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Showing papers on "Protoplast published in 1987"


Journal ArticleDOI
TL;DR: This paper analyses numerous transformation parameters in a comparative study on SR1Nicotiana tabacum and N. plumbaginifolia, and reports on a simple chemical technique for very efficient protoplast transformation based on the synergistic interaction of MgCl2 and PEG.
Abstract: Direct gene transfer into plant protoplasts has been recently developed, and conditions for high frequency transformation of SR1 tobacco protoplasts established. In this paper we analyse numerous transformation parameters in a comparative study on SR1 Nicotiana tabacum and N. plumbaginifolia, and report on a simple chemical technique for very efficient protoplast transformation. It is based on the synergistic interaction of MgCl2 and PEG. The technique yielded up to 1400 transformants per 3×105 treated N. tabacum protoplasts (up to 4.8% of the survivors, late selected clones). Using N. plumbaginifolia, the frequencies were 10-fold lower, indicating that the ‘competence’ for transformation has a species-specific component.

539 citations


Journal ArticleDOI
01 Jan 1987-Nature
TL;DR: This work reports an alternative approach to transformation of cereal plants which does not involve tissue culture techniques and can be used with the agriculturally important cereals.
Abstract: The most successful method currently used to produce transgenic plants is based on the ability of Argobacterium tumefaciens to infect a number of higher plant species and transfer a defined DNA fragment (T-DNA) to the genome of the infected cells1. This procedure cannot be used with the agriculturally important cereals, although preliminary data have suggested2 infection of maize seedlings by A. tumefaciens strains. Purified exogenous DNA can be taken up, integrated and expressed in cells of a variety of plant species including some cereals following direct gene transfer into isolated protoplasts3–7. However, although it is possible to grow isolated cereal protoplast into unorganized tissue (calli)5,7,8, only rice protoplasts have so far been shown to regenerate mature plants9–11. Here we report an alternative approach to transformation of cereal plants which does not involve tissue culture techniques.

269 citations


Journal ArticleDOI
TL;DR: Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses, suggesting karyotypic stability and various plant phenotypes.
Abstract: Novel nurse culture methods have been developed for plant regeneration from protoplasts of rice (Oryza sativa). The nurse culture methods use the agarose-bead type culture in combination with actively growing nurse cells that are either in the liquid part of the culture or inside a culture plate insert placed in the centre of the dish. Protoplasts isolated from either primary seed calluses or suspension cultures of various callus origins, divided and formed colonies with a frequency of up to 10% depending on the protoplast source and the genotype. The presence of nurse cells was absolutely required for the induction of protoplast division. Plants were regenerated from protoplast-derived calluses of five tested cultivars with a frequency of 17%–50%. Close examination of the plant regeneration process suggested that plants are regenerated through somatic embryogenesis from protoplast-derived calluses. Over 300 protoplast-derived plants were transferred to either pots or the field and are being examined for karyotypic stability and various plant phenotypes.

265 citations


Journal ArticleDOI
TL;DR: Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding.
Abstract: Tomato genotypes superior in regenerating plants from protoplast and callus cultures were obtained by transferring regeneration capacity from Lycopersicon peruvianum into L. esculentum by classical breeding. The genetics of regeneration and callus growth have been studied in selfed and backcross progenies of a selected plant (MsK93) which has 25% L. peruvianum in its ancestry. Segregation data showed that the favourable cell culture traits of L. peruvianum are dominant. Regeneration capacity from established callus cultures was controlled by two dominant genes. Callus growth on primary expiants, callus growth of established cultures and shoot regeneration from explants had high heritabilities (0.47, 0.78, 0.87, respectively). Callus growth and regeneration capacity were not correlated within the populations studied.

