Use of the protoplast fusion method in the selection of Streptomyces griseus--the producer of the streptothricin antibiotic grisin

An effective method for protoplast fusion in S. griseus producing grisin was developed. The method requires the use of polyethylene glycol with a molecular weight of 1000. It was demonstrated that protoplasts formed most effectively in this organism, when the mycelium of the streptomycete previously treated with ultrasound in the process of its growth was used for the treatment with lysozyme. The efficacy of protoplast regeneration in the strains with the use of the modified hypertonic medium R2MD was 25-75 per cent. The possibility of using the protoplast fusion method for constructing phage resistant strains producing kormogrisin was shown.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus.

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

An efficient and rapid procedure for plantlet regeneration from chicory mesophyll protoplasts

An efficient procedure for plantlet regeneration from chicory mesophyll protoplasts has been developed in order to perform protoplast fusion experiments. Protoplasts were isolated from a genotype of Italian red chicory (CH 363) and purified by centrifugation in a solution containing 13% (w/v) sucrose to collect uniform protoplasts in size. After 2 days culture at a density of 2×10⁴ protoplasts ml⁻¹ of liquid medium, protoplasts were cultured following three different procedures: in liquid medium, stratified in semi-solid medium, and embedded in Ca-alginate droplets. Four different media were used and culture procedures were evaluated recording the protoplast viability, protoplast division frequency and plating efficiency for each experiment. The embedding of protoplasts in Ca-alginate droplets enhanced both division frequency and plating efficiency for chicory mesophyll cells. Furthermore, this procedure shortened the cycle of plant regeneration from protoplasts, which could be completed in eight weeks. This revised version was published online in June 2006 with corrections to the Cover Date.     

Efficient plasmid transformation of the beta-lactam producer Streptomyces clavuligerus

The conditions for optimal formation and regeneration of protoplasts of Streptomyces clavuligerus were established. The optimal temperature for regeneration of protoplasts and for transformation was 26 degrees C in three different regeneration media. The best efficiency of transformation was obtained with 40% polyethylene glycol 1000. The efficiencies of regeneration and transformation increased greatly when protoplasts were obtained from cultures in the early stationary phase of growth. The number of transformants per assay increased linearly with rising concentrations of protoplasts. However, the number of transformants per protoplast decreased at concentrations of protoplasts above 1.5 X 10(9). The total number of transformants rose linearly at increasing plasmid DNA concentrations, but the number of the transformants per microgram of DNA became constant at concentrations above 1 microgram of DNA. Transformation frequencies as high as 5 X 10(5) transformants per microgram of DNA were obtained when plasmid pIJ702 was isolated from S. clavuligerus but not when isolated from Streptomyces lividans.     

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.     

Cytologic control of protoplast formation and regeneration in Streptomyces erythraeus, strain BTCC-2

Experiments on protoplast formation and regeneration in S. erythraeus, strain BTCC-2 (Saccharopolyspora erythrae) were performed under microscopic control at all the stages. It was shown that the highest protoplast titer was provided by the mycelium grown in one step in the absence of glycine. For characterizing the protoplasts formed by the mycelium grown under different conditions, their regeneration capacity was estimated by microscopic examination of the protoplasts after 15-20-hour growth in microchambers and evaluation of the regeneration efficiency 7-10 hours later. Of interest was the fact of spontaneous development of colonies consisting of the protoplast-like cells (L-cells) in 15-20 hours. Such colonies were formed only by the protoplasts grown from the mycelium incubated in one step in the absence of glycine or in the presence of 0.1 per cent of glycine. Such conditions provided also the maximum efficiency of the protoplast regeneration. The long-term storage of protoplasts led to a decrease in their viability.     

