Table I. Transgene integration and fatty acid contents of the first-round SSI events
EventaGene Copy No. by qPCRbFatty Acid Content by GCc
SSIDonorTargetFLP16:018:018:118:218:3
B511.00.00.00.24.02.150.825.617.6
B520.80.00.00.912.63.218.047.119.1
B530.80.00.00.03.81.451.727.315.8
B541.10.00.00.03.71.257.320.117.8
B53-11.90.00.00.02.42.590.41.03.5
B53-21.90.00.00.02.23.789.30.83.8
B53-30.90.00.00.02.52.390.60.93.4
B53-40.90.00.00.02.82.489.51.73.4
B53-50.00.00.00.013.03.414.455.513.6
B53-60.00.00.00.012.13.411.354.019.1
  • a Events B51 to B54 are representative hygromycin-resistant events selected from the retransformation of homozygous target QC288A329A with the first donor QC436 and FLP QC292 DNA. B53-1 to B53-6 are segregating T1 plants derived from event B53.

  • b Embryogenic callus or T1 plant leaf samples were analyzed by qPCR specific to the SCP1 and HPT junction of QC288A436A (SSI), a QC436-specific region (Donor), the SCP1 and ALS junction of QC288A329A (Target), and a QC292-specific region (FLP). A heat shock protein gene, HSP, was used as an endogenous control in duplex qPCR. A genomic DNA sample containing one copy of the respective transgene was used as the calibrator to calculate relative transgene copies in other samples using the Applied Biosystems 7500 system software. A value of less than 0.3 or between 0.4 and 1.3 was considered as zero or one copy. A value between 1.4 and 2.3 was considered as two copies.

  • c Fatty acids were determined on the bulk of 10 mature somatic embryos for B51 to B54 or on T1 seed chips for B53 by GC and expressed as the percentage of total fatty acids. Fatty acid measurements by GC as described here are reproducible to approximately 3% of total fatty acids. Untransformed control somatic embryos typically contain 12.6% to 20.8% 16:0, 4.2% to 6.6% 18:0, 12.3% to 22.9% 18:1, 39.3% to 46.9% 18:2, and 12.4% to 23.5% 18:3 (Meyer et al., 2008). Increases in 18:1 to greater than 30% and decreases in 16:0 to less than 10% are indicative of successful FAD2 and FATB gene down-regulation, respectively. The chipped B53 seeds later germinated to T1 plants that provided leaf DNA for the qPCR assay.