2.7 Evaluation of biuret tolerance in transgenic rice plants over expressing biuret hydrolase
Seeds from each line were sown onto two nylon-mesh floats on the culture solution in the absence of biuret and grown for two days, to achieve uniform germination. Then, one of the two floats were transferred into a new 2-L container that did not contain biuret and the other was transferred into another 2-L container containing 0.3 mmol L-1 biuret. Plants were harvested 7 days after the onset of the biuret treatment. After determining plant heights, leaf blades from the third leaf were used to determine the chlorophyll content, and leaf blades from the second leaf were used for DNA extraction. Chlorophyll was extracted from tissues using 80% aqueous acetone buffered with 2.5 mmol L-1 sodium phosphate buffer (pH 7.8), and its levels were determined according to the method described by Porra, Thompson & Kriedmann (1989). The presence of the transgene was confirmed by the PCR method, using Blend Taq polymerase (Toyobo, Osaka, Japan) and the primers 5´-ATGAAGACACTTTCCAGCGC-3´ and 5´-TGGCAAATGCCTCTCAAGG-3´, and the data for plants not carrying the transgene were omitted from the analysis.