于质膜上,随后在液泡内和细胞核中都出现ATP酶活性的分布;随着胚性细胞壁的加厚,细胞壁加厚处和细胞间隙中也出现ATP酶活性反应;当多细胞原胚形成后,ATP酶活性反应主要定位于液泡膜上。由此分析了结构特征、ATP酶活性定位变化与胚性细胞分化的关系。