Ihuoma Okwuonu
Ihuoma Okwuonu, Head Tissue culture & genetic transformation unit, Biotechnology programme, NRCRI Umudike, Nigeria
The effect of transgene copy number on vector backbone (VBB) integration in transgenic cassava genome was evaluated using Dot blot hybridization and confirmed by PCR analysis. Transgenic cassava events produced by transforming cassava friable embryogenic callus (FEC) with Agrobacterium LBA4404 strains independently carrying three different constructs p8016, p8052 and p900 were randomly selected and DNA samples extracted from these events were probed with specific neomycin phosphotransferase (nptII) probe. Copy number was measured based on density of the blot signal using Image J software. The occurrences of low and high copy number events were correlated with the total number of VBB integrated and VBB free events. Correlation coefficient of the data revealed that the number of VBB integrated events and the occurrences of low copy and high copy number events were less significant (r=0.396, n =5, P<0.05) and the correlation coefficient between number of VBB free events and occurrences of low and high copy number shows a high positive significance (r = 0.7708, n =5, p<0.05) for low copy number and a high negative significance on (r = -0.891, n =5, p<0.05) for high copy number. This study shows that the recovery of low copy number events increases the chances of recovering VBB free events. This means that strategies that favors recovery of low copy number events invariable favors the recovery of VBB free events.
Keywords: Vector backbone, transgenic cassava, copy number, Agrobacterium transformation.