172 citations


Journal ArticleDOI
TL;DR: An improved method is described which allows the storage of the protoplasts deep frozen (at − 70°C) for several months without affecting their transforming capability.
Abstract: Protoplast transformation is so far the only method to transfer plasmids into Staphylococcus carnosus. However, the preparation of new protoplasts for each transformation is time-consuming and the quality of the protoplasts may also vary each time. An improved method is described which allows the storage of the protoplasts deep frozen (at − 70°C) for several months without affecting their transforming capability. The possibility of keeping protoplasts competent for a long period opens several advantages: the transformation can be carried out immediately, the transformation procedure is reduced to only a couple of minutes, and the protoplasts of one batch have the same quality, yielding a more constant transformation frequency.

160 citations


Journal ArticleDOI
TL;DR: The availability of somatic plant protoplasts, which can be fused together and suitably cultured to produce somatic hybrid plants, is enabling the effects of such hybrid cytoplasms to be investigated in higher plants exhibiting maternal inheritance.
Abstract: In the majority of higher plants there is maternal inheritance of cytoplasmic organelles and, as a consequence, there are few opportunities for the study of the effects on plant phenotype of having cytoplasm initially containing organelles of both parents. Now the availability of somatic plant protoplasts, which can be fused together and suitably cultured to produce somatic hybrid plants, is enabling the effects of such hybrid cytoplasms to be investigated in higher plants exhibiting maternal inheritance. A very wide range of cytoplasmic genetic diversity, including mitochondrial and chloroplast recombinants, can be produced by such somatic hybridizations, and a theoretical model is presented to show the origins of this wide range of cytoplasmic diversity. Cybrids produced by such protoplast fusions have been shown to be of importance in plant breeding especially in relation to transfer of cytoplasmic male sterility and herbicide resistance. Protoplast fusion, including the fusion of gametic and somatic protoplasts, is also enabling the study of the inheritance of cytoplasmic controlled traits in higher plants. ORGANELLE GENETICS in an historical sense has

125 citations


Journal ArticleDOI
05 Jun 1987-Science
TL;DR: By means of direct interaction of cereal protoplast with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.
Abstract: Until recently, gene transfer in plants was achieved only by sexual hybridization. Now, in addition, plant genetic manipulation, with the use of both recombinant DNA and protoplast fusion technology, is being applied to an increasing range of plants. The soil bacterium Agrobacterium tumefaciens, with its associated plasmid, is used as a vector for introducing DNA into the genomes of dicotyledonous plants, but it has not proved suitable for cereals. Instead, the direct uptake of plasmid DNA into cereal protoplasts is being used for the transformation of cells in rice, wheat, and maize. Transformation efficiencies, in some cases, are becoming comparable to those obtained in dicotyledons with Agrobacterium. In rice it is now possible to regenerate efficiently whole plants from protoplasts, and this capability may soon be extended to the other cereals. By means of direct interaction of cereal protoplasts with plasmids, coupled with improved procedures for the regeneration of plants from their protoplasts, gene transfer in the cereals is becoming established at the frontiers of recombinant DNA technology.

100 citations


Journal ArticleDOI
TL;DR: Intergeneric hybrid plants were obtained through protoplast fusion between Brassica oleracea L. and Moricandia arvensis (L.)DC and the genetic regulation of photosynthetic systems in the hybrids were studied.

99 citations


Journal ArticleDOI
TL;DR: Physical mapping revealed recombination in a region which is not normally involved in the formation of subgenomic mtDNA circles in Brassica cybrids, and the role of treatments used to facilitate the recovery of cy hybrids, and of organelle compatibility in hybrid cytoplasm formation is discussed.
Abstract: Brassica cybrids were obtained after fusing protoplasts of fertile and cytoplasmic male sterile (CMS) B. napus lines carrying the original b. napus, and the Ogura Raphanus sativus cytoplasms, respectively. Iodoacetate treatment of the fertile line and X-irradiation of the CMS line prevented colony formation from the parental protoplasts. Colony formation, however, was obtained after protoplast fusion. Hybrid cytoplasm formation was studied in 0.5 g to 5.0 g calli grown from a fused protoplast after an estimated 19 to 22 cell divisions. Chloroplasts and mitochondria were identified in the calli by hybridizing appropriate DNA probes to total cellular DNA. Out of the 42 clones studied 37 were confirmed as cybrids. Chloroplast segregation was complete at the time of the study. Chloroplasts in all of the cybrid clones were found to derive from the fertile parent. Mitochondrial DNA (mtDNA) segregation was complete in some but not all of the clones. In the cybrids, mtDNA was different from the parental plants. Physical mapping revealed recombination in a region which is not normally involved in the formation of subgenomic mtDNA circles. The role of treatments used to facilitate the recovery of cybrids, and of organelle compatibility in hybrid cytoplasm formation is discussed.

