Lateral diffusion of proteins and lipids in the plasma membrane of rose protoplast

Summary Lateral diffusion measurements have been made on lipids and proteins in the plasma membrane of live protoplasts derived from rose (Rosa sp. Paul's Scarlet) suspension-cultured cells. Two different fluorescent lipid probes exhibited markedly different diffusion rates, indicating possible heterogeneity in the lipid domain of the membrane. Membrane proteins were labeled directly with covalently-reactive fluorophores, and factors that might perturb the lateral diffusion of these labeled proteins were investigated. Treatment of the protoplasts with various cytoskeleton-disrupting drugs generally had little effect on protein diffusion, although treatment with oryzalin, a microtubule-disrupting drug, did slightly reduce the mobile fraction of membrane proteins. Elevation of the CaCl₂ concentration in the medium from 1 mM to 10 mM significantly reduced the mobile fraction of membrane proteins and also increased the fraction of protoplasts that were able to regenerate cell walls and divide in culture. These results are discussed in relation to reported evidence of lipid domains in the plasma membranes of other cells and protoplasts. The relative importance of lipid domains and membrane-cytoskeleton interaction in governing protein diffusion is considered.Abbreviations D lateral diffusion coefficient - RCA Ricinus communis agglutinin - BPA Bauhinia purpurea agglutinin - DTAF dichlorotriazinylaminofluorescein - FTSC fluorescein-5-thiosemicarbazide - C₁₈-Fl 5-(N-octadecanoyl)aminofluorescein - LY-Chol Lucifer yellow conjugate of cholesterol, i.e., dilithium 4-amino-N-[(-(carbo(5-cho-lesten-3-yl)oxy)hydrazinocarbonyl)amino]-1,8-naphthalimide-3,6-disulfonate - APM amiprophosmethyl - DMSO dimethylsulfoxide - FPR fluorescence photobleaching recovery - sd standard deviation - FRAF fluorescence redistribution after fusion - M mobile fraction     

Structural analysis of the cell walls regenerated by carrot protoplasts

A procedure was developed to isolate protoplasts rapidly from carrot (Daucus carota L. cv. Danvers) cells in liquid culture. High purity of cell-wall-degrading enzymes and ease of isolation each contributed to maintenance of viability and initiation of regeneration of the cell wall by a great majority of the protoplasts. We used this system to re-evaluate the chemical structure and physical properties of the incipient cell wall. Contrary to other reports, callose, a (1 3)-d-glucan whose synthesis is associated with wounding, was not a component of the incipient wall of carrot protoplasts. Intentional wounding by rapid shaking or treatment with dimethyl sulfoxide initiated synthesis of callose, detected both by Aniline blue and Cellufluor fluorescence of dying cells and by an increase in (1 3)-linked glucan quantified in methylation analyses. Linkage analyses by gas-liquid chromatography of partially methylated alditol-acetate derivatives of polysaccharides of the incipient wall of protoplasts and various fractions of the cell walls of parent cells showed that protoplasts quickly initiated synthesis of the same pectic and hemicellulosic polymers as normal cells, but acid-resistant cellulose was formed slowly. Complete formation of the wall required 3 d in culture, and at least 5 d were required before the wall could withstand turgor. Pectic substances synthesized by protoplasts were less anionic than those of parent cells, and became more highly charged during wall regeneration. We propose that de-esterification of the carboxyl groups of pectin uronic-acid units permits formation of a gel that envelops the protoplast, and the rigid cellulose-hemicellulose frame-work forms along with this gel matrix.Abbreviations DEAE Diethylaminoethyl - DMSO dimethyl sulfoxide - ECP extracellular polymers - EDTA ethylenediaminetetraacetic acid - HGA nomogalacturonan - RG rhamnogalacturonan - Tes N-tris(hydroxymethyl)methyl-2-amino-ethanesufonic acid - TFA trifluoroacetic acid Journal paper No. 11,776 of the Purdue University Agriculture Experiment Station     

