Vector backbone integration in transgenic cassava is significantly correlated to T-DNA copy number

Abstract

Multiple T-DNA integrations often occur with transgenic technology, resulting in complex integration patterns and transgene silencing. This study, investigates the correlation coefficient of T-DNA copy number on vector backbone (VBB) integration in transgenic cassava using Dot blot and PCR analysis. Thirty-nine, fifty-one and thirty-eight transgenic cassava plant lines recovered from transformations of cassava friable embryogenic callus with A. tumefaciens strain LBA4404 independently carrying p8016, p8052, and p900 were randomly selected and evaluated for VBB integration and T-DNA copy number. The occurrences of events with low (1-2) and high (≥ 3) T-DNA copy numbers were correlated with the presence and absence of VBB integration. Seventy-two to ninety-eight percent of VBB-free events were low copy number events while 2 to 28% of same where high copy number events. Correlation coefficient of the data revealed that the number of VBB-free events showed a significant positive correlation (r = 0.821, n = 9, p = 0.01) for events with low T-DNA copy number and a significant negative correlation (r = -0.739, n =9, p = 0.02) for high copy number events. This shows that the recovery of events with low T-DNA copy number increases the chances of recovering VBB-free events thereby enhancing the production of quality transgenic events.
 Key words: Copy number, DsRed, T-DNA, transformation, transgenic cassava, vector backbone integration.