Binary Bacterial Artificial Chromosome Vector Based Gene Transformation in Arabidopsis Plants: A Technique to Generate Transgenic Plants With Single-Copy Insertion

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

1. Prepare the Arabidopsis plants for transformation.
   1. Grow Arabidopsis plants in a greenhouse or climate-controlled growth chamber until they are flowering (12 pots with 9 plants each per dipping).
   2. 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 siliques.
2. Floral dipping
   1. Dip inflorescences for 5–10s in Agrobacterium suspension carrying T-DNA cloned with binary bacterial ....

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 sh.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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