Showing papers on "Protoplast published in 1983"

Transformation of intact yeast cells treated with alkali cations.

Abstract: Intact yeast cells treated with alkali cations took up plasmid DNA. Li+, Cs+, Rb+, K+, and Na+ were effective in inducing competence. Conditions for the transformation of Saccharomyces cerevisiae D13-1A with plasmid YRp7 were studied in detail with CsCl. The optimum incubation time was 1 h, and the optimum cell concentration was 5 x 10(7) cells per ml. The optimum concentration of Cs+ was 1.0 M. Transformation efficiency increased with increasing concentrations of plasmid DNA. Polyethylene glycol was absolutely required. Heat pulse and various polyamines or basic proteins stimulated the uptake of plasmid DNA. Besides circular DNA, linear plasmid DNA was also taken up by Cs+-treated yeast cells, although the uptake efficiency was considerably reduced. The transformation efficiency with Cs+ or Li+ was comparable with that of conventional protoplast methods for a plasmid containing ars1, although not for plasmids containing a 2 microns origin replication. Images

Plasmid complements of Streptococcus lactis NCDO 712 and other lactic streptococci after protoplast-induced curing.

Abstract: The production and regeneration of bacterial protoplasts promoted the loss of three different plasmid-specified traits in Streptococcus lactis subsp. diacetylactis strains. The loss of five different plasmids, including small multicopy molecules, was readily detected in Streptococcus lactis 712 by screening lysates of random protoplast regenerants on agarose gels. In this strain sequential rounds of protoplast regeneration were used to produce a plasmid-free strain and derivatives carrying only single molecules from the plasmid complement. During these experiments a 33-megadalton plasmid, pLP712, was found to encode genes for lactose and protein utilization. Only this plasmid was required for normal growth and acid production in milk; the remaining four plasmids appeared to be cryptic. Lactose-defective derivatives of a strain carrying only pLP712 were readily isolated. Although these derivatives included instances of plasmid loss, deletions of pLP712 were frequently found. Many different deleted derivatives of pLP712, including some in which the lactose or protein utilization determinant or both were lost, were isolated. The molecular instability of pLP712 largely accounted for previous observations of plasmid complements in S. lactis 712 after lactose determinant curing or transfer by conjugation and transduction. Curing of cryptic molecules from multiple plasmid complements by protoplast regeneration may prove to be generally valuable in lactic streptococci and other gram-positive species.

Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion

Abstract: Rapeseed plants have been regenerated after fusion between protoplasts bearing cytoplasms of different genera Cybrids combine, in a first experiment Brassica napus chloroplasts and a cytoplasmic male sterility (cms) trait coming from Raphanus sativus, in a second experiment chloroplasts of a triazine resistant Brassica campestris and cms trait from Raphanus sativus Transfer of chloroplasts has been confirmed by restriction cp DNA analysis and two dimensional thylakoid protein electrophoresis These plants may be very useful for Brassica hybrid seed production

Agarose plating and a bead type culture technique enable and stimulate development of protoplast-derived colonies in a number of plant species

Abstract: Two novel techniques improve division and colony formation from protoplasts: 1) Plating in agarose stimulates colony formation of protoplasts from a wide range of species Protoplasts from Nicotiana tabacum developed to colonies from lower initial population densities in agarose than in agar or liquid Protoplasts from Hyoscyamus muticus which do not divide in agar divided and formed colonies in agarose at higher efficiencies than in liquid medium 2) Culture of gel embedded protoplasts in large volumes of liquid medium on a gyrotatory shaker (‘bead culture’) further improved plating efficiencies in some species (eg Lycopersicon esculentum and Crepis capillaris) and enabled sustained proliferation of protoplasts which had not previously developed beyond the few cell colony stage (Brassica rapa and a mutator gene variety of Petunia hybrida)

Intergeneric cytoplasmic hybridization in cruciferae by protoplast fusion

Abstract: SummaryRapeseed plants have been regenerated after fusion between protoplasts bearing cytoplasms of different genera. Cybrids combine, in a first experiment Brassica napus chloroplasts and a cytoplasmic male sterility (cms) trait coming from Raphanus sativus, in a second experiment chloroplasts of a triazine resistant Brassica campestris and cms trait from Raphanus sativus. Transfer of chloroplasts has been confirmed by restriction cp DNA analysis and two dimensional thylakoïd protein electrophoresis. These plants may be very useful for Brassica hybrid seed production.

