Kajiyama Shin'ichiro, Joseph Benesh, Inoue Fumihide, Shimamura Masaki, Fukusaki Eiichiro, Tomizawa Kenichi, Kobayashi Akio
Department of Biotechnology, Graduate School of Engineering, Osaka University, Suita, Osaka, Japan.
J Biosci Bioeng. 2008 Aug;106(2):194-8. doi: 10.1263/jbb.106.194.
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.
