Fast-track transformation and genome editing in Brachypodium distachyon.

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.

One-Week Scutellar Somatic Embryogenesis in the Monocot Brachypodium distachyon.

Plant somatic embryogenesis (SE) is a natural process of vegetative propagation. It can be induced in tissue cultures to investigate developmental transitions, to create transgenic or edited lines, or to multiply valuable crops. We studied the induction of SE in the scutellum of monocots with Brachypodium distachyon as a model system. Towards the in-depth analysis of SE initiation, we determined the earliest stages at which somatic scutellar cells acquired an embryogenic fate, then switched to a morphogenetic mode in a regeneration sequence involving treatments with exogenous hormones: first an auxin (2,4-D) then a cytokinin (kinetin). Our observations indicated that secondary somatic embryos could already develop in the proliferative calli derived from immature zygotic embryo tissues within one week from the start of in vitro culture. Cell states and tissue identity were deduced from detailed histological examination, and in situ hybridization was performed to map the expression of key developmental genes. The fast SE induction method we describe here facilitates the mechanistic study of the processes involved and may significantly shorten the production of transgenic or gene-edited plants.