94 citations


Journal ArticleDOI
TL;DR: An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed, showing the occurrence of multiple fusion and chromosome loss during the culture.
Abstract: An efficient procedure for obtaining somatic hybrids between B. oleracea and B. campestris has been developed. Hypocotyl protoplasts of B. oleracea were fused with mesophyll protoplasts from three different varieties of B. campestris by the polyethylene glycoldimethylsulfoxide method. The selection of somatic hybrids utilized the inactivation of B. oleracea protoplasts by iodoacetamide (IOA) and the low regeneration ability of B. campestris. The efficiency of recovery of somatic hybrids depended upon the IOA concentration, and when 15 mM IOA was used, 90% of the regenerated plants were found to be hybrid. The somatic hybrids were examined for i) leaf morphology, ii) leucine aminopeptidase (LAP) isozyme and iii) chromosome number. All the hybrids had intermediate leaf morphology and possessed LAP isozymes of both parental species. The chromosome analysis revealed a considerable variation in chromosome number of somatic hybrids, showing the occurrence of multiple fusion and chromosome loss during the culture. Some of the hybrids flowered and set seeds.

93 citations


Journal ArticleDOI
TL;DR: Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system.
Abstract: Factors influencing the transient expression of introduced foreign DNA in electroporated protoplasts and intact cells of sugar beet were determined by assaying for the activity of chloramphenicol acetyltransferase (CAT), using a rectangular pulse generating system. Extractable CAT activity depended upon 1) applied plasmid DNA concentration, 2) protoplast density, 3) the interaction between pulse field strength, duration, number, time interval between pulses and the resultant effect on culture viability, and 4) the physiological state of the protoplasts. Mesophyll protoplasts were more susceptible to damage by electroporation, and were more specific in their requirement for electroporations which allowed CAT expression, than were protoplasts derived from suspension culture cells. CAT activity was also demonstrated, at low levels, after electroporation of intact suspension culture cells, and could be increased by pectinase treatment of the cells before electroporation.

Journal ArticleDOI
TL;DR: Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion and Restriction enzyme analysis of chloroplast-DNA showed that either B.campestris or B. Oleracea chloroplasts were present in the somatic hybrid plants.
Abstract: Fertile somatic hybrids between Brassica campestris and B. oleracea have been produced by protoplast fusion. Fusion products were identified by their intermediate protoplast morphology. Heterokaryons were isolated either with micropipettes using a micromanipulator or by flow sorting. About 2% of the obtained calli differentiated to shoots. Of the shoots obtained from manually selected heterokaryons, 100% were true hybrids as confirmed by isozyme analysis while 87% of the flow sorted ones showed a hybrid pattern. Ploidy level of the hybrid plants was determined by chromosome counting and relative DNA-content analysis. The sum of the chromosome number (38) from the two fusion partners were found in 30% of the hybrids; 9% had fewer and 61% had more chromosomes. Pollen viability and seed set varied with ploidy level. Compared to natural B. napus, a pollen viability of 52%–93% and a fertility of 1%–40% was found for the somatic hybrids with normal chromosome number. Restriction enzyme analysis of chloroplast-DNA showed that either B. campestris or B. oleracea chloroplasts were present in the somatic hybrid plants. Of 11 hybrid plants 5 had the campestris and 6 had the oleracea type (1∶1 ratio).

