Detection of transgene copy number by analysis of the T1 generation of tobacco plants with introduced P3 gene of potato virus A.
Real-time PCR, namely the deltadeltaCt method was used to determine the relative copy number of Potato virus A (PVA) P3 gene in the genome of the T1 generation of 18 transgenic tobacco lines. These results were compared with segregation ratios of kanamycin (Km)-resistant phenotype in T1 plants of each line and were found to be, in general, concordant. All the five lines with the Mendelian segregation ratio of 3:1 carried one gene copy. In 12 of 13 lines with uneven segregation more inserted gene copies were detected. Only for one line the real-time PCR and phenotype segregation differed. According to our results the real-time PCR of T1 generation may be used as supplementary method of estimation of the number of transgene copies in the case of nonavailability of the original T0 plants.
Subr
Z
Z
Institute of Virology, Slovak Academy of Sciences, Bratislava, Slovak Republic. virusubr@savba.sk
Nováková
S
S
Drahovská
H
H
D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D018628:Gene Dosage; D005814:Genes, Viral; D030821:Plants, Genetically Modified / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods; D017800:Potyvirus / Q000235:genetics*; D014026:Nicotiana / Q000235:genetics*; D019076:Transgenes; D017361:Viral Nonstructural Proteins / Q000235:genetics
D004279:DNA, Viral; D017361:Viral Nonstructural Proteins