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.