A process for the in-vitro transformation of plant protoplasts with plasmid dna

Abstract: Genetic properties of higher plants are transformed by incubating protoplasts of the plants together with plasmid DNA. The incubation is effected in the presence of polyethylene glycol and calcium ions. Subsequently--during after-incubation--the calcium ion concentration in the incubation medium is gradually increased and at the same time the polyethylene glycol concentration is decreased. The resulting aggregates are separated, grown further separately, and examined for modification of their genetic properties.

A Pathway for Photosynthetic Carbon Flow to Mannitol in Celery Leaves: Activity and Localization of Key Enzymes

Abstract: In the polyol producing plant, celery ( Apium graveolens L.), mannitol is a major photosynthetic product and a form in which carbohydrate is translocated. Measurements of whole leaf extracts of celery indicated substantial activity of the following enzymes: mannose-6-P reductase, mannose-6-P isomerase, mannitol-1-P phosphatase, and nonreversible glyceraldehyde-3-P dehydrogenase. The activities of these enzymes were either undetectable or very low in the nonpolyol producing plants, Secale cereale L. (rye) and Vigna mungo (L.) Hepper (black gram). Mesophyll protoplasts were enzymically isolated from celery leaves, broken with a Yeda press and the intracellular localization of the above enzymes for mannitol synthesis studied following differential and/or sucrose density gradient centrifugation of the protoplast extract. These data suggested the enzymes involved in mannitol synthesis are exclusively localized in the cytoplasm. Ninety-five to 100% of the activity of these enzymes, along with the cytoplasmic marker enzyme phosphoenolpyruvate carboxylase, was found in the cytosolic fraction. We propose the pathway of photosynthetic carbon flow from triose-P to mannitol in celery occurs via fructose-6-P, mannose-6-P, and mannitol-1-P; these final reactions being catalyzed by the cytoplasmic enzymes, mannose-6-P isomerase, NADPH-dependent mannose-6-P reductase, and mannitol-1-P phosphatase, respectively. The requirement for NADPH may be met via the cytoplasmically located NADP-linked nonreversible glyceraldehyde-3-P dehydrogenase.

Phenotypic variation and ploidy level of plants regenerated from protoplasts of tetraploid potato (Solanum tuberosum L. cv. 'Bintje').

Abstract: A wide range of phenotypic variation occurred among protoplast — derived plants of tetraploid potato cultivar ‘Bintje’. The variant plants had alterations in growth and vigour, and in leaf and stem characteristics. The results suggest that the altered morphologies are caused predominantly by changes in ploidy levels. Some alterations could be attributed typically to octoploidy and aneuploidy. The occurrence of mixoploidy indicates that at least part of the observed variation arose during culture stage. The exogeneous cytokinin or auxin level and their combination during in vitro phase influenced the frequency of the variants observed. The origin of variation is discussed.

Confirmation of protoplast fusion-derived linkages in Staphylococcus aureus by transformation with protoplast DNA.

Abstract: Transformation provided definitive evidence for linkage between tyrB282::Tn551 ermB321 and omega (Chr::Tn551)34, and thus between the separate large linkage groups containing these markers, in Staphylococcus aureus NCTC 8325 Transformation also defined the chromosomal loci for the purC193::Tn551 and omega (Chr::Tn551)42 markers and the linkage of a tetracycline resistance marker (tet-3490) with a fusidic acid resistance marker (fus-149) The use of DNA isolated from protoplasts under conditions that reduced hydrodynamic shear greatly facilitated the demonstration of most of these linkages These results provide direct evidence confirming several of the linkages predicted by a microcomputer-assisted protoplast fusion analysis in a previous study (M L Stahl and P A Pattee, J Bacteriol 154:395-405, 1983); those markers whose predicted linkages were not confirmed by transformation are probably separated by chromosomal distances that exceed the limits of detection by transformation, even with protoplast DNA

