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Barley microspore embryogenesis represents an attractive system to study stress-induced cell differentiation and is a valuable tool for efficient plant breeding. In contrast to zygotic embryogenesis, all developmental stages are freely accessible at a large scale for observation, molecular analysis and manipulation techniques. In barley, there is a high percentage of microspores that become embryogenic after stress treatment in a mannitol solution. These microspores have the capacity to follow an embryogenic route in both liquid and solid cultures, yielding up to 10% of embryos. In this protocol, we describe three different culture systems for obtaining barley microspore-derived embryos, where embryos develop in liquid medium, on top of a solid medium layer or immobilized in a thin layer of agarose. While liquid culture systems allow the generation of large amounts of embryos for molecular analysis, solid culture systems are the ultimate tool for probing embryo development.
