Showing papers on "Protoplast published in 1977"
TL;DR: Shooting bud development within p-calli was controlled by a number of factors including light, temperature, basic medium composition, nature and source of phytohormones, the continued presence of an osmoticum, low concentrations of a utilizable carbohydrate, and the developmental stage of the p- callus.
Abstract: Mesophyll cell protoplasts were isolated from potato (Solanum tuberosum L. cv. Russet Burbank) leaves and induced to proliferate in culture. Protoplast division was observed only among preparations isolated from plants previously conditioned under short periods of low intensity illumination. Sustained growth and development of protoplast-derived calli (p-calli) occurred when they were maintained on defined media at 24 C under 500 lux lighting. Shoot bud development within p-calli was controlled by a number of factors including light, temperature, basic medium composition, nature and source of phytohormones, the continued presence of an osmoticum, low concentrations of a utilizable carbohydrate, and the developmental stage of the p-callus.
266 citations
TL;DR: Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.
Abstract: Protoplasts of methionine-and lysine-requiring h- mutants isolated from the L972 h- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl2, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h+. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.
167 citations
TL;DR: Somatic hybrids selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids.
Abstract: A technique has been developed for the isolation of large numbers of protoplasts from protonemal tissue of Physcomitrella patens, and for their regeneration to give whole plants. Somatic hybrids have been selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH. The hybrids have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids. The progeny resulting from selffertilisation of the hybrids show a segregation which is consistent with their being the products of meioses in an autotetraploid.
132 citations
TL;DR: Intact vacuoles were isolated from petals of Hippeastrum and Tulipa and the ATPase activity of fresh vacuole suspensions was found to be 2--3 times that of protoplasts from the same tissue.
117 citations
TL;DR: The cytological analysis of parasexual hybrids revealed that the chromosome number ranged from 34 to 54, and the most frequent chromosome number was 2n = 36.
Abstract: Protoplasts isolated from cultured cells of albino carrot (Daucus carota) and normal green D. capillifolius were fused by polyethylene glycol. Selection of somatic hybrid plants was based on the restoration of photosynthetic function in hybrids. Green plantlets selected from embryo cultures were characterized on the basis of leaf morphology. The interspecific protoplast fusion resulted in green plants with leaves which were intermediate between those of the parents. The somatic hybrids between orange rooted carrot variety and D. capillifolius with long white roots produced long, white and fleshy roots. The cytological analysis of parasexual hybrids revealed that the chromosome number ranged from 34 to 54. The most frequent chromosome number was 2n = 36. Hybrids were also found with 34 and 35 chromosomes. The somatic hybrid showed the same isoenzyme pattern of leaf peroxidase as D. carota.
102 citations
TL;DR: Phenotypic expression of different genes controlling resistance to tobacco mosaic virus (TMV) in tomato was analysed in the protoplast system using otherwise isogenic breeding lines and homozygous gene Tm-1 was able to express its effect in protoplasts as well as in leaf discs.
Abstract: Summary Phenotypic expression of different genes controlling resistance to tobacco mosaic virus (TMV) in tomato was analysed in the protoplast system using otherwise isogenic breeding lines. Genes Tm-2 and Tm-22 were not expressed and did not prevent TMV-L, a common tomato strain of TMV, infecting and multiplying. By contrast, homozygous gene Tm-1 was able to express its effect in protoplasts as well as in leaf discs; no virus progeny were detected by fluorescent antibody staining or by infectivity assay up to 3 days after inoculation with TMV-L. Protoplasts and leaf discs homozygous for Tm-1, however, became infected with TMV-CH2, a tomato strain able to overcome the effects of Tm-1 in intact plants. Protoplasts from Lycopersicon peruvianum P. I. 128650, known to have a high level of resistance to TMV, were as readily infected with TMV-L, and synthesized progeny virus as rapidly as protoplasts from susceptible tomato. This genotype seems to have no resistance expressible in isolated protoplasts.
82 citations
TL;DR: The transfer of mitochondrial genome using the protoplast fusion technique is described, which results in successful complementation of auxotrophic yeast cells of the same mating-type after protoplasts fusion.