Efficient protoplast regeneration for some homofermentative lactobacilli and pediococci

Conditions for protoplast regeneration were examined for several strains of homofermentative lactobacilli and pediococci isolated from silage. Attempts to regenerate protoplasts using previously published agar regeneration media for lactobacilli were unsuccessful for most of the strains. Replacing or increasing colloidal substances in a medium containing raffinose and MgCl(2) as osmotic stabilizers enabled efficient regeneration of the protoplasts at a frequency of 10-99%. A medium containing gelatin, polyvinylpyrrolidone (PVP) and no agar was effective for Lactobacillus plantarum, Lactobacillus pentosus and Lactobacillus rhamnosus protoplasts. An agar medium containing PVP (PVP medium) was effective for Pediococcus sp. protoplasts, and addition of agarose to the PVP medium enabled regeneration of Lactobacillus casei protoplasts. A medium containing calcium alginate gel and no agar was effective for Lactobacillus curvatus protoplasts. The type of colloidal substance required for protoplast regeneration varied from species to species. This result suggested that several kinds of media may be necessary to regenerate protoplasts for all the genera of lactobacilli and pediococci.     

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.     

Characteristic features of protoplast formation and regeneration in Fusidium coccineum

The methods for preparation and regeneration of protoplasts were tested with respect to the strains of F. coccineum markedly differing in their capacity for antibiotic production, sporulation and the growth rate. It was found that the substrate used for the culture growth had a significant effect on the cell wall and sensitivity of the mycelium to lytic enzymes. An enzyme from Hellix pomatia and its combination with lysozyme were used for lysing the culture. The cytological investigation of the time course of the culture lysis revealed a stage-by-stage pattern of protoplast formation by means of fragmentation of the hyphal contents till a ball was formed. Two to 4 protoplasts differing in their size and structure were formed within a cell. The pH value and osmotically stabilizing component had some effect on the rate of protoplast formation. Highly productive strains were characterized by formation of protoplasts heterogenous in their size and by decreased frequency of regenerations. The enzyme-free protoplasts preserved their viability and capacity for germination in osmotically stabilizing media for 72-96 hours of storage at 4 degrees C. On solid media the regeneration frequency reached 38 per cent. The regenerated cells formed colonies morphologically similar to those of the intact culture.     

Isolation and Identification of Ciliatine (2-Aminoethylphosphonic Acid) from Lipids of Edible Antarctic Krill,Euphausia superba

An improved method for regenerating Bacillus subtilis protoplasts at the frequency of 92～100% on a semi-synthetic medium was found. Protoplasts were preincubated in HCP-3 medium, an isotonic semi-synthetic medium supplemented with polyvinylpyrrolidone, and then plated on HCP-1.5 agar medium by overlaying. By this method, even on the regeneration medium supplemented with minimal nutritional requirements protoplasts regenerated at a frequency of as high as 20%. The modified method was applicable to the direct-selection of prototrophic recombinants after fusion (the highest recombination yield from the input protoplasts was 1.3%) and to protoplast transformation with plasmid DNA.     

Improved efficiency of genotype-dependent regeneration from protoplasts of important potato cultivars

The regeneration of protoplasts from potato (Solanum tuberosum L.) cvs. Desiree and King Edward has been significantly improved. Different shoot culture media were required for the release of viable protoplasts from cvs. Maris Piper and Desiree, and the response of protoplasts to different culture conditions depended upon the cultivar genotype of the protoplast source. Using protoplast isolation media containing 6mM CaCl₂ improved protoplast viability and culture in enriched media lead to the reproducible and relatively efficient recovery of colonies from protoplasts of these cultivars. Over 70% of protoplast-derived calli from King Edward and Desiree regenerated shoots. Many shoots were grown to mature plants in soil. This is the first report of the regeneration of mature Desiree plants from protoplasts.Abbreviations NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - 2,4-D 2,4-dichlorophenoxyacetic acid - MES 2-(N-Morpholino)ethanesulphonic acid - CH Casein hydrolysate - CW Coconut water - Inos myo-Inositol - PABA p-Aminobenzoic acid     

