Comparative histology of microprojectile-mediated gene transfer to embryogenic calli in japonica rice (Oryza sativa L.): influence of the structural organization of target tissues on genotype transformation ability

摘要

The histology and time course of hygromycin-resistant callus formation in bombarded, seed embryo scutellum-derived embryogenic nodular units (ENUs) were investigated in 4 japonica rice cultivars (Taipei309, IDSA6, IR47686, IRAT361) exhibiting varying responses to microprojectile-mediated transformation systems. ENUs exhibited contrasting organization in their peripheral regions. This organization influenced the hygromycin concentration required to stop ENU growth, along with the amenability of transformed cells to evolve into isolated, clonal, transgenic calli. Organized globular proembryos located at the periphery of ENUs were tolerant to antibiotic selection. They also developed in selective induction medium, irrespective of whether they had been sectorially transformed or not, and produced chimaeric or clonal, non-transgenic proliferations. In contrast, the presence of a single layer or small clusters of embryogenic cells at the periphery of the bombarded ENU, whose growth was fully inhibitedunder a selective regime, generally led to the formation of clonal transgenic calli. An average of 2.5, 4.3, and 3.2 hygromycin resistant calli were produced per responding ENU in Taipei 309, IDSA6 and IR47686, respectively. Histological and molecular evidence was obtained that the hygromycin resistant calli arising from a single bombarded ENU may have been formed through independent transformation events.