Abstract: INTRASPECIFIC and interspecific transfer of nuclear genes of fungi has recently been achieved by fusion of protoplasts1–5. We describe here the transfer of mitochondrial genome using the protoplast fusion technique. The experiments were carried out with strains of identical mating-type, so we also report successful complementation of auxotrophic yeast cells of the same mating-type after protoplast fusion.
74 citations
TL;DR: It is suggested that the ethylene-synthesizing enzyme system is highly structured in the apple cell and is localized in a cell wall-cell membrane complex.
Abstract: Apple (Malus sp.) slices gradually lost the ability to synthesize ethylene when incubated with a mixture of enzymes that digest cell walls. The released protoplasts did not produce ethylene. The release of protoplasts was faster from climacteric fruit slices than from preclimacteric tissue. In protoplast suspension culture, as new cell wall was deposited (as judged by the intensity of fluorescence of regenerating protoplasts stained with Calcofluor White and the incorporation of labeled myo-inositol into their ethanol-insoluble residue), ethylene synthesis was gradually regained. Restored ethylene synthesis reached a maximum after 80 hours in protoplasts from preclimacteric fruit and in 120 hours in those from climacteric tissue. Addition of methionine (1 mm) to the culture medium was essential for appreciable synthesis of ethylene; and this synthesis was inhibited by the aminoethoxy analogue of rhizobitoxine and by propyl gallate, inhibitors of ethylene synthesis in higher plants. We suggest that the ethylene-synthesizing enzyme system is highly structured in the apple cell and is localized in a cell wall-cell membrane complex.
72 citations
TL;DR: Findings suggest that cell turgor affects membrane components that determine cellular potential, and both Elodea leaf cells and tobacco protoplasts with regenerated cell walls became more electronegative during deplasmolysis.
Abstract: The internal electrical potential of protoplasts from six different plant species was positive. Plasmolyzed cells of leaves had positive voltages of similar magnitude. Both Elodea leaf cells and tobacco protoplasts with regenerated cell walls became more electronegative during deplasmolysis. These findings suggest that cell turgor affects membrane components that determine cellular potential.
68 citations
TL;DR: The nutrient requirements of mesophyll protoplasts from Pisum sativum L. cv.
Abstract: The nutrient requirements of mesophyll protoplasts from Pisum sativum L. cv. Timo have been investigated and a synthetic and completely defined medium has been designed. A high calcium concentration (12 mM) stimulated both protoplast survival and cell division. The content of iron and zinc was also critical. Additions of nicotinic acid, pyridoxine and thiamine were necessary. The protoplast growth was enhanced when some amino acids were included in the medium. An absolute requirement for auxin and cytokinin was shown.
In the revised medium about 90% of the isolated protoplasts survived and formed a cell wall. The first divisions were observed after 5 days and after 1 week 10–20% of the cells had divided at least once.
68 citations
TL;DR: It is reasonable to expect that PEG will induce the fusion of almost any two biological membranes and become an increasingly useful tool in their investigation.
Abstract: SOMATIC cell fusion has become a powerful tool in cell biology and genetics1–3 Until recently the standard reagent for increasing the rate of fusion in cultures of animal cells was inactivated Sendai virus4. In 1974 polymers of ethylene glycol (polyethylene glycol, PEG), over a wide range of molecular weights, were shown to be very effective, non-toxic chemical ‘fusogens’ for higher plant protoplasts5. They were then applied to fuse hen erythrocytes6 and these with yeast protoplasts7. Applied to mammalian, including human, cultured somatic cells they yielded hybrid cells capable of indefinite multiplication8,9. Applied to somatic cell protoplasts of two species of higher plants, they yielded hybrid protoplasts which regenerated, multiplied and developed into whole hybrid plants10. The list of cells and cell organelles fused has now expanded to include bacterial and fungal protoplasts and chloroplasts. It is reasonable to expect that PEG will induce the fusion of almost any two biological membranes and become an increasingly useful tool in their investigation6,11.
