Articoli di riviste: "Protoplast fusion"

1

Navrátilová, B. "Protoplast cultures and protoplast fusion focused on Brassicaceae: A review". Horticultural Science 31, No. 4 (25 novembre 2011): 140–57. http://dx.doi.org/10.17221/3809-hortsci.

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The subjects of this article are protoplast isolations and protoplast fusions, in particular their history, a review of factors influencing the protoplasts isolation and fusion, selection of hybrid plants and utilization of somatic hybrids in plant breed-ing. Somatic hybridization through protoplast fusion can overcome sexual incompatibility among plant species or genera; transfer genes of resistance to diseases (viral, bacterial, fungal), pests, herbicides and others stress factors; obtain cybrid plants; transfer cytoplasmic male sterility or incease content of secondary metabolites in hybrid plants. The article is focussed mainly on the family Brassicaceae because among representatives are significant crops for the human population. Various successful combination of intraspecific, interspecific and intergeneric protoplast fusion were reported between representatives of the family Brassicaceae with the genus Brassica which belonged to the first agricultural crops used for the isolation of protoplast.  

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Perera, Srini C., e Peggy Ozias-Akins. "Regeneration from Sweetpotato Protoplasts and Assessment of Growth Conditions for Flow-sorting of Fusion Mixtures". Journal of the American Society for Horticultural Science 116, n. 5 (settembre 1991): 917–22. http://dx.doi.org/10.21273/jashs.116.5.917.

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Petiole protoplasts of the sweetpotato [Ipomoea batatas (L.) Lam.] cultivars Red Jewel and Georgia Jet formed cell walls within 24 hours and divided in 2 to 3 days. Pretreating enzyme solutions with activated charcoal increased the viability and division frequency of protoplasts. Culture of protoplast-donor plants in a medium containing STS did not affect plant growth, protoplasm yield, or viability, but did increase the division frequency. Culture of protoplasts for 24 hours in a medium containing DB, a cell wall synthesis inhibitor, or staining of protoplasts with FDA did not significantly affect division frequency. The division frequency of protoplasts cultured in liquid medium was significantly higher than that of protoplasts cultured in agarose-solidified medium. Cell cycle analysis of petioles and freshly isolated protoplasts showed that the latter has a significantly higher proportion of nuclei in G1 phase. Protoplasts did not initiate DNA synthesis or mitosis within the first 24 hours of culture. Low-frequency regeneration of shoots from protoplast-derived callus was accomplished on MS medium containing 1.0 mg ldnetin/liter when preceded by MS medium modified to contain only (in mg·liter-1) 800 NH4NO3, 1400 KNO3, 0.5 2,4-D, 0.5 kinetin, and 1.0 ABA. Roots produced from protoplast-derived callus formed adventitious shoots after 4 weeks on MS medium containing 2% sucrose, 0.02 mg kinetin/liter and 0.2% Gelrite. Secondary shoot formation from regenerated roots will be a more effective means of obtaining plants from protoplasts than direct shoot regeneration from callus. Chemical names used: silver thiosulfate (STS): 2.6-dichlorobenzonitrile (DB); fluorescein diacetate (FDA): 2.4-diacetate (FDA); 2.4 dichlorophenoxyacetic acid (2,4-D); abscisic acid (ABA).

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3

Armita, Devi. "Plant Breeding Through Protoplast Fusion". Jurnal Biologi UNAND 8, n. 2 (31 dicembre 2020): 42. http://dx.doi.org/10.25077/jbioua.8.2.42-47.2020.

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Protoplast culture (protoplast fusion) is one method of tissue culture that is widely used in plant breeding programs in a relatively short time. This method is used to overcome the problem of plants that are difficult or impossible to cross conventionally as well as used for species improvement by transferring the desired gene from the donor plant to the target plant via protoplast fusion. Protoplast fusion makes it possible to produce plants that are resistant to a disease and various abiotic stresses, rapid growth rates and have a better quantity and quality of metabolites than their parents. Various factors affect the success of fusion and regeneration of protoplasts into whole plants, including the source of explants, the composition of the enzyme solution and the duration of incubation, fusagen type and culture media for regeneration.

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4

Ahmed, Mohamed A. A., Miao Miao, Emmanouil D. Pratsinakis, Hongliang Zhang, Wei Wang, Yuan Yuan, Meiling Lyu et al. "Protoplast Isolation, Fusion, Culture and Transformation in the Woody Plant Jasminum spp." Agriculture 11, n. 8 (26 luglio 2021): 699. http://dx.doi.org/10.3390/agriculture11080699.

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Plant protoplasts are significant for plant cell culture, somatic cell fusion, genetics, and breeding studies. In addition, in vitro plant regeneration has great importance for developmental biology, manifesting potential applications in agriculture and biotechnology. In this regard, we present a well-established protocol regarding protoplast isolation, cell culture and protoplast fusion of Jasminum spp. In particular, different tissues of Jasminum samab L. and Jasminum mesnyi were employed for protoplast isolation, and stem explants provided a high callus induction rate in a short period of time. The best source for protoplast isolation was calli tissues. The optimized isolation protocol consisted of digesting callus in an enzyme solution containing 0.4 M mannitol, 0.2 M MES, 1 M CaCl2, 0.2 M KCL and 1 M NaH2PO4, 1.5% Cellulases onozuka R-10, 0.4% Macerozyme R-10 and 0.8% Pectinase for 4 h at 26 °C in the dark, providing a yield of 23.8 × 106 Protoplast/gFW with 88% viability. Protoplasts were cultured both in liquid and agarose medium under optimum conditions, leading to microcalli formation after eight weeks. A 5% protoplast-fusion rate can be achieved when cultured in 40% (w/v) PEG-MW6000 supplemented with 0.1 M CaCl2, 0.1 M sorbitol and 1 M Tris for 20 min. Furthermore, we developed an efficient PEG-mediated transformation protocol for jasmine protoplasts. The best results regarding protoplast transformation were obtained when the protoplast concentration was 4 × 105 cells/mL and the exogenous plasmid DNA added had a concentration of 10 µg DNA/100 µL protoplast solution, followed by the application of 40% PEG-4000 for 10 min.

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5

Liu, Donglong, e Nancy A. Reichert. "PROTOPLAST ISOLATION AND CULTURE OF KENAF (HIBISCUS CANNABINUS L.)". HortScience 29, n. 7 (luglio 1994): 729e—729. http://dx.doi.org/10.21273/hortsci.29.7.729e.