Microcallus formation from maize protoplasts prepared from embryogenic callus

Conditions have been developed that induce maize (Zea mays L.) protoplasts to re-synthesize cell walls and to initiate cell divisions. Two types of embryogenic maize callus were used as a source of protoplasts: a heterogeneous callus (Type I) derived from immature embryos after three weeks in culture, and a friable, rapidly growing callus (Type II) selected from portions of the Type I callus. Many variables in the growth conditions of the donor tissue (type of medium, transfer schedule, age of callus), protoplast isolation solutions (pH, osmolarity, type and concentration of cell wall hydrolyzing enzymes, addition of polyamines) and conditions (amount of time in enzyme, amount of tissue per volume of enzyme incubation medium, agitation, preplasmolysis of source tissue, type of callus), and purification procedures (filtration and-or flotation), were found to affect both yield and viability of protoplasts (based upon fluorescein-diacetate staining). Our isolation procedure yielded high numbers of viable, uninucleated maize callus protoplasts which were densely cytoplasmic and varied in size from 20 to 50 m in diameter. Protoplasts plated in solid medium formed walls and divided several times. Of several gelling agents tested for protoplast propagation, only agarose resulted in protoplasts capable of sustained divisions leading to the formation of microcalli. Plating efficiency was established over a wide range of protoplast densities (10³–10⁷ protoplasts/ml). Highest plating efficiency (25%) was obtained at 1·10⁶ protoplasts/ml). The resulting microcalli grew to be dense clusters of about 0.1–0.5 mm in diameter and then stopped growing. Nurse cultures of maize and carrot (Daucus carota L.), were used to establish that individual protoplasts (not contaminating cells or cell clusters) formed walls and divided. Nurse cultures also increased the efficiency of microcallus formation from protoplasts.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - MS Murashige and Skoog (1962) salts - MS 1D Murashige and Skoog salts with 1 mg/l 2,4-D - MS 2D Murashige and Skoog salts with 2 mg/l 2,4-D - N₆ medium of Chu et al. (1975) - NN67-mod medium of Nitsch and Nitsch (1967) as modified in the present paper - FDA fluorescein diacetate - LMP low melting point     

The role of bacterial attachment in the transformation of cell-wall-regenerating tobacco protoplasts by Agrobacterium tumefaciens

The presence of a newly formed primary cell wall was shown to be required for attachment and subsequent transformation of tobacco leaf protoplasts by Agrobacterium tumefaciens in cocultivation experiments. In these experiments both protoplasts at different stages after their isolation and cell-wall inhibitors were used. The specificity of Agrobacterium attachment was shown by using other kinds of bacteria that did not attach. By diminishing the concentration of divalent cations using ethylenediaminetetraacetic acid, neither attachment nor transformation was found; however, when more specifically the Ca²⁺concentration was lowered by ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid, both phenomena occurred. Commercial lectins had no effect on binding, but this observation does not exclude the involvement of other lectins. Protoplasts isolated from various crown-gall callus tissues also developed binding sites, but when they were at the stage of dividing cells, attachment of agrobacteria was no longer observed. In this respect, cells from protoplasts of normal tobacco leaves behaved differently. Even 16 d after protoplast isolation, the dividing cells were still able to bind A. tumefaciens, while transformation was not detected. For transformation of 3-d-old tobacco protoplasts, a minimal co-cultivation period of 24 h was required, while optimal attachment took place within 5 h. It is concluded that the primary cell wall was sufficiently well formed that certain functional receptor molecules were available for attachment of Agrobacterium as the first step of a multistep process leading to the transformation of cells. The expression of bacterial functions required for attachment, moreover, was independent of the presence of Ti-plasmid.Abbreviations ConA concanavalin A - CW calcofluor white - EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis (-aminoethyl ether)-N,N,N,N-tetraacetic acid - -Man -methyl-d-mannoside     

