Transient gene expression in guard cell chloroplasts of tobacco [Nicotiana tabacum] using ArF excimer laser microablation
2008
Kajiyama, S.(Osaka Univ., Suita (Japan)) | Joseph, B. | Inoue, F. | Shimamura, M. | Fukusaki, E. | Tomizawa, K. | Kobayashi, A.
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 micro square m 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.
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