A selection system for transgenic Arabidopsis thaliana using potassium thiocyanate as the selective agent and AtHOL1 as the selective marker

Concern about the use of bacterial antibiotic resistance genes as selectable markers is considered to be one of the factors in public resistance towards genetically modified crops. We had previously shown that the Arabidopsis AtHOL1 protein had high S-adenosyl-L-methionine-dependent methyltransferase activity toward the thiocyanate ion (NCS-), which is toxic to plants beyond certain concentrations. Contrary to our expectations regarding the usability of AtHOL1 as a selectable marker, our initial trial screenings for AtHOL1-overexpressing Arabidopsis on 1/2 MS agar medium containing various concentrations of potassium thiocyanate under normal growth conditions had been unsuccessful. In order to explore the possibility of using AtHOL1 in Arabidopsis transformation, the screening conditions for AtHOL1-overexpressing Arabidopsis were further examined. We found that the transgenic seedlings Could be screened when the seeds were germinated and grown under conditions of 7-day darkness followed by a 12-h light/12-h dark cycle for 3-7 days oil a medium containing 3.0-5.0 mM potassium thiocyanate. Analyses of transgene insertions into the genomes and AtHOL1 mRNA accumulation in the screened seedlings were also performed. Neither escaped seedlings nor altered growth was observed with the seedlings screened under the conditions reported here.