Nuclear fusion leads to chromosome doubling during mannitol pretreatment of barley (Hordeum vulgare L.) microspores

Abstract

A cytological study of barley microspores during pretreatment of the uninucleate stage to the early culture stage was conducted utilizing six genotypes. Among the three main pretreatments investigated, microspores completed the first mitotic division during 28 d cold pretreatment of spikes, with or without leaf sheath attached, and during 0.3 M mannitol pretreatment of anthers at 25 degrees C. However, during a 4 d pretreatment in 0.3 M mannitol at 4 degrees C this first mitotic division was blocked or delayed and subsequently most often occurred during the first day on culture medium. The first mitotic division of most microspores pretreated in 0.3 M mannitol was mostly symmetrical (55-60%), whereas it was asymmetric (94%) during the 28 d cold pretreatment of spikes. Following the first mitotic division during the mannitol pretreatment at 25 degrees C, closely associated daughter nuclei often appeared to fuse via membrane coalescence, leading to a high frequency of large uninucleate microspores. Based upon nuclear size, the frequencies of fused uninucleate microspores in genotypes GBC 778, GBC 777 and Igri were estimated to be 87%, 54% and 75%, respectively, after a 4 d mannitol pretreatment at 25 degrees C. Chromosome numbers in dividing nuclei and relative densitometry measurements of nuclear DNA in microspores from cv. Igri confirmed the apparent fused nature of large nuclei in uninucleate microspores. The high frequency of fused nuclei indicates that nuclear fusion occurred between both symmetric and asymmetric nuclei. Microspores of cv. Igri cultured on filter paper following three different pretreatments provided an average of about 12 000 embryo-like structures (ELS) per plate. In samples, 85-97% of these ELS regenerated green shoots. The frequency of doubled haploids (74-83%) following all pretreatments was similar to the frequencies of fused nuclei. The pretreatment of spikes in 0.3 M mannitol at 4 degrees C for 4 d is preferred as it appears to provide genotype independent induction and suspension of nuclear division, as well as regenerating green plants in a shorter time than cold alone.