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.     

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.     

Regeneration of cells from protoplasts ofClostridium acetobutylicum B643

Summary Conditions that allow regeneration of cells fromClostridium acetobutylicum strain B643 protoplasts were studied. Protoplast formation and stabilization in minimal media with 50 mM CaCl₂, 50 mM MgCl₂ and 0.3 M sucrose were crucial to subsequent regeneration on soft yeast extract agar containing 25 mM CaCl₂ and 25 mM MgCl₂. A regeneration frequency of 8–25% was consistently obtained.     

利用荧光染色和流式细胞技术辅助卵孢小奥德蘑原生质体制备与再生研究

本研究以卵孢小奥德蘑液体培养菌丝作为实验材料,利用单因子变量法探索研究了菌丝培养时间、酶浓度、酶解时间、酶解温度、稳渗剂类型对卵孢小奥德蘑原生质体制备的影响,并对原生质体再生培养基进行选择和优化。通过荧光染色,利用激光共聚焦显微镜和流式细胞仪对原生质体的制备过程、得率和活力进行研究。结果表明,将卵孢小奥德蘑菌丝在液体培养基中培养5d收集菌丝体,以甘露醇作为渗透压稳定剂,在溶壁酶浓度2%、30℃条件下酶解5h,获得的原生质体得率最高,达2.0×10 ⁷个/mL;通过流式细胞仪分析,约57.69%的原生质体细胞为活细胞;在RM培养基中再生效果最好,再生率为(0.103±0.025)%。研究结果可以为卵孢小奥德蘑育种与食用菌原生质体制备再生提供研究基础。     

Protoplasts formation inFusarium species

Formation of protoplasts from four species ofFusarium genus is described. Protoplasts were isolated from mycelium by enzymatic digestion of the cell wall in the presence of an osmotic stabilizer. The results obtained differed between the studied species. Best yields of protoplasts were obtained fromF. moniliforme (90 % cells as protoplasts).     

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.     

Preparation of protoplasts from <Emphasis Type="Italic">Chlorella protothecoides</Emphasis>

This paper describes an enzymatic method for yielding protoplasts from the microalga Chlorella protothecoides. Four kinds of commercially available enzymes were tested. The enzymatic digestion was optimal with 2% cellulase R-10 and 1% snailase prepared in 25 mM Tris buffer (pH 6.0) containing 0.6 M D-mannitol, and the protoplast density could reach the peak after treatment at 30°C for 16 h. Nearly all liberated protoplasts were green in the presence of 0.01% phenosafranin, indicating their high viability. The regeneration rate was about 70% when 0.6 M D-mannitol was used as an osmotic stabilizer in the regeneration medium. This protocol will find useful applications in genetic studies of this algal species.     

Protoplasts from Aspergillus nidulans.

A very effective lytic enzyme system for massive micro/macro-scale production of protoplasts from the filamentous fungus Aspergillus nidulans is described. A striking coincidence was observed between maximal lytic activity towards Aspergillus mycelium and the presece of both chitinase and alpha-(1 leads to 3)-glucanase activities. The release of protoplasts was greatly enhanced by preincubating the mycelium with 2-deoxy-D-glucose. Furthermore, protoplast formation was influenced by fungal age, culture conditions, pH of incubation and the osmotic stabilizer used. From 40 mg of fresh mycelium, grown for 14--16 h on 1% glucose in a low phosphate-citrate medium, preincubated with 2-deoxy-D-glucose for 45 min, and then incubated with the lytic enzyme mixture at pH 6.5 in the presence of 0.3--0.4 M (NH4) SO4, 2.5 x 10(8) stable protoplasts were produced within 3 h of incubation at 30 degrees C. Comparable results were obtained with 40--50 g of mycelium. At low osmotic stabilizer concentrations a peculiar type of regeneration was observed in the presence of the lytic enzyme system; within 12 h of incubation aberrant hyphal structure emerged from the large vacuolated protoplasts.     

