Protoplast Formation Research Articles

Regeneration of Pathogenic Aphanomyces cochlioides and A. eutetches from Protoplasts

Enzyme mixtures were tested for the digestion of cultured Aphanomyces cochlioides and A. euteiches mycelia to promote the formation of protoplasts. Cell wall-digesting enzymes at 0.1% (w/v) in osmoticum were sufficient to convert the mycelia to protoplasts within 2 hr, similar to digestion conditions for other oomycetes. Protoplast integrity was maintained upon embedding in molten agar containing 1M mannitol. Within 4 days post-plating on potato dextrose agar, 10 to 20% of the embedded protoplasts of both fungal species formed germ tubes that subsequently formed mycelial colonies. Fungal isolates derived from regenerated protoplasts of A. cochlioides and A. euteiches retained the ability to induce black root disease in sugarbeet seedlings and water soaking in pea seedlings, respectively. The generalized protocol for production and regeneration of protoplasts for Aphanomyces species may be of use in the development of a gene transfer protocol for this important crop pathogen.

Formation of Protoplasts from Strain Streptomyces Albogriseolus 444

ABSTRACTCultural conditions for protoplast preparation of strain Streptomyces albogriseolus 444, producing antibiotic complex with wide antimicrobial spectrum were studied. Effective formation and stabilization of the protoplasts was accomplished by 24 h cultivation of the strain 444 in a medium, containing 0.5% glycine and 1% sucrose. After that the mycelia were treated with 4 mg per ml lysozyme. Under these optimal conditions it amounted up to 5.13x107 protoplasts per ml.

Characterization and applications of chitinases from Trichoderma harzianum – A review

Chitinases help degradation of cell walls containing chitin and thus accelerates protoplast formation. It indirectly helps strain improvement and development of new strains which are economically viable for industrial use. Chitinases consist of endochitinase, exochitinase, β-N-acetylglucosaminidases and chitobiases. The endochitinases can be further characterized for better understanding of mechanism of the enzyme reaction. This review covers the recent advances in isolation and characterization of endochitinases and its components, gene encoding sequences and cloning.

Screening of heterozygous DNA markers in shiitake (Lentinula edodes) using de-dikaryotization via preparation of protoplasts and isolation of four meiotic monokaryons from one basidium

A suitable screening method for heterozygous DNA markers in shiitake,Lentinula edodes (Berk.) Pegler, is reported. Monokaryons were derived from a dikaryon by de-dikaryotization via protoplast formation. Compatibility of the monokaryons was determined by pairwise culture on agar plates. We selected the primers to amplify polymorphic fragments among the original strain (Hokken600∶H600) and two monokaryons (H600PP-39 and H600PP-67) showing compatibility. A total of 135 fragments were selected as specific random amplified polymorphic DNAs (RAPDs) resulting from 56 primers of the 147 primers tested. Furthermore, we tested whether the polymorphic fragments segregated into 2∶2 among four strains isolated from a basidium. Most of the polymorphic fragments (about 97.8%) showed 2∶2 segregation among the four strains. We concluded that the polymorphic fragments were heterozygous if they were detected in either of the monokaryons (H600PP-39 and H600PP-67) and segregated to 2∶2 among four meiotic strains (H600B-1,-2, -3, and -4). A total of 132 heterozygous DNA markers were therefore selected from a dikaryon of shiitake (Hokken600∶H600).

Effect of Osmotic Stabilizers on Protoplast Generation of Chlorella ellipsoidea Yellow/White Color Mutants.

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 μE/m2/s and no reversion to normal cells at 0–26.8 μE/m2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.

Effect of osmotic stabilizers on protoplast generation of Chlorella ellipsoidea yellow/white color mutants

The effect of osmotic stabilizers on protoplast formation of yellow/white color mutants of Chlorella ellipsoidea was studied. The UF-1 strain, one of the mutants, had the same growth rate as the parent C. ellipsoidea at 4.02 μE/m 2/s and no reversion to normal cells at 0–26.8 μE/m 2/s. Therefore, protoplast formation from UF-1 was carried out, and it was found that the yield of protoplasts was 93% in the medium containing 1.0 M NaCl as an osmotic stabilizer.

