Formation and regeneration of Frankia protoplasts

A technique for forming protoplasts from Frankia cells and regenerating them to the normal hyphal mode of growth is described. Electron microscopy proved that protoplasts were studied and not spores or small hyphae. Regenerated colonies were investigated for genetic markers. One ArI3 colony had been cured of its plasmids without being affected in its symbiotic properties.     

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.     

Formation and regeneration of protoplasts of Streptomyces hygroscopicus

Techniques are described for the production and regeneration of protoplasts of Streptomyces hygroscopicus and related strains. No single technique was successful in producing protoplasts from all strains. Regeneration of protoplasts to the mycelial growth form was greatly improved by modifying the physical regeneration environment. Protoplast formation and regeneration was achieved in six out of the seven strains studied.     

Formation and regeneration of protoplasts in the moldRhizopus nigricans

Wall-less protoplasts of the moldRhizopus nigricans were obtained by means of enzymic digestion of the walls of vegetative hyphae by Helix pomatia digestive juice. After removing the enzymes the protoplasts transferred to a nutrition medium regenerate. In the first stage of this process the protoplasts from a firm wall and then the hyphae begin to grow. Regeneration results in the formation of a normal mycelium. If in the course of regeneration of the protoplasts snail enzymes are present, they partially block the synthesis of the wall and the protoplasts transform into incessantly growing giant formations. Re-establishment of morphogenesis begins only after eliminating the effect of the snail enzymes.     

Formation and regeneration of protoplasts in Sclerotium rolfsii ATCC 201126

AIMS: Different cultural conditions for forming and reverting protoplasts were systematically studied to establish a rapid and efficient protocol for Sclerotium rolfsii ATCC 201126. METHODS AND RESULTS: Osmotic stabilizer, lytic enzymes and mycelial age were the main factors influencing protoplast yields. An optimized protocol involving 1-h hydrolysis of 45-h-old mycelium with Trichoderma harzianum enzymes in a 1 : 1 (w/w) biomass : enzyme ratio and 0.6 mol l-1 MgSO4 as osmotic stabilizer was designed to produce approx. 2 x 109 protoplasts per gram biomass dry weight, with 99% viability. Differences on the lytic activity between batches of commercial enzymes were clearly evidenced. Protoplast release was highly efficient showing no remaining cell wall material as witnessed by fluorescent brightener 28. Up to 26% of purified protoplasts developed into the typical filamentous form after 50 h of incubation on 0.6 mol l-1 sucrose agar media. CONCLUSIONS: The methodology herein proposed allowed a rapid, inexpensive and efficient protoplast production. Optimum yields were higher or in the order of that elsewhere reported for other S. rolfsii strains and the required lytic time was significantly shorter. Purified protoplasts successfully reverted to the filamentous morphology. SIGNIFICANCE AND IMPACT OF THE STUDY: The present research reports the former protocol for the isolation and reversion of protoplasts in S. rolfsii ATCC 201126 providing key factors to ensure optimum results. In addition, the described procedure constitutes a starting point for downstream genetic manipulation.     

Formation, regeneration, and transfection of Lactobacillus plantarum protoplasts

Protoplasts of L. plantarum were produced by mutanolysin-lysozyme digestion at 50 degrees C and regenerated at a frequency of 1.6 to 3.8%. The addition of Tween 80 to the growth medium increased the length of time required for protoplast formation. When transfected with bacteriophage B2 DNA, transfection efficiencies ranged from 25 to 230 PFU/microgram of DNA and from 2.2 X 10(-5) to 4.7 X 10(-4) PFU per recovered protoplast. Total transfectant yield was 3.7 X 10(2) to 3.4 X 10(3) per treatment. Transformations with plasmid DNA were unsuccessful.     

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.     

Formation, regeneration, and transfection of Lactobacillus plantarum protoplasts.

Protoplasts of L. plantarum were produced by mutanolysin-lysozyme digestion at 50 degrees C and regenerated at a frequency of 1.6 to 3.8%. The addition of Tween 80 to the growth medium increased the length of time required for protoplast formation. When transfected with bacteriophage B2 DNA, transfection efficiencies ranged from 25 to 230 PFU/microgram of DNA and from 2.2 X 10(-5) to 4.7 X 10(-4) PFU per recovered protoplast. Total transfectant yield was 3.7 X 10(2) to 3.4 X 10(3) per treatment. Transformations with plasmid DNA were unsuccessful.     

