Showing papers on "Protoplast published in 2003"
TL;DR: The germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency, and Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.
Abstract: Four different transformation methods were tested and compared in an attempt to facilitate the genetic transformation of Aspergillus giganteus, the producer of an antifungal protein (AFP). The fungus was transformed to hygromycin B resistance, using the hph gene of Escherichia coli by protoplast transformation, electroporation, biolistic transformation, and Agrobacterium tumefaciens-mediated transformation. Electroporation and biolistic transformation were found to be inappropriate for transforming A. giganteus, due to a low transformation yield. The conventional transformation technique based on protoplasts yielded up to 55 transformants in 10(8) protoplasts/microg DNA and was enhanced to 140-fold by A. tumefaciens-mediated transfer of its T-DNA. Here, the germination time prior to cocultivation and the fungus:bacterium ratio were found to alter the transformation efficiency. Southern blot analysis revealed that the A. giganteus transformants contained a randomly integrated single T-DNA copy, whereas multiple integration events were frequent in transformants obtained by the protoplast method.
129 citations
TL;DR: In a protoplast system which models mesophyll, regulation of the minimal CP promoter involves both activation and derepression by TrAP.
77 citations
TL;DR: Density and survival rate of protoplasts were positively correlated, but the formation of long protonema filaments decreased markedly, the effect of different protoplast densities could be explained partly by physiologically active compounds excreted into the medium.
62 citations
TL;DR: An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija, and the absence of cytokinins had a positive effect on cell development.
Abstract: An efficient and fast regeneration system from cotyledon protoplasts was established for Arabidopsis thaliana accessions C24, Columbia, and Wassilewskija. Culture conditions and media compositions were optimised for the development of protoplasts embedded in thin alginate layers. Unexpectedly, the absence of cytokinins had a positive effect on cell development. Moreover, combined adjustment of α-naphthylacetic acid and dicamba concentrations resulted in high plating efficiencies of up to 30%, followed by shoot regeneration within only 19 days after protoplast isolation. The protocol is reproducible, efficient, extremely fast, and regenerated plants are fertile. Thus, this cotyledon-based system could prove useful for studying plant cell and molecular biology in A. thaliana.
58 citations
TL;DR: Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin, and strain FP-54 showed no protective effect against common scab and the disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.
Abstract: Intraspecific protoplast fusion was used to produce stable prototrophic recombinants of Streptomyces melanosporofaciens EF-76, a biocontrol agent of plant disease producing geldanamycin. Two recombinant strains (FP-54 and FP-60) that differed with regard to their antagonistic properties against Bacillus cereus ATCC 14579, Streptomyces scabies EF-35 and Phytophthora fragariae var. rubi 390 were characterized. FP-60 lost the ability to inhibit the in vitro growth of these microbial strains while FP-54 exhibited higher antagonistic activities against them. FP-60 was deficient in geldanamycin biosynthesis whereas FP-54 was shown to produce, in addition to geldanamycin, at least two other antimicrobial compounds that were absent in the culture supernatants of strain EF-76. Like the wild-type strain EF-76, strain FP-54 reduced common scab symptoms on potato tuber but no significant difference was observed between the disease index attributed to tubers treated with strain EF-76 or with strain FP-54. Strain FP-60 showed no protective effect against common scab. The disease index of tubers treated with this recombinant was worse than the index associated with potato tubers from control treatments.
49 citations
TL;DR: The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M. purpureus and the transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome.
