Showing papers on "Protoplast published in 1978"

Somatic hybrid plants of potato and tomato regenerated from fused protoplasts

Abstract: Mesophyll protoplasts of Lycopersicon esculentum Mill. var. cerasiforme (Dunal) Alef, mutant yellow green 6, Rick and protoplasts of a liquid callus culture of the dihaploid strain HH258 of Solanum tuberosum L. were prepared and many fusion products were visible after the protoplasts were incubated together first in the presence of polyethylene glycol and then with a high Ca2+ ion concentration. The protoplasts were transferred to a rich medium and the resultant calli were cultured. Some calli regenerated normal green shoots which were transferred to soil or grafted onto a tomato stock. The subunit polypeptide pattern of ribulose 1,5-bisphosphate carboxylase prepared from leaf material of four regenerated plants was analyzed by isoelectric focusing. The ribulose bisphosphate carboxylase enzyme oligomer in the four plants contained the small subunit products resulting from the expression of both tomato and potato nuclear genes proving these plants to be somatic hybrids between tomato and potato. In three of the four plants the large subunit polypeptides and hence the functional chloroplast DNA were from tomato whereas in the fourth the large subunit and therefore the chloroplast DNA was derived from potato. The plant material was insufficient to establish the chromosome numbers precisely, however counts close to 50 which is near to the expected 48 were obtained for three of the hybrids whereas in the fourth a number close to 72 was observed. In the absence of a selection system against the potato parent, the analysis of ribulose bisphosphate carboxylase provides a convenient marker to demonstrate the hybrid nature of the plants.

Photosynthesis by Isolated Protoplasts, Protoplast Extracts, and Chloroplasts of Wheat: Influence of Orthophosphate, Pyrophosphate, and Adenylates

Abstract: Protoplasts, protoplast extracts (intact chloroplasts plus extrachloroplastic material), and chloroplasts isolated from protoplasts of wheat ( Triticum aestivum ) have rates of photosynthesis as measured by light-dependent O 2 evolution of about 100 to 150 micromoles of O 2 per milligram of chlorophyll per hour at 20 C and saturating bicarbonate. The assay conditions sufficient for this activity were 0.4 molar sorbitol, 50 millimolar N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid KOH (pH 7.6), and 10 millimolar NaHCO 3 with protoplast, plus a requirement of 1 to 10 millimolar ethylenediaminetetraacetate (EDTA) and 0.2 to 0.5 millimolar inorganic orthophosphate (Pi) with protoplast extracts and chloroplasts. Protoplast extracts evolved approximately 6 micromoles of O 2 per milligram of chlorophyll before photosynthesis became largely dependent on exogenous Pi while photosynthesis by chloroplasts had a much stronger dependence on exogenous Pi from the outset. Photosynthesis by chloroplasts from 6-day-old wheat plants under optimum levels of Pi was similar to that with the addition of 5 millimolar inorganic pyrophosphate (PPi) plus 0.2 millimolar adenosine-5′-diphosphate (ADP). Either PPi or ADP added separately inhibited photosynthesis. When chloroplasts were incubated in the dark for 2 to 6 minutes, photosynthesis was strongly inhibited by 5 millimolar PPi and this inhibiting was relieved by including adenosine-5′-triphosphate (ATP) or ADP (0.2 to 0.6 millimolar). Chloroplasts from 9-day-old wheat leaves were slightly less sensitive to inhibition by PPi and showed little or no inhibition by ADP. Chloroplasts isolated from protoplasts and assayed with 0.3 millimolar Pi added before illumination have an induction time from less than 1 minute up to 16 minutes depending on the time of the assay after isolation and the components of the medium. In order to obtain maximum rates of photosynthesis and minimum induction time, NaHCO 3 and chelating agents, EDTA or PPi (+ATP), are required in the chloroplast isolation, resuspension and assay medium. With these inclusions in the isolation and resuspension medium the induction time decreased rapidly during the first 20 to 30 minutes storage of chloroplasts on ice. Requirements for isolating intact and photosynthetically functional chloroplasts from wheat protoplasts are discussed.

Genetic Recombination in Streptomyces fradiae by Protoplast Fusion and Cell Regeneration

Abstract: SUMMARY: Conditions for highly efficient genetic recombination in Streptomyces by protoplast fusion are described. Protoplasts of S. fradiae and S. griseofuscus were formed by a modification of the glycine-lysozyme-lytic enzyme method (Okanishi, Suzuki & Umezawa, 1974). Regeneration of cells from protoplasts was monitored throughout the growth cycle and was most efficient when cells of either S. fradiae or S. griseofuscus were taken from the transition phase between the exponential and stationary growth phases. Fusion of protoplasts carrying different auxotrophic or chromosomal drug-resistance markers was achieved by treatment with polyethylene glycol, and high frequencies of stable genetic recombinants were obtained.

