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Showing papers on "Protoplast published in 1979"


Journal ArticleDOI
TL;DR: The data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome.
Abstract: The hydrolase content of vacuoles isolated from protoplasts of suspension-cultured tobacco cells, of tulip petals, and of pineapple leaves, and the sedimentation behavior of tobacco tonoplasts were studied. Three precautions were found to be important for the analysis of vacuolar hydrolases and of the tonoplast. ( a ) Purification of protoplasts in a Ficoll gradient was necessary to remove cell debris which contained contaminating hydrolases adsorbed from the fungal cell-wall-degrading enzyme preparation. ( b ) Hydrolase activities in the homogenates of the intact cells or the tissue used and of the purified protoplasts had to be compared to verify the absence of contaminating hydrolases in the protoplast preparation. ( c ) Vacuoles obtained from the protoplasts by an osmotic shock had to be purified from the lysate in a Ficoll gradient. Since the density of the central vacuole approximates that of the protoplasts, about a 10% contamination of the vacuolar preparation by surviving protoplasts could not be eliminated and had to be taken into account when the distribution of enzymes and of radioactivity was calculated. The intracellular activities of the following acid hydrolases were primarily localized in the vacuole of tobacco cells: α-mannosidase, β- N -acetylglucosaminidase, β-fructosidase, nuclease, phosphatase, phosphodiesterase. A similar composition of acid hydrolases was found in vacuoles obtained from protoplasts of tulip petals. Proteinase, a hydrolase with low activity in tobacco cells and tulip petals and therefore difficult to localize unequivocally, was found to be vacuolar in pineapple leaves, a tissue containing high levels of this enzyme. Our data support the hypothesis that the central vacuole of higher plant cells has an enzyme composition analogous to that of the animal lysosome. None of the vacuolar enzymes investigated was found to be bound to the tonoplast. When vacuoles were isolated from cells labeled with radioactive choline, the vacuolar membrane was found to contain radioactivity. On sucrose gradients, the label incorporated into tonoplasts banded around a density of 1.10 grams per cubic centimeter (24% sucrose, w/w).

508 citations


Journal ArticleDOI
01 Oct 1979-Nature
TL;DR: It is reported here, however, that the mitochondrial (mt) DNAs of cybrids are different from those of the parents and from the mixture of the two.
Abstract: We have previously regenerated tobacco plants from fused protoplasts isolated from two varieties of Nicotiana tabacum. The parent cells had distinct morphological nuclear and cytoplasmic markers, enabling us to recognise, among the whole regenerated plants, those with a single parental nucleus and a hybrid cytoplasm produced by the mixing of the two parental cytoplasms1. Genetic analysis of their progeny has confirmed that the phenotypes of these cytoplasmic hybrids (or cybrids) are stable and maternally inherited. Analysis with restriction endonuclease has shown that only one or the other parental chloroplast DNA is present in the progeny of the cybrids2. We report here, however, that the mitochondrial (mt) DNAs of cybrids are different from those of the parents and from the mixture of the two. The new DNAs result from mitochondrial recombination.

373 citations


Journal ArticleDOI
TL;DR: The techniques of microspores and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A1 plant lines and of several hundred protoplasts derived plantlets has become possible.
Abstract: The techniques of microspore and protoplast regeneration starting from dihaploid Solanum tuberosum plants has been improved to such an extent that the production of more than 2000 microspore derived A1 plant lines and of several hundred protoplast derived plantlets has become possible. Further, from the dihaploid Solanum species S. phureja the regeneration of microspores to plants, and from the species S. infundibuliforme, S. sparsipilum and S. tarijense the regeneration of protoplasts to calluses, has been achieved. The plants descending from the two single cell culture systems are compared with reference to phenotypic markers and economic qualities. Some principles characteristic for either microspore or protoplast derived plants are examined and their significance is discussed. The results are compiled into an extended analytical synthetic breeding scheme based on a stepwise reduction of the autotetraploid to the monohaploid level and a subsequent controlled combination to a new synthetic completely heterozygous tetraploid potato.

