Antisense RNA Inhibition of uidA Gene Expression in Transgenic Plants: Evidence for Interaction Between First and Second Transformation Events

Abstract

The uidA reporter gene encoding β-glucuronidase (GUS) driven by the 35s promoter was introduced into Nicotiana tabacum using a bipartite vector system based on Agrobacterium tumefaciens strain A281. Four primary transformants showing high levels of uidA expression were transformed with a binary vector in which the uidA structural gene was in antisense orientation to the CaMV 35s promoter. The retransformants from each primary transformant showed reduction in GUS activity varying from 5 to 99 %. The average reduction between retransformants varied significantly from 44 to 93 %. Southern blot analysis showed the presence of the sense uidA gene in high copy number (10–20) in both primary and secondary transformants. The secondary transformants contained, in addition, 15–60 copies of the antisense uidA construct. Antisense uidA bands resulting from direct or inverted repeats and truncated forms could be provisionally identified. The banding pattern was characteristic for each family, indicating an interaction between the first and second transformation events. Seedlings raised by selling the primary transformants showed monohybrid segregation for the hygromycin resistance marker; seed set from the antisense plants was disturbed, and of the I1 plants setting sufficient seed for analysis, 6 showed dihybrid segregation for the kanamycin resistance marker.