Barley as a Producer of Heterologous Protein

Abstract

Recent developments in genetic engineering and molecular biology have enabled the production of transgenic plants with improved characteristics, or cell and tissue cultures with altered or improved metabolic activity. However, no universally applicable gene transfer method has hitherto been established for plant transformation. The monocotyledonous plants, including grasses and cereals, have proved to be recalcitrant to genetic engineering. They do not belong to the natural host range of the efficient gene vector Agrobacterium, which is widely used for transformation of dicotyledonous plants. Particle bombardment, developed by Sanford et al. (1), is a physical gene transfer method without target material limitations. Using this method, transgenic plants have been produced successfully from all major cereals (2-8). Protoplast-mediated gene transfer methods, e.g., electroporation or polyethelene glycol (PEG) treatment, have long been hampered by the recalcitrance of important cereal crop species to the regeneration of plants from these protoplasts. However, success has recently been reported in this field, even with elite cultivars of barley (9). In this chapter, particle bombardment is used as a means to obtain stable transgenic barley cultures, which can be used for production of a heterologous enzyme, i.e., the endo-[3-glucanase (EGI) of Trichoderma reesei, in amounts sufficient to conduct studies on its properties. Particle bombardment results in a mixed population of transgenic and untransformed cells, which leads to the need for efficient selection and screening in order to isolate a pure and stable clone. In this respect, a protoplast-mediated gene transfer method would offer an attractive alternative. All regenerated callus clusters would originate from a