226 citations


Journal ArticleDOI
TL;DR: The protoplast Fusion and the Hybridization of Fungal Species and Transformation in Fungi by Using Protoplasts by using Enzymatic and Nonenzymatic methods are studied.
Abstract: INTRODUCTION 21 ISOLATION OF PROTOPLASTS AND THEIR P ROPERTIES 22 Enzymatic Methods for Protoplast Isolation 22 Nonenzymatic Procedures for Protoplast Release 26 Properties of Protopiasts 27 WALL REGENERATION AND REVERSION OF PROTOPLASTS 28 Frequency of Protoplast Reversion 28 Morphology of Protoplast Reversion 29 Ultrastructure and Composition of the Regenerated Wall ..... 30 Biochemical Aspects oj Wall Biogenesis 31 PROTOPLAST FUSION AND GENETIC MANIPULATION 33 Protoplast Fusion and the Hybridization of Fungal Species 33 Transformation in Fungi by Using Protoplasts . .. ........ 36 CONCLUDING REMARKS 36

187 citations


Journal ArticleDOI
TL;DR: Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed.
Abstract: Routine procedures for the isolation of large numbers of protoplasts from an established cell culture of Zea mays and for the induction of sustained divisions leading to secondary cell cultures have been developed. The critical factors seem to be associated with neither specific enzymatic conditions for the isolation nor specific culture conditions for the protoplasts but with the ‘quality’ of the culture used for protoplast isolation.

172 citations


Journal ArticleDOI
TL;DR: Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases.
Abstract: Lecithin and lecithin/cholesterol liposomes formed in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases. The sequestered DNA molecules are efficiently protected against exogenous deoxyribonuclease action although nicking and linearization of circular DNA can be observed. The size of these liposomes ranges from approximately 0.5 to 7.5 mu with an average of 2.5--4 mu. DNA filled liposomes strongly interact with plant protoplasts under conditions inducing protoplast fusion. Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei.

95 citations


Journal ArticleDOI
TL;DR: Intergeneric gene transfer by fusion of protoplasts from dividing and mitotically inactive somatic cells is discussed as a possible method in genetic manipulation of higher plants.

89 citations


Journal ArticleDOI
TL;DR: Nicotine was shown to be associated with mature vacuoles isolated from protoplasts of Nicotiana rustica, and the vacuolar preparations also contained high levels of acid phosphatase, ATPase, and approximately 30% of the soluble protoplastic protein.
Abstract: Nicotine was shown to be associated with mature vacuoles isolated from protoplasts of Nicotiana rustica. The vacuolar preparations also contained high levels of acid phosphatase, ATPase, and approximately 30% of the soluble protoplastic protein. The contamination of the vacuolar isolate by chlorophyll, succinate dehydrogenase, and NADPH cytochrome c reductase (markers for chloroplasts, mitochondria, and endoplasmic reticulum) was low. The enzymic activity associated with the vacuoles was not due to the exogenously supplied digestive enzymes used in the preparation of the protoplast. The relatively easy isolation of tobacco vacuoles makes this an excellent system for biochemical investigations of the vacuole.

85 citations


Journal ArticleDOI
TL;DR: Results concerning the analysis and genesis of these plants are used to discuss the question whether such monster plants are of any importance.
Abstract: After fusion of somatic cells (isolated protoplasts) of Arabidopsis thaliana and Brassica campestris, plants could be regenerated in which genetic material from parents of taxonomically different tribes is combined. Between these regenerants asymmetric hybrids have also been obtained, in which one parental genome is represented by reduced chromosome numbers. Results concerning the analysis and genesis of these plants are used to discuss the question whether such monster plants are of any importance.

81 citations


Journal ArticleDOI
TL;DR: It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population, and fusion events are always adequate to produce substantially more potential recombinants than are registered.
Abstract: Bacterial protoplast fusion, induced by polyethylene glycol, has been made more regular and convenient by further specification and improvement of various steps in the previously used procedure. These have made it possible to obtain regularly 100% regeneration of Bacillus subtilis cells from protoplasts before treatment with polyethylene glycol and yields of 10 to 75% from polyethylene glycol-treated protoplasts. Genetic recombination frequencies do not increase correspondingly. Also, when regeneration is reduced by various experimental conditions, recombination does not decrease in proportion. It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population. Kinetic studies with varying individual parental or total protoplast concentrations strongly indicate that protoplast collision and contact is not the limiting factor determining the number of genetic recombinants obtained. Recombination approximates a linear, rather than quadratic, function of the total or of the majority protoplast population present, from which it is concluded that fusion events are always adequate to produce substantially more potential recombinants than are registered. The strong effect of the majority/minority ratio upon the number of minority cells that become recombinant is independent of which parent is in excess. This shows in a direct and physiological way that both parents are equivalent partners in their genetic contributions.

