Poster: Vegetative Development

Abs # 422: Reverse genetic approach that introduces plasmids and double-stranded RNAs into zinnia suspension-cultured cells for analysis of tracheary element differentiation

Presenter:	Endo, Satoshi , endoo@psc.riken.go.jp
Authors	Endo, Satoshi  (A)   Kuriyama, Hideo  (A)   Tashiro, Gen  (A)   Fukuda, Hiroo  (A) (B)  Demura, Taku  (A)
Affiliations:	(A): Plant Science Center, RIKEN (B): Department of Biological Sciences, Graduate School of Science, University of Tokyo

A cell culture system in which plant cell differentiation can be monitored is invaluable in revealing differentiation processes in detail. In the zinnia system, isolated mesophyll cells of Zinnia elegans can transdifferentiate into tracheary elements (TEs). Application of DNA microarray technology to the zinnia system has recently provided fundamental information on gene expression profiles during TE differentiation. Therefore, we tried to develop a gene transfer method with which to analyze the roles of specific genes in the zinnia system, in combination with RNA silencing technology. We successfully used an electroporation-based method to introduce DNA/RNA into isolated zinnia mesophyll cells. After partial digestion of the cell walls, the cells were transiently transformed with plasmid DNA, and transdifferentiated into TEs as frequently as cells of the conventional zinnia system. We then examined the usefulness of introducing double-stranded RNA (dsRNA) directly into the cells. A decrease in the number of GFP-expressing cells was observed after co-introduction of P35S::GFP plasmid and dsRNA corresponding to GFP mRNA, suggesting that RNA silencing functions in the zinnia system. We next investigated the effects on TE differentiation of dsRNAs corresponding to endogenous cellulose-synthase-encoding genes. These dsRNAs were prepared simply from zinnia cDNAs that had been constructed previously for zinnia microarray analysis. When cells were electroporated with dsRNAs corresponding to genes with TE morphogenesis-specific expression profiles, the frequency of abnormal TEs increased, demonstrating the validity of this reverse genetic approach in the zinnia system.