Preparation and Regeneration of Mycelial Protoplasts of Alternaria eichhomiae

Preparation 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.     

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.     

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.     

Production and regeneration of protoplasts from Cryptococcus

Protoplasts were quickly and efficiently produced from both varieties of Cryptococcus neoformans and from C. laurentii by use of the multi-enzyme product Novozym 234. Conditions for regeneration of protoplasts are described. DNA yield from the Novozym-produced protoplasts was superior to that from snail gut enzyme-derived protoplasts.     

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.     

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.     

Funcelase — An efficient preparation for the isolation of reversion competent protoplasts from yeasts

Summary The lytic preparation Funcelase was shown to be capable of releasing protoplasts from exponential phase cells ofCandida albicans, Kluyveromyces lactis, Saccharomyces cerevisiae, Saccharomycopsis fibuligera andSchizosaccharomyces pombe. The protoplasts so produced displayed reversion frequencies far superior to those isolated by treatment with Novozym 234 or Suc d'Helix pomatia.     

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%.     

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     

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.     

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.     

Kinetics of mycelial protoplast formation inGibberella fujikuroi andTrichoderma reesei

Mycelial protoplasts ofGibberella fujikuroi andTrichoderma reesei were prepared using Novozym 234 and linear relationship between the yield of the protoplasts and mycelial concentration up to 2 mg dry mass per mL was observed. The optima for concentration of the enzyme, the age of the culture and the contact time of the enzyme with mycelial cells were 2 g/L, 25h and 10 h, respectively, for both cultures. The frequency of reversion of the individual protoplasts was about 80%. The authors are thankful to Dr. A. Kama, Dr. K. Joseph, and Dr. N.G. Karanth for helpful suggestions and critical comments, and to Dr. B.L. Amla for constant encouragement. P.K.R. Kumar is grateful toCSIR, New Delhi, for the award of a research fellow-ship.     

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 isolation from cultured lichen <Emphasis Type="Italic">Usnea ghattensis</Emphasis>, their fusion with protoplasts of <Emphasis Type="Italic">Aspergillus nidulans</Emphasis>, fusant regeneration and production of usnic acid

Protoplasts isolated from the mycobiont of a cultured lichen Usnea ghattensis were fused with protoplasts of the fungus Aspergillus nidulans in order to increase the growth rate of the cultured lichen mycobiont in vitro. The maximum protoplast yield (102 × 10⁴/g fresh cell mass) was reached in citrate buffer with 50 mmol/L 2-sulfanylethanol (‘2-mercaptoethanol’) containing 0.1 % Novozym after 1.5 h at pH 5 and ≤25 °C. The increase in the concentration of the above effectors or the addition of others (e.g., MgSO₄) as well as increase in time, shaking frequency, etc. caused the lower yield of protoplasts. The fused protoplasts were regenerated after transfer to malt extract-yeast extract medium and produced, after a 45-d cultivation, a fresh cell mass of 0.232 g (from starting 0.3 g) along with the lichen substance usnic acid.     

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.     

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.     

Parameters influencing the liposome-mediated insertion of fluorescein diacetate into plant protoplasts

Maximum uptake of liposome-encapsulated fluorescein diacetate by Daucus carota protoplasts was observed when 6 × 10⁶ protoplasts per milliliter were incubated with 2.4 × 10⁷ liposomes per milliliter for 1 hour. In the case of Nicotiana glutinosa protoplasts, optimum ratio of protoplasts to liposomes was 1:10, where 2.3 × 10⁵ protoplasts per milliliter were provided. Neutral and positive liposomes were found to be efficient vehicles to transfer their contents into plant protoplasts. When protoplasts treated with liposomes were cultured in a synthetic medium for 1 week, 20% resumed cell divisions.     

Laboratory-scale biosynthesis ofTrichoderma mycolytic enzymes for protoplast release fromCochliobolus lunatus

Summary Microorganism useful for the induction of enzymes lytic towards walls of filamentous fungusCochliobolus lunatus were studies. Production of specificTrichoderma viride mycolytic enzymes was studied in a laboratory fermentor. The product with high chitinase and relatively low protease activity gave better yields ofC. lunatus protoplasts than commercial Novozym 234.     

Presence of the cyanogenic glucoside dhurrin in isolated vacuoles from sorghum

Large numbers of vacuoles (10⁶-10⁷) have been isolated from Sorghum bicolor protoplasts and analyzed for the cyanogenic glucoside dhurrin. Leaves from light-grown seedlings were incubated for 4 hours in 1.5% cellulysin and 0.5% macerase to yield mesophyll protoplasts which then were recovered by centrifugation, quantitated by a hemocytometer, and assayed for cyanogenic glucosides. Mature vacuoles, released from the protoplasts by osmotic shock, were purified on a discontinuous Ficoll gradient and monitored for intactness by their ability to maintain a slightly acid interior while suspended in an alkaline buffer as indicated by neutral red stain. Cyanide analysis of the protoplasts and the vacuoles obtained there from yielded equivalent values of 11 μmoles of cyanogenic glucoside dhurrin per 10⁷ protoplasts or 10⁷ vacuoles. This work supports an earlier study from this laboratory which demonstrated that the vacuole is the site of accumulation of the cyanogenic glucoside in Sorghum.     

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.