74 citations


Journal ArticleDOI
TL;DR: Callus and regenerated plants (thalli) from protoplasts were obtained by gradually reducing the osmolarity in the medium and subsequently culturing regenerated cells on an inorganic phytohormone-free medium.

Journal ArticleDOI
TL;DR: The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.
Abstract: Summary: The optimum concentration of polyethylene glycol 1000 (PEG) for the production of recombinants through protoplast fusion in Streptomyces coelicolor was about 50% (w/v). The addition of 14% (v/v) dimethyl sulphoxide to the fusion mixture enhanced recombination frequencies, but only at sub-optimal PEG concentrations. After treatment of protoplasts with 50% PEG for 1 min, the frequency of recombinants in a multi-factor ‘cross’ sometimes exceeded 20% of the total progeny. The frequency of recombinants in the progeny could be significantly enhanced by ultraviolet irradiation of the parental protoplast suspensions immediately before fusion.

Journal ArticleDOI
TL;DR: Nicotiana sylvestris cell lines resistant to the amino acid analogues S-2-aminoethyl-cysteine (AECR), or 5-methyl-tryptophan (5MTR), were isolated in suspension culture and determined as hybrids on the basis of resistance level, chromosome number, and chlorophyll content.
Abstract: Nicotiana sylvestris cell lines resistant to the amino acid analogues S-2-aminoethyl-cysteine (AECR), or 5-methyl-tryptophan (5MTR), were isolated in suspension culture. Assuming these resistances to be dominant, we have attempted to determine if such variant cell lines can be used to select double resistant somatic cell hybrids. A total of 1.8 × 104 control calli from mixed AECR and 5MTR protoplasts, and AECR and 5MTR homokaryotic fusions were placed on double analogue selection, but none survived. Eight somatic hybrid calli (0.8%), able to grow without inhibition on the double analogue selection medium, were obtained after AECR + 5MTR protoplast fusion. These were further determined as hybrids on the basis of resistance level, chromosome number, and chlorophyll content, all characteristics differing in the parental cell lines.

Journal ArticleDOI
TL;DR: The high regeneration capacity of H. muticus in culture suggests it to be a good system for Studies in morphogenesis and possibly for studies in genetic modification and secondary product biosynthesis.

Journal ArticleDOI
TL;DR: Protoplasts cultured on a fabric tissue support saturated with a liquid nutrient medium containing a high concentration (10 mM) of glutamine were capable of undergoing rapid cell division leading to colony formation.

Journal ArticleDOI
01 Dec 1979-Planta
TL;DR: Protoplasts have been obtained from vegetative thallus of the green seaweed Enteromorpha following enzymic digestion with driselase and pectinase and measurements of O2 uptake and evolution were taken.
Abstract: Protoplasts have been obtained from vegetative thallus of the green seaweed Enteromorpha following enzymic digestion with driselase and pectinase. The viability of purified protoplast fractions was assessed by staining and measurements of O2 uptake and evolution.

Journal ArticleDOI
01 Mar 1979-Nature
TL;DR: The isolation of stable fusion products obtained from cell wall haploid mutants of Chlamydomonas reinhardi having the same mating type (mt+) and bearing genetic markers of auxotrophy are reported.
Abstract: SOMATIC FUSION has recently been used to isolate hybrids from auxotrophic mutants of fungi1,2, yeast3 and mosses4,5. These fusions were performed by using polyethylene glycol (PEG) and/or CaCl2 solutions on protoplasts which were prepared from cells of tissues treated with enzyme mixtures to dissolve the cell wall. In the unicellular green alga Chlamydomonas mutant strains unable to make a cell wall have been isolated6,7. Such mutants, similar to plant protoplasts prepared by enzymatic digestion, can be easily grown in defined liquid or agar media8. However, they differ from true protoplasts in that they do not burst in hypotonic medium or even in distilled water, indicating that some mechanism of osmoregulation is present6. It was thus of interest to determine whether these cell wall mutants could be used as plant protoplasts in fusion experiments. We report here the isolation of stable fusion products obtained from cell wall haploid mutants of Chlamydomonas reinhardi having the same mating type (mt+) and bearing genetic markers of auxotrophy.

