Showing papers on "Protoplast published in 1982"
TL;DR: A reproducible method is reported for the stable transformation of tobacco protoplasts with Ti-plasmid DNA, using a similar selection procedure to select transformants by their ability to divide and grow in tissue culture without the addition of plant phytohormones to the synthetic culture medium.
Abstract: Genetic engineering requires a procedure for introducing DNA into host cells, followed by integration into the host genome and gene expression. Although several procedures for DNA-medi-ated gene transfer in mammalian cells, yeast and bacteria have been reported, no such methods are yet available for plant cells. The major obstacle to DNA uptake in plant cells is the cell wall, but this can be circumvented by using plant protoplasts, cells freed of their cell walls by enzymatic digestion. However, none of the reports on the uptake of DNA into plant protoplasts1,2 has produced conclusive evidence for the integration of DNA into the host genome, that is, that stable transformation occurs. The tumour-inducing bacterium Agrobacterium tumefaciens, which causes crown gall disease, is a natural system for the introduction of foreign DNA into plants. This bacterium introduces part of its tumour-inducing (Ti) plasmid, called T-DNA, into plant cells, where it becomes integrated into the nuclear DNA of the host3,4 and is transcribed into mRNA5,6. T-DNA encodes tumour-specific enzymes responsible for the formation of amino acid derivatives such as octopine or nopaline7, which the bacterium can use as a sole source of carbon and nitrogen. The transformed cells have also acquired the ability to grow in the absence of phytohormones (autotrophy). An in vitro system for infection of Nicotiana tabacum protoplasts by A. tumefaciens has already been reported8. Transformants are selected by their ability to divide and grow in tissue culture without the addition of plant phytohormones to the synthetic culture medium. Here, we report a reproducible method for the stable transformation of tobacco protoplasts with Ti-plasmid DNA, using a similar selection procedure.
472 citations
Book•
01 Jan 1982
TL;DR: This work presents a meta-analysis of cryopreservation of germplasm and somatic hybridization of parenchyma cells to tracheary elements in the context of callus-Derived Systems and its applications in organogenesis and tissue culture.
Abstract: Part I. Background Information: 1. Culture of plant cells, tissues, and organs 2. Laboratory facilities 3. Aseptic techniques 4. Media composition and preparation Part II. Experimental: Callus and Callus-Derived Systems: 5. Initiation and maintenance of callus 6. Organogenesis 7. Cell suspensions 8. Somatic embryogenesis Part III. Experimental: Culture of Organs and Organized Systems: 9. Isolated roots 10. Micropropagation by bud proliferation 11. Anther and pollen cultures Part IV. Experimental: Isolated Cells: 12. Transdifferentiation of parenchyma cells to tracheary elements 13. Isolation and culture of protoplasts 14. Protoplast fusion and somatic hybridization Part V. Supplementary Topics: 15. Cryopreservation of germplasm 16. Production of secondary metabolites 17. Quantitation of procedures 18. Formulations of tissue culture media 19. Commercial sources of supplies Author index Subject index.
376 citations
TL;DR: In this paper, a modified transformation procedure was devised which increased transformation frequency more than 20-fold (allowing up to 10(7) primary transformants per microgram of SLP1.2 covalently closed circular DNA) and greatly facilitated the cloning of drug resistance genes and biosynthetic genes.
Abstract: Methodology which allows consistent shotgun cloning of streptomycete genes is presented. Parameters that increase transformation efficiency of Streptomyces lividans 66 were adjusted to generate reproducibly a population of cloned genes likely to represent the entire genome. Factors which influence the recovery of viable transformants include: growth phase of the mycelium, ionic and osmotic characteristics of the medium during protoplast formation and transformation, and moisture content and protoplast density during regeneration. A modified transformation procedure was devised which increased transformation frequency more than 20-fold (allowing up to 10(7) primary transformants per microgram of SLP1.2 covalently closed circular DNA) and greatly facilitated the cloning of drug resistance genes and biosynthetic genes, using one of two plasmid vectors. Viomycin resistance genes on BamHI or PstI fragments were cloned from S. vinaceus genomic DNA into S. lividans, using the SLP1.2 vector. At least three different S. vinaceus BamHI fragments (1.9, 5.8, or 8.5 kilobases) confer viomycin resistance; only one PstI fragment (4.3 kilobases) was found. Recombinant plasmids were all able to produce lethal zygosis and to be transferred by conjugation within S. lividans. SCP2 was used to clone S. coelicolor A3(2) genes that "complemented" the auxotrophic mutation hisD3, argA1, or guaA1. Recombinant DNA technology can now be applied to economically and academically interesting problems unique to streptomycete molecular biology.