Abstract: The high-level pigment-producing Monascus strain IBCC1 was characterized by random amplification of polymorphic DNA as M. purpureus. This technique allowed us to distinguish between M. purpureus and M. ruber strains. Transformation of Monascus species has not been previously reported. Protoplast formation and regeneration from M. purpureus IBCC1 was optimized by modification of growth media, lytic enzyme mixture, osmotic stabilizer and regeneration media. Of the Monascus transformants, 60% were found to be mitotically stable and retained the plasmid inserted in the chromosome after repeated sporulation cycles. Additionally, an Agrobacterium-mediated DNA transfer system was developed. The transformants obtained by Agrobacterium-mediated DNA transfer remained fully stable (98%) after four sporulation rounds and showed bands of hybridization corresponding to integration of the plasmid in different sites of the genome. The green fluorescent protein marker was well expressed in the M. purpureus transformants. The development of transformation systems is a basic tool for advanced genetic manipulation of the natural pigment producers, M. purpureus and M. ruber.
48 citations
TL;DR: Calli derived from the R-biotype of E. indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants.
Abstract: Efficient methods in totipotent callus formation, cell suspension culture establishment and whole-plant regeneration have been developed for the goosegrass [Eleusine indica (L) Gaertn] and its dinitroaniline-resistant biotypes The optimum medium for inducing morphogenic calli consisted of N6 basal salts and B5 vitamins supplemented with 1–2 mg l–1 2,4-dichlorophenoxyacetic acid (2,4-D), 2 mg l–1 glycine, 100 mg l–1 asparagine, 100 mg l–1 casein hydrolysate, 30 g l–1 sucrose and 06% agar, pH 57 The presence of organogenic and embryogenic structures in these calli was histologically documented Cell suspension cultures derived from young calli were established in a liquid medium with the same composition Morphogenic structures of direct shoots and somatic embryos were grown into rooted plantlets on medium containing MS basal salts, B5 vitamins, 1 mg l–1 kinetin (Kn) and 01 mg l–1 indole-3-acetic acid (IAA), 3% sucrose, 06% agar, pH 57 Calli derived from the R-biotype of E indica possessed a high resistance to trifluralin (dinitroaniline herbicide) and cross-resistance to a structurally non-related herbicide, amiprophosmethyl (phosphorothioamidate herbicide), as did the original resistant plants Embryogenic cell suspension culture was a better source of E indica protoplasts than callus or mesophyll tissue The enzyme solution containing 15% cellulase Onozuka R-10, 05% driselase, 1% pectolyase Y-23, 05% hemicellulase and N6 mineral salts with an additional 02 M KCl and 01 M CaCl2 (pH 54–55) was used for protoplast isolation The purified protoplasts were cultivated in KM8p liquid medium supplemented with 2 mg l–1 2,4-D and 02 mg l–1 Kn
43 citations
TL;DR: Few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l−1 myo-inositol was replaced with the same osmolarity of 90 g l −1 mannitol, indicating that myo, inositol is essential for sustained cell division of protoplast.
Abstract: Culture conditions are described for sustained cell division and plant regeneration from protoplasts of rose (Rosa hybrida L. `Sumpath'). Protoplasts were enzymatically isolated from 2-week-old embryogenic cell suspension cultures. Freshly isolated protoplasts were plated as a thin layer onto protoplast culture medium (half-strength 21 Murashige and Skoog's medium containing 60 g l−1 myo-inositol, 4.4 μM BA, and 1.4 μM 2,4-D) at a density of 5×104 protoplasts ml−1. The plating efficiency reached 3.9% after 2 weeks of culture. However, few protoplasts underwent cell division when cultured in protoplast culture medium in which 60 g l−1 myo-inositol was replaced with the same osmolarity of 90 g l−1 mannitol, indicating that myo-inositol is essential for sustained cell division of protoplasts. Colonies were formed after 8 weeks of culture at a frequency of 0.2%. Colonies were then transferred to colony culture medium (0.4% Gelrite-solidified protoplast culture medium) and maintained by subculturing at 4-week intervals to form embryogenic calluses. Upon transfer to half-strength MS basal medium, embryogenic calluses gave rise to numerous somatic embryos. Somatic embryos were transferred to half-strength MS basal medium containing 48 mg l−1 ferric ethylenediamine di-(o-hydroxyphenylacetate), where they subsequently developed into plantlets at a frequency of 30.9%. The plantlets had the same chromosome number of 2n=3x=21 as the source plant. They were successfully transplanted to potting soil and grown to maturity in a greenhouse.
