An evaluation of feed-back insensitive aspartate kinase as a selectable marker for barley (Hordeum vulgare L.) transformation

In the present study we have evaluated the use of feedback insensitive aspa rtate kinase as a selectable marker for barley transformation. Immature emb ryos were bombarded with plasmids containing an Escherichia coli mutant lys C gene encoding a lysine-threonine feedback insensitive form of aspartate k inase (AK). The heterologous gene was fused to the sequence coding for the transit peptide of the small subunit of ribulose 1,5-bisphosphate carboxyla se of pea in order to target AK into the chloroplast. Either the cauliflowe r mosaic virus (CaMV) 35S or the maize ubiquitin promoter directed expressi on of lysC. Transgenic, regenerable cell cultures were obtained after bomba rdment with both constructs and selection on MS medium with millimolar conc entrations of lysine plus threonine. Transgenic regenerants were, however, only obtained from cultures where the ubiquitin promoter directed expressio n of the lysC gene. Additional parameters of importance were omission of gl utamine from the selection medium, a small embryo size and the application of selection immediately following isolation of embryos. From a total of 72 0 embryos bombarded with the ubiquitin-lysC construct, 13 transgenic cell l ines were selected. Two of these regenerated green plants, while 8 lines pr oduced only albino plants. All regenerated plants were transgenic as eviden ced 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 incre ase in AK activity in the leaves, respectively while in two independent tra nsgenic albino plants, the increase in AK activity was 6.5 and 11.5-fold, r espectively. The potential of this selection system for barley transformati on is discussed.