Regeneration of Plants from Single Cells of Cereals and Grasses

Abstract: A variety of novel methods have been developed in recent years for the genetic modification of plant cells These include the techniques of protoplast and cytoplast fusion, uptake of cell organelles, and molecular techniques involving the uptake and integration of foreign DNA Each of these aspects is adequately-covered elsewhere in this volume

Cell wall composition and protoplast regeneration in Candida albicans.

Abstract: The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.

Cybrids containing mixed and sorted-out chloroplasts following interspecific somatic fusions in Nicotiana

Abstract: Streptomycin resistance was transferred by “donor-recipient” protoplast fusion from Nicotiana tabacum (SR-1) protoplasts into Nicotiana tabacum (cytoplasmic male sterile — Line 92) protoplasts in one case and into Nicotiana sylvestris protoplasts in another. It is demonstrated that streptomycin resistance (SR-1) is a chloroplast marker which segregates independently from a mitochondrial marker. In the fusion experiment where Nicotiana tabacum (Line 92) was the recipient, microcalli were plated in the presence of streptomycin. In this case, chloroplast sorting out occurred at a stage preceeding plant regeneration, producing stable streptomycin resistant cybrids. In the fusion whre Nicotiana sylvestris was the recipient, no direct selection for streptomycin resistance was performed. In this case chloroplast sorting out was incomplete, thus producing cybrid plants with a mixed chloroplast population. In some plants, sorting out of streptomycin resistant and sensitive chloroplasts was still apparent in the second generation progeny.

A heteroplasmic state induced by protoplast fusion is a necessary condition for detecting rearrangements in Nicotiana mitochondrial DNA.

Abstract: Mitochondrial DNA (mtDNA) restriction patterns were studied in mutant, cybrid and somatic hybrid plants regenerated from Nicotiana protoplasts. It has been shown that neither components of the culture media used for protoplast culture and plant regeneration, nor the antibiotics streptomycin and lincomycin used for the mutant selection induced alterations in the mtDNA. No rearrangements were detected in the mtDNA of plants derived from homoplasmic fusions where the mtDNA of the parents was identical as judged by mtDNA restriction patterns. There were rearrangements, however, in the mtDNA of each of the cybrid plants derived from heteroplasmic fusions. Restriction patterns generated by BamHI and SalI restriction endonucleases were different from those of both parents, and were composed of parental and non-parental fragments.

Low density growth of cells derived from Nicotiana and Petunia protoplasts: Influence of the source of protoplasts and comparison of the growth‐promoting activity of various auxins

Abstract: Nicotiana tabacum (cv. Xanthi), N. plumbaginifolia Viviani, N. sylvestris Speg. and Comes and Petunia axillaris× (P. axillaris × P. hybrida) (cv. Mitchell) mesophyll protoplast-derived cells were able to grow at low densities in chemically-defined media. Protoplasts of different origins (mesophyll, epidermis, pith, suspension culture) also gave rise to protoplast-derived cells that were able to grow at low densities. When auxin requirements at low densities were compared for different sources of auxins, IAA was found to be efficient in the same range of concentrations as NAA. This was unexpected since tobacco mesophyll protoplasts cannot be induced to divide when plated at high density in the presence of IAA. Optimal 2,4-D concentrations for low density growth were higher and clearly pH-dependent. On the contrary, picloram induced low density growth over a wide range of concentrations suggesting a distinct mechanism of action. These results confirm and extend previous observation on the tobacco mesophyll protoplast and show that the low-density growth technique has a potential use for the study of the action of phytohormones.

