Protoplast

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

A transient gene expression system using barley protoplasts to evaluate microRNAs for post-transcriptional regulation of their target genes

Abstract: Transient gene expression assays using protoplasts have been frequently used in high-throughput screening and functional characterization of plant genes. In barley, however, very few studies have explored the use of protoplasts isolated from green tissues. In this study, a reliable and efficient transient gene expression system has been established using barley green tissue protoplasts. Due to the importance of osmolarity in maintaining protoplast viability and transfection efficiency, different mannitol concentrations were tested to determine the optimal osmolarity suitable for barley protoplast preparation. The method and conditions were also described for efficient isolation of protoplasts from barley leaf and stem tissues and transient expression of exogenous gene constructs. This transient expression system has been successfully demonstrated for protein immunoblot analysis, subcellular protein localization and quantitative analysis of gene expression. Furthermore, a simplified method has been described to quickly evaluate microRNAs for post-transcriptional regulation of their target genes in barley protoplasts.

Isolation of protoplasts and vacuoles from storage tissue of red beet.

Abstract: A fast and efficient method is described for the isolation of protoplasts and vacuoles from storage tissue of Beta vulgaris L. The viability of the isolated protoplasts is indicated by the development within a few hours of plasma strands with active cyclosis as well as by transport activity.The effect of aging of the tissue on the yield and on the properties of the protoplasts is examined. Adding 1 millimolar dithiothreitol to the water during the aging of the tissue prevents a change of the cell wall structure which otherwise reduces the protoplast yield within 2 days to zero. When protoplasts are isolated from tissue after different times of aging, they show an increase in transport activity which parallels that in the intact tissue.

Differences in Protein Synthesis and Peroxidase isoenzymes between recalcitrant and regenerating ProtoPlasts

Abstract: The reasons for the inability of recalcitrant mesophyll protoplasts to divide and re-enter the cell cycle are unknown. Changes in protein profile, indole-3-acetic acid (IAA)-oxidase and peroxidase activities, and isoenzymes were compared in protoplasts of recalcitrant grapcvine (Vitis vinifera) L. cv. Sultanina) and regenerating tobacco (Nicotiana tabacum) L. cv. Xanthi). Using [35S]-methionine. SDS-PAGE and two-dimensional separation of proteins, differences in protein profile during protoplast culture were assessed. The changes in the de novo synthesized proteins were both qualitative and quantitative between the two species. The number of proteins which changed was double in tobacco compared to grapevine protoplasts. Peroxidase and IAA-oxidase activities increased significantly in tobacco protoplasts during culture whereas in grapevine they remained low. In tobacco protoplasts. 3 and 7 basic and acidic peroxidases, respectively, were induced during protoplast culture. which were not detected in the intact leaf, whereas in grapevine no new peroxidases were induced during protoplast culture.