Abstract
BackgroundThe 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.ResultsA 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).ConclusionThe 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.
Highlights
The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation
We have developed a method of seedling selection that results in rapid, easy identification of transformants; the protocol presented works well for screening for resistance to kanamycin, phosphinothricin and hygromycin B
To increase hypocotyl length and so facilitate easier seedling removal, we placed seedlings in the dark for 48 h before illumination. This produced a clear cut difference between resistant and nonresistant seedlings and allowed the development of a growth regime than can distinguish seedlings resistant to kanamycin, phosphinothricin or hygromycin B from nonresistant seedlings in 3.25 d, as opposed to the 7–10 d required in previously reported selection procedures (Figure 1)
Summary
The floral dip method of transformation by immersion of inflorescences in a suspension of Agrobacterium is the method of choice for Arabidopsis transformation. 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. Arabidopsis transformation mediated by vacuum infiltration of inflorescences with an Agrobacterium suspension was first introduced by Bechtold et al [1]. This method has been widely used in preference to tissue culture techniques as it directly produces transformed seed and negates lengthy and complicated tissue culturing procedures. This transformation method was further modified (page number not for citation purposes)
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