Mazarei%20TBJ%20protoplast. Isolation and Whole-Cell Patch Clamping of Arabidopsis Guard Cell Protoplasts. Equipment Agar bridge (3 M KCl) Balance (analytical, 0.01-mg; e.g., Mettler) Blender (e.g., Waring) Burner (alcohol) Capillary tubes (glass, 1.5-mm outer diameter [o.d.], 10-cm length; e.g., Beijing ZhengTianYi Science and Technology Trade Co.) Cell chamber Centrifuge (clinical; e.g., International Equipment Co.) Dish (covered, with clay strip for storing pipettes) Electrodes (Ag/AgCl wire) Filter paper (autoclaved) Filters (0.45-μm; Corning) Forceps (sharp-tip) Glass capillary pipette puller (two-step; e.g., Narishige) Growth chamber Hood (laminar flow) Ice Manipulator (three-axis coarse and fine micromanipulator; e.g., Narishige) Mesh (nylon, 30- and 100-μm pore size, 15 cm × 15 cm; Spectra/Mesh) Microelectrode amplifier and data acquisition system (e.g., Axon 200B with CV 201AU headstage, DigiData 1200 interface and computer with pCLAMP software installed) Microforge (e.g., Narishige) Micropipettors (10- and 200-μL, and 1-mL) Microscope (bright-field; e.g., Nikon) Microscope (inverted; e.g., Zeiss) Spatula.
Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana. This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts. Date Published: 8/17/2009, Issue 30; doi: 10.3791/1149 Keywords: Plant Biology, Issue 30, protoplasts, isolation, Arabidopsis, seedlings Cite this Article Zhai, Z., Jung, H., Vatamaniuk, O. Protoplasts are plant cells that have had their cell walls enzymatically removed. Part 1. We typically culture plants on concentrated Murasige and Skoog (MS) medium supplemented with 1% sucrose and 0.7% agar. For preparing 1L of MS, media pH 5.7 *Tip 1: Do not attempt to dissolve agar.
Part 2. Sterilize seeds of Arabidopsis thalianaPlace 100 mg of seeds into eppendorf microcentrifuge tubes and add 1 ml of 70% ethanol. Part 3. Part 4. Part 5: Representative Results: Figure 1. Cocking, E. Metrics. 5383. Plant Methods | Full text | Tape-Arabidopsis Sandwich - a simpler Arabidopsis protoplast isolation method. Tobacco protoplast culture in a polydimethylsiloxane-based microfluidic channel. Formation of Protoplasts from Cultured Tobacco Cells and Arabidopsis thaliana by the Action of Cellulosomes and Pectate Lyase from Clostridium cellulovorans. Plant Methods | Full text | A highly efficient rice green tissue protoplast system for transient gene expression and studying light/chloroplast-related processes.