Binary Bacterial Artificial Chromosome Vector Based Gene Transformation in Arabidopsis Plants: A Technique to Generate Transgenic Plants With Single-Copy Insertion
Published: April 30, 2023
Abstract
Source: Tark-Dame, M., et al. Generating Transgenic Plants with Single-copy Insertions Using BIBAC-GW Binary Vector. J. Vis. Exp. (2018).
In this video, we demonstrate generation of transgenic Arabidopsis plants expressing stable transgene through Agrobacterium-mediated binary bacterial artificial chromosome-based gene transformation.
Protocol
1. Arabidopsis Transformation Prepare the Arabidopsis plants for transformation. Grow Arabidopsis plants in a greenhouse or climate-controlled growth chamber until they are flowering (12 pots with 9 plants each per dipping). Clip the first bolts to allow more secondary bolts to emerge. Plants are ready for dipping 4–6 days after clipping, when the plants have many immature flower heads and not many fertilized si…
Representative Results
Figure 1: Tray filled with Arabidopsis seedlings before and after Glufosinate-ammonium treatment. Seedlings not expressing the bar gene that is present in the pBIBAC-BAR-GW T-DNA die after being sprayed with Glufosinate-ammonium solution. The photos show the same tray of seedlings (A) before spraying with Glufosinate-ammonium, 14 days after sowing, and (B) 10 days later, after being spra…
Disclosures
The authors have nothing to disclose.
Materials
| Kanamycin sulphate monohydrate | Duchefa | K0126 | |
| Gateway LR clonase enzyme mix | Thermo Scientific – Invitrogen | 11791-019 | |
| Murashige Skoog medium | Duchefa | M0221 | |
| Agar | BD | 214010 | |
| Glufosinate-ammonium (Basta) | Bayer | 79391781 | |
| Phosphor imager | GE Healthcare Life Sciences | Typhoon FLA 7000 | |
| cling film (Saran wrap) | Omnilabo | 1090681 | |
| Agarose | Thermo Scientific – Invitrogen | 16500 |
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Cite This Article
Binary Bacterial Artificial Chromosome Vector Based Gene Transformation in Arabidopsis Plants: A Technique to Generate Transgenic Plants With Single-Copy Insertion. J. Vis. Exp. (Pending Publication), e20980, doi: (2023).