A bidirectional enhancer cloning vehicle for higher plants.

We have constructed an enhancer cloning vehicle for higher plants, which is based on Agrobacterium tumefaciens T-DNA-mediated transformation of Nicotiana tabacum leaf disk segments. The binary vector plasmid, pROA97, contains two genes in a divergent transcription pattern, which are separated by a unique Bg/II cloning site. Both genes carry abbreviated TATA regions from the cauliflower mosaic virus 35S promoter region. The neomycin phosphotransferase (NPT II) gene is used as a selectable marker in callus tissue cultured cells. Expression from the other gene encoding β-glucuronidase (GUS) can be monitored in regenerated plant tissues by fluorimetric and histochemical assays. Here we describe the transformation properties of the empty vector and control constructs with either the 35S enhancer or the nopaline synthetase promoter region inserted into the unique Bg/II site. A construct carrying the 35 S − 46 to + 8 TATA region is incapable of allowing callus production on selectable media; a construct carrying the 35 S −90 to + 8 TATA region linked to the NPT II gene coding sequence is sufficient to give transformed callus cells and regenerated transgenic plants. There is a high correlation between expression levels of the two genes in this bidirectional system. The vectors were designed so that the enhancer sequences could be rescued from the plant genome by a rapid marker rescue protocol.