Simplified selection of transgenic Arabidopsis thaliana seed in liquid culture.

We have developed a technique that simplifies the process of screening large quantities of Arabidopsis thaliana seeds to identify antibiotic-resistant individuals. We use this technique in our laboratory for screening seed populations after transforming by vacuum infiltration (1). Conventional screening methods are materialand labor-intensive, particularly if transformation frequencies are low, due to the necessity of plating large amounts of seeds at densities low enough to observe antibiotic-resistant plants. We minimize these difficulties by germinating seeds in liquid media. Germinating seeds in liquid culture makes it fairly simple for us to sterilize and grow a large amount of seeds. However, in standard liquid culture media, seeds aggregate rapidly. As they germinate, a jumble of plant tissue results, making it impossible to remove plants of interest for further growth on soil. The key to the success of our technique is the supplementation of the growth media with 0.1% agar before autoclaving. Upon cooling, a suspension of fine agar particles forms, and this suspension interferes with the seeds’ natural tendency to aggregate. Eighteen thousand A. thaliana ecotype Wassilewskija (WS) seeds (0.36 g) were surface-sterilized (30-min imbibition with water, followed by 5 min in 95% ethanol, 5 min in 10% bleach and 5 rinses with sterile water) as described in (2) and added to each of three 2-L flasks with 4 mL of germination media [1× MS salts (3) plus 1% (wt/vol) sucrose, 100 mg/L myoinositol, 1.0 mg/L thiamine, 0.5 mg/L pyridoxine, 0.5 mg/L nicotinic acid, 0.5 g/L 2-(morpholino)ethanesulfonic acid (MES), pH 5.65–5.8 with potassium hydroxide]. Ten seeds carrying an insert conferring kanamycin resistance (pBI121; CLONTECH Laboratories, Palo Alto, CA, USA) were added to one of the flasks. After a two-day cold treatment at 4°C, 250 mL of germination media were added to each flask. One of the flasks containing only wild-type WS seeds received standard germination media; the other two flasks received germination media supplemented with 0.1% agar (Bacto-Agar; Difco Laboratories, Detroit, MI, USA). Kanamycin (75 μg/mL) was added to the flask containing transgenic seeds. As an extra precaution, Timentin® (100 μg/mL, 30:1 ticarcillin:clavulanic acid; SmithKline Beecham, Philadelphia, PA, USA) was added to reduce bacterial contamination, but it may not be strictly necessary. Flasks were placed on a rotating platform and shaken at 70 rpm under fluorescent lights (40 μE m-2 s-1) and photographed after seven days of growth. Figure 1A shows the effect of agar