Fusion characteristics of plant protoplasts in electric fields

The electrical parameters important in the fusion of plant protoplasts aligned dielectrophoretically in high-frequency alternating electric fields have been established. Protoplasts were aligned in an alternating electric field between two relatively distant (1 mm) electrodes, by dielectrophoresis induced by field inhomogeneities caused by the protoplasts themselves. This arrangement allowed ease of manipulations, large throughput and low loss of protoplasts. In analytical experiments, sufficiently large samples could be used to study pulse duration-fusion response relations at different pulse voltages for protoplasts of different species, tissues and size (mesophyll protoplasts of Solanum brevidens, Triticum aestivum, Hordeum vulgare; suspension-culture protoplasts of Nicotiana sylvestris, N. rustica, Datura innoxia and S. brevidens; root-tip protoplasts of Vicia faba, hypocotyl protoplasts of Brassica napus). The percentage of aligned protoplasts that fused increased with increasing pulse parameters (pulse duration; voltage) above a threshold that was dependant on pulse voltage. The maximum fusion values obtained depended on a number of factors including protoplast origin, size and chain length. Leaf mesophyll protoplasts fused much more readily than suspension-culture protoplasts. For both types, there was a correlation of size with fusion yield: large protoplasts tended to fuse more readily than small protoplasts. In short chains (five protoplasts), fusion frequency was lower, but the proportion of one-to-one products was greater than in long chains (ten protoplasts). In formation by electrofusion of heterokaryons between mesophyll and suspension-culture protoplasts, the fusion-frequency response curves reflected those of homofusion of mesophyll protoplasts rather than suspension-culture protoplasts. There was no apparent limitation to the fusion of the smallest mesophyll protoplast with the largest suspension-culture protoplasts. Based on these observations, it is possible to direct fusion towards a higher frequency of one-to-one (mesophyll/suspension) products by incorporating low densities of mesophyll protoplasts in high densities of suspensionculture protoplasts and by using a short fusion pulse. The viability of fusion products, assessed by staining with fluorescein diacetate, was not impaired by standard fusion conditions. On a preparative scale, heterokaryons (S. brevidens mesophyll-N. sylvestris or D. innoxia suspension-culture) were produced by electrofusion and cultured in liquid or embedded in agar, and were capable of wall formation, division and growth. It is concluded that the electrode arrangement described is more suitable for carrying out directed fusions of plant protoplasts than that employing closer electrodes.     

Effects of low temperature on lateral diffusion in plasma membranes on maize (Zea mays L.) root cortex protoplasts: relevance to chilling sensitivity

The effects of temperature on the lateral diffusion of fluorescent phospholipids, sterols and proteins in the plasma membranes of maize root cortex protoplasts were monitored using fluorescence photobleaching recovery (FPR). Diffusion parameters were measured in two cultivars of maize having different chilling tolerance. Hydrodynamic theory predicts that the diffusion coefficient, D, should increase with increasing temperature. In the more chilling-tolerant cultivar, however, D for all three probes was nearly insensitive to temperature. In the more chilling-sensitive cultivar, D was also insensitive to temperature over the range from 12 to 21°C, but D for the lipid probes tended to be higher and more variable at lower temperatures. The proportion of probe molecules free to diffuse in the membrane was less than 1 for all probes, and increased significantly with increasing temperature for the protein probe. These results, taken together, support the concept that the plasma membrane contains domains having differing diffusional characteristics. Temperature effects on membrane diffusion are moderated by the existence of these domains to limit significant changes. The observed tendency for higher diffusion coefficients at low temperatures in the chilling-sensitive cultivar may correlate to morphological changes observed with protoplasts of that cultivar at low temperatures.     

Genotypic variability for protoplast regeneration in Saintpaulia ionantha (H. Wendl.)

