2.5 Transgene expression analysis
Total RNA was extracted from the youngest leaf blade of regenerated T0 plants at their vegetative growth stage, using the Plant Total RNA Extraction Miniprep System (Viogene, Taipei, Taiwan). First-strand cDNA was synthesized from total RNA using an oligo dT primer and ReverTra Ace polymerase (Toyobo, Osaka, Japan). Quantitative real-time RT-PCR was performed in duplicate with the TP850 thermal cycler dice real time system single (Takara Bio, Shiga, Japan), using the THUNDERBIRD® SYBR qPCR Mix (Toyobo, Osaka, Japan) and primers 5´-AGCCGATCAAAAAGGTGCTGTC-3´ and 5´-AATGATATCCCAGCCAGGTTCTCC-3´. The relative expression level was calculated as a ratio to a geometric mean of the expression ofubiquitin and actin . The sequences of primers were 5´-AGAAGGAGTCCACCCTCCACC-3´ and 5´-GCATCCAGCACAGTAAAACACG-3´ forubiquitin and 5´-ATCCTTGTATGCTAGCGGTCGA-3´ and 5´-ATCCAACCGGAGGATAGCATG-3´ for actin.