This is part of the Leiboff Lab maize transformation protocol for somatic embryogenesis of B104 immature embryos. This protocol is a combination of Chen et al. 2022 and Kang et al. 2022 with some modifications based on material availability. This protocol is intended for the GRF-GIF/BBM somatic embryogenesis transformation strategy with the LBA4404 Met- auxotrophic Agrobacterium strain.
Embryos will dissected and transferred to 700 prior to infection with 700 + L-Methionine (50mg/L) and Acetosyringone (100uM) with Agrobacterium at 0.35-0.4 OD600. Infection Medium 700 is used for 5 minutes before transferring infected embryos to 562V-MSM Co-cultivation medium. Infection Medium contains added synthetic auxin (2,4-D), a high level of sucrose, and glucose to encourage rapid plant growth. 700AMet is supplemented with Acetosyringone tpB contains 5 mg/L of bialaphos (preferred for basta selection in maize over glufosinate) as a plant selective agent, and uses both Cefotaxime and Timentin to control Agrobacterium contamination. The antibiotic concentrations used here are sufficient to control the LBA4404 Met- auxotrophic strain, but were not sufficient to control wild-type LBA4404 in 3 prior trials.
700 liquid media should be prepared in standard glass bottles, planning for 50 mL per construct. Pelleted Agrobacterium will be resuspended in 700 + L-Methionine (50mg/L) and Acetosyringone (100uM) at 0.35-0.4 OD600 and shaken in the dark for 2-6 hours at RT. Dissected embryos will be briefly stored in 700, rinsed in 700, then combined with infection media with Agrobacerium. Material grown on 605CefTB will be sealed with micropore tape and be incubated at 28C in the dark. Embryos are ready to move off 605CefTB after 1 week. There should be noticeable growth on the scutellum side of the embryo at this time and somatic embryos may be established, but do not be alarmed if this is not obvious.