Inheritance of a codon-optimized transgene expressing heat stable (1,3-1,4)-β-glucanase in scutellum and aleurone of germinating barley

Availability of transformation systems for barley (Hordeum vulgare) provides a means to characterize the developmental regulation and inheritance of transgenes. We report here the generation of 14 primary transformants and a detailed analysis of one of the derived homozygous Lines which transmitted over 3 generations to all progeny plants a transgene construct in which a barley alpha-amylase gene promoter directs expression of a sequence specifying a secreted, heat stable hybrid (1,3-1,4)-beta-glucanase, denoted H(A12-M)Delta Y13. Segregation analysis demonstrated Mendelian inheritance of the transgene, and using competitive PCR it was estimated that about 6 and 12 copies were present in heterozygous and homozygous plants, respectively. Immunofluorescence microscopy revealed appearance of H(A12-M)Delta Y13 in the scutelum of germinating transgenic grains of the homozygous line two days after imbibition, and was also synthesized later in the aleurone tissue during germination. Examination of adjacent grain sections for the distribution of homologous alpha-amylases disclosed a similar expression pattern. Accordingly, transgene expression reflected the spatial and temporal characteristics of genes for high-pi alpha-amylase in the aleurone and scutellum tissues during germination. Secreted H(A12-M)Delta Y13 amounts to 20 mg/kg of transgenic grain, corresponding to 0.3-0.5 mu g/grain. This result underlines the value of transformation not only to improve barley grain quality, but also for development of barley as a bioreactor for producing proteins of nutritional and pharmaceutical interest.