Development of an efficient marker‐free soybean transformation method using the novel bacterium Ochrobactrum haywardense H1

• Published in: Plant biotechnology journal Vol. 20; no. 5; pp. 977 - 990
• Main Authors: Cho, Hyeon‐Je, Moy, York, Rudnick, Nathan A., Klein, Theodore M., Yin, Jiaming, Bolar, Joy, Hendrick, Carol, Beatty, Mary, Castañeda, Leandro, Kinney, Anthony J., Jones, Todd J., Chilcoat, N. Doane
• Format: Journal Article
• Language: English
• Published: England John Wiley & Sons, Inc 01.05.2022; John Wiley and Sons Inc
• Subjects: Agrobacterium; Agrobacterium tumefaciens - genetics; Antibiotics; Backbone; Bacteria; Deoxyribonucleic acid; DNA; Drug resistance; Fatty acids; Flowers & plants; Genes; Genetic transformation; Genetic Vectors; Germplasm; Glycine max - genetics; Heat shock; heat shock promoters; high‐throughput soybean transformation; Markers; marker‐free events; Maximum likelihood method; non‐Agrobacterium‐mediated transformation; Ochrobactrum; Ochrobactrum - genetics; Ochrobactrum haywardense H1; Pathogens; Phylogenetics; Plant bacterial diseases; Plants, Genetically Modified; Product development; Sorghum; Soybeans; Spectinomycin; Statistical analysis; Tobacco; Transformation, Genetic; Transformations; Transgenic plants; Virulence; high-throughput soybean transformation; non-Agrobacterium-mediated transformation; heat shock promoters; Ochrobactrum haywardense H1; marker-free events; Ochrobactrum
• ISSN: 1467-7644; 1467-7652
• DOI: 10.1111/pbi.13777

Summary We have discovered a novel bacterium, Ochrobactrum haywardense H1 (Oh H1), which is capable of efficient plant transformation. Ochrobactrum is a new host for Agrobacterium‐derived vir and T‐DNA‐mediated transformation. Oh H1 is a unique, non‐phytopathogenic species, categorized as a BSL‐1 organism. We engineered Oh H1 with repurposed Agrobacterium virulence machinery and demonstrated Oh H1 can transform numerous dicot species and at least one monocot, sorghum. We generated a cysteine auxotrophic Oh H1‐8 strain containing a binary vector system. Oh H1‐8 produced transgenic soybean plants with an efficiency 1.6 times that of Agrobacterium strain AGL1 and 2.9 times that of LBA4404Thy‐. Oh H1‐8 successfully transformed several elite Corteva soybean varieties with T0 transformation frequency up to 35%. In addition to higher transformation efficiencies, Oh H1‐8 generated high‐quality, transgenic events with single‐copy, plasmid backbone‐free insertion at frequencies higher than AGL1. The SpcN selectable marker gene is excised using a heat shock‐inducible excision system resulting in marker‐free transgenic events. Approximately, 24.5% of the regenerated plants contained only a single copy of the transgene and contained no vector backbone. There were no statistically significant differences in yield comparing T3 null‐segregant lines to wild‐type controls. We have demonstrated that Oh H1‐8, combined with spectinomycin selection, is an efficient, rapid, marker‐free and yield‐neutral transformation system for elite soybean.