A rapid and robust method of identifying transformed Arabidopsis thaliana seedlings following floral dip transformation

Abstract

Background: The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. The presence of a marker, usually antibiotic- or herbicide-resistance, allows identification of transformed seedlings from untransformed seedlings. Seedling selection is a lengthy process which does not always lead to easily identifiable transformants. Selection for kanamycin-, phosphinothricin- and hygromycin B-resistance commonly takes 7-10 d and high seedling density and fungal contamination may result in failure to recover transformants.

Results: A method for identifying transformed seedlings in as little as 3.25 d has been developed. Arabidopsis T1 seeds obtained after floral dip transformation are plated on 1% agar containing MS medium and kanamycin, phosphinothricin or hygromycin B, as appropriate. After a 2-d stratification period, seeds are subjected to a regime of 4-6 h light, 48 h dark and 24 h light (3.25 d). Kanamycin-resistant and phosphinothricin-resistant seedlings are easily distinguished from non-resistant seedlings by green expanded cotyledons whereas non-resistant seedlings have pale unexpanded cotyledons. Seedlings grown on hygromycin B differ from those grown on kanamycin and phosphinothricin as both resistant and non-resistant seedlings are green. However, hygromycin B-resistant seedlings are easily identified as they have long hypocotyls (0.8-1.0 cm) whereas non-resistant seedlings have short hypocotyls (0.2-0.4 cm).

Conclusion: The method presented here is an improvement on current selection methods as it allows quicker identification of transformed seedlings: transformed seedlings are easily discernable from non-transformants in as little as 3.25 d in comparison to the 7-10 d required for selection using current protocols.

Figure 1

Time-line comparison of the current selection procedure and the rapid selection procedure.

Figure 2

Rapid selection of kanamycin-resistant Arabidopsis thaliana Col-0 seedlings. a. Seedlings from Arabidopsis thaliana Col-0 T1 seed obtained from plants subjected to floral dip transformation using Agrobacterium tumefaciens strain GV3101 harbouring the binary plasmid pBINPLUS. Seeds were plated on 1% agar containing MS medium (including vitamins) and kanamycin at a concentration of 50 μg.ml^-1. Following a stratification period of 2 d at 4°C in the dark, seedlings were incubated for 6 h at 22°C in white light, followed by incubation at 22°C in the dark for 2 d and continuous white light for a further 24 h. b. Kanamycin-resistant Arabidopsis seedling after rapid selection procedure (left); kanamycin-susceptible seedling after rapid selection procedure (right). Size bars 1 cm.

Figure 3

Rapid selection of phosphinothricin-resistant Arabidopsis thaliana Ws seedlings. a. Seedlings from Arabidopsis thaliana Ws T1 seed obtained from plants subjected to floral dip transformation using Agrobacterium tumefaciens strain GV3101 harbouring the binary plasmid pSKI015. Seeds were plated on 1% agar containing MS medium (including vitamins) and phosphinothricin at a concentration of 50 μM. Following a stratification period of 2 d at 4°C in the dark, seedlings were incubated for 6 h at 22°C in white light, followed by incubation at 22°C in the dark for 2 d and continuous white light for a further 24 h. b. Phosphinothricin-resistant Arabidopsis seedling after rapid selection procedure (left); phosphinothricin-susceptible seedling after rapid selection procedure (right). Size bars 1 cm.

Figure 4

Rapid selection of hygromycin B-resistant Arabidopsis thaliana Col-0 seedlings. a. Seedlings from Arabidopsis thaliana Col-0 T1 seed obtained from plants subjected to floral dip transformation using Agrobacterium tumefaciens strain GV3101 harbouring the binary plasmid pBIG-HYG. Seeds were plated on 1% agar containing MS medium (including vitamins) and hygromycin B at a concentration of 15 μg.ml^-1. Following a stratification period of 2 d at 4°C in the dark, seedlings were incubated for 6 h at 22°C in white light, followed by incubation at 22°C in the dark for 2 d and continuous white light for a further 24 h. b. Hygromycin B-resistant Arabidopsis seedling after rapid selection procedure (left); kanamycin-susceptible seedling after rapid selection procedure (right). Size bars 1 cm.