Improved protoplast culture and agarose media

Abstract: Media solidified with agarose resulted in higher plating efficiency of protoplasts than commonly used agar media. Improved culture efficiency was observed with mesophyll protoplasts of Nicotiana tabacum and N. plumbaginifolia and with protoplasts isolated from cell lines of Daucus carota, Hyoscyamus muticus and two lines of N. tabacum. The improvement with agarose was consistent over a wide cell density range and also for different media. The positive effect was not due to the lower temperature at which the protoplasts could be plated. Culture experiments with mixtures of different agar types generally gave intermediate division frequencies. There was no obvious effect on plating efficiency in experiments where diffusion was permitted between agar and other agar types.

Plant Regeneration from 'Trovita' Orange Protoplasts

Abstract: Protoplasts isolated from nucellar callus of 'Trovita' orange (Citrus sinensis Osbeck) wvere cultured in a MURASHIGE and TUCKER'S medium containing 0.15 M sucrose, 0.45 M glucose, and 0.6% agar, but lacking phytohormones. Colonies with 26.3% plating efficiency were observed after 6 weeks culture. When the protoplasts were cultured in the presence of exogenous phytohormones, reduction in colony formation was observed. Some colonies developed to green embryoids, which eventually formed plants.

Culture and selection of somatic hybrids using an auxotrophic cell line.

Abstract: Protoplast fusions between Nicotiana tabacum and N. paniculata and between N. tabacum and N. sylvestris were obtained by polyethylene glycol and Ca(NO3)2 treatment. The protoplasts of one parent originated from cell suspensions, while the protoplasts of the other originated from leaf mesophyll. The heterokaryons were detectable by their intermediate phenotype, namely the green chloroplasts from mesophyll and the dense cytoplasm from suspension cells. They were isolated with micropipettes immediately after fusion using a micromanipulator and were transferred into a protoplast suspension of an auxotrophic cell line serving as a nursery. This mutant is not able to utilize nitrate and had to be supplemented with amino acids. The somatic hybrids were selected by a stepwise reduction of the supplements, which caused the death of the mutant cell colonies, while the autotrophic somatic hybrids continued to grow. The hybrid character of the selected colonies was confirmed by isoenzyme investigations.

Improved culture ability of potato protoplasts by use of activated charcoal.

Abstract: Protoplasts were obtained from in vitro grown plants of Solanum tuberosum L. The protoplasts were cultured in X-plate petri dishes with the culture medium joined to a reservoirmedium. When activated charcoal was added to the reservoirmedium the culture ability of the protoplasts was significantly increased. The effect of activated charcoal was mainly due to a less pronounced browning of the developing protoplasts and this technique might be of help in protoplast cultures where browning is a problem.

Genetic transfer systems in lactic acid bacteria.

Abstract: Gene transfer processes (transfuction, conjugation, protoplast fusion mediated exchange, transformation in protoplasts) in lactic acid bacteria are reviewed in this paper. Besides, the detailed molecular nature of lactose plasmids in the Streptococcus lactis C2, 712 and ML3 strain complex is discussed.

Transfer of cytoplasmic male sterility by selection for streptomycin resistance after protoplast fusion in Nicotiana

Abstract: Cytoplasmic male sterility (CMS) in Nicotiana is located on mitochondrial DNA (mtDNA) on which no selectable mutation has been isolated. The possibility of co-transfer of CMS and a selectable chloroplast trait, streptomycin resistance, was investigated by marker rescue from irradiated protoplasts, a method described from this laboratory (Menczel et al. 1982). As the source of the CMS factor, the Nicotiana tabacum St-R701 line was chosen. This line is male sterile due to replacement of its original cytoplasm by that of N. megalosiphon, and carries a cytoplasmic streptomycin resistance mutation (Umiel 1979). Following fusion of irradiated (lethal dose) St-R701 protoplasts with untreated N. plumbaginifolia protoplasts, streptomycin-resistant clones were selected. Regenerated plants representing 27 clones were studied in detail. These plants have a N. plumbaginifolia nuclear genome and the resistance factor from the St-R701 parent. In each clone transfer of streptomycin resistance resulted in the transfer of St-R701 chloroplasts suggesting the involvement of chloroplast DNA in determining streptomycin resistance. All regenerated streptomycin resistant N. plumbaginifolia plants were male sterile. In plants from three clones, in addition to male sterile flowers, a few male fertile flowers were obtained.

