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Two virulent phages of Streptococcus faecium SF 77R were used in DNA-free liposome assisted polyethylene-glycol-mediated protoplast transfection. This procedure, established to develop an effective gene-cloning system, optimized the conditions for DNA uptake by the protoplasts of Str. faecium. Transfection frequencies of up to 10(7)-10(8) P.F.U./microgram of DNA were achieved.
A procedure was optimized for the formation and regeneration of protoplasts in a Clostridium acetobutylicum strain isolated from cassava roots in Misiones, Argentina. The regeneration frequency was calculated as the ratio of regenerants (colony forming units on regeneration plates minus colony forming units from osmotically resistant cells) per initial cell number (colony forming units before protoplast formation). The percentage regeneration varied from 10 to 15%.
Conditions for protoplast formation and cell wall regeneration in the genus Bifidobacterium are described. The ability to form protoplast in high percentage varied according to the species considered. High reversion levels were obtained with the species B. bifidum var. pennsylvanicus, B. thermophilum and B. boum.
Polyethyleneglycol induced fusion of Bacillus cereus protoplasts and its genetic consequences have been investigated. The technique used allows the transfer of a small plasmid pBC16 between Bacillus cereus cells. Fusion has resulted in isolation of hybrid cells having acquired TcR phenotype (harbouring pBC16) with high frequencies (10(-2)-10(-3)). However, the genetic instability of hybrid cells and segregation effects have influenced dramatically the final results of plasmid transfer. Nevertheless, the fusion of protoplasts proves to be useful in construction of BAcillus cereus strains inheriting plasmid determinants.
Tobacco leaf mesophyll protoplasts exhibit low incorporation of [3H]uridine and 32P into RNA, up to 12 h of cultivation, irrespective of the presence of phytohormones. After 24 h of cultivation a dramatic increase in RNA synthesis is observed; it is the highest in the heterogeneous nucleoplasmic RNA fraction. The protoplasts cultivated in the absence of phytohormones show lower incorporation of precursors.
Lytic enzymes from the edible snail--Helix pomatia--were used to obtain protoplasts of yeast Saccharomyces vini K-42. The yeast grown in aerobic conditions proved more sensitive. The resultant protoplasts oxidized glucose and sucrose and did not oxidize maltose.
Spherical and osmotically sensitive protoplasts were released from cultures of the yeast-like form of Paracoccidioides basilienisis strain IVIC Pb9 through the action of a mixture of crude enzyme preparations: alpha and beta-glucanases and chitinase, obtained from culture filtrates of Cladosporium resinae, Basidiomycete QM 806 and Streptomyces sp respectively. The highest efficiency of protoplast liberation was achieved when each crude enzyme preparation was used at 1 mg/ml.
Freeze-etching and ultrathin section techniques were used to demonstrate ultrastructural change in plasma membranes during fusion of Saccharomyces cerevisiae protoplasts. Incubation of protoplasts in 30% (w/v) polyethylene glycol fed to strong reduction in the number of particles in areas of close membrane contact and disappearance of invaginations. On sections, neighbouring plasma membranes merged to pentalaminar or trilaminar structures. After incubation in regeneration medium, these membrane changes progressed to form rounded bulging areas with crater-like openings which are thought to be channels through which coalescence of partner's cytoplasms may occur.
Protoplasts have been prepared from the mycelium of Aspergillus funigatus, using a lytic enzyme mixture from Trichoderma harzianum. A variety of experimental conditions were investigated in order to achieve optimal conditions for protoplast production. Electron micrographs showed the preparations to be free of cell-wall. Material obtained by this procedure can be used as a source of cytoplasmic antigens for further analysis.
The paper describes how to prepare osmotically sensitive forms from the fungus Oospora lactis. The lytic enzyme from actinomycetes at a concentration of 25 mg/ml causes abundant formation of protoplasts at 36 degrees C and pH 7.5 within 10--15 min. The best stabilizing agent is 1 M NaCl solution or a mixture of 1 M NaCl with 1 M mannitol solution (1:1). In order to induce lysis of cell walls by the enzyme from Helix pomatia, the cells must be pretreated with a solution containing 40 mM tris (hydroxymethyl)-aminomethane, 5 mM EDTA and 0.2 M cysteine. The production of Oospora lactis protoplasts was accompanied with the liberation of lipase from the periplasmic space. The activity of lipase was 90% of the overall intracellular activity as was confirmed by the results of differential centrifugation of a homogenate prepared by mechanical disintegration of the cells. Since the Rf in disc electrophoresis, the pH optimum and the substrate specificity of exocellular lipase were identical with those of periplasmic lipase, the enzyme must be liberated without a change in the molecular weight.
A simple method for large-scale isolation and regeneration of Arabidopsis thaliana green protoplasts utilizing a novel starting material is presented. This technique takes advantage of shooty/embryogenic masses regenerating from fresh or cultured roots. There is no time-consuming mechanical isolation of starting material. Economical Bacto agar is used as an embedding agent. Plating efficiencies are between 5% and 10%. Seventy-six percent of protoplast-derived colonies produce shoots.