Plant regeneration from mesophyll protoplasts of Solanum commersonii dun

Somatic fusion of Solanum commersonii, a frost tolerant wild potato species not crossable with Solanum tuberosum, relies on the possibility to isolate and culture protoplasts. This study was conducted to determine whether protoplasts could be isolated and plants regenerated in three S. commersonii accessions. Shoot cultures for protoplast isolation were maintained on Murashige and Skoog medium. Mesophyll protoplasts were isolated and cultured using a protocol originally described for S. tuberosum with some modifications. Differences were evident among the three accessions for protoplast yield, plating efficiency and regeneration frequency. Protoplast yield ranged from 3.0 to 8.5 × 10⁶ protoplasts per g of fresh tissue. At 1–2 × 10⁴ protoplasts ml⁻¹, which was the optimal plating density, 10–20% of plated protoplasts gave multicellular colonies. Regeneration of shoots was observed in two accessions only, the maximum regeneration frequency being 66%. In one of these accessions the reduction of sucrose concentration in regeneration media improved the regeneration frequency from 14 to 35%. About three hundred plants were rooted in vitro and successfully transferred to soil.     

Effect of cellulase fractionation on the viability of cultured barley (Hordeum vulgare) protoplasts

The age of the stock plants was important for the barley ( Hordeum vulgare L. cv. Perth) protoplast viability. Light conditions under which the stock plants were grown also affected the viability of the protoplasts. Greenhouse-grown plants yielded much higher number of protoplasts than dark-grown plants, but protoplast viability was better when protoplasts were isolated from etiolated plants. Light supplied during protoplast culture affected protoplast viability within the first 24 h of culture. Cellulase R-10 (Onozuka) was better than Cellulysin (Calbiochem) and Cellulase ⁺ Macerozyme R-10 (Onozuka) for barley mesophyll protoplast isolation. Cellulase R-10 (Onozuka) was fractionated on a G-75 Sephadex column. The eluted fractions were tested for their ability to release barley mesophyll protoplasts and for their toxicity towards the protoplasts. Only a small part of the Cellulase R-10 was necessary for protoplast isolation from barley leaves. When the fractionated cellulase was analysed by isoelectric focusing, this part of the cellolase appeared as a single band.     

Efficient plasmid transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts.

A procedure for efficient transformation of Streptomyces ambofaciens and Streptomyces fradiae protoplasts with plasmid DNA was developed. Transformation frequencies with S. fradiae protoplasts were strongly influenced by the temperatures for cell growth, protoplast formation, and protoplast regeneration. Transformation frequencies for both species were also influenced by the culture age before protoplast formation, the source and concentration of polyethylene glycol, the transformation-inducing agent, the concentration of protoplasts used in the transformation procedure, and the number of protoplasts added to regeneration plates. Transformation frequencies were substantially higher for both species when calf thymus DNA and protamine sulfate were added to the transformation mix. With S. fradiae, transformation frequencies were much lower with plasmid DNA prepared from other species than with the same plasmids prepared from S. fradiae, suggesting that S. fradiae expresses restriction and modification. With the modified transformation procedures using DNA prepared from homologous hosts, S. ambofaciens and S. fradiae are now transformed routinely at frequencies of 10(6) to 10(7) transformants per micrograms of plasmid DNA.     

Regulatory mechanisms controlling antibiotic production in Streptomyces clavuligerus

Streptomyces clavuligerus produces a large array of natural compounds with antibiotic, antitumor, beta-lactamase inhibition or inmunomodulating activities. The production of cephamycin C, clavulanic acid and other compounds with a clavam structure has been studied for many years. A network of regulatory mechanisms is present in S. clavuligerus to control the formation of different compounds by pathway-specific regulators or pleiotropic regulators. The possible existence of a gamma-butyrolactone signaling system in this streptomycete is emerging. In addition, S. clavuligerus possesses a stringent control mechanism somehow different from those previously reported in other Streptomyces species.     

Genetic mapping in Streptomyces clavuligerus by protoplast fusion

The development of a protoplast manipulation protocol for the industrially important bacterium Streptomyces clavuligerus, which produces the beta-lactamase inhibitor clavulanic acid, made possible a preliminary genetic mapping study based on protoplast fusion crosses. A preliminary position for 11 markers on the S. clavuligerus genetic map is proposed. Fusion progeny were characterized by random spore analysis because the markers present in the strains were not amenable to the conventional four-on-four selection procedure. Whilst the resulting map is similar to that derived by conjugation for S. clavuligerus and S. coelicolor, further analysis of the markers is required to confirm these observations.     

