This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::β-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.
I. Floral-dip transformation of Arabidopsis thaliana
II. Examining pTSO2::GUS expression patterns
Representative Results
When done correctly, transformation efficiency should be approximately 0.1-0.2%. In another word, one should get 5-10 transgenic seedlings by screening every 5000 seeds. On average, about 100 transgenic lines can be obtained by transforming 4 pots of wild-type plants.
For transgenic seedlings containing the pTSO2::GUS construct3, dark blue color reflecting GUS activity is found in actively dividing cells including young leaves, shoot apex, root tip, and lateral root primordia (Figure 1). The non-uniform sporadic pattern is characteristic of cell cycle phase-specific expression.
The efficiency of transformation is determined by many different factors, which are discussed below:
Depending on specific plasmid construct, the selection methods of transgenic plants may vary. If a plasmid vector carries the Bar (bialaphos acetyltransferase) marker gene conferring resistance to glufosinate ammonium, one can directly plant the seeds in soil without any sterilization of seeds. One then sprays the seedlings with 1:1000 diluted glufosinate ammonium (commercial name: Finale, Liberty, or Ignite) once every few days to select for resistant seedlings.
For GUS staining, it s important to harvest tissue in acetone on ice and keep all solutions cold to avoid any degradation. The staining buffers with and without X-Gluc needs to be made fresh, although the stock solution for each component can be made and stored ahead of time (see below). Potassium Ferrocyanide and Ferricyanide are toxic and should be handled with care. Additionally, it s important not to incubate the tissue at 37°C in the staining buffer with X-Gluc for longer than 1-2 days because the tissue can begin to deteriorate and/or the staining pattern may become too diffuse during the long incubations. Finally, higher ferri and ferrocyanide concentrations give lower overall staining level, but more specificity. 2mM X-Glux works well for most applications, but the concentrations may need to be adjusted for certain needs. X-Gluc is expensive. The staining should be carried out in minimal volumes.
Stock solutions that can be made ahead of time:
10% Triton X-100 (stored at room temperature for several months)
0.5 M NaHPO4 Buffer (pH7.2) (stored at room temperature for several months)
100 mM Potassium Ferrocyanide (store in the dark at 4°C for several months)
100 mM Potassium Ferricyanide (Store in the dark at 4°C for several months)
100 mM X-Gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexamine salt) is made in Dimethylformamide (DMF) and stored in -20°C in tinfoil wrapped tubes. It lasts for several months.
The authors have nothing to disclose.
We thank Chunxin Wang for constructing pTSO2::GUS and for photos in Figure 1, Detlef Weigel for sharing the GUS-foolproof protocol, Paja Sijacic, and Courtney Hollender for helpful comments. Research in Z. L s laboratory is supported by the US National Science Foundation (IOB0616096 and MCB0744752). Z.L. is partially supported by the University of Maryland Agricultural Experiment Station.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
6-benzyladenine | Sigma | 852430 | ||
Silwet-77 | Crompton Corp. | Toxic, wear gloves | ||
Murashige and Skoog Basal medium (MS) | Sigma | M5519 | ||
Gamborg’s vitamin solution 1000X | Sigma | G1019 | ||
X-Gluc (5-bromo-4-chloro-3-indolyl β-D-glucuronide cyclohexamine salt | Biosynth | B-7300 | Toxic, light sensitive keep in the dark | |
Micropore 3M Tape | Fisher | 19027761 |