A convenient set of vectors for expression of multiple gene combinations in plants

A set of vectors has been developed that simplify shuttling expression cassettes between small plasmids of high copy number ideal for experiments involving biolistic transient expression and a binary transformation plasmid. Three cassettes for the expression of a cloned coding sequence behind different promoters have been modified; combinations of these cassettes can be excised with Not I, and sequentially cloned into the transformation vector in a procedure that removes the first cloning site. The system is demonstrated by inducing anthocyanin synthesis with paired regulatory genes of maize biolistically delivered to a maize cell suspension, and then expressed in transformed tobacco. Acommon problem encountered in assembling chimeric genes in plasmids is finding enough unique restriction sites for cloning. If an expression cassette needs to be excised and introduced into another vector (such as a binary vector) for transfer to plant cells via Agrobacterium, the procedure can become complicated with the possibility of restricting the gene during the cloning process. These problems are compounded if a binary vector carrying t wo expression cassettes is constructed, with the added possibility of restricting within the first introduced cassette during the second cloning. One solution has been to engineer chimeric genes into separate Ti binary vectors, transform plants separately, and then perform crosses with the different transgenic plants (Lloyd et al., 1992). Another approach created a family of combinatorial cloning vectors that facilitate the multimerization of expression cassettes into a modified pBluescript vector (Lonsdale et al., 1995). We have developed an approach in which combinations of chimeric genes constructed in a family of expression cassettes can be directly assembled into a modified binary vector with minimal risk of restriction.