Summary The behaviour of eleven Saintpaulia ionantha (H. Wendl.) genotypes in protoplast culture was compared. Isolation of protoplasts from young shootlets regenerated in vitro on leaf explants, yielded 0.7 to 1.8 × 10⁶ protoplasts per gram fresh weight. In all cultivars and breeding lines tested, cell divisions were observed. The mean division frequencies varied between 1.0 and 5.0% after 14 days, and between 6.4 and 13.8% after 24 days of culture. In ten genotypes callussing and shoot regeneration were achieved. The difference between the genotypes in shoot regeneration rate, between 2 and 68%, was more pronounced. The comparison of four cytokinins indicated hat thidiazuron was most effective for shoot regeneration, but often resulted in poorer shoot quality than benzylaminopurine.Abbreviations BAP (6-Benzylaminopurine) - IAA (In-dole-3-acetic acid) - NAA (-Naphthaleneacetic acid) - TDZ (Thidiazuron = 1-Phenyl-3-(1,2,3-thiadiazol-5-yl)-urea) - 2iP (6-(,-dimethylallylamino)-purine)     

Chitin and β(1–3) glucan synthases in the protoplastic entomophthorales

(1–3) glucan and chitin synthases were studied in spontaneously produced protoplasts and in the mycelium (hyphal body) of the entomopathogenic Entomophthorale species Entomophaga aulicae, Conidiobolus obscurus and Entomophthora muscae. The absence of wall in protoplasts was correlated to an absence of chitin synthase and to a very low (1–3) glucan synthase activity, whereas these two polysaccharide synthases were present and active in the walled hyphal bodies. Physicochemical properties of chitin and (1–3) glucan synthases such as localization, optimum pH and temperature, activation by disaccharides and proteases were similar to those found in other fungi unable to spontaneously produce protoplasts and could not be related to the ability for protoplastic Entomophthorale species to produce and proliferate under a protoplast form. The absence or the low chitin and glucan synthase activites in Entomophthorale protoplasts was not due to an absence of proteolytic activation of the enzyme. However, all protoplast fractions contained inhibitory substances of glucan and chitin synthase activities. These inhibitors were stable and specific of the protoplast stage. They were not glucanase nor chitinase. These results suggest that the absence of wall synthesis in Entomophthorale protoplasts is due to a continuous inhibition of (1–3) glucan and chitin synthase activities by intracellular compounds and also for glucan synthase by protoplast medium constituents such as NaCl and fetal calf serum.Abbreviations BSA bovine serum albumin - DFP diisopropylfluorophosphate - EDTA ethylenediamine tetraaoetic acid - FCS fetal calf serum - GlcNAc N-acetylglucosamine - TCA trichloroacetic acid - 2 k pellet 2,000 g wall fraction - 140 k pellet 140,000 g particulate fraction - 140 k supernatant 140,000 g soluble fraction     

β-1-4-and β-1-3-glucan synthases are associated with the plasma membrane of the fungus Saprolegnia

Cytoplasmic membranes from mycelium or protoplasts of Saprolegnia monoica (a cellulosic cell-wall fungus) were separated by continuous sucrose-density-gradient centrifugation. Glucan synthases assayed at low (micromolar uridine 5-diphosphate (UDP) glucose for -1-4-glucan synthase) and high (millimolar UDP glucose for -1-3-glucan synthase) substrate concentrations were associated with membranes exhibiting vanadate-sensitive, oligomycin-insensitive ATPase and equilibrating at density 1.16 g cm^-3. Synthase activities were also bound to membranes of lower density (1.10 and 1.145 g cm^-3). Plasma membranes were stabilized by coating protoplasts with concanavalin A. After lysis of the protoplasts, plasma membranes recovered by low centrifugal forces were isolated in continuous isopycinic gradients. Both synthase activities peaked with [³H]concanavalin A and Na-vanadate ATPase indicating that the synthetases are located at the plasma membrane. Treatments of intact protoplasts with cold glutaraldehyde or proteases before disruption lead to a diminution of glucan-synthase activities indicating that at least part of the enzymes of plasma membrane face the outside of the cell.Abbreviations ConA concanavalin A - ER endoplasmic reticulum - GSI -1,4-glucan synthase - GSH -1,3-glucan synthase - UDP uridine 5-diphosphate     

