Effective Use of Novel Auxotrophic Agrobacterium tumefaciens Strains for Transformation and Biocontainment

Auxotrophic strains of <i>Agrobacterium tumefaciens</i> have been developed to address the <i>Agrobacterium</i> overgrowth issue in plant genetic transformation; however, their application remains limited. Here, we generated novel histidine and leucine auxotrophic strains of <i>A. tumefaciens</i> EHA105, namely EHA105hisD- and EHA105leuA-, as well as a dual auxotrophic strain EHA105hisD-leuA-, through gene deletion. The transient expression efficiency and survival rate of these three auxotrophic strains in <i>Nicotiana benthamiana</i> were significantly impaired but could be restored to wild-type EHA105 levels by supplementation with appropriate concentrations of the corresponding amino acids (CAAs). The use of these three auxotrophic strains for the genetic transformation of <i>N. benthamiana</i> resulted in a significant reduction in <i>Agrobacterium</i> overgrowth and achieved transformation efficiency comparable to wild-type EHA105, when appropriate exogenous concentrations of the CAAs were supplied during the co-cultivation stage. Furthermore, through incubation experiments on various plants and soil, it was confirmed that the incidence of surviving cells from these three auxotrophic strains was much lower than that observed for the wild-type EHA105. In summary, this study reports on the characteristics of the novel auxotrophic strains of <i>A. tumefaciens</i> along with the effective use of such auxotrophic <i>A. tumefaciens</i> strains in plant genetic transformation.