Mycelial protoplast isolation and regeneration of Lentinus lepideus

Generation of fungal protoplast is essential for fusion and transformation systems. Protoplast fusion offers great potential for the improvement of industrially important microorganisms. To establish conditions for the protoplast isolation and regeneration of the mycelia of Lentinus lepideus, various enzymes and osmotic stabilizers were examined. To investigate suitable medium for the culture of L. lepideus, the mycelia were grown in ten different media at 28 degrees C for 10 days. Among them potato dextrose agar (PDA) medium was found to be the best for colony growth. When Novozym 234, cellulase and beta-glucuronidase were added to the mycelia in combination or alone, Novozym 234 alone at the concentration of 10 mg/ml was the most effective for the protoplast yield. Purified spherical protoplasts of the mycelia were osmotically hypersensitive and further incubation of the mycelia with the lytic enzyme resulted in the older parts of the hyphae swollen. When we applied various osmotic stabilizers at the fixed concentration of 0.6 M on the protoplasts, the yields of protoplasts were increased until 4-hr incubation. However application of sucrose or MgSO4 led to further protection of protoplasts after that time and reached a plateau on 5- and 7-hr incubations, respectively. The suitable incubation time and optimal pH with the lytic enzyme for the maximum release of protoplasts were 6 hrs of incubation and pH 5, respectively. When we examined various osmotic stabilizers for the regeneration of the protoplast, the complete medium containing 0.6 M sucrose induced highest hyphal growth with regeneration frequency of 3.28%.     

Optimum conditions for the release and regeneration of protoplasts of Alternaria alternata

Best release of Alternaria alternata protoplasts was obtained when 20 h old mycelia were incubated in a hydrolytic enzyme mixture of Novozym 234, Driselase, and β -glucuronidase. Numbers of nuclei/protoplast varied but generally decreased with increased time of incubation. While salts, were better osmotic stabilizers for protoplast release, sucrose and sugar alcohols were better for regeneration.     

Regeneration and molecular characterization of an intergeneric hybrid between Graphium putredinis and Trichoderma harzianum by protoplasmic fusion

The fungal strains Graphium putredinis and Trichoderma harzianum were selected as parents for fusant development. Protoplasts were isolated using the combination of lysing enzymes Novozym 234 and cellulase with 0.6 M KCl as osmotic stabilizer. The optimum conditions for release of viable protoplasts from the fungal mycelium viz. age of the mycelium, lytic enzymes, osmotic stabilizers, pH, incubation period and regeneration medium were determined. Intergeneric protoplast fusion was carried out using 50% polyethylene glycol with calcium chloride (CaCl₂) and glycine buffer and the conditions for effective protoplast fusion, viz. fusogen, osmotic stabilizer, pH, incubation period and regeneration medium were optimized. At optimum conditions, the regeneration frequency of the fused protoplasts on potato dextrose agar (PDA) medium and fusion frequency were calculated. The regeneration frequency on non-selective (PDA) and selective media (PDA amended with starch) was determined for the parental and fusant strains in which, fusant showed a higher rate of regeneration. Fusant formation was confirmed by morphological markers (colony morphology and spore size and shape) and genetical markers like, mycelial protein pattern, restriction digestion pattern and random amplified polymorphic DNA (RAPD) analysis. The efficiency of these parental strains and their intergeneric fusant in the production of hydrolytic enzymes – amylases (treatment plant for sago factory effluent), cellulases (bioethanol), xylanases (bleaching agents for waste paper pulp) and proteases (additives in commercial detergents) – have probable applications in various industrial processes.     

Optimization of formation and regeneration of protoplasts from biocontrol agents ofTrichoderma species

The optimal conditions necessary for a large yield and a high frequency of regeneration of protoplasts isolated from the biocontrol agentsTrichoderma koningii andT. harzianum were investigated. Protoplast yields were 1.2×10⁸/ml fromT. koningii and 6×10⁷/ml fromT. harzianum when 20-h mycelial culture was treated with a lytic enzyme solution containing Novozym 234 (15 mg/ml), sucrose (0.6 M) and citrate phosphate buffer (0.02 M), pH 5.6 at 31°C. When the protoplasts were grown in the regeneration medium containing yeast extract (1.5%), 1 I of Mandel's salts, pH 5.6, and glucose (0.8 M), a high frequency of regeneration of the protoplast was obseved: 66% forT. koningii and 45% forT. harzianum. Two patterns of regeneration were observed. First, the hyphae arose directly from the regenerated protoplast mother cell. Second, a chain of bud cells developed from the protoplast and subsequently generating hyphae generally protruded from the terminal bud cells.     

