Showing papers on "Protoplast published in 1993"

Method for transforming monocotyledons

Abstract: A method for transforming a monocotyledon by which the time required from transformation to regeneration of a plant is shorter so that the frequency of emergence of mutants is smaller than the conventional methods, which may be generally applied even to the plants for which the regeneration method from a protoplast to a plant has not been established, and with which the preparation of the material to be subjected to the method is easy. That is, the present invention provides a method for transforming a monocotyledon comprising transforming a cultured tissue during dedifferentiation process or a dedifferentiated cultured tissue of said monocotyledon with a bacterium belonging to genus Agrobacterium containing a desired gene.

Method of transforming monocotyledon.

Abstract: A method of transforming monocotyledon which necessitates only a short period from the transformation to the generation of a plant body as compared with the conventional methods, thus reducing the frequency of occurence of mutants, and can be generally applied to the plant for which any system of regenerating the plant body from the protoplast has not been established, and in which the material to be used can be readily prepared. The method comprises transforming cultured tissues of a monocotyledon under or after dedifferentiation with a bacterium of the genus (Agrobacterium) containing desired genes.

Activity of a chimeric promoter with the doubled CaMV 35S enhancer element in protoplast-derived cells and transgenic plants in maize

Abstract: A reproducible and efficient transformation system has been developed for maize that is based on direct DNA uptake into embryogenic protoplasts and regeneration of fertile plants from protoplast-derived transgenic callus tissues. Plasmid DNA, containing the β-glucuronidase (GUS) gene, under the control of the doubled enhancer element (the −208 to −46 bp upstream fragment) from CaMV 35S promoter, linked to the truncated (up to −389 bp from ATG) promoter of wheat, α-amylase gene was introduced into protoplasts from suspension culture of HE/89 genotype. The constructed transformation vectors carried either the neomycin phosphotransferase (NPTII) or phosphinothricin acetyltransferase (PAT) gene as selective marker. The applied DNA uptake protocol has resulted at least in 10–20 resistant calli, or GUS-expressing colonies after treatment of 106 protoplasts. Vital GUS staining of microcalli has made possible the shoot regeneration from the GUS-stained tissues. 80–90% of kanamycin or PPT resistant calli showed GUS activity, and transgenic plants were regenerated from more than 140 clones. Both Southern hybridization and PCR analysis showed the presence of introduced foreign genes in the genomic DNA of the transformants. The chimeric promoter, composed of a tissue specific monocot promoter, and the viral enhancer element specified similar expression pattern in maize plants, as it was determined by the full CaMV 35S promoter in dicot and other monocot plants. The highest GUS specific activity was found in older leaves with progressively less activity in young leaves, stem and root. Histochemical localization of GUS revealed promoter function in leaf epidermis, mesophyll and vascular bundles, in the cortex and vascular cylinder of the root. In roots, the meristematic tip region and vascular tissues stained intensively. Selected transformants were grown up to maturity, and second-generation seedlings with segregation for GUS activity were obtained after outcrossing. The GUS-expressing segregants carried also the NPTII gene as shown by Southern hybridization.

Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA

Abstract: Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.

Use of bar as a selectable marker gene and for the production of herbicide-resistant rice plants from protoplasts

Abstract: We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2-4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was verified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T0 plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T0 plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.

Selection of transformed protoplast-derived Zea mays colonies with phosphinothricin and a novel assay using the pH indicator chlorophenol red

Abstract: Phosphinothricin (PPT) inhibits glutamine synthetase in plant cells, resulting in an accumulation of ammonia that can be toxic. Bar, from Streptomyces hygroscopicus is a gene that codes for phosphinothricin acetyl-transferase, an enzyme that detoxifies PPT by acetylation. We used polyethylene-glycol-mediated transformation to introduce bar into protoplasts of maize (Zea mays L.). With a novel assay utilizing the pH indicator chlorophenol red we identified transformants after only two to four weeks of selection on PPT.

Foreign gene expression in chloroplasts of higher plants mediated by tungsten particle bombardment.

