A simple method for the isolation of plant protoplasts

A simple protoplast isolation protocol that was designed to recover totipotent plant protoplasts with relative ease has been described. The key elements of the protocol are, tissue digestion at slightly elevated temperatures and use of protoplast-releasing enzymes that are stable and efficient at higher temperatures. Besides enzymes, the protoplast isolation cocktail consisted of an osmoticum (mannitol or MgSO₄), and a protectant (CaCl₂ 2H₂O), all dissolved in distilled water. The protocol has ensured reproducibility, higher yields and is gentle on protoplasts as the protoplasts obtained were amenable to cell wall regeneration and cell division. Plant regeneration was demonstrated forNicotiana tabacum cv. Thompson from protoplasts isolated by this method. Wall regeneration and cell division were obtained in other species. The merits of the protocol are, simple and easy-to-handle procedure, non-requirement of preconditioning of donor plant and explants, incubation without agitation, satisfactory yields, culturability of the protoplasts isolated and applicability of the protocol to a large number of species including mucilage-containing plants.     

Formation,growth, and regeneration of protoplasts of the green alga,Uronema gigas

Summary Separate protoplasts were obtained by the action of snail gut juice enzymes on the cell walls of the green algaUronema gigas. The cultivation of the protoplasts in mineral media caused only their enormous growth; in the presence of glucose a fibrillar network was formed on the surfaces of the growing protoplasts. Only after the addition of pectin the regeneration of the cell wall and the renewal of their morphogenesis could be observed.     

Seaweed protoplasts: status,biotechnological perspectives and needs

Protoplasts are living plant cells without cell walls which offer a unique uniform single cell system that facilitates several aspects of modern biotechnology, including genetic transformation and metabolic engineering. Extraction of cell wall lytic enzymes from different phycophages and microbial sources has greatly improved protoplast isolation and their yield from a number of anatomically more complex species of brown and red seaweeds which earlier remained recalcitrant. Recently, recombinant cell wall lytic enzymes were also produced and evaluated with native ones for their potential abilities in producing viable protoplasts from Laminaria. Reliable procedures are now available to isolate and culture protoplasts from diverse groups of seaweeds. To date, there are 89 species belonging to 36 genera of green, red and brown seaweeds from which successful protoplast isolation and regeneration has been reported. Of the total species studied for protoplasts, most belonged to Rhodophyta with 41 species (13 genera) followed by Chlorophyta and Phaeophyta with 24 species each belonging to 5 and 18 genera, respectively. Regeneration of protoplast-to-plant system is available for a large number of species, with extensive literature relating to their culture methods and morphogenesis. In the context of plant genetic manipulation, somatic hybridization by protoplast fusion has been accomplished in a number of economically important species with various levels of success. Protoplasts have also been used for studying foreign gene expression in Porphyra and Ulva. Isolated protoplasts are also exploited in numerous miscellaneous studies involving membrane function, cell structure, bio-chemical synthesis of cell walls etc. This article briefly reviews the status of various developments in seaweed protoplasts research and their potentials in genetic improvement of seaweeds, along with needs that must to be fulfilled for effective realization of the objectives envisaged for protoplast research.     

Cellulase secretion by immobilized protoplasts of Trichoderma reesei

Protoplasts from Trichoderma reesei were immobilized in alginate and induced to produce cellulase (endoglucanase and β-glucosidase) enzymes. The specific activities of the synthesized enzymes were higher in immobilized protoplasts than in both free and immobilized mycelia. Immobilized protoplasts show an enhanced rate of exocellular β-glucosidase production compared to intact mycelia due to the lack of cell wall. The ratio of the exocellular/intracellular β-glucosidase was 5.9 for immobilized protoplasts and 0.32 for free mycelia.     

Regenerated cell wall of carrot protoplasts isolated from suspension-cultured cells

Protoplasts of Daucus carota L. cultured in a synthetic liquid medium resumed cell division after about 4 days of cultivation. During this lag period, nucleic acid and protein showed only slight increases but the protoplasts commenced cell-wall regeneration soon after the removal of lytic enzymes. The originally spherical protoplasts became ellipsoidal before they underwent division. Radioactive glucose and myo-inositol were readily utilized by the protoplasts. Most of the radioactivity, however, appeared in extracellular polysaccharides and only a small portion was deposited in the regenerated wall. The sugar composition of new cell wall, as studies by chemical analysis and incorporation of labelled precursors, was shown to be considerably different from that of normal cell wall.     

