Release of Pollen Protoplasts in Large Quantities in Brassica napus and B. compestris var. purpurea

Protoplasts were released in large quantities from mature pollen of Brassica napus L. and B. campestris var. purpurea for the first time, with a yield up to 66.7% and 70.4% respectively. Most of the pollen protoplasts were viable as tested by fluorochromatic reaction with fluorescein diacetate. The success of isolation of pollen protoplasts in these two Brassica species relied on a technique modified from the previous method developed for several monocotyledonous flowers. The procedure included two steps: First, the pollen was hydrated in 1 mol/L of sucrose solution at 28–30℃ for ca. 9h. During this process, the exine of most pollen dehisced and was detached from the pollen grains which were then covered by intine alone. Second, the hydrated pollen was transferred into an enzyme solution containing 1% cellulase, 1% pectinase, I mol/L mannitol, 0.5% potassium dextran sulphate and Ks medium salts. After 4–6 of enzymatic maceration, the intine was degradated resulting in the release of protoplasts. Factors affecting the two steps have been investigated.     

The isolation of coated vesicles from protoplasts of soybean

Fractions enriched in coated vesicles were obtained from protoplasts derived from suspension cultured Glycine max (L.) Merr. cells. Initial enrichment was achieved by isopycnic centrifugation of a protoplast homogenate through a linear sucrose gradient in a vertical rotor. The coated-vesicle fractions from this gradient were pooled and centrifuged through a second linear sucrose gradient in a rate zonal fashion to remove the larger contaminating membrane vesicles. The most prominent polypeptide in the coated-vesicle fractions, plant clathrin, had a relative molecular mass of approx. 190 kdalton as determined by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. Other enriched polypeptides included bands at 105, 100, 96, 64, 50, 38 and 32 kdalton. This method was compared with a procedure utilizing sucrose step gradients for preparing coated vesicles from soybean protoplasts. The effectiveness of the isopycnic-rate zonal centrifugation procedure was also tested for the preparation of bovine-brain coated vesicles.NRCC No. 23142     

Preparation and fusion properties of protoplasts from mature pollen of Nicotiana tabacum

Summary A method to remove the exine from mature tobacco pollen and to release numerous intact pollen protoplasts has been developed. Post-anthesis binucleate pollen was treated with water, buffered with MES at pH 5.5, for two hours. Rupture of the exine was caused by the force of pollen hydration exposing the intine to subsequent enzymatic maceration. The high osmotic pressure (1000 mOsm·kg^-1 H₂O) of pollen protoplasts required a special maceration medium, 4% KCl (w/v). Action of an enzyme solution containing 1% (w/v) Macerozyme and 1% (w/v) Cellulase gave rise to viable protoplasts within 4 hours. When cultured in a tobacco mesophyll protoplast culture medium, the pollen protoplasts underwent regeneration of a cell wall, formation of various tube-shaped structures, and division of the generative nucleus into two nuclei. Using a PEG/Ca²⁺ method pollen protoplasts were fused with diploid mesophyll protoplasts. Evidence of transfer of chloroplasts into the pollen protoplasts was observed after one day of culture.Abbreviations BCP bromocresol purple - FDA fluoresceindiacetate - MES 2-(N-morpholino) ethanesulfonic acid - PEG polyethyleneglycol     

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation

Summary A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H₂O, the gradients provide a density range from 1.017 to 1.069 g/cm³ at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm³, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.     

Isoelectric focusing of plant cell protoplasts: separation of different protoplast types

The surface charge of plant protoplasts has been measured by a new technique, isoelectric focusing. The protoplasts were loaded in a dextran density gradient over which a pH gradient was superimposed. When voltage was applied, protoplasts moved to a point in the gradient corresponding to their isoelectric point (pI). The pI of the protoplasts varied with the compounds used for pH gradient generation. Using commercial ampholytes for pH gradient formation, the pI of all protoplasts tested was 4.4 ± 0.2, and viability following electrophoresis was low. Using an acetate/acetic acid mixture to generate the pH gradient, the pI of protoplasts varied from 3.7 to 5.3 depending on the species and tissue type of the parental cells. Postelectrophoresis viability was high. Using isoelectric focusing techniques, it was possible to separate mixtures of protoplasts derived from different species of plants.     