239 citations
TL;DR: The establishment of eight new protoplast systems in Citrus paves the way for cell genetics studies and for novel breeding approaches in these economically important orchard trees.
Abstract: Nucellar callus lines were established from two orange cultivars (‘Nucellar Shamouti’, ‘Shamouti Landau’), three mandarin cultivars (‘Murcott’, ‘Dancy’, ‘Ponkan’) one grapefruit cultivar (‘Duncan’) and sour orange (Citrus aurantium). These callus lines were initiated from in vitro cultured ovules of young fruits and maintained an embryogenic capacity. The plating efficiencies of protoplasts derived from these calli, as well as those of protoplasts from lemon (cv. ‘Villafranca’) nucellar callus were differentially affected by the maceration enzymes and by the sugars used as osmotic stabilizers. Plants with normal morphological features were regenerated from cultured protoplasts derived from each of the nucellar callus lines. The establishment of eight new protoplast systems in Citrus paves the way for cell genetics studies and for novel breeding approaches in these economically important orchard trees.
170 citations
TL;DR: The increase in total activity of the latter three enzymes following exposure of plants to 400 millimolar NaCl and the development of CAM was due to specific increases in the levels of activity in the cytoplasm.
Abstract: Mesembryanthemum crystallinum , a halophilic, inducible Crassulacean acid metabolism (CAM) species, was grown at NaCl concentrations of 20 and 400 millimolar in the rooting medium. Plants from the low salinity treatment showed exclusively C 3 -photosynthetic net CO 2 fixation, whereas plants exposed to the high salinity level exhibited net CO 2 dark fixation involving CAM. Mesophyll protoplasts, isolated from both tissues, were gently ruptured, and the intracellular localization of enzymes was studied following differential centrifugation and Percoll density gradient centrifugation of protoplast extracts. Both centrifugation techniques resulted in the separation of intact chloroplasts, with up to 90% yield, from other organelles and the nonparticulate fraction of cells. Enzymes were identified by determination of activity and by sodium dodecyl sulfate gel electrophoresis of enzyme protein. Experiments established the extraorganellar (cytoplasmic) location of phosphoenolpyruvate carboxylase, enolase, phosphoglyceromutase, and NADP-malic enzyme; the mitochondrial location of NAD-malic enzyme; and the chloroplastic location of pyruvate, Pi dikinase. NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphohexose isomerase, and phosphoglycerate kinase were associated with both cytoplasm and chloroplasts. NADP-dependent malate dehydrogenase activity was found in both the chloroplastic and extrachloroplastic fractions; the activity in the chloroplast showed an optimum at pH 8.0 and was dependent upon preincubation of enzyme with dithiothreitol. The extrachloroplastic activity showed an optimum at pH 6.5 and was independent of pretreatment with dithiothreitol. Protoplast extracts of M. crystallinum performing CAM exhibited higher activities (expressed per mg chlorophyll per min) of phosphoenolpyruvate carboxylase, pyruvate, Pi dikinase, NADP-malic enzyme, NAD-malic enzyme, NADP-malate dehydrogenase, enolase, phosphoglyceromutase, NAD-glyceraldehyde-3-phosphate dehydrogenase, phosphoglycerate kinase, and phosphohexose isomerase than protoplast extracts from M. crystallinum not exhibiting CAM. The increase in total activity of the latter three enzymes following exposure of plants to 400 millimolar NaCl and the development of CAM was due to specific increases in the levels of activity in the cytoplasm.
144 citations
TL;DR: A large discrepancy was found between the partial recovery of protoplast volumes and the complete absence of a recovery of photosynthesis in hygrophytes, and the response of photosynthetic enzymes to increased salt concentrations in the reaction medium.
