Estimating the number of integrations in transformed plants by quantitative real-time PCR

Abstract

Background: When generating transformed plants, a first step in their characterization is to obtain, for each new line, an estimate of how many copies of the transgene have been integrated in the plant genome because this can deeply influence the level of transgene expression and the ease of stabilizing expression in following generations. This task is normally achieved by Southern analysis, a procedure that requires relatively large amounts of plant material and is both costly and labour-intensive. Moreover, in the presence of rearranged copies the estimates are not correct. New approaches to the problem could be of great help for plant biotechnologists.

Results: By using a quantitative real-time PCR method that requires limited preliminary optimisation steps, we achieved statistically significant estimates of 1, 2 and 3 copies of a transgene in the primary transformants. Furthermore, by estimating the copy number of both the gene of interest and the selectable marker gene, we show that rearrangements of the T-DNA are not the exception, and probably happen more often than usually recognised.

Conclusions: We have developed a rapid and reliable method to estimate the number of integrated copies following genetic transformation. Unlike other similar procedures, this method is not dependent on identical amplification efficiency between the PCR systems used and does not need preliminary information on a calibrator. Its flexibility makes it appropriate in those situations where an accurate optimisation of all reaction components is impossible or impractical. Finally, the quality of the information produced is higher than what can be obtained by Southern blot analysis.

Figure 1

Real-time PCR amplification and standard curve of apx gene. Upper panel: real-time PCR logarithmic plot resulting from the amplification of four three-fold serial dilutions of a tomato standard DNA (see Methods for details) using the apx-specific primers and probe described in Table 5. Each sample was run in triplicate. Lower panel: Standard curve obtained for the same samples. Correlation coefficient and slope values are indicated. The calculated TC values were plotted versus the log of each starting quantity.

Figure 2

Real-time PCR amplification and standard curve of TSWV-N transgene. Upper panel: real-time PCR logarithmic plot resulting from the amplification of four three-fold serial dilutions of a tomato standard DNA (see Methods for details) using the TSWV-N-specific primers and probe described in Table 5. Each sample was run in triplicate. Lower panel: Standard curve obtained for the same samples. Correlation coefficient and slope values are indicated. The calculated TC values were plotted versus the log of each starting quantity.

Figure 3

Real-time PCR amplification and standard curve of nptII transgene. Upper panel: real-time PCR logarithmic plot resulting from the amplification of four three-fold serial dilutions of a tomato standard DNA (see Methods for details) using the nptII-specific primers and probe described in Table 5. Each sample was run in triplicate. Lower panel: Standard curve obtained for the same samples. Correlation coefficient and slope values are indicated. The calculated TC values were plotted versus the log of each starting quantity.