Journal ArticleDOI
TL;DR: Electric field pulses caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency, and a higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplast.
Abstract: Electric field pulses, ranging from 250 to 2000 V and of 10 to 50 μsec duration, were assessed for their effect on the growth in culture of isolated protoplasts ofGlycine canescens, Prunus avium × pseudocerasus, Pyrus communis, Solanum dulcamara andSolanum viarum. Three successive voltage pulses between 250 and 1000 V caused a small decrease in protoplast viability, but promoted cell division and enhanced significantly the plating efficiency. A higher percentage of electro-pulsed protoplasts showed sustained growth in culture to the microcallus stage compared to untreated protoplasts. The rate of cell division was also stimulated in electro-treated protoplasts. These observations are discussed in relation to present knowledge of the effects of electrical treatments on plant and animal cells.

Journal ArticleDOI
TL;DR: Complementation of two metabolic deficiences — nitrate reductase and tryptophan synthase — was used to select for somatic fusion hybrids between tobacco and henbane with prior X-irradiation of one partner.
Abstract: Complementation of two metabolic deficiences — nitrate reductase and tryptophan synthase — was used to select for somatic fusion hybrids between tobacco (Nicotiana tabacum) and henbane (Hyoscyamus muticus) with prior X-irradiation of one partner. Using species specific, radioactively labelled DNA probes it could be shown that a) irradiation significantly reduced the amount of chromosomal DNA of the irradiated fusion partner in the somatic hybrid, b) irradiation with doses which completely inhibit protoplast division did not pevent transfer of substantial amounts of chromosomal DNA into the fusion hybrids (so called ‘cybrids’) and c) this method transfers functional nuclear genes together with the partial genome from the irradiated partner.

Journal ArticleDOI
TL;DR: Plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker, the latter of which showed the highest regeneration frequency.
Abstract: Conditions were established for efficient plant regeneration from four freshmarket cultivars of Lycopersicon esculentum. In order to increase the yield of viable protoplasts which are able to sustain cell divisions, the donor plants are preconditioned by incubation at 25°C in the dark for 18 hours, followed by a cold treatment at 4°C in the dark for the last 6 hours, prior to protoplast isolation. Browning of the dividing cell colonies can be prevented by culturing protoplasts in 100 μl droplets of low-melting agarose, surrounded by liquid medium. Alternatively, protoplasts can be cultured in liquid medium. In both procedures the plating efficiencies and percentage of shoot regeneration are increased, only when dilutions were performed with auxin-free culture medium. Shoot regeneration is obtained by using a two step procedure: initiation of greening of microcalli on a medium containing 0.2 M mannitol and 7.3 mM sucrose, which is followed by shoot development on a mannitol-free medium containing 0.5 M sucrose. In this way, plants can be regenerated within 3 months from the hybrid cultivars Bellina, Abunda, Sonatine and also from the true seedline Moneymaker. The latter one showed the highest regeneration frequency (30%).

Journal ArticleDOI
TL;DR: A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed and 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics.
Abstract: A modified protoplast isolation technique, applicable to a range of dihaploidSolanum tuberosum genotypes, has been developed. A combination of high calcium and high pH was used to fuse mesophyl protoplasts of dihaploidS. tuberosum (PDH40) and the diploid wild speciesS. brevidens. Large numbers of colonies were obtained after fusion and putative hybrids selected on the basis of phenotype from regenerated shoots. From these, 11 somatic hybrid plants have been identified by their isoenzyme patterns and morphologic characteristics. Four of these hybrids had the expected chromosome number of 48. The approach of mass culture after fusion followed by selection of hybrids from regenerated shoots and the application of somatic hybridization to potato breeding are discussed.

