Isolation and cultivation of embryogenic microspores from barley (Hordeum vulgare L.)

Abstract

Two different techniques for mechanical isolation of microspores from the barley cultivar 'Igri' have been evaluated. The anthers were subjected to mannitol pretreatment prior to microspore isolation, which was performed either by maceration with a pestle or by blending of the excised anthers. The microspores were purified by centrifugation and washing and cultured in liquid medium on a membrane support. In the following four weeks the microspores developed into embryoids, which were subsequently regenerated to plants on solid medium. Microblending of the anthers was found to be more reproducible than pestle maceration, and the yield of large microspores was 100% higher using this method. With the microblending technique a mean of 9.4 green plants and 0.4 albino plants were regenerated per plated anther while a mean of only 2.8 green and 0.17 albino plants per anther were regenerated from microspores isolated after pestle maceration of the anthers. Microspores isolated from mass cultures were also cultured as single cells in microdroplets, and it was shown that microspores isolated from 3-5 days old mass cultures could develop into plants although at a low frequency (0.3%). Finally, the potential of using microinjection for transforming embryogenic microspores has been evaluated.