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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: Somatic hybrids selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids.
Abstract: A technique has been developed for the isolation of large numbers of protoplasts from protonemal tissue of Physcomitrella patens, and for their regeneration to give whole plants. Somatic hybrids have been selected following treatment of mixtures of protoplasts from complementary auxotrophic strains with 50 mM CaCl2 at high pH. The hybrids have a morphology different from that of normal haploid strains, but similar to that of aposporously produced diploids. The progeny resulting from selffertilisation of the hybrids show a segregation which is consistent with their being the products of meioses in an autotetraploid.

132 citations

Journal ArticleDOI
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

Journal ArticleDOI
TL;DR: The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice byculturing in NO3 medium that contained nitrate as the sole nitrogen source.
Abstract: The regeneration of haploid and diploid plants was demonstrated from protoplasts that were isolated from cell suspensions of anther callus in rice. The cell suspension in the AA medium that contained 4 amino acids as the sole nitrogen source was friable, finely dispersed, and readily released a large number of protoplasts. These protoplasts, subsequently cultured in NO3 medium that contained nitrate as the sole nitrogen source, formed compact calli. The compact calli produced green plants with a frequency of 24%. Out of 15 flowering plants, 4 were haploids, the others were diploids which showed a uniform morphology but varied in seed fertility from 95 to 0%.

129 citations

Journal ArticleDOI
TL;DR: This is the first study presenting detailed results on biolistic transformation of a filamentous fungus, and southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated.
Abstract: Biolistic (biological ballistic) and protoplast-mediated procedures were compared as methods for transforming strains of Gliocladium virens and Trichoderma harzianum. For biolistic transformation, conidia were bombarded using a helium-driven biolistic device to accelerate M5 tungsten particles coated with plasmid or genomic DNA. DNA from either source contained a bacterial hygromycin B resistance gene (hygB) as a dominant selectable marker. The same sources of DNA were also used to transform protoplasts using a standard polyethylene glycol-CaCl2 protoplast fusion protocol. Hygromycin B-resistant (HygBR) transformants were recovered from all strains, methods, and DNA sources except for genomic DNA used with the protoplast method. The biolistic procedure was technically simpler, and increased transformation frequency and genetic stability in the progeny as compared with the protoplast-mediated transformation. Southern analysis of homokaryotic HygBR progenies showed that the transforming sequences were integrated into the genome of the recipient strains, and apparently were methylated. This is the first study presenting detailed results on biolistic transformation of a filamentous fungus.

129 citations

Journal ArticleDOI
TL;DR: The bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.
Abstract: We have used the bar gene in combination with the herbicide Basta to select transformed rice (Oryza sativa L. cv. Radon) protoplasts for the production of herbicide-resistant rice plants. Protoplasts, obtained from regenerable suspension cultures established from immature embryo callus, were transformed using PEG-mediated DNA uptake. Transformed calli could be selected 2-4 weeks after placing the protoplast-derived calli on medium containing the selective agent, phosphinothricin (PPT), the active component of Basta. Calli resistant to PPT were capable of regenerating plants. Phosphinothricin acetyltransferase (PAT) assays confirmed the expression of the bar gene in plants obtained from PPT-resistant calli. The only exceptions were two plants obtained from the same callus that had multiple copies of the bar gene integrated into their genomes. The transgenic status of the plants was verified by Southern blot analysis. In our system, where the transformation was done via the protoplast method, there were very few escapes. The efficiency of co-transformation with a reporter gene gusA, was 30%. The T0 plants of Radon were self-fertile. Both the bar and gusA genes were transmitted to progeny as confirmed by Southern analysis. Both genes were expressed in T1 and T2 progenies. Enzyme analyses on T1 progeny plants also showed a gene dose response reflecting their homozygous and heterozygous status. The leaves of T0 plants and that of the progeny having the bar gene were resistant to application of Basta. Thus, the bar gene has proven to be a useful selectable and screenable marker for the transformation of rice plants and for the production of herbicide-resistant plants.

127 citations


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