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


CONCLUSIONS
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.
Harrison
Samuel J
SJ
Department of Plant Sciences, University of Cambridge, Downing Street, Cambridge, CB2 3EA, UK. Sam_Harrison@cantab.net
Mott
Ellie K
EK
Parsley
Kate
K
Aspinall
Sue
S
Gray
John C
JC
Cottage
Amanda
A