Basta tolerance as a screening marker for transgenic 35S-OsMST6 Arabidopsis plants

As an alternative to in vitro screening, primary transformants carrying the bar selection marker can be directly screened on soil in a greenhouse by spraying with the herbicide Basta. The presence of selective marker allows the identification of transformants from the non-transformants. Transformed seedlings showed resistant to herbicide (Basta) because of bar gene on T-DNA construct. Therefore, it is most likely that these herbicide resistance seedlings contained candidate gene. In this study, transformation vector harboring OsMST6 (encoding monosaccharide transporter 6) driven by 35S promoter was constructed using Gateway technology and transformed into Arabidopsis thaliana via floral dip technique. To find the optimum concentration of Basta for T1 screening, 100% minimal lethal dose (MLD100) test was performed. For this purpose, wild type Arabidopsis was sampled by 0, 50, 100, 150, 200, 250 mg/L of Basta. According to the results obtained from this study, 150 mg/L Basta was the minimum dose that kills 100% of the wild type Arabidopsis plants. Primary transformants (T1) were directly screened on soil in growth chamber by spraying with the herbicide. Non-sterile seeds were spread onto moistened soil under normal conditions into a tray. The herbicide spraying was performed when the cotyledons were visible, normally around 8–10 d after sowing. The seedlings were sprayed by 150 mg/L of herbicide Basta every 2-3 d until transformants were easily recognizable among nontransfomed dead/dying plants, approximately 3–4 weeks after herbicide treatment. Then, from initial screening under 150 mg/L Basta, forty-two independent transgenic lines were obtained and verified by PCR.