Mechanical release and lectin labeling of maize root protoplasts

Summary An improved method for the mechanical release of protoplasts from plant tissues is described. The historically-low yield of mechanically-released protoplasts is greatly increased by use of a simple electrically-driven tissue sheer and by optimization of various other steps in the procedure. As counted by light microscopy of a purified preparation, the number of mechanically-released protoplasts obtained is about 6×10⁴ per gram fresh weight of cortical tissue from the primary root of maize (Zea mays L. WF9×Mo 17) seedlings. Nuclear staining of the preparation, however, shows that about half of these protoplasts lack a nucleus and thus are actually subprotoplasts. Comparison of lectin binding to the plasma membranes of mechanically-and enzymatically-released protoplasts shows that both types contain binding sites forRicinus communis agglutinin. Binding sites for peanut (Arachis hypogaea) agglutinin are not naturally present on mechanically-released protoplasts but are generated by exposure to a mixture of Cellulysin and Pectolyase Y-23, the cell wall-degrading enzymes used to prepare enzymatically-released protoplasts.Abbreviations BSA bovine serum albumin - DDT dithiothreitol - gfw gram fresh weight - Mes 2-(N-morpholino) ethanesulfonic acid - PNA peanut (Arachis hypogaea) agglutinin - RCA Ricinus communis agglutinin - Tris tris(hydroxymethyl)aminomethane     

Karyokinesis in growing and reverting protoplasts ofSchizosaccharomyces pombe

Division of nuclei without cytokinesis proceeds in growing protoplasts ofSchizosaccharomyces pombe. Prior to regeneration of the complete cell wall and reversion the protoplasts contain 1–7 nuclei, protoplasts with 1–2 nuclei are most frequent. When regeneration of the wall is postponed by adding snail enzymes to the growth medium, protoplasts with a higher number of nuclei (2–4) occur. Multinuclear protoplasts can revert to cells. During the first cytokinesis the protoplast with the regenerated cell wall is divided into two cells by a septum, distribution of nuclei between the two cells being probably incidental. More than only a single nucleus can pass to the revertants even during the second cytokinesis. Septation of protoplasts occurs also during a partial blockage of the wall formation by the snail enzyme preparation, however, reversion to cells can never be observed here (it occurs only after transfer of protoplasts to the medium without the enzyme preparation). The growing and reverting protoplasts represent a very good model system for studying relations among individual processes of the cell cycle, primarily growth of the cell, nuclear cycle and cytokinesis. Yeast protoplasts are often utilized as models for studying morphogenic processes, relations among regeneration of the cell wall, including division of the nucleus (karyokinesis) and cytokinesis.     

Plant regeneration from protoplasts of Enteromorpha intestinalis (Chlorophyta, Ulvophyceae) as seedstock for macroagal culture

A continuous micropropagation was established from protoplasts of thegreen alga Enteromorpha intestinalis. The effects of two differentcrude enzymes and the osmolarity at different concentrations of the enzymesolution on algal protoplast yields were tested. The optimal enzymecomposition for cell wall digestion and protoplast viability was 2%cellulase R 10 Onozuka and 2% Aplysie with 0.5 m mannitol. Largenumbers of Enteromorpha protoplasts were released (10.0 × 10⁶protoplasts from 1 g fresh thalli) and settled on a rangeof substrata. Regeneration of the protoplasts followed the normal patternfor this species. Conditions for pure cultures and efficient systems offloating supports with nets were determined to optimise the product qualityof plantlets of Enteromorpha. A promising storage process has beendeveloped which involves including protoplasts in beads of alginic acid gel.Plants regenerated from protoplasts may also be used as seedstock tofacilitate propagation for macroalgal culture.     

Formation and regeneration of protoplasts in the moldRhizopus nigricans

Wall-less protoplasts of the moldRhizopus nigricans were obtained by means of enzymic digestion of the walls of vegetative hyphae by Helix pomatia digestive juice. After removing the enzymes the protoplasts transferred to a nutrition medium regenerate. In the first stage of this process the protoplasts from a firm wall and then the hyphae begin to grow. Regeneration results in the formation of a normal mycelium. If in the course of regeneration of the protoplasts snail enzymes are present, they partially block the synthesis of the wall and the protoplasts transform into incessantly growing giant formations. Re-establishment of morphogenesis begins only after eliminating the effect of the snail enzymes.     

