Generation of Doubled Haploid Barley by Interspecific Pollination with Hordeum bulbosum

The generation of doubled haploid barley plants by means of the so-called "Bulbosum" method has been practiced for meanwhile five decades. It rests upon the pollination of barley by its wild relative Hordeum bulbosum. This can result in the formation of hybrid embryos whose further development is typically associated with the loss of the pollinator's chromosomes. In recent years, this principle has, however, only rarely been used owing to the availability of efficient methods of anther and microspore culture. On the other hand, immature pollen-derived embryogenesis is to some extent prone to segregation bias in the resultant populations of haploids, which is due to its genotype dependency. Therefore, the principle of uniparental genome elimination has more recently regained increasing interest within the plant research and breeding community. The development of the present protocol relied on the use of the spring-type barley cultivar Golden Promise. The protocol is the result of a series of comparative experiments, which have addressed various methodological facets. The most influential ones included the method of emasculation, the temperature at flowering and early embryo development, the method, point in time and concentration of auxin administration for the stimulation of caryopsis development, the developmental stage at embryo dissection, as well as the nutrient medium used for embryo rescue. The present protocol allows the production of haploid barley plants at an efficiency of ca. 25% of the pollinated florets.