Isolation of Guard Cell Protoplasts from Mechanically Prepared Epidermis of Vicia faba Leaves

A method for isolating guard cell protoplasts (GCP) from mechanically prepared epidermis of Vicia faba is described. Epidermis was prepared by homogenizing leaves in a Waring blender in a solution of 10% Ficoll, 5 millimolar CaCl₂, and 0.1% polyvinylpyrrolidone 40 (PVP). Attached mesophyll and epidermal cells were removed by shaking epidermis in a solution of Cellulysin, mannitol, CaCl₂, PVP, and pepstatin A. Cleaned epidermis was transferred to a solution of mannitol, CaCl₂, PVP, pepstatin A, cellulase “Onozuka” RS, and pectolyase Y-23 for the isolation of GCP. Preparations made by this method included both adaxial and abaxial GCP and contained ≤0.017% mesophyll protoplasts, ≤0.6% mesophyll fragments, and no epidermal cell contaminants. Yields averaged 9 × 10⁴ protoplasts/leaflet and 98 to 100% of the GCP excluded trypan blue, concentrated neutral red, and hydrolyzed fluorescein diacetate. Isolated GCP increased in diameter by 2.2 micrometers after incubation in darkness in 10 micromolar fusicoccin, 0.4 molar mannitol, 5 millimolar KCl, and 1 millimolar CaCl₂. Illumination of GCP with 800 micromoles per square meter per second of red light resulted in alkalinization of their suspension medium. When 10 micromolar per square meter per second of blue light was superimposed onto the red light background, the medium acidified. Measurements of chlorophyll a fast fluorescence transients from isolated GCP indicated that GCP were capable of electron transport, and slow transients contained the “M” peak usually associated with a functional photosynthetic carbon reduction pathway.     

Separation of mesophyll protoplasts and bundle sheath cells from maize leaves for photosynthetic studies

Mesophyll protoplasts and bundle sheath strands of maize (Zea mays L.) leaves have been isolated by enzymatic digestion with cellulase. Mesophyll protoplasts, enzymatically released from maize leaf segments, were further purified by use of a polyethylene glycol-dextran liquid-liquid two phase system. Bundle sheath strands released from the leaf segments were isolated using filtration techniques. Light and electron microscopy show separation of the mesophyll cell protoplasts from bundle sheath strands. Two varieties of maize isolated mesophyll protoplasts had chlorophyll a/b ratios of 3.1 and 3.3, whereas isolated bundle sheath strands had chlorophyll a/b ratios of 6.2 and 6.6. Based on the chlorophyll a/b ratios in mesophyll protoplasts, bundle sheath cells, and whole leaf extracts, approximately 60% of the chlorophyll in the maize leaves would be in mesophyll cells and 40% in bundle sheath cells. The purity of the preparations was also evident from the exclusive localization of phosphopyruvate carboxylase (EC 4.1.1.31) and NADP-dependent malate dehydrogenase (EC 1.1.1) in mesophyll cells and ribulose 1,5-diphosphate carboxylase (EC 4.1.1.39), phosphoribulokinase (EC 2.7.1.19), and “malic enzyme” (EC 1.1.1.40) in bundle sheath cells. NADP-glyceraldehyde 3-phosphate dehydrogenase (EC 1.2.1.13) was found in both mesophyll and bundle sheath cells, while ribose 5-phosphate isomerase (EC 5.3.1.6) was primarily found in bundle sheath cells. In comparison to the enzyme activities in the whole leaf extract, there was about 90% recovery of the mesophyll enzymes and 65% recovery of the bundle sheath enzymes in the cellular preparations.     

Protoplast culture and plant regeneration ofPinellia ternata

A study was undertaken to develop a protoplast regeneration system for pinellia. A yield of 19 29 x 10⁵ protoplasts/g F. W. could be obtained from cell suspension cultures incubated in a digestion enzyme solution with 2% cellulase Onzuka R-10, 10% pectinase (Sigma), 0.01% pectolyase Y23. K8P and modified MS media were used to culture protoplasts in: a) liquid, b) liquid-solid double layer, or c) agarose embedded protoplast culture. The former two were conducive to colony formation from protoplast-derived cells. The frequency of cell division was about 8% after 3 days in culture. Gradually adding fresh medium of lower osmotic pressure into the medium for protoplast culture favored cell division. Calli (1–2 mm in diameter) formed after 30–40 days in culture. The calli transferred onto medium supplemented with KT (0.5 mg 1^–1) and NAA (0.2 mg 1)^–1) could regenerate plants after 40–50 days. Of 47 plantlets transplanted into plots, 29 flowered and were fertile.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - KT kinetin - CH casein hydrolysate     

