Improvement of β-amylase thermostability in transgenic barley seeds and transgene stability in progeny

The genetic improvement of enzymes important in the brewing process is one of the main goals of barley biotechnology. For the improvement of β-amylase thermostability in barley seeds, we have already constructed a mutant thermostable β-amylase gene, using site-directed mutagenesis and random mutagenesis to achieve the substitution of seven amino acids of the original barley β-amylase. This sevenfold-mutant barley β-amylase showed a thermostability increased by 11.6 °C compared to the original enzyme. In the present article, a thermostable β-amylase gene under the control of the barley β-amylase promoter was introduced to barley protoplasts, and fertile plants were generated from 9 independent transgenic lines. Subsequent analyses indicated that the thermostable β-amylase gene was expressed and β-amylase activity derived from both native and modified genes was detected in the seeds of 6 transgenic lines. The transgene was stably transmitted to progeny, and thermostable β-amylase was synthesized in T4seeds, demonstrating that our strategy is applicable for the improvement of seed quality for industrial utilization.