Transformation of barley by microinjection into isolated zygote protoplasts

Barley zygote protoplasts were mechanically isolated, embedded in agarose d roplets, and microinjected with a rice actin promoter Act1-gusA-nos gene co nstruct. On average 62% of the cells survived the injection and of these 55 % continued development into embryo-like structures and eventually to plant s. PCR screening for the presence of a 307-bp fragment in the middle of the gusA gene showed that on average 21% of the derived structures contained t his fragment. However, among the hundreds of injected zygotes, derived stru ctures and regenerants we only found significant GUS expression in two case s (embryo-like structures nine days after injection). Two lines of green pl ants, derived from zygotes microinjected with linearized plasmid (line A147 -1) or an isolated Act1-gusA-nos gene cassette (line A166-h) proved to be t ransgenic. Line A147-1 appeared to contain a single and intact copy of the expression cassette but a PCR based progeny analysis indicated the presence of additional shorter fragments of the cassette. Line A166-h appeared to c ontain a single fragment of the gusA gene that was transferred to the proge ny as a single Mlendelian trait. One additional fragment of the gusA gene w as identified in this line. The present data show that transformation of ba rley by microinjection of DNA into isolated zygotes is feasible but also th at gene expression rarely is achieved, possibly due to degradation of the i ntroduced DNA.