Abstract: Publisher Summary Several approaches have been used in the past to manipulate genes in chloroplasts—that is, in the generation of chloroplast mutants, protoplast fusion, organelle inactivation, and chloroplast recombination Reports of the introduction of chloroplasts into albino protoplasts and the observation of variegated progeny with the transfer of only two chloroplasts open up the possibilities of introduction of transformed chloroplasts into recipient protoplasts Transient expression of foreign genes in chloroplasts of tobacco, sugar beet, and wheat cells, and in leaves or calli has been observed It is clear that chloroplast promoters are interchangeable among monocots and dicots; the cat gene driven by the maize rbcL promoter functions in tobacco chloroplasts and the uidA gene driven by the pea psbA promoter functions in wheat chloroplasts In addition, chloroplast vectors introduced into isolated protoplasts by the electroporation or polyethylene glycol (PEG)-mediated DNA uptake do not express foreign genes in the nuclear compartment

Plant regeneration through direct somatic embryogenesis from protoplasts of banana (Musa spp.).

Abstract: We report the isolation and regeneration of protoplasts from an embryogenic banana (Musa spp.) cell suspension culture initiated from in vitro proliferating meristems. A high yielding isolation method (up to 6×107 protoplasts.ml−1 packed cells) is discussed. Optimal regeneration, with more than 50% of the protoplasts showing initial cell division, occurred when high inoculation densities (106 protoplasts.ml−1) or nurse cultures were applied. Under these conditions, the frequency of microcolony formation was 20–40%. These microcolonies developed directly, without an intervening callus phase, into somatic embryos which later germinated and formed plantlets.

Interspecific somatic hybridization between Solanum tuberosum L. and S. bulbocastanum Dun. as a means of transferring nematode resistance.

Abstract: Interspecific somatic hybrids were produced between tetraploid Solanum tuberosum and a nematode-resistant accession of the diploid species So/ahum bulbocastanum by protoplast fusion. Hybrid cells were selected using dual fluorescent labeling of protoplast preparations prior to fusion. Hybridity of regenerated plants was confirmed with a combination of morphological assessment, chromosome counting and isozyme analysis. Somatic hybrids had the same level of resistance to infection by race 1 of the nematode Meloidogyr~ chitwoodi as the S. bulbocastanum parent used in the fusion. Some of the somatic hybrids were fertile as females when crossed with tetraploid S. tuberosum breeding lines. Thus, these hybrids can be used in a potato improvement program to incorporate a valuable pest resistance.

Production and characterization of asymmetric somatic hybrids between Arabidopsis thaliana and Brassica napus.

Abstract: Cell suspension-derived protoplasts of a chlorsulfuron-resistant (GH50) strain of Arabidopsis thaliana cv Columbia were X-irradiated at 60 or 90 krad, to facilitate the elimination of GH50 donor chromosomes in fusion products. Irradiated GH50 protoplasts were fused, with polyethylene glycol, to protoplasts derived from stem epidermal strips of Brassica napus cv Westar. Chlorsulfuron-resistant colonies were selected in vitro and then transferred to shoot and root regeneration medium. Seventeen hybrid lines were regenerated in vitro, and eight were successfully established in the greenhouse, where they flowered. These eight asymmetric hybrids were intermediate in vegetative morphology between Arabidopsis and Brassica. The flowers from these hybrids were male-sterile with abnormal petal and pistil structures. Zymograms for phosphoglucomutase, esterase, and peroxidase showed the presence of all parental isozymes in each of the hybrids tested. Nuclear hybridity was also confirmed for the ribosomal RNA genes using a wheat rDNA probe; however, the chloroplast genome in each of the hybrids was derived solely from the Brassica parent. All selected somatic hybrids were capable of rooting at levels of chlorsulfuron which were inhibitory to unfused Brassica plantlets. The degree of herbicide resistance in the hybrid shoots is presently being evaluated.

Organellar segregation, rearrangement and recombination in protoplast fusion-derived Brassica oleracea calli.

Abstract: Cauliflower protoplasts were fused to determine the effect of protoplast source and pretreatment on organellar segregation in fusion products. Mitochondrial and chloroplast type were determined for over 250 calli from eight fusions between iodoacetate-treated or γ-irradiated leaf or hypocotyl protoplasts with fertile or Ogura cytoplasms. Organelles in fusion-derived calli were identified with five mitochondrial probes and one chloroplast probe. Mitochondrial and chloroplast segregation were independent but biased. Most calli had B. oleracea chloroplasts, but more calli had Ogura mitochondria than B. oleracea ones. Neither protoplast source nor pretreatment alone affected organelle segregation. However, iodoacetate treatment of hypocotyl protoplasts reduced their mitochondrial contribution to the fusion products although it did not affect chloroplast segregation. Over half of the calli had mitochondrial genomes distinct from those of either fusion partner; many of these contained the complete mitochondrial genome of one partner along with some mitochondrial DNA from the other. Out of 258 calli, 83 showed evidence of mitochondrial recombination, most commonly by formation of a novel 11-kb PstI fragment near the atp9 region.