Barley scutellum protoplasts: Isolation, culture and plant regeneration

Many applications of cereal protoplast culture systems are still limited by the difficulties of regeneration from suspension cells which are the usual protoplast source. The objective of the present study therefore was to investigate the conditions for the development of a culture system for protoplasts capable of plant regeneration isolated directly from immmature scutella of barley. The procedure developed involves a two-stage pre-culture of scutellar tissue, followed by vacuum infiltration with cell wall degrading enzymes and the culture of alginate-embedded protoplasts. The pre-culture of the scutella and the co-cultivation of protoplasts with nurse cells were the most important factors for the success of the culture system, but several other parameters affecting protoplast yield, viability and sustained division were identified, including the developmental stage of the embryo, the use of cold conditioning periods during pre-culture, the composition of the pre-culture and protoplast culture medium, and the embedding matrix. Protoplasts isolated from scutellar tissues of barley cvs Dissa, Clipper, Derkado and Puffin were capable of sustained division in culture. Macroscopic protoplast-derived tissues were obtained in all cultivars, except ev. Puffin, and fertile plants were regenerated from cvs Dissa and Clipper 3–4 months after protoplast isolation. The procedure described provides a novel approach for the isolation of totipotent protoplasts in barley which avoids the need for suspension cultures.     

草木樨状黄芪甲硫氨酸抗性系的原生质体培养及植株再生

建立了草木樨状黄芪(Astragalus melilotoides Pall．)甲硫氨酸抗性系原生质体再生植株的实验体系。以茎切段诱导的松软愈伤组织为材料,通过酶法分离出大量有活力的原生质体。原生质体经培养持续分裂形成了愈伤组织,并高频率地分化出再生苗。比较了不同培养基、培养方法和培养密度对原生质体分裂和再生的影响。结果表明,原生质体以3×10⁵/mL的植板密度,采用琼脂糖岛法培养在附加1．0mg／L 2,4-二氯苯氧乙酸(2,4-D)、0．5mg／L 6-苄氨基嘌呤(6BA)、500mg／L水解酪蛋白、3％蔗糖、0．3mol／L甘露醇的KM_8p培养基中,可获得最佳效果,其细胞分裂频率达38％左右。原生质体培养后仍然保持对甲硫氨酸的抗性,同时对乙硫氨酸表现交叉抗性。     

Inhibition of restriction in Streptomyces clavuligerus by heat treatment

Inefficient transformation of Streptomyces clavuligerus protoplasts by DNA from the plasmid pIJ702, isolated from S. lividans, was attributed to restriction in view of the observation that efficient transformation was observed using modified pIJ702 (isolated from S. clavuligerus). The restriction system could be partially inhibited by treating protoplasts at 45 degrees C prior to transformation. This treatment increased the transformation frequencies of pIJ702 DNA by 100-fold and was used to introduce other plasmids into S. clavuligerus.     

Improvement of regeneration of Lycopersicon pennellii protoplasts by decreasing ethylene production

Summary Lycopersicon pennellii shoots, cultured in vitro for more than a year (type I plants) produced few viable protoplasts in contrast to shoots cultured in vitro for less than five months (type II plants). Ethylene production of both plant types was compared. The low viability of plant type I protoplasts could be correlated with high ethylene production and an increased cell sap osmolality. The ethylene action inhibitor silver thiosulphate improved protoplast yield and viability, especially when using donor tissue, germinated and cultured on medium containing silver thiosulphate (type III plants). Moreover, the choice of cell wall degrading enzymes influenced protoplast viability, since ethylene release was significantly lower using Cellulase R 10 than Cellulysin. All improvements together resulted in an efficient protocol for the isolation and regeneration of Lycopersicon pennellii protoplasts.Abbrevations ACC 1-Aminocyclopropane-1-carboxylic acid - FW Fresh Weight - Mes -Morpholino ethane sulphonic acid - NMU N-Nitroso-N-Methyl-Urea - PE Plating Efficiency = Number of calli / number of protoplasts x 100% - Pps protoplasts - STS Silver thiosulfate