Protoplast fusion was applied to obtain intraspecific fusion in yeast strains in order to overcome restrictions imposed by the natural mating system. Acriflavine was used to construct petite mutants from non-flocculent industrial Saccharomyces cerevisiae saké strain. UV radiation was used to construct ura- mutants from a respiratory competent and highly flocculent S. cerevisiae NCYC869 strain. Fusion products were selected by complementation on minimal medium. The frequency of appearance of prototrophic hybrids was five per 10(5) protoplasts. The stable hybrids showed not only the S. cerevisiae saké characteristics but also flocculation ability, thereby confirming the success of the fusion.
Optimal conditions for the formation of stable protoplasts of S. recifensis subsp. lyticus 2435 were defined and the use of the protoplasting procedure to increase the activity of the synthesis of extracellular enzymes was shown in principle possible. The level of the synthesis of glycosidases and lytic endopeptidases by the isolated variant II-29 was higher than that the initial strain by 40-45 per cent which was due to the enrichment of the population with more active variants.
We have achieved successful transformation of Solanum dulcamara protoplasts by direct DNA uptake and regeneration of transgenic plants. The plasmids pDW2 carrying CAT gene and pCaMVNEO carrying NPTII gene were used. The electroporation voltage was 1500 V, which gave a field strength of 1500 V/cm with a time decay constant of 59.4 sec. The concentration of plasmid was 20 micrograms/2 x 10(6) protoplasts. Under these conditions, a very high transformation efficiency was obtained, with relative transformation frequency being up to 12.4% and absolute transformation frequency 2.4 x 10(-4). The activity of NPTII was detected in 75% of the kanamycin resistant calli and all of the plants regenerated from resistant calli. Southern blot analysis showed that the DNA sequence of NPTII gene derived from pCaMVNEO plasmid existed in the transformed plants of S. dulcamara.
Electric fields are now used extensively for the genetic manipulation of plant cells through protoplast fusion and direct gene uptake. The cost of commercially available electrofusion and electroporation equipment remains prohibitive for many laboratories. This paper describes an electronic apparatus, suitable for the large-scale electrofusion and electroporation of plant protoplasts that is compatible in both function and cost with commercially available equipment.
The hypocotyl protoplasts of Lycium barbarum L. CV. Ningji No. 1 were isolated in an enzyme mixture solution containing 1% cellulase and 1% pectinase. The protoplasts were cultured in KM 8 p liquid medium containing 0.3 mg/L 2,4-D and 0.3 mg/L BA, The first division of regenerated cells occurred 3 days after culture. The small cell clumps could be observed by naked eyes 20 days after culture. 40 days after culture, microcalli of 1-2 mm in size were transferred to MS solid differentiation medium containing 3% sugar, 0.1-0.4 mg/L BA and 0.05-0.1 mg/L NAA. About 30 days after culture on MS solid medium, the calli with shoots were transferred to 1/2 MS medium containing 0.1 mg/L BA and 0.2 mg/L IBA, from which 80% of them differentiated roots and regenerated whole plantlets.
From the suspension cultures of Gynostemma pentaphyllum (Thumb) Mak. protoplasts were isolated and cultured in a different medium with liquid and nurse culture method. Cell division was observed within 4 days and microcalli were formed within 4 weeks. On an MS solid medium with 1 mg/L of KT and 0.5 mg/L of IAA, protoplast-derived calli differentiated into embryos. Stems and leaves were formed on the MS medium with 1 mg/L of 6-BA and 0.5 mg/L of IAA. Finally, complete plantlets were obtained on a hormone-free MS solid medium.
B. pertussis suspension was tested by De Voe et al. method (1970) and its modification with the solutions of a definite ionic composition and a lysozyme. The best results were obtained by the following modification elaborated by the authors. The microbes were grown on the casein-carbon agar for 36 hours and were washed with chilled 0.5 M NaCl. The suspension was washed 4 times with the same solution and then the precipitate was suspended in saccharose solution (0.5 M). In 2 hours the saccharose was replaced by a solution of salts with lysozyme. After a 2-hour incubation at 35 degrees C the substance was centrifugated for 20 minutes and the precipitate suspended in the tris-buffer at pH 7.8. The following changes were observed: after the washing and incubation with saccharose there was seen a strong stretching and separation of the cell wall (CW) from the cytoplasmic membrane (CPM); cells without the CW were rarely revealed; 2) after the lysozyme treatment there were many cells of spherical shape (phasic-contrast microscopy) without any CW, limited by the CPM only. Morphologically they were no different from the true protoplasts of the Gram-positive bacteria. The chemical analysis also confirmed a possibility of obtaining the true protoplasts of the Gram-negative bacteria.