Rice Genetics IV - IRRI books - International Rice Research Institute

Materials and methodsPlant materials and growth conditionsSeeds of two rice varieties (Pokkali and IR29) were obtained from the InternationalRice Research Institute, Los Baños, Philippines. Maize (Zea mays L., B73) was providedby Dr. Vicky Chandler (University of Arizona) and barley (Hordeum vulgareL., Tokak) by Dr. Nermin Gozukirmizi (Marmara Research Center, Turkey). Mesembryanthemumcrystallinum L. was grown as described (Adams et al 1998). Maizeplants were grown on a potting mixture for 4 wk and then treated with 150 mM NaClusing water as a control.After imbibition, rice seeds were transplanted into Hoagland’s solution, with theamount of iron doubled, in hydroponic tanks. Plants were grown at 28/25 °C (50%humidity, 12 h light/dark at 700 µmol photons m –2 s –1 ). Plants were used when theroots and shoot measured approximately 7 and 10 cm, respectively. Salt stress began3 h after the start of the light period by adding 150 mM NaCl in diluted (1/4) Hoagland’ssolution, which provided external calcium at 1 mM. Roots and leaves were harvested,frozen in liquid nitrogen, and kept at –80 °C.Barley seeds were germinated in sand supplied with 0.3X Hoagland’s nutrientsolution. The plants were grown at 28/23 °C (80–85% relative humidity, 12 h dark/light at 500 µmol photons m –2 s –1 ). At age 3 wk, plants were removed from the sandand placed on a bench for drying at 80–85% relative humidity and light intensity asabove. Leaf and root samples were collected after 6 and 10 h of drying. Control plantsgrown in sand were harvested at the same times.Physiological analysisNet CO 2 assimilation, stomatal conductance, and transpiration rates were measuredwith attached leaves at saturating light intensity (1,000 µmol photons m –2 s –1 ) at28 °C using an infrared gas analyzing system (Li-6400, Li-cor). Data were collectedtwice for each time-course experiment. Physiological parameters were measured beforeand after salt additions.RNA isolation and cDNA library constructioncDNA libraries were constructed from the plant species and tissues listed in Table 1.Total root RNA and poly(A + ) RNA were isolated and cDNA libraries were generated(Stratagene) with Escherichia coli XL1-Blue MRF as the host. Inserts cloned intoBluescript SK+ were sequenced from the 5’ ends. Sequences were annotated acceptingrice ESTs included in public databases for transcripts that showed more than 95%identity to the Pokkali ESTs.Preparation of DNA microarraysPCR amplification (40 cycles, annealing at 55 °C) was performed in a 96-well formatwith individual colonies or 1 µL of plasmid DNA as templates, using T3 and T7primers with amino-modified ends. PCR products were combined with 100 µL ofbinding solution (150 mM potassium acetate, pH 4.8, 7 M guanidine hydrochloride)Isolation of candidate genes for tolerance of abiotic stresses 347