Transient Expression Following Electroporation into Immature Zygotic Embryos of Wheat

Abstract

The following protocol is designed for checking constructs containing visible markers as the ß-glucuronidase (Jefferson 1987) or the genes coding for the anthocyanin regulatory proteins Cl (Cone et al. 1986) and B-peru (Goff et al. 1990) in differentiated cells. It describes the electroporation-mediated transformation of wheat immature embryos (Triticum aestivum, spring wheat cultivar Sonora) and the following transient expression of the transformed genes. The same protocol can be used for electroporation of other tissues, like immature embryos of rice and maize. No special treatment of the tissue before electroporation is required. The described electroporation-chamber setup has been constructed for this experiment. It allows easy orientation of the embryos in the electroporation chamber. This orientation is an important factor as (1) only the parts of the tissue will be transformed that are facing the cathode (due to the electrophoretic nature of DNA movement during electroporation), and (2) different parts of the material (e.g., different cell types of the embryo) are not equally susceptible to electroporation-mediated DNA uptake. The scutellum cells of wheat immature embryos, for example, are far more susceptible than the cells on the coleoptile side. With rice immature embryos, good transient expression can be obtained in cells on the coleoptile side, whereas the scutellum cells of the same embryos are recalcitrant under these conditions (Klöti et al. 1993). In experiments where frequency and intensity of transient gene expression following electroporation are investigated, this has to be considered.