ISHS Acta Horticulturae 689: VII International Symposium on Grapevine Physiology and Biotechnology
EVALUATION OF EXOGENOUS DNA BY QUANTITATIVE REAL-TIME PCR IN TRANSGENIC GRAPE |
| Authors: | F. Savazzini, L. Dalla Costa, L. Martinelli |
| Keywords: | genetic transformation, Real-time PCR, Vitis |
| Abstract:
Quantitative Real-time PCR has proven to be a powerful tool to accurately estimate transgene copy number and exogenous gene expression in plants. Compared to Southern and Northern blot analysis, accuracy, rapidity and low-cost are the main advantages. However, this technique requires important preliminary work for standardizing and optimizing the many parameters involved in the analysis. Grapevine data on this topic are not presently available in the literature. We have developed a method for properly performing quantitative assays in grapes where the genes for the β-glucoronidase (GUS) under the control of the 35S promoter and for the neomicin phosphotransferase (NPTII) were transferred. Validation of our method was verified with Southern blot analysis comparison. The optimal endogenous genes to be applied as referee on grape were exploited and nine-cis-epoxycarotenoid dioxygenase2 (NCED2) resulted the most amenable. For transgene copy number detection, the approach based on synthetic hybrid amplicons proved to be promising. | |
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