Formation and regeneration of protoplasts and spheroplasts of gastrointestinal strains of lactobacilli

Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.     

Formation,regeneration, and transformation of protoplasts of Streptomyces diastatochromogenes 1628

Toyocamycin exhibits effective biological activities for use against plant pathogenic fungi thanks to its structural similarity to nucleoside. It has been recognized as a promising agricultural antibiotic utilized in controlling the occurrence of plant diseases. In our previous study, a strain that was isolated was identified and designated as Streptomyces diastatochromogenes whose major secondary metabolite was toyocamycin, but the production was largely limited. Protoplast transformation is a useful technique in the improvement of streptomycete. In this study, we optimized some key factors necessary for protoplast formation, regeneration, and transformation of S. diastatochromogenes. When mycelium was cultivated in CP medium with 1 % glycine, harvested at 48 h old, and then treated with 3 mg lysozyme/mL in P buffer for 1 h, the greatest regeneration frequency (42.5 %) of protoplasts was obtained. By using 1?×?10⁹/mL protoplasts with polyethylene glycol 1000 at a concentration of 30 % (w/v), the best performance of protoplast transformation efficiency was 4.8?×?10³/μg DNA transformants.     

Formation and regeneration of protoplasts and spheroplasts of gastrointestinal strains of lactobacilli.

Methods were developed for the formation of protoplasts and spheroplasts of gastrointestinal strains of Lactobacillus reuteri, Lactobacillus gasseri, and Lactobacillus salivarius. Attempts to regenerate vegetative cells from protoplasts were not successful, but spheroplasts could be regenerated consistently for five of six strains.     

Formation,growth, and regeneration of protoplasts of the green alga,Uronema gigas

Summary Separate protoplasts were obtained by the action of snail gut juice enzymes on the cell walls of the green algaUronema gigas. The cultivation of the protoplasts in mineral media caused only their enormous growth; in the presence of glucose a fibrillar network was formed on the surfaces of the growing protoplasts. Only after the addition of pectin the regeneration of the cell wall and the renewal of their morphogenesis could be observed.     

Isolation and regeneration of Micromonospora olivoasterospora protoplasts

The conditions for preparation and regeneration of the protoplasts of M. olivoasterospora were developed. It was found that effective formation of the protoplasts required preliminary cultivation of M. olivoasterospora in the medium containing glycine in a concentration inhibiting its growth at least by 60-80 per cent. The strains studied markedly differed in their sensitivity to glycine and were highly sensitive to it. The efficacy of the protoplast formation depended on the culture age and increased with the use of the lytic enzyme 3 of Cytophaga dissolvens. The possibility and advisability of the use of prolonged lysis of the Micromonospora cell walls were shown. A rich organic medium was used for regeneration of the protoplasts.     

Plant regeneration from Carica protoplasts

Summary Rapidly proliferating and highly regenerable suspension cultures of somatic embryos of Carica papaya x C. cauliflora were used for protoplast isolation. On average, protoplast yield was 1.5×10⁶/g fresh weight of somatic embryos. Protoplasts were first cultured in liquid KM8P-S medium for 2 weeks and then plated in the same medium solidified with 1% agarose. About 1.4% of the protoplasts developed directly into somatic embryos. Protoplast-derived somatic embryos proliferated rapidly through direct embryogenesis on modified MS medium supplemented with 1 mg/1 ABA, and developed into plantlets upon transfer to MS medium devoid of plant growth regulators. The plantlets were successfully transplanted to soil.Abbreviations MS Murashige and Skoog medium (1962) - KM8P Kao and Michayluk medium (1975) - NAA -naphthaleneacetic acid - BAP 6-benzylaminopurine - ABA abscisic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - FDA fluorescein diacetate - CPW Frearson et al. medium (1973)     

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.     

Plant regeneration fromArabidopsis thaliana protoplasts

Summary Arabidopsis thaliana protoplasts, isolated from a cell suspension, regenerated cell walls and then exhibited sustained cell divisions. Within 10 days after isolation as many as 40% of the protoplast-derived cells divided. Protoplast-derived calli exhibited a wide range of developmental responses, including apparent embryogenesis. The cell suspension has been cryopreserved.