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Protoplast isolation and culture protocols were developed for leaf tissue from 6 kenaf cultivars [Everglades 41 (E41), E71, Guatemala 4 (G4), G45, G51, and Tainung 1]. For protoplast isolation, the best combination of hydrolytic enzymes was cellulysin (1% w/v; Calbiochem) plus macerase (0.5% w/v; Calbiochem), with a 24 hour digestion at 30°C in the dark. Yields reached 7.2 (10)6 protoplasts/g leaf tissue. Protoplast viabilities ranged from 65% to 96%. Minor cultivar differences were observed related to protoplast yield, but all viability estimates were in an acceptable range. Greatest cell division frequencies and plating efficiencies were obtained when protoplasts were initially cultured in liquid medium at a density of 1.0 (10)5 protoplasts/ml. Electrofusion protocols were developed for kenaf protoplasts testing the range from 1200 to 3000 V/cm. A fusion voltage of 2000 V/cm yielded the highest fusion frequency and retained viability above 80%.

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Djajanegara, Ira, e Agus Masduki. "PROTOPLAST FUSION BETWEEN WHITE AND BROWN OYSTER MUSHROOMS". Indonesian Journal of Agricultural Science 11, n. 1 (8 luglio 2013): 16. http://dx.doi.org/10.21082/ijas.v11n1.2010.p16-23.

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Genetic crossing of white oyster mushroom (Pleurotus floridae) to introduce longer storage life trait can only be done within individuals in this particular species. However, longer storage life trait is possessed by brown oyster mushroom (Pleurotus cystidiosus). Therefore, a protoplast fusion experiment between white and brown oyster mushrooms was conducted to obtain an oyster mushroom strain showing high productivity and long storage life. The experiment was done at the biology laboratory of the University of Al Azhar Indonesia from May 2008 to August 2009. Protoplast fusion was done by isolating protoplast from 5-day old monokaryotic mycelia grown in potato dextrose broth (PDB). Around 3.15 x 105 protoplasts ml-1 were harvested using mixture of cellulase Onozuka R-10 (1%) and macerozyme R-10 (1%) from brown oyster mushroom with 80.61% viability. Similarly, 3.71 x 105 protoplasts ml-1 were harvested using lysing enzyme (2%) from white oyster mushroom with 83.68% viability. Protoplast fusions were conducted using 40% PEG6000 for 10 minutes. The candidate fusants were then screened using minimum regeneration media (MRM). There were 22 colonies grew on MRM media and four colonies (FS1, FS2, FS3, and FS4) showed clamp connection as well as primordia formation to be chosen as candidate fusants. However, isozyme studies using malate dehydrogenase and acid phosphatase as marker enzymes confirmed that only FS1 and FS2 were the hybridized products. The two colonies showed different mycelia growth patterns and hyphae sizes, fruit body morphology and productivity compared to their parents. These two fusants, however, did not indicate the presence of longer storage life trait as expected despite a higher productivity achieved by FS1. In this study, the protoplast fusion only yielded higher productivity strain of mushroom with different colors without any changes in storage life.

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Nolan, Richard A. "Stage-specific changes in cytoplasmic protein synthesis in Entomophaga aulicae protoplasts". Canadian Journal of Microbiology 35, n. 3 (1 marzo 1989): 373–78. http://dx.doi.org/10.1139/m89-057.

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The patterns of protein synthesis associated with three sequential stages in protoplast morphogenesis (spindle-shaped, early fusion sphere, and late fusion sphere protoplasts) of the fungus Entomophaga aulicae were studied using both one-dimensional gels with general protein staining and two-dimensional gels with [35S]methionine protein labelling and fluorography. A total of 332 proteins were observed with 63.5% (211) common to all three developmental stages. Of the individual totals, 3.3% (8 out of 245), 7.3% (22 out of 301), and 4.5% (13 out of 286) of the proteins were unique to the spindle-shaped, early fusion sphere, and late fusion sphere protoplasts, respectively. The molecular mass and pI distribution profiles for early fusion sphere protoplast proteins are discussed.Key words: protein synthesis, stage-specific proteins, fungal protoplasts, Entomophaga aulicae.

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8

Pauls, K. P., e P. V. Chuong. "Flow cytometric identification of Brassica napus protoplast fusion products". Canadian Journal of Botany 65, n. 5 (1 maggio 1987): 834–38. http://dx.doi.org/10.1139/b87-113.

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A flow cytometric method to identify Brassica somatic hybrids has been developed. The procedure is based on the use of fluorescein isothiocyanate stained hypocotyl protoplasts and unstained mesophyll protoplasts as partners for polyethylene glycol induced protoplast fusion. The fluorescein isothiocyanate stained hypocotyl protoplasts and unstained mesophyll protoplasts were passed through the flow cytometer – cell sorter separately to maximize the sensitivity of the red and green detectors to chlorophyll and fluorescein isothiocyanate fluorescence, respectively, and to ensure that there was no spillover of chlorophyll fluorescence into the green detector or fluorescein isothiocyanate fluorescence into the red detector. When adjusted properly, suspensions of a single protoplast type gave populations that fell on either the green or red axis of a two-dimensional green versus red fluorescence plot. With the same machine settings three populations of protoplasts could be identified in fusion mixtures of the two types of protoplasts: namely, the parental protoplasts that fell on the green or red axis as well as a third that had significant green and red fluorescence.

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Chen, Qin, H. Y. Li, Y. Z. Shi, D. Beasley, B. Bizimungu e M. S. Goettel. "Development of an effective protoplast fusion system for production of new potatoes with disease and insect resistance using Mexican wild potato species as gene pools". Canadian Journal of Plant Science 88, n. 4 (1 luglio 2008): 611–19. http://dx.doi.org/10.4141/cjps07045.

