High-frequency linkage of co-expressing T-DNA in transgenic Arabidopsis thaliana transformed by vacuum-infiltration of Agrobacterium tumefaciens

The efficiency of co-expression and linkage of distinct T-DNAs present in s eparate Agrobacterium tumefaciens was analysed in Arabidopsis thaliana tran sformed by the vacuum infiltration method. Co-expression was monitored by t he synthesis of three bacterial proteins involved in the production of poly hydroxybutyrate (PHB) in the plastids. Out of 80 kanamycin-resistant transg enic plants analysed, 13 plants were co-transformed with the two distinct T -DNAs and produced PHB. Of those, 7 lines had a kanamycin-resistance segreg ation ratio consistent with the presence of a single functional insert. Gen etic linkage between the distinct T-DNAs was demonstrated for all 13 PHB-pr oducing lines, while physical linkage between the distinct T-DNAs was shown for 12 out of 13 lines. T-DNAs were frequently linked in an inverted orien tation about the left borders. Transformation of A. thaliana by the co-infi ltration of two A. tumefaciens containing distinct T-DNAs is, thus, an effi cient approach for the integration and expression of several transgenes at a single locus. This approach will facilitate the creation and study of nov el metabolic pathways requiring the expression of numerous transgenes.