Simple methods for selection of T-DNA-free segregants from offspring of gene-edited Solanum nigrum

Plant genome editing has been mainly mediated by T-DNA transformation to study plant biology and improve crop quality and quantity. The T-DNA must eventually be removed (called T-DNA-free) in siblings via genetic segregation after completing editing using the CRISPR/cas9 mechanism. Here, we developed an easy and efficient method to select T-DNA-free segregants with kanamycin treatments at 10 days after germination (DAG) onto seedlings of T1 siblings propagated from transgenic plants in Solanum nigrum harboring NPTII on the CRISPR/cas9 vector. To quantify the responses in root and shoot growth and leaf color, three types of kanamycin treatments were applied as follows: (1) submerging germinated seeds in kanamycin liquid (Submerged), (2) growing seeds on agar MS media containing kanamycin (MS agar media), and (3) spraying kanamycin onto seedlings at 10 DAG (Sprayed). Optimal kanamycin concentrations were determined as 200 mg/L for “Submerged,” 50 mg/L for “MS agar media,” and 400 mg/L for “Sprayed” to distinguish wild type (WT) seedlings treated with kanamycin from untreated seedlings. Furthermore, T-DNA-free plants were selected from T1 siblings of genome-edited S. nigrum plants using the same methods of distinguishing untreated WT plants, followed by confirmation of T-DNA-free status using polymerase chain reaction. Notably, the efficiency of T-DNA-free selection was 100% with the “Sprayed” method, and after 2 weeks, all the selected plants recovered from the strong stress. However, although “Submerged” and “MS agar media” methods were useful for removing mutants harboring T-DNA from the population, the methods showed relatively low accuracy in selection of T-DNA-free mutants with efficiencies of 5% and 17%, respectively. This indicates that in addition to stress from kanamycin treatment, plant growth is likely controlled by many factors owing to which mutations often occur during the regeneration process; however, photosynthesis is often not affected by such mutations. Therefore, we suggest that the “Sprayed” method is an easy, simple, and accurate method for isolating genome-edited S. nigrum plants that do not contain T-DNA in the genome and could be generally applicable to breed T-DNA-free variants in other crops such as tomato.