Endogenous elicitor-like effects of alginate on physiological activities of plant cells

The effects of alginate on the physiological activities of plant cells were studied. Addition of alginate oligomer (AO) to the suspension culture of Catharanthus roseus L. or Wasabia japonica cells promoted the production of antibiotic enzymes such as 5′-phosphodiesterase or chitinase respectively. Ajmalicine (a secondary metabolite) production by C. roseus CP3 cells was also promoted when AO was added to the suspension culture. On the basis of these results, we assumed that alginate is an elicitor-like substance. We therefore compared the effect of AO on C. roseus L. and W. japonica cells with those of chitosan oligomer (CO) and oligo-galacturonic acid (OGA), which are well known as an exogenous elicitor and endogenous elicitor respectively. The effects of various concentrations of AO, OGA, and CO on the physiological activities, membrane permeability and protoplast formation of C. roseus L. or W. japonica cells were investigated. AO and OGA showed similar physiological effects, which were quite different from those of CO. Since alginate appeared to have similar effects to galacturonic acid, we concluded that alginate acts as an endogenous elicitor. Both alginate and galacturonic acid are uronic acids, and we considered their structural similarity. The effects of esterification of the carboxylic groups of alginate by propylene oxide were also studied. The greater the degree of esterification, the less the secretion of 5′-phosphodiesterase. Hence we assumed that carboxylic groups have an important role in the initiation of the elicitation reaction in plant cells, as shown in the case of galacturonic acid.

Influence of manganese on cell morphology, protoplasts formation and β- d-glucosidase activity in Phaffia rhodozyma

Among several potential cell wall lytic enzymes, such as β- d-glucosidase, β- d-galactosidase, β- N-acetyl- d-glucosaminidase, β- N-acetyl- d-galactosaminidase, and β- d- N, N′-diacetylchitinase, Phaffia rhodozyma showed only β- d-glucosidase activity. MnCl 2 increased the activity, but not the production of the enzyme. When the yeast was grown with 4.0 mM MnCl 2, the cells were bigger and rounder than those obtained in its absence, suggesting a weakening of the cell wall. Protoplasts were obtained from these cells, using 0.3 M KCl as the osmotic stabiliser.

Influence of manganese on cell morphology, protoplasts formation and β-d-glucosidase activity inPhaffia rhodozyma

Among several potential cell wall lytic enzymes, such as β-d-glucosidase, β-d-galactosidase, β-N-acetyl-d-glucosaminidase, β-N-acetyl-d-galactosaminidase, and β-d-N,N′-diacetylchitinase, Phaffia rhodozyma showed only β-d-glucosidase activity. MnCl2 increased the activity, but not the production of the enzyme. When the yeast was grown with 4.0 mM MnCl2, the cells were bigger and rounder than those obtained in its absence, suggesting a weakening of the cell wall. Protoplasts were obtained from these cells, using 0.3 M KCl as the osmotic stabiliser.

Protoplast formation in yeasts by the lytic enzyme of Streptomyces rochei DB-34 and their regeneration : Kazuhiko Ohbuchi,* Akiko Nishi, Masaaki Hamachi, and Chieko Kumagai ( General Research Laboratory, Ozeki Corp., Nishinomiya 663-8227) Seibutsu-kogaku 77: 137–146. 1999

Protoplast formation in yeasts by the lytic enzyme of Streptomyces rochei DB-34 and their regeneration : Kazuhiko Ohbuchi,* Akiko Nishi, Masaaki Hamachi, and Chieko Kumagai ( General Research Laboratory, Ozeki Corp., Nishinomiya 663-8227) Seibutsu-kogaku 77: 137–146. 1999

Patch Clamp Studies on Ion Pumps of the Cytoplasmic Membrane ofEscherichia coli: FORMATION, PREPARATION, AND UTILIZATION OF GIANT VACUOLE-LIKE STRUCTURES CONSISTING OF EVERTED CYTOPLASMIC MEMBRANE

Formation of giant protoplasts from normal Escherichia coli cells resulted in the formation of giant vacuole-type structures (which we designate as provacuoles) in the protoplasts. Electron microscopic observation revealed that these provacuoles were surrounded by a single membrane. We detected inner (cytoplasmic) membrane proteins in the provacuolar membrane but not outer membrane proteins. Biochemical analyses revealed that the provacuoles consist of everted cytoplasmic membranes. We applied the patch clamp method to the giant provacuoles. We have succeeded in measuring current that represents inward movement of H+ because of respiration and to ATP hydrolysis by the FoF1-ATPase. Such current was inhibited by inhibitors of the respiratory chain or FoF1-ATPase. This method is applicable for analyses of ion channels, ion pumps, or ion transporters in E. coli or other microorganisms.

Varietal Variations in Plant Regenerative Potential from Protoplasts in Spinach (Spinacia oleracea L.).