Journal ArticleDOI
01 Mar 1987-Planta
TL;DR: The results indicate that the double-mutant cell line established here will be able to serve as a universal hybridizer to select somatic hybrids after protoplast fusion with any other wild-type partner.
Abstract: If a double-mutant can be established with two markers that are dominant and counter-selectable, it can be serve as a universal hybridizer in selecting somatic hybrids exclusively after protoplast fusion with any other wild-type partner. Such a universal hybridizer was selected from the cell suspension of Sinapis turgida Del., members of the Brassicaceae. In a preliminary survey on tissue culture for about 50 species in Brassica and its wild allies, we found that S. turgida delivered cell suspension in a liquid culture (Toriyama et al 1987b). Following protoplast fusion between S. turgida and B. oleracea L., and between S. turgida and B. nigra (L.) Koch, somatic hybrids were selected and plantlets were obtained (Toriyama et al 1987a).

Journal ArticleDOI
TL;DR: ‘Polima’ cytoplasmic male sterility was transferred in one step from spring to winter lines of oilseed rape (Brassica. napus L.) by protoplast fusion by restriction fragment length polymorphism (RFLP) analysis of cytopLasmic DNA.

Journal ArticleDOI
TL;DR: Two cultivars of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts and plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.
Abstract: This is the first report on successful plant regeneration from protoplasts of sweet potato. Two cultivars (Guyana and Duclos XI) of sweet potato plants propagated under in vitro conditions were used as the source of protoplasts. Green compact calli with meristematic areas were induced in the medium supplemented with 2mg1−1 zeatin, and plant regeneration occurred when these calli were transferred onto the medium with zeatin level reduced to 0.25mg1−1. Plant regeneration was found to be genotype-dependent, since it was only obtained for cultivar Duclos XI.

Journal ArticleDOI
TL;DR: A simplified protoplast regeneration system for Vigna aconitifolia was developed and the plant cultivar used was an important factor in determining transformation frequencies since one of the cultivars had an 85 fold higher transformation rate than the other.
Abstract: A simplified protoplast regeneration system for Vigna aconitifolia was developed. A plating efficiency of 60% was obtained using mesophyll protoplasts from 10-day-old seedlings. By co-cultivation of protoplasts with Agrobacterium tumefaciens containing the Ti plasmid derivative pGV 3850∶1103 neo kanamycin-resistant colonies were obtained; 23% of the transformed lines showed expression of the nonselected co-transferred nopaline synthase gene. Transformation was confirmed by Southern blot analysis using a nonradioactive detection system. The plant cultivar used was an important factor in determining transformation frequencies since one of the cultivars had an 85 fold higher transformation rate than the other.

Journal ArticleDOI
01 Oct 1987-Planta
TL;DR: Maize (Zea mays L.) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos which can be induced to form roots and small leaf-like structures.
Abstract: Maize (Zea mays L) protoplasts have been obtained which divide rapidly and produce a callus that differentiates to form somatic embryos The somatic embryos can be induced to form roots and small leaf-like structures The genotype was the hybrid A188xBlack Mexican Sweet Protoplasts were prepared from an embryogenic suspension culture derived from a Type II callus which had been selected from Type I callus produced by immature zygotic embryos The basal medium for the suspension culture was N6 (CC Chu et al, 1975, Scientia Sinica 18, 659–668) The 2,4-dichlorophenoxyacetic acid concentration of the suspension culture was critical for subsequent protoplast growth and was optimal at 40 mgl Protoplasts had to be cultured in a low-osmoticum medium (03 M mannitol) for subsequent cell divisions to occur The protoplasts have been transformed transiently with the gene chloramphenicol acetyltransferase (CAT) containing the 35S promoter obtained from cauliflower mosaic virus (CaMV-35S)

Journal ArticleDOI
TL;DR: Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions.
Abstract: Suspension cultures have been initiated from embryogenic callus of hexaploid wheat (Triticum aestivum L.). Most commonly, these "suspensions" are composed of callus-like clusters (up to 2 mm in diameter). Two rapidly-growing lines (MBE6 and C82d) have been obtained, which consist of smaller aggregates of cytoplasmic cells, and these have been maintained for more than 4 years. These lines show very limited morphogenetic capacity and only a single plantlet has been regenerated, from line MBE6, after 9 months in culture. Protoplasts isolated from line MBE6 are unable to divide, but protoplasts from line C82d consistently undergo sustained divisions to form callus or secondary cell suspensions.

