Analysis of microspore embryogenesis by exhaustive isolation of cDNA and development of useful tool collecting embryogenic microspores

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Effective embryogenesis from isolated microspores has been established in Brassica spp. This system has great importance as a model in developmental study as well as in production of homozygous lines in practical breeding. To understand molecular mechanism underlying microspore embryogenesis, we have attmpted to exhaustively isolate genes related to induction of embryogenesis, to analyze an embryogenesis-specific promoter P22a1, and to develop a useful tool collecting embryogenic microspores in rapeseed.1.The 136 genes were isolated from early stage of microspore embryogenesis of rapeseed by suppression subtractive hybridization. BLASTX homology search for these 136 genes revealed that 87 genes were homologous to known genes, 23 ones were homologous unknown genes, and 26 ones had no mach in the database. When 15 genes selected from these 136 genes were examined their expression level by real-time RT-PCR, 14 genes showed the high expression in early stage of embryogenesis. Promoter anal … More ysis using Arabidopsis homologues indicated that the genes were classified into two types, (1)genes expressed in only microspore embryogeneis, but not in zygotic embryogenesis, (2)genes commonly expressed in both embryogenesis. The genes classified in former type are considered to be candidate genes related to induction of androgenesis.2.An embryogenesis-specific promoter P22a1 was fused to GUS and GFP reporter genes and introduced into Arabidopsis and rapeseed to examine spatial and temporal specificity of the promoter. The specific GFP accumulation was observed in young embryo sacs before fertilization, zygotes, young embryos and endosperm cells. A cis-acting enhancer-like region was identified in the promoter from -353 to -249, but other cis-acting element(s) would be necessary in addition to the 104 by region for complete promoter activity. Microspores isolated from P22a1 : : GFP transgenic rapeseed plants developed GFP-expressing pro embryonic cells in 36 hrs after culture initiation. The result suggested that the promoter : : reporter gene would be a useful tool to concentrate cells that switched their developmental course from sporogenesis to embryogenesis before they could be morphologically distinguished. Less