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Preparation of Leaf Mesophyll Protoplasts for Transient Gene Expression in Brachypodium distachyon

Cited 19 time in Web of Science Cited 22 time in Scopus
Authors
Hong, Shin-Young; Seo, Pil Joon; Cho, Shin-Hae; Park, Chung-Mo
Issue Date
2012-10
Citation
Journal of Plant Biology(한국식물학회지), Vol.55 No.5, pp.390-397
Keywords
Brachypodium GUS PEG-mediated transformationProtoplastTransient gene expression
Abstract
Transient gene expression systems using protoplasts have been widely used for rapid functional characterization of genes in many plant species. Brachypodium distachyon has recently been employed as a model plant for studies on biofuel grass species and grass crops because of its small genome size, short life-span, and availability of efficient transformation systems. Here, we report the an efficient protocol for the preparation of leaf mesophyll protoplasts from Brachypodium seedlings. We also modified the polyethylene glycol (PEG)-mediated transformation procedure to optimize experimental conditions, such as duration of enzyme digestion, PEG incubation time, and plasmid DNA concentration and size. The green fluorescence protein (GFP)- and beta-glucuronidase (GUS)-coding genes were used as reporters to evaluate the feasibility of this transient expression system. We found that the yield of viable protoplasts was highest after 3 h of enzymatic digestion. Viability of enzyme-digested protoplasts was moderately maintained up to 24 h in Mmg preincubation solution. In addition, the highest transient expression of reporter genes was obtained when protoplasts were transformed with 20 mu g of plasmid DNA and incubated for 16 h. Together with the recent completion of the Brachypodium genome sequence, the Brachypodium transient expression system using leaf mesophyll protoplasts can be widely used for cellular, molecular, and biochemical studies of genes involved in carbon metabolism and signaling pathways mediating intrinsic and environmental cues.
ISSN
1226-9239
URI
https://hdl.handle.net/10371/171995
DOI
https://doi.org/10.1007/s12374-012-0159-y
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College of Natural Sciences (자연과학대학)Dept. of Chemistry (화학부)Journal Papers (저널논문_화학부)
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