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Somatic hybridization through protoplast fusion is an important alternative approach for overcoming sexual incompatibility between diploid Solanum species and cultivated potatoes. However, compared with other potato species, methods for protoplast isolation and plant generation for several Mexican wild diploid potato species are not well established. In this study, a systematic procedure was designed for the isolation of a large number of high-quality protoplasts from various Mexican wild species that carry high levels of disease (late blight) and insect [Colorado potato beetle (CPB)] resistance. Using this procedure, an effective potato protoplast fusion system was developed to produce new somatic hybrids between two Mexican, one Argentina wild species, and cultivated potato clones. Regenerated plants and somatic hybrids were obtained at a high frequency from the protoplasts of the diploid wild species and their fused cells with S. tuberosum. Morphological, cytological and molecular marker analyses demonstrated that somatic hybrids were successfully obtained from the cell fusion of S. tuberosum and the diploid species S. pinnatisectum, S. cardiophyllum, and S. chacoense. Assessment of disease and insect reactions demonstrated that several of the protoplast-derived clones and somatic hybrids showed a higher level of resistance to both late blight and CPB than was found in S. tuberosum, confirming that the protoplast system is a powerful tool in potato breeding programs for the development of disease and insect resistance. This new fusion system provides breeders with opportunities to transfer disease and insect resistance genes from Mexican wild species into cultivated potato. Key words: Somatic hybrid, protoplast, fusion, potato, Solanum, late blight, disease resistance, Colorado potato beetle insect resistance

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10

Nolan, Richard A. "Influence of a negatively charged surface (Teflon disk) on Entomophaga aulicae protoplast morphogenesis under mass fermentation conditions". Canadian Journal of Botany 69, n. 11 (1 novembre 1991): 2578–81. http://dx.doi.org/10.1139/b91-321.

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The effects of a negatively charged surface (Teflon disk) on protoplast morphogenesis for the fungus Entomophaga aulicae under mass fermentation conditions were determined. The control consisted of a vessel lacking such a disk. In the presence of the disk the initial three and sequentially produced protoplast stages (spindle shaped, early fusion sphere, and late fusion sphere protoplasts) recycled with the early fusion sphere predominating. The production of the subsequent and walled stage (i.e., hyphal body) was suppressed. The results are in contrast with those obtained in a previous study using a neutral (Mylar) and a positively charged (polypropylene) disk in which hyphal body production was enhanced. This technique provides a new and subtle approach for altering protoplast developmental patterns which avoids the use of mutagens or added chemical metabolic inhibitors. Key words: Entomophaga aulicae, fungal protoplast morphogenesis, negatively charged surface, mass fermentation.

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Wang, Chao, XiaoLin Zhang, Zhi Chen, Ying Wen e Yuan Song. "Strain construction for enhanced production of spinosad via intergeneric protoplast fusion". Canadian Journal of Microbiology 55, n. 9 (settembre 2009): 1070–75. http://dx.doi.org/10.1139/w09-064.

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Spinosad is a new class of insecticides produced by Saccharopolyspora spinosa . The aim of this study was to construct a starch-utilizing strain that overproduced spinosad by intergeneric fusion between S. spinosa and Streptomyces avermitilis . Protoplast fusion is an important technique for engineering microbial strains, especially for microorganisms with few available molecular genetic tools. Protoplast fusion was conducted with UV-irradiated protoplasts of S. spinosa and S. avermitilis. Among 76 recombinants screened by ESI-MS and HPLC, a starch-utilizing strain F17, identified as S. spinosa, was obtained. The yield of spinosad in F17 was increased by 447.22%, compared with the yield of the wild-type strain. This is the first report of intergeneric protoplast fusion between S. spinosa and S. avermilitis, which shows great potential for industrial applications.

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12

Djajanegara, Ira, e Agus Masduki. "PROTOPLAST FUSION BETWEEN WHITE AND BROWN OYSTER MUSHROOMS". Indonesian Journal of Agricultural Science 11, n. 1 (8 luglio 2013): 16. http://dx.doi.org/10.21082/ijas.v11n1.2010.16-23.

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Genetic crossing of white oyster mushroom (Pleurotus floridae) to introduce longer storage life trait can only be done within individuals in this particular species. However, longer storage life trait is possessed by brown oyster mushroom (Pleurotus cystidiosus). Therefore, a protoplast fusion experiment between white and brown oyster mushrooms was conducted to obtain an oyster mushroom strain showing high productivity and long storage life. The experiment was done at the biology laboratory of the University of Al Azhar Indonesia from May 2008 to August 2009. Protoplast fusion was done by isolating protoplast from 5-day old monokaryotic mycelia grown in potato dextrose broth (PDB). Around 3.15 x 105 protoplasts ml-1 were harvested using mixture of cellulase Onozuka R-10 (1%) and macerozyme R-10 (1%) from brown oyster mushroom with 80.61% viability. Similarly, 3.71 x 105 protoplasts ml-1 were harvested using lysing enzyme (2%) from white oyster mushroom with 83.68% viability. Protoplast fusions were conducted using 40% PEG6000 for 10 minutes. The candidate fusants were then screened using minimum regeneration media (MRM). There were 22 colonies grew on MRM media and four colonies (FS1, FS2, FS3, and FS4) showed clamp connection as well as primordia formation to be chosen as candidate fusants. However, isozyme studies using malate dehydrogenase and acid phosphatase as marker enzymes confirmed that only FS1 and FS2 were the hybridized products. The two colonies showed different mycelia growth patterns and hyphae sizes, fruit body morphology and productivity compared to their parents. These two fusants, however, did not indicate the presence of longer storage life trait as expected despite a higher productivity achieved by FS1. In this study, the protoplast fusion only yielded higher productivity strain of mushroom with different colors without any changes in storage life.

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Binding, Horst, Monika Zuba, Joachim Rudnick e Gudrun Mordhorst. "Protoplast Gel Fusion". Journal of Plant Physiology 133, n. 4 (novembre 1988): 409–13. http://dx.doi.org/10.1016/s0176-1617(88)80027-0.

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14

Gleddie, Stephen, e Wilfred A. Keller. "Protoplast fusion technology". Journal of Tissue Culture Methods 12, n. 4 (dicembre 1989): 157–61. http://dx.doi.org/10.1007/bf01404443.

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Kurita-Tashiro, Asami, Noriko Hayashi, Tomoya Oyanagi e Hamako Sasamoto. "New Factors for Protoplast-Callose-Fiber Formation in Salt-Tolerant Mangrove Plants, Avicennia alba and Bruguiera sexangula and Analysis of Fiber Substructures". Journal of Plant Studies 9, n. 2 (2 maggio 2020): 1. http://dx.doi.org/10.5539/jps.v9n2p1.