Efficient plant regeneration from hairy root-derived protoplasts of Hyoscyamus muticus

Summary Successful plant regeneration was achieved for the first time from hairy root-derived protoplasts of Hyoscyamus muticus. High yields (7 × 10⁶ / g fresh weight) of protoplasts were isolated directly from the transformed roots of Hyoscyamus muticus using an enzyme mixture comprising 1 % macerozyme and 2 % cellulase in an osmoticum consisting of 0.2 M CaCl₂ and 0.6 M mannitol. Protoplasts were first cultured in liquid NT/PRO I medium and further on semi-solid NT/PRO II agar medium. The procedure permits highly efficient formation of colonies. The plating efficiency varied from 1–9 %. The small individual colonies regenerated easily into shoots and roots at frequencies of 18 % and 70 %, respectively. The time required for the development of small plantlets from protoplasts was 8–11 weeks. The regenerated plants contained rolB from Ri-T-DNA and exhibited an altered phenotype compared to the control plants.Abbreviations BAP benzylaminopurine - NAA naphthaleneacetic acid - PCR Polymerase Chain Reaction - fw fresh weight     

Protoplast preparation and polyethylene glycol (PEG)-mediated transformation of <Emphasis Type="Italic">Candida glycerinogenes</Emphasis>

The regeneration of Candida glycerinogenes protoplasts is a major step following genetic manipulations such as fusion and DNA-mediated transformation. An investigation of protoplast formation and cytological examination was used to gain further insight into the loss of protoplast viability in osmotically stabilized support media. Protoplasts with the highest regeneration frequency (98.6% protoplasts/mL) were isolated, using lysozyme dissolved in 1M sorbitol osmoticum. The commercial enzyme preparations, osmotic stabilisers, and growth phase were effective in raising the protoplast yield. Sodium chloride was effective for protoplast preparation; however, sugars and sugar alcohols were better for protoplast regeneration. Sorbitol at a concentration of 1 M was used in regeneration agar for further studies. Regeneration of colonies from protoplasts was maximal (11 ~ 15%) when protoplasts were incorporated in cooled agar containing 0.5% glucose, supplemented with 1M sorbitol as osmotic stabilizer. C. glycerinogenes strain was highly sensitive to zeocin, so transformation of protoplasts and PEG-mediated was achieved with an improved transformation system, using plasmid pURGAP-gfp containing zeocin gene driven by a PCgGAP promoter from C. glycerinogenes to express gfp gene and be transformed into the 5.8S rDNA site of C. glycerinogenes in order to test the system for studying the yeast osmoregulation. We developed an efficient method for transformation of C. glycerinogenes, and parameters involved in transformation efficiency were optimized. Expressions of gfp at different levels were conducted under osmotic stress containing NaCl, KCl, sorbitol or glycerol for the recombinant strains. These improved procedures for protoplast isolation, regeneration and transformation proved to be useful applications in genetic studies for other Candida species and industrial yeast.     

Formation and Regeneration of Protoplasts of the Actinorhizal Nitrogen-Fixing Actinomycete Frankia

Procedures for forming and regenerating protoplasts of four Frankia strains are described. Cells obtained from growth medium containing 0.1% glycine were digested with lysozyme (250 μg/ml) in a medium containing 0.5 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂. Protoplasts were formed during 15 to 120 min of digestion at 25°C. Optimum conditions for protoplast regeneration involved placing protoplasts on a layer of complex growth medium containing 0.3 M sucrose, 5.0 mM CaCl₂, and 5.0 mM MgCl₂ which was overlaid with a layer of 0.8% low-melting-point agarose containing 0.5 M sucrose, 5.0 mM MgCl₂, and 5.0 mM CaCl₂. The maximum regeneration efficiency was 36.9% for strain CpI1, 1.3% for strain ACN1^AG, 27% for strain EAN1pec, and 20% for strain EuI1c.     

蓝色犁头霉原生质体的制备与再生

研究了氢化可的松生产菌蓝色犁头霉原生质体的形成与再生。通过对溶解酶系统的选择,影响原生质体形成的因素如渗透压稳定剂、酶浓度、菌龄、菌丝培养基和培养方式等因素进行考察,发现以0．4mol/L NH4Cl做为稳定剂、2．5mg/mL溶壁酶和5mg/mL纤维素酶组成的混合酶液溶解菌丝,4h后原生质体量可达10^6cell/mL。通过显微镜观察原生质体的形成过程以及在高渗培养基上的再生情况,再生率为15．6％。     

Isolation and regeneration of protoplasts from the ectomycorrhizal ascomycete Cenococcum geophilum Fr.