41 citations
TL;DR: The protoplast to plant regeneration protocols developed in this study may provide the basis to investigate cell physiology and biochemistry of desert medicinal plants.
40 citations
TL;DR: This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplast per gram of fresh tissue was obtainable.
Abstract: Several tissue types of Lupinus albus L. were investigated as sources for the isolation of protoplasts. Cotyledons from in vitro seedlings were found to yield the highest number of protoplasts compared with leaves, hypocotyls and roots. A combination of the protoplast isolation enzymes, cellulase and Pectolyase Y23, was capable of releasing the highest number of protoplasts compared with a combination of cellulase and Macerase. Protoplast yield increased with increasing cotyledon age but was accompanied by a progressive decline in protoplast viability. The optimal combination of protoplast yield and viability occurred when the protoplasts were isolated from 14- to 18-day-old cotyledons. The ratio between the volume of enzyme solution and the tissue biomass did not affect the protoplast production significantly. This is the first report of the isolation of protoplasts from a lupin cotyledon and, following the procedure described in this paper, an average yield of 1.2 × 106 protoplasts per gram of fresh tissue was obtainable.
38 citations
TL;DR: Southern blot analysis indicated that transforming DNA was integrated into the oomycete genome and that the DNA was stably inherited through sporogenesis, and application of the technique to studies on the biochemical basis for pathogenesis in this agriculturally important group of fungi is discussed.
Abstract: Conditions for the production of protoplasts and gene transfer in Pythium aphanidermatum were investigated. Efficient protoplast generation was possible after culture of mycelium in potato dextrose broth followed by digestion with 0.5% (w/v) each of cellulase and β-d-glucanase. Plasmid pHAMT35N/SK encoding the nptII gene under control of the Ham34 promoter from the oomycete Bremia lactucae was used to define electroporation parameters for gene transfer. A square-wave electroporation pulse of 2500 V/cm at 50 μF capacitance reproducibly produced transformants, albeit at low efficiency (0.1–0.4 transformants from ~105 regenerable protoplasts per microgram of DNA). Thirty-two independent transformants exhibited wild-type growth on potato dextrose agar amended with geneticin at 50 μg/ml, a concentration that near completely inhibited the growth of untransformed P. aphanidermatum. Southern blot analysis indicated that transforming DNA was integrated into the oomycete genome and that the DNA was stably inherited through sporogenesis. Growth on geneticin-free media, the ability to form zoospores or oospores, and the ability to cause disease in sugarbeet seedlings in the laboratory were indistinguishable between a subset of the transformed isolates and the progenitor isolate 898B. Co-electroporation of pHAMT35N/SK with plasmid pACT-GUS encoding the Escherichia coli gusA gene controlled by oomycete transcriptional promoter and terminator sequences or with pEGFP encoding enhanced green fluorescent protein under the control of the immediate early promoter from the mammalian cytomegalovirus produced, respectively, stable β-glucuronidase and transient expression of blue-green fluorescence. Application of the technique to studies on the biochemical basis for pathogenesis in this agriculturally important group of fungi is discussed.
TL;DR: Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and β-glucanase (s) in KA-prep, suggesting that the component was an indispensable factor for protoplasts formation.
