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PubMed2000

[Direct embryogenesis from protoplast of winter wheat].

Friable embryogenic calli were obtained on a modified N6 medium (NBD medium) from a winter wheat cultivar "Jinghua No. 1" (Triticum aestivum L. cv. Jinghua No. 1) and were transferred to a modified MS liquid medium (MSDL medium) to initiate embryogenic suspension cultures. Protoplasts were isolated from the suspensions and cultured on a modified MS medium (MSDP medium). The somatic embryoids were formed directly from the protoplasts and germinated into entire plants. The development of the somatic embryoids was very similar to that of zygotic embryos of wheat.

Protoplasts↗
PubMed2001

In vitro Arabidopsis pollen germination and characterization of the inward potassium currents in Arabidopsis pollen grain protoplasts.

The focus of this study is to investigate the regulatory role of K(+) influx in Arabidopsis pollen germination and pollen tube growth. Using agar-containing media, in vitro methods for Arabidopsis pollen germination have been successfully established for the first time. The pollen germination percentage was nearly 75% and the average pollen tube length reached 135 microm after a 6 h incubation. A decrease in external K(+) concentration from 1 mM to 35 microM resulted in 30% inhibition of pollen germination and 40% inhibition of pollen tube growth. An increase in external K(+) concentration from 1 mM to 30 mM stimulated pollen tube growth but inhibited pollen germination. To study how K(+) influx is associated with pollen germination and tube growth, regulation of the inward K(+) channels in the pollen plasma membrane was investigated by conducting patch-clamp whole-cell recording with pollen protoplasts. K(+) currents were first identified in Arabidopsis pollen protoplasts. The inward K(+) currents were insensitive to changes in cytoplasmic Ca(2+) but were inhibited by a high concentration of external Ca(2+). A decrease of external Ca(2+) concentration from 10 mM (control) to 1 mM had no significant effect on the inward K(+) currents, while an increase of external Ca(2+) concentration from 10 mM to 50 mM inhibited the inward K(+) currents by 46%. Changes in external pH significantly affected the magnitude, conductance, voltage-independent maximal conductance, and activation kinetics of the inward K(+) currents. The physiological importance of potassium influx mediated by the inward K(+)-channels during Arabidopsis pollen germination and tube growth is discussed.

Arabidopsis↗
PubMed1996

Effects of fast clinostat treatment and microgravity on Vicia faba L. mesophyll cell protoplast ubiquitin pools and actin isoforms.

With parabolic rocket flights and fast clinostat treatments, the effect of microgravity on ubiquitin, ubiquitin-protein conjugates, and actin isoforms of Vicia faba mesophyll protoplasts was studied. Western immunoblotting with ubiquitin antibodies revealed that simulated and particularly, real microgravity influenced the amount of free ubiquitin and of 18, 19, and 40 kD ubiquitin conjugates by inducing strong oscillations in the proteins concentrations over time. Simulated microgravity and microgravity-phase during parabolic rocket flights resulted in a decrease of actin isoforms. Results obtained support the assumption, that microgravity and fast clinostat treatment have a direct effect on Vicia faba mesophyll protoplast metabolic activities.

Actins↗
PubMed2001

Signal transduction in maize and Arabidopsis mesophyll protoplasts.

Plant protoplasts show physiological perceptions and responses to hormones, metabolites, environmental cues, and pathogen-derived elicitors, similar to cell-autonomous responses in intact tissues and plants. The development of defined protoplast transient expression systems for high-throughput screening and systematic characterization of gene functions has greatly contributed to elucidating plant signal transduction pathways, in combination with genetic, genomic, and transgenic approaches.

Arabidopsis↗
PubMed2001

Regeneration from leaf protoplasts of Arabidopsis thaliana ecotype estland.

Protoplasts (2 x 10(7)/g fresh wt) were isolated from leaves of A. thaliana ecotype estland, with a viability of more than 90%. Protoplasts cultured in calcium alginate beads or layers showed division while culture in liquid or agarose beads failed to elicit any division. Effect of culture density showed highest frequency of division occurring at 5 x 10(5) while no division was seen when cultured at a density of 5 x 10(4). Culture in MS medium resulted in higher division frequency and better sustenance of microcolonies as compared to B5 medium. Under optimized conditions, macrocolonies were formed at a frequency of 1.8%. Shoot regeneration was seen in 50% of microcalli transferred to shoot induction medium for regeneration. Shoots were rooted and plantlets transferred to pots. The plants produced flowers and were fertile.

