The codon usage of a hybrid bacterial gene encoding a thermostable (1,3-1,4)-13-glucanase was modified to match that of the barley (1,3-1,4)-p-glucanase isoenzyme (4) and their degradation is a prerequisite for the enzymatic mobilization of endosperm storage components, which serve as nutrients for the growing embryo. Efficient degradation of endosperm cell walls is also important for utilization of barley as a monogastric animal feed (5, 6) and in industrial processes such as malting and brewing (7). Furthermore, extraction of non-food products deposited in the endosperm of transgenic barley would be facilitated by the action of highly efficient, heat stable cell wall-degrading enzymes. The (1,3-1,4)-3-glucanases (EC 3.2

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Availability of transformation systems for barley (Hordeurn 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 α‐amylase gene promoter directs expression of a sequence specifying a secreted, heat stable hybrid (1,3‐1,4)‐β‐glucanase, denoted H(A12‐M)ΔY 13. 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(A 12‐M)ΔY 13 in the scutellum 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 α‐amylases disclosed a similar expression pattern. Accordingly, transgene expression reflected the spatial and temporal characteristics of genes for high‐pI α‐amylase in the aleurone and scutellum tissues during germination. Secreted H(A12‐M)ΔY 13 amounts to 20 mg/kg of transgenic grain, corresponding to 0.3–0.5 μ/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.

Mobilization of stored reserves in the endosperm of barley grains involves the interaction of enzymes and other proteins synthesized during grain development and those synthesized during germination. In order to obtain information on this interaction, localization of a-amylase, the bifunctional subtilisinla-amylase inhibitor (BASI), the unspecific lipid transfer protein (LTP), and the antimicrobial proteins chitinase and ribosome inactivating protein (RIP) were determined by immunofluorescence microscopy in the different tissues of resting and germinating grains of the Hordeum uulgare L. genotypes Alexis, Klages, and Steptoe. Targeting of a-amylase from the proximal to the distal part of the endosperm progresses with different rates in the three genotypes. LTP is localized in the endosperms, the aleurone layers and in the embryo of the ungerminated grains. LTP is depleted during the first days of germination but then re-synthesized in the embryo and in the aleurone during the following phases of germination and released to the endosperm independent of cell wall hydrolysis. Chitinase is present in the aleurone layers and, depending on variety, in variable amounts in the endosperm of the mature and the germinating grains. Secretion of chitinase from the aleurone and scutellum is indicated by the increasing amount found in the endosperm during germination. Chitinase accumulates in the distal aleurone layers before a-amylase IS detected and thus must be able to penetrate the intact endosperm 0-glucan walls. BASI and RIP are localized in all parts of the mature grain and at later stages of germination the amount in the scutellum and aleurone layer increases. The analysed enzymes and proteins fulfill different functions during germination and are of importance for the industrial use of the barley crop. The efficient immunohistochemical demonstration of the genotypic bility in these characteristics provides a possibility to identify important breeding targets.