Analysis of induction mechanism of desiccationtolerance of embryos and production of osmotic tolerantplants

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1. ME-leaN4 is late embryogenesis abundant (Lea) gene isolated from microspore-derived embryos of Brassica napus which have been induced desiccation tolerance by ABA.In situ hybridization and immunocytochemistry showed that expression of the ME-leaN4 mRNA and protein was found on desiccation tolerant embryos but not on desiccation sensitive ones.2. Scanningelectron microscopy was employed to compare desiccation-tolerant and -sensitive microspore-derived embryos. The external surface of the desiccation-tolerant embryos was uniformly shriveled by desiccation and their internal tissue system was well preserved. In contrast, in desiccation-sensitive embryos, dehydration caused tearing of the epidermis and collapse of the internal tissue system.3. T1_1 plants of transgenictobacco having ME-leaN4 which was regulated by 35SCaMV or pMAT promoter were investigated about the osmotic stress tolerance. A part of T_1 plants showed slightly increase of desiccation tolerance, but most of T_1 plants did not show. When Nail tolerance was investigated, growth of non-transgenic plants was inhibited by 1% Nail treatment, but the inhibition of growth of T1 plants was slight.4. Promoter region(323bp) of ME-leaN4 was isolated. The expression analysis using chimeric gene constructs containing ME-leaN4 promoter fragment fused to GUS gene indicated that the promoter is activated by ABA, sorbitol or Nail. Mutation analysis of the promoter ascertained the presence of two ABRE (ABA response element) in this region.