Time saving method for protoplast isolation, transformation and transient gene expression assay in barley

Abstract

This study was conducted to establish a rapid method for barley (Hordeum vulgare L.) protoplast isolation to provide an easy-to-use procedure for the transformation and primary investigation of new gene constructs by transient gene expression assays. Protoplasts were successfully isolated from the chopped embryo and scutellum parts of mature barley seeds by digesting three hours with an enzyme mixture. Isolated protoplasts were washed in W5 washing solution, sieved through plastic meshes and then cleaned on sucrose gradient. The suitability of these directly from embryo-scutellum complexes derived protoplasts for transient gene expression studies was determined by transforming the protoplasts using the PEG (polyethylene glycol) method. Plasmid pAct1-F containing the rice Act1 promoter linked with the gus coding sequences and the nos polyadenylation signal was used in the transformation. After the PEG treatment protoplasts were cultured on KPR culture medium and the transient gus expression was assayed 24-36 hours after transformation. Up to 6% of the transformed protoplasts showed gus expression after treating the protoplasts with X-gluc. The results of this study show that the protoplasts isolated directly from dissected mature barley scutellum-embryo complexes could be used to investigate transient gene expressions in barley. This procedure requires negligible time prior the transformation experiment and so can be done in a very short time compared to the protoplast system based on a suspension culture.

Gene specificity is maintained in transient expression assays with protoplasts derived from different tissues of barley

“…Rapidity of analysis is the major advantage of the transient assay system, particularly when compared to the time-consuming regeneration of transgenic plants . In the case of barley, regarded until very recently as recalcitrant for stable transformation (only a few references reporting fertile transgenic plants obtained by bombardment of immature embryos : Wan & Lemaux, 1994 ;Ritala et al ., 1994), the transient transformation of protoplasts has been the method of choice for the functional analysis of gene promoters (Teen et al ., 1989 ;Salmenkallio et al ., 1990 ;Dfaz & Carbonero, 1992 ;Dfaz et al ., 1993 ;Jenes et al ., 1994) . However, transient expression data in cereal protoplasts derived from certain tissues such 203 Gene specificity is maintained in transient expression assays with protoplasts derived from different tissues of barley I. Dfaz', J .…”