The production of callus capable of plant regeneration from immature embryos of numerous Zea mays genotypes

In the summer of 1983, immature embryos from 101 selfed inbred lines and germplasm stocks of Zea mays L. were examined for their ability to produce callus cultures capable of plant regeneration (regenerable cultures) using a medium with which some limited success had previously been obtained. Forty-nine of the genotypes (49%) produced callus which visually appeared similar to callus previously cultured and shown to be capable of plant regeneration. After five months, 38 of these genotypes were alive in culture and plants were subsequently regenerated from 35 (92%) of them. No correlation was observed between plant regeneration and callus growth rate, the vivipary mutation (genes vp1, 2, 5, 7, 8 and 9), or published vigor ratings based on K⁺ uptake by roots. When F₁ hybrid embryos were cultured, 97% of the hybrids having at least one regenerable parent also produced callus capable of plant regeneration. No regenerable cultures were obtained from any hybrid lacking a parent capable of producing a regenerable callus culture.In the summer of 1984, immature embryos from 218 additional inbred lines and germplasm stocks were plated and examined for their ability to produce regenerable callus cultures on media containing altered micronutrient concentrations, 3,6-dichloro-o-anisic acid (dicamba), glucose, and elevated levels of vitamin-free casamino acids and thiamine. Of these genotypes 199 (91%) produced callus that was regenerable in appearance. In the 1984 study, plant regeneration was noted in many commercially important inbreds, including B73, Mo17, B84, A632, A634, Ms71, W117, H99³H95 and Cm105. Thus tissue-culture techniques are now available to obtain callus cultures capable of plant regeneration from immature embryos of most maize genotypes.Abbreviations trade names 2,4-D 2,4-dichlorophenoxyacetic acid - dicamba 3,6-dichloro-o-anisic acid     

Properties of the purified APS-kinase from Escherichia coli and Saccharomyces cerevisiae

Adenylylsulphate kinase (EC 2.7.1.25, ATP:adenylylsulphate 3-phosphotransferase) has been isolated from Escherichia coli and from Saccharomyces cerevisiae. As major steps of purification, affinity chromatography on Sepharose CL 6B (blue or red) and chromatofocusing on polybuffer PBE 94^tm were employed. The proteins were obtained in nearly homogeneous state after five chromatographic steps.The isolated enzymes from both sources appeared predominantly to exist as dimers. Upon reduction of the protein with dithiothreitol, it desintegrated into assumingly identical smaller subunits (E. coli rom M_r 90-85000 to 45-40000 and s. cerevisiae from 52-49500 to 28-29500). Both forms, dimer and monomer were found catalytically active.Preincubation of the isolated enzyme from either source in the presence of thioredoxin plus DTT, reduced glutathione or DTT increased the activity significantly. Treatment of the enzyme with SH-blocking reagents inactivated the enzyme irreversibly as compared to the inactivation caused by oxidants (2,6-dichlorophenol-indophenol, ferricyanide or oxydized glutathione). This oxidant induced inactivation was less pronounced for the fungal enzyme than for the bacterial protein. The enzyme from E. coli required thioredoxin in order to alleviate the GSSG-induced inactivation.Abbreviations APS adenylylsulphate - APS kinase - ATP adenylylsulphate 3-phosphotransferase - DCPIP 2,6-dichlorophenol indophenol - DTT dithiothreitol - GSH reduced glutathione - GSSG oxidized glutathione - HPLC high performance liquid chromatography - -MSH -mercaptoethanol - PAPS 3-phosphoadenylylsulphate - TNBS 2,4,6 tri-nitrobenzenesulphonic acid     

Comparison of cell wall regeneration on maize protoplasts isolated from leaf tissue and suspension cultured cells