An Ultrastructural Study on Pollen Protoplasts and Pollen Tubes Germinated From Them in Gladiolus gandavensis

Large quantities of protoplasts were isolated enzymatically from the mature pollen grains in Gladiolus gandavensis. Regeneration of cell wall and germination of pollen tubes were performed during culture of purified pollen protoplasts in Ks medium supplemented with 32% sucrose, 0.1 mg/1 2,4-D, 1 mg/1 NAA and 0.2 mg/1 6-BA, with a germination rate up to 47.7%. The materials were fixed gently with gradually increasing concentration of glutaraldehyde, followed by osmium, then preembedded in a thin layer of agar and surveyed under an inverted microscope so as to select desired specimens for subsequent procedure. Small agar blocks containing specimens were dehydrated through ethanal-propylene oxide series, embedded in Araldite and ultratomed. Electron microscopic observations show that the pollen protoplasts are surrounded by a smooth plasma membrane and with ultrastructurally intact cytoplasm, a vegetative nucleus and a generative cell. After 8h of culture, wall regeneration commences resulting in a multilayered, fibrillar wall structure which is different from the intine. No exine is formed. Numerous vesicles participate actively in the wall formation. The wall is uneven in thickness around its periphery; a thickened area somewhat resembling to germ furrow is formed, from which pollen tube emerges. The tubes contain abundant plastids, mitochondria and dictyosomes. Vesicles are released out of the plasma membrane and involved in tube wall formation. After 18h of culture, the vegetative nucleus and generative cell have migrated into the tube. Technical points of preparing pollen protoplast specimens for ultastructural studies and the fearnres of wall regeneration in pollen protoplast culture are discussed.     

Amino acid export from infected cells of Vicia faba root nodules: Evidence for an apoplastic step in the infected zone

In root nodules of leguminous plants, such as Vicia faba L., N₂ is fixed by rhizobial bacteroids within infected cells. These cells are located in the centre of the nodule, whereas the vascular system serving import and export of solutes is located in the periphery. Within the infected central tissue, metabolites may travel symplastically by using bands of interconnected uninfected cells. Structural evidence, however, speaks against symplastic movement between infected cells themselves. The present work examined the possibility of an apoplastic step in amino acid export from infected cells. Incubation experiments with dissected central tissue demonstrated the release of amino compounds by infected cells. The predominant compound released was asparagine, which is also the major amino acid in xylem sap of legumes forming indeterminate nodules. During incubation of central infected tissue, medium acidification by plasma membrane H⁺-ATPase quickly turned into slight alkalization, probably caused by the released amino acids. In vivo, this process would lead to an increased apoplastic pH with consequences for processes relying on the proton gradient across the plasma membrane. Uptake of ¹⁴C-labelled amino acids by uninfected and infected cells was studied using protoplasts isolated from the central nodule tissue. Uninfected protoplasts accumulated amino acids with low specificity in a ΔpH-dependent, bi-phasic manner, whereas infected protoplasts did not absorb amino acids from the medium. This indicates that uninfected protoplasts not only function in metabolite transport, but also in collection of amino acids from the apoplast. Taken together, both experimental approaches demonstrate the possibility of an apoplastic export step for amino acids in the central tissue of indeterminate legume nodules.     

The Apertural Wall in Pollen of Linum grandiflorum

The apertural wall in tricolpate pollen of Linum grandiflorumwas investigated in order to understand its functioning duringdesiccation and rchydration. Whole and sectioned pollen grainswere studied with light or electron microscopy and by cytochemicalmeans. The areas of the apertures were examined in fresh drypollen, in grains moistened on agar gel or removed from compatiblestigmas, and in pollen from mature undehisced anthers The intine was found to consist of an inner ß-glucanlayer and an outer pectic layer. At the apertures the pecticlayer is thickened and overlaid by a ß-glucan layer.The pectinaceous intine stains red with basic fuchsin. The presenceof a third wall layer, the medine, was not confirmed. The aperturalintine thickenings possess considerable imbibitional capacityand at rehydration they appear as swollen lenticular bodies A procedure is described for obtaining intact exine free grains(EFG's) and whole, separated exines of L. grandiflorum. Invariably,the released EFG's consisted of protoplasts encased in the cellulosicintine. In most grains the outer intine remained attached tothe separated exine In L. grandiflorum the outer wall of the aperture expands whilethe protoplast and endintine are still infolded. Apparently,the exintine becomes detached from the endintine during desiccationand re-attaches at rehydration. It is suggested that the transientdetachment controls the influx of water into the vegetativecell Except for morph-specific exine processes no differences instructure of the aperture wall or its functioning at rehydrationwere observed between pin and thrum grains Pollen wallM, apertures, exintine, exine free grains, rehydration, desiccation, Linum grandiflorum     

Isolation and identification of the cytoplasmic membrane from Saccharomyces carlsbergensis by radioactive labeling.

