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.
Freshly dissected immature embryos will be transferred from 700A liquid infection medium (containing induced Agrobacterium) to Co-cultivation Medium 562V-MSM. After draining excess infection medium and orienting embryos scutellum side up, plates are sealed with micropore tape and embryos and Agrobacterium will be co-cultured for 3 days in the dark at room temperature. Plan to promptly transfer from 562V-MSM to 605CefT to help suppress Agrobacterium contamination, which is extremely difficult to combat once it is noticeable. Co-Cultivation Medium contains added synthetic auxin (2,4-D) to encourage callus and embryogenic shoot growth. 562V-MSM is high in sucrose to encourage rapid plant growth. 562V-MSM contains no selective agents, and provides L-Methionine to permit LBA4404 Met- Agrobacterium growth. After 3 days of co-cultivation, all prior trials exhibited a minor, white film of bacterial growth on the plate surface, with little to no detectable growth on embryos themselves.
562V-MSM solid media should be prepared in 15x100 (standard) petri plates, planning for ~20 embryos per plate. Material grown on 562V-MSM will be sealed with micropore tape and be incubated at 20-25C (room temperature) in the dark. Embryos are ready to move off 562V-MSM after 3 days. There may be noticeable growth/swelling on the scutellum side of the embryo at this time, but do not be alarmed if this is not obvious.