2024-03-29T12:42:18Zhttp://digital.csic.es/dspace-oai/requestoai:digital.csic.es:10261/336572021-12-28T16:36:51Zcom_10261_72com_10261_6col_10261_325
2011-03-22T09:17:12Z
urn:hdl:10261/33657
Using quantitative real-time PCR to detect chimeras in transgenic tobacco and apricot and to monitor their dissociation
Faize, Mohamed
Faize, Lydia
Burgos Ortiz, Lorenzo
Gobierno de la Región de Murcia
Comisión Interministerial de Ciencia y Tecnología, CICYT (España)
Ministerio de Educación (España)
8 páginas, 6 figuras.-- Metodología.
[Background]: 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.
[Results]: 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 (nptII) transgene as well as of an internal control (β-actin), used to normalise the amount of target DNA at each reaction, allowed detection of chimeras at unexpected rates. The amount of the nptII 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 gus 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.
[Conclusions]: 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.
2011-03-22T09:17:12Z
2011-03-22T09:17:12Z
2010-07-16
artículo
BMC Biotechnology 10: 53 (2010)
1472-6750
http://hdl.handle.net/10261/33657
10.1186/1472-6750-10-53
http://dx.doi.org/10.13039/501100007273
http://dx.doi.org/10.13039/501100009569
20637070
eng
Publisher’s version
http://dx.doi.org/10.1186/1472-6750-10-53
openAccess
BioMed Central