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Elongated and spiral β-1,3-glucan (callose) fibers were obtained by new factors from protoplasts cultured in liquid medium from suspension cultured cells of two salt-tolerant mangrove species; Avicennia alba and Bruguiera sexangula. Differences in salt factor for protoplast-fiber formation were compared with those of the callose fibers developed from protoplasts of non-mangrove tree plants, Larix leptolepis and Betula platyphylla, which high concentrations of divalent cations, Mg2+ (50 mM) or Ca2+ (100 mM), were stimulatory. In the halophilic A. alba protoplasts, whose cell division was stimulated by up to 400 mM NaCl, addition of Mg2+, Ca2+, K+ ions inhibited protoplast-fiber formation. In B. sexangula, protoplast-fibers were rapidly and efficiently formed only by another new factor, electric cell fusion treatment of protoplasts. Spiral fibers developed from mangrove protoplasts were detected under an inverted microscope, and their specific blue-green color for callose after staining with Aniline Blue dye was detected under a fluorescence microscope. Enzymatic certification of callose was further performed with laminarinase, specific for callose, in comparison with cellulase CBH1, specific for cellulose. Differences in sub-structures, fibrils and sub-fibrils of two mangrove protoplast-fibers were analyzed using laser confocal scanning microscopy, atomic force microscopy and image J analysis. Tube-like fine structure was observed using transmission electron microscopy in single protoplast-fiber of B. sexangula selected with a micromanipulator.

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Tusa, N., J. W. Grosser e F. G. Gmitter. "Plant Regeneration of `Valencia' Sweet Orange, `Femminello' Lemon, and the Interspecific Somatic Hybrid following Protoplasm Fusion". Journal of the American Society for Horticultural Science 115, n. 6 (novembre 1990): 1043–46. http://dx.doi.org/10.21273/jashs.115.6.1043.

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Protoplasm culture following the chemical fusion of `Valencia' sweet orange [Citrus sinensis (L.) Osb.] protoplasts, isolated from an embryogenic suspension culture, with `Femminello' lemon [Citrus limon (L.) Burro. f.] leaf protoplasts resulted in the regeneration of an interspecific allotetraploid somatic hybrid plant, two autotetraploid lemon plants, and diploid plants from both parents. The regeneration of plants from lemon leaf protoplasts is an example of protoplast-to-plant regeneration from non-nucellus-derived tissue for Citrus. Regenerated plants were classified according to leaf morphology, chromosome number, and analyses of phosphohexose isomerase (PHI), peroxidase (PER), and 6-phosphoglucose dehydrogenase (PGD) zymograms. The somatic hybrid plant was vigorous, with leaves morphologically intermediate to the parents. The tetraploid lemon plants were similar to diploids, although less vigorous and with thicker leaves. The tetraploid lemon and somatic hybrid plants, if fertile, could be used in interploid sexual crosses to breed triploid seedless lemon cultivars with tolerance of mal secco disease from sweet orange. Further investigation of plant regeneration from leaf protoplasts could increase the number of totipotent Citrus clones amenable to somatic hybridization and genetic transformation experiments.

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Kucsera, Judit, Ilona Pfeiffer e Lajos Ferenczy. "A novel method for hybridization of Saccharomyces species without genetic markers". Canadian Journal of Microbiology 44, n. 10 (1 ottobre 1998): 959–64. http://dx.doi.org/10.1139/w98-093.

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Protoplasts of Saccharomyces cerevisiae were inactivated by treatment with different concentrations of antifungal compounds for various periods. Of the 14 compounds tested, N-ethylmaleimide proved to be the most efficient. The inactivation effect was fully reproducible. The inactivated protoplasts could be reactivated and still function as fusion partners. They were fused with untreated protoplasts by polyethylene glycol treatment and produced viable hybrid cells. Nuclear and extrachromosomal genetic analysis and chromosome separation of the fusion products from fusion experiments involving inactivated and non-inactivated protoplasts revealed that N-ethylmaleimide did not affect either of the genomes and hence it was perfectly suited for the hybridization of any type yeast cells without genetic markers.Key words: yeast protoplast fusion, chemical inactivation, chromosomal polymorphism, interspecies hybrid.

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Satpute, Aditi D., Chunxian Chen, Fredrick G. Gmitter, Peng Ling, Qibin Yu, Melinda R. Grosser, Jude W. Grosser e Christine D. Chase. "Cybridization of Grapefruit with ‘Dancy’ Mandarin Leads to Improved Fruit Characteristics". Journal of the American Society for Horticultural Science 140, n. 5 (settembre 2015): 427–35. http://dx.doi.org/10.21273/jashs.140.5.427.

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In cybridization, new combinations of nuclear and cytoplasmic genes result in a unique genotype that may bring cellular, physical, physiological, and biochemical changes to the plant. This has been demonstrated in the unexpected cybrids generated from the fusion of citrus (Citrus sp.) protoplasts in two independent experiments. The first experiment was conducted to generate potentially seedless triploids by fusing diploid protoplasts of embryogenic ‘Dancy’ mandarin (Citrus reticulata) suspension culture cells with haploid ‘Ruby Red’ grapefruit (C. paradisi) protoplasts derived from tetrad-stage microspores. After multiple attempts, only one triploid was recovered, but several diploid plants with typical grapefruit morphology were also regenerated. In the second experiment, protoplasts derived from embryogenic ‘Dancy’ mandarin suspension culture were fused with nonembryogenic protoplasts from ‘Duncan’ grapefruit leaves in an effort to produce an allotetraploid somatic hybrid. The fruit from the resulting trees resembled grapefruit in morphology and type, and maintained excellent quality throughout the summer, when commercial grapefruit rapidly loses quality. Fruit on these trees remained firm with exceptional sweetness and good flavor into August, and without seed germination. The regenerants obtained in the protoplast fusion experiments were confirmed as cybrids by genetic marker analyses. The test grapefruit were identical to commercial ‘Ruby Red’ grapefruit at six nuclear simple sequence repeat (SSR) marker loci, but identical to ‘Dancy’ with respect to a mitochondrial intron marker. The plastid genomes of individual trees originated from either fusion partner. In the first experiment, haploid ‘Ruby Red’ protoplast preparations must have also contained contaminant diploid protoplasts. Apart from the value of altered fruit quality attributes in the marketplace, these plants provide an opportunity to understand the contributions of cytoplasmic organelle genetics to important citrus fruit-breeding objectives.

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Al-Nema, Qutaiba, e Mozahim AL-Mallah. "Electrofusion of mesophyll protoplasts from two varieties of sugar beet, (Beta vulgaris L.)". Journal of Life and Bio Sciences Research 1, n. 1 (23 aprile 2020): 22–25. http://dx.doi.org/10.38094/jlbsr117.