Summary The cell wall regeneration on protoplasts derived from maize mesophyll cells was compared with wall regeneration on protoplasts derived from suspension cultured cells using light microscopy, transmission electron microscopy, and mass spectrometry. The time course of cell wall regeneration has shown that the mesophyll protoplasts regenerated walls much slower than the protoplasts derived from cultured cells. Moreover, cell wall materials on the mesophyll protoplasts were often unevenly distributed. Electron microscopy has further demonstrated that the mesophyll protoplasts have less organized and compact walls than the protoplasts from cultured cells. Chemical analysis revealed that the mesophyll protoplasts had a lower ratio ofβ-(1–3)-glucan toβ-(1–4)-glucan than protoplasts from cultured cells. The significance of these results for the viability and development of protoplasts in culture is discussed. National Research Council of Canada paper no. 32458.     

DNA replication in maize leaf protoplasts

Maize leaf protoplasts were investigated for their metabolic competence and capacity to synthesize DNA. When protoplasts were incubated at elevated temperatures, they exhibited a heat shock response with specific proteins being preferentially synthesized. This indicated that the protoplasts were fully metabolically functional and capable of responding to environmental stimuli. Significant DNA synthesis was observed in these protoplasts after incorporation of ³H-thymidine into chromatin by trichloroacetic acid precipitation and by incorporation of 5-bromo-2-deoxyuridine (BrdU), an analog of thymidine, detected by immunofluorescence. The immunocytochemical method revealed that about 50% of nuclei in the maize leaf protoplasts were labelled after 3 days of culture and that most of these nuclei were labelled as intensely as normal mitotic cells. Aphidicolin, an inhibitor of DNA polymerase-, decreased the percentage of labelled nuclei, demonstrating that the labelling was substantially due to replicative DNA synthesis. However, chromosome condensation was not observed. It is proposed that these protoplasts are capable of DNA synthesis, but incapable of nuclear division. Effects of media additives on the number of nuclei entering S phase in these protoplasts were also assessed by the immunocytochemical method. Inclusion of 80mM Ca²⁺ in the enzyme solution increased protoplast yield and also appeared beneficial to DNA synthesis. The antioxidant, n-propyl gallate, which was used to stabilize the protoplasts, delayed the onset of DNA synthesis. Arginine and spermidine produced a slight increase in DNA synthesis.Abbreviations BrdU 5-bromo-2-deoxyuridine - DMSO dimethyl sulfoxide - n-PG n-propyl gallate - PBS phosphate-buffered saline Dedicated to Dr. Friedrich Constabel on the occasion of his 60th birthday     

Genetic control and racial variation of β-glucosidase isozymes in maize (Zea mays L.)

-Glucosidase (-D-glucoside glucohydrolase, E.C. 3.2.1.21, -Glu) isozyme variants were studied in a large number of inbred lines, crosses, and races of maize (Zea mays L.). The pattern of Mendelian inheritance demonstrated for -GLU variants indicated that they are under nuclear gene control. Twenty-two allelic forms at a single locus were identified in the materials studied by starch gel electrophoresis. Genetic data indicate that -GLU in maize is functionally a dimer. Variation of -GLU isozymes in 51 racial collections of maize from Mexico showed little correlation with morphological or geographical data. In 39 collections from Central America, variation patterns appeared to have some association with altitude.This work was supported in part by NIH Research Grant GM 11546.Paper No. 5040 of the Journal Series of the North Carolina Agricultural Experiment Station, Raleigh, North Carolina.     

Somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (<Emphasis Type="Italic">Secale cereale</Emphasis> L.)