The cytoplasmic membrane of Saccharomyces carlsbergensis was isolated by enzymatic digestion of the yeast cell wall, followed by lysis of the protoplasts and fractionation by ultracentrifugation in a discontinuous sucrose density gradient. Location of the cytoplasmic membrane fraction on the sucrose gradient was made by labeling intact protoplasts with [G-3H]dansyl chloride, and was settled at the 50% (wt/vol) sucrose gradient (d = 1.186 g/cm3). Approximately 80% of the radioactivity was found in the membrane fraction prepared in the presence of Mg2+ ions. However, when protease inhibitors were used in the preparation step, the membrane fraction contained over 90% of the total radioactivity. The presence of Mg2+ ions during membrane isolation and purification enhanced the aggregation of membrane components but, at higher concentrations, as well as in the prolonged presence of Mg2+ ions in the membrane suspension, it caused the breakdown of membrane components. The membrane preparation contained Mg2+-adenosine triphosphatase, which was insensitive to oligomycin and ouabain. The distribution of Mg2+-adenosine triphosphatase in different fractions during sucrose gradient is reported.     

Isolation and identification of the cytoplasmic membrane from Saccharomyces carlsbergensis by radioactive labeling.

The cytoplasmic membrane of Saccharomyces carlsbergensis was isolated by enzymatic digestion of the yeast cell wall, followed by lysis of the protoplasts and fractionation by ultracentrifugation in a discontinuous sucrose density gradient. Location of the cytoplasmic membrane fraction on the sucrose gradient was made by labeling intact protoplasts with [G-3H]dansyl chloride, and was settled at the 50% (wt/vol) sucrose gradient (d = 1.186 g/cm3). Approximately 80% of the radioactivity was found in the membrane fraction prepared in the presence of Mg2+ ions. However, when protease inhibitors were used in the preparation step, the membrane fraction contained over 90% of the total radioactivity. The presence of Mg2+ ions during membrane isolation and purification enhanced the aggregation of membrane components but, at higher concentrations, as well as in the prolonged presence of Mg2+ ions in the membrane suspension, it caused the breakdown of membrane components. The membrane preparation contained Mg2+-adenosine triphosphatase, which was insensitive to oligomycin and ouabain. The distribution of Mg2+-adenosine triphosphatase in different fractions during sucrose gradient is reported.     

Simplified procedure for transient transformation of plant protoplasts using polyethylene glycol treatment

A modification of the polyethylene glycol-mediated transformation procedure which eliminates the manual polyethylene glycol dilution step is presented. A transformation mixture of protoplasts, DNA and polyethylene glycol was plated directly onto agarose blocks after incubation. The procedure was simple and fast, thereby suitable for screening the gene activity of large numbers of plasmid constructions. It has been tested for both maize and rice protoplasts.     

Isolation of mesophyll protoplasts from mature leaves of soybeans

A procedure based on a combined cellulase-Pectolyase Y-23 enzyme digestion and metrizamide-sorbitol gradient purification protocol was developed for isolating mesophyll protoplasts from mature leaves of soybean (Glycine max L. Merr.). Based on chlorophyll content, this procedure results in a 10 to 15% protoplast yield from fully expanded mature leaves and a 20 to 30% yield from young (expanding) leaves within 3 hours. Isolated protoplasts displayed high rates of HCO₃⁻-dependent photosynthesis; greater than 75 micromoles O₂ evolved per milligram chlorophyll per hour at 25°C. This photosynthetic rate is comparable to that of mesophyll cells isolated mechanically from the same leaves.     

An improved procedure for the isolation and purification of protoplasts from carrot suspension culture

A procedure is reported for the rapid and highly reproducible isolation of protoplasts from carrot suspension culture. The method utilizes Onozuka R 10 cellulase which has been purified by chromatography on Sephadex G75. Protoplast isolation, using this procedure, is quantitative and complete within 1 to 1.5 h. Intact protoplasts were separated from broken ones and other cellular debris by application of a polyethylene glycol/dextran two-phase system. The protoplasts isolated in this manner lack any detectable cell wall and are greater than 95% viable when assayed using fluorescein diacetate. It is concluded that such protoplasts are highly suitable for biochemical studies.Abbreviation PCM protoplast culture medium     

POLLEN WALL AND TAPETAL ORBICULAR WALL DEVELOPMENT IN SORGHUM BICOLOR (GRAMINEAE)

Pollen wall development in Sorghum bicolor is morphologically and temporally paralleled by the formation of a prominent orbicular wall on the inner tangential surface of the tapetum. In the late tetrad stage, a thin, nearly uniform primexine forms around each microspore (except at the pore site) beneath the intact callose; concurrently, small spherical bodies (pro-orbicules) appear between the undulate tapetal plasmalemma and the disappearing tapetal primary wall. Within the primexine, differentially staining loci appear, which only develop into young bacula as the callose disappears. Thus, microspore walls are devoid of a visible exine pattern when released from tetrads. Afterwards, sporopollenin accumulates simultaneously on the primexine and bacula, forming the exine, and on the pro-orbicules, forming orbicules. Channels develop in the tectum and nexine, and both layers thicken to complete the microspore exine. Channeled sporopollenin also accumulates on the orbicules. A prominent sporopollenin reticulum interconnects the individual orbicules to produce an orbicular wall; this wall persists even after the tapetal protoplasts degenerate and after anthesis. While the pollen grains become engorged with reserves, a thick intine, containing conspicuous cytoplasmic channels, forms beneath the exine. Fibrous material collects beneath the orbicular wall. The parallel development and morphological similarities between the tapetal and pollen walls are discussed.     

