Effect of tryptophan on the production of mucidin in cultures of basidiomyceteOudemansiella mucida

Addition ofL-tryptophan to cultures of the basidiomyceteOudemansiella mucida brought about a pronounced increase of production of the antibiotic mucidin. The highest increase was reached in the presence of 0.15–0.20 % tryptophan and after its addition to a 1-d culture. The methyl ester of tryptophan exhibited the same effect. Mycelium growing during the initial phases in the presence of tryptophan synthesized mucidin powerfully during later phases of the fermentation. Part VII of the series Antifungal antibiotic of the basidiomyceteOudemansiella mucida; part VI:Folia Microbiol. 27, 35 (1982).     

Preparation,reversion, mutagenesis and intra-species fusion of protoplasts ofOudemansiella mucida

A technique was developed for preparation and reversion of protoplasts of the mucidin-producing fungusOudemansiella mucida. The protoplasts can be obtained in sufficient amounts from an exponentially growing young mycelium treated with snail digestive juice in the presence of an osmotic stabilizer. The resulting protoplasts readily regenerate their cell walls and, at a frequency of 10-20%, reverse into mycelium. InO. mucida, which does not form asexual spores, protoplasts are a suitable starting material for mutagenesis. Auxotrophic and higher-producing mutants were obtained in this way. Fusion of protoplasts of two compatible monokaryotic auxotrophic isolates, induced by polyethylene glycol, yielded nutritionally complemented dikaryotes forming clamp connections and producing mucidin.     

Shoot regeneration of mesophyll protoplasts transformed by Agrobacterium tumefaciens,not achievable with untransformed protoplasts

Summary Alternative methods for shoot regeneration in protoplast derived cultures were developed in Nicotiana paniculata and Physalis minima. In both species protoplast derived callus is not regeneratable to shoots by conventional methods, e.g. hormone treatment. Leaf discs and stem segments of N. paniculata and P. minima were incubated with Agrobacterium tumefaciens shooter strains harbouring pGV 2215 or pGV 2298 or wildtype strain B6S3. After 36 h of co-incubation protoplasts were prepared. (Leaf disc and stem segment cloning). Co-cultivation experiments were also undertaken with protoplasts of both species. Transformed clones, characterized by their hormone independent growth and octopine production, could be isolated after about two months. Transformation frequencies of leaf disc and stem segment cloning and co-cultivation experiments varied from 5×10^–3 to 5×10^–5. After about one year of cultivation on hormone-free culture medium, shoots could be recovered from colonies of N. paniculata, transformed by the strain harbouring pGV 2298. In protoplast derived colonies of P. minima, shoot induction was obtained only after transformation by bacteria carrying pGV 2215. This demonstrates the importance of the particular shooter mutant, as well as the response of the host plant. Transformed shoots of P. minima produced octopine, whereas octopine production in transformed shoots and callus of N. paniculata was undetectable after one year of cultivation, though T-DNA was still present in the plant genome. Transformed shoots of N. paniculata and P. minima do not produce any roots. Shoots of N. paniculata have an especially tumerous phenotype. Shoots of both species were successfully grafted to normal donor plants of N. tabacum.Abbreviations B5-h Gamborg medium without hormones (Gamborg 1968) - V47 protoplast medium (Binding 1974) - D2a protoplast medium (Li et al. 1980) - MS-h Murashige and Skoog medium without hormones (Murashige and Skoog 1962) Dedicated to Professor Dr. G. Melchers in occasion of his 80th birthday     

Metabolism of aromatic acids in the antibiotic-producing basidiomyceteOudemansiella mucida

Aromatic acids were determined in the mycelium and fermentation medium ofOudemansiella mucida. Coumaric acids (bothm- andp-),p-hydroxybenzoic acid (salicylic acid) and benzoic acid were found to predominate in the mycelium. Phenylacetic acid represents the main component in the medium. Phenylalanine ammonia-lyase catalyzing conversion of phenylalanine to cinnamie acid which is further metabolized to benzoic acid was detected in the mycelium. The results are discussed with respect to the synthesis of the antibiotic mucidin.     

Composition of lipids and production of mucidin in a submerged culture of the basidiomyceteOudemansiella mucida

Mycelial lipids of the submerged culture ofOudemansiella mucida contain acylglycerols, free and esterified sterols. Free fatty acids are not present. Development of the culture is associated with an increased content of unsaturated fatty acids and, on the contrary, with a decreased content of saturated fatty acids. Content of total lipids depends on age of the culture and is inversely related with production of the antibiotic mucidin.     

