Selection of Arabidopsis transformants containing AtZAT12

Abstract

This study aimed to select Arabidopsis seeds after transformation with the AtZAT12 gene via Agrobacterium tumerfaciens. ZAT12 is a transcription factor that inhibits other transcription factors FIT through the EAR motif. FIT itself is a central transcription factor that controls Fe uptake under Fe-deficient conditions. However, if Fe is absorbed excessively, it will produce reactive oxygen species that could damage cells. That is the reason why AtZAT12 transcription is elevated and FIT transcription is inhibited under Fe deficiency for 10 days. To investigate the function, the AtZAT12 gene was inserted into the pMDC107 vector for transformation into Arabidopsis. The T0 Arabidopsis seeds obtained after floral dip transformation were placed on 1% MS agar supplemented with 15 μg/mL Hygromycin B. Beforehand, the T0 seeds were sterilized and kept in the dark for stratification. Subsequently, the seed plates were subjected to a regime of 6 h of light, 48 h of dark and 24 h of light (3.25 d). The hygromycin B-resistant seedlings had long hypocotyls (~ 1.0 cm), while the non-resistant seedlings had short (~ 0.3 cm) hypocotyls. This method took only 3.25 days to identify transgenic Arabidopsis seedlings. After that, positive transgene lines were examined by PCR method for AtZAT12, and the expression of AtZAT12 was observed under microscope.