Abstract: KA-prep, a culture filtrate of Bacillus circulans KA-304 grown on a cell-wall preparation (CWP) of Schizophyllum commune, has been reported to have an activity to form protoplasts from S. commune mycelia. The SDS-polyacrylamide gel electrophoreses described here demonstrated that a specific proteinous component (molecular weight: 150,000) occurred in KA-prep. The protein (P150T) was also formed in culture filtrates with CWP of several basidiomycetes, which could release the protoplasts, suggesting that the component was an indispensable factor for protoplast formation. P150T, isolated from an ammonium sulfate fraction of KA-prep (0-30% saturation), did not have any protoplast-forming activity. Results were obtained indicating that P150T participates in protoplast formation together with chitinase(s) and beta-glucanase(s) in KA-prep. The N-terminal amino acid sequence indicated an analogy of P150T to mutanase (alpha-1,3-glucanase) from Bacillus sp. RM1, and actually P150T hydrolyzed mutan as well as S-(alpha-1,3) glucan from S. commune.
TL;DR: Higher plants contain biologically active molecules that are recognized by anti-human atrial natriuretic polypeptide rabbit serum (anti-ANP), and it is demonstrated that the relative amounts of irPNP expressed as a percentage of total water are increased when plants are exposed to 300 mM NaCl.
Abstract: Higher plants contain biologically active molecules that are recognized by anti-human atrial natriuretic polypeptide rabbit serum (anti-ANP). These molecules are termed immunoreactant plant natriuretic peptides (irPNPs) and have previously been shown to be associated with conductive tissue and to affect ion fluxes, protoplast volume regulation and stomatal guard cell responses. Herein an irPNP from the brassicaceus weed Erucastrum strigosum is identified and it is demonstrated that the relative amounts of irPNP expressed as a percentage of total water : methanol (50 : 50) extracted proteins are increased when plants are exposed to 300 mM NaCl. Since 100 and 200 mM NaCl reduce dry and fresh mass as well as increase total tissue NaCl load, it is hypothesized that irPNP up-regulation is a late and possibly adaptive response. IrPNP is also significantly up-regulated in Arabidopsis thaliana suspension culture cells in response to 150 mM NaCl and even more so in response to iso-osmolar amounts of sorbitol. Finally, a recombinant A. thaliana irPNP (AtPNP-A) promotes net water-uptake into the protoplast and thus volume increases. This response is dependent on de novo protein synthesis and may suggest a complex and possibly regulatory function for irPNP-like molecules in plant homeostasis.
TL;DR: It is proposed that a great number of receptors to EPS Crass are present in the plasma membranes of potato cells of susceptible cultivars which contain a small but sufficient quantity of receptor to EPS able to induce defensive responses in plants.
Abstract: The interaction of extracellular polysaccharides (EPS) of the potato ring rot bacterial pathogen Clavibacter michiganensis ssp. sepedonicus (Spieck. et Kott.) Skaptason et Burkh. (Cms) with protoplasts isolated both from leaf cells of plants grown in vitro and microsomal membrane fractions obtained from cell suspension cultures of two potato (Solanum tuberosum L.) cultivars contrasted by their resistance to this pathogen was studied. The EPS intensively bind to protoplast surfaces and microsomal membranes of the susceptible cultivar but not to those of the resistant cultivar. Treatment with protease, excess of unlabelled EPS, and with dextran, did not lead to the binding of fluorochrome-labelled EPS to protoplasts and microsomal membranes (from both cultivars). It is proposed that (a) a great number of receptors to EPS Crass are present in the plasma membranes of potato cells of susceptible cultivars, (b) these receptors contain proteinaceous sites exposed on the external side of the plasma membrane which participate in EPS binding, and (c) the plasma membranes of cells of resistant cultivars contain a small but sufficient quantity of receptors to EPS able to induce defensive responses in plants.
TL;DR: To the authors' knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet and highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants.
Abstract: The successful application of recombinant DNA technology for crop plants requires efficient regeneration systems. A detailed study on the regeneration potential of callus and callus-derived protoplasts of a recalcitrant species, sugarbeet, was performed. A reproducible and highly efficient method for induction of regenerable friable callus was established from etiolated hypocotyl explants. A reduced sucrose concentration proved beneficial. Successful shoot regeneration could be demonstrated in 10 out of 12 tested lines. Seed germination, followed by callus induction and shoot regeneration required only a single culture medium. Additionally, the regeneration capacity of roots and root-derived callus was demonstrated. Highly efficient plant regeneration was also achieved when using protoplasts isolated from regenerable friable callus induced on etiolated hypocotyls explants. To our knowledge this represents the first report on callus protoplast to plant regeneration in sugarbeet.