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Somatic hybridization between different plants through protoplast fusion represent an efficient experimental approach to produce genetically transformed plant species. Electrofution of mesophyll protoplasts in sugar beet was occurred to overcome the barriers faced breeding program of this economically industrial crop Protoplasts were successfully isolated from leave's mesophyll of two varieties of sugar beet (Beta vulgaris L.). Various enzyme solutions were assessed for the cell wall degrading ability. They express different efficiency in isolation of mesophyll protoplasts of var. Baraka. The protoplasts yield was 18 × 104 cell ml-1 using the mixture consisting of 0.5% Cellulase RS, 1.0% Hemicellulase and 0.1% Pectolyase Y-23 with 13% mannitol. A total of 16 hrs. for cell wall digestion, and protoplast viability approached 93%. Protoplasts were isolated from leaf mesophyll of var. Carola using the same enzymatic mixtures. High protoplasts yield 20 × 104 cell ml-1 was obtained, requiring the same period 16 hrs. to approach viability 96%. The protoplasts were spherical in shape, varied in chloroplast distribution, having size ranged 12 – 52 µm. The present study succeeded in electrofusion between Baraka × Carola mesophyll protoplasts, producing somatic hybrid cells under conditions of 1MHz, 1000 Vcm-1, 2 pulses, 1.5 msec./pulse with fusion percent of 73%.

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Hassan, Mohamed M., Ismail A. Ismail e Abd El-Latif A. Sorour. "Phylogeny and antagonistic activity of some protoplast fusants in Trichoderma and Hypocrea". International Journal of Applied Sciences and Biotechnology 2, n. 2 (25 giugno 2014): 146–51. http://dx.doi.org/10.3126/ijasbt.v2i2.10113.

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The present work aimed to apply mutagenesis and inter-specific protoplast fusion techniques of two locally isolated Hypocrea and Trichoderma to enhancement their biocontrol abilities against some important plant fungal pathogens which cause damping-off diseases that attacking different crops. The mutants were selected after EMS/UV treatment of Trichoderma isolates. The obtained protoplasts were fused by polyethylene glycol, and six fusants were selected for further studies. The phylogeny of the parental strains was carried out using sequence of ITS region. The BLAST of the obtained sequence was identified these isolates as H. koningii and T. asperellum. The fused protoplasts of the two mutant strains have been regenerated on PDA medium supplemented with the two fungicides. Most of the fusants exhibited fast mycelial growth on PDA as compared to parent strains. The obtained results indicated that partial or incomplete genetic recombination may be possible during nuclear and cytoplasmic protoplast fusion. Most of the fusants have shown powerful antagonistic activity against the grapevine pathogens as Pythium ultimum and Fusarium roseum as shown in dual culture and observed by SEM technique. Results of the present study demonstrated the scope and significance of the protoplast fusion technique, which can be used to develop superior hybrid strains of filamentous fungi that absent sexual stages in Trichoderma enhance biological control activity.DOI: http://dx.doi.org/10.3126/ijasbt.v2i2.10113 Int J Appl Sci Biotechnol, Vol. 2(2): 146-151

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Zhao, Lihong, Wenli Yin e Lele Wang. "High-Yield Laccase-Producing Strains Constructed by Protoplast Fusion Between Bacterium and Fungus". Open Biotechnology Journal 9, n. 1 (2 novembre 2015): 221–24. http://dx.doi.org/10.2174/1874070701509010221.

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This study is based on the construction of high-yield laccase-producing fusant achieved by inter-kingdom protoplast fusion between Pleurotus ostreatus and Escherichia coli. The optimized protoplasts formation and regeneration conditions were demonstrated with the presence of 1.5% cellulase +1.0% snailase and 0.6M mannitol at 30°C for 3h. The fusants were screened for different characteristics between two parental strains and further identified by laccase activity, offering one of the genetically stable fusants, Strain F. The fusant F produced the highest yield of laccase, being about 22% higher than that of the parental strain. The results suggest that the protoplast fusion technique can be considered as a promising technique for the control of environmental pollution.

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Gyuris, J., e E. G. Duda. "High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion." Molecular and Cellular Biology 6, n. 9 (settembre 1986): 3295–97. http://dx.doi.org/10.1128/mcb.6.9.3295.

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After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.

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23

Gyuris, J., e E. G. Duda. "High-efficiency transformation of Saccharomyces cerevisiae cells by bacterial minicell protoplast fusion". Molecular and Cellular Biology 6, n. 9 (settembre 1986): 3295–97. http://dx.doi.org/10.1128/mcb.6.9.3295-3297.1986.

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After a new transformation procedure, 10% of Saccharomyces cerevisiae cells were found to contain transforming DNA sequences. We used direct transfer of plasmid molecules by fusing bacterial minicell protoplasts to yeast protoplasts. Since the procedure significantly reduces the toxic effect of procaryotic protoplasm on the eucaryotic organism, it might be generally applicable in other systems in which transformation is inefficient or impossible.

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24

Suryati, Emma, Andi Tenriulo e Sri Rejeki Hesti Mulyaningrum. "ISOLASI DAN KULTUR PROTOPLAS RUMPUT LAUT Kappaphycus alvarezii DI LABORATORIUM". Jurnal Riset Akuakultur 2, n. 3 (30 dicembre 2007): 399. http://dx.doi.org/10.15578/jra.2.3.2007.399-405.

