A method combining TA cloning and fluorescence screening for rapid acquisition of transgenic seeds

Abstract

The establishment of transgenic plants has greatly promoted the progress of plant research. However, traditional selection methods using antibiotics or herbicides may miss any positive transformants with growth defects. Additionally, screening with antibiotics/herbicides requires a huge amount of seeds, sterile work conditions and a large amount of space to germinate plants, making the selection process time- and labor-consuming. In this study, we constructed a novel stable transformation vector, plasmid of OLE1-GFP T-DNA vector (pOGT), which can shorten the steps of cloning foreign genes into expression vectors by using TA cloning. Additionally, selection of transformed seeds with fluorescence overcomes the difficulties of conventional selection with antibiotics/herbicides and simplifies the screening process for transgenic plants.

METHOD SUMMARY

We constructed a stable transformation vector, plasmid of OLE1-GFP T-DNA vector (pOGT) with an OLE1-GFP fusion gene driven by the OLE1 promoter ( ProOLE1) as a selection marker. The OLE1 promoter controls the expression of an OLE1-GFP fusion protein and makes the transformed seeds fluorescent. Digestion of pOGT with XcmI generates a single thymidine (T) overhang at the 3′ end which enables the introduction of foreign genes into pOGT through one-step TA cloning. We generated transgenic Arabidopsis lines transformed with the pOGT-exogenous gene and screened transformed seeds using fluorescence.