High frequency regeneration of barley doubled haploid plants from isolated microspore culture

Abstract

The development of a useful procedure for barley isolated microspore culture is described. This method resulted in not only efficient plant regeneration but also very high frequency of doubled haploid (DH) induction. With four cultivars investigated, a range of 469–534 embryos and 32–160 green plants (GPs) per 4×10 4 microspores were obtained. Moreover, a combination of cold pretreatment of spikes with mannitol treatment of microspores led to high frequencies of spontaneous chromosome doubling in all the tested genotypes: rates of DH plants ranged from 88.3 to 93.5%. In contrast, only 79.4% of DHs were produced by using spike cold pretreatment alone (cultivar ‘Igri’). Statistical analysis indicated that difference in these rates was significant. After extensive studies on factors affecting microspore culture efficiency, purification of microspores and induction culture method were found to be critical for embryo development and GP regeneration. Major differences between the present procedure and previously reported ones are discussed. To our knowledge, this is the first report using such a pretreatment to improve DH regeneration in barley. The percentages of DH plants described here are higher than those reported earlier. This high frequency of spontaneous chromosome doubling (>90%) has been confirmed with a total of nine genotypes.