Journal ArticleDOI
TL;DR: Protoplasts were isolated from cotyledons of pine and exhibited a high frequency of spontaneous fusion, which was reduced by overnight incubation in low concentration of enzymes, which resulted in sustained division of the regenerated cells.

Journal ArticleDOI
TL;DR: Cytology of callus, cell suspension cultures, and regenerated plants indicated a strong prevalence of polyploidization.

Journal ArticleDOI
TL;DR: Tobacco mesophyll protoplasts were successfully inoculated with tobacco necrotic dwarf virus (TNDV), a virus closely related to potato leafroll virus that should be greatly facilitated by using the mesophyLL protoplast system for studies on the replication of aphidborne, phloem-limited viruses.
Abstract: Summary Tobacco mesophyll protoplasts were successfully inoculated with tobacco necrotic dwarf virus (TNDV), a virus closely related to potato leafroll virus. The optimum conditions for infection as assessed by staining with fluorescent antibody to virus particles were 1 µg/ml TNDV, 1 µg/ml poly-l-ornithine, 0.025 m-phosphate buffer, pH 5.4, and 1 to 2 × 105/ml protoplasts: this combination resulted in 70 to 80% infection. Accumulation of virus antigen was first detected 12 h after inoculation, and both the number of fluorescing protoplasts and intensity of fluorescence increased markedly during the following 20 h. Virus antigen was distributed throughout the cytoplasm. Electron microscopy of thin sections revealed groups of virus-like particles in the cytoplasm and a few similar particles in the nucleus. Virus yield was estimated to be 7.8 × 105 particles per infected protoplast at 30 h after inoculation. Accumulation of virus antigen was completely inhibited by cycloheximide, but was not affected by actinomycin D or chloramphenicol, when they were applied 2 h after inoculation. Studies on the replication of aphidborne, phloem-limited viruses should be greatly facilitated by using the mesophyll protoplast system.

Posted ContentDOI
TL;DR: Topics include plant propagating and hybridizing; cytogenetical techniques; organ, tissue, cell and protoplast culture; protoplas fusion and organelle transfer; selection of biochemical cell varients; virology; disease resistance; and bacterial hypersensitivity.
Abstract: Background information and procedures for using the genus Nicotiana as experimental subjects are presented. Each chapter first surveys a topic, then, in detail, presents the associated experimental techniques .. The topics include plant propagating and hybridizing; cytogenetical techniques; organ, tissue, cell and protoplast culture; protoplast fusion and organelle transfer; selection of biochemical cell varients; virology; disease resistance; and bacterial hypersensitivity. In addition, a genetical overview is given of the genus.

Journal ArticleDOI
TL;DR: A method of protoplast isolation and culture from in vitro grown haploid Nicotiana sylvestris plants has been developed for application to mutant research and the effect of increasing doses of gamma rays from "Co on plating efficiency was studied.

Journal ArticleDOI
TL;DR: The findings from these experiments suggest that a high degree of chromosomal homology may exist between Aspergillus nidulans and As pergillus rugulosus, and that the two parents are closely related taxonomically.
Abstract: Interspecific hybrids produced by polyethylene glycol induced fusion of protoplasts from auxotrophic mutants of Aspergillus nidulans and Aspergillus rugulosus were grown in the presence of the recombinogens benomyl and chloral hydrate to stimulate segregation. The A. nidulans parental strains used had a known genetic marker in each linkage group. Hybrids grown on complete medium containing benomyl yielded more segregants. Analysis of the segregants showed that the distribution of A. nidulans linkage groups was random. No specific linkage group appeared in all the segregants. The two parents are closely related taxonomically and the findings from these experiments suggest that a high degree of chromosomal homology may exist between them.