Protoplasts from Phytolacca dodecandra L'Herit (endod) and P. americana L. (pokeweed)

Summary Pokeweed (Phytolacca americana L.) and endod (P. dodecandra L'Herit) produce ribosome-inactivating proteins which are sequestered in leaf cell walls. These proteins display strong antiviral activity. To aid in studying the antiviral mechanism, we developed protocols to isolate protoplasts from suspension culture cells and leaves. Ninety-five percent of pokeweed or endod culture cells were converted to protoplasts using 2% cellulase, 0.25% pectinase, 0.2 M mannitol, 2% sucrose, 15 mM CaCl₂ Murashige and Skoog salts, pH 5.7. Viability was >85% after 24 h. Culture-derived protoplasts were purified by centrifugation through a 15% sucrose pad. Protoplasts collected from the supernatant were then pelleted in 0.3 M mannitol. Pokeweed leaves provided respectable yields (4×10⁶ protoplasts/g f w) of partially-purified viable protoplasts when digested in solution containing 1% cellulase, 0.2% Pectolyase, 0.4 M mannitol, CPW salts, 0.5 mM MES, pH 5.6. We were unable to completely separate cell debris from mesophyll protoplasts, which were small and easily damaged by centrifugation. Endod leaves were found to be resilient to several digestion enzymes tested.     

Lokalisation der Peroxidase-Isoenzyme in Protoplasten und Zellwänden von Nicotiana tabacum L.

Summary Upon disk-electrophoresis with guaiacol as a substrate the peroxidase-isoenzymes of Nicotiana tabacum (L.) were localized on the gels in two anodic and two cathodic groups. By preparation of protoplasts and isolation of cell walls it was possible to show that only cathodic enzymes are located in the protoplasts in measurable amounts, whereas all the isoenzymes, anodic and cathodic, can be found associated with cell walls. The different groups of isoenzymes are bound to the cell wall in different ways as evidenced by differences in their extration. It seems possible that different biological functions are associated with the different groups of isoenzymes.The isoenzyme patterns of different organs and tissues of tobacco show qualitative differences only in the anodic (i.e. wall located) isoenzymes. It is suggested that the ontogenetic change in peroxidase-patterns is direct evidence of biochemical differences in the cell walls of the different tissues and organs.

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Lokalisation der Peroxidase-Isoenzyme in Protoplasten und Zellwänden von Nicotiana tabacum L.

     

Protoplast production fromPorphyra linearis using a simplified agarase procedure capable of commercial application

Abalone enzymes, Cellulase R-10, Macerozyme and agarase fromPseudomonas atlantica were tested for activity on agarose, cellulose, xylan, the cell wall matrix and porphyran isolated fromPorphyra linearis. Agarase, and to a lesser extent Macerozyme, digested both agarose and porphyran. Abalone enzymes and Cellulase R-10 reacted only weakly with porphyran. A simple standardized protocol for making protoplasts fromPorphyra linearis was developed using 0.025% agarase in seawater without added organic osmoticants. Protoplasts prepared with agarase remained viable for at least 24 h in the digestion medium. Regeneration of the protoplasts followed the normal pattern for this species. Agarase can be used to obtain large number of protoplasts which could substitute for conchospores in seeding nets for the aquaculture ofP. linearis Author for correspondenceIssued as NRCC no. 34897     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate     

Chloroplastic NADPH oxidase-like activity-mediated perpetual hydrogen peroxide generation in the chloroplast induces apoptotic-like death of <Emphasis Type="Italic">Brassica napus</Emphasis> leaf protoplasts

Despite extensive research over the past years, regeneration from protoplasts has been observed in only a limited number of plant species. Protoplasts undergo complex metabolic modification during their isolation. The isolation of protoplasts induces reactive oxygen species (ROS) generation in Brassica napus leaf protoplasts. The present study was conducted to provide new insight into the mechanism of ROS generation in B. napus leaf protoplasts. In vivo localization of H₂O₂ and enzymes involved in H₂O₂ generation and detoxification, molecular antioxidant-ascorbate and its redox state and lipid peroxidation were investigated in the leaf and isolated protoplasts. Incubating leaf strips in the macerating enzyme (ME) for different duration (3, 6, and 12 h) induced accumulation of H₂O₂ and malondialdehyde (lipid peroxidation, an index of membrane damage) in protoplasts. The level of H₂O₂ was highest just after protoplast isolation and subsequently decreased during culture. Superoxide generating NADPH oxidase (NOX)-like activity was enhanced, whereas superoxide dismutase (SOD) and ascorbate peroxidase (APX) decreased in the protoplasts compared to leaves. Diaminobenzidine peroxidase (DAB-POD) activity was also lower in the protoplasts compared to leaves. Total ascorbate content, ascorbate to dehydroascorbate ratio (redox state), were enhanced in the protoplasts compared to leaves. Higher activity of NOX-like enzyme and weakening in the activity of antioxidant enzymes (SOD, APX, and DAB-POD) in protoplasts resulted in excessive accumulation of H₂O₂ in chloroplasts of protoplasts. Chloroplastic NADPH oxidase-like activity mediated perpetual H₂O₂ generation probably induced apoptotic-like cell death of B. napus leaf protoplasts as indicated by parallel DNA laddering and decreased mitochondrial membrane potential.     