Regeneration of Mesophyll Protoplasts Isolated from Dihaploid Clones of Solanum tuberosum

Abstract: Protoplasts have been isolated from leaves of shoot cultures of six dihaploid clones of Solanum tuberosum L. (2n = 2x = 24). In the KM medium (Kao and Michayluk 1975), sustained cell divisions were obtained in up to 50% of the plated protoplasts of four clones, whereas only a few divisions occurred in the other two clones. The first mitosis appeared 2–8 days after plating, dependent on the clones. In the clones showing sustained cell divisions, a protoplast titre of about 5 × 103 per ml turned out to be optimal. The culture conditions for protoplasts of one of the poorly growing clones, clone H2 140, have been improved using modified KM media, plating at a concentration of as high as 5 × 104 cells per ml, and subsequent diluting at intervals 5 days. The dilutions were carried out with media containing 0.25% agar. Up to 60% of the plated protoplasts underwent divisions within 10 days under these conditions. After about 15 days, the regenerants were transferred onto media inducing organogenesis. Shoots and roots were formed on modified media MS (Murashige and Skoog 1962) and B5 (Gamborg et al. 1968). Plants have been regenerated in four of the investigated clones. Countings of chromosomes revealed a satisfactory stability of the karyotype in shoot culture and protoplast regeneration.

Bacterial protoplast fusion: Recombination in fused protoplasts of Streptomyces coelicolor

Abstract: Numerous recombinants arose when protoplasts of S. coelicolor were treated with polyethylene glycol and regenerated on non-selective solid medium. In six-factor crosses, recombination frequencies of more than 10% (up to 17%) were routinely observed. This recombination did not require either of the known sex factors, SCP1 and SCP2. The proportion of multiple crossover classes was much higher than amongst recombinants produced by conjugation between mycelia. Analysis of the spatial distribution of crossovers in double and quadruple crossover recombinants showed only a slight tendency for crossovers to occur closer together than randomly on the complete linkage group. This suggests that genomes brought together by protoplast fusion are complete, or nearly so (in conjugation, in contrast, one genome is represented by a comparatively short fragment). Individual colonies arising from fused protoplasts did not contain different parental genomes without recombinants, but recombinants often occurred without parentals. Several recombinant genotypes often occurred in the same colony, showing a segregation of some, only, of the parental alleles. Complementary genotypes, parental or recombinant, did not occur in the same colony. It is postulated that complete genomes of fused protoplasts usually become fragmented and that crossing-over, often repeated, occurs between the fragments, to generate haploid recombinants. Analysis of fusions between protoplasts of four different genotypes indicated that the average number of protoplasts fusing together was low, but nevertheless appreciable numbers of fusions involved three or four genomes. Crossing-over between them produced recombinants inheriting markers from three or four parents. The generation of nearly random populations of recombinants between two or more parent strains by protoplast fusion under the conditions described appears to have simple applications in industrial and academic strain construction.

Somatic Hybridization of Nitrate Reductase Deficient Mutants of Nicotiana tabacum by Protoplast Fusion

Abstract: Protoplasts were isolated from two mutant cell lines of Nicotiana tabacum L. cv. Gatersleben and fused with the aid of polyethylene glycol. Both mutants lacked nitrate reductase and were thus auxotrophic for reduced nitrogen. The fusion resulted in a high frequency of hybrid cells which were detected by their regained ability to grow in media containing nitrate as sole nitrogen source. Thus, the two mutants were found to complement each other in the hybrids. In control experiments, back mutation and cross-feeding were excluded as possible explanations for the occurrence of cell lines utilizing nitrate. A total of 1061 hybrid lines capable of sustained proliferation were isolated. Some of them were further characterized with respect to nitrate reductase activity, chlorate sensitivity, chromosome number, and shoot formation. The results demonstrate that protoplast fusion can be used for the genetic analysis of cell variants of higher plants and that nitrate reductase-deficient mutants provide efficient selective systems for hybrid cells.

Freeze-thaw injury to isolated spinach protoplasts and its simulation at above freezing temperatures.