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Isolasi protoplas rumput laut K. alvarezii, telah dilakukan dalam rangka penyiapan protoplas untuk penyilangan melalui fusi protoplas. Metode yang digunakan antara lain melalui cara kimia dengan melisis tallus rumput laut dengan campuran enzim komersial, kemudian enzim yang berasal dari viscera keong mas baik yang segar maupun yang beku, dengan media kultur yang digunakan pada pemeliharaan makro algae antara lain Conwy, PES, dan air laut steril. Tallus rumput laut yang digunakan berasal dari bagian pangkal, tengah dan ujung. Protoplas yang hidup diuji menggunakan evans blue 0,1%, hormon perangsang tumbuh yang digunakan pada media pertumbuhan antara lain auxin, IAA, dan Kinetin. Pengamatan dilakukan terhadap jumlah protoplas hidup, pertumbuhan, dan sintasan. Hasil percobaan memperlihatkan bahwa enzim yang paling baik digunakan adalah campuran enzim komersial dengan media kultur Conwy dengan jumlah protoplas mencapai 19,8 x 106 sel/mL, bagian tallus yang paling baik adalah bagian pangkal berkisar antara 8,1x106 hingga 18,8 x 106 sel/ mL. Perangsang tumbuh yang paling baik adalah auxin. Filamen terbentuk setelah 5 hari dengan fotoperiod L:D=12:12.Isolation of seaweed’s protoplast Kappaphycus alvarezii had been done to provide protoplast for crossbreeding purpose by protoplast fusion. The method was chemically done by lyses of tallus used commercial enzyme mixture, enzyme from viscera of snail both fresh and frozen, culture media were Conwy (CW), PES, and sterile sea water (SSW) which were used to maintain the macro algae. Part of used tallus were upper, middle and tip of tallus. The viable protoplast was examined by using 0.1% evans blue and the growth-stimulating hormone were auxin, IAA, and Kinetin. Observation was concerned to the amount of viable protoplast, the growth, and the long live. Result showed that the best enzyme was commercial enzyme mixture with Conwy as the best culture media, provided protoplast until 19.8 x 106 cell/mL. The greatest protoplast content was in upper part of tallus, it could provide protoplast about 8.1 x 106 cell/mL until 18.8 x 106 cell/mL, and the best growth-stimulating hormone was auxin. Filament was formed after 5 days with photoperiod L: D=12:12.

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25

Kang, Yup, Jung H. Kim e Dewey D. Y. Ryu. "Protoplast Fusion ofLactobacillus casei". Agricultural and Biological Chemistry 51, n. 8 (agosto 1987): 2221–27. http://dx.doi.org/10.1080/00021369.1987.10868376.

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26

Kohler, Joyce, e Gary Darland. "Protoplast fusion inStreptomyces avermitilis". Journal of Industrial Microbiology 3, n. 5 (agosto 1988): 311–20. http://dx.doi.org/10.1007/bf01569532.

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27

Gu, Yu-Huan, e Wen-Hsiung Ko. "Creation of hybrid vigor through nuclear transplantation in Phytophthora". Canadian Journal of Microbiology 47, n. 7 (1 luglio 2001): 662–66. http://dx.doi.org/10.1139/w01-074.

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When isolated nuclei of a diploid oomycete, Phytophthora parasitica, were fused with protoplasts of another strain of the same species, the regenerated nuclear hybrids grew faster than the parental isolates. Such a phenomenon did not occur in hybrids regenerated from mitochondrionprotoplast or protoplastprotoplast fusion products between these two strains. These results indicate that hybrid vigor is the result of the interaction between two different kinds of nuclei, but not between mitochondria, and they suggest that the presence of mitochondria from nuclear donor cells represses the expression of increased vigor. The nuclear hybrids also expressed increased fungicide resistance and propagule production. Increased vigor in growth was also observed in the interspecific nuclear hybrids when isolated nuclei of P. parasitica were transferred into protoplasts of Phytophthora capsici, and vice versa. This phenomenon may have potential applications, such as the creation of superior fungal strains and plant cultivars with improved commercial traits for usage in industry and agriculture.Key words: hybrid vigor, nuclear transplantation, Phytophthora parasitica, Phytophthora capsici.

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28

Burza, W., B. Woźniak, J. A. Tarkowska e S. Malepszy. "Cytohistological analysis of somatic embryogenesis in cucumber (Cucumis sativus L). II. Natural fluorescence and direct somatic embryogenesis from protoplasts". Acta Societatis Botanicorum Poloniae 63, n. 3-4 (2014): 265–68. http://dx.doi.org/10.5586/asbp.1994.035.

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The development of protoplast derived from somatic embryos and some of their characteristics were compared with embryos from suspension and in vivo in the same B line. Embryos formed in a protoplast culture differed from others that their younger stages contained vacuolated cells, and older ones had altered morphological and histological structure. Somatic embryogenesis is more regular from suspension then from protoplasts. No distinct differences were observed in the rate of embryo development in vivo and in vitro, and in vitro embryos show a larger variation in size at the same stage. Embryos in vitro with fluorescence are generally larger than zygotic ones at each stage. The use of fluorescence is suggested for the selection of heterokariocytes after protoplast fusion.

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29

Ftouhi, N., e N. Guillén. "Genetic analysis of fusion recombinants in Bacillus subtilis: function of the recE gene." Genetics 126, n. 3 (1 novembre 1990): 487–96. http://dx.doi.org/10.1093/genetics/126.3.487.

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Abstract Bacillus subtilis protoplast fusion allows the study of the genetic recombination of an entire procaryotic genome. Protoplasts from bacterial strains marked genetically by chromosomal mutations were fused using polyethylene glycol and the regenerated cells analyzed. Recombinants represent 19.3% of heterozygotic cells; they are haploids. Individual characterization of clones show a unique particular phenotype in each colony suggesting that recombination takes place immediately after fusion, probably before the first cellular division. Recombination occurs in the whole chromosome; in one-third of the cases both reciprocal recombinants could be shown in the colony. The genetic interval that includes the chromosome replication origin shows the highest recombination level. Our results suggest that the RecE protein accounts for most of the fused protoplast recombination; however, some "replication origin-specific" recombination events were independent of the recE gene product.

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30

GLORIA, FERNANDA JANUZZI MENDES DA, FRANCISCO DE ASSIS ALVES MOURÃO FILHO e BEATRIZ MADALENA JANUZZI MENDES. "Plant regeneration from protoplast of Brazilian citrus cultivars". Pesquisa Agropecuária Brasileira 35, n. 4 (aprile 2000): 727–32. http://dx.doi.org/10.1590/s0100-204x2000000400008.

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A procedure is described to regenerate plants from protoplasts of Brazilian citrus cultivars, after isolation, fusion and culture. Protoplasts were isolated from embryogenic cell suspension cultures and from leaf mesophyll of seedlings germinated in vitro. The enzyme solution for protoplast isolation was composed of mannitol (0.7 M), CaCl2 (24.5 mM), NaH2PO4 (0.92 mM), MES (6.15 mM), cellulase (Onozuka RS - Yakult, 1%), macerase (Onozuka R10 - Yakult, 1%) and pectolyase Y-23 (Seishin, 0.2%). Protoplast culture in liquid medium after chemical fusion lead to the formation of callus colonies further adapted to solid medium. Somatic embryo formation occurred spontaneously after two subcultures, on modified MT medium supplemented with 500 mg/L of malt extract. Well defined embryos were germinated in modified MT medium with addition of GA3 (2.0 muM) and malt extract (500 mg/L). Plant regeneration was also achieved by adventitious shoots obtained through direct organogenesis of not well defined embryos in modified MT medium with addition of malt extract (500 mg/L), BAP (1.32 muM), NAA (1.07 muM) and coconut water (10 mL/L). Plantlets were transferred to root medium. Rooted plants were transferred to a greenhouse for further adaptation and development.

