title: A novel system for gene silencing using siRNAs in rice leaf and stem-derived protoplasts.
abstract: BACKGROUND
  Transient assays using protoplasts are ideal for processing large quantities of genetic data coming out of hi-throughput assays. Previously, protoplasts have routinely been prepared from dicot tissue or cell suspension cultures and yet a good system for rice protoplast isolation and manipulation is lacking.
  
  
  RESULTS
  We have established a rice seedling protoplast system designed for the rapid characterization of large numbers of genes. We report optimized methods for protoplast isolation from 7-14 day old etiolated rice seedlings. We show that the reporter genes luciferase GL2 and GUS are maximally expressed approximately 20 h after polyethylene glycol (PEG)-mediated transformation into protoplasts. In addition we found that transformation efficiency varied significantly with plasmid size. Five micrograms of a 4.5 kb plasmid resulted in 60-70% transformation efficiency. In contrast, using 50 microg of a 12 kb plasmid we obtained a maximum of 25-30% efficiency. We also show that short interfering RNAs (siRNAs) can be used to silence exogenous genes quickly and efficiently. An siRNA targeting luciferase resulted in a significant level of silencing after only 3 hours and up to an 83% decrease in expression. We have also isolated protoplasts from cells prepared from fully green tissue. These green tissue-derived protoplasts can be transformed to express high levels of luciferase activity and should be useful for assaying light sensitive cellular processes.
  
  
  CONCLUSIONS
  We report a system for isolation, transformation and gene silencing of etiolated rice leaf and stem-derived protoplasts. Additionally, we have extended the technology to protoplasts isolated from fully green tissue. The protoplast system will bridge the gap between hi-throughput assays and functional biology as it can be used to quickly study large number of genes for which the function is unknown.
authors.affiliation: Department of Plant Pathology, University of California at Davis, Davis, California, USA. rbart@ucdavis.edu
authors.forename: Rebecca
authors.initials: R
authors.lastname: Bart
authors.forename: Mawsheng
authors.initials: M
authors.lastname: Chern
authors.forename: Chang-Jin
authors.initials: CJ
authors.lastname: Park
authors.forename: Laura
authors.initials: L
authors.lastname: Bartley
authors.forename: Pamela C
authors.initials: PC
authors.lastname: Ronald