REDUCED VARIATION IN TRANSGENE EXPRESSION FROM A BINARY VECTOR WITH SELECTABLE MARKERS AT THE RIGHT AND LEFT T-DNA BORDERS

A new binary vector for Agrobacterium-mediated plant transformation wa s constructed, in which two selectable markers, for kanamycin and hygr omycin resistance, were placed next to the right and left T-DNA border s, respectively, and a CaMV 35S promoter-driven beta-glucuronidase (GU S) gene was placed between these markers as a reporter gene (transgene ). Using double antibiotic selection, all transgenic tobacco plants ca rrying at least one intact copy of the T-DNA expressed the transgene, and this population exhibited reduced variability in transgene express ion as compared with that obtained from the parent vector pBI121. Abse nce of the intact transgene was the major reason for transgenic plants with little or no transgene expression. Integration of truncated T-DN As was also observed among transgenic plants that expressed the transg ene and carried multiple T-DNA inserts. The copy number of fully integ rated T-DNAs was positively associated with transgene expression level s in R(0) plants and R(1) progeny populations. Variability due to posi tion effect was determined among 17 plants carrying a single T-DNA ins ert. The coefficient of variability among these plants was only 35.5%, indicating a minor role for position effects in causing transgene var iability. The new binary vector reported here can therefore be used to obtain transgenic populations with reduced variability in transgene e xpression.