Generation of thymidine auxotrophicAgrobacterium tumefaciensstrains for plant transformation

Abstract

Agrobacterium-mediated transformation is a widely used gene delivery method for fundamental researches and crop trait improvement projects. Auxotrophic Agrobacterium tumefaciens strains are highly desirable for plant transformation because they can be easily removed from the explants after co-cultivation due to their dependence on essential nutrient supplementation. The thymidine auxotrophic A. tumefaciens strain LBA4404Thy- has been successfully used for plant transformation, however, auxotrophic version of other commonly used strains are not available yet to public laboratories. Here we report the generation of EHA101, EHA105 and EHA105D thymidine auxotrophic strains. These strains exhibited thymidine-dependent growth in the bacterial medium, and the transient GUS expression assay using Arabidopsis seedling showed that they retain the equivalent T-DNA transfer capability as the original strains thus are suitable for plant transformation.

Genetic Transformation of Recalcitrant Upland Switchgrass Using Morphogenic Genes

“…The auxotrophic Agrobacterium strains rely on thymidine for growth and are thus highly advantageous because it allows for much better control of Agrobacterium overgrowth after the co-cultivation stage ( Aliu et al, 2020 ). Agrobacterium will also less likely persist in callus or regenerated plants because of its dependence on thymidine for survival which is not supplemented in plant culture media.…”

“…A thymidine auxotrophic (Thy-) version of the Agrobacterium strains of LBA4404 ( Lowe et al, 2016 ; kindly provided by Dr. William Gordon-Kamm of Corteva Agriscience) and EHA105 ( Aliu et al, 2020 ) were used to harbor the binary vector construct PHP93739. Both Agrobacterium strains contain the ternary helper plasmid PHP71539 to increase virulence ( Anand et al, 2018 ; kindly provided by Dr. William Gordon-Kamm of Corteva Agriscience).…”

An Agrobacterium strain auxotrophic for methionine is useful for switchgrass transformation

“…Despite the potential benefits of using auxotrophic strains of Agrobacterium , such strains are generally not available in the public sector. Towards that end, Aliu et al (2020) used homologous recombination to develop thymidine auxotrophs of strains EHA101, EHA105, and EHA105D. Transient GUS expression assays using hyper-transformable seedlings (Wu et al, 2014) of arabidopsis showed that, except for EHA105D, auxotrophic strains can deliver T-DNA at comparable levels to the parental strains.…”

“…In addition to being a thymidine auxotroph, EHA105D has a deleted atsD gene, which is implicated in plant cell attachment. However, the LBA4404Thy-auxotroph remained the most effective strain (Aliu et al, 2020).…”

“…In addition to being a thymidine auxotroph, EHA105D has a deleted atsD gene, which is implicated in plant cell attachment. However, the LBA4404Thyauxotroph remained the most effective strain (Aliu et al, 2020). This work was started to develop additional auxotrophic strains of Agrobacterium and make them available in the public sector.…”