Distribution of 64Cu in Saccharomyces cerevisiae: cellular locale and metabolism.

Abstract: Summary: The metabolism of copper in the yeast Saccharomyces cerevisiae has been studied with respect to the distribution and stability to exchange of newly arrived 64Cu. Cells pre-incubated with 10 μm-Cu2+ accumulated 64Cu into two pools distinguishable by cellular locale and lability to exchange with extracellular cold copper. One pool was non-exchangeable and was localized to protoplasts. Size-exclusion chromatography of a soluble cell (protoplast) extract showed that this 64Cu was associated with up to four species. Two were identified as copper metallothionein and Cu,Zn superoxide dismutase based on comparisons of chromatograms derived from strains in which the genes for these two proteins had been deleted. A third species was identified as copper-glutathione based on chromatographic and biochemical assays. A second pool was exchangeable and was localized to the cell wall. In contrast to its rapid copper-stimulated exchange (t1/2 % 1 min), this pool exhibited only slow efflux (10% 64Cu loss per 60 min). Zn2+ did not stimulate the loss of 64Cu from this pool indicating that it was selective for copper. This pool was released into the supernatant upon protoplast formation and was found in the cell wall debris obtained when cells were mechanically disrupted. This 64Cu eluted in the void volume (peak Pv) of the column used to size-fractionate copper-binding species. The metal in Pv was exchangeable in vivo and in vitro. However, the corresponding chromatographic fraction obtained from copper-naive cells when labelled in vitro could bind less than 20 % of the 64Cu bound to it in vivo indicating that the deposition of copper in this pool was primarily cell-dependent. In fact, this deposition was shown to be dependent on the cellular reduction of medium sulphate or sulphite to the level of sulphide, or on the addition of sulphide to the 64Cu uptake buffer. 64Cu in the non-exchangeable protoplast pool was not mobilized by cellular sulphide generation, indicating that cellular sulphide generation did not causally lead to the partitioning of 64Cu to the cell wall pool. The data indicate that the appearance of copper sulphide(s) on the cell wall in S. cerevisiae is gratuitous and does not represent a sulphide-based mechanism of copper resistance in this yeast.

Intergeneric somatic hybridization of sexually incompatible parents: Citrus sinensis and Atalantia ceylanica

Abstract: Protoplast fusion using polyethylene glycol (PEG) was conducted to combine Citrus sinensis (L.) Osbeck cv. ‘Hamlin’ sweet orange protoplasts, isolated from nucellus-derived embryogenic callus with Atalantia ceylanica (Arn.) Oliv, leaf protoplasts. Five plants regenerated from independent fusion events following protoplast culture were identified as intergeneric allotetraploid somatic hybrids of ‘Hamlin’ sweet orange and A. ceylanica, and confirmed by isozyme analysis and chromosome number determination in root tip cells (2n=4x=36). Two different types of leaf morphology were observed among the hybrids (normal and narrow), although no differences in chromosome number nor isozyme banding patterns were observed. This is the first report of the production of hybrid plants between these sexually incompatible genera.

Male-sterile chicory cybrids obtained by intergeneric protoplast fusion.

Abstract: Male-sterile chicory plants were obtained by fusion of chicory mesophyll protoplasts and hypocotyl protoplasts derived from male-sterile sunflower plants. The protoplasts of both species were fused by the PEG method and the products were selected manually and cultivated at very low density in a liquid medium. Three to twenty percent of the heterokaryocytes divided and evolved into microcalli, then into calli where budding could be induced. The mitochondrial genome of ten male-sterile or totally sterile plants was studied. Restriction endonuclease profiles of mitochondrial DNA and molecular hybridization with specific genes of the mitochondrial genome used as probes indicated that mitochondrial DNA rearrangement had occurred between sunflower and chicory and the intensity of the rearrangements correlated with the degree of sterility of the different plants.