TL;DR: This is the first report to describe the successful realization of Lilium somatic hybridization via protoplast fusion, and the stability of the morphological features of the hybrids in the next generations is examined.
Abstract: Somatic hybridizations via electrofusion were performed in combinations of Oriental hybrid lilies (cvs. Acapulco and Shirotae) and Liliumxformolongi hort. (cv. Hakucho). Electrofusion-treated protoplasts divided only under nurse culture. The divided protoplasts grew into calli on the culture medium containing picloram, and the calli were then transferred to the hormone-free culture medium for induction of plant regeneration. The regenerants were transferred to a greenhouse, and were grown until the flower stage. In the fusion combinations of Acapulco + Hakucho and Shirotae + Hakucho, four regenerants apparently showed different morphological features compared with their parents. The results of molecular analyses by means of cleaved amplified polymorphic sequences markers and flow cytometry confirmed that these regenerants were somatic hybrid plants. Furthermore, we examined the stability of the morphological features of the hybrids in the next generations. This is the first report to describe the successful realization of Lilium somatic hybridization via protoplast fusion.
TL;DR: Flow cytometry analysis showed a low variability (8%) in the relative DNA content between mother plants and protoplast-derived plants, and the concentrations of ammonium nitrate and calcium chloride in the culture medium appeared as a crucial factor for successful protoplasts cultures.
TL;DR: Regeneration of protoplasts from extruded cytoplasm and successive development of aplanospores within regenerated cells are described in the marine green alga Chaetomorpha aerea (Dillwyn) Kützing.
Abstract: Regeneration of protoplasts from extruded cytoplasm and successive development of aplanospores within regenerated cells are described in the marine green alga Chaetomorpha aerea (Dillwyn) Kutzing (Cladophorales, Cladophoraceae). Agglutination of cell organelles in seawater seemed to be mediated by a complementary lectin-carbohydrate system. Three carbohydrates – D-galactosamine, D-glucosamine and α-D-mannose – inhibited agglutination of cell organelles. The presence of these sugar moieties on the surface of cell organelles was verified with their complementary fluorescein isothiocyanate lectins. Agglutination assays using human erythrocytes showed the presence of lectins specific for the above sugars in the protoplasm. The fluorescent probe l-(4-trimethylammoniumphenyl)-6-phenyl-a,3,5-hexatriene revealed that the envelope initially surrounding protoplasts was not a lipid-based cell membrane. Fluorescein diacetate staining showed esterase activity in the protoplasts from the beginning of the regeneration p...
TL;DR: The walls of mesophyll cells and newly formed walls of protoplasts were similar with respect to the presence of large amounts of pectins recognized by JIM7 antibodies, the scarcity of JIM5-pectins and the complete absence of LM5-responding pectin molecules.
Abstract: We have investigated the possible relation between plant cell-wall constituents and the recalcitrance of the cell to regenerate organs and whole plants in vitro. A temporal and spatial expression of several carbohydrate epitopes was observed both within leaf tissue used for protoplast isolation and within new walls reformed by recalcitrant mesophyll protoplasts of sugar beet (Beta vulgaris L.); these include four pectic epitopes, one xyloglucan (rhamnogalacturonan I) epitope, two carbohydrate motifs of arabinogalactan proteins (AGPs) and callose. The walls of mesophyll cells and newly formed walls of protoplasts were similar with respect to the presence of large amounts of pectins recognized by JIM7 antibodies, the scarcity of JIM5-pectins and the complete absence of LM5-responding pectin molecules. Their main differences were the significantly higher accumulation of LM6-recognizing pectins and the very conspicuous greater accumulation of AGPs and callose in walls deposited by protoplasts than in those synthesized by donor cells.