Abstract: Possibilities to account for the mechanism of freeze-thaw injury to isolated protoplasts of Spinacia oleracea L. cv. Winter Bloomsdale were investigated. A freeze-thaw cycle to −3.9 C resulted in 80% lysis of the protoplasts. At −3.9 C, protoplasts are exposed to the equivalent of a 2.1 osmolal solution. Isolated protoplasts behave as ideal osmometers in the range of concentrations tested (0.35 to 2.75 osmolal), arguing against a minimum critical volume as a mechanism of injury. Average protoplast volume after a freeze-thaw cycle was not greatly different than the volume before freezing, arguing against an irreversible influx of solutes while frozen. A wide variety of sugars and sugar alcohols, none of which was freely permeant, were capable of protecting against injury which occurred when protoplasts were frozen in salt solutions. The extent of injury was also dependent upon the type of monovalent ions present, with Li = Na > K = Rb = Cs and Cl ≥ Br > I, in order of decreasing protoplast survival. Osmotic conditions encountered during a freeze-thaw cycle were established at room temperature by exposing protoplasts to high salt concentrations and then diluting the osmoticum. Injury occurred only after dilution of the osmoticum and was correlated with the expansion of the plasma membrane. Injury observed in frozen-thawed protoplasts was correlated with the increase in surface area the plasma membrane should have undergone during thawing, supporting the contention that contraction of the plasma membrane during freezing and its expansion during thawing are two interacting lesions which cause protoplast lysis during a freezethaw cycle.

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation.

Abstract: A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H2O, the gradients provide a density range from 1.017 to 1.069 g/cm3 at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm3, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.

Growth of Avena Coleoptiles and pH Drop of Protoplast Suspensions Induced by Chlorinated Indoleacetic Acids

Abstract: Several indoleacetic acids, substituted in the benzene ring, were compared in the Avena straight growth bioassay. 4-Chloroindoleacetic acid, a naturally occurring plant hormone, is one of the strongest hormones in this bioassay. With an optimum at 10-6 mol l-1, it is more active than indoleacetic acid, 2,4-dichlorphenoxyacetic acid and naphthaleneacetic acid. 5-Chloro- and 6-chloroindoleacetic acids are very strong auxins as well. Other derivatives tested have a lower activity. 5,7-Dichloro- and 5-hydroxyindoleacetic acids have very low auxin activity at 10-4 mol l-1 and may be anti-auxins. Some of the derivatives were compared for their effect on pH decline in stem protoplast suspensions of Helianthus annuus L. and Pisum sativum L. The change of pH occurs without a lag period or with only a very short one. Derivatives which are very active in the Avena straight growth assay cause a larger pH decline than indoleacetic acid, while inactive derivatives cause effectively no pH decline.

Dual Mechanisms in Polyamine-mediated Control of Ribonuclease Activity in Oat Leaf Protoplasts

Abstract: Dibasic amino acids and polyamines added to oat (Avena sativa L.) leaf protoplast isolation media decrease the RNase activity of extracted protoplasts relative to controls. This effect, which is manifested even when the added polyamine is removed by exhaustive dialysis prior to assay, is due to a prevention of the rise in RNase activity which usually follows protoplast isolation. Polyamines, but not dibasic amino acids, also decrease RNase activity in vitro. This in vitro effect seems to result from electrovalent attachment of the polyamine to the RNA, because the greater the net positive charge on the polyamine, the greater is its inhibitory effect in vitro. The activity of dibasic amino acids when added during protoplast isolation probably results from their conversion to polyamines.

Surface charge of protoplasts and their significance in cell-cell interaction.

Abstract: ζ-potential of mesophyll protoplasts of tobacco (Nicotiana tabacum L.), petunia (Petunia hybrida Hort.), turnip (Brassica rapa L.) and cowpea (Vigna unguiculata L. Walpers) was determined by use of a cell electrophoresis apparatus. All protoplasts examined showed a constant negative value of-10 to-35 mV. The addition of CaCl2 nullified the ζ-potential of tobacco protoplasts. This phenomenon is explained by DLVO theory of colloid science, which has been successfully applied to animal cells. Furthermore, positively charged polymers reversed the ζ-potential to positive values. Treatment of the protoplast surface with several enzymes was carried out to characterize the chemical nature of suface charges. The removal of surface charges was most conspicuous by the treatment of acid phosphatase (EC 3.1.3.2), but did not occur upon treatment with α-neuraminidase (EC 3.2.1.18) or Streptomyces griseus pronase. Thus a major part of the surface charge originates from the phosphate groups at the cell membrane. The significance of these studies for the properties of the protoplast surface in cell adhesion is discussed.