A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N₆ regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.Abbreviations 2,4-D 2,4-Dichorophenoxyacetic acid - FDA Fluorescein diacetate - FW Fresh weight - GA₃ Gibberellic acid - Kinetin 6-Furfurylaminopurine - IAA Indole-3-acetic acid - NAA -Naphthaleneacetic acid     

Regeneration from Laminaria japonica Areschoug (Laminariales, Phaeophyceae) protoplasts isolated with bacterial alginase

Summary The conditions for effective isolation of viable protoplasts from Laminaria japonica with an alginase produced by marine bacterium Alteromonas sp. and a commercially available cellulase were investigated. The highest yields of viable protoplasts (7.910.4x10⁶ cells g^–1 FW) were obtained with a hypertonic solution containing 50 % seawater, 25 mM MgCl₂, 5 mM HEPES buffer system, and 0.5 M mannitol. Protoplasts were not obtained from thalli of L. japonica when an abalone alginase (abalone acetone powder; AAP: Sigma) was used instead of the bacterial alginase. The isolated protoplasts were cultured in an PESI medium at 5 °C. Complete cell wall formation was observed within 7 days, and dividing cells were first observed in a 9-day-old culture. Some protoplasts regenerated into sheet-shaped thalli and rhizoid structures were also observed on some thalli after 30 to 40 days in culture. This is the first report of protoplast regeneration into plantlets of L. japonica Areschoug (Laminariales, Phaeophyceae).Abbreviations FW Flesh weight - AAP Abalone acetone powder - HEPES N-2-hydroxy-ethylpiperazine-N-2-ethanesulfonic acid - Tris Tris(hyrdoxymethyl)aminomethane - PESI Provasoli's enriched seawater with iodine     

Studies of cotyledon protoplast cultures from Brassica napus,B. campestris and B. oleracea. I: Cell wall regeneration and cell division

Protoplasts isolated from cotyledons of a number of cultivars of Brassica napus, B. campestris and B. oleracea were cultured in different media to study the characteristics of cell wall regeneration and cell division at early stages of culture. Time course analysis using Calcolfluor White staining indicated that cell wall regeneration began in some protoplasts 2–4 h following isolation in all cultivars. 30–70% of cultured cotyledon protoplasts exhibited cell wall regeneration at 24 h and about 60–90% at 72 h after the initiation of culture. Results also indicated that a low percentage (0.4–5.4%) of cultured cotyledon protoplasts entered their first cell division one day after initial culture in all twelve cultivars. The percentage of dividing cells increased linearly up to 40% from 1 to 7 day, indicating that cotyledon protoplasts of Brassica had a high capacity for cell division. Factors that influence the level of cell wall regeneration and cell division during cotyledon protoplast culture have been investigated in this study. Cotyledons from seedlings germinated in a dark/dim light regime provided a satisfactory tissue source for protoplast isolation and culture for all Brassica cultivars used. The percentages of protoplasts exhibiting cell wall regeneration and division were significantly influenced by cultivar and species examined, with protoplasts from all five cultivars of B. campestris showing much lower rates of cell wall regeneration than those of B. napus and B. oleracea over 24–120 h, and with the levels of cell division in B. napus cultivars being much higher than those in B. campestris and B. oleracea over 1–9 days. The capacity of cell wall regeneration and cell division in cotyledon protoplast culture of the Brassica species appears under strong genetic control. Cell wall regeneration in protoplast culture was not affected by the culture medium used. In contrast, the composition of the culture medium played an important role in determining the level of cell division, and the interaction between medium type and cultivars was very significant.Abbreviations BA benzylaminopurine - CPW Composition of Protoplast Washing-solution - CW Calcolfluor White - EDTA ethylenediamine-tetraacetic acid - KT Kinetin - Md MS modified Murashige and Skoog medium - 2,4-d 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - IAA indole-3-acetic acid - PAR photosynthetically active radiation - SDS sodium dodecyl sulfate     