SPORE WALL FORMATION AND CHLOROPLAST DEVELOPMENT DURING SPOROGENESIS IN THE MOSS FISSIDENS LIMBATUS

The sequence of wall formation in spores of Fissidens limbatus Sullivant is as follows: The exine is formed around the protoplasts after the sporocyte has undergone meiosis. The fully enlarged spores then become coated by the perine; this is followed by intine formation. The source of the intine and exine appears to be from within the spore, but the perine is of an apparent exogenous origin. Ornamentation of the spore is due solely to deposition of the perine. Each spore originally has a single plastid. Plastids increase in number by fission, resulting in mature spores with numerous plastids with well differentiated lamellae.     

云南松小孢子发生的超微结构研究

云南松(Pinus yunnanensis Fr.)小孢子囊发育早期,原生质体发生收缩、同时形成胼胝质壁。小孢子母细胞减数分裂前互相分离,并被胼胝质壁包围。在壁上有约0.2微米的小孔。四分体小孢子形成后,核被膜及高尔基体显得非常地活跃,他们可能与外壁的沉积有关。这个现象在早期四分体阶段出现。内壁的形成是在自由小孢子时期,开始高尔基体和核被膜仍较活跃,但随之下降。内质网和线粒体增多,许多来自内质网的泡囊通过质膜被排放到内壁中去。乌氏体与花粉外壁之间观察到了孢粉素带。     

Light-dependent rubidium uptake into isolated mesophyll protoplasts from leaves of Pisum sativum

A method is described for the isolation of large amounts of physiologically active protoplasts from leaves of Pisum sativum L. Rubidium uptake was determined after separation of the intact protoplasts from the loading medium by rapid centrifugation through a phthalate step gradient. In freshly isolated mesophyll protoplasts of Pisum sativum , rubidium uptake was carbonylcyanide- p -trifluoromethoxyphenylhydrazone reduced by metabolic inhibitors such as 5 μ M , 0.1 mW cyanide, 2 μ M DCMU and 5 m M arsenate and by dark incubation. Reduction of rubidium uptake by inhibition of aerobic respiration or the photosynthetic electron transport system demonstrates that both processes play a role in the energy supply for membrane transport in these protoplasts.     

Ultrastructural investigations on sporogenesis inEquisetum fluviatile

Summary The spore mother cells ofEquisetum fluviatile undergo meiotic division, each forming a tetrad of spores. The spore protoplasts are separated from each other by an accumulation of mitochondria (organellar plate) at first and later on by plasma membranes, no cell wall is formed. The first layer of the sporoderm, the exine, originates from the plasmodial tapetum and is deposited at the outer side of the plasmalemma of the young spore. The exine reaches a thickness of about 330 nm. In the phase of spore greening the so-called perine, originating from the tapetum, is placed onto the exine and the inner layer of the sporoderm, the intine, is formed from the spore protoplast. The mature spore, about 40 m in diameter, does not enter dormancy and remains viable only for a few days.Member of the Study group on electron microscopy at the TierÄrztliche Hochschule Hannover.     

The isolation and culture of lily pollen protoplasts

Methods for the enzymatic isolation of lily protoplasts and their successful culture are described. When pre-anthesis binucleate pollen (immature pollen grains) was treated in enzyme solution containing macerozyme and cellulase, up to 80% lost their exine and gave rise to intact protoplasts within 1 h. These pollen protoplasts were uniform in size and densely cytoplasmic with two prominent generative and vegetative nuclei. The isolated pollen protoplasts regenerated a cell wall within 1 day of culture and produced a structure resembling a pollen tube after 10–12 days of culture. During this culture period, dividing generative nuclei or 2 sperm nuclei were observed in many protoplasts with regenerated cell walls.     

Enrichment of heterokaryocytes between mesophyll and epidermis protoplasts by density gradient centrifugation after electric fusion

Summary Mesophyll protoplasts from Nicotiana glauca were fused with epidermal protoplasts from N. langsdorffii by an electric pulse. After the fusion products were centrifuged on stepwise density gradient centrifugation using Percoll and sea water, somatic hybrids were observed at 70%–80% in the fraction recovered from the intermediate specific gravity fraction between epidermis and mesophyll protoplasts. From offsprings of these somatic hybrids, teratomatous plants were regenerated. Since the difference of specific gravity between mesophyll and epidermis protoplasts is inherent, this procedure can be essentially applied to obtain somatic hybrids between any combination of plants. The significance of this study is discussed in relation to obtaining somatic hybrids between plant materials without any appropriate genetic markers.