Genetic instability in somatic hybrids of Nicotiana tabacum and Nicotiana knightiana

Abstract: Somatic hybrids of Nicotiana knightiana (2n=2X=24) and an albino mutant of Nicotiana tabacum (2n=4X=48) were selected after polyethylene glycol induced protoplast fusion. Three lines were selected on the basis of the simultaneous expression of shoot inducibility and green pigmentation, traits originally separated in the parental species. The hybrid nature of the lines was confirmed by their characteristic isoenzyme patterns, the morphology of the regenerated plants, and by the appearance of heterochromatic blocks in the interphase nuclei. Chromosome numbers in the somatic hybrids varied greatly within individual plants. Variegation in leaf and flower colour and segregation for morphological traits in vegetatively multiplied plants are attributed to segregation of chromosomes in the somatic cells, a consequence of the numerical instability. Hybridity, caryotypic changes induced by tissue culture, and high chromosome numbers, are discussed as possible reasons for the observed genetic instability.

Fusion of Yeast Protoplasts Induced by Polyethylene Glycol

Abstract: SUMMARY: Protoplasts, prepared from auxotrophic strains of Saccharomyces cerevisiae, Schizosac-charomyces pombe and Hansenula wingei, were mixed to give intraspecific complementary combinations. Polyethylene glycol (PEG) was added to induce agglutination and fusion. Some of the fused products grew on the surface of solid minimal medium forming large vacuolated bodies. Others reverted to hybrid cells when embedded in solid minimal regeneration medium. The cytological and preliminary genetical analyses suggest a synkaryon formation and integration of genetic markers from parental strains. The frequency of intrageneric fusions assessed from the number of protoplasts growing on the surface of minimal agar was estimated to be 1 to 3%, while the frequency of hybrid colony formation in regeneration medium was less than 1%.

Viability of Rhizobium bacteroids isolated from soybean nodule protoplasts.

Abstract: Bacteriods isolated from protoplasts taken from Rhizobium japonicum induced root nodule of Glycine max L. showed complete viability when plated onto a conventional rhizobial growth medium supplemented with 0.2 M Mannitol. The same medium but without extra mannitol resulted in the absence of colony formation. The protoplast isolation method eliminated the possibility of contaminant bacteria from infection threads to be scored. The redifferentiated bacteroid clones have the same genetical characteristics as the orginal inoculum strain. This and other recent findings of bacteroid viability are discussed in the light of the existing belief that bacteroids are non-viable.

Somatic hybrids of plants by fusion of protoplasts

Abstract: Fusions of protoplasts from Nicotiana langsdorffii and Nicotiana glauca were induced using polyethylene glycol. Parasexual hybrid colonies were selected for their ability to grow without growth substances. Hybrid plants, regenerated after grafting, were all tumorous and exhibited morphological and chromosome number variations. Out of 48 colonies selected in vitro only 6 regenerated flowering plants. Two of these plants had 42 chromosomes and were morphologically identical to the sexual amphidiploid Nicotiana glaucaxlangsdorffii.

Genetic analysis of products of protoplast fusion in saccharomyces cerevisiae

Abstract: Protoplasts of Saccharomyces cerevisiae were prepared from two different haploid strains both of mating type a, which carried different nuclear (ade1, ura1, his4, leu2 and thr4) and mitochondrial (ρ, ω, CR, ER and OR) markers, and were fused with the aid of polyethylene glycol. Cells of fused products (prototrophs) displayed phenotype of mating type a and were crossed to mating type α/α diploids having auxotrophic markers, e.g. trp1. On sporulation of the resulting hybrid clones, as a rule, there were three tetrad types for mating types, i.e. (I) 4non-maters, (II) 2a:2α and (III) a:α: 2non-maters. The relative frequencies of these three tetrad types were close to the ones theoretically predicted from a/a/α/α tetraploids, suggesting that the fusion products were a/a diploids. Auxotrophic markers involved in these crosses, which were located on four different chromosomes, were also segregated to yield the tetrad distributions expected from the parentages. Consequently, it was concluded that the protoplast fusion proceeded to karyogamy to produce stable diploids. A study of mitochondrial recombination demonstrated that the fusion products accepted the mitochondrial genome (the polar gene ω as well as the drug resistance genes) from one parent of ρ+, but not from another of neutral petite.

Plant protoplast agglutination by artificial carbohydrate antigens

Abstract: The existence of beta-lectins on protoplast surfaces is confirmed by the agglutination of protoplasts by those Yariv antigens that have sugar specificities which also interact with isolated beta-lectins. Agglutination by beta-maltosyl but not by beta-D-mannosyl Yariv antigens is used to identify some of the structural features required of the ligand for beta-lectin binding. Inhibition of agglutination by phenolic glycosides and the effect of protoplast fixation are also investigated.

