Agrobacterium tumefaciens-mediated genetic transformation of barley ( Hordeum vulgare L.)

Abstract

A critical step in the development of a robust Agrobacterium tumefaciens-mediated transformation system for a recalcitrant species, like barley, is the establishment of optimal conditions for efficient T-DNA delivery into target tissue from which plants can be regenerated. We used Agrobacterium strain LBA4404 harbouring either binary vector pUGAB7, which contains a T-DNA incorporating the bar gene and uidA gene, or pYF133, which contains hpt gene and gfp gene. These were used to investigate and optimize major T-DNA delivery and tissue culture variables in the immature embryos of barley (cv. Golden Promise). A number of factors produced significant difference in T-DNA delivery. These included pre-culture of immature embryos, co-cultivation, presence of acetosyringone and sonication and vacuum filtration assisted inoculation of 1-day precultured immature embryos. Plant regeneration was achieved via somatic embryogenesis. Regeneration media showed significant difference in their capacity to support regeneration of transgenic plants. Optimizing factors for T-DNA delivery resulted in bialaphos and hygromycin resistant barley transgenic plants with transformation efficiencies ranging from 2.6 to 5.6% and 3.3 to 6.7%, respectively. Molecular analysis of transgenic plants demonstrated that transgenes were integrated into the barley genome and subsequently transmitted into progeny at Mendelian ratios. The developed protocol will facilitate the insertion of desirable traits into barley via Agrobacterium-mediated transformation.