Protoplast

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

Quantitative characterization of protoplasts and vacuoles from suspension-cultured cells of Catharanthus roseus

Abstract: A simple and efficient procedure for isolation of protoplasts and then vacuoles from cultured cells of Catharanthus roseus (L.) G. Don is presented. Protoplasts were disrupted by an osmotic shock and the vacuoles vere purified by flotation on a single-step gradient. A comparison of the content and concentration of solutes (proteins, sugars, organic acids, alkaloids, mineral ions) in protoplasts and cells showed that massive and selective losses occur for most solutes during protoplast preparation. These are attributed to the osmotic adjustment and changes of membrane permeabilities occurring during plasmolysis. Data concerning the size, yield and purity of the isolated vacuoles are discussed. By analysis of isolated vacuoles, the vacuolar concentration and localization of solutes within protoplasts have been determined. The limits of this latter approach are stressed, however. Some evidence in favour of the selection of a special class of vacuoles during isolation is reported and discussed.

High plating efficiency and plant regeneration frequency in low density protoplast cultures derived from an embryogenic Brassica napus cell suspension

Abstract: Protoplasts were isolated from an embryogenic cell suspension culture derived from microspores of Brassica napus cv. Jet Neuf. Protoplast yield varied with the cell suspension growth medium. Optimization of protoplast plating density, manipulation of culture medium, carbon source and medium matrix, and inclusion of Ficoll resulted in protoplast plating efficiencies close to 30%. Placement of the protoplasts close to the gas interface contributed greatly to the elevated plating efficiency. Low density cultures could be induced to regenerate calli at optimum plating efficiencies if grown in the presence of nurse culture. This is of great advantage for manipulation of individual protoplasts or for microinjection. Plants were regenerated directly from the cell suspension or from the protoplast cultures.

A highly efficient maize nucellus protoplast system for transient gene expression and studying programmed cell death-related processes.

Abstract: Conditions for the isolation and transfection of maize nucellus protoplasts were established. We demonstrated its utilization for protein expression, localization, protein–protein interaction, and the investigation of PCD-related processes. Plant protoplasts are an important and versatile cell system that is widely used in the analysis of gene characterization and diverse signaling pathways. Programmed cell death (PCD) occurs throughout the life of plants from embryogenesis to fertilization. The maize nucellus undergoes typical PCD during development of the embryo sac. The nucellus protoplast shows potential for use in research of PCD-related processes. No studies have reported previously the isolation and transfection of nucellus protoplasts. In this study, conditions for the isolation and transfection of maize nucellus protoplasts were established. The maize protoplast system can be used for protein expression, localization, and protein–protein interaction. We applied this system to investigate PCD-related processes. Quantitative real-time PCR analysis revealed that transient expression of MADS29 in the maize nucellus protoplast increases Cys-protease gene transcript level. In addition, β-glucuronidase and luciferase activity assays showed that MADS29 could enhance the promoter activities of the Cys-protease gene. Thus, we demonstrated the potential of a highly efficient maize nucellus protoplast system for transient gene expression and investigation of PCD-related processes.