Fast-track transformation and genome editing in Brachypodium distachyon

Abstract

Background Even for easy-to-transform species or genotypes, the creation of transgenic or edited plant lines remains a significant bottleneck. Thus, any technical advance that accelerates the regeneration and transformation process is welcome. So far, methods to produce Brachypodium distachyon (Bd) transgenics span at least 14 weeks from the start of tissue culture to the recovery of regenerated plantlets. Results We have previously shown that embryogenic somatic tissues grow in the scutellum of immature zygotic Bd embryos within 3 days of in vitro induction with exogenous auxin and that the development of secondary embryos can be initiated immediately thereafter. Here, we further demonstrate that such pluripotent reactive tissues can be genetically transformed with Agrobacterium tumefaciens right after the onset of somatic embryogenesis. In brief, immature zygotic embryos are induced for callogenesis for one week, co-cultured with Agrobacterium for three days, then incubated on callogenesis selective medium for three weeks, and finally transferred on selective regeneration medium for up to three weeks to obtain plantlets ready for rooting. This 7-to-8-week procedure requires only three subcultures. Its validation includes the molecular and phenotype characterization of Bd lines carrying transgenic cassettes and novel CRISPR/Cas9-generated mutations in two independent loci coding for nitrate reductase enzymes (BdNR1 and BdNR2). Conclusions With a short callogenesis stage and streamlined in vitro regeneration following co-cultivation with Agrobacterium, transgenic and edited T0 Bd plantlets can be produced in about 8 weeks, a gain of one to two months compared to previously published methods, with no reduction in transformation efficiency and at lower costs.