Plant Physiol. 2007 February; 143(2): 745–758
Germination and Root Elongation Tests For germination tests, seeds harvested from the same batches were cold pretreated and then grown on modified Murashige and Skoog medium supplemented with Glc, Suc, NaCl, ABA, glufosinate ammonium, or fluridone at various concentrations listed in the “Results.” The medium was autoclaved and cooled to 50°C to 60°C prior to the addition of filter-sterilized ABA, glufosinate ammonium, or fluridone. For root elongation experiments, cold-pretreated seeds from different genotypes were first grown on agar plates with 1% Suc for 4 or 5 d, then uniform seedlings of similar size and primary root length were transferred to appropriate fresh medium and grown vertically for another 6 or 7 d.
Plant Physiol. 2007 February; 143(2): 745–758
Preparation of Low-Water-Potential Medium Plates One-half-strength modified Murashige and Skoog medium (pH 5.7) with 0.7% Phyto agar (Duchefa Biochemie B.V.) was autoclaved. The sterilized medium was cooled to 50°C to 60°C and then aliquoted into petri dishes (100- × 20-mm depth), 20 mL each, for solidification. PEG-infused plates were made by dissolving PEG-8000 (Sigma) powder into one-half-strength Murashige and Skoog solution (pH 5.7) with the above-mentioned components, except phyto agar, and then filter sterilized; this PEG solution was then overlaid on agar-solidified medium at a ratio of 3:2 (v/v) and equilibrated overnight (≥12 h). The excess PEG solution was then removed. The procedure essentially followed the protocol of Verslues and Bray (2004).
Plant Physiol. 2007 February; 143(2): 745–758
NaCl and Dehydration TreatmentsWild type, aba2, and ABA2 overexpression seeds with cold pretreatment were grown on agar plates with various NaCl concentrations for 18 or 28 d. The ratios of bleached to total plants were counted to define tolerance to salinity. Seeds with cold pretreatment were also grown in soil for 3 weeks; then plants were subjected to watering with solutions containing four gradually increased concentrations of NaCl (50, 100, 150, and 200 mm), each for 4 d for a total of 16 d (Shi et al., 2003). For dehydration, seeds with cold pretreatment were grown in soil for 21 d and then plants were excised. The aerial parts of tissues were placed on plastic weighing boats and kept in an electronic dry box (model-DX-76; Taiwan Dry Tech Corp.) with approximately 40% relative humidity. The fresh weights of tissues were measured at 0-, 1.5-, and 3-h time points.
Plant Physiol. 2007 February; 143(2): 745–758
GUS Activity AssayCold-pretreated transgenic seeds harboring the ABA2GUS transgene were grown on agar plates or in soil for different time periods, depending on experiments, and then subjected to various stresses (cold, drought, or salinity). The treated plants were harvested and ground with extraction buffer (50 mm NaHPO4 [pH 7.0], 10 mm β-mercaptoethanol, 10 mm Na2EDTA, 0.1% sodium lauryl sarcosine, 0.1% Triton X-100). After centrifugation at 13K rpm for 10 min at 4°C, the supernatants were removed for further GUS assay, essentially according to the protocol of Jefferson et al. (1987), using a DyNA Quant 200 fluorometer (Amersham-Pharmacia Biotech).
Plant Physiol. 2007 February; 143(2): 745–758
RT-PCRTotal RNA was extracted from wild type, aba2, and ABA2 overexpression lines using TRIzol reagent (Invitrogen). Six micrograms of total RNA of each genotype with 1 μg oligo(dT) primer (Invitrogen) were heated at 70°C for 5 min and then chilled on ice immediately. RNA was then subjected to RT with reverse-transcriptase Avian myeloblastosis virus (Roche) at 42°C for 1 h according to the manufacturer's protocol. Synthesized cDNA was used as a template for PCR.
Plant Physiol. 2006 January; 140(1): 302–310
Germination AssayGermination rates were compared between seed lots that were produced, harvested, and stored under identical conditions. Before planting, seeds were surface sterilized with 70% ethanol for 1 min, then with 2% hypochlorite for 5 min, and rinsed five times with sterile deionized water. Fifty to 100 seeds from wild type (Col-0), rgs1-2, gpa1-3, and 35S-RGS1 seeds were stratified at 4°C for 48 h and planted in petri dishes on half-strength Murashige and Skoog (MS) 0.8% phytoagar medium lacking Suc and Gamborg vitamins at 22°C under continuous white light in triplicate. Seeds were considered germinated when the radicles completely penetrated the seed coat.
