title: A simple and effective method to encapsulate tobacco mesophyll protoplasts to maintain cell viability.
abstract: Protoplasts have been widely used for genetic transformation, cell fusion, and somatic mutation due to the absence of a cell wall. However, without the protection of a cell wall, protoplasts are easy to rupture and aggregate during washing, collecting, and gene transfection. In this work, we propose a simple and effective silica/alginate two-step method to immobilize protoplasts with advantages in experimental manipulation and microscopic imaging, as well as in potentially studying cell biological processes such as secretion and metabolism. The proposed two-step immobilization method adopts Transwell with clear tissue culture-treated membrane to support protoplasts in the form of uniform thin layer, which has three unique properties. •The tissue culture-treated membrane has a good affinity for the plant cell; thus, protoplasts can spread evenly and form a very thin layer.•There are more choices for membrane pore size, depending on the application.•It is very convenient to change or collect the solution without mechanically disturbing the protoplasts. This simple and effective silica sol-gel/alginate two-step immobilization of protoplasts in Transwell has great potential for applications in genetic transformation, metabolite production, and migration assays.
authors.affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.
authors.forename: Rong
authors.initials: R
authors.lastname: Lei
authors.affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China ; Department of Entomology, College of Agronomy and Biotechnology, China Agricultural University, Beijing, China.
authors.forename: Wenjie
authors.initials: W
authors.lastname: Qiao
authors.affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.
authors.forename: Fan
authors.initials: F
authors.lastname: Hu
authors.affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.
authors.forename: Hongshan
authors.initials: H
authors.lastname: Jiang
authors.affiliation: Institute of Plant Quarantine, Chinese Academy of Inspection and Quarantine, Beijing, China.
authors.forename: Shuifang
authors.initials: S
authors.lastname: Zhu
keywords: Alginate; CLSM, confocal laser scanning microscope; ETAF, extra thin alginate film; FDA, fluorescein diacetate; Immobilization; MES, 2-(N-morpholino)ethanesulfonic acid; PET, polyethylene terephthalate; Protoplast immobilization; Protoplasts; Silica sol–gel; TAL, thin alginate layer; Thin layer; Tris, tris(hydroxymethyl)aminomethane