To create transgenic plants overexpressing BrGRF8, the full-length cDNA was amplified by PCR with the primers BrGRF8-forward (5′-CGGGATCCATGATGAACCTAAGTGGAACTAGTG-3′) and BrGRF8-reverse (5′-TAGTCGACTCAGCTACCAGTGTCGAGTCTTGAC-3′). The underlined sequences represent restriction sites for BamHI and SalI, respectively. The amplified DNA fragments were cloned into the pGEM-T easy vector (Promega, Madison, WI, USA) and sequenced using an ABI 3730 × l DNA Sequencer (Applied Biosystems, Foster City, CA, USA). Then, the BrGRF8 cDNA in the pGEM-T easy vector was double-digested with BamHI and SalI and subcloned downstream of the CaMV 35S promoter into the binary vector pCAMBIA2300-35S-OCS to construct the plasmid CaMV35S:BrGRF5 (35S:BrGRF8). The pCAMBIA2300-35S-OCS vector without the BrGRF8 cDNA insert was used as a negative vector control. The 35S:BrGRF8 and vector control constructs were transformed separately into Agrobacterium tumefaciens LBA4404 and then the A. tumefaciens-mediated transformation of Arabidopsis was performed via vacuum infiltration
[30 ]. The transgenic plants were screened on 1/2 MS agar plates containing 50 mg/ml kanamycin sulfate, and further verified by reverse transcription PCR. Homozygous T3 generation transgenic plants were used for subsequent analyses.
[30 ]. The transgenic plants were screened on 1/2 MS agar plates containing 50 mg/ml kanamycin sulfate, and further verified by reverse transcription PCR. Homozygous T3 generation transgenic plants were used for subsequent analyses.