TL;DR: Fusion of protoplasts from the moss, Physcomitrella patens, was induced using polyethyleneglycol and three nonoverlapping complementation groups were identified.
Abstract: Fusion of protoplasts from the moss, Physcomitrella patens, was induced using polyethyleneglycol. Protoplasts were isolated from six nicotinic acid auxotrophic strains of independent origin and fusion was induced in all possible pairwise combinations. Complementation was detected by the ability to recover hybrids able to grow without nicotinic acid supplement. On the basis of the results presented, three nonoverlapping complementation groups were identified.
TL;DR: Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill, it was possible to select 33 green hybrid calli on agar culture medium and 20 somatic hybrids gave rise to leaves and some to shoots.
Abstract: Following fusion between protoplasts from two different chlorophyll-deficient diploid mutants of Datura innoxia Mill. it was possible to select 33 green hybrid calli on agar culture medium. Half of the somatic hybrids gave rise to leaves and some to shoots. The chromosome number of 20 somatic hybrids was determined: five were tetraploid, eight hexaploid, three octoploid, and four showed an aneuploid chromosome number. After transfer of the shoots of the five tetraploid hybrids to soil they developed roots. In control experiments in which protoplasts of the two mutants were cultured either as a mixture without being treated with the fusion agent, or cultured separately, no green callus could be obtained. Similar experiments involving protoplasts from one chlorophyll-deficient mutant of Datura innoxia, on the one hand, and those from similar mutants of Nicotiana sylvestris Spegazz. et Comes and Petunia hybrida, on the other, yielded no green somatic hybrid although hybrid protoplasts could be detected.
TL;DR: Cowpea mesophyll protoplasts were shown to bind irreversibly up to 3% input radioactive pBR313 plasmid DNA after 15 min of contact and the presence of donor DNA within the nuclear fraction is most probably not due to an artifactual redistribution of adsorbed plasmID DNA.
Abstract: Cowpea mesophyll protoplasts were shown to bind irreversibly up to 3% input radioactive pBR313 plasmid DNA after 15 min of contact. Maximum uptake occurred in the presence of 5mM ZnSO4 and 5 μg/ml poly-L-ornithine. Under these conditions about one half of the TCA precipitable radioactivity was associated with the nuclear fraction and behaved as linear plasmid molecules. These could not be chased from the protoplasts upon further incubation with unlabeled plasmid DNA. The presence of donor DNA within the nuclear fraction is most probably not due to an artifactual redistribution of adsorbed plasmid DNA. Prolonged incubation periods resulted in extensive degradation of plasmid in the incubation medium but little degradation occurred in the protoplasts. The donor DNA was not covalently associated with the protoplast nuclear DNA.
TL;DR: A critical influence of temperature was observed for the initiation and maintenance of protoplast division in LyCopersicon esculentum and Lycopersicon peruvianum.
TL;DR: Using leaf protoplasts of several highly inbred lines of petunia, it was possible to identify genetic variations with respect to their potential for growth on media containing specific hormone combinations, and it was concluded that only a few genes may control such differences.
TL;DR: The chapter describes mutant isolation and selection as mutants are utilized in understanding biochemical and developmental processes in microorganisms for their potential value in plant biology.
Abstract: Publisher Summary This chapter examines recent developments in plant cell culture and genetics, particularly in the context of their possible role in plant improvement. The potential value of these developments is found on the basis that plant cells can be cultured under defined conditions, biochemical mutants can be isolated, somatic hybrids can be obtained by protoplast fusion, haploid cell lines and/or plants can be obtained, and cell cultures can be induced to regenerate fertile plants. The chapter discusses plant cell tissue culture and examines the nutritional and environmental requirements of plant cells in culture. Plant tissue culture media consists of inorganic salts, trace elements, vitamins, a carbon source for energy, and plant growth regulators. A technique known as “freeze-preservation” is discussed to store plant cell cultures. This technique is of particular value to cell culture genetics, because any genetic program will sooner or later have a battery of cell culture mutants that need to be stocked. The chapter describes mutant isolation and selection as mutants are utilized in understanding biochemical and developmental processes in microorganisms for their potential value in plant biology.