Somatic hybridization between Dianthus chinensis and D. barbatus through protoplast fusion

Abstract: Protoplasts isolated from leaf mesophyll cells of Dianthus chinensis and D. barbatus were fused by polyethylene glycol (PEG). Calli exhibiting vigorous growth were selected from the PEG-treated protoplasts and shoots were regenerated from one of these calli after 5 months of culture. These shoots readily rooted and continuously produced flowers in the in-vitro condition. The data on flower color, chromosome number, and esterase isozyme patterns indicated that this plantlet was an interspecific somatic hybrid. The hybridity of the plantlet was also confirmed by nuclear rDNA analysis. This report provides the possibility of applying the somatic hybridization technique for the genetic improvement of the genus Dianthus.

Plant regeneration from mesophyll protoplasts of rice (Oryza sativa L.)

Abstract: We report induction of sustained divisions, colony formation and plant regeneration from mesophyll protoplasts isolated from the leaf base and sheath of rice seedlings. Protoplasts were purified on sucrose density gradients and cultured in modified liquid N6 medium in the presence of a feeder layer prepared from young rice-cell suspension cultures. Protoplasts formed cell walls and divided after 6 to 7 days giving rise to microcolonies which became macroscopic after 25 to 30 days. Nine to ten week old protocalli, on transfer to regeneration medium, formed somatic embryos that developed into shoots which were rooted on MS medium. Plantlets were recovered from four varieties, which on subsequent transfer to Yoshida's culture solution developed into healthy plants that were transferred to pots for further growth. Our results show that contrary to popular belief, mesophyll protoplasts of rice and perhaps other gramineous plants are capable of dedifferentiation and re-entry into the cell cycle, and are totipotent.

Production of somatic hybrids between frost tolerantSolanum commersonii andS. Tuberosum: Protoplast fusion, regeneration and isozyme analysis

Abstract: Mesophyll protoplasts ofSolanum commersonii, a frost tolerant wild species not crossable with the cultivated potato, were fused with either dihaploid or tetraploid S.tuberosum. Protoplasts were aggregated by means of alternating current (AC) or polyethylene glycol (PEG), and electrofused with three direct current (DC) pulses. The treatments with PEG/DC generally resulted in very low heterofusion frequency and protoplast viabiity. On the other hand, AC/DC fusion conditions were optimized by increasing the fusion density of protoplasts and adding CaCl2 to fusion medium. When a density of 4.8 × 105 protoplasts ml−1 was used in the fusion medium containing 0.2 mM Ca++, AC/DC treated protoplasts showed heterofusion frequencies and plating efficiencies of about 10 and 3%, respectively. Fast growing calli from AC/DC fusion experiments were further cultured for regeneration. Fifty-seven plants were regenerated and clonedin vitro as shoot cultures. Compared to parents they showed heterotic vigor and could be identified as hybrids, based on isozyme analysis.

Rapid isolation and sequencing of purified plasmid DNA from Bacillus subtilis.

Abstract: We report two methods for isolation of plasmid DNA from the gram-positive bacterium Bacillus subtilis. The protoplast alkaline lysis procedure was developed for general use, and the protoplast alkaline lysis magic procedure was developed for isolation of DNA for sequencing. Both procedures yielded large amounts of high-quality DNA in less than 1 h, while current protocols require 4 to 7 h to perform and give lower yields and quality. Plasmid DNA was obtained from strains containing either high- or low-copy-number plasmids. In addition, the procedures were easily adapted to yield large amounts of plasmid DNA suitable for sequencing from another gram-positive organism, Staphylococcus aureus. Further, we demonstrated that neither chloramphenicol, used for plasmid selection, nor the mutation recE4 reduced plasmid DNA yield from the strains we examined.

Effects of chlorhexidine diacetate and cetylpyridinium chloride on whole cells and protoplasts of Saccharomyces cerevisiae.

Abstract: Chlorhexidine diacetate (CHA) and the quaternary ammonium compound, cetylpyridinium chloride (CPC), were fungicidal to Saccharomyces cerevisiae A364A and CHA to its mannoprotein mutant LB6-5D. Both CHA and CPC induced leakage of K+ and pentose material from both strains and both agents induced protoplast lysis as well as interacting with crude cell sap. Differences were observed between CHA and CPC in protoplast lysis and in cell sap interaction. Q25 values for A364A with CHA as test agent were 5.6 and 1.6 (fungicidal activity, depending on method of calculation), 1.46 (cell sap interaction) and 0.77 (leakage of pentoses). A sub-lytic concentration of CHA reduced considerably protoplast regeneration. Although the plasma membrane is an important target site for both agents, interaction with other cellular components also contributes to fungal death.