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31

Pe'er, S., e I. Chet. "Trichoderma protoplast fusion: a tool for improving biocontrol agents". Canadian Journal of Microbiology 36, n. 1 (1 gennaio 1990): 6–9. http://dx.doi.org/10.1139/m90-002.

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Protoplasts from two auxotrophic mutants of Trichoderma harzianum Rifai (ATCC 32173), obtained from young thalli following cell wall digestion by NovoZym 234, were fused in 33% PEG suspended in 10 mM Tris-HCl and 10 mM CaCl2, pH 7.5. The frequency of fusion between lysine- and arginine-requiring auxotrophs resulting in prototrophic strains was about 5%. These prototrophic strains were classified into parental and nonparental types. Colonies developed from single conidia of the nonparental phenotype exhibited prototrophic parental or recombinant phenotypes. The ability of both prototrophic and parental strains to overgrow the soil-borne pathogenic fungi Rhizoctonia solani, Sclerotium rolfsii, and Pythium aphanidermatum in dual cultures was used to evaluate their antagonistic capability. The antagonistic abilities of the prototrophic strains were found to vary with each pathogenic fungus. The prototrophic strain A2 overgrew all the pathogenic fungi more rapidly than the parental strains. Strain A2 effectively controlled Rhizoctonia damping-off of cotton seedlings, in the greenhouse, when compared with the parental strains. Protoplast fusion appears to be a useful tool for combining desirable traits from parental strains to produce improved biocontrol strains. Key words: Trichoderma harzianum, biocontrol, protoplast fusion.

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32

Sukmadjaja, Deden, Novianti Sunarlim, Endang G. Lestari, Ika Roostika e Tintin Suharlini. "Teknik Isolasi dan Kultur Protoplas Tanaman Padi". Jurnal AgroBiogen 3, n. 2 (9 agosto 2016): 60. http://dx.doi.org/10.21082/jbio.v3n2.2007.p60-65.

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Protoplast fusion or somatic hybridization technology is an alternative technology for production hybrids of plants that are difficult to be produced by conventional methods due to their sexual incompatibility. An experiment was conducted to develop techniques for isolation, purification, and culture of rice protoplasts of cultivar IR64 and a wild rice species (Oryza officinalis). Optimization of protoplast isolation and purification methods from both rice genotypes were successfully done. The highest protoplast density was obtained by digesting embryonic callus or stems of young seedling in an enzyme solution containing of 2% cellulose, 0.1% pectolyase, 0.5% macerozyme, 0.5% driselase, 5 mM ES, and 13% mannitol in CPW solution. The protoplast digestion was done for three hours by soaking in the enzyme solution followed by shaking at 50 rpm under a room temperature. Purification of the protoplasts were done by separating them from plant debris using a 25% sucrose solution. Protoplast regeneration was not successful using although different media compositions and conditions. Growth process from cell division to cell aggregate was only successful on IR64 protoplast culture on a medium that contained AgNO3.

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33

Rahmawati, Novia, Muhammad Zainuri e Hermin Pancasakti Kusumaningrum. "Aplikasi Pakan Kaya Karotenoid Hasil Fusi ProtoplasmIntergenera Dunaliella salina dan Chlorella vulgaris pada Udang Windu (Penaeus monodon F.) Stadia PL-20 Di Desa Asempapan, Pati, Jawa Tengah". Bioma : Berkala Ilmiah Biologi 15, n. 2 (27 dicembre 2013): 46. http://dx.doi.org/10.14710/bioma.15.2.46-52.

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Dunaliella salina and Chlorella vulgaris is a natural feed microalgae with high carotenoid content that can be increased using protoplast fusion technique. Protoplast fusion as one of the application fields of genetic engineering is a method for obtaining recombinant with the desired properties and profitable in a short time. This study aimed to see the effect of the addition of carotenoid-rich feed results from protoplast fusion recombinant D. salina and C. vulgaris on the survival rate and weight of shrimp post larvae. Mixed fusion results feed and artificial feed needed for the growth of post-larval shrimp, moulting and skin pigmentation. The results showed that the recombinant from protoplast fusion intergenera D. salina and C. vulgaris contains carotenoid pigments higher, reaching 124.6 mg / g bks from the second parent, namely D. salina reached 101.83 mg / g bks, while C. vulgaris 97.18 ug / g bks. Feed manufacturing is done by mixing pellets and 80-100x103 cells per 0.0225 g of feed. Application of feed carried on Penaeus monodon F. (tiger prawn) stadia PL-20 for a month. The results of weight measurements showed the highest prawn post larvae reached at artificial feeding plus recombinant protoplast fusion results intergenera D. salina and C. vulgaris and was able to raise the level of post-larval shrimp survival rate reached 88%. Keywords: D. salina, C. vulgaris, Protoplast Fusion, Carotenoid, Penaeus monodon F.

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34

Cho, Kwang-Soo, e Tae-Ho Park. "Potato breeding via protoplast fusion". Journal of Plant Biotechnology 41, n. 2 (30 giugno 2014): 65–72. http://dx.doi.org/10.5010/jpb.2014.41.2.65.

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35

Arnau, JosÃ©, Antonio Ortiz, Juan C. Gomez-FernÃ¡ndez, Francisco J. Murillo e Santiago Torres-MartÃnez. "Liposome-protoplast fusion inPhycomyces blakesleeanus". FEMS Microbiology Letters 51, n. 1 (giugno 1988): 37–40. http://dx.doi.org/10.1111/j.1574-6968.1988.tb02964.x.

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36

KANG, Yup, Jung H. KIM e Dewey D. Y. RYU. "Protoplast fusion of Lactobacillus casei." Agricultural and Biological Chemistry 51, n. 8 (1987): 2221–27. http://dx.doi.org/10.1271/bbb1961.51.2221.

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37

Ohgawara, T., e S. Kobayashi. "Application of protoplast fusion tocitrusbreeding". Food Biotechnology 5, n. 2 (gennaio 1991): 169–84. http://dx.doi.org/10.1080/08905439109549800.

