Rapid and highly efficient morphogenic gene-mediated hexaploid wheat transformation.

The successful employment of morphogenic regulator genes, Zm-Baby Boom (ZmBbm) and Zm-Wuschel2 (ZmWus2), for Agrobacterium-mediated transformation of maize (Zea mays L.) and sorghum (Sorghum bicolor L.) has been reported to improve transformation by inducing rapid somatic embryo formation. Here, we report two morphogenic gene-mediated wheat transformation methods, either with or without morphogenic and marker gene excision. These methods yield independent-transformation efficiency up to 58% and 75%, respectively. In both cases, the tissue culture duration for generating transgenic plants was significantly reduced from 80 to nearly 50 days. In addition, the transformation process was significantly simplified to make the procedure less labor-intensive, higher-throughput, and more cost-effective by eliminating the requirement for embryonic axis excision, bypassing the necessity for prolonged dual-selection steps for callus formation, and obviating the prerequisite of cytokinin for shoot regeneration. Furthermore, we have demonstrated the flexibility of the methods and generated high-quality transgenic events across multiple genotypes using herbicide (phosphinothricin, ethametsulfuron)- and antibiotic (G418)-based selections.

Genotype-Independent Transformation and Genome Editing of Brassica napus Using a Novel Explant Material.

Agrobacterium-mediated transformation of canola (Brassica napus) via hypocotyl segments has been a commonly used method for the past 30 years. While the hypocotyl-based method is well-established, it is not readily adapted to elite germplasm and the prolonged process is not ideal for a production transformation setting. We developed an Agrobacterium-mediated transformation method based on epicotyl and higher stem (internodal) segments that is efficient, rapid and amenable for high-throughput transformation and genome editing. The method has been successfully implemented in multiple canola genotypes. The method appears to be genotype-independent, with varying transformation efficiencies. Internodal segment transformation was used to generate transgenic events as well as CRISPR-Cas9-mediated frameshift gene knockouts.