Isolation,culture, and cell division in cotyledon protoplasts of cotton (Gossypium hirsutum and G. barbadense)

Protoplasts were isolated from cotyledons and foliage leaves of cotton (Gossypium hirsutum and G. barbadense). Cotyledon protoplasts were larger and responded to culture better than leaf protoplasts. Cotyledon derived protoplasts regenerated cell walls and formed microcolonies of 2–3 cells in G. hirsutum and 5–8 cells in G. barbadense. However, the microcolonies did not grow beyond this stage. Protoplast yield and viability, cell wall regeneration and cell division were influenced by several factors, e.g., genotype, age, tissue and growth condition of donor plant, enzyme mixture and concentration, preplasmolysis period, incubation period, and culture medium.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - GA₃ gibberellic acid - p CPA p-chlorophenoxyacetic acid - MES 2[N-morpholino]ethanesulfonic acid     

Isolation,culture and division of olive (Olea europaea L.) protoplasts

Protoplasts from Olea europaea L. have been compared in terms of their yield, viability, cell division and callus differentiation. Viable protoplasts were isolated from in vitro cultured leaves and cotyledons by an overnight incubation in an enzyme solution containing 1–1.5% driselase and 0.5M sucrose. This method allowed high yield of purified protoplasts, which floated and formed a dark green band at the meniscus, after centrifugation. Purified protoplasts were diluted to 3×10⁴ protoplasts·ml^–1 in culture medium. After cell wall regeneration, protoplasts gradually increased their volumes under appropriate conditions. The first divisions occurred during the second week in culture. Division efficiency ranged from 5.2 to 9.8% after 20 days in culture. Two weeks later visible microcolonies developed only from cotyledon protoplasts. After 6 weeks in culture, the microcalli were transferred to a solidified culture medium with 0.6% agarose, which induced active callus growth.Abbreviations OM olive proliferation medium, Rugini 1984 - Omg OM for the germination of olive embryos - OMr=OM for root induction - OMp=OM for protoplasts - OMc=OM for callus - BN Bourgin and Nitsch medium 1967 - IBA indol-3-butyric acid - NAA naphthalene acetic acid - 2,4-D dichlorophenoxyacetic acid.     

Use of lipophilic cations to measure the membrane potential of oat leaf protoplasts

Uptake of the lipophilic cation triphenylmethylphosphonium into mesophyll protoplasts of oat (Avena sativa L. cv. “Garry”) approaches equilibrium at 3 to 4 hours. The resulting external and internal concentrations are then used with the Nernst equation to obtain a membrane potential of −62 millivolts, inside negative. Potentials calculated in this manner are depolarized by adding 2 mm sodium azide and 50 μm carbonyl cyanide m-chlorophenylhydrazone as well as by increasing the external proton and potassium concentrations. The depolarizations are qualitatively similar to those seen when oat mesoyphll cells are measured in situ with microelectrodes. It is concluded that due to the lack of turgor and fragility of protoplasts, estimations of their membrane potential may be made more reliably, under some conditions, with lipophilic cations than with microelectrodes.     

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.)

Summary We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×10⁷ protoplasts.ml^–1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (10⁶ protoplasts.ml^–1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.     

Protoplast isolation for species in the Chamelaucium group and the effect of antioxidant enzymes (superoxide dismutase and catalase) on protoplast viability