Abstract: Rates of photosynthesis of leaf slices from various hygro-, meso- and xerophytes were measured in the absence of stomatal control in various stages of osmotic dehydration The external osmotic potential π° for a 50% inhibition of photosynthesis varied between 20 bar in some hygrophytes up to 50 bar in xerophytes The response of photosynthetic enzymes to increased salt concentrations in the reaction medium was similar in leaf extracts from hygro-, meso- and xerophytes The total protoplast volume in vacuum-infiltrated leaf discs from various plants was measured as the difference between 3H2O-labeled space and [14C]sorbitol-labeled space In all plants, the protoplast volume could be reduced to about 55% of the maximum volume of tissue in equilibrium with water, without decreasing photosynthesis Reduction of the maximal protoplast volume below 55% decreased photosynthesis in all tissues to the same decreased photosynthesis in all tissues to the same degree At 20% maximal volume, photosynthesis of all plants was completely inhibited The differential decrease of protoplast volumes of various leaf tissues in response to changes in π° was mainly due to the different osmotic potential of the cell sap (πcs) The relative contribution of sugars to the overall osmolarity of the cell sap was up to nineteen times higher in xerophytes than in hygrophytes Short-term recovery of photosynthesis after hypertonic stress was good in xerophytes, incomplete in mesophytes and absent in hygrophytes There was also a large discrepancy between the partial recovery of protoplast volumes and the complete absence of a recovery of photosynthesis in hygrophytes
133 citations
TL;DR: The suitability of root protoplasts for genetic manipulations in the genus Brassica is discussed and plant regeneration was obtained from protoplast derived tissues of B. napus and B. oleracea, but only rhizogenesis was observed with B. campestris.
116 citations
TL;DR: Chloroplasts of N. tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion as discussed by the authors, and the results indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts.
Abstract: Chloroplasts of Nicotiana tabacum SR1 were transferred into Nicotiana plumbaginifolia by protoplast fusion. The protoplasts of the organelle donor were irradiated with different lethal doses using a (60)Co source, to facilitate the elimination of their nuclei from the fusion products. After fusion induction, clones derived from fusion products and containing streptomycin-resistant N. tabacum SR1 chloroplasts were selected by their ability to green on a selective medium. When N. tabacum protoplasts were inactivated by iodoacetate instead of irradiation, the proportion of N. plumbaginifolia nuclear segregant clones was low (1-2%). Irradiation markedly increased this value: Using 50, 120, 210 and 300 J kg(-1) doses, the frequency of segregant clones was 44, 57, 84 and 70 percent, respectively. Regeneration of resistant N. plumbaginifolia plants with SR1 chloroplasts indicated that plastids can be rescued from the irradiated cells by fusion with untreated protoplasts. Resistant N. plumbaginifolia plants that were regenerated (43 clones studied) had diploid (2n = 2X = 20) or tetraploid chromosome numbers and were identical morphologically to parental plants. The absence of aneuploids suggests that in these clones irradiation resulted in complete elimination of the irradiated N. tabacum nuclei. Resistance is inherited maternally (five clones tested). The demonstration of chloroplast transfer and the presence of N. tabacum plastids in the N. plumbaginifolia plants was confirmed by chloroplast DNA fragmentation patterns after EcoRI digestion.
106 citations
TL;DR: SR1 protoplasts, originally present as “contaminants” in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment, but the majority (about 80%) of the recovered resistant clones recovered after fusion-induction acquired the streptomycin resistance factor from the rare ‘contaminating’ SR1 protplasts.
Abstract: Protoplasts of Nicotiana tabacum (SR1), carrying a maternally-inherited streptomycin resistance mutation, were enucleated by centrifugation through a Percoll gradient. The resulting cytoplasts containing resistant plastids, were fused with sensitive Nicotiana plumbaginifolia protoplasts. The SR1 cytoplasts, having no nuclei, were unable to form calli. All resistant clones recovered after fusion-induction were therefore supposed to be derived from interspecific cytoplast-protoplast fusion.
N. plumbaginifolia plants regenerated in 17 out of the 75 resistant clones studied. Plants obtained from eight of these clones were resistant to streptomycin and inherited the resistance maternally, as expected when transferring SR1 plastids into the N. plumbaginifolia nuclear background. Plastid transfer in these plants has been confirmed by the EcoRI restriction pattern of the chloroplast DNA. In nine clones N. plumbaginifolia plants were sensitive although obtained from initially resistant clones. This phenomenon is explained by the maintenance of plastid heterogeneity on the selective streptomycin medium, and formation of plants from sensitive sectors on the non-selective regeneration medium. SR1 protoplasts, originally present as “contaminants” in the cytoplast preparation (2–7%) did not form colonies (or very rarely) after polyethylene glycol treatment. The nuclei from such protoplasts were recovered, however, in the interspecific somatic hybrids (56 clones), and in segregants having the SR1 nucleus but some cytoplasm from N. plumbaginifolia (2 clones). The majority (about 80%) of the recovered resistant clones therefore acquired the streptomycin resistance factor from the rare (2–7%) “contaminating” SR1 protoplasts. This is explained by the protoplasts being more stable during fusion induction.