Journal ArticleDOI
TL;DR: Protoplasts of auxotrophic mutants isolated from Schizosaccharomyces pombe and octosporus were fused, and their asymmetric segregation indicated thatSchizosACCharomyces octospora might be dominant in the fused cells.
Abstract: Protoplasts of auxotrophic mutants isolated fromSchizosaccharomyces pombe andSchizosaccharomyces octosporus were fused. The fusion products were capable of complementation and growth on minimal medium, but the morphological observations suggested a disturbed physiological balance, which was reflected in low viability, osmotic sensitivity, and the formation of incomplete cell wall. Their asymmetric segregation indicated thatSchizosaccharomyces octosporus might be dominant in the fused cells.

Journal ArticleDOI
TL;DR: The results demonstrate that miniprotoplasts, which are characterized by a dense cytoplasm free from large vacuoles, are favourable material for fusion experiments and that fusion using minipra toplasts is an alternative way of transferring nuclear material to both nuclear transplantation and protoplast fusion.

Journal ArticleDOI
TL;DR: Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer, and 2.0 M KCl; the possible association of the cryptic enzyme with periplasmic bodies is discussed.
Abstract: Cells of the osmotolerant yeast Saccharomyces rouxii were transformed to protoplasts in good yield (85%) by digesting cell walls with snail-gut enzyme in the presence of 10 mM dithioerythritol, 0.1 M sodium phosphate buffer (pH 6.8), and 2.0 M KCl. The requirement for 2.0 M KCl compares with that for S. bisporus var. mellis (another osmotolerant species) and contrasts with the 0.3 to 0.8 M KCl concentrations used in the preparation of most yeast protoplasts. Short digestions (60 min or less) produced mostly spheroplasts; longer incubations (90 min or more) yielded mostly protoplasts as judged by electron micrographs. These protoplasts could be transferred to 1.0 M KCl or 2.0 M sorbitol without lysing, but lysis was pronounced in 0.5 M KCl or 1.0 M mannitol and complete in 0.02 M KCl. Protoplasts were separated from isolated cell wall remnants and debris by centrifugation on a linear gradient of Ficoll 400 (35 to 17.5%, wt/vol) containing 2.0 M KCl. Both crude and fractionated protoplast preparations contained vesicles which were identified with the periplasmic bodies of whole cells. Some of the periplasmic bodies were connected to protoplasts by fine pedicels; others appeared free. Independent degeneracy of periplasmic bodies was occasionally observed. beta-Fructofuranosidase (EC 3.2.1.26) activity is cryptic (physically) in cells of S. rouxii in contrast to the expressed enzyme (periplasmic space) of other Saccharomyces species. This enzyme remains cryptic in protoplast preparations of S. rouxii but is expressed upon lysis. The same specific activities were found per unit cell or protoplast. The possible association of the cryptic enzyme with periplasmic bodies is discussed.

Journal ArticleDOI
TL;DR: All of the mature flowering plants showed to be perfectly somatic hybrid, and the shape of the leaves and flowers indicated that many somatic hybrids were of an intermediate characteristic between N. tabacum and N. glutinosa.
Abstract: Mesophyll protoplasts of Nicotiana tabacum. cv. Bright Yellow mutant Aurea and N. glutinosa and of N. tabacum. cv. Burley 21 and N. alata were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polycthylcne glycol. When the fused protoplasts were platcd on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the cell division could be seen beginning on the 14th day of culture in the combination of N. tabacum and N. glutinosa, whereas in that of N. tabacum and N. alata 10th day of culture. Cell division process took place over again after that. After one month culture, almost all of survived protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were placed on an agar medium containing 3.0 mg/l l-Naphthalenacetic acid and 1.0 mg/l 6-Benzylaminopurine in order to induce callus formation. On the medium, they grew vigorously and formed calli of 2 cm in diametcr after 2 months culture. However, it was too difficult to select the hybrid callus. They were trans-fered to a modified LINSMAIER and SKOOG medium containing 1.0 mg/l kinetine and 1.0 mg/l indoleacetic acid. After one month of culture on this mcdium, the hybrid calli, showing greenish white with a compact appearance werc selectcd in contrast to the parental type calli tinged with a white or green color. Thirty-three somatic hybrid calli of the combination of N. tabacum and N. glutinosa and 2 calli of the combination of N. tabacum and N. alata were obtained. Many plantlets were differentiated from these calli when transfered to enriching zeatin. All of the mature flowering plants showed to be perfectly somatic hybrid, and the shape of the leaves and flowers indicated that many somatic hybrids were of an intermediate characteristic between N. tabacum and N. glutinosa and between N. tabacum and N. alata. A few plants had a strong resemblance to a parent or a scxual hybrid. The plant height of the many somatic hybrids were higher than that of the parental plants, and the male sterility of the greater part of the hybrid plants was visible under investigation, and it was possible to back cross. They were highly resistant to the tobacco mosaic virus. The chromosomc number of the somatic hybrids of the combination of N. tabacum and N. glutinosa was determined: fivc were amphidiploid, twenty-eight showed aneuploid with 50 to 88 chromosomes. On the other hand, thc chromosome number of the somatic hybrids of the combination of N. tabacum and N. alata also was determined : one was amphidiploid and the other aneuploid.

