Transgene Homozygosity Following Particle Bombardment of Haploid Barley Microspores

Information from previously published studies that are basic to this study is： 1） Following isolated barley microspore culture, around 80% of the resulting barley plants are completely fertile and genetically homozygous doubled haploids （DH） （Kasha et al., 2003）. 2） Chromosome doubling occurred during early stages of microspore culture, mainly by nuclear fusion （Kasha et al., 2001）. 3） A fairly synchronous population of microspores was selected to determine the cell cycle stages （G1, S, G2） by relative DNA density measurements （Shim and Kasha, 2003a）. 4） The pretreatments of spikes used to induce microspore embryogenesis influenced the cell cycle progression during pretreatment. （i） Using a combination of cold （4℃） and 0.3 mol/L mannitol for 4 days, the microspores were held at the G1 uninucleate stage. （ii） The often used pretreatment of cold （4℃ for 21 to 28 d） permitted slow microspore stage progression so that most cells were at the G2 stage or had become binucleate by the end of the pretreatment. （iii） Pretreatment with mannitol for 4 days at 25 ℃ did not block cell cycle progression but prevented cell wall formation after the 1^st mitotic division, leading to nuclear fusion and chromosome doubling.

Transgene Homozygosity Following Particle Bombardment of Haploid Barley Microspores

Information from previously published studies that are basic to this study is: 1) Following isolated barley microspore culture, around 80% of the resulting barley plants are completely fertile and genetically homozygous doubled haploids (DH) (Kasha et al., 2003).