ANTISENSE RNA INHIBITION OF UIDA GENE-EXPRESSION IN TRANSGENIC PLANTS- EVIDENCE FOR INTERACTION BETWEEN 1ST AND 2ND TRANSFORMATION EVENTS

The uidA reporter gene encoding beta-glucuronidase (GUS) driven by the 35S promoter was introduced into Nicotiana tabacum using a bipartite vector system based on Agrobacterium tumefaciens strain A281. Four pri mary transformants showing high levels of uidA expression were transfo rmed with a binary vector in which the uidA structural gene was in ant isense orientation to the CaMV 35S promoter. The retransformants from each primary transformant showed reduction in GUS activity varying fro m 5 to 99 %. The average reduction between retransformants varied sign ificantly 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 ad dition, 15-60 copies of the antisense uidA construct. Antisense uidA b ands resulting from direct or inverted repeats and truncated forms cou ld be provisionally identified. The banding pattern was characteristic for each family, indicating an interaction between the first and seco nd transformation events. Seedlings raised by selfing the primary tran sformants showed monohybrid segregation for the hygromycin resistance marker; seed set from the antisense plants was disturbed, and of the 1 1 plants setting sufficient seed for analysis, 6 showed dihybrid segre gation for the kanamycin resistance marker.