Agricultural Applications of Plant Protoplast Fusion

Abstract: Plant protoplasts can be fused and the fusion products cultured to produce somatic hybrid plants. This technique has been used to produce germplasm previously unavailable to the plant breeder. Several researchers have emphasized production of hybrids between distantly related, sexually incompatible species, but many of these hybrids are sterile, precluding incorporation into a breeding program. Hence, to transfer traits such as disease and herbicide resistance, emphasis has shifted to production of hybrids between more closely related species. Novel variation has been observed in such somatic hybrids due to segregation of mixed organelles, cytoplasmic and nuclear gene recombination, and somaclonal variation. The unique gene combinations made possible by protoplast fusion ensure that new plant varieties will soon be derived from somatic hybridization.

Genetic variability induced in Nicotiana sylvestris by protoplast culture.

Abstract: Plants were regenerated from axenic plantlets by mesophyll protoplast culture, without mutagenic treatment. Two different lines of Nicotiana sylvestris were used: an original line, and a diploid androgenetic line derived from it. The regenerated plants were either diploid and phenotypically similar to their respective protoplast source line, or tetraploid. Genetic studies carried out on several diploid regenerated plants revealed genetic variability. Eight of 13 selfed progenies of plants regenerated from the original line, and 1 of 8 selfed progenies of plants regenerated from the androgenetic line, produced new mutant phenotypes never observed in the protoplast source lines. Two plants regenerated from the same protoplast-derived callus produced different mutations. Selfed progenies without a recognizable mutant phenotype were also different from their respective protoplast source line for quantitative characters; protoplast culture induced a depressive effect on the size of plants derived from protoplasts at younger and older stages of development. The origin of this depression and of the mutations is discussed.

Protoplast transformation of Staphylococcus carnosus by plasmid DNA

Abstract: Several coagulase-negative staphylococci were investigated for their ability to undergo polyethylene glycolinduced protoplast-transformation with plasmid DNA. Among this group of bacteria it was found that S. carnosus TM300 was superior in this respect. Several plasmids isolated from different species were readily transformed with an efficiency ranging between 4×106 to 2×103 transformants per μg plasmid DNA. Since S. carnosus is apathogenic and an important organism in food technology, this organism could be a suitable gram-positive strain for molecular cloning.

Genetic construction of yeast strains for high ethanol production

Abstract: To improve ethanol production capability, a flocculent yeast,Saccharomyces cerevisiae TJ1, was hybridized with other high-alcohol producing strains by protoplast fusion. A fusant strain of strain TJ1 hybridized with an Awamori-brewing strain, N1, produced 12.4 (w/v)% of ethanol even at 40°C. The fusant showed good flocculence and good sedimentation. In a repeated batch culture with cell reuse at 37°C, an average productivity of 13 g/l·h was attained.

Plastid number and plastid structural changes associated with tobacco mesophyll protoplast culture and plant regeneration.

Abstract: Mesophyll protoplasts were isolated from Nicotiana tabacum L cv Xanthi, and cell-colony formation induced in liquid culture The plastid changes associated with the morphogenetic sequence from mesophyll protoplast to whole plant were examined Minor ultrastructural changes in the plastids were evident after 1 d of culture, but by 8 d (four-to-eight-cell stage) the plastids were small, there was much less thylakoid membrane appression, and many prominent plastoglobuli were also present Plastid-division figures were evident at this point of time and it was common to find plastids clustered around the nucleus A typical proplastid was the dominant plastid type in the cultured cells from about 11 d until about five weeks when large amyloplasts and pregranal plastids were observed Normally structured chloroplasts were present in the regenerated plant There was no plastid division until the four-cell stage, with plastid numbers per cell approximately halving at each cell division, then stabilising around 12 per cell during cell-colony development, a number typical of meristematic cells Though nucleoids were always present, their numbers in the plastids were reduced by the eight-cell stage