Characterization of the epidermis from barley primary leaves

A method is described for isolating epidermal protoplasts from the primary leaves of barley (Hordeum vulgare L.). Epidermal protoplasts are lighter than mesophyll protoplasts because of their smaller ratio of cytoplasm to vacuole, and can be separated from the latter by density-gradient centrifugation after complete digestion of the leaves. We have started a basic characterization of the epidermal protoplast fraction in comparison with mesophyll protoplasts. Epidermal protoplasts had a mean diameter of 63.5 m, whereas that of mesophyll protoplasts was 35.7 m. Their respiratory oxygen consumption was not influenced by light. They contained acid hydrolases and cytoplasmic enzymes in relative activities different from those of mesophyll protoplasts. Their polypeptide pattern as judged from two-dimensional separations was, in principle, similar to that of mesophyll cells after elimination of the plastids from the latter by the preparation of vacuoplasts. However, in addition, a considerable number of epidermis-specific polypeptides were observed. Isolated epidermal protoplasts were viable and efficiently incorporated [³⁵S]methionine into newly synthesized proteins. The results show that epidermal protoplasts are suitable for the investigation of the physiological and molecular properties of epidermal cells in leaves.Abbreviation SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis We are grateful to Professor U. Heber (Lehrstuhl Botanik 1, Würzburg) for his continuous support. This work was supported by the DFG and the University of Würzburg within the Sonderforschungsbereich 176.     

Increased totipotency of protoplasts from Brassica oleracea plants previously regenerated in tissue culture

Factors affecting the division of cells derived from leaf and cotyledon protoplasts from Brassica oleracea L. var. italica (Green Comet hybrid broccoli) were examined to optimize conditions for plant regeneration and to determine whether there was a genetic basis for improved regeneration from protoplasts derived from plants previously regenerated from tissue cultures [15]. When leaf protoplasts from different plants grown from hybrid seed were isolated and cultured simultaneously, division efficiencies of 1–95% were obtained. Cells from some plants showed high division efficiencies in consecutive experiments while cells from other plants had consistently low division rates. More plants from hybrid seed gave high division efficiencies when cotyledon protoplasts were used. However, cotyledon or leaf protoplasts from selfed progeny of regenerated plants produced more vigorous calli and more shoots than protoplasts from hybrid seed. These results suggest that there may be a genetic component to the increased totipotency of Brassica oleracea protoplasts.     

Production and characterization of antibodies to the β-(1→6)-galactotetraosyl group and their interaction with arabinogalactan-proteins

Rabbit antisera were raised against -(16)-galactotetraose coupled to bovine serum albumin (Gal₄-BSA). The antisera reacted with arabinogalactan-proteins (AGPs) isolated from seeds, roots, or leaves of radish (Raphanus sativus L.) as revealed by immunodiffusion analysis. Extensive removal of -l-arabinofuranosyl residues from these AGPs enhanced the formation of precipitin with the antisera. The antisera did not react with such other polysaccharides as soybean arabinan-4-galactan, -(14)-galactan, and -(13)-galactan, indicating their high specificity toward the consecutive -(16)-galactosyl side chains of AGPs. The antibodies were purified by affinity chromatography on a column of immobilized -(16)-galactotetraose as ligand. The specificity of the antibodies toward consecutive (16)-linked -galactosyl residues was confirmed by enzyme-linked immunosorbent assay for hapten inhibition against Gal₄-BSA as antigen, which revealed that -(16)-galactotriose and-tetraose were potent inhibitors, while -(13)-or -(14)-galactobioses and -trioses were essentially unreactive. Electron-microscopic observation of immunogold-stained tissues demonstrated that AGPs were localized in the middle lamella as well as at the plasma membrane of primary roots of radish. Agglutination of protoplasts prepared from cotyledons occurred with the antibodies, supporting the evidence for localization of AGPs in the plasma membrane. The antibody-mediated agglutination was inhibited by addition of AGPs or -(16)-galactotetraose.Abbreviations AGP arabinogalactan-protein - BSA bovine serum albumin - ELISA enzyme-linked immunosorbent assay - FITC fluorescein isothiocyanate - Gal₃-BSA -(16)-galactotriose coupled to BSA - Gal₄-BSA -(16)-galactotetraose coupled to BSA - Ig immunoglobulin - 4-Me-GlcpA 4-O-methyl-d-glucopyranosyluronic acid - M_r relative molecular mass The authors wish to thank Dr. J. Ohnishi of Department of Biochemistry, Saitama University, for his help in preparing protoplasts.