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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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TL;DR: Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.
Abstract: Protoplasts of methionine-and lysine-requiring h- mutants isolated from the L972 h- strain of Schizosaccharomyces pombe were fused. The protoplasts were obtained from the cells with enzymes produced by Trichoderma viride. When a mixture of the protoplasts was treated with 30% PEG 4000 solution containing 10 mM CaCl2, cell fusion and complementation was attained with a frequency of 0.17%. Both fusion partners were recovered among the spores after crossing of the fusion products with the strain M210 ade6 h+. Cytological and haploidization examinations showed that the fusion cells are not heterokaryons, and that the increased amount of genetic material is situated in one nucleus.

167 citations

Journal ArticleDOI
TL;DR: The regeneration of plants from protoplasts of wheat isolated from a regenerable embryogenic suspension culture that was initiated from immature embryo-derived ‘aged’ compact callus produced healthy plants that were successfully transferred to soil and grown in the greenhouse.
Abstract: We describe the regeneration of plants from protoplasts of wheat isolated from a regenerable embryogenic suspension culture that was initiated from immature embryo-derived ‘aged’ compact callus. Proto-colonies were also obtained from a non-regenerable cell line established from friable callus that arose spontaneously on the aged callus, but did not give rise to any plants. Plating efficiencies well in excess of twenty percent were routinely obtained from protoplasts isolated from the regenerable as well as the non-regenerable cell lines in Kao and Michyaluk, as well as Murashige and Skoog based, nutrient media. Four to five week old protocolonies, derived from regenerable suspension culture protoplasts, formed distinct somatic embryos and shoots upon transfer to various regeneration media. Shoots were rooted on transfer to rooting media. Successive transfer of the plantlets to MS or half-strength MS medium, with or without activated charcoal, produced healthy plants that were successfully transferred to soil and grown in the greenhouse.

164 citations

Journal ArticleDOI
TL;DR: An improved method is described which allows the storage of the protoplasts deep frozen (at − 70°C) for several months without affecting their transforming capability.
Abstract: Protoplast transformation is so far the only method to transfer plasmids into Staphylococcus carnosus. However, the preparation of new protoplasts for each transformation is time-consuming and the quality of the protoplasts may also vary each time. An improved method is described which allows the storage of the protoplasts deep frozen (at − 70°C) for several months without affecting their transforming capability. The possibility of keeping protoplasts competent for a long period opens several advantages: the transformation can be carried out immediately, the transformation procedure is reduced to only a couple of minutes, and the protoplasts of one batch have the same quality, yielding a more constant transformation frequency.

160 citations

Journal ArticleDOI
TL;DR: It is demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.
Abstract: Site-directed mutagenesis of nitrate reductase genes using direct delivery of purified Cas9 protein preassembled with guide RNA produces mutations efficiently in Petunia × hybrida protoplast system. The clustered, regularly interspaced, short palindromic repeat (CRISPR)-CRISPR associated endonuclease 9 (CRISPR/Cas9) system has been recently announced as a powerful molecular breeding tool for site-directed mutagenesis in higher plants. Here, we report a site-directed mutagenesis method targeting Petunia nitrate reductase (NR) gene locus. This method could create mutations efficiently using direct delivery of purified Cas9 protein and single guide RNA (sgRNA) into protoplast cells. After transient introduction of RNA-guided endonuclease (RGEN) ribonucleoproteins (RNPs) with different sgRNAs targeting NR genes, mutagenesis at the targeted loci was detected by T7E1 assay and confirmed by targeted deep sequencing. T7E1 assay showed that RGEN RNPs induced site-specific mutations at frequencies ranging from 2.4 to 21 % at four different sites (NR1, 2, 4 and 6) in the PhNR gene locus with average mutation efficiency of 14.9 ± 2.2 %. Targeted deep DNA sequencing revealed mutation rates of 5.3–17.8 % with average mutation rate of 11.5 ± 2 % at the same NR gene target sites in DNA fragments of analyzed protoplast transfectants. Further analysis from targeted deep sequencing showed that the average ratio of deletion to insertion produced collectively by the four NR-RGEN target sites (NR1, 2, 4, and 6) was about 63:37. Our results demonstrated that direct delivery of RGEN RNPs into protoplast cells of Petunia can be exploited as an efficient tool for site-directed mutagenesis of genes or genome editing in plant systems.

158 citations

Journal ArticleDOI
TL;DR: Fusions Involving Nonviable Protoplasts, Genetic Mapping by Protoplast Fusion, and Detection of extrachromosomal inheritance are studied.
Abstract: Gram-poSlllve bacteria . Gram-negative bacteria . Protoplast Regeneration . GENETIC INTERACTIONS IN INTRASPECIFIC PROTOPLAST FUSIONS ..... ... . . . . . . . . ... . . ... . . . . ... ......... . . ... ... ... . . . . . . . . . . . . ....... . . . . . The Measurement of Fusion . Optim!!l Conditions for Protoplast F.usio� : .. : : .. GenetIc Events After Protoplast FUSIOn In Gram-Poslflve BacteTla .. Streptomyces . Bacillus . ComparisollS between Streptomyces and Bacillus . Genetic Mapping by Protoplast Fusion .. Chromosomal linkage . Detection of extrachromosomal inheritance . Fusions Involving Nonviable Protoplasts .. Protoplast Fusion in Gram-Negative Bacteria . INTERSPECIFIC PROTOPLAST FUSION . PLASMID TRANSFER BY PROTOPLAST FUSION ... . . . . . . . . . . . .. LOSS OF PLASMIDS BY PROTOPLASTING .. . . . . . . . . . . . . . . . .. . . . ........ .. PLASMID TRANSFORMATION AND TRANSFECTION OF PROTOPLASTS .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . The Techniques .

158 citations


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