An Improved Agrobacterium-Mediated Transformation and Genome-Editing Method for Maize Inbred B104 Using a Ternary Vector System and Immature Embryos.

For maize genome-editing and bioengineering, genetic transformation of inbred genotypes is most desired due to the uniformity of genetic background in their progenies. However, most maize inbred lines are recalcitrant to tissue culture and transformation. A public, transformable maize inbred B104 has been widely used for genome editing in recent years. This is primarily due to its high degree of genetic similarity shared with B73, an inbred of the reference genome and parent of many breeding populations. Conventional B104 maize transformation protocol requires 16-22 weeks to produce rooted transgenic plants with an average of 4% transformation frequency (number of T0 plants per 100 infected embryos). In this Method paper, we describe an advanced B104 transformation protocol that requires only 7-10 weeks to generate transgenic plants with an average of 6.4% transformation frequency. Over 66% of transgenic plants carried CRISPR/Cas9-induced indel mutations on the target gene, demonstrating that this protocol can be used for genome editing applications. Following the detailed and stepwise procedure described here, this quick and simplified method using the Agrobacterium ternary vector system consisting of a T-DNA binary vector and a compatible helper plasmid can be readily transferable to interested researchers.

Agrobacterium-Mediated Transformation of Setaria viridis, a Model System for Cereals and Bioenergy Crops.

Setaria viridis is an emerging model system for the genetic and molecular characterization of cereals and bioenergy crops. Here, we describe a detailed procedure for genetic transformation of the S. viridis accession line ME034V-1. This method utilizes callus generated from mature seeds for infection with Agrobacterium tumefaciens strain AGL1 to regenerate hygromycin-resistant stable transgenic plants. It takes approximately 7 weeks to generate callus from mature seeds, 11-17 weeks from infection to the regeneration of transgenic lines, and an additional 3-4 weeks for plant growth in the greenhouse for seed collection. The protocol as presented consistently results in transformation frequency of approximately 25% for the generation of transgenic plants, with fewer escapes and higher survivability in soil for optimal seed collection. © 2021 Donald Danforth Plant Science Center. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Generation of S. viridis (Accession ME034V-1) callus from mature seeds Basic Protocol 2: Agrobacterium-mediated transformation of callus to generate transgenic plants Basic Protocol 3: Plantlet transplantation in soil, plant growth in greenhouse, and seed collection Support Protocol: Preparation of Agrobacterium culture for infection.