Protoplast

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

Stable co-transformation of maize protoplasts with gusA and neo genes

Abstract: An efficient co-transformation protocol using polyethylene glycol was developed for Zea mays L. (cv. A188 x BMS) protoplasts isolated from suspension culture cells. Co-transformation was accomplished by using plasmid constructions containing beta-glucuronidase (gusA) or neomycin phosphotransferase (neo) gene coding sequences; both were under control of the CaMV 35S promoter. Protoplast culture and transformation conditions were optimized to assure efficient recovery of transformed cells. The overall efficiency of transformation was 1 x 10(-4) (calculated per viable protoplast plated). Among kanamycin-resistant lines, 50% showed a high level of GUS activity (above one unit). Southern blot hybridization confirmed the presence of numerous gusA and neo coding sequences in the maize genome. In two analyzed lines, integrated sequences appeared to be organized in tandem head-to-tail repeats. Results also indicated that the integrated sequences were partially methylated.

Fractionation of plant protoplast types by iso-osmotic density gradient centrifugation.

Abstract: A simple effective technique for the fractionation of protoplast populations is described. Protoplasts are separated by low-speed centrifugation in an iso-osmotic, discontinuous density gradient system on the basis of differences in their buoyant densities. At a constant osmolality of 660±20 mOs/kg H2O, the gradients provide a density range from 1.017 to 1.069 g/cm3 at 20 °C which corresponds to the buoyant densities of most protoplast types studied. Characteristics of the KMC/S-density gradient system and factors affecting the fractionation were investigated. Protoplasts were isolated from various tissues and cultivars of tobacco, barley, wheat, rye, oat and maize. Their density-dependent distribution profiles in KMC/S-gradients and their average buoyant densities were determined under standardized conditions. Great differences in the buoyant densities were found between protoplasts of different tissues. Mixed populations of two types of protoplasts, differing in buoyant density by about 15–20 mg/cm3, were separated to give highly purified fractions. Factors affecting the buoyant densities of protoplasts have been investigated. Ploidy level and species differences did not significantly affect the fractionation profiles. However, an age-dependent variation in the average buoyant density of tobacco mesophyll protoplasts was observed. Fractionation of tobacco mesophyll protoplasts and their subsequent regeneration to plants demonstrates the practicability and physiological compatibility of the KMC/S-density gradient system under sterile conditions. The morphogenetic potential of protoplasts was not affected by the separation procedure or the gradient components.

Light-Induced Conversion of Nicotinamide Adenine Dinucleotide to Nicotinamide Adenine Dinucleotide Phosphate in Higher Plant Leaves

Abstract: Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.Sucrose density gradient centrifugation of wheat protoplast extracts and differential centrifugation of protoplast extracts from various plants showed that most of the NAD kinase was localized in the chloroplasts. Therefore, the conversion of NAD to NADP is considered to occur in the chloroplasts. However, with extracts of maize mesophyll protoplasts, the enzyme was localized in the extrachloroplast fraction. The NAD kinase was activated some 30% by illumination of leaves or protoplasts of pea and wheat after preincubation in the dark.These results suggest that, in general, the light-induced conversion of NAD to NADP occurs in the chloroplast and is catalyzed by photoactivated NAD kinase using photochemically produced ATP.

Cell wall composition and protoplast regeneration in Candida albicans.

Abstract: The transition of blastospores to the mycelial phase in Candida albicans was induced after the blastospores were kept at 4 degrees C for several hours and then transferred to a fresh medium prewarmed at 37 degrees C. Glucan was the most abundant polymer in the wall in the two morphogenetic forms but the amount of chitin was higher in the mycelial form than in blastospores. Efficient protoplasting required reducing agents and proteases together with beta-glucanases (zymolyase). Protein synthesis in regenerating protoplasts was initiated after about 30 min. Chitin synthetase, initially very low, was incorporated in important amounts into cell membranes mainly in a zymogenic state. After a few hours chitin was the most abundant polymer found in the aberrant wall of the regenerating protoplast.

Highly efficient isolation of Populus mesophyll protoplasts and its application in transient expression assays.

Abstract: Background: Populus is a model woody plant and a promising feedstock for lignocellulosic biofuel production. However, its lengthy life cycle impedes rapid characterization of gene function. Methodology/Principal Findings: We optimized a Populus leaf mesophyll protoplast isolation protocol and established a Populus protoplast transient expression system. We demonstrated that Populus protoplasts are able to respond to hormonal stimuli and that a series of organelle markers are correctly localized in the Populus protoplasts. Furthermore, we showed that the Populus protoplast transient expression system is suitable for studying protein-protein interaction, gene activation, and cellular signaling events. Conclusions/Significance: This study established a method for efficient isolation of protoplasts from Populus leaf and demonstrated the efficacy of using Populus protoplast transient expression assays as an in vivo system to characterize genes and pathways.