Estimation of transgene copy number in transformed citrus plants by quantitative multiplex real-time PCR

Quantitative real-time PCR(qRT-PCR) was adapted to estimate transgene copy number of the rice Xa21 gene in transgenic citrus plants. This system used TaqMan qRT-PCR and the endogenous citrus gene encoding for lipid tansfer protein(LTP). Transgenic 'Hamlin' sweet orange plants were generated using two different protoplast-GFP transformation systems: cotransformation and single plasmid transformation. A dilution series of genomic DNA from one of the transgenic Xa21. This standard curve was used for relative quantification of the endogenous gene and the transgene. Copy number of the transgene Xa21 detected from qRT-PCR analysis correlated with that from Southern blot analysis (r=0.834). Thus, qRT-PCR is an efficient means of estimating copy number in transgenic citurs plants. This analysis can be performed at much earlier stages of transgenic plant development than southern blot analysis, which expedites investigation in slow-growing woody plants.