Selectable marker-free transgenic barley producing a high level of cellulase (1,4-β-glucanase) in developing grains

The use of barley grains as bioreactors for high-level production of cellulase (1,4-β-glucanase) was investigated. A hybrid cellulase gene, cel-hyb1, driven by the rice GluB-1 promoter was expressed specifically in developing endosperm. Codon usage optimisation of cel-hyb1 increased its expression in barley grains 527-fold and led to cellulase production of up to 1.5% of total grain protein. CEL-HYB1 enzyme in barley grains was highly stable during post-harvest storage. Selectable marker gene (hph) was subsequently eliminated from transgenic lines through segregation of hph from synthetic cel-hyb1 (syn.cel-hyb1) in T1 progeny, using a binary plasmid containing hph and syn.cel-hyb1 in separate T-DNAs. These data suggest that barley grains can potentially be used for the commercial production of cellulase