An Evaluation of Feed-Back Insensitive Aspartate Kinase as a Selectable Marker for Barley (Hordeum Vulgave L.) Transformation

Abstract

In the present study we have evaluated the use of feedback insensitive aspartate kinase as a selectable marker for barley transformation. Immature embryos were bombarded with plasmids containing an Escherichia coli mutant lysC gene encoding a lysine—threonine feedback insensitive form of aspartate kinase (AK). The heterologous gene was fused to the sequence coding for the transit peptide of the small subunit of ribulose 1,5-bisphosphate carboxylase of pea in order to target AK into the chloroplast. Either the cauliflower mosaic virus (CaMV) 35S or the maize ubiquitin promoter directed expression of lysC. Transgenic, regenerable cell cultures were obtained after bombardment with both constructs and selection on MS medium with millimolar concentrations of lysine plus threonine. Transgenic regenerants were, however, only obtained from cultures where the ubiquitin promoter directed expression of the lysC gene. Additional parameters of importance were omission of glutamine from the selection medium, a small embryo size and the application of selection immediately following isolation of embryos. From a total of 720 embryos bombarded with the ubiquitin—lysC construct, 13 transgenic cell lines were selected. Two of these regenerated green plants, while 8 lines produced only albino plants. All regenerated plants were transgenic as evidenced by PCR amplification of the heterologous gene. In two individual lines of green transgenic plants, enzyme assays revealed a 3.5 and 4.0-fold increase in AK activity in the leaves, respectively while in two independent transgenic albino plants, the increase in AK activity was 6.5 and 11.5-fold, respectively. The potential of this selection system for barley transformation is discussed.