Journal
PLANT CELL REPORTS
Volume 20, Issue 10, Pages 948-954Publisher
SPRINGER
DOI: 10.1007/s00299-001-0432-x
Keywords
quantitative real-time polymerase chain; reaction; transgene copy number; maize transformation; WHISKERS (TM) transformation; transgene insertion complexity
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Quantitative real-time PCR (qRT-PCR) was adapted to estimate transgene copy number in transgenic maize callus and plants. WHISKERS(TM)-derived transgenic callus lines and plants were generated using two different gene constructs. These transgenic materials represented a range of copy number. A 'standard curve' was established by mixing plasmid DNA with non-transgenic genomic maize DNA using a calculated ratio of target gene to host genome size. 'Estimated' copy number in the callus lines and plants using qRT-PCR was correlated with the 'actual' copy number based on Southern blot analysis. The results indicated that there was a significant correlation between the two methods with both gene constructs. Thus, qRT-PCR represents an efficient means of estimating copy number in transgenic maize.
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