The formation of protoplasts from Beauveria bassiana

Summary Beauveria bassiana protoplast formation from blastospores, conidia and mycelia was studied. The method of protoplast formation involves preincubation of the fungal cells with dithiothreitol and subsequent treatment with an enzyme mixture consisting of: cellulase, chitinase, -glucuronidase and lysozyme. Using this procedure protoplasts were formed from blastospores and mycelia but not conidia. Formation of protoplasts from 24 hour old mycelia was 100% efficient using the above conditions. A number of ionic and osmotic protoplast stabilizing agents were tested. Ammonium sulfate was shown to be the stabilizer of choice. Protoplasts were stable when stored at 4° C with a loss of only 17% in 6 days. We suggest that this procedure of protoplast production will allow a gentler method for the extraction and isolation of intact high molecular weight DNA from B. bassiana.     

Recovery of transgenic trees after electroporation of poplar protoplasts

Protoplasts from leaflets ofin vitro cuttings were electroporated in osmotically adjusted and buffered solutions containing plasmid DNA: pABD1, carrying thenptII gene for resistance to neomycin; pGH1, carrying a mutant acetolactate synthase gene,als, for resistance to sulfonylurea; and pGSFR781A, carrying a synthetic phosphinothricin acetyltransferase (pat) for resistance to phosphinothricin (Basta). Gene transfer was repeatedly efficient, without use of carrier DNA, in the range of one transformant for 10⁵ to 10⁴ protoplast-derived cell colonies. This was probably due to the high plating efficiency (30%) of protoplasts in our culture process. Selection for expression of foreign genes was applied in liquid medium and repeatedly achieved with 30 M paromomycin for NPTII, 200 nM chlorsulfuron for the mutant ALS ofArabidopsis and 25 M phosphinothricin for PAT expression. Integration of foreign genes into genomic DNA of resistant poplar trees was demonstrated by Southern blot hybridizations, which revealed that for some transformants practically no other part of the vector plasmid than the selected gene was integrated.Effective processes for protoplast culture, efficient selection at the cell colony stage and gene transfer will provide new possibilities in poplar breeding.     

Cell cycle related changes in the quantity of TMV virions bound to protoplasts ofNicotiana sylvestris

Summary Attachment of virions of tobacco mosaic virus to protoplasts isolated from dividing suspension cultured cells ofNicotiana sylvestris was estimated using quantitative autoradiography of individual protoplasts. Additionally, the position of each protoplast in the cell cycle was assessed by Feulgen microspectrophotometry. At pH 5.6, after preincubation with 4 g 1^–1 poly-L-ornithine, protoplasts in the G₁ and G₂ phases bound more virions than protoplasts in the S-phase. The possibility that such differential binding was caused by cyclical variation in the net charge on the protoplast membrane has been investigated. It was found that S-phase protoplasts ofN. sylvestris can be separarated from protoplasts of other cycle stages by partition in aqueous, two-phase, immiscible polymer systems, presumably because they differ in charge. Also, electrophoretic studies suggest that G₁ phase protoplasts bear higher surface charge than some non-G₁ protoplasts.     

Optimum conditions for efficient transformation ofStreptomyces venezuelae protoplasts

Summary Optimum conditions for protoplast regeneration and transformation ofStreptomyces venezuelae ETH 14630 have been established. Protoplasts from mycelium grown to the stationary phase and treated with lysozyme in P medium under mild conditions gave the best regeneration frequency. Transformation of protoplasts with naked DNA was very efficient using either polyethylene glycol of mol. wt. 4000 or 6000, at concentrations of 28.5% or 36% (w/v) respectively. About 10⁵ transformants/g DNA could be isolated using protoplasts derived from cells cultivated to the early exponential growth phase in LB medium containing 0.2%-0.6% glycine and subsequently treated at 30°–32°C with 20 mg lysozyme/ml in P medium for 30 min. Selection of the transformants occurred on MRYE plates containing less than 10⁵ regenerating protoplasts per plate. Higher protoplast densities considerably decreased the regeneration frequency of the transformants.     

Preparation and regeneration of protoplasts and spheroplasts for fusion and transformation ofSchizosaccharomyces pombe

Preparation and regeneration of protoplasts is essential for somatic hybridization and transformation of yeasts. We present conditions that were found to be optimal for preparing and regeneratingSchizosaccharomyces pombe protoplasts for cell fusion. In contrast to these conditions, genetic transformation ofS. pombe requires spheroplasts that are osmotically sensitive, but still have some wall material attached to the cell. The main finding were as follows: (a) For protoplast formation with Novozym SP234, 0.9M sorbitol was found to be the optimal osmotic milieu and -mercaptoethanol is not necessary. (b) Embedding in soft agar yields considerably better regeneration frequencies than direct plating. (c) Cell fusion is optimal when both fusion partners are fully protoplasted, although considerable fusion occurs between spheroplasted cells as well. (d)Schizosaccharomyces pombe transformation frequencies are much higher with spheroplasts than with protoplasts. Inclusion of -mercaptoethanol did not enhance transformation frequency.     