The regenerative potential of seven Spinacia oleracea L. varieties in protoplast culture was compared. Although protoplasts were isolated from all cultivars, the yield of protoplast, plating efficiency (PE), and colony formation ratio were remarkably different among them. The highest PE and colony formation ratio were obtained from protoplasts of 'Ujo', followed by 'Jiromaru', 'Hoyo', and 'Minsterland', which exceeded those of 'Sapporo-oba', 'Viroflay', and 'Viking'. Shoots were regenerated by 'Ujo', 'Jiromaru', 'Hoyo', and 'Minsterland'. 'Hoyo' and 'Jiromaru' had the highest frequency ; no shoot developed from the calli of 'Sapporo-oba', 'Viroflay', and 'Viking'. The highest frequency of shoot regeneration was obtained in a media containing 1.0 or 10.0mg·liter-1 IBA, but at 10.0mg·liter-1 IBA or IAA, the shootlets were often vitrified and fasciated.

Phytosulfokine-alpha, a sulfated pentapeptide, stimulates the proliferation of rice cells by means of specific high- and low-affinity binding sites.

Peptide growth factors were isolated from conditioned medium derived from rice (Oryza sativa L.) suspension cultures and identified to be a sulfated pentapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-Gln-OH] and its C-terminal-truncated tetrapeptide [H-Tyr(SO3H)-Ile-Tyr(SO3H)-Thr-OH]. These structures were identical to the phytosulfokines originally found in asparagus (Asparagus officinalis L.) mesophyll cultures. The pentapeptide [phytosulfokine-alpha (PSK-alpha)] very strongly stimulated colony formation of rice protoplasts at concentrations above 10(-8) M, indicating a similar mode of action in rice of phytosulfokines. Binding assays using 35S-labeled PSK-alpha demonstrated the existence of both high- and low-affinity specific saturable binding sites on the surface of rice cells in suspension. Analysis of [35S]PSK-alpha binding in differential centrifugation fractions suggested association of the binding with a plasma membrane-enriched fraction. The apparent Kd values for [35S]PSK-alpha binding were found to be 1 x 10(-9) M for the high-affinity type and 1 x 10(-7) M for the low-affinity type, with maximal numbers of binding sites of 1 x 10(4) sites per cell and 1 x 10(5) sites per cell, respectively. Competition studies with [35S]PSK-alpha and several synthetic PSK-alpha analogs demonstrated that only peptides that possesses mitogenic activity can effectively displace the radioligand. These results suggest that a signal transduction pathway mediated by peptide factors is involved in plant cell proliferation.

Electrorotational spectra of protoplasts generated from the giant marine algaValonia utricularis

Protoplasts ofValonia utricularis lacking the large central vacuole can be generated by cutting multi-nucleated, giant “mother” cells into small pieces after short exposure to air. When the protoplasmic content was squeezed out into sea water, irregularly shaped, green coloured aggregates were formed which changed into spherical protoplasts (radius of 20–60 μm) after about 2 h. In these protoplasts the dense internal material (consisting mainly of organelles) was separated from the plasmalemma by a thin transparent layer containing a large number of small lipid vesicles. Cell wall regeneration occurred rapidly after protoplast formation. A central vacuole developed after about 10h. The regenerated cells continued to grow and were viable for several months. Electrorotation studies on 2–3 h old protoplasts at pH 7 in low- and fairly high-conductivity solutions showed one or two anti-field rotation peaks (depending on medium conductivity) between 10 kHz to 1 MHz as well as one cofield rotation peak between 10 MHz to 100 MHz. The rotation spectra could not be fitted on the basis of the single- (or multi-) shell model (i.e., by modelling the cells as a homogeneous sphere surrounded by one or more layers). However, fairly good agreement between the experimental data and theory could be obtained by assuming that the rotational behaviour of the protoplasts depends not only on passive electrical properties of the plasmalemma but is influenced by “mobile charges” of carrier transport systems and/or the dielectric behaviour of the aggregated chloroplasts and vesicles.

Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

AbstractPreparation and regeneration of mycelial protoplasts from Alternaria eichhorniae were examined. A commercially available muralytic enzyme, Novozym 234, was used for isolation of protoplasts. The mycelial age and the pH of the stabilized buffer affected the formation of protoplasts. The maximum production of protoplasts (3,9 × 108/g fresh weight mycelia) was obtained from 24‐h‐old mycelia digested with Novozym 234 (20 mg/ml) in a stabilized buffer of pH 6.4 and incubated in the dark at 30°C on a rotary shaker (90 r.p.m.) for 6 h. Morphological characteristics of the protoplasts varied and depended on the age of the mycelia used in protoplast production. Moreover, mycelial age had a highly significant influence (P = 0.0001) on the frequency of protoplast regeneration.

Preparation of mitochondrial DNA from fungal protoplasts

A rapid and simple method based on protoplast formation is presented to prepare mitochondrial DNA (mtDNA) from filamentous fungi. This protocol can be applied to prepare mtDNA from numerous strains in relatively short time.

