Optimization of regeneration and Agrobacterium tumefaciens-mediated transient transformation systems for Australian native extremophile, Tripogon loliiformis

Abstract

Tripogon loliiformis, an Australian native resurrection grass having an abundant gene pool for combating desiccation, can be the putative model system for functional characterization of stress tolerance genes due to its diploid genome and being a monocotyledonous plant and member of the grass family (Poaceae), like many important cereal crops. For developing callus mediated regeneration from mature grains of Tripogon, Murashige and Skoog medium containing growth regulator, 2,4-dichlorophenoxyacetic acid (2,4-D) at 1.0mgL−1 was the optimum concentration for induction and proliferation of healthy cream calli. Successful regeneration of shoots from callus clumps in MS medium supplemented with 2.0mgL−1 6-benzylaminopurine (BAP) and 0.5mgL−1 α-naphthalene acetic acid (NAA) was obtained from 2 consecutive rounds subculturing of the calli at 3 weeks interval. In addition, rooting needed another 2 rounds within the same media with 2.0mgL−1 BAP but with 0.25mgL−1 NAA. The transient expression of UidA gene at 3 days after Tripogon callus transformation, performed with Agrobacterium tumefaciens strains AGL1 and LBA4404 following rice and Brachypodium distachyon transformation protocols, indicates successful Agrobacterium infection and gene delivery in calli. A stable transformation system for Tripogon loliiformis can be developed near future following the protocols in this study.