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

The histology and time course of hygromycin-resistant callus formation from bombarded, seed embryo scutellum-derived embryogenic nodular units (ENUs) were investigated in four japonica rice cultivars (Taipei309, IDSA6, IR47686, IRAT361) exhibiting contrasting amenabilies in a microprojectile-mediated transformation system. ENUs from the above genotypes collected at a stage considered suitable for bombardment, exhibited contrasted organization in their peripheral regions. This organization was found to influence the hygromycin concentration requested to stop ENU growth, along with the amenability of transformed cells to evolve into isolated, clonal, transgenic resistant calli, from the rest of the ENU. Organized globular proembryos located at the periphery of ENUs proved to be quite 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 inhibited under 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. We obtained histological and molecular evidence that the hygromycin resistant calli arising from a single bombarded ENU may have been formed through independent transformation events.