Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation
<p>Abstract</p> <p>Background</p> <p>The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the prese...
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BMC
2010-07-01
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Series: | BMC Biotechnology |
Online Access: | http://www.biomedcentral.com/1472-6750/10/53 |
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author | Burgos Lorenzo Faize Lydia Faize Mohamed |
author_facet | Burgos Lorenzo Faize Lydia Faize Mohamed |
author_sort | Burgos Lorenzo |
collection | DOAJ |
description | <p>Abstract</p> <p>Background</p> <p>The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.</p> <p>Results</p> <p>We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (<it>npt</it>II) transgene as well as of an internal control (β-<it>actin</it>), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the <it>npt</it>II transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the <it>gus </it>marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus.</p> <p>Conclusions</p> <p>We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be extended to monitor the dissociation of chimeras and the recovery of uniformly-transformed plants.</p> |
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spelling | doaj.art-7500e5a022084dbf933a9c3ef69bee4e2022-12-22T03:06:26ZengBMCBMC Biotechnology1472-67502010-07-011015310.1186/1472-6750-10-53Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociationBurgos LorenzoFaize LydiaFaize Mohamed<p>Abstract</p> <p>Background</p> <p>The routine generation of transgenic plants involves analysis of transgene integration into the host genome by means of Southern blotting. However, this technique cannot distinguish between uniformly transformed tissues and the presence of a mixture of transgenic and non-transgenic cells in the same tissue. On the other hand, the use of reporter genes often fails to accurately detect chimerical tissues because their expression can be affected by several factors, including gene silencing and plant development. So, new approaches based on the quantification of the amount of the transgene are needed urgently.</p> <p>Results</p> <p>We show here that chimeras are a very frequent phenomenon observed after regenerating transgenic plants. Spatial and temporal analyses of transformed tobacco and apricot plants with a quantitative, real-time PCR amplification of the neomycin phosphotransferase (<it>npt</it>II) transgene as well as of an internal control (β-<it>actin</it>), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the <it>npt</it>II transgene differed greatly along with the sub-cultivation period of these plants and was dependent on the localisation of the analysed leaves; being higher in roots and basal leaves, while in the apical leaves it remained at lower levels. These data demonstrate that, unlike the use of the <it>gus </it>marker gene, real-time PCR is a powerful tool for detection of chimeras. Although some authors have proposed a consistent, positive Southern analysis as an alternative methodology for monitoring the dissociation of chimeras, our data show that it does not provide enough proof of uniform transformation. In this work, however, real-time PCR was applied successfully to monitor the dissociation of chimeras in tobacco plants and apricot callus.</p> <p>Conclusions</p> <p>We have developed a rapid and reliable method to detect and estimate the level of chimeras in transgenic tobacco and apricot plants. This method can be extended to monitor the dissociation of chimeras and the recovery of uniformly-transformed plants.</p>http://www.biomedcentral.com/1472-6750/10/53 |
spellingShingle | Burgos Lorenzo Faize Lydia Faize Mohamed Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation BMC Biotechnology |
title | Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation |
title_full | Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation |
title_fullStr | Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation |
title_full_unstemmed | Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation |
title_short | Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation |
title_sort | using quantitative real time pcr to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation |
url | http://www.biomedcentral.com/1472-6750/10/53 |
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