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Protoplast

About: Protoplast is a research topic. Over the lifetime, 5474 publications have been published within this topic receiving 122468 citations.


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Journal ArticleDOI
TL;DR: The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.
Abstract: Resistance to streptomycin and lincomycin in plant cell culture is used as a color marker: resistant cells are green whereas sensitive cells are white on the selective medium. Streptomycin and lincomycin at appropriate concentrations do not kill sensitive Nicotiana cells. The selective value of plastid ribosomal DNA mutations, conferring resistance to streptomycin and lincomycin, was investigated by growing heteroplastidic cells on a selective medium. The heteroplastidic cells were obtained by protoplast fusion, and contained a mixed population of streptomycin resistant plastids from the N. tabacum line Nt-SR1-Kan2, and lincomycin resistant plastids from the N. plumbaginifolia line Np-LR400-Hyg1. Clones derived from protoplast fusion were selected by kanamycin and hygromycin resistance, transgenic nuclear markers. Somatic hybrids were then grown on a selective streptomycin or lincomycin medium, or in the absence of either drug to a 50 to 100 mg size callus. Southern analysis of a polymorphic region of plastid DNA (ptDNA) revealed that somatic hybrids grown on streptomycin contained almost exclusively ptDNA from the streptomycin resistant parent, somatic hybrids grown on lincomycin contained almost exclusively ptDNA from the lincomycin resistant parent whereas somatic hybrids grown in the absence of either drug contained mixed parental plastids. Sensitive ptDNA was below detection level in most clones on selective medium, but could be recovered upon subsequent culture in the presence of the appropriate drug. The drugs streptomycin and lincomycin provide a powerful selection pressure that should facilitate recovery of plastid transformants.

38 citations

Journal ArticleDOI
TL;DR: Limited compatibility may reduce the likelihood of parasexual recombination but does not preclude the possibility of genetic manipulation of Trichoderma strains by protoplast fusion.

38 citations

Journal ArticleDOI
01 Aug 1987-Planta
TL;DR: A sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed and identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplast and cells.
Abstract: Murine monoclonal antibodies to protoplast membrne antigens were generated using mouse myelomas and spleen cells from mice immunized with Nicotiana tabacum L. leaf protoplasts. For selecting antibody-secreting clones, a sensitive and rapid enzyme-linked immunosorbent assay (ELISA) for monoclonal antibody binding to immobilized cellular membrane preparations or immobilized protoplasts was developed. With intact protoplasts as immobilized antigen, the ELISA is selective for antibodies that bind to plasma-membrane epitopes present on the external surface of protoplasts. Using the membrane ELISA, a total of 24 hybridoma lines were identified that secreted antibodies to plant membrane epitopes. The protoplast ELISA and subsequent immunofluorescence studies identified four hybridoma lines as secreting antibodies which bound to the external surface of protoplasts and cells. The corresponding antigens were not species- or tissue-specific, were periodatesensitive, and were located in membranes which equilibrated broadly throughout a linear sucrose gradient. When protein blots of electrophoretically separated membrane proteins were probed with these antibodies, a band of Mr 14 kilodaltons (kDa) and a smear of bands of Mr 45–120 kDa were labeled. An additional set of three antibodies appeared by immunofluorescence to bind to the plasma membrane of broken but not intact protoplasts and labeled membranes equilibrating at a density of approx. 1.12 kg·l-1 in a linear sucrose density gradient. These classes of monoclonal antibodies enlarge the library of monoclonal antibodies (Norman et al. 1986, Planta 167, 452–459) available for the study of plant plasma-membrane structure and function.

38 citations

Journal ArticleDOI
TL;DR: The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.
Abstract: Protoplast fusion can be used to produce somatic hybrids of species that cannot be obtained by sexual hybridization. The possibility to introgress genes from Solanum species into the cultivated tomato species Lycopersicon esculentum, and to obtain novel cytoplasm-nucleus combinations (cybrids) was considered as an important strategy to extend the genetic variation available for tomato breeding. Somatic hybrids between L. esculentum and other Lycopersicon species, as well as between L. esculentum and Solanum or Nicotiana species, have been produced. Specific mutants, genotypes with antibiotic resistances, and metabolic inhibition by iodoacetate or iodoacetamide and irradiation were used for the selection of hybrids. In addition, the improvement of protoplast culture techniques and the use of the favourable tissue culture traits derived from species such as L. peruvianum, which have been introduced into tomato by classical breeding, allowed the efficient recovery of somatic hybrids. However, the occurrence of somatic incongruity in fusion combinations of L. esculentum and Solanum and even more in L. esculentum and Nicotiana, did not allow the production of true cybrids and/or fertile hybrids, indicating the importance of both cytoplasm-nucleus and nucleus-nucleus interactions in somatic incongruity. Another problem with fusions between distantly related species is the strongly reduced fertility of the hybrids and the very limited homoeologous recombination between chromosomes of the parental species. Partial genome transfer from donor to recipient through microprotoplast (+) protoplast fusion, and the production of monosomic or disomic chromosome addition lines, light overcome some of these problems. In symmetric somatic hybrids between L. esculentum and S. tuberosum the occurrence of limited somatic and meiotic recombination was demonstrated. Fertile progeny plants could be obtained, though at a low frequency, when embryo rescue was performed on a large scale after backcrossing hexaploid somatic tomato (+) potato hybrids with a tetraploid potato genotype. The potential value of genomic in situ hybridization (GISH) and RFLPs for the analysis of the genome/chromosome composition of the hybrids has been demonstrated for intergeneric somatic hybrids between Lycopersicon and Solanum.

37 citations

Journal ArticleDOI
TL;DR: Methods were developed for the reproducible production of high yields of stable protoplasts from mesophilic and thermophilic strains of Mucor, of special importance in these species since conventional genetic analysis by recombination studies is not feasible.
Abstract: Methods were developed for the reproducible production of high yields of stable protoplasts from mesophilic and thermophilic strains of Mucor. This is a pre-requisite for the genetic analysis and manipulation of Mucor by cell fusion or transformation and is of special importance in these species since conventional genetic analysis by recombination studies is not feasible. The cell wall lytic enzyme used was the concentrated culture fluid of Streptomyces sp no. 6 grown on chitosan and chitin. Using germlings of M. circinelloides f. lusitanicus the optimum conditions for protoplast formation were determined as: 0.5–1.0 mg·ml−1 streptozyme (1.2 units chitosanase·ml−1) plus 2.0 mg·ml−1 novozym 234 or 1%(v/v) helicase in 0.5m-sorbitol, 0.01m-sodium phosphate buffer, pH 6.5 for 3–4 hours at 23°C. Treatment of 107·ml−1 germlings gave yields of up to 3×107·ml−1 protoplasts with regeneration and fusion frequencies of up to 40% and 1.7%, respectively. With the thermophilic strains growth at low temperature (25°C) was critical for the subsequent formation of stable protoplasts. Synchronous growth and germination was obtained at this temperature by a short pre-incubation (1–3 hours) of the sporangiospores at 40°C.

37 citations


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Performance
Metrics
No. of papers in the topic in previous years
YearPapers
202358
2022153
202160
202060
201978
201855