TL;DR: The somatic hybrids are expected to be used as bridging plant material to introduce the C3-C4 intermediate trait into Brassica crop species.
Abstract: Somatic hybrids were produced between Moricandia arvensis (MaMa, 2n= 28) and Brassica oleracea (CC, 2n= 18) through cell fusion and then characterized by analysing their morphology, cytology, DNA constitution, leaf anatomy and seed fertility Cell fusion was carried out between greenish protoplasts isolated from the mesophyll of M arvensis and colourless ones from hypocotyls of B oleracea Three plants were generated from one shoot via cuttings and acclimatized in vivo They closely resembled each other in morphology, exhibiting traits intermediate between the parental species They were confirmed to be amphidiploids by mitotic and meiotic analyses, being 2n= 46 (MaMaCC), with pollen fertility of about 50%, which was enough to develop the subsequent progenies Anatomical analysis of the for leaf tissue showed that the bundle sheath cells of the somatic hybrids contained some centripetally arranged organelles, like those of M arvensis The hybridity was also confirmed by random amplified polymorphic DNA analysis Both chloroplast DNA and mitochondrial DNA of the somatic hybrids were estimated to be derived from M arvensis In leaf anatomy, the somatic hybrid showed the C3-C4 intermediate trait as in M arvensis Many progenies resulted from backcrossing with parental species The somatic hybrids are expected to be used as bridging plant material to introduce the C3-C4 intermediate trait into Brassica crop species
TL;DR: Data show for the first time that the actomyosin system is involved in the mechanism of volume regulation during the plasmolytic cycle and that it underlies the gliding movement of the deplasmolyzing protoplast.
Abstract: The leaf cells of Chlorophytum comosum seem to have the ability to regulate their protoplast volume and shape during the plasmolytic cycle. This phenomenon was morphologically expressed by the stabilization of the plasmolyzed protoplast volume and shape within 1-5 min after the immersion of the leaf segments in the plasmolytic fluid and temporarily at the onset of deplasmolysis. During the latter stage the plasmolyzed protoplast rounded up and assumed a perfectly convex shape and glided into the cell lumen along the cell axis. This gliding movement was active, nonsaltatory, and conducted with a constant velocity and lasted for a short time. During this movement the protoplast volume did not change appreciably. As far as we know, this movement has not been described so far. Deplasmolysis proceeded and was rapidly completed when the protoplast stopped moving. Leaf cells which have been affected by an antiactin filament drug or myosin inhibitors lost their ability to regulate the volume and shape of the plasmolyzing protoplast. In addition, the gliding protoplast movement was also inhibited in the treated cells. These data show for the first time that the actomyosin system is involved in the mechanism of volume regulation during the plasmolytic cycle and that it underlies the gliding movement of the deplasmolyzing protoplast.
TL;DR: Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium and showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.
Abstract: A method for somatic embryogenesis and fertile green plant regeneration from suspension cell-derived protoplasts of rye (Secale cereale L. cv. Auvinen) was developed. Fast-growing and friable embryogenic calli with a high regeneration capacity were induced from immature rye inflorescences using modified MS medium. These friable embryogenic calli were used for suspension culture initiation in liquid AA medium. A high yield of protoplasts was obtained from suspension cell clumps after 3–5 days of subculture. Isolated protoplasts were cultured in KM8p medium. The frequency of protoplast cell divisions and colony formations in liquid culture medium were similar to those on agarose-solidified medium. Compact embryogenic calli were developed from protoplast-derived microcalli in growth medium mMS. Approximately 7% of the transferred embryogenic calli produced green shoots on N6 regeneration medium. Of 33 green plants, 28 were fertile with normal flowering and seed set. The ratio of green and albino plantlets was 1:4. Rye protoplast-derived green plants showed normal diploid characters as determined by flow cytometer analysis and chromosome counting.
