PCR Verification and Quantification of Transgenic Citrus

Initial verification of transgenic status was performed on in vitro leaves. Leaves were harvested from putative transgenic plants and non-transgenic control and verified by PCR utilizing the Extract-N-Amp™ Plant PCR Kit (Sigma-Aldrich, St. Louis, MO) and gene-specific primers [42 ]. Total RNA was isolated utilizing the RNeasy Mini Kit (Qiagen Inc. Valencia, CA) according to the manufacturer’s protocol. A 1-μg aliquot of RNA was reverse transcribed using RevertAid First-Strand cDNA Synthesis Kit (Thermo Fisher Scientific Inc). Real-time quantitative PCR (qPCR) was performed using the StepOne Plus system (Thermo Fisher Scientific Inc) utilizing SYBR Green reagents. The relative mRNA levels were compared to those of the Citrus sinensis glyceraldehyde-3-phosphate dehydrogenase (GAPC) gene [43 (link)] and calculated using the 2^-ΔΔCT method [44 ]. Citrus genomic DNA from one-year-old transgenic trees and non-transgenic control was isolated using the PureLink™ Genomic Plant DNA Purification Kit (Thermo Fisher Scientific Inc). Transgene presence was re-confirmed by PCR. The transgene copy number in the selected lines was also validated using qPCR, essentially as previously described [45 , 46 ]. All primer sequences used in this study are listed in Table 1.