An effective protocol for protoplast isolation from young leaves and somatic embryogenic cells of species in the Chamelaucium group and the use of superoxide dismutase (SOD) and catalase (CAT) to enhance protoplast viability are described. Mesophyll protoplasts were isolated from young leaves of a white Geraldton waxflower (Chamelaucium uncinatum) line 583, using a mixture of 1% (w/v) cellulase R10, 0.5% (w/v) macerozyme R10, and 0.1% (w/v) pectolyase. Viability of isolated mesophyll protoplasts increased dramatically when SOD and CAT were added. The highest increase of 7.61-fold in viability and 4.34-fold of viable protoplast yield were achieved when a combination of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 was added to the enzyme mixture. Somatic embryogenic cell-derived protoplasts were isolated from embryogenic suspension cells of C. uncinatum line 583 when 1% (w/v) hemicellulase was added to a combination of 2% (w/v) cellulase R10, and 1% (w/v) macerozyme R10. Addition of SOD at 500 units mL^?1 and CAT at 2,000 units mL^?1 to the enzyme mixture improved viability only slightly, to above 90%, but improved yield significantly (6.6-fold). This combination of enzymes was also used to isolate protoplasts from embryogenic suspension cells of Chamelaucium repens and from young leaves of C. uncinatum, Actinodium calocephalum, Verticordia etheliana, Verticordia grandis, Verticordia hughanii, and Verticordia mitchelliana successfully with viability >80% and viable yield >7?×?10⁵ cells g^?1 fresh weight (or per milliliter packed cell volume in the case of suspension cells).     

Protoplasts of Gelidium robustum (Rhodophyta)

Viable protoplasts were isolated from apices of the agarophyte Gelidium robustum (Gardn.) Hollenb. & Abb. using a combination of commercial cell-wall degrading enzymes and extracellular wall-degrading enzymes isolated from a marine bacterium. The protoplasts were approximately 8–15 µm in diameter, liberated mainly from the surface cell layers and from cells at the distal ends of medullary filaments. The bacterial enzyme alone was not sufficient to liberate significant numbers of protoplasts. Maximum yield was 9 × 10⁵ protoplasts/g tissue (wet wt.). Optimum osmolality occurred between 1750–1950 mOs kg^–1; yield and viability were severely diminished at osmolalities less than 1350 mOs kg^–1. Viability, as determined by flurorescein diacetate staining and Evans Blue exclusion 1 hr after removal from the enzyme solution, was approximately 80–95%. Roughly 80% of the cells did not show Calcofluor fluorescence, while 40% stained positively for the presence of sulfated polysaccharides. Cell wall regeneration was observed with inconsistent reproducibility, and no cell division was observed when the protoplasts were placed in culture medium.Dedicated to the memory of Professor Michael Neushul.     

A novel method for regenerating plants from mesophyll protoplasts of Arabidopsis thaliana line WS

A procedure has been developed for the successful regeneration of plants from mesophyll protoplasts of Arabidopsis thaliana line WS. The protocol is an improved version of that of Damm and Willmitzer (1988). The main changes in original procedure are as follows:

1. A mixture of Cellulase Y-C (0.5%) and Pectolyase Y-23 (0.05%) is used for the isolation of protoplasts. Use of these enzymes reduces the incubation time to 50 min.
2. α-Naphthaleneacetic acid is used as the auxin throughout cultures of protoplasts and calli.
3. Protoplasts and calli are incubated under dim white light (0.8–8 μW/cm²) during culture.

With these modifications, we were able consistently to obtain regenerated shoots from about 70% of calli that had been transferred to shoot-forming medium even though the plating efficiency was rather low (about 0.5–1.5%).     

Synchronization of somatic embryogenesis in a carrot cell suspension culture

Synchronization of somatic embryogenesis was achieved in a carrot (Daucus carota L. cv. “Kurodagosun”) suspension culture by sieving the initial heterogeneous cell population, by density gradient centrifugation in Ficoll solutions, and by subsequent repeated centrifugations at a low speed (50g) for a short time (5 seconds), followed by transferring the cell clusters obtained, which were composed of 3 to 10 cells, to a medium containing zeatin (0.1 micromolar) but no auxin. The frequency of embryo formation reached more than 90%, and synchrony of the embryogenetic process was observed at least in the early stages of the process. The system established in the present work provides a useful system for biochemical research into the mechanisms of somatic embryogenesis.     