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38

Iwata, M., M. Mada e H. Ishiwa. "Protoplast fusion of Lactobacillus fermentum." Applied and Environmental Microbiology 52, n. 2 (1986): 392–93. http://dx.doi.org/10.1128/aem.52.2.392-393.1986.

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39

Kanatani, Kazuo, Kazushi Yoshida, Takatsugu Tahara, Masaru Sakamoto e Masao Oshimura. "Intraspecific Protoplast Fusion ofLactobacillus plantarum". Agricultural and Biological Chemistry 54, n. 1 (gennaio 1990): 225–27. http://dx.doi.org/10.1080/00021369.1990.10869927.

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40

Olivares-Fuster, O., N. Duran-Vila e L. Navarro. "Electrochemical protoplast fusion in citrus". Plant Cell Reports 24, n. 2 (10 febbraio 2005): 112–19. http://dx.doi.org/10.1007/s00299-005-0916-1.

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41

Kanchanapoom, Kamnoon, e Wendy F. Boss. "Osmoregulation of fusogenic protoplast fusion". Biochimica et Biophysica Acta (BBA) - Biomembranes 861 (1986): 429–39. http://dx.doi.org/10.1016/0005-2736(86)90451-7.

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42

Li, Yinmei, Lijie Guan, Liren Lou, Guoqiang Cui, Yuan Yao, Haowei Wang, Chuanshun Cao, Runlong Lu e Xi Chen. "Laser-induced tobacco protoplast fusion". Science in China Series C: Life Sciences 42, n. 2 (aprile 1999): 122–27. http://dx.doi.org/10.1007/bf02880046.

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43

Muralidhar, R. V., e T. Panda. "Fungal protoplast fusion – a revisit". Bioprocess Engineering 22, n. 5 (4 maggio 2000): 429–31. http://dx.doi.org/10.1007/s004490050755.

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44

Fo, Francisco A. A. Mourão, Jude W. Grosser e Frederick G. Gmitter. "362 PRODUCTION OF SEVEN NEW INTERGENERIC SOMATIC HYBRIDS FOR CITRUS ROOTSTOCK IMPROVEMENT". HortScience 29, n. 5 (maggio 1994): 482g—483. http://dx.doi.org/10.21273/hortsci.29.5.482g.

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Abstract (sommario):

Protoplast culture following polyethylene glycol (PEG)-induced fusion resulted in the regeneration of somatic hybrid plants from the following combinations: `Succari' sweet orange (C. sinensis L. Osbeck) + Severinia disticha; `Hamlin' sweet orange (C. sinensisj + S. disticha: `Valencia' sweet orange (C. sinesis) + S. disticha; `Nova' tangelo (C. reticulata hybrid) + S. disticha; `Succari' sweet orange + S. buxifolia; `Nova' tangelo + Citropsis gilletiana; and `Succari' sweet orange + Atlantia ceylanica. `Succari', `Hamlin', `Valencia', and `Nova' protoplasts were Isolated from ovule-derived embryogenic callus and/or suspension cultures whereas protoplasts of S. disticha, S. buxifolia, C. gilletiana, and A. ceylanica were isolated from leaves of potted trees in a greenhouse. Plants were regenerated via somatic embryogenesis and somatic hybrids were identified on the basis of leaf morphology. Electrophoretic analysis of isozyme banding patterns and root tip chromosome counts are being performed. Somatic hybrids with S. disticha are apparently weak whereas the other somatic hybrid plants with S. buxifolia, C. gilletiana, and A. ceylanica exhibit adequate vigor. These are more examples that the the techique of protoplast fusion can be an important tool in overcoming barriers to hybridization of sexually incompatible species.

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45

Perronnet, C., J. C. Chenieux e M. Rideau. "Protoplast fusion ofCatharanthus roseus cells by electrofusion of chemically-agglutinated protoplasts". Biologia plantarum 36, n. 1 (1 gennaio 1994): 1–8. http://dx.doi.org/10.1007/bf02921259.

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46

Grimes, Howard D., Robert D. Slocum e Wendy F. Boss. "α-Difluoromethylarginine treatment inhibits protoplast fusion in fusogenic wild-carrot protoplasts". Biochimica et Biophysica Acta (BBA) - Molecular Cell Research 886, n. 1 (aprile 1986): 130–34. http://dx.doi.org/10.1016/0167-4889(86)90218-1.

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47

Andrade, Carolina M. M., Flavia M. Oliveira e Valter R. Linardi. "Candida fennica: enhancement of protoplast formation and fusion". Canadian Journal of Microbiology 38, n. 8 (1 agosto 1992): 807–10. http://dx.doi.org/10.1139/m92-132.

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Abstract (sommario):

Auxotrophic mutants were obtained by exposing Candida fennica FTPT/CCT-8903 to ultraviolet light and treating the cells with nystatin in an enrichment procedure. The mutants were used for protoplast fusion. The highest protoplast regeneration frequency was obtained when cells were treated with 10 mg NovoZym 234/mL in the presence of 0.8 M KCl for 60 min. Fusion frequency was in the range 5.84 × 10−5 to 7.2 × 10−5. Five prototrophic hybrid colonies, designated S9, S15, S17, S18, and S25, were isolated, and their DNA contents were measured. These measurements indicated that the S17 hybrid is haploid, S9 and S18 are polyploid, and S15 and S25 are both aneuploid. Key words: Candida fennica, nystatin, auxotrophic mutants, protoplast fusion.

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48

Grosser, J. W., e J. L. Chandler. "NEW CITRUS ROOTSTOCKS VIA PROTOPLAST FUSION". Acta Horticulturae, n. 622 (agosto 2003): 491–97. http://dx.doi.org/10.17660/actahortic.2003.622.54.

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49

Kim, Byong Kak, Mi Ja Shim e Ha Won Kim. "Studies on Protoplast Fusion of Basidiomycetes". International Journal of Medicinal Mushrooms 3, n. 2-3 (2001): 1. http://dx.doi.org/10.1615/intjmedmushr.v3.i2-3.120.

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50

KANATANI, Kazuo, Kazushi YOSHIDA, Takatsugu TAHARA, Masaru SAKAMOTO e Masao OSHIMURA. "Intraspecific protoplast fusion of Lactobacillus plantarum." Agricultural and Biological Chemistry 54, n. 1 (1990): 225–27. http://dx.doi.org/10.1271/bbb1961.54.225.

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