99 citations
TL;DR: Nitrate reductase deficient (NR-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia by tentatively classified as defective in the apoenzyme or the cofactor by the xanthine dehydrogenase activity and in vitro enzyme complementation.
Abstract: Nitrate reductase deficient (NR-) cell lines were selected indirectly by their resistance to 40 mM chlorate in protoplast cultures of haploidNicotiana plumbaginifolia. Frequency of the chlorate resistant clones was 5.8×10-5 in non-mutagenized cultures, which could be increased up to 25 times by treatment with N-ethyl-N-nitrosourea (NEU) or gamma irradiation.
97 citations
TL;DR: A hypothesis that the extent of heat resistance is based in whole or in part on the extentof dehydration and diminution of the protoplast in the dormant spore is supported, without implications about physiological mechanisms for attaining this state.
Abstract: Five types of dormant Bacillus spores, between and within species, were selected representing a 600-fold range in moist-heat resistance determined as a D100 value. The wet and dry density and the solids and water content of the entire spore and isolated integument of each type were determined directly from gram masses of material, with correction for interstitial water. The ratio between the volume occupied by the protoplast (the structures bounded by the inner pericytoplasm membrane) and the volume occupied by the sporoplast (the structures bounded by the outer pericortex membrane) was calculated from measurements made on electron micrographs of medially thin-sectioned spores. Among the various spore types, an exponential increase in the heat resistance correlated directly with the wet density and inversely with the water content and with the protoplast/sporoplast volume ratio. Altogether with results supported a hypothesis that the extent of heat resistance is based in whole or in part on the extent of dehydration and diminution of the protoplast in the dormant spore, without implications about physiological mechanisms for attaining this state. Images
TL;DR: It is concluded that the variation is not a consequence of using protoplasts and that the expression or induction of variation is controllable.
Abstract: Plants were obtained from protoplasts of shoot cultures of potato (Solarium tuberosum L. cv. 'Maris Bard') and from in situ calluses upon plants of cv. 'Majestic'. None of the protoplast-derived plants resembled each other in all of ten morphological characteristics scored and only one resembled the parental 'Maris Bard' type. As there were a number of plants regenerated from each of ten protoplast-derived calluses it is concluded that variation arose after protoplast isolation during the cell culture phase. Plants regenerated from in situ calluses of cv. 'Majestic' were quite uniform. Reported cases of variation and uniformity from cultured potato tissues are discussed. It is concluded that the variation is not a consequence of using protoplasts and that the expression or induction of variation is controllable.
TL;DR: Plant regeneration from protoplast derived colonies of B. oleracea, D. innoxia, M. sativa and N. tabacum confirmed the totipotency of cotyledon protoplasts.
TL;DR: The results indicated that, over the initial period of culture, DB largely inhibited cellulose production at the cell surface, but did not inhibit DNA synthesis or protein accumulation by the protoplasts.
Abstract: The effect of 2,6-dichlorobenzonitrile (DB) and coumarin on tobacco protoplast development has been studied using a combination of flow microfluorimetry and quantitative fluorescence microscopy. The results indicated that, over the initial period of culture, DB largely inhibited cellulose production at the cell surface, but did not inhibit DNA synthesis or protein accumulation by the protoplasts. Continuous culture of protoplasts in DB resulted in the accumulation of an increased capacity for cellulose synthesis as expressed following the removal of inhibitor. The possible sites of the specific inhibitory action of DB are discussed.
TL;DR: Methanobacterium bryantii was found to undergo rapid lysis when grown in a prereduced chemically defined medium under H2-CO2 (4:1, vol/vol).
Abstract: Methanobacterium bryantii was found to undergo rapid lysis when grown in a prereduced chemically defined medium under H2-CO2 (4:1, vol/vol). The addition of 20 mM MgCl2 to the medium gave, rather than rapid lysis, a gradual formation of phase-dark spherical bodies which in thin section appeared as true protoplasts. In general, the protoplasts were stabilized by divalent but not monovalent cations and, unlike whole cells, were sensitive to lysis by Triton X-100. Electron microscopic examination revealed that protoplast formation was preceded by a general breakdown of the cell wall with an apparent squeezing out of the protoplast through the degraded wall. The growth of cells was greatly increased and not accompanied by detectable lysis in a medium modified by elevating the levels of nickel and ammonium.