Formation and regeneration of protoplasts of Sclerotium glucanicum

Summary Protoplast yields from Sclerotium glucanicum using Novozym 234 as the lytic enzyme were affected by the osmotic stabilizers selected, the incubation conditions used for wall degradation, and culture age. Scanning electron microscopic observations revealed that protoplast release from all hyphal regions gradually followed random wall attack, and nuclear staining showed that some protoplasts contained as many as eight nuclei. Their regeneration involved germ tube production on solid media, but formation of chains of buds and possibly cytoplasmic cleavage in liquid medium. Regenerated protoplasts gave similar exopolysaccharide yields to those of the parent culture.     

金龟子绿僵菌原生质体的制备和再生及其羟化酶活性研究

对金龟子绿僵菌(Metarhizium anisopliae)原生质体的制备和再生的影响因素进行实验,并在此基础上考察了甾体底物对原生质体羟化酶的诱导作用。结果表明,原生质体制备的合适条件是:42 h的菌丝体用纤维素酶(10 mg/mL)和蜗牛酶(5 mg/mL)的混合酶在含有0.8 mol/L甘露醇的pH 5.8磷酸缓冲液中,28℃震荡(80 r/min)酶解3 h,原生质体产量可达到6.12×10~7/mL,在含有0.6 mol/L KCl的双层马铃薯培养基上再生率达到7.79%。经过6 h底物诱导的菌丝体制备的原生质体细胞色素P450的表达量比没经过诱导的菌丝体制备的原生质体高约40%,证明该菌羟化酶系统的可诱导性。由于没有细胞壁的阻碍经过底物诱导的原生质体能够高效的将底物转化为产物,且副产物相对较少。     

The isolation and regenaration of protoblast from Lipomyces starkeyi: an oleaginous yeast

The isolation and regenration of prostoplasts from Lipomyces starkeyi have been optimised. Snail enzyme (12 mg·ml⁻¹) proved to be the most effective lytic enzyme although treatment with Novozym 234, Cellulase CP and β-glucanase also resulted in protoplast formation. Magnesium sulphate (0.55 M) was shown to be the best fro protoplast isolation. Exponential phase cells were most susceptible to the lytic enzyme, stationary phase cells appeared to be resistant. 2-Mercaptoethanol or dithiothreitol did not enahance the isolation of protoplasts in this yeast. The optimum pH for protoplast isolation was 5.8. Ultrastructural observations were made on cells during lytic digestion and revealed that the cell wall and capsule are stripped away from the protoplast.Protoplast synthesised new cell wall material when cultured on osmotically stabilised medium, regeneration was not oberved in liquid medium. Optimum regeneration occured when protoplasts were embedded in a thin layer of minimal medium osmotically stabilised with mannitol (0.6M) and solidified with 1.5–2.0% agar. A basal layer of medium was also stabilised with mannitol (0.6 M) but contained 3% agar. The lytic enzyme used for protoplast isolation did not appear to effect the regeneration of protoplasts.     

Protoplasts fromPodospora anserina: Isolation,purification, and transformation

Protoplasts fromPodospora anserina mycelium were produced using the commercially available enzyme Novozym 234. Different parameters involved in protoplast isolation were analyzed in order to establish optimal conditions, and protoplast production was notably increased. For the purification of protoplasts, several techniques based on both centrifugation and filtration were assayed, with filtration yielding the best results. Regeneration of protoplasts was studied on different media and osmostic stabilizers, and about 80% regeneration was obtained. The good physiological condition of the protoplasts produced with this method is demonstrated by the lack of cell wall and high regeneration rate and transformation frequencies.     

Protoplast formation and regeneration in <Emphasis Type="Italic">Lactobacillus delbrueckii</Emphasis>

Method for production and regeneration of Lactobacillus delbrueckii protoplasts are described. The protoplasts were obtained by treatment with a mixture of lysozyme and mutanolysin in protoplast buffer at pH 6.5 with different osmotic stabilizers. The protoplasts were regenerated on deMan, Rogosa and Sharpe (MRS) with various osmotic stabilizers. Maximum protoplast formation was obtained in protoplast buffer with sucrose as an osmotic stabilizer using a combination of lysozyme (1 mg/ml) and mutanolysin (10 μg/ml). Maximum protoplast regeneration was obtained on MRS medium with sucrose (0.5 M) as an osmotic stabilizer. The regeneration medium was also applicable to other species of lactobacilli as well. This is, to our knowledge, the first report on protoplast formation and efficient regeneration in case of L. delbrueckii.     

