Abstract
Plants transformed via Agrobacterium tumefaciens are prone to regenerate a chimera, or mixture, of transformed and non-transformed cells. In this study, we developed a method to identify putative chimeras in transgenic tobacco and apricot plants using quantitative real-time polymerase chain reaction (qPCR). The qPCR assay is based on the assumption that the amount of nptII DNA relative to an internal control (actin) DNA should be constant for solid, non-chimerical transgenics but will vary among tissue samples and over time for a chimerical clone. qPCR was used to estimate the amount of nptII DNA and actin control DNA that was used to normalize the amount of target DNA in each reaction. This method was able to detect putative chimeras when multiple tissue samples were evaluated from the same line. The method was successfully used to monitor the dissociation of chimeras in tobacco plants and apricot callus. Although Southern blot analysis is widely used to confirm transgene integration, we demonstrated here that it cannot detect T-DNA insertion chimeras.
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Claims 4
Claim · model-written summary
qPCR detected putative chimeras when multiple samples from the same line were tested.
- Taxon
- tobacco
Nicotiana tabacume1 - Outcome
- detect putative chimerase0
- Comparator
- multiple tissue samplese0
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“This method was able to detect putative chimeras when multiple tissue samples were evaluated from the same line”
e1“transgenic tobacco and apricot plants”
Claim · model-written summary
qPCR monitored chimera dissociation in tobacco plants.
- Taxon
- tobaccoNicotiana tabacume0
- Outcome
- monitor the dissociation of chimerase0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The method was successfully used to monitor the dissociation of chimeras in tobacco plants”
Claim · model-written summary
qPCR monitored chimera dissociation in apricot callus.
- Taxon
- apricot
Prunus armeniacae0 - Organ / stage
- calluse0
- Outcome
- monitor the dissociation of chimerase0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“The method was successfully used to monitor the dissociation of chimeras in tobacco plants and apricot callus”
Claim · model-written summary
Southern blot analysis did not detect T-DNA insertion chimeras.
- Taxon
- tobaccoNicotiana tabacume1
- Outcome
- cannot detect T-DNA insertion chimerase0
Not reportedComparatorValue
Organisms pictured · Representative pictures of the matched species, not figures from the paper.
Verbatim
e0“we demonstrated here that it cannot detect T-DNA insertion chimeras”
e1“transgenic tobacco and apricot plants”