title: A novel microsurgery method for intact plant tissue at the single cell level using ArF excimer laser microprojection.
abstract: A novel microsurgery technique for the partial removal of rigid cell-walls in intact plant tissue is established. Using a size-variable slit, an ArF excimer laser was microprojected on the surface of the targeted cell, and this method enabled the area- and depth-controllable processing of the cortical structure of plant cells including the cuticle and cell wall layer. In epidermal cells of all tested plants, viabilities of more than 90% were retained 24 h after irradiation. Scanning electron microscope (SEM) observation revealed that the cuticle layer of the irradiated region was completely ablated, and the cellulose microfibrils of the secondary cell wall were partially removed; furthermore, 4 days after laser treatment, the regeneration of cell wall fibrils was observed. As a model experiment, the transient expression of synthetic green fluorescent protein (sGFP) was performed by the microinjection of cauliflower mosaic virus (CMV) 35S promoter-derived sGFP gene through an "aperture" in the treated cell surface. Moreover, micron-sized fluorescent beads were successfully introduced by the same method into the onion cells indicating that this method can be used to introduce foreign materials as large as organelles.
authors.affiliation: Department of Biotechnology Graduate School of Engineering, Osaka University 2-1 Yamadaoka, Suita, Osaka 565-0871, Japan. kaiyama@bio.eng.osaka-u.ac.jp
authors.forename: Shin'ichiro
authors.initials: S
authors.lastname: Kajiyama
authors.forename: Takeshi
authors.initials: T
authors.lastname: Shoji
authors.forename: Shinya
authors.initials: S
authors.lastname: Okuda
authors.forename: Yoshihiro
authors.initials: Y
authors.lastname: Izumi
authors.forename: Ei-ichiro
authors.initials: E
authors.lastname: Fukusaki
authors.forename: Akio
authors.initials: A
authors.lastname: Kobayashi
mesh_terms: D017796:Caulimovirus / Q000235:genetics; D018014:Gene Transfer Techniques; D049452:Green Fluorescent Proteins / Q000032:analysis / Q000235:genetics; D007834:Lasers; D008845:Microinjections / Q000379:methods*; D008855:Microscopy, Electron, Scanning; D019697:Onions / Q000737:chemistry / Q000235:genetics / Q000528:radiation effects; D010944:Plants / Q000528:radiation effects* / Q000648:ultrastructure; D011401:Promoter Regions, Genetic / Q000235:genetics
chemical_list: D049452:Green Fluorescent Proteins