Journal ArticleDOI
01 Jan 1979-Planta
TL;DR: A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured.
Abstract: A mutant of Chlamydomonas reinhardii which lacks a cell wall was fused with Daucus carota protoplasts using polyethylene glycol and the resulting fusion products were cultured. Fusion involved integration of Chlamydomonas and carrot plasma membranes and the release of algal organelles into the carrot cytoplasm. Chlamydomonas basal bodies, nuclei and chloroplasts were frequently observed in the fusion products. Cultured fusion products regenerated cell walls and divided; most Chlamydomonas organelles degenerated during culture but chloroplasts were still recognizable in the carrot cytoplasm after 10.

Journal ArticleDOI
TL;DR: Evidence is presented that fusion occurs in two steps, one polyethylene glycol dependent, the other energy requiring, the bacterial growth medium affects the ability of the protoplasts to fuse and to regenerate a cell wall.
Abstract: In previous studies of bacterial protoplast fusion, only the frequencies of cell wall regeneration and of bacterial recombination were determined. In this work the frequency of the heterozygous fusion products is measured by prophage complementation. Two multiply marked nonsuppressing strains of Bacillus subtilis, each lysogenic for a different Sus mutant of the phage phi 105, were induced by mitomycin C, protoplasted, fused, and, after dilution in hypertonic broth, incubated until plating with phi 105-sensitive indicator bacteria. When cell lysis was avoided, the frequency of the heterozygous fused cells could be determined from the number of infectious centers produced. The very high frequencies observed are in good agreement with those determined directly, with nonlysogenic strains, by electron microscopic examination of the fused protoplasts (C. Frehel, A. M. Lheritier, C. Sanchez-Rivas, and P. Schaeffer, J. Bacteriol. 137:1354--1361, 1979). Evidence is presented that fusion occurs in two steps, one polyethylene glycol dependent, the other energy requiring. The bacterial growth medium affects the ability of the protoplasts to fuse and to regenerate a cell wall. When experiments using different growth media were compared, an inverse relationship between these abilities was observed, and a direct relationship appeared between the heterozygotes (corrected for wall regeneration) and the recombinant bacteria that were found.

Journal ArticleDOI
TL;DR: After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered.
Abstract: When protoplasts derived from sporulating cells of Bacillus subtilis were fused by exposure to polyethylene glycol (PEG) and fixed immediately thereafter, protoplasts with two enclosed prespores could be seen by electron microscope The number of fusion events was greatly increased, and multiply fused protoplasts appeared, when the PEG-treated suspension was diluted in hypertonic broth and reincubated before fixation This post-PEG incubation effect is taken to indicate a fusion mechanism of two steps: a short, PEG-dependent step of membrane activation, followed by a slow, metabolism-requiring step completing fusion When prespore-bearing protoplasts from two genetically different strains were mixed and fused, the extent of fusion could also be followed by counting clones of recombinant bacteria Maximal from the start, their number (1% of each parent type protoplast present) was unaffected by post-PEG incubation Fusion in this case is apparently completed after plating on the wall-regeneration medium After optimal post-PEG incubation, the majority of the protoplasts were seen to participate in fusion, and the cytological fusion observed, corrected for wall-regeneration frequency, accounted quantitatively for the prototrophic bacteria eventually recovered These results are in good agreement with those obtained independently by Sanchez-Rivas and Garro (J Bacteriol 137:1340--1345, 1979)