Isolation of protoplasts from developing xylem of Pinusbanksiana and Pinusstrobus

Abstract: Protoplasts were isolated from developing xylem of Pinusbanksiana Lamb, and Pinusstrobus L. by incubating freshly harvested tissue in a cellulose–pectinase mixture having mannitol as osmoticum. Protoplasts were then purified using a discontinuous sucrose–mannitol gradient. More than 70% of the isolated protoplasts were of small diameter (12–27 μm) and had dense cytoplasm and many small vacuoles, suggesting that they originated from ray cells. Larger protoplasts constituted about 25% of the protoplast population; these contained single large vacuoles and only parietal cytoplasm, suggesting that they originated from fusiform cells. Using combined gas chromatography–mass spectrometry, coniferin was confirmed to be present in protoplast preparations. By high-performance liquid chromatography (258 nm UV detection), coniferin was readily detected in protoplasts and in extracts of developing xylem from both species. On a fresh-weight basis, coniferin occurred at 1.0–1.6 mM in protoplasts. In late June, coniferin...

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

Abstract: The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine (Vitis vinifera) L. cv. Sultanina) and regenerating tobacco (Nicotiana tabacum) L. cv. Xanthi). Using [35S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.

Regeneration from protoplasts-a supplementary literature review

Abstract: Protoplast technology is important in, on the one hand, the application of various cellular methods in plant breeding (i.e. somatic hybridization, cybridization, direct DNA transfer via polyethylene glycol (PEG), electroporation, micro-injection), and various fundamental studies (e.g. on membrane transport, cell compartmentation, the cytoskeleton in relation to the cell cycle and cell division) on the other hand. For practical applications, plant regeneration from protoplasts is a prerequisite. In 1989, regeneration from protoplasts was listed (Roest & Gilissen 1989) for 214 higher plant species (Spermatophyta), representing 97 genera and 31 families. Since then, regeneration procedures for more than 100 higher plant species have been reported. These include many economically important agricultural and horticultural crops, as well as woody plant species. In this paper these new species are listed, supplemented with specific information on the donor tissue used, the culture technique applied, and the type of development of the regenerants. In addition, recent achievements in fundamental aspects of protoplast research, which gradually provide further insight into the genetical, physiological and ultrastructural background of the phenomenon of totipotency of plant cells, will be briefly reviewed.

Improved conditions for protoplast formation and transformation of Pleurotus ostreatus

Abstract: Conditions suitable for the production and regeneration of Pleurotus ostreatus protoplasts from dikaryotic mycelia were examined. Three commercially available muralytic enzymes, including Sigma lysing enzyme, Novozym 234 and Novozym 234 LP, were used for production of protoplasts. Over 2 × 107 protoplasts per gram fresh weight mycelia were obtained within 1.5 h by using each of these three enzymes. The colony regeneration rate was up to 13% on potato-dextrose-agar medium containing 0.8 m mannitol. Genetic transformation was based on positive selection for resistance to hygromycin B (HmB) using the plasmid vector pAN7-1 and accomplished by either electroporation or a polyethylene glycol (PEG)-divalent cation method. P. ostreatus strains used in this study have innate sensitivity to HmB at a critical inhibitory concentration of between 40–50 μg/ml. Selection for HmB resistance of this fungus, indicative of transformation, resulted in 3–48 HmB-resistant colonies per microgram of pAN7-1 per 107 viable protoplasts. No significant differences were apparent when either transformation protocol or either P. ostreatus strain was used. The best electrical condition found for the electrotransformation of P. ostreatus is at a field strength of 2.6–2.8 kV/cm with a capacitance of 25μF and a parallel resistance of 800 ohms, corresponding to a time constant range of 10–14 ms.

Characterization of hybrids obtained by protoplast fusion, between Pachysolen tannophilus and Saccharomyces cerevisiae

Abstract: The genes for utilization of xylose were transferred from Pachysolen tannophilus to Saccharomyces cerevisiae. The hybrids resembled the S. cerevisiae parent morphologically and in sugar assimilation. Pulsed field gel electrophoresis showed that the chromosome banding pattern was intermediate between the two parental species.