Journal ArticleDOI
TL;DR: Protoplasts isolated from a totipotent embryogenic cell suspension culture of Zea mays L. (cultivar ‘Dekalb XL82’) underwent sustained cell divisions when cultured in liquid as well as agarose media and clusters of somatic embryos germinated precociously but no plants were recovered.
Abstract: Protoplasts isolated from a totipotent embryogenic cell suspension culture of Zea mays L. (cultivar ‘Dekalb XL82’) underwent sustained cell divisions when cultured in liquid as well as agarose media. Optimal colony formation (5%) occurred in a liquid medium containing 0.5 mg/l 2,4-dichlorophenoxyacetic acid (2,4-D). A soft and unorganized callus was formed when the protocolonies were transferred to agar solidified suspension maintenance medium. Compact, organized and yellow to pale green folded structures and somatic embryos were formed upon subsequent transfer of this callus to a low 2,4-D medium. Clusters of somatic embryos germinated precociously but no plants were recovered.

Journal ArticleDOI
Horst Binding1, Dagmar Witt1, J. Monzer1, Gudrun Mordhorst1, Rainer Kollmann1 
TL;DR: Plants obtained by cell grafting in protoplast co-cultures and plantlets of all chimeral lines were grown to flowering under tissue culture conditions and some also in the greenhouse, discussing aspects of organogenesis and interspecific cooperation.
Abstract: Heterospecific chimeralSolanum nigrum (+)Solanum tuberosum plants were obtained by cell grafting in protoplast co-cultures. Periclinal, sectorial, and mericlinal chimeras have been identified by various morphological and cytological characteristics. Morphogenesis predominantly began in periclinal chimeral organization. Cells of different species have been found to be interconnected by secondary plasmodesmata. Plantlets of all chimeral lines were grown to flowering under tissue culture conditions and some also in the greenhouse. Aspects of organogenesis and interspecific cooperation are discussed.

Journal ArticleDOI
TL;DR: The hypothesis that superior clones result from heterokaryons after protoplast fusion or that they arise from other in vitro events such as somaclonal variation is discussed.
Abstract: Tetraploid potato plants were regenerated after polyethylene-glycol-induced protoplast fusion between dihaploids. Hybrid vigour of the regenerated calli was used for preselection of fusion products. Nearly all the selected vigorous clones possessed chromosome counts at the tetraploid level. Fusion products were compared to the parental material to auto-fused plants of and to three protoclones expressing different degrees of somaclonal variation. The selected clones, where grown in vitro in growth rooms and in pots in the glasshouse, showed increased vigour compared to their parents, to auto-fused and to 4x protoclones. Plants of clones from very vigorous calli, when assessed by height, the number of nodes per plant, leaf morphology and tuber production, showed hybrid vigour. The hypothesis that superior clones result from heterokaryons after protoplast fusion or that they arise from other in vitro events such as somaclonal variation is discussed.

