A red fluorescent protein (DsRED) from Discosoma sp. as a reporter for gene expression in walnut somatic embryos

An improved scorable marker was developed for somatic embryo transformation. This method is more reliable than GFP and provides more efficient embryo selection than β-glucuronidase assays (GUS, MUG). Reporter genes are widely used to select transformed cells and tissues. Fluorescent proteins have become an integral part of live-cell imaging research over the past 10 years. DsRED is an ideal reporter for avoiding plant chlorophyll autofluorescence and for double labeling in combination with other scorable markers. In this study, we transformed walnut somatic embryos with a construct containing the DsRED-expressing binary vector pKGW-RR to assess the effect of this red fluorescent protein visual reporter on both embryos and regenerated plants. Results showed that DsRED expression was apparent with maximum brightness at 7-10 days after initiation. Fourteen of twenty-four surviving somatic embryos were bright red. These E0 embryos generated 25 wholly fluorescent E1 embryos and 43 wholly fluorescent E2 embryos at 2 weeks intervals. The germination percentage of DsRED-positive embryos was greater than 80% and gave rise to 45 fluorescent transgenic walnut plants. The regenerated transgenic plants expressed DsRED in all tissues examined including transverse sections of vegetative organs. The percentage of transformed plants that developed roots (48.3%) was similar to control shoots (53%). For transformation of walnut somatic embryos, the DsRED-based reporter system is more stable and reliable than green fluorescent protein (GFP) and, since it is a directly read and non-destructive assay, it provides a more efficient means of monitoring transformation than β-glucuronidase (GUS).