TL;DR: Restoration of nitrate reductase and xanthine dehydrogenase activities confirm the presence of an active form of the molybdenum-cofactor by the expression of introduced genes of Physalis and Datura into the genome of cnx-68 Nicotiana.
Abstract: Nitrate reductase-deficient cells of Nicotiana tabacum cv Gatersleben (coded cnx-68) lacking active molybdenum-cofactor were corrected by introducing the genes from Physalis minima and Datura innoxia into NR- genomes In these itergeneric reconstruction experiments, X-irradiated inactive mesophyll protoplasts of Physalis and Datura were fused separately with the cultured cell protoplasts of cnx-68 Nicotiana A total of 45 cell colonies, 37 transformed by Physalis and 8 by Datura, were selected from about 17×103 heteroplasmic fusion products The selection of transformants was made by their ability to grow on a medium containing nitrate as the sole nitrogen source Some of these transformants were further characterized with respect to nitrate reductase, xanthine dehydrogenase and glutamate dehydrogenase activities, chlorate sensitivity, and chlorophyll synthesis The restoration of nitrate reductase and xanthine dehydrogenase activities confirm the presence of an active form of the molybdenum-cofactor by the expression of introduced genes of Physalis and Datura into the genome of cnx-68 Nicotiana Such stable transformations via fusion of normal and highly irradiated protoplasts may have a considerable application in higher plants for introducing desirable characters from diverse genomes
TL;DR: Techniques and media for the production and regeneration of stable Clostridium acetobutylicum protoplasts are described.
Abstract: Techniques and media for the production and regeneration of stable Clostridium acetobutylicum protoplasts are described.
TL;DR: Mesophyll and cell suspension protoplasts of Medicago coerulea and M. glutinosa divided to form callus and the potential of protoplast systems for transfer of characteristics from M. coerceda to M. sativa is discussed.
TL;DR: Restoration of NR activity was accompanied by the restoration of plant regeneration ability and gave somatic hybrids with restored nitrate reductase activity.
Abstract: Allelism of nine nitrate reductase deficient (NR−) Nicotiana plumbaginifolia cell lines was tested by complementation after protoplast fusion. Complementation was recognized by the appearance of somatic hybrid colonies growing on a selective NH4+/NO3− medium which cannot support the growth of NR− lines. All five apoenzyme defective (NA) lines were non-complementing and therefore allelic. The apoenzyme and the cofactor defective (NX) lines were complementing, as expected, and gave somatic hybrids with restored nitrate reductase activity. The four cofactor defective lines were found to belong to three complementation groups (NX1 and NX9; NX21; NX24). Two of these (NX21 and NX24) are of new types which have not been previously described in flowering plants. In the somatic hybrids restoration of NR activity was accompanied by the restoration of plant regeneration ability.
TL;DR: By rigorously adhereing to the method detailed in this paper, an average of one hundred Lukullus plants can be regenerated from one million protoplasts.
TL;DR: Studies on the biochemical composition of the isolated vacuoles indicated that amino acids, organic acids and protein contents varied with the cell culture cycle, emphasizing the dynamic status of the vacuolar system in cell suspension cultures of Acer pseudoplatanus.
TL;DR: The data obtained are interpreted as new evidence for the possibility of using non-sexual hybridization for the production of intergeneric, intertribal plant hybrids which cannot be obtained by sexual crossing.
Abstract: After fusion of isolated mesophyll protoplasts of belladonna (Atropa belladonna) with callus protoplasts of Chinese tobacco (Nicotiana chinensis) followed by mechanical isolation and cloning of individual heteroplasmic fusion products, 13 cell clones were obtained. The hybrid nature of most of the clones has been confirmed by biochemical (studies of amylase isozymes), cytogenetic (size and morphology of chromosomes) and physiological (peculiarities of cell-growth in vitro) analyses. Study of chromosomes and isozyme patterns in the hybrid cell lines revealed the presence of both parental genomes, without an indication of chromosome elimination, six months after hybridization. In 4 cell lines shootlike structures and plantlets have been produced by means of transfer to organogenesis-inducing media. The data obtained are interpreted as new evidence for the possibility of using non-sexual hybridization for the production of intergeneric, intertribal plant hybrids which cannot be obtained by sexual crossing. From these results the potential of Atropa (X) Nicotiana hybrids as a model system for genetic studies of distantly related plant species is discussed.
TL;DR: Protoplasts from 8- to 9-day-old wheat leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases.