Plasmalemma patch clamp experiments in plant root cells: procedure for fast isolation of protoplasts with minimal exposure to cell wall degrading enzymes

Summary A convenient and rapid isolation procedure for root cell protoplasts suitable for patch clamp experiments, was developed for root cells of tomato (Lycopersicon esculentum) andPlantago species, grown on hydroculture. The procedure is based on a minimal exposure of cells to cell wall degrading enzyme mixtures. After an incubation period of 30 min in a cell wall degrading enzyme mixture all free floating cells were discarded. Subsequently the root material was rinsed and a second group of cells, still present inside the tissue, was freed by application of mechanical pressure. The newly released protoplasts were filtered and collected on the glass bottom of a patch clamp dish. The bathing medium was rinsed extensively removing cellulose fibrils and protoplasts not attached to the glass. Removal of these cellulose fibrils significantly improved the seal success ratio. The isolated protoplasts were suitable for patch clamp experiments in the cell-attached patch, the whole cell and the isolated patch configuration.Abbreviations BSA bovine serum albumin - BTP bis-tris propane - CAP cell-attached patch - OOP outside out patch - PEG polyethylene glycol - WC whole cell     

Cytological study on wheat (Triticum timopheevi Zhuk.) protoplasts

Protoplasts were obtained from tetraploid wheat (Triticum timopheevi Zhuk.) suspension culture by incubation in solution of 1 % pectinase 500, 1 % driselase and 1 % cellulase and cultivated in Schenk and Hildebrandt medium. Freshly isolated protoplasts contained dense cytoplasm and constricted organellae exhibited negative contrast of their membranes. Together with normal protoplasts huge multinucleate protoplasts were present in the population. 3 h after plating, the cytoplasm showed normal appearance, the negative contrast of membranes was not evident any longer. Cisternae of endoplasmic reticulum and Golgi apparatus were numerous. There were some vesicles and fibres on the protoplast surface. 8 d after plating, many dividing cells were found out and cell clumps arosen in this way were present in the culture. Some of the protoplasts particularly those originally multinucleate ones were upset.     

Protoplast isolation optimization and regeneration of cell wall in Gracilaria gracilis (Gracilariales, Rhodophyta)

This paper reports the first successful isolation and cell wall regeneration of Gracilaria gracilis (Stackhouse) Steentoft, Irvine et Farnham protoplasts. These results form an important foundation for the development of a successful tissue culture system for G. gracilis. Initially, an isolation protocol was optimized by investigation of the effects of the enzyme constituents and concentrations, the pre-treatment of thalli, the incubation period and temperature, and the pH of the enzymatic medium on protoplast yields. A pre-treatment of G. gracilis thalli with 1 % (w/v) papain for 30 min followed by a 3-h enzymatic digestion of thalli with an enzymatic mixture containing 2 % (w/v) cellulase Onozuka R-10, 1 % (w/v) macerozyme R-10, and 10 U mL^?1 agarase at pH 6.15 was found to produce the highest yield of protoplasts at 22 °C. Reliably high yields (20–30?×?10⁵ protoplasts g^?1 f.wt) of protoplasts could be obtained from G. gracilis thalli when this optimized protocol was used. Cell wall re-synthesis by G. gracilis protoplasts, which constitutes the first step towards whole plant regeneration, was followed using calcoflour staining and scanning electron microscopy. Protoplasts were shown to complete the initial stages of cell wall re-synthesis within the first 24 h of culturing.     

Response of Nicotiana mesophyll protoplasts of normal and tumorous origin to indoleacetic Acid in vitro

Enzymatically isolated mesophyll protoplasts of the two normal, nontumor-forming parent species Nicotiana glauca and N. langsdorffii and two of their tumor-prone interspecific hybrids were maintained in a 0.5 m mannitol solution supplemented with various concentrations of auxin (indoleacetic acid) and the growth inhibitor abscisic acid. The bursting response of protoplasts in medium containing indoleacetic acid in physiological concentrations showed that protoplasts from the tumorous hybrids tolerate auxin in up to 30 times higher concentrations than protoplasts from parent plants. The “survival” of all protoplast preparations in comparable abscisic acid containing media was significantly greater than that in the indoleacetic acid supplemented solutions. Protoplasts in vitro respond with bursting only after the external indoleacetic acid concentrations reach levels comparable to those of endogenous auxins present in these cells. The data are discussed in conjunction with previous observations on uptake and maintenance of indoleacetic acid levels in tumorous Nicotiana tissues.     