Conditions for Efficient Isolation and Regeneration of Protoplasts from Fulvia fulva

A procedure for producing high yields and high regeneration frequencies of protoplasts from Fulvia fulva is described. This procedure was devised by systematic trials of various parameters, such as culture age and osmotic support, which are known to affect protoplast yield and regeneration. Mycelium from liquid cultures of 24–48 h, incubated with Novozym 234 in buffered 1.0 M MgSO₄, gave the best conditions for protoplast release. Regeneration frequencies up to 50% were obtained with a complete medium containing 0.8 M sucrose for osmotic support.     

Mutagenesis of Protoplasts and Regeneration of Mycelium in the Mushroom Volvariella volvacea

Regenerating protoplasts were obtained from mycelial culture of the mushroom Volvariella volvacea by the action of the lytic enzyme Novozym 234 in the presence of 0.01 M phosphate buffer (pH 6.0) containing 0.6 M NaCl. Regeneration was found to be poor in liquid medium, but more than 50% regeneration was achieved on solid 2% agar medium overlaid with 0.5% agar. Protoplasts of V. volvacea were found to be highly sensitive to the killing action of both UV irradiation and N-methyl-N′-nitro-N-nitrosoguanidine. However, no morphological or auxotrophic mutants could be obtained from protoplasts by chemical mutagenesis. Four types of morphological mutants and one auxotrophic (adenine-negative) mutant were obtained from UV-irradiated protoplasts. The adenine-negative mutant of V. volvacea was found to be stable, not losing auxotrophy on repeated subculture.     

Factors affecting protoplast formation and regeneration by four species of Streptomyces

Four species of Streptomyces, Streptomyces canescens, S. limosus, S. griseus and S. griseolus , were used to study the effects of glycerine and gelatin on the formation and regeneration of protoplasts. For each species efficient protoplast formation with high protoplast concentrations and low levels of non-protoplast units was obtained with mycelia grown in medium without glycerine. The low regeneration frequencies of protoplasts of S. canescens and S. limosus on R2 medium were increased substantially by the addition of 1% gelatin. The use of single colonies, rather than spores, to establish mycelial cultures was found routinely to produce good protoplast preparations.     

Production and regeneration of protoplasts from the mycorrhizal fungus Suillus granulatus

Experiments were performed with the mycorrhizal fungus Suillus granulatus to define the parameters for production and regeneration of protoplasts. Protoplasts were released at frequencies between 1 and 3×10⁷/ml from mycelium 3 to 7 days old. The best osmotic stabilizer for protoplast release was MgSO₄ (0.7 m). To optimize protoplast release and regeneration an enzyme (Novozym 234) concentration 1.7 mg/ml was chosen, with a digestion time of 1 to 2 h. Regenerated colonies formed mycorrhizae within 60 days after inoculation in Pinus caribaea var. hondurensis seedlings.     

Ultrastructure of protoplasts from mycelium and microconidia of Trichophyton mentagrophytes

Protoplast formation from mycelium and microconidia of Trichophyton mentagrophytes was achieved with Novozym 234. Pretreatment procedures with dithiothreitol or urea mercaptoethanol sodium lauryl sulphate before digestion with Novozym 234 greatly reduced protoplast yield from mycelium. Snail gut enzyme did not protoplasts in good yield. Scanning electron microscopy of mycelium protoplasts showed the acquired spherical shape. The plasma membrane appeared finely granular although remnants of cell wall could sometimes be observed. Transmission electron microscopy showed the cell interior of these protoplasts was plasmolysed. Microconidia treated with Novozym 234 displayed a range of cell wall digestion, with intact protoplasts showing distinct cytoplasmic organelles.     

