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

Availability of transformation systems for barley (Hordeum vulgare) provide s a means to characterize the developmental regulation and inheritance of t ransgenes. 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 b arley alpha-amylase gene promoter directs expression of a sequence specifyi ng 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 c opies were present in heterozygous and homozygous plants, respectively. Imm unofluorescence microscopy revealed appearance of H(A12-M)Delta Y13 in the scutelum of germinating transgenic grains of the homozygous line two days a fter imbibition, and was also synthesized later in the aleurone tissue duri ng germination. Examination of adjacent grain sections for the distribution of homologous alpha-amylases disclosed a similar expression pattern. Accor dingly, transgene expression reflected the spatial and temporal characteris tics of genes for high-pi alpha-amylase in the aleurone and scutellum tissu es during germination. Secreted H(A12-M)Delta Y13 amounts to 20 mg/kg of tr ansgenic 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 nu tritional and pharmaceutical interest.