Production and seeding of protoplasts of <Emphasis Type="Italic">Porphyra okhaensis</Emphasis> (Bangiales,Rhodophyta) in laboratory culture

High yields of viable protoplasts were produced from Porphyra okhaensis H. Joshi, Oza & Tewari following two-step enzymatic digestion (protease pretreatment and cell wall polysaccharides-degrading enzyme treatment) of the thallus. Pretreatment of the tissues with 1% Protease P6 at 20± 1 ^°C for 30 min prior to digestion with cell wall polysaccharide-degrading enzymes increased the protoplast yield two fold compared to tissues that were digested with polysaccharide-degrading enzyme mixture. The polysaccharide-degrading enzymes employed for protoplast isolation from P. okhaensis were Cellulase Onozuka R-10, Macerozyme R-10, abalone acetone powder and agarase. Suitable pH, temperature and duration of enzyme treatment for optimal production of viable protoplasts were pH 6, 20± 1 ^°C and 3 h, respectively. Mannitol (0.8 M) was found to be an excellent osmotic stabilizer. When the tissue of P. okhaensis pretreated with 1% protease solution was digested with commercial enzyme mixture consisting of 2% Cellulase Onozuka R-10, 2% Macerozyme R-10, 1% abalone acetone powder, 50 units of agarase and 0.8 M mannitol in 1% NaCl (adjusted to pH 6.0 with 25 mM MES buffer) with gentle agitation for 3 h at 20± 1 ^°C, 23.2± 0.24× 10⁶ protoplasts g⁻¹ fresh wt. were obtained. The regeneration rate of protoplasts isolated in the present study was found to be 79%. Protoplasts that regenerated cell walls underwent regular cell divisions and developed into leafy gametophytic thallus in the laboratory cultures. Further, the seeding of nylon threads with partially developed protoplasts of P. okhaensis was successful in the laboratory conditions and germlings as long as 3–4 cm were obtained from such seeded threads in one month period in aerated cultures.     

Impact of Temperature on the Physiological Status of a Potential Bioremediation Inoculant, Arthrobacter chlorophenolicus A6

Arthrobacter chlorophenolicus A6 (A6) can degrade large amounts of 4-chlorophenol in soil at 5 and 28°C. In this study, we investigated the effects of temperature on the physiological status of this bacterium in pure culture and in soil. A derivative of A6 tagged with the gfp gene (encoding green fluorescent protein [GFP]) was used to specifically quantify A6 cells in soil. In addition, cyano-ditolyl-tetrazoliumchloride was used to stain GFP-fluorescent cells with an active electron transfer system (“viable cells”) whereas propidium iodide (PI) was used to stain cells with damaged membranes (“dead cells”). Another derivative of the strain (tagged with the firefly luciferase gene [luc]) was used to monitor the metabolic activity of the cell population, since the bioluminescence phenotype is dependent on cellular energy reserves. When the cells were incubated in soil at 28°C, the majority were stained with PI, indicating that they had lost their cell integrity. In addition, there was a corresponding decline in metabolic activity and in the ability to be grown in cultures on agar plates after incubation in soil at 28°C, indicating that the cells were dying under those conditions. When the cells were incubated in soil at 5°C, by contrast, the majority of the cells remained intact and a large fraction of the population remained metabolically active. A similar trend towards better cell survival at lower temperatures was found in pure-culture experiments. These results make A. chlorophenolicus A6 a good candidate for the treatment of chlorophenol-contaminated soil in cold climates.     

The Structure of Plant Cell Walls: II. The Hemicellulose of the Walls of Suspension-cultured Sycamore Cells

The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed “amyloid” xyloglucans.     

Somatic proembryo formation and transient expression of a luciferase gene in Douglas fir and loblolly pine protoplasts

Somatic pro-embryos were regenerated from morphogenic protoplasts of cell suspension cultures of Douglas fir and loblolly pine. Morphogenic protoplasts were obtained from suspension cultures of embryonal cells grown in a modified basal medium high in myo-inositol. After 11–12 weeks, microcolonies of embryonal-suspensor masses (ESMs) were recovered from agarose-embedded protoplasts. Somatic pro-embryos were regenerated after 18–20 weeks by somatic polyembryogenesis from new ESMs on agar plates. The luciferase (luc) gene was successfully introduced into fir and pine protoplasts by electroporation. While viability of protoplasts was reduced from 90% to 45–55% by electroporation, the transient expression of the luc gene was detected in protoplasts surviving 36 h after electroporation. Gene expression was improved by the addition of polythylene glycol (PEG) to the electroporation mixture.