Agrobacterium-mediated transformation of mature embryos of Triticum aestivum and Triticum durum

Abstract

Plant regeneration studies in cereals have been undertaken in immature embryos, scutellum and also in immature inflorescence tissue. The wheat mature embryos can also be employed for callusing and regeneration, as they are available throughout the year and have presently been employed for transformation studies. An efficient and reproducible method for Agrobacterium- mediated transformation of mature embryos of hexaploid bread wheat (Triticum aestivum) and tetraploid pasta wheat (Triticum durum) is reported. Presence of acetosyringone at 200 μ M concentration in the bacterial growth medium, inoculation medium and cocultivation medium was essential for achieving a 1.5- 2.0 fold increase in transient expression of the introduced gus gene. Successful generation of T. aestivum and T. durum transgenic plants at a transformation frequency ranging from 1.28 to 1.77% has been achieved following 2-3 days co-cultivation using mature embryos and also mature embryo-derived calluses with binary Agrobacterium strain LBA4404 (pBI101 :: Act1) and LBA4404 (p35SGUSINT) respectively. Paromomycin and phosphinothricin served as effective selection agents as they did not adversely affect plantlet regeneration. Successful integration as well as inheritance of the transgene was confirmed by Southern hybridization and PCR amplification in T₀ as well as T₁ generation. Optimization of this method facilitated the introduction of bar gene as a selectable marker conferring herbicide resistance as well as potato proteinase inhibitor gene (pin2) for insect resistance into wheat.