TL;DR: Evidence is presented that the new class of lectins, called all-beta lectins by previous authors, are present in protoplast membranes and responsible for both forms of agglutination.
Abstract: Plant protoplast agglutination caused by normal and immune serum and by artificial carbohydrate antigens is described. Evidence is presented that the new class of lectins, called all-beta lectins by previous authors, are present in protoplast membranes and responsible for both forms of agglutination. Some non-specific serum component and the artificial antigens are the 2 respective passive ‘bridge’ molecules between agglutinating protoplasts.
TL;DR: Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root, but in the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.
Abstract: Zea mays inbred W64A in Texas (T, toxin sensitive) male sterile and non-male sterile (N, toxin resistant) cytoplasms were utilized. Roots of freshly germinated seeds were treated for 15 min of 2 hr with culture filtrate from liquid grown Helminthosporium maydis Race T, or with a chloroform extractable purified fraction from the culture filtrate. In the susceptible W64A T line, toxin treatment, both crude and purified, caused swelling and loss of matrix densiy in mitochondria of root cap and vacuolated cells in the region of elongation. One hour treatment with the chloroform extractable toxin fraction caused similar effects or mitochondria of isolated leaf protoplasts. This is the first report of such rapid in vivo effects of HmT toxin on mitochondria. Difficulty in obtaining consistent preservation of meristem mitochondria precluded drawing firm conclusions concerning that region of the root. In the resistant W64A N line, protoplast and root mitochondria were unaffected by the toxin.
TL;DR: It is concluded that the nutritional complementation in Aspergillus nidulans mutants may be due to interspecific aneuploidy.
Abstract: Protoplast fusion and nutritional complementation between auxotrophic mutants ofAspergillus nidulans andAspergillus fumigatus has been achieved. It is concluded that the nutritional complementation may be due to interspecific aneuploidy.
TL;DR: It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml, and models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior.
Abstract: When the cell wall of Bacillus subtilis is removed by lysozyme and the resultant protoplasts are plated on hypertonic soft agar medium, each protoplast forms an L colony L bodies from such L colonies again plate as L-colony-forming units (CFU) However, if protoplasts or L bodies are "conditioned" by 1 h of incubation in 04% casein hydrolysate medium and then incubated in 25% gelatin medium for 1 h, 60 to 100% of the formerly naked cells give rist to bacillary colonies The present experiments largely explain the mechanism responsible for the "heritable" persistence of the wall-less state in B subtilis It is shown that protoplasts produce a reversion inhibitory factor (RIF) which blocks reversion when the cell concentration exceeds 5 x 105 CFU/ml This inhibitor is nondialyzable and sensitive to trypsin, heat, and detergent Efficient reversion at 2 x 107 CFU/ml is obtained if the protoplasts are treated with trypsin after conditioning and chloramphenicol is incorporated into the gelatin reversion medium In the presence of 500 mug of trypsin per ml, the requirement for gelatin is sharply reduced, and reversion occurs rapidly in liquid medium containing only 10% gelatin Trypsin also stimulates reversion in L colonies growing on soft agar Latent RIF is activated by beta-mercaptoethanol This reagent blocks reversion of protoplast suspensions at densities of 5 x 105 CFU/ml Comparison of the autolytic behavior of B subtilis and of the RIF revealed that several or the properties of the two activities coincide: both are inhibited by high concentrations of gelatin, both are activated by beta-mercaptoethanol, and both have high affinity for cell wall Going on the assumption that RIF is autolysin, models for protoplast reversion is suggested by the finding that mutants with altered teichoic acid show altered reversion behavior
TL;DR: Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicilium cyaneo-fulvum and on transfer gave genetically stable colonies.
Abstract: Slow-growing interspecific heterokaryons were isolated on minimal medium following the induced fusion of protoplasts from auxotrophic mutants of Penicillium chrysogenum and Penicillium cyaneo-fulvum. After 5–7 days cultivation the heterokaryons produced vigorously growing sectors which on transfer gave genetically stable colonies. Cultivation of these colonies on a complete medium supplemented with p-fluorophenylalanine or benomyl broke down this stability and several different prototrophic and auxotrophic colony types were isolated. Many of these behaved as diploids or aneuploids showing sectoring either spontaneously, or in the presence of an haploidizing agent. Some of the latter isolates were recombinants for parental spore colour and auxotrophic markers.
