title: Transient gene expression in guard cell chloroplasts of tobacco using ArF excimer laser microablation.
abstract: In this paper, we report a novel method for delivering genes into chloroplasts of tobacco cells using laser microablation. The plasmid pLD200-GFP was introduced into chloroplasts of Nicotiana tabacum cv. Xanthi guard cells and transient GFP expression was detected in the chloroplasts after 2-3 d of incubation. The technique uses an argon fluoride (ArF) excimer laser to perforate the cell surface in a 4 mum(2) area in the presence of plasmid coated gold microparticles. Pretreatment of guard cells to promote stomatal closure prior to laser ablation resulted in a significant increase in the survival rate of cells and a transient expression rate of 2-3% in trial number basis was archived. Our method has unique advantages such as avoiding laborious pretreatments that adversely affect cell viability and specific delivery of transgenes into a desired cell in complex leaf tissue. This technique is a potential tool for cell specific transient gene expression studies for elucidation of gene regulation and expression.
authors.affiliation: Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
authors.forename: Shin'ichiro
authors.initials: S
authors.lastname: Kajiyama
authors.forename: Benesh
authors.initials: B
authors.lastname: Joseph
authors.forename: Fumihide
authors.initials: F
authors.lastname: Inoue
authors.forename: Masaki
authors.initials: M
authors.lastname: Shimamura
authors.forename: Eiichiro
authors.initials: E
authors.lastname: Fukusaki
authors.forename: Kenichi
authors.initials: K
authors.lastname: Tomizawa
authors.forename: Akio
authors.initials: A
authors.lastname: Kobayashi
mesh_terms: D002736:Chloroplasts / Q000378:metabolism*; D015870:Gene Expression; D006046:Gold / Q000737:chemistry; D054018:Lasers, Excimer; D010316:Particle Size; D014026:Nicotiana / Q000648:ultrastructure*
chemical_list: D006046:Gold