Improved conditions for protoplast formation and transformation of Pleurotus ostreatus

Conditions suitable for the production and regeneration of Pleurotus ostreatus protoplasts from dikaryotic mycelia were examined. Three commercially available muralytic enzymes, including Sigma lysing enzyme, Novozym 234 and Novozym 234 LP, were used for production of protoplasts. Over 2 × 10⁷ protoplasts per gram fresh weight mycelia were obtained within 1.5 h by using each of these three enzymes. The colony regeneration rate was up to 13% on potato-dextrose-agar medium containing 0.8 m mannitol. Genetic transformation was based on positive selection for resistance to hygromycin B (HmB) using the plasmid vector pAN7-1 and accomplished by either electroporation or a polyethylene glycol (PEG)-divalent cation method. P. ostreatus strains used in this study have innate sensitivity to HmB at a critical inhibitory concentration of between 40–50 g/ml. Selection for HmB resistance of this fungus, indicative of transformation, resulted in 3–48 HmB-resistant colonies per microgram of pAN7-1 per 10⁷ viable protoplasts. No significant differences were apparent when either transformation protocol or either P. ostreatus strain was used. The best electrical condition found for the electrotransformation of P. ostreatus is at a field strength of 2.6–2.8 kV/cm with a capacitance of 25F and a parallel resistance of 800 ohms, corresponding to a time constant range of 10–14 ms. Correspondence to: P. A. Lemke     

Lipase production by free and immobilized protoplasts of Sporotrichum (Chrysosporium) thermophile Apinis

Summary Production of lipase by free and alginate-entrapped protoplasts was studied in batch culture. Cell-wall-degrading enzymes Novozym 234 and cellulase CP improved lipase secretion of normal mycelium by 25%–100%. The protoplast-regenerated mycelium exhibited several-fold higher lipase activity in batch replacements in TRIS buffer over normal spore-derived mycelium. The specific lipase activity of immobilized protoplasts was about four times higher than normal mycelial beads. Protoplasts beads were stable and retained high enzyme activity even after three buffer replacements lasting 120 h; TRIS buffer was better than acetate or normal glucose medium. A minimum of 8 h regeneration period was necessary for lipase synthesis. Triolein, olive oil, tributyrin and oleic acid butylester were able to induce lipase in immobilized protoplasts. Tween 80 enhanced lipase activity of the immobilized protoplasts. Partially degraded immobilized mycelium was nearly as effective as normal immobilized protoplasts for lipase secretion. Both free and immobilized protoplasts could be reused for up to 200 h with some loss in enzyme activity.     

Release and regeneration of protoplasts from the fungus Trichothecium roseum

A protocol for isolating and regenerating protoplasts from Trichothecium roseum has been described. Protoplasts from T. roseum were isolated using (i) a lytic enzyme combination composed of Novozym 234, chitinase, cellulase, and pectinase at a 5-mg/mL concentration and (ii) 0.6 M KCl as an osmotic stabilizer. A maximum number of 28 x 10(4) protoplasts/mL were obtained at pH 5.5. Experiments on the regeneration and reversion of protoplasts revealed a maximum regeneration (60.8%) in complete medium (potato dextrose--yeast extract agar) amended with 0.6 M KCl. The regenerated protoplasts were similar to the original parent strain in morphology, pigmentation, growth, and sporulation.     

灭蚊真菌——大链壶菌原生质体形成和再生的研究

采用1％纤维素酶与1％真菌脱壁酶混合液作脱壁酶,0.6mol/L山梨醇为渗透压稳定剂,从摇床培养的12—14小时菌龄的大链壶菌(Lagenidium giganteum)菌丝体获得原生质体。酶解3—5小时后,产量可达1.4—2.0×10~6/mL。并在双层培养基上初步实现了原生质体再生。     

A method for isolation of protoplasts from dermatophytes

A method has been developed to isolate protoplasts from dermatophytes using Novozym 234. A simple technique of flotation in MgSO4 has been adapted to separate protoplasts from incubation mixture. Electron microscopic studies confirmed the absence of cell wall material on these protoplasts. The recovery of DNA from protoplasts was higher than from mycelia.     

银耳原生质体分离与再生条件优化研究*

应用正交设计,研究不同菌株(Tr01、Tr21)、材料(芽孢、菌丝体、子实体)、溶壁酶浓度和酶解温度对原生质体产量的影响。实验结果表明,实验材料对原生质体产量影响最大,以芽孢为材料原生质体产量可达到2.75×107个/ml,而菌丝体和子实体的原生质体产量仅为2.5×106个/ml 和1.0×106个/ml;在35℃下酶解,原生质体产量高;溶壁酶浓度在1%~3%范围内对原生质体产量影响不大;不同菌株原生质体产量差异不显著。本实验还研究了稳渗剂浓度对原生质体再生率的影响,结果表明,0.5 mol/L~0.7 mol/L的KCl 对原生质体再生没有显著差异,再生率最高可达32.3%。