Abstract: Protoplasts from 8- to 9-day-old wheat (Triticum aestivum L.) leaves were used to isolate organelles which were examined for their contents of peptide hydrolase enzymes and, in the case of vacuoles, other acid hydrolases. High yields of intact chloroplasts were obtained using both equilibrium density gradient centrifugation and velocity sedimentation centrifugation on sucrose-sorbitol gradients. Aminopeptidase activity was found to be distributed, in approximately equal proportions, between the chloroplasts and cytoplasm. Leucyltyrosine dipeptidase was mainly found in the cytoplasm, although about 27% was associated with the chloroplasts. Vacuoles shown to be free from Cellulysin contamination contained all of the protoplast carboxypeptidase and hemoglobin-degrading activities. The acid hydrolases, phosphodiesterase, acid phosphatase, α-mannosidase, and β-N-acetylglucosamidase were found in the vacuole to varying degrees, but no β-glucosidase was localized in the vacuole.
TL;DR: Protoplasts were isolated from seedling roots of Medicago sativa and Trigonella foenumgraecum using a simple enzyme mixture and plantlets were recovered via somatic embryogenesis from protoplast derived tissues.
TL;DR: Protoplasts isolated enzymatically from roots of germinating soybean seeds divide to form callus and the ability to culture protoplasts of a more meristematic type is discussed with respect to genetic manipulations in this crop species.
TL;DR: The method described represents a highly efficient and reliable technique for protoplast production from Aspergillus nidulans and corresponded to both the point of maximum autolysis and maximum lytic enzyme activity.
Abstract: The rate and degree of autolysis of Aspergillus nidulans was investigated during a 16 day incubation period. Changes in the activity of chitinase, B -glucanase, α-glucanase, invertase, esterase, acid and alkaline phosphatase and protease liberated into the culture fluid were monitored during growth and autolysis. Protoplasts were released from A. nidulans mycelium by lytic enzymes present in autolytic-phase culture filtrates of the same organism. Maximum protoplast-liberating activity corresponded to both the point of maximum autolysis and maximum lytic enzyme activity. The method described represents a highly efficient and reliable technique for protoplast production from Aspergillus nidulans.
TL;DR: Protoplasts of lettuce, Lactuca sativa L. sativa can be obtained from greenhouse and growth chamber grown plants, in vitro grown shoots and roots and cotyledons of seedlings and were most readily obtained with protoplasts from seedling plant material.
TL;DR: In this article, a series of intra-and interspecific crosses as well as protoplast fusions were performed to reveal the relationship between the various taxa of the yeast genus Schizosaccharomyces Lindner.
Abstract: Summary: Hybridization studies based on the use of crossing and the protoplast fusion technique revealed the relationship between the various taxa of the yeast genus Schizosaccharomyces Lindner. From a series of intra- and interspecific crosses as well as protoplast fusions, we concluded that a revision of the genus is required. Being interfertile, S. pombe and S. malidevorans have to be included in one species S. pombe. From the protoplast fusion results, this new taxon seems to be closely related to S. octosporus. The results obtained are discussed in connection with the characteristics of the low temperature spectra of the cytochromes.
TL;DR: Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number and could be regenerated to recover plants that retained the somatic chromosome number during culture.
Abstract: Cell suspension cultures of Nicotiana were initiated using conditions designed to selectively favor stable chromosome number. These conditions included use of leaf explants to initiate cultures, growth of cells in culture medium containing 2,4-D, and transfer of cells with short subculture intervals. Four cell lines derived from Nicotiana tissue with 2n=24, 48, or 72 were established and retain stable chromosome number. Each line could be regenerated to recover plants that retained the somatic chromosome number during culture. Establishment of haploid and diploid cell lines with stable chromosome number is important for mutant isolation and protoplast fusion.
TL;DR: The LR400 line was isolated by its ability to form a green callus on selective medium (RMOP medium containing 1,000 µg ml−1 lincomycin hydrochloride) and regenerated from this line inherited the resistance maternally.
Abstract: Lincomycin resistant cell lines were screened in monoploid (X = 10 chromosomes) protoplast cultures of Nicotiana plumbaginifolia. Lincomycin is an inhibitor of protein synthesis on plastid ribosomes and normally inhibits greening of cultured cells on RMOP medium. The LR400 line was isolated by its ability to form a green callus on selective medium (RMOP medium containing 1,000 µg ml(-1) lincomycin hydrochloride). Diploid plants regenerated from this line inherited the resistance maternally. The LR400 line is cross-resistant to cleocin (clindamycin), but is sensitive to streptomycin.