Regeneration of plantlets fromEnteromorpha (Ulvales,Chlorophyta) protoplasts in axenic culture

High yields of protoplasts have been obtained from vegetative thalli of three species ofEnteromorpha by enzymatic degradation of the cell wall. Several commercial and crude enzymes prepared from the digestive system and hepatopancrease of abalone and top-shell were tested at different concentrations and combinations to evaluate the yield. Commercial enzymes in combination with either abalone or top-shell crude enzymes, consistently produced a high yield of protoplasts from all three species. High regeneration rate (85–95%) occurred in the protoplasts cultured at a density greater than 1.72 × 10³ cells cm⁻² at 20 and 25°C. Light intensities tested in the present study did not affect protoplast wall formation and regeneration. Protoplasts, after regenerating the cell wall, followed different types of developmental patterns under identical culture conditions. In one type some cells underwent repeated cell divisions and formed a round and oval shaped hollow thallus with a single layer of cells. In the second type many cells underwent one or two cell divisions (occasionally no division) and soon matured and discharged many motile spores, which on germination grew into normal plantlets. In the third type some cells divided irregularly to form a mass of callus-like cells (exceptE. prolifera). Culture medium supplemented with either mannitol, sorbitol, dextrose, saccharose or NaCl at higher concentrations (> 0.4 M) inhibited cell division and further differentiation in all species. author for correspondence     

Inhibition of the cell wall regeneration and reversion in protoplasts ofRhizopus nigricans

During cultivation in a nutrient medium with snail gastric juice the protoplasts ofRhizopus nigricans produce an incomplete cell wall and grow. A true growth, associated with nuclear division, is involved. Morphology of growth of the formations is determined by the structure of the incomplete cell wall. When the incomplete wall is formed by a thin fibrillar net the growing formation assumes the physically optimal shape—i.e. a sphere; when the net is dense polar growth predominates. The degree of construction of the new wall depends on the activity of snail gastric juice enzymes which decreases during the cultivation. When fresh snail enzymes were added at certain intervals, only a fine fibrillar net was formed on the surface of growing protoplasts. The formations grew for up to 8 d under these conditions, reached several hundred μm in size and the number of nuclei increased up to 80-fold. When the blocking of the wall synthesis was interrupted, a complete cell wall regenerated on the surface of these giant formations and a reversion to hyphae was observed. The incomplete cell wall functions as a passive morphogenetic factor: It can influence the morphology of the growing protoplasts but it cannot induce reversion to hyphase and secure the permanent existence of these structures.     

Protoplasts of Pyricularia oryzae P2 : Preparation and Regeneration into Hyphal Form

Protoplasts of Pyricularia oryzae P₂, a rice blast mold, were prepared in high yield from the young mycelium of the fungus using lytic enzymes from Bacillus circulans WL 12. The majority of the protoplasts had one nucleus per cell. The protoplasts formed a cell wall and eventually reverted to normal mycelial form in liquid medium. The process of regeneration was studied under phase-contrast and electron microscopes. The protoplast built a very thick wall prior to the protrusion of a germ-tube like hypha. Golgi apparatus-like structures appeared in the early stage of regeneration and disappeared later. Electron-transparent amorphous structures accumulated during regeneration. Lomasomes were observed in the regenerated cell walls.     

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.     

The use of metabolic inhibitors for the selection of fusion products of higher plant protoplasts

Summary Protoplasts of Solanum nigrum and Petunia hybrida have been treated with lethal doses of different inhibitors irreversibly affecting cell metabolism. Survival was expected in fusion products by complementation of the inhibited enzymes. After fusion, three protoplasts survived, formed cell walls and two of them underwent mitoses. One of the multicellular regenerants could be examined cytologically and revealed to be a heterokaryon.     

Studies on the fungitoxicity of captan

The cytological changes in Neurospora crassa conidia following treatment with captan at the ED ₅₀ fungicidal dose have been examined by electron microscopy. With dormant conidia the only observable effect of captan was to produce a characteristic convoluted form of the nuclear membrane. It is suggested that this effect may be due to the reaction of captan with the sulphydryl groups of the nuclear proteins leading to an inhibition of cell division. The cytoplasmic membrane was unaffected by captan, confirming that this fungicide does not destroy cell permeability barriers. After incubation in Fries medium, captan-treated spores showed an almost complete loss of internal fine structure. Similar results were obtained with ‘protoplasts’ of N. crassa which showed disorganization of mitochondrial structure after captan treatment.