Protoplast

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

Somatic hybridisation of Daucus carota and D. capillifolius by protoplast fusion

Abstract: Protoplasts isolated from cultured cells of albino carrot (Daucus carota) and normal green D. capillifolius were fused by polyethylene glycol. Selection of somatic hybrid plants was based on the restoration of photosynthetic function in hybrids. Green plantlets selected from embryo cultures were characterized on the basis of leaf morphology. The interspecific protoplast fusion resulted in green plants with leaves which were intermediate between those of the parents. The somatic hybrids between orange rooted carrot variety and D. capillifolius with long white roots produced long, white and fleshy roots. The cytological analysis of parasexual hybrids revealed that the chromosome number ranged from 34 to 54. The most frequent chromosome number was 2n = 36. Hybrids were also found with 34 and 35 chromosomes. The somatic hybrid showed the same isoenzyme pattern of leaf peroxidase as D. carota.

Preparation by two-dimensional electrophoresis of proteins for antibody production: Antibodies against proteins whose synthesis is reduced by auxin in tobacco mesophyll protoplasts

Abstract: Using phenol extraction from tobacco callus, we have prepared extracts with a high protein content. These proteins were separated in cylindrical non-equilibrium pH gradient gels and visualized by dipping in sodium dodecyl sulfate (SDS)-containing solution. Three gel sections, each containing proteins previously detected as abundantly synthesized in tobacco mesophyll protoplasts and whose synthesis is reduced by auxin application, were excised from each gel and collected. These proteins were further separated on slab SDS gels and protein bands were excised after Coomassie Brilliant Blue R-250 staining and used to inject three rabbits. After one booster, highly specific antibodies were detected in their sera by ELISA and immunoblotting. Using these sera we have confirmed that the corresponding proteins are identical in callus and mesophyll protoplast and demonstrated that they are abundantly accumulated in tobacco roots but are undetectable in aerial organs and seeds.

A mannose selection system for production of fertile transgenic maize plants from protoplasts.

Abstract: Maize (Zea mays L.) callus cultures cannot use mannose as a sole carbohydrate source, but can utilize fructose for that purpose. Phosphomannose isomerase (PMI) can convert mannose to fructose. Transgenic maize plants were obtained by selecting polyethylene glycol (PEG)-mediated transformed protoplasts on mannose (20 g/l) containing medium. Transgenic calluses and plants carrying the PMI structural gene, manA, were able to convert mannose to fructose. The PEG-mediated protoplast transformation frequency was 0.06%. Stable transformation was confirmed by PCR, PMI activity, germination tests, and by histochemical staining with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc). Stable integration of the transgenes into the maize genome was demonstrated in T1 and T2 plants. Results indicate that the mannose selection system can be used for maize PEG-mediated protoplast transformation.

A periplasm in Bacillus subtilis.

Abstract: The possibility of there being a periplasm in Bacillus subtilis, in the distinct cell compartment bounded by the cytoplasmic membrane and the thick cell wall, has been investigated quantitatively and qualitatively. Cytoplasmic, membrane, and protoplast supernatant fractions were obtained from protoplasts which were prepared isotonically from cells grown under phosphate limitation. The contents of the protoplast supernatant fraction represent an operational definition of the periplasm. In addition, this cell fraction includes cell wall-bound proteins, exoproteins in transit, and contaminating cytoplasmic proteins arising through leakage from, or lysis of a fraction of, protoplasts. The latter, measured by assay of enzyme markers and by radiolabeled RNA and protein, was found to represent 7.6% of total cell protein, yielding a mean of 9.8% +/- 4.8% for B. subtilis 168 protein considered periplasmic. Qualitatively, after subjection of all cell fractions to polyacrylamide gel electrophoresis, RNase and DNase, zymographs revealed that (i) each cell fraction had a unique profile of nucleases and (ii) multiple species and a major fraction of both nucleases were concentrated in the periplasm. We conclude that the operationally defined periplasmic fraction corresponds closely, both quantitatively and qualitatively, to the contents of the periplasm of Escherichia coli. We discuss evidence that the maintenance of the components of this surface compartment in B. subtilis is compatible with the thick negatively charged cell wall acting as an external permeability barrier.