Protoplast

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

Transfer of mitochondria by protoplast fusion in Saccharomyces cerevisiae

Abstract: INTRASPECIFIC and interspecific transfer of nuclear genes of fungi has recently been achieved by fusion of protoplasts1–5. We describe here the transfer of mitochondrial genome using the protoplast fusion technique. The experiments were carried out with strains of identical mating-type, so we also report successful complementation of auxotrophic yeast cells of the same mating-type after protoplast fusion.

Ubiquitin genes are differentially regulated in protoplast-derived cultures of Nicotiana sylvestris and in response to various stresses.

Abstract: Four ubiquitin mRNA size classes were found to be differentially regulated in mesophyll protoplast-derived cultures of Nicotiana sylvestris. Three mRNA families of 1.9, 1.6 and 1.35 kb were expressed as soon as protoplasts were isolated. The 1.9 and 1.6 kb size classes were transiently expressed during the first hours of culture, whereas the level of expression of the 1.35 kb size class was maintained as long as cells kept dividing. A 0.7 kb mRNA size class started to be expressed just before the first divisions were observed. cDNAs corresponding to each of these families were isolated from a 6-h-old protoplast cDNA library and characterized. The 1.9, 1.6 and 1.35 kb mRNAs thus encode 7- or more, 6- and 5- mers, respectively, of ubiquitin whereas the 0.7 kb mRNAs encode a monomer of ubiquitin fused to a carboxyl extension protein of 52 amino acids. The expression of ubiquitin genes was studied, using probes specific for each of these transcript families, during protoplast culture and, for comparison, after various stresses including heat shock, HgCl2 treatment, a viral infection giving rise to a hypersensitive reaction, and an Agrobacterium tumefaciens infection which resulted in tumour formation. The 1.9 and 1.6 kb mRNA size classes were found to be stress-regulated, the 0.7 kb mRNA size class developmentally regulated and the 1.35 kb size class both stress- and developmentally regulated.

Parameters governing bacterial regeneration and genetic recombination after fusion of Bacillus subtilis protoplasts.

Abstract: Bacterial protoplast fusion, induced by polyethylene glycol, has been made more regular and convenient by further specification and improvement of various steps in the previously used procedure. These have made it possible to obtain regularly 100% regeneration of Bacillus subtilis cells from protoplasts before treatment with polyethylene glycol and yields of 10 to 75% from polyethylene glycol-treated protoplasts. Genetic recombination frequencies do not increase correspondingly. Also, when regeneration is reduced by various experimental conditions, recombination does not decrease in proportion. It is concluded that regeneration of recombinant-forming cells is independently determined and not closely related to the average regeneration for the population. Kinetic studies with varying individual parental or total protoplast concentrations strongly indicate that protoplast collision and contact is not the limiting factor determining the number of genetic recombinants obtained. Recombination approximates a linear, rather than quadratic, function of the total or of the majority protoplast population present, from which it is concluded that fusion events are always adequate to produce substantially more potential recombinants than are registered. The strong effect of the majority/minority ratio upon the number of minority cells that become recombinant is independent of which parent is in excess. This shows in a direct and physiological way that both parents are equivalent partners in their genetic contributions.

Growth of Avena Coleoptiles and pH Drop of Protoplast Suspensions Induced by Chlorinated Indoleacetic Acids

Abstract: Several indoleacetic acids, substituted in the benzene ring, were compared in the Avena straight growth bioassay. 4-Chloroindoleacetic acid, a naturally occurring plant hormone, is one of the strongest hormones in this bioassay. With an optimum at 10-6 mol l-1, it is more active than indoleacetic acid, 2,4-dichlorphenoxyacetic acid and naphthaleneacetic acid. 5-Chloro- and 6-chloroindoleacetic acids are very strong auxins as well. Other derivatives tested have a lower activity. 5,7-Dichloro- and 5-hydroxyindoleacetic acids have very low auxin activity at 10-4 mol l-1 and may be anti-auxins. Some of the derivatives were compared for their effect on pH decline in stem protoplast suspensions of Helianthus annuus L. and Pisum sativum L. The change of pH occurs without a lag period or with only a very short one. Derivatives which are very active in the Avena straight growth assay cause a larger pH decline than indoleacetic acid, while inactive derivatives cause effectively no pH decline.

X-ray irradiation promoted asymmetric somatic hybridisation and molecular analysis of the products.

Abstract: Complementation of two metabolic deficiences — nitrate reductase and tryptophan synthase — was used to select for somatic fusion hybrids between tobacco (Nicotiana tabacum) and henbane (Hyoscyamus muticus) with prior X-irradiation of one partner. Using species specific, radioactively labelled DNA probes it could be shown that a) irradiation significantly reduced the amount of chromosomal DNA of the irradiated fusion partner in the somatic hybrid, b) irradiation with doses which completely inhibit protoplast division did not pevent transfer of substantial amounts of chromosomal DNA into the fusion hybrids (so called ‘cybrids’) and c) this method transfers functional nuclear genes together with the partial genome from the irradiated partner.