Nuclear behaviour of colonies and regenerated buds from protoplasts ofNicotiana plumbaginifolia Viviani haploids

The ploidy level variations of protoplast cultures ofNicotiana plumbaginifolla Viviani (n=10) were investigated from protoplast isolation until regenerated buds, using cytophotometric measurements of nuclear DNA content and chromosome counting. An increase in the average nuclear DNA amount has been found to occur in freshly isolated protoplasts after 15 hours of maceration. Cytological abnormalities like nuclear fragmentation, chromatin connections between interphasic nuclei and micronuclei were observed during the following days. Chromosome counting in 15, 30 and 50-day-old calli and in regenerated buds revealed that nuclei are haploid, diploid or aneuploid.Abbreviations p-cells, p-calli or p-colonies protoplast-derived cells, calli or colonies - BAP 6-benzylaminopurine - NAA 1-naphtaleneacetic acid - 2iP ²-isopentyl-aednine     

An assessment of the cultural capabilities of protoplasts of some wild species of linum

Protoplasts of several wildLinum species were isolated enzymatically from roots, hypocotyls and cotyledons of seedlings, and also from theirin vitro grown shoots and cell suspension cultures. When cultured all these protoplasts divided to produce callus but only good plant regeneration capability was evident in the case ofLinum lewissii and to a much lesser extent forL. strictum. Only rhizogenesis was observed withL. alpinum, L. narbonense, L. grandiflorum andL. altaicum. The high plant regeneration capacity ofL. lewissii from protoplast -derived tissues ofin vitro shoots and cell suspension cultures makes this species an attractive experimental system for somatic genetic manipulation.Abbreviations BAP benzylaminopurine - NAA -naphthaleneacetic acid - CPW cell and protoplast wash solution - gFW gram fresh weight On leave from Department of Crop Sciences University of Alexandria Egypt     

Breeding anl-isoleucine producer by protoplast fusion ofCorynebacterium glutamicum

Summary Corynebacterium glutamicum R-18 is a strain forl-isoleucine production. Polyethylene glycol (PEG)-induced protoplast fusion was applied to improve the strain of thisl-isoleucine producer. Strain R-18 was fused with anl-lysine producerC. glutamicum S-37, becausel-isoleucine andl-lysine are synthesized from a common intermediate, aspartate--semialdehyde. Two thousand fusants were checked for their phenotypes. Most of the fusants accumulatedl-lysine, and only 0.9% of the fusants accumulatedl-isoleucine. Two strains, F-28 and F-91, were selected and cultivated in production medium. Fusant F-28 accumulated 12.1 g/l ofl-isoleucine and 4.8 g/l ofl-lysine, and fusant F-91 accumulated 4.8 g/l ofl-isoleucine and 13.0 g/l ofl-lysine, while the parental strains R-18 and S-37 accumulated 9.5 g/l ofl-isoleucine and 26.8 g/l ofl-lysine, respectively. Sugar consumption activity was improved by protoplast fusion, and thel-isoleucine production rate of F-28 was 2.4 times higher than that of R-18.     

Surface charge of protoplasts and their significance in cell-cell interaction

-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl₂ nullified the -potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the -potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with -neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.     

Regeneration of Medicago truncatula from protoplasts isolated from kanamycin-sensitive and kanamycin-resistant plants

Medicago truncatula (barrel medic) is an annual legume of agricultural and biological interest. In this report regeneration from isolated mesophyll protoplasts is described. A specifically developed, highly regenerable seed line is essential for regeneration. Other critical requirements for regeneration are the starting plant material, the use of agarose droplets incubated in a shallow layer of liquid medium, and protoplast density. Plants are grown in controlled environment conditions. Protoplasts are purified using a Percoll-based flotation procedure, then embedded in 100 l agarose droplets containing a basal medium plus 25 M NAA and 4 M BAP (the same medium as in the surrounding shallow liquid layer) to induce protoplast division. A protoplast density of 6–8×10⁵ ml^–1 is required for maximum colony formation. M. truncatula plants previously transformed for kanamycin resistance yielded embryogenic callus and also regenerated plants. Protoplasts from other annual Medicago (M.intertexta and M.scutellata) species readily form calli by the procedure we have described.Abbreviations BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - NAA 1-naphthaleneacetic acid     

Uptake of protoplasts from the filamentous fungiAspergillus nidulans andFusarium oxysporum into protoplasts from celery (Apium graveolens L.)