Arabidopsis↗
PubMed1999

Regeneration of Astragalus adsurgens via somatic embryogenesis from cell suspension protoplasts.

Protoplasts from 4-day-old embryogenic cell suspension cultures of Astragalus adsurgens, when cultured in KM8P medium which ammonium concentration was reduced to 2.5 mmol/L and supplemented with 0.5 mg/L NAA, 1.0 mg/L 2, 4-D, 0.7 mg/L BA and 0.4 mol/L glucose, underwent cell sustained divisions and formed cell colonies at a frequency of 16%-20%. Preplasmolysis or low temperature treatment of suspension cells prior to enzyme incubation enhanced colony formation. Following proliferation on MS medium containing 1.0 mg/L 2, 4-D and 0.5 mg/L BA, cell colonies were cultured on MS medium containing 0.1 mg/L NAA and 1.0 mg/L BA, where approximately 40% of colonies produced somatic embryos ranging in number from 20 to 40 per colony. No significant decrease was found in the potential of somatic embryogenesis when protoplast colonies were obtained from long-term cell suspensions. On hormone-free 1/2 MS medium, somatic embryos developed into intact plants, which showed normal morphology and stable chromosome number.

Astragalus Plant↗
PubMed2000

[Plant regeneration from protoplast culture in flax (Linum usitatissimum)].

Shoot protoplasts of four fiber flax (Linum usitatissimum) varieties (7309, 948, Belinka and Viking) were isolated and cultured. The optimal condition for higher protoplast yield 1.8 x 10(6)/gFW and activity 85.5% (c.v. 948) were from 10 day old seedings. Culture in V-KM Agroase-island medium led to first divisions after 3 days (c. v. 948), and after twenty days with an efficiency of 36% of divided cells and 5.2% in plating efficiency. Plant regeneration was obtained in 7309 and Belinka on agar media B5-2, MS3 containing 0.6 mg/L 6-BA and 0.1 mg/L NAA. Roots and leaves regeneration were observed in Viking and 948 respectively.

Cell Division↗
PubMed2000

[Production of intergeneric somatic hybrid plants via protoplast electrofusion in citrus].

Leaf derived protoplasts of Sour orange (Citrus aurantium L.) were fused electrically with embryogenic protoplasts of Microcitrus papuana Swingle. Plants were regenerated from the fusion products, which were characteristic with three types of leaf morphology. Most of the plants were identical to Sour orange (namely, Leaf-parent-type plant) and two plants had large and thick leaves whereas one plant had bifoliate and trifoliate leaves. Chromosome examination showed that these plants were diploid with 18 chromosomes (2n = 2x = 18). RAPD analysis was employed to verify the hybrid characteristics of the plants in the first two types. Four 10-mer arbitrary primers with polymorphism were chosen. Band pattern of the plants was similar with the leaf parent (Sour orange) for the primer OPAA-17. Band pattern of the plants was similar with either Sour orange or M. papuana for OPA-08. As for OPA-07 and OPA-04 three kinds of band profiles were detected. Results of RAPD marker, together with chromosome determination, indicated that all of the analyzed plants were intergeneric diploid somatic hybrids between Sour orange and M. papuana.

Cell Fusion↗
PubMed1999

[Breeding of yeast for beer manufacturing by inactivated protoplast fusion].

QSB-XI6, one of fusants, was obtained by PEG-induced fusion between UV-inactivated protoplasts of Saccharomyces cerevisiae LQ16 (Ile-, Datr) and heat-inactivated protoplasts of S. cerevisiae QSB7(Ala-, H2S-). The cell volume, bicmass, genotype and DNA content of the fusant were measured in comparison with of its parents and the results showed that QSB-XI6 was a fusant. The fermentation ability and the flocculent capacity of this fusant were higher than those of its parents. The diacetyl content of the beer produced by the fusant was measured and some other compositions of the beer was also analysed by gas chromatography. The sensory evaluation of the beer was much better and the flavour was distinctive. The results of many production trials in succession showed that all good characteristics of QSB-XI6 were stable.

