Proceedings of the XLV Italian Society of Agricultural Genetics - SIGA Annual Congress

Salsomaggiore Terme, Italy - 26/29 September, 2001

ISBN 88-900622-1-5

Poster Abstract

REAL-TIME QUANTITATIVE PCR: A NOVEL TENTATIVE APPROACH FOR EVALUATING THE NUMBER OF INTEGRATION EVENTS IN TRANSFORMED PLANTS EARLY AFTER REGENERATION

MASON G.*, ACCOTTO G.P.*, NERVO G.**, VAIRA A.M.*

*Istituto di Fitovirologia Applicata, CNR, Strada delle Cacce 73, 10135 Torino, Italy

a.vaira@ifa.to.cnr.it

**Istituto Sperimentale per l’Orticoltura, MIPAF, Via Paullese 28, 26836 Montanaso Lombardo (LO), Italy

Real-Time PCR, transgenic plants, copy number, quantitative PCR

When new transgenic plants are obtained following genetic transformation, the putative transformed lines must be checked for the presence of the new genes and, as soon as possible, the number of integrated copies must be estimated. This is normally achieved by Southern analysis, a time-consuming technique which requires several days and several micrograms of plant DNA. In order to overcome these two major constraints and therefore obtain information on the number of integration events in each line early after regeneration, we have tested a novel approach by using a quantitative Real-Time PCR assay. The rationale of the approach consists in the possibility of estimating the transgene copy number of a new plant line by comparison with another line which contains a known number of integrated copies. Two genes must be quantified in parallel experiments, a plant gene and the transgene. This is achieved using PCR reagents (primers and probe) specific for the plant gene, and others specific for the transgene.

The plant material used consisted of tomato lines previously transformed with the N gene of Tomato spotted wilt virus: primers and probe specific for a tomato gene (APX), and for the N transgene were designed. As an alternative to the APX-specific reagents, generic plant reagents (t-Leu) were also tested for the quantification of the plant DNA. Preliminary results showed that discrimination between one and two or more integration events is possible, when accurate data are obtained. To improve the accuracy of the measurements a duplex approach, where the two genes are simultaneously quantified in the same reaction tube, is under investigation and data will be presented. The Real-Time quantitative PCR technique requires much less plant DNA than Southern analysis, and therefore it can be performed early after regeneration, when the availability of plant material is limited. Furthermore, it is completed in a few hours. At the present time, however, it cannot be considered as a complete substitute of Southern blots. This new technique can be used for selecting early and rapidly the lines which are probably more interesting than others in the breeding procedures.