Summary Protoplasts isolated from celery cell suspension cultures, were mixed with fungal protoplasts, from either the saprophytic speciesAspergillus nidulans or the pathogenic speciesFusarium oxysporum. The incubation of protoplast mixtures with PEG caused close adhesion between plant and fungal protoplasts. Subsequent dilution of PEG resulted in the uptake of protoplasts from either fungal species into the plant protoplast cytoplasm. A range of PEG concentrations, incubation times and dilution rates were tested to maximise adhesion and uptake frequencies. Identification of uptake was achieved either by fluorescent staining of nuclei or by electron-microscopy. A maximum of 10% celery protoplasts had taken upA. nidulans protoplasts after PEG treatment. Fungal protoplasts were taken up into celery protoplast cytoplasm by endocytosis, and were maintained within vesicles; two bounding membranes were observed by electron microscopy. Plant protoplast viability was determined during prolonged incubation following fungal protoplast uptake. The presence ofA. nidulans protoplasts tended to maintain celery protoplast viability and although some morphological disintegration occurred intact celery protoplasts remained for at least 92 h after uptake. The uptake ofF. oxysporum protoplasts markedly depressed celery protoplast viability after 24 h incubation and greater celery protoplast disintegration occurred.Abbreviations PEG Polyethylene glycol - DAPI 4,6-diaminido-2-phenylindole - 2,4-D 2,4-dichlorophenoxyacetic acid     

Sporulation of protoplasts

Vegetative cells ofBacillus megaterium formed protoplasts in a sucrose-stabilized medium under the influence of lysozyme. The protoplasts sporulated during subsequent incubation. The morphological properties, germination, resistance to u. v. irradiation and thermo-resistance of protoplast spores were the same as with normal cells. It thus appears that in the later sporulation stages the spore formation occurs, without participation of the sporangium cell wall.     

Plant cell graft chimeras obtained by co-culture of isolated protoplasts

Summary Heterospecific chimeralSolanum nigrum (+)Solanum tuberosum plants were obtained by cell grafting in protoplast co-cultures. Periclinal, sectorial, and mericlinal chimeras have been identified by various morphological and cytological characteristics.Morphogenesis predominantly began in periclinal chimeral organization. Cells of different species have been found to be interconnected by secondary plasmodesmata. Plantlets of all chimeral lines were grown to flowering under tissue culture conditions and some also in the greenhouse. Aspects of organogenesis and interspecific cooperation are discussed.Abbreviations B 5 culture medium (Gamborg et al. 1968) supplemented with 2.5M 6-benzyladenin - L1, L2, L3 epidermis (L1), subepidermal layer (L2), core (L3) - MS culture medium (Murashige andSkoog 1962) - n, n _w symbols used for the indication ofSn-F (n) orSn-F-W2 (n _w ) tissue in L1, L2 or L3 - Sn Solanum nigrum - Sn-F is an atrazine-resistant biotype - Sn-F-W2 is a plastid mutant malbino derivative ofSn-F - St Solanum tuberosum - St-H²258 is a dihaploid clone - t symbol used the indication ofSt-H²258 tissue in L1, L2 or L3 - V-KM culture medium (Binding and Nehls 1977) Dedicated to Professor Dr.Josef Straub, late Director of the Max-Planck-Institute für Züchtungsforschung at Cologne, who was the first to study the production of chimeras by callus association in 1972.     

Selection methods in bacilli for recombinants and transformants of intra- and interspecific fused protoplasts

The intraspecific fusion frequencies obtained with the direct selection method on a semi-synthetic regeneration medium between strains of B. subtilis and B. licheniformis were distribution from 9.9×10^-2 to 4.5×10^-3, which was one or two orders higher than those of interspecific recombinations between B. subtilis and B. licheniformis.The regeneration media were also useful for selecting interspecific transformants from plasmid carrier to non-carrier. This selection could be used as a primary selection method for inter-and intraspecific recombinants obtained by protoplast fusion.Abbreviations NTG N-methyl-N-nitro-N-nitrosoguanidine - Cm chloramphenicol - SMM 0.5 M sucrose-0.02 M MgCl₂-0.02 M maleate buffer, pH 6.5     

Absence of glucose-stimulated Transport in yeast protoplasts

Protoplasts ofSaccharomyces cerevisiae prepared by snail-gut juice treatment were compared in their transport properties with intact cells. 1. Constitutive monosaccharide transport (D-xylose, 6-deoxy-D-glucose), as well as inducible transport of D-galactose, were unaltered. 2. Phosphorylation-associated transport of 2-deoxy-D-glucose was enhanced in protoplasts, possibly as a consequence of removal of the unstirred layer of the cell wall. 3. Proton-driven transports of trehalose, L-leucine, L-proline and monophosphate could not be activated by preincubation with D-glucose, apparently owing to lack of proton —solute coupling in transport. Utilization of glucose was not depressed but respiration was reduced by about 50% while acidification of the external medium after glucose addition was inhibited by more than 90%. This may be related to the inability of protoplast plasma membrane H-ATPase to be activated by glucose and hence to impaired proton-translocating capacity. Uranyl ions inhibited generally much less in protoplasts than in intact cells although their binding to protoplasts was greater (maximum 0.68 fmol per cell but 3.2 fmol per protoplast).