Journal ArticleDOI
TL;DR: The degree of genome separation calculated for different cell clones remained constant during in vitro propagation of cells but was significantly lower for subclones derived from colchicine-treated cells, concluding that spatial chromosome arrangement in metaphase is epigenetically controlled.
Abstract: Chromosome spatial arrangements on metaphase plates of intergeneric intertribal cell hybrids of Nicotiana chinensis and Atropa belladonna as well as interspecific somatic hybrid plants of Nicotiana plumbaginifolia and Nicotiana sylvestris were analyzed. In the metaphases of the first divisions of protoplast fusion products, chromosomes of the two parents were spatially separated (segmented metaphase). In long-term cultured somatic hybrids, the topology of genome separation pattern in both callus cells and plants showed changes in form from “segmental” to “radial.” Growing the hybrid cells in the presence of colchicine resulted in random chromosome arrangement both in cells directly exposed to different colchicine concentrations and in colchicine-treated cells grown in colchicine-free media. The degree of genome separation calculated for different cell clones remained constant during in vitro propagation of cells but was significantly lower for subclones derived from colchicine-treated cells. Therefore, it is concluded that spatial chromosome arrangement in metaphase is epigenetically controlled.

Journal ArticleDOI
01 Jan 1987-Plasmid
TL;DR: A method for the transformation of Lactobacillus protoplasts by plasmid DNA is reported, which involves polyethylene glycol treatment of protoplast to induce DNA uptake.

Journal ArticleDOI
TL;DR: Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts.
Abstract: Conditions were standardized for the isolation and culture of protoplasts from an embryogenic cell suspension culture of Picea glauca. A combination of 0.5% Cellulase R-10, 0.25% Macerozyme, 0.25% Driselase, 0.25% Rhozyme HP-150 with 0.5M mannitol and 5 mM CaCl2.2H2O produced an average of 4.5 × 106 protoplasts per gram fresh weight of cells. Of the several protoplast culture media tested, von Arnold and Eriksson and Kao and Michayluk (KM8P) media best supported mitotic divisions of protoplasts. A density of 105 protoplasts per ml and the addition of 5 mM glutamine to the culture medium was necessary to induce sustained divisions and microcallus formation. Microcalli grew into subculturable callus using a nurse culture technique.

Journal ArticleDOI
TL;DR: Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance, which resulted in a much faster and 3 orders of magnitude more efficient method.
Abstract: Plasmid DNA-mediated transformation of vegetative cells of Bacillus thuringiensis was studied with the following two plasmids: pBC16 coding for tetracycline resistance and pC194 expressing chloramphenicol resistance. A key step was the induction of competence by treatment of the bacteria with 50 mM Tris hydrochloride buffer (pH 8.9) containing 30% sucrose. Transformation frequency was strongly influenced by culture density during the uptake of DNA and required the presence of polyethylene glycol. Growth in a minimal medium supplemented with Casamino Acids gave 35 times more transformants than growth in a rich medium. The highest frequencies were obtained with covalently closed circular DNA. With all parameters optimized, the frequency was 10(-3) transformants per viable cell or 10(4) transformants per microgram of DNA. Cells previously frozen were also used as recipients in transformation experiments; such cells gave frequencies similar to those obtained with freshly grown cells. The procedure was optimized for B. thuringiensis subsp. gelechiae, but B. thuringiensis subsp. kurstaki, B. thuringiensis subsp. galleriae, B. thuringiensis subsp. thuringiensis, and B. thuringiensis subsp. israelensis were also transformed. Compared with protoplast transformation, our method is much faster and 3 orders of magnitude more efficient per microgram of added DNA.

Journal ArticleDOI
TL;DR: Somatic embryogenesis was achieved in protoplast cultures of coffee by transferring microcalli from cell suspension-derived somatic embryos to a medium lacking growth regulators, which resulted in the formation of globular embryos.
Abstract: Somatic embryogenesis was achieved in protoplast cultures of coffee. Protoplasts were isolated from cell suspension-derived somatic embryos of Coffea canephora. After repeated subculture in a medium supplemented with 0.5 mg/l of each of kinetin, 2,4-dichlorophenoxyacetic acid (2,4-D), and naphthaleneacetic acid (NAA), microcalli developed. Transfer of these microcalli to a medium lacking growth regulators resulted in the formation of globular embryos. Upon subculture without growth regulators they grew to well-differentiated embryos, Eventually some of them developed to plantlets which were transferred to the greenhouse for further observation.