The 2012 ASHS Annual Conference

Numerous 2-4 year old transgenic scion grapefruit and sweet orange and rootstock carrizo transgenic lines obtained via Agrobacterium-mediated transformation were evaluated by a Taqman based real-time polymerase chain reaction (qPCR) assay. We evaluated the expression levels of the inserted transgene (putative disease resistance or insecticidal gene) by quantifying messenger RNA (mRNA) levels and also estimated the copy number in each on these lines. The transcript levels from both the gene of interest and egfp reporter gene was determined. Expression levels varied in the individual lines and could be grouped into high, medium, and low levels of mRNA expression. There was no variation in mRNA expression levels from numerous clones of several selected transgenic lines. The copy number was calculated by comparing threshold cycle (CT) values of the gene of interest and egfp genes with those of the endogenous reference genes (18S rRNA or cytochrome oxidase (COX)). In most cases the copy number of the gene of interest was identical to that of egfp. A majority of the lines contained 1–4 copies of the transgene. No direct relationship between copy number and expression level of transgenes was obvious in a majority of the transgenic lines evaluated, suggesting that differential expression could be due to factors like rearrangements of the T-DNA in the genome, DNA methylation or position effects. Results obtained from qPCR were in agreement with that observed via Southern blotting indicating its potential for the rapid estimation of copy numbers from a large number of putative transgenic lines. Our results indicated that the transgenes were stable even after 4 years in the greenhouse.