Liquid culture enhances protoplast formation from the auxotroph (ser−) ofLentinula edodes

The optimal conditions for the production and regeneration of the protoplasts fromLentinula edodes were studied. Protoplast formation from the mycelia ofL. edodes which were cultured in liquid medium showed a significantly high yield compared with that of the mycelia which were cultured on cellophane covered agar media. A mixture of Novozyme 234 (15 mg/ml) and Cellulase Onozuka R10 (10 mg/ml) in 0.6 M mannitol (pH 4) was optimal lytic enzyme for the protoplast release. The optimal incubation time and mycelia age were 3.5-4 hours at 30 degrees C and 6-8 days, respectively. Regeneration frequency was 0.18% plated onto a medium containing 0.6 M sucrose, and 0.08% plated onto a medium containing mannitol. But hardly any regeneration was observed in the media containing NaCl, KCl, or MgSO(4). More than 90% of the protoplasts contianed nuclei and the nucleus number per protoplast was 1.1. The DNA content per nucleus was 5.1 pg. The diameter of the protoplast was 3-5 mum and it had a well defined cell structure.

4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by N-ethylmaleimide-reductase of Yarrowia lipolytica.

We studied the steric course of the reaction catalyzed by the N-ethylmaleimide (NEM) reductase of Yarrowia (Candida) lipolytica (Y. lipolytica), using 4R-[4-2H1]NADPH and 4S-[4-2H1]NADPH as cofactors and N-ethylcitraconimide as substrate. Active substrates and inhibitors of NEM reductase and its subcellular distribution were also investigated to clarify the biochemical properties of this enzyme. NEM reductase catalyzes the reduction of N-ethylmaleimide to N-ethylsuccinimide with NAD(P)H as the cofactor. Several maleimide and cyclopentenone derivatives tested were also active substrates for NEM reductase of Y. lipolytica. Some pyrazolone derivatives, particularly 1-phenyl-5-pyrazolone, were found to be effective inhibitors of NEM reductase. Subcellular localization of NEM reductase was carried out using protoplast formation and differential centrifugation. Ninety-eight percent of the NEM reductase activity was recovered in the cytosolic fraction, indicating that NEM reductase in Y. lipolytica was the cytosolic enzyme. We also determined the stereochemical specificity of the reduction of N-ethylcitraconimide by NEM reductase in Y. lipolytica, showing that 4 Pro-R hydrogen of NADPH was abstracted for enzymatic hydride transfer by NEM reductase, and two hydrogen atoms from NADPH and H2O added to opposite faces of the double bond of N-ethylcitraconimide.

Effects of some factors on protoplast formation of a microalga, Prototheca zopfii

The effects of experimental factors on protoplast formation of Prototheca zopfii Kru¨ger in 0.85 m NaCl using Macerozyme R-200 were studied based on a fractional factorial experimental design. The rate of protoplast formation was mainly affected by the incubation temperature and the age of algal cells. The optimal condition for the maximum protoplast yield was determined based on a response surface model. These were: mid-logarithmic phase cells and Macerozyme concentration of 4% at a temperature of 35°C.

Protoplast Formation from Schizophyllum commune by a Culture Filtrate of Bacillus circulans KA-304 Grown on a Cell-wall Preparation of S. commune as a Carbon Source.

From microorganisms growing on a cell-wall preparation (CWP) of Schizophyllum commune as a carbon source, Bacillus circulans KA-304 was isolated on the bases of activities in culture filtrate to decrease turbidity of the CWP-suspension and to form protoplasts from S. commune. The culture filtrate was also active in hydrolyzing p-nitrophenyl (p-NP)-α-d-glucoside, p-NP-β-d-glucoside, and p-ΝΡ-β-d-Ν-acetylglucosaminide. The protoplast-forming and the p-NP-glycoside-hydrolyzing activities were increased by the addition of CWP of S. commune to the culture medium, and this was not observed for other bacteria tested (15 genera, 80 species). B. circulans KA-304 was shown on gel filtration to produce at least two enzyme species for hydrolyzing both p-NP-α-d-glucoside and p-NP-α-d-glucoside. The protoplast-forming activity was retained for at least 6 months at 5°C as an ammonium sulfate (90% saturation) precipitate or at -20°C as a freeze-dried preparation (KA-preparation). The activity was stable at pH 6.5-7.0, and remained after 10 min of treatment at 40°C. Protoplast formation proceeded optimally at pH 6.5 with 50 mm potassium phosphate buffer and 0.5 m mannitol as an osmotic stabilizer. B. circulans KA-304 seems to be a suitable strain producing enzyme(s) to prepare protoplasts from S. commune.