Protoplasts of the ectomycorrhizal ascomycete Cenococcum geophilum were isolated from mycelium grown in liquid medium. The method was optimized with regard to culture conditions, preincubation, lytic enzyme system, pH value of the incubation medium, osmotic buffer and incubation temperature for C. geophilum strains SIV and 1448. The yields were 1-3·10⁸ and 7·10⁶ protoplasts per gram fresh weight for C. geophilum SIV and C. geophilum 1448, respectively. Protoplasts from C. geophilum SIV exhibited plasma membrane integrity close to 100% (fluorescein diacetate staining). At least 50% of the protoplasts contained a nucleus (staining with acridine orange). The regeneration of protoplasts from C. geophilum is described for the first time. The regeneration frequency was up to 13%, and, dependent on the conditions of culture (liquid medium, agarose, agar), four types of regeneration patterns could be distinguished Regenerated protoplasts of C. geophilum were capable of forming mycorrhizas with spruce (Picea abies) seedlings.     

Improved technique on reversion of mycelial protoplast of Trichoderma reesei into mycelia

Important parameters in the regeneration of protoplasts are viability, the capacity to synthesize cell walls and the retention of properties of the parent cell. Mycelial protoplasts of Trichoderma longibrachiatum (Trichoderma reesei) have been regenerated. Factors influencing the regeneration of protoplasts of T. longibrachiatum QM 9414 were found to be, the nature of osmotic stabilizer, the concentration of osmotic stabilizer, pH, temperature, and the composition of regeneration medium. With glucose-mineral regeneration medium, the optimum conditions for protoplasts regeneration were 0.5 M KCl, pH 6.0 and temperature 30°C. With Czapek-Dox medium, the optimum conditions were 0.7 M mannitol, pH 6.0 and temperature 30°C. Maximum regeneration frequency of T. longibrachiatum protoplasts were obtained using glucose-mineral medium.     

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.     

Characterization of protoplasts prepared from the edible fungus, Stropharia rugoso-annulata

Protoplast preparation and regeneration conditions of the edible fungus, Stropharia rugoso-annulata Farlow apud Murrill were studied, and the regenerated progenies were characterized in this study. The optimal condition for protoplast preparation was incubation of young mycelia with gentle shaking in 1.5%(w/v) Lywallzyme at 30 °C for 3 h. PGPM (potato/glucose/peptone/mannitol) was the most suitable regeneration medium. Served as osmotic stabilizer, sugars (mannitol and sucrose) were better than inorganic salts (MgSO₄) for clone development and growth. Pre-incubation of protoplasts in liquid regeneration medium resulted in a significantly decreased regeneration rate. Both dikaryotic isolates and monokaryotic isolates could be identified from protoplast-regenerated progenies, with a much higher frequency of monokaryotic isolates identified from the early-developed and fast-growing regenerated clones. Two parental mating types were also identified from protoplasted monokaryotic isolates, but not segregated by 1:1. The mycelial growth rate of protoplasted monokaryotic isolates showed a mating type-dependent model when cultured at different incubation temperatures and pH values, with A2B2 mating type monokaryotic isolates growing faster than those of A1B1 mating type monokaryotic isolates.     

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.     

Preparation and regeneration of protoplasts of three fungi of Boletaceae

Protoplasts of three fungi of Boletaceae,Suillus luteus, S. grevillei, andBoletinus cavipes, were prepared with yields of 45, 8.0, and 1.8×10⁷/g fresh mycelia under the optimal conditions, respectively. Nucleate protoplasts accounted for 42% of the whole preparation ofS. luteus and 32% of that ofS. grevillei, and 21% of the nucleate protoplasts ofS. luteus and 35% of those ofS. grevillei possesed two nuclei. Regeneration efficiency of protoplasts was 0.4% forS. luteus and 0.05% forS. grevillei. The regeneration ofB. cavipes protoplasts was also confirmed. Optimal conditions for regeneration were determined. Addition of gellan gum instead of agar to the medium and activated charcoal treatment of agar medium increased the regeneration efficiency significantly.     

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.     

Formation,regeneration, and fusion of protoplasts in aCellulomonas strain

The effect of different conditions on protoplast formation was studied in the streptomycin-resistant strainCellulomonas sp.M32Bo. The greatest efficiency (75% protoplasts) was achieved by use of 0.5M sodium succinate as osmotic stabilizer, supplemented with 20 mM MgCl₂, 200 µg/ml of lysozyme, and 0.01M EDTA at pH 7.4. Cells harvested at the midexponential growth phase were more suitable for protoplast formation than those of the stationary phase. Electron microscopy observations showed the presence of both protoplasts and spheroplasts in the treated samples, some of them still showing a rod shape. Two regeneration media were developed that showed similar regeneration frequencies (52%). StrainM32Bo was fused with a tetracycline-resistant strain (Cellulomonas sp. Sz). Segregation analysis of fusant colonies suggested the existence of a temporary diploid stage in which both parental genotypes were expressed.