TL;DR: The ready availability of isolated plant protoplasts, and the ability of these naked plant cells to fuse together, has greatly stimulated interest in the production of plant somatic hybrids, and this new protoplast technology may enable interspecies hybrids to be obtained which are otherwise not possible.
Abstract: The ready availability of isolated plant protoplasts, and the ability of these naked plant cells to fuse together, has greatly stimulated interest in the production of plant somatic hybrids. This new protoplast technology may enable interspecies hybrids to be obtained which are otherwise not possible. Many such interspecies hybrids are required amongst the crop plants, but application of this new technology to these species in presently prevented by our inability to culture cells of many of these species in vitro, and to regenerate plants reproducibly from cultured tissue.
TL;DR: The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids, and large multinucleate fusion products developed cell walls but failed to divide.
Abstract: Protoplasts from pea (Pisum sativum L.) leaves and cultured soybean (Glycine max L.) cells were fused by means of polyethylene glycol and subsequently cultured for one week. Both agglutinated protoplasts and cultured fusion products were examined by electron microscopy. Agglutination occurred over large areas of the plasma membranes. The membrane contanct was discontinuous and irregularly spaced. Many cultured fusion products regenerated cell walls and divided to form cell clusters. Fusion of pea and soybean interphase nuclei occurred in some cells. The detection of heterochromatin typical of pea in the synkaryon, even after division, suggests the cells were hybrids. The cytoplasm of the cells from the fusion products contained both soybean leucoplasts and pea chloroplasts. The chloroplasts had apparently ceased dividing and some showed signs of degenerating. Large multinucleate fusion products developed cell walls but failed to divide.
TL;DR: During culture, protoplasts regenerated a cell wall and subsequently divided with the formation of multicellular colonies, requiring a temperature higher than 25°C and the presence of ammonium nitrate and calcium chloride for protoplast survival and division.
TL;DR: Biochemical localization of the enzyme as a function of age of cell culture showed the alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.3.1) activity of Bacillus licheniformis MC14 predominantly in the particulate cell fraction in early- and mid-log cells, however, in late-log and stationary cells, increasing amounts of activity were found in the soluble fraction of lysed cells.
Abstract: Biochemical localization of the enzyme as a function of age of cell culture showed the alkaline phosphatase (orthophosphoric monoester phosphohydrolase, EC 3.1.3.1) activity of Bacillus licheniformis MC14 predominantly in the particulate cell fraction in early- and mid-log cells. However, in late-log and stationary cells, increasing amounts of activity were found in the soluble fraction of lysed cells. Upon protoplast formation of these cells, the activity was released into the soluble fraction. No alkaline phosphatase activity was found in either the cytoplasmic fraction or in the cell medium during any phase of cell growth. The soluble fraction released on protoplast formation that contained alkaline phosphatase activity showed immunological cross-reactivity with antibody to the purified heat--salt-solubilized membrane alkaline phosphatase (F. M. Hulett-Cowling and L. L. Campbell, 1971). Theparticulate membrane fraction containing a firmly associated alkaline phosphatase also showed similar cross-reactivity. Further, the effectiveness of nonionic detergents, ionic detergents, bile salts, and various concentrations of magnesium and sodium as solubilizing agents for this membrane-bound alkaline phosphatase was investigated. Hexadecyl pyridinium chloride (0.03 M) and magnesium and sodium salts (above 0.2 M) were effective solubilizing agents. The substrate specificities of the various fractions were determined and compared to the substrate specificities of the purified membrane alkaline phosphatase.
TL;DR: Cells of the unicellular green alga Chlorella vulgaris formed osmotically fragile protoplasts when incubated for 4–6 h in a defined medium plus 0.4 M mannitol plus0.5% Cellulysin.