Optimization of Agrobacterium-mediated transformation conditions in mature embryos of elite wheat

Immature embryos have been used frequently as target tissues in the genetical transformation of wheat. However, obtaining a large number of high quality immature embryos throughout the year is a laborious and delicate process, because of the need to cultivate the plants under controlled conditions. To circumvent this, we have employed mature embryos rather than immature ones as starter explants for Agrobacterium-mediated transformation of an elite wheat (Triticum aestivum L.) cultivar EM12. The neomycin phosphotransferase II, (npt II) and beta-glucuronidase (gus) genes were used as selectable and screenable marker genes, respectively, to assess and optimize the performance of T-DNA delivery. With the aid of an orthogonal design, the effect of four factors in combination on transfer DNA (T-DNA) delivery was studied. These factors were preculture duration, different kinds of inoculation, length of inoculation and co-culture condition. Optimal conditions for T-DNA delivery were obtained for mature embryos precultured for 14 days, followed by immersing in inoculation suspension with full strength Murashige and Skoog (MS) salts in darkness at 23-25 degrees C for 3 h, and then co-culturing with Agrobacterium under desiccating condition in the dark at 23-24 degrees C for 2-3 days. Complete analysis of transgene insertion demonstrated that the optimized method for Agrobacterium-mediated transformation of mature embryos of wheat was efficient and practicable.