Somatic Hybridization by Fusion of Protoplasts : 1. The combination of Nicotiana tabacum and Nicotiana rustica

Abstract: Mesophyll protoplasts of Nicotiana tabacum. cv. Bright Yellow mutant Aurea and Burley 21, and of N. rustica. cv. Rustica were prepared from leaf tissue by enzymatic digestion and were fused with the aid of polyethylene glycol. When the fused protoplasts were plated on an agar medium or in a liquid medium for cell culture, new cell walls were regenerated from them and the first cell division could be seen beginning on the 12th day of culture, and cell division process took place on and after the 21th day (Fig. 1). After one month culture, almost all of the surviving fused protoplasts grew to cell colonies of 1-2 mm in diameter. The cell colonies were subcultured on an agar medium for callus culture. On the medium, they grew vigorouly and formed calli of 2 cm in diameter after 2 months culture. At this stage, the hybrid calli showing greenish white with a compact appearance were selected in contrast to parental type calli tinged with white or green color (Fig. 2). Ten somatic hybrid calli of the combination of Aurea and Rustica and 3 calli of the combination of Burley 21 and Rustica were obtained. Many plantlets were differentiated from these calli when transfered to a medium enriching zeatin (Fig. 3). All of the mature flowering plants showed to be perfectly hybrid, and the shape of the leaves and flowers indicated an intermediate between N. tabacum and N. rustica (Fig. 5, 6). The plant height of the normal hybrid morphologically was higher than that of the parent plant and all of the hybrid plants were highly resistant to the tobacco mosaic virus (Fig. 4, Table 1, 2). The chromosome numbers of the somatic hybrids showed to be the aneuploid type having from 60 to 91 chromosomes (Table 1, 2). The normal hybrid plants morphologically, however, have a pollen fertility, and seeds and progenies were obtained from 4 of the Aurea-Rustica hybrids and 3 of the Burley 21-Rustica hybrids (Table 1, 2).

Interspecies matings of Streptomyces fradiae with Streptomyces bikiniensis mediated by conventional and protoplast fusion techniques.

Abstract: A genetic recombination system for an interspecies mating of Streptomyces bikiniensis with Streptomyces fradiae is demonstrated both by conventional and protoplast fusion methods.

Polyethylene glycol-induced fusion of heat-inactivated and living protoplasts of Bacillus megaterium.

Abstract: Protoplasts of Bacillus megaterium, incubated at 50 degrees C for 120 min, lost the ability to revert to bacillary form. Such heat-inactivated protoplasts, however, produced recombinants when fused by polyethylene glycol treatment with normal protoplasts. Although this differential inactivation effect is not yet fully reproducible, reciprocal inactivations of the parental protoplasts in genetic crosses have clearly shown that for protoplast fusion (i) either of the parents may serve as the viable recipient for markers coming from the heated parental protoplasts, and (ii) either of the parents may be rendered nonviable and yet, when fused with a viable partner, contribute to formation of a recombinant. Heat inactivation seems to provide a way to counterselect when few markers are available and one of the parents is prototrophic.

Fine structure and cytochemical properties of tobacco leaf protoplasts and comparison with the source tissue

Abstract: Protoplasts isolated from tobacco leaves showed the development of crystalline bodies in the chloroplasts and osmiophilic droplets in the chloroplasts and cytoplasm. Microbodies stained for catalase with the diaminobenzidine (DAB) reaction although, in the absence of H2O2, mitochondrial cristae also showed a dense reaction. Similar changes and DAB staining were observed in intact leaf cells aged in media similar to that used for protoplast isolation. The cell wall and plasma membrane of fresh leaf cells stained inconsistently with silicotungstic acid (STA). In comparison, the plasma membrane of freshly isolated protoplasts stained very poorly with STA although more intense staining was found in aged protoplasts. The surface of protoplasts showed heavy staining when incubated in lanthanum nitrate or cationized ferritin though not with ruthenium red. The potential of these surface stains as plasma membrane markers in isolated membrane fractions is discussed.

A Comparison of Regenerated Cell Walls in Tobacco and Cereal Protoplasts

Abstract: Four independent kinds of observations indicate that the cell wall regenerated by oat (Avena sativa L.) and corn (Zea mays L.) protoplasts in culture is less well developed than that regenerated by tobacco (Nicotiana tabacum L.) protoplasts. Following wall regeneration the cereal protoplasts remained susceptible to osmotic shock upon transfer to water, showed great enlargement, stained poorly with calcofluor white, and maintained a positive internal electrical potential. The development of a negative membrane potential by tobacco protoplasts in culture often occurred simultaneously with the onset of cell division. Since division was observed only in protoplasts which had regenerated good cell walls and had re-established negative membrane potentials it is suggested that culture conditions which favor these two processes should improve protoplast viability.