Cell Fusion↗
PubMedDate not supplied

[Activity of the corn Spm transposon system in transgenic plants Orychophragmus violaceus (L.) O.E. Schulz obtained by both direct transfer of DNA to protoplasts and agrobacterial transformation of root explants].

Transposon mediated insertional mutagenesis is one of the approaches for the unique gene cloning. A wild species of Cruciferae family Orychophragmus violaceus (L.) O.E. Schulz, which is of interest for practical breeding as a donor of improved plant oil, was an object of the investigation. Plasmid construction used in the experiments included selective NPT II gene, reported GUS gene serving as an excision marker, structural BAR gene located within the dSpm element and Spm transposase. The GUS gene of this plasmid had not his own promoter and became functional only after Spm-transposition. Transformed Orychophragmus violaceus (L.) O.E. Schulz. plants were obtained by direct mesophyll protoplast transformation as well as Agrobacterium tumefaciens-mediated root explant transformation. Gene transfer and the transposition event were confirmed by the GUS activity and the PCR analysis. Relative transformation efficiency using protoplasts was 5.8%.

Agrobacterium tumefaciens↗
PubMedDate not supplied

Cryopreservation of carrot (Daucus carota l.) cell suspensions and protoplasts by vitrification.

Carrot cell suspensions and protoplasts were successfully cryopreserved by vitrification. Cells were precultured in liquid Murashige and Skoog medium containing 0.175 M sucrose for 3 d and then in liquid MS medium containing 0.4 M sorbitol for 1 d. After loading of the precultured carrot cells in 25 % PVS2 at room temperature for 5 min and treatment with 100 % PVS2 at 0 degrees C for 7.5 min, they were quenched in liquid nitrogen. Optimal survival was 83.3 % (based on the triphenyl tetrazolium chloride reduction assay) following warming and unloading. Recovered cells retained the ability to regenerate plantlets in vitro. In the case of vitrification of protoplasts isolated from carrot cell suspensions, the optimal loading and dehydration durations were 5 min in 25% PVS2 and 3 min 100 % PVS2 respectively. Survival of 47 % of the untreated control (based on the FDA-PI (fluorescein diacetate-propidium iodide) staining) was achieved after cryopreservation.

Cryopreservation↗
PubMed1992

Utilization of the protoplast fusion technique to explore directional altering lincomycin producing microorganism.

Interspecific protoplast fusion between Streptomyces lincolnensis var. lincolnensis (LM gamma, CTC gamma, producing lincomycin) protoplast and Streptomyces aureofaciens (LM gamma, CTC gamma, producing chlorotetracycline) protpolast which had been treated with UV radiation 40 min for inactivation was performed with PEG 6000, the fusants were obtained by directly selecting from the regeneration plates containing CTC 50 micrograms/ml, the fusion frequency was about 9.05 x 10(-5). From many fusants, only 4 stable recombinants were obtained. These species produced antibiotics which are different from lincomycin and chlorotetracycline. Preliminary identification of the antibiotic synthesized by one of the recombinants suggests that its basic structure might be similar to that of lincomycin. The fermentation product of recombinant No. 2 showed new chromatographic spot which is similar to that of clindamycin. Though the products remain to be identified further, this strategy seems to be worthy of exploring for screening new antibiotics.

Anti-Bacterial Agents↗
PubMed1992

[Preparation and regeneration of protoplasts of the strain Streptomyces hygroscopicus 155].

Protoplasts of Streptomyces hygroscopicus 155 producing nigericin, a polyether antibiotic, were prepared. Conditions for protoplasting and optimal concentrations of lysozyme and glycine, and the age of mycelium were studied. With the method of the experiment design, concentrations of four components: CaCl2, MgCl2, KH2PO4 and TES buffer in the regeneration medium were determined. The levels of CaCl2 and TES buffer proved to be the most important parameters.

Culture Media↗
PubMed1992

[Comparative study of Streptomyces--producer of aminoglycoside antibiotics, monomycin and kanamycin, and the strain 344 obtained by fusion of their protoplasts by the method of restrictive total DNA fingerprinting].

