Topic
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: 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: Somatic hybrid plants produced after protoplast electrofusion with the objective of transferring insect resistance, which is associated with the presence of glandular hairs, into potato lines exhibited several morphological traits intermediate to both parents including plant habit, leaf form, and particularly short four-lobed glandular trichomes inherited from the wild parent.
48 citations
TL;DR: Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days and viable protoplasts could be obtained 15–21 days after thawing.
Abstract: A method is described for cryopreservation of cell suspension lines of rice (Oryza sativa L.) for use in protoplast research and as a way of retaining desirable characteristics of cell lines. The procedure involves pre-culture with mannitol, addition of a cryoprotectant solution of sucrose, dimethyl sulfoxide, glycerol and L-proline, two step freezing and storage in liquid nitrogen. Cells have been preserved for up to 14 months (the longest period tried in these experiments). Cryopreserved cells proliferated after plating on solid medium and new cell suspensions could be initiated within 15 days. Viable protoplasts, capable of divisions and callus formation, could be obtained 15-21 days after thawing. Variation between cell lines in terms of recovery rate after cryopreservation occurred. Differences between cell lines in plating efficiencies on solidified medium, however, contributed to this variation. Protoplasts from cryopreserved regenerable cell lines gave rise to embryogenic callus from which plants could be regenerated. These plants developed to maturity. A transformed cell line was also cryopreserved and it had retained the hygromycin resistance and regenerative capacity of the original cell line.
48 citations
TL;DR: Intact protoplasts show no oxygen evolution with 3-phosphoglycerate or with oxalacetate plus pyruvate as substrates, even when these substances are provided at high concentrations, while protoplast extracts and chloroplasts display rates of oxygen evolution of 2-3 µmol min-1 (mg Chl)-1 with the same substances.
Abstract: A procedure is described for the preparation of metabolically active mesophyll protoplasts from maize, and of functional, intact chloroplasts from these protoplasts. Intact protoplasts show no oxygen evolution with 3-phosphoglycerate or with oxalacetate plus pyruvate as substrates, even when these substances are provided at high concentrations. On the other hand, protoplast extracts and chloroplasts display rates of oxygen evolution of 2-3 µmol min-1 (mg Chl)-1 with the same substances. Pyruvate stimulates oxalacetate-dependent oxygen evolution substantially, indicating good coupling between non-cyclic electron flow and phosphorylation. Low PI concentrations stimulate 3-phosphoglycerate-dependent oxygen evolution; high PI concentrations, and pyridoxal phosphate, inhibit this activity, suggesting a common carrier for 3-phosphoglycerate and PI.
48 citations
01 Mar 2016
TL;DR: A rice protoplast system is established about 10 years ago, which has been recently used in many laboratories and is useful for protein expression, subcellular localization, bimolecular fluorescence complementation, and co-immunoprecipitation assays.
Abstract: Rice (Oryza sativa) is not only the staple food for half of the world's population but also a model monocot plant for molecular biology studies. Although rice genes have been extensively investigated in the last two decades, the functions of many genes in the rice genome are still not known. One of the rapid and efficient approaches for determining gene function in vivo is protoplast-based transient expression analysis. We established a rice protoplast system about 10 years ago, which has been recently used in many laboratories. This protocol is useful for protein expression, subcellular localization, bimolecular fluorescence complementation, and co-immunoprecipitation assays. © 2016 by John Wiley & Sons, Inc.
48 citations