TL;DR: Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplast shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of celldivision.
Abstract: Activation T-DNA tagging was used to generate four cytokinin-independent (cyi1-4) tobacco cell lines. Plants regenerated from the mutant lines displayed similar phenotypes: reduced apical dominance, poorly developed roots, delayed growth and flowering, and male and female sterility. Tissue culture experiments demonstrated that the mutations in the different lines uncouple cell proliferation from the effects of both cytokinin and auxin. No significant increase of cytokinin or auxin was found in transgenic calli in comparison with untransformed callus. The functional plant sequence tagged in one of the mutant lines, cyi1, was used to isolate an active cDNA, cyi1a, that was able to trigger cytokinin- and auxin-independent protoplast division. Northern analysis shows that the transcript corresponding to cyi1a accumulates to high levels in the untransformed protoplasts shortly before the onset of cell division, and that these levels decrease when protoplasts reach maximum rates of cell division. A small putative open reading frame, starting with the first ATG in cyi1a and encoding a 22 amino acid peptide, has the same activity in tobacco protoplasts as the whole cDNA. This activity is destroyed by a frame shift mutation. Apparently cyi1a encodes a peptide which participates in the events downstream of a joint point of cytokinin and auxin action leading to cell division.
TL;DR: The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system.
Abstract: The influence, was investigated, of abiotic parameters on the isolation of protoplasts from in vitro seedling cotyledons of white lupin. The protoplasts were found to be competent in withstanding a wide range of osmotic potentials of the enzyme medium, however, -2.25 MPa (0.5 M mannitol), resulted in the highest yield of protoplasts. The pH of the isolation medium also had a profound effect on protoplast production. Vacuum infiltration of the enzyme solution into the cotyledon tissue resulted in a progressive drop in the yield of protoplasts. The speed and duration of orbital agitation of the cotyledon tissue played a significant role in the release of protoplasts and a two step (stationary-gyratory) regime was found to be better than the gyratory-only system.
TL;DR: Yields of 106–107 apricot mesophyll protoplasts g fw–1 were obtained depending on factors such as plasmolysing pretreatment, digesting enzymes and digestion time, while Onozuka R-10 in combination with Pectolyase Y-23 and Hemicellulase was found best for protoplast isolation among several enzyme combinations tested.
Abstract: SummaryYields of 106–107 apricot mesophyll protoplasts g fw–1 were obtained depending on factors such as plasmolysing pretreatment, digesting enzymes and digestion time. Onozuka R-10 (1%) in combination with Pectolyase Y-23 (0.1%) and Hemicellulase (1%) was found best for protoplast isolation among several enzyme combinations tested. Viability was 83% with this enzyme combination. Plasmolysis of leaves for 90 min in a 13% sorbitol solution greatly increased the number of protoplasts obtained. Optimum incubation time of 13–16 h produced the best combination of yield and viability. During the purification of protoplasts a critical step was the centrifugation of the sucrose gradient at 75 g with a dramatic decrease in the recovery of protoplasts at lower and higher centrifugation speeds. Protoplasts were cultured under different media composition and growth regulator combinations. Limited growth and division of protoplasts embedded in agarose drops were obtained.
TL;DR: Electron microscopy showed that fiber bundles of Larix fibers had helical substructures, and single fiberpurification and micro-staining methods using a micromanipulator were developed, suggesting that they were composed of cell wall component(s), including callose.