The method of total DNA restriction finger prints was applied to the study of Streptomyces monomycini INA 1465 producing monomycin, Streptomyces kanamyceticus INA K-13 producing kanamycin and strain 344 isolated after fusion of the protoplasts of strain 1465 and K-13, which produced albofungin and chloralbofungin, aminoglycoside antibiotics. For preparing the finger prints of the strains splitting by endonucleases BamHI, PstI, PvuII, and BgIII was used. The finger prints showed that strain 344 was related to the strain of S. monomycini and markedly differed from the strain of S. kanamyceticus. Strain 344 was likely to result from reconstruction (probably 20-kb deletion) of the genome of S. monomycini INA 1465 induced by the preparation and regeneration of its protoplasts. The reconstruction could affect the genome area with localization of the genes involved in monomycin biosynthesis and monomycin resistance genes.

Cell Fusion↗
PubMed1990

The application of fungal protoplasts in biotechnology.

Fungi are organisms of enormous industrial importance, and are used for the production of compounds such as antibiotics, organic acids and immunosuppressants. Many studies have suggested that their industrial value may in some cases be increased by exploitation of technologies involving fungal protoplasts, where the cell wall has been removed, usually enzymically. This article critically examines what the advantages of using protoplasts are, the current methodology for the preparation, and how they behave subsequently.

Biotechnology↗
PubMed1990

Protoplast transfection of Lactobacillus casei by phage PL-1 DNA.

Polyethylene glycol (PEG) mediated transfection of Lactobacillus casei ATCC 27092 protoplasts by phage PL-1 DNA was done. The protoplasts were obtained by treatment with purified PL-1 phage N-acetylmuramidase in the presence of citrate. Optimum conditions for transfection were 50% PEG 4,000, 15 micrograms protamine sulfate/ml, 0.15 M sucrose, and 10 mM MgSO4 in MR medium (pH 6.0). The extent of transfection was proportional to the amounts of DNA added, and the greatest efficiency of transfection after a 10-min incubation was about 3.3 x 10(5) PFU/micrograms DNA. The eclipse period of growth of progeny phages in the transfectants was 3 hr and the average burst size was 200.

Bacteriophages↗
PubMed1977

Trypsin-induced changes in the orientation of latent ATPase in protoplast ghosts from Mycobacterium phlei.

Latent ATPase, located on the inner surface of protoplast ghosts of Mycobacterium phlei, was unmasked either by trypsin or an impermeable form of trypsin, ethylene maleic anhydride-trypsin. Density gradient experiments showed that the ghost preparations remained intact following trypsin treatment. Evidence was obtained that 125I-trypsin failed to penetrate the ghost membranes. Thus, attempts were made to determine whether the ATPase molecule in the ghost membranes is accessible from the outer surface. Treatment of protoplast ghosts and trypsin-treated ghosts with 125I by the lactoperoxidase method resulted in the labeling of ATPase only in the trypsin-treated ghost preparations. The antibody to latent ATPase inhibited ATPase activity in trypsin-treated ghosts. The changes in the fluorescence polarization of diphenyl hexatriene indicated that trypsin treatment of the ghost membranes resulted in an increase in membrane fluidity. These studies suggest that the latent ATPase moiety has undergone translocation to the outer surface or it became accessible to trypsin digestion from the outer surface of the membranes as a result of removal of some proteins covering ATPase molecule in the membranes.

Adenosine Triphosphatases↗
PubMed2004

Intensification of lipase biosynthesis as a result of electrofusion of Rhizopus cohnii protoplasts.

Our objective was to obtain products of fusion of the filamentous fungus Rhizopus cohnii Rh.c./1 with an increased capacity for lipase biosynthesis in comparison with the original strain. Protoplasts of auxotrophic mutants of the parent strain Rh.c./1 obtained after UV irradiation of the spores were subjected to electrofusion. We found that the largest number of electrofusion products could be obtained with the use of the following process parameters: 1 or 2 impulses immediately following one another with a field intensity of 200 V/cm and an exposition time of 1000 ms at the stage of dielectrophoresis, 1 impulse with a field intensity of 500 V/cm and an exposition time of 10 ms or 20 ms at the stage of fusion, regulated temperature of 4 degrees C before and after the process, rounding time of ca 20 min. Electrofusion of protoplasts of auxotrophic mutants of the Rh.c./1 strain produced 19 fusion products whose lipase biosynthesis capacity in a liquid medium culture was higher than that of the parent strains. The fusion product labelled XIII-21 was selected as the best strain. Lipase activity obtained after its culture in the liquid medium was ca 3.5 times higher than that obtained after the culture of the original strain Rh.c./1.

Electricity↗