Abstract: Novel elongated fiber-structures were repeatedly found both in leaf protoplast culture of two clones of Betula platyphylla and in protoplast culture of embryogenic cells of Larix leptolepis. Suboptimum culture conditions for cell division appeared to lead to fiber formation when using multi-well plate culture with varying medium compositions The suboptimum conditions for cell divisions were brought about by (1) plant growth regulators: auxins and cytokinins; (2) pH: 3.5, 4.5, 5.8; (3) divalent cations: CaCl2 and MgCl2; and (4) sugars: sucrose and mannitol. Divalent cations had the most profound effect on fiber formation. Calcium ions were preferred by Betula and magnesium ions were preferred by Larix. Single fiberpurification and micro-staining methods using a micromanipulator were developed. The fibers fluoresced when stained with Calcofluor White and Aniline Blue, which suggested that they were composed of cell wall component(s), including callose (β-1,3-glucan). Electron microscopy showed that fiber bundles of Larix fibers had helical substructures.
TL;DR: In this article, the peroxidase activity in a single protoplast of alga Bryopsis plumosa was quantitatively characterized by scanning electrochemical microscopy, which showed the generation of ferriceniummethanol (FMA+) at the alga surface.
Abstract: The peroxidase activity in a single protoplast of alga Bryopsis plumosa is quantitatively characterized by scanning electrochemical microscopy. The generation of ferriceniummethanol (FMA+) at the protoplast surface is directly detected by the microelectrode tip scanned close to the sample surface in seawater containing ferrocenemethanol (FMA) and hydrogen peroxide, an electron mediator and an enzyme-substrate, respectively. The oxidation reaction requires hydrogen peroxide, which clearly shows FMA+ generation due to the peroxidase (POD) catalytic reaction occurring in the protoplast. The FMA+ generation and the FMA accumulation rates at a single alga protoplast were equivalent. A plot of the FMA+ generation rates according to the hydrogen peroxide concentrations was well allowed with a Michaelis–Menten-type reaction. An estimation of the mass-transfer rate and a determination of the Km are quite important advantages of the SECM technique that cannot be realized using other techniques. The POD activity has...
TL;DR: Investigations of the organelles in the hybrids revealed that the chloroplast genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids.
Abstract: Somatic hybrids were produced by protoplast fusion between Arabidopsis thaliana ecotype Columbia and a male-sterile radish line MS-Gensuke (Raphanus sativus) with the Ogura cytoplasm. Forty-one shoots were differentiated from the regenerated calli and established as shoot cultures in vitro. About 20 of these shoots were judged to be hybrids based on growth characteristics and morphology. Molecular analyses of 11 shoots were performed, confirming the hybrid features. Of these 11 shoots, eight were established as rooted plants in the greenhouse. Polymerase chain reaction and randomly amplified polymorphic DNA analyses of the nuclear genomes of all analyzed shoots and plants confirmed that they contained hybrid DNA patterns. Their chromosome numbers also supported the hybrid nature of the plants. Investigations of the organelles in the hybrids revealed that the chloroplast (cp) genome was exclusively represented by radish cpDNA, while the mitochondrial DNA configuration showed a combination of both parental genomes as well as fragments unique to the hybrids. Hybrid plants that flowered were male-sterile independent of the presence of the Ogura CMS-gene orf138.
01 Jan 2003
TL;DR: Application of protoplast to genetic improvement and basic study of woody plants are discussed herein, such as cryopreservation, somatic hybridization, genetic transformation and other basic researches.
Abstract: Traditional breeding of most woody plants is more or less confronted with certain barriers. Protoplast isolation and culture pave way for genetic improvement of woody plants. Since the first try on protoplast isolation and culture was conducted much progress has been made in woody plants, covering most of the important species including fruit trees, such as citrus, apple, pear, mango, kiwifruit, litchi, and forestry trees, such as sandalwood, poplar, eucalyptus, mulberry, conifer, etc. The present paper reviews the general protocols concerning protoplast isolation and culture, summarizes the factors affecting protoplast isolation and culture, introduces the variations, inclusive of chromosome number, morphology and resistance, occurring in protoplast-derived plants. In addition, application of protoplast to genetic improvement and basic study of woody plants are discussed herein, such as cryopreservation, somatic hybridization, genetic transformation and other basic researches.